7^th Euro-Global Summit on Toxicology and Applied Pharmacology October 24-26, 2016 Rome, Italy

Vanda Maria Falcão Espada Lopes de Andrade graduated in Biology in 1992 and obtained a Master’s degree in Animal Biodiversity Conservation in 1998, both from Faculdade de Ciências da Universidade de Lisboa. She has completed her PhD in Pharmacy/Toxicology from Faculdade de Farmácia da Universidade de Lisboa, Portugal in 2014. She is Assistant Professor in Escola Superior Agrária de Santarém, Instituto Politécnico de Santarém since 2013, where she coordinates the curricular units of Toxicology since 2014; and since 2015, Pollution and Ecotoxicology. She published 9 papers in international journals, performed 15 communications (4 oral presentations and 11 poster presentations) and 8 seminars.

Lead (Pb) and arsenic (As) and manganese (Mn) are ubiquitous neurotoxic metals/metalloids present as mixtures in environmental settings. Nucleus accumbens is preferentially susceptible to Pb, while As accumulates mainly in the pituitary and Mn in the striatum. Cholinergic (Ch) and dopaminergic (DA) outputs to these areas are important to brain functions such as motor activity (MA). Competition among metals within a mixture may occur in vivo modifying the mechanisms through which toxicity is exerted. These work aimed to asses if the exposure to a Pb/As/Mn mixture would change the contribution of Ch and DA disarrays to MA toxicity. Wistar rats were exposed to Pb, As or Mn and its mixture. MA was evaluated; serum and brains were obtained; brain metal levels were determined; Ch function was assessed through brain acetylcholinesterase (AChE-Br) activity and DA functions through serum prolactin (PRL-S) levels. Factor analyses (FA) allowed to group similar variables and evaluate AChE-Br and PRL-S proximity to MA parameters. The metal mixture induced decreased MA and higher Pb accumulation in brain, compared with all the other groups. FA showed that in Pb treated rats PRL-S was the parameter closer to MA counts, while AChE-Br and PRL-S were similarly distant to MA upon exposure to As; AChE-Br activity exhibited the lower distance to MA in Mn- and mixture-treated groups. DA modifications seem to trigger Pb-induced MA changes more than Ch. In mixture exposures, Ch pathways sensitivity to the conjunct action of Pb/As/Mn appears to overwhelm DA effects of Pb higher accumulation in brain.

M Ömer Bostanci has completed his PhD from Ondokuz Mayis University and Post-doctoral studies from Ondokuz Mayis University School of Medicine. He is the Head of Department of Physiology and Deputy Dean of Medical Faculty of Hitit University. He has published more than 20 papers in reputed journals.

The common features of all types of epilepsy are synchronized and uncontrolled discharges of nerve cell ensembles. The reason underlying the pathologically synchronized discharges of the neurons is not precisely known yet. Recent studies asserted that gap junctional intercellular communication has a critical role in epileptic neuronal events. The aim of present study is to investigate the effects of glycyrrhetinic acid derivatives, gap junctional blocker, on picrotoxin-induced epileptiform activity. In the present study, 4-month-old male Sprague Dawley rats were used for this purpose. Permanent screw electrodes allowing EEG monitoring from conscious animals and guide cannula providing the infusion of the substance to the brain ventricle were placed in to the cranium of rats under anesthesia. At the end of the postoperative recovery period, epileptiform activity was induced by injecting picrotoxin (1 μg) through the ventricular guide cannula. Epileptiform activity monitored from data acquisition system, when it reached its peak level, 200 nmol dose of 18α-glycyrrhetinic acid, 18β-glycyrrhetinic acid or carbenoxolone (succinyl ester of glycyrrhetinic acid) was applied in the same way with picrotoxin. The effects of glycyrrhetinic acid derivatives on epileptiform activity were assessed by electrophysiological analysis. Within 30 min after application, 18α-glycyrrhetinic acid, 18β-glycyrrhetinic acid and carbenoxolone decreased mean spike frequency to 49%, 49% and 59%, respectively. At the 60 min of recording, spike amplitudes were 32%, 29% and 30% less than control values, respectively. Eventually, 18α-glycyrrhetinic acid, 18β-glycyrrhetinic acid and carbenoxolone attenuated epileptiform activity by decreasing spike frequency and amplitude of epileptiform activity.

Khelifi Touhami Fatima has completed his PhD at Université Constantine 1, Algeria. She is the Director of Research Laboratory of Ethno-botany-palynology & Ethno-pharmacology-toxicology at Université de Constantine1. She has published more than 20 papers in reputed journals. She has many research projects on “Endemic Algerian medicinal plants and their activities (antioxidant, preventive effects, anti-diabetic, antitumor and anti-hyperthyroidism).

The liver is a target organ for many chemical products. During normal cellular activities, various processes inside of cells produce reactive oxygen species (ROS). Some of the most common ROS are hydrogen peroxide (H2O2), superoxide ion (O2°-) and hydroxide radical (OH°). These compounds, when present in a high enough concentration, can damage cellular proteins and lipids or form DNA adducts. Injection of carbon tetrachloride (CCl4) intraperitoneally into model animals induces acute liver injury mediated by trichloromethyl radical (CCl3°) as reactive metabolites in hepatocytes. In this study, the cellular process in this type of liver injury was analyzed from the aspect of liver function and the antioxidant activity of methanolic extracts of Matricaria pubescens. Results obtained shows that the carbon tetrachloride on dose 3 ml/kg is responsible for hepatotoxicity. On the contrary, their association to methanolic extracts cancel their toxic effects on the tissues by the decrease of serum transaminases levels (TGO, TGP) and reinfence the antioxidants capacities of defense by decrease of cytosolic MDA, this will prove the preventive effect of methanolic extracts on the toxicity of the CCl4.

The present survey is centered on protective effect of Matricaria pubescens against liver injury induced by carbon tetrachloride in rats. In our study, we indicated that these plants treatment have a potent protective effect as revealed by remarkable decrease in MDA content, additionally, methanolic extract could ameliorate acute liver damage to a high degree, as demonstrated by reduction of serum AlT levels. Introduction: The liver is a target organ for the many chemical products. CCL4 is a classically known compound that causes hepatotoxicity by an acute exposer (Recknagel, 1967 ). Matricaria pubescens plant of family compositea used in algeria for the treatment of hepatic diseases. The main objective of the present work was to evaluate the hepatoprotective and antioxidant activity of Matricaria pubescens against liver injury induced by carbon tetrachloride in rats. Materials and methods: 1. Extraction of methanolic extract: Th e aerial parts (soft twigs and leaves) of Matricaria pubescens is air-dried and extracted with methanol. 2. Determination of free radical scavenging activity: The free radical scavenging activity of the extract was determined by the method described by (Burits and Bucar, 2000). 3. Determination of total phenolic content and fl avonoids: Total soluble phenolics in the methanol extract of Matricaria pubescens were determined with Prussian bleau according to the method of Price and Butler (1977), with slight modifications of (Graham 1992), using gallic acid as a standard compound. 4. The flavonoids content in the methanol extract of Matricaria pubescens was determined by ( Boharun et al., 1996) method using quercetin and rutin as a reference compound. 4. Pharmacological essays (Study carry out on rats ) Conclusion: Our investigation provided convincing data that Matricaria pubescens have an impressive hepatoprotective effects on acute liver injuries induced by CCL4. The mechanisms underlying hepatoprotection of the methanolic extract of Matricaria pubescens may be related to both its radical scavenging properties and indicate effects as a regulator of antioxidative systems, which might be considered to be therapeutic in clinical situations.

Elnaz Erfanifar has completed her MSc from Gorgan University of Agricultural Sciences and Natural Resource, Iran. She is Ecology Expert in the Offshore Fisheries Research Center, Chabahar, Iran. She is now researching on sea cucumber species of Iran’s southern waters in the Persian Gulf and Oman Sea. She has published 1 paper in reputed journal and has been serving as an Editorial Board Member of repute. She is in progress with about 22 research projects at the research center.

