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Supplementary Table 1 from Enhanced Spontaneous and Benzo(a)pyrene-Induced Mutations in the Lung of Nrf2-Deficient gpt Delta Mice

The lung is an organ that is sensitive to mutations induced by chemicals in ambient air, and transgenic mice harboring guanine phosphoribosyltransferase (gpt) gene as a target gene are a well-established model system for assessing genotoxicity in vivo. Transcription factor Nrf2 mediates inducible and constitutive expression of cytoprotective enzymes against xenobiotics and mutagens. To address whether Nrf2 is also involved in DNA protection, we generated nrf2+/−::gpt and nrf2−/−::gpt mice. The spontaneous mutation frequency of the gpt gene in the lung was approximately three times higher in nrf2-null (nrf2−/−) mice than nrf2 heterozygous (nrf2+/−) and wild-type (nrf2+/+) mice, whereas in the liver, the mutation frequency was higher in nrf2−/− and nrf2+/− mice than in nrf2+/+ wild-type mice. By contrast, no difference in mutation frequency was observed in testis among the three genotypes. A single intratracheal instillation of benzo(a)pyrene (BaP) increased the lung mutation frequency 3.1- and 6.1-fold in nrf2+/− and nrf2−/− mice, respectively, compared with BaP-untreated nrf2+/− mice, showing that nrf2−/− mice are more susceptible to genotoxic carcinogens. Surprisingly, mutation profiles of the gpt gene in BaP-treated nrf2+/− mice was substantially different from that in BaP-untreated nrf2−/− mice. In nrf2−/− mice, spontaneous and BaP-induced mutation hotspots were observed at nucleotides 64 and 140 of gpt, respectively. These results thus show that Nrf2 aids in the prevention of mutations in vivo and suggest that Nrf2 protects genomic DNA against certain types of mutations. [Cancer Res 2007;67(12):5643–8]

Supplementary Table 4 from Enhanced Spontaneous and Benzo(a)pyrene-Induced Mutations in the Lung of Nrf2-Deficient gpt Delta Mice

Supplementary Table 2 from Enhanced Spontaneous and Benzo(a)pyrene-Induced Mutations in the Lung of Nrf2-Deficient gpt Delta Mice

Supplementary Table 3 from Enhanced Spontaneous and Benzo(a)pyrene-Induced Mutations in the Lung of Nrf2-Deficient gpt Delta Mice

Introduction To examine whether the mutagenic potential of lung exposure to air-borne environmental mutagens is age dependent, we administered 1 mg of benzo[a]pyrene intratracheally to 11- and 24-month old (middle-aged and old, respectively) gpt delta transgenic mice that harbor gpt (guanine phosphoribosyltransferase) genes integrated in the genomic DNA as a target for mutation detection, and then analyzed the benzo[a]pyrene-induced and spontaneous in vivo mutations and mutation spectrum in the lungs. Results The mutant frequencies in the lungs of the 11- and 24-month–old control (vehicle-treated) gpt delta mice were 1.14 ± 0.22 × 10−5 and 1.00 ± 0.20 × 10−5, respectively, which are significantly higher than that observed for the control 3-month–old (young) mice (0.59 ± 0.13 × 10−5) in our previous studies, indicating that spontaneous mutation in the lung increases with age. The mutant frequencies in 11- and 24-month–old mice treated with benzo [a] pyrene were 1.5- and 2.3-fold, respectively, that of the age-matched control mice, and 4.3-fold that of the 3-month–old mice in our previous studies. Analysis of mutation spectra showed that both G:C to A:T transitions and G:C to T:A transversions were predominant in the lungs of control mice at all ages. In benzo [a] pyrene-treated mice in our previous studies, G:C to T:A transversions were the predominant type of mutation (55 %) at 3 months. Here we found that their frequency was dramatically reduced to 18 % by 24 months, and the G:C to A:T transitions became the predominant type of mutation in 24-month–old mice (41 % [16 % at CpG sites]). Conclusions Our findings suggest that susceptibility to benzo[a]pyrene is highest in young mice and is elevated again in old age. The elevation of G:C to A:T transitions was observed following benzo [a] pyrene administration in the lungs of aged mice, and accelerated cytidine deamination is speculated to contribute to this elevation.

