ARTICLE NO. TX982489 Differential Sensitivity of Chromium-Mediated DNA Interstrand Crosslinks and DNA-Protein Crosslinks to Disruption by Alkali and EDTA (original) (raw)
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Some compounds of hexavalent chromium are well-established carcinogens. Chromium enters mammalian cells in the hexavalent form and is reduced to chromium(III). Treatment of purified DNA with chromium(III) produces DNA-DNA interstrand crosslinks (DOC) which obstruct the progression of DNA polymerases in vitro. DDC were also detected in chromate-treated cultured normal human lung cells using the renaturing agarose gel electrophoresis (RAGE) assay and correlated with base-specific inhibition of DNA replication. Curiously, DDC have gone undetected in studies of cultured cells using the alkaline elution (AE) technique, whereas chromium-mediated DNA-protein crosslinks (DPC) were readily detected by AE. We tested the hypothesis that AE conditions [60 mM tetraethyl ammonium hydroxide (TEA), 20 mM EDTA, pH 12.6, for 16 h at room temperature] dissociate DDC but not DPC using chromium(III)-treated plasmid DNA and the RAGE assay. Dose dependent chromium-induced DOC were unaffected by TEA (pH 11.8) alone or by more rigorous alkaline denaturation conditions (200 mM NaOH, pH 13.5, for 16 h). DDC were, however, completely disrupted by EDTA (pH 12.6) alone or the combination of TEA and EDTA (pH 12.6). In contrast, DPC remained largely intact under these conditions. Therefore, past AE-based studies which have failed to detect chromium-induced DOC do not prove the absence of this lesion. AE may not be suitable for detecting DOC induced by EDTA-chelatable agents such as metals.
Journal of Biological Chemistry, 1996
DNA-protein complexes (DPCs) were induced in human leukemic T-lymphocyte MOLT4 cells by treatment with potassium chromate. DPCs were isolated by ultracentrifugal sedimentation in the presence of 2% SDS and 5 M urea. The complexes were analyzed by two-dimensional SDS-polyacrylamide gel electrophoresis. Three acidic proteins of 74, 44, and 42 kDa and a basic protein of 51 kDa were primarily complexed to DNA following 25 M chromate treatment. Higher concentrations of chromate cross-linked many other proteins to DNA. Amino acid sequencing and immunoblotting studies indicated that the acidic 44-kDa protein could be nuclear -actin. Lectin and aminoglycoside nucleotidyltransferase were also found to cross-link with DNA by chromate treatment. The composition and stability of the DPCs were studied using nucleases, proteinase K, and disruptive chemicals. Pretreatment of cells with antioxidants inhibited the formation of DPCs, measured as K ؉-SDS precipitable DPCs, indicating the involvement of oxidative mechanisms. Because chromate causes certain nuclear proteins to form complexes with DNA and the complexes are resistant to treatments such as 2% SDS and 5 M urea, but disruptable under gel electrophoretic conditions, chromium could be used as a cross-linking agent for the identification of other proteins, such as transcription factors, that transiently interact with DNA.
Chromium(VI)-induced DNA lesions and chromium distribution in rat kidney, liver, and lung
PubMed, 1983
DNA lesions were detected in rat organ nuclei following an i.p. injection of sodium dichromate. Kidney, liver, and lung nuclei were examined for DNA interstrand cross-links, strand breaks, and DNA-protein cross-links using the alkaline elution technique. The time course for formation of cross-links in kidney nuclei revealed the presence of DNA interstrand and DNA-protein cross-links 1 hr after injection of sodium dichromate. By 40 hr in kidney, DNA interstrand cross-links had been repaired, but DNA-protein cross-links persisted. In liver nuclei, the time course for formation of cross-links after injection of dichromate showed a maximum in DNA-protein cross-linking at 4 hr and a maximum in DNA interstrand cross-linking at 2 hr. By 36 hr, in the liver, both types of lesions had been repaired. In lung nuclei, both DNA interstrand and DNA-protein cross-links were observed 1 hr after dichromate injection; however, by 36 hr, only DNA-protein cross-links persisted. No DNA lesions were detectable in kidney 1 hr after an i.p. injection of chromium(III) chloride. Chromium distribution in rat kidney, liver, and lung was measured and is discussed with respect to the observed DNA lesions. The lung and kidney may be more sensitive than liver to chromium-induced DNA damage, an observation which correlates with the reported toxicity and carcinogenicity data for chromium(VI) in both animals and humans.
Hexavalent chromium (Cr) compounds are respiratory carcinogens in humans and animals. Treatment of Chinese hamster ovary cells with 150 and 300 µM sodium chromate (Na2Cr04) for 2 hr decreased colony-forming efficiency by 46 and 92%, respectively. These treatments induced dose dependent internucleosomal fragmentation of cellular DNA beyond 24 hr after chromate treatment. This fragmentation pattern is characteristic of apoptosis as a mechanism of cell death. These treatments also induced an immediate inhibition of macromolecular synthesis and delayed progression of cells through S-phase of the cell cycle. Cell growth (as evidenced by DNA synthesis) was inhibited for at least 4 days and transcription remained suppressed for at least 32 hr. Many of the cells that did progress to metaphase exhibited chromosome damage. Chromate caused the dose-dependent formation of DNA single-strand breaks and DNA-protein cross-links, but these were repaired 8 and 24 hr after removal of the treatment, respectively. In contrast, Cr-DNA adducts (up to 1/100 base-pairs) were extremely resistant to repair and were still detectable even 5 days after treatment. Compared with other regions of the genome, DNA-protein cross-links and Cr adducts were preferentially associated with the nuclear matrix DNA of treated cells, which was 4.5-fold enriched in actively transcribed genes. Chromium adducts, formed on DNA in vitro at a similar level to that detected in nuclear matrix DNA, arrested the progression of a DNA polymerase in a sequence-specific manner, possibly through the formation of DNA-DNA cross-links. Total RNA and mRNA synthesis and induction of expression of the inducible GRP78 gene were suppressed in a concentration- and time-dependent manner by chromate. The effects of chromate on GRP78 induction correlated most closely with the presence of DNA-protein cross-links but suppression of total RNA and mRNA synthesis correlated with the presence of DNA-Cr adducts in cells. These results suggest that the persistent Cr-DNA adducts may be responsible for the protracted cell cycle delay and transcriptional inhibition caused by chromate. Escape from apoptosis may be one of the steps involved in chromate-induced carcinogenesis.
