2-chloro-s-triazine herbicides induce aromatase (CYP19) activity in H295R human adrenocortical carcinoma cells: a novel mechanism for estrogenicity?

Abstract

There is increasing concern that certain chemicals in the environment can cause endocrine disruption in exposed humans and wildlife. Investigations of potential effects on endocrine function have been limited mainly to interactions with hormone receptors. A need exists for the development of alternate in vitro methods to evaluate chemicals for their potential to disturb various endocrine functions via other mechanisms. Our laboratory is using the human H295R adrenocortical carcinoma cell line to examine chemicals for their potential to interfere with the activity and/or expression of several key cytochrome P450 (CYP) enzymes involved in the biosynthesis of steroid hormones. In this report we demonstrated that the commonly used 2-chloro-s-triazine herbicides atrazine, simazine, and propazine dose-dependently (0 -30 M) induced aromatase (CYP19) activity to an apparent maximum of about 2.5-fold in H295R cells. Basal-and triazine-induced aromatase activity was completely inhibited by the irreversible aromatase inhibitor 4-hydroxyandrostenedione (100 M). The triazines increased levels of CYP19 messenger ribonucleic acid (mRNA) between 1.5-and 2-fold. The time-response profile of the induction of aromatase activity and CYP19 mRNA by the triazines was similar to that by 8-bromo-cyclic adenosine monophosphate, a known stimulant of the protein kinase-A pathway that mediates the induction of aromatase in these cells. The observed induction of aromatase, the rate-limiting enzyme in the conversion of androgens to estrogens, may be an underlying explanation for some of the reported hormonal disrupting and tumor promoting properties of these herbicides in vivo. Key Words: aromatase; atrazine; simazine; propazine; triazines; induction; adrenocortical carcinoma; CYP19; mRNA. In recent years, there has been growing concern that certain environmental contaminants and commercial products have the potential to disturb endocrine functions in exposed humans and wildlife. Disturbances by these "endocrine disrupters" may lead to impaired reproductive capacity and other toxicities related to sexual differentiation, growth, and development. Current research has focused on potential interactions with the sex hormone receptors, particularly the estrogen receptor The H295 and H295R (a subpopulation of H295 that forms a monolayer in culture) human adrenocortical carcinoma cell lines have been characterized in detail and shown to express most of the key enzymes necessary for steroidogenesis The triazine family of broad-leaved herbicides has been used increasingly since the 1960s to control weeds, particularly in maize crops, in North America and Europe. The estimated use 1 To whom correspondence should be addressed. Fax: 011-31-30 -253-5077. E-mail: [email protected]. TOXICOLOGICAL SCIENCES 54, 121-127 (2000) Copyright © 2000 by the Society of Toxicology 121 of atrazine in the United States was almost 35,000 tons in 1993 (U.S. MATERIALS AND METHODS Cell culture conditions. H295R cells were obtained from the American Type Culture Collection (ATCC # CRL-2128) and grown in 75-cm 2 flasks (Greiner, Germany) under culture conditions published previously Isolation and amplification of RNA. RNA was isolated using the RNA Insta-Pure System (Eurogentec, Belgium) according to the manufacturerЈs instructions and stored at -70°C. RT-PCRs were performed using the Access RT-PCR System (Promega, U.S.). Northern blotting was not considered as an option because of the low basal expression of aromatase, which could not be detected by other investigators using this technique Aromatase assay. The catalytic activity of aromatase was determined based on the method of RESULT