ANALYTICAL
BIOCHEMISTRY
87, 267-27
1 (1978)
Monooxygenase and Epoxide High-Performance Liquid
Hydratase Analysis Chromatography
by
A simple HPLC method for the estimation of monooxygenase and epoxide hydratase activity is described using rrans-stilbene and rrans-stilbene oxide as substrates. The analysis method also allows the determination of benzoin, benzil, and benzoic acid, which have been proposed as other metabolites of [runs-stilbene outside the traditional epoxide-diol pathway. We report the application of this method in an investigation of the biochemical potential of cell suspension cultures of Phaseolus vulgaris.
The detection and quantitation of monooxygenase and epoxide hydratase enzymes in both plant and animal systems have attracted considerable attention recently. Monooxygenase enzymes have been invoked in explaining the carcinogenic properties of aromatic hydrocarbons, which are converted into “K-region” epoxides more carcinogenic than the parent compounds (1). The examination of both monooxygenase and epoxide hydratase is important because, although appropriate substrates are metabolized via the conventional epoxide-diol pathway, a number of drugs have been demonstrated to produce the epoxide as the major metabolite (2). A similar situation appears in the metabolism of cyclodiene insecticides in plants (3). A number of simple substrates have been utilized for the assay of these enzymes. Epoxide hydratase has been determined using [7-3H]styrene oxide as substrate followed by radiometric assay (4). More simply, the synchronous monitoring of monooxygenase and epoxide hydratase activity was achieved using styrene and styrene oxide as substrates with gas-liquid chromatographic (GLC) analysis of styrene oxide (indicating monooxygenase activity) and phenyl(ethylene glycol) after conversion to its n-butyl boronate ester (indicating epoxide hydratase activity) (5). The carcinogenic compound 3-methylcholanthrene-l l , 12-oxide has been used as a substrate in two procedures for the estimation of epoxide hydratase activity. Stoming and Bresnick monitored diol production by GLC after silylation (6), and Nesnow and Heidelberger used a rapid high-performance liquid chromatographic method (HPLC) which was capable of monitoring metabolite production at the picomole level (7). During studies on the metabolism of cyclodiene insecticides in plant cell suspension cultures (8) it became necessary to demonstrate the presence of both monooxygenase and epoxide hydratase enzymes. Recent work on the metabolism of stilbene and analogs suggested the use of trans-stilbene and trans-stilbene 267
0003-2697/78/0871-0267$02.00/O Copyright All rights
Q 1978 by Academic Press. Inc. of reproduction in any form reserved.
268
SHORT
COMMUNlCATlONS
oxide as substrates for this work. These compounds and their metabolites are readily available and possess suitable chromophoric properties for detection by uv spectroscopy after HPLC. The use of HPLC for monitoring diol production avoids the derivatization required in GLC methods (5,6). RESULTS
AND DISCUSSION
Watabe and Akamatsu have investigated the metabolism of cis-stilbene and a number of rrans-stilbene derivatives (9-11) using rabbit liver microsomal systems with preparative thin-layer chromatography followed by GLClMS to confirm the nature of the metabolites. Our initial experiments utilizing GLC proved unsuccessful since trans-stilbene oxide rearranges to afford two peaks on analysis. A similar observation has been made by Metzler and Neumann in a study of the metabolism of cis- and trans-dimethylaminostilbene (12). Consequently, analysis by HPLC was investigated. As Akamatsu and Watabe have reported benzoin, benzil, and benzoic acid produced from truns-stilbene oxide, we sought an HPLC system which would include these compounds in addition to trans-stilbene, truns-stilbene oxide, and meso-1,2-diphenylethane 1,2-diol. Truns-stilbene, truns-stilbene oxide, and benzil proved amenable to analysis on a lo-pm Spherisorb column with 0.5% acetonitrile in iso-octane as eluent (Eluent 1). Benzoic acid was also assayable in this system after methylation with an ethereal solution of diazomethane. The separation of these compounds, together with benzaldehyde, which can be used as an
C B E
D
Id
Time
Onin)
FIG. IA. HPLC separation of (A) truns-stilbene, benzoate, (D) benzaldehyde. and(E) benzil using0.5%
(B) truns-stilbene oxide, (C) methyl acetonitrile in iso-octafie as eluent (1).
