Enzyme from Padina arborescens Holmes Synthesizes Parinaric Acid, a Conjugated Tetraenoic Fatty Acid, from α-Linolenic Acid.

Conjugated fatty acids have anticancer effects. Therefore, the establishment of a synthetic method for conjugated fatty acids is important for overcoming cancer. Here, we attempted to synthesize conjugated fatty acids using enzymes extracted from seaweeds containing these fatty acids. Lipids from 12 species of seaweeds from the seas around Japan were analyzed, and Padina arborescens Holmes was found to contain conjugated fatty acids. Then, we synthesized parinaric acid, a conjugated tetraenoic acid, from α-linolenic acid using the enzyme of P. arborescens. This method is expected to have a variety of potential applications for overcoming cancer.

Nobiletin enhances plasma Interleukin-6 and C-X-C motif chemokine ligand 1 levels that are increased by treadmill running.

Exercise increases the muscular secretion of Interleukin-6 (IL-6), which is partially regulated by ß2-adrenergic receptor signaling. Nobiletin is a polymethoxyflavone (PMF) found in citrus fruits that induces the secretion of IL-6 from C2C12 myotubes, but it remains unclear whether nobiletin promotes IL-6 secretion during exercise. The aim of this study was to clarify the effects of nobiletin on IL-6 secretion during exercise. Nobiletin and epinephrine were found to synergistically increase IL-6 secretion from differentiated C2C12 cells, which was suppressed by the inhibition of adenylyl cyclase (AC) or protein kinase A (PKA). Treadmill running for 60 min increased plasma levels of IL-6, epinephrine, and norepinephrine in rats. Nobiletin (5 mg/kg) orally administered 30 min before running increased plasma IL-6 levels further, although it did not increase plasma epinephrine and norepinephrine. In a similar manner to IL-6, nobiletin and epinephrine synergistically increased the secretion of C-X-C motif chemokine ligand 1 (CXCL-1) from C2C12 cells, or the increase in plasma CXCL-1 was enhanced by nobiletin after treadmill running of rats. Our results suggest that nobiletin promotes IL-6 and CXCL-1 secretion from skeletal muscle by synergistic enhancement of the PKA pathway in ß2-adrenergic receptor signaling.

Orange peel extract reduces the inflammatory state of skeletal muscle after downhill running via an increase in IL-1RA.

It has been reported that orange peel extract (OPE) and the 4 major polymethoxyflavones (PMFs) in OPE have a protective effect against downhill running (DR)-induced skeletal muscle inflammation. However, the mechanism is not well understood. We investigated the potential of OPE and PMF compounds for increasing anti-inflammatory cytokine levels. The plasma interleukin-1 receptor antagonist (IL-1RA) level was increased 1 and 8 h after OPE administration in rats. Nobiletin induced the secretion of IL-1RA from C2C12 myotubes. In the inflammatory state of skeletal muscle after DR, OPE administration reduced nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) expression, NF-κB-DNA binding, and monocyte chemotactic protein-1 mRNA levels, but these effects were all abrogated by the intravenous administration of IL-1RA neutralizing antibody. These results indicated that OPE reduces skeletal muscle inflammatory state after DR via an increase in IL-1RA, and that IL-1 receptor signaling is important for skeletal muscle inflammation after DR.

Polymethoxyflavones in orange peel extract prevent skeletal muscle damage induced by eccentric exercise in rats.

Polymethoxyflavones (PMFs) contained in the peel of citrus fruits have anti-inflammatory, anticancer, and antidepressant effects. However, their effects on skeletal muscle are unknown. We investigated whether PMFs could prevent skeletal muscle damage induced by eccentric exercise in rats. Downhill running for 90 min increased the levels of the inflammatory cytokines, monocyte chemotactic protein-1 (MCP-1), and interleukin-1ß (IL-1ß) in skeletal muscles, especially in vastus lateralis, and the plasma creatine kinase levels. These increases were attenuated by a single oral administration of orange peel extract (OPE) 30 min before downhill running. A mixture of nobiletin, sinensetin, 3,5,6,7,8,3',4'-heptamethoxyflavone, and tangeretin, which are the major PMFs of OPE, also showed similar effects on muscle damage. These results suggest that OPE has a protective effect against eccentric exercise-induced skeletal muscle damage, and that the effects may be attributed to the 4 major PMFs.

