Temporal Changes in K-ras Mutant Fraction in Lung Tissue of Big Blue B6C3F1 Mice Exposed to Ethylene Oxide

Abstract

Ethylene oxide (EO) is a genotoxicant and a mouse lung carcinogen, but whether EO is carcinogenic through a mutagenic mode of action remains unclear. To investigate this question, 8-week-old male Big Blue B6C3F₁ mice (10 mice/group) were exposed to EO by inhalation-6 h/day, 5 days/week for 4 weeks (0, 10, 50, 100, or 200 ppm EO) and 8 or 12 weeks (0, 100, or 200 ppm EO). Lung DNA samples were analyzed for levels of 3 K-ras codon 12 mutations (GGT→GAT, GGT→GTT, and GGT→TGT) using ACB-PCR. No measureable level of K-ras codon 12 TGT mutation was detected (ie, all lung mutant fractions [MFs] ≤ 10⁻⁵). Four weeks of inhalation of 100 ppm EO caused a significant increase in K-ras codon 12 GGT→GTT MF relative to controls, whereas 50, 100, and 200 ppm EO caused significant increases in K-ras codon 12 GGT→GAT MF. In addition, significant inverse correlations were observed between K-ras codon 12 GGT→GTT MF and cII mutant frequency in the lungs of the same mice exposed to 50, 100, or 200 ppm EO for 4 weeks. Surprisingly, 8 weeks of exposure to 100 and 200 ppm EO caused significant decreases in K-ras MFs relative to controls. Thus, the changes in K-ras MF as a function of cumulative EO dose were nonmonotonic and were consistent with EO causing early amplification of preexisting K-ras mutations, rather than induction of K-ras mutation through genotoxicity at codon 12. The possibility that these changes reflect K-ras mutant cell selection under varying degrees of oxidative stress is discussed.

Quantification of Kras mutant fraction in the lung DNA of mice exposed to aerosolized particulate vanadium pentoxide by inhalation

“…Finally, experience with the ACB-PCR approach suggests that the assay has sufficient sensitivity to detect early induction of mutation following carcinogen exposures. In two cases, ACB-PCR detected a significant induction of Kras mutation after 4 weeks of exposure using doses of carcinogens below that which produced a significant tumor response in a 2-year bioassay [8,10]. In one of these studies, an inhalation study quite similar in design to the present study, a significant induction of Kras codon 12 GAT and GTT mutation was detected after 4 weeks of exposure to 50 ppm ethylene oxide, when 100 ppm was the lowest positive dose in the NTP bioassay [10].…”

“…In two cases, ACB-PCR detected a significant induction of Kras mutation after 4 weeks of exposure using doses of carcinogens below that which produced a significant tumor response in a 2-year bioassay [8,10]. In one of these studies, an inhalation study quite similar in design to the present study, a significant induction of Kras codon 12 GAT and GTT mutation was detected after 4 weeks of exposure to 50 ppm ethylene oxide, when 100 ppm was the lowest positive dose in the NTP bioassay [10]. The relatively slow in vivo turn-over times of mouse lung epithelial and mesenchymal cell populations have been estimated as 28-35 days and approximately 7 days, respectively, although cell turn-over has been reported to increase significantly following chemical induced lung injury [15,16].…”

“…First-round PCR products were prepared from standards and unknowns as previously described [10]. A first-round PCR amplification, employing the PfuUltra hotstart high-fidelity DNA polymerase (Stratagene, La Jolla, CA) amplified a Kras gene sequence from linearized plasmid DNAs, in order to synthesize the MF standards.…”

“…ACB-PCR has been used successfully to detect the shortterm effects of mutagenic carcinogens and to characterize the dose-response relationship for the induction of mutations that drive cancer [6][7][8][9][10]. ACB-PCR detected significant increases in Ras mutations following in vivo exposures to azoxymethane and benzo[a]pyrene 1 and 4 weeks after acute exposures [8,11], and after 3-or 4-week exposures to aristolochic acid or ethylene oxide, respectively [9,10].…”

“…ACB-PCR detected significant increases in Ras mutations following in vivo exposures to azoxymethane and benzo[a]pyrene 1 and 4 weeks after acute exposures [8,11], and after 3-or 4-week exposures to aristolochic acid or ethylene oxide, respectively [9,10]. In addition, ACB-PCR data can be integrated with other measured endpoints (e.g., DNA adducts and neutral reporter gene mutations) to assess whether data across endpoints support a particular MOA [7][8][9][10]. Studies documenting that ACB-PCR can detect early effects of mutagenic carcinogens are quite important because temporality of key events is a major consideration in establishing MOA.…”