Best Practice & Research Clinical Endocrinology & Metabolism Vol. 20, No. 1, pp. 45–61, 2006 doi:10.1016/j.beem.2005.09.003 available online at http://www.sciencedirect.com

4

Endocrine-disrupting chemicals as modulators of sex steroid synthesis

Saffron A. Whitehead* PhD Professor of Reproductive Endocrinology

Suman Rice PhD Post doctorate Yellow Department of Basic Medical Sciences, St George’s University of London, Cranmer Terrace, London SW17 ORE, UK

Endocrine-disrupting chemicals (EDCs) are typically identified as compounds that can interact with oestrogen or androgen receptors and thus act as agonists or antagonists of endogenous hormones. Growing evidence shows that they may also modulate the activity/expression of steroidogenic enzymes. These are expressed not only in the adrenal glands and gonads but also in many tissues that have the ability to convert circulating precursors into active hormones. In this way, EDCs may impact both on sexual differentiation and development and on hormone- dependent cancers. This review summarizes the evidence for EDCs as modulators of steroidogenic enzymes, identifies the structure/activity relationship in terms of inhibiting specific enzyme activity, questions whether experimental observations can equate with natural in vivo exposure or dietary intake of EDCs, and finally looks at the mechanisms through which these chemicals may disrupt normal steroidogenesis. In summarizing the evidence, the question of whether or not the dietary intake of these endocrine disrupters could pose a threat to human sexual development and health will be addressed.

Key words: phyto-oestrogens; xeno-oestrogens; aromatase; hydroxysteroid dehydrogenases; steroidogenesis.

The actions of plant-derived phyto-oestrogens and the agricultural and industrial chemicals xeno-oestrogens go beyond their ability to bind to oestrogen receptors and either stimulate or inhibit the activity of endogenous oestrogens. Many studies have demonstrated the effect of EDCs on other target molecules and signalling pathways through which they may exert diverse actions.1 These include anti-androgenic properties, anti-oxidant actions, inhibition of cell cycles and cell differentiation, modulation of

* Corresponding author. Tel.: C44 208 8725 5360; fax: C44 208 8725 2993. E-mail address: [email protected] (S.A. Whitehead).

1521-690X/$ - see front matter Q 2006 Elsevier Ltd. All rights reserved. 46 S. A. Whitehead and S. Rice angiogenesis and modulators of the activity or expression of steroidogenic enzymes. It is the latter action of the EDCs that will form the focus of this review.

STEROIDOGENESIS

The synthesis of sex steroids from requires trafficking between mitochondria and smooth endoplasmic reticulum and involves many enzymatic steps. Most of these steps use cytochrome P450 haem-containing enzymes, and the genes coding for these enzymes are abbreviated to CYP. The enzyme that has received the most attention with regard to enzyme modulation and endocrine disrupters has been aromatase (CYP 19) that converts androgens to oestrogens (Figure 1). Other enzymes have included 3b-hydroxysteroid dehydrogen- ases (HSDs) and 17b-HSDs (Figure 1). There are two known isoforms of 3b-HSD, type 1 being found in the placenta, skin, mammary gland, prostate and endometrium, and type 2 in the adrenals and gonads.2 This particular enzyme is not a P450 enzyme but converts pregnenolone to progesterone and (DHEA) to androstenedione. 17b-HSDs are a relatively large family of steroidogenic enzymes of which types 1, 3, 5 and 7 catalyse a reductive reaction, generally converting biologically weaker steroids into more biologically active steroids. On the other hand, types 2, 4, 6 and 8 are oxidative enzymes and convert more active steroids into less biologically active steroids.3 To date the effects of phyto-oestrogens on this family of enzymes has not been studied extensively, and types 1 and 5 have received the greatest attention. 17b-HSD type 1 converts oestrone to the more potent 17b-oestradiol, whilst type 5 converts androstenedione to testosterone and DHEA to androstenediol. The latter can also catalyse 5a-dihydrotestosterone/androstanedione and androstenediol/androster- one interconversion. Finally, there are the sulphotransferases (mainly SULT1E1) and steroid sulphatase that interconvert active oestrone and DHEA into inactive oestrogen and androgen sulphates (Figure 1).