1,6-Dinitropyrene (1,6-DNP) is a ubiquitous airborne pollutant found in diesel exhaust. In this study, mutagenesis was examined in the lungs of gpt-delta transgenic mice after intratracheal instillation of 0-0.1 mg 1,6-DNP. In addition, the 1,6-DNP-induced gpt mutation spectrum was compared with that of control mice. A single intratracheal injection of 0-0.05 mg 1,6-DNP resulted in significant dose-dependent increases in mutant frequency; the induced mutant frequency declined at the 0.1 mg dose. The average lung mutant frequencies at doses of 0.025, 0.05, and 0.1 mg 1,6-DNP were 2.9-, 4.1-, and 1.9-times higher than for control mice ((0.50+/-0.16)x10(-5)). The major mutations induced by 1,6-DNP included G:C-->A:T transitions, G:C-->T:A transversions, and 1-base deletions. Among the G:C-->A:T transitions isolated from 1,6-DNP-treated mice, five (at nucleotide positions 64, 110, 115, 116, and 418) were observed in four or more animals. These positions therefore are potential hotspots for 1,6-DNP mutation. The predominant frameshift mutations following 1,6-DNP treatment included single base pair deletions at G:C (9/13=69%). The results of this study indicate that 1,6-DNP is mutagenic for the lungs of mice.

Benzo[a]pyrene (B[a]P) is a ubiquitous airborne pollutant whose mutagenicity has been evaluated previously by oral and intraperitoneal administration to experimental animals. In this study, mutagenesis in the lungs, the target organ of air pollutants, was examined after a single intratracheal instillation of 0-2 mg B[a]P into gpt delta transgenic mice. Intratracheal injection of B[a]P resulted in a statistically significant and dose-dependent increase in gpt mutant frequency as measured by 6-thioguanine selection. The mutant frequencies at B[a]P doses of 0.5, 1, and 2 mg were 2.8, 4.2, and 6.8 times higher than the frequency seen in nontreated mice (0.60 +/- 0.13 x 10(-5)). The most frequent mutations induced by B[a]P treatment were G:C-->T:A transversions, which are characteristic of B[a]P mutagenesis in other models, and single-base deletions of G:C base pairs. To characterize the hotspots of B[a]P-induced mutations in the gpt gene, we analyzed sequences adjacent to the mutated G:C base pairs. Guanine bases centered in the nucleotide sequences CGT, CGA, and CGG were the most frequent targets of B[a]P. Our results indicate that intratracheal instillation of B[a]P into gpt delta mice causes a dose-dependent increase in gpt mutant frequency in the lung, and that the predominant mutation induced is G:C-->T:A transversion.

We developed a method for enumerating trichloroethylene (TCE) degrading Mycobacterium sp. TA27 by means of PCR. The PCR primers and probe were designed based on the 16S rRNA sequences of Mycobacterium sp. TA27, compared with corresponding regions of 8 phylogenetically related strains. The total DNAs of strain TA27 and the other 8 mycobacteria were extracted and used for PCR amplification by a 5'-nuclease real-time PCR assay. The highest specificity for strain TA27 was observed at an annealing temperature of 62°C and at forward and reverse primer concentrations of 0.2 μM each. The other 8 mycobacteria were not detected under these conditions. The detection limits for the DNA and cells of strain TA27 were 0.05 pg/tube and 25 cells/tube, respectively. The utility of the PCR assay for the quantification of strain TA27 cells was demonstrated in TCE-polluted groundwater.

Mycobacterium spp. strains TA5 and TA27 (ethane-utilizing bacteria), which can degrade trichloroethylene (TCE) and 1,1,1-trichloroethane (1,1,1-TCA), were isolated from soil. Both bacteria could cometabolically degrade dichloromethane, chloroform, 1,1-dichloroethane, 1,2-dichloroethane, 1,1,1-TCA, 1,1,2-TCA, 1,1,1,2-tetrachloroethane, 1,1,2,2-tetrachloroethane, 1,1-dichloroethylene, cis-1,2-dichloroethylene, trans-1,2-dichloroethylene, and TCE with ethane as a carbon source. They could not degrade carbon tetrachloride, freon 113, or tetrachloroethylene. The TCE degradation characteristics of strain TA27 were determined. Under a head-space gas containing 3% ethane, strain TA27 degraded more than 95% of TCE at an initial concentration of 1 mg l–1 within 3 days. We observed good growth and TCE degradation between 25 and 35 °C. At an initial TCE concentration of 30 mg l–1, it degraded 30% of TCE within 7 days. Although growth was inhibited for more than 50 mg l–1 TCE at 3% ethane concentration, good growth and 50% degradation of TCE were observed at 12% ethane concentration within 14 days. High ethane concentration may mitigate the toxicity of TCE.

