Inorganic arsenic trioxide induces gap junction loss in association with the downregulation of connexin43 and E-cadherin in rat hepatic "stem-like" cells.

Chronic exposure to inorganic arsenic trioxide causes tumors of the skin, urinary bladder, lung, and liver. Several cancer initiators and promoters have been shown to alter cell-cell signaling by interference with gap junction intercellular communication (GJIC) and/or modulation of cell adhesion molecules, such as connexin43 (Cx43), E-cadherin, and ß-catenin. The aim of this study was to determine whether the disruption of cell-cell interactions occurs in liver epithelial cells after exposure to arsenic trioxide. WB-F344 cells were treated with arsenic trioxide (6.25-50 µM) for up to 8 hours, and gap junction function was analyzed using the scrape-load/dye transfer assay. In addition, the changes in mRNA and protein levels of Cx43, E-cadherin, and ß-catenin were determined. A significant dose- and time-dependent decrease in GJIC was observed when WB-F344 cells were exposed to arsenic trioxide (p < 0.05). Consistent with the inhibition of GJIC, cells' exposure to arsenic trioxide resulted in dose- and time-dependent decreases in Cx43 and E-cadherin mRNA expression and protein levels. However, arsenic trioxide did not alter the mRNA or protein levels of ß-catenin. In an immunofluorescence study, nuclei were heavily stained with anti-ß-catenin antibody, indicating significant nuclear translocation. In this study, we also demonstrated that arsenic trioxide-induced GJIC loss was a reversible process. Taken together, these data support the hypothesis that disruption of cell-cell communication may contribute to the tumor-promoting effect of inorganic arsenic trioxide.

Effects of cultured human adipose-derived stem cells transplantation on rabbit cornea regeneration after alkaline chemical burn.

Ocular chemical burn is a severe injury with poor outcomes. Immediate and appropriate management is highly related to prognosis. We studied the effect of cultured human adipose tissue-derived stem cells on the regeneration of the rabbit cornea after alkaline chemical burn, using used human adipose tissue-derived stem cells as the source material. Immediately after the chemical burn, the experimental eye received a single subconjunctival injection of a stem cell suspension (1.3 × 10(5) cells/0.2 mL), with the other eye serving as control. Rabbits were sacrificed and specimens taken 30 days after injection. The experimental group showed faster wound healing than the control group, and the result for the experimental group was clearer cornea medium. Histologically, there were five to six epithelial cell layers on the corneas of the experimental group as compared to two to three cell layers on the corneas of the control group. Wilcoxon signed rank test showed a significant difference in the epithelial cell layers between the two groups. Surface markers for connexin 43 (Cx43), ß-catenin, E-cadherin, and P63 were analyzed. Cx43 and ß-catenin showed significant change, as determined by the Wilcoxon signed rank test, which indicated good cell renewal during repair of the corneal epithelium damaged by the chemical burn. E-cadherin and P63 showed no significant change during the epithelium healing process. Transplantation of cultured human adipose tissue-derived stem cells as a treatment for a corneal chemical burn promotes cell renewal and assists in damage repair.

Childhood blood lead levels and intellectual development after ban of leaded gasoline in Taiwan: a 9-year prospective study.

