Comparative investigations of sodium arsenite, arsenic trioxide and cadmium sulphate in combination with gamma-radiation on apoptosis, micronuclei induction and DNA damage in a human lymphoblastoid cell line

In the field of radiation protection the combined exposure to radiation and other toxic agents is recognised as an important research area. To elucidate the basic mechanisms of simultaneous exposure, the interaction of the carcinogens and environmental toxicants cadmium and two arsenic compounds, arsenite and arsenic trioxide, in combination with gamma-radiation in human lymphoblastoid cells (TK6) were investigated. Gamma-radiation induced significant genotoxic effects such as micronuclei formation, DNA damage and apoptosis, whereas arsenic and cadmium had no significant effect on these indicators of cellular damage at non-toxic concentrations. However, in combination with gamma-radiation arsenic trioxide induced a more than additive apoptotic rate compared to the sum of the single effects. Here, the level of apoptotic cells was increased, in a dose-dependent way, up to two-fold compared to the irradiated control cells. Arsenite did not induce a significant additive effect at any of the concentrations or radiation doses tested. On the other hand, arsenic trioxide was less effective than arsenite in the induction of DNA protein cross-links. These data indicate that the two arsenic compounds interact through different pathways in the cell. Cadmium sulphate, like arsenite, had no significant effect on apoptosis in combination with gamma-radiation at low concentrations and, at high concentrations, even reduced the radiation-induced apoptosis. An additive effect on micronuclei induction was observed with 1 μM cadmium sulphate with an increase of up to 80% compared to the irradiated control cells. Toxic concentrations of cadmium and arsenic trioxide seemed to reduce micronuclei induction.

The results presented here indicate that relatively low concentrations of arsenic and cadmium, close to those occuring in nature, may interfere with radiation effects. Differences in action of the two arsenic compounds were identified.     

Relation between rate of cell proliferation and formation of micronuclei after combined treatment with X-rays and caffeine

We studied the effects of caffeine (2 mM), X-rays (1 Gy) and the combination of both agents on cell proliferation and formation of micronuclei in the early stages of preimplantation mouse embryos in vitro. Two-cell embryos were exposed to the agents shortly before division to the 4-cell stage. Proliferation and micronucleus production was monitored every 2 h in the 4- and 8-cell stages. A rather peculiar pattern of micronucleus formation after radiation exposure alone was observed for 8-cell embryos: those embryos that were the first to enter the 8-cell stage showed two to three times higher numbers of micronuclei per cell when compared with those embryos that entered the 8-cell stage some hours later. Studies of the kinetics of cell proliferation and of micronucleus formation in 4- and 8-cell embryos and exposure to caffeine revealed that this result could be explained by two factors: a slight asynchrony in the developmental stage at the time of exposure and the length of the interval being available for repair processes. When caffeine was present, a third factor had to be taken into consideration: direct inhibition of repair by caffeine.Dedicated to Prof. W. Jacobi on the occasion of his 65th birthday     

Dietary exposure of Finnish children to heavy metal mixture – a cumulative assessment

A preliminary assessment of the cumulative exposure to heavy metals among Finnish preschool children is reported. Cadmium, lead, arsenic,and mercury affect many of the same organs in the human body. The effects are mostly caused by oxidative stress or disruption of enzyme function. The cumulative effects of the heavy metals on the central nervous system and on the kidneys are determined based on national concentration and consumption data, and comparison of the relative toxicity of the heavy metals is based on dose–response values found in the literature. The cumulative effects were assumed to be additive. The main contributors to kidney toxicity among the studied population groups were cadmium and lead, while lead was the main contributor to neurotoxic effects.     