269
SHORT COMMUNICATIONS F
FIG. 1B. HPLC separation of(F) benzoin and (G) mew-1,2-diphenylethane 30% ethyl acetate in cyclohexane as eluent (2).
1,2-diol using
internal standard, is illustrated in Fig. 1A. Assay of epoxide hydratase based on the production of meso- 1,2-diphenylethane 1,2-diol requires a more polar eluent (30% ethyl acetate in cyclohexane) (Eluent 2). Benzoin is also assayable using this eluent (Fig. 1B). Calibration curves based on peak heights were linear over the concentration ranges studied, and minimum detectable levels and retention times are given in Table 1. Using this system, we have demonstrated the presence of both monooxygenase and epoxide hydratase activity for stilbene-based substrates in cell suspension cultures derived from Phaseolus vulgaris root TABLE HPLC
RETENTION
TIMES AND
Compound
AND
MINIMUM
ITS POSSIBLE
1
DETECTABLE DEGRADATION
Corrected retention time (min)
trans-Stilbene” truns-Stilbene oxide* Methyl benzoate* (from benzoic acid) Benzaldehyde BenziP Benzoin’ mew- 1,2-Diphenylethane 1.2-diolc (1As determined at 0.02 AUF. Ir Eluent, 0.5% acetonitrileiiso-octane. I’ Eluent, 30% ethyl acetateicyclohexane.
LIMITS
OF TRANS-STILBENE
PRODUCTS
Minimum detectable limit (ng)”
0.20 0.95
2 25
1.30 2.00 3.30 0.45
20 60 10 15
1.25
100
270
SHORT
COMMUNICATIONS
tissue. Such cultures demonstrated greater epoxide hydratase activity than monooxygenase activity (13,14). The only metabolite that could be detected outside the traditional epoxide-diol pathway was benzoin, an hydroxy ketone. MATERIALS
AND METHODS
trans-Stilbene, benzoin, benzil, benzaldehyde, and benzoic acid were purchased from British Drug Houses, trans-Stilbene oxide from Aldrich, and meso-l ,Zdiphenylethane 1,2-diol from Koch Light. High-performance liquid chromatography. All separations were performed with a 10 cm x 0.18 in. stainless steel column packed with lo-pm Spherisorb S 1OW (Jones Chromatography). The solvents were delivered by a Waters 6000M solvent delivery system, and the flow rate was 2 mhmin in all cases. Detection was with a Cecil CE 212 spectrophotometer, with a lo-p1 flow cell, monitoring at 264 nm. Eluent 1 was 0.5% acetonitrile in iso-octane; Eluent 2 was 30% ethyl acetate in cyclohexane. Assay. Cell suspension cultures of Phaseolus vulgaris var. Canadian Dwarf were grown under the previously described conditions (8) and were treated with either trans-stilbene or trans-stilbene oxide (as 5% solutions in diethyl ether, 25 mg/lOO ml of culture) 20 days after subculture. The level of cells at the 20-day stage was 1 g dry wt/lOO ml of culture. Cultures were maintained at 23°C on an orbital shaker (120 rpm) for 3 days, and the reaction was then stopped by the addition of cold hexane. Cells, obtained by filtration, were hot-extracted with methanol, while the medium was cold-extracted with 25% ethyl acetate/hexane. After evaporation, the samples were dissolved in cyclohexane for assay with Eluent 1 or ethyl acetate for assay with Eluent 2. Materials.
ACKNOWLEDGMENTS We wish to thank
the Science
Research
Council
for financial
support
to D.S.L.
