preferential overexpression of a class mu glutathiones-transferase subunit in mouse liver by...

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 236, 825–828 (1997)ARTICLE NO. RC977053

Preferential Overexpression of a Class MU GlutathioneS-Transferase Subunit in Mouse Liver by Myristicin

Hassan Ahmad,1 Maria T. Tijerina, and Amy S. TobolaDepartment of Chemistry, University of Texas-Pan American, Edinburg, Texas 78539

Received June 26, 1997

Thus, various compounds that induce GST activityThe present studies were undertaken to elucidate have been found to inhibit chemical carcinogenesis (3-

the mechanism of induction of glutathione S-trans- 6). Most of the cytosolic mammalian GST isoenzymesferase (GST) in mouse liver by myristicin, an active characterized to date are grouped into four species-constituent of parsley leaf. A/J albino mice, given 5 to independent classes (a, m, p, and u) on the basis of their50 mg doses of myristicin, showed 4- to 14-fold increase structural, immunological, kinetic, and physicochemi-in liver GST specific activity over the control. GST pu-

cal properties (11,12). The isoenzymes belonging torified from equal amounts of control and myristicin-these classes have been shown to exhibit overlappingtreated livers indicated a marked increase in the GSTyet discrete substrate specificities (11-13).activity. A relatively higher increase in GST activity

Epidemiological studies have demonstrated an in-towards 2,4-dichloronitrobenzene and a profound in-verse relationship between dietary intake of variouscrease in the levels of GST m on Western blot analysisvegetables and likelihood of contracting cancer (14-16).of the myristicin-treated mouse liver suggest a prefer-Myristicin (1-allyl-5-methoxy-3,4-methylenedioxyben-ential induction of GST m. Results of the study alsozene), an active constituent of parsley (Petroselinumindicate that out of the two m class GST subunits (Mr.sativum Hoffm, an edible Umbelliferae family plant)26,500 and Mr. 25,000) expressed in liver only one (Mr.

26,500) is significantly elevated. Myristicin treatment has been reported to be an inducer of GST activity incaused a slight change in the GST p levels while the mouse tissues (17) and an inhibitor of benzo(a)pyrene-levels of GST a showed a modest increase. These re- induced tumorigenesis in mouse lung (18). In previoussults suggest that myristicin could be an effective che- studies, however, only the myristicin-induced increasesmopreventive agent, particularly for carcinogens that in GST activity against 1-chloro-2,4-dinitrobenzeneare detoxified by the m class GST. q 1997 Academic Press (CDNB) have been determined (17,18). Since the GST

isoenzymes of a, m, and p classes exhibit substrate pref-erence in glutathione (GSH) conjugation to differentelectrophilic substrates, it is necessary to investigateA number of studies strongly suggest that elevation the changes in isoenzyme patterns by various GST in-of the levels of phase II enzymes, particularly glutathi- ducers. This would also establish the mechanism ofone S-transferase (GST; EC 2.5.1.18), is of critical im- regulation of the genes for the GST isoenzymes, as theyportance in achieving chemoprotection against chemi- are encoded by several genes. Therefore, this study wascal carcinogens (1-6). GSTs are ubiquitous enzymes undertaken to investigate the effect of myristicin onthat protect cells against a wide spectrum of toxic com- the differential induction of GST isoenzymes in mousepounds including various carcinogens (7-10). Different liver. Our results have indicated, for the first time, thatisoenzymes of GST either remove the carcinogens by myristicin preferentially and profoundly induces thenon-catalytic binding or conjugate the carcinogens with levels of GST m isoenzyme as compared to the a and pglutathione (GSH) catalytically, yielding less toxic hy- classes of GSTs.drophilic conjugates which are readily excreted (11).

