The genotoxicity of lucidin,a natural component of Rubia tinctorum L., and lucidinethylether,a component of ethanolic Rubia extracts

The genotoxic activity of lucidin (1,3-dihydroxy-2-hydroxymethyl-9,10-anthraquinone), a natural component of Rubia tinctorum L., was tested in a battery of short-term tests. The compound was mutagenic in five Salmonella typhimurium strains without metabolic activation, but the mutagenicity was increased after addition of rat liver S9 mix. In V79 cells, lucidin was mutagenic at the hypoxanthine-guanine phosphoribosyl transferase gene locus and active at inducing DNA single-strand breaks and DNA protein cross-links as assayed by the alkaline elution method. Lucidin also induced DNA repair synthesis in primary rat hepatocytes and transformed C3HI M2-mouse fibroblasts in culture. We also investigated lucidinethylether, which is formed from lucidin by extraction of madder roots with boiling ethanol. This compound was also mutagenic in Salmonella, but only after addition of rat liver S9 mix. Lucidinethylether was weakly mutagenic to V79 cells which were cocultivated with rat hepatocytes. The compound did not induce DNA repair synthesis in hepatocytes from untreated rats, but positive results were obtained when hepatocytes from rats pretreated with phenobarbital were used. We conclude that lucidin and its derivatives are genotoxic.Abbreviations DMBA 7,12-dimethylbenz(a)anthracene - HA hydroxyanthraquinones - LUE lucidinethylether - PRH primary rat hepatocytes - UDS unscheduled DNA synthesis     

Genetic toxicity of the benzene metabolite trans, trans-muconaldehyde in mammalian and bacterial cells

Previous studies in our laboratory identified trans,trans-muconaldehyde (MUC), a six-carbon diene dialdehyde, as a microsomal metabolite of benzene. This ring-opened metabolite of benzene was also shown to be hematotoxic in mice in a manner similar to benzene. To further explore the role of MUC in relation to benzene toxicity, a number of test systems were utilized to determine its genotoxic potential. In B6C3F1 mice, MUC induced a highly significant increase in sister-chromatid exchange (SCE), the lowest effective dose being 3 mg/kg, but failed to induce any micronuclei (MN). In Chinese hamster ovary (CHO) cells, MUC at concentrations up to 0.8 micrograms/ml was negative in the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) assay. Dose-related increases in the percentage of cells with MN were observed in CHO cells treated with 0.4-0.8 micrograms/ml MUC. MUC did not-cause unscheduled DNA synthesis in rat primary hepatocytes. Treatment of Salmonella typhimurium TA97 with MUC induced a low level of mutations at concentrations ranging from 10 to 70 micrograms/ml with or without S9 activation. MUC was inactive in strains TA1535, TA100, TA1538 and TA98. In CHO cells and rat primary hepatocytes, MUC was cytotoxic at 0.4 and 4.0 micrograms/ml, respectively. Concentrations of 100 micrograms/plate MUC were toxic for bacterial cells. The present findings indicate that MUC is nonmutagenic or minimally mutagenic in bacterial and mammalian in vitro systems. In mammalian cells, MUC is highly cytotoxic and genotoxic.     

Lack of DNA damage induction by okadaic acid, a marine toxin, in the CHO-Hprt and the in vitro UDS assays

Okadaic acid (OA) is a marine toxin produced by dinoflagellates and responsible for human intoxications. OA is a specific inhibitor of serine/threonine protein phosphatases PP1 and PP2A and a potent tumor promoter in mouse skin and rat glandular stomach. In a previous study, we demonstrated that OA induced aneuploidy in CHO-K1 cells using the cytokinesis-block micronucleus (CBMN) assay coupled to FISH and concluded that OA was not a direct mutagen. As some previous in vitro mutagenicity studies had given positive results with OA, we decided to perform two additional in vitro mutagenicity assays in accordance with the OECD guidelines: (i) the CHO/Hprt test, which provides end points about locus-specific gene mutation; (ii) the in vitro unscheduled DNA synthesis (UDS) assay in rat hepatocytes, which measures [(3)H]thymidine incorporation into DNA undergoing excision repair. In the CHO/Hprt assay, there was no significant increase in the number of mutants for doses ranging from 5 to 5000 nM in the presence or absence of rat liver S9 fraction. In the in vitro UDS assay, OA did not induce primary DNA damages in rat hepatocytes following 18 h exposure at concentrations between 1.32 and 100 nM. As OA could affect the DNA repair systems via the inhibition of protein phosphatases, its effects on the repair kinetic of 2AAF-induced DNA damage were also investigated with the UDS assay. The results showed that OA did not interact with the DNA-repair process involved in in vitro UDS in rat hepatocytes. We concluded that OA failed to induce direct DNA damage but acted principally by altering the chromosome number, which could contribute to its carcinogenic effect.     