In this study, 96 h LC50 values of copper sulphate (CuSO4.5H2O), a highly toxicant heavy metal, on gray mullet, Mugil cephalus of average weight mean weight 7.42 g; mean length 6.51 cm was determined. The LC50 values for CuSO4 at 24, 48, 72, and 96 h of exposure, were 1, 5, 10, 15, 20 and 30 ppm (range finding test) then fish were subjected to 7 concentrations of CuSO4 (control, 35, 40, 45, 50, 55 and 60 ppm), respectively. LC50 values were determined with probite analysis. The 96 hour LC50 value of CuSO4 to the fish was found to be 39.68 ppm. In addition, behavioral changes increased with increased concentration. The results obtained in this study clearly revealed the fact that it is necessary to control the use of a heavy metal such as copper.

Jee Sun Min is a graduate student with major in Pharmacology/Pharmacokinetics of The Catholic University of Korea.

Cilostazol is a phosphodiesterase III inhibitor with antiplatelet activity and mainly metabolized to 3,4-dehydrocilostazol (OPC-13015) and 4’-trans hydroxycilostazol (OPC-13213) via CYP3A4/5 and CYP2C19. Our previous IC50 shift assay revealed that both cilostazol and OPC-13015 were CYP3A time-dependent inhibitors in vitro and inhibitory effect on CYP3A4 in humans was further investigated. In this study, a randomized, open-label, multiple-dose, parallel study was conducted with 7 healthy Korean subjects and all subjects received the single oral dose of 40 mg simvastatin, a CYP3A4 probe substrate, on day 1. From day 2 to 7, the subjects were given 100 mg cilostazol 2 times per day with 12 hours interval. On day 8, the subjects received the single oral dose of 40 mg simvastatin again and plasma concentration of simvastatin on day 1 and 8 were measured using LC-MS/MS for pharmacokinetic assessment. AUC and Cmax ratio of simvastatin concentration in human plasma on Day 1 and Day 8 were 3.56 and 4.3, respectively, suggesting that cilostazol is a moderate inhibitor of CYP3A4. In conclusion, inhibitory effects of cilostazol and OPC-13015 on CYP3A4 in vitro were in line with clinical study and it is expected that significant drug-drug interaction between other CYP3A4 substrates and cilostazol may be possible.

Jiseon Kim is a graduate student with major in Pharmacology/Pharmacokinetics at The Catholic University of Korea.

Herbal and dietary supplements are commonly used throughout the world. Dietary supplements include amino acids, botanicals, herbals, vitamins and other products has accelerated research on herb-drug interactions with increasing the amount of consumption spending for healthy supplies. Therefore, co-administrations will have potentially dangerous side effects as well as herb-drug interactions; thus the regulating activity of co-administered dietary supplements has been shown to result in pronounced increase or decrease in the blood levels of the affected drugs. In addition to the potential for hepatotoxicity, some of herbal and dietary supplements may have interactions with certain prescription medications by various mechanisms leading to adverse events. The aim of this study was to investigate the inhibitory effect of commonly used herbal and dietary supplements on 10 UDP-glucuronosyltransferase (UGT) enzyme activities and evaluate their herb-drug interaction potential due to UGT inhibition. Th e extracts screened were artichoke, ashwagandha, burdock root, butcher’s broom, cascara sagrada, fennel seed, hawthorn, horse chestnut, horsetail, maitake mushroom extract, reishi and stinging nettle. The inhibitory effects of 12 herbal and dietary supplements on UGTs were determined using high-performance liquid chromatography by measuring the remaining activities with a probe substrate using recombinant human UGT isoforms and human liver microsomes in the absence or presence of extracts. Our data suggests that 12 herbs are unlikely to cause clinically significant herb-drug interactions mediated via inhibition of UGT enzymes involved in drug metabolism. These findings should enable an understanding of herb-drug interactions for the safety use of herb.

Mustafa Canli has completed his PhD from Glasgow University, UK and Post-doctoral studies from Ghent University, Belgium. He is a Full Time Professor in Department of Biology, Cukurova University, Turkey. He has published more than 40 papers in reputed journals and has attended many international congresses concerning ecotoxicology.

Nanoparticles (NPs) have potential to cause adverse effects on organ, tissue and cells due to their unusual physicochemical properties, because of their physical and chemical characteristics. NPs are being used increasingly in nanotechnology and data regarding their toxicities to animals are unsatisfactory. Titanium dioxide (TiO2), one of the most widely used nanoparticle, was administered to mature female rats (Rattus norvegicus var. albinos) via oral gavage (0, 0.5, 5, 50 mg/kg b.w./day) for 14 days. Then, the levels of 19 serum biomarkers (glucose, cholesterol, creatinine, urea, triglycerides, bilirubin, protein, ALP, ALT, AST, cortisol, T3, T4, estradiol, prolactin, IgG, IgM, total oxidant, total antioxidant) belonging to different metabolic systems and the activities of osmoregulation enzymes (Na,K-ATPase, Mg-ATPase, Ca-ATPase) in the erythrocyte were measured. TiO2 nanoparticles altered significantly all ATPase activities in the erythrocyte, except the lowest dose. Na,K-ATPase activity in the erythrocyte decreased following TiO2 exposures (up to 80%), but the activities of Ca-ATPase (up to 274%) and Mg-ATPase (up to 290%) increased. TiO2 nanoparticles increased significantly the levels of all the liver enzymes (ALP, ALT, AST) in the serum (up to 84%). However, the other serum parameters did not change significantly following TiO2 administration, except bilirubin levels (81% increase). The present study demonstrated that oral administration of TiO2 mostly affected the liver parameters in the serum and all ATPases in the erythrocyte. This study suggests carrying out further research to enlighten better the environmental fate of nanoparticles.

Tetradenia riparia (Hochst.) Codd, Lamiaceae a traditionally used medicinal plant is indigenous to Southern Africa. Studies have shown anti-bacterial and anti-helminthic properties in extracts and compounds from this species. However, cytotoxic assessment of nanoparticles has not been reported. This study investigated the in vitro screening of the nanoparticles for potential cytotoxic activity against the human breast cancer (MCF7) cell line. Data generated were used to plot a dose-response curve of which the concentration of extract required to kill 50% of MCF7 cell population (IC50) was determined to be 5 and 375 μg ml-1 for AgNPs and AuNPs, respectively, aft er 96 hrs. Gold and silver nanoparticles were also tested for anti-bacterial activity on five pathogenic bacteria. AgNPs were active and showed MIC against E. coli (1.56 μl ml-1), E. faecalis (1.56 μl ml-1), K. pneumonia (1.56 μl ml-1), P. aeruginosa (12.5 μl ml-1) and S. aureus (50 μl ml-1). MIC using AuNPs was as follows: K. pneumonia (1.56 μl ml-1), E. faecalis (1.56 μl ml-1), E. coli (6.25 μl ml-1), P. aeruginosa (12.5 μl ml-1) and S. aureus (50 μl ml-1). In order to fully extrapolate and reveal the full potential that may be attributed to these nanoparticles, further and more intensive studies must be undertaken.

Sara Damiano is a Biotechnology-Veterinary graduate and has done her PhD in Nephrology Science. She is specialized in in vivo studies of Renal Physiology and Pathophysiology in animal models. Her principal research interests are the studies of hypertension in spontaneously hypertensive animal model and the development of nephrotoxicity induced by immunosuppressive drugs. She has published more than 16 international papers and 2 national papers. She has received a Fellowship for the study and research on Renal Physiology by FROMO; and in 2010 Fellowship for study and research on Renal Physiology by SIN (Italian Society of Nephrology). She is the Principal Investigator in a project STAR 2014.

Cyclosporine A (CsA) has provided new perspective to prevent organ transplant rejection. However, the use of CsA is limited by several chronic renal diseases, including hypertension. The development of the hypertension is related to sodium and water retention in the kidney, regulated by the sodium transporters along the nephron: the sodium/hydrogen exchanger isoform 3 (NHE3) in the proximal tubule (PT) and the apical sodium two-chloride potassium co-transporter (NKCC2) in the thick ascending limb of the Henle loop (TAL). In this work we have investigated, in a rat animal model and in human transplant patients treated with CsA, the role of the sodium transporters. After the rats were treated for 21 days with CsA, we have measured the renal sodium and water reabsorption in PT and in the TAL by in vivo micro-puncture experiments showing a significant reduction of the PT reabsorption and an increase of the NKCC2 in the TAL. The molecular biological data, obtained by Western blot and RT-PCR, have confirmed the micro-puncture data showing a clear increase in NKCC2. In human transplant patient treated with CsA, we investigated the excretion of NKCC2 in the urinary exosomes and we observed a higher amount of NKCC2 than in non-CsA-treated patients. Our results suggested a key role of NKCC2 in the TAL as an important site of sodium retention in cyclosporine-induced hypertension. This data may have potential clinical implications for the treatment of chronic renal diseases like CsA- hypertension.

