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Effect of ethanol on glutathione S-transferase expression in co-cultured rat hepatocytes
Toxic. in VitroVol. 9, No. 4, pp. 467-471, 1995
Pergamon
Copyright 0 1995 Elxvier Science Ltd
0887~2333(%poo=9
Printedin Great Britain. All rights resewed M187-2333/95 $9.50 + 0.00
Effect of Ethanol on Glutathione S-Transferase Expression in Co-cultured Rat Hepatocytes G. DEBAST, S. COECKE, M. AKRAWI*, A. FORIERS, A. VERCRUYSSE, I. PHILLIPSt, E. SHEPHARD* and V. ROGIERS Department of Toxicology, Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090 Brussels, Belgium, *Department of Biochemistry and Molecular Biology, University College London, Gower Street, London WCIE 6BT and tDepartment of Biochemistry, Queen Mary and Westfield College, University of London, Mile End Road, London El 4NS, UK Abstract-To examine the effects of ethanol (EtOH) on rat liver glutathione S-transferase (GST, EC. 2.5.1.18), a key phase 2 biotransformation enzyme, an in vitro system consisting of rat hepatocytes co-cultured with rat epithelial cells derived from primitive biliary duct cells was used. Cells were either untreated or treated for 10 days with 0.1 or 0.4% EtOH. Cytosolic GST activities were assayed spectrophotometrically using l-chloro-2,4dinitrobenxene (CDNB) or 1,2-dichloro-4nitrobenzene (DCNB) as substrates. When cultures were exposed to 0.4% EtOH the total GST activity towards the substrate CDNB increased by 35%. However, the activity of GSTs towards DCNB did not increase significantly in the cells treated with this concentration of EtOH. Afhnity chromatography followed by reversed phase HPLC was used to separate the various GST subunits. EtOH was found to a&t the GST subunit pattern. Total GST proteins increased after exposure to 0.4% EtOH; this was largely due to a three-fold and 25fold increase in the GST p-class subunits 3 and 4 respectively. Since EtOH induces GST expression in liver parenchymal cells, particularly the p-class GSTs, its addition as a solvent to cultured hepatocytes or its use in viuo may result in important changes in the metabolism and toxicity of the xenobiotics under study.
IntrodoetIon
Xenobiotics are metabolized in the liver by phase 1 and phase 2 reactions, usually leading to derivatives that are less toxic and more water-soluble and thus ready for excretion. Biotransformation, however, does not always lead to detoxification. The balance between formation and detoxification of toxic metabolites determines whether toxic effects will occur or not. Since this balance is affected not only by endogenous variables but also by exogenous factors such as external inducers and inhibitors, it is of importance to know if a commonly used compound such as ethanol (EtOH) could represent such a factor. EtOH is also often used as a solvent for waterinsoluble substances in pharmacotoxicological research and testing. The effects of this compound on phase 2 biotransformation proteins, glutathione Stransferase (GST) in the intact organism and in cell culture has not been studied extensively. The available data concerned with the effect of EtOH on GST activity and amounts is conflicting. Long-tenn treatment of both male and female rats with up to 15% EtOH in their drinking water had little effect on either GST activity or the total amount of GST protein (van Abbreviations: GST = glutathione S-transferase; EtOH = ethanol; CDNB = I-chloro-2, Cdinitrobenxene; DCNB = I, 2-dichloro&titrobenzene.
de Wiel et al., 1993), although the amount of GST subunit 1 was significantly reduced. Gonzalez et al. (1988) reported a decrease in GST activity following acute exposure of rats to EtOH. In contrast, Munoz et al. (1987) found that total GST activity was maximally increased in rats after 2 wk of drinking a 36% solution of EtOH. However, after 6 wk of this treatment there was no difference in GST activity between the treated and control groups. In this paper the effect of EtOH treatment on the activity and isoenzyme pattern of the GST (E.C.2.5.1.18.) protein family is investigated using an in vitro system comprising adult rat hepatocytes co-cultured for 10 days with rat liver epithelial cells (Guguen-Guillouzo et al., 1983). This in vitro model has been chosen because the activities of most phase 1 and phase 2 biotransformation enzymes remain constant for at least 1 wk of culture (Rogiers and Vercruysse, 1993). The GST protein family comprises several isoenzymes that have been placed into the a-, p-. n- or fl classes depending on their amino acid sequence similarities (Mannervik et al., 1985; Meyer et al., 1991). These proteins catalyse the nucleophilic attack of an S-atom of the glutathione on the electrophilic centre of a substrate, the resulting product being a protein-glutathione conjugate. The major GST subunits expressed in the adult liver are a-class subunits 1 and 2, and the p-class subunits 3 and 4.