Unscheduled DNA synthesis in rat tracheal epithelial cells, hepatocytes and spermatocytes following exposure to methyl chloride in vitro and in vivo

Measurement of DNA repair as unscheduled DNA synthesis (UDS) in vitro following exposure in vivo in multiple tissues from the same treated animal can provide valuable information relating to the tissue- and organ-specificity of chemically induced DNA damage. UDS was evaluated in primary cultures of rat tracheal epithelial cells, hepatocytes and pachytene spermatocytes after exposure in vitro to methyl chloride (MeCl), and after isolation from the same treated animal following inhalation exposure in vivo. Concentrations of 1-10% MeCl in vitro induced UDS in hepatocytes and spermatocytes, but not in tracheal epithelial cells. Inhalation exposure to MeCl in vivo (3000-3500 ppm 6 h/day for 5 successive days) failed to induce DNA repair in any cell type. In vivo exposure to 15 000 ppm MeCl for 3 h also failed to induce UDS in tracheal epithelial cells and spermatocytes, but did cause a marginal increase in UDS in hepatocytes. Thus, MeCl appears to be a weak, direct-acting genotoxicant. While activity could be measured in hepatocytes and spermatocytes directly in vitro, only extremely high concentrations of MeCl elicited a response in the whole animal, and then only in hepatocytes.     

Induction of unscheduled DNA synthesis in primary rat hepatocytes by benzidine-congener-derived azo dyes in the in vitro and in vivo/in vitro assays

The genotoxicity of the benzidine-congener-derived azo dyes. Direct Blue 1 ( DB1 ), Direct Blue 14 ( DB14 ), Direct Brown 95 ( DB95 ), and Direct Red 46 ( DR46 ) was studied in the in vitro and in vivo/in vitro unscheduled DNA synthesis (UDS) assays in primary rat hepatocytes to determine if in vivo metabolism of these compounds was required for induction of UDS. Hepatocytes were isolated, cultured, and treated with the azo dyes and [3H]thymidine (in vitro assay); alternatively, in the in vivo/in vitro assay, rats were intubated with the azo dyes, the hepatocytes isolated at 17 h after dosing and incubated in a medium containing [3H]thymidine. UDS was quantified by an autoradiographic method. None of the azo dyes induced UDS in the in vitro assay. However, DR46 did induce marginal, but significant UDS in 1 experiment (1.2 net grains at 500 micrograms/ml media). No significant UDS was observed when DR46 was tested in a subsequent in vitro assay. In the in vivo/in vitro assay, DB95 (100 mg/kg), DB14 (125 mg/kg), and DR46 (100 mg/kg) induced significant UDS (12, 2.1, and 3.5 net grains, respectively). None of the azo dyes tested was mutagenic in the Salmonella/microsome assay in the presence and absence of rat liver enzymes. Therefore, in vivo reduction of azo dyes, presumably by the gut microflora, is a requirement for the genotoxicity of these azo dyes in the primary rat hepatocyte UDS assay.     

Activities of chlorinated ethane and ethylene compounds in the Salmonella/rat microsome mutagenesis and rat hepatocyte/DNA repair assays under vapor phase exposure conditions

Three chlorinated ethane and ethylene solvent products were examined for their genotoxicity in the Salmonella/microsome mutagenesis and hepatocyte primary culture DNA repair assays using vapor phase exposures. The positive control in this study, monochloroethylene (vinyl chloride), induced reversion mutation of Salmonella tester strains TA100 and TA1535 with enhancement by an exogenous activation system and elicited unscheduled DNA synthesis in rat hepatocytes in culture. Exposures to 1,1,1-trichloroethane (methyl chloroform) or 1,1,2-trichloroethylene samples which contained stabilizers resulted in increased recovery of revertant colonies of Salmonella at concentrations causing greater than 96% cell killing. However, these stabilized materials did not induce DNA repair and low-stabilized trichloroethylene did not induce reversion mutation or DNA repair. Exposure of Salmonella tester strains and hepatocytes to highly toxic vapor concentrations of technical grade 1,1,2,2-tetrochloroethylene, low-stabilized and stabilized, increased reversion mutation and elicited DNA repair. Tetrachloroethylene of high purity was not genotoxic. With all of these test products, the presence of an Aroclor-induced rat liver subcellular enzyme preparation in the mutagenesis assay did not have any effect on the results. These observations suggest that stabilizers or unknown impurities normally present at low concentrations in these products are responsible for the positive responses observed at the high exposure concentrations achievable under in vitro test conditions.Abbreviation HPC hepatocyte primary culture     

