Predictive modeling of a mixture of thyroid hormone disrupting chemicals that affect production and clearance of thyroxine.

Thyroid hormone (TH) disrupting compounds interfere with both thyroidal and extrathyroidal mechanisms to decrease circulating thyroxine (T(4)). This research tested the hypothesis that serum T(4) concentrations of rodents exposed to a mixture of both TH synthesis inhibitors (pesticides) and stimulators of T(4) clearance in the liver (polyhalogenated aromatic hydrocarbons, PHAHs) could be best predicted by an integrated addition model. Female Long-Evans rats, 23 days of age, were dosed with dilutions of a mixture of 18 PHAHs (2 dioxins, 4 dibenzofurans, and 12 PCBs, including dioxin-like and non-dioxin like PCBs) and a mixture of 3 pesticides (thiram, pronamide, and mancozeb) for four consecutive days. Serum was collected 24 hours after the last exposure and T(4) concentrations were measured by radioimmunoassay. Animals exposed to the highest dose of the mixture experienced a 45% decrease in serum T(4). Three additivity model predictions (dose addition, effect addition, and integrated addition) were generated based on single chemical data, and the results were compared. Effect addition overestimated the effect produced by the combination of all 21 chemicals. The results of the dose- and integrated-addition models were similar, and both provided better predictions than the effect-addition model. These results support the use of dose- and integrated additivity models in predicting the effects of complex mixtures.

Reversal of multidrug resistance in vivo by dietary administration of the phytochemical indole-3-carbinol.

A major obstacle to successful chemotherapy is the development of multidrug resistance (MDR) by cancer cells. MDR is characterized by enhanced cellular efflux of many structurally and functionally diverse compounds, including many anticancer drugs, due to overexpression of the MDR-1 gene product, P-glycoprotein. We hypothesized that the phytochemical, indole-3-carbinol (I3C), and some of its acid-condensation derivatives may inhibit P-glycoprotein-mediated transport due to their aromatic and nitrogen components, thus increasing the accumulation and efficacy of anticancer drugs and acting as a dietary adjuvant to conventional chemotherapy. I3C was subjected to acid conditions similar to those occurring in the stomach following ingestion and three acid-condensation products; a dimer, a noncyclic trimer, and a cyclic trimer were isolated and purified by high-performance liquid chromatography. The ability of I3C and its acid-condensation derivatives to reverse MDR was investigated using murine B16 melanoma cells that were transfected with the human MDR-1 gene (B16/hMDR-1 cells) and were cross-resistant to vinblastine and doxorubicin. The I3C acid-condensation product mixture, but not I3C, sensitized B16/hMDR-1 transfectants to the toxicity of vinblastine and doxorubicin. All three I3C acid-condensation products also increased the accumulation of the P-glycoprotein substrate, doxorubicin, in B16/hMDR-1 transfectants to levels comparable to parental B16 cells. The I3C acid-condensation product mixture competed with azidopine for binding to P-glycoprotein, suggesting that the observed MDR-reversing effect of the acid-condensation products was due to direct interaction with P-glycoprotein. The ability of p.o. administered I3C to reverse MDR was also tested in vivo. The resistance of B16/hMDR-1 transfectants to vinblastine and doxorubicin was preserved after i.p. injection and growth in nude mice. Tumor mass in mice that were provided with 333 or 500 mg/kg mouse/day I3C in their diet and injected s.c. with the anticancer drugs doxorubicin or vinblastine was significantly reduced as compared to tumor mass in mice provided with standard diet and injected with these anticancer drugs or mice provided with 500 mg/kg mouse/day I3C and not injected with anticancer compound. The concentrations of I3C used had no effect on survival or the general appearance and behavior of the mice. Collectively, these results indicate that ingestion of the common dietary constituent I3C results in its conversion to acid-condensation derivatives that sensitized MDR tumors to chemotherapeutic drugs without eliciting direct toxicity to the host.

Mechanisms of cyclophosphamide action on hepatic P-450 expression.

