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Glutathione S-Transferases and Esterases in Placenta after Normal and Pre-eclamptic Pregnancies
Placenta (2004), 25, 331–336 doi:10.1016/j.placenta.2002.12.001
Glutathione S-Transferases and Esterases in Placenta after Normal and Pre-eclamptic Pregnancies M. del Carmen Terrones Saldı´ var a,1, F. J. Jua´rez a, J. L. Viramontes a, M. L. Rodrı´ guez Va´zquez a and F. A. Posadas del Rı´ o b,* a
Departamento de Fisiologı´ a y Farmacologı´ a, Centro de Ciencias Ba´sicas, Universidad Auto´noma de Aguascalientes, Av. Universidad 940, 20100 Aguascalientes, Ags, Mexico; b Seccio´n Externa de Farmacologı´ a, CINVESTAV-IPN, Ap. Postal 14-740, 07000 Me´xico, DF, Mexico Paper accepted 11 December 2002
Severe pre-eclampsia reduced significantly (P<0.05) by 686 per cent (mean, n=10) the maximal velocity (Vmax) and, consequently, reduced significantly by 607 per cent the catalytic efficiency (CE) of placental glutathione transferase , assayed with ethacrynic acid. Mild and severe pre-eclampsia reduced significantly by 825 per cent (mean, n=5) and by 415 per cent (mean, n=10), respectively, the Vmax and, consequently, reduced significantly by 727 and by 3313 per cent, respectively, the CE of esterase, assayed with p-nitrophenyl acetate. Furthermore, severe pre-eclampsia increased significantly by 29678 per cent the Michaelis–Menten constant (Km) of total GST, assayed with chlorodinitrobenzene and, consequently, decreased significantly the CE by 833 per cent. On the other hand, the concentrations of total and non-protein thiols did not change significantly in placental homogenates from patients with mild or severe pre-eclampsia compared to normal pregnancies. These findings would indicate a decreased capacity of the glutathione transferases and esterase detoxification systems to protect the fetus from drugs prescribed to pregnant women suffering pre-eclampsia, mainly in the severe phase. Placenta (2004), 25, 331–336 2003 Elsevier Ltd. All rights reserved.
INTRODUCTION Pre-eclampsia is considered one of the most important health problems in human pregnancy because it complicates from 5 to 7 per cent of pregnancies and is a leading cause of fetal growth retardation, indicated premature delivery, and fetal and maternal death [1]; pre-eclampsia is a multisystem disorder of human pregnancy with a genetic predisposition [2]. Pre-eclampsia is usually diagnosed in late pregnancy by proteinuria, oedema and increased vasoconstriction leading to maternal hypertension and reduced uteroplacental blood flow. Drug biotransformation in placenta was reviewed by Juchau [3,4] and more recently by Harbison et al. [5] and Pasanen [6]. The glutathione S-transferases (GST; EC 2.5.1.18) are a family of soluble enzymes that play a key role in the biotransformation and detoxification of a wide number of endogenous [7–9] and exogenous compounds in liver, kidneys and intestine [10–15]. Glutathione S-transferase activity has been detected in human placenta from normal pregnancies [16–25]. GST have also been studied in pre-eclamptic pregnancies [25–27]. On the other hand, carboxylic esters are hydrolysed by preparations of placental tissues [3]; placental cytosol exhibits
1 In partial fulfilment of Doctorate’s degree in Pharmacology. * To whom correspondence should be addressed: Tel.: +52-4-499108424; Fax: +52-4-49910-8401; E-mail: [email protected]
0143-4004/$–see front matter
cholesterol ester hydrolase activity [28] whereas placental microsomes hydrolyse carboxylic esters of p-nitrophenol [29].
MATERIALS AND METHODS Chloro-2,4-dinitrobenzene, ethacrynic acid, reduced glutathione (GSH), p-nitrophenyl acetate and 5,5#-dithio-bisnitrobenzoic acid were purchased from Sigma Chemical (St Louis, MO, USA). All other reagents were of purest analytical grade commercially available. Subjects were recruited at the time of admittance for labour and delivery at the Gynecology and Obstetrics State Hospital, Jesu´s Marı´a, Aguascalientes, Mexico. The use of human placentae for this study was approved by the Hospital Ethic Committee. All participants were informed of the experimental procedure and gave written consent prior to delivery. Fifteen subjects with uncomplicated pregnancies throughout gestation with age of 26.16 years (means.d.) were selected as the control group; they were normotensive and had no proteinuria (Table 1); inclusion criteria considered subjects having no known medical illnesses, absence of tobacco or alcohol abuse, and abstinence from use of drugs known to be either inducers or inhibitors, over the preceding 3 months. Five patients with mild preeclampsia (age of 24.45 years; means.d.) and eleven patients with severe pre-eclampsia (age of 24.25 years; 2003 Elsevier Ltd. All rights reserved.
