Estrogen metabolism in primary kidney cell cultures from Syrian hamsters

Estrogen metabolism was evaluated in freshly isolated kidney and liver microsomes and in primary kidney cell cultures from Syrian hamsters, a potential experimental model for examining the possible role(s) of estrogens in tumor initiation and development. Initial velocity studies of the conversion of estradiol to 2-hydroxyestradiol, as determined by the 3H2O release assay with the substrate [2-3H]estradiol, resulted in similar apparent Kms of estrogen 2-hydroxylase of 2.85 and 6.25 microM for liver and renal microsomes, respectively. The apparent Vmax for freshly prepared liver microsomes was 0.13 nmol.mg-1.min-1, while that for renal microsomes was 0.040 nmol.mg-1.min-1. Evaluation of estrogen metabolism was also performed in primary cell cultures of hamster kidney cells, consisting of 75% epithelial cells. [6,7-3H]Estradiol (10 microM) was incubated for 0, 24 and 48 h in primary kidney cell cultures, and the organic soluble metabolites analyzed by reverse-phase HPLC. The cultures from untreated, castrated hamsters metabolize [3H]estradiol to yield small quantities of estrone and significant amounts of polar metabolites, while no catechol estrogens were isolated. Estrogen metabolism by diethylstilbestrol-treated (DES-treated) hamster kidney cell cultures also provided small quantities of estrone and no evidence of catechol estrogens. Additionally, larger amounts of additional polar metabolites were isolated in the cultures from DES-treated hamsters. Finally, levels of estrogen 2-hydroxylase were detected in these cultures using the 3H2O release assay. Thus, the short-term primary kidney cell cultures from the Syrian hamster are capable of metabolizing estrogens. Furthermore, the enzymatic processes appear to be available for the conversion of any catechol estrogens formed into more polar metabolites. These investigations in intact cells, capable of performing all biochemical processes, complement both in vivo and subcellular biochemical studies and may aid in elucidating the roles of estrogens and estrogen metabolism in the initiation and development of estrogen-induced, estrogen-dependent kidney tumors in the Syrian hamster.     

Multiple pathways for the oxidative metabolism of estrogens in Syrian hamster and rabbit kidney

Microsomal preparations from hamster kidney, a target tissue for the carcinogenic action of stilbene-type and steroidal estrogens, catalyze the oxidative metabolism of diethylstilbestrol (DES). The formation of the major metabolite Z,Z-dienestrol and of reactive intermediates capable of protein binding were mediated by enzyme activities requiring nicotinamide-adenine dinucleotide phosphate (reduced form-NADPH), cumene hydroperoxide, or arachidonic acid (ARA). In addition, hydroxylated DES metabolites were detected in NADPH-supplemented incubations. The NADPH-dependent oxidation of DES was inhibited by SKF 525A and metyrapone. Monooxygenase-catalyzed metabolism was apparently responsible for the majority of DES oxidation in microsomes from whole hamster kidneys in vitro and this activity is preferentially localized in the kidney cortex. However, ARA-dependent, i.e., prostaglandin H synthase (PHS) mediated oxidation of DES and of the catechol estrogen 2-hydroxyestrone was demonstrated as well in the medulla of both rabbit and hamster kidney. It is proposed that monooxygenase and PHS activities act in concert in the metabolic activation of carcinogenic estrogens. This appears to apply in particular to steroidal estrogens, since catechol estrogens formed by monooxygenases are further oxidized to reactive intermediates by PHS and other peroxidatic enzymes.     

Distribution of prostaglandin ω-hydroxylases in different tissues

The conversion of prostaglandins E₂ and F_2α to their 19- and 20-hydroxy metabolites by various tissues has been measured by gas chromatography-mass spectrometry (GC-MS) using selected ion monitoring. A number of different tissues of the pregnant rabbit possess prostaglandin 20-hydroxylase activity (lung > liver > fetal placenta > maternal placenta ≈ uterus > renal cortex > renal medulla ≈ placental membranes). With the exception of the liver, prostaglandins E₂ and F_2α are metabolized at equal rates by the 20-hydroxylases of different tissues. Only lung and liver microsomes possess high levels of prostaglandin 20-hydroxylase in non-pregnant rabbits and only liver microsomes have appreciable 19-hydroxylase activity. Pulmonary prostaglandin 20-hydroxylase is induced in male rabbits by treatment with progesterone. On the basis of substrate specificity studies and the effects of a cytochrome P-450 inhibitor, SKF-525A, the prostaglandin 20-hydroxylases of lung and liver microsomes from pregnant rabbits appear to be different enzymes. In pregnant rats and hamsters, liver and kidney are the only tissues in which we detected prostaglandin ω-hydroxylase activity.     

