Regular Article
Influences of cytochrome b5 expression and its genetic variant on the activity of CYP2C9, CYP2C19 and CYP3A4

https://doi.org/10.1016/j.dmpk.2019.03.001Get rights and content

Abstract

The objective of the present study was to investigate the effects of cytochrome b5 (cytb5) on the drug metabolism catalyzed by CYP2C9, CYP2C19 and CYP3A4. Activities of CYP2C9, CYP2C19, and CYP3A4 were determined by using the prototypical substrates tolbutamide, omeprazole and midazolam, respectively. Cytb5 protein and mRNA contents showed large inter-individual variations with 11- and 6-fold range, respectively. All of three P450s showed an increased activity in proportion to the amount of cytb5 expression. Particularly, CYP3A4 showed the strongest correlation between cytb5 protein amount and the activity, followed by CYP2C9 and CYP2C19. The putative splicing variant, c.288G>A (rs7238987) was identified and was screened in 36 liver tissues by direct DNA sequencing. Liver tissues having a splicing variant exhibited unexpected sizes of cytb5 mRNA and a decreased expression tendency of cytb5 protein compared to the wild-type. A decreased activity in the metabolism of the CYP2C19 substrate omeprazole was observed in liver tissues carrying the splicing variant when compared to the wild-type Cytb5 (P < 0.05). The present results propose that different expression of cytb5 can cause variations in CYP mediated drug metabolism, which may explain, at least in part, the inter-individual difference in drug responses in addition to the CYP genetic polymorphisms.

Introduction

Human cytochrome b5 (Cytb5, gene name; CYB5A) is involved in various biological metabolic pathways as an electron transfer component. The function of cytb5 includes CYP-catalyzed drug metabolism, fatty acid desaturation, methemoglobin/hemoglobin cycling [1], [2], [3], as well as steroid hormone biosynthesis [4]. Furthermore, cytb5 plays a role in the reduction of heavy metals, carcinogenic arylhydroxylamines, and in the biosynthesis of glycerol-based phospholipid in heart and neuronal tissues [5], [6], [7], [8].

The Cytochrome P450 (CYP) superfamily is involved in the oxidative metabolism of a variety of endogenous and exogenous compounds [9], [10]. Human CYPs are mainly expressed in liver and also exist in the lung, kidney and small intestine [11], [12]. Most of the CYPs have similar mechanism of action in their metabolism [9], [10]. Therefore, it has been suggested that cytb5 may contribute the second electron in some systems and thereby also influence CYP activity. The role of cytb5 in CYPs monooxygenase reaction has been reported in in vitro and in vivo studies. CYP2A6, CYP3A4, CYP3A5, CYP2B6, CYP2C8, CYP2D6 and CYP2E1 showed enhanced activities by presence of cytb5 in both enzyme reconstitution and CYP/NADPH-cytochrome P450 reductase (POR) membrane systems [13], [14]. In case of CYP2C9 and CYP2C19, cytb5 was required for catalytic activities in the reconstitution system [13]. In in vivo situations, apparent reduction of CYP-mediated metabolism has been observed in several drugs in the hepatic deletion of cytb5 in the mouse model including midazolam, metoprolol, tolbutamide, phenacetin and chlorzoxazon as well as some anticancer drugs, cyclophosphamide, tamoxifen and anastrozole [4], [15]. Moreover, genetic variability in cytb5 has been reported in multiple studies. Nonsynonymous mutations, T60A and S5A, in cytb5 caused the altered affinity for hydroxylamine and expressed the lower levels of cytb5 protein [8].

Phenotypic outcomes were observed from the decreased function of cytb5 in humans. It is known that 17, 20-lyase activity is modulated by activity of cytb5. Missense mutations (H44L) or nonsense mutations (W27X) in CYB5A revealed significantly impaired 17, 20-lyase activity, resulting in sex steroid deficiency, for example, male with abnormal masculinization, 46, XY disorder of sex development [16]. Furthermore, 16-bp deletion in CYB5A mRNA as an alternative splice site mutation exhibited severe cyanosis 7 days after birth and also in case of certain patient, cytb5 deficiency caused type IV methemoglobinemia, which indicated 12–19% of methemoglobin concentration in total hemoglobin level [17], [18].

Multiple studies reported cytb5 contents in African-American, Caucasian-American and also in Chinese by spectral or Western blot analysis [1], [5], [19], accounting for the large variations in its expression. However, there has been lack of a combined investigation using cytb5 protein contents, cytb5 genetics, and the CYP metabolism in the same human liver tissues. CYP2C9, CYP2C19, and CYP3A4 were investigated as examples of drug metabolizing P450s to estimate the contribution of cytb5 to the P450 activities.

Therefore, specific aims in the present study were to investigate the variations in cytb5 expression, to determine the relationship between cytb5 content and major CYP activity (CYP2C9, 2C19, and CYP3A4), and to analyze the influences of genetic polymorphisms in cytb5 gene on the corresponding P450 activities.

Section snippets

Human liver samples

Thirty-six human liver tissues originated from Koreans were obtained, with informed consent, from the Tissue Repository Biobank at Inje Pharmacogenomics Research Center (Inje University College of Medicine, Busan, Korea) as reported previously [20]. Human liver microsomes (HLM) were prepared by institutional guidelines and the study was approved by the Institutional Review Board of Busan Paik Hospital (Busan, Korea).

Quantification of cytochrome b5 protein level in HLMs

Protein contents of cytb5 in human liver were determined by Western blot

Cytochrome b5 protein expression levels in human liver tissue

Protein contents of cytb5 were determined by Western analysis using 36 human liver microsomes and the band densities were normalized to the β -actin protein level. To confirm the cytb5 protein contents in the studies, immunoblottings were conducted twice independently in the same experimental schemes to confirm inter-experimental variations. Firstly measured protein contents of cytb5 were highly correlated with the second measured results, showing high coefficient of correlation (r) value (r

Discussion

In this study, the expression levels of cytb5 protein and mRNA were determined using liver tissues and the effect of cytb5 contents on CYP2C9, CYP2C19 and CYP3A4 activities were analyzed by using their prototype substrates. Moreover, a splicing variant (c.288G>A, rs7238987) in cytb5 was identified and its influence on cytb5 expression and CYP activities were studied in the liver tissues with the genotyping information. There were large inter-individual variations in cytb5 protein and mRNA

Conflicts of interest

The authors report no declaration of interest.

Authorship contributions

Sung-Eun Yoo, Jae-Gook Shin, and Su-Jun Lee are participated in research design. Sung-Eun Yoo, MyeongJin Yi, Woo-Young Kim, Sun-A Cho, and Sang Seop Lee conducted experiments. Data were analyzed by Sung-Eun Yoo, Su-Jun Lee, and Jae-Gook Shin. Sung-Eun Yoo and Su-Jun Lee wrote the manuscript.

Acknowledgements

This research was supported by a grant of Korea Health Technology R&D Project through the Korea Health Industry Development Institute, funded by the Ministry of Health & Welfare, Republic of Korea (Grant number: HI15C1537). This work was supported by the National Research Foundation of Korea grant funded by the Korea government (MSIT) (No. 2018R1A5A2021242).

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