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Hepatic messenger RNA contents of cytochrome P4502E1 in patients with different P4502E1 genotypes
Intemutiorud
Hepatology communications ELSEVIER
Int Hepatol Commun, 2 (1994) 135-138
Hepatic messenger RNA contents of cytochrome P4502E 1 in patients with different P4502El genotypes Mikihiro
Tsutsumi,
Jian-Song
Wang, Akira Takada’
Division of Gastroenterology, Department of Internal Medicine, Kanazawa Medical University, Vchinada. Ishikawa 92042, Japan
(Received 12 May 1993;accepted 12 November 1993)
Abstract Since heavy drinkers do not always develop alcoholic liver disease (ALD), genetic factors may be involved. Cytochrome P4502El (P4502El) is the main enzyme that oxidizes ethanol in the non-alcohol dehydrogenase pathway. Recently, the presence of genetic polymorphisms at the 5’-flanking region of this enzyme was confirmed. We recently detected the c2 gene of P4502El in all patients with ALD, suggesting that development of ALD may be genetically controlled. High transcriptional activity of the c2 gene was contirrned in studies using the Hep G2 cell by Hayashi et al. (J Biochem 1991;110:559-565), suggesting that polymorphisms of P4502El may be linked to the development of ALD through enhancement of ethanol metabolism in the non-alcohol dehydrogenase pathway. However, little is known about transcriptional activity in human beings with the c2 gene. Therefore, messenger RNA (mRNA) contents of P4502El in hepatic biopsy specimens from patients with different P4502El genotypes were measured. Type A of P4502El was not found in any patients with ALD, suggesting that development of ALD is controlled genetically and that the genotype of P4502El is one of the most important determining factors for its development. Hepatic mRNA contents in type B were 3-times higher than in type A, suggesting that transcriptional activity of the c2 gene of P4502El is stronger than that of the cl gene even in human liver. The results of the present study are quite compatible with those by Hayashi et al. using the Hep G2 cell. These results suggest that polymorphisms of the P4502El gene may be linked to the development of ALD through enhancement of ethanol metabolism in the non-alcohol dehydrogenase pathway. Key words: Alcoholic liver disease; Cytochrome scriptional activity; Genetic control of ALD
P4502El;
P4502El gene; Genotype;
Tran-
1. Introduction
Since heavy drinkers do not always develop alcoholic liver disease (ALD), genetic factors may involved in its development. Cytochrome P4502El (P4502El) is the main *Corresponding
author.
0928-4346/94/$07.00 0 1994 Elsevier Science B.V. All rights reserved SSDZ 0928-4346(93)E0065-X
136
M. Tsutsumi et al. I Int Hepatol Commun, 2 (1994) 135-138
enzyme that oxidizes ethanol in the non-alcohol dehydrogenase pathway [l]. Recently, the presence of genetic polymorphisms at the 5’-flanking region of this enzyme was confirmed by Hayashi et al. [2]. We recently detected the c2 gene of P4502El in all patients with ALD [3]. These results suggest that development of ALD may be genetically controlled and that the c2 gene may be one of the most important determining factors. High transcriptional activity of the c2 gene was confirmed in studies with the chloramphenicol acetyltransferase (CAT) assay using the human hepatoma cell line Hep G2, which expresses the albumin gene, by Hayashi et al. [2]. These results strongly suggest the possibility that polymorphisms of P4502El may be linked to the development of ALD through enhancement of ethanol metabolism in the non-alcohol dehydrogenase pathway. However, evidences that transcriptional activity is high in human beings possessing the c2 gene, are needed to be ascertained. In the present study, messenger RNA (mRNA) contents of P4502El in hepatic biopsy specimens from patients with different P4502El genotypes were measured.
