Folic Acid Inhibits Aging-Induced Telomere Attrition and Apoptosis in Astrocytes In Vivo and In Vitro.

Folic acid (FA) has been reported to inhibit astrocyte apoptosis and improve aging-induced disorders; however, its role in telomere attrition remains unclear. In present study, 4-month-old senescence-accelerated mouse prone 8 (SAMP8) mice were assigned to four treatment groups for the in vivo experiment: FA-deficient diet (FA-D) group, FA-normal diet (FA-N) group, low FA-supplemented diet (FA-L) group, and high FA-supplemented diet (FA-H) group. These mice were euthanized when 10 months old. There was also a young SAMP8 (4 months old) control group (Con-Y) fed with FA-normal diet. In in vitro study, primary cultures of astrocytes from hippocampus and cerebral cortex were incubated for five generations with various concentrations of FA (0-40 µM) and were assigned to five groups: FA 0 µM (generation 5), FA 10 µM (generation 5), FA 20 µM (generation 5), FA 40 µM (generation 5), and FA 10 µM (generation 1). The results showed that FA supplementation inhibited aging-induced astrocytosis, astrocyte apoptosis, neurodegeneration, and prevented telomere attrition in hippocampus and cortex of SAMP8 mice. FA supplementation also decreased apoptosis and telomere attrition, and increased telomerase activity, in primary cultures of astrocytes. These results showed that it may be one of the mechanisms that FA inhibiting aging-induced apoptosis of astrocyte by alleviating telomere attrition.

Maternal Folic Acid Supplementation During Pregnancy Promotes Neurogenesis and Synaptogenesis in Neonatal Rat Offspring.

Maternal folic acid supplementation during pregnancy is associated with improved cognitive performances in offspring. However, the effect of supplementation on offspring's neurogenesis and synaptogenesis is unknown, and whether supplementation should be continued throughout pregnancy is controversial. In present study, 3 groups of female rats were fed a folate-normal diet, folate-deficient diet, or folate-supplemented diet from 1 week before mating until the end of pregnancy. A fourth group fed folate-normal diet from 1 week before mating until mating, then fed folate-supplemented diet for 10 consecutive days, then fed folate-normal diet until the end of pregnancy. Offspring were sacrificed on postnatal day 0 for measurement of neurogenesis and synaptogenesis by immunofluorescence and western blot. Additionally neural stem cells (NSCs) were cultured from offspring's hippocampus for immunocytochemical measurement of their rates of proliferation and neuronal differentiation. The results demonstrated that maternal folic acid supplementation stimulated hippocampal neurogenesis by increasing proliferation and neuronal differentiation of NSCs, and also enhanced synaptogenesis in cerebral cortex of neonatal offspring. Hippocampal neurogenesis was stimulated more when supplementation was continued throughout pregnancy instead of being limited to the periconceptional period. In conclusion, maternal folic acid supplementation, especially if continued throughout pregnancy, improves neurogenesis and synaptogenesis in neonatal offspring.

Folic Acid Decreases Astrocyte Apoptosis by Preventing Oxidative Stress-Induced Telomere Attrition.

Astrocytes are the most widely distributed cells in the brain, and astrocyte apoptosis may play an important role in the pathogenesis of neurodegenerative diseases. Folate is required for the normal development of the nervous system, but its effect on astrocyte apoptosis is unclear. In this study, we hypothesized that folic acid (the therapeutic form of folate) decreases astrocyte apoptosis by preventing oxidative stress-induced telomere attrition. Primary cultures of astrocytes were incubated for 12 days with various concentrations of folic acid (0-40 µmol/L), then cell proliferation, apoptosis, intracellular folate concentration, intracellular homocysteine (Hcy) concentration, intracellular reactive oxygen species (ROS) levels, telomeric DNA oxidative damage, and telomere length were determined. The results showed that folic acid deficiency decreased intracellular folate, cell proliferation, and telomere length, whereas it increased Hcy concentration, ROS levels, telomeric DNA oxidative damage, and apoptosis. In contrast, folic acid dose-dependently increased intracellular folate, cell proliferation, and telomere length but it decreased Hcy concentration, ROS levels, telomeric DNA oxidative damage, and apoptosis. In conclusion, folic acid inhibited apoptosis in astrocytes. The underlying mechanism for this protective effect may be that folic acid decreased oxidative stress and thereby prevented telomeric DNA oxidative damage and telomere attrition.

