LETTERS TO THE EDITOR
408 HEPAT 00996
Toxicity of diclofenac to isolate
atocytes
Berry and Friend (2) and modified by Seglen (10) and Baur et al. (1). In one series of experiments 1.10-6 cells/ml were incubated at 37’C, pH 7.4, in MEM-Earle medium supplemented with 20 mM Hepes. Diclofenac sodium solubilized with 1% DMSO was added to the cell suspensions at various concentrations for up to 90 min. In a second series of experiments hepatocytes in primary monolayer cultures according to Bissell and Guzelisn (3) were exposed to 0.1 or 0.5 mM diclofenac for 24 and 48 h. At the end of the incubation period aliquots were taken to measure the cell viability using the Trypan blue staining and LDH-leakage as indicators. Details of the procedure are described in an earlier paper (11). Mean values and standard deviations were calculated from 12 measurements. For statistical analyses of the data oneway analysis of variance or, if appropriate, Student’s t-test have been applied. Differences between two mean values are regarded significant, if p I 0.05. DMSO at the concentration used did not significantly affect the viability of the cells, whereas diclofenac was hepatotoxic and markedly reduced the number of viable
Diclofenac (l-10 mM) reduced the viability of isolated rat hepatocytes in a dose- and time-dependent manner which shows that diclofenac, like other NSAIDs, has direct hepatotoxic properties. Diclofenac, 2(2,6-dichloroanilino)phenyl acetate, is a nonsteroidal antiinflammatory agent which has been widely used for almost two decades. According to standard reference works (5) diclofenac may cause liver dysfunction, but liver damage is rare. However, during the last few years there has been an increasing number of reports of hepatitis associated with diclofenac use, some with fatal cases (for review see Ref. 8). The mechanism of diclofenac-induced hepatotoxicity is unclear. Hypersensitivity has been suggested as well as direct toxic effects mediated through the metabolization of diclofenac. In order to find out whether diclofenac is directly hepatotoxic we tested to see whether diclofenac had a toxic effect on isolated hepatocytes in vitro. Hepatocytes were isolated from male Wistar rats (190230 g) that had free access to standard diet (Herilan@) and water. The isol.ation was performed as described by A. Contmlsc
Diclolenac 0 0.5mM 0 1 mM q 2mM 6813 mM LDH-leokoge[%I
05
mM
10mM
loo-
80. 60.
40.
20.
O-l 2min
30min 8: Controls:
LDH-leakage p/J
24 hr
Diclofenac
’
60min
90min
.
q 0 0.1 mM
Ed 0.5 mM
48 hr
Fig. 1. LDH-leakage from isolated hepatocytes incubated with diclofenac. (A) Experiments with hepatocytes in cell suspension; (B) experimentswith hepatocytesin primary monolayer culture. Percentage of total cellular LDH released into the medium. Mean values & S.D. *p vs. controls 5 0.05.