467
G. Debast er al.
468
14
10
7
Culture time (days) Fig. 1. GST activity towards CDNB (@) and DCNB (1:) in untreated co-cultures of rat hepatocytes. Each value represents the mean (expressed as a percentage of the value obtained for freshly isolated hepatocytes) & SD (n = 3). Asterisks indicate significant difference from freshly isolated hepatocytes (*P < 0.05; Student’s f-test).
GST subunit 7, a member of the K class, is not expressed in adult liver; however, this protein is present in foetal hepatocytes, in cultured adult rat hepatocytes and during early stages of hepatocarcinogenesis (Vandenberghe et al., 1988a). Materials and Methods Chemicals
Crude collagenase type I, bovine serum albumin (fraction V), bovine insulin, I-chloro-2,4-dinitrobenzene (CDNB), glutathione and epoxy-activated sepharose 6B were obtained from Sigma Chemical Co. (St Louis, MO, USA). All culture media, foetal calf serum and trypsin-EDTA solution were purchased from Gibco (Brussels, Belgium). Hydrocortisone hemisuccinate was from Roussel (Paris, France). 1,2-Dichioro-4-nitrobenzene (DCNB) and absolute ethanol came from Merck-Schuchardt (Darmstadt, Germany). All other chemicals were of reagent grade and were purchased from general commercial sources.
350 g, obtained by Iffa Credo, Brussels, Belgium) and co-cultured for 10 days with rat liver epithelial cells of primitive biliary origin as described by Guguen-Guillouzo et al. (1983). Co-cultured cells remained either untreated or were continuously exposed to 0.1 or 0.4% EtOH. The medium was renewed every day. Cytosolic fractions from freshly isolated hepatocytes (To) and IO-day old co-cultures were prepared as described by Johnson et al. (1992). GSTs were isolated from cytosolic fractions by affinity chromatography as previously described (Vandenberghe ef al., 1988a). The separation and quantification of GST subunits was carried out by HPLC on a 10 x 0.8 cm Waters FBondapak C-18 reversed phase column in a Z-module using a Shimadzu LCIO
Table
2. Effect
of ethanol
on the concentration
co-cultured Subunit
of GST
cone”
(pg/mg
cytosolic Control
Preparation of hepatocyte cultures, cytosolic fractions and isolation and separation of GSTs
Subunits
I.
Effect
of ethanol DCNB
on GST
in co-cultured
activity
towards
Enzymatic (percentage Ethanol
concn
(%)
0.1
124+
0.4
135f6’
concentrations
Values
in
obtained
mean is derived culture
from from
epithelial
12
125 f
19
two-way
2.7 k 2.3
6.4 * 5.6
2.3 f
3.9 k 2.6
6.8 f 9.1 5.1 k 3.2
I.4
12.5 k 13.4
2.5 k 2.1
0.8 f 0.2
0.8 f 0.6
1.4*
8b
2.6 f 0.2
0.7 f 0.4
0.8 f 0.6
I .9 f 2.3
0.0 + 0.0
0.5 f 0.5
0.0 + 0.0
9.5 ? 3.5
20.7 it 8.4
21.9?
40.9 f
3.5’
I.3
0.4 f 0.7 21.0
12.9 i. I.1
Il.4
f 4.7
22.0 f
I I.5
33.6 + 8.6*
21.6+2.1
22. I k 4.0
36.4 f
16.3
52.0 k 7.8*
0.8 * 0.7
0.8 f
0.8 f 0.2 35.2+3.1
34.2k8.8
I.1 f
I .O
I.1
59.1 t27.4
86.8+
14.9’
7
value, that
E1OH) f SD. Each isolation
and
0.0 + 0.0’
concentration
hepatocytes (To) the absence
15.4 f
of individual
6.3
GST
18.3 + 5.4
14.6 + 2.3
and in hepatocytes
(control)
or presence
EtOH were determined by HPLC
in freshly isolated
subunits
co-cultured
for
IO days in
of various
concentrations
as described
in Materials
of and
Methods. analysis
cells were negligible
the co-cultures
2.3 k 0.7
n class
experiments.
