Wojciech Wąsowicz

Polish mother and child cohort study — defining the problem, the aim of the study and methodological assumptions

OBJECTIVES Exposures during prenatal period have implications for pregnancy outcome as well as for childrens health, morbidity and mortality. Prospective cohort study design allows for the identification of exposures that may influence pregnancy outcome and childrens health, verification of such exposures by biomarker measurements and notification of any changes in exposure level. MATERIALS AND METHODS Polish Mother and Child Cohort Study (REPRO_PL) is multicenter prospective cohort study conducted in 8 different regions of Poland. The final cohort is intended to comprise 1300 mother-child pairs to be recruited within 4-year period (2007-2011). The recruitment and all scheduled visits are conducted in maternity units or clinics in the districts included in the study. The women are followed-up 3 times in pregnancy (once in each trimester) and after delivery for the notification of pregnancy outcome. During each visit, detailed questionnaire and biological samples are collected including saliva, urine, hair, maternal blood and cord blood. About 6 weeks postpartum, breast milk from part of the women is collected. The study concentrates on the identification and evaluation of the effects of prenatal environmental exposure on pregnancy outcome and childrens health. Specific research hypotheses refer to the role of heavy metals, exposure to polycyclic aromatic hydrocarbons (PAHs) and environmental tobacco smoke (ETS) in the aetiology of small-for-gestational-age (SGA) and preterm delivery (PD). The role of oxidative stress putative mechanism and pregnant women nutritional status will be investigated. Based on questionnaire data, the impact of occupational exposures and stressful situations will be evaluated. RESULTS The results of the study will become available within the next few years and will help to determine levels of child prenatal exposure in several areas of Poland and its impact on course and outcome of pregnancy.

Hypermethylation of p16 and DAPK promoter gene regions in patients with non-invasive urinary bladder cancer

Introduction The aim of the study was to examine the frequency of methylation status in promoter regions of p16 and DAPK genes in patients with non-invasive bladder cancer. Material and methods Forty-two patients (92.9% men, 73.8% smokers, 71.4% T1G1, 19.1% T1G2, 9.5% T1G3) and 36 healthy controls were studied. Isolation of genomic DNA from blood serum and methylation-specific PCR (MSP) were applied. Methylation status – methylated and unmethylated promoter regions of p16 and DAPK genes were analysed. Results Seventeen out of 42 patients (40.5%) had the methylated p16 gene, while methylation of the DAPK gene was seen in 27 of 42 cases (64.3%). In 12 patients (28.6%) both analysed genes were methylated. A statistically significant (p = 0.046) higher frequency of DAPK gene methylation (71.4%) was observed in patients with lower grade (G1) bladder cancer. Conclusions Detection of the aberrant hypermethylation of DAPK and p16 genes in blood DNA from non-invasive bladder cancer patients might offer an effective means for earlier auxiliary diagnosis of the malignancy.

Today’s oxidative stress markers

Oxidative stress represents a situation where there is an imbalance between the reactive oxygen species (ROS) and the availability and the activity of antioxidants. This balance is disturbed by increased generation of free radicals or decreased antioxidant activity. It is very important to develop methods and find appropriate biomarkers that may be used to assess oxidative stress in vivo. It is significant because appropriate measurement of such stress is necessary in identifying its role in lifestyle-related diseases. Previously used markers of oxidative stress, such as thiobarbituric acid reactive substances (TBARS) or malondialdehyde (MDA), are progressively being supplemented by new ones, such as isoprostanes (IsoPs) and their metabolites or allantoin. This paper is focusing on the presentation of new ones, promising markers of oxidative stress (IsoPs, their metabolites and allantoin), taking into account the advantage of those markers over markers used previously.

DNA damage induced by nitrous oxide: study in medical personnel of operating rooms.

Occupational exposure to anaesthetics such as nitrous oxide (N(2)O) and halogenated hydrocarbons has been suggested to increase risk of genetic damage. However, the dose-dependency of genotoxic effects has not been unequivocally established and their relation to occupational exposure limit (OEL) remain obscure. In this study, the genotoxicity associated with occupational exposure to anaesthetics has been investigated in a group of 55 female nurses and 29 male anaesthesiologists active for at least 5 years in a working environment containing variable concentrations of N(2)O and halogenated hydrocarbons. 83 unexposed health care workers (52 female nurses and 31 male doctors) matched for age, gender, smoking habit and employment duration were included in the control group. Genotoxicity has been assessed using comet test. Concentrations of nitrous oxide, sevoflurane and isoflurane monitored by gas chromatography and mass spectrometry made possible to relate the extent of DNA damage to the level of exposure. Our results for the first time document a positive correlation between the DNA damage and the N(2)O levels in the ambient air. By contrast, no correlation has been observed between genotoxic effects and concentrations of sevoflurane and isoflurane. The extent of genetic injury was especially aggravated among nurses and anaesthesiologists exposed to N(2)O in concentrations exceeding OEL (180 mg/m(3)). We conclude that occupational exposure to N(2)O is associated with increased DNA damage and that the level of exposure plays a critical role in this regard.

