RESUMO
Currently, the increasing pollution of the environment by heavy metals is observed, caused both by natural factors and those related to human activity. They pose a significant threat to human health and life. It is therefore important to find an effective way of protecting organisms from their adverse effects. One potential product showing a protective effect is green tea. It has been shown that EGCG, which is found in large amounts in green tea, has strong antioxidant properties and can therefore protect cells from the adverse effects of heavy metals. Therefore, the aim of the study was to investigate the effect of EGCG on cells exposed to Cd. In the study, CHO-K1 cells (Chinese hamster ovary cell line) were treated for 24 h with Cd (5 and 10 µM) and EGCG (0.5 and 1 µM) together or separately. Cell viability, ATP content, total ROS activity, mitochondrial membrane potential and apoptosis potential were determined. The results showed that, in tested concentrations, EGCG enhanced the negative effect of Cd. Further analyses are needed to determine the exact mechanism of action of EGCG due to the small number of publications on the subject and the differences in the results obtained in the research.
Assuntos
Apoptose , Cádmio , Catequina , Sobrevivência Celular , Cricetulus , Potencial da Membrana Mitocondrial , Estresse Oxidativo , Espécies Reativas de Oxigênio , Catequina/análogos & derivados , Catequina/farmacologia , Animais , Células CHO , Apoptose/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Cádmio/toxicidade , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Antioxidantes/farmacologia , Cricetinae , Trifosfato de Adenosina/metabolismoRESUMO
European bison is a species for which health monitoring is essential in conservation activities. So far, little research has been carried out on the concentration of elements in this species. Most previous studies did not associate the concentration of elements with susceptibility to diseases. In this study we investigate the relationship between comorbidities in European bison and concentrations of a wide spectrum of elements in the liver. Samples were collected during the monitoring of the European bison population in Bieszczady (southeast Poland) over the 2020-2022 period. Each individual was also visually inspected by a veterinarian in the field for the presence of lesions as a part of a post-mortem examination. The animals were divided into 3 groups: group A-one type of clinical sign; group B-two types of clinical signs; group C-three or more types of clinical signs. The ICP-OES method was applied to assess the concentration of 40 elements in livers. Discriminant analysis showed clear differences between the mineral status of individuals in the groups with one, two, and at least three types of clinical signs. Detailed analysis of selected elements showed that, in the case of eight elements, there was a relationship with age, sex, or comorbidities. Cu, Se, and Zn showed significant differences in relation to comorbidities, but only Cu concentration was lower when the frequency of lesions was higher. We concluded that in research on the mineral status of the population, apart from the availability of trace elements in the environment, the health condition of the studied individuals should also be considered. However, inferring the mineral status of the population on the basis of randomly obtained samples from dead individuals may give an incomplete view of the population, especially in the case of species susceptible to diseases, such as European bison.
Assuntos
Bison , Oligoelementos , Animais , Oligoelementos/análise , Polônia/epidemiologia , Minerais/análise , Fígado/químicaRESUMO
We investigated vanadium, i.e., a redox-active heavy metal widely known for the generation of oxidative stress in cultured mammalian cells, to determine its ability to interfere with common oxidative stress-related bioassays in cell-free conditions. We first assessed the prooxidant abilities (H2O2 level, oxidation of DHR 123, and DCFH-DA dyes) and antioxidant capacity (ABTS, RP, OH, and DPPH methods) of popular mammalian cell culture media, i.e., Minimal Essential Medium (MEM), Dulbecco's Minimal Essential Medium (DMEM), Dulbecco's Minimal Essential Medium-F12 (DMEM/F12), and RPMI 1640. Out of the four media studied, DMEM has the highest prooxidant and antioxidant properties, which is associated with the highest concentration of prooxidant and antioxidant nutrients in its formulation. The studied vanadium compounds, vanadyl sulphate (VOSO4), or sodium metavanadate (NaVO3) (100, 500, and 1000 µM), either slightly increased or decreased the level of H2O2 in the studied culture media. However, these changes were in the range of a few micromoles, and they should rather not interfere with the cytotoxic effect of vanadium on cells. However, the tested vanadium compounds significantly stimulated the oxidation of DCFH-DA and DHR123 in a cell-independent manner. The type of the culture media and their pro-oxidant and antioxidant abilities did not affect the intensity of oxidation of these dyes by vanadium, whereas the vanadium compound type was important, as VOSO4 stimulated DCFH-DA and DHR oxidation much more potently than NaVO3. Such interactions of vanadium with these probes may artefactually contribute to the oxidation of these dyes by reactive oxygen species induced by vanadium in cells.
