[61]
EXTRAHEPATICP450
623
[61] C h a r a c t e r i z a t i o n of C y t o c h r o m e P 4 5 0 in Extrahepatic Tissues
By RICHARD M. PHILPOT
Introduction Since the liver is the primary "drug metabolism" tissue, it is not surprising that our understanding of mammalian cytochrome P450 and the P450 monooxygenase system is based chiefly on hepatic studies. This emphasis on liver is abetted by a number of characteristics, such as availability, size, high concentrations of P450, cellular homogeneity, ease of subcellular fractionation, and suitability for enzyme purification, that are not exhibited by extrahepatic tissues. However, it is clear that P450 and P450 systems are not confined to the liver and, more important, that extrahepatic P450 systems are not simply attenuated versions of hepatic systems. Each tissue appears to contain a unique complement of P450 isozymes, and each isozyme appears to have a unique pattern of distribution and regulation. This variability is further complicated by differences related to species, age, responses to inducers, and, in some cases, gender. Moreover, within a given tissue, P450 systems with distinct characteristics can be present in different cell types. The potential for this diversity to have a profound effect on the metabolism of xenobiotics offers a plausible explanation for the species-, tissue-, and cell-selective effects of many carcinogens and other toxic agents. Exploration of this possibility requires that extrahepatic P450 systems be examined thoroughly, and not treated as afterthoughts in hepatic studies. Although virtually every extrahepatic tissue has been the subject of at least one "P450 study," until recently only the pulmonary system has been dealt with in detail. Now, as presented in this volume, cytochrome P450 isozymes in nasal epithelium, renal tubular cells, gut, and brain, are being characterized. Our experience with kidney, aorta, smooth muscle, bladder, skin, and, in particular, lung provides the basis for this chapter in which general problems associated with the characterization of P450 in extra.hepatic tissues are discussed. The references given provide detailed methods developed specifically for work with extrahepatic tissues. METHODS IN ENZYMOLOGY, VOL. 206
624
CHARACTERIZATION OF EXTRAHEPATIC P450S
[61]
Characterization of P450 in Microsomal Preparations from Extrahepatic Tissues
Defining "Microsomal" The term microsomes was applied to the 100,000 g particulate fraction derived from tissue homogenates by differential centrifugation before the composition of the fraction was known. Later, the relationship between hepatic microsomes and endoplasmic reticulum (ER) was established, and the two became loosely equated. This was fine for liver but highly misleading for most extrahepatic tissues. In fact, even the term microsomes is a misnomer when applied to the I00,000 g particulate fraction from many extrahepatic sources; there are few "little bodies" in these preparations. However, we have used sucrose density centrifugation (20-40%) to obtain a pulmonary fraction that resembles the 100,000 g pellet from liver when examined by electron microscopy (R. Vanderslice and R. M. Philpot, unpublished, 1985). The concentrations of P450 (and ER) in these preparations are over 4 times higher than in the pulmonary I00,000 g pellet and nearly equal to those of hepatic samples. Isolation of such fractions is consistent with our estimation that the ER contributes only about 20% of the protein found in the pulmonary microsomal preparation. Morphological considerations suggest that the contributions of ER to the microsomal fractions of most extrahepatic tissues should also be fairly low. The consequences of this are two: first, little useful information is provided by comparisons of microsomal P450 concentrations from different tissues; second, concentrations of P450 will appear to change following any treatment that alters the contribution of ER to the microsomal preparation. For example, we have found that the pulmonary microsomal concentrations of P450IIB, P450IVB, and NADPH-cytochrome-P450 reductase increase up to 1.5-fold following treatment of rabbits with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 1 a compound that does not induce the synthesis of these enzymes. This effect could be accounted for by a 1.5fold increase in the 20% contribution of ER to the microsomal fraction, an increase that simply could not occur with liver where the contribution is already as high as 80%. In any case, marginal (less than 2-fold) changes in extrahepatic microsomal P450 concentrations should not automatically be labeled induction or repression without first considering this alternative explanation. Lastly, anyone wishing a more detailed explanation for why "microsomar' should be used only a s an operational term, and why it is inappropriate to equate the microsomal fractions from different tissues, I B. A. Domin, T. R. Devereux, and R. M. Philpot, Mol. Pharmacol. 30, 296 (1986).
