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Protection against hydroxyurea-induced cytotoxic effects in L5178Y cells by free radical scavengers
Cancer Letters, 17 (1982) 223-228 Elsevier Scientific Publishers Ireland Ltd.
223
PROTECTION AGAINST HYDROXYUREA-INDUCED CYTOTOXIC EFFECTS IN L5178Y CELLS BY FREE RADICAL SCAVENGERS
WALDEMAR M. PRZYBYSZEWSKI Department (Poland)
of Biochemistry,
Institute
and JANINA MALEC* of Haematology,
Chocimska
5, Warsaw 00-957
(Received 2 July 1982) (Revised version received 3 August 1982) (Accepted 25 August 1982)
SUMMARY
Exposure of L5178Y cells in culture to 1 mM hydroxyurea (HU) for 3 h followed by 24 h incubation in an HU-free medium induced an abnormal enlargement of about 40% of the cells in the population and post-treatment reduction of DNA synthesis in comparison with control cells. These effects were used to examine the protection afforded by free radical scavengers against HU-induced cytotoxicity. It has been found that with careful choice of conditions (suitable concentration of the protective agent, pretreatment of cells) substantial protective effect of a-tocopherol, sodium benzoate, acetylosalicylic acid, catalase, peroxidase or superoxide dismutase can be achieved.
INTRODUCTION
The pharmacologic actions of HU include the inhibition of the synthesis of DNA and rapid killing of cells in the S-phase of the cell cycle [ 91. Although inhibition of ribonucleotide reductase by HU could explain the inhibition of DNA synthesis, leading in consequence to a state of ‘unbalanced growth’ [6], some authors were hesitant to relate causally the inhibition of DNA synthesis to rapid cell death. To explain the mechanisms of rapid cell killing and teratogenic effects in embryos, De&so [ 71 hypothesized that HU exerts its rapid cytotoxic action through radical reactions initiated by the hydroxylamine group of HU. To form free radicals relatively few HU molecules are necessary while the majority of them are engaged in ribonucleotide inhibition. Amelioration of HU-induced teratogenesis in rabbits by the antioxidant propyl gallate [8] seems to be in line with this hypothesis. *To whom all correspondence
should be addressed.
0304-3835/82/0000-0000/$02.75 0 1982 Elsevier Scientific Publishers Ireland Ltd. - Published and Printed in Ireland.
224
Here, we attempted to protect L5178Y cells in culture against HUinduced rapid cytotoxicity through treatment with free radical scavengers. As an ‘index of protection’ 2 kinds of cytotoxic effects previously observed by us (unpublished data) were chosen: (1) abnormal enlargement of cells (‘giant’ cells formation) after short exposure to HU followed by 24 h incubation in an HU-free medium; (2) inhibition of net DNA synthesis in cells incubated in an HU-free medium after the end of HU treatment. a-Tocopherol, sodium benzoate, acetylosalicylic acid, cat&se, peroxidase or superoxide dismutase were used as the protecting agents. The extent of lipid peroxidation measured by malondialdehyde formation in HU-treated cells was also determined. MATERIALS
AND METHODS
The L5178Y cells were grown in culture as previously described [3]. HU, sodium benzoate and acetylosalicylic acid were dissolved in Eagle’s medium, cY-tocopherol acetate in absolute ethanol. All solutions were prepared just before use. Control cultures with corresponding concentration of absolute ethanol were simultaneously carried on. Cell number, cellular diameters, incorporation of [ 3H] thymidine and deoxy [ 14C] cytidine into DNA were measured (GoYos and Malec, unpublished data). DNA was estimated by the method of Burton [2]. In assessing lipid peroxidation, the thiobarbituric acid test [ 11 was used. Thiobarbituric acid was obtained from Calbiochem, [3H] thymidineand deoxy[ 14C] cytidine from UVVR (Prague). The remaining materials were of Polish production. RESULTS
AND DISCUSSION
Figures l-3 show that with a suitable choice of conditions substantial protective effect can be achieved by several free radical scavengers. The protection of HU-treated cells against post-treatment abnormal enlargement and reduction of DNA synthesis suggests that rapid cytotoxic effects of HU include DNA damaging action (Gores and Malec, unpublished) which is due to some mechanism unconnected with the inhibition of ribonucleotide reductase. To find further evidence for this suggestion we performed the test proposed by Painter [5] to detect agents that damage DNA. As shown in Fig. 4, the inhibition of DNA synthesis in the presence of HU is not affected by the presence of cY-tocopherol or catalase. Instead, the course of post-treatment recovery of DNA synthesis is different in cells treated with HU alone or with a protecting agent. According to Painter such a difference represents the discrimination between agents that damage DNA, and agents that inhibit DNA synthesis but do not damage DNA. The protection afforded by free radical scavengers supports the hypothesis on the role of free radical formation in HU-induced cytotoxic effects. The question arises as to what is the molecular mechanism of HU-induced free
