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Induction of DNA strand breaks by nitrosocimetidine
Cancer Letters, 10 (1980) 223-228 o Elsevier/North-Holland Scientific Publishers Ltd.
INDUCTION
OF DNA STRAND
223
BREAKS BY NITROSOCIMETIDINE
M. SCHWARZO, J. HUMMELa and G. EISENBRANDb ‘Institute Research
of Biochemistry, bZnstitute of Toxicology Center, Heidelberg (F.R. G.)
and Chemothempy,
German Cancer
(Received 25 April 1980) (Revised version received 13 June 1980) (Accepted 13 June 1980)
SUMMARY
The DNA of a transformed epithelial mouse cell line was studied by means of the alkaline filter elution test. Addition of N-nitrosocimetidine (NC) to the cells in vitro resulted in a concentration-dependent increase in DNA damage. In contrast cimetidine itself had no effect. The capacity of NC to generate DNA strand breaks was found to be smaller by comparison with the potent gastric carcinogen N-methyl-N’-nitro-N-nitrosoguanidine (MNNG).
INTRODUCTION
The carcinogenic potential of N-nitroso compounds is well recognised [3]. N-nitroso compounds are widely distributed in the environment, and have been detected in foodstuffs, cosmetics and drugs. It is also established that they can be formed in the gastrointestinal tract under acidic conditions from nitrosatable amines and nitrite [IO]. Cimetidine, a drug which is used in high doses over long periods of time in the therapy of stomach and duodenal ulcers, is suspected to be nitrosated in vivo. The resulting Nnitrosoderivative might possibly act as a carcinogen and induce gastric cancer in man [4,5]. Chemical carcinogenesis is believed to be initiated by the interaction of a carcinogen or an active metabolite with target cell DNA. In principle measurement of DNA alterations may be used to detect chemical carcinogens or mutagens. DNA damage induced in vivo or in vitro by chemical carcinogens has been demonstrated by means of alkaline sucrose gradient Address all correspondence to: Michael Schwarz, Institute of Biochemistry, German Cancer Research Center, Im Neuenheimer Feld 280, D-6900 Heidelberg, F.R.G. Abbreviations: MNNG, N-methyl-N’-nitro-N-nitrosoguanidine; NC, nitrosocimetidine.
224
sedimentation analysis of DNA [2] and by the alkaline filter elution test [ 12,17,18]. These 2 techniques where shown to produce very similar results with a variety of carcinogenic agents. For our experiments, we have used the filter elution test, which requires less complicated analytical procedures. In this paper we report the effect on alkaline elution characteristics of DNA, when NC, cimetidine and MNNG were incubated in vitro with transformed epithelial cells. MATERIALS
AND METHODS
Chemicals
Cimetidine was a gift from Smith Kline & French Laboratories Ltd., Welwyn Garden City, Hertfordshire, U.K. NC was prepared by nitrosation of cimetidine with NaNO, in hydrochloric acid following a standard procedure [3,6,13]. The compound (m.p. lo&110°C) was homogenous by thin-layer chromatography (solvent dichloromethane/methanol, 9 + l), and corresponded in its spectral properties (UV, NMR, MS) with the pastulated structure; details will be reported elsewhere (Eisenbrand, G., in preparation). MNNG was obtained from Serva, Heidelberg, F.R.G. Tetraethylammonium hydroxide was purchased from Merck-Schuchardt, Miinchen, F.R.G.; polyvinyl-filters (BSWP 02500) were obtained from the Millipore Corporation, Bedford, MA, U.S.A. [Methyl-3H]thymidine (45 Ci/mmol) and [ 214C] thymidine (56 Ci/mol) were obtained from Amersham Buchler, Braunschweig, F.R.G. Cell culture
and treatment
DNA of a mouse epidermal cell line, transformed in vitro by dimethylbenz[a]anthracene [7], was labelled by the addition of [methyl-3H]thymidine (0.2 pCi/ml) or [2-14C]thymidine (0.1 r_lCi/ml).Twenty-four hours later the cells were incubated for a further 2 h without serum, and then used. Cimetidine and NC were dissolved in dilute HCl, and required amounts of the solutions (approx. pH 6) were added to the cultures. The pH was kept constant by the addition of dilute NaOH. MNNG was dissolved in acetone; up to 20 ~1 (0.4% acetone) of this solution were used for the experiments. Acetone alone (0.4%) was found to have no effect. Filter elution
