Vanadium is generally known to be toxic to animals and humans (Domingo et al. 1995; Domingo 2000, 2002; Anke et al. 2003). Pentavalent compounds are the most toxic. This element is widely used in industry; it is emitted during combustion of fossil fuels (Barceloux 1999). Its compounds have been proven to be associated with various implications in the pathogenesis of some human diseases. It causes irritation of respiratory tracts, nasal catarrh, stinging pain in the throat, bronchial asthma, and pneumonia. It also leads to the development of gastrointestinal symptoms, green tongue, and focal neurological lesions (Barceloux 1999; Anke et al. 2003). Most clinically significant exposures to vanadium occur when workers inhale vanadium pentoxide fumes. In particular, workers have developed V poisoning while cleaning oil- or gas-fired boilers because V is a natural component of fuel oils (Barceloux 1999). Moreover, this element occurs in cigarettes (range 0.49–5.33 μg/g) and it is released into the atmosphere together with smoke (Adachi et al. 1998). It is well known that vanadium generates free radicals and lipid peroxides both in vitro and in vivo (Sheriff 1991; Younes and Strubelt 1991; Younes et al. 1991; Thompson and McNeill 1993) and diminishes the antioxidant system of organism (Oster et al. 1993; Zaporowska et al. 1997; Ścibior 1999). Instead, magnesium is the most abundant intracellular cation, which has a fundamental role as a co-factor in more than 300 enzymatic reactions involving energy metabolism and nucleic acid synthesis. Moreover, it is also involved in several processes like hormone-receptor binding, gating of calcium channels, muscle contraction, neuronal activity, neurotransmitter release, and other processes (Fawcett et al. 1999; Romani and Scarpa, 2000; Saris et al. 2000; Hartwig, 2001; Vormann, 2003). In the literature, some papers about vanadium-magnesium interaction were found. One of them described the synergistic interaction of magnesium and vanadate on glucose metabolism in diabetic rats (Matsuda et al. 1999), and another one showed the effect of vanadium on magnesium homeostasis in vitro conditions (Barbagallo et al. 2001). The other two articles presented the effect of magnesium and vanadate on the activity of Ca2+-ATPase from human red cell membranes (Romero 1993; Romero and Rega 1995). Instead, there are not any data about this interaction and its effect on haematological and biochemical blood parameters in mammals’ organisms. In the literature, only single reports about the protective role of magnesium against oxidative stress were found. It was described that magnesium reduces free radical production in vivo (Garcia et al. 1998; Zhang et al. 2003) and in vitro conditions (Kostellow and Morrill 2004). Furthermore, it was also described that magnesium may have an inhibitory effect on lipid peroxidation (Yamaguchi et al. 1994; Regan et al. 1998; Dumont et al. 2001; Peker et al. 2004) suggesting thereby the antioxidative action of this element. It was also found that Mg deficiency has been implicated in the initiation of oxidative damage (Franz 2004). Moreover, Hans et al. (2003) demonstrated that Mg supplementation can in part restore the antioxidant parameters and decrease the oxidative stress in experimental diabetic rats. It was also reported that Mg-gluconate may serve as a more advantageous Mg-salt for clinical use due to its additional anti-radical and cytoprotective activities (Mak et al. 2000). Other studies that were carried out on rat hearts showed the protective effect of Mg-gluconate against postischemic dysfunction and oxidative injury (Murthi et al. 2003). Furthermore, as some authors demonstrated, there is evidence to suggest that in nervous tissues Mg deprivation decreases reduced glutathione concentration, an effect accompanied by oxidative neuronal death. Similar results have been also obtained in endothelial cells and in the cardiomyocytes (Wofl et al. 2003). Weglicki et al. (1996) also observed that Mg-deficient tissues were less tolerant to oxidative stress. Instead, Shivakumar and Kumar (1997) reported that in rodents magnesium deficiency enhances circulating levels of factors that promote free radical generation.

Therefore, in the present study we have investigated if magnesium given to rats together with vanadium in drinking water for a period of 6 weeks will be able to limit its toxicity.

