In vitro assessment of the toxicity of metal compounds

A review of the activity of metal compounds in mammalian cell transformation assays has been completed. Results from these assays appear to correlate well with the known carcinogenic activity displayed by specific metal compounds in vivo. Studies of cell transformation in vitro may provide information pertaining to the mechanism of the induction of carcinogenesis by certain metals.     

Mechanisms of metal carcinogenesis

Experimental observations that pertain to mechanisms of metal carcinogenesis are summarized, with emphasis upon (a) interactions of metals with nucleic acids in vitro; (b) impairment by metals of the fidelity of DNA replication by DNA polymerase in vitro; (c) mutagenicity of metals in microorganisms; (d) cytogenetic aberrations induced by metals in tissue culture cells; (e) induction by metals of neoplastic transformation of tissue culture cells; and (f) nuclear uptake of metals in vivo and concomitant inhibitory effects of metals on synthesis of nucleic acids. Considered in toto, the experimental data support the somatic mutation hypothesis of chemical carcinogenesis. Sufficient experimental evidence is available regarding four carcinogenic metals (As, Be, Cr, and Ni) to permit speculations about the molecular reactions whereby these metals may induce somatic mutations. This article is an updated outgrowth of a review presented at the A. O. Beckman Conference on the Biochemistry of Cancer that was held in San Antonio, Texas, on September 6–8, 1978. The earlier draft of this article is being published in the proceedings volume.     

Occurrence, use and potential toxic effects of metals and metal compounds

Metals and metal compounds are constituents of our natural environment. Their distribution depends on the existence of natural sources (e.g. volcanoes or erosion) and their use in human’s activity. They are transformed naturally (e.g. by bacterial activity) with formation of organic species that influence their mobility and accumulation in abiotic as well as biotic systems. Up to date metal species are released into the environment questioning their influence on human health. Due to their widespread use in human activities such as industry, agriculture and even as medicine (e.g. As, Se, Pt), numerous health risks may be associated with exposure to these substances. Different reports on metal intoxication are documented and studies especially on neurotoxicity, genotoxicity, or carcinogenicity, are previously published in numerous articles. This mini-review gives an overview on the use and the actions of selected metal species of actual scientific concern, with a focus on neuronal cells.     

Effect of zinc on cadmium influx and toxicity in cultured CHO cells

Addition of zinc lowers the toxicity level of cadmium in cultured CHO cells. Cell survival and protein synthesis were used to measure the cellular toxicity of cadmium.¹⁰⁹Cd was used to measure cadmium uptake by the cells. The results suggest that these class IIB transition metals, zinc and cadmium, share a common transport mechanism. Thus, the antagonism appears to involve a reduction in the influx of cadmium due to the presence of zinc.     

Role in hemolysis of the interaction of tellurium compounds with glutathione

We have shown that tellurite and tellurate require the interaction with reduced glutathione (GSH) to hemolyze human erythrocytes. The study of the nature of this interaction is the main object of this paper. The degree of hemolysis was determined by the method of Angelone. The addition of extracellular 1 mM GSH or cysteine increased the rate of hemolysis. Concanavalin A (0.3 mg/mL) and/or 4 mg/mL adenosine did not affect the hemolysis by 0.1 mM tellurite. One tenth to 1 mM 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonate (SITS) inhibited this hemolysis by 60–100%. Millimolar GSH released this inhibition. Incubation of 0.1 mM tellurite with 1 mM GSH for 90 min at 37°C, produced a hemolytic agent when prepared and tested under nitrogen, but one that was not active when prepared in air. The hemolysis byp-hydroxymercuribenzoate orp-hydroxymercuriphenylsulfonate did not involve GSH. Scanning electron micrographs showed a sphero-echinocyte transformation, in the pre-hemolytic stage, with all the agents tested. The rate of penetration of tellurite plays a role in determining the rate of hemolysis, as shown by the effect of SITS. The release by GSH of the inhibition by SITS poses questions concerning the site of action and cell membrane penetration of the hemolytic agent. Telluride or some intermediate in the interaction of GSH with tellurite is the actual hemolytic agent.     

Protective effects of selenium against mercury toxicity as studied in the rat liver and kidney by nuclear analytical techniques

Mercuric chloride and sodium selenite were separately administered to male rats in the drinking water or in a combination (2.5 mmol Hg/L and 0.1 mmol Se/L). The mercuric chloride group showed histopathological lesions, as evidenced by cell necrosis in the liver and tubular necrosis in the kidney. The sodium selenite group showed some depression in growth, but pathological changes were found neither in the liver nor in the kidney. Simultaneous administration of both compounds produced a protective effect on weight loss and histopathology. These effects were associated with some small structures in the kidney proximal tubules and to some structure in the extracellular space in the liver. Thin, unstained cryosections were freeze-dried and examined in the Studsvik Nuclear Microprobe. The structures observed in the liver and the kidney were shown to contain both selenium and mercury.     

