Comparing the genotoxic sensitivities of human peripheral blood lymphocytes and mucosa cells of the upper aerodigestive tract using the Comet assay.

Carcinogenesis in the upper aerodigestive tract is influenced by multiple factors. Besides tobacco and alcohol consumption, specific pollutants such as phthalates, nitrosamines, and polycyclic aromatic carbohydrates may be important in tumor initiation. Genetic factors related to mutagen sensitivity and DNA repair capacity also play a role. The aim of this study was to investigate whether human peripheral blood lymphocytes and mucosal epithelium of the upper aerodigestive tract, the target for volatile and liquid xenobiotics, are equally sensitive to genotoxic agents. The Comet assay was used to detect for DNA damage induced by genotoxic agents in mucosal epithelial cells and peripheral blood lymphocytes of 60 volunteers. Mucosa was harvested from larynx, oropharynx, and inferior nasal turbinates. Xenobiotics investigated were dibutylphthalate (DBP), diisobutylphthalate (DiBP), N'-nitrosodiethylamine (NDELA), benzo[a]pyrene (B[a]P), and N'-methyl-N'-nitro-N-nitrosoguanidine (MNNG). DBP, DiBP, B[a]P, NDELA and MNNG induced a significant increase in DNA migration in both cell populations. Peripheral blood lymphocytes were more sensitive than mucosal cells to DBP and DiBP, but not to NDELA and B[a]P. The correlation, in terms of DNA migration, between lymphocytes and mucosal cells among volunteers was relatively poor. Based on the poor correlation in response between the two cell types, the sensitivity of peripheral blood lymphocytes to genotoxic agents appears to be a poor predictor of sensitivity in the target cells of the upper aerodigestive tract. Further attention should be focused on intra-individual mutagen sensitivities and inter-individual genetic differences as regards susceptibility to upper aerodigestive tract cancer.

Mutagen sensitivity of nasopharyngeal cancer patients.

Primary nasopharyngeal carcinomas (NPCs) may be of various types, including squamous cell carcinomas, undifferentiated carcinomas, and lymphoepitheliomas. Tumor initiation has been linked to the Epstein-Barr virus and, in some geographical regions, to alimentary factors. Possible hereditary components for the appearance of NPCs have not yet been clearly identified. In this study, genetic sensitivity to the genotoxic effects of carcinogenic xenobiotics as an endogenous risk factor of tumor initiation was investigated. The single cell microgel electrophoresis assay was used to quantify chemically-induced DNA damage in lymphocytes of 30 NPC patients and 30 non-tumor donors. The xenobiotics investigated were N'-nitrosodiethylamine, sodium dichromate, and nickel sulphate, with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and dimethyl sulfoxide (DMSO) as positive and negative controls, respectively. The extent of DNA migration in the solvent control cultures was not significantly different between the two groups (1.2+/-0.5 mean Olive tail moment and standard deviation of 30 individuals for NPC patients; 1.1+/-0.4 for non-tumor donors). With constant exposure and electrophoretic conditions, genotoxic effects of varying degrees were induced by the different xenobiotics in tumor and non-tumor patients (nickel sulphate: 7.1+/-2.5 for NPC patients and 5.9+/-1.6 for non-tumor donors; sodium dichromate: 18.1+/-5.3 for NPC patients and 16.2+/-5.4 for non-tumor donors; MNNG: 47.8+/-13.3 for NPC patients and 52.7+/-13.6 for non-tumor donors). Only N'-nitrosodiethylamine proved to induce significantly more DNA migration in lymphocytes of tumor patients (9.8+/-3.1) as compared to non-tumor patients (8.2+/-2.3). Although for sodium dichromate the degree of DNA migration did not significantly differ, variability in migration patterns proved to be lower in the tumor group. Mutagen sensitivity of NPC patients was shown to be elevated for a selected xenobiotic, whereas a general elevation of DNA fragility was not present. Further studies on mutagen sensitivity as an endogenous risk factor influencing the susceptibility of patients at the time of first diagnosis of nasopharyngeal carcinomas are warranted.

[Carcinogenic and co-carcinogenic effects of metals and ethanol on human salivary gland tissue]. / Karzinogene und kokarzinogene Effekte von Metallen und Ethylalkohol in humanen Speicheldrüsenzellen.

