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Biomedical subjects

J G Liehr

Publications and source records attributed to J G Liehr.

At least 55 records · Page 3Linked to original sources
PubMed1996

Influence of route of administration on [3H]-camptothecin distribution and tumor uptake in CASE-bearing nude mice: whole-body autoradiographic studies.

Camptothecin (CPT) inhibits the growth of a wide variety of experimental tumors. As a part of our exploration of this drug for use as a cancer chemotherapeutic agent, we studied the effect of route of administration on the absorption, distribution and tumor uptake of [3H]-CPT. The rate of disappearance of [3H]-CPT-derived radioactivity from blood during the first 48 h was highest following oral than following intravenous (i.v.) administration. Thereafter blood levels were low irrespective of route of administration. Considerable [3H]-CPT-derived radioactivity was detected in urine and feces up to 48 h after dosing. Distribution studies were conducted using quantitative whole-body autoradiography (WBA). These studies revealed that independent of the route of administration, [3H]-CPT was rapidly excreted in the bile (gallbladder) followed by elimination into the small and large intestinal tract. Levels of CPT-derived radioactivity in the kidneys were minimal and mostly localized in the renal pelvis. Hepatic concentrations of CPT were low and were almost equal to those of the tumor. The lungs of animals treated i.v. showed higher uptake of radioactivity than those treated intramuscularly or orally. Tumor/blood ratios were slightly higher following oral administration than following administration by other routes. This study indicates that CPT is primarily eliminated via the bile. The gastrointestinal tract is the major site of accumulation and excretion of CPT.

Administration, Oral↗
PubMed1996

Molecular mechanisms of estrogen carcinogenesis.

In western society, the causes of several cancers--including breast, endometrium, ovary, liver, and prostate--have been linked to inappropriate and/or prolonged exposure to synthetic or endogenous steroidal hormones. In this review, we discuss the mechanisms of estrogen carcinogenesis with a focus on estrogen metabolism to 16 alpha-hydroxy estrone and 2- and 4-hydroxy catechol estrogens and the potential effects of these metabolites in vitro and in vivo on hamster liver and kidney and rat liver carcinogenesis models. The examples demonstrate that the parent compounds and their metabolites cause both nongenotoxic cell proliferative effects as well as direct and indirect genotoxic effects, which illustrates the complex nature of estrogen carcinogenesis. These effects, in combination with the metabolic state of the tissue and the timing of its exposure, may determine the cell type (organ) of tumor development and the severity of disease.

Animals↗
PubMed1996

Lipid peroxidation-induced putative malondialdehyde-DNA adducts in human breast tissues.

The etiology of the majority of human breast cancers is unknown; however, oxidative stress and lipid peroxidation have been suggested to play a role in breast carcinogenesis. To address this possibility, DNA adducts induced by malondialdehyde (MDA), an end product of lipid peroxidation, were analyzed in surgical specimens of normal breast tissues of 51 breast cancer patients using the nuclease P1-enhanced version of the 32P-postlabeling assay. Normal breast tissue samples from 28 noncancer patients receiving reduction mammoplasty served as controls. Two previously characterized putative MDA-deoxyadenosine (dA) and one MDA-deoxyguanosine adduct were detected in all tissue samples examined. Normal breast tissues from cancer patients exhibited significantly higher levels of the putative MDA adducts [median (42.5) and range (2.2-202.8) of relative adduct labeling x 10(9) values] than those found in noncancer controls (median, 15.67; range, 2.4-382.1; P = 0.0001, Mann-Whitney U test). Ten of the 51 cancer patients and 1 of the 28 controls were found to contain the putative MDA adducts at the level of > 1/10(7) nucleotides, a frequency comparable to that found in human liver. Age and body mass did not significantly influence the levels of these adducts. However, the presence of a previously detected benzo(a)pyrene-like DNA adduct in the breast tissues was associated with higher levels of the putative MDA-dA adducts in cancer patients (P = 0.012). The level of the putative MDA-dA adducts was significantly lower in smokers and former smokers compared to nonsmokers among cases after adjusting for age, body mass index, and status of the benzo(a)pyrene-like adduct (P = 0.009). Tumor tissues (n = 11) displayed significantly lower levels of the putative MDA adducts (median, 10.2; range, 5.3-20.6) than their corresponding normal adjacent tissues (median, 25.5; range, 10.5-138; P < 0.01). These findings provide evidence that lipid peroxidation products can accumulate in human breast tissues and reach relatively high levels in the breast tissues of women with breast cancer. There seems to be an interaction between these endogenous DNA modifications and carcinogen exposure-induced DNA adducts. Detection and quantitation of the putative MDA-DNA adducts may potentially be a useful tool in the understanding of breast cancer etiology.

