648
CHARACTERIZATION OF EXTRAHEPATIC P450S
[64]
detecting low levels of mRNA in a tissue, should be used. This method is described in [62] in this volume. Acknowledgments This work was supported by a grant from the Swedish Medical Research Council (No. 03X-06807).
[64] Identification a n d Localization of C y t o c h r o m e s P 4 5 0 in G u t
By HENRY W. STROBEL, DIANNE K. HAMMOND,TEl~Y B. WHITE, and JAMES W. WHITE Introduction For any tissue, an array of methods is available for the detection of cytochromes P450 and cytochrome P450-dependent activity. First, activity assays using various substrates can suggest the general range of cytochromes P450 present in a tissue, but usually do not define the precise forms. These metabolic assays can be conducted with whole cells, tissue homogenates, 9000 g supernatant fractions, microsomes, or purified enzymes. The use of whole cells in metabolic assays provides a convenient, nondestructive way to survey for the presence of cytochromes P450. Second, immunological probes allow more specificity in defining which cytochromes P450 are present in a tissue. This approach is limited by the availability of antibodies to the P450 forms purified from other tissues, but it has the advantages of sensitivity and specificity greater than that available for the metabolic assays. Third, the use of cDNA probes in Northern blot analyses allows the assessment of mRNAs encoding specific forms of cytochrome P450. With appropriate standards (e.g.,/3-actin) quantitation of mRNA levels is possible with greater sensitivity than either of the preceding methods. As a variation on this theme, the polymerase chain reaction (PCR) technique with selected oligonucleotides can permit detection of low abundance mRNAs by specific amplification of the message of interest. However, the nature of the PCR as an amplification procedure makes it less useful as a quantitative methodology. Fourth, purification techniques allow the isolation of forms of P450 which are expressed or are uniquely expressed in a particular tissue. While this approach provides an absolute mode for METHODS IN ENZYMOLOGY,VOL. 206
Copyright © 1991by AcademicPress, Inc. All fightsof reproductionin any form reserved.
[64]
CYTOCHROMES P450 IN GUT
649
determining the presence of a form in a particular tissue, difficulties in yield, recovery, and the ability to account for all the P450 isozymes present at the outset of the procedure limit the utility of this approach as a method of specifying which forms are present in a tissue. Because no single approach satisfactorily addresses the issues of quantitation, accuracy and accountability, we have used all of these approaches in the study of P450s present in the gut. We have focl, sed our studies on the presence and role of cytochromes P450 in human and rat colon. Metabolic Activity in Whole Cell Assay System Metabolic activity with a wide range of cytochrome P450 substrates has been determined in microsomal preparations, ~ broken cells 2 and in reconstituted systems 3 consisting of purified enzymes. We have developed a method for assaying cytochrome P450-dependent metabolic activity using fluorescent substrates in unbroken colon tumor cells in culture. LS174T, 4 HT-29: and DiFi 6,7 colon tumor cell lines are grown in GIBCO (Grand Island, NY) minimum essential media (MEM) or Dulbecco's MEM with 2 mM glutamine and 10% fetal calf serum (Hyclone, Logan, UT). All enzyme measurements are made after varying lengths of time in culture such that the cells are at or near confluence. Some of the cells are induced by treatment overnight with 10 tiM benzanthracer e (BA). The tissue cultures to be tested are grown in 100-mm Coming (Cornin~, NY) plates and washed with saline to remove media and loose cells. The substrate, 3.4 ktM ethoxyresorufin (ER), is then added in Dulb:~cce~s phosphate-buffered saline (PBS), at 3 ml per plate, and the reaction is run for 10 rain (although the reaction approximates linearity for 30 min under these conditions). At the end of the specified time, 2 ml of the reaction mix is withdrawn from the cells, and fluorescence of the resorufin produced is measured using a Perkin-Elmer LS-5 fluorescence spectrophotometer, with 522 nm I W. F. Fang and H. W. Strobel, Arch. Biochem. Biophys. 186, 128 (1978). 2 j. A. Bradlaw, Fundam. Appl. Toxicol. 6, 598 (1982). 3 T. Saito and H. W. Strobel, J. Biol. Chem. 256, 984 (1981). 4 B. H. Tom, L. P. Rutsky, M. M. Jakstyo, R. Oyasu, C. J. Kaye, and B. D. Kahan, In Vitro 12, 180 (1976). 5 j. Fogh and G. Trompe, in "Human Tumor Cells in Vitro" (J. Fogh, ed.), p. 115. Plenum, New York, 1975. 6 S. Untawale, "Cytogenetie and Molecular Analysis of Colon Cancer." Masters Thesis, University of Texas Graduate School of Biomedical Sciences, Houston, Texas, 1987. 7 B. M. Bowman, M. Olive, S. Untawale, M. Blick, S. North, G. Gallick, G. Dolf, M. J. Siciliano, L. D. Roubein, H. Fritche, S. Pathak, D. M. Wildrick, and B. Levin, Fourth International Symposium on Colorectal Cancer, Springer-Vedag, Tokyo, 1989.
