(<em>S</em>)-α-Chlorohydrin Inhibits Protein Tyrosine Phosphorylation through Blocking Cyclic AMP - Protein Kinase A Pathway in Spermatozoa

<div><p>α-Chlorohydrin is a common contaminant in food. Its (<em>S</em>)-isomer, (<em>S</em>)-α-chlorohydrin (SACH), is known for causing infertility in animals by inhibiting glycolysis of spermatozoa. The aim of present work was to examine the relationship between SACH and protein tyrosine phosphorylation (PTP), which plays a critical role in regulating mammalian sperm capacitation. In vitro exposure of SACH 50 µM to isolated rat epididymal sperm inhibited PTP. Sperm-specific glyceraldehyde 3-phosphate dehydrogenase (GAPDS) activities, the intracellular adenosine 5′-triphosphate (ATP) levels, 3′-5′-cyclic adenosine monophosphate (cAMP) levels and phosphorylation of protein kinase A (PKA) substrates in rat sperm were diminished dramatically, indicating that both glycolysis and the cAMP/PKA signaling pathway were impaired by SACH. The inhibition of both PTP and phosphorylation of PKA substrates by SACH could be restored by addition of cAMP analog dibutyryl-cAMP (dbcAMP) and phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). Moreover, addition of glycerol protected glycolysis, ATP levels, phosphorylation of PKA substrates and PTP against the influence of SACH. These results suggested SACH inhibited PTP through blocking cAMP/PKA pathway in sperm, and PTP inhibition may play a role in infertility associated with SACH.</p> </div

The model by which SACH inhibits cAMP/PKA mediated protein phosphorylation through blocking glycolysis in spermatozoa during capacitation.

<p>The inhibition of GAPDS by SACH results in depletion of ATP and then depresses of P-PKAs and PTP. Glycerol overcomes these effects through restoring glycolysis, and IBMX and dbcAMP, the activators of PKA, reverse in part the inhibitions of P-PKAs and PTP.</p

Organic Extract Contaminants from Drinking Water Activate Nrf2-Mediated Antioxidant Response in a Human Cell Line

Traditional risk assessment methods face challenges in estimating risks from drinking waters that contain low-levels of large numbers of contaminants. Here, we evaluate the toxicity of organic contaminant (OC) extracts from drinking water by examining activation of nuclear factor E2-related factor 2 (Nrf2)-mediated antioxidant response. In HepG2 cells, the Nrf2-mediated antioxidant responsemeasured as Nrf2 protein accumulation, expression of antioxidant response element (ARE)-regulated genes and ARE-luciferase reporter gene assays were activated by OC extracts from drinking water sources that detected 25 compounds in 9 classification groups. Individual OCs induced oxidative stress at concentrations much higher than their environmental levels; however, mixtures of contaminants induced oxidative stress response at only 8 times the environmental levels. Additionally, a synthetic OC mixture prepared based on the contamination profiling of drinking water induced ARE activity to the same extent as the real-world mixture, reinforcing our conclusion that these mixture exposures produce responses relevant for human exposure situations. Our study tested the possibility of assessing toxicity of OCs of drinking water using a specific ARE-pathway measurement. This approach should be broadly useful in assisting risk assessment of mixed environmental exposure

Glycerol rescues glycolysis, PTP and PKA after inhibition by SACH.

<p>Rat spermatozoa were exposed to a series of concentrations of SACH in capacitating conditions for 6 hours and both GAPDS activity (A) and ATP level (B) were significantly reduced. n = 3, * <i>p</i><0.05 vs. control. (C and D) Rat sperm were exposed to a serial of concentrations of glycerol in the presence of 100 µM SACH for 6 hours and GAPDS activity and ATP were measured. n = 3, <i>a p</i><0.01, <i>b p</i><0.001 vs. the 100 µM SACH group. (E, F, G and H) With the same treatment as in (C and D), sperm were subjected to Western Blot analyses with anti-PTP (E and F) and anti-P-PKAs (G and H) antibodies. n = 2, * <i>p</i><0.05, ** <i>p</i><0.01 vs. control.</p

The role of depletion of cAMP in inhibition of PTP and P-PKAs by SACH.

