Tohoku
J. Exp.
Analysis Activities
Med.,
1992, 168, 67-72
of Human and
Cytochrome
P450
Catalytic
Expression
FRANKJ. GONZALEZ,CHARLESL. CRESPI*,MACEIJ CZERWINSKIand HARRYV. GELBOIN Laboratory of Molecular Carcinogenesis, National Cancer Institute, National Institutes of Health, * Gentest Corporation
GONZALEZ, F.J., CRESPI,C.L., CZERWINSKI, M. and GELBOIN, H.V. Analysis of Human CytochromeP450 Catalytic Activities and Expression. Tohoku J. Exp. Med., 1992, 168 (2), 67-72 Cytochromes P450 are a large group of membraneassociated heme protein monooxygenases, most of which are responsible for metabolizing foreign compounds. Chemical carcinogens, which are ingested or absorbed into the body as inert forms, are metabolically activated by P450s to electrophilic metabolites capable of binding to and mutating DNA. Different P450 forms are responsible for activation of the various classes of chemical carcinogens including the arylamines, polycyclic aromatic hydrocarbons, nitrosamines and afiatoxins. Thus, the cellular constituency and levels of P450s could determine the fate of a particular carcinogen and the risk of humans to exposure. To study the catalytic activities of human P450s, human P450 cDNAs were cloned and expressed into active enzymes using cultured cells. By both transient and stable cDNA expression systems, several human P450s were found to be capable of metabolically-activating the human hepatocarcinogen afiatoxin B1. These cDNA expression systems can also be used to determine whether an unknown chemical will be activated by a human P450 and thus be toxic or mutagenic in humans. To assess the extent of interindividual variation in P450 expression, probes developed from P450 cDNAs are being used to quantify levels of P450 mRNAs in various human tissues. Studies using RNase protection revealed that the closely related CYP2B6 and CYP2B7 mRNAs could be independently quantified in liver and lung, respectively. This procedure can be used to examine expression of different P450 genes in banks of human tissue specimens. human cytochrome P-450; catalytic activity ; cDNA expression ; chemical carcinogen ; RNase protection
Mammalian cytochromes P450 consist of ten gene families (Nebert et al. 1991), six of which encode enzymes involved in steroid and bile acid biosynthetic pathways. One gene family encodes P450s that hydroxylate fatty acids including prostaglandins and arachidonic acid. Three families, designated CYP1, CYP2 and CYP3, encompass the major foreign compound-metabolizing P450s responsible for converting chemicals into forms that can easily be eliminated from Address da, Maryland
for reprints : Building 20892, USA.
37, Room 67
3E24,
National
Institutes
of Health
Bethes-
68
F.J. Gonzalez
et al.
the body. Many of the P450s within these families are also capable of metabolically activating procarcinogens and promutagens. Marked species differences occur in the catalytic activities and regulation of P450s. This diversity is believed due to environmental and habitat constraints that determined the exposure of a foreign compound or toxin to particular populations of animals (Gonzalez and Nebert 1990). The principal driving force behind P450 evolution could have been plant toxins that needed to be inactivated by the P450s for the organism to survive and fluorish. The consequence of P450 evolution is that a large degree of species differences occur in expression and catalytic activities of the foreign compound-metabolizing P450s, particularly those in the CYP2 family. These differences suggest that caution be exercised when using rodents or rodent-derived testing systems to determine whether a particular chemical such as a drug, pesticide or herbicide under development, will present a potential risk to humans. In an effort to develop more reliable systems to evaluate chemical exposure and human risk assessment, human P450s are being directly examined for their abilities to activate known chemical carcinogens. This is being carried out through cDNA expression of P450s in cultured cells (Gonzalez et al. 1990). cDNA-expressed P450s can be used to predict how humans will metabolize a particular chemical and, most importantly, whether any metabolites will be harmful. In the present report, we demonstrated the utility of cDNA expression in analysis of the metabolic activation of the human hepatocarcinogen aflatoxin B1. It is well established from studies in rodents, that P450s are expressed to varying degrees in different tissues that are susceptible to cancer initiation, although the principal organ of expression is liver. In addition, a large degree of interindividual differences in expression of P450s are known to exist in humans as best illustrated by the known genetic polymorphisms in drug oxidation (Gonzalez and Meyer 1991). To determine P450 expression in humans, total RNA isolated from tissue specimens, was assayed for levels of different P450 mRNAs using a highly sensitive and specific RNase protection assay. This assay was validated by analyzing expression of two closely related P450 mRNAs in the CYP2B family in liver and lung. MATERIALS AND METHODS Poly (A) RNA was isolated from human lung and liver tissue specimens and used to prepare cDNA libraries in the vector ~1, gt 11. These libraries were screened using antibodies prepared from rat P450s and available cDNA probes against both rat and human P450s. Twelve full-length human P450 cDNAs were isolated from lung and liver libraries and sequenced. The cDNAs were inserted into vaccinia virus (Gonzalez et al. 1991) and B-lymphoblastoid cells (Crespi 1991) as described previously. P450 expression was analyzed using Western immunoblotting and standard enzyme assays. Aflatoxin B 1 activation by vaccinia-expressed human P450s was determined using in situ DNA binding and the Ames test (Aoyama et al. 1990). In the latter case, the common rat 89 preparations, used
Human
Fig. 1.
