Toxicological Sciences 54, 81-87 (2000)
Copyright © 2000 by the Society of Toxicology
Sex-Dependent Regulation of Hepatic Cytochrome P-450 by DDT
* 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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Dichlorodiphenyltrichloroethane (DDT) is a well-known inducer of microsomal monooxygenase systems in rodent liver. However, little information is available on its effects on the sex-dependent regulation of CYPs preferentially affected. Therefore, our objective was to evaluate the effects of DDT on the sexual expression pattern of some hepatic P-450 isozymes. Single doses of technical DDT (0, 0.1, 1, 5, 10, or 100 mg/kg body wt) were administered by gavage to Wistar rats. The effects on CYPs 1A1, 2B1/2B2, 2C11, 2E1, 3A1, and 3A2, were assessed 24 h later by means of CYP protein content determined by Western blotting and/or enzyme activities participating in alkoxyresorufin and p-nitrophenol metabolism. The highest dose induced 18-fold the expression of CYP3A2 in female rats without producing significant induction (< 3-fold) in males. The effects on this isozyme, which is not normally expressed in females, suggest that DDT is able to modulate sexual metabolic dimorphism, as 3A2 expression is androgen dependent. DDT did not significantly alter CYP3A1 in males, suggesting that DDT is not a pure phenobarbital (PB)-type inducer. The effects on CYP2B1/2B2 protein (19-fold) and associated enzyme activities indicated that males had a lower response threshold than females, but that the latter were able to reach a higher relative induction. The preferential induction of CYPs 2B and 3A by DDT in a sex-related manner suggest that CYP regulation could play an important role in endocrine disruption.
Key Words: DDT; cytochrome P450; sex-dependent regulation; pesticides; endocrine disrupters..
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Dichlorodiphenyltrichloroethane (DDT) is an organochlorine pesticide still widely used in developing countries for the control of malaria and other vector-transmitted diseases. The technical grade DDT used for this purpose is a mixture of p,p'-DDT (~85%), o,p'-DDT (~15%) and o,o'-DDT (trace amounts). The chemical characteristics of DDT compounds favor its accumulation and bioconcentration in lipid systems of all species, leading to continuous exposure and potential adverse effects to humans and wild animals. Exposure to technical DDT still represents a risk to human health in Mexico, as sprayers working in vector control campaigns in malaria-endemic areas are exposed to this pesticide.
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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Reagents.
DDT technical grade (80% p,p'-DDT and 20% o,p'-DDT) was obtained from CIBA-Geigy (Basel, Switzerland). Methoxy-, ethoxy-, pentoxy-, and benzyloxy-resorufin and resorufin were purchased from Molecular Probes, Inc. (Eugene, OR). NADPH, ß-naftoflavone (BNF), 4-nitrophenol and p-nitrocathechol were bought from Sigma Chemical Co. (St. Louis, MO). Phenobarbital was obtained from BDH Chemicals (Poole, U.K.) and pyridine from J. T. Baker (Mexico). Nitrocellulose paper and other chemicals used for Western blotting were obtained from BIO-RAD (Richmond, CA). Monoclonal anti-rat CYP3A1 was purchased from Oxford Biochemicals Research, Inc. (Oxford, MI) and monoclonal anti-rat CYP2E1 and 3A2 from Gentest Corp. (Woburn, MA). Goat anti-mouse and anti-rabbit IgG conjugated to horseradish peroxidase were obtained from PIERCE (Rockford, IL). All other chemicals were of the highest analytical grade available. The monoclonal anti-rat CYP1A1 and 2B1/2B2 were a kind gift of Dr. Colin Jefcoate (University of Wisconsin-Madison, Dept. of Pharmacology, Madison, WI) and anti-rat CYP2C11 was a kind gift of Dr. S. M. Bandiera (University of British Columbia, Vancouver).
Animal treatments.
Adult male and female Wistar rats (200–250 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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Liver weight and CYP content.
The highest dose of DDT (100 mg/kg body wt) significantly increased the liver relative weight (~15%) in both sexes. Total CYP content increased significantly in a dose- and sex-dependent manner. In males, significant increases were observed starting from 5 mg/kg, whereas significant increases were only observed at 100 mg/kg in females. However, at the highest dose, CYP content increase was ~1.8-fold in both sexes (Table 1
).
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CYP activities.
DDT also increased PROD, BROD, EROD, and PNPH microsomal enzyme activities at doses above 0.1 mg/kg body wt in a dose- and sex-dependent manner (Figs. 1 and 2
), whereas no significant effects on MROD activity were observed in either sex (Fig. 2). In absolute terms, the activities of PROD and BROD were about 4-fold higher in males (Fig. 2). However, in relative terms (treated/control), the maximum effect in males was observed at 10 mg/kg for both enzyme activities, reaching increases of 38- and 19-fold. In contrast, the maximum effect was observed at 100 mg/kg in females; PROD and BROD activities were 78-fold and 117-fold higher. EROD activity showed its maximal effect at 10 mg/kg, being 2.4- and 3-fold higher than their respective male and female control values; however, only this dose produced significant effects in females (Fig. 1). PNPH activity was also slightly increased in both sexes. In males, significant increases started from 1 mg/kg and the maximum effect (2.5-fold) occurred at 10 mg/kg, whereas the effect (1.8-fold) occurred at the 100 mg/kg dosage in females (Fig. 2).
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Immunoreactive CYP proteins.
