Cell and species differences in metabolic activation of chemical carcinogens

Metabolic activation and DNA binding of aflatoxin B1 (AFB1), N-nitrosodimethylamine (DMN) and benzo[a]pyrene (B[a]P) were compared in human, rat and mouse hepatocytes and human pulmonary alveolar macrophages (PAM). The degree of carcinogen activation by hepatocytes and PAM was measured by cell-mediated mutagenesis assays in which co-cultivated Chinese hamster V79 cells were used to monitor mutagenic metabolites. Hepatocytes from human, mouse and rat metabolized DMN and released the active metabolites to induce either ouabain- or 6-thioguanine-resistant mutation. The mutation frequencies mediated by hepatocytes of the 3 animal species were approximately 3-9 mutants/10(5) survivors at a concentration of 0.2 mM DMN. The variations of radioactivity bound to liver cell DNA were relatively small in cultured mouse, rat, and human hepatocytes exposed to 14C label DMN (0.5 mM) and the binding values were in a range of 6-12 X 10(3) pmoles/mg DNA. However, rat hepatocytes were at least 10-fold more effective than either human or mouse hepatocytes in generating mutagenic metabolites of AFB1 and also had a much higher AFB1 metabolite DNA-binding value. The AFB1 DNA-binding levels were 4.1, 12-27 (range), 120 pmoles/mg DNA respectively in mouse, human, and rat liver cells following AFB1 (3.3 microM) exposure for 20 h. Hepatocytes from the 3 animal species were unable to mediate mutation in the presence of 4 microM B[a]P; PAM activated B[a]P and effectively mediated mutation in the co-cultivated V79 cells. In contrast to results with hepatocytes, PAM failed to generate enough mutagenic metabolites of AFB1 (3.3 microM) and the mediation of mutations was seen only at very high concentration of DMN (80 mM). The genotoxic effects of the 3 carcinogens on hepatocytes from different species in vitro were in agreement with the in vivo animal experiments in that mice are relatively resistant to AFB1 carcinogenesis whereas rats are sensitive; B[a]P is not effective as a complete liver carcinogen in adult rat and mouse whereas DMN induces liver cancer.     

Evaluation of hexamethylphosphoramide for gene mutations in Salmonella typhimurium using plate incorporation, preincubation, and suspension assays

Hexamethylphosphoramide (HMPA), a potent rat nasal carcinogen by inhalation, and three of its metabolites, pentamethylphosphoramide (PMPA), trimethylphosphoramide (TriMPA), and formaldehyde (HCHO), were assessed in Salmonella typhimurium gene mutation assays using various protocols, including plate incorporation, preincubation and suspension assays. HMPA (tested up to 15 000 μg/plate) was not mutagenic in plate incorporation or preincubation assays with or without metabolic activation. HCHO was mutagenic in the plate incorporation and preincubation assays (tested up to 150 μg/plate). In suspension assays, however, HMPA (tested up to 40 mg/ml), PMPA (up to 44 mg/ml) and HCHO (up to 45 μg/ml), but not TriMPA (up to 29 mg/ml), were mutagenic. HMPA and PMPA were positive only with activation. HMPA's mutagenicity was optimized using a relatively high level of rat liver S9 protein (3.5 mg/plate) in the metabolic activation mixture. Semicarbazide, an HCHO trapping agent, added at concentrations up to 167 μg/ml, markedly inhibited the mutagenic activities of HMPA and PMPA suggesting that HCHO generation may play a role in their mutagenicity. These studies show that HMPA is mutagenic in a modified Salmonella typhimurium reverse mutation assay with metabolic activation. Successive N-demethylation of HMPA eventually eliminates the mutagenic activity which further suggests that HMPA's mutagenic activity is related to the release of HCHO.     

