FIGURE 1 - uploaded by Linda M Sargent
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Ratios of individual phenotypes of AHF compared with the "any" phenotype in male and female rats as shown after (A) promotion with phenobarbital or (B) initiation with diethylnitrosamine and promotion with phenobarbital. The marker symbols employed are: S placental form of glutathione S-transferase; R, y-glutamyl transpeptidase; Y, canalicular ATPase; G, glucose 6-phosphatase; and the combinations as shown. The reader is referred to the original reference for further details (36).
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Specific biochemical changes occurring during hepatocarcinogenesis have been sought by many investigators. The development of multistage models for hepatocarcinogenesis in the rodent has renewed interest in such marker alterations in preneoplastic as well as neoplastic hepatocytes. Preneoplastic altered hepatic foci (AHF) exhibit specific histomorp...
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Context 1
... order to study in a quantitative manner the dis- tribution of phenotypic markers of AHF when multiple markers are used for analysis, suitable methodologies are necessary. Campbell et al. (53) developed such a technique for use with three markers, and recently Xu et al. (36) have extended this to four markers and po- tentially even more. Earlier studies by Peraino et al. (34) had demonstrated that, when as many as six mark- ers were used, the phenotypic heterogeneity was such that virtually any of the more than 100 combinations of these markers could be qualitatively identified within one or more AHF. Using the quantitative technology described, Xu et al. (36) described the variation in the distribution of four markers in AHF of livers of animals subjected to an initiation-promotion protocol, with di- ethylnitrosamine (DEN) as initiator and phenobarbital (PB) as promoting agent and with age and sex as var- iables. The distribution ratio of phenotypes in this ex- periment, based on the "any" category (36,53), may be seen in Figure 1 for both males and females in animals that had been initiated and promoted compared with those that received no initiation with DEN but were given PB to promote endogenous or fortuitous AHF. As can be seen, the distributions are significantly dif- ferent between those focal lesions initiated with DEN, which includes spontaneous AHF as well, but the latter are considerably fewer in number than the former. Fur- thermore, the distribution changes with age in both sexes. These quantitative changes argue strongly that tumor promotion, as exemplified by the development of AHF in multistage hepatocarcinogenesis, is quite de- pendent on the environment as well as on the initiating agent itself, on the assumption that spontaneously ini- tiated cells that developed into AHF resulted from agents other than ...
Citations
... In mammals, AHF as a pre-neoplastic lesion has been recognized for many years as part of the general etiology of HCC (Goldsworthy et al. 1986;Beer and Pitot 1987;Sargent et al. 1989;Pitot 1990;Pitot et al. 1990Pitot et al. , 1991Dragan et al. 1995). In rats, the presence of AHF with altered growth characteristics has been observed in a number of studies (Newberne 1976;Nishizumi et al. 1977;Manson et al. 1984;Bannasch et al. 1985;Godlewski et al. 1985;Fischer 1986;Fischer et al. 1987;Gil et al. 1988;Harada et al. 1989b;Roebuck et al. 1991;Campbell 1992a, 1992b). ...
Adverse outcome pathways (AOPs) are frameworks starting with a molecular initiating event (MIE), followed by key events (KEs) linked by KE relationships (KERs), ultimately resulting in a specific adverse outcome. Relevant data for the pathway and each KE/KER are evaluated to assess biological plausibility, weight-of-evidence, and confidence. We aimed to describe an AOP relevant to chemicals directly inducing mutation in cancer critical gene(s), via the formation of chemical-specific pro-mutagenic DNA adduct(s), as an early critical step in tumor etiology. Such chemicals have mutagenic modes-of-action (MOA) for tumor induction. To assist with developing this AOP, Aflatoxin B1 (AFB1) was selected as a case study because it has a rich database and is considered to have a mutagenic MOA. AFB1 information was used to define specific KEs, KERs, and to inform development of a generic AOP for mutagen-induced hepatocellular carcinoma (HCC). In assessing the AFB1 information, it became clear that existing data are, in fact, not optimal and for some KEs/KERs, the definitive data are not available. In particular, while there is substantial information that AFB1 can induce mutations (based on a number of mutation assays), the definitive evidence – the ability to induce mutation in the cancer critical gene(s) in the tumor target tissue – is not available. Thus, it is necessary to consider the patterns of results in the weight-of-evidence for KEs and KERs. It was important to determine whether there was sufficient evidence that AFB1 can induce the necessary critical mutations early in the carcinogenic process, which was the case.
