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Identification of Cytochrome P450 1A1 in Human Brain
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
243, 808–810 (1998)
RC988171
Identification of Cytochrome P450 1A1 in Human Brain Chul-Ho Yun,*,1 Hee-Jin Park,* Sung-Jin Kim,† and Ha-Kun Kim† *Department of Biochemistry and †Department of Genetic Engineering, Pai-Chai University, Taejon 302-735, Korea
Received January 7, 1998
We have isolated cDNA fragments that were originated from P450 1A1 in human brain by polymerase chain reaction (PCR) analysis. Their size matched up to what we expected based on the reported P450 1A1 mRNA sequence of human breast carcinoma cell line MCF-7. To confirm proper PCR products, they were subcloned and their nucleotide sequences were determined. This result showed that PCR products were actually derived from P450 1A1 mRNA in human brain. We demonstrated for the first time that human brain contains a P450 1A1-dependent metabolism system which seems to be important for understanding the metabolism of endogenous and exogenous compounds. q 1998 Academic Press
Cytochrome P450 (P450), a superfamily of heme-containing enzymes, plays an important role in the oxidative metabolism of both endogenous and exogenous (xenobiotic) compounds (1). Although P450 occurs mainly in liver, evidence has accumulated for the presence of P450s in extrahepatic tissues including brain (2). Immunohistochemical studies and the detection of drugmetabolizing activities suggest that the brain may also contain several types of P450, although at extremely low levels (3). Several forms of P450 have recently purified from rat brain (4) and partially purified from human brain (5). Despite recent advances in our understanding of the existence of several P450s, little is known of their physiological functions. Brain P450 would likely be involved in the metabolism of neurosteroids (6), and pharmacological modulation of psychoactive drugs (7). It was demonstrated that human P450 1A1 is present in the lung (8) and in the lymphocytes (9). P450 1A1 catalyzes in the conversion of many environmental carcinogens such as benzo(a)pyrene to their ultimate DNA-binding, carcinogenic form (8, 10). A positive cor1
To whom correspondence should be addressed at Department of Biochemistry, Pai-Chai University, 439-6, Doma-dong, Seo-ku, Taejon 302-735, Korea. Fax: (82-42) 533-7354/ 520-5666. E-mail: chyun@ woonam.paichai.ac.kr. 0006-291X/98 $25.00
relation was also observed between P450 1A1 mRNA expression and lung cancer susceptibility (11, 12). In the present study, P450 1A1 was identified in human brain cDNA libraries by PCR. This demonstration will enable functional studies of P450 1A1 in human brain using PCR. MATERIALS AND METHODS Materials. Human brain cDNA libraries were from Clontech (Palo Alto, CA) and Stratagene (La Jolla, CA). DIG DNA labeling and detection kit was purchased from Boehringer Mannheim (Indianapolis, IN). Other chemicals were of the highest grade commercially available. PCR. Four oligonucleotides (P1, P2, P3, and P4) were designed based on the sequence of previously reported human breast P450 1A1 mRNA (13) as described in Table 1. Two pairs (P1-P2 and P3P4) of the primers have been used for the RT-PCR previously (9, 14). Oligonucleotide primers were synthesized and purified by Integrated DNA Technologies, Inc. (Coralville, IA). The PCR mixture contained 100 ng template DNA, 125 mM of each dNTP, 10 pmol of each primer, 1 unit of Taq DNA polymerase, and 0.1 unit of Pfu DNA polymerase in a total 20 ml reaction mixture. Amplification was performed with a DNA thermal cycler (Perkin-Elmer Cetus, Norwalk, CT) with initial denaturation at 957C for 5 min, followed by 30 cycles of 947C for 1 min, 557C for 1 min, and 727C for 1 min, and final extension at 727C for 10 min. PCR products were subjected to electrophoresis on a 1.5% agarose gel. P1-P2 PCR product was transferred onto nylon membrane (Nytran, Schleicher & Schuell, Keene, NH) for Southern blot analysis if necessary. Subcloning and nucleotide sequencing. The PCR products were purified by electroelution, subcloned into pBluescript SK(/) vector (Stratagene, La Jolla, CA), and sequenced using Sequenase ver 2.0 (Amersham Life Science, Arlington Heights, IL). Southern blot analysis. Membrane was prehybridized at 607C for 1 h, then hybridized with DIG-labeled P1-P2 PCR product probe at 657C for 16 h. Procedures for prehybridization, hybridization, DNA labeling, and color detection were carried out according to the manufacturer’s directions.