650
CHARACTERIZATION OF EXTRAHEPATIC P450S
[64]
excitation and 586 nm emission wavelengths and 5-ram slits, s In order to determine the disappearance of ER, the same sample is subjected to a spectral scan from 620 to 380 nm using a Beckman (Fullerton, CA) ACTA II spectrophotometer. Ethoxyresorufin standards in PBS are also scanned, and the values, which are linear in this range, are used to construct a standard curve using 482-620 n m v a l u e s . 9 The remaining reaction mix is also removed from the cells, and the cells are either returned to culture or tested for protein content. The protein content is determined by scraping the cells from the culture dish and suspending the cells in 5 ml of Dulbecco's PBS, which is then sampled after thorough suspension. Alternatively, the plates are treated with 10 ml of 0.2 N NaOH to solubilize the protein, and then the protein concentration is determined. Each plate normally contains 3-10 mg of cellular protein, depending on the cell line and the number of days in culture. Ethoxyresorufin dealkylation activity is assayed using the method of Burke 8 at a protein concentration of 0.4-1.0 mg/ml. The lysed cell assay is run according to the method of Blank and co-workers using 1.0-2.0 mg/ ml.l° Cells are counted using a hemocytometer, after treatment of the cells with 1% crystal violet for I hr to lyse the cells and allow counting of stained nuclei.ll At the end of all assay procedures used in this study, cell viability is routinely greater than 90% as assessed by trypan blue exclusion. Protein determinations are made using the Pierce (Rockford, IL) BCA protein assay kit utilizing bovine serum albumin as a standard. Ethoxyresorufin, pentoxyresorufin, and resorufin are purchased from Pierce, suspended in dimethyl sulfoxide (DMSO), and utilized such that less than 4% DMSO is present in any assay. Metabolic activities for ethoxyresorufin dealkylation in the whole cell assay system for LS174T cells are comparable to the values obtained for the lysed cell assay system (14.5 --+ 4.5 and 12.5 - 1.5 pmol/min/mg, respectively). Microsomes prepared from these cells show an activity of 23.5 --+ 1.6 pmol/min/mg. The whole cell assay system thus compares quite favorably when one considers the percentage of the total cell protein attributable to microsomes and the yield of microsomal preparations (20-25%). The fluorimetric assay is linear for at least 20 min and has the advantage of not disrupting the cells. Induction can be monitored and s M. D. Burke, S. Thompson, C. R. Elcomb, J. Halpert, T. Haaparanta, and R. R. Mayer, Biochemistry 34, 3337 (1985). 9 A. V. Klotz, J. J. Stegemaan, and C. Walsh, Anal. Biochem. 140, 138 (1984). x0j. A. Blank, A. N. Tucher, J. Sweafliek, T. A. Gasiewicz, and M. I. Luster, Mol. Pharmacol. 32, 168 (1987). II R. 1. Freshney, "Culture of Animal Cells: A Manual of Basic Techniques," p. 199. Alan R. Liss, New York, 1983.
