Mutation Research, 244 (1990) 227-231
227
Elsevier MUTLET0367
The effect of paracetamol on oxidative damage in human peripheral lymphocytes w
•
B. Binkovfi, J. Topinka and R.J. Sram Psychiatric Research Institute, Prague (Czechoslovakia)
(Accepted3 February1990)
Keywords: Lipidperoxidation;DNA repair; Free radicals;Paracetamol
Summary Unscheduled DNA synthesis (UDSox) and lipid peroxidation (LPO) induced by non-enzymatic activation of molecular oxygen (Fe2+ + H 2 0 2 ) were measured in human peripheral lymphocytes from healthy volunteers. The effect of paracetamol (PC) in a final concentration range of 0.05-10 mmole/l on these oxidative processes and on DNA repair induced by MNNG (UDSmut) was investigated. The level of induced LPO was measured by the thiobarbituric acid assay, UDSox and UDSmut were determined by scintillometric measurement of incorporated [methyl-3H]thymidine into damaged DNA. PC at concentrations lower than 1 mmole/l significantly potentiates the non-enzymatically induced LPO and UDSox with the maximum of the activation being around 0.1 mmole/l. In contrast, PC at concentrations higher than 1 mmole/l exhibits an inhibitory effect on both LPO and UDSox. On the other hand, concentrations higher than 1 mmole/l significantly suppressed DNA-repair synthesis induced by MNNG.
The molecular mechanisms causing the hepatotoxic effects of the widely used analgesic drug paracetamol (N-acetyl-p-aminophenol, PC) have been the subject of many investigations (Rosen et al., 1983; West et al., 1983; Koymans et Correspondence: Dr. B. Binkovtt, Psychiatric Research Institute, Ostavni91, 18103 Prague 8 (Czechoslovakia). Abbreviations: PC, paracetamol; LPO, lipid peroxidation;
UDS, unscheduledDNA synthesis;MNNG, 1-methyl-3-nitrol-nitrosoguanidine;DMSO, dimethylsulfoxide;TBA,thiobarbituric acid; T, treated samples; C, controls.
al., 1989). It was reported that oxidation of PC can lead to coproduction of reactive oxygen species (superoxide radical and hydrogen peroxide) through redox cycling between the PC semiiminoquinone radical and the PC metabolite Nacetyl-p-benzoquinone imine (NAPQI). Some investigators suggest that this oxidative stress rather than covalent binding of highly electrophilic NAPQI to bioactive cellular proteins is the cause of the cell injury (Wendel et al., 1982; Gerson et al., 1985). On the other hand it has been demonstrated that PC protects human erythrocytes against in
0165-7992/90/$ 03.50 © 1990ElsevierSciencePublishers B.V. (BiomedicalDivision)
228 vitro induced oxidative damage as an effective free radical scavenger (Van der Zee et al., 1988). In our laboratory the possible genotoxicity of PC was investigated in doses therapeutic for man in a group of healthy volunteers (Ko~igovfi et al., 1988; Topinka et al., 1989a). In these studies an increased frequency of chromosome aberrations and decreased unscheduled DNA synthesis (UDS) in peripheral lymphocytes, as well as an increased frequency of micronuclei in the buccal mucosa cells were observed. The present paper deals with the effect of PC on in vitro induced oxidative damage of cell membranes and genetic material as well as on the DNA-repair system in human peripheral lymphocytes.
Materials and methods
Chemicals Ficoll 400 (Pharmacia, Sweden), 60070 Verografin, paracetamol (Spofa, Czechoslovakia), MNNG, hydroxyurea, 5-fluoro-2'-deoxyuridine, sodium ascorbate (all Serva, F.R.G.), thiobarbituric acid and dimethyl sulfoxide (Merck, F.R.G.), 1,1,3,3-tetraethoxypropane (Sigma, U.S.A.), RPMI 1640 medium (USOL, Czechoslovakia), scintillation cocktail SLD 31 (Spolana, Czechoslovakia), all other chemicals had purity p.a.
