RKIP expression of liver and kidney after arsenic exposure.

Arsenic is associated with cancers of kidney and liver. Raf kinase inhibitor protein (RKIP) has been identified as a member of a novel class of molecules that suppress the metastatic spread of tumors. In order to investigate the effect of arsenic to RKIP of liver and kidney, the expression of RKIP of liver and kidney with As (III) was explored in this study. Thirty male mice were chronically fed with 42.5 ppm, 85 ppm NaAsO2 and water for 180 days. The kidney and liver accumulation levels of As (III) in mice were determined by electro-thermal atomic absorption spectrometry. The method of RT-PCR, Western blotting analysis and immunohistochemistry were used to determine gene expression and protein expression of RKIP. The results showed that arsenic level was significantly increased in kidney and liver of As (III)-exposed mice as compared with control group. The gene expression and protein expression of RKIP was significantly decreased in kidney and liver of As (III)-exposed mice in comparison with these of control mice. These data suggested that RKIP decrease of liver and kidney with As (III) may be dangerous index in formation of cancer. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1079-1082, 2017.

Recent trends in nanomaterial-based microanalytical systems for the speciation of trace elements: A critical review.

Trace element speciation in biomedical and environmental science has gained increasing attention over the past decade as researchers have begun to realize its importance in toxicological studies. Several nanomaterials, including titanium dioxide nanoparticles (nano-TiO2), carbon nanotubes (CNTs), and magnetic nanoparticles (MNPs), have been used as sorbents to separate and preconcentrate trace element species prior to detection through mass spectrometry or optical spectroscopy. Recently, these nanomaterial-based speciation techniques have been integrated with microfluidics to minimize sample and reagent consumption and simplify analyses. This review provides a critical look into the present state and recent applications of nanomaterial-based microanalytical systems in the speciation of trace elements. The adsorption and preconcentration efficiencies, sample volume requirements, and detection limits of these nanomaterial-based speciation techniques are detailed, and their applications in environmental and biological analyses are discussed. Current perspectives and future trends into the increasing use of nanomaterial-based microfluidic techniques for trace element speciation are highlighted.

Selective and eco-friendly method for determination of mercury(II) ions in aqueous samples using an on-line AuNPs-PDMS composite microfluidic device/ICP-MS system.

In this study we developed an on-line, eco-friendly, and highly selective method using a gold nanoparticle (AuNP)-coated polydimethylsiloxane (PDMS) composite microfluidic (MF) chip coupled to inductively coupled plasma mass spectrometry (ICP-MS) to separate trace Hg(2+) ions from aqueous samples. Because Hg(2+) ions interact with AuNPs to form Hg-Au complexes, we were able to separate Hg(2+) ions from aqueous samples. We prepared the AuNPs-PDMS composite through in situ synthesis using a PDMS cross-linking agent to both reduce and embed AuNPs onto PDMS microchannels so that no additional reductants were required for either AuNP synthesis or the PDMS surface modification (2% HAuCl4, room temperature, 48 h). To optimize the proposed on-line system, we investigated several factors that influenced the separation of Hg(2+) ions in the AuNPs-PDMS/MF, including adsorption pH, adsorption and elution flow rates, microchannel length, and interferences from coexisting ions. Under optimized conditions (pH 6.0; adsorption/elution flow rates: 0.05/0.5 mL min(-1); channel length: 840 mm), we evaluated the accuracy of the system using a standard addition method; the measured values had agreements of ≥ 93.0% with certified values obtained for Hg(2+) ions. The relative standard deviations of the proposed method ranged from 2.24% to 6.21%. The limit of detection for Hg(2+) for the proposed on-line AuNPs-PDMS/MF/ICP-MS analytical method was as low as 0.07 µg L(-1).

Nickel (II)-induced cytotoxicity and apoptosis in human proximal tubule cells through a ROS- and mitochondria-mediated pathway.