The effect of exposure to radiofrequency LTE signal and coexposure to mitomycin-C in Chinese hamster lung fibroblast V79 cells

This study aims to investigate the cellular effects of radiofrequency exposure, 1950 MHz, long-term evolution (LTE) signal, administered alone and in combination with mitomycin-C (MMC), a well-known cytotoxic agent. Chinese hamster lung fibroblast (V79) cells were exposed/sham exposed in a waveguide-based system under strictly controlled conditions of both electromagnetic and environmental parameters, at specific absorption rate (SAR) of 0.3 and 1.25 W/kg. Chromosomal damage (micronuclei formation), oxidative stress (reactive oxygen species [ROS] formation), and cell cycle progression were analyzed after exposure and coexposure. No differences between exposed samples and sham-controls were detected following radiofrequency exposure alone, for all the experimental conditions tested and biological endpoints investigated. When radiofrequency exposure was followed by MMC treatment, 3 h pre-exposure did not modify MMC-induced micronuclei. Pre-exposure of 20 h at 0.3 W/kg did not modify the number of micronuclei induced by MMC, while 1.25 W/kg resulted in a significant reduction of MMC-induced damage. Absence of effects was also detected when CW was used, at both SAR levels. MMC-induced ROS formation resulted significantly decreased at both SAR levels investigated, while cell proliferation and cell cycle progression were not affected by coexposures. The results here reported provide no evidence of direct effects of 1950 MHz, LTE signal. Moreover, they further support our previous findings on the capability of radiofrequency pre-exposure to induce protection from a subsequent toxic treatment, and the key role of the modulated signals and the experimental conditions adopted in eliciting the effect.     

Enhancement of radiation-induced DNA double-strand breaks and micronuclei in human colon carcinoma cells by N-methylformamide

The differentiation-inducing agent N-methylformamide (NMF) enhances the sensitivity of some cell lines to ionizing radiation. To elucidate the mechanism of NMF-mediated radiosensitization, we examined the effects of this agent on gamma-ray-induced DNA double-strand breaks and micronuclei in two cell lines, clone A (human colon carcinoma) and HCA-1 (murine hepatocarcinoma). Both cell lines form a better differentiated phenotype upon exposure to NMF, yet only clone A is radiosensitized. The neutral (pH 9.6) elution assay was used to evaluate the effects of this maturational agent on radiation-induced double-strand breaks in these cell lines. Exposure of HCA-1 cells to NMF had no effect on the level of DNA double-strand breaks induced by gamma rays. In clone A cells, however, exposure to NMF enhanced the initial formation of gamma-ray-induced double-strand breaks at each dose tested. The repair of double-strand breaks in both cell lines was not influenced by NMF. As a measure of chromosome fragmentation after irradiation, we evaluated micronuclei using the cytokinesis block method. Exposure to NMF had no effect on radiation-induced micronuclei formation in HCA-1 cells yet significantly enhanced the frequency of micronuclei induced by radiation in clone A cells. In clone A cells, the increases in radiation-induced double-strand breaks and micronuclei as a function of NMF exposure time reached maximums by approximately 72 h. These data suggest that NMF-mediated radiosensitization is the result of an increase in the initial level of radiation-induced DNA double-strand breaks.     

Influence of antimonite, selenite, and mercury on the toxicity of arsenite in primary rat hepatocytes

The long-term toxicity of arsenic (As) as a result of exposure to contaminated drinking water might be modified by coinciding exposures to elements like selenium, antimony, or mercury. In this study the influence of tetravalent selenite, trivalent antimonite, and divalent mercury was investigated in vitro using cultured primary rat hepatocytes. The cell vitality was assessed in the 3-[4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide] (MTT), assay with concurrent exposures of the cells to up to 50 microM sodium arsenite(III) and a potential modifier [50 microM sodium(IV) selenite, 10 microM antimony(III) chloride, 25 microM mercuric(II) chloride], which indicated an additive increase in the combined cytotoxicity. Sodium arsenite was tested for genotoxicity in the micronucleus test in a concentration range of 0.25 up to 7.5 microM. In this range, the MTT conversion was at least 80%, indicating high cell viability. Adose-dependent induction of micronuclei was observed. The lowest concentration causing a significantly elevated frequency of micronuclei was 1 microM As (p < 0.05). A significant influence (i.e., reduction of the combined genotoxicity as a result of the presence of a potential modifier) was only observed for 10 and 25 microM antimony chloride (p < 0.05, Fisher's exact test). The metabolic methylation of arsenite was not affected by concurrent incubation with any of the potential modifiers.     

