Chromosomal segregation in sperm of the Robertsonian translocation (21;22) carrier and its impact on IVF outcome.

PURPOSE: To assess the variability of meiotic segregation patterns in sperm of Robertsonian translocation (RobT) carrier t(21;22) and present effect on reproductive outcome. METHODS: Infertile couple enrolled in IVF/ICSI program. Sperm chromosomal segregation analysis was done using FISH; preimplantation genetic testing for aneuploids (PGT-A) was performed by NGS. RESULTS: Patients had a low fertilization rate and a negative outcome after the first IVF/ICSI cycle, so they were advised to do chromosomal aberration analysis before their next attempt. The second IVF/ICSI procedure resulted in pregnancy, and two blastocysts were cryopreserved. The NIFTY test has shown low risk for all tested trisomies, sex chromosomes aneuploidis, and deletion syndromes, so a healthy female child was born. During pregnancy, karyotypisation results revealed that the male partner is a RobT carrier t(21;22). Sperm segregation analysis of chromosomes 21 and 22 has shown six types of sperm chromosome sets. The majority of sperm cells had a normal/balanced RobT form of a haploid set of chromosomes (68.5-76%) called an "alternate." Sperm cells that had additional chromosome 21 or 22, or lack of chromosome 21 or 22, were present in 4-12%. PGT-A performed on two cryopreserved blastocysts revealed one embryo euploid and the other with the mosaic aneuploidy of chromosome 7 present in 50% of the cells. CONCLUSION: Infertile couples with a RobT male carrier who have semen comprising of normal/alternate form in the majority have a good prognosis of IVF/ICSI outcome. PGT is recommended because of the possible occurrence of viable trisomic embryos and potential interchromosomal effect.

Biochemical responses of Lemna minor experimentally exposed to cadmium and zinc.

The effects of 5 µM cadmium (Cd), a non-essential toxic element and 25 and 50 µM zinc (Zn), an essential micronutrient, were investigated in aquatic plant Lemna minor L. after 4 and 7 days of exposure to each metal alone or to their combinations. Both metals showed tendency to accumulate with time, but when present in combination, they reduced uptake of each other. Cd treatment increased the lipid peroxidation and protein oxidation indicating appearance of oxidative stress. However, Zn supplementation in either concentration reduced values of both parameters, while exposure to Zn alone resulted in elevated level of lipid peroxidation and protein oxidation but only on the 7th day. Enhanced DNA damage, which was found on the 4th day in plants treated with Cd alone or in combination with Zn, was reduced on the 7th day in combined treatments. Higher catalase activity obtained in all treated plants on the 4th day of experiment was reduced in Zn-treated plants, but remained high in plants exposed to Cd alone or in combination with Zn after 7 days. Cd exposure resulted in higher peroxidase activity, while Zn addition prominently reduced peroxidase activity in the plants subjected to Cd stress. In conclusion, Cd induced more pronounced oxidative stress and DNA damage than Zn in applied concentrations. Combined treatments showed lower values of oxidative stress parameters--lipid peroxidation, protein oxidation and peroxidase activity as well as lower DNA damage, which indicates alleviating effect of Zn on oxidative stress in Cd-treated plants.

DNA integrity of chub erythrocytes (Squalius cephalus L.) as an indicator of pollution-related genotoxicity in the River Sava.

An alkaline comet assay and a micronucleus test were carried out on erythrocytes of the European chub, Squalius cephalus L., collected in spring and autumn in 2005 and 2006 at three sampling sites in River Sava, near Zagreb, Croatia. The results of comet assay showed the lowest genotoxic influence at the least polluted site, while higher DNA damage was observed at the polluted sites. Although the basal levels of DNA damage were elevated, a clear gradation of DNA damage was found due to pollution intensity in all sampling periods. The lowest cytogenetic damage as revealed by the micronucleus test (MNT) was observed as well at the least polluted site. High variations in MN frequency were observed between sampling periods, although the number of micronucleated erythrocytes was consistently the highest one at the polluted site. The comet assay as a biomarker of genotoxic effect exhibited higher sensitivity in discriminating the genotoxic capacity of studied polluted sites while the MNT was less sensitive. However, both tests should be used together in biomonitoring studies because they can reveal different aspects of DNA damage; comet assay, the early event of genotoxic exposure, and MNT, its final result as a mutagenic potential.

