Elements of informed consent for pharmacogenetic research; perspective of the pharmacogenetics working group.
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Clinical Trials as Topic↗
Biomedical subjects
P M Shaw
Publications and source records attributed to P M Shaw.
A fluorescent assay amenable to measuring production of beta-D-glucuronides produced from recombinant UDP-glycosyl transferase enzymes.
Beta-glucuronidase cleavage of 4-methylumbelliferyl beta-D-glucuronide generates the highly fluorescent compound, 4-methylumbelliferone. We show that other beta-D-glucuronide compounds act as competitors in this assay. The 4-methylumbelliferyl beta-D-glucuronide cleavage assay can easily be adapted to high throughput formats to detect the presence of beta-D glucuronides generated using recombinant glycosyl transferase preparations.
Binding, Competitive↗
Induction of CYP1A1, but not CYP1A2, in adrenals of 3, 3'-methylcholanthrene-treated guinea pigs.
To test the inducibility of CYP1A homologs in guinea pig adrenal, the effects of 3,3'-methylcholanthrene, an archetypal inducer of CYP1A, were compared in guinea pig adrenal and liver. Western blot analysis showed that levels of both CYP1A1 (53 kDa) and CYP1A2 (56 kDa) increasedin liver microsomes of 3,3'-methylcholanthrene-treated guinea pigs. In adrenals, an immunoreactive protein comigrating with liver CYP1A1 was detected only after 3,3'-methylcholanthrene treatment. Protein comigrating with CYP1A2 was never detected in adrenal microsomes. A third inducible immunoreactive protein (57 kDa) was seen in liver, but not adrenal, after 3, 3'-methylcholanthrene treatment. Another immunoreactive protein (52 kDa), present constitutively in liver and adrenal microsomes, was not induced in either tissue by 3,3'-methylcholanthrene. The precise identities of the inducible 57-kDa and the noninducible 52-kDa proteins remain to be determined. However, the identity of the 53-kDa protein in the adrenal as CYP1A1 was confirmed by RT-PCR, Northern blot, and sequence analysis. Similar analyses demonstrated that, despite the fact that the 56-kDa protein was not detectable in adrenal microsomes, CYP1A2 mRNA was present in adrenals of control animals. Strikingly, CYP1A2 mRNA decreased in adrenal, but increased in liver, following 3,3'-methylcholanthrene treatment, underscoring differences in the regulation of CYP1A expression in the two tissues. Levels of ethoxyresorufin and methyoxyresorufin metabolism correlated with levels of CYP1A1 and CYP1A2 protein, respectively.
Adrenal Glands↗
Roles of cytochromes P450 1A2 and 3A4 in the oxidation of estradiol and estrone in human liver microsomes.
Of seven cDNA-expressed human cytochrome P450 (P450) enzymes (P450s 1A2, 2B6, 2C9, 2C19, 2D6, 2E1, and 3A4) examined, P450 1A2 was the most active in catalyzing 2- and 4-hydroxylations of estradiol and estrone. P450 3A4 and P450 2C9 also catalyzed these reactions although to lesser extents than P450 1A2. P450 1A2 also efficiently oxidized estradiol at the 16alpha-position but was less active in estrone 16alpha-hydroxylation; the latter reaction and also estradiol 16alpha-hydroxylation were catalyzed by P450 3A4 at significant levels. Anti-P450 1A2 antibodies inhibited 2- and 4-hydroxylations of these two estrogens catalyzed by liver microsomes of some of the human samples examined. Estradiol 16alpha-hydroxylation was inhibited by both anti-P450 1A2 and anti-P450 3A4, while estrone 16alpha-hydroxylation was significantly suppressed by anti-P450 3A4 in human liver microsomes. Fluvoxamine efficiently inhibited the estrogen hydroxylations in human liver samples that contained high levels of P450 1A2, while ketoconazole affected these activities in human samples in which P450 3A4 levels were high. alpha-Naphthoflavone either stimulated or had no effect on estradiol hydroxylation catalyzed by liver microsomes; the intensity of this effect depended on the human samples and their P450s. Interestingly, in the presence of anti-P450 3A4 antibodies, alpha-naphthoflavone was found to be able to inhibit estradiol and estrone 2-hydroxylations catalyzed by human liver microsomes. The results suggest that both P450s 1A2 and 3A4 have major roles in oxidations of estradiol and estrone in human liver and that the contents of these two P450 forms in liver microsomes determine which P450 enzymes are most important in hepatic estrogen hydroxylation by individual humans. P450 3A4 may be expected to play a more important role for some of the estrogen hydroxylation reactions than P450 1A2. Knowledge of roles of individual P450s in these estrogen hydroxylations has relevance to current controversies in hormonal carcinogenesis [Service, R. F. (1998) Science 279, 1631-1633].
