Exposure to endosulfan can cause long term effects on general biology, including the reproductive system of mice

Abstract

Increased infertility in humans is attributed to the increased use of environmental chemicals in the last several decades. Various studies have identified pesticides as one of the causes of reproductive toxicity. In a previous study, infertility was observed in male mice due to testicular atrophy and decreased sperm count when a sublethal dose of endosulfan (3 mg/kg) with a serum concentration of 23 μg/L was used. However, the serum concentration of endosulfan was much higher (up to 500 μg/L) in people living in endosulfan-exposed areas compared to the one used in the investigation. To mimic the situation in an experimental setup, mice were exposed to 5 mg/kg body weight of endosulfan, and reproductive toxicity and long-term impact on the general biology of animals were examined. HPLC analysis revealed a serum concentration of ∼50 μg/L of endosulfan after 24 h endosulfan exposure affected the normal physiology of mice. Histopathological studies suggest a persistent, severe effect on reproductive organs where vacuole degeneration of basal germinal epithelial cells and degradation of the interstitial matrix were observed in testes. Ovaries showed a reduction in the number of mature Graafian follicles. At the same time, mild vacuolation in liver hepatocytes and changes in the architecture of the lungs were observed. Endosulfan exposure induced DNA damage and mutations in germ cells at the molecular level. Interestingly, even after 8 months of endosulfan exposure, we observed increased DNA breaks in reproductive tissues. An increased DNA Ligase III expression was also observed, consistent with reported elevated levels of MMEJ-mediated repair. Further, we observed the generation of tumors in a few of the treated mice with time. Thus, the study not only explores the changes in the general biology of the mice upon exposure to endosulfan but also describes the molecular mechanism of its long-term effects.

Keywords: DNA repair; double-strand break; endosulfan; genomic stability; infertility; oogenesis; reproductive toxicity; spermatogenesis.

FIGURE 1

Evaluation of bioavailability of ES in mice when exposed to 5 mg/kg body weight of ES. (A) Schematic of the experimental strategy. HPLC analysis was performed using serum samples collected at different time points 4, 8, 10, 12, 24, and 48 h after oral ingestion of ES (5 mg/kg body weight, n = 3). (B) In vivo bioavailability analysis of ES in the serum of Balb/c mice at 4, 8, 10, 12, 24, and 48 h after exposure with ES (5 mg/kg body weight). Three animals were sacrificed for each time point, sera were collected and subjected to HPLC analysis at 214 nm wavelength. (C) Pharmacokinetics of ES in mice serum. The area under the curve (AUC) was calculated to obtain the maximum circulating concentration (C_max) and maximum time (T_max) to reach C_max.

FIGURE 2

Evaluation of physiological effects of ES in mice. (A) Schematic representation of ES-treatment in mice. Mice were exposed to 5 mg/kg body weight of ES through oral gavage every alternate day for 10 doses. (B,C) Liver and kidney function test of ES-treated mice. Bar graphs indicating enzymatic activities reflective of liver and kidney function at 31st day after ES treatment (5 mg/kg, n = 2). (D) Blood parameter analysis following oral administration of ES in mice after 31st day (n = 2). Analysis of red blood cells (RBC), white blood cells (WBC), haemoglobin (HGB), platelets, MCHC, MCV, MCH, PCV, neutrophils, monocytes, eosinophils, and lymphocytes counted among control and treated after 31 days of administration. Error bars denote mean ± SEM (ns: not significant, *p < 0.05).

FIGURE 3

Evaluation of the effect of ES on fertility in mice. (A) Schematic is showing mating groups following exposure to endosulfan (5 mg/kg body weight); Group 1 (male treated), Group 2 (female treated), Group 3 (male and female treated), and Group 4 (untreated control). (B) Bar graphs show the difference in fertility levels when ES was given only to males (n = 5), only to females (n = 10), or to both males and females (n = 15). Mating was in the ratio of 1:2 of male to female. Experiments were repeated 3 times. Error bars denote mean ± SEM.

FIGURE 4

Histopathological examination of reproductive organs from ES-exposed mice. (A) Histopathology of testis of mice following ES administration. Control indicates testis tissue from mice with no treatment, and ES represents tissue from ES-treated mice (5 mg/kg, 10 doses; n = 2; Magnification: ×5, ×10, and ×20). To evaluate long-lasting effects of exposure to ES, tissue samples were collected after 8 months following exposure. Nuclear components, including heterochromatin and nucleoli were stained by haematoxylin as deep blue or purple while eosin-stained cytoplasmic components like collagen, elastic fibers, muscle fibres and red blood cells. The blue arrows indicate the spermatogonia cells in basal epithelial layer, black arrow in ES indicates the interstitial space between the seminiferous tubules. (B) Histopathology of the ovary of mice following ES administration (5 mg/kg, 10 doses) after 8 months of exposure. The experiments were repeated two independent time for each tissue. Control indicates ovary tissue from mice with no ES treatment and ES represents tissue from ES exposed mice (Magnification: ×5, ×10, and ×20; Scale bar: 100 µm).