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Table 1. Data of obstetric parameters from the patients participating in our study Parameter
Control
Mild pre-eclampsia
Severe pre-eclampsia
Maternal age (years) Maternal weight (kg) Gestation time (weeks) Parity Caesarean delivery Sex of newborn Weight of newborn (kg) Apgar
26.16 778 37.71 Multiparous (8/15) 13/15 Feminine (11/15) 3.00.44 8–9
24.45 97.63 37.61 Second (3/5) 5/5 Masculine (3/5) 3.10.3 9–9
24.25 85.214.4 35.04a Multiparous (8/11) 11/11 Masculine (8/11) 2.40.9a 7–8
Values represent mean from control (n=15), mild (n=5) and Severe (n=10–11) pre-eclamptic patients. P>0.01.
a
means.d.) were also included in this study. Mild preeclampsia was defined as a blood pressure of R140/90 mm Hg, on two separate readings at least 6 h apart, proteinuria <2+ or pathologic oedema; severe pre-eclampsia was defined as a blood pressure of R160/110 mm Hg, on two separate readings at least 6 h apart, proteinuria R2+ and pathologic oedema. The diagnosis was based on hypertension, proteinuria and oedema in the late stages of their pregnancies. Hypertension was defined as an increase of 30 mm Hg systolic or 15 mm Hg diastolic blood pressure or an absolute blood pressure of >140/90 mm Hg after 20 weeks of gestation, if earlier blood pressures were not known (measured on two occasions, at least 6 h apart). Proteinuria was defined as >2+ in random urine specimens. Oedema in lower members is present in mild pre-eclampsia and generalized oedema in severe pre-eclampsia. Placentae were obtained within 20 min after vaginal or Caesarean delivery and suitable amounts were immediately and repeatedly washed with ice-cold physiological saline, to eliminate as much blood as possible; fragments were weighted, usually 5 g, minced and homogenized in 20 ml 50 m potassium phosphate, pH 7.5, with a Potter–Elvejhem homogenizer and the resultant homogenates were centrifuged at 12 100 g for 10 min, in a refrigerated Sorvall Super T centrifuge; the supernatants were placed in clean tubes, added enough CaCl2 to reach final concentration of 8 m [30–33] and centrifuged at 27 000 g for 20 min to obtain the microsomal and cytosolic fractions. All procedures were carried out at 42(C. The kinetics of total glutathione S-transferase activity was measured in the cytosolic fraction with 50 m potassium phosphate, pH 6.5, 0.5 m reduced glutathione (GSH) and 0.0125, 0.025, 0.05, 0.1, 0.2 and 0.4 m chlorodinitrobenzene (CDNB) as substrate [34], at 340 nm; we used 0.5 m GSH instead of 1.0 m in order to diminish the nonenzymatic conjugation of GSH with CDNB. The kinetics of glutathione S-transferase P () activity was measured in the cytosolic fraction with 50 m potassium phosphate, pH 6.5, 0.25 m reduced glutathione (GSH) and 0.0125, 0.025, 0.05, 0.1 and 0.2 m ethacrynic acid as substrate [34], at 270 nm. The kinetics of total esterase activity was also determined in the cytosolic fraction using 50 m Tris–Cl, pH 8.0, and 0.0125,
0.025, 0.05, 0.1, 0.2 and 0.4 m of p-nitrophenyl acetate as substrate, at 405 nm [35–37]. All the enzymatic assays were performed at room temperature (241(C) and in 1.0 ml final incubation volume. The initial enzymatic rates were calculated from 1 min readings, usually in duplicate, in a Hach DR/4000U UV-VIS spectrophotometer. Total and non-protein thiols or sulphydryl groups were determined in placental homogenates, as described by Sedlak and Lindsay [38]. Protein was measured with the Folin phenol reagent using bovine serum albumin as standard [39]. We calculated the kinetic constants (Vmax, Km) and the catalytic efficiency (CE) as well as the significance of differences between group means for the three variables with the twotailed Student’s t-test for grouped data using the program GraphPad Prism, v. 3.0 (GraphPad Sofware Inc). The catalytic efficiency (EC) or intrinsic metabolic clearance of enzymes is defined as the ratio of the maximal velocity (Vmax) of the reaction to the Michaelis–Menten constant (Km), that is Vmax/Km [40,41] or as the relative intrinsic clearance [42].
RESULTS Figure 1 shows that the maximal velocity (Vmax) of total glutathione S-transferases (T-GSH), assayed with chlorodinitrobenzene as substrate, were not significantly modified in placentae from the three groups; however, the Michaelis– Menten constant (Km) was significantly increased (P>0.05) by 396175 per cent (mean, n=5) and by 29678 per cent (mean, n=11) in placentae from mild pre-eclamptic patients and in placentae from severe pre-eclamptic patients, respectively, compared with placenta from control patients (mean, n=15). The catalytic efficiency (CE) was significantly reduced (833 per cent) only in placentae from severe pre-eclamptic patients. When we used ethacrynic acid as substrate, the Vmax of glutathione S-transferase P () was significantly reduced by 686 per cent (mean, n=10) only in placentae from severe pre-eclamptic patients (Figure 2). There were not significant differences in the Km among the three groups but the CE was significantly decreased (607 per cent and
M. del Carmen Terrones Saldı´var et al.: Glutathione S-Transferases and Esterases in Human Placenta
333
Figure 1. Relative values (mean) of the kinetic constants (Vmax, Km and CE) of total glutathione S-transferase (T-GST) in placentae from control patients and from mild and severe pre-eclamsia. The actual control values are: Vmax=11824 nmol GS-DNB/min1mg1, Km=23470 µ and Vmax/Km=0.77 140 ml/min1mg1. Vmax is actually five times lower, and CE (Vmax/Km) is 1000 times lower. PE=Pre-eclampsia. GS-DNB=Glutathione–dinitrobenzene conjugate.