RESULTS AND DISCUSSION To clarify the expression of P450 1A1 in human brain, we applied PCR method for the screening of human brain cDNA libraries. Template DNA from each human brain cDNA library was prepared from more
808
Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.
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BBRC 8171
/
6949$$$621
02-04-98 23:51:41
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AP: BBRC
Vol. 243, No. 3, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 1
Oligonucleotides Used for Human Brain P450 1A1 cDNA Amplification Primer
Sequence
Localization (number)a
Reference
P1 P2 P3 P4
TCA CAG ACA GCC TGA TTG AG GAT GGG TTG ACC CAT AGC TT TAG ACA CTG ATC TGG CTG CAG GGG AAG GCT CCA TCA GCA TC
Sense, 928-947 Antisense, 1341-1360 Sense, 1727-1747 Antisense, 1853-1872
14 14 9 9
a Numbers represent the relative positions of primers when compared to the nucleotide sequence of human breast P450 1A1 mRNA (13). Oligonucleotide sequences are listed in the 5* to 3* direction.
than 108 independent phages. Combinations of sense and antisense primers, which were P1-P2 and P3-P4, were used for amplification and results are summarized in Fig. 1A. DNA bands were amplified and they were 433 bp and 146 bp in size as expected based on the reported human breast P450 1A1 mRNA (13). PCR products (Fig. 1A, lanes 1, 2, 4, and 5) were then subcloned into pBluescript SK(/) vector and their nucleotide sequences were determined using dideoxynucleotide method (15). The nucleotide sequences of cDNA P1-P2 and cDNA P3-P4 are shown in Fig. 2. The nucleotide sequences of P1-P2 and P3-P4 PCR products from human brain hippocampus and whole cerebral brain cDNA libraries were exactly same, indicating that nucleotide substitutions did not occur during PCR amplification. Each nucleotide sequence of P1-P2 and P3-P4 DNA fragments of PCR were 99.7% and 98.1%, respectively, identical to the cDNA sequence obtained from a human breast cell line cDNA library (GenBank accession number K03191) (13). The predicted amino acid sequence of the DNA fragment (P1-P2) was exactly identical with that of a previous report (13). The nucleotide difference of G in place of A does not change the
FIG. 1. Electrophoresis (A) and Southern blot analysis (B) of PCR products. Templates were phage DNA from human brain hippocampus Lambda Zap II cDNA library (Stratagene) (lanes 1 and 4), human whole cerebral brain cDNA library (Clontech) (lanes 2 and 5), and human brain substantia nigra cDNA library (Clontech) (lanes 3 and 6). PCR products were run on 1.5% agarose gels and stained by ethidium bromide. Specific primers (Table 1) were used to amplify P450 1A1 cDNA fragments. Primer pairs were P1-P2 and P3-P4 for lanes 1-3 and 4-6, respectively. The 1 kb ladder (Gibco BRL, Bethesda, MD) was used as a size marker lane (M) and was also used as a negative control for Southern blot analysis.
amino acid, arginine. The nucleotide sequence of P3-P4 was from noncoding sequence region after stop codon. Southern blot analysis was also performed to confirm the expression of P450 1A1 in several human brain cDNA libraries (Fig. 1B). Signals were strongly detected from all PCR (P1-P2) bands except for the size marker. These experimental evidences clearly show that amplified cDNA fragments correspond to P450 1A1 in human brain. These results demonstrate the expression of P450 1A1 in human brain and suggest the important role of P450 1A1 in the metabolism of endogenous and exogenous compounds. It is hoped that this report facilitates further studies on the functions of P450 1A1 in brain cells.