Enhancement of DNA damage and involvement of reactive oxygen species after exposure to bitumen with UVA irradiation.

This study was carried out to evaluate whether bitumen cytotoxicity is enhanced when bitumen treatment is combined with UVA exposure. We also evaluated the oxidative processes in bitumen-induced DNA damage, and attempted to identify the DNA damage caused by bitumen and UVA exposures, either alone or in combination. The effects of bitumen and UVA on cell proliferation were examined using HL 60 cells. DNA-protein crosslinks (DPCs) were assessed using a K-SDS assay, and reactive oxygen species formation was detected by 8-OH-dG formation. We evaluated the formations of double-strand breaks (DSB) using lambdaDNA/HindIII and single-strand breaks (SSB) using PM2 DNA. The cytotoxicity assay showed enhanced suppression of cell proliferation when bitumen exposure and UVA exposure were combined. Combined exposure caused significant increases in DPCs over either exposure alone. Incubation of deoxyguanosine (dG) with bitumen or UVA showed an increase in 8-hydroxy-2'-deoxyguanosine (8-OH-dG) levels when compared with controls, and combined exposure enhanced this effect. An evaluation of agarose gel bands showed that DSB and SSB were not formed following exposure to bitumen and UVA. This fact indicates that bitumen and UVA may be involved in genotoxic processes by producing oxygen free radicals and that combined exposure enhances these effects.     

Synergistic effects of arsenic trioxide and radiation in osteosarcoma cells through the induction of both autophagy and apoptosis

Osteosarcoma is the most common primary malignant bone tumor, occurring mainly in children and adolescents, and survival largely depends on their response to chemotherapy. However, the risk of relapse and adverse outcomes is still high. We investigated the synergistic anti-cancer effects of ionizing radiation combined with arsenic trioxide (ATO) and the mechanisms underlying apoptosis or autophagy induced by combined radiation and ATO treatment in human osteosarcoma cells. We found that exposure to radiation increased the population of HOS cells in the G(2)/M phase within 12 h in a time-dependent manner. Radiation combined with ATO induced a significantly prolonged G(2)/M arrest, consequently enhancing cell death. Furthermore, combined treatment resulted in enhanced ROS generation compared to treatment with ATO or radiation alone. The enhanced cytotoxic effect of combined treatment occurred from the increased induction of autophagy and apoptosis through inhibition of the PI3K/Akt signaling pathway in HOS cells. The combined treatment of HOS cells pretreated with Z-VAD, 3-MA or PEG-catalase resulted in a significant reduction of cytotoxicity. In addition, G(2)/M arrest and ROS generation could be involved in the underlying mechanisms. The data suggest that a combination of radiation and ATO could be a new potential therapeutic strategy for the treatment of osteosarcoma.     

Time factors in combined exposures of mouse embryos to radiation and mercury

Summary There are situations in which the exposure to more than one agent results in an enhanced risk for the exposed organism, that is in which the observed effect exceeds the effect expected from the addition of the individual effects. Our knowledge of such hazards is rather limited, in particular for those agents that occur in the environment of man. When early mouse embryos in vitro were exposed to ionizing radiation and mercuric chloride, the observed risk was higher than expected from the individual effects. This increase in risk was due to an interaction between mechanisms induced by ionizing radiation and mercury. To gain some more insight into the mode of interaction, the time requirements of mercury exposure were studied. The amount of interaction did not depend on mercury exposure before or during irradiation. However, to achieve an enhanced risk, exposure had to start as soon as possible after irradiation and had to last as long as possible. This time dependence suggests that if inhibition of DNA-repair is involved in the mechanism of interaction at all, then there must be an additional late process that is also impaired by mercury.Dedicated to Prof. W. Jacobi on the occasion of his 60th birthday     

Combined treatment of preimplantation mouse embryos in vitro with sodium nitrite and X-rays

Summary Man takes up nitrite in a considerable amount. Effects of nitrite on DNA have been reported; therefore, interaction between nitrite and radiation might be possible.Preimplantation mouse embryos in vitro were treated with a combination of sodium nitrite (1 mM or 2.5 mM) and X-rays (0.94 Gy) in order to obtain some information whether radiation risk is influenced by the presence of nitrite. The microscopic visible development up to 144 h post conceptionem (h p.c.), the number of cell nuclei, and the number of micronuclei were determined.None of the experimental results gives any indication that radiation risk is influenced by nitrite. All effects after combined treatment correspond to the sum of the single effects.     