Sample preparation The experiments were carried out on peripheral lymphocytes from the blood of healthy volunteers of both sexes with an average age of 43.1 years (SD 8.0). The lymphocytes were isolated on Ficoll 400-Verografin gradient (Harris et al., 1970). The protein content in the suspension of isolated cells was determined according to Bradford (1976). The samples from each donor were diluted into RPMI 1640 medium at a cell density of 0.2 mg of protein per ml and divided into several aliquots. In treated samples (T) molecular oxygen was activated by adding Fe 2 ÷ (20 #mole/l) and hydrogen peroxide (1 mmole/1). The effect of paracetamol on the oxidative processes induced in this way was investigated at a final concentration range of 0.05-10
mmole/l. PC dissolved in DMSO was added to the lymphocyte suspension prior to the oxygenactivating agents. The DMSO content was the same in all samples (2°70 of the total sample volume). Samples with the same concentration of PC but without any induction of molecular oxygen were used as a control (C). After incubation of all samples for 30 min at 37°C lipid peroxidation (LPO) and unscheduled DNA synthesis (UDSox) were determined.
Lipid peroxidation The LPO level in lymphocytes was determined by the modified thiobarbituric acid (TBA) assay (Ohkawa et al., 1976). TBA-active products of LPO were extracted into butanol and measured spectrophotometrically at 532 nm on a Shimadzu UV-2100 (high concentration) or fluorophotometrically with ~x = 515 nm and ~m = 546 nm on a Shimadzu RF 5000. The obtained values were expressed as nmole MDA per mg of protein using 1,1,3,3-tetraethoxypropane as a standard for all treated (T) and control (C) samples. The ratio T/C gives information about the in vitro induced LPO level in the suspension of lymphocytes under the given conditions.
Unscheduled DNA synthesis UDSox induced by non-enzymatic activation of molecular oxygen was determined by the scintillometric measurement of incorporated [methyl3H]thymidine. DNA from cells was isolated according to Martin et al. (1978). The incorporated radioactivity was measured on a Beckman LS 5801 liquid scintillation counter. The concentration of DNA was evaluated by the diphenylamine method on the spectrophotometer UV-2100 (Shimadzu). The specific activity of the samples was expressed as cpm//zg DNA calculated for all treated and control samples. UDSmut was induced by the alkylating agent 1-methyl-3-nitro-l-nitrosoguanidine (MNNG) with incubation of treated samples (100 tzmole/l MNNG) for 3 h at 37°C (Topinka et al., 1989b). All other procedures were the same as used for the determination of UDSox.
229 TABLE 1
o f m o l e c u l a r oxygen. As c a n be seen, P C a l o n e
EFFECT OF PARACETAMOL ON IN VITRO INDUCED (Fe2+ 20 ttmole/l + H202 1 mmole/l) OXIDATIVE DAMAGE OF BIOMEMBRANES (LPO) AND DNA (UDSox) AND DNA-REPAIR SYNTHESIS INDUCED BY 100 #mole/l MNNG (UDSmut) IN HUMAN PERIPHERAL LYMPHOCYTES
slightly i n d u c e d U D S , which is in a c c o r d a n c e with the d a t a o f D y b i n g et al. (1984). N o free-radical
Paracetamol concentration
Lipid peroxidation
