Histopathological evaluation of tested zebrafish. (A–D) Exemplificative micrographs of alterations in the gills after 14 days of exposure (AB and Van Gieson’s elastic). (A) Control fish. (B) Fish exposed to Cd, evidencing focal interlamellar hyperplasia (hp) and hypertrophy of pavement cells (hy) as main alterations, together with localised hyperaemia, likely responsible for aneurysms (an) in lamellar capillaries. Inset. Evident aneurysms in the lamellae of fish exposed to B[a]P for 14 days as well. Note vessel engorgement, which implies blood pressure, therefore excluding post-mortem blood stasis. (C) Fish exposed to combined Cd and B[a]P showing severe interlamellar hyperplasia. Amidst hyperplasic cells, which included goblet cells as well (stained blue), are here found macrophages (ma) and lacunae (lc) holding mucus, plasma exudate and inflammatory cells. (D) Gills of a fish after the one-week recovery, following exposure to Cd. Lamellae present a condition similar to that of controls, albeit some evidence of hyperplasia (hp) persisting. Apoptotic epithelial cells (arrowhead) were noted in gill epithelia at hyperplastic foci. Compare to panel A. Note normal pavement cells of lamellae. cp) blood capillary; fl) filament; gc) goblet cell (mucocyte); lm) lamellae, pv) pavement cell. Scale bars: 25 µm. Panels below: fluorescence immunolocalization of tp53 (red fluorescent probe) in gills of treated fish after the one-week recovery period, counterstained with DAPI. The signal for tp53 protein in Cd-exposed animals (arrowhead) after recovery is essentially similar to that of controls. The response was higher in animals previously exposed to B[a]P or to the mixture treatment. This was reflected in larger and denser staining spots, as well as in a broader distribution of tp53-positive cells.

Common type of cells (a-e) and erythrocytic nuclear abnormalities (ENA) (f-i) observed in exposed Danio rerio: (a) reticulocyte , (b) mature erythrocyte, (c) lymphocyte, (d) neutrophil, (e) thrombocyte, (f) bilobed nucleus, (g) budding nucleus, (h) micronucleus (arrow), (i) kidney-shaped nucleus.