Cyclophosphamide was administered to adult male rats (130 mg/kg, single i.p. injection) and its effects on the P-450 enzymes that contribute to the activation of this drug in rat liver were then assessed. P-450-mediated cyclophosphamide 4-hydroxylase activity in isolated rat liver microsomes decreased by approximately 70% over a 9-day period following drug treatment. This decrease was due to the loss of cytochrome P-450 form 2c (IIC11), a major contributor to cyclophosphamide 4-hydroxylation in untreated male rat liver, while the other major hepatic cyclophosphamide 4-hydroxylase, P-450 PB-1 (IIC6), was largely unaffected. The loss of P-450 2c activity did not result from a decrease in P-450 reductase or from direct inactivation of the P-450 protein by cyclophosphamide or its metabolites, but rather was due to a reduction in hepatic P-450 2c protein and mRNA levels. Hepatic P-450 2a (IIIA2) and P-450 RLM2 (IIA2) were also suppressed by cyclophosphamide treatment. Serum testosterone, which contributes to the expression of P-450s 2c, 2a, and RLM2, was severely depleted in the cyclophosphamide-treated rats; however, this loss was not the direct cause of the decreases in these hepatic P-450s, since the decreases were not reversed upon restoration of normal testosterone levels by human chorionic gonadotropin stimulation of testicular androgen production. In contrast to the suppression of these testosterone-dependent P-450s, P-450 3 (IIA1), P-450j (IIE1), and the P-450-independent microsomal enzyme steroid 5 alpha-reductase were each elevated in rat liver following cyclophosphamide administration. In contrast to P-450 3 and steroid 5 alpha-reductase, however, the elevation of P-450j protein was transient and was not accompanied by an increase in P-450j-associated hepatic microsomal aniline hydroxylase activity. In vitro experiments revealed that P-450j was severalfold more susceptible to inactivation by the cyclophosphamide metabolite acrolein as compared with P-450 3. These observations suggest that P-450j protein is induced by cyclophosphamide treatment but that the protein is inactivated by the cyclophosphamide metabolite acrolein. These findings establish that cyclophosphamide treatment can modulate hepatic P-450 activities through multiple mechanisms and in a manner that may alter P-450 metabolism of cyclophosphamide and perhaps other anticancer drugs that undergo bioactivation in the liver.

Platinum anticancer drugs modulate P-450 mRNA levels and differentially alter hepatic drug and steroid hormone metabolism in male and female rats.

Treatment of male rats with the anticancer drug cisplatin leads to feminization of the profile of cytochrome P-450 and other microsomal enzymes involved in steroid hormone and drug metabolism (G.A. LeBlanc, and D.J. Waxman, J. Biol. Chem., 263: 15732-15739, 1988). The present study uses the rat model to evaluate the differential effects of cisplatin treatment on liver microsomal enzymes between genders, and also examines whether the modulation of enzyme activities by cisplatin and its analogues involves changes in P-450 gene expression. While cisplatin treatment of male rats caused a severalfold increase in female-predominant hepatic enzymes, including testosterone 5 alpha-reductase and testosterone 7 alpha-hydroxylase (P-450 form 2A1), it partially decreased the expression of these enzymes in females. The reduced expression of these estrogen-dependent enzymes in females may derive from the loss of circulating estradiol that was shown to occur in response to cisplatin treatment. Analysis of mRNA levels of individual P-450 forms revealed that the effects of cisplatin on P-450-catalyzed steroid hydroxylase activities in both male and female rats are primarily operative through the drug's effects on P-450 mRNA expression. P-450-dependent cyclophosphamide activation was significantly compromised in male rats after cisplatin administration; however, this activity was not altered in cisplatin-treated females. This sex-dependent effect of cisplatin was due to its suppression of P-450 form 2C11, a male-specific P-450 that is a major contributor to microsomal cyclophosphamide bioactivation in male rat liver. The clinically active cisplatin analogue iproplatin elicited effects very similar to those of cisplatin, while carboplatin and transplatin did not have significant effects on hepatic P-450 expression. Together, these findings demonstrate that the response of rat liver to cisplatin-induced changes in hepatic P-450 enzyme profiles and cyclophosphamide bioactivation capacity differs between the sexes, and in addition, these effects can be minimized by use of carboplatin in place of cisplatin.

Androgen metabolism in invertebrates and its modulation by xenoandrogens: a comparative study.