225
Control v=l.oo
40
All v=1.30
Al2 v=1.03 i
30
B/2 , V=1.27
B/3 V=l.lS
Cl2 v=1.31
c/3 V=l.lS
20
10
0
C/l
kl.52
20
10
300 !!J 0
100
300
--
1 500
0
100
300
500
i
0
-100
‘)
300
!m
Fig. 1. Comparison of cell volume distribution and average cell volume (V) of cells treated for 3 h with 1 mM HU alone or HU with indicated radical scavenger then incubated for a further 24 h in an HU- and scavenger-free medium. Ordinate: number of cells (% of total); Abscissa: relative cell volume. (A) 1, HU; 2, HU with 0.060 mM cr-tocopherol (preincubation of cells in the medium with a-tocopherol, 60 min). (B) 1, HU; 2, HU with 4 mM sodium benzoate; 3, HU with 5 mM sodium benzoate. (C) 1, HU; 2, HU with 0.05 mM acetylosalicylic acid; 3, HU with 0.15 mM acetylosalicylic acid. Each point represents the mean of 2 experiments performed in duplicate.
226
40
013
D/2
D/l
V=1.06
v=l.o3
v=l.41
DI4 v=1.09
30
30
30
30
20
20
20
20
10
10
10
10
0:r,.L 0 100
0 3oo-a"o
100
300 O 0 100
300
0
loo
300
Fig. 2. Comparison of cell volume distribution and average cell volume (V) of cells treated for 3 h with 1 mM HU alone or HU with indicated enzyme (preincubation of cells in the medium with the enzyme, 20 min), then incubated for a further 24 h in an HU- and enzyme-free medium. Ordinate: number of cells (7%of total); Abscissa: relative cell volume. (D) 1, HU; 2, HU with 14 &ml catalase; 3, HU with 10 @g/ml peroxidase; 4, HU with 5 rg/ml superoxide dismutase. Each point represents the mean of 2 experiments performed in duplicate.
Fig. 3. Comparison of net DNA synthesis in cells treated with 1 mM HU alone or HU with indicated protecting agent, then incubated in an HU- and scavenger-free medium. Condition8 as for Figs. 1 and 2. The extent of DNA synthesis is expressed in percent of control. (A) 1, HU; 2, HU with 0.06 mM a-tocopherol. (B) 1, HU; 2, HU with 4 mM sodium benzoate; 3, HU with 5 mM sodium benxoate. (C) 1, HU, 2, HU with 0.05 mM acetylosalicylic acid; 3, HU with 0.15 mM acetylo8alicylic acid. (D) 1, HU; 2, HU with 14 fig/ml cataiaee; 3, HU with 10 rg/ml peroxidase; 4, HU with 5 pg/ml superoxide dismutase. Each value represents the mean of 2 experiments. Bar8 denote ranges.
227
cc-tot
cat.
Fig. 4. Comparison of the rate of DNA synthesis measured as percentage of the control incorporation of [‘H] thymidine into DNA at various times after the exposure of the cells to HU alone, HU with cat&se or HU with a-tocopherol. Ordinate: [“HI thymidine incorporation into DNA. Abscissa: time after the end of exposure (h). The cells prelabeled with deoxy[‘V]cytidine for about 2 generation times were treated with HU or HU with indicated protecting agent in conditions as for Fig. 1 or 2. After washing and resuspending the cells in an HU- and scavenger-free medium, the aliquots of cell suspensions were pulse-labeled for 30 min with [‘Hlthymidine (0.01 rCi/ml) at time 0 and 0.5, 1.5 or 2.5 h after the end of treatment. Afterwards the cells were collected and ‘H/l% estimation carried out. Each point represents the mean of 2 experiments Bars denote ranges.
radical action in the cell. kee radicals are known DNAdamaging agents, considered to be mimetic chemicals of ionizing radiation [4]. On the other hand, it is well known that pathologic radical reactions can initiate lipid peroxidation in cell membranes. Thus, we determined the extent of lipid peroxidation in cells treated with 1 mM HU for 3 h in the presence or absence of several protecting scavengers. The data obtained revealed no difference between control and HU-treated non-protected and protected cells. In conclusion, we think that the results of this study provide some evidence on HU-induced free radical formation and their cytotoxic role in HUtreated cells. Although the molecular mechanism of such effects remains to be elucidated we feel that some experiments in vivo should be done employing radical scavengers against HU-induced cytotoxic side effects.