Materials and Methods

Reagents

L-ascorbic acid, sodium metavanadate, magnesium sulphate, saccharose, sodium tungstate, and thiobarbituric acid (TBA) were obtained from Sigma Chemicals (St. Louis, MO); di-sodium hydrogen phosphate anhydrous and trichloroacetic acid were obtained from POCh, Gliwice, Poland; PBS: buffered solution of physiological salt was obtained from Serum and Vaccine Factory (BIOMED, Lublin, Poland); oxalic acid was obtained from Chemical Reagents, Lublin, Poland. All other chemicals and reagents used were of analytical grade. Deionized water was used throughout.

Animals and Diet

This study was performed on adult, outbred 2-month-old, albino male WISTAR rats weighing about 244 g at the beginning of the experiment. During the 6 weeks of the experimental period, each of these animals was individually kept in stainless steel cages in a room under standard laboratory conditions (12 h light/12 h dark cycle at 20–21°C controlled temperature and relative air humidity 55 ± 5%). The size of used cages was in accordance with the size of each animal and it was allowed to receive a relatively constant level of motor activity of all rats throughout the whole experiment. All animals were fed with the same standard granulated rodent laboratory chow (Labofeed B; Fodder and Concentrate Factory, Kcynia, Poland). The vanadium and magnesium concentrations in the Labofeed B laboratory chow were determined by Inductively Coupled Plasma-Atomic Emission Spectrometry (ICP-AES) using the Liberty II AX plasma emission spectrometer (VARIAN) with the Ultrasonic Nebulizer (U-5000 AT+, CETACV) and with the Glass Concentric Nebulizer, respectively. The concentration of vanadium in this chow was 0.45 μg/g and the concentrations of magnesium was 1479.95 μg/g. Food, fluids, and water intakes were monitored daily over the experimental period and they were available ad libitum. Rats were weighted using weight Navigator (OHAUS, Switzerland) at the beginning of the treatment and then once per week during the study and again when sacrificed. Throughout the whole experiment, animal behavior was also observed. Animal care and all experimental procedures were used in accordance with the experimental protocol approved by the 1st Local Ethical Committee for Experiments with the Use of Laboratory Animals, Lublin.

Treatment of Rats

All animals were randomly distributed into 4 groups. The untreated group (control) received daily deionized water to drink from deionizer ARIES (Resin Tech., Inc.) over a 6-week time. The treatment groups at this time were as follows: group II received the water solution of NaVO3 (SMV; Sigma, St. Louis, MO) at a concentration of 0.125 mg V/mL; group III the water solution of MgSO4 (MS; Sigma, St. Louis, MO) at a concentration of 0.06 mg Mg/mL; group IV the water solution of SMV-MS at a concentration of 0.125 mg V and 0.06 mg Mg/mL. The experiment was terminated at the end of 6 weeks for haematological and biochemical studies. Vanadium and magnesium concentrations in drinking water were chosen on the basis of our previous studies (Zaporowska and Ścibior 1998) and studies of other authors (Russanov et al. 1994; Matsuda et al. 1999). In our experiment, a 10 mg higher Mg concentration (60 mg Mg/L) was used from the Maximal Admissible Concentration of Mg for potable water in Poland, which is 50 mg Mg/L (Zerbe and Siepak 2001; Bill Journal No 82, pos. 937, Schedule No 2). The vanadium and magnesium intakes were calculated based on the amount of SMV, MS, or SMV-MS solutions consumed by the rats.

Haematological Parameters Determination

Following 6 weeks of vanadium and/or magnesium treatment, from all anesthetized and treated as well as control rats, whole blood was collected into the plastic tubes with K2EDTA as an anticoagulant. RBC and WBC count, Hb level, Ht index, MCV, MCH, MCHC, and RDW were measured by using the automatic haematological analyzer MEDONIC CA 620-20 BALDER (Stockholm, Sweden).

NBT and Leukocytes Composition Determination

The phagocytic activity of neutrophils was determined in the whole blood by the nitroblue tetrazolium (NBT) reduction test of Raman and Poland (1975). Peripheral blood smears stained by the Pappenheim method by using Biochemtest (POCh, Gliwice, Poland) were analysed for the percentage composition of leukocytes.