BubR1 Is Essential for Thio-Dimethylarsinic Acid-Induced Spindle Assembly Checkpoint and Mitotic Cell Death for Preventing the Accumulation of Abnormal Cells.

Thio-dimethylarsinic acid (thio-DMA) was detected in human urine after exposure to inorganic arsenic and arsenosugars consumed by marine algae. Our previous studies have shown that thio-DMA disturbed the cell cycle progression and arrested cells in mitosis, though the biological significance or the mechanism by which thio-DMA-induced mitotic phase accumulation occurs is yet to be understood. In this study, we showed that thio-DMA promotes the phosphorylation of BubR1 protein, which is one of the constituents of the spindle assembly checkpoint (SAC) complex and accumulates in the cell in mitotic phase. Binding of Mad2 to CDC20, also known as the marker of the mitotic checkpoint complex (MCC) formation during the activation of SAC, was enhanced and mitotic associated cell death by apoptosis was promoted in HeLa cells but not in HepG2 cells. Basal BubR1 protein level in HepG2 was 10-times lower than that of HeLa cells. Consequently, BubR1 knockdown HeLa cells were generated by small interfering RNA (siRNA) technique. The MCC formation and mitotic arrest induced by thio-DMA were completely inhibited in BubR1 knockdown cells. Moreover, BubR1 knockdown cells could survive in the medium containing higher concentrations of thio-DMA with some abnormalities such as larger cell size, huge nucleus, multiple nuclei, and abnormal DNA contents. Especially, cyclin B1 negative tetraploid cells, which signify interphase cells with tetraploid, increased and survived after 48-72 h treatment with thio-DMA. Thus, these results suggest that BubR1-mediated SAC activation and MCC formation are one of the defense systems for preventing the accumulation and survival of abnormal cells induced by thio-DMA.

Seeds of Centranthus ruber and Valeriana officinalis Contain Conjugated Linolenic Acids with Reported Antitumor Effects.

Conjugated linolenic acids (CLNs) are naturally occurring fatty acids that are believed to have anticancer properties. In this study, we examined various plant seeds from herbs to discover seed oils containing CLNs. The ultraviolet spectra of total lipids from these seeds were measured. An absorption maximum around 270 nm was observed in seed oils belonging to the Valerianaceae family (Centranthus ruber and Valeriana officinalis). When the fatty acid compositions of these seed oils were measured, CLNs were detected. By silica column chromatography, neutral lipids (NLs), glycolipids, and phospholipids were eluted from seed oils of C. ruber and V. officinalis. Then, fatty acid compositions of these fractions were measured. This revealed that most of the CLNs in these seed oils existed in the NL fraction. When the NL fractions of these seed oils were reacted with lipase, CLNs showed good sensitivity to lipase hydrolysis. This suggested that the CLNs in the seed oils of C. ruber and V. officinalis existed predominantly at the sn-1,3 position of triacylglycerol and less at the sn-2 position. These results suggested that the CLNs from the seed oils of C. ruber and V. officinalis could easily be taken up by cancer cells as free fatty acids and had good potential as antitumor substances.

Arsenite-induced histone H3 modification and its effects on EGR1 and FOS expression in HeLa cells.

