Alteration of testicular steroidogenesis and histopathology of reproductive system in male rats treated with triclosan

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Abstract

Triclosan (TCS), a chlorophenol, is widely used as a preservative in different types of commercial preparations. The reports on TCS-mediated endocrine disruption are controversial and the present study aimed to elucidate the probable mode of action of TCS as an antiandrogenic compound using a robust study design. Male albino rats, Rattus norvegicus, were treated with three doses of triclosan for a period of 60 days followed by the analysis of various biochemical parameters. RT-PCR analysis demonstrated a significant decrease in mRNA levels for testicular steroidogenic acute regulatory (StAR) protein, cytochrome P450SCC, cytochrome P450C17, 3β-hydroxysteroid dehydrogenase (3β-HSD), 17β-hydroxysteroid dehydrogenase (17β-HSD) and androgen receptor (AR) in TCS treated rats (p < 0.05). TCS also induced a perturbed translation of testicular StAR, and AR proteins as shown by Western blot analysis in treated groups of rats. A reduced level of StAR was further indicated by immunohistochemistry in testicular Leydig cells. Further, there was a significant decrease (p < 0.05) in the level of serum lutenizing hormone (LH), follicle stimulating hormone (FSH), cholesterol, pregnenolone, and testosterone. In vitro assays demonstrated more than 30% decrease in testicular 3β-HSD and 17β-HSD enzyme activities in treated group of animals. Extensive histopathological malformations were observed in the testis and sex accessory tissues of the treated rats. Overall this study showed that TCS decreased the synthesis of androgens followed by reduced sperm production in treated male rats which could be mediated by a decreased synthesis of LH and FSH thus involving hypothalamo–pituitary–gonadal axis.

Introduction

Endocrine-disrupting chemicals (EDC) comprise a category of environmental contaminants that interferes with the function of endocrine system [1]. An increasing body of evidence reveals an association between various environmental compounds that act as EDC and lead to sex hormone-sensitive disease/disorders [2], [3]. Chemicals that mimic the structure of the natural hormones found in the animal/human body may pose species-specific risks that are difficult to investigate because of latent adverse effects [4]. A body of literature exists for various EDC demonstrating potential estrogenic activities which have been identified and classified [2]. Although there are similar health concerns regarding (anti)androgenic EDC that interfere with sperm production, alter genital development and contribute to neurological syndromes in males, the identification and classification of these putative health hazards have progressed comparatively slowly [3]. Recent reports of several non-steroidal compounds that have the ability to alter the androgen dependent functions are of particular concern because many of them are ubiquitously used in our daily life.

A number of antimicrobial agents and preservatives are commonly used in the personal care products such as soaps, shampoos, detergents, disinfectants, cosmetics and pharmaceutical products [5], [6], [7]. The continuous use of these chemicals results in their accumulation at detectable concentrations within different parts of our body like blood, milk, and various organs and tissues [5], [8], [9], [10]. Triclosan (TCS; 2,4,4′-trichloro-2′-hydroxydiphenyl ether; a chlorophenol) is an antimicrobial agent widely used as preservative in toothpastes, soaps, shampoos, and cosmetics [11]. The chemical structure is shown in Fig. 1. In general, TCS has been known to be a highly toxic chemical for aquatic flora and fauna [12] and thus has been included in the probable list of endocrine disruptors on account of its resemblance with known non-steroidal estrogens or its mimetic (e.g. diethylestradiol, bisphenol A). Further, TCS and its chlorinated derivatives are readily converted into various chlorinated dibenzo-p-dioxins by heat and ultraviolet irradiation which may also be harmful for biological systems [13], [14], [15]. The mode of action of TCS as an EDC is controversial and various studies indicate it to be of different nature, viz. estrogenic or weak androgenic or anti-androgenic. Fourteen days TCS exposure in Japanese medaka fry (Oryzias latipes) showed a weak androgenic effect [16]. Another study reported that the metabolite of TCS may be a weak estrogenic compound with the potential to induce vitellogenin in male medaka while decreasing the hatchability, as well as delaying the hatching in females [17]. TCS has also been shown to function as an anti-androgen since it inhibits testosterone-induced transcriptional activity [2]. Exposure of TCS to the human may be a consequence of its presence in the cosmetics and other human use products. This chemical has been reported to be absorbed mainly by two routes: either across the skin or through the gastrointestinal tract [8].