-z 0.05;
+
EtOH
2.8 f 0.7
p subtotal
presence of various
the results of three separate
Asterisk indicates a significant difference from (*P
107 *
of the control
in 0%
Control 0.4%
12.6 + 2.5’
6
The
cells cultured
EtOH
protein)
8a
3 4
of EtOH.
are mea” (expressed as a percentage
is that
the
0.1%
+
a class
100+6
Hepatocytes were co-cultured for IO days
4.8 f 4.5
IO
DCNB
14
18.5 * 3.8’
lb
OLsubtotal
of control)
IOOfll
la 2
and
activity
CDNB
0.0
CDNB
rat hepatocytes
in
x class
Hepatocytes were isolated from untreated adult male OFA Sprague-Dawley rats (I.O.P.S. caw, 15wk, Table
Control
TO
subunits
hepatocytes
(less than
of variance). in comparison
3.5%).
the control The with
values
value for
the
the values for
Values
are
isolation Asterisk
mean f
indicates
control
SD
and culture value
derived
significant (*P
from
the results
of
four
separate
experiments.
< 0.05;
difference two-way
from analysis
the
corresponding
of variance).
Efrect of ethanol on GST expression system with a Prolinea 4/33 HPLC dedicated integrator (Compaq, Houston, USA). The solvents were water and acetonitrile, each containing 0.06% (w/w) trifluoroacetic acid as described by Vandenberghe et al. (1990). The samples were injected at 36% acetonitrile. During a run, a linear gradient of acetonitrile, from 36 to 53%, was used over a 60-min period. The flow rate was 1.5 ml/min and UV absorbance at 214nm was monitored. Recovery of GSTs ranged from 90 to 100%.
469
tein Assay (Bio-Rad, Brussels, Belgium) with bovine serum albumin as a standard. Statistical analysis
Statistical evaluation of the differences obtained was achieved by general ANOVA procedures and Student’s t-test using the Software Package of the Social Sciences, SPSS/PC+ (Norusis, 1986). Results Total GST activity in the co-cultured cells remained constant for up to 14 days when CDNB was used as the substrate (Fig. 1). An increase in GST activity was observed in these cells after 7 days of co-culture if DCNB was used as a substrate. This increased activity was maintained for at least 7 days
Enzyme and protein assays
Cytosolic GST activities were assayed spectrophotometrically using CDNB and DCNB as substrates (Vandenberghe et al., 1988b). Cytosolic protein concentrations were determined using the Bio-Rad Prohl214nm
(a) 100
la
1
$ 50
lb
Chl 214nm (b)
3 A
I
n
I
I
20
40 min
Fig. 2(a and b)-Caption overleaf.
G. Debast
470
ef al.
30 Chl 214nm 4 7 20 3 2
?
E
I 0
/
I 20
I 40 min
Fig. 2. Composition of GST subunits present in rat hepatocytes. GST subunits were separated by reversed phase HPLC of extracts of freshly isolated rat hepatocytes (a), IO-day-old co-cultured hepatocytes (control) (b) and IO-day-old co-cultured hepatocytes treated with 0.4% ethanol (c). The conditions were as described in Materials and Methods. Numbers refer to GST subunits.
(Fig. 1). These findings are in agreement with our earlier observations (Rogiers et al., 1990). The maintenance of GST activity in this culture system makes it suitable for examining the effects of foreign compounds, such as EtOH, on the expression of GST proteins. In freshly isolated hepatocytes, GST activities for the substrates CDNB and DCNB were 1.2 pmol/ min/mg cytosolic protein and 35 nmol/min/mg cytosolic protein, respectively. These results are very similar to those reported by Vandenberghe et a/. (1988b). When IO-day-old co-cultures were exposed to EtOH, GST activity tended to increase in a dose-dependent way (Table 1). Total GST activity towards the substrate CDNB increased by 35% in cells treated with 0.4% EtOH. However, this concentration of EtOH did not significantly increase total GST activity when DCNB was used as a substrate (Table 1). The metabolism of DCNB or CDNB by GSTs was negligible in the helper epithelial cells and did not increase on treatment of these cells with EtOH. The effect of EtOH treatment on the amounts of individual GST subunits in co-cultured cells was investigated using HPLC. Figure 2 shows the separation pattern of GST subunits present in freshly isolated hepatocytes and in co-cultured cells that were treated for 10 days with 0.4% EtOH or remained untreated. The data presented in Table 2 show that the overall content of GST proteins decreases when hepatocytes are co-cultured. This is largely due to a reduction in the amounts of ~-class subunits. Total GST protein content decreased from 76.1 f 6.6 pg/mg cytosolic protein in freshly isolated hepatocytes to 59.1 + 18.6 pg/mg cytosolic protein in lo-day-old co-cultures. During this culture period,
r-class subunits decreased from 40.9 k 3.5 to 9.5 f 3.5 pg/mg cytosolic protein. Members of the r-class were not significantly induced by treatment of the cells with EtOH. In contrast, the amounts of the p-class protein subunits were maintained in the culture conditions used, and subunits 3 and 4 were increased about three-fold and 2.5-fold, respectively, in response to 0.4% EtOH compared with the control value without EtOH. As shown previously (Vandenberghe et al., 1990) the n-class subunit 7, which was absent from freshly isolated hepatocytes, was present in co-cultured cells at a concentration of 15.4 + 6.3 pg/mg cytosolic protein. The concentration of this subunit was not increased in response to EtOH treatment.
