Structure-activity relationships of epoxides: induction of sister-chromatid exchanges in V79 cells by enantiomeric epoxides.

Analysis of SCE frequencies in Chinese hamster V79 cells was used to investigate the influence of the stereoisomeric forms of epoxides in mammalian genotoxicity tests. The SCE-inducing potency of 12 pairs of (R)- and (S)-enantiomeric epoxides which differed in the degree of substitution of the oxirane ring was determined. Of these, 2 pairs of epoxides failed to induce SCE. Different SCE-inducing potencies between the (R)- and (S)-enantiomers were shown for 5 epoxides. This study demonstrates that stereoselectivity might play an important role in genotoxicity testing of chemicals with asymmetric C atoms.     

The genotoxic potential of glutaraldehyde in mammalian cells in vitro in comparison with formaldehyde

Glutaraldehyde (GA) induces DNA-protein crosslinks (DPX), but conflicting results have been reported with regard to other genotoxic and mutagenic effects in mammalian cells in vitro. We, therefore, characterized the genotoxic and mutagenic potential of GA in V79 cells. Using the alkaline comet assay we demonstrated the induction of DPX by GA (reduction of gamma ray-induced DNA migration) at a concentration of 10 microM and above. The standard comet assay did not reveal a significant DNA strand-breaking activity of GA. Cross-linking concentrations of GA were also cytotoxic, i.e. inhibited cell growth of treated V79 cultures. Interestingly, a small but statistically significant increase in sister chromatid exchange (SCE) and micronuclei (MN) was already measured at lower concentrations (2 and 5 microM). FISH analysis revealed that the majority of GA-induced MN was due to chromosome breaks. We also compared the genotoxic activity of GA to that of formaldehyde (FA). Similar to GA, FA-induced DPX, SCE and MN, but distinct differences exist with regard to the sensitivity of the endpoints and the relationship between genotoxicity and cytotoxicity. However, the differences in genotoxicity cannot readily explain the different carcinogenic activities of the two compounds.     

Quantitative comparative mutagenesis in bacteria, mammalian cells, and animal-mediated assays A convenient way of estimating genotoxic activity in vivo?

The accumulation of environmental compounds which exhibit genotoxic properties in short-term assays and the increasing lag of time for obtaining confirmation or not in long-term animal mutagenicity and carcinogenicity tests, makes it necessary to develop alternative, rapid methodologies for estimating genotoxic activity in vivo. In the experimental approach used here, it was assumed that the genotoxic activity of foreign compounds in animals, and ultimately humans, is determined among others by exposure level, organ distribution of (DNA) dose, and genotoxic potency per unit of dose, and that knowledge about these 3 parameters may allow to rapidly determine the expected degree of genotoxicity in various organs of exposed animals. In view of the high degree of qualitative correlation between mutagenic activity of chemicals in bacteria and in cultured mammalian cells, and their mutagenic and carcinogenic properties in animals, and in order to be able to distinguish whether mutagenic potency differences were due to differences in (DNA) dose rather than other physiological factors, the results of mutagenicity tests obtained in the present experiments using bacteria and mammalian cells were compared on the basis of DNA dose rather than exposure concentrations, with the following questions in mind: Is there an absolute or a relative correlation between the mutagenic potencies of various ethylating agents in bacteria (E. coli K12) and in mammalian cells (V79 Chinese hamster) after treatment in standardized experiments, and can specific DNA adducts be made responsible for mutagenicity? Is the order of mutagenic potency of various ethylating agents observed in bacteria in vitro representative of the ranking of mutagenic potency found in vivo? Since the answer to this last question was negative, a further question addressed to was whether short-term in vivo assays could be developed for a rapid determination of the presence (and persistence) of genotoxic factors in various organs of mice treated with chemicals. In quantitative comparative mutagenesis experiments using E. coli K12 and Chinese hamster cells treated under standardized conditions in vitro with 5 ethylating agents, there was no indication of an absolute correlation between the number of induced mutants per unit of dose in the bacteria and the mammalian cells. The ranking of mutagenic potency was, however, identical in bacteria and mammalian cells, namely, ENNG greater than ENU greater than or equal to DES greater than DEN congruent to EMS, the mutagenic activity of DEN being dependent on the presence of mammalian liver preparations.(ABSTRACT TRUNCATED AT 400 WORDS)     

