The role of oxidative stress in hormesis induced by sodium arsenite in human embryo lung fibroblast (HELF) cellular proliferation model

Hormetic dose-response relationships induced by environmental agents are often characterized by a low-dose stimulation and a high-dose inhibition. The mechanisms underlying hormesis induced by environmental agents still remain an enigma; however, hormetic consequences may have significant implications for health risk assessments. To investigate the role of oxidative stress in hormetic phenomena associated with cell proliferation induced by sodium arsenite, the levels of reactive oxygen species (ROS), lipid peroxidation (LPO), and heat-shock proteins (HSP) and the activities of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) were measured in human embryo lung fibroblast (HELF) cells after treatment with sodium arsenite at various concentrations for differing times. Results showed that sodium arsenite induced significant cell proliferation at low concentrations (0.5 microM for 12, 24, and 48 h), but inhibited cell growth at high amounts (5 and 10 microM for 24 and 48 h), reflected as a beta concentration-response curve. Data indicated that the relationship between ROS levels and sodium arsenite exposure concentration displayed a positive correlation. It was found out that sodium arsenite at high concentrations induced LPO damage. The activities of SOD were enhanced at low metal concentrations but inhibited with high amounts in a concentration-dependent manner. Similarly, heat-shock protein 27 (HSP27) levels were increased by sodium arsenite of low concentrations with early exposure time (3, 6, and 12 h), but decreased with high metal concentrations with greater exposure time (24 and 48 h).Sodium arsenite decreased HSP70 expression at lower concentrations, but increased HSP70 expression at higher concentration. The results indicated that this cellular hormetic model of cell proliferation induced by sodium arsenite occurred in HELF cells, which may explain contradictory effects seen with this metal. Sodium arsenite at low concentrations induced enhanced ROS generation without cytotoxicity and a cellular protective effect. In contrast, sodium arsenite at high concentrations produced marked ROS formation, marked oxidative stress, and cellular damage, as evidenced by LPO.     

Hormesis phenomena under Cd stress in a hyperaccumulator—Lonicera japonica Thunb

A hydroponic experiment was carried out to investigate possible hormetic response induced by cadmium (Cd) in a potential hyperaccumulator-Lonicera japonica Thunb. The results showed that Cd at low concentrations induced a significant increase in plant growth, leaf water content and content of photosynthetic pigments in L. japonica, but decreased them at high concentrations, displayed inverted U-shaped dose response curves, confirming a typical biphasic hormetic response. The U-shaped dose response curves were displayed in malondialdehyde (MDA) and electrolyte leakage in leaves at low doses of Cd, indicating reduce oxidative stress and toxic effect. The increase of superoxide dismutase (SOD) and catalase (CAT) activities was observed along with the increased Cd concentration, indicative of increase in anti-oxidative capacity that ensures redox homeostasis is maintained. After 28 days exposure to 10 mg L^?1 Cd, stem and leaf Cd concentrations reached 502.96 ± 28.90 and 103.22 ± 5.62 mg kg^?1 DW, respectively and the plant had high bioaccumulation coefficient (BC) and translocation factor (TF′). Moreover, the maximum TF value was found at 2.5 mg L^?1 Cd treatment, implying that low Cd treatment improved the ability to transfer Cd from medium via roots to aerial structures. Taking together, L. japonica could be considered as a new plant to investigate the underlying mechanisms of hormesis and Cd tolerance. Our results suggest that hormetic effects should be taken into consideration in phytoremediation of Cd-contaminated soil.     

Study on PCB 101, PCB 153, mercury and methyl mercury content in blue crab Portunus Pelagicus from Khuzestan shore (Persian Gulf)

Distribution of polychlorinated biphenyl (PCB 101, PCB 153), Mercury (Hg) and methyl mercury (MMHg) in muscle, gill and hepatopancreas of blue swimming crab Portunus segnis from Persian Gulf were investigated. In addition, the relationships between crab size (carapace width) and PCBs, Hg, MMHg levels in tissues were investigated by linear regression analysis. There were significant differences in crab PCB 101, PCB 153, Hg and MMHg concentrations were detected between different tissues (ANOVA, P<0.05). The pollutants concentrations were highest in hepatopancreas whereas lowest in the muscle of all crab species. The recorded mean concentrations were 0.485 μg g^?1 PCB 101, 1.89 μg g^?1 PCB 0.463, 0.815 μg g^?1 Hg and 0.471 μg g^?1 MMHg. The results showed that significant relationships between PCB and Hg-MMHg levels and crab size were positive. Comparison between male and female indicated that the average PCBs and Hg-MMHg concentrations in tissues of male crab were found to be significantly higher than those found in the female crab.     

