Biomedical subjects

J G Liehr

Publications and source records attributed to J G Liehr.

At least 91 records · Page 5Linked to original sources

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↗

PubMed1990

Genotoxic effects of estrogens.

Estrogens are associated with several cancers in humans and are known to induce tumors in rodents. In this review a mechanism of carcinogenesis by estrogens is discussed which features the following key events: (1) Steroid estrogens are metabolized by estrogen 2- and 4-hydroxylases to catecholestrogens. Target organs of estrogen-induced carcinogenesis, hamster kidney or mouse uterus, contain high levels of estrogen 4-hydroxylase activity. Since the methylation of 4-hydroxyestradiol by catechol-O-methyltransferase is inhibited by 2-hydroxyestradiol, it is proposed that a build up of 4-hydroxyestrogens precedes estrogen-induced cancer. (2) The catecholestrogen or diethylstilbestrol (DES) are oxidized to semiquinones and quinones by the peroxidatic activity of cytochrome P-450. The quinones are proposed to be (the) reactive intermediates of estrogen metabolism. (3) The quinones may be reduced to catecholestrogens and DES and redox cycling may ensue. Redox cycling of estrogens has been shown to generate free radicals which may react to form the organic hydroperoxides needed as cofactors for oxidation to quinones. (4) The quinone metabolites of catechol estrogens and of DES bind covalently to DNA in vitro whereas DNA binding in vivo has only been examined for DES. When DES is administered to hamsters, the resulting DES-DNA adduct profile in liver, kidney, or other organs closely matches that of DES quinone-DNA adducts in vitro. In vitro, DES-DNA adducts are chemically unstable and are generated in incubations with organic hydroperoxide as cofactor. It is proposed that the instability of adducts and the lower sensitivity of previous assay methods contributed to the reported failures to detect adducts. Steroid estrogen-DNA adducts in vivo are currently under investigation. (5) Tumors are postulated to arise in cells rapidly proliferating due to the growth stimulus provided by the estrogenic activity of the primary estrogen or of hormonally potent metabolites such as 4-hydroxyestradiol. The covalent modification of DNA in these cells is temporary because of the chemical instability of adducts and will result in altered genetic messages in daughter cells, whereas in non-proliferating cells there may be no lasting genetic damage. The sequence of events described above is a plausible mechanism for tumor initiation by estrogens and is partially substantiated by experimental evidence obtained in vitro and/or in vivo.

Animals↗

PubMed1990

Free radical generation by redox cycling of estrogens.

Natural and synthetic estrogens elicit normal hormonal responses in concentrations in a clearly defined yet low range. At elevated doses, metabolic reactions of the phenolic moiety, while harmless at low levels, may become the predominant biochemical activity and may exert deleterious effects. These metabolic pathways, such as i) oxidation of estrogens to catechol estrogens and further to their respective quinones, and ii) free radical generation by redox cycling between catechol estrogens or diethylstilbestrol and their quinones, are investigated for their influence in physiological or pathophysiological processes. In this review, the in vitro capacity of various enzymes to oxidize estrogen hydroquinones to quinones or to reduce corresponding quinones to hydroquinones is evaluated. The in vivo activities of enzymes supporting redox cycling of estrogens and free radical generation is correlated with induction of kidney tumors in Syrian hamsters. Concomitant changes in activities in quinone reductase and other detoxifying enzymes in kidneys of hamsters treated with estrogen support a role of free radicals in the induction of tumors by estrogen. Free radical damage to protein and possibly to DNA in kidneys of estrogen-treated hamsters may be used as markers of free radical action in vivo.

Animals↗

PubMed1990

The O-methylation of 4-hydroxyestradiol is inhibited by 2-hydroxyestradiol: implications for estrogen-induced carcinogenesis.