Some substrates and inhibitors of cytosolic epoxide hydrolase induce sister-chromatid exchanges in mammalian cells, but do not induce gene mutations in Salmonella typhimurium and V79 cells

Trans-stilbene oxide, trans-β-methylstyrene, 7,8-oxide, trans-β-ethylstyrene, 7,8-oxide, trans-β-propylstyrene 7,8-oxide and 4-fluorochalcone oxide were investigated for genotoxic activity in bacterial and mammalian cells, in the absence of external xenobiotic-metabolising systems. All compounds strongly enhanced the frequency of sister-chromatid exchanges (SCE) in cultured human lymphocytes. None of them was mutagenic in Salmonella typhimurium (reversion of the his⁻ strains TA98, TA100 and TA104). The limit of detection was 1/20,000 to 1/10⁶ of the activity of the positive control, benzo[a]pyrene 4,5-oxide, depending on the compound and the bacterial strain. Trans-β-methylstyrene 7,8-oxide and 4-fluorochalcone oxide were additionally tested for induction of SCE and gene mutations in the same target cells, namely Chinese hamster V79 cells. Their influence on the level of SCE was similar to that observed in human lymphocytes, whilst gene mutations (at the hprt locus) were not induced. The four investigated styrene oxide derivatives are known to be excellent substrates for a mammalian enzyme, cytosolic epoxide hydrolase (cEH). 4-Fluorochalcone oxide is a potent selective inhibitor of this enzyme and is structurally similar to the investigated styrene oxide derivatives. These properties of the test compounds however cannot explain the observed discrepancies in the results, since the genetic end point (SCE versus gene mutations) was decisive, and SCE were induced in cEH-proficient human lymphocytes as well as in cEH-deficient V79 cells.     

Mutagenic activity of arylnitrenium ions from arylazides--induction of sister chromatid exchange in mammalian (V79 Chinese hamster) cells.

Photolysis of arylazides produces short-lived reactive species, very likely arylnitrenium ions which bind to nucleotides and DNA and produce mutations in Salmonella. The present report shows that arylazides can be photo-activated in mammalian (V79 Chinese hamster) cells and that sister chromatid exchange can thus be induced. Arylazides studied are (in order of decreasing SCE-inducing potency) azido-isoIQ, azido-MeIQ, azido-IQ, azido-MeIQx, azido-PhIP, 6-azido-chrysene, 2-azidofluorene, 4-azidofluorene, 2-azido-naphthalene, 4-azidobi-phenyl, 2-azidobiphenyl, 2,4,6-trimethylphenylazide, phenylazide (inactive). The structure-activity relationships emerging from the data are the same as those found previously in Salmonella. In line with this, a clearcut positive linear correlation was seen between the logarithm of the SCE-inducing potency in V79 cells and the logarithm of the mutagenic potency in Salmonella (r = 0.955). Therefore, the ultimate reactive species derived from IQ and related heterocyclic aromatic amines are extremely potent genotoxins, not only in a bacterial but also in a mammalian cell. Previous findings of only weak genotoxic activity of IQ and related food mutagens in certain cultured mammalian cells must therefore be reinterpreted as the result of an insufficient activation of these amines in the cells used, possibly because of insufficient acetylation competence.     

Mutagenic effects of TPA-induced clastogenic factor in Chinese hamster cells

Previous work in our laboratory has shown that the clastogenic and SCE-inducing effects of 12-O-tetradecanoylphorbol 12-acetate (TPA) are mediated by secondary products formed by the cell in response to the tumor promoter. A low-molecular-weight clastogenic factor (CF) was isolated from the medium of TPA-treated human leukocytes and caused chromosome aberrations and sister-chromatid exchanges (SCE) in fresh cultures not exposed to TPA itself. In the present study, we show that Chinese hamster fibroblasts (V79 cells) also produce CF when exposed to TPA. CF from V79 cells induced SCE not only in hamster cells, but also in human lymphocytes. Vice versa, CF from human leukocyte cultures induced SCE in hamster cells. It also increased the frequency of 6-thioguanine-resistant mutants in this cell system. All cyto- and geno-toxic effects of TPA-induced CF were prevented if the cells were treated with superoxide dismutase before exposure. The lipophilic CF seems to be derived from arachidonic acid of cell membranes released as a consequence of oxidative damage and subsequently degraded to genotoxic aldehydes in an autoxidative process. CF is formed only under culture conditions with low antioxidant content in culture media and sera. This may explain the discordant results obtained by different laboratories with regard to the genotoxic effects of TPA.     

