A Physiologically based Approach to Access the Hazardous Effects of Carcinogen Food Contaminant Furan as an Endocrine Disruptor on Reproduction

Abstract

Various chemicals or toxicants have been classified as endocrine-disrupting chemicals (EDCs) because of their ability to alter animal pathophysiology. EDCs exogenously enters the body and make alterations in the normal functioning of endocrine systems and also caused adverse health effects in an organism or its offspring. EDCs target every endocrine axis, their action is not limited to some single-axis or organ, are hypothalamus-pituitary -thyroid (HPT), hypothalamus-pituitary-gonads (HPG), hypothalamus-pituitary-adrenal (HPA) axis and associated organ systems are the main targets of EDCs. These chemicals may also affect the central nervous system (CNS) and hypothalamic and pituitary functions. Many harmful compounds or EDCs are produced during food processing including furan and acrylamide. Furan (C4H4O) is a colourless volatile liquid, which is used in several industries and produced in a variety of food items during heating processes such as coffee, sauces, soups, canned, jarred foods, infant formulae, and baby foods, exerting adverse effect to human health including reproductive health. It is also present in the environment as the main constituent of cigarette smoke, wood smoke, and exhaust gases from engines. The Food and Drug Administration (FDA) and the European Food Safety Authority (EFSA) described the carcinogenic and genotoxic mechanism of furan. Previous studies reported that furan caused various types of adverse carcinogenic effects on the biological system of humans and animals. Furan caused significant changes in histological and DNA structure of ovarian cells, malondialdehyde levels, and antioxidant enzyme activities. An in vitro study showed that furan incorporates chromosomal aberrations and sister chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) cells and Chinese hamster V79-derived cell line. The previous investigation showed that furan has antiandrogenic activity. In a human study, furan exposure generated reactive oxygen species and affected the lymphocytes and sperm cells. However, data regarding the adverse effect of furan on the mammalian reproductive system is still scant. Objectives: Present study was designed for a comprehensive assessment of the food based toxicant furan on the mammalian reproductive system. The main objectives of the study are: To investigate the toxicological and endocrine disrupting potentials of furan in rats, an in vitro approach was used to target the process of androgenesis and oxidative stress in testis and epididymal sperms. • To determine the possible in vivo toxic effects of furan experience on the reproductive system of the male through 28 days of study. • To examine the effects of furan on sperm production, maturation, and functions to regulate or inhibit specific targets that are involved in infertility. • To evaluate the effect of furan on the development of gonads, exposed during the neonatal period. • To study effects of furan exposure on HPG and HPA-axis by determining plasma T, LH, FSH, cortisol concentrations, and the brain monoaminergic activity i.e., 5HIAA/5HT, DOPAC/DA, HVA /DA ratios, NE level. • To establish a link between furan and endocrine disruption, a bivariant correlation between T, LH, FSH concentrations, brain monoaminergic activity, 5HIAA/5HT, DOPAC/DA, HVA/DA ratios, NE level, and cortisol were performed with the aim of the effect of stress on reproduction. • To find out the potential toxic effect of furan on reproductive performance of rats in F0 (parental) and F1 (offspring). A frequent oral dose based extended one generation reproductive toxicity study was performed. Materials and Methods; Stock solution of furan (Cat#185922, 99% purity) was prepared in corn oil. Testis tissues and sperm of male adult rats were properly incubated with artificial media having various concentrations of furan (0, 1, 100, 500, and 1000 μg mL-1) for 2 hrs at 37 ºC with 5 % CO2 and 95 % air. The different concentrations of Furan were dissolved in dimethyl sulfoxide (DMSO) and further dilution was made through media of cell culture. The overall concentration of DMSO in the cell culture media was less than 1 per cent. Oxidative stress in the testicular sperm and tissues were concluded through antioxidant enzyme activity. Testicular testosterone (T) concentration was determined through the ELISA technique. Whereas the DNA damage was measured through comet assay procedure in sperm cells. To achieve the second objective, based on in vitro study an in vivo study was performed. In this part of the experiment different doses (5-40 mg kg-1 ) of furan were used through an oral route for 28 days. For hormones analysis blood was collected. Brain tissues were used for the analysis of monoamine, while for sperm analysis, histopathology, and oxidative stress determination testicular and epididymis tissues were used. In the third