[61]
EXTRAHEPATICP450
625
should refer to the work of DePierre and Dallner. z The same work provides methods for subfractionation of the 100,000 g pellet and the use of NADPH-cytochrome-P450 reductase as a marker enzyme for the endoplasmic reticulum. The investigator should be prepared to modify these methods, which were developed for use with liver, each time a different tissue is examined. Spectral Analysis
Relatively low extrahepatic microsomal P450 concentrations create two problems for the spectral determination of total P450 that are not usually encountered when working with liver. The first problem stems from attempts to overcome low content by using large samples. This can result in nonlinear responses owing to excessive absorbance and in spectral distortions caused by baseline instability, a problem exacerbated by the improper use of automatic baseline correct. When using concentrated samples (>2 mg/ml) the absolute absorbance of the sample should be determined and compared with specifications on instrument thresholds, and the stability of the baseline should be examined by making multiple "real-time" recordings. The second problem arises from the unfavorable ratio of P450 to chromophores, primarily hemoglobin (Hb) and cytochrome oxidase, that interfere with the P450 assay. The problem with Hb can be solved in two ways: (1) spectral interference by Hb can be avoided with a modified P450 assay3; (2) contamination of microsomal preparations with Hb can be avoided by vasculature perfusion of tissues. The latter approach is preferable because it also serves to remove any methemoglobin that might be present and to prevent formation of methemoglobin during subcellular fractionation. Spectral interference by methemoglobin cannot be eliminated in the same fashion as interference by Hb, and we have found that methods devised to cancel methemoglobin absorbance, which rely on selective reduction, are not effective with concentrations of methemoglobin high enough to actually cause problems. Although not as common as contamination with Hb, contamination with cytochrome oxidase at an equal concentration causes more interference, both in the reduction of peak height and in the shifting of the peak to longer wavelengths. Examples of this, such as the discovery of kidney cytochrome "P454," are to be found in the literature. Unfortunately, there is no good method for canceling cytochrome oxidase while assaying for 2j. DePierre and G. Dallner, in "BiochemicalAnalysis of Membranes" (A. H. Maddy, ed.), p. 79. Chapman & Hall, London, 1976. 3R. W. Estabrook, J. Peterson, J. Baron, and A. Hildebrandt, in "Methods of Pharmacology" (C. Chignell, ed.), p. 303. Appleton-Century-Crofts, New York, 1972.
626
CHARACTERIZATION OF EXTRAHEPATIC P450s
[61]
P450. However, even with samples where the P450 spectrum is completely obliterated by the cytochrome oxidase spectrum, a spectrum showing the presence of P450 can be resolved following selective solubilization of the microsomal pellet and removal of the undissolved mitochondrial fragments by centrifugation.4 Briefly, our recommendations for obtaining spectra of P450 from extrahepatic tissues are as follows: (1) remove hemoglobin and methemoglobin by perfusion in situ if possible; (2) if perfusion is not possible, reduce hemoglobin levels by avoiding homogenization in solutions containing sucrose and by washing the 100,000 g pellet with a pyrophosphate buffer; (3) reduce mitochondrial (cytochrome oxidase) contamination of the 100,000 g pellet by using glass on glass or Teflon on glass homogenization methods, avoiding procedures calling for the use of Polytron homogenizers or blenders; (4) remove unavoidable cytochrome oxidase contamination by treatment of the 100,000 g pellet with sodium cholate (1 mg/mg protein) and removal of unsolubilized mitochondrial fragments by centrifugation. If these precautions are followed, spectra indicative of the presence of P450 can be obtained from most tissues. However, calling these recordings P450 spectra is seldom justified. Likewise, using such spectra for calculating P450 concentrations is inappropriate unless the peak created by CO is centered at the near (---2 nm) 450 nm and is symmetrical above half-peak height. Finally, the question of P420 content needs to be addressed. In most studies of extrahepatic P450, P420 content is either dismissed as negligible or is ignored all together. Indeed, this is common even in the face of spectral evidence that sizable amounts of this breakdown product are present. Cytochrome P420 content can be assessed from the normal carbon monoxide difference spectrum used to assay P450, 5 provided that spectral interference in the area of 420 nm is at a minimum. In any case, claims of trace amounts of P420, along with reports of exceptionally low extrahepatic P450 concentrations (
EXTRAHEPATICP450
623
[61] C h a r a c t e r i z a t i o n of C y t o c h r o m e P 4 5 0 in Extrahepatic Tissues