Factors affecting tissue distribution of heavy metals

Metal distribution in an organism at any one time is a resultant of many extrinsic and intrinsic factors. Improved analytic methodology has made it feasible to obtain highly sensitive determinations for many metals in a single sample. Thus, it is now feasible to examine patterns of metal distribution. We report on a study of the effect of age on metal patterns in the common tern,Sterna hirundo, a fisheating seabird. We contrast the levels of nine metals in the liver of adult and young terns and compared these with levels in tern eggs. Unlike many previous studies, adults did not have significantly higher levels of metals, although for most metals, levels in eggs were significantly lower than liver levels of young and adults. The intermetal correlations showed more significant positive correlations for adults and eggs than for chicks, the latter showing instead a correlational chaos, probably reflecting the immaturity of their physiologic defense mechanisms and the absence of dynamic equilibrium.     

Macromolecular interactions with cadmium and the effects of zinc,copper, lead,and mercury ions

Interactions of cadmium (Cd) ions with bovine serum albumin (BSA), bovine hepatic metallothionein (MT), calf thymus histone and deoxyribonucleic acid (DNA), and bovine hepatic chromatins were studied in the presence and absence of divalent zinc (Zn), copper (Cu), mercury (Hg), or lead (Pb) ions, using equilibrium dialysis at pH 7 and at 37°C. The BSA had 3.5 Cd-binding sites with an apparent affinity constant of 1×10⁵. The other metal ions inhibited the binding by reducing the affinity constant and the number of Cd-binding sites in BSA. There were 6 high affinity and 13 low affinity Cd-binding sites in the MT. Zinc ions had poor efficacy in reducing the binding of Cd to the MT. However, the Cu²⁺ and Hg²⁺ ions inhibited the Cd binding to a considerable extent, the former ions being more potent in this respect. Histone did not bind Cd. There were two kinds of Cd-binding sites in DNA: One mole of Cd per four moles DNA-phosphorus at low affinity sites, and one mole of Cd per 6.7 moles DNA-phosphorus at high affinity sites. Their apparent association constants were 8.3×10⁵ and 4.4×10⁶ M, respectively. The other metal ions had inhibitory effects on the binding of Cd to DNA. Histone reduced the Cd-DNA interactions to only a minor extent. The other metal ions reduced the binding of Cd to DNA-histone complex to a small extent. Cadmium binds to the euchromatin (Euch), heterochromatin (Het), and Euch-Het mixture almost equally. The other metal ions reduced the binding maximally in Euch-Het followed next in order by Het and Euch. Cupric ions were the most potent inhibitors of the interactions of Cd with the nuclear materials.     

In vivo and in vitro effects of aluminum treatment on rat liver mitochondrial function

This study examines the effect on mitochondrial respiration and permeability of in vivo and in vitro aluminium (Al) exposure. Rats were treated intraperitoneally with AlCl₃ to achieve serum and liver Al concentrations comparable to those seen in Al-related disorders. Mitochondria isolated from Al-treated rats had higher (p<0.01) Al concentration, lower (p<0.05) state 3 respiration, respiratory control (RCR), and ADP/O ratio (succinate substrate), and greater passive swelling in 100 mM KCl or 200 mM NH₄NO₃ than controls. The in vitro addition of Al (0–180 μM) to mitochondria from normal rats also decreased (p<0.01) state 3 respiration, RCR, and ADP/O and stimulated passive swelling in KCl and NH₄NO₃ at 42–180 μM Al. These studies show that Al depresses mitochondrial energy metabolism and increases membrane permeability. The toxicity associated with Al may be related to its effect on mitochondria.     

Simultaneous measurement of the trace elements Al, As, B, Be, Cd, Co, Cu, Fe, Li, Mn, Mo, Ni, Rb, Se, Sr, and Zn in human serum and their reference ranges by ICP-MS

The goal of this article was to establish reference ranges of the concentration of trace elements in human serum and to compare these results with those reported by other authors. We describe the sample preparation and measurement conditions that allow the rapid, precise, and accurate determination of Al, As, B, Be, Cd, Co, Cu, Fe, Li, Mn, Mo, Ni, Rb, Se, Sr, and Zn in human serum samples (n=110) by inductively coupled plasma-mass spectrometry (ICP-MS). Accuracy and precision were determined by analyzing three reconstituted reference serum samples by comparison with other methods and by the standard addition procedure. The advantages of the ICP-MS method include short time of analysis of the elements mentioned, low detection limit, high precision, and high accuracy. Disadventages include a high risk of contamination due to the presence of some of the elements of interest in the environment, the relatively delicate sample handling, and the high cost of the equipment.     

