* Research Institute of Toxicology, University of Utrecht, P.O. Box 80176, 3508 TD Utrecht, The Netherlands; and
Department of Zoology, National Food Safety and Toxicology Center, Institute of Environmental Toxicology, Michigan State University, East Lansing, Michigan 48824
Received May 25, 1999; accepted September 7, 1999
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ABSTRACT |
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Key Words: aromatase; atrazine; simazine; propazine; triazines; induction; adrenocortical carcinoma; CYP19; mRNA.
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INTRODUCTION |
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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 (Gazdar et al., 1990; Rainey et al., 1993
, 1994
; Staels et al., 1993
). These include CYP11A (cholesterol side-chain cleavage), CYP11B1 (steroid 11ß-hydroxylase), CYP11B2 (aldosterone synthetase), CYP17 (steroid 17
-hydroxylase and/or 17,20 lyase), CYP19 (aromatase), CYP21B2 (steroid 21-hydroxylase) and 3ß-hydroxysteroid dehydrogenase. The cells have the physiological characteristics of zonally undifferentiated human fetal adrenal cells, with the ability to produce the steroid hormones of each of the three phenotypically distinct zones found in the adult adrenal cortex (Gazdar et al., 1990
; Staels et al., 1993
). To elucidate an underlying mechanism of action for some of the reported endocrine-disrupting properties of the 2-chloro-s-triazine herbicides (Elridge et al., 1994; Stevens et al., 1994
), we examined the effects of atrazine (2-chloro-4-ethylamino-6-isopropyl-amino-s-triazine), simazine (2-chloro-4,6-bis[ethylamino]-s-triazine), and propazine (2-chloro-4,6-bis[isopropylamino]-s-triazine) on mRNA expression and catalytic activity of CYP19 in the H295R cell line.
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 of atrazine in the United States was almost 35,000 tons in 1993 (U.S. EPA, 1994). Consequently, it is found in relatively high concentrations in surface waters in large parts of the North American continent (Solomon et al., 1996
). It is relatively resistant to abiotic and biotic breakdown (Khan and Foster, 1976
; Solomon et al., 1996
). Epidemiological studies have associated long-term exposures to triazine herbicides with increased risk of ovarian cancer in female farm workers in Italy (Donna et al., 1989
) and increased risk of breast cancer in the general population of Kentucky in the United States (Kettles et al., 1997
). In experiments with female F344 rats, atrazine has been shown to induce tumors of the mammary gland and reproductive organs (Pinter et al., 1990
). In female Sprague-Dawley rats, atrazine caused lengthening of the estrous cycle and a dose-dependent increase in plasma levels of estradiol (Wetzel et al., 1994
). Atrazine also resulted in an earlier onset of the incidence of mammary and pituitary tumors (Wetzel et al., 1994
), responses typical of exposure to exogenously administered estrogens (Brawer and Sonnenschein, 1975
; Geschickter and Byrnes, 1942
). Investigations into the mechanism of the observed pro- and/or antiestrogenic effects of triazine herbicides have been limited to interactions with the estrogen receptor and effects on receptor-mediated responses (Connor et al., 1996
; Tennant et al., 1994a
,b
). These studies have not been able to provide a consistent explanation for the observed responses and any spurious effects on estrogen receptor function occurred at extremely high concentrations. Our study provides a mechanistically plausible explanation for the estrogen-related endocrine-disrupting effects of the family of 2-chloro-striazine herbicides by demonstrating their common ability to induce the catalytic activity and mRNA levels of CYP19 using the H295R cell line as a steroidogenic model system.
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MATERIALS AND METHODS |
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Isolation and amplification of RNA.
RNA was isolated using the RNA Insta-Pure System (Eurogentec, Belgium) according to the manufacturer's 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 (Staels et al., 1993). RNA preparations were considered acceptable for RT-PCR when their A260 (nm)/A280 (nm) ratios were greater than 1.8. The purity of the RNA preparations was verified by denaturing agarose gel electrophoresis. Suitable primer pairs were obtained by entering the human CYP19 cDNA sequence obtained from the European Molecular Biology Laboratories database into the software program Geneworks (version 2.4; IntelliGenetics, U.S.). The primer pair used for CYP19 mRNA amplification was: 5'-TTA-TGA-GAG-CAT-GCG-GTA-CC-3'; 5'-CTT-GCA-ATG-TCT-TCA-CGT-GG-3', resulting in an amplification product of 314 base pairs. PCR conditions, such as annealing temperature and Mg2+ concentration were optimized empirically. The conditions of the RT-PCR using the Access RT-PCR system kit were adapted as follows: RT-PCR for CYP19 mRNA (100 ng/reaction) was performed in the presence of 0.75 mM MgSO4. After a reverse transcription step at 48°C for 45 min, the resultant cDNA underwent 40 cycles of denaturation at 94°C for 30 s, annealing at 57°C for 1 min and extension at 68°C for 2 min. A final extension of 7 min completed the amplification. As reference, RT-PCR was performed on ß-actin mRNA using a commercially available primer pair (Promega, U.S.). Beta-actin mRNA (200 ng/reaction) was amplified according to the procedure above with slight modifications. RT-PCR was performed in the presence of 1 mM MgSO4. The reverse transcription step took place at 55°C and the annealing and extension temperatures were 70 and 72°C, respectively, during the 40 cycles. The final extension took place at 68°C. Serial dilutions of total RNA concentrations were amplified using each primer pair to determine the linear range of the PCR reaction, so semi-quantitative inferences could be made. Furthermore, beta-actin mRNA was found not to be affected by any of the treatments (triazines or 8Br-cAMP) and could be used reliably as a reference amplification response. To further enhance the reproducibility and comparability of the RT-PCR method, we included in each triazine-exposure experiment, apart from a vehicle control (cells exposed to DMSO), also a positive control (cells exposed to 8Br-cAMP). Within a typical experiment, amplification response ratios of CYP19/beta-actin obtained from RT-PCR of 3 different RNA preparations would result in an average value with a standard deviation between 10 and 20%. Triplicate amplification response ratios obtained from single RNA preparations resulted in standard deviation less than 5%. Amplification products were detected using agarose gel electrophoresis and ethidium-bromide staining. Intensities of the ethidium bromide stains were quantified using a FluorImager (Molecular Dymanics, U.S.).
