CYP2E1 has significance in chemical toxicity and carcinogenicity so it has been studied for long years as of now. Despite the fact that CYP2E1 gene has number of SNPs (single nucleotide polymorphisms), it is well preserved in mammals and there are no polymorphism that is known because of CYP2E1 gene inactivity in human or animal models. Moreover, there is not much distinctions in the enzymatic activity among different species like humans, rabbits, rats, and mice which leads us to thought of CYP2E1 having an important functional activity in mammals.
Leiber et al. found that the oxidation of ethanol was catalyzed by the membrane fractions of microsomes which lead to the designation of new term i.e. 'Microsomal ethanol-oxidizing system (MEOS) since this was bound to membrane, required NADPH and was inactivated by Carbon monoxide which is quite uncommon to alcohol dehydrogenase. From this experiment they also obtained result showing that MEOS activity was induced by administering ethanol to rats and this lead to increased clearance of alcohol that is no blocked by pyrazole (Pyrazole is an inhibitor of ADH). These results lead to major advancement in alcohol field with substantial clinical implications.
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The first P450 which was capable of ethanol oxidation was obtained and purified from rabbit by Koop et al. Following this, the rat ortholog was extracted and was found to have enzymatic activity towards aniline p-hydroxylation by Ryan et al. After some time, experiment done using purified recombinant P450 showered that CYP2E1 metabolizes a many no of low molecular weight chemicals many of which are of low molecular weight and are carcinogenic agents. Therefore, we can speak that CYP2E1 is important for toxicological and carcinogenic properties.
CYP2E1 Transcriptional Control
Ueno and Gonzalez (1990) suggested the factors that regulate the developmental activation of CYP2E1 gene transcription and the position at which these transcription factors bind to the promoter region in this gene. Further studies in this direction has shown that rodent and homo sapiens CYP2E1 genes are under controlled regulation of liver reached transcription factor which is also known as HNF1-alpha (Hepatocyte nuclear factor 1 alpha). Despite these research evidences recently there are outcomes obtained that shows that regulation of CYP2E1 is not solely Hnf1α dependent as the deletion of β-catenin gene in mouse showed almost complete deletion of CYP2E1 in hepatic tissues without changing the level of Hnf1α. The principal action of catenin on a transcriptional co activator is always required for CYP2E1 activation by Hnf1α is among the available probabilities.
Substrates like acetone, ethanol and pyrazole are all inducers of CYP2E1 activity and proteins via a post translational mechanism revealed by in vivo labeling of presence of substrate. Others also discovered that electron transfer may actually increase the degration of CYP2E1 by proteasomes.
There are two pathways known: lysosomal pathway and the proteosome pathway that can participate in the degradation proteins from membranes. Lysomes degrades membrane proteins through fusion of Endoplasmic reticulum with membranes of lysosomes and this process is slow than that of the proteosome degradation pathway. It has also been evident from experiments on cell lines that degradation of CYP2E1 can occur via an ubiquitin-independent proteosomal pathway. Due to the biphasic nature of CYP2E1 that is observed in vivo, it is alluring to guess that CYP2E1 may not be focus to the additional rapid phase of turnover in the presence of chemical substrates via proteosomes due either to confiscation of the enzyme in various regions of the Endoplasmic reticulum or to altered protein conformation due in a part of a result of substrate binding, or may be simultaneous effect of both combinations. Lysosomal pathway would follow to function in the presence of substrate. The prompt for degradation via latter pathway is not known but could due to phosphorylation or alteration in the folding pattern of the protein. It should also be considered that substrate-induced stabilization has not been confirmed for other P450s. Long-term in vivo exposure to the substrate is required for a condition that could be achieved with chronic drug treatment to achieve significant P450 stabilization.
