Sulfotransferases in the bioactivation of xenobiotics

Conjugation of xenobiotics is often associated with detoxification. However, this traditional view is one-sided. In particular, numerous compounds are known that are metabolized to chemically reactive metabolites via sulfation (O-sulfonation). This can be rationalized by the fact that the sulfate group is electron-withdrawing and may be cleaved off heterolytically in appropriate molecules, thus leading to the formation of a strongly electrophilic cation. The heterologous expression of sulfotransferases in indicator cells of standard mutagenicity tests has substantially improved the accessibility of this activation pathway. The use of this technology is important, since many reactive sulfate conjugates only show strong toxicological effects if they are generated directly within the indicator cell, due to their insufficient penetration of cell membranes. Xenobiotic-metabolizing sulfotransferases are cytosolic enzymes, which form a superfamily (SULT). Eleven distinct human SULT forms are known, which strongly differ in their tissue distribution and their substrate specificity. Common functionally relevant genetic polymorphisms of the transcribed region are known for two of the forms, SULT1A1 and 1A2. Studies using recombinant test systems demonstrate that many promutagens are activated with high selectivity by an individual SULT form. Pronounced differences in promutagen activation were detected between the different human forms, including their allelic variants, and also between orthologous SULTs from different species. Therefore, SULTs may be involved in the individual genetic disposition, species differences, and organotropisms for toxicological effects of chemicals. Activation by SULTs differs from other activation pathway in its cyclic nature: reaction of a sulfuric acid ester with water usually regenerates the hydroxylated compound, which becomes available for a new cycle of activation. SULT-mediated reactivation may even occur if another initial reactive species, e.g. an epoxide, has reacted with water.     

Chemical models for cytochrome P450 as a biomimetic metabolic activation system in mutation assays

DNA damage is a critical factor in carcinogenesis. The Ames assay is a short-term test that screens for DNA-damaging agents. To be detected in the assay, most carcinogens require oxidation by cytochrome P450, a component of the liver homogenate preparation (S9 mix) that is traditionally used to metabolize promutagens to an active form in vitro. A combination of iron(III) porphyrin plus an oxidant activates many promutagens by mimicking cytochrome P450 metabolism. We previously reported that the mutagenicity of the N-nitrosodialkylamines was detected following reaction with tetrakis(pentafluorophenyl)porphyrinatoiron(III) chloride (Fe(F(5)P)Cl) plus tert-butyl hydroperoxide (t-BuOOH), which yielded the same alcohols and aldehydes as the enzymatic reaction. In the present study, to extend the scope of biomimetic models, we tested the mutagenicity of other carcinogens exposed to chemical oxidation systems.We investigated the optimal assay conditions for the models in Salmonella typhimurium TA1538, a strain sensitive to frame-shift mutagens. We activated 2-aminofluorene (AF), benzo[a]pyrene (B[a]P), a tryptophane pyrolysate 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), and 2-acetylaminofluorene (AAF) with Fe(F(5)P)Cl plus an oxidant-t-BuOOH, m-chloroperoxybenzoic acid (mCPBA), or magnesium monoperoxyphthalate (MPPT)-and we noted the effect of three solvents-acetonitrile (CH(3)CN),1,4-dioxane, and N,N-dimethylformamide (DMF)-on AF activation.All the promutagens became mutagenic in the presence of Fe(F(5)P)Cl plus an oxidant, with the effectiveness of the oxidant varying with the chemical. Aromatic amines, for example, showed the strongest mutagenicity with t-BuOOH whereas polycyclic hydrocarbons showed the strongest mutagenicity with mCPBA. All the promutagens were mutagenic in the presence of Fe(F(5)P)Cl plus MPPT. For AF activation, the order of effectiveness of the solvents was CH(3)CN>1,4-dioxane>DMF. The results suggested that these systems would serve as useful models for microsomal activating systems.     

