Comparison of delta-aminolaevulinic acid dehydratase activity in chick liver during sex steroid hormone dependent primary and secondary stimulation

1. Comparative study on primary and secondary stimulation of hepatic delta-aminolaevulinic acid dehydratase (ALAD) (EC 4.2.1.24) was carried out after oestradiol-17 beta and/or testosterone administration in immature female chicken. 2. When 2 mg/day oestradiol was administered to birds for 15 days successively, hepatic total ALAD activity increased to 170% by day 15 of primary stimulation, whereas a more rapid increased rate was observed within day 3 of secondary stimulation and thereafter the hepatic ALAD activity maintained the same high level from day 3 to day 15. 3. Testosterone (2 mg/day) alone decreased hepatic total ALAD activity during both primary and secondary stimulation. 4. When testosterone (0.25-10 mg/day) was injected into birds in combination with 2 mg oestradiol for 15 days during primary and secondary stimulation, only an antagonistic effect of testosterone on oestradiol-stimulated total ALAD activity in liver was observed independently of the testosterone amount administered. However, the extent of suppression of hepatic ALAD activity by testosterone during primary stimulation was markedly different from that of secondary stimulation.     

Comparison of sex steroid hormone-dependent induction of chick oviduct delta-aminolaevulinic acid dehydratase during primary and secondary stimulation.

1. A comparative study on primary and secondary stimulation of oviduct delta-aminolaevulinic acid dehydratase (ALAD) (EC 4.2.1.24) was carried out with oestradiol-17 beta and/or testosterone administration in immature female chickens during 15-day-primary stimulation, 20-day-withdrawal and 15-day-secondary stimulation periods. 2. Compared with primary stimulation in oestrogenized birds, synthesis and degradation rates of oviduct ALAD molecule during secondary stimulation increased 3.4- and 1.8-fold respectively, resulting in a rapid induction of the enzyme. 3. Specific activity of oviduct ALAD in oestradiol-plus-testosterone treated birds became significantly higher than that of oestradiol alone during secondary stimulation, whereas no significant changes were observed during primary stimulation.     

Metal constitution of metallothionein influences inhibition of delta-aminolaevulinic acid dehydratase (porphobilinogen synthase) by lead.

This study was undertaken to evaluate the effect of Zn and Cd pretreatment on the inhibition of delta-aminolaevulinic acid dehydratase (ALAD; porphobilinogen synthase, EC 4.2.1.24) by Pb. Male CD rats were pretreated with 200 mumol of Zn/kg s.c. (subcutaneously) or 18 mumol of Cd/kg s.c., 48 and 24 h before assay of ALAD. Pretreatment with Zn resulted in activation of hepatic and renal ALAD and attenuated the inhibition of this enzyme by Pb in vitro. Pretreatment with Cd increased hepatic ALAD activity, and the inhibitory effect of Pb on the hepatic enzyme was attenuated in this group. In contrast with the situation in liver, pretreatment with Cd did not affect the activity of renal ALAD and did not alter the inhibitory effect of Pb on the renal enzyme. The Pb IC50 (concentration causing half-maximal inhibition) values for hepatic and renal ALAD in Zn-pretreated rats and for hepatic ALAD in Cd-pretreated rats were increased above control, whereas the IC50 for renal ALAD in Cd-pretreated rats was unchanged. Cytosolic binding patterns for the three metals were assessed by gel-filtration chromatography and disclosed that 203Pb was co-eluted with Zn and Cd bound to liver and kidney Zn-thioneins and liver Cd,Zn-thionein, although minimal binding of 203Pb to kidney Cd,Zn-thionein was observed. Estimation of the molar ratio of metals bound revealed Cd/Zn ratios of 2 and 5 for Cd,Zn-thioneins from liver and kidney respectively. The inhibition of purified ALAD by Pb was also attenuated by addition of purified Zn-thioneins and Cd,Zn-thioneins from liver and kidney in the following order: liver Zn-thionein = kidney Zn-thionein greater than liver Cd,Zn-thionein much greater than kidney Cd,Zn-thionein. Thus liver and kidney Zn-thioneins and liver Cd,Zn-thionein with a low Cd/Zn ratio readily decrease the free pool of Pb available to interact with ALAD. These data also demonstrate that the capacity of metallothionein to alter the intracellular distribution of Pb and mediate the inhibition of ALAD by Pb is dependent on the tissue source and relative metal constitution of the metallothionein.     