The effects of DDT on the microsomal protein immunoreaction to monoclonal antibodies revealed a sex- and dose-dependent effect on CYP protein content (Table 2
and Fig. 3). Western blots showed increased levels of the male-specific CYP 3A2 in females; the highest dose (100 mg/kg) produced an 18-fold induction, whereas a ~3-fold induction was observed in males (Table 2). CYP3A1 reached its maximum induction (32-fold) at the highest dose, whereas no effects were observed in males. In the case of CYP2B1/2B2, a dose-dependent induction was observed in both sexes, starting from 1 mg/kg in males and from 5 mg/kg in females. The maximum relative effect was observed at 100 mg/kg, reaching 19-fold in both sexes. There was a good correlation between PROD and BROD activities and CYP2B1/2B2 content in both sexes. In males the values were r = 0.885 and 0.940, respectively (p < 0.05), whereas in females values were r = 0.960 and 0.941 (p < 0.05) (Fig. 4). CYP2C11 was induced 2.2-fold at 0.1 mg/kg in males but no response was observed at higher doses, whereas the protein was not detected in either treated or untreated female rats. Regarding CYP1A1 in males, doses below 10 mg/kg showed a tendency to decrease protein content; in females CYP1A1 was increased (1.8-fold) at the highest dose. No effects on CYP2E1 expression were observed below 10 mg/kg. The highest dose produced increases of 1.5- and 2-fold in males and females, respectively.
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) enzyme activities in rats treated with DDT. See Materials and Methods section for experimental details. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The main findings of this study were the induction by DDT of CYP3A2, an isoform normally not expressed in adult females, and the sex-related magnitude of the response in other types of CYP subfamilies induced. Technical grade DDT preferentially induced CYP3A in females, although CYP3A1 was more induced than 3A2. CYP3A1 is induced by dexamethasone and PB in male and female adult rats. Dexamethasone is more potent than PB, whereas 3A2 is only induced by PB (Ghosal et al., 1996
; Gonzalez et al., 1986). In contrast, DDT induced CYP3A1 in females but not in males, suggesting that its action is sex dependent and occurs at different regulation sites. Notwithstanding that CYPs 3A1 and 3A2 share a high degree of structural homology, they are regulated independently (Telhada et al., 1992). An interesting finding was that high DDT doses induced 18-fold the expression of CYP3A2 in female rats and only ~3-fold induction in males. CYP3A2 has been described to be normally expressed only in males (Gonzalez et al., 1986). The rationale for such a selective induction of CYP3A2 by DDT is unclear, but is not without precedent. For example, oxandrolone, a synthetic anabolic steroid displaying androgenic properties, has been reported to induce this isoform (6-fold) in female but not in male rats (Waskiewicz et al., 1995). Phenytoin, an anticonvulsant used to treat epilepsy, has also been reported to be a potent inducer of CYP3A2 in the female rat (Ghosal et al., 1996) and to preferentially induce CYP2B subfamily in the rat (Nims et al., 1994). The effects on this isozyme suggest that DDT is able to modulate metabolic sexual dimorphism by affecting regulatory sites of hepatic metabolism, as 3A2 expression is androgen dependent. In contrast to oxandrolone, marginal effects of DDT on CYP2C11, another male-specific and androgen-dependent isozyme, were also observed in males but not in females. The effects on 3A here described were relatively higher than the 2-fold increase reported by Li et al. (1995), who measured testosterone 6ß-hydroxylation and total 3A protein in female Sprague-Dawley CD rats. However, our results were not consistent with those of Henneman et al. (1994), who reported no significant effects on female cotton rat CYP3A total protein content and a 50% decrease in that of males. This diversity in results suggest that hepatic CYP induction by DDT is sex-, strain and administration route-dependent.
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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This work was partially supported by The Fund for Leadership Development, MacArthur Foundation, and CONACYT grant no. 28403-M. The authors also acknowledge Rosalinda Flores for secretarial help.
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NOTES |
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1 To whom correspondence should be addressed at Sección de Toxicología Ambiental, Centro de Investigación y Estudios Avanzados, Ave. Instituto Politécnico Nacional #2508, Col. San Pedro Zacatenco, G. A. Madero, México DF, Mexico 07300. Fax (5) 747-7095. E-mail: mcebrian{at}mail.cinvestav.mx.
Presented in part at the 36th annual meeting of the Society of Toxicology in Cincinnati, OH (1997).
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  C. Y. Ahn, S. K. Bae, Y. S. Jung, I. Lee, Y. C. Kim, M. G. Lee, and W. G. Shin Pharmacokinetic Parameters of Chlorzoxazone and Its Main Metabolite, 6-Hydroxychlorzoxazone, after Intravenous and Oral Administration of Chlorzoxazone to Liver Cirrhotic Rats with Diabetes Mellitus Drug Metab. Dispos., July 1, 2008; 36(7): 1233 - 1241. [Abstract] [Full Text] [PDF]
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I. M. Medina-Diaz, G. Arteaga-Illan, M. B. de Leon, B. Cisneros, A. Sierra-Santoyo, L. Vega, F. J. Gonzalez, and G. Elizondo Pregnane X Receptor-Dependent Induction of the CYP3A4 Gene by o,p'-1,1,1,-Trichloro-2,2-Bis (p-Chlorophenyl)ethane Drug Metab. Dispos., January 1, 2007; 35(1): 95 - 102. [Abstract] [Full Text] [PDF]
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Y. J. Moon, A. K. Lee, H. C. Chung, E. J. Kim, S. H. Kim, D. C. Lee, I. Lee, S. G. Kim, and M. G. Lee EFFECTS OF ACUTE RENAL FAILURE ON THE PHARMACOKINETICS OF CHLORZOXAZONE IN RATS Drug Metab. Dispos., June 1, 2003; 31(6): 776 - 784. [Abstract] [Full Text] [PDF]
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 M. E. Knuckles, F. Inyang, and A. Ramesh Acute and Subchronic Oral Toxicities of Benzo[a]pyrene in F-344 Rats Toxicol. Sci., June 1, 2001; 61(2): 382 - 388. [Abstract] [Full Text] [PDF]
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