Enhancing effect of tetrandrine on sister-chromatid exchanges induced by mitomycin C and cigarette-smoke condensate in mammalian cells

The enhancing effect of tetrandrine, an antisilicosis, antitumor and antiinflammatory drug, on the genotoxic activity of two known mutagens, mitomycin C (MMC) and cigarette-smoke condensate (CSC), has been studied using cultured Chinese hamster lung (V79) cells. The sister-chromatid exchange (SCE) was used as genetic endpoint to measure genotoxicity. One-day cultured cells were exposed to the test chemicals for 3 h with or without metabolic activation. The results show that the frequencies of SCE induced by MMC or CSC were enhanced by tetrandrine. The percent of enhancement was dependent on the concentration of tetrandrine.     

Evaluation of genotoxicity of tert.-butylhydroquinone in an hepatocyte-mediated assay with V79 Chinese hamster lung cells and in strain D7 of Saccharomyces cerevisiae.

tert.-Butylhydroquinone (TBHQ) has been reported to be genotoxic in some short-term assays but non-genotoxic in others. We have examined cytotoxicity and genotoxicity of TBHQ, a principal metabolite of the phenolic antioxidant 2(3)-tert.-butyl-4-hydroxyanisole (BHA), in an hepatocyte-mediated assay with V79 Chinese hamster lung cells including both sister-chromatid exchange (SCE) and thioguanine-resistance (TGR) endpoints. The ability of BHA and of TBHQ to elicit a genotoxic response in Saccharomyces cerevisiae strain D7 was also investigated. In V79 cytotoxicity tests, TBHQ without hepatocytes produced a 50% reduction in colony formation at 4.2 micrograms/ml and was lethal to 100% of the cells at concentrations above 5 micrograms/ml. At partially cytotoxic dose levels, (0.17-3.4 micrograms/ml of medium), TBHQ sometimes increased significantly the frequency of SCE. TBHQ also produced sporadic statistically significant increases in the mutation frequency at the HGPRTase (TGR) gene locus when tested alone or with activation by rat or hamster hepatocytes. Mitotic gene conversion and reverse mutation were not induced in strain D7 of Saccharomyces cerevisiae by exposure to BHA or to TBHQ for 4 h at concentrations as high as 200 micrograms/ml for BHA or 500 micrograms/ml for TBHQ, either alone or with activation by rat-liver S9. Incubation of the yeast cells with BHA or TBHQ for 24 h in growth medium without activation also did not induce genotoxic activity. The slight and sporadic response to TBHQ in the V79 test system may indicate weak genotoxicity which is sensitive to slight differences in test conditions. The classification and test strategies adopted for compounds such as TBHQ could have important implications for regulatory decisions and for the validation of short-term tests.     

Genotoxicity of 1,3- and 1,6-dinitropyrene: induction of micronuclei in a panel of mammalian test cell lines.

1,3-Dinitropyrene (1,3-DNP) and 1,6-dinitropyrene (1,6-DNP) were assessed for their potential to increase the frequencies of micronuclei in a panel of test cell lines consisting of H4IIEC3/G-, 5L, 5L/r-1,3-DNP1, 208F, V79, V79/r-1,6-DNP1, HepG2 and BWI-J cells, which have been partially characterized for their expression of xenobiotic metabolising enzymes. The micronuclei were analyzed for the presence or absence of kinetochores indicating the occurrence of aneuploidy or chromosome breakage, respectively. 1,3-DNP caused a substantial increase in the frequency of micronuclei only in V79 cells. 1,6-DNP was strongly genotoxic in lines H4IIEC3/G-, 208F, V79 and, to a minor degree, in 5L/r-1,3-DNP1. It caused the formation of kinetochore-positive as well as kinetochore-negative micronuclei in V79 cells but only of kinetochore-negative micronuclei in H4IIEC3/G- and 208F cells. 1,6-DNP-induced formation of micronuclei was paralleled by the appearance of multinucleated cells. Treatment of V79 cells with 1,3-DNP resulted in the same types of damage as treatment with 1,6-DNP, although considerably higher concentrations were required. The results show that 1,6-DNP can be highly genotoxic in mammalian cells, whereas, at least in the panel of test cell lines used, 1,3-DNP possesses only a low genotoxic activity. 1,3-DNP appears to be activated to genotoxic products in V79 cells by the same pathway(s) as 1,6-DNP.     