Folic acid attenuates the effects of amyloid ß oligomers on DNA methylation in neuronal cells.

PURPOSE: Alzheimer's disease (AD) is a highly prevalent type of dementia. The epigenetic mechanism of gene methylation provides a putative link between nutrition, one-carbon metabolism, and disease progression because folate deficiency may cause hypomethylation of promoter regions in AD-relevant genes. We hypothesized that folic acid supplementation may protect neuron cells from amyloid ß (Aß) oligomer-induced toxicity by modulating DNA methylation of APP and PS1 in AD models. METHODS: Primary hippocampal neuronal cells and hippocampal HT-22 cells were incubated for 24 h with a combination of folic acid and either Aß oligomers or vehicle and were then incubated for 72 h with various concentrations of folic acid. AD transgenic mice were fed either folate-deficient or control diets and gavaged daily with various doses of folic acid (0 or 600 µg/kg). DNA methyltransferase (DNMT) activity, cell viability, methylation potential of cells, APP and PS1 expression, and the methylation of the respective promoters were determined. RESULTS: Aß oligomers lowered DNMT activity, increased PS1 and APP expression, and decreased cell viability. Folic acid dose-dependently stimulated methylation potential and DNMT activity, altered PS1 and APP promoter methylation, decreased PS1 and APP expression, and partially preserved cell viability. Folic acid increased PS1 and APP promoter methylation in AD transgenic mice. CONCLUSION: These results suggest a mechanism by which folic acid may prevent Aß oligomer-induced neuronal toxicity.

Folic Acid Inhibits Amyloid ß-Peptide Production through Modulating DNA Methyltransferase Activity in N2a-APP Cells.

Alzheimer's disease (AD) is a common neurodegenerative disease resulting in progressive dementia, and is a principal cause of dementia among older adults. Folate acts through one-carbon metabolism to support the methylation of multiple substrates. We hypothesized that folic acid supplementation modulates DNA methyltransferase (DNMT) activity and may alter amyloid ß-peptide (Aß) production in AD. Mouse Neuro-2a cells expressing human APP695 were incubated with folic acid (2.8-40 µmol/L), and with or without zebularine (the DNMT inhibitor). DNMT activity, cell viability, Aß and DNMTs expression were then examined. The results showed that folic acid stimulated DNMT gene and protein expression, and DNMT activity. Furthermore, folic acid decreased Aß protein production, whereas inhibition of DNMT activity by zebularine increased Aß production. The results indicate that folic acid induces methylation potential-dependent DNMT enzymes, thereby attenuating Aß production.

Short-term effect of ascorbate on bacterial content, plasminogen activator inhibitor-1, and myeloperoxidase in septic mice.

BACKGROUND: Sepsis, a potential risk associated with surgery, leads to a systemic inflammatory response including the plugging of capillary beds. This plugging may precipitate organ failure and subsequent death. We have shown that capillary plugging can be reversed rapidly within 1 h by intravenous injection of ascorbate in mouse skeletal muscle. It is unknown whether, in parallel with this effect, ascorbate negatively affects the protective responses to sepsis involving the fibrinolytic and immune systems. We hypothesized that treatment with ascorbate for 1 h does not alter bacterial content, plasminogen activator inhibitor 1 (PAI-1), and neutrophil infiltration in lung, kidney, spleen, and liver (organs with high immune response) of septic mice. MATERIALS AND METHODS: Sepsis was induced by feces injection into the peritoneum. Mice were injected intravenously with ascorbate at 6 h (10 mg/kg), and samples of peritoneal fluid, arterial blood, and organs collected at 7 h were subjected to analyses of bacterial content, PAI-1 messenger RNA and enzymatic activity, and myeloperoxidase (MPO) (a measure of neutrophil infiltration). RESULTS: Sepsis increased bacterial content in all fluids and organs and increased PAI-1 messenger RNA and enzymatic activity in the lung and liver. Sepsis increased the myeloperoxidase level in the lung and liver, and lowered it in the spleen. Except for decreasing the bacterial content in blood, these responses to sepsis were not altered by ascorbate. CONCLUSIONS: The rapid effect of ascorbate against capillary plugging in the septic mouse skeletal muscle is not accompanied by alterations in PAI-1 or myeloperoxidase responses in the organs with high immune response.

