Oxidative Stress and Neurotoxicity of Cadmium and Zinc on Artemia franciscana.

Despite not being redox-active metals, Cd and Zn can disrupt cellular redox homeostasis by acting pro-oxidatively. The aim of this study was to examine the effects of exposure to Zn (14 and 72 mg/L) and Cd (7.7 and 77 mg/L) for 24 and 48 h on oxidative and antioxidative parameters and the activity of glutathione-S-transferase in Artemia franciscana tissue. In addition, the neurotoxicity of the metals was examined by determining the activity of acetylcholinesterase (AChE). In A. franciscana tissue, Cd (0.0026 ± 0.0001 mg/L) was detected only after 48 h of exposure to 77 mg/L Cd. After 24 h, the 14- and 72-mg/L Zn treatments resulted in significant increases in the Zn concentration (0.54 ± 0.026 mg/L (p < 0.01) and 0.68 ± 0.035 (p < 0.0001), respectively) in A. franciscana tissue compared with the control level, and significant increases were also detected after 48 h (0.59 ± 0.02 (p < 0.0001) and 0.79 ± 0.015 (p < 0.0001), respectively). The malondialdehyde (MDA) concentration in the metal-treated samples was increased after 24 h of exposure, whereas after 48 h, an increase in the MDA concentration was detected only with 7.7. mg/L Cd. A significant increase in the H2O2 concentration after 24 h was measured only after treatment with 72 mg/L Zn. The treatment with 7.7 mg/L Cd for 24 h induced a significant increase in the AChE activity, whereas 48 h of treatment with 77 mg/L Cd and 14 mg/L Zn significantly inhibited AChE. The results indicate that lipid peroxidation resulting from metal toxicity may constitute the basis of neurotoxicity.

Using Chemometric Analyses for Tracing the Regional Origin of Multifloral Honeys of Montenegro.

This is the first study of mineral content and basic physicochemical parameters of honeys of Montenegro. We examined honey samples from eight different micro-regions of Montenegro, and the results confirm that, with the exception of cadmium in samples from two regions exposed to industrial pollution, none of the 12 elements analyzed exceeded the maximum allowable level. The samples from areas exposed to industrial pollution were clearly distinguished from samples from other regions of Montenegro in the detectable contents of Pb, Cd, and Sr. This study showed that chemometric techniques might enhance the classification of Montenegrin honeys according to their micro-regional origin using the mineral content. Linear discriminant analysis revealed that the classification rate was 79.2% using the cross-validation method.

Environmental factors affecting water mite assemblages along eucrenon-hypocrenon gradients in Mediterranean karstic springs.

Springs are often recognized as biodiversity hotspots on the regional scale but at the same time they are among the most endangered freshwater habitats. Water mites are among the aquatic animal groups with highest share of crenobiotic (= spring-dwelling) species and, therefore, are possibly the best indicators of the ecological status of spring habitats. We studied water mites and environmental factors correlated with their distribution pattern along a eucrenon-hypocrenon gradient. The sampling was conducted in 14 karstic springs located in the Mediterranean part of Montenegro. We collected 17 water mite species of which four species were crenobiotic. We did not find significant differences between the water mite assemblages from the source and springbrook. Similarly, there were no significant differences in number of species and abundance between the studied spring sections, neither for crenobiotic taxa nor for non-crenobiotic taxa. We found that the number of non-crenobiotic taxa was predicted mainly by water depth, whereas the abundance of crenobionts was most strongly associated with temperature. No significant predictors for the number of crenobiotic species in spring habitats were found. Our results revealed also that distance from the nearby water body was the main driver of the crenobiotic species abundance in eucrenon suggesting the large effects of the local flooding events on crenobiotic species. Water mites may help in assessing the response of crenobiotic assemblages in those spring habitats which likely to be flooded in future as the results of ongoing climatic changes.

An environmentally relevant concentration of titanium dioxide (TiO2) nanoparticles induces morphological changes in the mouthparts of Chironomus tentans.

The present study was carried out in order to assess the influence of environmentally relevant concentrations of TiO2 nanoparticles (E171 human food grade) toxicity on the freshwater midge Chironomus tentans. Tested concentrations were 125, 250, 500, 1000, 2000 and 4000 mg of E171 TiO2 per 1 kg of sediment, for the experiment aiming at life trait toxicity observation; and 2.5, 25 and 250 mg of E171 TiO2 per 1 kg of sediment for the experiment aiming at mouthpart deformity observation. The experimental design was constructed for the sediment dwelling chironomid larvae according to OECD guidelines. For the first time, a geometric morphometric approach was used to assess the deformities in chironomid larvae as sublethal implications of nanoparticle exposure. The present study showed a concentration-response relationship between the TiO2 concentration in the substrate and the TiO2 intake. The mortality and emergence ratio was affected at concentrations >1000 mg/kg. Geometric Morphometrics revealed the tendency of the mentum teeth to narrow and elongate and the mandibles to widen, as well as the loss of the first inner tooth, with a TiO2 concentration rise. The variability of morphological changes observed in the mouthparts indicates that C. tentans could be used as a bioindicator in nano-TiO2 monitoring.

