Lung permeability, antioxidant status, and NO2 inhalation: a selenium supplementation study in rats.

Little is known about antioxidant status, selenium status in particular, and lung response to NO2, which acts as a proinflammatory air pollutant. The effects of a low selenium diet (1.3 microg Se/d) with or without selenium supplementation were therefore studied in 128 Wistar rats, 2 mo old, male exposed to either acute (50 ppm, 30 min), intermittent subacute (5 ppm, 6 h/d, 5 d), intermittent long-term NO2 (1 ppm, 10 ppm, 6 h/d, 5 d/wk, 28 d), or normal atmospheric air (controls). Following sacrifice, measurements of lipid peroxidation (thiobarbituric acid-reactive substances, chemiluminescence), antioxidative protective enzymes (glutathione peroxidase [GPx], superoxide dismutase [SOD], glutathione S-transferase [GST], ceruloplasmin), lung damage (lactate dehydrogenase, alkaline and acid phosphatases), lung permeability (total protein, albumin), and inflammation (cell populations), along with the determination of new biomarkers such as CC16 (Clara-cell protein), were performed in serum and bronchoalveolar lavage fluid (BALF). While selenium-supplemented animals had increased GPx activity in serum prior to inhalation experiments, they also had decreased BALF CC16, blood SOD, and GST levels. Nevertheless, the protective role of normal selenium status with respect to NO2 lung toxicity was evident both for long-term and acute exposures, as the increase in BALF total proteins and corresponding decrease in serum (indicating increased lung permeability) was significantly more pronounced in selenium-deficient animals. During the various inhalation experiments, serum CC16 demonstrated its key role as an early marker of increased lung permeability. These findings corroborate the important role of selenium status in NO2 oxidative damage modulation, but also indicate, in view of its negative impact on CC16, a natural anti-inflammatory and immunosuppressor, that caution should be used prior to advocating selenium supplementation.

Assessment of usefulness of J774A.1 macrophages for the assay of IL-1beta promoter activity.

Existing data indicate that the increase of il-1beta gene expression can be a promising marker of Langerhans cells activation after exposure to contact sensitizers. In this study, we were interested in development of an alternative in vitro screening test detecting such sensitizers. Two IL-1beta reporter constructs containing the enhanced green fluorescent protein (GFP) gene and mouse IL-1beta promoter fragments of varying lengths (-500 bp and -4093 bp) were used for transient transfections of J771A.1 murine monocyte-macrophage cells. As a result of the transfections performed using Lipofectamine reagent we did not observe any GFP fluorescence after stimulation of the cells with LPS as well as known sensitizers (potassium tetrachloroplatinate, dinitrochlorobenzene and nickel sulfate). Low transfection efficiency of J774A.1 cells (less than 0.1%) was confirmed using control plasmid containing GFP gene under the control of cytomegalovirus promoter. The fact that, using the same conditions, we were able to transfect murine fibroblasts 3T3-L1 with the control plasmid very efficiently, may support the theory of high metabolic activity of macrophages being responsible for the extremely low transfection efficiency. These data suggest limited suitability of J774A.1 cell line for transient transfections using cationic liposomes.

Prognostic significance of low serum levels of Clara cell phospholipid-binding protein in occupational aluminium neurotoxicity.

The relationship between respiratory and neurological effects of exposure to aluminium (Al) was investigated in a group of foundry workers exposed to Al at concentrations below the threshold limit value (TLV) binding in Poland (2.0 mg Al2O3 m(-3)). Neurological and neurophysiological parameters indicated subclinical effects of Al exposure on the nervous system. The measurement of serum anti-inflammatory Clara cell protein (CC16) was employed as a peripheral marker of the lung epithelium function. There was a strong inverse relationship between serum Al (Al-S) and CC16 concentrations (p = 0.006). The lowest CC16 concentrations were found in serum of workers characterised by subjective symptoms of the central nervous system (CNS) and abnormal results of neurophysiological examinations (EEG and VEP). Low serum CC16 concentrations and enhanced Al and iron (Fe) levels were also observed in the younger age group of workers with the subjective CNS symptoms and abnormal VEP results, which suggests that Fe is implicated in strengthening of the neurotoxic Al potential. The results of our study support the hypothesis that subclinical neurological symptoms (especially abnormal VEP) are most likely associated with internalisation of Al ions with lipid fractions of the lung epithelium, which in turn may help Al ions overcome the blood-brain barrier. Low serum CC16 concentrations (<10 microg L(-1)) were noted in workers with the abnormal results of neurological (CNS) and neurophysiological (EEG and VEP) examinations as well as with Al body burden manifested by urinary excretion (Al-U) below 60 microg L(-1) and Al-S concentration of 2 microg L(-1). This concentration may be considered as a threshold allowable biological concentration of aluminium.

