Bacteria associated with Zn-hyperaccumulators Arabidopsis halleri and Arabidopsis arenosa from Zn-Pb-Cd waste heaps in Poland as promising tools for bioremediation.

To identify metal adapted bacteria equipped with traits positively influencing the growth of two hyperaccumulator plant species Arabidopsis arenosa and Arabidopsis halleri, we isolated bacteria inhabiting rhizosphere and vegetative tissues (roots, basal and stem leaves) of plants growing on two old Zn-Pb-Cd waste heaps in Boleslaw and Bukowno (S. Poland), and characterized their potential plant growth promoting (PGP) traits as well as determined metal concentrations in rhizosphere and plant tissues. To determine taxonomic position of 144 bacterial isolates, 16S rDNA Sanger sequencing was used. A metabolic characterization of isolated strains was performed in vitro using PGP tests. A. arenosa and A. halleri accumulate high amounts of Zn in their tissues, especially in stem leaves. Among in total 22 identified bacterial taxa, the highest level of the taxonomical diversity (H' = 2.01) was revealed in A. halleri basal leaf endophytes originating from Bukowno waste heap area. The 96, 98, 99, and 98% of investigated strains showed tolerant to Cd, Zn, Pb and Cu, respectively. Generally, higher percentages of bacteria could synthesize auxins, siderophores, and acetoin as well as could solubilize phosphate. Nine of waste heap origin bacterial strains were tolerant to toxic metals, showed in vitro PGP traits and are potential candidates for bioremediation.

An Alliance of Trifolium repens-Rhizobium leguminosarum bv. trifolii-Mycorrhizal Fungi From an Old Zn-Pb-Cd Rich Waste Heap as a Promising Tripartite System for Phytostabilization of Metal Polluted Soils.

The Boleslaw waste heap in South Poland, with total soil Zn concentrations higher than 50,000 mg kg-1, 5,000 mg Pb kg-1, and 500 mg Cd kg-1, is a unique habitat for metallicolous plants, such as Trifolium repens L. The purpose of this study was to characterize the association between T. repens and its microbial symbionts, i.e., Rhizobium leguminosarum bv. trifolii and mycorrhizal fungi and to evaluate its applicability for phytostabilization of metal-polluted soils. Rhizobia originating from the nutrient-poor waste heap area showed to be efficient in plant nodulation and nitrogen fixation. They demonstrated not only potential plant growth promotion traits in vitro, but they also improved the growth of T. repens plants to a similar extent as strains from a non-polluted reference area. Our results revealed that the adaptations of T. repens to high Zn-Pb-Cd concentrations are related to the storage of metals predominantly in the roots (excluder strategy) due to nodule apoplast modifications (i.e., thickening and suberization of cell walls, vacuolar storage), and symbiosis with arbuscular mycorrhizal fungi of a substantial genetic diversity. As a result, the rhizobia-mycorrhizal fungi-T. repens association appears to be a promising tool for phytostabilization of Zn-Pb-Cd-polluted soils.

Trifolium repens-Associated Bacteria as a Potential Tool to Facilitate Phytostabilization of Zinc and Lead Polluted Waste Heaps.

Heavy metals in soil, as selective agents, can change the structure of plant-associated bacterial communities and their metabolic properties, leading to the selection of the most-adapted strains, which might be useful in phytoremediation. Trifolium repens, a heavy metal excluder, naturally occurs on metal mine waste heaps in southern Poland characterized by high total metal concentrations. The purpose of the present study was to assess the effects of toxic metals on the diversity and metabolic properties of the microbial communities in rhizospheric soil and vegetative tissues of T. repens growing on three 70-100-years old Zn-Pb mine waste heaps in comparison to Trifolium-associated bacteria from a non-polluted reference site. In total, 113 cultivable strains were isolated and used for 16S rRNA gene Sanger sequencing in order to determine their genetic affiliation and for in vitro testing of their plant growth promotion traits. Taxa richness and phenotypic diversity in communities of metalliferous origin were significantly lower (p < 0.0001) compared to those from the reference site. Two strains, Bacillus megaterium BolR EW3_A03 and Stenotrophomonas maltophilia BolN EW3_B03, isolated from a Zn-Pb mine waste heap which tested positive for all examined plant growth promoting traits and which showed co-tolerance to Zn, Cu, Cd, and Pb can be considered as potential facilitators of phytostabilization.

