Does granulocyte colony stimulating factor have protective effects against carbon monoxide-induced cardiomyocyte apoptosis?

S310

Abstracts / Toxicology Letters 238S (2015) S56–S383

vorinostat was 5.1 ␮M, while the clinically relevant concentrations are between 1 and 2 ␮M. In combination studies, HT-29 cells were treated with 1 ␮M and 2 ␮M vorinostat, 30 min after being treated with 10 mM NAC and 3 ␮M TOC that displayed negligible antiproliferative effects. Both NAC and TOC managed to sensitize cells towards activity of vorinostat, especially in a concentration of 2 ␮M. Calculated CIs of 0.1981 and 0.0803 for NAC, and CIs of 0.3566 and 0.0774 for TOC, in combination with 1 ␮M and 2 ␮M vorinostat respectively, suggest their synergistic effects in concentrations that can be achieved in vivo. The effect of NAC pretreatment was more pronounced than that of TOC for a lower concentration of vorinostat, while it was similar for a higher concentration of vorinostat. Conclusion: Response to vorinostat could be improved by combining it with antioxidants. The mechanisms responsible for this synergistic effect should be more in-depth investigated. Acknowledgement: This work is supported by Ministry of Education, Science and Technological Development of Serbia, Grant No. 41012. http://dx.doi.org/10.1016/j.toxlet.2015.08.885

P13-106 An in vitro comparative study of the neurotoxicological effects of tramadol and tapentadol in SH-SY5Y cells J. Faria 1,2,3 , J. Barbosa 1,2,3 , F. Carvalho 1 , O. Queirós 2,4 , R. Moreira 2,4 , R.J. Dinis-Oliveira 1,2,3,∗ 1 REQUIMTE, Laboratório de Toxicologia, Departamento de Ciências Biológicas, Porto, Portugal 2 IINFACTS – Instituto de Investigac¸ão e Formac¸ão Avanc¸ada em Ciências e Tecnologias da Saúde, CESPU, Gandra PRD, Portugal 3 Faculdade de Medicina da Universidade do Porto, Departamento de Medicina Legal e Ciências Forenses, Porto, Portugal 4 CBMA – Centro de Biologia Molecular e Ambiental, Departamento de Biologia, Universidade do Minho, Braga, Portugal

Tramadol and tapentadol are opioids clinically used in the treatment of moderate to severe pain, for which drug abuse and neurotoxic effects have been reported. As the neurotoxic mechanisms of these opioids are not fully understood, the present work aims to study the in vitro toxic effects of tramadol and tapentadol in a neuronal model, namely by assessing oxidative stress, mitochondrial and metabolic alterations. Human cell line SH-SY5Y was exposed to tramadol and tapentadol concentrations up to 600 ␮M. Upon exposure, toxic effects were evaluated through MTT and SRB assays. The results obtained suggest that, within the concentrations and exposure period tested, tapentadol shows higher toxicity than tramadol, which is reflected in its lower IC50 value. The analysis of ATP levels and biochemical parameters allows the evaluation of energetic metabolism changes. According to the results, tramadol and tapentadol promote a decrease in ATP levels, and alterations in metabolic parameters such as glucose and lactate levels. Nonetheless, considering the same concentrations and exposure times, tapentadol caused more effects compared to the non-treated control. Cell viability was also determined, and decreased after exposure to tramadol and tapentadol. The results obtained are relevant since the use of these opioids is increasing nowadays. Thus, it is important to understand their toxic effects in order to optimize therapeutic strategies and prevent adverse effects. http://dx.doi.org/10.1016/j.toxlet.2015.08.886

P13-108 Mechanistic evaluation of protective effects of granulocyte colony stimulating factor on carbon monoxide poisoning by measuring jak2, STAT3 and Akt1 proteins M. Hashemzaei 1,∗ , A.H. Mohamadpour 2 , M. Imen Shahidi 1,2 , S.A. Moallem 2 , K. Abnous 2 1