O-Methylation of catecholestrogens catalyzed by catechol-O-methyltransferase provides a major route for the rapid metabolic clearance of these steroids. However, the metabolic clearance rate of 4-hydroxyestradiol (4-OH-E2) is considerably lower than that of 2-hydroxyestradiol, although 2- and 4-hydroxycatecholestrogens (2- and 4-OH-CE) have similar apparent affinities for the enzyme. To determine the reason for this apparent paradox we have examined whether the efficiency of O-methylation of 4-OH-E2 could be affected by other catecholestrogens or their O-methyl ethers. The ratio of 4-methoxyestradiol:4-hydroxyestradiol 3-methyl ether was 2.6 at pH 8.5, the pH optimum for the reaction. The O-methylation of 4-OH-E2 (apparent Km 10 microM) was inhibited by 2-hydroxyestradiol (2-OH-E2) but not by 2- or 4-methyoxyestrogens. The values for Km, Vmax as well as the slope for the methylation of 4-OH-E2 were altered by 2-OH-E2 indicating a mixed inhibition. The inhibition constant for the intercept 1/V'max versus 2-OH-E2 concentrations and the inhibition constant for the slope versus 2-OH-E2 concentrations were 35 and 5.7 microM, respectively. The inhibition of O-methylation of 4-OH-E2 by 2-OH-E2 increased with the pH. In target tissues of the carcinogenic action of estrogens such as the rat pituitary, hamster kidney, or mouse uterus in which 2- and 4-OH-CE are both generated in almost equal amounts, the inactivation of 4-OH-CE by O-methylation may be impeded. Consequently, 4-OH-E2 would remain available as substrate for redox cycling, generation of active radicals and DNA damage.

Animals↗

PubMed1990

Inhibition of estrogen-induced kidney carcinogenesis in Syrian hamsters by modulators of estrogen metabolism.

Quinone metabolites of catechol estrogens have been postulated to mediate estradiol-induced carcinogenesis. In this study, this postulate was examined by investigating the effect of modulators of quinone metabolism on estradiol-induced tumor incidence in male Syrian hamsters. 2(3)-t-Butyl-4-hydroxyanisol (BHA) and dicumarol which are known to stimulate or inhibit respectively, the activity of quinone reductase, lowered tumor incidence by 33 and 42% respectively (3/9 and 5/12 tumor-free animals/total respectively), from 100% (13/13) observed with 17 beta-estradiol (E2) treatment only. Ebselen, a substance with glutathione peroxidase-like activity, and sodium 2-mercaptoethanesulfonate (Mesna), a cytoprotective thiol-containing agent, were only marginally effective in decreasing the estradiol-induced kidney tumor incidence (3/11 and 4/19 tumor-free animals/total respectively). The lowering of tumor incidence by BHA and dicumarol correlated well with a 40-45% decrease in renal peroxidatic activity of cytochrome P450 in hamsters treated with these substances plus estradiol for 1 month. In addition, these compounds also inhibited the oxidation of diethylstilbestrol to its corresponding quinone in vitro. An influence on quinone reductase or other detoxifying enzymes in chronically treated male Syrian hamsters could not be detected. These data support a mediation of estradiol-induced carcinogenesis by quinones formed by metabolic oxidation of catechol estrogens.

Animals↗

PubMed1989

Mechanism of genotoxicity of diethylstilbestrol in vivo.

Diethylstilbestrol (DES) is a carcinogen in humans and rodents which has eluded mechanistic clarification of its carcinogenic action. In vitro and in vivo, binding of DES to DNA has been found previously, but covalent DNA adducts could not be identified. In this study, the nature of binding was investigated by 32P-postlabeling, a rapid and highly sensitive assay for covalent DNA damage, to distinguish between a genotoxic or epigenetic mechanism of carcinogenesis by DES. A unique and distinct DNA adduct pattern was observed in kidney, liver, uterus (or testes) of female (or male, respectively) Syrian hamsters treated with a single injection of DES (200 mg/kg body weight). This set of DNA adducts closely matched patterns generated in vitro by reaction of diethylstilbestrol-4',4''-quinone with DNA or 2'-deoxyguanosine 3'-monophosphate. The major and several minor DES-DNA adducts in vivo had identical chromatographic mobilities in 11 different solvent systems with corresponding adducts obtained in vitro. The major adduct spot, generated in vitro by reaction of diethylstilbestrol-4',4''-quinone and DNA, was chemically unstable (half-life at 37 degrees C: 4-5 days). The persistence in vivo of these DNA modifications was low (biological half-life: 14 h) presumably because of chemical instability in concert with DNA repair. After injection of identical dosages of DES, adduct concentrations were 4-6-fold higher in females than in males. These results demonstrate that DES is capable of covalently modifying DNA. Moreover, diethylstilbestrol-4',4"-quinone is the major reactive metabolic intermediate responsible for the genotoxic activity of DES. Tumors are expected to arise only in rapidly dividing cells due to the short biological lifetimes of DES-DNA adducts.