Regulatory effects of testosterone and 17 beta-oestradiol on the metabolism of dimethylnitrosamine by renal and hepatic microsomal enzymes from BALB/c mice

Previous investigations with BALB/c mice have demonstrated that no sex-related differences exist in the ability of liver microsomal fractions (S-9) to biotransform dimethylnitrosamine (DMN) to its active mutagenic metabolites as evidenced by bacterial screening assays. In contrast, kidney microsomal enzymes from adult male BALB/c mice and not from females, castrates, and immature animals, were capable of activating DMN. The present study was designed to test the effects of testosterone and oestradiol on DMN bioactivation by hepatic or renal microsomal enzymes. Mutagenic assays were performed using liver and kidney microsomal enzymes with the histidine deficient mutant Salmonella typhimurium TA100. Results indicate that testosterone treatment of female BALB/c mice resulted in an increase in the ability of their renal microsomal enzymes to metabolize DMN to its active mutagenic intermediates. Renal microsomal enzymes from female mice treated with 17 beta-oestradiol had no effect on DMN metabolism. However, the ability of the renal microsomal enzymes treated with 17 beta-oestradiol to bioactivate DMN was significantly decreased in males.     

Dimethylnitrosamine metabolism: I. In vitro activation of dimethylnitrosamine to mutagenic substance(s) by hepatic and renal tissues from three inbred strains of mice

The potential of hepatic and renal homogenates from three inbred strains of mice (BALB/c, C57BL and DBA) to activate dimethylnitrosamine (DMN) was investigated. Microsomal enzyme (S-9) preparations of liver and kidney from mature and immature mice were used in the Ames Salmonella mutagenicity assay. No age or sex-related differences in the formation of active mutagenic DMN Metabolites by liver microsomal enzymes were observed within any of the three inbred strains. In contrast, mature male kidney S-9 fractions from all three strains had a significantly greater potential to activate DMN than mature female and immature animals. Testosterone treatment resulted in no apparent changes in the ability of hepatic tissue to biotransform DMN to its mutagenic metabolites among age and sex classes. However, after testosterone treatment, renal microsomal fractions from mature female mice of all three strains did not differ significantly from their male counterparts in their ability to transform DMN to mutagenic metabolites.     

Mutagenic profiles of carbazole in the male germ cells of Swiss albino mice

Mutagenic effect of carbazole was evaluated by employing dominant lethal mutation and sperm head abnormality assays in male Swiss albino mice. For the dominant lethal mutation assay, adult male mice were treated for five consecutive days either with 30 or 60 mg/kg body weight (b.w.) of carbazole by single intraperitoneal (i.p.) injection. For the sperm head abnormality assay mice were treated with 50, 100, 150, 200 and 300 mg/kg b.w as a single i.p. injection. Treatment of adult male mice with carbazole resulted in induction of dominant lethal mutation and abnormal sperm heads. The results show that carbazole is mutagenic in male germ cells of mice.     

Cytogenetic and dominant lethal studies on captan.

Possible mutagenic activity of captan was investigated by in vitro and in vivo cytogenetic studies and by the dominant lethal study in mice. In vitro cytogenetic study with cultured human diploid cells revealed a significant increase in the frequency of cells showing stickiness and a severe mitotic inhibition at concentrations of 3.0 and 4.0 microgram of captan per ml. although no chromosomal aberrations were observed. In in vivo cytogenetic study, no chromosomal aberrations were induced in the bone marrow cells of rats treated orally with captan at a single dose of 500, 1000 or 2000 mg/kg or at five consecutive doses of 200, 400 or 800 mg/kg/day. Dominant lethal study also failed to show any mutation induction after treatment of male mice with daily oral dose of 200 or 600 mg of captan per kg bw for five days.     

