Biomedical subjects

J G Liehr

Publications and source records attributed to J G Liehr.

At least 19 recordsLinked to original sources

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↗

PubMed1991

Vitamin C reduces the incidence and severity of renal tumors induced by estradiol or diethylstilbestrol.

The chronic administration of estradiol or diethylstilbestrol to male Syrian hamsters induces kidney tumors. The effect of vitamin C treatment on estrogen-induced carcinogenesis has been studied to elucidate the mechanism of tumor induction by estrogen. Vitamin C decreases the tumor incidence by approximately 50% but does not influence hormone-dependent growth of kidney tumors. Moreover, vitamin C lowers the concentration of diethylstilbestrol-4',4"-quinone, the genotoxic metabolite of diethylstilbestrol, in vitro and in Syrian hamsters treated with stilbene. Vitamin C also decreases the levels in hamsters of diethylstilbestrol-DNA adducts formed by the quinone metabolite. Estrogens may thus initiate tumors by their metabolic oxidation to corresponding quinone metabolites, which bind covalently to cellular macromolecules. Vitamin C may inhibit tumorigenesis by decreasing concentrations of quinone metabolites and their DNA adducts. Lowering quinone metabolite concentrations may also inhibit free radical generation by decreasing redox cycling between estrogens and their corresponding quinones.

Animals↗

PubMed1991

DNA adduct formation in liver and kidney of male Syrian hamsters treated with estrogen and/or alpha-naphthoflavone.

Chronic administration of estrogens to male Syrian hamsters induces kidney tumors. Co-administration of estrogen plus alpha-naphthoflavone (ANF) suppresses this kidney carcinogenesis but induces liver tumors instead. In an attempt to elucidate the mechanism of the switch from estrogen-induced kidney to liver carcinogenesis in response to ANF treatment, patterns of kidney and liver DNA adducts were investigated by 32P-postlabeling analysis and compared to controls. Chronic treatment of hamsters with ANF alone or in combination with estradiol resulted in a flavone-specific DNA adduct pattern in livers of these animals. These spots, adducts 1 and 2, on 32P-postlabeling maps were taken as evidence of covalent ANF-DNA modifications. The kidney-specific estrogen-induced indirect DNA adducts, observed previously in hamsters treated chronically with estrogen, occurred in renal but not hepatic DNA of animals treated with estradiol alone or in combination with ANF. Pretreatment of hamsters with ANF for 3 days decreased by 75-80% the hepatic and renal diethylstilbestrol (DES)-DNA adducts, which are formed after injection of a single large dose of DES. It is concluded from these changes in DNA adduct patterns and levels that estrogen quinone-DNA adduction may play an etiological role in estrogen-induced kidney cancer. The prevention of estrogen-induced kidney tumors by ANF co-treatment may be a consequence of the decrease in renal concentrations of these adducts in response to ANF. Hepatic concentrations of estrogen quinone-DNA adducts also decrease, but ANF-DNA adducts, observed only in liver, may assume an etiological role in the induction of hepatomas.

Animals↗

PubMed1991

Transplacental genotoxicity of diethylstilbestrol.

Diethylstilbestrol (DES) is a transplacental carcinogen in humans and in rodents. As part of an attempt to examine the mechanism of transplacental carcinogenesis, the transplacental genotoxity of this stilbene estrogen has been investigated. Pregnant hamsters received single injections of 200 mg/kg DES on the 10th day of gestation and were killed 5 or 24 h after treatment. The maternal organs were found to contain the same DES-DNA adduct patterns observed previously by 32P-postlabeling analysis in female hamsters. These adduct patterns, generated by the genotoxic metabolite diethylstilbestrol-4',4h'-quinone (DES Q), were also observed in fetal heart and kidney DNA. In fetal liver DNA, this modified nucleotide, generated by the quinone, was only the minor adduct. The major DNA adduct in this organ was not observed previously and may have been generated by an unknown DES metabolite. The data demonstrate that DES is a transplacentally active genotoxic agent. They also provide evidence for fetal metabolism of DES to DES Q and to other unknown genotoxic intermediates.

Animals↗

PubMed1991

Control of cell death (apoptosis) by diethylstilbestrol in an estrogen-dependent kidney tumor.

The role of cell death as a determinant for tumor growth and regression was studied using an estrogen-dependent, transplantable kidney tumor designated H301. H301 cells were injected s.c. into diethylstilbestrol(DES)-treated male Syrian hamsters and developed solid tumors of 1-2 g within 2-3 weeks. Upon withdrawal of estrogen the tumors regressed by 80-90% within 4 days. Mitoses, necrotic areas and single-cell death indicated by small, condensed cell residues, were counted in hematoxylin and eosin stained histological sections of the tumors. Coincident with tumor regression after DES withdrawal, mitotic activity decreased by approximately 90%; the rate of single-cell death increased (by approximately 2-fold at its maximum). The incidence of necrotic areas was not affected by DES withdrawal. DES re-treatment resulted in reduction of single-cell death by 80% within 8 h. Mitotic activity increased within 24 h to the level observed before DES withdrawal. Again, the incidence of necrotic areas did not change. As a result, tumors re-grew to their previous size within 2 days after resumption of DES treatment. These results led to the following conclusions: (i) DES treatment inhibits and DES withdrawal enhances single-cell death of H301 tumor cells. (ii) Both this functional property and its morphology characterize single-cell death in the tumors as apoptosis. (iii) Estrogen-dependent cell death determines, in addition to mitosis and necrosis, the growth rate of H301 tumors. (iv) This experimental model may provide a useful tool to study the interaction of potential anti-tumor drugs with apoptosis in neoplasia.

