Differentiating between micronucleus dose-responses induced by whole cigarette smoke solutions with Benchmark Dose potency ranking.

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.

In vitro mammalian cell mutation assays based on the Pig-a gene: A report of the 7th International Workshop on Genotoxicity Testing (IWGT) Workgroup.

Pig-a gene mutation assays enumerate cells with the glycosylphosphatidylinositol (GPI) anchor-deficient phenotype as a reporter of mutation in the endogenous Pig-a gene. Methods for measuring mutation in this gene are quite well established for in vivo systems. This approach to mutagenicity assessment has now been extended to in vitro mammalian cell-based systems. An expert workgroup from the 7th International Workshop on Genotoxicity Testing tasked with assessing the status of in vitro mammalian cell mutation assays has investigated the merits and limitations of in vitro Pig-a gene mutation assays. A review of the current status of these developing methodologies and the formation of consensus statements on the utility and application of these assays were performed to provide guidance for their potential use in genotoxicity hazard identification and risk assessment.

Derivation of point of departure (PoD) estimates in genetic toxicology studies and their potential applications in risk assessment.

Genetic toxicology data have traditionally been employed for qualitative, rather than quantitative evaluations of hazard. As a continuation of our earlier report that analyzed ethyl methanesulfonate (EMS) and methyl methanesulfonate (MMS) dose-response data (Gollapudi et al., 2013), here we present analyses of 1-ethyl-1-nitrosourea (ENU) and 1-methyl-1-nitrosourea (MNU) dose-response data and additional approaches for the determination of genetic toxicity point-of-departure (PoD) metrics. We previously described methods to determine the no-observed-genotoxic-effect-level (NOGEL), the breakpoint-dose (BPD; previously named Td), and the benchmark dose (BMD10 ) for genetic toxicity endpoints. In this study we employed those methods, along with a new approach, to determine the non-linear slope-transition-dose (STD), and alternative methods to determine the BPD and BMD, for the analyses of nine ENU and 22 MNU datasets across a range of in vitro and in vivo endpoints. The NOGEL, BMDL10 and BMDL1SD PoD metrics could be readily calculated for most gene mutation and chromosomal damage studies; however, BPDs and STDs could not always be derived due to data limitations and constraints of the underlying statistical methods. The BMDL10 values were often lower than the other PoDs, and the distribution of BMDL10 values produced the lowest median PoD. Our observations indicate that, among the methods investigated in this study, the BMD approach is the preferred PoD for quantitatively describing genetic toxicology data. Once genetic toxicology PoDs are calculated via this approach, they can be used to derive reference doses and margin of exposure values that may be useful for evaluating human risk and regulatory decision making.

Report of the IWGT working group on strategies and interpretation of regulatory in vivo tests I. Increases in micronucleated bone marrow cells in rodents that do not indicate genotoxic hazards.

In vivo genotoxicity tests play a pivotal role in genotoxicity testing batteries. They are used both to determine if potential genotoxicity observed in vitro is realised in vivo and to detect any genotoxic carcinogens that are poorly detected in vitro. It is recognised that individual in vivo genotoxicity tests have limited sensitivity but good specificity. Thus, a positive result from the established in vivo assays is taken as strong evidence for genotoxic carcinogenicity of the compound tested. However, there is a growing body of evidence that compound-related disturbances in the physiology of the rodents used in these assays can result in increases in micronucleated cells in the bone marrow that are not related to the intrinsic genotoxicity of the compound under test. For rodent bone marrow or peripheral blood micronucleus tests, these disturbances include changes in core body temperature (hypothermia and hyperthermia) and increases in erythropoiesis following prior toxicity to erythroblasts or by direct stimulation of cell division in these cells. This paper reviews relevant data from the literature and also previously unpublished data obtained from a questionnaire devised by the IWGT working group. Regulatory implications of these findings are discussed and flow diagrams have been provided to aid in interpretation and decision-making when such changes in physiology are suspected.

Report of the IWGT working group on strategy/interpretation for regulatory in vivo tests II. Identification of in vivo-only positive compounds in the bone marrow micronucleus test.

