Two pathways for chromium(VI)-induced DNA damage in 14 day chick embryos: Cr--DNA binding in liver and 8-0X0-2'-deoxyguanosine in red blood cells

Previous studies have shown that chromium(VI) induced cell-specifictypes of DNA damage, i.e. DNA cross-links in liver and DNA strandbreaks in red blood cells, in 14 day chick embryos. Direct andindirect pathways for chromium(VI)-mediated DNA damage, in theform of Cr-DNA binding and 8-oxo-2’-deoxyguanosine (8-oxo-dG)respectively, were examined in liver and red blood cells of14 day chick embryos. Levels of hepatic Cr-DNA binding increasedin a Cr(VI) dose-dependent manner. Cr-DNA binding in red bloodcells was 10-fold lower than in liver, although the Cr-uptakein red blood cells was only 2-fold lower than in liver. Thelevel of 8-oxo-dG formation in red blood cells increased atall Cr(VI) doses tested but peaked at 0.10 mmol Cr(VI)/kg, whereasno increase hi 8- oxo-dG levels over background levels was observedin liver of Cr(VI)-treated embryos. The possible role of glutathionehi modulating Cr(VI)-induced DNA damage was examined by usingL-buthionine-R,S-sulfoximine (BSO) to deplete glutathione. Nochanges in glutathione levels were observed in either liveror red blood cells of embryos treated with Cr(VI) in the presenceor absence of BSO pretreatment Ascorbate levels in liver andred blood cells were not affected by treatment of embryos withchromium(VI), BSO or Cr(VI) and BSO. Depletion of glutathioneby BSO resulted in a small increase of chromium uptake in liverof embryos treated with 0.050 and 0.10 mmol Cr(VI)/kg, but hadno effect on hepatic chromium uptake at 0.20 mmol Cr(VI)/kg.BSO had no effect on chromium uptake in red blood cells. Depletionof glutathione had no effect on hepatic or red blood cells Cr-DNAbinding in embryos treated with Cr(VT). However, depletion ofglutathione significantly decreased the 8-oxo-dG levels in redblood cells at all Cr(VI) doses tested. Levels of 8-oxo-dG inliver of Cr(VI)-treated embryos remained at background in thepresence or absence of BSO pretreatment These results indicatethat Cr(VI)-induced DNA damage in 14 day chick embryos is througha direct interaction of chromium with DNA in liver, but is throughan indirect oxidative pathway in red blood cells. It appearsthat glutathione plays an important role in chromium(VI)-inducedformation of 8-oxo-dG in red blood cells.     

Enhancement of cisplatin sensitivity in high mobility group 2 cDNA-transfected human lung cancer cells.

To elucidate the role of high mobility group 2 protein (HMG2) in cis-diamminedichloroplatinum (II) (cisplatin, CDDP) sensitivity, we constructed a human HMG2-transfected human non-small cell lung cancer cell line, PC-14/HMG2. The HMG2 mRNA expression level was approximately twice those of parental PC-14 and mock-transfected PC-14/CMV. Gel mobility shift assay revealed a CDDP-treated DNA-protein complex in the nuclear extract of PC-14/HMG2, which was not found in the extracts of PC-14 and PC-14/CMV. This complex formation was subject to competition by CDDP-treated non-specific salmon sperm DNA, indicating that ectopic HMG2 recognizes CDDP-damaged DNA. PC-14/HMG2 showed more than 3-fold higher sensitivity to CDDP than PC-14 and PC-14/CMV. The intracellular platinum content of PC-14/HMG2 after exposure to 300 microM CDDP was 1.1 and 1.5 times that of PC-14 and PC-14/CMV, respectively. Cellular glutathione levels were not different in these cell lines. Repair of DNA interstrand cross-links determined by alkaline elution assay was decreased in PC-14/HMG2. These results suggest that HMG2 may enhance the CDDP sensitivity of cells by inhibiting repair of the DNA lesion induced by CDDP.     