Adult↗
PubMed1995

4-Hydroxylation of estradiol by human uterine myometrium and myoma microsomes: implications for the mechanism of uterine tumorigenesis.

Estradiol is converted to catechol estrogens via 2- and 4-hydroxylation by cytochrome P450 enzymes. 4-Hydroxyestradiol elicits biological activities distinct from estradiol, most notably an oxidant stress response induced by free radicals generated by metabolic redox cycling reactions. In this study, we have examined 2- and 4-hydroxylation of estradiol by microsomes of human uterine myometrium and of associated myomata. In all eight cases studied, estradiol 4-hydroxylation by myoma has been substantially elevated relative to surrounding myometrial tissue (minimum, 2-fold; mean, 5-fold). Estradiol 2-hydroxylation in myomata occurs at much lower rates than 4-hydroxylation (ratio of 4-hydroxyestradiol/2-hydroxyestradiol, 7.9 +/- 1.4) and does not significantly differ from rates in surrounding myometrial tissue. Rates of myometrial 2-hydroxylation of estradiol were also not significantly different from values in patients without myomata. We have used various inhibitors to establish that 4-hydroxylation is catalyzed by a completely different cytochrome P450 than 2-hydroxylation. In myoma, alpha-naphthoflavone and a set of ethynyl polycyclic hydrocarbon inhibitors (5 microM) each inhibited 4-hydroxylation more efficiently (up to 90%) than 2-hydroxylation (up to 40%), indicating > 10-fold differences in Ki (<0.5 microM vs. > 5 microM). These activities were clearly distinguished from the selective 2-hydroxylation of estradiol in placenta by aromatase reported previously (low Km, inhibition by Fadrozole hydrochloride or ICI D1033). 4-Hydroxylation was also selectively inhibited relative to 2-hydroxylation by antibodies raised against cytochrome P450 IB1 (rat) (53 vs. 17%). These data indicate that specific 4-hydroxylation of estradiol in human uterine tissues is catalyzed by a form(s) of cytochrome P450 related to P450 IB1, which contribute(s) little to 2-hydroxylation. This enzyme(s) is therefore a marker for uterine myomata and may play a role in the etiology of the tumor.

Benzoflavones↗
PubMed1995

Lipid hydroperoxide-induced endogenous DNA adducts in hamsters: possible mechanism of lipid hydroperoxide-mediated carcinogenesis.

