N-nitrososdimethylamine is activated in microsomes from hepatocytes to reactive metabolites which damage DNA of non-parenchymal cells in rat liver.

The liver carcinogen N-nitrosodimethylamine (NDMA) has to be metabolically activated by specific cytochromes before it can react with cellular macromolecules (e.g. proteins or DNA). Although hepatocytes are believed to be responsible for this activation, the liver tumours originate mainly from non-parenchymal cells (NPC). To investigate their activation capacity we determined NDMA-demethylase activity in isolated microsomes from both liver cell types. The results demonstrate that only hepatocytes have activation capacity. Additional experiments were performed with hepatocytes and NPC using the single cell microgel electrophoresis assay (MGE). DNA damage appears in both cell types following in vivo exposure. Tested in vitro, however, the carcinogens induce DNA damages only in hepatocytes (the cells which activate these compounds). N-nitroso-hydroxymethyl-methylamine could be the responsible metabolite as it is stable enough to be transported from hepatocytes to NPC in an intact liver.

[Genotoxic effect on human mucous membrane biopsies of the upper aerodigestive tract]. / Genotoxische Wirkung auf menschliche Schleimhautbiopsien des oberen Aerodigestivtraktes.

BACKGROUND: In numerous epidemiologic studies, environmental and occupational substances such as sodium dichromate (Na2Cr2O7), benzo[a]pyren (B(a)P), and N'nitroso-diethanolamine (NDELA) have been shown to be of potential carcinogenic risk on human epithelial cells in the upper aerodigestive tract. METHODS: Using the alkaline microgel electrophoresis technique (comet assay). mucosal cells isolated from biopsies of the upper aerodigestive tract (nose, paranasal sinuses, mouth, pharynx, larynx, and tonsils) were used to analyze target sites for different genotoxic substances and specific sensitivities of each donor. The cells were freshly isolated by enzymic digestion. 0.5-1 x 10(6) cells per donor were obtained with viabilities between 80-100%. After in vitro incubation, the cells were subsequently subjected to the single cell microgel electrophoresis assay. Results were evaluated regarding the personal history of each donor, focusing on previous exposure to tobacco, alcohol, and occupational compounds. RESULTS: Na2Cr2O7 induced strong genotoxic damage in the nasal and paranasal sinus epithelia as well as in mucosa cells of the larynx. NDELA caused significant damage in mouth cavity epithelia and showed also to be harmful towards mucosa of pharynx and larynx. B(a)P induced fewer DNA strand breaks in mucosal cells of mouth, pharynx and larynx. Significant differences between individuals were apparent for tissue samples from different donors. The genotoxic damage induced in cells of donors with a history of chronic alcohol consumption was significantly higher than in cells of patients without chronic abuse of alcohol. CONCLUSIONS: The data shows that DNA damage in human epithelial cells of the upper aerodigestive tract induced by environmental and occupational substances can be demonstrated using the microgel electrophoresis technique. The influence of chronic alcohol consumption on the genotoxic effects of substances such as NDELA and B(a)P showed the importance of evaluating preexisting compounding factors.

Genotoxic bifunctional aldehydes produce specific images in the comet assay.

Seven genotoxic aldehydes (acrolein, chloroacetaldehyde, crotonaldehyde, formaldehyde, glutardialdehyde, glyoxal, and methylacrolein) have been studied in vitro using the alkaline version of the comet assay (or single cell microgel electrophoresis assay) in freshly isolated rat hepatocytes. Chloroacetaldehyde, glyoxal and methylacrolein treatment resulted in an elevated tail moment (TM), used as indicator for an DNA damaging activity and formation of comet like structures. In addition, this treatment also caused characteristic DNA spot images with small, highly condensed areas within the otherwise circular DNA spots. These were not seen in solvent and N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG)-treated control cells. Treatment of hepatocytes with acrolein, crotonaldehyde, formaldehyde and glutardialdehyde resulted in an TM which did not differ from those of control values although 86-95% of the cells showed characteristic changes of their DNA spot images. The condensed areas are probably the consequence of the known DNA and protein crosslinking activities of these bifunctional aldehydes. It is suggested that using the alkaline comet assay both TM (or overall comet length) as well as changes in the DNA spot image should be evaluated.

In vivo studies on genotoxicity of pure and commercial linuron.

The ureic herbicide linuron [3-(3, 4-dichlorophenyl)-1-methoxy-1-methylurea] (CAS 330-55-2) was investigated for genotoxicity in a series of in vivo experiments. Since human exposure to herbicides is not only to the active principles, but also to all the chemicals present in the commercial formulation, we tested both pure and commercial linuron. Groups of rats were treated with gavage containing different doses of the herbicide (pure compound or commercial formulation) for 14 days. The doses were 150, 300 and 450 mg/kg b.wt. for the pure compound and 315.8, 631.6 and 947.4 mg/kg b.wt. for the commercial formulation (47.5% of linuron). Faeces and urine were collected at regular intervals. Urine specimens were analysed for their mutagenic metabolites, thioethers and D-glucaric acid content. Faeces extracts were tested for mutagenicity. Linuron's ability to cause DNA damage and cytogenetic effects was also investigated after treating groups of rats once with different doses of pure or commercial linuron. DNA single-strand breaks were assessed in rat liver using the alkaline elution technique and the single-cell microgel electrophoresis assay (SCGE: 'comet' assay), and in rat testes cells with the SCGE assay. Micronuclei induction was analysed in rat bone marrow erythrocytes. Results obtained were mainly negative when the excretion of mutagenic metabolites in urine and faeces of animals treated with the pure compound or with the linuron-based commercial formulation were monitored, whereas an increase in the urinary excretion of thioethers and D-glucaric acid was observed in rats treated with the commercial formulation. No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the treated animals. However, linuron affected the viability of hepatocytes isolated from animals treated with higher doses. This cytotoxicity was accompanied by the induction of DNA single-strand breaks in the liver, as seen by the alkaline elution assay. The potential of pure linuron to induce in vivo DNA damage was confirmed with the microgel-electrophoresis technique ('comet' assay). Cytotoxicity was also seen in rat testes cells. However, no indication of DNA damage was visible.