BACKGROUND: The etiology of malignomas of human salivary glands is examined. MATERIAL AND METHODS: Macroscopic, healthy salivary gland tissue from 46 donors was harvested during surgery. Single cells were isolated by enzymatic digestion. These were then incubated for 60 min with Na(2)Cr(2)O(7), NiSO(4), CdSO(4), ZnCl(2) and ethanol. Additionally, incubation with Na(2)Cr(2)O(7) was combined with NiSO(4), CdSO(4), ZnCl(2) and ethanol. The influence of CdSO(4) was analyzed by altered combinations with Na(2)Cr(2)O(7) during incubation and by the DNA-repair period. Evaluation was performed using fluorescent staining and digital analysis. RESULTS: Of all of the substances tested, only Na(2)Cr(2)O(7) showed genotoxic effects. NiSO(4), ZnCl(2) and ethanol had neither genotoxic nor cofactorial impacts. CdSO(4), however, caused additional genotoxic effects in combination with Na(2)Cr(2)O(7), although it lacked direct genotoxic potential. A reduction of DNA-repair of Na(2)Cr(2)O(7)-induced oxidative damage by CdSO(4) could be demonstrated. CONCLUSIONS: In this investigation, sodium dichromate was identified as genotoxic in association with human salivary gland tissue. These effects could be increased by CdSO(4), reinforcing DNA damage based on oxidative stress.

[Mini-organ cultures of human nasal mucosa. A model for eco-genotoxicological investigations]. / Miniorgankulturen humaner nasaler Mukosa. Ein Modell für ökogenotoxikologische Untersuchungen.

BACKGROUND: Volatile and ingestive xenobiotics may induce cancer in the mucosa of the upper aerodigestive tract. A new model is presented combining mini-organ cultures of human mucosa and the Comet assay that allows investigation of tumor initiation steps in vitro. METHOD: Specimens of human mucosa of the inferior nasal turbinates were cultured as mini-organs and exposed to xenobiotics once, twice or three times with consecutive repair intervals. The cultures were monitored for structural integrity (inverse microscopy, histology), DNA fragmentation and repair activity (Comet assay), induction of apoptosis (annexin V assay), and production of IL-8 and GM-CSF (ELISA). RESULTS: Mini-organ cultures showed a good structural integrity during the whole culture period. Exposure to N-nitrosodiethylamine (NDEA) and benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) induced significant DNA fragmentation. Sodium dichromate (Na2Cr2O7) had an additive DNA fragmentation effect with repetitive exposure. Significant DNA repair was seen after strand break induction by Na2Cr2O7, only. Apoptosis was seen after three exposures to BPDE und Na2Cr2O7, but not NDEA. Inflammatory cytokine release was unaltered by NDEA. However, BPDE and Na2Cr2O7 reduced GM-CSF and Na2Cr2O7 reduced IL-8 excretion. CONCLUSION: This three dimensional mini-organ culture system proved to be very helpful in characterizing volatile and ingestive xenobiotics potentially hazardous to humans. Beside the information concerning genotoxicity, it allows cytological and immunological studies. In contrast to investigations with fresh specimens, repetitive or chronic exposure to xenobiotics is possible in mucosal cells with their epithelial structural integrity. Therefore, mini-organ cultures of human upper aerodigestive tract epithelia represent a model closely resembling the in vivo situation.

[DNA repair if mucous membrane cells and lymphocytes with the comet assay]. / Untersuchungen zur DNA-Reparatur von Schleimhautzellen und Lymphozyten mit dem Comet Assay.

BACKGROUND: Exogenous and endogenous risk factors are involved in human carcinogenesis of the head and neck. Noteworthy hereditary factors include mutagen sensitivity and the individual's capacity for DNA repair. Repair mechanisms influence different phases of mutation and malignant transformation. The present study introduces a highly sensitive method for evaluating the repair capacity of human mucosal cells and lymphocytes. METHOD: Human epithelia of the nose and peripheral lymphocytes were incubated with the tobacco-related carcinogen N'nitrosodiethylamine (NDEA). The solvent dimethylsulfoxide (DMSO) served as negative control. Following repair times of 0 min, 15 min and 30 min, the cells were subjected to a modified version of the alkaline microgel electrophoresis technique (Comet assay). The data were digitally analyzed after fluorescent staining. RESULTS: Using the Comet assay, DNA repair could be quantified in human mucosal cells and in lymphocytes. The majority of DNA strand breaks induced by NDEA were repaired within 15 min in both cell types. CONCLUSIONS: Up to now, the Comet assay has been the preferred method for demonstrating substance-induced DNA damage. It has been used in repair studies involving lymphocytes, bacterial systems and animal-derived cells. A modified version of this method, however, can be used to quantify DNA repair in human mucosal cells and peripheral lymphocytes targeted by carcinogens. It is thus possible to evaluate an endogenous factor involved in the development of malignant transformations in mucosal cells of the upper aerodigestive tract.