Schematic
mRNA
Cytochrome
representation
P450
Catalytic
of the
Activities
RNAse
and E xpression
protection
method
69
to quantify
levels.
for metabolic activation of chemicals, were replaced by vaccinia virus-expressed human P450s. Toxicity and mutagenicity of aflatoxin B 1 in B-lymphoblastoid cells containing human P450 cDNAs was carried out as detailed earlier (Crespi et al. 1991; Crespi 1991). Mutagenicity was measured at the hypoxanthine-quanine phosphoribosyl transferase (HGPRT) gene by survival of cells in the presence of 6-thioguanine. To quantify P450 mRNA, RNase protection vas performed as shown in Fig. 1. P450 cDNAs are inserted into vectors containing the T7 and SP6 RNA polymerase promoters. These are used to synthesize 32P-labeled antisense RNA probes which are hybridized with total human RNA. The hybrids are treated with ribonucleases to remove single-stranded material and the protected RNA fragments are analyzed by gel electrophoresis. The gels are exposed to autoradiographic film and the images are quantified by photographic densitometry.
RESULTS ANDDISCUSSION cDNA-directedexpressionof human cytochromeP450s Vaccinia virus is a lytic DNAvirus that is capable of producing thousands of progeny in any mammalian cell. cDNAs inserted into the vaccinia virus genome are efficiently expressed within one to two days after viral infection at which time the cells are harvested and the expressed enzyme analyzed. cDNAswere inserted into vaccinia virus to create a panel of recombinant viruses each containing a unique human cytochrome P450 cDNA. The viruses were used to infect human hepatoma HepG2 cells grown to confluency in culture. These cells contain ample
F.J. Gonzalez
70
et al.
intracellular membrane and NADPH-P450 oxidoreductase to support P450 catalytic activity. Infected cells were then analyzed for their capacities to activate the procarcinogen aflatoxin B1 (Aoyama et al. 1990). Out of twelve P450s analyzed, CYP1A2, CYP2A6, CYP2B6, CYP3A3 and CYP3A4 all converted aflatoxin B1 to metabolites capable of binding to DNA and producing histidine revertants in the Ames test. B-lymphoblastoid cells, stably expressing human P450s were also tested for their sensitivities to aflatoxin B1. These cells were developed for rapid growth and ease in determining genotoxic and mutagenic endpoints after exposure to test chemicals. Cells were constructed that stably express human P450s from cDNAcontaining episomally-replicating plasmids. As shown in Fig. 2, cells containing CYP1A2 and CYP3A4 were all sensitive to aflatoxin B1 as assessed by toxicity and mutagenicity at the HGPRT gene (Crespi et al. 1991). Control cells lacking expression of P450 were not killed or mutagenized by this compound. These studies suggest that since multiple human P450s can activate aflatoxin B1, interindividual variation in expression of one particular P450 form may not play a role in aflatoxin-induced hepatocarcinogenesis. For example, if a major aflatoxin-activating P450 is expressed at a low level in certain individuals other P450s could compensate and activate aflatoxin B1. It should be recognized, however, that levels of conjugating enzymes, capable of rapidly inactivating the mutagenic epoxide of this compound, may also be important in determining cancer susceptibility.
Fig. 2. Toxicity lymphoblastoid CYP3A4.
and mutagenicity cells and the
elicited by aflatoxin same cells expressing
B 1 of control P450s CYP1A2
Band
Human
Cytochrome
P450
Fig. 3. RNase protection quantification and lung, respectively.