DNA of livers or kidneys of rodents contain endogenously modified nucleotides of as yet unknown structure or function. As part of our examination of the origin of endogenous DNA modifications, we examined polar adducts of hamster liver and kidney DNA by 32P-postlabeling analysis (central and upper cuts) and compared them to adducts generated by incubation of DNA with [9Z,11E,(13S)]-13-hydroperoxyoctadecadienoic acid or with malondialdehyde. The central and upper cuts of DNA adduct maps of intact female retired breeder hamsters contained one and three adducts called C1, U1, U2, and U3, respectively, which cochromatographed with similar adducts in DNA incubated with the hydroperoxide or with malondialdehyde. Concentrations of adducts C1, U1, and U2 in organs of 1-month-old hamsters were lower than those in the 5-month-old female retired breeder hamsters. Adduct concentrations increased in tandem with lipid peroxide concentrations in livers and kidneys of hamsters treated with carbon tetrachloride compared to controls. A linear dependence of the concentrations of adducts on those of lipid peroxides was demonstrated independent of the treatment conditions or the organ examined. The polar endogenous adducts U1, U2, and U3 were identified by cochromatography in five different chromatographic systems with adducts induced by linoleic hydroperoxide or malondialdehyde. Adducts U1, U2, or U3 were also formed by incubating malondialdehyde with dAMP or with dGMP, respectively. The dependence of adduct concentrations in vivo on lipid peroxide levels are taken as evidence that decomposition products of lipid peroxides such as malondialdehyde induced endogenous DNA adducts in intact animals. Moreover, these adducts may mediate carcinogenic processes induced by chemicals which raise lipid peroxide levels.

Age Factors↗
PubMed1995

Induction of benign prostatic hyperplasia in intact dogs by near-physiological levels of 5 alpha-dihydrotestosterone and 17 beta-estradiol.

Benign prostatic hyperplasia was induced in mongrel dogs treated for 60 days with one silastic implant containing 17 beta-estradiol and four containing 5 alpha-dihydrotestosterone. The condition was characterized by (1) a marked increase of the stromal elements, particularly the stromal septa between the individual glands, (2) a slight increase in prostatic volume, and (3) a morphology that resembled spontaneous complex benign prostatic hyperplasia in the dog. Other groups of animals that remained untreated or received only 17 beta-estradiol or only 5 alpha-dihydrotestosterone did not develop this condition. Prostate volumes decreased by 14% in the estrogen-treated dogs, whereas they increased in the androgen-treated animals by 6% compared to pretreatment prostate volumes. The morphology of the epithelium of the prostates of androgen-treated animals was not different from that of controls despite the increase in prostate volume. The serum 17 beta-estradiol and 5 alpha-dihydrotestosterone concentrations were increased from 25 +/- 2 (mean +/- SEM) and 256 +/- 42 pg/mL, respectively, in control dogs to 52 +/- 37 and 562 +/- 37 pg/mL, respectively, in the dogs treated with the hormone combination. Thus, hormone concentrations were two- to three-fold higher than control values, and the ratio of estradiol-17 beta to 5 alpha-dihydrotestosterone was increased by up to 19%. These data demonstrate that treatment of dogs with low levels of estrogen and androgen may be an excellent model for the study of spontaneous complex benign prostatic hyperplasia in aging men.

Analysis of Variance↗
PubMed1995

DNA adducts induced by lipids and lipid peroxidation products: possible relationships to I-compounds.

A methanol-extractable lipid fraction of oats has been found previously to induce three specific I-compounds (age-dependent covalent DNA modifications) in female rat liver DNA, as detected by the 32P-postlabeling assay. The current report used an in vitro system to explore the possible mechanisms involved in the formation of these DNA derivatives. Ground oats or commercial oatmeal were extracted with methanol, and the extracts were incubated with rat lung DNA in vitro. DNA was recovered and analyzed by the nuclease P1-enhanced version of the 32P-postlabeling assay. A number of adducts were induced by the in vitro reaction but none of them was identical by chromatographic analysis to oats-specific I-compounds detected in vivo. Addition of rat liver microsomes and cofactors (NADPH or cumene hydroperoxide) to the in vitro reaction also failed to induce any of the oats-specific I-compounds. Pretreatment of oat lipids with soybean lipoxidase and oxygen enhanced formation of most adducts formed in vitro in a dose- and time-dependent manner. Several of these adducts were related to peroxide derivatives of linoleic acid. Chromatographic evidence suggests that one of the major adducts is derived from 4-hydroxynonenal, a reactive intermediate lipid peroxidation product. This adduct was detectable in liver and kidney DNA of untreated rats and its level increased with age. These results were in line with previous in vivo results, suggesting that the oats-specific I-compounds are presumably formed via an indirect mechanism rather than by direct binding of oats components to DNA.

