Elevated levels of DNA-protein crosslinks and micronuclei in peripheral lymphocytes of tannery workers exposed to trivalent chromium.

DNA-protein crosslinks (DPC) are a promising biomarker of exposure to hexavalent chromium, a known human carcinogen. Although trivalent chromium is considered to have much lower toxicity, the risk involved in chronic exposure is uncertain. DPC may be a useful tool in clarifying this risk, by signaling an exposure of body tissues to biologically active forms of chromium. DPC quantification was carried out in lymphocytes of a group of tannery workers exposed to trivalent chromium, a small group of manual metal arc stainless steel welders exposed to hexavalent chromium and a control group. This biomarker was compared with the frequency of micronuclei in cytokinesis blocked peripheral lymphocytes as a biomarker of cytogenetic lesions and total plasma and urine chromium levels as an index of exposure. The results indicate a significant increase in the formation of DPC in tannery workers compared with controls (0.88 +/- 0.19 versus 0.57 +/- 0.21%, P < 0.001, Mann-Whitney test) and an even higher level of DPC in welders (2.22 +/- 1.12%, P = 0.03). Tanners showed a significant increase in micronucleated cells compared with controls (6.35 +/- 2.94 versus 3.58 +/- 1.69 per thousand, P < 0.01), whereas in welders this increase was not significant (5.40 +/- 1.67 per thousand ). Urinary chromium was increased in both groups, with a greater increase observed in tanners compared with controls (2.63 +/- 1.62 versus 0.70 +/- 0.38 microg/g creatinine, P < 0.001) than in welders (1.90 +/- 0.37 microg/g creatinine, P < 0.005). Plasma chromium was also increased in both groups (tanners 2.43 +/- 2.11 microg/l, P < 0.001, welders 1.55 +/- 0.67 microg/l, P < 0.005 versus controls 0.41 +/- 0.11 microg/l). In summary, chronic occupational exposure to trivalent chromium can lead to a detectable increase in lymphocyte DNA damage which correlates with a significant exposure of the cells to the metal.

Reduction of Cr (VI) by cysteine: significance in human lymphocytes and formation of DNA damage in reactions with variable reduction rates.

The induction of genotoxicity by Cr (VI) is dependent on its reductive activation inside the cell. Our recent studies have found that reduction of Cr (VI) by cysteine resulted in the formation of mutagenic Cr (III)-DNA adducts in the absence of oxidative DNA damage. In this work, we examined the formation of oxidative and Cr (III)-dependent types of DNA damage under a broader range of Cr (VI) and cysteine concentrations and investigated a potential role of this reducer in intracellular metabolism of Cr (VI). Peripheral lymphocytes from unexposed humans had 7.8-fold excess of glutathione over cysteine, whereas lymphocytes from stainless steel welders contained only 3 times higher amount of glutathione (p = 0.0009) which was entirely caused by the decrease in the concentration of glutathione. A strong correlation (r = 0.72) between the levels of both thiols was found in lymphocytes from controls. The number of DNA-protein crosslinks in lymphocytes from welders was 4.1 times higher than among controls, indicating the presence of Cr (VI)-dependent DNA damage. The average rate of Cr (VI) reduction by cysteine was approximately 5 times faster than that by glutathione. Higher reduction rate combined with the decrease in the intracellular concentration of glutathione should make cysteine a predominant Cr (VI)-reducing thiol in lymphocytes of welders. Analysis of the initial rates of Cr (VI) reduction by different concentrations of cysteine suggested the presence of one- and two-electron pathways, with one-electron mechanism dominating in the physiological range of concentrations. There was no detectable formation of DNA breaks or abasic sites under a broad range of Cr (VI) and cysteine concentrations, resulting in up to 68-fold differences in the rates of reduction and the production of as many as 3 Cr (III)-DNA adducts per 10 bp. The reactions with slow reduction rates (low concentrations of cysteine) led to the most extensive formation of Cr (III)-DNA adducts. In summary, these results further establish Cr (III)-DNA adducts as the major form of DNA damage resulting from Cr (VI) metabolism by cysteine. The role of cysteine in reduction of Cr (VI) becomes more significant under conditions of occupational exposure to Cr (VI)-containing welding fumes.

