Molecular analysis of lacI mutants from bone marrow of B6C3F1 transgenic mice following inhalation exposure to 1,3-butadiene

Chronic exposure to 1,3-butadiene (BD) results in early occurrenceand high incidence of lethal lymphomas in male B6C3F1 mice.Male B6C3F1 lacI transgenic mice (BigBlue^TM) were exposed byinhalation to 0, 62.5, 625 or 1250 p.p.m. BD for 4 weeks (6h/day, 5 days/week). The lacI^- mutant frequency and mutationalspectrum were evaluated in DNA isolated from bone marrow cells.Two weeks after exposure the lacI^- mutant frequency in bonemarrow from BD-exposed mice had increased 2- to 3.5-fold overair control mice. DNA sequence analysis of 56 and 54 lacI^- mutantsfrom the air control and butadiene-exposed groups, respectively,demonstrated that there was a shift in the spectrum of basesubstitution mutations at A:T sites in BD-exposed mice (6/26)compared to the air control mice (2/45). A:T     

Mutagenicity of butadiene and its epoxide metabolites: II. Mutational spectra of butadiene, 1,2-epoxybutene and diepoxybutane at the hprt locus in splenic T cells from exposed B6C3F1 mice

The mutagenic potential and mutational spectra of butadiene(BD), 1,2-epoxybutene (EB), and diepoxybutane (DEB) were determinedin splenic T cells from exposed B6C3F1 mice. Mice exposed byinhalation to 625 p.p.m. BD for 2 weeks displayed an averagehprt^– mutation frequency of 6.2 x10^–6 compared to1.2x10^–6 in controls. Mice were also given three dailyi.p. doses of 60, 80 and 100 mg EB/kg or 7, 14 and 21 mg DEB/kg.Average hprt^– frequencies of 5.4x10^–6, 4.lx10^–6and 8.6x10^–6 were seen in the EB groups, respectively,while average frequencies of 4.6x10^–6, 9.4x10^–6and 13x10^–6 were seen in the DEB groups. DNA sequencingrevealed that approximately half of the mutations induced invivo by BD, EB and DEB were frameshift mutations. A +1 frameshift‘hotspot’ in six consecutive guanine bases in exon3 was observed with all three compounds. The remaining mutationsproduced by BD, EB and DEB were transition and transversionmutations at both AT and GC base pairs. Base pair substitutionsinduced by BD were biased in favor of mutation at AT base pairs.The mutational spectra produced by BD, EB and DEB were verysimilar to that observed previously with ethylene oxide, suggestingthat these epoxide agents may be working through a similar mutagenicmechanism.     

Mutations induced by alpha-hydroxytamoxifen in the lacI and cII genes of Big Blue transgenic rats

The antiestrogen tamoxifen is widely used for the treatment of breast cancer and more recently for the prevention of breast cancer. A concern over the use of tamoxifen as a chemopreventive agent is its carcinogenicity in rat liver, through a genotoxic mechanism involving alpha-hydroxylation, esterification, and DNA adduct formation, primarily by reaction with dG. In a recent study [Gamboa da Costa et al., Cancer Lett., 176, 37-45 (2002)], we demonstrated a significant increase in the mutant frequency in the lacI gene of Big Blue rats treated with tamoxifen, and a further increase in rats administered alpha-hydroxytamoxifen. In the present study, we have assessed mutation induction by tamoxifen and alpha-hydroxytamoxifen in the liver cII gene of Big Blue rats and have characterized the types of mutations induced by alpha-hydroxytamoxifen in the liver lacI and cII genes. The mutant frequencies in the liver cII gene were 80 +/- 13 x 10(-6) in the control, 112 +/- 13 x 10(-6) in the tamoxifen-treated group (P < 0.01 vs. control), and 942 +/- 114 x 10(-6) in the alpha-hydroxytamoxifen-treated animals (P < 0.001 vs. control; P < 0.001 vs. tamoxifen). Molecular analysis of the mutants indicated that the alpha-hydroxytamoxifen-induced mutational spectrum differed significantly from the control spectrum, but was very similar to the spectrum induced by tamoxifen for both the lacI and cII genes [Davies et al., ENVIRON: Mol. Mutagen., 28, 430-433 (1996); Davies et al., Carcinogenesis, 20, 1351-1356 (1999)]. G:C --> T:A transversion was the major type of mutation induced by alpha-hydroxytamoxifen and tamoxifen, while G:C --> A:T transition was the main type of mutation in the control. These results support the hypothesis that alpha-hydroxytamoxifen is a major proximate tamoxifen metabolite causing the initiation of tumors in the liver of rats treated with tamoxifen.     

