SULT1E1 inhibition leads to Increased E2 availability

The biological plausibility of the current KERs is related to the physiological role of SULT1E1 in estrogen metabolism.

Estrogen sulfotransferase (EST) is a cytosolic sulfotransferase that transfers a sulfuryl group from the ubiquitous sulphate donor 3´-phosphoadenosine 5´-phosphosulfate (PAPS) to the 3-hydroxyl on 17β-oestradiol (E2). Sulfation of E2 by SULT1E1 represent one of the pathways for the inactivation of this hormone. The sulfonation of E2 makes the compound more soluble for renal excretion as well as for the creation of inactive stores of sulphated E2 that can be de-sulphated by steroid sulfatases. Both the estrogen receptor (ER) and SULT1E1 have affinities for E2 in the nanomolar range (Km approximately 5nM Falany et al 1998), thus suggesting that SULT1E1 may play an important role in the regulation of estrogenic effects by controlling the levels of E2 (Shevtsov et al., 2003).

SULT1E1 readily sulphates E2 at concentrations at which it binds to the ER suggesting that it has a significant physiological role in modulating the response of ER-positive tissues to estrogenic stimulation. High levels of SULT1E1 activity in a tissue would render the tissue less responsive to estrogenic stimulation because the sulfated estrogens are not able to bind to the estrogen receptor and/or initiate a cellular response (Falany et al., 1998).

The inhibition of SULT1E1 has consequently the potential to increase the bioavailability of E2, thereby causing an estrogenic effect (e.g., changes in metabolism of estrogens resulting in higher oestradiol levels that interact with ER receptors in target tissues (Wang and James, 2006).

Only in the last decades increase knowledge of the intracrine (or local) regulation of estrogen and other steroid synthesis and degradation has been expanded. From a physiological point of view, it has been found that estrogen responsive tissues and organs are not passive receivers of the pool of steroids present in the blood, but they can actively modify the intra-tissue steroid concentrations. This allows fine-tuning the exposure of responsive tissues and organs to estrogens and other steroids in order to best respond to the physiological needs of each specific organ (Konings et al., 2018).

At first Brooks et al., in 1978 investigated the uterine metabolism of E2 throughout the porcine oestrous cycle underlined a fluctuation of the estrogen sulphate in the different phase of the cycle (Fig. 9).

Figure 9. Pattern of uterine estrogen sulfation and oxidation throughout the days of the porcine estrous cycle. Each incubation lasted 2 h and contained 400 mg gilt uterine minces, [6.73H] - 17β- estradiol {2.5 - 6.6 x 10-9 M} and Na235 SO4 (0.8-1.5 x 10-4 M). (*) Percentage sulfation of total 3H-labeled estrogens in the incubate; (O) percentage estrone sulfate; and (Δ) percentage estrone. An indication of the reproducibility of the results is given by the repeat experiments carried out on d 4, 13, and 14 uteri (adapted from Brooks et al., 1978)

Few years later, the ability of SULT1E1 to mediate local estrogen concentration has been also demonstrated in mice by gene disruption studies (Qian et al., 2001; Tong et al., 2005) and also in human proliferative and secretory endometrium obtained from pre-menopausal women (Falany et al., 1998).

Deviations in such intracrine control can lead to unbalanced steroid hormone exposure and disturbances. SULT1E1 is suggested to play an important role in protecting peripheral tissues from extreme estrogenic effects. Inhibition of SULT1E1 activity by Endocrine Disruptive Chemicals (EDCs) or lower SULT1E1 expression levels may lead to a higher in situ availability of biologically active estrogens, which can result in a higher cell proliferation or estrogen stimulated DNA synthesis (Reinen and Vermeulen 2015). In fact, the expression of SULT1E1 was significantly downregulated (p = 0.0392) in cancer tissue from premenopausal women, with significantly lower levels seen in cancer and adjacent control tissue from postmenopausal women as compared to premenopausal women (Sinhrein et al., 2017).

Utsunomya et al., 2004 and more recently Cornel et al., 2019, investigated the role of intratumorally metabolism and synthesis of estrogen. In line with the results from other experiments (Chetrite et al., 2000, Pasqualini and Chetrite, 1999; Dao et al., 1974; Pasqualini et al., 1986), the studies demonstrated that increased steroid sulfatase and decreased estrogen sulfotransferase expression in human endometrial carcinomas may result in increased availability of biologically active estrogens in situ and may be related to estrogen-dependent biological features of carcinoma.

Empirical evidence may be extrapolated from different studies investigating the MoA of specific stressors. In the context of the current AOP, evidence have been collected for two stressors known to inhibit the SULT1E1: an environmental contaminant belonging to PHAH (TBBPA) and a consumer product Triclosan.

No direct evidence evaluating in the same experiment inhibition of SULT1E1 and increase of estrogen bioavailability in estrogen responsive tissues is currently available.

