Carcinogenic Cr Research Articles

Abstract 1003: S-nitrosylation of Bcl-2 negatively affects autophagy in malignantly transformed lung epithelial cells

Abstract Purpose The purpose of this study is to identify the key autophagic biomarkers involved in Cr(VI)-induced malignant transformation of non-tumorigenic Beas-2B cells and to investigate the underlying mechanisms. Experimental Methods The non-tumorigenic (Beas-2B) and malignantly transformed (B-Cr) lung epithelial cell lines were used in all experiments. Cells were exposed to hexavalent chromium (Cr (VI)) doses of 20 and 50μM for 6h time points. Separate experiments were conducted using inhibitors for the anti-apoptotic protein Bcl-2 (ABT-737), nitric oxide (AG (Aminoguanidine)), and p38 (SB203580) for 1hr before Cr treatment as detailed above. Cells were transfected with myc-tagged wild-type (WT) Bcl-2 (WT Bcl-2), double-mutant (C158A/C229A) Bcl-2 (DM Bcl-2), or control (pcDNA3) plasmid DNA (5 μg) using Lipofectamine 2000 transfecting agent. S-nitrosylation of Bcl-2 was measured using the S-Nitrosylation Western Blot kit. For autophagy and apoptosis analysis, cell vacuoles and nuclei were stained using the Cyto-ID dye kit and Hoechst dye respectively, and scored by fluorescence microscopy. Results Here we show that Cr(VI)-induced carcinogenesis triggers autophagy in non-tumorigenic human lung epithelial cells, while malignantly-transformed lung cells are resistant to autophagy. We previously reported that chronic exposure of human lung epithelial cells to the carcinogenic hexavalent chromium Cr(VI) caused malignant transformation and Bcl-2 upregulation. In this study we report that nitric oxide (NO), a potent cellular messenger, has a negative effect on autophagy initiation by nitrosylating and stabilizing the Bcl-2 protein, thereby preserving the anti-autophagic Bcl-2-Beclin-1 interaction. We demonstrate that p38 MAPK is also critical in the autophagic response along with LC3 and Beclin-1. Further results also link apoptosis, analyzed by cell staining and measuring caspase-9 protein levels, with the autophagic proteins, suggesting a possible autophagy-apoptosis pathway crosstalk. The effect of Bcl-2 nitrosylation on autophagy was corroborated using a Bcl-2 inhibitor (ABT-737) and by transfecting cells with non-nitrosylable Bcl-2 double mutant (DM) plasmid, in which the two cysteine residues (Cys158 and Cys229) critical for nitrosylation are mutated. Both ABT-737 and transfection with Bcl-2 DM plasmid resulted in decreased S-nitro-cysteine expression and increased autophagy in lung cells. Conclusions We report a novel mechanism involving nitric oxide (NO)-mediated post-translational modification of Bcl-2 protein as being responsible for this resistance to autophagy induction. This Bcl-2 S-nitrosylation was observed to affect the Bcl-2-Beclin-1 interaction which is key for autophagy initiation. This novel regulation of autophagy may be important in cancer development and progression, as well as in carcinogenesis and its prevention. Citation Format: Clayton A. Wright, Anand Krishnan V. Iyer, Yogesh Kulkarni, Neelam Azad. S-nitrosylation of Bcl-2 negatively affects autophagy in malignantly transformed lung epithelial cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1003. doi:10.1158/1538-7445.AM2015-1003

Biogenic sulfides for sequestration of Cr (VI), COD and sulfate from synthetic wastewater

The aim of this work is to explore the possibility of using mixed culture of mesophilic sulfate-reducing bacteria (SRB) for retrieval of toxic and carcinogenic Cr (VI) from synthetic solution. In order to treat Cr (VI) containing wastewater effectively, SRB culture was adapted to 50 mg/L Cr (VI) and maintained through repeated sub-culturing to enhance the growth and activity of SRB. Batch biosorption experiments were carried out in glass serum vials by cultured SRB, accomplishing the removal of 82.1% Cr (VI), 76.9% sulfate, 85.7% COD under the following optimized conditions: pH 7, hydraulic retention time (HRT) 7 days, temperature 37 °C and initial Cr (VI) concentration of 50 mg/L. Further sorption experiments were conducted on synthetic wastewater under optimal operational conditions and resulted in 89.2% Cr (VI), 81.9% COD and 95.3% sulfate reduction from simulated wastewater. The results of this work contributed to a better understanding of metal uptake by biogenic sulfides and would be beneficial in the development of potential biosorbents that possess high capacities for Cr (VI) uptake from aqueous environments.