Since the ultimate carcinogens are electrophilic spe-MATERIALS AND METHODScies, induction in GST activity is believed to be the

principal mechanism of carcinogen detoxification.Most of the reagents used in this study including myristicin were

obtained from Sigma Chemicals Co., St. Louis, MO. Reagents forenzyme purification were obtained from Pharmacia Biotech, Piscata-1 To whom correspondence should be addressed. Fax: (210) 384-

5006. E-mail: [email protected]. way, NJ, whereas the reagents for electrophoresis and Western blot-

0006-291X/97 $25.00Copyright q 1997 by Academic PressAll rights of reproduction in any form reserved.

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AID BBRC 7053 / 6933$$1121 07-11-97 22:43:41 bbrcgs AP: BBRC

Vol. 236, No. 3, 1997 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ting were purchased from Bio-Rad Laboratories, Pasadena, CA. The RESULTS AND DISCUSSIONpolyclonal antibodies against the isoenzymes of glutathione S-trans-ferase were generously provided by Professor Y. C. Awasthi, Univer- Female Albino A/JOlaHsd mice treated with myris-sity of Texas Medical Branch, Galveston, TX.

ticin showed a dose dependent increase in liver GSTTreatment of animals. Female Albino A/JOlaHsd mice, 5-6 activity against CDNB. The specific GST activity

weeks old, were purchased from Harlan Sprague Dawley, Inc., India- showed a 4-fold increase in animals treated with 5 mgnapolis, IN. Guidelines approved by the Institutional Review Com- myristicin compared to control animals, while 10- andmittee for animal use and care were followed. The animals were

14-fold increases were observed in animals treatedacclimatized at our animal care facility for two weeks prior to start ofwith 20 and 50 mg myristicin, respectively (data notthe experiments. The animals were housed under controlled lighting

conditions (12 hours light and 12 hours dark cycle) and were fed presented). These increases in the levels of GST specificsemi-purified diet (AIN-76, ICN Laboratories, Cleveland, OH) and activities are consistent with earlier observations (17).water ad libitum. The experimental groups received varying concen- In order to determine the effect of myristicin on thetrations of myristicin dissolved in corn oil, while the control animals

specific induction of GST isoenzymes, the total GSTreceived only appropriate amounts of corn oil. A total of three dosesactivity and GST protein in liver from the control andof myristicin were administered by gavage every other day, and the

animals were sacrificed 48 hours after the last dose. In order to the treated animals were quantified after purificationinvestigate the dose dependency of myristicin, the three experimen- by GSH-affinity chromatography. For these studies,tal groups (groups 2, 3, and 4) were given 5, 20, and 50 mg of myris- equal weights of tissues from the animals within eachticin per animal, respectively. The animals were sacrificed by cervi- group were pooled, and purification was carried outcal dislocation; the livers were harvested and rinsed with cold saline,

under the same conditions as described under ‘‘Materi-and a small amount of freshly excised tissue from each group wasals and Methods’’. The yields of GSH-affinity purifiedused for GST activities. The remaining tissues were frozen at 0207C

until used. GST in all the samples representing the four groupswere comparable and were consistent with earlier stud-

Enzyme activity. GST activity against 1-chloro-2,4-dinitroben-ies. We have successfully used this procedure pre-zene (CDNB), 2,4-dichloronitrobenzene (DCNB), and ethacrynic acidviously to obtain apparently homogenous preparations(EA) were determined as described by Habig et al. (19). The Beck-

man-DU640 UV/Visible spectrophotometer equipped with enzyme of total GST from several mammalian tissues (21,22).kinetics software was programmed to directly calculate the enzyme The determination of total GST protein content in theunits. One unit of GST activity is defined as the amount of enzyme affinity purified fractions revealed 1.5-fold and 2.9-foldthat utilized 1 mmole of substrate per minute at 257C. Protein was increases over the controls in samples of tissues treateddetermined by Bradford’s dye binding method (20) using bovine se-

with 5 mg and 20-50 mg of myristicin, respectivelyrum albumin as a standard.(data not presented). Figure 1 summarizes the enzyme