Genotoxicity, metabolism and blood kinetics of epichlorohydrin in mice

Epichlorohydrin (ECH), a direct mutagen in vitro, did not induce chromosomal aberrations in bone-marrow cells of CD1 mice given single oral doses of 50 and 200 mg/kg in water. The ECH diol derivative (3-chloro-1,2-propanediol) was tested in vitro by a forward-mutation assay on the yeast Schizosaccharomyces pombe and showed a weak but significant mutagenic effect. The failure of ECH to induce mutagenic effects appears to be due to the rapid metabolic clearance of the compound in vivo. ECH blood kinetics at both doses, and at the same time the concentration of the diol, were determined. ECH rapidly disappeared from mouse blood, being no longer detectable 20 min after treatment. In contrast, 3-chloro-1,2-propanediol was measurable up to 5 h after dosage. No difference was observed in the kinetic and metabolic behavior of ECH after single and repeated doses (50 and 200 mg/kg/day for 7 days). When 3-chloro-1,2-propanediol was tested, neither glutathione depletion nor epoxide hydrolase inhibition (evaluated with both styrene-7,8-oxide and ECH as substrates) could be detected in mouse liver. Finally, no difference in ECH blood kinetics or metabolism were observed in experiments in which the compound was administered (200 mg/kg) intraperitoneally in water or orally as a solution in dimethyl sulfoxide.     

Evaluation of the genotoxicity of the imidazole antifungal climbazole: comparison to published results for other azole compounds

Climbazole is an imidazole antifungal agent that can provide anti-dandruff benefits when incorporated into a shampoo matrix. A series of genotoxicity studies were performed to support the human safety of this azole antifungal drug. Climbazole was not mutagenic in the Salmonella typhimurium or Escherichia coli Ames assay and did not induce micronuclei in human lymphocytes. In the mouse lymphoma assay (MLA), climbazole was negative (non-mutagenic) with and without metabolic (S9) activation after a 4 h exposure; an increase in small colony mutants was observed without metabolic activation after a 24 h exposure at concentrations of 15 and 17.5 μg/mL. An in vivo mouse micronucleus test was negative up to a maximum tolerated dose (MTD) of 150 mg/kg climbazole administered orally. In the in vivo/in vitro unscheduled DNA synthesis assay, climbazole showed no evidence of DNA damage in the livers of rats at doses up to the MTD of 200 mg/kg orally. A toxicokinetic study was performed in mice with oral administration of [14C]-climbazole (150 mg/kg). Radioactivity (20.42 μg-equiv./g plasma) was detected 15 min after oral administration of [14C]-climbazole, and the peak concentration was 62.96 μg-equiv./g plasma at 8 h after dosing. The measured amounts of radioactivity in plasma, at all sample times from 15 min up to 24 h, exceeded the concentrations that induced increases in mutation frequency after 24 h exposure of mouse lymphoma cells in vitro (15 and 17.5 μg/mL). These observations lend support to the conclusion that climbazole does not present a genotoxic risk in vivo. Furthermore, these data are consistent with the published data for other azole antifungals that work by preventing the synthesis of ergosterol and, as a class, are generally non-genotoxic, except some isolated positive results of questionable significance. Collectively, these data are supportive of the view that climbazole does not present a genotoxic or carcinogenic risk to humans.     

A comparative study of hepatic DNA repair, DNA replication and hepatotoxicity in the CD-1 mouse following multiple administrations of dimethylnitrosamine

The objective of this study was to quantify hepatic DNA repair and DNA replication following multiple administrations of dimethylnitrosamine (DMN) and to determine if these events were correlated with hepatotoxicity. Male CD-1 mice, 50-100 days old, were dosed daily, p.o., with DMN in water at dose levels of 2, 4, 7 and 10 mg/kg for 2 weeks. After 2, 7 and 14 days of dosing, hepatocytes were isolated by an in situ perfusion procedure, incubated in the presence of [3H] thymidine, and fixed. Unscheduled as well as scheduled DNA synthesis were assessed by quantitative autoradiography. Unscheduled DNA synthesis (UDS) represents DNA repair while scheduled DNA synthesis (S phase) represents DNA replication. In addition, the animals' serum was examined for enzymes which indicate hepatic toxicity. After 1, 7 and 14 days of dosing, animals were orbital-bled and the serum was analyzed for serum glutamic pyruvic transaminase (SGPT), serum glutamic oxalacetic transaminase (SGOT), alkaline phosphatase (AP) and gamma-glutamyl transpeptidase (GGT). No morbidity or mortality was observed at dose levels of 2 and 4 mg/kg, but all animals receiving 7 and 10 mg/kg died after 4-6 days of dosing. GGT or AP were not elevated at any dose level or at any time point examined. At 4 mg/kg only a slight increase (less than or equal to 2 X) in the concentration of SGOT and SGPT was observed but a sharp increase (greater than 20 X) in replicative DNA synthesis was seen. The 2 mg/kg dose level of DMN did not increase replicative DNA synthesis and SGOT and SGPT were not elevated above control values at any time point following dosing at 2 mg/kg. A weakly positive DNA repair response was observed for dose levels of 4, 7 and 10 mg/kg DMN after two consecutive days of dosing. No DNA repair was observed after either 7 or 14 days of dosing at the 2 and 4 mg/kg/day levels. These results indicate that hepatic toxicity is associated with the induction of replicative DNA synthesis (S phase) but not with the induction of DNA repair. The results also confirm and extend a previous study (Doolittle et al., 1987b) indicating that a significant elevation in hepatic DNA replication is induced by hepatocarcinogens after multiple administrations of dose levels which do not alter hepatic DNA replication after a single administration. This finding indicates that the utility of the in vivo-in vitro hepatocyte assay may be enhanced by using a multi-dose protocol.     