Figure 2. Relative values (mean) of the kinetic constants (Vmax, Km and CE) of glutathione S-transferase P or (GST P or ) in placentae from control patients and from mild and severe pre-eclamsia. The actual control values are: Vmax=72.412 nmol GS–ethacrynic acid/min1mg1, Km=58.917 µ and Vmax/Km=1.630.2 ml/min1mg1. CE (Vmax/Km) is actually 10 times lower. PE=Pre-eclampsia.
637 per cent; mean, n=5 and n=10) in placentae from mild pre-eclamptic patients and in placentae from severe pre-eclamptic patients, respectively, as compared with control patients (mean, n=15). To our surprise, we found that esterase activity was higher in the cytosolic fraction than in the microsomal fraction. The kinetic constants calculated in the placental cytosolic fraction showed that the Vmax of the total esterase activity was significantly reduced by 825 per cent (mean, n=5) in placentae from mild pre-eclamptic patients and by 415 per cent (mean, n=11) in placentae from severe preeclamptic patients as compared with control patients (n=15), with p-nitrophenyl acetate as substrate (Figure 3); there were
not significant differences in the Km (µ) among the three groups but the catalytic efficiency (CE) was significantly reduced by 727 per cent in placentae from mild pre-eclamptic patients and by 3313 per cent in placentae from severe pre-eclamptic patients as compared with control patients. Total and non-protein thiol concentrations did not show (Figure 4) significant differences among the three groups.
DISCUSSION We determined the enzyme kinetics of total glutathione S-transferases (T-GST), glutathione S-transferase P () and of
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Figure 3. Relative values (mean) of the kinetic constants (Vmax, Km and CE) of esterase in placentae from control patients and from mild and severe preeclamsia. The actual control values are: Vmax=43.85.5 nmol p-nitrophenol/min1mg1, Km=443 82 µ and Vmax/Km=0.110.012 ml/min1mg1. Vmax is actually 10 times lower, and CE (Vmax/Km) is 5000 times lower. PE=Pre-eclampsia.
Figure 4. Concentrations of total thiols and non-protein thiols (mean) in placental homogenates from control patients and from mild and severe pre-eclampsia. The actual control values are: total thiols=17617 nmol/g1 and non-protein thiols=8.011.1 nmol/g1. Non-protein thiols are actually 10 times lower. PE=Pre-eclampsia.
esterase in the cytosolic fraction of human placenta from normal and from mild and severe pre-eclamptic pregnancies. T-GST showed the highest maximal velocity (Vmax) but glutathione transferase P () was 2.1 and 14.8 times catalytically more efficient than T-GST and esterase, respectively, in placenta from normal pregnancies, mainly due to its lower Michaelis–Menten constant (Km). Polidoro et al. [16] found a Km of 0.67 m in placenta cytosols from healthy pregnancies, using chlorodinitrobenzene as substrate; this value is 2.8 times higher than the value we found in the control patients. Guthenberg and Mannervik [17] reported a Km of 2.1 m for purified glutathione transferase (GST-) from full term human placenta. Pacifici and Rane [18] reported a mean Vmax
of 11.76 nmol/min1 mg1 and a mean Km of 4.13 m (n=3) in human placental cytosols from normal deliveries, using styrene oxide as substrate; these values give a mean catalytic efficiency (CE) of 0.0028 ml/min1 mg1. In placenta from mild and severe pre-eclamptic patients only the Km of T-GST was significantly increased when compared to placenta from normal pregnancies and the CE was significantly decreased only in placenta from severe preeclamptic patients. In contrast, we found that pre-eclampsia did not significantly change the Km of GST-; however, pre-eclampsia diminished the Vmax of GST- but this decrease was significantly different only in placenta from severe preeclamptic patients compared to normal pregnancies; these
M. del Carmen Terrones Saldı´var et al.: Glutathione S-Transferases and Esterases in Human Placenta
results agree with the lower concentration of glutathione transferase P () described by Zusterzeel et al. [25] in placenta of pre-eclamptic pregnancies compared to normal pregnancies. The lack of significance in the Vmax of placenta from mild pre-eclamptic patients might be due to the comparatively low number of placenta studied (n=5). Glutathione transferase is the main isoform in normal placental tissue and comprises 36 per cent of total GST activity [43] and 67 per cent [25] of the T-GST concentration. Furthermore, placenta from mild and severe pre-eclamptic patients were catalytically less efficient than placenta from normal pregnancies. On the other hand, it was recently shown [24] than human term placental lipoxygenase, purified by affinity chromatography, catalyse GSH conjugate formation with ethacrynic acid, in the presence of linoleic acid; this reaction has a catalytic efficiency of 35.5 ml/ min1 mg1 and did not show detectable conjugation with chlorodinitrobenzene as substrate. Placental cytosolic esterase had the lowest Vmax, the highest Km and, consequently, the lowest CE of the activities tested; the cytosolic fractions used have slight cholinesterase activity with 0.8 m acetylthiocholine as substrate. The CE of microsomal Carboxylesterases from human term placenta has been reported to be approximately 10 times higher than our values, also using p-nitrophenyl acetate as substrate [29]. Mild and
335
severe pre-eclampsia diminished significantly the CE of cytosolic esterases mostly due to decreases in Vmax, when compared to normal pregnancies. Finally, the concentration of total and non-protein thiols (GSH represents more than 95 per cent; [44,45]) did not change significantly in placental homogenates from patients with mild or severe pre-eclampsia compared to normal pregnancies. Mutlu-Turkoglu et al. [22] reported that GSH concentrations are decreased in placenta from pre-eclamptic pregnant woman when compared to normal pregnant woman. In summary, pre-eclampsia reduced the Vmax and, consequently, the catalytic efficiency of placental glutathione transferase and esterase(s) most dramatically in patients with severe pre-eclampsia; these findings would indicate a decreased capacity of the glutathione transferases and esterase detoxification systems to protect the fetus from drugs prescribed to pregnant women. The reduction in drug biotransformation efficiency could be due to placental hypoperfusion as a result of vasoconstriction, intravascular coagulation and decreased uteroplacental blood flow. Moreover, the drugs used to treat pre-eclampsia might also participate in the decreased capacity of the drug metabolism systems; in this regard, there is little information except for the reported in vitro inhibition of commercial GST from bovine liver by dihydropyridines [46] and of hepatic GST activities from male rats by diuretics [47].
