The Radioprotective Effect of Procaine and Procaine-Derived Product Gerovital H3 in Lymphocytes from Young and Aged Individuals.

Ionizing radiation induces genomic instability in living organisms, and several studies reported an ageing-dependent radiosensitivity. Chemical compounds, such as scavengers, radioprotectors, and modifiers, contribute to reducing the radiation-associated toxicity. These compounds are often antioxidants, and therefore, in order to be effective, they must be present before or during exposure to radiation. However, not all antioxidants provide radioprotection. In this study, we investigated the effects of procaine and of a procaine-based product Gerovital H3 (GH3) on the formation of endogenous and X-ray-induced DNA strand breaks in peripheral blood mononuclear cells (PBMCs) isolated from young and elderly individuals. Interestingly, GH3 showed the strongest radioprotective effects in PBMCs from young subjects, while procaine reduced the endogenous amount of DNA strand breaks more pronounced in aged individuals. Both procaine and GH3 inhibited lipid peroxidation, but procaine was more effective in inhibiting mitochondria free radicals' generation, while GH3 showed a higher antioxidant action on macrophage-induced low-density lipoprotein oxidation. Our findings provide new insights into the mechanisms underlying the distinct effects of procaine and GH3 on DNA damage.

Early mitochondrial fragmentation is a potential in vitro biomarker of environmental stress.

Mitochondria are essential dynamic organelles that ordinarily balance between fragmentation and fusion. Under stress conditions, a shift toward fragmentation or hyper-fusion is observed as a pro-survival reaction. Fragmentation of mitochondria occurs within minutes or hours after the beginning of the stress and occurs in response to a large number of stress stimuli, including those triggered by environmental contaminants. In this study, we tested whether the change in the mitochondrial phenotype, from tubular to fragmented, could be used as a potential environmental stress biomarker in cells and compared this response with the standard MTT-based viability assay. Firstly, we show that mitochondrial fragmentation induced by selected stressors not only increases with concentrations, but also correlates positively with the cytotoxicity. Secondly, we found that the mitochondrial fragmentation that occurs in the first hour of stress correlated with the viability measured after a 24-h stress, allowing the establishment of a linear relation between mitochondrial fragmentation at 1 h and the predictable associated cytotoxicity of environmental contaminants alone or in mixture. In conclusion, we have succeeded in developing a model of predictable 24 h-cytotoxicity given mitochondrial fragmentation at 1 h with a set of chemicals. This model has been successful applied to three environmental toxicants and to a set of two chemical mixtures. We thus propose that mitochondrial fragmentation is a response that could be used as an early in vitro biomarker of environmental stress for toxicants alone or in mixture.

Application of a multidisciplinary and integrative weight-of-evidence approach to a 1-year monitoring survey of the Seine River.

Quality assessment of environments under high anthropogenic pressures such as the Seine Basin, subjected to complex and chronic inputs, can only be based on combined chemical and biological analyses. The present study integrates and summarizes a multidisciplinary dataset acquired throughout a 1-year monitoring survey conducted at three workshop sites along the Seine River (PIREN-Seine program), upstream and downstream of the Paris conurbation, during four seasonal campaigns using a weight-of-evidence approach. Sediment and water column chemical analyses, bioaccumulation levels and biomarker responses in caged gammarids, and laboratory (eco)toxicity bioassays were integrated into four lines of evidence (LOEs). Results from each LOE clearly reflected an anthropogenic gradient, with contamination levels and biological effects increasing from upstream to downstream of Paris, in good agreement with the variations in the structure and composition of bacterial communities from the water column. Based on annual average data, the global hazard was summarized as "moderate" at the upstream station and as "major" at the two downstream ones. Seasonal variability was also highlighted; the winter campaign was least impacted. The model was notably improved using previously established reference and threshold values from national-scale studies. It undoubtedly represents a powerful practical tool to facilitate the decision-making processes of environment managers within the framework of an environmental risk assessment strategy.

First characterization of the endocrine-disrupting potential of indoor gaseous and particulate contamination: comparison with urban outdoor air (France).