Non-oxidative mechanisms are responsible for the induction of mutagenesis by reduction of Cr(VI) with cysteine: role of ternary DNA adducts in Cr(III)-dependent mutagenesis.

Intracellular reduction of carcinogenic Cr(VI) generates Cr-DNA adducts formed through the coordination of Cr(III) to DNA phosphates (phosphotriester-type adduct). Here, we examined the role of Cr(III)-DNA adducts in mutagenesis induced by metabolism of Cr(VI) with cysteine. Reduction of Cr(VI) caused a strong oxidation of 2', 7'-dichlorofluoroscin (DCFH) and extensive Cr-DNA binding but no DNA breakage. Cr-DNA adducts induced unwinding of supercoiled plasmids and structural distortions in the DNA helix as detected by decreased ethidium bromide binding. Propagation of Cr-treated pSP189 plasmids in human fibroblasts led to a dose-dependent formation of the supF mutants and inhibition of replication. Blocking of Cr(III)-DNA binding by occupation of DNA phosphates with Mg(2+) or by sequestration of Cr(III) by inorganic phosphate or EDTA eliminated mutagenic responses and restored a normal yield of replicated plasmids. Dissociation of Cr(III) from DNA by a phosphate-based reversal procedure returned mutation frequency to background levels. The mutagenic responses at the different phases of the reduction reaction were unrelated to the amount of reduced Cr(VI) but reflected the number and the spectrum of Cr(III)-DNA adducts that were formed. Ternary cysteine-Cr(III)-DNA adducts were approximately 4-5 times more mutagenic than binary Cr(III)-DNA adducts. Although intermediate reaction products (CrV/IV, thiyl radicals) were capable of oxidizing DCFH, they were insufficiently reactive to damage DNA. Single-base substitutions at G/C pairs were the predominant type of Cr-induced mutations. The majority of mutations occurred at the sites where G had adjacent purine in the 3' or 5' position. Overall, our results present the first evidence that Cr(III)-DNA adducts play the dominant role in the mutagenicity caused by the metabolism of Cr(VI) by a biological reducing agent.

Reductive metabolism of Cr(VI) by cysteine leads to the formation of binary and ternary Cr--DNA adducts in the absence of oxidative DNA damage.

Carcinogenic chromium(VI) compounds require reduction for the induction of genotoxicity. In this work, we examined a spectrum of DNA damage produced in Cr(VI)-cysteine reactions at neutral pH. Cr(VI) reduction followed single-component kinetics and led to a significant oxidation of 2',7'-dichlorofluoroscein (DCFH). The presence of residual Fe and/or Cu resulted in an increased level of oxidation of DCFH, and the removal of adventitious metals required rigorous purification of cysteine. DNA breakage and abasic sites were not detected, suggesting that DNA is much less susceptible to oxidation than DCFH. Cr(VI) reduction led to the extensive formation of Cr-DNA adducts and Cys-Cr-DNA and interstrand DNA-DNA cross-links. Cr-DNA binding resulted in unwinding of supercoiled DNA and a greater stability of the DNA duplex to denaturation. Ionically bound Cr comprised 40-60% of the total DNA-bound Cr, while the remaining Cr-DNA complexes represented stable Cr-DNA adducts that exhibited significant resistance to dissociation by EDTA. The yield of Cr-DNA adducts was strongly influenced by the nature of the buffer that was used. Phosphate buffer completely blocked Cr-DNA binding, whereas adduct formation in organic buffers was largely dependent on the extent of buffer ionization. The level of formation of Cr-DNA adducts was several times higher at pH 6 which resulted from lower levels of buffer ionization and diminished competition from hydroxyl ions. Yield of a number of Cr-DNA and Cys-DNA adducts increased linearly as a function of Cr(VI) concentration, whereas formation of interstrand DNA cross-links exhibited exponential dose dependence. Approximately 60-90 min was required to convert a Cr-DNA monoadduct into a DNA-DNA cross-link. Prolonged incubations at alkaline conditions led to a selective cleavage of cross-linked DNA. The alkali sensitivity of Cr-adducted DNA suggests that results of alkaline elution analysis of DNA damage in cells require a more cautious interpretation. Overall, a spectrum of DNA damage derived from Cr(VI)-cysteine reactions was similar to that found in exposed cells.