二甲双胍联合放射线照射对鼻咽癌细胞CNE-1增殖的影响

目的:探讨二甲双胍联合放射线照射对鼻咽癌细胞CNE-1增殖的影响。方法:分别给予鼻咽癌细胞CNE-1二甲双胍(5m M)、2Gy放射线照射、二甲双胍(5 m M)联合2Gy放射线照射处理后,采用MTT实验、克隆形成实验检测和比较其细胞增殖抑制率和克隆形成抑制率。结果:MTT实验结果显示:与二甲双胍组或2Gy放射线照射组相比,二甲双胍联合放射线照射组细胞增殖抑制率显著升高,差异具有统计学意义(P0.05);克隆形成实验结果显示,与二甲双胍组或2Gy放射线照射组相比,二甲双胍联合放射线照射组细胞克隆形成抑制率显著升高,差异具有统计学意义(P0.05)。结论:二甲双胍联合放射线照射能够有效的抑制鼻咽癌细胞CNE-1的增殖。     

On the interaction of UV-B radiation (280–315 nm) with water stress in crop plants

Cowpea ( Vigna unguiculata L. Walp.) seedlings (3-day-old) were subjected to 4 kinds of experimental treatments: (1) control without exposure to any stress (−D-UV), (2) moderate water stress with no UV-B irradiation (+D-UV), (3) no water stress but to UV-B radiation (−D+UV). and (4)moderate water stress and exposure to UV-B (+D+UV). UV-B and drought stress in the combined form elicited beneficial effects on the morphological and growth characteristics, and a few additive inhibitory effects in some functional processes. An increase in the specific leaf weight (SLW) was observed in the combination of stresses, which could be a defence mechanism against UV-B. The combination of stresses promoted the synthesis of anthocyanins and phenolic compounds. The responses of plants to the combination of stresses indicate that during simultaneous exposure of plants to multiple stresses, one form of stress could minimize the damage by the other. The enhancement of superoxide dismutase (SOD) and catalase activities appear to serve as acclimation mechanisms to scavenge the toxic, free radicals of oxygen produced under stress conditions. However, the inhibition in nitrate metabolism was greater in the combined stresses than in either of the stresses imposed separately. The results of this study illustrate that the interaction of stresses during simultaneous multiple stress conditions brings out certain beneficial effects.     

Oleic acid and adipokines synergize in inducing proliferation and inflammatory signalling in human vascular smooth muscle cells

In the context of obesity, perivascular fat produces various adipokines and releases free fatty acids, which may induce inflammation and proliferation in the vascular wall. In this study we investigated how adipokines, oleic acid (OA) and the combined treatment regulate human vascular smooth muscle cell (hVSMC) proliferation and migration and the underlying signalling pathways. Adipocyte‐conditioned media (CM) generated from human adipocytes induces a prominent proliferation and migration of hVSMC. Autocrine action of adiponectin totally abolishes CM‐induced proliferation. Furthermore, OA but not palmitic acid induces proliferation of hVSMC. CM itself does not contain fatty acids, but CM in combination with OA markedly enhances proliferation of hVSMC in a synergistic way. Both the nuclear factor (NF)‐κB and the mammalian target of rapamycin (mTOR) pathway were synergistically activated under these conditions and found to be essential for hVSMC proliferation. Expression of iNOS and production of nitric oxide was only enhanced by combined treatment inducing a marked release of VEGF. Combination of OA and VEGF induces an additive increase of hVSMC proliferation. We could show that the combination of CM and OA led to a synergistic proliferation of hVSMC. Expression of iNOS and production of nitric oxide were only enhanced under these conditions and were paralleled by a marked release of VEGF. These results suggest that the combined elevated release of fatty acids and adipokines by adipose tissue in obesity might be critically related to hVSMC dysfunction, vascular inflammation and the development of atherosclerosis.     

