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Effect of histamine H2-receptor antagonist therapy on the mutagenic activity of gastric juice
201
Mutation Research, 188 (1987) 201-208 Elsevier
MTR 01189
Effect of histamine H2-receptor antagonist therapy on the mutagenic activity of gastric juice H.J. O’Connor a, S.E. Riley b, A.T.R. Axon a and R.C. Garner a Gastroenterologv Unit, General Infirmary at Lee&, Lee&, LSl 3EX, b Microtest Research Ltd, and’ Cancer Research Unit, University of York, York YOI 500
’ York
(Great Britain)
(Received 13 October 1986) (Revision received 9 February 1987) (Accepted 12 February 1987)
Keywords: Histamine HZ-receptor
antagonist therapy; Gastric juice; N-Nitroso compounds, intragastric formation.
There is concern at present that treatment with histamine H2-receptor antagonists might promote the development of gastric cancer by producing conditions which favour intragastric formation of N-nitroso compounds. If H2-receptor antagonist therapy causes increased intragastric levels of N-nitroso compounds, an issue not yet resolved by analytical studies, corresponding changes in the mutagenic activity of gastric juice might be anticipated. In this study mutagenic activity and pH were measured in fasting gastric aspirate from 18 peptic ulcer patients before and during the final week of therapy with ranitidine (n = 10) or cimetidine (n = 8). Mutagenic activity was assessed using Salmonella typhimurium TA98 and TAlOO in a modified pre-incubation “fluctuation” test. No significant change in mutagenic activity was detected after therapy. Of 15 patients found to have significant mutagenic activity in their fasting gastric juice before treatment, 14 remained mutagenic following treatment. Mutation frequencies (sum of positive wells in duplicate 96-well microtitre plates, mean + SD) for TA98 and TAlOO were respectively, 20 * 34 and 100 + 64 before compared with 10 f 6 and 102 k 65 after therapy (p > 0.05). Changes in mutagenic activity were similar in both treatment groups and unrelated to duration of therapy, changes in gastric pH or ulcer healing. In vitro, neither cimetidine in aqueous solution, nor gastric juice preincubated with cimetidine showed significant mutagenic activity. These results provide no evidence that increased intragastric levels of genotoxic chemicals, such as N-nitroso compounds, occur during H2-receptor antagonist therapy.
The histamine H2-receptor antagonists (H2RA) cimetidine and ranitidine are currently among the most widely prescribed drugs in the world. Though the short-term safety profile of both drugs is good, concern has been expressed Correspondence: Dr. H.J. O’Connor, Senior Medical Registrar, Queen Elizabeth Hospital, Birmingham B15 2TH (Great Britam). 0165-7992/87/$03.50
that treatment might promote the development of gastric cancer (Elder et al., 1982). This concern was fuelled initially by case reports of gastric cancer detected after treatment with cimetidine including instances where gastroscopy and biopsy before treatment found no evidence of malignancy and where cancer apparently complicated simple duodenal ulceration, ordinarily a rare occurrence (Elder et al., 1979; Taylor et al., 1978; Hawker et
0 1987 Elsevier Science Publishers B.V. (Bitamedical Division)
202
al., 1980). Also cimetidine and ranitidine can be nitrosated under acidic conditions in vitro to produce N-nitrosocimetidine and N-nitrosoranitidine respectively, both of which are mutagenic in bacterial and mammalian cell test systems (Ichinotsubo et al., 1981; De Flora et al., 1983) and produce genotoxic effects in gastric mucosal cell DNA in the intact rodent (Gombar and Magee, 1982; Brambilla et al., 1983). Inhibition of gastric acid secretion by H2RA therapy allows colonisation of the stomach by bacteria (Ruddell et al., 1980), some of which are capable of reducing salivary and dietary nitrate to nitrite (Draser and Hill, 1974). Whether a general increase in intragastric concentrations of N-nitroso compounds occurs as a result of these changes in gastric pH and microflora remains controversial and analytical studies on human gastric contents have yielded conflicting results (Reed et al., 1981; Milton-Thompson et al., 1982). If H2RA therapy causes increased intragastric levels of N-nitroso compounds, corresponding changes in the mutagenicity of gastric juice might be anticipated. Morris et al. (1984) using the Ames test, have reported a transient increase in mutagenic activity after H2RA therapy but unfortunately, their pre- and post-therapy gastric aspirates were obtained from different patient groups and experimental procedures to control prospectively for the effects of extraneous histidine were not used. This study was designed therefore to assess prospectively, for the first time, the effect of HZRA therapy on the mutagenic activity of fasting gastric juice in individual patients. Materials and methods
18 patients (8 male, 10 female), mean age 52 years (range 23-77), with endoscopically proven peptic ulceration (5 duodenal ulcer, 9 gastric ulcer, 4 combined duodenal and gastric ulcer) were allocated to oral treatment with either cimetidine (400 mg twice daily) or ranitidine (150 mg twice daily). Treatment started within 24 h of diagnosis. All patients who had taken either drug in the 3 months before diagnosis were excluded, as were patients