A survey conducted as part of an International Workshop on Genotoxicity Testing (IWGT) has identified a number of compounds that appear to be more readily detected in vivo than in vitro. The reasons for this property varies from compound to compound and includes metabolic differences; the influence of gut flora; higher exposures in vivo compared to in vitro; effects on pharmacology, in particular folate depletion or receptor kinase inhibition. It is possible that at least some of these compounds are detectable in vitro if a specific in vitro test is chosen as part of the test battery, but the 'correct' choice of test may not always be obvious when testing a compound of unknown genotoxicity. It is noted that many of the compounds identified in this study interfere with cell cycle kinetics and this can result in either aneugenicity or chromosome breakage. A decision tree is outlined as a guide for the evaluation of compounds that appear to be genotoxic agents in vivo but not in vitro. The regulatory implications of these findings are discussed.

Mutagenicity of comfrey (Symphytum Officinale) in rat liver.

Comfrey is a rat liver toxin and carcinogen that has been used as a vegetable and herbal remedy by humans. In order to evaluate the mechanisms underlying its carcinogenicity, we examined the mutagenicity of comfrey in the transgenic Big Blue rat model. Our results indicate that comfrey is mutagenic in rat liver and the types of mutations induced by comfrey suggest that its tumorigenicity results from the genotoxicity of pyrrolizidine alkaloids in the plant.

Analysis of mutations and bone marrow micronuclei in Big Blue rats fed leucomalachite green.

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.

Mutagenicity and carcinogenicity in relation to DNA adduct formation in rats fed leucomalachite green.

Leucomalachite green is a persistent and prevalent metabolite of malachite green, a triphenylmethane dye that has been used widely as an antifungal agent in the fish industry. Concern over the use of malachite green is due to the potential for consumer exposure, evidence suggestive of tumor promotion in rodent liver, and suspicion of carcinogenicity based on structure-activity relationships. Our previous study indicated that feeding rodents malachite or leucomalachite green resulted in a dose-related increase in liver DNA adducts, and that, in general, exposure to leucomalachite green caused an increase in the number and severity of changes greater than was observed following exposure to malachite green. To characterize better the genotoxicity of leucomalachite green, female Big Blue rats were fed leucomalachite green at doses of 0, 9, 27, 91, 272, or 543 ppm for up to 32 weeks. The livers were analyzed for lacI mutations at 4, 16, and 32 weeks and DNA adducts at 4 weeks. Using a 32P-postlabeling assay, we observed a dose-related DNA adduct in the livers of rats fed 91, 272, and 543 ppm leucomalachite green. A approximately 3-fold increase in lacI mutant frequency was found in the livers of rats fed 543 ppm leucomalachite green for 16 weeks, but significant increases in mutant frequencies were not found for any of the other doses or time points assayed. We also conducted 2-year tumorigenesis bioassays in female and male F344 rats using 0, 91, 272, and 543 ppm leucomalachite green. Preliminary results indicate an increasing dose trend in lung adenomas in male rats treated with leucomalachite green, but no increase in the incidence of liver tumors in either sex of rat. These results suggest that the DNA adduct formed in the livers of rats fed leucomalachite green has little mutagenic or carcinogenic consequence.

Prospects for applying genotypic selection of somatic oncomutation to chemical risk assessment.

Genotypic selection methods detect rare sequence changes in populations of DNA molecules. These methods have been used to investigate the chemical induction of mutation and for the detection and diagnosis of cancer. The possible use of genotypic selection for improving current risk assessment practices is based on the premise that the frequency of somatic mutation is of critical importance in understanding and modeling carcinogenesis. If genotypic selection can measure the induction of specific mutations that disrupt normal cell/tissue homeostasis, then it could provide key mechanistic information for cancer risk assessment. For example, genotypic selection data might support a particular low-dose extrapolation method or characterize the relationship between rodent and human cancer risk. Strategies for evaluating the use of genotypic selection in cancer risk assessment include the concept of developing a battery of targets that detect a range of agent-specific effects. Ideal targets to examine by genotypic selection are the oncogene and tumor suppressor gene mutations frequently detected in human tumors because these are thought to represent tumor-initiating events. The most commonly occurring basepair (bp) substitutions within the ras and p53 genes are identified. Also, the battery of genotypic selection methods is defined in terms of the most important mutational specificities to include. In theory, the major basepair substitution mutations induced by 29 of 31 chemical carcinogens could be detected by analyzing three different mutations: G:C-->T:A, G:C-->A:T, and A:T-->T:A. Genotypic selection will have the greatest impact on risk assessment if measurement of spontaneous mutation is possible. Data from phenotypic selection assays suggest this corresponds to detection of mutant fractions of approximately 10(-7), and this would necessitate examining DNA samples containing >10(7) target molecules. Despite its apparent potential, considerable development and validation is needed before genotypic selection data can be applied to cancer risk assessment.