Tissue Trace Elements and Lipid Peroxidation in Breeding Female Bank Voles Myodes glareolus.

Recent studies have demonstrated that reproduction reduces oxidative damage in various tissues of small mammal females. The present work was designed to determine whether the reduction of oxidative stress in reproductive bank vole females was associated with changes in tissue trace elements (iron, copper, zinc) that play an essential role in the production of reactive oxygen species. Lipid peroxidation (a marker of oxidative stress) and iron concentration in liver, kidneys, and skeletal muscles of reproducing bank vole females that weaned one litter were significantly lower than in non-reproducing females; linear regression analysis confirmed a positive relation between the tissue iron and lipid peroxidation. The concentrations of copper were significantly lower only in skeletal muscles of reproductive females and correlated positively with lipid peroxidation. No changes in tissue zinc were found in breeding females when compared with non-breeding animals. These data indicate that decreases in tissue iron and copper concentrations may be responsible for the reduction of oxidative stress in reproductive bank vole females.

Cadmium Accumulation and Pathological Alterations in the Midgut Gland of Terrestrial Snail Helix pomatia L. from a Zinc Smelter Area: Role of Soil pH.

The purpose of this study was to determine whether cadmium (Cd) accumulation and toxicity in the midgut gland of Helix pomatia snails living in a Cd-contaminated area were related to soil pH. Toxic responses in the midgut gland (i.e., increased vacuolization and lipid peroxidation) occurred in H. pomatia snails exhibiting the highest Cd levels in the gland (265-274 µg/g dry wt) and living on acidic soil (pH 5.3-5.5), while no toxicity was observed in snails accumulating less Cd (90 µg/g) and ranging on neutral soil (pH 7.0), despite the fact that total soil Cd was similar in the two cases. The accumulation of Cd in the gland was directly related to the water extractable Cd in soil, which in turn correlated inversely with soil pH, indicating that this factor had a significant effect on tissue Cd. It appeared further that the occurrence of Cd toxicity was associated with low levels of metallothionein in the gland of snails ranging on acidic soil.

Accumulation of cadmium in and its effect on the midgut gland of terrestrial snail Helix pomatia L. from urban areas in Poland.

The objectives of this study were (1) to determine cadmium (Cd) accumulation in the midgut gland of a land snail Helix pomatia L. inhabiting residential areas of the 14 largest cities in Poland, and (2) to examine whether the accumulated Cd exerted any toxic effects. The average accumulation of Cd in the midgut gland of snails, weighing 16-18 g, ranged from 7.00 to 87.3 µg/g dry weight (0.06-0.77 µmol/g) and differed significantly among animals from the various urban areas. This difference in Cd accumulation was not related to city population, but was associated with the topsoil Cd (R(2) = 0.868, p < 0.0001). The tissue Cd was not found to produce toxicity (histopathology, programmed cell death, lipofuscin formation or lipid peroxidation), probably due to the induction of sufficiently high quantities of metallothionein and glutathione, well-known protective molecules.

Differential susceptibility to cadmium-induced liver and kidney injury in wild and laboratory-bred bank voles Myodes glareolus.

The objective of the study was to compare the sensitivity of wild and laboratory-bred bank voles to cadmium (Cd)-induced histopathological changes in the liver and kidneys. For 4 weeks, the male bank voles-both wild and laboratory-bred-were provided with diet containing Cd in quantities <0.1 (control), 30, and 60 µg/g dry weight. At the end of exposure period, histopathology and analyses of Cd, metallothionein (MT), glutathione (GSH), zinc (Zn), copper (Cu), iron (Fe), and lipid peroxidation-all considered to be critical factors during the development of Cd toxicity in the liver and kidneys-were carried out. Histopathological changes (focal hepatocyte swelling, vacuolation and inflammation [leukocyte infiltration] in the liver, and focal proximal tubule degeneration [including epithelial cell swelling] in the kidneys) occurred only in the wild bank voles fed a diet containing 60 µg Cd/g. There were no differences in concentrations of Cd, MT, GSH, Zn, and Cu in liver and kidney between the respective groups of wild and laboratory-bred animals. However, a decrease of hepatic Fe and lipid peroxidation was observed in the wild voles exhibiting histopathological changes. These data indicate the following: (1) wild bank voles are more susceptible to Cd-induced liver and kidney injury than those bred and raised in the laboratory; (2) the difference in sensitivity may be associated with a distinct decrease of hepatic Fe in response to Cd exposure between the two groups of bank voles; and (3) dietary Cd may produce histopathological changes indirectly through decreasing the hepatic Fe and Fe-dependent oxidative processes. These results also suggest that histopathology in the liver and kidney of wild bank voles living in a contaminated environment may occur at relatively low levels of tissue Cd.