Metformin (dimethyl-biguanide) induced DNA damage in mammalian cells

Metformin (dimethyl-biguanide) is an insulin-sensitizing agent that lowers fasting plasma-insulin concentration, wherefore it's wide use for patients with a variety of insulin-resistant and prediabetic states, including impaired glucose tolerance. During pregnancy it is a further resource for reducing first-trimester pregnancy loss in women with the polycystic ovary syndrome. We tested metformin genotoxicity in cells of Chinese hamster ovary, CHO-K1 (chromosome aberrations; comet assays) and in mice (micronucleus assays). Concentrations of 114.4 μg/mL and 572 μg/mL were used in in vitro tests, and 95.4 mg/kg, 190.8 mg/kg and 333.9 mg/kg in assaying. Although the in vitro tests revealed no chromosome aberrations in metaphase cells, DNA damage was detected by comet assaying after 24 h of incubation at both concentrations. The frequency of DNA damage was higher at concentrations of 114.4 μg/mL. Furthermore, although mortality was not observed in in vitro tests, the highest dose of metformin suppressed bone marrow cells. However, no statistically significant differences were noted in micronuclei frequencies between treatments. In vitro results indicate that chronic metformin exposure may be potentially genotoxic. Thus, pregnant woman undergoing treatment with metformin should be properly evaluated beforehand, as regards vulnerability to DNA damage.     

In vivo repair of rat liver DNA damaged by dimethylnitrosamine or diethylnitrosamine.

Effects of hepatocarcinogens dimethylnitrosamine (DMN) and diethylnitrosamine (DEN) on the sedimentation pattern of rat liver DNA in alkaline sucrose gradients were studied with regard to time and dose dependency. Both DMN (10 mg/kg body weight) and den (13.4 or 134 mg/kg) induced appreciably decreased DNA sedimentation rates at 24 h after injection. DMN at 10 mg/kg was as effective in decreasing the DNA sedimentation rate at 24 h after injection as was the higher dose of DEN (134 mg/kg). Sedimentation patterns at 1, 6 and 14 days after injection indicated that damage induced by DEN (134 mg/kg) was repaired at a substantially lower rate than DMN (10 mg/kg) induced damage. When effects of equimolar doses of DMN (10 mg/kg) and DEN (13.4 mg/kg) were compared at 1, 6 and 14 days after injection, it was observed that the more pronounced damage of rat liver DNA induced by DMN was repaired at a faster rate than was the DEN-induced damage. At the molecular level this difference in repair between damage induced by the two nitrosamines is probably related to different DNA alkylation patterns. The relatively persistent nitrosamine-induced DNA lesions (observed especially after DEN administration) are thought to represent phosphotriesters which give rise to single strand DNA breaks at strongly alkaline conditions of lysis on top of the gradient. The results are discussed in relation to the possible significance of alkylation and repair of DNA in the formation of (pre)cancerous lesions in rat liver.     