Mutagenicity of butadiene and its epoxide metabolites: I. Mutagenic potential of 1,2-epoxybutene, 1,2,3,4-diepoxybutane and 3,4-epoxy-1,2-butanediol in cultured human lymphoblasts

The mutagenic potential of the epoxide metabolites of butadiene(BD) was measured at the tk and hprt loci in TK6 human lymphoblastoidcells. TK6 cells were exposed for 24 h to 0–400 µM1,2-epoxybutene (EB), 0–800 µM 3,4-epoxy-1,2-butanediol(EBD), or 0–6 µM 1,2,3,4-diepoxybutane (DEB). Treatedcells were allowed to grow for several days and then seededin medium containing either 6-thioguanine or trifluorothymidineto select for hprt^– or tk^–/– mutants, respectively.All three metaboiltes were mutagenic at both loci, with DEBexhibiting activity at concentrations approximately 100-foldlower than EB or EBD. At the hprt locus, an induced mutationfrequency of 5 x 10^–6 (approximately twice backgroundhprt^– frequency) was produced by treatment with 3.5 µMDEB, 150 µM EB and 450 µM EBD. At the tk locus,a similar increase in mutation frequency (total tk^–/–frequency) was produced by treatment with 1.0 µM DEB,100 µM EB and 350 µM EBD. Each epoxide tested wascapable of inducing slow growth tk^–/– mutants. Thismutant phenotype, as shown previously by others, results fromlarge alterations in the tk region which completely remove theactive tk allele. In addition, Southern blot analysis revealedthat approximately half of DEB-induced hprt^– mutants displayedloss of wild-type hprt restriction fragments. No statisticallysignificant increase in the fraction of hprt deletions amongEB mutants was observed. The ability of DEB to induce deletionsmay be related to its ability to form DNA-DNA and DNA-proteincross-links.     

Quantification of DNA and hemoglobin adducts of 3,4-epoxy-1,2-butanediol in rodents exposed to 3-butene-1,2-diol

1,3-Butadiene (BD) is a confirmed rodent carcinogen and a suspect human carcinogen that forms mutagenic epoxide metabolites during biotransformation. Species differences in the roles of individual DNA reactive intermediates in BD mutagenicity and carcinogenicity are not completely understood. Evidence suggests that 1,2:3,4-diepoxybutane (DEB) is responsible for the mutagenic effect induced by exposures to low concentrations of BD in mice and that metabolites of 3-butene-1,2-diol (BD-diol) are involved in the mutagenicity at high exposures in both mice and rats. Two reactive metabolites, 3,4-epoxy-1,2-butanediol (EB-diol) and hydroxymethylvinyl ketone (HMVK), are formed during the biotransformation of BD-diol and could potentially be involved in BD-diol associated mutagenicity. To examine the role of EB-diol in BD-diol mutagenicity we have evaluated the dosimetry of N7-(2,3,4-trihydroxybutyl)guanine (THB-Gua) and N-(2,3,4-trihydroxybutyl)valine (THB-Val) in female B6C3F1 mice and female F344 rats exposed by inhalation to 0, 6, 18 and 36 p.p.m. BD-diol for 4 weeks (6 h/day x 5 days/week). Results showed higher levels of both THB-Gua and THB-Val in mice than in rats. An evaluation of THB-Gua adducts showed virtually no differences between liver and lung for either species, suggesting that EB-diol is stable and is freely circulated. The data also indicated that THB adduct formation began to plateau around 18 p.p.m. in both species. Most importantly, the shape of the dose-response curve for THB adduct formation mimicked the one observed for hypoxanthine-guanine phosphoribosyltransferase (Hprt) mutation frequency. This showed that THB adducts, which are not thought to be responsible for causing the mutations, are good quantitative indicators of mutagenicity in rodents exposed to BD-diol. Although the potential contribution of HMVK still needs to be evaluated, the data suggest that EB-diol is responsible, at least in part, for BD-diol associated mutagenicity in rodents.     

