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Genotoxic effect of bile acids on human normal and tumour colon cells and protection by dietary antioxidants and butyrate
Abstract
Colorectal cancer is the second cause of death for tumour worldwide. Among the risk factors for this disease the dietary habits seem to have a pivotal role. An elevated intake of fats causes a high release in the gut lumen of bile acids that are positively correlated with colorectal cancer, since they act as detergents and proliferation promoters. Recently, it was evidenced that bile acids can also be able to induce DNA damage.
In this study the genotoxicity of deoxycholic acid (DCA) and chenodeoxycholic acid CDCA) has been evaluated in human normal colonocytes derived from 60 colon biopsies and in tumour cells. The involvement of reactive oxygen species (ROS) and the oxidative DNA damage was assessed. In addition, the protective effect exerted by both two well-known antioxidants commonly present in the diet, beta-carotene and alpha-tocopherol, and butyrate which is known to be involved in the regulation of several cellular functions, has also been tested.
The DNA damage was evaluated by the "comet assay" or single cell gel electrophoresis (SCGE) both in its conventional use and by the Endonuclease III modified method, which allow to detect the presence of oxidized pyrimidines.
Bile acids (CDA and CDCA) resulted genotoxic on both normal and tumour human colon cells. The inclusion of the endonuclease III digestion step in the comet assay demonstrated that bile acids induced an oxidative DNA damage. In addition, treatment of colonocytes with bile acids in the presence of the antioxidants (beta-carotene, alpha-tocopherol) and Na-butyrate caused a reduction of DNA damage.
Our results suggest that bile acids may be involved in the tumour initiation by inducing a DNA oxidative damage, and so add further evidences to the preventive properties of antioxidants present in the Mediterranean diet.
... However, for BA-FXR interaction the rank order of potency is estimated to be CDCA > LCA = DCA > CA both in the conjugated and unconjugated forms 18 and for BA-GPBAR-1 interaction the rank order of potency is estimated to TLCA > TDCA > TCDCA > TCA. 13 Thus, subtle quantitative or qualitative perturbations of the BA pool may greatly affect several BA physiological functions in the body. 1 Abnormalities in BA synthesis, secretion, absorption and local and systemic effects have been implicated during inflammation, 16,19 metabolic disorders, 16 liver diseases, 19,20 and many other conditions. 21,23 BAs play also a crucial role as potential cancer-promoting agents [24][25][26][27] and in regulating the proliferation of cancer cells of diverse origin. [28][29][30][31] A causal relationship between BAs (in particular DCA, one of the components of the human BA pool) and cancer was firstly proposed in 1940. ...
... While hydrophilic, less cytotoxic BAs play a protective role 71 on gastrointestinal [75][76][77][78][79] and liver 80,81 cells, hydrophobic BAs can be cytotoxic and can generate oxidative stress and DNA damage (genomic instability), which is a predisposing factor for cancer. 24 The main general mechanisms involved are the increased intracellular production of reactive oxygen and nitrogen species, 24,27,82 and the altered expression of tumour suppressor/promoting genes. 47,83,84 CDCA (chenodeoxycholic acid) and DCA are able to solubilise the cell membrane and to promote immuno-suppression and tissue damage. ...
... 87,88 Since unconjugated BAs are produced by intestinal microbiota, the direct negative effects are mainly due to the high concentrations reached in the gastrointestinal lumen. 27,39,82,83 For example, duodeno-gastro-oesophageal reflux of BA might play a cancer-promoting role both in the stomach 84,89,90 and in the oesophagus, 91,92 and local pH is involved in this process. ...
Bile acids (BAs) regulate the absorption of fat-soluble vitamins, cholesterol and lipids but have also a key role as singalling molecules and in the modulation of epithelial cell proliferation, gene expression and metabolism. These homeostatic pathways, when disrupted, are able to promote local inflammation, systemic metabolic disorders and, ultimately, cancer. The effect of hydrophobic BAs, in particular, can be linked with cancer in several digestive (mainly oesophagus, stomach, liver, pancreas, biliary tract, colon) and extra-digestive organs (i.e. prostate, breast) through a complex series of mechanisms including direct oxidative stress with DNA damage, apoptosis, epigenetic factors regulating gene expression, reduced/increased expression of nuclear receptors (mainly farnesoid X receptor, FXR) and altered composition of gut microbiota, also acting as a common interface between environmental factors (including diet, lifestyle, exposure to toxics) and the molecular events promoting cancerogenesis. Primary prevention strategies (i.e. changes in dietary habits and lifestyle, reduced exposure to environmental toxics) mainly able to modulate gut microbiota and the epigenome, and the therapeutic use of hydrophilic BAs to counterbalance the negative effects of the more hydrophobic BAs might be, in the near future, part of useful tools for cancer prevention and management.
... The inclusion of the endonuclease III digestion step in the comet assay demonstrated that bile acids induced an oxidative DNA damage. On the other side, treatment of colonocytes with bile acids in the presence of the antioxidants caused a reduction of DNA damage [7]. ...
... We can confirm that bile-acid-induced stresses cause cell death in susceptible cells contribute to genomic instability in surviving cells, impose Darwinian selection on survivors and enhance initiation and progression of colon cancer. The most likely major mechanisms by which hydrophobic bile acids induce stresses on cells are: the DNA damage, endoplasmic reticulum stress, and mitochondrial damage [6,7]. Persistent exposure of colon epithelial cells to hydrophobic bile acids can result in the activation of pro-survival stress-response pathways, and the modulation of numerous genes/proteins associated with chromosome maintenance and mitosis. ...
... Persistent exposure of colon epithelial cells to hydrophobic bile acids can result in the activation of pro-survival stress-response pathways, and the modulation of numerous genes/proteins associated with chromosome maintenance and mitosis. The multiple mechanisms by which hydrophobic bile acids contribute to genomic instability include: oxidative DNA damage, p53 and other mutations, micronuclei formation and aneuploidy [6,7]. A new research report suggests that antihistamines have significant anti-cancer properties because they interfere with the myeloid-derived suppressor cells (MDSC) which is known to reduce organism's ability to fight tumors. ...
... Additionally, it was reported that deoxycholic acid induces proteasomal degradation of p53 [84] and activates survival and proliferative pathways such as Wnt/β-catenin [83], PKC [85] and NFKB [85], which egress apoptosis-resistant clones [82,83]. ...
... Additionally, it was reported that deoxycholic acid induces proteasomal degradation of p53 [84] and activates survival and proliferative pathways such as Wnt/β-catenin [83], PKC [85] and NFKB [85], which egress apoptosis-resistant clones [82,83]. ...
The high incidence of colorectal cancer (CRC) in developed countries indicates a predominant role of the environment as a causative factor. Natural gut microbiota provides multiple benefits to humans. Dysbiosis is characterized by an unbalanced microbiota and causes intestinal damage and inflammation. The latter is a common denominator in many cancers including CRC. Indeed, in an inflammation scenario, cellular growth is promoted and immune cells release Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS), which cause DNA damage. Apart from that, many metabolites from the diet are converted into DNA damaging agents by microbiota and some bacteria deliver DNA damaging toxins in dysbiosis conditions as well. The interactions between diet, microbiota, inflammation, and CRC are not the result of a straightforward relationship, but rather a network of multifactorial interactions that deserve deep consideration, as their consequences are not yet fully elucidated. In this paper, we will review the influence of dysbiosis in the induction of DNA damage and CRC.
... L'étude de leur activité sur ce type de lignée n'est peutêtre pas appropriée. En revanche, une étude comparant les dommages à l'ADN de sels biliaires sur des colonocytes prélevés sur sujets sains versus des colonocytes cancéreux (lignée HT29) n'a pas montré de différence significative entre ces lignées (Rosignoli et al., 2008). (Fraser, 2013). ...
... L'augmentation de l'activité catalase observée dans mes travaux lors de l'enrichissement des nano-émulsions en rétinyl acétate pourrait avoir ce même fondement. Enfin, l'α-tocophérol (40 µM) et le β-carotène (3 µM) ont également montré des capacités protectrices contre les dommages à l'ADN induits par les SB sur des colonocytes issus de sujets sains et de la lignée cancéreuse HT29(Rosignoli et al., 2008), sans pour autant que le mécanisme ne soit clairement établit.En conclusion sur ce second chapitre l'étude du pouvoir antioxydant au niveau de la lumière et des cellules intestinales est en plein essor. Les modèles in vitro reproduisant les émulsions digestives se développent, mais très peu se concentrent sur les micelles mixtes intestinales. ...
Selon la FAO, deux milliards de personnes souffrent de la « faim cachée » qui correspond aux carences en micronutriments incluant la carence en vitamine A, principale cause de cécité dans le monde. Par ailleurs, une alimentation excédentaire est source de stress oxydant pour le corps, facteur de risque de nombreuses maladies non transmissibles comme le diabète et les maladies cardiovasculaires. Contenus dans les produits végétaux, certains caroténoïdes représentent une source indirecte de vitamine A et pourraient participer aux effets bénéfiques de la consommation de fruits et légumes via des propriétés antioxydantes. Dans cette thèse, le comportement in vitro des caroténoïdes et rétinoïdes pendant la digestion a été étudié, depuis leur libération de la matrice alimentaire jusqu'à leurs interactions avec les cellules intestinales. Mes recherches s'articulent autour de la micellisation : processus clé de l'absorption des caroténoïdes et rétinoïdes. Dans un premier temps, la stabilité et la bioaccessibilité en digestion in vitro (DIV) de ces composés purs ont été comparées à celles de leurs homologues issus d'un jus de carotte, d'épinards crus et cuits et d'une farine fortifiée. Dans un deuxième temps, un nouveau modèle d'oxydation de nano-émulsions intestinales à base de sels biliaires (SB) a été développé. Les relations entre structure des micelles mixtes, réactivité à l'oxydation et protection par des antioxydants ont été étudiées grâce notamment au suivi de la dégradation des acides gras. Enfin, des expériences préliminaires ont été menées sur la réactivité enzymatique de cellules coliques (Caco-2) à divers oxydants et aux micelles mixtes. Pendant la DIV, le β-carotène et palmitate de rétinyl purs étaient particulièrement instables contrairement à la lutéine et à l'acétate de rétinyl. De plus, la matrice alimentaire protège ces composés et favorise leur bioaccessibilité surtout lorsqu'elle est broyée ou cuite. En effet, les prétraitements ont un impact conséquent sur la libération des caroténoïdes et doivent être considérés comme des moyens sérieux pour optimiser les apports. Les nano-émulsions intestinales mises au point se composaient de micelles mixtes sphériques ou cylindriques, ces dernières étant les moins résistantes à l'oxydation. En effet, la distribution des molécules de SB doit être différente selon la morphologie de la micelle, impactant ainsi leur réactivité. Dans nos conditions expérimentales, l'AAPH fut le seul oxydant efficace. L'α-tocophérol et la lutéine ont significativement ralenti la dégradation des acides gras contrairement au β-carotène. Leur place au sein de la micelle (coeur ou bordure) pourrait expliquer ces observations. Enfin, aucun des oxydants testés n'a significativement modifié l'activité catalase des cellules Caco-2. En revanche, la mise en contact avec les micelles à base de SB ont significativement diminué l'activité des 3 enzymes suivies. L'effet était positivement corrélé à la concentration en SB dont la conjugaison fut un élément déterminant. Finalement, le β-carotène, la lutéine, et l'acétate de rétinyl présents dans ces micelles ont en partie rétabli l'activité de la catalase contrairement au rétinol, palmitate de rétinyl et à l'α-tocophérol.
... This may at least be partly due to the presence of secondary bile acids identified in the sample (Fig. 1). Van Munster et al. (1993) showed that faecal water induced toxicity correlated with the deoxycholate content of the faecal water and other researchers have reported a correlation between bile acid content and the cytotoxicity of faecal water (Barrasa, Santiago-Gómez, Olmo, Lizarbe, & Turnay, 2012;Bernstein et al., 2005;Rosignoli et al., 2008). Cytotoxicity induced by faecal water was significantly (p b 0.05) reduced by inulin (2%) with cell survival increasing by 20% whereas lactulose (2%) had no significant effect on faecal water induced cytotoxicity (Fig. 2c). ...
... The MALDI-TOF-MS spectra confirm the presence of secondary bile acids (lithocholic and deoxycholic) in the faecal water sample (Fig. 1). The presence of secondary bile acids in the faecal water as observed here is widely reported (Barrasa et al., 2012;Bernstein et al., 2005;Rosignoli et al., 2008;Venturi et al., 1997). However, in the present study using the SOS chromotest no genotoxicity was induced by either lithocholic or deoxycholic acids in the absence or presence of a metabolic activator (Fig. 3a-b). ...
... Hence, the gut microbiota profile is closely tied to the BA pool composition and secondary BA levels, which are associated with various diseases (8). For instance, in L2-IL1B mice, treatment with deoxycholic acid (DCA), a DNA-damaging (10) and carcinogenic secondary BA (11,12), induced substantial aggravation of the phenotype (13). ...
Background
The incidence of Barrett esophagus (BE) and Gastroesophageal Adenocarcinoma (GEAC) correlates with obesity and a diet rich in fat. Bile acids (BA) support fat digestion and undergo microbial metabolization in the gut. The farnesoid X receptor (FXR) is an important modulator of the BA homeostasis. The capacity of inhibiting cancer-related processes when activated, make FXR an appealing therapeutic target. In this work, we assess the role of diet on the microbiota-BA axis and evaluate the role of FXR in disease progression.
Results
Here we show that high fat diet (HFD) accelerated tumorigenesis in L2-IL1B mice (BE- and GEAC- mouse model) while increasing BA levels and enriching gut microbiota that convert primary to secondary BA. While upregulated in BE, expression of FXR was downregulated in GEAC in mice and humans. In L2-IL1B mice, FXR knockout enhanced the dysplastic phenotype and increased Lgr5 progenitor cell numbers. Treatment of murine organoids and L2-IL1B mice with the FXR agonist obeticholic acid (OCA) deacelerated GEAC progression.
Conclusion
We provide a novel concept of GEAC carcinogenesis being accelerated via the diet-microbiome-metabolome axis and FXR inhibition on progenitor cells. Further, FXR activation protected with OCA ameliorated the phenotype in vitro and in vivo, suggesting that FXR agonists have potential as differentiation therapy in GEAC prevention.
Statement of significance
If its inhibition is linked to disease progression and its activation to cancer prevention, exploring the potential of FXR as a therapeutic target has great clinical relevance in GEAC context.
... The consumption of pro-oxidants through diet, such as dietary iron, omega-6 fatty acids, and saturated fat, has been linked to an elevation in oxidative stress and DNA damage in the colon. This can be attributed to the increased production of free radicals in the colorectal region [22,[36][37][38][39][40][41][42][43][44]. The findings of a nested case-control study indicated a positive correlation between pre-diagnostic serum oxidized low density lipoprotein concentrations and the risk of colorectal cancer (CRC). ...
Background
The oxidative balance score (OBS) has been utilized to assess the overall pro- and antioxidant exposure status in various chronic diseases. The current meta-analysis was carried out to pool the association between OBS and the risk of cancer.
Methods
We systematically searched the Web of Science, PubMed, Scopus, Embase, and Google Scholar up to August 2023. All observational studies which evaluated the association of OBS with the risk of cancers were included. There was no time of publication or language restrictions. Heterogeneity between studies was assessed using the Chi-square-based Q-test and the I². A random-effects model meta-analysis was conducted to estimate the pooled effect sizes. Possible sources of heterogeneity were explored by subgroup and meta-regression analysis.
Results
Totally, 15 studies (9 case–control and 6 cohorts) were eligible for meta-analysis. Random effect model meta-analysis of case–control studies showed that higher OBS significantly decreases the odds of cancers (pooled OR: 0.64, 95% CI: 0.54, 0.74). In the cohort studies, the association of OBS with the risk of cancers was not significant (pooled HR: 0.97, 95% CI: 0.80,1.18). The subgroup analysis showed that cancer type and gender were the potential sources of heterogeneity.
Conclusion
Our results show an inverse and significant association between higher OBS and odds of colorectal cancers in case–control and cohort studies. In the case of prostate cancer in cohort studies, our results did not align with the hypothesis. Considering the importance of diet and antioxidant balance in the conditions of malignancy, it is suggested to conduct more comprehensive studies with standard measurement methods to obtain conclusive results.
... The genotoxic mechanism of BSs is not yet fully understood. It has been suggested that DNA damage can be caused by the increased production of ROS and RNS via stimulatory effect of BSs on the activation of LOX, cyclooxygenase (COX) and NADPH oxidase (Nguyen, Ung, Kim, & Jung, 2018;Rosignoli et al., 2008). Moreover, BSs due to their surfactant properties can cause mitochondrial damage, leading to the release of ROS generated in the mitochondrial electron transport chain (Nguyen et al., 2018). ...
