No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
CYP35: Xenobiotically induced gene expression in the nematode Caenorhabditis elegans
Abstract
Although over 80 cytochrome P450 (CYP) encoding genes have been identified in the genome of the nematode Caenorhabditis elegans very little is known about their involvement in biotransformation. This paper demonstrates a concentration-dependent relationship of C. elegans CYP35A1, A2, A5, and C1 gene expression in response to four organic xenobiotics, namely atrazine, PCB52, fluoranthene, and lansoprazole. The toxicity of these xenobiotics was determined using a reproduction assay. CYP-specific messenger RNA expression was analyzed by semi-quantitative RT-PCR resulting in a strongly increasing, concentration-dependent induction well below the EC50 for reproduction. For PCB52, approximately 0.5% of the EC50 induces a 2-fold increase of CYP35 gene expression. Using a double mutant and multiple RNAi of CYP35A/C it was possible to diminish the reproduction decline caused by PCB52 and fluoranthene.
... The CYP superfamily has been proved to be ubiquitous across living organisms, whose importance is associated with drug resistance in mammals, insects, and nematodes (Elzaki et al., 2016;Xu et al., 2021). The genome of C. elegans encodes more than 80 CYP proteins (Menzel et al., 2001;Menzel et al., 2005), where several subfamilies in C. elegans, including CeCYP13, CeCYP14, CeCYP35, CeCYP34, CeCYP33, and CeCYP31, have been suggested to be involved in detoxification of xenobiotics (Menzel et al., 2001;Menzel et al., 2005;Laing et al., 2010;Jones et al., 2013;Jones et al., 2015;Yilmaz et al., 2019;Liu et al., 2020). In addition, most studies on CYP-mediated metabolism of xenobiotics have been conducted in APNs, such as Haemonchus contortus (Yilmaz et al., 2017;Kellerováet al., 2019). ...
... The CYP superfamily has been proved to be ubiquitous across living organisms, whose importance is associated with drug resistance in mammals, insects, and nematodes (Elzaki et al., 2016;Xu et al., 2021). The genome of C. elegans encodes more than 80 CYP proteins (Menzel et al., 2001;Menzel et al., 2005), where several subfamilies in C. elegans, including CeCYP13, CeCYP14, CeCYP35, CeCYP34, CeCYP33, and CeCYP31, have been suggested to be involved in detoxification of xenobiotics (Menzel et al., 2001;Menzel et al., 2005;Laing et al., 2010;Jones et al., 2013;Jones et al., 2015;Yilmaz et al., 2019;Liu et al., 2020). In addition, most studies on CYP-mediated metabolism of xenobiotics have been conducted in APNs, such as Haemonchus contortus (Yilmaz et al., 2017;Kellerováet al., 2019). ...
... When nematodes are exposed to xenobiotics, the transcription levels of CYP genes are rapidly induced to initiate the detoxification processes. For instance, exposure of C. elegans to various xenobiotics, including pesticides, leads to a significant increase in the expression levels of some CYP gene subfamilies such as CeCYP13, CeCYP14, CeCYP35, CeCYP34, CeCYP33, and CeCYP31 (Menzel et al., 2001;Menzel et al., 2005;Jones et al., 2013;Yilmaz et al., 2019;Liu et al., 2020). In H. contortus, the induction of CYP1, CYP3, and CYP5 transcript levels by ivermectin has been reported (Kellerováet al., 2019). ...
The cytochrome P450 (CYP) genes of nematode play a crucial role in the metabolic detoxification of xenobiotics including pesticides. Heterodera glycines, also known as the soybean cyst nematode, is a sedentary endoparasite that infests plant roots, causing high annual economic losses in soybean production regions globally. In this study, we identified 36 CYP genes at a genome-wide level of the H. glycines isolate TN10 using all CYPs from Caenorhabditis elegans as queries. Subsequently, a full-length cDNA of HgCYP33E1 which was significantly up-regulated by the conventional nematicide abamectin was initially cloned from H. glycines. It presented significantly higher expressions in the second-stage juvenile (J2) compared to other parasitic stages of H. glycines. qRT-PCR analysis suggested that the expression of HgCYP33E1 was also xenobiotically induced by soybean root exudate and the metabolites of biocontrol agents. Using RNA interference (RNAi), we investigated the function of HgCYP33E1 in H. glycines parasitism and nematicide selectivity. Compared to the control and dsGFP-treated group, silencing of HgCYP33E1 did not affect the J2 behaviors and the early invasion ability, while it decreased the number of J4s in soybean roots after 18-d inoculation with the dsHgCYP33E1-treated nematodes. In addition, knockdown of HgCYP33E1 in H. glycines resulted in an increase in J2 mortality after 24-h incubation with abamectin compared to the GFP dsRNA-soaked and the control group. These findings revealed the potential role of HgCYP33E1 in the xenobiotic detoxification pathway of H. glycines. Moreover, our data also provided valuable gene information for studying the functions of the CYP family in H. glycines host adaption.
... Herbicide atrazine was able to induce expression of cyp-35A5, cyp-35C1, and cyp-35A1 (Menzel et al. 2001). Moreover, these inductions by atrazine were concentration-dependent in general (Menzel et al. 2005). Studies using green fluorescent protein (GFP) expression showed that C. elegans cyp-35A5 and cyp-35D1 were strongly induced by thiabendazole (fungicide), and cyp-35B1 and cyp-35B3 were strongly inducible by chloroquine (anti-malaria agent) (Jones et al. 2013). ...
... Fluoranthene demonstrated strong induction of cyp-35A1-5, and cyp-35C1 (Menzel et al. 2001). A subsequent study showed that these inductions followed concentrationdependent manner (Menzel et al. 2005 Besides, BaP was found to significantly increase cyp-35B1 expression in a dose-dependent manner (Brinkmann et al. 2021). However, a recent study found that C. elegans cyp-35D1 previously reported to not be regulated by inducers of mammalian CYP1 (Menzel et al. 2001) was unexpectedly significantly upregulated by BaP exposure to approximately 113-fold (Karengera et al. 2022); cyp-35A2 gene expression was induced by anthracene and alkyl-anthracenes (Roh et al. 2018). ...
... β-Naphthoflavone (a common CYP inducer) and polychlorinated biphenyl PCB52 and lansoprazole (a medication used to reduce stomach acid) were shown to potently induce cyp-35A1-5 and cyp-35C1 in a concentration-dependent order but partly induce cyp-35B1 and cyp-35B2 (Menzel et al. 2001(Menzel et al. , 2005. On the other hand, ethanol only induced cyp-35B1 and cyp-35B2 (Menzel et al. 2001). ...
With more than 80 cytochrome P450 (CYP) encoding genes found in the nematode Caenorhabditis elegans (C. elegans), the cyp35 genes are one of the important genes involved in many biological processes such as fatty acid synthesis and storage, xenobiotic stress response, dauer and eggshell formation, and xenobiotic metabolism. The C. elegans CYP35 subfamily consisted of A, B, C, and D, which have the closest homolog to human CYP2 family. C. elegans homologs could answer part of the hunt for human disease genes. This review aims to provide an overview of CYP35 in C. elegans and their human homologs, to explore the roles of CYP35 in various C. elegans biological processes, and how the genes of cyp35 upregulation or downregulation are influenced by biological processes, upon exposure to xenobiotics or changes in diet and environment. The C. elegans CYP35 gene expression could be upregulated by heavy metals, pesticides, anti-parasitic and anti-chemotherapeutic agents, polycyclic aromatic hydrocarbons (PAHs), nanoparticles, drugs, and organic chemical compounds. Among the cyp35 genes, cyp-35A2 is involved in most of the C. elegans biological processes regulation. Further venture of cyp35 genes, the closest homolog of CYP2 which is the largest family of human CYPs, may have the power to locate cyps gene targets, discovery of novel therapeutic strategies, and possibly a successful medical regime to combat obesity, cancers, and cyps gene-related diseases.
... Clans bring together families that belong to the same group (from the same ancestral gene) according to many phylogenetic trees established previously [15]. There are 11 animal P450s clans (clans 2, 3,4,7,19,20,26,46,51,74, and mitochondrial or mito). However, not all organisms own all 11 clans. ...
... Thus, ethanol acts as an inducer of P450s 13A12, 13B1, 25A1, 25A2, 29A2, 32B1, 33B1, 33C6, 34A4, 4A6, 35A3, 35A5, 35B1, 35B2, 35C1, 36A1 and 37B1 [70,71], and caffeine is an inducer of P450s 13A8, 13A12, 14A1, 14A2, 14A4, 14A5, 32A1, 33C3, 33C4, 33C6, 33C7, 33C9, 33E1, 33E2, 33E3, 33E4, 34A7, 34A9, 35A2, 35A3, 35A4, 35A5, 35B1, 35B2, and 43A1 [72]. Interestingly, drugs such as primaquine and lansoprazole are inducers of several members of family 35 (35A1, A2, A3, A4, A5, B1, B2, and C1) and P450 31A3 [73][74][75], whereas rifampicin is an inducer of P450 13A7 [76]. Many pesticides, such as atrazine, fenitrothion, DDT, dichlorvos, rotenone, and thiabendazole are inducers of members of family 35 [73][74][75][77][78][79], whereas endosulfan, cypermethrin, chlorpyrifos, rotenone, and dichlorvos are inducers of P450 34A9 [77], and glyphosate and paraquat of P450 29A2 [77]. ...
... Interestingly, drugs such as primaquine and lansoprazole are inducers of several members of family 35 (35A1, A2, A3, A4, A5, B1, B2, and C1) and P450 31A3 [73][74][75], whereas rifampicin is an inducer of P450 13A7 [76]. Many pesticides, such as atrazine, fenitrothion, DDT, dichlorvos, rotenone, and thiabendazole are inducers of members of family 35 [73][74][75][77][78][79], whereas endosulfan, cypermethrin, chlorpyrifos, rotenone, and dichlorvos are inducers of P450 34A9 [77], and glyphosate and paraquat of P450 29A2 [77]. Aromatic and polyaromatic molecules, such as beta-naphthoflavone, fluoranthene, bisphenol A, and polychlorinated biphenyls (PCBs), act as inducers of several members of family 35 [56,[73][74][75][80][81][82]. ...
Caenorhabditis elegans is an important model used for many aspects of biological research. Its genome contains 76 genes coding for cytochromes P450 (P450s), and few data about the biochemical properties of those P450s have been published so far. However, an increasing number of articles have appeared on their involvement in the metabolism of xenobiotics and endobiotics such as fatty acid derivatives and steroids. Moreover, the implication of some P450s in various biological functions of C. elegans, such as survival, dauer formation, life span, fat content, or lipid metabolism, without mention of the precise reaction catalyzed by those P450s, has been reported in several articles. This review presents the state of our knowledge about C. elegans P450s.
... For example, benzopyrene induced the expression of CYP14A3, an ortholog of the human CYP1A2 gene (Chakrapani et al., 2008). Four genes from the CYP35 family (CYP35A1, CYP35A2, CYP35A5, and CYP35C1) were induced in a concentration-dependent manner when C. elegans were exposed to some persistent organic pollutants, such as fluoranthene, PCB52, Lansoprazole, or atrazine (Menzel et al., 2005). The PAH compound, fluoranthene, at 0.25 mg/L significantly induced N2-fold upregulation of CYP35A1 and CYP35A5 (Menzel et al., 2005). ...
... Four genes from the CYP35 family (CYP35A1, CYP35A2, CYP35A5, and CYP35C1) were induced in a concentration-dependent manner when C. elegans were exposed to some persistent organic pollutants, such as fluoranthene, PCB52, Lansoprazole, or atrazine (Menzel et al., 2005). The PAH compound, fluoranthene, at 0.25 mg/L significantly induced N2-fold upregulation of CYP35A1 and CYP35A5 (Menzel et al., 2005). In addition, the model PAH compound benzo(a)pyrene was found to induce the expression of several other members of the CYP35 family: CYP35A5, CYP35B1, and CYP35C1 (Menzel et al., 2001). ...
... Although it is reasonable to assume that exposure to crude oil, a mixture that contains a number of PAHs, would induce the expression of PAH metabolizing enzymes, including CYP450; however, evaluation of this hypothesis in C. elegans following exposure to crude oil from DWH is lacking. Benzopyrene is known to induce the expression of CYP14A3 and fluoranthene induces expression of CYP35A1, A2, A5 and C1 (Chakrapani et al., 2008;Menzel et al., 2005). This study determined these four genes were all significantly unregulated following exposure to 500× dispersed oil (Fig. 6). ...
The Deepwater Horizon (DWH) oil spill marked the largest environmental oil spill in human history, where it was estimated a large amount of the polycyclic aromatic hydrocarbons (PAHs) were released with crude oil into the environment. In this study, common PAH compounds were quantitatively determined in crude oil from the DWH spill by gas chromatography-mass spectroscopy (GC–MS). Twelve PAH compounds were identified and quantified from a 100× dilution of DWH crude oil: naphthalene (7800 ng/mL), acenaphthylene (590 ng/mL), acenaphtehen (540 ng/mL), fluorene (2550 ng/mL), phenanthrene (2910 ng/mL), anthracene (840 ng/mL), fluoranthene (490 ng/mL), pyrene (290 ng/mL), benzo(k) fluoranthene (1050 ng/mL), benzo(b)fluoranthene (1360 ng/mL), dibenz(a,h)anthracene (2560 ng/mL), and benzo(g, h, i) perylene (630 ng/mL). Toxicity assays using the nematode, Caenorhabditis elegans (C. elegans), indicated a single PAH compound naphthalene, exposure increased C. elegans germ cell apoptosis which may adversely affect progeny reproduction. The number of apoptotic germ cells significantly increased from 1.4 to 2.5 when worms were treated with 10 μg/mL of naphthalene and from 1.3 to 2.5 and 3.5 cells in presence of 1 μg/mL and 5 μg/mL of benzo(a)pyrene, respectively. Five CYP450 genes (CYP14A3, CYP35A1, CYP35A2, CYP35A5, and CYP35C1) were significantly upregulated following 500× dilution of dispersed crude oil exposure (p < 0.05). These results suggest that CYP450s may play a role in bioactivation of PAHs in crude oil, resulting in DNA damage related germ cell apoptosis.
... The development of both taxa was delayed after the addition of benzo(a)pyrene, despite the fact they are classified as genera with well-developed physiological and behavioural adaptations to stress conditions. Aside from the direct, lethal effect on organisms, PAHs can also act as narcotics in lower dosages and affect essential physiological processes, including the growth, reproduction and development of organisms (Menzel et al., 2005). The development delay may be the result of interference between the PAHs and nutrient uptake and metabolism ultimately leading to the organism's "physiological starvation" (Postma & Davids, 1995). ...
... On the molecular level, PAHs induce multiple detoxification responses in nematodes, including the expression of cytochrome P450 genes responsible for the detoxification of xenobiotics. A concentration-dependent relationship was found between the intensity of expression of these genes and the benzo(a)pyrene added to the soil (Menzel et al., 2005). An even stronger induction of the P450 genes family was found in the use of fluoranthene (Menzel et al., 2001). ...
Polycyclic aromatic hydrocarbons (PAHs) are an important group of organic pollutants present in all parts of the environment, affecting ecosystems and human health. PAHs, which have a strong affinity for organic carbon, are found in large quantities in soil, which is one of the most important sinks for these contaminants. Their impact on the soil biotic compartments depends on a number of different factors in combination with PAH behaviour and can be assessed using soil monitoring. Soil fauna have already shown excellent properties for biomonitoring of contaminants with most promising indicator frameworks based on nematodes, which are involved in essential processes in this environment. Nematodes respond to PAHs at multiple levels, including molecular, individual and community levels. At the molecular level, this is associated with activation of metabolic pathways for xenobiotics and increased demand for energy and resources. At the individual level, this is reflected in the slowing down of various physiological processes, which has consequences at the individual and community level for sensitive taxa. In this review, the toxicity and the direct and indirect effects of PAHs on soil nematode communities are discussed. It also considers the perspectives and challenges in assessing the toxicity of PAHs and their indication using soil nematodes.
... For instance, the orthologs for many key mammalian redox systems have been reported in C. elegans including glutathione (GSH) and related systems, which are critical for detoxification of both xenobiotic and endogenous compounds in mammals (Ferguson and Bridge, 2019;Dancy et al., 2016). Furthermore, over 80 CYP genes encoding cytochrome P450 enzymes have been identified in the C. elegans genome (Menzel et al., 2005(Menzel et al., , 2001. Based on predicted amino acid sequences, the majority of C. elegans CYP genes were found to be closely related to the mammalian CYP2, CYP3, and CYP4 gene families (Gotoh, 1998). ...
... In comparison with literature, these LOTEL values were about 541-fold for AFB1, 2-fold for B(a)P, or 48-fold for PCB1254 smaller than the median effective concentrations (EC 50 ) for toxic effects on C. elegans reproduction. For 72-hour exposure, EC 50 that caused reproductive toxicity is equivalent to 5.41 µM for AFB1 (Yang et al., 2015), 0.23 µM for B(a)P (Sese et al., 2009), and 47.82 µM for PCB52 (Menzel et al., 2005). These results suggest that transcriptional effects in C. elegans are occurring at a concentration much lower than developmental effects, as is also the case in vertebrates like zebrafish embryotoxicity test (ZET) (Hermsen et al., 2012). ...
