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Dose-response modeling of in vitro micronucleus test (IVMNT) data was evaluated to determine if the approach has value in discriminating among different tobacco products. Micronucleus responses were generated in L5178Y/Tk +/- mouse lymphoma cells and TK6 human lymphoblastoid cells from a series of whole smoke solutions (WSSs) expected to have different levels of genotoxicity based on differences in their machine-generated smoke constituents. Eight WSSs were prepared by machine smoking different numbers (20 or 60) of two commercial cigarettes (Marlboro Silver or Red) under International Standardization Organization (ISO) or Health Canada Intense (HCI) smoking machine regimens and tested in the two cell lines with and without rat liver S9 activation. The S9-mediated IVMNT dose-response data from the WSSs were evaluated with PROAST software and Benchmark Doses (BMDs) and their upper and lower confidence intervals (CIs) were generated. IVMNT data differed based on the number and type of cigarettes smoked and smoking machine regimen. The IVMNT responses produced in mouse lymphoma cells generally were greater than in TK6 cells, but the ability of the two cell types to differentiate between WSSs was similar. The results indicate that BMD potency ranking was useful for differentiating between IVMNT responses., (Published by Elsevier B.V.)

Aristolochic acids (AAs) are carcinogenic plant toxins that are relatively strong gene mutagens, both in vitro and in vivo, but weak inducers of micronuclei in vivo. In order to clarify the reasons for these disparate responses, we evaluated the genotoxicity of AAs in F344 rats using several assays that respond to DNA damage in bone marrow. Groups of 7- to 8-week-old male rats (n=6) were gavaged with 0, 2.75, 5.5, and 11mg/kg AAs for 28 days or with 0, 11, 22, and 30mg/kg AAs for 3 days. Day 1 being the first day of treatment, Pig-a mutant frequencies (MFs) were assayed in peripheral blood erythrocytes up to Day 56 for the 28-day treatment or Day 42 for the 3-day treatment; micronuclei were assayed in peripheral blood reticulocytes on Day 4 (both treatment protocols) and on Day 29 of the 28-day treatment protocol; and at the final sampling times (Day 59 or Day 42), the animals were sacrificed and Hprt mutant lymphocytes were measured. In a separate study, the Comet assay was performed on liver, kidney, and bone marrow of animals gavaged with 0, 11, 22, and 30mg/kg AAs for 4 days and sacrificed 3h after the last treatment. While only weak increases in micronucleated reticulocyte frequency were observed in treated animals, Pig-a MFs increased in a dose- and time-dependent manner with both treatment schedules. Lymphocyte Hprt mutant frequencies also increased dose dependently in treated animals, and the Comet assay detected elevated levels of DNA damage in all the tissues evaluated. These findings indicate that the DNA damage produced by AAs in rat bone marrow is a weak inducer of micronuclei but a relatively strong inducer of gene mutation., (Published by Elsevier B.V.)

Furan is a multispecies liver carcinogen whose cancer mode of action (MOA) is unclear. A major metabolite of furan is a direct acting mutagen; however, it is not known if genotoxicity is a key step in the tumors that result from exposure to furan. In order to address this question, transgenic Big Blue rats were treated by gavage five times a week for 8 weeks with two concentrations of furan used in cancer bioassays (2 and 8mg/kg), and with two higher concentrations (16 and 30mg/kg). Peripheral blood samples taken 24h after the 5th dose (1 week of dosing) were used to assay for micronucleus (MN) frequency in normochromatic erythrocytes (NCEs) and reticulocytes (RETs), and Pig-a gene mutation in total red blood cells (RBCs). 24h after the last dose of the 8-week treatment schedule, the rats were euthanized, and their tissues were used to perform NCE and RET MN assays, the Pig-a RBC assay, Pig-a and Hprt lymphocyte gene mutation assays, the liver cII transgene mutation assay, and the liver Comet assay. The responses in the MN assays conducted at both sampling times, and all the gene mutation assays, were uniformly negative; however, the Comet assay was positive for the induction of liver DNA damage. As the positive responses in the Comet assay were seen only with doses in excess of the cancer bioassay doses, and at least one of these doses (30mg/kg) produced toxicity in the liver, the overall findings from the study are consistent with furan having a predominantly nongenotoxic MOA for cancer., (Published by Elsevier B.V.)