Induction of DNA repair in human and rat hepatocytes by 1,6-dinitropyrene

1,6-Dinitropyrene (DNP) was found to be an extremely potent genotoxicant in metabolically competent primary cultures of human and rat hepatocytes. Dose-dependent increases in DNA repair as measured by unscheduled DNA synthesis (UDS) were observed in the range from 0.05 to 5 microM 1,6-DNP for both species, indicating that the rat-hepatocyte assay is an appropriate model for assessing genotoxic potential in human hepatocytes for this class of compound. Unlike some nitroaromatic compounds, 1,6-DNP did not require gut flora for metabolic activation. No DNA repair was observed in hepatocytes isolated from rats treated with 50 mg/kg 1,6-DNP in corn oil by gavage 2, 12 or 24 h previously. The reason for the lack of a response in vivo is not known, but may relate to detoxification or distribution of the compound in the animal.     

DNA-repair studies with sodium fluoride: comparative evaluation using density gradient ultracentrifugation and autoradiography

Sodium fluoride (NaF) was assayed for the induction of DNA-repair synthesis in WI-38 human diploid fibroblasts and in primary cultures of rat hepatocytes. DNA-repair synthesis in non-replicating DNA was measured by ultracentrifugation of density-labeled DNA in CsCl gradients. When this method was used, NaF did not induce DNA-repair synthesis in either of these cell types. However, when NaF was assayed for induction of unscheduled DNA synthesis (UDS) in rat hepatocytes by autoradiography, an increased net nuclear grain count was observed. Because the autoradiographic results were not confirmed by density-gradient ultracentrifugation of hepatocyte DNA, which is a more definitive technique, it is doubtful whether the autoradiographic results actually represent DNA-repair synthesis. Modifications of the UDS/autoradiography protocol to include more extensive washing resulted in no UDS response. Published reports (Hellung-Larsen and Klenow, 1969; Srivastava et al., 1981) describe the formation of precipitable complexes of Mg2+, F-, and [3H]thymidine triphosphate which suggests that autoradiographic measurement of UDS may lead to artifacts when testing NaF unless extensive washing of the cultures is employed.     

Genotoxic activity of extractable organic matter from urban airborne particles in Shanghai, China

The aim of this research is to investigate the impact of air pollution on the population in Shanghai. The genotoxicity of extractable organic matter (EOM) from the air particles was investigated by the means of the Salmonella plate incorporation assay, rat hepatocyte unscheduled DNA repair assay, and mice micronuclei test. The airborne particles were collected in 13 locations during the summer of 1992 and winter of 1993. The crude extracts were fractionated by acid-base partitioning into acid, base and neutral fractions. The neutral fractions were further fractionated by resin-silica gel column chromatography into three subfractions. The induction of revertants with the crude extracts was higher in winter samples than in summer samples. Both indirect-acting and direct-acting mutagenicity were observed. The mutagenicity was detected with TA98, but was not detected with TA100. The mutagenic activity was the greatest in the acid, aromatic and polar fractions from summer samples. The fractions from the winter samples did not show clear differences. There was no substantial location-related variance in the mutagenic potencies of EOM, but substantial location- or time-related variances in the mutagenic potencies of the airborne particles per cubic meter air were found. While rat hepatocyte unscheduled DNA synthesis (UDS) assay revealed genotoxicity for all the samples, there was no big variance in the genotoxicity of the fractions. The mouse micronuclei test showed results similar to the UDS assay. The difference of locality did not have statistical significance.     