Assessment of the mutagenicity of dichloroacetic acid in lacI transgenic B6C3F1 mouse liver

Dichloroacetic acid (DCA) is a chlorination byproduct found in finished drinking water. When administered in drinking water this chemical has been shown to produce hepatocellular adenomas and carcinomas in B6C3F1 mice over the animal's lifetime. In this study, we investigated whether mutant frequencies were increased in mouse liver using treatment protocols that yielded significant tumor induction. DCA was administered continuously at either 1.0 or 3.5 g/l in drinking water to male transgenic B6C3F1 mice harboring the bacterial lacI gene. Groups of five or six animals were killed at 4, 10 or 60 weeks and livers removed. At both 4 and 10 weeks of treatment, there was no significant difference in mutant frequency between the treated and control animals at either dose level. At 60 weeks, mice treated with 1.0 g/l DCA showed a 1.3-fold increase in mutant frequency over concurrent controls (P = 0.05). Mice treated with 3.5 g/l DCA for 60 weeks had a 2.3-fold increase in mutant frequency over the concurrent controls (P = 0.002). The mutation spectrum recovered from mice treated with 3.5 g/l DCA for 60 weeks contained G:C-->A:T transitions (32.79%) and G:C-->T:A transversions (21.31%). In contrast, G:C-->A:T transitions comprised 53.19% of the recovered mutants among control animals. Although only 19.15% of mutations among the controls were at T:A sites, 32.79% of the mutations from DCA-treated animals were at T:A sites. This is consistent with the previous observation that the proportion of mutations at T:A sites in codon 61 of the H-ras gene was increased in DCA-induced liver tumors in B6C3F1 mice. The present study demonstrates DCA-associated mutagenicity in the mouse liver under conditions in which DCA produces hepatic tumors.      

Tamoxifen mutagenesis and carcinogenesis in livers of lambda/lacI transgenic rats: selective influence of phenobarbital promotion

Administration of tamoxifen (TAM) (20 mg/kg per day p.o.) for 6 weeks to female lambda/lacI transgenic rats caused a 4-fold increase in mutation frequency (MF) at the lacI gene locus in the livers of dosed animals compared with controls. After cessation of dosing, the MF showed a further increase with time at 2, 12 and 24 weeks, respectively. Phenobarbital promotion of similarly treated animals resulted in no increase in mutation frequency compared with TAM alone. Treatment with phenobarbital or TAM+phenobarbital resulted in time-dependent increases in liver weight compared with the corresponding controls. There was an increase in cell proliferation in the phenobarbital and TAM+phenobarbital groups, and at 24 weeks in the TAM dosed animals compared with controls. There was also a progressive increase in the number of GST-P expressing foci in the livers of TAM and TAM + phenobarbital rats compared with controls. The induction of cell proliferation and GSTP foci in the rat liver by phenobarbital is consistent with its ability to promote tamoxifen-initiated liver tumours in the rat. If the lacI gene is regarded as being representative of the rat genome in general (albeit that the gene is bacterial) the above observations suggest that promotion by tamoxifen confers selective advantage on mutated genes at loci that contribute to the tumour phenotype and that promotion of rat liver tumours by tamoxifen is not dependent simply upon the enhancement of cellular proliferation.     

Absence of specific chromosome alterations in bovine lung fibroblasts exposed to Rous sarcoma virus

Bovine lung fibroblasts from individual embryos were exposed to three different strains of Rous sarcoma virus (Bryan, Engelbreth-Holm, Schmidt-Ruppin) in vitro. Chromosomal analysis early as well as late after infection, including cultures with advanced morphologic cell transformation, failed to disclose any aberrations exceeding the findings in control cultures grown in parallel under identical conditions. Simian virus 40, on the other hand, caused pronounced changes with widely scattered heteroploid counts and various rearrangements.     

Oncogenes and Angiogenesis: down-regulation of thrombospondin-1 in normal fibroblasts exposed to factors from cancer cells harboring mutant ras