BACKGROUND: Lead (Pb) exposure is associated with children's neurodevelopment, even at low doses. Leaded gasoline was banned in Taiwan in 2000 to reduce environmental exposure to Pb. OBJECTIVES: To evaluate the neurodevelopmental effect of low-level Pb exposure in young children. METHODS: In 2001-2002, we have recruited 430 pregnant women in the third-trimester in Taichung, Taiwan who answered detailed questionnaires in the obstetric clinic. A total of 119, 76, and 66 children were followed up at 2-3, 5-6 and 8-9 years, respectively. We collected blood samples from pregnant women, Umbilical cord and children, and evaluated children's neurodevelopment and cognition function at all three time points using Bayley and Wechsler tests. Blood samples were analyzed for whole blood lead (BPb) levels. RESULTS: Geometric mean of BPb in pregnant women, cord blood and children at 2-3, 5-6 and 8-9 years old were 2.21, 1.30, 2.48, 2.49 and 1.97 µg/dl, respectively. Low-level postnatal Ln BPb was significantly associated with not only decreasing intelligence quotient (IQ), but also delayed cognitive function in children at 5-8 years (ß: -5.97, SE: 2.59, p=0.025), after adjustment for maternal education, maternal BPb exposure, Home Observation for Measurement of the Environment Inventory (HOME), and gender of child, using linear mixed models. No significant relation was observed between prenatal and cord blood Pb levels and children's cognitive function in children 2-8 years. CONCLUSIONS: Low-level postnatal BPb levels in children at 2-5 years may have lagged effects on neurodevelopment in those at 5 to 8 years. Action is warranted to reduce even very low environmental Pb levels to reduce the developmental burden of Pb on children.

Inhibition of gap junctional Intercellular communication in WB-F344 rat liver epithelial cells by triphenyltin chloride through MAPK and PI3-kinase pathways.

BACKGROUND: Organotin compounds (OTCs) have been widely used as stabilizers in the production of plastic, agricultural pesticides, antifoulant plaints and wood preservation. The toxicity of triphenyltin (TPT) compounds was known for their embryotoxic, neurotoxic, genotoxic and immunotoxic effects in mammals. The carcinogenicity of TPT was not well understood and few studies had discussed the effects of OTCs on gap junctional intercellular communication (GJIC) of cells. METHOD: In the present study, the effects of triphenyltin chloride (TPTC) on GJIC in WB-F344 rat liver epithelial cells were evaluated, using the scrape-loading dye transfer technique. RESULTS: TPTC inhibited GJIC after a 30-min exposure in a concentration- and time-dependent manner. Pre-incubation of cells with the protein kinase C (PKC) inhibitor did not modify the response, but the specific MEK 1 inhibitor PD98059 and PI3K inhibitor LY294002 decreased substantially the inhibition of GJIC by TPTC. After WB-F344 cells were exposed to TPTC, phosphorylation of Cx43 increased as seen in Western blot analysis. CONCLUSIONS: These results show that TPTC inhibits GJIC in WB-F344 rat liver epithelial cells by altering the Cx43 protein expression through both MAPK and PI3-kinase pathways.

Low-level laser irradiation promotes cell proliferation and mRNA expression of type I collagen and decorin in porcine Achilles tendon fibroblasts in vitro.

Achilles tendon problems are commonly encountered in sports medicine and low-level laser therapy (LLLT) is widely used in rehabilitative applications to decrease pain, reduce inflammatory processes, and promote tissue healing. This study examined the effects on the proliferation of porcine Achilles tendon fibroblasts and gene expression, using different doses of low-level laser irradiation (LLLI). Four groups of identically cultured fibroblasts were exposed to LLLI and harvested after 24 h. The control group (Group 1) was subjected to no LLLI. Other groups received 1 J/cm2 (Group 2), 2 J/cm2 (Group 3), and 3 J/cm2 (Group 4), respectively. Cell proliferation and mRNA expressions of type I collagen and decorin were then measured. When compared to the control group, the cell proliferation of irradiated Achilles tendon fibroblasts in the other three groups increased significantly by 13% +/- 0.8% (Group 2), 30% +/- 0.4% (Group 3), and 12% +/- 0.6% (Group 4) respectively. But progressively higher laser intensity did not achieve a correspondingly higher cell proliferation effect in Achilles tendon fibroblasts. The mRNA expressions of decorin and type I collagen in fibroblasts with LLLI were significantly higher (p < 0.05). Therefore, suitable dosages of LLLI may result in more effective tissue healing by promoting type I collagen and decorin synthesis. However, these positive effects of LLLI on the repair of the Achilles tendon in humans should be further investigated in clinic.

Common toxidromes of plant poisonings in Taiwan.

