A new static visual field test algorithm: the Ambient Interactive ZEST (AIZE).

Visual field (VF) test is one of the most vital tests in the diagnosis of glaucoma and to monitor the disease worsening. In the past couple of decades, the standard automated perimetry (SAP) test takes a major role in VF test for glaucoma patients. The SAP has been demanded to finish a test in short time without sacrificing accuracy. In this study, we developed and evaluated the performance of a new perimetric algorithm (ambient interactive zippy estimation by sequential testing (ZEST): AIZE) by computer simulation. AIZE is a modification of the ZEST procedure that utilizes the spatial information (weighted likelihood: WL) of neighboring test locations, which varies from the distance to the tested location, to estimate a visual threshold. Ten glaucomatous and 10 normal empirical visual field (VF) test results were simulated with five error conditions [(3% false positives (FP), 3% false negatives (FN)), (9% FP, 9% FN), (15% FP, 15% FN), (3% FP, 15% FN), (15% FP, 3% FN)]. The total number of test presentations and the root mean square error (RMSE) of the estimated visual sensitivities were compared among AIZE, the non-weighted test (WL = 0) and the fixed-weighted test (WL = 0.33). In both glaucomatous (G) and normal (N) VFs, the fixed-weighted test had the lowest number of test presentations (median G 256, N 139), followed by the AIZE (G 285, N 174) and the non-weighted test (G 303, N 195). The RMSE of the fixed-weighted test was lower (median 1.7 dB) than that of the AIZE (1.9 dB) and the non-weighted test (1.9 dB) for normal VFs, whereas the AIZE had a lower RMSE (3.2 dB) than the fixed-weighted test (4.5 dB) and the non-weighted test (4.0 dB) for glaucomatous VFs. Simulation results showed that AIZE had fewer test presentations than the non-weighted test strategy without affecting the accuracy for glaucomatous VFs. The AIZE is a useful time saving test algorithm in clinical settings.

Measurement of Fixational Eye Movements With the Head-Mounted Perimeter Imo.

Purpose: Although visual field testing is conducted with the subject gazing at a fixation target, constant minute eye movements, called fixational eye movements, do occur during fixation. We examined dynamic changes in fixational eye movements associated with stimulus presentation during visual field testing. Methods: We used the head-mounted perimeter imo, which is capable of measurement under binocular conditions, with the frame rate of its fixation monitoring camera improved to 300 Hz, to assess fixational eye movements in 18 healthy individuals. We measured changes in fixational eye movements during testing under monocular and binocular conditions and analyzed these changes based on the bivariate contour ellipse area (BCEA). We also assessed the changes in the horizontal and vertical microsaccade rates separately. Results: Both the BCEA and horizontal microsaccade rates were higher at 400 to 600 msec after stimulus presentation than during stimulus presentation (P < 0.01). Additionally, the BCEA and vertical microsaccade rates were significantly lower in the binocular condition than in the monocular condition (P < 0.01 and P < 0.05, respectively). We did not observe a significant correlation between the test locations and microsaccade direction during visual field testing. Conclusions: Fixational eye movements, especially vertical microsaccade rates, were lower in the binocular condition than in the monocular condition. Visual field testing under binocular conditions is a useful method for suppressing fixational eye movements and stabilizing the fixation during testing and may improve the reliability of the test results. Translational Relevance: Visual field testing under binocular conditions can make the fixation more stable during the testing compared with monocular conditions.

Apoptotic Effects of a Thioether Analog of Vitamin K3 in a Human Leukemia Cell Line.