Marisa cornuarietis (Mollusc), Hyalella azteca (Crustacean), and Paracentrotus lividus (Echinoderm) demonstrated the ability to metabolize androgens through different pathways catalyzed by 5alpha-reductases (5alpha-R), hydroxysteroid dehydrogenases (HSD), hydroxylases, sulfotransferases (SULT), and fatty-acid acyl-CoA acyltransferases (ATAT). Interspecies differences and tissue-specific distribution of those enzymatic activities were observed. Xenobiotics, such as triphenyltin, tributyltin, and fenarimol, interfered with some of the pathways studied, namely, testosterone sulfation, testosterone esterification, and 5alpha-R activity. The work evidenced different sensitivity of those pathways to androgenic compounds, together with interphyla differences in androgen metabolism.

Pituitary regulation of the male-specific steroid 6 beta-hydroxylase P-450 2a (gene product IIIA2) in adult rat liver. Suppressive influence of growth hormone and thyroxine acting at a pretranslational leve;.

Oligonucleotide probes that distinguish between two closely related mRNAs encoding steroid 6 beta-hydroxylases of rat P-450 gene family CYP3A were used to individually assess their responsiveness to pituitary hormone regulation. Northern blot analysis revealed that the elevation of immunoreactive P-450 IIIA2 in livers of hypophysectomized rats reflects an elevation of the constitutive, male-specific P-450 IIIA2 (P-450 2a) and not an induction of the drug-inducible P-450 IIIA1 (P-450p). P-450 IIIA2 mRNA levels in intact adult male rats were found to be markedly reduced by GH administered as a continuous infusion at levels as low as 1 mU/h, indicating that GH acts at a pretranslational step to suppress expression of this P-450 enzyme. In hypophysectomized male rats, however, this same hormone treatment was only partially effective at suppressing P-450 IIIA2 mRNA and protein, suggesting that other pituitary-dependent factors contribute to the suppression observed in the intact rats. Further analysis revealed that T4, but not ACTH or human CG, can act in concert with GH to effect a more complete suppression of hepatic P-450 IIIA2 mRNA and protein in hypophysectomized rats. T4 also suppressed the expression of another GH-regulated, male-specific hepatic enzyme, designated P-450 IIA2 (P-450 RLM2), particularly in hypophysectomized female rats. In contrast, the GH-responsive P-450 IIA1 (P-450 3) was much less affected by T4 treatment. Thus, while T4 can modulate P-450 IIIA2 expression, it does not serve as a universal regulator for hepatic expression of GH-responsive P-450s.

Testosterone conjugating activities in invertebrates: are they targets for endocrine disruptors?

Testosterone conjugation activities, microsomal acyltransferases and cytosolic sulfotransferases, were investigated in three invertebrate species, the gastropod Marisa cornuarietis, the amphipod Hyalella azteca, and the echinoderm Paracentrotus lividus. The goals of the study were to characterize steroid conjugation pathways in different invertebrate phyla and to assess the susceptibility of those processes to disruption by environmental chemicals. All three species exhibited palmitoyl-CoA: testosterone acyltransferase activity (ATAT) in the range of 100-510 pmol/min/mg protein. Despite similarities in specific activities, kinetic studies indicated that ATAT had a higher affinity for testosterone but a lower V(max) in M. cornuarietis than in P. lividus, and intermediate values were found for H. azteca. In contrast, the activity of testosterone sulfotransferase (SULT) was rather low (0.05-0.18 pmol/min/mg protein) in M. cornuarietis and H. azteca. The low activity precluded kinetic analyses and inhibition studies with these species. P. lividus digestive tube displayed high SULT activity (50-170 pmol/min/mg protein) at moderate testosterone concentrations, but was inhibited at high testosterone concentrations. The interference of model pollutants (triphenyltin (TPT), tributyltin (TBT), and fenarimol) with these conjugation pathways was investigated in vitro. Both TPT and TBT (100 microM) inhibited ATAT in P. lividus (68 and 42% inhibition, respectively), and appeared to act as non-competitive inhibitors. ATAT activity in M. cornuarietis was less affected by organotins, and a significant inhibition (20% inhibition) was detected only with TBT. Fenarimol (100 microM) did not affect ATAT in any of the species tested. Sulfation of testosterone was suppressed by the organotins as well as fenarimol when using cytosolic preparations from P. lividus. These results demonstrated the existence of interphyla differences in testosterone conjugation, and revealed that these processes can serve as targets for endocrine disrupting chemicals.

