Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.6.5.2 (NQO1)
 6,196 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Mitomycin C (MC), a clinically used natural antitumor agent, was shown to form three monoconjugates (11a-13a) and two bisconjugates (14a, 15a) with GSH upon reductive activation by rat liver microsomes, purified NADPH-cytochrome c reductase, or NADH-cytochrome c reductase or chemical reduction using H2/PtO2. Rat liver cytosol/NADH activated MC only at acidic pH (5.8), resulting in the formation of a single GSH-MC monoconjugate, 13a. The reductase responsible for cytosolic activation of MC to form this conjugate was DT-diaphorase. GSH itself did not reduce MC, and unreduced MC did not form conjugates with GSH. A moderate catalytic effect by glutathione S-transferase was demonstrated on the cytosol-activated reaction. Mercaptoethanol and N-acetylcysteine gave analogous sets of five MC-thiol conjugates under cytochrome c reductase or H2/PtO2 activation conditions. The structures of all 15 MC-thiol conjugates (five each with GSH, mercaptoethanol, and N-acetylcysteine, respectively) were determined, using 1H-NMR, UV, and mass spectroscopies, combined with analytical chemical and radiolabeling methods. The mechanism of formation of the conjugates features SN2 displacement of the carbamate of the reduced MC by GS-. The MC-GSH conjugates were noncytotoxic to the tumor cells tested. The conjugation of GSH with activated MC is likely to represent detoxication in mammalian cells. As another effect, GSH accelerates the rate of reduction of MC by "slow" reducing agents such as cytochrome c reductases and H2/PtO2. A mechanism is proposed to explain this effect, which involves further reduction of the initially formed MC semiquinone free radical by GSH.
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PMID:Conjugation of glutathione and other thiols with bioreductively activated mitomycin C. Effect of thiols on the reductive activation rate. 807 71

This study describes characteristics of a human bladder cancer cell line J82/MMC that is 6-fold more resistant to mitomycin C (MMC) than the parental cells. The J82/MMC subline was isolated by repeated continuous exposures of the J82/WT cells to increasing concentrations of MMC. The J82/MMC cell line showed (1) collateral sensitivity to taxol, 5-FU and topoisomerase II inhibitors; and (2) cross-resistance to cisplatin, melphalan and MMC analogues BMY 25282 and BMY 25067. Levels of two key MMC activation enzymes, NADPH cytochrome P450 reductase and DT-diaphorase, were significantly lower in J82/MMC cells compared with J82/WT, suggesting that lower sensitivity of J82/MMC cells to MMC may result from deficient drug activation. Further support is indicated by: 1) reduction in the differential in toxicity between the 2 cell lines by BMY 25282; and 2) a higher effect of DT-diaphorase inhibitor dicumarol on the wild-type cells compared with J82/MMC. Although glutathione (GSH) levels did not differ in these cells, a small but significant increase in GSH transferase (GST) activity was noticed in J82/MMC cells. GST inhibitor ethacrynic acid significantly enhanced MMC cytotoxicity in the J82/MMC cell line. A small but significant increase in the level of anti-oxidative enzyme catalase, but not GSH peroxidase, was also observed in J82/MMC cell line compared with J82/WT. Thus, the possibility that relatively lower sensitivity of J82/MMC cells to MMC may result from reduced oxygen radical generation cannot be ruled out. MMC-induced DNA interstrand cross-linking was markedly lower in the J82/MMC cell line compared with J82/WT. Our results suggest that the MMC resistance in the J82/MMC cell line may be multifactorial.
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PMID:Characterization of a human bladder cancer cell line selected for resistance to mitomycin C. 807 54