[64]
CYTOCHROMESP450 IN GUT
651
[ d 6 o
f
o et~ o !__
0 ..0 380
I
I
I
I
I
I
I
I
I
I
400
420
440
480
500
520
540
580
600
620
Wavelength FIG. 1. Absorption spectra of ethoxyresorufin (3.4/zM) disappearance over time. LS 174T cells (9 mg protein/plate) were treated for 0 ( X ~ 482 nm) and 30 (k~x 580 nrn) rnin.
continued as long as one chooses. Since these are intact cells, the normal pools of NADPH (and NADH) supply reducing equivalents to the system. As shown in Fig. I, the disappearance of ethoxyresorufin and the appearance of resorufin can be followed spectrophotometrically at 482 and 580 nm, respectively. At 0 time (curve with maximum at 482 nm) ethoxyresorufin is added to the cells, and by 30 min it is removed, with much having been converted into resorufin. These studies were conducted in the presence of dicumarol (10/zM), which inhibits the further metabolism of resorufin by quinone reductase.12 This method provides an easy and direct method of measuring activities and cytochrome P450 function. Its nondestructiveness makes it ideal for cells in culture. Western Analysis Immunological analysis of microsomes purified from the human colon tumor cell line is used to identify P450 isozymes present more specifically than could be determined by activity assay. Microsomes from cells treated beginning on the fourth day from passage are prepared by differential centrifugation 13and stored at - 78 ° in 20% glycerol or 0.25 M sucrose and 10 mM EDTA. Western analysis is performed by the method of Towbin 12 R. W. Nines, R. A. Prough, and R. A. Lubet, Arch. Biochem. Biophys. 2,29, 459 (1984). 13 S. N. Newaz, W. F. Fang, and H. W. Strobel, Cancer 52, 794 (1983).
652
CHARACTERIZATION OF EXTRAHEPATIC P450S
[64]
et al., 14except Carnation nonfat dry milk (1%) is used as a blocking agent 15 on the nitrocellulose. Antibodies to rat cytochrome P450IA1 are raised in New Zealand White rabbits as reported 16 and purified using DEAE AffiGel Blue columns [Bio-Rad (Richmond, CA) Bulletin 1092, 1982] in TrisHC1 buffer (20 mM, pH 8.0) containing 28 mM NaCl and 0.02% NAN3. Goat anti-rabbit immunoglobulin G (IgG) horseradish peroxidase (BioRad) is used as the second antibody, and 4-chloro-l-naphthol (Bio-Rad) as the staining reagent, according to the manufacturer's instructions. Using these procedures, we are able to detect proteins in microsomes which bind antibodies to a variety of different isozymes of cytochrome P450 including P450IIC9 and P450IA1. In the example shown, microsomes from benzanthracene-treated LS 174T ceils displayed proteins which bound antibodies to rat P450IAI (Fig. 2A).
RNA Analysis To increase the sensitivity of detection of P450 expression in the LS174T cell line, RNA is isolated and examined for the presence of message encoding various cytochrome P450 isozymes. The presence of P450encoding messenger RNA is determined by Northern. analysis or by the polymerase chain reaction (PCR). In the example given in Fig. 2B,C, benzanthracene (10/~M) was used as the P450 inducer. RNA is isolated from the LS 174T cell line by lysis of the cells in guanidine isothiocyanate followed by centrifugation through C s C l . 17 For some samples, poly(A) +enriched RNA is prepared by purification on oligo(dT)-cellulose Type 2 according to manufacturer's instructions (Collaborative Research, Bedford, MA). Northern analysis TMis performed following fractionation of the RNA through agarose in formaldehyde and transfer to Nytran nylon membranes by the protocol provided (Schleicher and Schuell, Keene, NH). RNA is fixed to the membrane with 1200 /.d UV irradiation in a Stratalinker (Stratagene, La Jolla, CA). In the example shown (Fig. 2B), the plasmid pA819 was used as a probe for the message for P450IAI. The plasmid is 14 H. Towbin, T. Staehelin, and J. Gordon, Proc. Natl. Acad. Sci. U.S.A. 76, 4350 (1979). is D. A. Johnson, J. W. Gantsch, J. R. Sportsman, and J. H. Elder, Gene Anal. Tech. 1, 3 (1984). I~ p. p. Lau and H. W. Strobel, J. Biol. Chem. 257, 5257 (1982). 17j. M. Chirgwin, A. E. Przybyla, R. J. MacDonald, and W. J. Rutter, Biochemistry 18, 5294 (1979). 18 p. S. Thomas, Proc. Natl. Acad. Sci. U.S.A. 77, 5201 (1980). 19 R. N. Hines, J. B. Levy, R. D. Conrad, P. L. Iversen, M.-L. Shen, A. M. Renli, and E. Bresnick, Arch. Biochem. Biophys. 237, 465 (1985).