REFERENCES 1. Grover, P. L., and Sims, P. (1973) Biochem. Pharmacol. 22, 661-666. 2. Pachecka, J., Salmona, M., Cantoni, L., Mussini, E., Pantarotto, C., Frigerio, A., and Belvedere, G. (1976) Xenobiorica 6, 593-598. 3. Keamey, P. C. (1976) /. AOAC 59, 866-881. 4. Oesch, F., Jerina, D. M., and Daly, J. (1971) Biochim. Biophys. Acta 227, 689-691. 5. Belvedere, G., Pachecka, J., Cantoni, L., Mussini. E., and Salmona. M. (1976) J. Chromatogr. 118, 387-393. 6. Stoming, T. A., and Bresnick, E. (1973) Science 181, 951-952. 7. Nesnow, S., and Heidelberger, C. (1975) Anal. Biochem. 67, 525-530. 8. Brain, K. R., and Lines, D. S. (1977) in Plant Tissue Culture and its Biotechnological
271
SHORT COMMUNICATIONS
9. 10. II. 12. 13. 14.
Applications (Barz, W.. Reinhard. E.. and Zenk, M. H., eds.). pp. 197-203. Springer-Verlag, Berlin. Watabe, T.. and Akamatsu, K. (1972) Biochitn. Biophys. Acta 279, 297-305. Watabe, T., and Akamatsu, K. (1974) B&hem. Pharmacof. 23, 1079-1085. Watabe, T., and Akamatsu, K. (1975) Biochem. Pharmacol. 24, 442-444. Metzler, M., and Newmann, H. G. (1977) Xenobiorica 7, 117-132. Lines, D. S. (1977) Ph.D. thesis, U.W.I.S.T., Cardiff. Ross. M. S. F., Lines. D. S.. Brain, K. R., and Stevens. R. G. (1978) Phyrochemistry Nov. 17, 45-48.
M. D. K. R. Deparment of Pharmacognosy Welsh School of Pharmacy u. W.I.S.T. King Edward VII Avenue Cardiff CFl 3NU, U. K. Received August 3, 1977; accepted
December
12, 1977
S. F. Ross S. LINES R. BRAIN G. STEVENS
BIOCHEMISTRY
87, 267-27
1 (1978)
Monooxygenase and Epoxide High-Performance Liquid
Hydratase Analysis Chromatography
by
A simple HPLC method for the estimation of monooxygenase and epoxide hydratase activity is described using rrans-stilbene and rrans-stilbene oxide as substrates. The analysis method also allows the determination of benzoin, benzil, and benzoic acid, which have been proposed as other metabolites of [runs-stilbene outside the traditional epoxide-diol pathway. We report the application of this method in an investigation of the biochemical potential of cell suspension cultures of Phaseolus vulgaris.
The detection and quantitation of monooxygenase and epoxide hydratase enzymes in both plant and animal systems have attracted considerable attention recently. Monooxygenase enzymes have been invoked in explaining the carcinogenic properties of aromatic hydrocarbons, which are converted into “K-region” epoxides more carcinogenic than the parent compounds (1). The examination of both monooxygenase and epoxide hydratase is important because, although appropriate substrates are metabolized via the conventional epoxide-diol pathway, a number of drugs have been demonstrated to produce the epoxide as the major metabolite (2). A similar situation appears in the metabolism of cyclodiene insecticides in plants (3). A number of simple substrates have been utilized for the assay of these enzymes. Epoxide hydratase has been determined using [7-3H]styrene oxide as substrate followed by radiometric assay (4). More simply, the synchronous monitoring of monooxygenase and epoxide hydratase activity was achieved using styrene and styrene oxide as substrates with gas-liquid chromatographic (GLC) analysis of styrene oxide (indicating monooxygenase activity) and phenyl(ethylene glycol) after conversion to its n-butyl boronate ester (indicating epoxide hydratase activity) (5). The carcinogenic compound 3-methylcholanthrene-l l , 12-oxide has been used as a substrate in two procedures for the estimation of epoxide hydratase activity. Stoming and Bresnick monitored diol production by GLC after silylation (6), and Nesnow and Heidelberger used a rapid high-performance liquid chromatographic method (HPLC) which was capable of monitoring metabolite production at the picomole level (7). During studies on the metabolism of cyclodiene insecticides in plant cell suspension cultures (8) it became necessary to demonstrate the presence of both monooxygenase and epoxide hydratase enzymes. Recent work on the metabolism of stilbene and analogs suggested the use of trans-stilbene and trans-stilbene 267
0003-2697/78/0871-0267$02.00/O Copyright All rights
Q 1978 by Academic Press. Inc. of reproduction in any form reserved.
