Hepatic P450 expression in hypothyroid rats: differential responsiveness of male-specific P450 forms 2a (IIIA2), 2c (IIC11), and RLM2 (IIA2) to thyroid hormone

Studies carried out in hypophysectomized adult rats have demonstrated that both thyroid hormone and GH can suppress hepatic expression of the steroid 6 beta-hydroxylase P450 2a (IIIA2). The present study further characterizes the influence of thyroid hormone on the expression of P450 2a and two other male-specific hepatic P450s, a steroid 2 alpha/16 alpha-hydroxylase, designated P450 2c (IIC11), and a steroid 15 alpha-hydroxylase, designated P450 RLM2 (IIA2). These studies were carried out in rats rendered hypothyroid by treatment with methimazole, which allows for the nonsurgical depletion of circulating T4, and in hypophysectomized rats. Hypothyroidism led to an increase in hepatic P450 2a (IIIA2) protein and mRNA in both male and female rats that was fully reversed by T4 replacement. In contrast, hypothyroidism decreased by 70-80% the expression of P450 2c (IIC11) activity and mRNA, but did not significantly alter the expression of P450 RLM2 (IIA2). The decrease in P450 2c (IIC11) was not reversed by T4 replacement, suggesting that it is a consequence of the loss of plasma GH pulses that occurs secondary to hypothyroidism. In agreement with these findings, T4 given to hypophysectomized rats partially suppressed the expression of P450 2a (IIIA2) mRNA, but not P450 2c (IIC11) or P450 RLM2 (IIA2) mRNA. A more complete suppression of P450 2a (IIIA2) mRNA as well as P450 2c (IIC11) mRNA was achieved when the hypophysectomized rats were treated with T3 at a supraphysiological, receptor-saturating dose. Although GH administered to intact male rats by continuous infusion fully suppressed all three male-specific P450 proteins and their mRNAs, the same treatment given to hypothyroid rats was only partially suppressive in the case of P450 2a (IIIA2) and P450 RLM2 (IIA2), unless combined with T4. In the case of P450 2c (IIC11), substantial suppression of the residual P450 present in hypothyroid rats was achieved by treatment with GH alone, despite persistent thyroid hormone deficiency. These studies demonstrate that while thyroid hormone is a negative regulator of P450 2a (IIIA2) expression and is required for the full suppression of that P450 and P450 RLM2 (IIA2) by the continuous plasma GH profiles associated with adult female rats, the suppression of P450 2c (IIC11) by continuous plasma GH is largely independent of the presence of thyroid hormone.     

Feminization of rat hepatic P-450 expression by cisplatin. Evidence for perturbations in the hormonal regulation of steroid-metabolizing enzymes

The biochemical basis for the complex effects of the anti-cancer drug cisplatin on hepatic cytochrome P-450 activity was studied in adult male rat liver using P-450 form-specific steroid hydroxylase assays and antibody probes. Cisplatin treatment of adult male rats resulted in a marked and prolonged feminization of the pattern of P-450 enzymes expressed in hepatic tissue. The adult male-specific cytochrome P-450 forms designated P-450 2c (P-450 gene IIC11), P-450 2a (gene IIIA2), and P-450 RLM2 were decreased by 70-90% after 7-14 days, with parallel decreases in their respectively associated microsomal steroid hydroxylase activities. Concomitantly, hepatic levels of the female-predominant enzymes P-450 3 (gene IIA1) and P-450j (gene IIE1) were elevated approximately 2-4-fold. The female-specific microsomal enzyme androstenedione 5 alpha-reductase was induced approximately 20-fold by cisplatin; however, no elevation of the female-specific P-450 2d was detected. The underlying hormonal basis for these effects of cisplatin was then examined. Serum testosterone levels were found to be depleted by cisplatin in a time- and dose-dependent manner which correlated with the observed changes in these hepatic enzymes. Furthermore, castration of adult rats altered the profile of these enzymes in a manner which resembled that observed with cisplatin treatment, suggesting that androgen depletion was the primary cause for the observed feminization of hepatic enzyme expression. Consistent with this possibility, the synthetic androgen methyltrienolone effectively blocked the changes in hepatic enzyme expression induced by cisplatin. Moreover, hepatic enzyme feminization was significantly reversed by chorionic gonadotropin, which fully restored serum testosterone levels in the cisplatin-treated rat. Luteinizing hormone-releasing hormone challenge experiments demonstrated that the responsiveness of the pituitary to this hypothalamic regulator of testicular androgen production was unimpaired by cisplatin treatment, indicating that hypothalamic production or secretion of luteinizing hormone-releasing hormone may be deficient in the cisplatin-treated animals. These studies establish that the effects of cisplatin on hepatic P-450 enzyme expression result from its interruption of the hypothalamic-pituitary stimulation of testicular androgen production and that this, in turn, leads to a depletion of circulating androgens required for maintenance of normal P-450 enzyme expression in adult male rats.     