Seasonal and photoperiodic effects on lipid droplet size and lipid peroxidation in the brown adipose tissue of bank voles (Myodes glareolus).

Seasonal changes in lipid droplet size and lipid peroxidation in the brown adipose tissue (BAT) of wild bank voles were examined. In addition, a role of photoperiod in these changes was studied; bank voles were held from the birth under long photoperiod (LP) for 12 weeks, and then half of them was transferred to short photoperiod (SP) for 6 weeks and another one remained under LP. In the wild bank voles the absolute BAT weight was seasonally constant, while the significant differences in the lipid droplet size were observed. The smallest lipid droplets (mean, 11 µm(2)) were seen in winter; they increased by 30 % in spring and reached the highest size (24 µm(2)) in summer. Lipid peroxidation in the BAT did not differ significantly between the seasons, although high intraseason variation of this process was noted. The laboratory experiment revealed that the size of lipid droplets was determined by photoperiod; SP induced 13-fold decrease, and continuous exposure to LP brought about a further 2.5-fold increase in the size of lipid droplets. Conversely, a significant decrease in lipid peroxidation was seen in LP bank voles in comparison with the SP animals. The data indicate that short photoperiod is responsible for the small size of lipid droplets in the BAT of bank voles during winter, which may be a necessary requirement for high thermogenic capacity of the tissue. Photoperiod appears also to affect lipid peroxidation in the BAT of these animals.

Effect of dietary cadmium and/or lead on histopathological changes in the kidneys and liver of bank voles Myodes glareolus kept in different group densities.

Bank voles free living in a contaminated environment are known to be more sensitive to cadmium (Cd) toxicity than the rodents exposed to Cd under laboratory conditions, but the reasons for this difference are poorly defined. The present work was designed to determine whether dietary lead (Pb), a common environmental co-contaminant, and/or animal density that affects various physiological processes, would influence susceptibility to Cd toxicity in the kidneys and liver of these animals. For 6 weeks, the female bank voles were kept individually or in a group of six and provided with diet containing environmentally relevant concentrations of Cd [<0.1 µg/g (control) and 60 µg/g dry wt] and Pb [<0.2 µg/g (control) and 300 µg/g dry wt] alone or in combination. At the end of exposure period, histopathology and analyses of metallothionein, glutathione and zinc that are linked to a protective effect against Cd toxicity, as well as Cd, Pb, copper, iron and lipid peroxidation were carried out. Histopathological changes in the kidneys (a focal glomerular swelling and proximal tubule degeneration) and liver (a focal hepatocyte swelling, vacuolation and inflammation) occurred exclusively in some bank voles kept in a group and exposed to Cd alone (2/6) or Cd + Pb (4/6). The observed toxicity in grouped bank voles appeared not to be based on altered (1) tissue disposition of Cd and/or Pb, (2) metallothionein, glutathione and zinc concentrations, or (3) tissue copper, iron and lipid peroxidation. The data indicate that high population density in combination with environmental Pb may be responsible for an increased susceptibility to Cd toxicity observed in bank voles free living in a contaminated environment; the mechanism by which animal density affects Cd toxicity deserves further study.

Combined effect of dietary cadmium and benzo(a)pyrene on metallothionein induction and apoptosis in the liver and kidneys of bank voles.