Purification of GST. Unless otherwise stated all purification activity data of the affinity purified GST against threesteps were carried out at 47C. Prior to homogenization, the liver was different substrates. The enzyme activity against allthoroughly washed with chilled phosphate-buffered saline (PBS). three substrates showed significant increases in myris-Liver homogenates (10% (w/v)) were prepared from each group in 10

ticin treated groups when compared to controls. SincemM phosphate buffer, pH 7.0, containing 1.4 mM b-mercaptoethanolthese activities are quantitated from equal weights of(buffer A) and centrifuged at 22,000 1 g for 45 min in a Beckman

J21 centrifuge. The supernatants were collected and used for purifi- the control and myristicin-treated mouse livers, the in-cation of GST by affinity chromatography. For comparative studies, creased enzyme activity in myristicin-treated samplesthe GSTs were purified by batch process as described earlier (21,22). appears to be a result of de novo synthesis of GST pro-Briefly, equal weights of the tissues from various groups were sepa-

tein. While the increase in activity against EA was therately homogenized in equal volumes of buffer; 1.0 ml of the superna-least (2.2- to 2.8-fold), the increase in activity againsttant from each of the four groups of liver homogenates were incu-

bated with 1.0 ml of GSH-linked epoxy-activated Sepharose-6B af- CDNB was 4- to 14-fold. The most remarkable increasefinity resin (23) for 3 hours at 47C on a rotator. The unabsorbed of 3- to 38-fold, however, was noted in GST activitysupernatant was removed and the resin was thoroughly washed with against DCNB. Since DCNB is a preferred substrate22 mM phosphate buffer, pH 7.0, containing 1.4 mM b-mercaptoetha-

for the m class of GSTs (11), these results suggest thatnol (buffer B) until the absorbance of the supernatant at 280 nmmyristicin preferentially induces GST m isoenzymes.came close to zero. The bound GST was finally eluted with 0.5 ml of

50 mM Tris-HCl, pH 9.6, containing 5 mM GSH and 1.4 mM b- To further support this observation we also investi-mercaptoethanol. This process was repeated with another 0.5 ml and gated the effect of myristicin on the induction of specificthe two eluates were combined. The affinity purified fraction of the isoenzymes of GST by SDS/PAGE and Western blot-GST was used for further studies. ting. When equal volumes of the affinity purified sam-

ples from the four groups of tissues were analyzed, anPolyacrylamide gel electrophoresis and Western blotting. To in-vestigate the effect of comparative induction of GST isoenzymes by expected increase in the intensities of the GST proteinmyristicin, equal volumes (10 ml) of affinity purified GST were ap- bands was observed (Fig. 2a) without a clear indicationplied to four identical SDS polyacrylamide gels. One gel was used to of specific GST isoenzyme induction. The Western blot-stain proteins and the other three were subsequently electroblotted

ting results, however, clearly indicated that GST m wasonto nitrocellulose papers in a Bio-Rad mini blotting system. Theinduced significantly (Fig. 2b). This observation is con-latter were then incubated against the anti-a anti-m and anti-p GST

antibodies and developed as described previously (21,24). sistent with the enzyme activity data (Fig. 1) which

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FIG. 1. Effect of myristicin administration on affinity purifiedglutathione S-transferase activity of mouse liver. The animals weretreated with, group 1: control; group 2: 5 mg myristicin; group 3: 20mg myristicin; group 4: 50 mg myristicin. Details are given in thetext. A, B, and C represent the GST activity against CDNB, DCNB,and EA, respectively. The values represent means { SD (nÅ4). Sig-nificantly different from control * p õ 0.002, ** p õ 0.001, *** p õ0.0001.

was also indicative of relatively much higher increasesof GST m compared to other GST isoenzymes. The West-ern blotting data also revealed that out of the two mclass GST subunits (Mr. 26,500 and Mr. 25,000) ex-pressed in liver only one (Mr. 26,500) was selectivelyinduced. Consistent with the enzyme activity data, the