Discussion In this paper we report the effects of EtOH on cytosolic GST enzyme activity and GST subunit composition in co-cultures of rat hepatocytes and rat liver epithelial cells. EtOH primarily increases the amount of p-class subunits. The effect of EtOH on the expression of these subunits was confirmed at the RNA level using Northern blot hybridization analysis (data not shown). The exact mechanism whereby EtOH acts to increase the expression of p subunits is not known. It is also not yet known whether the results we observe in vitro correspond to the in uivo situation. The available data concerning the effect of EtOH in rodents are conflicting (Gonzalez et al., 1988; Munoz et al., 1987; van de Wiel et al., 1993). Little information exists concerning the effects of this compound on GST activity and subunit composition in man. Human p-class GSTs are of particular
Effect of ethanol on GST expression
interest because about 45% of the European population are homozygous null for the GSTMl gene locus. ~-Class GSTs are very effective at deactivating mutagenic and carcinogenic epoxides. It is therefore reasonable to suppose that any induction of the p-class GST isoenzymes might result in a significant reduction in the toxicity of compounds such as polycyclic aromatic hydrocarbon epoxides present in tobacco smoke, and chemical compounds including styrene oxide and truns-stilbene oxide. EtOH also affects phase 1 xenobiotic metabolism in that it induces cytochrome P4502El in man and rodents (Gonzalez et al., 1991; Guengerich et al., 1990). Because of the ability of EtOH to induce different xenobiotic-metabolizing enzymes it is not clear whether the overall effect of the compound would increase the cells’ capacity for detoxification of foreign chemicals. Our data do, however, show that concentrations of EtOH greater than 0.1% should not be used in cultured cells, thus avoiding the effects of this compound on the metabolism of endogenous and exogenous compounds. REFERENCES Gonzalez F. J., Ueno T., Umeno M., Song B. J., Veech R. L. and Gelboin H. V. (1991) Microsomal ethanol oxidizing system: transcriptional and posttranscriptional regulation of cytochrome P450, CYPZEI. Alcohol and Alcoholism Suppl. 1, 97-101. Gonzalez J., Munoz M. E., Marin M. I., Collado P. S., Fermoso J., Esteller A. (1988) Influence of acute ethanol administration on hepatic glutathione metabolism in the rat. Alcohol 5, 103-106. Guengerich F. P., Kim D.-H. and Iwasaki M. (1990) Role of human cytochrome P-450 IIEl in the oxidation of many low molecular weight cancer suspects. Chemical Research in Toxicology 4, 1688179.
Guguen-Guillouzo C., Clement B., Baffet G., Beaumont C., Morel-Chany E., Glaise D. and Guillouzo A. (1983) Maintenance and reversibility of active albumin secretion by adult rat hepatocytes co-cultured with another liver epithelial ceil type. Experimental Cell Research 143, 4743.
471
Johnson J. A., Finn K. A. and Siegel F. L. (1992) Tissue distribution transferase ation of conjugation
of enzymic methylation of glutathione Sand its effects on catalytic activity. Methylglutathione S-transferase I l-l I inhibits activity towards I-chloro-2,4-dinitrobenzene.
Biochemical Journal 282, 279-289.
Mannervik B., Alin P., Guthenberg C., Jensson H., Tahir M. K.. Warholm H. and Jornvall H. (1985) Identification of three classes of cytosolic glutatdione S-transferases common to several mammalian species: correlation between structural data and enzymatic properties. Proceedings of the National Academy of Sciences of the U.S.A. 82, 7202-7206. Meyer D. J., Coles B., Pemble S. E., Gilmore K. S., Fraser G. M. and Ketterer B. (1991) Theta, a new class of glutathione transferases purified from rat and man. Biochemical Journal 214, 409414.
