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Stimulation of 3-benzo[a]pyrenyl glucuronide hydrolysis by calcium activation of microsomal β-glucuronidase
145
Cancer Letters, 26 (1985) 145-152 Elsevier Scientific Publishers Ireland Ltd.
STIMULATION OF 3-BENZO[a]PYRENYL GLUCURONIDE HYDROLYSIS BY CALCIUM ACTIVATION OF MICROSOMAL B-GLUCURONIDASE
MICHAEL WHITTAKER, PATRICIA M. SOKOLOVE, FREDERICK C. KAUFFMAN*
RONALD G. THURMAN’
and
Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Baltimore, MD 21201 and aDepartment of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, NC 27514 (U.S.A.) (Received 2 October 1984) (Revised version received 4 December 1984) (Accepted 12 December 1984)
SUMMARY
Rates of hydrolysis of 3-hydroxybenzo[a] pyrenyl glucuronide by microsomal /I-glucuronidase from rat liver were 397 + 17 nmol/min per g protein and were half-maximal with about 100 PM substrate. Treatment of rats with phenobarbital or 3_methylcholanthrene, which elevates activities of glucuronosyltransferase( s), lowered rates of hydrolysis of benzo [ a] pyrene glucuronide by 25%. Hydrolysis of the glucuronide by microsomal /3glucuronidase was stimulated by micromolar concentrations of calcium in the range existing in cytosol of hepatocytes (apparent Km - 0.2 MM). Thus, humoral factors that change intracellular concentrations of free calcium may alter the production and export of glucuronides of benzo[a] pyrene metabohtes from the liver.
INTRODUCTION
Recent studies [7,10] employing short-term bacterial bioassays for mutagens [l] demonstrated that conjugated mutagens of benzo[a] pyrene produced by the isolated perfused rat liver appear as phenolic glucuronides in both the bile and effluent perfusate [lo] . Data obtained from several laboratories [ 2,19,20] suggest that the formation of glucuronides from substrates generated via mixed-function oxidation or supplied to the liver from other sources is regulated, at least in part, by a conjugation-deconjugation cycle involving glucuronosyl transferase( s) and /3-glucuronidase located in *To whom correspondence should be addressed. o 1985 Elsevier Scientific Publishers Ireland Ltd. 0304-3835/85/$03.30 Published and Printed in Ireland
146
the hepatic endoplasmic reticulum. In accord with this possibility, we have found that chemical agents and hormones, which elevate cytosolic calcium and activate microsomal P-glucuronidase inhibit the production of p-nitrophenyl glucuronide by the perfused liver [ 2 ] . This activation of fi-glucuronidase by Ca*’ requires association of the enzyme with microsomal membranes and occurs via a calmodulin-independent mechanism [ 201. Since hormonal and nutritional factors influencing the activity of microsomal fl-glucuronidase may influence the export of mutagenic and carcinogenic conjugates from the liver, we examined the influence of micromolar concentrations of calcium on the hydrolysis of 3-hydroxybenzo[a] pyrenyl glucuronide (Fig. 1) by microsomes isolated from rat liver. MATERIALS
AND METHODS
Chemicals 3-Benzo[a] pyrenyl-fl-d-glucopyranosiduronic acid (3-hydroxybenzo[a] pyrenyl glucuronide) and 3-hydroxybenzo [a ] pyrene were obtained from the NC1 Chemical Carcinogen Reference Standard Depository (Illinois Institute of Technology, Chicago, IL). 3-Hydroxybenzo[a] pyrene was dissolved in acetone and stored as a 20 mM stock solution at -80°C; the glucuronide was dissolved in water to a concentration of 10 mM and stored at -80°C. All other chemicals were analyzed reagent grades from standard sources.