By RICHARD M. PHILPOT
Introduction Since the liver is the primary "drug metabolism" tissue, it is not surprising that our understanding of mammalian cytochrome P450 and the P450 monooxygenase system is based chiefly on hepatic studies. This emphasis on liver is abetted by a number of characteristics, such as availability, size, high concentrations of P450, cellular homogeneity, ease of subcellular fractionation, and suitability for enzyme purification, that are not exhibited by extrahepatic tissues. However, it is clear that P450 and P450 systems are not confined to the liver and, more important, that extrahepatic P450 systems are not simply attenuated versions of hepatic systems. Each tissue appears to contain a unique complement of P450 isozymes, and each isozyme appears to have a unique pattern of distribution and regulation. This variability is further complicated by differences related to species, age, responses to inducers, and, in some cases, gender. Moreover, within a given tissue, P450 systems with distinct characteristics can be present in different cell types. The potential for this diversity to have a profound effect on the metabolism of xenobiotics offers a plausible explanation for the species-, tissue-, and cell-selective effects of many carcinogens and other toxic agents. Exploration of this possibility requires that extrahepatic P450 systems be examined thoroughly, and not treated as afterthoughts in hepatic studies. Although virtually every extrahepatic tissue has been the subject of at least one "P450 study," until recently only the pulmonary system has been dealt with in detail. Now, as presented in this volume, cytochrome P450 isozymes in nasal epithelium, renal tubular cells, gut, and brain, are being characterized. Our experience with kidney, aorta, smooth muscle, bladder, skin, and, in particular, lung provides the basis for this chapter in which general problems associated with the characterization of P450 in extra.hepatic tissues are discussed. The references given provide detailed methods developed specifically for work with extrahepatic tissues. METHODS IN ENZYMOLOGY, VOL. 206
624
CHARACTERIZATION OF EXTRAHEPATIC P450S
[61]
Characterization of P450 in Microsomal Preparations from Extrahepatic Tissues
Defining "Microsomal" The term microsomes was applied to the 100,000 g particulate fraction derived from tissue homogenates by differential centrifugation before the composition of the fraction was known. Later, the relationship between hepatic microsomes and endoplasmic reticulum (ER) was established, and the two became loosely equated. This was fine for liver but highly misleading for most extrahepatic tissues. In fact, even the term microsomes is a misnomer when applied to the I00,000 g particulate fraction from many extrahepatic sources; there are few "little bodies" in these preparations. However, we have used sucrose density centrifugation (20-40%) to obtain a pulmonary fraction that resembles the 100,000 g pellet from liver when examined by electron microscopy (R. Vanderslice and R. M. Philpot, unpublished, 1985). The concentrations of P450 (and ER) in these preparations are over 4 times higher than in the pulmonary I00,000 g pellet and nearly equal to those of hepatic samples. Isolation of such fractions is consistent with our estimation that the ER contributes only about 20% of the protein found in the pulmonary microsomal preparation. Morphological considerations suggest that the contributions of ER to the microsomal fractions of most extrahepatic tissues should also be fairly low. The consequences of this are two: first, little useful information is provided by comparisons of microsomal P450 concentrations from different tissues; second, concentrations of P450 will appear to change following any treatment that alters the contribution of ER to the microsomal preparation. For example, we have found that the pulmonary microsomal concentrations of P450IIB, P450IVB, and NADPH-cytochrome-P450 reductase increase up to 1.5-fold following treatment of rabbits with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 1 a compound that does not induce the synthesis of these enzymes. This effect could be accounted for by a 1.5fold increase in the 20% contribution of ER to the microsomal fraction, an increase that simply could not occur with liver where the contribution is already as high as 80%. In any case, marginal (less than 2-fold) changes in extrahepatic microsomal P450 concentrations should not automatically be labeled induction or repression without first considering this alternative explanation. Lastly, anyone wishing a more detailed explanation for why "microsomar' should be used only a s an operational term, and why it is inappropriate to equate the microsomal fractions from different tissues, I B. A. Domin, T. R. Devereux, and R. M. Philpot, Mol. Pharmacol. 30, 296 (1986).
