The reported in vitro anti-estrogen pentachloronitrobenzene enhances the estrogenic activity of estradiol in vivo in the rat

Environmental Toxicology and Pharmacology 20 (2005) 199–208

The reported in vitro anti-estrogen pentachloronitrobenzene enhances the estrogenic activity of estradiol in vivo in the rat John Ashby∗ , Jenny Odum, Angela Burns, Paul Lefevre Syngenta Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire SK10 4TJ, UK Received 3 August 2004; accepted 19 December 2004 Available online 3 February 2005

Abstract Pentachloronitrobenzene (PCNB) has been shown to inhibit foci-formation for MCF-7 cells in vitro (Zou, E., Hatakeyama, M., Matsumra, F., 2002. Foci-formation of MCF-7 cells as an in vitro screening method for estrogenic chemicals. Environ. Toxicol. Pharmacol. 11, 71) This effect was referred to as representing an anti-estrogenic property of PCNB. However, we have found no evidence that PCNB acts as either an estrogen or an anti-estrogen, either in vitro or in vivo. The assays conducted were binding to human and rat estrogen receptors (ER), a hER yeast trans-activation assay, the immature rat uterotrophic assay and a pubertal female rat assay. Nonetheless, when PCNB was evaluated as a possible anti-estrogen against estradiol in the immature rat uterotrophic assay, it enhanced, rather than reduced the activity of estradiol. Absence of an effect by PCNB on the uterotrophic activity of diethylstilbestrol suggests that the effect with estradiol was related to alteration of its metabolism. However, PCNB was not hepatotoxic and failed to inhibit cytochrome P450 or estradiol sulphotransferase. Pentachlorophenol, a major metabolite of PCNB, was inactive as an estrogen and failed to enhance the uterotrophic activity of estradiol. © 2005 Elsevier B.V. All rights reserved. Keywords: Pentachloronitrobenzene; Pentachlorophenol; Endocrine disruptor; Uterotrophic assay; Estrogen receptor; Estradiol

1. Introduction Pentachloronitrobenzene (PCNB) has been suggested to be anti-estrogenic due to its ability to antagonize the proliferative effect of 17␤-estradiol (E2 ) in the foci-formation adaptation of the MCF-7 cell proliferation assay (Zou et al., 2002). Foci-formation was suggested to represent a convergence of several estrogenic pathways. This involves, in addition to the classical ligand-activated estrogen receptor (ER) transcriptional pathway, activation of c-Neu, phosphorylation of MAP kinase and ligand independent activation of ER. The anti-estrogenicity of PCNB was exhibited as a reduction in the number of both control and E2 -induced foci (reproduced from Zou et al. in Fig. 1). A number of derivatives of pentachlorobiphenyls were also shown to operate via the c-Neu pathway in this assay (Zou et al., 2002). ∗

Corresponding author. Fax: +44 1625 590249. E-mail address: [email protected] (J. Ashby).

1382-6689/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.etap.2004.12.047

The antagonistic activity of PCNB was weak, with 100 nM PCNB being required to produce a 40% reduction in fociformation, compared with a 50% reduction in foci observed for 10 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Zou et al. (2002) concluded PCNB to be a novel anti-estrogen whose mechanism of action warrants further study. Given the current need for a readily available anti-estrogen we decided to evaluate PCNB in a range of additional in vitro and in vivo assays for estrogenic/anti-estrogenic activity. These were binding to recombinant human ER␣ and ER␤ (hER␣ and hER␤) or rat uterine ER, a yeast-based hER assay (Routledge and Sumpter, 1996), the female pubertal rat assay for anti-estrogens (Ashby et al., 2002a) and weanling rat uterotrophic assays assessing both estrogenic and antiestrogenic activity (Kanno et al., 2001, 2003). Hydroxytamoxifen was used as a reference anti-estrogen in the yeast hER assays and the ER-antagonist Faslodex (FAS) (Wakeling et al., 1991) as a reference anti-estrogen in the rat pubertal and uterotrophic assays.

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2.3. ER binding assays

Fig. 1. The effect of PCNB on foci formation in MCF-7 cells. Columns sharing a common letter are significantly different from each other. Reproduced from Zou et al. (2002).

The early and unexpected finding that PCNB increased E2 -induced uterine growth in the immature rat then led us to investigate possible biochemical mechanisms for this activity. Pentachlorophenol (PCP), a metabolite of PCNB (O’Grodnick et al., 1981; Choudhury et al., 1987) was also evaluated to establish its possible role in the rodent activities observed for PCNB.

2. Materials and methods 2.1. Chemicals Pentachloronitrobenzene (PCNB) (99% pure), pentachlorophenol (PCP) (99% pure), hydroxytamoxifen, 17␤-estradiol (E2 ), diethylstilbestrol (DES) (>99% pure), niacinamide, Hepes, arachis oil (AO) and 31 (PAPS) were phosphoadenosine-51 -phosphosulphate obtained from Sigma Chemicals (Poole, Dorset, UK). Chlorophenol red-␤-d-galactopyranoside (CPRG) was obtained from Boehringer Mannheim, Frankfurt, Germany. Faslodex (FAS) was a gift from AstraZeneca, Alderley Park, Cheshire, UK. 3 H-(2,4,6,7)-17␤-Estradiol ([2,4,6,7-3 H]-E2 ) was obtained from Amersham Biosciences (Amersham, UK). 2.2. Animals Immature Alpk:APfSD (Wistar derived) rats were obtained from the AstraZeneca breeding unit (Alderley Park). Rats used in the pubertal rat anti-estrogen assay were 22–23 days old (body weights up to 62 g), rats used in the uterotrophic assays were 18–19 days old (body weights 37–45 g), on arrival and were acclimatized for 3 and 1 day, respectively, before dosing. Animal studies were performed in accordance with the UK “Animals (Scientific Procedures) Act”. Rats were housed (up to five per cage) in metal or polypropylene cages. Rat and Mouse No. 1 diet (Special Diet Services Ltd., Witham, Essex, UK) and water were available ad libitum. The phytoestrogen levels in the diet have been described earlier (Odum et al., 2001). Animal care and procedures were conducted according to in-house standards as described previously (Odum et al., 2001).