FIG. 2. Nucleotide sequences of cDNA P1-P2 (GenBank Accession No. AF040258) (A) and cDNA P3-P4 (GenBank Accession No. AF040259) (B). Only the nucleotides different from human breast P450 1A1 mRNA are shown as bold types. The primers used for PCR are represented as arrows.
809
AID
BBRC 8171
/
6949$$$621
02-04-98 23:51:41
bbrcg
AP: BBRC
Vol. 243, No. 3, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ACKNOWLEDGMENTS 6.
We thank Drs. Min Yoo (Keimyung University) and Kwan-Hee You (Chungnam National University) for providing human brain cDNA libraries and Mr. Sang-Chul Nam (Korea Advanced Institute of Science and Technology) for technical assistance. This work was supported by Basic Science Research Institute Program Grant 974431 (Ministry of Education, Korea) (to C.-H.Y.).
7. 8.
9.
REFERENCES 10. 1. Nelson, D. R., Koymans, L., Kamataki, T., Stegeman, J. J., Feyereisen, R., Waxman, D. J., Waterman, M. R., Gotoh, O., Coon, M. J., Estabrook, R. W., Gunsalus, I. C., and Nebert, D. W. (1996) Pharmacogenetics 6, 1–42.
11. 12.
2. Warner, M., Hellmold, H., Yoshida, S., Liao, D., Heldlund, E., and Gustafsson, J. A. (1997) Mutat. Res. 376, 79–85.
13.
3. Anandatheerthavarada, H. K., Shankar, S. K., and Ravindranath, V. (1990) Brain Res. 536, 339–343.
14.
4. Warner, M., and Gustafsson, J. (1994) Proc. Natl. Acad. Sci. USA 91, 1019–1023.
15.
5. Bhamre, S., Anandatheerthavarada, H. K., Shankar, S. K.,
Boyd, M. R., and Ravindranath, V. (1993) Arch. Biochem. Biophys. 301, 251–255. Stromstedt, M., Marner, M., Manner, C. D., Macdonald, P. C., and Gustafsson, J. A. (1993) Mol. Pharmacol. 44, 1077–1083. Sequeira, D. J., and Strobel, H. W. (1996) Brain Res. 738, 24– 31. Shimada, T., Yun, C.-H., Yamazaki, H., Gautier, J. C., Beaune, P. H., and Guengerich, F. P. (1992) Mol. Pharmacol. 41, 856– 864. Heuvel, J. P. V., Clark, G. C., Thompson, C. L., McCoy, Z., Miller, C. R., Lucier, G. W., and Bell, D. A. (1993) Carcinogenesis 14, 2003–2006. Kawajiri, K., Nakachi, K., Imai, K., Yoshii, A., Shinoda, N., and Watanabe, J. (1990) FEBS Lett. 263, 131–133. Omiecinski, C. J., Redlich, C. A., and Costa, P. (1990) Cancer Res. 50, 4315–4321. Hayashi, S., Watanabe, J., Nakachi, K., Eguchi, H., Gotoh, O., and Kawajiri, K. (1994) Carcinogenesis 15, 801–806. Jaiswal, A. K., Gonzalez, F. J., and Nebert, D. W. (1985) Science 228, 80–83. Hakkola, J., Pasanen, M., Hukkanen, J., Pelkonen, O., Kaenpaa, J., Edwards, R. J., Boobis, A. R., and Raunio, H. (1996) Biochem. Pharmacol. 51, 403–411. Sanger, F., Niklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. USA 74, 5467–5467.