The additive effects of progestins on testosterone-stimulated hepatic ethylmorphine metabolism and cytochrome P-450 content

The actions of several progestins on mouse liver were studied in terms of their inherent potency and for their ability to modify the biologic activity of testosterone. When hepatic ethylmorphine demethylase activity and cytochrome P-450 content were used as end points, the biological potency of progestins was ranked as follows: cyproterone acetate>progesterone>medroxyprogesterone acetate>hydroxyprogesterone caproate controls. The induced alterations of these parameters were, therefore, unrelated to reported progestational (cyproterone acetate medroxyprogesterone acetate>>hydroxyprogesterone caproate>progesterone) or androgenic (medroxyprogesterone acetate>cyproterone acetate = hydroxyprogesterone caproate = progesterone) actions of these steroids. A similar conclusion was reached when hepatic weight and microsomal protein content were used as end points.

When progestins (0.1–10 mg/day) were administered with testosterone (0.1 mg/day), the effect of both steroids were additive. This is in contrast to their actions on other tissues such as kidney and sub-maxillary gland where progestins potentiate and inhibit androgen action. We conclude from these studies that the mechanism of action of progestins on the liver differs from that on other tissues.     

Modulation of quail oviduct adenylate cyclase activity by estradiol and progesterone

Oviduct adenylate cyclase activity of the quail was measured by radiochemical analysis following different hormonal treatments. A single injection of estradiol benzoate (EB) to immature female quails resulted in a prereplicative surge of adenylate cyclase activity. A second surge of enzyme activity was observed during the proliferative phase induced by EB. Estradiol-17 alpha, estrone, estriol and testosterone were ineffective. Tamoxifen completely inhibits the growth-promoting effect of EB and the second surge of adenylate cyclase activity but does not inhibit the prereplicative increase of enzyme activity. This prereplicative increase of adenylate cyclase activity was also observed, even in the absence of increased plasma estradiol, when estradiol-17 beta (E2) was perfused through the hepatic portal vein. Moreover, E2 had no effect on enzyme activity when added directly to the oviduct homogenate preparation, at concentrations ranging from 10(-9) to 10(-7) M. In response to progesterone injection, oviduct adenylate cyclase activity followed a different pattern, beginning its increase after 3 h and remaining elevated up to 24 h. The activation by estradiol was independent of the presence of guanylylimidodiphosphate. Moreover, the enzyme was more sensitive to forskolin at submaximal concentration in estradiol treated birds than in control. These results demonstrate that transient activation of adenylate cyclase at the early stages of the action of estradiol does not occur through the classic nuclear receptor-gene activation pathway or a membrane receptor mediated process, but involves an indirect pathway, yet to be defined.     

Reactivity of the epithelial cells of the bovine oviduct in vitro on the exogenic gonadotropic and steroid hormones. Part I: The effect of gonadotropic and steroid hormones on the amount of lipids and activity of dehydrogenases.

Effects of 17 beta-estradiol, testosterone, progesterone, luteinizing hormone and a combined effect of estradiol and progesterone on the epithelial cells of the bovine oviduct cultured in vitro were investigated. It was found that these cells may transform under the effect of hormones. The effect of the applied hormones on the amount of lipids and activity of the dehydrogenases delta 5 3 beta-OH-SDH and G6P-DH was evident. Cells in vitro most intensely reacted on testosterone and estradiol: these hormones caused an increase of lipids and of enzymatic activity. The cells also reacted to progesterone and the luteinizing hormone which in turn decreased both activity and accumulation of lipids in cells.     

