Biliary excretion of arsenic by human HepaRG cells is stimulated by selenide and mediated by the multidrug resistance protein 2 (MRP2/ABCC2).

Millions of people worldwide are exposed to unacceptable levels of arsenic, a proven human carcinogen, in drinking water. In animal models, arsenic and selenium are mutually protective through formation and biliary excretion of seleno-bis (S-glutathionyl) arsinium ion [(GS)2AsSe]-. Selenium-deficient humans living in arsenic-endemic regions are at increased risk of arsenic-induced diseases, and may benefit from selenium supplementation. The influence of selenium on human arsenic hepatobiliary transport has not been studied using optimal human models. HepaRG cells, a surrogate for primary human hepatocytes, were used to investigate selenium (selenite, selenide, selenomethionine, and methylselenocysteine) effects on arsenic hepatobiliary transport. Arsenite + selenite and arsenite + selenide at different molar ratios revealed mutual toxicity antagonism, with the latter being higher. Significant levels of arsenic biliary excretion were detected with a biliary excretion index (BEI) of 14 ± 8%, which was stimulated to 32 ± 7% by selenide. Consistent with the formation and biliary efflux of [(GS)2AsSe]-, arsenite increased the BEI of selenide from 0% to 24 ± 5%. Arsenic biliary excretion was lost in the presence of selenite, selenomethionine, and methylselenocysteine. Sinusoidal export of arsenic was stimulated ∼1.6-fold by methylselenocysteine, but unchanged by other selenium forms. Arsenic canalicular and sinusoidal transport (±selenide) was temperature- and GSH-dependent and inhibited by MK571. Knockdown experiments revealed that multidrug resistance protein 2 (MRP2/ABCC2) accounted for all detectable biliary efflux of arsenic (±selenide). Overall, the chemical form of selenium and human MRP2 strongly influenced arsenic hepatobiliary transport, information critical for human selenium supplementation in arsenic-endemic regions.

Enzyme-assisted extraction and liquid chromatography mass spectrometry for the determination of arsenic species in chicken meat.

Chicken is the most consumed meat in North America. Concentrations of arsenic in chicken range from µg kg(-1) to mg kg(-1). However, little is known about the speciation of arsenic in chicken meat. The objective of this research was to develop a method enabling determination of arsenic species in chicken breast muscle. We report here enzyme-enhanced extraction of arsenic species from chicken meat, separation using anion exchange chromatography (HPLC), and simultaneous detection with both inductively coupled plasma mass spectrometry (ICPMS) and electrospray ionization tandem mass spectrometry (ESIMS). We compared the extraction of arsenic species using several proteolytic enzymes: bromelain, papain, pepsin, proteinase K, and trypsin. With the use of papain-assisted extraction, 10 arsenic species were extracted and detected, as compared to 8 detectable arsenic species in the water/methanol extract. The overall extraction efficiency was also improved using a combination of ultrasonication and papain digestion, as compared to the conventional water/methanol extraction. Detection limits were in the range of 1.0-1.8 µg arsenic per kg chicken breast meat (dry weight) for seven arsenic species: arsenobetaine (AsB), inorganic arsenite (As(III)), dimethylarsinic acid (DMA), monomethylarsonic acid (MMA), inorganic arsenate (As(V)), 3-nitro-4-hydroxyphenylarsonic acid (Roxarsone), and N-acetyl-4-hydroxy-m-arsanilic acid (NAHAA). Analysis of breast meat samples from six chickens receiving feed containing Roxarsone showed the presence of (mean±standard deviation µg kg(-1)) AsB (107±4), As(III) (113±7), As(V) (7±2), MMA (51±5), DMA (64±6), Roxarsone (18±1), and four unidentified arsenic species (approximate concentration 1-10 µg kg(-1)).

Plant Natural Products Calycosin and Gallic Acid Synergistically Attenuate Neutrophil Infiltration and Subsequent Injury in Isoproterenol-Induced Myocardial Infarction: A Possible Role for Leukotriene B4 12-Hydroxydehydrogenase?

