DDT increases hepatic testosterone metabolism in rats

Arch Toxicol (2005) 79: 7–12 DOI 10.1007/s00204-004-0603-y

T OX I C OK I N ET I C S A ND M ET A B OL I SM

Adolfo Sierra-Santoyo Æ Manuel Hernańdez Arnulfo Albores Æ Mariano E. Cebriań

DDT increases hepatic testosterone metabolism in rats

Received: 2 February 2004 / Accepted: 10 August 2004 / Published online: 15 September 2004 Springer-Verlag 2004

Abstract DDT and its metabolites are considered as endocrine disruptors able to promote hormone-dependent pathologies. We studied the effects of technicalgrade DDT on hepatic testosterone metabolism and testosterone hydroxylase activity ratios in the rat. Male and female Wistar rats were treated by gavage with a single dose of technical-grade DDT (0, 0.1, 1, 10, and 100 mg/kg body weight) and killed 24 h later. Hepatic microsomes were incubated with [4-14C]-testosterone and the metabolites were separated by thin-layer chromatography and quantified by radio scanning. DDT increased testosterone biotransformation and modified the profile of metabolites produced in a sex-dependent manner. Males treated with a representative dose (10 mg/kg) produced relatively less androstenedione (AD), 2a-hydroxytestosterone (OHT), and 16a-OHT but higher 6b-OHT whereas treated females produced less 7a-OHT and AD but higher 6b-OHT and 6a-OHT than their respective controls. In both sexes DDT decreased the relative proportion of AD and increased that of 6b-OHT suggesting that the androgen-saving pathway was affected. The testosterone 6a-/15a-OHT ratio, a proposed indicator of demasculinization, was increased in treated males. This effect was in agreement with the demasculinizing ability proposed for DDT. The effects on 6a-/16a-OHT and 6-dehydrotestosterone/16aOHT ratios followed a similar tendency, with the ratio 6a-/16a-OHT being the most sensitive marker. Interestingly, these ratios were reduced in treated females suggesting that technical-grade DDT shifted testosterone hydroxylations toward a more masculine pattern. Thus, technical-grade DDT altered the hepatic sexual dimorA. Sierra-Santoyo Æ A. Albores Æ M. E. Cebriań (&) Seccioń de Toxicologı´ a, Cinvestav-IPN, PO Box 14-740, Mexico DF, Mexico E-mail: [email protected] Tel.: +52-55-50613309 Fax: +52-55-57477111 M. Hernańdez Departamento de Biologı´ a Celular, Cinvestav-IPN, Mexico

phism in testosterone metabolism and decreased the metabolic differences between male and female rats. Keywords DDT Æ Endocrine disruption Æ Testosterone metabolism Æ Cytochrome P-450 Æ Sex-dependent regulation

Introduction Dichlorodiphenyltrichloroethane (DDT) is a restricted organochlorine pesticide still widely used in some 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 favor its accumulation and bioconcentration, leading to continuous exposure and potential adverse effects. DDT and its metabolites are endocrine disruptors able to promote hormone-dependent pathologies (Kavlock et al. 1996). Several effects have been explained on the basis of interactions between the o,p¢- or p,p¢-isomers and estrogen or androgen receptors. For example, female rats given o,p¢-DDT as neonates exhibited advanced puberty and persistent vaginal estrus in later life suggesting an estrogenic activity (Heinrichs et al. 1971). The most persistent metabolite of DDT, p,p¢-DDE, alters sexual differentiation in male rats probably due to its ability to block androgen action at its receptor and by altering the expression of androgen-dependent genes (Kelce et al. 1995, 1997). In addition, DDT and its metabolites were shown to affect androgen homeostasis by reducing serum testosterone levels in rats (Krause 1977) and in male alligators exposed to dicofol and DDT in Lake Apopka (Guillette et al. 1994). Recently, Gunderson et al. (2001) reported altered sexually dimorphic patterns of hepatic testosterone hydroxylase activities and phase II enzyme activities in juvenile alligators living in sites contaminated with DDT and other organochlorine compounds in Florida.

