Long-term, medium-term and acute stress response of urban populations of Eurasian red squirrels affected by different levels of human disturbance.

Animals in urban areas often encounter novel and potentially stressful conditions. It is important to understand how wildlife cope with anthropogenic disturbance. To investigate this specific adaptation we live-trapped squirrels in two study sites in Warsaw: a forest reserve and an urban park and we estimated stress responses at three levels: long-term and medium-term stress (the level of stress hormones, i.e. cortisol and cortisone concentrations, in hair and feces) and acute reaction to human-induced stress (measured during handling with the aid of the three indices: breath rate, struggle rate, and vocalization). According to GLMM models no difference in the stress hormones level was found between the two populations. The only differences in cortisol concentrations clearly depended on the season, i.e. being higher in autumn and winter comparying to other seasons. There was no influence of sex, or reproductive status on stress hormones. Forest squirrels had significantly higher breath rates, suggesting they were more stressed by handling. There was no difference in the struggle rate between study areas, this index was mostly affected by season (i.e. being highest in winter). First-trapped squirrels vocalized less than during the subsequent trappings. Assumingly, during the first, and more stressful trapping, squirrels used 'freezing' and/or little vocalization, while during next captures they used alarm calls to warn conspecifics. Overall, we showed that the two squirrel populations differed only in terms of their breath rate. This suggests that they did not differ in medium-term and long-term stress in general, but they can differ in acute response to handling. This also suggests that both populations were similarly affected by environmental factors. The lack of clear effects may also be due to population heterogeneity. Thus, in order to assess the effects of anthropogenic stressors a broader range of indicators and diverse analytical methods, including behavioral analyses, should be employed.

Stress hormone level and the welfare of captive European bison (Bison bonasus): the effects of visitor pressure and the social structure of herds.

BACKGROUND: Captive European bison (Bison bonasus) play an active role in conservation measures for this species; this includes education, which may conflict with these animals' welfare. The effect of the presence of visitors on the welfare of captive animals can be negative, positive or neutral. However, the response of a given species to visitors is difficult to predict, since even closely related species display varying levels of tolerance to captivity. The aim of the study was to compare immunoreactive fecal cortisol levels (regarded as an indicator of the level of physiological stress) in groups of captive European bison that differed in terms of their social structure and the level of visitor pressure. The second aim was to determine if there was a correlation between intestinal parasitic burden and immunoreactive fecal cortisol levels. RESULTS: Immunoreactive fecal cortisol levels were not influenced by sex or age. However, study site and the interaction between study site and visitor pressure were statistically significant. European bison in one enclosure presented higher levels of immunoreactive fecal cortisol on weekdays than at weekends. In the other two study sites, the levels did not differ between weekdays and weekends. No correlation was found between parasitological infestation and immunoreactive fecal cortisol levels. CONCLUSIONS: Measurement of fecal cortisol metabolites could be a valuable method for further research into the welfare of European bison in captivity. More subtle factors such as individual animal characteristics, feeding systems, and the arrangement of enclosures can be of great importance in terms of the effect of visitors on animals. The results of this study can be used in guidelines for the management of European bison populations.

Evaluation of lipid peroxidation and the level of some elements in rat erythrocytes during separate and combined vanadium and magnesium administration.

The impact of vanadium (V) and magnesium (Mg) as sodium metavanadate (SMV, 0.125 mg V/ml) and magnesium sulfate (MS, 0.06 mg Mg/ml) on lipid peroxidation (LPO) and selected elements in the rat erythrocytes (RBCs) was investigated. Relationships between some indices determined in RBC were also studied. SMV alone (Group II) elevated the malondialdehyde level (MDARBC) (by 95% and 60%), compared with the control (Group I) and MS-supplemented rats (Group III), respectively, reduced the concentration of CuRBC (by 23.5%), in comparison with Group I, but did not change the levels of NaRBC, KRBC, and CaRBC, whereas MS alone (Group III) only reduced the CuRBC concentration (by 22%), compared with Group I. The SMV + MS combination (Group IV) reduced and elevated the CuRBC (by 24%) and CaRBC (by 111%) concentrations, respectively, in comparison with Groups I and III, and these changes were induced by the V-Mg antagonistic and synergistic interaction, respectively. The combined SMV + MS effect also enhanced the MDARBC level, compared with Groups I (by 79%) and III (by 47%) and slightly limited its concentration, compared with Group II, which, in turn, resulted from the distinct trend toward the V-Mg antagonistic interaction. We can conclude that V (as SMV) is able to stimulate LPO in rat RBCs and that V-Mg interactive effects are involved in changes in CuRBC, CaRBC, and MDARBC. Further studies are needed to elucidate the exact mechanisms of the V-Mg antagonistic/synergistic interactions and to provide insight into the biochemical mechanisms of changes in rats suffering from anemia [1], characterized by a disrupted antioxidant barrier in RBCs [2] and an intensified free radical process in these cells.