The influence of atmospheric chromium on selenium content and glutathione peroxidase activity in blood of tannery workers.

The concentration of selenium and thiobarbituric acid reactive substances (TBARS) and activity of glutathione peroxidase (GSH-Px) were determined in blood of 34 workers of a tannery in Gniezno, Poland, who worked in an area containing chromium compounds. Fourteen workers were exposed to chromium compounds at concentrations of 0.11 +/- 0.07 mg Cr/m3 (mean +/- SD) and 20 at concentrations 5-10 times lower i.e., 0.022 +/- 0.009 mg Cr/m3. Excretion of Se in urine was measured in all of the investigated workers. Decreased Se concentration in whole blood and blood plasma and elevated TBARS concentration in blood plasma were found in the whole group of investigated tanners as compared to controls. Tanners working in areas with high chromium concentrations had a statistically significant decrease in Se concentration in blood and plasma and decreased urinary excretion of the microelement as compared with other tanners. TBARS concentration was 2.5 times lower in workers exposed to higher chromium concentrations (p < 0.005) than in other workers. Positive linear correlations were found between the concentration of Se in blood and the amount of the element excreted in urine (r = 0.48; p < 0.005), the concentration of Se in blood plasma and in urine (r = 0.46; p < 0.01), and the concentration of Se in blood and erythrocyte GSH-Px activity (r = 0.42; p < 0.02). The observed differences between Se concentration in blood and urine of tannery workers and people who are not employed in the industry may indicate a kind of specific adaptation of the body to the working environment containing chromium compounds.

The assessment of DNA damage in lymphocytes of wooden furniture workers.

Single-strand breaks (SSB) and DNA repair were detected in peripheral lymphocytes derived from workers of a furniture factory in a non-polluted region of Poland. The workers were exposed to wood dust (n = 19), or to the dust and varnishes or lacquers together (n = 5). Four groups were studied simultaneously: (a) exposed workers smokers of cigarettes (n = 14), (b) nonexposed smokers--control (n = 14), (c) exposed workers' nonsmokers (n = 14), (d) exposed nonsmokers (n = 10). In exposed workers DNA SSB and DNA repair were statistically significantly increased. DNA SSB was clearly higher in the smoking workers than in the smoking controls. Cigarette smoking itself has produced no evident increase in the frequency of DNA SSB in the control group. Occupational exposure had a significant effect on DNA repair in non stimulated lymphocytes both in smoking and nonsmoking workers.

Selenium status of low-selenium area residents: Polish experience.

The concentration of selenium (Se) in human organism varies widely between geographical areas depending on its content in soil and plants, dietary Se intake, bioavailability and retention, mineral interactions and other factors. The study includes healthy inhabitants of different regions of Poland; pregnant women, lactating women, children from 0 to 15 years of age and adults. Systematic determinations allow us to observe changes of the concentration of Se in time, which may be significant for developing preventive action. The results obtained confirm our thesis that Se concentration in the blood of the inhabitants of Poland depends on the region of the country. In recent years, in a considerable number of Polish inhabitants, the concentration of Se in blood plasma has been relatively low-about 50-55 microg/l, and the calculated daily dietary intake about 30-40 microg/day. The low levels of the element in the blood and urine are probably due to its deficiency in the diet.

DNA single-strand breaks and DNA repair in the lymphocytes of wooden furniture workers.

