Ciprofloxacin-induces free radical production in rat cerebral microsomes.

In the presence of ciprofloxacin (CPFX), free radical adduct formation was demonstrated in rat cerebral microsomes using a spin trap α-(4-pyridyl-1-oxide)-N-tert-butyl-nitrone by electron spin resonance spectroscopy. Active microsomes, dihydronicotinamide-adenine dinucleotide phosphate, and ciprofloxacin were necessary for the formation of a spin trap/radical adduct. Adduct formation increased dose-dependently at 0.5-1 mM CPFX concentration for 180 min, and 0.3-1 mM concentration level for 240 min. The addition of SKF 525A, ZnCl2 or desferrioxamine to the incubation system caused complete inhibition of the radical formation. However, pretreatment of microsomal system with superoxide dismutase (SOD) did not induce any protective effect. Induction of lipid peroxidation, and depletion of thiol levels by CPFX were also shown in the system. These results strongly suggested that CPFX produces free radical(s) in the cerebral microsomes of rats.

The effects of di(2-ethylhexyl) phthalate and/or selenium on trace element levels in different organs of rats.

Di(2-ethylhexyl)phthalate (DEHP), a widely used plasticizer for synthetic polymers, is known to have endocrine disruptive potential, reproductive toxicity, and induces hepatic carcinogenesis in rodents. Selenium (Se) is a component of several selenoenzymes which are essential for cellular antioxidant defense and for the functions of mammalian reproductive system. The present study was designed to investigate the effects of DEHP exposure on trace element distribution in liver, testis, and kidney tissues and plasma of Se-deficient and Se-supplemented rats. Se deficiency was produced by feeding 3-week old Sprague-Dawley rats with ≤0.05mg Se/kg diet for 5 weeks, and supplementation group were on 1mg Se/kg diet. DEHP treated groups received 1000mg/kg dose by gavage during the last 10 days of feeding period. Se, zinc (Zn), copper (Cu), iron (Fe) and manganese (Mn) levels were measured by inductively coupled plasma mass spectrometry (ICP-MS). Se supplementation caused significant increases in hepatic, renal, and testicular Se levels. With DEHP exposure, plasma Se and Zn, kidney Se, Cu and Mn levels were significantly decreased. Besides, liver Fe decreased markedly in all the DEHP-treated groups. Liver and kidney Mn levels decreased significantly in DEHP/SeD group compared to both DEHP and SeD groups. These results showed the potential of DEHP exposure and/or different Se status to modify the distribution pattern of essential trace elements in various tissues, the importance of which needs to be further evaluated.

The effects of di(2-ethylhexyl)phthalate on rat liver in relation to selenium status.

This study was performed to determine the hepatotoxicity of di(2-ethylhexyl)phthalate (DEHP) in relation to selenium status. In 3-week-old Sprague-Dawley rats, selenium deficiency was induced by a ≤0.05 selenium mg/kg. A selenium supplementation group was given 1 mg selenium/kg diet for 5 weeks. Di(2-ethylhexyl)phthalate-treated groups received 1000 mg/kg dose by gavage during the last 10 days of the experiment. Histopathology, peroxisome proliferation, catalase (CAT) immunoreactivity and activity and apoptosis were assessed. Activities of antioxidant selenoenzymes [glutathione peroxidase 1 (GPx1), glutathione peroxidase 4 (GPx4), thioredoxin reductase (TrxR1)], superoxide dismutase (SOD), and glutathione S-transferase (GST); aminotransferase, total glutathione (tGSH), and lipid peroxidation (LP) levels were measured. Di(2-ethylhexyl)phthalate caused cellular disorganization while necrosis and inflammatory cell infiltration were observed in Se-deficient DEHP group (DEHP/SeD). Catalase activity and immunoreactivity were increased in all DEHP-treated groups. Glutathione peroxidase 1 and GPx4 activities decreased significantly in DEHP and DEHP/SeD groups, while GST activities decreased in all DEHP-exposed groups. Thioredoxin reductase activity increased in DEHP and DEHP/SeS, while total SOD activities increased in all DEHP-treated groups. Lipid peroxidation levels increased significantly in SeD (26%), DEHP (38%) and DEHP/SeD (71%) groups. Selenium supplementation partially ameliorated DEHP-induced hepatotoxicity; while in DEHP/SeD group, drastic changes in hepatic histopathology and oxidative stress parameters were observed.

