Alkylphenol endocrine disrupters inhibit IP3-sensitive Ca2+ channels

BBRC Biochemical and Biophysical Research Communications 310 (2003) 261–266 www.elsevier.com/locate/ybbrc

Alkylphenol endocrine disrupters inhibit IP3-sensitive Ca2+ channels Shahla Zafar Khan, Christopher J. Kirk, and Francesco Michelangeli* School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK Received 1 September 2003

Abstract We have investigated the influence of alkylphenol endocrine disrupters and the synthetic estrogen diethylstilbestrol (DES) on inositol-1,4,5-trisphosphate (IP3 )-sensitive Ca2þ channels from porcine cerebellum and rat testicular membranes. All alkylphenols and DES inhibited the extent of IP3 -induced Ca2þ release (IICR) from both cerebellar and testicular microsomes. 4-n-nonylphenol was the most potent compound tested (IC50 , 8 lM). Inhibition of IICR was directly related to the length and hydrophobicity of the alkylphenol side chain. None of the alkylphenols or DES appeared to influence the concentration dependence of IICR nor did they have a significant effect on [3 H]IP3 binding to the membranes. An investigation of the effects of nonylphenol on the transient kinetics of IICR showed that it inhibited the rate constants for both the fast and the slow phases of IICR and also the extent of Ca2þ release. These results illustrate another mechanism by which these environmental pollutants can disrupt endocrine function without the involvement of estrogen receptors. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Endocrine disrupters; Alkylphenol; Nonylphenol; Diethylstilbestrol; IP3 -sensitive Ca2þ channels; IP3 receptors; Ca2þ homeostasis

A number of man-made pollutants present in the environment are capable of acting as “endocrine disrupters” (EDs), disturbing normal endocrine function in many species [1,2]. Examples include insecticides, herbicides, alkylphenols, and dioxins [2] which, being lipophilic, can bioaccumulate in living organisms [3–5]. Many EDs are estrogenic and exposure to these compounds is thought to be responsible for sexual dysfunction in fish [1], birds [6,7], reptiles [8], and mammals [9], possibly including humans [10]. Many EDs will bind to one of the two known classes of nuclear estrogen receptors [11] but it is not clear that this is their major or only mode of action. For example, considerable disparity between the biological potency and the binding affinity of EDs has been noted in some systems [12] and evidence against direct receptor involvement has been reported from experiments using receptor binding and reporter gene assays [13]. It has recently become clear that several other potential targets within cells may be directly affected by low concentrations of these pollutants without the involvement of * Corresponding author. Fax: +44-121-414-5925. E-mail address: [email protected] (F. Michelangeli).

0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2003.09.017

estrogen receptors. These targets include enzymes involved in the metabolism of endogenous steroid hormones including sulphotransferases [14,15] and cytochromes P450 [16,17], and Ca2þ signalling mechanisms [15,18,19,21]. Intracellular Ca2þ levels are precisely controlled by the interplay of a variety of Ca2þ pumps and Ca2þ channels [20]. If the activity of any of these Ca2þ transporters is impaired this may have drastic consequences for many cellular processes including: muscle contraction, neuronal function, hormonal regulation, fertilisation, growth, and development [20]. We have already demonstrated that some alkylphenols can inhibit the activity of the SERCA type Ca2þ pumps derived from muscle, neuronal, and testicular cells [15,18,19,21]. In addition, another study has shown that the muscle ryanodine receptor Ca2þ channel is activated by sub-lM concentrations of nonylphenol [22]. Elevation of intracellular [Ca2þ ] in non-muscle cells is typically caused by hormone/neurotransmitter stimulation of the phosphoinositide signalling pathway leading to the production of the second messenger inositol 1,4,5trisphosphate (IP3 ) and the activation of IP3 -sensitive Ca2þ channels located on the endoplasmic reticulum

262

S.Z. Khan et al. / Biochemical and Biophysical Research Communications 310 (2003) 261–266

membrane [20]. Here we investigate the effects of a number of alkylphenols and the synthetic estrogen diethylstilbestrol (DES) on this Ca2þ channel.

