scholarly journals Comparison of in Vitro Bioactivation of Flutamide and Its Cyano Analogue: Evidence for Reductive Activation by Human NADPH:Cytochrome P450 Reductase

2008 ◽  
Vol 21 (12) ◽  
pp. 2393-2406 ◽  
Author(s):  
Bo Wen ◽  
Kevin J. Coe ◽  
Peter Rademacher ◽  
William L. Fitch ◽  
Mario Monshouwer ◽  
...  
Keyword(s):  
Reductive Activation ◽  
P450 Reductase ◽  
Nadph:Cytochrome P450 Reductase

Reductive Activation of Carbon Tetrachloride by Human Haemoglobin

1993 ◽  
Vol 21 (1) ◽  
pp. 57-64
Author(s):  
Roberta Ferrara ◽  
Michela Rezzadore ◽  
Stefano Cazzaro ◽  
Roberto Tolando ◽  
Maurizio Manno
Keyword(s):  
Carbon Monoxide ◽  
Carbon Tetrachloride ◽  
Anaerobic Incubation ◽  
Reductive Activation ◽  
Anaerobic Conditions ◽  
Prosthetic Group ◽  
Experimental Conditions ◽  
Human Haemoglobin ◽  
Reductive Metabolism

The reductive metabolism of carbon tetrachloride (CC14) by human haemoglobin (Hb) was observed in vitro by absolute absorption spectra recorded under anaerobic conditions. The following results were obtained: 1) a decrease of the 430nm peak typical of free reduced Hb (Hb2+); 2) the formation of a shoulder of absorbance, attributable to the production of a complex between Hb2+ and a metabolite of CC14 carbon monoxide (Hb-CO); and 3) the oxidation of some Hb2+ to methaemoglobin (Hb3+). The concentration of these three forms — Hb2+, Hb-CO and Hb3+ — during anaerobic incubation of Hb with CC14 was calculated algebraically from the absolute spectra. CO production was then calculated from the concentration of Hb-CO, using a suitable calibration curve. Interestingly, under identical experimental conditions, a substrate-dependent loss of Hb-derived haem, but not of Hb itself nor of haem-derived porphyrin fluorescence, was measured. Preliminary HPLC studies to clarify the discrepancy and, in particular, the role and fate of the haem group, showed two substrate-dependent modified haem products. The results indicate that human Hb is able to catalyse the reductive activation of CCl4, and suggest that, during the process, its prosthetic group haem may be modified by CC14 metabolites to products which maintain a tetrapyrrolic structure but are unable to react with pyridine.

Treatment of mammalian cells with the endoplasmic reticulum-proliferator compactin strongly induces recombinant and endogenous xenobiotic metabolizing enzymes and 3-hydroxy-3-methylglutaryl-CoA reductase in vitro

1999 ◽  
Vol 112 (4) ◽  
pp. 515-523
Author(s):  
L. McLaughlin ◽  
B. Burchell ◽  
M. Pritchard ◽  
C.R. Wolf ◽  
T. Friedberg
Keyword(s):  
Endoplasmic Reticulum ◽  
Enzyme Activity ◽  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Reductase Activity ◽  
Cholesterol Biosynthesis ◽  
Metabolizing Enzymes ◽  
P450 Reductase ◽  
Coa Reductase

Some xenobiotics induce membrane-bound drug metabolizing enzymes (Xme) and a profound proliferation of the endoplasmic reticulum (ER) in vivo. However these effects are much weaker in vitro, possibly due to absence of certain transcription factors. We tested the possibility that ER proliferation can affect the level of ER-resident enzymes even in the absence of transcriptional activation. For this purpose we analysed the effects of compactin, which has been shown to induce ER proliferation in vitro, on recombinant Xme, which were expressed from a constitutive viral promoter. High levels of recombinant UDP-glucuronosyltransferase UGT1A6 were achieved by amplification of the UGT1A6 cDNA using the dihydrofolate reductase cDNA as selectable marker in DHFR- CHO cells. Treatment of the resulting cell lines with lipoprotein-deficient serum in the absence and presence of compactin for 5 days resulted in a 1.3- and 2.3-fold, respectively, increase of the UGT enzyme activity towards 4-methylumbelliferone, paralleled by an induction of immunoreactive UGT1A6 protein. Similarly, treatment with this 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor increased the endogenous P450 reductase activity 2.6-fold, concomitant with an increase of immunodetectable protein. As expected compactin induced the level of 3-hydroxy-3-methylglutaryl-CoA reductase. Increased levels of this protein have been associated with a proliferation of the ER. Compactin treatment of a separate cell line that expressed recombinant human P450 reductase increased this enzyme activity fivefold. Pulse-chase experiments revealed that the induction of the recombinant Xme by compactin was most likely due to decreased protein degradation. Our results show that enzyme systems unrelated to those involved in cholesterol biosynthesis are affected by compounds known to affect membrane biogenesis. Since this effect extends to heterologously expressed enzymes, it also provides an efficient means by which to increase the levels of recombinant ER proteins.