Antagonism of the estradiol-mediated repression of microsomal 3β-hydroxysteroid dehydrogenase activity in rat liver by antiestrogenic substances

5α-Dihydrotestosterone, 17-hydroxyprogesterone caproate, 2-methoxyestrone and a number of nonsteroidal antiestrogens (clomiphene citrate, nafoxidine hydrochloride, tamoxifen, MER-25) were tested for their ability to block estradiol-mediated repression of the androgen-dependent 3β-hydroxy-steroid dehydrogenase activity of male rat liver. With the exception of 5α-dihydrotestosterone, which induced activity in females, none of these substances affected 3β-hydroxy-steroid dehydrogenase activity when administered alone to otherwise untreated male and female rats. Tamoxifen (100 or 500 μg/day) was the only substance which prevented a decrease in enzyme activity when given simultaneously with estradiol (5 μg/day). The estradiol-mediated decrease in activity was not antagonized by a 100-fold higher dose of androgen (5α-dihydrotestosterone, 0.5 mg/day), demonstrating the potent antiandrogenic effect of estradiol on this hepatic androgen-dependent enzyme activity.     

Estradiol administration and the sexual activity of castrated male rhesus monkeys (Macaca mulatta)

To examine whether estradiol might be effective in maintaining sexual behavior after castration or after testosterone withdrawal, we have observed male rhesus monkeys during daily 1-hr tests alternately with each of two ovariectomized, estradiol-treated females (four males, four females, eight male-female pairs, 798 tests). Estradiol (2-5 micrograms/kg sc/day) or vehicle was administered in counterbalanced order immediately after castration and again immediately after withdrawal of testosterone propionate treatments (800 micrograms and 1.6 mg sc/day). There were no significant differences in behavior during vehicle and estradiol treatments to indicate that estradiol helped to maintain male sexual activity. Instead, estradiol treatment tended to interfere with the capacity to intromit. This supported the results of other studies, namely, that the systemic administration of estradiol does not enhance the sexual behavior of castrated male macaques, and raises questions about the role of both aromatization and estrogen receptors in the male primate brain.     

Effects of corticosterone on the negative feedback action of testosterone, 5 alpha-dihydrotestosterone and estradiol in the adult male rat

Corticosterone acetate (10 mg/day) was administered to gonadectomized and adrenalectomized male rats bearing 5, 10 or 15 mm long testosterone filled silicone elastomer capsules. It was found that the serum testosterone levels induced by these capsules were not influenced by corticosterone treatment and that the weights of the prostates in the corticosterone treated rats were not different from their controls. In contrast, corticosterone acetate increased markedly the LH and FSH inhibitory effects of testosterone. Since several brain structures are able to convert testosterone into 17-beta-hydroxy-5-alpha-androstan-3-one (5-alpha-dihydrotestosterone) and/or estradiol, and these metabolites are probably involved in mechanisms controlling gonadotropin secretion, we studied also the effects of corticosterone on the feedback action of dihydrotestosterone and estradiol. 5 alpha-Dihydrotestosterone was administered by 5, 10 or 20 mm long elastomere capsules whereas estradiol was given by daily s.c. injections of 0.125, 0.25 or 0.50 micrograms estradiol benzoate. In the presence of corticosterone acetate the gonadotropin inhibitory action of testosterone, 5 alpha-dihydrotestosterone and estradiol increased more than 2 times.     

New insights into the mechanism of imposex induction in the dogwhelk Nucella lapillus

In an attempt to clarify the mechanism(s) of tributyltin-mediated imposex induction in females of the neogastropod Nucella lapillus, dogwhelks collected in an almost imposex free population were exposed to several treatments for a 3 month-period, and the effects on imposex induction and testosterone/estradiol levels were evaluated. As a positive control, tributyltin (50 ng TBT Sn/L) clearly induced imposex and led to a significant increase in the severity of the phenomenon. In contrast, although a selective P450 aromatase inhibitor (formestane at 0.3 mg/L) was capable of imposex induction, it failed to increase its severity. A vertebrate androgen receptor (AR) antagonist (cyproterone acetate at 1.25 mg/L) in combination with TBT completely blocked the imposex induction capacity of TBT. On the other hand, an estrogen receptor antagonist (tamoxifen at 0.3 mg/L) rendered no effect. The determination of steroid levels in female specimens revealed that TBT induces an elevation of free testosterone (but not the total amount, free+esterified), while the co-administration of the anti-androgen and TBT was able to rescue the increase of free testosterone levels. Despite a minor decrease in the amount of testosterone-fatty acid esters in the TBT group, significant differences in esterified testosterone were not found among treatments. On the contrary, free estradiol levels were elevated in the TBT, anti-androgens and TBT plus anti-androgens groups. These results indicate that free estradiol biosynthesis in TBT-exposed females does not seem to be affected. Overall, our results demonstrate that a selective aromatase inhibitor can induce imposex in N. lapillus but not to a similar extent of TBT, which may suggest the involvement of other mechanism in imposex induction, besides aromatase inhibition. Additionally, the study points to the involvement of AR receptors in imposex induction.     