The onset of angiogenesis in cancer often involves down-regulation of endogenous angiogenesis inhibitors, of which thrombospondin-1 (TSP-1) is a paradigm. As this effect is thought to occur under the influence of transforming genetic lesions (e.g., expression of the mutant ras oncogene), its nature is regarded as intrinsic to cancer cells themselves. Here, we show that ras-transformed cancer cells can also induce TSP-1 down-regulation in their adjacent nontransformed stromal fibroblasts, but not in endothelial cells, in a paracrine and distance-dependent manner. Indeed, several H-ras-expressing fibrosarcoma (528ras1, B6ras, and NIH3T3Ras) and carcinoma (DLD-1 and IEC18Ras3) cells were found to release soluble factors capable of suppressing TSP-1 protein, mRNA, and promoter activity in nontumorigenic, immortalized dermal fibroblastic cell lines in culture (e.g., in fibroblasts expressing enhanced green fluorescent protein/TSP-1 reporter). This effect was abrogated in Id1-/- fibroblasts. At least two low molecular weight (<3 kDa), heat-labile, and trypsin-resistant mediators of TSP-1 suppression were found to be released from 528ras1 cells. Their effects on normal fibroblasts were inhibited (albeit to different extents) by pertussis toxin and, in one case, by dimethylsphingosine, none of which affected TSP-1 expression by 528ras1 cells. Collectively, our study suggests that the effect of mutant ras on tumor neovascularization is not limited to changes in angiogenic properties of cancer cells themselves. Rather, mutant ras, through a different signaling mechanism, may modulate the properties of the adjacent normal stroma, thus eliciting a proangiogenic field effect.     

Activation of homologous recombination DNA repair in human skin fibroblasts continuously exposed to X-ray radiation

Molecular and cellular responses to protracted ionizing radiation exposures are poorly understood. Using immunofluorescence microscopy, we studied the kinetics of DNA repair foci formation in normal human fibroblasts exposed to X-rays at a dose rate of 4.5 mGy/min for up to 6 h. We showed that both the number of γH2AX foci and their integral fluorescence intensity grew linearly with time of irradiation up to 2 h. A plateau was observed between 2 and 6 h of exposure, indicating a state of balance between formation and repair of DNA double-strand breaks. In contrast, the number and intensity of foci formed by homologous recombination protein RAD51 demonstrated a continuous increase during 6 h of irradiation. We further showed that the enhancement of the homologous recombination repair was not due to redistribution of cell cycle phases. Our results indicate that continuous irradiation of normal human cells triggers DNA repair responses that are different from those elicited after acute irradiation. The observed activation of the error-free homologous recombination DNA double-strand break repair pathway suggests compensatory adaptive mechanisms that may help alleviate long-term biological consequences and could potentially be utilized both in radiation protection and medical practices.     

Molecular dosimetry of O4-ethyldeoxythymidine in rats continuously exposed to diethylnitrosamine

There is considerable interest in incorporating mechanistically based biological data into the process of quantitative risk assessment. Presently, no adequate data bases for internal dosimeters, such as DNA adducts, exist for humans or experimental animals. Therefore, the major promutagenic ethyl adduct, O4-ethyldeoxythymidine (O4-EtdT), has been quantitated in liver DNA after continuous exposure of rats to drinking water containing 0.4, 1, 4, 10, 40, or 100 ppm diethylnitrosamine (DEN) for 1, 4, 7, 14, 28, 49, or 70 days. The rate of O4-EtdT accumulation was modeled as the difference between a DEN-dependent rate of formation and an O4-EtdT concentration-dependent rate of loss. In general, O4-EtdT concentrations increased rapidly during the first 7 days of exposure and by 7-28 days O4-EtdT had accumulated to apparent steady-state concentrations that were DEN concentration-dependent over the entire dose range. The concentration of the adduct increased with DEN concentration over the entire dose range for exposures of 28 days or less and for doses of 0.4 to 40 ppm DEN the adduct level increased with DEN concentration for exposures of 70 days or less. Although the dose response of O4-EtdT was relatively linear, with increasing DEN concentration a trend toward a less than linear relationship was observed. This suggests that there was a lower efficiency of formation and/or greater loss of O4-EtdT during high-dose exposures. This study provides a data base that can be used to qualitatively and quantitatively examine the relationship between external dose and O4-EtdT over a DEN dose range covering several orders of magnitude.     

