Marie-Jeanne Richard

Increased lipid peroxidation in pregnant women after iron and vitamin C supplementation

Iron overload could promote the generation of free radicals and result in deleterious cellular damages. A physiological increase of oxidative stress has been observed in pregnancy. A routine iron supplement, especially a combined iron and vitamin C supplementation, without biological justifications (low hemoglobin [Hb] and iron stores) could therefore aggravate this oxidative risk. We investigated the effect of a daily combined iron supplementation (100 mg/d as fumarate) and vitamin C (500 mg/d as ascorbate) for the third trimester of pregnancy on lipid peroxidation (plasma TBARS), antioxidant micronutriments (Zn, Se, retinol, vitaminE, (β-carotene) and antioxidant metalloenzymes (RBC Cu-Zn SOD and Se-GPX). The iron-supplemented group (n=27) was compared to a control group (n=27), age and number of pregnancies matched. At delivery, all the women exhibited normal Hb and ferritin values. In the supplemented group, plasma iron level was higher than in the control group (26.90±5.52 mmol/L) and TBARs plasma levels were significantly enhanced (p<0.05) (3.62±0.36 vs 3.01±0.37 mmol/L). No significant changes were observed in plasma trace elements and red blood cell antioxidant metalloenzymes. Furthermore, the α-tocopherol plasma level was lowered in the iron-supplemented groups, suggesting an increased utilization of vitamin E.These data show that pharmalogical doses of iron, associated with high vitamin C intakes, can result in uncontrolled lipid peroxidation. This is predictive of adverse effects for the mother and the fetus. This study illustrates the potential harmful effects of iron supplementation when prescribed only on the assumption of anemia and not on the bases of biological criteria.

Selenium and training effects on the glutathione system and aerobic performance.

Changes in blood glutathione antioxidant system in response to exercise and training, and aerobic performance, were investigated. Selenium (Se) supplementation effects on these changes were evaluated. The study was double blind. Both groups selenium (Sel, N = 12) and placebo (Pla, N = 12), followed a 10-wk endurance training program, with a prolonged exhaustive exercise bout performed (Cap Max), before (Pre) and after (Post) training. Blood was sampled before (Bef) and after (Aft) Cap Max. The oxidation of blood glutathione after Cap Max exercise showed a reactive oxygen species production. Training developed maximal aerobic power and capacity, significantly increased (P < 0.001) plasma and erythrocyte glutathione peroxidase (GPx) activity, and decreased (P < 0.001) erythrocyte glutathione reductase activity. The Se supplementation caused an increase in the basal plasma GPx level (P < 0.05). There was also a correlation (r = 0.66, P < 0.05) between the variation in VO2max and that of erythrocyte GPx only in supplemented subjects. Our results confirm that blood glutathione remains a sensitive marker of oxidative stress induced by exhausting submaximal exercise and that the antioxidant potential of GPx can be developed by endurance training. Se supplementation at the dose used had no effect on physical performance.

Pyrosequencing method to detect KRAS mutation in formalin-fixed and paraffin-embedded tumor tissues.

KRAS mutation status has been reported to be a predictive marker of tumor response to epidermal growth factor receptor (EGFR) inhibitors. We have designed a pyrosequencing assay based on nested polymerase chain reaction (PCR) to characterize KRAS mutation status using formalin-fixed and paraffin-embedded tumor tissues. Mutant and wild-type KRAS cell lines were used to determine the specificity and sensitivity (detection limit approximately 5% mutant alleles) of the method. The results obtained for tumor samples were 95% comparable to those obtained by dideoxy sequencing. Analysis of KRAS mutation using nested PCR and pyrosequencing is a simple, robust, fast, and sensitive method that can be used with formalin-fixed and paraffin-embedded tissues.

Induction of thioredoxin by ultraviolet-A radiation prevents oxidative-mediated cell death in human skin fibroblasts

The present study analyzes the expression of the thioredoxin/thioredoxin reductase (Trx/TR) system in UVA-irradiated human skin fibroblasts. Irradiation increases the intracellular level of Trx and a time-dependent increase of Trx mRNA is observed. Our data indicate that Trx translocates from the cytoplasm to the nucleus. In addition, UV exposure results in an increase in TR synthesis. In order to evaluate the function of Trx/TR system, we investigated the antioxidant role of Trx in transient transfected cells. The ROS accumulation in UVA irradiated cells was assessed using flow cytometry. A 3-fold decrease in ROS production was observed in transiently transfected fibroblasts. These results indicate that Trx acts as an antioxidant protein in UVA irradiated fibroblasts. As ROS are inducers of cell death, this raises the question as to whether Trx is able to protect cells from apoptosis and/or necrosis induced by UVA. Six hours after UVA-irradiation, 29.92% of cells were annexin-V positive. This population was significantly reduced in Trx-transfected cells (8.58%). Moreover, this work demonstrates that Trx prevents the loss of the membrane potential of the mitochondria, the depletion of cellular ATP content, and the loss of cell viability induced by irradiation.