Materials and methods Fluo-3 and ATP were obtained from Sigma. Alkylphenols (minimum purity 96%) were purchased from the Aldrich Chemical Company with the exception of 4-n-amylphenol (4-n-pentylphenol), 4-n-butylphenol, and 4-n-nonylphenol (Lancaster Synthesis, UK). IP3 was purchased from Alexis Corporation (UK) and [3 H]IP3 was obtained from Amersham Life Science. The alkylphenols were dissolved in dimethyl sulphoxide (DMSO). The concentration of DMSO was always 61% (v/v) and was shown to have no effect on the parameters studied in control experiments. Porcine cerebellar microsomes and rat testis microsomes were prepared as described by Tovey et al. [23]. Ca2þ uptake and release from cerebellar and testis microsomes was measured by following the change in fluorescence of the Ca2þ indicator dye fluo-3 as described by Michelangeli and co-workers [23–25]. Fluorescence changes were monitored in a Perkin–Elmer LS50B spectrofluorimeter, using excitation and emission wavelengths of 506 nm and 526 nm, respectively. Ca2þ concentrations were determined using the following equation: ½Ca2þ
¼ Kd ððF  Fmin Þ=ðFmax  F ÞÞ; where Kd is the dissociation constant for Ca2þ binding to fluo-3 (900 nM at 37 °C, pH 7.2, in 100 mM Kþ ), F is the fluorescence intensity of the sample, and Fmin and Fmax are the fluorescence intensities in the presence of 1.25 mM EGTA and 2 mM CaCl2 , respectively. Microsomal membranes (0.75–1.5 mg) were added to a stirred cuvette containing 2 ml of 40 mM Tris/phosphate, 100 mM KCl (pH 7.2) in the presence of 1.25 lM fluo-3, 10 lg/ml creatine kinase, and 10 mM phosphocreatine. Ca2þ uptake was then initiated by the addition of 1.5 mM Mg-ATP. Following sufficient Ca2þ accumulation, further uptake was halted by the addition of 0.25 mM orthovanadate which inhibits >90% of Ca2þ pump activity. After the fluorescence intensity had reached a plateau, indicating an equilibrium between Ca2þ efflux and influx, IP3 was added. Total Ca2þ accumulation was measured by the addition of 12.5 lg/ml Ca2þ ionophore (A23187). The extent of Ca2þ release induced by IP3 was expressed as a percentage or fraction of the Ca2þ mobilised by a saturating concentration of IP3 (20 lM). Rapid measurements of IICR from cerebellar microsomes were performed as described by Mezna and Michelangeli [24,25]. Microsomal membranes were loaded with Ca2þ and their Ca2þ pumps were inactivated by the addition of sodium orthovanadate (0.25 mM) before being introduced into the large syringe of a stopped-flow spectrofluorimeter (Applied Photophysics SX-17MV). IP3 (final concentration after mixing, 20 lM) was added from the smaller volume syringe (drive ratio 10:1; 2.5 ml:0.25 ml). The contents of the two syringes were rapidly mixed and changes in the fluorescence were recorded (monitored by exciting the reactants at 506 nm and detecting emission above 515 nm using a cut-off filter). The fluorescence data from the stoppedflow apparatus were compared with those of identical experiments undertaken in a conventional fluorimeter so that the traces could be used to determine fractional Ca2þ release. Typically the traces from between 8 and 10 experiments were averaged and the data were analysed by non-linear least-squares fitting (Fig. P, Biosoft). Data were then fitted to a biexponential equation: Ca2þ release ¼ A1 ð1  expk1t Þ þ A2 ð1  expk2t Þ; where A1 , A2 , k1, and k2 are the amplitudes and rate constants describing fractional Ca2þ release for the fast and slow components, respectively, and t is time (s). The goodness-of-fit of each data set using this equation was assessed by determining the v2 value, which was less