Three related Chinese hamster ovary (CHO) cell lines derived from CHO-K1R cells (MMC3-A2, 21-1 and G1B) previously shown to differ in their sensitivity to mitomycin C (MMC), were investigated in more detail to determine the factors controlling this sensitivity. A separately maintained wild type cell line (CHO-K1TOR) was included in this study for comparison. Continuous (chronic) exposure of the five cell lines to MMC during the 10-day colony forming assay demonstrated a 15-fold range in MMC sensitivity between the most sensitive cell line (MMC3-A2) and the most resistant cell line (G1B) with CHO-K1R, 21-1 and CHO-K1TOR falling at intermediate levels. Acute aerobic exposure (0-5 h) to MMC resulted in a reduced fivefold range of sensitivities, which was further reduced to a three-fold range under hypoxic exposure conditions. These results were suggestive of differences in the aerobic enzymatic activation of MMC as a possible mechanism contributing to the varying sensitivities. There was no correlation between the one-electron reducing enzyme NADPH:cytochrome P-450 oxidoreductase (P450R) activity and cellular sensitivity to MMC. The five cell lines had similar levels of reduced glutathione (GSH), suggesting that oxygen homeostasis was not correlated with the cells, differing sensitivity to MMC. A correlation did exist between NAD(P)H:quinone oxidoreductase (DT-diaphorase) activity and cellular sensitivity to MMC under chronic exposure conditions for the cell lines. High DT-diaphorase levels were also correlated with a reduced ability of oxygen to modulate MMC toxicity. Levels of P450R and DT-diaphorase were not altered significantly during five-hour aerobic or hypoxic exposures of control cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characterization of a set of Chinese hamster ovary variant cell lines demonstrating differing sensitivity to mitomycin C. 812 41

7-N-((2-([2-(gamma-L-Glutamylamino)ethyl]dithio)ethyl))mitomycin C (KW-2149) is an analogue of mitomycin C (MMC) and has prominent activities against various tumors. We studied the antitumor effects of KW-2149 in MMC-resistant variants of human colon carcinoma HT-29 (HT-29/MMC) and mouse hepatoma Hepa-I (C4, B13NBii1) cells, which are deficient in DT-diaphorase and cytochrome P450 reductase, respectively. These enzymes mediate the reductive activation of MMC in the cells. Although HT-29/MMC and C4, B13NBii1 cells showed significant resistance to MMC, they showed sensitivity tl KW-2149 comparable to their parental tumors, indicating that DT-diaphorase and cytochrome P450 reductase could not be involved in the activation of KW-2149. In studying the activation mechanism of KW-2149, we found that glutathione (GSH) and cysteine significantly enhanced the cytotoxicity of KW-2149 in HT-29 cells. The DNA adduct of KW-2149 was increased when HT-29 cells or the isolated nuclei of the cells were incubated with KW-2149 in the presence of physiological concentrations of GSH and cysteine. KW-2149 alkylated calf thymus DNA in the presence of GSH and cysteine in vitro. These results indicate that activation of KW-2149 by thiol molecules, unlike MMC, could be an important activation mechanism of KW-2149 to form DNA adduct and to exert its cytotoxicity. This is the reason why KW-2149 is effective against MMC-resistant tumors with deficiencies in the MMC activation enzymes.
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PMID:Nonenzymatic reductive activation of 7-N-((2-([2-(gamma-L-glutamylamino)ethyl]dithio)ethyl))mitomycin C by thiol molecules: a novel mitomycin C derivative effective on mitomycin C-resistant tumor cells. 816 87

Established cell lines derived from newborn livers of c14CoS/c14CoS and cch/cch mice have been shown to be genetically resistant (14CoS/14CoS cells) or susceptible (ch/ch cells) to menadione toxicity. These differences are due in part to relatively higher levels of reduced glutathione (GSH) and NAD(P)H:menadione oxidoreductase (NMO1) activity in the 14CoS/14CoS cells. The indolic membrane-stabilizing antioxidant 5,10-dihydroindeno[1,2-b]indole (DHII) was shown previously to protect against various hepatotoxicants in vivo and in primary rat hepatocytes. This report describes how the 14CoS/14CoS and ch/ch cell lines provide a valuable experimental system to distinguish the mechanism of chemoprotection by DHII from menadione toxicity. The addition of 25 microM DHII produced a time-dependent decrease in menadione-mediated cell death in 14CoS/14CoS cells, with little effect on ch/ch cell viability. The maximum protective effect occurred at 24 hr, although the concentration of DHII remained constant for 48 hr. The protective effect of DHII correlated with enhanced glutathione levels (234% increase at 24hr), as well as induction of four enzymes involved in the detoxification and excretion of menadione: NAD(P)H:menadione oxidoreductase (NMO1, quinone reductase), glutathione reductase, glutathione transferase (GST1A1), and UDP glucuronosyltransferase (UGT1*06), with 24-hr maximum induction of 707, 201, 171 and 198%, respectively. Other biotransformation enzymes not directly involved in menadione metabolism (glutathione peroxidase, cytochromes P4501A1 and P4501A2, copper-, zinc-dependent superoxide dismutase, and NADPH cytochrome c oxidoreductase) were not induced by DHII. Menadione-stimulated superoxide production was inhibited 50% by DHII only in 14CoS/14CoS cells, and the inhibition required 24-hr preincubation. Pretreatment with DHII also protected both cell types against the menadione-mediated depletion of GSH, and the increase in percent (oxidized glutathione GSSG), an indicator of oxidative stress. These results suggest that DHII does not protect against menadione toxicity by virtue of its antioxidant or membrane-stabilizing properties. Rather, it acts by inducing a protective enzyme profile that migates redox cycling and facilitates excretion of menadione.
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PMID:Mechanisms of protection from menadione toxicity by 5,10-dihydroindeno[1,2,-b]indole in a sensitive and resistant mouse hepatocyte line. 824 Apr 1

