Arsenite malignantly transforms human prostate epithelial cells in vitro by gene amplification of mutated KRAS.

Inorganic arsenic is an environmental human carcinogen of several organs including the urinary tract. RWPE-1 cells are immortalized, non-tumorigenic, human prostate epithelia that become malignantly transformed into the CAsE-PE line after continuous in vitro exposure to 5µM arsenite over a period of months. For insight into in vitro arsenite transformation, we performed RNA-seq for differential gene expression and targeted sequencing of KRAS. We report >7,000 differentially expressed transcripts in CAsE-PE cells compared to RWPE-1 cells at >2-fold change, q<0.05 by RNA-seq. Notably, KRAS expression was highly elevated in CAsE-PE cells, with pathway analysis supporting increased cell proliferation, cell motility, survival and cancer pathways. Targeted DNA sequencing of KRAS revealed a mutant specific allelic imbalance, 'MASI', frequently found in primary clinical tumors. We found high expression of a mutated KRAS transcript carrying oncogenic mutations at codons 12 and 59 and many silent mutations, accompanied by lower expression of a wild-type allele. Parallel cultures of RWPE-1 cells retained a wild-type KRAS genotype. Copy number analysis and sequencing showed amplification of the mutant KRAS allele. KRAS is expressed as two splice variants, KRAS4a and KRAS4b, where variant 4b is more prevalent in normal cells compared to greater levels of variant 4a seen in tumor cells. 454 Roche sequencing measured KRAS variants in each cell type. We found KRAS4a as the predominant transcript variant in CAsE-PE cells compared to KRAS4b, the variant expressed primarily in RWPE-1 cells and in normal prostate, early passage, primary epithelial cells. Overall, gene expression data were consistent with KRAS-driven proliferation pathways found in spontaneous tumors and malignantly transformed cell lines. Arsenite is recognized as an important environmental carcinogen, but it is not a direct mutagen. Further investigations into this in vitro transformation model will focus on genomic events that cause arsenite-mediated mutation and overexpression of KRAS in CAsE-PE cells.

Effects of Chronic Exposure to Sodium Arsenite on Expressions of VEGF and VEGFR2 Proteins in the Epididymis of Rats.

OBJECTIVE: To study the expressions of VEGF and VEGFR2 at protein level in the epididymis of rats with arsenism. METHODS: Forty male Sprague-Dawley rats were randomly divided into four groups: the high dose arsenic infected group (60.0 mg/L in water), the middle dose arsenic infected group (12.0 mg/L in water), the low dose arsenic infected group (2.4 mg/L in water), and the control group (distilled water). Rats were treated with arsenic through drinking water for 6 consecutive months. At the end of the experiment, the average densitometry values of apoptotic cells in epididymis tubules were determined by TUNEL method; the protein and mRNA levels of VEGF and VEGFR2 were observed by immunohistochemistry, Western blot, and real time fluorescent quantitative PCR, respectively. RESULTS: Compared with the control group, in each infected group, the average densitometry values of apoptotic cells in the epididymis tubules were significantly lower. Compared with control group, protein and mRNA levels of VEGF and VEGFR2 in each infected group were obviously declined. The correlations between protein and mRNA levels of VEGF and VEGFR2 were positively exhibited (r = 0.843, 0.869, p < 0.05). CONCLUSIONS: Arsenism affects the expressions of VEGF and VEGFR2 in the epididymis of rats and results in apoptosis of pathophysiology of male infertility.

Fowler's Solution and the Evolution of the Use of Arsenic in Modern Medicine.

While arsenic has been used medicinally and as a poison for thousands of years, Fowler's solution, an arsenic compound, has had a fascinating history in medicine during the past 200 years. The use of Fowler's solution was first described and published as a treatment for malaria and syphilis in the late 1700s. Many clinical applications for Fowler's solutions have been studied and utilized over the years, but toxicities have limited its utility. Even so, arsenic trioxide, chemically related to Fowler's solution, was approved by the US Food and Drug Administration for treating acute promyelocytic leukemia. The history of Fowler's solution, its applications and uses, and benefits and risks are discussed.

The metabolomic profiling of serum in rats exposed to arsenic using UPLC/Q-TOF MS.