The alkaline filter elution test was carried out essentially according to the method of Kohn and Grimek-Ewig [ 71, and Kohn et al. [ 81, with certain modifications. Controls and treated cells, the DNA of which had been prelabelled with either [ 14C]thymidine or [ 3H] thymidine, were mixed to give a final suspension of about 10’ cells/ml. Cells were lysed on top of the filters in 2 ml 2 M NaCl (pH 8.2), containing 0.2% Triton X-100 and 0.02 M EDTA and the lysate was passed through the filter at a flow rate
225
of 0.2 ml/min. The filters were further eluted with 4 ml of the lysis solution, after which the medium was washed out with 5 ml 1 mM EDTA (pH 7.8). The flow rate was then changed to 0.1 ml/min, and 13 ml of 0.02 M EDTA solution adjusted to pH 12.2-12.3 with tetraethylammonium hydroxide were added. Fractions were collected at lo-min intervals, and the radioactivity therein was determined by liquid scintillation counting after addition of 4 ml Aqua Luma (Baker, Gross-Gerau, F.R.G.). DNA remaining on the filters was hydrolysed in 0.4 ml 1 N HCl at 70°C for 15 min; 0.6 ml 1 N NaOH was added, and the radioactivity was measured in 10 ml of the same scintillation mixture as mentioned above. There was no loss of radioactivity from the filters before starting the elution with EDTA/tetraethylammonium hydroxide solution. RESULTS
About 90% of control DNA from untreated cells remained on the filters under the experimental conditions used (Fig. 1). There was no isotope effect when the cells were prelabelled with either [ 3H]- or [ 14C]thymidine prior to elution. Addition of NC to the cultures resulted in increases in alkaline elution which depended on the concentration of the compound in the medium (Fig. la). A further increase in elution was observed when NC was allowed to remain in contact with the cells for 1 h instead of 30 min (data not shown). In contrast, cimetidine itself failed to alter the elution characteristics of DNA at the highest concentration (5 mM) tested. Control IO- _ *.~0~000000~00 . - .
Control ‘PPy400000000
Cn-netldme 15d4
AA
_.
.
. E z
.
.
-'A
0.5-
z !Yl
9 “. . . .
rY
,
i=
AA IA..
E
01
.
.
.
.
. .
. .
.
. .
.
3.0mt-i :
.
0.1d-3
l0mt-i .
6
.
.
. '
A
B
2 L 6 8 10 12 I-'..
2
ALKALINE
l.=mmm..m 03 n9-t c
L
6 8 10 12 1L
ELUTION (ml1
Fig. 1, Effect of various concentrations of cimetidine, NC and MNNG on the alkaline elution characteristics of epidermal cell DNA. Cells were labelled with [3H]thymidine for 24 h, and incubated for 30 min with the respective compounds. As an internal standard, aliquots of untreated controls which had been prelabelled with [“Clthymidine were added to each assay. Lysis of the cells and alkaline elution were carried out as described in the text. A, NC; B, MNNG.
226
J 0.01
0.05
0,l
0,5
1.0
CONCENTRATION
5.0 (mM)
Fig. 2. Dependence of the apparent initial rate of elution on MNNG and NC concentration. Data were calculated from experiments similar to those shown in Fig. 1.
A concentration-dependent increase in DNA elution was also obtained when MNNG was added to the cultures instead of NC (Fig. lb). In order to compare quantitatively the DNA damage produced by MNNG with that of NC a common parameter of efficacy was chosen. Assuming first order kinetics for the initial rate of elution [1,8], the apparent initial elution constants were determined as functions of concentration. Concentrationdependent increases in the values of these constants were observed both for NC and for MNNG (Fig. 2). However, at a give concentration of NC the extent of DNA breakage was about one-tenth of that induced by an equimolar amount of MNNG. DISCUSSION
The role of NC as a potential carcinogen producing gastric cancer is at present under discussion [4,5,11,14,15,16,19,20]. The methylnitrosoguanidine moiety of NC resembles that of MNNG, a well known mutagen and agent causing tumours of the gastrointestinal tract.
