Cadmium as an ovarian toxicant: A review.

Cadmium (Cd) is a ubiquitous heavy metal toxicant with no biological function in the human body. Considerably, because of its long biological half-life and very low excretion rate, Cd is inclined to accumulate and cause deleterious effects on various body organs (e.g., liver, kidney, and ovary) in humans and animals. Ovaries are the most vulnerable targets of Cd toxicity. Cd has been shown to induce oxidative stress, follicular atresia, hormonal imbalance, and impairment of oocyte growth and development. Moreover, Cd toxicity has been associated with increasing incidences of menstrual disorders, pregnancy loss, preterm births, delayed puberty, and female infertility. Therefore, it is crucial to understand how Cd poisoning impacts specific ovarian processes for the development of preventive interventions to enhance female fertility. The current review attempts to collate the recent findings on Cd-induced oxidative stress, follicular apoptosis, steroid synthesis inhibition, and teratogenic toxicity, along with their possible mechanisms in the ovarian tissue of different animal species. Additionally, the review also summarizes the studies related to the use of many antioxidants, medicinal herbs, and other compounds as remedial approaches for managing Cd-induced ovarian toxicity.

Quercetin Supplementation Alleviates Cadmium Induced Genotoxicity-Mediated Apoptosis in Caprine Testicular Cells.

Being a common environmental pollutant, cadmium causes detrimental health effects, including testicular injury. Herein, we document the ameliorative potential of quercetin, a potent antioxidant, against cadmium-induced geno-cytotoxicity and steroidogenic toxicity in goat testicular tissue. Cadmium induced different comet types (Type 0 - Type 4), indicating the varying degree of DNA-damage in testicular cells. The quantitative analysis at 50 and 100 µM cadmium concentration revealed the DNA damage with per cent tail DNA as 75.78 ± 1.49 and 94.65 ± 0.95, respectively, in comparison to the control group (8.87 ± 0.48) post 8 h exposure duration. Cadmium caused a substantial decrease in the activity of key steroidogenic enzymes' (3ß-HSD and 17ß-HSD) along with reduction of testosterone level in testicular tissue. Furthermore, cadmium treatment induced various types of deformities in sperm, altered the Bax/Bcl-2 expression ratio in testicular tissue and thus suggesting the apoptosis-mediated death of testicular cells. Simultaneous quercetin supplementation, however, significantly (p < 0.05) averted the aforementioned cadmium-mediated damage in testicular tissue. Conclusively, the cadmium-induced DNA-damage and decrease in steroidogenic potential results in death of testicular cells via apoptosis, which was significantly counteracted by quercetin co-supplementation, and thus preventing the cadmium-mediated cytotoxicity of testicular cells.

Tail approach synthesis of triazolylthiazolotriazole bearing benzenesulfonamides as carbonic anhydrase inhibitors capable of inducing apoptosis.

Inhibition of human carbonic anhydrase (hCA) isoform IX with concurrent induction of apoptosis is a promising approach for targeting cancer in humans. Prompted by the scope, novel benzenesulfonamides containing the 1,2,3-triazolylthiazolotriazole tail were synthesized and screened as inhibitors of hCA isoforms I, II, IV, and IX. The tumor-associated isoform hCA IX was strongly inhibited by the sulfonamides reported here with KI values ranging from 45 nM to 1.882 µM. Overall, nine compounds showed hCA IX inhibition with KI < 250 nM. The glaucoma-associated isoform hCA II was moderately inhibited while the cytosolic isoform hCA I and membrane-bound isoform hCA IV were weakly inhibited by the synthesized sulfonamides. Compound 6Ac (KI = 3.6 nM) was found to be an almost three times more potent inhibitor of hCA II as compared to the standard drug acetazolamide (KI = 12.1 nM). The selective hCA IX inhibitors were further studied for their apoptotic efficacy in goat ovarian cells and showed better results as compared to the control. A comparative study of previously synthesized compounds and molecular docking study of representative compounds revealed some important generalizations that could prove beneficial in further investigations of isoform-selective hCA inhibitors.