3-HYDROXYBENZO(a)PYRENYL
a9
GLUCURONIDE
00
000
OH
3-HYDROXYBENZO(a)PYRENE
Fig. 1. Structures of 3-hydroxybenzo[n]pyrene ronide.
and 3-hydroxybenzo[a]pyrenyl
glucu-
147
Animals and preparation of microsomes Male Sprague-Dawley rats (200-250 g) were anesthetized lightly with ether and decapitated. Livers were removed and homogenized in 9 ~01s. of isolation medium containing 70 mM sucrose, 230 mM mannitol, 2 mM TrisHCl (pH 8.0) and 0.1 mM EGTA. Microsomes were isolated by conventional differential centrifugation [18] and washed twice in the isolated medium without EGTA. Final pellets were resuspended to concentrations of about 20 mg protein/ml, stored at 5°C and used within 1 week of preparation. Protein was determined according to Lowry et al. [ 131. 3_Benzo[a]pyrenyl glucuronide hydrolysis Assays were performed at 37°C in 0.4 ml of reagent consisting of 75 mM HEPES (pH 7.3), 5 mM MgClt, 125 mM KCl, 0.02% bovine serum albumin, 1 mM EGTA and approximately 0.1 mg microsomal protein/ml. Samples were preincubated at 37°C for 15 min before starting reactions by addition of 3-hydroxybenzo [a] pyrenyl glucuronide. Ionized Ca2’ concentrations were adjusted using the calculations described by Fabiato and Fabiato [6] for Ca”-EGTA buffers. 3-Hydroxybenzo[a] pyrene liberated during the course of reactions was determined using a modification of the method described by Dehnen et al. [ 51. At various intervals 50-~1 aliquots of the reaction mixture were pipetted into 250 ~1 of reagent consisting of 1 mM EDTA, 10% triethylamine and 1% Triton X-100 contained in 6 X 50 mm disposable glass culture tube. Fluorescence (435 nm excitation, 522 nm emission) of 3-hydroxybenzo[a]pyrene was determined with an Aminco Bowman Spectrofluorometer. Fluorescence was calibrated using dilutions of known concentrations of 3-hydroxybenzo[a]pyrene. The assay was linear with time for at least 40 min and with microsomal protein ranging between 50 and 130 pg. Under the conditions of the assay, 3-hydroxybenzo[a] pyrene fluorescence was linear up to a concentration of at least 1.75 nmol/ml. RESULTS
Rates of hydrolysis of 3-hydroxybenzo[a] pyrenyl glucuronide by microsomes isolated from normal rats and rats treated with either 3-methylcholanthrene or phenobarbital are shown in Table 1. Treatment of rats with either of the 2 inducing agents decreased rates of hydrolysis by about 25%. These effects differ from the action of the 2 drugs on glucuronosyl transferase which is elevated about 2-fold by phenobarbital pretreatment and g-fold by pretreatment with 3methylcholanthrene [14,21]. When microsomes from normal rats were incubated with 5 PM free Ca2+, glucuronide hydrolysis was increased about 60%. A similar stimulation by calcium was noted in microsomes from phenobarbital-treated rats; however, the increase observed in microsomes from 3-methylcholanthrene-treated rats was less than 40% (Table 1).
148 TABLE 1 EFFECT OF CALCIUM ON HYDROLYSIS OF 3-HYDROXYBENZO[a]PYRENYL GLUCURONIDE BY LIVER MICROSOMES ISOLATED FROM RATS EXPOSED TO VARIOUS TREATMENTS Treatment
None 3-Methylcholanthrene Phenobarbital
Hydrolysis of 3-hydroxybenzo[a]pyrenyl (nmol min-’ g protein-’ )
glucuronide
Control
5 /IM Ca’+
% Stimulation
397 + 17 (8) 262 + 10 (3)** 274 + 7 (3)**
616 + 32 (8)* 394 + 35 (3)** 380 + 12 (3)**
55.2 38.7 50.4
Values are averages + S.E.M. of the number of microsomal preparations indicated in parentheses. Incubations were performed at 37°C using 50 MM 3-hydroxybenzo[a]pyrenyl glucuronide. Animals treated with 3-methylcholanthrene received a single intraperitoneal injection of the drug (80 mg/kg) dissolved in corn oil 3 days before measurement of enzyme activity. Phenobarbital (80 mg/kg) was administered intraperitoneally once daily for 3 days prior to experiments. *P < 0.001 (control vs. calcium). **P < 0.01 (treatment vs. none).