Protective activity of pyruvate against vanadium-dependent cytotoxicity in Chinese hamster ovary (CHO-K1) cells.

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.

Evaluation of lipid peroxidation and antioxidant defense mechanisms in the bone of rats in conditions of separate and combined administration of vanadium (V) and magnesium (Mg).

The impact of vanadium (V) and magnesium (Mg) applied as sodium metavanadate (SMV, 0.125 mg V/ml) and magnesium sulfate (MS, 0.06 mg Mg/ml) on oxidative stress markers in bone of male Wistar rats was investigated. Some of them were also measured in the liver, e.g. l-ascorbic acid (hepatic L-AA). Additionally, relationships between selected indices determined in bone were examined. SMV alone (Group II) did not significantly alter the level of TBARS and the activity of SOD, compared with the control (Group I), but it slightly reduced the GR activity (by 13%) and the L-AA level (by 15.5%). It also markedly lowered the activity of CAT and GPx (by 34% and 29%), and to some degree elevated the activity of GST (by 16%) and the hepatic L-AA level (by 119%). MS alone (Group III) decreased the TBARS level (by 49%), slightly lowered the L-AA concentration (by 14%), and reduced the SOD, GPx, and GR activities (by 31%, 40%, and 28%), but did not change the activity of CAT, compared with the control. Additionally, it elevated the GST activity (by 56%) and the hepatic L-AA level (by 40%). In turn, the SMV + MS combination (Group IV) reduced the TBARS level (by 38%) and the SOD, CAT, GPx, and GR activities (by 61%, 58%, 72%, and 40%) but elevated the GST activity (by 66%), compared with the control. The activity of SOD and GPx in the rats in Group IV was also reduced, compared with Group II (by 61% and 61%) and Group III (by 44% and 54%). In turn, the activities of CAT and GR were decreased, compared with Group III (by 55%) and Group II (by 31%), and the L-AA level was lowered, in comparison with Groups II and III (by 53% and 54%). Further, the concentration of V in the bone of rats in Groups II and IV increased, whereas the concentration of Mg decreased, compared with Groups I and III, in which the V and Mg levels dropped and were not altered, respectively, compared with Group I. The total content of Fe in the bone of rats in Groups II and IV increased, compared with Group III, in which the total Fe content did not change, compared with Group I. In turn, the total bone Cu content significantly decreased in the rats in Groups III and IV, compared with Groups I and II, whereas the total Zn content and the Ca concentration did not change markedly. The results provided evidence that the concentration of V used as SMV did not enhance LPO in bone, whereas Mg, at the selected level, markedly reduced LPO in this tissue. On the other hand, both elements administered separately and in combination disrupted the antioxidant defense mechanisms and homeostasis of some metals in bone tissue, which consequently may have contributed to disturbances in the balance in the activities of osteoblastic and osteoclastic cells, and thereby negatively affected bone health.

Effects of vanadium (V) and magnesium (Mg) on rat bone tissue: mineral status and micromorphology. Consequences of V-Mg interactions.