I
I H3C
k
H2C \,/H2C
\
H
2
C/N\C/N\CH II
3
N02
II
‘i’ CN Nitrosobimetidine
’
‘C/
‘CH II N
I H
N-Methyl-N’-nitro-N-nitrosoguanidine
Both NC and MNNG effectively generate DNA strand breakage upon
3
227
in vitro incubation with epidermal cells (Figs. 1, 2). MNNG, however, was found to be more efficient when compared on an equimolar basis. This result contrasts with data obtained in a chemical in vitro system using 3,4-dichlorobenzenethiol as a nucleophilic trapping agent, towards which the methylating abilities of NC and MNNG were about the same [ 61. However, since thiol groups are known to induce strongly the decomposition of nitrosoguanidines, [ 91, alkyhtion data obtained from chemical model reactions with high concentrations of thiols might not entirely reflect the situation in whole cells. It might also be that differences in lipophilicity between MNNG (partition coefficient (HzO/octanol), KD = 0.75) and NC (KD = 4) might account at least in part for the observed differences. NC has been found to be mutagenic in microbial mutagenicity test systems [ 131. The present finding that NC is also able to produce DNA damage in vitro in eucaryotic cells of epithelial origin further underlines the possible hazard associated with this compound. Since cimetidine itself gave negative results in the alkaline elution test, the question as to whether cimetidine is nitrosated in the human gastrointestinal tract or not becomes important. This matter is currently under investigation in an appropriate animal system. ACKNOWLEDGEMENTS
The authors gratefully acknowledge Professor Dr. N. Fusenig for the gift of cultured PDV cells and Dr. G.R.N. Jones for textual corrections of the manuscript. REFERENCES 1 Brambilla, G., Cavanna, M., Parodi, S., Sciaba, L., Pino, A. and Robbiano, L. (1978) DNA damage in liver, colon, stomach, lung and kidney of Balb/c mice treated with 1,2-dimethylhydraxine. Int. J. Cancer, 22, 174-180. 2 Cox, R., Damjanov, J., Abanobi, S.E. and Sarma, D.S.R. (1973) A method for measuring DNA damage and repair in the liver in do. Cancer Res., 33, 2114-2121. 3 Druckrey, H., Preussmann, R., Ivankovic, S. and Schmiihl, D. (1967) Organotrope carcinogene Wirkungen bei 65 verschiedenen N-Nitroso-Verbindungen an BD Ratten. Z. Krebsforsch., 69, 103-201. 4 Elder, J.B., Ganguli, P.C. and Gillespie, J.E. (1979) Cimetidine and gastric cancer. Lancet, 1, 1005-1006. 5 Elder, J.B., Ganguli, P.C. and Gillespie, J.E. (1979) Gastric cancer in patients who have taken cimetidine. Lancet, 2, 245. 6 Foster, A.B., Jarman, M., Manson, D., Schulten, H.R. (1980) Structure and reactivity of nitrosocimetidine. Cancer Letters, 9, 47-52. 7 Fusenig, N.E., Samsel, W., Thon, W. and Worst, P.K.M. (1973) Malignant transformation of epidermal cells in culture by DMBA. Inserm, 19, 219-228. 8 Kohn, K.W. and Grimek-Ewig, R.A. (1973) Alkaline elution analysis, a new approach to the study of DNA single-strand interruptions in cells. Cancer Res., 33, 1849-1853. 9 Kohn, K.W., Erickson, L.C., Ewig, R.A. and Friedman, C.A. (1976) Fractionation of DNA from mammalian cells by alkaline elution. Biochemistry, 15, 4629-4637. 10 Lawley, P.D. and Thatcher, C.J. (1970) Methylation of deoxyribonucleic acid in
228
11 12 13 14 15 16 17 18
19
20 21