Nickel compounds are known to be toxic and carcinogenic in kidney and lung. In this present study, we investigated the roles of reactive oxygen species (ROS) and mitochondria in nickel (II) acetate-induced cytotoxicity and apoptosis in the HK-2 human renal cell line. The results showed that the cytotoxic effects of nickel (II) involved significant cell death and DNA damage. Nickel (II) increased the generation of ROS and induced a noticeable reduction of mitochondrial membrane potential (MMP). Analysis of the sub-G1 phase showed a significant increase in apoptosis in HK-2 cells after nickel (II) treatment. Pretreatment with N-acetylcysteine (NAC) not only inhibited nickel (II)-induced cell death and DNA damage, but also significantly prevented nickel (II)-induced loss of MMP and apoptosis. Cell apoptosis triggered by nickel (II) was characterized by the reduced protein expression of Bcl-2 and Bcl-xL and the induced the protein expression of Bad, Bcl-Xs, Bax, cytochrome c and caspases 9, 3 and 6. The regulation of the expression of Bcl-2-family proteins, the release of cytochrome c and the activation of caspases 9, 3 and 6 were inhibited in the presence of NAC. These results suggest that nickel (II) induces cytotoxicity and apoptosis in HK-2 cells via ROS generation and that the mitochondria-mediated apoptotic signaling pathway may be involved in the positive regulation of nickel (II)-induced renal cytotoxicity.

The expression of RKIP, RhoGDI, galectin, c-Myc and p53 in gastrointestinal system of Cr(VI)-exposed rats.

Hexavalent chromium (CrVI) is considered to be a risk factor in the formation of human cancer. Raf kinase inhibitor protein (RKIP), Rho-GDIα, galectin, c-Myc and p53 play important roles in cancer formation. The purpose of this study was to determine if Cr(VI) induces the formation of gastrointestinal cancer. We explored the expression of RKIP, Rho-GDIα, galectin, c-Myc and p53 in the colon and stomach in rats exposed to chromium (CrVI). Thirty Wistar rats were divided into six groups which were chronically fed with 250, 500, 750, 1000 and 1250 ppm Na(2) Cr(2) O(7) and water for 60 days. The level of Cr(VI) was determined by electrothermal atomic absorption spectrometry. The expression of RKIP, Rho-GDIα, galectin, c-Myc and p53 of stomach and colon was measured by western blot. The gene expression of RKIP, Rho-GDIα, galectin, c-Myc and p53 of the stomach and colon was determined by RT-PCR. The results showed that the expression of p53 and Rho-GDIα was decreased in the stomach and colon of rats with Cr(VI) treatment. The expression of RKIP was decreased in the stomach and colon of rats treated with high-dose Cr(VI). The expression of c-Myc and gelectin-1 was increased in the stomach and colon of rats with Cr(VI) treatment. We concluded that the anomalous expression of RKIP, Rho-GDIα, galectin, c-Myc and p53 might be a dangerous index of cancer formation in the stomach and colon of rats with Cr(VI) exposure.

Hexavalent chromium inhibited the expression of RKIP of heart in vivo and in vitro.

BACKGROUND: Chromium (Cr) is considered to be a risk factor to the cardiovascular effects of fine particulate matter components to PM2.5 from traffic in highway patrol officers. RKIP (raf kinase inhibitor protein) is a physiological inhibitor of GRK-2 (G-protein-coupled receptor kinase 2) and affects ß-adrenergic signaling and contractile activity in cardiomyocytes. OBJECTIVES: In this study, we explored the change of RKIP in heart of chromium (VI)-exposed rats and cultured myocardial cells with chromium (VI) treatment. METHOD: Wistar rats were divided into six groups which were chronically fed with 250, 500, 750, 1000, and 1250 ppm Na(2)Cr(2)O(7) and water for 60 days, respectively. Na(2)Cr(2)O(7) dose of 0.25, 0.5, 1.5, 3, 4.5, and 0 ppm (control group) was applied in cultured myocardial cells. The level of heart Cr (VI) was determined by electrothermal atomic absorption spectrometry. The expression of RKIP was measured by Western blot method. The MTT assay was used to measure the toxicity of myocardial cells with Cr (VI) treatment. The apoptosis test of myocardial cells was determined by caspase-3 colorimetric assay kit. RESULT: The result showed that the expression of RKIP in heart (in vivo) and myocardial cells (in vitro) was decreased following Cr (VI) dose-dependent treatment. CONCLUSION: We suggested that the decrement of RKIP of heart and myocardial cells with Cr (VI) treatment resulted in the function of cardiovascular system decreased.

(-)-Anonaine induces apoptosis through Bax- and caspase-dependent pathways in human cervical cancer (HeLa) cells.

