- Email: [email protected]
Helicobacter pylori induces DNA damage in vitro
Cancer Letters 152 (2000) 145±149
www.elsevier.com/locate/canlet
Helicobacter pylori induces DNA damage in vitro Bernd Schmauûer a, Stefan O. Mueller b,1, Matthias Eck a, Marianne MoÈller b, HansKonrad MuÈller-Hermelink a, Helga Stopper b,* b
a Institute for Pathology, University of WuÈrzburg, D-97078 WuÈrzburg, Germany Department of Toxicology, University of WuÈrzburg, Versbacher Strasse 9, D-97078 WuÈrzburg, Germany
Received 5 October 1999; received in revised form 11 December 1999; accepted 11 December 1999
Abstract A close association between Helicobacter pylori infection and the development of gastric adenocarcinoma in humans has been demonstrated. Therefore, the direct induction of DNA damage by H. pylori was investigated here using the in vitro micronucleus assay. After 5 days of incubation with bacterial lysate a dose-dependent formation of micronuclei was found, which was not limited to cytotoxic protein concentrations and was not observed after treatment with Escherichia coli lysate (control). This induction of DNA damage may be a link between chronic H. pylori infection and development of adenocarcinoma of the stomach. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Helicobacter pylori; Micronuclei; L5178Y mouse lymphoma cells; DNA damage; Genotoxicity; Gastric carcinoma
1. Introduction Epidemiological studies have demonstrated a close association between Helicobacter pylori infection and the subsequent development of gastric adenocarcinoma in humans [1,2]. Therefore in 1994 the World Health Organization classi®ed H. pylori as a group I carcinogen in humans [3]. Recently the development of gastric carcinoma after infection with H. pylori has been demonstrated in the Mongolian gerbil [4], further supporting a role of H. pylori in carcinogenesis. Several mechanisms such as an increased proliferation of gastric epithelial cells during H. pylori in¯ammation [5] and lowered gastric ascorbic acid level [6] have been proposed in carcinogenesis. Recently an
increased level of oxidative damage, which was suggested to be induced by oxygen radicals during the persistent in¯ammation, was discussed as a link between H. pylori infection and gastric carcinogenesis [7]. In this study the direct induction of DNA damage by H. pylori was investigated using the in vitro micronucleus test, which is well established for genotoxicity testing [8,9]. In this test, microscopic observation enables detection of chromosomal fragments and maldistributed whole chromosomes, which are enclosed in a membrane and can be found in the cytoplasm of mammalian cells.
2. Materials and methods * Corresponding author. Tel.: 149-931-201-3427; fax: 149931-201-3446. E-mail address: [email protected] (H. Stopper) 1 Present address: NIEHS/NIH, RTP, NC, USA.
2.1. Cell culture Cells were cultured in suspension in RPMI-1640
0304-3835/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(99)00442-5
146
B. Schmauûer et al. / Cancer Letters 152 (2000) 145±149
supplemented with antibiotics, 0.25 mg/ml l-glutamine, 107 mg/ml sodium pyruvate, and 10% heat-inactivated horse serum (all from Sigma Chemie GmbH, Deisenhofen, Germany). Cell cultures were grown in a humidi®ed atmosphere with 5% CO2 in air at 378C. 2.2. Preparation of H. pylori and Escherichia coli lysate H. pylori strain 6650 was isolated from a patient with severe active gastritis on H. pylori agar (Biomerieux, France) microaerobically. The H. pylori strain was expanded microaerobically on Colombia blood agar (Oxoid, Basingstoke, UK) containing 10% inactivated de®brinated horse blood (30 min at 568C) for 96 h. The H. pylori strain was CagA positive as determined by PCR. E. coli was used as a control for a non-carcinogenic gram-negative intestinal bacterium. A urease-negative E. coli strain was isolated from human feces and grown on Colombia blood agar (Oxoid, Basingstoke, UK) for 24 h. Bacteria were harvested from agar plates and washed in 10 mmol Tris-buffered saline (TBS) (pH 7.4). Bacterial cells were sonicated for 3 £ 1 min on ice. Additional irradiation was required for the E. coli lysate to eliminate surviving bacteria. Total protein was determined by nephelometrie (Behring, Marburg, Germany). 