cultured mammalian cells by N-methyl-N’nitro-N-nitrosoguanidine. Biochem. J., 116,693-707. Mirvish, S.S. (1975) Formation of N-nitroso compounds. Chemistry, kinetics and in uiuo occurrence. Toxicol. Appl. Pharmacol., 31, 325-351. Mullen, P.W. (1979) Gastric cancer in patients who have taken cimetidine. Lancet, 2, 1406. Petzold, G.L. and Swenberg, I.A. (1978) Detection of DNA damage induced in uiuo following exposure of rats to carcinogens. Cancer Res., 38, 1589-1594. Pool, B.L. Eixenbrand, G. and Schmiihl, D. (1979) Biological activity of nitrosated cimetidine. Toxicology, 15, 69-7 2. Reed, P.I. Casell, P.G. and Walters, C.L. (1979) Gastric cancer in patients who have taken cimetidine. Lancet, 1.1235. Roe, F.J.C. (1979) Cimetidine and gastric cancer. Lancet, 1, 1039. Ruddell, W.S.J. (1979) Gastric cancer in patients who have taken cimetidine. Lancet, 1, 1234. Schwarz, M., Hummel, J., Appel, K.E., Rickart, R. and Kunz, W. (1979) DNA damage induced in vivo evaluated with a non-radioactive alkaline elution technique. Cancer letters, 6, 221-226. Swenberg, J.A., Petzold, G.L. and Harbach, P.R. (1976) In vitro DNA damagelalkaline elution assay for predicting carcinogenic potential. Biophys Biochem. Res. Commun., 72,732-738. Taylor, R.H., Lovell, D., Menzies-Gow, N., La Brooy, S.J. and Misiewicz, J.J. (1978) Misleading response of malignant gastric ulcers to cimetidine. Lancet, 1, 686-688. Taylor, T.V., Lee, D., Howatson, A.G., Anderson, J. and MacLeod, J.B. (1979) Gastric cancer in patients who have been taken cimetidine. Lancet, 1, 1235-1236.
INDUCTION
OF DNA STRAND
223
BREAKS BY NITROSOCIMETIDINE
M. SCHWARZO, J. HUMMELa and G. EISENBRANDb ‘Institute Research
of Biochemistry, bZnstitute of Toxicology Center, Heidelberg (F.R. G.)
and Chemothempy,
German Cancer
(Received 25 April 1980) (Revised version received 13 June 1980) (Accepted 13 June 1980)
SUMMARY
The DNA of a transformed epithelial mouse cell line was studied by means of the alkaline filter elution test. Addition of N-nitrosocimetidine (NC) to the cells in vitro resulted in a concentration-dependent increase in DNA damage. In contrast cimetidine itself had no effect. The capacity of NC to generate DNA strand breaks was found to be smaller by comparison with the potent gastric carcinogen N-methyl-N’-nitro-N-nitrosoguanidine (MNNG).
INTRODUCTION
The carcinogenic potential of N-nitroso compounds is well recognised [3]. N-nitroso compounds are widely distributed in the environment, and have been detected in foodstuffs, cosmetics and drugs. It is also established that they can be formed in the gastrointestinal tract under acidic conditions from nitrosatable amines and nitrite [IO]. Cimetidine, a drug which is used in high doses over long periods of time in the therapy of stomach and duodenal ulcers, is suspected to be nitrosated in vivo. The resulting Nnitrosoderivative might possibly act as a carcinogen and induce gastric cancer in man [4,5]. Chemical carcinogenesis is believed to be initiated by the interaction of a carcinogen or an active metabolite with target cell DNA. In principle measurement of DNA alterations may be used to detect chemical carcinogens or mutagens. DNA damage induced in vivo or in vitro by chemical carcinogens has been demonstrated by means of alkaline sucrose gradient Address all correspondence to: Michael Schwarz, Institute of Biochemistry, German Cancer Research Center, Im Neuenheimer Feld 280, D-6900 Heidelberg, F.R.G. Abbreviations: MNNG, N-methyl-N’-nitro-N-nitrosoguanidine; NC, nitrosocimetidine.