Ameliorative potential of vitamin C and E against Roundup-glyphosate induced genotoxicity triggering apoptosis in caprine granulosa cells.

The present study was aimed to investigate the genotoxic and apoptotic effects of glyphosate (GLP) in Roundup formulation along with mitigation of two potent antioxidants that is, vitamin C and E in caprine granulosa cells in vitro. The entire work was done in a dose and time dependent manner where different concentrations of GLP (0.1, 2.0, and 4.0 mg/ml) in Roundup and antioxidants (0.5 and 1.0 mM) were employed to culture of granulosa cells for exposure durations of 24, 48, and 72 h. Analysis of GLP-induced geno-toxicity was accomplished by using single cell gel electrophoresis (comet assay) assay. Results have shown increased incidences of DNA fragmentation, evidenced by presence of different types of comets (Type 1-Type 4) in Roundup-GLP- exposed groups in contrast to the control group (Type 0 comet). However, mitigation by both vitamin C and E was significant (p < .05) in combating the GLP-induced genotoxicity in granulosa cells in a concentration- and time-dependent manner. The results of our study provide a clear indication of the ameliorative actions of vitamin C and E against Roundup-GLP-induced genotoxicity that instigate apoptosis in ovarian granulosa cells of caprine.

Selective inhibition of carbonic anhydrase IX by sulphonylated 1,2,3-triazole incorporated benzenesulphonamides capable of inducing apoptosis.

In search of selective carbonic anhydrase (CA) IX inhibitors endowed with apoptotic inducing properties, we designed and synthesised two subsets of 4- and 3-(5-aryl-(4-phenylsulphonyl)-1H-1,2,3-triazol-1-yl)benzenesulphonamides. All compounds were assayed for human carbonic anhydrase (hCA) isoforms I, II, IV, and IX inhibition. Isoforms hCA I and hCA IV were weakly inhibited by most of the synthesised compounds. Many four-substituted benzenesulphonamides displayed low nanomolar inhibition against isoform hCA II, unlike the three-substituted analogues. All target compounds exhibited good inhibition profile with KI values ranging from 16.4 to 66.0 nM against tumour-associated isoform hCA IX. Some selective and potent inhibitors of hCA IX were assayed for in vitro apoptotic induction in goat testicular cells. Compounds 10d and 10h showed interesting apoptotic induction potential. The present study may provide insights into a strategy for the design of novel anticancer agents based on hCA inhibitors endowed with apoptotic interference.

Ultrastructural analysis of cadmium-induced toxicity and its alleviation by antioxidant quercetin in caprine testicular germ cells in vitro.

Increasing evidence has demonstrated that cadmium (Cd), a common environmental toxicant, has been associated with testicular toxicity. Quercetin, an efficient flavonoid, has been shown to exert cytoprotective effect in numerous pathological processes. The current study has employed ultrastructural analysis to examine the Cd-induced toxicity in goat testicular tissue along with the ameliorative action of quercetin in a dose- and time-dependent manner in-vitro. Results of transmission electron microscopy (TEM) revealed that at lower selected concentrations (10 and 50 µM), Cd induced apoptosis-mediated cytotoxicity in testicular tissue as supported by presence of various morphological attributes of apoptosis in testicular germ cells such as condensed and marginated chromatin followed by breakdown of chromatin material, swollen mitochondria, and vacuolization. At 100 µM concentration, along with apoptosis, Cd-induced cytotoxicity in testicular tissue was associated with induction of necrosis also. However, the simultaneous supplementation of antioxidant quercetin has markedly abrogated the testicular cytotoxicity as shown by restoration of Cd-evoked aberrant ultrastructure of testicular germ cells in a dose- and time-dependent manner, providing a basis for future studies to involve quercetin in management of Cd-induced reproductive toxicity in males.

Recent advances in microbial toxin-related strategies to combat cancer.

