Understanding the Long-term Impact and Mechanism of Action of Endosulfan, an Organochlorine Pesticide on Fertility, Development and Growth of Mice

Abstract

Endosulfan (ES) is one of the major broad-spectrum organochlorine pesticides categorized as Class II (moderately hazardous) by WHO and Class I (highly acutely toxic) by US Environment Protection Agency. Although the use and manufacture of Endosulfan is banned or phased out in Europe and the US since the Stockholm Convention in 2011, it is still used in developing countries, including most parts of India. Endosulfan is persistent in nature due to which it bioaccumulates in food chain and its residues and metabolites are detected in water, food, milk, tissues and human blood samples. Various epidemiological and animal studies have reported Endosulfan and its metabolites as neurotoxic, genotoxic, and harmful to reproductive organs. Previous studies from our laboratory have shown that exposure to Endosulfan can cause infertility in male mice due to testicular atrophy and reduced sperm count. Endosulfan treatment induced DNA damage, altered DNA damage response, and promoted the error-prone DNA repair pathway called microhomologymediated end joining (MMEJ) (Sebastian and Raghavan, 2016). Various studies from the literature showed that the serum concentration of Endosulfan in the affected human population is up to 547.6 μg/L and it was up to 700 μg/L when tested after 2 h of exposure, which is much higher than that was used in our previous study (20-25 μg/L) Therefore, to study the effect of Endosulfan in environmentally relevant concentration, we have treated mice with 5 mg/Kg body weight for 10 doses every alternate day. Bioavailability analysis in mice serum revealed a concentration of ~50 μg/L. Results revealed that Endosulfan affects the normal physiology of mice. Haematological analysis, liver function test, and kidney function test showed a decrease in the total bilirubin, creatinine, and blood urea nitrogen (BUN) in the treated mice. Besides an increase in neutrophils, monocytes, and total leukocyte count and decreased lymphocyte percentage were observed in treated mice. Furthermore, our results showed that Endosulfan treatment resulted in infertility in majority of female and male mice. While treatment with Endosulfan resulted in infertility in 55% of male mice, it was 62% in females. Besides compromised fertility, exposure to Endosulfan in mice resulted in abnormal growth and development. The progenies of the Endosulfan-treated mice were born with multiple developmental defects like delayed fur development, delayed eye-opening, abnormal walking patterns, difficulty in parturition, etc. A significant reduction in body weight of new-born from treated mice was also observed, which was more prominent when both parents were exposed to Endosulfan. The comparison of organ weights between the treated and control mice at postnatal day 21 showed a significant reduction in size and weight of spleen and thymus. In contrast, a significant increase in size and weight were observed in case of kidney, lungs, and heart. A significant decrease in CD19+ B cells and CD3+ T cells in bone marrow and thymus respectively indicates the Endosulfan-induced effects on immune system of mice. Histopathological examination of various tissues from Endosulfan exposed mice showed a severe effect on testis, ovary, liver, and lungs which include vacuolar degeneration of the basal germinal epithelial cells, degradation of the interstitial matrix in testes, reduction in number of mature Graafian follicles, mild vacuolation in hepatocytes all over the liver, etc. Histopathological analysis of testis from postnatal day 12 mice showed a reduction in the size and number of seminiferous tubules. Comprehensive histopathological analysis of vital organs of post-natal day 12 mice showed that the progenies from treated mice had severe effects on different organs such as brain, cerebellum, liver, kidney, heart, ovary, testis, lung, spleen when compared with age matched control organs. Further, detailed analyses through TUNEL assay revealed long-term testicular cell death, indicative of persistent damage. Evaluation of DNA breaks using 53BP1 staining in mice testis from postnatal day 12 showed several 53BP1 positive cells in seminiferous tubules, unlike control animals. Consistent to the observed increase in DNA breaks in cells from reproductive tissues, an increase in expression of DNA Ligase III was also observed, which was consistent with previously reported elevated levels of MMEJ mediated repair. Furthermore, transcriptome analysis of tissues from treated, control and progeny mice revealed deregulation of several genes associated with gametogenesis and DNA repair pathways both in the testis and ovary. We have also analysed the genes associated with learning and memory, apoptosis, eye development, particularly from mice brain and validated by behaviour studies in progeny mice. Contextual fear conditioning test of progenies from treated mice revealed loss of both newly acquired memory and remote memory particularly when both the parents were treated. Importantly, tumor development near lung and ovary was observed post Endosulfan treatment (12 months). Further abnormal regression of ovary, elevated skin infection etc. were also observed in few animals over the months. The mice with developmental defects survived only for short period while animals with normal phenotype survived just like control animals. Similar observations like developmental delay, mental retardation, increased incidence of cancer and infertility were reported on the people living in Endosulfan exposed regions. Thus, by using the mice model system, we could recapitulate several developmental defects which were observed in people living in Endosulfan exposed areas. The persistence of Endosulfan in water is a controversial area of research. The stability of Endosulfan in water depends on various parameters like pH and micro4 organisms. Although the half-life of Endosulfan in water is considered less, various studies from different parts of the world have reported the detection of Endosulfan in water resources like rivers, oceans, and underground water. Using HPLC analysis, we find that Endosulfan remains stable for >100 days in water collected from various sources like local pond water, rainwater, underground water collected in a container. In conclusion, our study is relevant to the present Indian and Asian scenario where pesticide usage is not tightly regulated even though the side effects possess significant risk. In the present study, several characteristic features of people living in Endosulfan exposed areas have been reported using mice model.