Spontaneous mutations in the Big Blue® transgenic system are primarily mouse derived

The Big Blue® transgenic mouse mutation detection system provides a powerful approach for measuring spontaneous and induced mutations in vivo. The observed mutations may contain a fraction of ex vivo or prokaryotic mutational events. Indeed, a modified, selectable form of the Big Blue® assay seem to generate artifactual mutants under certain circumstances. Herein we review the evidence that circular mutants (i.e., the plaque circumference is at least 50% blue) collected in the standard Big Blue® assay are derived primarily from the mouse. The most direct evidence is the similarity in the types of mutations found in jackpot and nonjackpot mutations. In addition, about half of the spontaneous mutations in the lacI transgene are transitions and transversions at CpG dinucleotides, a mammalian-specific feature. The mutation pattern observed at lacI is consistent with AT mutation pressure operating in a GC rich DNA and approaches that reported for observed germline human factor IX mutations. Furthermore, the spontaneous mutation pattern of circular Big Blue® mutants differs significantly from that of an endogenous lacI gene in E. coli. Pinpoint mutants (a dot of blue color peripherally located in a wild type plaque), which a priori were not expected to be mouse-derived, have a mutation pattern consistent with the mutation pattern of an endogenous E. coli lacI gene. Analysis of induced mutagenesis studies reveals mutation frequencies and patterns for the Big Blue® circular mutants which are comparable to endogenous genes. In reconstruction experiments, blue plaques derived from a superinfection with wild type and mutant phage produced approximately 50% blue and 50% clear plaques on replating. This phenomenon has not been seen when plaques derived from mouse were replated in the Big Blue® assay. Collectively, the evidence strongly supports a murine origin for circular mutants recovered in the standard Big Blue® assay. Validation of current assays is an essential step in determining the frequency and pattern of spontaneous murine-specific mutations. Defining this benchmark will be helpful in evaluating the next generation of transgenic mutation detection systems.     

A more efficient Big Blue protocol improves transgene rescue and accuracy in a adduct and mutation measurement

Transgenic mutational systems have provided researchers with an invaluable tool, allowing the measurement of both spontaneous and induced mutations. The Big Blue transgenic rodent mutagenesis system developed by Stratagene (La Jolla, CA) uses a lambda shuttle vector carrying lacI as the mutational target gene. A common criticism of the Big Blue system is that it relies on visual screening to detect mutants rather than positive selection, which is employed in more recently developed systems. The lack of positive selection, however, has provided the Big Blue system with a unique advantage, as it allows for the dynamic quantification of mutation fixation, repair, and adduct stability, since both pre-mutagenic DNA adducts and mutations can readily be quantified [Proc. Natl. Acad. Sci. U.S.A. 97 (2000) 11391]. Improvements to the standard Big Blue assay protocol are required for the visualization of mutant plaques resulting from pre-mutagenic damage, as these can appear much lighter in color than the lightest color control mutant (CM0). This increase in detection has been achieved by the development of a protocol that now permits the effective measurement of repair and mutation fixation utilizing the Big Blue system. This new protocol has also addressed efficiency, allowing for a two-fold increase in the number of plaques produced per packaging reaction and a decrease in both phage migration and plaque size, permitting a greater than three-fold increase in plating density. The implementation of this protocol will make the Big Blue assay more economical and less demanding than before, while providing researchers with an efficient means to measure both repair and mutation in this system.     

