1 Departments of Gastroenterology and 2 Obstetrics and Gynaecology, University Hospital St Radboud, Nijmegen, The Netherlands
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Abstract |
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Key words: cysteine/enzyme activity/glutathione/glutathione S-transferase/isoform expression
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Introduction |
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Embryonic and fetal growth and development depend on a constant flow of nutrients from the mother (Ronzoni et al., 1999). During the first weeks of development, the embryo exchanges nutrients and waste products by diffusion, followed by exchange via the placental intravillous space. Despite the large detoxification capacity of the placenta (Knapen et al., 1999b
; Zusterzeel et al., 1999
), almost every drug present in the maternal circulation is able to pass the placental barrier and may reach the fetal organs (Krauer and Dayer, 1991
). Therefore, cysteine and GSH may have a function in fetal detoxification in combination with GSTs, and this may be vital in the scavenging of toxic compounds that pass the placental barrier. Although concentrations of cysteine and GSH, together with the distribution of GST isoforms in combination with GST enzyme activity have been extensively studied in adult tissues, little is known at present about these compounds in both embryonic or fetal tissues; hence, the present study was initiated.
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Materials and methods |
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Tissue homogenates were prepared on ice by adding 4 volumes of ice-cold homogenization buffer (0.25 mol/l saccharose, 20 mmol/l Tris/HCl buffer pH 7.4 and 1 mmol/l dithiothreitol). After five strokes using a glassglass Potter tube, homogenates were centrifuged for 1 h at 100 000 g at 4°C. Immediately after centrifugation, the supernatant (cytosolic fraction) was removed and stored in aliquots at 80°C until analysed. Protein was determined using a published method (Lowry et al., 1951) using bovine serum albumin as a standard.
Quantification of GST isoforms
Concentrations of the GST isoforms were determined as described previously (Van Lieshout and Peters, 1998). Samples of the embryo and fetus were run in parallel on different blots. In brief, cytosolic samples were subjected to sodium dodecylsulphatepolyacrylamide gel electrophoresis (12% acrylamide/bisacrylamide w/v, 37.5:1; Bio-Rad Laboratories, Veenendaal, The Netherlands) and separated under standardized conditions. Subsequently, proteins were transferred to nitrocellulose membranes (Protran®; Schleicher and Schuell, s'Hertogenbosch, The Netherlands) using a semidry blotting system (Novablot II; Pharmacia, Uppsala, Sweden). After blocking with 1% gelatine (w/v) in phosphate-buffered salineTris (PBS-T) buffer, Western blots were incubated with monoclonal antibodies (ascites diluted 1:5000) against human GSTA (both A1 and A2) (Peters et al., 1992
), GSTM1 (Peters et al., 1990
), GSTP1 (Peters et al., 1989
) and GSTT1 (Juronen et al., 1996
) as described previously in detail. After three wash cycles with PBS-T, specific binding of monoclonal antibodies to the isoforms was detected by incubation with peroxidase-conjugated rabbit anti-mouse immunoglobulin (Dakopatts, Glostrup, Denmark) followed by subsequent development of the peroxidase label with 0.1% 3,3'-diaminobenzidine in PBS (Sigma Chemical Co., Zwijndrecht, The Netherlands) containing 0.01% hydrogen peroxide (Merck, Darmstadt, Germany), 0.34 g/l imidazole (Merck) and 0.26 g/l cobalt chloride÷6H2O (ICN Biomedicals B.V., Zoetermeer, The Netherlands). Staining intensity on the immunoblots was quantified using a laser densitometer (Ultroscan XL; LKB, Bromma, Sweden). Quantification of GST isoforms in the cytosolic fractions was performed with known amounts of purified GSTs, which were run in parallel with the samples. The detection limit of each GST isoform was ~50 nmol/mg protein (Van Lieshout and Peters, 1998
); the within-assay and day-to-day variations were 10 and 15% respectively.
Determination of GST enzyme activity
GST enzyme activity was determined in duplicate according to a published method (Habig et al., 1974). In brief, 10 µl of each cytosolic fraction was added to 2.0 ml potassium phosphate buffer, pH 6.5, containing 1.0 mmol/l 1-chloro-2,4-dinitrobenzene (CDNB; Sigma Chemical Co.) and 5.0 mmol/l GSH (Sigma Chemical Co.) at 25°C in a disposable cuvette. The change in absorbance at 340 nm was followed for 3 min using a Lambda 12 spectrophotometer (Perkin Elmer, Nieuwerkerk a/d IJssel, The Netherlands).