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The induction of hepatic cytochrome P-450 (CYP)dependent microsomal monooxygenases by chemicals is an alternative mechanism by which androgen homeostasis may be disrupted by increasing the rate of its metabolism and elimination (LeBlanc et al. 1997). DDT and its metabolites are well-known inducers of the microsomal oxidase system in rodent liver (Li et al. 1995; Nims et al. 1998). CYP2B1, 2B2, 3A1, and 3A2 are preferentially induced by DDT in a sex-related manner, suggesting that DDT alters sexual dimorphism of these isoforms (Sierra-Santoyo et al. 2000). CYPs catalyze testosterone hydroxylations in a regio- and stereo-specific manner allowing the simultaneous study of changes in the expression of multiple CYP isoforms. Androgen hydroxylation in rat liver is directed to the 6b-position but hydroxylation at 2a-, 7a-, 15a-, 16a- and 16b-positions are also important (Waxman et al. 1990). CYP2A2 efficiently catalyzes testosterone 15a-hydroxylation as well as hydroxylations in other positions whereas CYP2A1 preferentially catalyzes testosterone 7ahydroxylation (Thummel et al. 1988; Aoyama et al. 1990). CYP2A1 also carries out 6-desaturation and 6ahydroxylation of testosterone, albeit at lower rates (Waxman 1988; Aoyama et al. 1989). CYPs 2C11 and 2B1 are involved in 2a- and 16a-testosterone hydroxylation (Yamazaki and Shimada 1997). Sexual dimorphisms of testosterone hydroxylase activities have been well characterized in the rat and other species (Pampori and Shapiro 1993; Niwa et al. 1995). Testosterone 6ahydroxylation and 6-dehydrogenation are catalyzed by CYP2A1 female rats (Aoyama et al. 1989; Waxman et al. 1989) and testosterone 16a- and 15a- hydroxylase by CYP2C11 and 2B1, and CYP2A2, respectively, in males (Niwa et al. 1995). In addition to hydroxylation, testosterone is also susceptible to oxido-reduction to androstenedione (AD), 6-dehydrotestosterone (6-DHT), and androstanediols, and conjugation to either glucuronic acid or sulfate, all of these metabolic activities contributing to testosterone inactivation and excretion enhancement (Zimniak and Waxman 1993). Testosterone can be dehydrogenated to produce AD through CYP isoforms (Waxman 1988) or 17b-hydroxysteroid dehydrogenase (Bloomquist et al. 1977), contributing to the pool of androgen precursors within the body and constituting a saving pathway for AD (Wilson and LeBlanc 1998). Several studies have explored the usefulness of studying the activities of testosterone hydroxylases as indicators of endocrine disruption since testosterone 6ahydroxylase and 15a-hydroxylase activities are preferentially expressed in female and male mice, respectively. Based on observations in mice treated with the antiandrogens vinclozolin or ketoconazole showing negative effects of testosterone on 6a-hydroxylase activity and positive effects on that of 15a-hydroxylase, it was proposed that an increased testosterone 6a-/15a-hydroxylase ratio was indicative of metabolic demasculinization and that this ratio could be used as an indicator of androgen status after exposure to model compounds

(Wilson et al. 1999; Wilson and LeBlanc 2000). Therefore, our objectives were to study the effects of the acute administration of technical-grade DDT on hepatic testosterone metabolism and testosterone hydroxylase activity ratios in the rat.

