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Laryngeal mucosa of head and neck cancer patients shows increased DNA damage as detected by single cell microgel electrophoresis
Toxicology 144 (2000) 149 – 154 www.elsevier.com/locate/toxicol
Laryngeal mucosa of head and neck cancer patients shows increased DNA damage as detected by single cell microgel electrophoresis P. Schmezer a,*, T. Rupprecht a, M. Tisch b, H. Maier b, H. Bartsch a a
Di6ision of Toxicology and Cancer Risk Factors, German Cancer Research Centre (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany b ENT Clinic, Bundeswehrkrankenhaus Ulm, 89081 Ulm, Germany
Abstract Major risk factors for cancer of the oral cavity, pharynx and larynx are smoking and excess alcohol consumption. Since long-term survival rates of head and neck cancer patients have not substantially been improved, new preventive strategies including the use of cancer chemopreventive agents have to be developed. With the aim of developing biomarkers which can verify the efficacy of chemopreventive interventions, a standardised alkaline microgel electrophoresis (MGE) assay was applied as a sensitive and rapid tool to detect DNA damage on a single cell level. Macroscopically normal laryngeal mucosa biopsies obtained by surgery from head and neck cancer patients (n = 29) and from hospital controls (n= 22) were analysed by MGE in a pilot study. As compared to controls, cells from head and neck cancer patients showed a significantly elevated DNA damage without any further genotoxic treatment (PB 0.01). We conclude that this increased background DNA damage in laryngeal epithelia could result from genetic alterations caused by smoking and alcohol leading, in accord with the field cancerisation hypothesis, to a gradual decrease of genomic stability and malignancy. MGE should now be explored as a rapid screening method in larger clinical studies: (i) to identify high-risk subjects carrying cells with decreased genomic stability and (ii) to verify the efficacy of chemopreventive regimens to prevent or slow down the development of head and neck cancer in high-risk persons. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Microgel electrophoresis; Head and neck cancer; DNA damage
1. Introduction Cancers of the upper aerodigestive tract are paradigms of environmentally induced diseases, as * Corresponding author. Tel.: +49-6221-42-3309; fax: + 49-6221-42-3359. E-mail address: [email protected] (P. Schmezer)
smoking and alcohol abuse are major risk factors (Baron et al., 1993). Since long-term survival rates of these head and neck cancer patients have not substantially been improved (Devesa et al., 1987; Becker and Wahrendorf, 1997; Me´ne´goz et al., 1997), new preventive strategies including the use of cancer chemopreventive agents have to be developed (Kelloff et al., 1993; Lippman et al., 1994). For this purpose, it is necessary to apply
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suitable biomarkers in order to verify the efficacy of chemopreventive regimens that can slow down or prevent tumour development. Furthermore, such markers are needed for identifying high-risk individuals who may profit most from such interventions. Most agents carcinogenic to humans exhibit genotoxic properties and are thought to cause cancer by inducing multiple genetic alterations within the cellular DNA. The alkaline microgel electrophoresis (MGE) assay has proven to be a sensitive and rapid tool to detect DNA damage on a single cell level. (Fairbairn et al., 1995; Schmezer, 1997; McGregor and Anderson, 1999). We therefore conducted a pilot study in order to find out whether the MGE assay can (i) be applied for the analysis of cells isolated from biopsies taken during endoscopy and (ii) identify individual differences in mutagen sensitivity and DNA damage in macroscopically normal laryngeal mucosa cells from head and neck cancer patients versus those from hospital control subjects.
2. Methods
2.1. Human subjects The study group consisted of 29 patients with newly diagnosed, previously untreated head and neck cancer and 22 hospital controls having no malignant diseases. Upon consent of the study
subjects, laryngeal mucosa biopsies were obtained during diagnostic endoscopy from controls. In the case of cancer patients, the specimens were taken from macroscopically healthy laryngeal mucosa located clearly distant from the tumour area. The donors were males and females aged 27–68 years, with varying stages of diseases. Other characteristics are outlined in Table 1 and were collected by questionnaire (Maier et al., 1992) including information on smoking habit, alcohol consumption, and other life-style or occupational exposures. Tissue samples were transported to the laboratory in cooled Joklik medium at 4°C. Laboratory analyses were performed blindly with regard to patient and control status.