810
AID
BBRC 8171
/
6949$$$621
02-04-98 23:51:41
bbrcg
AP: BBRC
243, 808–810 (1998)
RC988171
Identification of Cytochrome P450 1A1 in Human Brain Chul-Ho Yun,*,1 Hee-Jin Park,* Sung-Jin Kim,† and Ha-Kun Kim† *Department of Biochemistry and †Department of Genetic Engineering, Pai-Chai University, Taejon 302-735, Korea
Received January 7, 1998
We have isolated cDNA fragments that were originated from P450 1A1 in human brain by polymerase chain reaction (PCR) analysis. Their size matched up to what we expected based on the reported P450 1A1 mRNA sequence of human breast carcinoma cell line MCF-7. To confirm proper PCR products, they were subcloned and their nucleotide sequences were determined. This result showed that PCR products were actually derived from P450 1A1 mRNA in human brain. We demonstrated for the first time that human brain contains a P450 1A1-dependent metabolism system which seems to be important for understanding the metabolism of endogenous and exogenous compounds. q 1998 Academic Press
Cytochrome P450 (P450), a superfamily of heme-containing enzymes, plays an important role in the oxidative metabolism of both endogenous and exogenous (xenobiotic) compounds (1). Although P450 occurs mainly in liver, evidence has accumulated for the presence of P450s in extrahepatic tissues including brain (2). Immunohistochemical studies and the detection of drugmetabolizing activities suggest that the brain may also contain several types of P450, although at extremely low levels (3). Several forms of P450 have recently purified from rat brain (4) and partially purified from human brain (5). Despite recent advances in our understanding of the existence of several P450s, little is known of their physiological functions. Brain P450 would likely be involved in the metabolism of neurosteroids (6), and pharmacological modulation of psychoactive drugs (7). It was demonstrated that human P450 1A1 is present in the lung (8) and in the lymphocytes (9). P450 1A1 catalyzes in the conversion of many environmental carcinogens such as benzo(a)pyrene to their ultimate DNA-binding, carcinogenic form (8, 10). A positive cor1
To whom correspondence should be addressed at Department of Biochemistry, Pai-Chai University, 439-6, Doma-dong, Seo-ku, Taejon 302-735, Korea. Fax: (82-42) 533-7354/ 520-5666. E-mail: chyun@ woonam.paichai.ac.kr. 0006-291X/98 $25.00
relation was also observed between P450 1A1 mRNA expression and lung cancer susceptibility (11, 12). In the present study, P450 1A1 was identified in human brain cDNA libraries by PCR. This demonstration will enable functional studies of P450 1A1 in human brain using PCR. MATERIALS AND METHODS Materials. Human brain cDNA libraries were from Clontech (Palo Alto, CA) and Stratagene (La Jolla, CA). DIG DNA labeling and detection kit was purchased from Boehringer Mannheim (Indianapolis, IN). Other chemicals were of the highest grade commercially available. PCR. Four oligonucleotides (P1, P2, P3, and P4) were designed based on the sequence of previously reported human breast P450 1A1 mRNA (13) as described in Table 1. Two pairs (P1-P2 and P3P4) of the primers have been used for the RT-PCR previously (9, 14). Oligonucleotide primers were synthesized and purified by Integrated DNA Technologies, Inc. (Coralville, IA). The PCR mixture contained 100 ng template DNA, 125 mM of each dNTP, 10 pmol of each primer, 1 unit of Taq DNA polymerase, and 0.1 unit of Pfu DNA polymerase in a total 20 ml reaction mixture. Amplification was performed with a DNA thermal cycler (Perkin-Elmer Cetus, Norwalk, CT) with initial denaturation at 957C for 5 min, followed by 30 cycles of 947C for 1 min, 557C for 1 min, and 727C for 1 min, and final extension at 727C for 10 min. PCR products were subjected to electrophoresis on a 1.5% agarose gel. P1-P2 PCR product was transferred onto nylon membrane (Nytran, Schleicher & Schuell, Keene, NH) for Southern blot analysis if necessary. Subcloning and nucleotide sequencing. The PCR products were purified by electroelution, subcloned into pBluescript SK(/) vector (Stratagene, La Jolla, CA), and sequenced using Sequenase ver 2.0 (Amersham Life Science, Arlington Heights, IL). Southern blot analysis. Membrane was prehybridized at 607C for 1 h, then hybridized with DIG-labeled P1-P2 PCR product probe at 657C for 16 h. Procedures for prehybridization, hybridization, DNA labeling, and color detection were carried out according to the manufacturer’s directions.
