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Biomedical subjects

J G Liehr

Publications and source records attributed to J G Liehr.

At least 19 recordsLinked to original sources
PubMed1999

Estrogen, DNA damage and mutations.

Estrogen administration to rodents results in various types of DNA damage and ultimately leads to tumors in estrogen-responsive tissues. Yet these hormones have been classified as nonmutagenic, because they did not induce mutations in classical bacterial and mammalian mutation assays. In this review, we have discussed the induction by estrogens of DNA and chromosomal damage and of gene mutations, because the classical assays were designed to uncover mutations only at one specific locus and could not have detected other types of mutations or changes in other genes. Various types of estrogen-induced DNA damage include: (a) direct covalent binding of estrogen quinone metabolites to DNA; (b) enhancement of endogenous DNA adducts by chronic estrogen exposure of rodents; (c) free radical generation by metabolic redox cycling between quinone and hydroquinone forms of estrogens and free radical damage to DNA such as strand breakage, 8-hydroxylation of purine bases of DNA and lipid hydroperoxide-mediated DNA modification. Two different types of chromosomal damage have also been induced by estrogen in vivo and in cells in culture such as numerical chromosomal changes and also structural chromosomal aberrations. Gene mutations have been induced in several cell types in culture either by the parent estrogen or by reactive estrogen quinone metabolites. Furthermore, in estrogen-induced kidney tumors in hamsters, several mutations have been observed in the DNA polymerase beta gene mRNA. Estradiol also induces microsatellite instability in these kidney tumors and in premalignant kidney exposed to estradiol. Although this work is still ongoing, it can be concluded that estrogens are complete carcinogens capable of tumor initiation by mutation potentially in critical genes. The hormonal effects of estrogens may complete the development of tumors.

Animals↗
PubMed1999

Lack of mutations in DNA polymerase beta of estradiol-induced hamster kidney tumors: sequence of hamster DNA polymerase beta cDNA.

We examined the effects of estradiol (E2), the natural estrogenic hormone, on the structure and expression of DNA polymerase beta (DNA pol beta), a DNA repair gene, from E2-induced primary kidney tumors of twelve Syrian hamsters, their metastases, and from kidney tissues surrounding the tumors. We sequenced the coding region of the hamster DNA pol beta and found it to differ from that of the human by 11%. No mutations were detected in the entire coding region including the catalytic domain of the DNA pol beta from E2-induced primary kidney tumors, their metastases, or from kidney tissues surrounding the tumors. The expression of the DNA pol beta mRNA was also not significantly altered in E2-induced kidney tumors or in kidney tissues surrounding the tumors compared to that of control kidney tissues. These results suggest that mutations in the DNA pol beta gene may not be involved in the induction or malignant progression of hamster kidney tumors induced by E2. The nucleotide sequence of the hamster DNA pol beta described here will be useful for the study of the structure and expression of this gene.

Amino Acid Sequence↗
PubMed1999

Differential regulation of c-fos expression in estrogen-induced hamster renal tumors compared with kidney not due to creation of an estrogen-response element by point mutation in the gene's flanking sequence.