Aryl Hydrocarbon Hydroxylases↗
Sulfation of alpha-hydroxytamoxifen catalyzed by human hydroxysteroid sulfotransferase results in tamoxifen-DNA adducts.
The formation of tamoxifen (TAM)-derived DNA adducts was investigated by incubation of DNA with (E)-alpha-hydroxytamoxifen [(E)-alpha-OHTAM], 3'-phosphoadenosine 5'-phosphosulfate (PAPS), and human recombinant sulfotransferase. Using 32P-post-labeling and HPLC analysis, two TAM-DNA adducts were detected in incubations that included the human hydroxysteroid sulfotransferase SULT2A1 (hHST). When compared with standards of stereoisomers of alpha-(N2-deoxyguanosinyl)tamoxifen 3'-monophosphate (dG3'P-N2-TAM), the major adduct was identified chromatographically as an epimer of the transform of dG-N2-TAM, and the minor adduct was identified as an epimer of the cis-form. The amount of TAM adducts formed by hHST was approximately three times less than that formed by an equivalent amount of rat hydroxysteroid (alcohol) sulfotransferase a. These results indicate that sulfation of alpha-OHTAM catalyzed by hHST results in the formation of dG-N2-TAMs, highly miscoding lesions, in human tissues.
Animals↗
Reconstitution premixes for assays using purified recombinant human cytochrome P450, NADPH-cytochrome P450 reductase, and cytochrome b5.
The development of enzyme and buffer premixes for in vitro biotransformation assays is described. The protein premixes contain a mixture of three recombinant human proteins, cytochrome P450 (P450) 3A4, NADPH-P450 reductase, cytochrome b5, and liposomes. The buffer premix contains reagents which, when diluted, provide for optimal metabolic activity with selected P450 3A4 substrates. P450 3A4 premixes were competent in the oxidation of known substrates including testosterone, midazolam, nifedipine, erythromycin, benzphetamine, and amitriptyline. Premixes stored at -80 degrees C for 2 months and those that underwent an additional five freeze/thaw cycles were able to hydroxylate testosterone at turnover rates similar to freshly prepared reconstitution mixes. In addition, premixes stored unfrozen at 4 degrees C for 2 weeks showed no significant loss in the rate of testosterone 6 beta-hydroxylation by P450 3A4. Premixes prepared with and without reduced glutathione, a component which had previously been found to be important for P450 3A4 reactions, were equally efficient at carrying out testosterone hydroxylation under these conditions. Kinetic parameters determined for the metabolism of testosterone, amitriptyline, nifedipine, and benzphetamine using P450 3A4 premixes were compared with human pooled microsomes and insect microsomes prepared from cells infected with a baculovirus containing two cDNA inserts coding for P450 3A4 and NADPH-P450 reductase. Each format gave different Vmax and K(m) values indicating different catalytic efficiencies. Analysis of P450 1A2 premixes which contained different lipid concentrations indicated that Vmax and K(m) could be altered. The availability of human P450 recombinant enzymes and the development of the P450 premixes that remain active after being stored frozen should allow for rapid identification of novel P450 substrates and inhibitors and the development of large-scale screening assays.