It is a major concern to treat cancer successfully, due to the distinctive pathophysiology of cancer cells and the gradual manifestation of resistance. Specific action, adverse effects and development of resistance has prompted the urgent requirement of exploring alternative anti-tumour treatment therapies. The naturally derived microbial toxins as a therapy against cancer cells are a promisingly new dimension. Various important microbial toxins such as Diphtheria toxin, Vibrio cholera toxin, Aflatoxin, Patulin, Cryptophycin-55, Chlorella are derived from several bacterial, fungal and algal species. These agents act on different biotargets such as inhibition of protein synthesis, reduction in cell growth, regulation of cell cycle and many cellular processes. Bacterial toxins produce actions primarily by targeting protein moieties and some immunomodulation and few acts through DNA. Fungal toxins appear to have more DNA damaging activity and affect the cell cycle. Algal toxins produce alteration in mitochondrial phosphorylation. In conclusion, microbial toxins and their metabolites appear to have a great potential to provide a promising option for the treatment and management to combat cancer.

Transmission electron microscopic analysis of glyphosate induced cytotoxicity and its attenuation by N-acetyl-L-cysteine in caprine testicular germ cells in vitro.

The agricultural pesticide poisoning is currently the most thrust area of human health concern. Pesticide-induced cytotoxicity and the corresponding reproductive toxicity in today's scenario is not a concealed reality that has to be considered for the continuation of respective race. Here, the transmission electron microscopy (TEM) technique was employed to investigate the adverse impact of glyphosate (GLY) and its mitigation by N-acetyl-L-cysteine (NAC) in goat testicular germ cells under in vitro conditions. The ultrastructural observations of testicular tissue from GLY-treated groups at different concentrations (0.1 and 4 mg/ml) and exposure durations (8 and 12 h) revealed that this organophosphate herbicide induced different apoptotic characteristics in testicular germ cells in a time- and dose-dependent manner. However, NAC (10 mM), being a potent antioxidant, was found to mitigate GLY-induced cytotoxicity in testicular cells as evidenced by fewer apoptotic characteristics in GLY plus NAC-treated groups, suggesting its beneficial potential in alleviating the GLY-induced gonadotoxicity in males.Abbreviations: GLY (Glyphosate), NAC (N-acetyl-L-cysteine), TEM (Transmission electron microscopic), GE (genetic engineered), Organophosphate (OPs).

Quercetin mediated attenuation of cadmium-induced oxidative toxicity and apoptosis of spermatogenic cells in caprine testes in vitro.

Cadmium (Cd), an environmental toxic heavy metal, has been reported to cause testicular toxicity, which contributes to the recent decline in male fertility worldwide. Quercetin (Qcn), a major dietary antioxidant, has been shown to have protective effects under various pathological conditions. However, whether Qcn provides protection against Cd-stimulated testicular toxicity remains obscured. The present study was therefore aimed at investigating the ameliorative effect of Qcn supplementation on Cd-induced toxicity in the goat testis in vitro in a dose-(10, 50, and 100 µM) and time-dependent (4 and 8 h) manner. Different cytotoxicity, genotoxicity, and biochemical analyses have been carried out using appropriate methods. Cytotoxicity in testicular cells induced by Cd treatment was apparently mitigated by Qcn treatment, evidenced by decreased apoptotic attributes or frequency in Qcn plus Cd-treated groups compared to the only Cd-treated groups. Qcn treatment provides substantial protection to the Cd-triggered aggression in oxidative (increased MDA levels) and total antioxidant capacity (reduced FRAP activity) in testicular tissue, indicating the anti-oxidative function of Qcn against Cd exposure. Moreover, Cd-induced decline in antioxidant status (CAT, SOD, and GST activity) was markedly restored by Qcn supplementation in testicular tissue. In conclusion, this study shows that Qcn treatment significantly attenuated the Cd-evoked testicular damage, suggesting its beneficial potential in preventing or at least in managing the gonadotoxicity in males induced by steadily increasing Cd contamination in the environment.

Cadmium as a testicular toxicant: A Review.