DNA single-strand breaks (DNA SSB) in peripheral lymphocytes of wooden furniture workers and a control group, including smokers and nonsmokers, were detected by the microfiltration method. Our results show that cigarette smoking significantly increases the fragmentation of DNA single strands in the wooden furniture workers (by nearly two times) but not in the control group. Moreover, occupational exposure to wood dust and other wooden plant substances significantly induced DNA SSB only in the lymphocytes of smokers (by about two times). DNA repair in the lymphocytes was investigated as 3H incorporation into DNA. High 3H incorporation in the unstimulated lymphocytes of both smoking and nonsmoking workers, as compared to the references, suggests that besides DNA SSB other DNA damage can be caused by occupational exposure in the wooden plant. Since DNA repair is not always perfect, the possibility is high that the low level of DNA repair in the study group may lead to irreversible DNA damage. We think that the workers exposed to wood dust and the substances emitted by furniture coating materials in the plant may be at higher risk for mutagenesis and/or carcinogenesis than the unexposed population.

DNA damage detected by the comet assay in the white blood cells of workers in a wooden furniture plant.

The study was aimed at the assessment of genotoxic effects in workers of a wooden furniture manufacture, based on the level of DNA damage in white blood cells (WBC). The alkaline single cell gel electrophoresis assay (known as the comet assay) in individual cells was adapted for detecting damaged DNA in WBC. The level of DNA damage was determined as the percentage of cells with comets. It was assessed in cells before and after incubation in RPMI 1640 medium and CO(2) at 37 degrees C for 1 h to repair DNA breaks. Thirty-five woodworkers and 41 control subjects were studied. In the woodworkers, significantly more cells with DNA damage (21.5%) were observed than in the control persons (9.7%). A slight but significant decrease in the level of DNA damage was found in the WBC of woodworkers after incubation (17.2%). Significantly higher levels of damaged DNA was observed in woodworkers who either smoked (22.1%) or did not smoke cigarettes (20.8%) than in smokers (13.2%) and non-smokers (7.0%) from the control group. After incubation, a slight decrease in the level of DNA damage was found in both smoking and non-smoking woodworkers compared to the respective subjects in the control group. The increased levels of DNA damage observed in the woodworkers could be associated with the occupational exposure to wood dust in the furniture manufacture.

Genotoxic effects of occupational exposure to lead and cadmium.

This study was designed to assess genotoxic damage in somatic cells of workers in a Polish battery plant after high-level occupational exposure to lead (Pb) and cadmium (Cd), by use of the following techniques: the micronucleus (MN) assay, combined with in situ fluorescence hybridization (FISH) with pan-centromeric probes, analysis of sister chromatid exchanges (SCEs), and the comet assay. Blood samples from 44 workers exposed to lead, 22 exposed to cadmium, and 52 unexposed persons were used for SCE and MN analysis with 5'-bromodeoxyuridine (BrdU) or cytokinesis block, respectively. In parallel, the comet assay was performed with blood samples from the same persons for detection of DNA damage, including single-strand breaks (SSB) and alkali-labile sites (ALS). In workers exposed mostly to lead, blood Pb concentrations ranged from 282 to 655 microg/l, while the range in the controls was from 17 to 180 microg/l. Cd concentration in lead-exposed workers fell in the same range as for the controls. In workers exposed mainly to cadmium, blood Cd levels varied from 5.4 to 30.8 microg/l, with respective values for controls within the range of 0.2-5.7 microg/l. Pb concentrations were similar as for the controls. The incidence of MN in peripheral lymphocytes from workers exposed to Pb and Cd was over twice as high as in the controls (P<0.01). Using a combination of conventional scoring of MN and FISH with pan-centromeric probes, we assessed that this increase may have been due to clastogenic as well as aneugenic effects. In Cd- and Pb-exposed workers, the frequency of SCEs as well as the incidence of leukocytes with DNA fragmentation in lymphocytes were slightly, but significantly increased ( P<0.05) as compared with controls. After a 3h incubation of the cells to allow for DNA repair, a clear decrease was found in the level of DNA damage in the controls as well as in the exposed workers. No significant influence of smoking on genotoxic damage could be detected in metal-exposed cohorts. Our findings indicate that lead and cadmium induce clastogenic as well as aneugenic effects in peripheral lymphocytes, indicating a potential health risk for working populations with significant exposures to these heavy metals.

Behavioral changes following 4-week inhalation exposure to pseudocumene (1,2,4-trimethylbenzene) in the rat.