Chronic arsenicosis induced by excessive arsenic intake can cause damages to multi-organ systems, skin cancer and various internal cancers. However, the key metabolic changes and biomarkers which can reflect these changes remain unclear resulting in a lack of effective prevention and treatments. The aim of this study is to determine the impact of chronic arsenic exposure on the metabolism of organism, and find the metabolites changes by using metabolomic techniques. Thirty male Wistar rats were randomly divided into three groups. The arsenite was administered in water, and the doses were 0, 10, and 50mg/L, respectively. The exposure lasted for 6 months. The endogenous metabolite profile of serum was investigated by ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry. Partial least squares discriminant analysis (PLS-DA) enabled clusters to be visualized. Nine serum principal metabolites contributing to the clusters were identified, which were CPA (18:2(9Z,12Z)/0:0), LysoPC (14:0), LysoPC (18:4 (6Z,9Z,12Z,15Z)), LysoPC (P-18:0), l-palmitoylcarnitine, LysoPC (20:2(11Z,14Z)) in positive ESI mode and deoxygcholylglycine, LysoPE (0:0/20:2(11Z,14Z)), 15(S)-hydroxyeicosatrienoic acid in negative ESI. These changes of metabolites in rats suggested the changed metabolism in rats exposed to arsenic. These findings may further aid diagnose and serve as targets for therapeutic intervention of arsenicosis.

Angiogenesis, mediated by miR-21, is involved arsenite-induced carcinogenesis.

Evidence for arsenite-induced lung cancer in humans is strong, but the molecular mechanisms by which arsenite causes cancer remain to be established. Angiogenesis is a fundamental characteristic of cancer and is necessary in its multi-step progression. In this investigation, the mechanism for arsenite-induced angiogenesis was evaluated. Tumors formed from human bronchial epithelial (HBE) cells transformed by arsenite developed new blood vessels, which were prevented by the knockdown of miR-21, and cultures of human umbilical vein endothelial cells (HUVEC) exposed to arsenite developed endothelial tubes, a characteristic of angiogenesis. Also found in transformed HBE cells were up-regulation of a microRNA, miR-21, and increased levels of vascular endothelial growth factor (VEGF), which promotes angiogenesis. Down-regulation of miR-21 in these cells inhibited the arsenite-induced increases of VEGF levels. Thus, we conclude that arsenite induces tumor angiogenesis through processes mediated by miR-21.

Expression of WWOX and FHIT is downregulated by exposure to arsenite in human uroepithelial cells.

Ecological studies in Taiwan, Chile, Argentina, Bangladesh, and Mexico have confirmed significant dose-dependent associations between ingestion of arsenic-contaminated drinking water and the risk of various human malignancies. The FHIT and WWOX genes are active in common fragile sites FRA3B and FRA16D, respectively. Reduced expression of FHIT or WWOX is known to be an early indicator of carcinogen-induced cancers. However, the effect of arsenite on the expressions and molecular mechanisms of these markers is still unclear. The aims of this study were (i) to observe the expression of ATR, WWOX and FHIT proteins in urothelial carcinoma (UC) between endemic and non-endemic areas of blackfoot disease (BFD) by immunohistochemical analyses; (ii) to compare expression of these genes between arsenite-treated SV-HUC-1 human epithelial cells and rat uroepithelial cells; and (iii) to determine the role of DNMT and MEK inhibitors on expressions of WWOX and FHIT in response to arsenite in SV-HUC-1. The experiments revealed that expressions of ATR, WWOX and FHIT in UC significantly differed between BFD areas and non-BFD areas (p=0.003, 0.009 and 0.021, respectively). In fact, the results for the arsenite-treated groups showed that ATR, WWOX and FHIT are downregulated by arsenite in SV-HUC-1. However, the inhibitors suppressed the effects of arsenite on WWOX and FHIT proteins and mRNA expression. In conclusion, arsenite decreased expressions of ATR, WWOX and FHIT via ERK1/2 activation in SV-HUC-1 cells. These findings confirm that dysregulations of these markers may contribute to arsenite-induced carcinogenesis.

Protective role of the methanolic extract of Icacina trichantha on sodium arsenite induced genotoxicity and hepatotoxicity.