Cadmium (Cd) is a toxic heavy metal with no known biological functions in the human body. Due to a considerably long biological half-life and very low rate of excretion, accumulation of Cd in different body organs (eg, liver, kidney, and testes) over time is associated with perturbed functioning of these organs. Recent studies have shown the extreme sensitivity of the testes to Cd toxicity. In testes, Cd has been reported to induce oxidative stress, apoptosis of spermatogenic cells, reduction in androgen production and sperm functions. Moreover, Cd in combination with other environmental toxicants may be responsible for the declining fertility of males in both animals and humans. Pinpointing how Cd toxicity affects various testicular processes will be imperative for the development of preventative measures to promote fertility among males. Therefore, in the present review, we summarize the recent findings related to the Cd-induced oxidative toxicity, apoptotic toxicity, steroidogenic toxicity, and spermatotoxicity, along with their possible mechanisms in testicular tissue of different animal species. In addition, the utilization of various antioxidant compounds, medicinal plants and other compounds for the management of Cd toxicity in testes is discussed.

Ameliorating Effects of Natural Antioxidant Compounds on Female Infertility: a Review.

The prevalence of female infertility cases has been increasing at a frightening rate, affecting approximately 48 million women across the world. However, oxidative stress has been recognized as one of the main mediators of female infertility by causing various reproductive pathologies in females such as endometriosis, PCOS, preeclampsia, spontaneous abortion, and unexplained infertility. Nowadays, concerned women prefer dietary supplements with antioxidant properties over synthetic drugs as a natural way to lessen the oxidative stress and enhance their fertility. Therefore, the current review is an attempt to explore the efficacy of various natural antioxidant compounds including vitamins, carotenoids, and plant polyphenols and also of some medicinal plants in improving the fertility status of females. Our summarization of recent findings in the current article would pave the way toward the development of new possible antioxidant therapy to treat infertility in females. Natural antioxidant compounds found in fruits, vegetables, and other dietary sources, alone or in combination with other antioxidants, were found to be effective in ameliorating the oxidative stress-mediated infertility problems in both natural and assisted reproductive settings. Numerous medicinal plants showed promising results in averting the various reproductive disorders associated with female infertility, suggesting a plant-based herbal medicine to treat infertility. Although optimum levels of natural antioxidants have shown favorable results, however, their excessive intake may have adverse health impacts. Therefore, larger well-designed, dose-response studies in humans are further warranted to incorporate natural antioxidant compounds into the clinical management of female infertility.

N-acetyl-l-cysteine mediated regulation of DNA fragmentation, an apoptotic event, against methoxychlor toxicity in the granulosa cells of ovarian antral follicles.

Methoxychlor (MXC), an organo-chlorine insecticide, is a reproductive toxicant in females, causing apoptosis-mediated follicular atresia. To elucidate the potentials of Methoxychlor as a geno-toxicant, granulosa cells of healthy antral follicles, exposed to MXC and antioxidant, N-acetyl-l-cysteine, were studied by the terminal deoxynucleotidyltransferase-dUTP nick end-labelling and single-cell gel electrophoresis (comet) assays. MXC caused DNA fragmentation, as revealed by the increased incidence of dark brown condensed TUNEL positive cells in contrast with lightly brown TUNEL negative cells with maximum TUNEL positive cells were observed in 100 µg/mL MXC treated groups. Quantitatively, maximum geno-toxicity was exhibited at highest MXC treatment with percent tail DNA as 17.87 ± 0.85, 41.16 ± 3.94, and 47.73 ± 3.71 in comparison with control (0.65 ± 0.03, 2.91 ± 0.27, and 7.16 ± 1.39) after 24, 48 and 72 h exposure duration, respectively. MXC treated groups exhibited Type 1-Type 3 comets as compared to Type 0 comets in control groups. Supplementation of NAC led to significant (p < 0.05) decline in geno-toxicity in MXC treated groups with maximum amelioration observed at 5 and 10 mM. Consequently, increased DNA damage attributed to the granulosa cells apoptosis in response to Methoxychlor exposure was significantly combated by NAC supplementation, preventing the geno-toxicity induced cyto-toxicity in GCs.

Aggregation, adhesion and efficacy studies of probiotic candidate Pediococcus acidilactici NCDC 252: a strain of dairy origin.

