Biomedical subjects

J G Liehr

Publications and source records attributed to J G Liehr.

At least 73 records · Page 4Linked to original sources

PubMed1993

Localization of estrogen receptors in interstitial cells of hamster kidney and in estradiol-induced renal tumors as evidence of the mesenchymal origin of this neoplasm.

The mechanism of estrogen-induced and -dependent kidney carcinogenesis in Syrian hamsters and the cell of origin of the tumor are not well understood; they have been investigated in this study by mapping the cellular locations of estrogen receptor (ER) in estrogen-dependent tumors, in kidney tissue of hamsters treated with estradiol for 0.5 and 5.5 months, and in kidneys of age-matched controls. To validate the methods used, receptors have also been localized in uteri of hamsters and rats and in female hamster kidneys. ERs have been identified in cryostat sections by immunocytochemical techniques using an affinity-purified ER antibody, ER-715. Nuclei of tumors were intensely stained for ERs. In estrogen-treated kidneys and in controls, ER protein was identified in interstitial cells and capillaries, in arteries, and in renal corpuscles, particularly in podocytes and in the parietal layers surrounding the renal corpuscles. There was no ER protein in tubular epithelia even when tubuli were surrounded by tumor cells. The ER distribution in female hamster kidneys closely matched that in male kidneys. However, the staining intensity was stronger in female than in male kidneys. In hamster uteri, there was an intense ER-positive reaction in the nuclei of stroma, in stromal vessels, and in the luminal epithelia as demonstrated previously by others in rat uteri. ER mRNA has also been demonstrated by Northern blot analysis in estrogen-treated kidneys which contained tumors but was undetectable in untreated kidneys. The localization of ERs in estrogen-dependent tumors and in interstitial cell types but not in tubular epithelia supports previous conclusions of an interstitial origin of estrogen-induced hamster kidney tumors.

Animals↗

PubMed1993

Inhibition of the catechol-O-methyltransferase-catalyzed O-methylation of 2- and 4-hydroxyestradiol by catecholamine: implications for the mechanism of estrogen-induced carcinogenesis.

Catechol estrogens have been postulated to mediate estrogen-induced carcinogenesis. As part of our examination of this hypothesis, we studied the catechol-O-methyltransferase-catalyzed O-methylation of 2- and 4-hydroxyestradiol and the inhibition of this reaction by catecholamines. Epinephrine, norepinephrine, or dopamine (2 microM) inhibited the methylation of 2- and 4-hydroxyestradiol (20 mM) catalyzed by porcine liver catechol-O-methyltransferase by approximately 28-46% and 37-57%, respectively. One millimolar concentrations of catecholamines also inhibited the methylation of 5 microM 2- and 4-hydroxyestradiol by hamster kidney cytosol catechol-O-methyltransferase by approximately 27-31% and 19-33%, respectively. At a 15 microM 4-hydroxyestradiol concentration, the IC50 values for epinephrine and for dopamine were approximately 1200 and 3000 microM, respectively. Kinetic analyses of the methylation of 4-hydroxyestradiol in the presence of epinephrine, norepinephrine, or dopamine all revealed a competitive mechanism of inhibition. In contrast, the methylation of 160 microM 2-hydroxyestradiol was enhanced by approximately 75% in the presence of 1600 microM epinephrine or 2400 microM norepinephrine, likely due to a strong positive allosteric effect. An analysis of the substrate concentration dependence of O-methylation of 2-hydroxyestradiol revealed that at low concentrations (< 15 microM) this reaction was inhibited by epinephrine or norepinephrine, whereas it was significantly increased by approximately 50-100% at high substrate concentrations (50-200 microM). In contrast, dopamine competitively inhibited the methylation of all concentrations of 2-hydroxyestradiol (5-160 microM) tested. High levels of catecholamines were measured in hamster kidney or mouse uterus (1041 +/- 204 or 882 +/- 214 ng norepinephrine/g wet tissue, respectively) and in Fisher 344 rat pituitary (9.4 +/- 1.6 ng dopamine/mg protein), target organs of estrogen-induced carcinogenesis. Values were much lower in other organs of the same animals or in kidney, uterus, or pituitary of other rodent strains or species, which do not develop tumors under these conditions. High levels of catecholamines in target organs of hormonal cancer, the inhibition of O-methylation of 4-hydroxyestradiol, and the differential regulation of O-methylation of 2-hydroxyestradiol by catecholamines all support a role of 4-hydroxyestrogen metabolites in estrogen-induced carcinogenesis.