who had previously undergone gastric surgery, who were pregnant, or had other serious illness, for example, hepatic or renal failure. At the time of diagnosis and during the final weak of treatment, a 5-10 ml sample of fasting gastric juice was aspirated at endoscopy (using a sterile Teflon cannula passed down the biopsy channel of the endoscope) for measurement of pH and mutagenic activity. Patients were asked to take their treatment the evening before the second endoscopy. The pH of the gastric aspirate was measured immediately with a combined glass electrode (Phillips) calibrated at pH 4 and 7 and then adjusted to pH 5.0 with 500 mM sodium phosphate buffer prior to storage at - 80 o C. Samples were thawed immediately before analysis and sterilized by positive pressure filtration through a Millipore filter (pore size, 0.22 pm). Mutation assay Mutagenic activity was assessed using a pre-incubation modification of the Salmonella “fluctuastringent experimental tion” test incorporating procedures to control for the effects of extraneous histidine. These measures included the use of aqueous histidine controls based on prospective measurement of the histidine content of each gastric juice sample by reverse-phase h.p.1.c. (Lindroth and Mopper, 1979), and a bacterial washing procedure after the pre-incubation step to remove excess histidine prior to final plating out. The adequacy of these steps in controlling for the effects of added histidine and the sensitivity, specificity and reproducibility of the modified fluctuation assay have been described in a previous publication (O’Connor et al., 1984). Each assay had duplicates of the following test substances: gastric juice (undiluted and diluted 50% with sterile distilled water), sterile distilled water, an aqueous solution of histidine equal in concentration to the juice sample being tested as negative control and solutions of the reference mutagens methylnitrosourea and 4-nitro-o-phenylenediamine as positive controls. S. typhimurium TAlOO and TA98 were the bacterial tester strains used and have been described by Maron and Ames (1983). The liquid growth medium contained sterile Davis-Mingoli Salts, glucose (14.4
203
/xg/ml), D-biotin (2.5 /~g/ml) and L-histidine hydrochloride (0.25/xg/rnl). The assay was performed as follows: refrigerated nutrient broth cultures of TA98 and TA100 were grown up for 3 h. The resultant log-phase cultures were then centrifuged at 15 000 r.p.m, for 10 min, the bacterial pellet resuspended in 0.1 M sodium phosphate buffer pH 7.4, centrifuged again at 15 000 r.p.m, for 10 rain and finally reconstituted in 0.1 M sodium phosphate buffer pH 7.4 to give a density of - 108 bacteria per ml. 200 /xl of each test substance and 200 btl of bacteria were pre-incubated in 5-ml aliquots of growth medium for 2 h in a water bath at 37 ° C and then centrifuged at 5000 r.p.m, for 5 min. The bacterial pellet was resuspended in 4 ml of 0.1 M sodium phosphate buffer pH 7.4, centrifuged again at 5000 r.p.m, for 5 min and finally reconstituted in 20 ml of growth medium prior to dispensing in 96-well Microtitre plates. These were incubated for 68 h at 37 o C and the wells then scored by the addition of 50 ptl of bromothymol blue pH indicator dye. The dye changed colour to yellow in those wells where bacterial growth had occurred. The results were analysed using published chisquared tables (Gilbert, 1980). A statistically significant increase in the number of positive wells compared to its negative control was taken to indicate that a gastric juice sample was mutagenic. The sum of positive wells in duplicate 96-well Microtitre plates was also used as an index of mutation frequency, and differences in mutation frequency before and after therapy were tested by the Wilcoxon rank sum test for statistical significance.
Results
Details of the patients in the cimetidine- and ranitidine-treated groups and their response to treatment are shown in Table 1. Valid TA100 mutagenicity data are not available for "after therapy" samples on two patients because of bacterial contamination of the fluctuation assay and an inadequate volume of gastric juice in each case to allow a repeat experiment. No significant change in mutagenic activity was detected following H2RA therapy. Of 15 patients found to have significant mutagenic activity in their fasting gastric juice before treatment, 14 remained mutagenic following treatment. Mutation frequencies of undiluted gastric juice for TA98 and TA100 in the patient population as a whole were respectively, 20 + 34 and 100 ___64 before compared with 10 4-6 and 102 + 65 following treatment ( p > 0.05). Mutation frequencies of the corresponding negative control solutions for TA98 and TA100 were respectively, 3 4- 3 and 78 4- 64 before compared with 3 4- 3 and 69 4- 62 following H2RA therapy (p > 0.05). Mutation frequencies of the positive control solutions for TA98 and TA100 were respectively, 116 4- 61 and 115 4- 61 before compared with 148 4- 54 and 109 + 59 after treatment (p > 0.05). Similar insignificant changes in mutation frequency were seen on testing dilute gastric juice.
