Kazimierz S. Kasprzak

Oxidative DNA and protein damage in metal-induced toxicity and carcinogenesis.

This review discusses the relevance of oxidative damage to metal-induced toxicity and carcinogenesis. Presented are important facts and mechanistic concepts on the capacity of selected transition metals, mainly Ni, but also Cu, Co, Cr, and briefly several others, to generate active oxygen species and other reactive intermediates under physiological conditions. These metals are known to be toxic and/or carcinogenic contaminants of the occupational and general environments. Their redox activity may underlay the mechanism of mediation of oxidative damage to cell constituents. The presentation is focused on selected issues relative to genetic and epigenetic toxicity and illustrated with examples of metal-mediated oxidative damage to the principal components of chromatin, i.e., DNA, histones, and protamines.This review discusses the relevance of oxidative damage to metal-induced toxicity and carcinogenesis. Presented are important facts and mechanistic concepts on the capacity of selected transition metals, mainly Ni, but also Cu, Co, Cr, and briefly several others, to generate active oxygen species and other reactive intermediates under physiological conditions. These metals are known to be toxic and/or carcinogenic contaminants of the occupational and general environments. Their redox activity may underlay the mechanism of mediation of oxidative damage to cell constituents. The presentation is focused on selected issues relative to genetic and epigenetic toxicity and illustrated with examples of metal-mediated oxidative damage to the principal components of chromatin, i.e., DNA, histones, and protamines.

Induction of oxidative DNA damage by carcinogenic metals.

The metal ions carcinogenic to humans are As, Be, Cd, Cr and Ni, and the candidates also include Co, Cu, Fe and Pt. A range of molecular mechanisms was proposed for these metals, reflecting their diverse chemical properties. The oxidative concept in metal carcinogenesis proposes that some complexes of the above metals (Co, Cr, Cu, Fe, Ni) formed in vivo undergo redox cycling, yielding reactive oxygen species and/or high valence metal ions which oxidize DNA. Some of the products of oxidative DNA damage, including 8-oxoguanine and strand breaks, induce mutations, which may lead to neoplastic transformation. The establishment of metal-binding modes in the cell nucleus and of their reactivity is crucial for the understanding of molecular events in metal carcinogenesis. We have proposed the binding sites for Ni(II) and Cu(II) in core histones (H3, H2A) and sperm protamines (HP2) and, using molecular models, provided evidence for the generation of promutagenic oxidative DNA damage by the bound metals.

Nickel induced lipid peroxidation in the rat : correlation with nickel effect on antioxidant defense systems

Lipid peroxidation (LPO) and alterations in cellular systems protecting against oxidative damage were determined in the liver, kidney and skeletal muscle of male F344/NCr rats, 1 h to 3 days after a single intraperitoneal (i.p.) injection of 107 mumol nickel(II)acetate per kg body weight. At 3 h, when tissue nickel concentrations were highest, the following significant (at least, P less than 0.05) effects were observed: in kidney, increased LPO (by 43%), increased renal iron (by 24%), decreased catalase (CAT) and glutathione peroxidase (GSH-Px) activities (both by 15%), decreased glutathione (GSH) concentration (by 20%), decreased glutathione reductase (GSSG-R) activity (by 10%), and increased glutathione-S-transferase (GST) activity (by 44%); the activity of superoxide dismutase (SOD) and gamma-glutamyl transferase (GGT), as well as copper concentration, were not affected. In the liver, nickel effects included increased LPO (by 30%), decreased CAT and GSH-Px activities (both by 15%), decreased GSH level (by 33%), decreased GSSG-R activity (by 10%) and decreased GST activity (by 35%); SOD, GGT, copper, and iron remained unchanged. In muscle, nickel treatment decreased copper content (by 43%) and the SOD activity (by 30%) with no effects on other parameters. In blood, nickel had no effect on CAT and GSH-Px, but increased the activities of alanine-(ALT) and aspartate-(AST) transaminases to 330% and 240% of the background level, respectively. In conclusion, nickel treatment caused profound cell damage as indicated by increased LPO in liver and kidney and leakage of intracellular enzymes, ALT and AST to the blood. The time pattern of the resulting renal and hepatic LPO indicated a possible contribution to its magnitude from an increased concentration of nickel and concurrent inhibition of CAT, GSH-Px and GSSG-R, but not from increased iron or copper levels. The oxidative damage expressed as LPO was highest in the kidney and lowest in the muscle, which concurs with the corresponding ranking of nickel uptake by these tissues.