Catalytic
Activities
of CYP2B6
and E xpression
and CYP2B7
mRNAs
71
in 1aver
Quantitation of P450 mRNA in human liver and lung tissue specimens As noted above, variability in expression of human P450 forms may determine susceptibility or resistance to chemical carcinogenesis. Levels of some P450s cannot be accurately measured in humans through standard clinical pharmacological analysis of urine metabolites of administered drugs. To quantify P450 expression in human tissues, RNase protection was used. This method is highly quantitative and allows discrimination of mRNAs displaying a high degree of sequence similarity. In addition, an internal control probe can be included in the reaction mixture with the P450 mRNA probe. To validate this system, we examined expression of the CYP2B6 and CYP2B7 genes in liver and lung tissues. These genes display 93% sequence similarity across their exonic regions and their mRNAs would not be distinguishable on Northern blots (Yamano et al. 1989). However, with RNase protection, mRNAs with differencesof only 1 base can be distinguished since the hybrids obtained will be cleaved by RNase (Fig. 1). Antisense probes were made that differed in size to distinguish the CYP2B6 and CYP2B7 mRNAs. A probe was also made against human fl-actin mRNA that yielded a shorter protected fragment that could be readily resolved from the P450 hybrids. This was used as an internal control. Results, shown in Fig. 3, demonstrate that CYP2B6 and CYP2B7 are preferentially expressed in liver and lung, respectively. Analysis of a large number of specimens indicate a high degree of interindividual variability in levels of these mRNAs in both tissues. The mechanism of this variability is currently under investigation.
72
F.J. Gonzalez
et al.
References 1) Aoyama, T., Yamano, S., Guzelian, P.S., Gelboin, H.V. & Gonzalez, F.J. (1990) Five of 12 forms of vaccinia virus-expressed human hepatic cytochrome P450 metabolically activate aflatoxin B1. Proc. Natl. Acad. Sci. USA, 87, 4790-4793. 2) Crespi, C.L. (1991) Expression of human cytochromes P450 using B-lymphoblastoid cells. Meth. Enzymol., 206, 123-129. 3) Crespi, C.L., Penman, B.W., Steimel, D.T., Gelboin, H.V. & Gonzalez, F.J. (1991) The development of a human cell line stably expressing human CYP3A4 : Role in the metabolic activation of aflatoxin B1 and comparison to CYP1A2 and CYP2A3. Carcinogenesis, 12, 335-359. 4) Gonzalez, F.J. & Nebert, D.W. (1990) Evolution of the P450 gene superfamily : Animal-plant warfare, molecular drive and human genetic differences in drug oxidation. Trends Genet.,6, 182-186. 5) Gonzalez, F.J. & Meyer, U.A. (1991) Molecular genetics of the debrisoquine/sparteine polymorphism. Gun. Pharmacol. Ther., 50, 233-238. 6) Gonzalez, F.J., Crespi, CL. & Gelboin, H.V. (1990) cDNA-expressed human cytochrome P450s : A new age of molecular toxicology and human risk assessment. Mutat. Res., 247, 113-127. 7) Gonzalez, F.J., Aoyama, T. & Gelboin, H.V. (1991) Expression of mammalian cytochrome P450 using vaccinia virus. Meth. Enzymol., 206, 85-91. 8) Nebert, D.W., Nelson, D.R., Coon, M.J., Estabrook, R.W., Feyereisen, R., FujiiKuriyama, Y., Gonzalez, F.J., Guengerich, F.P., Gunsalus, I.C., Johnson, E.F., Loper, J.C., Sato, R., Waterman, M.R. & Waxman, D.J. (1991) The P450 superfamily : Update on new sequences, gene mapping and recommended nomenclature. DNA Cell. Biol., 10, 1-14. 9) Yamano, S., Nhamburo, P.T., Aoyama, T., Meyer,U.A., Inaba, T., Kalow, W., Gelboin, H.V., McBride, OW. & Gonzalez, F.J. (1989) cDNA cloning and sequence and cDNA-directed expression of human P450 IIB 1: Identification of a normal and two variant cDNAs derived from the CYP2B locus on chromosome 19 and differentiation of the JIB mRNAs in human liver. Biochemistry, 28, 7340-7348.