... However, for rat liver Steinmetz et al. (1998) showed that the Gstp promoter was hypomethylated with overexpressed protein levels in liver neoplasms. Overexpression of Gstp was previously used as a prognostic marker for rat hepatocarcinogenesis (Dragan et al., 1994;Pitot et al., 1991). Since ZF-L used in the current experiment is a cell line originating from zebrafish liver, the observed demethylation of Gstp1 induced by EE2 and DES may be interpreted in line with a neoplastic mechanism of action, but neither EE2 nor DES was to our knowledge previously reported to induce a hypomethylation in Gstp1 promoter as that noted here. ...
Early molecular events with correlation to disease, such as aberrant DNA methylation, emphasize the importance of DNA methylation as a potential environmental biomarker. Currently, little is known regarding how various environmental contaminants and mixtures alter DNA methylation in aquatic organisms, and testing is both time- and labor-consuming. Therefore, the potential of an in vitro screening method was evaluated by exposing zebrafish liver cells (ZF-L) for 96 h to the nonmutagenic model substance 5'-azacytidine (AZA), as well as a selection of environmental pollutants such as sodium arsenite (NAS), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 17α-ethinylestradiol (EE2), and diethylstilbestrol (DES). Six single genes with reported and anticipated importance in cancer were selected for analysis. Methylation of gene promoter areas was monitored by bisulfite conversion and high-resolution melt (HRM) analysis after exposure to sublethal concentrations of the test compounds. Subsequently, results were validated with direct bisulfite sequencing. Exposure of ZF-L cells to 0.5 μM AZA for 96 h led to hypomethylation of genes with both low and high basal methylation indicating similarity to mechanism of action in mammals. Further, NAS, EE2, and DES were shown to induce significant alterations in methylation, whereas TCDD did not. It was concluded that cell line exposure in combination with HRM may provide an initial contaminant screening assay by quantifying DNA methylation alterations with high throughput capacity. In addition, the rapid determination of effects following contaminant exposure with this in vitro system points to the possibility for new in vivo applications to be useful for environmental monitoring.
... Although cancer promotion is a preneoplastic stage, it is important to discriminate between promotion and progression in hepatocellular carcinogenesis, because promotion is reversible but progression is irreversible, and after this stage the prognosis is not good. Altered hepatic foci (AHF) occur as focal lesions in liver, and changes in AFH phenotype are useful as an index that provides criteria for the diagnosis and staging of liver disease (Pitot et al., 1991). To confirm the stages of multistage hepatocarcinogenesis, AFH may be analyzed based on morphology (Ward, 1981), altered expression of mRNA (Beer and Pitot, 1987), and/or protein biomarkers such as-glutamyl-transpeptidase (GGT), canalicular adenosine triphosphatase (ATPase), glucose-6-phosphatase (G6Pase), or the placental isoenzyme of glutathione S-transferase (GST) (Xu et al., 1990). ...
Hepatocellular carcinoma (HCC) is the fifth most frequent cause of cancer deaths in males and was the third most frequent cause of cancer deaths in 2007 throughout the world. The incidence rate is 2-3 times higher in developing countries than in developed countries. Animal models have enabled study of the mechanism of HCC and the development of possible strategies for treatment. Diethylnitrosamine (DEN) is a representative chemical carcinogen with the potential to cause tumors in various organs, including the liver, skin, gastrointestinal tract, and respiratory system. Specifically in HCC, DEN is a complete carcinogen. Many lines of evidence have demonstrated a relationship between carcinogenesis and cell cycle regulation. In this study we found that cell cycle regulatory proteins were critically involved in cancer initiation and promotion by DEN. Cyclin D1, cyclin E, cdk4, and p21(CIP1/WAF1) are factors whose expression levels may be useful as criteria for the classification of hepatic disease. In particular, cdk4 had a pivotal role in the transition to the neoplastic stage. In conclusion, we suggest that changes in the level of cdk4 may be useful as a biomarker for detection of HCC.