Aging↗
PubMed1995

Microsome-mediated 8-hydroxylation of guanine bases of DNA by steroid estrogens: correlation of DNA damage by free radicals with metabolic activation to quinones.

Free radical generation by metabolic redox cycling between catechol estrogens and their quinones and subsequent hydroxyl radical damage to DNA have been proposed to mediate estrogen-induced renal carcinogenesis in the hamster. In this study the content of 8-hydroxy-2'-deoxy-guanosine (8-OHdG), a marker product of hydroxyl radical action, was examined in DNA incubated with a liver microsomal activating system and with catechol estrogens, equilenin-3,4-quinone or with parent estrogens. Equilenin-3,4-quinone increased the formation of 8-OHdG by 50% over control levels. 4-Hydroxyestrone and 4-hydroxy-estradiol raised 8-OHdG contents significantly, to 1.61 +/- 0.79 and 1.27 +/- 0.31 8-OHdG/10(5) deoxyguanosine (dG) respectively over controls (0.68 +/- 0.25 8-OHdG/10(5) dG). The corresponding 2-hydroxylated estrogens and the parent hormones estrone, estradiol and equilenin did not affect 8-hydroxylation of guanine bases of DNA. In incubations of catechol estrogens with microsomes and cumene hydroperoxide the 4-hydroxyestrogens were oxidized to quinones more rapidly than the 2-hydroxyestrogens. Our data support a mechanism of hydroxyl radical generation from estrogens by redox cycling between 4-hydroxylated metabolites and their quinones. The rapid oxidation of 4-hydroxylated estrogens to quinones, their redox cycling and hydroxyl radical damage to DNA is consistent with the previously reported carcinogenic activities of 4-hydroxylated, but not of 2-hydroxylated, catechol estrogens.

Animals↗
PubMed1995

Induction of a DNA adduct detectable by 32P-postlabeling in the dorsolateral prostate of NBL/Cr rats treated with estradiol-17 beta and testosterone.

Treatment with estradiol-17 beta and testosterone induces epithelial dysplasia and, subsequently, adenocarcinoma in the dorsolateral prostate of NBL rats. The purpose of this study was to determine whether this carcinogenic effect is mediated by genotoxicity. Analogous to adducts produced by estrogens in the male hamster kidney, a target of estrogen carcinogenicity, induction of DNA adducts detectable by 32P-postlabeling was investigated in the prostate target tissue. NBL rats were treated with separate Silastic tubing implants containing testosterone and estradiol-17 beta. Control animals received empty implants. Animals were killed at 8, 16 and 24 weeks after initiation of treatment, and accessory sex glands were sampled for adduct analysis. DNA of the dorsolateral and ventral prostate and the coagulating gland (= anterior prostate) was isolated and analyzed by nuclease P1-enhancement of the 32P-post-labeling assay. DNA adducts were quantitated by Cerenkov counting. An adduct occurred selectively in DNA of the dorsolateral prostate of rats treated with estradiol plus testosterone for 16 or 24 weeks with relative adduct level values of approximately 10 x 10(9), but not in DNA of the ventral or anterior prostate. The adduct was not present in DNA of prostate tissue of rats treated for 8 weeks or in DNA of control tissues. This adduct was unique with respect to chromatographic location and has not been observed before in any tissue of control or hormone-treated animals. Neither the structure of the treatment-induced adduct nor the mechanism of its formation is known. However, the selective occurrence of this adduct in the tissue of origin of the carcinomas and its appearance coinciding with putative preneoplastic lesions and preceding carcinoma development suggests a causal relation between adduct formation and prostate cancer development in testosterone plus estradiol-17 beta-treated rats.

Adenocarcinoma↗
PubMed1995

Induction by estrogens of lipid peroxidation and lipid peroxide-derived malonaldehyde-DNA adducts in male Syrian hamsters: role of lipid peroxidation in estrogen-induced kidney carcinogenesis.