Glutathione peroxidase activity, selenium, and lipid peroxide concentrations in blood from a healthy Polish population : I. Maternal and cord blood.

Selenium (Se) concentrations in whole blood and plasma of 19 nonpregnant women. 14 mothers at delivery, 14 neonates, and 13 infants, aged 2–12 mo, were evaluated. The activity of glutathione peroxidase (GSH-Px) in erythrocytes and plasma and the level of lipid peroxides in plasma were also analyzed. Selenium concentrations in whole blood and plasma in mothers at delivery were significantly lower compared to nonpregnant women. Selenium concentrations in cord blood components were lower compared to mothers, but the differences were not significant. The concentration of the element decreased in the first few months of life. Glutathione peroxidase activity in erythrocytes differed only slightly in the examined groups. In plasma, however, the enzyme activity was significantly lower in pregnant compared to nonpregnant women and in neonates compared to their mothers. Lipid peroxide concentrations in plasma differed only slightly in the examined groups. The results obtained are discussed in terms of the observations of other investigators.

Role of selenium and zinc in the pathogenesis of food allergy in infants and young children

Introduction Selenium and zinc are indispensable microelements for normal functioning and development of the human body. They are cofactors of many enzymes of the antioxidative barrier (selenium – glutathione peroxidase; zinc – superoxide dismutase). The aim of the study was to evaluate the importance of selenium and zinc in the pathogenesis of food allergy in small children. Material and methods The study was performed in 134 children with food allergy, aged 1 to 36 months. The control group was composed of 36 children at the same age, without clinical symptoms of food intolerance. Each child had estimated serum levels of zinc and selenium. Furthermore, the authors evaluated activity of glutathione peroxidase (GSH-Px) in erythrocyte lysates and serum. Tests were performed twice, before and after 6-month administration of elimination diet. Results The obtained results showed that children with food allergy had significantly lower concentrations of selenium, zinc and examined enzymes in comparison to children from the control group. Concentration of selenium and zinc as well as activity of examined enzymes increased after application of eliminative diet. Conclusions In children with allergy decreased concentrations of selenium and zinc, and lower values of glutathione peroxidase and superoxide dismutase which increased after elimination diet were affirmed. These observations suggest their role in pathogenesis of food allergy. Conducted observations indicate the need to monitor trace elements content in the diet in children with food allergy. The results showed that children with food allergy had a weakened antioxidative barrier.

The role of zinc, copper, plasma glutathione peroxidase enzyme, and vitamins in the development of allergic diseases in early childhood: The Polish mother and child cohort study.

It has been hypothesized that the increase in allergic disorders may, in part, be a consequence of changing diet. The primary aim of this study was to assess the associations between occurrence of atopic dermatitis; food allergy; the incidence of wheeze inhaled glucocorticosteroid use in children during the 1st year of life; and cord blood concentrations of copper, zinc, vitamins (A and E), and glutathione peroxidase activity. We evaluated 240 1-year-old children from the Polish Mother and Child Cohort Study. Women were interviewed during pregnancy to collect demographic and socioeconomic data and medical and reproductive history. Exposure to tobacco constituents was assessed based on questionnaire data. At delivery, umbilical cord blood plasma was sampled. One year after the birth, the childs exposure and health status were examined. In the analyses a multivariable model was used. Higher zinc and copper concentrations in cord blood were associated with increased likelihood of wheezing in 1-year-old children. This effect was seen only among children exposed to tobacco smoke at home. We also showed significantly lower activity of glutathione peroxidase enzyme 3 in umbilical cord blood plasma of children with atopic dermatitis during the 1st year of life. There were no significant associations between vitamin A and E concentrations in plasma and childrens health. We showed imbalance in the antioxidant defense system in cord blood, which may lead to development of atopic dermatitis or wheezing in infancy. The association between maternal nutrient status during pregnancy and childs health is complex and interacts with other environmental factors such as tobacco exposure. This study was a part of the clinical trial NCT01861548 registered at ClinicalTrials.gov.