Effects of structurally different noncoplanar and coplanar PCBs on HELF cell proliferation,cell cycle,and potential molecular mechanisms

Polychlorinated biphenyls (PCBs) are a group of chemicals that persist in the environment, indoors, and humans. Lung exposure to airborne and food contaminants, such as PCBs, may cause possible lung disorders, such as cancer. In the present study, we investigated the effects of structurally different lower chlorinated (≤4Cl), noncoplanar PCB40, and coplanar PCB77 on human lung fibroblast cell line (HELF) cell proliferation, cell cycle progression, and possible molecular mechanisms. Noncoplanar PCB40 and coplanar PCB77 exhibited concentration‐ and time‐dependent biphasic dose–response effects on HELF cell proliferation. Noncoplanar PCB40 and coplanar PCB77 induced 23 and 45% cytotoxicity at higher concentrations than the control. The flow cytometry analysis showed that exposure to PCB40 caused a significant increase in time spent in the G1 phase but decreased length of the S phase in a concentration‐ and time‐dependent manner, whereas PCB77 exposure decreased time spent in the G1 and S phases but increased time spent in the G2 phase. Western blot analysis indicated that PCB77 increased the expression of cyclin E, CDK2, p21, and caspase‐9, while PCB40 decreased the expression of these proteins (except CDK2 and p21). An increase in CDK expression after exposure to PCB77 suggests that it may cause carcinogenic effects on HELF cells at higher doses. Our results also demonstrate that the different cytotoxic effects induced by coplanar and nonplanar PCBs were correlated with their structural characteristics; the coplanar congener was more cytotoxic than the nonplanar congener. The study elaborates threshold levels for these chemicals and suggests that the cytotoxicity mechanisms by which PCB congeners act on HELF cells depend on their planarity and chemical structures. Furthermore, the study will be important for developing antidotes to the adverse effects and risk assessment practices for PCBs. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1183–1190, 2017.     

Natural Products: Anti-proliferative Effects of Lichen-derived Inhibitors of 5-Lipoxygenase on Malignant Cell-lines and Mitogen-stimulated Lymphocytes

Several lichen species have been used traditionally as medicinal plants. It has previously been shown that two low-molecular-weight lichen metabolites, lobaric acid isolated from Stereocaulon alpinum Laur. and protolichesterinic acid isolated from Cetraria islandica L. (Ach.), have in-vitro inhibitory effects on arachidonate 5-lipoxygenase. We have studied the effects of these compounds on cultured cells from man, including three malignant cell-lines (T-47D and ZR-75-1 from breast carcinomas and K-562 from erythro-leukaemia), as well as normal skin fibroblasts and peripheral blood lymphocytes. Both test substances caused a significant reduction in DNA synthesis, as measured by thymidine uptake, in all three malignant cell-lines; the dose inducing 50% of maximum inhibition (ED50) was between 1.1 and 24.6 μg mL^?1 for protolichesterinic acid and between 14.5 and 44.7 μg mL^?1 for lobaric acid. The breast-cancer cell-lines were more sensitive than K-562. The proliferative response of mitogen-stimulated lymphocytes was inhibited with a mean ED50 of 8.4 μg mL^?1 and 24.5 μg mL^?1 for protolichesterinic acid and lobaric acid, respectively. These concentrations are of the same order of magnitude as the IC50 values in the 5-lipoxygenase assay. Significant cell death (assessed by the MTS (3–4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay and trypan blue exclusion) occurred in the three malignant cell-lines at protolichesterinic acid and lobaric acid concentrations above 20 and 30 μg mL^?1, respectively. In K-562 morphological changes consistent with apoptosis were detected. Up to 38% cell death was observed at 20 μg mL^?1 for protolichesterinic acid and 15 μg mL^?1 for lobaric acid in mitogen-stimulated lymphocytes but unstimulated lymphocytes were clearly less sensitive. In contrast, the DNA synthesis, proliferation and survival of normal skin fibroblasts were not affected at doses up to 20 μg mL^?1 for protolichesterinic acid and 30 μg mL^?1 for lobaric acid. We conclude that the anti-proliferative and cytotoxic effects observed might be related to the 5-lipoxygenase inhibitory activity of protolichesterinic acid and lobaric acid. These results open up the opportunity for future studies of these lichen metabolites with regard to their anti-tumour and anti-inflammatory properties.     