than 0.1 in all cases. The fluorescence changes, when related to Ca2þ concentration, were around the Kd value for Ca2þ binding to fluo-3: hence, the fluorescence change was linearly related to Ca2þ concentration [24,25]. The binding of [3 H]IP3 to microsomal membranes was measured as described in [23]. Approximately 0.3–0.6 mg microsomal membranes were suspended in 0.5 ml of binding buffer (50 mM Tris/HCl, pH 8.3, 1 mM EGTA, and 100 mM KCl) containing between 16 and 32 nCi/ml [3 H]IP3 . Specific binding was measured at 40 nM IP3 (which is around the Kd value for IP3 binding to brain microsomes under our experimental conditions). Non-specific binding was measured in the presence of excess (10 lM) non-radioactive IP3 . Following addition of microsomal membranes, the assay mixture was incubated on ice for 10 min. Bound [3 H]IP3 was separated from free [3 H]IP3 by centrifugation of the samples at 18,000g for 20 min at 4 °C. The resulting supernatant was removed and the pellet was washed with distilled water three times. After air-drying for approximately 1 h, the pellets were solubilised with 150 ll of solvable tissue solubiliser (Dupont) and incubated for 3 h at 37 °C. Ultima flow scintillant was added to each sample to give a final volume of 1 ml. The radioactivity present in each sample was then determined using liquid scintillation spectrometry, counting each sample for 10 min. Specific [3 H]IP3 binding was expressed as pmol [3 H]IP3 bound per mg of microsomal protein.

Results All straight chain alkylphenols tested inhibited the extent of IP3 -induced Ca2þ release (IICR) in cerebellar and testicular microsomes. Those with the longest side chains (i.e., 4-n-nonylphenol and 4-n-octylphenol) were particularly potent (see Table 1 and Figs. 1A and B). 1,1,3,3-Tetramethylphenol (tert-octylphenol), a highly branched alkylphenol (Table 1 and Fig. 1C), was also a potent inhibitor of IICR as was the synthetic estrogen DES (Table 1 and Fig. 1D). Table 1 lists the IC50 values for inhibition of IICR in both cerebellar and testicular microsomes for a variety of alkylphenols and reveals that the relative potencies of these compounds are generally similar in the two systems. Although the extent of IICR was reduced, the concentration dependence of Ca2þ release induced by IP3 was not greatly affected by the presence of 4-n-octylTable 1 Inhibition of IICR by alkylphenols Compound

Inhibition in cerebellum IC50 (lM)

Inhibition in testis IC50 (lM)

4-n-Nonylphenol 4-n-Octylphenol 4-n-Amylphenol 4-n-Butylphenol 4-n-Propylphenol 4-n-Ethylphenol 4-tert-Butylphenol 1,1,3,3-Tetramethylbutylphenol Diethylstilbestrol

83 41  5 64  6 120  8 220  15 670  60 112  21 18  5

27  8 39  6 48  6 ND 201  14 ND ND 27  7

19  3

21  2

ND, not determined. Each IC50 value was determined from inhibition curves where each point was repeated in triplicate.

S.Z. Khan et al. / Biochemical and Biophysical Research Communications 310 (2003) 261–266

B release (%)

120 100 80

120 100 80

2+

60

IP3-induced Ca

IP3-induced Ca

2+

release (%)

A

40 20

60 40 20 0

0 0

5

10

15

0

20

25

75

100

125

release (%)

D

100 80

120 100 80

2+

release (%)

120

IP3-induced Ca

2+

IP3-induced Ca

50

[Octylphenol] (

[Nonylphenol] (

C

263

60 40 20

60 40 20 0

0 0

10

20

30

40

50

60

[Tetramethylbutylphenol] (

0

10

20

30

40

50

60

[Diethylstilbestrol] (

Fig. 1. The effects of alkylphenols and diethylstilbestrol on the extent of IICR from porcine cerebellar microsomes. The graphs show the effects of: (A) 4-n-nonylphenol; (B) 4-n-octylphenol; (C) 1,1,3,3-tetramethylbutylphenol; and (D) diethylstilbestrol (DES); on Ca2þ release induced by 20 lM IP3 . The extent of release is expressed as a percentage of that released by the saturating dose of IP3 (20 lM) alone. Each data point is the mean  SD of between 3 and 6 determinations.

phenol (Fig. 2A). In the absence of octylphenol, the concentration of IP3 required to induce half-maximal Ca2þ release (EC50 ) in this preparation of cerebellar microsomes was 0.25  0.05 lM and maximal IICR occurred in the presence of 3–20 lM IP3 . In the presence of 50 lM octylphenol, maximal IICR was reduced by more than 50%, but the EC50 value for IP3 was not greatly affected (0.37  0.09 lM). Other alkylphenols also failed to influence the EC50 for IICR. Thus, in the presence of concentrations of 4-n-nonylphenol, 4-n-butylphenol, and 1,1,3,3-tetramethylbutylphenol which caused 50% inhibition of IICR, the concentrations of IP3 required to provoke half-maximal IICR were 0.30  0.1 lM, 0.25  0.04 lM, and 0.40  0.15 lM, respectively (results not shown). We have previously shown that the extent of IICR as a function of IP3 concentration is cooperative [30]. The Hill coefficient ðnHill Þ for this preparation of cerebellar microsomes was 1.25  0.06 and this was unchanged in the presence of either nonylphenol, octylphenol, butylphenol or tetramethylbutylphenol (nHill ¼ 1:18  0:04, 1.20  0.05, 1.17  0.20, and 1.10  0.06, respectively, results not shown).

