Inhibition of prolidase activity by nickel causes decreased growth of proline auxotrophic CHO cells

Occupational exposure to nickel has been epidemiologically linked to increased cancer risk in the respiratory tract. Nickel-induced cell transformation is associated with both genotoxic and epigenetic mechanisms that are poorly understood. Prolidase [E.C.3.4.13.9] is a cytosolic Mn(II)-activated metalloproteinase that specifically hydrolyzes imidodipeptides with C-terminal proline or hydroxyproline and plays an important role in the recycling of proline for protein synthesis and cell growth. Prolidase also provides free proline as substrate for proline oxidase, whose gene is activated by p53 during apoptosis. The inhibition of prolidase activity by nickel has not yet been studied. We first showed that Ni(II) chloride specifically inhibited prolidase activity in CHO-K1 cells in situ. This interpretation was possible because CHO-K1 cells are proline auxotrophs requiring added free proline or proline released from added Gly-Pro by prolidase. In a dose-dependent fashion, Ni(II) inhibited growth on Gly-Pro but did not inhibit growth on proline, thereby showing inhibition of prolidase in situ in the absence of nonspecific toxicity. Studies using cell-free extracts showed that Ni(II) inhibited prolidase activity when present during prolidase activation with Mn(II) or during incubation with Gly-Pro. In kinetic studies, we found that Ni(II) inhibition of prolidase varied with respect to Mn(II) concentration. Analysis of these data suggested that increasing concentrations of Mn(II) stabilized the enzyme protein against Ni(II) inhibition. Because prolidase is an important enzyme in collagen metabolism, inhibition of the enzyme activity by nickel could alter the metabolism of collagen and other matrix proteins, and thereby alter cell-matrix and cell-cell interactions involved in gene expression, genomic stability, cellular differentiation, and cell proliferation.     

Molecular models in nickel carcinogenesis

Nickel compounds are known human carcinogens, but the exact molecular mechanisms of nickel carcinogenesis are not known. Due to their abundance, histones are likely targets for Ni(II) ions among nuclear macromolecules. This paper reviews our recent studies of peptide and protein models of Ni(II) binding to histones. The results allowed us to propose several mechanisms of Ni(II)-inflicted damage, including nucleobase oxidation and sequence-specific histone hydrolysis. Quantitative estimations of Ni(II) speciation, based on these studies, support the likelihood of Ni(II) binding to histones in vivo, and the protective role of high levels of glutathione. These calculations indicate the importance of histidine in the intracellular Ni(II) speciation.     

Nickel Ions Inhibit Histone Demethylase JMJD1A and DNA Repair Enzyme ABH2 by Replacing the Ferrous Iron in the Catalytic Centers

Iron- and 2-oxoglutarate-dependent dioxygenases are a diverse family of non-heme iron enzymes that catalyze various important oxidations in cells. A key structural motif of these dioxygenases is a facial triad of 2-histidines-1-carboxylate that coordinates the Fe(II) at the catalytic site. Using histone demethylase JMJD1A and DNA repair enzyme ABH2 as examples, we show that this family of dioxygenases is highly sensitive to inhibition by carcinogenic nickel ions. We find that, with iron, the 50% inhibitory concentrations of nickel (IC₅₀ [Ni(II)]) are 25 μm for JMJD1A and 7.5 μm for ABH2. Without iron, JMJD1A is 10 times more sensitive to nickel inhibition with an IC₅₀ [Ni(II)] of 2.5 μm, and approximately one molecule of Ni(II) inhibits one molecule of JMJD1A, suggesting that nickel causes inhibition by replacing the iron. Furthermore, nickel-bound JMJD1A is not reactivated by excessive iron even up to a 2 mm concentration. Using x-ray absorption spectroscopy, we demonstrate that nickel binds to the same site in ABH2 as iron, and replacement of the iron by nickel does not prevent the binding of the cofactor 2-oxoglutarate. Finally, we show that nickel ions target and inhibit JMJD1A in intact cells, and disruption of the iron-binding site decreases binding of nickel ions to ABH2 in intact cells. Together, our results reveal that the members of this dioxygenase family are specific targets for nickel ions in cells. Inhibition of these dioxygenases by nickel is likely to have widespread impacts on cells (e.g. impaired epigenetic programs and DNA repair) and may eventually lead to cancer development.     

