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T J Monks

Publications and source records attributed to T J Monks.

At least 37 records · Page 2Linked to original sources
PubMed1998

The pharmacology and toxicology of polyphenolic-glutathione conjugates.

Polyphenolic-glutathione (GSH) conjugates and their metabolites retain the electrophilic and redox properties of the parent polyphenol. Indeed, the reactivity of the thioether metabolites frequently exceeds that of the parent polyphenol. Although the active transport of polyphenolic-GSH conjugates out of the cell in which they are formed will limit their potential toxicity to those cells, once within the circulation they can be transported to tissues that are capable of accumulating these metabolites. There are interesting physiological similarities between the organs that are known to be susceptible to polyphenolic-GSH conjugate-mediated toxicity. In addition, the frequent localization of gamma-glutamyl transpeptidase to cells separating the circulation from a second fluid-filled compartment coincides with tissues that are susceptible either to polyphenolic-GSH conjugate-induced toxicity or to quinone and reactive oxygen species-induced toxicity. Polyphenolic-GSH conjugates therefore contribute to the nephrotoxicity, nephrocarcinogenicity, and neurotoxicity of a variety of polyphenols.

Animals↗
PubMed1997

DNA damage, gadd153 expression, and cytotoxicity in plateau-phase renal proximal tubular epithelial cells treated with a quinol thioether.

2-Bromo-bis-(glutathion-S-yl)hydroquinone [2-Br-bis-(GSyl)HQ] causes DNA single-strand breaks (SSB), causes growth arrest, induces the expression of gadd153 (a gene inducible by growth arrest and DNA damage), and decreases histone H2B mRNA in log-phase renal proximal tubular epithelial cells (LLC-PK1). Renal epithelial cells in vivo normally exhibit a low mitotic index, therefore experiments in both plateau- and log-phase cells are necessary for a comprehensive understanding of the stress response to 2-Br-bis-(GSyl)HQ. In the present article we demonstrate that not all features of the stress response in log-phase cells are reproduced in plateau-phase cells. Thus, although 2-Br-bis-(GSyl)HQ causes concentration and time-dependent increases in DNA SSB, and increases the expression of gadd153, histone H2B mRNA levels are unaltered in plateau-phase cells. The relationship between reactive oxygen species, DNA damage, gene expression, and cytotoxicity was also investigated. Our findings suggest that (i) 2-Br-bis-(GSyl)HQ-mediated DNA damage in LLC-PK1 cells is mediated by the generation of H2O2; (ii) DNA damage, either directly or indirectly, contributes to cell death; and (iii) DNA damage, either directly or indirectly, provides the initial signal for gadd153 expression. In addition, DNA repair is rapid in LLC-PK1 cells, and the DNA-repair inhibitors 1-beta-D-arabinofuranosylcytosine and hydroxyurea have no effect on the amount of DNA SSB. Although the addition of 3-aminobenzamide following 2-Br-bis-(GSyl)HQ exposure has no effect on the removal of DNA SSB, it causes a slight but significant increase in gadd153 expression and cell viability, indicating that activation of poly(ADP-ribose)polymerase may exacerbate toxicity. Finally, aurintricarboxylic acid did not prevent DNA SSB or cytotoxicity in 2-Br-bis-(GSyl) HQ-treated LLC-PK1 cells, implying that activation of endonucleases does not play a role in these processes.

Animals↗
PubMed1997

2,5-Bis-(glutathion-S-yl)-alpha-methyldopamine, a putative metabolite of (+/-)-3,4-methylenedioxyamphetamine, decreases brain serotonin concentrations.