OBJECTIVE: To describe the toxidromes associated with plant poisonings in Taiwan. METHODS: Retrospective review of acute single-plant exposures with clinical signs and symptoms reported between January 1987 and December 2006 by hospitals to the network of Taiwan Poison Control Centers. Recorded data included demographic data, intent of exposures, exposure routes, clinical findings, and therapeutic strategies. RESULTS: There were 389 cases that met the criteria. Each case was placed into one of the expected toxidromes: anticholinergic, mucosal inflammation, gastroenteritis, acute multisystem organ failure, delayed multisystem organ failure, cholinergic, cardiac dysrhythmia, hepatotoxicity, dermatitis, seizures, and dyspnea. Anticholinergic poisoning was the most common toxidrome. CONCLUSION: Plant poisonings can be classified into recognizable toxicologic syndromes. These toxidromes may guide a clinician's evaluation and management before a botanist can confirm the actual plant identity.

Epidemiology of organophosphate pesticide poisoning in Taiwan.

INTRODUCTION: The nationwide epidemiology of organophosphate pesticide (OP) poisoning has never been reported in detail for Taiwan. METHODS: This study retrospectively reviewed all human OP exposures reported to Taiwan's Poison Control Centers (PCCs) from July 1985 through December 2006. RESULTS: There were 4799 OP exposures. Most OP exposures were acute (98.37%) ingestions (74.50%) of a single OP (80.37%) to attempt suicide (64.72%) in adults (93.25%). Males were the most common gender (64.95%). Most patients (61.97%) received atropine and/or pralidoxime. The mortality rate for all 4799 OP exposures was 12.71%. Exposures to single OPs without co-intoxicants caused 524 deaths; of these, 63.36% were due to dimethyl OPs. CONCLUSION: Dimethyl OPs cause the majority of deaths in Taiwan.

Family, peer and individual factors related to methylenedioxymethamphetamine use in Taiwanese adolescents.

Examination of the correlates of methylenedioxymethamphetamine (MDMA) use is crucial for the development and implementation of effective prevention programs for adolescents. The aim of the present study was therefore to identify the family, peer and individual factors that were related to MDMA use in Taiwanese adolescents. Two hundred adolescents who used MDMA and 200 who did not use MDMA were recruited into the study. The family, peer and individual factors related to MDMA use were examined using chi(2) automatic interaction detection (CHAID) analysis. The results indicated that the adolescents who had more friends involved with substance use, disruptive family and attitude favoring MDMA use were more likely to use MDMA. Multiple factors of family, peer and individuals were related to MDMA use among Taiwanese adolescents. This knowledge may be helpful when designing and implementing preventive intervention programs.

Isolation and characterization of human gastric cell lines with stem cell phenotypes.