Animals↗

PubMed1991

Multistage prostate carcinogenesis: the role of hormones.

Prostate cancer is the most frequently occurring non-skin cancer in men in the U.S.A. and other Western countries, but its etiology is poorly understood. Human prostate carcinogenesis has been viewed as a multi-step process involving progression from low histologic grade, small latent carcinoma, to large, higher grade, metastasizing carcinoma. However, recent data suggest that a variety of pathogenetic pathways exist. The precise role of hormones in the genesis of human prostate cancer remains largely undefined. It is difficult to investigate stages in the development of human prostate cancer, but some animal models provide opportunities in this regard. Short-term treatment of rats with chemical carcinogens will produce a low incidence (5-15%) of prostate cancer, provided that prostatic cell proliferation is enhanced during carcinogen exposure. A high carcinoma incidence can only be produced by chronic treatment with testosterone following administration of carcinogens such as N-methyl-N-nitrosourea (MNU). Testosterone markedly enhances prostate carcinogenesis even at doses that do not measurably increase circulating testosterone. Thus, testosterone is a strong tumor promoter for the rat prostate. All such MNU-initiated, testosterone-promoted tumors are adenocarcinomas mostly originating from the dorsolateral and anterior, but not ventral, prostate lobes. A high frequency (70%) of activation of the K-ras gene by a G35 to A mutation occurs in these carcinomas. A variable frequency of activation of H-ras and K-ras genes occurs in human prostate carcinomas. Another rat model, representing a different pathogenetic pathway, involves chronic administration of estradiol-17 beta in combination with low-dose testosterone. The resulting carcinomas are low-grade and originate exclusively from periurethral ducts of the dorsolateral and anterior prostate. We recently found a major adduct by 32P postlabeling analysis in the tissue region that includes these ducts, but not in, e.g., the ventral prostate, of rats treated for 16-24 weeks. While it is unknown whether testosterone is a tumor promoter in this system, the presence of a DNA adduct suggests that estradiol-17 beta acts as a tumor-initiating agent in this system.

Animals↗

PubMed1990

Effect of chronic estrogen treatment of Syrian hamsters on microsomal enzymes mediating formation of catecholestrogens and their redox cycling: implications for carcinogenesis.

Estrogens have previously been shown to induce DNA damage in Syrian hamster kidney, a target organ of estrogen-induced cancer. The biochemical mechanism of DNA adduction has been postulated to involve free radicals generated by redox cycling of estrogens. As part of an examination of this postulate, we measured the effect of chronic estrogen treatment of hamsters on renal microsomal enzymes mediating catechol estrogen formation and free radical generation by redox cycling of catechol estrogens. In addition, the activities of the same enzymes were assayed in liver in which tumors do not develop under these conditions. At saturating substrate concentration, 2- and 4-hydroxyestradiol were formed in approximately equal amounts (26 and 28 pmol/mg protein/min, respectively), which is 1-2 orders of magnitude higher than reported previously. Estradiol treatment for 2 months decreased 2-hydroxylase activity per mg protein by 75% and 4-hydroxylase activity by 25%. Hepatic 2- and 4-hydroxylase activities were 1256 and 250 pmol/mg protein/min, respectively. Estrogen treatment decreased both activities by 40-60%. Basal peroxidatic activity of cytochrome P-450, the enzyme which oxidizes estrogen hydroquinones to quinones in the redox cycle, was 2.5-fold higher in liver than in kidney and did not change with estrogen treatment. However, when normalized for specific content of cytochrome P-450 the enzyme activity in kidney was 2.5-fold higher than in liver and increased further by 2-3-fold with chronic estrogen treatment. The activity of cytochrome P-450 reductase, which reduces quinones to hydroquinones in the estrogen redox cycle, was 6-fold higher in liver than in kidney of both control and estrogen-treated animals. When normalized for cytochrome P-450, the activity of this enzyme was similar in liver and kidney, but over 4-fold higher in kidney than liver after estrogen treatment. Basal concentrations of superoxide, a product of redox cycling, were 2-fold higher in liver than in kidney. Estrogen treatment did not affect this parameter in liver, but increased it in kidney by 40%. These data provide evidence for a preferential preservation of enzymes involved in estrogen activation.

Animals↗