Pregnenolone Dehydroepiandrosterone Androstenediol β 3β HSD 3β HSD 3 HSD

Progesterone 17α hydroxyprogesterone Androstenedione Testosterone 17α hydroxylase 17,20 lyase 17β HSD

Arom Arom

20α HSD Oestrone Oestradiol 17β HSD 20α dihydro- sulphatase progesterone sulphotransferase Oestrone sulphate

Figure 1. Steroid synthesis of the sex hormones and the steroidogenic enzymes that have been reported to be targets of endocrine disrupting chemicals. The conversion of androstenedione to testosterone is catalysed by 17b-hydroxysteroid dehydrogenase (HSD) types 3 and 5, and the reverse oxidative reaction is catalysed by 17b-HSD type 2. The reduction of oestrone to oestradiol is catalysed by 17b-HSD types 1 and 7, and the reverse reaction is catalysed by types 2 and 4. Expression of these enzymes is tissue-specific. Endocrine-disrupting chemicals and sex steroid synthesis 47

Whilst these enzymes are expressed in classical steroidogenic organs such as the gonads and adrenal gland, they are also expressed in a large number of other tissues, including the brain, liver, reproductive tracts, adipose tissue, skin and breast tissue. Thus, a variety of tissues can convert relatively inactive circulating steroid precursors into biologically active steroids, and so any modulation of these enzymes by EDCs could have important consequences for sexual differentiation and development and for endocrine-dependent cancers.

PHYTO-OESTROGENS AND ENZYME ACTIVITY

There are numerous classes of plant-derived substances, some of which show close structural similarity to androgens and oestrogens (Figure 2). Different classes of phyto- oestrogen are present in a wide range of different dietary products (Table 1), and thus, human (and animal) exposure to different classes of phyto-oestrogens is very much dependent on dietary, geographical and social factors.

OH OH

D C C D

A B A B O HO Testosterone 17β – Estradiol

3' 2' 4' 5 O 5 O 5 O B 63 63 5' A C 6 2 2' A C 2' 3 6' 7 7 2 A C O 3' O 3' 2 8 8 7 B B 8 O 6' 4' 6' 4' Flavone 5' 5'

O HO CH2OH HO OH O O CH2OH

HO O O OH OH

Figure 2. Basic chemical structures of testosterone and 17b-oestradiol and the major classes of phyto- oestrogens. Structure/activity relationships show that the A and C rings of the flavones and flavonones mimic the D and C rings of androgen substrates. Isoflavones, that preferentially inhibit hydroxysteroid dehydrogenases (HSDs), have the 40-hydroxyphenol group at C3 instead of C2, and this prevents binding to aromatase. Hydroxylation of C7 is important for inhibition of HSDs, but further hydroxylations at positions 5 and 40 increase potency. 48 S. A. Whitehead and S. Rice

Table 1. Classification of some of the more common phyto-oestrogens and their common dietary sources.

Flavones Flavonones Isoflavones Lignansa Mycotoxins Narigenin Coumestrol Enterolactone Flavonone Enterodiol 7-Hydroxyflavone Mostly red/yellow Citrus fruit Legumes, Bean shoots, Most cereals, Mould on fruits and particularly alfalfa, sun- fruits and bread and vegetables soybean and flower seeds, vegetables plants clover spinach

a Enterolactone and enterodiol are formed from the plant secoisolariciresinol and matairesinol by intestinal bacteria.

Isoflavones

In a survey of more than 30 isoflavones, daidzein, genistein, biochanin A and formononetin inhibited the activity of 3b-HSD purified from bovine adrenal microsomes, and the IC50 values for converting pregnenolone to progesterone ranged 4 from 0.5 to 3.7 mM. Interestingly, oestradiol also inhibited this enzyme with an IC50 of 0.9 mM. The same isoflavones were also shown to inhibit 3b-HSD type II in mitochondrial and microsomal preparations of the human adrenocortical H295R cell 5 line with estimated IC50 values between 1.3 and 2.7 mM , and subsequently a similar inhibition of the conversion of DHEA to androstenedione by these isoflavones was observed in total membrane fractions of Sf9 insect cells in which human 3b-HSD had 6 been over-expressed. In this study, IC50 values ranged from 0.25 to 3.18 mM, which compare with an IC50 value of 7.6 nM for cyanoketone, a known inhibitor of 3b-HSD. LeBail et al7 also reported that genistein, daidzein and biochanin A inhibited the activity of 3b-HSD type 1 in human placental microsomes, with IC50 values between 2.9 and 10 mM. In contrast, Mesiano et al8 showed that genistein and daidzein specifically inhibited the activity of 21-hydroxylase (P450c21/CYP 21) in H295 cells but had no effect on other steroidogenic enzymes, including 3b-HSD. A few studies have been carried out on intact cell preparations. An early study on isolated bovine granulosa cells reported biphasic effects of genistein and biochanin A on progesterone synthesis.9 At low doses (185 and 3.5 nM, respectively) progesterone synthesis was stimulated by 40–50%, whilst inhibition was observed at higher doses of 1.85 and 0.176 mmol. Similar effects were seen with oestradiol but at doses ten times lower. In a study of human granulosa luteal (GL) cells, biochanin A was the only phyto- oestrogen to show a dose-dependent inhibition of the conversion of pregnenolone to progesterone, with 50% inhibition occurring around 10 mM. Genistein and daidzein inhibited 3b-HSD activity only at dosesR10 mM10, as was also observed in rat granulosa cells.11 Inhibitory effects of isoflavones on 17b-HSDs have also been reported. In both purified preparations of 17b-HSD type 1 and wild-type T-47D breast cancer cells, the conversion of oestrone to oestradiol was significantly inhibited by 1.2 mM genistein but not by biochanin A.12 Le Bail et al7 also showed that genistein, daidzein and biochanin A inhibited 17b-HSD type 1 in human placental microsomes with IC50 values in the range Endocrine-disrupting chemicals and sex steroid synthesis 49