To assess whether the inhibition of IICR by alkylphenols was due to inhibition of IP3 binding to the receptor, [3 H]IP3 binding studies with cerebellar microsomes were performed in the presence of octylphenol (Fig. 2B). Octylphenol at concentrations up to 125 lM (a concentration which almost completely inhibits IICR) had only a very small effect upon IP3 binding to its receptor. Table 2 shows that the other alkylphenols also had little effect upon [3 H]IP3 binding to either cerebellar or testis microsomes, even at concentrations which cause almost complete inhibition of IICR. Hence these results suggest that inhibition of IICR by these compounds is mediated by modulation of the channel opening process rather than by changes in ligand binding. As shown previously, the time course of IICR from cerebellar microsomes is biphasic and best fits the equation describing a biexponential process [24,25]. We have previously suggested that this reflects a heterogeneity within the IP3 -sensitive Ca2þ stores, with two (or more) types of store releasing their Ca2þ either rapidly (fast phase component) or more slowly (slow phase

264

S.Z. Khan et al. / Biochemical and Biophysical Research Communications 310 (2003) 261–266 Table 2 Effects of Alkylphenols on [3 H]IP3 binding Compound

(%)[3 H]IP3 binding (cerebellar microsomes)

(%) [3 H]IP3 binding (testis microsomes)

4-n-Nonylphenol 0 lM 100  8 50 lM 97  4 100 lM 94  4

100  4 72  7 81  10

4-n-Butylphenol 0 lM 100  5 100 lM 91  5 250 lM 80  4

100  15 88  5 92  8

Tetramethylbutylphenol 0 lM 100  7 30 lM 92  5 70 lM 83  4

ND ND

Diethylstilbestrol 0 lM 100  4 30 lM 86  4 60 lM 78  8

100  10 93  5 83  12

ND, not determined. The values are means  SD of three determinations. The maximum [3 H]IP3 binding under the conditions used in these experiments (i.e., 40 nM IP3 and pH 8.3) was typically 9.0 pmol/ mg for cerebellar microsomes and 1.6 pmol/mg for testis microsomes.

phase component. The rate constant for the fast phase decreased by about 9-fold in the presence of 20 lM nonylphenol, whilst that for the slow phase decreased about 3-fold. However, the amplitudes of both the fast and slow phase components were similarly reduced by about 75%. Fig. 2. The effects of octylphenol on the extent of IICR as a function of [IP3 ] and on [3 H]IP3 binding to porcine cerebellar microsomes. (A) Shows the concentration dependence of IICR between 0.01 and 20 lM IP3 in the absence (circle) and presence (square) of 50 lM octylphenol. (B) Shows the effect of a range of octylphenol concentrations (0– 125 lM) on the extent of [3 H]IP3 binding to cerebellar microsomes in the presence of 40 nM [3 H]IP3 (Kd for the cerebellar IP3 -receptor). The data points represent means  SD of 3 to 6 determinations.

component). These differences in Ca2þ release rates may be due to differences in the mechanisms that control the gating properties of the IP3 -sensitive Ca2þ channels within these stores. The effect of increasing concentrations of nonylphenol (0–20 lM) on the time course of IICR in the presence of 20 lM IP3 was studied and shown to fit well to the biexponential equation (as illustrated by the solid lines through the data points in Fig. 3A). As can be seen in Fig. 3A, nonylphenol inhibited both the extent and rate of IICR. The rate constants and amplitudes for the two components of IICR generated from the traces in Fig. 3A are plotted in Figs. 3B and C, respectively. From these data it can be seen that the fast phase of this process was more dramatically affected by nonylphenol than was the slow