The composition of endocrine-disrupting compounds (EDCs) in the ambient air of indoor environments has already been described, but little is known about the inherent endocrine-disrupting potential of indoor air contamination. We therefore aimed to study the distribution of bioactive EDCs in the gaseous and particulate phases of indoor air using a cellular bioassay approach that integrates the interaction effects between chemicals. Organic air extracts, both gaseous and particulate, were taken from three indoor locations (office, apartment, and children's day care) in France and sampled in two different seasons in order to study their interference with the signaling of estrogen, androgen, and thyroid receptors. The experiments were also conducted on aerial extracts from an outdoor site (urban center). We found that gaseous and/or particulate extracts from all locations displayed estrogenicity, anti-androgenicity, and thyroidicity. Overall, indoor air extracts had a higher endocrine-disrupting potential compared to outdoor ones, especially during winter and in the day care. The biological activities were predominant for the gaseous extracts and tended to increase for the particulate extracts in cool conditions. In conclusion, our data confirmed the presence of bioactive EDCs in a gaseous state and highlighted their indoor origin and concentration, especially in the cold season.

A new analytical protocol for the determination of 62 endocrine-disrupting compounds in indoor air.

The objective of this study was to develop and validate a new analytical protocol for simultaneous determination of 62 semi-volatile organic compounds in both phases of indoor air. Studied compounds belong to several families: polybrominated diphenyl ethers, polychlorinated biphenyls, hexachlorobenzene, pentachlorobenzene, phthalates, polyaromatic hydrocarbons, parabens, tetrabromobisphenol A, bisphenol A, hexabromocyclododecane, triclosan, alkylphenols, alkylphenol ethoxylates, synthetic musks (galaxolide and tonalide) and pesticides (lindane and cypermethrin). A medium volume sampling system was used to collect simultaneously these endocrine-disrupting compounds (EDCs) from the gaseous and particulate phases. An accelerated solvent extraction method was optimized to obtain all EDCs in a single extract by atmospheric phase. Their extraction from the sorbents and their analysis by liquid and gas chromatography-mass spectrometry (LC/MS/MS, GC/MS and GC/MS/MS) was validated using spiked sorbents (recovery study and analytical uncertainty analysis by fully nested design). The developed protocol achieved low limits of quantification (<0.5ng m(-3)) and low uncertainty values (<5ng m(-3)) for all compounds. Once validated, the method was applied to indoor air samples from four locations (a house, an apartment, a day nursery and an office) and compared to literature to confirm its efficiency. All target EDCs were quantified in the samples and were primarily present in the gaseous phase. The major contaminants found in indoor air were, in descending order, phthalates, synthetic musks, alkylphenols and parabens.

First evidence of estrogenic potential of the cyanobacterial heptotoxins the nodularin-R and the microcystin-LR in cultured mammalian cells.

The estrogenic activity of cyanobacterial hepatotoxins microcystin-LR (MC-LR) and nodularin-R (NOD-R) was for the first time investigated invitro in a stably transfect cell line with an estrogen-regulated luciferase gene. Treatment of cells with NOD-R caused a dose-dependent increase in the luciferase activity. NOD-R gave rise to an induction of luciferase activity with an EC(50) value of 66.4 nM, whereas the positive control, 17beta-estradiol (E2) had an EC(50) of 9.6 pM. This indicates that NOD-R is a 6900-fold weaker inducer of luciferase than E2. In contrast, only a slight but significant activation of the luciferase gene was observed with MC-LR between 2.01 and 60.1 nM, and a maximal-induced response was observed with 10.1 nM, approximately 25% of the maximal effect obtained with 1 nM E2. The decrease in the luciferase activity at high MC-LR concentrations can be explained by a cytotoxic effect. No synergistic estrogenic effect was observed when each toxin was co-administrated with E2. However, the induction of the luciferase activity by NOD-R and MC-LR was inhibited by co-treatment with 1 microM of the pure estrogenic receptor (ER) antagonist ICI 182,780, thus proving the ER-dependency of the estrogenic effect.

Cell culture media are potent antioxidants that interfere during LDL oxidation experiments.