Competitive binding assays with immature rat uterine cytosolic ER were conducted as previously described (Ashby et al., 1999). The hER␣ and hER␤ binding assays were carried out in a similar manner to that described for the rat (Ashby et al., 1999). Recombinant hER␣ and hER␤ (Merck Biosciences, Nottingham, UK), were diluted in hER buffer (Merck Biosciences; KCl, 500 mM; Tris, 50 mM; EDTA, 1 mM; sodium vanadate, 1 mM; dithiothreitol, 2 mM; glycerol, 10% (w/v); NaN3 , 0.02% (w/v); pH 7.5). Duplicate aliquots (∼7.5 pmol) were incubated with DMSO (10 ml) or 10-fold dilutions of either E2 (in 10 ml DMSO; 5 × 10−10 –5 × 10−6 M) or PCNB (in 10 ml DMSO; 5 × 10−10 –5 × 10−4 M) in a final volume of 500 ml hER buffer containing [2,4,6,7-3 H]-E2 (∼0.22 mCi, 88Ci/mmol, final concentration 5 × 10−9 M E2 ) for 17 h at 4 ◦ C. Receptor–ligand complexes were precipitated with 0.25 ml high resolution hydroxylapatite (60% (v/v) in hER buffer). The precipitated complexes were sedimented by centrifugation (10,600 × g, 4 ◦ C, 10 min), the supernatant removed and the complexes washed three times with hER buffer, centrifuging and decanting the supernatant each time. The washed precipitates were resuspended in Optiphase “HiSafe” 3 scintillant (Perkin Elmer Life Sciences, Beaconsfield, UK) and the radioactivity determined in a liquid scintillation counter. 2.4. Yeast hER assays The recombinant hER yeast strain was obtained from Professor J. Sumpter (Brunel University, UK) and was as described earlier (Routledge and Sumpter, 1996). Briefly, the DNA sequence encoding the human ER was integrated into the genome of S. cerevisiaie with expression vectors in which an ER response element was cloned upstream of the reporter gene LacZ (encoding the enzyme ␤-galactosidase). The assay was performed as described by Routledge and Sumpter (1996). Test chemicals were serially diluted (in ethanol, except Hepes which was diluted in aqueous media) and 10 ␮l aliquots were then transferred to a 96-well flat bottom microtiter plate (Linbro/Titertek, ICN FLOW, Bucks, UK) and allowed to evaporate to dryness. To determine whether PCNB possessed anti-estrogenic activity, E2 was added at a concentration that produced a sub-optimal assay response (5 nM). Aliquots (200 ␮l) of assay medium (i.e. medium containing recombinant yeast and the chromogenic substrate, CPRG) were then dispensed to each sample well. The plates were sealed with autoclave tape and shaken vigorously for 2 min prior to incubation at 32 ◦ C for 3 days. At this point, colour development in the medium was measured at an absorbance of 540 nm using a Labsystems iEMS Reader MF plate reader. Absorbance at 620 nm was also measured to assess turbidity (growth/viability of the yeast). If the turbidity measurement was decreased >10% in any sample then that result was discarded (deleterious effects of the test chemical

J. Ashby et al. / Environmental Toxicology and Pharmacology 20 (2005) 199–208

on yeast growth and viability). Niacinamide was used as a negative control agent and hydroxytamoxifen as a positive control anti-estrogen. 2.5. Pubertal rat anti-estrogen assay PCNB was tested first in the pubertal rat anti-estrogen assay (Expt. 1), which was conducted as described previously (Ashby et al., 2002a,b). PCNB was administered by subcutaneous (sc) injection because it is extensively metabolized when administered orally (Choudhury et al., 1987). Preliminary toxicity studies of PCNB in pubertal rats failed to show any clinical or bodyweight effects up to 800 mg PCNB/kg/day. Higher doses could not be administered due to the viscosity of the test suspension. A dose of 800 mg/kg of PCNB in AO (5 ml/kg) was therefore selected for this study. FAS was administered by sc injection at 0.5 mg/kg/day, in AO (5 ml/kg), as described previously (Ashby et al., 2002a). Control animals received vehicle only. Compounds were administered once daily, beginning on pnd 25 or 26, until >95% of the rats in the control and PCNB groups had open vaginas (12 days of dosing). Vaginal opening was monitored daily. Necropsy of treated animals was carried out on pnd 37 or 38. An additional group of untreated rats was also killed on the first day of the experiment in order to determine initial uterine weight. Animals were killed 24 h after the final dose using over exposure to halothane (Concord Pharmaceuticals, Dunmow, Essex, UK) followed by cervical dislocation. Uteri were removed, blotted and weighed, as described earlier (Odum etal., 1997). A vaginal smear was taken at the time of death and the stage of the estrous cycle determined using the criteria described by Cooper et al. (1993). 2.6. Uterotrophic assays PCNB was tested for estrogenic and anti-estrogenic activity in five immature rat uterotrophic assays conducted as described by Kanno et al. (2001, 2003). All compounds were given by sc injection in AO, once daily, beginning on pnd 19 or 20, for 3 days. Dosing volumes were 5 ml/kg. Compounds that were co-administered were injected sc at different sites within 5 min of each other. In Expts. 2 and 3 PCNB was administered at 800 mg/kg/day (as above) in the presence and absence of E2 over the dose range 1–10 ␮g/kg/day. FAS was also administered at 0.5 mg/kg/day in the presence and absence of E2 (10 ␮g/kg/day). In Expt. 4 a dose response for PCNB was conducted from 100 to 800 mg/kg/day in the presence and absence of E2 (1 ␮g/kg/day). Expts. 5 and 6 utilized PCNB at 100 mg/kg/day, a concentration at which a clear inhibitory effect was observed and a dose at which PCNB remained soluble in AO. PCNB was then administered in the presence and absence of either E2 (0.1–5 ␮g/kg/day; Expt. 5) or DES (0.01–0.5 ␮g/kg/day; Expt. 6). The concentrations of E2 and DES were chosen to provide a dose–response for their activity in the uterotrophic assay—from a no-effect level to