Materials and methods Chemicals Technical-grade DDT (80% p,p¢-DDT and 20% o,p¢DDT) was obtained from Ciba Geigy (Basel, Switzerland). [4-14C]-Testosterone (1.8 lCi/lmol) was obtained from Dupont NEN (Boston, MA). The metabolites 16bOHT, 6-DHT, 7a-OHT, and AD were purchased from Steraloids (Wilton, NH). Testosterone, 16a-OHT, 6bOHT, 2a-OHT, 15a-OHT, and 6a-OHT were obtained from Sigma Chemicals (St. Louis, MO). Other reagents were of the highest quality available. Animal treatments and microsome preparation Adult Wistar rats (200–250 g) bred in house were placed in hanging steel cages, fed a standard diet (PMI Feed, St. Louis, MO), allowed tap water ad libitum, and maintained at 21±1C and 50% relative humidity with 12 h light-dark cycles starting at 0700 h. Animals received by gavage a single dose of DDT (0, 0.1, 1, 10, and 100 mg/ kg body weight) dissolved in corn oil. Food was withdrawn after treatment, and animals were killed by exsanguination under deep ethyl ether anesthesia 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 (USA). Testosterone hydroxylation CYP-catalyzed testosterone hydroxylations were assayed in microsomal suspensions obtained from treated and control rats according to Waxman (1991). Briefly, [4-14C]-testosterone (50 lM) and 15–30 lg of microsomal protein were preincubated for 5 min at 37C in a 0.2-ml total volume incubation solution of 0.1 M HEPES buffer (pH 7.4) containing 0.1 mM EDTA. CYP reactions were initiated by addition of 1 mM NADPH dissolved in 20 ll buffer. Reactions were terminated after 10 min by the addition of 1 ml ethyl acetate and vortexed for 30 s. The layers were separated by centrifugation at 1,600 g for 5 min and the organic layer was transferred into a clean test tube. After a second extraction, the organic extracts were combined and solvent evaporated under N2 at room temperature. Products were separated by thin-layer chromatography

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using two solvent systems (A: dichloromethane:acetone 4:1 and B: chloroform:ethyl acetate:ethanol 4:1:0.7) and identified by comparison with unlabelled standards. Radioactivity (cpm) of each metabolite was directly determined on the plate in a b emission counter AMBIS 4000, Scanalytics (Billerica, MA). Protein content was determined according to Lowry et al. (1951) using bovine serum albumin as standard.

Statistical analysis Analysis of variance (ANOVA) was used to assess differences among doses for each sex. Differences between treated and control groups were assessed by Dunnett’s t-test. Significance was set at P £ 0.05. All calculations were performed using SigmaStat version 1.0 software (Jandel, San Rafael, CA).

Results Microsomes from untreated female rats metabolized testosterone at lower rates than those from untreated males. However, DDT treatment increased testosterone metabolism in female rats to rates similar to those observed in treated males (Fig. 1). Seven hydroxytestosterone metabolites, 15a-, 16a-, 6a-, 6b-, l6b-, 2a-, and 7a, in addition to 6-DHT, AD, and four metabolites not identified were detected in untreated and treated ani-

Fig. 1 Effects of the acute administration of technical-grade DDT on liver microsomal testosterone metabolism in Wistar rats. Values shown are presented as means±SD (n=5). Data were analyzed by ANOVA and Dunnett’s t-test. Asterisk indicates statistically significant differences when compared to respective controls (P £ 0.05)

mals. The main differences in the production rate of testosterone metabolites between untreated males and females were in the production of 16a-OHT and 2aOHT, whose formation was, respectively, seven and four times higher in males. In contrast, 7a-OHT production was eight times greater in untreated females (Tables 1, 2). Other activities showed less pronounced differences. In both sexes DDT differentially increased the formation rate of at least seven hydroxytestosterone metabolites and 6-DHT but not that of AD, as compared to their respective control groups (Tables 1, 2). Female rats showed the maximum effects on these metabolites at low doses. The rate of 6-DHT formation was the most affected, since it increased 4.6-fold at low doses (0.1 and 1 mg/kg). The effect on 6b-OHT formation showed a dose-dependent response reaching its maximum effect (4.5-fold) at the dose of 10 mg/kg. Formation of 16a-OHT increased above 3.3-fold at all doses tested. Testosterone 16b-hydroxylation was significantly increased at all doses, but the maximum effects (over 3.6-fold) were observed at 0.1 and 1 mg/kg and maintained thereafter. The production of 6a-OHT, 7aOHT, and 2a-OHT was also increased by DDT but the effects were less pronounced and observed at the lower doses. At the highest dose tested (100 mg/kg), the production of 6a-OHT, 2a-OHT, and 6-DHT was not significantly different from control values (Table 1). In males, maximum effects were observed at different doses and were less pronounced than those observed in females (Table 2). The more pronounced effects were seen on 6b-OHT and 6a-OHT formation (3.7-fold) at doses of 0.1 and 10 mg/kg, respectively, whereas 6-DHT production increased 3.2-fold at the dose of 1 mg/kg. Less pronounced effects were observed on 15a-OHT, 16a-OHT, and 16b-OHT formation. At the highest dose, the rate of production of metabolites 16a-OHT and 2aOHT was lower than that observed in the control group. Interestingly, a significant increase in the testosterone 6a-/15a-hydroxylase ratio, a proposed indicator of demasculinization, was observed in male rats whereas a significant decrease was noted in females at doses of 0.1, 1, and 100 mg/kg. However, no significant differences were observed between untreated males and females (Table 3). Similarly, we calculated the testosterone 6a-/ 16a-hydroxylase and 6-DHT/16a-hydroxylase ratios, which were able to show differences between untreated male and female rats (Table 4). DDT treatment significantly increased the testosterone 6a-/16a-hydroxylase ratio in males at doses of 10 and 100 mg/kg, whereas significant decreases were observed at all doses tested in females. Regarding the 6-DHT/16a-hydroxylase ratio, there was a significant increase at all doses tested in male rats whereas this ratio only decreased significantly at the two highest doses in females (Table 4). The relative proportions of metabolites formed reflected the alterations observed in enzyme activities. The major testosterone metabolites produced in untreated males were AD, accounting for 36.6% of total testosterone metabolites, followed by 16a-OHT, 2a-OHT, and