DNA adduct formation and mutant induction in Sprague-Dawley rats treated with tamoxifen and its derivatives.

The non-steroidal anti-estrogen tamoxifen is used as an adjunct chemotherapeutic agent for the treatment of all stages of breast cancer and more recently as a chemoprotective agent in women with elevated risk of developing breast cancer. While beneficial for the treatment of breast cancer, tamoxifen increases the risk of endometrial cancer. In addition, it has been shown to induce liver and endometrial tumors in rats. Tamoxifen is genotoxic in rat liver, as indicated by the formation of DNA adducts, through a metabolic pathway involving the alpha-hydroxylation of tamoxifen and N-desmethyltamoxifen. Since the contribution of these alpha-hydroxy metabolites of tamoxifen to the induction of endometrial tumors is presently unknown, we compared the extent of DNA adduct formation in liver and selected non-hepatic tissues of female Sprague-Dawley rats treated by gavage with tamoxifen, alpha-hydroxytamoxifen, N-desmethyltamoxifen, alpha-hydroxy-N-desmethyltamoxifen and N,N-didesmethyltamoxifen, or intraperitoneal injection with tamoxifen, alpha-hydroxytamoxifen, 3-hydroxytamoxifen and 4-hydroxytamoxifen. In addition, spleen lymphocytes from rats treated by gavage with tamoxifen or alpha-hydroxytamoxifen were assayed for the induction of mutants in the hypoxanthine phosphoribosyl transferase (Hprt) gene. The relative levels of binding in rats treated by gavage were alpha-hydroxytamoxifen > tamoxifen approximately N-desmethyltamoxifen approximately alpha-hydroxy-N-desmethyltamoxifen > N,N-didesmethyltamoxifen. In rats dosed intraperitoneally, the relative order of binding was alpha-hydroxytamoxifen > tamoxifen > 3-hydroxytamoxifen approximately 4-hydroxytamoxifen. None of the compounds resulted in an increase in DNA adducts in uterus, spleen, thymus or bone marrow DNA from rats treated by gavage or in uterus DNA from rats injected intraperitoneally. Neither tamoxifen nor alpha-hydroxytamoxifen increased the Hprt mutant frequency in spleen T-lymphocytes. These results confirm previous observations that tamoxifen is activated to a genotoxic agent in rat liver through alpha-hydroxylation, and also suggest that endometrial tumors in rats do not arise from the formation of tamoxifen-DNA adducts.

Comparison of hprt and lacI mutant frequency with DNA adduct formation in N-hydroxy-2-acetylaminofluorene-treated Big Blue rats.

N-Hydroxy-2-acetylaminofluorene (N-OH-AAF) is the proximate carcinogenic metabolite of the powerful rat liver carcinogen 2-acetylaminofluorene. In this study, transgenic Big Blue(R) rats were used to examine the relationship between in vivo mutagenicity and DNA adduct formation by N-OH-AAF in the target liver compared with that in nontarget tissues. Male rats were given one, two, or four doses of 25 mg N-OH-AAF/kg body weight by i.p. injection at 4-day intervals, and groups of treated and control rats were euthanized up to 10 weeks after beginning the dosing. Mutant frequencies were measured in the spleen lymphocyte hprt gene, and lacI mutant frequencies were determined in the liver and spleen lymphocytes. At 6 weeks after beginning the dosing, the hprt mutant frequency in spleen lymphocytes from the four-dose group was 16.5 x 10(-6) compared with 3.2 x 10(-6) in control animals. Also at 6 weeks, rats given one, two, or four doses of N-OH-AAF had lacI mutant frequencies in the liver of 97.6, 155.6, and 406.8 x 10(-6), respectively, compared with a control frequency of 25.7 x 10(-6); rats given four doses had lacI mutant frequencies in spleen lymphocytes of 55.8 x 10(-6) compared with a control frequency of 20.4 x 10(-6). Additional rats were evaluated for DNA adduct formation in the liver, spleen lymphocytes, and bone marrow by (32)P-postlabeling. Adduct analysis was conducted 1 day after one, two, and four treatments with N-OH-AAF, 5 days after one treatment, and 9 days after two treatments. N-(Deoxyguanosin-8-yl)-2-aminofluorene was the major DNA adduct identified in all the tissues examined. Adduct concentrations increased with total dose to maximum values in samples taken 1 day after two doses, and remained essentially the same after four doses. In samples taken after four doses, adduct levels were 103, 28, and 7 fmol/microg of DNA in liver, spleen lymphocytes, and bone marrow, respectively. The results indicate that the extent of both DNA adduct formation and mutant induction correlates with the organ specificity for N-OH-AAF carcinogenesis in the rat. Environ. Mol. Mutagen. 37:195-202, 2001. Published 2001 Wiley-Liss, Inc.