Oxidation of food-derived phospholipids (PLs) can influence nutrient digestion and induce oxidative stress in gastrointestinal epithelium. In this study, hen egg yolk PL fraction was used to evaluate the effect of lipoxygenase (LOX)-induced PL oxidation on the hydrolysis of PLs catalyzed by pancreatic phospholipase A2 (PLA2) in the presence of bile salts (BSs). Then, PL/BS solutions containing native or oxidized PLs were used in in vitro intestinal digestion to assess the effect of PL oxidation and hydrolysis on the toxicity towards HT29 cell line. Based on the obtained results, we suggest that hexanal and (E)-2-nonenal, formed by the decomposition of PL hydroperoxides, inhibited PLA2 activity. The cell exposure to simulated intestinal fluid (SIF) containing BSs decreased HT29 cell viability and significantly damaged cellular DNA. However, the genotoxic effect was reversed in the presence of all tested PL samples, while the protective effect against the BS-induced cytotoxicity was observed for native non-hydrolyzed PLs, but was not clearly visible for other samples. This can result from an overlap of other toxic effects such as lipotoxicity or disturbance of cellular redox homeostasis. Taking into account the data obtained, it was proposed that the PLA2 activity decline in the presence of PL oxidation products may be a kind of protective mechanism against rapid release of oxidized FAs characterized by high cytotoxic effect towards intestinal epithelium cells.
... In addition, SCFAs upregulate the function of colonic regulatory T cells, which play a role in intestinal homeostasis and gut inflammation [116]. Notably, treatment of colonocytes with butyrate and antioxidants appears to reduce the genotoxicity and DNA damage induced by BAs [117]. ...
Obesity is considered a risk factor for different types of cancer, including colorectal cancer (CRC). Bariatric surgery has been associated with improvements in obesity-related co-morbidities and reductions in overall cancer risk. However, given the contradictory outcomes of several cohort studies, the impact of bariatric surgery on CRC risk appears controversial. Furthermore, measurement of CRC biomarkers following Roux-en-Y gastric bypass (RYGB) has revealed hyperproliferation and increased proinflammatory gene expression in the rectal mucosa. The proposed mechanisms leading to increased CRC risk are alterations of the gut microbiota and exposure of the colorectum to high concentrations of bile acids, both of which are caused by RYGB-induced anatomical rearrangements. Studies in animals and humans have highlighted the similarities between RYGB-induced microbial profiles and the gut microbiota documented in CRC. Microbial alterations common to post-RYGB cases and CRC include the enrichment of pro-inflammatory microbes and reduction in butyrate-producing bacteria. Lower concentrations of butyrate following RYGB may also contribute to an increased risk of CRC, given the anti-inflammatory and anti-carcinogenic properties of this molecule. Laparoscopic sleeve gastrectomy appears to have a more moderate impact than RYGB; however, relatively few animal and human studies have investigated its effects on CRC risk. Moreover, evidence regarding the impact of one anastomosis gastric bypass is even more limited. Therefore, further studies are required to establish whether the potential increase in CRC risk is restricted to RYGB or may also be associated with other bariatric procedures.
... DCA and CDCA induce oxidative DNA damage, resulting in their involvement in tumor initiation, and antioxidants reduce the carcinogenic effects [45]. In addition, DCA increases nitrosative stress, resulting in damage to the cellular membrane and DNA [46]. ...
Background:
Hepatocellular carcinoma (HCC) is one of the most common diseases that threaten millions of lives annually. Evidence supports that bile acid (BA) affects HCC through inflammation, DNA damage, or other mechanisms.
Methods:
A total of 127 BA-associated genes were analyzed in HCC tumor and nontumor samples using The Cancer Genome Atlas data. Genes correlated to the prognosis of patients with HCC were identified using univariate and multivariate Cox regression analyses. Furthermore, a prediction model with identified genes was constructed to evaluate the risk of patients with HCC for prognosis.
Results:
Out of 26 genes with differential expressions between the HCC and nontumor samples, 19 and 7 genes showed upregulated and downregulated expressions, respectively. Three genes, NPC1, ABCC1, and SLC51B, were extrapolated to construct a prediction model for the prognosis of patients with HCC.
Conclusion:
The three-gene prediction model was more reliable than the pathological staging characters of the tumor for the prognosis and survival of patients with HCC. In addition, the upregulated genes facilitating the transport of BAs are associated with poor prognosis of patients with HCC, and genes of de novo synthesis of BAs benefit patients with HCC.
... Secondary bile acids such as DCA are cytotoxic to colon epithelial cells because they inhibit apoptosis, thus increasing the likelihood of carcinogenesis (Bernstein et al., 2005). DCA or hydrophobic bile acid (HBA) may increase oxidative stress (e.g., in reactive oxygen and nitrogen species) and cause DNA damage through mechanisms related to apoptotic factors (Booth, Gilmore, & Bilton, 1997;Huo et al., 2011;Payne, Bernstein, Dvorak, & Bernstein, 2008;Rosignoli et al., 2008). Payne et al. (2008) showed that HBA can induce stress in cells, resulting in oxidative DNA damage, fewer DNA repair proteins such as p53 and BRCA1, other genetic mutations, increased genomic instability, and mitochondrial damage (Payne et al., 2008). ...
Gut microbiota comprise microorganisms residing in the gastrointestinal tract. Some of these microbiota are implicated in the progression of colorectal cancer (CRC). Here, we highlight studies on the effects of meat intake and fermented foods on characteristics of gut microbiota that can influence colitis-associated factors underlying CRC. Gut microbiota can influence the development and progression of CRC, through influencing factors such as secretion of toxins; enzymes for activating carcinogenesis (including β-glucuronidase, β-glucosidase, azoreductase, nitroreductase, and alcohol dehydrogenase); hydrogen sulfide generation; generation of reactive oxygen species and inflammation; secondary bile salt transformation; and products of protein fermentation. Additionally, some studies that the composition of gut microbiota (probiotics) or prebiotics plays an important role in the production of short chain fatty acids, inactivation enzymes for carcinogenesis, antioxidant activities, and inhibition of pathogen colonization. In this review, we discuss various explanatory mechanisms of the relationship between the multifactorial role of the gut microbiota and the development of CRC. Moreover, this review provides fundamental information on dietary fermented food and the gut microbiota, which is helpful for healthy people and those with CRC alike.
... Epidemiological studies, animal models, and clinical studies identify fecal bile acid as a risk factor for colon cancer [6]. High levels of bile acids elicit hazardous influence on colonic mucosa characterized by DNA oxidative damage [7], inflammation [8], and hyperproliferation [9]. Farnesoid X receptor (FXR, translated by the NR1H4 gene), a bile acid-activated nuclear receptor, regulates the expression of target genes involved in lipid, cholesterol, and glucose metabolism [10]. ...
Our previous study indicated that colon cancer cells varied in sensitivity to pharmacological farnesoid X receptor (FXR) activation. Herein, we explore the regulatory mechanism of FXR in colorectal cancer (CRC) development and aim to design effective strategies of combined treatment based on the regulatory axis. We found that the expression of FXR was negatively correlated with enhancer of zeste homolog 2 (EZH2) in colon cancer tissues. EZH2 transcriptionally suppressed FXR via H3K27me3. The combination of FXR agonist OCA plus EZH2 inhibitor GSK126 acted in a synergistic manner across four colon cancer cells, efficiently inhibiting clonogenic growth and invasion in vitro, retarding tumor growth in vivo, preventing the G0/G1 to S phase transition, and inducing caspase-dependent apoptosis. Benign control cells FHC were growth-arrested without apoptosis induction, but retained long-term proliferation and invasion capacity. Mechanistically, the drug combination dramatically accelerated FXR nuclear location and cooperatively upregulated caudal-related homeobox transcription factor 2 (CDX2) expression. The depletion of CDX2 antagonized the synergistic effects of the drug combination on tumor inhibition. In conclusion, our study demonstrated histone modification-mediated FXR silencing by EZH2 in colorectal tumorigenesis, which offers useful evidence for the clinical use of FXR agonists combined with EZH2 inhibitors in combating CRC.
... DCA and CDCA induce oxidative DNA damage, which can then be involved in tumor initiation, and antioxidants could reduce the carcinogenic effects [43]. Additionally, DCA also increases nitrosative stress, resulting in damage to the cellular membrane and DNA [44]. ...
Background. Hepatocellular carcinoma (HCC) is one of the most common diseases, threatening millions of patients annually. Increasing evidence supports that bile acid (BA) has an impact on HCC through inflammation, DNA damage or other mechanisms.
Methods. With the data from The Cancer Genome Atlas portal, a total of 127 BA-associated genes were analyzed in HCC tumor and non-tumor samples, and then, using univariate and multivariate Cox regression, genes with correlations to the prognosis of HCC patients were identified. Then, a prediction model with identified genes was constructed for evaluating the risk of HCC patients for prognosis.
Results. Twenty-six genes with differential expression between HCC and control tissue samples were identified, of which 19 genes had up-regulated expression and 7 genes had down-regulated expression in tumor samples. Three genes, NPC1, ABCC1 and SLC51B, were extrapolated to construct a prediction model for prognosis of HCC patients.
Conclusion. The three-gene prediction model was more reliable than the pathological staging characters of the tumor for the prognosis and survival of HCC patients. Additionally, the up-regulated genes facilitating the transport of BAs are associated with poor prognosis of HCC patients and genes of de novo synthesis of BAs benefits HCC patients.
... The pathogenic mechanism of colon carcinogenesis induced by BAs involves the genotoxic effect exerted by CDCA and DCA in normal human colonic epithelial cells (HCoEpiCs): oxidative stress in CRC cells has been associated with genotoxic alteration of the DNA helix, which transforms normal cells into cancer cells (70). Damages caused by a prolonged exposition to BAs have been observed in the liver as well, with disruption of the normal apoptosis of hepatocytes (71)(72)(73). ...
... Pro-oxidative factors, including iron [43], omega-6 fatty acids [37,44,45], and saturated fats [46,47] intakes, obesity [48], smoking [49,50], and alcohol intake [51,52], increase RONS production and accelerate cellular damage caused by oxidative stress. The rationale for inclusion of each of the components of the dietary and lifestyle OBS was reported previously [12,13] and is summarized in Supplemental Table 1. ...
PurposeSubstantial basic science evidence suggests that oxidative stress may play a role in aging-related health outcomes, including cardiovascular diseases (CVD) and cancer, and oxidative stress markers were linked with all-cause and cause-specific mortality in epidemiologic studies. However, the associations of many individual dietary and lifestyle anti-/pro-oxidant exposures with mortality are inconsistent. Oxidative balance scores (OBS) that incorporated multiple dietary and lifestyle factors were previously developed and reported to reflect the collective oxidative effects of multiple exposures.Methods
We investigated associations of 11-component dietary and 4-component (physical activity, adiposity, alcohol, and smoking) lifestyle OBS (higher scores were considered more anti-oxidative) with all-cause and cause-specific mortality among women 55–69 years of age at baseline in the prospective Iowa Women’s Health Study (1986–2012). We assessed OBS-mortality associations using multivariable Cox proportional hazards regression.ResultsOf the 34,137 cancer-free women included in the analytic cohort, 18,058 died (4521 from cancer, and 6825 from CVD) during a mean/median 22.0/26.1 person-years of follow-up. Among participants in the highest relative to the lowest lifestyle OBS quintiles, the adjusted hazards ratios and their 95% confidence intervals for all-cause, all-cancer, and all-CVD mortality were 0.50 (0.48, 0.53), 0.47 (0.43, 0.52), and 0.54 (0.50, 0.58) (all Ptrend < 0.001), respectively. The associations of the dietary OBS with mortality were close to null.Conclusion
Our findings, combined with results from previous studies, suggest that a predominance of antioxidant over pro-oxidant lifestyle exposures may be associated with lower all-cause, all-CVD, and all-cancer mortality risk.
... The increase of the two bile acids in feces is associated with elevated incidence of colorectal cancer 76 . DCA damages genomic DNA by oxidation 77 and deficiency in base excision repair of oxidative DNA damage is linked to increased risk of intestinal tumors in mice 78 . Future work is needed to investigate how DCA induces the chronic diseases but attenuates the infectious enteritis. ...
Necrotic enteritis (NE) caused by Clostridium perfringens infection has reemerged as a prevalent poultry disease worldwide due to reduced usage of prophylactic antibiotics. The lack of alternative antimicrobial strategies to control this disease is mainly due to limited insight into NE pathogenesis, microbiome relationships, and host responses. Here we reported that the metabolic byproduct of microbial metabolism of bile acids to deoxycholic acid (DCA), at as low as 50 μM, inhibited 82.8% of C. perfringens growth in Tryptic Soy Broth (P < 0.05). Sequential Eimeria maxima and C. perfringens challenge strongly induced NE, severe intestinal inflammation, and body weight (BW) loss in broiler chickens. These negative effects were diminished by 1.5 g/kg DCA diet. At the cellular level, DCA alleviated NE-associated ileal epithelial death and lamina propria immune cell apoptosis. Interestingly, DCA reduced C. perfringens invasion into villi without significantly altering the bacterial luminal colonization. Molecular analysis showed that DCA reduced inflammatory mediators of Infγ, Litaf (Tnfα), Il1β, and Mmp9 mRNA accumulation in ileal tissue. Mechanically, C. perfringens induced elevated expression of inflammatory cytokines of Infγ, Litaf, and Ptgs2 (COX-2 gene) in chicken splenocytes. Inhibiting the COX signaling by aspirin attenuated INFγ- or TNFa-induced inflammatory response in the splenocytes. Consistently, chickens fed 0.12 g/kg aspirin diet resisted against NE-induced BW loss, ileal inflammation, and villus apoptosis. In conclusion, microbial metabolic product DCA prevents NE-induced BW loss and ileal inflammation through curbing inflammatory response. These novel findings could serve as a stepping-stone for developing next generation antimicrobial alternatives against NE.
... Some authors demonstrated an inversely relation between w-3 or w-6 PUFA consumption and CRC risk (Chao et al., 2005;Norat et al., 2005), others indicated a lack of association (Sasazuki et al., 2011;Song et al., 2014), while some shown a positive relationship (Daniel et al., 2009;Shen et al., 2012). In the other hand, studies including saturated fat consumption and CRC, demonstrated that there is a direct relation between these two factors (Giovannucci and Goldin, 1997;Rosignoli et al., 2008) but other authors have not found any significant relation (Williams et al., 2010. Oleic acid, w-9 FA, is considered one of the healthier sources of fat in the diet. ...
Etiology of colorectal cancer (CRC) is related, at least in part, with nutritional profile and epidemiological data indicating a key role of dietary fat on CRC pathogenesis. Moreover, inflammation and eicosanoids produced from arachidonic acid might have a pivotal role in CRC development. However, the effect of specific fatty acids (FAs) on intestinal epithelial cell growth is not completely studied now. By this reason, the aim of this work is to unravel the effect of different saturated and unsaturated long-chain fatty acids (LCFA) and some LCFA metabolites on CRC cell line growth and their possible mechanisms of action. Our results demonstrated that oleic acid is a potent mitogenic factor to Caco-2 cells, at least in part, through 10-hydroxy-8-octadecenoic synthesized by lipoxigenase pathway, whereas polyunsaturated FAs such as eicosapentaenoic (EPA) acid has a dual behavior effect depending on its concentration. A high concentration, EPA induced apoptosis through intrinsic pathway, whereas at low concentration induced cell proliferation that could be related to the synthesis of eicosanoids such as prostaglandin E3 and 12-hydroxyeicosapentaenoic acid and the subsequent induction of mitogenic cell signaling pathways (ERK 1/2, CREB, p38α). Thus, this study contributes to understand the complicated relationship between fat ingest and CRC.
... Excessive hydrophobic BAs, especially LCAs, are cytotoxic, thus can disrupt the plasma and mitochondrial membranes, 26 disturb water and salt transportation 27 and promote oncogenesis. 28 This could explain why sulfated BAs are usually elevated in cholestatic liver disease as a compensational mechanism to promote excretion of BAs. 29 Moreover, the regulation of BA sulfation is related to the actions of various nuclear receptors such as the FXR 1 and TGR5, which may also participate in the improvement in metabolic health after bariatric surgery. 30 Sulfation of BAs can modulate TGR5 activity, 31 and it is possible that the increase in sulfated BAs concentration after LSG could lead to improvements in metabolic health. ...
Background
Bile acids (BAs) are traditionally associated with lipid absorption and phase II detoxification by forming various BA conjugates. Recently, it has been discovered that BAs also regulate glucose metabolism, and the increase in BAs in patients following bariatric surgery may contribute to the post-surgery improvement in insulin resistance (IR). However, while Roux-en-Y gastric bypass can increase BA concentrations post-surgery, this may not be the case after laparoscopic sleeve gastrectomy (LSG). We hypothesized that the profiling of BAs that include the conjugated BA species could detect post-surgery BA changes after LSG. To test our hypothesis, we performed comprehensive profiling of BAs in Asian individuals with morbid obesity at baseline, and at 6 months following LSG.
Methods
Fourteen subjects scheduled for LSG were recruited. Anthropometric measurements, oral glucose tolerance test, and biochemistry tests were performed at baseline and at 6 months after LSG. BAs were profiled using liquid chromatography–mass spectrometry.
Results
At 6 months, subjects lost significant weight from 117.4±5.4 to 92.1±3.8 kg and demonstrated significant improvement in IR. HOMA-IR decreased from 6.2±0.7 to 2.0±0.2 and the Matsuda index increased from 1.9±0.3 to 3.3±0.3. We did not detect any significant post-operative change in the levels of total BAs (5237.1±1219.4 vs. 3631.7±457.9, p=0.181) or non-sulfated BAs after LSG. However, sulfated BA species increased significantly after LSG.
Conclusion
Our study showed that the serum concentrations of sulfated BA species in morbidly obese Asian individuals increased significantly 6 months after LSG; the increase in sulfated BAs after LSG might contribute to the post-surgery improvement of metabolic health.