Antioxidant supplementation preserves antioxidant response in physical training and low antioxidant intake

The present controlled-training double-blind study (supplemented (S) group, n 7; placebo (P) group, n 10) was designed to investigate whether an antioxidant mixture (Se 150 microg, retinyl acetate mg, ascorbic acid 120 mg, alpha-tocopheryl succinate 20 mg) would allow overloaded triathletes to avoid adaptation failure in the antioxidant system [corrected]. Dietary intakes were recorded. The supplement of Se, and vitamins A and E provided 100 % of the French RDA. Four weeks of overloaded training (OT) followed 4 weeks of normal training (NT). After NT and OT, biological studies were conducted at rest and after a duathlon test (run 5 km, cycle 20 km, run 5 km). During the 4-week period of NT, blood levels of GSH levels increased in response to supplementation (P<0.05) and remained elevated during OT. Plasma glutathione peroxidase activity was significantly higher in the S group in all situations after NT and OT (P<0.01). The S group had increased erythrocyte Cu,Zn-superoxide dismutase activity in response to OT (P<0.05). Supplementation significantly reduced (P<0.05) the magnitude in duathlon-induced creatine kinase isoenzyme MB mass increase, which tended to be higher with OT (P=0.09). We conclude that the antioxidant mixture helped to preserve the antioxidant system during an OT-induced stress in subjects with initially low antioxidant intakes. Effects of supplementation during NT and/or OT are shown mostly by the alleviated muscle damage. The effects of the antioxidant mixture were observed for doses that can be provided by a diversified and well-balanced diet. The maintenance of normal nutritional status with regard to the antioxidant intake (Se, vitamins C and E) plays a key role in antioxidant adaptive effects during NT and OT.

THIOLS AND SELENIUM : PROTECTIVE EFFECT ON HUMAN SKIN FIBROBLASTS EXPOSED TO UVA RADIATION

The sensitivity of human dermal fibroblasts to UVA radiation has been linked to a decrease in intracellular glutathione (GSH) levels. GSH (gamma-glutamyl-cysteinyl-glycine) is a radical scavenger and a cofactor for protective enzymes such as selenium-dependent GSH peroxidases. In this study, we examine the possibility of a cooperative interaction between three cysteine delivery systems and selenium in protecting human cultured fibroblast exposed to UVA radiation. Cells were irradiated (9, 15 and 20 J cm-2) following incubation with N-acetyl-cysteine (NAC, 5 mM), N-acetyl-homocysteine-thiolactone (citiolone (CIT), 1 mM) or L-2-oxothiazolidine-4-carboxylate (OTC, 1 mM). The modulation of the intracellular GSH levels by the addition of the different compounds was determined by enzymatic and separative methods. Cells were harvested for survival analysis by measuring the ability of the cell to adhere and proliferate. Treatments with NAC and CIT resulted in a significant rise in GSH levels compared with control cells, with protection against UVA radiation. OTC did not induce any rise in GSH level; nevertheless, the protective effect afforded by OTC is similar to that observed with NAC and CIT. Moreover, selenium (0.1 mg 1-1), as sodium selenite, significantly increased the protective efficiency of NAC and CIT, but not of OTC. Although the precise mechanism is not known, thiol molecules can inhibit the deleterious effects of UVA radiation. These results provide evidence that compounds capable of inducing GSH synthesis can act with selenium to protect cells against UVA damage.

Effects of cadmium and zinc on solar-simulated light-irradiated cells: potential role of zinc-metallothionein in zinc-induced genoprotection.

Zinc is an essential oligoelement for cell growth and cell survival and has been demonstrated to protect cells from oxidative stress induced by UVA or from genotoxic stress due to UVB. In a recent work we demonstrated that the antioxidant role of zinc could be related to its ability to induce metallothioneins (MTs). In this study we identified the mechanism of zinc protection against solar-simulated light (SSL) injury. Cultured human keratinocytes (HaCaT) were used to examine MTs expression and localization in response to solar-simulated radiation. We found translocation to the nucleus, with overexpression of MTs in irradiated cells, a novel observation. The genoprotective effect of zinc was dependent on time and protein synthesis. DNA damage was significantly decreased after 48 h of ZnCl(2) (100 microM) treatment and is inhibited by actinomycin D. ZnCl(2) treatment (100 microM) led to an intense induction, redistribution, and accumulation of MT in the nucleus of irradiated cells. MT expression correlated with the time period of ZnCl(2) treatment. CdCl(2), a potent MT inducer, did not show any genoprotection, although the MTs were expressed in the nucleus. Overall our findings demonstrate that MTs could be a good candidate for explaining the genoprotection mediated by zinc on irradiated cells.

The dual effect of mesenchymal stem cells on tumour growth and tumour angiogenesis.