RESULTS AND DISCUSSION To clarify the expression of P450 1A1 in human brain, we applied PCR method for the screening of human brain cDNA libraries. Template DNA from each human brain cDNA library was prepared from more
808
Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.
AID
BBRC 8171
/
6949$$$621
02-04-98 23:51:41
bbrcg
AP: BBRC
Vol. 243, No. 3, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 1
Oligonucleotides Used for Human Brain P450 1A1 cDNA Amplification Primer
Sequence
Localization (number)a
Reference
P1 P2 P3 P4
TCA CAG ACA GCC TGA TTG AG GAT GGG TTG ACC CAT AGC TT TAG ACA CTG ATC TGG CTG CAG GGG AAG GCT CCA TCA GCA TC
Sense, 928-947 Antisense, 1341-1360 Sense, 1727-1747 Antisense, 1853-1872
14 14 9 9
a Numbers represent the relative positions of primers when compared to the nucleotide sequence of human breast P450 1A1 mRNA (13). Oligonucleotide sequences are listed in the 5* to 3* direction.
than 108 independent phages. Combinations of sense and antisense primers, which were P1-P2 and P3-P4, were used for amplification and results are summarized in Fig. 1A. DNA bands were amplified and they were 433 bp and 146 bp in size as expected based on the reported human breast P450 1A1 mRNA (13). PCR products (Fig. 1A, lanes 1, 2, 4, and 5) were then subcloned into pBluescript SK(/) vector and their nucleotide sequences were determined using dideoxynucleotide method (15). The nucleotide sequences of cDNA P1-P2 and cDNA P3-P4 are shown in Fig. 2. The nucleotide sequences of P1-P2 and P3-P4 PCR products from human brain hippocampus and whole cerebral brain cDNA libraries were exactly same, indicating that nucleotide substitutions did not occur during PCR amplification. Each nucleotide sequence of P1-P2 and P3-P4 DNA fragments of PCR were 99.7% and 98.1%, respectively, identical to the cDNA sequence obtained from a human breast cell line cDNA library (GenBank accession number K03191) (13). The predicted amino acid sequence of the DNA fragment (P1-P2) was exactly identical with that of a previous report (13). The nucleotide difference of G in place of A does not change the
FIG. 1. Electrophoresis (A) and Southern blot analysis (B) of PCR products. Templates were phage DNA from human brain hippocampus Lambda Zap II cDNA library (Stratagene) (lanes 1 and 4), human whole cerebral brain cDNA library (Clontech) (lanes 2 and 5), and human brain substantia nigra cDNA library (Clontech) (lanes 3 and 6). PCR products were run on 1.5% agarose gels and stained by ethidium bromide. Specific primers (Table 1) were used to amplify P450 1A1 cDNA fragments. Primer pairs were P1-P2 and P3-P4 for lanes 1-3 and 4-6, respectively. The 1 kb ladder (Gibco BRL, Bethesda, MD) was used as a size marker lane (M) and was also used as a negative control for Southern blot analysis.
amino acid, arginine. The nucleotide sequence of P3-P4 was from noncoding sequence region after stop codon. Southern blot analysis was also performed to confirm the expression of P450 1A1 in several human brain cDNA libraries (Fig. 1B). Signals were strongly detected from all PCR (P1-P2) bands except for the size marker. These experimental evidences clearly show that amplified cDNA fragments correspond to P450 1A1 in human brain. These results demonstrate the expression of P450 1A1 in human brain and suggest the important role of P450 1A1 in the metabolism of endogenous and exogenous compounds. It is hoped that this report facilitates further studies on the functions of P450 1A1 in brain cells.