Hydrolysis of 3-hydroxybenzo[a] pyrenyl glucuronide as a function of ionized calcium is shown in Fig. 2. Half-maximal stimulation occurred with about 0.2 PM Ca*‘, which is the range known to exist in the cytosol of hepatocytes [ 4 ,151. Stimulation of the enzyme appears to require association of p-glucuronidase with microsomal membranes. Solubilization of the enzyme from microsomal membranes increased the rate of hydrolysis of the glucuronide to 921 nmol/min per g protein. In contrast to the membrane bound enzyme, the solubilized enzyme was not stimulated by Ca2+ (5 MMand 100 PM). The mechanism by which calcium stimulates hydrolysis of benzo[a] pyrene glucuronide appears to involve an action on the affinity of the enzyme for the glucuronide rather than an effect pn V-m,. Rates of hydrolysis as a function of glucuronide concentration in the presence and absence of 5 PM Ca2+ are shown in Fig. 3. At concentrations above 200 PM 3-hydroxybenzo[a] pyrene glucuronide, stimulation by calcium was slight (
Rates of hydrolysis of 3-hydroxybenzo[a] pyrenyl glucuronide by rat liver microsomal p-glucuronidase are comparable to those noted previously for p-nitrophenyl glucuronide [ 21 and methylumbelliferyl glucuronide [ 201.
149
0-T 0
1
FFLca2+:rw* 1
1
5
Fig. 2. Effect of ionized calcium on hydrolysis of 3-hydroxybenzo[a]pyrenyl glucuronide. Assays were performed with microsomes isolated from normal rats as described in Methods. Initial rates of hydrolysis were calculated from linear regression analyses of data obtained with various calcium concentrations. The regression correlation coefficient (r) of the double-reciprocal plot (insert) is 0.924. Basal rates of hydrolysis in the absence of added calcium were 0.4 nmol mg protein-’ mine’ . Each point is the average of 2 replicate samples.
The hydrolysis of all 3 substrates by microsomal /3-glucuronidase is stimulated by ionized calcium in the physiological range (0.1-5 PM). Thus, a variety of physiological or pharmacological stimuli that cause changes in intracellular calcium may alter net rates of glucuronide production by the liver. This possibility is supported by our recent finding that ol-adrenergic agents, which increase cytosolic free calcium [8,15] and the calcium ionophore, A23187, inhibited the production of p-nitrophenylglucuronide by isolated perfused rat livers [ 21. Possible regulation of net glucuronide production via changes in cytosolic calcium in the liver may have special significance for the meta-
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*, I
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0.01
0.02
3-HYDROXYBENZO(a)PYRENYL
0.03 GLUCURONIDE
0.04
(vM)~’
Fig. 3. Double reciprocal plot of the effect of calcium on hydrolysis of 3-hydroxybenzo [alpyrenyl glucuronide at various substrate concentrations. Initial rates of hydrolysis were determined as described in Fig. 2. Each point is the average of 2 replicate samples. Closed circles are rates measured in the absence of Cal+, open circles are rates measured in the presence of 5 PM free Cal+.