Amitriptyline↗
Regulation of the major detoxication functions by phenobarbital and 3-methylcholanthrene in co-cultures of rat hepatocytes and liver epithelial cells.
In the present study, we analysed the expression of monooxygenase activities and mRNAs associated with cytochrome P-450 (CYP), including CYP1A1/2, CYP2B1/2, CYP2C6, CYP2E1, CYP3A1/2, glutathione transferase alpha (GST alpha), aldehyde dehydrogenase and epoxide hydrolase in co-cultures of primary rat hepatocytes and rat liver epithelial cells. We observed that pentoxyresorufin O-deethylation activity was well maintained and ethoxyresorufin O-deethylation activity gradually decreased during co-culture time. In addition, we showed that phenobarbital and 3-methylcholanthrene treatments resulted in a significant increase of these activities. Two general patterns of accumulation of liver-specific mRNAs were observed. CYP1A1/2, CYP2B1/2, CYP3A1/2, GST alpha, aldehyde dehydrogenase and epoxide hydrolase mRNAs were maintained at a stable level, whereas CYP2C6 and CYP2E1 mRNAs showed a continuous decline. In addition, we observed a strong increase of CYP1A1/2 (13.6-fold) and GST alpha (3.9-fold) mRNA expression in 3-methylcholanthrene-treated co-cultures and induction of CYP2B1/2 (19-fold), CYP2C6 (10-fold), CYP3A1/2 (11.2-fold), GST alpha (9-fold), aldehyde dehydrogenase (6-fold) and epoxide hydrolase (5-fold) mRNA expression in phenobarbital-treated co-cultures. Furthermore, we demonstrated that liver-specific gene expression was restricted to hepatocytes, with the notable exception of epoxide hydrolase and CYP2E1 which were expressed in both cell types during the co-culture, as shown by the selective recovery of both hepatocytes and rat liver epithelial cells. Finally, to investigate whether co-cultures could be used to study the molecular mechanisms regulating CYP transcription, we performed transfection of hepatocytes, before the establishment of the co-culture, with large CYP2B1 (3.9 kb) or CYP2B2 (4.5 kb) promoter chloramphenicol acetyltransferase constructs or with a construct containing a 163-bp DNA sequence element reported to confer phenobarbital responsiveness. A 2-3-fold increase over the basal level of chloramphenicol acetyltransferase activity was observed in phenobarbital-treated co-cultures transfected with the phenobarbital-responsive element construct, although phenobarbital had no effect on large CYP2B1 or CYP2B2 promoter fragments. Our results demonstrate that the co-culture system provides a good tool for studying drug metabolism, and shows promise as a new tool for analysing transcriptional regulation under the influence of xenobiotics within primary hepatocytes.
Animals↗
Adjuvant immunotherapy for patients with melanoma: are patients with melanoma of the head and neck candidates for this therapy?
BACKGROUND: Although a wealth of information is available on adjuvant immunotherapy for melanoma, little is known about adjuvant immunotherapy for head and neck melanoma. Interestingly, a few immunotherapy clinical trials report the observation of clinical responses in a subset of patients with head and neck melanoma. METHOD: An up-to-date literature search was performed to identify the current information on adjuvant immunotherapy for patients with melanoma, including head and neck melanoma. Moreover, a retrospective analysis of a subset of primary head and neck melanoma was performed using data from a phase III, randomized, double-blind, multi-institutinal, vaccinia melanoma oncolysate adjuvant immunotherapy trial that was performed in our laboratory for patients with stage III (AJCC) melanoma. RESULTS: In a passive immunotherapy trial with an antibody to melanoma ganglioside antigen GM2, a complete regression was observed in one patient with lesions of the right cheek. In three active specific immunotherapy trials, including our phase III trial, a subset of patients with head and neck primary melanoma showed a longer disease-free and overall survival with immunotherapy. Moreover, these clinical responses were correlated to the induction of immune response, delayed-type hypersensitivity response and melanoma-specific antibody response. CONCLUSIONS: The above results therefore suggest that patients with head and neck melanoma clinically respond to immunotherapy. However, these results need to be confirmed in a prospectively randomized trial for patients with head and neck melanoma.