Małgorzata Przybyła has completed her PhD from Medical University of Silesia, Poland in 2012. She works as a Head of the Laboratory of Medical Analytics in the Department of Toxicological studies. She has participated in approximately 70 unpublished toxicological studies. She is responsible for the Planning of Toxicological studies conducted according to the Principles of Good Laboratory Practice and OECD Guidelines. She was a Member of the Local Ethics Committee for Animal Experimentation.

OECD guidelines for the testing of chemicals are periodically reviewed in the light of scientific progress. OECD guideline No. 422: “Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test” was updated in 2015 by adding endocrine (total T4-thyroid hormone) disruption relevant endpoints. According to the guideline, blood samples from adult males and 13-day-old pups should be examined for serum levels of thyroid hormone (TT4). Further assessment of TT4 in blood samples from dams and 4-day-old pups is made if relevant (not specified in the guideline). In the study based on OECD guideline No. 422 a vehicle control was administered on mature male and female Wistar rats from control groups (Cmdb:Wi; outbred) via oral gavage for 28 days (males) or 51-60 days (females). Serum TT4 in the control groups determined using the ELISA was in the range of 2.041-5.096 g/dL in males and 1.947-4.658 g/dL in 13-day-old pups. We suggest that further measurement of TT4 in dams and 4-day-old pups in this type of study should be made if treated groups exhibit any statistically significant changes in the TT4 level (in adult males and 13-day-old pups) or the absolute/relative weight of the thyroid gland, or histopathological changes of the thyroid gland.

Yu Fen Zheng is a graduate student with major in Pharmacology/Pharmacokinetics of The Catholic University of Korea.

Objective: The aim of this study was to investigate the enantioselective differences of sibutramine enantiomers in the inhibition of human CYP enzymes in vitro Methods: Using a cocktail assay, the effects of sibutramine enantiomers (R/S- form) and 4-(4-chlorobenzyl) pyridine (CBP) on the 9 CYP isoform specific marker reactions were screened in human liver microsomes. According to USFDA guidelines, phenacetin (50 μM), coumarin (5 μM), bupropion (50 μM), rosiglitazone (1 μM), tolbutamide (100 μM), omeprazole (20 μM), dextrophan (5 μM), chlorzoxazone (50 μM) and midazolam (5 μM) were chosen as substrates of CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A, respectively. Cocktails, test compounds and NADPH regenerating system were incubated 15 min after 5 pre-incubation with human liver microsomes (HLMs) in vitro for competitive screening. The respective metabolites of the nine substrates and internal standard chlorpropamide (200 ng/mL) were measured by LC-MS/MS system. Results: CBP showed more potent inhibitory eff ect on CYP2B6 with IC50 values of 0.014 μM than sibutramine enantiomers (7.07 μM for R-form and 2.43 μM for S-form) based on bupropion-dependent inhibition. However, CBP also potently inhibited other CYPs except CYP3A4. In addition, R-sibutramine less slightly inhibited dextromethorphan mediated CYP2D6 activities relative to S-sibutramine, and the IC50 value was more than 50 μM for R-sibutramine and 24.21 μM for S-sibutramine, respectively. Conclusion: Based on these data, R-sibutramine was selective and potent inhibitor for CYP2B6 in vitro and S-sibutramine potently inhibited CYP2B6 and slight inhibited CYP2D6 when human liver microsomes were used as the enzyme source. Thus, the present findings signified that R-isomer of sibutramine is a potent stereoselective inhibitor of CYP2B6, whereas, inhibition for other CYPs was substantially negligible.

Background & Objective: The objective of the present study was to histometricl and histochemical changes of thymus and lymph nodes in mice after using Ritalin. Method: In the present study 24 adult male Balb/C mice were used. At first the male mice were weighed and randomized into three groups: Two experimental groups and one control group. The experimental groups were orally administered Ritalin (2 mg/kg and 10 mg/kg body weight,) respectively, once a day and water by gavages for 40 days. Thereafter, animals were weighed and anesthetized. The blood samples were collected through cardiac puncture for analysis of blood cells. Then the thymus and lymph nodes were dissected out and processed for light microscopy studies through hematoxylin and eosin staining. Results: The changes of thymus and lymph nodes provided morphological evidences for Ritalin induced immune suppression.The results showed that Ritalin with different doses increased the neutrophils of peripheral blood and thickness in capsule of thymus and lymph node. Also decrease in the medulla of thymus and decreasing lymphocytes in peripheral blood were significantly observed in the experimental groups. However, no significant histopathological changes were seen in the control specimens. The data were analyzed using analysis of variance (ANOVA) and p<0.05 was considered significant. Conclusion: Our findings demonstrated that Ritalin administration in adulthood should be prescribed with more cautions.

Halina Milnerowicz has completed her PhD in 1980 from Ludwik Hirszfeld Institute of Immunology and Experimental Therapy Polish Academy of Sciences and Postdoctoral studies from Wroclaw Medical University, Faculty of Pharmacy. Since 2004, she is the Head of Department of Biomedical and Environmental Analyses. In 2008, she has got the title of Professor of Pharmacy Sciences. She was also Vice-Rector for University Development and Deputy Dean of the Division of Laboratory Diagnostics in Faculty of Pharmacy. She has published more than 140 papers in reputed journals and has been awarded at numerous Polish and international conferences.

The aim of the study was to verify the influence of oral contraceptives (OCs) on the arylesterase, lactonase and phosphotriesterase activities of paraoxonase 1 (PON1), hepatic enzymes such as aspartate aminotransferase (AST) and alanine aminotransferase (ALT) as well as lipid profile including cholesterol, triglycerides (TG), high-density lipoprotein (HDL) and low-density lipoprotein (LDL) in young healthy women. Additionally, the de Ritis ratio (AST/ALT) was calculated. Blood samples were collected from 120 women in similar age (22.6±1.0 years) with similar BMI value (20.71±2.20 kg/m2). Participants were divided into 2 groups: 46 women taking (group A) and 74 females do not take oral contraceptives (group B). Our study showed increase in ALT activity in group A (27.35±11.18 U/l) when compared to group B (18.44±8.58 U/l), while the de Ritis ratio was lower in group A (0.71±0.35) than in group B (1.06±0.63). Higher TG concentration (73.06±15.44 mg/dl) was observed in group A (56.89±19.71 mg/dl) than in group B; while LDL level showed a decrease in group A (90.45±20.21 mg/dl) in comparison to group B (107.14±26.07 mg/dl). The phosphotriesterase activity in group A was lower (79.08±18.47 U/l) when compared to group B (125.54±58.24 U/l), while increase in both arylesterase (167.53±20.91 vs. 184.33±32.69 U/l) and lactonase (9.27±2.29 vs. 10.98±3.36 U/l) were found. The use of OCs changes the lipid profile, causing the increase in TG and decrease in LDL levels. Lactonase activity of paraoxonase can be a useful marker of liver function in women taking OCs.

Fatima Laraba-Djebari is Teacher-Researcher at the Faculty of Biological Sciences of USTHB. She completed her Doctorate in Cellular Biology and Microbiology in 1994 from Marseille University and Biochemistry in 1995 from USTHB. She is Head of team on Biomolecules of interest and Immunotherapy. After being Head of Department of Cellular and Molecular Biology, she serves as Dean of Faculty at USTHB. Her main topics focused on Biochemistry Immunology and Pharmacology in Toxinology She is invited as reviewer by several editors and is author of two chapters in Handbook and more than 60 papers (Web of Science, Scopus, NCBI).

