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Methyleugenol, present in herbs and spices, has demonstrated carcinogenic activity in the liver and, to a lesser extent, in extrahepatic tissues of rats and mice. It forms DNA adducts after hydroxylation and sulphation. As previously reported, hepatic DNA adduct formation by methyleugenol in mice is strongly affected by their sulphotransferase (SULT) 1A status. Now, we analysed the adduct formation in extrahepatic tissues. The time course of the adduct levels was determined in transgenic (tg) mice, expressing human SULT1A1/2, after oral administration of methyleugenol (50 mg per kg body mass). Nearly maximal adduct levels were observed 6 h after treatment. They followed the order: liver > caecum > kidney > colon > stomach > small intestine > lung > spleen. We then selected liver, caecum, kidney and stomach for the main study, in which four mouse lines [wild-type (wt), Sult1a1-knockout (ko), tg, and humanized (ko-tg)] were treated with methyleugenol at varying dose levels. In the liver, caecum and kidney, adduct formation was nearly completely dependent on the expression of SULT1A enzymes. In the liver, human SULT1A1/2 led to higher adduct levels than mouse Sult1a1, and the effects of both enzymes were approximately additive. In the caecum, human SULT1A1/2 and mouse Sult1a1 were nearly equally effective, again with additive effects in tg mice. In the kidney, only human SULT1A1/2 played a role: no adducts were detected in wt and ko mice even at the highest dose tested and the adduct levels were similar in tg and ko-tg mice. In the stomach, adduct formation was unaffected by the SULT1A status., In Conclusion: (i) the SULT1A enzymes only affected adduct formation in those tissues in which they are highly expressed (mouse Sult1a1 in the liver and caecum, but not in the kidney and stomach; human SULT1A1/2 in the liver, caecum and kidney, not in the stomach of tg mice and humans), indicating a dominating role of local bioactivation; (ii) the additivity of the effects of both enzymes in the liver and caecum implies that the enzyme level was limiting in the adduct formation; (iii) SULT1A forms dominated the activation of methyleugenol in several tissues, but non-Sult1a1 forms or SULT-independent mechanisms were involved in its adduct formation in the stomach.

Hydroxycinnamic acids are a class of phenolic antioxidants found widely in dietary plants. Their biotransformation in the human organism primarily involves Phase II conjugation reactions. In this study, activities of UDP-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs) towards major dietary hydroxycinnamic acids (caffeic, dihydrocaffeic, dihydroferulic, ferulic and isoferulic acids) were investigated. Conjugate formation was evaluated using human liver and intestinal S9 homogenates, and in vitro characterization was carried out using recombinant human UGTs and SULTs. Analysis of the kinetics of hydroxycinnamic acid conjugation in human S9 homogenates revealed that intrinsic clearance (V(max)/K(m)) is much greater for sulfation than for glucuronidation. Assessment of activity using a panel of recombinant human SULTs showed that SULT1A1 is most active in the sulfation of caffeic, dihydrocaffeic and isoferulic acids, while SULT1E1 is most active in the sulfation of ferulic and dihydroferulic acids. Only isoferulic acid was significantly glucuronidated by human liver S9 homogenates, explained by the high activity of liver-specific UGT1A9. Studies on the kinetics of active SULTs and UGTs demonstrated a markedly lower K(m) for SULTs. To further corroborate our findings, we carried out an intervention study in healthy humans to determine the hydroxycinnamic acid conjugates in urine after consumption of hydroxycinnamate-rich coffee (200 ml). Analysis showed that sulfates are the main conjugates in urine, with the exception of isoferulic acid, which is mainly glucuronidated. These data suggest that sulfates are the predominant hydroxycinnamic acid conjugates in humans, and that SULT mediated sulfation is a major factor determining the bioavailability of hydroxycinnamic acids in vivo., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Some endocrine disrupting compounds such as phthalates and phenols act non-genomically by inhibiting the sulfotransferase (SULT 1E1 and SULT 1A1) isoforms which inactivate estrogens by sulfonation. A range of environmental phenolic contaminants and dietary flavonoids was tested for inhibition of the human SULT 1A1, 1E1 and 2A1 isoforms. In particular, the plasticisers 4-n-octyl- and 4-n-nonyl-phenol inhibit SULT 1E1 with IC(50) values of 0.16 microM vs. 10nM estradiol while the 2-substituted chlorophenols show similar values. Flavonoids are also SULT inhibitors; tricin is a competitive inhibitor of SULT 1E1 with a K(i) of 1.5+/-0.8 nM. In a small pilot study to determine whether ingestion of soy flavonoids would affect SULT1A1 activity in vivo as well as in vitro, sulfonation of daidzein was reduced in a group of women 'at risk' of breast cancer, as compared with controls, although the SULT 1A1*1/SULT 1A1*2 allele ratio was not different. Endocrine disrupting effects in man may be multifactorial when components from both the diet and the environment act at the same point in steroid metabolism.

Aim of the present experiments was to study the genotoxic effects of coffee diterpenoids, namely cafestol palmitate and a mix of cafestol and kahweol (C+K) in human derived hepatoma (HepG2) cells. Furthermore, we investigated the potential protective properties of these substances towards carcinogens contained in the human diet, namely N-nitrosodimethylamine (NDMA) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). C+K and cafestol palmitate were tested over a broad dose range in micronucleus (MN) assays and no indication for genotoxic effects was seen. In combination experiments with PhIP (300 microM), pronounced inhibition (approximately 1.7-fold) of MN formation was observed with C+K and cafestol palmitate at dose levels > or = 0.9 and 1.7 microg/ml, respectively. Enzyme measurements indicate that the protection is due to inhibition of sulfotransferase, an enzyme involved in the activation of the amine, and/or to induction of UDP-glucuronosyltransferase which detoxifies the DNA-reactive metabolites of PhIP. Furthermore, a significant increase of glutathione-S-transferase was seen, whereas the activities of cytochrome P-450 1A1 and N-acetyltransferase 1 were not significantly altered. Also in combination experiments with C+K and NDMA, strong protective effects (50% reduction of genotoxicity) were seen at low dose levels (> or = 0.3 microg/ml). Since inhibition of MN was also observed when C+K were added after incubation with NDMA, it is likely that the chemoprotective effects are due to induction of DNA repair enzymes. Comparison of data on the effects of C+K on the cholesterol metabolism, which was investigated in earlier in vivo studies, with the present findings suggests that DNA-protective effects take place at exposure levels which are substantially lower than those which cause hypercholesterolemia.