Munoz M. E., Martin M. I., Fermoso J., Gonzalez J. and Esteller A. (1987) Effect of chronic ethanol feeding on glutathione and glutathione-related enzyme activities in rat liver. Drug and Alcohol Dependence 20, 221-226.
Norusis M. J. (1986) SPSS-PC+ Advanced Statistics. pp. Bl52-B181. SPSS, Chicago. Rogiers V., Vandenberghe Y., Callaerts A., Verleye G., Comet M., Mertens K., Sonck W. and Vercruysse A. (1990) Phase I and Phase II xenobiotic biotransformation in cultures and co-cultures of adult rat hepatocytes. Biochemical Pharmacology 40, 1701-1706.
Rogiers V. and Vercruysse A. (1993) Rat hepatocyte cultures and co-cultures in biotransformation studies of xenobiotics. Toxicology 82, 193-208. Vandenberghe Y., Foriers A., Rogiers V. and Vercruysse A. (1990) Changes in expression and de novo synthesis of GST subunits in cultured adult rat hepatocytes. Biochemical Pharmacology 39, 685690.
_
Vandenberehe Y.. Glaise D.. Mever D. J.. Guillouzo A. and KettererB. (1988a) Glutathione transferase isoenzymes in cultured rat hepatocytes. Biochemical Pharmacology 37, 2482-2485.
Vandenberghe Y., Ratanasavanh D., Glaise D. and Guillouzo A. (1988b) Influence of various soluble factors on glutathione S-transferase activity in adult rat hepatocytes during culture. In Vitro Cellular and Developmental Biology 24, 281-288.
van de Wiel J. A. G., Fijneman P. H. S., Teeuw K. B., van Ommen B, Noordhoek J. and Bos R. P. (1993) Influence of long-term ethanol treatment on rat liver biotransformation enzymes. Alcohol 10,397402.
Pergamon
Copyright 0 1995 Elxvier Science Ltd
0887~2333(%poo=9
Printedin Great Britain. All rights resewed M187-2333/95 $9.50 + 0.00
Effect of Ethanol on Glutathione S-Transferase Expression in Co-cultured Rat Hepatocytes G. DEBAST, S. COECKE, M. AKRAWI*, A. FORIERS, A. VERCRUYSSE, I. PHILLIPSt, E. SHEPHARD* and V. ROGIERS Department of Toxicology, Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090 Brussels, Belgium, *Department of Biochemistry and Molecular Biology, University College London, Gower Street, London WCIE 6BT and tDepartment of Biochemistry, Queen Mary and Westfield College, University of London, Mile End Road, London El 4NS, UK Abstract-To examine the effects of ethanol (EtOH) on rat liver glutathione S-transferase (GST, EC. 2.5.1.18), a key phase 2 biotransformation enzyme, an in vitro system consisting of rat hepatocytes co-cultured with rat epithelial cells derived from primitive biliary duct cells was used. Cells were either untreated or treated for 10 days with 0.1 or 0.4% EtOH. Cytosolic GST activities were assayed spectrophotometrically using l-chloro-2,4dinitrobenxene (CDNB) or 1,2-dichloro-4nitrobenzene (DCNB) as substrates. When cultures were exposed to 0.4% EtOH the total GST activity towards the substrate CDNB increased by 35%. However, the activity of GSTs towards DCNB did not increase significantly in the cells treated with this concentration of EtOH. Afhnity chromatography followed by reversed phase HPLC was used to separate the various GST subunits. EtOH was found to a&t the GST subunit pattern. Total GST proteins increased after exposure to 0.4% EtOH; this was largely due to a three-fold and 25fold increase in the GST p-class subunits 3 and 4 respectively. Since EtOH induces GST expression in liver parenchymal cells, particularly the p-class GSTs, its addition as a solvent to cultured hepatocytes or its use in viuo may result in important changes in the metabolism and toxicity of the xenobiotics under study.
IntrodoetIon
Xenobiotics are metabolized in the liver by phase 1 and phase 2 reactions, usually leading to derivatives that are less toxic and more water-soluble and thus ready for excretion. Biotransformation, however, does not always lead to detoxification. The balance between formation and detoxification of toxic metabolites determines whether toxic effects will occur or not. Since this balance is affected not only by endogenous variables but also by exogenous factors such as external inducers and inhibitors, it is of importance to know if a commonly used compound such as ethanol (EtOH) could represent such a factor. EtOH is also often used as a solvent for waterinsoluble substances in pharmacotoxicological research and testing. The effects of this compound on phase 2 biotransformation proteins, glutathione Stransferase (GST) in the intact organism and in cell culture has not been studied extensively. The available data concerned with the effect of EtOH on GST activity and amounts is conflicting. Long-tenn treatment of both male and female rats with up to 15% EtOH in their drinking water had little effect on either GST activity or the total amount of GST protein (van Abbreviations: GST = glutathione S-transferase; EtOH = ethanol; CDNB = I-chloro-2, Cdinitrobenxene; DCNB = I, 2-dichloro&titrobenzene.