The conversion of a palindromic sequence, GGTCTnnnAGACC, in the 5'-flanking region of the murine c-fos proto-oncogene into a functional estrogen-response element by a single base change into GGTC(A/G)nnnAGACC has previously been postulated [Nawaz et al., 1993] as a possible mechanism of the induction of tumors by estrogens. This attractive hypothesis has been investigated in estradiol-induced Syrian hamster kidneytumors, in H-301 kidney tumor cells (a cell line derived from the Syrian hamster tumor), and in normal kidney tissue. The c-fos gene is differentially regulated by a classical estrogen receptor-mediated process in tumors, whereas in the acutely treated kidney, estradiol induces c-fos expression independent of estrogen-receptor function. In this study, we identified in the 5'-flanking region of the hamster kidney c-fos gene the sequence AGTCCnnnAGACC, which closely resembled but did not appear to function as an estrogen-response element. No mutations were detected in this sequence or in the 5'-flanking region of c-fos genes from three different primary tumors and from H-301 tumor cells. To rule out the possibility of a low copy number of mutant alleles in a tumor sample, polymerase chain reaction-based single-strand conformation polymorphism analysis was performed on 372 base pairs of the 5'-flank of the c-fos gene (-367 to +5 base pairs relative to the transcription start point). Nine different kidney tumor DNA samples and five normal kidney tissue samples (controls) produced an identical pattern of DNA bands, suggesting a lack of natural polymorphisms and mutations in this region of the c-fos gene. Acute treatment of hamsters with 17beta-estradiol for 6 h significantly induced renal c-fos mRNA expression, whereas control levels of c-fos were restored by co-treatment with estradiol and either N-acetyl-L-cysteine or alpha-naphthoflavone. We concluded that the previously observed change in regulatory control of c-fos expression in kidney versus estradiol-induced tumors does not involve the creation of a functional estrogen-response element by single point mutation in the 5'-flanking region of the gene. Additionally, c-fos expression in estradiol-treated hamster kidneys appears to be mediated by free radicals generated by the catechol metabolites of estradiol and not by the activation of any estrogen receptor.

Animals↗
PubMed1998

Concentration dependence of prooxidant and antioxidant properties of catecholestrogens.

Estradiol is an established antioxidant in vitro and in vivo. In contrast, prooxidant effects such as 8-hydroxylation of guanine bases of DNA have been induced by various estrogens in hamsters and by 4-hydroxyestradiol or -estrone and a microsomal activating system in vitro. As part of an examination of these conflicting reports, we studied the enhancement or inhibition of lipid peroxidation (conjugated diene formation monitored at 240 nm) by catecholestrogens in human low-density lipoprotein (LDL) incubated with cupric sulfate in phosphate buffer. Addition of 2- or 4-hydroxyestradiol, 2- or 4-methoxyestradiol, or estradiol or estriol (0.5-50 microM) increased lag times for diene formation by 30 to <300% over control values in the absence of estrogens (lag time, 1.6 h). In contrast, low concentrations (5 pM-100 nM) of catecholestrogens decreased lag times by about 40-50%, demonstrating their prooxidant activities. The prooxidant capabilities of catecholestrogens were examined by assaying the reduction by estrogens of Cu(II) to Cu(I) and of Fe(III) to Fe(II). Both 2- and 4-hydroxyestradiol and 2- and 4-methoxyestradiol reduced Cu(II) and Fe(III) ions to their lower oxidation state. In conclusion, the reduction of Cu(II) to Cu(I) by catecholestrogens is proposed to initiate lipid peroxidation and thus oxidation of LDL. In contrast, at high concentrations of catecholestrogens, the scavenging of oxygen radicals may predominate over lipid peroxidation and free radical generation by analogy to the action of similar phenolic antioxidants. With estradiol, estriol, and the methoxyestrogen metabolites, only antioxidant effects were observed.

Animals↗
PubMed1998

Induction of monoamine oxidase B by 17 beta-estradiol in the hamster kidney preceding carcinogenesis.

Estrogen-induced kidney tumorigenesis in the male Syrian hamster has been postulated to be mediated by free radicals generated by metabolic redox cycling of catecholestrogen intermediates. This tissue and other rodent tissues in which tumors develop in response to estrogen treatment have been shown to contain high levels of the catecholamine norepinephrine. In this study, we have thus examined the hypothesis that an additional source of free radicals may be hydrogen peroxide formed by the monoamine oxidase (MAO)-catalyzed deamination of catecholamines. We have studied the effect of 17beta-estradiol (25-mg pellet, sc) on MAO activity in the hamster kidney (a target organ) and in the hamster liver and the rat kidney and liver, organs which do not develop tumors under these conditions. 17beta-Estradiol treatment for 2 weeks significantly increased (P < 0.01) MAO activity in the hamster kidney (76.7 +/- 10.0 and 113.0 +/- 10.8% over controls for the substrates tyramine and kynuramine, respectively). MAO activity remained elevated after 4 weeks of 17beta-estradiol treatment. No significant changes were observed in the MAO activity of hamster liver or rat kidney and liver. The addition of Tamoxifen to 17beta-estradiol restored control levels of renal MAO activity. The use of selective MAO A and MAO B inhibitors (clorgyline and deprenyl, respectively) identified the B form as the major component of hamster kidney MAO activity and its hormonal regulation. In conclusion, the estrogen receptor-mediated activation of MAO in conjunction with high catecholamine concentrations in the hamster kidney as previously reported may significantly increase the production of hydrogen peroxide and hydroxyl radicals which are postulated to contribute to tumor initiation.