... In addition to BA concentration, primary and secondary BA as proportion of total BA were not affected over time (P > 0.05; Table 3). The level of hydrophobicity of BA is positively associated with its cytotoxic potential (BA hydrophobicity scale: ursodeoxycholic acid < cholic acid < chenodeoxycholic acid < deoxycholic acid < lithocholic acid; Hofmann,1999), with deoxycholic acid and lithocholic acid known to induce oxidative damage of DNA in vitro (Booth et al., 1997;Bernstein et al., 1999;Glinghammar, 2002;Payne, 2008;Rosignoli et al., 2008). In contrast, ursodeoxycholic acid is believed to have chemoprotective potential (Alberts et al., 2005;Akare et al., 2006). ...
Grain-free diets tend to have greater inclusions of pulses in contrast to grain-based diets. In 2018 the Food and Drug Administration (FDA) released a statement that grain-free diets may be related to the development of canine dilated cardiomyopathy (DCM). However, all dog foods met regulatory minimums for nutrient inclusion recommended by the Association of American Feed Controls Official (AAFCO). In some FDA case reports, but not all, dogs diagnosed with DCM also had low concentrations of plasma or whole blood taurine, as such we hypothesized that feeding these diets will result in reduced taurine status from baseline measures. The objective of this study was to determine the effects of feeding a grain-free diet to large breed dogs on taurine status and overall health. Eight Labrador Retrievers (4 males, 4 females; Four Rivers Kennel, MO) were individually housed and fed a commercial complete and balanced grain-free diet (Acana Pork and Squash formula; APS) for 26 wk. Fasted blood samples were collected prior to the start of the trial (baseline; wk 0), at wk 13 and wk 26 for analyses of blood chemistry, hematology, plasma amino acids (AA), and whole blood taurine. Urine was collected by free catch at wk 0 and 26 for taurine and creatinine analyses. Fresh fecal samples were collected at wk 0 and 26 for bile acid analyses. Data were analyzed using the GLIMMIX procedure with repeated measures in SAS (v. 9.4). Plasma His, Met, Trp, and taurine and whole blood taurine concentrations increased over the course of the study (P < 0.05). Urinary taurine to creatinine ratio was not affected by diet (P>0.05). Fecal bile acid excretion increased after 26 wk of feeding APS to dogs. Despite the higher fecal excretion of bile acids, plasma and whole blood taurine increased over the 26-wk feeding study. These data suggest the feeding APS, a grain-free diet, over a 26-wk period improved taurine status in Labrador Retrievers and is not the basis for incidence of DCM for dogs fed APS. Other factors that may contribute to the etiology of DCM should be explored.
... Uric acid is excreted in the gut and rapidly degraded by gut bacteria 37 . Further studies are needed to clarify the relative antioxidant power and role in microbiota maintenance of glutathione, ascorbic acid, uric acid, proteins, including glutathione-associated enzymes (glutathione-S-transferase (GST) and glutathione peroxidase (GPX)), alpha-tocopherol 38 , beta-carotene 38 and gut bilirubin derivatives (urobilin, stercobilin and protoporphyrin) 39 in the gut. ...
Uncontrolled oxidative stress, reported in Salmonella and HIV infections, colorectal cancer or severe acute malnutrition, has been associated with anaerobic gut microbiome alteration, impaired butyrate production, mucosal immunity dysregulation and disruption of host-bacterial mutualism. However, the role of major antioxidant molecules in the human body, such as glutathione, ascorbic acid and uric acid, has been neglected in this context. Here, we performed an in vitro metabolomics study of the 3 most odorous anaerobic microbes isolated from the human gut in our laboratory (Clostridium sporogenes, Clostridium subterminale and Romboutsia lituseburensis) when grown in anaerobiosis or in aerobiosis with these 3 antioxidant molecules via gas and liquid chromatography-mass spectrometry (GC/MS and LC/MS). There was no growth or volatile organic compound production in aerobic cultures without the 3 antioxidant molecules. In anaerobiosis, the major metabolic products of the bacteria were thiols, alcohols and short-chain fatty acid esters. The production of alkanes, cycloheptatriene and, paradoxically, increased butyrate production, was observed in the cultures grown in aerobiosis with the 3 antioxidant molecules. The qualitative shift suggests specific molecular mechanisms that remain to be elucidated. The increased production of butyrate, but also isobutyrate and isovalerate in vitro suggests that these 3 antioxidant molecules contributed to the maintenance and active resilience of host-bacterial mutualism against mucosal oxygen and uncontrolled oxidative stress in vivo.
... The increase of the two bile acids in feces is associated with elevated incidence of colorectal cancer 76 . DCA damages genomic DNA by oxidation 77 and deficiency in base excision repair of oxidative DNA damage is linked to increased risk of intestinal tumors in mice 78 . Future work is needed to investigate how DCA induces the chronic diseases but attenuates the infectious enteritis. ...
Abstract Necrotic enteritis (NE) caused by Clostridium perfringens infection has reemerged as a prevalent poultry disease worldwide due to reduced usage of prophylactic antibiotics under consumer preferences and regulatory pressures. The lack of alternative antimicrobial strategies to control this disease is mainly due to limited insight into the relationship between NE pathogenesis, microbiome, and host responses. Here we showed that the microbial metabolic byproduct of secondary bile acid deoxycholic acid (DCA), at as low as 50 µM, inhibited 82.8% of C. perfringens growth in Tryptic Soy Broth (P
... This is attributed to the hyperactivation of the EGFR-MAPK pathway and the concomitant activation of the AP-1 proto-oncogene and suppression of the p53 tumor-suppressor gene. These hydrophobic bile acids are also known to induce ER stress (Payne et al. 2005), oxidative stress (Washo-Stultz et al. 2002;Sokol et al. 2005;Jenkins et al. 2007;Payne et al. 2007), mitochondrial stress (Washo-Stultz et al. 2002Rolo 2004;Sokol et al. 2005;Payne et al. 2005Payne et al. , 2007, and DNA damage (Glinghammar 2002;Bernstein et al. 2006;Jenkins et al. 2007;Rosignoli et al. 2008). Naked mole-rats seem to have once again circumvented toxic consequences associated with high levels of secondary bile metabolites, showing surprisingly high levels of resilience reminiscent of their cells' high levels of resistance to cytotoxin exposure in vitro (Lewis et al. 2012). ...
Mouse-sized naked mole-rats (Heterocephalus glaber), unlike other mammals, do not conform to Gompertzian laws of age-related mortality; adults show no age-related change in mortality risk. Moreover, we observe negligible hallmarks of aging with well-maintained physiological and molecular functions, commonly altered with age in other species. We questioned whether naked mole-rats, living an order of magnitude longer than laboratory mice, exhibit different plasma metabolite profiles, which could then highlight novel mechanisms or targets involved in disease and longevity. Using a comprehensive, unbiased metabolomics screen, we observe striking inter-species differences in amino acid, peptide, and lipid metabolites. Low circulating levels of specific amino acids, particularly those linked to the methionine pathway, resemble those observed during the fasting period at late torpor in hibernating ground squirrels and those seen in longer-lived methionine-restricted rats. These data also concur with metabolome reports on long-lived mutant mice, including the Ames dwarf mice and calorically restricted mice, as well as fruit flies, and even show similarities to circulating metabolite differences observed in young human adults when compared to older humans. During evolution, some of these beneficial nutrient/stress response pathways may have been positively selected in the naked mole-rat. These observations suggest that interventions that modify the aging metabolomic profile to a more youthful one may enable people to lead healthier and longer lives.
Electronic supplementary material
The online version of this article (10.1007/s11357-018-0014-2) contains supplementary material, which is available to authorized users.
... A modulation of the cyto-and genotoxicity of the FW after calcium and phosphorus supplementation is discussed since ACP are known to precipitate secondary bile acids in the gut [29,43]. Secondary bile acids showed cyto-and genotoxic effects on human normal and tumour colon cells [44][45][46]. Our results indicate that the phosphorus supplementation with and without calcium did not affect the genotoxicity of FW and did not markedly effect the vitality of HT29 cells. ...
Background:
In recent years, high phosphate intakes were discussed critically. In the small intestine, a part of the ingested phosphate and calcium precipitates to amorphous calcium phosphate (ACP), which in turn can precipitate other intestinal substances, thus leading to a beneficial modulation of the intestinal environment. Therefore, we analysed faecal samples obtained from a human intervention study regarding gut-related parameters.
Methods:
Sixty-two healthy subjects (men, n = 30; women, n = 32) completed the double-blind, placebo-controlled and parallel designed study (mean age: 29 ± 7 years; mean BMI: 24 ± 3 kg/m2). Supplements were monosodium phosphate and calcium carbonate. During the first 2 weeks, all groups consumed a placebo sherbet powder, and afterwards a sherbet powder for 8 weeks according to the intervention group: P1000/Ca0 (1000 mg/d phosphorus), P1000/Ca500 (1000 mg/d phosphorus and 500 mg/d calcium) and P1000/Ca1000 (1000 mg/d phosphorus and 1000 mg/d calcium). After the placebo period and after 8 weeks of intervention faecal collections took place. We determined in faeces: short-chain fatty acids (SCFA) and fat as well as the composition of the microbiome (subgroup) and cyto- and genotoxicity of faecal water (FW). By questionnaire evaluation we examined tolerability of the used phosphorus supplement.
Results:
Faecal fat concentrations did not change significantly due to the interventions. Concentrations of faecal total SCFA and acetate were significantly higher after 8 weeks of P1000/Ca500 supplementation compared to the P1000/Ca0 supplementation. In men, faecal total SCFA and acetate concentrations were significantly higher after 8 weeks in the P1000/Ca1000 group compared to the P1000/Ca0 one. None of the interventions markedly affected cyto- and genotoxic activity of FW. Men of the P1000/Ca1000 intervention had a significantly different gut microbial community compared to the men of the P1000/Ca0 and P1000/Ca500 ones. The genus Clostridium XVIII was significantly more abundant in men of the P1000/Ca1000 intervention group compared to the other groups. Supplementations did not cause increased intestinal distress.
Conclusions:
The used high phosphorus diet did not influence cyto- and genotoxicity of FW and the concentrations of faecal fat independent of calcium intake. Our study provides first hints for a potential phosphorus-induced modulation of the gut community and the faecal total SCFA content.
Trial registration:
The trial is registered at ClinicalTrials.gov as NCT02095392 .
... For example, in LS174T colon cancer cells, butyrate decreased cell proliferation and rescued apoptosis-associated speck-like protein (ASC), a proapoptotic protein silenced by DNA methylation in CRC (151). In HT-29 colon cancer cells, butyrate was found to protect against genotoxicity induced by the secondary bile DCA (152), and lower DNMT1 levels (153). There was also evidence to suggest a synergistic effect between butyrate and DHA in regard to modulation of DNA methylation. ...
Colorectal cancer (CRC) is the third most common cancer diagnosis and the second and third leading cause of cancer mortality in men and women, respectively. However, the majority of CRC cases are the result of sporadic tumorigenesis via the adenoma–carcinoma sequence. This process can take up to 20 years, suggesting an important window of opportunity exists for prevention such as switching toward healthier dietary patterns. The Mediterranean diet (MD) is a dietary pattern associated with various health benefits including protection against cardiovascular disease, diabetes, obesity, and various cancers. In this article, we review publications available in the PubMed database within the last 10 years that report on the impact of a MD eating pattern on prevention of CRC. To assist the reader with interpretation of the results and discussion, we first introduce indexes and scoring systems commonly used to experimentally determine adherence to a MD, followed by a brief introduction of the influence of the MD pattern on inflammatory bowel disease, which predisposes to CRC. Finally, we discuss key biological mechanisms through which specific bioactive food components commonly present in the MD are proposed to prevent or delay the development of CRC. We close with a discussion of future research frontiers in CRC prevention with particular reference to the role of epigenetic mechanisms and microbiome related to the MD eating pattern.
... The presence of CRC has been associated with a higher fecal LCA/DCA ratio and with elevated fecal secondary bile acids levels [53][54][55][56]. Although the precise mechanism(s) by which bile acids induce colon carcinogenesis is poorly understood, DCA and chenodeoxycholic acid (CDCA)mediated genotoxicity in normal human colonic epithelial cells as well as in tumor cells has been found to be due to DNA oxidative damage [57]. Increased apoptosis and other forms of toxicity have been observed in the liver following prolonged exposure to high levels of bile acids [58][59][60][61]. ...
Background
Although the unconjugated secondary bile acids, specifically deoxycholic acid (DCA) and lithocholic acid (LCA), are considered to be risk factors for colorectal cancer, the precise mechanism(s) by which they regulate carcinogenesis is poorly understood. We hypothesize that the cytotoxic bile acids may promote stemness in colonic epithelial cells leading to generation of cancer stem cells (CSCs) that play a role in the development and progression of colon cancer. Methods
Normal human colonic epithelial cells (HCoEpiC) were used to study bile acid DCA/LCA-mediated induction of CSCs. The expression of CSC markers was measured by real-time qPCR. Flow cytometry was used to isolate CSCs. T-cell factor/lymphoid-enhancing factor (TCF/LEF) luciferase assay was employed to examine the transcriptional activity of β-catenin. Downregulation of muscarinic 3 receptor (M3R) was achieved through transfection of corresponding siRNA. ResultsWe found DCA/LCA to induce CSCs in normal human colonic epithelial cells, as evidenced by the increased proportion of CSCs, elevated levels of several CSC markers, as well as a number of epithelial–mesenchymal transition markers together with increased colonosphere formation, drug exclusion, ABCB1 and ABCG2 expression, and induction of M3R, p-EGFR, matrix metallopeptidases, and c-Myc. Inhibition of M3R signaling greatly suppressed DCA/LCA induction of the CSC marker ALDHA1 and also c-Myc mRNA expression as well as transcriptional activation of TCF/LEF. Conclusions
Our results suggest that bile acids, specifically DCA and LCA, induce cancer stemness in colonic epithelial cells by modulating M3R and Wnt/β-catenin signaling and thus could be considered promoters of colon cancer.
... First, it is the preferred energy source for colonocytes 51 . Second, it has a remarkably wide variety of antineoplastic properties, best exemplified by its function as a histone deacetylase inhibitor 52 , its capacity to downregulate the key canonical Wnt-signalling pathway linked to colonic carcinogenesis 53 and its ability to reduce the burden of carcinogens, such as bile acids 28,29,54 and red meat products 26 . Further studies are needed to explore the intriguing possibility that increased saccharolytic fermentation and butyrogenesis may counter the recognized proliferative properties of T-helper type 17 T cells 55 . ...
Rates of colon cancer are much higher in African Americans (65:100,000) than in rural South
Africans (o5:100,000). The higher rates are associated with higher animal protein and fat,
and lower fibre consumption, higher colonic secondary bile acids, lower colonic short-chain
fatty acid quantities and higher mucosal proliferative biomarkers of cancer risk in otherwise
healthy middle-aged volunteers. Here we investigate further the role of fat and fibre in this
association. We performed 2-week food exchanges in subjects from the same populations,
where African Americans were fed a high-fibre, low-fat African-style diet and rural Africans a
high-fat, low-fibre western-style diet, under close supervision. In comparison with their usual
diets, the food changes resulted in remarkable reciprocal changes in mucosal biomarkers of
cancer risk and in aspects of the microbiota and metabolome known to affect cancer risk, best
illustrated by increased saccharolytic fermentation and butyrogenesis, and suppressed
secondary bile acid synthesis in the African Americans.
... First, it is the preferred energy source for colonocytes 51 . Second, it has a remarkably wide variety of antineoplastic properties, best exemplified by its function as a histone deacetylase inhibitor 52 , its capacity to downregulate the key canonical Wnt-signalling pathway linked to colonic carcinogenesis 53 and its ability to reduce the burden of carcinogens, such as bile acids 28,29,54 and red meat products 26 . Further studies are needed to explore the intriguing possibility that increased saccharolytic fermentation and butyrogenesis may counter the recognized proliferative properties of T-helper type 17 T cells 55 . ...
Rates of colon cancer are much higher in African Americans (65:100,000) than in rural South Africans (<5:100,000). The higher rates are associated with higher animal protein and fat, and lower fibre consumption, higher colonic secondary bile acids, lower colonic short-chain fatty acid quantities and higher mucosal proliferative biomarkers of cancer risk in otherwise healthy middle-aged volunteers. Here we investigate further the role of fat and fibre in this association. We performed 2-week food exchanges in subjects from the same populations, where African Americans were fed a high-fibre, low-fat African-style diet and rural Africans a high-fat, low-fibre western-style diet, under close supervision. In comparison with their usual diets, the food changes resulted in remarkable reciprocal changes in mucosal biomarkers of cancer risk and in aspects of the microbiota and metabolome known to affect cancer risk, best illustrated by increased saccharolytic fermentation and butyrogenesis, and suppressed secondary bile acid synthesis in the African Americans.
... We found eleven cell culture studies [287,303304305306307308309310311312 with provitamin A carotenoids ( and carotene, cryptoxanthin, retinoic acid, retinal and retinol), and eight with nonvitamin A carotenoids (lycopene, lutein, astaxanthin or zeaxanthin) [294,304,313314315316317318. Experiments in most cases involved cotreatment with DNA damaging agent and carotenoid. ...