Chromium (VI)-induced transformation is enhanced by Zn deficiency in BALB/c 3T3 cells.

Hexavalent chromium [Cr(VI)] is a carcinogenic heavy metal that is reduced to intermediate oxidation states, such as Cr(V) and Cr(IV), in the process of forming stable Cr(III) forms; it is these intermediate forms that are thought to be responsible for much of the DNA damage and mutations that are induced by Cr(VI). Metallothionein (MT), a heavy metal-binding protein, is induced by zinc and other heavy metals and protects cells from the toxic effects of these metals by sequestering them. MT cannot bind Cr, but by scavenging reactive oxygen species through its cysteine residues, it may act as a protective factor against Cr(VI)-induced DNA lesions by reducing Cr(VI) directly to Cr(III), thereby avoiding the creation of the toxic intermediates. Here, we showed that Zn deficiency decreased MT expression in BALB/3T3 clone A31-1-1 cells and caused them to become highly susceptible to Cr(VI)-induced transformation. To obtain Zn-deficient cultures, cells were cultured in medium supplemented with 10% Chelex(®)-100 chelating resin-treated FBS. The increase in susceptibility to transformation was abolished by culturing the cells with supplemental Zn (50 µM). Previously, we reported that Cr(VI) inhibits MT transcription by preventing the zinc-dependent formation of a complex of metal response element-binding transcription factor-1 (MTF-1) and the co-activator p300. Our results suggest that the carcinogenicity of Cr(VI) is enhanced by MTF-1 dysfunction.

Trivalent chromium solubility and its influence on quantification of hexavalent chromium in ambient particulate matter using EPA method 6800

Measurement of carcinogenic Cr(VI) in ambient PM is challenging due to potential errors associated with conversion between Cr(VI) (a carcinogen) and Cr(III) (an essential nutrient). Cr(III) conversion is a particular concern due to its >80% atomic abundance in total Cr. U.S. Environmental Protection Agency (EPA) method 6800 that uses water-soluble isotope spikes can be used to correct the interconversion. However, whether the enriched Cr(III) isotope spikes can adequately mimic the Cr(III) species originally in ambient PM is unknown. This study examined the water solubility of Cr(III) in ambient PM and discussed its influence on Cr(VI) measurement. Ambient PM10 samples were collected on Teflon filters at four sites in New Jersey that may have different Cr emission sources. The samples were ultrasonically extracted with 5 mL DI-H2O (pH 5.7) at room temperature for 40 min, and then analyzed by ion chromatography–inductively coupled plasma mass spectrometry (IC-ICPMS). Cr(III) was below detection limit (0.06 ng/m3) for all samples, suggesting water-soluble Cr(III) species, such as CrCl3, Cr(NO3)3, and amorphous Cr(OH)3, in the ambient PM were negligible. Therefore, the enriched 50Cr(III) isotope spike (in the form of Cr(NO3)3) could not mimic the original ambient Cr(III). Only the conversion of 53Cr(VI) (in the form of K2CrO4) was taken into account when correcting the interconversion. We then used NaHCO3-pretreated MCE filters (prespiked with enriched isotope species) to measure Cr(VI) in the ambient PM10. The samples were ultrasonically extracted at 60 C pH 9 solutions for 40 min followed by IC-ICPMS analysis. Due to the correction of Cr(VI) reduction, the Cr(VI) concentrations determined by EPA method 6800, 0.26 ± 0.16 (summer) and 0.16 ± 0.11(winter) ng/m3 (n = 64), were significantly greater than those by the external standard curve, 0.21 ± 0.17 (summer) and 0.10 ± 0.07 (winter) ng/m3 (n = 56) (p < 0.01, Student’s t-test). Our study revealed that appropriate application of EPA method 6800 is important because it only applies to soluble fraction of Cr species in ambient PM.ImplicationsAccurate measurement of carcinogenic Cr(VI) in ambient PM is challenging due to conversion between Cr(VI) (a human carcinogen) and Cr(III) (a human essential nutrient). The conversion of Cr(III) is of particular concern due to its dominant presence in total Cr (>80%). This study examined the water solubility of Cr(III) in ambient PM that was collected at four locations in New Jersey. Then we discussed the influence of Cr(III) solubility on the application of EPA method 6800, which utilizes enriched isotope spikes to correct the interconversion. Our results suggested that appropriate application of EPA method 6800 is important because it only applies to soluble fraction of Cr species.