Dependence on intact cells for the in vitro activation of dimethylnitrosamine to an immunosuppressive form

The in vitro activation of dimethylnitrosamine (DMN) to an immunosuppressive form was studied utilizing liver-enzyme fractions and intact hepatocytes. The N-demethylation of DMN by mouse S9 and microsome preparations was confirmed by determination of formaldehyde generation. S9 fractions from both phenobarbital(PB)- and isopropanol(iso)-pretreated mice displayed significantly greater demethylase activity than uninduced S9 fractions. However, when incubated with spleen cells, neither S9 preparation was capable of activating DMN to a form capable of suppressing antibody responses by recovered spleen cells. In contrast, the positive control, cyclophosphamide, was activated to a markedly immunosuppressive form. S9 fractions failed to activate DMN to an immunosuppressive form regardless of S9 concentration, time of preincubation, or rocking speed. Liver microsomes from PB-pretreated mice displayed significantly greater N-demethylase activity than S9 fractions yet were unable to activate DMN to an immunosuppressive form. In contrast, the addition of DMN to mixed cultures of mouse hepatocytes and mouse spleen cells resulted in activation of DMN and marked suppression of antibody responses. The separation of spleen cells from the hepatocyte monolayer by an agar layer less than 1 mm thick resulted in complete reversal of the immunosuppressive effect of DMN. Unlike the metabolism of DMN to a mutagenic form, the in vitro activation of DMN to an immunosuppressive form was therefore dependent on intact cells. Furthermore, the activation by intact hepatocytes was shown to be dependent on cell-cell contact or close proximity of activating and target cells.     

Assessment of genotoxic potential of ethylenediamine: in vitro and in vivo studies

Ethylenediamine (EDA) was evaluated for potential genotoxic activity using a battery in vitro and in vivo mammalian tests. The tests employed were the Chinese hamster ovary (CHO) gene mutation assay, the sister-chromatid exchange (SCE) test with CHO cells, unscheduled DNA synthesis (UDS) assays with primary rat hepatocytes and a dominant lethal study with Fischer 344 rats. EDA did not produce a positive, dose-related, mutagenic effect in either the CHO mutation assay or in the SCE test when evaluated both with and without the addition of a rat-liver S9 activation system. With hepatocytes, no positive effects of EDA upon UDS values were noted in 2 separate studies using either a scintillation counting procedure or an autoradiographic method to determine UDS activity. In a dominant lethal study, male rats fed for 23 weeks with dietary levels of EDA X 2HCl of 0, 0.05, 0.15 or 0.50 g/kg/day, and mated with 1 virgin female/week for 3 consecutive weeks, showed no dose-related or statistically significant effects upon fertility, total number of implantations/female, or the number of living and dead implants per female; marked effects upon the incidence of dominant lethal mutations were noted in the positive control group injected intraperitoneally with one dose of 0.25 mg/kg triethylenemelamine. We conclude that EDA was not genotoxic in the in vitro and in vivo mammalian test systems employed.     

Mutagenic activity of 2-amino-N6-hydroxyadenine in the mouse spot test.

The base analogue 2-amino-N6-hydroxyadenine (AHA) was mutagenic in the spot test in (T x HT)F1 mouse embryos. Females were injected with single doses of 20 or 40 mg AHA per kg body weight on the 9th day of pregnancy. To rank the mutagenic potency of different compounds, the frequencies of genetically relevant spots induced by 1 mg/kg body weight were calculated. The observed somatic mutation frequency for 1 mg/kg AHA was lower (1.95 x 10(-3)) spots of genetic relevance) than that of mitomycin C (16 x 10(-3)), ethylnitrosourea (6.8 x 10(-3)) and cyclophosphamide (6.4 x 10(-3)) and therefore AHA was not classified as a very potent mutagen in this test system. The doubling dose to induce genetically relevant spots was calculated to be 20 mg/kg b.w. Based on these data, AHA is suggested to be a candidate to induce recessive specific-locus mutations in germ cells of mice.     