Genotoxicity of 1,3- and 1,6-dinitropyrene: induction of micronuclei in a panel of mammalian test cell lines

1,3-Dinitropyrene (1,3-DNP) and 1,6-dinitropyrene (1,6-DNP) were assessed for their potential to increase the frequencies of micronuclei in a panel of test cell lines consisting of H4IIEC3/G, 5L, 5L/r-1,3-DNP1, 208F, V79, V79/r-1,6-DNP1, HepG2 and BWI-J cells, which have been partially characterized for their expression of xenobiotic metabolising enzymes. The micronuclei were analyzed for the presence or absence of kinetochores indicating the occurrence of aneuploidy or chromosome breakage, respectively. 1,3-DNP caused a substantial increase in the frequency of micronuclei only in V79 cells. 1,6-DNP was strongly genotoxic in lines H4IIEC3/G⁻, 208F, V79 and, to a minor degree, in 5L/r-1,3-DNP1. It caused the formation of kinetochore-positive as well as kinetochore-negative micronuclei in V79 cells but only of kinetochore-negative micronuclei in H4IIEC3/G⁻ and 208F cells. 1,6-DNP-induced formation of micronuclei was paralleled by the appearance of multinucleated cells. Treatment of V79 cells with 1,3-DNP resulted in the same types of damage as treatment with 1,6-DNP, although considerably higher concentrations were required.The results show that 1,6-DNP can be highly genotoxic in mammalian cells, whereas, at least in the panel of test cell lines used, 1,3-DNP possesses only a low genotoxic activity. 1,3-DNP appears to be activated to genotoxic products in V79 cells by the same pathway(s) as 1,6-DNP.     

Comparative genotoxic effects of IQ and MeIQ in Salmonella typhimurium and cultured mammalian cells

The food mutagens 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) were studied for their genotoxic potential using hepatocytes isolated from untreated and Aroclor 1254 (PCB) pretreated rats as an activation system. Monolayers of hepatocytes co-incubated with Salmonella typhimurium TA98 activated IQ and MeIQ to bacterial mutagens, with MeIQ being about twice as potent as IQ. The mutagenic activities of IQ and MeIQ were increased by using hepatocytes from PCB-pretreated rats. IQ and MeIQ also caused primary DNA damage in the hepatocytes as determined by increases in the rate of alkaline elution of DNA, as well as increases in DNA-repair synthesis. Furthermore, exposure of V79 cells co-cultured with PCB-pretreated hepatocytes to IQ and MeIQ showed evidence of increased sister-chromatid exchanges and a low and variable increase in the number of 6-thioguanine-resistant mutants. The genotoxic potency of IQ and MeIQ in mammalian cells was low or virtually absent compared to their extreme potency in bacteria. This could be due to a lower capacity of mammalian cells to further metabolize the so-called directly acting bacterial mutagens, formed by a cytochrome P-450 dependent N-hydroxylation, to their ultimate reactive forms.     

V79-hCYP2E1-hSULT1A1, a cell line for the sensitive detection of genotoxic effects induced by carbohydrate pyrolysis products and other food-borne chemicals

We recently constructed a Chinese hamster V79-derived cell line that stably expresses human cytochrome P450 (CYP) 2E1 and human sulphotransferase (SULT) 1A1. These enzymes are involved in the bioactivation of numerous promutagens/procarcinogens, but are not taken into account in standard in vitro mutagenicity assays. Various carbohydrate pyrolysis products and other food contaminants that induce tumours or preneoplastic lesions in laboratory animals are inactive or only weakly active in standard in vitro genotoxicity assays. This is the case for acrylamide, furan, 5-hydroxymethylfurfural, nitrofen and N-nitrosodimethylamine. These compounds were investigated for induction of sister chromatid exchange (SCE) in V79-hCYP2E1-hSULT1A1 cells. All test compounds showed positive results over a wide concentration range, starting at 0.01 microM for N-nitrosodimethylamine, 3 microM for furan, 12.5 microM for nitrofen, 20 microM for 5-hydroxymethylfurfural, and 200 microM for acrylamide. The concentration-response curve of furan was unusual, as this compound induced a statistically significant, but rather constant and weak increase in SCE over an extremely wide concentration range (3-16,000 microM). Furan was slightly less active, whereas the remaining compounds were much less active in the parental V79 cell line than in V79-hCYP2E1-hSULT1A1 cells. Compared to many other genotoxic effects, the study of SCE only requires small numbers of cells (and incubation volumes) and usually is detected even at low concentrations of the genotoxicant. Therefore, induction of SCE in V79-hCYP2E1-hSULT1A1 cells may be useful in the genotoxicity testing of preparations of heated food and in their bioassay-directed fractionation.     