2.3. In vitro micronucleus test We used the mammalian L5178Y mouse lymphoma cell line, the most frequently used cell line in routine genotoxicity testing [10]. Exponentially growing L5178Y mouse lymphoma cells (doubling time about 11 h) were seeded at a density of 2 £ 10 5/ml. Lysates of H. pylori and E. coli were added. After 24 h cell numbers were counted for assessment of cytotoxicity. Then cells were centrifuged, medium was replaced and cell density was again adjusted to 2 £ 10 5/ml to ensure ability for exponential cell growth. Fresh lysates were added. Negative controls were treated alike, but without addition of bacterial lysates. This was repeated four times simulating a chronic process. On the ®fth day cells were harvested. For this purpose, cells were brought onto glass slides by cytospin-centrifugation and ®xed with methanol (2208C for 1 h). To stain
nuclei and micronuclei, the slides were incubated with Hoechst 33258 (5 mg/ml in phosphate-buffered saline (PBS) for 3 min). Slides were washed with buffer and mounted for microscopy, using mounting medium (Linaris, Germany). Numbers of nuclei and micronuclei were scored at a magni®cation of 500£. One thousand nuclei were evaluated on each slide and two slides were evaluated per experiment. Objects were classi®ed as micronuclei, if they appeared to be separated from the nuclei, were round or oval, had an area of less than 1/4 of the area of a nucleus, and showed staining characteristics similar to those of the nuclei. 2.4. Cell viability (FDA/EB-method) Cell viability was measured at the time of cell harvesting in experiment no. 3. For this, a staining solution of 0.6% (v/v) ¯uorescein diacetate (FDA; 5 mg/ml in acetone) and 1.6% ethidium bromide (EB; 1 mg/ml in H2O) was prepared. Cells were stained by adding 15 ml of this solution to 35 ml of cell suspension. After 3 min at least 200 cells were evaluated using a ¯uorescence microscope at a magni®cation of 500£. Viable cells, containing active esterases, appeared green, whereas red nuclei indicated dead cells after uptake of ethidium bromide. 3. Results Micronucleus-induction is a well established method for the assessment of genotoxic damage. In this test, microscopic analysis of mammalian cells after DNA-speci®c staining reveals structures in the cytoplasm which resemble the main nucleus, but are smaller. They are formed by the cell during mitosis from chromosome fragments or whole displaced chromosomes which are enclosed in a separate nuclearlike membrane. Incubation of the cells with lysate from H. pylori for 5 days resulted reproducibly in the dose-dependent formation of micronuclei (Table 1). The frequency was elevated 3.5-fold (experiment 1), 3-fold (experiment 2) and 3.8-fold (experiment 3) over the respective control. The control experiments using lysate from E. coli revealed no induction of micronuclei over the control. Cytotoxicity induced by the lysates was measured by counting cell number on the day of cell harvest (Table 2). Cell number was
B. Schmauûer et al. / Cancer Letters 152 (2000) 145±149
147
Table 1 Induction of micronuclei in L5178Y mouse lymphoma cells treated with lysates of H. pylori and E. coli a Concentration (mg/ml protein)
0 5 10 15 20 25 30
H. pylori MN-cells/1000 cells
E. coli MN-cells/1000 cells
Experiment 1
Experiment 2
Experiment 3
Experiment 1
Experiment 2
Experiment 3
7.0 5.5 12.5 15.5 18.5 24.5 n.d.
6.7 10.3 13.3 13.0 20.3 n.d. n.d.
6.4 11.7 14.1 18.1 23.4 24.2 n.d.
13.5 6.0 13.0 7.0 8.0 7.0 11.0
6.3 7.7 13.0 8.3 13.2 8.8 6.6
7.8 9.2 13.2 13.9 8.4 5.8 5.9
a Each data point represents the mean of two slides from one culture dish with 1000 nuclei being evaluated on each slide. MN-cells, micronucleus-containing cells.
reduced after treatment with H. pylori lysate to 44% (experiment 1), 66% (experiment 2) and 78% (experiment 3) of the control. At the most toxic doses cells were still able to perform one cell doubling within the last day before harvest. In contrast, E. coli lysate induced little or no cytotoxicity. Cell viability as measured by the FDA/EB-method uptake was slightly reduced after treatment with lysate from H. pylori (78% of control at the highest tested concentration) and not reduced after treatment with E. coli lysate (96% of control at the highest tested concentration). Overall, micronuclei were not only induced at toxic ($20 mg/ml protein), but also at non- or slightly toxic doses (5±15 mg/ml protein) by H. pylori lysate, whereas the E. coli lysate did not induce micronuclei at the same and higher (30 mg/ml protein) protein concentrations.