Animals↗

PubMed1989

Characterization of semiquinone free radicals formed from stilbene catechol estrogens. An ESR spin stabilization and spin trapping study.

Electron spin resonance spectroscopy has been used to detect, characterize, and to infer structures of o-semiquinones derived from stilbene catechol estrogens. Radicals were generated enzymatically using tyrosinase and were detected as their Mg2+ complexes. It is suggested that initial hydroxylation of stilbene estrogen gives a catechol estrogen in situ; subsequent two-electron oxidation of the catechol to the quinone, followed by reverse disproportionation, leads to the formation of radicals. Consistent with this mechanism, o-phenylenediamine, a quinone trapping agent, inhibits formation of o-semiquinones. A competing mechanism of radical production involves autoxidation of the catechol. Hydroxyl radicals are shown to be produced in this system via a mechanism involving reduction of iron and copper complexes by stilbene catechols. Possible differences in the reactivity of stilbene ortho- and para-semiquinones are discussed.

Benzoquinones↗

PubMed1989

Changes in activities of free radical detoxifying enzymes in kidneys of male Syrian hamsters treated with estradiol.

Target organ-specific estrogen-induced DNA adducts were previously shown to precede renal carcinogenesis in Syrian hamsters. Because estrogens induced these DNA modifications, but were not part of the adduct structure, free radical activation of endogenous electrophiles was postulated as a mechanism of tumor induction by estrogens. In the present study, the activities of enzymes which detoxify reactive intermediates were studied in liver and kidney of hamsters treated with estradiol for 1, 2, and 4 mo and in untreated controls. These studies were done to detect oxidative stress in the target organ of carcinogenesis. In the estrogen-exposed hamster kidney (1, 2, and 4 mo), activities of glutathione peroxidases I and II were significantly increased. The activity of catalase was decreased compared to those in untreated controls. In livers which are not the target organ of carcinogenesis, treatment of hamsters with estrogen for 1, 2, and 4 mo resulted in changes of activities of glutathione peroxidases I and II and catalase, which were opposite to the pattern found in the kidney. Activities of superoxide dismutase, glutathione reductase, glucose-6-phosphate dehydrogenase, gamma-glutamyl transpeptidase, and glutathione transferase in estradiol-treated hamster liver and kidney did not differ significantly from those in either liver or kidney of untreated age-matched controls. Fluorescent products of lipid peroxidation more than doubled in the kidney, but not in the liver of hamsters treated with estradiol for 1 mo. It is concluded that the increases in glutathione, in the activity of glutathione peroxidase, and in products of lipid peroxidation in the kidneys of hamsters treated chronically with estrogen all point towards elevated levels of oxidative stress.

Animals↗

PubMed1989

Comparison of assays for catechol estrogen synthase activity: product isolation vs radioenzymatic catechol-O-methyltransferase-coupled procedures.

Reported values for the activity of enzymes mediating catechol estrogen formation by hamster kidney and liver, measured by catechol-O-methyltransferase-coupled radioenzymatic assay, have been uniformly low and there have been marked discrepancies in values reported from different laboratories. Therefore, we examined the validity of the radioenzymatic assay used in these studies. NADPH-dependent estrogen 2- and 4-hydroxylase activity of hamster liver microsomes measured by radioenzymatic assay was comparable to that reported in the literature but at least one order of magnitude lower than that obtained with a direct product isolation assay. Several features of the radioenzymatic assay were identified which, together, contribute to the underestimation of enzyme activity. They include, incomplete protection from oxidative degradation of both the catechol estrogens generated and of the catechol-O-methyltransferase and assay conditions which are suboptimal for O-methylation of the catechol estrogens. We conclude that results obtained using the catechol-O-methyltransferase-based radioenzymatic assay can only be considered valid if a consistent stoichiometric relationship can be demonstrated between the amounts of catechol estrogens and their O-methylated products.

Animals↗

PubMed1989

Age-dependent covalent DNA alterations (I-compounds) in rodent tissues: species, tissue and sex specificities.