LETTERS
TO THE EDITOR
409
hepatocytes (Fig. 1). This effect was clearly dose- and time-dependent. With 10 mM of diclofenac, cells were destroyed within 15 min or less (data not shown), with 5 mM most of the cells were killed within 30 min, whereas 2 and 3 mM was effective after 30-90 min and 1 only a slight effect after SO min. Still lower con tions (0.1-0.5 mM) required at least 24 h exposure to diclofenac to monolayer cultures (Fig. 1B). Experiments using Trypan blue staining as an indicator of cell death gave essentially the same results. Our finding that diclofenac is toxic to isolated hepatocytes clearly suggests that the hepatotoxic reactions observed in patients treated with diclofenac are due to its cytotoxic effects and not to hypersensitivity as suggested by Breen et al. (4) and Dunk et al. (6). The molecular mechanism of the toxic effects remains, however, unclear. The slow onset of the cytotoxic effects at lower concentrations is compatible with the view that the cy-
totoxicity is associated with the me!abolization of diclofenac and possibly brought about by a reactive metabolite as suggested for other NSAIDs (9). The very rapid, almost instantaneous effects of the highest doses are probably due to additional effects, e.g., on the cell membrane with permeability changes. Since our prt:paration was essentially free of Kupffer cells which are the main source of these eicosanoids (7), it is improbable that the effect is mediated by leukotricnes which are generated as a consequence of inhibition of cyclooxygenase. This study was supported by the Wilhelm-SanderStiftung, Munich, F.R.G., grant No. 89-013.1. G. Schmitz, I. Stauffert, H. Sippel, H. Lepper and C.-J. Estler Instihtte for Pharmacology and Toxicology, University of ErlungenNiirnberg, Erlangen, Federai Republic of Germany
7 Hagman
rences 1 Baur M. Kasparek S, Pfaff E. Criteria of viability of isolated liver cells. Hoppe-Seyler’s Z Physiol Chem 1975; 356: 827-38. 2 Berry MN, Frieud DS. High yield preparation of rat liver parenchymal cells. J Cell Biol 1969; 43: 506-20. 3 Bissell DM, Guz’elian PS. Phenotypic stability of adult rat hepatocytes in primary monolayer culture. Ann NY Acad Sci 1980; 349: 85-98. 4 Breen E, McNicholl J, Cosgrove E, McCabe J, Stevens F. Fatal hepatitis associated with diclofenac. Gut 1986; 27: 1390-l. 5 Dukes MNG. Meylrr’s Side Etfects of Drugs. 11th Edn. Amsterdam: Elsevier, 1988. 6 Dunk A, Walt R, Jenkins W, Sherlock S. Diclofenac hepatitis. Br Med J 1982; 289: 1605-06.
8 9
10 11
W, Kepler D. Leukotrienes and other eicosanoids in liver pathology. In: Arias J&l, Jacoby WB, Popper H, Schachter D, Sbafritz DA. eds. The j,iver. Biology and Pathobiology. New York: Raven Press, 1988; 793-806. Helfgott SM, Sandberg-Cook J, Zahim D, Nestler J. Diclofenacassociated hepatotoxicity. J Am Med Ass 1990; 264: 2660-2. Mitchell MC. Lietman PS. Evidence for the role of reactive metabolites in hepatotoxicity of benoxaprofen and other NSAIQs. Hepatology 1983: 3: 808. Seglen PO. Preparation of rat liver cells. Exp Cell Res 1972; 74: 450-4. Sippel H, Estler C-J. Comparative evaluation of hepatotoxic effects of various ilew trypanocidal diamidines in rat hepatocytes and mice. Arzneim Forsch 1990: 40: 290-3.
HEPAT 00997
ow rate of
atitis
The mode of contamination of HCV-infected patients without previous exposure to blood is unknown. HCV transmission to patient’s close contacts, possibly by sexual practice has been suggested. Previous reports of HCV transmissibility in sexual partners range from 0% to 26% using anti-clOO-3 antibodies (1,2). To further investigate non-parenteral HCV transmission, we studied the families of 53 anti-clOO-3 ELISA-positive patients. We determined serum antibodies against different viral coded proteins using the recently described second generation recombinant immune-binding assay (RIBA2, Chiron/ Ortho Diagnostic Systems). Fourteen patients were asymptomatic with several normal alanine aminotransferase (ALT) determinations. In contrast, 39 patients had liver disease: cirrhosis (n = 14).
