Ecophysiology, genotoxicity, histopathology, and gene responses of naphthalene injected Colossoma macropomum (Cuvier, 1818) exposed to hypoxia

Abstract

The present study aimed to evaluate the biological responses of Colossoma macropomum to naphthalene injection and subsequent hypoxia exposure, emphasizing the expression of the tumor suppressor gene tp53. Tambaquis were intraperitoneally injected with naphthalene (50 mg/kg) and, after 96 hours, the fish were transferred to respirometry chambers and, submitted to progressive hypoxia for the determination of critical PO2. In a subsequent experiment, the fish received an intraperitoneal injection of naphthalene and were kept for 96 hours under normoxia. Successively, fish were challenged with acute hypoxia (PO2<PO2 crit) during 6 hours. We observed that the PO2 crit was not affected by naphthalene injection. Moreover, hematological parameters were modulated only in response to hypoxia. Fish with naphthalene injection plus hypoxia exposure presented altered activity of the GST and CAT enzymes. Exposure to naphthalene also resulted in DNA damages, which was not influenced by hypoxia. Hypoxia accentuated the hepatic lesions caused by naphthalene, as well as it also impaired the transcription of tp53 in naphtalene injected fish, demonstrating the risks of contaminating aquatic environments, especially environments where hypoxic conditions are common and occur on a daily or on seasonal basis, as in the Amazon basin.

Figure 1. Class of DNA damages (comet assay) observed in blood cells of C. macropomum in the three experimental groups: Group Without Injection (GWI), Group with corn oil injection (Sham); and Group with naphthalene injection (Naph) after 96 h normoxia exposure followed by 6 h of hypoxia. Class 0, without damages (A), class 1 (B), class 2 (C), class 3 (D), and class 4, maximum damages (E). The images were captured with 400 x magnication.

Figure 2. GST (A) and CAT (B) activity and lipoperoxidation (LPO) (C) in the liver of C. macropomum in the three experimental groups: Group Without Injection (GWI), Group with corn oil injection (Sham); and Group with naphthalene injection (Naph) after 96 h normoxia exposure followed by 6 h of hypoxia. Small caps letters (a and b) indicate differences among treatments in normoxia. Capital letters (A and B) indicate differences among treatments in hypoxia. *Indicates differences between normoxia and hypoxia at the same treatment. Significance level of the Tukey test was p <0.05.

Figure 3. Distribution of classes of DNA damage in C. macropomum blood cells in the three experimental groups: Group Without Injection (GWI); Group with corn oil injection (Sham); and Group naphthalene injection (Naph) after 96 h normoxia exposure (N) followed by 6 h of hypoxia (H). The Genetic Damage Index (GDI) is identified in each treatment (on the bars). Small cap letters (a and b) indicate statistical difference among treatments in normoxia. Capital letters (A and B) indicate statistical difference among hypoxia treatments. The statistical significance value was p <0.05.

Figure 4. Liver histopathology of C. macropomum in the three experimental groups: Group Without Injection (GWI); Group with corn oil injection (Sham); and Group with naphthalene injection (Naph) after 96 h normoxia exposure followed by 6 h of hypoxia. (A) normal C. macropomum liver standing out the hepatopancreas. (B, C) after Naph injection in normoxia. Arrows indicate nuclei hypertrophy (B) and nuclei vacuolization (C). (D, E) after Naph injection and subsequent hypoxia exposure. Arrows indicate hepatocytes with nuclei degeneration (D) and nuclei vacuolization (E); N shows focal necrosis. Hematoxylin and eosin stain. (F) C. macropomum liver of the Sham group in normoxia demonstrating a strong positive reaction to PAS. (G, H) C. macropomum liver after hypoxia exposure: GWI (G) and Naph groups (H) demonstrating a weak reaction to PAS. Asterisks indicate areas with higher glycogen concentration. PAS corresponds to Periodic Acid Schiff Stain.

Figure 5. Relative tp53 gene expression in C. macropomum liver in the the three experimental groups: Group Without Injection (GWI); Group with corn oil injection (Sham); and Group with naphthalene injection (Naph) after 96 h normoxia exposure followed by 6 h of hypoxia. Small cap letters (a and b) indicate differences among treatments in normoxia. Capital letters (A and B) indicate differences among treatments in hypoxia. *Indicates differences between normoxia and hypoxia of the same treatment (p <0.05).

Figure 6. Biplot representing the distribution of PCA values for the variables analyzed in C. macropomum in the three experimental groups: Group Without Injection (GWI); Group with corn oil injection (Sham); and Group with naphthalene injection (Naph) after 96 h normoxia exposure followed by 6 h of hypoxia. All groups are compared and variation among variables is explained by p1 = 50.0% and p2 = 24.0%.