It is evident that trivalent arsenicals do not have mutagenicity, but they are human carcinogens. Recently, epigenetic modification has been considered as one of the important causes of arsenical carcinogenicity. Here we examined global histone H3 modification by trivalent inorganic arsenite (iAs(III)) and its contribution to gene expression in HeLa cells. iAs(III) induced histone H3K9 dimethylation (H3K9me2) and trimethylation (H3K9me3), histone H3S10 phosphorylation (H3S10p), histone H3T11 phosphorylation (H3T11p) and histone H3K9S10 trimethyl-phosphorylation (H3K9me3S10p). Among these modifications, H3S10p, H3T11p and H3K9me3S10p were observed as a punctate signal in interphase cells, which seems to associate with remodeling of the chromatin structure at the specific locus. A chromatin immunoprecipitation assay was performed to examine histone H3 modifications around the FOS, EGR1 and IL8 promoters, as previous studies revealed some relation between histone H3 modification and induction of these genes. iAs(III) increased H3S10p and H3K9me3S10p in the FOS promoter around the SRE/ELK1 binding site (-400 to -200) and CRE-binding site (-50). In contrast, histone H3 around the EGR1 promoter of SRE/CRE-binding site (-200 to -50) was modified to H3S10p and H3K9me3S10p by iAs(III). Reporter gene assays with deletion mutants of the FOS and EGR1 promoters revealed that the around SRE/ELK1 site is important for iAs(III)-mediated FOS induction, and SRE/CRE site for EGR1 induction. Collectively, these results demonstrate that iAs(III) induces histone H3 modifications around the transcription factor binding sites of the FOS and EGR1 promoter, and these modifications seem to be important in transcriptional activation of these genes.

Arachidonic acid intake and asthma risk in children and adults: a systematic review of observational studies.

The effect of arachidonic acid (ARA) intake on asthma risk is unclear. The objective of the present review was to systematically evaluate available observational studies on the relationship between ARA exposure and asthma risk in children and adults. A PubMed search was conducted on 22 October 2013 and seventy-three publications were checked against predefined criteria for eligibility. To identify additional eligible publications, potentially relevant articles were searched from bibliographies of articles on ARA and asthma. A total of 2924 citations were scrutinised. Finally, fourteen articles were included. A quality assessment was conducted based on the reporting and methodological quality. A meta-analysis was not conducted; therefore, a qualitative assessment is presented. Three high-, two medium- and ten low-quality studies were reviewed. Eleven studies, including two high- and two medium-quality studies, did not find a significant association between ARA exposure and asthma risk. In contrast, one high-quality study indicated a significant trend toward reducing asthma risk in children with decreasing maternal ARA intake (P trend = 0·025), and one low-quality study reported a significant trend of increasing asthma risk with higher blood ARA levels (P trend = 0·007). In two low-quality studies, asthma patients had significantly lower blood ARA levels than controls (both P < 0·05). These studies did not sufficiently demonstrate any relationships between ARA exposure and asthma risk because of the limited number of studies and their methodological limitations. They seem to suggest that ARA exposure is not consistently associated with asthma risk. Nevertheless, further evidence is required to prove or disprove the association.

Arachidonic Acid and Cerebral Ischemia Risk: A Systematic Review of Observational Studies.