Ecotoxicological assessment of industrial effluent using duckweed (Lemna minor L.) as a test organism.

This study aimed at assessing the toxic effects of industrial effluents using duckweed (Lemna minor L.) plants as a test system. Growth inhibition test according to standardized protocol (ISO 20079) was performed. The suitability of the Comet assay (indicates DNA damage) and certain parameters such as peroxidase activity and lipid peroxidation level, as biomarkers for environmental monitoring was evaluated. The water samples were collected monthly over a 3-month period from the stream near the industrial estate of Savski Marof, Croatia. All samples caused inhibition of growth rates based on frond number and biomass as well as decrease of chlorophylls content. In contrast, peroxidase activity, malondialdehyde content and tail extent moment (measure of DNA strand breaks) markedly increased. Obtained data demonstrate the relevance of duckweed as sensitive indicators of water quality as well as the significance of selected biological parameters in the reliable assessment of phyto- and genotoxic potential of complex wastewaters.

Genotoxicity monitoring of freshwater environments using caged carp (Cyprinus carpio).

The present study deals with genotoxicity assessment of freshwaters using caged carp (Cyprinus carpio). Carps were transplanted from a fish-farm to three differently polluted sites in eastern Croatia. Two polluted sites were situated in the river Drava, downstream from the cities of Belisce and Osijek, while the reference site was in the Nature Park Kopacki rit, a preserved wetland area with limited anthropogenic influence. Exposure lasted for 3 weeks and was repeated for 3 years (2002-2004). DNA damage was assessed in erythrocytes of the exposed animals by the Comet assay and micronucleus test (MNT). In order to evaluate possible differences in stress responses to polluted water in situ and in aquaria a laboratory exposure was performed with water from the studied location in the second year of the study. Carp from the sites with high anthropogenic influence (Belisce and Osijek) had higher average DNA damage as expressed in both the MNT and Comet assay. Of the two, the Comet assay appeared to be more sensitive following both caging and aquaria exposures. The results from this study suggest that 3 weeks caging exposure of C. carpio may be a useful strategy to monitor for genotoxic agents in freshwater ecosystems.

miRNAs: EBV Mechanism for Escaping Host's Immune Response and Supporting Tumorigenesis.

Epstein-Barr virus (EBV) or human herpesvirus 4 (HHV-4) is a ubiquitous human oncogenic virus, and the first human virus found to express microRNAs (miRNAs). Its genome contains two regions encoding more than 40 miRNAs that regulate expression of both viral and human genes. There are numerous evidences that EBV miRNAs impact immune response, affect antigen presentation and recognition, change T- and B-cell communication, drive antibody production during infection, and have a role in cell apoptosis. Moreover, the ability of EBV to induce B-cell transformation and take part in mechanisms of oncogenesis in humans is well known. Although EBV infection is associated with development of various diseases, the role of its miRNAs is still not understood. There is abundant data describing EBV miRNAs in nasopharyngeal carcinoma and several studies that have tried to evaluate their role in gastric carcinoma and lymphoma. This review aims to summarize so far known data about the role of EBV miRNAs in altered regulation of gene expression in human cells in EBV-associated diseases.

Effects of radiofrequency electromagnetic fields on seed germination and root meristematic cells of Allium cepa L.