Regulatory effects of testosterone and 17 beta-oestradiol on the metabolism of dimethylnitrosamine by renal and hepatic microsomal enzymes from BALB/c mice

Previous investigations with BALB/c mice have demonstrated that no sex-related differences exist in the ability of liver microsomal fractions (S-9) to biotransform dimethylnitrosamine (DMN) to its active mutagenic metabolites as evidenced by bacterial screening assays. In contrast, kidney microsomal enzymes from adult male BALB/c mice and not from females, castrates, and immature animals, were capable of activating DMN. The present study was designed to test the effects of testosterone and oestradiol on DMN bioactivation by hepatic or renal microsomal enzymes. Mutagenic assays were performed using liver and kidney microsomal enzymes with the histidine deficient mutant Salmonella typhimurium TA100. Results indicate that testosterone treatment of female BALB/c mice resulted in an increase in the ability of their renal microsomal enzymes to metabolize DMN to its active mutagenic intermediates. Renal microsomal enzymes from female mice treated with 17 beta-oestradiol had no effect on DMN metabolism. However, the ability of the renal microsomal enzymes treated with 17 beta-oestradiol to bioactivate DMN was significantly decreased in males.     

Metabolism of progesterone by hamster blastocysts and the ontogeny of progesterone metabolic capability

Progesterone (P) is required for the differentiation of reproductive tracts and maintenance of pregnancy. This study investigates whether the hamster blastocyst is capable of metabolizing P and, if so, at what stage of preimplantation development such capability becomes detectable. When the blastocysts collected from superovulated hamsters on Day 4 of pregnancy were cultured in 0.4 microM P medium, P metabolism was easily detectable at 1.25 h of culture and over half was metabolized by 7.5 h. Two major metabolites were generated: 5 alpha-pregnane-3,20-dione (or 5 alpha-dihydroprogesterone; 5 alpha-DHP) and 5 alpha-pregnane-3 beta-ol-20-one (or allopregnanolone; AP), about 90-95% and 5-10%, respectively. This indicates the activity of two enzymes: delta 4-5 alpha-reductase and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD). The rate of P metabolism increased with P concentration (0.4-6.4 microM), indicating a high capacity of the enzymes. Studies of embryos collected on Days 1-3 showed that P metabolism was not detectable up to 0100 h of Day 3 (2-4-cell), but was detectable with two metabolites, 5 alpha-DHP and AP, at 1515 h of Day 3 (morula) and thereafter. This indicates that, by the morula stage, the hamster embryo has already acquired the enzymatic capability (5 alpha-reductase and 3 beta-HSD) to metabolize P. These results, together with our earlier finding of 17 beta-hydroxysteroid dehydrogenase activity in Days 1-4 embryos, suggest that hamster preimplantation embryos can metabolize both P and estrogens, thus possibly modulating local actions of these hormones and causing local effects in the reproductive tract.     

The metabolism of nonane, a JP-8 jet fuel component, by human liver microsomes, P450 isoforms and alcohol dehydrogenase and inhibition of human P450 isoforms by JP-8

Nonane, a component of jet-propulsion fuel 8 (JP-8), is metabolized to 2-nonanol and 2-nonanone by pooled human liver microsomes (pHLM). Cytochrome P450 (CYP) isoforms 1A2, 2B6 and 2E1 metabolize nonane to 2-nonanol, whereas alcohol dehydrogenase, CYPs 2B6 and 2E1 metabolize 2-nonanol to 2-nonanone. Nonane and 2-nonanol showed no significant effect on the metabolism of testosterone, estradiol or N,N-diethyl-m-toluamide (DEET), but did inhibit carbaryl metabolism. JP-8 showed modest inhibition of testosterone, estradiol and carbaryl metabolism, but had a more significant effect on the metabolism of DEET. JP-8 was shown to inhibit CYPs 1A2 and 2B6 mediated metabolism of DEET, suggesting that at least some of the components of JP-8 might be metabolized by CYPs 1A2and/or 2B6.     