Animals↗
PubMed1998

Estrogen-induced microsatellite DNA alterations are associated with Syrian hamster kidney tumorigenesis.

Exposure to estrogens is associated with an increase in cancers, including malignancies of the breast and uterus in humans, and of the kidney in hamsters. DNA damage induced by metabolic activation of estrogen has been postulated to result in gene mutations critical for the development of estrogen-induced kidney tumors in hamsters. As part of our examination of the genetic consequences of estrogen-induced DNA damage, we searched for estrogen-induced alterations in microsatellite DNA, a frequent site of mutation in tumors. Genomic DNA isolated from kidney of hamsters treated with estradiol, from estrogen-induced kidney tumors and from untreated age-matched controls, was examined by Southern blot analysis with three multi-locus oligonucleotide probes: (GACA)4, (CAC)6 and (CAG)6. Alterations in DNA fragments containing GACA and CAC tandem repeats were detected in kidney DNA of hamsters treated with hormone for 3 and 4 months, whereas no such effects were seen in control animals. In estrogen-induced tumors, microsatellite alterations were observed in fragments that contain these same two repeat sequences and also CAG repeat sequences. The induction of microsatellite alterations by estradiol in kidney DNA preceding estrogen-induced renal malignancy may play a role in hormone-induced tumorigenesis.

Animals↗
PubMed1998

Enhancement of estrogen-induced renal tumorigenesis in hamsters by dietary iron.

Iron participates in the generation of hydroxyl radicals by the iron-catalyzed Fenton reaction. Its role in estrogen-induced carcinogenesis has been examined in this study by investigating the effects of iron content of hamster diets on tumor induction by estradiol. The renal tumor incidence and number of tumor nodules in hamsters treated with estradiol plus a diet enriched with iron (384 p.p.m. Fe as ferric citrate) for 5 months were 2- and 4-fold higher, respectively, than those observed in animals on an iron-poor diet plus estradiol (3.9 p.p.m. Fe, as ferric citrate). Tumor incidence and number of tumor nodules in estradiol-treated hamsters on the iron-deficient diet were not different from those of animals on a normal rodent chow. No tumors were detected in hamsters treated only with the low or high iron diets. Total serum iron was significantly increased in animals treated with the high iron diet plus estradiol compared with the low iron diet plus estradiol group and the high and low iron controls. Estrogen treatment increased non-heme iron in liver of both high and low iron treatment groups and in kidney of the hamsters on the low iron diet. It is concluded that dietary iron enrichment enhances the incidence and severity of estrogen-induced tumor induction.

Animals↗
PubMed1998

Accumulation of albumin in renal cytosol of hamsters treated with estradiol and in estrogen-induced hamster kidney tumors.

The intracellular accumulation of albumin has been observed in cytosols of benign and malignant human breast tumors and in mammary tumors of rodents induced by carcinogens. Additionally, cellular uptake of albumin has been detected in MCF-7 human breast cancer cells in culture. The clinical relevance of the albumin accumulation in human and rodent mammary tumors is not clear. In this study, we investigated the accumulation of albumin in an estrogen-induced and -dependent hamster kidney tumor model to understand the mechanisms and the role of hormones in this process. Protein accumulation patterns were examined by Western blot analyses in kidney homogenates of hamsters treated with 17beta-estradiol for various lengths of time and in kidney tumors which are induced with 100% incidence by this treatment for at least six months. Such analyses were also carried out in tissues of hamsters treated with the weakly carcinogenic estrogen 17alpha-ethinylestradiol (10% tumor incidence after nine months of treatment). Our data demonstrate the accumulation of albumin in kidney of hamsters treated with 17beta-estradiol but not with 17alpha-ethinylestradiol. Albumin accumulates specifically in the target organ of carcinogenesis, the kidney, however, with no increase in the serum concentrations or in the liver. Tumors do not develop in the livers of hamsters under these conditions of 17beta-estradiol treatment. This accumulation of albumin in hamster kidney may be the result of damage to the glomerulum which may be compromised by estradiol-induced toxicity and therefore unable to filter out excess albumin.