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.     

Modulation of hepatic level of microsomal testosterone 7 alpha-hydroxylase, P-450a (P450IIA), by thyroid hormone and growth hormone in rat liver

The effects of thyroid hormone and growth hormone on microsomal testosterone 7 alpha-hydroxylase, P-450a, were studied to understand the interaction of these hormone-mediated regulations in rats. In Western blots using anti-P-450a IgG, 1.7-fold higher content of P-450a was observed in livers of female than male adult rats, while no appreciable sex-related difference was detected in prepubertal rats and rats of 24 months of age. Treatment with n-propyl-2-thiouracil or thyroidectomy of male rats increased by 2-fold the hepatic content of P-450a, but neither regimen had a significant effect on the content in female rats. Levels of P-450a in both sexes of thyroidectomized rats were decreased by the supplementation of triiodothyronine (T3, 50 micrograms per kg, i.p. for 7 days) to levels similar to that observed in normal male rats. Hypophysectomy also caused an increase in microsomal P-450a content in male rats. Continuous infusion of human growth hormone, which mimicked the female secretion, further significantly increased the content in hypophysectomized rats to a level similar to that observed in normal female rats. In contrast, hepatic level of P-450a in hypophysectomized male and female rats was reduced by intermittent injection, which mimicked the male secretion. Clear suppression on the level of hepatic P-450a was also observed by the treatment of hypophysectomized rats with 5 or 50 micrograms/kg of T3 and of hGH-infused hypophysectomized rat with 50 micrograms/kg of T3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interacting role of thyroxine and growth hormone in the hepatic synthesis of alpha 2u-globulin and its messenger RNA

Hypophysectomy completely abolishes and thyroidectomy results in a 90% reduction in the hepatic content of alpha 2u-globulin and its mRNA in the male rat. Thyroid hormone is also known to be required for the synthesis and secretion of pituitary growth hormone. In the hypothyroid rat either thyroxine or growth hormone was found to increase the activity and number of sequences of the mRNA for alpha 2u-globulin (measured by translational assay and hybridizational analysis with a cloned cDNA probe) to the euthyroid level. Treatment of hypophysectomized rats with a hormone combination containing growth hormone but not thyroxine increased the hepatic level of the mRNA for alpha 2u-globulin to that of normal animals. From these results we conclude that thyroxine indirectly influences the hepatic concentration of the mRNA for alpha 2u-globulin through its effect on pituitary growth hormone. Although administration of growth hormone to hypothyroid animals raised the hepatic concentration of alpha 2u-globulin mRNA to the euthyroid level, synthesis of alpha 2u-globulin remained low (50% of the normal). Complete recovery of alpha 2u-globulin synthesis required thyroxine. Therefore, in addition to an indirect effect on the hepatic level of alpha 2u-globulin mRNA, thyroxine also directly influences the synthesis of this protein. This direct effect of thyroxine on alpha 2u-globulin synthesis seems to be exerted at a step distal to the formation of mature mRNA.     

Regulation of the expression of the sex-specific isoforms of cytochrome P-450 in rat liver