Rats retain chromium in tissues following chronic ingestion of drinking water containing hexavalent chromium.

Humans have sometimes been exposed to as much as 10 ppm Cr(VI) in drinking water from contaminated wells. The risks to these individuals are not well understood because the digestive tract reduces some of the Cr(VI) to the less bioavailable Cr(III) prior to absorption, and the disposition of the remaining Cr(VI) has not been well studied. We determined tissue Cr concentrations in rats after chronic ingestion of Cr(VI) in drinking water at concentrations relevant to human exposure levels. Adult male and female Fischer 344 rats consumed ad libitum 0, 0.5, 3, or 10 ppm Cr(VI) as K2CrO4 in drinking water for 44 wk. Rats then were given deionized water 4-6 d prior to sample collection. Females given 3 or 10 ppm Cr(VI) consumed more Cr(VI) per unit of body weight than did males. Bone Cr concentrations were significantly elevated in rats that drank 10 ppm Cr(VI). Renal Cr concentrations were significantly elevated in male rats that drank 3 or 10 ppm Cr(VI) and in female rats dosed with 10 ppm Cr(VI). Female rats had elevated liver Cr concentrations after drinking 3 or 10 ppm Cr(VI). Testicular Cr concentrations were slightly elevated in rats that drank 10 ppm Cr(VI). Brain, ovarian, and whole-blood Cr concentrations were below detection limits in all exposure groups. Although tissue Cr accumulation may have resulted from absorption of Cr(III), it is poorly absorbed. Therefore, the increased tissue retention may also have resulted, in part, from increased absorption of Cr(VI) and its subsequent uptake from the systemic circulation.

Loss of DNA-protein crosslinks from formaldehyde-exposed cells occurs through spontaneous hydrolysis and an active repair process linked to proteosome function.

DNA-protein crosslinks (DPC) involving all major histones are the dominant form of DNA damage in formaldehyde-exposed cells. In order to understand the repair mechanisms for these lesions we conducted detailed analysis of the stability of formaldehyde-induced DPC in vitro and in human cells. DNA-histone linkages were found to be hydrolytically unstable, with t(1/2) = 18.3 h at 37 degrees C. When histones were allowed to remain bound to DNA after crosslink breakage, the half-life of DPC increased to 26.3 h. This suggests that approximately 30% of spontaneously broken DPC could be re-established under physiological conditions. The half-lives of DPC in three human cell lines (HF/SV fibroblasts, kidney Ad293 and lung A549 cells) were similar and averaged 12.5 h (range 11.6-13.0 h). After adjustment for spontaneous loss, an active repair process was calculated to eliminate DPC from these cells with an average t(1/2) = 23.3 h. Removal of DPC from peripheral human lymphocytes was slower (t(1/2) = 18.1 h), due to inefficient active repair (t(1/2) = 66.6 h). This indicates that the major portion of DPC is lost from lymphocytes through spontaneous hydrolysis rather than being actively repaired. Depletion of intracellular glutathione from A549 cells had no significant effect on the initial levels of DPC, the rate of their repair or cell survival. Nucleotide excision repair does not appear to be involved in the removal of DPC, since the kinetics of DPC elimination in XP-A and XP-F fibroblasts were very similar to normal cells. Incubation of normal or XP-A cells with lactacystin, a specific inhibitor of proteosomes, caused inhibition of DPC repair, suggesting that the active removal of DPC in cells may involve proteolytic degradation of crosslinked proteins. XP-F cells showed somewhat higher sensitivity to formaldehyde, possibly signaling participation of XPF protein in the removal of residual peptide-DNA adducts.

Analysis of DNA-protein crosslinking activity of malondialdehyde in vitro.