Strain differences in in vitro rat hepatocyte unscheduled DNA synthesis (UDS): effect of UV is independent of strain while increased sensitivity is apparent using Fischer-344 instead of Sprague-Dawley rats

The unscheduled DNA synthesis (UDS) assay measures DNA repair following in vitro treatment of rat primary hepatocytes. This report compares the UDS response of primary hepatocytes from 2 widely used rat strains, the Fischer-344 (F344) and Sprague-Dawley (SD) strains. Ultraviolet (UV) light and 5 known genotoxic chemicals were evaluated in each strain in parallel experiments. The chemicals tested were 2-acetylaminofluorene (2-AAF), 4-aminobiphenyl (4-AB), benzidine, dimethylnitrosamine (DMN) and N-propyl-N'-nitro-N-nitrosoguanidine (PNNG). Four of these compounds (2-AAF, 4-AB, benzidine and DMN) require metabolic activation. Benzidine and PNNG were both negative using SD rat hepatocytes, but were weakly positive using F344 rat hepatocytes. In the first of 2 experiments, 4-AB was inconclusive in SD hepatocytes, but strongly positive in F344 cells. In the second experiment, 4-AB was positive in hepatocytes from both strains. 2-AAF was more strongly positive in F344 cells than in SD cells. DMN and UV light induced positive dose responses with little or no differences between strains. It is concluded that hepatocytes from F344 rats may be more sensitive, qualitatively and quantitatively, than hepatocytes from SD rats as indicators of UDS. This difference is not due to intrinsic differences in DNA repair mechanisms but is probably due to differences in drug-metabolizing enzymes between these strains. Thus, for routine screening, F344 rats are preferable for measurement of the in vitro UDS-inducing potential of compounds.     

Synthesis and genotoxicity of acetoxyoxirane, the epoxide of vinyl acetate

Acetoxyoxirane, the epoxide of vinyl acetate and a potential reactive intermediate, was synthesized and characterized by 13C-nuclear magnetic resonance (13C-NMR) and mass spectroscopy. The compound induced lesions (endonuclease-sensitive and alkali-labile sites) in supercoiled PM2 DNA in vitro and was directly mutagenic toward Salmonella typhimurium TA100. The mutagenicity of the epoxide in phosphate buffer (pH 7.4, 37 degrees C) decreased, with an initial half-life of 2.8 minutes, and mutagenicity was completely abolished by addition of S-9 mix. Acetoxyoxirane did not induce unscheduled DNA synthesis on incubation with Syrian hamster embryo fibroblasts (SHE cells). These findings may possibly be explained by an effective inactivation of acetoxyoxirane by esterases when these are present in the biological system. This view is consistent with the lack of acetoxyoxirane detected in rat liver microsomal incubations of vinyl acetate.     

Bacterial mutagenesis and hepatocyte unscheduled DNA synthesis induced by chrysoidine azo-dye components

5 azo dye components of Gurr chrysoidine 'Y' have been separated, synthesised and identified. Dyes with a methyl substitution (particularly between the two amino groups) were more mutagenic in Salmonella typhimurium strain TA100 with control rat liver S9 than the non-methylated counterpart (range 66-1992 revertants at 50 micrograms/plate). Mutagenicity was also catalysed by human-liver S9 and pre-treatment of rats with either phenobarbitone or beta-naphthoflavone enhanced the activation ability of S9 by greater than 4-fold. Using the most potent promutagenic component (2,4-diamino-3-methylazobenzene), the use of inhibitors of cytochrome P450 (metyrapone: 1.0 mM; alpha-naphthoflavone: 0.075 mM; DPEA: 0.125 mM) and of the flavin monooxygenase (methimazole: 0.75 mM) suggested a major role for cytochrome P448 in the activation of chrysoidine to mutagens. The ability of chrysoidine components to induce unscheduled DNA synthesis in rat hepatocytes in vitro was demonstrated and ranged between 11.92 and 23.5 net nuclear grains at a dose level of 2.5 micrograms/incubation. Since each dye was equi-potent, methyl substitution had little influence on genetic toxicity in hepatocytes.     

Unscheduled DNA synthesis in human hair follicles after in vitro exposure to 11 chemicals: comparison with unscheduled DNA synthesis in rat hepatocytes.

A new method is described to investigate unscheduled DNA synthesis (UDS) in human tissue after exposure in vitro: the human hair follicle. A histological technique was applied to assess cytotoxicity and UDS in the same hair follicle cells. UDS induction was examined for 11 chemicals and the results were compared with literature findings for UDS in rat hepatocytes. Most chemicals inducing UDS in rat hepatocytes raised DNA repair at comparable concentrations in the hair follicle. However, 1 of 9 chemicals that gave a positive response in the rat hepatocyte UDS test, 2-acetylaminofluorene, failed to induce DNA repair in the hair follicle. Metabolizing potential of hair follicle cells was shown in experiments with indirectly acting compounds, i.e., benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene and dimethylnitrosamine. The results support the conclusion that the test in its present state is valuable as a screening assay for the detection of unscheduled DNA synthesis. Moreover, the use of human tissues may result in a better extrapolation to man.     