Inhibition of core histones acetylation by carcinogenic nickel(II)

Nickel, a well-established human carcinogen, was shown to decrease acetylation of histones H4 and H3 in cultured cells. Such a decrease is expected to suppress gene expression. However, nickel is known to not only suppress but also enhance the expression of many genes. So, perhaps, nickel can alter histone acetylation in a more complex way? In a first step of testing this presumption, we examined acetylation status of histones H2A, H2B, H3 and H4, in human (HAE) and rat (NRK) cells exposed to nickel(II) under various conditions. In both cell lines, acetylation of all four histones was down-regulated by nickel(II) in a concentration- and time-dependent manner. Acetylation of histone H2B was suppressed to greater extent than that of the others, with histone H3 being relatively least affected. The analysis of acetylation status of each of the four lysine sites at the N-terminal tail of histone H2B revealed decreases consistent with those observed in the total acetylation patterns, with the K12 and K20 residues being markedly more affected than K5 and K15 residues. Thus, the decrease in acetylation was to some degree site specific. In NRK cells, the observed uniform down-regulation of histone acetylation was consistent with a marked suppression of global gene transcription measured as [3H]-uridine incorporation into mRNA. However, in HAE cells, global RNA expression was transiently increased (in 24 h) before dropping below control after longer exposure (3 days). In conclusion, the effects of Ni(II) on histone acetylation are inhibitory, with their extent depending on the dose and exposure time. This uniform inhibition, however, is not consistently reflected in global RNA expression that in HAE cells may include both increase and decrease of the expression, clearly indicating the involvement of factors other than histone acetylation. The observed effects may contribute to neoplastic transformation of Ni(II)-exposed cells.

Low level, long-term inorganic arsenite exposure causes generalized resistance to apoptosis in cultured human keratinocytes: Potential role in skin co-carcinogenesis†

Inorganic arsenic is a human carcinogen that targets the skin. Carcinogenesis is a multistep process in which acquired apoptotic resistance is a common event and prior work in non‐skin cells shows acquired resistance to apoptosis occurs with chronic arsenite exposure. In the present study, when HaCaT cells, an immortalized, non‐tumorigenic human keratinocyte cell line, were continuously exposed to low‐level inorganic arsenite (as sodium arsenite; 100 nM) for 28 weeks, the cells acquired a generalized resistance to apoptosis. This included resistance to apoptosis induced by acute high concentrations of arsenite, ultraviolet A (UVA) irradiation, and several chemotherapeutic compounds (cisplatin, etoposide and doxorubicin). These arsenite‐tolerant (As‐TL) cells showed similar levels of UVA‐induced reactive oxygen species (ROS) and oxidative DNA damage when compared to passage match control cells. Because cellular apoptosis is dependent on the balance between proapoptotic and survival pathways, the roles of protein kinase B (PKB), a key antiapoptotic molecule, in this acquired apoptotic resistance were investigated. Stimulation of apoptosis markedly decreased nuclear phosphorylated PKB (P‐PKB) levels in control cells, but As‐TL cells showed greatly increased stability of nuclear P‐PKB. Pretreatment of the As‐TL cells with LY294002 or Wortmannin, which specifically inhibit PKB phosphorylation, completely blocked apoptotic resistance in As‐TL cells, indicating acquired apoptotic resistance is associated with increased stability of nuclear P‐PKB. Because arsenic and UV irradiation are co‐carcinogenic in mouse skin, resistance to UV‐induced apoptosis in As‐TL cells may allow UV‐damaged cells to escape normal cell population controls and initiate the carcinogenic cascade. The observation that As‐TL cells show no lessening of UV‐induced genotoxicity supports this possibility.

Antagonism by essential divalent metals and amino acids of nickel(II)-DNA binding in vitro.

