Increased frequency of chromatid breaks in lymphocytes of heterozygotes of ataxia telangiectasia after in vitro treatment with caffeine

The frequencies of caffeine-induced chromosomal aberrations (CA), mainly chromatid (CdB) and chromosome (CB) breaks, were studied in lymphocyte cultures derived from 6 obligatory heterozygotes and 1 homozygote of ataxia telangiectasia (AT), and from 4 control adult healthy persons. Caffeine (CF, 1 mM) was added at the beginning of the cultures exposed to CF the frequency of CB was 1.9% and of CdB 1.3%. In cells of the AT homozygote, the frequency of CdB was 6.8% in the absence and 8.7% in the presence of caffeine, the frequencies of CB being 3.4 and 10.9%, respectively. In AT heterozygous cells treated with CF, CdB increased 13-fold as compared to a less than 3-fold increase in control cells. Comparing the frequencies of CF-induced chromosomal lesions in control and AT heterozygous cells, potentiation factors (Pf) for the effect of 1 AT gene on cell sensitivity of CF (Pf [AT]) were 3.5 for CB, 6.6 for CdB and 5.5 for CA. These data demonstrate that lymphocytes of AT heterozygotes are significantly more sensitive to caffeine treatment in vitro in terms of increased frequency of CdB than normala cells, which may be useful for the diagnosis of carriers of this defective gene.     

Functionalized fullerenes mediate photodynamic killing of cancer cells: Type I versus Type II photochemical mechanism

Photodynamic therapy (PDT) employs the combination of nontoxic photosensitizers (PS) and harmless visible light to generate reactive oxygen species (ROS) and kill cells. Most clinically studied PS are based on the tetrapyrrole structure of porphyrins, chlorines, and related molecules, but new nontetrapyrrole PS are being sought. Fullerenes are soccer-ball shaped molecules composed of 60 or 70 carbon atoms and have attracted interest in connection with the search for biomedical applications of nanotechnology. Fullerenes are biologically inert unless derivatized with functional groups, whereupon they become soluble and can act as PS. We have compared the photodynamic activity of six functionalized fullerenes with 1, 2, or 3 hydrophilic or 1, 2, or 3 cationic groups. The octanol-water partition coefficients were determined and the relative contributions of Type I photochemistry (photogeneration of superoxide in the presence of NADH) and Type II photochemistry (photogeneration of singlet oxygen) were studied by measurement of oxygen consumption, 1270-nm luminescence and EPR spin trapping of the superoxide product. We studied three mouse cancer cell lines: (J774, LLC, and CT26) incubated for 24 h with fullerenes and illuminated with white light. The order of effectiveness as PS was inversely proportional to the degree of substitution of the fullerene nucleus for both the neutral and the cationic series. The monopyrrolidinium fullerene was the most active PS against all cell lines and induced apoptosis 4-6 h after illumination. It produced diffuse intracellular fluorescence when dichlorodihydrofluorescein was added as an ROS probe, suggesting a Type I mechanism for phototoxicity. We conclude that certain functionalized fullerenes have potential as novel PDT agents and phototoxicity may be mediated both by superoxide and by singlet oxygen.     

Interactions of plasmalogens and their diacyl analogs with singlet oxygen in selected model systems

Plasmalogens are phospholipids containing a vinyl-ether linkage at the sn-1 position of the glycerophospholipid backbone. Despite being quite abundant in humans, the biological role of plasmalogens remains speculative. It has been postulated that plasmalogens are physiological antioxidants with the vinyl-ether functionality serving as a sacrificial trap for free radicals and singlet oxygen. However, no quantitative data on the efficiency of plasmalogens at scavenging these reactive species are available. In this study, rate constants of quenching of singlet oxygen, generated by photosensitized energy transfer, by several plasmalogens and, for comparison, by their diacyl analogs were determined by time-resolved detection of phosphorescence at 1270nm. Relative rates of the interactions of singlet oxygen with plasmalogens and other lipids, in solution and in liposomal membranes, were measured by electron paramagnetic resonance oximetry and product analysis using HPLC-EC detection of cholesterol hydroperoxides and iodometric assay of lipid hydroperoxides. The results show that singlet oxygen interacts with plasmalogens significantly faster than with the other lipids, with the corresponding rate constants being 1 to 2 orders of magnitude greater. The quenching of singlet oxygen by plasmalogens is mostly reactive in nature and results from its preferential interaction with the vinyl-ether bond. The data suggest that plasmalogens could protect unsaturated membrane lipids against oxidation induced by singlet oxygen, providing that the oxidation products are not excessively cytotoxic.     