Animals↗

PubMed1993

Covalent modification of DNA by daunorubicin.

Daunorubicin, a clinically useful antitumor agent, induces mammary adenocarcinoma in Sprague-Dawley rats. As part of an investigation of the mechanism of tumor induction by daunorubicin, the formation of daunorubicin-DNA adducts has been investigated by 32P-postlabeling assay. Rat-liver DNA incubated with either 0.05 or 0.1 mM daunorubicin, rat-liver microsomes, and 5 mM reduced nicotinamide adenine dinucleotide phosphate (NADPH) for 1 h contained covalent DNA adducts in addition to the endogenous adduct profile present in control DNA. With 1.5 mM cumene hydroperoxide serving as a cofactor, higher levels of these two adducts and two additional adducts were formed, all of which most likely were daunorubicin-DNA adducts. This latter treatment also resulted in an intensification of three endogenous DNA modifications over levels occurring in control DNA. Covalent DNA alterations in vivo were studied in rats treated with 20 mg/kg daunorubicin for 2 days and 200 mg/kg on the 3rd day. Daunorubicin-DNA adducts as observed in vitro could not be detected in DNA of liver or mammary epithelial cells. The levels of endogenous modifications in drug-treated rats were increased by 200% in mammary DNA and by 50% in hepatic DNA as compared with controls. It was concluded from these experiments that daunorubicin may be metabolically activated to a reactive metabolite that binds covalently to DNA. These daunorubicin-DNA adducts may not play a role in tumor induction because they were not detectable in vivo. However, the increase in levels of endogenous DNA modifications induced by daunorubicin both in vitro and in vivo is consistent with a role of this class of DNA modification in the carcinogenic process.

Animals↗

PubMed1993

The carcinogenic activity of ethinyl estrogens is determined by both their hormonal characteristics and their conversion to catechol metabolites.

Estrogens induce kidney tumors in Syrian hamsters. The mechanism of carcinogenesis is unknown and has been investigated in this study using a weak carcinogen, 17 alpha-ethinyl estradiol (EE), and a strongly carcinogenic estrogen, 17 alpha-ethinyl-11 beta-methoxyestradiol [moxestrol (MOX)]. We investigated rates of conversion of estrogens to catechol metabolites and rates of their methylation to methyl ethers in order to examine the hypothesis that catechol metabolites mediate estrogen-induced carcinogenesis. Rates of conversion of MOX to catechol metabolites by hamster liver or kidney cortex microsomes were 40-50% of those with estradiol as substrate. However, the rate of catechol-O-methyltransferase-catalyzed methylation of 2-hydroxy-MOX was the least of the catechol metabolites examined when incubated with cytosol of hamster kidney. In contrast, EE was converted to catechol metabolites by hamster liver and kidney microsomes at rates 25-35% of those obtained with estradiol. These catechol metabolites of EE were methylated by catechol-O-methyltransferase of hamster kidney cytosol at rates slightly lower than those observed with catechols of estradiol. The progesterone receptor binding of EE and MOX was investigated, because progesterone is known to inhibit estrogen-induced carcinogenesis in the hamster kidney. Neither estradiol nor MOX inhibited the binding of progesterone to its receptor in hamster kidney cytosol. However, in the presence of 20 nM EE, the binding affinity of radiolabeled progesterone to receptor was inhibited (increase in Kd from 0.98 nM in controls to 3.02 nM in the presence of EE). Maximum binding values (5.0 fmol/mg protein in controls and 6.0 fmol/mg protein in the presence of EE) were not significantly altered. These results support the hypothesis that estrogen-induced carcinogenesis is mediated by catechol estrogen metabolites. The carcinogenic estrogen MOX is converted to catechol metabolites at lower rates than estradiol, but their methylation may be sterically hindered by the 11 beta-methoxy substituent. In contrast, the rates of conversion of the weakly carcinogenic EE to catechol metabolites are low, whereas their methylation rates are only marginally lower than those of 2- and 4-hydroxyestradiol. The decreased capacity of EE to form catechol metabolites in conjunction with its partial progestin agonist activity in the target organ of hamsters may contribute to the low tumor incidence.