Induction of reactive oxygen species and algal growth inhibition by tritiated water with or without copper

Tritium (³H) is a radioactive element of ecological concern because of its release into aquatic ecosystems from nuclear power plants. However, the acute and chronic effects of tritiated water (HTO) on aquatic organisms are poorly documented, as are its effects on oxidative stress. In addition, the effects of HTO in combination with other contaminants remain largely unexamined. Herein, we document the effect of HTO on a primary aquatic producer (Chlamydomonas reinhardtii) by measuring growth and oxidative stress using fluorimetric (H₂DCF‐DA) determination of Reactive Oxygen Species (ROS) production. The maximum cell density of the alga (1.65 × 10⁶ cells mL^‐1) was reduced by 23% (1.27 × 10⁶ cells mL^‐1) at the highest exposure tested (59 MBq mL^‐1 HTO), whereas cells exposed to 0.9 MBq mL^‐1 showed a significantly enhanced maximum cell density of 1.90 × 10⁶ cells mL^‐1, an increase of 15%. With regard to oxidative stress, exposure to HTO (0.04, 0.16, and 2.8 MBq mL^‐1) induced an early dose‐dependent peak in ROS production after 14–15 min of exposure, followed by a slow decrease in ROS which stabilized after 60 min. Moreover, this study showed that the presence of HTO may influence the impact of other conventional, nonradioactive contaminants, such as copper, a well known oxidizing trace metal for aquatic organisms. A significant synergic effect of copper and HTO on ROS production was observed. This synergic effect on oxidative stress was shown to be linked to an enhanced copper uptake rate measured in the presence of HTO (> 4 times). We conclude that HTO should be considered as a sensitizer when in a mixture with other contaminants, especially through interactions on the antioxidant system of algae. © 2010 Wiley Periodicals, Inc. Environ Toxicol, 2012.     

Effects of embryonic exposure to polychlorinated biphenyls on zebrafish (Danio rerio) retinal development

Polychlorinated biphenyls (PCBs) are persistent environmental pollutants that affect embryonic development. The purpose of this study was to examine the effects of embryonic exposure to PCBs on early retinal development in zebrafish, Danio rerio. Zebrafish embryos were immediately exposed to different concentrations (0, 0.125, 0.25, 0.5, 1.0 and 2.0 mg) of PCBs per liter of medium at 28.5 °C. Embryos were assessed at 30, 48, 72 and 96 h post‐fertilization (hpf) for changes in embryonic survival rate, development, larval retinal morphology and ultrastructure of the retina. The results show that PCB exposure decreased the survival rate of embryos in a time‐ and dose‐dependent manner. Embryos exposed to the higher concentrations of PCBs (0.5, 1.0 and 2.0 mg l^?1) displayed obvious gross morphological deformities. At 72 hpf, the retinal layer development of zebrafish was delayed at higher PCB concentrations (1.0 mg l^?1). At 96 hpf, irregularity of photoreceptor cells arrangement and thickening of photoreceptor and ganglionic layers were observed in PCB‐treated larvae at concentrations of 0.25–1 mg l^?1. Ultrastructural examination showed signs of growth inhibition of the photoreceptor outer segment at 0.25–1 mg l^?1 PCB exposure at 72 hpf, as well as the appearance of massive vacuoles and holes inside the outer segments in the PCB exposure group at 96 hpf. These results suggest that embryonic exposure to moderate and high levels of PCBs induced developmental deficits in zebrafish retinas, particularly in photoreceptor cells. Copyright © 2011 John Wiley & Sons, Ltd.     

Assessment of the genotoxic effects of organophosphorus insecticides phorate and trichlorfon in human lymphocytes

In vitro genotoxic effects of organophosphorus insecticides Phorate (PHR) and Trichlorfon (TCF) were investigated using four genotoxicity endpoints. Different concentration ranges between 0.25–2.00 μg mL^?1 of PHR and 2.34–37.50 μg mL^?1 of TCF were applied to lymphocytes. PHR and TCF significantly increased the frequency of chromosomal aberrations (except 2.34 μg mL^?1 for TCF) and sister chromatid exchanges at all treatment times and concentrations. Most of the used concentrations induced a significant increase in the frequency of micronuclei. Furthermore, PHR and TCF significantly decreased the mitotic index at the higher concentrations after 24‐ and 48‐h treatments. In the comet assay, PHR and TCF significantly increased the comet tail at all concentrations. However, the comet tail intensity was significantly increased at only the highest concentration of PHR and at all concentrations of TCF. According to these results, PHR and TCF possess clastogenic, mutagenic, and DNA damaging effects in human lymphocytes in vitro. © 2012 Wiley Periodicals, Inc. Environ Toxicol 29: 577–587, 2014.     