Pseudocumene (1,2,4-trimethylbenzene, TMB) is a component of several solvent mixtures. During recent studies on rats we investigated the effect of a 4-week (6 h/day, 5 days/week) inhalation exposure to TMB at concentrations of 0, 25, 100, or 250 ppm on radial maze performance, open field activity, passive avoidance, active two-way avoidance, and shock-induced changes in the pain sensitivity reflecting the magnitude of the shock-induced fear response (hot plate test). The tests were performed between days 14 and 54 after the last exposure. The radial maze performance was not disturbed in any dose group. During testing in the open field grooming was significantly increased in rats exposed to 100 ppm TMB. In rats exposed to 100 and 250 ppm TNB, a foot shock applied after stepping off an elevated platform (a safe area) resulted in a significantly smaller increase in the step-down latency (i.e., passive avoidance, on days 3 and 7 after the foot shock) than in sham-exposed animals. Learning of a two-way active avoidance was slightly retarded in rats exposed to 250 ppm of TMB. Results of the hot plate test revealed no differences between groups in the paw sensitivity to heat (54.5 degrees C) before a 2-min intermittent food shock, but in rats exposed to 100 and 250 ppm of TMB the foot shock-induced fear response persisted apparently longer. These results suggest that inhalation exposure to TMB may lead to long-lasting disturbances in CNS functions.

Effect of sodium butyrate treatment on the granule morphology, histamine level and elemental content of the bone marrow-derived mast cell.

Mast cells derived from the bone marrow of BALB/mice (BMMC) were cultured and their growth ceased with sodium butyrate. Sodium butyrate treatment (1mM, 4 days) caused maturation of the granules, an increased histamine content from approx. 1 pg/cell to 4 pg/cell. X-ray microanalysis revealed that maturation of the granules was accompanied by the increase in relative weight percent of sodium, phosphorus and sulphur, with concomitant decrease in chloride. The sulphur to potassium ratio increased three-fold in butyrate-treated mast cells. The existence of a different elemental composition during mast cell maturation may provide additional parameter for rapid discrimination of mast cell subpopulations.

The mast cells derived from mouse bone marrow grow in close apposition with the reticular cells.

Cultures of mast cells of more than 95% purity were grown from bone marrow of BALB/c mice, and examined with various morphological methods. The presence of elongated, reticular cells was documented in the adherent layer on day 7 of the culture. The committed stem cells as well as immature bone marrow-derived mast cells (BMMCs) growing in clusters over the reticular cells were observed. After 14 days of cultivation BMMC harvested from the medium showed extensive plasma membrane ridges and numerous immature granules in their cytoplasm. These BMMCs increased their histamine to 0.7-1.1 pg/cell as compared to 0.1-0.2 pg/cell on the day 7. In the adherent layer BMMCs were seen in close apposition to the reticular cells. Their microvilli interdigitated with one another, forming end-to-end contracts. Our findings provide the evidence that for differentiation and proliferation of BMMCs in vitro close contacts with reticular cells in the adherent layer are necessary.

Glutathione S-transferase M1 and P1 metabolic polymorphism and lung cancer predisposition.

Individual susceptibility to different environmental agents is expected to be associated with alterations in metabolism of xenobiotics. Thus, genetic polymorphism of glutathione S-transferase (GST) can be recognized as a potential risk modifier in lung cancer development. The distribution of GSTM1 and GSTP1 genotypes was studied in a group of 138 diagnosed lung cancer patients and in 165 controls living in central Poland and RFLP-PCR technique was applied. The frequency of GSTM1 null genotype and GSTP1 Val single and duplicated alleles was similar among patients and controls. GSTM1 homozygous deletion was most prevalent in small-cell carcinoma groups (adjusted odds ratio (OR): 2.32, 95% confidence interval (CI): 0.98-5.52). In patients and controls, GSTM1A genotype was most frequent (34.1% vs. 37.0%). The estimated lung cancer risk for GSTM1 null, GSTP1 Ile/Val and GSTP1 Val/Val combined genotype was 1.44 (95% CI: 0.73-2.83), suggesting the absence of modifying effect of defective GSTM1 and GSTP1 alleles on lung cancer predisposition.

Immunohistochemical evaluation of P21ras and P53 proteins expression in human non-small-cell lung cancers.