Comparative and interactive effects of mercury, cadmium and lead on tissue GSH levels in Oreochromis aureus (Steindachner): implications for monitoring heavy metal pollution

The effects of 45 days exposure to mercury, cadmium and lead on tissue GSH levels were studied in Oreochromis aureus (Steindachner). Liver, brain, gill filaments, intestine and caudal muscle were assayed after exposure to these heavy metals singly, or in combination. Significant increases in intestinal GSH concentrations consistently occurred after exposure to mixtures of heavy metals. Exposure to cadmium or lead did not change hepatic GSH levels, while exposure to two different concentrations of mercury caused significant increases in hepatic GSH.     

Influence of high light and UV-B radiation on photosynthesis and D1 turnover in atrazine-tolerant and -sensitive cultivars of Brassica napus

An atrazine-tolerant mutant and an atrazine-sensitive cultivar of Brassica napus L. were grown under visible radiation (400 mumol m-2 s-1, photosynthetically active radiation, PAR) and then subjected to treatment conditions. These included short-term high PAR (1600 mumol m-2 s-1) which was given for 4 h either alone or in combination with an enhanced level of UV-BBE radiation (4.6 kJ m-2 h-1 biologically effective UV-B, 280-320 nm). Recovery from the radiation treatment was studied for 4 h under the light conditions for growth. Since it is known that the atrazine-tolerant mutant is susceptible to photoinhibition, one of the aims of the present study was to determine the effects of a supplemental, enhanced level of UV-B radiation with regard to the mutant. The results indicate an additive effect of UV-B radiation on Fv/Fm, photochemical yield and photosynthetic oxygen evolution during both exposure and recovery, and also a higher susceptibility of the mutant to photoinhibitory PAR conditions alone and in combination with UV-B, which may have implications in a changing environment. Both cultivars also showed a higher D1 turnover during the radiation stress than during recovery, as shown by immunoblotting and 35S-methionine incorporation measurements.     

Combined effects of gamma radiation and arsenite on the proteome of human TK6 lymphoblastoid cells

Arsenic present in drinking water and mining environments in some areas has been associated with an increased rate of skin and internal cancers. Contrary to the epidemiological evidence in humans, arsenic does not induce cancer in animal models, but is able to enhance the mutagenicity of other agents. In order to achieve a better understanding of the interaction between arsenic and ionising radiation, an investigation was conducted to detect differences at the proteome level of human TK6 lymphoblastoid cells exposed to these agents. Cells were exposed to either a single dose of 1-Gy 137Cs-gamma-rays or to 1 microM arsenite (As(III)) or to both agents in combination. Two-dimensional (2D) electrophoresis and matrix-assisted laser desorption/ionisation-time of flight (MALDI-TOF) were employed for the screening and identification of proteins, respectively. It proved possible to identify seven proteins with significantly affected abundance, three of which showed increased levels and the remaining four showed decreased levels under at least one of the exposure conditions. Following arsenite treatment or irradiation, a significant increase compared with that of the control was observed for glutathione (GSH) transferase omega 1 and proteasome subunit beta type 4 precursor. The combined exposure did not result in an induction of the enzymes. The expression of electron-transfer flavoprotein subunit alpha was found to be enhanced under all three-exposure conditions. Ubiquinol-cytochrome C reductase complex core protein I, adenine phosphoribosyl transferase and endoplasmic reticulum protein hERp29 showed decreased levels after irradiation or arsenite treatment, but not after the combined exposure. The level of serine/threonine protein phosphatase 1 alpha decreased with all treatments. The main conclusions are that both arsenite and gamma-radiation influence the levels of several proteins involved in major metabolic and regulatory pathways, either directly or by triggering the defence mechanisms of the cell. The combined effect of both exposures on the level of some essential proteins such as glutathione transferase, proteasome or serine/threonine phosphatase may contribute to the co-carcinogenic effect of arsenic.     

