Both K-ras and H-ras protooncogene mutations are associated with Harderian gland tumorigenesis in B6C3F1 mice exposed to isoprene for 26 weeks

Isoprene is the 2-methyl analog of 1,3-butadiene, a genotoxic andcarcinogenic compound in rats and mice. Male B6C3F1 mice were exposed to 0,2200 or 7000 ppm isoprene by inhalation (6 h/day; 5 days/week) for 26weeks. Following a 26-week recovery period, an increased incidence ofHarderian gland (HG) neoplasms was observed at both concentrations. Thepresent study was designed to characterize genetic alterations in the K-rasand H-ras protooncogenes in HG neoplasms. Mutations in K-ras and H-ras wereidentified by single-strand conformational analysis and direct sequencingof polymerase chain reaction (PCR) amplified DNA, isolated fromparaffin-embedded sections of HG neoplasms. A higher frequency of rasmutations, in particular K- ras mutations, was detected in isoprene-inducedneoplasms than in 1,3- butadiene-induced or control HG neoplasms. All ofthe isoprene-induced HG neoplasms exhibited activated K-ras (60%) or H-ras(40%) mutations. In contrast, ras mutations were detected in 69% of HGneoplasms from 1,3-butadiene exposed mice (14% K-ras and 55% H-ras) and in56% of HG neoplasms obtained from control B6C3F1 mice (8% K-ras and 48%H-ras). The predominant mutations in isoprene-induced HG neoplasms, but notin previously or newly analysed 1,3-butadiene-induced HG neoplasms,consisted of A-->T transversions (CAA-->CTA) at K-ras codon 61(15/30) and C-->A transversions (CAA-->AAA) at H-ras codon 61 (8/30).Two- thirds of the K-ras CTA mutations were detected in HG neoplasms fromthe 2200 ppm exposure group while one-third was present in the 7000 ppmgroup. Isoprene-induced HG neoplasms with K-ras or H-ras mutations had anelevated proliferating cell nuclear antigen (PCNA) index, compared tospontaneous HG neoplasms without ras mutations. The high frequency andspecificity of the ras mutation profile suggest that ras protooncogeneactivation contributes to isoprene-induced HG tumorigenesis.