The hepatocyte primary culture/DNA repair assay using mouse or hamster hepatocytes

The hepatocyte primary culture (HPC)/DNA repair assay using rat hepatocytes was developed to identify genotoxic chemicals. Since there are species differences in susceptibility to chemical carcinogens, it was desirable to extend the assay to other species. Carcinogens and noncarcinogens from six structural classes were tested with hepatocytes from B6C3F1 mice or Syrian hamsters. In hepatocytes from both species, DNA repair was elicited by the carcinogens methyl methanesulfonate, aflatoxin B1, 2-acetylaminofluorene, benzo(a)pyrene, dimethylnitrosamine, nitrosopyrrolidine, 3'-methyl-4-dimethylaminoazobenzene, and p-dimethylaminoazobenzene. With aflatoxin B1, mouse hepatocytes required a dose of 10(-4) M for a maximum response while only 10(-6) was needed for hamster hepatocytes. All the presumed noncarcinogens, except 4-dimethylaminoazobenzene-4'-sulfonyl chloride, were negative in mouse hepatocytes. This chemical, as well as aflatoxin G2 and pyrene, which have not been tested for carcinogenicity in the hamster, were positive in hamster hepatocytes. These findings demonstrate that genotoxic chemicals can be identified by the HPC/DNA repair assay using hamster or mouse hepatocytes. Furthermore, in vivo differences in susceptibility to chemical carcinogens such as aflatoxin B1 are reflected in the in vitro assay.     

Aroclor 1254 pretreatment effects on DNA repair in rat hepatocytes elicited by in vivo or in vitro exposure to various chemicals

Inducers of liver mixed function oxidase (MFO) activities have profound effects on the genotoxicity of substances that undergo metabolic activation by the MFO system. The polychlorinated biphenyl mixture Aroclor 1254 is a broad-spectrum inducer of liver MFO activities that has been employed as a pretreatment to augment the metabolic activation capabilities of rat liver fractions used in a number of short-term tests for genotoxicity, including the Ames Salmonella/bacterial mutagenicity assay. The present study was designed to characterize the effects of Aroclor pretreatment of rats on the DNA repair responses elicited by various chemicals in the in vitro hepatocyte primary culture/DNA repair (HPC/DR) assay as well as the in vivo/in vitro HPC/DR assay. The amount of DNA repair produced in vitro by diethylnitrosamine (DEN), benzo(a)pyrene (B(a)P), 3-methylcholanthrene (3-MC), 2-acetylaminofluorene (2-AAF), o-aminoazotoluene (o-AT), and aflatoxin B1 (AFB1) was significantly greater in hepatocytes derived from Aroclor-pretreated rats than in control rat hepatocytes; in vitro responses to dimethylnitrosamine (DMN), 7,12-dimethylbenzanthracene (DMBA), benzidine (BZ), and 2-naphthylamine (2-NA) were not significantly affected by Aroclor pretreatment. DNA repair elicited by the direct-acting alkylating agents methyl methanesulfonate and N-methyl-N'-nitro-N-nitrosoguanidine was also not increased by Aroclor pretreatment, which indicated that Aroclor does not exert a general stimulatory effect on the hepatocellular DNA repair capacity. Therefore, the pretreatment-related potentiation of DNA repair observed for 6 out of 12 compounds tested in vitro was considered to be due to enhanced metabolic activation. These results suggested that pretreatment with Aroclor may increase the sensitivity of the in vitro HPC/DR assay to certain compounds. In contrast, Aroclor pretreatment had little effect on the amount of hepatocellular DNA repair elicited by in vivo administration of DMN, DEN, o-AT, 2-AAF, 3-MC, or AFB1, which indicated that this pretreatment regimen may have little utility for improving the sensitivity of the in vivo/in vitro HPC/DR assay.     

