Biochemical speciation in chromium genotoxicity

The biochemical speciation of chromium compounds in mammalian cells is discussed with respect to uptake, metabolism, DNA binding and damaging. Whereas soluble hexavalent chromium is taken up rapidly and accumulated intracellularly after its reduction, compounds of trivalent chromium penetrate biomembranes about three orders of magnitude slower. Cr(VI) after its uptake is metabolised by electron donating compounds via Cr(V) to Cr(III) compounds. Chromium from various Cr(III) compounds, but not chromate, binds to chromatin in isolated cell nuclei. The DNA‐protein crosslinks and DNA strand breaks observed in rat liver and kidney after chromate administration are also found in vitro, when Cr(III) compounds (but not chromate) interacts with isolated nuclei. In the Chinese Hamster cell HGPRT mutation assay, three out of four tested Cr(III) complexes were found to be mutagenic. In a direct DNA strand break assay with supercoiled bacteriophage PM 2 DNA, neither chromate nor the four Cr(III) compounds tested caused nicks. However, the combined action of chromate plus glutathione as well as the isolated complex of pentavalent chromium, Na₄Cr(glutathione)₄, did cause DNA breaks. Reactive oxygen species are inferred to be the ultimate DNA nicking agents in this assay. In conclusion there appear to be two mechanisms of chromate genotoxicity; one with direct DNA damage caused by Cr(V) species and one via DNA‐protein crosslinks formed with Cr(III), the final reduction state of chromate.     

Genotoxicity of chromium in vitro on yeast: Interaction with DNA†

The genotoxicity of chromium chloride was investigated in cells of D₇ strain of Saccharomyces cerevisiae harvested either from logarithmic or stationary growth phase.

A weak induction of mifotic gene conversion and point reverse mutation was obtained when the incubations were performed using phosphate buffer. No genetic effect was observed when the incubations were performed using Tris‐HCl buffer.

The experiments with ⁵¹Cr radiotracer demonstrated that Cr³⁺ ion enters the yeast cells and binds to DNA even if the incubation mixture was performed with Tris HCl buffer. This behaviour could be due to the highest concentration of CrCl₃ that cause some damages to cytoplasmatic membrane.     

The cytotoxicity of nici2 for mammalian cells in culture†

The effects of NiCl₂ were studied in two human cell lines, HeLa and diploid embryonic fibroblasts as well as in V79 Chinese hamster cells and in L‐A mouse fibroblasts. NiCl₂ produces a dose‐dependent depression of proliferation, mitotic rate, and viability, accompanied by an increasing release of lactic dehydrogenase and stimulation of lactic acid production. The plating efficiency is reduced, as are DNA and protein synthesis and, to a lesser degree, RNA synthesis.

The cytotoxicity of NiCl₂ is comparable in degree to those of PbCl₂ and MnCl₂, but is weaker than those of HgCl₂ and CdCl₂. However, the different sensitivities of different cell lines must also be considered.

NiCl₂ effects are more severe in serum‐free medium than in medium containing serum or serum albumin indicating that serum constituents, notably albumin, bind the metal effectively and inhibit cellular uptake; this confirms earlier reports on the serum binding and slow uptake of NiCl₂.

Synchronized cells are most sensitive in the Gl and early S phases of the cell cycle. In the Painter test the depression of DNA synthesis persists following cessation of exposure to NiCI₂. These findings contribute an explanation for the known genotoxic effects of nickel.     

Model reactions of chromium compounds with mammalian and bacterial cells†

Chromate uptake, reduction, cytotoxicity and mutagenicity were studied with human red blood cells, Chinese hamster ovary (CHO) cells and/or Salmonella typhimurium mutant cells. All cell types rapidly took up chromates whereas chromium(III) salts were excluded under the experimental conditions. Red blood cells reduced and accumulated chromium from chromate. At concentrations above 0.1 mM, chromate inactivated the red cell chromate carrier. Chromate above 0.01 mM inhibited CHO cell proliferation irrespective of the cations present. Chromate and two chromium(III) complexes were mutagenic with Salmonella mutants in the Ames’ assay. A model for chromate metabolism and genotoxicity is proposed.     