In vitro binding of nickel(II) to DNA and the effects of divalent essential metals calcium, magnesium, manganese, copper, and zinc, and of amino acids histidine, cysteine, glutamine, arginine, lysine, alanine, and glycine upon that binding were investigated. Samples of 0.156 mg of calf thymus DNA (0.078 mg/ml in 5 mM ammonium acetate buffer, pH = 7.4) were incubated for 1 hr at 24 degrees C with various concentrations of nickel(II)acetate labeled with 63Ni (0.1 to 250 microM) in the absence or presence of 50 microM concentrations of the essential metal acetates, or with 100 microM concentrations of the amino acids. Free and DNA-bound nickel(II) fractions were separated by gel filtration on Sephadex G-25 and quantified by liquid scintillation counting. Scatchard analysis revealed more than two types of nickel(II)-binding sites and a positive cooperativity of binding at the bound-Ni concentrations below 0.35 microM. The high-affinity nickel(II)-binding sites at DNA were identified as phosphate groups. Their binding capacity equalled 0.043 mumol/mg DNA (approx. 1 mol Ni/70 mol of DNA bases). The apparent dissociation constant of nickel(II) from the high-affinity sites was 5.35 microM. Double reciprocal plots showed the essential divalent metals to be competitive antagonists of nickel(II)-binding to the high-affinity sites, ranking Mg(II) greater than or equal to Mn(II) greater than or equal to Ca(II) greater than or equal to Cu(II) = Zn(II). Similarly, the amino acids antagonized nickel binding to DNA with a relative strength of His greater than Gln greater than or equal to His/Cys greater than Arg greater than Cys greater than or equal to Gly = Ala greater than or equal to Lys. The strongest inhibitors of nickel(II)-DNA binding in vitro appear to be magnesium and manganese, i.e., the same metals that are capable of attenuating nickel carcinogenicity in vivo.

Ascorbate Depletion: A Critical Step in Nickel Carcinogenesis?

Nickel compounds are known to cause respiratory cancer in humans and induce tumors in experimental animals. The underlying molecular mechanisms may involve genotoxic effects; however, the data from different research groups are not easy to reconcile. Here, we challenge the common premise that direct genotoxic effects are central to nickel carcinogenesis and probably to that of other metals. Instead, we propose that it is formation of metal complexes with proteins and other molecules that changes cellular homeostasis and provides conditions for selection of cells with transformed phenotype. This is concordant with the major requirement for nickel carcinogenicity, which is prolonged action on the target tissue. If DNA is not the main nickel target, is there another unique molecule that can be attacked with carcinogenic consequences? Our recent observations indicate that ascorbate may be such a molecule. Nickel depletes intracellular ascorbate, which leads to the inhibition of cellular hydroxylases, manifested by the loss of hypoxia-inducible factor (HIF)-1α and - 2α hydroxylation and hypoxia-like stress. Proline hydroxylation is crucial for collagen and extracellular matrix assembly as well as for assembly of other protein molecules that have collagen-like domains, including surfactants and complement. Thus, the depletion of ascorbate by chronic exposure to nickel could be deleterious for lung cells and may lead to lung cancer.

Ascorbate depletion mediates up‐regulation of hypoxia‐associated proteins by cell density and nickel

Exposure of human lung cells to carcinogenic nickel compounds in the presence of oxygen up‐regulated carbonic anhydrase IX (CA IX) and NDRG1/Cap43, both known as intrinsic hypoxia markers and cancer‐associated genes. This suggests that factors other than a shortage of oxygen may be involved in this induction. Both proteins can also be induced in the presence of oxygen by culturing these cells to a high density without medium change. The intracellular ascorbate measurements revealed its rapid depletion in both metal‐ and density‐exposed cells. Nickel exposure caused strong activation of HIF‐1α and HIF‐2α proteins, underscoring activation of HIF‐1‐dependent transcription. In contrast, cell density‐dependent transcription was characterized by minor induction of HIF‐1α or HIF‐2α. Moreover, the up‐regulation of NDRG1/Cap43 in HIF‐1α deficient fibroblasts suggested the involvement of different transcription factor(s). The repletion of intracellular ascorbate reversed the induction of CA IX and NDRG1/Cap43 caused by cell density or nickel exposure. Thus, the loss of intracellular ascorbate triggered the induction of both tumor markers by two different conditions in the presence of oxygen. Ascorbate is delivered to lung cells via the SVCT2 ascorbate transporter, which was found to be sensitive to nickel or cell density. Collectively these findings establish the importance of intracellular ascorbate levels for the regulation of expression of CA IX and NDRG1/Cap43. We suggest, that, in addition to low oxygenation, insufficient supply of ascorbate or its excessive oxidation in tumors, can contribute to the induction of hypoxia‐associated proteins via both HIF‐dependent and independent mechanisms. J. Cell. Biochem. 97: 1025–1035, 2006.