In silico Protein Structure Modeling and Conservation Analysis of ChrR, a Class-I Chromate Reducing Flavoenzyme from Pseudomonas putida

Hexavalent chromium [Cr(VI)] is a widespread environmental pollutant, arising as a by-product of numerous industrial processes. Bacteria can reduce toxic and carcinogenic Cr(VI) to insoluble and less toxic Cr(III), offering promise for an environmental friendly and affordable solution to chromate pollution. ChrR, a class I chromate-reducing flavoenzyme from Pseudomonas putida is an efficient chromate reducer. The crystal structure of ChrR is yet unknown to the scientific community, hence a three-dimensional (3D) structure is very essential for structural studies, protein - ligand interaction simulations and designing novel bioremediation strategies. The 3D model of the P. putida ChrR protein was predicted using in silico approach. Due to low percentage of sequence identity for homology modeling, I-TASSER was used for structure prediction which combines the methods of threading, ab initio modeling and structural refinement. The stereo chemical quality of the best model was validated with 90.6% residues under favored region from Ramachandran plot. The modeled protein was submitted to Protein Model Database and can be downloaded with the ID PM0077375. The degree of conservation was mapped onto the predicted model and ligand binding sites were found. The results of conservation analysis and binding site prediction were combined to show several highly conserved binding sites. Altogether, the structure for ChrR has been predicted. The work reveals novel universally conserved residues. These residues could be candidates for binding interactions and provide the basis for designing advanced chromium bioremediation strategies.

Isolation, Characterization, and Nuclease Activity of Biologically Relevant Chromium(V) Complexes with Monosaccharides and Model Diols. Likely Intermediates in Chromium-Induced Cancers

The stabilization of Cr(V) by biological 1,2-diolato ligands, including carbohydrates, glycoproteins, and sialic acid derivatives, is likely to play a crucial role in the genotoxicity of Cr(VI) and has also been implicated in the antidiabetic effect of Cr(III). Previously, such complexes have been observed by electron paramagnetic resonance (EPR) spectroscopy in living cells or animals, treated with carcinogenic Cr(VI), as well as in numerous model systems, but attempts to isolate them have been elusive. Recently, the first crystal structure of a Cr(V) complex with cis-1,2-cyclohexanediol (1, a close structural analogue of carbohydrates) has been reported. In this work, Cr(V) complexes of the general formula [Cr(V)OL2](-) [where LH2 = 1, cis-1,2-cyclopentanediol (2), D-glucose (3), D-mannose (4), D-galactose (5), and D-ribose (6)] have been isolated from light-catalyzed reactions of Cr(VI) (anhydrous Na2Cr2O7) with slight molar excesses of the corresponding ligands in N,N-dimethylformamide. The complexes were characterized by elemental analyses, electrospray mass spectrometry (ESMS), and EPR spectroscopy. Studies by electronic absorption spectroscopy have shown that the solids isolated from reactions of Cr(VI) with 3-6 contained mixtures of Cr(V) complexes (40-65 mol %) and Cr(III) species (probably complexes with oxidized ligands), while those from reactions with 1 and 2 were practically pure Cr(V). The first isolation of solids containing significant proportions of chromium(V) monosaccharide complexes led to the definitive assignment of their general formula ([Cr(V)OL2](-), based on ESMS), in agreement with the earlier EPR spectroscopic data. The first direct comparison of the decomposition rates of Cr(V) complexes with 1-6, made possible by isolation of the solids, have shown that the complexes with five-membered-ring ligands (2 and 6) are more stable at pH ∼ 7 compared with their six-membered-ring counterparts (1 and 3-5). This finding emphasizes the likely biological roles of chromium(V) pentose complexes, e.g., those with sugar residues of RNA, ATP, or NAD(P)H. Finally, the first direct evidence for the ability of these Cr(V) complexes to cause oxidative DNA damage in the absence of added reductants or oxidants has been obtained. These data support significant roles for chromium(V) 1,2-diolato complexes in the diverse biological activities of Cr(VI) and Cr(III).

