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brought to I ml with homogenization buffer. To initiate the reaction, 3.3 pmol of 1,25-(OH)2126,27-3H]D3 is added in 2/xl of ethanol. Metabolism was not observed in the microsomal or cytosolic fractions but was observed with the homogenate, nuclei]plasma membrane (P2), and mitochondrial fractions (P3). No qualitative differences were observed after analysis with HPLC-I among the fractions that produced metabolites. The mitochondrial or nuclei]plasma membrane fractions were equally effective at converting 1,25-(OH)2126,27-aH]D3 to the metabolites shown in Fig. 1 (Table II). It is not clear whether this represents metabolism by the nuclei and/or plasma membrane, or contamination by whole cells and/or mitochondria, but it is clear that microsomes are not a major locus of 1,25(OH)2D3 catabolism. Acknowledgments This work was supported by National Institutes of Health Grant DK36870.
[48] M e a s u r e m e n t o f C y t o c h r o m e P 4 5 0 A c t i v a t i o n o f X e n o b i o t i c s U s i n g t h e A m e s Salmonella T e s t
By M. E. MCMANUS and R. A. MCKINNON Introduction The toxicity of many chemicals is due to their metabolic conversion to reactive electrophiles which interact irreversibly with critical nucleophilic sites on cellular macromolecules. 1In both laboratory animals and humans the cytochrome P450 system has been shown to be the major enzyme system involved in the activation of most xenobiotics. 2 It is generally accepted that for the vast majority of carcinogens, the initiation stage of carcinogenesis involves a critical activation step. This metabolic activation results in the formation of an electrophile capable of causing an inheritable alteration in the DNA of a cell (mutation)) This link between metabolic activation and mutational events has guided much of the work aimed at establishing the role of the cytochrome P450 system in the carcinogenic process. J. A. Miller, Cancer Res. 30, 559 (1970). z F. P. Guengerich, Cancer Res. 48, 2946 (1988). 3 j. McCann, E. Choi, E. Yamasaki, and B. N. Ames, Proc. Natl. Acad. Sci. U.S,A. 72, 5135 (1975).
METHODS 1N ENZYMOLOGY, VOL, 206
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
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Of all the tests used for determining the potential mutagenicity of a chemical, the Ames test has received the greatest attention. 4 This test measures the ability of a xenobiotic following activation to revert histidinerequiring strains of Salmonella typhimurium back to the wild type which thereby regains the ability to grow in a histidine-deficient environment. The popularity of the Ames test stems from the fact that it is (1) simple to perform, (2) relatively inexpensive, (3) very sensitive, and (4) able to detect both base-pair substitution and frameshift mutations. The Ames test in chemical genotoxicity studies has generally been used to answer two questions. First, the Ames test can determine whether following metabolism a chemical causes genetic damage. In this regard the test has been quite successful, and its utility has been increased by additional mutations to the tester strains enhancing their sensitivity. 4 Second, the test has been used to predict the potential carcinogenicity of chemicals, and in this regard it has not always been possible to show a direct relationship between mutagenicity and carcinogenicity.5,6 Traditionally, the Ames Salmonella test is carried out by combining the test compound, the bacterial tester strain, the $9 mix, and soft top agar. This mixture is then poured onto a minimal agar plate. 4 However, because of the physicochemical properties of different chemicals, it has not always been possible to detect a mutagenic response using the standard plate incorporation test. This has prompted various modifications of the initial test such as a preincubation procedure to detect the mutagenicity of azo dyes 7 and aliphatic nitrosamines,8 a chamber incubation for volatile gases, 9 and an anaerobic preincubation for detecting the mutagenicity of nitroaromatic compounds requiring reduction.l° Thus, a negative result in the plate incorporation test should be investigated further by manipulating the procedure before concluding that a chemical is nonmutagenic in the Ames test. Although the $9 mix has been used most extensively as the metabolic activation source in the Ames test, various workers have also employed
4 D. M. Maron and B. N. Ames, Mutat. Res. 113, 173 (1983). S. J. Rinkus and M. S. Legator, Cancer Res. 39, 3289 (1979). 6 j. Ashby, R. W. Tennant, E. Zeigler, and S. Stasiewicz, Murat. Res. 223, 73 (1989). 7 T. Yahagi, M. Degawa, Y. Senio, T. Matsushima, M. Nagao, T. Sugimura, and Y. Hashimoto, Cancer Lett. 1, 91 (1975). 8 T. Yahagi, M. Nagao, Y. Senio, T. Matsushima, T. Sugimura, and M. Okada, Mutat. Res. 48, 121 (1977). 9 U. Rannung, A. Johansson, C. Ramel, and C. A. Wachmeister, Ambio 3, 194 (1974). l0 M. J. Prival and V. D. Mitchell, Murat. Res. 97, 103 (1982).