Caenorhabditis elegans is a well-established model organism for toxicity testing of chemical substances. We recently demonstrated its potential for bioanalysis of the toxic potency of chemical contaminants in water. While many detoxification genes are homologues to those in mammalians, C. elegans is reported to be deficient in cytochrome CYP1-like P450 metabolism and that its aryl hydrocarbon receptor (AhR) homolog encoded by ahr-1 purportedly does not interact with dioxins or any other known xenobiotic ligand. This suggests that C. elegans is insensitive for compounds that require bioactivation (indirectly acting compounds) and for dioxins or dioxin-like compounds. This study analysed genome-wide gene expression of the nematode in response to 30 μM of aflatoxin B1 (AFB1), benzo(a)pyrene (B(a)P), Aroclor 1254 (PCB1254), and 10 μM of 2,3,7,8-tetrachlorodibenzodioxin (TCDD). After 24 h of exposure in the early L4 larval stage, microarray analysis revealed 182, 86, and 321 differentially expressed genes in the nematodes treated with 30 μM of AFB1, B(a)P, and PCB1254, respectively. Among these genes, many encode xenobiotic-metabolizing enzymes, and their transcription levels were among the highest-ranked fold-changed genes. Interestingly, only one gene (F59B1.8) was upregulated in the nematodes exposed to 10 μM TCDD. Genes related to metabolic processes and catalytic activity were the most induced by exposure to 30 μM of AFB1, B(a)P, and PCB1254. Despite the genotoxic nature of AFB1 and B(a)P, no differential expression was found in the genes encoding DNA repair and cell cycle checkpoint proteins. Analysis of concentration–response curves was performed to determine the Lowest Observed Transcriptomic Effect Levels (LOTEL) of AFB1, B(a)P, and PCB1254. The obtained LOTEL values showed that gene expression changes in C. elegans are more sensitive to toxicants than reproductive effects. Overall, transcriptional responses of metabolic enzymes suggest that the nematode does metabolize AFB1, B(a)P, and PCB1254. Our findings also support the assumption that the transcription factor AhR homolog in C. elegans does not bind typical xenobiotic ligands, rendering the nematode transcriptionally insensitive to TCDD effects.
... Several studies have investigated the upregulation of P450 enzymes in C. elegans in response to various xenobiotic compounds [36][37][38][39][40] . The CYP-35 genes in particular have been shown to be strongly inducible 38 . ...
... They found that the enzymes cyp-14A and cyp-34A6 metabolised PCB52 33 . In earlier studies PCB52 had induced the expression of many different P450s including cyp-14A3, cyp-34A10, cyp-35A and cyp-35C1 [36][37][38] but no induction of expression of cyp-34A6 has been reported. Therefore in this case the induction of cytochrome P450 genes including cyp-35C1 was not indicative of them being involved in the metabolism of the compound. ...
To determine whether a C. elegans bioassay could predict mammalian developmental activity, we selected diverse compounds known and known not to elicit such activity and measured their effect on C. elegans egg viability. 89% of compounds that reduced C. elegans egg viability also had mammalian developmental activity. Conversely only 25% of compounds found not to reduce egg viability in C. elegans were also inactive in mammals. We conclude that the C. elegans egg viability assay is an accurate positive predictor, but an inaccurate negative predictor, of mammalian developmental activity. We then evaluated C. elegans as a tool to identify mechanisms affecting toxicological outcomes among related compounds. The difference in developmental activity of structurally related fungicides in C. elegans correlated with their rate of metabolism. Knockdown of the cytochrome P450s cyp-35A3 and cyp-35A4 increased the toxicity to C. elegans of the least developmentally active compounds to the level of the most developmentally active. This indicated that these P450s were involved in the greater rate of metabolism of the less toxic of these compounds. We conclude that C. elegans based approaches can predict mammalian developmental activity and can yield plausible hypotheses for factors affecting the biological potency of compounds in mammals.
... C. elegans is a popular living model for examining the toxicological relevance of chemical-induced toxicity at the molecular level [34,35]. In this study, LC50 (50% lethal concentration) values of paraquat and dicamba were determined for C. elegans. ...
Paraquat and dicamba are chemicals commonly used in agriculture for plant control. The US Environmental Protection Agency (EPA) has classified paraquat into a restricted use class for use only by practitioners, as it is highly toxic. In this study, the effects of different concentrations of paraquat and dicamba toxic substances on C. elegans were studied. In tests, C. elegans were directly exposed to different concentrations of paraquat and dicamba for 6h, 12h, 18h, 24h. In particular, it was determined at which paraquat and dicamba doses that half of the C. elegans individuals (LC50) died. In the analysis results, paraquat LC50 values were found as LC50 6h= LC50 6h= 7412 µM, LC50 12h= 459 µM, LC50 18h= 123 µM, LC50 24h= 61 µM. Similarly, dicamba LC50 values were found as LC50 6h= 14610 µM, LC50 12h= 1404 µM, LC50 18h= 906 µM, LC50 24h= 463 µM.
... Additionally, some studies suggest the opposite. For example, some proteins of C. elegans, especially members of the CYP35 family, have been shown to be inducible by exogenous organisms [52,53]. ABZ can induce the expression of multiple CYP genes in C. elegans [54], while the inhibitor of CYP, piperonyl butoxide (PBO), increases the toxicity of the insecticide rotenone to H. contortus larvae and adults [55]. ...
A major problem faced by the agricultural industry is the resistance of Haemonchus contortus to anthelmintic drugs. For a better understanding of the response of H. contortus to IVM and for the screening of drug-resistance-related genes, we used RNA sequencing and isobaric tags for relative and absolute quantification (iTRAQ) technology to detect the transcriptomic and proteomic changes in H. contortus after ivermectin treatment. An integrated analysis of the two omics showed that the differentially expressed genes and proteins were significantly enriched in the pathways of amino acid degradation, the metabolism of xenobiotics by cytochrome P450, the biosynthesis of amino acids, and the tricarboxylic acid cycle. We found that the upregulated UDP-glycosyltransferases (UGT), glutathione S-transferase (GST), cytochrome P450 (CYP), and p-glycoprotein (Pgp) genes play important roles in drug resistance in H. contortus. Our work will help in the understanding of the transcriptome and proteome changes in H. contortus after IVM and will facilitate the discovery of genes related to drug resistance. This information can be further applied to increase the understanding of the response of IVM in relation to H. contortus.
... Dissolved organic matter (DOM) is widely distributed in terrestrial and aquatic environments, and it has great signi cance in the fate, transport, and toxicity of environmental pollutants (Steinberg et al., 2007;Yang et al., 2014;Yu et al., 2018). DOM is recognized as environmental signaling chemical, since organisms could take up DOM and then interact with molecular substances in nematodes (Menzel et al., 2005a(Menzel et al., and 2005b(Menzel et al., and 2011. Researchers found C. elegans tend to prefer living in the DOM-rich environment both in nature and laboratory, and DOM is bene cial for them to live longer and increase their reproductive ability (Menzel et al. 2005a and b). ...
As a type of effective soil amendment, biochars require comprehensive ecological assessment before their widespread agricultural applications, since the endogenous contaminants, especially environmentally persistent free radicals (EPFRs) in biochars certainly pose an ecological risk to soil invertebrates. In this study, Caenorhabditis elegans ( C. elegans ) was chosen as a model organism to assess the neurotoxicity of three rice straw biochars pyrolyzed at 500, 700, and 1000°C. After 24 h-exposure to unwashed biochars, washed biochars, and leaching liquids (supernatants), neurobehavioral parameters of L4 C. elegans larvae were determined in a liquid toxicity test. The results showed that the washed 700°C biochar particles significantly impaired the locomotion and prolonged the defecation interval at biochar concentration of 4 g·well − 1 , whereas unwashed biochars and supernatants did not cause obvious impairment. At the same time, the electron paramagnetic resonance (EPR) results showed that the intensity of EPFRs in unwashed 700°C biochar was stronger than that of corresponding washed particles. It suggests that, in the liquid test, EPR signal alone does not indicate the toxicity of the particle. The accessibility and activity of EPFRs should be considered. Dissolved organic matter (DOM) has great significance in the fate, transport, and toxicity of environmental pollutants, which is beneficial for C. elegans to live longer and increase their reproductive ability. In this paper, the neurobehaviors of washed biochar were alleviated after adding supernatant back, which indicated that DOM could modulate the reactivity of EPFRs in the liquid phase.
... Nevertheless, the significance of stress-induced changes in gene expression will depend on the downstream biological effects elicited (Feder and Walser, 2005). The biological function of stress responsive genes has been tested using loss of function mutants and RNAi (Menzel et al., 2005(Menzel et al., , 2007Cui et al., 2007). Once a physiological role has been established, transgenic strains can be generated containing reporter genes under control of the regulatory regions of stress inducible genes. ...
This chapter focuses on transgenic Caenorhabditis elegans developed for ecotoxicology purposes to the end of 2007. This chapter discusses how transgenic Caenorhabditis elegans strains are generated and reviews literature concerning their use in biomonitoring. Developments in the understanding of stress responses is also briefly explored as they have the potential to be translated into novel C. elegans biosensor strains.
... Phase I enzymes include the cytochrome P450 family (CYP), short-chain dehydrogenase/reductase (SDR) superfamily and flavin containing monooxygenase (FMO) superfamily. The C. elegans genome contains over 80 CYP genes, of which several have been shown to be inducible by xenobiotic substances including benzimidazoles (Menzel et al. 2001;Menzel et al. 2005;Laing et al. 2010;Stasiuk et al. 2019). Several CYP genes have also been identified in H. contortus (Laing et al. 2015), and a slight elevation of the expression of a CYP gene was found in a highly benzimidazoleresistant isolate of H. contortus in comparison to isolates susceptible and with moderate resistance to benzimidazoles (Yilmaz et al. 2017). ...
... The cyp-35s are thought to be part of the first stage xenobiotic detoxification response in C. elegans and were shown to be induced by β-naphthoflavone, PCB52, atrazine and lansoprazole. (Menzel et al. 2001(Menzel et al. , 2005(Menzel et al. , 2007Lindblom and Dodd 2006;Harlow et al. 2018). BaP is metabolised in Homo sapiens mainly by the action of CYP1′s (CYP1A1, CYP1A2, and CYP1B1) (Shimada et al. 1997;Gotoh 1998;Xue and Warshawsky 2005;Shimada 2006;Luch and Baird 2010). ...
Benzo[ a ]pyrene (BaP) is bioactivated in most organisms by the cytochrome P450 (CYP) enzymes, mainly CYP1A1, ultimately resulting in the reactive metabolite BaP-7,8-dihydrodiol-9,10-epoxide (BPDE) capable of covalently binding to DNA and forming adducts. This step has been defined as the key process in cancer initiation in humans. However, limited knowledge is available about the consequences of BaP exposure in organisms lacking this classical CYP1A1 pathway, one example is the model nematode Caenorhabditis elegans. The aim of this study was to define the genotoxic potential of BaP in C. elegans and to advance our understanding of xenobiotic processing in the absence of the CYP1A1 pathway. Exposure to high concentrations of BaP (0–40 µM) significantly affected life cycle endpoints of C. elegans , which were manifested by a reduced reproductive output and shortened life span. An optimised comet assay revealed that DNA damage increased in a dose-dependent manner; however, no bulky DNA adducts (dG- N 2 -BPDE) were observed by ³² P-postlabelling. Global transcriptomic analysis by RNA-Seq identified responsive transcript families, most prominently members of the cyp-35 and UDP-glucuronosyltransferases (UGTs) enzyme families, both of which are linked to xenobiotic metabolism. Strains harbouring mutations in the cyp-35A2 and cyp-35A3 genes were notably less prone to BaP-mediated toxicity, and BaP led to longevity in cyp-35A5 mutants. In summary, BaP induces transcriptional, genotoxic and phenotypic responses in C. elegans , despite the absence of the classical CYP1A1 bioactivation pathway. This provides first evidence that parallel pathways are implicated in BaP metabolism in C. elegans and this seems to be mediated via the cyp-35 pathway.
... While CYPs are widely distributed in nature, their presence in helminths is ignored for a long time [83,84]. However, this view has been changed with the discovery of CYPs-encoding genes in C. elegans, where more than 80 CYP proteins are present [85,86], and these have been shown to be xenobiotics inducible [85,[87][88][89][90]. The CYPs family have also been characterized recently in H. contortus [91,92]. ...
The most important and broad-spectrum drug used to control the parasitic worms to date is ivermectin (IVM). Resistance against IVM has emerged in parasites, and preserving its efficacy is now becoming a serious issue. The parasitic nematode Haemonchus contortus (Rudolphi, 1803) is economically an important parasite of small ruminants across the globe, which has a successful track record in IVM resistance. There are growing evidences regarding the multigenic nature of IVM resistance, and although some genes have been proposed as candidates of IVM resistance using lower magnification of genome, the genetic basis of IVM resistance still remains poorly resolved. Using the full magnification of genome, we herein applied a population genomics approach to characterize genome-wide signatures of selection among pooled worms from two susceptible and six ivermectin-resistant isolates of H. contortus, and revealed candidate genes under selection in relation to IVM resistance. These candidates also included a previously known IVM-resistance-associated candidate gene HCON_00148840, glc-3. Finally, an RNA-interference-based functional validation assay revealed the HCON_00143950 as IVM-tolerance-associated gene in H. contortus. The possible role of this gene in IVM resistance could be detoxification of xenobiotic in phase I of xenobiotic metabolism. The results of this study further enhance our understanding on the IVM resistance and continue to provide further evidence in favor of multigenic nature of IVM resistance.
... Additionally, CYP oxidase presence has been demonstrated in some parasitic nematodes (Kotze 1997;Cvilink et al. 2008;Laing et al. 2015). For example, the genome of Caenorhabditis elegans encodes 80 CYPs (Menzel et al. 2001(Menzel et al. , 2005. Cvilink et al. (2008) showed that CYP activity is higher in the larval stages than in adult stages of Haemonchus contortus, which reflects an increased metabolic activity in response to exposure of free-living stages to environmental toxins. ...
The concentrations of polycyclic aromatic hydrocarbon metabolites (PAHm) and their bioconcentration factors (BCF) were determined in the larval stages of the cestode Oncomegas wageneri, recovered from the intestine of the Mexican flounder Cyclopsetta chittendeni, in the southern Gulf of Mexico. The PAHm concentrations in O. wageneri were measured using fixed-wavelength fluorescence spectrometry and compared with PAHm concentrations in host bile. Oncomegas wageneri PAHm concentrations were markedly higher than those in host tissues. The highest BCF values were obtained for 1-hydroxypyrene (OHP) and benzo(a)pyrene (BaP). Using a General Linear Model, a significant negative relationship was found between O. wageneri PAHm concentrations (as response variable) and the number of O. wageneri and oil well proximity. Low BCF values and PAHm concentrations in C. chittendeni correlated positively with O. wageneri PAHm concentrations. In contrast, high BCF values for PAHm concentrations in C. chittendeni had a negative association with O. wageneri PAHm concentrations. This study provides the first evidence of the presence of PAHm in intestinal larval cestodes of marine flatfishes, demonstrating levels of PAHm that were higher than levels in their hosts.
... In mammals, the synthesis of eicosanoids requires the participation of cyclooxygenase, lipoxygenase, and cytochrome P450 enzymes [29]. The cyp-gene family is reported to be responsible for encoding cytochrome P450s, NADPH-dependent monooxygenases that metabolize endogenous and exogenous compounds [30]. Sucrose treatment, stearic acid treatment, and sucrose-stearic acid co-treatment of nematodes downregulated cyp-gene expression, such as cyp-29A3, cyp-14A3, and cyp-35A4 and interfered with metabolism of nematodes. ...
Background:
Excessive intake of carbohydrates and fats causes over-nutrition, leading to a variety of diseases and complications. Here, we characterized the effects of different types of sugar and lipids on the growth and development of Caenorhabditis elegans.
Methods:
We measured the lifespan, reproductive capacity, and length of nematodes after sugars and lipids treatment alone and co-treatment of sugars and lipids. Furthermore, we studied the mechanisms underlying the damage caused by high-sucrose and high-stearic acid on C.elegans by using transcriptome sequencing technology.
Results:
The results showed that a certain concentration of sugar and lipid promoted the growth and development of nematodes. However, excessive sugars and lipids shortened the lifespan and length of nematodes and destroyed their reproductive capacity. Based on the results of the orthogonal test, we selected 400 mmol/L sucrose and 500 μg/mL stearic acid to model a high-sugar and high-lipid diet for C. elegans.
Conclusion:
High-sugar and high-lipid intake altered the expression of genes involved in biofilm synthesis, genes that catalyze the synthesis and degradation of endogenous substances, and genes involved in innate immunity, resulting in physiological damage. Furthermore, we explored the protective effect of resveratrol on high-sugar and high-lipid damage to nematodes. Resveratrol plays a role in repairing by participating in the metabolism of foreign substances and reducing cellular oxidative stress.
... Similarly, CYP-13A6, 14A4, 34A9, 35A1-5, 35B2, and 35C1 may be involved in the oxidation/reduction biotransformation reactions. Interestingly, the CYP-35 group has been shown in several other studies to be a core xenobiotic response group in C. elegans references ( Menzel et al., 2001Menzel et al., , 2005Reichert and Menzel, 2005;Roh et al., 2007). Several members of the core group of BZ xenobiotic metabolizing genes which we identified (cyp-35a5, ugt-8, ugt-25 and ugt-37), were also found to be up-regulated in response to several xenobiotics, including thiabendazole ( Jones et al., 2013), which further Table 3 Summary of Complete Gene Ontology (GO Slim) biological process analysis of the C. elegans CB3474 ben-1(e1880)III for differentially expressed genes, significantly up-regulated greater than 2-fold using Panther Overrepresentation Test (release 20190711) with FDR correction. ...