High plating density improves Big Blue system efficiency without loss of sensitivity

To increase efficiency in the Big Blue system, the plating density was increased from 15000 to 30000 or 45000 plaque forming units (pfus) per plate by increasing the density of the E. coli lawn and decreasing individual plaque size. Small plaque size ensured minimal overlap of the plaques. Liver from one 3- and one 25-month-old mouse (low and high mutation frequencies, respectively) was analyzed and neither plating density nor plaque size affected mutant/mutation frequency and pattern. The color intensity of particular mutant plaques was not affected by plaque size or plating density. Optimal sensitivity is achieved by sequencing mutants to calculate the mutation frequency from the mutant frequency and to identify altered patterns of mutation. Detailed effort and cost accounting of the Big Blue system (including mouse handling, DNA extraction, plaque screening, plaque purification, and DNA sequencing) reveals that one-quarter of the total effort is devoted to plating and screening of plates. This effort is reduced two fold with high plating density. The total cost of the Big Blue system is reduced by 17%. The total cost of the High Plating Density Big Blue system is now only 12% more costly than a selectable assay and offers an extensively validated system with a large mutation database representing a decade of effort.     

iMARS--mutation analysis reporting software: an analysis of spontaneous cII mutation spectra

The sensitivity of any mutational assay is determined by the level at which spontaneous mutations occur in the corresponding untreated controls. Establishing the type and frequency at which mutations occur naturally within a test system is essential if one is to draw scientifically sound conclusions regarding chemically induced mutations. Currently, mutation-spectra analysis is laborious and time-consuming. Thus, we have developed iMARS, a comprehensive mutation-spectrum analysis package that utilises routinely used methodologies and visualisation tools. To demonstrate the use and capabilities of iMARS, we have analysed the distribution, types and sequence context of spontaneous base substitutions derived from the cII gene mutation assay in transgenic animals. Analysis of spontaneous mutation spectra revealed variation both within and between the transgenic rodent test systems Big Blue Mouse, MutaMouse and Big Blue Rat. The most common spontaneous base substitutions were G:C-->A:T transitions and G:C-->T:A transversions. All Big Blue Mouse spectra were significantly different from each other by distribution and nearly all by mutation type, whereas the converse was true for the other test systems. Twenty-eight mutation hotspots were observed across all spectra generally occurring in CG, GA/TC, GG and GC dinucleotides. A mutation hotspot at nucleotide 212 occurred at a higher frequency in MutaMouse and Big Blue Rat. In addition, CG dinucleotides were the most mutable in all spectra except two Big Blue Mouse spectra. Thus, spontaneous base-substitution spectra showed more variation in distribution, type and sequence context in Big Blue Mouse relative to spectra derived from MutaMouse and Big Blue Rat. The results of our analysis provide a baseline reference for mutation studies utilising the cII gene in transgenic rodent models. The potential differences in spontaneous base-substitution spectra should be considered when making comparisons between these test systems. The ease at which iMARS has allowed us to carry out an exhaustive investigation to assess mutation distribution, mutation type, strand bias, target sequences and motifs, as well as predict mutation hotspots provides us with a valuable tool in helping to distinguish true chemically induced hotspots from background mutations and gives a true reflection of mutation frequency.     

Evidence that proximal multiple mutations in Big Blue transgenic mice are dependent events

To characterize the nature of multiple mutations in the tissues of an intact animal, the Big Blue transgenic mouse mutation detection system was used to examine 1459 mutants from eight normal tissues and 507 mutants from 11 tumors. Multiple mutations occurred and predominantly doublet mutants were identified (i.e. two mutations within one mutant lacI gene), but multiplets of up to five mutations were observed. The frequency of doublets in normal tissues and spontaneous tumors from p53-deficient mice was enhanced to the same degree (660 and 667 fold, respectively) over that expected for two independent mutational events. Doublets, multiplets and singlets have similar patterns of mutation. The distance between mutations in doublets fits an exponential distribution, not that expected for randomly spaced events, suggesting that many doublets occur in rapid succession within the same cell cycle.     