L-Ascorbic Acid Determination in Plasma and in Erythrocytes

The plasma was separated by centrifugation of whole blood (10 min., 1.500 × g, 4°C) by using centrifuge UNIVERSAL 32 (Hettich, Germany). Erythrocytes were washed 3 times in ice cold 0.9% NaCl. Then plasma and RBC were used for biochemical investigations. In obtained plasma and erythrocytes, the L-ascorbic acid concentration was estimated according to the method described by Kyaw (1978). Briefly, into plasma and washed erythrocytes 2 mL of coloured reagent (Na2WO4 x 2H2O + Na2HPO4 + H2SO4) were added. After 30 min of incubation in the room temperature all samples were centrifuged (3,000g for 10 min.) by using centrifuge UNIVERSAL 32 (Hettich, Germany). The supernatants were read at A700 in the UV-Vis BioMate spectrophotometer. The results were expressed in μM/L.

Malondialdehyde (MDA) Determination in Erythrocytes

The MDA concentration in RBC was determined as thiobarbituric acid reactive substances (TBARS) according to the method of Stocks and Dormandy (1971) modified by Gilbert et al. (1984). Briefly, erythrocytes were washed 3 times in the cold PBS and then 5% solution of RBC in PBS was prepared. In the obtained solution, the level of Hb was determined and 0.5% Hb solution was prepared and then followed according to the above-mentioned method. After 60 min of incubation at 37°C, one of the reagents (trichloroacetic acid) dissolved in 0.1 M solution of sodium arsenite was added to all samples and then all samples were centrifuged (5 min., 1.500g, 4°C). Next, 0.5 mL of TBA was added to 1 mL of supernatant. All samples were incubated 15 min at 100°C. After incubation, the supernatants were read at A532 and A453 in the UV-Vis BioMate spectrophotometer. The MDA formed was calculated using the molar extinction coefficient 1.56 × 105 M−1 cm−1. The results were expressed in nM/mg Hb.

Total Antioxidant Status (TAS) Determination in Plasma

The plasma Total Antioxidant Status was determined using a Calbiochem® assay kit (Calbiochem-Novabiochem Corporation, San Diego, CA). Briefly, in this method ABTS is incubated with metmyoglobin (a peroxidase) and H2O2 to produce the radical cation (ABTS*+), which can be monitored by reading the absorbance at 600 nm. The results were expressed in mmol/L.

Determination of Magnesium, Calcium, and Vanadium Concentrations in Plasma

Magnesium and calcium concentrations in plasma were determined using POCh kits (Gliwice, Poland). The results were expressed in mmol/L. Instead, vanadium concentration in plasma was measured by Inductively Coupled Plasma-Atomic Emission Spectrometry (ICP-AES) using the Liberty II AX plasma emission spectrometer (VARIAN) with the Ultrasonic Nebulizer (U-5000 AT+, CETACV). The results were expressed in μg/mL.

Statistical Analysis

The control and three experimental groups were compared using the ANOVA test. To discern the possible interactions between vanadium and magnesium, two-way analysis of variance (two-way ANOVA) was used. The differences at P < 0.05 were considered significant. All results were expressed as means ± S.E.M. The statistical calculations were carried out with SPSS (version 12.0 PL for Windows, SPSS Inc.).

Results

General Observations

No distinctive differences were observed in physical appearance and in motor behavior in all rats exposed to vanadium and/or magnesium compounds compared to the controls during the whole period of the experiment. Only one rat exposed to SMV solution had diarrhea, and in the 30 days of the experimental period it died.

It was calculated that rats from the particular experimental groups took up about 10.7 mg V/kg body weight/24 h (group II), 6 mg Mg/kg body weight/24 h (group III), 9 mg V and 4.5 mg Mg/kg body weight/24 h (group IV) (Table 1).

Table 1 Number of animals and intake of vanadium and magnesium with drinking water by the tested rats
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The Food and Fluid Intake, and Body Weight Gain

In rats exposed to vanadium alone (group II), body weight gain, food, and fluid intakes decreased (by 15%, P < 0.05; 6%, P < 0.05; and 30%, P < 0.05, respectively) compared to the controls (Table 2). Moreover, the administration of magnesium alone (group III) resulted in a decrease in fluid intake (by 16%, P < 0.05) (Table 2). Two-way analysis of variance (ANOVA) revealed that changes in body weight gain, food and fluid intakes resulted from an independent effect of vanadium. Moreover, the ANOVA test also revealed that the changes in fluid intake resulted from an independent effect of magnesium. An interaction between V and Mg was not noted (Table 2).