The fungal toxin, Ochratoxin A (OTA), is a common contaminant in human food and animal feed. The present study evaluated micronucleus (MN) induction by OTA in comparison with its ability to induce cytotoxicity and DNA damage in two mammalian cell lines, CHO-K1-BH(4) Chinese hamster ovary cells and TK6 human lymphoblastoid cells. Micronuclei were evaluated by flow cytometry, cytotoxicity was estimated by relative population doubling (RPD), while direct DNA damage and oxidative DNA damage were measured with the Comet assay, performed without and with digestion by formamidopyrimidine-DNA glycosylase (fpg). For the MN and cytotoxicity measurements, the cell lines were treated for 24h (CHO cells) or 27h (TK6 cells) with 5-25μM OTA in the absence of exogenous metabolic activation. The OTA treatments resulted in concentration-responsive increases in cytotoxicity, with higher concentrations of the agent being more cytotoxic in CHO cells than TK6 cells. 15μM OTA produced positive responses for MN induction and hypodiploid events (a measure of aneugenicity) in both cell lines; this concentration of OTA also produced cytotoxicity near to the recommended limit for the assay (45±5% RPD). A time course assay with TK6 cells indicated that at least 4h of OTA treatment were required to produce a positive MN response. For the Comet assay DNA damage assessments, the cell lines were treated with 5-50μM OTA for 4h. Direct DNA damage was detected in TK6 cells, but not CHO cells, while concentration-related increases in fpg-sensitive sites were detected for both cell lines. The consistent association of oxidative DNA damage with OTA exposure suggests its involvement in producing OTA-induced clastogenicity and aneugenicity; however, based on its detection in TK6 cells direct DNA damage could be involved in any human risk posed by OTA exposure., (Published by Elsevier B.V.)

Methylphenidate hydrochloride (MPH), a widely prescribed pediatric drug for attention deficit hyperactivity disorder, induced liver adenocarcinomas in B6C3F1 mice exposed to 500 ppm in feed for 2 years (Dunnick and Hailey (1995) [14]). In order to determine if the induction of liver tumors was by a mutagenic mode of action, groups of male Big Blue (BB) mice (B6C3F1 background) were fed diets containing 50-4000 ppm MPH for 4, 12, or 24 weeks. At sacrifice, the livers were removed and the cII mutant frequency (MF) and spectrum of cII mutations were determined. In addition, the frequencies of micronucleated reticulocytes (MN-RETs) and normochromatic erythrocytes (MN-NCEs) were measured in peripheral blood erythrocytes as was the Hprt MF in splenic lymphocytes. Food consumption and body weight gain/loss were recorded weekly for each animal. The levels of MPH and RA were determined immediately before sacrifice in the serum of mice fed MPH for 24 weeks. A significant loss in body weights (p