BACKGROUND: Arsenic is a toxic metalloid, whose toxicity has raised a lot of concern. Humans are exposed to this metalloid through contaminated water, air and even foods. As a sulfhydryl reactive metal, arsenic has been found to cause extensive damage to organs such as the liver. It has also been found to be a potent clastogen, causing DNA damage leading to both benign and malignant tumors. OBJECTIVES: The anti-hepatotoxic and anti-genotoxic effects of methanolic leaf extract of Icacina trichantha on sodium arsenite induced toxicity in rats were determined. METHODS: Oral gavage of sodium arsenite was used to induced genotoxicity in rats and micronucleus assay was used to measure the number of micronucleated polychromatophilic erythrocytes. The determination of activities of serum alanine amino transferase (ALT), aspartate amino transferase (AST) and gamma glutamyl transferase (GGT) activities were used for the hepatotoxicity assay.. RESULTS: The mean number of micronucleated polychromatophilic erythrocytes (MPCE) per 1000 cells +/- SEM from the bone marrow smear was 57.50 +/- 9.71 in rats treated with both arsenite and extract compared to 129.00 +/- 16.34 in rats treated with arsenite alone. The serum ALT, AST and GGT activities +/- SEM were 67.04 + 3.71, 39.12 +/- 3.45 and 11.54 +/- 0.42 lu respectively for the rats treated with arsenite alone. Combined treatment of arsenite and the extract significantly decrease (p<0.05) in the activity of the enzymes, 29.75 +/- 3.43, 15.8 +/- 4.42, 6.87 +/- 0.433 lu for serum ALT, AST and GGT respectively. CONCLUSION: The methanolic leaf extract of I. trichantha modulated both the hepatotoxic and genotoxic effect induced by sodium arsenite in rats, which suggest that Icacina trichantha may serve as a hepatoprotective and anti-tumor agent.

Activation of Hedgehog signaling by the environmental toxicant arsenic may contribute to the etiology of arsenic-induced tumors.

Exposure to the environmental toxicant arsenic, through both contaminated water and food, contributes to significant health problems worldwide. In particular, arsenic exposure is thought to function as a carcinogen for lung, skin, and bladder cancer via mechanisms that remain largely unknown. More recently, the Hedgehog signaling pathway has also been implicated in the progression and maintenance of these same cancers. Based on these similarities, we tested the hypothesis that arsenic may act in part through activating Hedgehog signaling. Here, we show that arsenic is able to activate Hedgehog signaling in several primary and established tissue culture cells as well as in vivo. Arsenic activates Hedgehog signaling by decreasing the stability of the repressor form of GLI3, one of the transcription factors that ultimately regulate Hedgehog activity. We also show, using tumor samples from a cohort of bladder cancer patients, that high levels of arsenic exposure are associated with high levels of Hedgehog activity. Given the important role Hedgehog signaling plays in the maintenance and progression of a variety of tumors, including bladder cancer, these results suggest that arsenic exposure may in part promote cancer through the activation of Hedgehog signaling. Thus, we provide an important insight into the etiology of arsenic-induced human carcinogenesis, which may be relevant to millions of people exposed to high levels of arsenic worldwide.

Protective effects of the dietary supplementation of turmeric (Curcuma longa L.) on sodium arsenite-induced biochemical perturbation in mice.

The present study was undertaken to evaluate the protective effect of turmeric powder on arsenic toxicity through mice model. Swiss albino male mice were divided into four groups. The first group was used as control, while groups 2, 3, and 4 were treated with turmeric powder (T, 50 mg/kg body weight/day), sodium arsenite (Sa, 10 mg/kg body weight/day) and turmeric plus Sa (T+Sa), respectively. Results showed that oral administration of Sa reduced the weight gain of the mice compared to the control group and food supplementation of turmeric prevented the reduction of weight gain. Turmeric abrogated the Sa-induced elevation of serum urea, glucose, triglyceride (TG) level and alanine aminotransferase (ALT) activity except the activity of alkaline phosphatase (ALP). Turmeric also prevented the Sa-induced perturbation of serum butyryl cholinesterase activity (BChE). Therefore, ameliorating effect of turmeric on Sa-treated mice suggested the future application of turmeric to reduce or to prevent arsenic toxicity in human.

Effects of leaves extract of Ocimum sanctum L. on arsenic-induced toxicity in Wistar albino rats.