224
sedimentation analysis of DNA [2] and by the alkaline filter elution test [ 12,17,18]. These 2 techniques where shown to produce very similar results with a variety of carcinogenic agents. For our experiments, we have used the filter elution test, which requires less complicated analytical procedures. In this paper we report the effect on alkaline elution characteristics of DNA, when NC, cimetidine and MNNG were incubated in vitro with transformed epithelial cells. MATERIALS
AND METHODS
Chemicals
Cimetidine was a gift from Smith Kline & French Laboratories Ltd., Welwyn Garden City, Hertfordshire, U.K. NC was prepared by nitrosation of cimetidine with NaNO, in hydrochloric acid following a standard procedure [3,6,13]. The compound (m.p. lo&110°C) was homogenous by thin-layer chromatography (solvent dichloromethane/methanol, 9 + l), and corresponded in its spectral properties (UV, NMR, MS) with the pastulated structure; details will be reported elsewhere (Eisenbrand, G., in preparation). MNNG was obtained from Serva, Heidelberg, F.R.G. Tetraethylammonium hydroxide was purchased from Merck-Schuchardt, Miinchen, F.R.G.; polyvinyl-filters (BSWP 02500) were obtained from the Millipore Corporation, Bedford, MA, U.S.A. [Methyl-3H]thymidine (45 Ci/mmol) and [ 214C] thymidine (56 Ci/mol) were obtained from Amersham Buchler, Braunschweig, F.R.G. Cell culture
and treatment
DNA of a mouse epidermal cell line, transformed in vitro by dimethylbenz[a]anthracene [7], was labelled by the addition of [methyl-3H]thymidine (0.2 pCi/ml) or [2-14C]thymidine (0.1 r_lCi/ml).Twenty-four hours later the cells were incubated for a further 2 h without serum, and then used. Cimetidine and NC were dissolved in dilute HCl, and required amounts of the solutions (approx. pH 6) were added to the cultures. The pH was kept constant by the addition of dilute NaOH. MNNG was dissolved in acetone; up to 20 ~1 (0.4% acetone) of this solution were used for the experiments. Acetone alone (0.4%) was found to have no effect. Filter elution
The alkaline filter elution test was carried out essentially according to the method of Kohn and Grimek-Ewig [ 71, and Kohn et al. [ 81, with certain modifications. Controls and treated cells, the DNA of which had been prelabelled with either [ 14C]thymidine or [ 3H] thymidine, were mixed to give a final suspension of about 10’ cells/ml. Cells were lysed on top of the filters in 2 ml 2 M NaCl (pH 8.2), containing 0.2% Triton X-100 and 0.02 M EDTA and the lysate was passed through the filter at a flow rate
225
of 0.2 ml/min. The filters were further eluted with 4 ml of the lysis solution, after which the medium was washed out with 5 ml 1 mM EDTA (pH 7.8). The flow rate was then changed to 0.1 ml/min, and 13 ml of 0.02 M EDTA solution adjusted to pH 12.2-12.3 with tetraethylammonium hydroxide were added. Fractions were collected at lo-min intervals, and the radioactivity therein was determined by liquid scintillation counting after addition of 4 ml Aqua Luma (Baker, Gross-Gerau, F.R.G.). DNA remaining on the filters was hydrolysed in 0.4 ml 1 N HCl at 70°C for 15 min; 0.6 ml 1 N NaOH was added, and the radioactivity was measured in 10 ml of the same scintillation mixture as mentioned above. There was no loss of radioactivity from the filters before starting the elution with EDTA/tetraethylammonium hydroxide solution. RESULTS
About 90% of control DNA from untreated cells remained on the filters under the experimental conditions used (Fig. 1). There was no isotope effect when the cells were prelabelled with either [ 3H]- or [ 14C]thymidine prior to elution. Addition of NC to the cultures resulted in increases in alkaline elution which depended on the concentration of the compound in the medium (Fig. la). A further increase in elution was observed when NC was allowed to remain in contact with the cells for 1 h instead of 30 min (data not shown). In contrast, cimetidine itself failed to alter the elution characteristics of DNA at the highest concentration (5 mM) tested. Control IO- _ *.~0~000000~00 . - .