In vivo effects of ascorbate and glutathione on the uptake of chromium, formation of chromium(V), chromium-DNA binding and 8-hydroxy-2'-deoxyguanosine in liver and kidney of osteogenic disorder shionogi rats following treatment with chromium(VI).

Several previous in vitro studies have indicated that ascorbate and glutathione are the major reductants of Cr(VI) in cells. In order to evaluate the in vivo effects of ascorbate and glutathione on Cr(VI)-induced carcinogenesis, Cr uptake and the formation of Cr(V), Cr-DNA adducts and 8-hydroxy-2'-deoxyguanosine (8-OH-dG) were measured in the liver and kidney of Osteogenic Disorder Shionogi (ODS) rats that lack the ability to synthesize ascorbate. Despite a 10-fold difference in tissue ascorbate levels among different dietary ascorbate groups, the Cr(V) signal intensity, Cr uptake and total Cr-DNA binding were not affected in either organ. Treatment of ODS rats with Cr(VI) (10 mg/kg) had no substantial effect on the levels of ascorbate and glutathione in these tissues. The levels of Cr(V) and Cr-DNA binding were approximately 2-fold higher in the liver than in the kidney, although the levels of total Cr uptake were similar in both tissues. Cr uptake levels were significantly lower in the liver and kidney of ODS rats treated with high levels of ascorbate and a high dose of Cr(VI) (40 mg/kg), suggesting a detoxifying role played by plasma ascorbate. Similarly, modulation of glutathione levels by N-acetyl-L-cysteine, L-buthionine-S, R-sulfoximine or phorone in these animals by up to 2-fold had little or no consistent effect on Cr uptake, Cr-DNA binding, Cr(V) levels or 8-OH-dG formation in either organ. One possible explanation is that reduction of ascorbate and glutathione concentration to <10 and 50%, respectively, of normal in these two organs still provides threshold levels of these two reductants that are in excess of what is needed for significant reductive activation of Cr(VI). Alternatively, it is possible that ascorbate and glutathione do not play a major role in the formation of Cr(V), Cr-DNA binding or 8-OH-dG and that other cellular reductants, such as cysteine or other amino acids, might be more important reductants of Cr(VI) in vivo.     

Analysis of HMG protein binding to DNA modified with the anticancer drug cisplatin

 Cisplatin (CDDP) is an effective and widely used cancer chemotherapy drug. High mobility group (HMG) proteins 1 and 2 have been shown to bind with high affinity to CDDP-DNA. In this study we analyzed the interaction of HMG proteins with CDDP-DNA. We demonstrate that after binding, HMG proteins can be removed from CDDP-DNA leaving the Pt adducts intact and capable of rebinding HMG proteins. Furthermore, the very HMG proteins that have been removed remain functionally viable and capable of rebinding CDDP-DNA. We also investigated the role that Cys residues play in protein binding. Replacement of Cys 45 or Cys 106 with a Ser residue reduced HMG2 protein binding to CDDP-DNA. These results indicate that Cys residues play a critical role in the high affinity binding of this protein to CDDP-DNA. From these findings, we speculate that the intracellular oxidative environment could affect the redox state of protein thiols in HMG1 and HMG2 and in addition, regulate the ability of these proteins to recognize cis-Pt-DNA adduct formation in tumor cells. Received: 16 May 1995 / Accepted: 9 October 1995     

Enhancement of Cisplatin Sensitivity in High Mobility Group 2 cDNA-transfected Human Lung Cancer Cells