Transport of reactive metabolites of procarcinogens between different liver cell types, as demonstrated by the single cell microgel electrophoresis assay.

Procarcinogens have to be activated by specific cytochromes before showing adverse effects. Freshly isolated hepatocytes (parenchymal liver cells, PC) are characterized by a high content of such xenobiotic enzymes and are widely used to investigate chemically induced DNA damage. But in many cases liver tumors caused by indirect acting carcinogens can also originate from non-parenchymal liver cells (NPC). We used freshly isolated rat PC and NPC to demonstrate that only PC have activation capacity when treated in vitro with different genotoxic procarcinogens (N-nitrosodimethylamine, NDMA; vinyl chloride, VC). The alkaline single cell microgel electrophoresis assay was applied to measure the genotoxic activity of the activated compounds. In order to test the hypothesis that reactive metabolites can be transported from PC to NPC, we performed additional in vivo studies as well as studies in which PC were incubated together with NPC, only separated by a dialysis tube (in vitro coincubation). The results indicate that reactive metabolites of both NDMA and VC are stable enough to be transported intercellularly from PC to NPC.

Detection of genotoxic effects in human gastric and nasal mucosa cells isolated from biopsy samples.

To assess genotoxic burdens from chemicals, it is necessary to relate observations in experimental animals to humans. The success of this extrapolation would be increased by including data on chemical activities in human tissues. Therefore, we have developed techniques to assess DNA damage in human gastric and nasal mucosa (GM, NM) cells. Biopsy samples were obtained during gastroscopy from macroscopically healthy tissue of the stomach or from healthy nasal epithelia during surgery. The specimens were incubated for 30-45 min at 37 degrees C with a digestive solution. We obtained 1.5-8 x 10(6) GM cells and 5-10 x 10(5) NM cells per donor, both with viabilities of 80-95%. The cells were incubated in vitro for 1 hr at 37 degrees C with the test compounds added in their appropriate solvents. In GM cells, we studied N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), sodium dichromate (Na2Cr2O7), nickel sulphate (NiSO4), cadmium sulphate (CdSO4), and lindane. In NM cells, lindane was investigated. Each compound was assessed for DNA damaging activity in cells of at least three different human donor samples using the microgel single cell assay. Similar studies were performed with GM and NM cells obtained from Sprague-Dawley rats. We have found human GM cells to be more sensitive to the genotoxic activity of MNNG than rat GM cells (low effective concentration [LEC] = 0.16 and 0.625 micrograms/ml for human and rat, respectively). Human cells were also more sensitive to the cytotoxic/genotoxic activity of NiSO4 (LEC = 5 and 19 mumoles/ml for human and rat, respectively). CdSO4 was genotoxic in human GM cells (LEC = 0.03-0.125 mumoles/ml), whereas no dose-related genotoxicity was observed in rat GM at concentrations up to 0.5 mumoles/ml. In contrast, approximately equal responses regarding genotoxicity and cytotoxicity were observed in rat and human GM for Na2Cr2O7 (0.25-1 mumoles/ml). Lindane, however, was genotoxic in three out of four rat GM but not in human GM cells (0.5-1 mumoles/ml), whereas it was active in both rat and human NM cells. Together with other recently published in vivo findings, our results with lindane can be interpreted according to a parallelogram approach. In view of possible human exposure situations and the sensitivities of the two target tissues from both species, the data imply that lindane will pose a health risk to humans by inhalation but not by ingestion.

Systemic genotoxic effects of tobacco-related nitrosamines following oral and inhalational administration to Sprague-Dawley rats.

An ex vivo model to detect nonspecific DNA damage in different rat tissues has been developed and employed to study systemic properties of tobacco-specific N-nitrosamines. One hour after treatment of rats with the carcinogens, primary, intact cells were isolated from various organs. Viability of the cells was monitored by trypan blue exclusion. Genotoxicity was determined by alkaline elution, in situ nick translation or microgel electrophoresis. We found that oral application of 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induces genotoxic effects in the liver (3.125-50 mg/kg), whereas N-nitrosonornicotine (NNN) is only moderately active (50-100 mg/kg). Furthermore, oral administration of NNK, NNN, and of N-nitrosodimethylamine (NDMA), induces DNA damage in the nasal cavity. In peripheral blood lymphocytes genotoxicity of NDMA (less than 2 mg/kg), but not of NNK (50 mg/kg), was observed. NDMA and NNK are just as genotoxic in the liver when administered by inhalation as orally (effective doses: 0.1-1 and 50 mg/kg, respectively). For human cancer, these results indicate that in addition to the susceptibilities in local organs (oral cavity after snuff dipping and lung after tobacco smoke inhalation), these nitrosamines also pose a risk systemically for more remote organs.