Estragole: a weak direct-acting food-borne genotoxin and potential carcinogen

We evaluated the genotoxicity of the food-flavouring agent estragole in V79 cells using the sister chromatid exchange (SCE) assay and the alkaline comet assay. Unexpectedly, we observed an increase in SCE without an exogenous biotransformation system (S9) and a decrease in its presence. Positive results were also observed in the alkaline comet assay without S9, indicating DNA strand breakage. To ascertain repair of damage, we performed the comet assay in V79 cells after two hours of recovery, and observed a reduction of the genotoxic response. Estragole did not produce strand breaks in plasmid DNA in vitro. We then evaluated the formation of DNA adducts in V79 cells by use of the (32)P-postlabelling assay and detected a dose-dependent formation of DNA adducts, which may be responsible for its genotoxicity. We then assayed estragole in the comet assay with two CHO cell lines, a parental AA8 cell line, and an XRCC1-deficient cell line, EM9. Results confirmed the genotoxicity of estragole without biotransformation in both cell lines, although the genotoxicity in EM9 cells compared with that in AA8 cells was not significantly different, suggesting that the XRCC1 protein is not involved in the repair of estragole-induced lesions. Estragole induces apoptosis, but only with high doses (2000μM), and after long treatment periods (24h). Overall, our results suggest that estragole, besides being metabolized to genotoxic metabolites, is a weak direct-acting genotoxin that forms DNA adducts.     

Genotoxic effects of paracetamol in V79 Chinese hamster cells

Paracetamol was studied for possible genotoxic effects in V79 Chinese hamster cells. Paracetamol (0.5 mM for 30 min) reduced the rate of DNA synthesis in exponentially growing V79 cells to about 50% of control. A further decrease in the DNA synthesis was seen during the first 30 min after termination of paracetamol exposure. Paracetamol (3 and 10 mM for 2 h) caused a small increase in DNA single-strand breaks, as measured by the alkaline elution technique. After 16 h elution, the amount of DNA retained on the filters was 79 and 70% of controls in cells treated with 3 and 10 mM paracetamol respectively. No indication of DNA damage was seen in measuring the effect of paracetamol (0.25-10 mM for 2 h) on unscheduled DNA synthesis in growth-arrested cultures of V79 cells. At the highest concentrations (3 and 10 mM paracetamol), decreased unscheduled DNA synthesis was observed. Also UV-induced DNA-repair synthesis was inhibited by 3 and 10 mM paracetamol. DNA-repair synthesis was, however, inhibited at a much higher concentration than that inhibiting replicative DNA synthesis. The number of sister-chromatid exchanges (SCE) increased in a dose-dependent manner on 2 h exposure to paracetamol from 1 mM to 10 mM. At the highest dose tested (10 mM), the number of SCE increased to 3 times the control value. Co-culturing the V79 cells with freshly isolated mouse hepatocytes had no further effect on the paracetamol induced sister-chromatid exchanges. The present study indicates that paracetamol may cause DNA damage in V79 cells without any external metabolic activation system added.     

Hepatocyte-mediated SCE induction by indirect mutagens: importance of hepatocyte density and cell-to-cell contact

