Role of NADPH-Cytochrome P450 Reductase and Cytochrome-b5/NADH-b5 Reductase in Variability of CYP3A Activity in Human Liver Microsomes

Gan, Lu; Moltke, Lisa L. von; Trepanier, Lauren A.; Harmatz, Jerold S.; Greenblatt, David J.; Court, Michael H.

doi:10.1124/dmd.108.023424

Cited by 37 publications

(38 citation statements)

References 30 publications

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“…3A). However, we could detect increased expression of the P450 electron donor POR and cytochrome b 5 (CYB5A), an important modulator for CYP3A4 activity (Schenkman and Jansson, 2003;Gan et al, 2009). Both proteins gradually increased with increased time of confluence, but CYB5A could not be detected until 2 to 3 weeks of confluence (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…It is believed that under certain conditions, POR can be the rate-limiting factor for P450 activity, because the POR protein is expressed at much lower stoichiometric amounts than the P450s in the liver (Schmucker et al, 1990). Like POR, CYB5A, another well known modulator of CYP3A4 activity (Gan et al, 2009), also gradually increased with time of confluence, although it could not be detected until 3 weeks of confluence.…”

Section: Discussionmentioning

confidence: 99%

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CYP3A4 Catalytic Activity Is Induced in Confluent Huh7 Hepatoma Cells

Sivertsson¹,

Ek²,

Darnell³

et al. 2010

Drug Metabolism and Disposition

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ABSTRACT:Drug-induced hepatotoxicity is an important cause for disapproval, limitations of use, or withdrawal of drugs, and there is a high need for reproducible in vitro systems that can predict such toxicity. In this study, we show that confluent growth of the human hepatoma cell line Huh7 up to 5 weeks results in increased gene expression of several cytochromes P450 (P450s), UDP-glucuronosyltransferases, transporters, transcription factors, and several liver-specific genes, as measured by low-density array. The most striking effect was seen for CYP3A4 expression. Western blot analysis revealed increased amounts of CYP3A4 together with increased levels of NADPH-P450 reductase, cytochrome b 5 , and albumin with prolonged time of confluence. By using the CYP3A4-specific substrates luciferin 6 benzyl ether, testosterone, and midazolam, we could confirm that the increased CYP3A4 gene expression also was accompanied by a similar increase in catalytic activity, inhibitable by the CYP3A4-selective inhibitor ketoconazole. The CYP3A4 activity in confluent cells was also inducible by rifampicin. Finally, the cell system could support the CYP3A4-dependent hepatotoxic activation of aflatoxin B 1 , which was effectively inhibited by ketoconazole. Our results show that Huh7 cells grown confluent differentiate into a more metabolically competent cell line, especially with regard to CYP3A4.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

CYP3A4 Catalytic Activity Is Induced in Confluent Huh7 Hepatoma Cells

Sivertsson¹,

Ek²,

Darnell³

et al. 2010

Drug Metabolism and Disposition

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show abstract

“…The activity of the enzymes mentioned above in adult animals changes to a greater degree compared to the base level of activity in younger animals. The results presented by Court et al [47] indicate that hepatic cyt b5 reductase, as well as cyt b5 activity and content, decrease with age, particularly in the livers of males. Changes in the expression and activity of cyt b5 reductase are probably not sufficient to account for age-related decreases in CYP3A activity in vitro [48]; however, the authors' conclusion may have been different if the effects of lipid-radical cycles were considered in this process.…”

Section: Iron and Iron Homeostasismentioning

confidence: 79%

Biological Membranes Are Nanostructures that Require Internal Heat and Imaginary Temperature as New, Unique Physiological Parameters Related to Biological Catalysts

2010

Cell Biochem Biophys

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In 1961, Peter Mitchell advanced a new idea for solving the problem of coupling between oxidation and phosphorylation, but some aspects of the relationship between the redox-chain as a potential energy donor and different energy acceptors remain largely unknown. The main structure-function relationships behind catalytic rate optimization in membrane enzymes are highly important, and comparative analyses of the energetics of catalytic reactions from membrane proteins of different destination are needed to advance our understanding. Moreover, the mode of control of primary radicals, such as reactive oxygen species (ROS), should be considered. For example, iron is essential for most organisms because it serves as an electron donor and acceptor in various metabolic processes. However, these chemical properties also allow iron to participate in the formation of ROS that cause substantial damage to lipids; iron can contribute to excess production of damaging ROS through Fenton chemistry. The evidence that iron contributes to various diseases of ageing is to be examined along with the need for low or moderate levels of iron, depending on homeostasis level. If this level in the organs and tissues is close to the optimal amount needed for an initiation of lipid-radical cycles, which may be responsible for the effectiveness of some membrane enzymes, this might minimize the ROS production and retard the processes related to ageing. To my mind, biological membranes possess an internal heat and imaginary temperature that are new, unique physiological parameters related to a role as factors of biological catalysis. This is speculation and additional studies will be needed to determine whether the imaginary temperature has an equal importance with the real temperature in cellular metabolism, membrane energetics (microsomal monooxygenase and ATP synthesis) and ageing.

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“…The contents of cytochrome-b 5 (Cyb5) in hepatic S9 fractions were determined by measuring the differential absorbance between NADH-reduced and oxidized S9 fractions as previously described (Venkatakrishnan et al, 2000;Gan et al, 2009).…”

Section: Methodsmentioning

confidence: 99%

Cholic Acid Feeding Leads to Increased CYP2D6 Expression in CYP2D6-Humanized Mice

Pan

Kent

Won

et al. 2017

Drug Metabolism and Disposition

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Cytochrome P450 2D6 (CYP2D6) is a major drug-metabolizing enzyme, but the factors governing transcriptional regulation of its expression remain poorly understood. Based on previous reports of small heterodimer partner (SHP) playing an important role as a transcriptional repressor of CYP2D6 expression, here we investigated how a known upstream regulator of SHP expression, namely cholestasis triggered by cholic acid (CA) feeding in mice, can lead to altered CYP2D6 expression. To this end, CYP2D6-humanized (Tg-CYP2D6) mice were fed with a CA-supplemented or control diet for 14 days, and hepatic expression of multiple genes was examined. Unexpectedly, CA feeding led to insignificant changes in SHP mRNA but also to significant (2.8-fold) decreases in SHP protein levels. In silico analysis of the SHP gene regulatory region revealed a putative binding site for a microRNA, miR-142-3p. Results from luciferase reporter assays suggest that miR-142-3p targets the SHP gene. Hepatic expression of miR-142-3p was significantly increased in CA-fed mice (∼5-fold), suggesting a potential role of miR-142-3p in the regulation of SHP expression in cholestasis. The decreased SHP protein levels were accompanied by increased expression and activity of CYP2D6 in the liver of CA-fed mice. These results suggest potential roles of differential hepatic levels of bile acids in the transcriptional regulation of CYP2D6 expression.

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Role of NADPH-Cytochrome P450 Reductase and Cytochrome-b5/NADH-b5 Reductase in Variability of CYP3A Activity in Human Liver Microsomes

Cited by 37 publications

References 30 publications

CYP3A4 Catalytic Activity Is Induced in Confluent Huh7 Hepatoma Cells

CYP3A4 Catalytic Activity Is Induced in Confluent Huh7 Hepatoma Cells

Biological Membranes Are Nanostructures that Require Internal Heat and Imaginary Temperature as New, Unique Physiological Parameters Related to Biological Catalysts

Cholic Acid Feeding Leads to Increased CYP2D6 Expression in CYP2D6-Humanized Mice

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