Research suggests that thioether analogs of vitamin K3 (VK3) can act to preserve the phosphorylation of epidermal growth factor receptors by blocking enzymes (phosphatases) responsible for their dephosphorylation. Additionally, these derivatives can induce apoptosis via mitogen-activated protein kinase and caspase-3 activation, inducing reactive oxygen species (ROS) production, and apoptosis. However, vitamin K1 exhibits only weak inhibition of phosphatase activity, while the ability of VK3 to cause oxidative DNA damage has raised concerns about carcinogenicity. Hence, in the current study, we designed, synthesized, and screened a number of VK3 analogs for their ability to enhance phosphorylation activity, without inducing off-target effects, such as DNA damage. 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay revealed that each analog produced a different level of cytotoxicity in the Jurkat human leukemia cell line; however, none elicited a cytotoxic effect that differed significantly from that of the control. Of the VK3 analogs, CPD5 exhibited the lowest EC50, and flow cytometry results showed that apoptosis was induced at final concentrations of ≥10 µM; hence, only 0.1, 1, and 10 µM were evaluated in subsequent assays. Furthermore, CPD5 did not cause vitamin K-attributed ROS generation and was found to be associated with a significant increase in caspase 3 expression, indicating that, of the synthesized thioether VK3 analogs, CPD5 was a more potent inducer of apoptosis than VK3. Hence, further elucidation of the apoptosis-inducing effect of CPD5 may reveal its efficacy in other neoplastic cells and its potential as a medication.

Sustained high expression of NRF2 and its target genes induces dysregulation of cellular proliferation and apoptosis is associated with arsenite-induced malignant transformation of human bronchial epithelial cells.

In arsenic toxicity, activation of the erythroid 2-related factor 2 (NRF2) pathway is regarded as a driver of cancer development and progression; however, the mechanisms by which NRF2 gene expression regulates cell cycle progression and mediates pathways of cellular proliferation and apoptosis in arsenic-induced lung carcinogenesis are poorly understood. In this study, we explored the regulatory functions of NRF2 expression and its target genes in immortalized human bronchial epithelial (HBE) cells continuously exposed to 1.0 µM sodium arsenite over approximately 43 passages (22 weeks). The experimental treatment induced malignant transformation in HBE cells, characterized by increased cellular proliferation and soft agar clone formation, as well as cell migration, and accelerated cell cycle progression from G0/G1 to S phase with increased levels of cyclin E-CDK2 complex,decreased cellular apoptosis rate. Moreover, we observed a sustained increase in NRF2 protein levels and those of its target gene products (NQO1, BCL-2) with concurrently decreased expression of apoptosis-related proteins (BAX, Cleaved-caspase-3/Caspase-3 and CHOP) and increased expression of the anti-apoptotic protein MCL-1. Silencing NRF2 expression with small interfering RNA (siRNA) in arsenite-transformed (T-HBE) cells was shown to reverse the malignant phenotype. Further, siRNA silencing of NQO1 significantly decreased levels of the cyclin E-CDK2 complex, inhibiting G0/G1 to S phase cell cycle progression and transformation to the T-HBE phenotypes. This study demonstrated a novel role for the NRF2/NQO1 signaling pathway in mediating arsenite-induced cell transformation by increasing the expression of cyclin E-CDK2, and accelerating the cell cycle and cell proliferation. Arsenite promotes activation of the NRF2/BCL-2 signaling pathway inhibited CHOP increasing cellular resistance to apoptosis and further promoting malignant transformation.

Role of GH/IGF axis in arsenite-induced developmental toxicity in zebrafish embryos.

Growth hormone (GH)/insulin-like growth factor (IGF) axis plays a critical role in fetal development. However, the effect of arsenite exposure on the GH/IGF axis and its toxic mechanism are still unclear. Zebrafish embryos were exposed to a range of NaAsO2 concentrations (0.0-10.0 mM) between 4 and 120 h post-fertilization (hpf). Development indexes of survival, malformation, hatching rate, heart rate, body length and locomotor behavior were measured. Hormone levels, GH/IGF axis-related genes, and nerve-related genes were also tested. The results showed that survival rate, hatching rate, heart rate, body length and locomotor behavior all decreased, while deformity increased. At 120 hpf, the survival rate of zebrafish in 1.5 mM NaAsO2 group was about 70%, the deformity rate exceeded 20%, and the body length shortened to 3.35 mm, the movement distance of zebrafish decreased approximately 63.6% under light condition and about 52.4% under dark condition. The level of GH increased and those of IGF did not change significantly, while the expression of GH/IGF axis related genes (ghra, ghrb, igf2r, igfbp3, igfbp2a, igfbp5b) and nerve related genes (dlx2, shha, ngn1, elavl3, gfap) decreased. In 1.5 mM NaAsO2 group, the decrease of igfbp3 and igfbp5b was almost obvious, about 78.2% and 72.2%. The expression of nerve genes in 1.5 mM NaAsO2 group all have declined by more than 50%. These findings suggested that arsenite exerted disruptive effects on the endocrine system by interfering with the GH/IGF axis, leading to zebrafish embryonic developmental toxicity.