Vitamin C in sepsis.

Bacterial bloodstream infection causes septic syndromes that range from systemic inflammatory response syndrome (SIRS) and encephalopathy to severe sepsis and septic shock. Microvascular dysfunction, comprising impaired capillary blood flow and arteriolar responsiveness, precedes multiple organ failure. Vitamin C (ascorbate) levels are low in critically ill patients. The impact of ascorbate administered orally is moderate because of its limited bioavailability. However, intravenous injection of ascorbate raises plasma and tissue concentrations of the vitamin and may decrease morbidity. In animal models of polymicrobial sepsis, intravenous ascorbate injection restores microvascular function and increases survival. The protection of capillary blood flow and arteriolar responsiveness by ascorbate may be mediated by inhibition of oxidative stress, modulation of intracellular signaling pathways, and maintenance of homeostatic levels of nitric oxide. Ascorbate scavenges reactive oxygen species (ROS) and also inhibits the NADPH oxidase that synthesizes superoxide in microvascular endothelial cells. The resulting changes in redox-sensitive signaling pathways may diminish endothelial expression of inducible nitric oxide synthase (iNOS), tissue factor and adhesion molecules. Ascorbate also regulates nitric oxide concentration by releasing nitric oxide from adducts and by acting through tetrahydrobiopterin (BH4) to stimulate endothelial nitric oxide synthase (eNOS). Therefore, it may be possible to improve microvascular function in sepsis by using intravenous vitamin C as an adjunct therapy.

Ascorbate protects against vascular leakage in cecal ligation and puncture-induced septic peritonitis.

Vascular leakage in multiple organs is a characteristic pathological change in sepsis. Our recent study revealed that ascorbate protects endothelial barrier function in microvascular endothelial cell monolayers through inhibiting serine/threonine protein phosphatase 2A (PP2A) activation (Han M, Pendem S, Teh SL, Sukumaran DK, Wu F, Wilson JX. Free Radic Biol Med 48: 128-135, 2010). The present study addressed the mechanism of protection by ascorbate against vascular leakage in cecal ligation and puncture (CLP)-induced septic peritonitis in mice. CLP caused NADPH oxidase activation and endothelial nitric oxide synthase (eNOS) uncoupling to produce superoxide, increased NO production by inducible NOS (iNOS) and neuronal NOS (nNOS) activity, and elevated 3-nitrotyrosine (a product of peroxynitrite) formation and PP2A activity in the hindlimb skeletal muscles at 12 h after CLP. The increase in PP2A activity was associated with decreased levels of phosphorylated serine and threonine in occludin, which was immunoprecipitated from freshly harvested endothelial cells of the septic skeletal muscles. Moreover, CLP increased the vascular permeability to fluorescent dextran and Evans blue dye in skeletal muscles. An intravenous bolus injection of ascorbate (200 mg/kg body wt), given 30 min prior to CLP, prevented eNOS uncoupling, attenuated the increases in iNOS and nNOS activity, decreased 3-nitrotyrosine formation and PP2A activity, preserved the phosphorylation state of occludin, and completely inhibited the vascular leakage of dextran and Evans blue. A delayed ascorbate injection, given 3 h after CLP, also prevented the vascular permeability increase. We conclude that ascorbate injection protects against vascular leakage in sepsis by sequentially inhibiting excessive production of NO and superoxide, formation of peroxynitrite, PP2A activation, and occludin dephosphorylation. Our study provides a scientific basis for injection of ascorbate as an adjunct treatment for vascular leakage in sepsis.

Systems biology of antioxidants.

Understanding the role of oxidative injury will allow for therapy with agents that scavenge ROS (reactive oxygen species) and antioxidants in the management of several diseases related to free radical damage. The majority of free radicals are generated by mitochondria as a consequence of the mitochondrial cycle, whereas free radical accumulation is limited by the action of a variety of antioxidant processes that reside in every cell. In the present review, we provide an overview of the mitochondrial generation of ROS and discuss the role of ROS in the regulation of endothelial and adipocyte function. Moreover, we also discuss recent findings on the role of ROS in sepsis, cerebral ataxia and stroke. These results provide avenues for the therapeutic potential of antioxidants in a variety of diseases.