TABLE 1 DETAILS OF PATIENTS IN THE CIMETIDINE- AND RANITIDINE-TREATED GROUPS AND THEIR RESPONSE TO TREATMENT
Mutagenic activity of cimetidine The mutagenic activity of cimetidine in sterile aqueous solution (concentration range, 1.0-20.0 m g / m l ) was assessed using the same assay procedures as for gastric juice. In addition, 200-/~1 aliquots of cimetidine in aqueous solution (concentrations, 1.0, 5.0, 10.0 and 20.0 mg per ml) were preincubated in 1 ml volumes of gastric juice (pH 2.7, obtained from a patient with normal oesophagus, stomach and duodenum at endoscopy) for 1 h in a shaking water bath at 37 ° C and the mutagenic response then compared with that of cimetidine and gastric juice alone.
Number of patients Mean age (range) Sex M : F Ulcer site Duodenal Gastric Combined duodenal and gastric Mean duration of therapy (range) Dose Ulcers healed (%)
Cimetidine
Ranitidine
8 52 (28-70) yrs 2:6
10 54 (23-77)yrs 6:4
1 4
4 5
3
1
8.9 (6-20) wks 8.1 (4-20) wks 400 nag twice daily. 150 nag twice dally. 5 (63) 8 (80)
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Pre Post Cimetidine
Post
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b
Ranitidine b
Fig. 1. Changes in mutagenic activity following therapy with cimetidine or ranitidine. (a) TA98; (b) TA100.
Fig. 2. Mutagenic activity and duration of H2-receptol antagonist therapy. (a) TA98; (b) TA100; O, cimetidine; O ranitidine.
205 192192 P>O.05 110
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0
Pre
Healed
Pre Post Not healed
Post b
Fig. 4. Mutagenic activity and ulcer healing. (a) TA98; (b) TA100. O, cimetidine; O, ranitidine.
206 TABLE 2 C H A N G E S IN M U T A G E N I C A C T I V I T Y D U R I N G H 2 - R E C E P T O R A N T A G O N I S T THERAPY: RELATIONSHIP TO T R E A T M E N T TYPE, D U R A T I O N OF THERAPY, CHANGES IN GASTRIC pH A N D ULCER HEALING n
Mutagenic samples a Before therapy
H2 receptor antagonist type Cimetidine 8 8 Ranitidine 10 7
After therapy
Statistical significance
5 9
P > 0.05
Duration of therapy (wks) 8 13 11 >8 5 4
11 3
P > 0.05
Change in pH b $ 11 1,~-~ 7
10 5
10 4
P > 0.05
Ulcer healing Healed Not healed
11 4
12 2
P > 0.05
13 5
" Mutagenic for TA98 a n d / o r TA100. b T, increased; $, decreased; ~ , no change.
Marked variations in mutagenic activity were noted between subjects and in individual subjects before and after treatment. Changes in mutagenicity were similar in the cimetidine- and ranitidinetreated groups and unrelated to duration of treatment, changes in pH or ulcer healing (Table 2; Figs. 1-4). As changes in mutagenicity were similar in undiluted and dilute gastric juice, Table 2 and Figs 1-4 show data for undiluted gastric juice only. In experiments designed to assess the mutagenic activity of cimetidine in vitro, both cimetidine in sterile water solution, and gastric juice preincubated with cimetidine were nonmutagenic for TA98 and TA100 (data not shown). Discussion
No significant change was detected in the mutagenic activity of fasting gastric juice following treatment for peptic ulceration with either cimetidine or ranitidine.