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CYP2E1 Null mice
Past evidences suggests that CYP2E1 has an important role in mammals. It is expressed in hepatic tissues and participates in gluconeogenesis process of the body. The enzyme and its activities are well preserved in humans and other experimental animals such as rats and mice and rabbits. No functional polymorphism is found in Homo sapiens or any animal model that alters enzymatic activity of CYP2E1. Thus, it was thought that any important function for this P450 would be revealed by phenotypes obtained from gene knockout mice. However, this model has been of great value in shaping the role that CYP2E1 is involved in chemical toxicity and carcinogenicity studies.
CYP2E1 role in Carcinogenesis
Azoxymethane is colon carcinogen that is activated by CYP2E1 to its metabolite that forms addcuts in colon and extrahepatic tissues. In extrahepatic tissues like colon, non-P450 enzymes such as ADH can trigger MAM. CYP2E1 is a highly liver-enriched enzyme but is not significantly expressed in the colon. There is a belief that MAM, produced in the liver, is transferred to the colon where it is then converted to the methyl diazonium-alkylating agent that is responsible for generating the DNA adducts which are precursors of the colon cancer.
CYP2E1 role in Alcohol metabolism
Gong et al. showed that CYP2E1 plays a vital role in alcohol metabolism and liver damage due to alcohol by producing O2-· and H2O2 which deplete the intracellular glutathione levels and causes cell damage. Moreover, these reactive oxygen species produces radicals like hydroxyl that are powerful oxidants. Also lipid peroxides formed from ROS form adducts with nucleophiles in cell environment like protein and nucleic acids resulting in cell damage. Some research in this field states that cell lines like HepG2 which overexpresses this enzyme cause elevation in oxidative stress and also activation of p38 mitogen pathway and induces erythroid like factor 2. Although of little consequence to liver toxicity there are subtle difference observed in DNA damage. Because of difference in liver architecture it is accepted that alcohol induced hepatic damage differs in mice and other rodent models.
CYP2E1 role in Acetaminophen toxicity
Raucy et al showed that CYP2E1 is important enzyme that plays a role in activation of Acetaminophen conversion to NAPQI which is an active metabolite responsible for acetaminophen toxicity. This evidence agrees with the fact that ethanol enhances the acetaminophen induced hepatotoxicity in rats and also in humans the drug disulfiram which is CYP2E1 inhibitor decreases the clearance of acetaminophen.
CYP2E1: future study prospects
Metabolomics: The analysis of small molecules in biological fluids to determine the outcome of altered metabolism or the response to pathological trigger such as xenobiotic. These measurements made on small molecules are carried out by chromatographic methods which are methods that are sensitive for molecules below 800 Da. 1H NMR has been widely used method to study because it allows the accurate determination of the structure and amount of chemicals in a biological samples. Biomarkers can be identified that helps in determining the organ-specific drug toxicity by use of animal models. 1H NMR metabolomics may also be useful to diagnose diseases like cancer and neurological disorders and also atherosclerosis.
Metabolomics is only possible when we combine it with powerful data analysis machine. Multiple samples is subjected to data sourcing which is then followed by chemo metric and multivariate analysis to extract relevant information that shows the differences between samples or groups. Principal components analysis (PCA) is generally used in metabolomics studies. The concurrent analysis and assessment of thousands of ions from two or more groups is thinkable only due to PCA.
1H NMR yields has less sensitivity and resolution power that are needed to study low-abundance metabolites in complex combinations. Metabolomics studies can also be conceded out by using more accessible and more advanced LC-MS technology which does not give precise chemical structures and quantification in the absence of standards but allows comparison of thousands of compounds as well as its analysis.
An ultra-performance liquid chromatography-coupled time-of-flight mass spectrometry (UPLC-TOFMS) has been recently developed for use in metabolomics studies. This instrumentation lets the resolution and determines the exact mass of thousands of molecules in a biological sample where the quantities of biological materials are scarce and is preferably matched for studies in rodent model systems. A urine specimen can typically give 5 to 10,000 ions that can be analyzed by PCA and are detected by the MS.
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There are many more things that remains to be derived or developed to understand the tools of regulation of the CYP2E1 genes although there have been a considerable progress made in recognizing the role of CYP2E1 in alcohol and chemical toxicity.