Effects of di(2-ethylhexyl)phthalate on testicular oxidant/antioxidant status in selenium-deficient and selenium-supplemented rats.

Di(ethylhexyl)phthalate (DEHP), the most widely used plasticizer, was investigated to determine whether an oxidative stress process was one of the underlying mechanisms for its testicular toxicity potential. To evaluate the effects of selenium (Se), status on the toxicity of DEHP was further objective of this study, as Se is known to play a critical role in testis and in the modulation of intracellular redox equilibrium. Se deficiency was produced in 3-weeks-old Sprague-Dawley rats feeding them ≤0.05 mg Se /kg diet for 5 weeks, and Se-supplementation group was on 1 mg Se/kg diet. DEHP-treated groups received 1000 mg/kg dose by gavage during the last 10 days of the feeding period. Activities of antioxidant selenoenzymes [glutathione peroxidase 1 (GPx1), glutathione peroxidase 4 (GPx4), thioredoxin reductase (TrxR)], catalase (CAT), superoxide dismutase (SOD), and glutathione S-transferase (GST); concentrations of reduced glutathione (GSH), oxidized glutathione (GSSG), and thus the GSH/GSSG redox ratio; and thiobarbituric acid reactive substance (TBARS) levels were measured. DEHP was found to induce oxidative stress in rat testis, as evidenced by significant decrease in GSH/GSSG redox ratio (>10-fold) and marked increase in TBARS levels, and its effects were more pronounced in Se-deficient rats with ∼18.5-fold decrease in GSH/GSSG redox ratio and a significant decrease in GPx4 activity, whereas Se supplementation was protective by providing substantial elevation of redox ratio and reducing the lipid peroxidation. These findings emphasized the critical role of Se as an effective redox regulator and the importance of Se status in protecting testicular tissue from the oxidant stressor activity of DEHP.

Thyroidal effects of di-(2-ethylhexyl) phthalate in rats of different selenium status.

This study was designed to investigate the effects of di-(2-ethylhexyl) phthalate (DEHP) on thyroid hormone levels and oxidant/antioxidant parameters in the rat and to evaluate the effects of selenium status. Selenium deficiency was produced by feeding 3-week-old Sprague-Dawley rats with <0.05 mg selenium/kg body weight for 5 weeks, and the supplementation group received a diet of 1 mg selenium/kg body weight. DEHP-treated groups received the compound at a dose of 1000 mg/kg by gavage during the last 10 days of the feeding period. Levels of thyroid hormone levels as well as selenoenzyme (glutathione peroxidase 1, thioredoxin reductase), catalase, and superoxide dismutase (SOD) activity and thiobarbituric acid reactive substance (TBARS) were measured. Total thyroxine (TT4) levels decreased significantly with DEHP exposure (~25%), whereas TT3 level was not altered. The TT4 lowering effect of DEHP exposure was not affected by selenium deficiency but was observed when animals exposed to DEHP received a selenium supplement. DEHP was found to alter the antioxidant status and induce oxidative stress in rat thyroid by increasing SOD activity (~30%) and TBARS levels (~35%). The effects of DEHP were much more pronounced in selenium-deficient rats, as evidenced by significant increases in SOD activity (~65%) and TBARS levels (~55%) compared with the control levels. Thus, these results show the thyroid-disrupting effect of DEHP in rats and protection by selenium.

Di(2-ethylhexyl)phthalate-induced renal oxidative stress in rats and protective effect of selenium.