4. Discussion A close association between H. pylori and gastric adenocarcinoma was demonstrated in several studies [1,2]. However, the mechanisms leading to carcinogenesis are not well understood. H. pylori infection leads to chronic antrum gastritis preceding the development of carcinoma. Therefore, oxidative DNA damage by oxygen radicals in chronic in¯ammation has been suggested [7]. As H. pylori persists in mucous overlaying the gastric epithelium, the question is arising as to whether H. pylori might directly be involved in the DNA damage process. In this study an induction of DNA damage (genotoxicity) by H. pylori was demonstrated in the micronucleus test, which may be a link between chronic H. pylori infection and development of adenocarcinoma of the stomach.
Table 2 Induction of cytotoxicity in L5178Y mouse lymphoma cells treated with lysates of H. pylori and E. coli a Concentration (mg/ml protein)
0 5 10 15 20 25 30 a
H. pylori cell number ( £ 10 5/ml)
E. coli cell number ( £ 10 5/ml)
Experiment 1
Experiment 2
Experiment 3
Experiment 1
Experiment 2
Experiment 3
9 10 7 7 4 4 n.d.
6 6 7 7 4 n.d. n.d.
9 8 8 8 7 7 n.d.
10 8 9 7 7 8 8
9 11 9 9 9 9 9
9 8 9 10 10 9 10
Given are cell numbers on day 5 (harvest). Cells were seeded on day one with a density of 2 £ 10 5 and diluted every day to this number.
148
B. Schmauûer et al. / Cancer Letters 152 (2000) 145±149
Our data are in line with an earlier publication by Ning et al. [11], who also found the formation of micronuclei in another cell line after treatment with H. pylori lysate. However, we have now shown that this micronucleus induction was not limited to cytotoxic protein concentrations, whereas these authors had not included cytotoxicity measurements. Additionally, we supported the relevance of the H. pylori-induced micronuclei by comparison with E. coli, another gastrointestinal bacterium, which did not induce cytotoxicity or genotoxicity at the same protein concentrations. Chromosome breakage leading to fragments as well as whole chromosomes, which are not correctly distributed during mitosis, give rise to the formation of micronuclei in the next interphase. Micronuclei are thus an indicator for DNA-damaging effects. The micronucleus test has been used extensively to assess DNA damage in humans after environmental exposure [12]. An international guideline for the use of the in vitro micronucleus test for assessment of DNA damage in the process of licensing a pharmaceutical or chemical is under development [13]. The formation of micronuclei may itself be involved in the carcinogenic process [9]. For example, the inclusion of a tumor suppressor gene in a micronucleus and the subsequent inactivation or loss of that micronucleus could lead to a transformed cell and therefore be a step in carcinogenesis. Even if micronuclei are not themselves involved in the carcinogenic process they are a sensitive indicator for DNA damage preceding the development of cancer. Usually incubation in the micronucleus test is performed for several hours (up to 1.5 cell cycle durations). With such a protocol we did not see any clear genotoxicity (data not shown). Therefore we extended the incubation period to several days, mimicking the biological in vivo situation (chronic process). Now, we observed a dose-dependent induction of micronuclei by lysate of H. pylori, but not by equal and higher protein concentrations of E. coli lysate. These results suggest that a factor contained in the lysate of H. pylori may exert genotoxic effects. It will be interesting to determine the chemical nature of this factor. Only a factor with small molecular size can be expected to diffuse into the cell for induction of DNA damage. For larger sized factors (e.g. proteins) the question is arising as to whether they are active