Pretreatment with mixed-function oxidase inducers increases the sensitivity of the hepatocyte/DNA repair assay

A recent National Toxicology Program evaluation indicates that the rat hepatocyte/DNA repair assay has a high false-negative rate and that it is insensitive to some genotoxic hepatocarcinogens as well as other species and organ-specific carcinogens. In this study, we examined whether the sensitivity of the hepatocyte/DNA repair assay might be increased through animal pretreatment with various hepatic mixed-function oxidase inducers, i.e., Aroclor 1254, phenobarbital, and 3,3',4,4'-tetrachloroazobenzene (TCAB). The effects on unscheduled DNA synthesis (UDS), a measure of DNA damage and repair, were studied in cultures exposed to known and/or potential carcinogens that had been evaluated as negative or questionable or that produced conflicting results with hepatocytes isolated from uninduced animals. 4,4'-Oxydianiline, 1-nitropyrene, and TCAB produced concentration-dependent increases in UDS in hepatocytes from rats pretreated with Aroclor 1254. 4,4'-Oxydianiline and TCAB also induced a dose-dependent increase in DNA repair in hepatocytes from rats pretreated with phenobarbital, whereas 1-nitropyrene was negative. 4,4'-Methylenedianiline produced a marginal response, and 3,3',4,4'-tetrachloroazoxybenzene (TCAOB) was negative in Aroclor- and phenobarbital-induced hepatocytes; however, TCAOB, as well as TCAB, produced concentration-dependent increases in UDS in TCAB-induced hepatocytes. These data indicate that the limited sensitivity to chemical carcinogens displayed by the hepatocyte/DNA repair assay may be increased by using hepatocytes isolated from animals exposed to hepatic mixed-function oxidase inducers.     

Chemically-induced unscheduled DNA synthesis in primary rat hepatocyte cultures: A comparison with bacterial mutagenicity using 218 compounds

The autoradiographic identification of unscheduled DNA synthesis (UDS) in primary cultures of adult rat hepatocytes (HPC) has been proposed as a predictive test for mutagens/carcinogens. To assess the predictive value of this test, results in the hepatocyte UDS assay were compared with data for bacterial mutagenicity using a modified Ames test. Over 200 compounds representing a variety of chemical classes consisting of procarcinogens, ultimate carcinogens, and noncarcinogens were tested in each system. The accurate discrimination of many carcinogens/noncarcinogens was demonstrated by both systems. The induction of UDS in hepatocytes showed an excellent correlation with bacterial mutagenesis in response to polycyclic aromatic hydrocarbons, aromatic amines, biphenyls, nitrosamines, carbamates, azo-compounds, acridines, halogenated compounds, nitrosureas, quinolines, pyridines, purines, pyrimidines, esters and carbamates. Nitrocompounds, although active in bacteria, were poor inducers of UDS. The results support the complementary and confirmatory nature of these tests for genotoxic chemicals and indicate the usefulness of the hepatocyte UDS system as a component in a battery of short-term predictive tests for mutagens/carcinogens.     

Effects of aging and caloric restriction on the genotoxicity of four carcinogens in the in vitro rat hepatocyte/DNA repair assay

The effects of aging and chronic caloric restriction (CR) on the genotoxicity of four carcinogens, representing four different classes of chemicals, in the in vitro rat hepatocyte/DNA repair assay were investigated. Hepatocyte cultures were isolated from young, middel-aged, and old male Fischer (F344) rats which were maintained on either an ad libitum (AL) or a CR diet (60% of AL). Hepatocyte cultures from old AL rats, treated with 2-acetylaminofluorene (2-AAF), aflatoxin B₁ (AFB₁), 7,12-dimethylbenz[a]anthracene (DMBA) and dimethylnitrosamine (DMN), exhibited age-related decreases in DNA repair as compared to young AL rats. By contrast, cultures from young CR rats exhibited significant diet-related decreases in DNA repair with 2-AAF, AFB₁, DMBA and DMN, when compared to results from young AL diet-fed rats. Old CR F344 rat derived cultures exhibited no significant age-related dose-dependent decrease in the DNA repair response with any of the chemicals tested. However, in cultures from old CR rats 10.0 μM AFB₁ produced an age-related decrease in DNA repair from the response observed in young CR rats. When hepatocytes were isolated from Aroclor 1254-induced rats, increases in DNA repair were observed. These data indicate an age- and diet-related decrease in DNA repair and/or DNA damage and suggest that this decrease is due to a decrease in metabolic activation of these carcinogens to genotoxic species.     