Therapeutic efficacy of oral administration of Ocimum sanctum (200mg/kg, once daily) post arsenic exposure (100 ppm in drinking water for 4 months) was investigated in rats. Animals exposed to arsenic (III) showed a significant inhibition of delta-aminolevulinic acid dehydratase (ALAD) activity, decrease in reduced glutathione (GSH) level and an increase in reactive oxygen species (ROS) in blood. On the other hand, a significant decrease in hepatic ALAD, and increase in delta-aminolevulinic acid synthetase activity were noted after arsenic exposure. These changes were accompanied by an increase TBARS level in liver and kidney. Activities of liver, kidney and brain superoxide dismutase and catalase also showed a decrease on arsenic exposure. Administration of O. sanctum post arsenic exposure, exhibited significant recovery in blood ALAD activity while, it restored blood GSH and ROS levels. Other blood biochemical variables remained unchanged on O. sanctum supplementation. Interestingly, there was a marginal, but significant depletion of arsenic from blood, liver and kidneys. The results conclude that post arsenic administration of O. sanctum has significant role in protecting animals from arsenic-induced oxidative stress and in the depletion of arsenic concentration. Further studies thus can be recommended for determining the effect of co-administrating of O. sanctum during chelating therapy with a thiol chelator.

Transporters of arsenite in rice and their role in arsenic accumulation in rice grain.

Arsenic poisoning affects millions of people worldwide. Human arsenic intake from rice consumption can be substantial because rice is particularly efficient in assimilating arsenic from paddy soils, although the mechanism has not been elucidated. Here we report that two different types of transporters mediate transport of arsenite, the predominant form of arsenic in paddy soil, from the external medium to the xylem. Transporters belonging to the NIP subfamily of aquaporins in rice are permeable to arsenite but not to arsenate. Mutation in OsNIP2;1 (Lsi1, a silicon influx transporter) significantly decreases arsenite uptake. Furthermore, in the rice mutants defective in the silicon efflux transporter Lsi2, arsenite transport to the xylem and accumulation in shoots and grain decreased greatly. Mutation in Lsi2 had a much greater impact on arsenic accumulation in shoots and grain in field-grown rice than Lsi1. Arsenite transport in rice roots therefore shares the same highly efficient pathway as silicon, which explains why rice is efficient in arsenic accumulation. Our results provide insight into the uptake mechanism of arsenite in rice and strategies for reducing arsenic accumulation in grain for enhanced food safety.

Inhibition of function of T cell subpopulations and decrease in cytokine production during subacute poisoning with various toxicants.

Experiments on outbred albino rats showed that subacute intoxication with organophosphorus compounds dimethyldichlorovinyl phosphate and malathion primarily decreased functional activity of Th1 lymphocytes, immune reactions associated with these cells, and interferon-g production compared to that of Th2 lymphocytes and interleukin-4 synthesis. Acrylic acid nitrile and methanol produced the opposite effect. Sulfur mustard and sodium arsenite were equally potent in reducing the function of Th1 and Th2 lymphocytes and production of cytokines.

Bovine arsenic toxicosis.

A ranch in central South Dakota had a number of dead calves because of arsenic poisoning. The clinical picture included diarrhea, central nervous system signs, and death. Gross necropsy findings included adequate body fat, stomachs full of normal-appearing ingesta, and large amounts of greenish brown watery fluid in the intestine and colon. Microscopically there was severe lymphoid tissue necrosis in the mesenteric lymph nodes and gut-associated lymphoid tissue. Chemical analysis of kidneys showed no significant amounts of lead; however, kidney arsenic concentrations were 25 to 44 ppm. The source was a small pile of Paris Green (common name for cupric acetoarsenite) found in an old dump site in the pasture.

[Molecular genetic mechanism of sodium arsenite on growth and development].

OBJECTIVE: To study the effects of sodium arsenite on gene expression related to growth and development and explored the molecular mechanism of arsenic effects using gene chips. METHODS: Normal human hepatic cells were dripped on chips and then hybrided with the first strand of cDNA from hepatic cell exposed to different concentration of sodium arsenite. Gene sequence of clone differently expressed was determined and then defined which gene it was and finally those genes which associated with growth and development were identified. RESULTS: The p55 gene expression level of two experimental groups was severaly 2.21 and 2.93 times as the control group. The PL gene level of two experimental groups were 0.13 and 0.27 times as the control group, and the HOXA10 gene level was 0.22 and 0.35 times of the control group. These results indicated that sodium arsenite increase p55 gene expression, and inhibited PL and HOXA10 gene expression. CONCLUSIONS: The sodium arsenite could affect the gene expression related to growth and development and it is shown that the molecular genetic mechanism of sodium arsenite is related to growth and development.

Hydrogeological investigation of ground water arsenic contamination in south Calcutta.