BACKGROUND: Arachidonic acid (ARA) is a precursor of various lipid mediators. ARA metabolites such as thromboxane A2 cause platelet aggregation and vasoconstriction, thus may lead to atherosclerotic disease. It is unclear whether dietary ARA influences the ARA-derived lipid mediator balance and the risk for atherosclerotic diseases, such as cerebral ischemia. Considering the function of ARA in atherosclerosis, it is reasonable to focus on the atherothrombotic type of cerebral ischemia risk. However, no systematic reviews or meta-analyses have been conducted to evaluate the effect of habitual ARA exposure on cerebral ischemia risk. We aimed to systematically evaluate observational studies available on the relationship between ARA exposure and the atherothrombotic type of cerebral ischemia risk in free-living populations. SUMMARY: The PubMed database was searched for articles registered up to June 24, 2014. We designed a PubMed search formula as follows: key words for humans AND brain ischemia AND study designs AND ARA exposure. Thirty-three articles were reviewed against predefined criteria. There were 695 bibliographies assessed from the articles that included both ARA and cerebral ischemia descriptions. Finally, we identified 11 eligible articles and categorized them according to their reporting and methodological quality. We used the Strengthening the Reporting of Observational Studies in Epidemiology Statement (STROBE) checklist to score the reporting quality. The methodological quality was qualitatively assessed based on the following aspects: subject selection, ARA exposure assessment, outcome diagnosis, methods for controlling confounders, and statistical analysis. We did not conduct a meta-analysis due to the heterogeneity among the studies. All eligible studies measured blood ARA levels as an indicator of exposure. Our literature search did not identify any articles that evaluated dietary ARA intake and tissue ARA as assessments of exposure. Seven of the 11 eligible articles were considered to be of low quality. No articles reported a dose-dependent positive association between an increased cerebral ischemia risk and ARA exposure. However, most studies did not assess the risk in each subtype of cerebral ischemia, thus various etiological types of cerebral ischemia risk were involved in their results. KEY MESSAGES: We did not find a positive association between ARA exposure and cerebral ischemia risk. Eligible studies reported inconsistent findings: cerebral ischemia risk did not change or significantly decreased. We could not draw any conclusions due to the limited number of eligible high-quality studies. Further evidence from well-designed observational studies is required. Simultaneously, in order to develop effective preventive measures against cerebral ischemia, it is imperative to establish standardized definitions, nomenclatures, classifications, and diagnostic procedures.

Phosphorylation of histone H3 at serine 10 has an essential role in arsenite-induced expression of FOS, EGR1 and IL8 mRNA in cultured human cell lines.

Trivalent inorganic arsenite [iAs(III)] is known to alter the expression of a number of genes associated with transcription and cell proliferation, which was thought to be one of the possible mechanisms of arsenical carcinogenesis. However, the detailed mechanisms underlying iAs(III) induction of changes in gene expression are not fully understood. Here we examine the role of histone H3 phosphorylation at serine 10 (Ser(10) ) in gene regulation when the cells were treated with iAs(III). Among the 34 genes tested, iAs(III) induced mRNA expression of JUN, FOS, EGR1, HMOX1, HSPA1A, IL8, GADD45A, GADD45B and GADD153. Phosphorylation of histone H3 Ser(10) was induced by iAs(III) in interphase cells, and was effectively blocked by the ERKs pathway inhibitor (U0126). U0126 treatment significantly reduced constitutive mRNA expression of FOS and EGR1, and dramatically suppressed the induction of FOS, EGR1 and IL8 mRNA in iAs(III)-treated cells. The other genes, which were induced by iAs(III), were not affected by U0126 treatment. When the histone H3 nonphosphorylatable mutant of serine 10 (S10A) was overexpressed in cells, iAs(III) induction of FOS, EGR1and IL8 expression was significantly decreased as compared with wild-type cells. The other genes induced by iAs(III) were not changed in S10A cells nor by U0126 treatment. In addition, S10A cells were more resistant to iAs(III) cytotoxicity. These results indicated that the phosphorylation of histone H3 at Ser(10) through the ERKs pathway in interphase cells is an important regulatory event for iAs(III)-mediated gene expression. Aberrant gene expression seems to be an important cause of cytotoxicity and may have some relation to iAs(III) carcinogenicity.

Diphenylarsinic acid promotes degradation of glutaminase C by mitochondrial Lon protease.