FIG. 2. (a) SDS/PAGE of affinity purified mouse liver GST. LaneWestern blotting results also showed only slight1 contains the standard molecular weight markers. Lanes 2-5 containchanges in the levels of GST p protein while proteinequal volumes of affinity purified GST from groups 1-4, respectively.levels of GST a showed a modest increase (Fig. 2b).(b) Western blot analysis of affinity purified GST of mouse liver.Thus, the major effect of myristicin is the selective ele-Lane 1 contains the purified antigens against which the antibodies

vation of GST m levels in mouse liver. This is evident are raised. Lanes 2-5 represent equal volumes of the affinity purifiedfrom the Western blotting (fig. 2b) and the enzyme ac- total GST from groups 1-4, respectively. The nitrocellulose paperstivity towards GST m characteristic substrate, DCNB. were incubated with polyclonal antibodies raised against GST a (i),

GST m (ii), and GST p (iii). Details are given in the text.Other studies also seem to indicate a somewhat prefer-

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5. Prochaska, H. J., and Talalay, P. (1988) Cancer Res. 48, 4776–ential induction of GST m with GST inducers like, t-4782.butylhydroxyanisol (BHA) and phenobarbital (25).

6. Talalay, P., De Long, M. J., and Prochaska, H. J. (1988) Proc.Pearson et al. (26) showed that BHA dramatically in-Natl. Acad. Sci. 85, 8261–8265.

creases both GST m and GST a in mouse liver. However,7. Jakoby, W. B. (1978) Adv. Enzymol. Relat. Areas Mol. Biol. 46,

other studies showed administration of BHA and b- 383–414.naphthoflavone in mice cause preferential induction 8. Mannervik, B., and Danielson, H. U. (1988) CRC Crit. Rev. inonly for a class GST in liver. The reason for this dis- Biochem. 23, 283–337.crepancy is not clear. The current report is the first 9. Chasseaud, L. F. (1979) Adv. Cancer Res. 29, 175–274.

10. Singh, S. V., Srivastava, S. K., and Awasthi, Y. C. (1985) FEBSstudy that indicates the induction of one subunit of theLetters 179, 111–114.two GST m subunits. Further studies to isolate and

11. Mannervik, B., Alin, P., Guthenberg, C., Jensson, H., Tahir,characterize these two m subunits are needed to under-M. K., Warholm, M., and Jornvall, H. (1985) Proc. Natl. Acad.stand the significance of this selective induction.Sci. 82, 7702–7706.

Since GST isoenzymes of various classes exhibit lim- 12. Meyer, D. J., Coles, B., Pemble, S. E., Gilmore, K. S., Fraser,ited GSH conjugating activities towards electrophilic G. M., and Kettrer, B. (1991) Biochem. J. 274, 409–412.carcinogens or their metabolites, it is important to test 13. Hayes, J. D., and Pulford, D. J. (1995) Crit. Rev. Biochem. Mol.the conjugating activity of the myristicin-induced GST Biol. 30, 445–600.

14. Beecher, C. (1995) in Chemoprevention of Cancer (Nixon, D. W.,m against these carcinogens or their metabolites. Sev-Ed.), pp. 21–62, CRC Press, Boca Raton, FL.eral studies suggest that GST m isoenzymes are most

15. Sparnins, V. L., Barany, G., and Wattenberg, L. W. (1988) Carci-efficient in conjugating the carcinogen, 4-nitroquino-nogenesis 9, 131–134.line 1-oxide, with GSH (27,28). Epoxides, such as sty-

16. You, Y. C., Blot, W. J., Chang, Y. S., Ershow, A. G., Yang, Z. T.,rene 7,8-oxide and benzo(a)pyrene 4,5-oxide, represent An, Q., Henderson, B. E., Fraumeni, J. F., Jr., and Wang, T. G.another class of carcinogenic compounds which are se- (1989) J. Natl Cancer Inst. 81, 162–164.lectively metabolized by GST m (29,30). Like other ep- 17. Zheng, G.-q., Kenney, P. M., and Lam, L. K. T. (1992) J. Agric.oxides, benzo(a)pyrene-7,8-diole-9,10-oxide, a potent Food Chem. 40, 107–110.