Assuntos
Compostos de Vanádio , Vanádio , Animais , Espécies Reativas de Oxigênio/metabolismo , Vanádio/toxicidade , Antioxidantes , Peróxido de Hidrogênio/toxicidade , Artefatos , Compostos de Vanádio/toxicidade , Meios de Cultura/química , Corantes , Mamíferos/metabolismoRESUMO
Vanadium is ranked as one of the world's critical metals considered important for economic growth with wide use in the steel industry. However, its production, applications, and emissions related to the combustion of vanadium-containing fuels are known to cause harm to the environment and human health. Pyruvate, i.e., a glucose metabolite, has been postulated as a compound with multiple cytoprotective properties, including antioxidant and anti-inflammatory effects. The aim of the present study was to examine the antioxidant potential of sodium pyruvate (4.5 mM) in vanadyl sulphate (VOSO4)-exposed CHO-K1 cells. Dichloro-dihydro-fluorescein diacetate and dihydrorhodamine 123 staining were performed to measure total and mitochondrial generation of reactive oxygen species (ROS), respectively. Furthermore, mitochondrial damage was investigated using MitoTell orange and JC-10 staining assays. We demonstrated that VOSO4 alone induced a significant rise in ROS starting from 1 h to 3 h after the treatment. Additionally, after 24 and 48 h of exposure, VOSO4 elicited both extensive hyperpolarisation and depolarisation of the mitochondrial membrane potential (MMP). The two-way ANOVA analysis of the results showed that, through antagonistic interaction, pyruvate prevented VOSO4-induced total ROS generation, which could be observed at the 3 h time point. In addition, through the independent action and antagonistic interaction with VOSO4, pyruvate provided a pronounced protective effect against VOSO4-mediated mitochondrial toxicity at 24-h exposure, i.e., prevention of VOSO4-induced hyperpolarisation and depolarisation of MMP. In conclusion, we found that pyruvate exerted cytoprotective effects against vanadium-induced toxicity at least in part by decreasing ROS generation and preserving mitochondrial functions.
RESUMO
In this review, we highlight the effects of epigallocatechin gallate (EGCG) against toxicities induced by heavy metals (HMs). This most active green tea polyphenol was demonstrated to reduce HM toxicity in such cells and tissues as testis, liver, kidney, and neural cells. Several protective mechanisms that seem to play a pivotal role in EGCG-induced effects, including reactive oxygen species scavenging, HM chelation, activation of nuclear factor erythroid 2-related factor 2 (Nrf2), anti-inflammatory effects, and protection of mitochondria, are described. However, some studies, especially in vitro experiments, reported potentiation of harmful HM actions in the presence of EGCG. The adverse impact of EGCG on HM toxicity may be explained by such events as autooxidation of EGCG, EGCG-mediated iron (Fe3+) reduction, depletion of intracellular glutathione (GSH) levels, and disruption of mitochondrial functions. Furthermore, challenges hampering the potential EGCG application related to its low bioavailability and proper dosing are also discussed. Overall, in this review, we point out insights into mechanisms that might account for both the beneficial and adverse effects of EGCG in HM poisoning, which may have a bearing on the design of new therapeutics for HM intoxication therapy.