Genomic instability can initiate cancer, augment progression, and influence the overall prognosis of the affected patient. Genomic instability arises from many different pathways, such as telomere damage, centrosome amplification, epigenetic modifications, and DNA damage from endogenous and exogenous sources, and can be perpetuating, or limiting, through the induction of mutations or aneuploidy, both enabling and catastrophic. Many cancer treatments induce DNA damage to impair cell division on a global scale but it is accepted that personalized treatments, those that are tailored to the particular patient and type of cancer, must also be developed. In this review, we detail the mechanisms from which genomic instability arises and can lead to cancer, as well as treatments and measures that prevent genomic instability or take advantage of the cellular defects caused by genomic instability. In particular, we identify and discuss five priority targets against genomic instability: (1) prevention of DNA damage; (2) enhancement of DNA repair; (3) targeting deficient DNA repair; (4) impairing centrosome clustering; and, (5) inhibition of telomerase activity. Moreover, we highlight vitamin D and B, selenium, carotenoids, PARP inhibitors, resveratrol, and isothiocyanates as priority approaches against genomic instability. The prioritized target sites and approaches were cross validated to identify potential synergistic effects on a number of important areas of cancer biology.
Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.
... We found eleven cell culture studies [287,303304305306307308309310311312 with provitamin A carotenoids ( and carotene, cryptoxanthin, retinoic acid, retinal and retinol), and eight with nonvitamin A carotenoids (lycopene, lutein, astaxanthin or zeaxanthin) [294,304,313314315316317318. Experiments in most cases involved cotreatment with DNA damaging agent and carotenoid. ...
Genomic instability can initiate cancer, augment progression, and influence the overall prognosis of the affected patient. Genomic instability arises from many different pathways, such as telomere damage, centrosome amplification, epigenetic modifications, and DNA damage from endogenous and exogenous sources, and can be perpetuating, or limiting, through the induction of mutations or aneuploidy, both enabling and catastrophic. Many cancer treatments induce DNA damage to impair cell division on a global scale but it is accepted that personalized treatments, those that are tailored to the particular patient and type of cancer, must also be developed. In this review, we detail the mechanisms from which genomic instability arises and can lead to cancer, as well as treatments and measures that prevent genomic instability or take advantage of the cellular defects caused by genomic instability. In particular, we identify and discuss five priority targets against genomic instability: (1) prevention of DNA damage; (2) enhancement of DNA repair; (3) targeting deficient DNA repair; (4) impairing centrosome clustering; and, (5) inhibition of telomerase activity. Moreover, we highlight vitamin D and B, selenium, carotenoids, PARP inhibitors, resveratrol, and isothiocyanates as priority approaches against genomic instability. The prioritized target sites and approaches were cross validated to identify potential synergistic effects on a number of important areas of cancer biology.
... The expression system was used to characterize and compare the sulfation kinetics of DHEA and 15 human bile acids by SULT2A1. 2. Formation of DHEA sulfate demonstrated Michaelis–Menten kinetics with apparent K m and V max values of 3.8 μM and 130.8 pmol min −1 mg by BAs, including cholestasis, bile duct infarction, liver fibrosis, liver cirrhosis, and colon cancer, were demonstrated in several species (Fickert et al. 2006; Hofmann 1999b; Palmer 1972; Rosignoli et al. 2008). Therefore, BA homeostasis must be tightly controlled by regulating BA synthesis and elimination to prevent their accumulation and toxicity. ...
Bile acids (BAs) play a variety of physiological functions. However, BAs are also cytotoxic and cancer promoters. In humans, sulfation by sulfotransferase 2A1 (SULT2A1) is the dominant pathway of BA elimination and detoxification. In this study, we have constructed a stable cell line expressing SULT2A1 by transfection into HEK293 cells. The expression system was used to characterize and compare the sulfation kinetics of DHEA and 15 human BAs by SULT2A1. Formation DHEA-sulfate also demonstrated Michaelis-Menten kinetics with apparent Km and Vmax values of 3.8 μM and 130.8 pmol/min/mg protein, respectively. Sulfation kinetics of BAs demonstrated Michaelis-Menten kinetics with a marked variation in apparent Km and Vmax values between individual BAs. Sulfation affinity was inversely proportional to the number of hydroxyl groups of BAs. The monohydroxy- and most toxic BA (lithocholic acid) had the highest affinity, whereas the trihydroxy- and least toxic BA (cholic acid) had the lowest affinity to sulfation by SULT2A1, which support the major role of SULT2A1 in the elimination and detoxification of BAs in humans. Intrinsic clearance (CLint) of ursodeoxycholic acid (UDCA) was 1.5 and 9 fold higher than that of deoxycholic acid and chenodeoxycholic acid, respectively, despite the fact that all three are dihydroxy-BAs.
Diet and nutrition play key roles in the maintenance of genomic stability. Obesity is the result of an excess of caloric intake in comparison with energy expenditure. In itself, this leads to oxidative stress and DNA damage. DNA-reactive mutagens including those formed during high temperature–cooking processes of red meats or from other processing methods are still common, albeit unintentional, components of the diet of many individuals. Improved food storage technologies may be leading to a reduction in fungal contaminants such as aflatoxin B1, but endogenously or exogenously formed reactive species, inhibitors of DNA repair, or of the mitotic spindle occur in the human diet, and may also lead to genomic instability. Some of these may become incorporated into the diet through the uptake of environmental contaminants by food plants and animals. Conversely, various nutrients and phytochemicals may be protective. Carotenoids, vitamin D, and selenium are among the best studied nutrients here. Various phytochemicals, including isothiocyanates such as sulforaphane or polyphenols such as genistein or curcumin, have differing mechanisms of promoting genomic stability. Polymorphisms in genes for nutrient uptake, metabolism, and excretion will determine the optimal dietary intake for an individual. Human studies are essential to quantifying and overcoming diet-related genomic instability.
Apart from their commonly known, almost exclusive role in nutrient absorption and cholesterol homeostasis, there is now empirical evidence to suggest that bile acids exert pleiotropic effects on the host immune system. Here, bile acids serve as ligands for various evolutionarily conserved nuclear hormone and G-protein-coupled receptors. The wide distribution of these bile acid-activated receptors among tissues and cells, especially the immune cells of the intestine, facilitates the influence of bile acids on host gut immune responses. Concurrently, these immune cells are bound to experience endoplasmic reticulum (ER) stress due to dynamic cellular functions, resulting in the activation of a salvage pathway, the unfolded protein response (UPR). Severely dysregulated ER stress signaling cascade is found to be associated with many debilitating inflammatory disorders. Finally, there is growing evidence of the interplay between bile acids and ER stress signaling pathways gearing towards inflammation-driven intestinal disorders. This review expounds on the enigmatic networking of the triad: bile acids, ER stress and inflammation—emphasizing on molecular players of the signaling cascades involving the trio, which may offer a promising therapeutic approach for inflammation-driven disorders in the intestine.
Farnesoid X receptor (FXR, encoded by NR1H4), a bile acid-activated nuclear receptor, is widely implicated in human tumorigenesis. The FXR agonist obeticholic acid (OCA) has preliminarily displayed tumour suppressor potential. However, the anticancer effects of this agent on colorectal cancer (CRC) remain unclear. In this study, the treatment of colon cancer cells with OCA inhibited cell proliferation and invasion in vitro, retarded tumour growth in vivo and prevented the G0 /G1 to S phase transition. Moreover, the expression of active caspase-3, p21 and E-cadherin was up-regulated and the expression of cyclin D1, c-Myc, vimentin, N-cadherin and MMP9 was down-regulated in OCA-treated colon cancer cells. Mechanistic studies indicated that OCA treatment suppressed the activity of JAK2/STAT3 pathway by up-regulating SOCS3 expression. Colivelin, an agonist of JAK2/STAT3 pathway, antagonized the tumour-suppressive effect of OCA on colon cancer cells. Dual-luciferase reporter and quantitative chromatin immunoprecipitation (qChIP) assays further confirmed that OCA promoted SOCS3 transcription by enhancing the binding of FXR to the FXRE/IR9 of the SOCS3 promoter. In conclusion, our study demonstrates that targeting FXR and improving its function might be a promising strategy for CRC treatment.
Colorectal cancer (CRC) risk is predominantly driven by environmental factors, in particular diet. A high intake of dietary fat has been implicated as a risk factor inducing the formation of pre-neoplastic lesions (e.g., adenomatous polyps) and/or exacerbating colonic tumorigenesis. Recent data attributed the tumor-promoting activity of high-fat diets to their effects on gut microbiota composition and metabolism, in particular with regard to bile acids. Bile acids are synthesized in the liver in response to dietary fat and facilitate lipid absorption in the small intestine. The majority of bile acids is re-absorbed during small intestinal transit and subjected to enterohepatic circulation. Bile acids entering the colon undergo complex biotransformation performed by gut bacteria, resulting in secondary bile acids that show tumor-promoting activity. Excessive dietary fat leads to high levels of secondary bile acids in feces and primes the gut microbiota to bile acid metabolism. This promotes an altered overall bile acid pool, which activates or restricts intestinal and hepatic cross-signaling of the bile acid receptor, farnesoid X receptor (FXR). Recent studies provided evidence that FXR is a main regulator of bile acid-mediated effects on intestinal tumorigenesis integrating dietary, microbial and genetic risk factors for CRC. Selective FXR agonist or antagonist activity by specific bile acids depends on additional factors (e.g., bile acid concentration, composition of bile acid pool, genetic instability of cells) and, thus, may differ in healthy and tumorigenic conditions in the intestine. In conclusion, fat-mediated alterations of the gut microbiota link bile acid metabolism to CRC risk and colonic tumorigenesis, exemplifying how gut microbial co-metabolism affects colon health.
Background:
Basic science literature strongly supports a role of oxidative stress in colorectal cancer (CRC) etiology, but in epidemiologic studies, associations of most individual exposures with CRC have been weak or inconsistent. However, recent epidemiologic evidence suggests that the collective effects of these exposures on oxidative balance and CRC risk may be substantial.
Methods:
Using food frequency and lifestyle questionnaire data from the prospective Iowa Women's Health Study (1986-2012), we investigated associations of 11-component dietary and 4-component lifestyle oxidative balance scores (OBS) with incident CRC using multivariable Cox proportional hazards regression.
Results:
Of the 33,736 cancer-free women aged 55-69 years at baseline, 1,632 developed CRC during follow-up. Among participants in the highest relative to the lowest dietary and lifestyle OBS quintiles (higher anti-oxidant relative to pro-oxidant exposures), the adjusted hazard ratios (HRs) and their 95% confidence intervals (CI) were, respectively, 0.77 (0.63, 0.94) (Ptrend =0.02) and 0.61 (0.52, 0.71) (Ptrend <0.0001). Among those in the highest relative to the lowest joint lifestyle/dietary OBS quintile, the HR was 0.45 (95% CI 0.26, 0.77).
Conclusions:
Our findings suggest that a predominance of antioxidant over pro-oxidant dietary and lifestyle exposures-separately and especially jointly-may be inversely associated with CRC risk among older women.
Purpose of Review
To review recent data on the role and interactions of fiber and fat as dietary risk factors associated with colorectal cancer (CRC) risk in humans.
Recent Findings
Fiber intake shows convincing and linear dose-response negative correlation with CRC risk. Dietary fiber stimulates butyrogenic activity of the gut microbiota, providing high amounts of butyrate that shows extensive anti-neoplastic effects. A high-fat diet promotes CRC risk through stimulated bile acid metabolism, facilitating bile acid conversion by the gut microbiota to tumor-promoting deoxycholic acid. Comprehensive interactions of these microbial metabolites are likely to underlie mechanisms driving diet-dependent CRC risk in different populations, but require further experimental investigation.
Summary
Dietary fiber and fat shape the composition and metabolic function of the gut microbiota, resulting in altered amounts of butyrate and deoxycholic acid in the colon. Fiber supplementation and restriction of fat intake represent promising strategies to reduce CRC risk in healthy individuals.
Background
A method for bile acid profiling measuring 21 primary and secondary bile acids in serum samples was developed and validated with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Sample preparation included spiking with internal standards followed by protein precipitation, centrifugation, drying under nitrogen gas and reconstitution. Extracted samples were injected onto a Phenomenex Kinetex C18 column (150 × 4.60 mm, 2.6 μm).
Methods
Data was collected with LC-MS/MS operated in negative ion mode with multiple reaction monitoring (MRM) and single reaction monitoring (SRM). The analytical run time was 12 min.
Results
The method showed excellent linearity with high regression coefficients (>0.99) over a range of 0.05 and 25 μM for all analytes tested. The method also showed acceptable intra-day and inter-day accuracy and precision. As a proof of concept, the analytical method was applied to patients with neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD), biliary atresia (BA), and necrotizing enterocolitis (NEC), and distinct bile acids profiles were demonstrated.
Conclusions
The method could be poised to identify possible biomarkers for non-invasive early diagnosis of these disorders.
Introduction. Cholesterol gallstone disease have relationships with various conditions linked with insulin resistance, but also with heart disease, atherosclerosis, and cancer. These associations derive from mechanisms active at a local (i.e. gallbladder, bile) and a systemic level and are involved in inflammation, hormones, nuclear receptors, signaling molecules, epigenetic modulation of gene expression, and gut microbiota. Despite advanced knowledge of these pathways, the available therapeutic options for symptomatic gallstone patients remain limited. Therapy includes oral litholysis by the bile acid ursodeoxycholic acid (UDCA) in a small subgroup of patients at high risk of post-dissolution recurrence, or laparoscopic cholecystectomy, which is the therapeutic radical gold standard treatment. Cholecystectomy, however, may not be a neutral event, and potentially generates health problems, including the metabolic syndrome.
Areas covered. Several studies on risk factors and pathogenesis of cholesterol gallstone disease, acting at a systemic level have been reviewed through a PubMed search. Authors have focused on primary prevention and novel potential therapeutic strategies.
Expert commentary. The ultimate goal appears to target the manageable systemic mechanisms responsible for gallstone occurrence, pointing to primary prevention measures. Changes must target lifestyles, as well as experimenting innovative pharmacological tools in subgroups of patients at high risk of developing gallstones.
Background:
Dogs are fed various diets, which also include components of animal origin. In humans, a high-fat/low-fibre diet is associated with higher faecal levels of bile acids, which can influence intestinal health. It is unknown how an animal-based diet high in fat and low in fibre influences the faecal bile acid levels and intestinal health in dogs. This study investigated the effects of high intake of minced beef on the faecal bile acid profile in healthy, adult, client-owned dogs (n = 8) in a 7-week trial. Dogs were initially adapted to the same commercial dry food. Thereafter, incremental substitution of the dry food by boiled minced beef over 3 weeks resulted in a diet in which 75% of each dog's total energy requirement was provided as minced beef during week 5. Dogs were subsequently reintroduced to the dry food for the last 2 weeks of the study. The total taurine and glycine-conjugated bile acids, the primary bile acids chenodeoxycholic acid and cholic acid, and the secondary bile acids lithocholic acid, deoxycholic acid (DCA) and ursodeoxycholic acid (UDCA) were analysed, using liquid chromatography-tandem mass spectrometry.
Results:
The faecal quantities of DCA were significantly higher in dogs fed the high minced beef diet. These levels reversed when dogs were reintroduced to the dry food diet. The faecal levels of UDCA and taurine-conjugated bile acids had also increased in response to the beef diet, but this was only significant when compared to the last dry food period.
Conclusions:
These results suggest that an animal-based diet with high-fat/low-fibre content can influence the faecal bile acids levels. The consequences of this for canine colonic health will require further investigation.
Bile acid homeostasis is maintained by liver synthesis, bile duct secretion, microbial metabolism and intestinal reabsorption into the blood. When drug insults result in liver damage, the variances of bile acids (BAs) are related to the physiological status of the liver. Here, we established a method to simultaneously quantify 19 BAs in rat plasma, liver, bile and different intestinal section contents (duodenum, jejunum, ileum, cecum and colon) using high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) to reveal the pattern of bile acid homeostasis in the enterohepatic circulation of bile acids in physiological situations. Dynamic changes in bile acid composition appeared throughout the enterohepatic circulation of the BAs; taurine- and glycine-conjugated BAs and free BAs had different dynamic homeostasis levels in the circulatory system. cholic acid (CA), beta-muricholic acid (beta-MCA), lithocholic acid (LCA), glycocholic acid (GCA) and taurocholic acid (TCA) greatly fluctuated in the bile acid pool under physiological conditions. Taurine- and glycine-conjugated bile acids constituted more than 90% in the bile and liver, whereas GCA and TCA accounted for more than half of the total bile acids and the secretion of bile mainly via conjugating with taurine. While over 80% of BAs in plasma were unconjugated bile acids, CA and HDCA were the most abundant elements. Unconjugated bile acids constituted more than 90% in the intestine, and CA, beta-MCA and HDCA were the top three bile acids in the duodenum, jejunum and ileum content, but LCA and HDCA were highest in the cecum and colon content. As the main secondary bile acid converted by microflora in the intestine, LCA was enriched in the cecum and DCA mostly in the colon. As endogenous substances, the concentrations of plasma BAs were closely related to time rhythm and diet. In conclusion, analyzing detailed BA profiles in the enterohepatic circulation of bile acids in a single run is possible using LC-MS/MS. Based on the physiological characteristics of the metabolic profiling of 19 BAs in the total bile acid pool and the time rhythm variation of the endogenous bile acids, this study provided a new valuable method and theoretical basis for the clinical research of bile acid homeostasis.