ACKNOWLEDGEMENTS
The authors thank Dr. T. Wronowski, Department of Radiobiology and Health Protection, Institute of Nuclear Research in Warsaw, for supplying tumor bearing DBA/2 mice. This work was supported by a grant of the Polish National Cancer Programme PR-6 (contract 2501).
228 REFERENCES 1 Asakawa, T. and Matsushita, S. (1980) Coloring conditions of thiobarbituric acid test for detecting lipid hydroperoxides. Lipids, 15,137-140. 2 Burton, K. (1956) A study of the conditions and mechanism of the diphenylamine reaction for the calorimetric estimation of the deoxyribonucleic acid. B&hem. J., 62.315-323. 3 Kornacka, L., Sawecka, J. and Malec, J. (1977) Comparison of hydroxyurea and excess thymidine as synchronizing agents in cultures of L5178Y mouse lymphoma cells. Folia Biol. (Praha), 23,299-304. 4 Meneghini, R. and Hoffmann, M.E. (1980) The damaging action of hydrogen peroxide on DNA of human fibroblasts is mediated by a nondialyzable compound. Biochim. Biophys. Acta, 608,167173. 5 Painter, R.B. (1977) Rapid test to detect agents that damage human DNA. Nature, 265,650-651. 6 Ross, D.W. (1981) The nature of unbalanced cell growth caused by cytotoxic agents Virchows Arch. B., 37,225-235. 7 DeSesso, J.M. (1979) Cell death and free radicals: A mechanism for hydroxyurea teratogenesis. Med. Hypotheses, 5, 937-951. 8 DeSesso, J.M. (1981) Amelioration of teratogenesis. I. Modification of hydroxyureainduced teratogenesis by the antioxidant propyl gallate. Teratology, 24,19-35. 9 Timson, J. (1975) Hydroxyurea. Mutat. Res., 32,115-132.
223
PROTECTION AGAINST HYDROXYUREA-INDUCED CYTOTOXIC EFFECTS IN L5178Y CELLS BY FREE RADICAL SCAVENGERS
WALDEMAR M. PRZYBYSZEWSKI Department (Poland)
of Biochemistry,
Institute
and JANINA MALEC* of Haematology,
Chocimska
5, Warsaw 00-957
(Received 2 July 1982) (Revised version received 3 August 1982) (Accepted 25 August 1982)
SUMMARY
Exposure of L5178Y cells in culture to 1 mM hydroxyurea (HU) for 3 h followed by 24 h incubation in an HU-free medium induced an abnormal enlargement of about 40% of the cells in the population and post-treatment reduction of DNA synthesis in comparison with control cells. These effects were used to examine the protection afforded by free radical scavengers against HU-induced cytotoxicity. It has been found that with careful choice of conditions (suitable concentration of the protective agent, pretreatment of cells) substantial protective effect of a-tocopherol, sodium benzoate, acetylosalicylic acid, catalase, peroxidase or superoxide dismutase can be achieved.
INTRODUCTION
The pharmacologic actions of HU include the inhibition of the synthesis of DNA and rapid killing of cells in the S-phase of the cell cycle [ 91. Although inhibition of ribonucleotide reductase by HU could explain the inhibition of DNA synthesis, leading in consequence to a state of ‘unbalanced growth’ [6], some authors were hesitant to relate causally the inhibition of DNA synthesis to rapid cell death. To explain the mechanisms of rapid cell killing and teratogenic effects in embryos, De&so [ 71 hypothesized that HU exerts its rapid cytotoxic action through radical reactions initiated by the hydroxylamine group of HU. To form free radicals relatively few HU molecules are necessary while the majority of them are engaged in ribonucleotide inhibition. Amelioration of HU-induced teratogenesis in rabbits by the antioxidant propyl gallate [8] seems to be in line with this hypothesis. *To whom all correspondence
should be addressed.
0304-3835/82/0000-0000/$02.75 0 1982 Elsevier Scientific Publishers Ireland Ltd. - Published and Printed in Ireland.