Estrogen-induced kidney carcinogenesis in male Syrian hamsters has previously been postulated to be mediated by free radicals generated by redox cycling of catecholestrogen metabolites. As part of our examination of this hypothesis, we have studied the induction of lipid peroxidation and lipid peroxide-derived malondialdehyde (MDA)-DNA adducts in kidney and liver of hamsters treated with single injections of diethylstilbestrol (DES) or with estradiol (E2) implants for various lengths of time. Treatment of hamsters with 50 and 100 mg/kg DES increased concentrations of both lipid hydroperoxides and of MDA-DNA adducts. In hamsters treated with E2 implants for up to 50 days, lipid peroxide levels in liver were double control values 3 h after hormone implantation, and then decreased to plateau values of 30% over controls. Those in kidney rose to 2- to 3-fold above controls 3 days after hormone implantation and then decreased to plateau values of 51% above controls. MDA-DNA adduct levels were two or three times higher than those of controls in liver and kidney of hamsters treated with hormone implants for 3 and 7 days. Renal lipid peroxide concentrations were raised by chronic treatment with E2, but not by weakly carcinogenic estrogens ethinylestradiol or 2-fluoroestradiol. In contrast, MDA-DNA adduct levels were raised by all three steroidal estrogens 3 days after estrogen implantation. The increases in lipid peroxides and in MDA-DNA adducts in estrogen-treated hamsters support a mechanism of carcinogenesis by free radical generation via redox cycling of catcholestrogen metabolites. Lipid peroxides are postulated to play a dual role in estrogen-induced carcinogenesis, (i) as cofactors for cytochrome P450-mediated formation of catecholestrogen metabolites and their redox cycling, and (ii) as precursors of MDA, a DNA adduct-forming endogenous electrophile.

Animals↗
PubMed1994

8-Hydroxylation of guanine bases in kidney and liver DNA of hamsters treated with estradiol: role of free radicals in estrogen-induced carcinogenesis.

The chronic administration of estradiol induces a high incidence (80-100%) of renal tumors in male Syrian hamsters. As part of our examination of a mechanism of carcinogenesis by free radicals generated during redox cycling of catecholestrogen metabolites, we assayed levels of 8-hydroxy-2'-deoxyguanosine (8-OHdGua), a marker product of hydroxy radical interaction with DNA, in livers and kidneys of hamsters treated with estradiol. Injections of 50 and 100 mg/kg estradiol doubled renal 8-OHdGua levels over controls [10.0 +/- 0.1 (SD) and 5.4 +/- 0.4 8-OHdGua/10(5) dGua, respectively] and raised hepatic 8-OHdGua levels almost 4-fold over control values, respectively. These changes were observed in kidney 4 h and in liver 1 or 2 h after treatment of hamsters with estradiol. Estradiol implants administered to hamsters for 3 days raised renal levels of 8-OHdGua by 50% over control values. Six days after 17 beta-estradiol implantation, 8-OHdGua levels returned to near-normal values. Liver DNA was not affected by estradiol implants. These data support a mechanism of estrogen-induced carcinogenesis by free radicals generated via redox cycling of catecholestrogen metabolites.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1994

Identification of fatty acid hydroperoxide cofactors in the cytochrome P450-mediated oxidation of estrogens to quinone metabolites. Role and balance of lipid peroxides during estrogen-induced carcinogenesis.

