Wing membrane and Fur as indicators of metal exposure and contamination of internal tissues in bats.

All European bats are protected by the EU and Associated Members legal regulations. Being insectivorous and top predators, they can be particularly exposed to persistent organic and inorganic pollutants. It is surprising how little is known about the impact of environmental pollutants on bats from physiological to populational levels. In this study we focused on contamination with trace metals of first-year bats from Kharkiv city, NE Ukraine. Tissues from the carcasses of two species, Nyctalus noctula (n = 20) and Eptesicus serotinus (n = 20), were used for metal analysis. The samples of external (wing membrane, fur) and internal (liver, lung, kidney, bones) tissues were analysed for contents of Pb, Cu, Zn, and Cd to see whether fur or wing membrane can be used as proxies for metal contamination of the vital internal tissues. In E. serotinus, significant positive correlations in Pb concentrations were found between all external and internal tissues. For Cd only, correlation between the fur and lung was found, for Cu between the fur and liver, and for Zn between the fur and kidney. In contrast, for N. noctula, only one such correlation was found - between Zn concentrations in the fur and kidney. The tissues differed significantly in concentrations of all studied metals, with no difference between the species. The results showed that the fur and wing membrane can be used as good proxies for Pb concentrations in internal organs of E. serotinus, but not necessarily for other metals or for N. noctula. The results for Pb are, however, encouraging enough to conclude that the topic is worth further studies, covering more species, a wider age range and more diverse environments.

DNA damage in a liver tissue of metal exposed Clethrionomys glareolus.

It is widely known that some toxic agents may act on DNA strand resulting in its damages. One of the possible impairments is formation of abasic sites in DNA. The aim of this study was to indicate a presence of these DNA sites in the liver tissue of bank voles inhabiting the vicinity of zinc/lead smelters. Samples that were used originated from animals collected from unpolluted (Niepolomice, Telesnica Oszwarowa, Mikolajki) and polluted (Miasteczko Slaskie, Katowice, Olkusz) populations. They significantly differed in terms of tissue lead concentrations in the kidney and liver. The means of detected AP sites per 105 bp ranged between 3.39 (Telesnica Oszwarowa) to 5.13 (Miasteczko Slaskie). Statistical analysis (ANOVA) showed no difference in terms of number of the AP sites between single populations. However, t-test showed significant difference between the unpolluted and polluted populations. Factorial ANOVA indicated that sex is not a factor influencing the number of AP sites. The analyses revealed statistically significant relationships between the number of AP sites and Cu concentrations in the liver, and also Pb and Cd concentrations in the kidney.

Variation of Metallothionein I and II Gene Expression in the Bank Vole (Clethrionomys glareolus) Under Environmental Zinc and Cadmium Exposure.

The main idea of the study was to assess how environmental metal pollution activates defence responses at transcription levels in the tissues of bank voles (Clethrionomys glareolus). For this purpose, the metallothioneine (MT) genes expression (a well known biomarker of exposure and response to various metals) was measured. The real-time PCR method was used for relative quantification of metallothionein I and metallothionein II expressions in the livers, kidneys and testes of bank voles from six populations exposed to different contaminants, mainly zinc, cadmium and iron. The assessment of Zn, Cu and Fe concentrations in the tissues allowed to study the MTs gene expression responses to these metals. ANOVA analysis showed differences between populations in terms of metal concentration in tissues, livers and kidneys. Student T test showed significant differences in metal concentration between unpolluted and polluted sites only for the liver tissue: significantly lower Zn levels and significantly higher Fe levels in the unpolluted sites. Kruskal-Wallis test performed on C T data shows differences in the gene expressions between populations for both MT genes for liver and testes. In the liver metallothionein I gene expression was upregulated in populations considered as more polluted (up to 7.5 higher expression in Miasteczko Slaskie comparing to Mikolajki). Expression of metallothionein II revealed a similar pattern. In kidneys, differences in expression of both MT genes were not that evident. In testes, MT upregulation in polluted sites was noted for metallothionein II. For metallothionein however, we found downregulation in populations from more contaminated sites. The expressions of both MTs were positively influenced by cadmium in kidney (concentration data from the previous study) and zinc and copper in liver, while cadmium had effects only on the liver MT II gene expression. Positive relationship was obtained for lead and metallothionein II expression in the liver.

Modification of nickel accumulation in the tissues of the bank vole Myodes glareolus by chemical and environmental factors.