(-)-Anonaine has been shown to have some anticancer activities, but the mechanisms of (-)-anonaine inducing cell death of human cancer cells is not fully understood. We investigated the mechanisms of apoptosis induced by (-)-anonaine in human HeLa cancer cells. Treatment with (-)-anonaine induces dose-dependent DNA damage that is correlated with increased intracellular nitric oxide, reactive oxygen species, glutathione depletion, disruptive mitochondrial transmembrane potential, activation of caspase 3, 7, 8, and 9, and poly ADP ribose polymerase cleavage. Our data indicate that (-)-anonaine up-regulated the expression of Bax and p53 proteins in HeLa cancer cells. The apoptosis and expression of Bax induced by (-)-anonaine could be inhibited when the HeLa cells were pretreated with Boc-Asp(OMe)-fmk, which is a broad caspases inhibitor. There was no obvious DNA damage in the (-)-anonaine-treated Madin-Darby canine kidney and Vero cell lines. Both Madin-Darby canine kidney and Vero cell lines are kidney epithelial cellular morphology. These results suggest that (-)-anonaine might be considered a potent compound for chemotherapy against cervical cancer or a health food supplement for cancer chemoprevention.

6-shogaol (alkanone from ginger) induces apoptotic cell death of human hepatoma p53 mutant Mahlavu subline via an oxidative stress-mediated caspase-dependent mechanism.

Mahlavu cells, poorly differentiated and p53 mutants of a human hepatoma subline, are known to be highly refractory to a number of chemotherapeutic agents and radiotherapy due to their high expressions of multidrug resistance gene-1 (MDR-1) and Bcl-2 proteins. Thus, it is desirable to search for an alternative strategy for effective eradication of this type of cancer cells. We present evidence here for the first time that 6-shogaol (6-SG), an alkanone isolated from the rhizomes of ginger, can effectively induce apoptotic cell death of Mahlavu cells via an oxidative stress-mediated caspase-dependent mechanism. The cascade of events in 6-SG-induced apoptosis of these cells involved an initial overproduction of reactive oxygen species (ROS) followed by a severe depletion of intracellular glutathione (GSH) contents. Both events consequently entailed a significant drop in mitochondrial transmembrane potential (DeltaPsim), which ultimately activated the activities of caspases 3/7 resulting in the DNA fragmentation. Interestingly, we also found that N-acetylcysteine (NAC), an antioxidant and a precursor of GSH biosynthesis, could offer a near complete protection of apoptotic cell death exerted by 6-SG. Similarly, exogenously added GSH could also provide protection with an equal efficacy. However, it was paradoxical that both Boc-Asp(OMe)-fmk (a broad caspases inhibitor) and cyclosporin A (an mitochondrial permeability transition opening inhibitor) could only partially protect these cells from 6-SG-induced apoptosis. Taking these data into consideration, it is obvious that GSH depletion is the major contributing factor in arbitrating 6-SG-induced apoptosis of Mahlavu cells. In conclusion, we provide here a novel modality that can help to eradicate a p53 mutant of human hepatoma cells by using a natural consistent isolated form of ginger. These data also provide evidence to reaffirm the notion that consumption of certain foodstuffs can be beneficial to health because some of the constituents contained in them may be anticarcinogenic.

Real-time dynamic monitoring of nonprotein-bound copper in the blood.

To obtain real-time dynamic changes of non-protein-bound copper in the blood, we have developed an online microdialysis sampling system coupled with a flow-injection graphite furnace-atomic absorption spectrometer (FI-GFAAS). The analytical performances of the online system such as linearity, limit of detection, precision, and spiked recoveries were validated. Before the in vivo experiments, the in vivo recovery was conducted. The levels of non-protein-bound Cu in the blood of living rabbits were evaluated before and after administering them with 5 mg/kg body weight of CuSO4 by the online microdialysis-FI-GFAAS system. The results showed that the average basal concentration of non-protein-bound Cu in the blood of living rabbits was 16.2 microg/L (n = 3). Furthermore, the levels of non-protein-bound Cu in the blood of living rabbits were observed after a long delay following intravenous injection of CuSO4. The non-protein-bound Cu reached the maximum value at 125 min after injection. Our present study might provide the in vivo, direct observation that different metals have their own binding characteristics with proteins when transported into the blood of living organisms.

Genetic and cellular characterizations of human TCF4 with microsatellite instability in colon cancer and leukemia cell lines.