Female-predominant rat hepatic P-450 forms j (IIE1) and 3 (IIA1) are under hormonal regulatory controls distinct from those of the sex-specific P-450 forms.

Hepatic expression of cytochrome P-450j (alcohol-inducible nitrosamine demethylase; P-450 gene IIE1) and P-450 3 (testosterone 7 alpha-hydroxylase; P-450 gene IIA1) is female predominant in adult rats [female/male greater than or equal to 1.5-2 (P-450j) or 3-4 (P-450 3)]. This sex difference emerges during the postsuckling period, when P-450 3 declines significantly (60-70% decrease) in male, but not female, rats, and P-450j declines in both sexes, but to a lower constitutive level in males than in females. The biochemical factors and regulatory events that control these developmental changes were investigated and found to be distinct from those that regulate expression of the female-specific hepatic enzymes P-450 2d (P-450 gene IIC12) and steroid 5 alpha-reductase. Immunoquantitation of the changes in P-450j and P-450 3 levels in hormonally altered rats established that both P-450s are under gonadal control. However, while androgen and estrogen both suppress P-450j expression, estrogen stimulates and androgen suppresses the expression of P-450 3. Since many of the effects of gonadal hormones on hepatic enzyme levels are mediated by the pituitary, the contribution of pituitary hormones to P-450j and P-450 3 expression was evaluated. Hypophysectomy of adult rats of either sex elevated P-450j to the levels found in immature rats (3- to 5-fold increase). This elevation was largely reversed in both sexes by GH administered either intermittently or continuously, demonstrating that P-450j is under the suppressive control of this pituitary hormone. The regulation of P-450 3 was more complex. Hypophysectomy elevated P-450 3 to prepubertal levels in adult male rats, but had no effect on P-450 3 levels in adult females. In both sexes GH suppressed P-450 3 expression when administered to hypophysectomized rats intermittently, but stimulated P-450 3 expression when infused continuously. Corresponding changes in hepatic microsomal P-450 3-dependent testosterone 7 alpha-hydroxylase and P-450j-dependent aniline hydroxylase activities were observed in response to hypophysectomy and GH replacement, but only after supplementation of microsomal NADPH P-450 reductase [which was 63-77% suppressed by hypophysectomy] with exogenous purified NADPH P-450 reductase. These studies demonstrate that these female-predominant hepatic P-450 enzymes are regulated by different mechanisms, and that both are under hormonal regulatory controls distinct from those that govern expression of the female-specific hepatic enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)

Genetics of xenobiotic metabolism in Drosophila. I. Genetic and environmental factors affecting glutathione-S-transferase in larvae.

The enzyme glutathione-S-transferase, which plays a crucial role in xenobiotic detoxification, was investigated in Drosophila melanogaster. Based upon examination of substrate specificities and pH optima, it was observed that the enzyme in Drosophila is considerably more restricted in its activities than in mammals. The effects of various xenobiotics on activities in third instar larvae were examined. While beta-naphthoflavone and phenobarbital had no effect, pentamethyl benzene (PMB) administration resulted in a 50% increase in enzyme activity. Comparison of lines of known genetic composition indicates that the degree of response to PMB is modulated by genes on chromosome II, and that differences exist with respect to the patterns of response of activities towards the substrates 1-chloro-2, 4-dinitrobenzene and ethacrynic acid. Results obtained suggest the existence of at least two loci on chromosome II that code for glutathione S-transferase isozymes.

Elevation of serum cholesterol levels in mice by the antioxidant butylated hydroxyanisole.

The food antioxidants butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) are structurally related to the hypocholesterolemic drug probucol. The purpose of this study was to determine if BHA can lower serum cholesterol levels as is observed with probucol. Treatment of mice with 0.75% BHA in their feed for 10 days resulted in a significant (P < or = 0.01) elevation of serum cholesterol levels. This effect contrasts with the cholesterol-lowering effect of probucol. Further, the degree of cholesterol elevation was comparable to that observed in mice administered 3% cholesterol in their feed for 7 days. The enzyme acyl CoA:cholesterol acyltransferase (ACAT) was decreased significantly (P < or = 0.01) in liver microsomes from BHA-treated mice. In contrast, hepatic microsomal ACAT activity was increased significantly (P < or = 0.01) in cholesterol-fed mice. These results suggested that the increased serum cholesterol observed in BHA-treated mice was not accompanied by an increase in hepatic cholesterol levels. Indeed, hepatic microsomal cholesterol levels were reduced in BHA-treated mice, but were increased significantly (P < or = 0.01) in cholesterol-fed mice. These results demonstrate that the common food additive BHA elevates serum cholesterol levels by a mechanism that apparently involves the decreased uptake of cholesterol by the liver.