Leukotriene B4 12-hydroxydehydrogenase (LTB4DH) catalyzes the oxidation of proinflammatory LTB4 into less bioactive 12-oxo-LTB4. We recently discovered that LTB4DH was induced by two different natural products in combination. We previously isolated gallic acid from Radix Paeoniae through a bioactivity-guided fractionation procedure. The purpose of this study is to test the hypothesis that LTB4DH inducers may suppress neutrophil-mediated inflammation in myocardial infarction. We first isolated the active compound(s) from another plant, Radix Astragali, by the similar strategy. By evaluating LTB4DH induction, we identified calycosin and formononetin from Radix Astragali by HPLC-ESI-MS technique. We confirmed that gallic acid and commercial calycosin or formononetin could synergistically induce LTB4DH expression in HepG2 cells and human neutrophils. Moreover, calycosin and gallic acid attenuated the effects of LTB4 on the survival and chemotaxis of neutrophil cell culture. We further demonstrated that calycosin and gallic acid synergistically suppressed neutrophil infiltration and protected cardiac integrity in the isoproterenol-induced mice model of myocardial infarction. Calycosin and gallic acid dramatically suppressed isoproterenol-induced increase in myeloperoxidase (MPO) activity and malondialdehyde (MDA) level. Collectively, our results suggest that LTB4DH inducers (i.e., calycosin and gallic acid) may be a novel combined therapy for the treatment of neutrophil-mediated myocardial injury.

N-Propargyl Caffeate Amide (PACA) Potentiates Nerve Growth Factor (NGF)-Induced Neurite Outgrowth and Attenuates 6-Hydroxydopamine (6-OHDA)-Induced Toxicity by Activating the Nrf2/HO-1 Pathway.

Insufficient production of neurotrophic factors is implicated in the pathogenesis of various neurodegenerative disorders. The aim of the present study was to evaluate the potential of N-propargyl caffeate amide (PACA) to enhance nerve growth factor (NGF)-induced neurite outgrowth and the underlying mechanisms. We discovered that PACA not only potentiated NGF-induced neurite outgrowth but also attenuated 6-hydroxydopamine (6-OHDA) neurotoxicity in dopaminergic PC12 cells and primary rat midbrain neurons. To identify the PACA-binding proteins, we introduced a biotin tag to the covalent PACA-protein adducts via "click chemistry" alkyne-azido cycloaddition. As a result, kelch-like ECH-associated protein 1 (Keap1) was isolated as the predominant protein from PACA treated PC12 cells. We demonstrated that the formation of PACA-Keap1 conjugates induced the nuclear translocation of transcription factor Nrf2 and the expression of antioxidant heme oxygenase-1 (HO-1). Importantly, specific HO-1 inhibitor SnPP diminished the neuroprotective and neuritogenic activities of PACA. Moreover, PACA attenuated 6-OHDA-induced production of neurotoxic reactive oxygen species and reactive nitrogen species. PACA also preserved mitochondrial membrane integrity and enhanced the cellular resistance against 6-OHDA neurotoxicity. These results suggest that PACA may exhibit neuroprotective and neuritogenic activities via activating the Nrf2/HO-1 antioxidant pathway.

Uptake and speciation of vanadium in the benthic invertebrate Hyalella azteca.

Vanadium has the potential to leach into the environment from petroleum coke, an oil sands byproduct. To determine uptake of vanadium species in the biota, we exposed the benthic invertebrate Hyalella azteca with increasing concentrations of two different vanadium species, V(IV) and V(V), for seven days. The concentrations of vanadium in the H. azteca tissue increased with the concentration of vanadium in the exposure water. Speciation analysis revealed that V(IV) in the exposure water was oxidized to V(V) between renewal periods, and therefore the animals were mostly exposed to V(V). Speciation analysis of the H. azteca tissue showed the presence of V(V), V(IV), and an unidentified vanadium species. These results indicate the uptake and metabolism of vanadium by H. azteca. Because H. azteca are widely distributed in freshwater systems and are an important food supply for many fish, determining the uptake and metabolism of vanadium allows for a better understanding of the potential environmental effects on invertebrates.

Pharmacological induction of leukotriene B4-12-hydroxydehydrogenase suppresses the oncogenic transformation of human hepatoma HepG2 cells.