It might be argued that insignificant change in mutagenic activity merely reflects failure to achieve a significant overall increase in gastric pH particularly as bacterial overgrowth and increased concentrations of nitrite in fasting gastric juice are reported effects of H2RA-induced hypochlorhydria (Ruddell et al., 1980). This seems an unlikely explanation especially as the subset of patients whose pH increased significantly failed to show a corresponding increase in mutagenic activity. Further studies with more powerful secretory inhibitors, such as the proton pump inhibitor omeprazole (Langman, 1985), combined with serial sampling of gastric contents over 24 h would further clarify the relationship between antisecretory drugs, gastric pH and mutagenic activity. No significant relationship was found between changes in mutagenic activity and whether cimetidine or ranitidine was used in therapy. This finding suggests that neither drug has any effect on mutagenicity in gastric juice under the conditions tested, despite the fact that, on a molar basis, ranitidine produces more profound acid inhibition than cimetidine (Konturek et al., 1980). Although duration of therapy had no significant effect on mutagenic activity, the possibility of transient changes in mutagenicity, as suggested by Morris et al. (1984), in the hours immediately following ingestion of either drug cannot be excluded. However, we found gastric juice preincubated in vitro for 1 h with increasing concentrations of cimetidine had insignificant mutagenic activity. In contrast, De Flora and Picciotto (1980) obtained a mutagenic response in the Salmonella/ microsome reversion test by preincubating sodium nitrite and cimetidine in human gastric juice or by adding nitrite to gastric juice samples from patients receiving cimetidine. Whether ulcers healed or not following treatment also had no significant effect on mutagenic activity suggesting that active gastroduodenal disease may not be a prerequisite for the occurrence of mutagens in the stomach. The large number of patients, 15 of 18 (83%), found to have mutagenic gastric juice is also noteworthy. Though this high figure may reflect factors such as patient selection and the known sensitivity of the fluctuation assay for the detection of low levels of mutagenic activity (Gatehouse and Wedd, 1984), nevertheless, it suggests that
207
there may be widespread, and hitherto largely unrecognised, human intragastric exposure to potentially genotoxic substances. This is hardly surprising when one considers the many natural mutagens, teratogens and carcinogens encountered in the human diet (Ames, 1983) coupled with the potential for endogenous synthesis of mutagens after ingestion of appropriate dietary precursors (Carr, 1985). Pronounced variations in mutagenic activity were detected both between subjects and in individual subjects before and following H2RA therapy. Though the negative and positive control solutions also gave a variable mutagenic response, such large variations between individuals are unlikely to be accounted for solely by variability in assay conditions. Variable concentrations of nitrite are present in fasting gastric juice (Muscroft et al., 1981) and nitrite per se is mutagenic for TA100 (Sugimura et al., 1976; De Flora and Picciotto, 1980). Hence, the variable mutagenic response in TA100, but not in TA98, might be accounted for, at least in part, by variations in intragastric nitrite following H2RA therapy (Milton-Thompson et al., 1982). Recent epidemiological, analytical and clinical evidence suggests that the genotoxic substances producing mutagenic activity in gastric juice are likely to be N-nitroso compounds (Mirvish, 1983). If this is so, our results suggest that H2RA therapy has no significant effect on their intragastric concentrations. On the contrary, the marked variations in mutagenic activity suggest that intragastric concentrations of N-nitroso compounds may vary widely and bear little relationship to cimetidine- or ranitidine-induced changes in the intragastric milieu. Obviously, these conclusions are for the present tentative, pending isolation and precise characterisation of the mutagenic constituents of gastric juice.
Acknowledgements This work was supported by a research grant from Smith Kline and French Research Ltd., Welwyn (Great Britain). RCG is partly supported by a grant from the Yorkshire Cancer Research Campaign.
References Ames, B.N. (1983) Dietary carcinogens and anticarcinogens: oxygen radicals and degenerative diseases, Science, 221, 1256-1264. Brambilla, G., M. Cavanna, P. Faggin et al. (1983) Genotoxic effects in rodents given high oral doses of ranitidine and sodium nitrite, Carcinogenesis, 4, 1281-1285. Carr, B.I. (1985) Chemical carcinogens and inhibitors of carcinogenesis in the human diet, Cancer, 55, 218-224. De Flora, S., and A. Picciotto (1980) Mutagenicity of cimetidine in nitrite-enriched human gastric juice, Carcinogenesis, 1,925-930. De Flora, S., C. Bennicelli, A. Camoirano et al. (1983) Genotoxicity of nitrosated ranitidine, Carcinogenesis, 4, 255-260. Draser, B.S., and M.J. Hill (1974) Human intestinal flora, Academic Press, New York, pp. 72-102. Elder, J.B., P.C. Ganguli and I.E. Gillespie (1979) Cimetidine and gastric cancer, Lancet, i, 1005-1006. Elder, J.B., P.C. Ganguli, C.G. Koffman et al. (1982) Possible role of cimetidine and its nitrosation products in human stomach cancer, in: P.N. Magee (Ed.), Banbury Report 12, Nitrosamines and human cancer, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp. 335-349. Gatehouse, D., and D.J. Wedd (1984) The differential mutagenicity of isoniazid in fluctuation assays and Salmonella plate tests, Carcinogenesis, 5, 391-397. Gilbert, R.I. (1980) The analysis of fluctuation tests, Mutation Res., 74, 283-289. Gombar, C.T., and P.N. Magee (1982) DNA-methylation by nitrosocimetidine and N-methyl-N-nitro-N-nitrosoguanidine in the intact rat, Chem.-Biol. Interact., 40, 149-157. Hawker, P.C., T.J. Muscroft and M.R.B. Keighley (1980) Gastric cancer after cimetidine in patient with two negative pre-treatment biopsies, Lancet, i, 709-710. Ichinotsubo, D., E.A. MacKinnon, C. Liu et al. (1981) Mutagenicity of nitrosated cimetidines, Carcinogenesis, 2, 261-264. Konturek, S.J., W. Obtulowicz, N. Kwiecien et al. (1980) Comparison of ranitidine and cimetidine in the inhibition of histamine, sham-feeding and meal-induced gastric secretion in duodenal ulcer patients, Gut, 21, 181-186. Langman, M.J.S. (1985) Antisecretory drugs and gastric cancer, Br. Med. J., 290, 1850-1852. Lindroth, P., and K. Mopper (1979) High performance liquid chromatographic determination of subpicomole amounts of amino acids by precolumn fluorescence derivatization with o-phthaldialdehyde, Anal. Chem., 51, 1667-1674. Maron, D.M., and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215. Milton-Thompson, G.J., N.F. Lightfoot, Z. Ahmet et al. (1982) Intragastric acidity, bacteria, nitrite, and N-nitroso compounds before, during, and after cimetidine treatment, Lancet, i, 1091-1095. Mirvish, S.S. (1983) The etiology of gastric cancer: intragastric nitrosamide formation and other theories, J. Natl. Cancer Inst., 71,629-647.