In an attempt to identify endogenous chemicals producing DNA-protein crosslinks, we have studied in vitro crosslinking potential of malondialdehyde, a bifunctional chemical that is ubiquitously formed as a product of lipid peroxidation of polyunsaturated fatty acids. We have found that malondialdehyde readily forms crosslinks between DNA and histones under physiological ionic and pH conditions. Formation of DNA-protein crosslinks was limited to proteins that were able to bind to DNA. Malondialdehyde failed to form DNA-protein crosslinks when histone binding to DNA was prevented by elevated ionic strength or when bovine serum albumin was used in the reaction mixture. Malondialdehyde-produced DNA-histone crosslinks were relatively stable at 37 degrees C with t1/2=13.4 days. Crosslinking of histones to DNA proceeds through the initial formation of protein adduct followed by reaction with DNA. Modification of DNA by malondialdehyde does not lead to a subsequent crosslinking of proteins. Significant formation of DNA-protein crosslinks was also registered in isolated kidney and liver nuclei treated with malondialdehyde. Based on its reactivity and stability of the resulting crosslinks, it is suggested that malondialdehyde could be one of the significant sources of endogenous DNA-protein crosslinks.

Utilization of DNA-protein cross-links as a biomarker of chromium exposure.

Human exposure to carcinogenic Cr(VI) compounds is found among workers in a large number of professional groups, and it can also occur through environmental pollution. A significant number of toxic waste sites contain Cr as a major contaminant. In this paper we summarize our efforts to apply measurements of DNA-protein cross-links (DPC) as test for biologically active doses of Cr(VI). DPC were found at elevated levels in lymphocytes in several human populations with low to medium Cr exposures. At high exposure to Cr(VI), exemplified by a group of Bulgarian chromeplaters, DPC plateaued and adducts' levels were similar to those found in environmentally exposed individuals. Lymphocytic DPC correlated strongly with Cr levels in erythrocytes that are indicative of Cr(VI) exposure. DPC in lymphocytes were not confounded by such variables as smoking, age, body weight, gender, or ethnicity. A new version of the cross-link assay offers improved sensitivity and requires a small amount of biologic material. Preliminary results indicate that the ability of DPC to reach detectable levels at low levels of Cr exposure could be related to a lack of repair of these lesions in lymphoid cells. Cr(III)-mediated cross-links of DNA with peptide glutathione or single amino acids were mutagenic in human cells, with a relationship of higher molecular weight of the peptide/amino acid correlating with a more potent mutagenic response. We speculate that bulky DPC could also have a significant promutagenic effect. The current methodology does not allow specific determination of Cr-induced DPC; however, demonstrated sensitivity of DPC measurements and the assay's large sample capacity may allow this assay to be used as the initial screening test for the occurrence of DNA damage in Cr(VI)-exposed populations.

Cr(III)-mediated crosslinks of glutathione or amino acids to the DNA phosphate backbone are mutagenic in human cells.

Carcinogenic Cr(VI) compounds were previously found to induce amino acid/glutathione-Cr(III)-DNA crosslinks with the site of adduction on the phosphate backbone. Utilizing the pSP189 shuttle vector plasmid we found that these ternary DNA adducts were mutagenic in human fibroblasts. The Cr(III)-glutathione adduct was the most potent in this assay, followed by Cr(III)-His and Cr(III)-Cys adducts. Binary Cr(III)-DNA complexes were only weakly mutagenic, inducing a significant response only at a 10 times higher number of adducts compared with Cr(III)-glutathione. Single base substitutions at the G:C base pairs were the predominant type of mutations for all Cr(III) adducts. Cr(III), Cr(III)-Cys and Cr(III)-His adducts induced G:C-->A:T transitions and G:C-->T:A transversions with almost equal frequency, whereas the Cr(III)-glutathione mutational spectrum was dominated by G:C-->T:A transversions. Adduct-induced mutations were targeted toward G:C base pairs with either A or G in the 3' position to the mutated G, while spontaneous mutations occurred mostly at G:C base pairs with a 3' A. No correlation was found between the sites of DNA adduction and positions of base substitution, as adducts were formed randomly on DNA with no base specificity. The observed mutagenicity of Cr(III)-induced phosphotriesters demonstrates the importance of a Cr(III)-dependent pathway in Cr(VI) carcinogenicity.

DNA-protein cross-links produced by various chemicals in cultured human lymphoma cells.