[Cytotoxicity and genotoxicity of fluorides in human mucosa and lymphocytes]. / Zytotoxizität und Genotoxizität von Fluoriden in humaner Mukosa und Lymphozyten.

BACKGROUND: Fluorides are widely used in dental health products and drinking water, due to their beneficial effects in caries-prophylaxis and -treatment. Nevertheless, irritation of the gingiva and oropharyngeal mucosa as well as in gastric mucosa is observed since neither local nor systemic application is restricted to the teeth. These effects may partly be attributed to a known cytotoxicity of fluorides. Whether fluorides also have genotoxic effects on human mucosa or lymphocytes as a possible factor in tumor initiation was investigated in this study. MATERIAL AND METHODS: Human oropharyngeal epithelial cells and peripheral lymphocytes were incubated after single cell preparation with the aminefluoride Olaflur at concentrations of 2 ppm, 21 ppm, 35 ppm, 71 ppm and 213 ppm. The extent of cytotoxicity was investigated using the trypan blue exclusion test. Following incubation, electrophoresis for migration of DNA fragments, fluorescence staining and digital image analysis according to a standard protocol of the single cell microgel electrophoresis assay (Comet assay) followed. DNA damage was characterized using the Olive Tail Moment (OTM). RESULTS: For fluoride concentrations of 2 ppm to 35 ppm, non vital cells of less than 10% could be shown. After incubation with 71 ppm and 213 ppm Olaflur, there were 15% and 43% of damaged cells, respectively. Weak genotoxic effects on mucosal cells as well as on lymphocytes could be demonstrated at all concentrations tested. In fluoride concentrations of 213 ppm genotoxicity increased to max. OTM-levels of 23. CONCLUSIONS: Beside the cytotoxic effect of fluorides, also a minor genotoxic impact on human mucosa and on peripheral lymphocytes could be demonstrated using the Comet assay. Further investigations are warranted to examine fluorides in a model allowing for repeated or long term incubations on structurally intact human mucosa in vitro. Such a model will help to distinguish between DNA damage that may be repaired successfully and other impairments that may show an additive character in repetitive or chronic exposure in vivo.

[Genotoxicity of phthalates. On the discussion of plasticizers in children's toys]. / Genotoxizität von Phthalaten. Zur Diskussion über Weichmacher in Kinderspielzeug.

BACKGROUND AND OBJECTIVE: Recently, health hazards caused by phthalates, which are added as softeners to plastic materials, have been subject to discussion. The aim of the present study was to measure possible genotoxic impacts on mucosal cells of the upper aerodigestive tract. PATIENTS AND METHODS: Genotoxicity tests for dibutyl phthalate (DBP) and diisobutyl phthalate (DiBP) on human oropharyngeal mucosa in vitro were performed using the alkaline comet assay. Specimens (n = 50) were harvested from the surface of ectomized tonsils. RESULTS: DBP and DiBP caused significant DNA damage in human mucosal cells of the upper aerodigestive tract. The impact of DiBP was higher than that of DBP. CONCLUSIONS: A genotoxic impact of phthalates on human epithelial cells as a hazard to babies and children chewing these materials cannot be excluded and demands further investigation. The DNA damage measured in this study may represent one factor in the complex genesis of neoplasms in the upper aerodigestive tract.

Genotoxicity of di-butyl-phthalate and di-iso-butyl-phthalate in human lymphocytes and mucosal cells.

The genotoxicity of phthalates, widely used plasticizers, has been shown previously for di-butyl-phthalate (DBP) and di-iso-butyl-phthalate (DBP) in human mucosal cells of the upper aerodigestive tract in a previous study using the Comet assay. Furthermore, higher genotoxic sensitivities of patients with squamous cell carcinomas of either the larynx or the oropharynx compared to non-tumor patients were described. Other authors have demonstrated DNA damage by a different phthalate in human lymphocytes. It was the aim of the present study to determine whether there is a correlation between the genotoxic sensitivities to DBP and its isomer DiBP in either mucosal cells or lymphocytes. The single-cell microgel electrophoresis assay (Comet assay) was applied to detect DNA strand breaks in human epithelial cells of the upper aerodigestive tract (n=132 specimens). Human mucosa was harvested from the oropharynx in non-tumor patients and patients with squamous cell carcinomas of the oropharynx. Laryngeal mucosa of patients with laryngeal squamous cell carcinomas was harvested as well. Peripheral lymphocytes (n=49 specimens) were separated from peripheral blood. Xenobiotics investigated were DBP, DiBP, and N'methyl-N'-nitro-N-nitrosoguanidine (MNNG) as positive control, respectively. For statistical analysis, the SPSS correlation analysis according to Pearson and the Wilcoxon test were performed. Genotoxicity was found for DBP and DiBP in epithelial cells and lymphocytes (P<0.001). MNNG caused severe DNA damage. In analyzing DBP and DiBP results, genotoxic impacts in mucosal cells showed an intermediate correlation (r=0.570). Correlation in lymphocytes was the same (r=0.570). Phthalates have been investigated as a potential health hazard for a variety of reasons, including possible xenoestrogenic impact, peroxisome proliferation, and membrane destabilization. The present investigation suggests a correlated DNA-damaging impact of DBP and DiBP in human mucosal cells and in lymphocytes, respectively.