Gene- and tissue-specificity of mutation in Big Blue rats treated with the hepatocarcinogen N-hydroxy-2-acetylaminofluorene.

In a previous study, we found that treating transgenic Big Blue rats with the hepatocarcinogen N-hydroxy-2-acetylaminofluorene (N-OH-AAF) produced the same major DNA adduct in the target liver and the nontarget spleen lymphocytes and bone marrow cells, induced lacI mutants in the liver, and induced much lower frequencies of lacI and hprt mutants in spleen lymphocytes. In the present study, sequence analysis was conducted on lacI DNA and hprt cDNA from the mutants, to determine the mutational specificity of N-OH-AAF in the rat. All the mutation spectra from N-OH-AAF-treated rats differed significantly from corresponding mutation profiles from untreated animals (P = 0.02 to P < 0.0001). Although there were similarities among the mutational patterns derived from N-OH-AAF-treated rats (e.g., G:C --> T:A transversion was the most common mutation in all mutation sets), there were significant differences in the patterns of basepair substitution and frameshift mutation between the liver and spleen lymphocyte lacI mutants (P = 0.02) and between the spleen lymphocyte lacI and hprt mutants (P = 0.04). Also, multiplex PCR analysis of genomic DNA from the hprt mutants indicated that 12% of mutants from treated rats had major deletions in the hprt gene; no corresponding incidence of large deletions was evident among lacI mutations. All the mutation profiles reflect the general mutational specificity of the major DNA adduct formed by N-OH-AAF. The differences between N-OH-AAF mutation in the endogenous gene and transgene can be partially explained by the structures of the two genes. The tissue-specificity of the mutation spectra may contribute to targeting tumor formation to the liver. Environ. Mol. Mutagen. 37:203-214, 2001. Published 2001 Wiley-Liss, Inc.

Comparison of the mutations at Hprt exon 3 of T-lymphocytes from B6C3F1 mice and F344 rats exposed by inhalation to 1,3-butadiene or the racemic mixture of 1,2:3,4-diepoxybutane.

Experiments were conducted to define the spectra of mutations occurring in Hprt exon 3 of T-cells isolated from spleens of female B6C3F1 mice and F344 rats exposed by inhalation to 1,3-butadiene (BD) or its reactive metabolite, (+/-)-diepoxybutane (DEB). Hprt mutant frequencies (Mfs) in BD-exposed (1250 ppm for 2 weeks or 625 ppm for 4 weeks; 6 h/day, 5 days/week) and DEB-exposed (2 or 4 ppm for 4 weeks or 5 ppm for 6 weeks; 6 h/day, 5 days/week) mice and rats were significantly increased over concurrent control values. Mutant T-cell colonies from control and treated animals were screened for mutations in Hprt exon 3 using PCR amplification of genomic DNA and denaturing gradient gel electrophoresis, followed by sequence analysis. Exon 3 mutations were found at the following frequencies: 20/394 (5%) in control mice, 56/712 (8%) in BD-exposed mice, 59/1178 (5%) in BD-exposed rats, 66/642 (10%) in DEB-exposed mice, and 51/732 (7%) in DEB-exposed rats. Mutations in exposed animals included base substitutions, small deletions (1 to 74 bp), and small insertions (1 to 8 bp), with base substitutions predominating. Among the types of base substitutions observed in mice, the proportions of G.C-->A.T transitions (p=0.035, Fisher's Exact Test) and G.C-->C.G transversions (p=0.05) were significantly different in control vs. BD-exposed animals. Given the small number of exon 3 mutants analyzed, there was a high degree of overlap in the mutational spectra between BD-exposed mice and rats, between BD- and DEB-exposed mice, and between BD- and DEB-exposed rats in terms of the sites with base substitutions, the mutations found at those mutated sites, the relative occurrence of the most frequently observed base substitutions, and the occurrence of a consistent strand bias for the most frequently observed base substitutions. The spectra data suggest that adduction of both G.C and A.T bps is important in the induction of in vivo mutations by BD metabolites in exposed mice and rats.