Unscheduled DNA synthesis
(mmole/l)
C LPO (nmolea) T/C
C (cpm/
UDSox UDSmut T/C T/C
39.6 8.4 44.2 4.1 44.3 5.2 45.5 4.6 51.7 6.8 48.8 5.9 46.2 9.4 5
1.83 0.26 2.22 0.18 2.81" 0.27 2.67* 0.18 2.10 0.21 1.96 0.17 1.57 0.08 5
0 0.05 0.10 0.50 1.0 5.0 10.0 N
1.14 SD 0.11 1.15 SD 0.12 1.15 SD 0.09 1.10 SD 0.15 1.06 SD 0.10 1.11 SD 0.12 1.02 SD 0.16 8
3.78 0.23 6.39* 0.42 6.93* 0.43 4.91" 0.33 3.90 0.11 2.71' 0.11 2.30* 0.27 8
/zgb)
4.86 0.40 4.67 0.48 4.65 0.36 4.72 0.47 4.62 0.33 3.43* 0.15 2.86* 0.46 5
scavenging effect o f D M S O was observed in o u r experimental conditions. P C at c o n c e n t r a t i o n s lower t h a n 1 m m o l e / l significantly potentiates non-enzymatically (Fe 2 + + H202) i n d u c e d L P O with m a x i m u m activat i o n a r o u n d 0.1 mmole/1. I n contrast, P C at conc e n t r a t i o n s higher t h a n 1 m m o l e / 1 exhibits the inh i b i t o r y effect o n L P O (see Fig. 1). This fact leads to the a s s u m p t i o n that the P C - o x i d i z e d int e r m e d i a t e p h e n o x y radical at low c o n c e n t r a t i o n s participates i n the 1-electron t r a n s f e r in the redox system Fe 2 + / F e 3 ÷ in the u p w a r d direction, producing Fe 2 + ions, which take part i n the f o r m a t i o n o f free oxygen radicals ( M i n o t t i et al., 1987). W i t h increasing levels o f P C its a n t i o x i d a n t properties u n d e r o u r e x p e r i m e n t a l c o n d i t i o n s b e c o m e evident. The same effect o n in vitro experiments with h u m a n l y m p h o c y t e s has been observed with s o d i u m ascorbate ( T o p i n k a et al., 1989b). O u r results c o r r e s p o n d with the o b s e r v a t i o n s o f P C ' s i n h i b i t o r y effect on H202and t-butyl-
N, number of experiments (each in duplicate). a nmole MDA per mg of protein in the cell suspension. b cpm/t~g DNA. * P
227
Elsevier MUTLET0367
The effect of paracetamol on oxidative damage in human peripheral lymphocytes w
•
B. Binkovfi, J. Topinka and R.J. Sram Psychiatric Research Institute, Prague (Czechoslovakia)
(Accepted3 February1990)
Keywords: Lipidperoxidation;DNA repair; Free radicals;Paracetamol
Summary Unscheduled DNA synthesis (UDSox) and lipid peroxidation (LPO) induced by non-enzymatic activation of molecular oxygen (Fe2+ + H 2 0 2 ) were measured in human peripheral lymphocytes from healthy volunteers. The effect of paracetamol (PC) in a final concentration range of 0.05-10 mmole/l on these oxidative processes and on DNA repair induced by MNNG (UDSmut) was investigated. The level of induced LPO was measured by the thiobarbituric acid assay, UDSox and UDSmut were determined by scintillometric measurement of incorporated [methyl-3H]thymidine into damaged DNA. PC at concentrations lower than 1 mmole/l significantly potentiates the non-enzymatically induced LPO and UDSox with the maximum of the activation being around 0.1 mmole/l. In contrast, PC at concentrations higher than 1 mmole/l exhibits an inhibitory effect on both LPO and UDSox. On the other hand, concentrations higher than 1 mmole/l significantly suppressed DNA-repair synthesis induced by MNNG.