3,4-(+/-)-Methylenedioxyamphetamine (MDA) and 3,4-(+/-)-methylenedioxymethamphetamine (MDMA) are serotonergic neurotoxicants. However, when injected directly into brain, MDA and MDMA are not neurotoxic, suggesting that systemic metabolism plays an important role in the development of neurotoxicity. The nature of the metabolite(s) responsible for MDA- and MDMA-mediated neurotoxicity is unclear. alpha-Methyldopamine is a major metabolite of MDA and is readily oxidized to the o-quinone, followed by conjugation with glutathione (GSH). Because the conjugation of quinones with GSH frequently results in preservation or enhancement of biological (re)activity, we have been investigating the role of quinone-thioethers in the acute and long-term neurochemical changes observed after administration of MDA. Although intracerebroventricular (i.c.v.) administration of 5-(glutathion-S-yl)-alpha-methyldopamine (4 x 720 nmol) and 5-(N-acetylcystein-S-yl)-alpha-methyldopamine (1 x 7 nmol) to Sprague-Dawley rats produced overt behavioral changes similar to those seen following administration of MDA (93 mumol/kg, s.c.) they did not produce long-term decreases in brain serotonin (5-hydroxytryptamine, 5-HT) concentrations. In contrast, 2,5-bis-(glutathion-S-yl)-alpha-methyldopamine (4 x 475 nmol) decreased 5-HT levels by 24%, 65% and 30% in the striatum, hippocampus and cortex, respectively, 7 days after injection. The relative sensitivity of the striatum, hippocampus and cortex to 2,5-bis-(glutathion-S-yl)-alpha-methyldopamine was the same as that observed for MDA; the absolute effects were greater with MDA. The effects of 2,5-bis-(glutathion-S-yl)-alpha-methyldopamine were also selective for serotonergic nerve terminal fields, in that 5-HT levels were unaffected in regions of the cell bodies. Because 2,5-bis-(glutathion-S-yl)-alpha-methyldopamine caused long-term depletion in 5-HT without adversely affecting the dopaminergic system, it also mimics the selectivity of MDA/MDMA. The data imply a possible role for quinone-thioethers in the neurobehavioral and neurotoxicological effects of MDA/MDMA.

3,4-Methylenedioxyamphetamine↗
PubMed1997

The response of renal tubular epithelial cells to physiologically and chemically induced growth arrest.

Cells respond to a variety of stresses by activating the transcription of a battery of "acute phase" or "stress response" genes. The nature of this response is tailored to the nature of the stress. The extent to which physiologically and pathophysiologically induced growth arrest share common genomic responses is unclear. We therefore compared the effects of a physiologically induced (serum and nutrient depletion) and a chemically induced (2-Br-bis-(GSyl)HQ and 2-Br-6-(GSyl)HQ) stress in renal tubular epithelial cells (LLC-PK1). The response to physiological stress, induced by serum depletion, involves growth arrest characterized by an inhibition of DNA synthesis that occurs in the absence of a decrease in histone mRNA or an increase in gadd153 mRNA, one of the growth arrest and DNA damage inducible genes. In contrast, the chemical-induced stress involves growth arrest accompanied by a decrease in histone mRNA, particularly core histone H2B and H2A mRNA, and the induction of gadd153. Chemical-induced changes in histone mRNA inversely correlate to changes in the expression of a stress gene, hsp70, whose expression is dependent upon the maintenance of appropriate nucleosomal structure.

Animals↗
PubMed1997

Identification of quinol thioethers in bone marrow of hydroquinone/phenol-treated rats and mice and their potential role in benzene-mediated hematotoxicity.