inside the cells (endocytosis) or whether they interact with the cellular outside (membrane or receptors). It will be of further interest whether this factor is identical with known virulence factors in H. pylori such as CagA, which is associated with gastric cancer or Malt-type lymphoma [14,15]. In our experiments micronucleus induction was not limited to concentrations with high cytotoxicity. Cytotoxicity can contribute to or mediate genotoxic effects. For example, one mechanism might be the formation of DNA strand breaks by DNA-degrading enzymes which have been released from cellular organelles destroyed by cytotoxic effects. On the other side, cytotoxicity in itself is a factor that can contribute to cell transformation if compensatory growth of surrounding cells is induced [16]. Investigation of the mechanism of genotoxicity exerted by H. pylori lysate is necessary. Furthermore, it remains to be determined whether such DNA damage can also be found in vivo during H. pylori infection, possibly preceding carcinogenesis. Acknowledgements This study was supported by the `Deutsche Forschungsgemeinschaft' grant GRK 73/3-97 to B.S. We thank Mrs M. Ruppert and Mrs M. Simet for their skilled technical assistance. References [1] J. Parsonnet, G.D. Friedmann, D.P. Vandersteen, Y. Chang, J.H. Vogelman, N. Orentreich, R.K. Sibley, Helicobacter pylori infection and the risk of gastric carcinoma, N. Engl. J. Med. 325 (1991) 1127±1131. [2] D. Foreman, Eurogast Study Group, An international association between Helicobacter pylori infection and gastric cancer, Lancet 3421 (1993) 359±362. [3] IARC Working Group on the Evaluation of Carcinogenic Risks to Humans. Helicobacter pylori. In: Schistosomes, Liver Flukes, and Helicobacter pylori: Views and Expert Opinions of an IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, IARC, Lyon, 1994, pp.177± 240. [4] T. Watanabe, M. Tada, H. Nagai, S. Sasaki, M. Nakao, Helicobacter pylori infection induces gastric cancer in Mongolian gerbils, Gastroenterology 115 (1998) 642±648. [5] M. Anti, A. Armuzzi, A. Gasbarrini, G. Gasbarrini, Importance of changes in epithelial cell turnover during Helicobac-
B. Schmauûer et al. / Cancer Letters 152 (2000) 145±149
[6]
[7]
[8]
[9] [10]
ter pylori infection in gastric carcinogenesis, Gut 43 (1998) 27±32. Z.W. Zhang, S.E. Patchett, D. Perrett, P.H. Katelaris, P. Domizio, M.J.G. Farthing, The relation between gastric vitamin C concentrations, mucosal histology, and CagA seropositivity in the human stomach, Gut 43 (1998) 322±326. K.B. Hahm, K.J. Lee, J.H. Kim, S.W. Cho, M.H. Chung, Helicobacter pylori infection, oxidative DNA damage, gastric carcinogenesis, and reversibility by rebamipide, Dig. Dis. 43 (1998) 72±77. B. Miller, F. Locher, A. Seelbach, H. Stopper, D. Utesch, S. Madle, Evaluation of the in vitro micronucleus test (MNT) as an alternative to the in vitro chromosomal aberration assay: position of the GUM working group ``in vitro MNT'', Mutat. Res. 410 (1998) 81±116. H. Stopper, S.O. MuÈller, Micronuclei: biological endpoint for genotoxicity, Toxicol. In Vitro 11 (1997) 661±667. R.D. Combes, H. Stopper, W.J. Caspary, The use of the L5178Y mouse lymphoma cells to assess mutagenic, clastogenic and aneugenic activity of chemicals, Mutagenesis 10 (5) (1995) 403±408.
149
[11] T. Ning, H. Ma, J. Zhou, Micronucleus effect induced by Helicobacter pylori on human gastric mucous epithelial membrane, Chung Hua Yu Fang I Hsueh Tsa Chih. 30 (3) (1996) 139±140. [12] M. Fenech, The cytokinesis-block micronucleus technique: a detailed description of the method and its application to genotoxicity studies in human populations, Mutat. Res. 285 (1993) 35±44. [13] J.M. Parry, A proposal for a new OECD guideline for the in vitro micronucleus test, Eur. J. Genet. Toxicol. 6 (1998) http:// www.swan.ac.uk/cget/jounal/ejgt15.htm. [14] A.C. Wotherspoon, C. Ortiz-Hidalgo, M.R. Falzon, P.G. Isaacson, Helicobacter pylori- associated gastritis and primary B-cell gastric lymphoma, Lancet 338 (1991) 1175±1176. [15] M. Eck, B. Schmauûer, R. Haas, A. Greiner, S. Czub, H.K. MuÈller-Hermelink, MALT-type lymphoma of the stomach is associated with Helicobacter pylori strains expressing the CagA protein, Gastroenterology 112 (5) (1997) 1482±1486. [16] J.C. Barrett, Cellular and molecular mechanisms of asbestos carcinogenicity: implications for biopersistence, Environ. Health Perspect 102 (Suppl. 5) (1994) 19±23.