In situ effects of titanium dioxide nanoparticles on community structure of freshwater benthic macroinvertebrates.

For the first time in the current literature, the effect of titanium dioxide (TiO2) nanoparticles on the community structure of macroinvertebrates has been investigated in situ. Macroinvertebrates were exposed for 100 days to an environmentally relevant concentration of TiO2 nanoparticles, 25 mg kg(-1) in sediment. Czekanowski's index was 0.61, meaning 39% of the macroinvertebrate community structure was affected by the TiO2 treatment. Non-metric multidimensional scaling (NMDS) visualized the qualitative and quantitative variability of macroinvertebrates at the community level among all samples. A distance-based permutational multivariate analysis of variance (PERMANOVA) revealed the significant effect of TiO2 on the macroinvertebrate community structure. The indicator value analysis showed that the relative frequency and abundance of Planorbarius corneus and Radix labiata were significantly lower in the TiO2 treatment than in the control. Meanwhile, Ceratopogonidae, showed a significantly higher relative frequency and abundance in the TiO2 treatment than in the control.

Telomere shortening and immune activity in war veterans with posttraumatic stress disorder.

BACKGROUND: There is increasing evidence that chronic stress accelerates telomere erosion in leukocytes/peripheral blood mononuclear cells (PBMCs). However, functional changes associated with telomere shortening are poorly understood. We hypothesized that war veterans with PTSD would have shorter telomeres in PBMCs and that these cells might exhibit changes in measures of immune reactivity such as proliferation, cytokine production and expression of regulators of immune responses. METHODS: We measured relative telomere length and basal telomerase activity in PBMCs of 62 individuals (PTSD patients (N=30); age-matched healthy controls (N=17), elderly volunteers (N=15)). In parallel, we have assessed proliferation of activated T cells, interferon (IFN)-γ, interleukin (IL)-2, IL-4, tumor necrosis factor (TNF)-α and IL-6 cytokine production and expression of programmed death 1 (PD-1) receptor and its ligand PD-L1 on activated T cells. RESULTS: Middle-aged war veterans with current PTSD had shorter PBMC telomere length than their age-matched healthy controls while the elderly had the shortest telomeres. There was no difference in telomerase activity between PTSD patients and healthy controls while telomerase activity was significantly lower in the elderly. While the elderly group exhibited robust changes in immune activity such as increased production of proinflammatory cytokines (TNF-α, IL-6) and reduced proliferation of all T cells, the PTSD group showed reduced proliferative response of CD8(+) T cells to high concentrations of mitogen and reduced spontaneous production of IL-2 and IFN-γ. CONCLUSIONS: This study adds to the accumulating evidence that psychological trauma and chronic stress are associated with accelerated telomere attrition. However, changes in immune function associated with stress-related telomere shortening are not well understood. Although much less pronounced in PTSD patients than in elderly persons, reduced proliferative responses of T cells accompanied by shorter telomeres might be a sign of early immunosenescence. Together with reduced production of Th1 cytokines, observed immune changes may contribute to health risks associated with PTSD.

Patients with posttraumatic stress disorder exhibit an altered phenotype of regulatory T cells.

BACKGROUND: Regulatory T cells (Tregs) play a key role in immune homeostasis in vivo. Tregs have a critical role in preventing the development of autoimmune diseases and defects in Treg function are implicated in various autoimmune disorders. Individuals with posttraumatic stress disorder (PTSD) have higher prevalence of autoimmune disorders than the general population. We hypothesized that war veterans with PTSD would exhibit a decreased number and/or altered phenotype of Tregs. METHODS: We analyzed peripheral blood mononuclear cells (PBMCs) of patients with PTSD (N = 21) (mean age = 45.9) and age-matched healthy controls (N = 23) (mean age = 45.7) to determine the proportion of Tregs and their phenotype according to the expression of CD127 and HLA-DR markers which describe the differentiation stages of Tregs. In addition, we analyzed the expression of membrane ectoenzyme CD39 on Tregs of the study groups, an important component of the suppressive machinery of Tregs. RESULTS: We found no differences in the proportion of Tregs between PTSD patients and controls, but PTSD patients had a higher percentage of CD127(-)HLA-DR(-) Tregs and a lower percentage of CD127(lo)HLA-DR(+) Tregs compared to controls. There was no difference in expression of CD39 on Tregs of the study groups. CONCLUSIONS: Although the proportions of Tregs in PTSD patients were unchanged, we found that they exhibit a different phenotype of Tregs that might be less suppressive. Impaired differentiation and function of Tregs is likely involved in disruption of immune homeostasis in PTSD.