Glutaminase C (GAC), a splicing variant of the kidney-type glutaminase (KGA) gene, is a vital mitochondrial enzyme protein that catalyzes glutamine to glutamate. Earlier studies have shown that GAC proteins in the human hepatocarcinoma cell line, HepG2, were down-regulated by diphenylarsinic acid (DPAA), but the mechanism by which DPAA induced GAC protein down-regulation remained poorly understood. Here, we showed that DPAA promoted GAC protein degradation without affecting GAC transcription and translation. Moreover, DPAA-induced GAC proteolysis was mediated by mitochondrial Lon protease. DPAA insolubilized 0.5% Triton X-100-soluble GAC protein and promoted the accumulation of insoluble GAC in Lon protease knockdown cells. DPAA destroyed the native tetrameric GAC conformation and promoted an increase in the unassembled form of GAC when DPAA was incubated with cell extracts. Decreases in the tetrameric form of GAC were observed in cells exposed to DPAA, and decreases occurred prior to a decrease in total GAC protein levels. In addition, decreases in the tetrameric form of GAC were observed independently with Lon protease. Mitochondrial heat shock protein 70 is known to be an indispensable protein that can bind to misfolded proteins, thereby supporting degradation of proteins sensitive to Lon protease. When cells were incubated with DPAA, GAC proteins that can bind with mtHsp70 increased. Interestingly, the association of mtHsp70 with GAC protein increased when the tetrameric form of GAC was reduced. These results suggest that degradation of native tetrameric GAC by DPAA may be a trigger in GAC protein degradation by Lon protease.

Significance of lutein in red blood cells of Alzheimer's disease patients.

Red blood cells (RBC) of Alzheimer's disease (AD) patients are known to be in an excessively oxidized state (i.e., with a high accumulation of peroxidized phospholipids (PLOOH)). Previously we confirmed in vitro, in vivo murine, and in human studies that carotenoids can effectively inhibit accumulation of RBC PLOOH. Thus, the relationship between RBC carotenoids and PLOOH concentrations in AD patients is of interest. In this study, RBC carotenoids and PLOOH were evaluated in 28 normal control subjects (age: 74.1 ± 1.3 years) and 28 patients with AD (age: 72.5 ± 1.4 years). The concentrations of RBC carotenoids, especially lutein, in AD patients were significantly lower than in control subjects. An inverse relationship was seen between RBC carotenoids, especially lutein, and PLOOH concentrations in AD patients. These results suggest that RBC lutein, in particular, may contribute to suppression of PLOOH accumulation in RBC of AD patients.

Trivalent dimethylarsenic compound induces histone H3 phosphorylation and abnormal localization of Aurora B kinase in HepG2 cells.

Trivalent dimethylarsinous acid [DMA(III)] has been shown to induce mitotic abnormalities, such as centrosome abnormality, multipolar spindles, multipolar division, and aneuploidy, in several cell lines. In order to elucidate the mechanisms underlying these mitotic abnormalities, we investigated DMA(III)-mediated changes in histone H3 phosphorylation and localization of Aurora B kinase, which is a key molecule in cell mitosis. DMA(III) caused the phosphorylation of histone H3 (ser10) and was distributed predominantly in mitotic cells, especially in prometaphase cells. By contrast, most of the phospho-histone H3 was found to be localized in interphase cells after treatment with inorganic arsenite [iAs(III)], suggesting the involvement of a different pathway in phosphorylation. DMA(III) activated Aurora B kinase and slightly activated ERK MAP kinase. Phosphorylation of histone H3 by DMA(III) was effectively reduced by ZM447439 (Aurora kinase inhibitor) and slightly reduced by U0126 (MEK inhibitor). By contrast, iAs(III)-dependent histone H3 phosphorylation was markedly reduced by U0126. Aurora B kinase is generally localized in the midbody during telophase and plays an important role in cytokinesis. However, in some cells treated with DMA(III), Aurora B was not localized in the midbody of telophase cells. These findings suggested that DMA(III) induced a spindle abnormality, thereby activating the spindle assembly checkpoint (SAC) through the Aurora B kinase pathway. In addition, cytokinesis was not completed because of the abnormal localization of Aurora B kinase by DMA(III), thereby resulting in the generation of multinucleated cells. These results provide insight into the mechanism of arsenic tumorigenesis.