[61]
EXTRAHEPATICP450
625
should refer to the work of DePierre and Dallner. z The same work provides methods for subfractionation of the 100,000 g pellet and the use of NADPH-cytochrome-P450 reductase as a marker enzyme for the endoplasmic reticulum. The investigator should be prepared to modify these methods, which were developed for use with liver, each time a different tissue is examined. Spectral Analysis
Relatively low extrahepatic microsomal P450 concentrations create two problems for the spectral determination of total P450 that are not usually encountered when working with liver. The first problem stems from attempts to overcome low content by using large samples. This can result in nonlinear responses owing to excessive absorbance and in spectral distortions caused by baseline instability, a problem exacerbated by the improper use of automatic baseline correct. When using concentrated samples (>2 mg/ml) the absolute absorbance of the sample should be determined and compared with specifications on instrument thresholds, and the stability of the baseline should be examined by making multiple "real-time" recordings. The second problem arises from the unfavorable ratio of P450 to chromophores, primarily hemoglobin (Hb) and cytochrome oxidase, that interfere with the P450 assay. The problem with Hb can be solved in two ways: (1) spectral interference by Hb can be avoided with a modified P450 assay3; (2) contamination of microsomal preparations with Hb can be avoided by vasculature perfusion of tissues. The latter approach is preferable because it also serves to remove any methemoglobin that might be present and to prevent formation of methemoglobin during subcellular fractionation. Spectral interference by methemoglobin cannot be eliminated in the same fashion as interference by Hb, and we have found that methods devised to cancel methemoglobin absorbance, which rely on selective reduction, are not effective with concentrations of methemoglobin high enough to actually cause problems. Although not as common as contamination with Hb, contamination with cytochrome oxidase at an equal concentration causes more interference, both in the reduction of peak height and in the shifting of the peak to longer wavelengths. Examples of this, such as the discovery of kidney cytochrome "P454," are to be found in the literature. Unfortunately, there is no good method for canceling cytochrome oxidase while assaying for 2j. DePierre and G. Dallner, in "BiochemicalAnalysis of Membranes" (A. H. Maddy, ed.), p. 79. Chapman & Hall, London, 1976. 3R. W. Estabrook, J. Peterson, J. Baron, and A. Hildebrandt, in "Methods of Pharmacology" (C. Chignell, ed.), p. 303. Appleton-Century-Crofts, New York, 1972.
626
CHARACTERIZATION OF EXTRAHEPATIC P450s
[61]
P450. However, even with samples where the P450 spectrum is completely obliterated by the cytochrome oxidase spectrum, a spectrum showing the presence of P450 can be resolved following selective solubilization of the microsomal pellet and removal of the undissolved mitochondrial fragments by centrifugation.4 Briefly, our recommendations for obtaining spectra of P450 from extrahepatic tissues are as follows: (1) remove hemoglobin and methemoglobin by perfusion in situ if possible; (2) if perfusion is not possible, reduce hemoglobin levels by avoiding homogenization in solutions containing sucrose and by washing the 100,000 g pellet with a pyrophosphate buffer; (3) reduce mitochondrial (cytochrome oxidase) contamination of the 100,000 g pellet by using glass on glass or Teflon on glass homogenization methods, avoiding procedures calling for the use of Polytron homogenizers or blenders; (4) remove unavoidable cytochrome oxidase contamination by treatment of the 100,000 g pellet with sodium cholate (1 mg/mg protein) and removal of unsolubilized mitochondrial fragments by centrifugation. If these precautions are followed, spectra indicative of the presence of P450 can be obtained from most tissues. However, calling these recordings P450 spectra is seldom justified. Likewise, using such spectra for calculating P450 concentrations is inappropriate unless the peak created by CO is centered at the near (---2 nm) 450 nm and is symmetrical above half-peak height. Finally, the question of P420 content needs to be addressed. In most studies of extrahepatic P450, P420 content is either dismissed as negligible or is ignored all together. Indeed, this is common even in the face of spectral evidence that sizable amounts of this breakdown product are present. Cytochrome P420 content can be assessed from the normal carbon monoxide difference spectrum used to assay P450, 5 provided that spectral interference in the area of 420 nm is at a minimum. In any case, claims of trace amounts of P420, along with reports of exceptionally low extrahepatic P450 concentrations (