[64]
CYTOCnROMES P450 IN GUT
1
v
w
653
2
+
0, " ,
A
CHARACTERIZATION OF EXTRAHEPATIC P450S
[64]
detecting low levels of mRNA in a tissue, should be used. This method is described in [62] in this volume. Acknowledgments This work was supported by a grant from the Swedish Medical Research Council (No. 03X-06807).
[64] Identification a n d Localization of C y t o c h r o m e s P 4 5 0 in G u t
By HENRY W. STROBEL, DIANNE K. HAMMOND,TEl~Y B. WHITE, and JAMES W. WHITE Introduction For any tissue, an array of methods is available for the detection of cytochromes P450 and cytochrome P450-dependent activity. First, activity assays using various substrates can suggest the general range of cytochromes P450 present in a tissue, but usually do not define the precise forms. These metabolic assays can be conducted with whole cells, tissue homogenates, 9000 g supernatant fractions, microsomes, or purified enzymes. The use of whole cells in metabolic assays provides a convenient, nondestructive way to survey for the presence of cytochromes P450. Second, immunological probes allow more specificity in defining which cytochromes P450 are present in a tissue. This approach is limited by the availability of antibodies to the P450 forms purified from other tissues, but it has the advantages of sensitivity and specificity greater than that available for the metabolic assays. Third, the use of cDNA probes in Northern blot analyses allows the assessment of mRNAs encoding specific forms of cytochrome P450. With appropriate standards (e.g.,/3-actin) quantitation of mRNA levels is possible with greater sensitivity than either of the preceding methods. As a variation on this theme, the polymerase chain reaction (PCR) technique with selected oligonucleotides can permit detection of low abundance mRNAs by specific amplification of the message of interest. However, the nature of the PCR as an amplification procedure makes it less useful as a quantitative methodology. Fourth, purification techniques allow the isolation of forms of P450 which are expressed or are uniquely expressed in a particular tissue. While this approach provides an absolute mode for METHODS IN ENZYMOLOGY,VOL. 206
Copyright © 1991by AcademicPress, Inc. All fightsof reproductionin any form reserved.
[64]
CYTOCHROMES P450 IN GUT
649
determining the presence of a form in a particular tissue, difficulties in yield, recovery, and the ability to account for all the P450 isozymes present at the outset of the procedure limit the utility of this approach as a method of specifying which forms are present in a tissue. Because no single approach satisfactorily addresses the issues of quantitation, accuracy and accountability, we have used all of these approaches in the study of P450s present in the gut. We have focl, sed our studies on the presence and role of cytochromes P450 in human and rat colon. Metabolic Activity in Whole Cell Assay System Metabolic activity with a wide range of cytochrome P450 substrates has been determined in microsomal preparations, ~ broken cells 2 and in reconstituted systems 3 consisting of purified enzymes. We have developed a method for assaying cytochrome P450-dependent metabolic activity using fluorescent substrates in unbroken colon tumor cells in culture. LS174T, 4 HT-29: and DiFi 6,7 colon tumor cell lines are grown in GIBCO (Grand Island, NY) minimum essential media (MEM) or Dulbecco's MEM with 2 mM glutamine and 10% fetal calf serum (Hyclone, Logan, UT). All enzyme measurements are made after varying lengths of time in culture such that the cells are at or near confluence. Some of the cells are induced by treatment overnight with 10 tiM benzanthracer e (BA). The tissue cultures to be tested are grown in 100-mm Coming (Cornin~, NY) plates and washed with saline to remove media and loose cells. The substrate, 3.4 ktM ethoxyresorufin (ER), is then added in Dulb:~cce~s phosphate-buffered saline (PBS), at 3 ml per plate, and the reaction is run for 10 rain (although the reaction approximates linearity for 30 min under these conditions). At the end of the specified time, 2 ml of the reaction mix is withdrawn from the cells, and fluorescence of the resorufin produced is measured using a Perkin-Elmer LS-5 fluorescence spectrophotometer, with 522 nm I W. F. Fang and H. W. Strobel, Arch. Biochem. Biophys. 186, 128 (1978). 2 j. A. Bradlaw, Fundam. Appl. Toxicol. 6, 598 (1982). 3 T. Saito and H. W. Strobel, J. Biol. Chem. 256, 984 (1981). 4 B. H. Tom, L. P. Rutsky, M. M. Jakstyo, R. Oyasu, C. J. Kaye, and B. D. Kahan, In Vitro 12, 180 (1976). 5 j. Fogh and G. Trompe, in "Human Tumor Cells in Vitro" (J. Fogh, ed.), p. 115. Plenum, New York, 1975. 6 S. Untawale, "Cytogenetie and Molecular Analysis of Colon Cancer." Masters Thesis, University of Texas Graduate School of Biomedical Sciences, Houston, Texas, 1987. 7 B. M. Bowman, M. Olive, S. Untawale, M. Blick, S. North, G. Gallick, G. Dolf, M. J. Siciliano, L. D. Roubein, H. Fritche, S. Pathak, D. M. Wildrick, and B. Levin, Fourth International Symposium on Colorectal Cancer, Springer-Vedag, Tokyo, 1989.