Iron- and 2-oxoglutarate-dependent Dioxygenases: an emerging group of molecular targets for nickel toxicity and carcinogenicity

Nickel compounds are important occupational and environmental pollutants. Chronic exposure to these pollutants has been connected with increased risks of respiratory cancers and cardiovascular diseases. However, it is still not clear what are the specific molecular targets for nickel toxicity and carcinogenicity. Here, we propose that the iron- and 2-oxoglutarate-dependent dioxygenase family enzymes are important intracellular targets that mediate the toxicity and carcinogenicity of nickel. In support of this hypothesis, our data show that three different classes of enzymes in this iron- and 2-oxoglutarate-dependent dioxygenase family, including HIF-prolyl hydroxylase PHD2, histone demethylase JHDM2A/JMJD1A, and DNA repair enzyme ABH3, are all highly sensitive to nickel inhibition. Inactivation of these enzymes accounts for a number of deleterious effects caused by nickel in cells, namely hypoxia-mimic stress and aberrant epigenetic changes. Future studies on nickel’s effects on these iron- and 2-oxoglutarate-dependent dioxygenases would deepen our understanding on nickel toxicity and carcinogenicity.     

Effect of soluble nickel on cellular energy metabolism in A549 cells

Iron is an essential nutrient to most organisms, and is actively involved in oxygen delivery, electron transport, DNA synthesis, and many other biochemical reactions important for cell survival. We previously reported that nickel (Ni) ion exposure decreases cellular iron level and converts cytosolic aconitase (c-aconitase) to iron-regulatory protein-1 in A549 cells (Chen H, Davidson T, Singleton S, Garrick MD, Costa M. Toxicol Appl Pharmacol 206:275-287, 2005). Here, we further investigated the effect of Ni ion exposure on the activity of mitochondrial iron-sulfur (Fe-S) enzymes and cellular energy metabolism. We found that acute Ni ion treatment up to 1 mM exhibits minimal toxicity in A549 cells. Ni ion treatment decreases the activity of several Fe-S enzymes related to cellular energy metabolism, including mitochondrial aconitase (m-aconitase), succinate dehydrogenase (SDH), and NADH:ubiquinone oxidoreductase (complex I). Low doses of Ni ion for 4 weeks resulted in an increased cellular glycolysis and NADH to NAD+ (NADH/NAD+) ratio, although glycolysis was inhibited at higher levels. Collectively, our results show that Ni ions decrease the activity of cellular iron (Fe)-containing enzymes, inhibit oxidative phosphorylation (OxPhos), and increase cellular glycolytic activity. Since increased glycolysis is one of the fundamental alterations of energy metabolism in cancer cells (the Warburg effect), the inhibition of Fe-S enzymes and subsequent changes in cellular energy metabolism caused by Ni ions may play an important role in Ni carcinogenesis.     

Metal selectivity of the Escherichia coli nickel metallochaperone, SlyD

SlyD is a Ni(II)-binding protein that contributes to nickel homeostasis in Escherichia coli. The C-terminal domain of SlyD contains a rich variety of metal-binding amino acids, suggesting broader metal binding capabilities, and previous work demonstrated that the protein can coordinate several types of first-row transition metals. However, the binding of SlyD to metals other than Ni(II) has not been previously characterized. To improve our understanding of the in vitro metal-binding activity of SlyD and how it correlates with the in vivo function of this protein, the interactions between SlyD and the series of biologically relevant transition metals [Mn(II), Fe(II), Co(II), Cu(I), and Zn(II)] were examined by using a combination of optical spectroscopy and mass spectrometry. Binding of SlyD to Mn(II) or Fe(II) ions was not detected, but the protein coordinates multiple ions of Co(II), Zn(II), and Cu(I) with appreciable affinity (K(D) values in or below the nanomolar range), highlighting the promiscuous nature of this protein. The order of affinities of SlyD for the metals examined is as follows: Mn(II) and Fe(II) < Co(II) < Ni(II) ~ Zn(II) ? Cu(I). Although the purified protein is unable to overcome the large thermodynamic preference for Cu(I) and exclude Zn(II) chelation in the presence of Ni(II), in vivo studies reveal a Ni(II)-specific function for the protein. Furthermore, these latter experiments support a specific role for SlyD as a [NiFe]-hydrogenase enzyme maturation factor. The implications of the divergence between the metal selectivity of SlyD in vitro and the specific activity in vivo are discussed.     

