Effects of Glutathione on Chromium-induced DNA Crosslinking and DNA Polymerase Arrest

Abstract

Hexavalent chromium (Cr (VI» is reduced intracellularly to Cr (V), Cr (IV) and Cr (III) by ascorbate (Asc), cysteine and glutathione (GSH). These metabolites induce a spectrum of genomic DNA damage resulting in the inhibition of DNA replication. Our previous studies have shown that treatment of DNA with Cr (III) or Cr (VI) plus Asc results in the formation ofDNA-Cr-DNA crosslinks (Cr-DDC) and guanine-specific arrests of both prokaryotic and mammalian DNA polymerases. GSH not only acts as a reductant ofCr (VI) but also becomes crosslinked to DNA by Cr, thus, the focus of the present study was to examine the role of GSH in Cr-induced DNA damage and polymerase arrests. Co-incubation of Cr (III) with plasmid DNA in the presence ofGSH led to the crosslinking ofGSH to DNA. GSH co-treatment with Cr (III) also led to a decrease in the degree ofCrinduced DNA interstrand crosslinks relative to Cr (III) alone, without affecting total Cr DNA binding. DNA polymerase arrests were observed following treatment of DNA with Cr (Ill) alone, but were markedly reduced when GSH was added to the reaction mixture. Pre-formed polymerase-arresting lesions (Cr-DDC) were not removed by subsequent addition of GSH. Treatment of DNA with Cr (VI), in the presence of GSH, resulted in crosslinking of GSH to DNA, but failed to produce detectable DNA interstrand crosslinks or polymerase arrests. The inhibitory effect of GSH on Cr-induced polymerase arrest was further confirmed in human genomic DNA using quantitative PCR (QPCR) analysis. Treatment of genomic DNA with Cr (III) resulted in a marked inhibition of the amplification of a 1.6 kb target fragment of the p53 gene by Taq polymerase. This was almost completely prevented by co-treatment with GSH and Cr (III). These results indicate that Cr-induced DNA interstrand crosslinks, and not DNA-Cr-GSH crosslinks, are the principal lesions responsible for blocking DNA replication. Moreover, the formation ofDNA-Cr-GSH crosslinks may actually preclude the formation of the polymerase arresting lesions. (Mol Cell Biochem 222: 173-182, 2001)

Genotoxicity and mutagenicity of water contaminated with tannery effluents, as evaluated by the micronucleus test and comet assay using the fish Oreochromis niloticus and chromosome aberrations in onion root-tips

“…Milli-Q water was the negative control and an aqueous solution of 0.089 mgL We also observed that clastogenic and aneugenic frequencies were higher for water from the collection sites where total chromium concentration was highest (Table 3) and this supports the view of Sahi et al (1998) and Matsu- Matsumoto et al 155 Table 2 ( The strongest mutagenic effect detected by us were recorded during the dry period from autumn and winter when the water flow is usually low, which results in a higher concentration of chromium. It is known that tannery effluents have the potential to damage the DNA of test organisms (Zhitkovich et al, 1996;O'Brien et al, 2001;Matsumoto et al, 2003 andMatsumoto et al, 2004) and as such may compromise the quality of the water in the Corrego dos Bagres stream. Our data agree with that of Matsumoto et al (2003) who used the comet assay and CHO-K1 cells to detect genotoxicicity in water from the Corrego dos Bagres stream collected during the winter dry period.…”

“…Several in vivo and in vitro studies have shown that chromium compounds damage DNA in a variety of ways, including DNA single and double-strand breaks (SDSBs) generating chromosomal aberrations, micronucleus formation, sister chromatid exchanges, formation of DNA adducts, and alteration in DNA replication and transcription (Zhitkovich et al, 1996;O'Brien et al, 2001;Matsumoto, 2003;Matsumoto and Marin-Morales, 2004).…”

Oxidative stress-related mechanisms are associated with xenobiotics exerting excess toxicity to Fanconi anemia cells.

“…These events include both direct and indirect mechanisms related to oxidative stress, for example, Cr-induced depletion of GSH and other thiol-based antioxidants (Nguyen-nhu and Knoops 2002;O'Brien et al 2001;Quievryn et al 2001); protective effects of catalase, melatonin, or NAC (Cabrer et al 2001;Izzotti et al 1998;Shi et al 1999); ROS formation (Bagchi et al 2001;Hassoun and Stohs 1995;Izzotti et al 1998;Tsou et al 1996); nitric oxide production ; and lipid peroxidation (Bagchi et al , 1997. The eventual formation of Cr adducts to DNA, or to proteins and DNA, may be viewed as an ultimate step in a set of redox-dependent mechanisms (Blasiak and Kowalik 2000;Hodges et al 2001).…”

Hexavalent Chromium Induced Alteration of Carbohydrate Bioenergetics: A Dose-dependent Study

“…Chromate and dichromate are highly soluble in the water and thus readily absorbed by the gastrointestinal tract due to its high diffusional capacity through all types of the cell membrane [7]. Once inside the cell, Cr (VI) is metabolized to trivalent chromium, either enzymatically (via microsomal enzymes), or non-enzymatically (via ascorbate and glutathione) [8]. This intracellular reduction yields reactive intermediates, chromium (V), and chromium (IV).…”