Mechanisms of DNA damage by chromium(V) carcinogens
Nucleic acids …, 1998
Reactions of bis(2-ethyl-2-hydroxy-butanato)oxochromate(V) with pUC19 DNA, single-stranded calf thymus DNA (ss-ctDNA), a synthetic oligonucleotide, 5′-GATCT-ATGGACTTACTTCAAGGCCGGGTAATGCTA-3′ (35mer), deoxyguanosine and guanine were carried out in Bis-Tris buffer at pH 7.0. The plasmid DNA was only nicked, whereas the single-stranded DNA suffered extensive damage due to oxidation of the ribose moiety. The primary oxidation product was characterized as 5-methylene-2-furanone. Although all four bases (A, C, G and T) were released during the oxidation process, the concentration of guanine exceeds the other three. Orthophosphate and 3′-phosphates were also detected in this reaction. Likewise, the synthetic oliogomer exhibits cleavage at all bases with a higher frequecncy at G sites. This increased cleavage at G sites was more apparent after treating the primary oxidation products with piperidine, which may indicate base oxidation as well. DNA oxidation is shown to proceed through a Cr(V)-DNA intermediate in which chromium(V) is coordinated through the phosphodiester moiety. Two alternative mechanisms for DNA oxidation by oxochromate(V) are proposed to account for formation of 5-methylene-2-furanone, based on hydrogen abstraction or hydride transfer from the C1′ site of the ribose followed by hydration and two successive β-eliminations. It appears that phosphate coordination is a prerequisite for DNA oxidation, since no reactions between chromium(V) and deoxyguanosine or guanine were observed. Two other additional pathways, hydrogen abstraction from C4′ and guanine base oxidation, are also discussed.
Metallomics, 2023
Epidemiological and animal studies have supported the carcinogenicity of hexavalent chromium [Cr(VI)]; however, molecular changes responsible for the induction of cancer by Cr(VI) are not entirely understood. Numerous mechanistic studies suggested the role of oxidative stress and genotoxicity in Cr(VI)-mediated carcinogenesis; however, specific types of DNA damage have not yet been conclusively attributed to specific chromium species or other reactive byproducts generated in biological systems exposed to Cr(VI). Due to the remarkably complex chemistry and biological effects of chromium species generated through the intracellular reduction of Cr(VI), their relevance for Cr(VI)-mediated carcinogenesis has not yet been fully elucidated and continues to be a subject of ongoing discussions in the field. In this report, we describe a complex world of chromium species and their reactivity with DNA and other biologically relevant molecules in vitro to inform a more complete understanding of Cr(VI)-mediated toxicity. In addition, we discuss previous results in the context of in vitro models and analytical methods to reconcile some conflicting findings on the biological role of chromium species.
Chromium(III) Decreases the Fidelity of Human DNA Polymerase β
Biochemistry, 1998
Certain particulate compounds of hexavalent chromium are well-known occupational and environmental human carcinogens. Hexavalent chromium primarily enters the cells and undergoes metabolic reduction; however, the ultimate trivalent oxidation state of chromium, Cr(III), predominates within the cell. DNA-bound Cr(III) has been previously shown to decrease the fidelity of replication in the M13 phage mutation assay. This study was done to understand how Cr(III), in the presence of physiological concentrations of magnesium, affects the kinetic parameters of steady-state DNA synthesis in vitro across site-specific O 6-methylguanine (m 6 dG) residues by DNA polymerase (pol). Cr(III) binds to the short oligomer templates in a dose-dependent manner and stimulates the activity of pol. Cr(III) stimulates the mutagenic incorporation of dTTP opposite m 6 dG more than the nonmutagenic incorporation of dCTP, and thereby Cr(III) further decreases the fidelity of DNA synthesis across m 6 dG by pol. In contrast, Cr(III) does not affect the fidelity of DNA synthesis across the normal template base, dG. Both the enhanced activity and the mutagenic lesion bypass in the presence of Cr(III) may be associated with Cr(III)-dependent stimulation of pol binding to DNA as reported here. This study shows some of the mechanisms by which mutagenic chromium affects DNA synthesis.
Biological Trace Element Research, 2002
We are trying to understand individual differences in susceptibility to chromate toxicity by comparing three different lymphoblastic cell lines derived from three different individuals. We have compared the uptake of CrO 4 2-, the release of LDH from cells, the proliferation ability of the cells, and the DNA-protein crosslinks in these lymphoblastic cell lines exposed to chromate. We report here that one lymphoblastic cell line, GM0922B, appears to be considerably less sensitive than the other two cells lines to the cytotoxic effects of hexavalent chromium. The diminished sensitivity is almost twofold and can be accounted for by the decreased uptake of hexavalent chromium, which results in less lactate dehydrogenase release, and greater tolerance to chromate inhibition of cell proliferation and less DNA-protein crosslinking. This lower uptake of chromate combined with interindividual differences in extracellular Cr(VI) reducing capacity are probably the two most important determinants of genetic susceptibility to chromate toxicity.