... However, this possibility appears to be highly unlikely in view of the following: (1) treatment with T3 for 1 week showed no inhibitory effect on GSTP expression of bile ducts, as shown by immunohistochemical staining of livers from CD ϩ T3-fed rats (Fig. 3A), and, (2) T3 treatment for 1 week did not modify the expression of GSTP in rat kidney, an organ that expresses a large amount of this protein (Fig. 3B). Finally, feeding a CD ϩ GC-1 diet caused a striking decrease in the number of preneoplastic lesions identified by their positivity for GGT, the second-best marker for preneoplasia, 25 compared with the animals fed the CD diet alone (11.6 Ϯ 2 foci/cm 2 versus 37.8 Ϯ 4 foci/cm 2 ). Taken together, these findings seem to rule out the possibility that the decrease in the number of GSTPpositive preneoplastic foci caused by TR ligands is merely a consequence of a loss of staining due to their inhibitory effect on GSTP gene expression or protein degradation. ...
Unlabelled:
Triiodothyronine (T3), through interaction with its intracellular thyroid hormone receptors (TRs), influences various physiological functions, including metabolism, development, and growth. We investigated the effect of T3 and the selective TR-beta agonist GC-1 in two models of hepatocarcinogenesis. Preneoplastic lesions were induced in F-344 rats via a single dose of diethylnitrosamine, followed by a choline-deficient (CD) diet for 10 weeks. Rat subgroups were then fed the CD diet or a CD diet containing either 4 mg/kg T3 or 5 mg/kg GC-1 for another week. Rats fed a CD diet alone showed a large number (65/cm(2)) of preneoplastic lesions positive for the placental form of glutathione S-transferase (GSTP). Coadministration of T3 for the last week caused an almost complete disappearance of the foci (3/cm(2)). A reduction of GSTP-positive foci was also observed in rats fed a CD + GC-1 diet (28/cm(2) versus 75/cm(2) of rats fed a CD diet alone) in the absence of significant differences in labeling or apoptotic index of preneoplastic hepatocytes between the two groups. An antitumoral effect of GC-1 was also observed with the resistant hepatocyte model of hepatocarcinogenesis. Nodule regression was associated with a return to a fully differentiated phenotype, indicated by the loss of the fetal markers GSTP and gamma glutamyl transpeptidase, and reacquisition of the activity of glucose 6-phosphatase and adenosine triphosphatase, two enzymes expressed in normal hepatocytes.
Conclusion:
Our results indicate that activated TRs negatively influence the carcinogenic process through induction of a differentiation program of preneoplastic hepatocytes. The results also suggest that TRs could be a meaningful target in liver cancer therapy.
... Histological presence of GGT or GSTP positive hepatocyte foci usually represents an end point marker for several xenobiotics carcinogenicity (Ito et al., 2003;Pitot et al., 1991Pitot et al., , 1996. However, chemicals such as proliferators of peroxisome induce GSTP-negative foci in rodents that must be identified with other markers. ...
The purpose of this investigation was to evaluate the possible influence of a mixture of pesticides on medium-term carcinogenesis using improved hepatocarcinogenesis protocols. We performed a 12 commercially available pesticides combination with alachlor, atrazine, carbofuran, chlorpyrifos, diazinon, dicofol, endosulfan, iprodione, mancozeb, maneb, procymidone and rotenone. The mixture was given at 1-fold and 10-fold the acceptable daily intake (ADI) level in a set of Solt–Farber-derived protocols involving diethylnitrosamine, 2-acetylaminofluorene treatments and a partial hepatectomy. Co-carcinogenic effect and promoting activity were evaluated using γ-glutamyl transpeptidase (GGT) positive altered hepatocyte foci, as well, protein and mRNA levels of glutathione S-transferase P (GSTP) in liver extracts as molecular biomarkers of carcinogenic effects. The pesticide treatments when compared to vehicle treatments always produced the same number of hepatocyte lesions and an equal GSTP expression on liver extracts independently of carcinogenic-protocol utilized. On this base, we concluded that the pesticide mixture evaluated in this report does not have tumor promoting activity or co-carcinogenic effect in the rat medium-term liver carcinogenesis. Altogether these data contribute to the confidence that the ADI represents a safe intake level to mixture of pesticides at dietary exposure.
... Based on the wide therapeutic use of a-LA in several diseases (5-7), including conditions related to liver diseases (8)(9)(10), and since no data are available on the effect of a-LA on liver cancer development, the aim of the present study was to investigate the effect of a-LA on the occurrence of putative pre-neoplastic lesions induced by a short-term in vivo assay consisting of a single dose of the hepatocarcinogen diethylnitrosamine (DENA) and promotion with the CMD. We report here that addition of a-LA to the CMD strongly promoted the growth of hepatic pre-neoplastic foci positive for the placental form of glutathione S-transferase (GSTP) and gamma glutamyl transpeptidase (GGT), the two most reliable markers for rat pre-neoplasia (24,25), and potentiates accumulation of triglycerides in the liver and hepatocellular damage, suggesting caution about its use in conditions associated with pre-existing liver damage. ...