2.2. Isolation of laryngeal mucosa cells Primary cell suspensions were prepared from laryngeal mucosa biopsies by treatment with proteolytic enzymes (Pool-Zobel et al., 1994). Cells were obtained by incubating the biopsy samples (pieces of B 2 mm diameter) at 37°C in a digestion mixture containing 5 mg protease, 1 mg collagenase, and 1 mg hyaluronidase in 1 ml Joklik medium. Cells were subsequently centrifuged at 400 rpm, and the pellet was resuspended in Joklik medium. Cell aliquots were analysed for viability using Trypan blue staining. The remaining cells were distributed into tubes for the in vitro incubation with mutagens.
Table 1 Characteristics of unaffected controls and patients with head and neck cancer Variables
Control subjects (n = 22) %
Cancer patients (n =29) %
P-value of comparison
Gender Male Female
73 27
93 7
Age Mean 9 S.D.
479 12
549 8
B0.05
Smoking status Mean pack-years 9 S.D.
22 9 22
409 19
B0.01
Alcohol consumption Mean daily intake in g 9 S.D.
609 70
125 9 80
B0.01
P. Schmezer et al. / Toxicology 144 (2000) 149–154
2.3. In 6itro treatment of laryngeal mucosa cells with mutagens The isolated cells were adjusted to 0.5 – 1× 105/ml Joklik medium. They remained either untreated (solvent control) or were treated with 50 mM N-nitrosodimethylamine (NDMA) or with 14 mM N-methyl-N%-nitro-N-nitrosoguanidine (MNNG) in a shaking water bath at 37°C for 1 h. Both mutagens were dissolved in DMSO (10 ml/1 ml Joklik medium). Following incubation, the cells were resuspended in fresh medium, and the viability was verified by Trypan blue staining prior to analysis of DNA damage.
2.4. Alkaline microgel electrophoresis assay The MGE assay was essentially performed as described by Singh et al. (1988) and Kuchenmeister et al. (1998) with minor modifications. Fully frosted microscope slides were covered with a bottom layer of normal melting agarose. 6 – 8 ×105 cells were mixed with 65 ml of a 0.7% low-melting agarose at 37°C and placed on top of the bottom layer. The slides were put on an ice-tray for 10 min to allow solidification of the agarose. Finally, the cell-containing middle layer was covered with a top layer of 75 ml low-melting agarose. The slides carrying the solidified cell containing gels were immersed in a lysing solution (100 mM Na-EDTA, 10 mM Tris, 2.5 mM NaCl, 1% Na-sarcosinate, 1% Triton-X100 and 10% DMSO, at pH 10) and kept at 4°C for at least 1 h. Following cell lysis, the slides were placed into a horizontal gel electrophoresis unit filled with alkaline electrophoresis buffer (1 mM Na-EDTA, 300 mM NaOH, at pH 13). After 20 min of alkali pre-treatment to allow DNA unwinding, electrophoresis was performed for 20 min at 25 V and 300 mA. Thereafter, the slides were rinsed with neutralisation buffer (0.4 M Tris, pH 7.5), stained with 20 mg/ml ethidium bromide, and kept in a humidified box. In order to prevent additional DNA damage all steps described were conducted under red light. The coded slides were evaluated using a fluorescence microscope coupled with an image analysis system (Perceptive Instruments, Essex, UK). The
151
DNA of an individual cell is seen as a cometlike spot with a head and a tail region. The more DNA damage has been introduced, the more DNA will migrate into the tail region during electrophoresis. The results are expressed either as overall comet length or percentage of undamaged cells (comet length 5 35 mm). Comet length and percentage of undamaged cells were determined for 51 randomly selected cells per slide with usually three parallel slides per data point.
2.5. Statistical analysis Samples were decoded and data analysed using the non-parametric Mann–Whitney U test. Effects of possible confounding factors were analysed for quantitative data by using the Student’s t-test, and for qualitative data by using the Fisher’s exact test. P values below 0.05 were regarded as statistically significant.
