Importance of DNA-Adduct Formation and Gene Expression Profiling of Disease Candidate Genes in Rats Exposed to Bitumen Fumes

Abstract

The equivocal experimental and epidemiological evidence of bitumen fumes and the possible mechanisms of toxicity remain uncertain. This study therefore aimed at investigating the genotoxicity of bitumen fumes, the biotransformation and urinary excretion of PAHs, and altered expression of a selected number of genes in lung, nasal epithelium, and white blood cells of rats. Animals were exposed to three different concentrations (low: 4 mg/m 3 ; medium: 20 mg/m 3 ; high: 100 mg/m 3 ) of bitumen fume condensate for 5 days, 30 days, and 12 months (6 hours per day) or ambient air. Notably, no dose-related signs of intolerance were observed throughout the inhalation period but dose dependent uptake of bitumen fumes was observed based on urinary excretion of PAHs and metabolites. At best, measurements of naphthols enabled an estimate of dose-dependent body burden. Excretion of 1-hydroxy- and 2-hydroxyphenanthrene was dose dependent and their production is catalyzed by the CYP1A1 monooxygenase which we found to be strongly induced upon exposure to bitumen fumes. Furthermore, pyrene, a minor component in bitumen fumes, produced hydroxypyrene levels close to the detection limit in rat urine. We additionally determined DNA adduct formation by the 32 P-postlabelling assay and observed a dose and time dependent increase of 3 to 4 stable DNA adducts in lung, nasal, and alveolar epithelium. DNA adduct levels were highest in nasal epithelium, the relative adduct level (RAL) being 450 adducts per 10 9 nucleotides. For lung and alveolar epithelium the RAL was 114 and 76 adducts per 10 9 nucleotides, respectively. However, we did not observe micronucleated red blood cells of the peripheral blood or with polychromatic erythrocytes of the bone marrow (after 12 months). It is important to note that erythrocyte cell count in bone marrow smears was reduced in four out of six animals after 12 months of exposure, clearly demonstrating that components of bitumen fume had reached the bone marrow. Finally, we investigated by reverse transcription polymerase chain reaction regulation of genes with known functions in inflammation, asthma and other pulmonary diseases. Gene expression changed during the time of exposure. With the monooxygenases CYP1A1 and CYP2G1 we observed dose dependent regulation in nasal and lung tissue. We also observed significant, but dose independent, regulation of cathepsin K and D, cadherin 22, platelet activating factor acetylhydrolase isoform 1b alpha subunit and the regulator of G-protein signalling in nasal epithelium of male rats after exposure to bitumen fumes. We found bitumen fumes to be genotoxic in target tissue of exposure and observed altered regulation of genes involved in the metabolic activation of polycyclic aromatic hydrocarbons and cellular inflammatory processes. These findings are consistent with the histopathology observed in the respiratory tract of rats chronically exposed to bitumen fume. An understanding of the regulation of suspected disease candidate genes in target tissues of exposure will be an interesting objective for further research into the mechanisms of toxicity.