Metabolism of benzene is required to produce the classical hematological disorders associated with its exposure. After coadministration of hydroquinone (0.9 mmol/kg, ip) and phenol (1.1 mmol/kg, ip) to male Sprague-Dawley rats and DBA/2 mice, 2-(glutathion-S-yl)hydroquinone was identified in the bone marrow of both species. 2,5-Bis(glutathion-S-yl)hydroquinone, 2,6-bis(glutathion-S-yl)hydroquinone, and 2,3,5-tris(glutathion-S-yl)hydroquinone were also observed in the bone marrow of rats but were detected only sporadically in mice. Both species produced 2-(cystein-S-ylglycinyl)hydroquinone, 2-(cystein-S-yl)hydroquinone, and 2-(N-acetylcystein-S-yl)hydroquinone, indicating the presence of a functional mercapturic acid pathway in bone marrow. The ability of bone marrow to acetylate 2-(cystein-S-yl)hydroquinone and deacetylate 2-(N-acetylcystein-S-yl)hydroquinone was confirmed in vitro. Total quinol thioether concentrations were higher in, and eliminated more slowly from, the bone marrow of mice. Intravenous injection of 100 micromol/kg 2-(glutathion-S-yl)hydroquinone to rats gave rise to substantially lower bone marrow C(max) and AUC values compared to values found following coadministration of hydroquinone/phenol, suggesting that the major fraction of the GSH conjugates present in bone marrow are formed in situ. Finally, the erythrotoxicity of several of these conjugates was determined in rats using the erythrocyte 59Fe incorporation assay. Administration of 2,3,5-tris(glutathion-S-yl)hydroquinone (17 micromol/kg, iv), 2,6-bis(glutathion-S-yl)hydroquinone (50 micromol/kg, iv), and benzene (11 mmol/kg, sc) significantly decreased 59Fe incorporation into reticulocytes to 45 +/- 6%, 28 +/- 3%, and 20 +/- 9% of control values, respectively. Although the doses of 2,3,5-tris(glutathion-S-yl)hydroquinone and 2,6-bis(glutathion-S-yl)hydroquinone represented only 0.2% and 0.4% of the dose of benzene, both conjugates reduced 59Fe incorporation to the same degree as benzene. 2-(Glutathion-S-yl)hydroquinone had no effect at the dose tested (200 micromol/kg, iv). In summary, these data suggest that hydroquinone-glutathione conjugates are erythrotoxic and may contribute to benzene-mediated hematotoxicity.

Animals↗
PubMed1997

Cytotoxicity and cell-proliferation induced by the nephrocarcinogen hydroquinone and its nephrotoxic metabolite 2,3,5-(tris-glutathion-S-yl)hydroquinone.

Hydroquinone, an intermediate used in the chemical industry and a metabolite of benzene, is a nephrocarcinogen in the 2-year National Toxicology Program bioassay in male Fischer 344 rats. Current evidence suggests that certain chemicals may induce carcinogenesis by a mechanism involving cytotoxicity, followed by sustained regenerative hyperplasia and ultimately tumor formation. Glutathione (GSH) conjugates of a variety of hydroquinones are potent nephrotoxicants, and we now report on the effect of hydroquinone and 2,3,5-(tris-glutathion-S-yl)hydroquinone, on site-selective cytotoxicity and cell proliferation in rat kidney. Male Fischer 344 rats (160-200 g) were treated with hydroquinone (1.8 mmol/kg or 4.5 mmol/kg, p.o.) or 2,3,5-(tris-glutathion-S-yl)hydroquinone (7.5 micromol/kg; 1.2-1.5 micromol/rat, i.v.), and blood urea nitrogen (BUN), urinary gamma-glutamyl transpeptidase (gamma-GT), alkaline phosphatase (ALP), glutathione-S-transferase (GST) and glucose were measured as indices of nephrotoxicity. Hydroquinone (1.8 mmol/kg, p.o.) is nephrotoxic in some rats, but not others, but cell proliferation (BrDU incorporation) in proximal tubular cells of the S3M region correlates with the degree of toxicity in individual rats. At 4.5 mmol/kg, hydroquinone causes significant increases in the urinary excretion of gamma-GT, ALP and GST. Pretreatment of rats with acivicin prevents hydroquinone-mediated nephrotoxicity, indicating that toxicity is dependent on the formation of metabolites that require processing by gamma-GT. Consistent with this view, 2,3,5-(tris-glutathion-S-yl)hydroquinone, a metabolite of hydroquinone, causes increases in BUN, urinary gamma-GT and ALP, all of which are maximal 12 h after administration of 2,3,5-(tris-glutathion-S-yl)hydroquinone. In contrast, the maximal excretion of GST and glucose occurs after 24 h. By 72 h, BUN and glucose concentrations return to control levels, while gamma-GT, ALP and GST remain slightly elevated. Examination of kidney slices by light microscopy revealed the presence of tubular necrosis in the S3M segment of the proximal tubule, extending into the medullary rays. Cell proliferation rates in this region were 2.4, 6.9, 15.3 and 14.3% after 12, 24, 48 and 72 h, respectively, compared to 0.8-2.4% in vehicle controls. Together with the metabolic data, the results indicate a role for hydroquinone-thioether metabolites in hydroquinone toxicity and carcinogenicity.