201

75% maximum response, as shown previously (Odum et al., 1997, 2002). PCP (a metabolite of PCNB; O’Grodnick et al., 1981; Choudhury et al., 1987) was also evaluated in immature rat uterotrophic assays in the presence and absence of E2 . Preliminary toxicity studies with PCP indicated that 10 mg/kg/day by sc injection was the maximum dose tolerated by the immature rats. PCP was also administered in AO (5 ml/kg), it was soluble at the doses used. Uterotrophic assays were conducted as described above. In Expt. 7 a dose–response for PCP was conducted from 1 to 10 mg/kg/day in the presence and absence of E2 (1 ␮g/kg/day). PCP (5 and 10 mg/kg/day) was then administered in the presence and absence of E2 (0.5–5 ␮g/kg/day; Expt. 8) to encompass the range of E2 concentrations used with PCNB (above). A final experiment (Expt. 9) utilized PCP at 10 mg/kg and E2 at 1 ␮g/kg/day (the dose of E2 which gave the maximum enhancement by PCNB). Animals were killed (as described above) 24 h after the final dose. Uteri were removed, blotted and weighed. In Expt. 5 blood was taken by cardiac puncture into heparinised tubes and plasma prepared by centrifugation. Livers from animals in Expt. 5 were removed and weighed and used to prepare microsomes and cytosol as described by Elcombe et al. (2002). Liver preparations were stored at −70 ◦ C. 2.7. Biochemical assays Plasma alkaline phosphatase (ALP), alanine transaminase (ALT), aspartate transaminase (AST) and glutamate dehydrogenase (GLDH) were determined on plasma from Expt. 5 using a Kone 60i analyzer and proprietary kits (Labmedics, Salford Quays, Manchester, UK). The protein content of liver microsomes and cytosol was determined by the method of Lowry et al. (1951). Cytochrome P450 (CYP) content of liver microsomes was determined on selected groups from Expt. 5. Total CYP was determined by the method of Omura and Sato (1964). CYP 2B1/2 isoenzyme profiles were determined in microsomal fractions by SDS-gel electrophoresis and Western Blotting using a “Novex” gel system with 4–12% Tris–glycine gels supplied by Invitrogen (Paisley, UK). Microsomes from rats treated with phenobarbitone (a gift from K. Frost, CTL) were used as a positive control for CYP 2B1/2 (Soucek and Gut, 1992). Gels were loaded with 7.5 ␮g protein/lane. Anti-rabbit antibody to rat CYP 2B1/2 was purchased from Xenotech via Tebu-Bio (Peterborough, Cambridgeshire, UK). Donkey anti-rabbit IgG-HRP linked secondary antibody was purchased from Amersham Biosciences (Amersham, UK). Proteins were detected by chemiluminescence using the ’ECL Western Blotting Analysis System’ from Amersham Biosciences. CYP 2B1/2 was quantified by densitometry. Estrogen sulphotransferase (EST) activity was determined on selected groups from Expt. 5, using liver cytosol and the method of Kester et al. (2002). The effect of PCNB treatment on EST was determined at two concentrations of the substrate

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E2 : 5 ␮M (a saturating concentration to determine maximum enzyme activity; Kauffman et al., 1998) and 5 nM (a limiting substrate concentration to determine enzyme activity in the presence of possible inhibitors; Kester et al., 2002). Total reaction volumes of 0.2 ml contained: 5 nM (0.1 ␮Ci) or 5 ␮M (0.2 ␮Ci) 3 H-E2 , 0.1 mg cytosolic protein, 5 ␮M PAPS (used to start the reaction) in 0.1 M sodium phosphate buffer pH 7.2 containing 2 mM EDTA and 1 mM dithiothreitol. Reactions were incubated at 37 ◦ C for 30 min and stopped with 2 ml icecold water. Unreacted E2 was extracted with dichoromethane (2 ml) and sulphate formation, in 1 ml of the aqueous phase, quantified in a liquid scintillation counter after addition of scintillant. 2.8. Statistical methods Organ weights were analyzed by covariance with the terminal body weights. Analysis of variance was determined as described in SAS Institute and Inc. (1996). Differences from control values in all cases were assessed statistically using a two-sided Student’s t-test based on the error mean square from the analysis of variance.

3.2. Yeast hER assay The anti-estrogen hydroxytamoxifen gave the expected inhibition of E2 -induced activation of ␤-galactosidase, and the negative controls chemicals niacinamide and Hepes showed no effect on E2 -induced activation of ␤-galactosidase. PCNB showed no anti-estrogenic activity (Fig. 3). 3.3. Pubertal rat anti-estrogen assay PCNB (800 mg/kg) had no effect on peri-pubertal uterine growth in rats (Expt. 1). Uterine blotted weights for animals at the start of the experiment (pnd 25–26) were 40 mg and had increased 4.5-fold to 179 mg by pnd 37–38 at termination of the experiment. PCNB treatment had no effect on this increase, determined either as overall mean blotted weight or divided into subgroups according to stage of the estrous cycle (Table 1). The anti-estrogen FAS inhibited uterine growth as reported previously (Ashby et al., 2002a). Age and weight at vaginal opening was also unaffected by PCNB. FAS delayed vaginal opening, as reported previously (Ashby et al., 2002a), such that only 20% of rats had open vaginas by pnd 37/38 (Table 1). Body weights were unaffected by any of the treatments (Table 1).

3. Results 3.4. Uterotrophic assays with PCNB 3.1. ER binding assays Equivocal evidence was observed that PCNB competed with E2 in binding to hER␣ and that PCP competed with E2 in binding to rat uterine ER (Fig. 2). Although both binding curves were reproducible, no more than ∼25% displacement could be achieved. As discussed by Tinwell et al. (2002), this apparent displacement could be an artefact of the diminished aqueous solubility of PCNB and PCP. No binding of PCNB to hER␤ or rat uterine ER was observed (Fig. 2). Overall it was concluded that PNCB and PCP do not show biologically relevant binding to ER.

PCNB (800 mg/kg) failed to antagonize the uterotrophic activity of E2 (Expts. 2 and 3, Table 2) while FAS abolished it (Table 2), as shown previously (Odum et al., 1997). Unexpectedly, PCNB potentiated the uterotrophic activity of E2 at all dose levels of E2 evaluated (Table 2; Fig. 4). This potentiation by 800 mg/kg PNCB was maintained down to 100 mg/kg PNCB when evaluated against a fixed dose of 1 ␮g/kg E2 (Table 2; Fig. 5). This lower dose of 100 mg/kg PCNB was then used to challenge a dose–response of E2 in the uterotrophic assay (Table 3; Expt. 5). E2 increased uterine weight at doses of 0.5 ␮g/kg and above (as shown

Fig. 2. Assessment of the binding of (A) PCNB to recombinant hER␣ and hER␤ or (B) PCNB and PCP to rat uterine ER. Values are the means of duplicate determinations from a representative experiment.