... Although GSTP is considered to be the most reliable marker for pre-neoplastic lesions induced by genotoxic carcinogens (24), it does not identify all putative pre-neoplastic lesions. Therefore, pre-neoplastic lesions were also identified by their positivity for GGT, the second best marker for pre-neoplasia (25). The results confirmed that feeding a CMD þ a-LA diet for 10 weeks caused a striking increase in the number of pre-neoplastic lesions compared with the animals fed the CMD alone (72.1 ± 13 foci/cm 2 versus 25.4 ± 7 foci/ cm 2 ), although, in agreement with the literature (25), the number of lesions detected by GGT staining was lower than that determined by GSTP staining. ...
alpha-lipoic acid (alpha-LA) is an antioxidant used in a number of conditions related to liver diseases. Herein, we investigated the effect of alpha-LA on the development of rat pre-neoplastic lesions generated by a model of hepatocarcinogenesis, which has similarities in its histopathological sequence to human hepatocellular carcinoma development with cirrhosis. Initiation of hepatocytes was achieved by treatment with a single dose of diethylnitrosamine and promotion by feeding a choline-methionine-deficient diet (CMD), with or without alpha-LA. Pre-neoplastic lesions were identified by their positivity to the placental form of glutathione S-transferase (GSTP) or to gamma glutamyl transpeptidase. alpha-LA given to rats fed a CMD for 6 weeks dramatically increased the number of GSTP-positive foci as compared with rats fed a CMD alone (96/cm(2) versus 7/cm(2)), the mean foci area (0.033 versus 0.008 mm(2)) and the percentage of GSTP-positive liver tissue (3.01 versus 0.07%). Essentially similar results were obtained after 10 weeks of treatment. Co-treatment with CMD + alpha-LA also resulted in the enhancement of fat accumulation, lipid peroxidation and hepatocyte death; increased expression of tumor necrosis factor-alpha, cytochrome 2E1 and cyclooxygenase-2, enhanced activation of c-jun N-terminal kinase and signal transducer activator of transcription 3, and chronic hepatocyte proliferation was also observed. No such effects were observed when alpha-LA was added to a choline-supplemented diet. In conclusion, administration of alpha-LA in conditions associated with hepatic damage aggravates liver injury and stimulates the development of pre-neoplastic lesions; the results also suggest caution in its use in the presence of chronic liver injury.
... Based on the wide therapeutic use of a-LA in several diseases (5-7), including conditions related to liver diseases (8)(9)(10), and since no data are available on the effect of a-LA on liver cancer development, the aim of the present study was to investigate the effect of a-LA on the occurrence of putative pre-neoplastic lesions induced by a short-term in vivo assay consisting of a single dose of the hepatocarcinogen diethylnitrosamine (DENA) and promotion with the CMD. We report here that addition of a-LA to the CMD strongly promoted the growth of hepatic pre-neoplastic foci positive for the placental form of glutathione S-transferase (GSTP) and gamma glutamyl transpeptidase (GGT), the two most reliable markers for rat pre-neoplasia (24,25), and potentiates accumulation of triglycerides in the liver and hepatocellular damage, suggesting caution about its use in conditions associated with pre-existing liver damage. ...
... Although GSTP is considered to be the most reliable marker for pre-neoplastic lesions induced by genotoxic carcinogens (24), it does not identify all putative pre-neoplastic lesions. Therefore, pre-neoplastic lesions were also identified by their positivity for GGT, the second best marker for pre-neoplasia (25). The results confirmed that feeding a CMD þ a-LA diet for 10 weeks caused a striking increase in the number of pre-neoplastic lesions compared with the animals fed the CMD alone (72.1 ± 13 foci/cm 2 versus 25.4 ± 7 foci/ cm 2 ), although, in agreement with the literature (25), the number of lesions detected by GGT staining was lower than that determined by GSTP staining. ...
... These three stages of tumor formation have been characterized in many mammalian tissues, particularly in the liver [2,3] . According to the concept of multistage carcinogenesis, clones of cells arise with increasing autonomy from normal growth regulation at each stage of development, and from these selected populations of cells, neoplasms ultimately develop [4] . The placental form of rat glutathione S-transferase (rGST P) positive foci is thought to be preneoplastic lesions of the liver [5][6][7][8][9][10][11][12] . ...