7,12-dimethylbenz[a]anthracene-induced mutation in the Tk gene of Tk(+/-) mice: automated scoring of lymphocyte clones using a fluorescent viability indicator.

7,12-Dimethylbenz[a]anthracene (DMBA) is a rodent carcinogen and a potent in vivo mutagen for the X-linked hypoxanthine guanine phosphoribosyl transferase (hprt) gene of rats and for the lacI transgene of Big Blue mice and rats. Although DMBA is also a powerful clastogen, molecular analysis of these DMBA-induced hprt and lacI mutations indicates that most are single base-pair (bp) substitutions and 1- to 3-bp frameshifts. In the present study, we evaluated the types of mutations induced by DMBA in the autosomal thymidine kinase (Tk) gene of Tk(+/-) mice. Male and female 5- to 6-week-old animals were injected i.p. with DMBA at a dose of 30 mg/kg. Five weeks after the treatment, hprt and Tk mutant frequencies were determined using a limiting dilution clonal assay in 96-well plates. We established conditions for the automated identification of wells containing expanded lymphocyte clones using the fluorescent indicator alamarBlue. This procedure allowed the unbiased identification of viable clones and calculation of mutant frequencies. In male mice, DMBA treatment increased the frequency of hprt mutants from 1.8 +/- 1.1 to 34 +/- 9 x 10(-6), and Tk mutants from 33 +/- 12 to 78 +/- 26 x 10(-6); treated female mice had a significant but lower increase in hprt mutant frequency than did males. Molecular analysis of DMBA-induced Tk mutants revealed that at least 75% had the entire wild-type Tk allele missing. The results indicate that the predominant types of DMBA-induced mutation detected by the autosomal Tk gene are different from those detected by the X-linked hprt gene. The Tk gene mainly detects loss of heterozygosity mutation, whereas the majority of mutations previously found in the hprt gene were point mutations.

Development of a mouse cell line containing the PhiX174 am3 allele as a target for detecting mutation.

Transgenic mice containing multiple copies of the PhiX174 am3 allele are being developed as a model for detecting tissue-specific in vivo mutation. In order to derive an analogous system for measuring am3 mutation in vitro, cells were cultured from 15-day-old C57Bl/6J mouse embryos that were homozygous for the transgene and these cells were transfected with a plasmid expressing the SV40 large T-antigen. Two G418-resistant colonies were isolated from this culture and expanded to continuously proliferating cell lines (PX-1 and PX-2). Line PX-2 was treated with up to 1.0 mg/ml of N-ethyl-N-nitrosourea (ENU), assayed for survival by cloning efficiency after overnight culture, and assayed for am3 mutations after 5 days of culture. Survival decreased to 31% at the highest dose of ENU, while mutant frequency increased with dose from approximately 2 x 10(-7) in the untreated cells to 13 x 10(-7) in cultures treated with 0.6 mg/ml of ENU. PX-2 cells also were treated with 0 and 0.6 mg/ml of ENU and mutant frequency assays were performed after 5, 24, 48 and 72 h of growth. The mutant frequency in the treated culture increased to 20 x 10(-7) at 48 h and remained approximately the same at 72 h. These results indicate that PX-2 cells should be a useful resource for developing the in vivo am3 mutant assay and for evaluating the sensitivity of the am3 allele to various classes of mutagens.

Hprt lymphocyte mutant frequency in relation to DNA adduct formation in rats fed the hepatocarcinogen 2-acetylaminofluorene.