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Fig. 3. Assessment of the anti-estrogenic potential of PCNB in the hER yeast assay. Closed squares: hydroxytamoxifen and 5 nM 17␤-estradiol; open squares: niacinamide and 5 nM 17␤-estradiol; open circles: Hepes and 5 nM 17␤-estradiol; closed triangles: PCNB and 5 nM 17␤-estradiol. Values are the means of triplicate determinations from a representative experiment.

previously, Tinwell and Ashby, 2004), and co-administration of PCNB potentiated this effect (Table 3, Fig. 6). In contrast, PCNB (100 mg/kg) had no effect on the uterotrophic activity of DES over the dose range 0.01–0.5 ␮g/kg (Expt. 6, Table 3, Fig. 7). In the DES study the no-effect level was 0.05 ␮g DES/kg, similar to that observed previously (Odum et al., 2002). A small difference in uterine weight occurred with PCNB at 0.25 ␮g DES/kg, but the dose–response curves for DES in the presence and absence of PCNB appeared identical (Fig. 7). The absence of a potentiating effect of PCNB on the uterotrophic activity of DES indicated that the potentiation by PCNB of the uterotrophic activity of E2 was specific to E2 .

dose of 10 mg/kg (Expt. 7; Table 4). When this dose range of PCP was used to challenge the uterotrophic activity of a fixed dose of E2 a small apparent potentiation of activity was observed for the 5 and 10 mg/kg doses of PCP (Expt. 7; Table 4). However this effect was not confirmed in two extended repeat studies (Expts. 8 and 9), the latter of which had increased group sizes to enhance the chance of detecting a weak effect (Table 4). It is concluded that PCP is inactive as an uterotrophic agent and is unable to potentiate the uterotrophic activity of E2 . The potentiating activity of PCNB is therefore not associated with its metabolite PCP.

3.5. Uterotrophic assays with PCP

The animals from Expt. 5 were used to assess a range of biochemical parameters of the plasma and liver. PCNB had no effect on liver weight or the plasma markers of hepatotoxicity ALP, AST and GLDH. A small reduction in ALT (∼75%

PCP, a metabolite of PCNB, was not uterotrophic when dosed alone by sc injection up to its maximum tolerated

3.6. Biochemical assays

Table 1 The effect of PCNB and FAS on uterine weight and vaginal opening in pubertal rats (Expt. 1) Treatment

Uterine blotted weight (mg)

No. of rats with open vaginas at term

Age at VO (pnd)

Body weight at VO (g)

Terminated at start of experiment AO 5 ml/kg/day; sc

39.9 ± 8.9

0/10a

–

–

178.8 ± 47.5 (M + D: 144.2 ± 21.0; P + E: 224.0 ± 31.1b ) 176.4 ± 49.3 (M + D: 147.9 ± 28.0; P + E: 213.5 ± 38.5b ) 45.5 ± 13.7**

29/30

32.1 ± 2.3

109.2 ± 15.2

147.9 ± 14.8

25–26/37–38

29/30

32.4 ± 2.6

110.9 ± 15.9

148.5 ± 16.5

25–26/37–38

2/10

–

–

145.7 ± 11.7

25–26/37–38

PCNB 800 mg/ kg/day; sc FAS 0.5 mg/kg/day; sc a b ∗∗

Body weight at term (g) 67.8 ± 6.1

Data are mean ± S.D., number of animals per group are shown as the denominator. Uterine weights grouped according to the stage of the estrous cycle, M + D: metestrus and diestrus; P + E: proestrus and estrus. Significantly different from the appropriate vehicle control group (P < 0.01).

Age at start/term (days) 25–26

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Table 2 The effect of PCNB and FAS on the uterotrophic activity of E2 Treatmenta Expt. 2 AO + AO PCNB 800 mg/kg/day + AO FAS 0.5 mg/kg/day + AO E2 1 ␮g/kg/day + AO E2 1 ␮g/kg/day + PCNB 800 mg/kg/day E2 10 ␮g/kg/day + AO E2 10 ␮g/kg/day + PCNB 800 mg/kg/day E2 10 ␮g/kg/day + FAS 0.5 mg/kg/day Expt. 3 AO + AO PCNB 800 mg/kg/day + AO FAS 0.5 mg/kg/day + AO E2 1 ␮g/kg/day + AO E2 1 ␮g/kg/day + PCNB 800 mg/kg/day E2 5 ␮g/kg/day + AO E2 5 ␮g/kg/day + PCNB 800 mg/kg/day E2 10 ␮g/kg/day/AO E2 10 ␮g/kg/day + PCNB 800 mg/kg/day E2 10 ␮g/kg/day + FAS 0.5 mg/kg/day Expt. 4 AO + AO PCNB 100 mg/kg/day + AO PCNB 200 mg/kg/day + AO PCNB 400 mg/kg/day + AO PCNB 800 mg/kg/day + AO E2 1 ␮g/kg/day + AO E2 1 ␮g/kg/day + PCNB 100 mg/kg/day E2 1 ␮g/kg/day/PCNB 200 mg/kg + day E2 1 ␮g/kg/day/PCNB 400 mg/kg + day E2 1 ␮g/kg/day/PCNB 800 mg/kg + day

Table 3 The effect of PCNB on the uterotrophic activity of E2 and DES

Uterine blotted weight (mg)

Final body weight (g)

n

22.5 ± 1.3 23.6 ± 3.1 16.3 ± 1.4** 47.3 ± 9.1** 85.9 ± 8.3**,++

54.6 ± 3.3 54.2 ± 3.5 55.3 ± 4.0 54.7 ± 4.7 54.2 ± 3.1

10 10 10 10 10

80.4 ± 10.9** 103.3 ± 14.2**,++

53.7 ± 3.1 54.0 ± 3.0

10 10

24.2 ± 3.3++

53.5 ± 3.5

10

20.4 ± 0.7 24.4 ± 3.1 16.9 ± 1.3* 48.6 ± 18.1** 87.0 ± 15.6**,++

54.7 ± 3.0 54.2 ± 3.9 53.5 ± 4.8 54.9 ± 5.2 53.3 ± 5.7

10 10 10 10 10

74.4 ± 11.5** 94.2 ± 8.4**,++

55.7 ± 3.7 53.5 ± 5.9

10 10

74.2 ± 15.1** 94.1 ± 13.5**,++

53.1 ± 5.0 53.2 ± 3.5

10 10

29.0 ± 6.0**,++

53.8 ± 5.4

10

21.4 ± 2.5 24.5 ± 4.4 23.6 ± 4.1 25.7 ± 6.1 25.7 ± 5.0 47.5 ± 9.5**,++ 73.3 ± 10.7**,++