IntroductionUnderstanding the multiple biological functions played by human mesenchymal stem cells (hMSCs) as well as their development as therapeutics in regenerative medicine or in cancer treatment are major fields of research. Indeed, it has been established that hMSCs play a central role in the pathogenesis and progression of tumours, but their impact on tumour growth remains controversial.MethodsIn this study, we investigated the influence of hMSCs on the growth of pre-established tumours. We engrafted nude mice with luciferase-positive mouse adenocarcinoma cells (TSA-Luc+) to obtain subcutaneous or lung tumours. When tumour presence was confirmed by non-invasive bioluminescence imaging, hMSCs were injected into the periphery of the SC tumours or delivered by systemic intravenous injection in mice bearing either SC tumours or lung metastasis.ResultsRegardless of the tumour model and mode of hMSC injection, hMSC administration was always associated with decreased tumour growth due to an inhibition of tumour cell proliferation, likely resulting from deep modifications of the tumour angiogenesis. Indeed, we established that although hMSCs can induce the formation of new blood vessels in a non-tumoural cellulose sponge model in mice, they do not modify the overall amount of haemoglobin delivered into the SC tumours or lung metastasis. We observed that these tumour vessels were reduced in number but were longer.ConclusionsOur results suggest that hMSCs injection decreased solid tumour growth in mice and modified tumour vasculature, which confirms hMSCs could be interesting to use for the treatment of pre-established tumours.

L-arginine increases UVA cytotoxicity in irradiated human keratinocyte cell line: potential role of nitric oxide.

Human fibroblasts and keratinocytes possess nitric oxide synthases (NOS), which metabolize L‐arginine (L‐Arg) for producing nitric oxide (NO•). This report delineates the relations between NO• and UVA in the human keratinocyte cell line HaCaT. NOS activity was stimulated by exposure of cells to L‐Arg just after irradiation. L‐Arg (5 mM) supply led to an increase in UVA (25.3 J/cm2) cytotoxicity (% of viability 18 ± 3%) whereas neither L‐Arg itself nor UVA irradiation induced cell death at the doses used in this study. Cells were also treated either with L‐thiocitrulline (L‐Thio), an irreversible inhibitor of NOS, or with exogenous superoxide dismutase (SOD) and catalase. L‐Thio and SOD prevented L‐Arg‐mediated deleterious effects in irradiated cells, whereas catalase was ineffective. Intracellular antioxidant enzyme activities were also determined. UVA/L‐Arg stress altered catalase (66% decrease) and glutathione peroxidase (83% decrease). DNA damage was evaluated using the ‘comet assay’ and quantified using the ‘tail moment’. UVA alone was genotoxic (mean tail moment: 25.43 ± 1.23, P<0.001 compared control cells). The addition of L‐Arg potentiated DNA damage (mean tail moment: 41.05±3.9) whereas L‐Thio prevented them (mean tail moment 9.86 ± 0.98). We attempted to assess the effect of poly(ADP‐ribose) polymerase (PARP) inhibition on cell death. Using the PARP inhibitor 3‐aminobenzamide, we established that PARP determines both cell lysis and DNA damage induced by UVA and/or L‐Arg. Our findings demonstrated that L‐Arg was able to increase UVA‐mediated deleterious effects in keratinocytes (both DNA damage and cytotoxicity) and that the ratio NO•/O2•‐plays a key role in these processes.—Didier, C., Emonet‐Piccardi, N., Béani, J.‐C., Cadet, J., Richard, M.‐J. L‐arginine increases UVA cytotoxicity in irradiated human keratinocyte cell line: potential role of nitric oxide. FASEB J. 13, 1817–1824 (1999)

Effect of Okadaic Acid, a Protein Phosphatase Inhibitor, on Heat Stress-Induced HSP72 Synthesis and Thermotolerance

Heat stress proteins (HSPs), in particular HSP72, seem to play a major role in cell protection against lethal stresses such as hyperthermia or ischemia. HSP synthesis is negatively regulated by protein phosphatases, which are implicated in dephosphorylation processes. In the present study, we have investigated the effect of okadaic acid (OA, a protein phosphatase inhibitor) on heat stress-induced HSP72 synthesis and thermotolerance in smooth muscle cells (SMC).SMC were heat stressed (42°C for 20 minutes) in the presence of 250 nM OA (HS+OA cells) or its vehicle (HS+V cells). Control (OA or V) cells were not heat stressed. HSP72 mRNA expression was determined 1, 1.5, 3, and 6 hours after heat stress by RT-PCR, and HSP72 synthesis was determined 6, 12, 24, 48, and 72 hours after heat stress by Western blotting. SMC survival of lethal hyperthermia (47°C for 90 minutes) was assessed 6, 24, and 48 hours after heat stress by a tetrazolium assay.The maximal expression of HSP72 mRNA was markedly prolonged in HS+OA cells (until 6 hours after heat stress) compared to HS+V cells (1 hour after heat stress). The kinetics of HSP72 synthesis and thermotolerance of SMC were not different between HS+OA and HS+V cells. Baseline HSP72 mRNA and protein expression were similar in control V and OA cells.In conclusion, okadaic acid treatment of SMC potentiated HSP72 mRNA expression without affecting heat stress-induced HSP72 synthesis and thermotolerance.