I-compounds are non-polar covalent DNA modifications of as yet undetermined structure that tend to accumulate in an age-dependent manner in tissues of untreated animals. They are detectable by 32P-postlabeling assay because of their adduct-like properties and chromatographically resemble DNA nucleotides containing bulky/hydrophobic moieties. To determine which factors may be involved in their formation, I-compounds were examined by 32P-postlabeling in liver and kidney DNA of female and male Sprague-Dawley rats and Syrian hamsters of different ages (1, 4 and 10 months and 1, 2.5 and 9.5 months, respectively). The following results were obtained: (i) Every tissue DNA studied contained characteristic I-compounds. (ii) Patterns and amounts of I-compounds were reproducible among animals of the same kind. (iii) There were pronounced organ and species differences. (iv) I-compound patterns were sex-dependent. (v) I-compound levels increased with age in all tissues studied, except in male hamster kidney, a target organ of estrogen-induced carcinogenesis. The highest levels were observed in liver and kidney of 10-month-old female rats. (vi) The rise of I-compound levels was less steep during the later part of the observation period for female but not male animals. (vii) Gonadectomy decreased I-compound levels in female hamster kidney DNA, while causing a slight increase in male animals later in life. These I-compounds were identical to previously reported DNA modifications that increased in male hamster kidneys after prolonged estrogen treatment. Points, iv, vi and vii strongly implicated sex hormones in I-compound formation. The qualitative effects of species, tissue differentiation, gender and sex hormones on these DNA modifications support the hypothesis that I-compounds are formed by the binding of endogenous electrophiles to DNA. As persistent DNA alterations, they are likely to affect DNA replication and to play a role in spontaneous and chemically induced carcinogenesis and in aging.

Aging↗

PubMed1989

Mechanism of inhibition of estrogen-induced renal carcinogenesis in male Syrian hamsters by vitamin C.

Dietary supplementation of vitamin C to diethylstilbestrol (DES)- or estradiol-treated male Syrian hamsters is known to inhibit renal carcinogenesis by approximately 50%. To elucidate the mechanism of inhibition, the influence of administration of vitamin C on a series of previously described biochemical markers of kidney carcinogenesis was investigated. Hamsters were stratified into four groups: (i) untreated controls; (ii) vitamin C-treated; (iii) estrogen-treated; and (iv) estrogen plus vitamin C-treated animals. Concomitant administration of vitamin C and diethylstilbestrol (DES) decreased concentrations of the major DES-DNA adduct by 70-90% in liver, kidney and testis than those receiving DES only. Diethylstilbestrol-4',4"-quinone has previously been shown to be the genotoxic metabolite of DES responsible for DNA adduct formation in vivo. In vitro, vitamin C reduced diethylstilbestrol-4',4"-quinone to cis- and trans-diethylstilbestrol in a dose-dependent fashion. Changes in activities of quinone reductase, catalase, superoxide dismutase and of glutathione metabolizing enzymes (glutathione peroxidase, glutathione reductase, gamma-glutamyl transpeptidase and glucose-6-phosphate dehydrogenase) in response to vitamin C were not observed or not sufficiently large to account for the 50% decrease in tumor incidence. No differences were detected in indirect estrogen-induced kidney DNA adducts in response to vitamin C treatment. It is concluded that vitamin C inhibits estrogen-induced carcinogenesis by reducing concentrations of estrogen quinone metabolites and their DNA adducts.

Animals↗

PubMed1989

Metabolic oxidation of diethylstilbestrol to diethylstilbestrol-4',4"-quinone in Syrian hamsters.