Aromatase assay.
The catalytic activity of aromatase was determined based on the method of Lephart and Simpson (1991) with minor modifications. Cells were exposed to 54 nM 1ß-3H-androstenedione (New England Nuclear Research Products, U.S.) dissolved in serum-free (Ultroser SF-free) culture medium and incubated for 1.5 h at 37°C in an atmosphere of 5% CO2 and 95% air. All further steps were as reported previously (Letcher et al., 1999). The release of tritiated water was quantified by liquid scintillation counting after quench correction using an internal quench correction curve for 3H. The activity of aromatase was corrected for the distribution of the 3H-label on the androstenedione molecule, which was 25.7% at the 1
position and 74.2 % at the 1ß-position (Krekels et al., 1991
). A small volume (20 µl) of reaction medium directly added to liquid scintillation cocktail (total counts) verified the concentration of tritiated androstenedione in the reaction medium. Incubations in the absence of cells (blanks or background counts) were included as negative controls. The release of tritiated water by the cells was linear over time and led to about 26% conversion of androstenedione after 1.5 h. Aromatase activity was expressed in pmol of androstenedione converted per h per milligram protein. The specificity of the aromatase assay, based on the release of tritiated water, was verified by measuring the production of estrone (the aromatization product of androstenedione), using a 125I-labeled double-antibody radioimmunoassay kit (DSL-8700; Diagnostic Systems, Inc, U.S.), and by using 4-hydroxyandrostenedione, an irreversible inhibitor of the catalytic activity of aromatase, to block the formation of tritiated water (Brodie et al., 1977
).
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RESULTS |
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DISCUSSION |
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This is the first report to demonstrate consistently that several members of the class of 2-chloro-s-triazine herbicides induce the human aromatase enzyme in vitro at relatively low concentrations. Although the observed 2-fold induction is relatively weak, aromatase is the rate-limiting enzyme in the conversion of androgens to estrogens. Induction of this rate-limiting enzyme in vivo would be expected to lead to increased local production of estrogens with the potential to cause or contribute to estrogen-mediated pathologies, such as the tumor promotion observed experimentally in atrazine-exposed rats (Elridge et al., 1994; Pinter et al., 1990; Stevens et al., 1994
; Wetzel et al., 1994
). Furthermore, elevated expression of aromatase has been associated with increased risk of breast cancer (Bulun and Simpson, 1994
; Lu et al., 1996
; Reed et al., 1993
) and endometrial cancer (Bulun et al., 1994
) in female humans. A study examining the effects of aromatase inhibition on embryonic development found that exposure in ovo of chickens to non-steroidal aromatase inhibitors led to "masculinization" of females (Elbrecht and Smith, 1992
). Furthermore, co-administration of exogenous estrogen prevented the observed masculinization of females but led to "feminization" of males, which is the "default" sex in avian species (Elbrecht and Smith, 1992
). Estrogens appear to be the key hormones that lead to feminization of the central nervous system in birds, while they lead to defeminization and masculinization of the mammalian central nervous system (Jost, 1983
). Thus, it can be suggested that during critical (irreversible) developmental periods, such as embryonic, perinatal, and pubertal development, aromatase induction may result in inappropriate (de)feminizing responses, depending on the tissues in which local estrogen concentrations have been increased. A logical concern would be that exposure of wildlife and humans to triazine herbicides, which are produced and used in large quantities, and are ubiquitous environmental contaminants, may similarly contribute to estrogen-mediated toxicities and inappropriate sexual differentiation.
The H295R cell system deployed in the present study has proven useful in identifying a potential essential target for the endocrine-disrupting effects of several 2-chloro-s-triazine herbicides. If future studies in vivo support our hypothesis linking aromatase induction in specific target tissues to the endocrine toxicities of the triazines, this may have implications for the regulation of this class of herbicides as potential endocrine disrupters and tumor promoters.
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ACKNOWLEDGMENTS |
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NOTES |
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