[Mutagen sensitivity of patients with laryngeal and oropharyngeal carcinoma]. / Mutagensensitivität von Patienten mit Larynx- bzw. Oropharynxkarzinomen.

BACKGROUND: Carcinogenesis in the larynx and oropharynx is often associated with excessive exposure to tobacco smoke and alcohol. However, attention is increasingly being focused on genetically determined mutagen sensitivities and on the mutagenic impact of xenobiotics. The purpose of this study was to evaluate the genotoxicity of phthalates (plasticizers widely used in synthetic materials), as well as nitrosamines and polycyclic aromatic carbohydrates, on laryngeal and oropharyngeal epithelia and peripheral lymphocytes of patients with laryngeal and oropharyngeal carcinomas. METHODS: The comet assay was used to detect induced DNA strand breaks. Macroscopically healthy supraglottic and oropharyngeal epithelia of patients with laryngeal and oropharyngeal tumors, respectively, and lymphocytes were investigated with dibutyl phthalate (DBP), diisobutylphthalate (DiBP). N'nitrosodiethylamine (NDELA), and benzo[a]pyrene (BaP). The Olive Tail Moment (OTM) was used to quantify genotoxicity. RESULTS: For the first time, the genotoxicity of DBP and DiBP was demonstrated in laryngeal and oropharyngeal epithelia, as well as in peripheral lymphocytes, of patients suffering from laryngeal and oropharyngeal carcinomas. OTM levels for NDELA were higher than for phthalates; levels for BaP were lower. Testing of lymphocytes and mucosa showed no significant differences among the various substances. CONCLUSIONS: Phthalates show a genotoxic impact on epithelia of tumor patients. OTM levels were higher than in nasal and oropharyngeal mucosa of healthy donors in results reported earlier. Thus, specific susceptibilities to these xenobiotics need to be discussed. No such effect was demonstrated for NDELA and BaP. In tumor patients, no significant differences could be shown in mutagenic sensitivities in mucosal cells and lymphocytes.

[The single cell microgelelectrophoresis technique in ecogenotoxicology]. / Stellenwert der Einzelzell Mikrogelelektrophorese in der Okogenotoxikologie.

BACKGROUND: The development of carcinoma in the upper aerodigestive tract is often associated with exposure to xenobiotics. Therefore, the identification of such tumor initiating substances is relevant. Most genotoxicity test systems require mammalian cells, human lymphocytes or cell cultures to detect genotoxicity caused by carcinogens. The single cell microgelelectrophoresis technique (Comet assay) is presented, being a sensitive method, identifying DNA strand breaks, alkali labile sites and DNA repair in human epithelial cells of the upper aerodigestive tract. It is compared to other common techniques for the identification of genotoxic damage. Future applications and contributions of the method are introduced. GENOTOXICITY TEST SYSTEMS: Using the alkaline microgel electrophoresis assay, freshly isolated single epithelial cells are incubated with xenobiotics causing DNA strand breaks and alkali labile sites. Data are examined using a digital computer analysis. The method is described for the application of epithelial cells of the upper aerodigestive tract and compared to other procedures for the monitoring of genotoxicity. These are the Ames test identifying mutagenicity in bacteria, the sister chromatid exchange and the micronucleus test demonstrating genomic instability in lymphocytes and cultured mammalian cells. CONCLUSIONS: The microgel electrophoresis technique is a sensitive method to detect genotoxic effects and DNA repair in human epithelia of the upper aerodigestive tract. The assay offers considerable advantages to other common genotoxicity tests. However, combining of the Comet assay with mini organ cultures allows to use repetitive incubations with xenobiotics. Furthermore, signalling selected chromosomal material by the combination of the assay with the fluorescence in situ hybridisation, DNA-damage and -repair mechanisms within comets can be identified.