Amino Acid Sequence↗
PubMed1997

Release of iron from ferritin storage by redox cycling of stilbene and steroid estrogen metabolites: a mechanism of induction of free radical damage by estrogen.

Estrogens induce hydroxyl radical-mediated DNA and protein damage and lipid peroxidation. As part of a study of the mechanism of hydroxyl radical generation by estrogens, we investigated the in vitro mobilization of Fe2+ from ferritin by redox cycling of the stilbene or steroid estrogen metabolites diethylstilbestrol-4',4"-quinone (DESQ), equilenin-3,4-quinone (EQ), or estrone-3,4-quinone (3,4EQ). Aerobic cytochrome P450 reductase-mediated redox cycling of 35.50 microM DESQ, 0.35 microM EQ, or 3.55 microM 3,4EQ increased the reduction of succinoylated cytochrome c, a measure of superoxide radical formation, by 19-20% over control values (24.5+/-0.3 microM) in the absence of estrogen quinone substrate. Rates of Fe2+ release from horse spleen ferritin by cytochrome P450 reductase-mediated redox cycling of 35.50 microM DESQ, 0.35 microM EQ, or 3.55 microM 3,4EQ were 94.4+/-0.6, 117.2+/-9.4, or 137.7+/-19.9 pmol Fe2+/min, respectively, compared to 67.3 + 2.3 pmol Fe2+/min in the absence of estrogen substrates. Redox cycling of 35.5 microM DESQ, EQ, or 3,4EQ mediated by microsomes of hamster kidney, a target organ of estrogen-induced carcinogenesis, released 511+/-30.10, 516.91+/-22.90, or 410.27+/-28.49 pmol Fe2+/min, respectively. Corresponding values with microsomes of hamster liver, where tumors do not develop by estrogen treatment, were 272.27+/-43.10, 222.25+/-21.78, or 91.36+/-8.54 pmol Fe2-/min, respectively. Diethylstilbestrol, equilenin, and 4-hydroxyestrone do not induce detectable iron release from ferritin under these conditions. The cytochrome P450 reductase-mediated redox cycling of DESQ, EQ, or 3,4EQ in the presence of iron resulted in the hydroxylation of benzoic acid by hydroxyl radical attack. These data demonstrate that redox cycling of estrogen metabolites releases Fe2+ from ferritin, which in turn generates hydroxyl radicals by a Fenton reaction. This estrogen-induced hydroxyl radical damage may contribute to tumor initiation in hormone target tissues, including breast cancer.

Animals↗
PubMed1997

Cytochrome P450 metabolism of estradiol in hamster liver and kidney.