Subchronic administration of phenobarbital alters the mutation spectrum of lacI in the livers of Big Blue transgenic mice

Phenobarbital (PHE) is a liver carcinogen in B6C3F1 mice and a weak mutagen that does not appear to form DNA adducts. To investigate PHE mutagenicity in vivo, B6C3F1 Big Blue(R) male transgenic mice harboring the lambdaLIZ shuttle vector containing the lacI target gene were fed PHE at 2500 ppm for 180 days. A modest increase in the mutant frequency (MF) from 5.02+/-2.4x10(-5) in the control group to 6.88+/-0.754x10(-5) in the PHE-treated group, which was marginally different (p<0.05), was obtained. To better assess the relevance of this increase in MF, a random collection of mutants from each PHE-exposed mouse was sequenced. After correcting for clonal expansion, which is the most conservative approach, the MF in the PHE-treated mice decreased to 6.39+/-1.02x10(-5), an insignificant difference (p=0.10) from that in control group. Despite this modest increase in MF, the mutation spectrum obtained from the PHE-exposed group was significantly different (pA:T transitions remained the same in the two spectra. It is postulated that the increase in transversions at G:C base pairs found in the PHE-derived spectrum is likely due to oxidative damage as a result of induction of CYP2B isozymes by the chronic administration of PHE. Results from this study demonstrate that PHE alters the spectrum of mutations, rather than inducing a significant global increase in the MF. The PHE-derived spectrum of lacI mutants from the liver of Big Blue(R) B6C3F1 male mice was remarkably similar (p=0.8) to that generated by oxazepam (OX), a compound which also induces CYP2B isozymes following chronic administration of the drug.     

Mutation spectrum of 4-nitroquinoline N-oxide in the lacI transgenic Big Blue Rat2 cell line.

This paper describes the spectrum of mutations induced by 4-nitroquinoline N-oxide (4-NQO) in the lacI target gene of the transgenic Big Blue Rat2 cell line. There are only a few report for the mutational spectrum of 4-NQO in a mammalian system although its biological and genetic effects have been well studied. Big Blue Rat2 cells were treated with 0.03125, 0.0625 or 0.125 microg/ml of 4-NQO, the highest concentration giving 85% survival. Our results indicated that the mutant frequency (MF) induced by 4-NQO was dose-dependent with increases from three- to seven-fold. The DNA sequence analysis of lacI mutants from the control and 4-NQO treatment groups revealed an obvious difference in the spectra of mutations. In spontaneous mutants, transition (60%) mutations, especially G:C-->A:T transition (45%), were most frequent. However, the major type of base substitution after treatment of 4-NQO was transversions (68.8%), especially G:C-->T:A (43.8%), while only 25% of mutants were transitions. These results are consistent with those produced by 4-NQO in other systems and the transgenic assay system will be a powerful tool to postulate more accurately the mechanism of chemical carcinogenesis involved.     

A random mutation capture assay to detect genomic point mutations in mouse tissue

Herein, a detailed protocol for a random mutation capture (RMC) assay to measure nuclear point mutation frequency in mouse tissue is described. This protocol is a simplified version of the original method developed for human tissue that is easier to perform, yet retains a high sensitivity of detection. In contrast to assays relying on phenotypic selection of reporter genes in transgenic mice, the RMC assay allows direct detection of mutations in endogenous genes in any mouse strain. Measuring mutation frequency within an intron of a transcribed gene, we show this assay to be highly reproducible. We analyzed mutation frequencies from the liver tissue of animals with a mutation within the intrinsic exonuclease domains of the two major DNA polymerases, δ and ε. These mice exhibited significantly higher mutation frequencies than did wild-type animals. A comparison with a previous analysis of these genotypes in Big Blue mice revealed the RMC assay to be more sensitive than the Big Blue assay for this application. As RMC does not require analysis of a particular gene, simultaneous analysis of mutation frequency at multiple genetic loci is feasible. This assay provides a versatile alternative to transgenic mouse models for the study of mutagenesis in vivo.     

