Interleukin-1beta expression in murine J774A.1 macrophages exposed to platinum compounds: the role of p38 and ERK 1/2 mitogen-activated protein kinases.

Although skin and respiratory sensitizing properties of platinum compounds have been proved in humans and mice, little is known about signal transduction pathways leading to cytokine production in the induction phase. It is generally assumed that induction of skin sensitization, but not skin irritation, is associated with a rapid increase in the IL-1beta mRNA expression. In this study, IL-1beta expression and a role of mitogen-activated protein kinases (MAPKs) in this process were investigated in murine macrophages J774A.1 exposed to four platinum compounds. Potassium tetrachloroplatinate (K(2)PtCl(4); TCPP), ammonium tetrachloroplatinate ((NH(4))(2)PtCl(4); TCPA), ammonium hexachloroplatinate ((NH(4))(2)PtCl(6); HCPA) showed a very similar range of cytotoxic concentrations (IC(50) values: 238 microM+/-30; 269 microM+/-39 and 245 microM+/-31, respectively) as assessed in the 24-h MTT reduction test. Cytotoxicity of cis-diammineplatinum dichloride (cisplatin) was considerably higher (IC(50) of 23 microM+/-4). While increased expression of IL-1beta mRNA was observed in the macrophages exposed to each test compound, IL-1beta protein production was detected in cell lysates after treatment with TCPP, TCPA and HCPA for 24h (concentration range of 150-350 microM) as well as for 2h (450-650 microM). The treatment with each compound resulted in the phosphorylation of both p38 MAPK and ERK 1/2 (p44/42). Blocking the activation of p38 MAPK as well as ERK 1/2 with specific inhibitors (SB203580 and U0126, respectively) down-regulated the IL-1beta expression. Interestingly, the skin irritant sodium dodecyl sulfate did not trigger phosphorylation of these kinases, nor induced IL-1beta production. These data suggest that p38 MAPK and ERK 1/2 play an important role in induction of IL-1beta expression in J774A.1 macrophages exposed to test platinum compounds.

Biomonitoring of cyanobacterial blooms in Polish water reservoir and the cytotoxicity and genotoxicity of selected cyanobacterial extracts.

OBJECTIVES: Water pollution with toxic cyanobacterial blooms is a worldwide problem. Cyanobacteria species that mainly produce microcystins predominate in Polish water reservoirs. MATERIALS AND METHODS: In our study, cyanobacterial blooms were monitored during summer of 2004 in the Sulejów reservoir. The concentration of microcystins in water and cyanobacterial cells were determined using liquid chromatography and immunobiotests, while the biological activity of microcystic cyanobacterial extracts was assessed using bacterial tests (SOS Chromotest, UMU test), the comet assay and micronucleus test with human lymphocytes. RESULTS: It was revealed that cyanobacterial bloom was most intensive in mid August and lasted until the end of September. Microcystis aeruginosa and Aphanizomenon flos-aquae dominated in the blooms. The highest concentration of microcystins in cyanobacterial cells was also observed at that time. The concentration of microcystins in water did not exceed 1 microg/l. All cyanobacterial extracts showed weak genotoxicity only for Escherichia coli PQ37. The cyanobacterial extracts prepared at the beginning of September were most toxic to human lymphocytes, the effective microcystin extracts (EC50) concentration was about two or three times lower compared to the other extracts. The level of DNA damage in lymphocytes after short exposure to microcystic extracts (3 and 6 h) was significantly higher than respective levels after longer exposure. The microcystins of cyanobacterial blooms induced a slight increase in micronuclei frequencies in human lymphocytes. CONCLUSION: Phytoplankton biomass and the genotoxicity of massive cyanobacterial blooms should be assessed for eucariotic cells in the Sulejów reservoir to avoid the hazard induced by cyanobacterial blooms.

Genotoxic effects in C57Bl/6J mice chronically exposed to arsenate in drinking water and modulation of the effects by low-selenium diet.

In C57Bl/6J mice chronically exposed to arsenate in drinking water at 50, 200, or 500 microg As/L, genotoxic effects in bone-marrow cells using micronucleus test and in peripheral blood leukocytes using the comet assay were determined after 3, 6 or 12 mo. To assess the modulating role of selenium in development of the effects, the animals were fed a specially prepared low-selenium diet and were supplemented with sodium selenite (200 microg Se/L) in drinking water (supplemented groups) or were without Se supplementation (nonsupplemented groups). Measurements of glutathione peroxidase activity in erythrocytes and plasma as well as selenium concentration in plasma were performed after 3, 6, and 12 mo and showed a marked decrease in values in animals in non-Se supplemented compared to Se-supplemented groups. After 3 mo of arsenic exposure in the Se-supplemented animals the level of DNA fragmentation (without Endo III and Fpg enzymes) did not differ from the control; however, increased oxidative damage of purine and pyrimidine bases was observed. In groups not supplemented with Se, an increase of DNA fragmentation was observed; however, the levels of oxidative DNA damage in these groups did not differ from the control. None of the positive effects observed in the comet assay after 3 mo was related to arsenate concentration. The levels of DNA damage after 6 and 12 mo of exposure to arsenic as well as the frequency of micronuclei after 3, 6, and 12 mo did not differ significantly between exposed and control animals, irrespective of Se supplementation status.