52.8 ± 3.7 53.5 ± 4.9 54.9 ± 4.2 53.8 ± 5.4 53.1 ± 4.6 52.7 ± 3.0 54.3 ± 4.2

8 8 8 7 7 8 8

79.8 ± 22.3**,++

53.7 ± 3.3

8

90.7 ± 14.1**,++

55.1 ± 5.3

7

87.4 ± 8.2**,++

54.6 ± 4.0

7

Treatmenta Expt. 5 AO + AO PCNB 100 mg/kg day + AO E2 0.1 ␮g/kg/day + AO E2 0.1 ␮g/kg/day + PCNB 100 mg/kg/day E2 0.2 ␮g/kg/day + AO E2 0.2 ␮g/kg/day + PCNB 100 mg/kg/day E2 0.5 ␮g/kg/day + AO E2 0.5 ␮g/kg/day + PCNB 100 mg/kg/day E2 1 ␮g/kg/day + AO E2 1 ␮g/kg/day + PCNB 100 mg/kg/day E2 5 ␮g/kg/day + AO E2 5 ␮g/kg/day + PCNB 100 mg/kg/day Expt. 6 AO + AO PCNB 100 mg/kg/day + AO DES 0.01 ␮g/kg/day + AO DES 0.01 ␮g/kg/day + PCNB 100 mg/kg/day DES 0.025 ␮g/kg/day + AO DES 0.025 ␮g/kg/day + PCNB 100 mg/kg/day DES 0.05 ␮g/kg/day + AO DES 0.05 ␮g/kg/day + PCNB 100 mg/kg/day DES 0.25 ␮g/kg/day + AO DES 0.25 ␮g/kg/day + PCNB 100 mg/kg/day DES 0.5 ␮g/kg/day + AO DES 0.5 ␮g/kg/day + PCNB 100 mg/kg/day

Uterine blotted weight (mg)

Final body weight (g)

n

22.6 ± 3.0 26.2 ± 3.5 23.7 ± 3.8 27.0 ± 4.6

54.4 ± 2.1 54.9 ± 2.7 55.3 ± 4.5 55.6 ± 3.0

8 7 8 8

21.3 ± 2.9 30.7 ± 4.7+

54.6 ± 4.0 55.5 ± 3.3

8 8

31.0 ± 5.8* 44.2 ± 8.6**,++

52.7 ± 5.0 55.6 ± 2.8

7 8

43.7 ± 8.1** 67.4 ± 12.0**,++

53.4 ± 2.1 54.1 ± 3.9

7 8

72.8 ± 11.0** 86.1 ± 16.8**,++

54.9 ± 3.5 54.9 ± 2.8

8 8

21.6 ± 3.2 25.3 ± 2.2 22.2 ± 4.9 24.3 ± 4.6

57.1 ± 5.4 55.6 ± 3.9 54.7 ± 4.4 55.0 ± 4.2

8 8 8 8

20.6 ± 1.2 22.8 ± 2.6

54.7 ± 3.5 55.3 ± 4.1

8 8

24.5 ± 5.5 26.1 ± 2.8*

54.9 ± 5.1 54.3 ± 3.3

8 8

36.2 ± 3.9** 40.8 ± 4.8**,+

55.4 ± 2.8 55.3 ± 3.9

8 8

62.7 ± 8.1** 64.4 ± 7.4**

54.0 ± 4.1 55.3 ± 3.3

8 8

a All compounds were administered sc. Values are mean ± S.D. Statistically significant changes compared with AO + AO control group (*: P < 0.05, **: P < 0.01), or E2 + AO group at the same E2 concentration (+: P < 0.05, ++: P < 0.01).

a All compounds were administered sc. Values are mean ± S.D. Statistically significant changes compared with AO + AO control group (**: P < 0.01, *: P < 0.05), or E2 + AO group at the same E2 concentration (++: P < 0.01, Expts. 2 and 3) or PCNB + AO group at the same PCNB concentration (++: P < 0.01, Expt. 4).

Fig. 4. The effect of PCNB on E2 -induced uterine growth (Expt. 2). Values are mean + S.D. Statistically significant changes compared with arachis oil minus PCNB control group (**: P < 0.01), or E2 minus PCNB group at the same E2 concentration (++: P < 0.01).

Fig. 5. The effect of different doses of PCNB on the uterotrophic activity of a fixed dose of 1 ␮g/kg E2 (Expt. 4). Values are mean + S.D. Statistically significant changes compared with control minus E2 group (**: P < 0.01), or PCNB minus E2 group at the same PCNB concentration (++: P < 0.01).

J. Ashby et al. / Environmental Toxicology and Pharmacology 20 (2005) 199–208

Fig. 6. The effect of PCNB on the uterotrophic activity of E2 (Expt. 5). Values are mean + S.D. Statistically significant changes compared with control minus PCNB group (*: P < 0.05, **: P < 0.01), or E2 minus PCNB group at the same E2 concentration (+: P < 0.05, ++: P < 0.01).

of control values) was consistently observed in all groups receiving PCNB (Table 5). The reason for this isolated effect is not known, but is probably biologically insignificant given the other data shown in Table 5 and the fact that hepatotoxicity would lead to increases in these plasma markers. Table 4 The effect of PCP on the uterotrophic activity of E2 Treatmenta Expt. 7 AO + AO PCP 1 mg/kg/day + AO PCP 5 mg/kg/day + AO PCP 10 mg/kg/day + AO E2 1 ␮g/kg/day + AO E2 1 ␮g/kg/day + PCP 1 mg/kg/day E2 1 ␮g/kg/day + PCP 5 mg/kg/day E2 1 ␮g/kg/day + PCP 10 mg/kg/day Expt. 8 AO + AO PCP 10 mg/kg/day + AO E2 0.5 ␮g/kg/day + AO E2 0.5 ␮g/kg/day + PCP 10 mg/kg/day E2 1 ␮g/kg/day + AO E2 1 ␮g/kg/day + PCP 5 mg/kg/day E2 1 ␮g/kg/day + PCP 10 mg/kg/day E2 5 ␮g/kg/day + AO E2 5 ␮g/kg/day + PCP 10 mg/kg/day Expt. 9 AO + AO PCP 10 mg/kg/day + AO E2 1 ␮g/kg/day + AO E2 1 ␮g/kg/day + PCP 10 mg/kg/day

Uterine blotted weight (mg)