[Preservation of mucosal specimens before processing in the alkaline single cell microgel electrophoresis assay]. / Lagerung humaner nasaler Mukosa für die Einzelzell-Mikrogelelektrophorese.

BACKGROUND: Human mucosal biopsies are established in ecogenotoxicological studies, but up until now they have demanded immediate processing after harvesting. We report our experience with the preservation of specimens either for 24 h at 4 degrees C or for longer periods at -80 degrees C and compare the results with fresh specimens using the alkaline single cell microgel electrophoresis assay. PATIENTS AND METHODS: Nasal mucosa was harvested from ten patients, transferred to the laboratory and divided into groups for immediate processing,24 h preservation at 4 degrees C and cryopreservation at -80 degrees C. Alkaline single cell microgel electrophoresis assays were performed after separating the specimens into single cells and after exposure to benzo[a]pyrene,benzo[a]pyrene-diolepoxide, N-nitrosodiethylamine, or sodium dichromate. The trypan blue exclusion test was used to assess cytotoxicity. RESULTS: Despite of the fact that cell viability remained stable, after cryopreservation DNA-migration increased significantly for the negative control and benzo[a]pyrene. Although an increase was also seen for sodium dichromate, this was not significant. For benzo[a]pyrene-diolepoxide, N-nitrosodiethylamine and N-methyl-N'-nitro-N-nitrosoguanidine there were no significant changes in DNA-migration. After 24 h in cell medium at 4 degrees C,DNA-migration did not rise compared to the samples which were immediately processed. CONCLUSIONS: Preservation of mucosal specimens at 4 degrees C for 24 h may be legitimate in order to facilitate laboratory practice. However, cryopreservation should not be applied because it leads to higher rates of DNA migration in some tested substances in the alkaline single cell microgel electrophoresis assay.

Altered genotoxicity in mucosal cells of head and neck cancer patients due to environmental pollutants.

The complexity of carcinogenesis in squamous cell cancer (SCC) of the upper aerodigestive tract requires examining environmental risk factors, including mutagen sensitivities to xenobiotics. Three environmental, occupational, and habitual pollutants - dibutylphthalate (DBP), diisobutylphthalate (DiBP), and N'nitrosodiethylamine (NDELA) - were submitted to genotoxicity testing on mucosal biopsy specimens of tumor and nontumor patients in vitro. The single-cell microgel electrophoresis (Comet) assay was applied to detect DNA strand breaks in human epithelial cells of the pharynx and larynx from nontumor patients, patients with SCC of the oropharynx and patients with SCC of the larynx. Genotoxicity was found for DBP, DiBP, and NDELA in cells derived from nontumor and tumor patients. With respect to phthalates, Olive tail moment (OTM) levels were higher in patients with SCC of the oropharynx and SCC of the larynx (P < 0.01), the latter showing even more pronounced genotoxicity for DiBP. Testing epithelial cells of the patients with either oropharyngeal or laryngeal SCC for NDELA demonstrated results similar to the nontumor patients. Present findings indicate heterogeneous mutagen sensitivities to some but not all xenobiotics.

Genotoxicity of nitroso compounds and sodium dichromate in a model combining organ cultures of human nasal epithelia and the comet assay.

Genotoxic effects of xenobiotics are a possible step in tumor initiation in the mucosa of the upper aerodigestive tract. Using the comet assay, detecting genotoxicity in human tissue has been restricted to single incubations in vitro, but in vivo most xenobiotics harm their target in a repetitive or chronic manner. Therefore, we propose a model, which provides repetitive incubations in human upper aerodigestive tract mucosa cultures. Samples of human inferior nasal turbinate mucosa (n = 25) were cultured according to a modified version of a technique originally described by Steinsvåg. On day 1 fresh samples and on days 7, 9 and 11 organ cultures were incubated with N-nitrosodiethylamine (NDEA), sodium dichromate (Na2Cr2O7) and N'-methyl-N-nitro-N-nitrosoguanidine (MNNG). Mucosa samples and organ cultures, respectively, underwent a modified comet assay on days 1, 7 and 11. Genotoxicity could be shown for NDEA, Na2Cr2O7 and MNNG on days 1, 7 and 11. Duration of tissue culture and repetitive incubations did not significantly influence the results for NDEA. Nevertheless, Na2Cr2O7 and MNNG caused higher genotoxic effects on cultures subjected to the comet assay on day 11. This model may help to assess genotoxic hazards posed by environmental pollutants that have a cumulative character in repetitive or chronic exposure in vivo.