The extent of ferrihemoglobin formation in human erythrocytes by 4-nitrosophenetol and its metabolisation rate strongly depended on the availability of cellular GSH. Ferrihemoglobin formation rate was increased by inhibition of the red cell glutathione reductase, and 4-nitrosophenetol disappeared more slowly. When red cells were completely depleted from SH groups, ferrihemoglobin formation was retarded, despite 4-nitrosophenetol was hardly metabolized. In turn, the glutathione status of human red cells was strongly affected by 4-nitrosophenetol. GSSG, which was produced in large amounts, was reduced, as long as the reducing system was intact. The decreased total glutathione content, however, did not recover completely, indicating formation of stable glutathione S-conjugates. The active export of the stable model glutathione thioether S-(2,4-dinitrophenyl)glutathione was strongly inhibited by 4-nitrosophenetol. A Lineweaver-Burk plot of the transport data suggested a competitive inhibition mechanism, presumably caused by glutathione adducts. The results indicate that the strong pi-donor substituent in 4-nitrosophenetol enables metabolic reactions with glutathione, producing biological effects hitherto not observed with nitrosobenzene. Bicyclic arylamines and glutathione S-conjugates may cause ferrihemoglobin formation that is not brought about by the diaphorase reaction. The latter may be responsible for transport inhibition of GSSG and other glutathione S-conjugates.
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PMID:Effects of the phenacetin metabolite 4-nitrosophenetol on the glutathione status and the transport of glutathione S-conjugates in human red cells. 843 97

One mechanism by which chemicals cause cellular injury is the formation of reactive oxygen species. In vitro studies have shown that metallothionein (MT), a small metal-binding, sulfhydryl-rich, readily inducible protein, can scavenge reactive oxygen species, especially hydroxyl radicals. Nevertheless, whether or not MT protects against oxidative stress in the intact animal is not known. Experimental induction of MT could help to clarify this question, however, it is unclear whether agents that induce MT also influence known antioxidant systems. Therefore, the present study was designed to determine whether the well-known MT inducers are specific for induction of MT or whether they might also influence other hepatic systems that protect against oxidative stress. Male rats were administered cadmium chloride (Cd; 30 mumol/kg, s.c.), zinc chloride (Zn; 1000 mumol/kg, s.c.), alpha-hederin (alpha-H, 30 mumol/kg, s.c.) or lipopolysaccharide (LPS; 1 mg/kg, s.c.) 24 h prior to measurement of antioxidant systems. Zn and alpha-H increased hepatic GSH concentration 20% and 55%, respectively. Cd significantly increased, whereas LPS reduced, the activities of selenium-dependent glutathione peroxidase and glutathione reductase. Glutathione S-transferases were not altered by any of the inducers. Cd also increased DT-diaphorase activity. Cd, Zn and alpha-H all decreased catalase activity 20-35%, while the activity of superoxide dismutase was unaffected by the inducers. The amount of total cytochrome P450 enzymes and cytochrome b5 were decreased by LPS, Cd and alpha-H, while Zn appeared to have no effect. The activities of P450 enzymes towards testosterone oxidation were also decreased by LPS, Cd and alpha-H. In conclusion, all four MT inducers examined affect systems known to protect cells against oxidative stress. Therefore, using these chemicals to determine the in vivo role of MT in protecting against oxidative stress poses difficulties.
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PMID:Effect of several metallothionein inducers on oxidative stress defense mechanisms in rats. 856 Apr 99