TBBPA (see Table 2)

In vitro

The potential of TBBPA to interfere with estrogen metabolism by competing with the same enzyme systems involved in the conjugation of estrogens has been investigated by different authors to support the hypothesis that TBBPA exerts estrogenic activity by trough this mechanism. In recombinant human systems, TBBPA inhibits SULT1E1 with an IC50 in the range of 12-33 nM (Kester et al., 2002; Hamers et al., 2006). In both the experiments the IC50 is near the Km of 5 nM for E2, suggesting a high affinity of TBBPA for the SULT1E1.

Crystallography studies (Gosavi et al., 2013) showed that hydroxyl moiety, bromine atoms and non-planar structure of TBBPA contribute to stable binding of TBBPA to SULT1E1. Gosavi and colleagues demonstrated that TBBPA is able to mimic oestradiol binding to the active site of the enzyme.

In vitro

• Kester et al., 2002. Observed a marked inhibition of SULT1E1 by various PHAH-OHs, in particular by compounds with two adjacent halogen substituents around the hydroxyl group that were effective at (sub)nanomolar concentrations. Depending on the structure, the inhibition is primarily competitive or non-competitive. TBBPA was one of the compounds tested in the study.
• Hamers et al., 2006. Investigate the In Vitro Profiling of the Endocrine-Disrupting Potency of Brominated Flame Retardants. The author found that TBBPA is a potent inhibitor of SULT1E1 and therefore that the ED potency of such BFR is not directly linked to estrogen receptor (in)activation.

In vivo

• Borghoff et al., 2016, investigated the ratio of TBBPA-S/TBBPA GA in liver, plasma and uterus. The ratio is decrease in uterus indicating, according to the author a potential saturating/limitating effect of SULT on oestradiol sulfation.
• Sanders et al., 2016. Observed a change in genes indicated to be target of estrogen stimulation. However, is not clear if TBPPA act directly on these genes or whether the effect is mediated by E2 increase bioavailability.

Others (indicative of a potential link between KE up and KE down)

Indirect correlation may be extrapolated from different studies investigating the toxicological profile of TBBPA and/or its mode of action in the induction of uterine adenocarcinoma in rats, that in humans is usually associated to high estrogen concentrations (ECHA, 2006). In its review, Wikoff and co-workers observed that serum glutamate is inversely associated to increase of circulating levels of estrogen; this is also in line with the neuroprotective effect of estrogens against various neurodegenerative conditions.  The EU risk assessment report on TBBPA (ECB, 2006), reported an increase of 17% in serum glutamic pyruvic transaminases at the high – dose (100 mg/kg day) in female Sprague Dawley rats administered dietary TBBPA for 90-day (Table 2).

Table 2. Empirical evidence table assembled for KER1, TBBPA

Triclosan (Table 3)

In vitro

• James et al., 2010. Investigated the effect of Triclosan on oestradiol and estrone sulfation in sheep placenta. Triclosan is a substrate of SULT1E1 with Km in the range of 1.14 uM (compared to the 0.27 uM of oestradiol). Triclosan inhibit both oestradiol and estrone sulfation with competitive/uncompetitive type of inhibition

In vivo

• Jung et al., 2012. Investigate the potential estrogenic activity of Triclosan in the uterus of immature rats and rat pituitary GH3 cells. The study author reported an increase uterine weight together with increase expression of Calbidin-d9k and complement C3 that are reversed by steroid antagonists.

• Louis et al., 2013 investigate the effect of Triclosan on the Uterotrophic response to EE. The evidence of an estrogenic activity was reported only when rats are treated with Triclosan and EE. 

Table 3. Empirical evidence table assembled for KER1, Triclosan

Dose and temporal concordance

In accordance with the OECD handbook for the AOP developers this section should include the extent of the evidence that KE upstream is generally impacted at doses (or stressor severities) equal to or less than those at which KE downstream is impacted.

The dose and temporal concordance tables for stressors TBBPA and Triclosan, were included in Annex A.3 of the Scientific Opinion.  

In the case of the current KER the evidence is poor and many inconsistencies were identified. In the following lines general (not applicable to specific stressors) uncertainties and inconsistencies were reported.  

Evidence of Perturbation by Stressor 

Overview for Molecular Initiating Event

The PAPS binding region and substrate binding region are two essential components of SULT enzymes: occupation of one or both of these regions may cause inhibition of SULT activity.

Different types of inhibition of SULT activity, competitive, non-competitive and mixed-type inhibition, have been reported for different inhibitors in the different tissues (Wang and James, 2006).

• Competitive inhibition

If inhibitors occupy one or both of the binding sites of the cofactor or the substrate of the SULT, the inhibition type will be competitive.

• Non-competitive Inhibition

If the inhibitor does not occupy the binding region of the substrate, the inhibition is likely to be non-competitive.

• Mixed type Inhibition

There are two types of mixed-type inhibition, competitive- non-competitive inhibition and non-competitive-uncompetitive inhibition.

Stressors

Various xenobiotics including dietary and environmental chemicals, some therapeutic drugs, PAPS analogues, derivatives of substrate, some library compounds, were shown to inhibit one or more SULTs. Some inhibitors were isoform selective, while others inhibited all forms of SULT.