We have undertaken a detailed analysis of the biotransformation of five of the most therapeutically important benzimidazole anthelmintics - albendazole (ABZ), mebendazole (MBZ), thiabendazole (TBZ), oxfendazole (OxBZ) and fenbendazole (FBZ) - in Caenorhabditis elegans and the ruminant parasite Haemonchus contortus. Drug metabolites were detected by LC-MS/MS analysis in supernatants of C. elegans cultures with a hexose conjugate, most likely glucose, dominating for all five drugs. This work adds to a growing body of evidence that glucose conjugation is a major pathway of xenobiotic metabolism in nematodes and may be a target for enhancement of anthelmintic potency. Consistent with this, we found that biotransformation of albendazole by C. elegans reduced drug potency. Glucose metabolite production by C. elegans was reduced in the presence of the pharmacological inhibitor chrysin suggesting that UDP-glucuronosyl/glucosyl transferase (UGT) enzymes may catalyze benzimidazole glucosidation. Similar glucoside metabolites were detected following ex vivo culture of adult Haemonchus contortus. As a step towards identifying nematode enzymes potentially responsible for benzimidazole biotransformation, we characterised the transcriptomic response to each of the benzimidazole drugs using the C. elegans resistant strain CB3474 ben-1(e1880)III. In the case of albendazole, mebendazole, thiabendazole, and oxfendazole the shared transcriptomic response was dominated by the up-regulation of classical xenobiotic response genes including a shared group of UGT enzymes (ugt-14/25/33/34/37/41/8/9). In the case of fenbendazole, a much greater number of genes were up-regulated, as well as developmental and brood size effects suggesting the presence of secondary drug targets in addition to BEN-1. The transcriptional xenobiotic response of a multiply resistant H. contortus strain UGA/2004 was essentially undetectable in the adult stage but present in the L3 infective stage, albeit more muted than C. elegans. This suggests that xenobiotic responses may be less efficient in stages of parasitic nematodes that reside in the host compared with the free-living stages.
... In the eggs and larvae of the gastro-intestinal nematodes Cooperia oncophora and Ostertagia ostertagia, increased toxicity to thiabendazole (TBZ) and ivermectin (IVM) after exposure to the CYP inhibitor piperonyl butoxide (PBO) were shown (AlGusbi et al., pesticide rotenone (AlGusbi et al., 2014;Kotze et al., 2006). In Caenorhabditis elegans, the genome encodes 80 CYPs, including four large xenobiotic-induced enzyme families: CYP35, CYP34, CYP33, CYP31 (Laing et al., 2010;Menzel et al., 2001Menzel et al., , 2005. The CYPs of H. contortus cluster with members of all of these families; however, the parasite is apparently lacking the extensive duplications of genes as appear in C. elegans . ...
Haemonchus contortus, one of the most pathogenic of all small ruminant parasites, have developed resistance to all used anthelmintics. Detoxification enzymes, e.g. cytochromes P450 (CYPs) and efflux transporters P-glycoproteins (P-gps), which represent the main defense system against harmful xenobiotics, have been suggested to contribute to drug resistance development. The present study was designed to compare the constitutive expression of individual CYPs and P-gps in females and males of H. contortus adults and to follow up on the changes in expression of these genes in nematodes exposed to sub-lethal concentrations of ivermectin (IVM), which might occur during inaccurate treatment. The adults of inbred susceptible-Edinburgh strain (ISE, MHco3) of H. contortus were used for this purpose. The nematodes were incubated ex vivo with or without IVM (1, 10 and 100 nM) in culture medium for 4, 12 and 24 h. After incubation, total RNA was isolated and expression levels of individual CYPs and P-gps were analyzed using qPCR. Our results showed a great variability in the constitutive expression of individual CYPs and P-gps in H. contortus adults. The constitutive expression as well as the inducibility of CYPs and P-gps significantly differed in males and females. Contact of adult nematodes with sub-lethal IVM concentrations led to only minor changes in expression of CYPs, while expression of several P-gps, particularly pgp-9.2 in males and pgp-10, pgp-11 in females was increased significantly in IVM-exposed nematodes. In conclusion, inaccurate treatment of sheep with IVM might contribute to drug resistance development via increased expression of efflux transporters in H. contortus adults.
... Moreover, cyp14A, cyp35A and cyp37B isoforms were constitutively and inversely regulated in nhr-8(ok186) and IVM-resistant worms. In line with this, members of the C. elegans cyp14A and cyp35A subfamily respond to a variety of xenobiotic stressors [27,[60][61][62]. In addition, the cyp14, cyp33, cyp34, and cyp35 families in C. elegans are orthologs of mammalian xenobiotic inducible genes CYP1A2 [61] and the CYP2 family [63], which are both induced by IVM [21][22][23]. ...
Resistance to the anthelmintic macrocyclic lactone ivermectin (IVM) has a great impact on the control of parasitic nematodes. The mechanisms by which nematodes adapt to IVM remain to be deciphered. We have identified NHR-8, a nuclear hormone receptor involved in the xenobiotic response in Caenorhabditis elegans, as a new regulator of tolerance to IVM. Loss-of-function nhr-8(ok186) C. elegans mutants subjected to larval development assays and electropharyngeogram measurements, displayed hypersensitivity to IVM, and silencing of nhr-8 in IVM-resistant worms increased IVM efficacy. In addition, compared to wild-type worms, nhr-8 mutants under IVM selection pressure failed to acquire tolerance to the drug. In addition, IVM-hypersensitive nhr-8(ok186) worms displayed low transcript levels of several genes from the xenobiotic detoxification network and a concomitant low Pgp-mediated drug efflux activity. Interestingly, some pgp and cyp genes known to impact IVM tolerance in many nematode species, were down regulated in nhr-8 mutants and inversely upregulated in IVM-resistant worms. Moreover, pgp-6 overexpression in nhr-8(ok186) C. elegans increased tolerance to IVM. Importantly, NHR-8 function was rescued in nhr-8(ok186) C. elegans with the homolog of the parasitic nematode Haemonchus contortus, and silencing of Hco-nhr-8 by RNAi on L2 H. contortus larvae increased IVM susceptibility in both susceptible and resistant H. contortus isolates. Thus, our data show that NHR-8 controls the tolerance and development of resistance to IVM in C. elegans and the molecular basis for this relates to the NHR-8-mediated upregulation of IVM detoxification genes. Since our results show that Hco-nhr-8 functions similarly to Cel-nhr-8, this study helps to better understand mechanisms underlying failure in drug efficacy and open perspectives in finding new compounds with NHR-8 antagonist activity to potentiate IVM efficacy.
... Incidentally, a reduction in the expression of cyp-35C1 seemed to restore the fertility of F3 ash-2 and hrde-1 worms with sanguinarine-treated ancestors but not that of berberine-treated worms. This result supports the finding of a previous report that the cyp-35A/C gene reduces the reproduction of C. elegans exposed to xenobiotics (Menzel et al., 2005). This result also suggests that cyp-35C1 is a useful indicator of the worm's xenobiotic response for transgenerational inheritance experiments. ...
Epigenetic mechanisms allow for transgenerational memory of an ancestor's environment and can affect the gene expression, physiology and phenotype of that ancestor's descendants, independent of DNA sequence alteration. Among many model organisms, Caenorhabditis elegans has been instrumental in studies of transgenerational inheritance, most of which have focused on the effects of external stressors of the parent worm on the lifespan and stress resistance of future generations. In this work, we used Nile red staining of accumulated lipids in C. elegans to investigate the transgenerational effect of two benzylisoquinoline alkaloids, namely, berberine and sanguinarine. Our results showed that a reduction in Nile red fluorescence can be propagated to subsequent worm generations. Using mutant worms, we found that the transgenerational effect requires the ASH‐2 component of the histone H3K4me3 complex and the HRDE‐1 worm Argonaute protein. Ash‐2 is also required for transgenerational inheritance of the xenobiotic response in the worm. Our study offers new insights into transmissible drug effects across multiple generations and suggests the importance of such analyses in the drug development process.
This article is protected by copyright. All rights reserved.
... 35 Bioavailable PAHs have been shown to strongly induce cyp35 genes, coding for NADPH-dependent monooxygenases involved in bioactivation and detoxification of hydrophobic xenobiotics. 58,59 Toxic concentration, however, was generally above 0.25 mg·L −1 and thereby more than 2 orders of magnitude higher than PAH concentration measured in a comparable biochar. Therefore, it is unlikely that direct toxicity of PAHs are the reason for the observed effects on neurobehavior of C. elegans; this argument gets supported by the fact, that the bioavailable share of the PAHs is usually much lower than the total concentration. ...
In recent years, biochars have gained increasing interest in mitigating climate changes and revitalizing contaminated or drained soil. Studies determining their impact on the ecosystem, especially on soil invertebrates, however, are still scarce and the neurotoxic potential of biochars has never been evaluated before. Using the model organism Caenorhabditis elegans we determined the neurotoxic effect of biochar produced from rice straw by pyrolysis at 500 °C at concentrations ranging from 0 to 2000 mg C∙L-1. Biochar had a hormetic effect on locomotion behavior. Furthermore, high concentrations impaired defecation as well as the recognition and response to a chemical attractant. None of the potential toxic chemicals in the biochar had sufficient high concentrations to explain the detected neurotoxic effect. Using electron paramagnetic resonance (EPR) spectroscopy, we detected free radicals in the biochar. Detrimental reaction of free radicals with biotic macromolecules can induce oxidative stress responses and are a potential reason for the evaluated neurotoxic effect of biochar. Overall, we were able to prove that biochars have the potential to act as weak neurotoxins to soil organisms and effects of persistent free radicals should be investigated further.
... Markedly, β-NF strongly induced all cyp-35A subfamily members and several other CYPs (Menzel et al. 2001). Menzel et al. (2005) knocked down cyp-35A subfamily members, which decreased the reproductive toxicity of PCB52 and fluoranthene. Inokuchi et al. (2014) suggested a role for CYPs (including CYP-35A3) in the tolerance against triclosan and trichlocarban. ...
Anthropogenic micropollutants and transformation products (TPs) negatively affect aquatic ecosystems and water resources. Wastewater treatment plants (WWTP) represent major point sources for (micro)pollutants and TPs in urban water cycles. The aim of the current study was to assess the removal of micropollutants and toxicity during conventional and advanced wastewater treatment. Using wild-type and transgenic Caenorhabditis elegans, the endpoint reproduction, growth, and cytochrome P450 (CYP) 35A3 induction (via cyp-35A3::GFP) were assessed. Samples were collected at four WWTPs and a receiving surface water. One WWTP included the advanced treatments: ozonation followed by granular activated carbon (GAC) or biological filtration (BF), respectively. Relevant micropollutants and WWTP parameters (n = 111) were included. Significant reproductive toxicity was detected for one WWTP effluent (31–83% reduced brood size). Three of four effluents significantly promoted the growth of C. elegans larvae (49–55% increased lengths). This effect was also observed for the GAC (34–41%) and BF (30%) post-treatments. Markedly, significant cyp-35A3::GFP induction was detected for one effluent before and after ozonation, being more pronounced for the ozonated samples (5- and 7.4-fold above controls). While the advanced treatments decreased the concentrations of most micropollutants, the observed effects may be attributed to effects of residual target compounds and/or compounds not included in the target chemical analysis. This highlights the need for an integrated assessment of (advanced) wastewater treatment covering both biological and chemical parameters.
... However, this general assumption has been weakened by a number of reports and attention devoted to CYPs is gradually increasing. The genome of C. elegans encodes more than 80 CYP proteins and several of these, in particular members of the family CYP35, have been shown to be inducible by xenobiotics (Menzel et al., 2001(Menzel et al., , 2005. Increased expression of cyp35C1, cyp35A5 and cyp35A2 mRNAs has been reported in C. elegans upon albendazole exposure (Laing et al., 2010) and recently TBZ has been shown to induce cyp35A3, cyp35A5, cyp35C1 and cyp35D1 (Jones et al., 2013) and to be metabolised by CYP35D1 in C. elegans (Jones et al., 2015). ...
Benzimidazoles (BZs) remain amongst the most widely used anthelmintic drug classes against gastro-intestinal nematode infections, although their efficacy is increasingly compromised by resistance. The primary underlying mechanisms for BZ resistance are single-nucleotide polymorphisms (SNPs) in the isotype 1 β-tubulin gene causing the substitutions F167Y, E198A or F200Y. However, resistance is believed to be multi-genic and previous studies have shown that isolates carrying 90–100% F200Y can vary considerably in their resistance level in the egg hatch assay (EHA). Cytochrome P450 monooxygenases (CYPs) are associated with drug resistance in mammals and arthropods and have been considered as mediators of anthelmintic resistance. In Caenorhabditis elegans, several members of the CYP34/35 and CYP31 families are BZ and/or xenobiotic inducible and thiabendazole (TBZ) is metabolised by CYP35D1. Here, expression of all 5 CYPs closely related to the C. elegans CYP34/35 and CYP31 families was investigated in fourth-stage larvae of two susceptible and three BZ-resistant Haemonchus contortus isolates following in vitro exposure to TBZ for 3 and 6 h using real-time RT-PCR. The resistance status of all isolates was determined using EHAs and quantification of resistance-associated β-tubulin SNPs using pyrosequencing. While none of the CYPs was TBZ inducible, constitutive expression of CYP34/35 family member HCOI100383400 was significantly 2.4–3.7-fold higher in the multi-drug resistant WR isolate with the strongest BZ resistance phenotype compared to susceptible and intermediate-level BZ-resistant isolates. Although this increase is only moderate, HCOI100383400 might still be involved in high-level BZ resistance by further decreasing susceptibility in isolates already carrying 100% of a β-tubulin SNP causing BZ resistance. Lower transcript levels were observed for all CYPs in the intermediately resistant IRE isolate in comparison to the susceptible HcH isolate, which, except for CYP HCOI01579500, were statistically non-significant. This suggests that none of the investigated CYPs may contribute to protection against TBZ in this particular isolate.
... Conversely, increased PTQ toxicity in gst-4 mutants was found (Fig. 4b), reinforcing its detoxifying role. A systematic screen with different xenobiotics found that GST-4, as many other GSTs, possesses a wide range of different substrates [49], thus we cannot state whether our observations are due to direct interaction between PTQ and GST-4 or between ROS sub products and GST-4. As GST-4 consumes GSH, it was not surprising that the levels of GCS-1::GFP, the limiting step on glutathione synthesis, were increased following PTQ exposure at LC (Table 3). ...
Organic selenium and tellurium compounds are known for their broad-spectrum effects in a variety of experimental disease models. However, these compounds commonly display high toxicity and the molecular mechanisms underlying these deleterious effects have yet to be elucidated. Thus, the need for an animal model that is inexpensive, amenable to high-throughput analyses and feasible for molecular studies is highly desirable to improve organochalcogen pharmacological and toxicological characterization. Herein, we use Caenorhabdtis elegans (C. elegans) as a model for the assessment of pharmacological and toxicological parameters following exposure to two 4-phenylchalcogenil-7-chloroquinolines derivatives (PSQ for selenium and PTQ for tellurium-containing compounds). While non-lethal concentrations (NLC) of PTQ and PSQ attenuated paraquat-induced effects on survival, lifespan and oxidative stress parameters, lethal concentrations (LC) of PTQ and PSQ alone are able to impair these parameters in C. elegans. We also demonstrate that DAF-16/FOXO and SKN-1/NRF2 transcription factors underlie the mechanism of action of these compounds, as their targets sod-3, gst-4 and gcs-1 were modulated following exposures in a daf-16- and skn-1-dependent manner. Finally, in accordance with a disturbed thiol metabolism in both LC and NLC, we found higher sensitivity of trxr-1 worm mutants (lacking the selenoprotein thioredoxin reductase 1) when exposed to PSQ. Finally, our study suggests new targets for the investigation of organochalcogen pharmacological effects, reinforcing the use of C. elegans as a powerful platform for preclinical approaches.
... Many studies have investigated the up-regulation of P450 enzymes in C. elegans in response to various xenobiotics, such as PCB52, fluoranthene, benzo[a]pyrene, fenitrothion, and CP, and cyp35 genes have been shown to be strongly inducible by them. 23,44 Among cyp35 genes, cyp35a2 and cyp35a3 genes acted to metabolize the organophosphate pesticides, CP and fenitrothion, to a more toxicologically active metabolite as shown in our previous studies. 17,18 Although no increase in the activation of CP catalyzed by CYPs was measured, this could be inferred by changes in the expression of CYP metabolic-related mRNA over time. ...
The internal concentration of chemicals in exposed organism changes over time due to absorption, distribution, metabolism, and excretion processes, since chemicals are taken up from the environment. Internal concentration and time are very important modifiers of toxicity when biomarkers are used to evaluate the potential hazards and risks of environmental pollutants. In this study, the responses of molecular biomarkers, and the fate of chemicals in the body, were comprehensively investigated to determine cause-and-effect relationships over time. Chlorpyrifos (CP) was selected as a model chemical, and Caenorhabditis elegans was exposed to CP for 4 h using the passive dosing method. Worms were then monitored in fresh medium during a 48-h recovery regime. The mRNA expression of genes related to CYP metabolism (cyp35a2 and cyp35a3) increased linearly during the constant exposure phase. The body residue of CP decreased once it reached a peak level during the early stage of exposure, indicating that the initial uptake of CP rapidly induced biotransformation with the synthesis of new CYP metabolic proteins. The residual chlorpyrifos-oxon concentration, an acetylcholinesterase (AChE) inhibitor, continuously increased even after the recovery regime started. These delayed toxicokinetics seem to be important for the extension of AChE inhibition for up to 9 h after the start of the recovery regime. Comprehensive investigation into the molecular initiation events and changes in the internal concentrations of chemical species provide insight into response causality within the framework of an adverse outcome pathway.
