Effects of Benzo[a]pyrene on Gilthead Sea Bream (Sparus Aurata L.) Hepatocytes Exposed in Vitro to Short and Long Term Trials

Figures & data

Figure 1. Dose-response curves obtained in the MTT assay after treatment of S. aurata hepatocytes with increasing B[a]P concentrations for 24, 48 and 72 h. Data are expressed as percentage of the unexposed controls.

Figure 2. Immunofluorescence analysis of B[a]P induced apoptotic damage on hepatocytes primary culture after 24 and 72 h exposure: original magnification 200x (A, B, C, E, F, G, I, L); 100x (D, H). A-F) Normal control without B[a]P exposure (annexin V-/6-CFDA+; living cells green). Hepatocytes cultured in 96-well plates were exposed to B[a]P at doses of 100 µg/mL for 24 and 72 h (B-G) (annexin V+/6-CFDA-; necrotic cells red); 1 μg/mL (C-H), 1 ng/mL (D-I) and 1 pg/mL (E-L) for 24 h and 72 h (annexin V+, 6-CFDA+; early apoptotic cells yellow-orange). Methanol-fixed cells were stained with annexin V-Cy3.18.

Figure 3. Contrast-phase analysis of S. aurata hepatocytes in primary culture exposed to 1 ng/mL of B[a]P after 72 h: uncontrolled cell proliferation observed in correspondence of the small nests of actively proliferating hepatocytes indicating the formation of neoplastic foci. Original magnification 400x. *Immunofluorescent labeling of DNA (PCNA antigen +).

Table 1. Statistical analysis of variance; two way ANOVA, followed by Bonferroni’s multiple comparison post-hoc test.

Source of variation	Df	MQ	F	P value	% Tot. of variation
Treatment time	2	5971	43.53	<0.0001	51.99
B[a]P doses	10	828.6	6.042	0.0003	36.07

Df, degree of freedom; MQ, mean square due to error; F, variance ratio for tests; P is the estimated probability of rejecting the null hypothesis; P values ≤0.05 indicate significant difference from control.