Developmental toxicity,EROD, and CYP1A mRNA expression in zebrafish embryos exposed to dioxin‐like PCB126

Dioxin‐like PCB126 is a persistent organic pollutant that causes a range of syndromes including developmental toxicity. Dioxins have a high affinity for aryl hydrocarbon receptor (AhR) and induce cytochrome P4501A (CYP1A). However, the role of CYP1A activity in developmental toxicity is less clear. To better understand dioxin induced developmental toxicity, we exposed zebrafish (Danio rerio) embryos to PCB126 at concentrations of 0, 16, 32, 64, and 128 μg L^?1 from 3‐h post‐fertilization (hpf) to 168 hpf. The embryonic survival rate decreased at 144 and 168 hpf. The fry at 96 hpf displayed gross developmental malformations, including pericardial and yolk sac edema, spinal curvature, abnormal lower jaw growth, and non‐inflated swim bladder. The pericardial and yolk sac edema rate significantly increased and the heart rate declined from 96 hpf compared with the controls. PCB126 did not alter the hatching rate. To elucidate the mechanism of PCB126‐induced developmental toxicity, we conducted ethoxyresorufin‐O‐deethylase (EROD) in vivo assay to determine CYP1A enzyme activity, and real‐time PCR to study the induction of CYP1A mRNA gene expression in embryo/larval zebrafish at 24, 72, 96, and 132 hpf. In vivo EROD activity was induced by PCB126 at 16 μg L^?1 concentration as early as 72 hpf but significant increases were observed only in zebrafish exposed to 64 and 128 μg L^?1 doses (p < 0.005) at 72, 96, and 132 hpf. Induction of CYP1A mRNA expression was significantly upregulated in zebrafish exposed to 32 and 64 μg L^?1 at 24, 72, 96, and 132 hpf. Overall, the severe pericardial and yolk sac edema and reduced heart rate suggest that heart defects are a sensitive endpoint, and the general trend of dose‐dependent increase in EROD activity and induction of CYP1A mRNA gene expression provide evidence that the developmental toxicity of PCB126 to zebrafish embryos is mediated by activation of AhR. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 201–210, 2016.     

Effects of exposure to six chemical ultraviolet filters commonly used in personal care products on motility of MCF‐7 and MDA‐MB‐231 human breast cancer cells in vitro

Benzophenone (BP)‐1, BP‐2, BP‐3, octylmethoxycinnamate (OMC), 4‐methylbenzilidenecamphor and homosalate are added to personal care products to absorb ultraviolet light. Their presence in human milk and their oestrogenic activity suggests a potential to influence breast cancer development. As metastatic tumour spread is the main cause of breast cancer mortality, we have investigated the effects of these compounds on migration and invasion of human breast cancer cell lines. Increased motility of oestrogen‐responsive MCF‐7 human breast cancer cells was observed after long‐term exposure (>20 weeks) to each of the six compounds at ≥10^?7 m concentrations using three independent assay systems (scratch assay, live cell imaging, xCELLigence technology) and increased invasive activity was observed through matrigel using the xCELLigence system. Increased motility of oestrogen‐unresponsive MDA‐MB‐231 human breast cancer cells was observed after 15 weeks of exposure to each of the six compounds by live cell imaging and xCELLigence technology, implying the increased migratory activity was not confined to oestrogen‐responsive cells. Molecular mechanisms varied between compounds and cell lines. Using MCF‐7 cells, reduction in E‐cadherin was observed following 24 weeks' exposure to 10^?5 m BP‐1 and 10^?5 m homosalate, and reduction in β‐catenin was noted following 24 weeks' exposure to 10^?5 m OMC. Using MDA‐MB‐231 cells, increased levels of matrix metalloproteinase 2 were observed after 15 weeks exposure to 10^?7 m OMC and 10^?7 m 4‐methylbenzilidenecamphor. Although molecular mechanisms differ, these results demonstrate that exposure to any of these six compounds can increase migration and invasion of human breast cancer cells.     