Hypocholesterolemic properties of plant indoles. Inhibition of acyl-CoA:cholesterol acyltransferase activity and reduction of serum LDL/VLDL cholesterol levels by glucobrassicin derivatives.

Studies were undertaken to investigate the effects of the plant compound indole-3-carbinol (I3C) and its acid condensation products, which are generated in the stomach following ingestion of I3C, on cholesterol homeostasis in mice. Individual acid condensation products were synthesized and purified by HPLC. In vitro experiments revealed that several of the acid condensation products effectively inhibited the enzyme acyl-CoA:cholesterol acyltransferase (ACAT), which is responsible for the conversion of free cholesterol to the cholesteryl ester, at micromolar concentrations. Since the inhibition of ACAT in vivo should reduce serum cholesterol levels, I3C was administered to mice, and the effects on serum cholesterol levels were evaluated. Total serum cholesterol levels were elevated by 29% in mice provided a 3% cholesterol-supplemented diet, but this elevation was attenuated significantly (P < or = 0.05) by approximately 50% when I3C (100 mg/kg/day) was added to this diet. This effect of I3C was entirely on low density lipoprotein (LDL)/very low density lipoprotein (VLDL) cholesterol, which was lowered significantly (P < or = 0.05) by approximately 30%. In summary, I3C lowered serum LDL/VLDL cholesterol levels in mice, and this effect was likely mediated by the inhibition of ACAT by some of the acid condensation products of I3C. These results provide a possible mechanism by which I3C-rich vegetables lower serum cholesterol levels.

Are environmental sentinels signaling?

There is an increasing perception that environmental contamination by chemicals no longer poses a significant health threat and that relaxation of environmental regulations is warranted. However, many wildlife populations are showing signs of developmental, behavioral, and reproductive dysfunction due to environmental contamination by endocrine-disrupting chemicals. Scientists, regulators, and legislators must mobilize to identify current health threats posed by environmental pollutants, develop testing protocols that will detect such properties of new chemicals, and strengthen legislation designed to protect environmental health.

Assessing deleterious ecosystem-level effects of environmental pollutants as a means of avoiding evolutionary consequences.

Recent interest has been expressed in the possible need to develop ways to detect and quantify pollutants that affect evolution. Although environmental pollutants clearly can affect evolutionary processes, the evolutionary changes are a response to ecosystem-level toxicity elicited by the pollutant, rather than a direct effect of the pollutant on evolution. Accordingly, emphasis needs to be placed on assessing the adverse effects of environmental pollutants on ecosystem structure and function in order to avoid subsequent evolutionary consequences.

Chronic toxicity of environmental contaminants: sentinels and biomarkers.

Due to the use of a limited number of species and subchronic exposures, current ecological hazard assessment processes can underestimate the chronic toxicity of environmental contaminants resulting in adverse responses of sentinel species. Several incidences where sentinel species have responded to the effects of chronic exposure to ambient levels of environmental contaminants are discussed, including the development of neoplasia in fish, immunosuppression in marine mammals, pseudohermaphrodism in invertebrates, teratogenicity in amphibians, and aberrations in the sexual development of fish and reptiles. Biomarkers of chronic toxicity, including DNA mutations, alterations in specific protein and mRNA levels, and perturbations in metabolism, are presented. The incorporation of appropriate surrogate species and biomarkers of chronic toxicity into standard toxicity characterizations is proposed as a means of significantly refining the ecological hazard assessment process.

Embryotoxicity of the alkylphenol degradation product 4-nonylphenol to the crustacean Daphnia magna.