The SCE-inducing effects of the indirectly acting mutagens cyclophosphamide (CP), dimethylnitrosamine (DMN) and aflatoxin B1 (AFB1) were analysed in hepatocyte (hpc)/mammalian cell coculture systems with regard to the importance of the hpc density. V79 cells and human lymphocytes served as target cells. For all 3 compounds steadily increasing genetic effects were observed when the hpc density was increased from 3.2 X 10(4) up to 3.2 X 10(6) viable hpc per culture (25-cm2 flask), i.e. the more hpc available for metabolisation, the more genetic effects induced. The frequency distributions of the CP-induced SCE values were clearly different from those obtained with DMN, especially when high hpc densities were used: distribution patterns obtained for the mutagen with stable metabolites (CP) are characterized by the presence of distinct maxima and the absence of cells with SCE control values, whereas distribution patterns for the mutagen with very short-lived metabolites (DMN) can be described by the absence of maxima and the presence of cells with SCE control values. The frequency distributions of the AFB1-induced SCE values were more similar to the CP type than to the DMN type. From these results it is deduced that close contact between metabolising and target cells is necessary for the detection of the genotoxic effect of DMN. For CP and AFB1 a direct contact seems not to be essential, i.e. reactive intermediates may also be transported via the culture medium to the target cells.     

The genotoxicity of enantiomeric aliphatic epoxides

The (R)- and (S)-optical isomers of 9 epoxides, benzyloxymethyloxirane, epichlorohydrin, glycidol, glycidyl 3-nitrobenzenesulfonate, glycidyl 4-nitrobenzoate, glycidyl tosylate, styrene oxide, glycidyl 1-naphthyl ether and glycidyl 4-nitrophenyl ether, have been compared for their in vivo and in vitro genotoxicity. The in vitro short-term test employed was the Ames mutagenicity assay with Salmonella strain TA100. The in vivo tests were chromosomal aberrations (CA) as well as sister-chromatid exchange (SCE) in bone-marrow cells of mice following intraperitoneal administration of these epoxides. Differences in mutagenicity between isomers were established with TA100 for all the compounds. While 13 of the isomers were genotoxic compared to a negative control by CA measurements, only in the case of glycidyl 4-nitrobenzoate could a significant difference be found between isomers by this test. However, with SCE evaluations, differences were detected between the (R)- and (S)-isomers for all the pairs of compounds with the exception of those for benzyloxymethyloxirane and glycidyl 4-nitrophenyl ether. At least in part, differences in the patterns of genotoxicity among compounds can be related to their differences in reaction pathways.     

Studies by means of the SCE assay in V79-E Chinese hamster cells on the mode of action of tri-substituted nitrosoureas

The genotoxic activity of 3,3-diethyl-1-methyl-1-nitrosourea ( DEMNU ), 1,3-dimethyl-3-phenyl-1-nitrosourea ( DMPNU ) and 1-chloroethyl-3-methyl-3-phenyl-1-nitrosourea ( CEMPNU ) was studied in the SCE assay in V79-E cells in vitro. These compounds are very stable in aqueous solutions, but are directly acting genotoxins . The SCE rates increase linearly with the length of the incubation period. This direct activity is presumably due to an intracellular catalytic decomposition. Whereas the SCE-inducing effect of DMPNU and CEMPNU is not influenced by addition of S9 mix, that of DEMNU is strongly potentiated by rat and Syrian hamster S9 mix. This DEMNU activation is an NADPH-dependent enzymatic reaction and is inducible by phenobarbital. The absence of a direct mutagenic effect of DEMNU in the Ames test, as reported by other authors, is probably caused by a striking insensitivity to tri-substituted nitrosoureas of the Salmonella assay. This assumption was substantiated by long-term application of very low DMPNU doses to V79-E. Long-term simultaneous treatment with DMPNU and bromodeoxyuridine (BUdR) significantly diminished the rate of SCE induction.     