de Wiel et al., 1993), although the amount of GST subunit 1 was significantly reduced. Gonzalez et al. (1988) reported a decrease in GST activity following acute exposure of rats to EtOH. In contrast, Munoz et al. (1987) found that total GST activity was maximally increased in rats after 2 wk of drinking a 36% solution of EtOH. However, after 6 wk of this treatment there was no difference in GST activity between the treated and control groups. In this paper the effect of EtOH treatment on the activity and isoenzyme pattern of the GST (E.C.2.5.1.18.) protein family is investigated using an in vitro system comprising adult rat hepatocytes co-cultured for 10 days with rat liver epithelial cells (Guguen-Guillouzo et al., 1983). This in vitro model has been chosen because the activities of most phase 1 and phase 2 biotransformation enzymes remain constant for at least 1 wk of culture (Rogiers and Vercruysse, 1993). The GST protein family comprises several isoenzymes that have been placed into the a-, p-. n- or fl classes depending on their amino acid sequence similarities (Mannervik et al., 1985; Meyer et al., 1991). These proteins catalyse the nucleophilic attack of an S-atom of the glutathione on the electrophilic centre of a substrate, the resulting product being a protein-glutathione conjugate. The major GST subunits expressed in the adult liver are a-class subunits 1 and 2, and the p-class subunits 3 and 4.
467
G. Debast er al.
468
14
10
7
Culture time (days) Fig. 1. GST activity towards CDNB (@) and DCNB (1:) in untreated co-cultures of rat hepatocytes. Each value represents the mean (expressed as a percentage of the value obtained for freshly isolated hepatocytes) & SD (n = 3). Asterisks indicate significant difference from freshly isolated hepatocytes (*P < 0.05; Student’s f-test).
GST subunit 7, a member of the K class, is not expressed in adult liver; however, this protein is present in foetal hepatocytes, in cultured adult rat hepatocytes and during early stages of hepatocarcinogenesis (Vandenberghe et al., 1988a). Materials and Methods Chemicals
Crude collagenase type I, bovine serum albumin (fraction V), bovine insulin, I-chloro-2,4-dinitrobenzene (CDNB), glutathione and epoxy-activated sepharose 6B were obtained from Sigma Chemical Co. (St Louis, MO, USA). All culture media, foetal calf serum and trypsin-EDTA solution were purchased from Gibco (Brussels, Belgium). Hydrocortisone hemisuccinate was from Roussel (Paris, France). 1,2-Dichioro-4-nitrobenzene (DCNB) and absolute ethanol came from Merck-Schuchardt (Darmstadt, Germany). All other chemicals were of reagent grade and were purchased from general commercial sources.
350 g, obtained by Iffa Credo, Brussels, Belgium) and co-cultured for 10 days with rat liver epithelial cells of primitive biliary origin as described by Guguen-Guillouzo et al. (1983). Co-cultured cells remained either untreated or were continuously exposed to 0.1 or 0.4% EtOH. The medium was renewed every day. Cytosolic fractions from freshly isolated hepatocytes (To) and IO-day old co-cultures were prepared as described by Johnson et al. (1992). GSTs were isolated from cytosolic fractions by affinity chromatography as previously described (Vandenberghe ef al., 1988a). The separation and quantification of GST subunits was carried out by HPLC on a 10 x 0.8 cm Waters FBondapak C-18 reversed phase column in a Z-module using a Shimadzu LCIO
Table
2. Effect
of ethanol
on the concentration
co-cultured Subunit
of GST
cone”
(pg/mg
cytosolic Control
Preparation of hepatocyte cultures, cytosolic fractions and isolation and separation of GSTs
Subunits
I.
Effect
of ethanol DCNB
on GST
in co-cultured
activity
towards
Enzymatic (percentage Ethanol
concn
(%)
0.1
124+
0.4
135f6’
concentrations
Values
in
obtained
mean is derived culture
from from
epithelial
12
125 f
19
two-way
2.7 k 2.3
6.4 * 5.6
2.3 f
3.9 k 2.6
6.8 f 9.1 5.1 k 3.2
I.4
12.5 k 13.4
2.5 k 2.1
0.8 f 0.2
0.8 f 0.6
1.4*
8b
2.6 f 0.2
0.7 f 0.4
0.8 f 0.6
I .9 f 2.3
0.0 + 0.0
0.5 f 0.5
0.0 + 0.0
9.5 ? 3.5
20.7 it 8.4
21.9?