Comparative genotoxicity studies of the flame retardant tris(2,3-dibromopropyl)phosphate and possible metabolites

Tris(2,3-dibromopropyl)phosphate (Tris-BP) was activated to mutagens in the Salmonella/microsome quantitative test system. Liver microsomes from rats pretreated with phenobarbital (PB) increased the mutagenicity of 0.05 mM Tris-BP to 186% of the activity obtained with liver microsomes from untreated rats. The addition of 0.02 mM Tris-BP to V79 Chinese hamster cells co-incubated with liver microsomes from PB-pretreated rats increased the number of mutants by a factor of 9.7. Tris-BP also caused genotoxic and cytotoxic responses in primary monolayers of rat hepatocytes. The relative increase in unscheduled DNA synthesis after treatment with 0.05 mM Tris-BP was 2.3-fold as measured by scintillation counting of radiolabelled thymidine incorporated into DNA of isolated nuclei. The use of hepatocytes isolated from PB-pretreated rats reduced the increases in DNA repair synthesis relatively to that in control cells. Monolayers of hepatocytes from untreated rats co-cultured with Salmonella typhimurium TA100 activated Tris-BP to mutagenic intermediates which were released into the culture medium. The studies with the V79 and liver-cell systems indicate that the reactive intermediates formed from Tris-BP are sufficiently stable and lipophilic to traverse the various membranes from the site of generation to the respective cellular targets. The relative degree of genotoxic responses of bis(2,3-dibromopropyl)phosphate, 2,3-dibromopropylphosphate, tris(2,3-bromopropyl)phosphate, tris(2-bromopropyl)phosphate and 2,3-dibromopropanol in the systems studied did not indicate that these compounds were proximate or ultimate reactive metabolites of Tris-BP in liver-derived activation systems.     

Stimulation of DNA synthesis but not of peroxisomal beta-oxidation by nafenopin in primary cultures of marmoset hepatocytes

The effects of the peroxisome proliferator nafenopin upon primary cultures of marmoset hepatocytes have been investigated and compared to those on cultured rat hepatocytes. Nafenopin did not induce peroxisomal beta-oxidation or peroxisome proliferation but did induce replicative DNA synthesis. These findings demonstrate that peroxisome proliferation and mitogenicity are two independent properties of nafenopin and question the widely held view that primates are generally insensitive to the effects of peroxisome proliferators.     

Mutagenicity of methyl bromide in a series of short-term tests

Methyl bromide is commonly used as a soil fumigant in greenhouses. In the framework of a toxicological evaluation, it was tested for possible genotoxic properties in two bacterial test systems (the fluctuation test using Klebsiella pneumoniae and the plate test using Salmonella typhimurium TA100 and TA98), two systems using mammalian cells in vitro (forward mutations at the TK and HPRT loci in L5178Y mouse lymphoma cells and unscheduled DNA synthesis in primary rat-liver cells) and in the sex-linked recessive lethal test using Drosophila melanogaster. Methyl bromide was active in all tests except the DNA-repair assay. The results indicate a relatively low mutagenic efficiency of the compound, as expected from its alkylating properties.     

Mechanism of stimulation of DNA synthesis induced by epinephrine in primary culture of adult rat hepatocytes

Addition of epinephrine to primary cultured adult rat hepatocytes stimulated their DNA synthesis dose-dependently, especially in presence of insulin and epidermal growth factor. This effect of epinephrine was strongly inhibited by an alpha 1-antagonist, prazosin, but not by a beta-antagonist, propranolol, and was also slightly inhibited by an alpha 2-antagonist, yohinbin. These results indicate that the stimulation of DNA synthesis of hepatocytes by epinephrine is mediated predominantly by an alpha 1-action. 12-o-Tetradecanoylphorbol-13-acetate (TPA) or Ca2+-ionophore A-23187 stimulated DNA synthesis of Swiss 3T3 cells, but did not induce DNA synthesis of hepatocytes either singly or in combination. The fact that pretreatment of hepatocytes with TPA caused down-regulation of the stimulatory effect of epinephrine on DNA synthesis of hepatocytes within 15 min suggested that the effect of epinephrine on hepatocytes is mediated by its alpha 1 receptor and that TPA activated protein kinase c in the hepatocytes. Addition of dibutyryl cGMP did not induce DNA synthesis of hepatocytes. Therefore, the alpha 1-action of epinephrine that induce stimulation of DNA synthesis of primary cultured adult rat hepatocytes was apparently not mediated by either activation of phospholipid-dependent protein kinase or Ca2+ mobilization. Possible alternative mechanism was discussed.     