University of Medical Sciences, Zabol, Iran, Department of Pharmacology and Toxicology, Faculty of Pharmacy, Zabol, Islamic Republic of Iran 2 Mashhad University of Medical Sciences, Pharmacodynamy and Toxicology, Mashhad, Islamic Republic of Iran Question: Carbon monoxide is an odorless, colorless, tasteless and non-irritating gas that is by-product of inefficient combustion of hydrocarbon fuels such as motor vehicle exhausted gases. It is leading cause of mortality in the USA amongst all unintentional toxicants. CO poisoning has many cardiovascular effects such as, cardiomyopathy, tachycardia, arrhythmias, and ischemia and in severe cases, myocardial infarction (MI) and cardiac arrest. Cardiomyocyte apoptosis is one of most frequent effect in the heart. G-CSF is a cytokine that mobilize and differentiate granulocytes from stem cells. It can stimulate many anti apoptotic pathways such as JAK2-STAT3 and PI3-Akt kinases following cardiac ischemia. GCSF exerts its anti-apoptotic effects by binding to its specific cell surface receptor. The purpose of this study was to elucidate the mechanism of anti-apoptotic effect of G-CSF following CO poisoning. Methods: Rats were intoxicated with 1500 (n = 5) or 3000 (n = 5) ppm CO levels. Animals received G-CSF100 ␮g/kg for five consecutive days after CO intoxication. Western blot analysis was used to evaluate expression of 6 proteins including JAK2, p-JAK2, STAT3, p-STAT3, Akt1 and p-Akt1 using G-CSF 100 ␮g/kg multiple dose following CO poisoning. Results: There were a significant difference between phosphorylated proteins including p-JAK2, p-STAT3 and p-Akt1 in the G-CSF groups in comparison to control groups and there weren’t any significant differences at total proteins amongst groups. Conclusion: The results showed that G-CSF ameliorate cardiomyocytes apoptosis via JAK2/STAT3 and Akt/PI3 pathways following CO poisoning in rat. http://dx.doi.org/10.1016/j.toxlet.2015.08.887

P13-109 Does granulocyte colony stimulating factor have protective effects against carbon monoxide-induced cardiomyocyte apoptosis? M. Hashemzaei 1,∗ , S.A. Moalem 2 , A.H. Mohamadpour 2 , M. Imen Shahidi 2 1 University of Medical Sciences, Zabol, Iran, Department of Pharmacology and Toxicology, Faculty of Pharmacy, Zabol, Islamic Republic of Iran 2 Mashhad University of Medical Sciences, Pharmacodynamy and Toxicology, Mashhad, Islamic Republic of Iran

Purpose: Carbon monoxide (CO) which is produced by incomplete combustion of hydrocarbons has many toxic effects on different organs especially the heart and the brain that have greater demands for oxygen. The present study aimed to evaluate the pro-

Abstracts / Toxicology Letters 238S (2015) S56–S383

tective effects of granulocyte colony stimulating factor (G-CSF) on cardiomyocyte apoptosis after CO poisoning in rats. Methods: Male Wistar albino rats were exposed to CO 1500 or 3000 ppm for 60 min. Single and multiple doses of G-CSF (10, 50, and 100 ␮g/kg) were administered to 17 groups (N = 5) following CO 1500 ppm exposure and 17 groups (N = 5) following CO 3000 ppm exposure. After CO poisoning, carboxyheamoglobin concentration were measured for all groups. Also, cardiomyocyte apoptosis in all groups were assessed via determination of caspase 3 activity by immunofluorescence and for the group poisoned with CO 3000 ppm and treated with G-CSF 100 ␮g/kg apoptosis was determined byterminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Results: Concentration of carboxyheamoglobin in blood significantly increased following exposure to CO 1500 ppm (19–47%) and 3000 ppm (60–76%). However, carboxyheamoglobin levels were significantly higher in CO 3000 ppm group as compared to CO 1500 ppm (p < 0.05). Caspase 3 activity differences between G-CSF and control groups were significant and G-CSF could decrease apoptosis following CO 3000 ppm poisoning (p < 0.001). The greatest decrease in caspase 3 activity was seen in animals exposed to CO 3000 ppm and treated with multiple doses of G-CSF 100 ␮g/kg. TUNEL assay showed that in G-CSF 100 ␮g/kg multiple doses treated group, apoptosis was significantly ameliorated in comparison to control and sham groups (p < 0.05). Conclusions: In this study, we confirmed that CO poisoning leads to cardiomyocytes apoptosis which could be significantly reduced by G-CSF treatment.

S311

administration at 200 mg/kg. Serum was prepared and analyzed by immunoassay. The following evaluations were performed during the study: clinical signs, body weights, clinical pathology, and pharmacokinetic analysis. Results: There were no test item-related deaths, and no clinical signs of local or systemic toxicity observed during the study. The No Observed Adverse Effect Level was considered to be 200 mg/kg for both the sc and iv routes of administration. Following sc and iv administrations, elimination of RPH-104 was complete by 504 h post-dose. The mean half-life of RPH-104 was 28–87 h and 52–70 h for sc and for iv administration respectively, with no trend relating to dose level or animal gender. Following both sc and iv administrations of RPH-104, increases in maximum concentrations and mean exposures to RPH-104 were essentially proportional with respect to dose level, indicating that there was no saturation of routes of absorption or elimination with increasing dose level. http://dx.doi.org/10.1016/j.toxlet.2015.08.889