We have investigated the use of peripheral blood from the nonhuman primate (NHP) rhesus monkey (Macaca mulatta) as a model system for mutation detection. The rhesus monkey is metabolically closer to humans than most common laboratory animals, and therefore may be a relevant model for hazard identification and human risk assessment. To validate the model, conditions were determined for in vitro selection and expansion of 6-thioguanine-resistant (6-TGr) HPRT mutant and proaerolysin-resistant (ProAERr) PIG-A mutant lymphocytes from peripheral blood obtained by routine venipuncture. Also, flow cytometric methods were developed for the rapid detection of PIG-A mutant erythrocytes. The flow cytometric analysis of PIG-A mutant erythrocytes was based on enumerating cells deficient in surface markers attached to the cellular membrane via glycosylphosphatidyl inositol (GPI) anchors. Mutant cells were enumerated over an extended period of time in peripheral blood of male monkeys receiving daily doses of the electrolyte replenisher Prangtrade mark (a common carrier for oral delivery of drugs in NHPs), and in the blood of one male monkey treated with a single i.p. dose of 50mg/kg of N-ethyl-N-nitrosourea at approximately 2 years of age and another similar injection at approximately 3.5 years of age. The spontaneous PIG-A and HPRT T-cell mutant frequency (MF) was low in animals receiving Prang (0-8x10(-6)), and treatment with ENU resulted in a clearly detectable increase in the frequency of ProAERr and 6-TGr lymphocytes (up to approximately 28x10(-6) and approximately 30x10(-6), respectively). Also, the ENU-treated animal had higher frequency of GPI-deficient erythrocytes (46.5x10(-6) in the treated animal vs. 7.8+/-4.2x10(-6) in control animals). Our results indicate that the rhesus monkey can be a valuable model for the identification of agents that may impact upon human health as mutagens and that the PIG-A gene can be a useful target for detection of mutation in both white and red blood cells.

The studies presented in this work were designed to evaluate the genetic toxicity of methylphenidate hydrochloride (MPH) in non-human primates (NHP) using a long-term, chronic dosing regimen. Thus, approximately two-year old, male rhesus monkeys of Indian origin were orally exposed to MPH diluted in the electrolyte replenisher, Prang, five days per week over a 20-month period. There were 10 animals per dose group and the doses were (1) control, Prang only, (2) low, 0.15 mg/kg of MPH twice per day increased to 2.5mg/kg twice per day and (3) high, 1.5 mg/kg of MPH twice per day increased to 12.5 mg/kg twice per day. Blood samples were obtained from each animal to determine the base-line serum levels of MPH and the major metabolite of MPH in NHP, ritalinic acid (RA). In addition, the base-line frequency of micronucleated erythrocytes (MN-RETs) by flow cytometry, HPRT mutants by a lymphocyte cloning assay, and chromosome aberrations by FISH painting were determined from peripheral blood samples. Once dosing began, the serum levels of MPH and its major metabolite, RA, were determined monthly. The MN-RET frequency and health parameters (CBC, serum chemistries) were also determined monthly. HPRT mutant and chromosome aberration frequencies were measured every three months. CBC values and serum chemistries, with the exception of alanine amino transferase, were within normal limits over the course of drug exposure. The final plasma levels of MPH were similar to those produced by the pediatric dose of 0.3 microg/ml. No significant increases in the frequencies of MN-RETs, HPRT mutants, or chromosome aberrations were detected in the treated animals compared to the control animals over the 20-month exposure period.

Deficiencies in DNA mismatch repair (MMR) result in predisposition to neoplasia in both rodents and humans. Pms2 is one of the several proteins involved in the eukaryotic MMR system. In order to determine the effect of Pms2-deficiency on mutation, we measured mutant frequencies in the endogenous Hprt gene of lymphocytes from male Pms2(-/-), Pms2(+/-), and Pms2(+/+) mice. Spleens were removed from mice of various ages and lymphocytes isolated from spleens were cultured to determine the frequency of 6-thioguanine-resistant mutants. Mean mutant frequencies in Pms2(-/-) mice at 6, 10, 18, and 34 weeks of age [42.6 x 10(-6) (n=6), 38.5 x 10(-6) (n=6), 58.2 x 10(-6) (n=9), and 49.1 x 10(-6) (n=5), respectively] were significantly higher than those of comparably aged Pms2(+/+) and Pms2(+/-) mice (all less than 3 x 10(-6)). Mutant clones from the mice were expanded, RNA extracted, and Hprt cDNA amplified by RT-PCR. DNA sequencing analysis of 221 mutant cDNAs from the three different Pms2 genotypes identified 182 clones with independent mutations, including five clones that contained multiple mutations. When compared to the mutational spectrum observed in Pms2(+/+) and Pms2(+/-) mice, the mutational spectrum for Pms2(-/-) mice was significantly different. The Pms2(-/-) mutational analysis indicated that loss of the Pms2 protein causes increases in the frequencies of strand-slippage-type frameshift mutations and of A:T --> G:C transitions in the Hprt gene. The absolute frequencies of A:T --> G:C transitions in MMR-deficient mice suggest increases in this mutation may be a common feature of MMR-deficient mice, not just of Pms2-deficient mice, and may be related to the cancer predisposition that results from loss of MMR function.