1–10 mM, and in human GL cells genistein inhibited 17b-HSD type 1 but only at high doses (R10 mM), whilst biochanin A had no effect.13,14 In contrast, biochanin A was shown to be a relatively potent inhibitor of recombinant 17b-HSD type 5 in both its oxidative and reductive actions.15 Finally, isoflavones have been reported to inhibit both sulphotransferases and sulphatases in liver microsomes and cytosol preparations.16,17 In general, experiments have shown that isoflavones are weak inhibitors of aromatase in both cell-free preparations7 and whole-cell preparations.10 Some recent studies, however, provide interesting but conflicting outcomes. Almstrup et al developed an assay that simultaneously detected oestrogenicity and aromatase activity in MCF-7 cells, and they showed that the isoflavones formononetin, biochanin A and an extract of red clover flowers, but not genistein (see Figure 3), inhibited aromatase activity at low doses (!1 mM) but had oestrogenic activity at higher doses.18 In contrast, other studies have reported an increase of aromatase activity by genistein in the H295R cell line19 and in K K isolated rat immature follicles20 at doses between 10 7 and 10 5 M. The former observation was correlated with increased promoter-specific aromatase transcripts. An increase in 17b-HSD type 2 activity by genistein has also been reported.21 Hormone- dependent MCF-7 and hormone-independent MBA-MB-231 breast cancer cell lines exposed to 100 nM genistein for 5 days increased the oxidation of oestradiol to oestrone, and Western blots confirmed an increased expression of this enzyme. Overall evidence shows that isoflavones have weak or negligible inhibitory activity on aromatase but are more potent inhibitors of 3b-HSD and 17b-HSD. Recent studies showing that genistein may stimulate aromatase and 17b-HSD type 2 activity requires further investigation.

U-shaped dose-response from 160

140

120

100 Anast + T 80 Bioch A 60 BiochA + T Gen 40 Gen + T 20 Red Clov % induction (100% set to 100nMT) Red Clov + T 0 0.1nM 1nM 10nM 100nM 1µM10µM Concentration

Figure 3. U-shaped dose-response curves of MCF-7 cells incubated for 24 hours with the following compounds: anastrazole (Anast), biochanin A (Bioch A), genistein (Gen), and extracts of red clover (Red Clov) with or without 100 nM testosterone (T). Values represent the expression levels of the oestrogen- induced pS2 mRNA and are shown as a percentage relative to the response to 100 nM T set at 100%. Error bars represent SD of four cell culture replicates. 50 S. A. Whitehead and S. Rice

Flavones and flavonones

High doses (R10 mM) of flavonoids such as apigenin and quercetin are required to inhibit 3b-HSD5,6,13 or have no detectable effects13,22, although 6-hydroxyflavone was 5 reported to inhibit this enzyme in H295R cells with an IC50 of 1.3 mM. One recent study investigated the effects of 15 flavonoids on the reduction of progesterone to 20a- hydroxyprogesterone in the cytosolic fraction of rat hepatocytes23, a reaction catalysed by 20a-HSD. The flavones and flavonones—including apigenin, luteolin, kaemferol and quercetin—were the most potent phyto-oestrogens in this respect, with IC50 values in the range of 8–30 mM. Isoflavones had negligible activity. Flavones and flavonones appear to be more potent in inhibiting the family of 17b- HSDs than 3b-HSD. Apigenin, but not quercetin, significantly inhibited 17b-HSD type 1 in breast cancer cells and a purified enzyme preparation at a dose of 1.2 mM12, and similar observations were made with chrysin, apigenin and narigenin on placental microsomes.7 In human granulosa luteal cells, apigenin inhibited 17b-HSD type 1, but only at micromolar doses, whilst quercetin is without effect.13 In contrast, quercetin was a relatively potent inhibitor of recombinant 17b-HSD type 515 and also of oestrone sulphatase in human liver microsomes.17 Compared with isoflavones, flavones are relatively potent inhibitors of aromatase. Early studies by Kellis and Vickery24 showed that apigenin and quercetin were the most potent inhibitors of aromatase in human placental microsomes, and subsequently Le Bail et al22 reported that apigenin as well as chrysin and 7-hydroxyflavone were the most potent inhibitors of aromatase, with over 60% inhibition observed at doses between 1 and 10 mM. These flavones inhibited aromatase with IC50 values in the range 0.3–3.0 mM, whilst the isoflavones investigated were inactive.7 In another study, 28 flavonoids were screened for their effects on purified aromatase prepared from human placenta. Over 50% of the compounds significantly inhibited aromatase activity, and whilst apigenin, chrysin and were the most potent, their IC50 values were still in the order of 1 mM.25 More recently Sanderson et al19 reported that in H295R cells flavones were consistently more potent inhibitors than flavonones, with IC50 values for 7-hydroxy- flavone, chrysin and apigenin being 4, 7 and 20 mM, respectively. This compared with the aromatase inhibitor 4-hydroxyandrostenedione that had an IC50 of 20 nM in this model. In human GL cells apigenin, but not quercetin, significantly and dose-dependently inhibited aromatase. Significant inhibition occurred at 0.1 mM, but only when testosterone was the substrate. Higher doses of apigenin were required to inhibit the conversion of androstenedione to oestradiol10, and this may reflect the higher affinity of aromatase for androstenedione compared with testosterone.26