FIGURE 5

Evaluation of ES induced DNA breaks in mice reproductive organs. To evaluate the long-term effects of ES, mice (n = 2) were exposed to ES (5 mg/kg, 10 doses), and tissue samples were collected after 8 months and examined for 53BP1 foci, a hallmark of DSBs. (A) Immunofluorescence for 53BP1 expression in testis of ES treated mice. Control indicates sections of testes from untreated control mice, while treated refers to sections from testes of ES-exposed mice. (B) Bar graph indicates the average number of 53BP1 positive cells in control and treated mice seminiferous tubules. (C) Immunofluorescence of 53BP1 expression in ovary of control and ES treated mice. Control indicates ovary sections from untreated mice, whereas ES is sections from treated mice. The experiments were repeated two independent time. (D) Bar graph indicates number of 53BP1 positive cells in control and treated mice nucleus calculated as Mander’s coefficient on ImageJ (ns: not significant, **p < 0.005). In panel (B) at least 25 seminiferous tubules and in panel (D) at least 20 fields each were analyzed.

FIGURE 6

Evaluation of ES induced DNA repair through Ligase III in reproductive organs. (A) Immunofluorescence of DNA Ligase III expression in testes of ES-treated mice (5 mg/kg, 10 doses, n = 2). Tissue samples were collected after 8 months of exposure to ES. Control indicates sections of testes from untreated mice, whereas, ES is sections from treated mice. The nucleus is counterstained with DAPI. The merged image shows localisation of DNA Ligase III positive cells. (B) Bar graph shows localization of DNA Ligase III positive cells in control and treated mice testis calculated as Mander’s coefficient on ImageJ. (C) Evaluation of ES induced DNA Ligase III in mice ovary. Immunofluorescence staining of DNA Ligase III expression in ovary of control and ES treated mice. (D) Bar graph indicates localization of DNA Ligase III positive cells in control and treated mice ovary calculated as Mander’s coefficient on ImageJ. In panels (B,D) at least 20 fields each were analyzed from two independent batches (ns: not significant, *p < 0.05, **p < 0.005, ***p < 0.0001).

FIGURE 7

Histopathological examination of vital organs of mice post 8 months of ES treatment. (A) Histopathology of liver and lung of mice following ES administration (5 mg/kg, 10 doses). Tissue samples were collected after 8 months of completion of the dose. Control indicates tissue from untreated mice and ES represents tissue from ES treated mice. Hepatocytes and central vein are marked using blue arrows in case of liver. Changes in the architecture of alveoli in lungs is also indicated using arrows. The magnification shown is ×20. (B) Histopathology of spleen, kidney, intestine, and cerebellum of mice following ES administration (5 mg/kg, 10 doses). Control indicates tissue sections from untreated mice and ES represents tissue from ES-treated mice. The experiments were repeated two independent time for each tissue. Magnification shown is of ×20. Scale bar: 100 µm.

FIGURE 8

Histopathological examination of abnormal growth in lungs of mice post treatment with ES. (A) Abnormal growth was observed in the lungs following 16 months of ES exposure (5 mg/kg, 10 doses) in male mice. The white arrow indicates abnormal growth. (B) Histopathological analysis of abnormal growth in lungs from ES exposed male mice. The red arrow indicates the infiltrating cells (×5, ×10, and ×20). (C) Immunohistochemical analysis of abnormal growth in lungs of ES exposed mice. Immunohistochemical analysis of control mice lung with Ki67 (top panel) and treated mice lung with Ki67 and p53 (bottom panel) (×20). In panels (B,C) experiment was repeated three independent times from the tissues of same animal. Control indicates lung tissue from untreated mice and treated indicates ES-treated tissue from male mice. Scale bar: 100 μm.

FIGURE 9

Histopathological examination of abnormal growth in ES-exposed mice post treatment. (A) Abnormal growth in neck region observed after 3 months of ES exposure indicated by black arrow in ES-exposed female mice (5 mg/kg, 10 doses). (B) Histology of abnormal tissue from the neck region of the ES-exposed female mice (×20). Experiment was repeated three independent times from the tissues of same animal. Scale bar: 100 μm.