Estrogen receptor expression in the facial nucleus of adult hamsters: does axotomy recapitulate development?

Testosterone propionate (TP) augments hamster facial motoneuron regeneration following axonal injury by an androgen-mediated mechanism. Although many of the trophic properties of TP are androgenic, TP can be metabolized to estradiol (E). We have recently shown that E administered in supraphysiological doses can also enhance facial nerve regeneration. The mechanism by which E alters nerve regeneration is unknown. The recent discovery of transient estrogen receptor (ER) expression in the developing rat facial motor nucleus (FMN), coupled with the concept that regeneration may recapitulate development, has led to the hypothesis that facial nerve injury may transiently induce expression of ER in the adult hamster FMN or one of its chief afferents, the principal nucleus of the trigeminal nerve (Nu5). In the present study, this hypothesis was tested using steroid hormone autoradiographic procedures. The right facial nerve was injured in castrated or castrated plus TP adult hamsters. A gonadally intact, nonaxtomized group of hamsters was also included to examine constitutive expression of ER in the FMN or Nu5. The paraventricular nucleus of the hypothalamus (PVN; positive control), FMN, and Nu5, were qualitatively and quantitatively examined for the presence of ER. As expected, ER were present in the PVN-positive control in all groups. ER were neither present nor induced with facial nerve injury or TP administration in either the FMN or Nu5. Alternate mechanisms by which E enhancement of facial nerve regeneration without ER might be explained are discussed.     

Metabolism of benzo(a)pyrene by microsomes from tissues of pregnant and fetal hamsters

Pretreatment of hamsters with benzo (a) pyrene (BaP) greatly increased the $\text{in vitro}$ metabolism of BaP by lung microsomes from pregnant hamsters, and had less effect on the metabolism of BaP by liver microsomes. The production of various metabolites of BaP by lung microsomes was increased to different extents: 3-hydroxy-BaP (3-OH-BaP) was one of the major metabolites; the metabolic yields of 9, 10-dihydrodihydroxy-BaP (9, 10-diol) and 7,8-diol were increased more than that of the 4,5-diol. In the case of liver microsomes, only the yields of 9,10-diol and 7,8-diol were increased over the control levels. The presence of cyclohexene oxide in the incubation mixtures decreased the production of the diols. Basal-level enzyme activities in placental, fetal liver, and fetal skin microsomes in metabolizing BaP were very low. Pretreatment of pregnant hamsters with BaP induced BaP-metabolizing enzymes in fetal tissue 2–3 fold.     

Hormonal regulation of male-specific 20beta-hydroxysteroid dehydrogenase with carbonyl reductase-like activity present in kidney microsomes of rats.

Progesterone, 17alpha-hydroxyprogesterone, cortisone and cortisol, which are C(21)-steroids with a ketone group at the 20-position, potently inhibited the activity of enzyme acetohexamide reductase (AHR) responsible for the reductive metabolism of acetohexamide in kidney microsomes of male rats. Furthermore, progesterone was a competitive inhibitor of AHR. In the case of progesterone usage as the substrate, 20beta-hydroxysteroid dehydrogenase (20beta-HSD) activity was much higher than 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) activity in kidney microsomes of male rats. These results indicate that AHR present in kidney microsomes of male rats, functions as 20beta-HSD with carbonyl reductase-like activity. In male rats, both testectomy and hypophysectomy decreased the renal microsomal 20beta-HSD activity, but the decreased enzyme activities were increased by the treatment with testosterone propionate (TP). We propose the possibility that TP treatment regulates the renal microsomal 20beta-HSD activity by acting directly on the kidney of male rats. This is supported from the fact that when TP was given to ovariectomized and hypophysectomized female rats, the male-specific 20beta-HSD activity was detected in their kidney microsomes.     