A variety of carcinogenic heterocylic amines (HAs) are found in cooked food. They can be metabolised to reactive intermediates via N-hydroxylation catalysed by cytochrome P450 1A2, followed by conjugation of the resulting N-hydroxyl group by N-acetyltransferase (NAT) or sulfotransferase (SULT). In order to compare the role of O-acetylation and O-sulfonation by human enzymes in the activation of HAs, we have introduced the cDNAs for wild-type forms of human NAT1, NAT2 and SULT1A1 in the acetyltransferase-deficient Salmonella typhimurium strain TA1538/1,8-DNP. Functional expression of recombinant proteins was demonstrated using immunoblot analysis and determination of enzyme activity with characteristic substrates. The established strains were used to study the mutagenicity of the N-hydroxy derivatives of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). The results demonstrate that N-hydroxy-HAs are activated by different human enzymes. At the concentrations used in the mutagenicity assay, N-hydroxy-IQ was activated by human NAT2, but not by NAT1 or SULT1A1. In contrast, N-hydroxy-PhIP was activated specifically by human SULT1A1, but not by NAT1 or NAT2. Therefore, both O-acetylation and O-sulfonation by human enzymes have to be regarded as important determinants for HA genotoxicity in humans.

During the frying of meat and fish, genotoxic heterocyclic amines (HCAs) are formed. The dietary exposure to HCAs may be implicated in the aetiology of human cancer, but there may be other factors in our diet that prevent the genotoxic effects of these compounds. Within the project described here, we plan to identify regional and individual cooking habits that affect HCA-levels in our food. These are determined with a validated analytical method and the exposure to HCAs is estimated by dietary assessment. Biomarker analysis will be employed to estimate recent or long-term exposure to HCAs. In order to identify genetically determined risk factors in humans, cell lines are genetically engineered expressing allelic variants of acetyl- and sulfotransferases implicated in HCA metabolism. Species differences of metabolism and toxicity of HCAs are assessed and the influence of the intestinal microflora on HCA-induced toxicity is evaluated. Dietary constituents that may reduce the genotoxicity of HCAs are screened for potential protective effects in in vitro and in vivo model systems. Finally, we will aim at human intervention studies to investigate if these protective factors are relevant for man. The objectives of this project are to estimate and possibly reduce the exposure levels to HCAs in Europe, to identify populations highly susceptible to HCA toxicity, and to reduce the toxic effects of HCAs by protective factors.

Most environmental carcinogens require metabolic activation to reactive intermediates and are mutagenic in appropriate test systems. During the last decade, the cDNAs of numerous xenobiotic-metabolizing enzymes have been cloned. The individually expressed enzymes were used to study their substrate specificities and their inhibition by other compounds. Various enzymes were expressed directly in target cells of in vitro mutagenicity tests. This is illustrated in the present study for rat and human sulphotransferases (SULTs) expressed in Salmonella typhimurium TA1538. Numerous compounds were mutagenic in the new test system. Some of these promutagens were activated by several different SULT forms, whereas many other promutagens were activated with high selectivity by a specific enzyme form, but not by genetically closely related forms from the same species (e.g. allelic variants) or orthologous enzymes from other species. Similar findings have been made using recombinant test systems for specific forms of other classes of enzymes (e.g. cytochromes P450). This high selectivity in activation (and inactivation) may explain some organotropisms as well as species and inter-individual differences in the action of carcinogens. Many carcinogen-metabolizing enzymes are induced or inhibited by other xenobiotics. Such interactions can be exploited for chemo-prevention, which however may be carcinogen- and tissue-dependent.

The LC(50) of duroquinone was about 25 times lower in a Chinese hamster V79-derived cell line which was genetically engineered for the expression of human cytochrome P-450 reductase (V79-MZ-hOR) than in the parental V79-MZ cell line. Reduction of the O(2) concentration in the atmosphere from 21 to 5% decreased the cytotoxicity of duroquinone by a factor of 4. The addition of duroquinone to the homogenate of V79-MZ-hOR cells led to the formation of Superoxide radicals, as demonstrated by the formation of formazan from nitroblue tetrazolium, and inhibition of this reaction in the presence of Superoxide dismutase. Taken together, these results indicate that the cytotoxicity of duroquinone in V79-MZ-hOR cells is caused by Superoxide [and/or other reactive oxygen species (ROS) formed from Superoxide], In this system, ROS can be formed continuously in a controlled manner within the indicator cells (e.g. by varying the concentrations of O(2) and duroquinone), and the parental V79-MZ cell line can be used as a control.

We found that different ploidy effects were induced in four Chinese hamster-derived cell lines (V79-MZ, V79, CHL and CHO-K1) treated through two cell cycles with polycyclic aromatic hydrocarbons in the absence of a metabolic activation system. 5-Bromodeoxyuridine was used to investigate cell cycle delay and sister chromatid exchanges (SCE) induced by the chemicals. Benzo[a]pyrene (BP) induced aneuploidy at 2.5-10 micrograms/ml in V79-MZ cells. 7,12-Dimethylbenz[a]anthracene (DMBA) induced polyploidy at 3.125-6.25 and 6.25-1.25 micrograms/ml in V79-MZ and V79 cells respectively. Higher concentrations caused cell cycle delay and, therefore, did not affect ploidy. BP and DMBA did not induce a significant increase in SCE frequency at the above doses. 3-Methylcholanthrene tested up to its solubility limit (10 micrograms/ml) did not induce numerical aberrations in any cell line. The clastogen mitomycin C, tested up to 0.01 microgram/ml, did not produce numerical aberrations but did significantly increase SCE frequency in all cell lines. The spindle poison colchicine, tested up to 0.1 microgram/ml, induced ploidy changes in the four cell lines that showed different sensitivities. Four cell lines showed no arylhydrocarbon hydroxylase activity, and V79-MZ, but not the other cells lines, showed high glutathione S-transferase activity. Aneuploidy induction by BP and polyploidy induction by DMBA in the absence of S9 mix in vitro have not been described before, and the finding might be due to the effect on tubulin. Due to their specificity and high sensitivity, the V79-MZ and V79 cell lines might be good systems for detecting aneuploidogens.

Four regioisomeric phenanthrene (PH) quinones (Q) were investigated for their ability to induce chromosomal damage and spindle disturbances. PH 1,4-Q and PH 1,2-Q induced structural as well as numerical chromosomal aberrations, whereas the isomers PH 9,10-Q and PH 3,4-Q were virtually inactive in this respect, However, all four compounds enhanced the frequency of c-mitoses.

To investigate whether hydroxylated metabolites of benzene may be responsible for the amplification of granulocyte-macrophage progenitor cells (GM-CFC) observed in mice that inhale benzene, groups of six C57BL6 mice were injected with hydroquinone (HQ) (75 mg/kg) or HQ (50 mg/kg) plus phenol (PHE) (50 mg/kg) twice daily for 11 days. Deviations in blood leukocyte and erythrocyte levels by up to one-third were noted in the treated groups; however, the peripheral blood differential counts were unchanged. Although no changes in bone marrow cellularity were observed in mice treated with HQ, cellularity was decreased by a factor of two in the mice that had received HQ plus PHE. The number of GM-CFC per femur was doubled in both treated groups. In vitro experiments using the murine multipotent hematopoietic progenitor cells FDCP mix also showed a duplication of GM-CFC formation in the presence of HQ at concentrations between 10(-6) M and 10(-10) M. When HQ and PHE were present at equimolar concentrations, significantly increased colony formation was still observed with 10(-12) M of metabolites. The effect was independent of the concentration of GM-colony-stimulating factor used. We suggest that HQ is a major mediator of the stimulatory effect of benzene on GM-CFC in mice. In addition, the in vitro data indicate that a direct effect of GM-CFC is involved.