www.elsevier.com/locate/canlet
Helicobacter pylori induces DNA damage in vitro Bernd Schmauûer a, Stefan O. Mueller b,1, Matthias Eck a, Marianne MoÈller b, HansKonrad MuÈller-Hermelink a, Helga Stopper b,* b
a Institute for Pathology, University of WuÈrzburg, D-97078 WuÈrzburg, Germany Department of Toxicology, University of WuÈrzburg, Versbacher Strasse 9, D-97078 WuÈrzburg, Germany
Received 5 October 1999; received in revised form 11 December 1999; accepted 11 December 1999
Abstract A close association between Helicobacter pylori infection and the development of gastric adenocarcinoma in humans has been demonstrated. Therefore, the direct induction of DNA damage by H. pylori was investigated here using the in vitro micronucleus assay. After 5 days of incubation with bacterial lysate a dose-dependent formation of micronuclei was found, which was not limited to cytotoxic protein concentrations and was not observed after treatment with Escherichia coli lysate (control). This induction of DNA damage may be a link between chronic H. pylori infection and development of adenocarcinoma of the stomach. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Helicobacter pylori; Micronuclei; L5178Y mouse lymphoma cells; DNA damage; Genotoxicity; Gastric carcinoma
1. Introduction Epidemiological studies have demonstrated a close association between Helicobacter pylori infection and the subsequent development of gastric adenocarcinoma in humans [1,2]. Therefore in 1994 the World Health Organization classi®ed H. pylori as a group I carcinogen in humans [3]. Recently the development of gastric carcinoma after infection with H. pylori has been demonstrated in the Mongolian gerbil [4], further supporting a role of H. pylori in carcinogenesis. Several mechanisms such as an increased proliferation of gastric epithelial cells during H. pylori in¯ammation [5] and lowered gastric ascorbic acid level [6] have been proposed in carcinogenesis. Recently an
increased level of oxidative damage, which was suggested to be induced by oxygen radicals during the persistent in¯ammation, was discussed as a link between H. pylori infection and gastric carcinogenesis [7]. In this study the direct induction of DNA damage by H. pylori was investigated using the in vitro micronucleus test, which is well established for genotoxicity testing [8,9]. In this test, microscopic observation enables detection of chromosomal fragments and maldistributed whole chromosomes, which are enclosed in a membrane and can be found in the cytoplasm of mammalian cells.
2. Materials and methods * Corresponding author. Tel.: 149-931-201-3427; fax: 149931-201-3446. E-mail address: [email protected] (H. Stopper) 1 Present address: NIEHS/NIH, RTP, NC, USA.
2.1. Cell culture Cells were cultured in suspension in RPMI-1640
0304-3835/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(99)00442-5
146
B. Schmauûer et al. / Cancer Letters 152 (2000) 145±149
supplemented with antibiotics, 0.25 mg/ml l-glutamine, 107 mg/ml sodium pyruvate, and 10% heat-inactivated horse serum (all from Sigma Chemie GmbH, Deisenhofen, Germany). Cell cultures were grown in a humidi®ed atmosphere with 5% CO2 in air at 378C. 2.2. Preparation of H. pylori and Escherichia coli lysate H. pylori strain 6650 was isolated from a patient with severe active gastritis on H. pylori agar (Biomerieux, France) microaerobically. The H. pylori strain was expanded microaerobically on Colombia blood agar (Oxoid, Basingstoke, UK) containing 10% inactivated de®brinated horse blood (30 min at 568C) for 96 h. The H. pylori strain was CagA positive as determined by PCR. E. coli was used as a control for a non-carcinogenic gram-negative intestinal bacterium. A urease-negative E. coli strain was isolated from human feces and grown on Colombia blood agar (Oxoid, Basingstoke, UK) for 24 h. Bacteria were harvested from agar plates and washed in 10 mmol Tris-buffered saline (TBS) (pH 7.4). Bacterial cells were sonicated for 3 £ 1 min on ice. Additional irradiation was required for the E. coli lysate to eliminate surviving bacteria. Total protein was determined by nephelometrie (Behring, Marburg, Germany). 