Bank voles free living in a contaminated environment have been shown to be more sensitive to cadmium (Cd) toxicity than the rodents exposed to Cd under laboratory conditions. The objective of this study was to find out whether benzo(a)pyrene (BaP), a common environmental co-contaminant, increases Cd toxicity through inhibition of metallothionein (MT) synthesis-a low molecular weight protein that is considered to be primary intracellular component of the protective mechanism. For 6 weeks, the female bank voles were provided with diet containing Cd [less than 0.1 µg/g (control) and 60 µg/g dry wt.] and BaP (0, 5, and 10 µg/g dry wt.) alone or in combination. At the end of exposure period, apoptosis and analyses of MT, Cd, and zinc (Zn) in the liver and kidneys were carried out. Dietary BaP 5 µg/g did not affect but BaP 10 µg/g potentiated rather than inhibited induction of hepatic and renal MT by Cd, and diminished Cd-induced apoptosis in both organs. The hepatic and renal Zn followed a pattern similar to that of MT, attaining the highest level in the Cd + BaP 10-µg/g group. These data indicate that dietary BaP attenuates rather than exacerbates Cd toxicity in bank voles, probably by potentiating MT synthesis and increasing Zn concentration in the liver and kidneys.

Tissue cadmium accumulation is associated with basal metabolic rate in mice.

The objective of this study was to examine relations between basal metabolic rate (BMR) and cadmium (Cd) accumulation in the liver, kidneys, and duodenum in mice. The 5-month-old mice selected for high (H) and low (L) BMR were exposed for 8 weeks to 0, 10, and 100 µg Cd/mL of drinking water. The H-BMR mice showed significantly higher concentrations of Cd in the liver (47-79%), kidneys (61-70%), and duodenum (74-100%) than L-BMR animals. The tissue Cd accumulation also positively correlated with the duodenal iron which, in turn, was positively associated with BMR (Spearman R (s) = 0.81, P = 0.0004). The data indicate that tissue accumulation of Cd in mice is linked to BMR; the correlation between tissue Cd and duodenal iron suggests an involvement of iron transport pathway in the accumulation of Cd.

Cadmium accumulation, metallothionein and glutathione levels, and histopathological changes in the kidneys and liver of magpie (Pica pica) from a zinc smelter area.

The objective of this study was to examine a relationship between cadmium (Cd) accumulation and histopathological changes in the kidneys and liver of magpies (Pica pica) from a zinc smelter area. The concentrations of metallothionein (MT) and glutathione (GSH) that are linked to a protective effect against Cd toxicity were also determined. There was a positive correlation between the concentration of Cd (2.2-17.9 microg/g) and histopathological changes (interstitial inflammation and tubular cell degeneration) in the kidneys (R (s) = 0.87, P = 0.0000). The renal Cd also positively correlated with apoptosis (R (s) = 0.72, P = 0.0005) but the metal did not affect lipid peroxidation. Notably, the average concentration of Cd in the kidneys exceeded MT capacity by about 7 microg/g which is thought to produce renal injury. Importantly, GSH level in the kidneys of magpies from the polluted area dropped to 38% of that observed in the reference birds, probably potentiating Cd toxicity. On the contrary, the liver accumulation of Cd was relatively small (0.88-3.38 microg/g), the hepatic MT capacity exceeded the total concentration of Cd and no association between the hepatic Cd and histopathology was found despite the fact that GSH level was only half that observed in the reference birds. The data suggest that Cd intoxication may be responsible for histopathological changes occurring in the kidneys of free-ranging magpies and that the pathology may be associated with inappropriate amount of renal MT and GSH.

Seasonal changes of body iron status determine cadmium accumulation in the wild bank voles.

The objective of this study was to examine relations between body iron (Fe) status and cadmium (Cd) accumulation in a small rodent, the bank vole, caught from the wild population in late autumn (November) and early spring (March). The concentrations of Fe in the liver, kidneys, and duodenum in the bank voles from the spring were only 30%, 60%, and 70%, respectively, of those found in the animals from the autumn. An analysis of hematocrit and hemoglobin content of blood showed no significant effect of the season, suggesting that the animals from the spring were not anemic. The exposure to dietary Cd (10 microg/g) for 7 days resulted in 70% higher accumulation of Cd in the liver and kidneys of the spring than autumn bank voles, and the concentration of Cd in the duodenum was 3.5 times higher in the spring animals, despite the fact that relative Cd intake was significantly higher in the autumn bank voles. The data indicate that seasonal changes of body Fe status occurring in the wild bank voles may influence tissue accumulation of Cd.