Hepatic effects of yttrium oxide nanoflowers: in vitro risk evaluation

Yttrium oxide nanoflowers were prepared by a hydrothermal technique, and X-ray diffraction and scanning electron microscopy were used to determine their structures. The cytotoxic and genotoxic potentials of aqueous dispersions of the nanoflowers to cultured primary rat hepatocytes were examined at concentrations up to 500 mg L^?1 for 72 h. Cell viability was determined by monitoring the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, release of lactate dehydrogenase, and uptake of neutral red. Genotoxicity was assessed by the liver micronucleus assay. Exposure to Y₂O₃ nanoflowers at concentrations lower than 100 mg L^?1 did not lead to any cytotoxicity or genotoxicity. At higher concentrations (200, 400, and 500 mg L^?1), cell viability decreased and induction of micronuclei increased (400 and 500 mg L^?1).     

Chromium binding by calf thymus nuclei and effects on chromatin

1. No binding of chromium was detected after incubation of calf thymus nuclei with hexavalent chromium up to 0.5 mM.

2. Chromium was readily taken up and tightly bound after incubation with trivalent chromium.

3. In a DNA‐filter binding assay, increasing amounts of chromium and DNA were bound with increasing chromium trichloride concentrations incubated with the nuclei.

4. Treatment with proteinase K abolished the increase in DNA retention induced by trivalent chromium.

5. It is concluded that trivalent chromium is the ultimate genetoxic agent after chromate uptake by living cells.

     

人血淋巴细胞检测浊漳河地表水的遗传毒性

为评价浊漳河水体中有机污染物的致突变性,采用单细胞凝胶电泳(SCGE)技术,研究了浊漳河水体3个不同样点的有机提取物对人外周血淋巴细胞DNA损伤效应。结果显示,各水样中的有机提取物均能引起DNA损伤,且随剂量的增高,有机物对DNA的损伤加重;细胞DNA损伤与对照组相比均有显著性差异(p<0.01)。这表明人外周血淋巴细胞对水体有机污染物的毒作用非常敏感。细胞彗星尾部DNA含量(%DNAT)和染毒剂量呈极显著正相关(R2>0.8547,p<0.01)。以上结果表明,浊漳河水体受到一定程度的有机物污染,这些有机污染物可诱导淋巴细胞DNA损伤,具有遗传毒性。SCGE技术作为一种简便、快速和灵敏的检测方法在水环境遗传毒性监测方面具有较大的应用价值。     

Voltammetric studies of damages in DNA by mutagenic and carcinogenic alkylating chemicals

Voltammetry provides new insights into the effects exerted in vitro by methylation of native DNA. Applying single sweep voltammetry at a stationary mercury electrode the successive steps of the destabilization of alkylated DNA are investigated. The methylation of the nucleic acids is manifested by a specific electrochemical response, due to the 7‐methylguanine, a major product of the methylation of DNA. Short time effects of the methylation include the labilization of 4 to 5 base pairs per methylated guanine base. Furthermore, uncommon protonation properties of the base 7‐methylguanine‐cytosine have been detected. Long term effects of the methylation are ultimately spontaneous hydrolytic strand breaks induced by the prior depurination connected with the release of the 7‐methylguanine from the methylated DNA. A half time t_1/2 of 102 h for the depurination at 37°C has been determined. The depurination and the subsequent strand breaks alter the hydrodynamic properties of the damaged DNA, an effect which can be sensitively followed with voltammetry via the resulting changes in the diffusion coefficient of the damaged biopolymer.     

Potentially harmful elements in house dust from Estarreja,Portugal: characterization and genotoxicity of the bioaccessible fraction

Due to their behavioral characteristics, young children are vulnerable to the ingestion of indoor dust, often contaminated with chemicals that are potentially harmful. Exposure to potentially harmful elements (PHEs) is currently exacerbated by their widespread use in several industrial, agricultural, domestic and technological applications. PHEs cause adverse health effects on immune and nervous systems and can lead to cancer development via genotoxic mechanisms. The present study is an integrated approach that aims at assessing the genotoxicity of bioaccessible PHEs following ingestion of contaminated house dust. A multidisciplinary methodology associating chemical characterization of five house dust samples, extraction of the bioaccessible PHEs in gastric extracts by the unified BARGE method, determination of the bioaccessible fraction and in vitro genotoxicity of gastric extracts in adenocarcinoma gastric human (AGS) cells was developed. The five gastric extracts induced dose-dependent genotoxicity in AGS cells. Copper (bioaccessible concentration up to 111 mg/kg) was probably the prevalent PHE inducing primary DNA damage (up to 5.1-fold increase in tail DNA at 0.53 g/l of gastric extract). Lead (bioaccessible concentration up to 245 mg/kg) was the most prevalent PHE inducing chromosome-damaging effects (r = 0.55; p < 0.001 for micronucleated cells induction). The association of principal component analysis and Spearman’s correlations was decisive to understand the chromosome-damaging properties of the bioaccessible PHEs in AGS cells. This methodology could be used on a larger-scale study to provide useful information for science-based decision-making in regulatory policies, and a better estimation of human exposure and associated health risks.     