of aromatic hydrocarbons, since evidence exists that mutagenic metabolites of these environmental pollutants are exported from the liver as glucuronides [ 7,101 . Although dihydrodiols and epoxide derivatives of benzo[a] pyrene are more than lOO-fold more mutagenic toward Salmonella typhimurium than 3-hydroxybenzo[a] pyrene [22], consideration of events regulating the formation of 3-hydroxybenzo[a] pyrenyl glucuronide is important. The more reactive dihydrodiol epoxides are not released from the liver either because they are not formed in vivo [3] or because they bind to cellular constituents before leaving the liver. In contrast, glucuronides are readily released, and their transport in blood and bile and subsequent hydrolysis at peripheral tissues [ 171 may be a common mechanism involved in chemical carcinogenesis. The formation of 3 -hydroxybenzo [a] pyrenyl glucuronide bolism
151
and subsequent enzymatic hydrolysis of this glucuronide by p-glucuronidase in liver and other tissues yields metabolites which bind to DNA to a significantly greater extent than 3-hydroxybenzo[a] pyrene, the major end product [ 111. Mechanisms responsible for the production of alternative hydrolysis products are not clear but could involve rearrangement to yield a mixture of yet undefined structures of varying biological activity [ 12,161. Invdvement of glucuronide formation has been suggested for at least one other chemical carcinogen. N-Hydroxynaphthylamine is oxidized via hepatic mixed-function oxidation and conjugated via glucuronosyltransferase. The resulting naphthylarnine glucuronide is transported from the liver to the kidney where it is subsequently hydrolysed by the low pH of urine to an arylnitrenium residue that binds to nucleic acids and other macromolecules within the bladder epithelium [ 91 . The possibility that production of carcinogenic glucuronides might be modulated by agents that influence cytosolic free calcium within the liver and thereby change the activity of P-glucuronidase warrants further study. ACKNOWLEDGMENT
This work was supported ES 02759.
in part by NIH grants CA-20807,
CA 23080 and
REFERENCES 1 Ames, B.N., Durston, W.E., Yamasaki, E. and Lee, F.D. (1973) Carcinogens‘are mutagens: a simple test system combining liver homogenates for activation and bacteria for detection. Proc. Natl. Acad. Sci. U.S.A., 70,2281-2285. 2 Belinsky, S.A., Kauffman, F.C., Sokolove, P.M., Tsukuda, T. and Thurman, R.G. (1984) Calcium-mediated inhibition of glucuronidation by epinephrine in the perfused rat liver. J. Biol. Chem., 259,7705-7711. 3 Boroujerdi, M., Kung, H.-C., Wilson, A.G.E. and Anderson, M.W. (1981) Metabolism and DNA binding of benzo(a)pyrene in uiuo in the rat. Cancer Res., 41, 951-957. 4 Charest, R., Blackmore, P.F., Berthon, B. and Exton, J.H. (1983) Changes in free cytosolic Caz+ in hepatocytes following Q!I -adrenergic stimulation. J. Biol. Chem., 258,8769-8773. 5 Dehnen, W., Tomingas, R. and Roos, J. (1973) A modified method for the assay of benzo[a]pyrene hydroxylase. Anal. Biochem., 53, 373-383. 6 Fabiato, A. and Fabiato, F. (1979) Calculator programs for computing the composition of the solutions containing multiple metals and ligands used for experiments in skinned muscle cells. J. Physiol. (Paris), 75, 463-505. 7 Forti, G.C. and Trieff, N.M. (1980) Kinetics of uptake and biliary excretion of benzo(a)pyrene and mutagenic metabolites in isolated perfused rat liver. Teratogenesis, Carcinogenesis and Mutagenesis, 1,269-282. 8 Joseph, S.K. and Williamson, J.R. (1983) The origin, quantitation, and kinetics of intracellular calcium mobilization by vasopressin and phenylephrine in hepatocytes. J. Biol. Chem., 258, 10425-10432. 9 Kadlubar, F.F., Miller, J.A. and Miller, E.C. (1977) Hepatic microsomal N-glucuronidation and nucleic acid binding of N-hydroxyarylamines in relation to urinary bladder carcinogenesis. Cancer Res., 37, 805-814.
