POTENTIAL OF KISS-1 PEPTIDE IN MITIGATING SODIUM ARSENITE-INDUCED TESTICULAR TOXICITY IN ADULT MICE AND ITS COMPARISON WITH N-ACETYL CYSTEINE

Abstract

Contamination of soil and water reservoirs with arsenic has severely affected human health. Arsenic which is a naturally found metalloid has been enlisted among such contaminants that impose toxic effects on animal and human health upon exposure to above the safe levels (<10 ppb) recommended by World Health Organization. Besides other pathophysiological changes, trivalent form of arsenic is specifically found to cause andrological dysfunctions in several animal species. Arsenic-induced reproductive toxicity has been previously evaluated through spermatological, biochemical, hormonal, histological and molecular parameters. Redox imbalance leading to oxidative damage resulting in apoptosis is now considered the main underlying mechanism mediating the deleterious effects of arsenic. Considering the rapid increase in the prevalence of andrological disorders and exposure to various pollutants like arsenic, potential therapeutics have also been investigated. These include several naturally found compounds like vitamins and other plant based chemicals etc. N-acteyl cysteine (NAC) is one such antioxidant that has well established role as an antidote against several oxidizing agents including the trivalent form of arsenic as sodium arsenite (NaAsO2). Recent clinical research on finding the endogenous regulators of reproductive functions which could be used for treatment of several fertility disorders with minimum un desired effects have highlighted the kisspeptin which is a neuropeptide initially discovered as “metastatin”. Considering its therapeutic potential in regulating hormonal dysfunctions and recently reported anti-oxidative properties against oxidizing stressors, present study was designed to comprehensively evaluate and compare the mitigating role of Kiss-1 peptide (KP-10) along with NAC (well-known antioxidant) against NaAsO2-induced testicular toxicity at seminal, biochemical, hormonal, histological and genetic level through a series of in vivo and in vitro experiments on adult albino mice. In vivo study comprised of following experiments. An initial pilot study on adult male mice evaluated the efficacy of KP-10 (50 nmol) intraperitoneal dosing regimens (continuous and intermittent) upon exposure to low (4 ppm) and high (10 ppm) doses of NaAsO2 in drinking water for the whole length of mice spermatogenic cycle (35 xi days). Later, taking into account the findings of first experiment, similar experiment was conducted to compare the efficacies of both KP-10 (50 nmol) and NAC (for 5 alternative days) against arsenic toxicity in adult mice. However, only once a week intermittent dosing of KP-10 was selected for this experiment to avoid the extraneous stress caused by overhanding. Outcomes of these in vivo experimentations revealed that KP-10 has the efficacy comparable to NAC in mitigating the toxic effects of NaAsO2 on male reproductive system. Additionally, when provided in combination both therapeutics produce synergistic effect which is depicted by all investigated parameters. Such as, spermatological parameters including the sperm count, motility and viability were significantly improved upon supplementation both KP-10 and NAC along with NaAsO2 exposure at both 4 and 10 ppm doses, when compared to NaAsO2 alone treatment group. Whereas, testicular oxidative stress level was also decreased as depicted by significantly decreased ROS and TBARS levels, while elevating the activities of antioxidant enzymes including SOD, POD, CAT and non enzymetic reduced glutathione GSH. Moreover, testicular lactate dehydrogenase LDH levels were also found to be significantly decreased, when compared to NaAsO2 alone treatment group thus indicating prevention of tissue damage. Circulating testosterone levels were also increased to near control level (tap water provided) in supplemented groups as compared to NaAsO2 alone treatment groups, thus indicating the protection against arsenic-induced endocrine disruption. Histological evaluation also supported the protective effects of the respective therapeutics as germinal layers in seminiferous tubules showed normal arrangement and replenished sperm cells which were prominently lost upon NaAsO2 alone exposure at both 4 ppm and 10 ppm doses. Therefore, current substantial in vivo study provides evidence of spermatological, biochemical, hormonal and histoprotective effects of KP-10 against NaAsO2-induced testicular damage for the first time. It also suggests the potential of Kiss-1 peptide to be used in combination with other known therapeutics such as NAC for treatment of andrological disorders. To further verify these in vivo findings and evaluate the direct effect of KP-10 treatment, organ culture technique was employed in the current study. It involved 2 and xii 24 h incubation of testicular fragments with culture media containing respective doses of KP-10 (1 µM), NaAsO2 (100 µM) and NAC (1 µM) according to study design. Whereas, treatment free media served as control for these in vitro experiments. Evaluation of biochemical, hormonal and histological parameters also revealed the protective effects of KP-10 and NAC supplementation when compared to NaAsO2 alone treated cultures following both 2 and 24 h incubation. It was found that both therapeutics equally prevented the testicular damage by significantly decreasing the ROS, TBARS and LDH levels while increasing the activities of antioxidant enzymes including SOD, POD, CAT and non enzymetic GSH. Additionally, histoprotective effects of these therapeutics were demonstrated by significant recovery in germ cells within seminiferous tubules which were depleted in NaAsO2 alone incubated cultures. Intra-testicular testosterone levels also demonstrated the significant increase upon KP 10 and NAC supplementation in a time dependent manner which was reduced upon NaAsO2 alone incubation for both 2 and 24 h. These observations also suggest the direct effect of KP-10 on testicular steroidogenesis which is compromised due to arsenic toxicity. It is concluded therefore that KP-10 protects against oxidizing and endocrine disrupting effect of NaAsO2 by involving indirect and direct mediatory pathways. Considering the outcomes of in vivo and in vitro evaluation of biochemical, hormonal and histological parameters which suggest the anti-apoptotic and steroidogenic effect of KP-10, expression levels of mRNAs for genes involved in apoptosis and steroidogenesis were further evaluated by real time PCR. The target genes included Caspase 3, StAR and Cyp11a1. The results demonstrated the significantly increased expression of Caspase 3 upon NaAsO2 alone exposure in tissue samples from both in vivo and in vitro experiments in a dose and time dependent manner respectively, when compared to respective controls. Whereas, both KP-10 and NAC supplementation significantly decreased the mRNA expression of Caspase 3 as compared to NaAsO2 alone treatment. Moreover, expression of mRNAs for both StAR and Cyp11a1 were also found to be significantly elevated upon supplementation with KP-10 and NAC when compared to NaAsO2 alone treated groups thus indicating the steroidogenic effect of these therapeutics. Additionally, synergistic effect of both KP-10 and NAC was observed when given in combination in both experimental regimens. Therefore, these xiii findings suggest the protective effect of both KP-10 and NAC against NaAsO2-induced testicular damage at genetic level. Current study is novel in the aspect that for the first time it evaluates Kiss-1 peptide both alone and in combination with a well-known therapeutic agent NAC through spermatological, biochemical, hormonal, histological and genetic parameters in both in vivo and in vitro conditions. The outcomes from both regimens conclude that as a potent endocrine regulator KP-10 effectively mitigates the endocrine disruption caused by arsenic. Moreover, it is also suggested that it can counteract the oxidizing effect of trivalent arsenite by promoting testicular oxidant antioxidant balance thus preventing cellular apoptosis which is depicted at both biochemical and genetic level. Additionally, it promotes steroidogenesis by involving either indirect or direct intra- testicular signalling pathways. Furthermore, synergistic effect of both KP-10 and NAC strongly suggest that combination of these therapeutics can be used as a potential infertility treatment