Testing of 24 food, drug, cosmetic, and fabric dyes in the in vitro and the in vivo/in vitro rat hepatocyte primary culture/DNA repair assays

Twenty-four dyes currently or previously used in the food, drug, cosmetic, and textile industries were tested in the in vitro rat hepatocyte primary culture/DNA repair (HPC/DR) assay and, to a limited extent, in the in vivo/in vitro HPC/DR assay. The positive control, Solvent Yellow 3 (o-aminoazotoluene), and five other dyes (4-dimethylaminobenzeneazo-1-naphthalene, 4-dimethylaminobenzeneazo-2-naphthalene, Direct Blue 53, Acid Blue 9, and 4-dimethylaminostilbene) induced DNA repair in rat hepatocytes both in vitro and in vivo, while 13 of the dyes (Food Red 1, Food Red 5, Food Orange 4, Food Red 7, Acid Red 14, Acid Red 27, Pigment Red 53, Acid Yellow 23, Food Black 1, Food Green 3, Acid Red 51, Acid Blue 74, and Natural Red 4) did not produce any detectable DNA repair in either the in vitro or in vivo/in vitro assays. Direct Blue 14 had weak activity in vitro but none was detected in vivo. In contrast, Solvent Yellow 5 was not active in vitro, but produced a weak positive response in vivo. Negative responses were also obtained for Solvent Yellow 14 and Acid Green 5 in the in vitro assay, whereas the responses produced by these dyes in the in vivo/in vitro assay were judged to be equivocal. An equivocal response was also obtained for Direct Red 28 in the in vivo/in vitro assay as well as in the in vitro assay. These findings provide information about the potential genotoxicity of a number of dyes for which previous genotoxicity data has been inconsistent or inadequate. For some dyes (eg, Solvent Yellow 5), discrepancies between the results obtained in the in vitro and in vivo/in vitro assays may implicate a role for intestinal microflora in their metabolic activation.     

Genotoxicity of the cooked-food mutagens IQ and MeIQ in primary cultures of rat, hamster, and guinea pig hepatocytes

To investigate possible interspecies differences in the hepatocellular genotoxicity of the food-borne mutagens 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ) and 2-amino-3,4-dimethyl-imidazo[4,5-f]quinoline (MeIQ), primary cultures of rat, hamster, and guinea pig hepatocytes were established. The induction of DNA repair activity in cultures exposed to various concentrations of IQ and MeIQ was determined by liquid scintillation spectrometry. DNA repair responses to MeIQ were, in general, greater than those elicited by IQ. In all three preparations of rat and hamster hepatocytes and in two of three preparations of guinea pig cells, MeIQ produced statistically significant (p less than 0.05) repair responses. IQ stimulated significant levels of repair in all three rat hepatocyte preparations and in two of three hamster cell preparations. In guinea pig cells exposed to IQ, no significant repair activity was observed. These results indicate that the genotoxicity of IQ and MeIQ in hepatic cells in species-dependent.     

Effect of pretreatment with hepatic mixed-function oxidase inducers on the genotoxicity of four rat carcinogens in the hepatocyte/DNA repair assay

Four genotoxic rat carcinogens, previously reported as negative in the standard hepatocyte/DNA repair assay, were studied in hepatocyte cultures derived from the livers of rats pretreated with the hepatic mixed-function oxidase (MFO) inducers, Aroclor (ARO) and 3-methylcholanthrene (3-MC). Cytembena and dimethyl hydrogen phosphite induced positive dose-dependent DNA repair responses in ARO- and 3-MC-pretreated cultures, while 2,4-, 2,6-toluene diisocyanate, and ziram were negative for unscheduled DNA synthesis in cultures from both pretreatments. The results from this study indicate that pretreatment with hepatic MFO inducers lowers the false-negative detection rate of various genotoxic rat carcinogens and other organ-specific genotoxins and significantly increases the sensitivity of the in vitro rat hepatocyte/DNA repair assay.     

Ultraviolet light-induced DNA repair synthesis in hepatocytes from species of differing longevities

The DNA repair capabilities of cultured hepatocytes derived from five mammalian species in response to a broad range of ultraviolet light exposures were measured. Differences in the induction of DNA repair synthesis and the proportion of responding cells were noted only for the lowest fluences. These differences appeared to be positively correlated with the potential lifespans of the species involved; mouse and rat hepatocytes displayed less repair than those from guinea pig or rabbit. At higher fluence levels, however, there were no differences in the amount of repair induced. Thus the maximal repair potential of hepatocytes derived from the rat, mouse, hamster, guinea pig and rabbit were the same.     