Gene expression dose-response changes in microarrays after exposure of human peripheral lung epithelial cells to nickel(II)

Occupational exposure to nickel compounds is associated with lung cancer risk; both genotoxic and epigenetic mechanisms have been proposed. For comprehensive examination of the acute effects of nickel(II) acetate on gene expression in cultured human peripheral lung epithelial HPL1D cells, microarray analyses were carried out with cDNA chips (approximately 8000 cDNAs). Cells were exposed for 24 h to nontoxic (50, 100, and 200 microM) or toxic (400, 800, and 1600 microM) nickel(II) concentrations. Cluster analysis was applied to the 868 genes with > or = 2-fold change at any concentration. Two main clusters showed marked up- or down-regulation at the highest, toxic concentrations. The data further subdivided into 10 highly cohesive clusters with high probability, and of these only 2 had the same response trend at low nontoxic as at high concentrations, an observation of clear relevance to the process of high- to low-dose extrapolation in risk assessment. There were 113 genes showing > or = 2-fold change at the three lower nontoxic concentrations, those most relevant to in vivo carcinogenesis. In addition to expected responses of metallothionein, ferritin, and heat-shock proteins, the results revealed for the first time changed expression of some potential cancer-related genes in response to low-dose Ni(II): RhoA, dyskerin, interferon regulatory factor 1, RAD21 homologue, and tumor protein, translationally controlled. Overall, most of the genes impacted by nontoxic concentrations of nickel(II) acetate related to gene transcription, protein synthesis and stability, cytoskeleton, signaling, metabolism, cell membrane, and extracellular matrix.

Nickel-induced lipid peroxidation in the liver of different strains of mice and its relation to nickel effects on antioxidant systems

After a single intraperitoneal injection of 170 mumol nickel(II)acetate/kg body wt., the activity of hepatic catalase (CAT) decreased by 25-56% in a strain- and time-dependent manner, the most susceptible being C57BL/6NCr greater than C3H/HeNCr-MTV- greater than B6C3F1 greater than or equal to BALB/cAnNCr mice. The glutathione (GSH) levels in all 4 strains were inhibited by nickel with the C57BL/6NCr mice showing the biggest decrease (68%) followed by BALB/cAnNCr (46%) greater than or equal to B6C3F1 (42%) greater than C3H/HeNCr-MTV- (22%). The response of hepatic glutathione peroxidase (GSH-Px) to nickel was variable and included 30% enhancement in C3H/HeNCr-MTV- or lack of biologically significant effect (max. +/- 10% variations in time) in the remaining strains. The activity of glutathione reductase (GSSG-R) increased gradually by up to 30% (48 h post-injection) in B6C3F1 and C3H/HeNCr-MTV- mice or, transiently, by 15-18% (3 h), in C57BL/6NCr and BALB/cAnNCr mice. Also, in some strains, nickel significantly affected superoxide dismutase (SOD) (14-19% loss in C57BL/6NCr and B6C3F1 mice, respectively), and GSH-S-transferase (GST) (26% loss in C3H/HeNCr-MTV- mice). Lipid peroxidation (LPO) in the liver reached its highest value 24 h after nickel treatment in C57BL/6NCr (549% over the control) greater than or equal to BALB/cAnNCr (519%) greater than B6C3F1 (426%) much greater than C3H/HeNCr-MTV- (39%). In conclusion, the magnitude of nickel-induced LPO shows a reverse correlation with the extent and direction of nickel effect on GSH, GSH-Px and GSSG-R, but not on CAT, SOD or GST.