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Effect of blood on styrene oxidation in perfused rat liver
261
Toxicology Letters, 23 (1984) 261-265 Elsevier
TOXLett. 1315
EFFECT OF BLOOD ON STYRENE OXIDATION IN PERFUSED RAT LIVER (Styrene; styrene oxide; oxyhemoglobin)
G. BELVEDEREas*, L. TALVEb, E. HIETANENb and H. VAINIO’ aLaboratory for Enzyme Research, Istituto di Ricerche Farmacologiche ‘Mario Negri’, Via Eritrea 62, 20157Milan (Italy); bDepartment of Physiology, University of Turku, SF-20520 Turku 52, and ‘Institute of Occupational Health, Haartmaninkatu I, 00290 Helsinki 29 (Finland) (Received April 3Oth, 1984) (Revision received July 16th, 1984) (Accepted July 26th, 1984)
SUMMARY The oxidation of styrene to styrene oxide was studied in the isolated perfused rat liver in the presence and absence of blood at styrene concentrations of 2.5 and 50 mM. Erythrocytes contained in whole blood increased the levels of styrene glycol about 5 times after a short perfusion time with both concentrations. This increase was observed up to 1 h with 2.5 mM styrene. At both styrene concentrations styrene oxide was not detectable, either in the presence or absence of blood indicating that the liver was able completely to detoxify the styrene oxide produced by the mixed-function oxidases (MFO) and the oxyhemoglobin in the erythrocytes.
INTRODUCTION
Styrene is metabolized to styrene glycol through an epoxide-diol pathway catalyzed by microsomal MFO and epoxide hydrolase, mainly in the liver [ 1, 21. The toxicity and cytogenetic damage caused by styrene have been related to the formation of its reactive metabolic intermediate styrene-7,8-oxide (styrene oxide) [3-51. Findings such as the failure of MFO inhibition to prevent the hepatotoxic effect of styrene and to modify the urinary excretion profile of styrene metabolites [6], and the observation of the same number of SCE in cells of different mouse tissues [7] *Supported by an EEC fellowship. Abbreviations:
MFO, mixed-function
oxidases; SCE, sister chromatid exchange.
0378-4274/84/% 03.00 0 Elsevier Science Publishers B.V.
262
suggested that metabolic activation pathways not depending on MFO or not occurring in the liver were responsible for styrene toxicity. Recently it was found that the oxidation of styrene to styrene oxide could be catalyzed by the oxyhemoglobin in erythrocytes [8, 91. The presence of blood was also necessary for the induction of SCE in cultured human lymphocytes [5]. The formation of styrene oxide was studied in the perfused liver to establish whether the activation of styrene catalyzed by oxyhemoglobin could be relevant in conditions similar to those in vivo and might thus explain the metabolic and genotoxic findings previously observed. METHODS
Liver was perfused either with Tyrode buffer (100-120 ml) containing 5% glucose or with Tyrode buffer, 5% glucose and blood (40 ml buffer and 80 ml blood). In each case the perfusion medium also contained 500 IU heparin and 3% (w/v) bovine serum albumin. Blood was collected from rats by heart puncture with a heparinized needle and syringe. Styrene was dissolved in the perfusion buffer on the previous day at the perfusion concentrations or at appropriate concentrations when blood was to be added. Perfusion buffer was left to circulate about 5 min before adding the liver in the perfusion apparatus. To take the liver, adult male rats (Wistar), 200-250 g, were anesthetized with ether, the abdominal cavity was opened and 500 IU heparin were injected. The portal vein was cannulated with a thin teflon cannula (filled with Tyrode solution), ligated tightly and the liver was carefully dissected. The liver was lightly rinsed with Tyrode solution and installed in the perfusion chamber, connected to the recirculating perfusion medium. The hydrostatic pressure for perfusion was kept at 25 cm (HzO). The whole perfusion apparatus was maintained at 37°C with the aid of an all-glass water jacket. The liver was visually monitored for even perfusion. 