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 and carc inogenic 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 2 6 weeks, Following a 26-week recovery period, an increased incidence o f 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 an d direct sequencing of polymerase chain reaction (PCR) amplified DNA, isolated from paraffin-embedded sections of HG neoplasms, A higher fre quency of ras mutations, in particular K-ras mutations, was detected i n isoprene-induced neoplasms than in 1,3-butadiene-induced or control HG neoplasms, All of the isoprene-induced HG neoplasms exhibited activ ated 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 cont rol B6C3F1 mice (8% K-ras and 48% H-ras), The predominant mutations in isoprene-induced HG neoplasms, but not in previously or newly analyse d 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 w ere detected in HG neoplasms from the 2200 ppm exposure group while on e-third was present in the 7000 ppm group, Isoprene-induced HG neoplas ms with K-ras or H-ras mutations had an elevated proliferating cell nu clear antigen (PCNA) index, compared to spontaneous HG neoplasms witho ut ras mutations, The high frequency and specificity of the pas mutati on profile suggest that ras protooncogene activation contributes to is oprene-induced HG tumorigenesis.