Evidence for trichloroethylene bioactivation and adduct formation in the rat epididymis and efferent ducts

Recent studies indicate that trichloroethylene (TCE) may be a male reproductive toxicant. It is metabolized by conjugation with glutathione and cytochrome p450-dependent oxidation. Reactive metabolites produced along both pathways are capable of forming protein adducts and are thought to be involved in TCE-induced liver and kidney damage. Similarly, in situ bioactivation of TCE and subsequent binding of metabolites may be one mechanism by which TCE acts as a reproductive toxicant. Cysteine-conjugate beta-lyase (beta-lyase) bioactivates the TCE metabolite dichlorovinyl cysteine (DCVC) to a reactive intermediate that is capable of binding cellular macromolecules. In the present study, Western blot analysis indicated that the soluble form of beta-lyase, but not the mitochondrial form, was present in the epididymis and efferent ducts. Both forms of beta-lyase were detected in the kidney. When rats were dosed with DCVC, no protein adducts were detected in the epididymis or efferent ducts, although adducts were present in the proximal tubule of the kidney. Trichloroethylene can also be metabolized and form protein adducts through a cytochrome p450-mediated pathway. Western blot analysis detected the presence of cytochrome p450 2E1 (CYP2E1) in the efferent ducts. Immunoreactive proteins were localized to efferent duct and corpus epididymis epithelia. Metabolism of TCE was demonstrated in vitro using microsomes prepared from untreated rats. Metabolism was inhibited 77% when efferent duct microsomes were preincubated with an antibody to CYP2E1. Dichloroacetyl adducts were detected in epididymal and efferent duct microsomes exposed in vitro to TCE. Results from the present study indicate that the cytochrome p450-dependent formation of reactive intermediates and the subsequent covalent binding of cellular proteins may be involved in the male reproductive toxicity of TCE.     

Estradiol metabolism by liver homogenates and microsomes of normal and cirrhotic male rats.

Incubation experiments show that homogenates and microsomes obtained from CCl4-induced cirrhotic livers of male rats metabolize an estradiol load at a much slower rate than preparations from normal livers. The decreased metabolic capacity results in a slower disappearance of estradiol from the incubation medium, and in a slower transformation of metabolized estradiol to polar extractable and to highly polar nonextractable metabolites.     

Sexual dimorphism in N-acetyltransferase activity, hydroxyindole-O-methyltransferase activity, and melatonin content in the Harderian gland of Syrian hamsters: changes following gonadectomy

The activities of N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT) and the melatonin content of the Harderian glands of intact and gonadectomized male and female Syrian hamsters were studied. NAT activity in intact male Harderian glands was twice that of the female. Prepubertal or adult castrated males exhibited a decrease in NAT activity to a level comparable to that seen in the female. Testosterone implants in the castrated males led to a recovery of the original male NAT levels. Intact male hamsters had very low levels of Harderian HIOMT activity and melatonin content in comparison with the glands of the females. Prepubertal gonadectomy but not castration of adult males raised the levels of HIOMT activity and the melatonin content to those of the females. Bilateral ovariectomy had no effect on melatonin content, NAT activity, or HIOMT activity in the female hamster Harderian gland.     

Aflatoxin B1 metabolism by 3-methylcholanthrene-induced hamster hepatic cytochrome P-450s

We have studied the activation of aflatoxin B1 by hamster liver microsomes and purified hamster cytochrome P-450 isozymes using a umu mutagen test. The hamster liver microsomes or S-9 fractions were much more active than rat liver microsomes or S-9 fractions in the activation of umu gene expression by aflatoxin B1 metabolites. 3-Methyl-cholanthrene treatment increased aflatoxin B1 activation by hamster liver microsomes. Two major 3-methylcholanthrene-inducible cytochrome P-450 isozymes, P-450 MC1 (IIA) and P-450 MC4 (IA2), were purified from 3-methylcholanthrene-treated hamster liver microsomes, and the metabolism of aflatoxin B1 by these two cytochromes was studied. In the reconstituted enzyme system, both P-450 MC1 and P-450 MC4 were highly active in the activation of aflatoxin B1, and antibodies against these P-450s specifically inhibited these activities. Antibody against P-450 MC1 inhibited the activation of aflatoxin B1 by 20% in the presence of 3-methyl-cholanthrene-treated hamster liver microsomes. In contrast, antibody against P-450 MC4 stimulated the activity by 175%. These results indicated that hamster P-450 MC1 might convert aflatoxin B1 to more toxic metabolite(s), whereas P-450 MC4 might convert aflatoxin B1 to less toxic metabolite(s), than aflatoxin B1 in liver microsomes. The metabolite(s) produced by both hamster cytochrome P-450 MC1 and MC4 were genotoxic in the umu mutagen test.     