Final body weight (g)

n

23.4 ± 4.6 22.7 ± 4.5 20.8 ± 2.9 23.3 ± 3.3 41.8 ± 7.5** 40.4 ± 8.7**

55.8 ± 4.3 54.7 ± 3.5 54.7 ± 5.0 54.8 ± 3.5 55.5 ± 4.6 54.3 ± 3.2

8 8 8 8 8 8

48.0 ± 3.9**,+

54.9 ± 3.8

8

51.7 ± 8.1**,++

54.8 ± 4.6

8

24.6 ± 5.5 22.6 ± 4.3 30.6 ± 5.5 36.2 ± 6.8**

53.5 ± 6.2 53.9 ± 6.3 53.3 ± 6.4 54.4 ± 6.0

10 10 10 9

42.9 ± 14.0** 46.3 ± 17.0**

53.4 ± 7.6 53.7 ± 6.2

10 10

43.4 ± 12.6**

53.1 ± 6.0

10

64.7 ± 10.9** 67.7 ± 8.4**

52.4 ± 5.7 52.2 ± 5.5

10 10

24.2 ± 4.7 23.6 ± 4.3 45.3 ± 9.0** 47.9 ± 13.1**

54.6 ± 5.6 56.4 ± 5.3 56.3 ± 4.9 55.6 ± 7.4

25 15 25 25

All compounds were administered sc. Values are mean ± SD. Statistically significant changes compared with AO + AO control group (**: P < 0.01) or E2 + AO group at the same E2 concentration (+: P < 0.05, ++: P < 0.01). a

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The enhancement by PCNB of the uterotrophic activity of E2 , but not of DES, suggested the possibility that PCNB was inhibiting the metabolism of E2 , and thereby increasing its bioavailability. The possible effects of PCNB on CYP and EST, two of the major routes of metabolism of E2 (Zhu and Conney, 1998; Strott, 1996), were therefore investigated (Table 5). PCNB, in the presence and absence of E2 , had no effect on total cytochrome P450 levels (Table 5). Further, no changes in CYP2B1/2 levels (determined on western blots) were observed (Table 5). EST was determined at both saturating and limiting concentrations of the substrate E2 . In the former case to investigate whether PCNB treatment would reduce EST activity when assayed under optimal conditions, and in the latter case, to reduce the chance that inhibition of EST by weak binding of a PCNB metabolite was overwhelmed by competition for the active site. PCNB treatment (in the presence and absence of E2 ) did not inhibit EST when determined at either concentration of the substrate E2 (Table 5). 4. Discussion Zou et al. (2002) classified PCNB as a weak anti-estrogen based on its ability to inhibit foci formation in E2 -stimulated MCF-7 cells in vitro (Fig. 1) and suggested the need for further studies to evaluate the mechanism of action of this effect. The present experiments were designed to this end, and in particular, to determine if PCNB was anti-estrogenic in vivo. We were stimulated to undertake these studies by the fact PCNB is a small aromatic monocycle—molecular features that are unprecedented for an anti-estrogen and rare for an estrogen. PCNB failed to bind significantly to hER␣ and hER␤ or rat uterine ER (Fig. 2) and was devoid of anti-estrogenic activity in the yeast hER assay (Fig. 3). It also failed to delay the onset of puberty in the female pubertal rat assay (Table 1), an assay known to be sensitive to receptor-mediated anti-estrogens, the aromatase inhibitor Anastrozole, and the GnRH antagonist Antarelix (Ashby et al., 2002a). PCNB was also inactive in the immature rat uterotrophic assay and failed to inhibit the uterotrophic activity of E2 in that same assay (Fig. 5). We therefore conclude from the present experiments that PCNB is neither an estrogen nor an anti-estrogen, either in vitro or in vivo. The inhibition of foci-formation in MCF-7 cells by PCNB (Zou et al., 2002) is therefore unlikely to be ERmediated and other cellular pathways are probably involved. As a result of the above studies we were faced with the unexpected observation that PCNB potentiated the uterotrophic activity of E2 in the uterotrophic assay (Tables 2 and 3; Figs. 4 and 5). This effect was specific to E2 , there being no enhancement of the uterotrophic activity of DES (Fig. 7). This suggested that PCNB was interfering with the biochemical formation or elimination of E2 . The ability to enhance the uterotrophic action of both E2 (and estrone) has been described previously for carbon tetrachloride (CTC) and the CYP inhibitor SKF 525A (Levin et al., 1970).

206

Treatmenta

Liver wt. (g)

ALPb (U/l)

ALTb (U/l)

ASTb (U/l)

GLDHb (U/l)

P450c (mol/mg protein)

CYP 2B1/2c fold change vs. control

ESTd (SS) (mol/ min/mg protein)

ESTd (LS) (mol/ min/mg protein)

AO + AO PCNB 100 mg/kg day + AO E2 0.1 ␮g/kg/day + AO E2 0.1 ␮g/kg/day + PCNB 100 mg/kg/day E2 0.2 ␮g/kg/day + AO E2 0.2 ␮g/kg/day + PCNB 100 mg/kg/day E2 0.5 ␮g/kg/day + AO E2 0.5 ␮g/kg/day + PCNB 100 mg/kg/day E2 1 ␮g/kg/day + AO E2 1 ␮g/kg/day + PCNB 100 mg/kg/day E2 5 ␮g/kg/day + AO E2 5 ␮g/kg/day + PCNB 100 mg/kg/day

2.34 ± 0.14 (8) 2.60 ± 0.20* (7) 2.51 ± 0.28 (8) 2.51 ± 0.23 (8)

868 ± 64 (8) 823 ± 65 (7) 850 ± 130 (8) 821 ± 98 (8)

76 ± 17 (8) 63 ± 12* (7) 86 ± 9 (8) 54 ± 10** (8)

115 ± 6 (8) 113 ± 8 (7) 121 ± 12 (8) 112 ± 7 (8)

8.9 8.8 9.0 8.6

± ± ± ±

0.36 ± 0.07 (6) 0.44 ± 0.14 (6) ND ND

1.0 ± 0.3 (4) 1.0 ± 0.3 (4) ND ND

6.85 ± 0.67 (6) 7.29 ± 0.91 (6) ND ND

0.091 ± 0.012 (6) 0.107 ± 0.009* (6) ND ND

2.32 ± 0.19 (8) 2.46 ± 0.20 (8)

805 ± 78 (8) 798 ± 71 (8)

72 ± 11 (8) 59 ± 9** (8)

113 ± 7 (8) 114 ± 10 (8)