J. Exp.
Analysis Activities
Med.,
1992, 168, 67-72
of Human and
Cytochrome
P450
Catalytic
Expression
FRANKJ. GONZALEZ,CHARLESL. CRESPI*,MACEIJ CZERWINSKIand HARRYV. GELBOIN Laboratory of Molecular Carcinogenesis, National Cancer Institute, National Institutes of Health, * Gentest Corporation
GONZALEZ, F.J., CRESPI,C.L., CZERWINSKI, M. and GELBOIN, H.V. Analysis of Human CytochromeP450 Catalytic Activities and Expression. Tohoku J. Exp. Med., 1992, 168 (2), 67-72 Cytochromes P450 are a large group of membraneassociated heme protein monooxygenases, most of which are responsible for metabolizing foreign compounds. Chemical carcinogens, which are ingested or absorbed into the body as inert forms, are metabolically activated by P450s to electrophilic metabolites capable of binding to and mutating DNA. Different P450 forms are responsible for activation of the various classes of chemical carcinogens including the arylamines, polycyclic aromatic hydrocarbons, nitrosamines and afiatoxins. Thus, the cellular constituency and levels of P450s could determine the fate of a particular carcinogen and the risk of humans to exposure. To study the catalytic activities of human P450s, human P450 cDNAs were cloned and expressed into active enzymes using cultured cells. By both transient and stable cDNA expression systems, several human P450s were found to be capable of metabolically-activating the human hepatocarcinogen afiatoxin B1. These cDNA expression systems can also be used to determine whether an unknown chemical will be activated by a human P450 and thus be toxic or mutagenic in humans. To assess the extent of interindividual variation in P450 expression, probes developed from P450 cDNAs are being used to quantify levels of P450 mRNAs in various human tissues. Studies using RNase protection revealed that the closely related CYP2B6 and CYP2B7 mRNAs could be independently quantified in liver and lung, respectively. This procedure can be used to examine expression of different P450 genes in banks of human tissue specimens. human cytochrome P-450; catalytic activity ; cDNA expression ; chemical carcinogen ; RNase protection
Mammalian cytochromes P450 consist of ten gene families (Nebert et al. 1991), six of which encode enzymes involved in steroid and bile acid biosynthetic pathways. One gene family encodes P450s that hydroxylate fatty acids including prostaglandins and arachidonic acid. Three families, designated CYP1, CYP2 and CYP3, encompass the major foreign compound-metabolizing P450s responsible for converting chemicals into forms that can easily be eliminated from Address da, Maryland
for reprints : Building 20892, USA.
37, Room 67
3E24,
National
Institutes
of Health
Bethes-
68
F.J. Gonzalez
et al.
the body. Many of the P450s within these families are also capable of metabolically activating procarcinogens and promutagens. Marked species differences occur in the catalytic activities and regulation of P450s. This diversity is believed due to environmental and habitat constraints that determined the exposure of a foreign compound or toxin to particular populations of animals (Gonzalez and Nebert 1990). The principal driving force behind P450 evolution could have been plant toxins that needed to be inactivated by the P450s for the organism to survive and fluorish. The consequence of P450 evolution is that a large degree of species differences occur in expression and catalytic activities of the foreign compound-metabolizing P450s, particularly those in the CYP2 family. These differences suggest that caution be exercised when using rodents or rodent-derived testing systems to determine whether a particular chemical such as a drug, pesticide or herbicide under development, will present a potential risk to humans. In an effort to develop more reliable systems to evaluate chemical exposure and human risk assessment, human P450s are being directly examined for their abilities to activate known chemical carcinogens. This is being carried out through cDNA expression of P450s in cultured cells (Gonzalez et al. 1990). cDNA-expressed P450s can be used to predict how humans will metabolize a particular chemical and, most importantly, whether any metabolites will be harmful. In the present report, we demonstrated the utility of cDNA expression in analysis of the metabolic activation of the human hepatocarcinogen aflatoxin B1. It is well established from studies in rodents, that P450s are expressed to varying degrees in different tissues that are susceptible to cancer initiation, although the principal organ of expression is liver. In addition, a large degree of interindividual differences in expression of P450s are known to exist in humans as best illustrated by the known genetic polymorphisms in drug oxidation (Gonzalez and Meyer 1991). To determine P450 expression in humans, total RNA isolated from tissue specimens, was assayed for levels of different P450 mRNAs using a highly sensitive and specific RNase protection assay. This assay was validated by analyzing expression of two closely related P450 mRNAs in the CYP2B family in liver and lung. MATERIALS AND METHODS Poly (A) RNA was isolated from human lung and liver tissue specimens and used to prepare cDNA libraries in the vector ~1, gt 11. These libraries were screened using antibodies prepared from rat P450s and available cDNA probes against both rat and human P450s. Twelve full-length human P450 cDNAs were isolated from lung and liver libraries and sequenced. The cDNAs were inserted into vaccinia virus (Gonzalez et al. 1991) and B-lymphoblastoid cells (Crespi 1991) as described previously. P450 expression was analyzed using Western immunoblotting and standard enzyme assays. Aflatoxin B 1 activation by vaccinia-expressed human P450s was determined using in situ DNA binding and the Ames test (Aoyama et al. 1990). In the latter case, the common rat 89 preparations, used
Human
Fig. 1.