ACKNOWLEDGEMENTS The authors acknowledge the medical and nurse personnel of the Gynecology and Obstetrics State Hospital, Jesu´s Marı´a, Aguascalientes, Me´xico, for their valuable help and support, and Mr Alfredo Padilla for the art work.
REFERENCES [1] Chesley LC. Hypertensive disorders in pregnancy. New York: Appleton-Century-Crofts, 1978. [2] Brown MA. The physiology of pre-eclampsia. Clin Exp Pharmacol Physiol 1995;22:781–91. [3] Juchau MR. Drug biotransformation reactions in placenta. In: Mirkkin B, editor. Perinatal pharmacology and therapeutics. New York: Academic Press; 1976, p. 71–118. [4] Juchau MR. Drug biotransformation in placenta. Pharmacol Ther 1980; 8:501–24. [5] Harbison RD, Borgert CJ, Teaf CM. Placental metabolism of xenobiotics. In: Rama Sastry BV, editor. Placental toxicology. Boca Raton: CRC Press; 1995, p. 197–238. [6] Pasanen M. The expression and regulation of drug metabolism in human placenta. Adv Drug Deliv Res 1999;38:81–97. [7] Mannervik B, Jensson H, Alin P, Orning L, Hammarstrom S. Transformation of leukotriene A4 methylester to leukotriene C4 monomethylester by cytosolic glutathione transferases. FEBS Lett 1984;174:289–94. [8] Soderstro¨m M, Mannervik B, Orning L, Hammastro¨m S. Leukotriene C4 formation by three distinct forms of human glutathione transferase. Biochem Biophys Res Commun 1985;128:265–70. [9] Tsuchida S, Izumi T, Shimizu T, Ishikawa T, Satoh K, Sato K. Purification of a new acidic glutathione S-transferase, GST-YniYni, with high leukotriene-C4 synthase activity from rat brain. Eur J Biochem 1987; 170:159–64. [10] Boyland E, Chasseaud LF. The role of glutathione and glutathione S-transferase in mercapturic acid biosynthesis. Adv Enzymol 1969; 32:172–219. [11] Kaplowitz N, Clifton C, Kuhlenkamp J, Wallin JD. Comparison of renal and hepatic glutathione S-transferases in the rat. Biochem J 1976; 158:243–8.
[12] Clifton C, Kaplowitz N. The glutathione S-transferases of small intestine in the rat. Cancer Res 1977;37:788–91. [13] Pinkus LM, Netley JN, Jakoby WB. The glutathione S-transferases as a possible detoxification system in rat intestinal epithelium. Biochem Pharmacol 1977;26:2359–63. [14] Jakoby WB, Habig WH. Glutathione transferases. Enzymatic basis of detoxication. New York: Academic Press; 1980;II:63–94. [15] Mannervik B. The isoenzymes of glutathione transferase. Adv Enzymol Relat Areas Mol Biol 1985;57:357–414. [16] Polidoro G, Di Ilio C, Del Boccio G, Zulli P, Federici G. Glutathione S-transferase activity in human placenta. Biochem Pharmacol 1980; 29:1677–80. [17] Guthenberg C, Mannervik B. Glutathione S-transferase (transferase ) from human placenta is identical or closely related to Glutathione S-transferase (transferase ) from erythrocytes. Biochim Biophys Acta 1981;661:255–60. [18] Pacifici GM, Rane A. Glutathione S-epoxidetransferase in human placenta at different stages of pregnancy. Drug Metab Dispos 1981;9:472–5. [19] Di Ilio C, Polidoro G, Arduini A, Muccini A, Federici G. Glutathione peroxidase, glutathione reductase, glutathione S-transferase, and -glutamyltranspeptidase activities in the human early pregnancy placenta. Biochem Med 1983;29:143–8. [20] McRobie DJ, Glover DD, Tracy TS. Effect of gestational and overt diabetes on human placental cytochromes 450 and glutathione S-transferase. Drug Metab Dispos 1996a;26:367–71. [21] Obolenskaya MY, Tschaikovskaya TL, Lebedeva LM, Macewicz LL, Didenko LV, Decker K. Glutathione status of placenta from differently polluted regions of Ukraine. Eur J Obstet Gynecol Obstet Reprod Biol 1997;71:23–30. [22] Mutlu-Turkoglu U, Ademoglu E, Ibrahimoglu L, Aykac-Toker G, Uysal M. Imbalance between lipid peroxidation and antioxidant status in preeclampsia. Gynecol Obstet Invest 1998;46:37–40.