152 10
11 12
13 14
15 16
17 18
19 20
21
22
Kari, F.W., Thurman, R.G. and Kauffman, F.C. (1985) Characterization of mutagenic glucuronide formation from benzo( a)pyrene in the perfused rat liver. Cancer Res., in press. Kinoshita, N. and Gelboin, H. (1978) p-Glucuronidase catalyzed hydrolysis of benzo(a)pyrene-3-glucuronide and binding to DNA. Science, 199, 307-309. Lorentzen, R., Caspary, W., Lesho, S. and Ts’o, P.O.P. (1975) The autoxidation of 6-hydroxybenzo( a)pyrene and Goxabenzo(a)pyrene radical, reactive metabolites of benzo(a)pyrene. Biochemistry, 14, 3970-3977. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the folin phenol reagent. J. Biol. Chem., 193, 265-275. Mackenzie, P.I., Gonzalez, F.J. and Owens, I.S. (1984) Cell-free translation of mouse liver mRNA coding for two forms of UDP glucuronosyltransferase. Arch. Biochem. Biophys., 230, 676-680. Murphy, E., Coll, K., Rich, T.L. and Williamson, J.R. (1980) Hormonal effects on calcium homeostasis in isolated hepatocytes. J. Biol. Chem., 255, 6600-6608. Owens, I.S., Legraverand, C. and Pelkonen, 0. (1979) Deoxyribonucleic acid binding of 3-hydroxyand 9-hydroxybenzo( a)pyrene following further metabolism by mouse liver microsomal cytochrome P-450. Biochem. Pharmacol., 28, 1623-1629. Paigen, K. (1979) Acid hydrolases as models of genetic control. Annu. Rev. Genet., 13,417-466. Remmer, H., Schenkman, J., Estabrook, R.W., &same, H., Gillette, J., Narasimhulu, S., Cooper, D.Y. and Rosenthal, 0. (1966) Drug interaction with hepatic microsomal cytochrome. Mol. Pharmacol., 2, 187-190. Schollhammer, I., Poll, D.S. and Bickel, M.H. (1975) Liver microsomal p-glucuronidase and UDP-glucuronyltransferase. Enzyme, 20, 269-275. Sokolove, P.M., Wilcox, M.A., Thurman, R.G. and Kauffman, F.C. (1984) Stimulation of hepatic microsomal p-glucuronidase by calcium. B&hem. Biophys. Res. Commun., 121,897-993. Tsukuda, T., Thurman, R.G. and Kauffman, F.C. (1983) Effect of inducing agents on the distribution and kinetic properties of UDP-glucuronyltransferase in periportal and pericentral zones of rat liver. Fed. Proc., 42, 912 (abstr. no. 3642). Wislocki, P.G., Wood, A., Chang, R., Levin W., Yagi, H., Hernandez, O., Donsette, P., Jerina, D. and Conney, A. (1976) Mutagenicity and cytotoxicity of benzo(a)pyrene arene oxides, phenol, quinones and dihydrodials in bacterial and mammalian cells. Cancer Res., 36, 3350-3357.
Cancer Letters, 26 (1985) 145-152 Elsevier Scientific Publishers Ireland Ltd.
STIMULATION OF 3-BENZO[a]PYRENYL GLUCURONIDE HYDROLYSIS BY CALCIUM ACTIVATION OF MICROSOMAL B-GLUCURONIDASE
MICHAEL WHITTAKER, PATRICIA M. SOKOLOVE, FREDERICK C. KAUFFMAN*
RONALD G. THURMAN’
and
Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Baltimore, MD 21201 and aDepartment of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, NC 27514 (U.S.A.) (Received 2 October 1984) (Revised version received 4 December 1984) (Accepted 12 December 1984)
SUMMARY
Rates of hydrolysis of 3-hydroxybenzo[a] pyrenyl glucuronide by microsomal /I-glucuronidase from rat liver were 397 + 17 nmol/min per g protein and were half-maximal with about 100 PM substrate. Treatment of rats with phenobarbital or 3_methylcholanthrene, which elevates activities of glucuronosyltransferase( s), lowered rates of hydrolysis of benzo [ a] pyrene glucuronide by 25%. Hydrolysis of the glucuronide by microsomal /3glucuronidase was stimulated by micromolar concentrations of calcium in the range existing in cytosol of hepatocytes (apparent Km - 0.2 MM). Thus, humoral factors that change intracellular concentrations of free calcium may alter the production and export of glucuronides of benzo[a] pyrene metabohtes from the liver.