40.9 f
3.5’
I.3
0.4 f 0.7 21.0
12.9 i. I.1
Il.4
f 4.7
22.0 f
I I.5
33.6 + 8.6*
21.6+2.1
22. I k 4.0
36.4 f
16.3
52.0 k 7.8*
0.8 * 0.7
0.8 f
0.8 f 0.2 35.2+3.1
34.2k8.8
I.1 f
I .O
I.1
59.1 t27.4
86.8+
14.9’
7
value, that
E1OH) f SD. Each isolation
and
0.0 + 0.0’
concentration
hepatocytes (To) the absence
15.4 f
of individual
6.3
GST
18.3 + 5.4
14.6 + 2.3
and in hepatocytes
(control)
or presence
EtOH were determined by HPLC
in freshly isolated
subunits
co-cultured
for
IO days in
of various
concentrations
as described
in Materials
of and
Methods. analysis
cells were negligible
the co-cultures
2.3 k 0.7
n class
experiments.
-z 0.05;
+
EtOH
2.8 f 0.7
p subtotal
presence of various
the results of three separate
Asterisk indicates a significant difference from (*P
107 *
of the control
in 0%
Control 0.4%
12.6 + 2.5’
6
The
cells cultured
EtOH
protein)
8a
3 4
of EtOH.
are mea” (expressed as a percentage
is that
the
0.1%
+
a class
100+6
Hepatocytes were co-cultured for IO days
4.8 f 4.5
IO
DCNB
14
18.5 * 3.8’
lb
OLsubtotal
of control)
IOOfll
la 2
and
activity
CDNB
0.0
CDNB
rat hepatocytes
in
x class
Hepatocytes were isolated from untreated adult male OFA Sprague-Dawley rats (I.O.P.S. caw, 15wk, Table
Control
TO
subunits
hepatocytes
(less than
of variance). in comparison
3.5%).
the control The with
values
value for
the
the values for
Values
are
isolation Asterisk
mean f
indicates
control
SD
and culture value
derived
significant (*P
from
the results
of
four
separate
experiments.
< 0.05;
difference two-way
from analysis
the
corresponding
of variance).
Efrect of ethanol on GST expression system with a Prolinea 4/33 HPLC dedicated integrator (Compaq, Houston, USA). The solvents were water and acetonitrile, each containing 0.06% (w/w) trifluoroacetic acid as described by Vandenberghe et al. (1990). The samples were injected at 36% acetonitrile. During a run, a linear gradient of acetonitrile, from 36 to 53%, was used over a 60-min period. The flow rate was 1.5 ml/min and UV absorbance at 214nm was monitored. Recovery of GSTs ranged from 90 to 100%.
469
tein Assay (Bio-Rad, Brussels, Belgium) with bovine serum albumin as a standard. Statistical analysis
Statistical evaluation of the differences obtained was achieved by general ANOVA procedures and Student’s t-test using the Software Package of the Social Sciences, SPSS/PC+ (Norusis, 1986). Results Total GST activity in the co-cultured cells remained constant for up to 14 days when CDNB was used as the substrate (Fig. 1). An increase in GST activity was observed in these cells after 7 days of co-culture if DCNB was used as a substrate. This increased activity was maintained for at least 7 days
Enzyme and protein assays
Cytosolic GST activities were assayed spectrophotometrically using CDNB and DCNB as substrates (Vandenberghe et al., 1988b). Cytosolic protein concentrations were determined using the Bio-Rad Prohl214nm
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Fig. 2(a and b)-Caption overleaf.
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Fig. 2. Composition of GST subunits present in rat hepatocytes. GST subunits were separated by reversed phase HPLC of extracts of freshly isolated rat hepatocytes (a), IO-day-old co-cultured hepatocytes (control) (b) and IO-day-old co-cultured hepatocytes treated with 0.4% ethanol (c). The conditions were as described in Materials and Methods. Numbers refer to GST subunits.