One-pot microwave mediated growth of heterostructured ZnO@AlSi as a potential dual-function eco-catalyst for treating hazardous pollutants in water resources

Dual function, eco-catalyst material had been designed with nano ZnO@aluminosilicate mineral (ZnO@AlSi) and processed via microwave assisted in situ intercalation and crystal growth reactions. Dual function of ZnO@AlSi was confirmed by the simultaneous adsorption and UV photoreduction of carcinogenic Cr(VI) to non-toxic Cr(III) and photooxidation of phenol and methylene blue dye (MB); a co-existed model system was simulated and prepared together in laboratory conditions. Single phase nano ZnO and ion exchanged AlSi layered silicates were also separately prepared for comparisons with ZnO@AlSi system. All these materials were characterized for BET, DLS, TGA, SEM, TEM, UV–visible and fluorescence spectrophotometry to analyze structural, thermal, morphological, and optical properties. It was seen that the inherent silicate layered structure significantly restricted the growth of ZnO, resulting in mesoporous, UV-active ZnO@AlSi heterogeneous nanocatalyst. An effective adsorption of Cr(VI), phenol and MB to the extent of 99% and the transformation of Cr(VI) to Cr (III) through photoreduction, and decomposition of phenol and MB by photooxidation followed by mineralization were achieved at once. This synergic adsorption and photodecomposition effects could not be found together in single phase nano ZnO as well as ion exchanged AlSi system. The kinetic photo-experiments indicated that reduction of Cr(VI) and degradation of phenol and MB by ZnO@AlSi followed Langmuir–Hinshelwood first-order kinetic model. The investigation of ZnO@AlSi at Cr(VI)/phenol and Cr(VI)/MB binary systems displayed high decontamination performance of ZnO@AlSi than in single pollutant system due to the arresting of electron–hole pair recombination via consuming both the members in separate redox half-reactions. Alternatively, life time of catalyst was found decreased in organic–inorganic pollutant system compared to single component systems. In summary, a dual function catalyst was designed through a facile technique and validated for the cost-effective removal and decomposition of organic and inorganic pollutants simultaneously in the complex wastewater.

Metal-based anti-diabetic drugs: advances and challenges

The current status and likely future directions of complexes of V(V/IV), Cr(III), Mo(VI), W(VI), Zn(II), Cu(II), and Mn(III) as potential oral drugs against type 2 diabetes are reviewed. We propose a unified model of extra- and intracellular mechanisms of anti-diabetic efficacies of V(V/IV), Mo(VI), W(VI), and Cr(III), centred on high-oxidation-state oxido/peroxido species that inhibit protein tyrosine phosphatases (PTPs) involved in insulin signalling. The postulated oxidative mechanism of anti-diabetic activity of Cr(III) via carcinogenic Cr(VI/V) (which adds to safety concerns) is consistent with recent clinical trials on Cr(III) picolinate, where activity was apparent only in patients with poorly controlled diabetes (high oxidative stress), and the correlation between the anti-diabetic activities and ease of oxidation of Cr(III) supplements and their metabolites in vivo. Zn(II) and Cu(II) anti-diabetics act via different mechanisms and are unlikely to be used as specific anti-diabetics due to their diverse and unpredictable biological activities. Hence, future research directions are likely to centre on enhancing the bioavailability and selectivity of V(V/IV), Mo(VI), or W(VI) drugs. The strategy of potentiating circulating insulin with metal ions has distinct therapeutic advantages over interventions that stimulate the release of more insulin, or use insulin mimetics, because of many adverse side-effects of increased levels of insulin, including increased risks of cancer and cardiovascular diseases.