3. Results
3.1. General characteristics of control and cancer patients Macroscopically normal laryngeal mucosa cells from 22 hospital controls and 29 patients with head and neck cancer were analysed. The control subjects underwent endoscopy because of having either laryngeal polyps or cysts. These disorders had no influence on the observed amount of DNA damage present in the isolated cells. The cancer patients had a confirmed diagnosis of squamous-cell carcinoma of the oral cavity (24%), the oropharynx (14%), oro- and hypopharynx (7%), hypopharynx (14%), esophagus (3%), and larynx (38%). No significant influence of tumour localisation on the observed amount of DNA damage could be found, although the group size for some cancer patients (by site) was too small for a statistical analysis. Selected variables of control and tumour patients are given in Table 1. Male donors were more frequent in both groups. The mean age was significantly (PB 0.05) higher in head and
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P. Schmezer et al. / Toxicology 144 (2000) 149–154
Table 2 In vitro treatment of isolated laryngeal mucosa cells with the two mutagenic compounds NDMA and MNNG Treatmenta
Control subjects (n= 22)
Cancer patients (n = 29)
P-value of comparison
Sol6ent control (1% DMSO) Overall comet length (mm) Undamaged cells (%)c
479 16 45925
63 918 27 9 20
B0.05 B0.01
NDMA (50 mM) Increased comet length (mm)b Undamaged cells (%)c
26927 21919
16 9 18 15 9 14
MNNG (14 mM) Increased comet length (mm)b Undamaged cells (%)c
55920 49 12
35 9 22 091
B0.05
Mean cell viability was \90% in all groups. Values for treated cells minus solvent control are given. c Cells are designated as undamaged when overall comet length was 535 mm. a
b
neck cancer patients (5498 years) than in control subjects (479 12 years). All cancer patients and 81% of the controls were smokers, and on the basis of mean pack-years, there was a significantly (PB 0.01) higher exposure to tobacco smoke in cancer patients (40 versus 22 packyears). Furthermore, the cancer group showed daily intake values for alcohol which were more than twice as high as for the control subjects (125 versus 60 g; PB 0.01).
3.3. Differences in the spontaneous le6el of DNA damage The strongest difference in effect between groups of patients and control subjects was, however, observed in mucosa cells which received no mutagen treatment (solvent control). The amount of DNA damage, measured as background level, was clearly elevated in cells isolated from the laryngeal mucosa of head and
3.2. Measurement of DNA damage In order to assess differences in mutagen sensitivity, laryngeal mucosa cells were treated with two mutagens, NDMA or MNNG, resulting in a clear increase in DNA damage without affecting cell viability (Table 2). There was a trend for cells from control subjects to be more sensitive to the mutagenic challenge in comparison to cells isolated from tumour patients. With MNNG treatment, this effect reached statistical significance (PB 0.05) for the parameter ‘increased comet length’ but there was no difference when looking at the distribution of the cells into undamaged (overall comet length 5 35 mm) or damaged. This unexpected finding that control cells were apparently more sensitive may be related to the methodology used (Section 4).
Fig. 1. DNA damage expressed as overall comet length in macroscopically normal laryngeal mucosa cells; *adjusted for age, tobacco and alcohol consumption.
P. Schmezer et al. / Toxicology 144 (2000) 149–154
Fig. 2. Distribution of subjects according to their percentage prevalence of undamaged (overall comet length 5 35 mm) laryngeal mucosa cells. At a cut-off point of 49%, 12/22 control subjects but only 4/29 head and neck cancer patients showed high numbers of undamaged cells (P B 0.01; adjusted for age, tobacco and alcohol consumption).
neck cancer patients versus cells derived from control subjects (Fig. 1). The mean overall comet length was 63 and 47 mm, respectively. This difference between the two groups was highly significant when dichotomising at a value of 49% undamaged cells (close to the median value in control subjects): 12/22 controls but only 4/29 head and neck cancer patients were above this level (Fig. 2).