[48]
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a Salrnonella/hepatocyte system. 11,12This test system enables the simultaneous determination of the mutation frequency in the bacteria and the genotoxic effects in the hepatocytes. In this system the hepatocytes are carrying out the metabolic activation of the chemical, and only the electrophiles that escape the detoxification mechanisms will induce bacterial mutagenesis. This approach has proved valuable in demonstrating how the modulation of specific cytochrome P450 isozymes can affect the genotoxicity of a compound at the cellular level when all the defense mechanisms are presentJ 3
Metabolic Activation Source In the majority of studies the $9 fraction from Aroclor 1254-pretrcated rats has been used as the metabolic activation source. Aroclor 1254 is a mixture of polychlorinated biphenyls which have inducing properties similar to a combination of phenobarbital and 3-methylcholanthrene. 14 Thus, an array of cytochrome P450 isozymes are being induced in Aroclor 1254treated animals, and this is reflected by a marked induction of total liver microsomal cytochrome P450. It is probable that one of the induced isozymes will be responsible for the activation of the chemical under study. It is important to realize, however, that not all inducing agents cause an increase in total cytochrome P450 content of microsomes. For example, isoniazid induces P450IIE1 in rat liver microsomes without any increase in total cytochrome P450 content, suggesting that other isozymes are repressed. 14 As a crude indicator of what isozyme is responsible for the activation of a chemical, one should first determine the activity of a range of liver microsomes prepared from animals pretreated with different inducing agents. It is preferable to use microsomes rather than the $9 fraction as this circumvents the problems associated with detoxification of electrophiles by nucleophiles such as glutathione. The following agents have been shown to induce specific P450 isozymes: 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD) and 3-methylcholanthrene, P450IA1 and P450IA2; phenobarbital, P450IIB 1 and P450IIB2 in the rat and P450IIB4 and P450IVB 1 in the rabbit; rifampicin, P450IIIA6 in the rabbit; dexamethasone, P450IIIA1 II N. Staiano, L. C. Erickson, and S. S. Thorgeirsson, Biochem. Biophys. Res. Comrnun. 94, 837 (1980). 12 K. Williams, J. Inmon, and J. Lewtas, Teratog. Carcinog. Mutagen 3, 367 (1983). 13 N. Staiano, L. C. Erickson, C. L. Smith, E. Marsden, and S. S. Thorgeirsson, Carcinogenesis 4, 161 (1983). 14 D. E. Ryan and W. Levin, Pharmacol. Ther. 45, 153 (1990).
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in the rat; and ethanol, P450IIE1.14,15 Studies utilizing microsomes from the above species as the enzyme source will indicate the likely isozyme of P450 responsible for the activation of a particular compound. The result should then be confirmed using purified cytochrome P450 isozymes as the activation s o u r c e . 16'17 To negate criticism of the reconstituted system producing an artifact, an antibody raised against the active isozyme should be tested for its ability to inhibit mutagenesis when microsomes are used as the activation source. If the antibody is noninhibitory, it is possible to confirm the degree of involvement of an isozyme by correlating its level in control and a range of pretreated microsomes with the number of revertants formed in the Ames test. ~6-18 These studies can be complemented by incorporating into the incubation mixture known inhibitors and activators of particular isozymes.18 Because of the existence of multiple forms of cytochrome P450, their overlapping substrate specificities, and the microheterogeneity that exists within many subfamilies, it has not always been possible to definitively assign an activity to a particular isozyme. This ambiguity may occur despite using purified proteins as the activation source. Recently, however, the cDNAs for at least 75 cytochrome P450 genes and pseudogenes have been isolated and characterized. 19.20These cDNAs may be expressed in cytochrome P450-deficient cells, thereby providing an unambiguous system for determining the catalytic function of an isozyme. ~8'2L22 It is pertinent to remember, however, that such expression systems are artificial, and results should be confirmed at the microsomal or cellular level. In addition, when comparing the relative abilities of different expressed cytochromes P450 to activate a xenobiotic, it is important to determine the amount of isozyme present in the cell lysates so activities can be related to enzyme content. This can be achieved using immunochemical techniques such as the Western blot procedure, zl
15G. E. Schwab and E. F. Johnson, in "Mammalian Cytochromes P450" (F. P. Guengerich, ed.), p. 55. CRC Press, Boca Raton, Florida, 1987. 16M. E. McManus, W. Burgess, E. Snyderwine, and I. Stupans, Cancer Res. 48, 4513 (1988). 17 M. E. McManus, J. S. Felton, M. G. Knize, W. M. Burgess, S. Roberts-Thomson, S. M. Pond, I. Stupans, and M. E. Veronese, Carcinogenesis 10, 357 (1989). 18 M. E. McManus, W. M. Burgess, M. E. Veroncse, A. Huggett, L. C. Quattrochi, and R. H. Tukey, Cancer Res. 50, 3367 (1990). t9 F. J. Gonzalez, Pharmacol. Rev. 40, 243 (1989). 2o D. W. Nebert, D. R. Nelson, M. Adesnik, M. J. Coon, R. W. Estabrook, F. J. Gonzalez, F. P. Guengerich, I. C. Gunsalus, E. F. Johnson, B. Kemper, W. Levin, I. R. Phillips, R. Sato, and M. R. Waterman, D N A 8, 1 (1989). 2t E. G. Snyderwine and N. Battula, J. Natl. Cancer Inst. 81, 223 (1989). 22 T. Aoyama, F. J. Gonzalez, and H. V. Gelboin, Mol. Carcinog. 1, 252 (1989).