Defect delineation by chemical etching techniques in silicon and engineered silicon substrates: Novel chemistries and basic aspects

AbstractPreferential etching techniques in combination with light optical microscopy are still the workhorse for the evaluation of defect types and area densities in engineered silicon substrates such as silicon‐on‐insulator (SOI) wafers. Most etching recipes are based on chromium (VI) compounds as oxidizing agent. Since in the meantime the use of carcinogenic Cr (VI) compounds is restricted by law there is a need for Cr‐free recipes with adequate or superior defect delineation performance. We present novel Cr‐free etching chemistries based on organic peracids or on organic oxidizing agents (OOE) such as benzoquinone and derivatives in organic solvents. Some of these have very low removal rates (nm/min at 25 °C), are very defect sensitive and are especially suitable for the application on thin (&lt;50 nm) SOI films. Preferential etching solutions can be characterized by parameters like removal rate, activation energy for the etching process and selectivity. The selectivity describes the ratio of the removal at the crystal defect and at the perfect crystal. In this work the selectivity was determined experimentally on dislocations for various defect etching chemistries. Dislocations were generated by damaging silicon substrates by controlled indentation with a diamond tip and subsequent annealing at 1000 °C. After preferential etching the depth of etch pits was measured by an atomic force microscope. The activation energy was evaluated via an Arrhenius plot of the temperature dependent etch rates. At the dislocations the activation energies are reduced by about 5%. For the various defect etching chemistries selectivities in the range of 1.5–2.5 were found.

A quantitative assessment of the carcinogenicity of hexavalent chromium by the oral route and its relevance to human exposure

Background Hexavalent chromium (Cr 6+) has long been recognized as an inhalation carcinogen. Useful data on its carcinogenicity by ingestion have been slower to develop. The 2008 NTP chronic bioassay of sodium dichromate dihydrate in drinking water found clear evidence of carcinogenicity in rodents and allows a generalizable estimate of the human ingestion cancer potency of Cr 6+. Objectives To estimate the human ingestion cancer potency of Cr 6+ and evaluate its relevance for human exposure. Methods Tumors of the small intestine in male mice were selected as the critical endpoint, for the derivation of cancer potency. Following the 2005 USEPA Cancer Risk Assessment Guidelines, the point of departure in the dose–response data was defined using benchmark–dose modeling. Linear extrapolation was carried out from the point of departure and interspecies dose conversion was based on allometric scaling of body weight. Results Small intestine tumor incidence in male mice provided a robust and nearly identical fit with most available dose–response models using benchmark–dose modeling. A human equivalent cancer slope factor of 0.5 (mg/kg/day) −1 was derived based on linear extrapolation from the point of departure. Statistical and kinetic analysis carried out on the NTP data as well as data reported in other studies support that the carcinogenicity of Cr 6+ did not result from an exceedance of the reduction capacity of the mouse gastrointestinal tract at the doses in the NTP study. Conclusions Mouse gastric emptying time and human Cr 6+ dosing studies strongly suggest that even at doses considerably lower than those in the NTP study, Cr 6+ escapes reduction in both the mouse and human stomachs due to kinetic competition from Cr 6+ absorption and gastric emptying. The cancer potency derived from the NTP data is, therefore, deemed to be relevant and applicable to human exposure. Cr 6+ is, therefore, identified as “likely to be carcinogenic to humans” in accordance with the USEPA’s cancer characterization rubric.

Formation and Reactivity of Chromium(V)−Thiolato Complexes: A Model for the Intracellular Reactions of Carcinogenic Chromium(VI) with Biological Thiols

The nature of the long-lived EPR-active Cr(V) species observed in cells and biological fluids exposed to carcinogenic Cr(VI) has been definitively assigned from detailed kinetic and spectroscopic analyses of a model reaction of Cr(VI) with p-bromobenzenethiol (RSH) in the presence or absence of cyclic 1,2-diols (LH(2)) in aprotic or mixed solvents. The first definitive structures for Cr(V) complexes with a monodentate thiolato ligand, [Cr(V)O(SR)(4)](-) (g(iso) = 1.9960, A(iso) = 14.7 x 10(-4) cm(-1)), [Cr(V)OL(SR)(2)](-) (g(iso) = 1.9854, A(iso) = (15.8-16.2) x 10(-4) cm(-1)) and [Cr(V)(O)(2)(SR)(2)](-) (g(iso) = 1.9828, A(iso) = 6.8 x 10(-4) cm(-1)) were assigned by EPR spectroscopy and electrospray mass spectrometry. The unusually low A(iso) ((53)Cr) value for the latter species is consistent with its rare four-coordinate, bis-oxido structure. The [Cr(V)OL(SR)(2)](-) species are responsible for the transient g(iso) approximately 1.986 EPR signals observed in living cells and animals treated with Cr(VI) (where RSH and LH(2) are biological thiols and 1,2-diols, respectively). For the first time, concentrations of Cr(V) intermediates formed during the reduction of Cr(VI) were determined by quantitative EPR spectroscopy, and a detailed reaction mechanism was proposed on the basis of stochastic simulations of the kinetic curves for Cr(V) species. A key feature of the proposed mechanism is the regeneration of Cr(V) species in the presence of Cr(VI) through the formation of organic free radicals, followed by the rapid reactions of the formed radicals with Cr(VI). The concentration of Cr(V) grows rapidly at the beginning of the reaction, reaches a steady-state level, and then drops sharply once Cr(VI) is spent. Similar mechanisms are likely to operate during the reduction of Cr(VI) in biological environment rich in reactive C-H bonds, including the oxidative DNA damage by Cr(V) intermediates.