Macrofilaricidal activity of the plant Plumbago indica/rosea in vitro

The macrofilaricidal property of the plant, Plumbago indica/rosea, was investigated in vitro against Setaria digitata, a filarial parasite of cattle. Adult worms were incubated in a medium containing crude extract at concentrations 0.05, 0.04, 0.02, and 0.01 mg mL^?1 for various incubation periods of 30 min, 1 h, 2 h, and 6 h, respectively, at 37°C and the worm motility was compared with that of solvent control worms kept in drug‐free medium. Complete inhibition of motility was observed for concentrations ranging from 0.02 to 0.05 mg mL^?1, whereas in the control all the worms were active. Bioassay‐guided fractionation of the crude extract by silica gel column chromatography resulted in the identification of a very active fraction. The activity of this fraction against adult worms was further confirmed by 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyl tetrazolium bromide–formazan colorimetric assay, which gave >50% inhibition for the active fraction at concentrations 0.05, 0.002, 0.005, and 0.0006 mg mL^?1 at 30 min, 1 h, 2 h, and 6 h incubation periods, respectively. The physical and chemical data obtained from melting point determination, thin‐layer chromatography and high‐performance liquid chromatography analysis and IR, ¹HNMR, ¹³CNMR, and gas chromatography‐mass spectrometry spectral analysis have indicated the chemical structure of the active molecule as 5‐hydroxy‐2‐methyl‐1,4‐naphthalenedione (plumbagin). Drug Dev. Res. 56:33–39, 2002. © 2002 Wiley‐Liss, Inc.     

Polychlorinated biphenyl quinone induces mitochondrial‐mediated and caspase‐dependent apoptosis in HepG2 cells

Polychlorinated biphenyl (PCB) quinones are known to cause toxic effects, but their mechanisms are quite unclear. In this study, we examined whether 2,3,5‐trichloro‐6‐phenyl‐[1,4]benzoquinone, PCB29‐pQ, induces cell death via apoptosis pathway. Our result showed PCB29‐pQ exposure decreased HepG2 cell viability in a time‐dependent manner. Lactate dehydrogenase leakage assay also implied the cytotoxicity of PCB29‐pQ. 4′,6‐Diamidino‐2‐phenylindole dihydrochloride staining and flow cytometry assays both confirmed PCB29‐pQ caused dose‐dependent apoptotic cell death in HepG2 cells. Furthermore, we found that PCB29‐pQ exposure increased cellular reactive oxygen species (ROS) level, decreased mitochondrial membrane potential and induced the translocation of cytochrome c from mitochondria into cytosol in HepG2 cells. Moreover, PCB29‐pQ exposure induced B‐cell lymphoma 2 (Bcl‐2) downregulation and Bcl‐2‐associated X (Bax) upregulation, poly(ADP‐ribose) polymerase cleavage, accompanied with the increased caspase‐3/9 and p53 expressions. Taking together, these results suggested PCB29‐pQ induced HepG2 cells apoptosis through a ROS‐driven, mitochondrial‐mediated and caspase‐dependent pathway. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 1063–1072, 2015.     

Involvement of oxidative stress in methyl parathion and parathion‐induced toxicity and genotoxicity to human liver carcinoma (HepG2) cells

Methyl parathion (C₈H₁₀NO₅PS) and parathion (C₁₀H₁₄NO₅PS) are both organophosphate insecticides (OPI) widely used for household and agricultural applications. They are known for their ability to irreversibly inhibit acetylcholinesterase which often leads to a profound effect on the nervous system of exposed organisms. Many recently published studies have indicated that human exposure to OPI may be associated with neurologic, hematopoietic, cardiovascular, and reproductive adverse effects. Studies have also linked OPI exposure to a number of degenerative diseases including Parkinson's, Alzheimer's, and amyotrophic lateral sclerosis. Also, oxidative stress (OS) has been reported as a possible mechanism of OPI toxicity in humans. Hence, the aim of the present investigation was to use human liver carcinoma (HepG₂) cells as a test model to evaluate the role of OS in methyl parathion‐ and parathion‐induced toxicity. To achieve this goal, we performed the MTT [3‐(4, 5‐dimethylthiazol‐2‐yl)‐2, 5‐diphenyltetrazolium bromide] assay for cell viability, lipid peroxidation assay for malondialdehyde (MDA) production, and Comet assay for DNA damage, respectively. Results from MTT assay indicated that methyl parathion and parathion gradually reduce the viability of HepG₂ cells in a dose‐dependent manner, showing 48 h‐LD₅₀ values of 26.20 mM and 23.58 mM, respectively. Lipid peroxidation assay resulted in a significant increase (P < 0.05) of MDA level in methyl parathion‐ and parathion‐treated HepG₂ cells compared with controls, suggesting that OS plays a key role in OPI‐induced toxicity. Comet assay indicated a significant increase in genotoxicity at higher concentrations of OPI exposure. Overall, we found that methyl‐parathion is slightly less toxic than parathion to HepG₂ cells. The cytotoxic effect of these OPI was found to be associated, at least in part, with oxidative cell/tissue damage. © 2011 Wiley Periodicals, Inc. Environ Toxicol, 2013.     