208 Morris, D.L., D. Youngs, T.J. Muscroft et al. (1984) Mutagenicity in gastric juice, Gut, 25, 723-727. Muscroft, T.J., S.A. Dearie, D. Youngs et al. (1981) The microflora of the postoperative stomach, Br J. Surg., 68, 560-564. O'Connor, H.J., A.T.R. Axon, S.E. Riley et al. (1984) Mutagenicity of gastric juice: the importance of controlling histidine concentration when using Salmonella tester strains, Carcinogenesis, 5, 853-856. Reed, P.I., P.L.R. Smith, K. Haines et al. (1981) Gastric juice N-nitrosamines in health and gastroduodenal disease, Lancet, ii, 550-552.
Ruddell, W.S.J., A.T.R. Axon, J.M. Findlay et al. (1980) Effect of cimetidine on the gastric bacterial flora, Lancet, i, 672-674. Sugimura, T., S. Sato, M. Nagao et al. (1976) Overlapping of carcinogens and mutagens, in: P.N. Magee et al. (Eds.), Fundamentals in Cancer Prevention, University of Tokyo Press/University Park Press, Tokyo/Baltimore, pp. 191-215. Taylor, T.V., D. Lee, A.G. Howatson et al. (1979) Gastric cancer in patients who have taken cimetidine, Lancet, i, 1235-1236.
Mutation Research, 188 (1987) 201-208 Elsevier
MTR 01189
Effect of histamine H2-receptor antagonist therapy on the mutagenic activity of gastric juice H.J. O’Connor a, S.E. Riley b, A.T.R. Axon a and R.C. Garner a Gastroenterologv Unit, General Infirmary at Lee&, Lee&, LSl 3EX, b Microtest Research Ltd, and’ Cancer Research Unit, University of York, York YOI 500
’ York
(Great Britain)
(Received 13 October 1986) (Revision received 9 February 1987) (Accepted 12 February 1987)
Keywords: Histamine HZ-receptor
antagonist therapy; Gastric juice; N-Nitroso compounds, intragastric formation.
There is concern at present that treatment with histamine H2-receptor antagonists might promote the development of gastric cancer by producing conditions which favour intragastric formation of N-nitroso compounds. If H2-receptor antagonist therapy causes increased intragastric levels of N-nitroso compounds, an issue not yet resolved by analytical studies, corresponding changes in the mutagenic activity of gastric juice might be anticipated. In this study mutagenic activity and pH were measured in fasting gastric aspirate from 18 peptic ulcer patients before and during the final week of therapy with ranitidine (n = 10) or cimetidine (n = 8). Mutagenic activity was assessed using Salmonella typhimurium TA98 and TAlOO in a modified pre-incubation “fluctuation” test. No significant change in mutagenic activity was detected after therapy. Of 15 patients found to have significant mutagenic activity in their fasting gastric juice before treatment, 14 remained mutagenic following treatment. Mutation frequencies (sum of positive wells in duplicate 96-well microtitre plates, mean + SD) for TA98 and TAlOO were respectively, 20 * 34 and 100 + 64 before compared with 10 f 6 and 102 k 65 after therapy (p > 0.05). Changes in mutagenic activity were similar in both treatment groups and unrelated to duration of therapy, changes in gastric pH or ulcer healing. In vitro, neither cimetidine in aqueous solution, nor gastric juice preincubated with cimetidine showed significant mutagenic activity. These results provide no evidence that increased intragastric levels of genotoxic chemicals, such as N-nitroso compounds, occur during H2-receptor antagonist therapy.