INTRODUCTION
Recent studies [7,10] employing short-term bacterial bioassays for mutagens [l] demonstrated that conjugated mutagens of benzo[a] pyrene produced by the isolated perfused rat liver appear as phenolic glucuronides in both the bile and effluent perfusate [lo] . Data obtained from several laboratories [ 2,19,20] suggest that the formation of glucuronides from substrates generated via mixed-function oxidation or supplied to the liver from other sources is regulated, at least in part, by a conjugation-deconjugation cycle involving glucuronosyl transferase( s) and /3-glucuronidase located in *To whom correspondence should be addressed. o 1985 Elsevier Scientific Publishers Ireland Ltd. 0304-3835/85/$03.30 Published and Printed in Ireland
146
the hepatic endoplasmic reticulum. In accord with this possibility, we have found that chemical agents and hormones, which elevate cytosolic calcium and activate microsomal P-glucuronidase inhibit the production of p-nitrophenyl glucuronide by the perfused liver [ 2 ] . This activation of fi-glucuronidase by Ca*’ requires association of the enzyme with microsomal membranes and occurs via a calmodulin-independent mechanism [ 201. Since hormonal and nutritional factors influencing the activity of microsomal fl-glucuronidase may influence the export of mutagenic and carcinogenic conjugates from the liver, we examined the influence of micromolar concentrations of calcium on the hydrolysis of 3-hydroxybenzo[a] pyrenyl glucuronide (Fig. 1) by microsomes isolated from rat liver. MATERIALS
AND METHODS
Chemicals 3-Benzo[a] pyrenyl-fl-d-glucopyranosiduronic acid (3-hydroxybenzo[a] pyrenyl glucuronide) and 3-hydroxybenzo [a ] pyrene were obtained from the NC1 Chemical Carcinogen Reference Standard Depository (Illinois Institute of Technology, Chicago, IL). 3-Hydroxybenzo[a] pyrene was dissolved in acetone and stored as a 20 mM stock solution at -80°C; the glucuronide was dissolved in water to a concentration of 10 mM and stored at -80°C. All other chemicals were analyzed reagent grades from standard sources.
3-HYDROXYBENZO(a)PYRENYL
a9
GLUCURONIDE
00
000
OH
3-HYDROXYBENZO(a)PYRENE
Fig. 1. Structures of 3-hydroxybenzo[n]pyrene ronide.
and 3-hydroxybenzo[a]pyrenyl
glucu-
147
Animals and preparation of microsomes Male Sprague-Dawley rats (200-250 g) were anesthetized lightly with ether and decapitated. Livers were removed and homogenized in 9 ~01s. of isolation medium containing 70 mM sucrose, 230 mM mannitol, 2 mM TrisHCl (pH 8.0) and 0.1 mM EGTA. Microsomes were isolated by conventional differential centrifugation [18] and washed twice in the isolated medium without EGTA. Final pellets were resuspended to concentrations of about 20 mg protein/ml, stored at 5°C and used within 1 week of preparation. Protein was determined according to Lowry et al. [ 131. 3_Benzo[a]pyrenyl glucuronide hydrolysis Assays were performed at 37°C in 0.4 ml of reagent consisting of 75 mM HEPES (pH 7.3), 5 mM MgClt, 125 mM KCl, 0.02% bovine serum albumin, 1 mM EGTA and approximately 0.1 mg microsomal protein/ml. Samples were preincubated at 37°C for 15 min before starting reactions by addition of 3-hydroxybenzo [a] pyrenyl glucuronide. Ionized Ca2’ concentrations were adjusted using the calculations described by Fabiato and Fabiato [6] for Ca”-EGTA buffers. 