Evaluation of genotoxicity of N-nitrosodibenzylamine in Chinese hamster V79 cells and in Salmonella

Health concerns have arisen due to the formation of N-nitrosodibenzylamine (NDBzA; CAS No. 5336-53-8) in pork processed in a new type of rubber netting. In view of the potent carcinogenicity of related nitrosamines (e.g. N-nitroso-n-dibutylamine and N-nitrosodiethylamine), NDBzA was evaluated for genotoxicity in vitro in both Chinese hamster V79 cells and in Salmonella. In V79 cells, concentrations up to 25 micrograms/ml were tested with and without activation by rat or hamster hepatocytes. Significant elevation of SCE frequency was seen only at 25 micrograms/ml in the presence of uninduced hamster hepatocytes. Mutation to 6-thioguanine resistance was observed at 25 micrograms/ml, in the absence of hepatocytes and in the presence of induced (Aroclor 1254) or uninduced hamster hepatocytes, but not with rat hepatocytes. With uninduced rat hepatocytes, a small but significant (p less than 0.05) increase in the mutation frequency was seen with 10 micrograms/ml NDBzA. In the Salmonella assay, using a pre-incubation protocol and concentrations up to 1000 micrograms/ml, NDBzA was negative in strain TA98, and in TA100 with rat S9, but was positive at the highest dose in TA100 with hamster S9, and more strongly with Aroclor 1254-induced hamster S9. When activated by uninduced rat or hamster hepatocytes, as opposed to S9, NDBzA was negative with all tester strains. Hamster hepatocytes activated more than rat in the V79 studies, and hamster S9 was more strongly activating in the Salmonella assay. These results indicate that NDBzA is weakly mutagenic to both Salmonella and V79 cells.     

Genotoxic activity of m-nitrobenzaldehyde

m-Nitrobenzaldehyde (MNB) was evaluated for mutagenic activity using the Ames microbial mutagenicity test and for its ability to induce DNA single-strand breaks in rat hepatocytes as measured by alkaline elution. MNB was tested in S. typhimurium strains TA1535, TA1537, TA1538, TA98, and TA100, both with and without pretreatment with liver microsomes (S9) isolated from rats pretreated with Aroclor 1254. MNB produced 2-fold or greater increases in revertants in TA1538, both with and without S9, and in TA100 with S9 only. A 2-fold increase in revertants was seen in TA98, but only at the highest dose tested which did not produce inhibition of background growth. MNB caused a greater than 3-fold increase in elution slope, with DNA alkaline elution assay, but only at highly cytotoxic doses and, therefore, is not considered genotoxic in this system. It is concluded that MNB possesses weak genotoxic activity.     

The early effects of chemical carcinogens on adult rat hepatocytes in primary culture. II. Effects on unscheduled DNA synthesis, cell division and alpha-fetoprotein production.

The effects of exposure of rat hepatocytes in primary maintenance culture to chemical carcinogens has been studied with respect cytotoxicity and alterations in mitotic index, unscheduled DNA synthesis and alpha-fetoprotein (AFP) production. All compounds tested produced cytotoxicity. Increases in mitotic index and unscheduled DNA synthesis and the production of AFP were observed after treatment of the cells with the carcinogens but not after treatment with the non-carcinogenic isomers. These increases were dose-dependent and depended on the time of exposure and the time incubated postexposure. The patterns of the increase in mitotic index and AFP production after cessation of carcinogen exposure were very similar, with the increase in mitotic index occurring slightly before that for the AFP production and it is suggested from this and other data that the production of AFP is dependent on the generation of a cell species functionally distinct from the non-dividing hepatocytes. It is also suggested that measurement of unscheduled DNA synthesis in conjunction with that of AFP production in cultured hepatocytes may be useful as part of a screening programme for chemical carcinogens.