The molecular mechanisms causing the hepatotoxic effects of the widely used analgesic drug paracetamol (N-acetyl-p-aminophenol, PC) have been the subject of many investigations (Rosen et al., 1983; West et al., 1983; Koymans et Correspondence: Dr. B. Binkovtt, Psychiatric Research Institute, Ostavni91, 18103 Prague 8 (Czechoslovakia). Abbreviations: PC, paracetamol; LPO, lipid peroxidation;
UDS, unscheduledDNA synthesis;MNNG, 1-methyl-3-nitrol-nitrosoguanidine;DMSO, dimethylsulfoxide;TBA,thiobarbituric acid; T, treated samples; C, controls.
al., 1989). It was reported that oxidation of PC can lead to coproduction of reactive oxygen species (superoxide radical and hydrogen peroxide) through redox cycling between the PC semiiminoquinone radical and the PC metabolite Nacetyl-p-benzoquinone imine (NAPQI). Some investigators suggest that this oxidative stress rather than covalent binding of highly electrophilic NAPQI to bioactive cellular proteins is the cause of the cell injury (Wendel et al., 1982; Gerson et al., 1985). On the other hand it has been demonstrated that PC protects human erythrocytes against in
0165-7992/90/$ 03.50 © 1990ElsevierSciencePublishers B.V. (BiomedicalDivision)
228 vitro induced oxidative damage as an effective free radical scavenger (Van der Zee et al., 1988). In our laboratory the possible genotoxicity of PC was investigated in doses therapeutic for man in a group of healthy volunteers (Ko~igovfi et al., 1988; Topinka et al., 1989a). In these studies an increased frequency of chromosome aberrations and decreased unscheduled DNA synthesis (UDS) in peripheral lymphocytes, as well as an increased frequency of micronuclei in the buccal mucosa cells were observed. The present paper deals with the effect of PC on in vitro induced oxidative damage of cell membranes and genetic material as well as on the DNA-repair system in human peripheral lymphocytes.
Materials and methods
Chemicals Ficoll 400 (Pharmacia, Sweden), 60070 Verografin, paracetamol (Spofa, Czechoslovakia), MNNG, hydroxyurea, 5-fluoro-2'-deoxyuridine, sodium ascorbate (all Serva, F.R.G.), thiobarbituric acid and dimethyl sulfoxide (Merck, F.R.G.), 1,1,3,3-tetraethoxypropane (Sigma, U.S.A.), RPMI 1640 medium (USOL, Czechoslovakia), scintillation cocktail SLD 31 (Spolana, Czechoslovakia), all other chemicals had purity p.a.
Sample preparation The experiments were carried out on peripheral lymphocytes from the blood of healthy volunteers of both sexes with an average age of 43.1 years (SD 8.0). The lymphocytes were isolated on Ficoll 400-Verografin gradient (Harris et al., 1970). The protein content in the suspension of isolated cells was determined according to Bradford (1976). The samples from each donor were diluted into RPMI 1640 medium at a cell density of 0.2 mg of protein per ml and divided into several aliquots. In treated samples (T) molecular oxygen was activated by adding Fe 2 ÷ (20 #mole/l) and hydrogen peroxide (1 mmole/1). The effect of paracetamol on the oxidative processes induced in this way was investigated at a final concentration range of 0.05-10
mmole/l. PC dissolved in DMSO was added to the lymphocyte suspension prior to the oxygenactivating agents. The DMSO content was the same in all samples (2°70 of the total sample volume). Samples with the same concentration of PC but without any induction of molecular oxygen were used as a control (C). After incubation of all samples for 30 min at 37°C lipid peroxidation (LPO) and unscheduled DNA synthesis (UDSox) were determined.
Lipid peroxidation The LPO level in lymphocytes was determined by the modified thiobarbituric acid (TBA) assay (Ohkawa et al., 1976). TBA-active products of LPO were extracted into butanol and measured spectrophotometrically at 532 nm on a Shimadzu UV-2100 (high concentration) or fluorophotometrically with ~x = 515 nm and ~m = 546 nm on a Shimadzu RF 5000. The obtained values were expressed as nmole MDA per mg of protein using 1,1,3,3-tetraethoxypropane as a standard for all treated (T) and control (C) samples. The ratio T/C gives information about the in vitro induced LPO level in the suspension of lymphocytes under the given conditions.