Molecular analysis of occupational cancer: infrequent p53 and ras mutations in renal-cell cancer in workers exposed to gasoline

Occupational exposure to gasoline has been identified in several studies as a risk factor for renal-cell cancer. Cases of renal-cell cancer with and without work-related exposure to gasoline or gasoline and diesel fuel were studied for the presence of mutations in the tumour-suppressor gene p53 (n = 23 exposed and 30 non-exposed cases studied) and ras oncogene (n = 30 exposed and 36 non-exposed cases studied). An average cumulative exposure was estimated at 10 ppm-years benzene among the exposed. Three p53 mutations were detected by denaturing-gradient gel electrophoresis (DGGE) among the 23 exposed cases (3/23, 13%). Of the non-exposed referent cases, 4 had a mutation (4/30, 13%). All but one of the cases with a p53 mutation had smoked. A ras gene (K-ras or N-ras) mutation was found in 3 (3/66, 4.5%) cases, all of whom were smoker referents. We conclude that p53 and ras mutations are infrequent in renal-cell cancer associated with occupational exposure to gasoline. However, the majority of the mutations (6/7 for p53, and 3/3 for ras genes) were seen in smokers. Int. J. Cancer 73:492–496, 1997. © 1997 Wiley-Liss, Inc.     

Tamoxifen induces G:C-->T:A mutations in the cII gene in the liver of lambda/lacI transgenic rats but not at 5'-CpG-3' dinucleotide sequences as found in the lacI transgene.

Tamoxifen, a rat liver carcinogen, can induce mutations in the lacI gene in the livers of lambda/lacI transgenic rats. However, the presence of persistent tamoxifen adducts on the liver DNA raises the possibility that some contribution to the mutagenesis from ex vivo mutations during the in vitro lacI assay cannot be ruled out. To address this issue, mutagenesis at the cII gene of the transgenic shuttle vector was determined using a selection based assay which is unaffected by the presence of tamoxifen-DNA adducts. Female lambda/lacI transgenic rats were dosed orally with tamoxifen (20 mg/kg body wt) daily for 6 weeks, causing a 3.2-fold increase in the mutant frequency (MF) in the cII gene compared with that obtained with solvent treated animals. This was similar to the MF found previously at the lacI gene and confirms that tamoxifen is mutagenic in vivo. The major class of mutation induced by tamoxifen in the cII gene was G:C-->T:A transversions as was found previously in the lacI gene. However, in the one unreplicated study of mutations in the p53 gene of liver tumours induced by tamoxifen, no G:C-->T:A transversions were found; possible differences between mutagenesis in normal and tumour tissues are explored. The major proportion of the G:C-->T:A transversions occurred at 5'-CpG-3' dinucleotide (CpG) sites in the lacI gene, but not at such sites in the cII gene. The methylation of CpG sites greatly enhances the targeting of deoxyguanosine by carcinogens, thus this finding might be explained by differences in the methylation patterns at their respective CpG sites; however, nothing is known about the methylation status of either the lacI nor the cII gene in this transgenic rat. This study raises the important issue of which target genes (mammalian or transgenic) should be used as endpoints in mammalian mutagenesis assays.     

Elevation of 1,2-diacylglycerol in ras-transformed neonatal liver and pancreas of transgenic mice

Expression of the activated Harvey-ras (H-ras) oncogene in cultured cells is associated with an elevated steady-state concentration of 1,2-diacylglycerol (DG), an intracellular second messenger capable of promoting cell division. To explore the biochemistry of ras expression in vivo, we measured DG in ras-transformed neonatal liver and pancreas of transgenic mice. DG was elevated over 2-fold in these tissues compared to controls, but was not elevated in transgenic neonatal liver expressing normal H-ras, the nuclear oncogene myc, or the Simian Virus 40 T-antigens. DG was also not elevated in ras-induced lung adenomas in transgenic mice. These findings demonstrate an association between activated ras expression and DG concentration in neonatal tissue, but suggest that marked elevation of DG is not necessary for the development of ras-induced tumors in lung.     

Gene amplification in fibroblasts from ataxia telangiectasia (AT) patients and in X-ray hypersensitive AT-like Chinese hamster mutants

In search of functions involved in the regulation of gene amplification, and given the relevance of chromosome breakage in initiating the process, we analyzed the gene amplification ability of cells hypersensitive to inducers of DNA double-strand breaks and defective in cell cycle control: two human fibroblast strains derived from patients affected by ataxia telangiectasia (AT) and two hamster mutant cell lines belonging to complementation group XRCC8 of the rodent X-ray-sensitive mutants. These mutants are considered hamster models of AT cells. To measure gene amplification, the frequency and the rate of occurrence of N-(phosphonacetyl)-L-aspartate resistant cells were determined. In both hamster mutants, these two parameters were increased by about an order of magnitude compared with parental cells, suggesting that amplification ability was increased by the genetic defect. In primary AT fibroblasts, as in normal human fibroblasts, gene amplification was undetectable and a block in the G(1) phase of the cell cycle was induced upon PALA treatment. These results suggest that in AT fibroblasts, where only the ATM gene is mutated, ATM-independent mechanisms prevent gene amplification, while, in the immortalized hamster cell lines, which are already permissive for gene amplification, the AT-like defect increases the probability of gene amplification.     