Animals↗

PubMed1993

32P-postlabelling in studies of hormonal carcinogenesis.

The nature of the covalent modification of DNA by estrogens has been investigated by 32P-postlabelling and other techniques. Three different classes of covalent DNA alteration are induced by estrogens in vivo and are evaluated in this review for their potential to play a role in hormonal carcinogenesis: (a) estrogen-DNA adducts formed by reactive estrogen quinone metabolites; (b) enhancement of endogenous DNA modification by estrogen specifically in the target organ for cancer; (c) 8-hydroxyguanine bases formed by free radicals generated by redox cycling of estrogen. Additional studies are required to identify the class or classes of DNA adducts that are crucial for the induction of hormonal cancer.

Animals↗

PubMed1993

Microsomal hydroxylation of 2- and 4-fluoroestradiol to catechol metabolites and their conversion to methyl ethers: catechol estrogens as possible mediators of hormonal carcinogenesis.

In male Syrian hamsters, an animal model for estradiol-induced carcinogenesis, 2-fluoroestradiol was not carcinogenic, whereas 4-fluoroestradiol induced kidney tumors after a prolonged latency period, compared with estradiol (100% tumor incidence), when the compounds were administered to hamsters in hormonally equipotent doses. Catechol estrogen metabolites have previously been postulated to mediate this estrogen-induced kidney carcinogenesis. To examine this proposed mechanism of tumor induction by estrogens, we investigated the conversion of 2- and 4-fluoroestradiol to catechol metabolites by kidney and liver microsomes of hamsters and the further conversion to methyl ethers by catechol-O-methyltransferase, and we compared the values with those obtained with nonfluorinated estrogens as substrates. The rates of conversion of 2-fluoroestradiol to 2-hydroxyestradiol and 2-fluoro-4-hydroxyestradiol by hepatic microsomes were 30-50% lower than corresponding rates with estradiol as substrate. With renal microsomes the rate of 4-hydroxylation was 10 times faster than that of estradiol, whereas 2-hydroxylation was at best marginal. With 4-fluoroestradiol as substrate the rate of 2-hydroxylation by hepatic microsomes was enhanced 5-fold, compared with values for estradiol, but 4-hydroxyestradiol formation was almost eliminated. In contrast, the conversion of this substrate to 4-fluoro-2-hydroxyestradiol by kidney microsomes occurred at a rate 15 times faster than 2-hydroxylation of estradiol, whereas 4-hydroxyestradiol formation proceeded at a rate of 315 pmol/mg of protein/min. Except for the decrease in both 2- and 4-hydroxylation of 2-fluoroestradiol by liver microsomes, fluorine substitution of estrogenic phenols enhanced microsome-mediated aromatic hydroxylation at sites unoccupied by substituents. At pH 7.5, the highest rates of catechol-O-methyltransferase-mediated methylation were observed with the catechol metabolites of 2-fluoroestradiol, 2-fluoro-4-hydroxyestradiol and 2-hydroxyestradiol (3780 and 2960 pmol/mg of protein/min, respectively). Lower rates were found with those of 4-fluoroestradiol, 4-fluoro-2-hydroxyestradiol and 4-hydroxyestradiol (1670 and 470 pmol/mg of protein/min, respectively). These data are consistent with the postulate that catechol metabolites of estrogens are reactive intermediates in estrogen-induced carcinogenesis. For the noncarcinogenic 2-fluoroestradiol, a high metabolic flux was observed through a pathway of renal 2-fluoro-4-hydroxyestradiol formation and further conversion to methyl ethers. This flux likely results in low steady state concentrations of catechol metabolites in kidneys of hamsters treated with this modified estrogen and therefore in its lack of carcinogenic activity. In contrast, the carcinogenic activity of 4-fluoroestradiol is consistent with its rapid conversion in the kidney to 2- and 4-hydroxylated metabolites and a less rapid methylation of these catechols.

Animals↗

PubMed1992

Catalysis of the oxidation of steroid and stilbene estrogens to estrogen quinone metabolites by the beta-naphthoflavone-inducible cytochrome P450 IA family.