Genetic toxicology of metal compounds: An examination of appropriate cellular models †

Bacterial systems have not had success predicting metal carcinogenicity. Hypotheses explaining this failure are examined. Using a broad genetic endpoint, λ prophage induction, under sub‐toxic growing conditions, genotoxicity is seen for compounds of chromium, manganese, lead, molybdenum and tungsten. Copper, manganese, arsenic and molybdenum compounds enhanced UV mutagenesis in E. coli WP2.

The toxicity of metal compounds to cultured mammalian cells correlates well with rat oral LD₅₀ values. Whereas insolubility can present problems in bacterial studies, concentrations of metal compounds toxic to mammalian cells can be determined even in the presence of precipitate, and sometimes [Pb(NO₃)₂, BaCl₂ and BeCl₂] occurs only in its presence. PbS and MnS, which are insoluble, are much more toxic than the more soluble compounds Pb(NO₃)₂ and MnCl₂. These results demonstrate the importance of cellular phagocytosis of insoluble metal compounds as a factor in studying the toxicity and genotoxicity of metal compounds.     

气态甲醛对卡氏毛园蛛细胞遗传毒性的研究

为了研究气态甲醛对蜘蛛的遗传毒性,采用单细胞凝胶电泳实验及KCl-SDS沉淀法检测了卡氏毛园蛛(Eriovixia cavaleriei)暴露于气态甲醛(0、0.5、1.0、3.0mg·m-3)后的细胞DNA分子断裂、DNA-DNA交联、DNA-蛋白质交联状况,并以甲醛的水溶性实验为基础,总结了甲醛对不同动物细胞的遗传毒性.结果显示,中、低浓度甲醛(0.5、1.0mg·m-3)可引起卡氏毛园蛛细胞DNA链断裂,高浓度甲醛(3.0mg·m-3)除引起DNA链断裂外,还可诱导核内交联物的形成;随着甲醛染毒浓度的升高,DNA的损伤程度逐渐加重,显示出一定的剂量-效应关系.通过总结甲醛对不同动物的遗传毒性发现,随着甲醛浓度的升高(5～625μmol·L-1,生理盐水溶液),动物细胞DNA损伤基本表现为由DNA链断裂(DSB)→DNA-DNA交联(DDC)→DNA-蛋白质交联(DPC)的过渡;甲醛所致的DNA损伤在不同动物细胞中也存在差异,反映了不同动物细胞对甲醛的敏感性不同。     

Assessment of DNA damage and cytotoxicity of palmatine on human skin epithelial carcinoma cells

The present investigation was carried out to examine the cytotoxic and genotoxic effects of a Tinospora cordifolia crude methanolic extract (palmatine) on human skin epithelial carcinoma cells (A431). T. cordifolia is one of the indispensable medicinal plants used in Ayurvedic medicine for treatment of various diseases and recommended for improving the immune system. Cytotoxicity and genotoxicity evaluation was carried out using A431 cells treated with different concentrations of palmatine. The duration of the treatment was 24 and 48 hr. A cellular proliferative capacity test showed that palmatine produced cytotoxicity in concentration- and time-dependent manner. Further, palmatine induced significant intracellular reactive oxygen species generation and elevated lipid peroxidation, as well as activities of catalase and superoxide dismutase. DNA fragmentation analysis using the comet assay showed that palmatine induced genotoxicity in a concentration- and time-dependent manner. Evidence indicates palmatine is capable of induction of oxidative stress resulting in cell death and genomic instability.     

The synergistic interaction of Nickel(II) with dna damaging agents

In this report we present several examples in which nickel(II) in combination with DNA damaging agents caused an enhanced or synergistic biological response using several different endpoints. These examples of Ni(II) toxicity represent several approaches designed to understand the genotoxicity of Ni(II) as well as several other metal ions. They are discussed in this report as a partial basis for our hypothesis that Ni(II) may alter the cellular processing of DNA damage at some common point in the pathway for DNA repair of several different agents. In cultured Chinese hamster cells DNA damage by Ni(II) ions was not readily demonstrated by the method of alkaline elution, but pretreatment of cells with Ni(II) before X‐irradiation produced an enhanced amount of strand breaks compared to the amount produced by X‐rays alone. A synergistic enhancement of cell transformation of Syrian hamster embryo cells was observed for combined treatments of Ni(II) and benzo(a)pyrene. The nickel enhancement of mutagenesis by ultraviolet light was demonstrated for the bacterial gene gpt stably integrated into the Chinese hamster V79 genome.     