Lipid hydroperoxide-supported metabolic redox cycling between diethylstilbestrol (DES) or catechol-estrogens and their corresponding quinones has been postulated previously to play a role in estrogen-induced toxicity and carcinogenesis. As part of an examination of this postulate, we investigated (a) the effectiveness of various lipid hydroperoxides as oxidants in microsome-mediated quinone formation, and (b) the dependence of DES oxidation to quinone on lipid hydroperoxide levels in liver and kidney of hamsters. Kinetic analyses of the lipid hydroperoxide-mediated conversion of DES to DES quinone revealed that of the peroxides tested (9Z,11E,13(S))-13-hydroperoxyoctadecadien-1-oic acid was the most powerful oxidant (Vmax = 10.5 nmol min-1; Km = 21.3 microM) followed by (5(S),6E,8Z,11Z,14Z)-5-hydroperoxyeicosatetra en-1-oic acid (Vmax = 1.0 nmol min-1; Km 10.0 microM). The other pure fatty acid hydroperoxide isomers or mixtures had a lower affinity for the enzyme (Km values ranging from 23.1 to 130 microM) and/or lower maximal velocity (Vmax values ranging from 0.2 to 7.0 nmol min-1). Levels of Z,Z-dienestrol, which arises from spontaneous rearrangement of DES quinone, were not affected in liver of hamsters treated with estradiol implants for 9 days and an injection of 20 mg/kg DES but increased in kidney by 120% over levels observed in animals without implants (5.5 and 12.1 nmol/g of wet tissue, respectively). Z,Z-Dienestrol concentrations correlated directly with lipid hydroperoxide concentrations in liver and kidney of control and estradiol-treated hamsters. The increased conversion of DES to quinone in kidney compared with that in liver was catalyzed by comparable activities of cytochrome P450 IA, the enzyme family catalyzing this oxidation. These results demonstrated that the oxidation of DES to quinone was directly dependent on the lipid hydroperoxide levels in the organ of metabolic conversion in addition to the enzyme activity catalyzing this reaction. The elevated lipid hydroperoxide levels in kidney of estrogen-treated hamsters indicate enhanced estrogen quinone formation and therefore enhanced generation of free radicals by metabolic redox cycling of estrogens in this organ prior to the appearance of malignancy.

Animals↗
PubMed1994

Catechol-O-methyltransferase-catalyzed rapid O-methylation of mutagenic flavonoids. Metabolic inactivation as a possible reason for their lack of carcinogenicity in vivo.

Quercetin is highly mutagenic in vitro, yet is not carcinogenic when administered chronically at large doses to rodents for 12 months. We hypothesized that catechol-O-methyltransferase-catalyzed O-methylation of quercetin and other mutagenic catechol-containing flavonoids may provide an efficient inactivation in vivo and may therefore prevent tumor induction by these flavonoids. After one intraperitoneal administration of 50 mg/kg quercetin to hamsters, a urinary ether extract contained 2% quercetin and 97% 3'-O-methylquercetin. When the urine was treated first with beta-glucuronidase and sulfatase, 13% quercetin and 87% 3'-O-methylquercetin were recovered. Quercetin was rapidly O-methylated by either porcine liver or hamster kidney catechol-O-methyltransferase, with Km values of 6.1 and 6.9 microM and Vmax values of 14,870 and 200 pmol/mg of protein/min, respectively. S-Adenosyl-L-homocysteine exhibited a potent feedback inhibition of the catechol-O-methyltransferase-catalyzed O-methylation of quercetin by a competitive mechanism with respect to S-adenosyl-L-methionine and by a competitive plus noncompetitive mechanism with respect to the substrate. A comparison of the O-methylation rates and kinetic characteristics (Km, Vmax, and Vmax/Km) demonstrated that rates of O-methylation of quercetin and fisetin were up to three orders of magnitude higher than those of catechol estrogens and catecholamines. In conclusion, the rapid metabolic inactivation of mutagenic flavonoids catalyzed by catechol-O-methyltransferase may be a major reason for the lack of their carcinogenic activities in vivo.

Animals↗
PubMed1994

Quercetin increases the severity of estradiol-induced tumorigenesis in hamster kidney.