The histamine H2-receptor antagonists (H2RA) cimetidine and ranitidine are currently among the most widely prescribed drugs in the world. Though the short-term safety profile of both drugs is good, concern has been expressed Correspondence: Dr. H.J. O’Connor, Senior Medical Registrar, Queen Elizabeth Hospital, Birmingham B15 2TH (Great Britam). 0165-7992/87/$03.50
that treatment might promote the development of gastric cancer (Elder et al., 1982). This concern was fuelled initially by case reports of gastric cancer detected after treatment with cimetidine including instances where gastroscopy and biopsy before treatment found no evidence of malignancy and where cancer apparently complicated simple duodenal ulceration, ordinarily a rare occurrence (Elder et al., 1979; Taylor et al., 1978; Hawker et
0 1987 Elsevier Science Publishers B.V. (Bitamedical Division)
202
al., 1980). Also cimetidine and ranitidine can be nitrosated under acidic conditions in vitro to produce N-nitrosocimetidine and N-nitrosoranitidine respectively, both of which are mutagenic in bacterial and mammalian cell test systems (Ichinotsubo et al., 1981; De Flora et al., 1983) and produce genotoxic effects in gastric mucosal cell DNA in the intact rodent (Gombar and Magee, 1982; Brambilla et al., 1983). Inhibition of gastric acid secretion by H2RA therapy allows colonisation of the stomach by bacteria (Ruddell et al., 1980), some of which are capable of reducing salivary and dietary nitrate to nitrite (Draser and Hill, 1974). Whether a general increase in intragastric concentrations of N-nitroso compounds occurs as a result of these changes in gastric pH and microflora remains controversial and analytical studies on human gastric contents have yielded conflicting results (Reed et al., 1981; Milton-Thompson et al., 1982). If H2RA therapy causes increased intragastric levels of N-nitroso compounds, corresponding changes in the mutagenicity of gastric juice might be anticipated. Morris et al. (1984) using the Ames test, have reported a transient increase in mutagenic activity after H2RA therapy but unfortunately, their pre- and post-therapy gastric aspirates were obtained from different patient groups and experimental procedures to control prospectively for the effects of extraneous histidine were not used. This study was designed therefore to assess prospectively, for the first time, the effect of HZRA therapy on the mutagenic activity of fasting gastric juice in individual patients. Materials and methods
18 patients (8 male, 10 female), mean age 52 years (range 23-77), with endoscopically proven peptic ulceration (5 duodenal ulcer, 9 gastric ulcer, 4 combined duodenal and gastric ulcer) were allocated to oral treatment with either cimetidine (400 mg twice daily) or ranitidine (150 mg twice daily). Treatment started within 24 h of diagnosis. All patients who had taken either drug in the 3 months before diagnosis were excluded, as were patients
who had previously undergone gastric surgery, who were pregnant, or had other serious illness, for example, hepatic or renal failure. At the time of diagnosis and during the final weak of treatment, a 5-10 ml sample of fasting gastric juice was aspirated at endoscopy (using a sterile Teflon cannula passed down the biopsy channel of the endoscope) for measurement of pH and mutagenic activity. Patients were asked to take their treatment the evening before the second endoscopy. The pH of the gastric aspirate was measured immediately with a combined glass electrode (Phillips) calibrated at pH 4 and 7 and then adjusted to pH 5.0 with 500 mM sodium phosphate buffer prior to storage at - 80 o C. Samples were thawed immediately before analysis and sterilized by positive pressure filtration through a Millipore filter (pore size, 0.22 pm). Mutation assay Mutagenic activity was assessed using a pre-incubation modification of the Salmonella “fluctuastringent experimental tion” test incorporating procedures to control for the effects of extraneous histidine. These measures included the use of aqueous histidine controls based on prospective measurement of the histidine content of each gastric juice sample by reverse-phase h.p.1.c. (Lindroth and Mopper, 1979), and a bacterial washing procedure after the pre-incubation step to remove excess histidine prior to final plating out. The adequacy of these steps in controlling for the effects of added histidine and the sensitivity, specificity and reproducibility of the modified fluctuation assay have been described in a previous publication (O’Connor et al., 1984). Each assay had duplicates of the following test substances: gastric juice (undiluted and diluted 50% with sterile distilled water), sterile distilled water, an aqueous solution of histidine equal in concentration to the juice sample being tested as negative control and solutions of the reference mutagens methylnitrosourea and 4-nitro-o-phenylenediamine as positive controls. S. typhimurium TAlOO and TA98 were the bacterial tester strains used and have been described by Maron and Ames (1983). The liquid growth medium contained sterile Davis-Mingoli Salts, glucose (14.4
203
/xg/ml), D-biotin (2.5 /~g/ml) and L-histidine hydrochloride (0.25/xg/rnl). The assay was performed as follows: refrigerated nutrient broth cultures of TA98 and TA100 were grown up for 3 h. The resultant log-phase cultures were then centrifuged at 15 000 r.p.m, for 10 min, the bacterial pellet resuspended in 0.1 M sodium phosphate buffer pH 7.4, centrifuged again at 15 000 r.p.m, for 10 rain and finally reconstituted in 0.1 M sodium phosphate buffer pH 7.4 to give a density of - 108 bacteria per ml. 200 /xl of each test substance and 200 btl of bacteria were pre-incubated in 5-ml aliquots of growth medium for 2 h in a water bath at 37 ° C and then centrifuged at 5000 r.p.m, for 5 min. The bacterial pellet was resuspended in 4 ml of 0.1 M sodium phosphate buffer pH 7.4, centrifuged again at 5000 r.p.m, for 5 min and finally reconstituted in 20 ml of growth medium prior to dispensing in 96-well Microtitre plates. These were incubated for 68 h at 37 o C and the wells then scored by the addition of 50 ptl of bromothymol blue pH indicator dye. The dye changed colour to yellow in those wells where bacterial growth had occurred. The results were analysed using published chisquared tables (Gilbert, 1980). A statistically significant increase in the number of positive wells compared to its negative control was taken to indicate that a gastric juice sample was mutagenic. The sum of positive wells in duplicate 96-well Microtitre plates was also used as an index of mutation frequency, and differences in mutation frequency before and after therapy were tested by the Wilcoxon rank sum test for statistical significance.