2-8 ml samples were taken for the analysis of styrene metabolites during perfusion. The hemoglobin concentration was monitored in most of the perfusions when blood was added (101.3 f 6.2 g/100 ml perfusate) and the hematocrit value (25 + 1(r/o)was recorded at the end of perfusion. The perfusion rate was normally 20-25 ml/min but in some cases at 50 mM styrene concentration it was reduced to 6-8 ml/min. The amount of styrene oxide formed during liver perfusion was measured by a method previously described [lo]. The accumulated styrene oxide was quantitatively hydrated chemically to the glycol by overnight incubation with acid, this substance being more suitable for GC analysis [l 11. RESULTS
AND DISCUSSION
Styrene oxide formation in the perfusion medium was measured by GC assay of the styrene glycol formed after acid hydrolysis of the epoxide. The glycol formed enzymically by epoxide hydrolase was measured by the same assay but without acid
263
hydrolysis. The amounts of glycol formed by the epoxide hydrolase were not significantly different (PcO.05) from those formed in the presence of acid, indicating the absence of detectable levels of unhydrated styrene oxide in the perfusate, both in the presence and absence of blood. In the liver perfusion system, in the absence of blood, a linear relationship was found between styrene concentration and styrene glycol accumulation between 2.5 and 50 mM (Fig. 1). To study the effect of erythrocytes on styrene oxidation the same two concentrations were used: 2.5 mM, close to levels of styrene to which workers are exposed [ 121, and 50 mM in the range of saturation for styrene oxidation catalyzed by oxyhemoglobin [8]. At the 50 mM concentration styrene oxide formation was 7 times higher in the presence of blood after 5 min of perfusion (Fig. 2). After 30 min, however, the rate was similar to that observed in the absence of blood. The smaller increase in styrene oxidation in samples containing blood after 30 min of perfusion, might be due to the toxic effects of complete hemolysis of erythrocytes at this styrene concentration (not shown). With the 2.5 mM concentration, that does not cause hemolysis, accumulation of styrene glycol in the perfusate occurred at a 4 times higher rate in the presence of blood, even after 1 h of perfusion (Fig. 2). The perfused liver can completely detoxify 100 pmol of styrene oxide/h/liver and the rate of styrene oxide formation is half that of its detoxification [13]. Therefore, although blood may contribute to the formation of styrene oxide in vivo, the levels of this metabolite are probably mainly regulated by the detoxifying activity of the liver.
0
I
I
2.5
5
I
10
50
I
mM
Fig. 1. Styrene oxide formed by the perfused liver system with different styrene concentrations. glycol was measured after acid hydrolysis. Bars represent the mean f SE.
Styrene
264 A
0
150
15
.-f
2
i
0 " 1 cn
100
0, : L a ;; -d E c
50
,P’. 0
_p” I
5
1
15
30
( 60
0
r
5
I
15
30
I
60min
Fig. 2. Time course of styrene oxide formation in the perfused liver: styrene 50 mM (A) and 2.5 mM (B). Rat liver was perfused with blood (M) and without blood (O---O). Styrene glycol was measured after acid hydrolysis. Bars represent the mean f S.E.