4-chloro-o-phenylenediamine induces a dose-related increase in G:C > T:A transversions and one major DNA adduct in the liver of Big Blue mice after 26 weeks in feed treatment

The monocyclic aromatic amine 4-chloro-o-phenylenediamine (4-C-o-PDA), a known mutagen and mouse hepatocarcinogen, was tested for its in vivo mutagenic potential in the Big Blue transgenic mouse assay system. Genomic DNA was isolated from liver tissue of control and treated animals and lacI mutants were recovered. In an initial 2-week study 4-C-o-PDA was administered daily per os to groups of male and female C57BL/6 Big Blue mice at doses of 0 and 200 mg/kg for 2 weeks (on working days) followed by a treatment free expression time of 10 days. Only a weak increase in the mutant frequencies in females was observed. In a 26-week study, where 4-C-o-PDA was given to groups of male and female Big Blue mice in feed at dietary concentrations of 0, 5,000 and 10,000 ppm, 4-C-o-PDA was found to induce a pronounced dose-dependent increase in mutant frequencies in either sex. In the present work, we analyzed the mutation spectrum by automated DNA sequencing of lacI mutants from both studies. Following the 2-week administration of 4-C-oT:A transversions in both sexes. In addition, upon 26-week treatment with 4-C-o-PDA, one major DNA adduct was detected by 33P postlabelling and subsequent multidimensional thin layer chromatography. It is concluded that 4-C-oT:A transversions after 26 weeks in feed treatment. This result indicates that the sensitivity of the Big Blue transgenic assay system, in detecting a unique chemically induced mutation spectrum, is dependent on experimental parameters, such as treatment time. The data suggest that the formation of one major DNA adduct upon 4-C-o-PDA treatment may be critical for its mutagenicity.     

Mutagenic properties of a nitrofuran, 7-methoxy-2-nitronaphtho[2, 1-b]furan (R7000), in lacI transgenic mice

The in vivo mutagenic properties of a 5-nitrofuran, the 7-methoxy-2-nitronaphtho[2,1-b]furan (R7000), already well known in bacteria, was evaluated in lacI transgenic mice (Big Blue). The mutation frequency was determined in various organs of i.p. - treated mice and the nature of induced mutations was determined for the target organs in which mutation induction was significant. It was found that R7000 is mutagenic in mice, although, on the basis of the number of induced mutants per unit mass in comparison with other known mutagenic chemicals, R7000 appears to be considerably less mutagenic in mice than in bacteria. The most affected organs, small intestine, caecum and colon organs belong to the digestive apparatus. The distribution of R7000-induced mutations in the lacI gene recovered from small intestine of transgenic mice was very similar to that which had been found in E. coli. The difference between mouse and E. coli in the R7000 induced mutational spectra are mainly in the proportion of single base frameshifts versus base substitutions. Since R7000 induced mutations seemed to arise in the population of stem cells and that the stem cells are important for carcinogenesis, our results are compatible with a possible carcinogenic effect of R7000 and other nitrofurans.     

Recent advances in the protocols of transgenic mouse mutation assays

Transgenic mutation assays were developed to detect gene mutations in multiple organs of mice or rats. The assays permit (1) quantitative measurements of mutation frequencies in all tissues/organs including germ cells and (2) molecular analysis of induced and spontaneous mutations by DNA sequencing analysis. The protocols of recently developed selections in the lambda phage-based transgenic mutation assays, i.e. cII, Spi(-) and 6-thioguanine selections, are described, and a data set of transgenic mutation assays, including those using Big Blue and Muta Mouse, is presented.     