The effect of acrylamide on hepatocellular DNA repair

Acrylamide has recently been reported to induce tumors in laboratory animals. The effect of acrylamide on unscheduled DNA synthesis using the hepatocyte primary culture (HPC)/DNA repair test was examined. Isolated hepatocytes were exposed to acrylamide and [3H]thymidine ( [3H]TdR) for 18 hr. Incorporation of [3H]TdR into DNA was determined by autoradiography. No DNA repair was observed at acrylamide concentrations up to 10(-2) M. These findings were confirmed using density gradients. Acrylamide concentrations exceeding 10(-2) M were cytotoxic to hepatocytes. Because both autoradiography and density gradients measure DNA repair as an endpoint, the ability of acrylamide to inhibit these repair processes was also determined. Acrylamide had no effect on the repair of UV-damaged DNA. These results show that acrylamide is not genotoxic in isolated hepatocytes.     

Sex and strain differences in the hepatocyte primary culture/DNA repair test

The hepatocyte primary culture (HPC)/DNA repair test was developed using hepatocytes isolated from male F-344 rats. A number of genetic polymorphisms have been shown to occur in inbred strains of rats, which may lead to variation in biotransformation of xenobiotics resulting in differences in susceptibility to genotoxins. The effect of the strain utilized as a source of hepatocytes was investigated with female Lewis, F-344, and DA rats. Variation was observed when hepatocytes from the three strains were exposed to aflatoxin B₁ (AFB₁). Hepatocytes from female Lewis rats were more sensitive to AFB₁ genotoxicity than cells from F-344 or DA rats. DNA repair was induced by a concentration as low as 10⁸ M AFB ₁ in the Lewis strain while 10⁴ M was needed for hepatocytes from female F-344 or DA rats. Sex-related differences were also observed with AFB₁; DNA repair was induced at 10⁸ M in hepatocytes from male F-344 rats. No clearcut strain differences were seen when cells were exposed to diethylnitrosamine (DEN) or 2-acetylaminofluorene, although 10² M DEN was only cytotoxic to hepatocytes from DA rats. These results demonstrate that both the strain and the sex of the animal used as a source of hepatocytes can affect the HPC/DNA repair test.     

Evaluation of the genotoxic potential of glutaraldehyde

The cytotoxic and genotoxic effects of glutaraldehyde were studied in vitro in the human TK6 lymphoblast cell line and in primary cultures of rat hepatocytes. TK6 lymphoblasts were exposed to glutaraldehyde for 2 hr in serum-free GSH-free media. Cytotoxic effects were observed at concentrations as low as 10 microM with only 10% cell survival at 20 microM. Alkaline elution studies indicated that glutaraldehyde-induced DNA-protein crosslinking increased linearly over the concentration range from 0 to 25 microM. Glutaraldehyde-induced mutations were assessed at the thymidine kinase locus over the same concentration range and reached a plateau at 10 microM of about six times the background mutant frequency. At equivalent levels of DNA-protein crosslinks and cytolethality, glutaraldehyde was mutagenic at approximately a one-seventh lower concentration than the rodent nasal carcinogen formaldehyde (Craft et al.; Mutation Research 176:147-155, 1987). Glutaraldehyde induced a marginal increase in unscheduled DNA synthesis in the in vitro hepatocyte DNA repair assay, but only at the two highest concentrations of 50 and 100 microM, indicating the induction of some DNA excision-repair activity. These data demonstrate that glutaraldehyde exhibits DNA-reactive genotoxic activity that may involve, at least in part, DNA-protein crosslinking in these cell culture models. These findings suggest the need to examine the potential carcinogenic activity of glutaraldehyde in appropriate inhalation studies.     

