Cellular biomarkers of exposure and biological effect in hepatocytes of turbot (Scophthalmus maximus) exposed to Cd, Cu and Zn and after depuration

Cellular biomarkers of exposure and biological effects were measured in hepatocytes of turbot exposed to either Cd, Cu or Zn at concentrations of 1 and 10 mg/l seawater for 7 days and after depuration for 14 days. Metal content in hepatocyte lysosomes was determined by image analysis after autometallography (AMG) as volume density of autometallographed black silver deposits (VvBSD). Metallothionein (MT) levels were quantified on liver sections by microdensitometry after immunohistochemical staining with a polyclonal anti cod-MT antibody (MT-OD), and in the cytosolic fraction of hepatocytes by difference pulse polarography (MT-DPP). Lysosomal structural changes (lysosomal volume, surface and numerical densities-VvLYS, SvLYS and NvLYS−, and surface-to-volume ratio S/VLys) were quantified by image analysis after demonstration of β-glucuronidase activity on liver cryotome sections. Vacuolisation produced by metal-exposure in hepatocytes was quantified by stereology as volume density of vacuoles (VvVAC). Exposure time and metal concentrations significantly affected VvBSD in lysosomes, MT levels and the degree of vacuolisation after 1 h and 1 day exposure to the three metals. The highest VvBSD, MT and VvVAC values were recorded after 7 days exposure in all cases. MT-OD and MT-DPP were significantly correlated with VvBSD. VvLYS in hepatocytes increased significantly after exposure to the metals. Exposure biomarkers returned to control values after depuration with the exception of those turbots that had been exposed to 10 mg Cd/l. Alike, VvLYS and Svlys (Cu exposure) and NvLYS (Cd and Zn exposures) returned to control values after depuration. It has been therefore demonstrated that the biomarkers used are reversible and return towards control levels once metal exposure ceases. Overall, it is concluded that VvBSD, MT-levels and lysosomal responses are valuable biomarkers to assess metal exposure and its effects in turbot, although in quantitative terms the biomarker response varied between metals.