Aggregation and adhesion capability and survival efficacy of candidate probiotic strain Pediococcus acidilactici NCDC 252 under simulated gastric, intestinal and vaginal conditions was studied. The strain exhibited strong autoaggregation phenotype and coaggregation with other Lactic acid bacteria (LAB) and E. coli. The adhesion studies of NCDC 252 to pig's intestinal epithelial cells showed its adhesive ability. Aggregation and adhesiveness were related through cell surface proteins as removal/extraction of surface proteins resulted in altered aggregation and no adhesiveness. Cell surface proteins were analysed by SDS-PAGE and also in silico analysed from its genome. SDS-PAGE analysis of cell surface proteins of NCDC 252 revealed two potential proteins of approximately 74.3 and 53.6 kDa to be involved in host-probiotic interaction. Removal of cell surface proteins by LiCl-treatment (5 mol l-1) resulted in loss of aggregation and adhesiveness. Further survival of NCDC 252 under simulated gastrointestinal and vaginal conditions in terms of high viable counts confirmed its efficacy for its survival under gut and urogenital conditions. These observations suggest that it can be used further in functional foods, nutraceuticals and in combating urogenital infections. As NCDC 252 was able to survive in intestinal conditions, interaction of its cell surface proteins with intestinal mucins was studied in silico by docking. Highest affinity of adhesion was observed for MUC3B. In conclucion, NCDC 252, exhibited aggregation phenotype and adhesion capability. Survivability of NCDC 252 under simulated conditions and its interaction with human mucins confirms its efficacy to be used as probiotic.

Effective attenuation of glyphosate-induced oxidative stress and granulosa cell apoptosis by vitamins C and E in caprines.

Pesticides are known to cause a wide range of reproductive problems that possess degenerative effects on mammalian fertility. Glyphosate (GLP), a broad-spectrum organophosphate herbicide, is known to be a potent mammalian toxicant. The present study aims at assessing the GLP-induced (0.1, 2.0, and 4.0 mg/ml) granulosa cells toxicity and evaluating the mitigating effects of vitamins C and E (0.5 mM and 1.0 mM) in healthy caprine antral follicles, cultured in vitro in a dose- and time-dependent manner (24, 48, and 72 hr) and subjected to various cytotoxic and geno-toxic analysis, namely, classic histology, EB/AO differential staining, oxidative stress parameters, and antioxidant enzymatic activity. The histomorphological analysis and EB/AO staining elucidated increase in the incidence of apoptotic attributes within granulosa cells with increasing dose and duration of the GLP treatment. The highest apoptotic frequency was observed at 4.0 mg/ml GLP after 72-hr exposure duration in comparison with the control. GLP exposure also led to a significant decline in the antioxidant enzymes' activity, namely, SOD, catalase, and GST along with enhanced lipid peroxidation and reduced FRAP activity in a dose- and time-dependent manner. Vitamins C and E supplementation decreased oxidative stress-mediated granulosa cells apoptosis, suggesting its efficiency to diminish GLP-mediated GCs cytotoxicity and thereby, preventing associated fertility disorders.

Antiapoptotic effects of vitamins C and E against cypermethrin-induced oxidative stress and spermatogonial germ cell apoptosis.

Toxicological studies have demonstrated the relation between use of agrochemicals and fertility issues within males. Thus, the present study aimed to elucidate the propensity of cypermethrin (CYP) in bringing testicular germ cell apoptosis and effective attenuation by vitamins C and E in caprines. Reproductive toxicity of CYP was evaluated using histomorphological, cytological, and biochemical changes in the testicular germ cells in dose-dependent (1, 5, 10 µg/mL) and time-dependent (4, 6, 8 h) manner. Histological and ethidium bromide/acridine orange fluorescence staining exhibited that vitamins C and E (0.5 and 1.0 mM) successfully diminished the CYP-induced testicular germ cells apoptosis. CYP exposure along with vitamins C and E supplementation also resulted in significantly increased ferric reducing antioxidant power activity along with the antioxidant enzymes, namely catalase, superoxide dismutase, and glutathione-s-transferase, and decreased lipid peroxidation in testicular germ cells. Thus, vitamins C and E ameliorated CYP-induced testicular germ cell apoptosis, thereby preventing spermatogonial cells degeneration and male infertility.