The lymphocyte hypoxanthine-guanine phosphoribosyltransferase (Hprt) assay is frequently used as a biomarker for the exposure of both humans and laboratory animals to potentially carcinogenic agents. To obtain information concerning the sensitivity of the rat Hprt lymphocyte assay toward aromatic amine carcinogens, male F344 rats were fed 0.02% 2-acetylaminofluorene (2-AAF) for 1 month and then returned to control diet for 2 months. At 4, 27, 48, 62, and 90 days after the initiation of 2-AAF-feeding, the frequency of mutants in the Hprt gene was determined. In addition, DNA was isolated from liver nuclei, spleen lymphocytes, bone marrow, and thymus, and DNA adducts were analyzed by 32P-postlabeling. 2-AAF feeding resulted in a significant induction of 6-thioguanine-resistant T-lymphocytes and the mutant frequency continued to increase after the 2-AAF feeding was stopped. The same major DNA adduct, N-(deoxyguanosin-8-yl)-2-aminofluorene, was detected in liver, spleen lymphocytes, bone marrow, and thymus. DNA adduct levels were greatest in the tumor target tissue (liver) but occurred in all T-lymphocyte compartments, being highest in spleen lymphocytes. The DNA adduct levels were highest at the end of the 1-month 2-AAF feeding period and decreased rapidly in all tissues. The data indicate that the Hprt lymphocyte mutagenesis assay detects arylamine carcinogens, but with relatively low sensitivity.

Evaluation of the transgenic p53+/- mouse for detecting genotoxic liver carcinogens in a short-term bioassay.

The transgenic p53-deficient heterozygous (p53+/-) mouse is prone to both spontaneous and induced tumors and has been proposed for use in a sensitive, short-term (6 months) assay for identifying genotoxic, multispecies carcinogens. It is not clear, however, if a short-term assay with p53+/- mice detects agents that target certain organs, in particular, the liver. In this study, we treated neonatal male p53+/- and p53+/+ mice with the genotoxic carcinogens dimethylnitrosamine (DMN), 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP), and 6-nitrochrysene (6-NC). In keeping with the methodology of the proposed short-term assay, the p53+/- mice were evaluated for tumors 7 months after treatment. Wild-type neonatal mice treated with genotoxic carcinogens are known to develop tumors within 1 year; hence, the p53+/+ animals used as controls were subjected to pathological examination at 1 year of age. Our results showed that PhIP was not tumorigenic in either group of mice. Liver tumor incidence increased significantly in the p53+/+ mice treated with DMN and 6-NC, indicating that the conditions of the bioassay were conducive to the promotion of liver tumorigenesis. On the other hand, these two chemicals failed to induce a significant increase in liver tumors in the p53+/- mice by seven months. This result suggests that a deficiency in the amount of p53 protein does not lead to accelerated liver tumorigenesis in mice, and contrasts with previous reports that show a decreased latency of tumors in non-liver targets.

Mutagenicity of 1,3-butadiene at the Hprt locus of T-lymphocytes following inhalation exposures of female mice and rats.

The species specific response to 1,3-butadiene (BD), an important industrial chemical, was investigated by determining the influence of exposure duration and exposure concentration on the mutagenicity of BD in mice and rats and by defining the spectra of mutations in the Hprt gene T-cell mutants from control and BD-exposed mice. Female B6C3F1 mice and F344 rats (4-5 weeks old) were exposed by inhalation to 0, 20, 62.5, or 625 ppm of BD for up to 4 weeks (6 h/day, 5 days/week). Groups of control and exposed animals (n=4-12/group) were necropsied at multiple time points after exposure and the T-cell cloning assay was used to measure Hprt mutant frequencies in lymphocytes isolated from spleen. Mutant clones collected from control and BD-exposed mice were propagated and analyzed by RT-PCR to produce Hprt cDNA for sequencing. In animals necropsied 4 weeks after 2 or 4 weeks of BD exposure (0 or 625 ppm), the rate of accumulation of mutations was greater in mice than in rats. Supra-linear dose-response curves were observed in BD-exposed mice, indicating a higher efficiency of mutant induction at lower concentrations of BD. The mutagenic potency estimates (represented by the differences in the areas under the mutant T-cell 'manifestation' curves of treated vs. control animals) in mice were 11 and 61 following 4 weeks of exposures to 62.5 and 625 ppm of BD, respectively, while mutant frequencies (Mfs) in rats were significantly increased only at 625 ppm BD (mutagenic potency of 7). Molecular analysis of Hprt cDNA from expanded T-cell clones from control and BD-exposed mice demonstrated an increased frequency of mutants in exposed animals that likely contain large deletions in the Hprt gene (P=0.016). These data indicate that both exposure duration and exposure concentration are important in determining the magnitude of mutagenic response to BD, and that mutagenic and carcinogenic properties of BD in mice may be related more to the ability of its metabolites to cause chromosomal deletions than to produce point mutations.