4. Discussion The MGE assay used was found to be a sensitive and rapid tool to detect DNA damage on a single cell level. This study demonstrated that it is suitable to analyse laryngeal mucosa cells which have been isolated from small biopsy samples taken during endoscopy. Similar results were obtained in cells isolated from other aerodigestive tract tissues such as nasal (Pool-Zobel et al., 1994; Harre´us et al., 1999) and buccal mucosa (Stone et al., 1995; Rojas et al., 1996). The evaluation of questionnaire data revealed that significant differences existed between control subjects and head and neck cancer patients with regard to age,
153
smoking habits, and alcohol consumption, all known risk factors for head and neck cancers such as laryngeal carcinomas (Maier et al., 1992; Cattaruzza et al., 1996). The MGE results therefore had to be adjusted for these confounding factors. Following adjustment, significant differences between tumour and control subjects were observed for mutagen sensitivity to MNNG and for the background level of DNA damage present in the isolated cells. The latter effect was stronger than the former, and this could at least in part be responsible for the difference in MNNG sensitivity. As the background level of DNA damage was already profoundly higher in the cells of cancer patients, an additional induction of DNA damage might not result in the same net increase as for lower background levels. This idea of a non-linear dose-response curve is supported by data from Fairbairn et al. (1995) who showed that for radiation-induced DNA damage, a linear relationship of the comet length with dose was only seen in small to moderate comet lengths. The major finding of our pilot study was that cells isolated from macroscopically normal laryngeal mucosa of head and neck cancer patients carried a significantly higher amount of background DNA lesions in comparison to cells from control subjects without cancer at this site. The increased level of damage could be the result of genetic alterations caused by smoking and alcohol leading to a gradual decrease of genomic stability, and is therefore in line with the theory of field cancerisation of aerodigestive-tract epithelia (Papadimitrakopoulou et al., 1996). Several other studies already indicated that many different types of genetic and tissue alterations, which could be induced by smoking and alcohol exposure, were found more frequently in macroscopically normal tissue of the upper aerodigestive tract in cancer patients, e.g. p53 mutations (Nees et al., 1993; Gallo and Bianchi, 1995; Waridel et al., 1997) and chromosomal aberrations (Voravoud et al., 1993; Smith et al., 1996). As the MGE assay described herein is more rapid and easier in analysing large sample numbers than most of the methods used so far, we propose MGE as a screening method to be explored in larger clinical studies: (a) to identify high-risk individuals carrying cells in the upper aerodigestive tract with decreased genomic stabil-
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ity, (b) to verify the efficacy of chemopreventive regimens which are aimed to prevent or to slow down the development of head and neck cancer from pre-damaged cells in these high-risk persons.
Acknowledgements We are thankful to Dr L. Edler and R. Rausch for their help and advice with the statistical evaluation.
References Baron, A.E., Franceschi, S., Barra, S., Talamini, R., La Vecchia, C., 1993. A comparison of the joint effects of alcohol and smoking on the risk of cancer across sites in the upper aerodigestive tract. Cancer Epidemiol. Biomark. Prev. 2, 519 – 523. Becker, N., Wahrendorf, J., 1997. Krebsatlas der Bundesrepublik Deutschland. Springer, Heidelberg. Cattaruzza, M.S., Maisonneuve, O., Boyle, P., 1996. Epidemiology of laryngeal cancer. Eur. J. Cancer B. Oral Oncol. 32, 293 – 305. Devesa, S.S., Silverman, D.T., Young, J.L., Pollack, E.S., Brown, C.C., Horm, J.W., Percy, C.L., Myers, M.H., McKay, F.W., Fraumeni, J.F., 1987. Cancer incidence and mortality trends among whites in the United States, 1947– 84. J. Natl. Cancer Inst. 79, 701–716. Fairbairn, D.W., Olive, P., O’Neill, K.L., 1995. The comet assay: a comprehensive review. Mutat. Res. 339, 37–59. Gallo, O., Bianchi, S., 1995. p53 expression: a potential biomarker for risk of multiple primary malignancies in the upper aerodigestive tract. Eur. J. Cancer B. Oral. Oncol. 31, 53 – 57. Harre´us, U., Schmezer, P., Kuchenmeister, F., Maier, H., 1999. Genotoxic effects in human epithelial cells of the upper aerodigestive tract. Laryngol. Rhinol. Otol. 78, 176– 181. Kelloff, G.J., Boone, C.W., Steele, V.K., Perloff, M., Crowell, J., Doody, L.A., 1993. Development of chemopreventive agents for lung and upper aerodigestive tract cancers. J. Cell. Biochem. Suppl. 17F, 2–17. Kuchenmeister, F., Schmezer, P., Engelhardt, G., 1998. Genotoxic bifunctional aldehydes produce specific images in the comet assay. Mutat. Res. 419, 69–78. Lippman, S.M., Benner, S.E., Hong, W.K., 1994. Retinoid chemoprevention studies in upper aerodigestive tract and lung carcinogenesis. Cancer Res. (Suppl.) 54, 2025s–2028s. Maier, H., Gewelke, U., Dietz, A., Heller, W.-D., 1992. Risk factors of cancer of the larynx: results of the Heidelberg