Diethylstilbestrol (DES) or catecholestrogens are metabolized by microsomal enzymes to quinones, DES Q or catecholestrogen quinones, respectively, which have been shown to bind covalently to DNA and to undergo redox cycling. The isoforms of cytochrome P450 catalyzing this oxidation of estrogens to genotoxic intermediates were not known and have been identified in this study by (a) using microsomes of rats treated with various inducers of cytochrome P450; (b) using purified cytochrome P450 isoforms; and (c) examining the peroxide cofactor concentrations necessary for this oxidation by microsomes or pure isoenzymes. The highest rate of oxidation of DES to DES Q was obtained using beta-naphthoflavone-induced microsomes (14.0 nmol DES Q/mg protein/min) or cytochrome P450 IA1 (6.4 pmol DES Q/min/pmol P450). Isosafrole-induced microsomes or cytochrome P450 IA2 oxidized DES to quinone at one-third or one-fifth of that rate, respectively. Low or negligible rates of oxidation were measured when oxidations were catalyzed by microsomal rat liver enzymes induced by phenobarbital, ethanol, or pregnenolone-16 alpha-carbonitrile or by pure cytochromes P450 IIB1, IIB4, IIC3, IIC6, IIE1, IIE2, IIG1, or IIIA6. Cytochrome P450 IA1 also catalyzed the oxidation of 2- or 4-hydroxyestradiol to their corresponding quinones. The beta-naphthoflavone-induced microsomes and cytochrome P450 IA1 had the highest "affinity" for cumene hydroperoxide cofactor (Km = 77 microM). Cofactor concentrations above 250 microM resulted in decreased rates of oxidation. The other cytochrome P450 isoforms required much higher cofactor concentrations and were not inactivated at high cofactor concentrations. The data demonstrate that beta-naphthoflavone-inducible cytochrome P450 IA family enzymes catalyze most efficiently the oxidation of estrogenic hydroquinones to corresponding quinones. This oxidation may represent a detoxification pathway to keep organic hydroperoxides at minimal concentrations. The resulting quinone metabolites may be detoxified by other pathways. However, in cells with decreased detoxifying enzyme activities, quinones metabolites may accumulate and initiate carcinogenesis or cell death by covalent arylation of DNA or proteins.

Animals↗

PubMed1992

Lack of effectiveness of antiestrogens RU 39,411 or keoxifene in the prevention of estrogen-induced tumors in Syrian hamsters.

As part of a search for an effective and safe antiestrogen to be used as adjunct therapy in the treatment of breast cancer, we examined the potential of RU 39,411 and keoxifene to inhibit the incidence of estradiol-induced kidney tumors in Syrian hamsters. Groups of 10 hamsters were chronically treated with implants of either keoxifene, RU 39,411, estradiol plus keoxifene, or estradiol plus RU 39,411 for 8 months. Five hamsters received only estradiol and 5 control animals remained untreated. There was a 100% kidney tumor incidence in estradiol-treated hamsters, which was not statistically different from that in animals co-treated with estradiol plus keoxifene (3 of 4 hamsters with tumors) or estradiol plus RU 39,411 (7 of 8 hamsters with tumors). Rodents treated only with antiestrogen remained tumor free. In addition to kidney tumors, testicular cancer was also found in animals cotreated with either estradiol plus keoxifene (2 of 4 hamsters with tumors) or estradiol plus RU 39,411 (3 of 8 hamsters with tumors). Two animals of this latter group also developed liver tumors. Testicular or liver neoplasms were not observed in hamsters implanted only with estradiol or only with antiestrogen. The lack of inhibition of estrogen-induced carcinogenesis in hamsters by RU 39,411 or keoxifene suggests that these two antiestrogens are not as effective as previously tested substances in inhibiting the appearance of this cancer. However, their concentrations were sufficient to induce, in combination with estradiol, the development of testicular tumors in these hamsters.

Animals↗

PubMed1992

Target organ-specific inactivation of drug metabolizing enzymes in kidney of hamsters treated with estradiol.