chronic active hepatitis (n = 121, chronic persistent hepatitis (n = 5) and elevation of ALT in eight patients. Criteria for RIBA positivity% for 37 patients, were met in 31 cases. Qne hundred and six household sera (48 sexual partners and 58 children) were tested. Three couples had reproductible ELISA anti-clOO-3 reactive sera. By testing these six sera with the RIBA2, HCV infection could only be confirmed in one couple (Fig. 1). The two partners in this couple were previous drug addicts. For the two other couples, only one of the two partners was infected according to RIBA criteria. None of the other sexual partners had detectable antibodies in their sera against structural (~22-3) or non-structural (c33c, ~100-3, 5-l-l) viral proteins. Thus. using the R!BA2, we could
408 HEPAT 00996
Toxicity of diclofenac to isolate
atocytes
Berry and Friend (2) and modified by Seglen (10) and Baur et al. (1). In one series of experiments 1.10-6 cells/ml were incubated at 37’C, pH 7.4, in MEM-Earle medium supplemented with 20 mM Hepes. Diclofenac sodium solubilized with 1% DMSO was added to the cell suspensions at various concentrations for up to 90 min. In a second series of experiments hepatocytes in primary monolayer cultures according to Bissell and Guzelisn (3) were exposed to 0.1 or 0.5 mM diclofenac for 24 and 48 h. At the end of the incubation period aliquots were taken to measure the cell viability using the Trypan blue staining and LDH-leakage as indicators. Details of the procedure are described in an earlier paper (11). Mean values and standard deviations were calculated from 12 measurements. For statistical analyses of the data oneway analysis of variance or, if appropriate, Student’s t-test have been applied. Differences between two mean values are regarded significant, if p I 0.05. DMSO at the concentration used did not significantly affect the viability of the cells, whereas diclofenac was hepatotoxic and markedly reduced the number of viable
Diclofenac (l-10 mM) reduced the viability of isolated rat hepatocytes in a dose- and time-dependent manner which shows that diclofenac, like other NSAIDs, has direct hepatotoxic properties. Diclofenac, 2(2,6-dichloroanilino)phenyl acetate, is a nonsteroidal antiinflammatory agent which has been widely used for almost two decades. According to standard reference works (5) diclofenac may cause liver dysfunction, but liver damage is rare. However, during the last few years there has been an increasing number of reports of hepatitis associated with diclofenac use, some with fatal cases (for review see Ref. 8). The mechanism of diclofenac-induced hepatotoxicity is unclear. Hypersensitivity has been suggested as well as direct toxic effects mediated through the metabolization of diclofenac. In order to find out whether diclofenac is directly hepatotoxic we tested to see whether diclofenac had a toxic effect on isolated hepatocytes in vitro. Hepatocytes were isolated from male Wistar rats (190230 g) that had free access to standard diet (Herilan@) and water. The isol.ation was performed as described by A. Contmlsc
Diclolenac 0 0.5mM 0 1 mM q 2mM 6813 mM LDH-leokoge[%I
05
mM
10mM
loo-
80. 60.
40.
20.
O-l 2min
30min 8: Controls:
LDH-leakage p/J
24 hr
Diclofenac
’
60min
90min
.
q 0 0.1 mM
Ed 0.5 mM
48 hr
Fig. 1. LDH-leakage from isolated hepatocytes incubated with diclofenac. (A) Experiments with hepatocytes in cell suspension; (B) experimentswith hepatocytesin primary monolayer culture. Percentage of total cellular LDH released into the medium. Mean values & S.D. *p vs. controls 5 0.05.
