A new technique for long preservation of 14C-labelled Cladocerans

Three ways of preserving labelled Cladocerans fed with ¹⁴C-Chlorella for 7.5–10 min were tested. Tracer leakage in 4% formalin at room temperature is rapid and extensive (half of the label was found in the animals after 1 hour of preservation). Even when individuals are frozen and sorting is made quickly in a liquid, losses nevertheless occur (substantial decrease of animal activity after only 4–5 min in the water in one of the two experiments performed). Results obtained after freezing in 4% formalin and sorting exactly 2 hours after thawing gave consistent losses: 16 separate experiments with Daphnia pulex, Ceriodaphnia spp. and Diaphanosoma brachyurum gave apparent filtering rates underestimated from 35% to 63% for freezing periods of up to 45 days. The good agreement in in situ community filtering rates between measured values and estimated ones from individual data confirmed the validity of a correction factor of x 2 applied to animals frozen in formalin.     

Enhancement by methylxanthines of sister-chromatid exchange frequency induced by cytostatics in normal and leukemic human lymphocytes

The effect of theobromine (TB) and diphylline (DP) or (1,2-dihydroxy-3-propyl)theophylline on SCE rates induced in vitro by mitomycin C (MMC), and the effect of caffeine on SCE rates induced in vitro by cytosine arabinoside (Ara-C) was studied. The combined treatments with MMC plus TB or DP showed the potentiating ability of the latter drugs. Caffeine also enhanced SCEs induced by Ara-C in cultured human lymphocytes. Caffeine and adriamycin (ADR) did not act synergistically on induction of SCEs. In a combined study, in vivo and in vitro, lymphocytes taken from 2 leukemic patients who had been given chlorambucil (CBC) or Ara-C by injection 3 h before, and then treated with caffeine in vitro, were found to have synergistically increased exchange frequencies.     

Induction of sister-chromatid exchanges in heroin-cannabis, heroin and cannabis addicts

The aim of this study was to determine the frequency of sister-chromatid exchanges (SCEs) in heroin-cannabis, heroin and cannabis addicts. The group of 84 subjects consisted of 42 controls, 16 heroin-cannabis addicts, 12 heroin addicts and 14 cannabis addicts. The mean number of SCEs/cell was 12.95 in heroin-cannabis addicts, 12.05 in heroin addicts and 11.99 in cannabis addicts. These values are significantly (P less than 0.002) higher than the mean values found in controls. This increase in SCEs may be related to reduced DNA repair in chronic drug addicts, which would allow the fixation or retention of a greater fraction of the DNA lesions caused by normal environmental exposure.     

Enhancement and attenuation of cytogenetic damage by vitamin C in cultured human lymphocytes exposed to thiotepa or L-ethionine

Vitamin C (vit C) at 2 mM enhanced sister chromatid exchange (SCE) frequencies induced by Thiotepa (THIO) or L-ethionine (L-ETH) in cultured human lymphocytes. However, when vit C was tested at 0.02 mM and 0.2 mM a rather protective effect on SCE rates induced by THIO or L-ETH was identified. Vit C (2 mM) caused a cell division delay in cultures treated with THIO or L-ETH. Division delays caused by THIO or L-ETH were reversed in the presence of 0.02 mM or 0.2 mM vit C. Mitotic indices (MIs) in cultures treated with THIO or L-ETH continued to be suppressed in the presence of 2 mM vit C. However, vit C at 0.02 mM reversed suppression of MIs caused by L-ETH or THIO. These findings illustrate the complexity of the interactions of vit C in biological systems and indicate that with different concentrations vit C can cause or prevent genetic toxicity.     

Precursor microRNA-programmed silencing complex assembly pathways in mammals

Assembly of microRNA ribonucleoproteins (miRNPs) or RNA-induced silencing complexes (RISCs) is essential for the function of miRNAs and initiates from processing of precursor miRNAs (pre-miRNAs) by Dicer or by Ago2. Here, we report an in?vitro miRNP/RISC assembly assay programmed by pre-miRNAs from mammalian cell lysates. Combining in?vivo studies in Dicer Knockout cells reconstituted with wild-type or catalytically inactive Dicer, we find that the miRNA loading complex (miRLC) is the primary machinery linking pre-miRNA processing to?miRNA loading. We show that a miRNA precursor deposit complex (miPDC) plays a crucial role in Dicer-independent miRNA biogenesis and promotes?miRNP assembly of certain Dicer-dependent miRNAs. Furthermore, we find that 5'-uridine, 3'-mid base pairing, and 5'-mid mismatches within pre-miRNAs promote their assembly into miPDC. Our studies provide a comprehensive view of miRNP/RISC assembly pathways in mammals, and our assay provides a versatile platform for further mechanistic dissection of such pathways in mammals.     

Frequencies of chromosomal aberrations in pesticide sprayers working in plastic green houses.