Mutagenicity of ultraviolet A radiation in the lacI transgene in Big Blue mouse embryonic fibroblasts

Sunlight ultraviolet A (UVA) irradiation has been implicated in the etiology of human skin cancer. A genotoxic mode of action for UVA radiation has been suggested that involves photosensitization reactions giving rise to promutagenic DNA lesions. We investigated the mutagenicity of UVA in the lacI transgene in Big Blue mouse embryonic fibroblasts. UVA irradiation of these cells at a physiologically relevant dose of 18J/cm(2) caused a 2.8-fold increase in the lacI mutant frequency relative to control, i.e., 12.12+/-1.84 versus 4.39+/-1.99 x 10(-5) (mean+/-S.D.). DNA sequencing analysis showed that of 100 UVA-induced mutant plaques and 54 spontaneously arisen control plaques, 97 and 51, respectively, contained a minimum of one mutation along the lacI transgene. The vast majority of both induced- and spontaneous mutations were single base substitutions, although less frequently, there were also single and multiple base deletions and insertions, and tandem base substitutions. Detailed mutation spectrometry analysis revealed that G:C-->T:A transversions, the signature mutations of oxidative DNA damage, were significantly induced by UVA irradiation (P<0.003). The absolute frequency of this type of mutations was 7.4-fold increased consequent to UVA irradiation as compared to control (3.38 versus 0.454 x 10(-5); P<0.00001). These findings are in complete agreement with those previously observed in the cII transgene of the same model system, and reaffirm the notion that intracellular photosensitization reactions causing promutagenic oxidative DNA damage are involved in UVA genotoxicity.     

Functional consequences of the G235R mutation in liver arginase leading to hyperargininemia

Hyperargininemia is a rare autosomal disorder that results from a deficiency in hepatic type I arginase. This deficiency is the consequence of random point mutations that occur throughout the gene. The G235R patient mutation has been proposed to affect the catalytic activity and structural integrity of the protein [D. E. Ash, L. R. Scolnick, Z. F. Kanyo, J. G. Vockley, S. D. Cederbaum, and D. W. Christianson (1998) Mol. Genet. Metab. 64, 243-249]. The G235R (patient) and G235A (control) arginase mutants of rat liver arginase have been generated to probe the effects of these point mutations on the structure and function of hepatic type I arginase. Both mutant arginases were trimeric by gel filtration, but the control G235A mutant had 56% of wild-type activity and the G235R mutant had less than 0.03% activity compared to the wild-type enzyme. The G235R mutant contained undetectable levels of tightly bound manganese as determined by electron paramagnetic resonance, while the G235A mutant had a Mn(II) stoichiometry of 2 Mn/subunit. Molecular modeling indicates that the introduction of an arginine residue at position 235 results in a major rearrangement of the metal ligands that compromise Mn(II) binding.     

Mutagenicity testing with transgenic mice. Part II: Comparison with the mouse spot test

The mouse spot test, an in vivo mutation assay, has been used to assess a number of chemicals. It is at present the only in vivo mammalian test system capable of detecting somatic gene mutations according to OECD guidelines (OECD guideline 484). It is however rather insensitive, animal consuming and expensive type of test. More recently several assays using transgenic animals have been developed. From data in the literature, the present study compares the results of in vivo testing of over twenty chemicals using the mouse spot test and compares them with results from the two transgenic mouse models with the best data base available, the lacI model (commercially available as the Big Blue(R) mouse), and the lacZ model (commercially available as the Mutatrade mark Mouse). There was agreement in the results from the majority of substances. No differences were found in the predictability of the transgenic animal assays and the mouse spot test for carcinogenicity. However, from the limited data available, it seems that the transgenic mouse assay has several advantages over the mouse spot test and may be a suitable test system replacing the mouse spot test for detection of gene but not chromosome mutations in vivo.     