We present here the results obtained within the framework of an EU funded project aimed to develop and validate alternative metabolic activating systems to be used in short-term mutagenicity assays, in order to reduce the use of laboratory animals for toxicology testing. The activating systems studied were established cell lines (Hep G2, CHEL), genetically engineered V79 cell lines expressing specific rat cytochromes P450, erythrocyte-derived systems, CYP-mimetic chemical systems and plant homogenates. The metabolically competent cell lines were used as indicator cells for genotoxic effects as well as for the preparation of external activating systems using other indicator cells. The following endpoints were used: micronuclei, chromosomal aberrations and sister chromatid exchanges, mutations at the hprt locus, gene mutations in bacteria (Ames test), unscheduled DNA synthesis and DNA breaks detected in the comet assay. All metabolic systems employed activated some promutagens. With some of them, promutagens belonging to many different classes of chemicals were activated to genotoxicants, including carcinogens negative in liver S9-mediated assays. In other cases, the use of the new activating systems allowed the detection of mutagens at much lower substrate concentrations than in liver S9-mediated assays. Therefore, the alternative metabolizing systems, which do not require the use of laboratory animals, have a substantial potential in in vitro toxicology, in the basic genotoxicity testing as well as in the elucidation of activation mechanisms. However, since the data basis is much smaller for the new systems than for the activating systems produced from subcellular liver preparations, the overlapping use of both systems is recommended for the present and near future. For example, liver S9 preparations may be used with some indicator systems (e.g., bacterial mutagenicity), and metabolically competent mammalian cell lines may be used with other indicator systems (e.g., a cytogenetic endpoint) in a battery of basic tests.

The ability of various metabolites of benzene to regulate the expression of cytochrome P-450 (CYP)1A1 mRNA in human haemopoietic cells was investigated. CYP1A1 mRNA was quantified using a Northern blot technique and high stringency hybridization with a (32)P-labelled cDNA probe. Benz[a]anthracene (BA, 1 or 10 mum), used as a positive control, induced CYP1A1 mRNA in two out of three human leukaemic haemopoietic stem cell lines (positive: KG-1, U937; negative: HL-60), as well as in long-term bone marrow cultures established from healthy volunteers. In KG-1 and U937 cells, CYP1 Al mRNA induction was studied in the presence of the benzene metabolites, hydroquinone (HQ), p-benzoquinone (BQ), phenol (PHE) and catechol (CAT). HQ and BQ induced CYP1A1 mRNA when added at concentrations of 100 nm or more; CAT was active at a concentration of 1 mum, whereas PHE had almost no effect, even at the highest concentration used (1 mum). Maximum mRNA levels induced by 1 mum HQ were seen at 6 and 12 hr after addition of inducers, and induction was detectable for at least 48 hr. Little, if any, cellular toxicity was seen in clonogenic assays of KG-1 cells at concentrations of maximum induction. In conclusion, CYP1A1 mRNA induction was demonstrated in haemopoietic cells; inducers for CYP1A1 were not only a polycyclic aromatic hydrocarbon (BA), but also, unexpectedly, hydroxylated metabolites of benzene.

The objective of the present study was to investigate the cytotoxicity of Adriamycin (ADR) and mitomycin C (MMC) in tumor and non-tumor cells with respect to the role of cytochrome P450 (P450). Therefore, genetically engineered V79 Chinese hamster fibroblasts expressing only single enzymes of P450 were used. SD1 and XEM2 cells expressed rat P450IIB1 and P450IA1, respectively, whereas the V79 parental cells contained no detectable P450 levels. The cytotoxicity of ADR and MMC in the V79 cell system was compared with that in freshly isolated hepatocytes from phenobarbital (PB-hepatocytes)- and beta-naphthoflavone (beta NF-hepatocytes)-induced rats. Following 24 h of exposure to ADR equal cytotoxicity was observed in V79, SD1 and XEM2 cells. Addition of metyrapone (MP, an inhibitor of P450IIB1) and alpha-naphthoflavone (alpha NF, an inhibitor of P450IA1) had no effect on the ADR-induced cytotoxicity in SD1 and XEM2 cells, respectively. Likewise, MMC was equitoxic in V79 and SD1 cells. Co-incubation of SD1 cells with MP did not alter MMC-induced cytotoxicity. MMC, however, showed a decreased cytotoxicity in XEM2 cells when compared to the parental V79 cells. Unexpectedly, the cytotoxicity of MMC in XEM2 cells was increased by alpha NF to the same level as observed in the parental V79 cells. In contrast to V79- and V79-derived cells, in freshly isolated hepatocytes from PB or beta NF-induced rats, MMC was cytotoxic (measured as lactate dehydrogenase leakage) within 3 h of incubation. ADR, however, was only cytotoxic to the hepatocytes when intracellular glutathione was first depleted by diethylmaleate. The MMC- and ADR-induced cytotoxicity was found to be more pronounced in PB-hepatocytes than in beta NF-hepatocytes. Contrary to the findings in the V79-derived cells, MP afforded complete protection against both MMC- and ADR-induced cytotoxicity in PB-hepatocytes, whereas alpha NF only partially inhibited the cytotoxicity of MMC in beta NF-hepatocytes. In conclusion, we have demonstrated that PB-inducible P450s play a role in the cytotoxicity of both MMC and ADR in freshly isolated PB-hepatocytes but that P450IIB1 does not in genetically reconstituted SD1 cells. P450IA1, however, decreased the cytotoxicity of MMC in the XEM2 cells. The ADR-induced cytotoxicity, which was observed in XEM2 cells, was not mediated by P450IA1. The present study underscores the complexity in the comparison of ADR- and MMC-induced cytotoxicities in normal and tumor cells.

A genetically engineered V79 cell line expressing rat CYP1A2 and another cell line expressing rat CYP1A2 as well as endogenous acetyltransferase activity, as well as CYP-deficient parental V79 cell lines, were used to assess the genotoxicity of the aromatic amines and amides 2-aminoanthracene, 2-aminofluorene, 2-acetylaminofluorene, 4-acetylaminofluorene and 2-amino-3-methylimidazo[4,5-f]quinoline, with chromosomal aberrations and sister chromatid exchanges as the end-points. None of the test compounds showed a clear effect on the frequency of chromosomal aberrations in any cell line used. Sister chromatid exchanges, however, were induced by 2-aminoanthracene, 2-aminofluorene and 2-acetylaminofluorene in the CYP1A2-proficient cells, but not in the CYP1A2-deficient cells. The presence of acetyltransferase activity enhanced the effect of 2-aminoanthracene, 2-aminofluorene and 2-acetylaminofluorene. 4-Acetylaminofluorene and 2-amino-3-methylimidazo[4,5-f]quinoline did not induce sister chromatid exchanges in the investigated cell lines. The use of cell lines with defined metabolic capabilities seems to be a valuable tool to study specific metabolic pathways important in the activation of procarcinogens.