To elucidate the role of high mobility group 2 protein (HMG2) in cis -diamminedichloroplatinum (II) (cisplatin, CDDP) sensitivity, we constructed a human HMG2 -transfected human non-small cell lung cancer cell line, PC-14/HMG2. The HMG2 mRNA expression level was approximately twice those of parental PC-14 and mock-transfected PC-14/CMV. Gel mobility shift assay revealed a CDDP-treated DNA-protein complex in the nuclear extract of PC-14/HMG2, which was not found in the extracts of PC-14 and PC-14/CMV. This complex formation was subject to competition by CDDP-treated non-specific salmon sperm DNA, indicating that ectopic HMG2 recognizes CDDP-damaged DNA. PC-14/HMG2 showed more than 3-fold higher sensitivity to CDDP than PC-14 and PC-14/CMV. The intracellular platinum content of PC-14/HMG2 after exposure to 300 μM CDDP was 1.1 and 1.5 times that of PC-14 and PC-14/CMV, respectively. Cellular glutathione levels were not different in these cell lines. Repair of DNA interstrand cross-links determined by alkaline elution assay was decreased in PC-14/HMG2. These results suggest that HMG2 may enhance the CDDP sensitivity of cells by inhibiting repair of the DNA lesion induced by CDDP.     

Lower mutagenicity but higher stability of Cr-DNA adducts formed during gradual chromate activation with ascorbate

Recent epidemiological and risk assessment studies have found a very high risk of lung cancer among chromium(VI)-exposed workers even at permissible levels of exposure. However, mechanistic views on the key genotoxic role of transient Cr(V) intermediates were more consistent with the threshold or highly non-linear (heavy dose) models of genetic damage by intracellular Cr(VI). In this work, we examined the production of mutagenic DNA lesions during metabolism of Cr(VI) by its dominant reducer ascorbate (vitamin C) under conditions promoting increased yield of transient Cr forms. We found that slow reductive activation of Cr(VI) by limited concentrations of ascorbate resulted in a greater yield of DCFH-oxidizing Cr intermediates but these species were unable to cause DNA strand breaks. Cr(VI)-ascorbate reactions generated a high number of Cr-DNA adducts that were responsible for all mutagenic responses detected in Cr(VI)-treated pSP189 shuttle plasmids following their replication in human cells. Mutagenicity of DNA damage resulting from the reactions with increased stability of Cr intermediates was approximately four times lower relative to the conditions lacking detectable Cr(V) formation. Unlike other reactions, slow reduction of Cr(VI) with ascorbate produced Cr-DNA adducts that were more resistant to dissociation by chelators, suggesting multicoordinate binding of Cr(III) to DNA. Overall, our findings do not support the possibility that increased Cr(V) formation at depleted ascorbate levels modeling heavy dose exposures causes higher levels of mutagenic DNA damage.     

High mobility group 1 and 2 proteins bind preferentially to DNA that contains bulky adducts induced by benzo[a]pyrene diol epoxide and N-acetoxy-acetylaminofluorene

High mobility group (HMG) proteins 1 and 2 are abundant non-histone chromosomal proteins that bind preferentially DNA that is bent or underwound. Previous studies have shown that these proteins preferentially bind to DNA damaged by the crosslinking agents cis-diammine-dichloro-platinum(II), chromium(III) and UV-C radiation. Here we have studied the binding of HMG-1/2 proteins to a duplex oligonucleotide damaged by benzo(a)pyrene diol epoxide or N-acetoxy-acetylaminofluorene using an electrophoretic mobility shift assay. Both chemicals induce monoadducts that are known to distort DNA structure. The affinities of HMG-1/2 for DNA damaged by benzo[a]pyrene diol epoxide or N-acetoxy-acetylaminofluorene were similar to that for UV-irradiated DNA, which were an order of magnitude higher than for undamaged DNA. In contrast, DNA modified by dimethyl sulfate was not preferentially recognised by HMG-1/2.     