The hypothalamo-pituitary-thyroid (HPT) axis: a target of nonpersistent ortho-substituted PCB congeners.

Coplanar polychlorinated biphenyls (PCBs) cause adverse effects in developing and adult animals. Less is known about the effects of nonplanar ortho-substituted PCBs. We investigated the effects of 2 nonplanar PCB congeners, 95 (2,3,6-2',5'-penta CB) or 101 (2,4,5-2',5'-penta CB), and estradiol on selected endocrine parameters. In Study 1, weanling female Sprague-Dawley (S-D) rats were given a single dose of PCB 95 ip at 4, 8, 16, and 32 mg/kg/day for 2 consecutive days and killed 24 h after the last dose. PCB 95 exposure caused a dose-dependent (p < 0.001) decrease in serum thyroxine (T4) levels. Serum thyroid stimulating hormone (TSH) concentrations did not change, but prolactin (PRL) levels increased in a nonlinear (with dose) manner. No significant changes were seen in thyroid gland morphology and pituitary lactotroph number. In Study 2, progression or regression of effects was assessed by lengthening the time and a second congener was tested. Weanling female S-D rats received a single dose of PCB 95 or PCB 101 ip at 16 and 32 mg/kg/day for 2 days and were killed 48 h after the last dose. PCB 95 and PCB 101 both decreased serum T4 (p < 0.001) and hypothalamic dopamine (DA; p < 0.05) levels. No changes were seen in serum triiodothyronine (T3), TSH, and PRL concentrations. Morphological analysis of the thyroid gland showed a decrease (p < 0.05) in colloid area in rats treated with PCB 95 or 101. However, the epithelial cell height increased only in PCB 95 treated rats. Thyroid epithelial cell proliferation increased (p < 0.05) following exposure to estradiol and PCB 95. The results suggest that the HPT axis appears to be a target of ortho-substituted PCBs. PCB 95 was more effective than PCB 101 in causing these changes.     

In vitro evaluation of the cytotoxic and genotoxic effects of artemether,an antimalarial drug,in a gastric cancer cell line (PG100)

Artemisinin is a sesquiterpene lactone endoperoxide, obtained from Artemisia annua, and extensively used as an antimalarial drug. Many studies have reported the genotoxic and cytotoxic effects of artemisinins; however, there are no studies that compare such effects between cancer cell lines and normal human cells after treatment with artemether, an artemisinin derivative. Gastric cancer is the fourth most frequent type of cancer and the second highest cause of cancer mortality worldwide. Thus, the aim of this study was to evaluate the in vitro genotoxic and cytotoxic effects induced by artemether in gastric cancer cell line (PG100) and compare them with the results obtained in human lymphocytes exposed to the same conditions. We used MTT (3‐(4,5‐methylthiazol‐2‐yl)‐2, 5‐diphenyl‐tetrazolium bromide) assay, comet assay and ethidium bromide/acridine orange viability staining to evaluate the cytotoxic and genotoxic effects of artemether in PG100. MTT assay showed a decrease in the survival percentages for both cell types treated with different concentrations of artemether (P < 0.05). PG100 also showed a significant dose‐dependent increase in DNA damage index at concentrations of 119.4 and 238.8 µg ml^?1 (P < 0.05). Our results showed that artemether induced necrosis in PG100 at concentrations of 238.8 and 477.6 µg ml^?1, for all the tested harvest times (P < 0.05). In lymphocytes, artemether induced both apoptosis and necrosis at concentrations of 238.8 and 477.6 µg ml^?1, for all the tested harvest times (P < 0.05). In conclusion, human lymphocytes were more sensitive to the cytotoxic effects of the antimalarial drug than the gastric cancer cell line PG100. Copyright © 2011 John Wiley & Sons, Ltd.     