Because colorectal cancer (CRC) remains a leading cause of cancer mortality worldwide, more accessible screening tests are urgently needed to identify early stage lesions. We hypothesized that highly sensitive, metabolic profile analysis of stool samples will identify metabolites associated with early stage lesions and could serve as a noninvasive screening test. We therefore applied traveling wave ion mobility mass spectrometry (TWIMMS) coupled with ultraperformance liquid chromatography (UPLC) to investigate metabolic aberrations in stool samples in a transgenic model of premalignant polyposis aberrantly expressing the gene encoding the high mobility group A (Hmga1) chromatin remodeling protein. Here, we report for the first time that the fecal metabolome of Hmga1 mice is distinct from that of control mice and includes metabolites previously identified in human CRC. Significant alterations were observed in fatty acid metabolites and metabolites associated with bile acids (hypoxanthine xanthine, taurine) in Hmga1 mice compared to controls. Surprisingly, a marked increase in the levels of distinctive short, arginine-enriched, tetra-peptide fragments was observed in the transgenic mice. Together these findings suggest that specific metabolites are associated with Hmga1-induced polyposis and abnormal proliferation in intestinal epithelium. Although further studies are needed, these data provide a compelling rationale to develop fecal metabolomic analysis as a noninvasive screening tool to detect early precursor lesions to CRC in humans.
Diet and nutrition play key roles in the maintenance of genomic stability. Obesity is the result of an excess of caloric intake in comparison with energy expenditure. In itself, this leads to oxidative stress and DNA damage. DNA-reactive mutagens including those formed during high temperature–cooking processes of red meats or from other processing methods are still common, albeit unintentional, components of the diet of many individuals. Improved food storage technologies may be leading to a reduction in fungal contaminants such as aflatoxin B1, but endogenously or exogenously formed reactive species, inhibitors of DNA repair, or of the mitotic spindle occur in the human diet, and may also lead to genomic instability. Some of these may become incorporated into the diet through the uptake of environmental contaminants by food plants and animals. Conversely, various nutrients and phytochemicals may be protective. Carotenoids, vitamin D, and selenium are among the best studied nutrients here. Various phytochemicals, including isothiocyanates such as sulforaphane or polyphenols such as genistein or curcumin, have differing mechanisms of promoting genomic stability. Polymorphisms in genes for nutrient uptake, metabolism, and excretion will determine the optimal dietary intake for an individual. Human studies are essential to quantifying and overcoming diet-related genomic instability.
Introduction:
Deoxycholic acid (DOCA) is involved in many physiological functions and has been used in various fields of pharmaceutical formulations as a natural active solubilizing and permeation-enhancing agent. Although DOCA has been suggested to be a promoter of colon cancer, it has also been used extensively as a starting material to obtain new derivatives for potential therapeutic applications. Area covered: In this review, we focus on patents and research reports from 2011 to 2014 related to pharmaceutical formulations and therapeutic applications using DOCA and its derivatives as surfactants or absorption enhancers, drug delivery carriers, and anti-cancer agents. Expert opinion: In recent few years, DOCA and its derivatives have been used mostly as pharmaceutical excipients for solubilizing lipophilic compounds to improve their bioavailability. Other studies have expanded its applications to include enhanced drug permeability and have designed more effective drug carriers by conjugation with polymeric materials. Recently, a synthetic DOCA injection, ATX-101, has shown long-term efficacy in the non-surgical treatment of unwanted submental fat and acceptable tolerability in humans. Thus, it may be used for reducing specific localized fat accumulations. Additionally, DOCA has been a starting material for anti-cancer drugs, and some derivatives showed strong inhibitory activities against several carcinoma cells.
Although oxidative stress is implicated in colorectal carcinogenesis, human studies on associations of individual prooxidants and antioxidants with colorectal cancer (CRC) have been inconclusive. We incorporated individual environmental factors known to affect oxidative stress into 4 oxidative balance scores (OBS) and investigated their associations with CRC in the Cancer Prevention Study II Nutrition Cohort. During 1999-2009, a total of 1,109 incident CRC cases were identified among 80,063 participants in the Nutrition Cohort who had completed detailed questionnaires. Four OBS with different weighting methods (equal weights, literature review-based, a posteriori data-based, and weights based on Bayesian analysis) were created by combining 16 dietary and nondietary lifestyle factors. Higher values for all 4 OBS, representing more antioxidant exposures than prooxidant exposures, were associated with 41%-53% lower risks of CRC; for example, the relative risk for the highest OBS quartile versus the lowest in the Bayesian analysis was 0.50 (95% confidence interval: 0.41, 0.61). The associations were more modest when OBS was restricted to either dietary or nondietary components. These results, obtained using comprehensive summary measures of oxidative balance-especially considering the similarity of the findings derived using the different weighting methods-support the hypothesis that a predominance of antioxidant lifestyle exposures (both dietary and nondietary) over prooxidant lifestyle exposures reduces risk of CRC.
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The southern Africa region is a region of unique floral biodiversity; this study was undertaken to address the limited knowledge regarding the physiochemical, antioxidant activity and the ability of these honeys to protect biomolecules and cells against oxidative damage. The physicochemical properties, total polyphenolic and flavonoid content (TPC and TFC), catalase and antioxidant activity (DPPH, TEAC, and ORAC assays) of 13 representative honey samples was determined. Biological and cellular protection was investigated using the erythrocyte haemolysis, the pBR322 plasmid, as well as the dichlorofluorescein diacetate (DCFH-DA) assays in SC-1 and Caco-2 cells. High TPC, TFC, catalase and antioxidant activity was obtained, and all honeys protected DNA, erythrocytes and cells in vitro. Colour, TPC, TFC and antioxidant activity correlated well but no correlation was seen between these parameters and catalase activity, biological and cellular effects. Nevertheless, honeys with high catalase activity and/or are dark in colour with high TPC, TFC and/or antioxidant activity did show the highest degree of biological and cellular protection.
The aim of this study was to investigate the ability of epoxides of styrene (styrene-7,8-oxide; SO) and 1,3-butadiene (3,4-epoxy-1-butene; 1,2:3,4:-diepoxybutane) to cause oxidative stress and oxidative DNA damage on human peripheral blood mononuclear cells (PBMCs) and whether a complex mixture of olive oil phenols (OOPE) could prevent these effects. The DNA damage was measured by the single-cell gel electrophoresis (SCGE; comet assay). We found that the DNA damage induced by alkene epoxides could be prevented by N-acetyl-cysteine (10 mM) and catalase (100 U/ml). Alkene epoxides caused a significant (P < 0.05) increase of both peroxide concentration in extra- and intracellular environment and formamidopyrimidine DNA glycosylase (FPG)- and Endonuclease III (ENDO III)-sensitive sites in PBMCs, demonstrating the presence of oxidized bases. OOPE (1 μg of total phenols/ml) was able to prevent the alkene epoxide induced DNA damage both after 2 and 24 h of incubation. In addition, OOPE completely inhibited the SO-induced intracellular peroxide accumulation in PBMCs and prevented the oxidative DNA damage induced by SO, as evidenced by the disappearance of both FPG- and ENDO III-sensitive sites. This is the first study demonstrating the ability of OOPE to prevent the DNA damage induced by alkene epoxides providing additional information about the chemopreventive properties of olive oil.
The repeated dose liver micronucleus (RDLMN) assay using young adult rats has the potential to detect genotoxic hepatocarcinogens that can be integrated into a general toxicity study. The assay methods were thoroughly validated by 19 Japanese facilities. Methapyrilene hydrochloride (MP), known to be a non-genotoxic hepatocarcinogen, was examined in the present study. MP was dosed orally at 10, 30 and 100 mg/kg/day to 6-week-old male Crl:CD (SD) rats daily for 14 days. Treatment with MP resulted in an increase in micronucleated hepatocytes (MNHEPs) with a dosage of only 100 mg/kg/day. At this dose level, cytotoxicity followed by regenerative cell growth was noted in the liver. These findings suggest that MP may induce clastogenic effects indirectly on the liver or hepatotoxicity of MP followed by regeneration may cause increase in spontaneous incidence of MNHEPs.
The hepatoprotective action of ursodeoxycholic acid (UDCA) was previously suggested to be partially dependent on its antioxidative effect. Doxorubicin (DOX) and reactive oxygen species have also been implicated in the overexpression of P-glycoprotein (P-gp), which is encoded by the MDR1 gene and causes antitumor multidrug resistance. In the present study, we assessed the effects of UDCA on the expression of MDR1 mRNA, P-gp, and intracellular reactive oxygen species levels in DOX-treated HepG2 cells and compared them to those of other bile acids. DOX-induced increases in reactive oxygen species levels and the expression of MDR1 mRNA were inhibited by N-acetylcysteine, an antioxidant, and the DOX-induced increase in reactive oxygen species levels and DOX-induced overexpression of MDR1 mRNA and P-gp were inhibited by UDCA. Cells treated with UDCA showed improved rhodamine 123 (Rho123) uptake, which was decreased in cells treated with DOX alone. Moreover, cells exposed to DOX for 24h combined with UDCA accumulated more DOX than that of cells treated with DOX alone. Thus, UDCA may have inhibited the overexpression of P-gp by suppressing DOX-induced reactive oxygen species production. Chenodeoxycholic acid (CDCA) also exhibited these effects, whereas deoxycholic acid and litocholic acid were ineffective. In conclusion, UDCA and CDCA had an inhibitory effect on the induction of P-gp expression and reactive oxygen species by DOX in HepG2 cells. The administration of UDCA may be beneficial due to its ability to prevent the overexpression of reactive oxygen species and acquisition of multidrug resistance in hepatocellular carcinoma cells.
Unlabelled:
Bile acid accumulation in liver with cholangiolar neoplastic lesions may occur before cholestasis is clinically detected. Whether this favors intrahepatic cholangiocarcinoma development has been investigated in this study. The E. coli RecA gene promoter was cloned upstream from Luc2 to detect in vitro direct genotoxic ability by activation of SOS genes. This assay demonstrated that bile acids were not able to induce DNA damage. The genotoxic effect of the DNA-damaging agent cisplatin was neither enhanced nor hindered by the hepatotoxic and hepatoprotective glycochenodeoxycholic and glycoursodeoxycholic acids, respectively. In contrast, thioacetamide metabolites, but not thioacetamide itself, induced DNA damage. Thus, thioacetamide was used to induce liver cancer in rats, which resulted in visible tumors after 30 weeks. The effect of bile acid accumulation on initial carcinogenesis phase (8 weeks) was investigated in bile duct ligated (BDL) animals. Serum bile acid measurement and determination of liver-specific healthy and tumor markers revealed that early thioacetamide treatment induced hypercholanemia together with upregulation of the tumor marker Neu in bile ducts, which were enhanced by BDL. Bile acid accumulation was associated with increased expression of interleukin (IL)-6 and downregulation of farnesoid X receptor (FXR). Bile duct proliferation and apoptosis activation, with inverse pattern (BDL > thioacetamide + BDL > thioacetamide vs. thioacetamide > thioacetamide + BDL > BDL), were observed. In conclusion, intrahepatic accumulation of bile acids does not induce carcinogenesis directly but facilitates a cocarcinogenic effect due to stimulation of bile duct proliferation, enhanced inflammation, and reduction in FXR-dependent chemoprotection.
Implications:
This study reveals that bile acids foster cocarcinogenic events that impact cholangiocarcinoma.
Human lymphocytes were either exposed to X-irradiation (25 to 200 rads) or treated with H2O2 (9.1 to 291 μM) at 4 °C and the extent of DNA migration was measured using a single-cell microgel electrophoresis technique under alkaline conditions. Both agents induced a significant increase in DNA migration, beginning at the lowest dose evaluated. Migration patterns were relatively homogeneous among cells exposed to X-rays but heterogeneous among cells treated with H2O2. An analysis of repair kinetics following exposure to 200 rads X-rays was conducted with lymphocytes obtained from three individuals. The bulk of the DNA repair occurred within the first 15 min, while all of the repair was essentially complete by 120 min after exposure. However, some cells demonstrated no repair during this incubation period while other cells demonstrated DNA migration patterns indicative of more damage than that induced by the initial irradiation with X-rays. This technique appears to be sensitive and useful for detecting damage and repair in single cells.
Bile acids, endogenous promoters of gastrointestinal cancer, activate protein kinase C (PKC) and the activator protein-1 (AP-1) transcription factor. Because other activators of PKC and AP-1 induce cyclooxygenase-2 (COX-2), we determined the effects of bile acids on the expression of COX-2 in human esophageal adenocarcinoma cells. Treatment with the dihydroxy bile acids chenodeoxycholate and deoxycholate resulted in an approximately 10-fold increase in the production of prostaglandin E2 (PGE2). Enhanced synthesis of PGE2 was associated with a marked increase in the levels of COX-2 mRNA and protein, with maximal effects at 8-12 and 12-24 h, respectively. In contrast, neither cholic acid nor conjugated bile acids affected the levels of COX-2 or the synthesis of PGE2. Nuclear run-off assays and transient transfections with a human COX-2 promoter construct showed that induction of COX-2 mRNA by chenodeoxycholate and deoxycholate was due to increased transcription. Bile acid-mediated induction of COX-2 was blocked by inhibitors of PKC activity, including calphostin C and staurosporine. Treatment with bile acid enhanced the phosphorylation of c-Jun and increased binding of AP-1 to DNA. These data are important because dihydroxy bile acid-mediated induction of COX-2 may explain, at least in part, the tumor-promoting effects of bile acids.
Human faecal waters from 35 healthy non-smoking volunteers (23 from England and 12 from Sweden) consuming their habitual diet were screened for genotoxicity by the single-cell gel electrophoresis (comet) assay using a human colon adenocarcinoma cell line (CACO-2) as the target. Hydrogen peroxide induced DNA damage was categorized as low, intermediate or high for tail moments greater than 5, 17 and 32, respectively: 11 samples were highly genotoxic, four were intermediate, one was low and 19 showed no activity. Endonuclease III treatment significantly increased DNA damage for all except the non-genotoxic faecal waters, suggesting that faecal water genotoxicity may be due, at least in part, to oxidative damage. Faecal water cytotoxicity has previously been attributed to the bile and fatty acid content. In the comet assay no DNA damage was induced by deoxycholate or lithocholate at normal physiological concentrations, suggesting that the genotoxicity of faecal water was due to other substances. Both bile acids induced DNA damage above 300 microM, levels often found in patients with colonic polyps and there was a significant increase in genotoxicity after endonuclease III treatment indicative of oxidative DNA damage.
Bile salts induce apoptosis and are implicated as promoters of colon cancer. The mechanisms by which bile salts produce these effects are poorly understood. We report that the cytotoxic bile salt, sodium deoxycholate (NaDOC), activates the key stress response proteins, NF-kappaB and poly(ADP-ribose) polymerase (PARP). The activation of NF-kappaB and PARP, respectively, indicates that bile salts induce oxidative stress and DNA damage. The pre-treatment of cells with specific inhibitors of these proteins [pyrrolidine dithiocarbamate (NF-kappaB inhibitor) and 3-aminobenzamide (PARP inhibitor)] sensitizes cells to the induction of apoptosis by NaDOC, indicating that these stress response pathways are protective in nature. Colon cancer risk has been reported to be associated with resistance to apoptosis. We found an increase in activated NF-kappaB at the base of human colon crypts that exhibit apoptosis resistance. This provides a link between an increased stress response and colon cancer risk. The implications of these findings with respect to apoptosis and to colon carcinogenesis are discussed.
Epidemiological studies support the involvement of short-chain fatty acids (SCFA) in colon physiology and the protective role of butyrate on colon carcinogenesis. Among the possible mechanisms by which butyrate may exert its anti-carcinogenicity an antioxidant activity has been recently suggested. We investigated the effects of butyrate and mixtures of SCFA (butyrate, propionate and acetate) on DNA damage induced by H(2)O(2) in isolated human colonocytes and in two human colon tumour cell lines (HT29 and HT29 19A). Human colonocytes were isolated from endoscopically obtained samples and the DNA damage was assessed by the comet assay. H(2)O(2) induced DNA damage in normal colonocytes in a dose-dependent manner which was statistically significant at concentrations over 10 microM. At 15 microM H(2)O(2) DNA damage in HT29 and HT29 19A cells was significantly lower than that observed in normal colonocytes (P < 0.01). Pre-incubation of the cells with physiological concentrations of butyrate (6.25 and 12.5 mM) reduced H(2)O(2) (15 microM) induced damage by 33 and 51% in human colonocytes, 45 and 75% in HT29 and 30 and 80% in HT29 19A, respectively. Treatment of cells with a mixture of 25 mM acetate + 10.4 mM propionate + 6.25 mM butyrate did not induce DNA damage, while a mixture of 50 mM acetate + 20.8 mM propionate + 12.5 mM butyrate was weakly genotoxic only towards normal colonocytes. However, both mixtures were able to reduce the H(2)O(2)-induced DNA damage by about 50% in all cell types. The reported protective effect of butyrate might be important in pathogenetic mechanisms mediated by reactive oxygen species, and aids understanding of the apparent protection toward colorectal cancer exerted by dietary fibres, which enhance the butyrate bioavailability in the colonic mucosa.
Oxidative stress is believed to be an important mediator of neurodegeneration. However, the transcriptional pathways induced in neurons by oxidative stress that activate protective gene responses have yet to be fully delineated. We report that the transcription factor Sp1 is acetylated in response to oxidative stress in neurons. Histone deacetylase (HDAC) inhibitors augment Sp1 acetylation, Sp1 DNA binding, and Sp1-dependent gene expression and confer resistance to oxidative stress-induced death in vitro and in vivo. Sp1 activation is necessary for the protective effects of HDAC inhibitors. Together, these results demonstrate that HDAC inhibitors inhibit oxidative death independent of polyglutamine expansions by activating an Sp1-dependent adaptive response.