Animals↗
PubMed1997

Formation of catechol estrogen glutathione conjugates and gamma-glutamyl transpeptidase-dependent nephrotoxicity of 17beta-estradiol in the golden Syrian hamster.

In an animal model of hormone-mediated carcinogenesis, male golden Syrian hamsters develop renal carcinoma following prolonged exposure to 17beta-estradiol. The basis for the species and tissue specificity is unclear. Detailed information on the disposition of 17beta-estradiol in this model is lacking. Because catechol estrogens have been implicated in this model of carcinogenesis, we investigated the metabolism and nephrotoxicity of 17beta-estradiol in golden Syrian hamsters, with emphasis on the formation of catechol estrogen thioethers. 17beta-Estradiol (50 micromol/kg, i.p.) is a mild nephrotoxicant, causing significant elevations in the urinary excretion of gamma-glutamyl transpeptidase (gamma-GT), alkaline phosphatase, glutathione S-transferase (GST) and glucose. Increases in renal protein carbonyls and lipid hydroperoxides, which are markers of oxidative damage, also occur after administration of 17beta-estradiol (50 micromol/kg, i.p.). 17beta-Estradiol-mediated nephrotoxicity is reduced by treating animals with acivicin, an inhibitor of gamma-GT, implying that toxicity is mediated by metabolites requiring metabolism by this enzyme. Following administration of 17beta-[14C]estradiol (100 micromol/kg) to hamsters, 9.7% of the dose is recovered in bile after 5 h, the majority (7.9%) representing aqueous metabolites. Seven catechol estrogen GSH conjugates were identified, 2-hydroxy-1,4-bis-(glutathion-S-yl)-17beta-estradiol, 2-hydroxy-4-(glutathion-S-yl)-17beta-estradiol, 2-hydroxy-4-(glutathion-S-yl)-estrone, 4-hydroxy-1-(glutathion-S-yl)-estrone, 2-hydroxy-1-(glutathion-S-yl)-estrone, 4-hydroxy-1-(glutathion-S-yl)-17beta-estradiol, and 2-hydroxy-1-(glutathion-S-yl)-17beta-estradiol. At 5.4 micromol/kg of 17beta-estradiol, a dose-reflective of daily exposure levels in the hamster model of nephrocarcinogenicity, 12% of the dose is recovered within 5 h as a combination of GSH conjugates of 2- and 4-hydroxy-17beta-estradiol and 2- and 4-hydroxyestrone. In summary, oxidation of catechol estrogens, followed by GSH conjugation, occurs in vivo and 17beta-estradiol is a mild nephrotoxicant in a manner dependent on the activity of gamma-GT.

Alkaline Phosphatase↗
PubMed1997

PGE2-mediated cytoprotection in renal epithelial cells: evidence for a pharmacologically distinct receptor.

Although the exact mechanism of prostaglandin E2 (PGE2)-mediated cytoprotection has not been elucidated, its ability to induce cytoprotection in cell culture suggests this action occurs at the cellular level. The present studies were conducted to determine whether PGE2 induces protection against 2,3,5-(trisglutathion-S-yl)-hydroquinone [2,3,5-(trisglutathion-S-yl)-HQ]-mediated cytotoxicity in a renal proximal tubule epithelial cell line (LLC-PK1) and to delineate the cellular and molecular mechanisms associated with this response. Pretreatment of LLC-PK1 cells with 0.01-40 microM PGE2 for 24 h fully protects against a moderately toxic concentration of 2,3,5-(trisglutathion-S-yl)-HQ. PGE2-mediated cytoprotection is observed in cells pretreated at pH 7.4 but not at pH 7.8. However, cytoprotection is observed in LLC-PK1 cells pretreated with the PGE2 analog, 11-deoxy-16,16-dimethyl PGE2 (DDM-PGE2) but not with the PGE2 receptor [E-prostanoid (EP)] agonists 17-phenyltrinor PGE2 (EP1), 11-deoxy PGE1 (EP2/EP4), sulprostone (EP1/EP3), PGE1, or PGA2. 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent activator of protein kinase C (PKC), also induces cytoprotection, supporting a role for this pathway in the cytoprotective response. PGE2, DDM-PGE2, and TPA all induce the binding of nuclear proteins to a TPA responsive element (TRE), whereas analogs that did not induce cytoprotection (PGE1, 17-phenyltrinor PGE2, sulprostone) were without effect. DDM-PGE2- and TPA-mediated cytoprotection and TRE binding activity are inhibited by N-(2[[3-(4-bromophenyl)-2-propenyl]-amino]-ethyl)-5-isoquinolinesulfonam ide (H-89), a PKC inhibitor. These data suggest that cytoprotection by PGE2 and DDM-PGE2 in LLC-PK1 cells is mediated by a PKC-coupled receptor, which is pharmacologically distinct from the presently classified EP receptor subtypes.

