In vivo mutagenesis induced by benzo[a]pyrene instilled into the lung ofgpt delta transgenic mice

Abstract

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.

In vivo mutagenesis in the lungs of gpt‐delta transgenic mice treated intratracheally with 1,6‐dinitropyrene

“…The mutant frequency in the lungs of control mice was (0.5 6 0.2) 3 10 À5 (Table I). The mutant frequencies in control mice in this study were similar to those observed previously in several tissues of gpt-delta mice [Masumura et al, 1999[Masumura et al, , 2003Hashimoto et al, 2005]. Single injections of 0.025, 0.05, and 0.1 mg 1,6-DNP resulted in 2.9-, 4.1-, and 1.9-Environmental and Molecular Mutagenesis.…”

“…After purification, amplified products were sequenced with a Big Dye Terminator v3.1 Cycle sequencing kit (Applied Biosystems, Foster City, CA) and an Applied Biosystems model 3730xl DNA analyzer. The sequencing oligonucleotides, primer-A and primer-C, are given in earlier reports Hashimoto et al, 2005].…”

“…The animal was placed on a restraining board with linen threads to hold the mouth open. The 1,6-DNP solution was instilled into the trachea via a polyethylene tube [Takano et al, 2002;Hashimoto et al, 2005]. Two doses (0.025 and 0.05 mg) of 1,6-DNP were dissolved in 50 ll tricaprylin, while the 0.1 mg dose was dissolved in 100 ll tricaprylin.…”

“…A 739-bp DNA fragment containing the gpt gene was amplified by PCR using primer-1 and primer-2, as described previously Hashimoto et al, 2005]. The reaction mixture contained 5 pmol of each primer, and was 200 mM for each dNTP.…”

Spontaneous Mutagenesis in Rodents: Spontaneous Gene Mutations Identified by Neutral Reporter Genes in gpt Delta Transgenic Mice and Rats

“…The gpt and Spi − assay systems have been validated primarily in mice with many chemical mutagens/carcinogens, UV and ionizing radiation, for which mutagenicity, organ specificity and mutation spectrum have been thoroughly characterized. 1,[10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28] Recently, the outbred gpt delta SD rat was backcrossed with Fisher 344 (F344) rat, to establish an inbred gpt delta rat (F344). In this review, we focus on the spontaneous mutations detected by the gpt and Spi − assays in gpt delta mice and rats and discuss possible mechanisms underlying these in vivo mutations.…”