Detection of the genotoxicity of air pollutants in and around the city of São Paulo (Brazil) with the Tradescantia-micronucleus (Trad-MCN) assay

Tradescantia pallida cv. purpurea, a popular garden plant in Brazil, was used for the Tradescantia micronucleus (Trad-MCN) assay. In situ monitoring of the genotoxicity of air pollutants was carried out by sentinel approach, using the plant grown in the field or using the plants in pots which were carried to the monitoring sites. Two highly polluted sites, in S?o Paulo city (Cerqueira Cesar and Congonhas) and two rural sites (the cities of Pirassununga, 200 km and Caucaia do Alto, 50 km from S?o Paulo, respectively) were chosen for this study, in order to determine the gradient difference of the air pollution levels. Sentinel plants in Congonhas site presented the highest frequency of micronuclei (4.4%), in comparison with 2.2 and 2.3% found in plants from Pirassununga and Cerqueira Cesar sites, respectively (Kruskal-Wallis; P<0.020). Significant increases (F test; P<0.0001) in the frequency of micronuclei were observed in plants exposed in the polluted urban sites (Cerqueira Cesar: 5.7%; Congonhas: 7.1% and Caucaia do Alto: 2.3%). The increase in the frequency of micronuclei observed indicates the potential risk of mutagenicity in presence of high concentrations of pollutants.     

Inhibition of PARP activity by PJ‐34 leads to growth impairment and cell death associated with aberrant mitotic pattern and nucleolar actin accumulation in M14 melanoma cell line

The capability of PARP activity inhibitors to prevent DNA damage recovery suggested the use of these drugs as chemo‐ and radio‐sensitisers for cancer therapy. Our research, carried out on cultured human M14 melanoma cells, was aimed to examine if PJ‐34, a potent PARP activity inhibitor of second generation, was per se able to affect the viability of these cancer cells without any DNA damaging agents. Using time‐lapse videomicroscopy, we evidenced that 10 µM PJ‐34 treatment induced severe mitotic defects leading to dramatic reduction of cell proliferation and to cell death. PJ‐34 cytotoxic effect was further confirmed by analysis of cell viability and clonogenic assay. Absence of canonic apoptosis markers allowed us to exclude this kind of cell death. No single and/or double stranded DNA damage was evidenced. Immunofluorescence analysis showed an aberrant mitotic scenario in several cells and subsequent multinucleation suggesting an atypical way for cells to die: the mitotic catastrophe. The detection of aberrant accumulation of polymerised actin inside the nucleolus was noteworthy. Taken toghether, our results demonstrate that, targeting PARP activity by PJ‐34, cancer cell survival is affected independently of DNA damage repair. Two findings are remarkable: (a) cisplatin concentration can be reduced by three quarters if it is followed by treatment with 10 µM PJ‐34 for 24 h to obtain the same citotoxic effect; (b) effects dependent on PJ‐34 treatment are reversible. Our data suggest that, to reduce the harm done to non‐tumour cells during chemotherapy with cisplatin, the latter could be coupled with PJ‐34 treatment. J. Cell. Physiol. 222: 401–410, 2010. © 2009 Wiley‐Liss, Inc.     

The respiration-linked limiting step of tumor cell transition from the non-cycling to the cycling state: Its inhibition by oxidizable substrates and its relationships to purine metabolism

The recruitment into the cycling state of resting Yoshida AH 130 hepatoma cells was studied with respect to its dependence on respiration in an experimental system wherein the overall energy requirement for this recruitment can be supplied by the glycolytic ATP. The G₁-S transition of these cells, unaffected by 2,4-dinitrophenol (DNP) at concentrations which uncouple the respiratory phosphorylation, is impaired either by blocking the electron flow to oxygen by antimycin A or by adding an excess of some oxidizable substrates, chiefly pyruvate and oxalacetate. An experimental analysis, focused on pyruvate activity, showed that the inhibition of cell recruitment into S is not related to the depressing effects of this substrate on aerobic glycolysis of tumor cells, nor is it modified by forcing, in the presence of DNP, pyruvate oxidation through the tricarboxylic acid cycle as well as the overall oxygen consumption. Addition of suitable concentrations of preformed purine bases (mainly adenine), completely removes the block of the G₁-S transition produced either by the excess of oxidizable substrates or by antimycin A. These findings indicate the existence of a respiration-linked step in purine metabolism, which restricts the above transition and is equally impaired by blocking the respiratory chain or by saturating it with an excess of reducing equivalents derived from unrelated oxidations. The inhibitory effects of pyruvate and antimycin A can be largely removed by the addition of folate and tetrahydrofolate, suggesting that the respiration-linked restriction point of tumor cell cycling involves the folate metabolism and its connections to purine synthesis.     

beta-N-acetylhexosaminidases from Serratia marcescens.

Two forms of beta-N-acetylhexosaminidase from Serratia marcescens with an optimum pH of 5.0 and 6.5, respectively, to 4-methylumbelliferyl-2-acetamido-2-deoxy-beta-D-glucopyranoside were separated by DEAE-cellulose chromatography and Sephacryl S-200 chromatography. On the basis of their molecular weights, thermal stability, substrate specificity and isoelectric points, the form with an acidic pH optimum resembled hexosaminidase B, whereas the form with a neutral pH optimum resembled hexosaminidase C. Lectin binding studies showed that the acidic form does not bind to concanavalin-A-Sepharose, Tetragonolobus purpurea-agarose, wheat germ-agglutinin-Sepharose or Ricinus communis-agglutinin-agarose, whereas the neutral form binds to the last two lectin columns.