2.3. In vitro micronucleus test We used the mammalian L5178Y mouse lymphoma cell line, the most frequently used cell line in routine genotoxicity testing [10]. Exponentially growing L5178Y mouse lymphoma cells (doubling time about 11 h) were seeded at a density of 2 £ 10 5/ml. Lysates of H. pylori and E. coli were added. After 24 h cell numbers were counted for assessment of cytotoxicity. Then cells were centrifuged, medium was replaced and cell density was again adjusted to 2 £ 10 5/ml to ensure ability for exponential cell growth. Fresh lysates were added. Negative controls were treated alike, but without addition of bacterial lysates. This was repeated four times simulating a chronic process. On the ®fth day cells were harvested. For this purpose, cells were brought onto glass slides by cytospin-centrifugation and ®xed with methanol (2208C for 1 h). To stain
nuclei and micronuclei, the slides were incubated with Hoechst 33258 (5 mg/ml in phosphate-buffered saline (PBS) for 3 min). Slides were washed with buffer and mounted for microscopy, using mounting medium (Linaris, Germany). Numbers of nuclei and micronuclei were scored at a magni®cation of 500£. One thousand nuclei were evaluated on each slide and two slides were evaluated per experiment. Objects were classi®ed as micronuclei, if they appeared to be separated from the nuclei, were round or oval, had an area of less than 1/4 of the area of a nucleus, and showed staining characteristics similar to those of the nuclei. 2.4. Cell viability (FDA/EB-method) Cell viability was measured at the time of cell harvesting in experiment no. 3. For this, a staining solution of 0.6% (v/v) ¯uorescein diacetate (FDA; 5 mg/ml in acetone) and 1.6% ethidium bromide (EB; 1 mg/ml in H2O) was prepared. Cells were stained by adding 15 ml of this solution to 35 ml of cell suspension. After 3 min at least 200 cells were evaluated using a ¯uorescence microscope at a magni®cation of 500£. Viable cells, containing active esterases, appeared green, whereas red nuclei indicated dead cells after uptake of ethidium bromide. 3. Results Micronucleus-induction is a well established method for the assessment of genotoxic damage. In this test, microscopic analysis of mammalian cells after DNA-speci®c staining reveals structures in the cytoplasm which resemble the main nucleus, but are smaller. They are formed by the cell during mitosis from chromosome fragments or whole displaced chromosomes which are enclosed in a separate nuclearlike membrane. Incubation of the cells with lysate from H. pylori for 5 days resulted reproducibly in the dose-dependent formation of micronuclei (Table 1). The frequency was elevated 3.5-fold (experiment 1), 3-fold (experiment 2) and 3.8-fold (experiment 3) over the respective control. The control experiments using lysate from E. coli revealed no induction of micronuclei over the control. Cytotoxicity induced by the lysates was measured by counting cell number on the day of cell harvest (Table 2). Cell number was
B. Schmauûer et al. / Cancer Letters 152 (2000) 145±149
147
Table 1 Induction of micronuclei in L5178Y mouse lymphoma cells treated with lysates of H. pylori and E. coli a Concentration (mg/ml protein)
0 5 10 15 20 25 30
H. pylori MN-cells/1000 cells
E. coli MN-cells/1000 cells
Experiment 1
Experiment 2
Experiment 3
Experiment 1
Experiment 2
Experiment 3
7.0 5.5 12.5 15.5 18.5 24.5 n.d.
6.7 10.3 13.3 13.0 20.3 n.d. n.d.
6.4 11.7 14.1 18.1 23.4 24.2 n.d.
13.5 6.0 13.0 7.0 8.0 7.0 11.0
6.3 7.7 13.0 8.3 13.2 8.8 6.6
7.8 9.2 13.2 13.9 8.4 5.8 5.9
a Each data point represents the mean of two slides from one culture dish with 1000 nuclei being evaluated on each slide. MN-cells, micronucleus-containing cells.
reduced after treatment with H. pylori lysate to 44% (experiment 1), 66% (experiment 2) and 78% (experiment 3) of the control. At the most toxic doses cells were still able to perform one cell doubling within the last day before harvest. In contrast, E. coli lysate induced little or no cytotoxicity. Cell viability as measured by the FDA/EB-method uptake was slightly reduced after treatment with lysate from H. pylori (78% of control at the highest tested concentration) and not reduced after treatment with E. coli lysate (96% of control at the highest tested concentration). Overall, micronuclei were not only induced at toxic ($20 mg/ml protein), but also at non- or slightly toxic doses (5±15 mg/ml protein) by H. pylori lysate, whereas the E. coli lysate did not induce micronuclei at the same and higher (30 mg/ml protein) protein concentrations.