part of the experiment, a neonatal exposure study was performed during the gonads' development period. In this study, the male rat pups were subcutaneously injected with various concentrations of furan (1 - 20 mg kg-1 furan in 50 μL corn oil) after PND 4 to PND 15 and were kept under observation till adulthood to inspect any amendments in the reproductive systems. Different parameters were monitored and compared with the control group. One-generation reproduction toxicity study was performed. In this study parents F0 (for male rats 70 days and female rats 14 days) were exposed to furan at a concentration of 1, 2.5, 5, and 10 mg kg-1 furan dissolved in 50 μL corn oil respectively before mating. Male rat’s dose exposure continued during mating whereas female rats received doses of furan during pregnancy and lactation. Complete monitoring of male rats (pre breed and during mating periods) and female rats during (pre breed, gestation, and lactation period) for complete physical examination. The number of living and dead pups, their weights, sex ratio, anogenital distance, nipple retention, and survival rate, etc were recorded during physical examination. Monitor all the reproductive parameters of F1 generation animals. At the age of 10 weeks, the F1 rats were properly marked for different reproductive examinations (body weights, hormonal profile, and histopathology). Results; In the in vitro study after exposing the testicular tissues and epididymal sperms to furan, oxidative stress was increased along with a decrease in antioxidant activity. At the maximum concentration, furan tempted the formation of reactive oxygen species and increased DNA fragmentation in the rat sperm cells. Also, furan cause reduced testosterone (T) secretion by the testes. The sub-chronic study of Lipid Peroxidation (LPO) and Reactive Oxygen Species (ROS) were significantly increased in the higher doses treated groups, whereas the activity of the antioxidant enzyme was reduced significantly in testicular tissues. The sub-chronic furan further decreased the plasma and intra-testicular T, LH, and FSH although, the level of cortisol increased in the groups treated with higher doses of furan. Brain monoaminergic activity was also interrupted in higher dose treatment groups. A significantly elevated oxidative stress was observed in testes while sperm parameters were reduced. Testicular and epididymis morphology results also revealed significant alterations after treatment with higher concentrations of furan. However, not a highly significant delay in puberty onset was reported in the groups treated with higher doses. Plasma hormonal analysis demonstrated a significant reduction in T, LH, FSH, GH, and a significantly elevated level of cortisol was observed in the high dose groups as compared to the control. Brain monoaminergic activity was also interrupted by exposure to furan. Histopathological variations involved a drop in epithelial height and diameter of the seminiferous tubules. Sperm parameters were affected by the high dose treated group than the control. However, F1, an extended one-generation reproductive toxicity study was accomplished to observe the adverse effects of furan in the next generation. After exposure, we observed significant changes in the weight of parental F0 animals. We found no significant change in the Parental F0 reproductive parameters. We also observed F1 litters parameters during the lactation period. At the pubertal stage, we observe a decline DSP in F1 generation male rats. Plasma hormonal concentration of testosterone (T) and Luteinizing hormone (LH) was decreased in F1 males respectively. Some histopathological changes were also observed in the F1 generation, whose parents were previously exposed to the high dose of furan. Conclusion; It is concluded that furan exposure caused reproductive toxicity by generating ROS in in vitro and in vivo studies. Besides its effect in adult animals, exposure during the neonatal stage of development we find clear evidence that exposure to the two highest doses broadly and consistently affects the development of the male reproductive system, which ultimately leads to a reduction in fertility rate. Furan can cause organizational effects on the development of male reproductive systems. This study provides important preclinical data on the minimal dose of furan at which endocrine disruption can occur. Otherwise in F1, extended generation the reproductive toxicity investigation a prominent change in sex hormone concentrations was evident, and slight histological alterations were also seen in testis. These findings suggest that furan has detrimental effects on the developmental stages of life because the developmental period is the most sensitive period of life. Due to these findings, it is concluded here that the use of furan may be a matter of high concern for human life and health. The toxicological profile of furan presented in these studies supports the identification of furan as an endocrine disruptor.