Lignans

The lignans enterodiol and enterolactone exert weak inhibitory effects on aromatase. In human placental microsomes and human pre-adipocytes, enterolactone and enterodiol inhibited aromatase at 10–100 mM.27,28 This inhibition was competitive with androgens, and the Ki value for enterolactone was 14.4 mM.28 Similar weak inhibitory effects of these lignans on aromatase activity were observed in human GL cells.10 Saarinen et al further showed that aromatase inhibition by enterolactone was associated with a reduction in anthracene-induced mammary carcinomas in rats when serum concentrations were maintained at 0.4 mM by oral administration of 10 mg/kg.29 Endocrine-disrupting chemicals and sex steroid synthesis 51

Coumestans

Few studies have investigated the effects of coumestans on enzyme activity. Coumestrol significantly inhibited the activity of 17b-HSD type 1 in purified enzyme preparations at doses in the micromolar range12,23, and inhibition of recombinant 17b-HSD type 5 by this phyto-oestrogen has also been reported.15 Coumestrol also inhibited aromatase in human pre-adipocytes with a Ki value of 1.3 mM.28 This compared with a Ki value of 0.5 mM for aminoglutethimide, a pharmaceutical aromatase inhibitor.

Mycotoxins

Overall these have been the least investigated phyto-oestrogens despite the fact that they have been shown to exert potent effects. Krazeisen et al showed the zearalenone was the most potent phyto-oestrogen in inhibiting the activity of both the reductive and oxidative activity of 17b-HSD type 5.15 In human GL cells, it was a potent aromatase K inhibitor with significant inhibition observed at 10 7 M, but only after the cells had been exposed to the compound for 48 hours.10 In the same cells, zearalenone had no effect on 3b-or17b-HSD activities, and Western blots did not reveal any significant reduction in expression of either aromatase or 3b-HSD.10 In contrast, two derivatives of zearalenone, a- and b-zearalenol, inhibited both the forskolin- and FSH-induced increase in progesterone synthesis in cultured porcine granulosa cells at doses between 15 and 30 mM. These derivatives also inhibited RNA transcripts of both P450scc and 3b- 30 HSD, protein expression of P450scc, and activity of 3b-HSD.

Structure/function relationships of phyto-oestrogens as inhibitors of enzyme activity

Overall evidence shows that flavones are more potent and specific inhibitors of aromatase, whilst the isoflavones that have a hydroxyphenol group on C3 instead of C2 preferentially inhibit 3b- and 17b-HSDs (Figure 2). Comparative studies on the potency of different isoflavones has revealed that hydroxylation on C7 of the A ring is important for anti-HSD activity, and that compounds hydroxylated at both positions 5 and 7 on the A ring and position 40 on the B ring were the most potent inhibitors of 17b-HSD type 1.7,31 Krazeisen et al further confirmed the importance of these hydroxylations and showed that the number of hydroxylations was positively correlated with the inhibitory potency of the phyto-oestrogens on the activity of 17b-HSD type 5.5 Some studies have shown that the isoflavones act as competitive inhibitors of 3b- K HSD, with Ki values in the range of 10 7 M or less.6,32 These inhibitors have a common structural feature in the hydroxyl group at C7, and it was subsequently shown that the steric conformation between the deflection of the B ring and the C7 hydroxy group on the A ring is similar to the 3b-hydroxy group on the A ring and deflection of the CD rings of steroid substrates such as DHEA and progesterone.32 An explanation as to why flavones are more potent aromatase inhibitors than isoflavones was provided by studies with wild-type- and mutant-expressing Chinese hamster ovary cells together with computer modelling. In this site-directed mutagenesis study, Kao et al showed that flavonoids bind to the active site of aromatase in an orientation in which rings A and C of the phyto-oestrogens mimic rings D and C of the androgen substrates.33 The molecular basis for isoflavones such as genistein and daidzein being weaker inhibitors of aromatase than flavonones is 52 S. A. Whitehead and S. Rice because the 40-hydroxyphenyl group at C-3 greatly reduces the ability of these compounds to bind with aromatase. Interestingly, the reverse is true of the ability of isoflavones to bind with the oestrogen receptor. Some isoflavones have been found to bind 5–10 times more strongly with the oestrogen receptor compared with flavones34, and this is due not only to the ability of their A and C rings to mimic the A and B rings of oestrogen but also that the 40-hydroxy group is required for effective ER binding (Figure 2).23 With the exception of the findings of Almstrup and colleagues18, inhibition of enzyme activity is typically observed in the micromolar range. At concentrations in the range 50–100 mM, phyto-oestrogens can exert toxic effects on cultured cells, and this necessitates careful monitoring of cell viability—a parameter that is often lacking in many of these studies.