REFERENCES K.C. Leibman and E. Ortiz, Oxidation of styrene in liver microsomes, Biochem. Pharmacol., 18 (1969) 552-554. H. Ohtsuji and M. Ikeda, The metabolism of styrene in the rat and the stimulatory effect of phenobarbital, Toxicol. Appl. Pharmacol., 18 (1971) 321-328. J. Marniemi, E-M. Suolinna, N. Kaartinen and H. Vainio, Covalent binding of styrene oxide to rat liver macromol~ules in vivo and in vitro, in V. Ullrich, I. Roots, A. Hildebrandt, R.W. Estabrook and A.H. Conney @is.), Microsomes and Drug Oxidation, Pergamon, London, 1977, pp. 698-702. H. Vainio, R. Paakkonen, K. Ronnholm, V. Raunio and 0. Pelkonen, A study on the mutagenic activity of styrene and styrene oxide, Stand. J. Work Environ. Health, 3 (1976) 147-151. H. Norppa, H. Vainio and M. Sorsa, Metabolic activation of styrene by erythrocytes detected as increased sister chromatid exchanges in cultured human lymphocytes, Cancer Res., 43 (1983) 3579-3582. S. Chakrabarti and J. Brodeur, Effects of microsomal induction and inhibition on styrene-induced acute hepatotoxicity in rats, J. Toxicol. Environ. Health, 8 (1981) 599-607. M.K. Conner, Y. Alarie and R.L. Dombroske, Sister chromatid exchange in murine alveolar macrophages, bone marrow, and regenerating liver cells induced by styrene inhalation, Toxicol. Appl. Pharmacol., 55 (1980) 37-42. 8 G. Belvedere and F. Tursi, Styrene oxidation to styrene oxide in human blood erythrocytes and lymphocytes, Res. Commun. Chem. Pathol. Pharmacol., 33 (1981) 273-282. 9 F. Tursi, M. Samaia, M. Salmona and G. Belvedere, Styrene oxidation to styrene oxide in human erythrocytes is catalyzed by oxyhemoglobin, Experientia 39 (1983) 593-594. 10 G. Belvedere, J. Pachecka, L. Cantoni, E. Mussini and M. Salmona, A specific gas chromatographic
265 method for the determination of microsomal styrene monooxygenase and styrene epoxide hydratase activities, J. Chromatogr., 118 (1976) 387-393. 11 G. Gazzotti, E. Garattini and M. Salmona, Improved gas chromatographic method for measuring phenylethyiene glycol. Application to the determination of styrene monooxygenase and epoxide hydrase activities, J. Chromatogr., 188 (1980) 400404. 12 MS. Wolff, W.V. Lorimer, R. Lilis and I.J. Selikoff, Blood styrene and urinary metabolites in styrene polymerisation, Br. J. Ind. Med., 35 (1978) 318-329. 13 J. van Anda, B.R. Smith, J.R. Fouts and J.R. Bend, Concentration-dependent metabolism and toxicity and [‘%]styrene oxide in the isolated perfused rat liver, J. Pharmacol. Exp. Ther., 211 (1979) 207-212.
Toxicology Letters, 23 (1984) 261-265 Elsevier
TOXLett. 1315
EFFECT OF BLOOD ON STYRENE OXIDATION IN PERFUSED RAT LIVER (Styrene; styrene oxide; oxyhemoglobin)
G. BELVEDEREas*, L. TALVEb, E. HIETANENb and H. VAINIO’ aLaboratory for Enzyme Research, Istituto di Ricerche Farmacologiche ‘Mario Negri’, Via Eritrea 62, 20157Milan (Italy); bDepartment of Physiology, University of Turku, SF-20520 Turku 52, and ‘Institute of Occupational Health, Haartmaninkatu I, 00290 Helsinki 29 (Finland) (Received April 3Oth, 1984) (Revision received July 16th, 1984) (Accepted July 26th, 1984)
SUMMARY The oxidation of styrene to styrene oxide was studied in the isolated perfused rat liver in the presence and absence of blood at styrene concentrations of 2.5 and 50 mM. Erythrocytes contained in whole blood increased the levels of styrene glycol about 5 times after a short perfusion time with both concentrations. This increase was observed up to 1 h with 2.5 mM styrene. At both styrene concentrations styrene oxide was not detectable, either in the presence or absence of blood indicating that the liver was able completely to detoxify the styrene oxide produced by the mixed-function oxidases (MFO) and the oxyhemoglobin in the erythrocytes.
INTRODUCTION
Styrene is metabolized to styrene glycol through an epoxide-diol pathway catalyzed by microsomal MFO and epoxide hydrolase, mainly in the liver [ 1, 21. The toxicity and cytogenetic damage caused by styrene have been related to the formation of its reactive metabolic intermediate styrene-7,8-oxide (styrene oxide) [3-51. Findings such as the failure of MFO inhibition to prevent the hepatotoxic effect of styrene and to modify the urinary excretion profile of styrene metabolites [6], and the observation of the same number of SCE in cells of different mouse tissues [7] *Supported by an EEC fellowship. Abbreviations:
MFO, mixed-function
oxidases; SCE, sister chromatid exchange.