Cancer Vaccines↗
Different contributions of cytochrome P450 2C19 and 3A4 in the oxidation of omeprazole by human liver microsomes: effects of contents of these two forms in individual human samples.
Omeprazole 5-hydroxylation and sulfoxidation activities were determined in liver microsomes of different humans whose levels of individual forms of cytochrome P450 (P450 or CYP) varied. Correlation coefficients between omeprazole 5-hydroxylation activities (when determined at a substrate concentration of 10 microM) and S-mephenytoin 4'-hydroxylation and testosterone 6beta-hydroxylation activities were found to be 0.64 and 0.67, respectively, in liver microsomes of 84 human samples examined. Omeprazole sulfoxidation activities in these human samples were correlated with testosterone 6beta-hydroxylation activities (r = 0. 86). Omeprazole 5-hydroxylation by liver microsomes of a human sample that contained relatively high levels of CYP3A4 and low levels of CYP2C19 were inhibited very significantly by ketoconazole and anti-CYP3A4 antibodies, although a human sample having high in CYP2C19 and low in CYP3A4 was found to be sensitive toward fluvoxamine and anti-CYP2C9 antibodies. Sulfaphenazole (at 100 microM) did not affect the omeprazole 5-hydroxylation and sulfoxidation catalyzed by human liver microsomes. Both recombinant human CYP2C19 and CYP3A4 enzymes had activities for omeprazole 5-hydroxylation, with low Km and high Vmax values for the former enzyme and high Km and low Vmax values for the CYP3A4. These results suggest that contributions of CYP2C19 and CYP3A4 in the omeprazole 5-hydroxylation depend upon the ratio of these two P450 levels in human liver microsomes. Omeprazole 5-hydroxylation activities of different human samples were found to be related to predicted values calculated from the kinetic parameters of recombinant enzymes and the levels of liver microsomal CYP2C19 and CYP3A4 enzymes. Finally, when recombinant human CYP2C19 and CYP3A4 were mixed at levels found in different human samples, relatively similar profiles of omeprazole oxidation by the recombinant and microsomal enzyme systems were determined by analysis of high-performance liquid chromatography. These results suggest that both CYP2C19 and CYP3A4 are involved in the 5-oxidation of omeprazole (at a substrate concentration of 10 microM) in human liver microsomes and that contributions of these P450 enzymes depend on the compositions of CYP2C19 and CYP3A4 in liver.
Animals↗
Transcriptional regulation of the CYP2B1 and CYP2B2 genes by C/EBP-related proteins.
Cytochrome P450 (CYP) 2B1 and 2B2 are encoded by two closely related genes, CYP2B1 and CYP2B2, that are expressed at low levels in adult rat liver but are induced markedly by the administration of the drug phenobarbital (PB) or other structurally unrelated hydrophobic compounds to animals. Very little is understood about the molecular mechanisms that control both basal and induced transcription of these genes. We have identified two liver specific DNase I hypersensitive sites associated with the CYP2B1 and CYP2B2 (CYP2B) genes. One site, which maps to a region in the 5'-flanking region between -2.2 and -2.3 kb, became more resistant to DNase I cleavage in nuclei from PB-treated rats; the converse was true of the other hypersensitive site, which maps to the proximal promoter region between -0.05 and -0.15 kb. DNase I footprint analysis revealed three prominent and one weak footprinted regions in the promoter region in the vicinity of the proximal hypersensitive site. Using competitor oligonucleotides, we determined that one footprinted region (FT2), between -42 and -66 bp, is likely to represent a binding site for CCAATT enhancer binding protein (C/EBP) family members. Indeed, bacterial expressed recombinant C/EBP alpha bound at this site and formed a footprint pattern identical to the pattern observed with liver nuclear extract. In vitro transcription assays demonstrated that the FT2 site contributed strongly to promoter activity, since its mutation reduced transcription by 80%. Two other sites identified by footprint analysis (FT1 and FT3) are also required to maintain high basal transcription of CYP2B2 promoter constructs in an in vitro transcription assay. Transient transfection experiments confirmed the expectation that C/EBP alpha could activate the 1.4 kb CYP2B promoter constructs, with mutation of the FT2 site impairing both basal transcription and transactivation by exogenous C/EBP alpha.