P13-111 Dissolution of alkaline earth silicate wool and polycrystalline wool in saline solution A. Campopiano 1,∗ , A. Cannizzaro 1 , F. Angelosanto 1 , E. Barrese 2 , S. Iavicoli 1 1 2

INAIL, Monteporzio Catone (Rome), Italy INAIL, Lamezia Terme (CZ), Italy

http://dx.doi.org/10.1016/j.toxlet.2015.08.888

P13-110 Toxicity and Pharmacokinetics of novel IL-1 Trap heterodimeric fusion protein A. Krotkova 1,∗ , E. Shipaeva 1,∗ , C.M. Luetjens 2 , F.T. Ludwig 2 , T. Hardwick 3 , Y. Lavrovsky 4 , M. Samsonov 1 1

CJSC «R-Pharm», Mocow, Russian Federation Covance Preclinical Services GmbH, Münster, Germany 3 Covance Laboratories Ltd., Harrogate, United Kingdom 4 R-Pharm Overseas Inc., San Diego, United States 2

Signal transduction through the IL-1 receptor type I (IL-1RI) pathway has been shown to mediate a strong pro-inflammatory response, which contributes to a number of human autoinflammatory diseases such as diabetes, cryopyrin-associated periodic syndromes, Behc¸et Disease, Familial Mediterranean Fever, gout and certain types of arthritis. RPH-104 is a novel heterodimeric fusion protein capable of binding to human IL-1␤, IL-1a and IL-1 receptor agonist (IL-1RA). The RPH-104 heterodimer comprised of soluble portions of human extracellular portions of IL-1RI and IL-1 receptor accessory protein (IL-1RAcP) each linked to a mutant Fc portion of human IgG1 fragment. Objectives: This study aimed to assess the toxicity and pharmacokinetics of RPH-104 following a single subcutaneous (sc) or intravenous (iv) (bolus) administration in the cynomolgus monkey, followed by a 30-day recovery period. Methods: 24 naive male and female cynomolgus monkeys were allocated to 6 groups and dosed with either the vehicle, 5, 50 and 200 mg/kg RPH-104 (sc, Groups 1–4) or 5 and 200 mg/kg RPH-104 (iv, Groups 5 and 6) on Day 1. Following dosing, blood samples were collected pre-dose and at 0.5, 1, 4, 8, 24, 48, 96, 168, 336, 504 and 696 h post-dose. Additional blood samples were taken from animals at 0.083 and 0.25 h post-dose following both sc and iv

The possible pathogenicity of a fiber depend upon the extent to which the fibers can be inhaled and can persist in the lung. Biopersistence of fibers is determined by the rate at which they are physically cleared from the lung by mechanical action and the rate at which they dissolve in the lung (biodurability). Biodurability may involve processes occurring both within cells and in the extracellular fluids. Among man made mineral fibers, the alkaline earth silicate wool (AESW) and polycrystalline wool (PCW), new generation wools of high-temperature insulation materials having low biopersistence, are replacing ceramic fibers in some applications. For these materials, IARC decided not to make an overall evaluation because no human data were available, although such fibers appear to have low carcinogenic potential in experimental animals. The aim of this paper is to study the behaviour of AESW and PCW in saline solution simulating physiological fluids. AESW is composed principally of SiO2 (61–67%) and CaO (27–33%), and PCW of Al2 O3 > 72% and SiO2 . To simulate macrophage environment and extracellular environment of the lung, a Gamble’s solution to two different pH (4.5 and 7.4) was used. The stationary test lasted from 1 to 4 weeks. Morphological and physical changes of fibers that accompanied the leaching and dissolution process are studied by scanning electron microscopy (SEM). Each solution was analyzed by inductively coupled plasma atomic emission spectrometry (ICPOES) to quantify the concentration of the elements extracted from wools. The amount of silicon released in the solution at pH 7.4 rose during the four weeks of treatment, much more for AESW than PCW. The SEM analysis showed no variation of length-weighted fiber diameter (DLG ≈ 4 ␮m) for PCW in both environments; and an almost complete leaching of AESW in the pH 7.4 solution. For PCW, the dissolution seemed to produce uncorroded fibers with the same chemical composition as the untreated fibers. The dissolution fiber leads to breakage transverse of fiber and the DLG value is affected little of this change. AESW showed a dissolution highly non-congruent with rapid leaching of calcium. The chemical composition of the fibers and the pH environment have a strong