2. Materials and methods Genotypes of P4502El were determined according to the restriction fragments length polymorphisms technique described by Hayashi et al. [2]. DNA was extracted from the buffy coat of patients’ blood and DNA fragments of the P4502El gene were amplified by polymerase chain reactions (PCR). The DNA fragments amplified by PCR were treated with two types of restriction endonucleases, RraI and PstI. The cl gene was digested with &zI, but not with PstI. The c2 gene was digested with PstI, but not with J&I. Consequently, the genotypes of P4502El were separated into three types: type A, which is homozygous for the cl genes; type B, which is heterozygous for the cl and c2 genes; and type C, which is homozygous for the c2 genes. Total RNA was extracted from liver biopsy specimens according to the description of Chomczynski and Sacchi [4] and the RNA content was measured at the absorbance of 260 run. P4502El mRNA was amplified by the reverse transcription-PCR (RTPCR) technique, using the sense and antisense primers specific for P4502El cDNA [5]. The 800 base pair PCR products were labeled with digoxigenin-deoxyuridine triphosphate by PCR [6], for use in the hybridization cDNA probe. Slot-blot hybridization was performed using the PCR products and the digoxigenin-labeled cDNA probe as reported previously with some modifications [7]. Immunostaining of the hybridized cDNA probe was performed using alkaline phosphatase-conjugate antidigoxigenin IgG following the manufacturer’s instructions (Boehringer Mannheim). The staining intensity of the hybrids was determined by a scanning densitometer (Shimazu CS-900 dual wave length TLC Scanner). The content of mRNA of P4502El was expressed as the intensity of staining reactions (densitometer unit) per pg of total RNA. Student’s t test or chi-square test was used for statistical analysis.
M. Tsutsumi et al. I Int Hepatol Commun, 2 (1994) 135-138
137
uni t I5pgRNA
genotype
A B Fig. 1. Slot-blot analysis of hepatic mRNA of P4502El. The staining reactions were more intense and the densitometer counts were greater in type B than in type A.
3. Results
The prevalence of the P4502El genotypes in ALD and in healthy controls is shown in Table 1, In 3 1 patients with ALD (one case of minimal change, 15 cases of fibrosis, two cases of alcoholic hepatitis, two cases of chronic hepatitis and 11 cases of cirrhosis), the genotypes of P4502El were type B in 29 (93.5%) and type C in two (6.5%), and type A was not found. On the other hand, type A was found in 63.3% of healthy controls. The difference in prevalence of the P4502El genotypes between ALD and healthy controls was significant statistically. Slot-blot hybridizations of hepatic P4502El mRNA are shown in Fig. 1, The staining reactions were more intense in type B than in type A. Consequently, the densitometer counts were larger in type B than in type A. P4502El mRNA contents in livers with different genotypes are shown in Table 2. In four livers with type A, the mRNA content was 2.8 + 0.35 unitslpg RNA and was 8.9 + 0.22 unitslpg RNA in four livers with type B. The difference between type A and type B was significant statistically (P < 0.005)
Table 1 Prevalence of the genotypes of cytochrome P4502El. Groups
Cases
ALD
31
Healthy control
30
*P < 0.01 vs. healthy control.
_ Genotypes of P4502El A (cl/cl)
B (clk2)
c (c2k2)
0* (0%) 19 (63.3%)
29 (93.5%) 11 (36.7%)
2* (6.5%) 0 (0%)
_
138
M. Tsutsumi et al. IInt Hepatol Commun, 2 (1994) 135-138
Table 2 Hepatic mRNA contents in different P4502El genotypes. Genotypes
Number
mRNA (unit&g RNA)
A B
4 4
2.8* f 0.35 8.9 -+ 0.22
*P < 0.005 vs. type B.