10 Table 1 Effects of acute administration of technical-grade DDT on liver microsomal testosterone hydroxylation in female rats. Rats were treated with DDT and liver microsomes were incubated with [14C]-testosterone. Testosterone metabolites were resolved by thin-

layer chromatography (TLC) and b emissions were quantified. Values shown are presented as means±SD (n=5). Data were analyzed by ANOVA and Dunnett’s t-test. (OHT Hydroxytestosterone,6-DHT 6-dehydrotestosterone,AD androstenedione)

DDT 15a-OHT 16a-OHT 6a-OHT 6b-OHT doses (mg/kg (pmol of product/min per mg protein) body weight)

16b-OHT

2a-OHT

6-DHT

7a-OHT

AD

0 0.1 1 10 100

49.9±14.6 182.1±6.6* 196.6±52.1* 94.8±13.8* 120.3±19.3*

58.2±10.6 102.1±17.3* 97.3±8.2* 71.1±14.2 69.6±13.2

88.1±20.6 395.1±45.1* 407.8±73.8* 118.5±34.6 126.6±34.3

1,845.2±92.7 2,912.4±477.6* 2,102.2±525.2 2,429.8±362.8* 2,373.1±318.5*

930.6±208.2 856.3±216.1 901.7±285.6 1,011.6±174.4 888.8±205.2

25.6±5.6 60.8±7.2* 56.6±7.6* 44.1±3.9* 45.8±6.6*

31.1±5.3 131.4±11.2* 119.4±13.5* 101.3±10.4* 106.4±15.2*

87.8±17.4 165.6±18.7* 133.3±29.1* 129.3±21.8* 115.9±22.4

277.5±79.4 779.3±74.2* 568.2±115.1* 1,251.3±211.8* 503.4±70.8*

*Statistically significant differences when compared to their respective control (P £ 0.05)

Table 2 Effects of acute administration of technical-grade DDT on liver microsomal testosterone hydroxylation in male rats. Rats were treated with DDT and liver microsomes were incubated with [14C]testosterone. Testosterone metabolites were resolved by TLC and

b emissions were quantified. Values shown are presented as means±SD (n=5). Data were analyzed by ANOVA and Dunnett’s t-test

DDT doses 15a-OHT 16a-OHT 6a-OHT 6b-OHT (mg/kg body (pmol of product/min per mg protein) weight)