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Preincubation Procedure In this modification of the original A m e s test,4'vthe activation source, bacteria, and test chemical arc preincubatcd in a finalvolume of 0.5-0.75 ml for varying times prior to the addition of the top agar.16-IsThis brings the bacterial target into close proximity of thc cytochromc P450 isozymcs producing the reactive elcctrophilcs and increases the chance of causing D N A damage. Although a prcincubation step lengthens the procedure, it is used routinely in our laboratory because of thc enhanced sensitivity it produccs. It has also proved valuable for detecting thc mutagcnicity of some chemicals that arc negative in the standard plate incorporation tcst.4 Other advantages of this procedure arc that (1) smaller amounts of purified and expressed proteins arc required owing to the increased sensitivity, and (2) the reaction time can bc tightlycontroUcd by stopping itat the end of the incubation time with a cycling agent such as mcnadionc (Sigma, St. Louis, MO). The prcincubation step is no different to a normal cnzymatic reaction, but instead of measuring mctabolitc production wc arc determining mutagenie hits on bacterial D N A , being expressed as bacterial growth. As with other enzymatic assays, the reaction conditions should bc clearly dcfincd. Thc rcaction rates should be zero ordcr with respect to cofactors and linear with rcspcct to protein concentration and time. If only single point determinations arc being carried out, maximal reaction ratcs should bc determined at a substratc concentration approximately 4 times the apparent Kin. Howcvcr, owing to bacterial toxicity or solubilityproblems with many chemicals, itis not always possible to adhere to the above guidelines. Nonetheless, it is important to clearly record the substratc concentration uscd when comparing thc ability of different cytochromc P450 isozymcs to activatc promutagens. Other factors which prevent us from applying strict Michaclis-Mentcn kinetics to the A m e s test arc (I) the possibility that the bacteria may further process the mctabolitc produced by thc cytochromc P450 isozymes23; (2) differences in the rcactivity of mctabolitcstoward bactcrial D N A bases; and (3) the rclativc stabilityof different clcctrophilcs. Salmonella Tester Strains
If the appropriate bacterial strain has not been determined for a particular xenobiotic, a series of tester strains sensitive to base-pair mutations (TA1535) and frameshift mutations (TA98 and TA1537) should be used in 23 R. Kato, Crit. Rev. Toxicol. 16, 307 (1986).
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the preincubation test with the metabolic activation source. The tester strains TA100, TA102, and TA104 are now sensitive to frameshift as well as base-pair substitution mutagens because of the introduction of the pKM 101 (R factor) plasmid. The R factor increases the incidence of spontaneous as well as chemical mutagenesis by enhancing an error-prone DNA repair system. 4 Regardless of the type of tester strain being used, it is prudent to always run the bacteria in each experiment with and without the activation source, solvent, and xenobiotic. The number of background revertants should remain constant from experiment to experiment, providing a measure of the stability of the tester strain. Should the background vary significantly from one batch of xenobiotic to the next, it is important to look very closely at the purity of the compound. The phenotype of each tester strain should be confirmed: (1) on receipt of the cultures; (2) if a new frozen batch is used; and (3) if the number of spontaneous revertants varies or the sensitivity of a strain to a known mutagen is altered. A detailed description of all the tester strains available and the composition of the different media and reagents is beyond the scope of this chapter. For a detailed treatise of these, one should consult the excellent review by Maron and Ames. 4 Test Procedure
Preparation of Metabolic Activation Source. Sterile preparations of most animal tissues can be obtained by removing the tissue aseptically and carrying out all steps at 00-4 ° using sterile solutions and glassware. In the case of human tissues, which are usually obtained at best 30 min after death and handled by pathology staff before being available for research purposes, it is necessary to filter sterilize the $9 or microsomal fractions before use. Purified cytochrome P450 isozymes and gastrointestinal tract and lung tissue from animals are also routinely filter sterilized in our laboratory before use in the Ames test. Cell cultures in which cytochrome P450 cDNAs have been expressed are processed aseptically. The difficulty with many filters is the retention of a considerable amount of protein. In addition, we have found that when preparing microsomes from the gastrointestinal tract of humans, some particulate matter is retained, To overcome these difficulties we routinely prefilter the subcellular fraction through a 5/zm Millex-SV filter (Millipore, Bedford, MA, catalog number SLSV025LS) and then sterilize the preparation by passing it through a 0.22/.~m Millex-GV (Millipore, catalog number SLGV025LS) or Millex-HV (Millipore, catalog number SLHV025LS) filter. In the case of human liver microsomes only a 0.22 or 0.45/zm filtration step is required. All the above filters are listed in the Millipore catalog as being low protein
[48]
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retention units. When we sterilize a 2 mg/ml human liver microsomal solution through a 0.22 ~m filter we lose approximately 20% of the protein. Because many of our purified cytochrome P450 isozyme preparations are not as concentrated as the above, we generally lose less than 5% of the protein on filtration. The yield of purified isozymes following filtration can be enhanced by using a 0.22 tzm Miilex-GV (Millipore, catalog number SLGV013OS) instead of the SLGV025LS filter. The former filter possesses a surface area 3 times smaller than the latter. Microsomes and purified cytochrome P450 isozymes are stable for prolonged periods of time (e.g., 3 years) when stored at - 8 0 ° in 0.1 M potassium phosphate buffer (pH 7.4) containing 20% glycerol. Maron and Ames 4 also report that $9 fractions are stable for up to 2 years when stored at - 80°. We routinely sterilize a vial of the activation source on the day of the experiment and determine the protein concentration of the filtrate before carrying out the assay. This has proved satisfactory for small assays up to 50 plates, but alternatively one may wish to filter a stock of microsomes before aliquoting and storing them at - 8 0 °. Regardless of when the filtration step is carried out, the protein concentration of the filtrate should always be determined. As a source of reducing equivalents we have usually used N A D P H made up immediately prior to the assay and filter sterilized using a 0.22/zm Millex GS unit (Millipore, catalog number SLGS025OS). It is important to establish that the amount of N A D P H added to the incubation mixture is not rate limiting. Preparation of Tester Strain. The appropriate tester strain is grown up overnight in sterile Oxoid nutrient broth No. 2 (Oxoid, Basingstoke, England) to a density of 1-2 × 109 cells/ml. For a 50-plate assay we routinely scrape a sterile wooden stick across the frozen culture ( - 80°) and add the bacteria to 10 ml of Oxoid broth. The bacteria are then grown up overnight in a loosely capped flask incubated in a Ratek Instruments (Melbourne, Australia) bacterial shaker (37°) set at 100 rpm. One hundred microliters of the culture is added to each assay. Dilution ofXenobiotic Substrates. Unless a xenobiotic is soluble and stable in aqueous solution we have used dimethyl sulfoxide (DMSO) as a universal solvent for dissolving substrates. The advantage of using organic solvents such as DMSO is that it avoids the necessity of filter sterilizing the xenobiotic-containing solutions. It is important to determine that the solvent used does not have an inhibitory effect on the cytochrome P450 system. Indeed, it is important to test the effect of the solvent on each isozyme since the degree of inhibition may vary depending on the isozyme used. In general we have always restricted the amount of DMSO to 1 to 2% of the incubation mixture. Incubation Mixture for Preincubation Test. A standard 0.5 ml incuba-