3-Hydroxybenzo[a] pyrene liberated during the course of reactions was determined using a modification of the method described by Dehnen et al. [ 51. At various intervals 50-~1 aliquots of the reaction mixture were pipetted into 250 ~1 of reagent consisting of 1 mM EDTA, 10% triethylamine and 1% Triton X-100 contained in 6 X 50 mm disposable glass culture tube. Fluorescence (435 nm excitation, 522 nm emission) of 3-hydroxybenzo[a]pyrene was determined with an Aminco Bowman Spectrofluorometer. Fluorescence was calibrated using dilutions of known concentrations of 3-hydroxybenzo[a]pyrene. The assay was linear with time for at least 40 min and with microsomal protein ranging between 50 and 130 pg. Under the conditions of the assay, 3-hydroxybenzo[a] pyrene fluorescence was linear up to a concentration of at least 1.75 nmol/ml. RESULTS
Rates of hydrolysis of 3-hydroxybenzo[a] pyrenyl glucuronide by microsomes isolated from normal rats and rats treated with either 3-methylcholanthrene or phenobarbital are shown in Table 1. Treatment of rats with either of the 2 inducing agents decreased rates of hydrolysis by about 25%. These effects differ from the action of the 2 drugs on glucuronosyl transferase which is elevated about 2-fold by phenobarbital pretreatment and g-fold by pretreatment with 3methylcholanthrene [14,21]. When microsomes from normal rats were incubated with 5 PM free Ca2+, glucuronide hydrolysis was increased about 60%. A similar stimulation by calcium was noted in microsomes from phenobarbital-treated rats; however, the increase observed in microsomes from 3-methylcholanthrene-treated rats was less than 40% (Table 1).
148 TABLE 1 EFFECT OF CALCIUM ON HYDROLYSIS OF 3-HYDROXYBENZO[a]PYRENYL GLUCURONIDE BY LIVER MICROSOMES ISOLATED FROM RATS EXPOSED TO VARIOUS TREATMENTS Treatment
None 3-Methylcholanthrene Phenobarbital
Hydrolysis of 3-hydroxybenzo[a]pyrenyl (nmol min-’ g protein-’ )
glucuronide
Control
5 /IM Ca’+
% Stimulation
397 + 17 (8) 262 + 10 (3)** 274 + 7 (3)**
616 + 32 (8)* 394 + 35 (3)** 380 + 12 (3)**
55.2 38.7 50.4
Values are averages + S.E.M. of the number of microsomal preparations indicated in parentheses. Incubations were performed at 37°C using 50 MM 3-hydroxybenzo[a]pyrenyl glucuronide. Animals treated with 3-methylcholanthrene received a single intraperitoneal injection of the drug (80 mg/kg) dissolved in corn oil 3 days before measurement of enzyme activity. Phenobarbital (80 mg/kg) was administered intraperitoneally once daily for 3 days prior to experiments. *P < 0.001 (control vs. calcium). **P < 0.01 (treatment vs. none).
Hydrolysis of 3-hydroxybenzo[a] pyrenyl glucuronide as a function of ionized calcium is shown in Fig. 2. Half-maximal stimulation occurred with about 0.2 PM Ca*‘, which is the range known to exist in the cytosol of hepatocytes [ 4 ,151. Stimulation of the enzyme appears to require association of p-glucuronidase with microsomal membranes. Solubilization of the enzyme from microsomal membranes increased the rate of hydrolysis of the glucuronide to 921 nmol/min per g protein. In contrast to the membrane bound enzyme, the solubilized enzyme was not stimulated by Ca2+ (5 MMand 100 PM). The mechanism by which calcium stimulates hydrolysis of benzo[a] pyrene glucuronide appears to involve an action on the affinity of the enzyme for the glucuronide rather than an effect pn V-m,. Rates of hydrolysis as a function of glucuronide concentration in the presence and absence of 5 PM Ca2+ are shown in Fig. 3. At concentrations above 200 PM 3-hydroxybenzo[a] pyrene glucuronide, stimulation by calcium was slight (
Rates of hydrolysis of 3-hydroxybenzo[a] pyrenyl glucuronide by rat liver microsomal p-glucuronidase are comparable to those noted previously for p-nitrophenyl glucuronide [ 21 and methylumbelliferyl glucuronide [ 201.