It has been reported that the mutational inactivation of the adenomatous polyposis coli (APC) and beta-catenin genes play important roles in colorectal carcinogenesis. However, alteration of the components in the Wnt signaling pathway in colorectal cancer (CRC) with microsatellite instability (MSI) has been elucidated. To define the precise role of the Wnt signaling components in CRC and leukemia cell lines with MSI, mutational analyses of the T cell factor 4 (TCF4) genes were performed. Here we describe for the first time a TCF4 MSI+ phenotype in leukemia cell lines except in colon cancer cell lines. Moreover, we found that these cell lines exhibited deletion and insertion of 1-2A in an (A)9 repeat so as to result in (A)7, (A)8, (A)10 and (A)11 repeat, respectively. To characterize the cellular function of these special TCF4 mutant clones, transient transfection and fluorescent microscopy were analyzed and the results revealed that the TCF4 frameshift gene products all localized in nuclei. Surprisingly, these TCF4 frameshift mutants lost transcriptional activity with beta-catenin and down-regulate the target gene expression. These results delineate a novel role for MSI+TCF4 in leukemia and colon cancer progression.

Continuous multi-element (Cu, Mn, Ni, Se) monitoring in saline and cell suspension using on-line microdialysis coupled with simultaneous electrothermal atomic absorption spectrometry.

We have developed a microdialysis sampling technique coupled on-line with simultaneous electrothermal atomic absorption spectrometry (SIMAAS) for the continuous monitoring of copper (Cu), manganese (Mn), nickel (Ni), and selenium (Se) in saline solutions and in cell suspensions. These trace elements are considered to be those associated most significantly with oxidative stress in biological systems. We employed ultrapure saline (0.9% NaCl) as the perfusate and, thus, the dialysate samples contained a high concentration of salt in the matrix. The use of modifiers [Pd coupled with Mg(NO3)2] prevented the target elements from undergoing evaporation at a pyrolysis temperature of 1200 degrees C, a process that effectively eliminated interference from NaCl. The excellent linearity, detection limits, and precision of the SIMAAS technique allowed the Cu, Mn, Ni, and Se concentrations to be determined in saline. For the on-line microdialysis-SIMAAS system, the ultrapure saline was perfused at a flow rate of 1 microL/min. The probe recoveries of Cu, Mn, Ni, and Se in saline were 57.9, 65.0, 65.5, and 67.9%, respectively. A standard saline solution was measured continuously by the on-line system to ensure long-term stability; each measurement fell within a range of two standard deviations. We determined the on-line spiked recoveries of Cu, Mn, Ni, and Se (101.3, 88.8, 91.3, and 98.5%, respectively) by adding a spiking standard into the stirred saline. The spiked recoveries (Cu, 37.5%; Mn, 3.8%; Ni, 71.1%; Se, 33.8%) were also determined through on-line spiking of a standard into the stirred cell suspension; these values demonstrate that Cu, Mn, and Se were depleted in the cell suspension, but Ni was not. The use of this on-line microdialysis-SIMAAS system permitted the in situ, dynamic, and continuous monitoring of Cu, Mn, Ni, and Se in cell suspensions at a temporal resolution of 20 min.

Continuous in vivo monitoring of blood diffusible calcium using on-line microdialysis sampling coupled with flame atomic absorption spectrometry.

A direct, rapid and continuous in vivo monitoring of diffusible calcium in the blood of living rabbits has been developed using microdialysis sampling coupled on-line with flame atomic absorption spectrometry. Microdialysates perfused through implanted microdialysis probes were collected with a sample loop on an injection valve and directly introduced into the flame atomizer by a carrier solution. An ultrapure saline solution (0.9% NaCI, pH 7.2) was used as the perfusion solution at a flow rate of 20 microI min(-1) via the microdialysis probe. A 0.1% La solution in 0.5% HNO3 solution was employed as the carrier solution at a nebulizer uptake flow rate of 2.5 ml min(-1). The interval for each determination was 2.5 min (2 min of sampling time, 20 s of read time and 10 s of washing time). The performance characteristics of the on-line microdialysis-FAAS system were validated as follows: linearity range, 0 - 100 mg l(-1); detection limit (3a, n = 7), 3.66 mg l(-1); precision (RSD, n = 50), 6.2%. For the evaluation of analytical accuracy, the proposed on-line method was compared with the in vivo no net flux method. The use of an on-line microdialysis-FAAS system permitted the in situ, dynamic and continuous in vivo monitoring of diffusible calcium in the blood of the living rabbits after CaCl2 administration with a temporal resolution of 2.5 min.

On-line microdialysis sampling coupled with flame atomic absorption spectrometry for continuous in vivo monitoring of diffusible magnesium in the blood of living animals.