Nickel(II) acetate-treated Chinese hamster ovary cells differentially express Vimentin, hSNF2H homologue, and H ferritin.

In probing the possible non-genotoxic molecular mechanism(s) of nickel(II)-induced carcinogenesis, we performed a non-radioactive mRNA differential display analysis for nickel(II) acetate-treated Chinese hamster ovary cells (CHO-K1-BH4). Three out of thirty differentially expressed cDNAs had sequences highly similar to known genes. Down-regulation of vimentin and a hSNF2H homologue and up-regulation of ferritin heavy chain were confirmed by Northern blot analysis. The expression of these mRNAs was time- and nickel(II) concentration-dependent. For vimentin, the decrease in mRNA level was concurrent with a decrease in the protein level. For ferritin, the increase in mRNA had no effect on the protein level. Dysregulation of these gene products signifies their involvement in the epigenetic effects of carcinogenic nickel(II) compounds.     

Bioaccumulation of metals from nickel smelter waste in P and F1 generations of exposed animals. I. Dynamics of metal distribution in the organs and AM activity

A group of female Chinchilla rabbits was exposed respiratory to metal aerosol originating from dumped wastes of a nickel smelter. The experiment was carried out in a biomonitoring station situated 4 km away from the dump in the direction of prevailing winds. Metals (Fe, Cr, Ni) introduced by inhaled waste particles were found in the increased amount in the body organs of exposed (P and F1) animals. Following a 3-months exposure of female rabbits (P), the elevated level of Cr in liver and kidneys was found, and after 6-month exposure also in the heart and thigh muscles. The Ni content proved to be increased in the lungs, heart and thigh muscles following 3-month period and in the liver after the 6-month exposure. Concerning the Fe content, it was already elevated in all the organs investigated except the lungs after 3-month exposure. The alveolar macrophage (AM) count was increased significantly in the exposed female rabbits (P) only after 6 months (P less than 0.05) and rose continually till the end of 9-month exposure (P less than 0.01). The enzyme activity of lysosomes was enhanced after 9-month exposure. In F1 rabbits 6 weeks of age elevated levels of chromium were found in the liver, kidneys, heart and thigh muscles. The Fe content was increased in the liver and thigh muscle, while the Ni content was enhanced only in the brain. The increased mucus secretion was observed on the tracheal mucous relief of F1 rabbits in response to the noxious effect of needle-shaped metal particles using an electron scanning microscope, despite the short duration (6 weeks) of postnatal exposure. The reported findings emphasize the effects of metal particles originating from the waste of a nickel smelter in an environment where the mean value of the dust fallout was only 4 g.m-2.30 days-1.     

Ni-induced Changes in Nitrate Assimilation and Antioxidant Metabolism of Verbascum olympicum Boiss.: Could the Plant be Useful for Phytoremediation or/and Restoration Purposes?

Verbascum olympicum Boiss. (Scrophulariaceae) were studied as a candidate plant for remediating the Ni polluted soils. The metabolic responses, such as nitrate assimilation (nitrate reductase and glutamine synthetase activity) and antioxidant system activity [superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) activity], of this species exposed to nickel in Hoagland's nutrient medium were investigated as remediation performance parameters. The accumulation of nickel and the variations in the content of some elements (B, Cu, Fe, Mg, Mn, Mo and Zn) and some growth parameters, such as the water content, biomass production, and contents of chlorophyll and soluble protein, were also examined. The accumulation of Ni in both the roots and leaves varied depending on the exposure times and doses. Increased oxidative stress was suggested by the increases in the activities of SOD, CAT and APX. Although some element contents were inhibited by Ni treatments, these inhibitory effects was decreased depending on the time, and even these elements are accumulated in roots. These results are the novelties in the use of this species in biotechnology.     