N-Acetyl-cysteine mediated inhibition of spermatogonial cells apoptosis against malathion exposure in testicular tissue.

Toxicological studies so far suggest that excessive use of malathion, an organophosphate insecticide, causes serious ill-effects in mammalian reproductive physiology. The present study aims at assessing malathion-induced toxicity in a dose- and time-dependent manner with mitigating effects of N-acetyl-l-cysteine. The testicular germ cell viability was monitored using MTT assay, where NAC, being an antioxidant significantly reduced malathion-induced toxicity by enhancing the frequency of cell viability. The histomorphological analysis showed that NAC successfully diminished several apoptotic features in testicular cells, induced by malathion. The differential EB/AO staining revealed a significant decline in the percentage of apoptosis after NAC supplementation. NAC also diminished the malathion-induced DNA fragmentation along with significantly reduction in oxidative stress parameters causing decrease in lipid peroxidation and enhancement of ferric reducing antioxidant power within testicular germ cells. Thus, NAC mitigated the malathion-induced toxicity, proving its potential in infertility treatment.

Multicomponent Synthesis of Some Molecular Hybrid Containing Thiazole Pyrazole as Apoptosis Inducer.

The present study describes a multicomponent synthesis of molecular hybrid containing pyrazole, thiazole moiety using hydrazone as a linker, which have been synthesized by condensation of 1-phenyl-3-(aryl)-1H-pyrazole-4-carbaldehydes 1A-B: , thiosemicarbazide and α-bromoketones 2A-C: .The target hybrid compounds, 1-((1-phenyl-3-aryl-1H-pyrazole-4-yl)methylene)-2-(4-arylthiazole-2-yl)hydrazine 3A-F: are characterized by 1H-NMR, 13C NMR, FT-IR and mass. Apoptosis inducing ability and cytotoxic nature of all the hybrid compounds having thiazole, pyrazole and hydrazone were assessed by using biological assays viz morphological, fluorescence and tunel assays on granulosa cells of ovarian antral follicles of goat (Capra hircus) in vitro. Apoptosis was recognized and quantified using differential staining of ethidium bromide and acridine orange where apoptotic cells exhibited red fluorescence and live normal cells with intact cell membrane and normal nucleus displayed bright green fluorescence. Among the tested compounds, compound 3E: and 3B: showed the maximum potency to induce apoptosis with percentage of apoptosis 25.61±2.95and 23.45±1.46 respectively followed by 3F: (20.95±0.40) and 3D: (20.44±1.60) in comparison with control (5.14±0.44).

N-acetyl cysteine-mediated effective attenuation of methoxychlor-induced granulosa cell apoptosis by counteracting reactive oxygen species generation in caprine ovary.

Methoxychlor (MXC), an organochloride insecticide, is a potent toxicant-targeting female reproductive system and known to cause follicular atresia by inducing apoptosis within granulosa cells. Oxidative stress plays a pivotal role in apoptosis; thus, this study focuses on the ameliorative action of N-acetyl cysteine (NAC) on MXC-induced oxidative stress and apoptosis within granulosa cell of caprine ovary. Classic histology, fluorescence assay, and biochemical parameters were employed to evaluate the effect of varied concentration of NAC (1, 5, and 10 mM) on granulosa cell apoptosis after 24, 48, and 72 h exposure duration. Histomorphological studies revealed that NAC diminished the incidence of apoptotic attributes like condensed or marginated chromatin, pyknosis, crescent-shaped nucleus, empty cell spaces, and degenerated cellular structure along with the presence of cytoplasmic processes within granulosa cells in dose- and time-dependent manner. NAC significantly downregulated the percentage of MXC-induced granulosa cell apoptosis within healthy ovarian follicle with its increasing dose, maximum at 10 mM concentration. It also significantly (p < 0.05) upregulated the activity of antioxidant enzymes, namely catalase, superoxide dismutase, and glutathione-s-transferase, along with ferric reducing antioxidant power further declining lipid peroxidation in the MXC-treated caprine ovary. The results revealed a negative correlation between apoptosis frequency and antioxidant enzymes' activity (rCAT = -0.67, rSOD = -0.56, rGST = -0.31; p < 0.05) while a positive correlation was observed with lipid peroxidation (r = 0.63; p < 0.05) after NAC supplementation. Thus, NAC supplementation reduces the MXC-generated oxidative stress that perhaps declines the ROS generating signal transduction pathway of apoptosis, thereby preventing MXC-induced granulosa cell apoptosis and follicular atresia. © 2015 Wiley Periodicals, Inc. Environ Toxicol 32: 156-166, 2017.

