Toxicogenomic profiling of endocrine disruptor 4-Nonylphenol in male catfish Heteropneustes fossilis with respect to gonads

Abstract

Toxicogenomics study reveals information of gene activity and proteins within the particular cells or tissue of an organism in response to toxic substances. 4-Nonylphenol is a potent environmental contaminant and endocrine disruptor. This study elucidates the toxic and xeno-estrogenic effect of 4-Nonylphenol from the cellular level to the gene level by in vivo and in silico approach. In vivo, studies show that exposure of 4-Nonylphenol at a low dose 64µgL^{- 1} and a high dose of 160µgL^{- 1} for 30 days to 60 days of duration during pre-spawning to the spawning period in testes of Heteropneustes fossilis causes cellular level toxicity i.e., dose and duration dependent clumping of spermatocytes. Dose and duration-dependent decrease in superoxide dismutase(SOD), Catalase, glutathione peroxidase(GPx) and increase in lipid peroxidase (LPO) level in testes. There was a dose and duration-dependent decrease in total antioxidant status and increased level of total oxidant status in the testicular tissue of H. fossilis along with an increase in cortisol level 0.4-NP caused alteration in antioxidant enzyme levels impedes the first line of defense mechanism in the body of an organism. There was a dose-dependent increase in necrosis percentage in testicular cells, cell death, and an increase in total ROS (reactive oxygen species) in a dose-dependent manner in testicular cells of H. fossilis. 4-NP causes gene level toxicity i.e., increased DNA migration or DNA fragmentation. Upregulation of gene expression of gonadal aromatase (CYP19a1a) and downregulation of the 3-beta-hydroxysteroid dehydrogenase (3-β HSD) gene in testes were observed. In silico studies also confirmed the interacting potency of 4-NP with steroid enzyme 3- β HSD and CYP19a1a. Present investigations shows that exposure to water bodies contaminated with xenoestrogens like 4-NP has significantly reduced reproductive parameters like fertilization, fecundity, hatching, and larval survival in numerous fish species.4-NP causes alteration in gene expression of the proteins which are very crucial for reproduction and maintenance of maleness. Due to chronic exposure to 4-NP, it becomes a toxicant causing tissue cell death. So, the harmful impact of 4-NP on reproduction in teleost fish is concerning, not just for the fish themselves but for the entire ecosystem. Therefore, efforts should be made to reduce the contamination of water bodies with xenoestrogens.

Keywords: H. fossilis; 4-NP; COMET assay; Gene expression; SEM.

Fig. 1

Scanning electron microscope images showing the effect of 4-NP on the spermatocytes of testes of H. fossill is at low dose (64 µg/L) and high dose (160 µg/L) for 30 and 60 days of pre-spawning to spawning season. Spermatocytes containing cells in different stages of spermatogenesis; Scale bar 2 μm. Sc-spermatocytes.

Fig. 2

Effects of 4-NP on antioxidant defense system (SOD, CAT, GPx, LPO) in testes of catfish H. fossilis during pre-spawning and spawning phase of the reproductive cycle of low dose (64 µg/L) and high dose (160 µg/L) of exposure for 30 days and 60 days. Statistical significance is determined using two- way ANOVA and post-hoc test Bonferroni test. Different letters indicate significant differences with respective control (n = 15, P < 0.05).

Fig. 3

Effects of 4-NP on TAS (total antioxidant status) and TOS (total oxidant status) in testes of catfish H. fossilis during pre-spawning and spawning phase of the reproductive cycle of low dose (64 µg/L) and high dose (160 µg/L) of exposure for 30 days to 60 days. Statistical significance is determined using two-way ANOVA and post hoc test Bonferroni test. Different letters indicate significant differences with respective control (n = 15, P < 0.05).

Fig. 4

Effects of 4-NP on stress hormone (cortisol level) in testes of catfish H. fossilis during pre-spawning and spawning phase of the reproductive cycle of low dose (64 µg/L) and high dose (160 µg/L) of exposure for 30 days to 60 days. Statistical significance is determined using two-way ANOVA and post-hoc test Bonferroni test. Different letters indicate significant differences with respective control (n = 15, P < 0.05).

