Biochemical Responses of Fish Sac Fry and a Primary Cell Culture of Fish Hepatocytes Exposed to Polychlorinated Naphthalenes

Abstract

Chlorinated naphthalenes are planar halogenated aromatic compounds, which are widespread in the environment. Knowledge of their biochemical and toxicological actions in aquatic biota is, however, limited. The objective of this study was to assess the toxicity of highly chlorinated naphthalene congeners found in the aquatic environment on fish sac fry and to study their effects on xenobiotic metabolizing enzymes (CYP) using a short-term primary culture of fish hepatocytes and liver microsomes. A few days after hatching, rainbow trout sac fry were administered either Hallovax 1014, a mixture of 1,2,3,4,6,7-hexachloronaphthalene and 1,2,3,5,6, 7-hexachloronaphthalene (HxCN-mix), or 1,2,3,4,5,6, 7-heptachloronaphthalene (HpCN) (0.08, 0.8, and 4 microg/sac fry injected into the yolk sac). The exposure was terminated 2 weeks later. The naphthalene preparations did not cause any clinical signs of toxicity or difference in mortality rates between the control and treated groups. Immunohistochemical analysis of CYP1A expression in the treated sac fry revealed that staining was most pronounced in the hepatocytes and thereafter in kidney tubular epithelial cells. Moderate CYP1A staining was also seen in the mucosal epithelium of pyloric caecae and mild staining in the epithelium of olfactory organ. Staining in control sac fry was weak or absent. Exposure of the primary cell culture of trout hepatocytes to a low doses (</=10 ng/ml) of the chlorinated naphthalenes increased significantly CYP1A-associated EROD activity and CYP1A mRNA content, HxCN-mix being the most potent and thereafter HpCN and Hallovax 1014. The higher doses (50-100 ng/ml) of each naphthalene also inhibited EROD activity. However, the content of CYP1A mRNA or the intensity of the CYP1A protein band (58 kDa) recognized by anti-trout CYP1A peptide antibodies were not decreased with increasing polychlorinated naphthalene (PCN) concentration, indicating that the inhibition was not due to reduced protein synthesis. Furthermore, in vitro analyses of the inhibitory potential of PCNs on CYP1A activity with trout liver microsomes suggested that these naphthalene preparations may be CYP1A substrates and act as competitive inhibitors of CYP1A catalyst. Our results demonstrate that highly chlorinated naphthalenes are potent modulators of fish CYP1A enzyme and suggest that hepatocytes and tubular epithelial cells are the cell types that may be vulnerable to their metabolic products for cell injury in fish sac fry.