Chronic treatment of hamsters with estradiol for several months has previously been shown to decrease the specific content of cytochrome P450 in the kidney, a target of hormonal carcinogenesis, but not in liver. The reason for this decrease in metabolic enzyme activity is unknown and has been examined in this investigation. We now report that the decrease in specific content of renal cytochrome P450 by 73% in response to estradiol was not affected by co-treatment with tamoxifen for 1 month. The subcutaneous infusion of 250 micrograms/day estradiol for 7 days lowered renal cytochrome P450 by 71% from control values and was therefore used for further mechanistic studies. This treatment decreased renal activities of estradiol 2- or 4-hydroxylase by 77 to 80%, of 7-ethoxycoumarin-O-deethylase by 66% of control values, respectively, and completely eliminated aryl hydrocarbon hydroxylase activities, whereas liver enzymes remained unaffected. After 7 days of infusion of estradiol, fluorescent products of lipid peroxidation were more than doubled in hamster kidney but remained unchanged in liver. The possibility of enzyme destruction by binding of estradiol 2,3-quinone to metabolizing enzymes was investigated in vitro. In the presence of 2-hydroxyestradiol, cumene hydroperoxide, and microsomes, conditions known to favor the oxidation of the steroid to quinone, the binding of catechol estrogen metabolite to microsomal protein increased 60 fold over control values in the absence of cofactor. Purified rat liver cytochrome P450c also oxidized 2-hydroxyestradiol to 2,3-estradiol quinone. The rate of oxidation was linear for the first 2-3 min, but thereafter decreased with time.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Induction of covalent DNA adducts in rodents by tamoxifen.

The antiestrogen tamoxifen, increasingly used as adjuvant treatment for breast cancer, has been found to covalently modify DNA of rodents. For instance, the liver DNA of female Sprague-Dawley rats treated with a single injection of tamoxifen contained two DNA adducts. Four additional DNA adducts were formed and adduct concentrations increased 5- 7- and 10-15-fold after three and six tamoxifen injections, respectively, from levels observed after a single dose. The accumulation of DNA adducts with repeated administrations of tamoxifen to rodents may make this drug a poor choice for the chronic preventative treatment of breast cancer.

Acetylcysteine↗

PubMed1992

Elevation of protooncogene messenger RNAs in estrogen-induced kidney tumors in the hamster.

Cellular oncogenes such as c-fos, c-jun and c-myc are expressed prior to estrogen-induced growth of normal target tissues such as rodent uterus. Transient increases in the levels of these genes are induced by the administration of estradiol and are followed by DNA replication. In this study, we examined the expression of these three oncogenes in estradiol-induced kidney tumors in Syrian hamsters in order to understand mechanistic aspects of hormonal carcinogenesis. Kidney tumors were induced in all male Syrian hamsters treated chronically with estradiol for 7 or 9 months, whereas neoplasms were not detected in animals treated for 5 months. mRNA levels of fos, myc and jun were elevated 15-, 4- and 6-fold respectively in kidney tumors of estradiol-treated hamsters (9 months) compared with age-matched untreated control kidneys. The expression of all three protooncogenes was also increased in the kidney tissue surrounding tumors, though there was no consistent pattern in the ratios of transcripts in the tumor and kidney tissues. After 7 months of estrogen treatment, kidney tumors also contained elevated amounts of c-fos, c-jun and c-myc transcripts at levels comparable with older tumors. In abdominal metastases of hamster kidney tumors, mRNA levels of fos, myc and jun were elevated 9-, 12- and 3-fold respectively over control levels. In kidneys of hamsters treated with estradiol for 5 months, in which tumors were not yet detected, the expression of protooncogenes was slightly increased. Ratios of c-fos, c-myc and c-jun in estrogen-treated (5 months) over control tissue were 1.4, 1.1 and 1.3 respectively. Overexpression of cellular oncogenes such as c-fos, c-jun and c-myc may have played a role in the induction and growth of kidney tumors by estradiol in hamsters.

Animals↗

PubMed1992

Elevated 4-hydroxylation of estradiol by hamster kidney microsomes: a potential pathway of metabolic activation of estrogens.