Animals↗
Sequence of the rat PB-inducible CYP2B1 promoter.
The 5' flanking region of the rat PB-inducible CYP2B1 gene was isolated and the sequence from +27 to -3878 was determined. This sequence contains several putative binding sites for the liver-enriched factors HNF3 as well as an AP1, two NF-kappa B and several possible STAT sites. The promoter sequence of the CYP2B1 gene was compared to that of the CYP2B2 sequence, published by Hoffman et al. ((1992) Gene Exp. 2, 353-362) and was found to be almost identical up to -2300 bp, beyond which it diverges significantly from the remaining published sequence of CYP2b2 gene. Transient transfection experiments in the differentiated hepatoma cell line, FGC4, showed that the 3.9 kb promoter was expressed, however, an increase in reporter activity was not observed in the presence of phenobarbital.
Animals↗
Percutaneous technique for management of persistent airspace with prolonged air leak using fibrin glue.
A 68-year-old white man with lung carcinoma underwent removal of the lower lobe of the left lung. Four months later, the patient developed a hemothorax requiring exploratory thoracotomy, evacuation of hemothorax, and decortication. Postoperatively a persistent airspace with prolonged air leak developed. Bronchoscopic application of fibrin glue failed to seal the leak. Percutaneous transthoracic application of fibrin glue with CT scan guidance partially obliterated the space and completely sealed the leak. We describe this simple and effective technique for management of this special problem.
Adenocarcinoma↗
Occult inguinal hernia, a cause of rapid onset of penile and scrotal edema in patients on chronic peritoneal dialysis.
From 1976 to 1993 we inserted 160 chronic peritoneal dialysis catheters for renal failure patients. Three of these patients developed sudden onset of penile and scrotal edema after the catheter had been in place for several months. The first patient was diagnosed by instilling technetium sulfur colloid in the peritoneal cavity, which showed the radioisotope flowing via the right inguinal canal. He was operated on and the processus vaginalis was tied off and the scrotal and penile edema resolved. Subsequently, two more patients were seen with similar problems and had their inguinal canals explored and the processus vaginalis in one and the hernia sac in the other were found and tied off, which resulted in resolution of the problem. This is an uncommon complication, reported to occur in 3 to 4% of patients.
Adult↗
Sequence and activity of the rat PB-inducible aldehyde dehydrogenase promoter.
The 5' flanking region of the rat PB inducible aldehyde dehydrogenase gene was isolated and the sequence from +42 to -1339 was determined. This sequence contains several putative binding sites for the liver-enriched factors HNF3 and DBP as well as a GRE and several possible AP1 sites. A TATA and CCAAT motif were assigned at positions -26 and -53. A promoter construct containing the -1339 bp of the aldehyde dehydrogenase 5' flanking region was active when transfected into both H411 and HepG2 liver cell lines.
Aldehyde Dehydrogenase↗
Optimization of chest films of equalization radiography (advanced multiple beam equalization radiography). Comparison by means of a receiver operating characteristic study of simulated nodular interstitial disease.