Effects of chromium supplementation on food energy utilization and the trace-element composition in the liver and heart of glucose-exposed young mice

Essentiality of selenium (Se) for Japanese quail,Coturnix coturnix japonica, was confirmed using a formulated semipurified low-Se diet (basal) (0.05 ppm). Selenium-deficiency symptoms appeared in quails on this diet within 15 d, which corresponded to low levels of hemolysate glutathione peroxidase (GSH-Px) activity. Selenium administration at 0.05 and 2.0 ppm levels resulted in an increase of hemolysate GSH-Px activity by 64 and 116%, respectively, in both short- and long-term experiments. Growth over a 2-mo period increased the hemolysate GSH-Px activity by 120% at each level of dietary Se. A differential response was exhibited by hepatic mitochondrial and soluble GSH-Px activity to Se supplementation, the former increasing progressively with increments of Se at 0.05, 2.0, and 4.0 ppm by 45, 70 and 150%, respectively. The soluble GSH-Px activities of tissues, such as liver, kidney, and testis, and RBC membrane-bound activity remained unchanged in long-term studies at different levels of Se. Replenishment of Se to quails maintained on low-Se diets reflected no change in RBC membrane-bound and liver-soluble GSH-Px activities, although the activity in hemolysate increased consistently with Se. The GSH-Px activity in hemolysate was restored to the levels comparable to those of long-term studies only at Se administration at the 2.0-ppm level. The differential response of mitochondrial and soluble GSH-Px activities to Se and other related observations on mitochondrial functions suggest an additional role for Se in mitochondrial membrane processes and glutathione-related metabolic regulations.     

Assessment of heavy metal toxicity related with human health risk in the surface water of an industrialized area by a novel technique

This study innovates an assessment technique to evaluate heavy metal toxic load (HMTL). Assessment of surface water in Durgapur industrial area, West Bengal India illustrates that heavy metals in majority of sampling locations exceed the drinking water quality standards. Therefore, pollution status and health impact were predicted by heavy metal pollution index (HPI) and hazard index (HI). More than 50% of sampling locations were marked as polluted. Moreover, 5% and 90% of sampling locations near the industries have health risk to adult and child on ingestion. The study of specific exposure-time and exposure-duration affirms the suitability of water for fishing and regular activities. However, HPI or HI does not quantify heavy metal concentrations that pose threat to human health. Therefore, the proposed assessment technique, HMTL, determines the concentration of heavy metals responsible for health hazard. HMTL identifies Mn, Pb, and Co as toxic metals and estimates 85%, 63%, and 70% removal of these metals from surface water to limit pollution. Origins of metals were also investigated through statistical techniques, which revealed that Fe has geogenic and anthropogenic source, while other metals originate by anthropogenic activities solely. This study demonstrates that HMTL will help the planning authority to document effective water quality management plan.     

Concentration of 17 trace elements in serum and whole blood of plains viscachas (Lagostomus maximus) by ICP-MS, their reference ranges, and their relation to cataract

The reference ranges of the trace elements Al, As, Be, B, Cd, Co, Cu, Fe, Mn, Mo, Ni, Pb, Li, Rb, Se, Sr, and Zn were determined by inductively coupled plasma-mass spectrometry (ICP-MS) in sera of a group of free-ranging plains viscachas of the pampa grasslands of Argentina. The values were compared with those of a small group of captive plains viscachas of the Zurich Zoo with diabetes and bilateral cataracts. In addition, a method for digestion of whole-blood samples is described for the trace element determination. Significant differences in the concentration of trace elements in the two groups of animals are discussed. No correlation was found between the levels of selenium and of other trace elements compared to the formation of cataracts.     