... Nevertheless, the significance of stress-induced changes in gene expression will depend on the downstream biological effects elicited (Feder and Walser, 2005). The biological function of stress responsive genes has been tested using loss of function mutants and RNAi (Menzel et al., 2005(Menzel et al., , 2007Cui et al., 2007). Once a physiological role has been established, transgenic strains can be generated containing reporter genes under control of the regulatory regions of stress inducible genes. ...
... ). Diese nahe Verwandtschaft zwischen den bei Säugern am Metabolismus beteiligten Isoformen und den CYP in C. elegans lassen ihre Beteiligung an der Biotransformation von Xenobiotika in C. elegans annehmen. Außerdem wurden den Säugern homologe Proteine AHR (aryl hydrocarbon receptor) und ARNT (AHR nuclear transporter)(Powell-Coffmann et al. 1998) und die Induktion der CYP in C. elegans durch Xenobiotika in Screeningversuchen nachgewiesen.Menzel et al. (2001) inkubierten C. elegans mit PAK (β-Naphthoflavon, Benz(a)pyren, Fluoranthen), PCB52, Pharmaka (Lansoprazol, Phenobarbital, Clofibrat) und dem Pestizid Atrazin und weisen mit "Northern blot"-Experimenten die Regulation von CYP-Genen in C. elegans durch Xenobiotika nach.Die Ergebnisse des Screeningtests werden vonMenzel et al. (2005) für CYP35A1, A2, A5, und C1, deren verstärkte Expression durch PCB52, Fluoranthen, Atrazin und Lansoprazol nachgewiesen wird, bestätigt. Wie die Daten der gemessenen Kupfer(II)-Verdünnungsreihe zeigen, stimmen die Ergebnisse für die ROS-Bildung nach Inkubation mit Kupfer(II) gut mit Messwerten aus in vitro Modellen überein, was auf nah verwandte Mechanismen der intrazellulären Redoxprozesse und der antioxidativen Schutzmechanismen zurückzuführen ist. ...
Caenorhabditis elegans Modelsysteme für Freiland und Labor
Caenorhabditis elegans ist eines der am intensivsten untersuchten Tiermodelle, dabei weisen die nur unzureichenden Informationen über sein ursprüngliches Habitat eine Vielzahl offener Fragen auf. Deshalb beschäftigt sich der erste Teil vorliegender Arbeit mit einer Überprüfung der Freilandökologie und Biogeographie des Taxons.
Nach den Untersuchungen spricht vieles dafür, dass die Ursprungsart der C. elegans Gruppe ein Begleiter von Gastropoden war, welche auch für ihre Ausbreitung häufig verantwortlich sind. Als Folge der Verschleppung durch Wirtstiere (u.a. die Weinbergschnecke Helix aspersa) erreichte sie ihr heutiges Areal, in allen Kontinenten.
Ein nicht weniger wichtiges Ziel der vorliegenden Arbeit war die Etablierung von C. elegans als Biotest-System für die Indikation von Chemikalien-Wirkungen.
In dieser Arbeit wurden ein ökotoxikologisches Testsystem entwickelt mit den C. elegans als Modellorganismus, das durch Messung von umweltbedingten intrazellulär gebildeten ROS im Gesamtorganismus eine Einschätzung der biologischen Aktivität von Chemikalien und Umweltmedien ermöglichen sollte. Als grundlegender biochemischer Parameter ist eine, die Kapazität der antioxidativen Schutzmechanismen übersteigende, ROS-Bildung ursächlich für oxidative Schädigungen der Zelle verantwortlich und damit Basis für zahlreiche pathologische Effekte. Aus den Resultaten als Bewertungsgrundlage lässt sich folgern, dass sich das erarbeitete Biotestsystem als adäquates Verfahren zur Messung von oxidativem Stress, als Folge exogener Belastung, einsetzen lässt. Damit leistet es einen Beitrag zur Beurteilung des Toxizitätspotentials von Chemikalien und Umweltproben.
Zur Eignung des Einsatzes von C. elegans für Toxizitätstest wurden weitere Untersuchungen mit verschiedenen Chemikalien durchgeführt. Dabei ergibt sich bezogen auf molare Einheiten eine immer gleich bleibende Reihenfolge der Sensitivitäten gegenüber BaP > Cu2+ > AcL, ein Ergebnis welches mit Testergebnissen von anderen Versuchsorganismen zu vergleichen war.
Die Wirkung von Atrazin auf die Entwicklung von C. elegans wurde auch untersucht.
Es wurde festgestellt, dass Atrazin auf Wachstum und Reproduktion von C. elegans konzentrationsabhängig toxisch wirkt.
Die bisherigen Ergebnisse zeigen jedoch, dass das Testsystem C. elegans hervorragend geeignet ist, um Chemikalienwirkungen frühzeitig sichtbar zu machen.
... Several studies have validated classification systems (Burczynski et al., 2000) and large amounts of gene expression data are accumulating in dedicated databases, such as Chemical Effects in Biological Systems (CEBS) (Waters et al., 2003). Gene expression measured by DNA microarrays is believed to provide a more comprehensive , sensitive and characteristic insight into toxicity than typical toxicological parameters such as morphological changes, altered reproductive capacity or mortality (Hamadeh et al., 2001; Menzel et al., 2005a). In addition to these classical ecotoxicological parameters, ecotoxicogenomics is a powerful tool that unravels mechanistic processes, reveals novel modes of action, and provides the opportunity to get a dynamic picture of biological systems. ...
The book comprises four parts. The first part (Chapters 1-3) deals with background issues relating to nematodes themselves and to bioindicators in general. Part II (Chapters 4-8) describes practical aspects of using nematode assemblages as bioindicators in real world settings. Part III (Chapters 9-11) describes the potential of nematodes, particularly the model organisms Caenorhabditis elegans , to be used in laboratory-based toxicity assays. It is precisely the same features (short life span, ease of culture, transparent cuticle) that made C. elegans the first model animal that make it so suitable for laboratory-based bioassays. Furthermore, because of the vast amount of knowledge and technical know-how generated by the C. elegans community, data can be generated and interpreted from C. elegans assays better than from any other animal-based biosensor. The final part (Chapter 12) deals with commercial aspects of using nematodes as environmental bioindicators.
... The expression of cyp-35A5, a further transcript co-expressed with cyp-35A2, revealed a modest, yet statistically significant, up-regulation within the fat-5(tm420) and cyp-35A2(gk317) mutant backgrounds, and a striking induction in the fat-5(tm420);cyp-35A2(gk317) double mutant (Fig. 3E). As the cyp-35A/C gene members share protein domains [11,31], their interaction network is inter-woven. Previous reports have demonstrated that the RNAi of cyp-35A3 and cyp-35A5 affects the normal lipid regulatory pathways in C. elegans [9]. ...
An imbalance between energy uptake and energy expenditure can lead to obesity and increase the risk of coronary heart disease, high blood pressure, stroke, type II diabetes and some cancers. Given that key elements of the energy pathway are evolutionary conserved, invertebrate research is an attractive alternative that overcomes the many legislative, financial and experimental hurdles typical of research with higher metazoan animals. Recent studies have suggested that some members of the cytochrome P450 superfamily are involved in lipid metabolism in addition to the traditional xenobiotic activity. To investigate this notion in more detail, the present study aimed to pinpoint phenotypic, genetic and genomic-level responses of Caenorhabditis elegans using selected deletion mutants including fat-5 (a member of the Δ9 desaturases) and cyp-35A2 (a member of the cytochrome P450 family). The creation of a fat-5(tm420);cyp-35A2(gk317) mutant uncovered that the deletion of both genes resulted in a strain which is marked by an extended lifespan. Furthermore, it diminished the overall level of Nile Red positive compartments, which is indicative of a change in lipid metabolism. Comprehensive transcriptomics revealed that several genes involved in aging and lipid transport/homeostasis were modulated following the double deletion of fat-5 and cyp-35A2. Taken together, the results suggest the presence of a putative correlation between longevity and lipid regulation and given that both genes have human homologs, this finding may offer a new lead to investigate in higher organisms.
... Given that these proteins are known to be induced during stress (Urano et al. 2002 ), it suggests that the observed downregulation may reflect the stress-protective effect of MC-BA. On the other hand, the upregulation of several cyp-genes (cytochrome P450 family), known for their role in the metabolism of xenobiotics (Menzel et al. 2001(Menzel et al. , 2005Ioannides 2008 ), implies an increase in chemical stress. A similar pattern was recently described by Peltonen et al. ( 2013 ) who describe that chronic alcohol exposure can lead to the repression of several abu-genes and the induction of cyp-genes in C. elegans. ...
Elevated levels of adsorbable organic bromine compounds (AOBr) have been detected in German lakes, and cyanobacteria like Microcystis, which are known for the synthesis of microcystins, are one of the main producers of natural organobromines. However, very little is known about how environmental realistic concentrations of organobromines impact invertebrates. Here, the nematode Caenorhabditis elegans was exposed to AOBr-containing surface water samples and to a Microcystis aeruginosa-enriched batch culture (MC-BA) and compared to single organobromines and microcystin-LR exposures. Stimulatory effects were observed in certain life trait variables, which were particularly pronounced in nematodes exposed to MC-BA. A whole genome DNA-microarray revealed that MC-BA led to the differential expression of more than 2000 genes, many of which are known to be involved in metabolic, neurologic, and morphologic processes. Moreover, the upregulation of cyp- and the downregulation of abu-genes suggested the presence of chronic stress. However, the nematodes were not marked by negative phenotypic responses. The observed difference in MC-BA and microcystin-LR (which impacted lifespan, growth, and reproduction) exposed nematodes was hypothesized to be likely due to other compounds within the batch culture. Most likely, the exposure to low concentrations of organobromines appears to buffer the effects of toxic substances, like microcystin-LR.
Aquilaria crassna (AC) is a beneficial plant widely used to alleviate various health ailments. Nevertheless, the neuroprotection, antiaging, and xenobiotic detoxification against high benzo[a]pyrene induction have not been investigated. This study aimed to investigate the effects of ethanolic extract of AC leaves (ACEE) in vitro using SH-SY5Y cells and in vivo using Caenorhabditis elegans (C. elegans). Neuroprotective activities and cell cycle progression were studied using SH-SY5Y cells. Additionally, C. elegans was used to determine longevity, health span, and transcriptional analysis. Furthermore, ACEE possible active compounds were analyzed by gas chromatograph–mass spectrometry (GC-MS) analysis and the possible active compounds were evaluated using a molecular docking study. First, ACEE possessed neuroprotective effects by normalizing cell cycle progression via the regulation of AhR/CYP1A1/cyclin D1 pathway. Next, ACEE played a role in xenobiotic detoxification in high B[a]P-induced C. elegans by the amelioration of lifespan reduction, and body length and size decrease through the reduction in gene expression in hexokinase (hxk) and CYP35 pathway. Finally, phytochemicals of ACEE were identified and we uncovered that clionasterol was the possible active constituent in powerfully inhibiting both CYP1A1 and hexokinase II receptor. Essentially, ACEE was recognized as a potential alternative medicine to defend against high B[a]P effects on neurotoxicity and xenobiotic detoxification.
Pesticides are prevalent pollutants found in river sediments in agricultural regions worldwide, leading to environmental pollution and toxic effects on biota. In this study, twenty sediment samples were collected from the Magdalena River in Colombia and analyzed for forty pesticides. Methanolic extracts of the sediments were used to expose Caenorhabditis elegans for 24 h, evaluating the effects on its reproduction. The most abundant pesticides found in Magdalena River sediments were atrazine, bromacil, DDE, and chlorpyrifos. The concentrations of DDE and the sum of DDD, DDE, and DDT were above the Threshold Effect Concentration (TEC) values for freshwater sediments, indicating potential effects on aquatic organisms. The ratios of DDT/(DDE + DDD) and DDD/DDE suggest historical contributions of DDT and degradation under aerobic conditions. Several sampling sites indicated a moderate toxicity risk to biota, as calculated by the sediment quality guideline quotient (SQGQ). Nematode brood size was reduced by up to 37%. The presence of chlordane, DDT-related compounds, and chlorpyrifos in Magdalena River sediments was associated with reproductive toxicity among C. elegans.
Citation: Zhang, X.; Saul, N.; Lieke, T.; Chen, Y.; Wu, M.; Pan, B.; Steinberg, C.E.W. Biochar Extracts Can Modulate the Toxicity of Persistent Free Radicals in the Nematode Caenorhabditis elegans. Appl. Biosci. 2023, 2, 71-83. https:// Abstract: As an effective soil amendment, biochars require a comprehensive ecological evaluation before they can be widely used in agriculture because endogenous contaminants, such as environmentally persistent free radicals (EPFRs), certainly pose an ecological risk to soil invertebrates. In this study, Caenorhabditis elegans (C. elegans) was used as a model organism to investigate the neurotoxicity of two rice straw biochars pyrolyzed at 500 and 700 • C. After 24 h exposure to unwashed biochar, washed biochar, and leaching fluids (supernatants), the neurobehavioral parameters of C. elegans were determined in a liquid toxicity test. The results showed that the washed 700 • C biochar particles significantly impaired locomotion and prolonged the defecation interval at a biochar concentration of 4 g·well −1 , while the unwashed biochar and supernatants caused no apparent impairment. Supporting this, electron paramagnetic resonance (EPR) results showed that the intensity of EPFRs in unwashed 700 • C biochar was stronger than that of the corresponding washed particles. This indicates that, in the liquid test, the EPR signal alone is not indicative of particle toxicity. The accessibility and activity of the EPFRs should be considered. Dissolved organic matter (DOM) was observed in the leaching fluids. The neurotoxic activity of the washed biochar was alleviated after the re-addition of leaching fluids to the washed biochar, suggesting that the dissolved organic materials modulate the reactivity of the EPFRs in the liquid phase. This study suggests that the leaching process may increase the risk of biochar when used in the field environment.
Obesity is a global public health problem that is associated with oxidative stress. One of the strategies for the treatment of obesity is the use of drugs; however, these are expensive and have numerous side effects. Therefore, the search for new alternatives is necessary. Baccharis trimera is used in Brazilian folk medicine for the treatment of obesity. Here, B. trimera leaf extract (BT) showed antioxidant activity in seven in vitro tests, and it was not toxic to 3T3 murine fibroblasts or Caenorhabditis elegans. Furthermore, BT reduces the intracellular amount of reactive oxygen species and increases C. elegans survival. Moreover, these effects were not dependent on transcription factors. The inhibition of fat accumulation by BT in the C. elegans model was also investigated. BT reduced lipid accumulation in animals fed diets without or with high amount of glucose. Furthermore, it was observed using RNA interference (iRNA) that BT depends on the transcription factor NHR-49 to exert its effect. Phytochemical analysis of BT revealed rutin, hyperoside, and 5-caffeoylquinic acid as the main BT components. Thus, these data demonstrate that BT has antioxidant and anti-obesity effects. However, further studies should be conducted to understand the mechanisms involved in its action.
The number of reported macrocyclic lactones (ML) resistance cases across all livestock hosts is steadily increasing. Different studies in the parasitic nematode Haemonchus contortus assume the participation of cytochrome P450s (Cyps) enzymes in ML resistance. Still, functional data about their individual contribution to resistance or substrate specificity is missing. Via microinjection, transgenic Caenorhabditis elegans expressing HCON_00141052 (transgene-Hco-cyp-13A11) from extrachromosomal arrays were generated. After 24 h of exposure to different concentrations of ivermectin (IVM), ivermectin aglycone (IVMa), selamectin (SEL), doramectin (DRM), eprinomectin (EPR), and moxidectin (MOX), motility assays were performed to determine the impact of the H. contortus Cyp to the susceptibility of the worms against each ML. While transgene-Hco-cyp-13A11 significantly decreased susceptibility to IVM (four-fold), IVMa (2-fold), and SEL (3-fold), a slight effect for DRM and no effect for MOX, and EPR was observed. This substrate specificity of Hco-cyp-13A11 could not be explained by molecular modeling and docking studies. Hco-Cyp-13A11 molecular models were obtained for alleles from isolates with different resistance statuses. Although 14 amino acid polymorphisms were detected, none was resistance specific. In conclusion, Hco-cyp-13A11 decreased IVM, IVMa, and SEL susceptibility to a different extent, but its potential impact on ML resistance is not driven by polymorphisms.
Evidence about the adverse effects of methamphetamine (METH) on invertebrates is scarce. Hence, C. elegans, a representative invertebrate model, was exposed to METH at environmental levels to estimate chronic and transgenerational toxicity. The results of chronic exposure were integrated into an underlying toxicity framework of METH in invertebrates (e.g., benthos) at environmentally relevant concentrations. The induction of cellular oxidative damage-induced apoptosis and fluctuation of ecologically important traits (i.e., feeding and locomotion) might be attributed by the activation of the longevity regulating pathway regulated by DAF-16/FOXO, and detoxification by CYP family enzymes. The adverse effects to the organism level included impaired viability and decreased fecundity. The results from transgenerational exposure elucidated the cumulative METH-induced damage in invertebrates. Finally, a new risk assessment method named toxicity indicator sensitivity distribution (TISD) analysis was proposed by combining multiple toxicity indicator test data (ECx) to derive the hazardous concentration for 10% indicators (C10) of one species. The risk quotient (RQ) values calculated by measured environmental concentrations and C10 in southern China, southeastern Australia, and the western US crossed the alarm line (RQ = 5), suggesting a need for long-term monitoring.