Estradiol induces kidney tumors in Syrian hamsters. The elevated conversion of estradiol to 4-hydroxylated metabolites in kidney compared to the predominant 2-hydroxylation in liver and other organs, where tumors are not induced by this treatment, has been proposed to be the basis of estrogen-induced carcinogenesis. In this study, we examined the hepatic and renal enzymes catalyzing the formation of catecholestrogens to understand the differences in estrogen metabolism in these organs. In liver, 2-hydroxylation of estradiol is the major metabolic pathway with 4-hydroxylation a minor by-product and with the formation of both catechols responding coordinately to the same inhibitors. Western blot analysis and inhibition studies suggest that the major form catalyzing hepatic estrogen 2-hydroxylation is a member of the CYP3A family, as previously observed with rat liver microsomes, and that 4-hydroxylation is a by-product of this metabolism. In the kidney, 4-hydroxylation of estradiol appears to be catalyzed by more than one enzyme according to the Eadie-Hofstee analysis. Both 2- and 4-hydroxylation in the kidney are affected differentially by inhibitors and are induced by beta-napthoflavone. Western blots of renal microsomes reveal that CYP1A2 is induced whereas CYP1A1 is detectable in kidney, but not induced by this treatment. Finally, a part of the 2-hydroxylation and a small part of the 4-hydroxylation by kidney microsomes may be catalyzed by a member of the CYP3A family, since these reactions are partially inhibited by CYP3A inhibitors such as progesterone and other progestins, although renal enzyme levels are much lower than those in the liver as revealed by Western blot. Our data suggest that estrogen 2-hydroxylation in the hamster kidney is catalyzed by members of the CYP1A and CYP3A families, which also contribute to 4-hydroxylation. The majority of 4-hydroxyestradiol formation in the hamster kidney may be catalyzed by a form(s) of the newly discovered CYP1B family that has yet to be characterized.

Animals↗
PubMed1997

Comparative disposition of the antineoplastic agent 9-nitrocampotothecin and the inactive isomer 12-nitro camptothecin in CASE-bearing nude mice: effect of route of administration on tissue distribution.

PURPOSE: 9-Nitrocamptothecin (9-NC) and 12-nitrocamptothecin (12-NC) are synthetic structural analogues of camptothecin (CPT) which have been prepared to explore the structure/activity relationship of this group of compounds against a wide variety of experimental tumors. As part of our investigation of the pharmacology and the mechanism of tumor inhibition of these compounds, we examined the effect of route of administration on the distribution of tritium-labeled 9-NC and 12-NC, an active and a poor chemotherapeutic agent, respectively. METHODS: Quantitative whole-body autoradiography was used and our results were compared with previous results obtained with the parent compound CPT. RESULTS: These studies revealed that, independent of the route of administration, both CPT derivatives were rapidly distributed to gall bladder, gastrointestinal tract and kidney. The excretion from these organs was indicated by the high levels of radioactivity in urine (urinary bladder) and feces (large intestines). The studies also indicated that the distributions of 9-NC and 12-NC were qualitatively similar, but quantitatively higher uptake of radioactivity was observed in animals treated with 12-NC than in those treated with 9-NC at 30 min following treatment. With the exception of the late sampling time (12 h after administration), the accumulation of radioactivity in the lungs (bronchioles) of animals that received an intravenous (i.v.) dose of 9-NC or 12-NC was higher than those treated with an intramuscular (i.m.) dose. However, the retention of drug-derived radioactivity in the tumors of mice treated with an i.m. dose of 9-NC was higher than that in the tumors of i.v.-treated animals and was also higher than that in tumors of animals treated with 12-NC. CONCLUSIONS: These results suggest that higher accumulation of 9-NC in tumor tissues than of 12-NC may contribute to the more potent chemotherapeutic activity of the former agent. Our results also suggest that i.m. injection is a more effective route of administration than i.v. administration.

Animals↗
PubMed1997

Immunocytochemical localization of C-myc and C-jun oncoproteins in hamster kidney and estrogen-induced kidney tumors.

The chronic administration of 17beta-estradiol to male Syrian hamsters for 6-7 months induces kidney tumors which express high levels of c-fos, c-myc and c-jun mRNA compared to surrounding tissue or untreated controls. In this study, we have investigated, by immunocytochemical methods, the cellular localization of c-myc and c-jun oncoproteins in estrogen-dependent kidney tumors, in kidney tissue of hamsters treated with 17beta-estradiol for 6 months and in the kidneys of age-matched controls. The c-myc oncoprotein was strongly expressed in tumors, in smooth muscle layers of arteries and in parietal epithelial cells of the glomerulus. In age-matched untreated kidneys, there was little or no staining in the glomerulus, arteries or kidney tubular cells. The c-jun oncoprotein was detected in kidney tumors and in the tubular epithelium of surrounding tissue. The immunoreactivity for c-jun oncoprotein was highest in the tumor, intermediate in estrogen-treated kidney tissue and lowest in kidney tubular cells of controls. It is concluded that the high expression of c-myc in estrogen-induced kidney tumors, in the smooth muscle layer of arteries, and in glomerular parietal epithelial cells in the kidneys of 17beta-estradiol-treated hamsters, but poor expression in control kidneys indicate an involvement of this oncoprotein in the tumorigenic process. In contrast, c-jun is expressed in untreated, in 17beta-estradiol-treated kidneys and in tumors, and may not serve as a prognostic marker in the transformation of these cells to the malignant phenotype.