Folic acid enhances Notch signaling, hippocampal neurogenesis, and cognitive function in a rat model of cerebral ischemia.

Increasing neurogenesis may restore cognitive functions that are impaired in ischemia stroke. Folic acid has been reported to play an important role in neuronal development and reduce the risk of ischemic stroke in primary prevention. Folic acid supplementation stimulates Notch signaling and cell proliferation in neural progenitor cells cultured from neonatal brain. The present study determined whether folic acid supplementation stimulates Notch signaling and neurogenesis and improves cognitive function after ischemic stroke in adult brain. Rats were randomly assigned to four groups: sham operation plus vehicle (Sham), middle cerebral artery occlusion plus vehicle (MCAO), MCAO plus low-dose folic acid (4 mg/(kg day)), and MCAO plus high folic acid (12 mg/(kg day)). The vehicle and folic acid were administered by oral gavage for 28 days prior to sham or MCAO operation and up to 14 days after surgery. Newborn hippocampal neurons were detected at 3, 7, and 14 days post-MCAO. Cognitive function (learning and memory in Y-maze tests) and the protein expression levels of components of the Notch signaling system (Notch1, Hes1, and Hes5) were measured at 7 days post-MCAO. The results showed that MCAO impaired Y-maze performance and stimulated Notch signaling and hippocampal neurogenesis in brain. Folic acid prevented the impairment of Y-maze performance. The nutrient also increased further the expression of Notch1, Hes1, and Hes5 and the number of the newborn hippocampal neurons. Folic acid enhances the stimulation by ischemia of Notch signaling and hippocampal neurogenesis in adult brain and lessens the impairment of cognitive function that occurs after experimental stroke.

Alleviating Oxidative Damage-Induced Telomere Attrition: a Potential Mechanism for Inhibition by Folic Acid of Apoptosis in Neural Stem Cells.

DNA oxidative damage can cause telomere attrition or dysfunction that triggers cell senescence and apoptosis. The hypothesis of this study is that folic acid decreases apoptosis in neural stem cells (NSCs) by preventing oxidative stress-induced telomere attrition. Primary cultures of NSCs were incubated for 9 days with various concentrations of folic acid (0-40 µM) and then incubated for 24 h with a combination of folic acid and an oxidant (100-µM hydrogen peroxide, H2O2), antioxidant (10-mM N-acetyl-L-cysteine, NAC), or vehicle. Intracellular folate concentration, apoptosis rate, cell proliferative capacity, telomere length, telomeric DNA oxidative damage, telomerase activity, intracellular reactive oxygen species (ROS) levels, cellular oxidative damage, and intracellular antioxidant enzyme activities were determined. The results showed that folic acid deficiency in NSCs decreased intracellular folate concentration, cell proliferation, telomere length, and telomerase activity but increased apoptosis, telomeric DNA oxidative damage, and intracellular ROS levels. In contrast, folic acid supplementation dose-dependently increased intracellular folate concentration, cell proliferative capacity, telomere length, and telomerase activity but decreased apoptosis, telomeric DNA oxidative damage, and intracellular ROS levels. Exposure to H2O2 aggravated telomere attrition and oxidative damage, whereas NAC alleviated the latter. High doses of folic acid prevented telomere attrition and telomeric DNA oxidative damage by H2O2. In conclusion, inhibition of telomeric DNA oxidative damage and telomere attrition in NSCs may be potential mechanisms of inhibiting NSC apoptosis by folic acid.

The effects of hyperoxia exposure on lung function and pulmonary surfactant in a rat model of acute lung injury.