Evaluation of electrospray ionization and atmospheric pressure chemical ionization for simultaneous detection of estrone and its metabolites using high-performance liquid chromatography/tandem mass spectrometry

The levels of estrogens and/or their metabolites play important roles in carcinogenesis, reproductive function, and sexual development during perinatal and adolescence periods. The main purpose of this report was to investigate the applicability of high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) with electrospray ionization (ESI) and/or atmospheric pressure chemical ionization (APCI) for simultaneous detection of estrone (E1) and its six metabolites. Both positive and negative ionization modes in ESI and APCI were used to evaluate the signal responses of seven target analytes. Among the seven target analytes, five analytes, E1, 16alpha-hydroxyestrone, 2-methoxyestrone, 4-methoxyestrone, and 2-hydroxyestrone-3-methyl, produced signals with the best signal-to-noise (S/N) ratios in positive APCI-MS/MS mode, while the other two analytes, 2-hydroxyestrone and 4-hydroxyestrone, yielded the best S/N ratios in negative ESI-MS/MS mode. Based on the results of the evaluation, HPLC-APCI-MS/MS with switching between positive and negative modes was recommended for simultaneous detection of E1 and its six metabolites. The proposed analytical scheme was successfully applied in the analysis of cell culture medium of Human liver carcinoma cells treated with varying amounts of 2,3,7,8-tetrachlorodibenzo-p-dioxin.     

High-performance liquid chromatographic assay detects pentamidine metabolism by Fisher rat liver microsomes

Fisher rat liver microsomes metabolized the antimicrobial drug pentamidine to four new compounds detected by gradient elution reversed-phase high-performance liquid chromatography with variable wavelength detection. Coelution experiments with pentamidine metabolite standards determined the new peaks to be previously identified hydroxylated metabolites of pentamidine, with 1,5-bis(4′-amidinophenoxy)-3-pentanol and 1,5-di-(4′-amidinophenoxy)-2-pentanol formed in the greatest amount. The data contradict a previous report that Fisher rat liver homogenates do not metabolize pentamidine. Pentamidine and its known primary metabolites have almost identical absorption spectra; thus, pentamidine metabolism must be evaluated using gradient elution HPLC to resolve pentamidine from its metabolites. The current assay has now been used to demonstrate that Fisher and Sprague-Dawley rat, mouse, rabbit and human liver microsomes all metabolize pentamidine in vitro.     

P NMR study of phosphorus containing metabolites in the uterus of hamster: Changes during the estrous cycle and the effect of hormonal manipulation

Changes in the concentrations of phosphorus containing metabolites were monitored by ³¹P NMR in the uteri of hamsters during the estrous cycle. Concentrations of phosphocreatine (PCr) and ATP were significantly increased in estrus animals compared to diestrus animals. Concentrations of these metabolites were also increased in immature female hamsters and ovariectomized (OVX) adult hamsters treated with estradiol indicating that estradiol was responsible for this effect. However, the steroid hormones progesterone and testosterone did not increase the concentrations of the phosphorus containing metabolites. Further, immature female hamsters also following treatment with estradiol showed an initial decline in phosphomonoester (PME), PCr, ATP and inorganic phosphate but by 24 h of treatment the concentrations returned to control levels. The NMR study also revealed that the intracellular pH of the hamster uterus was around 7.4 all through the estrous cycle.     

Effects of various inducers on diethylstilbestrol metabolism, drug-metabolizing enzyme activities and the aromatic hydrocarbon (Ah) receptor in male Syrian golden hamster liver