A novel on-line microdialysis sampling coupled with flame atomic absorption spectrometry (FAAS) with an attractive application is reported. Microdialysates perfused through implanted microdialysis probes were directly introduced into the flame atomizer of a FAAS system using 0.2% HNO(3) as carrier solution at a nebulizer uptake flow rate of 6mlmin(-1). The interval for each determination was 90s (60s sampling time, 10s read time and 20s washing time). The analytical characteristics of the on-line microdialysis-FAAS system were validated as follows: linearity range, 0-300mgl(-1); detection limit (3sigma, n=7), 0.53mgl(-1); precision (R.S.D., n=50), 4.1%. By comparing Mg levels in the blood of living rabbits with the results obtained from in vivo no net flux (NNF) method, the accuracy of the proposed on-line method was found to be good. The present method can be successfully applied to the in vivo monitoring of diffusible Mg in the blood of living rabbits after magnesium sulfate (MgSO(4)) administration with a temporal resolution of 1.5min.

Spectrophotometric determination of a thiobarbituric acid-reactive substance in human hair.

A simple and sensitive spectrophotometric method for the determination of a thiobarbituric acid-reactive substance (TBARS) in human hair has been developed. The proposed method is based on the formation of a red-colored product by the reaction of products of lipid peroxidation with thiobarbituric acid in an acidic medium. The absorbance of the resulting red product was measured at 534 nm. The linear dynamic range was between 1.0 and 20 micromol/L. The recoveries were 98.3-105.0%, and the relative standard deviations (RSD) were 0.32-1.24, respectively. TBARS in digested hair sample was stable for 3 days at room temperature. It was found that, using this method, the hair TBARS concentration in smokers (0.116 +/- 0.030 micromol/g, n = 30) was significantly higher than that in non-smokers (0.096 +/- 0.015 micromol/g, n = 30) (p < 0.05).

Determination of thiobarbituric acid adduct of malondialdehyde using on-line microdialysis coupled with high-performance liquid chromatography.

An on-line analytical system for the continuous monitoring of malondialdehyde (MDA) was developed. This method involves the use of microdialysis perfusion, on-line derivatization and on-line HPLC analysis. This method gave a linear response for MDA concentrations and HPLC peak areas in the range from 0.051 microM to 2.43 microM. The intra-day (RSD = 1.6-10.5%) and inter-day (RSD = 1.1-9.3%) precisions were acceptable. The average in vitro probe recovery of MDA standard was 18.4 +/- 1.0%. The detection limit was 0.03 microM, corresponding to 0.6 pmol for an injection volume of 20 microl. This method was used for in vitro peroxidation investigations, which provided evidence for elevated MDA levels following the incubation of metal ions to a linoleic acid solution.

Automated, continuous, and dynamic speciation of urinary arsenic in the bladder of living organisms using microdialysis sampling coupled on-line with high performance liquid chromatography and hydride generation atomic absorption spectrometry.

An on-line and fully automated method was developed for the continuous and dynamic in vivo monitoring of four arsenic species [arsenite (AsIII), arsenate (AsV), monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA)] in urine of living organisms. In this method a microdialysis sampling technique was employed to couple on-line with high performance liquid chromatography (HPLC) and hydride generation atomic absorption spectrometry (HGAAS). Dialysates perfused through implanted microdialysis probes were collected with a sample loop of an on-line injector for direct and automated injection into HPLC system hyphenated with HGAAS. The saline (0.9% NaCl) solution was perfused at the rate of 1 microl min(-1) through the microdialysis probe and the dialysate was loaded into 50 microl of sample loop. The separation conditions were optimally selected to be in phosphate buffer solution at a pH 5.2 with a flow rate of 1.2 ml min(-1). The effluent from the HPLC was first mixed on-line at the exit of the column with HCl (1 M) solution and then mixed with a NaBH4 (0.2% m/v) solution. Based on the optimal conditions obtained, linear ranges of 2.5-50 ng ml(-1) for AsIII and 6.75-100 ng ml(-1) for the other three arsenic species were obtained. Detection limits of 1.00, 2.18, 1.03 and 2.17 ng ml(-1) were obtained for AsIII, DMA, MMA and AsV, respectively. Typical precision values of 3.4% (AsIII), 5.4% (DMA), 3.6% (MMA) and 7.5% (AsV) were obtained, respectively, at a 25 ng ml(-1) level. Recoveries close to 100%, relative to an aqueous standard, were observed for each species. The average in vivo recoveries of AsIII, DMA, MMA and AsV in rat bladder urine were 56+/-5%, 60+/-9%, 49+/-3% and 55+/-7%, respectively. The use of an on-line microdialysis-HPLC-HGAAS system permitted the determination of four urinary arsenic species in the bladder of an anesthetized rat with a temporal resolution of 50 min sampling.