Structure-effect relationship in the down-regulation of glutaminase in cultured human cells by phenylarsenic compounds.

Diphenylarsinic acid [DPAA(V)] was detected in ground water used as drinking water after a poisonous incident in Kamisu, Japan. An approach to define the target molecules of DPAA(V) with a high throughput analysis of proteins from cultured human cells demonstrated down-regulation of glutaminase C (GAC). GAC is a splicing variant of the kidney-type glutaminase (KGA) gene and has the enzyme activity of phosphate-activated glutaminase (PAG). To gain some insights into the mechanism of arsenic intoxication in Kamisu, the effects of various arsenic compounds, including arsenicals that were detected in ground water ([DPAA(V)], phenylarsonic acid [PAA(V)] and bis(diphenylarsine)oxide [BDPAO(III)]) and rice (phenylmethylarsinic acid [PMAA(V)]), were investigated for the expression of GAC and PAG activity. When cultured human HepG2 cells were incubated with arsenicals for 24h, the pentavalent phenylarsenic form of PAA(V) and PMAA(V) as well as DPAA(V) suppressed the expression of GAC protein and PAG activity in a concentration-dependent manner. On the other hand, the trivalent phenylarsenic form of BDPAO(III) had no suppressive effect on GAC and PAG. In addition, trivalent phenylarsenic compounds, such as the glutathione (GSH) conjugate of DPAA(V) [DPA-GS (III)] and triphenylarsine [TPA(III)], and the inorganic arsenics, iAs(V) and iAs(III), and methylated metabolites of inorganic arsenics, dimethylarsinic acid [DMA(V)] and dimethylarsinous acid [DMA(III)], had no suppressive effect on glutaminase. Likewise, methyl substituents of the hydroxyl groups of DPAA(V), PAA(V) and PMAA(V), diphenylmethylarsine oxide [DPMAO(V)] and phenyldimethylarsine oxide [PDMAO(V)], did not have any suppressive effects. These results suggest that pentavalent arsenic compounds with both phenyl groups and hydroxyl groups are effective in the suppression of glutaminase. In addition, the fact that it was only the arsenicals detected in Kamisu that were effective in suppressing glutaminase provides insights into the cause of the arsenic intoxication at Kamisu.

Product chain-length determination mechanism of Z,E-farnesyl diphosphate synthase.

cis-Prenyltransferases catalyze the consecutive condensation of isopentenyl diphosphate (IPP) with allylic prenyl diphosphates, producing Z,E-mixed prenyl diphosphate. The Mycobacterium tuberculosis Z,E-farnesyl diphosphate synthase Rv1086 catalyzes the condensation of one molecule of IPP with geranyl diphosphate to yield Z,E-farnesyl diphosphate and is classified as a short-chain cis-prenyltransferase. To elucidate the chain-length determination mechanism of the short-chain cis-prenyltransferase, we introduced some substitutive mutations at the characteristic amino acid residues of Rv1086. Among the mutants constructed, L84A showed a dramatic change of catalytic function to synthesize longer prenyl chain products than that of wild type, indicating that Leu84 of Rv1086 plays an important role in product chain-length determination. Mutagenesis at the corresponding residue of a medium-chain cis-prenyltransferase, Micrococcus luteus B-P 26 undecaprenyl diphosphate synthase also resulted in the production of different prenyl chain length from the intrinsic product, suggesting that this position also plays an important role in product chain-length determination for medium-chain cis-prenyltransferases.

Preparation of pure lipid hydroperoxides.