In vitro cell-induced low-density lipoprotein (LDL) oxidation is a model frequently used for studies on antioxidant compounds which may be potentially antiatherogens. Using Cu2+ or the free radical generator 2,2'-azobis-[2-amidinopropane] dihydrochloride (AAPH) to oxidize human LDL, we showed that the cell culture media Ham's F10 and RPMI are potent antioxidants which reduce LDL-protective effect of various thyroid compounds. The culture media interfered with the compounds depending on their mechanism of action, and RPMI had the greatest antioxidant effect, completely hiding antioxidant efficiency of the compounds whatever the prooxidant agent was. We suggest some recommendations for study of antioxidant compounds using cell-induced LDL oxidation models.

Thyroid hormone (T3) and its acetic derivative (TA3) protect low-density lipoproteins from oxidation by different mechanisms.

Triiodothyronine (T3) and triiodothyroacetic acid (TA3) are thyroid compounds that similarly protect low-density lipoprotein (LDL) against oxidation induced by the free radical generator 2,2'-azobis-[2-amidinopropane] dihydrochloride (AAPH). However, TA3 is more antioxidant than T3 on LDL oxidation induced by copper ions (Cu2+), suggesting that these compounds act by different mechanisms. Here we measured conjugated diene production kinetics during in vitro human LDL (50 mg LDL-protein per l) oxidation induced by various Cu2+ (0.5-4 microM) or AAPH (0.25-2 mM) concentrations in the presence of T3, TA3, butylated hydroxytoluene (BHT) (a free radical scavenger) or ethylenediaminetetracetic acid (EDTA) (a metal chelator). From the kinetics were estimated: length of the lag phase (Tlag), maximum velocity of conjugated diene production (Vmax), and maximum amount of generated dienes (Dmax). Thyroid compound effects on these oxidation parameters were compared to those of the controls BHT and EDTA. In addition we measured by atomic absorption spectrometry copper remaining in LDL after a 30 min incubation of LDL with Cu2+ and the compounds followed by extensive dialysis, i.e. copper bound to LDL. As expected, LDL-copper was decreased by EDTA in a concentration-dependent manner, whereas it was not affected by BHT. T3 increased LDL-copper whereas TA3 slightly decreased it. The whole data suggest that T3 and TA3 are free radical scavengers that also differently disturb LDL-copper binding, an essential step for LDL lipid peroxidation. The most likely mechanisms are that T3 induces new copper binding sites inside the LDL particle, increasing the LDL-copper amount but in a redox-inactive form, whereas TA3 blocks some redox-active copper binding sites highly implicated in the initiation and the propagation of lipid peroxidation. Alternatively, we also found that a little amount of copper is tightly bound in LDL, which may be essential for the propagation of lipid peroxidation induced by free radical generators.

9-cis-Retinoic acid regulation of four UGT isoforms in hepatocytes from rats with various thyroid states.

PURPOSE: To investigate the influence of thyroid hormone status on the regulation of UGTs expression by 9-cis-retinoic acid in cultured rat primary hepatocytes. METHODS: Hepatocytes from rats with various thyroid states were isolated and treated with 9-cis retinoic acid (1 x 10(-6) M). mRNA was amplified by reverse transcription and polymerase chain reaction (RT-PCR) and quantified by UV light densitometry. Variations in the expression levels of four different UGT isoforms (UGT1A1, 1A2, 1A5, and 1A6) that are involved in the glucuronication of bilirubin and phenols were determined by comparison with those of an internal standard, beta-actin, which is known to be insensitive to nutritional and hormonal conditions. RESULTS: Primary hepatocyte cultures from rats with various thyroid states present similar metabolite characteristics to those from hypo- or hyperthyroid animals. The treatment of hepatocytes from hypothyroid rats with 9-cis-retinoic acid (1 x 10(-6) M) did not significantly modify bilirubin and phenol-UGT isoform expression. In contrast, in hepatocytes from normal and specially hyperthyroid rats treated with 9-cis-retinoic acid, UGT mRNA levels were modified. This suggests that the effect of retinoic acid on UGT mRNA expression requires the presence of thyroid hormone. This was confirmed by the treatment of cultured hepatocytes from hypothyroid rats with both retinoic acid and L-T3. CONCLUSIONS: This study demonstrates that in cultured hepatocytes, the thyroid status can differentially modulate the expression of four UGT isoforms, and the regulation of their expression can be affected by 9-cis-retinoic acid.