XENO-OESTROGENS AND STEROIDOGENIC ENZYME ACTIVITY

There is a huge range of xeno-oestrogens in the environment (Table 2) that have the ability to bind, albeit weakly, to steroid receptors35, and their chemical structures are diverse. Some xeno-oestrogens such as p,p0-DDE and vincolzolin have also been shown to possess affinity for the androgen receptor.36 Compared with the phyto-oestrogens, there have been relatively few studies examining the direct action of xeno-oestrogens on steroidogenic enzyme activity in vitro, even though in vivo studies suggest they are more potent endocrine disrupters than phyto-oestrogens in relation to sexual differentiation and development.37 Furthermore, to date most attention has been directed towards the effects of these xenobiotics on aromatase.

Pesticides

1,1-Dichloro-2,2-bis(p-chlorophenyl)ethylene (p,p0-DDE) is a stable metabolite of the pesticide DDT; despite its use being banned in many countries, it is a persistent environmental pollutant and is found in human serum. At a dose of 100 ng/mL, p,p0- DDE significantly stimulated aromatase activity in human granulosa luteal cells and had synergistic effects with FSH on enzyme activity.38 Yo u e t a l 39 failed to show any

Table 2. Common agricultural and industrial chemicals that may be important modulators of hormone synthesis.

Pesticides Herbicides Fungicides Plasticizers/ Other industrial surfactants chemicals p,p0-DDT Vinclozolin Polychlorinated biphenyls (PCBs) p,p0-DDE Simazine Prochloraz Propazine Imizalil Octylphenols Alachlor Bifonazole Di- and tri-butyltin Miconazole Clotrimazol

Use of many chemicals, including DDT and PCBs, are banned in many countries. Endocrine-disrupting chemicals and sex steroid synthesis 53 induction of aromatase expression in cultured rat hepatocytes exposed to p,p0-DDE for 24 hours, although after oral treatment with this pesticide (100 mg/kg/day) induction of hepatic aromatase was observed by immunoblot and immunohistochemistry. In contrast, DDT and several metabolites inhibited aromatase activity in H295R adrenocortical carcinoma cells after 24 hours exposure, although p,p0-DDE was without effect. However, this inhibition was only observed at cyctotoxic concen- trations.40 Inhibition of aromatase by pp0-DDE in human placental, prostate and liver 41 tissue was also observed, with IC50 values around 10 mM. In countries where the use of DDT was prohibited it was replaced by its methoxylated analogue, methoxychlor. Chedrese and Feyles42 compared the activity of p,p0-DDE and methoxychlor on steroidogenesis in porcine granulosa cells and Chinese hamster ovary (CHO) cells stably transfected with the FSH receptor. Both p,p0-DDE and methoxychlor (10 mM) inhibited progesterone synthesis and, together with the results from experiments on the CHO cells, suggested that DDE inhibited the generation of cAMP, whilst the action of methoxychlor was distal to this part of the signalling pathway. In rat granulosa cells, 2,40-DDT stimulated progesterone synthesis at K K doses between 10 7 and 10 5 M.11 Organotin compounds are ubiquitous environmental contaminants and widely used in industry, agriculture and anti-fouling paints. They have been linked to imposex in molluscs and inhibition of CYP activities—including aromatase—in fish, but only at high and unrealistic micromolar ranges.43 In H295R cells dibutyltin (DTB), tributyltin (TBT) and triphenyltin (TPT) dose-dependently inhibited aromatase activity. TBT was the most potent inhibitor, but at the concentration at which enzyme inhibition wasR20% of control (300 nM), significant reductions in cell viability, proportional to the percentage aromatase inhibition, were observed.40 This inhibition occurred only after 24 hours exposure without any direct inhibition during a catalytic assay. In placental microsomes and JEG-3 cells, both DTB and TBT inhibited both aromatase and 5a- 41 reductase with IC50 values between 5 and 17 mM , and in porcine Leydig cells both TBT and TPT suppressed cAMP-and gonadotrophin-stimulated testosterone synthesis; this was due to an inhibition of 17b-HSD (IC50Z2.6 mM for TPT) and weaker inhibition of 17a-hydroxylase/C17-20 lyase.6 Lindane, the g-isomer of hexachlorocyclohexane (HCH), is one of the oldest synthetic pesticides still in use worldwide, and has also been linked with reproductive dysfunction. Lindane and other HCH isomers dose-dependently inhibited progesterone synthesis in the MA-10 mouse Leydig cell tumour line, but this was not paralleled by a reduction in steroidogenic enzyme expression.44 In contrast, these HCHs dramatically reduced dibutyrl cAMP-stimulated StAR (steroidogenic acute regulatory protein) expression. This protein mediates the rate-limiting and acutely regulated step of transferring cholesterol from the outer to inner mitochondrial membrane where P450scc initiates steroid synthesis. Lindane also inhibited aromatase activity in human placental JEG-3 cells and transfected human embryonal kidney 293 cells40, although this effect was only observed after long incubation times (18 hours). It was not associated with changes in CYP19 mRNA levels or cellular toxicity, despite the very high doses used (25–100 mM). Paradoxically, short-term exposure resulted in increased aromatase activity. An induction of aromatase activity in H259R cells was seen with vinclozolin, with a parallel increase in CYP19 mRNA levels and increased cAMP concentrations.40 Evidence suggested that this effect was mediated through inhibition of phosphodiester- ase activity. 54 S. A. Whitehead and S. Rice