336 [23] McRobie DJ, Glover DD, Tracy TS. Regiospecificity of placental metabolism by cytochromes 450 and glutathione S-transferase. Gynecol Obstet Invest 1996b;42:154–8. [24] Kulkarni AP, Sajan M. Lipoxygenase—another pathway for glutathione conjugation of xenobiotics: A study with human term placental lipoxygenase and ethacrynic acid. Arch Biochem Biophys 1999;371:220–7. [25] Zusterzeel PL, Peters WH, De Bruyn MA, Knapen MF, Merkus HM, Steegers EA. Glutathione S-transferase isoenzymes in decidua and placenta of pre-eclamptic pregnancies. Obstet Gynecol 1999;94:1033–8. [26] Poranen AK, Ekblad U, Uotila P, Ahotupa M. Lipid peroxidation and antioxidants in normal and pre-eclamptic pregnancies. Placenta 1996; 17:401–5. [27] Knapen MF, Peters WH, Mulder TP, Merkus HM, Jansen JB, Steegers EA. Glutathione and glutathione related enzymes in decidua and placenta of controls and women with pre-eclampsia. Placenta 1999;20:541–6. [28] Waterman IJ, Emmison N, Dutta-Roy AK. Characterisation of triacylglycerol hydrolase activities in human placenta. Biochim Biophys Acta 1998;1394:169–76. [29] Yan B, Matoney L, Yang D. Human carboxylesterases in term placentae: enzymatic characterization, molecular cloning and evidence for the existence of multiple forms. Placenta 1999;20:599–607. [30] Kamath SA, Kimmerow FA, Narayan KA. A simple procedure for the isolation of rat liver microsomes. FEBS Lett 1971;17:90–2. [31] Schenkman JB, Cinti DL. Hepatic mixed function oxidase activity in rapidly prepared microsomes. Life Sci 1972;11(Part II):247–57. [32] Cinti DL, Moldeus P, Schenkman JB. Kinetic parameters of drugmetabolizing enzymes in Ca2+-sedimented microsomes from rat liver. Biochem Pharmacol 1972;21:3249–56. [33] Schenkman JB, Cinti DL. Preparation of microsomes with calcium. Methods Enzymol 1978;52C:83–9. [34] Habig WH, Pabst MJ, Jakoby WB. Glutathione S-transferases. The first enzymatic step in mercapturic acid formation. J Biol Chem 1974; 249:7130–9. [35] Shirkey RS, Chakraborty J, Bridges JW. Comparison of the drug metabolizing activity of rat intestinal mucosal microsomes with that of liver. Biochem Pharmacol 1979;28:2835–9.
Placenta (2004), Vol. 25 [36] Mentlein R, Suttorp M, Heymann E. Specificity of monoacylglycerol lipase, palmitoyl-CoA hydrolase, palmitoyl-carnitine hydrolase and nonspecific carboxylesterase from rat liver microsomes. Arch Biochem Biophys 1984;228:230–46. [37] Morgan EW, Yan B, Greenway D, Petersen DR, Parkinson A. Purification and characterization of two rat liver microsomal carboxylesterases (Hydrolases A and B). Arch Biochem Biophys 1994;315:495–512. [38] Sedlak J, Lindsay RH. Estimation of total, protein bound, and non protein sulfhydryl groups in tissue with Ellman’s reagent. Anal Biochem 1968;25:192–205. [39] Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ. Protein measurements with the Folin phenol reagent. J Biol Chem 1951;193:265–75. [40] Wallace KB, Dargan JE. Intrinsic metabolic clearance of parathion and paraoxon by livers from fish and rodents. Toxicol Appl Pharmacol 1987; 90:235–42. [41] Kaminski LS, Zhang Z-Y. Human P-450 metabolism of warfarin. Pharmacol Ther 1997;73:67–74. [42] Birkkett DJ, Mackenzie PI, Veronese ME, Miners JO. In vitro approaches can predict human drug metabolism. Trends Pharmacol Sci 1993;14:292–4. [43] Steisslinger HW, Pfleiderer G. Acidic and basic forms of glutathione S-transferases from human placenta and comparison with human kidney glutathione S-transferase. Enzyme 1988;40:1–6. [44] Kosower NS, Kosower RJ. Glutathione status of cells. Int Rev Cytol 1978;54:109–60. [45] Meister A. New aspects of glutathione biochemistry and transportselective alterations of glutathione metabolism. Nutr Rev 1984; 42:397–410. [46] Tirzite D, Tirzites G, Vigante B, Duburs G. The effect of 1,4dihydropyridine and 1,4-dihydroindeno(1,2-b)pyridine derivatives on glutathione S-transferase in vitro. Biochem Pharmacol 1993;46:773–5. [47] Ahokas JT, Davis C, Ravenscroft PJ, Emmerson BT. Inhibition of soluble glutathione S-transferase by diuretic drugs. Biochem Pharmacol 1984;33:1629–932.
