133
Mutation Research, 263 (1991) 133-136 © 1991 Elsevier Science Publishers B.V. 0165-7992/91/$ 03.50 ADONIS 0165799291000556 MUTLET 0501
DNA-repair capacity and lipid peroxidation in chronic alcoholics J. Topinka, B. Binkovfi, R.J. Srfim and I. Fojtikovfi Psychiatric Research Institute, Prague (Czechoslovakia) (Received 12 September 1990) (Revision received 31 January 1991) (Accepted 9 February 1991)
Keywords: Unscheduled DNA synthesis; Lipid peroxidation; Alcoholism;Antioxidants
Summary The possible impact of long-term overexposure to ethanol was studied in a group of chronic alcoholics in the psychiatric hospital. The level of DNA methylation and unscheduled DNA synthesis (UDS) induced by N-methyl-N-nitrosourea (MNU) in lymphocytes and lipid peroxidation (LPO) in plasma were used as markers of injury caused by alcohol abuse. The data were correlated with plasma levels of some natural antioxidants (vitamins A, C and E) and vitamin B12. The following results were obtained. The degree of DNA methylation by MNU in lymphocytes was the same in the exposed and control groups under our experimental conditions. The DNA excision-repair capacity of lymphocytes measured as UDS was decreased in alcoholics (p < 0.01) and LPO in plasma was significantly higher (p < 0.01) as a consequence of alcohol overconsumption. By the simple regression method, a correlation was found between LPO and vitamin C levels (LPO = - 0.078 x vit. C + 1.9; p < 0.05) and between UDS and LPO values (UDS -- - 0.384 x LPO + 4.1; p < 0.05). These results support the hypothesis of a connection of cell membrane status and DNA damage and repair and the possible role of active oxygen species in cell damage caused by ethanol.
The pathological consequences of alcohol overconsumption are related either to ethanol or to its metabolic products, acetaldehyde (AA) and acetate (Nuutinen et al., 1985). Studies on the metabolites of ethanol have mainly been focused on AA which seems to be responsible for the mutagenic properties o f ethanol. Chronic ethanol treatment results
Correspondence: Jan Topinka, Psychiatric Research Institute, Ostavni 91, 181 03 Prague 8 (Czechoslovakia).
in membrane changes, primarily in the lipid fraction, that serve to increase the rigidity of the membrane (Chin et al., 1978). These changes include increases in membrane cholesterol content (Johnson et al., 1979) and in the saturation of fatty acids (Waring et al., 1981); both would serve to decrease the fluidity of the lipid fraction of the membrane. Ethanol could disrupt the function of membranebound enzymes by acting directly on the enzymes themselves or by disrupting membrane lipidprotein interactions (Collins et al., 1984).
134
The genetic risk of chronic alcoholism and DNA damage may be related to the mutagenic and carcinogenic activity of AA. AA is highly reactive: its electrophilic properties cause it to react with nucleophilic groups of various macromolecules, like DNA. Its condensation with nucleic acid bases may be related to effects on DNA synthesis (adducts, cross-links) or chromosomal aberrations. AA has been shown to be a clastogen, inducing chromosome aberrations in human peripheral lymphocytes and other cells in vitro and in vivo, as well as sister-chromatid exchanges (Obe and Anderson, 1987). In our study, we analyzed unscheduled DNA synthesis (UDS) induced by N-methyl-N-nitrosourea (MNU) in peripheral lymphocytes of chronic alcoholics and plasma levels of lipid peroxidation ( L P O ) a n d some vitamins (A, C, E and B~2) in the same group.
SLibjec[s
Measurement of UDS DNA excision-repair capacity was estimated by measuring UDS induced in vitro by MNU in peripheral lymphocytes (Martin et al., 1978) of alcoholics and controls. Cells were preincubated with 10 mmole/1 hydroxyurea and 2 #mole/l 5-fluoro-2'-deoxyuridine to inhibit semiconservative DNA synthesis. All cell cultures were then divided into 3 aliquots. The first aliquot of cells from each donor was incubated with 400 kBq/ml of [Me-~H]thymidine (spec. act. 2 TBq/mmole) and 1 mmole/1 MNU in DMSO for 3 h at 37°C (treated cells, T). The second aliquot was incubated under the same conditions but without MNU (control cells, C) and the third aliquot (radioactively methylated, M) was treated with 40 kBq/ml [Me-3H]MNU (spec. act. 740 GBq/mmole). DNA from cells was isolated by the method described by Martin et al. (1978). The activity incorporated into the DNA was measured using a liquid scintillation counter LS 5801 (Beckman, U.S.A.) and the concentration of DNA was determined by the diphenylamine method using a UV-2100 spectrophotometer (Shimadzu, Japan) at 600 nm. The specific activity ot' samples was expressed as cpm/#g of DNA and calculated for all treated, control and radioactively methylated samples. The ratio T / C gives information about increased incorporation of the radiolabeled nucleoside [3H]thymidine as a consequence of DNA damage by MNU (excision repair). This ratio is called unscheduled DNA synthesis. Specific activity in M samples is dependent on the degree of DNA methylation by MNU and is therefore connected with the extent of DNA damage that must be repaired.