Herbicides

The chloro-s-triazine herbicides, such as atrazine and propazine, were reported to induce aromatase expression, and the degree of induction was greater in H295R (adrenocortical carcinoma) cells compared with JEG-3 (placental choriocarcinoma), the latter having a much higher basal aromatase expression.45 This effect did not appear to be oestrogen-receptor-mediated since they did not induce vitellogenin production in primary cultures of male carp hepatocytes. Like the fungicide vinclozolin, 30 mM atrazine increased cAMP levels, possibly by inhibiting phosphodiesterase.

Fungicides

The anti-fungal properties of various azoles that are used widely in agriculture and medicine are due to their ability to inhibit ergosterol biosynthesis (an essential sterol component in membranes of fungi and yeast) by inhibiting the CYP enzyme sterol 14a- demethylase. Thus, it is not surprising to find that they also inhibit other CYP enzymes, including aromatase.46 Trotsken et al47 compared the activity of 22 azole compounds. The potencies of 13 fungicides used in agriculture and seven used in medicine ranged more than 700-fold and 7000-fold, respectively, with IC50 values starting from 0.047 and 0.019 mM. The most potent medicinal anti-fungals were bifonazole, miconazole and clotrimazole, whilst several studies have shown the agricultural fungicides prochloraz and imizalil to be potent inhibitors of aromatase.40,47,48 Other therapeutic azole drugs targeted for the treatment of breast cancer are anastrozole, fadrozole and letrozole, and in the rat ovary microsome they inhibited aromatase with IC50 values of 25, 7 and 7 nM, respectively.49 In contrast, the anti-androgenic fungicide vinclozolin induced aromatase mRNA and also increased cAMP levels intracellularly in H295R cells, but these effects were only observed at 10–100 mM.40

Plasticizers and surfactants

Bisphenol A (BPA) is one of many diphenylalkanes that are raw materials for the production of polymers; they are used commercially as plastics and coatings in the dental and food industry. BPA binds weakly to the oestrogen receptor (1000-fold less than 17b-oestradiol) and also has anti-androgenic activity.36 Short-term exposure (10 minutes) of JEG-3 cells to 25 mM BPA stimulated aromatase activity (inhibition at 100 mM), whilst longer exposure times (18 hours) inhibited aromatase activity without affecting CYP19 mRNA levels.50 Akingbemi et al51 recently showed that perinatal and chronic postnatal exposure to BPA reduced both basal and LH-stimulated testosterone synthesis in cultured Leydig cells, but when Leydig cells were cultured with BPA at doses ranging from 0.01 to 1000 nM, inhibition was observed only at the lowest dose, all other doses being the same as the control. No effects of BPA or other diphenylalkanes on aromatase activity in H295R cells were observed, even at concentrations as high as 100 mM. Phthalates are widely used in plastic products, and metabolites of phthalates can be measured in human urine. The female reproductive toxicity of di-(2-ethylhexyl) (DEHP) and its active metabolite mono-(2-ethylhexyl)phthalate (MEHP) has been attributed to its ability to suppress ovarian granulosa cell oestradiol production. Amongst several structurally related phthalates, MEHP was unique in inhibiting Endocrine-disrupting chemicals and sex steroid synthesis 55 oestradiol production in rat granulosa cells. This was paralleled by a reduction in aromatase mRNA and protein expression.52 4-tert-Octylphenol is a weakly oestrogenic alkylphenol used widely in surfactants and as a plastic additive. In rat granulosa cells, it induced a dose-dependent stimulation of progesterone secretion11, whilst a more recent study reported a dose-dependent decrease of oestradiol and testosterone secretion in FSH-maintained isolated immature rat ovarian follicles.20 This effect was not due to reduced aromatase activity, and results indicated that this was due to the inhibitory action of 4-tert-octylphenol on cAMP production.