RATIONALE AND OBJECTIVE: To optimize screen-film combinations for equalization radiography (advanced multiple beam equalization radiography [AMBER]), five different film-screen-technique combinations were compared by receiver operating characteristics study of simulated interstitial disease. MATERIALS AND METHODS: The Ortho C-Lanex Regular and the Insight Thoracic Imaging HC system were compared in conventional nonequalized technique; T-Mat G-Lanex Regular and T-Max L-Lanex Regular were compared in conventional, nonequalized, and AMBER technique; and an experimental high-contrast, low-noise, near-zero crossover film-screen combination was compared in AMBER technique. Interstitial disease was simulated by superimposing birdseed on the back of a humanoid phantom. Twenty-five posterior-anterior radiographs were made with each technique. Seven observers scored the presence of interstitial disease in each of the quadrants on a 5-point scale following receiver operating characteristic methodology. RESULTS: The highest performance was found with the experimental film-screen-AMBER combination (Az = 0.92) and the lowest with the T-Mat L-Lanex Regular-AMBER combination (Az = 0.83) and the Insight Thoracic Imaging HC system-conventional combination (Az = 0.85). T-Mat L-Lanex Regular-conventional ranked second (Az = 0.90) while T-Mat G-Lanex Regular-conventional (Az = 0.89), T-Mat L-Lanex Regular-AMBER (Az = 0.88) and Ortho-C-Lanex Regular-conventional (Az = 0.87) scored lower. CONCLUSION: Higher contrast films in AMBER improve diagnostic performance, whereas a loss of information is found if the AMBER system is combined with lower contrast films.
Humans↗
Hepatocyte nuclear factor 3 is a major determinant of CYP2C6 promoter activity in hepatoma cells.
Cytochrome P450 2C6 (CYP2C6) is a developmentally regulated, constitutively expressed form of rat liver microsomal cytochrome P450 that in the liver of adult male rats is induced to a limited extent by phenobarbital. The gene is not expressed at detectable levels in the lung, kidney, or brain. It is expressed and inducible by phenobarbital in differentiated Reuber hepatoma cells that express many hepatocyte-specific genes but not in dedifferentiated derivatives lacking the majority of hepatocyte-specific functions. A 505-base pair proximal segment of the CYP2C6 promoter is highly efficient in driving transcription of a linked chloramphenicol acetyltransferase reporter gene in the differentiated rat hepatoma cell line FGC4, is much less effective in a related dedifferentiated variant H5, and has no measurable activity in nonhepatic C33 human cervical carcinoma cells. The activity of the CYP2C6 promoter in the differentiated hepatoma cells is strongly dependent on hepatocyte nuclear factor (HNF)3, which acts at a complex site just upstream of the TATA motif. Transactivation experiments show that the D-site-binding protein (DBP) may also contribute to CYP2C6 promoter activity, via a site that is adjacent to the proximal HNF3 site. A substantial contribution to promoter activity by the base pair -505 to -316 segment is observed in FGC4 and H5 cells but not in HepG2 cells; deletion of this segment causes a marked diminution in promoter activity only in the former two cell lines. Although footprinting experiments have permitted the definition of three protein binding sites in this region (two HNF3 and one unidentified), mutation of these sites does not diminish promoter activity. The functionally important cis sequences in this region therefore remain to be defined. In HepG2 cells the distal region does not contribute to promoter activity. This most likely accounts for the low promoter activity in HepG2 and implies a deficiency in the relevant trans-acting factor(s).
Animals↗
The phenobarbital-induced transcriptional activation of cytochrome P-450 genes is blocked by the glucocorticoid-progesterone antagonist RU486.