18. Zheng, G.-q., Kenney, P. M., Zheng, J., and Lam, L. K. T. (1992)carcinogenic metabolite, is also a substrate for the mCarcinogenesis 13, 1921–1923.class GST (31). The over-expression of GST m by myris-

19. Habig, W. H., Pabst, M. J., and Jakoby, W. B. (1974) J. Biol.ticin in mouse liver, therefore, could be very significantChem. 249, 7130–7139.in chemoprevention against at least those carcinogens

20. Bradford, M. M. (1976) Anal. Biochem. 72, 248–254.that are preferentially metabolized by GST m. Studies21. Gupta, S., Ahmad, H., Haque, A. K., and Awasthi, Y. C. (1990)on the substrate specificities of the myristicin-induced Clin. Chem. Enzym. Comms. 3, 115–124.

GST m against the above mentioned carcinogens and 22. Sharma, R., Ahmad, H., Singhal, S. S., Saxena, M., Srivastava,possibly others are, however, needed to establish the S. K., and Awasthi, Y. C. (1993) Comp. Biochem. Physiol. 105C,

31–37.chemopreventive potential of myristicin. The cytotoxic-23. Simmons, P. C., and Vander Jagt, D. L. (1977) Anal. Biochem.ity studies indicated that myristicin, as oppose to other

82, 334–341.alkenylbenzene compounds, appears to be non-carcino-24. Ahmad, H., Singh, S. V., Medh, R. D., Ansari, G. A. S., Kurosky,genic in rodents (32,33).

A., and Awasthi, Y. C. (1988) Arch. Biochem. Biophys. 266, 416–426.

25. Di Simplicio, P., Jensson, H., and Mannervik, B. (1989) Biochem.ACKNOWLEDGMENTSJ. 263, 679–685.

26. Pearson, W. R., Reinhart, J., Sick, S. K., Anderson, K. S., andThis investigation was supported by the US PHS Grants GM08038Alder, P. N. (1988) J. Biol. Chem. 263, 13324–13332.and GM50080 awarded by the National Institutes of General Medical

27. Stanley, J. S., and Benson, A. M. (1992) Biochem. J. 256, 303–Sciences and the University of Texas-Pan American Faculty Re-306.search Council grant.

28. Aceto, A., Di Ilio, C., Lo Bello, M., Bucciarelli, T., Angelucci, S.,and Federici, G. (1990) Carcinogenesis 11, 2267–2274.

REFERENCES 29. Warholm, M., Guthenberg, C., and Mannervik, B. (1983) Bio-chemistry 22, 3610–3617.

1. Wattenberg, L. W. (1983) Cancer Res. 43, 2448–2452. 30. Dostal, L. A., Guthenberg, C., Mannervik, B., and Bend, J. R.2. Wattenberg, L. W. (1985) Cancer Res. 45, 1–18. (1988) Drug Metab. Dispos. 16, 420–424.

31. Robertson, I. G. C., Guthenberg, C., Mannervik, B., and Jern-3. Talalay, P. (1992) in Cancer Chemoprevention (Wattenberg, L.,strom, B. (1986) Cancer Res. 46, 2220–2224.Lipkin, M., Boone, C. W., and Kelloff, G. J., Eds.), pp. 469–478.

CRC Press, Boca Raton, FL. 32. Miller, E. C., Swanson, A. B., Phillips, D. J., Lloyd-Fletcher, T.,Leim, A., and Miller, J. A. (1983) Cancer Res. 43, 1124–1134.4. Wattenberg, L. W. (1992) in Cancer Chemoprevention (Watten-

berg, L., Lipkin, M., Boone, C. W., and Kelloff, G. J., Eds.), pp. 33. Hasheminejad, G., and Caldwell, J. (1994) Fd Chem. Toxic. 32,223–231.19–40. CRC Press, Boca Raton, FL.

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preferential overexpression of a class mu glutathiones-transferase subunit in mouse liver by...

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