The endocrine-disrupting activities of bisphenol A (BPA) and 19 related compounds were comparatively examined by means of different in vitro and in vivo reporter assays. BPA and some related compounds exhibited estrogenic activity in human breast cancer cell line MCF-7, but there were remarkable differences in activity. Tetrachlorobisphenol A (TCBPA) showed the highest activity, followed by bisphenol B, BPA, and tetramethylbisphenol A (TMBPA); 2,2-bis(4-hydroxyphenyl)-1-propanol, 1,1-bis(4-hydroxyphenyl)propionic acid and 2,2-diphenylpropane showed little or no activity. Anti-estrogenic activity against 17beta-estradiol was observed with TMBPA and tetrabromobisphenol A (TBBPA). TCBPA, TBBPA, and BPA gave positive responses in the in vivo uterotrophic assay using ovariectomized mice. In contrast, BPA and some related compounds showed significant inhibitory effects on the androgenic activity of 5alpha-dihydrotestosterone in mouse fibroblast cell line NIH3T3. TMBPA showed the highest antagonistic activity, followed by bisphenol AF, bisphenol AD, bisphenol B, and BPA. However, TBBPA, TCBPA, and 2,2-diphenylpropane were inactive. TBBPA, TCBPA, TMBPA, and 3,3'-dimethylbisphenol A exhibited significant thyroid hormonal activity towards rat pituitary cell line GH3, which releases growth hormone in a thyroid hormone-dependent manner. However, BPA and other derivatives did not show such activity. The results suggest that the 4-hydroxyl group of the A-phenyl ring and the B-phenyl ring of BPA derivatives are required for these hormonal activities, and substituents at the 3,5-positions of the phenyl rings and the bridging alkyl moiety markedly influence the activities.

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Parkinson's disease (PD) is an age-related neurodegenerative disorder characterized by progressive loss of dopaminergic neurons and the appearance of Lewy bodies in the substantia nigra, leading to clinical symptoms of rigidity, resting tremor, and bradykinesia. A few percent of PD is of genetic origin, and over ten genes have been identified as causes of familial PD so Received October 9, 2009; revised manuscript received November 27, 2009; accepted December 1, 2009. Address correspondence and reprint requests to Dr Y. Kotake, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan. E-mail: yaichiro@hiroshima-u.ac.jp Abbreviations used: 1BnTIQ, 1-Benzyl-1,2,3,4-tetrahydroisoquinoline; 1DAzBnTIQ, 1-(3-azido-5-azidomethylbenzyl)-1,2,3,4-tetrahydroisoquinoline; 1MeTIQ, 1-methyl-1,2,3,4-tetrahydroisoquinoline; 3¢,4¢ DHBnTIQ, 1-(3,4-dihydroxybenzyl)-1,2,3,4-tetrahydroisoquinoline; AR-JP, autosomal recessive juvenile parkinsonism; BSA, bovine serum albumin; CBB, Coomassie Brilliant Blue; GST, glutathione-S-transferase; IP, immunoprecipitation; N-Boc, N-tert-butoxycarbonyl; Pael-R, parkinassociated endothelin receptor-like receptor; PBS, phosphate-buffered saline; PD, Parkinson's disease; PVDF, polyvinylidene difluoride; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TIQ, 1,2,3,4-tetrahydroisoquinoline; TNE, tris-NaCl-EDTA; TOF, time of flight; Tris, tris(hydroxymethyl)aminomethane; TTBS, Tween 20-containing tris-buffered saline; WB, western blotting. AbstractSubstances that mimic the actions of causative gene products of familial Parkinson's disease (PD) are candidate as causative agents of idiopathic PD. 1-Benzyl-1,2,3,4-tetrahydroisoquinoline (1BnTIQ), an endogenous neurotoxin, is present at three times higher levels in CSF of PD patients than in CSF of control subjects. However, the mechanism of 1BnTIQ's neurotoxicity is unclear. In this study, we tried to identify 1BnTIQ-binding proteins by using a diazido-functionalized 1BnTIQ analog, 1-(3-azido-5-azidomethylbenzyl)-1,2,3,4-tetrahydroisoquinoline, designed and synthesized as a probe for radioisotope-free photoaffinity labeling. One major photolabeled protein identified using this probe was tubulin b, which has been reported to be a substrate of parkin, a ubiquitin E3 ligase and a causative gene product of familial PD. Loss of function mutation of parkin is reported to result in loss of tubulin b ubiquitination. Therefore, we examined the effect of 1BnTIQ on ubiquitination of tubulin b. The polyubiquitinated tubulin b level in human neuroblastoma SH-SY5Y cells was reduced in the presence of 1BnTIQ, even at concentrations as low as those detected in parkinsonian CSF. In vitro ubiquitination assay gave similar results. It is suggested that 1BnTIQ has the same effect on tubulin ubiquitination as does mutant parkin in familial PD. Taken together, substances which reduce polyubiquitination of tubulin such as 1BnTIQ are supposed to be candidates of etiological factors of PD.