Bacterial systems for carcinogenicity testing

During the past 30 years, bacterial test systems have been extensively refined in their ability to detect not only mutagenic agents but, in many cases, carcinogenic ones as well. Since many carcinogens are known to be activated within the mammalian body, major improvements in bacterial test systems were made when representative parts of mammalian metabolism were included as part of the test protocol. Presently, systems of great simplicity and convenience are available for the efficient detection of gene mutations, lysogenic induction of prophages, and differential DNA repair. These qualities render bacterial systems potentially useful in distinguishing between carcinogens and non-carcinogens, in characterizing induced mutation spectra, and possibly in quantifying mutagenic potency that may be used to predict tumor-initiating potency. Sensitive strains of Salmonella typhimurium. Escherichia coli and Bacillus subtilis with altered DNA-repair capacities have been constructed which accurately identify many carcinogens. Comparative studies have shown that techniques using these strains can be standardized to some extent and that the majority of carcinogens are active in all adequately sensitive genetic systems. Because of this redundancy, it may be sufficient to employ only one standardized set of tester strains and methodology. However, serveral classes of known carcinogens are undetected or underestimated when assayed in standard testing procedures. Some of these chemicals can be efficiently recognized as mutagens upon varying the methodology, the genetic endpoint, or the mammalian activation system. Thus, to modify and adjust the experimental protocol to the particular type of chemical under study and to calibrate the system with appropriate carcinogenic and non-carcinogenic reference compounds is advisable. It is noteworthy that chemical carcinogens which probably act by non-genotoxic mechanisms thus far remain undetected in bacterial tests. Newly developed systems which measure specific types of genetic events, such as transpositions of DNA segments and derepression of genes, presently are being tested for their ability to detect such carcinogens. A final matter of growing concern is the increasing number of environmental chemicals that are found to be mutagenic in bacteria but for which information about carcinogenic activity in vivo is insufficient. The possible use of bacteria for quantifying mutagenic potency and extrapolating this information to tumor-initiating potency can be envisaged in three ways: (i) direct extrapolation from standard in vitro tests, (ii) indirect extrapolation making use of an in vitro/in vivo comparison of induced effects (the parallelogram method) as devised by Sobels [138] on the basis of identical dose (to DNA), and (iii) host-mediated assays to assess mutagenic potency of carcinogens in selected organs of mammals...     

Flavonoids-potent and versatile biologically active compounds interacting with cytochromes P450.

Flavonoids represent a group of phytochemicals exhibiting a wide range of biological activities arising mainly from their antioxidant properties and ability to modulate several enzymes or cell receptors. Flavonoids have been recognized to exert anti-bacterial and anti-viral activity, anti-inflammatory, anti-angionic, analgesic, anti-allergic effects, hepatoprotective, cytostatic, apoptotic, estrogenic and anti-estrogenic properties. However, not all flavonoids and their actions are necessarily beneficial. Some flavonoids have mutagenic and/or prooxidant effects and can also interfere with essential biochemical pathways. Among the proteins that interact with flavonoids, cytochromes P450 (CYPs), monooxygenases metabolizing xenobiotics (e.g. drugs, carcinogens) and endogenous substrates (e.g. steroids), play a prominent role. Flavonoid compounds influence these enzymes in several ways: flavonoids induce the expression of several CYPs and modulate (inhibit or stimulate) their metabolic activity. In addition, some CYPs participate in metabolism of flavonoids. Flavonoids enhance activation of carcinogens and/or influence the metabolism of drugs via induction of specific CYPs. On the other hand, inhibition of CYPs involved in carcinogen activation and scavenging reactive species formed from carcinogens by CYP-mediated reactions can be beneficial properties of various flavonoids. Flavonoids show an estrogenic or anti-estrogenic activity owing to the structural similarity with the estrogen skeleton. Mimicking natural estrogens, they bind to estrogen receptor and modulate its activity. They also block CYP19, a crucial enzyme involved in estrogen biosynthesis. Flavonoids in human diet may reduce the risk of various cancers, especially hormone-dependent breast and prostate cancers, as well preventing menopausal symptoms. For these reasons the structure-function relationship of flavonoids is extensively studied to provide an inspiration for a rational drug and/or chemopreventive agent design of future pharmaceuticals.     