Analysis of thiols
For the analysis of cysteine, homocysteine, cysteinylglycine and GSH, cytosols were diluted six times with 12% (v/v) perchloric acid, centrifuged for 5 min at 10 000 g, and subsequently 10 µl 10% (w/v) Tris (2-carboxyethyl) phosphine (Fluka Chemie AG, Bornem, The Netherlands) was added to 100 µl of each sample. After reduction for 30 min at room temperature, samples were neutralized by adding 75 µl 2 mol/l NaOH. Subsequently, 100 µl of the neutralized sample was derivatized with 7-fluorobenzofurazane-4-sulphonic acid (SBDF; Fluka Chemie AG) for 1 h at 60°C by adding 60 µl of derivatization buffer containing 50 µl borate buffer (125 mmol/l K2B4O7÷4H2O and 4 mmol/l EDTA, pH 9.5), 5 µl SBDF (4 mg/ml borate buffer) and 5 µl NaOH (1.55 mol/l). Thiols were separated using high-performance liquid chromatography as described previously (Raijmakers et al., 2000). Thiol concentrations were determined using a calibration curve for all thiols which was run in parallel with the samples.
Statistical analysis
In order to determine any associations between GSH concentrations, cysteine concentrations, GST isoforms and GST enzyme activity, the Spearman Rank coefficient of correlation was calculated using Astute for Microsoft Excel 5.0. A P-value of 0.05 indicated an association to be statistically significant.
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Results |
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In the fetus of 13 weeks gestation, GSTP1 was also predominantly expressed in all organs, with highest concentrations in the small intestine, kidney and lung. Most other tissues had high GSTP1 expression, except for spleen which showed relatively low concentrations. Overall, GST was composed of 26% GSTA, 69% GSTP1 and 5% GSTM1. The highest expression of GSTA was seen in the liver, small intestine and adrenal gland, whilst moderate expression was seen in the oesophagus and low expression was seen in the spleen and lung. No GSTA was detected in the brain. GSTM1 was mainly present in kidney and in tissues exposed to the amniotic fluid, these being the oesophagus, small intestine and lung. In all other tissues only moderate expression was found, except for the heart where only faint expression of GSTM1 was seen. GSTT1 was not detected in any of the fetal organs examined, but was found in considerable amounts in the decidua. PCR analysis revealed that this fetus also bore the GSTT1 null genotype. GST enzyme activity was highest in the small intestine, with lower concentrations in the liver, adrenal gland, kidney and lung. In the other tissues only moderate enzyme activities were found, except for spleen where it was just measurable.
Both the embryo and fetus showed considerable and similar amounts of acid-soluble GSH and cysteine in all organs examined, whereas concentrations of cysteinylglycine and homocysteine were much lower (Table II). Overall, thiols were composed of 14% cysteine, 85% GSH, <1% cysteinylglycine and <1% homocysteine. Surprisingly, GSH concentrations in liver tissues were very low, whereas the cysteine concentration was high compared with other organs. In the embryo, the highest cysteine concentrations were found in the liver and testis, while low amounts were found in kidney and lung compared with other organs. GSH concentrations were highest in the stomach, lung, heart and kidney, but almost undetectable in the liver. In the fetus, the liver and small intestine showed the highest amounts of cysteine compared with other tissues, but almost none was detected in the oesophagus and spleen. High GSH concentrations were found in the adrenal gland, heart, brain and brainstem, whilst the lowest concentrations were in the liver, testis and spleen.
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Significant correlations between GST enzyme activity and expression of GSTA (rs = 0.61, P < 0.05), GSTP1 (rs = 0.48, P = 0.03) and the sum of all GST isoforms (rs = 0.59, P = 0.005) were found in the fetus, whereas in the embryo no such correlations could be found. A significant inverse correlation between cysteine and GSH was found in the embryo (rs = 0.74, P = 0.04).
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Discussion |
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In liver, a relatively high expression of GSTM1 was found, and this contrasted with data reported by others (Strange et al., 1984, 1985
), who found no or only faint GSTM1 expression in fetuses of 1020 weeks gestation using starch gel electrophoresis. In accordance with these studies, high concentrations of GSTA and GSTP1 were also found in the current study. A higher GST enzyme activity was also found in both the embryo and fetus compared with other data (Strange et al., 1984
), and this might be explained by the high expression levels of GSTM1. In adult liver, GSTA is predominantly expressed whereas GSTP1 is seen only in the bile duct epithelium and blood vessels (Mulder et al., 1994
). However, in embryonic liver GSTP1 is the most expressed GST isoenzyme, indicating that the expression of GSTM1 and GSTA increases during human development, whilst expression of GSTP1 decreases, as reported elsewhere (Beckett et al., 1990
). These changes in the expression of GST isoforms might also explain the higher GST enzyme activity in adult liver reported earlier (Mulder et al., 1994
). The different GST isoenzymes expressed in the developing liver, and the lower GST enzyme activity as compared with adult liver, are in agreement with the different functions of embryonic, fetal and adult liver. In the developing liver, the synthesis of erythrocytes is the main function, whereas in adults the biotransformation of toxic compounds is one of the primary functions.