650
CHARACTERIZATION OF EXTRAHEPATIC P450S
[64]
excitation and 586 nm emission wavelengths and 5-ram slits, s In order to determine the disappearance of ER, the same sample is subjected to a spectral scan from 620 to 380 nm using a Beckman (Fullerton, CA) ACTA II spectrophotometer. Ethoxyresorufin standards in PBS are also scanned, and the values, which are linear in this range, are used to construct a standard curve using 482-620 n m v a l u e s . 9 The remaining reaction mix is also removed from the cells, and the cells are either returned to culture or tested for protein content. The protein content is determined by scraping the cells from the culture dish and suspending the cells in 5 ml of Dulbecco's PBS, which is then sampled after thorough suspension. Alternatively, the plates are treated with 10 ml of 0.2 N NaOH to solubilize the protein, and then the protein concentration is determined. Each plate normally contains 3-10 mg of cellular protein, depending on the cell line and the number of days in culture. Ethoxyresorufin dealkylation activity is assayed using the method of Burke 8 at a protein concentration of 0.4-1.0 mg/ml. The lysed cell assay is run according to the method of Blank and co-workers using 1.0-2.0 mg/ ml.l° Cells are counted using a hemocytometer, after treatment of the cells with 1% crystal violet for I hr to lyse the cells and allow counting of stained nuclei.ll At the end of all assay procedures used in this study, cell viability is routinely greater than 90% as assessed by trypan blue exclusion. Protein determinations are made using the Pierce (Rockford, IL) BCA protein assay kit utilizing bovine serum albumin as a standard. Ethoxyresorufin, pentoxyresorufin, and resorufin are purchased from Pierce, suspended in dimethyl sulfoxide (DMSO), and utilized such that less than 4% DMSO is present in any assay. Metabolic activities for ethoxyresorufin dealkylation in the whole cell assay system for LS174T cells are comparable to the values obtained for the lysed cell assay system (14.5 --+ 4.5 and 12.5 - 1.5 pmol/min/mg, respectively). Microsomes prepared from these cells show an activity of 23.5 --+ 1.6 pmol/min/mg. The whole cell assay system thus compares quite favorably when one considers the percentage of the total cell protein attributable to microsomes and the yield of microsomal preparations (20-25%). The fluorimetric assay is linear for at least 20 min and has the advantage of not disrupting the cells. Induction can be monitored and s M. D. Burke, S. Thompson, C. R. Elcomb, J. Halpert, T. Haaparanta, and R. R. Mayer, Biochemistry 34, 3337 (1985). 9 A. V. Klotz, J. J. Stegemaan, and C. Walsh, Anal. Biochem. 140, 138 (1984). x0j. A. Blank, A. N. Tucher, J. Sweafliek, T. A. Gasiewicz, and M. I. Luster, Mol. Pharmacol. 32, 168 (1987). II R. 1. Freshney, "Culture of Animal Cells: A Manual of Basic Techniques," p. 199. Alan R. Liss, New York, 1983.