Table 2 Food and fluid intake and body weight gain in the tested animal groups
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Some Haematological Parameters of Rats After Vanadium and/or Magnesium Treatment

The exposure to vanadium alone (group II) resulted in a statistically significant decrease in RBC count, Hb concentration, MCV, and MCH (by 6%, P < 0.05; 10.6%, P < 0.05; 4%, P < 0.05; and 6%, P < 0.05, respectively) (Tables 3 and 4). Instead, in rats treated with magnesium alone, a significant increase in Hb level (by 2%, P < 0.05) was found (Table 3).

Table 3 Erythrocytes count, haematocrit values, haemoglobin concentration, and RDW in the tested animal groups
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Table 4 MCV, MCH, MCHC, and the phagocytic activity of neutrophils in the tested animal groups
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Moreover, in all tested animals statistically significant changes in the Ht values, MCHC, RDW, and in the phagocytic activity of neutrophils in comparison with the controls were not shown (Tables 3 and 4).

Two-way ANOVA analysis revealed that the changes in some haematological parameters resulted from an independent action of vanadium (RBC count, Hb level, MCV, and MCH) and magnesium (Hb level). An interaction between V and Mg affecting all the above-mentioned parameters was not shown (Tables 3 and 4). In none of the tested animal groups were statistically significant differences found in the number of leukocytes and in the leukocyte composition (Table 5).

Table 5 Leukocytes count and leukocytes composition of peripheral blood in the tested animal groups
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In the SMV-treated group, a decrease in the L-ascorbic acid concentration (by 26%, P < 0.05) in plasma was demonstrated. Moreover, in the same group of rats an increase in MDA concentration (by 78%, P < 0.05) in erythrocytes was found (Table 6). Furthermore, the administration to rats of magnesium alone (group III) resulted in a decrease in the L-ascorbic acid concentration (by 22%, P < 0.05) in plasma (Table 6). No significant differences in the L-ascorbic acid concentration in RBC of all tested groups were shown (Table 6). Two-way analysis of variance revealed that the changes in some blood biochemical parameters resulted from an independent vanadium and magnesium action. An interactive effect of vanadium and magnesium was not observed (Table 6).

Table 6 The L-ascorbic acid and malondialdehyde (MDA) concentration in blood in the tested animal groups
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The Plasma Calcium, Magnesium, and Vanadium Concentrations and the Plasma TAS Level in Rats After Vanadium and/or Magnesium Treatment

In group III, which was treated for 6 weeks with magnesium alone, a statistically significant increase in the magnesium concentration in plasma was demonstrated. This increase compared to the controls was higher by 85%, P < 0.05 (Table 7). In none of the tested animal groups were statistically significant differences shown in the calcium concentration in plasma. Similarly, no significant differences in the plasma TAS level in all groups were found but we observed a tendency of the parameter level to decrease in all tested rats (Table 7). Plasma vanadium concentrations in the control and in group III were below the determination limit whereas the exposure to vanadium resulted in a significant increase in V content in plasma in group II (Table 7).

Table 7 The calcium, magnesium, and vanadium concentration in plasma and the plasma Total Antioxidant Status (TAS) level in the tested animal groups
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Discussion

Vanadium, magnesium, and antioxidants are essential for insulin action (Paolisso and Barbagallo 1997; Gregory and Kelly 2000; Guerrero-Romero and Rodríguez-Morán 2005). This is why there has been a strong interest in these elements in the last years (Weglicki and Mak 1992; Bonnefont-Rousselot 2004; Soltani et al. 2005).

