Effect of organotins on human aromatase activity in vitro
Introduction
Organotin compounds have been used as heat stabilizers in vinyl chloride polymers for more than 50 years and they have also been used as effective biocides (bactericidal, fungicidal). Tributyltin (TBT) has been used in wood preservation, as an antifouling agent in marine paints, for slime control in paper mills and as a disinfectant in circulating industrial cooling waters. Some octyltins are used in food contact packaging and drink containers.
Organotin compounds contaminate human food and water, mainly through contributions from industrial effluents and from leaching of PVC water pipes (Snoeij et al., 1987). A tolerable daily intake level for humans of 0.25 μg/kg body weight has been proposed, based on immunotoxicity studies (Penninks, 1993). Mammalian species are prone to the toxic effects of TBT. Humans exposed accidentally or occupationally to organotins, develop seizures, episodes of violent pain and psychic disturbances (Fortemps et al., 1978, Ross et al., 1981). In animal studies, the effects of TBT were similar to those found for humans and at higher exposure levels, animals have exhibited permanent brain damage (Sloviter et al., 1986). TBT is a known endocrine disrupter in marine species, yet there are very few studies on the effects of TBT on mammalian reproductive endpoints (CEPA, 1993). Organotin compounds (TBT and triphenyltin) decreased litter size in rats following exposure on days 0–3, 4–7 and 7–15 of gestation (Harazono et al., 1996, Harazono et al., 1998, Ema et al., 1997a, Ema et al., 1997b) and TBT appeared to have its greatest effect during the pre-implantation period although early post-implantation losses were observed. Surviving pups exhibited low-body weights (at GD 20) and higher incidences of skeletal abnormalities (Noda et al., 1993).
Organotins, and especially TBT, are toxic to marine species at low concentrations. For example, acute mortality of macroinvertebrates (mussels) (Beaumont and Budd, 1984), zooplankton (copepods) (Hall et al., 1988) and early life stages of fish (rainbow trout fry) (Seinen et al., 1981) has been described following exposure to TBT at low concentrations (0.1–5 μg/l). Furthermore, limb regeneration in fiddler crabs (Weis et al., 1987) and brittle stars (Walsh et al., 1986) is impaired following exposure to TBT. The endocrine disruptive effects of TBT are clearly seen in some marine organisms, which develop ambiguous genitalia (female dogwhelks and periwinkles exhibit imposex, a condition where the females become sterile due to the development of non-functional secondary male characteristics) (Bailey and Davies, 1989). Female shellfish that have been exposed to TBT and, as a consequence develop ambiguous genitalia, demonstrate increased levels of androgens and decreased levels of estrogens in the tissues (Oehlman et al., 1996, Mesink et al., 1997, Morcillo and Porte, 2000). Consequently, it has been proposed that TBT causes abnormal sexual development in shellfish by inhibiting the aromatase enzyme (CYP 19) (Oehlman et al., 1996, Matthiessen and Gibbs, 1998), possibly, because the aromatase enzyme catalyzes the conversion of TBT to dibutyltin (Lee, 1985). In a similar manner, the human placental aromatase has been found to catalyze the O-deethylation of 7-ethoxycoumarin (Toma et al., 1996). Hence, from the existing evidence, it was proposed that TBT would be inhibiting aromatase activity through an active site oriented mechanism.
The importance of organotins as environmental endocrine disrupters and their potential to adversely affect human health, has prompted the European Commission to identify TBT as a ‘priority hazardous substance’ the commercial use of which should be phased out completely (European Commission, 2001).
In view of the fact that estrogens are of considerable importance to normal sexual development in human fetuses the following studies were done to determine the effects of TBT and some other chemically related organotins on the human aromatase activity in vitro. The results demonstrate that organotins exhibit a structure related inhibition of human aromatase activity.