Complexation of the fungal metabolite tenuazonic acid with copper (II), iron (III), nickel (II), and magnesium (II) ions

Tenuazonic acid (TA) is a phytotoxin produced by a fungal pathogen of rice, Pyricularia oryzae. We have synthesized and characterized the metal complexes of TA with copper (II), iron (III), nickel (II), and magnesium (II). The stoichiometry of the complexes determined by microanalysis and mass spectroscopy (D/CI) are Cu(II)TA2, Fe(III)TA3, Ni(II)TA2, and Mg(TA)2. Voltammograms of Fe(III)TA3, and Cu(II)TA2 in methanolic solutions confirmed this stoichiometry. Ni(II)TA2 paramagnetism and visible absorption data suggest an octahedral geometry. Fe(III)TA3 showed a characteristic visible absorption at 450 nm. Addition of Fe(III)Cl3 and Mg(II)Cl2 did not reverse the toxicity of NaTA to rice and bacterial cells, showing that this toxicity is not due to the privation of the cells of these metals essential for cell growth.     

Salivary histatin-5, a physiologically relevant ligand for Ni(II) ions

Histatins are a family of human salivary antimicrobial peptides. Histatin-5 (Hst-5, DSHAKRHHGYKRKFHEKHHSHRGY), a prominent member of this family contains an albumin-like, N-terminal Asp-Ser-His sequence, known to bind a Ni(II) ion in a square-planar geometry. Nickel is a strong allergen, and oral exposure to Ni(II) ions can elicit allergic reaction in sensitized persons. In contrast, prior oral exposure to nickel in non-sensitized persons can prevent sensitization. The fate of Ni(II) ions in saliva is obviously important for these processes, yet little is known about it. Using potentiometry, UV-visible titrations and circular dichroism, we determined stability constants for Ni(II) complexes of Hst-5 and two truncated analogs, 5Hst-5 (DSHAK) and 10Hst-5 (DSHAKRHHGY). The conditional binding constant at pH 7.4 for Hst-5 was 10(7.5±0.2), compared to the corresponding value for albumin, 10(6.8±0.3) (M. Soko?owska, A. Kr??el, M. Dyba, Z. Szewczuk, W. Bal, Eur. J. Biochem. 269 (2002) 1323-1331). These values indicate that Hst-5 binds Ni(II) five times stronger than HSA. The simulated competition for Ni(II) between Hst-5 and albumin shows that significant amounts of Ni(II) ions may be carried by Hst-5 in vivo. Therefore, Hst-5 and other histatins should be considered as factors in nickel allergy and other forms of nickel toxicity.     

Effect of metal ions on HIF-1alpha and Fe homeostasis in human A549 cells

Several metals are carcinogenic but little is known about the mechanisms by which they cause cancer. A pathway that may contribute to metal ion induced carcinogenesis is by hypoxia signaling, which involves a disruption of cellular iron homeostasis by competition with iron transporters or iron-regulated enzymes. To examine the involvement of iron in the hypoxia signaling activity of these metal ions we investigated HIF-1alpha protein stabilization, IRP-1 activity, and ferritin protein levels in human lung carcinoma A459 cells exposed to various agents in serum- and iron-free salt-glucose medium (SGM) or in normal complete medium. We also studied the effects of excess exogenous iron on these responses induced by nickel ion exposure. Our results show the following: (1) SGM enhanced metals-induced HIF-1alpha stabilization and IRP-1 activation (e.g., nickel and cobalt ions). (2) If SGM was reconstituted with a slight excess level (25 microM of FeSO(4)) of iron, this enhancing ability was significantly decreased. (3) The effect of a high level of exogenous iron (500 microM of FeSO(4)) on metal-induced hypoxia and iron metabolism was highly dependent on the order of addition. If treatment with the Fe and metal ions was simultaneous (co-treatment), the effects of nickel ion exposure were overwhelmed, since the added Fe reversed HIF-1alpha stabilization, decreased IRP-1 activity, and increased ferritin level. Pre-treatment with iron was not able to reverse the responses caused by nickel ion exposure. These results imply that it is important to consider the available iron concentration and suitable exposure design when studying metal-induced hypoxia or metal-induced disruption of Fe homeostasis.     