Human non-small-cell lung cancers (NSCLCs) of 48 patients were analyzed immunohistochemically to detect P21 ras and P53 proteins expression. The relationship between P21 ras and P53 proteins expression and clinicopathologic findings was also assessed. DAKO EnVision TM detection system was employed in the study. The P21 ras and P53 proteins expression was shown in 75% (36/48) and 33.3% (16/48) studied NSCLCs, respectively. In both cases the difference was significant when compared with adequate negative control. Simultaneous expression of both studied proteins was observed in all cases in which P53 expression was noticed. No significant association of P21 ras and P53 expression was found with age, histologic type, histologic grade, tumor size or lymph node metastasis of the studied NSCLCs. Therefore, our study suggests that P21 ras and P53 protein play a role in the pathogenesis of NSCLCs but they have no value as a prognostic markers in the case of lung cancers.

Oxidative-stress markers in blood of lung cancer patients occupationally exposed to carcinogens.

The study covered 152 lung cancer patients and 210 controls. The results of the study indicated decreased selenium (Se) concentrations and lowered activity of erythrocyte antioxidant enzymes (glutathione peroxidase, superoxide dismutase, glutathione-S-transferase) in the blood of lung cancer patients, as well as significantly increased concentrations of vitamin E in erythrocytes and thiobarbituric acid reactive substances in the plasma of the study population. Low plasma Se concentrations (< 45.7 microg/L) enhance the estimated risk of lung cancer (odds ratio = 3.047, p < 0.001). A more precise exposure assessment is required to identify the association between lung cancer incidence and occupational exposure to carcinogens.

Selenium, zinc, and copper concentrations in the blood and milk of lactating women.

The aim of the study was to determine Se, Zn, and Cu concentrations in blood plasma and milk of lactating women from central Poland who were in different stages of lactation and to investigate the relationship between the content of trace elements in mothers' blood and concentrations of microelements in their milk. Se and Zn concentrations in blood plasma of mothers were the lowest and Cu was the highest on the first 4 d of lactation (colostrum, n = 43) and were found to be 34.9 +/- 11.8 microg/L, 0.51 +/- 0.13 mg/L, and 1.70 +/- 0.55 mg/L, respectively. The highest plasma level of Se and Zn and the lowest content of Cu could be observed between d 10 and 30 of lactation (mature milk, n = 41), and were found to be 54.3 +/- 14.6 microg/L for Se (p < 0.001), 0.76 +/- 0.20 mg/L for Zn (p < 0.001), and 1.03 +/- 0.30 mg/L (p < 0.001) for Cu. The results of Se, Zn, and Cu determination in breast milk samples demonstrate a pattern of decline in their concentration with advancing stages of lactation. We found out that Se, Zn, and Cu concentrations were the highest in colostrum (n = 43) and amounted to 24.8 +/- 10.1 microg/L, 8.2 +/- 2.8 mg/L, and 0.45 +/- 0.11 mg/L, respectively. The content of all determined microelements declined significantly during the time of lactation. Statistically significant linear correlation was found between concentrations of Zn in blood plasma and milk in the first stage of lactation. Weak but statistically significant linear correlations were also found between plasma Se content in plasma and in transitional and mature milk of breast-feeding women.

Can Cytotoxic Effects Induced by Industrial Chemicals be Time-dependent?

Short-term and delayed cytotoxic effects of selected water-soluble and water-insoluble industrial chemicals (dimethyl sulphoxide, ethyl alcohol, methyl alcohol, ammonium nitrate, benzalkonium chloride, butoxyethanol and propylene glycol) were tested on confluent 3T3-L1 mouse fibroblasts by using the neutral red uptake (NRU) assay and the MTT assay. The NRU and MTT assays were performed after exposure to a chemical for 10 minutes and 3 hours and then again 7 days later. The results indicate that the system of testing used permits the assessment of both early and delayed cytotoxic effects of different classes of chemicals. Our experiments revealed that three out of the seven substances tested (butoxyethanol, dimethyl sulphoxide and propylene glycol) exhibited similar cytotoxic effects when assessed after exposure for 10 minutes and 7 days later. The results for ammonium nitrate, ethyl alcohol and methyl alcohol, tested after exposure for 10 minutes and 7 days later, did not significantly differ from each other. However, we noted that the cytotoxic effects observed 7 days after exposure were more pronounced than those found for the same concentration, after exposure for 3 hours. One chemical tested (benzalkonium chloride) had almost no cytotoxic effect after exposure for 10 minutes and 3 hours, but showed a strong cytotoxic effect 7 days after exposure. Our study indicated that the cytotoxic effect might be time-dependent in some chemicals, thus inducing a delayed effect in vitro. In our opinion, it is advisable to assess the cytotoxicity of a chemical after exposure for 3 hours (for immediate effects) and again 7 days later (for delayed effects).