Mutagenic action of cadmium on the sex cells of male mice

Possible mutagenic effect of cadmium chloride was studied by determining the frequency of dominant lethal mutations induced in germ cells of male mice. Water solution of CdCl2 was injected intraperitoneally to male mice at doses of 1.0, 2.0 and 4.0 mg/kg. The results obtained did not reveal any mutagenic effect of this compound. The dose of 4.0 mg/kg CdCl2 resulted in the death of spermatocytes and spermatogonia and the sterility of male mice. Cadmium chloride at a dose of 2.0 mg/kg did not affect the frequency of dominant lethal mutation induced by gamma-rays 60Co at a dose of 450 r in germ cells of male mice.     

Effects of pretreatment with pyrazole and inducers of mixed function oxidases on DNA repair elicited by dimethylnitrosamine in rat hepatocytes in vivo and in vitro

Experiments were performed to investigate the relationship between the rate of oxidative metabolism of dimethylnitrosamine (DMN) by rat liver microsomes (i.e., DMN demethylase activity, DMNd) and its genotoxicity in liver, as assessed by the in vitro and in vivo/in vitro rat hepatocyte primary culture/DNA repair (HPC/DR) assays. Pretreatment of rats with pyrazole (PYR) resulted in a 4-fold increase in DMNd and a 3-fold greater DNA repair response to in vivo administration of 5 mg DMN/kg body weight. Pretreatment with phenobarbital (PB), dichlorodiphenyltrichloroethane (DDT), 3-methylcholanthrene (3-MC), beta-naphthoflavone (beta-NF) or Aroclor 1254 (ARO) produced a variable degree of inhibition of DMNd and had no significant effects on the response to DMN in the in vivo/in vitro HPC/DR assay. DNA repair elicited by DMN in vitro was decreased in hepatocytes from rats pretreated with 3-MC, while PB, DDT, beta-NF and ARO pretreatments had little effect on the response. In contrast, PYR pretreatment produced a 4.5-6.7-fold increase in the in vitro DNA repair response to DMN, and extended detection of positive responses to lower concentrations. Most of the inducers had no effect on DNA repair elicited by the direct acting alkylator, methyl methanesulfonate (MMS). Thus, the pretreatment-related changes in DMN-induced DNA repair were probably due to alterations in DMNd rather than to effects on the DNA repair capacity of the hepatocytes.     

Assessment of the mutagenic activity of aversectin C]

Aversectin C was evaluated for mutagenic activity in the Ames test with Salmonella typhimurium TA 97, TA 98 and TA 100, in the dominant lethal assay on uninbred albino rats in a dose of 2.25 mg/kg body weight (1/40 of the LD50) and in the metaphase test on F1CBAxC57BI/6 mice in a dose of 8.2 mg/kg body weight (1/5 of the LD50). The agent showed no mutagenic activity in any of the tests. The anaphase test on F1CBAxC57BI/6 mice revealed no antimitotic activity of aversectin C.     

Role of dimethylnitrosamine-demethylase in the metabolic activation of dimethylinitrosamine.

In vivo administration to rats of the mixed-function oxidase modifiers 3-methylcholanthrene (MC), pregnenolone-16 alpha-carbonitrile (PCN) or beta-naphthoflavnoe (beta-f) inhibits the hepatic microsome-catalyzed in vitro binding of dimethylnitrosamine (DMN) to DNA. This parallels their effect on DMN-demethylase I, regarded to be the sole activating step in DMN carcinogenesis and fails to account for the previously observed anomaly that MC and PCN inhibit, while beta-NF enhances, the hepatocarcinogenic activity of DMN. The in vitro binding of DMN is clearly dependent on microsomes and NADPH, and is strongly enhanced by soluble cytoplasmic proteins; the presence of the latter has no effect. however, on the relative response to pretreatment by the modifiers. In mice beta-NF enhances and PCN inhibits DMN-demethylase I; beta-NF has no effect on either the cytochrome P-450 level or on the LD50, while PCN strongly increases the cytochrome P-450 level but without influencing the LD50. Neither of the two modifiers has any effect in mice on the host-mediated mutagenicity of DMN in a dose-response study, except for the highest dose of DMN (200 mg/kg) where PCN pretreatment significantly enhanced mutagenicity. To account for the anomalous observations, other potential pathways of DMN metabolism have been explored. Whole rat liver nuclei or isolated nuclear membrane fractions contain no DMN-demethylase or diethylnitrosamine-deethylase activity. In a microsomal mixed-function amine-oxidase assay system neither purified enzyme preparations nor whole microsomes catalyze NADPH oxidation in the presence of DMN as substrate. In addition, the purified enzyme does not catalyze formaldehyde production in the DMN-demethylase assay system. Benzylamine, a typical inhibitor of mitochondrial monoamine oxidase (MAO), is a potent inhibitor of DMN-demethylase activity, but microsomes are devoid of MAO activity. Furthermore, purified MAO has no DMN-demethylase activity. The differential effect of modifiers on the carcinogenicity of DMN probably involves pathways other than DMN metabolism.     