Several of the hepatic microsomal cytochromes P450 can be induced by various drugs and xenobiotics, among them the barbiturate phenobarbital. Rat hepatoma cells (Fao and its derivatives) respond to phenobarbital or dexamethasone treatment with an increased accumulation of CYP2C6 mRNA and thus provide a culture system to investigate the mechanisms involved. Examination of the kinetics of CYP2C6 mRNA induction revealed that the response to dexamethasone is rapid, whereas induction by phenobarbital occurs only slowly after an 8-10-hr lag. Run-on transcription measurements demonstrated that phenobarbital treatment led to a 3-4-fold increase in CYP2C6 gene transcription. Surprisingly, induction by phenobarbital of both accumulation of CYP2C6 mRNA and transcription of the gene was blocked by the antiprogestin-antiglucocorticoid RU486, suggesting the involvement of a steroid receptor in the induction process. Transfection of promoter constructs containing a reporter gene whose expression is driven by a 1.4-kilobase 5' flanking segment of the CYP2B1 or CYP2B2 genes, which are highly inducible by phenobarbital in rat liver, led to > 3-fold increases in reporter gene activity in the presence of the drug. Again, phenobarbital induction was prevented by RU486. The RU486 inhibition of the phenobarbital induction of both the endogenous CPY2C6 gene and the transfected CYP2B1 and CYP2B2 promoter constructs leads us to propose a model whereby the drug acts indirectly to cause the accumulation of an endogenous steroid, and this molecule, acting via its receptor, would be the direct inducer of cytochromes P450. Whether or not this model proves to be correct, the results presented here provide the first evidence of the involvement of a steroid receptor in phenobarbital induction.
Animals↗
The human dioxin-inducible NAD(P)H: quinone oxidoreductase cDNA-encoded protein expressed in COS-1 cells is identical to diaphorase 4.
NAD(P)H: quinone oxidoreductase (NQO1) is believed to be protective against cancer and toxicity caused by exposure to quinones and their metabolic precursors. This enzyme catalyzes the two-electron reduction of compounds, compared with one-electron reduction mediated by NADPH: cytochrome-P450 oxidoreductase which produces toxic and mutagenic free radicals. Recently we cloned and sequenced the cDNA encoding human 2.3,7,8-tetrachlorodibenzo-p-dioxin (dioxin)-inducible cytosolic NQO1 [Jaiswal et al. (1988) J. Biol. Chem. 263, 13572-13578] and provided preliminary evidence that this enzyme may correspond to diaphorase 4, an enzymatic activity present in various tissues that catalyzes the reduction of a variety of quinones by both NADH and NADPH [Edwards et al. (1980) Biochem. J. 187, 429-436]. In the present report we characterize the catalytic properties of the protein encoded by the NQO1 cDNA. The enzyme was synthesized in monkey kidney COS-1 cells transfected with a pMT2-based expression plasmid containing the NQO1 cDNA. Western blot analysis of the transfected cells using an antibody against rat liver cytosolic NQO1 revealed a 31-kDa band that was not detected in nontransfected cells. This band corresponded to a polypeptide with the same electrophoretic mobility as the endogenous NQO1 protein detected in the human hepatoblastoma (Hep-G2) cells with the same antibody. The immunoreactive protein detected in human Hep-G2 cells was induced approximately fourfold by exposure of the cultures to dioxin, an increase commensurate with the increased in quinone oxidoreductase activity. These studies suggest that the protein encoded by NQO1 cDNA is indeed similar, if not identical, to the dioxin-inducible protein band detected in human Hep-G2 cells. Further characterization of the product of NQO1 cDNA, which was present at approximately 20-30-fold higher levels in transfected COS cells than the endogenous product in uninduced human Hep-G2 cells indicated that it had very high capacity (greater than 1000-fold over background) to catalyze the reduction of 2.6-dichloroindophenol and menadione. Besides these two commonly used substrates for quinone reductase, the expressed NQO1 protein also effectively metabolized 2,6-dimethylbenzoquinone, methylene blue, p-benzoquinone, 1,4-naphthoquinone, 2-methyl-1,4-benzoquinone, with the latter being the most potent electron acceptor at 50 microM concentration of the substrate.
Animals↗