Channelopathies due to the brain ion channel dysfunction is considered to be an important mechanism involved in various neurodegenerative diseases such as epilepsy. In this study, a neurodegenerative model was developed using KTX a neuroexcitotoxic agent that blocks specifically Kv1 channels in the CNS, to explore the pathogenesis of excitotoxicity in neurodegenerative disorders. The detection of the KTX was analyzed by immunofl uorescence in various brain areas, neuroinflammatory parameters (i.e. levels of NO, MDA, GSH, catalase, proinflammatory cytokines IL-6 and TNFα), NF-κB expression and tissue analysis of neurodegenerative alterations were evaluated with or without pretreatment using an antioxidant (astaxantin). Results showed that KTX was detected only in cerebral cortex area aft er 1 day due to its binding to the specific receptors. It induced an activation of inflammatory cascade characterized by an increase of IL-6, TNFα, NO, MDA levels and NF- κB expression associated to a decrease of GSH level in cerebral cortex and hippocampus after 15 days. The neuroinflammation is associated with seizure and cerebral alterations in cortex observed at 1 and 15 days and in the hippocampus only at 15 days. The use of an antioxidant prior to the KTX exerts a preventive effect not only on the neuroinflammatiion but also on altered tissues and the BBB disruption. This model could be a useful tool to study neuronal degeneration, as well as to better understand the mechanisms underlying neurodegenerative diseases and to improve and design new therapeutic strategies.

Manool is a diterpene isolated from Salvia offi cinalis L., with antimicrobial activity against cariogenic microorganisms and chemo-preventive effect against methyl methanesulfonate in human hepatocellular carcinoma line cells. In this sense, the present study aimed to evaluate the anti-genotoxic potential of manool in Swiss mice using the peripheral blood micronucleus test. Additionally, the hepatotoxicity was assessed through dosing of aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Therefore, the animals were treated simultaneously with different doses of diterpene (1.25; 5.0 and 20.0 mg/kg b.w.) and chemotherapeutic agent doxorubicin (DXR; 10 mg/kg b.w.). The negative (no treatment), solvent (dimethyl sulfoxide- DMSO, 5 %) and positive (DXR) controls were also included. The treatments were carried out on two consecutive days, and euthanasia was performed 24 hours after the last treatment. The results revealed that animals treated with the lowest dose evaluated of manool (1.25 mg /kg b.w.) plus DXR showed frequencies of chromosomal damage significantly lower (4 micronucleus/1000 PCE) when compared with those observed in animals treated only with DXR (13 micronucleus/1000P CE). The ratio of polychromatic erythrocytes to total erythrocytes did not differ significantly between treated groups and negative control, revealing absence of cytotoxicity. The AST and ALT doses were not significantly different between the different groups. Therefore, manool showed no cytotoxicity and no hepatotoxicity, but revealed chemo-preventive effect on genotoxicity induced by DXR.

Jung Ho Kim is a Graduate student with major in Pharmacology/Pharmacokinetics of The Catholic University of Korea.

Objective: BST204, a fermented ginseng extract, shows anti-tumor effects and can be used as a supplement for cancer patients. This paper was to assess the potential drug-drug interaction of BST204 with 4 anti-cancer drugs (5-fl uorouracil, irinotecan, tamoxifen) in male Sprague–Dawley rats. Methods: The rats (9-11 weeks, 270–310 g) in every study were randomly separated into 4 groups: (1) anti-cancer drug alone; (2) single co-administration of BST204 and anti-cancer drug; (3) multiple dose of vehicle for 7 days+single anti-cancer drug on the 7th day; (4) multiple dose of BST204 for 7 days+single anti-cancer drug on the 7th day. 15 minutes prior to anti-cancer drug administration, BST204 at doses of 400 mg/kg or 1 g/kg (control) were orally administered by gastric gavage tube to the rats. Multi-dose BST204 (1 g/kg or 400 mg/kg) were administrated to rats once a day for continuous 6 days in the 3 and 4 groups. On the 7th day, rats were operated carotid artery surgery for cannulation and then received BST204 or vehicle before chemotherapeutic agents. 30 mg/kg 5-fluorouracil (i.v.), 20 mg/kg irinotecan (i.p.) and 30 mg/kg tamoxifen (p.o.) were administrated to rats and LC-MS/MS was used to detect the concentration of them and their main active metabolites. Results: There were no statistical significance for AUCinf, t1/2, MRT, CL and Vdss of 5-fluorouracil in without and with BST204 groups (P>0.05). For both irinotecan and its active metabolite (SN-38), the AUCt and Cmax in without BST204 groups were similar with them in with BST204 groups. AUC or Cmax of tamoxifen, 4-hydroxytamoxifen, endoxifen, and N-desmethyltamoxifen between BST204 and vehicle groups showed no statistical significance. In addition, the Cmax of tamoxifen were relatively higher in presence of BST204 than in absence of BST204 (P>0.05). It may be associated with P-gp inhibition of 20 (S)-ginsenoside Rh2, which is highly contained in BST204. But, the effect can be ignorable because there was no statistical significance compared with control groups. Conclusion: The pharmacokinetic interaction of BST204 with the 3 anti-cancer drugs in rats undefined us that the potential impact of BST204 on drug-drug interaction (5-fluorouracil, irinotecan, tamoxifen) can be ignorable.

Edson Luis Maistro has completed his PhD from Universidade Estadual Paulista (UNESP), São Paulo, Brazil and Post-doctoral studies from Universidade Estadual de Londrina (UEL), Brazil. He has published more than 70 papers in reputed journals and has been serving as an Editorial Board Member of several journals. He is a mentor of several students in their Master’s and PhD theses.

Medinal plants are widely consumed as home remedies and raw materials for the pharmaceutical industry. Crataegus oxyacantha L. (Rosaceae family) is a shrub native to Europe, being introduced in several countries, including Brazil. Its fruit has been used over the course of time as a diuretic, for dyspnea, and renal calculi. There are also studies that show its sedative and anxiolytic effects. Considering that plants can synthethise toxic substances to protect themselves wich can affect the organisms that feed of them, the present investigation was carried out to examine the genotoxic effects of a C. oxyacantha fruits extract on human lymphocytes and HepG2 (liver hepatocellular carcinoma), using comet and micronucleus assays. The cells were treated with five different concentrations (2,5, 5, 10, 50 and 100 μg/ml) of extract, selected through a preliminary trypan blue-staining and MTT assays. The positive control was methyl methane-sulfonate (MMS) to lymphocytes and Benzo(A)pyrene to HepG2 cells. The results showed that this fruits extract presented DNA and/or chromosome damage at concentrations of 5, 10, 50 and 100 μg/ml in both cell types. Despite the medicinal potential of the C. oxyacantha fruits extract, the results observed suggest caution on its use and indicate that are necessary in vivo studies to assess the safety of using this extract by humans.

Tatiana Matakova has completed her PhD from Comenius University in Bratislava in the field of Medical Biochemistry. She is the Head of Department of Molecular Analysis in the Division of Molecular Medicine, Martin Biomedical Center. Her research is focused on developing new markers for early cancer detection. She has published 2 university textbooks and 45 scientific papers in reputed journals.

People employed in the shoe manufacture and repair industries are at an increased risk for cancer. These people are routinely exposed to complex mixtures of solvents diluents in glues, primers and degreasers. The aim of this study was to determine the genotoxic effects in a group of footwear-workers occupationally exposed to solvent-based adhesive and solutions containing organic solvents, mainly toluene. Cytogenetic analysis in peripheral blood lymphocytes was used to compare 90 shoe workers and 131 control subjects. Frequencies of damaged cells, chromosomal aberrations, including breaks and rearrangements were scored for both groups. Polymorphisms in genes GSTT1, GSTM1, and GSTP1, determined by PCR-based methods, were used as biomarkers of susceptibility. The exposed group showed a significant increase of total chromosomal aberrations (CAs) in comparison to control (2.65±0.24% and 1.54+0.12, respectively). The frequency of chromosome-type aberrations (CSA) was 3.9 time higher (P+0.001) in the exposed than in the control group (2.07+0.19% and 1.52+0.12%, respectively). The frequency of chromatid-type aberrations (CTA) was significantly lower (P+0.05) in exposed group than in control (0.58+0.2% and 1.02+0.11%, respectively). GSTT1 null subjects exhibited significantly higher (P+0.05) frequency of CSAs than GSTT1 positive subjects (2.00+0.43% and 1.40+0.25%). Different genotype of GSTM1 and GSTP1 did not show any influence on frequency of evaluated parameters.