The extent to which the 12 week separate and combined administration of vanadium (as sodium metavanadate--SMV, 0.125 mg V per ml) and magnesium (as magnesium sulphate--MS, 0.06 mg Mg per ml) affects bone mineral status and micromorphology as well as the alkaline phosphatase (ALP) activity in femoral diaphysis (FD) was examined in male rats. The bone chemical composition of SMV-exposed rats was also investigated. SMV alone or in combination with MS (as SMV-MS) reduced the levels of MgFD (by 21% and 20%) and PFD (by 12% and 9%), lowered the CaFD content (by 7% and 10%), and caused a rise of the FeFD concentration (by 22.5% and 17%), compared with the control; SMV alone also reduced and enhanced the KFD and ZnFD concentrations (by 19% and 15%, respectively) but remained without significant effect on the femoral bone surface roughness (FBSR), whereas MS alone lowered the VFD, PFD, and CuFD levels (by 42%, 10%, and 20.6%), reduced FBSR, and created the regular femoral bone surface shape. The SMV-MS combination also induced a decline and rise in the levels of CuFD (by 30%) and NaFD (by 15%), respectively, compared with the control and the MS-supplemented rats; elevated ALPFD activity (by 24%, 35%, and 40%), compared with the control, SMV-exposed, and MS-supplemented animals; and increased FBSR. Relationships between the root mean square roughness (Sq) and skewness (Ssk): Sq [MS < SMV < Control < SMV-MS] ⇔ Ssk [SMV-MS > Control > SMV > MS], ALPFD and Sq: ALPFD⇔ Sq [SMV-MS > Control > SMV > MS], and between other variables were demonstrated. A partial limitation of the drop in the PFD and KFD levels and normalization of the ZnFD concentration were a consequence of the V-Mg antagonistic interaction whereas a consequence of the V-Mg synergistic interaction was the increase in the NaFD level, ALPFD activity, and FBSR. Ca10(PO4)5(SiO4)(OH) was part of the inorganic component of the bone of the SMV-exposed rats.

The renal effects of vanadate exposure: potential biomarkers and oxidative stress as a mechanism of functional renal disorders--preliminary studies.

The alterations in the levels/activities of selected biomarkers for detecting kidney toxicity and in the levels of some oxidative stress (OS) markers and elements were studied in male rats to evaluate biochemically the degree of kidney damage, investigate the role of OS in the mechanism of functional renal disorders, reveal potential biomarkers of renal function, and assess the renal mineral changes in the conditions of a 12-week sodium metavanadate (SMV, 0.125 mg V/mL) exposure. The results showed that OS is involved in the mechanism underlying the development of SMV-induced functional renal disturbances. They also suggest that the urinary cystatin C (CysCu) and kidney injury molecule-1 (KIM-1u) could be the most appropriate to evaluate renal function at the conditions of SMV intoxication when the fluid intake, excreted urinary volume (EUV), body weight (BW), and the urinary creatinine excretion (Creu) decreased. The use of such tests as the urinary lactate dehydrogenase, alkaline phosphatase, γ-glutamyltranspeptidase, and N-acetyl-ß-D-glucosaminidase (LDHu, ALPu, GGTPu, and NAGu) seems not to be valid given their reduced activities. The use of only traditional biomarkers of renal function in these conditions may, in turn, be insufficient because their alterations are greatly influenced by the changes in the fluid intake and/or BW.

The influence of combined magnesium and vanadate administration on the level of some elements in selected rat organs: V-Mg interactions and the role of iron-essential protein (DMT-1) in the mechanism underlying altered tissues iron level.

The effect of 12 week co-administration of sodium metavanadate (SMV) and magnesium sulfate (MS) on the levels of some elements in selected rats' organs and an attempt to elucidate a role of divalent metal transporter 1 (DMT-1) in the mechanism(s) of the SMV-induced disorders in some tissue Fe homeostasis were studied. SMV taken up separately or in combination with MS may pose a risk of the rise and shortage of the total hepatic and splenic Fe and Cu contents, respectively, cerebral Fe deficiency, splenic Ca deposition, and the hepatic, renal, and cerebral DMT-1 down-regulation. When administered alone, SMV may also cause the decrease in the total renal Fe and Cu contents. A visible protective effect of Mg against the renal and cerebral V accumulation and the decrease in the renal Fe and Cu contents during the SMV-MS co-administration together with our previous findings suggest a beneficial role of Mg at SMV exposure. Further, the SMV-induced fall in total iron binding capacity (TIBC), reported previously, and its correlations with the hepatic, splenic, and cerebral Fe levels allow us to suggest that diminished TIBC could be partly involved in the mechanism(s) responsible for the dramatic redistribution of Fe in those tissues. Finally, DMT-1, which potentially could participate in the hepatic non-transferrin Fe-bound uptake, does not play a significant role in this process indicating the need for studying other Fe transporters to more precisely elucidate molecular mechanism(s) underlying the hepatic Fe loading in our experimental conditions.