In a full factorial laboratory experiment, the effects of temperature and two chemical stressors (nickel and chlorpyrifos) on the accumulation of nickel in the liver and kidney of bank voles were studied. The nine-week experiment consisted of three periods: acclimatisation (3 days), intoxication (6 weeks) and elimination (3 weeks). During the main intoxication phase the animals were orally exposed for 42 days to different doses of nickel (Ni) (0, 300 and 800mg/kg food) or chlorpyrifos (CPF) (0, 50 and 350mg/kg food) or a mixture of both chemicals. Additionally, animals from each chemical treatment were divided into subgroups assigned to three temperatures: 10, 20 or 30°C. The highest concentrations of nickel were found in the testis, but there were no statistical effects of studied factors on this tissue. The nickel concentrations were higher in the kidney than in the liver of the bank voles. Nickel levels in the livers were influenced by Ni concentration in the food during intoxication time and additionally by interactions between Ni, temperature and day of exposure during elimination. The kidney concentrations of nickel depended on the level of nickel exposure but also on the interactions of the nickel with other factors: temperature, chlorpyrifos, day of exposure. This influence was observed only during the intoxication phase. The body mass and liver and kidney masses of the animals were affected both by the nickel concentration in the food and by the temperature. THE CAPSULE ABSTRACT: Ni in the tissues depended on the interactions between the factors: Ni, temperature and other. The body, liver and kidney masses were affected by both Ni in the food and by the temperature.

No ALAD Polymorphism in Bank Vole Populations from Unpolluted and Lead-Zinc Polluted Areas in Poland.

Although a variety of biomarkers has been developed to assess the adverse effect of lead on human and animal populations, studies show that the most important factor conditioning the response may be the genetic make-up of an individual. ALAD (delta aminolevulinic acid dehydratase) gene polymorphism found in human populations results in the existence of two alleles: ALAD1 and ALAD2. The difference between them is the result of one nucleotide change (G177C, rs1800435) which implicates different enzyme affinity for lead ions. As a result, carriers of these alleles respond differently to lead exposure in terms of tissue lead content, as well as neurobehavioral response. The aim of our study was to determine if such a phenomenon is present in wild animal populations. Two hypotheses were tested: (i) does the same ALAD polymorphism occur in lead exposed rodent species and is the frequency of different alleles similar to that of humans, (ii) if polymorphism exists, is the tissue lead content higher in individuals having ALAD2 alleles. We used bank voles (Myodes glareolus) inhabiting different lead contaminated sites for this purpose. The results obtained show no polymorphism in the bank vole populations that were studied. Contrary to the results obtained for humans, our study shows that all animals were found to have the C nucleotide. In human populations this nucleotide determines the ALAD2 allele and is much less frequent.

Effects of chemical and thermal stress on acetylcholinesterase activity in the brain of the bank vole, Myodes glareolus.

One of the most important issues in ecotoxicology is better understanding the effects of interactions between chemical pollutants and physical environmental factors on animals. To fill this knowledge gap, changes in the activity of acetylcholinesterase (AChE) in the brain samples of bank voles Myodes (Clethrionomys) glareolus due to temperature effects, and two chemical stressors were studied in a full factorial laboratory experiment (27 treatments). The experiment was divided into three phases: acclimatisation (3 days), intoxication (42 days) and elimination (21 days). During the intoxication phase, animals were orally exposed to different concentrations of either nickel (0, 300 or 800 mg Ni/kg food), chlorpyrifos (CPF) (0, 50 or 350 mg CPF/kg food) or a mixture of both chemicals. During the acclimatisation and elimination phases, the bank voles were given uncontaminated food. The experiment was conducted at three different temperatures (10, 20 or 30 °C), and a 12 h:12 h light:dark regime. The animals were sacrificed at 0, 5, 10, 20, 42, 49 and 63 days after the beginning of the intoxication, and brain samples were obtained for chemical analysis. The nickel accumulation in the brain depended on the level of nickel exposure and on interactions between the temperature and other factors. Nickel exhibited no effect on AChE activity. In contrast, AChE was drastically inhibited by chlorpyrifos and low temperature, but interactions between all factors significantly influenced the enzyme activity during the elimination phase of the experiment. High mortality was observed in the groups exposed to high concentrations of nickel and chlorpyrifos.

Genetic variation in bank vole populations in natural and metal-contaminated areas.

The effects of isolation and heavy-metal pollution on genetic diversity in Myodes (=Clethrionomys) glareolus populations were studied. Isolation and pollution are considered to have important effects on biodiversity. Animals were collected from ten populations in isolated (island), mainland, and metal-polluted areas. Three populations were in areas near zinc and lead smelters; four were on islands in the relatively unpolluted Mazurian Lake District and in the Bieszczady Mountains; and three were in clean-mainland areas in the Mazurian Lake District, the Niepolomice Forest, and the Bieszczady Mountains. Cadmium and lead concentrations in liver and kidney were measured to assess the animals' exposure to metals. The metal concentrations were greater in animals from areas classed as polluted than in animals from the clean-mainland areas and islands. The genetic diversity of each population was analyzed using eight microsatellite markers. The results confirmed that isolation adversely affects genetic diversity in M. glareolus populations (giving low heterozygosity and poor allelic richness), but the effect of metal exposure on genetic diversity was not strong. Of the samples from polluted areas, only the Katowice population, which is exposed to high levels of metal pollution and is also isolated because of human activity, showed genetic variation parameters that were similar to those for the island populations. Nei's genetic distances indicated that the island populations were genetically distant from each other and from the other populations, and there were noticeable inbreeding effects that would have been caused by the isolation of these populations.