16,16-Dimethylprostaglandin E2↗
PubMed1996

Glutathione conjugates of tert-butyl-hydroquinone, a metabolite of the urinary tract tumor promoter 3-tert-butyl-hydroxyanisole, are toxic to kidney and bladder.

3-tert-Butyl-4-hydroxyanisole and tert-butyl-hydroquinone (TBHQ) are antioxidants known to promote renal and bladder carcinogenesis in the rat, although the mechanisms of these effects are unclear. Because glutathione (GSH) conjugates of a variety of hydroquinones are nephrotoxic, and because 2-tert-butyl-5-(glutathion-S-yl)hydroquinone [5-(GSyl)TBHQ], 2-tert-butyl-6-(glutathion-S-yl)hydroquinone [6-(GSyl)TBHQ], and 2-tert-butyl-3,6-bis-(glutathion-S-yl)hydroquinone [3,6-bis-(GSyl)-TBHQ] have been identified recently as metabolites of TBHQ in the male rat, we investigated the effects of these metabolites in the male rat. At the highest dose tested (400 micromol/kg,i.v.) 5-(Gsyl)TBHQ and 6-(GSyl)TBHQ caused 2-fold increases in the urinary excretion of gamma-glutamyl transpeptidase and alkaline phosphatase, and pigments arising from the polymerization of metabolites were deposited in the kidney. 3,6-bis-(GSyl)TBHQ (200 micromol/kg) was the most potent of the GSH conjugates tested and produced significant increases in the urinary excretion of gamma-glutamyl transpeptidase, alkaline phosphatase, lactate dehydrogenase, and glucose (2-, 2-, 22-, and 11-fold increases, respectively). Alterations in the biochemical parameters correlated with the degree of single cell and tubular necrosis in the S(3)-M segment of the proximal tubule, as observed by light microscopy. In addition to nephrotoxicity, 3,6-bis-(GSyl)TBHQ increased the bladder wet weight 2-fold and caused severe hemorrhaging of the bladder. The half-wave oxidation potentials of 5-(Gsyl)TBHQ and 6-(GSyl)TBHQ were similar to that of TBHQ, whereas the half-wave oxidation potential of 3,6-bis-(Gsyl)TBHQ was approximately 100 mV higher than that of TBHQ. The TBHQ-GSH conjugates also catalyzed the formation of 8- hydroxydeoxyguanosine, indicating that GSH conjugation does not impair the redox activity of TBHQ. Because some chemicals may induce carcinogenesis by a mechanism involving cytotoxicity followed by sustained regenerative hyperplasia, our results suggest that the toxicity of GSH conjugates of TBHQ to kidney and bladder may contribute to the promoting effect of 3-tert-butyl-4-hydroxyanisole and TBHQ in these tissues.