Various benzylic alcohols are metabolically activated to electrophilic, potentially mutagenic and carcinogenic sulphuric acid esters. The involved sulphotransferases are not expressed in the cell lines in culture which are commonly used for mutagenicity testing. The liver of adult female rats is very efficient in the bioactivation of 1-hydroxymethylpyrene. The major enzyme involved was purified and identified as hydroxysteroid sulphotransferase a. Its cDNA was stably expressed in Chinese hamster V79 cells, which are particularly suited for the quantitative detection of various types of mutations and other genotoxic and cytotoxic effects. The mRNA, protein and enzyme activity levels in the constructed cell lines (V79rSTa-1 and V79rSTa-2) were measured, and the cells were also used in mutagenicity and cytotoxicity investigations with benzylic alcohols. 1-Hydroxymethylpyrene, 9-hydroxymethylanthracene and 6-hydroxymethylbenzo[a]pyrene showed enhanced cytotoxicity in V79rSTa-1 and V79rSTa-2 cells, as compared with sulphotransferase-deficient control cells. In addition, 1-hydroxymethylpyrene induced sister chromatid exchanges, and 6-hydroxymethylbenzo[a]pyrene induced gene mutations in V79rSTa-1 cells. We intend carrying out more investigations with other chemicals on these cell lines. Their advantages, as compared with systems with external metabolising systems, include the formation of the active metabolites within the target cell, as in ST-proficient cells in vivo, eliminating the problems which may result from restricted intercellular transport of reactive and ionized sulphuric acid conjugates. Furthermore, cells expressing other sulphotransferases, including human enzymes, may be constructed and used for comparative investigations.

As part of the International Workshop on Standardization of Genotoxicity Test Procedures, in Melbourne, 27-28 February 1993, various international guidelines were examined with respect to protocol issues in the area of mammalian cell gene mutation assays. The working group on mammalian cell gene mutation assays discussed a wide range of protocol issues related to study design; in most cases the recommendations are reasonably consistent with existing guidelines. Agreement was reached on several issues as follows. The upper limit of concentration for testing non-toxic substances should be 10 mM or 5 mg/ml, whichever is lower. For testing toxic substances the criteria of an acceptable upper limit of concentration should yield 10-20% survival. Any of several established mammalian cell mutation assays (L5178Y TK+/-, CHO/HPRT, AS52/XPRT, V79/HPRT) can be used to evaluate mutagenesis in mammalian cells; the ouabain (Na/K-ATPase) system is not an acceptable mutation assay for routine evaluation of mutagenesis in mammalian cells. Ability to recover small colonies must be convincingly demonstrated when using the L5178Y TK+/- mouse lymphoma assay. In the mouse lymphoma assay (L5178Y TK+/-), colonies in positive controls and at least two (if available) representative positive doses of the test compound should be sized if a positive response is seen; in the event of a negative response due to the test compound, colony sizing of the positive control is necessary to validate the conduct of the assay. Testing both in the presence and absence of S9 metabolic activation is necessary. It was not possible to come to a firm conclusion about the length of treatment. There was a general agreement that extended treatment times (> 2 cell cycles) often bear more disadvantages than advantages and should only be used with adequate justification. It is not necessary to repeat clear positive or clear negative tests when the assay has been adequately performed; this recommendation differs significantly from the UK guidelines. If treatment groups are not replicated, the numbers of doses tested should be increased; this recommendation differs significantly from the UK guidelines. Each laboratory should establish a historical database for the performance of a given assay in that laboratory.

V79 Chinese hamster cell lines genetically engineered to express rat CYP2B1, CYP1A1, CYP1A2, and their parental cell lines V79-MZ, without acetyltransferase, and V79-NH, with acetyltransferase, were studied for chromosome aberrations and sister chromatid exchange induced by aflatoxin B1, cyclophosphamide, benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene and dimethylnitrosamine. The parental V79 cell lines did not show clastogenic effects. Significant clastogenic effects were observed after an 18 h exposure to aflatoxin B1 and cyclophosphamide in CYP2B1 expressing cells, to benzo[a]pyrene in CYP1A1 and CYP1A2 expressing cells, to 7,12-dimethylbenz[a]anthracene and dimethylnitrosamine in cells, expressing CYP1A2 with or without acetyltransferase, and to cyclophosphamide in cells expressing both CYP1A2 and acetyltransferase. A significant sister chromatid exchange inducing effect was found after a 24 h exposure in each of the genetically engineered cell lines, except for benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene in CYP2B1 expressing cells, and for benzo[a]pyrene in cells expressing both CYP1A2 and acetyltransferase. Thus, a battery of cell lines genetically engineered for metabolic competence may serve as a tool for investigating chromosomal changes induced by activated xenobiotics.

The binding of 1-hydroxymethylpyrene (HMP) and its active metabolites, 1-hydroxymethylpyrene sulfate (SMP) and 1-chloromethylpyrene (CMP), to DNA was studied. In the liver of rats, maximum adduct levels were observed 1.5 h after i.p. injection of HMP, followed by a relatively rapid decrease. Separate exposure of different liver cell types in vitro to HMP led to high adduct levels in parenchymal cells, intermediate levels (1/10) in endothelial cells and low levels (1/200) in Kupffer cells. The adduct patterns were similar in the different cell types. The same pattern was also obtained when isolated DNA was incubated with SMP or CMP. One of the four major spots co-chromatographed on TLC with a dG adduct, one with a dA adduct and one with both dG and dA adducts. The fourth spot did not co-chromatograph with any of the adducts observed in reactions with any nucleic acid homopolymer.

Genotoxic species and metabolites are usually under the control of a complex set of activating, inactivating and precursor sequestering enzymes. These enzymes differ greatly between test systems, animal species and man. An adequate metabolic design of genotoxicity studies requires careful attention to factors such as: Dilution of cofactors in in vitro tests which are present in much higher concentrations in the intact cell; Induction in high dose carcinogenicity bioassays of enzymes, which are constitutively not expressed and not induced at such doses of the compound, which occur in the situations of the practical use of the compound; Modifications of control enzymes, which are effected by hormones or other endogenous factors, which are differently influenced by high dose (bioassay) versus moderate dose (real exposure) or by in vivo (endocrine regulation) versus in vitro (no endocrine regulation) conditions.