DNA damage in leukocytes of workers occupationally exposed to arsenic in copper smelters.

Inorganic arsenic (i-As) is a known human carcinogen; however, humans continue to be exposed to i-As in drinking water and in certain occupational settings. In this study, we used the Comet assay to evaluate DNA damage in the somatic cells of workers from three Polish copper smelters who were occupationally exposed to i-As. Blood samples were collected from 72 male workers and 83 unexposed male controls and used for the detection of DNA damage, oxidative DNA damage, and DNA damage after a 3-hr incubation in culture. Urine samples were collected to assess the level of exposure. The mean concentration of arsenic metabolites in urine [the sum of arsenite (AsIII), arsenate (AsV), monomethylarsenate (MMA) and dimethylarsenate (DMA)] and the concentrations of DMA (the main metabolite in urine) were higher in workers than in controls, but the differences were not statistically significant. By contrast, the level of DNA damage, expressed as the median tail moment, was significantly higher in the leukocytes of workers than in the controls. Comet assays conducted with formamidopyrimidine glycosylase (FPG) digestion to detect oxidative DNA damage indicated that oxidative lesions were present in leukocytes from both the exposed and control groups, but the levels of damage were significantly higher among the workers. Incubation of the cells in culture resulted in a significant reduction in the levels of DNA damage, especially among leukocytes from the workers, suggesting that the DNA damage was subject to repair. Our findings indicate that copper smelter workers have increased levels of DNA damage in somatic cells, suggesting a potential health risk for the workers. Although i-As was present in air samples from the smelters and in urine samples from workers, no clear association could be made between i-As exposure and the DNA damage.

Testing the immunosuppressive effects of cyclophosphamide in the popliteal lymph node assay in the modification of graft-vs-host reaction (PLNA-GvHR) in the rat.

We tested the hypothesis that popliteal lymph node assay (PLNA) in the modification of graft vs host reaction (GvHR) enabled to assess immunosuppressive potential of xenobiotics. We conducted PLNA in GvHR modification under two experimental conditions using cyclophosphamide (CY). In the first experiment average lymph node weight index (IW, weight ratio of popliteal lymph node of hind footpad injected with parental lymphocytes to lymph node of footpad injected with vehicle) in rats administered intraperitoneally with CY simultaneously with, or 3 days before local GvHR induction was 84% and 42%, respectively less than average IW in external control animals injected with parental lymphocytes into one footpad and vehicle into contralateral footpad. Average lymph node lymphocyte index (IL, ratio of cell number in the lymph node undergoing GvHR to control lymph node) in the tested animals was 92% and 86%, respectively less than in controls. In the second experiment in rats injected subcutaneously into one footpad with parental lymphocytes suspended in solution of CY at the concentrations of 4.6 mM or 18.6 mM and with parental lymphocytes into contralateral footpad, average IW was 88%and 92%, respectively less than average IW in the external control animals injected with parental lymphocytes into both footpads (average IL was 84 and 91%, respectively less than in controls). Our preliminary experiments showed a strong inhibitory effect of CY in both PLNA-GvHR models used and we believe that both models may be of value in designing future protocols aiming at prediction of immunosuppressive potential of chemicals.

Assessment of apoptosis in thymocytes and splenocytes from mice exposed to arsenate in drinking water: cytotoxic effects of arsenate on the cells in vitro.

This study provides an assessment of the level of apoptosis in thymocytes and splenocytes from mice exposed to arsenate in drinking water. To simulate the naturally occurring exposure conditions of humans, the animals were exposed to arsenate at the concentrations of 0.5, 5, and 50 mgAs/L. TUNEL method for staining of thymocytes and splenocytes isolated from the mice after 8 and 12 weeks revealed increased percentage of apoptotic cells in the exposed groups. Although statistically significant increases were observed only for the highest concentration of arsenate, the increases showed linear trend as a function of arsenate concentration in drinking water. In vitro experiments performed on isolated cells incubated for 24 hours with arsenate at 6.7-2000 microM showed very similar concentration-viability relationships for both cell populations (IC50 was 442+/-15 microM and 427+/-18 microM for thymocytes and splenocytes, respectively). Arsenate induced a concentration-dependent increase in the percentage of the cells undergoing apoptosis. At higher concentrations, apoptosis was the predominant mode of cell death. It can be speculated that proapoptotic effects of arsenate as observed in our in vivo study may contribute to some immunotoxic symptoms observed in people chronically exposed to arsenic in drinking water.