Animals↗
PubMed1996

Effects of intracerebroventricular administration of 5-(glutathion-S-yl)-alpha-methyldopamine on brain dopamine, serotonin, and norepinephrine concentrations in male Sprague-Dawley rats.

alpha-Methyldopamine (alpha-MeDA) is a metabolite of the serotonergic neurotoxicants 3,4-(+/-)-(methylenedioxy)amphetamine (MDA) and 3,4-(+/-)-(methylenedioxy)methamphetamine (MDMA). alpha-MeDA readily oxidizes, and in the presence of glutathione (GSH) it forms 5-(glutathion-S-yl)-alpha-methyldopamine [5-(glutathion-S-yl)-alpha-MeDA]. Since GSH conjugates of many polyphenols are biologically (re)active, we investigated the role of 5-(glutathion-S-yl)-alpha-MeDA in the acute and long-term neurochemical changes observed after administration of MDA. Intracerebroventricular (icv) administration of 5-(glutathion-S-yl)-alpha-MeDA (720 nmol) to male Sprague-Dawley rats produced behavioral changes similar to those reported after subcutaneous administration of MDA. Thus, animals became hyperactive and aggressive and displayed forepaw treading and Straub tails, behaviors usually seen after administration of serotonin (5-HT) releasers, and consistent with a role for 5-(glutathion-S-yl)-alpha-MeDA in some of the behavioral alterations seen after administration of MDA and MDMA. In addition to the behavioral changes, 5-(glutathion-S-yl)-alpha-MeDA also caused short-term alterations in the dopaminergic, serotonergic, and noradrenergic systems. An increase in dopamine synthesis appears to be a prerequisite for the long-term depletion of brain 5-HT following MDMA administration. However, although 5-(glutathion-S-yl)-alpha-MeDA reproduced some of the effects of MDA on the dopaminergic system and was capable of causing acute increases in 5-HT turnover, a single icv injection of 5-(glutathion-S-yl)-alpha-MeDA did not result in long-term serotonergic toxicity. Thus, although acute stimulation of dopamine turnover may be necessary for long-term serotonergic toxicity, such changes are not sufficient to produce these effects. The effects of a multiple dosing schedule of 5-(glutathion-S-yl)-alpha-MeDA will therefore require investigation before we can define a role for this metabolite in MDA and MDMA mediated neurotoxicity. MDA also produces a pressor response that is related to its ability to release neuronal norepinephrine stores, and 5-(glutathion-S-yl)-alpha-MeDA caused comparable depletions of brain norepinephrine concentrations, indicating that both compounds produce similar effects on the noradrenergic system.

Animals↗
PubMed1996

17 beta-estradiol metabolism by hamster hepatic microsomes: comparison of catechol estrogen O-methylation with catechol estrogen oxidation and glutathione conjugation.

Catechol estrogens, the cytochromes P450 mediated metabolites of 17 beta-estradiol, undergo further metabolism either via catechol O-methyltransferase (COMT) catalyzed methylation, or by oxidation and subsequent thioether formation with glutathione (GSH). Secondary metabolites of 17 beta-estradiol arising from both these metabolic pathways have been identified in vivo. However, the relative contribution of catechol O-methylation, and catechol oxidation followed by GSH conjugation, to the disposition of the catechol estrogens is unclear. We have therefore quantified both pathways of catechol estrogen disposition, generated in situ from 17 beta-estradiol, in hamster hepatic microsomes. 17 beta-Estradiol was readily converted to 2- and 4-hydroxy-17 beta-estradiol, both of which were effectively methylated in the presence of COMT (300 units/mL). Addition of GSH (50 microM-1 mM) to microsomal incubations resulted in the formation of four catechol estrogen-derived GSH conjugates. Three conjugates of 2-hydroxy-17 beta-estradiol were identified: 2-hydroxy-1,4-bis(glutathione-S-yl)-17 beta-estradiol, 2-hydroxy-1-glutathione-S-yl-17 beta-estradiol, and 2-hydroxy-4-glutathione-S-yl-27 beta-estradiol. In contrast, just one GSH conjugate of 4-hydroxy-17 beta-estradiol was identified: 4-hydroxy-1-glutathione-S-yl-17 beta-estradiol. When a combination of COMT and GSH were simultaneously added to microsomal incubations, both metabolic pathways competed for the same pool of catechol estrogens, and ascorbate dramatically influenced which of these two pathways predominate. In the presence of ascorbate, catechol estrogen methylation predominated over catechol estrogen oxidation and GSH conjugation. In the absence of ascorbic acid, catechol estrogen methylation, and catechol estrogen oxidation inked to GSH conjugation, contributed equally to the disposition of the catechol estrogens. 17 beta-Estradiol 2- and 4-hydroxylase activity was always higher in the absence of ascorbate, irrespective of whether GSH or COMT was used as the trapping agent. Thus, the usual method (COMT plus ascorbate) of determining 17 beta-estradiol 2- and 4-hydroxylase activity underestimates enzyme activity by approximately 50% when compared to the value obtained when GSH is used to trap the o-quinones in the absence of ascorbate. A reassessment of 17 beta-estradiol 2- and 4-hydroxylase activity in different species and tissues is required to permit a more informed evaluation of the role of catechol estrogens in estrogen-induced carcinogenesis.