Two strains of 1,1,1-trichloroethane (TCA)-degrading bacteria, TA5 and TA27, were isolated from soil and identified as Mycobacterium spp. Strains TA5 and TA27 could degrade 25 and 75 mg · liter of TCA −1 cometabolically in the presence of ethane as a carbon source, respectively. The compound 2,2,2-trichloroethanol was produced as a metabolite of the degradation process.

Diesel exhaust (DE) is a major airborne pollutant of urban areas. It contains various polycyclic aromatic hydrocarbons (PAH) and nitrated PAHs. In this study, gpt delta mice were treated with inhalation of 1 or 3 mg m(-3) DE, or a single intratracheal instillation of diesel exhaust particles (DEP) or DEP extract. In the lungs of mice treated with inhalation of 3 mg m(-3) DE for 12 weeks, the mutant frequency (MF) was 3.2-fold higher than that of the control group (1.90 x 10(-5) and 0.59 x 10(-5), respectively). An instillation of DEP and DEP extract resulted in a significant dose-dependent linear increase in MF. In mice treated with 0.5 mg DEP and 0.2 mg DEP extract, the MFs were 3.0- and 2.7-fold higher than that of the control group, respectively. The mutagenic potency (MF mg(-1)) of DEP extract (5.6 x 10(-5)) was double that of DEP (2.7 x 10(-5)), suggesting that the mutagenicity of the latter is derived primarily from compounds in the extract, which itself is responsible for ca. 50% of the weight of DEP. G:C-->A:T transitions were the predominant gpt mutation induced by all three treatments and G:C-->T:A transversions were induced by DEP and DEP extract. Guanine bases centered in nucleotide sequences such as GGA, TGA, CGG, and CGT were the major mutation targets of all three treatments. Thus, our results suggest that the mutagens contained in DEP such as PAH and nitrated PAHs induce mutations and may be responsible for carcinogenesis caused by inhalation of DE.

The lung is an organ that is sensitive to mutations induced by chemicals in ambient air, and transgenic mice harboring guanine phosphoribosyltransferase (gpt) gene as a target gene are a well-established model system for assessing genotoxicity in vivo. Transcription factor Nrf2 mediates inducible and constitutive expression of cytoprotective enzymes against xenobiotics and mutagens. To address whether Nrf2 is also involved in DNA protection, we generated nrf2+/−::gpt and nrf2−/−::gpt mice. The spontaneous mutation frequency of the gpt gene in the lung was approximately three times higher in nrf2-null (nrf2−/−) mice than nrf2 heterozygous (nrf2+/−) and wild-type (nrf2+/+) mice, whereas in the liver, the mutation frequency was higher in nrf2−/− and nrf2+/− mice than in nrf2+/+ wild-type mice. By contrast, no difference in mutation frequency was observed in testis among the three genotypes. A single intratracheal instillation of benzo(a)pyrene (BaP) increased the lung mutation frequency 3.1- and 6.1-fold in nrf2+/− and nrf2−/− mice, respectively, compared with BaP-untreated nrf2+/− mice, showing that nrf2−/− mice are more susceptible to genotoxic carcinogens. Surprisingly, mutation profiles of the gpt gene in BaP-treated nrf2+/− mice was substantially different from that in BaP-untreated nrf2−/− mice. In nrf2−/− mice, spontaneous and BaP-induced mutation hotspots were observed at nucleotides 64 and 140 of gpt, respectively. These results thus show that Nrf2 aids in the prevention of mutations in vivo and suggest that Nrf2 protects genomic DNA against certain types of mutations. [Cancer Res 2007;67(12):5643–8]