Thomas N. Müller

Cadmium/zinc-metallothionein induces DNA strand breaks in vitro

The in vitro DNA strand breaking activity of metallothionein (MT) containing Cd2+ and Zn2+ in a molar ratio of 5∶2 is described. Studies with radical scavengers and electron paramagnetic resonance spectroscopy indicate that the DNA damage might be caused by a radical species formed by the native protein (i.e., MT) charged with the heavy metal ions. No DNA strand breaks are detectable with the heat-denatured MT or with Cd2+ or Zn2+ alone. Inhibition studies using EDTA as a metal ion chelator orN-ethylmaleimide to alkylate sulfhydryl groups suggest that both the bound heavy metal ions as well as the SH groups of the various cysteine residues of MT may be involved in the MT-dependent DNA cleavage. Further characterization showed that the DNA cleavage is more likely random than sequence- or base-specific. These observations may provide a clue in the search for initial events in Cd-related carcinogenicity.

Evidence for radical species as intermediates in cadmium/zinc-metallothionein-dependent DNA damage in vitro.

Toxicologic data on cadmium (Cd) indicate that intracellular metallothionein (MT) is protective for Cd exposure, whereas extracellular Cd-containing MT might be toxic. Moreover, Cd is suspected to be a carcinogen though the underlying mechanism is not known. Here we report on the genotoxic activity of cadmium/zinc-metallothionein (Cd/Zn-MT) in a cell-free test system: a concentration-dependent increase in DNA strand breaks was detected with increasing doses of Cd/Zn-MT, whereas no DNA strand breaks were observed in the presence of heat-denatured MT or Cd or Zn ions alone. Modifications of native Cd/Zn-MT by the metal ion-chelating agent EDTA or the sulfhydryl group alkylating agents N-ethylmaleimide and iodoacetamide suggest that the various cysteine residues of MT, together with the attached heavy metal ions, may be involved in the DNA cleavage reaction. Furthermore, DNA strand breaks caused by Cd/Zn-MT seem more likely to be random than sequence- or base-specific. Results from experiments with radical scavengers and electron spin resonance spectroscopy point to radical species formed by Cd/Zn-MT as mediators of the DNA damage. Thus, the actual activity of Cd/Zn-MT--whether protective or damaging--appears to depend on various parameters governed by the extra- and intracellular environment. ImagesFigure 1.

Synthesis and characterization of lipid-linked mannosyl oligosaccharides in Volvox carteri f. nagariensis

Particulate membrane fractions from Volvox carteri catalyze the transfer of mannose from GDP-mannose to dolichyl diphosphate-[14C]chitobiose to form lipid-linked oligosaccharides up to a dolichyl diphosphate-chitobiose-(mannose)5 structure. Mannosylation of the chitobiosyl lipid requires divalent cations and detergents as solubilizing agents. Depending on the nature of the detergent, the oligosaccharide pattern differs markedly: With deoxycholate or the zwitterionic detergent 3-14 a lipid-linked trisaccharide accumulates. The nonionic Triton X-100, however, gives rise to a spectrum of compounds up to a heptasaccharide. Enzyme digestion of the tri- and pentasaccharide structure, obtained after mild acid hydrolysis of the corresponding [14C]glycolipids, revealed that the first mannose is bound via a beta-glycosidic linkage to the chitobiosyl core, whereas the outer mannose residues are linked as alpha-mannosides. Our studies indicate that, in agreement with recent findings in other organisms, the innermost alpha-mannosidic residues are donated directly from GDP-mannose. The structure of oligosaccharides synthesized by Volvox membranes is thus consistent with results from other eucaryotic species, suggesting a common pathway of N-glycosylation of glycoproteins.