Endogenous chemicals

• E2. Substrate inhibition of humSULT1E1 by E2 was observed with inhibition constants of 1.11 ± 0.04 µM (UniProt: Hempel et al., 2000). In other studies, maximal E2 sulfation was observed at concentration of 20 nM and substrate inhibition with higher E2 concentrations (Falany et al.,1995) whereas Kester et al., 2002 reported that IC50 of E2 is in the range between 3.8 – 7.1 nM with relative potency of 1. It has been thought that the binding of E2 to the allosteric site of SULT1E1 is responsible for this phenomenon.  
• Redox status. SULT1E1 is a cytosolic protein, and it is well-known that the cytoplasm is a highly reducing environment (containing millimolar levels of GSH) in which protein cysteine residues are maintained primarily in their thiol (-SH) state. Redox modification of Cys residues of an enzyme provides a mechanism for regulating enzyme ac DHEA tivity. Maiti and co-workers demonstrated that human liver cytosolic SULT1E1 is sensitive to glutathione (GSSG) treatment in a time and concentration-dependent manner. The inactivation was due to the ability of GSSG to modify the Cys active site of the enzyme (Maiti et al., 2007).

Environmental chemicals

• (Hydroxylated) Polychlorinated Biphenyls (PCB and OH-PCBs): PCBs and OH-PCBs have been shown to inhibit human SULT1E1 with IC50 values as low as 0.1 nM (Kester et al., 2000). The crystal structure of human SULT1E1 with the OH-PCB bound gives physical evidence that certain OH-PCBs can mimic binding of estrogenic compounds in biological systems (Shevtsov et al., 2003).
• Polyhalogenated aromatic hydrocarbons (PHAHs) such as polychlorinated dibenzo-p-dioxins and dibenzofurans, polybrominated diphenylethers, and bisphenol A derivatives are persistent environmental pollutants that induce a broad spectrum of effects in humans. Kester et al., 2002 investigated the possible inhibition of human SULT1E1 by hydroxylated PHAH metabolites. The inhibition of SULT1E1 was found by various PHAH-OHs, in particular by compounds with two adjacent halogen substituents around the hydroxyl group (Kester et al.,2002). Tetrabromobisphenol-A  (TBBPA) is one of the compounds tested by Kester and is a well-known brominated flame retardant used to improve the fire safety of consumer products. TBBPA is metabolised to sulphate and glucuronide conjugates and therefore is able to compete with the same enzyme systems involved in the estrogen metabolism. The IC50 is 12-33 nM and relative potency vs. E2 0.30 (Kester et al., 2002).
• N-ethyl-N-nitrosourea (ENU) is a carcinogen used in experimental animal models for tumorigenesis/carcinogenesis mainly to induce different types of gynaecological cancer. A recent study demonstrates that ENU induced a reduction of rSULT1E1 in liver tissues of female albino Wistar rats with a consequent increase of serum and liver oestradiol (Nazmeen and Maiti, 2018).

Consumer products  

• Triclosan. Triclosan  is a broad-spectrum antibacterial agent used in many household products. Triclosan  has been demonstrated to be a very potent inhibitor of both oestradiol and estrone sulfation suggesting competitive binding of Triclosan  for oestradiol sites on the sulfotransferase enzyme Competitive inhibitory constant Kic 0.09 nM, uncompetitive Kiu 5.2nM (James et al., 2010).

Drugs

• Cyclizin eantistaminic) (Konings et al., 2018)

• There are no studies investigating in the same experiment the KEupstream and KEdownstream.
• Some empirical evidence is showing changes in KEupstream that did not elicit alterations in KEdownstream.
• Some experiment test only one dose, so the dose-temporal concordance could not be extrapolated and their use in support to the empirical evidence is limited.
• It is well-known that there is large variability in the uterotrophic assay (Browne et al., 2015), this can be explained by the differences in the experimental design
• There are other factors that in target tissue i.e., uterus can contribute to the intra cellular metabolism of E2 e.g., STS, HSD17B (Konings et al.,2018). However, there are gaps in the biological understanding of the contribution of each of these factors.
• There are two other sulfotransferases in the SULT1 class that catalyse the sulfonation of estrogens: SULT1A1 and SULT1A3 (both EC 2.8.2.1 and phenol sulfotransferase enzymes). These enzymes have much lower affinities for estrogens, with maximal activity at about Km = 25 uM. However, their possible interaction in the current KER has not been investigated in the experiments.
• The internal quality of the primary research study used to substantiate the empirical evidence has not been evaluated in a systematic way (e.g., using tools to evaluate the risk of bias).
• Uncertainties and inconsistencies should be further explored.

There are only a limited number of studies where both SULT1E1 and increase E2 bioavailability in uterus have been measured in vivo, and there is not enough data available to make any definitive quantitative correlations. Overall, given that this is an MIE to KE relationship there is only one response to evaluate in the relationship: increase E2 bioavailability in uterus as a result of the SULT1E1 inhibition.

Measurable response in the KERs is not a discrete assessment of SULT1E1 inhibition. Indeed, a number of factors could contribute to increase the E2 bioavailability in uterus.