Attenuation of low dose phenobarbital induction of hepatic microsomal aminopyrine N-demethylase activity in rats by cimetidine

Cimetidine, a substituted imidazole, is an inhibitor of hepatic cytochrome P-450-mediated drug metabolism in rats and humans. We investigated the effect of cimetidine on phenobarbital induction of hepatic microsomal aminopyrine N-demethylase activity in the rat. Phenobarbital induction of aminopyrine N-demethylase was log-linear in the range of 1-6 mg/kg/day and the ED50 was approximately 3 mg/kg/day. Cimetidine 75 mg/kg (four times a day) attenuated the induction of aminopyrine N-demethylase activity by 58% in low dose (3 mg/kg/day) but not in high dose (40 mg/kg/day) phenobarbital treated rats. This result could not be explained by residual inhibition of enzyme activity by cimetidine and suggests that cimetidine affects the induction of hepatic cytochrome P-450 by low dose phenobarbital.     

Strain differences in the dose-response curves of adrenalectomized, starved-refed rats to dehydroepiandrosterone (DHEA)

The effects of adrenalectomy and dehydroepiandrosterone (DHEA) doses (0, 15, 30, 60, 120 and 240 mg/kg/day ip) on hepatic enzyme activity and lipid content and on the amount of epididymal fat pad lipid were studied in starved-refed BHE and Sprague-Dawley rats. BHE rats had significantly greater relative liver size, glucose-6-phosphate dehydrogenase (G6PD) and malic enzyme (ME) activities, and percentage liver lipid but less epididymal fat pad lipid than Sprague-Dawley rats. Adrenalectomized (ADX) rats consumed significantly less food, gained less weight per day, and had less lipid in their livers and fat pads than intact rats. As the level of DHEA increased from 0 to 240 mg/kg/day there was a significant linear decrease in average daily weight gain, food intake, G6PD activity, and percentage liver lipid. At the 15 mg/kg/day dose, G6PD activity was significantly reduced without reductions in the other parameters measured. At the 120 mg/kg/day dose, however, weight gain, food intake, G6PD activity, and percentage liver lipid were significantly lower than that of the controls. At this dose DHEA treatment reduced food intake by 17% whereas it diminished average daily weight gain and G6PD activity by 30 and 56%, respectively. The 240 mg/kg/day dose of DHEA significantly reduced food intake, weight gain, liver lipid, G6PD activity, and ME activity. Intact and ADX BHE rats reduced their G6PD activity and liver lipid more rapidly than Sprague-Dawley rats as the level of DHEA administered increased. ADX Sprague-Dawley rats receiving DHEA had greater liver lipid content and enzyme activity than their intact counterparts whereas the reverse situation was true in BHE rats. These data indicate that the effect of DHEA on body weight gain, food intake, and hepatic and peripheral adiposity are dependent on the strain of rat, the adrenal status, and the DHEA dose.     

The anti-carcinogenic plant compound indole-3-carbinol differentially modulates P450-mediated steroid hydroxylase activities in mice.

