Cytotoxic and Apoptotic Responses to Single and Mixture PAH Exposure in Brown Trout Primary Hepatocytes

Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental contaminants that pose a significant threat to aquatic ecosystems due to their persistence, bioaccumulation, and toxicity, occurring in the aquatic environments as both individual compounds and complex mixtures. Understanding the cellular effects of single and mixture PAH exposures has therefore become increasingly important to assess their ecological risk. Benzo[a]pyrene (BaP) and benzo[a]anthracene (BaA) are common priority pollutants that can be metabolically activated into reactive intermediates, leading to oxidative stress, cellular damage, and apoptosis. Primary fish hepatocytes represent a physiologically relevant in vitro model for investigating these effects while supporting the 3Rs principles (Replacement, Reduction, and Refinement).

The present study aimed to evaluate the cytotoxic and apoptotic effects of BaP and BaA, and their equimolar mixtures in primary hepatocytes isolated from brown trout (Salmo trutta). Hepatocytes were cultured at 18 °C and exposed for 72 h to control medium (supplemented Leibovitz's L-15 medium), solvent control (0.1% dimethyl sulfoxide in supplemented L-15 medium), or BaP and BaA individually or in equimolar mixtures at 1 and 10 µM. Cytotoxicity was assessed by lactate dehydrogenase (LDH) release, and apoptosis was evaluated by caspase-3 immunocytochemistry. Caspase-3 protein expression was quantified based on the intensity of cytoplasmic immunoreactivity relative to background area.

No significant differences in cytotoxicity were observed between exposure conditions. Exposure to the individual compounds did not significantly affect caspase-3 protein expression, whereas exposure to the higher mixture concentration caused a significant increase. These findings highlight the importance of considering the toxicity of PAH mixtures in aquatic organisms and suggest that mixture exposure of BaP and BaA may enhance apoptotic responses in brown trout hepatocytes.