In this work, Caenorhabditis elegans was employed as an in vivo model to determine the toxic effects of atrazine at different concentrations. After the exposure period from the larval stage L1 to adulthood day 1, atrazine (10 mg/L) significantly decreased the body length and lifespan of nematodes. In addition, exposure to ≥ 0.01 mg/L atrazine remarkably increased the intestinal reactive oxygen species (ROS) levels and reduced locomotion behavior of nematodes, while exposure to ≥ 1 mg/L atrazine decreased the brood size of nematodes. Moreover, atrazine (0.001 - 0.1 mg/L) upregulated the expression levels of hsp-6::GFP and hsp-6/60 in nematodes, indicating the activation of mitochondrial unfolded protein response (mtUPR). On the contrary, atrazine (1 - 10 mg/L) downregulated the expression levels of hsp-6::GFP and hsp-6/60 in nematodes. Furthermore, mtUPR induction governed by the RNAi knockdown of atfs-1 could increase the vulnerability of nematodes against atrazine toxicity. Overall, our findings highlighted the dynamic responses of nematodes toward different concentrations of atrazine, which could be monitored using different sublethal endpoints as bioindicators.
Anisakis simplex (s.l.) is a complex of three sibling (biological) species of parasitic nematodes of marine mammals, including A. berlandi, A. pegreffii and A. simplex (s.s.). It is characterized by a complex life cycle in which humans can become accidental hosts by consuming dishes made of raw or undercooked fish containing L3 larvae, which in many regions of the world is related to the national or regional culinary tradition. This has spurred scientific efforts to develop new methods for treating the disease, called anisakiasis, and to neutralize invasive L3. Thiabendazole (TBZ) is a wide-spectrum anthelminthic with a higher efficacy than albendazole, a drug whose long-term use induces resistance in many parasitic species. Cytochromes P450 participate in TBZ metabolism, and the expression of their genes is controlled by nuclear hormone receptors (NHR). This study aimed to examine the effects of TBZ on the above-described pathway in invasive larvae of A. simplex (s.l.). The efficacy of TBZ against A. simplex (s.l.) larvae was observed for the first time. Larvae were cultured in vitro for 72 h in a medium containing TBZ at five concentrations from 0.5 to 1.5 mM. However, the survival curves did not significantly differ from each other. This means that all of the concentrations of TBZ had a similar effect on the A. simplex (s.l.) L3 larvae during in vitro culture. Nevertheless, TBZ modified the expression of nhr-48, cyp13a3 and cyp1a1 genes in the L3 of A. simplex (s.l.).
Polycyclic aromatic hydrocarbons (PAHs) are environmental toxicants primarily produced during incomplete combustion; some are carcinogens. PAHs can be safely metabolized or, paradoxically, bioactivated via specific cytochrome P450 (CYP) enzymes to more reactive metabolites, some of which can damage DNA and proteins. Among the CYP isoforms implicated in PAH metabolism, CYP1A enzymes have been reported to both sensitize and protect from PAH toxicity. To clarify the role of CYP1A in PAH toxicity, we generated transgenic Caenorhabditis elegans that express CYP1A at a basal (but not inducible) level. Because this species does not normally express any CYP1 family enzyme, this approach permitted a test of the role of basally expressed CYP1A in PAH toxicity. We exposed C. elegans at different life stages to either the PAH benzo[a]pyrene (BaP) alone, or a real-world mixture dominated by PAHs extracted from the sediment of a highly-contaminated site on the Elizabeth River (VA, USA). This site, the former Atlantic Wood Industries, was declared a Superfund site due to coal tar creosote contamination that caused very high levels (in the [mg/mL] range) of high molecular weight PAHs within the sediments. We demonstrate that CYP1A protects against BaP-induced growth delay, reproductive toxicity, and reduction of steady state ATP levels. Lack of sensitivity of a DNA repair (Nucleotide Excision Repair)-deficient strain suggested that CYP1A did not produce significant levels of DNA-reactive metabolites from BaP. The protective effects of CYP1A in Elizabeth River sediment extract (ERSE)-exposed nematodes were less pronounced than those seen in BaP-exposed nematodes; CYP1A expression protected against ERSE-induced reduction of steady-state ATP levels, but not other outcomes of exposure to sediment extracts. Overall, we find that in C. elegans, a basal level of CYP1A activity is protective against the examined PAH exposures.
Caenorhabditis elegans has been an invaluable model organism in research fields such as developmental biology and neurobiology. Neurotoxicity is one of the subfields greatly profiting from the C. elegans model within biomedical context, while the corresponding potential of the organism applied to environmental studies is relevant but has been largely underexplored. Within the biomedical scope, the implication of metals and organic chemicals with pesticide activity (hereinafter designated as pesticides) in the etiology of several neurodegenerative diseases has been extensively investigated using this nematode as a primary model organism. Additionally, as a well-known experimental model bearing high sensitivity to different contaminants and representing important functional levels in soil and aquatic ecosystems, C. elegans has high potential to be extensively integrated within Environmental Risk Assessment (ERA) routines. In spite of the recognition of some regulatory agencies, this actual step has yet to be made. The purpose of this review is to discuss the major advantages supporting the inclusion of C. elegans in lower tiers of ERA. Special emphasis was given to its sensitivity to metals and pesticides, which is similar to that of other model organisms commonly used in ERA (e.g. Daphnia magna and Eisenia sp.), and to the large array of endpoints that can be tested with the species, both concerning the aquatic and the soil compartments. The inclusion of C. elegans testing may hence represent a relevant advance in ERA, providing ecologically relevant insights toward improvement of the regulatory capacity for establishing appropriate environmental protection benchmarks.
Oxidative stress was evaluated for anthracene (Ant) and alkyl-Ants (9-methylanthracene [9-MA] and 9,10-dimethylanthracene [9,10-DMA]) in Caenorhabditis elegans to compare changes in toxicity due to the degree of alkylation. Worms were exposed at 1) the same external exposure concentration and 2) the maximum water-soluble concentration. Formation of reactive oxygen species, superoxide dismutase activity, total glutathione concentration, and lipid peroxidation were determined under constant exposure conditions using passive dosing. The expression of oxidative stress-related genes (daf-2, sir-2.1, daf-16, sod-1, sod-2, sod-3 and cytochrome 35A/C family genes) was also investigated to identify and compare changes in the genetic responses of C. elegans exposed to Ant and alkyl-Ant. At the same external concentration, 9,10-DMA induced the greatest oxidative stress, as evidenced by all indicators, except for lipid peroxidation, followed by 9-MA and Ant. Interestingly, 9,10-DMA led to greater oxidative stress than 9-MA and Ant when worms were exposed to the maximum water-soluble concentration, although the maximum water-soluble concentration of 9,10-DMA is the lowest. Increased oxidative stress by alkyl-Ants would be attributed to higher lipid-water partition coefficient and the π electron density in aromatic rings by alkyl substitution, although this supposition requires further confirmation.
The mitochondrial genome (mtDNA) is intimately linked to cellular and organismal health, as demonstrated by the fact that mutations in and depletion of mtDNA result in severe mitochondrial disease in humans. However, cells contain hundreds to thousands of copies of mtDNA, which provides genetic redundancy, and creates a threshold effect in which a large percentage of mtDNA must be lost prior to clinical pathogenesis. As certain pharmaceuticals and genetic mutations can result in depletion of mtDNA, and as many environmental toxicants target mitochondria, it is important to understand whether reduced mtDNA will sensitize an individual to toxicant exposure. Here, using ethidium bromide (EtBr), which preferentially inhibits mtDNA replication, we reduced mtDNA 35-55% in the in vivo model organism Caenorhabditis elegans. Chronic, lifelong, low-dose EtBr exposure did not disrupt nematode development or lifespan, and induced only mild alterations in mitochondrial respiration, while having no effect on steady-state ATP levels. Next, we exposed nematodes with reduced mtDNA to the known and suspected mitochondrial toxicants aflatoxin B-1, arsenite, paraquat, rotenone or ultraviolet C radiation (UVC). EtBr pre-exposure resulted in mild sensitization of nematodes to UVC and arsenite, had no effect on AfB(1) and paraquat, and provided some protection from rotenone toxicity. These mixed results provide a first line of evidence suggesting that reduced mtDNA content may sensitize an individual to certain environmental exposures.
The toxicity of cadmium on Caenorhabditis elegans was investigated to identify sensitive biomarkers for environmental monitoring and risk assessment. Stress-related gene expression were estimated as toxic endpoints Cadmium exposure led to an increase in the expression of most of the genes tested. The degree of increase was more significant in heat shock protein-16.1, metallothionein-2, cytochrome p450 family protein 35A2, glutathione S-transferase-4, superoxide dismutase-1, catalase-2, C. elegans p53-like protein-1, and apoptosis enhancer-1 than in other genes. The overall results indicate that the stress-related gene expressions of C. elegans have considerable potential as sensitive biomarkers; for cadmium toxicity monitoring and risk assessment.
The nematode Caenorhabditis elegans can be a powerful model system for the discovery and characterization of new anthelmintics. C. elegans is free-living and its use therefore circumvents the complications of culturing parasitic nematodes that are invariably dependent on a host to complete their life cycle. C. elegans is also small (1mm in length), hermaphroditic, and has a 3-day life cycle. These features make C. elegans a seemingly ideal high-throughput platform with which to discover novel anthelmintics. However, C. elegans has formidable xenobiotic defenses that have likely evolved to protect it from a plethora of chemical insults in its native environment. In this chapter, we will discuss the extent of C. elegans xenobiotic resistance and review strategies to circumvent this resistance in order to take full advantage of one of the most powerful animal models available.
Usually, stress is considered adverse: too much work load, or, conversely, unemployment; lack of success; unsolved family problems, etc. More scientifically, Selye (1936) discovered in his fundamental study by challenging rats that “if the organism is severely damaged by acute non-specific nocuous agents such as exposure to cold, surgical injury, production of spinal shock (transcision of the cord), excessive muscular exercise, or intoxications with sublethal doses of diverse drugs (adrenaline, atropine, morphine, formaldehyde, etc.), a typical syndrome appears, the symptoms of which are independent of the nature of the damaging agent or the pharmacological type of the drug employed, and represent rather a response to damage as such”.
Usually, stress is considered adverse: too much work load, or, conversely, unemployment; lack of success; unsolved family problems, etc. More scientifically, Selye (1936) discovered in his fundamental study by challenging rats that “if the organism is severely damaged by acute non-specific nocuous agents such as exposure to cold, surgical injury, production of spinal shock (transcision of the cord), excessive muscular exercise, or intoxications with sublethal doses of diverse drugs (adrenaline, atropine, morphine, formaldehyde, etc.), a typical syndrome appears, the symptoms of which are independent of the nature of the damaging agent or the pharmacological type of the drug employed, and represent rather a response to damage as such”.
How do organisms cope with complex environmental stressors? How do they respond to environmental challenges? Transcription is the initial step in gene expression, thus a transcriptional response gives the first indication of cellular mechanisms that are affected by a stressor. As a consequence, it can provide a sensitive starting point to assess ecological stress responses assuming all ecologically relevant effects are indeed accompanied by alterations in gene expression profiles. Ecological transcriptomics is a powerful tool that unravels mechanistic processes, reveals novel modes of action and regulation pathways, and provides the opportunity to get a dynamic picture of biological systems and the ability to comprehensively dissect different states of biological activities in cells, tissues, or whole organisms.
Interactions between plants and herbivores are of major importance in natural ecosystems. The phytochemical co-evolution theory suggests that plant secondary metabolites are likely the most important mediators of plant-herbivore interactions, although their primary cause for production is the shielding effect against adverse environmental triggers. According to this theory, both plants and insect herbivores generate selective forces that lead to the evolution of plant defense (i.e., plant secondary metabolites, PSM) and herbivore offense (i.e., detoxification ability) in a so-called co-evolutionary arms race. These chemicals, although not required for primary plant metabolic processes such as respiration or growth, have been extensively recognized for their role in plant defense against herbivore and pathogen attack (Bidart-Bouzat and Imeh-Nathaniel 2008). There is still some concern, though, as to whether or not the primary reason for the production of PSM is protection from photodamage rather than from herbivory. Nevertheless, there is no doubt that these metabolites also function as food allelochemicals, that is plant defense.
IntroductionExperimental DesignsApplications of RNAi in Drug Metabolizing Enzymes and TransportersConclusions
AcknowledgmentReferences
The cytochrome P450 enzymes are of great importance and interest because they catalyze reactions which have profound effects on the biological activities of drugs, environmental chemistry, and endogenous compounds. As a consequence, the literature on these enzymes is too voluminous to read, scan, or even cite. And while the pace accelerates and the scope broadens to include more biochemical aspects such as amino acid sequencing and three-dimensional structural models of isoenzymes, the bench scientists struggles to perceive where the new information fits and what applicability it has, if any, zo his/her work. Pharmacologists and toxicologists also feel swamped by the exponential increase in cytochrome P450 publications. The present paper is designed to assist by providing a framework for the prediction and interpretation of new data. It is an effort to summarize the functions of known human cytochrome P450s in terms of their specific catalytic activities, substrates, inducers, and inhibitors. Summaries are presented in tables for ready access of information. (Tables 5-21) The literature through 1996 is covered in the reference section. In some instances, references are to reviews that should be consulted for citations of the original literature.
(Full text of the paper available through Informa Healthcare Publisher only, or download updated article: Summary of information on human CYP enzymes: human P450 metabolism data. Drug Metabolism Reviews 01/2002; 34(1-2):83-448.)
In this review we argue against two paradigms: (1) humic substances (HS) are not taken up by aquatic organisms; (2) HS are inert in aquatic systems, except for the release of reactive oxygen species after irradiation. In fact, these paradigms are recycled and do not apply any longer. We show that HS-like substances, such as caffeic acid oxidation products, are taken up by all aquatic organisms studied so far. Furthermore, we present phenomenological as well as mechanistic evidence that HS have direct effects on aquatic plants and animals. The effects may be categorized as non-specific, such as expression of heat shock proteins (hsp) and modulation of biotransformation enzymes, or specific, such as inhibition of photosynthetic oxygen release in plants. Basic ecotoxicological requirements are fulfilled: several mechanisms apply to a variety of aquatic organisms, dose-response relationships and quantitative structure effect relationships may be established where applicable. We conclude that HS are natural xenobiotics that exert a chemical stress and, thus, are able to structure aquatic guilds by various modes of action.
This paper reviews the current status of nematodes with stress-inducible transgenes as biosensors responsive to a range of
external stressors, e.g., soil or water pollution, microwave radiation or immunological attack. TransgenicCaenorhabditis elegans carrying reporter genes under heat shock promoter control express reporter products only under stressful conditions. Although
relatively insensitive to single metal ions, these worms respond to complex mixtures present in metal-contaminated watercourses
and to laboratory mixtures containing similar constituents, but not to any of their components singly at comparable concentrations.
Responses to metal mixtures are enhanced by a non-ionic surfactant, Pluronic F-127. Metals taken up by food bacteria and insoluble
metal carbonates can also evoke stress responses, both in soil and aqueous media. However, high concentrations of added metals
are needed to induce clear-cut responses in soil, owing to metal sorption onto clays and organic matter. Transgenic worms
are also stressed by exposure to microwave radiation; pulsed signals generate responses that diminish markedly with distance
from the source. Finally, stress responses are inducible by anti-epicuticle antisera and complement, suggesting that immune
attack can also activite the heat shock system. The development of rapid microplate toxicity assays based on transgenic nematodes
is discussed.
This study describes a new approach for assessing behavioral changes following toxicant exposure and compares the method to other common endpoints used in environmental toxicology. The nematode Caenorhabditis elegans was exposed to a range of ethanol concentrations to determine its effect on survival, reproduction and behavior. Each endpoint was evaluated for its sensitivity by comparing LC50, RC50 (concentration at which there is a 50% reduction in number of offspring as compared to controls), and BC50 (concentration at which there is a 50% reduction in movement as compared to controls) values for ethanol exposure. Worms showed 24-h lethality at concentrations of ethanol in the range of 83 g/L to 99 g/L. Reproduction in C. elegans was estimated by counting the number of off-spring after 3 d of exposure, which decreased with the increase in ethanol concentration from 8 g/L to 71 g/L. Behavior was quantified by using a new computer tracking method, which can simultaneously assess hundreds of nematodes and provides several behavioral parameters in real time. Worms showed some hyperactivity (increased movement) at very low ethanol concentrations (0.8 g/L and 2.4 g/L) and a decrease in movement at higher ethanol concentrations (4 g/L to 40 g/L). A comparison for sensitivity between the three endpoints was performed. Behavior and reproduction responses were found to be similar and, as expected, both are much more sensitive indicators of toxicity than lethality. The advantages and disadvantages of the computer tracking system are discussed.