Assuntos
Catequina/análogos & derivados , Metais Pesados/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Animais , Disponibilidade Biológica , Catequina/química , Catequina/farmacologia , Exposição Ambiental/análise , Humanos , Fatores de RiscoRESUMO
Vanadium (V) in its inorganic forms is a toxic metal and a potent environmental and occupational pollutant and has been reported to induce toxic effects in animals and people. In vivo and in vitro data show that high levels of reactive oxygen species are often implicated in vanadium deleterious effects. Since many dietary (exogenous) antioxidants are known to upregulate the intrinsic antioxidant system and ameliorate oxidative stress-related disorders, this review evaluates their effectiveness in the treatment of vanadium-induced toxicity. Collected data, mostly from animal studies, suggest that dietary antioxidants including ascorbic acid, vitamin E, polyphenols, phytosterols, and extracts from medicinal plants can bring a beneficial effect in vanadium toxicity. These findings show potential preventive effects of dietary antioxidants on vanadium-induced oxidative stress, DNA damage, neurotoxicity, testicular toxicity, and kidney damage. The relevant mechanistic insights of these events are discussed. In summary, the results of studies on the role of dietary antioxidants in vanadium toxicology appear encouraging enough to merit further investigations.
Assuntos
Antioxidantes/uso terapêutico , Dano ao DNA , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Vanádio/toxicidade , Animais , HumanosRESUMO
Exposure to vanadium has been associated with deleterious effects on the central nervous system in animals and humans. Although vanadium-derived pro-oxidant species were reported to be involved in vanadium-mediated neurotoxicity, the ability of this metal to induce oxidative stress markers in glial cells remains to be elucidated. In this study, we investigated the cytotoxicity and the generation of reactive oxygen species (ROS) and nitric oxide (NO) by mouse primary astrocytes after treatment with vanadyl sulfate (VOSO4 ) at concentrations of 20, 50, 100, 200, and 500 µM. The resazurin assay revealed that treatment with VOSO4 for 24 and 48 h at concentrations of 50 and 100 µM, respectively, or higher substantially induced astrocytic cytotoxicity. Intracellular ROS increased after 6-h exposure to the lowest concentration tested (20 µM VOSO4 ) and tended to intensify after 24- and 48-h treatments reaching significant values for 20 and 500 µM VOSO4 . In turn, NO production in the examined cells was elevated after exposure to all concentrations at the 6-, 24-, and 48-h incubation periods. Our study demonstrated the ability of VOSO4 to induce H2 O2 generation in cell-free DMEM/F12 medium. The H2 O2 levels were in the micromolar range (up to 5 µM) and were detected mostly during the first few minutes after VOSO4 addition, suggesting that the generated H2 O2 could not induce toxic effects on the cells. Taken together, these results show VOSO4 induced cytotoxicity in primary astrocyte cells, which may have resulted from vanadyl-stimulated intracellular ROS and NO generation in these cells.
Assuntos
Astrócitos/efeitos dos fármacos , Óxido Nítrico/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Compostos de Vanádio/toxicidade , Animais , Astrócitos/metabolismo , Astrócitos/patologia , Biomarcadores/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , CamundongosRESUMO
Arsenic (As) and cadmium (Cd) are elements arousing major public health concerns associated with environmental pollution, high toxicity potential, and carcinogenic nature. However, selenium (Se) at low doses and incorporated into enzymes and proteins has antioxidant properties and protects animals and humans from the risk of various diseases. It also has an exceptionally narrow range between necessary and toxic concentrations, which is a well-known hindrance in its use as a dietary supplement. The present article aims to update and expand the role of Se in As and Cd toxicity discussed in our earlier paper. In general, recent reports show that Se, regardless of its form (as selenite, selenomethionine, nanoSe, or Se from lentils), can reduce As- or Cd-mediated toxicity in the liver, kidney, spleen, brain, or heart in animal models and in cell culture studies. As was suggested in our earlier review, Se antagonizes the toxicity of As and Cd mainly through sequestration of these elements into biologically inert complexes and/or through the action of Se-dependent antioxidant enzymes. An increase in the As methylation efficiency is proposed as a possible mechanism by which Se can reduce As toxicity. However, new studies indicate that Se may also diminish As or Cd toxicity by activation of the Nrf2 pathway. In addition, this paper discusses possible signs of Se toxic effects, which may be a challenge for its future use in the therapy of As and Cd poisoning and provide future directions to address this issue.