Possible differences in the mechanism of malignant transformation of HaCaT cells by arsenite and its dimethyl metabolites, particularly dimethylthioarsenics.

BACKGROUND: As a confirmed human carcinogen, arsenic can cause skin cancer, lung cancer, etc. However, its carcinogenic mechanism is still unclear. In recent years, the oxidative stress hypothesis has become widely accepted. In mammals it has been found that arsenic can be converted to dimethylarsinous acid (DMAIII) and dimethylmonothioarsinic acid (DMMTAV) through a series of methylation and redox reactions. DMAIII and DMMTAV are highly toxic. METHODS: Human keratinocytes (HaCaT) were exposed to different concentrations of NaAsO2 (IAsIII), DMMTAV and DMAIII for 24 h. Reactive oxygen species (hydrogen peroxide and superoxide), oxidative damage markers (8-hydroxydeoxyguanosine and malondialdehyde), and antioxidant markers (glutathione and superoxide dismutase) were measured. In addition, sulfane sulfurs were measured in HaCaT cells and a cell-free system. RESULTS: In the DMMTAV and DMAIII treatment groups, the levels of hydrogen peroxide and superoxide in HaCaT cells were higher than in the IAsIII treatment groups at the same dose. Levels of 8-OHdG and MDA in the DMMTAV and DMAIII treatment groups were also higher than those in the IAsIII treatment groups at the same dose. However, in the DMMTAV and DMAIII treatment groups, the levels of GSH and SOD activity were lower than that in the IAsIII treatment groups. In DMMTAV-treated HaCaT cells, sulfane sulfurs were produced. Further, it was found that DMMTAV could react with DMDTAV to form persulfide in the cell-free system, which may explain the mechanism of the formation of sulfane sulfurs in DMMTAV-treated HaCaT cells. CONCLUSIONS: DMMTAV and DMAIII more readily induce reactive oxygen species (ROS) and cause oxidative damage in HaCaT cells than inorganic arsenic. Further, the persulfide formed by the reaction of DMMTAV and DMDTAV produced from the metabolism of DMMTAV may induce a stronger reductive defense mechanism than GSH against the intracellular oxidative stress of DMMTAV. However, the cells exposed to arsenite are transformed by the continuous nuclear translocation of Nrf2 due to oxidative stress, and the persulfide from dimethylthioarsenics may promote Nrf2 by the combination with thiol groups, especially redox control key protein, Keap1, eventually cause nuclear translocation of sustained Nrf2.

Long-term arsenite exposure decreases autophagy by increased release of Nrf2 in transformed human keratinocytes.

Autophagy dysfunction in arsenite toxicity plays critical roles in cancer development and progression. However, the precise mechanisms of arsenite-induced skin cancer by blocking autophagy remain uncertain. Herein, this study investigated molecular mechanisms of arsenite-induced autophagy dysfunction mediated by nuclear factor erythroid-2 related factor 2 (Nrf2) in human keratinocyte (HaCaT) cells. The effects of long-term arsenite exposure on Nrf2 activation and autophagy were established using a siRNA interference assay and western blots. A specific siRNA of Nrf2 was used to verify that autophagy induced by arsenite can be influenced by Nrf2. Specific inhibitors of PI3K (LY294002) and mTOR (Rapamycin) and siRNA of Nrf2 were employed to verify that upregulation of Nrf2 correlated with activating the PI3K/Akt pathway. Downstream mTOR and Bcl2 were upregulated by Nrf2 signaling, inhibiting autophagy initiation in arsenite-exposed HaCaT cells. In conclusion, our data suggest that long-term exposure to arsenite promotes Nrf2 upregulation via the PI3K/Akt pathway and, along with upregulation of downstream mTOR and Bcl2, contributes to autophagy dysfunction in transformed HaCaT cells. This work provides new insights into the mechanisms underlying arsenite-induced autophagy dysfunction in cancer promotion and malignancy progression.