Sensory irritating properties of cyanuric chloride as revealed with plethysmographic method.

Sensory respiratory irritation properties of cyanuric chloride (2,4,6-trichlorotriazine; CC) were studied in mice. For this purpose, the respiratory rate was measured in Balb/C male mice by means of the whole body plethysmographic method. Each animal was placed in a body plethysmograph attached to a small (0.25 m3) dynamic inhalation chamber and exposed to various concentrations (2.1, 6.7, 9.1, 11.7 and 14.6 mg/m3) of CC. Respiratory rates were recorded before, during and after termination of the exposure. It was found that exposure to CC caused a concentration-dependant decrease of respiratory rates in mice. After termination of exposure fast and full recovery of respiratory rates were observed within 5 minutes. RD50 value calculated with probit method was established as 5.9 (1.3-13.9 for 95% confidence limits) mg/m3. The slope of the dose response curve was 1.366 (0.78). It is concluded that cyanuric chloride is a strong respiratory irritant.

Effects of acute combined inhalation exposure to n-butyl alcohol and n-butyl acetate in experimental animals.

The effects of combined exposure to n-butyl alcohol and n-butyl acetate on rotarod performance and hot plate behaviour in rats and respiratory rate in mice were investigated in the condition of an acute inhalation experiment. Rotarod performance and hot-plate behaviour were tested in rats exposed to various concentrations of n-butyl alcohol, n-butyl acetate and their mixture consisting of 50 Vol-% n-butyl alcohol and 50 Vol-% n-butyl acetate immediately after termination of a 4-hour exposure period. The respiratory rate in mice was recorded continuously before the exposure to solvents, during 6 min of exposure and 6 min after termination of exposure using whole body plethysmographic method. Mice were exposed to vapours of single solvents and their 50:50 Vol-% mixture. Both solvents and their mixture caused concentration-dependent disturbances of rotarod performance in rat. The medial effective concentration (EC50) for the effect amounted to 7559 ppm, 8339 ppm and 10672 ppm for n-butyl alcohol, n-butyl acetate and their mixture, respectively. Both solvents and their mixture decreased sensitivity to the pain and changes were concentration-dependent. In condition of combined exposure the results obtained in rotarod and hot-plate behaviour test indicate the summation of individual solvent effects. The tested solvents resulted in concentration-dependent decrease in respiratory rate in mice. n-Butyl alcohol produced maximal decrease in respiratory rate in the first minute of exposure whereas n-butyl acetate in the sixth minute.(ABSTRACT TRUNCATED AT 250 WORDS)

Neurotoxic effects of acute and subchronic inhalation exposure to trimethylbenzene isomers (pseudocumene, mesitylene, hemimellitene) in rats.

Neurotoxic effects of trimethylbenzene isomers (pseudocumene, mesitylene and hemimellitene) in male rats were investigated in conditions of acute and subchronic inhalation exposure. Rotarod performance and pain sensitivity behaviour were tested in rats exposed to trimethylbenzenes at concentrations of 250-2,000 ppm immediately after termination of a 4-hour exposure. Exposure to each of trimethylbenzene isomers resulted in concentration-dependent disturbances in rotarod performance, and decrease in pain sensitivity in rats. Pseudocumene, mesitylene and hemimellitene EC50 values for rotarod performance behaviour disturbances were 954, 963, 768 ppm and for decreases in pain sensitivity EC50 were 1,115, 1,212, 848, ppm, respectively. In conditions of subchronic inhalation exposure, pseudocumene and hemimellitene at concentrations of 25, 100 and 250 ppm caused concentration-dependent disturbances in rotarod performance behaviour and decrease in pain sensitivity. Neurotoxic effect of hemimellitene was more pronounced than that of pseudocumene and mesitylene. Two weeks after cessation of inhalation exposure to pseudocumene or hemimellitene no recovery in rotarod performance behaviour was observed.