Unscheduled DNA synthesis UDSox induced by non-enzymatic activation of molecular oxygen was determined by the scintillometric measurement of incorporated [methyl3H]thymidine. DNA from cells was isolated according to Martin et al. (1978). The incorporated radioactivity was measured on a Beckman LS 5801 liquid scintillation counter. The concentration of DNA was evaluated by the diphenylamine method on the spectrophotometer UV-2100 (Shimadzu). The specific activity of the samples was expressed as cpm//zg DNA calculated for all treated and control samples. UDSmut was induced by the alkylating agent 1-methyl-3-nitro-l-nitrosoguanidine (MNNG) with incubation of treated samples (100 tzmole/l MNNG) for 3 h at 37°C (Topinka et al., 1989b). All other procedures were the same as used for the determination of UDSox.
229 TABLE 1
o f m o l e c u l a r oxygen. As c a n be seen, P C a l o n e
EFFECT OF PARACETAMOL ON IN VITRO INDUCED (Fe2+ 20 ttmole/l + H202 1 mmole/l) OXIDATIVE DAMAGE OF BIOMEMBRANES (LPO) AND DNA (UDSox) AND DNA-REPAIR SYNTHESIS INDUCED BY 100 #mole/l MNNG (UDSmut) IN HUMAN PERIPHERAL LYMPHOCYTES
slightly i n d u c e d U D S , which is in a c c o r d a n c e with the d a t a o f D y b i n g et al. (1984). N o free-radical
Paracetamol concentration
Lipid peroxidation
Unscheduled DNA synthesis
(mmole/l)
C LPO (nmolea) T/C
C (cpm/
UDSox UDSmut T/C T/C
39.6 8.4 44.2 4.1 44.3 5.2 45.5 4.6 51.7 6.8 48.8 5.9 46.2 9.4 5
1.83 0.26 2.22 0.18 2.81" 0.27 2.67* 0.18 2.10 0.21 1.96 0.17 1.57 0.08 5
0 0.05 0.10 0.50 1.0 5.0 10.0 N
1.14 SD 0.11 1.15 SD 0.12 1.15 SD 0.09 1.10 SD 0.15 1.06 SD 0.10 1.11 SD 0.12 1.02 SD 0.16 8
3.78 0.23 6.39* 0.42 6.93* 0.43 4.91" 0.33 3.90 0.11 2.71' 0.11 2.30* 0.27 8
/zgb)
4.86 0.40 4.67 0.48 4.65 0.36 4.72 0.47 4.62 0.33 3.43* 0.15 2.86* 0.46 5
scavenging effect o f D M S O was observed in o u r experimental conditions. P C at c o n c e n t r a t i o n s lower t h a n 1 m m o l e / l significantly potentiates non-enzymatically (Fe 2 + + H202) i n d u c e d L P O with m a x i m u m activat i o n a r o u n d 0.1 mmole/1. I n contrast, P C at conc e n t r a t i o n s higher t h a n 1 m m o l e / 1 exhibits the inh i b i t o r y effect o n L P O (see Fig. 1). This fact leads to the a s s u m p t i o n that the P C - o x i d i z e d int e r m e d i a t e p h e n o x y radical at low c o n c e n t r a t i o n s participates i n the 1-electron t r a n s f e r in the redox system Fe 2 + / F e 3 ÷ in the u p w a r d direction, producing Fe 2 + ions, which take part i n the f o r m a t i o n o f free oxygen radicals ( M i n o t t i et al., 1987). W i t h increasing levels o f P C its a n t i o x i d a n t properties u n d e r o u r e x p e r i m e n t a l c o n d i t i o n s b e c o m e evident. The same effect o n in vitro experiments with h u m a n l y m p h o c y t e s has been observed with s o d i u m ascorbate ( T o p i n k a et al., 1989b). O u r results c o r r e s p o n d with the o b s e r v a t i o n s o f P C ' s i n h i b i t o r y effect on H202and t-butyl-
N, number of experiments (each in duplicate). a nmole MDA per mg of protein in the cell suspension. b cpm/t~g DNA. * P