Barrett's oesophagus is an acquired precancerous condition that develops from mucosal injury incurred due to chronic gastro-oesophageal reflux. The aim of this study was to determine if bile and/or acid components of the refluxate can induce DNA damage in vitro. The oesophageal cell lines FLO-1 and HET1-A were exposed to primary bile salts, individually or as a mixture, and the secondary bile salt sodium deoxycholate, in neutral or acidified media. Cells were then examined in the comet assay to measure DNA strand breaks. Cell viability was also monitored. Acidified media induced DNA damage in a pH- and time-dependent manner. The primary bile compounds sodium glycocholate, glycocholic acid, sodium taurocholate and taurochenodeoxycholate, as an equimolar mixture (100 microM), caused a small but significant (P < 0.028) elevation in DNA damage, but only at neutral pH in FLO-1 cells. Sodium deoxycholate (100 microM) caused a significant (P < 0.008) elevation in DNA damage in both cell lines, but again only at neutral pH. These data suggest that specific components of gastro-oesophageal refluxate are capable of causing DNA damage and may participate in the genesis and progression of Barrett's oesophagus via this mechanism.
Epidemiologic studies have demonstrated variations in the incidence of colon cancer between populations and socioeconomic groups. Differences in dietary habits have been implicated in the risk of developing colon cancer. Diet appears to influence our colonic microflora. Such variations may allow for future utilization of the fecal flora as markers for screening and diagnosis of colon cancer. The composition of the diet not only dictates the available substrates for the flora but also helps to establish predictable and competitive relationships between intestinal bacteria. To appreciate the significance of populations deemed high and low risk based on host flora, an understanding of several dynamic microbial relationships and metabolites produced is necessary. In this review, we explore the critical relationships between bile acid 7 alpha-dehydroxylation, sulfidogenesis, methanogenesis, and how they relate to carbohydrate and bile acid metabolism. We summarize the chemopreventative, anticarcinogenic, and detoxifying activity of probiotics and prebiotics, as well as potential mechanisms for protection against colon cancer.
Butyrate, formed by bacterial fermentation of plant foods, has been suggested to reduce colon cancer risks by suppressing the proliferation of tumor cells. In addition, butyrate has been shown to induce glutathione S-transferases (GSTs) in tumor cell lines, which may contribute to the detoxification of dietary carcinogens. We hypothesize that butyrate also affects biotransformation in non-transformed colon cells. Thus, we have investigated the gene expression of drug metabolism genes in primary human colon tissue, premalignant LT97 adenoma and HT29 tumor cells cultured in an appropriate medium+/-butyrate. A total of 96 drug metabolism genes (including 12 GSTs) spotted on cDNA macroarrays (Superarray; n = 3) were hybridized with biotin-labeled cDNA probes. To validate the expression detected with Superarray, samples of LT97 cells were also analyzed with high density microarrays (Affymetrix U133A), which include biotransformation genes that overlap with the set of genes represented on the Superarray. Relative expression levels were compared across colon samples and for each colon sample+/-butyrate. Compared with fresh tissue, 13 genes were downregulated in primary cells cultivated ex vivo, whereas 8 genes were upregulated. Several genes were less expressed in LT97 (40 genes) or in HT29 (41 and 17 genes, grown for 72 and 48 h, respectively) compared with primary colon tissue. Butyrate induced GSTP1, GSTM2, and GSTA4 in HT29 as previously confirmed by other methods (northern blot/qPCR). We detected an upregulation of GSTs (GSTA2, GSTT2) that are known to be involved in the defence against oxidative stress in primary cells upon incubation with butyrate. The changes in expression detected in LT97 by Superarray and Affymetrix were similar, confirming the validity of the results. We conclude that low GST expression levels were favourably altered by butyrate. An induction of the toxicological defence system possibly contributes to reported chemopreventive properties of butyrate, a product of dietary fibre fermentation in the gut.
Evidence is accumulating that bile acids induce apoptosis in colonic cells. Therefore, it becomes important to study the underlying molecular mechanisms and the role of this phenomenon in tumor promotion. Minutes after exposure of HCT 116 and HT-29 cells to deoxycholate (DCA), DNA damage, measured using the COMET assay, was evident. Caspase-3 was rapidly activated in HCT 116 cells exposed to DCA, whereas in HT-29 cells, caspase-3 activation was delayed. Using transient transfections with reporter constructs, we showed that the transcription factors activator protein-1 (AP-1) and NF-kB were increased in HCT 116 cells, in a dose-dependent fashion, by DCA COX-2 promoter activity was also induced by DCA and using mutant COX-2 promoter plasmids, we showed that the ability of DCA to induce promoter activity was partly dependent upon a functional NF-kB and C/EBP site, and completely dependent on a functional c-AMP response element site. DNA damage thus appears to be the initiating event in DCA-induced apoptosis. In conclusion, the bile acid, DCA, has a major impact on apoptotic mechanisms in colonic cells and this may be contributing to its effect as a tumor promoter.
Epidemiological studies have clearly demonstrated a link between dietary carotenoids and the reduced incidence of certain diseases, including some cancers. However recent intervention studies (e.g. ATBC, CARET and others) have shown that β-carotene supplementation has little or no beneficial effect and may, in fact, increase the incidence of lung cancers in smokers. This presents a serious dilemma for the scientific community — are carotenoids at high concentrations actually harmful in certain circumstances?
Currently, a significant number of intervention studies are on-going throughout the world involving carotenoids (of both natural and synthetic origin). Our approach has been to study the ability of supplementary carotenoids in protecting cells against oxidatively-induced DNA damage (as measured by the comet assay), and membrane integrity (as measured by ethidium bromide uptake). Both lycopene and β-carotene only afforded protection against DNA damage (induced by xanthine/xanthine oxidase) at relatively low concentrations (1–3 μM). These levels are comparable with those seen in the plasma of individuals who consume a carotenoid-rich diet. However, at higher concentrations (4–10 μM), the ability to protect the cell against such oxidative damage was rapidly lost and, indeed, the presence of carotenoids may actually serve to increase the extent of DNA damage. Similar data were obtained when protection against membrane damage was studied.
This would suggest that supplementation with individual carotenoids to significantly elevate blood and tissue levels is of little benefit and, may, in fact, be deleterious. This in vitro data presented maybe significant in the light of recent intervention trials.
Toxic bile salts, retained within the liver because of impaired biliary excretion, are considered to play a major role in liver injury during cholestasis. Bile salts cause cellular stresses that may result in apoptosis. To better understand such cellular stresses, the effect of the bile salt sodium deoxycholate (NaDOC) on activation of 13 specific gene promoters or response elements associated with different cellular stresses was measured in the transformed human hepatoma line, HepG2. NaDOC was found to activate transcription factors and induce or activate the promoters of genes that respond to protein malfolding (grp78 and hsp70), DNA damage (gadd153, hsp70 and c-fos), oxidative stress (NF-κB, c-fos, hsp70 and gadd153), ER stress (grp78) and Ca++ imbalance (grp78).
We evaluated genotoxic and cytotoxic effects of the three non-mutagenic and non-carcinogenic compounds p-nitrophenol, d-menthol and sodium N-lauroyl sarcosine which have previously been shown to induce DNA double strand breaks (DNA dsb) secondary to induced cytotoxicity. We tested wheter genotoxic effects in the alkaline single cell gel test (comet assay) may be confounded by cytotoxicity-induced DNA dsb. Cell viability was determined at the end of the treatment using the fluorescein diacetate/ethidium bromide-assay and plating efficiency was used as an indicator of long-term survivability. Experiments with V79 Chinese hamster cells and human white blood cells revealed negative results in the comet assay despite strong cytotoxic effects. However, cells with extremely fragmented DNA (‘clouds’) occured but were excluded from the evaluation under the principle that they represent dead cells. We also noticed a significant loss of cells at cytotoxic concentrations that might be attributed to the induction of highly fragmented DNA which is lost during electrophoresis. Since the comet assay allows the determination of DNA effects on the single cell level, a confounding effect of cytotoxicity on test results can be avoided.
In gerontology, there are abundant data indicating that functional cell capacity decreases with age. Clinical practice and the results of several studies have demonstrated that a high (depending on the study) percentage of people over eighty are practically 'normal' (successful aging). The hypothesis is that they are in fact in much the same position as the normal population of middle age. The reason for this may be the genetic stability of these persons. With time, the amount of damage to DNA rises and accumulates, which leads to cancer, heart problems and getting old. In genetically stable individuals, that period of accumulation is longer so they live longer. In humans, there appear to be two subgroups that age at different rates. The slower-aging group have better DNA repair systems. Genome stability plays a fundamental role in such age-related differences. The evolution of the human race would have been much slower if it had involved only genetically stable individuals. From the individual's point of view, however, it is better to have genetic stability. Regardless of the classical theory of aging, the lifespan of a species is related to metabolic rate. The apes and the humans have practically the same rate of metabolism, but humans live twice as long as apes. A similar phenomenon has been observed in other species and other tissues. Modification of DNA repair capacity plays a fundamental role in determination of the life span of a species. Genetic instability could be one of the basic reasons for senescence. The rise of chromosomal aberrations in long-lived persons is attributable to their genetic stability. They are practically in the same position as a normal population at middle age. Genetic instability is often associated with cancer and many different disorders of the immune system, which are frequent in the elderly.
The promoting effect of sodium deoxycholate (DC) on colon carcinogenesis was studied in female F344 germfree rats. Animals received intrarectal (ir) instillations of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) for 4 weeks (total dose, 16 mg/rat), then weekly ir doses of DC (total dose, 3 g/rat); the rats were autopsied 52 weeks after the first injection. DC increased the number of MNNG-induced colon adenocarcinomas. No tumors were in the colons of germfree rats given DC alone. It was concluded that DC (present in high concentrations in human stools) had a promoting effect on colon carcinogenesis in rats.
Protein kinase C (PKC) is the target for a number of tumor promoters. The mechanism underlying the promoting effects of bile acids in colorectal cancer is not understood. We report that sodium deoxycholate (DOC) triggered activation of PKC in physiological conditions. The biphasic effects of DOC upon PKC activation were Ca(2+)-stimulated and did not require phosphatidylserine (PtdSer) as phospholipid co-factor. The optimal rate of activation was obtained at 0.4 mM DOC and reached approximately half the maximal rate of activation obtained in the presence of PtdSer. Similarly to PtdSer, DOC supported diacylglycerol- as well as phorbol-ester-mediated PKC activation. The reciprocal effects of PtdSer and DOC upon PKC in either 0.5 mM CaCl2 or 0.5 mM EGTA suggest that DOC interacts with the phospholipid-binding domain to elicit PKC activation. DOC-supported enzyme activation exhibited substrate specificity different from that of PtdSer-supported enzyme activation. All tested primary and secondary bile acids activated PKC to various extents, with DOC being the most potent. We suggest that amphipathic bile acids acting in a PtdSer-like manner provide the hydrophobic environment required for PKC activation. Treatment of 32P-labeled platelets and colonic cells HT29 Cl.19A with DOC enhanced the phosphorylation of endogenous substrates for PKC. Colonic cells responsive at 50 microM DOC, appeared to be 10-fold more sensitive than platelets. We suggest that direct or indirect activation of PKC by bile acids may account for the promoting effects of these non-phorbol-ester-type tumor promoters.
The effect of 2 bile acids in colon carcinogenesis was studied in rats. A single dose of N methyl N' nitro N nitrosoguanidine (MNNG) was instilled intrarectally. Subsequently, repeated intrarectal doses of lithocholic or taurodeoxycholic acid were administered. Both bile acids increased the frequency of MNNG induced colorectal neoplasms. Neither bile acid induced tumors when used alone. The lesions were like those induced by MNNG alone, and included adenomas and adenocarcinomas. The bile acids acted as promotors of carcinogenesis.
Analysis of the etiologic factors and relevant mechanisms involved in carcinogenesis leads to a classification of agents involved in the carcinogenic process as genotoxic or epigenetic. Their mode of action is distinct, especially with regard to dose-response effects and reversibility. The genotoxic carcinogens for colon cancer are unknown, but mutagenic components found in fried beef and fish are under study. Epigenetic agents as promoting factors play a major role in the development of cancer of the colon. Specific nutritional elements associated with colon cancer risk are high fat diets, high cholesterol intake, and low fiber intake. The role of micronutrients as modulators and inhibitors needs to be explored. Through metabolic studies in diverse populations and in reliable animal models, it is now clear that dietary fat and cholesterol control the total flow of bile acids in lumen and a high-fat, high-cholesterol diet increases the total of bile acids in the gut. Bile acids but not neutral sterols have promoting effects and are related to colon cancer risk although bile acids by themselves do not act as complete carcinogens. The effect of dietary fiber such as cereal bran is to increase stool bulk which dilutes the concentration of bile acids. Reducing the concentration of bile acids either by lower dietary fat and cholesterol or by increasing dietary fiber may effectively lower the risk for colon cancer.
Administration of cholic acid (1.0% of the diet) to male Fisher rats for 3 days resulted in increased numbers of DNA synthesizing epithelial cells per colonic crypt column as compared to those found in either control or 0.2% cholic acid-fed rats. The middle third of the crypt was the area stimulated to contribute the additional proliferating cells. The maximum number of 3H-TdR-labeled cells was doubled by 24 h and migration had processed further up the colonic crypt of the 1% cholic acid-fed rats than the 0.2% cholic acid or control animals. Compared with cholic acid-deprived rats, long-term dietary intake of 0.2% cholic acid (26 weeks) was found to heighten the numbers of labeled cells per column and expand the proliferative compartment. The enhanced manifestation of colonic neoplasia in MNU-induced rats consuming cholic acid (previously reported by us) appears related to the elevated levels of cell proliferation brought about in response to the deleterious action of the bile acid on the mucosa. Increased numbers of epithelial cells undergoing DNA synthesis in cholic acid-treated animals would allow the earlier expression of malignant transformation in the large intestine.
During the last 2 decades, substantial progress has been made in understanding the relationship between dietary constituents and the development of colon cancer in man. Unlike studies of cancer among smokers and nonsmokers, nutritional epidemiologic studies are confronted with the inherent difficulty of assessing reasonably precise exposures. The lack of consistency between international correlation studies and case-control studies does not necessarily negate a dietary etiology of colon cancer because these inconsistencies may have arisen, at least in part, from methodological limitations. Some of these deficiencies in epidemiological studies of diet and cancer have been corrected; recent case-control studies demonstrated that high dietary fat is a risk factor for colon cancer development and that an overall increase in intake of foods high in fiber might decrease the risk for colon cancer. The results of epidemiologic studies may be assumed to present conservative estimates of the true risk for cancer associated with diet. The populations with high incidences of colon cancer are characterized by high consumption of dietary fat, which may be a risk factor in the absence of factors that are protective, such as whole-grain cereals and of other high fiber. Laboratory-animal model studies have shown that certain dietary lipids and fibers influence tumorigenesis in the colon. The data of metabolic epidemiological and laboratory-animal model studies are sufficiently convincing with respect to the enhancement of colon cancer by type of fat and protection by certain dietary fibers.
Expression of protein kinase C (PKC) isoenzymes was determined in paired samples of normal mucosa and colorectal cancer tissue from 13 patients. Total PKC activity in cancer tissue was significantly decreased compared to that in normal mucosa. Western blotting, using PKC isoenzyme-specific antibodies, showed that two PKC isoenzymes, PKC beta and PKC epsilon, were significantly decreased in cancer tissue. The level of PKC delta was increased in cancer tissue and the expression of PKC alpha and zeta was not altered significantly. Primary bile acids--cholic acid (CA) and chenodeoxycholic acid (CDCA)--and the principal secondary bile acids--deoxycholic acid (DCA), lithocholic acid (LCA) and ursodeoxycholic acid (UDCA)--were found to be potent and selective activators of partially purified PKC isoenzymes. PKC beta 1 was the isoenzyme most effectively activated by secondary bile acids. Our data provide a model for the involvement of secondary bile acids in colorectal carcinogenesis through specific PKC isoenzyme modulation in colorectal mucosa.
The in vitro experiment was carried out following 32P-postlabeling analysis to determine the DNA-reactive bile acids present in the human body. The unconjugated and conjugated forms of cholic (CA), chenodeoxycholic (CDCA), deoxycholic (DCA) and lithocholic acid (LCA) were added to calf thymus DNA followed by 1 h of incubation at 37 degrees C. After the incubation the mixture was analyzed by the nuclease P1 modification of 32P-postlabeling. Among the 12 bile acids tested, our results showed that unconjugated CDCA and LCA and the glycine and taurine conjugates of LCA (g-LCA, t-LCA) were able to bind covalently with naked DNA in vitro without intervention of any catalyst. It was also shown that bile acid alone did not give any spot on TLC. These binding reactions depended on the bile acid concentration in a linear manner. The data on the extent of binding at a concentration of 0.1 mg/ml showed values of 28.5 (t-LCA), 23.7 (g-LCA), 3.47 (LCA) and 1.32 (CDCA) adducts per 10(8) nucleotides. These reactive bile acids were also incubated with COLO 205 human colon carcinoma cells and Hep G2 human hepatocellular carcinoma cells for 24 h, but no specific DNA adduct was formed. Further, when LCA or CDCA was administered into male Fischer 344 rats by gavage at a dose of 10 mg/rat every 8 h for 3 days, no specific DNA adduct was detected in their liver or colon. Covalent DNA adducts are believed to cause alteration of the primary structure of genes, which is potentially linked to carcinogenesis. Though our preliminary data failed to detect any bile acid-related DNA adducts in the cultured cells or in rats, the results may provide a basis for assuming some of these bile acids to be potential initiators of colon cancer.