This study was designed to examine the oxidative stress potential of di(2-ethylhexyl)phthalate (DEHP) on rat kidney and to evaluate possible protective effect of selenium (Se) status. Se deficiency (SeD) was produced in 3-week old Sprague-Dawley rats by feeding them ≤ 0.05 Se mg/kg diet for 5 weeks; Se supplementation group (SeS) was on 1 mg Se/kg diet. DEHP treated groups received 1000 mg/kg dose by gavage during the last 10 days of the feeding period. Activities of antioxidant selenoenzymes [glutathione peroxidase 1 (GPx1), glutathione peroxidase 4 (GPx4), thioredoxin reductase (TrxR)], catalase (CAT), superoxide dismutase (SOD), and glutathione S-transferase (GST); concentrations of total glutathione (GSH), thiols and thiobarbituric acid reactive substance (TBARS) levels were measured. DEHP treatment was found to induce oxidative stress in rat kidney, as evidenced by significant decreases in GPx1 (~20%) and SOD (~30%) activities and GSH levels (~20%), along with marked decrease in thiol content (~40%) and increase in TBARS (~30%) levels. The effects of DEHP was more pronounced in SeD rats, whereas Se supplementation was protective by providing substantial elevations of GPx1 and GPx4 activities and GSH levels. These findings emphasized the critical role of Se as an effective redox regulator and the importance of Se status in protecting renal tissue from the oxidant stressor activity of DEHP.

Oxidative stress induced by cadmium in the C6 cell line: role of copper and zinc.

In this report, we have investigated the role of copper (Cu) and zinc (Zn) in oxidative stress induced by cadmium (Cd) in C6 cells. Cells were exposed to 20 µM Cd, 500 µM Cu, and 450 µM Zn for 24 h. Then, toxic effects, cellular metals levels, oxidative stress parameters, cell death, as well as DNA damage were evaluated. Cd induced an increase in cellular Cd, Cu, and Zn levels. This results not only in the inhibition of GSH-Px, GRase, CAT, and SOD activities but also in ROS overproduction, oxidative damage, and apoptotic cell death not related to Cu and Zn mechanisms. The thiol groups and GSH levels decreased, whereas the lipid peroxidation and DNA damage increased. The toxicity of Zn results from the imbalance between the inhibition of antioxidant activities and the induction of MT synthesis. The increase in Cu and Zn levels could be explained by the disruption of specific transporter activities, Cd interference with signaling pathways, and metal displacement. Our results suggest that the alteration of Cu and Zn homeostasis is involved in the oxidative stress induced by Cd.

Low doses of selenium specifically stimulate the repair of oxidative DNA damage in LNCaP prostate cancer cells.

Epidemiological studies have demonstrated an inverse relationship between selenium (Se) intake and cancer incidence and/or mortality. However, the molecular mechanisms underlying the cancer chemopreventive activity of Se compounds remain largely unknown. The objective of this study was to investigate the effect of low doses of Se on the stimulation of DNA repair systems in response to four different qualities of DNA damage. P53-proficient LNCaP human prostate adenocarcinoma cells were grown either untreated or in the presence of low concentrations of two Se compounds (30° nM sodium selenite, or 10 µM selenomethionine) and exposed to UVA, H2O2, methylmethane sulfonate (MMS) or UVC. Cell viability as well as DNA damage induction and repair were evaluated by the alkaline Comet assay. Overall, Se was shown to be a very potent protector against cell toxicity and genotoxicity induced by oxidative stress (UVA or H2O2) but not from the agents that induce other types of deleterious lesions (MMS or UVC). Furthermore, Se-treated cells exhibited increased oxidative DNA repair activity, indicating a novel mechanism of Se action. Therefore, the benefits of Se could be explained by a combination of antioxidant activity, the reduction in DNA damage and the enhancement of oxidative DNA repair capacity.

The toxicity redox mechanisms of cadmium alone or together with copper and zinc homeostasis alteration: its redox biomarkers.