Results
Details of the patients in the cimetidine- and ranitidine-treated groups and their response to treatment are shown in Table 1. Valid TA100 mutagenicity data are not available for "after therapy" samples on two patients because of bacterial contamination of the fluctuation assay and an inadequate volume of gastric juice in each case to allow a repeat experiment. No significant change in mutagenic activity was detected following H2RA therapy. Of 15 patients found to have significant mutagenic activity in their fasting gastric juice before treatment, 14 remained mutagenic following treatment. Mutation frequencies of undiluted gastric juice for TA98 and TA100 in the patient population as a whole were respectively, 20 + 34 and 100 ___64 before compared with 10 4-6 and 102 + 65 following treatment ( p > 0.05). Mutation frequencies of the corresponding negative control solutions for TA98 and TA100 were respectively, 3 4- 3 and 78 4- 64 before compared with 3 4- 3 and 69 4- 62 following H2RA therapy (p > 0.05). Mutation frequencies of the positive control solutions for TA98 and TA100 were respectively, 116 4- 61 and 115 4- 61 before compared with 148 4- 54 and 109 + 59 after treatment (p > 0.05). Similar insignificant changes in mutation frequency were seen on testing dilute gastric juice.
TABLE 1 DETAILS OF PATIENTS IN THE CIMETIDINE- AND RANITIDINE-TREATED GROUPS AND THEIR RESPONSE TO TREATMENT
Mutagenic activity of cimetidine The mutagenic activity of cimetidine in sterile aqueous solution (concentration range, 1.0-20.0 m g / m l ) was assessed using the same assay procedures as for gastric juice. In addition, 200-/~1 aliquots of cimetidine in aqueous solution (concentrations, 1.0, 5.0, 10.0 and 20.0 mg per ml) were preincubated in 1 ml volumes of gastric juice (pH 2.7, obtained from a patient with normal oesophagus, stomach and duodenum at endoscopy) for 1 h in a shaking water bath at 37 ° C and the mutagenic response then compared with that of cimetidine and gastric juice alone.
Number of patients Mean age (range) Sex M : F Ulcer site Duodenal Gastric Combined duodenal and gastric Mean duration of therapy (range) Dose Ulcers healed (%)
Cimetidine
Ranitidine
8 52 (28-70) yrs 2:6
10 54 (23-77)yrs 6:4
1 4
4 5
3
1
8.9 (6-20) wks 8.1 (4-20) wks 400 nag twice daily. 150 nag twice dally. 5 (63) 8 (80)
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o
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.~_ >~ c ~
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Pre Post Cimetidine
Post
Post >8wk
b
Ranitidine b
Fig. 1. Changes in mutagenic activity following therapy with cimetidine or ranitidine. (a) TA98; (b) TA100.
Fig. 2. Mutagenic activity and duration of H2-receptol antagonist therapy. (a) TA98; (b) TA100; O, cimetidine; O ranitidine.
205 192192 P>O.05 110
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0
Pre
Healed
Pre Post Not healed
Post b
Fig. 4. Mutagenic activity and ulcer healing. (a) TA98; (b) TA100. O, cimetidine; O, ranitidine.
206 TABLE 2 C H A N G E S IN M U T A G E N I C A C T I V I T Y D U R I N G H 2 - R E C E P T O R A N T A G O N I S T THERAPY: RELATIONSHIP TO T R E A T M E N T TYPE, D U R A T I O N OF THERAPY, CHANGES IN GASTRIC pH A N D ULCER HEALING n
Mutagenic samples a Before therapy
H2 receptor antagonist type Cimetidine 8 8 Ranitidine 10 7
After therapy
Statistical significance
5 9
P > 0.05
Duration of therapy (wks) 8 13 11 >8 5 4
11 3
P > 0.05
Change in pH b $ 11 1,~-~ 7
10 5
10 4
P > 0.05
Ulcer healing Healed Not healed
11 4
12 2
P > 0.05
13 5
" Mutagenic for TA98 a n d / o r TA100. b T, increased; $, decreased; ~ , no change.