Catechol estrogens have been postulated to mediate estradiol-induced kidney tumorigenesis in Syrian hamsters. As part of an examination of this postulate, we studied the influence of quercetin, a polyphenolic flavonoid, on the incidence and severity of estrogen-induced kidney tumors, on the metabolic activation of estradiol to catechol estrogens, and on the inactivation of catechol estrogens by catechol-O-methyltransferase-mediated O-methylation. None of the hamsters treated with 0.3 or 3% quercetin in the diet for 5.7 or 6.5 months, respectively, developed tumors, whereas all animals treated with estradiol developed kidney tumors. The coadministration of estradiol plus 3% quercetin significantly (p < 0.05) increased the mean number of large tumor nodules and the incidence of abdominal metastases over values obtained with hormone treatment alone. The coadministration of estradiol plus 0.3 or 3% quercetin for 16 days increased renal NADPH-dependent estradiol-4- but not estradiol-2-hydroxylase activities by 91 or 73%, respectively, over values obtained with hormone treatment alone. In vitro, quercetin and its structural analog, fisetin, strongly inhibited the catechol-O-methyltransferase-catalyzed O-methylation of 60 microM 4-hydroxyestradiol, with IC50 values of approximately 2-4 microM. Kinetic analyses of the enzyme inhibition by quercetin and fisetin revealed a mixed-type of inhibition (competitive plus non-competitive). Combined treatment of estradiol plus 3% quercetin decreased the rates of kidney catechol-O-methyltransferase-catalyzed O-methylation of 2- and 4-hydroxyestradiol by 34 and 22%, respectively, from values obtained with hormone treatment alone. Rates of hamster liver and kidney microsome-mediated estrogen quinone formation and quinone reduction (redox cycling) in estradiol plus 3% quercetin-treated hamsters were not markedly altered compared to values obtained in estradiol-treated animals. It is concluded that increased rates of formation of 4-hydroxyestradiol combined with an inhibition of the inactivation of this catechol estrogen by catechol-O-methyltransferase may result in elevated levels of this estrogen metabolite, specifically in the hamster kidney, where it may undergo metabolic redox cycling and generate potentially mutagenic free radicals. Thus, the potentiation by quercetin of estradiol-induced tumorigenesis in hamster kidney supports a role of 4-hydroxyestradiol in estrogen-induced carcinogenesis in this species.

Animals↗
PubMed1994

Regulation of the formation of the major diethylstilbestrol-DNA adduct and some evidence of its structure.

Diethylstilbestrol (DES) induces kidney tumors in hamsters. In previous studies, DES has been shown by 32P-post-labeling analysis to bind covalently to DNA in vivo and in vitro and DES-DNA adduct formation has been suggested to play a key role in DES-induced carcinogenicity. In this study, we have examined the influence of the dose of DES, age of animals and organ specificity on adduct formation in hamsters. In addition, we examined the characteristics of DES-DNA adduct formation in vitro and the structure of the major adduct. DES-DNA adducts were detected in liver and kidney of hamsters treated with at least 20 mg/kg DES. Adduct concentrations were higher at higher doses or in older compared to younger animals. The covalent binding of DES to DNA catalyzed by hamster liver microsomes required cumene hydroperoxide as cofactor, whereas with NADPH, adducts were barely detectable, presumably because the reactive metabolic intermediate DES quinone was reduced to DES. The major DES-DNA adduct formed in vitro was purified by semipreparative and analytical high pressure liquid chromatography. It is concluded that DES-DNA adducts are formed from DES quinone at very low rates in vitro and occur at low levels in vivo, even when hamsters receive very large doses of DES. The dependence of DES-DNA adduct concentrations in vitro on organic hydroperoxide cofactors required for cytochrome P450-mediated DES quinone formation indicates that stilbene-DNA adduction may occur only under conditions of oxidative stress.

Animals↗
PubMed1994

DNA single-strand breaks in kidneys of Syrian hamsters treated with steroidal estrogens: hormone-induced free radical damage preceding renal malignancy.

