Isoprene is the 2-methyl analog of
1,3-butadiene, a genotoxic and carcinogenic 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 26 weeks. Following a 26-week recovery period, an increased incidence of Harderian gland (HG)
neoplasms was observed at both concentrations. The present study was designed to characterize genetic alterations in the K-ras and H-ras protooncogenes in HG
neoplasms. Mutations in K-ras and H-ras were identified by single-strand conformational analysis and direct sequencing of polymerase chain reaction (PCR) amplified
DNA, isolated from
paraffin-embedded sections of HG
neoplasms. A higher frequency of ras mutations, in particular K-ras mutations, was detected in
isoprene-induced
neoplasms than in 1,3-butadiene-induced or control HG
neoplasms. All of the
isoprene-induced HG
neoplasms exhibited activated K-ras (60%) or H-ras (40%) mutations. In contrast, ras mutations were detected in 69% of HG
neoplasms from
1,3-butadiene exposed mice (14% K-ras and 55% H-ras) and in 56% of HG
neoplasms obtained from control B6C3F1 mice (8% K-ras and 48% H-ras). The predominant mutations in
isoprene-induced HG
neoplasms, but not in 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 from the 2200 ppm exposure group while one-third was present in the 7000 ppm group.
Isoprene-induced HG
neoplasms with K-ras or H-ras mutations had an elevated
proliferating cell nuclear antigen (
PCNA) index, compared to spontaneous HG
neoplasms without ras mutations. The high frequency and specificity of the ras mutation profile suggest that ras protooncogene activation contributes to
isoprene-induced HG
tumorigenesis.