FIG. 2. Nucleotide sequences of cDNA P1-P2 (GenBank Accession No. AF040258) (A) and cDNA P3-P4 (GenBank Accession No. AF040259) (B). Only the nucleotides different from human breast P450 1A1 mRNA are shown as bold types. The primers used for PCR are represented as arrows.
809
AID
BBRC 8171
/
6949$$$621
02-04-98 23:51:41
bbrcg
AP: BBRC
Vol. 243, No. 3, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ACKNOWLEDGMENTS 6.
We thank Drs. Min Yoo (Keimyung University) and Kwan-Hee You (Chungnam National University) for providing human brain cDNA libraries and Mr. Sang-Chul Nam (Korea Advanced Institute of Science and Technology) for technical assistance. This work was supported by Basic Science Research Institute Program Grant 974431 (Ministry of Education, Korea) (to C.-H.Y.).
7. 8.
9.
REFERENCES 10. 1. Nelson, D. R., Koymans, L., Kamataki, T., Stegeman, J. J., Feyereisen, R., Waxman, D. J., Waterman, M. R., Gotoh, O., Coon, M. J., Estabrook, R. W., Gunsalus, I. C., and Nebert, D. W. (1996) Pharmacogenetics 6, 1–42.
11. 12.
2. Warner, M., Hellmold, H., Yoshida, S., Liao, D., Heldlund, E., and Gustafsson, J. A. (1997) Mutat. Res. 376, 79–85.
13.
3. Anandatheerthavarada, H. K., Shankar, S. K., and Ravindranath, V. (1990) Brain Res. 536, 339–343.
14.
4. Warner, M., and Gustafsson, J. (1994) Proc. Natl. Acad. Sci. USA 91, 1019–1023.
15.
5. Bhamre, S., Anandatheerthavarada, H. K., Shankar, S. K.,
Boyd, M. R., and Ravindranath, V. (1993) Arch. Biochem. Biophys. 301, 251–255. Stromstedt, M., Marner, M., Manner, C. D., Macdonald, P. C., and Gustafsson, J. A. (1993) Mol. Pharmacol. 44, 1077–1083. Sequeira, D. J., and Strobel, H. W. (1996) Brain Res. 738, 24– 31. Shimada, T., Yun, C.-H., Yamazaki, H., Gautier, J. C., Beaune, P. H., and Guengerich, F. P. (1992) Mol. Pharmacol. 41, 856– 864. Heuvel, J. P. V., Clark, G. C., Thompson, C. L., McCoy, Z., Miller, C. R., Lucier, G. W., and Bell, D. A. (1993) Carcinogenesis 14, 2003–2006. Kawajiri, K., Nakachi, K., Imai, K., Yoshii, A., Shinoda, N., and Watanabe, J. (1990) FEBS Lett. 263, 131–133. Omiecinski, C. J., Redlich, C. A., and Costa, P. (1990) Cancer Res. 50, 4315–4321. Hayashi, S., Watanabe, J., Nakachi, K., Eguchi, H., Gotoh, O., and Kawajiri, K. (1994) Carcinogenesis 15, 801–806. Jaiswal, A. K., Gonzalez, F. J., and Nebert, D. W. (1985) Science 228, 80–83. Hakkola, J., Pasanen, M., Hukkanen, J., Pelkonen, O., Kaenpaa, J., Edwards, R. J., Boobis, A. R., and Raunio, H. (1996) Biochem. Pharmacol. 51, 403–411. Sanger, F., Niklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. USA 74, 5467–5467.
810
AID
BBRC 8171
/
6949$$$621
02-04-98 23:51:41
bbrcg
AP: BBRC