Study of cytochrome P4502E1 mRNA level of mononuclear cells in patients with alcoholic liver disease.
BACKGROUND
Cytochrome P-4502E1 (CYP2E1) is an important enzyme because of its unique ability to convert many substrates to cytotoxins. The increased production of reactive intermediates by elevated enzyme concentrations leads to various pathological conditions. Therefore, it is important to detect induced CYP2E1 levels in alcoholic individuals to avoid xenobiotic-promoted liver injury. In the present investigation, we detected CYP2E1 mRNA levels of mononuclear cells obtained from 10 ml of blood by using competitive polymerase chain reaction (PCR) method.


METHODS
Mononuclear cells were obtained from healthy individuals who did and did not drink habitually and patients with alcoholic liver disease (ALD). Complementary DNA synthesis was performed with RNA obtained from mononuclear cells by reverse transcription-PCR. Competitive PCR of CYP2E1 was performed with the sense (5'-CTGCAACGTCATA-GCCGACA-3') and antisense (5'-TCCATTTCCACGAGCAGGCA-3') primer and competitor DNA. Competitive PCR of beta-actin also was performed. Electrophoresis was scanned, and each band was digitized. The concentration of CYP2E1 and beta-actin mRNA was calculated from the ratio of competitor DNA.


RESULTS
In healthy individuals who did and did not drink habitually, CYP2E1 mRNA levels were 103.3 copies/microl RNA and 101.7 copies/microl RNA, respectively. In actively drinking patients with ALD, CYP2E1 mRNA levels were 103.5 copies/microl RNA, but those levels decreased to 101.7 copies/microl RNA after 4 days of abstinence. No significant difference was observed in CYP2E1 mRNA levels between alcoholic fibrosis and cirrhosis. As control, we measured beta-actin mRNA levels in mononuclear cells in all samples. The mean value of beta-actin mRNA was 104.3 copies/microl RNA in all cases, which included patients with ALD.


CONCLUSIONS
The results demonstrated that it is possible to measure the CYP2E1 mRNA levels of mononuclear cells in a 10 ml blood sample. The CYP2E1 mRNA level in mononuclear cells increases during drinking and decreases in abstinence for a short period of 3 to 4 days. It is concluded that CYP2E1 mRNA level may be used as an effective marker for alcoholic intake.
Yano
H
H
Division of Gastroenterology, Department of Internal Medicine, Kanazawa Medical University, Uchinada, Ishikawa, Japan.
Tsutsumi
M
M
Fukura
M
M
Chen
W B
WB
Shimanaka
K
K
Tsuchishima
M
M
Takase
S
S
Imaoka
S
S
Funae
Y
Y
D000199:Actins / Q000235:genetics; D000328:Adult; D019392:Cytochrome P-450 CYP2E1 / Q000235:genetics*; D004247:DNA / Q000097:blood; D005260:Female; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000201:enzymology*; D008108:Liver Diseases, Alcoholic / Q000201:enzymology*; D008297:Male; D008875:Middle Aged; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000097:blood*; D012016:Reference Values
D000199:Actins; D012333:RNA, Messenger; D004247:DNA; D019392:Cytochrome P-450 CYP2E1