The quality of porcine oocytes is affected by sexual maturity of the donor gilt

Although differences in the quality of oocytes derived from young gilts and adult sows are well documented, evidence concerning gametes of pre-pubertal and cycling gilts is scarce and inconsistent. The aim of this work was to establish whether sexual maturity of gilts affects the quality of their oocytes with the use of the brilliant cresyl blue (BCB) test, oocyte diameter and apoptosis. Ovarian morphology was evaluated, and gonads with corpus luteum or albicans were recognized as originating form cycling gilts (C) and those with follicles as originating form pre-pubertal females (P). Altogether 952 cumulus-oocyte complexes (COCs; group P: 554; group C: 398) were examined, whereas 149 COCs, not subjected to BCB test, served as a control for TUNEL. COCs of proper morphology were evaluated by the BCB test which differentiated two categories of gametes: more competent, BCB+, and less competent BCB- oocytes. The control group comprised oocytes of proper morphology aspirated from ovaries of P and C gilts not subjected to BCB test. Finally five groups of COCs were matured in vitro: 1/P-BCB+, 2/P-BCB-, 3/C-BCB+, 4/ C-BCB- and 5/ control. Significantly more large oocytes (≥ 120 μm), more BCB+ oocytes and more high quality (both BCB+ and ≥ 120 μm) oocytes originated from ovaries of cycling gilts than pre-pubertal gilts (p<0.001). The rate of mature oocytes at the MII stage differed significantly between C-BCB+ (68.5%) and P-BCB+ (32.9%) oocytes. The incidence of apoptosis among BCB-treated oocytes after in vitro maturation was 21.4% and was similar to that observed in control oocytes (17.4%). BCB+ oocytes from cycling gilts showed significantly higher (28.7%) incidence of apoptosis than that of the group P (16.2%). Interestingly, high quality oocytes displayed a similar level of apoptosis regardless of the donor puberty. We demonstrated that C gilts provided more BCB+ oocytes as well as more large oocytes than P gilts, although C-BCB+ oocytes showed higher apoptotic rate. In conclusion, high incidence of apoptosis and a big variation in the diameter of more competent BCB+ oocytes make the BCB test a less effective selection tool than previously reported.     

Circulating beta-chemokines and matrix metalloproteinase-9/tissue inhibitor of metalloproteinase-1 system in hemodialyzed patients--role of oxidative stress

Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs), beta-chemokines, increased oxidative stress (SOX) and inflammation have been implicated as important factors in atherosclerosis and vascular remodeling. We hypothesized the possible roles of beta-chemokines [monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory proteins (MIP-1alpha, MIP-1beta) and regulated upon activation, normal T-cell expressed and secreted (RANTES)] as regulators of the metabolism of the vascular extracellular matrix in conditions of increased SOX in hemodialysis (HD) patients. We compared pre-dialysis levels of MMP-9/TIMP-1 system, beta-chemokines, Cu/Zn superoxide dismutase (Cu/Zn SOD) as a marker of SOX and C-reactive protein (CRP) as a marker of inflammation in HD patients with and without cardiovascular disease (CVD) to those of controls. HD patients, particularly those with CVD, showed a significant increase in values of Cu/Zn SOD, CRP, TIMP-1, TIMP-1/MMP-9 ratio, MCP-1 and MIP-1beta, whereas RANTES levels were lower than in the controls. The levels of MIP-1alpha as well as MMP-9 in all HD groups were similar to the controls. The positive correlations were observed between the MMP-9/TIMP-1 system and beta-chemokines, SOX and inflammation in whole HD group and in the subgroup with CVD. Multivariate analysis showed that the duration of dialysis followed by Cu/Zn SOD, MIP-1alpha and beta levels were the significant positive predictors of TIMP-1. In conclusion, our data show that MMP-9/TIMP-1 system and beta-chemokines could cooperate in conditions of elevated SOX, which ultimately predisposes hemodialysis patients to accelerated atherosclerosis.     

Antibiotic-resistant Escherichia coli in karstic systems: a biological indicator of the origin of fecal contamination?

Occurrences of antibiotic-resistant Escherichia coli in two springs of a karstic system (NW France) providing drinking water were determined to study the role of aquifers in the dissemination of the resistance genes. Water samples were collected during wet and dry periods and after a heavy rainfall event to investigate E. coli density, antibiotic resistance patterns, and occurrences of class 1, 2, and 3 integrons. By observing patterns of the resistant isolates (i.e. number and type of resistances) and their occurrences, we were able to define two resistant subpopulations, introduced in the aquifer via surface water: (1) R1-2, characterized by one or two resistance(s), essentially to chloramphenicol and/or tetracycline (96.5%), was always found during the heavy rainfall event; (2) R3-10, characterized by three or more resistances, mostly resistant to tetracycline (94.1%) and beta-lactams (86%), was found transiently. Class 1 and 2 integrons were detected, mostly in the R3-10 subpopulation for class 1 integrons. The characteristics of these two subpopulations strongly suggest that the contamination originates from pasture runoff for the R1-2 subpopulation and from wastewater treatment plant effluents for the R3-10 subpopulation. These two subpopulations of E. coli could be used as biological indicators to determine the origin of groundwater contamination.