[64]
CYTOCHROMESP450 IN GUT
651
[ d 6 o
f
o et~ o !__
0 ..0 380
I
I
I
I
I
I
I
I
I
I
400
420
440
480
500
520
540
580
600
620
Wavelength FIG. 1. Absorption spectra of ethoxyresorufin (3.4/zM) disappearance over time. LS 174T cells (9 mg protein/plate) were treated for 0 ( X ~ 482 nm) and 30 (k~x 580 nrn) rnin.
continued as long as one chooses. Since these are intact cells, the normal pools of NADPH (and NADH) supply reducing equivalents to the system. As shown in Fig. I, the disappearance of ethoxyresorufin and the appearance of resorufin can be followed spectrophotometrically at 482 and 580 nm, respectively. At 0 time (curve with maximum at 482 nm) ethoxyresorufin is added to the cells, and by 30 min it is removed, with much having been converted into resorufin. These studies were conducted in the presence of dicumarol (10/zM), which inhibits the further metabolism of resorufin by quinone reductase.12 This method provides an easy and direct method of measuring activities and cytochrome P450 function. Its nondestructiveness makes it ideal for cells in culture. Western Analysis Immunological analysis of microsomes purified from the human colon tumor cell line is used to identify P450 isozymes present more specifically than could be determined by activity assay. Microsomes from cells treated beginning on the fourth day from passage are prepared by differential centrifugation 13and stored at - 78 ° in 20% glycerol or 0.25 M sucrose and 10 mM EDTA. Western analysis is performed by the method of Towbin 12 R. W. Nines, R. A. Prough, and R. A. Lubet, Arch. Biochem. Biophys. 2,29, 459 (1984). 13 S. N. Newaz, W. F. Fang, and H. W. Strobel, Cancer 52, 794 (1983).
652
CHARACTERIZATION OF EXTRAHEPATIC P450S
[64]
et al., 14except Carnation nonfat dry milk (1%) is used as a blocking agent 15 on the nitrocellulose. Antibodies to rat cytochrome P450IA1 are raised in New Zealand White rabbits as reported 16 and purified using DEAE AffiGel Blue columns [Bio-Rad (Richmond, CA) Bulletin 1092, 1982] in TrisHC1 buffer (20 mM, pH 8.0) containing 28 mM NaCl and 0.02% NAN3. Goat anti-rabbit immunoglobulin G (IgG) horseradish peroxidase (BioRad) is used as the second antibody, and 4-chloro-l-naphthol (Bio-Rad) as the staining reagent, according to the manufacturer's instructions. Using these procedures, we are able to detect proteins in microsomes which bind antibodies to a variety of different isozymes of cytochrome P450 including P450IIC9 and P450IA1. In the example shown, microsomes from benzanthracene-treated LS 174T ceils displayed proteins which bound antibodies to rat P450IAI (Fig. 2A).
RNA Analysis To increase the sensitivity of detection of P450 expression in the LS174T cell line, RNA is isolated and examined for the presence of message encoding various cytochrome P450 isozymes. The presence of P450encoding messenger RNA is determined by Northern. analysis or by the polymerase chain reaction (PCR). In the example given in Fig. 2B,C, benzanthracene (10/~M) was used as the P450 inducer. RNA is isolated from the LS 174T cell line by lysis of the cells in guanidine isothiocyanate followed by centrifugation through C s C l . 17 For some samples, poly(A) +enriched RNA is prepared by purification on oligo(dT)-cellulose Type 2 according to manufacturer's instructions (Collaborative Research, Bedford, MA). Northern analysis TMis performed following fractionation of the RNA through agarose in formaldehyde and transfer to Nytran nylon membranes by the protocol provided (Schleicher and Schuell, Keene, NH). RNA is fixed to the membrane with 1200 /.d UV irradiation in a Stratalinker (Stratagene, La Jolla, CA). In the example shown (Fig. 2B), the plasmid pA819 was used as a probe for the message for P450IAI. The plasmid is 14 H. Towbin, T. Staehelin, and J. Gordon, Proc. Natl. Acad. Sci. U.S.A. 76, 4350 (1979). is D. A. Johnson, J. W. Gantsch, J. R. Sportsman, and J. H. Elder, Gene Anal. Tech. 1, 3 (1984). I~ p. p. Lau and H. W. Strobel, J. Biol. Chem. 257, 5257 (1982). 17j. M. Chirgwin, A. E. Przybyla, R. J. MacDonald, and W. J. Rutter, Biochemistry 18, 5294 (1979). 18 p. S. Thomas, Proc. Natl. Acad. Sci. U.S.A. 77, 5201 (1980). 19 R. N. Hines, J. B. Levy, R. D. Conrad, P. L. Iversen, M.-L. Shen, A. M. Renli, and E. Bresnick, Arch. Biochem. Biophys. 237, 465 (1985).
[64]
CYTOCnROMES P450 IN GUT
1
v
w
653
2
+
0, " ,
A