11-β-hydroxy-7-deoxy-rosenonolactone (TSS1), a mycotoxin of the rosenane class, was tested on cytotoxicity, induction of DNA single strand breaks and muta-genicity. Its effects were compared to those of rosenonolactone and trichothecin. TSS1 had stronger antibiotic activity againstEscherichia coli (EC 50: 10μg/mL) than rosenonolactone (EC 50: >200μg/mL) but weaker activity than trichothecin (EC 50: 3μg/mL). The same order of activity was found for the inhibition of yeast fermentation (EC 50 of TSS1: 45μg/mL; EC 50 of rosenonolactone: > 120μg/mL; EC 50 of trichothecin: 3.4μg/mL).In the trypan blue exclusion test using V79 Chinese hamster cells, TSS1 proved to be cytotoxic (EC50: 30μg/mL) at even lower doses than trichothecin (EC50: 200μg/mL). Rosenonolactone had no significant toxicity up to the highest soluble concentration (500μg/mL).DNA single strand breaks caused by TSS1 occurred at the same concentrations at which damage of the cell membrane became apparent. For trichothecin single strand breaks were detected only at concentrations at which the membrane was already highly damaged. No single strand breaks were observed in V79 cells after incubation with rosenonolactone up to the limit of solubility (500μg/mL).In the reversion assay withhis Salmonella typhimurium strains TA 98 and TA 100, no mutagenicity was observed for any of the examined mycotoxins up to 800μg/plate with and without the addition of a rat liver preparation for metabolism of the test compound.

1. Chinese hamster V79-derived cell lines, stably expressing cytochromes P4501A1, 1A2, and 2B1 activities, were constructed by genetic engineering in continuation of our work to establish a battery of V79 derived cell lines designed to study the metabolism of xenobiotics. 2. Cell lines XEM1 and XEM2, expressing cytochrome P4501A1, were capable of the O-dealkylation of 7-ethoxycoumarin and the hydroxylation of benzo[a]pyrene. 3. Cell lines XEMd.MZ and XEMd.NH, expressing P4501A2, were shown to hydroxylate 17 beta-estradiol and 2-aminofluorene. 4. Cell line SD1, expressing cytochrome P4502B1, was able to hydroxylate testosterone stereo- and regio-specifically at the 16 alpha and 16 beta positions. 5. Cell lines were validated in mutagenicity, cytotoxicity, and metabolism studies employing benzo[a]pyrene, trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene, cyclophosphamide, ifosfamide, and picene. 6. Construction of metabolically-competent V79-derived cell lines be recombinant DNA technology will be a fundamental improvement for the evaluation of the cytotoxic, genotoxic and pharmacological properties of a chemical.

A total of 78 polycyclic aromatic compounds (PAH), including pure hydrocarbons, PAH metabolites, aromatic amines and nitroarenes, were tested in the initiator tRNA acceptance assay (tR assay) for carcinogens. Among the pure hydrocarbons, all strong carcinogens were highly active in the tR assay. Some weak carcinogens showed moderate positive responses, others as well as all possible non-carcinogens were inactive. Various PAH metabolites, including phenols, dihydrodiols, arene oxides, dihydrodiol epoxides, quinones and benzylic sulfate esters, were positive as well. Strikingly, however, their effects rarely reached the levels observed with the strong carcinogens among the pure hydrocarbons. Moreover, the correlation with carcinogenicity was less clear, partially due to limitations in the available carcinogenicity data. The activities in the tR assay were also compared with the mutagenicity in Salmonella typhimurium. No appreciable correlation was observed. For example, trans-9,10-dihydroxy-9,10-dihydrobenzo[c]chrysene, in the absence of a mammalian metabolic system, was highly active in the tR assay, but non-mutagenic. Upon addition of rat liver enzymes, the reverse result was obtained. syn-Benzo[c]chrysene-9,10-dihydrodiol-11,12-oxide, on the other hand, was a potent direct mutagen, but required the presence of liver microsomes for a positive response in the tR assay. Thus, the metabolic basis for these two activities is different, and not yet understood for the tR assay. The partial correlations in the tR assay and in the Salmonella mutagenicity assay with carcinogenicity, and the pronounced discrepancies between these in vitro tests, may suggest that they detect different mechanisms involved in carcinogenicity. However, the tR assay was less predictive for the carcinogenicity of PAHs as compared to the previously investigated N-nitroso compounds and mycotoxins.

V79 cells, genetically engineered to express active cytochromes P450IIB1 and P450IA1, were used to study the cytotoxicity and mutagenicity of cyclophosphamide and ifosfamide. Cyclophosphamide, tested up to a concentration of 2 mM, was not cytotoxic in V79 nor in the P450IA1-expressing V79-derived cell line XEM2. Pronounced cytotoxicity was, however, observed in the P450IIB1-expressing V79-derived cell line SD1. Induction of gene mutations (acquisition of 6-thioguanine resistance) was observed in SD1 cells as well, but the effects were weak. Ifosfamide was inactive in V79 cells, but was cytotoxic in SD1 cells. Ifosfamide mustard, an active metabolite of ifosfamide, was equally cytotoxic and showed similar mutagenic effects in SD1 and parental V79 cells. The results indicate that cyclophosphamide and ifosfamide are metabolically activated by cytochrome P450IIB1. In contrast, cytochrome P450IA1 was not capable of activating cyclophosphamide. Thus, V79-derived cell lines defined for their expression of a specific form of cytochrome P-450 can be used as diagnostic tools to identify the cytochrome P-450 that is responsible for the metabolic activation of drugs.

Many foreign compounds are transformed into reactive metabolites, which may produce genotoxic effects by chemically altering critical biomolecules. Reactive metabolites are under the control of activating, inactivating and precursor sequestering enzymes. Such enzymes are under the long-term control of induction and repression, as well as the short-term control of post-translational modification and low molecular weight activators or inhibitors. In addition, the efficiency of these enzyme systems in preventing reactive metabolite-mediated toxicity is directed by their subcellular compartmentalization and isoenzymic multiplicity. Extrapolation from toxicological test systems to the human requires information of these variables in the system in question and in man. Differences in susceptibility to toxic challenges between species and individuals are often causally linked to differences in these control factors.

Mammals have at least two epoxide hydrolases (EHs) with a broad significance in drug metabolism. One enzyme is localized in the endoplasmic reticulum and other membranes (EHm), and the other is in the cytosol (EHc). In the present study we found that humans differ greatly in the activities of these enzymes in liver. The specific activities in microsomes from 166 subjects (most of them patients suffering from hepatic diseases), measured with benzo[a]pyrene 4,5-oxide as the substrate, varied by a factor of 63. The activities in the cytosol, determined with trans-stilbene oxide as substrate varied 539-fold among 135 subjects. A subdivision into different diagnostic groups showed an increase in EHm activity (1.7-fold control) but not EHc activity in tuberculosis patients treated with rifampicin, ethambutol and isoniazid. No other diagnostic group showed significantly altered EH activities. Furthermore the activities did not differ between females and males, alcoholics and non-alcoholics or smokers and non-smokers. In the 77 subjects where both EHc and EHm activities were determined, no correlation between them was observed, indicating separate biological control.