<p>(A) cAMP levels in rat sperm decreased by SACH after incubation in capacitating conditions for 6 hours, * <i>p</i><0.05 vs. control. (B C, D and E) Rat sperm were incubated in complete BWW supplemented with 50 µM SACH, 100 µM SACH and 10 µM H89 or in BWW-HCO₃⁻ in the presence and absence of 0.25 mM dbcAMP and 0.1 mM IBMX as indicated at the bottoms of the figures. After 6 hours of incubation, PTP (B) and P-PKAs (D) were identified by Western Blot, and levels of phosphorylation were quantitated by densitometry analysis of representative bands (C, E). n = 3, * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001 vs. control.</p

Vascular Dysfunction in Patients with Chronic Arsenosis Can Be Reversed by Reduction of Arsenic Exposure-0

<p><b>Copyright information:</b></p><p>Taken from "Vascular Dysfunction in Patients with Chronic Arsenosis Can Be Reversed by Reduction of Arsenic Exposure"</p><p>Environmental Health Perspectives 2004;113(3):339-341.</p><p>Published online 9 Dec 2004</p><p>PMCID:PMC1253762.</p><p>This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original DOI.</p

DataSheet1_Nrf2−/− regulated lung DNA demethylation and CYP2E1 DNA methylation under PM2.5 exposure.docx

Cytochrome P450 (CYP450) can mediate fine particulate matter (PM2.5) exposure leading to lung injury. Nuclear factor E2-related factor 2 (Nrf2) can regulate CYP450 expression; however, the mechanism by which Nrf2−/− (KO) regulates CYP450 expression via methylation of its promoter after PM2.5 exposure remains unclear. Here, Nrf2−/− (KO) mice and wild-type (WT) were placed in a PM2.5 exposure chamber (PM) or a filtered air chamber (FA) for 12 weeks using the real-ambient exposure system. The CYP2E1 expression trends were opposite between the WT and KO mice following PM2.5 exposure. After exposure to PM2.5,CYP2E1 mRNA and protein levels were increased in WT mice but decreased in KO mice, and CYP1A1 expression was increased after exposure to PM2.5 in both WT and KO mice. CYP2S1 expression decreased after exposure to PM2.5 in both the WT and KO groups. We studied the effect of PM2.5 exposure on CYP450 promoter methylation and global methylation levels in WT and KO mice. In WT and KO mice in the PM2.5 exposure chamber, among the methylation sites examined in the CYP2E1 promoter, the CpG2 methylation level showed an opposite trend with CYP2E1 mRNA expression. The same relationship was evident between CpG3 unit methylation in the CYP1A1 promoter and CYP1A1 mRNA expression, and between CpG1 unit methylation in the CYP2S1 promoter and CYP2S1 mRNA expression. This data suggests that methylation of these CpG units regulates the expression of the corresponding gene. After exposure to PM2.5, the expression of the DNA methylation markers ten-eleven translocation 3 (TET3) and 5-hydroxymethylcytosine (5hmC) was decreased in the WT group but significantly increased in the KO group. In summary, the changes in CYP2E1, CYP1A1, and CYP2S1 expression in the PM2.5 exposure chamber of WT and Nrf2−/− mice might be related to the specific methylation patterns in their promoter CpG units. After exposure to PM2.5, Nrf2 might regulate CYP2E1 expression by affecting CpG2 unit methylation and induce DNA demethylation via TET3 expression. Our study revealed the underlying mechanism for Nrf2 to regulate epigenetics after lung exposure to PM2.5.</p

Proteomic Characterization of the Cellular Response to Nitrosative Stress Mediated by S-Nitrosoglutathione Reductase Inhibition