The effects of dicoumarol, an inhibitor of DT-diaphorase, on the cytotoxicity of tert-butylhydroquinone (tBHQ) were studied in freshly isolated rat hepatocytes. Addition of tBHQ (0.5 mM) to hepatocytes resulted in a time-dependent cell death accompanied by depletion of intracellular ATP, glutathione (GSH), and protein thiols. Pretreatment of hepatocytes with dicoumarol (30 microM) did not affect cell viability or cellular levels of ATP, GSH, or protein thiols during the incubation period; however, dicoumarol did promote the appearance of cell blebs and the depletion of ATP and protein thiols induced by tBHQ and ultimately enhanced the cytotoxicity of tBHQ.
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PMID:Effects of dicoumarol on cytotoxicity caused by tert-butylhydroquinone in isolated rat hepatocytes. 861 6

This study describes characteristics of a mitomycin C (MMC)-resistant human bladder cancer cell line, J82/MMC-2, which was established by repeated in vitro exposures of a 6-fold MMC-resistant variant (J82/MMC) to 18 nM MMC. A 9.6-fold higher concentration of MMC was required to kill 50% of the J82/MMC-2 sub-line compared with parental cells (J82/WT). NADPH cytochrome P450 reductase and DT-diaphorase activities were significantly lower in J82/MMC-2 cells compared with J82/WT, suggesting that reduced sensitivity of J82/MMC-2 cells to MMC resulted from impaired drug activation. Consistent with this hypothesis, the formation of MMC-alkylating metabolites was significantly lower in J82/MMC-2 cells compared with J82/WT. Furthermore, DT-diaphorase activity in J82/MMC-2 cells was significantly lower compared with the 6-fold MMC-resistant variant. Glutathione (GSH) levels were comparable in all 3 cell lines. Although GSH transferase (GST) activity was significantly higher in the J82/MMC-2 cells compared with J82/WT, this enzyme activity did not differ between 6- and 9.6-fold MMC-resistant variants. Whereas DNA polymerase alpha mRNA expression was comparable in these cell lines, levels of DNA ligase I mRNA were slightly lower in both MMC-resistant variants relative to J82/WT. However, the DNA polymerase beta mRNA level was markedly higher in the J82/MMC-2 cell line compared with either J82/WT or J82/MMC. Thus, emergence of a higher level of resistance to MMC in J82/MMC-2 cells compared with J82/MMC may be attributed to (i) impaired drug activation through further reduction in DT-diaphorase activity and (ii) enhanced DNA repair through over-expression of DNA polymerase beta.
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PMID:Biochemical characterization of a mitomycin C-resistant human bladder cancer cell line. 863 3

Hepatic levels of GSH and Phase II detoxication enzymes were compared to biochemical and histological indices of hepatic damage in 4- to 76-week-old nontransgenic mice and their transgenic littermates that overexpress the hepatitis B virus large envelope protein. The mice were fed a low-sucrose AIN-76A diet ad libitum. Hepatic-specific activities of quinone reductase (QR) and glutathione S-transferase (GST) were increased 2- to 10-fold beginning at 12 weeks of age in transgenic mice and correlated with increases in serum alanine aminotransferase (ALT) (r = 0.84 and 0.59, respectively). Quantitative histological analysis demonstrated that apoptosis was the predominant feature in 4- to 12-week-old transgenic mice, whereas necrosis and inflammation predominated at later time points. Surprisingly, 3-fold elevations in ALT were observed beginning at 52 weeks of age in nontransgenic mice, and hepatic-specific activities of QR and GST were also modestly increased in elderly nontransgenic animals. In contrast to transgenic mice, apoptosis was not a prominent feature. The strongest histological correlates to ALT in 4- to 76-week-old nontransgenic mice were necrosis and inflammation (r > 0.96), which in turn may have been evoked by hepatic fat accumulation. Profiles of specific GST isoforms were quantitated chromatographically and identified by sequencing tryptic digests. The Ya1 subunit of alpha-class GST was markedly increased from undetectable levels in transgenic mice, while more modest increases were observed in nontransgenic mice more than 1 year old. Fivefold elevations of the Yb1 subunit, a constitutively expressed mu-class GST, were found in transgenic mice older than 4 weeks of age, while 2-fold increases were observed in nontransgenic animals that were more than 1 year old. These studies demonstrate that selected increases in Phase II detoxication enzymes are a stereotyped response to chronic hepatitis that is strikingly reminiscent of the treatment of mice with anticarcinogenic enzyme inducers.
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PMID:Elevations of hepatic quinone reductase, glutathione, and alpha- and mu-class glutathione S-transferase isoforms in mice with chronic hepatitis: a compensatory response to injury. 866 Jun 89


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