Environmental epigenetics in metal exposure

Although it is widely accepted that chronic exposure to arsenite, nickel, chromium and cadmium increases cancer incidence in individuals, the molecular mechanisms underlying their ability to transform cells remain largely unknown. Carcinogenic metals are typically weak mutagens, suggesting that genetic-based mechanisms may not be primarily responsible for metal-induced carcinogenesis. Growing evidence shows that environmental metal exposure involves changes in epigenetic marks, which may lead to a possible link between heritable changes in gene expression and disease susceptibility and development. Here, we review recent advances in the understanding of metal exposure affecting epigenetic marks and discuss establishment of heritable gene expression in metal-induced carcinogenesis.Key words: environmental metal, epigenetic, metal carcinogenesis, histone modification, DNA methylation, chromatin, gene expression     

Nickel(II) induces microsatellite mutations in human lung cancer cell lines

Nickel(II) is a human carcinogen causing respiratory cancers. The purpose of this study was to determine whether Ni(II) may induce microsatellite mutations in human cells. We transfected the three human lung tumor cell lines A427, HCC15 and NCI-H2009 with a mammalian expression vector containing a (CA)(13) repeat in the coding sequences of the reporter hygromycin gene (hyg). A total of 33 clones carrying the integrated vector derived from the three cell lines was investigated for spontaneous and Ni(II)-induced hygromycin-resistant (hyg(r)) reversion mutants. Significantly higher frequencies of hyg(r) reversion mutations were observed in Ni(II)-treated cells (NCI-H2009 and HCC-15) than control cells. In the majority of the colonies hyg(r) phenotype was due to mutations within the integrated (CA) repeat sequence. The type of mutations consisted of both contraction and expansion of the (CA) repeat unit. The finding that Ni(II) promotes microsatellite mutations raises the possibility that genetic instability may be a mechanism involved in nickel carcinogenesis.     

Environmental epigenetics in metal exposure

Although it is widely accepted that chronic exposure to arsenite, nickel, chromium and cadmium increases cancer incidence in individuals, the molecular mechanisms underlying their ability to transform cells remain largely unknown. Carcinogenic metals are typically weak mutagens, suggesting that genetic-based mechanisms may not be primarily responsible for metal-induced carcinogenesis. Growing evidence shows that environmental metal exposure involves changes in epigenetic marks, which may lead to a possible link between heritable changes in gene expression and disease susceptibility and development. Here, we review recent advances in the understanding of metal exposure affecting epigenetic marks and discuss establishment of heritable gene expression in metal-induced carcinogenesis.     

Interaction of nickel with mutagens in the induction of sister chromatid exchanges in human lymphocytes

In this study, individual treatments of human lymphocytes with Ni(II) [0.5–25 μM], Cr(VI) [0.65–1.30 μM], UV-light or X-rays induced SCEs in a dose-dependent fashion, and combined treatments of Ni(II) with Cr(VI), UV-light or X-rays interacted antagonistically. Nickel, at environmentaly relevant exposure levels, cna have the effect in complex mixtures of reducing an otherwise positive SCE response and could lead to underestimating human exposures to certain classes of chemicals or radiation. Furthermore, our data indicate that antagonism may occure when human lymphocytes are exposed simultaneously to Ni(II) and Cr(VI), suggesting an explanation for epidemiological studies reporting conflicting results for cytogenetic effects in lymphocytes of workers exposed to chromium and nickel.     