Assuntos
Arsênio/toxicidade , Cádmio/toxicidade , Selênio/farmacocinética , Selênio/uso terapêutico , Antioxidantes/metabolismo , Humanos , Fator 2 Relacionado a NF-E2/metabolismo , Especificidade de Órgãos/efeitos dos fármacosRESUMO
With increasing human exposure to vanadium-containing compounds and growing concern over their impact on human health, identification of safe methods for efficient treatment of vanadium poisoning may be of value. In this study, using Chinese hamster ovary (CHO-K1) cells, we show that the toxicity of vanadyl sulphate (VOSO4) is mitigated in the presence of sodium pyruvate. The exposure of CHO-K1 cells to 100 µM VOSO4 for 48 h induced significant cytotoxicity (measured with a resazurin assay) and elevation of the contents of malondialdehyde (MDA), a lipid peroxidation product, in the examined cells. When added simultaneously with VOSO4 to the culture medium, pyruvate (4.5 mM) reduced VOSO4-mediated cytotoxicity by twofold and inhibited MDA formation. Phase-contrast microscopy confirmed that the general morphology of cell cultures treated with 100 µM VOSO4 and 4.5 mM pyruvate was improved compared to VOSO4-only treated cells. The two-way analysis of variance revealed that the reduction of the adverse effects of VOSO4 in the presence of pyruvate was due to the independent action of pyruvate as well as antagonistic interaction between VOSO4 and pyruvate. From these data, it can be concluded that the pyruvate treatment may play a beneficial role in reducing vanadium-triggered health hazards.
Assuntos
Substâncias Protetoras/farmacologia , Ácido Pirúvico/farmacologia , Vanádio/toxicidade , Animais , Bioensaio , Células CHO , Cricetinae , Cricetulus , Peroxidação de Lipídeos/efeitos dos fármacos , Malondialdeído/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Compostos de Vanádio/toxicidadeRESUMO
Previously, evaluation of sodium metavanadate (NaVO3) cytotoxicity after 24 h exposure of Chinese hamster ovary K1 (CHO-K1) cells revealed different sensitivity of the in vitro assays used starting from the neutral red (NR, 3-amino-7-dimethylamino-2-methylphenazine hydrochloride) test (detecting lysosomal and possibly the Golgi apparatus damage) as the most sensitive followed by the 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide inner salt (XTT) and resazurin (7-hydroxy-3H-phenoxazin-3-one-10-oxide) tests (mitochondrial disruption). The trypan blue (TB) staining (plasma membrane permeability) showed cytotoxicity of NaVO3 at a much higher NaVO3 concentration than the above-mentioned assays. In the current study, using the same experimental approach, we have assessed the toxicity of vanadyl sulphate (VOSO4) and compared the obtained results with NaVO3 action. Unlike metavanadate, VOSO4 treatment at 24 h resulted in similar sensitivity of the NR and resazurin tests. Nevertheless, following the 48-h incubation with VOSO4, the NR test showed markedly higher sensitivity than the resazurin test when comparing the half maximal inhibitory concentration values (61 and 110 µM for the NR and resazurin test, respectively, p < 0.05). The TB staining method was the least susceptible for detecting vanadyl cytotoxicity at each exposure time point. In summary, both the NR and resazurin tests can be advocated as similarly sensitive in detection of VOSO4-induced cytotoxicity in the CHO-K1 cell line at 24 h. However, the longer incubation time with VOSO4 showed that the NR test is more sensitive than the resazurin assay. The differences in the results between the cytotoxicity tests employed probably arise from dissimilar susceptibility of the endpoints (targets) measured with these tests to the damage by vanadium. Considering this, the current and the previous studies highlight the role of lysosomes (and possibly the Golgi apparatus) apart from mitochondria in the toxicity mechanism induced by inorganic vanadium in mammalian cells.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Testes de Toxicidade/métodos , Compostos de Vanádio/toxicidade , Animais , Bioensaio , Células CHO , Cricetinae , Cricetulus , Complexo de Golgi/efeitos dos fármacos , Concentração Inibidora 50 , Lisossomos/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Vermelho Neutro/química , Oxazinas/toxicidade , Sensibilidade e Especificidade , Sais de Tetrazólio/toxicidade , Vanadatos/toxicidade , Xantenos/toxicidadeRESUMO
The effect of selenium applied as sodium selenite (Na2SeO3) on the cytotoxicity of vanadyl sulphate (VOSO4) was examined using CHO-K1 cells. From the resazurin-based assay, it appears that Na2SeO3 at low doses (0.5 and 1 µM) can enhance 100 µM VOSO4-induced cell damage. The two-way ANOVA analysis revealed that the increased cell damage was a consequence of a synergistic interaction of 0.5 µM Na2SeO3 with VOSO4 and 1 µM Na2SeO3 with VOSO4. Observations performed with a phase-contrast microscope showed most cells to be rounded upon treatment with VOSO4 alone. In turn, a majority of cells co-treated with VOSO4 and 1 µM Na2SeO3 were elongated, and exhibited cytoplasmic vacuolization. These results warn of the potential contribution of inorganic selenium to vanadium-induced toxicity.