Schematic
mRNA
Cytochrome
representation
P450
Catalytic
of the
Activities
RNAse
and E xpression
protection
method
69
to quantify
levels.
for metabolic activation of chemicals, were replaced by vaccinia virus-expressed human P450s. Toxicity and mutagenicity of aflatoxin B 1 in B-lymphoblastoid cells containing human P450 cDNAs was carried out as detailed earlier (Crespi et al. 1991; Crespi 1991). Mutagenicity was measured at the hypoxanthine-quanine phosphoribosyl transferase (HGPRT) gene by survival of cells in the presence of 6-thioguanine. To quantify P450 mRNA, RNase protection vas performed as shown in Fig. 1. P450 cDNAs are inserted into vectors containing the T7 and SP6 RNA polymerase promoters. These are used to synthesize 32P-labeled antisense RNA probes which are hybridized with total human RNA. The hybrids are treated with ribonucleases to remove single-stranded material and the protected RNA fragments are analyzed by gel electrophoresis. The gels are exposed to autoradiographic film and the images are quantified by photographic densitometry.
RESULTS ANDDISCUSSION cDNA-directedexpressionof human cytochromeP450s Vaccinia virus is a lytic DNAvirus that is capable of producing thousands of progeny in any mammalian cell. cDNAs inserted into the vaccinia virus genome are efficiently expressed within one to two days after viral infection at which time the cells are harvested and the expressed enzyme analyzed. cDNAswere inserted into vaccinia virus to create a panel of recombinant viruses each containing a unique human cytochrome P450 cDNA. The viruses were used to infect human hepatoma HepG2 cells grown to confluency in culture. These cells contain ample
F.J. Gonzalez
70
et al.
intracellular membrane and NADPH-P450 oxidoreductase to support P450 catalytic activity. Infected cells were then analyzed for their capacities to activate the procarcinogen aflatoxin B1 (Aoyama et al. 1990). Out of twelve P450s analyzed, CYP1A2, CYP2A6, CYP2B6, CYP3A3 and CYP3A4 all converted aflatoxin B1 to metabolites capable of binding to DNA and producing histidine revertants in the Ames test. B-lymphoblastoid cells, stably expressing human P450s were also tested for their sensitivities to aflatoxin B1. These cells were developed for rapid growth and ease in determining genotoxic and mutagenic endpoints after exposure to test chemicals. Cells were constructed that stably express human P450s from cDNAcontaining episomally-replicating plasmids. As shown in Fig. 2, cells containing CYP1A2 and CYP3A4 were all sensitive to aflatoxin B1 as assessed by toxicity and mutagenicity at the HGPRT gene (Crespi et al. 1991). Control cells lacking expression of P450 were not killed or mutagenized by this compound. These studies suggest that since multiple human P450s can activate aflatoxin B1, interindividual variation in expression of one particular P450 form may not play a role in aflatoxin-induced hepatocarcinogenesis. For example, if a major aflatoxin-activating P450 is expressed at a low level in certain individuals other P450s could compensate and activate aflatoxin B1. It should be recognized, however, that levels of conjugating enzymes, capable of rapidly inactivating the mutagenic epoxide of this compound, may also be important in determining cancer susceptibility.
Fig. 2. Toxicity lymphoblastoid CYP3A4.
and mutagenicity cells and the
elicited by aflatoxin same cells expressing
B 1 of control P450s CYP1A2
Band
Human
Cytochrome
P450
Fig. 3. RNase protection quantification and lung, respectively.