The hepatic metabolism of steroid hormones and of xenobiotics frequently depends on the expression of the sex-specific isoforms of cytochrome P-450 and on differences in sex hormones. Following biochemical, immunological and molecular biological investigations, it was shown that in adult rat liver there exist at least four male-specific and one female-specific isoforms of cytochrome P-450. The designation of these sex-specific genes is IIC11, IIIA2, IIC13 and IIA2 in males, and IIC12 in females. The irreversible programming of the expression of these isoforms of cytochrome P-450 in adulthood occurs during the perinatal period of life, and is named enzyme imprinting. One of the main factors that regulates the expression of the sex-specific isoforms of cytochrome P-450 is the level of androgens in the blood. Castration of adult rats decreased the level of the male isoforms of cytochrome P-450 and the activity of the monooxygenase enzyme system that remained higher than in intact females. The mechanism of enzyme imprinting can be explained as follows: neonatal androgens program the secretion of hypothalamic hormones, somatostatin and growth-hormone-releasing factor. These factors determine the type of growth hormone secretion in adult rats, and this controls the type of sex-specific isoforms of cytochrome P-450 expressed in adulthood. Metabolic regulation similar to that outlined above was shown to occur for several metabolism-dependent chemical carcinogens. Such a pathway may explain the different sensitivity displayed by male and female rats to treatment with these carcinogenic agents. One possible way of modulating the expression of some isoforms of cytochrome P-450 in adult rats is by treating neonates with specific xenobiotics that change the constitutive expression of neonatal androgens. It appears that this enzyme imprinting plays an important role in determining the individual sensitivity to the carcinogenic effects of chemicals.     

Effects of thyroid state on the expression of hepatic thyroid hormone transporters in rats

Liver uptake of thyroxine (T4) is mediated by transporters and is rate limiting for hepatic 3,3',5-triiodothyronine (T3) production. We investigated whether hepatic mRNA for T4 transporters is regulated by thyroid state using Xenopus laevis oocytes as an expression system. Because X. laevis oocytes show high endogenous uptake of T4, T4 sulfamate (T4NS) was used as an alternative ligand for the hepatic T4 transporters. Oocytes were injected with 23 ng liver mRNA from euthyroid, hypothyroid, or hyperthyroid rats, and after 3-4 days uptake was determined by incubation of injected and uninjected oocytes for 1 h at 25 degrees C or for 4 h at 18 degrees C with 10 nM [125I]T4NS. Expression of type I deiodinase (D1), which is regulated by thyroid state, was studied in the oocytes as an internal control. Uptake of T4NS showed similar approximately fourfold increases after injection of liver mRNA from euthyroid, hypothyroid, or hyperthyroid rats. A similar lack of effect of thyroid state was observed using reverse T3 as ligand. In contrast, D1 activity induced by liver mRNA from hyperthyroid and hypothyroid rats in the oocytes was 2.4-fold higher and 2.7-fold lower, respectively, compared with euthyroid rats. Studies have shown that uptake of iodothyronines in rat liver is mediated in part by several organic anion transporters, such as the Na+/taurocholate-cotransporting polypeptide (rNTCP) and the Na-independent organic anion-transporting polypeptide (rOATP1). Therefore, the effects of thyroid state on rNTCP, rOATP1, and D1 mRNA levels in rat liver were also determined. Northern analysis showed no differences in rNTCP or rOATP1 mRNA levels between hyperthyroid and hypothyroid rats, whereas D1 mRNA levels varied widely as expected. These results suggest little effect of thyroid state on the levels of mRNA coding for T4 transporters in rat liver, including rNTCP and rOATP1. However, they do not exclude regulation of hepatic T4 transporters by thyroid hormone at the translational and posttranslational level.     

Thioredoxin and glutaredoxin systems of rat liver cytosol are not influenced by thyroid dysfunction

The relation of thyroid hormone status to the function of hepatic cytosolic components activating microsomal reverse triiodothyronine (rT3) 5'-monodeiodination was studied in rats. Hyperthyroidism was induced by administration of thyroxine and hypothyroidism, by thyroidectomy. The DTT-stimulated microsomal rT3 5'-monodeiodination rate was increased by 125% in hyperthyroid rats and reduced to about 30% in hypothyroid rats (when compared with euthyroid animals). Thyroid status was unrelated to NADPH-dependent activation of microsomal 5'-deiodinase by cytosol components or to cytosolic concentrations of thioredoxin and glutaredoxin, which stimulate in vitro microsomal deiodination of thyroid hormone.     