High NF-E2-related factor 2 expression predicts poor prognosis in patients with lung cancer: a meta-analysis of cohort studies.

NF-E2-related factor 2 (Nrf2) plays an important role in tumour proliferation and chemoresistance. Nrf2 expression levels may be associated with prognosis of lung cancer, but previous results have been inconsistent. Pooled hazard ratios (HRs) and odds ratios were calculated to assess the prognostic value of the Nrf2 expression in this meta-analysis. Nine studies with 940 patients were included. A high Nrf2 expression level was significantly related to decreased overall survival (OS) (HR = 1.948, 95% CI = 1.564-2.427), lower response rate (HR = 2.675, 95% CI = 1.553-4.610), and poor progression-free survival (HR = 3.078, 95% CI = 1.791-5.293). Subgroup analysis demonstrated that high-Nrf2-expression was significantly correlated with worse OS of patients possessing epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) or undergoing chemotherapeutic treatments (HR = 2.500, 95% CI = 1.556-4.018). Conversely, this high expression was not significantly related to the OS of patients with surgical resection (HR = 1.750, 95% CI = 0.995-3.080, and p=.052). High Nrf2 expression was significantly correlated with worse OS of patients in advanced stage (HR = 2.500, 95% CI = 1.556-4.018), compared with early cancer stage (HR = 1.609, 95% CI = 0.675-3.835, and p=.283). This meta-analysis suggests that high Nrf2 expression may be a predictive factor of poor outcomes in lung cancer. Therefore, Nrf2 likely plays an important role in prognostic evaluation and may be a therapeutic target for EGFR-TKIs therapy and chemotherapy.

Metabolism of 3-[5'-deoxy-5'-(dimethylarsinoyl)-ß-ribofuranosyloxy]-2-hydroxypropylene glycol in an artificial digestive system.

Seaweeds contain large amounts of organoarsenic compounds, mostly arsenosugars (AsSug) and arsenolipids (AsLipid). AsSug is mainly metabolized into dimethylarsinic acid (DMA V ) in humans. However, this metabolic process is not well understood. We investigated the metabolism of an AsSug, 3-[5'-deoxy-5'-(dimethylarsinoyl)-ß-ribofuranosyloxy]-2-hydroxypropylene glycol (AsSug328), in the gastrointestinal tract using an in vitro artificial gastrointestinal digestion system. AsSug328 was incubated with gastric juice for 4 h, with bile-pancreatic juice for 0.5 h, and finally with enteric bacteria solution for 24 h. The conversion of arsenic compounds after artificial digestion was analyzed by HPLC-ICP-MS and HPLC-ESI-Q-TOF-MS. Our results show that artificial gastrointestinal digestion converted AsSug328 into thio-AsSug328. However, no formation of DMA V was detected. Under the artificial digestion system, the 5-deoxyribofuranose structure of AsSug was maintained. Therefore, AsSug should be absorbed in the intestinal tract after its sugar moiety is partially decomposed. They are then possibly metabolized to DMA V in the liver and subsequently excreted through urine.

The protective effect of silk fibroin on high glucose induced insulin resistance in HepG2 cells.

The therapeutic use of silk-derived materials such as fibroin in biomedicine is well-established in Southeast Asian countries. Studies indicated that silk fibroin (SF) peptide enhances insulin sensitivity and glucose metabolism phenomena associated with type 2 diabetes mellitus (T2DM) suggesting this peptide may be beneficial to treat this disease. However, the mechanisms underlying protective effect of SF in insulin-mediated hepatic metabolic dysfunction remains unclear. The aim of this study was to investigate the influence of SF on insulin resistant HepG2 cells which were used a model of T2DM. Treatment of cells with 30 mmol/L of glucose and 10-6 mol/L insulin for 48 h significantly reduced glucose consumptions and intracellular glycogen levels but increased triglyceride (TG) levels. SF or metformin alone elevated glucose consumptions and glycogen accumulation accompanied by lower TG content. Greater effects in these metabolic parameters were found when SF and metformin were combined. Treatment of insulin resistant cells with SF or metformin alone decreased levels of reactive oxygen species (ROS), malondialdehyde (MDA), tumor necrosis factor (TNF-α) and interleukin-6 (IL-6); whereas antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) activity, as well as total antioxidant capacity (T-AOC) ability increased. The combination of SF and metformin produced greater changes in these parameters compared to metformin alone. Data indicated that the protective effect of SF or metformin in insulin resistant HepG2 cells involves inhibition of oxidant processes and that the combination of agents may prove more effective therapeutically.