Cadmium (Cd) is a toxic metal and can induce and/or promote diseases in humans (cancer, aging diseases, kidney and bone diseases, etc.). Its toxicity involves many mechanisms including the alteration of copper (Cu) and zinc (Zn) homeostasis leading to reactive oxygen species (ROS) production, either directly or through the inhibition of antioxidant activities. Importantly, ROS can induce oxidative damages in cells. Cadmium, Cu and Zn are also able to induce glutathione (GSH) and metallothioneins (MT) synthesis in a cell-type-dependent manner. As a consequence, the effects induced by these three metals result simultaneously from the inhibition of antioxidant activities and the induction of other factors such as GSH and MT synthesis. MT levels are regulated not only by the p53 protein in a cell-type-dependent manner, or by transcription factors such as metal-responsive transcription factor 1 (MTF-1) and cellular Zn levels but also by cellular GSH level. As described in the literature, DNA damage, GSH and MT levels are sensitive biomarkers used to identify Cd-induced toxicity alone or together with Cu and Zn homeostasis alteration.

Selenium supplementation ameliorates static magnetic field-induced disorders in antioxidant status in rat tissues.

The aim of this study was to investigate the effect of selenium supplementation on the antioxidant enzymatic system (such as GPx, GR and SOD), GSH and selenium level in liver, kidney, muscle and brain of static magnetic field (SMF) exposed rats. Male adult rats were divided into control rats (n=6), SMF-exposed rats (128 mT; 1h/day for 5 days), selenium-treated rats (Na(2)SeO(3), 0.2mg/l, in drinking water for 4 weeks) and co-exposed rats (selenium for 4 weeks and SMF during the last 5 consecutive days). Sub-acute exposure to SMF induces a decrease of selenium levels in kidney, muscle and brain. Our results also revealed a decrease of GPx activities in kidney and muscle. By contrast, SMF exposure increased total GSH levels and total SOD activities in liver, while glutathione reductase activity is unaffected. Selenium supplementation in SMF-exposed rats restored selenium levels in kidney, muscle and brain and elevated the activities of GPx in kidney and muscle to those of control group. In the liver, selenium supplementation failed to bring down the elevated levels of total GSH and SOD activity. Our investigations suggested that sub-acute exposure to SMF altered the antioxidant response by decreasing the level of total selenium in kidney, muscle and brain. Interestingly, selenium supplementation ameliorates antioxidant capacity in rat tissues exposed to SMF.

Effect of selenium pre-treatment on plasma antioxidant vitamins A (retinol) and E (α-tocopherol) in static magnetic field-exposed rats.

In the present study, we evaluate the effect of the co-exposure to static magnetic field (SMF) and selenium (Se) on the antioxidant vitamins A and E levels and some other parameters of oxidative stress in rat. Sub-acute exposure of male adult rats to a uniform SMF (128 mT, 1 h/day during 5 consecutive days) increased plasma activity of glutathione peroxidase (+35%) but decreased α-tocopherol (-67%) and retinol levels (-41%). SMF exposure failed to alter the plasmatic thiobarbituric acid-reactive species (TBARs), total thiol groups and selenium concentrations. Sub-chronic administration of Se (Na(2)SeO(3), 0.2 mg/L, for 30 consecutive days, per os) ameliorated the antioxidant capacities in SMF-treated rats. Our investigation demonstrated that sub-acute exposure to SMF induced oxidative stress, which may be prevented by a pretreatment with selenium.

Effects of static magnetic field and cadmium on oxidative stress and DNA damage in rat cortex brain and hippocampus.

The present study was undertaken to determine the effect of co-exposure to static magnetic field (SMF) and cadmium (Cd) on the antioxidant enzymes activity and DNA integrity in rat brain. Sub-chronic exposure to CdCl (CdCl(2), 40 mg/L, per os) for 30 days resulted in a significant reduction in antioxidant enzyme activity such as the glutathione peroxidase (GPx), catalase (CAT) and superoxide dismutase (SOD) in frontal cortex and hippocampus. Total GSH were decreased in the frontal cortex of the Cd-exposed group. Cd exposure induced an increase in malondialdehyde (MDA) concentration in the frontal cortex and hippocampus. Moreover, the same exposure increased 8-oxo-7,8-dihydro-2-desoxyguanosine (8-oxodGuo) level in rat brain. Interestingly, the combined effect of SMF (128 mT, 1 hour/day for 30 consecutive days) and CdCl (40 mg/L, per os) decreased the SOD activity and glutathione level in frontal cortex as compared with the Cd group. Moreover, the association between SMF and Cd increased MDA concentration in frontal cortex as compared with Cd-exposed rats. DNA analysis revealed that SMF exposure failed to alter 8-oxodGuo concentration in Cd-exposed rats. Our data showed that Cd exposure altered the antioxidant enzymes activity and induced oxidative DNA lesions in rat brain. The combined effect of SMF and Cd increased oxidative damage in rat brain as compared with Cd-exposed rats.