EXPERIMENTAL DOSAGE VERSUS DIET OR NATURAL EXPOSURE—A THREAT TO HUMAN HEALTH?

The above discussion has shown that a variety of phyto-oestrogens and xeno- oestrogens can inhibit the activity of several key steroidogenic enzymes, although in a few cases such endocrine disrupters have been reported to increase activity. Either way this may be pertinent to the effects of exposure to endocrine disrupters on abnormal sexual differentiation and development. But an important question to ask is whether or not such in vitro effects and the doses of endocrine disrupters at which such effects are observed are relevant or applicable to in vivo human and animal exposure. Whilst relatively little is known about the absorption, metabolism and bioavailability of various phyto-oestrogens, they do not accumulate in tissues and are relatively rapidly excreted. In addition, many circulate in conjugated forms (e.g. as sulphates or glucuronides) and a smaller fraction in the free form. Mean plasma concentrations of phyto-oestrogens can range from 100 nmol/L to 1 mmol/L, but such high concen- trations are only measured in association with vegetarian diets or those rich in soy products.53 These are concentrations at which phyto-oestrogens have been shown to have oestrogenic effects.54 In this respect, soy supplements are indicated as a natural alternative to hormone replacement therapy. Paradoxically, epidemiological studies have shown that high-soy diets are also linked with a reduced incidence of hormone- dependent breast and prostate cancer.55 This could be due to an inhibition of the peripheral conversion of steroid precursors to active androgens and oestrogens. For example, substrates for oestradiol synthesis in breast tissue include oestrone, oestrone sulphate and androstenedione56, so that inhibition of 17b-HSD, sulphatase or aromatase could have important consequences in inhibiting oestradiol production and hence promotion of oestrogen-dependent breast cancer. The studies quoted above generally show that significant enzyme inhibition occurs in the micromolar range, above the circulating concentrations of unconjugated phyto- oestrogens57 even with a vegetarian or high-soy diet. There is, however, evidence that genistein, at least, can react with oxidants, forming brominated, chlorinated and/or nitrated genistein that may substantially increase the biological activity of the parent compound.57 Thus, there is a need to investigate the metabolism of phyto-oestrogens at tissue level and the effects of metabolites on enzyme activity. With regard to environmental exposure to xeno-oestrogens, these are typically associated with a disruption in sexual development both in humans and animals.37 Like phyto-oestrogens, the concentrations at which enzyme inhibition is observed is either in the cytotoxic range or far exceeds serum concentrations. For example p,p0-DDE has been measured in various biological fluids—including serum, breast milk, and follicular and seminal fluid—and reported concentrations, when detectable, are in the range of 56 S. A. Whitehead and S. Rice about 0.1–1 ng/mL.58 Similarly TPT concentrations in human serum ranged between 0.49 and 1.92 nmol cation/L, and yet inhibition of steroidogenic enzyme activity in vitro was in the micromolar range.59 Phthalate esters can be detected in serum, and levels of DEHP between approximately 200 and 2000 ng/mL and MEHP around 20 ng/mL have been measured in young Puerto Rican girls with premature thelarche.60 A more recent study showed that the geometric mean of urinary phthalate esters in American men ranged from 183 to 4.5 ng/mL.61 Bisphenol A has also been measured in human serum with concentrations in the range of !5 ng/mL, and there is evidence that this xeno- oestrogen accumulates in amniotic fluid in early pregnancy.62 Taken together, the data show that circulating concentrations of xeno-oestrogens are generally in the nanomolar range, but their effects on steroidogenesis in vitro are in the micromolar range. One may well ask: do serum concentrations reflect their accumulation and/or their metabolites in tissues? There are clearly many issues to be addressed.