Typical clinical symptoms of acute arsenic poisoning have been detected in 1000 residents near a factory in P.N. Mitra Lane, Behala, South Calcutta, located in a thickly populated area manufacturing copper acetoarsenite (Paris-Green) an arsenical pesticide for the past 25 years. Soil around the effluent dumping point of the factory was exceptionally contaminated, with arsenic, copper and chromium concentrations of 20,100-35,500 mg kg-1, 33,900-51,100 mg kg-1 and 5300-5510 mg kg-1. Arsenic and copper concentrations in bore-hole soils collected up to a depth of 24.4 m at the effluent dumping point, decreased with depth. Arsenous acid, arsenic acid, methylarsonic acid (MA) and dimethylarsinic acid (DMA) were detected in bore-hole soils up to a depth of 1.37 m, after which only inorganic arsenical compounds were present. A positive correlation was established between arsenic and copper authenticated the Paris-Green waste disposal site as the source of contamination. Mechanism of ground water contamination from this disposal site had been probed by a systematic hydrogeological survey and the arsenic content of the tube-well waters in the surrounding areas. Hydraulic conductivity was maximum in the central part. The site for disposal of the effluent was a ditch located in the zone of discharge. Sparingly soluble Paris-Green cumulatively deposited in the waste disposal site is decomposed by micro-organisms to water-soluble forms and finally percolated to underground aquifers along with rain water through the discharge zone. The contaminant is currently moving towards WNW with ground water flow and the residents in the direction of encroaching contamination are insecure due to penetration of the contaminant.

Selenium and arsenic in the environment in Finland.

A characteristic feature of glaciated Precambrian environments is their low selenium content, as a chalcophile element, Se, replaces sulfur in many of the sulfide minerals, for example, pyrite, chalcopyrite, pyrrhotite, and pentlandite. The average Se concentration in rocks and related till deposits in Finland is in the range of 0.01 to 0.2 mg/kg. Due to geological conditions, Se concentrations in surface and ground water are low in Finland compared with other countries. In a nationwide study dealing with the hydrogeochemistry of headwater streams, the median Se concentration in streams during August to September 1990 was 30 to 180 microg/L. For comparison, Se concentrations in shallow well waters are generally in the range of 50 to 1000 microg/L. The Se concentrations in stream sediments varied from 0.03 to 3.94 mg/kg. There was a highly significant correlation between the Se concentrations in stream water and in stream sediment. The streams with Se concentrations exceeding the general level in both water and sediment were most common in southern Finland. A speciation study on Finnish stream waters revealed that there were equal proportions of Se complexed with humic substances (36%) and Se as a selenate species (36%), whereas selenite accounted for less than 10% of total Se. About 8% of the Se in stream water occurred in particulate form. In an effort to enhance the Se intake of Finns through diet, Se-supplemented fertilizers have been used nationwide since 1985. While greatly improving Se levels in the population, the measure has raised concerns about undesirable environmental effects. Therefore, the amount of Se added to fertilizers has been reduced since 1991. Differing in behavior from Se, arsenic is considered one of the most toxic metals derived from the natural environment. Alarm has been triggered in Finland by the recent lowering from 50 microg/L to 10 microg/L of the upper level of As permissible in potable water, the recent information of high As concentrations in water from drilled bedrock wells, and the findings of international medical studies suggesting that As is a carcinogen. The most important source of As is arsenopyrite (FeAsS). Hence, high As concentrations most frequently occur in areas of sulfide mineralization, often in connection with occurrences of mafic rocks such as gabbros, amphibolites, and peridotites. The As concentrations in till fines, the most common glaciogenic soil type in Finland, reflect those in bedrock. The concentrations in groundwater are controlled by the chemical composition of the bedrock and the soil and prevailing hydrogeochemical conditions, for example, pH and Eh levels. Arsenic concentrations are lowest in surface water and swiftly flowing shallow ground water discharged by springs and are somewhat higher in shallow wells dug into overburden. By far, the highest As concentrations are to be found in wells drilled into bedrock (maximum 1 to 2 mg/L), although the concentrations vary by several orders of magnitude from well to well. The highest probability of encountering deleteriously arsenious well water is in areas with characteristic As anomalies in the till and bedrock. Hence, it is important to understand local geological conditions, particularly in the case of wells drilled into bedrock. The risk of deleteriously high As concentrations occurring in captured springs and shallow wells is slight.