Subjects for study were chosen from inpatient male alcoholics undergoing therapy at the psychiatric hospital. They were aged 38.6_+9.9 (SD) years. Healthy volunteers aged 39.0_+9.8 (SD) years served as the control group. Lymphocytes were isolated on Ficoll 400-Verografin gradients (Harris, 1970). Cells were washed in phosphate-buffered saline (PBS, pH 7.2) and resuspended in RPMI 1640 medium for determination of UDS. Separated plasma was used for LPO and vitamin analysis.
Measurement of LPO The LPO level in plasma was measured by a modification of the thiobarbituric acid (TBA) assay (Ohkawa et al., 1979). The TBA assay involves acid hydrolysis of lipoperoxides to malondialdehyde (MDA), which subsequently reacts with TBA producing MDA-TBA adducts suitable for spectrophotometric measurement. The obtained values are expressed as nmole M D A / m l plasma using 1,1,3,3-tetraethoxypropane as a standard.
Materials and methods
Chemicals Ficoll 400 (Pharmacia, Sweden); 60% Verografin (Spofa, Czechoslovakia); MNU, hydroxyurea; 5-fluoro-2'-deoxyuridine; D,L-oetocopherol (all Serva, F.R.G.); 1,1,3,3-tetraethoxypropane; ascorbic acid (Sigma, U.S.A.); [Me-3H]thymidine (UVVVR, Czechoslovakia), [Me-3H]MNU (Amersham, U.K.); medium RPMI 1640 (USOL, Czechoslovakia); SLD 31 scintillation cocktail (Spolana, Czechoslovakia); all other chemicals had the minimal degree of purity necessary for analysis.
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Vitamin analysis Ascorbic acid was determined by a colorimetric method at 500 nm using its reaction with 2,4dinitro-phenylhydrazine, c~-Tocopherol (vitamin E) and retinol (vitamin A) in plasma were determined after extraction into n-heptane by H P L C (Driskell et al., 1982). The plasma level of vitamin Bt2 was estimated using competitive protein binding analysis with a RIA set (Laborchemie, G.D.R.). Results The results of the UDS determination and DNA methylation in lymphocytes and LPO in plasma of chronic alcoholics and controls are summarized in Table 1. The UDS values were significantly decreased (p
Mutation Research, 263 (1991) 133-136 © 1991 Elsevier Science Publishers B.V. 0165-7992/91/$ 03.50 ADONIS 0165799291000556 MUTLET 0501
DNA-repair capacity and lipid peroxidation in chronic alcoholics J. Topinka, B. Binkovfi, R.J. Srfim and I. Fojtikovfi Psychiatric Research Institute, Prague (Czechoslovakia) (Received 12 September 1990) (Revision received 31 January 1991) (Accepted 9 February 1991)
Keywords: Unscheduled DNA synthesis; Lipid peroxidation; Alcoholism;Antioxidants
Summary The possible impact of long-term overexposure to ethanol was studied in a group of chronic alcoholics in the psychiatric hospital. The level of DNA methylation and unscheduled DNA synthesis (UDS) induced by N-methyl-N-nitrosourea (MNU) in lymphocytes and lipid peroxidation (LPO) in plasma were used as markers of injury caused by alcohol abuse. The data were correlated with plasma levels of some natural antioxidants (vitamins A, C and E) and vitamin B12. The following results were obtained. The degree of DNA methylation by MNU in lymphocytes was the same in the exposed and control groups under our experimental conditions. The DNA excision-repair capacity of lymphocytes measured as UDS was decreased in alcoholics (p < 0.01) and LPO in plasma was significantly higher (p < 0.01) as a consequence of alcohol overconsumption. By the simple regression method, a correlation was found between LPO and vitamin C levels (LPO = - 0.078 x vit. C + 1.9; p < 0.05) and between UDS and LPO values (UDS -- - 0.384 x LPO + 4.1; p < 0.05). These results support the hypothesis of a connection of cell membrane status and DNA damage and repair and the possible role of active oxygen species in cell damage caused by ethanol.
The pathological consequences of alcohol overconsumption are related either to ethanol or to its metabolic products, acetaldehyde (AA) and acetate (Nuutinen et al., 1985). Studies on the metabolites of ethanol have mainly been focused on AA which seems to be responsible for the mutagenic properties o f ethanol. Chronic ethanol treatment results
Correspondence: Jan Topinka, Psychiatric Research Institute, Ostavni 91, 181 03 Prague 8 (Czechoslovakia).
in membrane changes, primarily in the lipid fraction, that serve to increase the rigidity of the membrane (Chin et al., 1978). These changes include increases in membrane cholesterol content (Johnson et al., 1979) and in the saturation of fatty acids (Waring et al., 1981); both would serve to decrease the fluidity of the lipid fraction of the membrane. Ethanol could disrupt the function of membranebound enzymes by acting directly on the enzymes themselves or by disrupting membrane lipidprotein interactions (Collins et al., 1984).