Induction of gene mutations by 5-(2-chloroethyl)-2'-deoxyuridine (CEDU), an antiviral pyrimidine nucleoside analogue

5-(2-chloroethyl)-2'-deoxyuridine (CEDU) had been developed for the treatment of herpes simplex infections. In the Salmonella reverse mutation test, the compound was found to be mutagenic in strains TA1535 and TA102 at very high concentrations (> or =2500 micro g/plate), both with and without S9-mix. The mutagenic potential of CEDU was further investigated in vivo and in vitro. It did not induce DNA repair in rat hepatocyte primary cultures, and was negative in the micronucleus test in V79 cells and in the comet assay in human leukocytes. In vivo, CEDU was negative in the bone marrow micronucleus test in CD1 mice. The mouse spot test provided a clearly positive result. Treatment of mice on day 9 of pregnancy with 2000 mg/kg resulted in 5.9% of the F1 animals having genetically relevant spots, whereas the corresponding vehicle control group had a spot rate of 1.9%. Since these data clearly identified CEDU as an inducer of gene mutations in vivo, this potential was further investigated in lacZ transgenic Muta Mouse. Six female animals were treated daily on five consecutive days with 2000 mg/kg/day and sacrificed, after a treatment-free sampling time, 14 days later. The data showed a clear increase in the mutant frequency in the bone marrow, the lung and in the spleen. CEDU is an exception in the group of nucleoside analogues, because it was found to be a strong gene mutagen and, in contrast to the other compounds of this group investigated so far, had no considerable clastogenic effects.     

Mutagenic evaluation of ronidazole

Ronidazole was evaluated for mutagenic potential using in vitro microbial tests and in vivo studies in mice. The microbial test used the histidine requiring mutants of Salmonella typhimurium with and without a rat liver microsomal activation system (Ames test). The studies in mice included the dominant lethal test, micronucleus test and cytogenetic assays.Ronidazole was given orally in doses of 50, 100 and 200 mg/kg/day in the in vivo studies. In the dominant lethal test, groups of male mice were treated for five consecutive days before being mated with untreated females. In the micronucleus test, the mice were administered the compound for 2 or 5 consecutive days; they were killed 6 h after the last dose and bone marrow was examined for the presence of micronuclei in developing erythrocytes. In the cytogenetic assays, bone marrow cells in metaphase were examined for chromosome aberrations, 6, 24 and 48 h after mice were treated acutely with the test compound. In addition, similar examinations of chromosomes were made on mice given five consecutive dosages of ronidazole and killed 6 h after the last dose. The results of the various in vivo studies did not suggest that ronidazole would be mutagenic for the mammal.Ronidazole at concentrations of 10 and 50 μg/plate was found to increase the number of back mutations of missense mutants in the in vitro bacterial test. This finding confirms the results of Voogd et al. [19]. Incorporation of the microsomal activation system had no effect on the mutagenic capability of the test compound.In conclusion, although ronidazole was shown to be mutagenic in in vitro bacterial systems, the in vivo systems did not suggest that the compound would be mutagenic for the mammal.