Differences in apoptotic signaling and toxicity between dimethylmonothioarsinic acid (DMMTAV) and its active metabolite, dimethylarsinous acid (DMAIII), in HepaRG cells: Possibility of apoptosis cascade based on diversity of active metabolites of DMMTAV.

Dimethylmonothioarsinical acid (DMMTAV), a metabolite of arsenosugars (AsSug) and arsenolipids (AsLP), which are major organoarsenicals contained in seafoods, has been a focus of our attention due to its toxicity. It has been reported that the toxicity of DMMTAV differs according to the host cell type and that dimethylarsinous acid (DMAIII), which is a higher active metabolite of inorganic and organo arsenic compounds, may be the ultimate substance. To further elucidate the details of the mechanisms of DMMTAV, we carried out toxicological characterization by comparing DMMTAV and DMAIII using HepaRG cells, which are terminally differentiated hepatic cells derived from a human hepatic progenitor cell line that retains many characteristics, e.g, primary human hepatocytes including the morphology and expression of key metabolic enzymes (P450 s and GSTs, etc.) and complete expression of all nuclear receptors. HepaRG cells were induced to undergo differentiation by DMSO, which result red in increased levels of metabolic enzymes such as P450 and GST, in non-differentiated cells the cellular toxicities of DMMTAV and DMAIII were reduced and the induction of toxicity by DMMTAV was increased by GSH but not by DMAIII. Both DMAIII and DMMTAV induce apoptosis and increase caspase 3/7 activity. DMAIII exposure increased the activity of caspase-9. On the contrary, DMMTAV exposure resulted in markedly elevated activity of caspase-8 as well as caspase-9. These results suggest there are differences between the signaling pathways of apoptosis in DMAIII and DMMTAV and that between their active metabolites. Consequently, the ultimate metabolic substance of toxicity induction of DMMTAV may not only be DMAIII, but may also be partly due to other metabolic substances produced through the activation mechanism by GSH.

Total arsenic and speciation analysis of saliva and urine samples from individuals living in a chronic arsenicosis area in China.

BACKGROUND: It is generally acknowledged that the determination of harmful chemical compounds excreted into saliva is useful for assessing their exposure levels. The aim of the present study was to compare the total arsenic and its species in saliva and urine samples collected from the people residing in an arsenic-contaminated area of China and to further verify the feasibility of using salivary arsenic as a new biomarker of arsenic exposure. METHODS: Total arsenic and speciation analyses in urine and saliva samples among 70 residents exposed to arsenic from drinking water in Shanxi, China were carried out by high-performance liquid chromatography-inductively coupled plasma-mass spectrometry (HPLC-ICP/MS). RESULTS: The result showed that, total arsenic concentration in saliva was relatively lower than in urine samples, but it existed a strong positive correlation with total urinary arsenic, drinking water arsenic and different skin lesions. For arsenic metabolism analyses, AsIII, AsV, MMA, and DMA were detected in all of the urine samples with the dominating species of DMA (73.2%). Different with urinary arsenic species, most arsenic species in saliva were not methylated. The major species in saliva was iAs (AsIII + AsV, 76.18%), followed by DMA (13.08%) and MMA (9.13%). And the primary methylation index (PMI), second methylation index (SMI) and proportion of the four different species (AsIII, AsV, MMA, and DMA) in saliva showed no significant positive relationship with that of in urine. CONCLUSIONS: These findings indicated saliva may be used as a useful tool for biological monitoring of total arsenic exposure in the crowd rather than an efficient tool for assessing arsenic metabolism in human body after exposed to arsenic.

Effects of head tilt on visual field testing with a head-mounted perimeter imo.