High frequency of the R75Q CFTR variation in patients with chronic obstructive pulmonary disease.

We performed the complete screening of the CFTR gene in a group of 31 patients with COPD in order to investigate the impact of mutations and polymorphisms in the CFTR gene. The cumulative frequency of CFTR mutations (17.74%) was significantly higher than in our general population (P < 0.0001). The R75Q was significantly overrepresented in COPD patients (8.06%; P = 0.002). In all patients carrying the R75Q chronic bronchitis was a dominant symptom of COPD, and all were homozygous for the V470 allele. These findings suggest that R75Q mutation could be characteristic CFTR variant for COPD patients.

[Mutations and polymorphisms in CFTR genes in infertile men with oligospermia or azoospermia]. / Mutacije i polimorfizmi u genu CFTR kod infertilnih muskaraca s oligospermijom ili azospermijom.

INTRODUCTION: Impaired infertility of the male partner is causative or contributOry to in up to one half of all couples unable to conceive spontaneously. A considerable number of genes are now known that have an essential function in human reproduction and which, when deleted or mutated, can cause pathologic changes in the male reproductive system. Congenital bilateral absence of the vas deferens (CBAVD) is an important cause of obstructive azoospermia in otherwise healthy men. It is also present in 95% of men with an autosomal recessive systematic disease--cystic fibrosis. However, clinically affected CF patients present a spectrum of genital phenotypes ranging from normal fertility to severely impaired spermatogenesis and CBAVD. Cystic fibrosis and most cases of CBAVD are caused by mutations in CFTR (cystic fibrosis transmembrane conductance regulator) gene. The aim of this study was to test the possible involvement of the CFTR gene in the aetiology of male infertility other than CBAVD. METHODS: Twenty one infertile men with oligo or azoospermia were analysed for the presence of mutations and polymorphisms in the CFTR gene. Patients were divided in two groups according to the spermatogram: 1) patients with obstructive azoospermia (V < 2 mL, pH < 7.2, low level of a-glucosidase and fructose and absence of spermatozoa; 2) patients with impaired spermatogenesis or sperm maturation. We performed direct detection for the following mutations: delta F508 and delta 1507 (heteroduplex analysis), 621 + 1 G-->T, and N1303K (PSM--PCRmediated site-specific mutagenesis), A455E, 1717-1 G-->A, S549N, R560T, W1282X, R334W, R347P, R117H, 3849 + 10 kb C-->T and Tn, F508C, 1507V, 1506V polymorphisms (reverse dot blot method). G542X, R553X and GSS1D mutations were tested by SSCP (Single Strand Conformation Polymorphism). We also performed indirect detection of mutations and polymorphisms in 3, 5, 6a, 8, 9, 11, 12, 14a, 14b, 15, 17b, 18, 20, 21 and 23 exons by DGGE (Denaturant Gradient Gel Electrophoresis). Differences between frequencies were tested by chi-square statistic, p values of less than 0.05 were considered statistically significant. RESULTS: Among 42 chromosomes from infertile men with oligo or azoospermia we detected 7 mutations in CFTR gene (16.7%), which was significantly (p = 0.0319) more frequent than in general population (2%). Frequency of 5T allele in analysed group was high (11.9%) compared to general population (5%), but not statistically significant (0.0938). The most common mutation in the group of 10 men with obstructive azoospermia was delta F508. It was detected on one chromosome in five patients. In three of these patients with 4F508 mutation on the other chromosome we found 5T allele on polymorphic Tn locus. In one patient, heterozygous for delta F508 mutation, 711 + 3 A-->G mutation on the other chromosome was detected. In the group of 11 infertile men with impaired spermatogenesis or sperm maturation we detected one mutation--delta F508. Two patients from this group had 5T variant on one chromosome. DISCUSSION: We analysed 21 infertile men with oligo or azoospermia not caused by endocrine or inflammatory character, or chromosome mutations. Within this group frequency of CFTR mutations was increased compared to general population (p = 0.0319), suggesting that CFTR gene may be involved in the aetiology of infertility in men with oligo or azOospermia. In the group of patients with obstructive azoospermia 50% had at least one mutation, but only 10% had mutations in both chromosomes. One of the possible explanations would be that mutations are in the promoter region, introns or exons that were not included in analyses. The second explanation could be that some cases of obstructive azoospermia are only partially (or not) related to CFTR gene. In the group of patients with impaired spermatogenesis or sperm maturation, the frequencies of CFTR mutations and 5T allele were also increased compared to general population, but lower than in the group with obstructive azoospermia. This fact could mean that the influence of some other genes is higher in this condition than in the case of obstructive azoospermia. CONCLUSION: We concluded that CFTR gene plays a role in the aetiology of obstructive azoospermia and that is also could be involved in some cases of impaired spermatogenesis and sperm maturation. Due to the high incidence of CFTR mutations in patients with obstructive azoospermia we suggest screening of CFTR mutations before assisted reproduction.