Animals↗
PubMed1996

17 beta-Estradiol metabolism by hamster hepatic microsomes. Implications for the catechol-O-methyl transferase-mediated detoxication of catechol estrogens.

We have shown that the metabolism of 17 beta-estradiol in hamster liver microsomes is concentration-dependent. At low (< 25 microM) concentrations of 17 beta-estriol, 16 alpha-hydroxylase activity predominated, and estriol was the major metabolite. At higher concentrations (25-75 microM), 16 alpha-hydroxylation and aromatic hydroxylation at C2 contributed equally to 17 beta-estradiol metabolism. Aromatic C4-hydroxylation was maximal at 75 microM of 17 beta-estradiol and was always less than C2-hydroxylation. Dehydrogenation of the 17 beta-hydroxyl group to the ketone (estrone) was also observed, but both estrone and 2-hydroxyestrone were minor (approximately 3%) metabolites of 17 beta-estradiol, only detectable at concentrations of 50 microM and above. Catechol-O-methyl transferase (COMT) effectively converted both 2- and 4-hydroxyl-17 beta-estradiol to their corresponding monomethoxy metabolites. Effective reducing conditions are required for COMT activity, because catechol estrogens are readily oxidized to their corresponding ortho-quinones, and ascorbic acid is routinely added to assays of COMT activity. Interestingly, although ascorbic acid (1 mM) increased the recovery of 2- and 4-hydroxy-17 beta-estradiol from microsomal incubations, it decreased the recovery of the methoxy metabolites (approximately 40%). Since the enediol function of ascorbate resembles that of a catechol group, ascorbate is a substrate for COMT and probably competes with the catechol estrogens for methylation. Because previous studies describing the ability of COMT to inhibit the covalent binding of electrophilic reactive metabolites of [4-(14)C]17 beta-estradiol to microsomal protein were performed in the presence of high (100 mM) Mg2+ concentrations, we also investigated the effects of Mg2+ on 17 beta-estradiol metabolism. Concentrations of Mg2+ > 10 mM inhibited the metabolism of 17 beta-estradiol, as evidenced by i) the increased recovery of substrate; ii) a decrease in the formation of estriol, estrone, and 2-, and 4-hydroxy-17 beta-estradiol; iii) a decrease in the recovery of water-soluble metabolites when incubations were performed in the presence of glutathione (GSH) to trap the reactive electrophilic metabolites; and iv) a decrease in the amount of reactive electrophilic metabolites bound to microsomal protein. GSH also decreased the covalent binding of electrophilic metabolites of [4-(14)C]17 beta-estradiol to microsomal protein, with the concomitant formation of water-soluble metabolites. Thus, both COMT and GSH combine to limit the formation of electrophilic metabolites from 17 beta-estradiol. The relative importance of each of these pathways to the disposition of the catechol estrogens remains to be determined.

Animals↗
PubMed1996

Quinone thioether-mediated DNA damage, growth arrest, and gadd153 expression in renal proximal tubular epithelial cells.

