Evaluation of genotoxic and cytotoxic effects of H2O2 and DMNQ on freshly isolated rat hepatocytes; protective effects of carboxymethyl chitin-glucan.
OBJECTIVE
Utilizing primary rat hepatocytes we investigated the potential antimutagenic and anti-cytotoxic effects of carboxymethyl chitin-glucan (CM-CG) with respect to oxidative stress induced by the model free-radical-generating compounds hydrogen peroxide (H2O2) or 2,3-dimethoxy-1,4-naphthoquinone (DMNQ). Different kinds of CM-CG action were studied by two different treatment protocols: a. pre-incubation of freshly isolated hepatocytes with the potential anti-mutagen followed by exposure to the oxidant or b. simultaneous treatment of hepatocytes with the potential anti-mutagen and the oxidant.


METHODS
As a measure of genotoxicity, the percentages of DNA in tails of comets by single cell gel electrophoresis were evaluated. The cytotoxicological endpoints analysed were the cell density (number of cells/cm2), and the percentages of apoptotic and necrotic cells.


RESULTS
H2O2 and DMNQ, causing DNA single-strand breaks via the formation of *OH radicals, have been demostrated to induce both genotoxic and cytotoxic effects in primary rat hepatocytes resulting in increased percentages of DNA in tails of comets, and increased frequencies of apoptotic and necrotic cells accompanied by a decreased cell density. Further investigations were therefore focussed on possible modifications of these parameters by CM-CG. The results obtained clearly demonstrate that CM-CG (applied before and during treatment) protects primary rat hepatocytes against the genotoxic and cytotoxic effects of oxidative stress (H2O2 or DMNQ), whereas CM-CG itself has no effect on the endpoints of genotoxicity and cytotoxicity studied.


CONCLUSIONS
Our results indicate that carboxymethyl chitin-glucan represents a natural fungal polysaccharide that can inhibit the genotoxicity and cytotoxicity of experimentally induced oxidative stress in primary rat hepatocytes.
Horvathova
E
E
Cancer Research Institute, Slovak Academy of Sciences, Bratislava, Slovakia. eva.horvathova@savba.sk
Eckl
Peter M
PM
Bresgen
Nikolaus
N
Slamenova
Darina
D
D000818:Animals; D000975:Antioxidants; D017209:Apoptosis / Q000187:drug effects; D002452:Cell Count; D016923:Cell Death; D002469:Cell Separation; D002686:Chitin / Q000031:analogs & derivatives* / Q000494:pharmacology; D020552:Comet Assay; D004247:DNA / Q000187:drug effects / Q000235:genetics; D004249:DNA Damage / Q000187:drug effects; D005260:Female; D005936:Glucans / Q000494:pharmacology*; D022781:Hepatocytes / Q000187:drug effects*; D006861:Hydrogen Peroxide / Q000633:toxicity*; D009152:Mutagenicity Tests; D009153:Mutagens; D009285:Naphthoquinones / Q000633:toxicity*; D009336:Necrosis / Q000473:pathology; D016877:Oxidants / Q000633:toxicity*; D020011:Protective Agents; D051381:Rats; D011916:Rats, Inbred F344
D000975:Antioxidants; D005936:Glucans; D009153:Mutagens; D009285:Naphthoquinones; D016877:Oxidants; D020011:Protective Agents; C091649:carboxymethyl-chitin-glucan; D002686:Chitin; C063002:2,3-dimethoxy-1,4-naphthoquinone; D004247:DNA; D006861:Hydrogen Peroxide