TOX4 and its binding partners recognize DNA adducts generated by platinum anticancer drugs.

Platinating agents are commonly prescribed anticancer drugs damaging DNA. Induced lesions are recognized by a wide range of proteins. These are involved in cellular mechanisms such as DNA repair, mediation of cytotoxicity or chromatin remodeling. They therefore constitute crucial actors to understand pharmacology of these drugs. To expand our knowledge about this subproteome, we developed a ligand fishing trap coupled to high throughput proteomic tools. This trap is made of damaged plasmids attached to magnetic beads, and was exposed to cell nuclear extracts. Retained proteins were identified by nanoHPLC coupled to tandem mass spectrometry. This approach allowed us to establish a list of 38 proteins interacting with DNA adducts generated by cisplatin, oxaliplatin and satraplatin. Some of them were already known interactome members like high mobility group protein 1 (HMGB1) or the human upstream binding factor (hUBF), but we also succeeded in identifying unexpected proteins such as TOX HMG box family member 4 (TOX4), phosphatase 1 nuclear targeting subunit (PNUTS), and WD repeat-containing protein 82 (WDR82), members of a recently discovered complex. Interaction between TOX4 and platinated DNA was subsequently validated by surface plasmon resonance imaging (SPRi). These interactions highlight new cellular responses to DNA damage induced by chemotherapeutic agents.

Assessment of the photoprotection properties of sunscreens by chromatographic measurement of DNA damage in skin explants.

Evaluation of the photoprotection provided by sunscreens is performed either through the induction of erythema and expressed as the sun protection factor (SPF), or by the UVA-mediated persistent pigment darkening (PPD). None of these two endpoints has a link with skin cancer, the most deleterious consequence of excess exposure to solar UV radiation. We thus set up a complementary approach to evaluate the protection provided by sunscreens to the genome of human skin. This is based on the quantification of the thymine cyclobutane dimer (TT-CPD), the main DNA lesion induced by both UVB and UVA radiations. Irradiations were performed ex vivo on human skin explants and the level of TT-CPD in DNA was determined by HPLC associated with tandem mass spectrometry. The technique was first optimized and validated with three standard sunscreens. The study was then extended to the evaluation of a commercial high SPF sunscreen exhibiting efficient UVA photoprotection. The DNA protecting factor was found to reflect the ratio between UVB and UVA photoprotection, although the absolute values of the genomic protection were, as a general trend, lower than either SPF or PPD. These data show the usefulness of the proposed approach for the evaluation of the genoprotection afforded by sunscreens.

Evaluation of cytotoxicity and oxidative DNA damaging effects of di(2-ethylhexyl)-phthalate (DEHP) and mono(2-ethylhexyl)-phthalate (MEHP) on MA-10 Leydig cells and protection by selenium.