In this study, vanadium at the dose of 10.7 mg/kg b.w./24 h consumed by rats with their drinking water for 6 weeks caused a significant decrease in food and fluid intakes, body weight gain, RBC count, Hb level, and MCV and MCH values whereas no significant differences in WBC count were observed in these animals compared to the control group. Additionally, vanadium at the tested dose resulted in a significant decrease in L-ascorbic acid concentration in plasma and caused a significant increase in MDA concentration in RBC whereas no significant differences in TAS level and in Ca and Mg concentrations in plasma of rats were shown compared to the controls. Moreover, V-treated rats had a higher vanadium concentration in the plasma than the control animals. The changes (or lack of them) in the above-mentioned parameters were also found and discussed in our previous reports (Zaporowska et al. 1993, 1997; Ścibior 1999; Zaporowska and Ścibior 1999; Ścibior 2005). The decreased body weight gain resulted from the reduced food intake. The decrease in food or fluid intakes or in body weight gain in rats after V treatment was also observed by other authors (Domingo et al. 1991; Thompson and McNeill, 1993; Dai et al., 1995; De la Torre et al. 1999; Poggioli et al. 2001; Thompson et al. 2002; Mohamad et al. 2004).

It is well known that a decrease in RBC count after vanadium treatment is a result of hemolysis caused by pro-oxidative action of this element (Zaporowska and Ścibior 1998; Hogan 1990, 2000). An increase in MDA concentration in RBC, which was demonstrated in this experiment, clearly confirms it. The excess of vanadium diminishes the activity of Na+, K+-ATPase, which causes the disturbances of sodium and potassium gradient on both sides of the cell membrane (Ingbar and Wendt 1997). Furthermore, vanadium inhibits the plasma membrane Ca2+-ATPase activity (Tiffert and Lew, 2001) and may open an L type-like Ca2+ channel in human red cells (Varecka et al. 1997). This situation enhances erythrocytes swelling and leads to haemolysis. Instead, because of erythrocyte swelling, the decrease of the RBC count is not always accompanied by the marked decrease of Ht.

The vanadium-induced enhancement of lipid peroxidation in RBC and the decrease in the L-ascorbic acid concentration in plasma are probably a consequence of increased formation of free radicals. Ding et al. (1994) demonstrated in vitro a one-electron reduction of V (V) to V (IV) by ascorbate. This reaction may represent an important V (V) reduction pathway in vivo. Instead, reactive species generated by V (IV) from H2O2 and lipid hydroperoxide via a Fenton-like reaction play a significant role in the mechanism of V (V)-induced cellular injury (Ding et al. 1994). The changes in the L-ascorbic acid concentration in plasma might also result from a higher catabolism of this compound in the experimental conditions.

Other authors did not demonstrate any significant changes in RBC and WBC systems in male Wistar rats that received vanadyl sulphate (0.5 mg/ml) in drinking water for 52 weeks (Dai and McNeill 1994) or AMV (0.14 mg/ml) and VS (0.26 mg/ml) in drinking water for 12 weeks (Dai et al. 1995). The divergences between our results and the results that were obtained by the above cited authors may be due, for example, to the use of various forms of vanadium, lower doses, and longer times of treatment. Moreover, the age of the studied animals and their age-related ability to vanadium adaptation could also probably influence the results presented above (Bolzán et al. 1995; Erdincler et al. 1997).

In this experiment, no significant changes in the magnesium and calcium concentrations in rats’ plasma after vanadium treatment were observed. Any significant alterations in the serum Mg concentration were also observed by Karczewski (1999) in male Wistar rats that received by gavage an AMV solution (0.5 mg V/kg b. w./day) for 6 months. Similarly, Noda et al. (2003) did not find any significant changes in the serum Mg and Ca concentrations in Wistar rats after giving them vanadium (1.5 mM Na3VO4 dissolved in 0.5 % NaCl) in drinking water for 4 weeks.

The administration of magnesium alone (6 mg/kg b. w./24 h) led to a statistically significant decrease in fluid intake. Additionally, magnesium at the above-mentioned dose resulted in a significant increase in Hb level and caused a significant decrease in L-ascorbic acid concentration in plasma of rats compared to the controls. Moreover, Mg-treated rats had a higher magnesium concentration in the plasma than the control animals. Our results are in agreement with several reports in which a significant increase in plasma Mg concentration was demonstrated. This increase was found by Altura et al. (1990) in rabbits that were treated with Mg in drinking water, and by Riond et al. (2000) and Zimmermann et al. (2000) in rats that received Mg in the diet.