Benzo[a]pyrene 4,5-oxide was covalently bound to DNA of cultured human fibroblasts and caused sister chromatid exchange. The monooxygenase inhibitor alpha-napthoflavone suppressed this induction of sister chromatic exchange, but did not affect binding to DNA. Control experiments with 4-nitroquinoline 1-oxide showed that alpha-naphthoflavone does not inhibit sister chromatid exchange in general. A more likely explanation for the discrepancy between induction of sister chromatid exchange and binding to DNA is that benzo[a]pyrene 4,5-oxide itself can bind to DNA, but this binding does not lead to a significant increase in sister chromatid exchange. However benzo[a]pyrene 4,5-oxide can be oxidized by monooxygenase to yet unknown products which are potent inducers of sister chromatid exchange. An important conclusion from this is that a biological effect such as the induction of sister chromatid exchange may correlate with the exact nature of DNA binding rather than with total binding, to the point where just measuring total binding may be completely misleading if intended to detect the causes of the biological effect.

9-Hydroxy-trans-1,2-dihydro-1,2-dihydroxychrysene (9-hydroxychrysene-1,2-diol), which may be the triol involved in the formation of a chrysene triol-epoxide-DNA adduct in mouse skin, was not detected when chrysene was incubated with rat-liver microsomal preparations. In separate experiments an excess of synthetic 9-hydroxychrysene-1,2-diol was added during the incubation of 3H-labelled chrysene with rat-liver microsomes and was then re-isolated. The triol was found to contain a radioactive product that had chromatographic properties identical to those of 9-hydroxychrysene-1,2-diol when examined by reverse-phase h.p.l.c., both before and after acetylation, by normal-phase h.p.l.c. and by t.l.c. both before and after oxidation. When treated with m-chloroperoxybenzoic acid, the synthetic 9-hydroxychrysene-1,2-diol formed products that possessed alkylating activity and that reacted with DNA in vitro. Examination of the triol-epoxides produced by oxidation of a mixture of synthetic and metabolic 9-hydroxychrysene-1,2-diol by t.l.c. suggested that the anti-isomer was formed.

Results of various studies have shown that male Swiss Webster mice are more susceptible to toxic effects of vinylidene chloride (VDC) than are females of the same mouse strain, females and males of the C57BL mouse strain, Chinese hamsters and rats. The main targets of toxicity are kidney and liver. The kidney of male Swiss Webster mice is the only organ where VDC unambiguously induces tumours. In the present study we have investigated the ability of NADPH-foritifed postmitochondrial supernatant fractions (S-9 mix) of kidney and liver from susceptible and nonsusceptible animals to activate VDC to a bacterial mutagen. The following sequence of activating potencies was observed: mouse liver (both strains and sexes) and Chinese hamster liver greater than rat liver greater than human liver greater than Chinese hamster kidney greater than kidney from male mice of both strains greater than kidney from rats and female mice. The last two preparations only occasionally showed weak activation of VDC. Addition of purified microsomal epoxide hydrolase to S-9 mix did not affect the mutagenicity of VDC; addition of glutathione reduced the mutagenicity up to 50%. Pretreatment of animals (male rats, male and female Swiss Webster mice) with VDC did not potentiate the ability of the subcellular preparations to activate this compound. In fact, in some cases, a weaker activation was observed. Following this treatment, microsomal 7-ethoxy-coumarin O-dealkylase was decreased in mouse kidney and in rat liver. The enzyme was not affected in mouse liver and was not measurable in rat kidney. Microsomal epoxide hydrolase activity (with styrene 7,8-oxide as substrate) was not affected in mouse liver and rat kidney. In the kidney of male mice treated with a high concentration of VDC, epoxide hydrolase activity was decreased initially, but after longer treatment, in some cases a weak increase above control was noticed. A stronger increase in activity of epoxide hydrolase was observed in the rat liver and the kidney of female mice. Cytosolic glutathione transferase activity (with 2,4-dinitrochlorobenzene as substrate) was not affected by the VDC treatment in the liver of male mice, but was decreased in the kidney of male mice, and was elevated in the kidney and liver of rats and of female mice. The different effects of VDC on this enzyme may be one of the reasons for the differences in susceptibility towards the toxic and carcinogenic actions of this compound in different species, strains and sexes.

Epoxide hydrolase activity was determined in hepatic microsomes of adult males of 22 rat strains. The specific activity varied between 4.3 and 12.7 nmole styrene glycol/mg protein per min. The enzyme in F344, DA and Sprague--Dawley rats, strains with low, high and intermediate activity, respectively, was studied in more detail. No differences in substrate specificity and pH-dependence of the activity were observed between the strains with high and low activity, and immunoprecipitation by antibodies raised against microsomal epoxide hydrolase purified from Sprague--Dawley rats showed that the amounts of enzyme protein in microsomes from DA and F344 rats correlated with the activities. These results indicate quantitative rather than qualitative differences in epoxide hydrolase. The enzyme activity was inherited in an autosomal and codominant manner. The hepatic activity in females (about 78% of that in males) and, with the limitation that only few situations were studied, the trans-stilbene oxide-induced activity were under the same genetic control as the basal hepatic activity in males. In contrast, some extrahepatic tissues showed strain differences in epoxide hydrolase activity which contrasted with those found in liver. Hence, the enzyme activity in one tissue cannot serve as a reliable guide to the relative activity in another tissue, unless a specific correlation between the two tissues has been established. Although the strain differences in activity were not very large in themselves, in combination with inter-individual variation, sex differences and effects of the enzyme inducer transstilbene oxide they led to a 20-fold variation in hepatic epoxide hydrolase activity among the rats investigated in the present study.

Metabolic activation of chrysene in mouse skin appears to involve r-1,t-2-dihydroxy-t-3,4-oxy-1,2,3,4-tetrahydrochrysene (anti-chrysene-1,2-diol 3,4-oxide) and 9-hydroxy-r-1,t-2-dihydroxy-t-3,4-oxy-1,2,3,4-tetrahydrochrysene (anti-9-OH-chrysene-1,2-diol 3,4-oxide). The enzyme-catalysed conjugation of these epoxides with [35S]glutathione has been studied in experiments in which the glutathione conjugates were separated by h.p.l.c. and examined by fluorescence spectrophotometry. Both anti-chrysene-1,2-diol 3,4-oxide and anti-9-OH-chrysene-1,2-diol 3,4-oxide formed conjugates nonenzymically and both were shown to be substrates for rat liver glutathione transferases. When anti-chrysene-1,2-diol 3,4-oxide was incubated with [35S]glutathione and a rat liver microsomal metabolizing system, glutathione conjugates with h.p.l.c. and fluorescence spectral characteristics identical to those of conjugates formed from both anti-chrysene-1,2-diol 3,4-oxide and anti-9-OH-chrysene-1,2-diol 3,4-oxide were detected. This finding provides evidence that anti-chrysene-1,2-diol 3,4-oxide can be further metabolized to the triol-epoxide, anti-9-OH-chrysene-1,2-diol 3,4-oxide by rat liver microsomal systems.

