|Year : 2020 | Volume
| Issue : 1 | Page : 62-66
Assessment of cytochrome P450 2E1 activity in Hausa/Fulani of northwest Nigeria using chlorzoxazone as a probe determination of polymorphism
Muhammad T Umar1, Shaibu O Bello2, Aminu Chika2, Yakubu Abdulmumini3
1 Department of Pharmacology and Therapeutics, Faculty of Basic Clinical Sciences, College of Health Sciences Usmanu Danfodiyo University, Sokoto, Nigeria
2 Department of Pharmacology and Therapeutics, Faculty of Basic Clinical Sciences, College of Health Sciences Usmanu Danfodiyo University, Nigeria
3 Department of Internal Medicine, Faculty of Clinical Sciences, College of Health Sciences Usmanu Danfodiyo University/Teaching Hospital Sokoto, Sokoto, Nigeria
|Date of Submission||07-Nov-2019|
|Date of Decision||22-Jan-2020|
|Date of Acceptance||29-Jan-2020|
|Date of Web Publication||24-Mar-2020|
MBBS, MPH, MSc, PhD Muhammad T Umar
Department of Pharmacology and Therapeutics, Faculty of Basic Clinical Sciences, College of Health Sciences Usmanu Danfodiyo University, PMB 2346, Sokoto, Nigeria
Source of Support: None, Conflict of Interest: None
Background and objective High expression and activity of cytochrome P450 2E1 have been linked to non-alcoholic fatty liver disease; increased susceptibility to the gastric, nasopharyngeal, colorectal, urinary bladder and esophageal malignancies; and acetaminophen-induced hepatotoxicity. It plays key roles in activating procarcinogens to carcinogens, metabolism of xenobiotics, and hosts of endogenous compounds. This study aimed at determining the polymorphism of this highly polymorphic enzyme among Hausa/Fulani in northwest Nigeria.
Materials and methods A total of 20 nonrelated Hausa/Fulani from Sokoto metropolis were selected by convenient sampling. A tablet of 250 mg chlorzoxazone was administered orally to them with 100 ml of distilled water after an overnight fast, and 3 h after dosing, urine was collected. HPLC equipped with a UV detector was performed for simultaneous estimation of chlorzoxazone and its metabolite 6-hydroxychlorzoxazone. Metabolic ratio index method was used for each participant The data generated were analyzed using Statistical Package for the Social Sciences version 20 (SPSS-20) by constructing frequency histogram and probit plots. A trend line was added to the probit plot, and polynomial equation obtained was resolved to get antimode. Participants with antimode greater than or equal to value of intercept on logMR were regarded as poor metabolizers, whereas those with less were extensive metabolizers.
Result and conclusion Anti-mode was found to be −1.8, and only 7 of 20 participants were extensive metabolizers (35%, odds 0.54, 95% confidence interval: 0.22–1.3). Although convenience sampling was used, the findings are worrisome considering the highly polymorphic and the procarcinogenic nature of the enzyme.
Keywords: chlorzoxazone, cytochrome 2E1, Hausa/Fulani, polymorphism
|How to cite this article:|
Umar MT, Bello SO, Chika A, Abdulmumini Y. Assessment of cytochrome P450 2E1 activity in Hausa/Fulani of northwest Nigeria using chlorzoxazone as a probe determination of polymorphism. Egypt Pharmaceut J 2020;19:62-6
|How to cite this URL:|
Umar MT, Bello SO, Chika A, Abdulmumini Y. Assessment of cytochrome P450 2E1 activity in Hausa/Fulani of northwest Nigeria using chlorzoxazone as a probe determination of polymorphism. Egypt Pharmaceut J [serial online] 2020 [cited 2020 May 29];19:62-6. Available from: http://www.epj.eg.net/text.asp?2020/19/1/62/281021
| Introduction|| |
Cytochrome P450 2E1 is expressed mainly in the liver, nasal mucosa, kidney, lungs, and brain and serves as an important source of reactive oxygen species . It is mapped to chromosome10q24.3-qter, spans 11 kb containing 9 exons, and encodes a membrane-bound protein of 493 amino acids ,.