Maria Valeria Puzio is a Veterinarian Graduate with the highest marks. She is PhD student in the second year of Veterinary Science. Her studies focused on viral oncolysis and hypertension induced by drugs and environmental contaminants in animal model. She published three international papers and two poster communications.

Ochratoxin A (OTA) is a natural mycotoxin with important implications for animal and human health, such as nephrotoxicity and hypertension. Several mechanisms of OTA toxicity have been proposed, as well as the contribution of OTA to cause an imbalance between oxidant and antioxidant parameters. In an our previous paper we demonstrated, in rats treated with OTA, that the increased production of reactive oxygen species (ROS) is linked to a reduction in glomerular filtration rate (GFR) and with an increase in blood pressure. In addition, we have shown that treatment with recombinant mitochondrial manganese containing superoxide dismutase (rMnSOD) plus OTA significantly reduces hypertension and restores GFR levels. In this study, we have evaluated the involvement of the proximal tubule (PT) in the renal damage induced by OTA. The PT absorbs 60% to 70% of the filtered fluid and NaCl. Therefore, the changes in PT reabsorption can have profound effects on the kidney and fluid balance of the body and may contribute to the development of hypertension. Using in vivo micropuncture experiments, in OTA treated rats we have found that absoluted fluid reabsorption (Jv) in PT has been reduced, and also the expression of the principal sodium transporter in the PT: The sodium/hydrogen exchanger isoform 3 (NHE3). Instead, in the rat treated simultaneously with OTA and Apocynin, we have found a significant restore of Jv and NHE3 expression. Considering the O2 - scavenging activity of Apocynin, our data suggest an essential role of ROS in nephrotoxicity induced by OTA in PT.

J Döhring studied Cell Biology at the University of Osnabrück, Germany. Afterwards she attended the Master’s program “Animal Biology and Biomedical Sciences“ at the University of Veterinary Medicine Hannover, Germany. Since 2014, she is working as Doctoral student in the Institute of Food Toxicology and Analytical Chemistry at the University of Veterinary Medicine in Hannover. She investigates the influence of red meat consumption during colon cancer formation.

Colorectal cancer (CRC) is the third most common kind of cancer worldwide. Several environmental risk factors are known to contribute to the development of this disease. For example, the consumption of (processed) red meat has long been acknowledged as being involved in the pathogenesis of CRC, a fact recently confirmed by the International Agency for Research on Cancer (IARC). Consumption of red meat entails the endogenous formation of nitroso compounds (NOC´s), which are known to induce a variety of DNA lesions. Most of them are repaired by different repair mechanisms such as the removal of the damaged base by O6-MeG-methyltransferase (MGMT) or the base excision repair pathway. Even though MGMT repairs O6-methylguanine adducts, there is an ongoing discussion whether this enzyme is actually capable of repairing O6-carboxymethylguanine (O6-CMG), a specific DNA adduct induced by NOC´s. O6-CMG is known to be present in colon cancer cells after red meat consumption and is a highly mutagenic DNA lesion. It was therefore of great interest to clarify whether the O6-CMG adduct is a substrate for the MGMT and consequently to shed some light on the role of this enzyme in colorectal cancer protection. For this purpose, non-malignantly transformed human colon epithelial cells (HCEC) were transfected with shRNA in order to suppress MGMT expression, which was confirmed by an activity and expression analysis. Possible geno- and phenotypic changes of the transfected cell line were investigated by testing for anchorage-independent growth in the growth in low attachment (GILA) and soft agar assay and growth inhibition by testing the saturation density in cell culture. As no differences between the transfected and the parental cell line were observed, both cell lines were then treated with azaserine, a substance known to induce O6-CMG adducts. Potential DNA damage induced by this compound was detected via single gel electrophoresis, and it could be shown that MGMT-deficient HCEC cells were highly sensitive to the azaserine treatment. To further understand the role of MGMT in the repair of DNA lesions, a quantification of DNA adducts using LC-MS/MS techniques will be performed in order to investigate whether more O6-CMG adducts can be detected in MGMT-deficient cells and whether MGMT is responsible for the repair of the O6-CMG lesions.

Ksenia S Egorova completed her graduation from Lomonosov Moscow State University with a Master of Science in Biochemistry in 2006. In 2010, she completed her PhD in Molecular Biology at the Institute of Molecular Genetics RAS (Moscow, Russia). Since 2012, she has been a Researcher at N.D. Zelinsky Institute of Organic Chemistry. She is an author of 18 papers and three book chapters. Her research interests include “Biological activity, natural products, cancer proteomics, ionic liquids and carbohydrate research”.

Ionic liquids (ILs) are fascinating chemicals with unique physicochemical and biological properties. The major advantage of ILs is their high tunability which suggests that practically any desirable properties can be combined within one IL molecule. A novel active pharmaceutical ingredient- ionic liquid (API-IL) concept proposes using traditional drugs in the form of ILs for solving the issues of low solubility and polymorphism which are major drawbacks of many traditional medicines. There are three ways to introduce API into IL: As an anion or a cation; via a covalent linkage or; employing both ionic and covalent binding. We used salicylic acid (SA) as a model drug and synthesized ILs (SA-ILs) which contained SA in the cation 1-(2-((2-hydroxybenzoyl) oxy)ethyl)-3-methylimidazolium tetrafl uoroborate, 1-(2-((2 hydroxybenzoyl)oxy)ethyl)-3-methylimidazolium chloride, 1-(3-((2 hydroxybenzoyl)oxy)propyl)-3-methylimidazolium chloride), anion (1-ethyl-3-methylimidazolium salicylate, 1-butyl-3- methylimidazolium salicylate, 1-hexyl-3-methylimidazolium salicylate), or both cation and anion (1-(2-((2-hydroxybenzoyl)oxy) ethyl)-3-methylimidazolium salicylate) and studied their cytotoxicity in the human cell lines CaCo-2 (colorectal adenocarcinoma) and 3215 LS (normal fi broblasts), in comparison with conventional imidazolium ILs and SA. SA-ILs demonstrated signifi cantly higher cytotoxicity than conventional ILs and were comparable with the pure SA. Moreover, SA-ILs displayed much higher water solubility than SA. Th us, introduction of salicylic acid into an ionic liquid either via covalent or ionic bond increased the solubility of SA and did not disturb its biological activity. The results confirm the strategy of API-ILs to be a promising tool for pharmaceutical research.

Adenine-induced model of chronic kidney disease (CKD) is a widely used model especially in studies testing novel nephroprotective agents. We investigated the effects of adenine-induced CKD in rats on the activities of some xenobiotic metabolizing enzymes in liver and kidneys, and on some in vivo indicators of drug metabolism like: Pentobarbital sleeping time, and plasma concentration of theophylline 90 min post administration. CKD was induced by orally feeding adenine (0.25% w/w) for 35 days. Adenine induced all the characteristics of CKD, which was confirmed by biochemical and histological findings. Glutathione concentration and activities of some enzymes involved in its metabolism were reduced in kidneys and livers of rats with CKD. Renal CYP4501A1 activity was significantly inhibited by adenine, but other measured isoenzymes (1A2, 3A4 and 2E1) were not significantly affected. Adenine significantly prolonged pentobarbital-sleeping time and increased plasma theophylline concentration 90 min post administration. Adenine also induced a moderate degree of hepatic damages as indicated histologically and by significant elevations in some plasma enzymes. The results suggested that adenine-induced CKD is associated with significant in vivo inhibitory activities on some drug-metabolizing enzymes, with most of the effect on the kidneys rather than the liver.

Tarek M K Motawi is Professor of Biochemistry at Faculty of Pharmacy, Cairo University. He received PhD in Pharmaceutical Sciences in 1984; MSc in Pharmaceutical Sciences in 1979 and BSc in Pharmaceutical Sciences from Faculty of Pharmacy, Cairo University in 1976. His professional experience include: Instructor (1976), Lecturer Assistant (1980), Lecturer (1984), Assistant Professor (1989), Professor (1994) and Head of the Department of Biochemistry, Faculty of Pharmacy, Cairo (2008-2014).