Genotoxicity testing of extracts of a Swedish moist oral snuff

The present study was designed to investigate the potential genotoxicity of aqueous and methylene chloride extracts of Swedish moist oral snuff. The test systems were selected to provide optimal data for the prediction of carcinogenicity in rodents and included assays for the induction of mutation in bacteria, sister-chromatid exchanges (SCE) in human lymphocytes, of chromosome aberrations and gene mutations in V79 Chinese hamster cells and of micronuclei in mouse bone marrow cells. In addition, the methylene chloride extract was tested for the induction of sex-linked recessive lethal mutations in Drosophila melanogaster. The aqueous extract of 'Snus' induced SCE in human lymphocytes and chromosome aberrations in V79 cells, the latter effect being observed both with and without metabolic activation. No induction of point mutations was detected with the Ames test or in V79 cells and the micronucleus test in mice was negative. It was demonstrated that the induction of chromosome aberrations without metabolic activation may be due to a high salt concentration, indicating that the clastogenic agent(s) in this extract required metabolic activation. The methylene chloride extract showed genotoxicity in the Ames test, the SCE test and the chromosome aberration test, whereas no induction of gene mutations in V79 cells was observed. Once again, the results suggested that metabolism is required for genotoxicity. The methylene chloride extract did not cause induction of micronuclei in mice or of sex-linked recessive lethal mutations in Drosophila melanogaster. These combined data on genotoxicity were analyzed using various models for the prediction of carcinogenicity. In a sequential testing model, the probabilities that the aqueous and methylene chloride extracts of 'Snus' are carcinogenic due to a genotoxic mechanism were both predicted to be low. Using carcinogenicity prediction by battery selection (CPBS), the probabilities of the methylene chloride and aqueous extracts being correctly identified as non-carcinogens are 71 and 77%, respectively. Up to date, the CPBS approach has been validated primarily for individual compounds, so some caution should at present be exercised in interpreting the results using this method. Based on these results, the carcinogenic potential of Swedish 'Snus' should be considered to be low, a conclusion in agreement with the low incidence of oral cancer in Sweden compared to other countries.     

Rodent species and strain specificities for sister-chromatid exchange induction and gene mutagenesis effects from ethyl carbamated,ethyl N-hydroxycarbamate,and vinyl carbanate

Ethyl carbamate (EC) and two related carcinogens, ethyl N-hydroxycarbamate (ENHC) and vinyl carbamated (VC), caused species-specific increase in sister-chromatid exchange (SCE) formation in the bone marrow cells of rodents. Mice exposed to 400 mg/kg of EC had SCE increases of 6-times-baseline, while rats, Chines hamsters, and golden hamsters showed 3- to 4-times-baseline increases in response to this dose. Lesser, but still significant, differences were found for ENHC and VC; the severest effects consistently occured in mice. Control bone marrow cell-cycle kinetics among the rodent species were similar. Mouse strains A and C57BL/6, which have high and low susceptibilities to EC induction of lung adenomas, respectively, showed nearly identical levels of SCE induction after in vivo exposure to these carbamate. However, testing of VC, a possible metabolite of EC, in vitro revelaed strain-dependent liver enzyme (Aroclor-induced S-9 fraction) capabilities to convert VC to genotoxic products. SCE induction, gene mutation for 6-thioguanine and ouabain resistance, and cytotoxicity in Chinese hamster V79 cells were significantly greater when A strain S-9 enzymes were used as compared with C57BL/6 strain S-9 enzyme preparations. No effect of SCE of reseeding, compared with no reseeding, of VC-treated V79 cells was observed. At a concentration of 25 μg/ml, VC cause 6-times-baselin induction of SCE in the presence of A strain S-9 mix and 4-times-baseline induction in the presence of C57BL/6 strain S-9 mix. These in vitro strain-dependent patterns of response are relevant to the current theory that VC amy be a proximate carcinogenic metabolite of EC.     

Gamma-tocopherol is less effective than alpha-tocopherol in preventing oxidant-induced sister chromatid exchanges in Chinese hamster V79 cells.

Although alpha-tocopherol (alpha-TOC) is the most biologically active form of vitamin E and is found at high levels in plasma, gamma-tocopherol (gamma-TOC) has also been found to be a powerful antioxidant in vitro and constitutes up to 70% of the dietary intake of TOC. Low plasma levels of gamma-TOC and a high alpha-TOC:gamma-TOC ratio may be associated with coronary heart disease, suggesting that there may be a positive protective role for the gamma-form of TOC. In this study the ability of different forms of vitamin E to protect against sister chromatid exchanges (SCE) induced by either hydrogen peroxide or menadione was investigated. Chinese hamster V79 cells were pre-treated with 10 microM TOC for 24 h, and then challenged with a genotoxin. After a 24 h pre-treatment, there was a greater incorporation of gamma-TOC (319.8 +/- 66.2 ng/10(6) cells) into V79 cells compared to alpha-TOC (66.9 +/- 6.4 ng/10(6) cells). Gamma-TOC did not protect the cells against SCE induced by either hydrogen peroxide or menadione, alpha-TOC acetate was partially protective against both genotoxins, whereas alpha-TOC completely abolished the oxidant induced SCE. These results demonstrate that, despite a greater incorporation of gamma-TOC into V79 cells, alpha-TOC but not gamma-TOC was more effective at inhibiting oxidatively-induced SCE in V79 cells.