Role of valence state and solubility of chromium compounds on induction of cytotoxicity, mutagenesis, and anchorage independence in diploid human fibroblasts

We previously showed that carcinogenic nickel, arsenic, and chromium(VI) compounds induced anchorage independence (AI) in diploid human fibroblastic cells (HFC) derived from foreskins (K. A. Biedermann and J. R. Landolph, Cancer Res., 47: 3815-3823, 1987). To elucidate the role of the valence state of chromium and solubility of chromium compounds in inducing AI, we studied the ability of soluble and insoluble hexavalent [chromium(VI)] and trivalent [chromium(III)] chromium compounds to induce mutation and AI in HFC. Chromium(VI) compounds (PbCrO4, CaCrO4, Na2CrO4, and CrO3) were 1000-fold more cytotoxic to HFC (average 50% lethal dose 0.5 microM) than chromium(III) compounds (CrCl3, Cr2O3, Cr2S3; average 50% lethal dose 500 microM). However, equal concentrations (0.1-10.0 microM) of soluble or insoluble chromium compounds in either +6 or +3 valence states induced similar increases in frequencies of AI in HFC (100-200/10(5]. Chromium(VI)- and chromium(III)-induced AI was a stable phenotype. All soluble chromium(VI) and insoluble chromium(III) compounds studied induced mutation to 6-thioguanine resistance at cytotoxic concentrations in HFC. Insoluble PbCr(VI)O4 and a soluble form of Cr(III)Cl3 were inactive in this assay. Mutation induction by chromium(III) compounds only occurred at cytotoxic concentrations (100-1000 microM) 1000-fold greater than those concentrations of chromium(VI) compounds (0.25-1 microM) which were cytotoxic, mutagenic, and induced AI. Soluble hexavalent Na2(51)CrO4 was taken up facilely by cells at concentrations that induced cytotoxicity, mutation, and AI. At concentrations of 0.25-1.0 microM, which induced AI but were not cytotoxic or mutagenic, or concentrations of 1-1000 microM, which were cytotoxic and mutagenic, soluble trivalent 51CrCl3 was not taken up by cells. An insoluble form of CrCl3 was not taken up intracellularly but did avidly associate with cells over the concentration range 1 to 100 microM which induced AI, cytotoxicity, and mutagenicity. Therefore, both chromium(VI) and chromium(III) compounds induced genotoxic effects in human fibroblasts. Cellular uptake, cytotoxicity, mutagenicity, and AI induced by soluble chromium(VI) compounds all occurred at the low concentrations of 0.2 to 1.0 microM; hence mutagenicity and induction of AI may be coupled for soluble chromium(VI) compounds but not for insoluble PbCrO4, which induced AI but was not mutagenic. Cytotoxicity and mutagenicity of insoluble chromium(III) occurred at concentrations of 10-100 microM, but induction of AI occurred at concentrations of 0.1-10 microM, indicating that inductions of mutagenicity and AI were not coupled for chromium(III) compounds.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differential binding of chromium(VI) and chromium(III) complexes to salmon sperm nuclei and nuclear DNA and isolated calf thymus DNA

The binding of CrCl₃.6H₂O, Cr(NO₃)₃.9H₂O, [Cr(L-His)₂] (NO₃)₃.H₂O,[Cr(L-Cys)(L-His)].3.5H₂O, [Cr(L-His)(D-Pen)].H₂O, Na[Cr(L-Cys)₂].2H₂O,K₂[Cr(GS)₂].3H₂O, Na₂-CrO₄.4H₂O, and Na₂Cr₂O₇.2H₂O to salmonsperm nuclei and nuclear DNA was determined. The Cr(III)-aminoacid complexes and Cr(VI) exhibited significantly lower Cr-nucleiand Cr-DNA binding levels relative to the inorganic complexesCrCl₃.6H₂O and Cr(NO₃)₃.9H₂O. The binding of CrCl₃.6H₂O, Cr(NO₃)₃.9H₂Oand Na₂Cr₂O₇.2H₂O to salmon sperm nuclei and nuclear DNA inthe presence of rat lung cytosol was determined under the sameconditions. For those complexes studied in both buffer and cytosol,the Cr-DNA binding levels for Cr(III) complexes were higherin buffer than in cytosol, while a relatively higher bindinglevel was observed for Cr(VI) in cytosol than in buffer. Slightlylower nuclear protein levels were present in Cr(VI) incubationsthan in Cr(III) incubations with nuclei both in the presenceand the absence of cytosol. The relative binding of CrCl₃.6H₂O,Cr(NO₃)₃.9H₂O, [Cr(L-His)₂](NO)₃.H₂O, [Cr(L-Cys) (L-His)].3.5H₂O,[Cr(L-His)(D-Pen)].H₂O, Na[Cr(L-Cys)₂].2H₂O and Na₂CrO₄.4H₂Oto isolated calf thymus DNA in buffer was also determined. Positivelycharged, labile inorganic Cr(III) complexes, CrCl₃.6H₂O andCr(NO₃)₃.9H₂O, exhibited higher binding to DNA than [Cr(L-His)(D-Pen)].H₂O, and no binding to DNA was observed with Cr(VI)and the other neutral, positively and negatively charged, inertCr(III)-amino acid complexes. Although labile aquo chromium(III)complexesare quite reactive with DNA, the reactivity of chromium(III),formed upon intracellular reduction of carcinogenic chromium(VI),toward DNA will be diminished by complexation with cellularproteins, peptides and amino acids.     