Molecular analysis by electron microscopy of the removal of psoralen-photoinduced DNA cross-links in normal and Fanconi's anemia fibroblasts

The induction and fate of psoralen-photoinduced DNA interstrand cross-links in the genome of Fanconi's anemia (FA) fibroblasts of complementation groups A and B, and of normal human fibroblasts, were investigated by quantitative analysis of totally denatured DNA fragments visualized by electron microscopy. 8-Methoxypsoralen (5 x 10(-5) M) interstrand cross-links were induced as a function of the near ultraviolet light dose. With time of postexposure incubation, a fraction of interstrand cross-links disappeared in all cell lines. However, 24 h after treatment, this removal was significantly lower in the two FA group A cell lines examined (34-39%) than in the FA group B and normal cell lines (43-53 and 47-57%, respectively). These data indicate that FA cells are at least able to recognize and incise interstrand cross-links, as normal cells do, although group A cells seem somewhat hampered in this process. This is in accord with data obtained on the same cell lines using another biochemical assay (D. Papadopoulo, D. Averbeck, and E. Moustacchi. Mutat. Res., DNA Repair Rep., 184: 271-280, 1987). Since the fate of cross-links in FA constituted a controversial matter, it is important to stress that two different methodologies applied to genetically well defined cell lines led to the same conclusions.     

Molecular alterations and lung tumors in p53 mutant mice exposed to cigarette smoke

Mutations and deletions in p53 are the most common genetic lesions in human cancer,and an extraordinarily high incidence of lung cancer occurs in smokers suffering from Li-Fraumeni syndrome, which is characterized by germ-line inactivation of one p53 allele. In contrast, p53 mutations are infrequent in lung tumors formed in A/J mice. Moreover, despite the dominant role of cigarette smoke in the epidemiology of human lung cancer, it is very difficult to reproduce the lung tumorigenicity of this complex mixture in animal models. We used a transgenic mouse with a dominant-negative p53 mutation to examine the effects of a mutant p53 on smoke-induced lung carcinogenesis in mice. p53 mutant (UL53-3 x A/J)F(1) mice of both genders and their wild-type (wt) littermate controls were exposed whole-body to environmental cigarette smoke (ECS) for up to 9.5 months. Untreated mutant mice of both genders underwent an early stimulus of bronchial cell proliferation, and an age-related formation of DNA adducts in lung and heart. In males, there was an age-related increase of micronucleated normochromatic erythrocytes in peripheral blood and an impairment of body weight gain. These findings underscore a physiological protective role of p53 in wt A/J mice. The response of wt and mutant mice to ECS was similar in terms of oxidative DNA damage in lung and heart, proliferation of the bronchial epithelium, and levels of p53 oncoprotein, as assessed after exposure for 28 days. In contrast, ECS-exposed mutant mice underwent a lower induction of apoptosis in bronchial epithelium, a greater formation of DNA adducts in lung and heart, and a more intense cytogenetic damage, shown by a higher frequency of micronuclei in pulmonary alveolar macrophages and in peripheral blood normochromatic erythrocytes. Interestingly, at the end of the experiment, DNA adducts were not repaired in either wt or mutant mice after discontinuing exposure to ECS for 1 week. A weak but significant increase of lung tumor incidence and multiplicity was induced in p53 mutant (UL53-3 x A/J)F(1) mice after exposure to ECS for either 5 months, followed by recovery in air for 4.5 months, or 9.5 continuative months. Conversely, no tumorigenic effect was observed in their wt littermate controls, carrying a 99.9% A/J background and 5% FVB genome. This contrasts with the weakly positive results obtained in previous studies using wt A/J mice. Thus, in agreement with the results of previous lung tumorigenicity studies performed with the smoke carcinogens benzo(a)pyrene and 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone, (UL53-3 x A/J)F(1) mice carrying a mutant p53 transgene appear to be more sensitive to ECS than the corresponding wt littermate controls. These findings provide evidence that p53 mutations play a role in smoke-related carcinogenesis not only in humans but also in A/J mice.