* Department of Environmental Health and Center for Ecogenetics and Environmental Health, University of Washington, Seattle, Washington 98105; and
Center for Teaching and Learning, Swiss Federal Institute of Technology, Zurich, Switzerland
Received June 20, 2001; accepted October 8, 2001
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ABSTRACT |
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Key Words: aflatoxin B1; microsomal epoxide hydrolase; cytochrome P450; genotoxicity; Saccharomyces cerevisiae.
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INTRODUCTION |
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Because it is possible that enzymatic hydrolysis could be important in the lipid microenvironment, where the reactive epoxide is formed in the intact cell, we sought to re-examine the role of mEH in AFB genotoxicity by heterologous expression in the eukaryotic organism, S. cerevisiae. By reproducing the enzymatic pathway of bioactivation by human CYP1A enzymes coupled with coexpression of mEH, we could directly examine mEH function without membrane reconstitution, because both CYP1A and mEH enzymes colocalize in yeast endoplasmic reticulum (Eugster and Sengstag, 1993). In this study we were able to show that mEH does have a functional role in AFB detoxification, as measured by DNA adduct formation, mitotic recombination, and Ames assay mutagenicity. Our data provide an example of the strength of a eukaryotic yeast expression system for studying the role of biotransformation enzymes in the production and elimination of reactive intermediates in intact eukaryotic cells.
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MATERIALS AND METHODS |
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Yeast strains.
The parental S. cerevisiae strain YHE2 (MATa/MAT, ade2-40/ade2-119, trp5-12/trp5-27, ilv1-92/ilv1-92, ura3
5/ura3
5) and the transformed strains expressing human cDNAs have been previously described (Eugster and Sengstag, 1993
; Eugster et al., 1990
) (except for pHE13 and pEK30) and are listed as follows: pDP34-control strain with no cDNA; pHE5-mEH; pHE10-CYP1A1; pHE12-CYP1A1/mEH coexpression; pHE36-CYP1A2; pHE13-CYP1A2/mEH coexpression; and pEK30-CYP1A2/
mEH. The yeast were transformed by standard methods (Schiestl and Gietz, 1989
) and cultured in minimal synthetic media lacking uracil.
Plasmid constructs.
All the plasmids used in this study have been described (Eugster and Sengstag, 1993) except for pHE13 and pEK30. For pHE13, the vector containing mEH (pHE5) was linearized with Sac I and an expression cassette for CYP1A2 was excised from pHE36 as a 2-kb Sac-I fragment and ligated into the Sac I-site of pHE5. The plasmid pEK30 is identical to pHE13 except that residue 226 of mEH was mutagenized from aspartic acid to glycine; a mutation known to abolish activity in rat mEH (Arand et al., 1999
). The single base-pair change was introduced by site-directed mutagenesis using the QuikChange kit (Stratagene, La Jolla, CA) and the following primer, CATTCAAGGAGGGGgCTGGGGGTCCC, and its complement (mutant base shown in lower case). The mEH expression cassette was excised from pHE5 as a 2.2 Kb Sac I/Sal I fragment and ligated into these same sites in pBluescript (Stratagene). After site-directed mutagenesis, the entire cassette was resequenced to verify the presence of the mutation and absence of any other nucleotide changes. The mEH cassette was then ligated into the Sac I/Sal I sites of pDP34. The CYP1A2 cassette was then cloned into the Sac I site as previously described for pHE13.
Yeast microsomes.
Microsomes were prepared as previously described (Sengstag and Würgler, 1994) and aliquots snap frozen in liquid nitrogen and stored at -80°C prior to use. Protein concentrations were determined with Bradford reagent (BioRad) with bovine serum albumin as the standard. Heterologous microsomal P450 content was measured by the method of Omura and Sato (1964).
Enzyme assays.