Chemicals such as cis-platinum, formaldehyde, chromate, copper, and certain arsenic compounds have been shown to produce DNA-protein cross-links in human in vitro cell systems at high doses, such as those in the cytotoxic range. Thus far there have only been a limited number of other chemicals evaluated for their ability to produce cross-links. The purpose of the work described here was to evaluate whether select industrial chemicals can form DNA-protein cross-links in human cells in vitro. We evaluated acetaldehyde, acrolein, diepoxybutane, paraformaldehyde, 2-furaldehyde, propionaldehyde, chloroacetaldehyde, sodium arsenite, and a deodorant tablet [Mega Blue; hazardous component listed as tris(hydroxymethyl)nitromethane]. Short- and long-term cytotoxicity was evaluated and used to select appropriate doses for in vitro testing. DNA-protein cross-linking was evaluated at no fewer than three doses and two cell lysate washing temperatures (45 and 65 degrees C) in Epstein-Barr virus (EBV) human Burkitt's lymphoma cells. The two washing temperatures were used to assess the heat stability of the DNA-protein cross-link, 2-Furaldehyde, acetaldehyde, and propionaldehyde produced statistically significant increases in DNA-protein cross-links at washing temperatures of 45 degrees C, but not 65 degrees C, and at or above concentrations of 5, 17.5, and 75 mM, respectively. Acrolein, diepoxybutane, paraformaldehyde, and Mega Blue produced statistically significant increases in DNA-protein cross-links washed at 45 and 65 degrees C at or above concentrations of 0.15 mM, 12.5 mM, 0.003%, and 0.1%, respectively. Sodium arsenite and chloroacetaldehyde did not produce significantly increased DNA-protein cross-links at either temperature nor at any dose tested. Excluding paraformaldehyde and 2-furaldehyde treatments, significant increases in DNA-protein cross-links were observed only at doses that resulted in complete cell death within 4 d following dosing. This work demonstrates that DNA-protein cross-links can be formed in vitro following exposure to a variety of industrial compounds and that most cross-links are formed at cytotoxic concentrations.

Monitoring human lymphocytic DNA-protein cross-links as biomarkers of biologically active doses of chromate.

A simple and sensitive assay for DNA-protein cross-links has been used as a biomarker of chromate exposure and early carcinogenic effects. Pilot studies of DNA-protein cross-links in peripheral blood lymphocytes have been conducted with individuals who had higher exposure to chromate, including welders, and with individuals who had lower levels of exposure such as residents living in a chromium-contaminated area in Jersey City, New Jersey. Studies were also conducted in two Bulgarian cities (Jambol and Burgas) with different levels of air pollution and Cr(VI) exposure and in chrome platers in Bulgaria who had high exposure to chromate. DNA-protein cross-links in U.S. welders and in individuals living in Hudson County, New Jersey around chromium-contaminated areas were significantly higher compared to matched controls. Although blood and urinary levels of chromium were not extensively studied in these populations, we were able to obtain these measurements in the Bulgarian population. Chromium levels in red blood cells of controls living in Burgas were in the order of 1 to 2 ppb chromium, and these individuals had the lowest levels of DNA-protein cross-links. However, the chromium levels in Jambol ranged from about 2 to 7 ppb in red blood cells of city residents to about 22 ppb in chrome platers. DNA-protein cross-links were saturated at about 7 to 8 ppb chromium in the red blood cells, and cross-links correlated well only with chromium levels in red blood cells. Urinary chromium levels did not correlate well with either DNA-protein cross-links or chromium levels in with red blood cells.

Interlaboratory validation of a new assay for DNA-protein crosslinks.

In 1992, a simple and sensitive assay for detecting DNA-protein crosslinks was developed [1]. In an effort to facilitate the greater use of the assay, a number of studies were conducted to evaluate its reliability and reproducibility. During this work, the assay was used to assess whether various metals and other compounds could induce crosslinks in cultured human lymphocytes (Epstein-Barr virus-transformed Burkitt's Lymphoma cell line). Potassium permanganate, mercury chloride, lead nitrate, magnesium perchlorate, aluminum chloride, and cadmium chloride did not induce DNA-protein crosslinks at either cytotoxic or non-cytotoxic levels. Copper sulfate, arsenic trioxide, and potassium chromate induced DNA-protein crosslinks only at cytotoxic concentrations. Acute lethality of the cells was assessed immediately after exposure to metals by trypan blue exclusion while long-term lethality was assessed by cell proliferation and trypan blue exclusion following an incubation period of 5 days after exposure to the metal compound. All metals exhibited 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 sulfate, as well as cis-platinum and chromate, were sent to four different 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 various laboratories using the assay. However, all laboratories generally showed that cis-platinum, chromate, arsenic trioxide and copper sulfate induced DNA-protein crosslinks at levels that produced acute cytotoxicity, whereas cadmium chloride and lead acetate did not.