Increasing evidence of lipid peroxidation in food deterioration and pathophysiology of diseases have revealed the need for a pure lipid hydroperoxide (LOOH) reference as an authentic standard for quantification and as a compound for biological studies in this field. Generally, LOOH is prepared from photo- or enzymatically oxidized lipids; however, separating LOOH from other oxidation products and preparing pure LOOH is difficult. Early studies showed the usability of reaction between hydroperoxide and vinyl ether for preparation of pure LOOH. Because the reactivity of vinyl ether with LOOHs other than fatty acid hydroperoxides has never been reported, here, we employed the reaction for preparation of a wide variety of pure LOOHs. Phospholipid, cholesteryl ester, triacylglycerol, or fatty acid was photo- or enzymatically oxidized; the resultant crude sample containing hydroperoxide was allowed to react with a vinyl ether [2-methoxypropene (MxP)]. Liquid chromatography (LC) and mass spectrometry confirmed that MxP selectively reacts with LOOH, yielding a stable MxP adduct (perketal). The lipophilic perketal was eluted at a position away from that of intact LOOH and identified and isolated by LC. Upon treatment with acid, perketal released the original LOOH, which was finally purified by LC. Using our optimized purification procedures, for instance, we produced 75 mg of pure phosphatidylcholine hydroperoxide (>99%) from 100 mg of phosphatidylcholine. Our developed method expands the concept of the perketal method, which provides pure LOOH references. The LOOHs prepared by the perketal method would be used as "gold standards" in LOOH methodology.

Cytotoxic, genotoxic and cell-cycle disruptive effects of thio-dimethylarsinate in cultured human cells and the role of glutathione.

Thio-dimethylarsinate (thio-DMA), a recently discovered urine metabolite in humans, was investigated for its cytotoxic, genotoxic and cell-cycle disruptive effects in the cultured human hepatocarcinoma cell line, HepG2, and Syrian hamster embryo cells. In addition, the role of glutathione (GSH) on the cytotoxic effects of thio-DMA was investigated in terms of the effects of GSH depletion and the effects of exogenously added GSH. LC50 values of arsenicals for cells incubated for 48 h were 0.026 mM for thio-DMA, 0.343 mM for DMA and 3.66 mM for dithio-DMA. Depletion of cell GSH reduced the cytotoxic effects of thio-DMA. The cytotoxic effects of 0.02 mM and 0.05 mM thio-DMA were enhanced markedly when used in combination with 1 to 3 mM GSH, but decreased again when combined with 5 mM GSH. These results suggested that cytotoxic intermediates were generated by the interaction of thio-DMA with GSH, while an excessive amount of GSH suppressed the generation of these intermediates. Flow-cytometry showed that thio-DMA was an inducer of cells with 4N DNA and hypo 2N DNA. The results also demonstrated that cells arrested in the mitotic phase had abnormalities in their spindle organization and centrosome integrity. In addition, cells arrested in mitosis by thio-DMA had chromosome structural aberrations, such as chromatid gaps, chromatid breaks and chromatid exchanges. Moreover, the cytotoxic effects of thio-DMA may in part be associated with an apoptotic mode of cell death that was evaluated by the appearance of nucleosome level DNA fragmentations and an 85-kDa cleavage fragment of poly (ADP-ribose) polymerase. These findings suggest that the presence of thio-DMA in human urine has implications for human health in terms of arsenic metabolism and toxicity.

Efficacy of low-density lipoprotein apheresis in arteriosclerosis obliterans of the lower extremities: two cases with marked alleviation of clinical symptoms.

We have recognized marked effectiveness for intermittent claudication with low-density lipoprotein (LDL) apheresis in two cases of arteriosclerosis obliterans (ASO). The Case 1 was a 73-year-old man with intermittent claudication of both lower extremities (Fontaine class II), digital subtraction angiography (DSA) revealed complete obstruction of the left common iliac artery, formation of a collateral to the peripheral portion of the left common iliac artery, and diffuse stenosis of the peripheral portion of the right common iliac artery. Ten sessions of LDL apheresis (LDL-A) improved the walking distance from 100 m before to 600 m after LDL-A treatment. The Case 2 was a 61-year-old man with intermittent claudication of the left lower extremity (walking distance: 200 m) began at 59 years. DSA revealed diffuse stenosis of the peripheral portion of the left popliteal artery. Ten sessions of LDL-A improved the walking distance from 200 m before to 800 m after LDL-A. At one month after the end of LDL-A treatment, DSA revealed formation of collateral to the peripheral portion of the left popliteal artery. Our findings suggest that LDL-A combined with drug treatment is safe and useful for the treatment of ASO, especially walking distance improved with short term treatment.