Di(2-ethylhexyl)-phthalate (DEHP) is the most abundantly used phthalate derivative, inevitable environmental exposure of which is suspected to contribute to the increasing incidence of testicular dysgenesis syndrome in humans. Oxidative stress and mitochondrial dysfunction in germ cells are suggested to contribute to phthalate-induced disruption of spermatogenesis in rodents, and Leydig cells are one of the main targets of phthalates' testicular toxicity. Selenium is known to be involved in the modulation of intracellular redox equilibrium, and plays a critical role in testis, sperm, and reproduction. This study was aimed to investigate the oxidative stress potential of DEHP and its consequences in testicular cells, and examine the possible protective effects of selenium using the MA-10 mouse Leydig tumor cell line as a model. In the presence and absence of selenium compounds [30 nM sodium selenite (SS), and 10 µM selenomethionine (SM)], the effects of exposure to DEHP and its main metabolite mono(2-ethylhexyl)-phthalate (MEHP) on the cell viability, enzymatic and non-enzymatic antioxidant status, ROS production, p53 expression, and DNA damage by alkaline Comet assay were investigated. The overall results of this study demonstrated the cytotoxicity and genotoxicity potential of DEHP, where MEHP was found to be more potent than the parent compound. SS and SM produced almost the same level of protection against antioxidant status modifying effects, ROS and p53 inducing potentials, and DNA damaging effects of the two phthalate derivatives. It was thus shown that DEHP produced oxidative stress in MA-10 cells, and selenium supplementation appeared to be an effective redox regulator in the experimental conditions used in this study, emphasizing the critical importance of the appropriate selenium status.

Protective effect of selenium supplementation on the genotoxicity of di(2-ethylhexyl)phthalate and mono(2-ethylhexyl)phthalate treatment in LNCaP cells.

Selenium is an essential cofactor in the key enzymes involved in cellular antioxidant defense. It plays a critical role in testis and reproduction and regulates DNA damage within the prostate. Phthalates are ubiquitous environmental contaminants that cause alterations in endocrine and spermatogenic functions in animals. The objective of this study was to investigate the cytotoxicity and genotoxicity potentials of di(2-ethylhexyl)phthalate (DEHP), the most widely used phthalate and its primary toxic metabolite mono(2-ethylhexyl)phthalate (MEHP), and their effects on the antioxidant balance in the LNCaP human prostate adenocarcinoma cell line. Protection by selenium supplementation with either sodium selenite (SS, 30 nM) or selenomethionine (SM, 10 microM) was also investigated. Both DEHP (3mM) and MEHP (3 microM) caused significant decreases in cell viability; altered antioxidant status, particularly decreasing the GPx1 activity; and induced DNA damage as measured by the alkaline comet assay. Selenium supplementation was highly protective against cytotoxicity, partially prevented genotoxicity, and restored the antioxidant status. The results of this study suggested that the underlying mechanism of cytotoxicity and resulting disturbances produced by DEHP or MEHP was an an oxidative stress process and/or an effect on the expression of antioxidant enzymes, and accentuated the importance of selenium status, particularly with respect to the high probability of phthalate exposures and their adverse effects.

Trace elements status in multinodular goiter.

Importance of iodine and selenium in thyroid metabolism is well known, but the roles of other essential trace elements including copper, zinc, manganese and iron on thyroid hormone homeostasis remain unclear. The aim of this study was to investigate the status of those trace elements in benign thyroid diseases and evaluate possible links between trace element concentrations and thyroid hormones. The study group was composed of 25 patients with multinodular goiter. Concentrations of thyroid hormones (plasma-free thyroxine, FT(4); free triiodothyronine, FT(3); and thyrotropin, TSH), selenium, copper, zinc, manganese and iron in plasma, and urinary iodine were determined. The results were compared with those of a healthy control group (n=20) with no thyroid disorder. A mild iodine deficiency was observed in the patients with multinodular goiter whereas urinary iodine levels were in the range of "normal" values in healthy controls. All patients were euthyroid, and their thyroid hormone concentrations were not significantly different from the control group. Plasma selenium, zinc and iron concentrations did not differ from controls, while copper and manganese levels were found to be significantly higher in the patients with multinodular goiter indicating links between these trace elements and thyroid function and possibly in development of goiter. Besides iodine, there was a significant correlation between plasma copper concentration and FT(3)/FT(4) ratio.

Study of fibroblast gene expression in response to oxidative stress induced by hydrogen peroxide or UVA with skin aging.