Background: In vitro fertilization (IVF) is highly challenged by the erratic individual variability to controlled ovarian hyperstimulation (COH). This study aimed at implementing the role of pharmacogenetics in predicting the stimulation success and thus tailoring the treatment reaching advancement in patient care. The follicle stimulating hormone receptor (FSHR) and the estrogen receptors (ERs) are important genes in explaining the variability in COH results. Methods: We performed a prospective study on 216 young women with unexplained infertility. Ovarian stimulation was performed according to the GnRH antagonist protocol with a fixed daily morning dose of human menopausal gonadotropin (HMG) [Merional® 75 IU ampoules, IBSA institute] intra-muscular injection starting on cycle day 2. Based on the patient’s body mass index and hormonal profile, the daily dose of HMG was adjusted. The ESR2 (+1730G>A) (rs4986938), FSHR p.Th r307Ala (c.919A>G, rs6165) and FSHR p.Asn680Ser (c.2039A>G, rs6166) single nucleotide polymorphisms (SNPs) were detected by real-time polymerase chain reaction (RT-PCR). Serum FSH, estradiol (E2) and Anti-Müllerian Hormone (AMH) levels were measured by Enzyme-Linked Immunosorbent Assay (ELISA). Results: This study revealed that the low AMH level was highly significantly related to the poor ovarian response (p<0.001). Furthermore, the frequency of the ESR2 (AA) genotype and the FSHR (Ala307Ala), (Ser680Ser) genotypes were highly significantly associated with the poor ovarian response (p<0.001). In the combined analysis of FSHR and ESR2 genes, the more specific haplotypes were (AAG) for good responders and (GGA) for poor responders. Conclusion: The AMH level in combination with the ESR2 and the FSHR gene polymorphisms predict the poor ovarian response to COH in Egyptian women.

Xiomara Patricia Perea-Domínguez has completed her Master’s in Science from National Polytechnic Institute in Mexico. She is a PhD student at the National Polytechnic Institute in Mexico and has worked in the development of research projects in Environmental Toxicology.

Organophosphorus (OP) compounds are the most widely used class of pesticides for agricultural and landscape pest control. OP insecticides may induce oxidative stress leading to the generation of free radicals and alteration in the antioxidant system. Malathion is one of the most commonly used OP pesticide in the environment for agricultural and domestic purposes and oxidative stress has been reported as a possible mechanism of malathion toxicity. In this work, we studied the malathion effect in two of the main antioxidant enzymes (SOD and GPx) in human erythrocytes isolated from inhabitants of Northern of Sinaloa State, in Mexico. Results showed that high purity malathion at concentrations tested did not show an inductive effect of oxidative stress related to SOD and GPx activity. The oxidative effects observed for malathion in literature, at least in part, might arise from impurities, in the technical or commercial grade mixtures, therefore we consider necessary evaluation of oxidative stress induced by individual degradation products of malathion. Besides, the results obtained in this study may provide information of a change in the oxidative response of the inhabitants of the Northern of Sinaloa State compared with others regions, taking into consideration that it is a region with intensive use of wide range of pesticides.

Halina Milnerowicz has completed her PhD in 1980 years from Ludwik Hirszfeld Institute of Immunology and Experimental Therapy - Polish Academy of Sciences and Post-doctoral studies from Wroclaw Medical University, Faculty of Pharmacy. Since 2004, she is the Head of Department of Biomedical and Environmental Analysis. In 2008, she became the Professor of Pharmacy Sciences. She was also the Vice-rector for University Development and Deputy Dean of the Division of Laboratory Diagnostics on Faculty of Pharmacy. She has published more than 140 papers in reputed journals and has been awarded at numerous Polish and international conferences.

The aim of the study was to examine the effects of tobacco smoke on the lipid peroxidation, lipid profile and paraoxonase activities (arylesterase, lactonase and phosphotriesterase) in the blood of pregnant women aged ≥35 years. A total of 138 pregnant women were recruited and qualified for prenatal examination due to the mother’s age and were divided into 4 groups: Non-smoking, passively and actively smoking before pregnancy and during first trimester. Our data showed an increased lipid peroxidation in plasma of passive smokers (6.6±2.2 μmol/ml) as well as active tobacco smokers before (6.9±2.0 μmol/ml) and during first trimester (7.4±1.8 μmol/ml) of pregnancy when compared to non-smokers (5.4±1.2 μmol/ml). In the group of smokers before pregnancy, only higher LDL concentration was detected. In the plasma of passive smokers a combination of elevated LDL and decreased HDL concentrations were found. Higher value of LDL/HDL ratio was found in all studied groups when compared to non-smokers. Statistically significant decrease in all paraoxonase activities were observed in the group of smokers. In the group of passive smokers lower arylesterase and lactonase activities were observed, while in the group of smokers before pregnancy only arylesterase activity was decreased. Changes in analyzed parameters enhance the atherosclerotic risk. Lipid profile should be monitored during pregnancy, with respect of risks for mother and her fetus, especially in women, who are above 35 years old and are exposed to tobacco smoke. A normal reference standard for lipid profile for pregnant women should be estimated for the clinician.

Lingling Zhao has completed her PhD from Central South University. She is the Director of Pediatric of the Third Xiangya Hospital of Central South University. She has researched on the relation of environment and children’s health for more than ten years.

Increasing epidemiological and toxicological evidence suggests that pesticides and exposures may be associated with the development of Parkinson’s disease (PD). Chlorpyrifos (CPF) is a widely used organophosphorous pesticide with developmental neurotoxicity. Its neurotoxicity, notably on the monoamine system, suggests that exposure of CPF may induce dopaminergic neuronal injury. We investigated whether neonatal exposure to CPF contributes to initiation and progression of dopaminergic neurotoxicity and explored the possible underlying mechanisms. The newborn rats were administrated 5 mg/kg CPF subcutaneously from postnatal day (PND) 11 to PND 14 daily. The effect of CPF on dopaminergic neurons, microglia, astrocyte, nuclear factor-kB (NF-kB) p. 65 and p. 38 mitogen-activated protein kinase (MAPK) signaling pathways was analyzed in the substantia nigra of rats at 12 h, 24 h, 72 h, 16 d and 46 d after exposure. CPF-treated rats exhibited significant reduction of dopaminergic neurons at 16 d and 46 d after exposure, and a significant increase in the expression of microglia and astrocytes in the substantia nigra after CPF exposure. Intense activation of NF-kB p. 65 and p. 38 MAPK inflammatory signaling pathways was observed. Our findings indicate that neonatal exposure to CPF may induce long term dopaminergic neuronal damage in the substantia nigra mediated by the activation of inflammatory response via NF-kB p. 65 and p. 38 MAPK pathways in the nigrostriatal system.

Tae Jin Lee is an Associate Professor of Yeungnam University, Department of Anatomy, College of Medicine from 2008. He obtained his PhD degree from the Department of Microbiology of Kyungpook National University, South Korea in 2000. He had a Post-doctoral Training at Louisiana State University Health Sciences Center, Stanley S Scott Cancer Center. After relocation to Korea in 2005, he worked on mechanisms of novel TRAIL sensitizers, and role of c-FLIP in apoptotic pathways. Currently, he studies on the molecular mechanisms underlying regulating apoptosis-related genes by miRNA in cancer and human umbilical vein endothelial cells (HUVECs).

Methylglyoxal (MGO) is a reactive dicarbonyl metabolite of glucose and its levels are elevated in diabetic patient’s plasma. Some studies have demonstrated that MGO combined with amino and sulfhydryl groups of proteins to form stable advanced glycation end products (AGE), which was associated with vascular endothelial cells (ECs) injury and was responsible for atherosclerosis. In the present study, we found that MGO induced apoptosis in a dose-dependent manner in HUVECs, which was attenuated by pretreatment with z-VAD, pancaspase inhibitor. Treatments with MGO dose-dependently downregulated c-FLIPL expression. MGO-induced increase in ROS levels preceded the down-regulation of c-FLIPL, which was abrogated by the ROS scavengers NAC, indicating that MGO acts through ROS generation to repress c-FLIPL expression. The forced expression of c-FLIPL attenuated the MGO-mediated apoptosis in HUVECs. We also confirmed that MGO induced apoptosis in endothelium derived from mice. Collectively, this study demonstrates that MGO-induced apoptosis is dependent on the generation of ROS through the down-regulation of c-FLIP expression via repressing NFB pathways in HUVECs.