8.5 ± 1.1 (8) 9.0 ± 1.0 (8)

ND ND

ND ND

ND ND

ND ND

2.49 ± 0.28 (7) 2.58 ± 0.34 (8)

855 ± 160 (7) 870 ± 83 (8)

76 ± 18 (7) 62 ± 6** (8)

116 ± 13 (7) 121 ± 10 (8)

8.6 ± 1.5 (7) 10.3 ± 2.0* (8)

ND ND

ND ND

ND ND

ND ND

2.29 ± 0.23 (7) 2.49 ± 0.29 (8)

831 ± 82 (7) 862 ± 129 (8)

76 ± 11 (7) 58 ± 14** (8)

119 ± 7 (7) 117 ± 15 (8)

9.5 ± 1.6 (7) 9.0 ± 1.1 (8)

0.32 ± 0.06 (6) 0.41 ± 0.08 (6)

0.8 ± 0.1 (4) 1.5 ± 0.3 (4)

7.11 ± 0.43 (6) 7.94 ± 3.03 (6)

0.099 ± 0.008 (6) 0.101 ± 0.013 (6)

2.43 ± 0.25 (8) 2.29 ± 0.16 (8)

851 ± 135 (8) 852 ± 112 (8)

75 ± 14 (8) 58 ± 11** (8)

113 ± 11 (8) 111 ± 8 (8)

9.0 ± 1.5 (8) 9.1 ± 1.4 (8)

ND ND

ND ND

ND ND

ND ND

0.9 (8) 1.2 (7) 0.8 (8) 1.0 (8)

EST was determined at saturating substrate (SS) and limiting substrate (LS) concentrations. Values are mean ± S.D. (n). Statistically significant changes compared with AO + AO control group (*: P < 0.05, **: P < 0.01). a All compounds were administered by sc injection. b Determined in plasma. c Determined in liver microsomes. d Determined in liver cytosol.

J. Ashby et al. / Environmental Toxicology and Pharmacology 20 (2005) 199–208

Table 5 The effect of PCNB and E2 on liver weight and biochemical parameters (Expt. 5)

J. Ashby et al. / Environmental Toxicology and Pharmacology 20 (2005) 199–208

Fig. 7. The effect of PCNB on the uterotrophic activity of DES (Expt. 6). Values are mean + S.D. Statistically significant changes compared with control minus PCNB group (*: P < 0.05, **: P < 0.01) or DES minus PCNB group at the same DES concentration (+: P < 0.05).

The mechanism of this effect for CTC is a reduction in the metabolism of both E2 and estrone, caused by a general reduction in xenobiotic metabolising enzymes, consequent to CTCinduced liver damage. For example, administration of CTC at doses that potentiated E2 -induced uterine growth caused a 7-fold increase in blood markers of hepatotoxicity (Levin et al., 1970). SKF 525A, on the other hand, while failing to cause liver damage, inhibited cytochrome P450, an enzyme critically involved in E2 metabolism (Zhu and Conney, 1998). Either of these mechanisms could provide an explanation for the uterotrophic enhancing effects of PCNB. In addition, EST is a major phase 2 route of metabolism of E2 (Strott, 1996), and Kester et al. (2000, 2002) have demonstrated inhibition of EST in vitro by hydroxylated polyhalogenated aromatic hydrocarbons. Inhibition of EST by PCNB, or one of its metabolites, was therefore also worthy of evaluation. PCNB is extensively metabolized to various chlorinated phenols and thiophenols (Renner, 1981), and although few quantitative data exist, PCP is stated to be one of the major metabolites following oral administration of PCNB to rats (O’Grodnick et al., 1981; Choudhury et al., 1987) and this metabolite is a potent inhibitor of recombinant EST (Kester et al., 2000). The absence of liver damage induced by PCNB essentially eliminates a CTC-type mechanism of action (Table 5). It also failed to reduce the levels of total CYP or CYP2B1/2 (Table 5) indicating that selective inhibition of phase 1 E2 -metabolising enzymes is an unlikely mechanism for the uterotrophic enhancing effects observed. The absence of inhibition of EST by PCNB (Table 5) was not unexpected because Kester et al. (2000) showed that chlorinated phenols are transient non-competitive inhibitors of EST in vitro; thus any inhibition of EST by PCP or PCNB may no longer be evident 24 h after dosing. It is also possible that the unavoidable use of E2 as a substrate for the EST assay competed with the PCNB-metabolite for the allosteric site thus resulting in the loss of inhibition. Enzyme activity was determined at a low E2 substrate concentration as well as the usual high concentration (Kauffman et al., 1998) to allow for this possibility,

207

but no differences were observed between control and treated groups. While inhibition of EST, by PCNB, or one of its metabolites, remains the most likely mechanism for the uterotrophic enhancing activity seen for PCNB, determining which metabolite is responsible was outside the scope of the current experiments. The studies conducted on the major metabolite PCP indicated it to be devoid of estrogenic and anti-estrogenic activities and Kojima et al. (2004) have reported its inactivity in a hER reporter gene assay. Further, PCP was unable to potentiate the uterotrophic activity of E2 (Table 4). A complication when studying PCP, and perhaps other metabolites of PCNB, in vivo is that Arrehenius et al. (1977) have shown it to be an inhibitor of mitochondrial respiration, a property that probably accounted for its toxicity above the dose level of 10 mg/kg in the present experiments. A final complication in determining the active metabolite of PCNB is that its formation might be close to the site of EST and may not be reflected by its parenteral administration. We conclude from these studies that PCNB is not a functional anti-estrogen. However, PCNB clearly has the potential to act as an endocrine disrupting agent, possibly resulting from an ability to inhibit the metabolism of E2 .