224
Here, we attempted to protect L5178Y cells in culture against HUinduced rapid cytotoxicity through treatment with free radical scavengers. As an ‘index of protection’ 2 kinds of cytotoxic effects previously observed by us (unpublished data) were chosen: (1) abnormal enlargement of cells (‘giant’ cells formation) after short exposure to HU followed by 24 h incubation in an HU-free medium; (2) inhibition of net DNA synthesis in cells incubated in an HU-free medium after the end of HU treatment. a-Tocopherol, sodium benzoate, acetylosalicylic acid, cat&se, peroxidase or superoxide dismutase were used as the protecting agents. The extent of lipid peroxidation measured by malondialdehyde formation in HU-treated cells was also determined. MATERIALS
AND METHODS
The L5178Y cells were grown in culture as previously described [3]. HU, sodium benzoate and acetylosalicylic acid were dissolved in Eagle’s medium, cY-tocopherol acetate in absolute ethanol. All solutions were prepared just before use. Control cultures with corresponding concentration of absolute ethanol were simultaneously carried on. Cell number, cellular diameters, incorporation of [ 3H] thymidine and deoxy [ 14C] cytidine into DNA were measured (GoYos and Malec, unpublished data). DNA was estimated by the method of Burton [2]. In assessing lipid peroxidation, the thiobarbituric acid test [ 11 was used. Thiobarbituric acid was obtained from Calbiochem, [3H] thymidineand deoxy[ 14C] cytidine from UVVR (Prague). The remaining materials were of Polish production. RESULTS
AND DISCUSSION
Figures l-3 show that with a suitable choice of conditions substantial protective effect can be achieved by several free radical scavengers. The protection of HU-treated cells against post-treatment abnormal enlargement and reduction of DNA synthesis suggests that rapid cytotoxic effects of HU include DNA damaging action (Gores and Malec, unpublished) which is due to some mechanism unconnected with the inhibition of ribonucleotide reductase. To find further evidence for this suggestion we performed the test proposed by Painter [5] to detect agents that damage DNA. As shown in Fig. 4, the inhibition of DNA synthesis in the presence of HU is not affected by the presence of cY-tocopherol or catalase. Instead, the course of post-treatment recovery of DNA synthesis is different in cells treated with HU alone or with a protecting agent. According to Painter such a difference represents the discrimination between agents that damage DNA, and agents that inhibit DNA synthesis but do not damage DNA. The protection afforded by free radical scavengers supports the hypothesis on the role of free radical formation in HU-induced cytotoxic effects. The question arises as to what is the molecular mechanism of HU-induced free
225
Control v=l.oo
40
All v=1.30
Al2 v=1.03 i
30
B/2 , V=1.27
B/3 V=l.lS
Cl2 v=1.31
c/3 V=l.lS
20
10
0
C/l
kl.52
20
10
300 !!J 0
100
300
--
1 500
0
100
300
500
i
0
-100
‘)
300
!m
Fig. 1. Comparison of cell volume distribution and average cell volume (V) of cells treated for 3 h with 1 mM HU alone or HU with indicated radical scavenger then incubated for a further 24 h in an HU- and scavenger-free medium. Ordinate: number of cells (% of total); Abscissa: relative cell volume. (A) 1, HU; 2, HU with 0.060 mM cr-tocopherol (preincubation of cells in the medium with a-tocopherol, 60 min). (B) 1, HU; 2, HU with 4 mM sodium benzoate; 3, HU with 5 mM sodium benzoate. (C) 1, HU; 2, HU with 0.05 mM acetylosalicylic acid; 3, HU with 0.15 mM acetylosalicylic acid. Each point represents the mean of 2 experiments performed in duplicate.
226
40
013
D/2
D/l
V=1.06
v=l.o3
v=l.41
DI4 v=1.09
30
30
30
30
20
20
20
20
10
10
10
10
0:r,.L 0 100
0 3oo-a"o
100
300 O 0 100
300
0
loo
300
Fig. 2. Comparison of cell volume distribution and average cell volume (V) of cells treated for 3 h with 1 mM HU alone or HU with indicated enzyme (preincubation of cells in the medium with the enzyme, 20 min), then incubated for a further 24 h in an HU- and enzyme-free medium. Ordinate: number of cells (7%of total); Abscissa: relative cell volume. (D) 1, HU; 2, HU with 14 &ml catalase; 3, HU with 10 @g/ml peroxidase; 4, HU with 5 rg/ml superoxide dismutase. Each point represents the mean of 2 experiments performed in duplicate.