The effects of exposure to radiofrequency electromagnetic fields (RF-EMFs) on seed germination, primary root growth as well as mitotic activity and mitotic aberrations in root meristematic cells were examined in Allium cepa L. cv. Srebrnjak Majski. Seeds were exposed for 2h to EMFs of 400 and 900MHz at field strengths of 10, 23, 41 and 120Vm(-1). The effect of longer exposure time (4h) and field modulation was investigated at 23Vm(-1) as well. Germination rate and root length did not change significantly after exposure to radiofrequency fields under any of the treatment conditions. At 900MHz, exposures to EMFs of higher field strengths (41 and 120Vm(-1)) or to modulated fields showed a significant increase of the mitotic index compared with corresponding controls, while the percentage of mitotic abnormalities increased after all exposure treatments. On the other hand, at 400MHz the mitotic index increased only after exposure to modulated EMF. At this frequency, compared with the control higher numbers of mitotic abnormalities were found after exposure to modulated EMF as well as after exposure to EMFs of higher strengths (41 and 120Vm(-1)). The types of aberration induced by the EMFs of both frequencies were quite similar, mainly consisting of lagging chromosomes, vagrants, disturbed anaphases and chromosome stickiness. Our results show that non-thermal exposure to the radiofrequency fields investigated here can induce mitotic aberrations in root meristematic cells of A. cepa. The observed effects were markedly dependent on the field frequencies applied as well as on field strength and modulation. Our findings also indicate that mitotic effects of RF-EMF could be due to impairment of the mitotic spindle.

Oxidative stress and DNA damage in broad bean (Vicia faba L.) seedlings induced by thallium.

Thallium (Tl) is a metal of great toxicological concern because it is highly toxic to all living organisms through mechanisms that are yet poorly understood. Since Tl is accumulated by important crops, the present study aimed to analyze the biological effects induced by bioaccumulation of Tl in broad bean (Vicia faba L.) as well as the plant's antioxidative defense mechanisms usually activated by heavy metals. Thallium toxicity was related to production of reactive oxygen species in leaves and roots of broad bean seedlings following short-term (72 h) exposure to thallium (I) acetate (0, 0.5, 1, 5, and 10 mg/L) by evaluating DNA damage and oxidative stress parameters as well as antioxidative response. The possible antagonistic effect of potassium (K) was tested by combined treatment with 5 mg/L of Tl (Tl+) and 10 mg/L of potassium (K+) acetate. Accumulation of Tl+ in roots was 50 to 250 times higher than in broad bean shoots and was accompanied by increase in dry weight and proline. Despite responsive antioxidative defense (increased activities of superoxide dismutase, ascorbate peroxidase, and pyrogallol peroxidase), Tl+ caused oxidative damage to lipids and proteins as evaluated by malondialdehyde and carbonyl group levels, and induced DNA strand breaks. Combined treatment caused no oxidative alternations to lipids and proteins though it induced DNA damage. The difference in Tl-induced genotoxicity following both acellular and cellular exposure implies indirect DNA damage. Results obtained indicate that oxidative stress is involved in the mechanism of Tl toxicity and that the tolerance of broad bean to Tl is achieved, at least in part, through the increased activity of antioxidant enzymes.

Assessment of genotoxicity in polluted freshwaters using caged painter's mussel, Unio pictorum.

This study was undertaken to evaluate the applicability of caged painter's mussel, Unio pictorum for freshwater environmental genotoxicity assessment. Mussels in cages were exposed for 3 weeks in 2002-2004 to polluted sites in two large rivers in the Croatia, the Sava and Drava, and on the respective reference sites. DNA damage was assessed in haemocytes of the exposed mussels by the comet and micronucleus assays. Both assays provided good discriminative power between polluted and control sites and showed the same gradation of sites according to their genotoxic properties, with high concordance between investigated years. Background levels of the DNA damage in haemocytes of painter's mussels are defined for both assays for easier detection of contamination-related genotoxicity. U. pictorum is found to be a very suitable sentinel species, sufficiently sensitive to the impact of pollution but at the same time unsusceptible to stress caused by translocation or cage exposure.

Persistence of DNA damage in the freshwater mussel Unio pictorum upon exposure to ethyl methanesulphonate and hydrogen peroxide.