Formation of the amino acid-DNA complexes by hexavalent and trivalent chromium in vitro: importance of trivalent chromium and the phosphate group.

We have recently shown that a substantial fraction of all Cr-DNA adducts in chromate-exposed cells are represented by ternary complexes involving amino acids or glutathione bridged by Cr-(III) to DNA. The tridentate amino acids such as cysteine, glutamic acid, and histidine were predominantly found cross-linked to DNA. The mechanism by which Cr can cross-link these amino acids to DNA has been modeled by reacting DNA and trivalent and hexavalent chromium with cysteine and histidine. The formation of a Cr(III)-amino acid binary complex was required before Cr(III) reacted with DNA to yield a ternary complex. Cr(III)-pretreated DNA did not bind cysteine or histidine even after prolonged incubations. Reduction of Cr(VI) in the presence of DNA gave rise to an extensive cross-linking of cysteine and histidine. Addition of DNA to Cr(VI) mixtures at the start of reduction or after the reduction was complete had little effect on the level of ternary complexes indicating that Cr(III)-amino acid binary complexes were DNA-attacking species. In order to identify DNA groups involved in the ternary complex formation, pre-formed Cr(III)-histidine complexes were reacted with nucleosides and nucleotide monophosphates followed by separation and analysis of the products. The incubation of the Cr(III)-histidine complexes with nucleotide monophosphates but not with nucleosides gave rise to ternary complexes that contained both histidine and Cr, showing the primary importance of the phosphate group in this reaction. All four DNA nucleotides were capable of the ternary complex formation with Cr(III) and histidine. No apparent base preference in the amino acid cross-linking was also found in the reaction of Cr(III)/cysteine and Cr(VI)/cysteine mixtures with oligonucleotides of base-specific composition.

Occupational exposure to Cr(VI): comparison between chromium levels in lymphocytes, erythrocytes, and urine.

The relationships between chromium (Cr) levels in lymphocytes, erythrocytes, urine, and ambient air were compared among 14 chrome-platers from a metallurgic plant in Bulgaria and two groups of local controls, one from the same heavily polluted industrial town as the chrome-platers (n = 11) and one from a seaside resort town 100 km away (n = 6). Among the chrome-platers, the Cr concentration in peripheral lymphocytes was positively correlated with total Cr and Cr(VI) levels in ambient air and with Cr excretion in urine. As compared to the controls, the chrome-platers had mean Cr levels in lymphocytes twice as high, in erythrocytes ninefold higher, and in urine fourfold to eightfold higher. Although Cr levels in urine and lymphocytes were similar between the two control groups, levels in erythrocytes were 3 times higher among subjects from the industrial area than among those from the seaside town. The study suggests that lymphocyte Cr could be a good indicator of the Cr body burden caused by high exposures to Cr(VI), such as in electroplating operations. In these conditions, erythrocyte Cr may be less useful, possibly owing to increased toxicity due to the high affinity of erythrocytes for Cr. However, when exposure is lower, such as in most environmental situations, erythrocyte Cr should provide a better and more sensitive index than lymphocyte Cr. By contrast, urinary Cr, which provides information on total Cr exposure, including Cr(III) from dietary and environmental sources, does not seem to be of value for studying occupational exposure to Cr(VI).

DNA-protein crosslinks as a biomarker of cis-platinum activity in cancer patients.