Tae Hyun Choi has completed his PhD from Keimyung University School of Medicine, South Korea. He is currently working as an Assistant Professor and a Doctor in Seoul National University School of Medicine. He has published more than 95 papers in reputed journals and has been serving as an Editorial Board Member of repute.

In vivo animal testing was necessary for the evaluation of skin irritation and corrosion (OECD TG 404). With the increasing need of non-animal tests to predict acute skin irritation of chemicals, many in vitro alternative tests have been developed and recommended to be used for discriminating between irritant chemicals and non-irritant chemicals. In the present study, emerging organ on a chip technology was employed to develop reconstructed human skin on a chip which closely mimics the properties of the human skin, including the epidermis, dermis and blood vessels. The skin on a chip consists of top, middle and bottom layers representing the epidermis, dermis and blood vessels, respectively. The membrane between the top and middle layer in the skin on a chip was used to compare the results from the reconstructed human epidermis (RhE) by applying the chemicals and measuring the cell viability. More importantly, the membrane between the middle and bottom layer was used to observe the tight junction’s presence, using the confocal microscopy. Chemicals that were previously determined as irritant or non-irritant, according to OECD TG 439 and animal testing, were applied to the skin on a chip and the classification of irritants by measuring viability exhibited the same categorization of irritant and non-irritant chemicals. In addition, the absence of tight junction when irritant chemicals were applied was observed. These results suggest that the skin on a chip system can be used to predict skin irritating ability of chemicals as an in vitro skin irritation assay.

Syama S completed her PhD in Toxicology from Sree Chitra Tirunal Institute for Medical Sciences and Technology, Trivandrum (Govt. of India). Her thesis title is “Interfacing of nanographene with mouse bone marrow mesenchymal stem cells and its allied molecular toxicity using in vitro and in vivo methods”. She has published 17 research papers in National and International journals about nanomaterials toxicity. She is a recipient of Raman-Charpak fellowship from Indo French Centre for Promotion of Advanced Research (CEFIPRA). She has also participated in several national and international conferences.

Graphene, a new form of carbon with high scientific and technological impact belongs to 2D carbon based material. Graphene is going to revolutionize biomedical field in the forthcoming years. Increase in graphene production has created anxiety about their safety and toxicity towards human application. Present study focused on the bio-distribution and toxicokinetics of in house synthesized PEGylated reduced graphene oxide (PrGO), after intra-peritoneal and intravenous administration in mice. Bio-distribution analysis showed presence of PrGO in liver, brain, spleen and kidney even after 21 days of exposure. Blood was collected for biochemical and hematological analysis and no significant changes were observed in all the parameters. Excretion of PrGO was observed in urine. Presence of larger aggregates of PrGO inside the body suggested less clearance by kidney. Toxicokinetics study showed increased PrGO concentration in blood after 3 h of injection followed by time dependent decreases thereafter. Histopathological analysis revealed damage in liver cells after 7 days and 21 days of exposure. Immune response was evident by increased splenocytes proliferation during initial days of exposure and becomes normal.

Tembotrione is the youngest member of the triketone herbicide family, used for postemergence weed control in corn. Although tembotrione’s impacts on the environment and human and animal health have been determined to be acceptable with regard to regulations, the biomarkers of exposure to this compound have not been strictly defined. To our knowledge, no studies using molecular biology and biochemistry methods have been performed evaluating tembotrione’s cytotoxic and oxidative potential in mammals. Therefore, we evaluated the impact of acute (4 and 24 h) exposure to low concentrations of tembotrione corresponding to the occupational exposure limit (0.0012 μg/ml), residential exposure level (0.002 μg/ml), acceptable daily intake (0.17 μg/ml), and reference concentration (3 μg/ml) on human hepatocellular carcinoma cell line HepG2 by assessing the extent of lipid peroxidation and total anti-oxidant capacity as well as the DNA damage using the alkaline comet assay. Viability of HepG2 cells after treatment with tembotrione was assessed using CCK-8 colorimetric assay. Our results have shown that low concentrations of tembotrione possess DNA damaging potential even though the viability of HepG2 cells was not significantly impaired. Considering that biomarkers of oxidative stress were not significantly altered at the tested concentrations, the indirect effects mediated through free radicals probably did not play an important role in the formation of DNA damage. Taking into account that similar investigations on the toxicity of low pesticide doses are rare, the present study points to the use of other model systems and methods for clarifying a compound’s toxic mode of action.

In a series of experiments on rats we compared adverse health effects of spherical metallic particles in nanometer and, for some of them, also in micrometer range. We studied iron oxide (Fe3O4) nanoparticles produced by a chemical technique and nanoparticles of silver, gold, copper oxide, nickel oxide, manganese oxide, lead oxide and zinc oxide produced by laser ablation of respective pure metals in de-ionized water. In some experiments, we compared particles of one and the same composition having different diameters, while in others–equidimensional nanoparticles of different metals. For these comparisons we used 2 models: (a) a single intra-tracheal instillation in low doses 24 hrs before the broncho-alveolar lavage; (b) repeated intraperitoneal injections during 6-7 weeks in non-lethal doses. All comparative assessments were based on experiments conducted strictly in parallel. Besides, we carried out a 10 months inhalation experiment with iron oxide (Fe2O3) aerosol produced by a sparking nanoparticles generator and fed to rats in the “nose only” system. It was found that the investigated nanoparticles were much more noxious on both cellular and systemic levels as compared with their fine micrometric or even submicron counterparts. However, within the conventional nanometer range the dependence of the organ-systemic toxicity on particle size is intricate and non-unique. Our data testify to a high activity of at least one of the key physiological defense mechanisms (pulmonary phagocytosis of deposited particles) against nanoparticles deposited in lower airways, while their noticeable solubilization in the biological milieus plays a more or less important role in toxicokinetics and toxicodynamics, depending on the nanoparticles size and chemical nature. There are, however, some important patterns of the in vivo toxic action common for all nanometals investigated by us–such as high cytotoxicity; as hepatotoxicity and splenotoxicity associated with nanoparticles’ accumulation in RES-rich organs; as nephrotoxicity (mostly tubular) associated presumably with nanoparticles’ active dissolution; and the last but not the least– heir “in vivo” genotoxicity. Taken together, these facts suggest that safe levels of exposure to metallic nanoparticles are possible in principle but should be very low.

Ana Lucic Vrdoljak is appointed as a Scientific Advisor at the Institute for Medical Research and Occupational Health, Zagreb and as a Lecturer at the Faculty of Pharmacy and Biochemistry, Chair of Toxicological Chemistry, University of Zagreb. Her research interests are focused on experiments dealing anticholinesterase poisoning and search for more effective therapy. She also possess extensive experience in human biomonitoring using the acetylcholinesterase assay and cytogenetic techniques to assess and control the risk of long-term outcomes associated with exposure to organophosphate and carbamate pesticides. Her work has been presented through 56 scientific papers.

Terbuthylazine (TB) belongs to the chloro-triazine group of herbicides, and primarily acts as an inhibitor of photosynthesis. It is used as a substitute for atrazine that is banned in EU countries. Workers in industrial and residential settings could be exposed to TB, especially if pesticide is handled without appropriate safety precautions. Considering that its toxicity profile, especially genotoxic and carcinogenic potential is not fully described yet, this study focused on the evaluation of in vitro toxicity of TB on human peripheral blood lymphocyte model. Assessments were made using the biomarkers of lipid peroxidation and oxidative stress, quantitative fluorescent assay for simultaneous identification of apoptotic and necrotic cells, alkaline comet assay, and hOGG1-modified comet assay. Lymphocytes were treated for 4 h at doses of TB equal to its occupational exposure level, acceptable daily intake, and residential exposure level. Following TB treatment, we observed slight changes in the levels of thiobarbituric acid reactive substances, and total antioxidant capacity in lymphocytes. Cell viability decreased in a concentration-dependent manner. Apoptosis dominated over necrosis. The levels of primary DNA damage were concentration-dependent, both in terms of comet tail length and DNA % in comet tail. However, the results of hOGG1 comet assay did not indicate significant contribution of oxidative lesions to overall DNA damage. Taken together, our results suggest that TB has low cyto-/genotoxic potential to lymphocytes but to clarify the mechanisms involved in its toxicity further studies including more sensitive techniques to detect physiological and metabolic changes at the cell level should be undertaken.

