Cytochrome P4501A (CYP1A) induction and DNA adducts as biomarkers for organic pollution in the natural environment.: Evaluation and validation in bioassays and in a field laboratory experiment

Abstract

Due to the serious ecological consequences associated with exposure to environmental pollutants, considerable attention has been focused on the applicability of early warning signals, or biomarkers in environmental research and monitoring. Biomarkers have been defined as “any biological response to an environmental chemical at the individual level or below demonstrating a departure from normal status”. However, before biomarkers can be usefully employed, a critical evaluation of each of them is necessary. In this thesis, biomarker responses towards exposure to organic pollutants as polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) were evaluated through laboratory studies (bioassays) and in a field laboratory experiment. The biomarkers applied were induction of cytochrome P4501A (CYP1A) and formation of DNA adducts. CYP1A induction was measured in terms of CYP1A immunopositive protein (Western blotting). DNA adducts were analysed by the 32P-postlabeling technique.

The sensitivities and the concentration-dependent relationships of the biomarker responses were investigated in the rat hepatoma cell line, Fao, after exposure to environmentally relevant concentrations of selected organic compounds, singly and in combinations (paper I), and to organic extract of soil samples expected to be differentially exposed to environmental contamination (paper II). The supernatant fraction (S9 fraction) of Fao cells, pretreated with the most potent CYP1A inducing soil extract, was also used as an activation system of mutagens in the Ames Salmonella test. The applicability of the biomarkers was then evaluated in a field laboratory experiment (paper III), using glaucous gulls (Larus hyperboreus) which were fed with environmentally contaminated gull eggs (exposed) or hen eggs (controls).

In summary, the measurement of CYP1A immunopositive protein was proven a very sensitive response. The results mainly showed a concentration-dependent endpoint in vitro (paper I, II), although non-linearity of the concentration-response relationship was observed in certain cases. The differences observed between the inducing powers of the four soil sample extracts (paper II) are most probably explained by the different compositions and concentrations of CYP1A inducers in soil at these localities. The S9 fraction of the pretreated Fao cells activated benzo(a)pyrene (B(a)P) to mutagen(s) in a concentration-dependent relationship, although the response was weak. This may further support the CYP inducing capacity of this soil extract (paper II). The rat hepatoma cell line Fao may serve as a valuable system in the evaluation of environmental samples contaminated with CYP1A inducing compounds (paper II). DNA adduct formation was also shown to be a rather sensitive response in Fao cells (paper I), but failed to detect potentially harmful compounds in the environmental soil samples (paper II). The in vitro experiment (paper I) also demonstrated that mixtures of organic compounds may affect biomarker responses differently from exposure to single compounds. The field laboratory experiment (paper III) indicated a clear potential for applying CYP1A protein induction and DNA adduct formation as biomarkers in the monitoring of organic pollutant exposure, at least in glaucous gulls.