The objective of this study was to determine if prolonged hyperoxia exposure would deplete antioxidants, resulting in excessive oxidative stress that would lead to oxidation of pulmonary surfactant and contribute to lung dysfunction. Rats were exposed to either hyperoxic (> 95% O(2)) or normoxic (21% O(2)) oxygen concentrations for 48 or 60 hours. Pulmonary compliance, inflammatory cells, and total protein levels were measured as indicators of lung injury. Bronchoalveolar lavage (BAL) samples were analyzed for surfactant composition, antioxidant content, and markers of oxidative stress. Antioxidants were also measured in lung tissue and plasma samples. Hyperoxia exposure for 60 hours resulted in increased protein and inflammatory cells in BAL, and lower pulmonary compliance, compared to all other groups. Total surfactant and surfactant large aggregates were increased following 48 hours of hyperoxia exposure, with a further increase following 60 hours. Animals exposed to 60 hours of hyperoxia also demonstrated lower ascorbate levels in lung tissue, increased lipid peroxides in BAL, and increased oxidation of phosphatidylglycerol species in surfactant. This study demonstrates that the balance of oxidant/antioxidant components is disrupted within the lung during periods of hyperoxia, and that although surfactant lipids may be susceptible to oxidative damage, they do not likely represent a major mechanism for the lung dysfunction observed.

Folate stimulates ERK1/2 phosphorylation and cell proliferation in fetal neural stem cells.

Cellular events for neural progenitor cells, such as proliferation and differentiation, are regulated by multiple intrinsic and extrinsic cell signals. Folate plays central roles in central nervous system development, so folate, as an extrinsic signal, may affect neural stem cell (NSC) proliferation and differentiation. In this study, we have investigated the effect of folate on extracellular signal-regulated kinase (ERK1/2) phosphorylation, cell proliferation and apoptosis in fetal NSCs. The results showed that treatment of neurospheres with folate increased ERK1/2 phosphorylation and cell proliferation in a concentration-dependent manner. Folate also decreased the percentage of apoptotic cells. All of these effects of folate were prevented by a selective inhibitor (U0126) of mitogen-activated/ERK kinase 1/2. In conclusion, fetal NSCs respond to folate with ERKl/2 phosphorylation, cell proliferation and decreased apoptosis. This mechanism may mediate the regulation by folate of neurogenesis in the central nervous system.

Mechanism of action of vitamin C in sepsis: ascorbate modulates redox signaling in endothelium.

Circulating levels of vitamin C (ascorbate) are low in patients with sepsis. Parenteral administration of ascorbate raises plasma and tissue concentrations of the vitamin and may decrease morbidity. In animal models of sepsis, intravenous ascorbate injection increases survival and protects several microvascular functions, namely, capillary blood flow, microvascular permeability barrier, and arteriolar responsiveness to vasoconstrictors and vasodilators. The effects of parenteral ascorbate on microvascular function are both rapid and persistent. Ascorbate quickly accumulates in microvascular endothelial cells, scavenges reactive oxygen species, and acts through tetrahydrobiopterin to stimulate nitric oxide production by endothelial nitric oxide synthase. A major reason for the long duration of the improvement in microvascular function is that cells retain high levels of ascorbate, which alter redox-sensitive signaling pathways to diminish septic induction of NADPH oxidase and inducible nitric oxide synthase. These observations are consistent with the hypothesis that microvascular function in sepsis may be improved by parenteral administration of ascorbate as an adjuvant therapy.

Folic acid delays age-related cognitive decline in senescence-accelerated mouse prone 8: alleviating telomere attrition as a potential mechanism.

The occurrence of telomere attrition in brain may cause senescence and death of neurons, leading to cognitive decline. Folic acid (FA) has been reported to improve cognitive performance in mild cognitive impairment; however, its association with telomere remains unclear. The study aimed to investigate if alleviation of telomere attrition by FA supplementation could act as a potential mechanism to delay age-related cognitive decline in senescence-accelerated mouse prone 8 (SAMP8). Aged SAMP8 mice were assigned to four treatment groups: FAdeficient diet (FA-D) group, FA-normal diet (FA-N) group, low FA-supplemented diet (FA-L) group and high FAsupplemented diet (FA-H) group. There was also an age-matched senescence-accelerated mouse resistant 1 (SAMR1) control group (Con-R), and a young SAMP8 control group (Con-Y). The results demonstrated that FA supplementation delayed age-related cognitive decline and neurodegeneration in SAMP8 mice. Importantly, this effect could be attributed to the alleviated telomere attrition, which might be interpreted by the decreased levels of reactive oxygen species. Additionally, improved telomere integrity stimulated mitochondrial function via telomere-p53-mithondria pathway, consequently delayed neuronal degeneration. In conclusion, we demonstrate that FA supplementation delays age-related neurodegeneration and cognitive decline in SAMP8 mice, in which alleviated telomere attrition could serve as one influential factor in the process.

iNOS expression requires NADPH oxidase-dependent redox signaling in microvascular endothelial cells.