Ascorbate is the principal reductant of chromium(VI) in rat lung ultrafiltrates and cytosols, and mediates chromium-DNA binding in vitro.

Chromium(VI) reductase activity was measured in ultrafiltrates of rat lung after various pretreatments in vitro at 37 degrees C and pH 7.0. Pretreatment of ultrafiltrates with L-ascorbate oxidase (EC 1.10.3.3), which specifically eliminated ascorbate, blocked approximately 95% of chromium(VI) reductase activity in ultrafiltrates. Preincubation of ultrafiltrates with heat-denatured ascorbate oxidase or the sulfhydryl-blocking agent N-ethylmaleimide (NEM) had no significant effect on Cr(VI) reductase activity. In rat lung cytosols, L-ascorbate oxidase blocked approximately 95% and NEM blocked approximately 15% of Cr(VI) reductase activity. The extent of inhibition of Cr(VI) reductase activity in cytosols by L-ascorbate oxidase was significantly decreased to approximately 75% after addition of 1.0 mM NADPH. When Cr(VI) was incubated with salmon sperm nuclei suspended in rat lung cytosol for 15 min, Cr became bound to nuclear DNA. This Cr-DNA binding was completely inhibited by preincubation of rat lung cytosols with L-ascorbate oxidase and inhibited approximately 60% by preincubation with NEM. Taken together these data suggest that ascorbate and/or ascorbate-dependent factors are the principal reductants of Cr(VI) in both ultrafiltrates and cytosols prepared from rat lung and ascorbate-dependent metabolism of Cr(VI) results in Cr binding to nuclear DNA in vitro. Although sulfhydryl-containing factors and NADPH-dependent factors only make a minor contribution to Cr(VI) reduction in rat lung cytosols, sulfhydryls may be significantly involved in the binding of Cr to nuclear DNA.     

Inhibition of benzo(a)pyrene and benzo(a)pyrene-trans-7,8-diol metabolism and DNA binding in mouse lung explants by ellagic acid