Molecular analysis of in vivo mutations induced by N-ethyl-N-nitrosourea in the autosomal Tk and the X-linked Hprt genes of mouse lymphocytes.

The endogenous, autosomal Tk gene is a potentially useful reporter of in vivo mutation since it may recover a wider range of mutational events than the X-linked Hprt gene or bacterial transgenes. In this study, we characterized mutations produced in the Tk gene of Tk(+/-) mice and compared them with mutations induced in the Hprt gene. Treatment of Tk(+/-) mice with N-ethyl-N-nitrosourea (ENU) resulted in dose-related increases in Tk mutants, as measured by the frequency of 5-bromodeoxyuridine-resistant (BrdUrd(r)) spleen lymphocytes. ENU-induced mutant frequencies in the Hprt gene, determined by measuring 6-thioguanine-resistant (TG(r)) lymphocytes, were similar to the Tk mutant frequencies. Allele-specific PCR of DNA from BrdUrd(r) lymphocyte clones suggested that 35% of clones from mice treated with ENU and 65% of clones from untreated animals had loss of heterozygosity (LOH) of the Tk gene due to deletion of the functional Tk allele. Reverse transcriptase-PCR/sequencing analysis of BrdUrd(r) and TG(r) clones from ENU-treated mice indicated that point mutations in both genes predominantly occurred at A:T basepairs; however, A:T-->G:C transition was the most common mutation in the Tk gene, while A:T-->T:A transversion was the most frequent mutation in the Hprt gene. Substitution at A:T basepairs in the Hprt gene occurred disproportionately with the mutated dT on the nontranscribed DNA strand, while this strand bias for mutation was not seen in the Tk gene. The results indicate that the specificity of ENU-induced point mutation differs between the two endogenous genes and that the autosomal Tk gene of Tk(+/-) mice is capable of recovering mutations caused by LOH. Environ. Mol. Mutagen. 34:30-38, 1999 Published 1999 Wiley-Liss, Inc.

Tumorigenicity and liver tumor ras-protooncogene mutations in CD-1 mice treated neonatally with 1- and 3-nitrobenzo[a]pyrene and their trans-7,8-dihydrodiol and aminobenzo[a]pyrene metabolites.

The environmental pollutants 1- and 3-nitrobenzo[a]pyrene (1- and 3-NBaP) are metabolized by mammalian microsomes through ring oxidation to 1-NBaP trans-7,8-dihydrodiol and 3-NBaP trans-7,8-dihydrodiol, and by nitroreduction to 1- and 3-aminobenzo[a]pyrene. To determine if these compounds are tumorigenic, 1- and 3-NBaP, along with several of their metabolites and the parent benzo[a]pyrene (BaP) and its trans-7,8-dihydrodiol metabolite, were tested in the neonatal CD-1 mouse bioassay. Male mice were administered i.p. injections at a total dose of 100 or 400 nmol per mouse on 1, 8 and 15 days after birth. While the liver tumor incidences for BaP, BaP trans-7,8-dihydrodiol, and the positive control 6-nitrochrysene (6-NC) were significantly higher than in the solvent control animals, all the other tested compounds exhibited no tumorigenicity. The frequency of Ha- and Ki-ras mutations in liver tumors of mice treated with BaP, BaP trans-7,8-dihydrodiol, and 6-NC were higher than in the few liver tumors isolated from control mice or mice treated with the NBaPs or their metabolites. Since 1- and 3-NBaP and their metabolites are potent mutagens in the Salmonella assay and moderate mutagens in the Chinese hamster ovary (CHO) mammalian mutagenicity assay, our results indicate that the in vitro mutagenicity of these compounds does not correlate with their tumorigenicity.