title: Cumulative genotoxic and apoptotic effects of xenobiotics in a mini organ culture model of human nasal mucosa as detected by the alkaline single cell microgel electrophoresis assay and the annexin V-affinity assay.
abstract: Three-dimensional mini organ cultures of human inferior nasal turbinate epithelia have proved to be a useful tool in genotoxicology studies. They allow repetitive or chronic exposure of cells to xenobiotics in a well-preserved organ-specific mucosal architecture for an extended period of time. It is the aim of the present study to concurrently monitor cumulative genotoxic and apoptotic effects of sodium dichromate, N-nitrosodiethylamine (NDEA) and N-methyl-N-nitro-N-nitroso-guanidine (MNNG). Mini organs were raised by separating fresh specimens of human inferior nasal turbinates (n=11) into 1 mm3 sized pieces and culturing them on multiwell plates with bronchial epithelial basal medium for 6 days. Aliquots of the mini organs were subsequently exposed to sodium dichromate (1.0 mM, 1h), NDEA (50 mM, 1h) or MNNG (0.07 mM, 1h) on days 7, 9 and 11 versus a single exposure on day 11 only. DNA fragmentation and apoptotic events were assessed on day 11 using the alkaline single cell microgel electrophoresis assay (comet assay) and the annexin V-affinity assay. Significant DNA fragmentation could be demonstrated after a single exposure of the mini organs to sodium dichromate. Following three subsequent incubations, there was a further increase in the genetic damage observed, accompanied by an increase in the rate of apoptotic cells. In contrast, after single and triple incubation with NDEA there was neither an increase in genetic damage nor in the fraction of apoptotic cells detectable. Repetitive exposure to MNNG resulted in an accumulation of DNA damage without an observable increase in apoptosis. The results verify the need to assess apoptosis in genotoxicology research and to investigate cumulative effects of xenobiotics. Three-dimensional mini organ cultures of human upper aerodigestive tract epithelia have shown to be well-suited for improving the ability to distinguish between cumulative genotoxic and apoptotic effects.
authors.affiliation: Department of Internal Medicine, Munich-Neuperlach Hospital, Munich, Federal Republic of Germany. michael.buehrlen@web.de
authors.forename: Michael
authors.initials: M
authors.lastname: Buehrlen
authors.forename: Ulrich A
authors.initials: UA
authors.lastname: Harréus
authors.forename: Fernando
authors.initials: F
authors.lastname: Gamarra
authors.forename: Rudolf
authors.initials: R
authors.lastname: Hagen
authors.forename: Norbert H
authors.initials: NH
authors.lastname: Kleinsasser
mesh_terms: D000328:Adult; D017304:Annexin A5 / Q000737:chemistry; D017209:Apoptosis / Q000187:drug effects; D002274:Carcinogens, Environmental / Q000633:toxicity*; D002470:Cell Survival / Q000187:drug effects; D002840:Chromates / Q000633:toxicity*; D020552:Comet Assay; D004052:Diethylnitrosamine / Q000633:toxicity*; D005260:Female; D006801:Humans; D008297:Male; D008769:Methylnitronitrosoguanidine / Q000633:toxicity*; D008875:Middle Aged; D009297:Nasal Mucosa / Q000187:drug effects* / Q000473:pathology; D009924:Organ Culture Techniques; D017670:Sodium Compounds / Q000633:toxicity*
chemical_list: D017304:Annexin A5; D002274:Carcinogens, Environmental; D002840:Chromates; D017670:Sodium Compounds; D008769:Methylnitronitrosoguanidine; D004052:Diethylnitrosamine; C028982:sodium chromate(VI)