The S-nitrosoglutathione-metabolizing enzyme, GSNO reductase (GSNOR), has emerged as an important regulator of protein S-nitrosylation. GSNOR ablation is protective in models of asthma and heart failure, raising the idea that GSNOR inhibitors might hold therapeutic value. Here, we investigated the effects of a small molecule inhibitor of GSNOR (GSNORi) in mouse RAW 264.7 macrophages. We found that GSNORi increased protein S-nitrosylation in cytokine-stimulated cells, and we utilized stable isotope labeling of amino acids in cell culture (SILAC) to quantify the cellular response to this “nitrosative stress”. The expression of several cytokine-inducible immunomodulators, including osteopontin, cyclooxygenase-2, and nitric oxide synthase isoform 2 (NOS2), were decreased by GSNORi. In addition, selective targets of the redox-regulated transcription factor, nuclear factor (erythroid-derived 2)-like 2 (Nrf2)including heme oxygenase 1 (HO-1) and glutamate cysteine ligase modulatory subunitwere induced by GSNORi in a NOS2- and Nrf2-dependent manner. In cytokine-stimulated cells, Nrf2 protected from GSNORi-induced glutathione depletion and cytotoxicity and HO-1 activity was required for down-regulation of NOS2. Interestingly, GSNORi also affected a marked increase in NOS2 protein stability. Collectively, these data provide the most complete description of the global effects of GSNOR inhibition and demonstrate several important mechanisms for inducible response to GSNORi-mediated nitrosative stress

Iodoacetic Acid Activates Nrf2-Mediated Antioxidant Response <i>in Vitro</i> and <i>in Vivo</i>

Iodoacetic acid (IAA) is an unregulated drinking-water disinfection byproduct with potent cytotoxicity, genotoxicity, and tumorigenicity in animals. Oxidative stress is thought to be essential for IAA toxicity, but the exact mechanism remains unknown. Here we evaluated the toxicity of IAA by examining nuclear factor E2-related factor 2 (Nrf2)-mediated antioxidant response, luciferase antioxidant response element (ARE) activity, and intracellular glutathione (GSH) in HepG2 cells. IAA showed significant activation of ARE-luciferase reporter, mRNA, and protein expression of Nrf2 and its downstream genes (<i>GCLC</i>, <i>NQO1</i>, and <i>HO-1</i>). IAA also increased the intracellular GSH level in HepG2 cells in a time- and concentration-dependent manner. Moreover, we verified IAA induced Nrf2-mediated antioxidant response in rats. Subsequently, we confirmed the specific role of Nrf2 in IAA induced toxicity using <i>NRF2</i>-knockdown cells. Deficiency of <i>NRF2</i> significantly enhanced sensitivity to IAA toxicity and led to an increase of IAA induced micronulei. We also examined the effects of antioxidant on Nrf2-mediated response in IAA treated cells. Pretreatment with curcumin markedly reduced cytotoxicity and genotoxicity (micronuclei formation) IAA in HepG2 cells. Our work here provides direct evidence that IAA activates Nrf2-mediated antioxidant response <i>in vitro</i> and <i>in vivo</i> and that oxidative stress plays a role in IAA toxicity

Proteomic Characterization of the Cellular Response to Nitrosative Stress Mediated by S-Nitrosoglutathione Reductase Inhibition

The S-nitrosoglutathione-metabolizing enzyme, GSNO reductase (GSNOR), has emerged as an important regulator of protein S-nitrosylation. GSNOR ablation is protective in models of asthma and heart failure, raising the idea that GSNOR inhibitors might hold therapeutic value. Here, we investigated the effects of a small molecule inhibitor of GSNOR (GSNORi) in mouse RAW 264.7 macrophages. We found that GSNORi increased protein S-nitrosylation in cytokine-stimulated cells, and we utilized stable isotope labeling of amino acids in cell culture (SILAC) to quantify the cellular response to this “nitrosative stress”. The expression of several cytokine-inducible immunomodulators, including osteopontin, cyclooxygenase-2, and nitric oxide synthase isoform 2 (NOS2), were decreased by GSNORi. In addition, selective targets of the redox-regulated transcription factor, nuclear factor (erythroid-derived 2)-like 2 (Nrf2)including heme oxygenase 1 (HO-1) and glutamate cysteine ligase modulatory subunitwere induced by GSNORi in a NOS2- and Nrf2-dependent manner. In cytokine-stimulated cells, Nrf2 protected from GSNORi-induced glutathione depletion and cytotoxicity and HO-1 activity was required for down-regulation of NOS2. Interestingly, GSNORi also affected a marked increase in NOS2 protein stability. Collectively, these data provide the most complete description of the global effects of GSNOR inhibition and demonstrate several important mechanisms for inducible response to GSNORi-mediated nitrosative stress