The crude extract of Corni Fructus induces apoptotic cell death through reactive oxygen species‐modulated pathways in U‐2 OS human osteosarcoma cells

Crude extract of Corni Fructus (CECF) has been used in Traditional Chinese medicine for the treatment of different diseases for hundreds of years. The purpose of this study was to investigate the cytotoxic effects of CECF on U‐2 OS human osteosarcoma cells. Flow cytometry was used for measuring the percentage of viable cells, cell‐cycle distribution, apoptotic cells in sub‐G1 phase, reactive oxygen species (ROS), Ca²⁺ levels, and mitochondrial membrane potential (ΔΨ_m). Comet assay and 4′‐6‐diamidino‐2‐phenylindole staining were used for examining DNA damage and condensation. Western blotting was used to examine apoptosis‐associated protein levels in U‐2 OS cells after exposed to CECF. Immunostaining and confocal laser system microscope were used to examine protein translocation after CECF incubation. CECF decreased the percentage of viability, induced DNA damage and DNA condensation, G₀/G₁ arrest, and apoptosis in U‐2 OS cells. CECF‐stimulated activities of caspase‐8, caspase‐9, and caspase‐3, ROS, and Ca²⁺ production, decreased ΔΨ_m levels of in U‐2 OS cells. CECF increased protein levels of caspase‐3, caspase‐9, Bax, cytochrome c, GRP78, AIF, ATF‐6α, Fas, TRAIL, p21, p27, and p16 which were associated with cell‐cycle arrest and apoptosis. These findings suggest that CECF triggers apoptosis in U‐2 OS cells via ROS‐modulated caspase‐dependent and ‐independent pathways. © 2012 Wiley Periodicals, Inc. Environ Toxicol 29: 1020–1031, 2014.     

Evaluation of the urinary threshold concentration of formoterol in sports drug testing

The use of formoterol in sports is allowed by inhalation at the maximum recommended therapeutic dose. Recently, a threshold concentration of 30 ng.mL^‐1 was defined by the World Anti‐Doping Agency (WADA) to distinguish between therapeutic and forbidden use of formoterol. The objective of this work was to evaluate that threshold concentration. Concentrations of formoterol were measured in urine samples collected after administration of 18 µg of inhaled formoterol to five healthy volunteers, and in samples collected in routine doping tests belonging to athletes having declared inhaled formoterol use. Formoterol was detected up to 8 h after administration in all volunteers with concentrations up to 19.6 ng.mL^‐1. From 28 routine samples, 27 had less than 10 ng.mL^‐1 of formoterol and only in one of the samples the concentration was 25 ng.mL^‐1. Therefore, administration of formoterol by inhalation at the maximum dose allowed by WADA will not produce false positive results using a threshold concentration of 30 ng.mL^‐1, and the experience up to now in routine doping tests indicates that the probability of obtaining urines with concentrations greater than 30 ng.mL^‐1 is close to nil. For this reason, sports authorities should re‐evaluate the need of a threshold concentration for formoterol and its practical usefulness. Copyright © 2013 John Wiley & Sons, Ltd.     

Biochemical and genotoxic effect of triclosan on earthworms (Eisenia fetida) using contact and soil tests

Triclosan (TCS) is a broad‐spectrum bactericide that is used for a variety of antimicrobial functions. TCS is frequently detected in the terrestrial environment due to application of sewage sludge to agricultural land. In the present study, 48‐h paper contact and 28‐day spiked soil tests were conducted to examine the toxic effects of TCS on the antioxidative and genetic indices of earthworms (Eisenia fetida). The activity of antioxidative enzymes (superoxide dismutase, SOD; catalase, CAT) and the content of the lipid peroxidation product (malondialdehyde, MDA) were determined as biomarkers of oxidative stress in E. fetida. Moreover, single cell gel electrophoresis (SCGE) was used as a biomarker of genotoxicity. The results showed that triclosan induced a significant increase (P < 0.05) in antioxidative enzyme activities and MDA content. Of all of the biomarkers examined, CAT activity was most sensitive to TCS, and the CAT activity increased significantly (P < 0.05) at bactericidal concentrations of 7.86 ng cm^?2 in the contact test and 10 mg kg^?1 in the spiked soil test. The comet assay showed that TCS treatments significantly induced (P < 0.05) DNA damage in E. fetida, and that 78.6 ng cm^?2 caused significant genotoxic effects in the acute test (48 h). Clear dose‐dependent DNA damage to E. fetida was observed both in contact and spiked soil tests. These results imply that TCS may have potential biochemical and genetic toxicity toward earthworms (E. fetida). A battery of biomarkers covering multiple molecular targets of acute toxicity can be combined to better understand the impacts of TCS on E. fetida. © 2010 Wiley Periodicals, Inc. Environ Toxicol, 2012.     