The effect of ellagic acid, a naturally occurring plant phenol, on the binding to DNA and metabolism of benzo(a)pyrene (BP) and trans-7,8-dihydro-7,8-dihydroxybenzo(a)pyrene (BP 7,8-DHD) in cultured explants of strain A mouse lung was investigated. The explants were cultured in a rocking organ culture chamber for 16 h in the presence or absence of 10, 25, 50, and 100 microM ellagic acid. These concentrations of ellagic acid were nontoxic as determined by biochemical and histological methods. The ellagic acid was then removed from the cultures, and the explants were incubated with either 1 microM [3H]BP or [3H]BP 7,8-DHD for 24 h. Explant DNA was isolated using hydroxylapatite chromatography, and the BP metabolites in the medium were analyzed by high-pressure liquid chromatography. Ellagic acid (50 microM) inhibited the binding of BP and BP 7,8-DHD to lung DNA by 46 to 50% and 60 to 70%, respectively. High-pressure liquid chromatography analysis showed that ellagic acid (100 microM) inhibited the metabolism of BP by 20 to 40% and of BP 7,8-DHD by 20%, as indicated by the increased amounts of unmetabolized substrates and decreased amounts of metabolites in the medium. The major BP:DNA adduct in the explants was 7R-N2-[10 beta-[7 beta, 8 beta, 9 alpha-trihydroxy-7,8,9,10-tetrahydrobenzo(a)pyrene]yl: deoxyguanosine, and its formation was reduced by 60 to 65% in the presence of 100 microM ellagic acid. These data suggest that the reduction of BP and BP 7,8-DHD metabolite binding to DNA by ellagic acid may have been due to inhibition of the formation and/or removal of BP 7,8-diol-9,10-epoxide prior to its binding to DNA.     

Down-regulation of the DNA-repair endonuclease 8-oxo-guanine DNA glycosylase 1 (hOGG1) by sodium dichromate in cultured human A549 lung carcinoma cells.

Hexavalent chromium is a genotoxic human pulmonary carcinogen that elevates DNA oxidation, apparently through the generation of reactive DNA-damaging intermediates including Cr(V), Cr(IV) and reactive oxygen species. We tested the hypothesis that elevation of DNA oxidation may also be through inhibition of the expression of the repair glycosylase for 8-oxo deoxyguanine (hOGG1) in cultured A549 human lung epithelial cells. Treatment with sodium dichromate (0-100 microM, 16 h) resulted in a concentration-dependent decrease in the levels of OGG1 mRNA as measured by both RT-PCR and RNase protection assay. Sodium dichromate at 25 microM and above gave a marked reduction of OGG1 mRNA expression which was not seen at 1 microM and below. No effect on the expression of the apurinic endonuclease hAPE or the house-keeping gene GAPDH was observed at any of the concentrations of sodium dichromate investigated. Treatment of cells with the pro-oxidant H(2)O(2) (0-200 microM) for 16 h had no detectable effect on the levels of OGG1 mRNA or protein expression suggesting that the effect of sodium dichromate is not mediated by H(2)O(2). Western blotting demonstrated that sodium dichromate (100 microM; 16 h and >25 microM; 28 h) markedly reduced levels of OGG1 protein in nuclear cell extracts. Additionally, treatment of cells with sodium dichromate (>25 microM, 28 h) resulted in a concentration-dependent decrease in the ability of nuclear extracts to nick a synthetic oligonucleotide containing 8-oxo deoxyguanine (8-oxo dG). We conclude that the elevation of 8-oxo dG levels observed in A549 cells treated with sodium dichromate may be, at least in part, due to a reduced capacity to repair endogenous and hexavalent chromium-induced 8-oxo dG.     

Detection of proteins that recognize platinum-modified DNA using gel mobility shift assay

Using a gel mobility shift assay, we have identified proteins, in the nuclear extracts of a human lung cancer cell line, that recognize cis-diamminedichloroplatinum(II) (cis-DDP, CDDP)-modified DNA. A 158-base-pair double-stranded DNA fragment, derived from pBR322 plasmid DNA, was modified by either CDDP, tetrachloro(dl-trans)-1,2-diaminocyclohexaneplatinum(IV) (tetraplatin) or trans-DDP (the stereoisomer of CDDP and clinically ineffective). These platinum drug-modified probes were incubated with nuclear extracts and analyzed by gel mobility shift assay. Proteins in the extracts selectively recognized the clinically active platinum-modified DNA fragment. No binding to the trans-DDP-modified DNA fragment was observed. These proteins may play a role in the cytotoxicity or in a DNA repair process.     