The localisation of HSP70 and oxidative stress indices in heads of Spodoptera exigua larvae in a cadmium-exposed population.

The effects of cadmium toxicity may vary between animals with different history of metal exposure. The aim of our study was to examine HSP70, protein carbonyl levels, catalase activity and total antioxidant capacity in the heads of Spodoptera exigua (Hübner) larvae originated from undergoing 1- and 44-generational cadmium treatment and in control (those that were not exposed to cadmium). We also measured the cadmium concentration and DNA damage level in the larvae. We observed higher level of heat shock proteins (HSPs) in the heads of larvae derived from multi-generational metal treatment than in the heads of those from one-generational treatment (derived from the control rearing). Analysis of HSP localisation in the larval brain suggests that these changes could be important for protecting the neural function of larval mushroom bodies for animals selected during multigenerational metal exposure. Animals from one-generational treatment had, in turn, higher total antioxidant capacity than animals from multigenerational treatment. Anyway, animals from one- and 44-generational metal treatments did not differ in metal accumulation in the heads and the whole larval bodies, catalase activity or DNA damage level. All these measurements were higher than for control larvae and cadmium accumulation in the heads was much lower than in the whole bodies.

Expression of metallothionein genes I and II in bank vole Clethrionomys glareolus populations chronically exposed in situ to heavy metals.

The expression of two metallothionein genes (Mt-I and Mt-II) in the liver, kidney, and gonad of bank voles collected at four metal-contaminated sites (Cd, Zn, Pb, and Fe) were measured using the quantitative real-time PCR method (QPCR). Relative Mt gene expression was calculated by applying a normalization factor (NF) using the expression of two housekeeping genes, ribosomal 18S and beta-actin. Relative Mt expression in tissues of animals from contaminated sites was up to 54.8-fold higher than those from the reference site for Mt-I and up to 91.6-fold higher for Mt-II. Mt-II gene expression in the livers of bank voles from contaminated sites was higher than Mt-I gene expression. Inversely, Mt-II expression in the kidneys of voles was lower than Mt-I expression. Positive correlations between cadmium levels in the tissues and Mt-I were obtained in all studied tissues. Zinc, which undergoes homeostatic regulation, correlated positively with both Mt-I and Mt-II gene expression only in the kidney. Results showed that animals living in chronically contaminated environments intensively activate detoxifying mechanisms such as metallothionein expression. This is the first time that QPCR techniques to measure MT gene expression have been applied to assess the impact of environmental metal pollution on field collected bank voles.

Cadmium, zinc and iron interactions in the tissues of bank vole Clethrionomys glareolus after exposure to low and high doses of cadmium chloride.

In present study, bank voles Clethrionomys glareolus were peritioneally injected with different doses of cadmium, 0, 1.5, 3.0 mg Cd/kg body mass. Animals were sacrificed on the 21st day after cadmium exposure and the liver and kidney were obtained for cadmium, zinc and iron analysis using atomic absorption spectrometry. Results showed that cadmium had accumulated in the tissues according to dosage and sex. Cadmium affected the survival and body masses of dosed females. Cadmium decreased the iron concentrations in the liver of voles, whereas zinc concentrations increased in both the kidney and liver.

Characterisation of DNA probes for the analysis of metallothionein gene expression in the bank vole (Clethrionomys glareolus).

DNA probes have been developed for subsequent use in monitoring the exposure of animals to heavy metal pollution in terrestrial environments using metallothionein (MT) gene expression in the bank vole (Clethrionomys glareolus). Three different bank vole sequences were characterised corresponding to the cDNA and the genomic DNA for MT-I and the genomic DNA for MT-II. Nucleotide sequence analysis indicates that the coding sequences of the bank vole MT-I and MT-II genes exhibit a very high degree of similarity (greater than 92%) to the corresponding genes of the Chinese hamster, the mouse and the rat. In common with other mammalian MT genes, both the MT-I and MT-II genes in the bank vole are interrupted by two introns, which are at identical positions as those in other rodent MT genes; furthermore, the sizes of these introns are similar to those in other rodents with the first intron being larger than the second and those in the MT-I gene being larger than those in the MT-II gene. The predicted amino acid sequence for the proteins shows that both proteins contain 20 cysteine residues at positions identical to those in other known mammalian MTs. The availability of these DNA sequences now provides a good opportunity to investigate MT gene expression and possible gene amplification in bank voles exposed to metal pollution.