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tion mixture contains microsomal protein, 1-2 x 10s bacterial cells, 1.2 /zmol NADPH added in 50/zl of 0.1 M potassium phosphate buffer (pH 7.4), xenobiotic dissolved in 5-10/.d DMSO, and 50/zmol sodium phosphate (pH 7.4). For reconstitution experiments we have generally used I0 pmol of purified P450 isozyme, 0.5 units of NADPH-cytochrome-P450 reductase, and 75/~g of dilauroyl-L-a-lecithin. However, depending on the xenobiotic and isozyme, the composition of the reconstituted system will vary. When cytochrome P450 expression systems are used, cell lysate protein is substituted for the microsomal protein. The reaction mixture is preincubated at 37° for 3 min prior to adding the bacteria and NADPH. The reaction is terminated by the addition of 0. I ml of 0.12 M menadione solution and then placed on ice. The menadione stops the reaction by competing for electrons from NADPH. Following the addition of 2 ml of molten top agar at 45 °, the incubation mixtures are poured onto petri dishes containing 30 ml minimal top agar and a limited amount of L-histidine. At this step it is possible to process three tubes simultaneously before the top agar sets and prevents pouring of the plates. The colonies on each plate are scored after a 48-hr incubation period at 37°. When antibody inhibition studies are being conducted, the amount of protein in each tube should be kept constant by the addition of preimmune immunoglobulin G (IgG). This is important as the protein may react with the electrophiles, and variability in the number of revertants formed may only reflect the protein content. As an indication of the integrity of a bacterial strain and the stability of the enzyme system, we incorporate into each experiment various positive controls. For tester strain TA98 we have routinely used 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) as the model mutagen. When determining the capacity of extrahepatic tissues to metabolize a mutagen, liver microsomes are routinely run in parallel. The reproducibility of these standard controls is excellent and provides a useful means of evaluating the technique. Data Analysis. A less than clear-cut result for mutagenicity of a xenobiotic in the Ames test can be a product of the substrate concentration or the amount of activation source added to the incubation. This again highlights the need to determine the correct reaction conditions for each xenobiotic. In the case of a negative result, it is important to establish by inspecting the bacterial lawn that the result does not reflect that the chemical has been toxic to the bacteria. As a trace amount of histidine is added to the top agar, all the bacteria on the plate undergo several divisions. If a toxic response has been elicited the lawn becomes mottled in appearance. Results should be reported as the mean count of at least three plates plus or minus the standard deviation. If the data are to be expressed as the number of revertants per milligram of microsomal protein or microgram of chemical, it is advantageous to provide the raw plate data together with
[49]
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the relevant controls (e.g., minus generating system). Further, if expressing the effect of antibody inhibition on mutagenicity as a percentage of original activity, it is appropriate to give original activities. This ensures the reader can work backward from the transformation and accurately derive the original data. Biohazard Requirements. The wild-type Salmonella typhimurium strain can cause food poisoning. Although the genetically manipulated strains used in the Ames test are not very virulent, it is prudent to use care when handling these bacteria. 4 Since many of the xenobiotics used will be mutagenic and carcinogenic, extreme care should be taken to prevent human exposure. Workers should consult local Biohazard Committee guidelines before commencing an experiment. Ideally all laboratory materials used should be of the disposable type, and guidelines for the disposal of both biological and carcinogenic waste should be strictly followed.
[49] B u f u r a l o l , D e x t r o m e t h o r p h a n , a n d D e b r i s o q u i n e as P r o t o t y p e S u b s t r a t e s for H u m a n P 4 5 0 I I D 6
By
THOMAS
KRONBACH
Introduction The human debrisoquine/sparteine-type genetic polymorphism of drug oxidation affects the expression of cytochrome P450IID6,1,2 an enzyme which is involved in the metabolism of many drugs (reviewed in Ref. 3). Although the P450 enzymes are often believed to exhibit a broad and overlapping substrate specificity, many drugs which are substrates for P450IID6 are only marginally metabolized in individuals affected by this polymorphism (poor metabolizers). This indicates that, among the other hepatic P450s, P450IID6 has a unique substrate selectivity and is the major catalyst involved in the clearance of these drugs. This chapter describes assays for the metabolism of three prototype substrates for P450IID6. The experimental/t-blocking agent bufuralol is metabolized by P450IID6 by l'-hydroxylation, the antihypertensive drug debrisoquine is metabolized by 4-hydroxylation, and dextromethorphan is F. J. Gonzalez, R. C. Skoda, S. Kimura, M. Umeno, U. M. Zanger, D. W. Nebert, H. V. Gelboin, J. P. Hardwick, and U. A. Meyer, Nature (London) 331, 442 (1988). 2 U. M. Zanger, F. Vilbois, J. P. Hardwick,and U. A. Meyer,Biochemistry27, 5447(1988). 3 U. A. Meyer, R. C. Skoda, and U. M. Zanger, Pharmacol. Ther. 46, 297 (1990). METHODS IN ENZYMOLOGY, VOL. 206
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
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brought to I ml with homogenization buffer. To initiate the reaction, 3.3 pmol of 1,25-(OH)2126,27-3H]D3 is added in 2/xl of ethanol. Metabolism was not observed in the microsomal or cytosolic fractions but was observed with the homogenate, nuclei]plasma membrane (P2), and mitochondrial fractions (P3). No qualitative differences were observed after analysis with HPLC-I among the fractions that produced metabolites. The mitochondrial or nuclei]plasma membrane fractions were equally effective at converting 1,25-(OH)2126,27-aH]D3 to the metabolites shown in Fig. 1 (Table II). It is not clear whether this represents metabolism by the nuclei and/or plasma membrane, or contamination by whole cells and/or mitochondria, but it is clear that microsomes are not a major locus of 1,25(OH)2D3 catabolism. Acknowledgments This work was supported by National Institutes of Health Grant DK36870.