Influence of Chinoin-170, a Novel Antitussive,on the Mucociliary Activity in Respiratory Airways of Rats,Rabbits, Guinea-pigs and Man

Abstract— Chinoin-170 (Ch-170; 3,7-dihydro-1,3-dimethyl-7-[(5-methyl-1,2,4-oxadiazol-3-yl)methyl]1H-purine-2,6-dione) is a new antitussive with bronchodilating activity. Its effects on the ciliary beating frequency (CBF) and mucociliary clearance were studied. In tracheal explants of rats, Ch-170 dose-dependently at concentrations 2 and 5 mg mL^?1 depressed CBF by 24 and 33%, respectively. In human mucosal explants, however, no effects were seen at concentrations up to 5 mg mL^?1. In anaesthetized guinea-pigs, an intravenous 50 mg kg^?1 dose of Ch-170 caused no changes, and 100 mg kg^?1 increased the CBF by 15%. Intravenous Ch-170 dose-dependently increased by 93 (50 mg kg^?1), 179 (70 mg kg^?1) and 253% (100 mg kg^?1) the tracheobronchial mucociliary clearance in rabbits. The effect, studied using ^99mTc-labelled red blood cells as a marker, was of similar quantity to that brought about by administering 16, 25 and 40 mg kg^?1 doses of bromhexine. It is concluded that unlike many older antitussives, Ch-170 in-vitro only slightly decreases the CBF in rats and has no adverse effects on the CBF in human mucosal explants at concentrations up to 5 mg mL^?1. In-vivo, Ch-170 does not significantly alter the CBF in guinea-pigs, but dose-dependently increases the mucociliary clearance in rabbits. The increase is most probably a result of changes in the production and the properties of respiratory mucus.     

Pharmacokinetics of a novel retinoid AGN 190168 and its metabolite AGN 190299 after intravenous administration of AGN 190168 to rats

The pharmacokinetics of AGN 190168, a novel synthetic retinoid, and its major metabolite, AGN 190299, in rat blood after intravenous administration was investigated. Approximately 4.4 mg kg^?1 (high dose) or 0.49 mg kg^?1 (low dose) of AGN 190168 was administered to rats via the femoral vein. Blood was collected from the femoral artery at various time points during an 8 h period. Blood concentrations of AGN 190168 and AGN 190299 were determined by a specific and sensitive high-pressure liquid chromatographic (HPLC) method. AGN 190168 was rapidly metabolized in rats. The only detectable drug-related species in the blood was AGN 190299. Therefore, only pharmacokinetics of AGN 190299 were calculated. Elimination of AGN 190299 appeared to be non-linear after administration of the high dose, and linear after administration of the low dose. The maximum elimination rate (V_max) and the concentration at half of the V_max (k_m), as estimated by a Michaelis—Menten one-compartment model, were 7.58 ± 2.42 μg min^?1 (mean ± SD) and 6.10 ± 1.58 μg mL^?1, respectively. The value of the area under the blood concentration time curve (AUC) was 9.54 ± 1.68 μg h mL^?1 after administration of the high dose and 0.594 ± 0.095 μg h mL^?1 after administration of the low dose. The clearance value was 7.79 ± 1.20 mL min^?1 kg^?1 after the high dose, statistically significantly different from that after the low dose (p < 0.05), 14.0 ± 2.2 mL min^?1 kg^?1. The terminal half-life (t_1/2) was 1.25 ± 0.74 h for the high-dose group and 0.95 ± 0.16 h for the low-dose group. Study results demonstrate rapid systemic metabolism of AGN 190168 to AGN 190299, non-linear pharmacokinetics of AGN 190299 after the 4.4 mg kg^?1 dose, and the lack of difference in disposition profiles between sexes after intravenous administration of AGN 190168 to rats.