High expression and activity of cytochrome P450 2E1 have been linked to non-alcoholic fatty liver disease and increased susceptibility to the gastric, nasopharyngeal, colorectal, urinary bladder, and esophageal malignancies among a host of others ,. The activity of this enzyme undoubtedly also influences acetaminophen metabolism among different age groups. Ethnicity, apart from age, has been demonstrated to significantly determine cytochrome P450 2E1 gene expression. Higher level was found in Northern Europe-Americans and Hispanics than in African-Americans .
It plays key roles in activating procarcinogens to carcinogens, metabolism of xenobiotics, and hosts of endogenous compounds . It metabolizes acetaminophen, isonicotinic acid hydrazide, chlorzoxazone, acetone, alcohol, aniline, vinylchloride, benzene, N-nitrosodimethylamines, and styrene. CYP 2E1 belongs to phase I DMEs, and though it metabolizes only ∼3% of clinically used drugs, it is highly polymorphic, and this polymorphism corresponds to the replacement of cytosine and thymine at position −1019 ,,,,,,,.
Polymorphism of this enzyme and allelic distribution frequencies vary within and between populations. Increased susceptibility to breast cancer has been reported to be linked to the polymorphism of this enzyme (CYP 2E1*6) as well as coronary artery lesions in patients with Kawasaki disease ,. This study aimed at determining the polymorphism of this all-important drug-metabolizing enzyme among Hausa/Fulani in northwest Nigeria. To the best of our knowledge, this is the first attempt to assess cytochrome P450 2E1 activity among the Hausa/Fulani ethnic group, which forms the most populous ethnic group in Nigeria.
| Materials and methods|| |
Study population, exclusion criteria, and ethical consideration
Only participants who were declared of Hausa/Fulani descent and identified by one of the researchers (MTU) were involved in the study by convenient sampling from Sokoto metropolis in northwest Nigeria through their expressed consents. It was an exploratory study.
All volunteers who consumed alcohol, smoke tobacco, or with suspicion of hypersensitivity to chlorzoxazone (2E1 probe) were excluded. Other criteria were taking any prescription or herbal medicines within 2 weeks before the study or any over-the-counter medication within 1 week before the study. Similarly, all pregnant and lactating women, as well as children were excluded ,.
All participants read and signed a study-specific informed consent form before participating in the study procedures. The study was approved by the Ethics Committee of Sokoto State Ministry of Health.
Samples taking, preparation, and chromatography
Participants were asked to refrain from consuming caffeine 48 h before chlorzoxazone administration in addition to satisfying inclusion criteria. They fasted overnight for 11 h. At 8 a.m. on the day of the sample taking, the participants emptied their bladders, and a tablet of 250 mg chlorzoxazone was administered orally to them with 100 ml of distilled water. Three hours after dosing, urine was collected in a universal sample tube. The participants were observed for 8 h after the dose for any untoward effects and were discharged uneventfully.
Assay of chlorzoxazone and 6-hydroxychlorzoxazone in urine was carried out using Stiff et al.  method.
HPLC equipped with a UV detector was performed for the simultaneous estimation of chlorzoxazone and its metabolite 6-hydroxychlorzoxazone in urine using the method . Urine samples (diluted 1 : 500) were treated with β-glucuronidase before analysis. The detection of components was on the wavelength of 270 nm.