The endogenous production of oxidative damage in DNA by free radicals released as a by-product of respiration is a likely
cause of mutations which, if they occur in appropriate genes, may lead to cancer. Using an endonuclease specific for oxidized
pyrimidines, in conjunction with the highly sensitive method of single cell gel electrophoresis, we have detected significant
oxidative damage in untreated, freshly isolated lymphocytes from normal, healthy individuals.
A high-fat, low-fiber diet resulting in increased excretion of fecal secondary bile acids is regarded as a major risk factor for colon cancer. Incubation of human colonic biopsies with the secondary bile acid deoxycholic acid (DCA) leads to hyperproliferation with expansion of the proliferative zone, ie, a biomarker of increased cancer risk. Antiproliferative effects on various colon cancer cell lines, however, were reported for butyrate (BUT), a fermentation product of dietary fiber.
In the following in vitro study we incubated biopsies from the normal sigmoid colon of 12 patients (age 55.8 +/- 3.6 years) with 5 microM DCA or a combination of 5 microM DCA plus 10 mM BUT (DCA/BUT) and determined epithelial proliferation by bromodeoxyuridine immunohistochemistry. As a possible mediator for the DCA effects on colonic cell proliferation, mucosal prostaglandin E2 (PGE2) release into the incubation medium was measured by 125I-PGE2 radioimmunoassay.
Incubation with DCA alone revealed a significantly higher labeling index for the whole crypt (.17 vs .11, p < .01) and for the upper 40% of the crypt (.05 vs .01, p < .01) compared with DCA/BUT. Mucosal PGE2 release during DCA/BUT incubation showed a trend toward lower values compared with DCA incubation (357.07 vs 434.29 pg/mg per hour; p = .07).
The results indicate a normalization of DCA-induced hyperproliferation of colonic epithelium by butyrate that is not clearly mediated by PGE2. Considering that nutrition affects the luminal concentrations of DCA and butyrate, our findings may have implications for colonic carcinogenesis.
Studies on colon carcinogenesis suggest that the short-chain fatty acid butyrate may be protective, whereas the secondary bile acid deoxycholate may promote tumor development. Crypt surface hyperproliferation is regarded as a biomarker of colon cancer risk and can be modulated in vitro by the differentiation inducer butyrate and the tumor promoter deoxycholate. We hypothesized that butyrate decreases and deoxycholate increases crypt surface proliferation in vivo and that these effects are mediated by changes in the expression of the protooncogenes c-Fos and c-Jun, which are known to regulate proliferation and differentiation.
Twenty-five adult Sprague-Dawley rats underwent colonic isolation and 24-hour intraluminal instillation of 10 mmol/L sodium chloride, 10 mmol/ L sodium butyrate, or 10 mmol/L sodium deoxycholate. Proliferation of the whole crypt and five crypt compartments from base to surface was assessed by proliferating cell nuclear antigen immunohistochemistry. The øh value, an index of "premalignant" hyperproliferation, was calculated as the ratio of labeled cells in the two surface compartments divided by the labeled cells in the entire crypt. Expression of c-Fos and c-Jun was evaluated by Western blot.
Crypt surface proliferation and the øh value were significantly decreased by butyrate and increased by deoxycholate. Butyrate increased colonic expression of c-Jun, whereas deoxycholate significantly induced c-Fos.
The in vivo effects on surface proliferation are consistent with a potential protective [corrected] role for butyrate and a promotive role for deoxycholate in colon carcinogenesis. The concurrently observed effects on colonic c-Jun and c-Fos expression represent a novel finding and suggest that direct or indirect modulation of protooncogene expression may be the mechanism by which these dietary byproducts regulate proliferation in vivo.
In order to increase the understanding of the factors responsible for causing human colon cancer, a technique was developed to detect genotoxic effects of chemicals in human colon cells. Risk factors suspected to be associated with the aetiology of human colon cancer were subsequently investigated: the method is based on the measurement of DNA damage in primary cells freshly isolated from human colon biopsies with the single cell microgel ectrophoresis technique ('Comet Assay'). 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3-methyl-3H-imidazo[4,5f]quinoline (IQ), N-methyl-N-nitro-N-nitrosoguanidine (MNNG), dinitrosocaffeidine (DNC) lithocholic acid (LCA), hydrogen peroxide (H2O2) and benzo[a]pyrene (B[a]P) were investigated for their genotoxic and cytotoxic effects following 30 min incubation with colon cells of human, and for comparative purposes also of the rat colon. The nitrosamides (MNNG, DNC) were very genotoxic in human colon cells. MNNG was more genotoxic in human than in rat colon cells. In contrast, the rat colon carcinogens PhIP and IQ were not genotoxic in human colon cells. PhIP did induce DNA damage in rat colon cells, which correlates to its capacity of inducing tumors in this animal tissue. LCA was toxic (rat > human) and concomitantly caused DNA damage in higher concentrations. The widespread contaminant B[a]P was not genotoxic in colon cells of either species using this system. H2O2 was found to be a potent genotoxic agent to both rat and human colon cells (human > rat). In summary, those compounds chosen as representatives of endogenously formed risk factors (MNNG, H2O2, LCA) have a higher toxic and/or genotoxic potency in human colon tissue than in rat colon. They are also more effective in this system than the contaminants tested so far (B[a]P, PhIP, IQ). The newly developed technique is rapid and yields relevant results. It is a novel and useful approach to assess different chemical compounds for genotoxic activities in tumour target tissues of the human.
Increased bile acid secretion, as a consequence of a high fat diet, results in the increased production of bile acids that may escape the enterohepatic circulation, and be subsequently metabolised by the colonic micro-flora to form the co-mutagenic and co-carcinogenic secondary bile acids. The potential of the secondary bile acids lithocholate (LOC) and deoxycholate (DOC), to induce DNA damage, in the colonocyte cell line HT29, at physiological concentrations both individually and in a 2:1 ratio was assessed. Results indicated significant levels of DNA damage induced by both bile acids, with LOC having the greater DNA damaging capacity. The potential role of vitamin A, and the antioxidant vitamin E, in reducing this damage was determined, over a range of vitamin concentrations. Both vitamins reduced the bile acid induced DNA damage. Vitamin A displayed a dose response relationship, whereas vitamin E reduced DNA damage close to negative control values at all concentrations above 50 microM. These results indicate a protective role for Vitamins A and E, against the DNA damaging capacity of LOC and DOC.
Butyrate, a physiologically occurring agent, has been reported to decrease constitutively high expressed p53 levels in transformed cells. To elucidate whether butyrate also inhibits DNA-damage-induced p53 response we investigated the effects of butyrate and the anticancer drug mitomycin C in normal C3H10T1/2 cells harbouring wild-type p53. In comparison with p53-deficient fibroblasts we examined p53 protein level, cell cycle arrest, differentiation, and apoptosis. Butyrate induced G1 phase arrest, differentiation, and p53-independent increase in p21(waf1/cip1) protein. Moreover, butyrate induced p53-independent apoptosis, which was, as well as p53-mediated apoptosis, associated with a dose-dependent increase in Bax and c-Myc protein. Pretreatment with butyrate repressed dose-dependently mitomycin-C-induced p53 accumulation and interfered with p53-dependent cell cycle arrest. Butyrate further partially inhibited p53-mediated apoptosis, but low doses of butyrate were more effective than higher concentrations. This was reflected in an enhanced decrease in c-Myc and Bax protein in response to mitomycin C with low concentrations of butyrate. Our data indicate that the differentiation stimulus of butyrate, in association with p21(waf1/cip1) induction, and apoptosis, may explain antineoplastic effects of butyrate. Co-carcinogenic features of butyrate may result from inhibition of p53-mediated DNA damage response.
Crypt surface hyperproliferation is an intermediate biomarker of colon cancer risk. In vitro studies indicate that the short-chain fatty acid and antineoplastic agent butyrate may reverse the crypt surface hyperproliferation induced by the secondary bile acid and tumor promoter, deoxycholate. We hypothesized that butyrate may reverse deoxycholate-induced crypt surface proliferation in vivo.
Thirty-one Sprague-Dawley rats (250-300 g) underwent surgical isolation of the colon and 24-hour luminal instillation of either sodium chloride, butyrate, deoxycholate, or butyrate plus deoxycholate (all solutions, 2 ml; pH 7; total sodium = 20 mM). Study variables included colon weight, mucosal DNA, mucosal protein, and proliferating cell nuclear antigen immunohistochemistry, labeling of which was determined in five crypt compartments from base to surface (12 crypts per rat). Labeling indexes were calculated as proliferating cell nuclear antigen immunohistochemistry-labeled cells divided by total counted cells in the whole colonic crypt and each of five crypt compartments. The phi(h) value (an index of premalignant risk) was calculated as the ratio of labeled cells in the two surface compartments divided by the total labeled cells.
Deoxycholate significantly increased colon wet weight, mucosal protein, total crypt labeling indexes, crypt surface labeling indexes, and the phi(h) value and raised the mucosal DNA content. Butyrate alone slightly reduced total mucosal DNA and protein content. The combination of butyrate plus deoxycholate significantly decreased mucosal DNA and tended to reduce mucosal protein compared with deoxycholate alone. In contrast to prior in vitro findings, butyrate plus deoxycholate did not reverse the deoxycholate-induced surface hyperproliferative changes as measured by proliferating cell nuclear antigen labeling.
Because co-treatment with butyrate plus deoxycholate inhibits deoxycholate-induced increases in total mucosal DNA and protein content, we conclude that butyrate may play a role in maintaining the proliferative balance of the colonic mucosa, in vivo. However, co-treatment with butyrate plus deoxycholate does not reverse the deoxycholate-induced increases in colon weight and proliferating cell nuclear antigen labeling indexes under the studied experimental conditions.
Epidemiological studies have suggested that the concentration and composition of fecal bile acids are important determining factors in the etiology of colon cancer. However, the mechanism by which these compounds influence tumor development is not understood. To begin to elucidate their mechanism of action, four bile acids, cholic acid, chenodeoxycholic acid, deoxycholic acid (DCA), and ursodeoxycholic acid, were examined for their effects on the growth of several different tumor cell lines. We found that incubating cells with chenodeoxycholic acid or DCA caused morphological changes, seen by electron and light microscopy, that were characteristic of apoptosis, whereas incubating cells with ursodeoxycholic acid inhibited cell proliferation but did not induce apoptosis. Cholic acid had no discernible effect on cells. Notably, the apoptosis induced by DCA could be suppressed by inhibiting protein kinase C activity with calphostin C. These results indicate that different bile acids exhibit distinct biological activities and suggest that the cytotoxicity reported for DCA may be due to its capacity to induce apoptosis via a protein kinase C-dependent signaling pathway.
The balance of genetic damage and deactivating enzymes is decisive for cancer risk. To assess these factors in normal human colon cells, we determined background levels of DNA breaks or oxidized bases and of glutathione S-transferases (GSTs) as potential biomarkers of risk and chemoprevention, respectively. Also, genotoxicity by compounds involved in lipid peroxidation was determined to elucidate possible sources of damage. Cells were isolated from sigmoid biopsies of 51 donors and processed with the comet assay to reveal genetic damage. GST proteins were analyzed immunologically. HT29 clone 19A colon tumor cells, resembling primary cells, were treated with 2-trans-hexenal (400 microM) or hydrogen peroxide (75 microM) and processed for damage. Fifteen percent of primary colon cells contained strand breaks; 22% contained additional oxidized bases, with distinct sex differences. Similar damage was found in HT29 clone cells and is induced by both test compounds. GST levels were similar in both cell types. The comet assay is sufficiently sensitive to detect oxidative genetic damage in small amounts of cells from small amounts of biopsies. Lipid peroxidation is a possible risk factor. Together with GST as a potential biomarker of chemoprevention, the technique may serve as a valuable biomarker to assess exposure to risk factors.
Short chain fatty acids (SCFAs) are the end products of anaerobic bacteria break down of carbohydrates in the large bowel. This process, namely fermentation, is an important function of the large bowel; SCFAs, mainly acetate, propionate and butyrate account for approximately 80% of the colonic anion concentration and are produced in nearly constant molar ratio 60:25:15. Among their various properties, SCFAs are readily absorbed by intestinal mucosa, are relatively high in caloric content, are metabolized by colonocytes and epatocytes, stimulate sodium and water absorption in the colon and are trophic to the intestinal mucosa. While the fermentative production of SCFAs has been acknowledged as a principal mechanism of intestinal digestion in ruminants, the interest in the effects of SCFAs production on the human organism has been raising in the last ten years. SCFAs are of major importance in understanding the physiological function of dietary fibers and their possible role in intestinal neoplasia. SCFAs production and absorption are closely related to the nourishment of colonic mucosa, its production from dietary carbohydrates is a mechanism whereby considerable amounts of calories can be produced in short-bowel patients with remaining colonic function and kept on an appropriate dietary regimen. SCFAs enemas or oral probiotics are a new and promising treatment for ulcerative colitis. The effects have been attributed to the oxidation of SCFAs in the colonocytes and to the ability of butyrate to induce enzymes (i.e. transglutaminase) promoting mucosal restitution. Evidence is mounting regarding the effects of butyrate on various cell functions the significance of which needs further considerations. Up until now, attention has been related especially to cancer prophylaxis and treatment. This article briefly reviews the role of SCFAs, particularly butyrate, in intestinal mucosal growth and potential clinical applications in inflammatory and neoplastic processes of the large bowel.
Apoptosis is central to cell number regulation in the colonic epithelium, and interest in its role in colon carcinogenesis has been growing rapidly. It thus becomes of interest to characterize luminal components, possibly of dietary origin, that may influence this process. We have investigated the sensitivity of two human colonic cell lines, the human adenocarcinoma cell line (HT-29) and the human fetal colonic mucosa cell line (FHC), to induction of apoptosis by sodium butyrate, bile acids, and human fecal water fractions. The apoptotic effect has been studied by 1) morphological changes in cells examined by fluorescence microscopy, 2) DNA fragmentation analysis by gel electrophoresis, 3) flow cytometry analysis of DNA strand breaks assessed by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay (TUNEL), and 4) poly(ADP-ribose) polymerase cleavage by Western blot. Sodium butyrate and bile acids induced a time- and concentration-dependent apoptosis in both cell lines. Quantitation of this effect, by use of the TUNEL assay, indicated that deoxycholic acid was most effective in inducing this effect at lower concentrations and at shorter times. Apoptotic effects were also observed, in both cell lines, when the cells were exposed to intact human fecal waters (the fecal fraction in direct contact with the epithelium) and their lipid extracts, with the intact samples being more effective. Although all fecal waters examined induced apoptosis, quantitation of the effect by the TUNEL assay indicated that the ability to induce apoptosis differed markedly between samples. Induction of apoptosis by the fecal waters was not correlated to cytotoxicity but was negatively correlated to the pH of the samples. Interestingly, the cells derived from the fetal mucosa (FHC) were consistently less sensitive to apoptotic effects of the luminal components than the tumor-derived cells (HT-29). Thus human fecal water fractions induce apoptosis in colonic cells, and this effect is not due to lipid components alone.
Colorectal cancer is one of the most common internal malignancies in Western society. The cause of this disease appears to be multifactorial and involves genetic as well as environmental aspects. The human colon is continuously exposed to a complex mixture of compounds, which is either of direct dietary origin or the result of digestive, microbial and excretory processes. In order to establish the mutagenic burden of the colorectal mucosa, analysis of specific compounds in feces is usually preferred. Alternatively, the mutagenic potency of fecal extracts has been determined, but the interpretation of these more integrative measurements is hampered by methodological shortcomings. In this review, we focus on exposure of the large bowel to five different classes of fecal mutagens that have previously been related to colorectal cancer risk. These include heterocyclic aromatic amines (HCA) and polycyclic aromatic hydrocarbons (PAH), two exogenous factors that are predominantly ingested as pyrolysis products present in food and (partially) excreted in the feces. Additionally, we discuss N-nitroso-compounds, fecapentaenes and bile acids, all fecal constituents (mainly) of endogenous origin. The mutagenic and carcinogenic potency of the above mentioned compounds as well as their presence in feces, proposed mode of action and potential role in the initiation and promotion of human colorectal cancer are discussed. The combined results from in vitro and in vivo research unequivocally demonstrate that these classes of compounds comprise potent mutagens that induce many different forms of genetic damage and that particularly bile acids and fecapentaenes may also affect the carcinogenic process by epigenetic mechanisms. Large inter-individual differences in levels of exposures have been reported, including those in a range where considerable genetic damage can be expected based on evidence from animal studies. Particularly, however, exposure profiles of PAH and N-nitroso compounds (NOC) have to be more accurately established to come to a risk evaluation. Moreover, lack of human studies and inconsistency between epidemiological data make it impossible to describe colorectal cancer risk as a result of specific exposures in quantitative terms, or even to indicate the relative importance of the mutagens discussed. Particularly, the polymorphisms of genes involved in the metabolism of heterocyclic amines are important determinants of carcinogenic risk. However, the present knowledge of gene-environment interactions with regard to colorectal cancer risk is rather limited. We expect that the introduction of DNA chip technology in colorectal cancer epidemiology will offer new opportunities to identify combinations of exposures and genetic polymorphisms that relate to increased cancer risk. This knowledge will enable us to improve epidemiological study design and statistical power in future research.