The catalytic activity of the microsomes towards AFB was determined as described by Gallagher et al. (1994). Briefly, approximately 1 mg of yeast microsomal protein, 500 µg of a 2:1 (v:v) mixture of rat:mouse hepatic cytosol (to quantitatively capture the reactive AFB-epoxide as the glutathione conjugate, Gallagher et al., 1994), 1 mM NADPH and 5 mM reduced glutathione in 0.1 M potassium phosphate, pH 7.2 were mixed in a total volume of 240 µl. After a 5-min preincubation at 37°C, the reaction was started by the addition of 10 µl of AFB dissolved in dimethysulfoxide, for a final concentration of 128 µM as determined by UV spectrophotometric analysis of stock solutions (Busby and Wogan, 1984). The reactions were allowed to proceed at 37°C for 10 min and then terminated by addition of 250 µl ice-cold methanol containing 1% trifluoroacetic acid and AFG1 (10 µM) as an internal standard. AFB metabolites were separated by reversed-phased, high-performance liquid chromatography as previously described (Monroe and Eaton, 1987) and measured by UV detection. Microsomal epoxide hydrolase activity for cis-stilbene oxide (CSO) was performed as described by Gill et al. (1983) with the following modifications. Yeast microsomes were diluted in 10 mM potassium phosphate buffer, pH 7.4 for a final protein concentration of 1 mg/ml in a 100 µl reaction volume. The microsomes were incubated for 5 min at 37°C and then 1 µl of [3H] CSO stock solution was added for a final concentration of 50 µM [3H] CSO (specific activity
10 mCi/mmol). After 20 min at 37°C, the reactions were stopped by addition of 200 µl iso-octane followed by vortexing; aliquots of the aqueous phase were analyzed by liquid scintillation counting to quantitate the diol product.
Immunoblotting.
Yeast whole-cell extracts or microsomal protein fractions were separated by SDS-PAGE and transferred to Immobilon P membranes (Millipore Corp., Bedford, MA). Nonspecific binding was blocked with 5% BLOTTO dissolved in phosphate-buffered saline containing 0.1% TWEEN 20. Primary antibodies against either human mEH or rabbit CYP1A were diluted in blocking buffer 1:15,000 or 1:100, respectively. Binding of antibody to antigen was detected with alkaline phosphatase-conjugated secondary antibody and visualized by exposure to X-Omat ARTM film (Eastman Kodak, Rochester, NY) using chemiluminescent CSPD substrate.
Trp recombination test.
The homologous recombination between the trp5-12/trp5-27 alleles was performed as previously described (Sengstag and Würgler, 1994). Briefly, yeast were grown to the exponential phase, washed in sodium phosphate buffer (100 mM, pH 7.4) and then resuspended in buffer at a concentration of 108 cells/ml. Cells were then exposed to AFB or AFM in DMSO at a final concentration of 5% v/v DMSO for 4 h at 30°C, centrifuged and washed twice with tryptophan-deficient media before plating on agar plates lacking tryptophan. Trp+ convertants were scored after 3 days of growth at 30°C. The conversion frequency was calculated by comparing it to the number of colonies that grew on complete (YPD) media plates (after diluting the cell cultures 1:40,000 in media, to obtain plating densities easily counted).
AFB-DNA adduct formation.
Exponentially growing yeast were harvested and 0.05 OD units were inoculated into 0.5 ml 100 mM sodium phosphate buffer, pH 7.4 containing [3H] AFB (specific activity 18.6 Ci/mmol) dissolved in methanol for a final solvent concentration of 1.7% (v:v). The cells were incubated at 30°C for 6 h, protoplasts prepared as described (Sengstag and Würgler, 1994), and then total nucleic acid was isolated by alkaline lysis followed by ethanol precipitation. Cellular RNA was removed by treatment with 0.2 mg/ml RNase A and then extracted with phenol/chloroform, followed by ethanol precipitation. The DNA was quantified by UV absorption and adducts were measured by liquid scintillation counting.
Ames assay.
The Salmonella typhimurium strain TA98 (hisD3052, rfa, uvrB, pKM101) was used to assay the mutagenicity of AFB metabolized by yeast microsomes as described previously (Sengstag and Würgler, 1994
). In brief, an overnight culture of TA98 was harvested and resuspended in 150 mM potassium chloride, pH 7.4; 100 µl of bacteria were then added to 700 µl of an NADPH-regenerating system (containing 150 mM potassium phosphate, pH 7.4, 0.75 mM NADP+, 15 mM glucose 6-phosphate, 10 mM magnesium chloride and 10 units glucose 6-phosphate dehydrogenase), followed by 200 µg of yeast microsomes and subsequent addition of 30 µl of AFB. The cell mixture was incubated for 20 min at 37°C before 2 ml of top agar was added and the mixture poured onto minimal plates. The revertant colonies were then scored after incubation at 37°C for 2 days.
Statistical analysis.
All data are presented as mean ± SEM and are derived from triplicate cultures of at least duplicate experiments. Results were compared by one-way ANOVA and Tukey-Kramer multiple comparisons test, using the statistical program InStat for the Macintosh (Graphpad Software Inc., San Diego, CA). A p 0.05 was considered significant.