PURPOSE: A newly developed head-mounted perimeter termed "imo" enables visual field (VF) testing without a fixed head position. Because the positional relationship between the subject's head and the imo is fixed, the effects of head position changes on the test results are small compared with those obtained using a stationary perimeter. However, only ocular counter-roll (OCR) induced by head tilt might affect VF testing. To quantitatively reveal the effects of head tilt and OCR on the VF test results, we investigated the associations among the head-tilt angle, OCR amplitude and VF testing results. SUBJECTS AND METHODS: For 20 healthy subjects, we binocularly recorded static OCR (s-OCR) while tilting the subject's head at an arbitrary angle ranging from 0° to 60° rightward or leftward in 10° increments. By monitoring iris patterns, we evaluated the s-OCR amplitude. We also performed blind spot detection while tilting the subject's head by an arbitrary angle ranging from 0° to 50° rightward or leftward in 10° increments to calculate the angle by which the blind spot rotates because of head tilt. RESULTS: The association between s-OCR amplitude and head-tilt angle showed a sinusoidal relationship. In blind spot detection, the blind spot rotated to the opposite direction of the head tilt, and the association between the rotation angle of the blind spot and the head-tilt angle also showed a sinusoidal relationship. The rotation angle of the blind spot was strongly correlated with the s-OCR amplitude (R2≥0.94, p<0.0001). A head tilt greater than 20° with imo causes interference between adjacent test areas. CONCLUSIONS: Both the s-OCR amplitude and the rotation angle of the blind spot were correlated with the head-tilt angle by sinusoidal regression. The rotated VF was correlated with the s-OCR amplitude. During perimetry using imo, the change in the subject's head tilt should be limited to 20°.

Visual Field Testing with Head-Mounted Perimeter 'imo'.

PURPOSE: We developed a new portable head-mounted perimeter, "imo", which performs visual field (VF) testing under flexible conditions without a dark room. Besides the monocular eye test, imo can present a test target randomly to either eye without occlusion (a binocular random single eye test). The performance of imo was evaluated. METHODS: Using full HD transmissive LCD and high intensity LED backlights, imo can display a test target under the same test conditions as the Humphrey Field Analyzer (HFA). The monocular and binocular random single eye tests by imo and the HFA test were performed on 40 eyes of 20 subjects with glaucoma. VF sensitivity results by the monocular and binocular random single eye tests were compared, and these test results were further compared to those by the HFA. The subjects were asked whether they noticed which eye was being tested during the test. RESULTS: The mean sensitivity (MS) obtained with the HFA highly correlated with the MS by the imo monocular test (R: r = 0.96, L: r = 0.94, P < 0.001) and the binocular random single eye test (R: r = 0.97, L: r = 0.98, P < 0.001). The MS values by the monocular and binocular random single eye tests also highly correlated (R: r = 0.96, L: r = 0.95, P < 0.001). No subject could detect which eye was being tested during the examination. CONCLUSIONS: The perimeter imo can obtain VF sensitivity highly compatible to that by the standard automated perimeter. The binocular random single eye test provides a non-occlusion test condition without the examinee being aware of the tested eye.

A biological indicator of inorganic arsenic exposure using the sum of urinary inorganic arsenic and monomethylarsonic acid concentrations.

OBJECTIVES: The sum of urinary inorganic arsenic (iAs), monomethylarsonic acid (MMA), and dimethylarsinic acid (DMA) concentrations is used for the biological monitoring of occupational iAs exposure. Although DMA is a major metabolite of iAs, it is an inadequate index because high DMA levels are present in urine after seafood consumption. We estimated the urinary iAs+MMA concentration corresponding to iAs exposure. METHODS: We used data from two arsenic speciation analyses of urine samples from 330 Bangladeshi with oral iAs exposure and 172 Japanese workers without occupational iAs exposure using high-performance liquid chromatography with inductively coupled plasma mass spectrometry. RESULTS: iAs, MMA, and DMA, but not arsenobetaine (AsBe), were detected in the urine of the Bangladeshi subjects. The correlation between iAs+MMA+DMA and iAs+MMA was obtained as log (iAs+MMA) = 1.038 log (iAs+MMA+DMA) -0.658. Using the regression formula, the iAs+MMA value was calculated as 2.15 and 7.5 µg As/l, corresponding to 3 and 10 µg As/m(3) of exposures, respectively. In the urine of the Japanese workers, arsenic was mostly excreted as AsBe. We used the 95th percentile of iAs+MMA (12.6 µg As/l) as the background value. The sum of the calculated and background values can be used as a biological indicator of iAs exposure. CONCLUSION: We propose 14.8 and 20.1 µg As/l of urinary iAs+MMA as the biological indicators of 3 and 10 µg As/m(3) iAs exposure, respectively.