References Arrehenius, E., Renberg, L., Johansson, L., Zetterqvist, M.A., 1977. Disturbance of microsomal detoxification mechanisms in liver by chlorophenol pesticides. Chem. Biol. Interact. 18, 35. Ashby, J., Tinwell, H., Pennie, W., Brooks, A.N., Lefevre, P.A., Beresford, N., Sumpter, J.P., 1999. Partial and weak oestrogenicity of the red wine constituent resveratrol: consideration of its superagonist activity in MCF-7 cells and its suggested cardiovascular protective effects. J. Appl. Toxicol. 10, 39. Ashby, J., Owens, W., Deghenghi, R., Odum, J., 2002a. Concept evaluation: an assay for receptor-mediated and biochemical anti-estrogens using pubertal rats. Regul. Toxicol. Pharmacol. 35, 393. Ashby, J., Tinwell, H., Stevens, J., Pastoor, T., Breckenridge, C.B., 2002b. The effects of atrazine on the sexual maturation of female rats. Regul. Toxicol. Pharmacol. 35, 468. Choudhury, H., Coleman, J., Mink, F.L., de Rosa, C.T., Stara, J.F., 1987. Health and environmental effects profile for pentachloronitrobenzene. Toxicol. Ind. Health 3, 5. Cooper, R.L., Goldman, J.M., Stoker, T.E., 1993. Measuring sexual behaviour in the rat. In: Methods on Toxicology, vol. 38. Academic Press. Elcombe, C.R., Odum, J., Foster, J.R., Stone, S., Hasmall, S., Soames, A.R., Ashby, J., 2002. Predictive value for carcinogenicity of acute and sub-acute tissue changes in rodents following exposure to 9 nongenotoxic NTP rodent carcinogens. Environ. Health Perspect. 110, 363. Kanno, J., Onyon, L., Haseman, S., Fenner-Crisp, P., Ashby, J., Owens, W., 2001. The OECD program to validate the rat uterotrophic bioassay to screen compounds for in vivo estrogenic responses: phase 1. Environ. Health Perspect. 109, 785. Kanno, J., Onyon, L., Peddada, S., Ashby, J., Jacob, E., Owens, W., 2003. The OECD program to validate the rat uterotrophic bioassay: phase 2—dose–response studies. Environ. Health Perspect. 111, 1530. Kauffman, F.C., Sharp, S., Allan, B.B., Burchell, A., Coughtrie, M.W., 1998. Microsomal steroid sulfatase: interactions with

208

J. Ashby et al. / Environmental Toxicology and Pharmacology 20 (2005) 199–208

cytosolic steroid sulfotransferases. Chem. Biol. Interact. 109, 169. Kester, M.H., Bulduk, S., Tibboel, D., Meinl, W., Glatt, H., Falany, C.N., Coughtrie, M.W., Bergman, A., Safe, S.H., Kuiper, G.G., Schuur, A.G., Brouwer, A., Visser, T.J., 2000. Potent inhibition of estrogen sulfotransferase by hydroxylated PCB metabolites: a novel pathway explaining the estrogenic activity of PCBs. Endocrinology 141, 1897. Kester, M.H., Bulduk, S., van Toor, H., Tibboel, D., Meinl, W., Glatt, H., Falany, C.N., Coughtrie, M.W., Schuur, A.G., Brouwer, A., Visser, T.J., 2002. Potent inhibition of estrogen sulfotransferase by hydroxylated metabolites of polyhalogenated aromatic hydrocarbons reveals alternative mechanism for estrogenic activity of endocrine disrupters. J. Clin. Endocrinol. Metab. 87, 1142. Kojima, H., Katsura, E., Takeuchi, S., Niiyama, K., Kobayashi, K., 2004. Screening for estrogen and androgen receptor activities in 200 pesticides by in vitro reporter gene assays using Chinese Hamster ovary cells. Environ. Health Perspect. 112, 524. Levin, W., Welch, R.M., Conney, A.H., 1970. Effect of carbon tetrachloride and other inhibitors of drug metabolism on the metabolism and action of estradiol and estrone in the rat. J. Pharmacol. Exp. Ther. 173, 247. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. Biol. Chem. 193, 265. Odum, J., Lefevre, P.A., Tittensor, S., Paton, D., Harris, C.A., Beresford, N.A., Sumpter, J.P., Ashby, J., 1997. The rodent uterotrophic assay: critical protocol feature studies with nonyl phenols, comparison with a yeast oestrogenicity assay. Regul. Toxicol. Pharmacol. 25, 176. Odum, J., Tinwell, H., Jones, K., Van Miller, J.P., Joiner, R.L., Tobin, G., Kawasaki, H., Deghenghi, R., Ashby, J., 2001. Effect of rodent diets on the sexual development of the rat. Toxicol. Sci. 61, 115. Odum, J., Lefevre, P.A., Tinwell, H., Van Miller, J.P., Joiner, R.L., Chapin, R.E., Wallis, N.T., Ashby, J., 2002. Comparison of the developmental

and reproductive toxicity of diethylstilbestrol administered to rats in utero, lactationally, pre-weaning or post weaning. Toxicol. Sci. 68, 147. O’Grodnick, J.S., Adamovics, J.A., Blake, S.H., Wedig, J., 1981. The metabolic fate of 14 C-labelled pentachloronitrobenzene in OsbourneMendel rats. Chemosphere 10, 67. Omura, T., Sato, R., 1964. The carbon monoxide binding pigment of liver microsomes. J. Biol. Chem. 239, 2370. Renner, G., 1981. Biotransformation of the fungicides hexachlorobenzene and pentachloronitrobenzene. Xenobiotica 11, 435. Routledge, E.J., Sumpter, J.P., 1996. Estrogenic activity of surfactants and some of their degradation products assessed using a recombinant yeast screen. Environ. Toxicol. Chem. 15, 241. SAS Institute Inc., 1996. SAS/STATS Software: Changes and Enhancements through Release 6.11. SAS Institute Inc., Cary, NC. Soucek, P., Gut, I., 1992. Cytochromes P-450 in rats: structures, functions, properties and relevant human forms. Xenobiotica 22, 83. Strott, C.A., 1996. Steroid sulfotransferases. Endocrine Rev. 17, 670. Tinwell, H., Lefevre, P.A., Moffat, G.J., Burns, A., Odum, J., Spurway, T.D., Orphanides, G., Ashby, J., 2002. Confirmation of uterotrophic activity of 3-(4-methylbenzylidine) camphor in the immature rat. Environ. Health Perspect. 110, 533. Tinwell, H., Ashby, J., 2004. Sensitivity of the immature rat uterotrophic assay to mixtures of estrogens. Environ. Health Perspect. 112, 575. Wakeling, A.E., Dukes, M., Bowler, J., 1991. A potent specific pure antiestrogen with clinical potential. Cancer Res. 51, 3867. Zhu, B.T., Conney, A.H., 1998. Functional role of estrogen metabolism in target cells: review and perspectives. Carcinogenesis 19, 1. Zou, E., Hatakeyama, M., Matsumra, F., 2002. Foci-formation of MCF7 cells as an in vitro screening method for estrogenic chemicals. Environ. Toxicol. Pharmacol. 11, 71.