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case-control study. Otolaryngol. Head Neck Surg. 197, 577 – 582. McGregor, D.B., Anderson, D., 1999. DNA damage and repair in mammalian cells in vitro and in vivo as indicators of exposure to carcinogens. In: McGregor, D.B., Rice, J.M., Venitt, S. (Eds.), The Use of Short- and Mediumterm Tests for Carcinogens and Data on Genetic Effects in Carcinogenic Hazard Evaluation. IARC Scientific Publication No. 146, Lyon, pp. 309 – 354. Me´ne´goz, F., Black, R.J., Arveux, P., Magne, V., Ferlay, J., Bue´mi, A., Carli, P.M., Chapelain, G., Faivre, J., Gignoux, M., Grosclaude, P., Mace-Lesech, J., Raverdy, N., Schaffer, P., 1997. Cancer incidence and mortality in France in 1975 – 95. Eur. J. Cancer Prev. 6, 442 – 466. Nees, M., Homann, N., Discher, H., Andl, T., Enders, C., Herold-Mende, C., Schuhmann, A., Bosch, F.X., 1993. Expression of mutated p53 occurs in tumour-distant epithelia of head and neck cancer patients: a possible molecular basis for the development of multiple tumours. Cancer Res. 53, 4189 – 4196. Papadimitrakopoulou, V.A., Shin, C.M., Hong, W.K., 1996. Molecular and cellular biomarkers for field cancerisation and multistep process in head and neck cancer. Cancer Metastasis Rev. 15, 53 – 76. Pool-Zobel, B.L., Lotzmann, N., Knoll, M., Kuchenmeister, F., Lambertz, R., Leucht, U., Schro¨der, H.-G., Schmezer, P., 1994. Detection of genotoxic effects in human gastric and nasal mucosa cells isolated from biopsy samples. Environ. Mol. Mutagen. 24, 23 – 45. Rojas, E., Valverde, M., Sordo, M., Ostrosky-Wegman, P., 1996. DNA damage in exfoliated buccal cells of smokers assessed by the single cell gel electrophoresis assay. Mutat. Res. 370, 115 – 120. Schmezer, P., 1997. Mutation Research (Special Issue) The Comet Assay, Volume 375, No. 2. Singh, N.P., McCoy, M.T., Tice, R.R., Schneider, E.L., 1988. A simple technique for quantification of low levels of DNA damage in individual cells. Exp. Cell. Res. 175, 184 – 191. Smith, A.L., Hung, J., Walker, L., Rogers, T.E., Vuitch, F., Lee, E., Gazdar, A.F., 1996. Extensive areas of aneuploidy are present in the respiratory epithelium of lung cancer patients. Br. J. Cancer 73, 203 – 209. Stone, J.G., Jones, N.J., McGregor, A.D., Waters, R., 1995. Development of a human biomonitoring assay using buccal mucosa: comparison of smoking-related DNA adducts in mucosa versus biopsies. Cancer Res. 55, 1267 – 1270. Voravoud, N., Shin, D.M., Ro, J.Y., Lee, J.S., Hong, W.K., Hittelman, W.N., 1993. Increased polysomies of chromosomes 7 and 17 during head and neck multistage tumorigenesis. Cancer Res. 53, 2874 – 2883. Waridel, F., Estreicher, A., Bron, L., Flaman, J.-M., Fontolliet, C., Monnier, P., Frebourg, T., Iggo, L.R., 1997. Field cancerisation and polyclonal p53 mutation in the upper aerodigestive tract. Oncogene 14, 163 – 169.
Laryngeal mucosa of head and neck cancer patients shows increased DNA damage as detected by single cell microgel electrophoresis P. Schmezer a,*, T. Rupprecht a, M. Tisch b, H. Maier b, H. Bartsch a a
Di6ision of Toxicology and Cancer Risk Factors, German Cancer Research Centre (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany b ENT Clinic, Bundeswehrkrankenhaus Ulm, 89081 Ulm, Germany
Abstract Major risk factors for cancer of the oral cavity, pharynx and larynx are smoking and excess alcohol consumption. Since long-term survival rates of head and neck cancer patients have not substantially been improved, new preventive strategies including the use of cancer chemopreventive agents have to be developed. With the aim of developing biomarkers which can verify the efficacy of chemopreventive interventions, a standardised alkaline microgel electrophoresis (MGE) assay was applied as a sensitive and rapid tool to detect DNA damage on a single cell level. Macroscopically normal laryngeal mucosa biopsies obtained by surgery from head and neck cancer patients (n = 29) and from hospital controls (n= 22) were analysed by MGE in a pilot study. As compared to controls, cells from head and neck cancer patients showed a significantly elevated DNA damage without any further genotoxic treatment (PB 0.01). We conclude that this increased background DNA damage in laryngeal epithelia could result from genetic alterations caused by smoking and alcohol leading, in accord with the field cancerisation hypothesis, to a gradual decrease of genomic stability and malignancy. MGE should now be explored as a rapid screening method in larger clinical studies: (i) to identify high-risk subjects carrying cells with decreased genomic stability and (ii) to verify the efficacy of chemopreventive regimens to prevent or slow down the development of head and neck cancer in high-risk persons. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Microgel electrophoresis; Head and neck cancer; DNA damage