Chromium nitrate as a source of chromium added for nutritional purposes to food supplements

Chromium nitrate as a source of chromium added for nutritional purposes to food supplements

Hexavalent chromium is carcinogenic to F344/N rats and B6C3F1 mice after chronic oral exposure.

BackgroundHexavalent chromium [Cr(VI)] is a human carcinogen after inhalation exposure. Humans also ingest Cr(VI) from contaminated drinking water and soil; however, limited data exist on the oral toxicity and carcinogenicity of Cr(VI).ObjectiveWe characterized the chronic oral toxicity and carcinogenicity of Cr(VI) in rodents.MethodsThe National Toxicology Program (NTP) conducted 2-year drinking water studies of Cr(VI) (as sodium dichromate dihydrate) in male and female F344/N rats and B6C3F1 mice.ResultsCr(VI) exposure resulted in increased incidences of rare neoplasms of the squamous epithelium that lines the oral cavity (oral mucosa and tongue) in male and female rats, and of the epithelium lining the small intestine in male and female mice. Cr(VI) exposure did not affect survival but resulted in reduced mean body weights and water consumption, due at least in part to poor palatability of the dosed water. Cr(VI) exposure resulted in transient microcytic hypochromic anemia in rats and microcytosis in mice. Nonneoplastic lesions included diffuse epithelial hyperplasia in the duodenum and jejunum of mice and histiocytic cell infiltration in the duodenum, liver, and mesenteric and pancreatic lymph nodes of rats and mice.ConclusionsCr(VI) was carcinogenic after administration in drinking water to male and female rats and mice.

Proteomic analysis of chromium cytotoxicity in cultured rat lung epithelial cells

Chromium (Cr) has been widely used in industry for more than one century. Exposure to hexavalent Cr compounds is strongly associated with increasing risk of lung cancer. Extensive researches at DNA level indicated that generation of ROS from the reduction of Cr(VI) leading to DNA damage is the major cause of the toxicity and carcinogenicity of Cr(VI). The present study in cellular and protein levels confirmed that Cr(VI) induced apoptosis of lung epithelial cells (LEC) via ROS generation. To view the differentially expressed proteins in the process of Cr(VI) reduction, subcellular proteomics was applied and allowed the identification of more than 30 proteins with expression alteration. Most of those proteins are correlated with ROS-elicited responses, which were further validated by Western blotting analysis, induction of p53 pathway and antioxidative treatment. The current findings provided additional evidence in protein level to support the claim that ROS generated during the process of Cr(VI) reduction are involved in the Cr(VI)-induced toxicity and carcinogenesis.

A Novel Chromate Reductase from Thermus scotoductus SA-01 Related to Old Yellow Enzyme

Bacteria can reduce toxic and carcinogenic Cr(VI) to insoluble and less toxic Cr(III). Thermus scotoductus SA-01, a South African gold mine isolate, has been shown to be able to reduce a variety of metals, including Cr(VI). Here we report the purification to homogeneity and characterization of a novel chromate reductase. The oxidoreductase is a homodimeric protein, with a monomer molecular mass of approximately 36 kDa, containing a noncovalently bound flavin mononucleotide cofactor. The chromate reductase is optimally active at a pH of 6.3 and at 65 degrees C and requires Ca(2+) or Mg(2+) for activity. Enzyme activity was also dependent on NADH or NADPH, with a preference for NADPH, coupling the oxidation of approximately 2 and 1.5 mol NAD(P)H to the reduction of 1 mol Cr(VI) under aerobic and anaerobic conditions, respectively. The K(m) values for Cr(VI) reduction were 3.5 and 8.4 microM for utilizing NADH and NADPH as electron donors, respectively, with corresponding V(max) values of 6.2 and 16.0 micromol min(-1) mg(-1). The catalytic efficiency (k(cat)/K(m)) of chromate reduction was 1.14 x 10(6) M(-1) s(-1), which was >50-fold more efficient than that of the quinone reductases and >180-fold more efficient than that of the nitroreductases able to reduce Cr(VI). The chromate reductase was identified to be encoded by an open reading frame of 1,050 bp, encoding a single protein of 38 kDa under the regulation of an Escherichia coli sigma(70)-like promoter. Sequence analysis shows the chromate reductase to be related to the old yellow enzyme family, in particular the xenobiotic reductases involved in the oxidative stress response.