Discussion These results demonstrate that several alkylphenols which have been speculatively associated with diminished reproductive potential in animals including humans [1,2,6–10] are also able to inhibit IP3 -sensitive Ca2þ channels (IP3 receptors) in testis and cerebellar microsomes. We have previously used immuno-chemical and molecular biological methods to show that all three isoforms of the IP3 receptor are found in testis microsomes [23] and that these channels are particularly abundant in Sertoli cells. Sertoli cells nurture and support the developing spermatids as they undergo the process of maturation to become mature sperm cells [26]. We and others have speculated that, if this process is impaired, then this is likely to be reflected in poor semen quality [10,21]. It is known that Sertoli cells require numerous endocrine factors (including some signalling through Ca2þ signalling pathways) during development and for normal cellular function [26]. Hence any agent that interferes with normal Ca2þ signalling in these cells, for example by inhibiting IP3 receptors as shown here or by inhibiting Ca2þ pumps, as

S.Z. Khan et al. / Biochemical and Biophysical Research Communications 310 (2003) 261–266 1.2

0µM

1.0 0.8

Fractional Ca

2+

release

A

5µM

0.6

10µM 0.4

15µM

0.2

20µM

0 0

5

10

15

20

Time (Seconds)

-1

Rate constants (s )

B

1

0.8

0.6

0.4

0.2

0 0

5

10

15

20

[Nonylphenol] (

Relative Amplitudes

C

0.6 0.5 0.4 0.3 0.2 0.1 0 0

5

10

15

20

[Nonylphenol] ( Fig. 3. The effect of nonylphenol on the transient kinetics of Ca2þ release induced by 20 lM IP3 from cerebellar microsomes. (A) Shows the time course of IICR in the presence of 0–20 lM nonylphenol concentrations. The traces are averaged from 8 to 10 separate runs and the solid line through the data points represents the best fits assuming a biexponential process. The kinetic parameters for the fast phase (circle) and slow phase (square) components of the data from (A) are shown in (B) (rate constants) and (C) (amplitudes).

shown previously [15,18,19,21], might be expected to have serious consequences for Sertoli cell function and spermatogenesis.

265

The data presented in Fig. 1 and Table 1 indicate that the potency of straight chain alkylphenols as inhibitors of IICR is directly related to their chain length. However, the IC50 values for inhibition of IICR by the branched chain alkylphenols such as tert-butylphenol (4-carbons) and tetramethylbutyphenol (8-carbons) were not dissimilar to those of the corresponding, straight chain alkylphenols (i.e., 4-n-butylphenol and 4-n-octylphenol, respectively). Hence it may be that the hydrophobicity of the side chain, rather that its overall length, is the most important factor in determining the potency of these compounds as inhibitors of IICR. The IP3 receptor consists of a single polypeptide chain of around 300 kDa in size. The IP3 binding domain is located within the first 650 amino acids closest to the amino-terminal end of the protein while the six transmembrane helices, which form part of the channel domain, are located close to the carboxy-terminus [27]. An active channel consists of four of these polypeptides arranged in a ‘four-leaf clover’ assembly with the pore or channel located at the interface where the trans-membrane regions meet [28]. The alkylphenols that we have studied inhibit both the extent and rates of Ca2þ release rather than the IP3 -binding properties of the receptors, so it is likely that these compounds affect the channel gating mechanism itself. The pore of the channel is lined by hydrophobic trans-membrane helices [28], and these structures may be a potential site where alkylphenols could bind and interfere with channel gating. In a recent study we investigated the effects of PKA-mediated phosphorylation on the kinetics of IICR and compared the results with those observed with single channel electro-physiological recordings [29]. We found that changes in the rate constants for Ca2þ release correlated well with changes in the conductance state of the channel. Changes in the extent of Ca2þ release were believed to reflect changes in the periods of channel inactivity that we have previously referred to as the ‘occupied unproductive’ state [24,29]. The current results therefore suggest that the inhibition of IICR by endocrine disrupting alkylphenols may reflect a decreased conductance state of the channel and an increase in the refractory period where the channel binds IP3 but remains closed. In summary, this study has identified another mechanism by which alkylphenols and other endocrine disrupters may affect hormone signalling in a variety of tissues, including testis, via an estrogen receptor-independent mechanism. Large quantities of these endocrine disrupters continue to be released into the environment. They are lipophilic and can bio-accumulate to micromolar concentrations in living organisms, especially those living in polluted aquatic environments [3–5]. The effects upon cellular Ca2þ homeostasis which we report in this paper may be of significance to animals living in

266

S.Z. Khan et al. / Biochemical and Biophysical Research Communications 310 (2003) 261–266

such environments and to others potentially exposed via the food chain, possibly including human. Acknowledgments We thank the Government of Pakistan for a scholarship to S.Z.K. and Dr. Bob Harris for the gift of some alkylphenols. Luigi Michelangeli is greatly thanked for all his support and help throughout the years.