The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor, until now described only in vertebrates, that mediates many of the carcinogenic and teratogenic effects of certain environmental pollutants. Here, we describe orthologs of AHR and its dimerization partner AHR nuclear translocator (ARNT) in the nematode Caenorhabditis elegans, encoded by the genes ahr-1 and aha-1, respectively. The corresponding proteins, AHR-1 and AHA-1, share biochemical properties with their mammalian cognates. Specifically, AHR-1 forms a tight association with HSP90, and AHR-1 and AHA-1 interact to bind DNA fragments containing the mammalian xenobiotic response element with sequence specificity. Yeast expression studies indicate that C. elegans AHR-1, like vertebrate AHR, requires some form of post-translational activation. Moreover, this requirement depends on the presence of the domains predicted to mediate binding of HSP90 and ligand. Preliminary experiments suggest that if AHR-1 is ligand-activated, its spectrum of ligands is different from that of the mammalian receptor: C. elegans AHR-1 is not photoaffinity labeled by a dioxin analog, and it is not activated by beta-naphthoflavone in the yeast system. The discovery of these genes in a simple, genetically tractable invertebrate should allow elucidation of AHR-1 function and identification of its endogenous regulators.
Effect of cytochrome P450 (P450) inducers on liver microsomal metabolism of 3,4,3',4'-, 3,5,3',5'- and 2,5,2', 5'-tetrachlorobiphenyl (TCB) was studied using male Wistar rats, male Hartley guinea pigs and male Golden syrian hamsters. In metabolism of 3,4,3'4'- and 3,5,3',5'-TCB, liver microsomes from 3-methylcholanthrene (MC)- or 3,4,5,3',4'-pentachlorobiphenyl (PenCB)-treated hamsters showed hydroxylase activities for both TCB isomers, although the activities were much less than those of rats. In contrast, liver microsomes form untreated and phenobarbital (PB)-, MC- or PenCB-treated guinea pigs showed no hydroxylase activity. In 2,5,2',5'-TCB metabolism, 3-hydroxylase activity was observed in untreated guinea pigs and hamsters, but not in untreated rats. The activity pigs was induced by PB treatment in all three species, at rates of 324, 19 and 20 pmol/min/mg protein in rats, guinea and hamsters, respectively. This activity was not enhanced by treatment with either MC or PenCB. Only in hamsters was 4-hydroxylated metabolite formed in all microsomes used in addition to the 3-hydroxylated one, and the formation was accelerated 2.0-, 2.7- and 4.8-fold by treatment with PB, MC and PenCB, respectively. These results suggest that different P450 isoforms in hamster liver microsomes are involved in 3- and 4-hydroxylation of 2,5,2',5'-TCB. Thus, there are species differences in the basal ability to hydroxylate TCB isomers, and in the extent of effect of P450 inducers on the metabolism of these isomers among the three species.
The heteromeric unliganded aryl hydrocarbon receptor complex (AHRC) contains the aryl hydrocarbon receptor monomer (AHR). Binding of polycyclic or halogenated aromatic hydrocarbon (PAH and HAH) ligand causes release of AHR, which then associates with the AHR nuclear translocator protein (ARNT) to generate the heterodimeric "transformed" AHRC. AHR and ARNT belong to a novel subclass of basic helix-loop-helix-containing transcription factors. The transformed AHRC binds xenobiotic responsive elements in responsive genes and turns on their transcription. Certain of these genes encode enzymes involved in the metabolic activation of PAHs to mutagenic derivatives. HAHs are not genotoxic: Their pathogenicity depends on the AHRC but not on their metabolism. Current research includes investigations directed towards delineating the pathways of HAH pathogenesis, ascertaining whether AHR can mediate signal transduction independently of DNA binding, understanding the mechanism of transcriptional activation, and investigating the potential roles of AHR and ARNT in development.
The aim of this work was to identify the form(s) of human cytochrome P450 (P450) involved in the hepatic biotransformation of lansoprazole to its two main metabolites, i.e., the sulfone and the hydroxy derivative. In liver microsomes, the production of the sulfone of lansoprazole correlated with the level of P450 3A4, cyclosporin oxidase, and the production of the hydroxy derivative, as well as of omeprazole sulfone. The production of hydroxylansoprazole moderately correlated with the level of P450 3A4, cyclosporin oxidase, and (S)-mephenytoin 4'-hydroxylase. The production of the sulfone and of the hydroxy derivative of lansoprazole was significantly inhibited by anti-P450 3A4 antibodies, by cyclosporin and ketoconazole, and by tolbutamide. Anti-P450 2C8 and 2C3 antibodies moderately inhibited the biotransformation of lansoprazole, whereas they completely inhibited (S)-mephenytoin 4'-hydroxylase activity under the same conditions. In primary cultures of human hepatocytes, the biotransformation of lansoprazole and the oxidation of cyclosporin were strongly increased by rifampicin and phenobarbital, whereas (S)-mephenytoin 4'-hydroxylation was not. beta-Naphthoflavone did not induce the formation of the sulfones but stimulated the production of hydroxylansoprazole. Among several forms of cDNA-expressed human P450s, 3A4 generated significant amounts of the sulfones of lansoprazole and omeprazole and 2C18 was active for the production of hydroxylansoprazole but inactive in the 4'-hydroxylation of (S)-mephenytoin. We conclude that P450 3A4 is the major enzyme involved in the production of the sulfone of lansoprazole and that this P450, as well as P450 2C18 and/or another 2C-related form, could contribute to the production of hydroxylansoprazole.
This investigative study assesses the ease and usefulness of the nematode Caenorhabditis elegans for identifying contributors to effluent toxicity within an industrial and municipal wastewater treatment plant (WWTP) system. Several different types of industries, including fiberglass manufacturing, paper packaging, and yarn dyeing, discharge effluent into the municipal wastewater treatment plant, which in turn discharges into a local creek. A major objective of this study was to identify primary sources of toxicity throughout the system with a nematode toxicity test. Twenty-four-hour composite water samples were taken periodically over a ten-month period at five strategic points within the system: (1) at the point of discharge at each of the three industries, (2) at the combined industrial influent of the wastewater treatment plant, (3) at the effluent of the WWTP, (4) upstream of the WWTP discharge, and (5) downstream of the WWTP discharge. Samples were analyzed for basic water chemistry, and each sample was tested for whole effluent toxicity using a 72-h nematode test with mortality as the end point. Results suggest that interactions between the wastewaters of certain industries may increase the overall nematode toxicity in the wastewater treatment facility's composite influent and effluent. Nematode mortality trends indicate relatively high toxicity levels in wastewater entering the WWTP from contributing industries. High WWTP influent toxicity may potentially be due to varying flow rate ratios of industrial discharges, release of varying toxic constituents in wastewaters, and toxic interactions between chemical constituents of industrial wastewaters. The evaluation of toxicity within the treatment system may pinpoint locations where pollution prevention strategies may be implemented to reduce overall toxicity at the point of discharge.
The transition metal cadmium is a pervasive and persistent environmental contaminant that has been shown to be both a human toxicant and carcinogen. To inhibit cadmium-induced damage, cells respond by increasing the expression of genes encoding stress-response proteins. In most cases, the mechanism by which cadmium affects the expression of these genes remains unknown. It has been demonstrated in several instances that cadmium activates gene transcription through signal transduction pathways, mediated by protein kinase C, cAMP-dependent protein kinase, or calmodulin. A codicil is that cadmium should influence the expression of numerous genes. To investigate the ability of cadmium to affect gene transcription, the differential display technique was used to analyze gene expression in the nematode Caenorhabditis elegans. Forty-nine cDNAs whose steady-state levels of expression change 2-6-fold in response to cadmium exposure were identified. The nucleotide sequences of the majority of the differentially expressed cDNAs are identical to those of C. elegans cosmids, yeast artificial chromosomes, expressed sequence tags, or predicted genes. The translated amino acid sequences of several clones are identical to C. elegans metallothionein-1, HSP70, collagens, and rRNAs. In addition, C. elegans homologues of pyruvate carboxylase, DNA gyrase, beta-adrenergic receptor kinase, and human hypothetical protein KIAA0174 were identified. The translated amino acid sequences of the remaining differentially expressed cDNAs encode novel proteins.
The daf-9 gene functions to integrate transforming growth factor-beta and insulin-like signaling pathways to regulate Caenorhabditis elegans larval development. Mutations in daf-9 result in transient dauer-like larval arrest, abnormal reproductive development, molting defects and increased adult longevity. The phenotype is sterol-dependent, and dependent on the activity of DAF-12, a nuclear hormone receptor. Genetic tests show that daf-9 is upstream of daf-12 in the genetic pathways for larval development and adult longevity. daf-9 encodes a cytochrome P450 related to those involved in biosynthesis of steroid hormones in mammals. We propose that it specifies a step in the biosynthetic pathway for a DAF-12 ligand, which might be a steroid. The surprising cellular specificity of daf-9 expression (predominantly in two sensory neurons) supports a previously unrecognized role for these cells in neuroendocrine control of larval development, reproduction and life span.
The daf-9 gene functions to integrate transforming growth factor-β and insulin-like signaling pathways to regulate Caenorhabditis elegans larval development. Mutations in daf-9 result in transient dauer-like larval arrest, abnormal reproductive development, molting defects and increased adult longevity. The phenotype is sterol-dependent, and dependent on the activity of DAF-12, a nuclear hormone receptor. Genetic tests show that daf-9 is upstream of daf-12 in the genetic pathways for larval development and adult longevity. daf-9 encodes a cytochrome P450 related to those involved in biosynthesis of steroid hormones in mammals. We propose that it specifies a step in the biosynthetic pathway for a DAF-12 ligand, which might be a steroid. The surprising cellular specificity of daf-9 expression (predominantly in two sensory neurons) supports a previously unrecognized role for these cells in neuroendocrine control of larval development, reproduction and life span.
Humic isolates have previously been shown to modulate the reproduction of the nematode, Caenorhabditis elegans MAUPAS. The aim of this study was to find out if a hormone-like effect is an intrinsic feature of humic substances. Therefore, the same nematode bioassay was used with ten humic substances (XAD and/or reverse osmosis isolates). The results confirmed the hormone-like effect of humic substances. nine isolates significantly increased the numbers of offspring per worm, one isolate did not modulate the reproduction of C. elegans. This effect occurred with XAD isolates and with reverse osmosis isolates as well. Thus, this effect is an intrinsic feature of humic substances. This reproduction modulating potential of humic substances may be due to alkylaromatics that are major structural and photo stable structural components, rather than to sterols that are only minor structural components. This is a new view about the direct interactions of humic with aquatic organisms. The effects haven been mathematically modeled by a simple approach, considering a hormone-like effect (beta) and a non-hormone like effect, such as nutrient effect (alpha) on growth. The different effects observed can be interpreted in terms of trade-offs between these two competing mode of actions.
Arylhydrocarbon receptor knock-out, AhR(–/–), mice have recently been shown to be rather resistant to benzo[ a ]pyrene (B[ a ]P)-induced tumor formation, probably reflecting the inability of these mice to express significant levels of cytochrome P450 (P450 or CYP) 1A1 that activates B[ a ]P to reactive metabolites (Y.Shimizu, Y.Nakatsuru, M.Ichinose, Y.Takahashi, H.Kume, J.Mimura, Y.Fujii-Kuriyama and T.Ishikawa (2000) Proc. Natl Acad. Sci. USA , 97, 779–782). However, it is not precisely determined whether CYP1B1, another enzyme that is also active in activating B[ a ]P, plays a role in the B[ a ]P carcinogenesis in mice. To understand the basis of roles of CYP1A1 and CYP1B1 in the activation of chemical carcinogens, we compared levels of induction of liver and lung CYP1A1, 1A2, and 1B1 by various polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls in AhR(+/+) and AhR(–/–) mice. Liver and lung CYP1A1 and 1B1 mRNAs were highly induced in AhR(+/+) mice by a single intraperitoneal injection of each of the carcinogenic PAHs, such as B[ a ]P, 7,12-dimethylbenz[ a ]anthracene, dibenz[ a,l ]pyrene, 3-methylcholanthrene, 1,2,5,6-dibenzanthracene, benzo[ b ]fluoranthene, and benzo[ a ]anthracene and by a co-planar PCB congener 3,4,3′,4′-tetrachlorobiphenyl. We also found that 6-aminochrysene, chrysene, benzo[ e ]pyrene, and 1-nitropyrene weakly induced the mRNA expression of CYP1A1 and 1B1, whereas anthracene, pyrene, and fluoranthene that have been reported to be non-carcinogenic in rodents, were very low or inactive in inducing these P450s. The extents of induction of liver CYP1A2 by these chemicals were less than those of CYP1A1 and 1B1 in AhR(±/±) mice. In AhR(–/–) mice, there was no induction of these P450s by PAHs and polychlorinated biphenyls. Liver microsomal activities of 7-ethoxyresorufin and 7-ethoxycoumarin O -deethylations and of mutagenic activation of (±)- trans -7,8-dihydroxy-7,8-dihydro-B[ a ]P to DNA-damaging products were found to correlate with levels of CYP1A1 and 1B1 mRNAs in the liver. Our results suggest that carcinogenicity potencies of PAHs may relate to the potencies of these compounds to induce CYP1A1 and 1B1 through AhR-dependent manner and that these induced P450s participate in the activation of B[ a ]P and related carcinogens causing initiation of cancers in mice.
A possible role for metabolic activation of 2,2',5,5'-tetrachlorobiphenyl (TCB) to quinonoid metabolites was investigated in vitro in rat liver microsomes and in vivo in male Sprague-Dawley rats. Incubation of TCB with phenobarbital-induced rat liver microsomes resulted in metabolism of TCB to 3-hydroxy-TCB (3-OH-TCB) and 3,4-dihydroxy-TCB (3,4-diOH-TCB), which were further oxidized to form a reactive intermediate that bound Do liver proteins, The predominant species observed in the Raney nickel assay for cysteinyl adducts was identified as 3,4-diOH-TCB, consistent with an adduct having the structure 5-cysteinyl-3,6-dichloro-4-(2',5'-dichlorophenyl)-1,2-benzoquinone. This adduct may arise via the Michael addition of the sulfhydryl group of cysteine to 3,6-dichloro-4-(2',5'-dichlorophenyl)-1,2-benzoquinone, (Cl(4)PhBQ). Metabolism of 3-OH-TCB by phenobarbital-induced microsomes in the presence of either NADPH or cumene hydroperoxide as a cofactor resulted in the formation of adducts. Dose-dependent formation of cysteinyl adducts was observed in liver cytosolic protein from rats treated with a single dose of TCB (0-200 mg/kg) by gavage. By regression analysis, the TCB adducts decayed with a half-life of 2.03 +/- 0.131 days (mean +/- SE), which is similar to 2.5-fold shorter than the endogenous half-life for liver cytosolic protein in rat liver, suggesting adduct instability, Saturable formation of TCB adducts was observed in liver cytosolic protein of rats receiving multiple doses of TCB over 5 days, The levels of Cl(4)PhBQ-derived adducts were 2.1-fold greater than the estimated steady-state levels predicted by the single-dose treatment [97.7 +/- 13.2 vs 45.7 +/- 3.73 (pmol/g)/(mg/kg of body weight)], suggesting induction of metabolism, A single cysteinyl adduct, inferred to be 5-cysteinyl-3,6-dichloro-4-(2',5'-dichlorophenyl)-1,2-benzoquinone quinone, was detected in brain cytosolic protein of rats treated with multiple doses of TCB with levels of 15.2 (pmol/g)/(mg/kg of body weight). Implied involvement of a reactive quinone in the liver and brain of TCB-treated rats supports the idea that quinonoid metabolites may be important contributors to PCB-derived oxidative damage to genomic DNA.
Humic isolates have previously been shown to modulate the reproduction of the nematode, Caenorhabditis elegans MAUPAS. The aim of this study was to find out if a hormone-like effect is an intrinsic feature of humic substances. Therefore, the same nematode bioassay was used with ten humic substances (XAD and/or reverse osmosis isolates). The results confirmed the hormone-like effect of humic substances. nine isolates significantly increased the numbers of offspring per worm, one isolate did not modulate the reproduction of C. elegans. This effect occurred with XAD isolates and with reverse osmosis isolates as well. Thus, this effect is an intrinsic feature of humic substances. This reproduction modulating potential of humic substances may be due to alkylaromatics that are major structural and photo stable structural components, rather than to sterols that are only minor structural components. This is a new view about the direct interactions of humic with aquatic organisms. The effects haven been mathematically modeled by a simple approach, considering a hormone-like effect (beta) and a non-hormone like effect, such as nutrient effect (alpha) on growth. The different effects observed can be interpreted in terms of trade-offs between these two competing mode of actions.
We present a method using the free-living nematode Caenorhabditis elegans (Maupas, 1899) to assess toxicity in liquid medium and whole-sediment setups. Test duration is 72 h; endpoints are body length, number of eggs inside worms, percentage of gravid worms, and number of offspring per worm. The effect of CdCl2 on C. elegans in liquid-phase exposures is described as an example. Results from a field study with polluted sediments from the River Elbe (Germany) suggest that nematodes may be useful organisms in assessing toxicity of sediments in the whole phase.
Caenorhabditis elegans is a small, free-living hermaphroditic nematode that is widely used for the investigation of basic biological phenomena at the genetic and molecular levels. The hsp16 genes in this system encode a family of stress-inducible 16-kDa proteins. Stable transgenic nematode lines were derived that carry fusions of the hsp16 genes to the Escherichia coli lacZ reporter gene. These transgenic strains express high levels of β-galactosidase in the nucleus, in response to a heat shock or to a variety of chemical stressors. Agents tested to date that induce the stress response in these animals include Cd2+, Cu2+, Hg2+, Pb2+, Zn2+, AsO2−, and the herbicide paraquat. Some of these agents yield distinct tissue patterns of stress induction (e.g., Pb2+ in the posterior pharynx, Cd2+ throughout the pharynx, Hg2+ in intestine), suggesting that classification of stress agents in complex mixtures may be a useful feature of this biomonitoring system. Using a soluble β-galactosidase substrate, an assay was developed that allows the magnitude of the stress response to be measured. Stress reporter gene induction always occurred below the LC50 of the test substance, suggesting that this assay is a more sensitive and rapid indicator of stress than current LC50 assays using Caenorhabditis elegans.
