* Sección de Toxicología Ambiental, Departamento de Farmacología y Toxicología, and
Departamento de Biología Celular, CINVESTAV-IPN, Mexico DF 07000, Mexico
Received September 16, 1999; accepted November 12, 1999
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ABSTRACT |
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Key Words: DDT; cytochrome P450; sex-dependent regulation; pesticides; endocrine disrupters..
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INTRODUCTION |
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Although the more evident toxic signs during acute exposure to DDT occur in the central nervous system, recent studies have suggested that chronic exposure alters sexual steroidal hormone homeostasis in wild and experimental animals. For example, female rats given o,p'-DDT as neonates exhibited advanced puberty and persistent vaginal estrus in later life (Heinrichs et al., 1971). DDT was shown to induce masculinization in female rats (Bulger and Kupfer, 1985
) and p,p'-DDE, an antiandrogenic metabolite of DDT, alters sexual differentiation in male rat pups following exposure of pregnant females (Kelce et al., 1995
). Guillette et al. (1995) reported in juvenile alligators abnormal ovarian morphology and increased estradiol concentrations in females and abnormal testicular germ cells, decreased serum testosterone levels, and small phalli in males exposed to dicofol and DDT in Lake Apopka. These findings have led to DDT and its metabolites being considered as endocrine disruptors able to promote hormone-dependent pathology (Kavlock et al., 1996
).
Several studies have shown that DDT alters the activity of many microsomal enzyme activities, including those involved in phase I and phase II metabolism of xenobiotics (Lubet et al., 1992; Madhukar and Matsumura, 1979
). Different treatment schemes, most of them using relatively high doses in food or repeated administration by gavage during several days, have shown that DDT compounds induce hepatic CYP-dependent microsomal monooxygenases in different species (Abernathy et al., 1971
; Bunyan et al., 1972
) and in rats of different strains and sex (Henneman et al., 1994
; Li et al., 1995
; Lubet et al., 1990
). Pharmacodynamic studies on CYP2B induction indicated no important differences between the isomer p,p'-DDT and its metabolites DDE and DDD (Nims et al., 1998
). The effects, determined either by immunoreactive proteins or catalytic activities, consisted mainly of a preferential induction of CYP2B subfamily, a lesser induction on CYP3A, and minimal or no induction of CYP1A. On this basis, DDT has been considered a PB type of inducer (Okey, 1972
; Nims et al., 1998
).
Although there has been an active investigation on the inductive capacity of DDT compounds on hepatic cytochrome P-450, new information would contribute to an explanation of the endocrine disruptive properties attributed to those compounds, as the regulation of the CYPs preferentially affected by DDT is sex dependent. Therefore, the objective of this work was to evaluate the effects of DDT on the sexual expression pattern of some hepatic P-450 isozymes.
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MATERIALS AND METHODS |
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Animal treatments.
Adult male and female Wistar rats (200250 g) obtained from our animal house were fed a standard chow (PMI Feed Inc., St. Louis, MO) and tap water ad libitum, placed in steel cages at 21 ± 1°C, a relative humidity of 50% and 12 h light-dark cycles. Animals received by gavage a single dose of DDT (0, 0.1, 1.0, 5.0, 10, and 100 mg/kg body weight) dissolved in corn oil. Positive controls for CYPs were obtained by treating male rats with intraperitoneal injections of PB (80 mg/kg/day in saline for 3 days), BNF (50 mg/kg/d in corn oil for 2 days) and pyridine (100 mg/kg/day in saline for 3 days). Animals were sacrificed by exsanguination under deep anaesthesia with ethyl ether 24 h later. Livers were perfused with ice-cold saline (0.85% NaCl w/v) and microsomes were obtained as described by Mayer et al. (1990). Animals were treated according to the Guiding Principles in the Use of Animals in Toxicology adopted by the Society of Toxicology.
Total CYP and alkoxyresorufin metabolism assays.
Total CYP content of hepatic microsomes was measured according to Omura and Sato (1964). Microsomal O-dealkylation of 7-ethoxy- (EROD), 7-methoxy- (MROD), 7-pentoxy- (PROD), and 7-benzyloxy-resorufin (BROD) were assayed fluorimetrically at 37°C using excitation and emission wavelengths set at 530 and 585 nm, respectively (Burke et al., 1985; Lubet et al., 1985
; Nerurkar et al., 1993
). p-Nitrophenol hydroxylation (PNPH) activity was assayed by measuring the formation of 4-nitrocatechol, which was determined colorimetrically at 510 nm (Reinke and Moyer, 1985
). Protein content was determined by the method of Lowry et al. (1951) using bovine serum albumin (BSA) as standard.
Western immunoblots.