References [1] J. Sumpter, Xenoendocrine disrupters—environmental impacts, Toxicol. Lett. 102/103 (1998) 337–342. [2] J. Toppari, J. Larsen, P. Christiansen, A. Giwercman, P. Grandjean, L. Guillette, B. Jegou, T. Jensen, P. Jouannet, N. Keiding, H. Leffers, J. McLaclan, O. Meyer, J. Muller, E. RajpertDeMeyts, T. Scheike, R. Sharpe, J. Sumpter, N. Skakkebaek, Male reproductive health and environmental xenoestrogens, Environ. Health Perspect. 104 (Suppl. 4) (1996) 741–803. [3] M. Ahel, J. McEvoy, W. Giger, Bioaccumulation of the lipophilic metabolites of nonionic surfactants in freshwater organisms, Environ. Pollut. 79 (1993) 243–248. [4] S.A Snyder, T.L. Keith, S.L. Pierens, E.M. Snyder, J.P. Giesy, Bioconcentration of nonylphenol in fathead minnows (Pimephales promelas), Chemosphere 44 (2001) 1697–1702. [5] T. Tsuda, A. Takino, M. Kojima, H. Harada, K. Muraki, M. Tsuji, 4-Nonylphenols and 4-tert-octylphenol in water and fish from rivers flowing into Lake Biwa, Chemosphere 41 (2000) 757–762. [6] G.A. Fox, Wildlife as sentinels of human health effects in the Great Lakes—St. Lawrence basin, Environ. Health Perspect. 109 (Suppl. 6) (2001) 853–861. [7] L.E. Gray Jr., Xenoendocrine disrupters: laboratory studies on male reproductive effects, Toxicol. Lett. 102–103 (1998) 331–335. [8] L.J. Guillette Jr., J.W. Brock, A.A. Rooney, A.R. Woodward, Serum concentrations of various environmental contaminants and their relationship to sex steroid concentrations and phallus size in juvenile American alligators, Arch. Environ. Contam. Toxicol. 36 (1999) 447–455. [9] C.F. Facemire, T.S. Gross, L.J. Guillette Jr., Reproductive impairment in the Florida panther: nature or nurture? Environ. Health Perspect. 103 (Suppl. 4) (1995) 79–86. [10] N.E. Skakkebaek, E. Rajpert-De Meyts, K.M. Main, Testicular dysdegesis syndrome: an increasingly common developmental disorder with environmental aspects, Hum. Rep. 16 (2001) 972– 978. [11] B. Gutendorf, J. Westendorf, Comparison of an array of in vitro assays for the assessment of the estrogenic potential of natural and synthetic estrogens, phytoestrogens and xenoestrogens, Toxicology 166 (2001) 79–89. [12] M. Jorgensen, B. Vendelbo, N.E. Skakkebaek, H. Leffers, Assaying estrogenicity by quantitating expression levels of endogenous estrogen-regulated genes, Environ. Health Perspect. 108 (2000) 403–412. [13] K. Saitoh, Y. Tomigahara, N. Ohe, N. Isobe, I. Natatsuka, H. Kaneko, Lack of significant estrogenic or antiestrogenic activity of pyrethroid insecticides in three in vitro assays based on classic estrogen receptor a-mediated mechanisms, Toxicol. Sci. 57 (2000) 54–60.