Inhibition of the mutagenic activity of some heterocyclic dietary carcinogens and other mutagenic/carcinogenic compounds by rat organ preparations

The mutagenic activity of some dietary mutagens, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1) and 2-amino-dipyrido[1,2-a:3',2'-d]imidazole (Glu-P-2), was inhibited in the Salmonella-plate test preincubated with heat-inactivated rat intestinal preparations. A similar inhibition was observed by preincubating intestinal preparations with 2-acetylaminofluorene (AAF) and benzo[a]pyrene (B[a]P). The effect was not specific for small intestine and was also obtained with spleen, liver, lung, colon and stomach preparations. Mutagenic activity was not inhibited by beef muscle proteins. Lipids extracted from intestinal mucosa preparations were equally effective as inhibitors of the mutagenic activity. Lipid fractions from intestinal mucosa were capable of inhibiting the formation of activated IQ by mammalian S9, and other components of the intestinal preparations were able to bind the promutagens and their active metabolites. The mutagenic activity of 1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole (metronidazole) and of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was also inhibited by intestinal preparations, but not by their lipid fractions. A binding of IQ to intestinal preparations was also demonstrated with HPLC techniques. The data indicate that tissue components may reduce the mutagenic activity of chemicals by interfering with the activation process and by reducing the concentration of the promutagens and their active metabolites at target sites.     

Purified prostaglandin synthase activates aromatic amines to derivatives that are mutagenic to Salmonella typhimurium.

Prostaglandin H synthase (PHS) is widely distributed in mammalian tissues and has the ability to oxidize a variety of mutagens and carcinogens. It may therefore play a key role in the metabolic activation of xenobiotics. The present study documents that highly purified PHS can be used in conjunction with 5-phenyl-4-pentenyl-1-hydroperoxide (PPHP), a relatively stable and non-mutagenic hydroperoxide substrate, for the metabolic activation of aromatic amines to mutagenic derivatives that can be detected in short-term Salmonella typhimurium mutagenesis assays. The PHS-based activation system alone was not mutagenic for these tester strains, nor were the test compounds significantly toxic for the bacteria over the concentration range tested. When used in conjunction with Salmonella strains TA98 and TA100 in a modified Ames assay, this system should prove useful for screening of a wide range of compounds for metabolic activation by this mammalian peroxidase. The potential broad utility of this purified PHS-dependent metabolic activation system was investigated by evaluating the activation of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), which are representative of a group of mutagenic and carcinogenic heterocyclic arylamines to which humans are exposed via their diet. Both IQ and MeIQ were activated by PHS to potent mutagens and confirm the utility of the PPHP/PHS system for the activation of premutagens. Whereas the extent of activation of aromatic amines by S9-based systems is significantly greater than for the PHS activation system described herein, PHS may play a significant role in target tissues in which it is present at significantly greater levels than P450 isoenzymes. Moreover, it is likely that the substrate specificity of PHS differs sufficiently from that of P450 isoenzymes so that PHS may activate some compounds that are not efficiently activated by mixed-function oxidase based systems.     

Microbial mutagenicity of selected hydrazines

Selected hydrazines and related compounds were examined for their mutagenic activity in S. typhimurium strains TA1535 and TA1537. These in vitro assays were conducted with and without metabolic activation by Aroclor-induced rat-liver enzymes. Relatively high levels of mutagenicity were observed with phenylhydrazine X HCl, methylhydrazine, N'-acetyl-4-(hydroxymethyl)phenylhydrazine, and 4-(hydroxymethyl)benzenediazonium tetrafluoroborate, the stabilized salt of a carcinogenic metabolite of agaritine; only low levels of mutagenicity were observed with other compounds, although most are strong carcinogens. Several of the compounds were highly toxic to the bacteria, and detection of mutagenicity was enhanced by calculating the increase in mutagenic activity on the basis of the surviving fractions of bacteria.     

Drosophila wing-spot test: improved detectability of genotoxicity of polycyclic aromatic hydrocarbons

The somatic mutation and recombination tests (SMART) using eyes or wings in the fruit fly Drosophila melanogaster are flexible and sensitive systems for the detection of genotoxicity of individual chemical compounds and complex mixtures. It is of special interest that adults and larvae possess cytochrome P-450-dependent activation systems able to metabolize most promutagens, e.g., nitrosamines, aflatoxins, pyrrolizidine alkaloids, safrole, etc. The polycyclic aromatic hydrocarbons (PAHs) represent a class of promutagens poorly detectable in Drosophila genotoxicity systems. Therefore, new tester strains for the wing-spot test were constructed by introducing chromosomes 1 and 2 from a wild-type strain with increased cytochrome P-450-dependent metabolism linked to a gene on chromosome 2. Previous investigations with the new strains showed their increased detection capability for diethylnitrosamine. Comparative tests with the 3 PAHs benzo[a]pyrene, benz[a]anthracene and 7,12-dimethylbenz[a]anthracene demonstrate, in a reproducible way, that with the new strains all 3 can be detected as active genotoxic compounds. The dose-response curves for all compounds show a plateau with higher exposures. This is interpreted as indicative of a saturation of the cytochrome P-450-dependent activation systems.     