268
SHORT
COMMUNlCATlONS
oxide as substrates for this work. These compounds and their metabolites are readily available and possess suitable chromophoric properties for detection by uv spectroscopy after HPLC. The use of HPLC for monitoring diol production avoids the derivatization required in GLC methods (5,6). RESULTS
AND DISCUSSION
Watabe and Akamatsu have investigated the metabolism of cis-stilbene and a number of rrans-stilbene derivatives (9-11) using rabbit liver microsomal systems with preparative thin-layer chromatography followed by GLClMS to confirm the nature of the metabolites. Our initial experiments utilizing GLC proved unsuccessful since trans-stilbene oxide rearranges to afford two peaks on analysis. A similar observation has been made by Metzler and Neumann in a study of the metabolism of cis- and trans-dimethylaminostilbene (12). Consequently, analysis by HPLC was investigated. As Akamatsu and Watabe have reported benzoin, benzil, and benzoic acid produced from truns-stilbene oxide, we sought an HPLC system which would include these compounds in addition to trans-stilbene, truns-stilbene oxide, and meso-1,2-diphenylethane 1,2-diol. Truns-stilbene, truns-stilbene oxide, and benzil proved amenable to analysis on a lo-pm Spherisorb column with 0.5% acetonitrile in iso-octane as eluent (Eluent 1). Benzoic acid was also assayable in this system after methylation with an ethereal solution of diazomethane. The separation of these compounds, together with benzaldehyde, which can be used as an
C B E
D
Id
Time
Onin)
FIG. IA. HPLC separation of (A) truns-stilbene, benzoate, (D) benzaldehyde. and(E) benzil using0.5%
(B) truns-stilbene oxide, (C) methyl acetonitrile in iso-octafie as eluent (1).
269
SHORT COMMUNICATIONS F
FIG. 1B. HPLC separation of(F) benzoin and (G) mew-1,2-diphenylethane 30% ethyl acetate in cyclohexane as eluent (2).
1,2-diol using
internal standard, is illustrated in Fig. 1A. Assay of epoxide hydratase based on the production of meso- 1,2-diphenylethane 1,2-diol requires a more polar eluent (30% ethyl acetate in cyclohexane) (Eluent 2). Benzoin is also assayable using this eluent (Fig. 1B). Calibration curves based on peak heights were linear over the concentration ranges studied, and minimum detectable levels and retention times are given in Table 1. Using this system, we have demonstrated the presence of both monooxygenase and epoxide hydratase activity for stilbene-based substrates in cell suspension cultures derived from Phaseolus vulgaris root TABLE HPLC
RETENTION
TIMES AND
Compound
AND
MINIMUM
ITS POSSIBLE
1
DETECTABLE DEGRADATION
Corrected retention time (min)
trans-Stilbene” truns-Stilbene oxide* Methyl benzoate* (from benzoic acid) Benzaldehyde BenziP Benzoin’ mew- 1,2-Diphenylethane 1.2-diolc (1As determined at 0.02 AUF. Ir Eluent, 0.5% acetonitrileiiso-octane. I’ Eluent, 30% ethyl acetateicyclohexane.