MECHANISM OF ACTION

It has generally been assumed that phyto-oestrogens inhibit steroidogenic enzymes by competitive inhibition with natural substrates for a particular enzyme, and certainly some kinetic studies provide proof of this. There is also evidence that they may inhibit the generation of cAMP, the classic second messenger in the pathway for regulating aromatase expression and hence activity. This is of interest in view of the fact that both genistein and apigenin are inhibitors of cell signalling pathways and that steroidogenesis can be regulated by other signalling pathways apart from the cAMP/PKA pathway. These include the MEK/ERK 1/2 and phosphatylinositol kinase (PI 3-K)/AKT pathways that may be activated by gonadotrophin-activated cAMP and/or growth factors that also regulate steroidogenesis.63 Genistein and apigenin are inhibitors of protein tyrosine kinase and PI 3-K, respectively.64,65 Tyrosine kinase inhibitors inhibit steroidogenesis in rat granulosa cells66, and recent experiments in our laboratory have shown that inhibitors of both pathways inhibit aromatase activity, and that these effects are potentiated by genistein and apigenin but not quercetin or zearalenone (Figure 4). In contrast, coumestrol and some pesticides and plasticizers have been shown to induce a

800 1200 b 700 a 1000 600 a a 800 500 b 400 b c 600 b b b 300 c c 400 200 c b Oestradiol (pmol/L) c Oestradiol (pmol/L) 200 100 c c d c 0 * 0 Con10–5LY A A+LYZ Z+LYG G+LY Con PD A A+PDZ Z+PDG G+PD FSH FSH

Figure 4. Inhibition of FSH-induced aromatase activity by LY294002 (PI3-kinase inhibitor) and PD98059 (MEK/ERK1/2 inhibitor) in human granulosa luteal cells and the potentiation of this inhibition by genistein and apigenin, but not the mycotoxin zearalenone. The conversion of testosterone to oestradiol was measured over a 4 hours period after cells had been exposed to drugs for 48 hours. Endocrine-disrupting chemicals and sex steroid synthesis 57 rapid phosphorylation of ERK 1/2 in a pituitary tumour cell line.67 Along the same theme, endocrine disrupters may bind with intracellular receptors that control the expression of various enzymes. For example, the plasticizer MEHP may inhibit aromatase expression by activating peroxisome proliferator-activated receptors (PPARs).68 Thus, the action of endocrine disrupters on steroidogenic enzyme activity may involve complex intracellular signalling pathways apart from their ability to bind competitively with steroidogenic enzymes.

CONCLUSIONS

The potency of different EDCs in their ability to modulate enzyme activity varies according to the different experimental model, and this may reflect the different levels of enzyme concentration/activity in a particular preparation. One comparison would be between aromatase activity in human GL cells and that in MCF-7 cells. Human GL cells can convert approximately the same the amount of androstenedione to oestradiol in 4 hours as the same concentration of MCF-7 cells can in 48 hours (Whitehead and Lacey, unpublished observations). Further consideration must be made regarding the long-term ‘chronic’ effects of exposure to EDCs compared with the acute effects of these chemicals on enzyme activity. In this respect, human GL cells exposed to phyto- oestrogens for 48 hours showed a marked reduction in aromatase mRNA, quantified by real-time reverse transcriptase polymerase chain reaction (RT-PCR), prior to any measurable change in enzyme activity.69 Thus, whilst phyto-oestrogens and xeno- oestrogens may bind to steroidogenic enzymes they may also influence their expression by modulating cell signalling pathways. Further studies are required to investigate how EDCs modulate enzyme activity, and comparisons should be made between their acute effects on enzyme activity and the longer-term chronic effects that could alter steroidogenic enzyme expression.

SUMMARY

Experimental evidence shows that phyto-oestrogens and xeno-oestrogens generally inhibit key steroidogenic enzymes, although the literature is not always consistent. These enzymes include 3b-and17b-HSDs, aromatase, sulphatases and sulphotransferases. Kinetic studies have shown that some phyto-oestrogens are competitive inhibitors of enzyme activity, and structure/activity studies have elucidated why, for example, flavones are more potent aromatase inhibitors than isoflavones, whilst the latter are more potent inhibitors of 3b-and17b-HSDs. There is also evidence that both phyto- oestrogens and xeno-oestrogens can modulate intracellular signalling pathways through which they could inhibit the expression and subsequently activity of steroidogenic enzymes. Although the concentrations at which such experimental effects are observed are usually above those that have been measured in body fluids, our lack of information about tissue accumulation and metabolism into more active compounds cannot be ignored. Thus, like many other issues and questions surrounding the potential adverse effects of EDCs, the ability of these compounds to modulate enzyme activity could have important consequences for sexual differentiation and development and in the protection (or promotion) of hormone-dependent cancers. 58 S. A. Whitehead and S. Rice

Research agenda

Equating the results of controlled laboratory investigations with ‘natural’ in vivo exposure to EDCs is fraught with problems and frequently leads to misinterpretation of the data. There continues to be a need to address the following questions:

† do effective experimental doses of EDCs translate to concentrations that cells and tissues are exposed to and accumulate in vivo through normal environmental exposure and dietary intake? † how do different species metabolise ingested or absorbed EDCs, and can in vitro experiments reflect such metabolism? † can laboratory experiments ever investigate the broad cocktail of EDCs to which cells and tissues are potentially exposed in vivo?

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