Many patients undergoing chronic therapy with the purine analogue Azathioprine (Aza) have highly elevated HPRT lymphocyte mutant frequencies (MFs), and it is likely that these increases are due to selection of pre-existing HPRT mutant lymphocytes. A similar selection in germ cells might result in an increased frequency of the Lesch-Nyhan syndrome. In this study, a mouse model for Aza mutant selection was developed and Aza toxicity was evaluated in the germ cells of treated mice. Groups of 20 male C57BL/6 mice were treated by gavage three times/week with 0, 5, 10, 25, 50, or 100mg/kg Aza, and three to eight mice from each group were sacrificed at various times for up to 23 weeks. Mice treated with 25-100mg/kg Aza were all dead by 14 weeks of treatment. Hprt lymphocyte MF assays indicated that the treated mice had reduced numbers of spleen lymphocytes. Most treated mice had Hprt MFs similar to those of control mice (2.1+/-1.6 x 10(-6)), however, highly elevated MFs were detected in one out of three mice given 5mg/kg for 10 weeks, one out of three mice given 10mg/kg for 10 weeks, and one out of eight mice given 10mg/kg for 23 weeks (e.g., 233 x 10(-6) after 10 weeks of 5mg/kg). Sequence analysis of Hprt cDNA indicated that all mutant clones from one of these mice had a T-->A transversion in the initiation codon. Multiplex-PCR on mutant clones from the other two mice indicated that all the clones from one had a deletion of Hprt exons 2 and 3, while most of the mutants from the other had lost all of the Hprt exons. Measurements of testicular weight, and of sperm count, viability, morphology, and motility found that Aza produced low levels of toxicity in sperm, with the most consistent effect being a reduction in the testicular weight. The data suggest that mice chronically treated with 5 and 10mg/kg Aza (doses similar to those used in humans) have elevated Hprt MFs due to clonal amplification of selected Hprt mutants. The results also suggest that mice treated with these doses of Aza retain reasonable fertility, and will be useful for breeding experiments to examine the possibility of increasing the germ-line transmission of Hprt mutations.

Malachite green, a triphenylmethane dye used in aquaculture as an antifungal agent, is rapidly reduced in vivo to leucomalachite green. Previous studies in which female B6C3F1 mice were fed malachite green produced relatively high levels of liver DNA adducts after 28 days, but no significant induction of liver tumors was detected in a 2-year feeding study. Comparable experiments conducted with leucomalachite green resulted in relatively low levels of liver DNA adducts but a dose-responsive induction of liver tumors. In the present study, we fed transgenic female Big Blue B6C3F1 mice with 450 ppm malachite green and 204 and 408 ppm leucomalachite green (the high doses used in the tumor bioassays) and evaluated genotoxicity after 4 and 16 weeks of treatment. Neither malachite green nor leucomalachite green increased the peripheral blood micronucleus frequency or Hprt lymphocyte mutant frequency at either time point; however, the 16-week treatment with 408 ppm leucomalachite green did increase the liver cII mutant frequency. Similar increases in liver cII mutant frequency were not seen in the mice treated for 16 weeks with malachite green or in female Big Blue rats treated with a comparable dose of leucomalachite green for 16 weeks in a previous study [Mutat. Res. 547 (2004) 5]. These results indicate that leucomalachite green is an in vivo mutagen in transgenic female mouse liver and that the mutagenicities of malachite green and leucomalachite green correlate with their tumorigenicities in mice and rats. The lack of increased micronucleus frequencies and lymphocyte Hprt mutants in female mice treated with leucomalachite green suggests that its genotoxicity is targeted to the tissue at risk for tumor induction.