4. Discussion
In the present study, type A was not found in any patients with ALD. The prevalence of the c2 gene was significantly higher in patients with ALD than in healthy controls. These results suggest that the development of ALD may be controlled genetically and that the genotype of P4502El may be one of the most important determining factors for its development. Hepatic mRNA contents in type B were 3-times higher than in type A. These results suggest that transcriptional activity of the c2 gene of P4502El is stronger than that of the cl gene even in human liver. Hayashi et al. [2] reported that the transcriptional activity of the c2 gene was IO-times higher than that of the cl gene in studies with the CAT assay using the Hep G2 cell. The results of the present study are quite compatible with those with the Hep G2 cell by Hayashi et al. [2], suggesting that polymorphisms of the P4502El gene may be linked to the development of ALD through enhancement of ethanol metabolism in the non-alcohol dehydrogenase pathway. However, the number of livers examined is too small to obtain final conclusions. Since P4502El is induced by several conditions, such as alcohol drinking, starvation, diabetes, etc. [ 11,the backgrounds of patients examined should be carefully analyzed.
References
VI Koop DR, Coon MJ. Ethanol oxidation and toxicity: Roles of alcohol-P450 oxygenase. Alcoholism: Clin Exp Res 1986;10:44s49s.
121Hayashi S, Watanabe J, Kawajiri K. Genetic polymorphisms in the 5’-flanking region change transcriptional regulation of the human cytochrome P450IIEl. J Biochem 1991;110:559-565. of alcoholic liver disease. Acta Hepatol Jpn 1992;33:28 (Abstr., in Japanese). 141Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanatephenol-chloroform extraction. Anal Biochem 1987;162:156159. [51Umeno M, Mcbide OW, Yang CS, Gelboin HV, Gonzalez FJ. Human ethanol-inducible P450IIEl: Complete gene sequence, promoter characterization, chromosome mapping, and cDNA-directed expression. Biochemistry 1988;27:9006-9013. WI Lo YM, Mehal WZ, Fleming KA. Rapid production of vector-free biotinylate probes using the polymerase chain reaction. Nucleic Acids Res 1988;16:8719. [71Takada N, Takase S, Enomoto N, Takada A, Date T. Clinical backgrounds of the patients having different types of hepatitis C virus genomes. J Hepatology 1992;14:35-40. [31Takada A. Diagnosis and pathophysiology
Hepatology communications ELSEVIER
Int Hepatol Commun, 2 (1994) 135-138
Hepatic messenger RNA contents of cytochrome P4502E 1 in patients with different P4502El genotypes Mikihiro
Tsutsumi,
Jian-Song
Wang, Akira Takada’
Division of Gastroenterology, Department of Internal Medicine, Kanazawa Medical University, Vchinada. Ishikawa 92042, Japan
(Received 12 May 1993;accepted 12 November 1993)
Abstract Since heavy drinkers do not always develop alcoholic liver disease (ALD), genetic factors may be involved. Cytochrome P4502El (P4502El) is the main enzyme that oxidizes ethanol in the non-alcohol dehydrogenase pathway. Recently, the presence of genetic polymorphisms at the 5’-flanking region of this enzyme was confirmed. We recently detected the c2 gene of P4502El in all patients with ALD, suggesting that development of ALD may be genetically controlled. High transcriptional activity of the c2 gene was contirrned in studies using the Hep G2 cell by Hayashi et al. (J Biochem 1991;110:559-565), suggesting that polymorphisms of P4502El may be linked to the development of ALD through enhancement of ethanol metabolism in the non-alcohol dehydrogenase pathway. However, little is known about transcriptional activity in human beings with the c2 gene. Therefore, messenger RNA (mRNA) contents of P4502El in hepatic biopsy specimens from patients with different P4502El genotypes were measured. Type A of P4502El was not found in any patients with ALD, suggesting that development of ALD is controlled genetically and that the genotype of P4502El is one of the most important determining factors for its development. Hepatic mRNA contents in type B were 3-times higher than in type A, suggesting that transcriptional activity of the c2 gene of P4502El is stronger than that of the cl gene even in human liver. The results of the present study are quite compatible with those by Hayashi et al. using the Hep G2 cell. These results suggest that polymorphisms of the P4502El gene may be linked to the development of ALD through enhancement of ethanol metabolism in the non-alcohol dehydrogenase pathway. Key words: Alcoholic liver disease; Cytochrome scriptional activity; Genetic control of ALD
P4502El;
P4502El gene; Genotype;
Tran-
1. Introduction
Since heavy drinkers do not always develop alcoholic liver disease (ALD), genetic factors may involved in its development. Cytochrome P4502El (P4502El) is the main *Corresponding
author.
