Genetic Polymorphism And MRNA Levels Of CYP450IIE1 And GSTP1 In Alcoholic Liver Disease

BACKGROUND: Excessive alcohol intake can cause physical, psychological, and social problems. Alcohol abuse and dependence are major factors in the pathogenesis of alcoholic liver disease (ALD). Not all drinkers develop ALD. Approximately 20% of chronic alcoholics develop hepatic cirrhosis, while 20% tolerate the chronic toxic effects without developing any liver pathology.Cytochrome P450IIE1 (CYPIIE1), a member of the phase I family of detoxifying liver enzymes, is the main component of the microsomal ethanol oxidizing system. It plays a major role in the metabolic activation of environmental xenobiotics such as ethanol, benzene, and small-molecular-weight procarcinogens, including nitrosamines, or drugs such as acetaminophen, chlorzoxazone, and salicylate. This metabolic activation can give rise to carcinogenic intermediate compounds. However, phase II enzymes, primarily the glutathione S-transferases (GST), normally act on these electrophilic, toxic intermediates by conjugating them to reduced glutathione to produce less toxic or readily excretable metabolites.The level and persistence of bioactivated toxic or carcinogenic compounds in the liver thus depends on the interplay of both biotransforming enzyme systems. This raises the possibility that an increase in the activity of the phase I system, coupled with a decrease in phase II activity, might increase the level of toxic or carcinogenic intermediates and predispose an individual to developing diseases such as ALD.A likely contributor to such a potential imbalance between phase I and II enzymes is allelic variation in the genes that encode them. The genes encoding both CYPIIE1 and GSTP1 exist in multiple polymorphic forms, causing their expression level to vary widely among individuals.[6] CYPIIE1 is located on 10q2403-qter. It is 1104 kb gene, consisting of 9 exons and 8 introns, that encodes a 493 amino acid protein. CYPIIE1 contains six restriction fragment length polymorphisms (RFLP), of which the PstI/RsaI polymorphism in its 5'-flanking region, has been shown to affect its transcription level. The C2 allele can enhance the transcription and increases the level of CYPIIE1 enzymatic activity. Similarly, polymorphisms of the GSTP1 gene exist, and were first reported by Board and his colleagues. These consist of an A-to-G transition of nucleotide 313 in exon 5 (GSTP1*B) and a G-to-T transition of nucleotide 341 in exon 6 (GSTP1*C), leading to the substitution of two amino acids in the enzyme active site, Ile to Val and Val to Ala. These allele variants appear to reduce GSTP1 activity, potentially leading to genetic damage and increased cancer risk.Alcohol abuse is becoming an increasingly severe problem among the Han, Mongol, and Chaoxian nationalities in the northeast of China. The present study was conceived to determine whether variation in the relative frequencies of these polymorphisms might be correlated with the incidence of ALD in patients of these three nationalities.Methods: Patients were admitted to the study, between September 2006 and November 2007, from several large hospitals in Heilongjiang, Jilin, Liaoning and Neimenggu provinces. Patients were from the Han, Mongol, and Chaoxian nationalities and included, respectively, 126, 118, and 109 adults with ALD. Also included in the study were 100 patients of each nationality who were alcohol dependent without ALD (alcoholic), and 120 healthy people of each nationality as controls . Real-time polymerase chain reaction (RT-PCR) and PCR restriction fragment length polymorphism (PCR-RFLP) was used in this research.Results: The respective frequencies of the CYPIIE1 C2 and GSTP1 Val alleles in patients with ALD were: 50.00% and 26.98% in Han patients (Table 2); 31.36% and 22.87% in the Mongol group (Table 3); and 45.87% and 22.02% in the Chaoxian (Table 4). In contrast, the corresponding frequencies in control patients were 10.00% and 8.3% in the Han, 7.50% and 10.00% in the Mongol group, and 11.67% and 9.17% in the Chaoxian. These data revealed an increased frequency of the C2/C2 and Val/Val genotype in ALD patients, as compared to either controls or alcoholics. In each of the three nationalities, the frequency of the C2 allele was significantly higher in the ALD group than in either of the other two groups. The frequency of the C2 allele across all three nationalities included in our study was approximately 35%, which is in agreement with the frequency reported for other Asian populations. The frequency of the C2 and Val alleles did not differ among ALD patients in the three nationality groups. In contrast, for each nationality, the frequency of C2 and Val was higher in ALD patients than in controls.The average CYPIIE1 mRNA level was higher in ALD patients (10.05%) than in controls (2.21%) (P<0.001). In contrast, GSTP1 mRNA levels were lower in ALD patients (0.53%) than in controls (2.12%) (P<0.001).Discussion: We found that the relative frequencies of the two CYPIIE1 polymorphic alleles in ALD patients were similar in the three nationalities we studied. The relative frequency of the two GSTP1 alleles was also similar in ALD of all three nationalities, although there is little published data to which these can be compared. However, in each nationality, there was a clear and significant difference in the mRNA levels of CYPIIE1 GSTP1 between ALD patients and controls. Although the genetic and pathophysiological factors that lead to the development of ALD have not been definitively established, we believe the findings we have reported here provide a strong indication that polymorphisms in the genes encoding CYPIIE1 and GSTP1 may contribute significantly to these processes. Our findings indicate that individuals who are homozygous for the CYPIIE1 C2 and GSTP1 Val alleles have higher and lower expression, respectively, of the corresponding enzymes, and appear to have an increased incidence of ALD.Besides, through our experiment we found that no matter what stages have the ALD developed, the rule of polymorphisms of CYPIIE1 and GSTP1 and the m-RNA expression of the two enzymes are that individuals who are homozygous for the CYPIIE1 C2 and GSTP1 Val alleles have higher and lower expression, respectively. That is to say, there is no difference about polymorphisms of CYPIIE1,GSTP1 and the m-RNA expression of the two enzymes among hepatic adipose infiltration, alcoholic hepatitis, and alcoholic cirrhosis. The difference about polymorphisms of CYPIIE1,GSTP1 and the m-RNA expression of the two enzymes decides whether it is possible that someone who sufferes from ALD or not, and is nothing with what stages of ALD.The Han, Mongol, and Chaoxian are nationalities which are good at drinking in north-east of China. So we extensively collected the drinkers from the three nationalities to study the relation between the polymorphisms of CYPIIE1,GSTP1 and the m-RNA expression of the two enzymes and ALD in order to discover the pathogeny of ALD, and the rule of morbidity among different races further. Providing the foundation of molecular biology to prophylaxis. Doing some help to treatment about ALD in clinic.