GSTP1 mRNA expression in human circulating blood leukocytes is associated with GSTP1 genetic polymorphism

OBJECTIVES We explored association between GSTP1 Ile(105)Val (rs1695) polymorphism and GSTP1 mRNA expression in circulating blood leukocytes. DESIGN AND METHODS GSTP1 transcripts level and polymorphism were determined by Real-Time PCR in 51 bladder cancer and 90 healthy men. RESULTS Individuals with at least one GSTP1 Val(105) variant allele possessed higher GSTP1 mRNA level in blood leukocytes compared to GSTP1 Ile(105)Ile carriers. CONCLUSIONS GSTP1 Ile(105)Val gene polymorphism influences its expression in blood, regardless of cancer disease.

Pulmonary Irritation After Inhalation Exposure to Benzalkonium Chloride in Rats

BACKGROUND Benzalkonium chloride (BAC) is a quaternary ammonium compound (QAC) with a C8 to C18 chain length of alkyl groups. Since BAC exerts toxic effects on microorganisms, it has been used as an effective germicide and preservative, mostly in cosmetic industry and medicine. However, the toxic potential of BAC may be hazardous to humans, due to the common use of preparations containing BAC as a preservative. MATERIAL AND METHODS To assess the possible toxic effects of BAC, two-stage experiments were performed on female Wistar rats. At first, LC50 after a single exposure to BAC aerosol was determined. Then, the animals were exposed to BAC aerosol at 30 mg/m3 for 6 h, and for 3 days (6 h/day). The controls were unexposed rats. Directly after BAC exposure and 18 h afterwards, BALF concentrations were measured of total protein, Clara cell protein, matrix metalloproteinase-9 (MMP-9), hyaluronic acid (HA), immunoglobulin E (IgE) and cytokines (TF-alpha, IL-6 and MIP-20), lactate dehydrogenase (LDH) and GSH-S-transferase (GST). RESULTS The LC50 value for exposed rats was ca. 53 mg BAC in m3 air for 4 h. All the rats survived single and repeated inhalation exposure to 30 mg/m3 BAC. After single and repeated exposure, lung weight, total protein, HA and LDH activity in BALF of exposed rats were higher than in controls while CC16 levels were decreased. A significantly higher BALF concentration of IL-6 and IgE was noted in animals exposed to single and repeated doses. BALF concentrations of MMP-9, TNF-alpha, and MIP-2 in exposed rats were similar to those in control animals. CONCLUSION BAC may be classified to class I acute inhalation toxicity. It showed a strong inflammatory and irritant activity on the lungs after 6h inhalation and stimulated dynamic patterns of IL-6 and IgE production and protein infiltration from blood vessels to BALF. Continued exposure resulted in cellular destruction, a statistically significant increase in LDH activity and a continuous decrease in CC16 concentration in BALF.

Evaluation of biological effects of nanomaterials. Part I. Cyto- and genotoxicity of nanosilver composites applied in textile technologies

ObjectivesThe aim of this study was to investigate the cyto- and genotoxicity of nanocomposites (NCs) and generation of reactive oxygen species (ROS) as a result of particle-cell interactions.Materials and MethodsTitanium dioxide (TiO2-Ag) and ion-exchange resin (Res-Ag), both coated with silver (Ag), were examined. The murine macrophage J774A.1 cells were incubated in vitro with NC at different concentrations for 24 h. Cytotoxicity was analyzed by the methylthiazolyldiphenyl-tetrazolium bromide reduction test (MTT reduction test). ROS generation was assessed by incubation of cells with dichlorodihydrofluorescein diacetate (DCF) and flow cytometry. DNA damage was detected by comet assay and included single-strand breaks (SSB), alkali-labile sites (ALS) and oxidative DNA damage after formamidopyrimidine glycosylase (FPG) treatment. The tail moment was used as an indicator of DNA damage.ResultsTiO2-Ag was not cytotoxic up to 200 μg/ml, whereas IC50 for Res-Ag was found to be 23 μg/ml. Intracellular ROS levels were elevated after 4 h of exposure to Res-Ag at the concentration of 50 μg/ml. Both types of NC induced fragmentation of DNA strands, but only one of the composites caused damage to purine bases. TiO2-Ag induced SSB of DNA at concentrations of 10 and 5 μg/ml. For Res-Ag, a concentration-dependent increase in tail moments was observed.ConclusionsSilver-coated nanocomposites (both TiO2-Ag and Res-Ag) may cause genotoxic effects in murine macrophages J774A.1. Res-Ag increased generation of ROS which suggested that toxicity of Res-Ag in murine macrophages is likely to be mediated through oxidative stress. This paper will support industry and regulators alike in the assessment of hazards and risks and methods for their mitigation at the earliest possible stage in material and product development.