Glutathione S-Transferases and Esterases in Placenta after Normal and Pre-eclamptic Pregnancies M. del Carmen Terrones Saldı´ var a,1, F. J. Jua´rez a, J. L. Viramontes a, M. L. Rodrı´ guez Va´zquez a and F. A. Posadas del Rı´ o b,* a
Departamento de Fisiologı´ a y Farmacologı´ a, Centro de Ciencias Ba´sicas, Universidad Auto´noma de Aguascalientes, Av. Universidad 940, 20100 Aguascalientes, Ags, Mexico; b Seccio´n Externa de Farmacologı´ a, CINVESTAV-IPN, Ap. Postal 14-740, 07000 Me´xico, DF, Mexico Paper accepted 11 December 2002
Severe pre-eclampsia reduced significantly (P<0.05) by 686 per cent (mean, n=10) the maximal velocity (Vmax) and, consequently, reduced significantly by 607 per cent the catalytic efficiency (CE) of placental glutathione transferase , assayed with ethacrynic acid. Mild and severe pre-eclampsia reduced significantly by 825 per cent (mean, n=5) and by 415 per cent (mean, n=10), respectively, the Vmax and, consequently, reduced significantly by 727 and by 3313 per cent, respectively, the CE of esterase, assayed with p-nitrophenyl acetate. Furthermore, severe pre-eclampsia increased significantly by 29678 per cent the Michaelis–Menten constant (Km) of total GST, assayed with chlorodinitrobenzene and, consequently, decreased significantly the CE by 833 per cent. On the other hand, the concentrations of total and non-protein thiols did not change significantly in placental homogenates from patients with mild or severe pre-eclampsia compared to normal pregnancies. These findings would indicate a decreased capacity of the glutathione transferases and esterase detoxification systems to protect the fetus from drugs prescribed to pregnant women suffering pre-eclampsia, mainly in the severe phase. Placenta (2004), 25, 331–336 2003 Elsevier Ltd. All rights reserved.
INTRODUCTION Pre-eclampsia is considered one of the most important health problems in human pregnancy because it complicates from 5 to 7 per cent of pregnancies and is a leading cause of fetal growth retardation, indicated premature delivery, and fetal and maternal death [1]; pre-eclampsia is a multisystem disorder of human pregnancy with a genetic predisposition [2]. Pre-eclampsia is usually diagnosed in late pregnancy by proteinuria, oedema and increased vasoconstriction leading to maternal hypertension and reduced uteroplacental blood flow. Drug biotransformation in placenta was reviewed by Juchau [3,4] and more recently by Harbison et al. [5] and Pasanen [6]. The glutathione S-transferases (GST; EC 2.5.1.18) are a family of soluble enzymes that play a key role in the biotransformation and detoxification of a wide number of endogenous [7–9] and exogenous compounds in liver, kidneys and intestine [10–15]. Glutathione S-transferase activity has been detected in human placenta from normal pregnancies [16–25]. GST have also been studied in pre-eclamptic pregnancies [25–27]. On the other hand, carboxylic esters are hydrolysed by preparations of placental tissues [3]; placental cytosol exhibits
1 In partial fulfilment of Doctorate’s degree in Pharmacology. * To whom correspondence should be addressed: Tel.: +52-4-499108424; Fax: +52-4-49910-8401; E-mail: [email protected]
0143-4004/$–see front matter
cholesterol ester hydrolase activity [28] whereas placental microsomes hydrolyse carboxylic esters of p-nitrophenol [29].
MATERIALS AND METHODS Chloro-2,4-dinitrobenzene, ethacrynic acid, reduced glutathione (GSH), p-nitrophenyl acetate and 5,5#-dithio-bisnitrobenzoic acid were purchased from Sigma Chemical (St Louis, MO, USA). All other reagents were of purest analytical grade commercially available. Subjects were recruited at the time of admittance for labour and delivery at the Gynecology and Obstetrics State Hospital, Jesu´s Marı´a, Aguascalientes, Mexico. The use of human placentae for this study was approved by the Hospital Ethic Committee. All participants were informed of the experimental procedure and gave written consent prior to delivery. Fifteen subjects with uncomplicated pregnancies throughout gestation with age of 26.16 years (means.d.) were selected as the control group; they were normotensive and had no proteinuria (Table 1); inclusion criteria considered subjects having no known medical illnesses, absence of tobacco or alcohol abuse, and abstinence from use of drugs known to be either inducers or inhibitors, over the preceding 3 months. Five patients with mild preeclampsia (age of 24.45 years; means.d.) and eleven patients with severe pre-eclampsia (age of 24.25 years; 2003 Elsevier Ltd. All rights reserved.
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Table 1. Data of obstetric parameters from the patients participating in our study Parameter
Control
Mild pre-eclampsia
Severe pre-eclampsia
Maternal age (years) Maternal weight (kg) Gestation time (weeks) Parity Caesarean delivery Sex of newborn Weight of newborn (kg) Apgar
26.16 778 37.71 Multiparous (8/15) 13/15 Feminine (11/15) 3.00.44 8–9
24.45 97.63 37.61 Second (3/5) 5/5 Masculine (3/5) 3.10.3 9–9
24.25 85.214.4 35.04a Multiparous (8/11) 11/11 Masculine (8/11) 2.40.9a 7–8
Values represent mean from control (n=15), mild (n=5) and Severe (n=10–11) pre-eclamptic patients. P>0.01.