In vitro Methylation of Arsenite in Flemish giant rabbit Cytosol

The formation of ⁷⁴As‐monomethyIarsonic acid and ⁷⁴As‐dimethylarsinic acid from carrier‐free radiolabelled ⁷⁴As‐arsenite was evaluated in an assay mixture containing 17.6% liver cytosol from the Flemish Giant rabbit. The optimal incubation temperature and pH were respectively, 39°C and 7.6. After a 2h incubation about 90% of ⁷⁴As was protein bound. Protein bound ⁷⁴As was released by hot 2 M HNO₃ (1 min, 110°C). The treatment did not destroy methylated As‐species. Up to 70% of the carrier‐free ⁷⁴As‐arsenite was methylated. Liver cytosol was stored, without loss of activity, in liquid nitrogen in the presence of 2 mM glutathione. The optimal s‐adenosyl‐methionine concentration was 1.7mM. Formation of ⁷⁴As‐monomethylarsonic acid and ⁷⁴As‐dimethylarsinic acid increased up to a glutathione concentration of respectively 10 and 20 mM. Methylation also went through in the presence of other reducing agents and proved to be ATP dependent.     

Cadmium uptake by a phytoplankton species in the presence of different inhibitors&

The uptake of cadmium by the Haptophyceae Hymenomonas elongata was studied as regards the energetic processes and Ca‐transport. For this purpose, experiments were carried out in the presence of an uncoupler of the phosphorylation : CCCP and of an inhibitor of calcium channels and also of Ca²⁺‐ATPase : verapamil. To reduce the number of assays, a factorial experimental design was used, in which all variables:— concentration of Cd in the medium—incubation time and—the presence of the inhibitor, are at two levels. In the absence of inhibitor, Cd uptake was found to vary as a function of metal concentration in the medium and of incubation time. CCCP significantly increased Cd uptake by H. elongata at sufficient incubation time. Therefore Cd uptake seemed linked to an energy‐dependent process, involving an ATPase. Verapamil immediately increased Cd uptake, implying an interaction between Cd and Ca.     

Utilization of glutamate and glucose for heterotrophic growth by the marine pennate diatom Nitzschia laevis

Nitzschia laevis Hustedt grew in the dark in the presence of either glutamate or glucose as substrate. Complex mixtures of yeast extract or tryptone plus lactate also supported good heterotrophic growth, while tryptone alone only supported very slow growth in the dark. The observed growth rates of N. laevis in the dark at different concentrations of glutamate or glucose could be accounted for by the measured uptake rates of these compounds. The affinity of the uptake systems for glutamate and glucose (K _s =0.03 mM for each) was quite high, and similar for dark- and light-grown cells. The lack of a lag-phase when cells were transferred from photoautotrophic to heterotrophic growth conditions can be explained by the presence of uptake systems for glutamate and glucose in ligh--grown cells, as well as in dark-grown cells. However, the uptake capacity was generally higher in the latter than the former. N. laevis also took up alanine and lactate according to Michaelis-Menten kinetics, with a K _s for alanine of 0.02 mM and for lactate of 0.4 mM. Malate and glycerol were not taken up to a significant extent by the cells. Cells grown in continous light had a doubling time of 18 h. The shortest doubling time observed in the dark on glutamate was 48 h and on glucose 24 h. Glutamate was used for heterotrophic growth with an efficiency of 43% and glucose with an efficiency of 48%.Contribution No., 945 from the Department of Oceanography, University of Washington, Seattle, Washington 98195, USA.     

Detection of oxidative stress and DNA damage in freshwater snail <Emphasis Type="Italic">Lymnea leuteola</Emphasis> exposed to profenofos