Laboratory studies have suggested that some alkylphenols and pesticides elicit developmental toxicity to crustaceans. The purpose of the present study was to evaluate the possibility that the alkylphenol degradation product 4-nonylphenol is embryotoxic to the crustacean Daphnia magna through its known ability to interfere with the metabolic elimination of testosterone. Direct exposure of maternal daphnids to testosterone caused developmental abnormalities in neonates that consisted of partial arrest of early embryonic development and abnormalities in shell spine and first antennae development. Exposure of maternal daphnids to concentrations of 4-nonylphenol also produced developmental abnormalities though the profile of abnormalities was distinct from that observed throughout the testosterone concentration-response curve. Thus, 4-nonylphenol is a developmental toxicant in daphnids, but its toxicity is not consistent with that elicited by elevated testosterone accumulation. Further experiments demonstrated that testosterone was directly toxic to developing embryos, and the maternal organism can serve as the vector for this toxicity. In contrast, neither direct embryo exposure nor early maternal exposure to 4-nonylphenol elicited embryotoxicity consistent with that observed during continuous maternal and gestational exposure. Thus, 4-nonylphenol is not directly embryotoxic at these exposure levels, but rather toxicity is mediated by maternal influences during gestation. The threshold concentration for the occurrence of developmental abnormalities ( approximately 44 microg/L) indicates that typical environmental concentrations of 4-nonylphenol pose no imminent hazard with respect to developmental toxicity. However, these effects do occur at sufficiently low levels to warrant evaluation of the relative susceptibility of other crustacean species to this previously uncharacterized mode of toxicity.

Structure-activity relationships for xenobiotic transport substrates and inhibitory ligands of P-glycoprotein.

The multixenobiotic resistance phenotype is characterized by the reduced accumulation of xenobiotics by cells or organisms due to increased efflux of the compounds by P-glycoprotein (P-gp) or related transporters. An extensive xenobiotic database, consisting primarily of pesticides, was utilized in this study to identify molecular characteristics that render a xenobiotic susceptible to transport by or inhibition of P-gp. Transport substrates were differentiated by several molecular size/shape parameters, lipophilicity, and hydrogen bonding potential. Electrostatic features differentiated inhibitory ligands from compounds not catagorized as transport substrates and that did no interact with P-gp. A two-tiered system was developed using the derived structure-activity relationships to identify P-gp transport substrates and inhibitory ligands. Prediction accuracy of the approach was 82%. We then validated the system using six additional pesticides of which tow were predicted to be P-gp inhibitors and four were predicted to be noninteractors, based upon the structure-activity analyses. Experimental determinations using cells transfected with the human MDR1 gene demonstrated that five of the six pesticides were properly catagorized by the structure-activity analyses (83% accuracy). Finally, structure-activity analyses revealed that among P-gp inhibitors, relative inhibitory potency can be predicted based upon the surface area or volume of the compound. These results demonstrate that P-gp transport substrates and inhibitory ligands can be distinguished using molecular characteristics. Molecular characteristics of transport substrates suggest that P-gp may function in the elimination of hydroxylated metabolites of xenobiotics.

Pesticides: multiple mechanisms of demasculinization.

Many pesticides are known to produce reproductive and developmental effects in chronically exposed non-target organisms, including humans. Recent evidence suggests that demasculinization may be an important mechanism responsible for some of these effects. Some pesticides have been shown to interact with the androgen receptor and to act as antagonists, while others have been shown to interact with the estrogen receptor and function as estrogens in both in vitro and in vivo. Many pesticides can also lower serum androgen levels by altering rates of synthesis or metabolism. Given the ubiquity of pesticides in the environment and the multiple mechanisms whereby they can elicit demasculinizing effects, synergy between such compounds may produce clinical endocrine dysfunction at current human exposure levels.

Alterations in sexually dimorphic biotransformation of testosterone in juvenile American alligators (alligator mississippiensis) from contaminated lakes.

The goal of this study was to determine whether hepatic biotransformation of testosterone is normally sexually dimorphic in juvenile alligators and whether living in a contaminated environment affects hepatic dimorphism. Lake Woodruff served as our reference site. Moonshine Bay, located on the west side of Lake Okeechobee, served as an intermediate site. Lake Apopka, the Belle Glade area located at the south end of Lake Okeechobee, and Water Conservation Area 3A, in the northern Everglades, served as our contaminated sites (all lakes are in Florida). Normal testosterone hydroxylase activity exhibited sexually dimorphic patterns of expression, with reference animals from Lake Woodruff exhibiting a female:male ratio of 1.44. This pattern was perturbed in all of the intermediate and contaminated sites investigated. Normal testosterone oxido-reductase activity exhibited sexually dimorphic expression (Lake Woodruff female:male ratio of 1.45). This pattern was altered in all contaminated sites investigated. UDP-Glucuronosyltransferase activity exhibited no sexually dimorphic pattern in animals collected from our reference site, with Lake Woodruff animals exhibiting a female:male ratio of 1.06. This pattern was perturbed in animals from Water Conservation Area 3A, which exhibited a female:male ratio of 0.65. Sulfotransferase activity demonstrated no sexual dimorphism at any of the sites investigated, although elevated activity was observed in males from the Lake Okeechobee watershed compared to those from Lake Woodruff. These data demonstrate different patterns of hepatic androgen biotransformation in animals living in contaminated environments.