Metabolic activation of promutagens in the yeast-microsome test. II. Activation of cyclophosphamide and 2-aminofluorene

The effect of recombinogenic and mutagenic activation of promutagens, cyclophosphamide and 2-aminofluorene was studied in S9 mouse liver preparations. Cyclophosphamide was activated to induce reversions of an ochre mutation and heteroallelic reversion in yeast tester strains r2089-15V-P4 and P3288, respectively. Metabolic activation of this chemical was greatly enhanced by pretreatment of mice with AROGLOR--1254. 2-aminofluorene was a potent recombinogen after metabolic activation, but proved a poor inducer of reversions of the ochre mutation. For the stationary yeast cells, activated 2-aminofluorene was shown to be not recombinogenic, while for logarithmic cells grown in galactose medium it was moderately recombinogenic, being highly active in this respect for logarithmic cells grown in glucose medium. We recommend to use these compounds as positive controls for exogenous activation in the yeast/microsome test.     

Carcinogen activation by human liver enzymes in the Ames mutagenicity test.

Liver post-mitochondrial supernatants derived from 10 individuals were used as the source of metabolic activation for carcinogens in the Ames quantitative mutagenicity test using Salmonella typhimurium TA 100. The liver samples were obtained from brain-dead donors and autopsy cases. The ability of human enzymes to activate aromatic amines ranged from the undetectable to highly active for 2-acetylaminofluorene. None of the samples exhibited any ability to activate benzidine. A generally low activity was observed in the capability of human enzymes to activate the polynuclear aromatic hydrocarbons, 3-methylcholanthrene and benzo(a)pyrene. Most samples were positive for activating 4-nitrobiphenyl. However, the highest mutagenic activity in the presence of human enzymes was consistently observed for aflatoxin B1 and sterigmatocystin. These results indicated that (a) human enzyme systems, like rodent systems, are more effective in inducing mutagenic activity from mycotoxins than aromatic amines and polynuclear aromatic hydrocarbons, and (b) samples derived from different individuals exhibited considerable variation in the ability to activate carcinogens belonging to a same class of compound.     

Aliphatic halogenated hydrocarbons produce volatile Salmonella mutagens

Production of volatile mutagenic metabolites from 5 halogenated promutagens was examined by a simple modification of the conventional Salmonella/microsome mutagenicity assay. This method incorporates the taping together of 2 agar plates face to face during the initial portion of their incubation at 37 degrees C. By varying the contents of the soft agar in each of the two plates with respect to promutagen, S9 and tester strain cells, mutagenesis due to volatile promutagens and their metabolites could be quantitated separately. Using the taped plate assay, volatile mutagenic metabolites were detected from the promutagens 3-(2-chloroethoxy)-1,2-dichloropropene, the herbicides diallate, triallate and sulfallate, and the flame-retardant tris-(2,3-dibromopropyl) phosphate (Tris-BP). All compounds except Tris-BP were also found to be volatile promutagens. The mutagenic metabolites accounted for 50-80% of the activity of these compounds observed in the standard assay. Morever, our studies suggest that a small, but appreciable percentage of the mutagenic metabolites from all 5 compounds escaped detection in the conventional, untaped assay. Mutagenic activity of the volatile mutagenic metabolites from diallate was quenched by various Salmonella tester strains independent of their responsiveness to diallate mutagenesis. Detection of volatile mutagen formation from diallate was also prevented by cysteine and glutathione, but not by DNA or metyrapone. This taped plate method for the Salmonella assay should facilitate future investigations of the detection, isolation and identification of volatile mutagenic metabolites from other promutagenic compounds or mixtures.     

The evaluation of sixteen carcinogens in the rat using the micronucleus test.