The epoxide metabolites of two clinically used drugs and an experimental psychotropic agent, carbamazepine 10,11-oxide, cyproheptadine 10,11-oxide and cyclobenzaprine 10,11-oxide, were fully devoid of any mutagenic activity under conditions where K-region-epoxide metabolites of some known carcinogens, such as benzo(a)pyrene, proved to be potent frameshift mutational agents for Salmonella typhimurium TA 1537 and TA 1538. All epoxides tested were non-mutagenic for TA 1535, designed to detect substitution mutations. The 10,11-epoxides of the three drugs, carbamazepine, cyproheptadine and cyclobenzaprine, were not cytotoxic to any of the bacterial tester strains used, precluding that mutagenicity might have been overshadowed by cytotoxicity. When the mutagen, precursor, benzo(a)pyrene, was incubated together with TA 1537 and a mammalian microsomal preparation in the presence of a system generating the co-factor necessary for mono-oxygenase activity, activation to mutagenic species was observed which was dramatically increased in the presence of a potent epoxide hydratase inhibitor, 1,1,1-trichloropropene 2,3-oxide, suggesting epoxide(s) as the (or one of the) mutagenically active species metabolically produced in situ. None of these effects was observed with the three medical drugs. Moreover, the observation that the alkene oxide 4-phenylstyrene 7,8-oxide is mutagenic to the two strains TA 1537 and TA 1538 but the K-region arene oxide derived from 7,12-dimethylbenz(a)anthracene is inactive for the latter strain indicates that epoxidation of an aromatic double bond of a polycyclic hydrocarbon is neither a necessary nor a satisfying condition for frameshift mutagenesis to occur.

Diethylstilbestrol was tested for mutagenicity with his- S. typhimurium strains under 10 different matabolic situations (no exogenous metabolizing system; S9 mix from liver homogenate of rats induced with Aroclor 1254, with or without inhibition of epoxide hydratase; liver and/or kidney S9 mix from control or hamsters treated with Aroclor 1254; horse-radish peroxidase + H2O2). Under none of these conditions did diethylstilbestrol give any indication of a mutagenic effect. Furthermore, 11 metabolites and other derivatives of diethylstilbestrol, 2 of them potent inducers of sister-chromatid exchange in cultured fibroblasts, were not mutagenic with any of the 4 tester strains (S. typhimurium TA100, TA98, TA1537, TA1535) in the presence or absence of S9 mix from liver homogenate of rats induced with Aroclor 1254. Thus, one of the few known human carcinogens is very resistant to detection by the mammalian enzyme-mediated Salmonella typhimurium mutagenicity test (Ames test). This is especially remarkable since the metabolizing systems used included: (1) some of very high metabolic activity (S9 mix from liver homogenate of rats and hamsters induced with Aroclor 1254); (2) metabolizing systems from organs susceptible to the carcinogenic activity of diethylstilbestrol (hamster kidney); as well as (3) a mixture of (1) and (2) in case both activities are required for the carcinogenic effect in the whole animal.

Microsomal monooxygenases catalyze the biosynthesis of epoxides from olefinic and aromatic compounds whilst microsomal epoxide hydratase and cytoplasmic glutathione S-transferases are responsible for their further biotransformation. Although catalytically very efficient the cytoplasmic glutathione S-transferases play, due to their subcellular localization, a minor role in the inactivation of epoxides derived from large lipophilic compounds and were, therefore, not included in this study. It was shown with such a lipophilic compound, benzo(a)pyrene, as a model substance and with liver enzyme mediated bacterial mutagenesis as biological endpoint that species and strain differences in epoxide hydratase and monooxygenases are reflected in very dramatic differences in mutagenicity of benzo(a)pyrene which varied from extremely potent to a degree which could easily be overlooked. In order to investigate whether the differences in enzyme activities were causally linked to the observed differences in mutagenicity, the enzyme activities were modulated by inhibition and induction. These manipulations were always accompanied by the corresponding changes in mutagenicity. It is concluded that species such as mice which possess high monooxygenase activity but very low epoxide hydratase activity are much more susceptible than man to those toxic effects which are mediated by metabolically formed epoxides which are substrates of epoxide hydratase. In this regard, it is especially noteworthy that mice possess a much lower hepatic epoxide hydratase activity than man.

N-nitrodimethylamine is metabolized oxidatively to N-nitrohydroxymethylmethylamine, which decomposes to yield formaldehyde and N-nitromethylamine. All four compounds and N-nitromethylamine were tested for their ability to induce DNA single strand breaks in hepatocytes and in SV 40-transformed Chinese hamster embryo cell lines. Only the two monoalkylnitramines were positive. They induced single strand breaks in hepatocytes, but were not effective in the other cells. Formaldehyde and N-nitrohydroxymethylmethylamine were toxic to the cells. None of the compounds tested was able to induce selective DNA amplification in the two transformed cell lines. Enzymes involved in drug metabolism were assayed in the hamster cell lines. The activity of UDP-glucuronosyltransferase and cytosolic epoxide hydrolase were not detectable. N-nitrodimethylamine demethylation was low. The content of reduced glutathione and the activities of glutathione transferase and membrane bound epoxide hydrolase were comparable to values obtained in the rat liver.

The mutagenicities of 17 closely related oxiranes were determined in 4 tester strains (Salmonella typhimurium TA98, TA100, TA1535, TA1537). The test compounds comprised all possible oxides of benzene and its partially hydrogenated congeners. In TA100 and TA1535, 12 of the tested oxiranes were weak to moderate mutagens. 4 of these were also active in TA98. No mutagenicity was observed with the remaining 5 compounds in any of the 4 strains. The presence of a double bond in formal conjugation with the epoxide ring increased the mutagenicity relative to that of the saturated oxirane. Interestingly, additional epoxide rings within the same molecule did not markedly increase the mutagenic activity, and for the oxiranes that are not activated by a double bond, the relationship between mutagenic activity and the number of epoxide rings in the molecule was even inverse. The influence of bromo and hydroxyl substitution on oxirane mutagenicity is discussed. Most notably, a compound having a 4-hydroxyl group in syn position to a 1,2-epoxide ring fused to the cyclohexane ring, a structure which has been suggested to increase the electrophilic reactivity of dihydrodiol epoxides through hydrogen bonding, was almost inactive.