Down-regulation of glutaminase C in human hepatocarcinoma cell by diphenylarsinic acid, a degradation product of chemical warfare agents.

In a poisonous incident in Kamisu, Japan, it is understood that diphenylarsinic acid (DPAA) was a critical contaminant of ground water. Most patients showed dysfunction of the central nervous system. To understand the overall mechanism of DPAA toxicity and to gain some insight into the application of a remedy specific for intoxication, the molecular target must be clarified. As an approach, a high throughput analysis of cell proteins in cultured human hepatocarcinoma HpG2 exposed to DPAA was performed by two-dimensional electrophoresis (2-DE). Four proteins, which were up- and down-regulated by exposure of cultured HepG2 cells to DPAA, were identified. They were chaperonin containing TCP-1 (CCT) beta subunit, aldehyde dehydrogenase 1 (ALDH1), ribosomal protein P0 and glutaminase C (GAC). Of these, GAC was the only protein that was down-regulated by DPAA exposure, and cellular expression levels were reduced by DPAA in a concentration- and time-dependent manner. Decrease in cellular GAC levels was accompanied by decreased activity of the enzyme, phosphate-activated glutaminase (PAG). Decreased expression of GAC by DPAA was also observed in human cervical carcinoma HeLa and neuroblastoma SH-SY5Y cells. By contrast, no significant changes in GAC protein expression were observed when cells were incubated with arsenite [iAs (III)] and trivalent dimethylarsinous acid [DMA (III)]. In the central nervous system, GAC plays a role in the production of the neurotransmitter glutamic acid. Selective inhibition of GAC expression by DPAA may be a cause of dysfunction of glutamatergic neuronal transmission and the resultant neurological impairments.

Glutathione plays a role in regulating the formation of toxic reactive intermediates from diphenylarsinic acid.

The role of glutathione (GSH) in the cytotoxicity of diphenylarsinic acid [DPAA(V)], which was detected in drinking well water after a poisoning incident in Kamisu, Japan, was investigated in cultured human HepG2 cells. DPA-GS(III), which is the GSH adduct of DPAA, was synthesized and analyzed by HPLC/ESI-MS. DPA-GS(III) was highly toxic to cells and the potency was about 1000 times that of DPAA(V). DPAA(V) was stable in culture medium, while DPA-GS(III) was unstable and changed to protein-bound As (protein-As). By contrast, DPA-GS(III) remained stable with the addition of exogenous GSH, thereby reducing transformation to protein-As. In addition, DPA-GS(III) was transformed to bis(diphenylarsine)oxide [BDPAO(III)], which was observed under serum-free conditions. BDPAO(III) was very unstable and disappeared conversely with an increase in protein-As. In contrast, the presence of GSH suppressed the transformation of BDPAO(III) to protein-As while it enhanced the transformation of BDPAO(III) to DPA-GS(III). Depletion of cell GSH enhanced the cytotoxic effects of DPA-GS(III) and BDPAO(III). Moreover, exogenously-added GSH suppressed the cytotoxic effects of DPA-GS(III) and BDPAO(III). The dynamic behavior of arsenicals in the culture medium and the resultant cytotoxic effects suggested that GSH played a role in regulating the formation of toxic intermediates, such as DPA-GS(III) and BDPAO(III). Moreover, the results suggested that the formation of protein-As in culture medium was compatible with the cytotoxic effects and that GSH was a factor capable of regulating the formation of protein-As from either DPA-GS(III) or BDPAO(III).