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 a...

Assessing cancer hazards of bitumen emissions – a case study for complex petroleum substances

“…Wang et al 2003 also identified DNA adducts in the lungs of mice exposed to paving bitumen emissions representative of Midwest formulations generated at 180 C using the NIOSH/Heritage fume-generating system. However similar DNA adduct results from Halter et al 2007, in the absence of increased micronuclei in bone marrow erythrocytes and with no increase in 8-oxo-dG adducts from exposure to semiblown paving bitumen emissions, produced no respiratory tumors in rats after 2 years of exposure as discussed more thoroughly later. Studies performed with transgenic rodents exposed by nose-only inhalation to emissions from road paving bitumen [Venezuelan 50/70 pen generated at 170 C] for 6 h/day, 5 days produced no adducts in the lungs of male Lac1 mice and no change in the mutant frequency for lac 1 gene or CII, a neural reporter gene (Micillino et al 2002).…”

“…The authors explained this transitory inflammatory response as the result of sacrifice immediately after termination of exposure, allowing the observation of particles being cleared by alveolar macrophages. Halter et al 2007 also reported up regulation of CYP1A1 and CYP2G1 genes or actual isoforms (cytochrome P450 monooxygenase 1B1 and 2G1) in nasal and lung tissue of rats exposed for 1 year to partially oxidized bitumen fume condensate as well as up and downregulation of genes with known functions in inflammation/immune responses. However, no lung tumors were observed at the end of two years of exposure.…”

“…The applicability of this study for evaluating carcinogenic/genotoxic potential was enhanced by the inclusion of additional endpoints, including DNA adducts, urinary PAC metabolites, micronuclei formation and altered gene expression (Halter et al 2007). Despite evidence of PAC uptake and metabolism, altered gene expression, and DNA adducts (respiratory tract), no evidence of micronuclei formation, oxidative DNA damage or an increase in overall or organ-specific neoplasms was observed.…”

“…Urinary 8-hydroxy-2 0 -deoxyguanosine [8-0H-dG] is a by-product of DNA repair and oxidative damage. Halter et al (2007) reported no evidence of 8-OH-dG adducts in an extensive rat inhalation study, concluding that oxidative damage was not a component of DNA adduct or strand break induction. The Toraason et al (2001) study of roofing workers did not show an increased leukocyte DNA 8-OHdG/dG ratio and urinary 8-OHdG levels were comparable to those of controls.…”

“…In a parallel series of investigations, as part of the same chronic toxicity/carcinogenicity study, the following parameters were examined in groups of rats exposed to bitumen emissions (Halter et al 2007):…”