Fig. 3. Comparison of net DNA synthesis in cells treated with 1 mM HU alone or HU with indicated protecting agent, then incubated in an HU- and scavenger-free medium. Condition8 as for Figs. 1 and 2. The extent of DNA synthesis is expressed in percent of control. (A) 1, HU; 2, HU with 0.06 mM a-tocopherol. (B) 1, HU; 2, HU with 4 mM sodium benzoate; 3, HU with 5 mM sodium benxoate. (C) 1, HU, 2, HU with 0.05 mM acetylosalicylic acid; 3, HU with 0.15 mM acetylo8alicylic acid. (D) 1, HU; 2, HU with 14 fig/ml cataiaee; 3, HU with 10 rg/ml peroxidase; 4, HU with 5 pg/ml superoxide dismutase. Each value represents the mean of 2 experiments. Bar8 denote ranges.
227
cc-tot
cat.
Fig. 4. Comparison of the rate of DNA synthesis measured as percentage of the control incorporation of [‘H] thymidine into DNA at various times after the exposure of the cells to HU alone, HU with cat&se or HU with a-tocopherol. Ordinate: [“HI thymidine incorporation into DNA. Abscissa: time after the end of exposure (h). The cells prelabeled with deoxy[‘V]cytidine for about 2 generation times were treated with HU or HU with indicated protecting agent in conditions as for Fig. 1 or 2. After washing and resuspending the cells in an HU- and scavenger-free medium, the aliquots of cell suspensions were pulse-labeled for 30 min with [‘Hlthymidine (0.01 rCi/ml) at time 0 and 0.5, 1.5 or 2.5 h after the end of treatment. Afterwards the cells were collected and ‘H/l% estimation carried out. Each point represents the mean of 2 experiments Bars denote ranges.
radical action in the cell. kee radicals are known DNAdamaging agents, considered to be mimetic chemicals of ionizing radiation [4]. On the other hand, it is well known that pathologic radical reactions can initiate lipid peroxidation in cell membranes. Thus, we determined the extent of lipid peroxidation in cells treated with 1 mM HU for 3 h in the presence or absence of several protecting scavengers. The data obtained revealed no difference between control and HU-treated non-protected and protected cells. In conclusion, we think that the results of this study provide some evidence on HU-induced free radical formation and their cytotoxic role in HUtreated cells. Although the molecular mechanism of such effects remains to be elucidated we feel that some experiments in vivo should be done employing radical scavengers against HU-induced cytotoxic side effects.
ACKNOWLEDGEMENTS
The authors thank Dr. T. Wronowski, Department of Radiobiology and Health Protection, Institute of Nuclear Research in Warsaw, for supplying tumor bearing DBA/2 mice. This work was supported by a grant of the Polish National Cancer Programme PR-6 (contract 2501).
228 REFERENCES 1 Asakawa, T. and Matsushita, S. (1980) Coloring conditions of thiobarbituric acid test for detecting lipid hydroperoxides. Lipids, 15,137-140. 2 Burton, K. (1956) A study of the conditions and mechanism of the diphenylamine reaction for the calorimetric estimation of the deoxyribonucleic acid. B&hem. J., 62.315-323. 3 Kornacka, L., Sawecka, J. and Malec, J. (1977) Comparison of hydroxyurea and excess thymidine as synchronizing agents in cultures of L5178Y mouse lymphoma cells. Folia Biol. (Praha), 23,299-304. 4 Meneghini, R. and Hoffmann, M.E. (1980) The damaging action of hydrogen peroxide on DNA of human fibroblasts is mediated by a nondialyzable compound. Biochim. Biophys. Acta, 608,167173. 5 Painter, R.B. (1977) Rapid test to detect agents that damage human DNA. Nature, 265,650-651. 6 Ross, D.W. (1981) The nature of unbalanced cell growth caused by cytotoxic agents Virchows Arch. B., 37,225-235. 7 DeSesso, J.M. (1979) Cell death and free radicals: A mechanism for hydroxyurea teratogenesis. Med. Hypotheses, 5, 937-951. 8 DeSesso, J.M. (1981) Amelioration of teratogenesis. I. Modification of hydroxyureainduced teratogenesis by the antioxidant propyl gallate. Teratology, 24,19-35. 9 Timson, J. (1975) Hydroxyurea. Mutat. Res., 32,115-132.