Carcinogenic chromium (Cr6+) enters cells via the sulfate transport system and undergoes intracellular reduction to trivalent chromium, which strongly adducts to DNA. In this study, the effect of adducted trivalent chromium on in vitro DNA synthesis was analyzed with a polymerase-arrest assay in which prematurely terminated replication products were separated on a DNA sequencing gel. A synthetic DNA replication template was treated with increasing concentrations of chromium(III) chloride. The two lowest chromium doses used resulted in biologically relevant adduct levels (6 and 21 adducts per 1,000 DNA nucleotides) comparable with those measured in nuclear matrix DNA from cells treated with a 50% cytotoxic dose of sodium chromate in vivo. In vitro replication of the chromium-treated template DNA using the Sequenase version 2.0 T7 DNA polymerase (United States Biochemical Corp., Cleveland, OH) resulted in dose-dependent polymerase arrest beginning at the lowest adduct levels analyzed. The pattern of polymerase arrest remained consistent as chromium adduct levels increased, with the most intense arrest sites occurring 1 base upstream of guanine residues on the template strand. Replication by the DNA polymerase I large (Klenow) fragment as well as by unmodified T7 DNA polymerase also resulted in similar chromium-induced polymerase arrest. Interstrand cross-linking between complementary strands was detected in template DNA containing 62, 111, and 223 chromium adducts per 1,000 DNA nucleotides but not in template containing 6 or 21 adducts per 1,000 DNA nucleotides, in which arrest nevertheless did occur. Low-level, dose-dependent interstrand cross-linking between primer and template DNA, however, was detectable even at the lowest chromium dose analyzed. Since only 9% of chromium adducts resulted in polymerase arrest in this system, we hypothesized that arrest occurred when the enzyme encountered chromium-mediated interstrand DNA-DNA cross-links between either the template and a separate DNA molecule or the template and its complementary strand in the same molecule. These results suggest that the obstruction of DNA replication by chromium-mediated DNA-DNA cross-links is a potential mechanism of chromium-induced genotoxicity in vivo.
Metallothionein (MT) is a low-molecular-weight cysteine-rich protein with extensive metal binding capacity and potential nonenzymatic antioxidant activity. Despite the sensitivity of vascular endothelium to either heavy metal toxicity or oxidative stress, little is known regarding the role of MT in endothelial cells. Accordingly, we determined the sensitivity of cultured sheep pulmonary artery endothelial cells (SPAEC) that overexpressed MT to tert-butyl hydroperoxide ( t-BOOH), hyperoxia, or 2,2′-azobis(2,4-dimethylvaleronitrile) (AMVN; peroxyl radical generator). Nontoxic doses of 10 μM Cd increased MT levels from 0.21 ± 0.03 to 2.07 ± 0.24 μg/mg and resulted in resistance to t-BOOH and hyperoxia as determined by reduction of Alamar blue or [3H]serotonin transport, respectively. SPAEC stably transfected with plasmids containing either mouse or human cDNA for MT were resistant to both t-BOOH and hyperoxia. In addition, we examined transition metal-independent, noncytotoxic AMVN-induced lipid peroxidation after metabolic incorporation of the oxidant-sensitive fluorescent fatty acid cis-parinaric acid into phospholipids and high-performance liquid chromatography separation. SPAEC that overexpressed MT after gene transfer completely inhibited peroxyl oxidation of phosphatidylserine, phosphatidylcholine, and sphingomyelin (but not phosphatidylethanolamine) noted in wild-type SPAEC. These data show for the first time that MT can 1) protect pulmonary artery endothelium against a diverse array of prooxidant stimuli and 2) directly intercept peroxyl radicals in a metal-independent fashion, thereby preventing lipid peroxidation in intact cells.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.