(Fig. 1). These findings are in agreement with our earlier observations (Rogiers et al., 1990). The maintenance of GST activity in this culture system makes it suitable for examining the effects of foreign compounds, such as EtOH, on the expression of GST proteins. In freshly isolated hepatocytes, GST activities for the substrates CDNB and DCNB were 1.2 pmol/ min/mg cytosolic protein and 35 nmol/min/mg cytosolic protein, respectively. These results are very similar to those reported by Vandenberghe et a/. (1988b). When IO-day-old co-cultures were exposed to EtOH, GST activity tended to increase in a dose-dependent way (Table 1). Total GST activity towards the substrate CDNB increased by 35% in cells treated with 0.4% EtOH. However, this concentration of EtOH did not significantly increase total GST activity when DCNB was used as a substrate (Table 1). The metabolism of DCNB or CDNB by GSTs was negligible in the helper epithelial cells and did not increase on treatment of these cells with EtOH. The effect of EtOH treatment on the amounts of individual GST subunits in co-cultured cells was investigated using HPLC. Figure 2 shows the separation pattern of GST subunits present in freshly isolated hepatocytes and in co-cultured cells that were treated for 10 days with 0.4% EtOH or remained untreated. The data presented in Table 2 show that the overall content of GST proteins decreases when hepatocytes are co-cultured. This is largely due to a reduction in the amounts of ~-class subunits. Total GST protein content decreased from 76.1 f 6.6 pg/mg cytosolic protein in freshly isolated hepatocytes to 59.1 + 18.6 pg/mg cytosolic protein in lo-day-old co-cultures. During this culture period,
r-class subunits decreased from 40.9 k 3.5 to 9.5 f 3.5 pg/mg cytosolic protein. Members of the r-class were not significantly induced by treatment of the cells with EtOH. In contrast, the amounts of the p-class protein subunits were maintained in the culture conditions used, and subunits 3 and 4 were increased about three-fold and 2.5-fold, respectively, in response to 0.4% EtOH compared with the control value without EtOH. As shown previously (Vandenberghe et al., 1990) the n-class subunit 7, which was absent from freshly isolated hepatocytes, was present in co-cultured cells at a concentration of 15.4 + 6.3 pg/mg cytosolic protein. The concentration of this subunit was not increased in response to EtOH treatment.
Discussion In this paper we report the effects of EtOH on cytosolic GST enzyme activity and GST subunit composition in co-cultures of rat hepatocytes and rat liver epithelial cells. EtOH primarily increases the amount of p-class subunits. The effect of EtOH on the expression of these subunits was confirmed at the RNA level using Northern blot hybridization analysis (data not shown). The exact mechanism whereby EtOH acts to increase the expression of p subunits is not known. It is also not yet known whether the results we observe in vitro correspond to the in uivo situation. The available data concerning the effect of EtOH in rodents are conflicting (Gonzalez et al., 1988; Munoz et al., 1987; van de Wiel et al., 1993). Little information exists concerning the effects of this compound on GST activity and subunit composition in man. Human p-class GSTs are of particular
Effect of ethanol on GST expression
interest because about 45% of the European population are homozygous null for the GSTMl gene locus. ~-Class GSTs are very effective at deactivating mutagenic and carcinogenic epoxides. It is therefore reasonable to suppose that any induction of the p-class GST isoenzymes might result in a significant reduction in the toxicity of compounds such as polycyclic aromatic hydrocarbon epoxides present in tobacco smoke, and chemical compounds including styrene oxide and truns-stilbene oxide. EtOH also affects phase 1 xenobiotic metabolism in that it induces cytochrome P4502El in man and rodents (Gonzalez et al., 1991; Guengerich et al., 1990). Because of the ability of EtOH to induce different xenobiotic-metabolizing enzymes it is not clear whether the overall effect of the compound would increase the cells’ capacity for detoxification of foreign chemicals. Our data do, however, show that concentrations of EtOH greater than 0.1% should not be used in cultured cells, thus avoiding the effects of this compound on the metabolism of endogenous and exogenous compounds. REFERENCES Gonzalez F. J., Ueno T., Umeno M., Song B. J., Veech R. L. and Gelboin H. V. (1991) Microsomal ethanol oxidizing system: transcriptional and posttranscriptional regulation of cytochrome P450, CYPZEI. Alcohol and Alcoholism Suppl. 1, 97-101. Gonzalez J., Munoz M. E., Marin M. I., Collado P. S., Fermoso J., Esteller A. (1988) Influence of acute ethanol administration on hepatic glutathione metabolism in the rat. Alcohol 5, 103-106. Guengerich F. P., Kim D.-H. and Iwasaki M. (1990) Role of human cytochrome P-450 IIEl in the oxidation of many low molecular weight cancer suspects. Chemical Research in Toxicology 4, 1688179.
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