Assuntos
Selenito de Sódio/toxicidade , Compostos de Vanádio/toxicidade , Animais , Células CHO , Sobrevivência Celular/efeitos dos fármacos , Cricetulus , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Humanos , Microscopia de Contraste de FaseRESUMO
This investigation was undertaken to compare five different in vitro cytotoxicity assays for their power in revealing vanadium-mediated toxicity in Chinese hamster ovary (CHO)-K1 cells. The cells were exposed to sodium metavanadate (NaVO(3)) in the range of 10-1000 µM for 24 h and thereafter the cytotoxic effects of NaVO(3) were measured by colorimetric in vitro assays: the neutral red (NR) test, the 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide inner salt (XTT) assay, the resazurin assay, the sulforhodamine B (SR-B) assay, and by microscopic assessment of cell viability using the trypan blue (TB) staining method. Among the assays used, the NR test was the most sensitive, since it revealed metavanadate cytotoxicity at the lowest NaVO(3) dose (=50 µM). Also, NaVO(3) cytotoxicity expressed as inhibitory concentration (IC) showed the lowest values for the NR test. Three other tests XTT, resazurin, and SR-B assays showed intermediate sensitivity revealing the cytotoxicity of NaVO(3) at 100 µM. The corresponding IC10 and IC50 values calculated for the XTT, resazurin, and SR-B tests were similar. The TB staining method was the least sensitive, since it recorded metavanadate cytotoxicity at the highest NaVO(3) concentration tested (=600 µM). Based on the cytotoxicity end points measured with the above assays, it can be concluded that lysosomal/Golgi apparatus damage (measured by NR assay) may be the primary effect of NaVO(3) on CHO-K1 cells. The disintegration of mitochondria (assessed with the XTT and resazurin assays) probably follows lysosomal impairment. Plasma membrane permeability (staining with TB) occurs at a late stage of NaVO(3)-induced cytotoxicity on CHO-K1 cells. The results obtained in this research work show that the NR test can be recommended as a very sensitive assay for the assessment of NaVO(3) cytotoxicity in the CHO-K1 cell culture model. Considering the convenience of assay performance along with adequate sensitivity, the XTT and resazurin assays can also be advocated for NaVO(3) cytotoxicity assessment.
Assuntos
Sobrevivência Celular/fisiologia , Kit de Reagentes para Diagnóstico/normas , Animais , Células CHO , Membrana Celular/patologia , Cricetulus , Complexo de Golgi/patologia , Lisossomos/patologia , Microscopia , Vermelho Neutro , Rodaminas , Sensibilidade e Especificidade , Sódio , Sais de Tetrazólio , Azul Tripano , VanadatosRESUMO
Deleterious health effects induced by inorganic vanadium compounds are linked with carcinogenic, immunotoxic and neurotoxic insults. The goal of this review is to provide a summary of mammalian cell culture studies (from the 1990s to most recent) looking into the mode of the above-mentioned adverse actions of vanadium. Regarding the carcinogenicity potential, the key cell-based studies have evidenced the ability of vanadium to induce genotoxic lesions, cell morphological transformation and anti-apoptotic effects in a certain type of cells. Two contradictory effects of vanadium on the immune functions of cells have been observed in cell culture studies. The first effect involves reduction of cell immune responses such as vanadium-dependent inhibition of cytokine-inducible functions, which may underlie the mechanism of vanadium-induced immunosuppression. The second one involves stimulation of immune activity, for example, a vanadium-mediated increase in cytokine production, which may contribute to vanadium-related inflammation. So far, an in vitro evaluation of vanadium neurotoxicity has only been reported in few articles. These papers indicate probable cytotoxic mechanisms resulting from exposure of neurons and glial cells to vanadium. In summary, this literature review collects in vitro reports on adverse vanadium effects and thus provides vanadium researchers with a single, concise source of data.