Control ‘PPy400000000
Cn-netldme 15d4
AA
_.
.
. E z
.
.
-'A
0.5-
z !Yl
9 “. . . .
rY
,
i=
AA IA..
E
01
.
.
.
.
. .
. .
.
. .
.
3.0mt-i :
.
0.1d-3
l0mt-i .
6
.
.
. '
A
B
2 L 6 8 10 12 I-'..
2
ALKALINE
l.=mmm..m 03 n9-t c
L
6 8 10 12 1L
ELUTION (ml1
Fig. 1, Effect of various concentrations of cimetidine, NC and MNNG on the alkaline elution characteristics of epidermal cell DNA. Cells were labelled with [3H]thymidine for 24 h, and incubated for 30 min with the respective compounds. As an internal standard, aliquots of untreated controls which had been prelabelled with [“Clthymidine were added to each assay. Lysis of the cells and alkaline elution were carried out as described in the text. A, NC; B, MNNG.
226
J 0.01
0.05
0,l
0,5
1.0
CONCENTRATION
5.0 (mM)
Fig. 2. Dependence of the apparent initial rate of elution on MNNG and NC concentration. Data were calculated from experiments similar to those shown in Fig. 1.
A concentration-dependent increase in DNA elution was also obtained when MNNG was added to the cultures instead of NC (Fig. lb). In order to compare quantitatively the DNA damage produced by MNNG with that of NC a common parameter of efficacy was chosen. Assuming first order kinetics for the initial rate of elution [1,8], the apparent initial elution constants were determined as functions of concentration. Concentrationdependent increases in the values of these constants were observed both for NC and for MNNG (Fig. 2). However, at a give concentration of NC the extent of DNA breakage was about one-tenth of that induced by an equimolar amount of MNNG. DISCUSSION
The role of NC as a potential carcinogen producing gastric cancer is at present under discussion [4,5,11,14,15,16,19,20]. The methylnitrosoguanidine moiety of NC resembles that of MNNG, a well known mutagen and agent causing tumours of the gastrointestinal tract.
I
I H3C
k
H2C \,/H2C
\
H
2
C/N\C/N\CH II
3
N02
II
‘i’ CN Nitrosobimetidine
’
‘C/
‘CH II N
I H
N-Methyl-N’-nitro-N-nitrosoguanidine
Both NC and MNNG effectively generate DNA strand breakage upon
3
227
in vitro incubation with epidermal cells (Figs. 1, 2). MNNG, however, was found to be more efficient when compared on an equimolar basis. This result contrasts with data obtained in a chemical in vitro system using 3,4-dichlorobenzenethiol as a nucleophilic trapping agent, towards which the methylating abilities of NC and MNNG were about the same [ 61. However, since thiol groups are known to induce strongly the decomposition of nitrosoguanidines, [ 91, alkyhtion data obtained from chemical model reactions with high concentrations of thiols might not entirely reflect the situation in whole cells. It might also be that differences in lipophilicity between MNNG (partition coefficient (HzO/octanol), KD = 0.75) and NC (KD = 4) might account at least in part for the observed differences. NC has been found to be mutagenic in microbial mutagenicity test systems [ 131. The present finding that NC is also able to produce DNA damage in vitro in eucaryotic cells of epithelial origin further underlines the possible hazard associated with this compound. Since cimetidine itself gave negative results in the alkaline elution test, the question as to whether cimetidine is nitrosated in the human gastrointestinal tract or not becomes important. This matter is currently under investigation in an appropriate animal system. ACKNOWLEDGEMENTS