Role of transition metals present in air particulate matter on lung oxygen metabolism

Several epidemiological studies have shown a positive correlation between daily increases in airborne particulate matter (PM) concentration and the occurrence of respiratory and cardiovascular diseases. Transition metals present in air PM were associated with adverse health effects after PM exposure. The aim of this work was to study lung O₂ metabolism after an acute exposure to transition metal-coated nanoparticles (NPs). Female Swiss mice (25 g) were intranasally instilled with a suspension of silica NP containing Ni (II), Cd (II), Fe (III), or Cr (VI) at 0, 0.01, 0.05, 0.1, and 1.0 mg metal/kg body weight. Lung O₂ consumption was found to be significantly increased after the exposure to most doses of Ni-NP and Fe-NP, and the 0.05 mg metal/kg body weight dose of Cr-NP, while no changes were observed for Cd-NP. Lucigenin chemiluminescence (as an indicator of NADPH oxidase (NOX) activity) was evaluated in lung homogenates. Only Ni-NP and Fe-NP have shown the ability to induce a significant increase in lucigenin chemiluminescence. In order to establish the possible occurrence of pulmonary oxidative stress, TBARS levels and the GSH/GSSG ratio were determined. The higher doses of Ni-NP and Fe-NP were able to induce an oxidative stress condition, as shown by changes in both TBARS levels and the GSH/GSSG ratio. Taken together, the present results show differential effects for all the metals tested. These findings emphasize the importance of transition metals present air PM in PM adverse health effects, and contribute to the understanding of the pathological mechanisms triggered by the exposure to environmental PM.     

Kinetic and spectroscopic investigation of CoII, NiII, and N-oxalylglycine inhibition of the FeII/alpha-ketoglutarate dioxygenase, TauD

Co(II), Ni(II), and N-oxalylglycine (NOG) are well-known inhibitors of Fe(II)/alpha-ketoglutarate (alphaKG)-dependent hydroxylases, but few studies describe their kinetics and no spectroscopic investigations have been reported. Using taurine/alphaKG dioxygenase (TauD) as a paradigm for this enzyme family, time-dependent inhibition assays showed that Co(II) and Ni(II) follow slow-binding inhibition kinetics. Whereas Ni(II)-substituted TauD was non-chromophoric, spectroscopic studies of the Co(II)-substituted enzyme revealed a six-coordinate site (protein alone or with alphaKG) that became five-coordinate upon taurine addition. The Co(II) spectrum was not perturbed by a series of anions or oxidants, suggesting the Co(II) is inaccessible and could be used to stabilize the protein. NOG competed weakly (Ki approximately 290 microM) with alphaKG for binding to TauD, with the increased electron density of NOG yielding electronic transitions for NOG-Fe(II)-TauD and taurine-NOG-Fe(II)-TauD at 380 nm (epsilon380 90-105 M(-1) cm(-1)). The spectra of the NOG-bound TauD species did not change significantly upon oxygen exposure, arguing against the formation of an oxygen-bound state mimicking an early intermediate in catalysis.     

Effect of metal ions on HIF-1α and Fe homeostasis in human A549 cells

Several metals are carcinogenic but little is known about the mechanisms by which they cause cancer. A pathway that may contribute to metal ion induced carcinogenesis is by hypoxia signaling, which involves a disruption of cellular iron homeostasis by competition with iron transporters or iron-regulated enzymes. To examine the involvement of iron in the hypoxia signaling activity of these metal ions we investigated HIF-1α protein stabilization, IRP-1 activity, and ferritin protein levels in human lung carcinoma A459 cells exposed to various agents in serum- and iron-free salt–glucose medium (SGM) or in normal complete medium. We also studied the effects of excess exogenous iron on these responses induced by nickel ion exposure. Our results show the following: (1) SGM enhanced metals-induced HIF-1α stabilization and IRP-1 activation (e.g., nickel and cobalt ions). (2) If SGM was reconstituted with a slight excess level (25 μM of FeSO₄) of iron, this enhancing ability was significantly decreased. (3) The effect of a high level of exogenous iron (500 μM of FeSO₄) on metal-induced hypoxia and iron metabolism was highly dependent on the order of addition. If treatment with the Fe and metal ions was simultaneous (co-treatment), the effects of nickel ion exposure were overwhelmed, since the added Fe reversed HIF-1α stabilization, decreased IRP-1 activity, and increased ferritin level. Pre-treatment with iron was not able to reverse the responses caused by nickel ion exposure. These results imply that it is important to consider the available iron concentration and suitable exposure design when studying metal-induced hypoxia or metal-induced disruption of Fe homeostasis.