Ras oncogene and Hypoxia-inducible factor-1 alpha (hif-1α) expression in the Amazon fish Colossoma macropomum (Cuvier, 1818) exposed to benzo[a]pyrene

Abstract

Benzo[a]pyrene (B[a]P) is a petroleum derivative capable of inducing cancer in human and animals. In this work, under laboratory conditions, we analyzed the responses of Colossoma macropomum to B[a]P acute exposure through intraperitoneal injection of four different B[a]P concentrations (4, 8, 16 and 32 μmol/kg) or corn oil (control group). We analyzed expression of the ras oncogene and the Hypoxia-inducible factor-1 alpha (hif-1α) gene using quantitative real-time PCR. Additionally, liver histopathological changes and genotoxic effects were evaluated through the comet assay. Ras oncogene was overexpressed in fish exposed to 4, 8 of 16 μmol/kg B[a]P, showing 4.96, 7.10 and 6.78-fold increases, respectively. Overexpression also occurred in hif-1α in fish injected with 4 and 8 μmol/kg B[a]P, showing 8.82 and 4.64-fold increases, respectively. Histopathological damage in fish liver was classified as irreparable in fish exposed to 8, 16 and 32 μmol/kg μM B[a]P. The genotoxic damage increased in fish injected with 8 and 16 μmol/kg in comparison with the control group. Acute exposure of B[a]P was capable to interrupt the expression of ras oncogene and hif-1α, and increase DNA breaks due to tissue damage.

Figure 1. C. macropomum liver exposed to corn oil (control group) or doses of B[a]P. (A) Normal liver, hepatocytes are organized in one or two layers surrounded by sinusoids (black arrows). (B) Normal liver parenchyma, highlighting a blood vessel with red blood cells (asterisk). (C) Image of liver exposed to 8 μmol/kg B[a]P evidencing a hepatopancreas (asterisk) and sinusoid obstruction (white arrow). (D) Image of fish liver exposed to 8 μmol/kg B[a]P, showing necrotic area (asterisk). (E) Image of liver exposed to 16 μmol/kg B[a]P showing some hepatocytes without nucleus (white asterisk), sinusoidal dilatation (black arrows) and hemosiderin (white arrow). (F) Image of vacuolated hepatocytes of fish exposed to 32 μmol/kg B[a]P; cytoplasm degeneration (black asterisks) and pyknotic nuclei (black arrow) are evident. Slides were stained with hematoxylin and eosin.

Figure 2. Histopathological alteration index (HAI) of C. macropomum liver after exposure to different concentrations of B[a]P. Indexes are in accordance with Poleksic and Mitrovic-Tutundic (1994). *Indicates significant differences compared to control group (corn oil) (p < 0.05). Kruskal-Wallis test was used.

Figure 3. Genetic damage index (GDI) of erythrocytes of C. macropomum after 96 h of injection of different concentrations of B[a]P. *Indicates significant differences compared to control group (corn oil) (p < 0.05). Kruskal-Wallis test was used.

Figure 4. Relative expression of the oncogene ras in liver of C. macropomum after 96 h of injection of different concentrations of B[a]P. *Indicates significant difference in comparison to control group (p < 0.05). Kruskal-Wallis test was used.

Figure 5. Relative expression of gene hif-1α gene in C. macropomum after 96 h of injection of different concentrations of B[a]P. *Indicates significant difference in comparison to control group (corn oil) (p < 0.05). Kruskal-Wallis test was used.