Fig. 4

Protective effect of pravastatin towards membrane remodeling and hepatotoxicity-induced by B[a]P/ethanol in HFD zebrafish larvae. Membrane remodeling was assessed in the liver of HFD steatotic zebrafish larvae after exposure to ethanol and B[a]P for seven days with and without pravastatin (0.5 µM) from 5 to 12 dpf. Zebrafish larvae under four conditions, control (untreated (C) ± Pravastatin), or treated with combination of both toxicants (BE ± Pravastatin; 25 nM B[a]P and 43 mM ethanol) were stained with di-4-ANEPPDHQ—a membrane order-sensitive fluorescent probe—and analyzed on confocal fluorescence microscopy. Membrane order in membranes of zebrafish liver was measured by computing GP factor. (A) Changes in GP values were expressed as the difference between individual larva GP value and the mean of GP found in control larvae (ΔGP). Values are the mean ± SEM of at least eight larvae. (B) On the left, some representative liver images of each treatment have been selected according to the respective mean of delta GP (magnification ×400). Pixels with higher GP values (could be considered as lipid rafts) have been highlighted in yellow through membrane area of liver cells to pinpoint lipid raft spatial distribution. Liver area outlined in white square on left images are magnified on right side to show lipid raft spatial distribution in plasma membrane. (C) Liver damages were evaluated on zebrafish liver section after HES staining (magnification ×400). Black dotted line outlines liver. Images are representative of at least 3 larvae. (D) From images obtained in (C), histological count of damaged cells was realized. Values are the mean ± SEM of at least three larvae. * Significantly different from HFD control larvae; ^P Significant difference between larvae treated by pravastatin compared to untreated counterparts.