Cinzia Lucia Ursini has completed her PhD from University of L’Aquila. She worked as Research Contractor at the Department of Biochemistry of University of L’Aquila. She was awarded a Fellowship at CNR (National Research Council) Institute of Atmospheric Pollution, Rome. Currently, she is researcher of the Laboratory “Risk of Carcinogenic and Mutagenic Agents” at INAIL, Department of Occupational and Environmental Medicine, Epidemiology and Hygiene in Monte Porzio Catone, Rome, Italy. She has published more than 40 papers in reputed journals and 9 book chapters and has been Reviewer of several international journals.

Alkaline earth silicate wool(AESW) represents a new generation of high-temperature insulation material characterized by low bio-persistence and high bio-solubility and therefore considered not hazardous. However, at present, a few reliable toxicity studies are available. We evaluated cytotoxic, genotoxic-oxidative and inflammatory effects induced in human alveolar (A549) cells by 24 h exposure to 2-200 μg/ml of an AESW designed for high temperature (1260°C) usage and characterized by high magnesium oxide percentage. SEM analysis was performed to characterize fiber dimensions and to evaluate, on fibers treated with Gamble’s solution at pH neutral, fiber dissolution. Cytotoxic effects were studied evaluating cell viability by MTT assay and membrane damage by LDH release assay. The genotoxicity was evaluated by Fpg comet assay and inflammatory potential detecting IL-6 and IL-8 release by ELISA. SEM analysis found a length-weighted geometric mean fiber diameter(DLG) of 2.5 μm, 62% of respirable fibers (d<3 μm and l>5 μm) and a dissolution constant (Kdis) 70 ng/cm2/h. No viability reduction was found. Membrane damage was induced by the highest concentration. A dose-dependent moderate DNA damage and oxidative DNA damage associated to induction of IL-6 and IL-8 release were found at the highest concentrations. The findings show moderate membrane damage and genotoxic/oxidative effects associated to pro-inflammatory response at the highest concentrations of AESW. The effects could be related to the specific chemical composition and lower biosolubility of this AESW in comparison with other highly soluble AESWs (having Kdis>100 ng/cm2/h). These results suggest further investigations on this fibre which does not seem so harmless.

Delia Cavallo graduated in Biological Science from Sapienza University of Rome and worked as Researcher at ENEA (National Agency for New Technologies, Energy, Environment), Rome and at IRG (Institute of Genetic Researches), Naples. She is currently Head of Laboratory “Risk of Carcinogenic and Mutagenic Agents” at INAIL, Department of Occupational and Environmental Medicine, Epidemiology and Hygiene in Monte Porzio Catone, Rome, Italy. She has published more than 50 papers in reputed journals in the field of occupational toxicology, carcinogenesis, reproductive effects, genetic disease, safety and health.

The widespread use of silica nanoparticles (NPs) in consumer products with consequent increase of occupational and general exposure to such NM, raises concerns about their possible adverse effects. Potential genotoxicity is among the most critical health risks. We applied the cytokinesis block micronucleus (CBMN) and Fpg comet assays on BEAS-2B cells, a human lung cell model to test genotoxicity of two synthetic amorphous silica (JRC) synthesized by two different processes (precipitated NM200 and pyrogenic NM203) and with different surface properties. NM characterization has been performed by TEM analysis and DLS. The cells were treated with NP concentrations from 0.1 to 100 μg/ml for 24 h (Fpg comet assay) and for 48 h (CBMN) to detect direct/oxidative DNA damage and MN induction respectively. NM203 was better dispersed in water/BSA but showed higher aggregate/agglomerate sizes than NM200 in culture medium. Slight direct DNA damage at 10 and 100 μg/ml and oxidative DNA damage at the lowest concentration were induced by NM200. Otherwise dose-dependent direct DNA damage reaching 5.3 fold of control at 100 μg/ml and significant oxidative DNA damage up to 10 μg/ml were found for NM203. In the CBMN assay, a slight induction of MN and a reduced replication index were found only for NM203 indicating greater toxicity of NM203 in comparison with NM200 possibly due to surface differences. The findings show higher genotoxic effects for the pyrogenic NM203 in agreement with its higher capability to induce toxic and pro-inflammatory/ oxidative effects already demonstrated particularly on macrophages.

E Halasova has completed her PhD from Comenius University in Bratislava. In 2007, she habilitated in the field of Medical Biology. She is the Director of Division of Molecular Medicine, Martin Biomedical Center. Her research is focused on developing new markers for early detection of cancer. She has published 2 monographs, 6 university textbooks and 85 scientific papers in reputed journals. Since 2014, she has been serving as Editorial Board Member of the journal Scientific Reports.

Chromium is a well-known mutagen and carcinogen involved in lung cancer development. Biotransformation enzymes play an important role in elimination of oxidative changes caused by chromium exposure. In the present study, we investigated the polymorphisms of the following biotransformation genes GSTT, GSTP, GSTM and NAT2 and the risk they present towards the development of lung cancer with emphasis on effect of chromium exposure. We analyzed 106 individuals; 45 lung cancer patients exposed to chromium and 61 healthy controls. Genotypes were determined by PCR-RFLP method. Distribution of genotype´s frequencies in polymorphisms of observed genes in lung cancer patients and controls were analyzed in dominant, additive and recessive genetic models. Allelic distribution analysis did not show difference between exposed and control group in allelic frequency and distribution in any analyzed genes. Also we did not find out any effect of analyzed gene polymorphisms on risk of lung cancer development neither in dominant nor recessive models.

Porreca Immacolata has completed her PhD from University of Naples “Federico II” working in the developmental biology field. After that, she started her Postdoc in the Genes and Environment Laboratory of Biogem Research Institute. She is responsible of studies aiming to dissect the mechanisms of toxicity of pesticides on the endocrine system using in vitro and in vivo model systems. Combining toxicogenomics with classical toxicology approaches, she aims to generate new strategies for human risk assessment.

The recent advances in biological techniques, from the Omics technologies to the most innovative cellular models, are proposed as methods of modern toxicology. Particularly, the application of the toxicogenomic approach to cell cultures has been suggested for chemical testing to reduce animal use and to identify molecular targets extrapolable to humans. Despite this promise, the studies showing the validation of in vitro toxicogenomics results in animal models are limited. The present work is a proof of the concept that toxicogenomics data obtained in cellular models can recapitulate impairments occurring in vivo and predict cell-type unrelated outcomes. We generated gene expression profiles by multiple-dose and co-exposure testing of 2 pesticides, ethylene-thiourea (ETU) and chlorpyrifos (CPF), in immortalized rat follicular cells (PCCl3) by high-throughput RNA sequencing. The deregulated pathways/mechanisms were highlighted by a multi-leveled bioinformatics approach, based on gene clustering analyses. Different techniques were applied to confirm molecular data and bioinformatics suggestions in mice exposed in utero and lifelong to different dose of both pesticides and their mixtures. We highlighted specific and common mechanisms of toxicity for ETU and CPF, the last exerted throughout the inhibition of transcripts (Egr1, Hmga1, Zfp36l2) controlling proliferation/survival of thyroid and blood cells. In vitro expression data, mechanisms and unexpected outcomes were verifi ed in vivo. The yrocytes damage was confirmed by the reduction of circulating T4 and of Bcl2 transcript. Hematopoietic injury, involving several compartments, was confirmed with a more severe phenotype for the red cells. Indeed, a reduction of the reticulocyte count was retrieved in mouse co-exposed to CPF and ETU, both 0.1 mg/kg/die, below the CPF and ETU NOAEL. In conclusion, although in vitro systems cannot entirely replace the animal models because not fully recapitulating adaptation processes, our approach shows that in vitro toxicogenomics is a powerful tool in identification of signature/mechanisms of toxicity or unpredicted outcomes verifiable in rodents and hopefully, in humans, a point that should be further addressed.