1. Introduction Cancers of the upper aerodigestive tract are paradigms of environmentally induced diseases, as * Corresponding author. Tel.: +49-6221-42-3309; fax: + 49-6221-42-3359. E-mail address: [email protected] (P. Schmezer)
smoking and alcohol abuse are major risk factors (Baron et al., 1993). Since long-term survival rates of these head and neck cancer patients have not substantially been improved (Devesa et al., 1987; Becker and Wahrendorf, 1997; Me´ne´goz et al., 1997), new preventive strategies including the use of cancer chemopreventive agents have to be developed (Kelloff et al., 1993; Lippman et al., 1994). For this purpose, it is necessary to apply
0300-483X/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 0 - 4 8 3 X ( 9 9 ) 0 0 2 0 1 - 2
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P. Schmezer et al. / Toxicology 144 (2000) 149–154
suitable biomarkers in order to verify the efficacy of chemopreventive regimens that can slow down or prevent tumour development. Furthermore, such markers are needed for identifying high-risk individuals who may profit most from such interventions. Most agents carcinogenic to humans exhibit genotoxic properties and are thought to cause cancer by inducing multiple genetic alterations within the cellular DNA. The alkaline microgel electrophoresis (MGE) assay has proven to be a sensitive and rapid tool to detect DNA damage on a single cell level. (Fairbairn et al., 1995; Schmezer, 1997; McGregor and Anderson, 1999). We therefore conducted a pilot study in order to find out whether the MGE assay can (i) be applied for the analysis of cells isolated from biopsies taken during endoscopy and (ii) identify individual differences in mutagen sensitivity and DNA damage in macroscopically normal laryngeal mucosa cells from head and neck cancer patients versus those from hospital control subjects.
2. Methods
2.1. Human subjects The study group consisted of 29 patients with newly diagnosed, previously untreated head and neck cancer and 22 hospital controls having no malignant diseases. Upon consent of the study
subjects, laryngeal mucosa biopsies were obtained during diagnostic endoscopy from controls. In the case of cancer patients, the specimens were taken from macroscopically healthy laryngeal mucosa located clearly distant from the tumour area. The donors were males and females aged 27–68 years, with varying stages of diseases. Other characteristics are outlined in Table 1 and were collected by questionnaire (Maier et al., 1992) including information on smoking habit, alcohol consumption, and other life-style or occupational exposures. Tissue samples were transported to the laboratory in cooled Joklik medium at 4°C. Laboratory analyses were performed blindly with regard to patient and control status.
2.2. Isolation of laryngeal mucosa cells Primary cell suspensions were prepared from laryngeal mucosa biopsies by treatment with proteolytic enzymes (Pool-Zobel et al., 1994). Cells were obtained by incubating the biopsy samples (pieces of B 2 mm diameter) at 37°C in a digestion mixture containing 5 mg protease, 1 mg collagenase, and 1 mg hyaluronidase in 1 ml Joklik medium. Cells were subsequently centrifuged at 400 rpm, and the pellet was resuspended in Joklik medium. Cell aliquots were analysed for viability using Trypan blue staining. The remaining cells were distributed into tubes for the in vitro incubation with mutagens.
Table 1 Characteristics of unaffected controls and patients with head and neck cancer Variables
Control subjects (n = 22) %
Cancer patients (n =29) %
P-value of comparison
Gender Male Female
73 27
93 7
Age Mean 9 S.D.
479 12
549 8
B0.05
Smoking status Mean pack-years 9 S.D.
22 9 22
409 19
B0.01
Alcohol consumption Mean daily intake in g 9 S.D.
609 70
125 9 80
B0.01
P. Schmezer et al. / Toxicology 144 (2000) 149–154
2.3. In 6itro treatment of laryngeal mucosa cells with mutagens The isolated cells were adjusted to 0.5 – 1× 105/ml Joklik medium. They remained either untreated (solvent control) or were treated with 50 mM N-nitrosodimethylamine (NDMA) or with 14 mM N-methyl-N%-nitro-N-nitrosoguanidine (MNNG) in a shaking water bath at 37°C for 1 h. Both mutagens were dissolved in DMSO (10 ml/1 ml Joklik medium). Following incubation, the cells were resuspended in fresh medium, and the viability was verified by Trypan blue staining prior to analysis of DNA damage.