0378-4274/84/% 03.00 0 Elsevier Science Publishers B.V.
262
suggested that metabolic activation pathways not depending on MFO or not occurring in the liver were responsible for styrene toxicity. Recently it was found that the oxidation of styrene to styrene oxide could be catalyzed by the oxyhemoglobin in erythrocytes [8, 91. The presence of blood was also necessary for the induction of SCE in cultured human lymphocytes [5]. The formation of styrene oxide was studied in the perfused liver to establish whether the activation of styrene catalyzed by oxyhemoglobin could be relevant in conditions similar to those in vivo and might thus explain the metabolic and genotoxic findings previously observed. METHODS
Liver was perfused either with Tyrode buffer (100-120 ml) containing 5% glucose or with Tyrode buffer, 5% glucose and blood (40 ml buffer and 80 ml blood). In each case the perfusion medium also contained 500 IU heparin and 3% (w/v) bovine serum albumin. Blood was collected from rats by heart puncture with a heparinized needle and syringe. Styrene was dissolved in the perfusion buffer on the previous day at the perfusion concentrations or at appropriate concentrations when blood was to be added. Perfusion buffer was left to circulate about 5 min before adding the liver in the perfusion apparatus. To take the liver, adult male rats (Wistar), 200-250 g, were anesthetized with ether, the abdominal cavity was opened and 500 IU heparin were injected. The portal vein was cannulated with a thin teflon cannula (filled with Tyrode solution), ligated tightly and the liver was carefully dissected. The liver was lightly rinsed with Tyrode solution and installed in the perfusion chamber, connected to the recirculating perfusion medium. The hydrostatic pressure for perfusion was kept at 25 cm (HzO). The whole perfusion apparatus was maintained at 37°C with the aid of an all-glass water jacket. The liver was visually monitored for even perfusion. 2-8 ml samples were taken for the analysis of styrene metabolites during perfusion. The hemoglobin concentration was monitored in most of the perfusions when blood was added (101.3 f 6.2 g/100 ml perfusate) and the hematocrit value (25 + 1(r/o)was recorded at the end of perfusion. The perfusion rate was normally 20-25 ml/min but in some cases at 50 mM styrene concentration it was reduced to 6-8 ml/min. The amount of styrene oxide formed during liver perfusion was measured by a method previously described [lo]. The accumulated styrene oxide was quantitatively hydrated chemically to the glycol by overnight incubation with acid, this substance being more suitable for GC analysis [l 11. RESULTS
AND DISCUSSION
Styrene oxide formation in the perfusion medium was measured by GC assay of the styrene glycol formed after acid hydrolysis of the epoxide. The glycol formed enzymically by epoxide hydrolase was measured by the same assay but without acid
263
hydrolysis. The amounts of glycol formed by the epoxide hydrolase were not significantly different (PcO.05) from those formed in the presence of acid, indicating the absence of detectable levels of unhydrated styrene oxide in the perfusate, both in the presence and absence of blood. In the liver perfusion system, in the absence of blood, a linear relationship was found between styrene concentration and styrene glycol accumulation between 2.5 and 50 mM (Fig. 1). To study the effect of erythrocytes on styrene oxidation the same two concentrations were used: 2.5 mM, close to levels of styrene to which workers are exposed [ 121, and 50 mM in the range of saturation for styrene oxidation catalyzed by oxyhemoglobin [8]. At the 50 mM concentration styrene oxide formation was 7 times higher in the presence of blood after 5 min of perfusion (Fig. 2). After 30 min, however, the rate was similar to that observed in the absence of blood. The smaller increase in styrene oxidation in samples containing blood after 30 min of perfusion, might be due to the toxic effects of complete hemolysis of erythrocytes at this styrene concentration (not shown). With the 2.5 mM concentration, that does not cause hemolysis, accumulation of styrene glycol in the perfusate occurred at a 4 times higher rate in the presence of blood, even after 1 h of perfusion (Fig. 2). The perfused liver can completely detoxify 100 pmol of styrene oxide/h/liver and the rate of styrene oxide formation is half that of its detoxification [13]. Therefore, although blood may contribute to the formation of styrene oxide in vivo, the levels of this metabolite are probably mainly regulated by the detoxifying activity of the liver.