Characterization of enzymes mediating the formation of catecholestrogens (CE) by hamster kidney is of importance because of the proposed role of CE in renal cancer induced in this species by estrogens. We have reexamined the potential of hamster kidney to convert estradiol (E2) to 2- and 4-hydroxylated CE because of recent evidence of the limitations of assays used in previous studies, in particular in measuring 4-hydroxylation of estrogens. Under conditions optimized for NADPH-dependent activity, hamster kidney microsomes exhibited high levels of both E2-2- and E2-4-hydroxylase activities. Evidence that the two activities depend on different forms of cytochrome P-450 was obtained by the demonstration that 2- and 4-hydroxylation of E2 were affected differentially 1) by chronic treatment of hamsters with E2 and 2) by fadrozole hydrochloride, a selective cytochrome P-450 inhibitor. NADPH-dependent 2-hydroxylation of E2 from control and E2-treated hamsters, measured by a direct product isolation assay, was 1 order of magnitude higher (apparent maximum velocity, 24-32 and 6-12.5 pmol/mg protein.min in control and E2-treated hamsters, respectively) than that reported previously using radioenzymatic assays. NADPH-dependent 4-hydroxylation of E2 in controls approached and in E2-treated hamsters exceeded 2-hydroxylation of E2 (apparent maximum velocity, 17-21 and 7.5-19 pmol/mg protein.min in control and E2-treated hamsters, respectively). Thus, estrogen treatment reversed the ratios of NADPH-dependent E2-2-/4-hydroxylase activities by causing a much greater decline in 2- than 4-hydroxylation of E2 (P less than 0.007, by analysis of variance). Fadrozole hydrochloride caused a marked dose-dependent decrease in 2-hydroxylation of E2, in contrast to a small nondose-dependent inhibition of 4-hydroxylation. Under conditions optimized for peroxidatic organic hydroperoxide-dependent activity, hamster kidney microsomes generated 2- and 4-hydroxylated CE in similar amounts. The amounts of the two CE and, consequently, the ratios remained unaffected by estrogen treatment (1:0.9 and 1:1.0 in control and E2-treated hamsters, respectively). Thus, this study establishes that CE can be generated in the same tissue by three different pathways, i.e. NADPH-dependent E2-2-hydroxylase, NADPH-dependent E2-4-hydroxylase, and organic hydroperoxide-dependent E2-2/4-hydroxylase activities. We also show that these three activities can be regulated differentially and are, thus, probably mediated by different forms of cytochrome P-450. In hamster kidney, the potential to generate 4-hydroxylated CE metabolites with distinct properties could be a factor in this tissue's vulnerability to estrogen-induced carcinogenesis.

Animals↗

PubMed1991

Xanthine oxidase-catalyzed reduction of estrogen quinones to semiquinones and hydroquinones.

Metabolic redox cycling between the stilbene estrogen diethylstilbestrol (DES) and diethylstilbestrol-4',4"-quinone (DES Q) has been demonstrated previously. The xanthine and xanthine oxidase-catalyzed reduction of estrogen quinone has been studied in this work to understand the role of metabolic redox cycling in estrogen metabolism. Xanthine and xanthine oxidase catalyzed the reduction of DES Q to 44% Z-DES and 9% E-DES. This reaction was inhibited by the addition of superoxide dismutase or by a lack of oxygen (under anaerobic conditions). DES Q was also reduced in a non-enzymatic reaction by superoxide radicals generated by potassium superoxide and crown ether. The reaction between the O2-. and DES Q was also investigated by an electron spin resonance spin-trapping technique. The superoxide anion generated in an oxygen-saturated xanthine and xanthine oxidase system was detected as 5,5-dimethyl-1-pyrroline-1-oxide-superoxide adduct. The addition of DES Q or 2,3-estradiol quinone totally inhibited the formation of this adduct. The reduction of DES Q by superoxide radicals was taken as evidence that this reaction was one possible mechanism of xanthine and xanthine oxidase-mediated reduction. In addition, reduction of DES Q by direct electron transfer to quinone by the enzyme may also occur. The intermediate formation of semiquinone free radicals in the reduction is implied by the nature of the single electron transfer reactions and, in addition, has been demonstrated for the catechol estrogen by electron spin resonance measurements. It is concluded that the reduction of estrogen quinones to their hydroquinones by xanthine oxidase occurs by both one electron transfer to the quinone and by formation of superoxide which then reduces the quinone.

Diethylstilbestrol↗

PubMed1991

Free radical-induced carbonyl content in protein of estrogen-treated hamsters assayed by sodium boro[3H]hydride reduction.