[48] M e a s u r e m e n t o f C y t o c h r o m e P 4 5 0 A c t i v a t i o n o f X e n o b i o t i c s U s i n g t h e A m e s Salmonella T e s t
By M. E. MCMANUS and R. A. MCKINNON Introduction The toxicity of many chemicals is due to their metabolic conversion to reactive electrophiles which interact irreversibly with critical nucleophilic sites on cellular macromolecules. 1In both laboratory animals and humans the cytochrome P450 system has been shown to be the major enzyme system involved in the activation of most xenobiotics. 2 It is generally accepted that for the vast majority of carcinogens, the initiation stage of carcinogenesis involves a critical activation step. This metabolic activation results in the formation of an electrophile capable of causing an inheritable alteration in the DNA of a cell (mutation)) This link between metabolic activation and mutational events has guided much of the work aimed at establishing the role of the cytochrome P450 system in the carcinogenic process. J. A. Miller, Cancer Res. 30, 559 (1970). z F. P. Guengerich, Cancer Res. 48, 2946 (1988). 3 j. McCann, E. Choi, E. Yamasaki, and B. N. Ames, Proc. Natl. Acad. Sci. U.S,A. 72, 5135 (1975).
METHODS 1N ENZYMOLOGY, VOL, 206
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
502
ENZYME ASSAYS
[48]
Of all the tests used for determining the potential mutagenicity of a chemical, the Ames test has received the greatest attention. 4 This test measures the ability of a xenobiotic following activation to revert histidinerequiring strains of Salmonella typhimurium back to the wild type which thereby regains the ability to grow in a histidine-deficient environment. The popularity of the Ames test stems from the fact that it is (1) simple to perform, (2) relatively inexpensive, (3) very sensitive, and (4) able to detect both base-pair substitution and frameshift mutations. The Ames test in chemical genotoxicity studies has generally been used to answer two questions. First, the Ames test can determine whether following metabolism a chemical causes genetic damage. In this regard the test has been quite successful, and its utility has been increased by additional mutations to the tester strains enhancing their sensitivity. 4 Second, the test has been used to predict the potential carcinogenicity of chemicals, and in this regard it has not always been possible to show a direct relationship between mutagenicity and carcinogenicity.5,6 Traditionally, the Ames Salmonella test is carried out by combining the test compound, the bacterial tester strain, the $9 mix, and soft top agar. This mixture is then poured onto a minimal agar plate. 4 However, because of the physicochemical properties of different chemicals, it has not always been possible to detect a mutagenic response using the standard plate incorporation test. This has prompted various modifications of the initial test such as a preincubation procedure to detect the mutagenicity of azo dyes 7 and aliphatic nitrosamines,8 a chamber incubation for volatile gases, 9 and an anaerobic preincubation for detecting the mutagenicity of nitroaromatic compounds requiring reduction.l° Thus, a negative result in the plate incorporation test should be investigated further by manipulating the procedure before concluding that a chemical is nonmutagenic in the Ames test. Although the $9 mix has been used most extensively as the metabolic activation source in the Ames test, various workers have also employed
4 D. M. Maron and B. N. Ames, Mutat. Res. 113, 173 (1983). S. J. Rinkus and M. S. Legator, Cancer Res. 39, 3289 (1979). 6 j. Ashby, R. W. Tennant, E. Zeigler, and S. Stasiewicz, Murat. Res. 223, 73 (1989). 7 T. Yahagi, M. Degawa, Y. Senio, T. Matsushima, M. Nagao, T. Sugimura, and Y. Hashimoto, Cancer Lett. 1, 91 (1975). 8 T. Yahagi, M. Nagao, Y. Senio, T. Matsushima, T. Sugimura, and M. Okada, Mutat. Res. 48, 121 (1977). 9 U. Rannung, A. Johansson, C. Ramel, and C. A. Wachmeister, Ambio 3, 194 (1974). l0 M. J. Prival and V. D. Mitchell, Murat. Res. 97, 103 (1982).