Indole-3-carbinol (I3C), a component of cruciferous vegetables, exhibits anti-carcinogenic activity in a variety of model systems. This activity has been attributed in part to the induction of cytochrome P450 CYP1A subfamily members and the resulting increased metabolic inactivation of chemical carcinogens. The present study was undertaken to assess the effects of I3C on several constitutive P450 activities that contribute to both carcinogen and steroid hormone metabolism. Mice were administered I3C in their diet at estimated daily doses of 250, 500 and 750 mg/kg for 1 week. Liver microsomes from treated and untreated mice were subsequently assayed for CYP1A-mediated ethoxy-resorufin O-deethylase (EROD) activity, estradiol 2-hydroxylase activity and seven different testosterone hydroxylase activities. I3C elevated EROD, estradiol 2-hydroxylase and testosterone 6 alpha-hydroxylase activities in a dose-dependent manner. The other six testosterone hydroxylase activities were not significantly affected by in vivo treatment with I3C. In addition to its effects on steroid hydroxylase activities, I3C also elevated NADPH-cytochrome P450 reductase activity, a necessary component to the P450 monooxygenase system. We next examined the direct in vitro effects of I3C and its acid condensation products, as are generated in the stomach following ingestion, on the P450 catalytic activities. Testosterone 6 beta-hydroxylase, the major testosterone hydroxylase activity in untreated mice, was significantly inhibited (IC50 approximately 12 micrograms/ml) by the acid condensation products of I3C. In contrast, all other P450 activities were not appreciably affected by I3C or its acid condensation products. These results indicate that I3C can elicit both inductive and suppressive effects on the constitutive P450s that participate in carcinogen and steroid hormone metabolism. This pleiotropic effect on hepatic catalytic enzymes may contribute to the anti-carcinogenic properties of this compound.     

Coadministration of melatonin and estradiol in rats: effects on oxidant status.

This study was designed to investigate the effects of melatonin and estradiol (E2) on lipid peroxidation and antioxidant defense enzymes in blood and liver tissue when administered in vivo. Wistar albino rats were divided into three experimental groups and treated with either estradiol (25 mg/kg bw, s.c.), melatonin (i. p.), or melatonin plus E2, whereas control animals had diluent injections only. Melatonin was given 10 mg/kg bw x 2 intraperitoneally 30 min before and 60 min after E2 treatment to the melatonin plus E2 group. Animals were sacrificed three hours after the estradiol injection, and their blood and liver tissues were prepared for biochemical analyses. Tissue malondialdehyde (MDA) levels and antioxidant enzyme activities--superoxide dismutase (SOD) and glutathione peroxidase (GPx)--were determined in the postmitochondrial fraction, and the results were compared. Estradiol injection caused significant increases in both MDA levels and GPx activity in liver. When melatonin was administered in combination with E2, the effect of estradiol on MDA levels was abolished. A significant decrement in SOD activity occurred in melatonin-treated animals. GPx activity in the blood of E2 plus melatonin-injected animals was significantly higher than those in control animals. Melatonin-treated animals exhibited relatively lower levels of SOD activity than those from the control and E2 plus melatonin groups. This indicates that estradiol could exert oxidant action resulting in an increment in tissue malondialdehyde levels. Enhanced activity of GPx in both liver and blood following melatonin injection may indicate the contribution of this neurohormone on the antioxidant defense.     

Effect of a newly developed ketolide antibiotic, telithromycin, on metabolism of theophylline and expression of cytochrome P450 in rats

The effects of a newly-developed ketolide antibiotic, telithromycin, on the metabolism of theophylline and the expression of hepatic cytochrome P450 (CYP) 1A2 and CYP3A2 were investigated in rats. Telithromycin at a high dose (100 mg/kg of body weight) was injected intraperitoneally once a day for 3 days. Twenty-four hours (day 4) after the final administration of telithromycin, theophylline (10 mg/kg) was administered intravenously. The presence of telithromycin significantly delayed the disappearance of theophylline from plasma. Parameters related to the pharmacokinetic interaction between theophylline and telithromycin were examined by noncompartmental methods. A significant decrease in the systemic clearance of theophylline was observed in the presence of telithromycin. Pretreatment with telithromycin significantly decreased the metabolic clearance of the major metabolites, 1-methyluric acid and 1,3-dimethyluric acid, with no change in the renal clearance of theophylline, suggesting that the decreased systemic clearance of theophylline by telithromycin is due to reduction of their metabolic clearance. Pretreatment with telithromycin significantly decreased the activity of 7-ethoxyresorufin O-deethylation and testosterone 6 beta-hydroxylation, suggesting that telithromycin decreases the activity of hepatic CYP1A2 and CYP3A2. Western blot analysis revealed that telithromycin significantly decreased the protein levels of CYP1A2 and CYP3A2 in the liver, which could explain the observed decreases in the systemic clearance of theophylline and metabolic clearance of 1-methyluric acid and 1,3-dimethyluric acid. The present study suggests that telithromycin at the dose used in this study alters the pharmacokinetics and metabolism of theophylline, due to reductions in the activity and expression of hepatic CYP1A2 and CYP3A2.     