Oxidative damage to proteins is known to occur via conversion of side chain amino groups to corresponding carbonyl derivatives. Such damage to enzymes and purified proteins has been quantified previously by reduction with sodium boro[3H]hydride and subsequent measurement of the incorporation of 3H into amino acid fractions. In this study, the NaB3H4 reduction assay was modified to permit the quantitation of free radical-mediated oxidative damage to proteins obtained from animals. Modifications included additional extractions of protein isolates with organic solvents to remove lipids and with nitric acid to remove metal ions. The modified assay has first been validated in vitro by measuring changes in levels of oxidative damage to bovine serum albumin exposed to xanthine plus xanthine oxidase (2-fold increase), to hydrogen peroxide and iron(II) sulfate (5-fold increase), or to gamma radiation (30-fold increase over controls, respectively). gamma radiation of isolated hamster kidney protein also raised the carbonyl content in a dose-dependent manner. The modified assay has then been validated in vivo by measuring the changes in oxidative damage to lung tissue in animals exposed to approximately 85% oxygen (2-fold increase) or to different doses of paraquat (5-fold increase with the high dose over controls, respectively). The assay was then used to examine free radical-mediated oxidation introduced by acute or chronic treatment of hamsters with estrogens, since both synthetic and natural estrogens induce kidney tumors in this species. Priming of hamsters for 3 days with 20 mg/kg/day diethylstilbestrol and treatment with 100 mg/kg of this drug on the 4th day resulted in a 160% increase in free radical modification of renal proteins. Oxidative damage to kidney proteins was also assayed in hamsters treated with estradiol implants for up to 7 months, a regimen known to induce kidney tumors. Significant increases in covalent oxidative modification to renal proteins over values in age-matched controls were detected after 1, 2, and 7 months of continuous estradiol exposure. It is concluded that the modification of the NaB3H4 reduction assay is a useful postlabeling method for monitoring free radical action in vivo. Furthermore, it is postulated that free radical damage in estrogen-treated hamster kidney plays a role in estrogen-induced carcinogenesis.

Animals↗

PubMed1991

Elevated 8-hydroxydeoxyguanosine levels in DNA of diethylstilbestrol-treated Syrian hamsters: covalent DNA damage by free radicals generated by redox cycling of diethylstilbestrol.

The generation of free radicals by microsome-mediated redox cycling between catechol estrogens or diethylstilbestrol and their corresponding quinones has previously been demonstrated in vitro. However, the reaction of free radicals with DNA has not yet been detected in animals treated with estrogen and is the subject of this investigation. The reaction of guanine bases of DNA with hydroxyl radicals to form 8-hydroxydeoxyguanosine has been used as a monitor of free radical generation in kidney and liver of Syrian hamsters, a species prone to estrogen-induced carcinogenesis. Prior to in vivo measurements, the in vitro hydroxylation of guanine bases of DNA under conditions of redox cycling of estrogen was investigated. In incubations of DNA or deoxyguanosine with hamster kidney microsomes, NADPH, and diethylstilbestrol 4',4"-quinone, the hydroxylation of guanine bases of free deoxyguanosine or of DNA was 50 to 100% higher than in controls. When incubations were carried out in the presence of iron(III) chloride, the hydroxylation of guanine bases was 2.5- or 10-fold higher than control values. There was a 65% increase from control values in levels of 8-hydroxydeoxyguanosine in liver DNA of hamsters treated with 20 mg/kg/day diethylstilbestrol for 3 days and 100 mg/kg on the 4th day. In hamsters treated chronically with diethylstilbestrol implants for 15 days, 8-hydroxydeoxyguanosine levels more than doubled from control values in kidney but not liver DNA. Treatment of hamsters with estradiol for various time periods did not induce any changes in levels of hydroxylated guanine in either kidney or liver. It was concluded that in vitro and in vivo redox cycling of diethylstilbestrol hydroxylated guanine bases in DNA.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed1991

Determination of estradiol 2- and 4-hydroxylase activities by gas chromatography with electron-capture detection.

A highly sensitive assay has been developed for measuring the rate of formation of 2-hydroxyestradiol and 4-hydroxyestradiol from estradiol by microsomal preparations. Catechol estrogens were converted to heptafluorobutyryl esters, which were separated by capillary column gas chromatography and quantified using electron-capture detection. 2-Hydroxyestradiol 17-acetate was used as an internal standard. The identity of catechol estrogen derivatives was verified by gas chromatography-mass spectrometry using negative-ion chemical ionization. Estrogens were identified by negative molecular ions and/or by characteristic fragments. This procedure permits quantification of catechol estrogens at the subpicogram level. The assay was validated by comparing estrogen 2- and 4-hydroxylase activities in microsomes from hamster and rat liver with values reported previously.