Fig. 5

(A) Flow cytometry dot plots of the simultaneous binding of annexin V-fluorescein isothiocyanate (FITC) (FL-1) and propidium iodide uptake (FL-2) in testes of H.fossilis control, Low dose 64 µg/l and high dose 160 µg/l for 30 to 60 days exposed groups to 4-NP. The numbers represent the percentage (%) of cells. The cells from the control H.fossilis were mostly negative for both annexin V binding and uptake of propidium iodide (PI). In contrast, the cells from nonylphenol (NP)treated groups exhibited strong positive staining for annexin V or both annexin V and PI in dose and time-dependent manner, (B) and (C) are graphical representation of necrosis and apoptosis percentage of flow cytometry dot plots. Statistical significance is determined using two-way ANOVA and post hoc test Bonferroni test. Different letters indicate significant differences with respective control (n = 15, P < 0.05).

Fig. 6

(A) Cytofluorometric (ROS-FACS) analysis of the frequency histograms for total ROS (reactive oxygen species) generation percentage by flow cytometer (B) Showing graphical representation of Total ROS generation intensity in testicular cells showing the effect of different concentrations of 4-NP doses low dose 64 µg and high dose of 160 µg on testicular cells of H. fossilis duration of 30 to 60 days pre-spawning to spawning period. Statistical significance is determined using two-way ANOVA and post hoc test Bonferroni test. Different letters indicate significant differences with respective control (n = 15, P < 0.05).

Fig. 7

(A) Measuring DNA damage in cells using CASP lab software showing different degrees of DNA migration (DNA fragmentation) indicated by comet parameters as comet head and tail. (a) Control: No DNA migration in undamaged cell (b) Treated: Mild increase in DNA migration or tail length (c) Treated: Moderate increase in DNA migration; (d) Treated: Severe DNA damage with increased DNA migration or tail length. (B) Unprocessed comet assay image showing control and treated group (C) DNA migration (tail length, µm) in control, low dose 64 µg/l, and high dose 160 µg/l for 30 to 60 days exposed groups to 4-NP in the testes of H. fossilis Statistical significance is determined using Two-way ANOVA and post hoc test Bonefferoni test. Different letters indicate significant differences with respective control (n = 15, P < 0.05).

Fig. 7

(A) Measuring DNA damage in cells using CASP lab software showing different degrees of DNA migration (DNA fragmentation) indicated by comet parameters as comet head and tail. (a) Control: No DNA migration in undamaged cell (b) Treated: Mild increase in DNA migration or tail length (c) Treated: Moderate increase in DNA migration; (d) Treated: Severe DNA damage with increased DNA migration or tail length. (B) Unprocessed comet assay image showing control and treated group (C) DNA migration (tail length, µm) in control, low dose 64 µg/l, and high dose 160 µg/l for 30 to 60 days exposed groups to 4-NP in the testes of H. fossilis Statistical significance is determined using Two-way ANOVA and post hoc test Bonefferoni test. Different letters indicate significant differences with respective control (n = 15, P < 0.05).

Fig. 8

Effects of 4-NP on gene expression of Gonadal aromatase and 3-β HSD in the Testes of catfish H. fossilis of 30 to 60 days treatment of low dose (64 µg/L) and high dose (160 µg/L) during pre-spawning and spawning phase of the reproductive cycle. Data were expressed as mean ± SEM (n = 15). Assessment of data by two-way ANOVA followed by post hoc test Bonferroni test. Different letters denote significant changes from the control. (P < 0.05).

Fig. 9

Alpha fold PDB file of proteins of 3-β HSD from UniProt database.

Fig. 10

3-D form representation of docking complex of A-3β HSD, B-CYP19a1a with 4-Nonylphenol Amino-acid residues in the binding pocket of 3-βBHD and CYP19a1a involved in interactions with 4-Nonylphenol.