The skin aging process, implying oxidative stress, is associated with specific gene expression. Ultraviolet A (UVA) and hydrogen peroxide (H(2)O(2)) both generate reactive oxygen species (ROS) making them relevant in the study of skin cell responses to oxidative stresses. To investigate transcript expression associated with chronological skin aging and its modulation by two oxidative stresses, cDNA micro-arrays, composed of a set of 81 expressed sequence tag (EST) clones, were used to probe the patterns of transcript expression in human fibroblasts of five young (< 21 years-old) and five older (> 50 years-old) healthy females at basal levels and 24 h after exposure to UVA (7 J/cm2) and H(2)O(2) (20 mM). At the basal state, 22% of total genes were up-regulated in the older group. Although both stresses led to the same cell mortality, H(2)O(2) induced a stronger modulation of gene expression than UVA, with 19.5% of transcripts up-regulated versus 4%. The aging process affected the response to H(2)O(2) and even though cells from old donors presented higher basal levels of transcripts they were not able to regulate them in response to the stress. Interestingly, UVA had a specific strong inhibitory effect on the expression of chemokine (C-C) motif ligand 2 (CCL2) transcript, suggesting a possible mechanism for its anti-inflammatory and immunoregulatory roles.

Quantitative determination of plasma vitamin K1 by high-performance liquid chromatography coupled to isotope dilution tandem mass spectrometry.

Plasma vitamin K1 (phylloquinone) determination is commonly used for the diagnosis of vitamin K deficiency in patients suffering from lipid malabsorption. Moreover, current evidence that adequate vitamin K intake, and correspondingly adequate plasma vitamin K1 concentration, could also be of importance in relation to bone and brain diseases emphasizes the need to improve the current analytical methods. We developed a liquid chromatography coupled to tandem mass spectrometry method using a stable isotope ring-D4-labeled internal standard of vitamin K1 and operating in the multiple reaction monitoring mode by the selection of a precursor and product ions. The atmospheric pressure chemical ionization (APCI) method was shown to be more sensitive than electrospray ionization. After a single-step extraction with cyclohexane, chromatographic separation was performed on a C18 column with an isocratic mobile phase. The linearity was up to 5400ng/L, and the limit of detection was 14ng/L. Intra- and interrun precision were 2.4% and 8.3%, respectively, for the lower limit of the reference range. Recovery was better than 98%. The method is simple and reliable, allowing accurate vitamin K1 measurement in plasma samples from healthy subjects and patients suffering from vitamin K deficiency.

Incidence of cancers, ischemic cardiovascular diseases and mortality during 5-year follow-up after stopping antioxidant vitamins and minerals supplements: a postintervention follow-up in the SU.VI.MAX Study.

The Supplementation in Vitamins and Mineral Antioxidants Study was a double-blind, placebo-controlled, randomized trial, in which 12,741 French adults (7,713 women aged 35-60 years and 5,028 men aged 45-60 years) received a combination of ascorbic acid (120 mg), vitamin E (30 mg), beta-carotene (6 mg), selenium (100 microg) and zinc (20 mg), or placebo daily for a median follow-up time of 7.5 years [October 1994 to September 2002]. Antioxidant supplementation decreased total cancer incidence and total mortality in men. Postintervention follow-up assessment of total cancer incidence, ischemic cardiovascular disease incidence and total mortality was carried out for 5 years [September 1, 2002, to September 1, 2007]. No late effect of antioxidant supplementation was revealed 5 years after ending the intervention neither on ischemic cardiovascular disease incidence and mortality in both genders nor on cancer incidence in women. Regarding duration of intervention effects in men, the reduced risk of total cancer incidence and total mortality was no longer evident after the 5-year postintervention follow-up. During the postsupplementation period, the relative risk (RR) for total cancer incidence (n = 126) was 0.98 (95% confidence interval [CI], 0.75-1.27) among antioxidant recipients compared to nonrecipients. For total mortality (n = 90), the RR was 0.98 (95% CI, 0.75-1.26) for men receiving antioxidants compared to nonrecipients. In conclusion, beneficial effects of antioxidant supplementation in men disappeared during postintervention follow-up.