0928-4346/94/$07.00 0 1994 Elsevier Science B.V. All rights reserved SSDZ 0928-4346(93)E0065-X
136
M. Tsutsumi et al. I Int Hepatol Commun, 2 (1994) 135-138
enzyme that oxidizes ethanol in the non-alcohol dehydrogenase pathway [l]. Recently, the presence of genetic polymorphisms at the 5’-flanking region of this enzyme was confirmed by Hayashi et al. [2]. We recently detected the c2 gene of P4502El in all patients with ALD [3]. These results suggest that development of ALD may be genetically controlled and that the c2 gene may be one of the most important determining factors. High transcriptional activity of the c2 gene was confirmed in studies with the chloramphenicol acetyltransferase (CAT) assay using the human hepatoma cell line Hep G2, which expresses the albumin gene, by Hayashi et al. [2]. These results strongly suggest the possibility that polymorphisms of P4502El may be linked to the development of ALD through enhancement of ethanol metabolism in the non-alcohol dehydrogenase pathway. However, evidences that transcriptional activity is high in human beings possessing the c2 gene, are needed to be ascertained. In the present study, messenger RNA (mRNA) contents of P4502El in hepatic biopsy specimens from patients with different P4502El genotypes were measured.
2. Materials and methods Genotypes of P4502El were determined according to the restriction fragments length polymorphisms technique described by Hayashi et al. [2]. DNA was extracted from the buffy coat of patients’ blood and DNA fragments of the P4502El gene were amplified by polymerase chain reactions (PCR). The DNA fragments amplified by PCR were treated with two types of restriction endonucleases, RraI and PstI. The cl gene was digested with &zI, but not with PstI. The c2 gene was digested with PstI, but not with J&I. Consequently, the genotypes of P4502El were separated into three types: type A, which is homozygous for the cl genes; type B, which is heterozygous for the cl and c2 genes; and type C, which is homozygous for the c2 genes. Total RNA was extracted from liver biopsy specimens according to the description of Chomczynski and Sacchi [4] and the RNA content was measured at the absorbance of 260 run. P4502El mRNA was amplified by the reverse transcription-PCR (RTPCR) technique, using the sense and antisense primers specific for P4502El cDNA [5]. The 800 base pair PCR products were labeled with digoxigenin-deoxyuridine triphosphate by PCR [6], for use in the hybridization cDNA probe. Slot-blot hybridization was performed using the PCR products and the digoxigenin-labeled cDNA probe as reported previously with some modifications [7]. Immunostaining of the hybridized cDNA probe was performed using alkaline phosphatase-conjugate antidigoxigenin IgG following the manufacturer’s instructions (Boehringer Mannheim). The staining intensity of the hybrids was determined by a scanning densitometer (Shimazu CS-900 dual wave length TLC Scanner). The content of mRNA of P4502El was expressed as the intensity of staining reactions (densitometer unit) per pg of total RNA. Student’s t test or chi-square test was used for statistical analysis.
M. Tsutsumi et al. I Int Hepatol Commun, 2 (1994) 135-138
137
uni t I5pgRNA
genotype
A B Fig. 1. Slot-blot analysis of hepatic mRNA of P4502El. The staining reactions were more intense and the densitometer counts were greater in type B than in type A.