A novel metabolic activation associated with glutathione in dimethylmonothioarsinic acid (DMMTA(V))-induced toxicity obtained from in vitro reaction of DMMTA(V) with glutathione.

The purpose of the present study was to elucidate the metabolic processing of dimethylmonothioarsinic acid (DMMTA(V)), which is a metabolite of inorganic arsenic and has received a great deal of attention recently due to its high toxicity. The metabolites produced from an in vitro reaction with GSH were analyzed by high performance liquid chromatography-time of flight mass spectrometer (HPLC-TOFMS), HPLC with a photodiode array detector (PDA), and also gas chromatography-mass spectrometry (GC-MS) and GC with a flame photometric detector (FPD). The reaction of dimethylarsinic acid (DMA(V)) with GSH did not generate DMA(V)-SG but did generate dimethylarsinous acid (DMA(III)) or DMA(III)-SG. On the contrary, we confirmed that the reaction of DMMTA(V) with GSH directly produced the stable complex of DMMTA(V)-SG without reduction through a trivalent dimethylated arsenic such as DMA(III) and DMA(III)-SG. Furthermore, the present study suggests the production of hydrogen sulfide (H2S) and dimethylmercaptoarsine (DMA(III)-SH), a trivalent dimethylated arsenic, as well as DMA(III) and DMA(III)-SG in the decomposition process of DMMTA(V)-SG. These results indicate that the toxicity of DMMTA(V) depends not only on the formation of DMA(III) but also on at least those of H2S and DMA(III)-SH.

Continuous activation of Nrf2 and its target antioxidant enzymes leads to arsenite-induced malignant transformation of human bronchial epithelial cells.

Long-term exposure to arsenite leads to human lung cancer, but the underlying mechanisms of carcinogenesis remain obscure. The transcription factor of nuclear factor-erythroid-2 p45-related factor (Nrf2)-mediated antioxidant response represents a critical cellular defense mechanism and protection against various diseases. Paradoxically, emerging data suggest that the constitutive activation of Nrf2 is associated with cancer development, progression and chemotherapy resistance. However, the role of Nrf2 in the occurrence of cancer induced by long-term arsenite exposure remains to be fully understood. By establishing transformed human bronchial epithelial (HBE) cells via chronic low-dose arsenite treatment, we showed that, in acquiring this malignant phenotype, continuous low level of ROS and sustained enhancement of Nrf2 and its target antioxidant enzyme levels were observed in the later-stage of arsenite-induced cell transformation. The downregulation of Keap1 level may be responsible for the over-activation of Nrf2 and its target enzymes. To validate these observations, Nrf2 was knocked down in arsenite-transformed HBE cells by SiRNA transfection, and the levels of Nrf2 and its target antioxidant enzymes, ROS, cell proliferation, migration, and colony formation were determined following these treatments. Results showed that blocked Nrf2 expression significantly reduced Nrf2 and its target antioxidant enzyme levels, restored ROS levels, and eventually suppressed cell proliferation, migration, and colony formation of the transformed cells. In summary, the results of the study strongly suggested that the continuous activation of Nrf2 and its target antioxidant enzymes led to the over-depletion of intracellular ROS levels, which contributed to arsenite-induced HBE cell transformation.

Hypermethylation of the Keap1 gene inactivates its function, promotes Nrf2 nuclear accumulation, and is involved in arsenite-induced human keratinocyte transformation.