The authors gratefully acknowledge Professor Dr. N. Fusenig for the gift of cultured PDV cells and Dr. G.R.N. Jones for textual corrections of the manuscript. REFERENCES 1 Brambilla, G., Cavanna, M., Parodi, S., Sciaba, L., Pino, A. and Robbiano, L. (1978) DNA damage in liver, colon, stomach, lung and kidney of Balb/c mice treated with 1,2-dimethylhydraxine. Int. J. Cancer, 22, 174-180. 2 Cox, R., Damjanov, J., Abanobi, S.E. and Sarma, D.S.R. (1973) A method for measuring DNA damage and repair in the liver in do. Cancer Res., 33, 2114-2121. 3 Druckrey, H., Preussmann, R., Ivankovic, S. and Schmiihl, D. (1967) Organotrope carcinogene Wirkungen bei 65 verschiedenen N-Nitroso-Verbindungen an BD Ratten. Z. Krebsforsch., 69, 103-201. 4 Elder, J.B., Ganguli, P.C. and Gillespie, J.E. (1979) Cimetidine and gastric cancer. Lancet, 1, 1005-1006. 5 Elder, J.B., Ganguli, P.C. and Gillespie, J.E. (1979) Gastric cancer in patients who have taken cimetidine. Lancet, 2, 245. 6 Foster, A.B., Jarman, M., Manson, D., Schulten, H.R. (1980) Structure and reactivity of nitrosocimetidine. Cancer Letters, 9, 47-52. 7 Fusenig, N.E., Samsel, W., Thon, W. and Worst, P.K.M. (1973) Malignant transformation of epidermal cells in culture by DMBA. Inserm, 19, 219-228. 8 Kohn, K.W. and Grimek-Ewig, R.A. (1973) Alkaline elution analysis, a new approach to the study of DNA single-strand interruptions in cells. Cancer Res., 33, 1849-1853. 9 Kohn, K.W., Erickson, L.C., Ewig, R.A. and Friedman, C.A. (1976) Fractionation of DNA from mammalian cells by alkaline elution. Biochemistry, 15, 4629-4637. 10 Lawley, P.D. and Thatcher, C.J. (1970) Methylation of deoxyribonucleic acid in
228
11 12 13 14 15 16 17 18
19
20 21
cultured mammalian cells by N-methyl-N’nitro-N-nitrosoguanidine. Biochem. J., 116,693-707. Mirvish, S.S. (1975) Formation of N-nitroso compounds. Chemistry, kinetics and in uiuo occurrence. Toxicol. Appl. Pharmacol., 31, 325-351. Mullen, P.W. (1979) Gastric cancer in patients who have taken cimetidine. Lancet, 2, 1406. Petzold, G.L. and Swenberg, I.A. (1978) Detection of DNA damage induced in uiuo following exposure of rats to carcinogens. Cancer Res., 38, 1589-1594. Pool, B.L. Eixenbrand, G. and Schmiihl, D. (1979) Biological activity of nitrosated cimetidine. Toxicology, 15, 69-7 2. Reed, P.I. Casell, P.G. and Walters, C.L. (1979) Gastric cancer in patients who have taken cimetidine. Lancet, 1.1235. Roe, F.J.C. (1979) Cimetidine and gastric cancer. Lancet, 1, 1039. Ruddell, W.S.J. (1979) Gastric cancer in patients who have taken cimetidine. Lancet, 1, 1234. Schwarz, M., Hummel, J., Appel, K.E., Rickart, R. and Kunz, W. (1979) DNA damage induced in vivo evaluated with a non-radioactive alkaline elution technique. Cancer letters, 6, 221-226. Swenberg, J.A., Petzold, G.L. and Harbach, P.R. (1976) In vitro DNA damagelalkaline elution assay for predicting carcinogenic potential. Biophys Biochem. Res. Commun., 72,732-738. Taylor, R.H., Lovell, D., Menzies-Gow, N., La Brooy, S.J. and Misiewicz, J.J. (1978) Misleading response of malignant gastric ulcers to cimetidine. Lancet, 1, 686-688. Taylor, T.V., Lee, D., Howatson, A.G., Anderson, J. and MacLeod, J.B. (1979) Gastric cancer in patients who have been taken cimetidine. Lancet, 1, 1235-1236.