Transmission electron microscopic analysis of malathion-induced cytotoxicity in granulosa cells of caprine antral follicles.

Malathion, one of the most abundantly used organophosphate pesticides, has immoderate potency as a cytotoxic and genotoxic compound that induces toxicity in granulosa cells, resulting in its apoptosis. Thus, the present study aims to employ ultrastructural analysis for assessing the cytotoxicity of malathion at nanomolar concentrations (1 nM and 10 nM) in granulosa cells of caprine antral follicles at different exposure durations. Transmission electron microscopy revealed diminished cell-cell contact and cellular integrity, presence of crescent-shaped nucleus, chromatin condensation, and pyknosis with nuclear membrane folding, accumulation of lipid droplets with occurrence of cytoplasmic protrusions in granulosa cells treated with 1 nM malathion, whereas at 10 nM concentration, along with apoptotic attributes, prominent association of nucleus, endoplasmic reticulum, mitochondria and lipid droplets, nucleus invagination into lipid droplets, apical localization of lipid bodies, and occurrence of autophagic body were observed as compared to healthy granulosa cells in control with normal intact cellular integrity, well-developed cellular association, and doubled membrane nuclear lamina with homogenously dispersed chromatin surrounded by intact mitochondria with well-developed cristae. Thus, the results of ultrastructural analysis clearly suggest that nanomolar concentration of malathion induces apoptotic hallmarks within the granulosa cells of antral follicles that play a consequential role in increasing the incidence of follicular atresia, thereby affecting the overall fertility.

Granulosa cell apoptosis by impairing antioxidant defense system and cellular integrity in caprine antral follicles post malathion exposure.

Toxicological studies have demonstrated the exposure-risk relationship of several pesticides on reproduction of living organisms. To evaluate the role of malathion as a reproductive toxicant, this study aims at assessing the cytological and biochemical changes in the granulosa cells after malathion exposure in dose (1 nM, 10 nM, 100 nM) and time (4 h, 6 h, 8 h) dependent manner. Histomorphological analysis, fluorescence assay, apoptosis quantification, and terminal deoxynucleotidyl transferase d-UTP mediated nick end labeling (TUNEL) assay were done to determine cytological changes, whereas antioxidant enzyme assays were done to measure the oxidative stress in malathion treated ovarian antral follicles. Histological studies exhibited the occurrence of highly condensed or marginated chromatin with fragmented nucleus, pyknosis, loss of membrane integrity, increased empty spaces, and vacuolization in malathion treated granulosa cells. Ethidium bromide/acridine orange (EB/AO) fluorescence staining demonstrated a significant increase in incidence and percentage of apoptosis after malathion exposure (p < 0.001), both between and within the groups. Malathion exposure also resulted in increased DNA fragmentation and decline in both antioxidant enzymes activity namely catalase (CAT) and superoxide dismutase (SOD) in granulosa cells of antral follicles. Moreover, there was found a significant negative correlation between the apoptosis incidence and the level of antioxidant enzymes activity, SOD (r = -0.73 p < 0.01) and CAT (r = -0.80 p < 0.01), in malathion treated ovarian antral follicles. Thus, highlighting the role of DNA fragmentation and declining antioxidant level as a possible mechanism underlying malathion induced reproductive toxicity. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1944-1954, 2016.