Toxicity tests in invertebrates often use sublethal endpoints, which may exhibit different sensitivity for various toxicants. Our objective was to characterize the sensitivity of movement, feeding, growth, and reproduction as endpoints for heavy metal toxicity testing with Caenorhabditis elegans. Growth and feeding were assessed in the same nematode samples used to assess movement and reproduction. Median effective concentrations (EC50s) for 24-h exposures to Pb, Cu, and Cd were determined for movement, feeding, and growth and a 72-h EC50 was derived for reproduction. The order of toxicity was Cu > Pb > Cd for each endpoint, including lethality and movement. There were no differences in sensitivity among endpoints for any metal. When exposed for 4 h at (sublethal) concentrations that were 14 times the 24-h EC50 value, Pb and Cu reduced feeding to the same extent while movement was reduced significantly more by Pb than by Cu. Thus, a difference in sensitivity of endpoints was apparent at 4 h, which was not evident at 24 h. These observations suggest potentially different mechanisms of toxicity for 24- and 4-h tests.
Most pharmacologically active molecules are lipophilic and remain un-ionized or only partially ionized at physiological pH.
Biotransformation means that a lipid-soluble xenobiotic or endobiotic compound is enzymatically transformed into polar, water-soluble,
and excretable metabolites. The major organ for drug biotransformation is the liver. The metabolic products often are less
active than the parent drug or inactive. However, some biotransformation products (metabolites) may have enhanced activity
or toxic effects. Thus biotransformation may include both “detoxication” and “toxication” processes. One of the major enzyme
systems that determines the organism's capability of dealing with drugs and chemicals is represented by the cytochrome P450
monooxygenases. Studies in the last 15 years have provided evidence that cytochrome P450 occurs in many different forms or
“isozymes” which differ in spectral, chemical, and immunological properties and have different substrate affinities. These
isozymes also differ in their regulation and tissue distribution. Recombinant DNA studies indicate that between 40 and 60
structural genes code for different cytochrome P450 isozymes in a single organism. Other enzyme systems include dehydrogenases,
oxidases, esterases, reductases, and a number of conjugating enzyme systems including glucuronosyltransferases, sulfotransferases,
glutathione S-transferases, etc. Environmental and genetic factors cause interindividual and intraindividual differences in
drug metabolism and may alter the balance between toxification and detoxification reactions. Genetic polymorphisms lead to
subpopulations of patients with decreased, absent, or even increased activities of certain reactions (e.g., CYP2D6, CYP2C19,
N-acetyltransferase polymorphism). Environmental factors such as other drugs, steroids, dietary factors, alcohol, and cigarette
smoke can induce or inhibit drug-metabolizing enzymes and cause intraindividual variation.
In order to identify genes that are differentially expressed as a consequence of oxidative stress due to paraquat we used
the differential display technique to compare mRNA expression patterns in Caenorhabditis elegans. A C.elegans mixed stage worm population and a homogeneous larval population were treated with 100 mM paraquat, in parallel with controls.
Induction of four cDNA fragments, designated L-1, M-47, M-96 and M-132, was confirmed by Northern blot analysis with RNA from
stressed and unstressed worm populations. A 40-fold increase in the steady-state mRNA level in the larval population was observed
for the L-1/M-47 gene, which encodes the detoxification enzyme glutathione S-transferase. A potential stress-responsive transcription
factor (M-132) with C2H2-type zinc finger motifs and an N-terminal leucine zipper domain was identified. The M-96 gene encodes a novel stressresponsive
protein. Since paraquat is known to generate superoxide radicals in vivo, the response of the C.elegans superoxide dismutase (SOD) genes to paraquat was also investigated in this study. The steady-state mRNA levels of the manganese-type
and the copper/zinc-type SODs increased 2-fold in the larval population in response to paraquat, whereas mixed stage populations
did not show any apparent increase in the levels of these SOD mRNAs.
Pathways of biotransformation of the carcinogenic polycyclic hydrocarbon fluoranthene in individual isolated perifused liver cells were delineated using noninvasive microfluorescence spectroscopic techniques. An example of heterogeneity of coupling between phase I and phase II enzymes detected in a population of beta-naphthoflavone-induced rat liver cells demonstrates the usefulness of the experimental model of perifused liver cells.
The in vitro metabolism of fluoranthene (FA) was assessed by incubating 3-[3H]FA, the synthesis of which is described, with rat hepatic microsomal enzymes. Several metabolites including the FA 2,3-diol, FA 2-3,-quinone, 3-OH-FA, 1-OH-FA, and 8-OH-FA were isolated by high-pressure liquid chromatography and identified by comparison of chromatographic properties and uv-visible spectra with those of synthetic standards. The major metabolite produced over the FA concentration range studied (23-233 microM) was FA 2,3-diol, accounting for 29-43% of the total extractable metabolites. This diol was characterized further by high-resolution mass spectroscopy and H-NMR and determined to be identical in structure to the trans-2,3-dihydroxy-2,3-dihydrofluoranthene. The FA 2,3-diol, syn and anti 2,3-diol-1,10b-epoxides, FA 2,3-quinone, and FA 7,8-diol were all shown to be mutagenic toward Salmonella typhimurium TM677. The FA 1,10b-diol and syn and anti FA 1,10b-diol-2,3-epoxides were not mutagenic. The epoxide hydrolase inhibitor, 3,3,3-trichloropropylene oxide, markedly reduced the mutagenic potency of FA while concurrently inhibiting FA 2,3-diol production but not overall FA metabolism. These results suggests that a major metabolic activation pathway of FA resulting in the production of mutagenic species involves the formation of the FA 2,3-diol and the subsequent oxidation of this diol to a FA 2,3-diol-1,10b-epoxide. Another minor activation pathway with mutagenic endpoints may involve the formation of the 7,8-diol.
Methods are described for the isolation, complementation and mapping of mutants of Caenorhabditis elegans, a small free-living nematode worm. About 300 EMS-induced mutants affecting behavior and morphology have been characterized and about one hundred genes have been defined. Mutations in 77 of these alter the movement of the animal. Estimates of the induced mutation frequency of both the visible mutants and X chromosome lethals suggests that, just as in Drosophila, the genetic units in C. elegans are large.
Chemical analysis and a study of renaturation kinetics show that the nematode, Caenorhabditis elegans, has a haploid DNA content of 8 x 10(7) base pairs (20 times the genome of E. coli). Eighty-three percent of the DNA sequences are unique. The mean base composition is 36% GC; a small component, containing the rRNA cistrons, has a base composition of 51% GC. The haploid genome contains about 300 genes for 4S RNA, 110 for 5S RNA, and 55 for (18 + 28)S RNA.
One of the most useful theories to explain some mechanistic aspects of chemical carcinogenesis is the theory of toxification, i.e. the formation of reactive metabolites by enzymes and the covalent linkage of these activated intermediates with cellular macromolecules to initiate the carcinogenic process. With this theory as a background, the authors examine the formation of reactive polycyclic hydrocarbon intermediates and factors affecting their interactions with DNA, RNA, and proteins. They have surveyed the literature concerning the effects of covalent binding of such reactive intermediates on the structure and function of biological macromolecules. They summarize some studies about possible correlations between the binding of these chemicals to DNA and their mutagenicity and carcinogenicity. Although these kinds of correlative studies cannot prove that DNA is the critical target for the carcinogenic action of chemicals, positive correlations at least do not refute this hypothesis. During recent years, important experimental advances have revolutionized the study of the interactions of polycyclic hydrocarbons with macromolecules. First, the availability of compounds of high specific radioactivity has increased the sensitivity of detecting minute amounts of carcinogens bound to nucleic acids. Second, many different metabolites have been synthesized. Third, analytical instrumentation and methods for measuring specific nucleoside-hydrocarbon adducts and for measuring biochemical and biological activity of metabolites have been improved.
Tritiated 2,2'5,5'-tetrachlorobiphenyl (3H-TCB) was incubated with phenobarbital(PB)-induced rat liver microsomes in the presence of an epoxide hydrase inhibitor and brominated analog (BrAO) of the expected metabolic intermediate, 2,2',5,5'-tetrachlorobiphenyl-3,4-oxide (TCBAO). A putative arene oxide intermediate (3H-AO), which was radiolabeled, was separated from 3H-TCB and BrAO by column chromatography, high pressure liquid chromatography (HPLC) and thin-layer chromatography (TLC), and was analyzed for TCBAO by methods that were independent of radiometric techniques. The retention times (Rt's) of TCBAO and 3H-AO on two gas chromatography (GC) columns were the same, both before and after acid catalyzed rearrangement. 3H-AO was further characterized by rearrangement to a mixture of 3- and 4-hydroxy-TCB that was identified by gas chromatography-mass spectroscopy (GC-MS). The rate of TCB metabolism and the production of 3H-AO by liver microsomes from a PB-induced, adult male rhesus monkey was less than that observed with rat microsomes. The 3H-AO from the monkey was also characterized as TCBAO by rearrangement to the characteristic TCB phenols that were analyzed by GC-MS using selective ion monitoring. This study is the first in which an arene oxide of a polychlorinated biphenyl (PCB) was actually isolated as a mammalian metabolite and subjected to direct chemical analysis.
The aim of this study was to identify which human P450 enzymes are involved in the metabolism of lansoprazole. In the presence of NADPH and oxygen, human liver microsomes converted lansoprazole to lansoprazole sulfide, lansoprazole sulfone and 5-hydroxylansoprazole. Formation of lansoprazole sulfide occurred nonenzymatically. The formation of lansoprazole sulfone appeared to be catalyzed by a single, low-affinity enzyme (apparent Km approximately 100 microM). In contrast, lansoprazole 5-hydroxylation appeared to be catalyzed by two kinetically distinct enzymes (apparent Km approximately 100 microM and approximately 15 microM). When human liver microsomes (n = 16) were incubated with 100 microM lansoprazole, both the 5-hydroxylation and sulfoxidation of lansoprazole appeared to be catalyzed by CYP3A4/5 (based on correlation analyses). Antibodies against rat CYP3A enzymes inhibited the rate of both 5-hydroxylation (approximately 55%) and sulfoxidation (approximately 70%) and cDNA-expressed CYP3A4 catalyzed both the 5-hydroxylation and sulfoxidation of lansoprazole (apparent Km approximately 100 microM). However, at the pharmacologically relevant substrate concentration of 1 microM, lansoprazole sulfoxidation was still highly correlated with CYP3A4/5 activity (r2 = .905), but lansoprazole 5-hydroxylation appeared to be catalyzed by CYP2C19 (r2 = .875) rather than CYP3A4/5 (r2 = .113). Antibodies and chemical inhibitors of CYP2C enzymes preferentially inhibited the 5-hydroxylation of lansoprazole, whereas lansoprazole sulfoxidation was preferentially inhibited by antibodies and chemical inhibitors of CYP3A4/5. The cDNA expressed enzymes CYP2C8, CYP2C9 and CYP2C19 catalyzed varying rates of lansoprazole 5-hydroxylation at a substrate concentration of 50 microM, but only CYPC19 catalyzed this reaction at 1 microM. These results suggest that at pharmacologically relevant concentrations, the 5-hydroxylation of lansoprazole is primarily catalyzed by CYP2C19, whereas the sulfoxidation of lansoprazole is primarily catalyzed by CYP3A4/5. It is possible that individuals lacking CYP2C19 will be poor metabolizers of lansoprazole.
1. Induction of endogenous cytochrome P4501A1 (CYP1A1) by benzimidazole derivatives has been investigated in the human hepatoma cell line HepG2. 2. By Northern and Western blot analysis, omeprazole has been shown to be a more potent inducer of CYP1A1 than both lansoprazole and E3810, whereas pantoprazole did not induce CYP1A1. Similar results were obtained for the CYP1A1 enzyme-specific deethylation of 7-ethoxyresorufin. 3. The induction of CYP1A1 in the permanent cell line HepG2 corresponds to results observed in human hepatocytes in primary culture. 4. The results provide experimental evidence that HepG2 cells can be used as an appropriate tool to examine inducing effects of drugs on the expression of CYP1A1.
Concentration-dependent induction of cytochrome P4501A (CYP1A) and intracellular porphyrin accumulation were observed following treatment of chicken embryo hepatocyte (CEH) cultures with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 3,3',4,4'-tetrachlorobiphenyl (PCB 77, IUPAC nomenclature), 2,3',4,4',5-pentachlorobiphenyl (PCB 118), 3,3',4,4',5-pentachlorobiphenyl (PCB 126), 3,3',4,4',5,5'-hexachlorobiphenyl (PCB 169), and a commercial mixture of PCBs (Aroclor 1254). For these halogenated aromatic hydrocarbons (HAHs), or mixture, maximal CYP1A activity [measured as ethoxyresorufin-O-deethylase (EROD) activity] and immunodetectable protein were observed at concentrations just prior to, or coincident with, the concentrations at which porphyrin accumulation became evident. Both immunodetectable CYP1A protein and catalytic activity decreased at high concentrations of these compounds, but the rate and extent of decrease of immunodetectable CYP1A protein varied. Time-course studies with PCB 77 indicated a decrease in potency and an increase in maximal CYP1A induction between 24 and 48 hr of exposure which may indicate in vitro metabolism of this HAH. Intracellular accumulation of total porphyrins without CYP1A induction, was observed for 2,2',5,5'-tetrachlorobiphenyl (PCB 52), 2,2',6,6'-tetrachlorobiphenyl (PCB 54), 2,2',3,5',6-pentachlorobiphenyl (PCB 95), 2,2',4,5,5'-pentachlorobiphenyl (PCB 101), 2,2',3,3',6,6'-hexachlorobiphenyl (PCB 136), and 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153). Overall, these results are consistent with a role for CYP1A induction and/or Ah receptor activation in porphyrin accumulation mediated by HAHs with a planar configuration, whereas those that are not planar may mediate porphyrin accumulation by a mechanism not involving induction of CYP1A.
Biomarker assays that provide measures of the toxic effects of chemicals on key organisms are of particular interest in ecotoxicology and environmental risk assessment. Typically, such assays provide measures of the molecular mechanisms that underlie toxicity (e.g., inhibition of brain acetylcholinesterase activity by organophosphorus insecticides and retardation of the vitamin K cycle by anticoagulant rodenticides). They are particularly valuable for detecting and quantifying toxicity where organisms are exposed to mixtures of compounds and for identifying cases of potentiation. In birds, inhibition of brain acetylcholinesterase activity can provide an index of potentiation of organophosphorus and carbamate insecticides by other pesticides. Inhibition of serum butyrylcholinesterase also is very useful as a nondestructive assay but is not simply related to inhibition of brain acetylcholinesterase. Assays for DNA damage can indicate where there is an increase in the rate of activation of carcinogens and mutagens due to induction of the cytochrome P450 system. Assays for blood levels of retinol (vitamin A) and thyroxine can establish thyroxine antagonism by metabolites of 3,3,4,4-tetrachlorobiphenyl. Assays for changes in levels of clotting protein in serum can give an indication of the effect of mixtures of anticoagulant rodenticides on the vitamin K cycle. The interactive effects of mixtures of pesticides in the field are starting to be investigated by this approach (e.g., a recent study of the combined action of malathion and prochloraz in the red-legged partridge).
1. We examined the effect of two chloro-s-triazines (atrazine and simazine) on hepatic microsomal cytochrome P450 enzymes in rat. Rats were treated intraperitoneally with atrazine or simazine daily for 3 days with 100, 200 and 400 mumol/kg. 2. Among the P450-dependent monooxygenase activities, testosterone 2 alpha-hydroxylase (T2AH) activity in rat, which is associated with CYP2C11, was significantly decreased at all doses of atrazine and simazine. The levels relative to control activities were 59-46 and 60-32% respectively. Similarly, oestradiol 2-hydroxylase (ED2H) activity was also significantly decreased by 28-51% by atrazine and simazine at all doses. However, no change in CYP2C11 protein level by either chloro-s-triazine was observed. K(m) for T2AH was significantly increased only by simazine (200 mumol/kg), whereas the Vmax and Cl(int) for T2AH were significantly decreased by atrazine and simazine at all doses. 3. 7-Ethoxyresorufin O-deethylase (EROD), 7-methoxyresorufin O-demethylase (MROD) and 7-pentoxyresorufin O-depentylase (PROD) activities were significantly increased by 1.4-1.6-, 1.7-3.2- and 1.5-2.2-fold respectively, by both chloro-s-triazines at 200 or 400 mumol/kg. Lauric acid omega-hydroxylase (LAOH) was also increased by 1.4-fold by simazine at 200 and 400 mumol/kg. Immunoblotting showed that only simazine induces CYP1A2 and CYP4A1/2 protein expression. 4. The activities of 7-ethoxycoumarin O-deethylase (ECOD), bufuralol 1'-hydroxylase (BF1'H), chlorzoxazone 6-hydroxylase (CZ6H), testosterone 6 beta-hydroxylase (T6BH) and testosterone 7 alpha-hydroxylase (T7AH) were not affected by either chloro-s-triazine. 5. These results suggest that the pattern of changes in P450 isoforms by chloro-s-triazines differs between atrazine and simazine, that these herbicides change the constitutive and/or male specific P450 isoform(s) in rat liver, and that these changes closely relate to the toxicity of chloro-s-triazines.