134
The genetic risk of chronic alcoholism and DNA damage may be related to the mutagenic and carcinogenic activity of AA. AA is highly reactive: its electrophilic properties cause it to react with nucleophilic groups of various macromolecules, like DNA. Its condensation with nucleic acid bases may be related to effects on DNA synthesis (adducts, cross-links) or chromosomal aberrations. AA has been shown to be a clastogen, inducing chromosome aberrations in human peripheral lymphocytes and other cells in vitro and in vivo, as well as sister-chromatid exchanges (Obe and Anderson, 1987). In our study, we analyzed unscheduled DNA synthesis (UDS) induced by N-methyl-N-nitrosourea (MNU) in peripheral lymphocytes of chronic alcoholics and plasma levels of lipid peroxidation ( L P O ) a n d some vitamins (A, C, E and B~2) in the same group.
SLibjec[s
Measurement of UDS DNA excision-repair capacity was estimated by measuring UDS induced in vitro by MNU in peripheral lymphocytes (Martin et al., 1978) of alcoholics and controls. Cells were preincubated with 10 mmole/1 hydroxyurea and 2 #mole/l 5-fluoro-2'-deoxyuridine to inhibit semiconservative DNA synthesis. All cell cultures were then divided into 3 aliquots. The first aliquot of cells from each donor was incubated with 400 kBq/ml of [Me-~H]thymidine (spec. act. 2 TBq/mmole) and 1 mmole/1 MNU in DMSO for 3 h at 37°C (treated cells, T). The second aliquot was incubated under the same conditions but without MNU (control cells, C) and the third aliquot (radioactively methylated, M) was treated with 40 kBq/ml [Me-3H]MNU (spec. act. 740 GBq/mmole). DNA from cells was isolated by the method described by Martin et al. (1978). The activity incorporated into the DNA was measured using a liquid scintillation counter LS 5801 (Beckman, U.S.A.) and the concentration of DNA was determined by the diphenylamine method using a UV-2100 spectrophotometer (Shimadzu, Japan) at 600 nm. The specific activity ot' samples was expressed as cpm/#g of DNA and calculated for all treated, control and radioactively methylated samples. The ratio T / C gives information about increased incorporation of the radiolabeled nucleoside [3H]thymidine as a consequence of DNA damage by MNU (excision repair). This ratio is called unscheduled DNA synthesis. Specific activity in M samples is dependent on the degree of DNA methylation by MNU and is therefore connected with the extent of DNA damage that must be repaired.
Subjects for study were chosen from inpatient male alcoholics undergoing therapy at the psychiatric hospital. They were aged 38.6_+9.9 (SD) years. Healthy volunteers aged 39.0_+9.8 (SD) years served as the control group. Lymphocytes were isolated on Ficoll 400-Verografin gradients (Harris, 1970). Cells were washed in phosphate-buffered saline (PBS, pH 7.2) and resuspended in RPMI 1640 medium for determination of UDS. Separated plasma was used for LPO and vitamin analysis.
Measurement of LPO The LPO level in plasma was measured by a modification of the thiobarbituric acid (TBA) assay (Ohkawa et al., 1979). The TBA assay involves acid hydrolysis of lipoperoxides to malondialdehyde (MDA), which subsequently reacts with TBA producing MDA-TBA adducts suitable for spectrophotometric measurement. The obtained values are expressed as nmole M D A / m l plasma using 1,1,3,3-tetraethoxypropane as a standard.
Materials and methods
Chemicals Ficoll 400 (Pharmacia, Sweden); 60% Verografin (Spofa, Czechoslovakia); MNU, hydroxyurea; 5-fluoro-2'-deoxyuridine; D,L-oetocopherol (all Serva, F.R.G.); 1,1,3,3-tetraethoxypropane; ascorbic acid (Sigma, U.S.A.); [Me-3H]thymidine (UVVVR, Czechoslovakia), [Me-3H]MNU (Amersham, U.K.); medium RPMI 1640 (USOL, Czechoslovakia); SLD 31 scintillation cocktail (Spolana, Czechoslovakia); all other chemicals had the minimal degree of purity necessary for analysis.
135
Vitamin analysis Ascorbic acid was determined by a colorimetric method at 500 nm using its reaction with 2,4dinitro-phenylhydrazine, c~-Tocopherol (vitamin E) and retinol (vitamin A) in plasma were determined after extraction into n-heptane by H P L C (Driskell et al., 1982). The plasma level of vitamin Bt2 was estimated using competitive protein binding analysis with a RIA set (Laborchemie, G.D.R.). Results The results of the UDS determination and DNA methylation in lymphocytes and LPO in plasma of chronic alcoholics and controls are summarized in Table 1. The UDS values were significantly decreased (p
