Contributions of enzymes and gut microbes to biotransformation of perfluorooctane sulfonamide in earthworms (Eisenia fetida)

Highlights

• FOSA could be bioaccumulated and biotransformed to PFOS by earthworms in sand.

• CYP450 and GST activities were increased, while POD activity was not changed.

• ABT inhibited the transformation of FOSA in earthworms via the inhibition of CYPs.

• TLK199 decreased the formation of PFOS from FOSA in worms via the GST inhibition.

• The gut microbes did not contribute to FOSA biotransformation in earthworms.

Abstract

Perfluorooctane sulfonamide (FOSA) is known as a key intermediate of perfluorooctane sulfonic acid (PFOS) precursors, which can be frequently detected in the environment and biota. FOSA could be bioaccumulated in earthworms from soil, but the contributions of enzymes and gut microbes involved in the biotransformation of FOSA in earthworms have not been identified. Therefore, the effects of enzyme inhibitors and intestinal microflora on biotransformation of FOSA in earthworms were investigated in the present study. FOSA was biotransformed to form PFOS by earthworms obtained from in vivo and in vitro tests. The addition of FOSA had significantly positive effects on cytolchrome P450 (CYP450) and glutathione-s-transferase (GST) activities, suggesting CYP450 and GST are likely involved in the enzymatic transformation. In addition, both 1-Aminobenzotriazole (ABT) and ezatiostat hydrochloride (TLK199), which were selected to inhibit the CYP and GST enzymes, respectively, demonstrated inhibition effects on biotransformation of FOSA in earthworms with a dose-dependent relationship. However, the concentrations of FOSA weren't changed by the bacteria isolated from worm gut, suggesting that gut bacteria did not contribute to FOSA biotransformation in earthworms. The results of this study confirm that the transformation of FOSA in earthworms is mediated mainly by enzymes rather than by gut microbes.

Introduction

Poly - and per -fluoroalkyl substances (PFASs) have been widely used in industrial and commercial products (Begley et al., 2008). Perfluorooctane sulfonate (PFOS), which is one of the dominant PFASs, has been listed in Stockholm Convention Persistent Organic Pollutants (POPs) in 2009 as a result of its persistent and toxicological characteristics (Paul et al., 2009). Besides the direct release of PFOS, the biological and abiological transformation of PFOS precursors (PreFOS) lead to the presence of PFOS in the environment and biota (Chu and Letcher, 2014). Perfluorooctane sulfonamide (FOSA) is a vital and typical intermediate of the PreFOSs, which is widely detected in human, wildlife, consumption product, indoor air, water and soil (Ahrens et al., 2009; Fromme et al., 2007; Hart et al., 2009; Haug et al., 2011; Houtz et al., 2013; Kannan et al., 2004). Previous study has reported that FOSA is the most toxic PFASs (even stronger than PFOS) to pheochromocytoma (PC12) cells which is thought to be potential developmental neurotoxicant (Slotkin et al., 2008). Otherwise, FOSA was known as the biologically active form of the insecticide sulfluramid (Schnellmann and Manning, 1990). Plenty of studies have demonstrated the deaminization of FOSA to form PFOS in rat, fish, human and microorganisms (Avendañoa and Liu, 2015; Bizkarguenaga et al., 2016; Brandsma et al., 2011; Martin et al., 2006; Ross et al., 2012; Xu et al., 2004). FOSA is also formed from the degradation of other PreFOSs, such as N-ethyl perfluorooctane sulfonamido ethanol (EtFOSE), N-ethyl perfluorooctane sulfonamide (EtFOSA) and (N-ethyl perfluorooctanesulfonamido) ethanol-based phosphate diester (diPAP) in environment and living biotas (Peng et al., 2014; Tomy et al., 2004a; Wei et al., 2009).

Earthworms, which play a crucial role in soil nutrient cycling and soil food web in terrestrial ecosystems, are often used as OECD standard biological indicator of soil pollution (Butt and Briones, 2017; Wang et al., 2017). Earthworms accumulate and biotransform organic pollutants through ingestion, digestion, assimilation in the gut, transformation by enzymes in earthworms and microorganisms in the gut and then casting (Katagi and Ose, 2015). According to our previous study, FOSA could be bioaccumulated and biotransformed to terminal product PFOS in soil-earthworm systems (Zhao et al., 2018d). Phase I biological metabolism of xenobiotic pollutants in organism is mainly mediated by the CYP450-dependent monooxygenase system which exists generally in human, plants, animals and microbes (Yi et al., 2007; Zhang et al., 2006). Previous studies suggested that animal and human CYP450 were proposed as the enzymes metabolizing PreFOS, such as EtFOSE (Xu et al., 2004) and EtFOSA (Benskin et al., 2009; Fu et al., 2015) to form PFOS. GST is an inducible phase II enzyme that metabolize xenobiotics and detoxify pollutants in biotas (Huang et al., 2013; Lash et al., 2002). It was reported that GST were involved in 8:2 fluorotelomer alcohol (8:2 FTOH) metabolism in rat (Fasano et al., 2006) and soybean (Zhang et al., 2016a), and FOSA biotransformation in plants (Zhao et al., 2018a). 1-Aminobenzotriazole (ABT) has been extensively used in animals for studying metabolic mechanism of medicine and xenobiotics, which has no obvious toxicity and prohibitive effects on other functional enzyme (Mico et al., 1988; Mugford et al., 1995). Ezatiostat hydrochloride (TLK199) is a novel glutathione analog inhibitor of GST (Quddus et al., 2010). In order to explore the role of CYPs and GST in the biotransformation of FOSA to PFOS in earthworms, ABT and TLK199 were chosen as the inhibitor for CYPs and GST, respectively, to investigate the biotransformation behaviors of FOSA in earthworms.

It was speculated that the metabolism of PreFOSs in the earthworms was due to enzymes located in the earthworm tissue and/or by gut-associated bacteria. Moreover, bacterial communities in earthworm gut are different from the surrounding environment due to its gut unique microhabitat (Wang et al., 2019). There is a large number of aerobic and anaerobic bacteria which is known to have efficient detoxification capability in worm gut (Karsten and Drake, 1995). Some studies have reported that gut bacteria contributed to organic pollutant degradation in earthworms, such as hexachlorocyclohexane (HCH) (Ramteke and Hans, 1992) and endosulfan (Verma et al., 2006) could be biodegraded by the bacteria isolated from the earthworm gut. But, the effect of gut microbes on PreFOS degradation in earthworms has rarely been examined.

In the present study, the earthworms (Eisenia fetida) were exposed to the spiked sands to investigate the effects of enzyme inhibitor and gut microbiota on biotransformation of FOSA in earthworms. The CYP450, GST and peroxidase (POD) activities in earthworms over different exposure times exposed to FOSA were analyzed to explore the responses of biotransformation enzymes in earthworms. The inhibition tests of CYP450 and GST were used to further investigate their roles in metabolizing FOSA in earthworms. In addition, the ability of earthworm gut bacteria to transform FOSA by inoculating the bacterial colonies in simulative gut environment was tested.