A genetic interference phenomenon in the nematode Caenorhabditis elegans
has been described in which expression of an individual gene can be
specifically reduced by microinjecting a corresponding fragment of
double-stranded (ds) RNA. One striking feature of this process is a
spreading effect: interference in a broad region of the animal is
observed following the injection of dsRNA into the extracellular body
cavity. Here we show that C. elegans can respond in a gene-specific
manner to dsRNA encountered in the environment. C. elegans normally feed
on bacteria, ingesting and grinding them in the pharynx and subsequently
absorbing bacterial contents in the gut. We find that Escherichia coli
bacteria expressing dsRNAs can confer specific interference effects on
the nematode larvae that feed on them.
The biochemistry of foreign compound metabolism and the roles played by individual cytochrome P450 (CYP) enzymes in drug metabolism and in the toxification and detoxification of xenochemicals prevalent in the environment are important areas of molecular pharmacology and toxicology that have been widely studied over the past decade. Important advances in our understanding of the mechanisms through which foreign chemicals impact on these P450-dependent metabolic processes have been made during the past 2 years with several key discoveries relating to the mechanisms through which xenochemicals induce the expression of hepatic P450 enzymes. Roles for three "orphan" nuclear receptor superfamily members, designated CAR, PXR, and PPAR, in respectively mediating the induction of hepatic P450s belonging to families CYP2, CYP3, and CYP4 in response to the prototypical inducers phenobarbital (CAR), pregnenolone 16alpha-carbonitrile and rifampicin (PXR), and clofibric acid (PPAR) have now been established. Two other nuclear receptors, designated LXR and FXR, which are respectively activated by oxysterols and bile acids, also play a role in liver P450 expression, in this case regulation of P450 cholesterol 7alpha-hydroxylase, a key enzyme of bile acid biosynthesis. All five P450-regulatory nuclear receptors belong to the same nuclear receptor gene family (family NR1), share a common heterodimerization partner, retinoid X-receptor (RXR), and are subject to cross-talk interactions with other nuclear receptors and with a broad range of other intracellular signaling pathways, including those activated by certain cytokines and growth factors. Endogenous ligands of each of those nuclear receptors have been identified and physiological receptor functions are emerging, leading to the proposal that these receptors may primarily serve to modulate hepatic P450 activity in response to endogenous dietary or hormonal stimuli. Accordingly, P450 induction by xenobiotics may in some cases lead to a perturbation of endogenous regulatory circuits with associated pathophysiological consequences.
Transgenic Caenorhabditis elegans expressing jellyfish Green Fluorescent Protein under the control of the promoter for the inducible small heat shock protein gene hsp-16-2 have been constructed. Transgene expression parallels that of the endogenous hsp-16 gene, and, therefore, allows direct visualization, localization, and quantitation of hsp-16 expression in living animals. In addition to the expected upregulation by heat shock, we show that a variety of stresses, including exposure to superoxide-generating redox-cycling quinones and the expression of the human beta amyloid peptide, specifically induce the reporter transgene. The quinone induction is suppressed by coincubation with L-ascorbate. The ability to directly observe the stress response in living animals significantly simplifies the identification of both exogenous treatments and genetic alterations that modulate stress response, and possibly life span, in C. elegans.
Among 11 isoforms of the human cytochrome P450 enzymes metabolizing xenobiotics, CYP 1A1 and CYP 1A2 were major P450 species in the metabolism of the herbicides chlortoluron and atrazine in a yeast expression system. CYP1A2 was more active in the metabolism of both herbicides than CYP1A1. The fused enzymes of CYP1A1 and CYP1A2 with yeast NADPH-cytochrome P450 oxidoreductase were functionally active in the microsomal fraction of the yeast Saccharomyces cerevisiae and showed increased specific activity towards 7-ethoxyresorufin as compared to CYP1A1 and CYP1A2 alone. Then, both fused enzymes were each expressed in the microsomes of tobacco (Nicotiana tabacum cv. Samsun NN) plants. The transgenic plants expressing the CYP1A2 fusion enzyme had higher resistance to the herbicide chlortoluron than the plants expressing the CYP1A1 fusion enzyme did. The transgenic plants expressing the CYP1A2 fused enzyme metabolized chlortoluron to a larger extent to its non-phytotoxic metabolites through N-demethylation and ring-methyl hydroxylation as compared to the plants expressing the CYP1A1 fused enzyme. Thus, the possibility of increasing the herbicide resistance in the transgenic plants by the selection of P450 species and the fusion with P450 reductase is discussed.
We describe a novel approach to assess toxicity to the free-living nematode Caenorhabditis elegans that relies on the ability of firefly luciferase to report on endogenous ATP levels. We have constructed bioluminescent C. elegans with the luc gene under control of a constitutive promoter. Light reduction was observed in response to increasing temperature, concentrations of copper, lead and 3,5-dichlorophenol. This was due to increased mortality coupled with decreased metabolic activity in the surviving animals. The light emitted by the transgenic nematodes gave a rapid, real-time indication of metabolic status. This forms the basis of rapid and biologically relevant toxicity tests.
Toxicity tests in invertebrates often use sublethal endpoints, which may exhibit different sensitivity for various toxicants. Our objective was to characterize the sensitivity of movement, feeding, growth, and reproduction as endpoints for heavy metal toxicity testing with Caenorhabditis elegans. Growth and feeding were assessed in the same nematode samples used to assess movement and reproduction. Median effective concentrations (EC50s) for 24-h exposures to Pb, Cu, and Cd were determined for movement, feeding, and growth and a 72-h EC50 was derived for reproduction. The order of toxicity was Cu > Pb > Cd for each endpoint, including lethality and movement. There were no differences in sensitivity among endpoints for any metal. When exposed for 4 h at (sublethal) concentrations that were 14 times the 24-h EC50 value, Pb and Cu reduced feeding to the same extent while movement was reduced significantly more by Pb than by Cu. Thus, a difference in sensitivity of endpoints was apparent at 4 h, which was not evident at 24 h. These observations suggest potentially different mechanisms of toxicity for 24- and 4-h tests.
A variety of polycyclic aromatic hydrocarbons and their dihydrodiol derivatives, arylamines, heterocyclic amines, and nitroarenes, were incubated with cDNA-based recombinant (Escherichia coli or Trichoplusia ni) systems expressing different forms of human cytochrome P450 (P450 or CYP) and NADPH-P450 reductase using Salmonella typhimurium tester strain NM2009, and the resultant DNA damage caused by the reactive metabolites was detected by measuring expression of umu gene in the cells. Recombinant (bacterial) CYP1A1 was slightly more active than any of four CYP1B1 allelic variants, CYP1B1*1, CYP1B1*2, CYP1B1*3, and CYP1B1*6, in catalyzing activation of chrysene-1,2-diol, benz[a]anthracene-trans-1,2-, 3,4-, 5,6-, and 8,9-diol, fluoranthene-2,3-diol, dibenzo[a,l]pyrene, benzo[c]phenanthrene, and dibenz[a,h]anthracene and several arylamines and heterocyclic amines, whereas CYP1A1 and CYP1B1 enzymes had essentially similar catalytic specificities toward other procarcinogens, such as (+)-, (-)-, and (+/-)-benzo[a]pyrene-7,8-diol, 5-methylchrysene-1,2-diol, 7,12-dimethylbenz[a]anthracene-3,4-diol, dibenzo[a,l]pyrene-11,12-diol, benzo[b]fluoranthene-9,10-diol, benzo[c]chrysene, 5,6-dimethylchrysene-1,2-diol, benzo[c]phenanthrene-3,4-diol, 7,12-dimethylbenz[a]anthracene, benzo[a]pyrene, 5-methylchrysene, and benz[a]anthracene. We also determined activation of these procarcinogens by recombinant (T. ni) human P450 enzymes in S. typhimurium NM2009. There were good correlations between activities of procarcinogen activation by CYP1A1 preparations expressed in E. coli and T. ni cells, although basal activities with three lots of CYP1B1 in T. ni cells were very high without substrates and NADPH in our assay system. Using 14 forms of human P450s (but not CYP1B1) (in T. ni cells), we found that CYP1A2, 2C9, 3A4, and 2C19 catalyzed activation of several of polycyclic aromatic hydrocarbons at much slower rates than those catalyzed by CYP1A1 and that other enzymes, including CYP2A6, 2B6, 2C8, 2C18, 2D6, 2E1, 3A5, 3A7, and 4A11, were almost inactive in the activation of polycyclic aromatic hydrocarbons examined here.
The majority of cytochrome P450 (CYP)-dependent xenobiotic metabolism is carried out by polymorphic and inducible enzymes which can cause abolished, quantitatively or qualitatively altered or enhanced drug metabolism. Stable duplication, multi-duplication or amplification of active genes, most likely in response to dietary components causing a selection of alleles with multiple genes, has been described. Several examples exist where subjects carrying certain alleles suffer from a lack of drug efficacy due to ultra-rapid metabolism caused by multiple genes or by induction of gene expression or, alternatively, adverse effects from the drug treatment due to the presence of defective alleles. The polymorphism of CYP enzymes is expected to influence the individual sensitivity and toxicity for different environmental agents, although there is no real consensus in the literature about specific firm relationships in this regard. Dosage requirements for several commonly used drugs that have a narrow therapeutic range can differ more than 20-fold dependent on the genotype or the enzyme expression status. The incidence of serious and fatal adverse drug reactions has been found to be very high among hospitalised patients and causes over 100,000 deaths per year in the US, making it between the 4th and 6th leading cause of death. It is likely that predictive genotyping could avoid 10-20% of these deaths. In the present contribution, an overview is presented about our present knowledge about the polymorphism of xenobiotic metabolising CYPs and the importance for adverse effects of drugs and metabolic activation of xenobiotics.
The soil nematode Caenorhabditis elegans is one of the simplest animals having the status of a laboratory model. Its genome contains 80 cytochrome P450 genes (CYP). In order to study CYP gene expression in C. elegans mixed stages and synchronized hermaphrodites were exposed to 18 known xenobiotic cytochrome P450 inducers. Messenger RNA expression was detected by DNA arrays and semiquantitative RT-PCR. Using subfamily-specific primers, a pooled set of exon-rich CYP fragments could be amplified. In this way it was possible to systematically check the influence of different inducers on CYP expression at the same time. The well-known CYP1A inducers beta-naphthoflavone, PCB52, and lansoprazol were the most active and in particular they strongly induced almost all CYP35 isoforms. A few number of further CYP forms were found to be inducible by other xenobiotics like phenobarbital, atrazine, and clofibrate. In addition, a transgenic C. elegans line expressing GFP under control of the CYP35A2 promoter showed a strong induction of the fusion by beta-naphthoflavone in the intestine.
During C. elegans development, animals must choose between reproductive growth or dauer diapause in response to sensory cues. Insulin/IGF-I and TGF-beta signaling converge on the orphan nuclear receptor daf-12 to mediate this choice. Here we show that daf-9 acts downstream of these inputs but upstream of daf-12. daf-9 and daf-12 mutants have similar larval defects and modulate insulin/IGF-I and gonadal signals that regulate adult life span. daf-9 encodes a cytochrome P450 related to vertebrate steroidogenic hydroxylases, suggesting that it could metabolize a DAF-12 ligand. Sterols may be the daf-9 substrate and daf-12 ligand because cholesterol deprivation phenocopies mutant defects. Sensory neurons, hypodermis, and somatic gonadal cells expressing daf-9 identify potential endocrine tissues. Evidently, lipophilic hormones influence nematode metabolism, diapause, and life span.
The aim of toxicology is to study all possible harmful effects of a toxic substance that can represent a risk for humans. In order to establish the safest level of exposure for a certain chemical, a general and very broad screening process is required.
In order to assist in the identification of possible endocrine disrupting chemicals (EDC) in groundwater, we are developing Caenorhabolitis elegans as a high throughput bioassay system in which responses to EDC may be detected by gene expression using DNA microarray analysis. As a first step we examined gene expression patterns and vitellogenin responses of this organism to vertebrate steroids, in liquid culture. Western blotting showed the expected number and size of vitellogenin translation products after estrogen exposure. At 10(-9) M, vitellogenin decreased, but at 10(-7) and 10(-5), vitellogenin was increased. Testosterone (10(-5) M) increased the synthesis of vitellogenin, but progesterone-treated cultures (10(-5) M) had less vitellogenin. Using DNA microarray analysis, we examined the pattern of gene expression after progesterone (10(-5), 10(-7), and 10(-9) M), estrogen (10(-5) M), and testosterone (10(-9) M) exposure, with special attention to the traditional biomarker genes used in environmental studies [vitellogenin, cytochrome P450 (CYP), glutathione s-transferase (GST), metallothionein (MT), and heat shock proteins (HSP)]. GST and P450 genes were affected by estrogen (10(-5) M) and progesterone (10(-5) and 10(-7) M) treatments. For vitellogenin genes, estrogen treatment (10(-5) M) caused overexpression of the vit-2 and vit-6 genes (2.68 and 3.25 times, respectively). After progesterone treatment (10(-7) M), the vit-5 and vit-6 were down-regulated and vit-1 up-regulated (3.59-fold). Concentrations of testosterone and progesterone at 10(-9) M did not influence the expression of the vit, CYP, or GST genes. Although the analysis is incomplete, and low doses and combinations of EDC need to be tested, these preliminary results indicate C. elegans may be a useful laboratory and field model for screening EDC.
1. Xenobiotic-mediated regulation of mRNA expression of all members of the human cytochrome P450 (CYP) 1 family has been measured by RT-PCR in the hepatoma cell line, HepG2. Besides the positive control beta -naphthoflavone, the H(+)/K(+)-ATPase inhibitors omeprazole, lansoprazole, pantoprazole and rabeprazole and the anti-malaria drug primaquine were included in this study. 2. beta-Naphthoflavone, primaquine, omeprazole and lansoprazole increased mRNA levels of CYP1A1, CYP1A2 and CYP1B1. Induction by rabeprazole was significant only for CYP1A1 and CYP1A2, whereas none of the CYP1 mRNAs was induced by pantoprazole. This result was confirmed in primary human hepatocytes. 3. Transcriptional regulation was proved by inhibition of induction with actinomycin D. 4. Increase of CYP1 mRNA was significant after 1 h and maximal after 4 h. CYP1B1, but not CYP1A1 or CYP1A2, was dramatically down-regulated between 4 and 24 h. This decrease was prevented by treatment of cells with actinomycin D after induction, indicating an active transcription-dependent mechanism of CYP1B1 mRNA degradation. 5. In conclusion, xenobiotics inducing CYP1A1 mRNA expression have been shown also to induce CYP1A2 and CYP1B1, differing only with regard to level and time course of induction.
This study investigated the spectral interactions of hepatic microsomal cytochrome p450 (CYP) enzymes with four symmetrical polychlorinated biphenyls (PCBs): 2,2',4,4'-tetrachlorobiphenyl (PCB 47); 2,2',5,5'-tetrachlorobiphenyl (PCB 52); 2,2',6,6'-tetrachlorobiphenyl (PCB 54); and 3,3',4,4'-tetrachlorobiphenyl (PCB 77). The PCBs were selected to explore structure-activity relationships and the effect of the chlorination pattern on PCB-CYP spectral interactions. To examine CYP enzyme specificity, difference spectra were measured with hepatic microsomes prepared from control, phenobarbital (PB)-, 3-methylcholanthrene (MC)-, and dexamethasone (DEX)-treated rats in the absence and presence of CYP-specific antibodies. The four PCB congeners elicited a type I spectral change with all hepatic microsomal preparations. The binding efficiency of the PCBs was highest with microsomes from PB-treated rats. The largest absorbance change and highest binding efficiency were observed with PCB 54, the most non-coplanar congener tested. Antibody inhibition and CYP immunoquantitation data showed that the PCBs bind to CYP1A, CYP2B, CYP2C and CYP3A enzymes to varying degrees. For example, PCB 47, 52, and 54 bind preferentially to CYP2B and to a lesser extent to CYP3A enzymes in microsomes from PB-treated male rats; PCB 52 binds primarily to CYP3A enzymes in microsomes from DEX-treated female rats; and PCB 54 binds to CYP3A and to CYP2C enzymes in microsomes from control male rats. The study demonstrated that the extent of PCB-CYP binding interaction was dependent on the chlorination pattern of the PCB and on the relative abundance of individual CYP enzymes in hepatic microsomes.
The aryl hydrocarbon receptors (AHR) are bHLH-PAS domain containing transcription factors. In mammals, they mediate responses to environmental toxins such as 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD). Such functions of AHRs require a cofactor, the aryl hydrocarbon receptor nuclear translocator (ARNT), and the cytoplasmic chaperonins HSP90 and XAP2. AHR homologs have been identified throughout the animal kingdom. We report here that the C. elegans orthologs of AHR and ARNT, ahr-1 and aha-1, regulate GABAergic motor neuron fate specification. Four C. elegans neurons known as RMED, RMEV, RMEL and RMER express the neurotransmitter GABA and control head muscle movements. ahr-1 is expressed in RMEL and RMER neurons. Loss of function in ahr-1 causes RMEL and RMER neurons to adopt a RMED/RMEV-like fate, whereas the ectopic expression of ahr-1 in RMED and RMEV neurons can transform them into RMEL/RMER-like neurons. This function of ahr-1 requires aha-1, but not daf-21/hsp90. Our results demonstrate that C. elegans ahr-1 functions as a cell-type specific determinant. This study further supports the notion that the ancestral role of the AHR proteins is in regulating cellular differentiation in animal development.