LIMITS
OF TRANS-STILBENE
PRODUCTS
Minimum detectable limit (ng)”
0.20 0.95
2 25
1.30 2.00 3.30 0.45
20 60 10 15
1.25
100
270
SHORT
COMMUNICATIONS
tissue. Such cultures demonstrated greater epoxide hydratase activity than monooxygenase activity (13,14). The only metabolite that could be detected outside the traditional epoxide-diol pathway was benzoin, an hydroxy ketone. MATERIALS
AND METHODS
trans-Stilbene, benzoin, benzil, benzaldehyde, and benzoic acid were purchased from British Drug Houses, trans-Stilbene oxide from Aldrich, and meso-l ,Zdiphenylethane 1,2-diol from Koch Light. High-performance liquid chromatography. All separations were performed with a 10 cm x 0.18 in. stainless steel column packed with lo-pm Spherisorb S 1OW (Jones Chromatography). The solvents were delivered by a Waters 6000M solvent delivery system, and the flow rate was 2 mhmin in all cases. Detection was with a Cecil CE 212 spectrophotometer, with a lo-p1 flow cell, monitoring at 264 nm. Eluent 1 was 0.5% acetonitrile in iso-octane; Eluent 2 was 30% ethyl acetate in cyclohexane. Assay. Cell suspension cultures of Phaseolus vulgaris var. Canadian Dwarf were grown under the previously described conditions (8) and were treated with either trans-stilbene or trans-stilbene oxide (as 5% solutions in diethyl ether, 25 mg/lOO ml of culture) 20 days after subculture. The level of cells at the 20-day stage was 1 g dry wt/lOO ml of culture. Cultures were maintained at 23°C on an orbital shaker (120 rpm) for 3 days, and the reaction was then stopped by the addition of cold hexane. Cells, obtained by filtration, were hot-extracted with methanol, while the medium was cold-extracted with 25% ethyl acetate/hexane. After evaporation, the samples were dissolved in cyclohexane for assay with Eluent 1 or ethyl acetate for assay with Eluent 2. Materials.
ACKNOWLEDGMENTS We wish to thank
the Science
Research
Council
for financial
support
to D.S.L.
REFERENCES 1. Grover, P. L., and Sims, P. (1973) Biochem. Pharmacol. 22, 661-666. 2. Pachecka, J., Salmona, M., Cantoni, L., Mussini, E., Pantarotto, C., Frigerio, A., and Belvedere, G. (1976) Xenobiorica 6, 593-598. 3. Keamey, P. C. (1976) /. AOAC 59, 866-881. 4. Oesch, F., Jerina, D. M., and Daly, J. (1971) Biochim. Biophys. Acta 227, 689-691. 5. Belvedere, G., Pachecka, J., Cantoni, L., Mussini. E., and Salmona. M. (1976) J. Chromatogr. 118, 387-393. 6. Stoming, T. A., and Bresnick, E. (1973) Science 181, 951-952. 7. Nesnow, S., and Heidelberger, C. (1975) Anal. Biochem. 67, 525-530. 8. Brain, K. R., and Lines, D. S. (1977) in Plant Tissue Culture and its Biotechnological
271
SHORT COMMUNICATIONS
9. 10. II. 12. 13. 14.
Applications (Barz, W.. Reinhard. E.. and Zenk, M. H., eds.). pp. 197-203. Springer-Verlag, Berlin. Watabe, T.. and Akamatsu, K. (1972) Biochitn. Biophys. Acta 279, 297-305. Watabe, T., and Akamatsu, K. (1974) B&hem. Pharmacof. 23, 1079-1085. Watabe, T., and Akamatsu, K. (1975) Biochem. Pharmacol. 24, 442-444. Metzler, M., and Newmann, H. G. (1977) Xenobiorica 7, 117-132. Lines, D. S. (1977) Ph.D. thesis, U.W.I.S.T., Cardiff. Ross. M. S. F., Lines. D. S.. Brain, K. R., and Stevens. R. G. (1978) Phyrochemistry Nov. 17, 45-48.
M. D. K. R. Deparment of Pharmacognosy Welsh School of Pharmacy u. W.I.S.T. King Edward VII Avenue Cardiff CFl 3NU, U. K. Received August 3, 1977; accepted
December
12, 1977
S. F. Ross S. LINES R. BRAIN G. STEVENS