Activities of drug metabolizing enzymes were determined in subcellular fractions of lung biopsies from 12 human subjects and in liver biopsies from 15 other human subjects. Monooxygenase (MO) activity with 7-ethoxycoumarin as a substrate and epoxide hydrolase (EH) activity with benzo[a]pyrene 4,5-oxide as a substrate were measured in the microsomal fraction, glutathione S-transferase (GST) activity toward 2,4-dinitrochlorobenzene in the cytosolic fraction. MO activity was further characterized by the use of inhibitors known to act preferentially on different MO forms. To facilitate extrapolations from test results obtained in animals to man enzyme activities were also determined in corresponding fractions from commonly used laboratory animals, Sprague-Dawley rat, NMRI mouse, and Syrian golden hamster. All investigated specific activities were lower in lung than in liver preparations by factors ranging from 2.4 to 7 in the mouse, 4 to 18 in rat and hamster, and 11 to 697 in man. The high ratio between liver and lung activity in man occurred with MO and is due to an extremely low activity in human lung. It is not clear whether this low activity is predominantly due to low amounts of enzyme or to inhibitors known to be present in human lung preparations. With this exception of human lung MO, species differences in the investigated enzyme activities were moderate. Among the three rodent species, rat was most similar to man, with none of the investigated activities differing by a factor more than 2. The mouse differed from these two species by considerably higher MO and GST activities in both organs, and by a relatively low EH activity in liver for the investigated substrates, while the hamster displayed comparatively high lung GST and EH and liver GST and MO activities. MO inhibition patterns by different in vitro inhibitors were similar in the same organs of different species, but differed in lung and liver. Standard concentrations of the diagnostic inhibitors led to preferential inhibition of lung MO by metyrapone and considerably less by tetrahydrofuran, while for liver MO the reverse was true. In both organs, the standard concentration of alpha-naphthoflavone had only very weak effects. In conclusion, man and commonly used laboratory rodents are not grossly different with respect to the investigated enzyme activities with the possible exception of lung MO. For the substrates investigated, the rat represented clearly the best model for man among the studied animal species.

Mutation and malignant transformation were followed in the same cells. Mouse fibroblasts (C3H 10T 1/2) were mutated and transformed by 4-nitroquinoline-1-oxide with similar, approximately linear dose-responses. The presence of caffeine immediately after exposure to 4-nitroquinoline-1-oxide potently inhibited mutation and transformation at high but not at low doses of 4-nitroquinoline-1-oxide. Whilst the coordinate induction of mutation and transformation could be explained by both a common target (DNA) or a common reactive species hitting several targets, the identical modulation by a DNA repair inhibitor of both end points suggests fundamental similarities in the nature of the lesions leading to mutation and transformation and in the processing of these lesions, implying DNA as target and mutation as one (but not necessarily the sole) required step in transformation.

Epithelial liver cells of the Chinese hamster (CHEL cells) were propagated in culture for 35 passages. At favourable cell densities, the population doubling time in normal medium, was 20 h. L-Tyrosine amino transferase activity was retained at a measurable level, but its enhancement by dexamethasone was detected solely in cells of early passages. Pyruvate kinase was strongly activated by fructose-1,6-biphosphate at low substrate concentrations. These enzymatic properties suggest that the CHEL cells are derived from a sub-population of parenchymal hepatocytes or from cells closely related to parenchymal hepatocytes. With a lag period of a few hours, CHEL cultures metabolized benzo[a]pyrene. In cell homogenates the various monooxygenase activities investigated were below the detection limits. However, other xenobiotic-metabolizing activities, such as cytochrome P-450 reductase, glutathione transferase and UDP-glucuronosyl-transferase were high, with levels comparable to those observed in freshly isolated rat parenchymal cells. Epoxide hydrolase activity was also detected, but was lower than in the liver. The CHEL cells were able to activate benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene and aflatoxin B1 to mutagens, as shown in a co-culture assay with V79 cells, in which acquisition of resistance to 6-thioguanine was studied. At early passages, the CHEL cells had a near diploid set of chromosomes. Then, gradually the frequency of cells with slight changes in the number of chromosomes and the frequency of tetraploids were increased. During the observation period (up to passage 20) the modal number of chromosomes shifted from 22 to 23. No gross morphological changes in the cultures were noticed during the 20 passages.(ABSTRACT TRUNCATED AT 250 WORDS)

43 heteropolycyclic compounds belonging to a homologous series were investigated for mutagenicity. The results are compared with carcinogenicity data obtained with the same batches of compounds under conditions identical for all of them. Mutagenicity was tested in the Ames test with Salmonella typhimurium strains TA1535, TA1537 and TA100 in the presence and absence of liver 10 000 g supernatant from rats treated with Aroclor 1254. Carcinogenicity was tested by injection of the compounds into subcutaneous tissue of XVIInc/Z mice. 18 test compounds showed carcinogenic activity, some strongly, others only weakly. Of these, 17 were detected as mutagens: one weak carcinogen did not revert the Salmonella strains. No quantitative correlation was observed between the extents of the mutagenic and the carcinogenic effects. Of the 25 substances that did not produce tumours, 13 showed mutagenicity (12 in the presence, 2 in the absence, of the liver homogenate). The mutagenic effects of these compounds were quantitatively similar to those of the compounds that produced tumours. The most sensitive strain of Salmonella typhimurium was TA100. It detected all 30 mutagens. TA98 was mutated by 25 compounds, TA1537 by 16 compounds. No mutagenic effects were seen with TA1535. Possible reasons for the high percentage of apparently "false positives" in the Ames test and the lack of a quantitative correlation between the potency of the mutagenic and carcinogenic effects are discussed. It is suggested that the complexity of the metabolism of these heterocyclic compounds may lead to critical differences in metabolism in mouse subcutaneous tissue in vivo and in liver homogenates from rats treated with Aroclor. Therefore the present study will be extended to life-long oral and intrahepatic carcinogenicity tests leading to a higher proportion of metabolism in the liver.

Benzo(a)pyrene and benz(a) anthrancene which, in contrast to the K-region epoxides benzo(a)pyrene 4,5-oxide and benz(a)anthracene 5,6-oxide, are not mutagenic to Salmonella typhimurium TA 1537 in the absence of mammalian enzyme preparations, were activated by liver microsomes from C3H mice, which had not received any pretreatment, to mutagens reverting this tester strain to histidine prototrophy. Addition of epoxide hydratase inhibitors greatly increased this mutagenicity and addition of pure epoxide hydratase reduced it by more than 95% down to the range of spontaneous mutations as observed in absence of any added mutagen. This demonstrates than the metabolic pathway responsible for the mutagenicity of both polycyclic hydrocarbons observed in this system proceeds entirely via an epoxidation pathway and that the responsible metabolites are epoxides or species arising from them. Moreover, further metabolism by epoxide hydratase does not lead to produce contributing to the mutagenicity observed with the tester strain used. Finally, the epoxides relevant for the observed mutagenicity are substrates for epoxide hydratase; indeed, modest amounts of the pure enzyme can prevent the mutagenic effect.