Animals↗

PubMed1991

Changes in the cellular phenotype and extracellular matrix during progression of estrogen-induced mesenchymal kidney tumors in Syrian hamsters.

The cellular origin of estrogen-induced kidney tumors in male Syrian hamsters has been repeatedly the subject of controversy. Several authors have proposed that the tumors arise from proximal tubules, from a combination of tubular and interstitial stromal cells, or solely from interstitial cells. Because of the model character of this tumor for hormone-associated cancer, it was further investigated in this study with respect to morphology, enzyme and intermediate filament pattern, the expression of alpha-smooth muscle actin and the extracellular matrix proteins fibronectin and tenascin. These analyses were carried out with early and late tumors as well as metastases to determine possible changes in expression of biochemical parameters during the development and progression of this neoplasm. The enzyme histochemical and intermediate filament patterns were usually the same as those described previously for proliferative foci and early tumors, i.e. highly elevated activities of glucose-6-phosphate dehydrogenase, adenylate cyclase and alkaline phosphatase, a lack of glucose-6-phosphatase and gamma-glutamyltransferase and coexpression of vimentin and desmin, alpha-smooth muscle actin could not be detected in early lesions. In five of 24 advanced tumors inclusions of kidney tubules were found which showed various degrees of alteration in their morphology and enzyme histochemical pattern, but were often directly connected with tubular segments of normal appearance outside the tumor. Like the normal tubules, the enclosed tubular segments were strongly positive for cytokeratin but never expressed vimentin or desmin. Among the 24 tumors studied, two contained cysts which expressed cytokeratin and sometimes also vimentin but not desmin. The enzyme histochemistry of the cells lining the cysts was similar to that of the surrounding tumor mass, except adenylate cyclase was lacking and alkaline phosphatase was not uniformly distributed. In tumors containing cytokeratin-positive cysts, there often were cytokeratin-positive, vimentin-negative and desmin-negative tumor formations in close contact to these cysts. With the exception of cyst formation, the pattern of metastases were identical to that of the primary tumors. All large tumors and the main component of the metastases expressed vimentin, desmin and fibronectin. Mesothelia surrounding metastatic tumor complexes were positive for vimentin, desmin, alpha-smooth muscle actin, fibronectin, cytokeratin and tenascin. It was concluded from these and previous observations on early stages of tumor development that the estrogen-induced hamster kidney tumor originates from mesenchymal interstitial cells (probably pericytes) which may rarely acquire an epithelial phenotype by metaplastic transformation during tumor progression.

Adenylyl Cyclases↗

PubMed1991

Cytochrome b5-mediated redox cycling of estrogen.

Previously, we have demonstrated microsomal cytochrome P450-catalyzed redox cycling of estrogens. In this study, we investigated the role of cytochrome b5 in redox cycling in order to obtain a full understanding of enzymatic contributions to redox reactions of estrogens. Pure cytochrome P450c and hydrogen peroxide or cumene hydroperoxide oxidized diethylstilbestrol (DES) to diethylstilbestrol-4',4"-quinone (DES Q). This oxidation by H2O2 was doubled by addition of cytochrome b5 to cytochrome P450c (molar ratio of 1:4), but did not proceed with cytochrome b5 alone. The stimulation by cytochrome b5 of the cytochrome P450c-catalyzed oxidation of DES to DES Q occurred via modulation of the Vmax of cytochrome P450c rather than of the Km. DES Q was reduced to DES by purified cytochrome b5 and NADH-dependent cytochrome b5 reductase. Pretreatment of microsomes with an antibody to cytochrome b5 reductase inhibited microsomal NADH-dependent reduction of DES Q to DES by 55%. Cytochrome b5 likely participates in the oxidation of DES to DES Q by interacting with cytochrome P450c and in the reduction of DES Q to DES by interacting with cytochrome b5 reductase. Thus, the study demonstrates that cytochrome b5 plays an active role in biological oxidation and reduction reactions.

Animals↗