Priyanka Saraf

Malathion-induced granulosa cell apoptosis in caprine antral follicles: an ultrastructural and flow cytometric analysis.

Organophosphate pesticides (OPs) like malathion interfere with normal ovarian function resulting in an increased incidence of atresia and granulosa cell apoptosis that plays a consequential role in the loss of ovarian follicles or follicular atresia. The aim of present study was to assess malathion-induced (100 nM) reproductive stress, ultrastructural damage and changes in apoptosis frequency in ovarian granulosa cells of antral follicles. Transmission electron microscopy (TEM) was employed for ultrastructural characterization, oxidative stress was evaluated using thiobarbituric acid reactive substances (TBARS) assay to measure lipid peroxidation, and apoptosis was quantified via flow cytometry. By TEM, apoptosis was identified by the presence of an indented nuclear membrane with blebbing, pyknotic crescent-shaped fragmented nuclei, increased vacuolization, degenerating mitochondria, and lipid droplets. The results indicate a significant increase in malondialdehyde (MDA) level (nmols/g wet tissue) at a 100 nM dose of malathion i.e. 7.57±0.033*, 8.53±0.12*, and 12.87±0.78** at 4, 6, or 8 h, respectively, as compared with controls (6.07±0.033, p<0.01*, p<0.05**) showing a positive correlation between malathion-induced lipid peroxidation and percentage of granulosa cell apoptosis (r=1; p<0.01). The parallel use of these three methods enabled us to determine the role of malathion in inducing apoptosis as a consequence of cytogenetic damage and oxidative stress generated in granulosa cells of antral follicles.

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.

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.

Synthesis and biological evaluation of some functionalized 1H-1,2,3-triazole tethered pyrazolo[3,4-b]pyridin-6(7H)-ones as antimicrobial and apoptosis inducing agents

A series of novel molecular hybrids containing pyrazole, pyridinone and 1,2,3-triazoles have been synthesized by one-pot four-component reaction of Meldrum’s acid, substituted aryl azides, 4-(prop-2-yn-1-yloxy)aryl aldehyde and 3-methyl-1-phenyl-1H-pyrazol-5-amine using L-proline as a basic organocatalyst besides CuSO4.5H2O and sodium ascorbate as catalysts for click chemistry in PEG-400 as a highly efficient and green media. Apoptosis studies have been carried out on ovarian follicles of goat (Capra hircus) and in vitro antibacterial activity has been done against six strains namely Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, Bacillus cereus, Escherichia coli and Pseudomonas aeruginosa and antifungal activity against two yeast strains namely, Candida albicans and Saccharomyces cerevisiae.

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

ABSTRACT 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.

Influence of Toxic Chemicals on Female Reproduction: A Review

The female reproductive system is very complex and co-ordinated system that works well in the presence of hormonal and other signaling factors. Female reproduction is necessary for growth and maintenance of population. There has been a gradual increase in production and consumption of pesticides in agriculture to meet the population rising demands.

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).

Pesticides induced oxidative stress and female infertility: a review

Abstract Oxidative stress impairs the female reproductive physiology by altering the affectivity of antioxidant defense system in the individual. Several toxicants mainly pesticides alters this defense system producing various lifestyle-related diseases. The imbalance in antioxidant defense system, due to pesticidal toxicity-induced oxidative stress, is found to be associated with reproductive disorders like estrous cycle defects, impaired folliculogenesis, follicular atresia, implantation defects, spontaneous abortions, endometriosis, fetal, and other birth defects, thereby, directly affecting fertility and reproductive physiology of the organism. So, the present review emphasizes upon pesticides-mediated oxidative stress and its influence on female fertility.

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.

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.