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RESULTS |
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DISCUSSION |
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The kinetic studies of Guengerich et al. (1996) were conducted with bacterially expressed CYP3A4 and purified rat or human mEH that were subsequently reconstituted in vitro. This study found that only at very high ratios of mEH to CYP3A4 could mEH alter AFB genotoxicity as assessed by umu response in S. typhimurium tester strain TA1535. The experiments by Johnson et al. (1997b) found that the rate of hydrolysis of AFBO was unaffected by addition of purified human mEH while purified rat mEH was able to slightly increase the hydrolysis rate.
Significant differences exist between those experiments and this current study. First, we are using CYP1A2, not CYP3A4, and in our case the enzyme-coding sequence (i.e., deletion of N-terminal membrane anchor) has not been altered to facilitate expression in E. coli; however, these differences are not likely to be of major importance. Second, we are expressing mEH and CYP1A2 together so they colocalize without any membrane reconstitution utilizing synthetic lipids. Finally, in our recombinant yeast, the AFBO is generated in the endoplasmic reticulum (a hydrophobic environment) which may protect the CYP-generated AFBO from non-enzymatic hydrolysis.
Because mEH and CYP enzyme are localized to the same subcellular compartment, mEH may well be positioned to facilitate hydrolysis of AFBO near its intracellular site of formation. Although it is conceptually difficult to understand how the hydrophobic environment of the endoplasmic reticulum could effectively protect CYP-generated exo-AFBO from the high concentration of intracellular water present in the intact cell or sub-cellular fractions, this must indeed occur, because numerous studies have previously demonstrated the effectiveness of certain glutathione S-transferases with high efficiency toward AFBO to effectively compete with water for the highly reactive exo-AFBO (Buetler et al., 1992; Ch'ih et al., 1983b
; Guengerich et al., 1996
; Raney et al., 1992a
,b
; Gallagher et al., 1994
; Johnson et al., 1997a
). Thus, in the absence of an effective glutathione conjugation system such as occurs in the mouse, and to a lesser extent in rat liver, mEH activity may play a functional role in protection of DNA against the reactive AFB-exo-8,9-oxide. Because primate GSTs, including human, have very low AFBO-conjugating activity (Raney et al., 1992b
; Johnson et al., 1997a
; Wang et al., 2000
) relative to that seen in rodent liver (Buetler et al., 1992
; Van Ness et al., 1998
), mEH may provide important protection against AFB-induced DNA damage in the human liver.
The observation that CYP1A1 was capable of converting AFB to a mutagenic metabolite in both intact yeast (trp5 gene conversion) and in purified microsomes (Ames assay) is puzzling since CYP1A1 forms AFM at a high rate but generates little AFBO and forms no detectable DNA adducts. However, the acute toxicity of AFM in rats is actually close to what one sees with AFB (Pong and Wogan, 1971). We tested the possibility that CYP1A1 or an endogenous enzyme is further metabolizing AFM to a genotoxic compound. Exposure of yeast to purified AFM only induced a slight increase in trp5 gene conversion in CYP1A-expressing cells while incubation of AFM with microsomes expressing CYP1A1 or CYP1A2 yielded no detectable metabolites. The low rate of mitotic recombination may be a result of poor permeability of AFM through the yeast cell wall. Previously, it was shown that human liver microsomes were capable of bioactivating AFM to the corresponding epoxide (Neal et al., 1998
). In contrast to these findings, we were unable to detect such a metabolite, either with yeast-expressed CYP1A1 or CYP1A2 or with several different human liver microsome preparations. The reasons for this are not clear but may be due to sensitivity of the assay. The fact that CYP1A1 is capable of forming a mutagenic metabolite (as assessed by Ames assay mutagenicity and reversion of tryptophan prototrophy) and that mEH attenuates this response would seem to imply that an epoxide intermediate is involved, although quantity is very small, relative to the amount of AFM1 formed, and is below the limit of detection of our HPLC-based assay.
In conclusion, we have provided several lines of evidence that coexpression of mEH is capable of modifying the genotoxic properties of AFB, thus supporting the epidemiological correlation that differences in mEH activity due to genetic polymorphisms can alter the risk of AFB-induced hepatocellular carcinoma (McGlynn et al., 1995). The use of recombinant yeast to recreate a multi-step biotransformation pathway facilitated this finding in a manner not utilized in previous studies, which found no significant role for mEH in AFB metabolism. Finally, because human gene products are readily expressed and function in yeast in a manner similar to higher organisms, they provide a useful system for testing the functional significance of sequential human biotransformation enzymes involved in multistep xenobiotic activation and detoxification.
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ACKNOWLEDGMENTS |
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NOTES |
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