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.

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.

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.

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.

Arsine toxicity is induced by inhalation but not by percutaneous exposure in hairless mice.

Arsine (AsH3) is used in many industries, but there is insufficient knowledge about the potential for percutaneous absorption. In order to examine possible percutaneous absorption of arsine, we conducted inhalation studies. Arsine was generated by reducing arsenic trioxide with NaBH4. Male 5-week-old Hos:HR-1 hairless mice were subjected to a single percutaneous exposure or whole-body inhalation exposure of ca. 300 ppm arsine for 5 min. The examination was performed 0-6 hr after the exposure. Total arsenic in whole blood and hematocrit (Ht) values were measured. Generation of an arsenic-hemoglobin (As-Hb) adduct in the blood was detected using high-performance liquid chromatography with an inductively coupled plasma mass spectrometer (HPLC-ICP-MS). Ht values in the inhalation group significantly decreased after 3 hr, but those in the percutaneous exposure group did not. Total arsenic in the inhalation group was 9.0-14.2 mg/l, which was significantly higher than that in the percutaneous group. The As-Hb adduct was detected only in mice in the inhalation group. Histopathological changes were noted only in the inhalation group, with marked deposition of eosinophilic globules in the proximal convoluted tubules of the kidneys, the Kupffer cells of the liver, and the red pulp in the spleen, but not in the lungs. Immunohistochemically, these eosinophilic globules were stained positively by hemoglobin (Hb) antibody. In the present study, arsine-induced hemolysis and deposition of Hb occurred in the kidney via the inhalation route but not via percutaneous exposure. The presence of As-Hb adduct may be a useful indicator for confirming arsine poisoning.

Acute arsine poisoning confirmed by speciation analysis of arsenic compounds in the plasma and urine by HPLC-ICP-MS.

OBJECTIVES: Arsine, a potent hemolytic agent, is widely used in the semiconductor industry. We report a case of arsine poisoning confirmed by arsenic speciation analysis in serum and urine that occurred in a recycling factory. CASE: A male worker in his twenties noticed hematuria 3 h after finishing work and was admitted into our hospital 34 h later. Speciation analysis of arsenics in serum and urine samples was performed using HPLC-ICP-MS. On admission, anemia, hematuria, and renal and liver dysfunction were observed. His clinical condition had improved remarkably after 5-days of transfusion and 4 units of RBC transfusion. The total arsenic content in the serum was 244.8 µg/l at admission and 97.1 µg/l at discharge. In the speciation analysis, four kinds of As compounds derived from arsine metabolism were detected in serum and urine. The concentrations of arsenite (AsIII), arsenate (AsV), monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) in serum at admission were 45.8, 5.2, 17.9 and 9.3 µg/l, respectively. The concentrations of AsIII, AsV and MMA decreased with biological half-lives (BHL) of 30.1, 43.0, and 96.3 h, respectively, while that of DMA was increased towards discharge. The urinary AsIII, AsV, MMA, and DMA concentrations at discharge were 384.5, 20.8, 547.4 and 1816.3 µg/g creatinine, respectively. These concentrations in urine subsequently decreased and their BHL was 15 days. CONCLUSION: The results of the present study suggest that arsine is quickly metabolized to AsIII and then metabolized via MMA to DMA in humans.