Free-ranging European bisons accumulate more cadmium in the liver and kidneys than domestic cattle in north-eastern Poland.

It has been shown that free-ranging big game animals accumulate several-fold more cadmium (Cd) in the liver and kidneys than domestic animals. To examine possible reasons for this difference, in the present work we determined the concentrations of Cd in the liver and kidney cortex of European bisons (n=23) from Bialowieza Forest (north-eastern Poland) and domestic cattle (n=15) from the same region; in addition, analyses of Cd in the grasses and soil as well as of soil pH were carried out. The accumulation of Cd in liver and kidney cortex of the female bisons correlated significantly with the age up to 7 years, but stabilized thereafter. The 7-12-year-old bisons had 2.14- and 2.25-fold higher concentrations of Cd in the liver and kidney cortex, respectively, than the age-matched domestic cattle. Notably, the Cd levels in the liver and kidneys of the 8-12-year-old cattle were comparable to those found in the 2-year-old and 4-6-year-old bisons, respectively. The content of Cd in the grasses from Bialowieza Forest appeared to be 2.1-fold higher than that in the plants from the pastures. Similarly, the concentration of water-extractable Cd in the soil was 2.7-fold greater in Bialowieza Forest than in the pastures, despite the fact that nitric acid-extractable Cd (total Cd) was similar in the soils from the two sites. The concentration of water-extractable Cd in the soil as well as the content of Cd in the grasses inversely correlated with soil pH, which appeared to be significantly lower in Bialowieza Forest. These data indicate that soil pH is probably responsible for the higher concentrations of Cd in the feed and tissues of bisons as compared with those of domestic cattle.

The effect of orally administered melatonin on tissue accumulation and toxicity of cadmium in mice.

The aim of this study was to determine whether an oral administration of melatonin, a known antioxidant, free radical scavenger and metal chelator, influences tissue accumulation and toxicity of cadmium (Cd) in mice exposed subchronically to the metal. The animals received drinking water containing 50 microg Cd/mL only or with additional 2, 4 or 6 microg/mL melatonin for 8 weeks. Melatonin co-treatment brought about a dose-dependent decrease in the renal, hepatic and intestinal Cd concentrations, and the renal and hepatic metallothionein levels followed a pattern similar to that of the Cd accumulation. Histopathological changes occurred only in the kidneys (glomerular swelling and focal tubular degeneration) in all mice from the Cd alone group. In mice co-treated with melatonin, only slight (2 microg/mL melatonin) or no damage (4 and 6 microg/mL melatonin) was seen. The Cd and melatonin treatments did not affect renal lipid peroxidation and iron concentration. These data indicate that orally administered melatonin together with Cd reduces tissue accumulation of this metal; in particular, the reduction of renal Cd accumulation by melatonin is probably responsible for the prevention of Cd-induced injury in this organ.

Hepatic and renal cadmium accumulation is associated with mass-specific daily metabolic rate in the bank vole (Clethrionomys glareolus).

Previous study has shown that photoperiod and age affect tissue accumulation of cadmium (Cd) in a small rodent, the bank vole. Since the body mass is also influenced by these factors, the present study was designed to determine whether mass-specific daily metabolic rate might be responsible for differential accumulation of Cd in the liver and kidneys of the short- and long-photoperiod bank voles as well as of the young and old animals. One- and five-month old male bank voles were held under short (8 h light/16 h dark) or long (16 h light/8 h dark) photoperiods and exposed to dietary Cd (100 microg/g) for 6 weeks. The bank voles raised under the short photoperiod and those injected subcutaneously with melatonin (7 micromol/kg/day) under the long photoperiod showed significantly higher concentrations of Cd in the liver (43-60%) and kidneys (40-47%) than the age-matched long-photoperiod animals. The old bank voles accumulated significantly less Cd in both organs than the young animals. These differences in Cd accumulation appeared not to be associated with the relative Cd intake. However, the hepatic and renal Cd levels followed a pattern similar to that of the mass-specific daily metabolic rate (or energy expenditure) and energy assimilation efficiency. These data indicate that mass-specific daily metabolic rate and energy assimilation efficiency (an indicative of digestive and absorptive processes) may be responsible for differential tissue Cd accumulation in the bank vole.