Role of growth hormone in modulating the constitutive and phenobarbital-induced levels of two P-450(6)beta (testosterone 6 beta-hydroxylase) mRNAs in rat livers

The role of growth hormone in the expression of two forms of hepatic cytochrome P-450(P-450), P-450(6)beta-1(6 beta-3), and P-450(6)beta-4, was investigated using RNA blots. The level of P-450(6)beta-1(6 beta-3) mRNA was twenty times higher than that of P-450(6) beta-4 mRNAs in untreated male rat livers. The levels of P-450(6)beta-1(6 beta-3) and P-450(6)beta-4 mRNAs were increased two fold and three fold, respectively, by hypophysectomy of adult male rats. By intermittent injection of human growth hormone (hGH) into hypophysectomized male rats, both mRNAs were decreased to the level of normal rats, and almost disappeared after continuous infusion of hGH. In female rats, these two mRNAs were not detected, but were increased remarkably by hypophysectomy. The increases in these mRNAs were almost abolished after continuous infusion of hGH in hypophysectomized female rats. The effect of hGH on PB-mediated induction of P-450(6)beta-1(6 beta-3) and P-450(6)beta-4 mRNAs was also examined. The PB-mediated increases in P-450(6)beta-1(6 beta-3) and P-450(6)beta-4 mRNAs were higher in hypophysectomized male rats (2.5-fold and 10.9-fold, respectively) than in normal male rats (1.5-fold and 5.2-fold, respectively). Thus, the levels of P-450(6)beta-1(6-beta-3) and P-450(6)beta-4 mRNAs were 4.1-fold and 7.3-fold, respectively, higher in PB-induced hypophysectomized rats than in normal male rats. Concerning the postnatal developmental profiles, P-450(6)beta-1(6 beta-3) mRNA was detectable at neonate and reached a maximal level at around 17 days of age.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multihormonal regulation of hepatic sinusoidal Ntcp gene expression

Bile acids are efficiently removed from sinusoidal blood by a number of transporters including the Na+-taurocholate-cotransporting polypeptide (Ntcp). Na+-dependent bile salt uptake, as well as Ntcp, are expressed twofold higher in male compared with female rat livers. Also, estrogen administration to male rats decreases Ntcp expression. The aims of this study were to determine the hormonal mechanism(s) responsible for this sexually dimorphic expression of Ntcp. We examined castrated and hypophysectomized rats of both sexes. Sex steroid hormones, growth hormone, thyroid, and glucocorticoids were administered, and livers were examined for changes in Ntcp messenger RNA (mRNA). Ntcp mRNA and protein content were selectively increased in males. Estradiol selectively decreased Ntcp expression in males, whereas ovariectomy increased Ntcp in females, confirming the importance of estrogens in regulating Ntcp. Hypophysectomy decreased Ntcp mRNA levels in males and prevented estrogen administration from decreasing Ntcp, indicating the importance of pituitary hormones. Although constant infusion of growth hormone to intact males reduced Ntcp, its replacement alone after hypophysectomy did not restore the sex differences. In contrast, thyroid hormone and corticosterone increased Ntcp mRNA in hypophysectomized rats. Sex differences in Ntcp mRNA levels were produced only when the female pattern of growth hormone was administered to animals also receiving thyroid and corticosterone. Thyroid and dexamethasone also increased Ntcp mRNA in isolated rat hepatocytes, whereas growth hormone decreased Ntcp. These findings demonstrate the essential role that pituitary hormones play in the sexually dimorphic control of Ntcp expression in adult rat liver and in the mediation of estrogen effects.     

Hormonal regulation of rat liver microsomal enzymes. Role of gonadal steroids in programming, maintenance, and suppression of delta 4-steroid 5 alpha-reductase, flavin-containing monooxygenase, and sex-specific cytochromes P-450