It is well known that long-term exposure to arsenite leads to human skin cancer, but the underlying mechanisms of carcinogenesis remain obscure. The transcription factor Nrf2-mediated antioxidant response represents a critical cellular defense mechanism; however, emerging data suggest that constitutive activation of Nrf2 is associated with cancer development and chemotherapy resistance. The reasons Nrf2 continuously accumulates in cancer cells remain to be fully understood. By establishing transformed human keratinocyte cells via chronic arsenite treatment, we observed a continuous reduction in reactive oxygen species levels and enhanced levels of Nrf2 and its target antioxidant enzymes in the later stage of arsenite-induced cell transformation. We also revealed that hypermethylation of the Keap1 gene promoter region induced by DNA methyltransferase-3 leading to inactivation of its function was responsible for constitutive activation of Nrf2 and its target enzymes. To validate these observations, the expression of Keap1 protein was restored in arsenite-transformed cells by treatment with a DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5-Aza-dC), and protein levels of Nrf2 and colony formation were then determined after these treatments. Results showed that enhancement of Keap1 expression by 5-Aza-dC significantly reduced Nrf2 and its target antioxidant enzyme levels, and that in turn suppressed cell proliferation and colony formation of the transformed cells. Taken together, the present study strongly suggests that loss of Keap1 function by hypermethylation of its promoter region leading to Nrf2 nuclear accumulation appears to play a role in arsenite-induced human keratinocyte transformation.

Possible production of arsenic hemoglobin adducts via exposure to arsine.

OBJECTIVES: Arsine is an arsenic compound generated as a by-product in metal refineries. Accidental poisoning occurs sporadically; however, the administrative level for workers has not been established. Thus, it is essential to identify a highly specific biomarker for risk management in the workplace. The aim of this study was to identify an arsenic adduct, a potential biomarker, in the plasma. METHODS: Preserved mouse blood was exposed to arsine in vitro, and the plasma was separated. The residual clot of the control sample was hemolyzed using ultrapure water, and the supernatant was collected. Plasma from mice exposed to arsine in vivo was also separated from blood. Immunoprecipitation assays were conducted using all samples after ultrafiltration, and three fractions were collected. The total arsenic concentration in each fraction was quantified using inductively coupled plasma mass spectrometry (ICP-MS). The three in vitro samples and the eluate fraction from immunoprecipitation were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). RESULTS: In the exposed samples, the arsenic concentration in the fraction containing immunocomplexes was higher when immunoprecipitation was conducted with an anti-globin antibody. Three peaks were specifically observed in arsine-exposed samples after MALDI-TOF-MS analysis. Two of them were around m/z 15,000, and the other was m/z 15,700. The latter peak was confirmed even after immunoprecipitation. CONCLUSIONS: Globin forms an adduct with arsenic after both in vitro and in vivo exposure to arsine. This adduct together with hemoglobinuria could be a candidate biomarker of acute arsine poisoning in plasma.

Proposal for novel metabolic pathway of highly toxic dimethylated arsenics accompanied by enzymatic sulfuration, desulfuration and oxidation.

The International Agency for Research on Cancer (IARC) has concluded that dimethylarsinic acid [(CH3)2AsO(OH), DMA(V)], a main metabolite of inorganic arsenic, is responsible for carcinogenesis in urinary bladder and lung in rodents, and various modes of carcinogenic action have been proposed. One theory concerning the mode of action is that the biotransformation of dimethylarsinous acid [(CH3)2AsOH, DMA(III)] from DMA(V) plays an important role in the carcinogenesis by way of reactive oxygen species (ROS) production. Furthermore, dimethylmonothioarsinic acid [(CH3)2AsS(OH), DMMTA(V)], a metabolite of DMA(V), has also been noted because of its higher toxicity. However, the metabolic mechanisms of formation and disappearance of DMA(III) and DMMTA(V), and their toxicity are not fully understood. Thus, the purpose of the present study was to clarify the mechanism of metabolic formation of DMMTA(V) and DMA(V) from DMA(III). The in vitro transformation of arsenicals by treatment with liver homogenate from rodents and sulfur transferase was detected by HPLC-ICP-MS and HPLC-tandem MS. DMMTA(V) is produced from DMA(III) but not DMA(V) by cellular fractions from mouse liver homogenates and by rhodanese from bovine liver in the presence of thiosulfate, a sulfur donor. Not only DMMTA(V) thus produced but also DMA(III) are re-converted into DMA(V) by an in vitro addition of S9 mix. These findings indicate that the metabolic process not only of DMA(III) to DMA(V) or DMMTA(V) but also of DMMTA(V) to DMA(V) consists of a complicated mode of interaction between monooxygenase including cytochrome P450 (CYP) and/or sulfur transferase.