Marked variations in mutagenic activity were noted between subjects and in individual subjects before and after treatment. Changes in mutagenicity were similar in the cimetidine- and ranitidinetreated groups and unrelated to duration of treatment, changes in pH or ulcer healing (Table 2; Figs. 1-4). As changes in mutagenicity were similar in undiluted and dilute gastric juice, Table 2 and Figs 1-4 show data for undiluted gastric juice only. In experiments designed to assess the mutagenic activity of cimetidine in vitro, both cimetidine in sterile water solution, and gastric juice preincubated with cimetidine were nonmutagenic for TA98 and TA100 (data not shown). Discussion
No significant change was detected in the mutagenic activity of fasting gastric juice following treatment for peptic ulceration with either cimetidine or ranitidine.
It might be argued that insignificant change in mutagenic activity merely reflects failure to achieve a significant overall increase in gastric pH particularly as bacterial overgrowth and increased concentrations of nitrite in fasting gastric juice are reported effects of H2RA-induced hypochlorhydria (Ruddell et al., 1980). This seems an unlikely explanation especially as the subset of patients whose pH increased significantly failed to show a corresponding increase in mutagenic activity. Further studies with more powerful secretory inhibitors, such as the proton pump inhibitor omeprazole (Langman, 1985), combined with serial sampling of gastric contents over 24 h would further clarify the relationship between antisecretory drugs, gastric pH and mutagenic activity. No significant relationship was found between changes in mutagenic activity and whether cimetidine or ranitidine was used in therapy. This finding suggests that neither drug has any effect on mutagenicity in gastric juice under the conditions tested, despite the fact that, on a molar basis, ranitidine produces more profound acid inhibition than cimetidine (Konturek et al., 1980). Although duration of therapy had no significant effect on mutagenic activity, the possibility of transient changes in mutagenicity, as suggested by Morris et al. (1984), in the hours immediately following ingestion of either drug cannot be excluded. However, we found gastric juice preincubated in vitro for 1 h with increasing concentrations of cimetidine had insignificant mutagenic activity. In contrast, De Flora and Picciotto (1980) obtained a mutagenic response in the Salmonella/ microsome reversion test by preincubating sodium nitrite and cimetidine in human gastric juice or by adding nitrite to gastric juice samples from patients receiving cimetidine. Whether ulcers healed or not following treatment also had no significant effect on mutagenic activity suggesting that active gastroduodenal disease may not be a prerequisite for the occurrence of mutagens in the stomach. The large number of patients, 15 of 18 (83%), found to have mutagenic gastric juice is also noteworthy. Though this high figure may reflect factors such as patient selection and the known sensitivity of the fluctuation assay for the detection of low levels of mutagenic activity (Gatehouse and Wedd, 1984), nevertheless, it suggests that
207
there may be widespread, and hitherto largely unrecognised, human intragastric exposure to potentially genotoxic substances. This is hardly surprising when one considers the many natural mutagens, teratogens and carcinogens encountered in the human diet (Ames, 1983) coupled with the potential for endogenous synthesis of mutagens after ingestion of appropriate dietary precursors (Carr, 1985). Pronounced variations in mutagenic activity were detected both between subjects and in individual subjects before and following H2RA therapy. Though the negative and positive control solutions also gave a variable mutagenic response, such large variations between individuals are unlikely to be accounted for solely by variability in assay conditions. Variable concentrations of nitrite are present in fasting gastric juice (Muscroft et al., 1981) and nitrite per se is mutagenic for TA100 (Sugimura et al., 1976; De Flora and Picciotto, 1980). Hence, the variable mutagenic response in TA100, but not in TA98, might be accounted for, at least in part, by variations in intragastric nitrite following H2RA therapy (Milton-Thompson et al., 1982). Recent epidemiological, analytical and clinical evidence suggests that the genotoxic substances producing mutagenic activity in gastric juice are likely to be N-nitroso compounds (Mirvish, 1983). If this is so, our results suggest that H2RA therapy has no significant effect on their intragastric concentrations. On the contrary, the marked variations in mutagenic activity suggest that intragastric concentrations of N-nitroso compounds may vary widely and bear little relationship to cimetidine- or ranitidine-induced changes in the intragastric milieu. Obviously, these conclusions are for the present tentative, pending isolation and precise characterisation of the mutagenic constituents of gastric juice.
Acknowledgements This work was supported by a research grant from Smith Kline and French Research Ltd., Welwyn (Great Britain). RCG is partly supported by a grant from the Yorkshire Cancer Research Campaign.
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