In this study, liver microsome-mediated activation of diphenyl (DP), diphenylmethane (DPM) and 2,2-diphenylpropane (DPP) to estrogens was demonstrated. These three compounds were negative in estrogen reporter assay using estrogen-responsive human breast cancer cell line MCF-7. However, they exhibited estrogenic activity after incubation with liver microsomes of 3-methylcholanthrene-treated rats in the cases of DP and DPM, or of phenobarbital-treated rats in the cases of DP and DPP, in the presence of NADPH. When these compounds were incubated with liver microsomes in the presence of NADPH, monohydroxyl and dihydroxyl derivatives were formed. These hydroxylated metabolites, 4-hydroxydiphenyl, 3-hydroxydiphenyl, 2-hydroxydiphenyl, 4-hydroxydiphenylmethane, 2-(4-hydroxyphenyl)-2-phenylpropane (4-OH-DPP), 4,4′-dihydroxydiphenyl, 4,4′-dihydroxydiphenylmethane and 2,2-bis(4-hydroxyphenyl)propane (bisphenol A), all exhibited estrogenic activity in MCF-7 cells. Binding assay of these hydroxylated compounds with rat uterus estrogen receptor was also positive. These results suggest that the estrogenic activities of DP, DPM and DPP were due to the formation of hydroxylated metabolites by the liver cytochrome P450 system.Key words ---diphenyl, estrogenic activity, metabolic activation, human breast cancer cell line MCF-7, endocrine disruption, cytochrome P450 exert biological effects. Many so-called xenoestrogens produce a wide variety of toxic effects in animals. They may be playing a role in the increasing incidence of hormonally related cancers such as breast cancer and testicular cancer, and other problems of the reproductive system in humans. It is therefore important to screen environmental contaminants for estrogenic activity. Their metabolites also need to be identified and screened. We recently showed that trans-stilbene, which is the parent compound of diethylstilbestrol, and trans-stilbene oxide were not estrogenic, but exhibited a potent estrogenic activity after metabolic activation by a liver microsomal oxidation system. 4,5) In that report, we suggested that the estrogenic activity was due to the hydroxylated metabolites, trans-4-hydroxystilbene and trans-4,4′-dihydroxystilbene, formed by cytochrome P450 1A1/2. Furthermore, we demonstrated that styrene oligomers were metabolically activated to estrogens by rat liver microsomes, especially cytochrome P450 2B1.6) These results demonstrate the importance of considering proestrogenic potential, when screening for xenoestrogens in the environment.

trans-Stilbene exhibits estrogenic activity in an estrogen reporter assay after metabolic activation. In this study, uterotrophic assay was conducted using ovariectomized B6C3F1/Crj female mice treated with trans-stilbene, trans-4-hydroxystilbene, trans-4,4′-dihydroxystilbene and diethylstilbestrol. Administration of trans-4-hydroxystilbene, trans-4,4′-dihydroxystilbene and diethylstilbestrol elicited increases in absolute and relative uterus weight. Furthermore, the uterine response caused by the hydroxylated stilbenes was accompanied with an increase in the thickness of epithelial cell layers. trans-Stilbene itself also showed estrogenic activity, affecting uterine weight and causing histological changes of the uterus. This suggests that trans-stilbene is activated to hydroxylated metabolites to exhibit its estrogenic activity. Indeed, trans-hydroxystilbenes were detected in the urine and feces of mice dosed with trans-stilbene. The effect of stilbene derivatives on testis in newborn B6C3F1/Crj mouse was examined. Administration of diethylstilbestrol slightly decreased the testis weight and atrophy of seminiferous tubules. However, transstilbene and trans-4-hydroxystilbene affected neither testis weight nor the histological appearance of the testis.