The role of dietary copper,manganese, selenium,and vitamin E in lipid peroxidation in tissues of the rat

The role of dietary Cu and Mn in maintaining tissue integrity, through the effects of these metals on activity of the superoxide dismutase (SOD) enzyme, and their interactions in peroxidative pathways involving Se and vitamin E was investigated. Weanling rats were fed diets deficient in Mn, Cu, Se, and/or vitamin E for 35 days, in a factorial experimental design. Dietary effects on peroxidation, measured in mitochondrial fractions prepared from liver and heart tissue, were compared with changes in the activities of glutathione peroxidase and the Cu and MnSOD enzymes. Decreased heart MnSOD and CuSOD activities, resulting from dietary Mn and Cu deficiencies, were both associated with increased peroxidation. Adequate Se (and glutathione peroxidase activity) prevented the peroxidation associated with either of these deficiencies, but was ineffective with a combined Cu−Mn deficiency. These effects of Se were only observed in tissue lacking glutathione transferase activity. Effects of Cu, Mn, and Se on peroxidation appeared to be present at both levels of vitamin E, although in both tissues, vitamin E deficiency greatly increased the overall peroxidation. Comparison of these in vitro peroxidation results with the deficiency associated lesions observed in vivo indicates that changes in SOD activities and peroxidation pathways may be the dominant cause of these lesions in only some cases. In others, the roles of Cu and Mn in different metabolic pathways appear to be of greater importance.     

Inhibitory effect of ethylenediaminetetraacetate (EDTA) on the porcine lymphocyte response to mitogens and reversal of the effect with metal ions

The effect of ethylenediaminetetraacetate (EDTA) on the mitogen response of porcine lymphocytes and the role of metal ions in reversal of the inhibitory effect of EDTA were determined. Porcine lymphocyte responses to mitogens were totally suppressed when serum used to supplement Ca²⁺, Mg²⁺-free minimum essential medium (MEM) was dialyzed against saline or saline with 0.2 or 0.60 mM EDTA, but the responses were only partially reduced when the same serum was added to RPMI-1640 medium. The inhibition observed in MEM could be reversed by adding 1×10⁻³ M Ca²⁺ and 1×10⁻³ M Mg²⁺ to the dialyzed serum. Serum treated directly with 0.60 mM EDTA completely suppressed blastogenesis in lymphocyte cultures maintained in RPMI-1640 or Ca²⁺, Mg²⁺ free MEM. The inhibitory effect of EDTA-treated serum could be completely reversed by adding Zn²⁺ or a combination of Zn²⁺ with other cationic ions, or partially reversed by adding Ni²⁺ or Fe³⁺. Zn²⁺ was the most effective ion, in that it was the only ion that, when alone added to the serum, could completely restore lymphocyte responses to phytohemagglutinin (PHA) or pokeweed mitogen (PWM).     

Metal-directed affinity labeling of zinc(II), cobalt(II) and cadmium(II) horse liver alcohol dehydrogenases

The active site metal in horse liver alcohol dehydrogenase has been studied by metal-directed affinity labeling of the native zinc(II) enzyme and that substituted with cobalt(II) or cadmium(II). Reversible binding of bromoimidazolyl propionic acid to the cobalt enzyme blueshifts the visible absorption band originating from the catalytic cobalt atom at 655 to 630 nm. Binding of imidazole to the cobalt(II) enzyme redshifts the 655 nm band to 667 nm. Addition of bromoimidazolyl propionic acid blueshifts this 667 nm band back to 630 nm. This proves direct binding of the label to the active site metal in competition with imidazole. The affinity of the label for the reversible binding site in the three enzymes follows the order Zn ? Cd ? Co. After reversible complex formation, bromoimidazolyl propionic acid alkylates cysteine-46, one of the protein ligands to the active site metal. The nucleophilic reactivity of this metal-mercaptide bond in each reversible complex follows the order Co ? Zn ? Cd.     

Magnesium (and trace substance) deficiencies in the pathogenesis of cancer

Except for a few experimental models of magnesium (Mg)-deficiency-induced neoplasms, less attention has been paid in the past quarter century in the Western world to this macromineral than to the trace elements; e.g., selenium (Se) and zinc (Zn), and to vitamins, deficiencies of which are each considered probable factors in oncogenesis. Although early epidemiologic studies showed an inverse correlation between the amount of Mg in soil and water and the incidence of (gastric) cancer, and several animal studies supported the premise that Mg has a prophylactic effect against induction of cancer, other studies showed that Mg supplementation increased the growth of established experimental tumors. Thus, enthusiasm for this approach subsided. The early epidemiologic findings have since been confirmed, and there have been studies demonstrating the importance of Mg in maintaining immunocompetence, and others indicating that immunodeficiencies increase susceptibility to the development of cancer. Evidence has now accrued that indicates that Mg deficiency increases susceptibility to chemical oncogens. The abnormal metabolism of tryptophan (yielding a carcinogenic metabolite) that indicates functional or absolute pyridoxine deficiency is an indirect clue to Mg deficiency. Vitamin B₆-activated enzymes require Mg as a cofactor. However, the early warnings against the use of Mg as part of an antineoplastic program against established cancer were justified, since rapidly metabolizing cells (such as cancers) are dependent on Mg. There are similarities between experiences with Mg and with Se and Zn. All three are required for normal metabolism; Se also protects against free radicals in the environment. Mg and Zn have increased established tumor growth, and their depletion has been applied to antineoplastic programs, with risks comparable to those of using antimetabolic agents.     