2.4. Alkaline microgel electrophoresis assay The MGE assay was essentially performed as described by Singh et al. (1988) and Kuchenmeister et al. (1998) with minor modifications. Fully frosted microscope slides were covered with a bottom layer of normal melting agarose. 6 – 8 ×105 cells were mixed with 65 ml of a 0.7% low-melting agarose at 37°C and placed on top of the bottom layer. The slides were put on an ice-tray for 10 min to allow solidification of the agarose. Finally, the cell-containing middle layer was covered with a top layer of 75 ml low-melting agarose. The slides carrying the solidified cell containing gels were immersed in a lysing solution (100 mM Na-EDTA, 10 mM Tris, 2.5 mM NaCl, 1% Na-sarcosinate, 1% Triton-X100 and 10% DMSO, at pH 10) and kept at 4°C for at least 1 h. Following cell lysis, the slides were placed into a horizontal gel electrophoresis unit filled with alkaline electrophoresis buffer (1 mM Na-EDTA, 300 mM NaOH, at pH 13). After 20 min of alkali pre-treatment to allow DNA unwinding, electrophoresis was performed for 20 min at 25 V and 300 mA. Thereafter, the slides were rinsed with neutralisation buffer (0.4 M Tris, pH 7.5), stained with 20 mg/ml ethidium bromide, and kept in a humidified box. In order to prevent additional DNA damage all steps described were conducted under red light. The coded slides were evaluated using a fluorescence microscope coupled with an image analysis system (Perceptive Instruments, Essex, UK). The
151
DNA of an individual cell is seen as a cometlike spot with a head and a tail region. The more DNA damage has been introduced, the more DNA will migrate into the tail region during electrophoresis. The results are expressed either as overall comet length or percentage of undamaged cells (comet length 5 35 mm). Comet length and percentage of undamaged cells were determined for 51 randomly selected cells per slide with usually three parallel slides per data point.
2.5. Statistical analysis Samples were decoded and data analysed using the non-parametric Mann–Whitney U test. Effects of possible confounding factors were analysed for quantitative data by using the Student’s t-test, and for qualitative data by using the Fisher’s exact test. P values below 0.05 were regarded as statistically significant.
3. Results
3.1. General characteristics of control and cancer patients Macroscopically normal laryngeal mucosa cells from 22 hospital controls and 29 patients with head and neck cancer were analysed. The control subjects underwent endoscopy because of having either laryngeal polyps or cysts. These disorders had no influence on the observed amount of DNA damage present in the isolated cells. The cancer patients had a confirmed diagnosis of squamous-cell carcinoma of the oral cavity (24%), the oropharynx (14%), oro- and hypopharynx (7%), hypopharynx (14%), esophagus (3%), and larynx (38%). No significant influence of tumour localisation on the observed amount of DNA damage could be found, although the group size for some cancer patients (by site) was too small for a statistical analysis. Selected variables of control and tumour patients are given in Table 1. Male donors were more frequent in both groups. The mean age was significantly (PB 0.05) higher in head and
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P. Schmezer et al. / Toxicology 144 (2000) 149–154
Table 2 In vitro treatment of isolated laryngeal mucosa cells with the two mutagenic compounds NDMA and MNNG Treatmenta
Control subjects (n= 22)
Cancer patients (n = 29)
P-value of comparison
Sol6ent control (1% DMSO) Overall comet length (mm) Undamaged cells (%)c
479 16 45925
63 918 27 9 20
B0.05 B0.01
NDMA (50 mM) Increased comet length (mm)b Undamaged cells (%)c
26927 21919
16 9 18 15 9 14
MNNG (14 mM) Increased comet length (mm)b Undamaged cells (%)c
55920 49 12
35 9 22 091
B0.05
Mean cell viability was \90% in all groups. Values for treated cells minus solvent control are given. c Cells are designated as undamaged when overall comet length was 535 mm. a
b
neck cancer patients (5498 years) than in control subjects (479 12 years). All cancer patients and 81% of the controls were smokers, and on the basis of mean pack-years, there was a significantly (PB 0.01) higher exposure to tobacco smoke in cancer patients (40 versus 22 packyears). Furthermore, the cancer group showed daily intake values for alcohol which were more than twice as high as for the control subjects (125 versus 60 g; PB 0.01).