Species and sex differences in genotoxicity of heterocyclic amine pyrolysis and cooking products in the hepatocyte primary culture/DNA repair test using rat, mouse, and hamster hepatocytes

Eleven mutagenic heterocyclic amines, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]-indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido[4,3]indole (Trp-P-2), 2-amino-6-methyl-dipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1), 2-aminodipyrido[1,2-a:3',2'-d]imidazole (Glu-P-2), 2-amino-9H-pyrido[2,3-b]indole (A alpha C), 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeA alpha C), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethylimidazo [4,5-f]quinoline (MeIQX), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-diMeIQX), and 2-amino-3,7,8-trimethylimidazo[4,5-f]quinoxaline (7,8-diMeIQX), were studied for genotoxicity in the hepatocyte/DNA repair test employing hepatocytes of male rats, male and female mice, and male hamsters. In these four assay systems, all compounds elicited DNA repair in at least three systems, except Trp-P-2, which was uniformly inactive. However, there were several significant differences in the responses of different systems. Rat and hamster hepatocytes responded to nine of the ten genotoxic compounds with the exception of Glu-P-2. Male and female mouse hepatocytes responded to Glu-P-2, whereas female, but not male, mouse hepatocytes responded to MeIQX and 4,8-diMeIQX. These results illustrate species and sex differences in response to these heterocyclic amines and suggest that a number of these compounds are carcinogenic in hamsters, as they have been in rats and mice.     

Testing of 24 food,drug, cosmetic,and fabric dyes in the in vitro and the in vivo/in vitro rat hepatocyte primary culture DNA repair assays

Twenty-four dyes currently or previously used in the food, drug, cosmetic, and textile industries were tested in the in vitro rat hepatocyte primary culture/DNA repair (HPC/DR) assay and, to a limited extent, in the in vivo/in vitro HPC/DR assay. The positive control, Solvent Yellow 3 (o-aminoazotoluene), and five other dyes (4-dimethylaminobenzeneazo-1 -naphthalene, 4-dimethylaminobenzeneazo-2-naphthalene, Direct Blue 53, Acid Blue 9, and 4-dimethylaminostilbene) induced DNA repair in rat hepatocytes both in vitro and in vivo, while 13 of the dyes (Food Red 1, Food Red 5, Food Orange 4, Food Red 7, Acid Red 14, Acid Red 27, Pigment Red 53, Acid Yellow 23, Food Black 1, Food Green 3, Acid Red 51, Acid Blue 74, and Natural Red 4) did not produce any detectable DNA repair in either the in vitro or in vivo/in vitro assays. Direct Blue 14 had weak activity in vitro but none was detected in vivo. In contrast. Solvent Yellow 5 was not active in vitro, but produced a weak positive response in vivo. Negative responses were also obtained for Solvent Yellow 14 and Acid Green 5 in the in vitro assay, whereas the responses produced by these dyes in the in vivo/in vitro assay were judged to be equivocal. An equivocal response was also obtained for Direct Red 28 in the in vivo/in vitro assay as well as in the in vitro assay. These findings provide information about the potential genotoxicity of a number of dyes for which previous genotoxicity data has been inconsistent or inadequate. For some dyes (eg, Solvent Yellow 5), discrepancies between the results obtained in the in vitro and in vivo/in vitro assays may implicate a role for intestinal microflora in their metabolic activation.     

Tissue-specific genotoxic effects of acrylamide and acrylonitrile.

Acrylamide (AA) has been reported to induce dominant lethal mutations in male rat germ cells and tumors in a variety of organs, including the scrotum, thyroid and mammary glands, but not the liver of rats. The structurally similar vinyl monomer acrylonitrile (ACN) does not induce dominant lethal mutations but does induce tumors of the brain, Zymbal gland, forestomach and mammary gland, but not the liver of rats. Several in vitro and/or in vivo unscheduled DNA synthesis (UDS) assays were employed to examine the potential tissue-specific genotoxic activity of these compounds. Neither AA nor ACN induced DNA repair in either the in vitro or in vivo hepatocyte DNA repair assays. Glycidamide (GA), a mutagenic metabolite of AA, induced DNA repair in the in vitro hepatocyte DNA repair assay. Cyanoethylene oxide (CEO), a mutagenic metabolite of ACN, did not yield a DNA repair response in the in vitro hepatocyte DNA repair assay, but was highly toxic and could not be tested at doses equivalent to GA. AA, but not ACN, produced a DNA repair response in the in vivo spermatocyte DNA repair assay. AA produced a slight response in the in vitro human mammary epithelial cell (HMEC) DNA repair assay in normal cells derived from discarded surgical samples from five different women. GA produced a strong UDS response in all cases in the same assay. CEO, but not its parent compound ACN, produced a response in the HMEC DNA repair assay. These results show a highly tissue-specific pattern of genotoxic activity for AA and ACN that correlates, to the extent that it has been examined, with the tissue-specific pattern of carcinogenic and dominant lethal activity. The induction of DNA repair by GA and CEO confirms the genotoxic potential of these metabolites. While the observation of genotoxic activity of AA in the HMEC DNA repair assay suggests that mammary cells might be a target for carcinogenic activity of this compound in humans, other factors such as pharmacokinetics and epidemiology must be evaluated to establish that effect.     