Catalytic
Activities
of CYP2B6
and E xpression
and CYP2B7
mRNAs
71
in 1aver
Quantitation of P450 mRNA in human liver and lung tissue specimens As noted above, variability in expression of human P450 forms may determine susceptibility or resistance to chemical carcinogenesis. Levels of some P450s cannot be accurately measured in humans through standard clinical pharmacological analysis of urine metabolites of administered drugs. To quantify P450 expression in human tissues, RNase protection was used. This method is highly quantitative and allows discrimination of mRNAs displaying a high degree of sequence similarity. In addition, an internal control probe can be included in the reaction mixture with the P450 mRNA probe. To validate this system, we examined expression of the CYP2B6 and CYP2B7 genes in liver and lung tissues. These genes display 93% sequence similarity across their exonic regions and their mRNAs would not be distinguishable on Northern blots (Yamano et al. 1989). However, with RNase protection, mRNAs with differencesof only 1 base can be distinguished since the hybrids obtained will be cleaved by RNase (Fig. 1). Antisense probes were made that differed in size to distinguish the CYP2B6 and CYP2B7 mRNAs. A probe was also made against human fl-actin mRNA that yielded a shorter protected fragment that could be readily resolved from the P450 hybrids. This was used as an internal control. Results, shown in Fig. 3, demonstrate that CYP2B6 and CYP2B7 are preferentially expressed in liver and lung, respectively. Analysis of a large number of specimens indicate a high degree of interindividual variability in levels of these mRNAs in both tissues. The mechanism of this variability is currently under investigation.
72
F.J. Gonzalez
et al.
References 1) Aoyama, T., Yamano, S., Guzelian, P.S., Gelboin, H.V. & Gonzalez, F.J. (1990) Five of 12 forms of vaccinia virus-expressed human hepatic cytochrome P450 metabolically activate aflatoxin B1. Proc. Natl. Acad. Sci. USA, 87, 4790-4793. 2) Crespi, C.L. (1991) Expression of human cytochromes P450 using B-lymphoblastoid cells. Meth. Enzymol., 206, 123-129. 3) Crespi, C.L., Penman, B.W., Steimel, D.T., Gelboin, H.V. & Gonzalez, F.J. (1991) The development of a human cell line stably expressing human CYP3A4 : Role in the metabolic activation of aflatoxin B1 and comparison to CYP1A2 and CYP2A3. Carcinogenesis, 12, 335-359. 4) Gonzalez, F.J. & Nebert, D.W. (1990) Evolution of the P450 gene superfamily : Animal-plant warfare, molecular drive and human genetic differences in drug oxidation. Trends Genet.,6, 182-186. 5) Gonzalez, F.J. & Meyer, U.A. (1991) Molecular genetics of the debrisoquine/sparteine polymorphism. Gun. Pharmacol. Ther., 50, 233-238. 6) Gonzalez, F.J., Crespi, CL. & Gelboin, H.V. (1990) cDNA-expressed human cytochrome P450s : A new age of molecular toxicology and human risk assessment. Mutat. Res., 247, 113-127. 7) Gonzalez, F.J., Aoyama, T. & Gelboin, H.V. (1991) Expression of mammalian cytochrome P450 using vaccinia virus. Meth. Enzymol., 206, 85-91. 8) Nebert, D.W., Nelson, D.R., Coon, M.J., Estabrook, R.W., Feyereisen, R., FujiiKuriyama, Y., Gonzalez, F.J., Guengerich, F.P., Gunsalus, I.C., Johnson, E.F., Loper, J.C., Sato, R., Waterman, M.R. & Waxman, D.J. (1991) The P450 superfamily : Update on new sequences, gene mapping and recommended nomenclature. DNA Cell. Biol., 10, 1-14. 9) Yamano, S., Nhamburo, P.T., Aoyama, T., Meyer,U.A., Inaba, T., Kalow, W., Gelboin, H.V., McBride, OW. & Gonzalez, F.J. (1989) cDNA cloning and sequence and cDNA-directed expression of human P450 IIB 1: Identification of a normal and two variant cDNAs derived from the CYP2B locus on chromosome 19 and differentiation of the JIB mRNAs in human liver. Biochemistry, 28, 7340-7348.