3.3. Differences in the spontaneous le6el of DNA damage The strongest difference in effect between groups of patients and control subjects was, however, observed in mucosa cells which received no mutagen treatment (solvent control). The amount of DNA damage, measured as background level, was clearly elevated in cells isolated from the laryngeal mucosa of head and
3.2. Measurement of DNA damage In order to assess differences in mutagen sensitivity, laryngeal mucosa cells were treated with two mutagens, NDMA or MNNG, resulting in a clear increase in DNA damage without affecting cell viability (Table 2). There was a trend for cells from control subjects to be more sensitive to the mutagenic challenge in comparison to cells isolated from tumour patients. With MNNG treatment, this effect reached statistical significance (PB 0.05) for the parameter ‘increased comet length’ but there was no difference when looking at the distribution of the cells into undamaged (overall comet length 5 35 mm) or damaged. This unexpected finding that control cells were apparently more sensitive may be related to the methodology used (Section 4).
Fig. 1. DNA damage expressed as overall comet length in macroscopically normal laryngeal mucosa cells; *adjusted for age, tobacco and alcohol consumption.
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Fig. 2. Distribution of subjects according to their percentage prevalence of undamaged (overall comet length 5 35 mm) laryngeal mucosa cells. At a cut-off point of 49%, 12/22 control subjects but only 4/29 head and neck cancer patients showed high numbers of undamaged cells (P B 0.01; adjusted for age, tobacco and alcohol consumption).
neck cancer patients versus cells derived from control subjects (Fig. 1). The mean overall comet length was 63 and 47 mm, respectively. This difference between the two groups was highly significant when dichotomising at a value of 49% undamaged cells (close to the median value in control subjects): 12/22 controls but only 4/29 head and neck cancer patients were above this level (Fig. 2).
4. Discussion The MGE assay used was found to be a sensitive and rapid tool to detect DNA damage on a single cell level. This study demonstrated that it is suitable to analyse laryngeal mucosa cells which have been isolated from small biopsy samples taken during endoscopy. Similar results were obtained in cells isolated from other aerodigestive tract tissues such as nasal (Pool-Zobel et al., 1994; Harre´us et al., 1999) and buccal mucosa (Stone et al., 1995; Rojas et al., 1996). The evaluation of questionnaire data revealed that significant differences existed between control subjects and head and neck cancer patients with regard to age,
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smoking habits, and alcohol consumption, all known risk factors for head and neck cancers such as laryngeal carcinomas (Maier et al., 1992; Cattaruzza et al., 1996). The MGE results therefore had to be adjusted for these confounding factors. Following adjustment, significant differences between tumour and control subjects were observed for mutagen sensitivity to MNNG and for the background level of DNA damage present in the isolated cells. The latter effect was stronger than the former, and this could at least in part be responsible for the difference in MNNG sensitivity. As the background level of DNA damage was already profoundly higher in the cells of cancer patients, an additional induction of DNA damage might not result in the same net increase as for lower background levels. This idea of a non-linear dose-response curve is supported by data from Fairbairn et al. (1995) who showed that for radiation-induced DNA damage, a linear relationship of the comet length with dose was only seen in small to moderate comet lengths. The major finding of our pilot study was that cells isolated from macroscopically normal laryngeal mucosa of head and neck cancer patients carried a significantly higher amount of background DNA lesions in comparison to cells from control subjects without cancer at this site. The increased level of damage could be the result of genetic alterations caused by smoking and alcohol leading to a gradual decrease of genomic stability, and is therefore in line with the theory of field cancerisation of aerodigestive-tract epithelia (Papadimitrakopoulou et al., 1996). Several other studies already indicated that many different types of genetic and tissue alterations, which could be induced by smoking and alcohol exposure, were found more frequently in macroscopically normal tissue of the upper aerodigestive tract in cancer patients, e.g. p53 mutations (Nees et al., 1993; Gallo and Bianchi, 1995; Waridel et al., 1997) and chromosomal aberrations (Voravoud et al., 1993; Smith et al., 1996). As the MGE assay described herein is more rapid and easier in analysing large sample numbers than most of the methods used so far, we propose MGE as a screening method to be explored in larger clinical studies: (a) to identify high-risk individuals carrying cells in the upper aerodigestive tract with decreased genomic stabil-
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ity, (b) to verify the efficacy of chemopreventive regimens which are aimed to prevent or to slow down the development of head and neck cancer from pre-damaged cells in these high-risk persons.
Acknowledgements We are thankful to Dr L. Edler and R. Rausch for their help and advice with the statistical evaluation.
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