4. Discussion A close association between H. pylori and gastric adenocarcinoma was demonstrated in several studies [1,2]. However, the mechanisms leading to carcinogenesis are not well understood. H. pylori infection leads to chronic antrum gastritis preceding the development of carcinoma. Therefore, oxidative DNA damage by oxygen radicals in chronic in¯ammation has been suggested [7]. As H. pylori persists in mucous overlaying the gastric epithelium, the question is arising as to whether H. pylori might directly be involved in the DNA damage process. In this study an induction of DNA damage (genotoxicity) by H. pylori was demonstrated in the micronucleus test, which may be a link between chronic H. pylori infection and development of adenocarcinoma of the stomach.
Table 2 Induction of cytotoxicity in L5178Y mouse lymphoma cells treated with lysates of H. pylori and E. coli a Concentration (mg/ml protein)
0 5 10 15 20 25 30 a
H. pylori cell number ( £ 10 5/ml)
E. coli cell number ( £ 10 5/ml)
Experiment 1
Experiment 2
Experiment 3
Experiment 1
Experiment 2
Experiment 3
9 10 7 7 4 4 n.d.
6 6 7 7 4 n.d. n.d.
9 8 8 8 7 7 n.d.
10 8 9 7 7 8 8
9 11 9 9 9 9 9
9 8 9 10 10 9 10
Given are cell numbers on day 5 (harvest). Cells were seeded on day one with a density of 2 £ 10 5 and diluted every day to this number.
148
B. Schmauûer et al. / Cancer Letters 152 (2000) 145±149
Our data are in line with an earlier publication by Ning et al. [11], who also found the formation of micronuclei in another cell line after treatment with H. pylori lysate. However, we have now shown that this micronucleus induction was not limited to cytotoxic protein concentrations, whereas these authors had not included cytotoxicity measurements. Additionally, we supported the relevance of the H. pylori-induced micronuclei by comparison with E. coli, another gastrointestinal bacterium, which did not induce cytotoxicity or genotoxicity at the same protein concentrations. Chromosome breakage leading to fragments as well as whole chromosomes, which are not correctly distributed during mitosis, give rise to the formation of micronuclei in the next interphase. Micronuclei are thus an indicator for DNA-damaging effects. The micronucleus test has been used extensively to assess DNA damage in humans after environmental exposure [12]. An international guideline for the use of the in vitro micronucleus test for assessment of DNA damage in the process of licensing a pharmaceutical or chemical is under development [13]. The formation of micronuclei may itself be involved in the carcinogenic process [9]. For example, the inclusion of a tumor suppressor gene in a micronucleus and the subsequent inactivation or loss of that micronucleus could lead to a transformed cell and therefore be a step in carcinogenesis. Even if micronuclei are not themselves involved in the carcinogenic process they are a sensitive indicator for DNA damage preceding the development of cancer. Usually incubation in the micronucleus test is performed for several hours (up to 1.5 cell cycle durations). With such a protocol we did not see any clear genotoxicity (data not shown). Therefore we extended the incubation period to several days, mimicking the biological in vivo situation (chronic process). Now, we observed a dose-dependent induction of micronuclei by lysate of H. pylori, but not by equal and higher protein concentrations of E. coli lysate. These results suggest that a factor contained in the lysate of H. pylori may exert genotoxic effects. It will be interesting to determine the chemical nature of this factor. Only a factor with small molecular size can be expected to diffuse into the cell for induction of DNA damage. For larger sized factors (e.g. proteins) the question is arising as to whether they are active