Role of hepatic cytochromes P450 in bioactivation of the anticancer drug ellipticine: Studies with the hepatic NADPH:Cytochrome P450 reductase null mouse

2008 ◽  
Vol 226 (3) ◽  
pp. 318-327 ◽  
Author(s):  
Marie Stiborová ◽  
Volker M. Arlt ◽  
Colin J. Henderson ◽  
C. Roland Wolf ◽  
Věra Kotrbová ◽  
...  
Keyword(s):  
Anticancer Drug ◽  
Cytochromes P450 ◽  
Null Mouse ◽  
P450 Reductase ◽  
Nadph:Cytochrome P450 Reductase

Effect of Neurotransmitters on NADPH-Cytochrome P450 Reductase In Vitro Activity

2007 ◽  
Vol 1 (3) ◽  
pp. 172-175 ◽  
Author(s):  
Guillermo Gervasini ◽  
Carmen Martinez ◽  
Julio Benitez ◽  
Jose A.G. Agundez
Keyword(s):  
Cytochrome P450 ◽  
Vitro Activity ◽  
Cytochrome P450 Reductase ◽  
In Vitro Activity ◽  
Nadph Cytochrome ◽  
P450 Reductase ◽  
Nadph Cytochrome P450 Reductase

scholarly journals Bioreductive metabolism of small molecule nitroaromatics and N-oxides in hypoxia

2012 ◽  
Author(s):  
◽  
Anuruddha Rajapakse
Keyword(s):  
Dna Damage ◽  
Xanthine Oxidase ◽  
Tumor Therapy ◽  
Authentic Sample ◽  
Strong Impact ◽  
P450 Reductase ◽  
Hypoxic Conditions ◽  
Show Evidence ◽  
Dehydration Mechanism ◽  
Nadph:Cytochrome P450 Reductase

Hypoxia in tumors causes adverse effects to therapy and negatively impacts on patient prognosis. Identification and quantification of hypoxia is considered to have a strong impact on treatments in tumor therapy. Fluorescent-based detection to mark hypoxia may be vital to be used along with available methods such as radiochemical and immunohistochemical staining. In this work, the non-fluorescent 6-nitroquinoline (42) was used to investigate the production of a fluorescent 6-aminoquinoline (43) and other metabolites under bio-reducing hypoxic conditions. In the presence of the enzymatic reducing system NADPH:cytochrome P450 reductase/NADPH, 6-nitroquinoline (42) produced the fluorescent helicene (44), along with the non-florescent azo (45). An authentic sample of (44) was chemically synthesized and characterized and used to confirm the production of this molecule in the enzymatic process. Interestingly, the expected fluorophore (43) is not produced by NADPH:cytochrome P450 reductase/NADPH. In another study, the enzymatic reducing system xanthine/xanthine oxidase was used to reduce (42) under hypoxia to obtain (43). In these experiments (43) was produced and the yield is increased with xanthine concentration. Metabolic identification revealed that intermediates of typical nitro reduction pathway are present along with 6-nitroquinolone (51).which is formed by xanthine oxidase mediated oxidation of (42). The absence of (44) as a metabolite with xanthine/xanthine oxidase system highlights the complexity of bioreduction of nitroaromatics under hypoxia. In our laboratory, bio-activation of di-N-oxides such as tirapazamine (TPZ, 42) has been studied. TPZ undergoes one-electron bio-reduction to produce oxidizing radical, which causes DNA damage under hypoxia. In our laboratory, the mechanism by which TPZ mediated DNA damage has been investigated using TPZ and its analogs. Our evidence suggests that upon undergoing bio-reduction, TPZ produces hydroxyl radical as the DNA damaging radical species. Others have suggested another mechanism, which proposes the formation benzotriazine radical (38) upon dehydration process over the bio-reduction step. In the current work, TPZ analog 1,2,4-benzotraizine-1,4-dioxide (55) and deuterated (60) were used to test the dehydration mechanism. Isotopic content analysis of metabolites, derived from bio-reducing metabolism of (55) and its deuterated analog (60), using HRMS show evidence against the dehydration mechanism.

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