[48]
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a Salrnonella/hepatocyte system. 11,12This test system enables the simultaneous determination of the mutation frequency in the bacteria and the genotoxic effects in the hepatocytes. In this system the hepatocytes are carrying out the metabolic activation of the chemical, and only the electrophiles that escape the detoxification mechanisms will induce bacterial mutagenesis. This approach has proved valuable in demonstrating how the modulation of specific cytochrome P450 isozymes can affect the genotoxicity of a compound at the cellular level when all the defense mechanisms are presentJ 3
Metabolic Activation Source In the majority of studies the $9 fraction from Aroclor 1254-pretrcated rats has been used as the metabolic activation source. Aroclor 1254 is a mixture of polychlorinated biphenyls which have inducing properties similar to a combination of phenobarbital and 3-methylcholanthrene. 14 Thus, an array of cytochrome P450 isozymes are being induced in Aroclor 1254treated animals, and this is reflected by a marked induction of total liver microsomal cytochrome P450. It is probable that one of the induced isozymes will be responsible for the activation of the chemical under study. It is important to realize, however, that not all inducing agents cause an increase in total cytochrome P450 content of microsomes. For example, isoniazid induces P450IIE1 in rat liver microsomes without any increase in total cytochrome P450 content, suggesting that other isozymes are repressed. 14 As a crude indicator of what isozyme is responsible for the activation of a chemical, one should first determine the activity of a range of liver microsomes prepared from animals pretreated with different inducing agents. It is preferable to use microsomes rather than the $9 fraction as this circumvents the problems associated with detoxification of electrophiles by nucleophiles such as glutathione. The following agents have been shown to induce specific P450 isozymes: 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD) and 3-methylcholanthrene, P450IA1 and P450IA2; phenobarbital, P450IIB 1 and P450IIB2 in the rat and P450IIB4 and P450IVB 1 in the rabbit; rifampicin, P450IIIA6 in the rabbit; dexamethasone, P450IIIA1 II N. Staiano, L. C. Erickson, and S. S. Thorgeirsson, Biochem. Biophys. Res. Comrnun. 94, 837 (1980). 12 K. Williams, J. Inmon, and J. Lewtas, Teratog. Carcinog. Mutagen 3, 367 (1983). 13 N. Staiano, L. C. Erickson, C. L. Smith, E. Marsden, and S. S. Thorgeirsson, Carcinogenesis 4, 161 (1983). 14 D. E. Ryan and W. Levin, Pharmacol. Ther. 45, 153 (1990).
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ENZYME ASSAYS
[48]
in the rat; and ethanol, P450IIE1.14,15 Studies utilizing microsomes from the above species as the enzyme source will indicate the likely isozyme of P450 responsible for the activation of a particular compound. The result should then be confirmed using purified cytochrome P450 isozymes as the activation s o u r c e . 16'17 To negate criticism of the reconstituted system producing an artifact, an antibody raised against the active isozyme should be tested for its ability to inhibit mutagenesis when microsomes are used as the activation source. If the antibody is noninhibitory, it is possible to confirm the degree of involvement of an isozyme by correlating its level in control and a range of pretreated microsomes with the number of revertants formed in the Ames test. ~6-18 These studies can be complemented by incorporating into the incubation mixture known inhibitors and activators of particular isozymes.18 Because of the existence of multiple forms of cytochrome P450, their overlapping substrate specificities, and the microheterogeneity that exists within many subfamilies, it has not always been possible to definitively assign an activity to a particular isozyme. This ambiguity may occur despite using purified proteins as the activation source. Recently, however, the cDNAs for at least 75 cytochrome P450 genes and pseudogenes have been isolated and characterized. 19.20These cDNAs may be expressed in cytochrome P450-deficient cells, thereby providing an unambiguous system for determining the catalytic function of an isozyme. ~8'2L22 It is pertinent to remember, however, that such expression systems are artificial, and results should be confirmed at the microsomal or cellular level. In addition, when comparing the relative abilities of different expressed cytochromes P450 to activate a xenobiotic, it is important to determine the amount of isozyme present in the cell lysates so activities can be related to enzyme content. This can be achieved using immunochemical techniques such as the Western blot procedure, zl
15G. E. Schwab and E. F. Johnson, in "Mammalian Cytochromes P450" (F. P. Guengerich, ed.), p. 55. CRC Press, Boca Raton, Florida, 1987. 16M. E. McManus, W. Burgess, E. Snyderwine, and I. Stupans, Cancer Res. 48, 4513 (1988). 17 M. E. McManus, J. S. Felton, M. G. Knize, W. M. Burgess, S. Roberts-Thomson, S. M. Pond, I. Stupans, and M. E. Veronese, Carcinogenesis 10, 357 (1989). 18 M. E. McManus, W. M. Burgess, M. E. Veroncse, A. Huggett, L. C. Quattrochi, and R. H. Tukey, Cancer Res. 50, 3367 (1990). t9 F. J. Gonzalez, Pharmacol. Rev. 40, 243 (1989). 2o D. W. Nebert, D. R. Nelson, M. Adesnik, M. J. Coon, R. W. Estabrook, F. J. Gonzalez, F. P. Guengerich, I. C. Gunsalus, E. F. Johnson, B. Kemper, W. Levin, I. R. Phillips, R. Sato, and M. R. Waterman, D N A 8, 1 (1989). 2t E. G. Snyderwine and N. Battula, J. Natl. Cancer Inst. 81, 223 (1989). 22 T. Aoyama, F. J. Gonzalez, and H. V. Gelboin, Mol. Carcinog. 1, 252 (1989).
[48]
XENOBIOTIC ACTIVATION BY CYTOCHROMES P450
505
Preincubation Procedure In this modification of the original A m e s test,4'vthe activation source, bacteria, and test chemical arc preincubatcd in a finalvolume of 0.5-0.75 ml for varying times prior to the addition of the top agar.16-IsThis brings the bacterial target into close proximity of thc cytochromc P450 isozymcs producing the reactive elcctrophilcs and increases the chance of causing D N A damage. Although a prcincubation step lengthens the procedure, it is used routinely in our laboratory because of thc enhanced sensitivity it produccs. It has also proved valuable for detecting thc mutagcnicity of some chemicals that arc negative in the standard plate incorporation tcst.4 Other advantages of this procedure arc that (1) smaller amounts of purified and expressed proteins arc required owing to the increased sensitivity, and (2) the reaction time can bc tightlycontroUcd by stopping itat the end of the incubation time with a cycling agent such as mcnadionc (Sigma, St. Louis, MO). The prcincubation step is no different to a normal cnzymatic reaction, but instead of measuring mctabolitc production wc arc determining mutagenie hits on bacterial D N A , being expressed as bacterial growth. As with other enzymatic assays, the reaction conditions should bc clearly dcfincd. Thc rcaction rates should be zero ordcr with respect to cofactors and linear with rcspcct to protein concentration and time. If only single point determinations arc being carried out, maximal reaction ratcs should bc determined at a substratc concentration approximately 4 times the apparent Kin. Howcvcr, owing to bacterial toxicity or solubilityproblems with many chemicals, itis not always possible to adhere to the above guidelines. Nonetheless, it is important to clearly record the substratc concentration uscd when comparing thc ability of different cytochromc P450 isozymcs to activatc promutagens. Other factors which prevent us from applying strict Michaclis-Mentcn kinetics to the A m e s test arc (I) the possibility that the bacteria may further process the mctabolitc produced by thc cytochromc P450 isozymes23; (2) differences in the rcactivity of mctabolitcstoward bactcrial D N A bases; and (3) the rclativc stabilityof different clcctrophilcs. Salmonella Tester Strains
If the appropriate bacterial strain has not been determined for a particular xenobiotic, a series of tester strains sensitive to base-pair mutations (TA1535) and frameshift mutations (TA98 and TA1537) should be used in 23 R. Kato, Crit. Rev. Toxicol. 16, 307 (1986).