Section snippets
Materials
Human aromatase (CYP 19)+P450 reductase Supersomes™ were purchased from Gentest Corporation (Woburn, MA). The microsomes were obtained from baculovirus infected insect cells (BTI-TN-5B1-4) that were transfected with human CYP 19 cDNA and human P450 reductase and contained 1 nmol cytochrome P450 per ml (quoted aromatase activity was 5 pmol product/min/pmol P450). The microsomes were stored at −80 °C until required. Unlabeled steroids were purchased from Steraloids Inc. (Newport, RI) and [1,2,6,7-3
Aromatase assay
The radiometric assay for aromatase activity was done according to the modified methods of Cooke (1991). The microsomes were diluted with nine parts of Tris–HCl buffer (50 mM, pH 7.5, containing sucrose (0.25 M), KCl (25 mM), MgCl2 (5 mM) and mercaptoethanol (7 mM)). Microsomes were added to 3 ml Tris–HCl buffer containing testosterone (60 nM, 40 000 cpm 3H (1 cpm=0.0167 Bq)), and NADPH (0.25 mM) and incubated at 37 °C in a reciprocating water bath. After 15, 30 and 45 min, 1.0 ml was withdrawn
Statistics
Statistical comparisons of organotin effects on aromatase activity were made using one-way ANOVA. Identification of treatments that were significantly different from controls was achieved by Dunnet's multiple comparison procedure. (sigmastat Program version 1.0 for Windows, Jandel Scientific 1992–1994, San Rafael, CA). For each kinetic study, the apparent Michaelis constant (Km(app)) and apparent maximal velocity (Vmax(app)) were obtained using methods of analysis that differ in their criteria
Results
The effects of mono-, di- and tributyltin and mono-, di- and trioctyltin on human aromatase activity in vitro are shown in Fig. 2. TBT at 12 and 59 μM and dibutyltin at 74 μM were inhibitory to aromatase activity (P<0.05) but monobutyltin and the three octyltins were without effect. TBT at 59 μM caused aromatase activity to be decreased to about 50% of the control activity but dibutyltin was less inhibitory and at 74 μM decreased aromatase to about 75% of the control activity.
The mechanism of
Discussion
These studies have demonstrated that TBT is a competitive inhibitor of human aromatase activity. Others have demonstrated that several species of female shellfish, when exposed to TBT, undergo masculinization (Oehlman et al., 1996, Mesink et al., 1997, Morcillo and Porte, 2000). Furthermore, in these females, the tissue levels of testosterone are higher and those of estradiol are lower compared with controls to the extent that the androgen–estrogen ratio may be higher by an order of magnitude
Acknowledgements
The author wishes to thank the Toxic Substances Research Initiative (TSRI), a research program managed jointly by Health Canada and Environment Canada for financial support.
References (49)
- et al.
Equine cytochrome P450 aromatase exhibits an estrogen 2-hydroxylase activity in vitro
J. Steroid Biochem. Mol. Biol.
(1996) - et al.
High mortality of the larvae of the common mussel at low concentrations of tributyltin
Mar. Pollut. Bull.
(1984) - et al.
Effects of triphenyltin chloride on implantation and pregnancy in rats
Reprod. Toxicol.
(1997) - et al.
Estrogen synthesis in human colon cancer epithelial cells
J. Steroid Biochem. Mol. Biol.
(1999) - et al.
Evaluation of early embryonic loss induced by tributyltin chloride in rats: Phase- and dose-dependent antifertility effects
Toxicol. Lett.
(1996) - et al.
Inhibition of human cytochrome P450 aromatase activity by butyltins
Steroids
(2001) Aromatase: neuromodulator in the control of behaviour
J. Steroid Biochem. Mol. Biol.
(1993)- et al.
Characterization of oviductal aromatase in the northern leopard frog, Rana pipiens
Comp. Biochem. Physiol. Biochem. Mol. Biol.
(1996) - et al.
Aromatase in the human choriocarcinoma JEG-3: inhibition by R 76 713 in cultured cells and in tumors grown in nude mice
J. Steroid Biochem. Mol. Biol.
(1991) Metabolism of tributyltin oxide by crabs, oysters and fish
Mar. Environ. Res.
(1985)
Evidence of endocrine disruption in clams—Ruditapes decussata—transplanted to a tributyltin-polluted environment
Environ. Pollut.
Biological aromatization of delta 4,6- and delta 1,4,6-androgens and their 6-alkyl analogues, potent inhibitors of aromatase
J. Steroid Biochem. Mol. Biol.
Short term toxicity of tri-n-butyltinchloride in rainbow trout (Salmo gairdneri Richardson) yolk sac fry
Sci. Total Environ.
On the role of seizure activity in the hippocampal damage produced by trimethyltin
Brain Res.
Biological activity of organotin compounds—an overview
Environ. Res.
Novel aromatase transcripts from bovine placenta contain repeated sequence motifs
Gene
Inhibition of arm regeneration by Ophioderma brevispina (Echinodermata, Ophiuroidea) by tributyltin oxide and triphenyltin oxide
Ecotoxicol. Environ. Saf.
Localisation of aromatase activity in androgen target areas of the mouse brain
Neurosci. Lett.
Distribution of aromatase cytochrome P450 messenger ribonucleic acid in adult rhesus monkey brains
Biol. Reprod.
Functional domains of human aromatase cytochrome P450 characterized by linear alignment and site-directed mutagenesis
Mol. Endocrinol.
The effects of tributyltin on dogwhelks (Nucella lapillus) from Scottish coastal waters
J. Mar. Biol. Assoc. UK
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