Leucomalachite green (LMG) is the major metabolite of malachite green (MG), a triphenylmethane dye that has been used widely as an antifungal agent in the fish industry. Concern over MG and LMG is due to the potential for consumer exposure, suggestive evidence of tumor promotion in rodent liver, and suspicion of carcinogenicity based on structure-activity relationships. In order to evaluate the risks associated with exposure to LMG, female Big Blue rats were fed up to 543 ppm LMG; groups of these rats were killed after 4, 16, or 32 weeks of exposure and evaluated for genotoxicity. We previously reported that this treatment resulted in a dose-dependent induction of liver DNA adducts, and that the liver lacI mutant frequency (MF) was increased, but only in rats fed 543 ppm LMG for 16 weeks. In the present study, we report the results from lymphocyte Hprt mutant assays and bone marrow micronucleus assays performed on these same rats. In addition, we have determined the types of lacI mutations induced in the rats fed 543 ppm LMG for 16 weeks and the rats fed control diet. No significant increases in the frequency of micronuclei or Hprt mutants were observed for any of the doses or time points assayed. Molecular analysis of 80 liver lacI mutants from rats fed 543 ppm LMG for 16 weeks revealed that 21% (17/80) were clonal in origin and that most (55/63) of the independent mutations were base pair substitutions. The predominant type of mutation was G:C --> A:T transition (31/63) and the majority (68%) of these involved CpG sites. When corrected for clonality, the 16-week lacI mutation frequency (36 +/- 10) x 10(-6) in treated rats was not significantly different from the clonally corrected control frequency (17 +/- 9 x 10(-6); P = 0.06). Furthermore, the lacI mutational spectrum in treated rats was not significantly different from that found for control rats (P = 0.09). Taken together, these data indicate that the DNA adducts produced by LMG in female rats do not result in detectable levels of genotoxicity, and that the increase in lacI MF observed previously in the liver of treated rats may be due to the disproportionate expansion of spontaneous lacI mutations.

Mother-to-child transmission of the human immunodeficiency virus (HIV) is reduced by perinatal treatment with the antiretroviral nucleoside analogue 3'-azido-3'-deoxythymidine (AZT, zidovudine). AZT, however, is genotoxic and carcinogenic in mice when administered either transplacentally or neonatally, suggesting a possible cancer risk for children later in life. In a previous study we found that treating B6C3F1/Tk(+/-) mice on postnatal days 1 through 8 with intraperitoneal injections of 200 mg AZT per kg body weight per day significantly increased spleen lymphocyte mutant frequencies in the autosomal Tk gene. Allele-specific PCR of Tk mutants from treated mice indicated that 61% had lost the Tk(+) allele (loss of heterozygosity; LOH), compared with 35% of Tk mutants from control mice, a difference that was significant. In the present study, Tk mutant lymphocyte clones were analyzed further using polymorphic microsatellite markers that flank the Tk gene along the length of mouse chromosome 11. The analysis indicated that allele-loss mutations in control mice were due to either total chromosome loss, mitotic recombination, or both. The pattern of marker loss in mutants from AZT-treated mice differed significantly from the control mice and was consistent with chromosome loss, mitotic recombination, interstitial deletion, gene conversion, and an unusual discontinuous LOH. The results indicate that AZT induced a unique pattern of mutations in the Tk gene of mice and that the major mechanisms of mutation by AZT involved deletion and recombination.