LETTERS
TO THE EDITOR
409
hepatocytes (Fig. 1). This effect was clearly dose- and time-dependent. With 10 mM of diclofenac, cells were destroyed within 15 min or less (data not shown), with 5 mM most of the cells were killed within 30 min, whereas 2 and 3 mM was effective after 30-90 min and 1 only a slight effect after SO min. Still lower con tions (0.1-0.5 mM) required at least 24 h exposure to diclofenac to monolayer cultures (Fig. 1B). Experiments using Trypan blue staining as an indicator of cell death gave essentially the same results. Our finding that diclofenac is toxic to isolated hepatocytes clearly suggests that the hepatotoxic reactions observed in patients treated with diclofenac are due to its cytotoxic effects and not to hypersensitivity as suggested by Breen et al. (4) and Dunk et al. (6). The molecular mechanism of the toxic effects remains, however, unclear. The slow onset of the cytotoxic effects at lower concentrations is compatible with the view that the cy-
totoxicity is associated with the me!abolization of diclofenac and possibly brought about by a reactive metabolite as suggested for other NSAIDs (9). The very rapid, almost instantaneous effects of the highest doses are probably due to additional effects, e.g., on the cell membrane with permeability changes. Since our prt:paration was essentially free of Kupffer cells which are the main source of these eicosanoids (7), it is improbable that the effect is mediated by leukotricnes which are generated as a consequence of inhibition of cyclooxygenase. This study was supported by the Wilhelm-SanderStiftung, Munich, F.R.G., grant No. 89-013.1. G. Schmitz, I. Stauffert, H. Sippel, H. Lepper and C.-J. Estler Instihtte for Pharmacology and Toxicology, University of ErlungenNiirnberg, Erlangen, Federai Republic of Germany
7 Hagman
rences 1 Baur M. Kasparek S, Pfaff E. Criteria of viability of isolated liver cells. Hoppe-Seyler’s Z Physiol Chem 1975; 356: 827-38. 2 Berry MN, Frieud DS. High yield preparation of rat liver parenchymal cells. J Cell Biol 1969; 43: 506-20. 3 Bissell DM, Guz’elian PS. Phenotypic stability of adult rat hepatocytes in primary monolayer culture. Ann NY Acad Sci 1980; 349: 85-98. 4 Breen E, McNicholl J, Cosgrove E, McCabe J, Stevens F. Fatal hepatitis associated with diclofenac. Gut 1986; 27: 1390-l. 5 Dukes MNG. Meylrr’s Side Etfects of Drugs. 11th Edn. Amsterdam: Elsevier, 1988. 6 Dunk A, Walt R, Jenkins W, Sherlock S. Diclofenac hepatitis. Br Med J 1982; 289: 1605-06.
8 9
10 11
W, Kepler D. Leukotrienes and other eicosanoids in liver pathology. In: Arias J&l, Jacoby WB, Popper H, Schachter D, Sbafritz DA. eds. The j,iver. Biology and Pathobiology. New York: Raven Press, 1988; 793-806. Helfgott SM, Sandberg-Cook J, Zahim D, Nestler J. Diclofenacassociated hepatotoxicity. J Am Med Ass 1990; 264: 2660-2. Mitchell MC. Lietman PS. Evidence for the role of reactive metabolites in hepatotoxicity of benoxaprofen and other NSAIQs. Hepatology 1983: 3: 808. Seglen PO. Preparation of rat liver cells. Exp Cell Res 1972; 74: 450-4. Sippel H, Estler C-J. Comparative evaluation of hepatotoxic effects of various ilew trypanocidal diamidines in rat hepatocytes and mice. Arzneim Forsch 1990: 40: 290-3.
HEPAT 00997
ow rate of
atitis
The mode of contamination of HCV-infected patients without previous exposure to blood is unknown. HCV transmission to patient’s close contacts, possibly by sexual practice has been suggested. Previous reports of HCV transmissibility in sexual partners range from 0% to 26% using anti-clOO-3 antibodies (1,2). To further investigate non-parenteral HCV transmission, we studied the families of 53 anti-clOO-3 ELISA-positive patients. We determined serum antibodies against different viral coded proteins using the recently described second generation recombinant immune-binding assay (RIBA2, Chiron/ Ortho Diagnostic Systems). Fourteen patients were asymptomatic with several normal alanine aminotransferase (ALT) determinations. In contrast, 39 patients had liver disease: cirrhosis (n = 14).
chronic active hepatitis (n = 121, chronic persistent hepatitis (n = 5) and elevation of ALT in eight patients. Criteria for RIBA positivity% for 37 patients, were met in 31 cases. Qne hundred and six household sera (48 sexual partners and 58 children) were tested. Three couples had reproductible ELISA anti-clOO-3 reactive sera. By testing these six sera with the RIBA2, HCV infection could only be confirmed in one couple (Fig. 1). The two partners in this couple were previous drug addicts. For the two other couples, only one of the two partners was infected according to RIBA criteria. None of the other sexual partners had detectable antibodies in their sera against structural (~22-3) or non-structural (c33c, ~100-3, 5-l-l) viral proteins. Thus. using the R!BA2, we could