Butyrate is produced in the colon by fermentation of dietary fibre and induces apoptosis in colon adenoma and cancer cell lines, which may contribute to the protective effect of a high fibre diet against colorectal cancer (CRC). However, butyrate is present in the colon together with unconjugated bile acids, which are tumour promoters in the colon. We show here that bile acids deoxycholate (DCA) and chenodeoxycholate (CDCA), at levels present in the colon, gave a modest increase in cell proliferation and decreased spontaneous apoptosis in AA/C1 adenoma cells. Bile acids significantly inhibited the induction of apoptosis by butyrate in AA/C1 cells. However, the survival-inducing effects of bile acids on AA/C1 cells could be overcome by increasing the concentration of sodium butyrate. These results suggest that dysregulation of apoptosis in colonic epithelial cells by dietary factors is a key factor in the pathophysiology of CRC.
Diets rich in fat result in higher concentrations of secondary bile acids or their salts in the colon, which may adversely affect cells of the colonic epithelium. Because secondary bile acids are thought to be genotoxic, exposing colon epithelial cells to secondary bile acids may induce DNA damage that might lead to apoptosis. The requirement for the p53 tumor suppressor gene in such events is unknown. In particular, the effects of secondary bile acids on colon epithelial cells having different p53 tumor suppressor gene status have not been examined. Therefore, HCT-116 and HCT-15 human colon adenocarcinoma cells, which express the wild-type and mutant p53 genes, respectively, were exposed to physiological concentrations of deoxycholate. The cells were then analyzed for evidence of DNA damage and apoptosis. After 2 h of incubation with 300 microM deoxycholate, both cell lines had greater levels of single-strand breaks in DNA as assessed by the comet assay. After 6 h of exposure to deoxycholate, HCT-116 and HCT-15 cells showed morphological signs of apoptosis, i.e., membrane blebbing and the presence of apoptotic bodies. Chromatin condensation and fragmentation were also seen after staining DNA with 4',6-diamidino-2-phenylindole. Other apoptotic assays revealed greater binding of annexin V-fluorescein isothiocyanate, as well as greater post-enzymatic labeling with dUTP-fluorescein isothiocyanate, by both cell lines exposed to deoxycholate. These data suggest that deoxycholate caused DNA damage in colon epithelial cells that was sufficient to trigger apoptosis in a p53-independent manner.
Apoptosis after the loss of cell anchorage--"anoikis"--plays an important role in the life cycle of adherent cells. Furthermore, loss of anchorage dependency is believed to be a critical step in metastatic transformation. The aim of this study was to further characterize the sequence of intracellular events during anoikis in a nontransformed population of human intestinal epithelial cells (IECs). Purified human IECs were kept in suspension to induce anoikis in over 90% of IECs within 3 h. Two initiator caspases, caspase-2 and -9, are activated within 15 min, followed by the hierarchical activation of downstream caspases within 1 h. The activation of the caspase FLICE (caspase-8) does not contribute to the initiation of anoikis, and massive release of cytochrome c from mitochondria cannot be detected before 60 min, indicating that cytochrome c release does not play a role during initiation of anoikis. This study delineates the signaling cascade during anoikis of nontransformed cells. Future studies may identify alterations of this cascade in neoplastic cells, thereby possibly gaining insight into carcinogenesis and metastatic transformation.
Evidence is accumulating that bile acids induce apoptosis in colonic cells. Therefore, it becomes important to study the underlying molecular mechanisms and the role of this phenomenon in tumor promotion. Minutes after exposure of HCT 116 and HT-29 cells to deoxycholate (DCA), DNA damage, measured using the COMET assay, was evident. Caspase-3 was rapidly activated in HCT 116 cells exposed to DCA, whereas in HT-29 cells, caspase-3 activation was delayed. Using transient transfections with reporter constructs, we showed that the transcription factors activator protein-1 (AP-1) and NF-kB were increased in HCT 116 cells, in a dose-dependent fashion, by DCA COX-2 promoter activity was also induced by DCA and using mutant COX-2 promoter plasmids, we showed that the ability of DCA to induce promoter activity was partly dependent upon a functional NF-kB and C/EBP site, and completely dependent on a functional c-AMP response element site. DNA damage thus appears to be the initiating event in DCA-induced apoptosis. In conclusion, the bile acid, DCA, has a major impact on apoptotic mechanisms in colonic cells and this may be contributing to its effect as a tumor promoter.
Recent epidemiological evidence and animal studies suggest a relationship between the intake of olive oil and a reduced risk of several malignancies. The present study assesses the effect of hydroxytyrosol, a major antioxidant compound of virgin olive oil, on proliferation, apoptosis and cell cycle of tumour cells. Hydroxytyrosol inhibited proliferation of both human promyelocytic leukaemia cells HL60 and colon adenocarcinoma cells HT29 and HT29 clone 19A. The con-centrations of hydroxytyrosol which inhibited 50% of cell proliferation were approximately 50 and approximately 750 micromol/l for HL60 and both HT29 and HT29 clone 19A cells, respectively. At concentrations ranging from 50 to 100 micromol/l, hydroxytyrosol induced an appreciable apoptosis in HL60 cells after 24 h of incubation as evidenced by flow cytometry, fluorescence microscopy and internucleosomal DNA fragmentation. Interestingly, no effect on apoptosis was observed after similar treatment of freshly isolated human lymphocytes and polymorphonuclear cells. The DNA cell cycle analysis, quantified by flow cytometry, showed that the treatment of HL60 cells with hydroxytyrosol 50-100 micromol/l arrested the cells in the G0/G1 phase with a concomitant decrease in the cell percentage in the S and G2/M phases. These results support the hypothesis that hydroxytyrosol may exert a protective activity against cancer by arresting the cell cycle and inducing apoptosis in tumour cells, and suggest that hydroxytyrosol, an important component of virgin olive oil, may be responsible for its anticancer activity.
Epidemiologic studies indicate that environmental (smoking) and dietary factors (high fat) contribute to carcinogenesis in many organ systems. The aim of our study was to test the hypothesis that nicotine, a component of cigarette smoke, and sodium deoxycholate (NaDOC), a cytotoxic bile salt that increases in concentration in the gastrointestinal tract after a high fat meal, induce similar cellular stresses and that nicotine may enhance some of the NaDOC-induced stresses. We found that nicotine, at 0.8 microM, the very low sub-micromolar level occurring in the tissues of smokers: (1). increases oxidative stress; (2). activates NF-kappaB, a redox-sensitive transcription factor; (3). activates the 78 kD glucose regulated protein promoter, an indication of endoplasmic reticulum stress; (4). induces apoptosis; (5). enhances the ability of NaDOC to activate the 153 kD growth arrest and DNA damage promoter, an indication of increased genotoxic stress; and (6). enhances the ability of NaDOC to activate the xenobiotic response element. Our findings have applicability to G.I. cancer, in general, since smoking is a risk factor in the development of esophageal, pancreatic, gastric and colon cancer, and these cancers are also promoted by bile acids.
The free water phase of feces (fecal water) may mediate the effects of diet on colon carcinogenesis. We examined the effects of fecal water from adenoma patients and controls on three parameters in colonocytes believed to be relevant to tumorigenesis, i.e. genotoxicity in intact cells and on isolated DNA, proliferative activity and activator protein-1 (AP-1) activity.
Genotoxicity in intact colonic cells was assayed using the single-cell gel electrophoresis assay ('comet' assay) and on isolated DNA using double-stranded DNA from the X-174 RF plasmid. Cell proliferation was assessed using the commercially available 'alamar blue' proliferation kit and AP-1 activity using cells transiently transfected with an AP-1-luciferase reporter construct.
The data showed that lipid extracts of fecal water samples from the adenoma patients had a significantly higher capacity to induce cell proliferation than those from controls, and that this effect could be explained to a large extent by the concentrations of deoxycholic and chenodeoxycholic acids in the fecal water using regression models. No difference between patients and controls was observed for induction of AP-1 activity or induction of DNA strand breaks in intact cells. However, induction of DNA strand breaks in isolated DNA was significantly higher for the fecal waters from patients than for those from controls, which could be explained in part in a regression model by concentrations of lithocholic acid in fecal water and fecapentaene-12 in feces.
Our results support the hypothesis that the biochemistry of fecal waters from adenoma patients and controls differs.
The colonic epithelium is often exposed to high concentrations of secondary bile acids, which stresses the epithelial cells, leading potentially to activation of stress-response genes. To examine this possibility in vitro, the purpose of this study was to determine if expression of certain growth arrest and DNA damage-inducible genes (GADD) is upregulated in human colonic epithelial cells exposed to deoxycholate (DOC). DNA macroarray screening of a small cluster of stress/apoptosis-related genes in DOC-treated HCT-116 colonocytes revealed clearly higher expression of only GADD45, which was confirmed by gene-specific relative RT-PCR analysis. Subsequently, it was found that DOC also increased GADD34 mRNA expression. However, mRNA expression of GADD153 was increased most markedly in DOC-treated HCT-116 colonocytes, which express wild-type p53. However, the upregulation of GADD34, GADD45, and GADD153 mRNA expression apparently did not require p53, based on the finding that DOC increased expression of all three GADD genes in HCT-15 colonocytes, which express mutant p53. In further studying GADD153 in particular, the effect of DOC on GADD153 mRNA was prevented by actinomycin-D (Act-D), but not by antioxidants or MAPK inhibitors. DOC also caused GADD153 protein to be expressed in close parallel with increased GADD153 mRNA expression. Induction of GADD153 protein by DOC was prevented by either anisomycin or cycloheximide. These findings suggest that DOC-induced upregulation of GADD153 mRNA expression occurred at the level of transcription without involving reactive oxygen species and MAPK signaling, and that the expression of GADD153 protein was due also to translation of pre-existing, and not just newly synthesized, mRNA.
LT97 human colonic adenoma cells reflecting early premalignant genotype and growth characteristics have been posed to tumor promoting bile acids in order to identify marker genes that permit identification of tumor promoters in vitro. Physiologically relevant concentrations of desoxycholate (DOC) and chenodesoxycholate (CDC) upregulated expression of c-fos and COX-2 in a concentration- and time-dependent manner. Transient induction of c-fos was seen with the non-promoting taurodesoxycholate (TDOC) as well as DOC, however extended induction at 3 h was only achieved by DOC and CDC reaching 3-6-fold as compared to the control. Stimulation of COX-2 expression was completely specific for the tumor promoting analogs DOC and CDC. It was about 4-fold in the 80 microM DOC and CDC groups after 3 h and increased to 12- and 7-fold respectively after 6 h. Expression of VEGF was stimulated 4-5-fold in the tumor promoter (DOC and CDC) groups and about 2-fold in the non-promoting controls TDOC and GCDC. At later times the tumor promoter specific difference was lost. Our results show that all three genes are modulated in a tumor promoter dependent way and that their upregulation in LT97 adenoma cells can be used for in vitro testing of colon tumor promoters and chemopreventive compounds.
Bile acids were first proposed to be carcinogens in 1939 and 1940. On the basis of later work with rodent models, bile acids came to be regarded as cancer promoters rather than carcinogens. However, considerable indirect evidence, obtained more recently, supports the view that bile acids are carcinogens in humans. At least 15 reports, from 1980 through 2003, indicate that bile acids cause DNA damage. The mechanism is probably indirect, involving induction of oxidative stress and production of reactive oxygen species that then damage DNA. Repeated DNA damage likely increases the mutation rate, including the mutation rate of tumor suppressor genes and oncogenes. Additional reports, from 1994 through 2002, indicate that bile acids, at the increased concentrations accompanying a high fat diet, induce frequent apoptosis. Those cells within the exposed population with reduced apoptosis capability tend to survive and selectively proliferate. That bile acids cause DNA damage and may select for apoptosis-resistant cells (both leading to increased mutation), indicates that bile acids are likely carcinogens. In humans, an increased incidence of cancer of the laryngopharyngeal tract, esophagus, stomach, pancreas, the small intestine (near the Ampulla of Vater) and the colon are associated with high levels of bile acids. The much larger number of cell generations in the colonic (and, likely, other gastrointestinal) epithelia of humans compared to rodents may allow time for induction and selection of mutations leading to cancer in humans, although not in rodents.
Butyrate, the four-carbon fatty acid, is formed in the human colon by bacterial fermentation of carbohydrates (including dietary fiber), and putatively suppresses colorectal cancer (CRC). Butyrate has diverse and apparently paradoxical effects on cellular proliferation, apoptosis and differentiation that may be either pro-neoplastic or anti-neoplastic, depending upon factors such as the level of exposure, availability of other metabolic substrate and the intracellular milieu. In humans, the relationship between luminal butyrate exposure and CRC has been examined only indirectly in case-control studies, by measuring fecal butyrate concentrations, although this may not accurately reflect effective butyrate exposure during carcinogenesis. Perhaps not surprisingly, results of these investigations have been mutually contradictory. The direct effect of butyrate on tumorigenesis has been assessed in a number of in vivo animal models, which have also yielded conflicting results. In part, this may be explained by methodological differences in the amount and route of butyrate administration, which are likely to significantly influence delivery of butyrate to the distal colon. Nonetheless, there appears to be some evidence that delivery of an adequate amount of butyrate to the appropriate site protects against early tumorigenic events. Future study of the relationship between butyrate and CRC in humans needs to focus on risk stratification and the development of feasible strategies for butyrate delivery.
Interest has been recently rekindled in short chain fatty acids (SCFAs) with the emergence of prebiotics and probiotics aimed at improving colonic and systemic health. Dietary carbohydrates, specifically resistant starches and dietary fiber, are substrates for fermentation that produce SCFAs, primarily acetate, propionate, and butyrate, as end products. The rate and amount of SCFA production depends on the species and amounts of microflora present in the colon, the substrate source and gut transit time. SCFAs are readily absorbed. Butyrate is the major energy source for colonocytes. Propionate is largely taken up by the liver. Acetate enters the peripheral circulation to be metabolized by peripheral tissues. Specific SCFA may reduce the risk of developing gastrointestinal disorders, cancer, and cardiovascular disease. Acetate is the principal SCFA in the colon, and after absorption it has been shown to increase cholesterol synthesis. However, propionate, a gluconeogenerator, has been shown to inhibit cholesterol synthesis. Therefore, substrates that can decrease the acetate: propionate ratio may reduce serum lipids and possibly cardiovascular disease risk. Butyrate has been studied for its role in nourishing the colonic mucosa and in the prevention of cancer of the colon, by promoting cell differentiation, cell-cycle arrest and apoptosis of transformed colonocytes; inhibiting the enzyme histone deacetylase and decreasing the transformation of primary to secondary bile acids as a result of colonic acidification. Therefore, a greater increase in SCFA production and potentially a greater delivery of SCFA, specifically butyrate, to the distal colon may result in a protective effect. Butyrate irrigation (enema) has also been suggested in the treatment of colitis. More human studies are now needed, especially, given the diverse nature of carbohydrate substrates and the SCFA patterns resulting from their fermentation. Short-term and long-term human studies are particularly required on SCFAs in relation to markers of cancer risk. These studies will be key to the success of dietary recommendations to maximize colonic disease prevention.
Nuclear factor kappa B (NF-κB) is a redox-associated transcription factor that is involved in the activation of survival pathways.
We have previously shown that deoxycholate (DOC) activates NF-κB in hepatocytes and colon epithelial cells and that persistent
exposure of HCT-116 cells to increasing concentrations of DOC results in the constitutive activation of NF-κB, which is associated
with the development of apoptosis resistance. The mechanisms by which DOC activates NF-κB in colon epithelial cells, and whether
natural antioxidants can reduce DOC-induced NF-κB activation, however, are not known. Also, it is not known if DOC can generate
reactive oxygen species within mitochondria as a possible pathway of stress-related NF-κB activation. Since we have previously
shown that DOC activates the NF-κB stress-response pathway in HCT-116 cells, we used this cell line to further explore the
mechanisms of NF-κB activation. We found that DOC induces mitochondrial oxidative stress and activates NF-κB in HCT-116 cells
through multiple mechanisms involving NAD(P)H oxidase, Na+/K+-ATPase, cytochrome P450, Ca++ and the terminal mitochondrial respiratory complex IV. DOC-induced NF-κB activation was significantly (P < 0.05) inhibited by pre-treatment of cells with CAPE, EGCG, TMS, DPI, NaN3, EGTA, Ouabain and RuR. The NF-κB-activating pathways, induced by the dietary-related endogenous detergent DOC, provide mechanisms
for promotion of colon cancer and identify possible new targets for chemoprevention.
Polyunsaturated fatty acids (PUFAs) play an important role in both induction and prevention of carcinogenic process. It is well known that several types of neoplastic cells show decreased total PUFA content, contributing to their resistance to chemotherapy and lipid peroxidation. In the light of this, human lung cancer A549 cells, with low PUFA content, were exposed to arachidonic or docosahexaenoic acid to investigate the effect of n-6 and n-3 PUFAs on growth and elucidate underlying mechanisms. The bulk of the results showed that both n-6 PUFAs and n-3 PUFAs decrease human lung-tumor cell growth in a concentration-dependent manner, inducing cell death mainly evident at 100microM concentration. The mechanism underlying the antiproliferative effect of n-6 and n-3 PUFAs appeared to be the same, involving changes in fatty acid composition of biomembranes, production of cytostatic aldehydes derived from lipid peroxidation and able to affect DNA-binding activity of AP-1, and induction of PPAR. From these results it may be hypothesized that n-6 PUFAs, like n-3 PUFAs, are able to inhibit tumor growth.
Colonic health: fermentation and short chain fatty acids
- JMW Wong