Sixteen carcinogens were evaluated in rats for their ability to induce micronuclei. The direct acting agent, ethyl methanesulfonate and the procarcinogens/promutagens, cyclophosphamide and 4-nitroquinoline-1-oxide, induced dose-related increases in micronucleated polychromatophilic erythrocytes. Aflatoxin B1 also significantly increased the number of micronucleated polychromatophillic erythrocytes for 2 doses although no dose-response could be detected. Dimethylnitrosamine produced variable results. The remaining 11 compounds, 2-acetylaminofluorene, 4-aminobiphenyl, benzidine, diethylnitrosamine, dimethylbenzanthracene, 1,2-dimethylhydrazine, ethionine, ethyl carbamate, hexametapol, metronidazole, and beta-naphthylamine, failed to induce significantly increased numbers of micronuclei. The large number of false negative results obtained in the present investigations using the micronucleus test suggests that in vivo cytogenetic assays utilizing bone marrow may also lack the sensitivity needed to detect clastogenic effects of procarcinogens/promutagens which require tissue specific metabolic activation.     

Differential responses of N-nitrosoamines and aromatic amines in the plant cell/microbe coincubation assay

The plant cell/microbe coincubation assay is based on employing living tobacco cells in suspension culture as the activating system for promutagens and the Ames/Salmonella cells as the genetic indicator system. In contrast to aromatic amines(e.g. 2-aminofluorene andm-phenylenediamine) that were previously reported to be activated to products mutagenic in theS. typhimurium strains TA98 or YG1024 by tobacco cells, promutagenic N-nitrosoamines (N-nitrosodimethylamine, N-nitroso-morpholine, N-nitrosopiperidine, N-nitrosomethyl-2-hydroxypropylamine) were not activated to product(s) mutagenic inS. typhimurium TA 100.     

Mutagenicity of aliphatic nitrosamines in Salmonella typhimurium

25 aliphatic nitrosamines were examined in the Ames assay for bacterial mutagens, using rat liver “S-9” for activation. Of them, 8 carcinogens were mutagenic and 5 non-carcinogens were not mutagenic. However, 2 compounds not carcinogenic in rats were mutagenic and 9 carcinogens were not mutagenic, including 6 that are liver carcinogens in rats.     

Mutagenicity of aliphatic nitrosamines in Salmonella typhimurium.

25 aliphatic nitrosamines were examined in the Ames assay for bacterial mutagens, using rat liver "S-9" for activation. Of them, 8 carcinogens were mutagenic and 5 non-carcinogens were not mutagenic. However, 2 compounds not carcinogenic in rats were mutagenic and 9 carcinogens were not mutagenic, including 6 that are liver carcinogens in rats.     

Comparison of metabolic systems required to activate pro-mutagens/carcinogens in vitro for sister-chromatid exchange studies

Irradiated Syrian hamster fetal cells (feeder layer) and rat-liver homogenate (S9 mix) were used to compare their capacity to metabolize 3 known promutagens/carcinogens; BaP, 3-MC and DMBA. DNA-damaging potential was determined by the induction of SCE in V79 target cells. The S9 mix (1/20th strength) was toxic to the target cells and reduced the mitotic index by half with an exposure time of 2.5 h. The feeder layer was not toxic to the target cells and, therefore, was included for the duration of the Expt. The test chemicals elicited a dose-response with both activating systems. At similar concentrations of the test chemicals, the cells grown on the feeder layer showed a greater number of SCEs as compared to those activated by the S9 mix.     

Mutagenic activity of promutagens in plants: indirect evidence of their activation

This review summarizes data concerning mutagenic activity of promutagens in various plant in vivo assays. These data are compared with the present knowledge about the metabolism of xenobiotics and activation of promutagens in plants obtained by biochemical studies and by the separation of the activation process from the genetic endpoints assayed for the mutagenicity. The article documents a differential response of plant species in the endogenous transforming of various classes of promutagens into mutagens. Attention is devoted to the following types of promutagens: nitrosamines, polycyclic aromatic hydrocarbons and aromatic amines, aflatoxins, pyrrolizidine alkaloids, diallate, styrene, vinylchloride, ethanol, cycasin, nitrofurans, sodium azide, s-triazine herbicides, 1,2-dibromoethane and maleic hydrazide.     