Alteration in sexually dimorphic testosterone biotransformation profiles as a biomarker of chemically induced androgen disruption in mice.

Assessment of the impact of environmental chemicals on androgen homeostasis in rodent models is confounded by high intraindividual and interindividual variability in circulating testosterone levels. Our goal was to evaluate changes in testosterone biotransformation processes as a measure of androgen homeostasis and as a biomarker of exposure to androgen-disrupting chemicals. Sex-specific differences in hepatic testosterone biotransformation enzyme activities were identified in CD-1 mice. Gonadectomy followed by replacement of individual steroid hormones identified specific sex differences in biotransformation profiles that were due to the inductive or suppressive effects of testosterone. Notably, significant androgen-dependent differences in testosterone 6[alpha]- and 15[alpha]-hydroxylase activities were demonstrated, and the ratio of 6[alpha]- and 15[alpha]-hydroxylase activities proved to be an excellent indicator of the androgen status within the animal. The male or "masculinized" testosterone 6[alpha]/15[alpha]-hydroxylase ratio was significantly less than the female or "feminized" ratio. Male mice were exposed to both an antiandrogen, vinclozolin, and to a compound that modulates serum androgen levels, indole-3-carbinol, to test the utility of this ratio as a biomarker of androgen disruption. Treatment with the antiandrogen vinclozolin significantly increased the 6[alpha]/15[alpha]-hydroxylase ratio. Indole-3-carbinol treatment resulted in a dose-dependent, but highly variable, decrease in serum testosterone levels. The 6[alpha]/15[alpha]-hydroxylase ratio increased as serum testosterone levels decreased in these animals. However, the increase in the ratio was much less variable and more sensitive than serum testosterone levels. These investigations demonstrate that the 6[alpha]/15[alpha]-hydroxylase ratio is a powerful measure of androgen modulation and a sensitive indicator of exposure to androgen-disrupting chemicals in CD-1 mice.

The contribution of hepatic inactivation of testosterone to the lowering of serum testosterone levels by ketoconazole.

Hepatic biotransformation processes can be modulated by chemical exposure and these alterations can impact the biotransformation of endogenous substrates. Furthermore, chemically mediated alterations in the biotransformation of endogenous steroid hormones have been implicated as a mechanism by which steroid hormone homeostasis can be disrupted. The fungicide ketoconazole has been shown to lower serum testosterone levels and alter both gonadal synthesis and hepatic inactivation of testosterone. The present study examined whether the effects of ketoconazole on the hepatic biotransformation of testosterone contribute to its lowering of serum testosterone levels. Results also were used to validate further the use of the androgen-regulated hepatic testosterone 6alpha/15alpha-hydroxylase ratio as an indicator of androgen status. Male CD-1 mice were fed from 0 to 160 mg/kg ketoconazole in honey. Four h after the initial treatment, serum testosterone levels, gonadal testosterone secretion, and hepatic testosterone hydroxylase activity decreased, and the hepatic testosterone 6alpha/15alpha-hydroxylase ratio increased in a dose-dependent manner. Immunoblot analysis indicated that the transient decline in hepatic biotransformation was not due to reduced P450 protein levels. Rather, hepatic testosterone biotransformation activities were found to be differentially susceptible to direct inhibition by ketoconazole. Differential inhibition was also responsible for the increase seen in the 6alpha/15alpha-hydroxylase ratio. The changes in serum testosterone levels could be explained by decreased gonadal synthesis of testosterone and were not impacted by decreased hepatic biotransformation of testosterone. These results demonstrate that changes in the hepatic hydroxylation of testosterone by ketoconazole, and perhaps other chemicals, have little or no influence serum testosterone levels.