Redox regulation of inducible nitric oxide synthase (iNOS) expression was investigated in lipopolysaccharide and interferon-gamma (LPS + IFNgamma)-stimulated microvascular endothelial cells from mouse skeletal muscle. Unstimulated endothelial cells produced reactive oxygen species (ROS) sensitive to inhibition of NADPH oxidase (apocynin and DPI), mitochondrial respiration (rotenone) and NOS (L-NAME). LPS + IFNgamma caused a marked increase in ROS production; this increase was abolished by inhibition of NADPH oxidase (apocynin, DPI and p47phox deficiency). LPS + IFNgamma induced substantial expression of iNOS protein. iNOS expression was prevented by the antioxidant ascorbate and by NADPH oxidase inhibition (apocynin, DPI and p47phox deficiency), but not by inhibition of mitochondrial respiration (rotenone) and xanthine oxidase (allopurinol). iNOS expression also was prevented by selective antagonists of ERK, JNK, Jak2, and NFkappaB activation. LPS + IFNgamma stimulated activation/phosphorylation of ERK, JNK, and Jak2 and activation/degradation of IkappaB, but only the activation of JNK and Jak2 was sensitive to ascorbate, apocynin and p47phox deficiency. Ascorbate, apocynin and p47phox deficiency also inhibited the LPS + IFNgamma-induced DNA binding activity of transcription factors IRF1 and AP1 but not NFkappaB. In conclusion, LPS + IFNgamma-induced NFkappaB activation is necessary for iNOS induction but is not dependent on ROS signaling. LPS + IFNgamma-stimulated NADPH oxidase activity produces ROS that activate the JNK-AP1 and Jak2-IRF1 signaling pathways required for iNOS induction. Since blocking either NFkappaB activation or NADPH oxidase activity is sufficient to prevent iNOS expression, they are separate targets for therapeutic interventions that aim to modulate iNOS expression in sepsis.

Septic impairment of capillary blood flow requires nicotinamide adenine dinucleotide phosphate oxidase but not nitric oxide synthase and is rapidly reversed by ascorbate through an endothelial nitric oxide synthase-dependent mechanism.

OBJECTIVE: To determine the roles of nitric oxide synthase (NOS) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in the impairment of capillary blood flow in sepsis and in the reversal of this impairment by ascorbate. DESIGN: Prospective, controlled laboratory study. SETTING: Animal laboratory in research institute. SUBJECTS: Adult male wild type (WT), neuronal nitric oxide synthase (nNOS)-/-, inducible NOS (iNOS)-/-, endothelial NOS (eNOS)-/-, and gp91phox-/- mice. INTERVENTIONS: Sepsis was induced by feces injection into peritoneum (FIP). A bolus of ascorbate or NADPH oxidase inhibitor apocynin was injected intravenously at 6 hrs post-FIP. Alternatively, NOS cofactor (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4) or nitric oxide donor S-nitroso-N-acetylpenicillamine was superfused on the surface of the extensor digitorum longus muscle. MEASUREMENTS AND MAIN RESULTS: Capillary blood flow impairment and NOS activity in the extensor digitorum longus muscle were measured by intravital microscopy and by enzymatic assay, respectively. Sepsis at 6 hrs impaired flow in WT mice. Apocynin, and knockout of gp91phox but not of any NOS isoforms, rescued this impairment. Constitutive NOS activity was unaffected by sepsis, but it was abolished by nNOS knockout (iNOS activity was negligible in all mice). Ascorbate rapidly (10 mins) rescued impaired flow in WT, nNOS-/-, iNOS-/- but not eNOS-/- mice. Ascorbate also improved survival of WT mice after FIP. BH4 and SNAP rescued flow in WT mice, while BH4 failed to rescue it in eNOS-/- mice. CONCLUSION: Capillary blood flow impairment in septic skeletal muscle requires NADPH oxidase but not NOS, and it is rapidly reversed by ascorbate and BH4 through an eNOS-dependent mechanism.

Organochlorines inhibit acetaminophen glucuronidation by redirecting UDP-glucuronic acid towards the D-glucuronate pathway.

