Wortmannin enhances the induction of micronuclei by low and high LET radiation.

In mammalian cells, the repair of DNA double-strand breaks (DSBs) is mainly mediated by DNA non-homologous end joining. DNA-dependent protein kinase (DNA-PK), a nuclear serine-threonine kinase and a member of the phosphaditylinositol-3 kinase-related kinase family that is activated by DSBs, is a key component of this pathway. Wortmannin (WM) is known to be an irreversible and potent inhibitor of DNA-PK and has thus been proposed as an effective sensitizer for ionizing radiation and for radiomimetic compounds. The present study, using the cytokinesis block micronucleus assay, reports on the differential effect of WM on the repair of the DNA damage induced by low LET ((60)Co gamma-radiation) and high LET radiation by the boron neutron capture reaction (alpha and Li particles) in V79 Chinese hamster cells. Significant increases in the number of micronuclei per binucleated cell as well as in the frequency of micronucleated binucleated cells were observed in the presence of different concentrations of WM for high LET radiation from the boron neutron capture reaction. The increases observed reached a maximum of approximately 2-fold in comparison with the respective controls. WM, however, had a more pronounced effect on (60)Co gamma-radiation-induced micronuclei, increasing the genotoxic damage from this radiation by approximately 3- to 4-fold. These results are in general in agreement with the concept that DSBs induced by high LET radiation are not a more suitable substrate for the end joining processes mediated by DNA-PK, yet they do not preclude a role for DNA-PK in high LET-induced damage repair.

Evaluation of the genotoxic effects of the boron neutron capture reaction in human melanoma cells using the cytokinesis block micronucleus assay.

The present work reports on the genotoxicity of the boron neutron capture (BNC) reaction in human metastatic melanoma cells (A2058) assessed by the cytokinesis block micronucleus assay (CBMN) using p-borono-L-phenylalanine (BPA) as the boron delivery agent. Different concentrations of BPA (0.48, 1.2 and 2.4 mM) and different fluences of thermal neutrons were studied. Substantial genotoxic potential of alpha and lithium particles generated inside or near the malignant cell by the BNC reaction was observed in a dose-response manner as measured by the frequency of micronucleated binucleated melanoma cells and by the number of micronuclei (MN) per binucleated cell. The distribution of the number of MN per micronucleated binucleated cell was also studied. The BNC reaction clearly modifies this distribution, increasing the frequency of micronucleated cells with 2 and, especially, > or =3 MN and conversely decreasing the frequency of micronucleated cells with 1 MN. A decrease in cell proliferation was also observed which correlated with MN formation. A discrete genotoxic and anti-proliferative contribution from both thermal neutron irradiation and BPA was observed and should be considered secondary. Additionally, V79 Chinese hamster cells (chromosomal aberrations assay) and human lymphocytes (CBMN assay) incubated with different concentrations of BPA alone did not show any evidence of genotoxicity. The presented results reinforce the usefulness of the CBMN assay as an alternative method for assessment of the deleterious effects induced by high LET radiation produced by the BNC reaction in human melanoma cells.

Nitric oxide and human thermal injury short term outcome.

Nitric oxide (NO) is an important mediator in numerous physiological and pathophysiological events. After thermal injury an increase in plasma and urinary levels has been observed. The real importance of this fact is unknown. The stable NO derivatives (NO2-/NO3-) plasma concentrations were determined in 27 burned patients admitted to the Burn Unit at Santa Maria Hospital in Lisbon at first, third, fifth, seventh, ninth and 15th days and the values were compared with healthy controls (n=9). A significant increase (P<0.05) in burn patient determinations upon admission was found. The patients with inhalation injury revealed greater values compared to the other patients with statistical significance at 5th day (P<0.05). The patients who died showed a NO increase (0.397+/-0.138 vs. 0.267+/-0.017, P> 0.1, day 1) with significance at day 5 (0.615+/-0.223 vs. 0.154+/-0.048, P<0.05). The determinations in patients with sepsis were higher than in the other patients (P<0.01) at day 3. No relation with total burned surface area (TBSA) was found. For the first time, considering burned patients, a significant increase of NO was found in patients who died, in patients with inhalation injury and in patients in sepsis. The possible role of NO in burn injury is discussed. The authors suggest the possible role of NO determination as an indicator of sepsis. The role of NO synthesis inhibitors is discussed. Further studies are needed to clarify these questions.

Acrylic bone cement induces the production of free radicals by cultured human fibroblasts.

Arthroplasty with poly(methyl methacrylate) (PMMA) bone cement induces late loosening phenomena that compromise the prosthetic stability. As free radicals are inflammatory mediators and cytotoxic, it seemed useful to investigate whether PMMA induces the liberation of free radicals and/or cytotoxicity. The effect of PMMA interaction on cultured human fibroblasts was accessed by the cell viability test (MTT), and by the measurement of lipoperoxides in the incubation medium. The incubation with the medium exposed to PMMA induced a significant reduction in the viability and a significant increase in lipoperoxide liberation (vs control). These data suggest that PMMA is cytotoxic. This effect seems to be mediated by lipoperoxide and possibly by other free radicals, and may explain the peri-implant loosening phenomena that compromise the prosthetic stability.