Extensive production and use of organophosphate pesticide in agriculture, has risen concerned about its ecotoxicity and risk assessment of insecticides, which are more important. Therefore, the present investigation was aimed to study the induction of oxidative stress and DNA damage by organophosphate insecticide profenofos (PFF) in freshwater snail Lymnea luteola (L. luteola). The median lethal value (96 h LC₅₀) of PFF was estimated as 1.26 mg/L for L. luteola in a semi-static system and on the basis of LC₅₀ value three concentrations viz., 0.126 (1/10 of LC₅₀, Sublethal I), 0.63 (1/2 of LC₅₀, Sublethal II) and 0.84 mg/L (2/3 of LC₅₀, Sublethal III) were determined. Snails were exposed to above-mentioned concentrations of PFF along with solvent control (acetone) and negative control for 96 h. The haemolymph was collected at 24 and 96 h of after treatment. In heamolymph of PFF exposed snail, lipid peroxide, glutathione reduced glutathione S transferase and superoxide dismutase activities at the tested concentrations significantly differ from those in the control. The genotoxicity induced in hemocytes of treated snails was measured by alkaline single cell gel electrophoresis assay. The data of this experiment demonstrated significantly enhancement of oxidative stress and DNA damage in the treated snails as compared to controls. Also, we observed statistically significant correlations of ROS with DNA damage (% tail DNA) (R² = 0.9708) for 24 h and DNA damage (R2 = 0.9665) for 96 h.

Results of the current experiment can be useful in risk evaluation of PFF among aquatic organisms. The study confirmed the use of comet assay for in vivo laboratory experiments using freshwater snail for selecting the toxic potential of industrial chemicals and environmental contaminants.

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Study on phosphate uptake of the marine cyanophyte Synechococcus sp. NIBB 1071 in relation to oligotrophic environments in the open ocean

Inorganic phosphate (Pi) uptake by the marine cyanophyte Synechococcus sp. NIBB 1071 was studied using cells grown in an artificial seawater medium. The phosphate uptake was markedly enhanced in cells grown in the medium of low phosphate concentrations (phosphate-limited cells) than in cells grown in the phosphate-rich medium (phosphate-replete cells). The diagnosis of kinetics of instantaneous phosphate-uptake showed that V _max of the former was more than two orders of magnitude greater than that of the latter, and the k _m of the former was about 1/20 of that of the latter. The enhancement of the phosphate uptake was completed after a 40-h incubation of phosphate-replete cells in the phosphate-free medium. The activation was suppressed by chloramphenicol, an inhibitor of protein synthesis. The uptake developed in phosphate-limited cells was energy dependent and susceptive to osmotic shock, which suggests the involvement of a periplasmic phosphate-binding protein, analogous to that found in heterotrophic gram-negative eubacterial cells. The relationship between phosphate quota and growth rate, together with the kinetical data for phosphate uptake, predicted that ambient phosphate as low as 0.5 nM could support cell growth at a rate of one division per day. Results indicate that cells can grow rapidly even at phosphate concentrations as low as nanomolar levels. A possible regulatory mechanism of phosphate uptake in marine Synechococcus spp. is discussed in relation to a wide distribution of this picophytoplankton in the ocean environment. Received: 19 March 1997 / Accepted: 2 April 1997     

Uptake of aluminum by lipid vesicles

Aluminium uptake and tight binding were studied in multilayered phospholipid liposomes, as a model for cellular uptake of aluminum ions. Most of these studies were conducted with an initial aluminum concentration of 10 μM, while aluminum superficially bound to liposomes was removed by citrate chelation. Maximum uptake and tight binding of aluminium were pH‐dependent. In dimyristoyl phosphatidylcholine (DMPC) liposomes, this maximum occurred in the neutral pH region, while it was shifted towards more acidic pH values in DMPC liposomes containing 20% of acidic phosphatidylserine. The initial rate of aluminum uptake was apparently dependent on the physical state of the liposome membrane. Prior formation of an aluminum‐citrate chelate prevented aluminum uptake and tight binding to DMPC liposomes.     

Uptake and release of phosphorus by phytoplankton in the Chesapeake Bay estuary,USA

The phytoplankton uptake and release rates for inorganic phosphate, dissolved organic phosphate and polyphosphate were estimated during 5 cruises on the Chesapeake Bay over a 9-month period. Phosphorus in all pools turned over in several minutes to 100 h, and each soluble pool appeared to contain fractions which were metabolically useful to the phytoplankton. Maximal uptake rates (V _m ) for orthophosphate ranged from 0.02 to 2.95 μg-at P (1.h)^-1 with half saturation constants (K _s ) between 0.09 and 1.72 μg-at P l^-1. At low soluble reactive phosphorus concentrations, the uptake rate of trace ³²P orthophosphate was initially rapid, but declined after 15 to 60 min incubation. The data suggest that the initial uptake phase was dominated by exchange of ³²PO₄ ^≡ for ³¹PO₄ ^≡ in the membrane transport systems whereas the subsequent phase represented the net incorporation of orthophosphate into phytoplankton cells.