Neonatal gonadectomy studies and hormonal replacement regimens were employed to characterize the regulation of delta 4-steroid 5 alpha-reductase, microsomal flavin-containing monooxygenase, and several forms of rat hepatic microsomal cytochrome P-450, including three that are sexually differentiated. Rats of both sexes that had been gonadectomized at birth were either untreated or were administered testosterone propionate or estradiol benzoate neonatally (subcutaneous injection on days 1 and 3 of life), postpubertally (an implant of a hormone-packed capsule at 5 weeks of age), or both neonatally and postpubertally. At the age of 10 weeks, all rats were killed, and several liver microsomal enzymes were assayed using immunochemical and catalytic techniques. Expression in the 10-week-old male and female rats of two male-specific cytochrome P-450 forms, termed P-4502c/UT-A and P-4502a/PCN-E, and their associated respective 16 alpha- and 6 beta-steroid hydroxylase activities could either be imprinted (programmed) by androgen exposure during the early neonatal period or, alternatively, could be stimulated by continuous hormone treatment after the age of 5 weeks. By contrast, hepatic expression of two female-specific enzymes, P-4502d/UT-1 and delta 4-steroid 5 alpha-reductase, was only partially dependent on estradiol; birth-gonadectomized rats expressed as much as 30-50% of the enzyme levels present in untreated adult females. Expression of both female-specific enzymes was fully suppressed upon postpubertal exposure to testosterone. In another study, birth sham-operated female rats were administered testosterone using the same regimens described above for the birth-gonadectomized rats. Although neonatal testosterone treatment alone did not affect the expression in these females of the four sex-specific enzymes examined in this study, it did enhance significantly the masculinization effected by postpubertal androgen exposure. This resulted in expression of the male-specific enzymes P-4502c/UT-A and P-4502a/PCN-E in these females at levels comparable to those found in adult males, while simultaneously suppressing the two female-specific enzymes, P-4502d/UT-I and delta 4-steroid 5 alpha-reductase, by approximately 70-75% to levels characteristic of prepubertal rats of either sex. The levels of another microsomal enzyme, flavin-containing monooxygenase, were also measured and found to be regulated by testosterone, but the ontogenic profiles and the effects of gonadectomy and hormone replacement indicated clear differences in its regulation when compared to the other male-specific enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Hormonal and developmental regulation of expression of the hepatic microsomal steroid 16 alpha-hydroxylase cytochrome P-450 apoprotein in the rat

The hormonal regulation of the sexually differentiated cytochrome P-450 isozyme which catalyzes 16 alpha-hydroxylation of testosterone and 4-androstene-3,17-dione in male rat liver (P-450(16) alpha) was investigated. Estradiol valerate injection of male rats caused a decrease in P-450(16) alpha levels to almost the female level, while methyltrienolone injection had the reverse effect in female animals. Hypophysectomy abolished the sex difference in P-450(16) alpha levels. Human growth hormone infusion into male rats, mimicking the female pattern of growth hormone secretion, caused a feminization of P-450(16) alpha levels. The same effect was also seen in hypophysectomized rats of both sexes. In contrast, a different administration schedule involving 12 h injections of human growth hormone, mimicking the male pattern of growth hormone secretion, caused a masculinization of P-450(16) alpha levels in hypophysectomized rats, at a daily dose which causes feminization when given by infusion. Thus, the level of expression of P-450(16) alpha in the liver is dependent on the temporal pattern of blood growth hormone levels. While infusion of rat growth hormone into male rats also feminized the P-450(16) alpha levels, infusion of ovine prolactin had no effect. Ontogenic studies showed that the developmental pattern of P-450(16) alpha expression in the liver coincided with the known pattern of development of the sexual differentiation of hepatic steroid 16 alpha-hydroxylase activity and of the diurnal pattern of growth hormone secretion.     

Divergence of growth hormone actions on hepatic drug metabolism in the presence and absence of the pituitary gland.

Experiments were carried out to compare the effects of growth hormone on hepatic drug oxidation in normal and hypophysectomized rats. Administration of growth hormone to normal male rats lowered hepatic microsomal cytochrome P-450 content and decreased the rates of ethylmorphine n-demethylation and aniline hydroxylation. These effects were fully manifested in orchiectomized or adrenalectomized males, excluding a dependence upon endogenous steroids. Growth hormone was without effect on hepatic drug metabolism or cytochrome P-450 content in normal female rats. In contrast to its actions in animals with intact pituitary glands, administration of growth hormone to hypophysectomized rats of either sex increased the rate of ethylmorphine metabolism. Furthermore, in both males and females, aniline hydroxylation and microsomal cytochrome P-450 content were unaffected by growth hormone in the absence of the pituitary gland. Prolactin administration did not affect hypophysectomized or in normal rats of either sex. The results indicate that the nature of growth hormone actions on hepatic drug oxidation is pituitary-dependent and probably intertwined with the effects of other hormones. Furthermore, the direct physiological effects of growth hormone on hepatic mixed function oxidases seem to depend upon the substrate employed.