Although the conjugation of quinones with glutathione is associated with the process of detoxication, the reaction frequently facilitates quinone-induced toxicity. Thiol conjugates of quinones retain the ability to redox cycle and generate reactive oxygen species (ROS), contributing to the biological (re)activity of a variety of polyphenolic compounds. 2-Bromo-bis(glutathion-S-yl) hydroquinone (2-Br-bis(GSyl)HQ) and 2-bromo-6-(glutathion-S-yl) hydroquinone [2-Br-6-(GSyl)HQ] are potent nephrotoxicants in rats, inducing rapid karyolysis in vivo and DNA single-strand breaks in cultured renal proximal tubular epithelial cells (LLC-PK1). We investigated the cellular and molecular responses initiated after exposure of LLC-PK1 cells to 2-Br-bis(GSyl)HQ and 2-Br-6-(GSyl)HQ. Both quinone thioethers cause the concentration-dependent formation of DNA single-strand breaks, rapidly (2-10 min) inhibit DNA synthesis, and increase the expression of gadd153, a gene responsive to growth arrest and DNA damage. The addition of catalase to LLC-PK1 cells exposed to 2-Br-6-(GSyl)HQ or 2-Br-bis(GSyl)HQ effectively prevents gadd153 induction, which is consistent with findings that the gadd153 gene is subject to redox modulation and that ROS play an important role in quinone thioether-mediated cytotoxicity. Deferoxamine pretreatment also diminishes gadd153 induction, suggesting that in renal proximal tubular epithelial cells, decreased expression of gadd153 is not dependent on the removal of hydrogen peroxide per se but rather on preventing the generation of hydroxyl radical. Chelation of intracellular calcium with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid-acetoxy-methyl ester also reduces gadd153 induction by 2-Br-6-(GSyl)HQ and 2-Br-bis(GSyl)HQ, suggesting a role for calcium in the signaling process. Thus, 2-Br-6-(GSyl)HQ and 2-Br-bis(GSyl)HQ activate a genomic stress response via a signaling pathway that may include ROS, Ca2+, and DNA damage.

Animals↗
PubMed1995

Reactive oxygen species and DNA damage in 2-bromo-(glutathion-S-yl) hydroquinone-mediated cytotoxicity.

Exposure of renal proximal tubular epithelial cells (LLC-PK1) to the nephrotoxicants 2-bromo-6-(glutathion-S-yl)hydroquinone, 2-bromo-3-(glutathion-S-yl)-hydroquinone, and 2-bromo-(diglutathion-S-yl)hydroquinone caused DNA fragmentation and cytotoxicity. Viability measured by lysosomal neutral red accumulation was the most sensitive parameter of cytotoxicity, and preceded toxicity determined by either the mitochondrial MTT assay or by measuring intracellular lactate dehydrogenase activity. DNA fragmentation was detected as early as 15 min after exposure to 2-bromo-6-(glutathion-S-yl)hydroquinone (100 microM), 2-bromo-3-(glutathion-S-yl)hydroquinone (200 microM), and 2-bromo-(diglutathion-S-yl)hydroquinone (400 microM) and prior to other indices of toxicity. The ability of the cells to repair DNA damage was evident by the decrease in the extent of single strand breaks following removal of 2-bromo-3-(glutathion-S-yl)hydroquinone from the incubation medium. Moreover, inhibition of poly(ADP-ribose)polymerase with 3-amino-benzamide (10 mM), following exposure of LLC-PK1 cells to 0.5 mM 2-bromo-6-(glutathion-S-yl)hydroquinone or 2-bromo-(diglutathion-S-yl)hydroquinone, decreased cytotoxicity, indicating that DNA repair processes, activated in response to DNA damage, exacerbate toxicity. Treatment with the endonuclease inhibitor, aurintricarboxylic acid did not decrease cytotoxicity. A decrease in the cytotoxicity caused by 2-bromo-6-(glutathion-S-yl)hydroquinone and 2-bromo-(diglutathion-S-yl)hydroquinone was observed when cells were incubated with catalase or pretreated with deferoxamine (10 mM). The data suggest a mechanism whereby the conjugates generate hydrogen peroxide, and the subsequent iron-catalyzed generation of hydroxyl radicals causes DNA fragmentation and cytotoxicity.

Animals↗