We have developed a new method to assess the amount of DNA-protein crosslinks (DNA-PC) in peripheral lymphocytes, based on the selective precipitation of the DNA crosslinked to proteins. We assessed the amount of DNA-PC in peripheral lymphocytes of 18 cancer patients who underwent chemotherapy with cis-platinum for the first time. Since the chemotherapy was administered over a two-day period, blood samples were drawn at baseline (before starting the therapy), 4 h after the infusion of the first dose of cis-platinum, the next day (24 h after the first dose, and immediately before the infusion of the second dose), and 2 days later (48 h after the first dose). The mean change of DNA-PC 4 h after therapy was 0.8+/-0.8% (p=0.006), 0.5+/-0.6% after 1 day (p=0.007), and 0.1+/-0.5% after two days (ns). The correlation between DNA-PC changes and cumulative dose of cis-platinum was -0.22 at 4 h, -0.19 after 1 day, and -0.68 at 2 days (p=0.005). The crosslinking effect of cis-platinum seems to vary among individuals and with dose; the DNA-PC may be used to define sub-populations of patients with various degree of sensitivity to the pharmacologic action of this chemotherapeutic agent, and thus to adjust the dosage on an individual basis.

DNA-protein crosslinks in peripheral lymphocytes of individuals exposed to hexavalent chromium compounds.

Abstract DNA-protein crosslinks were measured in peripheral blood lymphocytes of chrome-platers and controls from Bulgaria in order to evaluate a genotoxic effect of human exposure to carcinogenic Cr(VI) compounds. Chrome-platers and most of the unexposed controls were from the industrial city of Jambol; some additional controls were recruited from the seaside town of Burgas. The chrome-platers had significantly elevated levels of chromium in pre- and post-shift urine, erythrocytes and lymphocytes compared with the control subjects. The largest differences between the two groups were found in erythrocyte chromium concentrations which are considered to be indicative of Cr(VI) exposure. Despite the significant differences in internal chromium doses, levels of DNA-protein crosslinks were not significantly different between the combined controls and exposed workers. Individual DNA-protein crosslinks, however, correlated strongly with chromium in erythrocytes at low and moderate doses but at high exposures, such as among the majority of chrome-platers, these DNA adducts were saturated at maximum levels. The saturation of DNA-protein crosslinks seems to occur at 7-8 µg I-(1) chromium in erythrocytes whereas a mean erythrocyte chromium among the chrome platers was as high as 22.8 µg l(-1). Occupationally unexposed subjects exhibited a significant variability with respect to the erythrocyte chromium concentration, however erythrocyte chromium levels correlated closely with DNA-protein crosslinks in lymphocytes. The controls from Jambol had higher chromium concentrations in erythrocytes and elevated levels of DNA-protein crosslinks compared with Burgas controls. Occupational exposure to formaldehyde among furniture factory workers did not change levels of DNA-protein crosslinks in peripheral lymphocytes. DNA-protein crosslink measurements showed a low intraindividual variability and their levels among both controls and exposed indivduals were not affected by smoking, age or weight.

Increased DNA-protein crosslinks in lymphocytes of residents living in chromium-contaminated areas.

It has been known for a number of years that chromium-containing mine slags were used as landfill in residential areas of Hudson County, New Jersey. Since one of the major lesions induced in intact cells by chromate is the DNA-Protein crosslink, we have used this lesion as a biomarker of biological effect of chromium (Cr) exposure. We have previously developed a sensitive and easy-to-perform assay to detect DNA-Protein crosslinks, based on the selective K SDS precipitation of DNA associated with protein. We examined the levels of DNA-Protein crosslinks in peripheral blood mononuclear cells of 33 individuals determined to be at risk for chromium exposure by virtue of their residence in Hudson County and their urinary Cr levels. These data were compared to the levels of DNA-Protein crosslinks among 49 controls who resided in noncontaminated areas. A complete clinical examination and urine analysis did not show any Cr-related abnormalities among the exposed population. The mean DNA-Protein crosslink level in the lymphocytes of the exposed group was 1.3 +/- 0.5% (SD), whereas the unexposed group had 0.8 +/- 0.4% (p < 0.001), after adjustment for age, gender, race, smoking, and weight. Further studies in this population are needed to confirm the possible association between the high levels of DNA-Protein crosslink and Cr exposure.

Heterochromatic proteins specifically enhance nickel-induced 8-oxo-dG formation.