The Rat2 cell line carries 50-70 stably integrated copies per cell of a lambda/lacI shuttle vector as a target for mutagenicity testing. Rat2 cells were exposed to 1 and 10 micrograms/ml of 7,12-dimethylbenz[a]anthracene (DMBA) for 24 h at 37 degrees C in the presence of primary rat hepatocytes, and grown to confluence. The shuttle vector was rescued from untreated and mutagen-treated cells and mutant frequencies were determined. The low and high doses of DMBA induced mutant frequencies that were 7-fold (25 +/- 4.9 x 10(-5)) and 33-fold (127 +/- 19.9 x 10(-5)) higher, respectively, than the spontaneous mutant frequency (3.8 +/- 0.7 x 10(-5)). DNA sequence analysis of the DMBA-induced lacI- mutants indicated that they contained mainly basepair substitution mutations at A:T and G:C, and that A:T-->T:A and G:C-->T:A transversions were the predominant types. In addition, 23 of 28 (82%) A:T basepair substitution mutations occurred with the mutated dA, the putatively adducted base, on the coding strand. Furthermore, 20 of the 28 (71%) A:T mutations had the mutated dA flanked 5' by a dC, and 17 of these were A:T-->T:A transversions, suggesting a sequence preference for this mutation. Except for a higher proportion of G:C-->A:T transitions in the low dose data, the mutational profiles from low and high doses of DMBA were similar. These results indicate that DMBA mutagenesis in the lacI gene of Rat2 cells displays distinct DNA sequence and DNA strand preferences.

Tacrine (1,2,3,4-tetrahydro-9-aminoacridine; THA), a reversible centrally acting anticholinesterase, has been shown to be potentially useful for treatment of patients with Alzheimer's disease. However, currently available forms of THA may be therapeutically limited by the fact that high doses have resulted in liver and kidney damage. To determine if THA is hepatotoxic via a genotoxic mechanism, we evaluated its ability to induce unscheduled DNA synthesis (UDS) in primary cultures of rodent hepatocytes. Positive dose-dependent increases in UDS were observed in hepatocytes derived from male B6C3F1 mice and from young, middle-aged, old, and old Aroclor-induced (ARO) male F344 rats maintained on either an ad libitum (AL) or a caloric restricted (CR) diet (60% of AL) and exposed to 0.05-1000.0 micrograms/ml of THA. Hepatocytes from old AL rats, treated with THA, exhibited significant age-related decreases in DNA repair compared to young and middle-aged AL rats. By contrast, cultures from CR rats exhibited age- and diet-related decreases in UDS from the AL and young CR animals, respectively. Moreover, ARO-induced old AL- and CR-derived hepatocytes exhibited significant increases in UDS compared to uninduced old AL and CR animals. No cytotoxicity was observed in the uninduced old AL- or any CR-derived hepatocytes. These data indicate that the aged and CR fed animal is less susceptible to the cytotoxic and genotoxic effects of THA; while the younger AL fed and enzyme induced old AL or CR fed animals were more susceptible. The data suggest that THA may be a genotoxic rodent carcinogen. At present, the relationship of these findings to the clinical use of THA are unclear and further study is required.

Pretreatment of animals with mixed-function oxidase inducers has been shown to increase the metabolic activation capacity of isolated hepatocytes resulting in an apparent increase in DNA repair. We recently reported decreases in chemically-induced DNA repair, measured as unscheduled DNA synthesis (UDS), in hepatocyte cultures isolated from aging ad libitum (AL) and caloric restricted (CR) diet-fed animals. In the present study, we evaluated the effects of pretreatment with Aroclor 1254 (ARO) on the genotoxicity of 4 carcinogens, from different chemical classes, in primary hepatocytes isolated from male Fischer 344 rats. ARO-induced old AL- and CR-derived cultures, treated with 2-acetylaminofluorene (2-AAF), aflatoxin B1 (AFB1), 7,12-dimethylbenz[a]anthracene (DMBA), and dimethylnitrosamine (DMN), exhibited significant induction-related increases in DNA repair in comparison to uninduced old AL and CR animals. These data indicate that the constitutive levels of specific cytochrome P450 decline with age and chronic caloric restriction, while the ability to respond to exogenous inducers is retained, and suggest that DNA repair may not be modified with age or diet restriction.