This report describes the various targets of TCS toxicity in an effort to help understand its probable mode of action as an (anti)androgenic endocrine disruptor using male albino rats as model. The rats were treated with three dose levels of TCS for a fixed period of time. On completion of treatment serum and tissue samples were analyzed for their (anti)androgenic effects in response to TCS. Further histopathological analysis of testis and sex accessory tissues (SATs) were performed to assess the action of this chemical at the cellular levels. The data presented here demonstrates that some widely used antimicrobial compounds like that of TCS have anti-androgenic properties and warrant further investigation to understand its impact on human reproductive health.

Section snippets

Animals

The study was carried out on the male Wistar rats, Rattus norvegicus, with the approval as well as guidelines of institutional ethical committee. Animals were purchased from All India Institute of Medical Sciences (New Delhi, India) and were in healthy condition at the time of purchasing. They were housed in a well-ventilated animal house with 12 h light:12 h dark schedule. The animals were fed with a balanced animal diet obtained commercially (Ashirwad Animal Feed Industries, Punjab, India) and

Body weight and weight of testis and SATs

Treatment of rats with test samples did not induce significant changes in the body weight at any of the test doses (Table 2). Whatever minimal increase in the body weight observed could be attributed to normal aging. Administration of TCS did not cause significant change in the weight of testis and SATs at the 5 mg/(kg day) dosage. In contrast, the higher test doses (10 and 20 mg/(kg day)) induced a significant decrease in the weight of testis and SATs. TCS exposure decreased the weights of testis,

Discussion

TCS is a synthetic chemical widely used as an antimicrobial agent in different commercial preparations [11]. Since TCS possess a phenolic moiety like many of the common EDC it could be presumed to display similar activities as demonstrated by other EDC of the same chemical family [31], [32]. The dosage for the test chemicals used in this study were selected based on LD50 values and also some earlier reports where a similar compound, triclocarbon (TCC), was used in rat models [33], [34]. In this

Conflict of interest

None declared.

Acknowledgements

Kind help of Prof. Ilpo Huhtaniemi, Imperial College London, UK with all steroidal test chemicals is greatly acknowledged. We would also like to thank Prof. D.M. Stocco, Texas Tech University, Lubbock, TX, and Dr. R.K. Tyagi, Jawaharlal Nehru University, New Delhi, India for kindly providing the StAR and androgen receptor antibodies, respectively. The study was supported by the Ministry of Human Resource and Development as fellowship to V.K., and by the Department of Biotechnology (DBT, no.

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      Citation Excerpt :

      In general, much discrepancy is observed between the results, but this discrepancy can be explained by the doses and the exposure times. For example, at low exposure doses (0.8–20 mg/kg/day), no significant variation is found by TCS exposure in the weight of rats at the end of the studies independent of sex, pregnancy or period of treatment (varying from 3 days to 13 weeks) (Garcia et al., 2018; Kumar et al., 2009; Ena et al., 2018; Rabaglino et al., 2016). In other studies where the dose is similar to that proposed here (37.5 mg/kg/day), a significant effect was found of weight loss after 32 weeks of exposure (Louis et al., 2017), which coincides with what was reported in our work but in a much shorter period (4 weeks).

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    1

    Present address: Department of Biotechnology, The ICFAI University, Dehradun, Rajawala Road, Dehradun 248197, Uttarakhand, India.

    2

    Contributed equally to this work.

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