Leukotriene B4-12-hydroxydehydrogenase (LTB4DH) is characterized as a chemopreventive and tumor suppressor gene. The aim of this study was to investigate the pharmaco-logical induction of LTB4DH and potential anticancer activity. Using HepG2 cells as a cellular detector, we successfully isolated the active compounds from the herbs Radix Astragali and Radix Paeoniae Rubra through a bioactivity-guided fractionation procedure. Using various analytical techniques including electronic spray ionization-mass spectrometry (ESI-MS) and nuclear magnetic resonance (NMR), gallic acid (GA) was identified as the active compound from Radix Paeoniae Rubra whereas the active compound from Radix Astragali, designated as RA-C, was also purified to the extent that it is now suitable for further identifi-cation. We found that the active compounds from these two different herbs synergistically induced LTB4DH expression in a dose- and time-dependent manner. A key finding was that commercial GA in combination with purified RA-C attenuated the focus formation and anchorage-independent growth, two indexes of in vitro oncogenic transformation, of HepG2 cells via the induction of LTB4DH expression. Moreover, the combination of GA and purified RA-C significantly induced G2/M cell cycle arrest in HepG2 cells. Our results demon-strated for the first time that GA and purified RA-C suppress the in vitro oncogenic transformation of HepG2 cells via the induction of LTB4DH expression. Importantly, pharmaco-logical induction of LTB4DH represents a potential alternative strategy for the therapy of hepatocellular carcinoma.

Effects of an epidermal growth factor receptor inhibitor on arsenic associated toxicity in the rat bladder epithelium.

Arsenite (As(III)), an inorganic arsenical, is a known human carcinogen, inducing tumors of the skin, urinary bladder and lung. It is known to be metabolized to organic methylated arsenicals in vivo. As(III) has been reported to have the ability to up-regulate the epidermal growth factor receptor (EGFR)-associated pathway in epithelial cells, including human urothelial cells in vitro. EGFR is a cell-surface receptor belonging to the ErbB family of receptor tyrosine kinases, and the EGFR-associated signaling pathway has been reported to play an important role in carcinogenesis and cancer progression, including in bladder cancer. In this study, we investigated the growth effects of As(III) and an organic trivalent arsenical, dimethylarsinous acid (DMA(III)), and the effects of co-exposure of gefitinib, an EGFR inhibitor, with As(III) to a rat urothelial cell line (MYP3). We also investigated the effects of co-administration of dietary As(III) and gefitinib in vivo. In vitro, concentrations of 1.0microM As(III) or 0.5microM DMA(III) induced cytotoxicity. However, lower concentrations of As(III) treatment had a slight mitogenic growth effect whereas lower concentrations of DMA(III) did not. Gefitinib blocked As(III)-induced cell growth in vitro. In vivo, a high dose of gefitinib alone induced slight urothelial cytotoxicity, and did not reduce cytotoxicity and regenerative cell proliferation when co-administered with As(III). The majority of arsenic metabolites present in the urine of As(III)-treated rats were organic arsenicals, mainly dimethylarsinic acid (DMA(V)). As(III) was also present, and its concentration was higher than the concentration required to produce cytotoxicity in vitro. These data suggest that an EGFR inhibitor has the ability to block As(III)-induced cell proliferation in vitro but not in vivo in a short-term study.

Chronic arsenic exposure and oxidative stress: OGG1 expression and arsenic exposure, nail selenium, and skin hyperkeratosis in Inner Mongolia.

Arsenic, a human carcinogen, is known to induce oxidative damage to DNA. In this study we investigated oxidative stress and As exposure by determining gene expression of OGG1, which codes for an enzyme, 8-oxoguanine DNA glycosylase, involved in removing 8-oxoguanine in As-exposed individuals. Bayingnormen (Ba Men) residents in Inner Mongolia are chronically exposed to As via drinking water. Water, toenail, and blood samples were collected from 299 Ba Men residents exposed to 0.34-826 microg/L As. RNA was isolated from blood, and mRNA levels of OGG1 were determined using real-time polymerase chain reaction. OGG1 expression levels were linked to As concentrations in drinking water and nails, selenium concentrations in nails, and skin hyperkeratosis. OGG1 expression was strongly associated with water As concentrations (p < 0.0001). Addition of the quadratic term significantly improved the fit compared with the linear model (p = 0.05) . The maximal OGG1 response was at the water As concentration of 149 microg/L. OGG1 expression was also significantly associated with toenail As concentrations (p = 0.015) but inversely associated with nail Se concentrations (p = 0.0095) . We found no significant differences in the As-induced OGG1 expression due to sex, smoking, or age even though the oldest group showed the strongest OGG1 response (p = 0.0001) . OGG1 expression showed a dose-dependent increased risk of skin hyperkeratosis in males (trend analysis, p = 0.02) , but the trend was not statistically significant in females. The results from this study provide a linkage between oxidative stress and As exposure in humans. OGG1 expression may be useful as a biomarker for assessing oxidative stress from As exposure.