AIM: The aim of this study was to develop an in vitro human gastric stem and/or progenitor cell model that may be used to study the mechanism of gastric carcinogenesis induced by Helicobacter pylori infection. METHODS: Human gastric biopsy was minced and digested with collagenase and dispase and cultured in a low-calcium medium (serum-free keratinocyte medium; keratinocyte-SFM) supplemented with N-acetyl-L-cysteine and L-ascorbic acid 2-phosphate. Actively proliferating epithelial colonies with sustained growth were isolated and characterized for karyotype and phenotypes related to stem cell characteristics including proliferation and differentiation potential, ability of anchorage-independent growth (AIG), gap junctional intercellular communication (GJIC) and the expression of Oct-4, a transcription factor previously shown to be expressed in embryonic stem cells, adult stem cells and undifferentiated tumor cells. To study the carcinogenic effect of H. pylori infection, gastric stem and/or progenitor cells were incubated with H. pylori culture products and/or N-methyl-N-nitro-N-nitrosoguanidine (MNNG), a chemical carcinogen, to see the telomerase activation. RESULTS: Multiple cell lines with stem cell features were isolated by this new cell culture method. The results based on detailed characterization of one cell clone, KMU-GI2, revealed stem cell features of these cells. The initial clone contained mostly undifferentiated epithelial-like cells, which, upon subculture and propagation, gave rise to a heterogeneous cell population. Single cell-derived subclones, similar to the parental population, retained high differentiation potential and were capable of giving rise to many morphologically different cell types (i.e. epithelial-like, glial or neuron-like, round and various peculiar-shaped cells). Although these cells were normal in karyotype and competent in GJIC, they had the ability to grow in soft agar. Cells expressing epithelial membrane antigen (EMA), mucin 5AC, glial fibrillary acidic protein (GFAP), cytokeratin-18 (CK-18), trefoil factor 1 (TFF-1) and Oct-4 were found in the cell culture, but not E-cadherin-, gastrin- or telomerase-expressing cells. Furthermore, spontaneously immortalized non-tumorigenic clones could be derived from the cell population. After treating these cell cultures with the chemical carcinogen, MNNG and H. pylori culture products for 5 days, telomerase activity and telomerase mRNA expression were significantly elevated, while treatment with either of them showed no effect. CONCLUSION: The new cell culture method can be used to develop gastric epithelial cell clones with sustained growth from endoscopic biopsy. The gastric cell clone showed several stem and/or progenitor cell phenotypes (i.e. the ability of AIG, high differentiation capacity, high susceptibility to spontaneous immortalization and the expression of Oct-4). The telomerase expression in these gastric stem and/or progenitor cells can be upregulated by exposure to H. pylori culture products and MNNG, an important step in neoplastic transformation. These results show that putative human gastric stem and/or progenitor cell clones can be developed by our method and these cells could be useful for studying the mechanisms of human gastric carcinogenesis including the mechanism of action of H. pylori, as well as the regulation of the proliferation and differentiation of human gastric mucosa.

Performance characteristics of ELISAs for monitoring ketamine exposure.

BACKGROUND: Growing misuse of ketamine necessitates the development of high throughput testing approaches. Two commercial enzyme-linked immunosorbent assays (ELISA) for ketamine have recently become available and were adapted for this development. METHOD: The newly available ketamine ELISA reagents were examined to better understand their cross-reacting, calibration and other performance characteristics. ELISA apparent analyte concentrations were also correlated against ketamine concentrations as determined by GC-MS to examine the relationship between these 2 parameters. RESULT: Both adapting ketamine as the targeted analyte, reagent from International Diagnostic Systems (IDS) also responded very significantly to the metabolites of ketamine (norketamine and dehydronorketamine), while the NEOGEN reagent responded very specifically to ketamine. CONCLUSION: NEOGEN ELISA test data exhibit better correlation with the ketamine concentration as determined by GC-MS. It can be more reliably used as the preliminary test method in the 2-step approach now routinely adapted in workplace drug testing programs. Using 100 ng/ml ketamine as the GC-MS cutoff, the corresponding ELISA cutoff value is approximately 110-120 ng/ml. With significantly higher responses to ketamine metabolites, IDS reagent can detect specimens with much lower ketamine/metabolites concentrations and can better meet other testing requirements.

Accelerated growth and prolonged lifespan of adipose tissue-derived human mesenchymal stem cells in a medium using reduced calcium and antioxidants.

Human mesenchymal stem cells (MSCs) have been isolated from bone marrow and other adult tissues and are potentially useful for tissue engineering. Adipose tissue has several clear advantages as a starting material for harvesting stem cells, as it is abundant and relatively easy to procure. However, existing methods to expand adipose-derived MSCs are less than optimal. Here we describe a new cell culture method that accelerates greatly the growth rate and prolongs the lifespan of adipose MSCs. This was accomplished by using a growth medium with low calcium and supplemented with N-acetyl-L-cysteine and L-ascorbic acid-2-phosphate. Cells produced early in these cultures displayed characteristics similar to those previously reported for multipotential stem cells, including a high frequency of anchorage- independent growth in soft agar, lack of gap junctional intercellular communication in a cell type with serpiginous morphology, and the expression of Oct-4. Furthermore, these cells could readily be induced to differentiate into adipocytes, osteoblasts, and chondrocytes. Thus, modification of growth medium by reduction of calcium and addition of antioxidants greatly enhanced the growth rate and extended the lifespan of adipose-derived multipotential human MSCs.