In lung, a similar expression of GST isoforms as found in the current study was described previously (Beckett et al., 1990); concentrations of GSTP1 were high in early gestation, but decreased during gestation, whereas GSTM1 and GSTA expression were moderate and weak respectively. During gestation, GST enzyme activity decreased in parallel with GSTP1 expression (Fryer et al., 1986
), the GST enzyme activity remaining low in samples obtained more than a year after birth. In adult tissue, GSTP1 still is by far the most predominant GST isoform (Antilla et al., 1993; Rowe et al., 1997
); however, the concentrations were approximately two-fold lower than in fetal tissue. GST enzyme activities were found to be much lower in adult than in fetal lung tissue (Mukhtar et al., 1981
; Clapper et al., 1991
). When fully functional, the placenta, which has a large detoxification capacity (Knapen et al., 1999b
; Zusterzeel et al., 1999
), removes toxic metabolites from the fetal circulation. Amniotic fluid may also contain toxic metabolites, and therefore tissues exposed to the amniotic fluid (e.g. lung and gastrointestinal tract) may need to adapt to such an environment. The high concentration of GSTP1 and high enzyme activity of lung tissue in early gestation, when compared with other tissues as well as with adult concentrations, may represent such an adaptation.
With the exception of the low GSTP1 expression in the embryo, the expression pattern of GST isoforms in kidney in the current study were in line with those reported previously (Beckett et al., 1990), these being high, moderate and faint expression of GSTP1, GSTA and GSTM1 respectively. In contrast to the results of others (Strange et al., 1985
) who found GSTA to be absent from fetal kidney, a moderate expression of this GST subclass was measured. In fetal kidney, concentrations of GST isoforms and enzyme activity were similar to those found in adult tissues, where the predominantly expressed GST isoform was shown to be GSTA (Howie et al., 1990
; Eickelmann et al., 1994
; Rowe et al., 1997
; Rodilla et al., 1998
). This similarity might indicate that GST subclass development is an early event of pregnancy.
Very similar results were found in the literature for the expression and activity of GST isoforms in adult tissues, including brain (Rowe et al., 1997), oesophagus and stomach (Peters et al., 1993
) compared with embryonic and fetal tissues (Table IV
). In contrast, values in adult bladder differ considerably compared with corresponding fetal values (Berendsen et al., 1997
). These discrepancies and similarities between adult and fetal GST capacity may indicate the different development patterns for the various fetal organs.
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As compared with adults, the GSH concentrations in embryonic and fetal lung and bladder were approximately six-fold (Cook et al., 1991) and two-fold (Giralt et al., 1993
; Singh et al., 1994
; Pendyala et al., 1997
) higher respectively. The results in lung might be explained by the finding that GSH concentrations are coupled to the expression of GST isoenzymes and enzyme activity, both of which are very high during early gestation but decrease two-fold in adults (Fryer et al., 1986
; Rowe et al., 1997
). This change in GSH concentration, GST expression and GST enzyme activity during gestation might be explained by the development of the placenta. In early gestation, the placenta is not fully developed, and subsequent adaptations must be made to prevent damage by toxic products, which are by preference excreted into the amniotic fluid. Thus, tissues exposed to the amniotic fluid most likely have higher concentrations of GSH, GSTs and a higher enzyme activity. When placental detoxification begins to function and waste products are removed from the fetal circulation, this adaptation is no longer necessary and consequently both the concentration of GST and the enzyme activity may be reduced.
Although the placenta and decidua are partly and totally from maternal origin respectively, both tissues most likely play an important role in fetal detoxification and protection during growth and development. In the current study, GST enzyme activity in the placenta and decidua was comparable with that in previous studies in early pregnancy (Di Ilio et al., 1983) and in term placenta and decidua (Polidoro et al., 1980
; Mutlu-Turkoglu et al., 1998
; Knapen et al., 1999b
). However, the expression of all GST isoforms found here was much higher compared with the concentrations reported in term placenta and decidua (Zusterzeel et al., 1999
), and this may point to another function of the placenta in early gestation as compared with third-trimester placenta. In placenta and decidua tissue, similar concentrations of GSH were found. Although placental GSH concentrations and GST enzyme activities were similar in the current study, the GSH concentration in term decidua appeared to be much higher than was reported earlier (Knapen et al., 1999b
). These authors found the GSH concentration in decidua to be about five-fold higher than in placenta, perhaps indicating that decidual GSTs found in early gestation have other functions or are less important than in third-trimester pregnancy.
In conclusion, it has been shown that during the early stages of embryonic and fetal development, cysteine, GSH, GST isoforms and GST enzyme activity were expressed in considerable amounts in most tissues examined. In contrast to adult tissue, GSTP1 was the predominant GST isoform in embryonic and fetal organs. However, only moderate concentrations of GSTA and GSTM1 were found compared with adults, and this might point to different functions of GSTs in the tissues of the embryo and/or fetus than in those of adults.
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Acknowledgements |
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Notes |
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References |
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Submitted on April 6, 2001; accepted on August 10, 2001.