The data were analyzed by obtaining urine chlorzoxazone and 6-hydroxychlorzoxazone (metabolite) concentrations from the chromatograms generated by the HPLC. The metabolic ratio (MR) was calculated for each participant from these concentrations and logarithmic values determined. Statistical Package for the Social Sciences IBM* version 25, Armonk, NY, IBM Corp. USA, 2017 (SPSS-25) was used to construct frequency histogram using number of participants and logMR. Probit values (standard normal deviates) were obtained from Z-table and were plotted on Y-axis against logMR on X-axis (Scatter chart). A trend line was added to the probit plot and polynomial equation obtained. Anti-mode was determined as the intercept of X-axis, where Y=0 from logMR. Participants with antimode greater than or equal to value of intercept on logMR regarded as poor metabolizers, whereas those with less than values were considered extensive metabolizers. The outcome was reported as proportions with 95% confidence intervals. Polymorphism was determined graphically as the deviation of the probits values from the line of fit on the graph ([Figure 1] and [Figure 2]).
|Figure 2 Urine probit vs LogMR plot of participants. Scatter (XY) chart showing the Trend line with the best linear fit to the data and polynomial equation. At X-intercept where Y=0, the equation becomes 0.083×2+0.373×+0.435=0. The values of X were −1.8 and −2.9 and therefore −1.8 was considered as the antimode. The probits that do not fit to the trend line are indicative of polymorphism.|
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| Results|| |
The age range and median are shown in [Table 1]. MRs for each participant and metabolic status are displayed in [Table 2]. Nonparametric statistics were used because the sampling was by convenience. Nonetheless, the means, standard deviations, and P values are also shown simply to identify trends, although they may be considered inappropriate for nonrandom data.
| Discussion|| |
Cytochrome P450 phenotype studies provide useful information on the instantaneous activity of drug-metabolizing enzymes by the use of specific probes of which chlorzoxazone is one . Most drugs in clinical use are efficacious in only ∼25–60% of patients mainly owing to polymorphism of cytochrome P450 enzymes, which may be up to 50-folds between individuals as previously cited .
Probe drug’s characteristics and MR measurements are critical in the evaluation of phenotype cocktails, which serve as a reservoir for future application in personalized therapy. Except for cumbersomeness and vulnerability to errors owing to multiple points of sample collection, the formation clearance method appeared to be the most suitable approach for the measurement of drug-metabolizing enzyme activity in vivo . However, metabolic indexes (MR) in which coefficients between probes and metabolites are calculated from probits and logarithms of MRs are generally used to circumvent drawbacks in the formation clearance method and achieve the desired outcome .
Chlorzoxazone hydroxylation to its metabolite, 6-hydroxychlorzoxazone, is a recognized measure of the in-vivo cytochrome P450 2E1 activity. Because the metabolism of this drug has fairly low interindividual variability at a dose of 250 mg, single sample taking is enough to assess cytochrome P450 2E1 activity in vivo ,. Chlorzoxazone is a standard probe for cytochrome 2E1 that is used in establishing phenotypes of individual subjects . Between-subjects variability in the enzyme’s activities and consistent ethnic variations in the gene expression have been demonstrated previously ,. The variability of cytochrome P450 2E1 expression between persons is noteworthy and is interrelated with its enzymatic activity .
The occurrence of deviations from the line of fitness observed in the probit plots in this study was suggestive of polymorphisms among the participants . This finding further support what was reported by Kim et. al. . The correlations of determination of the plots revealed an outstanding relationship between the variables of probits and logMR as only 14% variations were unexplainable from polynomial expressions studied.
Cytochrome P450 2E1 enzyme activity among the participants was categorized phenotypically into poor and extensive metabolism. A good number of the participants were classified as poor metabolizers based on the anti-mode derived from urine probit versus logMR plots. This observation may be worrisome, as poor activity of the enzyme results ultimately to the toxicity of the agents being metabolized by the enzyme with wider pathological implications.On a long-term basis, this is manifested in the population as vulnerability to pathogenesis of nasopharyngeal, colorectal, stomach, esophageal, liver, lungs, and bladder malignancies ,,,,,,,,,,,,,,,,,,. While on a short-term range, fulminant hepatic failure from acetaminophen ingestion affecting millions of people globally remained in focus ,,.
| Conclusion|| |
Although convenience sampling was used, the findings are worrisome considering the highly polymorphic and the procarcinogenic nature of the enzyme.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2]