Age-dependent sensitivity of Big Blue transgenic mice to the mutagenicity of N-ethyl-N-nitrosourea (ENU) in liver

The incidence of childhood cancer is increasing and recent evidence suggests an association between childhood cancer and environmental exposure to genotoxins. In the present study, the Big Blue transgenic mouse model was used to determine whether specific periods in early life represent windows of vulnerability to mutation induction by genotoxins in mouse liver. Groups of mice were treated with single doses of 120 mg N-ethyl-N-nitrosourea (ENU)/kg body weight or the vehicle either transplacentally to the 18-day-old fetus or at postnatal days (PNDs) 1, 8, 15, 42 or 126; the animals were sacrificed 6 weeks after their treatment. The cII mutation assay was performed to determine the mutant frequencies (MFs) in the livers of the mice. Liver cII MFs for both sexes were dependent on the age at which the animals were treated. Perinatal treatment with ENU (either transplacental treatment to the 18-day-old fetus or i.p. injection at PND 1) induced relatively high MFs. However, ENU treatment at PNDs 8 and 15 resulted in the highest mutation induction. The lowest mutation induction occurred in those animals treated as adults (PND 126). For instance, the cII MF for the PND 8 female group was 646 x 10(-6) while the MF for female adults was only 145 x 10(-6), a more than 4-fold difference. Molecular analysis of the mutants found that A:T-->T:A transversions and A:T-->G:C transitions characterized the pattern of mutations induced by ENU in both the neonate and adult mice, while the predominate type of mutation in the controls was G:C-->A:T. The results indicate that mouse liver is most sensitive to ENU-induced mutation during infancy. This period correlates well with the age-dependent sensitivity to carcinogenicity in mouse liver, suggesting that mutation is an important rate-limiting factor for age-related carcinogenesis.     

Mutagenic properties of a nitrofuran, 7-methoxy-2-nitronaphtho[2,1-b]furan (R7000), in lacI transgenic mice

The in vivo mutagenic properties of a 5-nitrofuran, the 7-methoxy-2-nitronaphtho[2,1-b]furan (R7000), already well known in bacteria, was evaluated in lacI transgenic mice (Big Blue). The mutation frequency was determined in various organs of i.p. — treated mice and the nature of induced mutations was determined for the target organs in which mutation induction was significant. It was found that R7000 is mutagenic in mice, although, on the basis of the number of induced mutants per unit mass in comparison with other known mutagenic chemicals, R7000 appears to be considerably less mutagenic in mice than in bacteria. The most affected organs, small intestine, caecum and colon organs belong to the digestive apparatus. The distribution of R7000-induced mutations in the lacI gene recovered from small intestine of transgenic mice was very similar to that which had been found in E. coli. The difference between mouse and E. coli in the R7000 induced mutational spectra are mainly in the proportion of single base frameshifts versus base substitutions. Since R7000 induced mutations seemed to arise in the population of stem cells and that the stem cells are important for carcinogenesis, our results are compatible with a possible carcinogenic effect of R7000 and other nitrofurans.     

Ethylnitrosourea-induced mutation and molecular analysis of transgenic mice containing the gpt shuttle vector