Diethylstilbestrol-4',4"-quinone (DES Q) has previously been postulated to be a reactive intermediate in diethylstilbestrol (DES) metabolism. DES is oxidized to DES Q in vitro, but the occurrence of the quinone metabolite in vivo has not yet been demonstrated due to its instability and chemical reactivity. In this report, the characteristics of in vitro formation of DES Q and the isolation of 3H-labeled DES Q from tissue extracts of hamsters injected with radiolabeled DES is described. In vitro, the time-dependent formation of DES Q as a function of microsomal protein, cofactor or substrate concentrations was demonstrated. The microsome-mediated oxidation of DES to quinone was inhibited by various compounds that also effectively inhibit the peroxidatic activity of cytochrome P-450. In vivo, the formation of DES Q occurred in all tissues investigated, livers and kidneys of male and female adult hamsters, neonates and fetuses, and in uterus and placenta. Concentrations of quinone metabolite in liver and kidney of adult hamsters after injection of 75 mumol/kg DES were 76 and 20 pmol/g tissue respectively. In neonates and fetus, concentrations of DES Q after the same dose of DES were markedly less than those in adults (0.026 and 0.047% of adult levels in neonatal liver and kidney and 0.013 and 0.016% of adult levels in fetal liver and kidney respectively). Since DES Q was also formed by fetal liver homogenate in vitro, fetal oxidizing enzymes appear to be the source of the quinone metabolite in this tissue. DES Q concentrations were also examined after injection of DES into hamsters pretreated with vitamin C or alpha-naphthoflavone, substances known to inhibit DES-induced renal carcinogenesis. Quinone metabolite levels were cut in half in response to vitamin C in correlation with the approximately 50% decrease in DES-induced renal tumors reported previously. alpha-Naphthoflavone pretreatment decreased renal and hepatic DES Q concentrations by 70 and 17% respectively, also in correlation with the known prevention of kidney tumors by this flavone. These data support a role of DES Q in DES-induced carcinogenesis. Since there is no correlation between DES Q concentrations and target site specificity of DES induced tumors, the oxidation of DES to DES Q and the genotoxicity of this metabolite may be a necessary but not sufficient event in tumor development. Hormone-dependent growth of initiated cells may also be necessary for the occurrence of cancers.

Aging↗

PubMed1989

Use of the 32P-postlabelling assay to study transplacental carcinogens and transplacental carcinogenesis.

The formation of DNA adducts represents a key step in the postnatal initiation of the carcinogenic process. Little is known as yet about the role of prenatally induced adducts in transplacental carcinogenesis in offspring. Measurement of transplacental DNA damage in fetal organs of experimental animals has been difficult in the past because of the small amounts of DNA available and low adduct levels. In principle, these difficulties have been overcome by the recent development of a highly sensitive 32P-postlabelling assay which can be applied to a large number of DNA adducts of diverse structure and requires only microgram amounts of DNA for analysis. In this assay, tissue DNA is degraded to mononucleotides; these are enzymatically 32P-labelled via T4 polynucleotide kinase-catalysed [32P]phosphate transfer from [gamma--32P]ATP, to form 5'--32P-labelled 3',5'-bisphosphate derivatives; the labelled products are separated into normal and adducted [32P]nucleotides and quantified by thin-layer chromatography, autoradiography and scintillation (Cerenkov) counting. This technique allows the detection and quantitation of one adduct in 10(8)-10(10) DNA nucleotides (approximately 1-100 adducts/mammalian genome) using a 10-micrograms DNA sample and has been applied in studies of adduct formation from transplacental carcinogens in fetal and adult rodent tissues. In this paper, we review application of 32P-postlabelling to DNA adducts formed with transplacental or suspected transplacental carcinogens in fetal and maternal tissues. The carcinogens studied include diethylstilboestrol (DES), benzo[a]pyrene, safrole, 4-aminobiphenyl and 4-nitroquinoline-1-oxide, as well as cigarette smoke condensate. In DNA of DES-exposed hamsters, one major and several minor adduct spots were observed, which were absent from vehicle controls. A characteristic adduct, which resembled the major hamster adduct chromatographically, was detected in all exposed mouse tissue, except fetal kidney. Chronic administration of low doses of DES to male Syrian hamsters led to an entirely different pattern of adducts in kidney DNA, the target organ of carcinogenesis. These adducts did not contain covalently bound oestrogen moieties and appeared to be formed indirectly: oestrogen appeared to induce or enhance the synthesis of an endogenous electrophilic metabolite reacting with DNA. Thus, multiple mechanisms exist by which DES can damage DNA. Additional work using 32P-postlabelling has shown that non-hormonal genotoxicants (e.g., benzo[a]pyrene, safrole, 4-aminobiphenyl, 4-nitroquinoline-1-oxide) and cigarette smoke condensate given to pregnant mice can induce specific DNA adduct profiles in fetal tissues.(ABSTRACT TRUNCATED AT 400 WORDS)

4-Nitroquinoline-1-oxide↗