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, middle-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 microM 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.

The effects of primary hepatocyte culture on the rat cytochrome P450-dependent monooxygenase system and several conjugating enzyme activities were examined using a culture system similar to those used for evaluation of chemicals as potential genotoxins. Cytochrome P450 and cytochrome b5 contents progressively decreased throughout the 72-h culture period to less than 25% of initial values, whereas cytochrome P450 reductase rapidly decreased by 50% during attachment, but then remained stable. Cytochrome P450-dependent testosterone hydroxylase activities decreased more rapidly in culture than did cytochrome P450 content reaching less than 50% of attachment levels by 24 h. Cytochrome P450IIIA immunoreactive protein decreased at a similar rate to testosterone-6 beta-hydroxylase. Activated UDP-glucuronyltransferase activities towards 1-naphthol and testosterone declined more slowly over the 72 h than cytochrome P450 and remained at 50-60% of initial values at 72 h. UDP-glucuronyltransferase activity towards digitoxigenin monodigitoxoside (DIG) did not decrease during culture. Glutathione-S-transferase and sulfotransferase activities also declined during the 72 h at rates which appeared to be isozyme-dependent. Addition of 1 microM dexamethasone (DEX) to the culture medium increased UDP-glucuronyltransferase activity towards DIG, cytochrome P450 reductase and testosterone-6 beta-hydroxylase activities up to 2.5-, 2.0- and 7-fold, respectively and induced cytochrome P450IIIA immunoreactive protein(s) in the hepatocytes after 24 and 48 h of culture; DEX was less effective at the 72 h time-point. DEX treatment also significantly accelerated the decreases in glutathione-S-transferase activities and in sulfotransferase activities towards 1-naphthol and estrone. Thus, it appears that primary rat hepatocytes cultured under standard conditions, not only rapidly lose their monooxygenase capabilities, but also some of their capacity for conjugation. Furthermore, the use of DEX in cell culture medium to enhance cell survival does not maintain total drug-metabolizing enzyme capability, but appears to transiently and selectively increase expression of certain isozymes at the expense of others.

Methapyrilene and four related antihistamines were evaluated for their ability to cause DNA repair measured autoradiographically as unscheduled DNA synthesis (UDS) in primary cultures of Fischer-344 rat hepatocytes. Methapyrilene failed to induce UDS at all doses tested while pyrilamine and tripelennamine induced a concentration-dependent increase in DNA repair. Doxylamine and thenyldiamine, previously untested in this system, induced a weak response at the highest non-toxic doses tested. Methapyrilene was clearly cytotoxic at doses of 100 microM and higher, as judged by morphology, and precursor incorporation into RNA and protein. Precursor incorporation into RNA was irreversibly inhibited 90% and 55% at 1000 microM and 100 microM methapyrilene, respectively, while precursor incorporation into protein was inhibited 80% and 60%. These data verify the genotoxicity of pyrilamine and tripelennamine and the failure of methapyrilene to elicit DNA repair, and suggest that doxylamine and thenyldiamine may be weak DNA-damaging agents.

The antihistamine methapyrilene hydrochloride has been shown to be a potent hepatocarcinogen in Fischer 344 rats. It has also been evaluated in a number of short-term in vitro genotoxicity assays resulting in conflicting reports. Short-term in vivo assays suggest that it may act as a promoter. We studied its ability to form DNA adducts in the target organ using the highly sensitive 32P-postlabeling technique. Methapyrilene failed to induce formation of DNA adducts in hepatocellular DNA at doses which induced S-phase DNA synthesis. These data suggest that methapyrilene does not induce the carcinogenesis process through a direct genotoxic mechanism.