An important endpoint in assessing pollution-related toxicity is genotoxicity. To obtain insight into the time-course of oxidative- and alkylation-induced DNA damage in the freshwater mussel, Unio pictorum, mussels were exposed for 24 hr to concentration gradients of pro-oxidant hydrogen peroxide (H(2)O(2)) and a mono-functional alkylating agent, ethyl methanesulfonate (EMS). DNA damage was assessed in haemocytes immediately upon exposure and over the recovery period of up to 72 days by means of comet and micronucleus assays. Following exposure to H(2)O(2), DNA damage as detected by the comet assay returned to control values after one day, except for the mussels exposed to the highest dose when damage was detectable for the next 3 days. In contrast, alkylation-induced DNA damage was detectable even after 72 days of recovery in de-chlorinated water, with a dose-response relationship observable throughout the whole recovery period. Micronucleus frequency was the highest on Day 3 after exposure to EMS; it decreased considerably by Day 7 and returned almost to the control levels 19 days after exposure, while no significant induction of micronuclei was observed in mussels exposed to H(2)O(2). Although the comet assay is considered a biomarker of recent genotoxic exposure, detecting DNA damage of shorter longevity than with the micronucleus assay, results presented here show that in the case of alkylation damage the comet assay reveals genotoxic exposure of U. pictorum in a dose-dependent manner even after 2 months.

Detection of DNA damage in haemocytes of Mytilus galloprovincialis in the coastal ecosystems of Kastela and Trogir bays, Croatia.

Coastal waters are burdened with different contaminants of anthropogenic origin due to intensive urbanisation and economical development. Bays, semi-enclosed areas with limited water renewal ability, are particularly endangered by contaminant inputs. Kastela Bay (Dalmatia, Eastern Adriatic) has earlier been identified as an area loaded with diffuse sources of pollution, including genotoxic agents. However, there is lack of data on the effects of these contaminants on the local marine fauna. The aim of this study was to assess genotoxic impacts in Kastela Bay and the neighbouring Trogir Bay using the micronucleus test and Comet assay with mussel (Mytilus galloprovincialis) haemocytes. Native and caged mussels were included in the studies. Our results confirmed that mussels in Kastela and Trogir Bays are affected by genotoxic contaminants. In addition to mussels from the most known polluted site (Vranjic), there was evidence for genotoxic effects in mussels collected at other locations. The response in the micronucleus test and the Comet assay differed somewhat between sites, the latter apparently being more sensitive, but the two methods complement each other and it is therefore desirable to use them both in monitoring the impacts of genotoxic pollution in coastal waters.

Application of the micronucleus and comet assays to mussel Dreissena polymorpha haemocytes for genotoxicity monitoring of freshwater environments.

Assessment of DNA damage is of primary concern when determining the pollution-related stress in living organisms. To monitor genotoxicity of the freshwater environments we used micronucleus (MN) and comet assay on Dreissena polymorpha haemocytes. Caged mussels, collected from the river Drava, were transplanted to four monitoring sites of different pollution intensity in the river Sava. Exposition lasted for a month. The baseline level of MN frequencies in the haemocytes of mussels from reference site (river Drava) was 0.5 per thousand. No increase in MN frequency was found in mussels from the medium-polluted site (Zagreb) in the river Sava while other, more polluted sites showed higher MN frequencies ranging from 2.7 per thousand (Lukavec) and 3.1 per thousand (Oborovo) to 5.2 per thousand (Sisak). Results from comet assay showed concordance with MN assay in indicating intensity of DNA damage. The use of haemocytes from caged, non-indigenous mussels in MN and comet assay proved to be a sensitive tool for the freshwater genotoxicity monitoring.

The effects of cadmium-zinc interactions on biochemical responses in tobacco seedlings and adult plants.

The objective of the present study was to investigate the effects of cadmium-zinc (Cd-Zn) interactions on their uptake, oxidative damage of cell macromolecules (lipids, proteins, DNA) and activities of antioxidative enzymes in tobacco seedlings as well as roots and leaves of adult plants. Seedlings and plants were exposed to Cd (10 µM and 15 µM) and Zn (25 µM and 50 µM) as well as their combinations (10 µM or 15 µM Cd with either 25 µM or 50 µM Zn). Measurement of metal accumulation exhibited that Zn had mostly positive effect on Cd uptake in roots and seedlings, while Cd had antagonistic effect on Zn uptake in leaves and roots. According to examined oxidative stress parameters, in seedlings and roots individual Cd treatments induced oxidative damage, which was less prominent in combined treatments, indicating that the presence of Zn alleviates oxidative stress. However, DNA damage found in seedlings, and lower glutathione reductase (GR) and superoxide dismutase (SOD) activity recorded in both seedlings and roots, after individual Zn treatments, indicate that Zn accumulation could impose toxic effects. In leaves, oxidative stress was found after exposure to Cd either alone or in combination with Zn, thus implying that in this tissue Zn did not have alleviating effects. In conclusion, results obtained in different tobacco tissues suggest tissue-dependent Cd-Zn interactions, which resulted in activation of different mechanisms involved in the protection against metal stress.