506
ENZYME ASSAYS
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the preincubation test with the metabolic activation source. The tester strains TA100, TA102, and TA104 are now sensitive to frameshift as well as base-pair substitution mutagens because of the introduction of the pKM 101 (R factor) plasmid. The R factor increases the incidence of spontaneous as well as chemical mutagenesis by enhancing an error-prone DNA repair system. 4 Regardless of the type of tester strain being used, it is prudent to always run the bacteria in each experiment with and without the activation source, solvent, and xenobiotic. The number of background revertants should remain constant from experiment to experiment, providing a measure of the stability of the tester strain. Should the background vary significantly from one batch of xenobiotic to the next, it is important to look very closely at the purity of the compound. The phenotype of each tester strain should be confirmed: (1) on receipt of the cultures; (2) if a new frozen batch is used; and (3) if the number of spontaneous revertants varies or the sensitivity of a strain to a known mutagen is altered. A detailed description of all the tester strains available and the composition of the different media and reagents is beyond the scope of this chapter. For a detailed treatise of these, one should consult the excellent review by Maron and Ames. 4 Test Procedure
Preparation of Metabolic Activation Source. Sterile preparations of most animal tissues can be obtained by removing the tissue aseptically and carrying out all steps at 00-4 ° using sterile solutions and glassware. In the case of human tissues, which are usually obtained at best 30 min after death and handled by pathology staff before being available for research purposes, it is necessary to filter sterilize the $9 or microsomal fractions before use. Purified cytochrome P450 isozymes and gastrointestinal tract and lung tissue from animals are also routinely filter sterilized in our laboratory before use in the Ames test. Cell cultures in which cytochrome P450 cDNAs have been expressed are processed aseptically. The difficulty with many filters is the retention of a considerable amount of protein. In addition, we have found that when preparing microsomes from the gastrointestinal tract of humans, some particulate matter is retained, To overcome these difficulties we routinely prefilter the subcellular fraction through a 5/zm Millex-SV filter (Millipore, Bedford, MA, catalog number SLSV025LS) and then sterilize the preparation by passing it through a 0.22/.~m Millex-GV (Millipore, catalog number SLGV025LS) or Millex-HV (Millipore, catalog number SLHV025LS) filter. In the case of human liver microsomes only a 0.22 or 0.45/zm filtration step is required. All the above filters are listed in the Millipore catalog as being low protein
[48]
XENOBIOTIC ACTIVATION BY CYTOCHROMES P450
507
retention units. When we sterilize a 2 mg/ml human liver microsomal solution through a 0.22 ~m filter we lose approximately 20% of the protein. Because many of our purified cytochrome P450 isozyme preparations are not as concentrated as the above, we generally lose less than 5% of the protein on filtration. The yield of purified isozymes following filtration can be enhanced by using a 0.22 tzm Miilex-GV (Millipore, catalog number SLGV013OS) instead of the SLGV025LS filter. The former filter possesses a surface area 3 times smaller than the latter. Microsomes and purified cytochrome P450 isozymes are stable for prolonged periods of time (e.g., 3 years) when stored at - 8 0 ° in 0.1 M potassium phosphate buffer (pH 7.4) containing 20% glycerol. Maron and Ames 4 also report that $9 fractions are stable for up to 2 years when stored at - 80°. We routinely sterilize a vial of the activation source on the day of the experiment and determine the protein concentration of the filtrate before carrying out the assay. This has proved satisfactory for small assays up to 50 plates, but alternatively one may wish to filter a stock of microsomes before aliquoting and storing them at - 8 0 °. Regardless of when the filtration step is carried out, the protein concentration of the filtrate should always be determined. As a source of reducing equivalents we have usually used N A D P H made up immediately prior to the assay and filter sterilized using a 0.22/zm Millex GS unit (Millipore, catalog number SLGS025OS). It is important to establish that the amount of N A D P H added to the incubation mixture is not rate limiting. Preparation of Tester Strain. The appropriate tester strain is grown up overnight in sterile Oxoid nutrient broth No. 2 (Oxoid, Basingstoke, England) to a density of 1-2 × 109 cells/ml. For a 50-plate assay we routinely scrape a sterile wooden stick across the frozen culture ( - 80°) and add the bacteria to 10 ml of Oxoid broth. The bacteria are then grown up overnight in a loosely capped flask incubated in a Ratek Instruments (Melbourne, Australia) bacterial shaker (37°) set at 100 rpm. One hundred microliters of the culture is added to each assay. Dilution ofXenobiotic Substrates. Unless a xenobiotic is soluble and stable in aqueous solution we have used dimethyl sulfoxide (DMSO) as a universal solvent for dissolving substrates. The advantage of using organic solvents such as DMSO is that it avoids the necessity of filter sterilizing the xenobiotic-containing solutions. It is important to determine that the solvent used does not have an inhibitory effect on the cytochrome P450 system. Indeed, it is important to test the effect of the solvent on each isozyme since the degree of inhibition may vary depending on the isozyme used. In general we have always restricted the amount of DMSO to 1 to 2% of the incubation mixture. Incubation Mixture for Preincubation Test. A standard 0.5 ml incuba-