The frequency of chromosome aberrations was studied in peripheral blood lymphocytes of 29 workers occupationally exposed to a mixture of pesticides and in 14 age- and sex-matched healthy controls. There was a significant increase in chromosome aberrations in sprayers when compared to unexposed persons (2.39% compared to 0.54%). No positive correlation between the frequency of chromosome aberrations and the duration of exposure was observed. No significant difference between smokers and non-smokers was found.     

Cytogenetic damage by melphalan and hyperthermia in patients with an initial epileptic attack.

Sister-chromatid exchanges (SCEs) and cell kinetics in cultured lymphocytes of patients with an initial epileptic attack, and prior to any anticonvulsant treatment, were studied. Spontaneous melphalan (MEL) and MEL-hyperthermia (MEL-HYP) induced SCE frequencies have been studied in 18 adults with an initial epileptic seizure. Fifteen age and sex matched healthy subjects were used as the control group. The incidence of spontaneous SCEs in lymphocytes from epileptics was not significantly greater than in those from the control subjects. However, when exposed to MEL in vitro, cells from both groups showed an increase in SCE frequency. When exposed to MEL and HYP (41 degrees C for 3 h) in vitro, cells from both groups showed a further increase in SCE frequency with yields from epileptics higher (P less than 0.05) than from controls. HYP in combination with MEL enhanced synergistically SCEs and cell division delays in both groups with synergistic effects in cells from epileptics (P less than 0.01 and P less than 0.01 respectively) higher than from controls (P less than 0.05 and P less than 0.05 respectively.     

Arginine methylation of Aubergine mediates Tudor binding and germ plasm localization

Piwi proteins such as Drosophila Aubergine (Aub) and mouse Miwi are essential for germline development and for primordial germ cell (PGC) specification. They bind piRNAs and contain symmetrically dimethylated arginines (sDMAs), catalyzed by dPRMT5. PGC specification in Drosophila requires maternal inheritance of cytoplasmic factors, including Aub, dPRMT5, and Tudor (Tud), that are concentrated in the germ plasm at the posterior end of the oocyte. Here we show that Miwi binds to Tdrd6 and Aub binds to Tudor, in an sDMA-dependent manner, demonstrating that binding of sDMA-modified Piwi proteins with Tudor-domain proteins is an evolutionarily conserved interaction in germ cells. We report that in Drosophila tud¹ mutants, the piRNA pathway is intact and most transposons are not de-repressed. However, the localization of Aub in the germ plasm is severely reduced. These findings indicate that germ plasm assembly requires sDMA modification of Aub by dPRMT5, which, in turn, is required for binding to Tudor. Our study also suggests that the function of the piRNA pathway in PGC specification may be independent of its role in transposon control.     

Sister-chromatid exchange, chromosomal aberrations and delays in cell-cycle kinetics in human lymphocytes induced by dental composite resin eluates

We have investigated eluates derived from commercially available composite resin-based materials used for direct (Tetric Ceram/Ivoclar-Vivadent, Simile/Pentron, Filtek Z-250/3M ESPE) and indirect (Adoro/Ivoclar-Vivadent and Conquest Sculpture/Pentron) dental restorations, with respect to their genotoxic effects on human peripheral lymphocytes. Primary lymphocyte cultures obtained from blood samples of three healthy donors were exposed to eluates of freshly cured specimens of all the materials tested. Metaphases were induced with phytohaemagglutinin, collected after a 72-h treatment using colchicine and stained with the Fluorescence Plus Giemsa (FPG) procedure. Preparations were scored for sister-chromatid exchange (SCE) and chromosomal aberrations (CAs). The proliferation rate index (PRI) and the mitotic index (MI) were also calculated. Our results show that eluates derived from the three direct composites (Filtek Z-250, Simile and Tetric Ceram) increased the frequencies of SCE and CAs and markedly reduced PRI and MI. Tetric Ceram's eluate, being the most genotoxic of all eluates tested, increased the frequencies of SCE up to 24.40 per cell (control, 9.87 per cell) and of CAs up to 424 per 100 metaphases scored (control, 5). Moreover, it caused a pronounced decrease of the PRI down to 1.31 (control, 2.44) and of the MI down to 9.8 per thousand (control, 19.2 per thousand). In contrast, eluates derived from the laboratory-processed composites (Adoro and Conquest Sculpture) induced much less cytogenetic damage. Overall, the degree of genotoxicity and cytotoxicity decreased as follows: Tetric Ceram>Filtek Z-250>Simile>Adoro=Conquest Sculpture. These results indicate that composite resins used for direct and indirect dental restorations differ extensively in their cytotoxic and genotoxic potential and in their ability to affect chromosomal integrity, cell-cycle progression, DNA replication and repair. This underlines the impact of improved polymerization with respect to their biological behavior.