Animals↗
PubMed1997

Mechanism of cytochrome P450-catalyzed aromatic hydroxylation of estrogens.

The mechanism of aromatic hydroxylation of estrogens by cytochrome P450 enzymes has been examined by comparing the oxidation of estrone with that of substrates carrying additional aromaticity such as equilenin and the structural analog 2-naphthol. Hamster liver microsomes preferentially catalyzed the conversion of estrone to 2-hydroxyestrone (Km = 30 and 25 microM and Vmax = 1497 and 900 pmol (mg of protein)-1 min-1 for 2- and 4-hydroxyestrone formation, respectively). In contrast, equilenin was hydroxylated exclusively at C-4 of the steroid ring system and 2-naphthol at the corresponding C-1 position (Km = 67 and 42 microM and Vmax = 2083 and 3226 pmol (mg of protein)-1 min-1 for 4-hydroxyequilenin and 1,2-dihydroxynaphthalene formation, respectively). This shift in the specificity of hydroxylation was due to the introduction of additional aromaticity at ring B of equilenin, because hamster liver microsomes are known not to contain any estrogen-4-hydroxylase, only estrogen-2-hydroxylase activity catalyzed by cytochrome P450 3A family enzymes. The exclusive 4-hydroxylation of equilenin is proposed to be due to a preferred delocalization of the naphthoxy radical an intermediate in the hydroxylation, to C-4, whereas delocalization to C-2 requires additional activation energy and is energetically not favored. Based on these electronic considerations, a mechanism of aromatic hydroxylation of estrogens is proposed which features hydrogen abstraction from the phenolic hydroxy group, electron delocalization of the phenoxy radical to a carbon-centered radical, and subsequent formation of catechol metabolites by hydroxy radical addition at C-2 or C-4 depending on steric or electronic constraints.

Animals↗
PubMed1997

Dual role of oestrogens as hormones and pro-carcinogens: tumour initiation by metabolic activation of oestrogens.

Epidemiological evidence increasingly points to exogenous or endogenous oestrogens as a risk factor for breast cancer. However, it is unlikely that induction of oestrogen-dependent tumour growth is the sole contribution of oestrogens to tumour development in the mammary gland, because oestrogen receptors are barely detectable in normal mammary epithelial cells. In this review, I examine a mechanism for mammary carcinogenesis, which emphasizes tumour initiation by metabolic activation of oestrogens in combination with cell transformation and growth stimulation by oestrogen receptor-mediated processes. Catecholestrogen metabolites are capable of metabolic redox cycling between quinone and hydroquinone forms, a mechanism of free radical generation. Several types of direct and indirect free radical-mediated DNA damage are induced by oestrogens in vitro and in vivo, such as DNA single strand breaks, 8-hydroxylation of guanine bases, and DNA adduct formation by malondialdehyde, a decomposition product of free radical-induced lipid peroxides. The substrate for redox cycling and free radical generation may be 4-hydroxoestradiol, because this metabolite is formed from oestradiol by a specific oestrogen 4-hydroxylase detected in several human organs including mammary tissue. It has also been formed in organs of rodents where oestrogens induce tumours, with the exception of the liver. 4-Hydroxyoestradiol is a potent, long-acting oestrogen and may complete the carcinogenic process by stimulating receptor-mediated proliferation. An understanding of a possible mechanism of mammary carcinogenesis as a result of oestrogen-mediated initiation means that several prevention strategies, based on inhibiting metabolic activation of oestrogens or free radical action, can be developed.

Animals↗