7,8-Dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG) was measured as an indicator of nickel-induced oxidative base damage in the presence of H2O2. Heterochromatic proteins isolated from Chinese hamster liver cells enhanced the formation of 8-oxo-dG induced by NiCl2 and H2O2 in vitro, whereas euchromatic proteins inhibited this reaction. The inhibitory effect of euchromatic proteins on dG oxidation may be due to the oxygen radical scavenging effects of low molecular weight protein-rich fractions. Gel electrophoresis confirmed that histone H1 was present at a higher concentration in heterochromatin than in euchromatin. It is believed that the presence of nickel-protein complexes in cells is crucial for the formation of reactive oxygen species (ROS). We found that Ni2+ binds to histone H1 and core histones as determined by 63Ni autoradiography of proteins on nitrocellulose membranes. In vitro studies showed that commercially purified histone H1, and to a considerably lesser extent core histones, enhanced the NiCl2 and H2O2 catalyzed formation of 8-oxo-dG in a reaction containing free dG base. Since histone H1 is a lysine- and alanine-rich protein, the levels of 8-oxo-dG induced by NiCl2 and H2O2 were studied in the presence of these amino acids and found to be enhanced by them. These results suggest that nickel may specifically produce oxidative DNA damage in heterochromatin because of the nature of its binding to histone H1 and core histones. This selective oxidation of genetically inactive heterochromatin may explain why nickel compounds which generate oxygen radicals and oxidize DNA bases are inactive in most gene mutation assays.

Carcinogenic nickel silences gene expression by chromatin condensation and DNA methylation: a new model for epigenetic carcinogens.

A transgenic gpt+ Chinese hamster cell line (G12) was found to be susceptible to carcinogenic nickel-induced inactivation of gpt expression without mutagenesis or deletion of the transgene. Many nickel-induced 6-thioguanine-resistant variants spontaneously reverted to actively express gpt, as indicated by both reversion assays and direct enzyme measurements. Since reversion was enhanced in many of the nickel-induced variant cell lines following 24-h treatment with the demethylating agent 5-azacytidine, the involvement of DNA methylation in silencing gpt expression was suspected. This was confirmed by demonstrations of increased DNA methylation, as well as by evidence indicating condensed chromatin and heterochromatinization of the gpt integration site in 6-thioguanine-resistant cells. Upon reversion to active gpt expression, DNA methylation and condensation are lost. We propose that DNA condensation and methylation result in heterochromatinization of the gpt sequence with subsequent inheritance of the now silenced gene. This mechanism is supported by direct evidence showing that acute nickel treatment of cultured cells, and of isolated nuclei in vitro, can indeed facilitate gpt sequence-specific chromatin condensation. Epigenetic mechanisms have been implicated in the actions of some nonmutagenic carcinogens, and DNA methylation changes are now known to be important in carcinogenesis. This paper further supports the emerging theory that nickel is a human carcinogen that can alter gene expression by enhanced DNA methylation and compaction, rather than by mutagenic mechanisms.

Glutathione and free amino acids form stable complexes with DNA following exposure of intact mammalian cells to chromate.

Exposure of cells to carcinogenic Cr(VI) compounds results in the formation of several types of DNA lesions such as strand breaks, DNA-protein crosslinks and uncharacterized DNA-Cr adducts. Hexavalent chromium compounds are positive in most bacterial and eukaryotic mutagenic systems, although the nature of DNA modifications underlying the chromium-induced mutagenesis is not known. Hexavalent chromate(VI) is very active in cellular systems because it is actively transported into cells, but intracellularly it is ultimately reduced to Cr(III). Here we show that exposure of Chinese hamster ovary (CHO) cells to potassium chromate(VI) leads to the formation of stable complexes between DNA and amino acids or glutathione. Cysteine, glutamic acid and histidine were the major amino acids crosslinked to DNA in chromate-treated cells. Incubation of purified DNA in the presence of EDTA dissociated SDS stable amino acid-DNA complexes, which indicates that these DNA adducts are most likely to represent ternary coordination complexes mediated by Cr(III) rather than covalent linkage between amino acids/glutathione and DNA. The amino acids that were found complexed with DNA purified from chromate-exposed cells did not orginate from previously crosslinked proteins during DNA isolation, but represented authentic reactions of free amino acids and glutathione with chromium and DNA in cells. Ternary complexes of glutathione or amino acids with Cr(III) and DNA were estimated to account for as much as 50% of DNA-bound chromium following exposure to < or = 25 microM chromate.