Assuntos
Carcinógenos/toxicidade , Doenças do Sistema Nervoso Central/induzido quimicamente , Vanádio/toxicidade , Animais , Carcinógenos/química , Poluentes Ambientais/química , Poluentes Ambientais/toxicidade , HumanosRESUMO
Selenium is an essential trace element for mammals. Through selenoproteins, this mineral participates in various biological processes such as antioxidant defence, thyroid hormone production, and immune responses. Some reports indicate that a human organism deficient in selenium may be prone to certain diseases. Adverse health effects following selenium overexposure, although very rare, have been found in animals and people. Contrary to selenium, arsenic and cadmium are regarded as toxic elements. Both are environmental and industrial pollutants, and exposure to excessive amounts of arsenic or cadmium can pose a threat to many people's health, especially those living in polluted regions. Two other elements, vanadium and chromium(III) in trace amounts are believed to play essential physiological functions in mammals. This review summarizes recent studies on selenium interactions with arsenic and cadmium and selenium interactions with vanadium and chromium in mammals. Human studies have demonstrated that selenium may reduce arsenic accumulation in the organism and protect against arsenic-related skin lesions. Selenium was found to antagonise the prooxidant and genotoxic effects of arsenic in rodents and cell cultures. Also, studies on selenium effects against oxidative stress induced by cadmium in various animal tissues produced promising results. Reports suggest that selenium protection against toxicity of arsenic and cadmium is mediated via sequestration of these elements into biologically inert conjugates. Selenium-dependent antioxidant enzymes probably play a secondary role in arsenic and cadmium detoxification. So far, few studies have evaluated selenium effects on chromium(III) and vanadium actions in mammals. Still, they show that selenium may interact with these minerals. Taken together, the recent findings regarding selenium interaction with other elements extend our understanding of selenium biological functions and highlight selenium as a potential countermeasure against toxicity induced by arsenic and cadmium.
Assuntos
Arsênio/toxicidade , Cádmio/toxicidade , Cromo/toxicidade , Selênio/metabolismo , Selênio/farmacologia , Vanádio/toxicidade , Animais , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Arsênio/farmacologia , Cádmio/farmacologia , Cromo/farmacologia , Interações Medicamentosas , Humanos , Inativação Metabólica , Selenoproteínas/fisiologia , Oligoelementos/metabolismo , Vanádio/farmacologiaRESUMO
In the present work, we investigated the cytotoxicity of vanadium and the influence of zinc and selenium on vanadium-dependent cell damage in the BALB/c 3T3 cell culture. Treatment of cells for 24 hours with medium containing 50, 100 and 200 microM NaVO3 caused a significant decrease in the cell viability as measured by MTT test. Furthermore, the assays for reactive oxygen species (NBT reduction and phenol red oxidation) demonstrated the increase in superoxide and hydrogen peroxide production. In the cotreatment studies, the cells were exposed to NaVO3 (50, 100 and 200 microM) in the presence of nontoxic concentrations of ZnCl2 (5 microM) or Na2SeO3 (0.5 microM). Following 24 h incubation, the cell viability (assessed in MTT assay) and reactive oxygen species generation were evaluated. Our data suggest that zinc and selenium, in the concentrations mentioned above, provide no protection against adverse actions induced by sodium metavanadate at concentration levels of 50, 100 and 200 microM. To our knowledge, this is the first report from in vitro studies on interaction between pentavalent vanadium and trace elements that function as antioxidants: zinc and selenium.