Microsomal proteins from each group (treated and untreated) were pooled and the same amounts of protein (10 µg/well for CYP1A1, 2B1/2B2, 2C11, 2E1, and 3A1, or 20 µg/well for CYP3A2) were electrophoretically separated in 10% SDS-PAGE as described by Laemmli, (1970). The resolved proteins were electrotransferred to nitrocellulose membranes and then blocked with 100 mM glycine, 1% BSA and 5% nonfat milk powder/PBS-1% Triton X-100 solution overnight at 4°C. Membranes were incubated with monoclonal anti-rat CYP1A1, 2B1/2B2, 2C11, 2E1, 3A1, or 3A2 antibodies for 1 h, followed by incubation for 1 h with horseradish peroxidase-conjugated secondary antibody. The specific protein bands in the blot were visualized by chemiluminescence using ECL detection reagents (Amersham Life Sci., Piscataway, NJ) followed by a brief exposure to Kodak XAR Scientific Imaging film. A Foto/Eclipse video camera (Fotodyne Inc.) and Collage ver. 3.0 software were used to scan blot negatives. Band intensities between control and treated groups were compared.
Statistical analysis.
Analysis of variance (ANOVA) was used to assess differences among doses for each sex. Differences between treated and controls groups were assessed by Dunnett's t test. Estimation of association between enzyme activities and CYP2B1/2B2 protein content was established by linear regression analysis. Significance was set at p < 0.05. All calculations were performed using SigmaStat Ver. 1.0 software (Jandel Corporation, San Rafael, CA).
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RESULTS |
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DISCUSSION |
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The effects on CYP2B1/2B2 protein and associated enzyme activities (PROD and BROD) indicated that males had a lower response threshold than females but that the latter were able to reach a higher relative induction. This suggest that these CYPs in females already had a starting lower activity by virtue of their sexual characteristics, hence higher doses of DDT were needed to produce a significant induction. These results were in general terms consistent with those reported when higher doses and/or longer periods of treatment were used (Henneman et al., (1994); Li et al., 1995; Lubet et al., 1992
). Based on the preferential induction of CYP2B and 3A subfamilies, DDT was classified as a PB-type inducer (Lubet et al., 1992
; Nims et al., 1998
; Okey, 1972
). However, this classification may be limited. This and previous published work (Fry et al., 1992
) have shown some differences between the hepatic CYP induction profiles of DDT and PB, suggesting that DDT is not a pure PB-type inducer. In our study, DDT did not significantly alter CYP3A1 in males, which has been shown to be affected by PB in this and previously published studies (Ghosal et al., 1996
; Waxman and Azaroff, 1992
). Further research is needed on the effects of DDT on the sexual dimorphism associated with CYPs 2B1, 2B2, 3A1, and 3A2, and on the molecular events involved in the activation of these CYPs.
Regarding CYP1A, there was no agreement between the effects on CYP1A1 protein content and EROD activity. These effects were consistent with earlier reports (Flodström et al., 1990; Henneman et al., 1994
; Lubet et al., 1992
). In our study, CYP2E1 expression was slightly affected by DDT exposure in both sexes, whereas no effect was reported by Li et al. (1995). On the other hand, PNPH activity associated with CYP2E1 was more responsive than immunoreactive protein analysis in both sexes. A similar effect has been reported after PB treatment (Reinke and Moyer, 1985
).
Possible explanations for the induction of CYPs could be related to the toxic effects of DDT on the neuroendocrine regulatory centers and/or to direct effects on liver. Pituitary hormones are important regulatory elements of liver CYP gene expression. For example, growth hormone (GH) and thyroxine (T4) play an important role in the regulation of sex-specific CYP expression and suppress the expression of several CYPs, among them 2B1, 2B2, and 3A2 (Kato and Yamazoe, 1993; Waxman et al., 1990
). Female rats have a relatively continuous level of GH in plasma, which effectively suppresses the expression of these male-specific isozymes. Thus, a pulsatile pattern of GH release could be responsible for the masculinization of the expression pattern of sex-specific isozymes such as CYP3A2. Although the DDT analog methoxychlor was shown to produce hypothyroidism, no information is available on the effects of DDT on GH or T4 (EPA, 1997). These observations suggest the need to clarify the effects of DDT on the hormones involved in the regulation of hepatic CYP expression.
The preferential induction of CYPs involved in steroid metabolism (2B1, 2B2, 3A1, and 3A2) suggests that DDT is able to increase steroid turnover. This is consistent with the increased catabolism of cortisol in DDT-exposed humans who significantly excreted increased amounts of 6ß-hydroxycortisol in urine (Nhachi and Loewenson, 1989; Poland et al., 1970
). In addition, phenytoin has also been reported to increase 6ß-hydroxycortisol urinary excretion in humans, reflecting an inductive effect on liver CYP3A (Fleishaker et al., 1995
). These findings suggest that induction of liver CYP isoforms plays an important role in the mechanisms by which DDT alters hormonal homeostasis. However, it remains to be established if this modulation of sexual dimorphism in rats has significance in exposed human populations.
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ACKNOWLEDGMENTS |
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NOTES |
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Presented in part at the 36th annual meeting of the Society of Toxicology in Cincinnati, OH (1997).
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