Chronic Exposure to Lead Chromate Causes Centrosome Abnormalities and Aneuploidy in Human Lung Cells

Hexavalent chromium [Cr(VI)] compounds are established human lung carcinogens. The carcinogenicity of Cr(VI) is related to its solubility, with the most potent carcinogens being the insoluble particulate Cr(VI) compounds. However, it remains unknown why particulate Cr(VI) is more carcinogenic than soluble Cr(VI). One possible explanation is that particulates may provide more chronic exposures to chromate over time. We found that aneuploid cells increased in a concentration- and time-dependent manner after chronic exposure to lead chromate. Specifically, a 24-hour lead chromate exposure induced no aneugenic effect, whereas a 120-hour exposure to 0.5 and 1 microg/cm2 lead chromate induced 55% and 60% aneuploid metaphases, respectively. We also found that many of these aneuploid cells were able to continue to grow and form colonies. Centrosome defects are known to induce aneuploidy; therefore, we investigated the effects of chronic lead chromate exposure on centrosomes. We found that centrosome amplification in interphase and mitotic cells increased in a concentration- and time-dependent manner with 0.5 and 1 microg/cm2 lead chromate for 120 hours, inducing aberrant centrosomes in 18% and 21% of interphase cells and 32% and 69% of mitotic cells, respectively; however, lead oxide did not induce centrosome amplification in interphase or mitotic cells. There was also an increase in aberrant mitosis after chronic exposure to lead chromate with the emergence of disorganized anaphase and mitotic catastrophe. These data suggest that one possible mechanism for lead chromate-induced carcinogenesis is through centrosome dysfunction, leading to the induction of aneuploidy.

Carcinogenic Cr(VI) and the Nutritional Supplement Cr(III) Induce DNA Deletions in Yeast and Mice

Industrial Cr(VI) emissions contaminate drinking water sources across the U.S., and many people take Cr(III) nutritional supplements. Cr(VI) is a human pulmonary carcinogen, but whether it is carcinogenic in the drinking water is not known. Due to widespread human exposure, it is imperative to determine the carcinogenic potential of Cr(VI) and Cr(III). DNA deletions and other genome rearrangements are involved in carcinogenesis. We determined the effects of Cr(VI) as potassium dichromate and Cr(III) as chromium(III) chloride on the frequencies of DNA deletions measured with the deletion assay in Saccharomyces cerevisiae and the in vivo p(un) reversion assay in C57BL/6J p(un)/p(un) mice. Exposing yeast and mice via drinking water to Cr(VI) and Cr(III) significantly increased the frequency of DNA deletions. We quantified intracellular chromium concentrations in yeast and tissue chromium concentrations in mice after exposure. Surprisingly, this revealed that Cr(III) is a more potent inducer of DNA deletions than Cr(VI) once Cr(III) is absorbed. This study concludes that both the environmental contaminant Cr(VI) and the nutritional supplement Cr(III) increase DNA deletions in vitro and in vivo, when ingested via drinking water.

Lead chromate-induced chromosome damage requires extracellular dissolution to liberate chromium ions but does not require particle internalization or intracellular dissolution.