Novel transgenic mice were developed in order to study the in vivo mutagenesis. The transgenic mice carried pCGK shuttle vector, which contained the Escherichia coli gpt gene as a mutational target, the kanamycin-resistant gene (Kanr) and cos region derived from bacteriophage lambda. The shuttle vector can be recovered from the transgenic mouse genome into the gpt-deficient E. coli by an in vitro packaging method and is selectable as a Kanr phenotype. Mutations induced at the gpt gene can be easily detected with a selective agent, 6-thioguanine (6-TG). In the previous study, the pCGK shuttle vector was incorporated into Chinese hamster CHL/IU cells and the resultant transgenic cell line was shown to be a useful system to study in vitro mutagenesis at the gpt gene. Therefore, an advantage of the shuttle vector is that in vivo mutational data obtained from the transgenic mouse can be compared with those of transgenic cell line in vitro. A transgenic CD-1 mouse line, designated as #128, that carried approximately 50 copies of pCGK shuttle vectors, was selected among 4 transgenic mouse lines. To investigate the sensitivity of the #128 line, the transgenic mice were treated with a single intraperitoneal injection of 250 mg/kg of N-ethyl-N-nitrosourea (ENU) or with 50 mg kg-1 day-1 of ENU for 5 consecutive days, and bone marrow, spleen and liver were dissected to investigate their mutational responses. The background mutant frequency was between 18x10(-6) and 75x10(-6) among all tissues tested. ENU induced significant increases in the mutant frequency above the background level in all three tissues at 14 days after single or 5-day treatment with the chemical. The increases in the mutant frequencies in bone marrow, spleen and liver were 6.4- to 6.8-fold, 3.0- to 5.6-fold and 3.0- to 3.3-fold, respectively. The shuttle vector DNA was recovered from the bone marrow of both spontaneous and ENU-treated mice and the gpt gene was amplified by polymerase chain reaction. The amplified DNA was subject to DNA sequence analysis. Out of 79 spontaneous and 52 ENU-induced mutants, the gpt gene could be amplified from 28 spontaneous and 46 ENU-induced mutants. DNA sequence analysis showed that predominant mutations were identified as A:T to T:A transversions (22 out of 46 sequenced mutants) and G:C to A:T transitions (9/46) in ENU-induced mutants, whereas G:C to T:A transversions (7 out of 28 sequenced mutants) were predominant in spontaneous mutants. These results demonstrate that this transgenic mouse, in combination with the transgenic CHL/IU cell line, is a useful system to study in vivo and in vitro mutational events at the same target gene.     

Cystic fibrosis mice carrying the missense mutation G551D replicate human genotype-phenotype correlations.

We have generated a mouse carrying the human G551D mutation in the cystic fibrosis transmembrane conductance regulator gene (CFTR) by a one-step gene targeting procedure. These mutant mice show cystic fibrosis pathology but have a reduced risk of fatal intestinal blockage compared with 'null' mutants, in keeping with the reduced incidence of meconium ileus in G551D patients. The G551D mutant mice show greatly reduced CFTR-related chloride transport, displaying activity intermediate between that of cftr(mlUNC) replacement ('null') and cftr(mlHGU) insertional (residual activity) mutants and equivalent to approximately 4% of wild-type CFTR activity. The long-term survival of these animals should provide an excellent model with which to study cystic fibrosis, and they illustrate the value of mouse models carrying relevant mutations for examining genotype-phenotype correlations.     

Genotoxic effects of bitumen fumes in Big Blue transgenic rat lung

Road paving workers are exposed to bitumen fumes (CAS No. 8052-42-4), a complex mixture of volatile compounds and particles containing carcinogenic and non-carcinogenic polycyclic aromatic hydrocarbons. However, epidemiological and experimental animal studies failed to draw unambiguous conclusions concerning their toxicity. In order to gain better insights on their genotoxic potential, we used an experimental design able to generate bitumen fumes at road paving temperature (temperature: 170 degrees C, total particulate matter: 100mg/m3) and perform a nose-only exposure of Big Blue transgenic rodents 6h/day for five consecutive days. The mutagenic properties of bitumen fumes were determined by analyzing the mutation frequency and spectrum of the neutral reporter gene cII inserted into the rodent genome. We previously observed in mouse lung, that bitumen fumes did not induce an increase of cII mutants, a modification of the mutation spectrum, nor the formation of DNA adducts. Since DNA adducts were found in the lungs of rats exposed to asphalt fumes in similar conditions, we decided to carry out an analogous experiment with Big Blue rats. A DNA adduct was detected 3 and 30 days after the end of treatment suggesting that these genetic alterations were quite steady. Thirty days after exposure, the cII mutant frequency was similar in control and exposed rats. In addition, a slight but not significant modification of the mutation spectrum associated with an increase of G:C to T:A and A:T to C:G transversions was noticeable in the treated animals. Then, these data failed to demonstrate a pulmonary mutagenic potential for bitumen fumes generated at road paving temperature in our experimental conditions despite the presence of a DNA adduct. These results may provide information concerning the pulmonary mechanism of action of this aerosol and may contribute to the occupational health hazard assessment.