The nucleotide sequence of metallothioneins (MT) in liver of the Kafue lechwe (Kobus leche kafuensis) and their potential as biomarkers of heavy metal pollution of the Kafue River

The study determined heavy metal concentrations and MT1 nucleotide sequence [phylogeny] in liver of the Kafue lechwe. Applicability of MT1 as a biomarker of pollution was assessed. cDNA-encoding sequences for lechwe MT1 were amplified by RT-PCR to characterize the sequence of MT1 which was subjected to BLAST searching at NCBI. Phylogenetic relationships were based on pairwise matrix of sequence divergences calculated by Clustal W. Phylogenetic tree was constructed by NJ method using PHILLIP program. Metals were extracted by acid digestion and concentrations of Cr, Co, Cu, Zn, Cd, Pb, and Ni were determined using an AAS. MT1 mRNA expression levels were measured by quantitative comparative real-time RT-PCR. Lechwe MT1 has a length of 183bp, which encode for MT1 proteins of 61AA, which include 20 cysteines. Nucleotide sequence of lechwe MT1 showed identity with sheep MT (97%) and cattle MT1E (97%). Phylogenetic tree revealed that lechwe MT1 was clustered with sheep MT and cattle MT1E. Cu and Ni concentrations and MT1 mRNA expression levels of lechwe from Blue Lagoon were significantly higher than those from Lochinvar (p<0.05). Concentrations of Cd and Cu, Co and Cu, Co and Pb, Ni and Cu, and Ni and Cr were positively correlated. Spearman's rank correlations also showed positive correlations between Cu and Co concentrations and MT mRNA expression. PCA further suggested that MT mRNA expression was related to Zn and Cd concentrations. Hepatic MT1 mRNA expression in lechwe can be used as biomarker of heavy metal pollution.     

Production of phenolics and the emission of volatile organic compounds by perennial ryegrass (Lolium perenne L.)/Neotyphodium lolii association as a response to infection by Fusarium poae

Grasses very often form symbiotic associations with Neotyphodium/Epichloë endophytic fungi. These endophytes often allow the host grass to be protected from different pathogens. However, there is little known about the mechanisms of such endophyte influence on the host. Thus, the purpose of this research was to examine the effect of the N. lolii endophyte on the total production of phenolic compounds, VOCs emission and the resistance of three perennial ryegrass genotypes infected by pathogenic Fusarium poae. Analyses of total phenolics content were performed in control (not inoculated) and inoculated plants after 1, 2, 3, 4, 5, and 6 days (DAI) and for VOCs after 0, 3, 6 and 12 DAI. The presence of endophytes significantly reduced the disease index in two of the three genotypes relative to that in E−. Plants infected by N. lolii exhibited higher production of phenolics relative to the E− plants. The highest amounts of phenolics were observed on the second and sixth DAI. Genotype Nl22 showed the strongest effect of the endophyte on the production of phenolics, which increased by over 61%. Both the endophyte infected and non-infected plants emitted most abundantly two GLVs ((Z)-3-hexenal, (Z)-3-hexen-1-yl acetate), three terpenes (linalool, (Z)-ocimene, β-caryophyllene) and three shikimic acid pathway derivatives (benzyl acetate, indole, and methyl salicylate). The endophyte presence and the intervals of VOCs detection were a highly significant source of variation for all emitted volatiles (P < 0.001). The genotype of the perennial ryegrass significantly affected only the emission of methyl salicylate (P < 0.05) and β-caryophyllene (P < 0.05). Most of the VOCs ((Z)-3-hexen-1-yl acetate, (Z)-3-hexenal, linalool and methyl salicylate) reached their highest levels of emission on the sixth DAI, when averaged over genotypes and endophyte status. The results highlight the role of Neotyphodium spp. in the mediation of quadro-trophic interactions among plants, symbiotic endophytes, invertebrate herbivores and plant pathogenic fungi. Our results also confirm the fact that symbiotic plants can activate a defense reaction faster than non-symbiotic plants after a pathogen attack. Thus, N. lolii can be involved in the defense of perennial ryegrass against pathogens and potentially could be central to the host plants’ protection.