Exposure to Metals through the Consumption of Fish and Seafood by the Population Living Near the Ebro River in Catalonia,Spain: Health Risks

The health risks due to metal exposure from consuming various fish and seafood species were assessed for the Catalan population living near the Ebro River (Spain). The concentrations of arsenic, cadmium, chromium, copper, mercury, manganese, nickel, and lead were determined in samples of mussel, clam, hake, sole, cuttlefish, sardine, and anchovy randomly acquired in various localities of the zone under evaluation. In general terms, metal concentrations were similar or lower than the levels recently reported in the literature. The current dietary intake of metals is analogous to that recently estimated for the non-exposed population of Catalonia. Metal exposure through fish and seafood consumption would only mean a slight increase of noncarcinogenic and carcinogenic risk for arsenic, whereas the remaining elements showed risk values below the corresponding threshold levels.     

Effect of alkylation and carbamoylation on the characteristics of genetic damage to mammalian somatic cells cultured in vitro

Genetic effects of alkylation alone and combined with carbamoylation were studied following treatment of CHO-AT3-2 Chinese hamster cell line with N-nitroso-N-methylurea for 7 and 60 min. Gene mutations at HGPRT and Na+/K+-ATPase loci, micronuclei, cells with fragmented nuclei and lethality caused by NMU were recorded. Prolonged exposure to the mutagen made these effects more pronounced, particularly the fragmented nuclei and cell death. The combined action of the two mechanisms, therefore, enhanced the mutagenic effects of alkylation and expanded the range of DNA lesions towards greater incidence of gross damage to chromosomes and chromatids.     

In Vitro Penetration of Pig Skin by Heavy Metals in Soil

The potential health risk from exposure to heavy metal contaminated soil is often based on the quantity of metal that can be removed from soil by vigorous extraction procedures. This approach can overestimate risk since it ignores complex interactions between metals and soil that can result in a reduction in the amount of metal that desorbs from soil and is subsequently absorbed by the body. The aim of this research was to determine the relative contribution of the soil matrix and heavy metal sequestration in soil with time (“aging”) on the dermal penetration of arsenic, mercury, and nickel, respectively, as arsenic acid, mercuric chloride, and nickel chloride. In vitro flow-through diffusion cell studies were performed utilizing dermatomed male pig skin and radioactive compounds to measure total penetration (the sum of each metal in receptor fluid and skin). For arsenic and nickel, the soil matrix produced a 78–87% reduction in dermal penetration compared to 12–19% after aging. A greater effect was observed with aged mercury (52–56% decrease in dermal penetration) than in freshly spiked soil (40–43%). The results indicate that the potential health risk from dermal exposure to the metals can be significantly reduced by soil and aging.     

A Dietary-Wide Association Study (DWAS) of Environmental Metal Exposure in US Children and Adults

Background

A growing body of evidence suggests that exposure to toxic metals occurs through diet but few studies have comprehensively examined dietary sources of exposure in US populations.

Purpose

Our goal was to perform a novel dietary-wide association study (DWAS) to identify specific dietary sources of lead, cadmium, mercury, and arsenic exposure in US children and adults.

Methods

We combined data from the National Health and Nutrition Examination Survey with data from the US Department of Agriculture''s Food Intakes Converted to Retail Commodities Database to examine associations between 49 different foods and environmental metal exposure. Using blood and urinary biomarkers for lead, cadmium, mercury, and arsenic, we compared sources of dietary exposure among children to that of adults.

Results

Diet accounted for more of the variation in mercury and arsenic than lead and cadmium. For instance we estimate 4.5% of the variation of mercury among children and 10.5% among adults is explained by diet. We identified a previously unrecognized association between rice consumption and mercury in a US study population – adjusted for other dietary sources such as seafood, an increase of 10 g/day of rice consumption was associated with a 4.8% (95% CI: 3.6, 5.2) increase in blood mercury concentration. Associations between diet and metal exposure were similar among children and adults, and we recapitulated other known dietary sources of exposure.

Conclusion

Utilizing this combination of data sources, this approach has the potential to identify and monitor dietary sources of metal exposure in the US population.