a
means.d.) were also included in this study. Mild preeclampsia was defined as a blood pressure of R140/90 mm Hg, on two separate readings at least 6 h apart, proteinuria <2+ or pathologic oedema; severe pre-eclampsia was defined as a blood pressure of R160/110 mm Hg, on two separate readings at least 6 h apart, proteinuria R2+ and pathologic oedema. The diagnosis was based on hypertension, proteinuria and oedema in the late stages of their pregnancies. Hypertension was defined as an increase of 30 mm Hg systolic or 15 mm Hg diastolic blood pressure or an absolute blood pressure of >140/90 mm Hg after 20 weeks of gestation, if earlier blood pressures were not known (measured on two occasions, at least 6 h apart). Proteinuria was defined as >2+ in random urine specimens. Oedema in lower members is present in mild pre-eclampsia and generalized oedema in severe pre-eclampsia. Placentae were obtained within 20 min after vaginal or Caesarean delivery and suitable amounts were immediately and repeatedly washed with ice-cold physiological saline, to eliminate as much blood as possible; fragments were weighted, usually 5 g, minced and homogenized in 20 ml 50 m potassium phosphate, pH 7.5, with a Potter–Elvejhem homogenizer and the resultant homogenates were centrifuged at 12 100 g for 10 min, in a refrigerated Sorvall Super T centrifuge; the supernatants were placed in clean tubes, added enough CaCl2 to reach final concentration of 8 m [30–33] and centrifuged at 27 000 g for 20 min to obtain the microsomal and cytosolic fractions. All procedures were carried out at 42(C. The kinetics of total glutathione S-transferase activity was measured in the cytosolic fraction with 50 m potassium phosphate, pH 6.5, 0.5 m reduced glutathione (GSH) and 0.0125, 0.025, 0.05, 0.1, 0.2 and 0.4 m chlorodinitrobenzene (CDNB) as substrate [34], at 340 nm; we used 0.5 m GSH instead of 1.0 m in order to diminish the nonenzymatic conjugation of GSH with CDNB. The kinetics of glutathione S-transferase P () activity was measured in the cytosolic fraction with 50 m potassium phosphate, pH 6.5, 0.25 m reduced glutathione (GSH) and 0.0125, 0.025, 0.05, 0.1 and 0.2 m ethacrynic acid as substrate [34], at 270 nm. The kinetics of total esterase activity was also determined in the cytosolic fraction using 50 m Tris–Cl, pH 8.0, and 0.0125,
0.025, 0.05, 0.1, 0.2 and 0.4 m of p-nitrophenyl acetate as substrate, at 405 nm [35–37]. All the enzymatic assays were performed at room temperature (241(C) and in 1.0 ml final incubation volume. The initial enzymatic rates were calculated from 1 min readings, usually in duplicate, in a Hach DR/4000U UV-VIS spectrophotometer. Total and non-protein thiols or sulphydryl groups were determined in placental homogenates, as described by Sedlak and Lindsay [38]. Protein was measured with the Folin phenol reagent using bovine serum albumin as standard [39]. We calculated the kinetic constants (Vmax, Km) and the catalytic efficiency (CE) as well as the significance of differences between group means for the three variables with the twotailed Student’s t-test for grouped data using the program GraphPad Prism, v. 3.0 (GraphPad Sofware Inc). The catalytic efficiency (EC) or intrinsic metabolic clearance of enzymes is defined as the ratio of the maximal velocity (Vmax) of the reaction to the Michaelis–Menten constant (Km), that is Vmax/Km [40,41] or as the relative intrinsic clearance [42].
RESULTS Figure 1 shows that the maximal velocity (Vmax) of total glutathione S-transferases (T-GSH), assayed with chlorodinitrobenzene as substrate, were not significantly modified in placentae from the three groups; however, the Michaelis– Menten constant (Km) was significantly increased (P>0.05) by 396175 per cent (mean, n=5) and by 29678 per cent (mean, n=11) in placentae from mild pre-eclamptic patients and in placentae from severe pre-eclamptic patients, respectively, compared with placenta from control patients (mean, n=15). The catalytic efficiency (CE) was significantly reduced (833 per cent) only in placentae from severe pre-eclamptic patients. When we used ethacrynic acid as substrate, the Vmax of glutathione S-transferase P () was significantly reduced by 686 per cent (mean, n=10) only in placentae from severe pre-eclamptic patients (Figure 2). There were not significant differences in the Km among the three groups but the CE was significantly decreased (607 per cent and
M. del Carmen Terrones Saldı´var et al.: Glutathione S-Transferases and Esterases in Human Placenta
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Figure 1. Relative values (mean) of the kinetic constants (Vmax, Km and CE) of total glutathione S-transferase (T-GST) in placentae from control patients and from mild and severe pre-eclamsia. The actual control values are: Vmax=11824 nmol GS-DNB/min1mg1, Km=23470 µ and Vmax/Km=0.77 140 ml/min1mg1. Vmax is actually five times lower, and CE (Vmax/Km) is 1000 times lower. PE=Pre-eclampsia. GS-DNB=Glutathione–dinitrobenzene conjugate.
Figure 2. Relative values (mean) of the kinetic constants (Vmax, Km and CE) of glutathione S-transferase P or (GST P or ) in placentae from control patients and from mild and severe pre-eclamsia. The actual control values are: Vmax=72.412 nmol GS–ethacrynic acid/min1mg1, Km=58.917 µ and Vmax/Km=1.630.2 ml/min1mg1. CE (Vmax/Km) is actually 10 times lower. PE=Pre-eclampsia.
637 per cent; mean, n=5 and n=10) in placentae from mild pre-eclamptic patients and in placentae from severe pre-eclamptic patients, respectively, as compared with control patients (mean, n=15). To our surprise, we found that esterase activity was higher in the cytosolic fraction than in the microsomal fraction. The kinetic constants calculated in the placental cytosolic fraction showed that the Vmax of the total esterase activity was significantly reduced by 825 per cent (mean, n=5) in placentae from mild pre-eclamptic patients and by 415 per cent (mean, n=11) in placentae from severe preeclamptic patients as compared with control patients (n=15), with p-nitrophenyl acetate as substrate (Figure 3); there were
not significant differences in the Km (µ) among the three groups but the catalytic efficiency (CE) was significantly reduced by 727 per cent in placentae from mild pre-eclamptic patients and by 3313 per cent in placentae from severe pre-eclamptic patients as compared with control patients. Total and non-protein thiol concentrations did not show (Figure 4) significant differences among the three groups.