Genotoxicity testing of potassium canrenoate in cultured rat and human cells

Potassium canrenoate (PC), a competitive aldosterone antagonistused as a diuretic and in the treatment of hypertension, wasexamined for its capacity to produce genotoxic effects in culturedrat and human cells. At subtoxic concentrations (10–90µM) PC was found to induce a dose-dependent degree ofDNA fragmentation, as detected by the Comet assay, and of DNArepair synthesis, as measured by quantitative autoradiography,in primary cultures of hepatocytes from rat and human donorsof both genders. In rat hepatocytes both DNA fragmentation andDNA repair were more marked after 3 h than after 20 h exposureand in cultures from females than from males. In human hepatocytesfrom one male and two female donors, PC caused a similar effectin terms of DNA fragmentation, whereas DNA repair was detectedin cultures from only two of the same three donors and was lessmarked than in rat hepatocytes. A modest but statistically significantincrease in micronucleated cells was present in primary culturesof replicating rat hepatocytes exposed to 10 or 30 µMPC for 48 h, the response being, in this case also, more evidentin females than in males. In contrast, PC did not induce micronucleusformation in human hepatocytes from two female donors. Any evidenceof DNA fragmentation and micronucleus formation was absent incultured human lymphocytes. Taken as a whole these findingssupport the hypothesis that hepatocytes activate PC to DNA-damagingreactive species. PC induced the observed genotoxic effectsat concentrations close to those produced in humans by the administrationof therapeutic doses, but these effects were as a whole moremarked in rat than in human hepatocytes. Since PC shares the17-hydroxy-3-oxopregna-4,6-diene structure with cyproteroneacetate, chlormadinone acetate and megestrol acetate, previouslyfound to be genotoxic to both rat and human hepatocytes, thepotential carcinogenic hazard of this type of steroids cannotbe neglected. ¹ To whom correspondence should be addressed. Tel: +39 010 3538800; Fax: +39 010 538232; Email: farmdimi{at}unige.it     

Employment of adult mammalian primary cells in toxicology: in vivo and in vitro genotoxic effects of environmentally significant N-nitrosodialkylamines in cells of the liver, lung, and kidney

This report focuses on the use of freshly isolated primary mammalian cells from different tissues and organs of the rat for the rapid and efficient analysis of toxic and genotoxic chemicals. The cells are either treated in vitro or they are isolated from treated animals. Viability by trypan blue exclusion and DNA damage as single-strand breaks are monitored in either case. Therefore, it is possible to compare in vitro and in vivo results directly. N-nitrosamines with unique organ-specific modes in carcinogenesis were studied in vitro using hepatocytes derived from three species (rat, hamster, and pig) and in rat lung and kidney cells. The sensitive detection of all carcinogenic nitrosamines was achieved, although a pattern of cell-specific activation was not observable. The new modification of the in vivo approach allowed the sensitive detection of NDMA genotoxicity in hepatic and in extrahepatic tissues. It is important to point out that the method is an efficient tool for toxicokinetic studies with genotoxic carcinogens in vivo.     