16b-OHT

2a-OHT

6-DHT

7a-OHT

AD

0 0.1 1 10 100

59.0±8.5 130.0±7.0* 91.6±11.4* 107.7±12.8* 66.7±7.8

217.9±13.4 338.5±60.9* 189.9±40.3 239.6±48.7 156.6±10.0*

112.6±8.4 350.1±69.5* 361.8±50.3* 325.0±105.6* 187.1±59.6*

236.7±66.1 319.4±113.7 184.0±12.0 361.8±92.8 228.9±34.2

659.8±57.6 837.6±193.4 862.9±59.4 772.0±135.9 721.6±172.8

15.4±2.5 30.2±2.2* 34.4±7.2* 35.4±2.0* 23.4±2.2*

218.2±43.9 382.8±86.8* 337.0±62.7* 287.6±69.5 158.1±14.9*

49.7±10.4 155.3±27.3* 112.0±15.9* 184.7±46.7* 76.0±24.9*

191.3±92.5 715.5±105.7* 420.5±114.4* 682.9±181.1* 570.4±81.6*


7a-OHT accounting each for 12%. In untreated females, 7a-OHT, AD, and 6b-OHT represented 54%, 28.5%, and 8.5%, respectively (Table 5). The main differences between untreated animals and those treated with a representative dose of DDT (10 mg/kg) were that treated males produced lower proportions of AD (24.1%), 2a-OHT (8%), and 16a-OHT (8.8%) but a higher proportion of 6b-OHT (22.1%). Treated females showed smaller proportions of 7a-OHT (41.3%) and AD (17.9%) but increased proportions of 6b-OHT (21.3%) and MNI (11.4%).

Discussion The main finding of this study was that technical-grade DDT increased testosterone metabolism and modified the profile of produced metabolites in a sex-dependent manner. Our results showed that a single dose of DDT (0.1 mg/kg) increased the activity of at least seven CYPdependent testosterone-hydroxylating activities contributing to testosterone inactivation in both sexes, yet the magnitude of the effect was sex-dependent. DDT also decreased the relative proportion of AD formed and increased that of 6b-OHT, suggesting that the androgen-

Table 3 Effects of acute administration of technical-grade DDT on the liver 6a-OHT/15a-OHT ratio. Values shown are presented as means±SD (n=5). Data were analyzed by ANOVA and Dunnett’s t-test

Table 4 Effects of acute administration of technical-grade DDT on liver 6a-OHT/16a-OHT and 6-DHT/16a-OHT ratios. Values shown are presented as means±S.D (n=5). Data were analyzed by ANOVA and Dunnett’s t-test

DDT doses(mg/kg)

6-DHT/16a-OHT ratio Males

Females

0.42±0.15 0.76±0.23* 1.11±0.28* 1.25±0.66* 1.20±0.40*

2.87±0.90 3.01±0.30 3.33±0.60 1.57±0.19* 1.20±0.34*

0 0.1 1 10 100

Males

Females

DDT doses 6a-/16a-OHT ratio (mg/kg) Males Females

3.3±0.6 5.2±0.9* 4.1±1.3 5.3±1.0* 3.3±1.2

3.5±0.6 2.5±0.4* 2.2±0.6* 2.9±1.1 2.6±0.5*

0 0.1 1 10 100

6a-OHT/15a-OHT ratio


0.23±0.09 0.34±0.10 0.34±0.08 0.76±0.26* 0.49±0.17*

2.87±0.63 1.16±0.14* 1.04±0.18* 1.31±0.21* 1.1±0.24*


11 Table 5 Effects of the acute exposure to technical-grade DDT on the relative profile of testosterone metabolites produced by rat liver microsomes. Values shown are the percentage of pmol of every metabolite with respect to total pmol of all metabolites of testosterone (n=5). Rats were treated with DDT and liver microsomes were incubated with [14C]-testosterone Metabolites Females

Males

Non-treated Treated Non-treated Treated (10 mg/kg) (10 mg/kg) 7a-OHT 2a-OHT 6b-OHT 6a-OHT 16a-OHT 15a-OHT 16b-OHT AD 6-DHT MNI