inside the cells (endocytosis) or whether they interact with the cellular outside (membrane or receptors). It will be of further interest whether this factor is identical with known virulence factors in H. pylori such as CagA, which is associated with gastric cancer or Malt-type lymphoma [14,15]. In our experiments micronucleus induction was not limited to concentrations with high cytotoxicity. Cytotoxicity can contribute to or mediate genotoxic effects. For example, one mechanism might be the formation of DNA strand breaks by DNA-degrading enzymes which have been released from cellular organelles destroyed by cytotoxic effects. On the other side, cytotoxicity in itself is a factor that can contribute to cell transformation if compensatory growth of surrounding cells is induced [16]. Investigation of the mechanism of genotoxicity exerted by H. pylori lysate is necessary. Furthermore, it remains to be determined whether such DNA damage can also be found in vivo during H. pylori infection, possibly preceding carcinogenesis. Acknowledgements This study was supported by the `Deutsche Forschungsgemeinschaft' grant GRK 73/3-97 to B.S. We thank Mrs M. Ruppert and Mrs M. Simet for their skilled technical assistance. References [1] J. Parsonnet, G.D. Friedmann, D.P. Vandersteen, Y. Chang, J.H. Vogelman, N. Orentreich, R.K. Sibley, Helicobacter pylori infection and the risk of gastric carcinoma, N. Engl. J. Med. 325 (1991) 1127±1131. [2] D. Foreman, Eurogast Study Group, An international association between Helicobacter pylori infection and gastric cancer, Lancet 3421 (1993) 359±362. [3] IARC Working Group on the Evaluation of Carcinogenic Risks to Humans. Helicobacter pylori. In: Schistosomes, Liver Flukes, and Helicobacter pylori: Views and Expert Opinions of an IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, IARC, Lyon, 1994, pp.177± 240. [4] T. Watanabe, M. Tada, H. Nagai, S. Sasaki, M. Nakao, Helicobacter pylori infection induces gastric cancer in Mongolian gerbils, Gastroenterology 115 (1998) 642±648. [5] M. Anti, A. Armuzzi, A. Gasbarrini, G. Gasbarrini, Importance of changes in epithelial cell turnover during Helicobac-
B. Schmauûer et al. / Cancer Letters 152 (2000) 145±149
[6]
[7]
[8]
[9] [10]
ter pylori infection in gastric carcinogenesis, Gut 43 (1998) 27±32. Z.W. Zhang, S.E. Patchett, D. Perrett, P.H. Katelaris, P. Domizio, M.J.G. Farthing, The relation between gastric vitamin C concentrations, mucosal histology, and CagA seropositivity in the human stomach, Gut 43 (1998) 322±326. K.B. Hahm, K.J. Lee, J.H. Kim, S.W. Cho, M.H. Chung, Helicobacter pylori infection, oxidative DNA damage, gastric carcinogenesis, and reversibility by rebamipide, Dig. Dis. 43 (1998) 72±77. B. Miller, F. Locher, A. Seelbach, H. Stopper, D. Utesch, S. Madle, Evaluation of the in vitro micronucleus test (MNT) as an alternative to the in vitro chromosomal aberration assay: position of the GUM working group ``in vitro MNT'', Mutat. Res. 410 (1998) 81±116. H. Stopper, S.O. MuÈller, Micronuclei: biological endpoint for genotoxicity, Toxicol. In Vitro 11 (1997) 661±667. R.D. Combes, H. Stopper, W.J. Caspary, The use of the L5178Y mouse lymphoma cells to assess mutagenic, clastogenic and aneugenic activity of chemicals, Mutagenesis 10 (5) (1995) 403±408.
149
[11] T. Ning, H. Ma, J. Zhou, Micronucleus effect induced by Helicobacter pylori on human gastric mucous epithelial membrane, Chung Hua Yu Fang I Hsueh Tsa Chih. 30 (3) (1996) 139±140. [12] M. Fenech, The cytokinesis-block micronucleus technique: a detailed description of the method and its application to genotoxicity studies in human populations, Mutat. Res. 285 (1993) 35±44. [13] J.M. Parry, A proposal for a new OECD guideline for the in vitro micronucleus test, Eur. J. Genet. Toxicol. 6 (1998) http:// www.swan.ac.uk/cget/jounal/ejgt15.htm. [14] A.C. Wotherspoon, C. Ortiz-Hidalgo, M.R. Falzon, P.G. Isaacson, Helicobacter pylori- associated gastritis and primary B-cell gastric lymphoma, Lancet 338 (1991) 1175±1176. [15] M. Eck, B. Schmauûer, R. Haas, A. Greiner, S. Czub, H.K. MuÈller-Hermelink, MALT-type lymphoma of the stomach is associated with Helicobacter pylori strains expressing the CagA protein, Gastroenterology 112 (5) (1997) 1482±1486. [16] J.C. Barrett, Cellular and molecular mechanisms of asbestos carcinogenicity: implications for biopersistence, Environ. Health Perspect 102 (Suppl. 5) (1994) 19±23.