Magnesium can protect against vanadium-induced lipid peroxidation in the hepatic tissue.

The protective effect of magnesium as magnesium sulfate (MS) on sodium-metavanadate- (SMV-) induced lipid peroxidation (LPO) under in vivo and in vitro conditions was studied. The 18-week SMV intoxication (Group II, 0.125 V(end)/mL) enhanced spontaneous malondialdehyde (MDA) generation in rat liver, compared with the control (Group I) and MS-supplemented animals (Group III, 0.06 Mg(end)/mL). Coadministration of SMV with MS (Group IV, SMV-MS) caused a return of the MDA level to the control value range. The effect seems to result from the Mg(end)-independent action and its antagonistic interaction with V(end). The in vitro treatment of liver supernatants (LS) obtained from all the tested animals groups with selected exogenous concentrations of Fe(exg) or V(exg) exhibited enhanced MDA production, compared with spontaneously formed MDA. It also showed Mg(exg)-stimulating effect on LPO (LS I, Group I) and revealed that the changes in the MDA generation in LS IV (Group IV) might have resulted from the synergistic interactions of V(end) with Fe(exg) and V(exg) and from the antagonistic interactions of Mg(end) with Fe(exg) and V(exg). The findings allow a suggestion that adequate Mg intake for a specific period in the conditions of SMV exposure may prevent V-induced LPO in the liver.

Inhibitory and stimulating effect of magnesium on vanadate-induced lipid peroxidation under in vitro conditions.

The behaviour of Mg related to vanadium(V)-induced lipid peroxidation (LPO) under in vitro conditions was examined. The studies performed on the liver supernatants (LS) obtained from control, sodium metavanadate-intoxicated, and sodium metavanadate-magnesium sulphate-administered male Wistar rats revealed and confirmed the pro-oxidative potential of V. Simultaneously, they indicated that the improved Mg status may be one of the mechanisms by which the treatment with this element may contribute to reduction of oxidative stress under the conditions of vanadate exposure. On the other hand, the results confirmed that Mg may also stimulate LPO and demonstrated that the incubation conditions and the experimental treatment of the rats from which the liver supernatants were obtained affect the intensity of the examined free radical process.

Effect of 12-week vanadate and magnesium co-administration on chosen haematological parameters as well as on some indices of iron and copper metabolism and biomarkers of oxidative stress in rats.

Changes in some blood parameters after 12-week administration of sodium metavanadate (SMV; 0.125mgV/ml) or/and magnesium sulphate (MS; 0.06mgMg/ml) in drinking water were studied in outbred male Wistar rats (16 rats/each group) to explore the probable mechanism(s) underlying SMV toxicity and check whether Mg at the level selected during SMV co-administration can protect, at least in part, from a possible deleterious action of SMV. Exposure to SMV alone and in combination with MS (a) led to a decrease in fluid and food intake and body weight gain; (b) predisposed the animals to the development of microcytic-hypochromic anaemia (with excessive liver and spleen Fe deposition, unaltered plasma Fe level and enhanced Zn concentration in the erythrocytes (RBCs) characterized by a reduced haematocrit (Ht) index and haemoglobin (Hb) level, unchanged erythrocyte and reticulocyte count, anisocytosis, lowered total iron binding capacity (TIBC) and elevated transferrin saturation (TS); (c) disturbed Cu homeostasis, but (d) did not influence the leukocyte count and the plasma total antioxidant status (TAS). We suggest that abnormal metabolism and accumulation of Fe as well as an altered Cu status and the RBC Zn level might lead to defective Fe utilization and be a factor promoting the development of Fe-utilization anaemia. The disturbances in the antioxidative capacity reported previously in rats' RBCs after SMV intoxication (Scibior, Zaporowska, Environ. Toxicol. Pharmacol. 30 (2010) 153-161) may suggest that oxidative stress (OS) could also be, in part, involved in the mechanism responsible for the development of anaemia. The Mg dose ingested in combination with V under SMV-MS co-administration (a) was able to decrease, to some extent, the V concentration in the blood, (b) normalized the RBC Mg and Fe levels and (c) restored the values of some parameters of the Fe status near the control values. These results allow a supposition that a higher Mg dose consumed during SMV exposure could have better protective potential and be more effective in limiting the SMV toxicity observed.