DISCUSSION We determined the enzyme kinetics of total glutathione S-transferases (T-GST), glutathione S-transferase P () and of
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Figure 3. Relative values (mean) of the kinetic constants (Vmax, Km and CE) of esterase in placentae from control patients and from mild and severe preeclamsia. The actual control values are: Vmax=43.85.5 nmol p-nitrophenol/min1mg1, Km=443 82 µ and Vmax/Km=0.110.012 ml/min1mg1. Vmax is actually 10 times lower, and CE (Vmax/Km) is 5000 times lower. PE=Pre-eclampsia.
Figure 4. Concentrations of total thiols and non-protein thiols (mean) in placental homogenates from control patients and from mild and severe pre-eclampsia. The actual control values are: total thiols=17617 nmol/g1 and non-protein thiols=8.011.1 nmol/g1. Non-protein thiols are actually 10 times lower. PE=Pre-eclampsia.
esterase in the cytosolic fraction of human placenta from normal and from mild and severe pre-eclamptic pregnancies. T-GST showed the highest maximal velocity (Vmax) but glutathione transferase P () was 2.1 and 14.8 times catalytically more efficient than T-GST and esterase, respectively, in placenta from normal pregnancies, mainly due to its lower Michaelis–Menten constant (Km). Polidoro et al. [16] found a Km of 0.67 m in placenta cytosols from healthy pregnancies, using chlorodinitrobenzene as substrate; this value is 2.8 times higher than the value we found in the control patients. Guthenberg and Mannervik [17] reported a Km of 2.1 m for purified glutathione transferase (GST-) from full term human placenta. Pacifici and Rane [18] reported a mean Vmax
of 11.76 nmol/min1 mg1 and a mean Km of 4.13 m (n=3) in human placental cytosols from normal deliveries, using styrene oxide as substrate; these values give a mean catalytic efficiency (CE) of 0.0028 ml/min1 mg1. In placenta from mild and severe pre-eclamptic patients only the Km of T-GST was significantly increased when compared to placenta from normal pregnancies and the CE was significantly decreased only in placenta from severe preeclamptic patients. In contrast, we found that pre-eclampsia did not significantly change the Km of GST-; however, pre-eclampsia diminished the Vmax of GST- but this decrease was significantly different only in placenta from severe preeclamptic patients compared to normal pregnancies; these
M. del Carmen Terrones Saldı´var et al.: Glutathione S-Transferases and Esterases in Human Placenta
results agree with the lower concentration of glutathione transferase P () described by Zusterzeel et al. [25] in placenta of pre-eclamptic pregnancies compared to normal pregnancies. The lack of significance in the Vmax of placenta from mild pre-eclamptic patients might be due to the comparatively low number of placenta studied (n=5). Glutathione transferase is the main isoform in normal placental tissue and comprises 36 per cent of total GST activity [43] and 67 per cent [25] of the T-GST concentration. Furthermore, placenta from mild and severe pre-eclamptic patients were catalytically less efficient than placenta from normal pregnancies. On the other hand, it was recently shown [24] than human term placental lipoxygenase, purified by affinity chromatography, catalyse GSH conjugate formation with ethacrynic acid, in the presence of linoleic acid; this reaction has a catalytic efficiency of 35.5 ml/ min1 mg1 and did not show detectable conjugation with chlorodinitrobenzene as substrate. Placental cytosolic esterase had the lowest Vmax, the highest Km and, consequently, the lowest CE of the activities tested; the cytosolic fractions used have slight cholinesterase activity with 0.8 m acetylthiocholine as substrate. The CE of microsomal Carboxylesterases from human term placenta has been reported to be approximately 10 times higher than our values, also using p-nitrophenyl acetate as substrate [29]. Mild and
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severe pre-eclampsia diminished significantly the CE of cytosolic esterases mostly due to decreases in Vmax, when compared to normal pregnancies. Finally, the concentration of total and non-protein thiols (GSH represents more than 95 per cent; [44,45]) did not change significantly in placental homogenates from patients with mild or severe pre-eclampsia compared to normal pregnancies. Mutlu-Turkoglu et al. [22] reported that GSH concentrations are decreased in placenta from pre-eclamptic pregnant woman when compared to normal pregnant woman. In summary, pre-eclampsia reduced the Vmax and, consequently, the catalytic efficiency of placental glutathione transferase and esterase(s) most dramatically in patients with severe pre-eclampsia; these findings would indicate a decreased capacity of the glutathione transferases and esterase detoxification systems to protect the fetus from drugs prescribed to pregnant women. The reduction in drug biotransformation efficiency could be due to placental hypoperfusion as a result of vasoconstriction, intravascular coagulation and decreased uteroplacental blood flow. Moreover, the drugs used to treat pre-eclampsia might also participate in the decreased capacity of the drug metabolism systems; in this regard, there is little information except for the reported in vitro inhibition of commercial GST from bovine liver by dihydropyridines [46] and of hepatic GST activities from male rats by diuretics [47].
ACKNOWLEDGEMENTS The authors acknowledge the medical and nurse personnel of the Gynecology and Obstetrics State Hospital, Jesu´s Marı´a, Aguascalientes, Me´xico, for their valuable help and support, and Mr Alfredo Padilla for the art work.
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