Detection of Proteins that Recognize Platinum-modified DNA Using Gel Mobility Shift Assay

Using a gel mobility shift assay, we have identified proteins, in the nuclear extracts of a human lung cancer cell line, that recognize cis -diamminedichloroplatinum(II) ( cis -DDP, CDDP)-modified DNA. A 158-base-pair double-stranded DNA fragment, derived from pBR322 plasmid DNA, was modified by either CDDP, tetrachloro( dl-trans )-1,2-diaminocyclohexaneplatinum(IV) (tetraplatin) or trans -DDP (the stereoisomer of CDDP and clinically ineffective). These platinum drug-modified probes were incubated with nuclear extracts and analyzed by gel mobility shift assay. Proteins in the extracts selectively recognized the clinically active platinum-modified DNA fragment. No binding to the trans -DDP-modified DNA fragment was observed. These proteins may play a role in the cytotoxicity or in a DNA repair process.     

Analysis of the binding sites of chromium to DNA and protein in vitro and in intact cells.

Previous studies have examined Cr(III), or CrO4 reduced to Cr(III), binding in vitro to DNA. However, there have been few studies examining chromate binding to DNA in intact cells. Treatment of intact cells with chromate (Na2(51)CrO4) resulted in chromium (Cr) binding to DNA. The binding of Cr to DNA was much more stable when more residual peptide/amino acids were associated with DNA. A substantial portion of the Cr bound to DNA was released by treatment with EDTA, suggesting that trivalent Cr was the major oxidation state of Cr bound to DNA. Cr(III) stimulated the formation of amino acid-DNA and protein-DNA complexes in vitro. Tyrosine and cysteine exhibited the highest activity in being complexed to DNA by Cr(III) in vitro, while histidine, methionine and threonine also exhibited more activity than any other amino acid. Similar results were found in intact cells. The activity of proteins complexed to DNA by trivalent Cr depended upon the content of these reactive amino acids. Thus, bovine serum albumin was more active than actin, which in turn was more active than histones. These and other studies presented suggested that Cr(III) was involved directly in the formation of DNA-protein complexes in intact cells, unlike other metals such as Ni(II), which are thought to form DNA-protein cross-links catalytically and not participate directly in the complex. The majority of trivalent Cr associated with DNA was bound to the phosphate backbone without exhibiting any base specificity. Collectively, these results indicate that trivalent Cr creates DNA protein crosslinks by binding with reactive amino acids (i.e. cysteine, tyrosine or histidine) and linking these to the phosphate backbone of DNA.     

Effect of transition metals on binding of p53 protein to supercoiled DNA and to consensus sequence in DNA fragments.

Recently we have shown that wild-type human p53 protein binds preferentially to supercoiled (sc) DNA in vitro in both the presence and absence of the p53 consensus sequence (p53CON). This binding produces a ladder of retarded bands on an agarose gel. Using immunoblotting with the antibody DO-1, we show that the bands obtained correspond to ethidium-stained DNA, suggesting that each band of the ladder contains a DNA-p53 complex. The intensity and the number of these hands are decreased by physiological concentrations of zinc ions. At higher zinc concentrations, binding of p53 to scDNA is completely inhibited. The binding of additional zinc ions to p53 appears much weaker than the binding of the intrinsic zinc ion in the DNA binding site of the core domain. In contrast to previously published data suggesting that 100 microM zinc ions do not influence p53 binding to p53CON in a DNA oligonucleotide, we show that 5-20 microM zinc efficiently inhibits binding of p53 to p53CON in DNA fragments. We also show that relatively low concentrations of dithiothreitol but not of 2-mercaptoethanol decrease the concentration of free zinc ions, thereby preventing their inhibitory effect on binding of p53 to DNA. Nickel and cobalt ions inhibit binding of p53 to scDNA and to its consensus sequence in linear DNA fragments less efficiently than zinc; cobalt ions are least efficient, requiring >100 microM Co2+ for full inhibition of p53 binding. Modulation of binding of p53 to DNA by physiological concentrations of zinc might represent a novel pathway that regulates p53 activity in vivo.