51.8 1.7 8.2 2.5 1.0 0.8 1.4 27.3 2.6 2.7

40.7 1.3 21.0 2.2 1.7 0.7 1.6 17.7 1.8 11.3

11.5 12.2 10.2 2.8 12.0 0.8 3.2 36.3 7.0 4.0

10.1 7.8 21.6 5.9 8.6 1.1 3.3 23.6 9.9 8.1

saving pathway was affected in both sexes. It is also possible that disruption of testosterone metabolism by DDT could have decreased testosterone transformation to dihydrotestosterone or to estradiol, reactions catalyzed by 5a-reductase and aromatase, respectively. However, little information is available on the effects of DDT on these enzymes. This study has also shown that DDT significantly increased 15a-, 6a-, 6b-, 16a-, and 2a-hydroxylase activities and the 6-desaturation of testosterone, suggesting that DDT induces CYP2A2, 2A1, 3A, and 2C11 in both sexes. These results are in agreement with previous findings showing in both sexes induction of CYPs 2B and 2C11, and 3A in female rats, all accompanied by increased CYP2B-dependent PROD and BROD enzyme activities in a sex-related manner (Sierra-Santoyo et al. 2000). These findings suggest that DDT modulates the expression of most hepatic CYP isoforms involved in testosterone metabolism. The sex-dependent modulation of these isoforms by DDT could explain the differences observed in the profile of testosterone metabolites. Our results are also in good agreement with the alterations in hepatic sexual dimorphism testosterone hydroxylase activities reported in juvenile alligators living in sites contaminated with DDT and other organochlorine compounds (Gunderson et al. 2001). Taken together, these findings suggest that DDT modulates metabolic sexual dimorphism by affecting regulatory sites of hepatic metabolism. However, no information is available on the effects of DDT on growth hormone or thyroid hormone metabolism, which play important roles in the regulation of sex-dependent CYP isoforms (Waxman et al. 1990; Pampori and Shapiro 1999). DDT treatment also altered the profile of testosterone metabolites in both sexes. The formation of 6b-OHT was greatly increased and was accompanied by concomitant decreases in AD, 7a-OHT, and 2a-OHT in both sexes. The preferential increase of 6b-hydroxylase activity by DDT observed in this study suggests an inductive effect on

hepatic CYP3A, despite that a significant induction was not observed in male rats (Sierra-Santoyo et al. 2000). The increase in 6b-hydroxylase activity is in agreement with the increased cortisol catabolism observed in DDTexposed humans excreting significant amounts of 6bhydroxycortisol in urine (Poland et al. 1970; Nhachi and Loewenson 1989). In our study, DDT treatment increased the testosterone 6a-/15a-hydroxylase ratio in males, which is in agreement with the feminizing or demasculinizing ability proposed for DDT (Guillette et al. 1995; LeBlanc et al. 1997). Interestingly, in our study the 6a-/15a-hydroxylase ratio was decreased in treated females, suggesting that technical-grade DDT shifted the hydroxylation pattern of testosterone toward a more masculine pattern, thus decreasing the metabolic differences between male and female rats. However, there were no significant differences between untreated male and female basal ratios. Therefore, testosterone 6a-/16a-hydroxylase and 6-DHT/16a-hydroxylase ratios appear to be more useful indicators in rats, since significant differences between male and female basal ratios were observed and both ratios were more sensitive to DDT than the 6a-/15ahydroxylase ratio. The ratio 6a-/16a-hydroxylase might be a better indicator of endocrine disruption in rats since its response to DDT was more pronounced. We have interpreted the increased 6a-/16a-hydroxylase ratio as indicative of metabolic demasculinization in the rat, similar to the interpretation of the 6a-/15a-hydroxylase ratio in mice (Wilson and LeBlanc 2000), whereas a decrease in the 6a-/16a-hydroxylase ratio in females suggested defeminization. Our proposal is based on the capability of DDT to induce CYP isoforms which are not normally expressed in a specific sex (Sierra-Santoyo et al. 2000) and on the effects reported here regarding the activity of enzymes involved in testosterone metabolism. In summary, technical-grade DDT increased testosterone biotransformation, altered the hepatic sexual dimorphism in testosterone metabolism, and decreased the metabolic differences between male and female rats. Thus, testosterone biotransformation profiles are potentially useful biomarkers of androgen status in rats exposed to DDT. However, further studies are needed to assess the value of ratios derived from other CYPdependent testosterone hydroxylase activities. Acknowledgements This work was partially supported by the MacArthur Foundation (The Fund for Leadership Development) and by CONACYT grant number 28403-M. The authors also acknowledge Dr. Jaime Garcı´ a Mena for advice on radiometric analysis. The experiments performed complied with the current Mexican laws on animal experimentation.

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