Potential long-term developmental toxicity of in utero and lactational exposure to Triclocarban (TCC) in hampering ovarian folliculogenesis in rat offspring

Abstract

Triclocarban (TCC), an antimicrobial compound commonly added to a wide range of household and personal hygiene care products, is one of the most prevalent endocrine-disrupting substances (EDS). This study was conducted to elucidate whether in utero and lactational exposure to TCC could adversely affect folliculogenesis and the onset of puberty in female rat offspring. Twenty pregnant Sprague Dawley rats were equally divided into Control and TCC dam groups (supplemented daily with drinking water enriched with 0.5 mg/L of TCC) from gestational day5 to postnatal day21 (PND21). Female offspring, 20 from control and 20 from TCC dams, were subdivided into 4 subgroups (PND21, PND28, PND35 & PND42). The day of vaginal opening and first estrous cycle were determined. Ovarian sections of the offspring were processed for H&E staining and for immunohistochemical expression of Ki67, Caspase-3 and androgen receptors (AR) on the granulosa cells of ovarian follicles. Follicular count and atretic index were assessed besides, serum estradiol, progesterone, FSH and LH, C-reactive protein (CRP), malondialdehyde (MDA) and total antioxidant capacity (TAC) were measured. TCC offspring exhibited a significant delay in the onset of puberty and impedance of normal transition of the primordial follicles to more developed ones with altered cyctoarchitecture. Also, TCC decreased follicular count, proliferation and gonado-somatic index while it increased atretic index, apoptosis and AR of the granulosa cells along with disturbance of the feminine hormonal profile and oxidant/antioxidant balance. This study highlighted the potential long-term consequences of in utero and lactational exposure to TCC on the postnatal development of the ovary in rat offspring.

Introduction

Ample evidence from experimental studies revealed that a variety of exogenous compounds, designated as endocrine-disrupting substances (EDS) having the ability to alter the signaling pathway and the functions of mammalian hormones, particularly the reproductive ones (Craig et al., 2011; Patel et al., 2015). Previous reports have argued a link between the exposure to these EDS and the onset of puberty (Blanck et al., 2000), ovarian dysfunction (Savabieasfahani et al., 2006), early onset of reproductive senescence (Louis et al., 2006), abnormal fetal development (Jefferson et al., 2007) and reduced fertility (Zama and Uzumcu, 2010).

One of these EDS is the Triclocarban (TCC; 3,4,4^\-trichlorocarbanilide) which is a broad-spectrum bacteriostatic antimicrobial agent that is primarily used for its sanitizing properties in many household and personal care products including bar soaps, detergents, body washes, cleansing lotions, toothpaste, shampoo and wipes (Sapkota et al., 2007; Rochester et al., 2017; Wei et al., 2018). Although, TCC- containing products have been marketed broadly and have a long history of use in Europe, USA and other countries for more than five decades (Kennedy et al., 2015), Nevertheless, worldwide concerns have recently raised about the potential risks of TCC ubiquitous exposure (FDA, 2016; Costa et al., 2020a).

Pharmaceutically, TCC is a halogenated hydrocarbon compound with a diphenyl urea moiety (two aromatic rings linked by urea) and this makes it to be hardly biodegradable and subsequently it is incompletely scavenged by the ordinary wastewater treatment processes (Furukawa and Fujihara, 2008; Costa et al., 2020b). Therefore, TCC is usually detected in the downstream wastewater and contaminates the water resources, where it is considered among the top ten most commonly wastewater contaminants and consequently it is toxic to the aquatic organisms (snails and fish), animals and humans (Brausch and Rand, 2011; Enright et al., 2017). Additionally, TCC has a robust propensity to accumulate in the sludge and amended soils following the sewage-contaminated water irrigation with subsequent transfer to the vegetables and plants then to the consumed individuals (Cha and Cupples, 2009; Wu et al., 2010; Snyder et al., 2011).

In view of the ubiquity of TCC-containing personal care products and the high background level of environmental contamination by this compound, human beings are continually exposed to TCC either directly or indirectly. A direct human exposure was inevitable, whereby a single shower of a liquid soap containing 1.5 % TCC caused approximately 0.6 % skin absorption where dermal contact is believed to be the main route of exposure during the use of personal care products. Consequently, TCC has been detected in urine and serum samples of humans (Zhou et al., 2012; Costa et al., 2020b). Indirect exposure to TCC usually commences via consumption of TCC-contaminated water or via consumption of polluted wastewater-irrigated crops (Wu et al., 2010; Schebb et al., 2012).

Also, indirect exposure of the fetus during development was settled down; however this was little traced in the literature so far. Yet, in two studies by Enright et al. (2014) and Enright et al. (2017), TCC has been shown to transfer to the fetus via maternal exposure in both human placental in vitro and rodent in vivo models. In the in vivo model, TCC was found in maternal and feto-placental tissue, as well as in the fetus. TCC also transfers to offspring during lactation in exposed mothers.

Biologically, perinatal exposure to subtle exogenous environmental factors during susceptive windows of reproductive development may detrimentally affect organogenesis and the postnatal biological and functional maturation of the most organs (Fudvoye et al., 2014). Therefore, maternal exposure to these factors either prenatally or postnatally up to the time of sexual maturation increased the propensity of offspring to the "developmental toxicity" that may be detected later in life reflecting the so called “embryo-fetal origins of adult disease’’ (Selevan et al., 2000).

Recently, it was reported that the transfer of TCC to offspring, during pregnancy and breast-feeding, might impose a high potential risk of developmental toxicity for the highly vulnerable fetal and neonatal outcomes (Kennedy et al., 2015; Costa et al., 2020a). TCC has been reported to enhance the action of endogenous hormones (androgens and estrogens) in vitro (Chen et al., 2008; Tarnow et al., 2013) and in vivo (Chen et al., 2008; Duleba et al., 2011) rather than directly activating hormone receptors. However, Cao et al. (2020) recently demonstrated that human exposure to TCC might disrupt the estrogen system via the estrogen-related receptor γ (ERRγ). Additionally, human exposure to a relevant dose of TCC was associated with a decrease in the gestational period (Aker et al., 2019). These drawbacks of TCC are mostly related to its endocrine disruption property (Costa et al., 2020b) and to TCC-induced oxidative stress and biological dysfunctions in both humans and animals (Watkins et al., 2015; Wei et al., 2018).

The wide use and environmental persistence of TCC and the paucity of its studies together with its aforementioned potential detrimental impacts warrants our attention in this study to evaluate in utero and lactational exposure to environmentally relevant concentrations of TCC-enriched drinking water on the postnatal development of the ovary together with disclosing folliculogenesis and the onset of puberty in female rat offspring at four consecutive postnatal developmental periods (PND21, PND28, PND35 & PND42).