... The expression of many enzymes including those involved in GSH metabolism is altered in AHF and persists during HC (31,55). The pattern of expression of many of them is similar to that found in fetal liver (56). Various hepatomas, transformed rat embryo fibroblasts and cells transformed by erbB, src, ras and raf are low in GGCS and GS with frequent concomitant high GGT activities (57)(58)(59)(60)(61)(62)(63)(64)(65)(66). ...
Glutathione synthesis and growth properties were studied in the gamma-glutamyl transpeptidase(GGT)-negative, non-tumorigenic rat liver oval cell line OC/CDE22, and in its GGT-positive, tumorigenic counterpart line M22. gamma-Glutamylcysteine synthetase (GGCS) activities were comparable. Growth rates of M22 cells exceeded those of OC/CDE22 cells at non-limiting and limiting exogenous cysteine concentrations. A monoclonal antibody (Ab 5F10) that inhibits the transpeptidatic but not the hydrolytic activity of GGT did not affect the growth rates of OC/CDE22, and decreased those of M22 to the OC/CDE22 level. In GSH-depleted M22, but not in OC/CDE22 cells, the rate and extent of GSH repletion with exogenous cysteine and glutamine exceeded those obtained with exogenous cysteine and glutamate. With Ab 5F10, repletion with cysteine/glutamine was similar to that obtained with cysteine/glutamate. Repletion with exogenous GSH occurred only in M22 cells, and was abolished by the GGT inhibitor acivicin. Repletion with gamma-glutamylcysteine (GGC) in OC/CDE22 was resistant to acivicin whereas that in M22 was inhibited by acivicin. Repletion with exogenous GSH or cysteinylglycine (CG) required aminopeptidase activity and was lower than that obtained with cysteine. Unless reduced, CG disulfide did not support GSH repletion. The findings are compatible with the notions that (i) GGT-catalyzed transpeptidation was largely responsible for the growth advantage of M22 cells at limiting cysteine concentration, and for their high GSH content via the formation of GGC from a gamma-glutamyl donor (glutamine) and cyst(e)ine, and (ii) aminopeptidase/dipeptidase activity is rate-limiting in GSH repletion when GSH or CG serve as cysteine sources.
... The expression of many enzymes including those involved in GSH metabolism is altered in AHF and persists during HC (31,55). The pattern of expression of many of them is similar to that found in fetal liver (56). Various hepatomas, transformed rat embryo fibroblasts and cells transformed by erbB, src, ras and raf are low in GGCS and GS with frequent concomitant high GGT activities (57)(58)(59)(60)(61)(62)(63)(64)(65)(66). ...
Glutathione synthesis and growth properties were studied in the γ-glutamyl transpeptidase(GGT)-negative, nontumorigenic rat liver oval cell line OC/CDE22, and in its GGT-positive, tumorigenic counterpart line M22. γ-Glutamylcysteine synthetase (GGCS) activities were comparable. Growth rates of M22 cells exceeded those of OC/CDE22 cells at non-limiting and limiting exogenous cysteine concentrations. A monoclonal antibody (Ab 5F10) that inhibits the transpeptidatic but not the hydrolytic activity of GGT did not affect the growth rates of OC/ CDE22, and decreased those of M22 to the OC/CDE22 level. In GSH-depleted M22, but not in OC/CDE22 cells, the rate and extent of GSH repletion with exogenous cysteine and glutamine exceeded those obtained with exogenous cysteine and glutamate. With Ab 5F10, repletion with cysteine/glutamine was similar to that obtained with cysteine/glutamate. Repletion with exogenous GSH occurred only in M22 cells, and was abolished by the GGT inhibitor acivicin. Repletion with γ-glutamylcysteine (GGC) in OC/CDE22 was resistant to acivicin whereas that in M22 was inhibited by acivicin. Repletion with exogenous GSH or cysteinylglycine (CG) required aminopeptidase activity and was lower than that obtained with cysteine. Unless reduced, CG disulfide did not support GSH repletion. The findings are compatible with the notions that (i) GGT-catalyzed transpeptidation was largely responsible for the growth advantage of M22 cells at limiting cysteine concentration, and for their high GSH content via the formation of GGC from a γ-glutamyl donor (glutamine) and cyst(e)ine, and (ii) aminopeptidase/dipeptidase activity is rate-limiting in GSH repletion when GSH or CG serve as cysteine sources.