Thallium toxicity in humans.

Thallium is a naturally occurring trace element, widely distributed in the earth's crust, but at very low concentrations. It does not have a known biological use and does not appear to be an essential element for life. It has been considered one of the most toxic heavy metals.Occasionally, there are reports on thallium poisoning as results of suicide or murder attempt or accident. The main threat to humans is through occupational exposure, environmental contamination, and accumulation in food, mainly in vegetables grown on contaminated soil. Increasing use in emerging new technologies and demanding high-tech industry constantly raise concern about exposure risk to all living organisms. Thallium is considered a cumulative poison that can cause adverse health effects and degenerative changes in many organs. The effects are the most severe in the nervous system. The exact mechanism of thallium toxicity still remains unknown, although impaired glutathione metabolism, oxidative stress, and disruption of potassium-regulated homeostasis may play a role. The lack of data about mutagenic, carcinogenic, or teratogenic effects of thallium compounds in humans calls for further research.

Cage exposure of European sea bass (Dicentrarchus labrax) for in situ assessment of pollution-related genotoxicity.

Genotoxic effects are often the earliest signs of pollution-related environmental disturbance. In this study, we used the comet assay and micronucleus test to assess DNA damage in the erythrocytes of the European sea bass (Dicentrarchus labrax) exposed to environmental pollution in situ. Fish were collected from a fish farm in the Trogir Bay and their cages placed at an unpolluted reference site Solta (Necujam Bay) and a polluted site Vranjic (Kastela Bay) for four weeks. A group of fish which remained at the fish farm Trogir Bay were used as the second control group. Fish exposed at the Vranjic site showed a significantly higher erythrocyte DNA damage, measured by the comet assay, than either control group. Micronucleus induction showed a similar gradient of DNA damage, but did not reach statistical significance. Our results show that cage exposure of a marine fish D. labrax can be useful in environmental biomonitoring and confirm the comet assay as a suitable tool for detecting pollution-related genotoxicity.

Effect of copper on the toxicity and genotoxicity of cadmium in duckweed (lemna minor L.).

We investigated interactions between copper (in the concentrations of 2.5 µmol L-1 and 5 µmol L-1) and cadmium (5 µmol L-1) in common duckweed (Lemna minor L.) by exposing it to either metal or to their combinations for four or seven days. Their uptake increased with time, but it was lower in plants treated with combinations of metals than in plants treated with either metal given alone. In separate treatments, either metal increased malondialdehyde (MDA) level and catalase and peroxidase activity. Both induced DNA damage, but copper did it only after 7 days of treatment. On day 4, the combination of cadmium and 5 µmol L-1 copper additionally increased MDA as well as catalase and peroxidase activity. In contrast, on day 7, MDA dropped in plants treated with combinations of metals, and especially with 2.5 µmol L-1 copper plus cadmium. In these plants, catalase activity was higher than in copper treated plants. Peroxidase activity increased after treatment with cadmium and 2.5 µmol L-1 copper but decreased in plants treated with cadmium and 5 µmol L-1 copper. Compared to copper alone, combinations of metals enhanced DNA damage after 4 days of treatment but it dropped on day 7. In conclusion, either metal given alone was toxic/genotoxic and caused oxidative stress. On day 4 of combined treatment, the higher copper concentration was more toxic than either metal alone. In contrast, on day 7 of combined treatment, the lower copper concentration showed lower oxidative and DNA damage. These complex interactions can not be explained by simple antagonism and/or synergism. Further studies should go in that direction.