In order to elucidate the role of metabolic activation of the synthetic estrogen, diethylstilbestrol (DES), in the mechanism of liver tumor formation in male Syrian golden hamsters observed after combined treatment with DES and 7,8-benzoflavone (7,8-BF), the metabolism of DES and the concentrations and activities of various drug-metabolizing enzymes were studied in hamster liver microsomes after various pretreatments. The levels of the hepatic aromatic hydrocarbon (Ah) receptor were also determined. Pretreatment with 7,8-BF increased both P450 and cytochrome b5 levels, whereas phenobarbital (PB) and 3-methylcholanthrene (MC) induced P450 but not cytochrome b5. 7,8-BF pretreatment increased 7-ethoxyresorufin-O-deethylase (EROD) 3-fold and 7-pentoxyresorufin-O-dealkylase (PROD) 2.5-fold, whereas aromatic hydrocarbon hydroxylase (AHH) and 7-ethoxycoumarin-O-deethylase (ECOD) activities were only slightly induced by 7,8-BF. MC pretreatment increased EROD 8-fold and PROD activity 7-fold, whereas PB pretreatment enhanced AHH 4.5-fold and PROD activity 4-fold. In contrast to PB, pretreatment with 7,8-BF and MC reduced the oxidative metabolism of DES in hepatic microsomes, but the pattern of metabolites was identical with that in untreated controls. Treatment of hamsters with the inducers changed the hepatic Ah receptor level. PB and MC-pretreatment resulted in an increase of the receptor level 1.5-fold and 1.3-fold, respectively, whereas 7,8-BF-pretreatment leads to a 1.5-fold decrease. The dissociation constant Kd is 170 nM for the reaction of 7,8-BF with the hamster Ah receptor compared to 70 nM for 5,6-BF and 38 nM for 2,3,7,8-tetrachlorodibenzofuran (TCDF). The Kd-value is 3.6 nM for TCDF with the rat receptor protein. It is concluded from these data that metabolic activation of DES is not involved in the mechanism of hepatocarcinogenesis in this animal tumor model.     

Modulation of CYP4502E1 and oxidative stress by testosterone in liver and kidney of benzene treated rats

Bilateral castration increased lipid peroxidation and consequently reduced glutathione in both liver and kidney. Testosterone administration reduced lipid peroxidation in the liver of castrated and benzene treated rats, however, reduced glutathione status could not be restored. Benzene depleted CYP4502E1 in castrated rats, however, the enzyme was restored in liver and kidney both after testosterone treatment. The results suggest that testosterone affects the metabolism and disposition of benzene by influencing CYP4502E1. Other hormonal and cellular/molecular factors may also alter the actions of testosterone. Testosterone dependent mechanism of toxicity of benzene in the liver and kidney has been discussed.     

Proteolysis of chloroplast thylakoid membranes. II. Evidence for the involvement of the light-harvesting chlorophyll a/b-protein complex in thylakoid stacking and for effects of magnesium ions on photosystem II-light-harvesting complex aggregates in the absence of membrane stacking

Experimental evidence indicates that the major pathway of retinoic acid metabolism in hamster liver microsomes follows the sequence: retinoic acid → 4-hydroxy-retinoic acid → 4-keto-retinoic acid → more polar metabolites. Using all-trans-[10-³H]retinoic acid, it can be shown by reverse-phase high pressure liquid chromatographic analysis that the first and last steps of this sequence require NADPH, whereas the oxidation of 4-hydroxy to 4-keto-retinoic acid is NAD⁺ (or NADP⁺) dependent. Both NADPH-dependent steps, but not the NAD⁺-dependent dehydrogenase reaction, are strongly inhibited by carbon monoxide. The metabolism of retinoic acid but not of 4-hydroxy-retinoic acid is highly dependent on the vitamin A regimen of the animal. Retinoic acid is rapidly metabolized by liver microsomes either from vitamin A-normal hamsters or from vitamin A-deficient hamsters that have been pretreated with retinoic acid, but not by microsomes from vitamin A-deficient animals; in direct contrast, the rate of metabolism of 4-hydroxy-retinoic acid is equivalent in each of these microsomal preparations. Analysis of the kinetics of these reactions yields the following Michaelis constants with respect to the retinoid substrates: retinoic acid, 1 × 10^?6m; 4-hydroxy-retinoic acid, 2 × 10^?5m; and 4-keto-retinoic acid, 1 × 10^?7m. The 4-hydroxy to 4-keto-retinoic acid oxidation has been shown to be experimentally irreversible, to have a K_mNAD⁺of 2 × 10^?5m, to be strongly inhibited by NADH, and to be unaffected by the presence of retinoic acid or its 4-keto-derivative in an equimolar ratio to the 4-hydroxy-substrate.