Determination of urinary trace elements (arsenic, copper, cadmium, manganese, lead, zinc, selenium) in patients with Blackfoot disease.

To determine the relationship of arsenic, copper, cadmium, manganese, lead, zinc and selenium to Blackfoot disease (BFD, a peripheral vascular disorder endemic to areas of Taiwan, which has been linked to arsenic in drinking water) the authors measured the amount of these substances in urine from BFD patients, using atomic absorption spectrometry. Results indicate significantly higher amounts of urinary arsenic, copper, cadmium, manganese, and lead for BFD patients than for normal controls, also significantly lower urinary zinc and selenium.

Acute poisonings with Breynia officinalis--an outbreak of hepatotoxicity.

BACKGROUND: In combination with other traditional Chinese medicines, Breynia officinalis, a species of Euphorbiaceae, has long been used to treat contusions, heart failure, venereal diseases, growth retardation, and conjunctivitis. B. officinalis, regarded as a poison, was mistaken for a similar plant, Securinega suffruticosa, and cooked in a soup used for the treatment of muscle soreness, lumbago, and as a tonic in this outbreak. CASE SERIES: Nineteen patients, 11 males and 8 females (average age 49.2 +/- 9.1 years) consumed an average of 130 mL (30-900 mL) of soup containing B. officinalis stems. Fourteen patients developed diarrhea, 10 experienced nausea and chilly sensations, 9 had sensations of abdominal fullness, and 7 suffered from vomiting. The results of liver function tests (LFTs) indicated that the observed maximum median level of alanine aminotransferase (ALT) was 647U/L (range 89-9440 U/L), aspartate aminotransferase (AST) 314 U/L (range 47-7756 U/L), alkaline phosphatase 251 U/L (range 224-278 U/L), and gamma glutamyl transpeptidase 106 U/L (range 84-313 U/L). The median time to the observed median peak levels was 3 days for ALT, 2 days for AST, 5 days for alkaline phosphatase, and 12 days for gamma glutamyl transpeptidase. With supportive treatment, the majority of abnormalities in 14 of the cases resolved within 6 months of exposure. CONCLUSIONS: The consumption of a soup containing B. officinalis Hemsley resulted in dose-related toxic effects. Clinical toxicity consisted primarily of gastrointestinal symptoms and signs and hepatotoxicity. Hepatocellular liver injury rather than cholestatic liver injury was observed. Marked jaundice did not develop.

Simultaneous determination of urinary cadmium, cobalt, lead, and nickel concentrations in steel production workers by differential pulse stripping voltammetry.

Screening for metals in urine is important because toxic levels of these elements are linked to disease. In the current study, the authors used differential pulse stripping voltammetry on a hanging mercury drop electrode for the simultaneous determination of cadmium, cobalt, lead, and nickel in the urine of 63 production and 63 quality control workers in a steel production plant, along with 63 matched normal controls. Urinary sampling is a noninvasive procedure, and in this study participants had good compliance. Metal levels for both the production and quality control workers were significantly higher than for controls. The results reveal the need for immediate improvements in workplace ventilation and industrial hygiene practices for this cohort.

Eight cases of acute renal failure from Vipera russelli formosensis venom after administration of antivenom.

The six-valent antivenom from Vipera russelli formosensis in Taiwan has recently become available in Taiwan. Between June 1998 and December 2001 8 Vipera russelli formosensis snakebite victims were treated with antivenom within 24 h of snakebite. Bleeding stopped and coagulopathy improved after antivenom use, and neurotoxic symptoms were mild. Prothrombin time was prolonged on the 1st day and thrombocytopenia changed within 2 d. In the majority of cases, renal dysfunction persisted for several days after coagulation abnormalities normalized: 3 cases required hemodialysis. Although Vipera russelli formosensis antivenom is capable of preventing hemorrhage, antivenom administration may not alter the course of severe renal damage.