The fact that the rats treated with Mg alone have a similar body weight gain as the control animals, in spite of reduced fluid intake, might result from unchanged food intake. No significant differences were observed by Matsuda et al. (1999) in food and fluid intake and also in body weight after giving magnesium (0.3 mg MgSO4 /mL) to male Sprague-Dawley rats in drinking water for 3 weeks. Brown et al. (2002) also did not show any significant differences in body weight gain in rats fed with a diet supplemented with magnesium for 12 weeks. Similarly, Altura et al. (1990) and Touyz and Milne (1999) did not find any significant changes in food and in body weight gain in male rabbits that received magnesium in drinking water (17.8 g Mg aspartate hydrochloride/L) for 8 weeks and in body weight gain in Wistar-Kyoto rats after magnesium supplementation (650 mg MgCl2/L, 17 weeks), respectively.

The results of this study indicate that following vanadium and magnesium co-administration, no significant differences in body weight gain were observed. Instead, other authors demonstrated that magnesium given to rats together with vanadium as a Mg-V solution caused a significant decrease in the body weight of rats (Matsuda et al. 1999). We may suppose that the differences between our results and the results obtained by Matsuda et al. may be due to the use of other rat chow and inbred (Sprague-Dawley) rats. However, those authors did not give the composition of the chow so it is difficult to compare the results.

According to the haematological parameters, Navas and Córdova (1996) did not show any significant changes in RBC count, Hb level, and Ht in male Wistar rats supplemented with MgCl2 (1,000 ppm of Mg) in drinking water for 21 days. Instead, Soltani et al. (2005) observed a significant increase in Hb level in diabetic Mg-treated rats (10 mg MgSO4/L in drinking water) for 8 weeks compared to the chronic diabetic control but these authors did not investigate the healthy control rats.

The increased MDA concentration and the decreased TAS level in a group of rats that were treated with magnesium alone (in spite of lack of significant differences compared to the controls) suggest the need to test for the combination of other vanadium and magnesium doses.

Moreover, it was demonstrated that separate administration to rats of vanadium or magnesium alone and also the co-exposure to these elements did not cause any significant differences in the plasma Total Antioxidant Status but we observed a tendency of the parameter level to decrease in all treatment rats. We decided to study TAS because it reflects the cumulative action of all the antioxidants present in plasma and in body fluids (Serafini and Del Rio 2004). The capacity of known and unknown antioxidants and their synergistic interaction was, therefore, assessed, thus giving an insight into the delicate balance in vivo between oxidants and antioxidants. The measurement of plasma TAS level may help in the evaluation of physiological, environmental, and nutritional factors of the redox status in humans (Ghiselli et al. 2000). Manuel y Keenoy et al. (2000) investigated Total Antioxidant Capacity in plasma in magnesium deficient patients and they observed that in these humans the above-mentioned parameter was lower.

Summary and Conclusions

The administration to rats of magnesium alone resulted in a significant decrease in the plasma L-ascorbic acid concentration. Moreover, in this study a visible tendency to an increase in the MDA concentration in RBC and to the decrease in the plasma TAS level was demonstrated. Therefore, we should be very careful when we use high magnesium doses as a therapeutic agent in the therapy of some human diseases. In addition, magnesium in mammal organisms may interact with other elements and in this way it may lead to the enhancement of the negative effects.

This study is the first, to our knowledge, to show the effect of vanadium–magnesium interaction on some blood parameters that were chosen for this experiment performed in in vivo conditions. An important and new finding of this study is that an interaction between vanadium and magnesium is not revealed in the investigated parameters. As determined by ANOVA, the significant differences that were demonstrated for some of the studied parameters were a result of an independent effect of vanadium or magnesium. An interactive effect of these two elements was not observed. The results of this report suggest testing TAS when other vanadium doses would be used, and also investigating a slightly lower magnesium doses that would be given to these animals in drinking water for 12 or even 18 weeks. Moreover, may be an experiment which would take a longer time (for example 18 weeks) in which not only other magnesium doses but also other magnesium compounds would be used, would allow elimination of the visible (in some cases) side effect of magnesium action, demonstrated in this study.