508
ENZYME ASSAYS
[48]
tion mixture contains microsomal protein, 1-2 x 10s bacterial cells, 1.2 /zmol NADPH added in 50/zl of 0.1 M potassium phosphate buffer (pH 7.4), xenobiotic dissolved in 5-10/.d DMSO, and 50/zmol sodium phosphate (pH 7.4). For reconstitution experiments we have generally used I0 pmol of purified P450 isozyme, 0.5 units of NADPH-cytochrome-P450 reductase, and 75/~g of dilauroyl-L-a-lecithin. However, depending on the xenobiotic and isozyme, the composition of the reconstituted system will vary. When cytochrome P450 expression systems are used, cell lysate protein is substituted for the microsomal protein. The reaction mixture is preincubated at 37° for 3 min prior to adding the bacteria and NADPH. The reaction is terminated by the addition of 0. I ml of 0.12 M menadione solution and then placed on ice. The menadione stops the reaction by competing for electrons from NADPH. Following the addition of 2 ml of molten top agar at 45 °, the incubation mixtures are poured onto petri dishes containing 30 ml minimal top agar and a limited amount of L-histidine. At this step it is possible to process three tubes simultaneously before the top agar sets and prevents pouring of the plates. The colonies on each plate are scored after a 48-hr incubation period at 37°. When antibody inhibition studies are being conducted, the amount of protein in each tube should be kept constant by the addition of preimmune immunoglobulin G (IgG). This is important as the protein may react with the electrophiles, and variability in the number of revertants formed may only reflect the protein content. As an indication of the integrity of a bacterial strain and the stability of the enzyme system, we incorporate into each experiment various positive controls. For tester strain TA98 we have routinely used 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) as the model mutagen. When determining the capacity of extrahepatic tissues to metabolize a mutagen, liver microsomes are routinely run in parallel. The reproducibility of these standard controls is excellent and provides a useful means of evaluating the technique. Data Analysis. A less than clear-cut result for mutagenicity of a xenobiotic in the Ames test can be a product of the substrate concentration or the amount of activation source added to the incubation. This again highlights the need to determine the correct reaction conditions for each xenobiotic. In the case of a negative result, it is important to establish by inspecting the bacterial lawn that the result does not reflect that the chemical has been toxic to the bacteria. As a trace amount of histidine is added to the top agar, all the bacteria on the plate undergo several divisions. If a toxic response has been elicited the lawn becomes mottled in appearance. Results should be reported as the mean count of at least three plates plus or minus the standard deviation. If the data are to be expressed as the number of revertants per milligram of microsomal protein or microgram of chemical, it is advantageous to provide the raw plate data together with
[49]
HPLC ASSAYSFOR HUMANP450IID6 SUBSTRATES
509
the relevant controls (e.g., minus generating system). Further, if expressing the effect of antibody inhibition on mutagenicity as a percentage of original activity, it is appropriate to give original activities. This ensures the reader can work backward from the transformation and accurately derive the original data. Biohazard Requirements. The wild-type Salmonella typhimurium strain can cause food poisoning. Although the genetically manipulated strains used in the Ames test are not very virulent, it is prudent to use care when handling these bacteria. 4 Since many of the xenobiotics used will be mutagenic and carcinogenic, extreme care should be taken to prevent human exposure. Workers should consult local Biohazard Committee guidelines before commencing an experiment. Ideally all laboratory materials used should be of the disposable type, and guidelines for the disposal of both biological and carcinogenic waste should be strictly followed.
[49] B u f u r a l o l , D e x t r o m e t h o r p h a n , a n d D e b r i s o q u i n e as P r o t o t y p e S u b s t r a t e s for H u m a n P 4 5 0 I I D 6
By
THOMAS
KRONBACH
Introduction The human debrisoquine/sparteine-type genetic polymorphism of drug oxidation affects the expression of cytochrome P450IID6,1,2 an enzyme which is involved in the metabolism of many drugs (reviewed in Ref. 3). Although the P450 enzymes are often believed to exhibit a broad and overlapping substrate specificity, many drugs which are substrates for P450IID6 are only marginally metabolized in individuals affected by this polymorphism (poor metabolizers). This indicates that, among the other hepatic P450s, P450IID6 has a unique substrate selectivity and is the major catalyst involved in the clearance of these drugs. This chapter describes assays for the metabolism of three prototype substrates for P450IID6. The experimental/t-blocking agent bufuralol is metabolized by P450IID6 by l'-hydroxylation, the antihypertensive drug debrisoquine is metabolized by 4-hydroxylation, and dextromethorphan is F. J. Gonzalez, R. C. Skoda, S. Kimura, M. Umeno, U. M. Zanger, D. W. Nebert, H. V. Gelboin, J. P. Hardwick, and U. A. Meyer, Nature (London) 331, 442 (1988). 2 U. M. Zanger, F. Vilbois, J. P. Hardwick,and U. A. Meyer,Biochemistry27, 5447(1988). 3 U. A. Meyer, R. C. Skoda, and U. M. Zanger, Pharmacol. Ther. 46, 297 (1990). METHODS IN ENZYMOLOGY, VOL. 206
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