Dysfunction of the mitochondrial respiratory chain in the rostral ventrolateral medulla during experimental endotoxemia in the rat.

We investigated the functional changes in the mitochondrial respiratory chain at the rostral ventrolateral medulla (RVLM), the medullary origin of sympathetic vasomotor tone, in an experimental model of endotoxemia that mimics systemic inflammatory response syndrome. In Sprague-Dawley rats maintained under propofol anesthesia, intravenous administration of Escherichia coli lipopolysaccharide (LPS; 30 mg/kg) induced a reduction (Phase I), followed by an augmentation (Phase II) and a secondary decrease (Phase III) in the power density of vasomotor components (0-0.8 Hz) in systemic arterial pressure signals. LPS also elicited progressive hypotension, and death ensued within 4 h. Enzyme assay revealed significant depression of the activity of nicotinamide adenine dinucleotide cytochrome c reductase (Complexes I + III) and cytochrome c oxidase (Complex IV) in the RVLM during all three phases of endotoxemia. On the other hand, the activity of succinate cytochrome c reductase (Complexes II + III) remained unaltered. We conclude that selective dysfunction of respiratory enzyme Complexes I and IV in the mitochondrial respiratory chain at the RVLM, whose neuronal activity is intimately related to the death process, is closely associated with fatal endotoxemia in the rat.

Relationship between blood lead concentrations and learning achievement among primary school children in Taiwan.

Over the past 20 years lead has been proven to exert an influence on the intelligence of children. Especially for children exposed to environmental lead, average blood lead was often lower than the officially recognized intoxication level. Because Kaohsiung is an industrial area in Taiwan and lead exposure is an important environmental issue, we attempted to ascertain the extent to which environmental lead influences the achievement of primary school children. We randomly selected 934 children from 32 primary schools in 11 districts of Kaohsiung City. Blood lead levels of the children were checked, and they were administered a questionnaire about their family information. Scores of several courses were used in this study on the relationship between a child's blood lead and his or her academic performance (Ranking with his or her classmates), including Chinese (reading and writing short Chinese articles), Mathematics, History and Society, and Natural Science. Multiple regression models were done with adjustments for the confounding effects of their parents' socioeconomic levels. The mean (SD) of 934 blood lead level was 5.50 (1.86) microg/dL. Spearman's coefficient showed that class rankings in Chinese, Mathematics, Natural Science, and History and Society were all strongly associated with blood lead levels (P<0.01). The multiple regression models revealed that blood lead level exerts a stronger influence on children's language ability (Chinese) than on their ability to calculate (Mathematics). Our results suggest that environmental lead exposure adversely affects a child's academic achievement, making a direct link between exposure to lead and academic attainment.

Determination of urinary lead, cadmium and nickel in steel production workers.

The determination of toxic metals in urine is an important clinical screening procedure. In this study, the aim of our investigation was to determine the concentrations of Pb, Cd, and Ni in urine samples under routine clinical laboratory conditions. To assess the reliability of these methods, critical factors such as detection limit(s), calibration range(s), cost, accuracy and precision were studied. The method was employed for the quantitative determination of lead, cadmium and nickel in urine samples from steel production and quality control (QC) workers and healthy unexposed controls. After pre-treatment with acids, the samples were digested via a microwave oven and the samples were determined by a graphite furnace atomic absorption spectrometry (GFAAS). Quality control for these procedures was established with the concurrent analysis of Standard Trace Metals 7879 Level II and NIST SRM 2670 (Toxic Elements in Freeze Dried Urine). The results indicate that urinary lead, cadmium and nickel levels of the exposed workers are significantly higher those of the controls. The possible connection of these elements with the etiology of disease is discussed. The results also show the need for immediate improvements in workplace ventilation and industrial hygiene practices.