INTERLABORATORY VALIDATION OF A NEW ASSAY FOR DNA-PROTEIN CROSS-LINKS

In 1992, a simple and sensitive assay for detecting DNA-protein crossl inks was developed [1]. In an effort to facilitate the greater use of the assay, a number of studies were conducted to evaluate its reliabil ity and reproducibility. During this work, the assay was used to asses s whether various metals and other compounds could induce crosslinks i n cultured human lymphocytes (Epstein-Barr virus-transformed Burkitt's Lymphoma cell line). Potassium permanganate, mercury chloride, lead n itrate, magnesium perchlorate, aluminum chloride, and cadmium chloride did not induce DNA-protein crosslinks at either cytotoxic or non-cyto toxic levels, Copper sulfate, arsenic trioxide, and potassium chromate induced DNA-protein crosslinks only at cytotoxic concentrations. Acut e lethality of the cells was assessed immediately after exposure to me tals by trypan blue exclusion while long-term lethality was assessed b y cell proliferation and trypan blue exclusion following an incubation period of 5 days after exposure to the metal compound. All metals exh ibited more toxicity in the long-term lethality assay compared to the short-term assay, The cultured human lymphocytes treated with various doses of lead acetate, cadmium chloride, arsenic trioxide and copper s ulfate, as well as cis-platinum and chromate, were sent to four differ ent laboratories to compare the reliability and reproducibility of the DNA-protein crosslink assay. Depending on the chemical studied, there were quantitative differences in the results observed among the vario us laboratories using the assay. However, all laboratories generally s howed that cis-platinum, chromate, arsenic trioxide and copper sulfate induced DNA-protein crosslinks at levels that produced acute cytotoxi city, whereas cadmium chloride and lead acetate did not.