成年去胸腺大鼠和老年大鼠肝微粒体混合功能氧化酶及血浆性激素水平的变化

成年去胸腺(ATx)大鼠和老年大鼠肝微粒体混合功能氧化酶(MFO,包括细胞色素P450、氨基比林-N-脱甲基酶)的活力比成年对照大鼠的低,且降低幅度雄性明显大于雌性。雄性ATx大鼠和老年大鼠血浆睾酮(T)水平降低,雌二醇(E_2)水平增高,E_2/T比值明显增高;雌性ATx大鼠和老年大鼠血浆E_2和T水平均降低,E_2/T比值无明显变化。给雄性ATx大鼠皮下注射丙酸睾丸素可使其肝微粒体MFO活力恢复。提示胸腺对肝脏MFO的影响可能是通过性激素介导的。     

Estrogen antagonism on T3 and growth hormone control of the liver microsomal low-affinity glucocorticoid binding site (LAGS)

Male rat liver microsomes contain a low-affinity glucocorticoid binding site (LAGS) capable of binding all natural glucocorticoids and progesterone with a K_d from 20 to 100 nM. The LAGS level is under endocrine control by T₃, glucocorticoids and GH. These hormones act synergistically at physiological concentrations to increase the LAGS level. Since female rats show a LAGS level that is much lower than the males (0.15 vs 23 pmol/mg protein, respectively), here we investigated whether estradiol could decrease the LAGS in the male rat. Orchiectomized (OX) male rats showed a higher LAGS level than intact rats. This effect was reversed by implanting a Sylastic capsule containing testosterone. When the OX rats were implanted for 20 days with estrogen capsules that provided an estradiol level in serum of 40 pg/ml, their LAGS level decreased from 23 to 0.2 pmol/mg protein. This effect was not observed in intact male rats and can be partially reversed by testosterone implants into OX rats. Both hypophysectomized male rats and hypothyroid-orchiectomized male rats showed very low levels of LAGS. Administration of physiological doses of GH and/or T₃ to these rats greatly increased their LAGS level (from 0.3 to 15 and 16 pmol/mg protein, respectively). Implantation of estrogen capsules to these rats two weeks prior to starting treatment completely inhibited the increase in the LAGS level in response to T₃, and significantly decreased the response to hGH, and to a combination of hGH and T₃. These results suggest that physiological estradiol levels can antagonize the LAGS induction by T₃ and hGH in the male rat, and could be responsible for the low level of LAGS in the female rat. Moreover, estrogen capsules also inhibited the increase in the body and hepatic weights observed after hGH treatment, which suggests a powerful inhibitory effect of low estradiol levels on the male rat liver functions under regulation by T₃ and/or GH.     

A reproductive phase-dependent effect of dietary L-tryptophan on pineal gland and gonad of a nocturnal bird, Indian spotted owlet Athene brama

Unlike other temperate owls, Indian spotted owlet Athene brama possesses a well-developed pineal gland that secrets moderate amount of hydroxy- (serotonin) and methoxy- (melatonin) indoles in circulation. However, in this study, we have reported the response of this endocrine gland to exogenous L-Tryptophan (precursor of the above indoles), and also its effect on gonads of this nocturnal bird. During breeding phase or pineal inactive phase (March), oral treatment of L-Trp (0.5 mg/100 g Bwt/day) significantly increased the pineal gland wt and plasma melatonin (MEL) level, while decreased the gonadal wt and plasma sex steroids levels (estradiol and progesterone in female and testosterone in male). Interestingly, during reproductively quiescent phase or pineal active phase (August), similar amount of L-Trp significantly decreased the plasma MEL level, while increased the above sex steroid levels in plasma. Finally, the results show a clear reproductive phase-dependent inverse effect of L-Trp on pineal gland and gonads for both sexes of the spotted owlets, and suggest that the therapeutic use of this amino acid would be a great advantage for controlling the reproduction of these economically important birds.