Industry-derived organochlorines are persistent environmental pollutants that are a continuing health concern. The effects of these compounds on drug metabolism are not well understood. In the current study we present evidence that the inhibition of acetaminophen (APAP) glucuronidation by minute concentrations of organochlorines correlates well with their ability to stimulate the d-glucuronate pathway leading to ascorbate synthesis. A set of 6 arylated organochlorines, including 5 PCB (polychlorinated biphenyl) congeners, were assessed for their effects on APAP glucuronidation in isolated hepatocytes from male Sprague-Dawley rats. The capacity of each organochlorine to inhibit APAP glucuronidation was found to be directly proportional to its capacity to stimulate ascorbate synthesis. PCB153, PCB28 and bis-(4-chlorophenyl sulfone) (BCPS) in increasing order were the most effective organochlorines for inhibiting APAP glucuronidation and stimulating the d-glucuronate pathway. None of the 3 inhibitors of APAP glucuronidation were able to alter the expression of UGT1A6, UGT1A7 and UGT1A8 (the major isoforms responsible for APAP glucuronidation in the rat), however, their efficacy at inhibiting APAP glucuronidation was proportional to their capacity to deplete UDP-glucuronic acid (UDPGA). BCPS-mediated inhibition of APAP glucuronidation in isolated hepatocytes had non-competitive characteristics and was insensitive to the inactivation of cytochrome P450. The effective organochlorines were also able to selectively stimulate the hydrolysis of UDPGA to UDP and glucuronate in isolated microsomes, but could not inhibit APAP glucuronidation in microsomes when UDPGA was in excess. We conclude that organochlorines are able to inhibit APAP glucuronidation in hepatocytes by depleting UDPGA via redirecting UDPGA towards the d-glucuronate pathway. Because the inhibition is non-competitive, low concentrations of these compounds could have long term inhibitory effects on the glucuronidating capacity of hepatocytes.

Effects of folate on notch signaling and cell proliferation in neural stem cells of neonatal rats in vitro.

The aim of the present study was to determine if folate alters Notch signaling and cell proliferation in neural stem cells (NSCs). NSCs were isolated from neonatal rats and grown in serum-free suspension culture. The cells were identified as NSCs by their expression of immunoreactive nestin. Individual cultures were assigned to one of three treatment groups: vehicle control, low-dose folate group (Folate-L, liquid media contained 4 mg/L folate), or high-dose folate group (Folate-H, liquid media contained 40 mg/L folate). Proliferating cells were identified by labeling with 5-bromo-2'-deoxyuridine (BrdU). Cell proliferation was quantitated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Gene expression of components of the Notch signaling system (Notch1, Hes1 and Mash1) was quantified by real-time polymerase chain reaction (PCR) assay. We observed that Nestin-positive NSCs grew as neurospheres in the serum-free suspension cultures. Folate increased the rate of cell proliferation compared to vehicle control (p<0.05). During cell proliferation, folate also increased Notch1 and Hes1 expression and decreased Mash1 expression compared to vehicle control (p<0.05). These results suggest that NSCs cultured from neonatal rats respond to folate with altered Notch signaling and increased cell proliferation.

Effects of genistein on oxidative injury in endothelial cells.

The aim of this study was to test the hypothesis that genistein protects vascular endothelial cells against the pro-atherosclerotic stressor, oxidized low-density lipoprotein (ox-LDL), by inducing antioxidant enzymes and preventing apoptosis. Human umbilical cord-derived endothelial cells (ECV 304) were incubated with genistein (10-100 micromol/L), the radical scavenging antioxidant vitamin E (alpha-tocopherol, 50 micromol/L), or vehicle for 24 h and then were incubated with ox-LDL for an additional 24 h. Subsequently, antioxidant enzyme activities, lipid peroxidation, adhesion to monocytes, cell morphology, viability and apoptotic index were assessed. Ox-LDL decreased superoxide dismutase and glutathione peroxidase activities in endothelial cells and caused lipid peroxidation, adhesion to monocytes, morphological injury and apoptosis (p<0.05). These effects were prevented by vitamin E and dose-dependently by genistein (p<0.05). Further, this effect of genistein is associated with maintenance of antioxidant enzyme activities and inhibition of lipid peroxidation.