Melatonin increases tissue accumulation and toxicity of cadmium in the bank vole (Clethrionomys glareolus).

Recent study has shown that a short photoperiod increases the accumulation and toxicity of cadmium (Cd) in the bank vole as compared to a long photoperiod. Since many of the effects of photoperiod on physiological processes in small mammals are transduced by the pineal gland and its hormone melatonin, in this study the effect of subchronic melatonin injection (7 micromol/kg/day for 6 weeks) on the hepatic, renal and intestinal Cd accumulation in the bank voles raised under a long photoperiod and exposed to dietary Cd (0.9 micromol/g) was examined. Simultaneously, histological examinations of the liver and kidneys, and analyses of metallothionein (MT) and lipid peroxidation were carried out. Melatonin co-treatment brought about a significant increase in the hepatic (61%), renal (79%) and intestinal (77%) Cd concentrations as compared to those in the Cd alone group. However, the concentrations of MT in the liver and kidneys of the Cd + melatonin co-treated bank voles did not differ from those in the Cd alone group. Also, histopathological changes in the liver (infiltration of leukocytes) and kidneys (glomerular swelling and a focal tubular cell degeneration) as well as an increase (2-fold) in the renal lipid peroxidation occurred only in animals from the Cd + melatonin group. These data indicate that (1) subchronic melatonin injection has similar effect on the tissue accumulation and toxicity of Cd to that produced by a short photoperiod and (2) the Cd-induced toxicity in the liver and kidneys of melatonin co-treated bank voles is probably due to increased Cd accumulation and decreased synthesis of MT.

Zinc protection from fluoride-induced testicular injury in the bank vole (Clethrionomys glareolus).

Previous work has shown that a high fluoride intake in rodents leads to histopathological changes in the germinal epithelium of testes that is associated with zinc deficiency. The purpose of this study was to determine whether supplemental dietary Zn would protect against testicular toxicity induced by fluoride in a small rodent, the bank vole. The 4-month exposure period to fluoride (200 microg/ml of drinking water) induced histopathological changes (hemorrhage in interstitium, necrosis and apoptosis in seminiferous tubule epithelium) which were accompanied by decreased testicular zinc concentration and increased lipid peroxidation. Supplemental dietary zinc (110-120 microg/g) together with fluoride treatment resulted in complete reversal of the fluoride-mediated effects. However, supplemented dietary Zn did not affect the accumulation of fluoride in the testes and bone. These data suggest that a zinc-enriched diet protects seminiferous tubules against fluoride toxicity by preventing the fluoride-induced testicular zinc deprivation.

Testicular toxicity induced by dietary cadmium is associated with decreased testicular zinc and increased hepatic and renal metallothionein and zinc in the bank vole (Clethrionomys glareolus).

Mechanism of testicular toxicity induced by dietary cadmium (Cd) has been less investigated than that following acute Cd injection. In the present study we characterized testicular injury in a small rodent, the bank vole, exposed subchronically to dietary Cd in a quantity of 0.9 micromol/g, and determined the importance of some factors (Cd accumulation, metallothionein (MT), oxidative stress, and zinc (Zn)) in the injury. Dietary Cd induced moderate histopathological changes (hemorrhage in interstitium, necrosis and apoptosis in seminiferous tubule epithelium) in young (1 month old) bank voles fed, for 6 weeks, Fe-adequate (1.1-1.4 micromol/g) and Fe-enriched (4.5-4.8 micromol/g) diets. In contrast, adult (5 months old) bank voles appeared to be resistant to the toxic effects of dietary Cd, despite the fact that testicular Cd contents were higher and MT levels lower than those in the young animals. The Cd-induced histopathological changes and apoptosis were accompanied by increased testicular lipid peroxidation, decreased testicular Zn concentration and elevated levels of hepatic and renal MT and Zn. Supplemental dietary Zn (1.7-1.8 micromol/g) prevented the Cd-induced testicular Zn depletion and injury. The data indicate that dietary Cd produces testicular lesions indirectly, through decreasing testicular Zn, which seems to be due to the sequestration of this element by the Cd-induced hepatic and renal MT.