Establishment of ten strains of genetically engineered Salmonella typhimurium TA1538 each co-expressing a form of human cytochrome P450 with NADPH-cytochrome P450 reductase sensitive to various promutagens

We newly developed 10 Salmonela typhimurium TA1538 strains each co-expressing a form of human cytochrome P450s (P450 or CYP) together with NADPH-cytochrome P450 reductase (CPR) for highly sensitive detection of mutagenic activation of mycotoxins, polycyclic aromatic hydrocarbons, heterocyclic amines, and aromatic amines at low substrate concentrations. Each form of P450 (CYP1A1, CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4 or CYP3A5) expressed in the TA1538 cells efficiently catalyzed the oxidation of a representative substrate. Aflatoxin B1 was mutagenically activated effectively by CYP1A1, CYP1A2, and CYP3A4 and weakly by CYP2A6 and CYP2C8 expressed in S. typhimurium TA1538. CYP1A1 and CYP1A2 were responsible for the mutagenic activation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-acetylaminofluorene. Benzo[a]pyrene was also activated efficiently by CYP1A1 and weakly by CYP1A2, CYP2C9, CYP2C19, and CYP3A4 expressed in TA1538. These results suggest that the newly developed S. typhimurium TA1538 strains are applicable for detecting the activation of promutagens of which mutagenic activation is not or weakly detectable with N-nitrosamine-sensitive YG7108 strains expressing human P450s.     

Understanding the role of xenobiotic-metabolism in chemical carcinogenesis using gene knockout mice

Most chemical carcinogens require metabolic activation to electrophilic metabolites that are capable of binding to DNA and causing gene mutations. Carcinogen metabolism is carried out by large groups of xenobiotic-metabolizing enzymes that include the phase I cytochromes P450 (P450) and microsomal epoxide hydrolase, and various phase II transferase enzymes. It is extremely important to determine the role P450s play in the carcinogenesis and to establish if they are the rate limiting and critical interface between the chemical and its biological activities. The latter is essential in order to validate the use of rodent models to test safety of chemicals in humans. Since there are marked species differences in expressions and catalytic activities of the multiple P450 forms that activate carcinogens, this validation process becomes especially difficult. To address the role of P450s in whole animal carcinogenesis, mice were produced that lack the P450s known to catalyze carcinogen activation. Mouse lines having disrupted genes encoding the P450s CYP1A2, CYP2E1, and CYP1B1 were developed. Mice lacking expression of microsomal epoxide hydrolase (mEH) and NADPH-quinone oxidoreductase (NQO1) were also made. All of these mice exhibit no gross abnormal phenotypes, suggesting that the xenobiotic-metabolizing enzymes have no critical roles in mammalian development and physiological homeostasis. This explains the occurrence of polymorphisms in xenobiotic-metabolizing enzymes among humans and other mammalian species. However, these null mice do show differences in sensitivities to acute chemical toxicities, thus establishing the importance of xenobiotic metabolism in activation pathways that lead to cell death. Rodent bioassays using null mice and known genotoxic carcinogens should establish whether these enzymes are required for carcinogenesis in an intact animal model. These studies will also provide a framework for the production of transgenic mice and carcinogen bioassay protocols that may be more predictive for identifying the human carcinogens and validate the molecular epidemiological studies ongoing in humans that seek to establish a role for polymorphisms in cancer risk.     

Metabolism of dietary genotoxins by the human colonic microflora; the fecapentaenes and heterocyclic amines

The microflora of the human colon is a complex ecosystem of anaerobic bacteria which have the capability of enzymatically transforming a variety of dietary (or biliary) compounds to genotoxic metabolites. In the past, most investigators studying the interplay between diet and colonic flora and its role in the etiology of cancers focused on the reductive and glycosidic potential of the bacterial enzymes--many of which reverse the oxidative and conjugative reactions performed by the liver. Recent work in our laboratory has focused on the metabolism of two relatively new classes of genotoxins, the fecapentaenes and the heterocyclic amines (pyrolysis carcinogens). The fecapentaenes (conjugated ether lipids) are produced in the colon by Bacteroides spp. from polyunsaturated ether phospholipids (plasmalogens) whose natural origin and function are unknown. The fecapentaenes are potent direct-acting genotoxins that are detected in the feces of most individuals on normal western diets. The heterocyclic amines, which originate from fried or broiled proteinaceous foods, normally require activation by the liver before being potent mutagens or carcinogens. However, the "IQ" subclass (e.g. IQ and MeIQ) can be activated in the colon by Eubacterium and Clostridium species to a 7-hydroxy form which is directly mutagenic in Salmonella. Although there is no direct evidence that the fecapentaenes or the 7-hydroxy "IQ" compounds influence risk for colon cancer, the potency and prevalence of these bacterial metabolites is cause for concern.