Hexavalent chromium [Cr(VI)] is a well-established human lung carcinogen. Water solubility has proven to be a key factor in the carcinogenicity of Cr(VI), with the water insoluble or "particulate" compounds the more potent carcinogens. Pathology studies indicate that chromates target cells at bronchial bifurcation sites in human lungs. However, it is uncertain what roles particle internalization and dissolution play in the genotoxicity of these compounds to human lung cells. We investigated these mechanisms in a human lung cell line after exposure to particulate lead chromate. We found that lead chromate was clastogenic in a concentration-dependent manner with 0.1, 0.5, and 1 microg/cm(2), while 5 and 10 microg/cm(2) caused complete cell cycle arrest. We also found concentration-dependent increases in intracellular and extracellular chromium ion levels. We investigated particle internalization by using transmission electron microscopy and found an apparent relative increase with concentration but no apparent particle internalization at the lowest concentration (0.1 microg/cm(2)) even after 24 h. Furthermore, we found no lysosomal association with the vacuoles containing particles, further suggesting that intracellular dissolution did not occur. Cotreating the cells with lead chromate and vitamin C eliminated both the uptake of ionic chromium and the clastogenic activity of lead chromate but had no effect on particle internalization. These data indicate that in human bronchial cells lead chromate clastogenesis is mediated by the extracellular dissolution of the particles and not their internalization. These findings have important implications for our understanding of the physicochemical mechanism of particulate chromates as they contradict previous indirect data from human bronchial epithelial cells, which suggest that particles dissolve inside those cells. Thus, these new data suggest that there may be different mechanisms of genotoxicity for epithelial cells and fibroblasts exposed to chromate particles.

Human Nucleotide Excision Repair Efficiently Removes Chromium-DNA Phosphate Adducts and Protects Cells against Chromate Toxicity

Intracellular reduction of carcinogenic Cr(VI) leads to the extensive formation of Cr(III)-DNA phosphate adducts. Repair mechanisms for chromium and other DNA phosphate-based adducts are currently unknown in human cells. We found that nucleotide excision repair (NER)-proficient human cells rapidly removed chromium-DNA adducts, with an average t((1/2)) of 7.1 h, whereas NER-deficient XP-A, XP-C, and XP-F cells were severely compromised in their ability to repair chromium-DNA lesions. Activation of NER in Cr(VI)-treated human fibroblasts or lung epithelial H460 cells was manifested by XPC-dependent binding of the XPA protein to the nuclear matrix, which was also observed in UV light-treated (but not oxidant-stressed) cells. Intracellular replication of chromium-modified plasmids demonstrated increased mutagenicity of binary Cr(III)-DNA and ternary cysteine-Cr(III)-DNA adducts in cells with inactive NER. NER deficiency created by the loss of XPA in fibroblasts or by knockdown of this protein by stable expression of small interfering RNA in H460 cells increased apoptosis and clonogenic death by Cr(VI), providing genetic evidence for the role of monofunctional chromium-DNA adducts in the toxic effects of this metal. The rate of NER of chromium-DNA adducts under saturating conditions was calculated to be approximately 50,000 lesions/min/cell. Because chromium-DNA adducts cause only small changes in the DNA helix, rapid repair of these modifications in human cells indicates that the presence of major structural distortions in DNA is not required for the efficient detection of the damaged sites by NER proteins in vivo.

Chromium is the proximate clastogenic species for lead chromate-induced clastogenicity in human bronchial cells

Hexavalent chromium (Cr(VI)) is a well-established human lung carcinogen with potentially widespread exposure. Solubility is a key factor in the carcinogenicity of Cr(VI), with the water-insoluble or ‘particulate’ compounds being the more potent carcinogens. Studies have indicated that the component ions are responsible for their clastogenicity, but it is uncertain whether chromium (Cr), lead (Pb) or some combination of the two is responsible for the clastogenic effects. Accordingly, we compared the clastogenicity of lead chromate (LC) with soluble sodium chromate (SC) and lead glutamate (LG) in WTHBF-6 human lung cells. We found that 1436 μM was the maximal intracellular level of Pb after exposure to clastogenic concentrations of LC. However, clastogenesis was not observed after exposure to LG, even when intracellular Pb concentrations reached 13,347 μM, indicating that intracellular Pb levels did not reach clastogenic levels in WTHBF-6 cells after LC treatment. By contrast, SC was clastogenic damaging 16 and 44% of metaphase cells at intracellular Cr doses of 312 and 1262 μM respectively, which was comparable to the clastogenesis observed after LC treatment. LC damaged 10, 27 and 37% of metaphases at intracellular Cr doses of 288, 926 and 1644 μM, respectively. These data indicate that with respect to LC-induced clastogenicity, Cr and not Pb is the proximate clastogenic species in human lung cells.