okadaic-acid and Chromosome-Deletion

Okadaic acid (OA), a major polyether toxin involved in diarrhetic shellfish poisoning (DSP), is a potent tumor promoter in rodent skin and glandular stomach and a specific inhibitor of the serine/threonine protein phosphatases PP1 and PP2A. A previous study, which used the cytokinesis-block micronucleus (CBMN) assay in CHO-K1 cells, showed that OA induced chromosome damage in the presence of a rat liver metabolic activation system (S9). To support OA biotransformation by S9, the same test system was performed, and DNA damage induced by OA was measured with and without metabolic activation as well as in the presence of heat-inactivated S9 fraction. The results showed that only in the presence of active S9 did OA significantly increased the frequency of micronucleated binucleated (MNBN) cells. After a 4-h treatment a 2- to 5-fold increase of MNBN cells was observed at 30 nM and at 50 nM of OA. However, without S9 or in the presence of heat-inactivated S9, OA did not induce any chromosome damage. We concluded that OA can be metabolically activated in vitro into metabolites that are more genotoxic. The CBMN assay coupled with fluorescence in situ hybridization (FISH) using a DNA probe for centromere detection was performed to discriminate between clastogenic (chromosome breakage) and aneugenic (chromosome loss) effects. FISH analysis showed that OA metabolites increased in a dose-dependent manner in centromere positive micronuclei (CEN+): 60% of CEN+ at 30 nM and 75% of CEN+ at 50 nM of OA. The uptake of OA into CHO-K1 cells and the biotransformation of the toxin are discussed.

Topics: Analysis of Variance; Aneuploidy; Animals; Cell Division; CHO Cells; Chromosome Breakage; Chromosome Deletion; Cricetinae; Cricetulus; DNA Damage; Dose-Response Relationship, Drug; In Situ Hybridization, Fluorescence; Liver; Micronuclei, Chromosome-Defective; Micronucleus Tests; Okadaic Acid; Rats; Rats, Sprague-Dawley

The amiloride-sensitive Na+/H+ exchanger (NHE1 human isoform) is activated in response to diverse mitogenic and oncogenic signals presumably through phosphorylation. To get insight into the activating mechanism, a set of deletion mutants within the C-terminal cytoplasmic domain of NHE1 has been generated. These mutant forms expressed in antiporter-deficient fibroblasts revealed that deletion of the complete cytoplasmic domain (i) preserves amiloride-sensitive Na+/H+ exchange and activation by intracellular H+, (ii) reduces the affinity of the internal "H(+)-modifier site" in a manner mimicked by cellular ATP depletion, and (iii) abolishes growth factor-induced cytoplasmic alkalinization. We conclude that NHE1 can be separated into two distinct functional domains. One is an N-terminal transporter domain (T) that has all the features required to catalyze amiloride-sensitive Na+/H+ exchange with a built-in H(+)-modifier site. The other is a C-terminal cytoplasmic regulatory domain (R) that (i) determines the set point value of the exchanger and (ii) mediates growth factor signals by interacting with the "H(+)-sensor" in a phosphorylation-dependent manner.

Topics: Animals; Blotting, Northern; Carrier Proteins; Cell Line; Chromosome Deletion; Cricetinae; Cytoplasm; DNA; Ethers, Cyclic; Growth Substances; Humans; Hydrogen-Ion Concentration; Kinetics; Okadaic Acid; Phorbol Esters; Platelet-Derived Growth Factor; RNA; Signal Transduction; Sodium; Sodium-Hydrogen Exchangers; Transfection