3. Results
The prevalence of the P4502El genotypes in ALD and in healthy controls is shown in Table 1, In 3 1 patients with ALD (one case of minimal change, 15 cases of fibrosis, two cases of alcoholic hepatitis, two cases of chronic hepatitis and 11 cases of cirrhosis), the genotypes of P4502El were type B in 29 (93.5%) and type C in two (6.5%), and type A was not found. On the other hand, type A was found in 63.3% of healthy controls. The difference in prevalence of the P4502El genotypes between ALD and healthy controls was significant statistically. Slot-blot hybridizations of hepatic P4502El mRNA are shown in Fig. 1, The staining reactions were more intense in type B than in type A. Consequently, the densitometer counts were larger in type B than in type A. P4502El mRNA contents in livers with different genotypes are shown in Table 2. In four livers with type A, the mRNA content was 2.8 + 0.35 unitslpg RNA and was 8.9 + 0.22 unitslpg RNA in four livers with type B. The difference between type A and type B was significant statistically (P < 0.005)
Table 1 Prevalence of the genotypes of cytochrome P4502El. Groups
Cases
ALD
31
Healthy control
30
*P < 0.01 vs. healthy control.
_ Genotypes of P4502El A (cl/cl)
B (clk2)
c (c2k2)
0* (0%) 19 (63.3%)
29 (93.5%) 11 (36.7%)
2* (6.5%) 0 (0%)
_
138
M. Tsutsumi et al. IInt Hepatol Commun, 2 (1994) 135-138
Table 2 Hepatic mRNA contents in different P4502El genotypes. Genotypes
Number
mRNA (unit&g RNA)
A B
4 4
2.8* f 0.35 8.9 -+ 0.22
*P < 0.005 vs. type B.
4. Discussion
In the present study, type A was not found in any patients with ALD. The prevalence of the c2 gene was significantly higher in patients with ALD than in healthy controls. These results suggest that the development of ALD may be controlled genetically and that the genotype of P4502El may be one of the most important determining factors for its development. Hepatic mRNA contents in type B were 3-times higher than in type A. These results suggest that transcriptional activity of the c2 gene of P4502El is stronger than that of the cl gene even in human liver. Hayashi et al. [2] reported that the transcriptional activity of the c2 gene was IO-times higher than that of the cl gene in studies with the CAT assay using the Hep G2 cell. The results of the present study are quite compatible with those with the Hep G2 cell by Hayashi et al. [2], suggesting that polymorphisms of the P4502El gene may be linked to the development of ALD through enhancement of ethanol metabolism in the non-alcohol dehydrogenase pathway. However, the number of livers examined is too small to obtain final conclusions. Since P4502El is induced by several conditions, such as alcohol drinking, starvation, diabetes, etc. [ 11,the backgrounds of patients examined should be carefully analyzed.
References
VI Koop DR, Coon MJ. Ethanol oxidation and toxicity: Roles of alcohol-P450 oxygenase. Alcoholism: Clin Exp Res 1986;10:44s49s.
121Hayashi S, Watanabe J, Kawajiri K. Genetic polymorphisms in the 5’-flanking region change transcriptional regulation of the human cytochrome P450IIEl. J Biochem 1991;110:559-565. of alcoholic liver disease. Acta Hepatol Jpn 1992;33:28 (Abstr., in Japanese). 141Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanatephenol-chloroform extraction. Anal Biochem 1987;162:156159. [51Umeno M, Mcbide OW, Yang CS, Gelboin HV, Gonzalez FJ. Human ethanol-inducible P450IIEl: Complete gene sequence, promoter characterization, chromosome mapping, and cDNA-directed expression. Biochemistry 1988;27:9006-9013. WI Lo YM, Mehal WZ, Fleming KA. Rapid production of vector-free biotinylate probes using the polymerase chain reaction. Nucleic Acids Res 1988;16:8719. [71Takada N, Takase S, Enomoto N, Takada A, Date T. Clinical backgrounds of the patients having different types of hepatitis C virus genomes. J Hepatology 1992;14:35-40. [31Takada A. Diagnosis and pathophysiology