[14] R.M. Harris, R.H. Waring, C.J. Kirk, P.J. Hughes, Sulfation of “estrogenic” alkylphenols and 17b-estradiol by human platelet phenol sulfotransferases, J. Biol. Chem. 275 (2000) 159–166. [15] C.J. Kirk, L. Bottomley, N. Minican, H. Carpenter, S. Shaw, N. Kohli, M. Winter, E.W. Taylor, R.H. Waring, F. Michelangeli, R.M. Harris, Environmental endocrine disrupters dysregulate estrogen metabolism and Ca2þ homeostasis in fish and mammals via receptor-independent mechanisms, Comp. Biochem. Physiol. 135 (2003) 1–8. [16] T. Niwa, Y. Maekawa, M. Fujimoto, K. Kishimoto, Y. Yabusaki, F. Ishibashi, M. Katagiri, Inhibition of human hepatic cytochrome P450s and steroidogenic CYP17 by nonylphenol, Biol. Pharm. Bull. 25 (2002) 235–238. [17] A. Sturm, J.P. Cravedi, E. Perdu, M. Baradat, H. Segner, Effect of prochloraz and nonylphenol diethoxylate on hepatic transformation enzymes in trout: a comparative in vitro/in vivo-assessment using cultured hepatocytes, Aquat. Toxicol. 53 (2001) 229–245. [18] F. Michelangeli, S. Orlowski, P. Champeil, J.M. East, A.G. Lee, Mechanism of inhibition of the (Ca2þ –Mg2þ )-ATPase by nonylphenol, Biochemistry 29 (1990) 3091–3101. [19] G.R. Brown, S.L. Benyon, C.J. Kirk, M. Wictome, J.M. East, A.G. Lee, F. Michelangeli, Characterisation of a novel Ca2þ pump inhibitor (Bis-phenol) and its effects on intracellular Ca2þ mobilization, Biochim. Biophys. Acta 1195 (1994) 252–258. [20] M.J. Berridge, M.D. Bootman, P. Lipp, Calcium: a life and death signal, Nature 395 (1998) 645–648. [21] P.J. Hughes, H. McLellan, D.A. Lowes, S.Z. Khan, J.G. Bilmen, S.C. Tovey, R.E. Godfrey, R.H. Michell, C.J. Kirk, F. Michelangeli, Estrogenic alkylphenols induce cell death by inhibiting testis endoplasmic reticulum Ca2þ pumps, Biochem. Biophys. Res. Commun. 277 (2000) 568–574. [22] T.J. Beeler, K.S. Gable, Activation of Ca2þ release from sarcoplasmic reticulum vesicles by 4-alkylphenols, Arch. Biochem. Biophys. 301 (1993) 216–220. [23] S.C. Tovey, R.E. Godfrey, P.J. Hughes, M. Mezna, S.D. Minchin, K. Mikoshiba, F. Michelangeli, Identification and characterization of inositol 1,4,5-trisphosphate receptors in rat testis, Cell Calcium 21 (1997) 311–319. [24] M. Mezna, F. Michelangeli, The effects of inositol 1,4,5-trisphosphate analogues on the transient kinetics of Ca2þ release from cerebellar microsomes, J. Biol. Chem. 271 (1996) 31818–31823. [25] M. Mezna, F. Michelangeli, Ca2þ release from cerebellar microsomes 1,4,5-trisphosphate-induce, Biochem. J. 325 (1997) 177– 182. [26] T. Meehan, S. Schlatt, M. O’Bryan, D. de Krester, K. Loveland, Regulation of germ cell and Sertoli cell development by activin, follistatin, and FSH, Dev. Biol. 220 (2000) 225–237. [27] F. Michelangeli, M. Mezna, S. Tovey, L.G. Sayers, Pharmacological modulators of the inositol 1,4,5-trisphosphate receptor, Neuropharmacology 34 (1995) 1111–1122. [28] P.C.A. Da Fonseca, S.A. Morris, E.P. Nerou, C.W. Taylor, E.P. Morris, Domain organisation of the type 1 inositol 1,4,5trisphosphate receptor as revealed by single particle analysis, Proc. Natl. Acad. Sci. USA 100 (2003) 3936–3941. [29] J.L. Dyer, H. Mobasheri, E.J.A. Lea, A.P. Dawson, F. Michelangeli, Differential effect of PKA on the Ca2þ release kinetics of the type I and III InsP3 receptors, Biochem. Biophys. Res. Commun. 302 (2003) 121–126. [30] G.R. Brown, L.G. Sayers, C.J. Kirk, R.H. Michell, F. Michelangeli, The opening of the inositol-1,4,5-trisphosphate-sensitive Ca2þ channel in rat cerebellum is inhibited by caffeine, Biochem. J. 282 (1992) 309–312.