Detection of genotoxic carcinogens in the in vivo-in vitro hepatocyte dna repair assay

The in vivo-in vitro hepatocyte DNA repair assay has been shown to be useful in the evaluation of the carcinogenic potential of chemicals. The purpose of this study was to apply this assay to determining the genotoxicity of compounds from a wide variety of structural classes. Male Fischer-344 rats were treated by gavage or ip injection with compounds dissolved in either corn oil, water, or dimethyl sulfoxide (DMSO). At selected times after treatment, hepatocytes were isolated by liver perfusion and cultured with ³H-thymidine. Unscheduled DNA synthesis (UDS) was measured by quantitative autoradiography as net grains/nucleus (NG); ≥ 5 NG was considered positive. Water, corn oil, or DMSO controls produced -3 to -6 NG with ≤ 6% of the cells in repair. All genotoxic hepatocarcinogens tested produced strong positive responses of > 15 NG including dimethylnitrosamine, 2-acetylaminofluorene (2-AAF), azoxymethane, 1,2-dimethylhydrazine, benzidine, aflatoxin B₁, 2,6-dinitrotoluene, and 2,4-diaminotoluene. The noncarcinogen, 2,6-diaminotoluene, was negative. The mutagen and rat brain carcinogen methyl methanesulfonate (MMS) and the rat pancreatic carcinogen azaserine were also positive. The carcinogens benzo(a)pyrene and 7,12-dimethylbenzen(a)anthracene yielded from -2 to -4 NG. This negative response is consistent with their lack of carcinogenic activity in rat liver. MMS produced the greatest amount of UDS 2 hr after treatment whereas 2-AAF did not induce its maximum response until 12 hr post-treatment. The potent hepatotoxin carbon tetrachloride induced a 40-fold elevation in DNA replication 48 hr after a 400 mg/kg dose, but no UDS was observed at 2, 12, 24, or 48 hr post-treatment. The weak hepatocarcinogen safrole induced no UDS suggesting that is is either nongenotoxic or is metabolized to an active form at an extremely slow rate following a single administration. These results demonstrate that this assay is valuable for the detection and study of a variety of genotoxic carcinogens.     

Genotoxic effects of {alpha}-hexachlorocyclohexane in primary cultures of rodent and human hepatocytes

The genotoxicity of -hexachlorocyclohexane (-HCH) was evaluatedin primary cultures of mouse, rat and human hepatocytes. DNAfragmentation was measured by the alkaline elution techniqueand DNA repair synthesis by quantitative autoradiography. A20 h exposure to subtoxic concentrations ranging from 0.056to 0.32 mM produced a dose-dependent frequency of DNA breaksin rat hepatocytes and in hepatocytes from four of five humandonors, but not in mouse hepatocytes. DNA repair induction wasabsent in hepatocytes from all three species. The reductionin the frequency of DNA breaks observed in rat hepatocytes simultaneouslyexposed to metyrapone suggests that -HCH is transformed intoreactive species by a cytochrome P450-dependent reaction. Thedetection of DNA fragmentation but not of DNA repair synthesismay be tentatively explained by assuming that -HCH behaves asa chemical eliciting short patch DNA repair, which is more easilyrevealed as genotoxic by the occurrence of DNA singlestrandbreaks. ³To whom correspondence should be addressed     

Nitroreduction is not involved in the genotoxicity of 2-nitropropane in cultured mammalian cells

We have investigated the importance of nitroreduction for the genotoxicity of the carcinogen 2-nitropropane (2-NP) in primary cultures of rat hepatocytes and in V79 Chinese hamster cells. Induction of DNA repair synthesis was used as an indicator of genotoxic effects in hepatocytes. Genotoxicity in V79 cells was determined as induction of DNA repair, micronuclei and mutations to 6-thioguanine (TG) resistance. Both hepatocytes and V79 cells were found capable of reducing and oxidizing 2-NP. Reduction of 2-NP was indicated by the formation of acetone oxime, the tautomeric form of 2-nitrosopropane, the first reduction product of 2-NP. Oxidation of 2-NP was indicated by the production of acetone and nitrite. 2-NP strongly elicited repair in hepatocytes, but acetone oxime and the products of a possible further nitroreduction, isopropyl hydroxylamine (IPHA) and 2-aminopropane did not. None of the reduction products caused repair synthesis in V79 cells. However, in these cells IPHA and 2-NP increased the frequency of TG-resistant mutants. IPHA also markedly induced micronuclei. This was not seen with 2-NP. Acetone oxime was not genotoxic in V79 cells. The observations suggest that reduced metabolites are responsible neither for the induction of DNA repair synthesis by 2-NP in hepatocytes nor for the induction of gene mutations by 2-NP in V79 cells.