The chronic administration of estradiol by subcutaneous (s.c.) implantation into male Syrian hamsters induces kidney tumors. Free radicals generated by redox cycling between catecholestrogens and their quinones have been proposed to damage DNA and to thus mediate renal hormone-induced carcinogenesis. As part of an examination of this postulate, we assayed by a filter elution technique DNA single-strand breaks in livers and kidneys of male hamsters treated with estrogen by single intraperitoneal (i.p.) injection, by s.c. implant or by continuous infusion and compared values to those in untreated controls. The DNAs of hamster liver and kidney were not affected by one i.p. injection of 5, 15 or 150 mg/kg estradiol. However, treatment of hamsters with one 25 mg estradiol implant/animal for 2 weeks elevated by 10% the levels of DNA single-strand breaks in kidney, but only to a minor extent in liver, which is not a target of estrogen-induced carcinogenesis. An infusion of 250 micrograms/day/animal of estradiol or 4-hydroxyestradiol for one week by osmotic pumps into hamsters resulted in a comparable increase of single-strand breaks in kidney DNA, whereas 2-hydroxyestradiol under these conditions had a negligible effect. It is concluded that the induction of DNA single-strand breakage by either estradiol or 4-hydroxyestradiol in hamster kidney supports a mechanism of estrogen-induced carcinogenesis by free radical generation via redox cycling between 4-hydroxyestradiol and its corresponding quinone.

Animals↗
PubMed1994

Conversion of estrone to 2- and 4-hydroxyestrone by hamster kidney and liver microsomes: implications for the mechanism of estrogen-induced carcinogenesis.

As part of an ongoing investigation of the role of metabolic activation of estrogens in the genesis of cancers such as estrogen-induced renal tumors in hamsters, we have 1) determined steroid-17 beta-oxidoreductase activity of microsomes and cytosol prepared from hamster kidney and liver; 2) compared the rates of 2-, 4-, and 16 alpha-hydroxylations of estrone by microsomes from hamster kidney and liver; and 3) determined the rates of inactivation of 2- and 4-hydroxyestrone by catechol-O-methyltransferase from hamster kidney and by purified enzyme. Microsomal steroid-17 beta-oxidoreductase activity in hamster kidney and liver was low and favored the conversion of estrone to estradiol. Cytosolic steroid-17 beta-oxidoreductase activity was only barely detectable in both liver and kidney. Using hepatic microsomes, the rate of 2-hydroxylation of estrone was comparable to that found previously using estradiol as substrate, whereas 4-hydroxylation of estrone was double that of estradiol. Using renal microsomes, the rates of 2- and 4-hydroxylation of estrone were 10- to 20-fold higher than those with estradiol as substrate, and the ratio of 2- to 4-hydroxylation was about 2:1. Fadrozole hydrochloride was an equally good inhibitor of rates of 2- and 4-hydroxylation of estrone (20 microM) by hepatic microsomes (IC50, approximately 25 microM). Corresponding IC50 values with renal microsomes were less than 2 microM, and 2-hydroxylation of estrone was inhibited by Fadrozole hydrochloride up to 15% more than 4-hydroxylation. Treatment of hamsters with estradiol for 2 months decreased rates of 2- and 4-hydroxylation of estrone by renal microsomes by approximately 95%. The rate of conversion of estrone to 16 alpha-hydroxyestrone by hepatic microsomes was 10-20% that of 2-hydroxylation. Renal microsomes catalyzed 16 alpha-hydroxylation of estrone at an even lower rate (approximately 5% of that of 2-hydroxylation). Rates of O-methylation of 2- and 4-hydroxyestrone by hamster kidney cytosol were comparable to those of 2- and 4-hydroxyestradiol. In conclusion, conversion of estrone to its catechol metabolites by microsomes of hamster kidney, a target organ of estrogen-induced carcinogenesis, is quantitatively more important than the conversion to 16 alpha-hydroxyestrone. The findings are consistent with the postulated role of catechol estrogens generated in situ in estrone-induced carcinogenesis.

Animals↗