149
0-T 0
1
FFLca2+:rw* 1
1
5
Fig. 2. Effect of ionized calcium on hydrolysis of 3-hydroxybenzo[a]pyrenyl glucuronide. Assays were performed with microsomes isolated from normal rats as described in Methods. Initial rates of hydrolysis were calculated from linear regression analyses of data obtained with various calcium concentrations. The regression correlation coefficient (r) of the double-reciprocal plot (insert) is 0.924. Basal rates of hydrolysis in the absence of added calcium were 0.4 nmol mg protein-’ mine’ . Each point is the average of 2 replicate samples.
The hydrolysis of all 3 substrates by microsomal /3-glucuronidase is stimulated by ionized calcium in the physiological range (0.1-5 PM). Thus, a variety of physiological or pharmacological stimuli that cause changes in intracellular calcium may alter net rates of glucuronide production by the liver. This possibility is supported by our recent finding that ol-adrenergic agents, which increase cytosolic free calcium [8,15] and the calcium ionophore, A23187, inhibited the production of p-nitrophenylglucuronide by isolated perfused rat livers [ 21. Possible regulation of net glucuronide production via changes in cytosolic calcium in the liver may have special significance for the meta-
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I
0.01
0.02
3-HYDROXYBENZO(a)PYRENYL
0.03 GLUCURONIDE
0.04
(vM)~’
Fig. 3. Double reciprocal plot of the effect of calcium on hydrolysis of 3-hydroxybenzo [alpyrenyl glucuronide at various substrate concentrations. Initial rates of hydrolysis were determined as described in Fig. 2. Each point is the average of 2 replicate samples. Closed circles are rates measured in the absence of Cal+, open circles are rates measured in the presence of 5 PM free Cal+.
of aromatic hydrocarbons, since evidence exists that mutagenic metabolites of these environmental pollutants are exported from the liver as glucuronides [ 7,101 . Although dihydrodiols and epoxide derivatives of benzo[a] pyrene are more than lOO-fold more mutagenic toward Salmonella typhimurium than 3-hydroxybenzo[a] pyrene [22], consideration of events regulating the formation of 3-hydroxybenzo[a] pyrenyl glucuronide is important. The more reactive dihydrodiol epoxides are not released from the liver either because they are not formed in vivo [3] or because they bind to cellular constituents before leaving the liver. In contrast, glucuronides are readily released, and their transport in blood and bile and subsequent hydrolysis at peripheral tissues [ 171 may be a common mechanism involved in chemical carcinogenesis. The formation of 3 -hydroxybenzo [a] pyrenyl glucuronide bolism
151
and subsequent enzymatic hydrolysis of this glucuronide by p-glucuronidase in liver and other tissues yields metabolites which bind to DNA to a significantly greater extent than 3-hydroxybenzo[a] pyrene, the major end product [ 111. Mechanisms responsible for the production of alternative hydrolysis products are not clear but could involve rearrangement to yield a mixture of yet undefined structures of varying biological activity [ 12,161. Invdvement of glucuronide formation has been suggested for at least one other chemical carcinogen. N-Hydroxynaphthylamine is oxidized via hepatic mixed-function oxidation and conjugated via glucuronosyltransferase. The resulting naphthylarnine glucuronide is transported from the liver to the kidney where it is subsequently hydrolysed by the low pH of urine to an arylnitrenium residue that binds to nucleic acids and other macromolecules within the bladder epithelium [ 91 . The possibility that production of carcinogenic glucuronides might be modulated by agents that influence cytosolic free calcium within the liver and thereby change the activity of P-glucuronidase warrants further study. ACKNOWLEDGMENT
This work was supported ES 02759.
in part by NIH grants CA-20807,
CA 23080 and
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