0
I
I
2.5
5
I
10
50
I
mM
Fig. 1. Styrene oxide formed by the perfused liver system with different styrene concentrations. glycol was measured after acid hydrolysis. Bars represent the mean f SE.
Styrene
264 A
0
150
15
.-f
2
i
0 " 1 cn
100
0, : L a ;; -d E c
50
,P’. 0
_p” I
5
1
15
30
( 60
0
r
5
I
15
30
I
60min
Fig. 2. Time course of styrene oxide formation in the perfused liver: styrene 50 mM (A) and 2.5 mM (B). Rat liver was perfused with blood (M) and without blood (O---O). Styrene glycol was measured after acid hydrolysis. Bars represent the mean f S.E.
REFERENCES K.C. Leibman and E. Ortiz, Oxidation of styrene in liver microsomes, Biochem. Pharmacol., 18 (1969) 552-554. H. Ohtsuji and M. Ikeda, The metabolism of styrene in the rat and the stimulatory effect of phenobarbital, Toxicol. Appl. Pharmacol., 18 (1971) 321-328. J. Marniemi, E-M. Suolinna, N. Kaartinen and H. Vainio, Covalent binding of styrene oxide to rat liver macromol~ules in vivo and in vitro, in V. Ullrich, I. Roots, A. Hildebrandt, R.W. Estabrook and A.H. Conney @is.), Microsomes and Drug Oxidation, Pergamon, London, 1977, pp. 698-702. H. Vainio, R. Paakkonen, K. Ronnholm, V. Raunio and 0. Pelkonen, A study on the mutagenic activity of styrene and styrene oxide, Stand. J. Work Environ. Health, 3 (1976) 147-151. H. Norppa, H. Vainio and M. Sorsa, Metabolic activation of styrene by erythrocytes detected as increased sister chromatid exchanges in cultured human lymphocytes, Cancer Res., 43 (1983) 3579-3582. S. Chakrabarti and J. Brodeur, Effects of microsomal induction and inhibition on styrene-induced acute hepatotoxicity in rats, J. Toxicol. Environ. Health, 8 (1981) 599-607. M.K. Conner, Y. Alarie and R.L. Dombroske, Sister chromatid exchange in murine alveolar macrophages, bone marrow, and regenerating liver cells induced by styrene inhalation, Toxicol. Appl. Pharmacol., 55 (1980) 37-42. 8 G. Belvedere and F. Tursi, Styrene oxidation to styrene oxide in human blood erythrocytes and lymphocytes, Res. Commun. Chem. Pathol. Pharmacol., 33 (1981) 273-282. 9 F. Tursi, M. Samaia, M. Salmona and G. Belvedere, Styrene oxidation to styrene oxide in human erythrocytes is catalyzed by oxyhemoglobin, Experientia 39 (1983) 593-594. 10 G. Belvedere, J. Pachecka, L. Cantoni, E. Mussini and M. Salmona, A specific gas chromatographic
265 method for the determination of microsomal styrene monooxygenase and styrene epoxide hydratase activities, J. Chromatogr., 118 (1976) 387-393. 11 G. Gazzotti, E. Garattini and M. Salmona, Improved gas chromatographic method for measuring phenylethyiene glycol. Application to the determination of styrene monooxygenase and epoxide hydrase activities, J. Chromatogr., 188 (1980) 400404. 12 MS. Wolff, W.V. Lorimer, R. Lilis and I.J. Selikoff, Blood styrene and urinary metabolites in styrene polymerisation, Br. J. Ind. Med., 35 (1978) 318-329. 13 J. van Anda, B.R. Smith, J.R. Fouts and J.R. Bend, Concentration-dependent metabolism and toxicity and [‘%]styrene oxide in the isolated perfused rat liver, J. Pharmacol. Exp. Ther., 211 (1979) 207-212.