Day :
Keynote Forum
Alexei Basnakian
Keynote: DNase/endonuclease network: a new target for mitigation of acute nephrotoxicity
Time : 10:00:10:55
Biography:
Alexei Basnakian received his PhD and DSc degrees from the Russian Academy of Medical Science, both in the field of DNA-degrading enzymes. He had postdoctoral trainings in molecular biology at the Harvard Medical School and in toxicology/cancer research at the National Center for Toxicological Research/U.S. Food and Drug Administration. Dr. Basnakian is a tenured Professor at the Department of Pharmacology and Toxicology, and Director of the DNA Damage and Toxicology Core Center at the University of Arkansas for Medical Sciences, and Research Career Scientist at the Veterans Hospital in Little Rock, Arkansas, USA. He is an author of 87 peer-reviewed papers and 14 reviews or book chapters. Dr. Basnakian is an Editorial Board member of four biomedical journals, and a member of NIH, AHA and VA grant study sections. His research interests are in DNases/endonucleases and DNA damage associated with toxicity, anti-cancer therapy, tissue injury and cell death.
Abstract:
Statement of the Problem: Deoxyribonucleases (DNases) universally induce irreversible cell death by fragmenting DNA in response to cell injury. All of the nine known cell death DNases are endonucleases. Despite that most of the DNase activity is used after cell death, a genetic inactivation of DNases provide protection of cells and tissues against DNA breaks induced by cytotoxic stimuli, and partially protect against tissue injury. Therefore, DNases act before the point-of-no-return in cell death, and can be potentially used as therapeutic targets for tissue protection against injury. Our studies identified two DNases, DNase I and EndoG, as being responsible for tubular epithelial toxicity during acute kidney injury induced by cisplatin, rhabdomyolysis or ischemia. However, inhibitors of DNases are not available. The purpose of this study was to identify DNase inhibitors, which might be used for mitigation of acute kidney injury.
Methodology & Theoretical Orientation: To identify DNase inhibitors, we have developed a high-throughput screening assay based on a proprietary fluorescent probe.
Findings: This assay allowed the identification of several new inhibitors of deoxyribonuclease I (DNase I), which were also active against two other DNases, endonucleases G (EndoG) and deoxyribonuclease II (DNase II). The DNase inhibitors were able to significantly protect kidney tubular epithelial cells in vitro and mouse kidneys in vivo against acute kidney toxicity induced by cisplatin, glycerol (rhabdomyolysis), or renal ischemia-reperfusion. The inhibitors showed no toxicity in vivo at 5x therapeutic doses.
Conclusion & Significance: DNases can be used as a therapeutic target for mitigation of toxic or hypoxic acute kidney injury. The identified DNase inhibitors or similar compounds have a great potential for tissue protection against toxic kidney failure. Considering that DNases are expressed and responsible for cell death in all tested cells, tissues and animal models, it is likely that the same compounds may be used for universal tissue protection against various injuries.
Keynote Forum
Stephanie Lora Sharp
Keynote: The Role of the Forensic Pharmacologist as an Expert Witness
Biography:
Dr. Sharp is a Forensic Pharmacologist. She has an M.Sci. degree in Pharmacology from the University of Glasgow, a Ph.D. in pharmacokinetics from the University of Dundee and Certificates in Civil and Criminal Law from the University of Cardiff. She specialises in drugs of abuse and the clearance of drugs from the body. She has prepared reports in many cases for a variety of legal firms in England, Wales, Ireland and Scotland She has been a research scientist for 8 years and has researched at the University of Cape Town in South Africa and the University of Dundee. Dr. Sharp is a co-director of the Glasgow Expert Witness Service Ltd. and is a registered expert witness with the Law Society of Scotland Directory of Expert Witnesses, a Professional Member of The Chartered Society of Forensic Sciences and a registered expert adviser on the National Crime Agency (NCA) database.
Abstract:
Dr. Sharp is a Forensic Pharmacologist. She has an M.Sci. degree in Pharmacology from the University of Glasgow, a Ph.D. in pharmacokinetics from the University of Dundee and Certificates in Civil and Criminal Law from the University of Cardiff. She specialises in drugs of abuse and the clearance of drugs from the body. She has prepared reports in many cases for a variety of legal firms in England, Wales, Ireland and Scotland She has been a research scientist for 8 years and has researched at the University of Cape Town in South Africa and the University of Dundee. Dr. Sharp is a co-director of the Glasgow Expert Witness Service Ltd. and is a registered expert witness with the Law Society of Scotland Directory of Expert Witnesses, a Professional Member of The Chartered Society of Forensic Sciences and a registered expert adviser on the National Crime Agency (NCA) database.
Biography:
Dr. Ming-Tsang Wu has completed his MD from Chung Shan Medical University in Taiwan and PhD from Harvard School of Public Health in the USA. He is a full professor in the Department of Public Health and the Director in Research Center for Environmental Medicine, Kaohsiung Medicine University, Taiwan. His major research interest is on the interactive effects of environmental and occupational exposures, genetic factors, and biomarkers on the health outcomes.
Abstract:
We are still exposed to low-dose melamine in daily-life environment, even after 2008 toxic milk food scandal. One of the main sources is the intake of melamine chemical from the migration of melamine-made tableware, when contacted with high-temperature soup/water. Our previous study has found that chronic low-dose melamine exposure is associated with the risk of renal stones in adults, but the data about the relationship between environmental melamine exposure and the risk of renal damage in humans is still lacking. In this talk, I will present our recent findings about that link from different susceptible populations and propose the mechanisms behind that.
- Toxicology and Pharmacology
Session Introduction
Mahmoud Said
Zagazig University, Egypt
Title: Ameliorative effect of Phoenix dactylifera on adverse effects of Linezolid in rats
Biography:
Mahmoud Said has his knowledge in monitoring and counteracting the adverse effects of anti-biotics by using different antioxidant natural plants to reduce the mortality rate and health hazards resulting from antibiotic use. He has five years of experience in research, teaching and administration in hospital. His current position is director of the blood bank and he has experience in performing serological tests.
Abstract:
Linezolid is one of the oxazolidinone anti-biotics that’s used to treat methicillin resistant staphylococcus aureus (MRSA) & vancomycin resistant staphylococcus aureus VRSA and was discovered in the 1990s and first approved for use in 2000. This study was aimed to investigate the adverse effects of linezolid on bone marrow, brain & kidney damaging effect of linezolid and counteracting these adverse effects using Phoenix dactylifera methanolic extract in rats. It was found that oral administration of linezolid (100 mg/kg body weight) given for 14 successive days induced a significant decrease in hemoglobin content (7.88±0.18 g/L) on the first day post-treatment, significant increase in serum urea (59.75±0.85) & serum creatinine (1.89±0.04) on the 14th day post- treatment and induced mild brain damage on the first day post treatment. The concurrent oral administration of Phoenix dactylifera methanolic extract (1000 mg/kg body weight) and linezolid (100 mg/kg body weight) for the same period corrected the damaging effects of linezolid of the hemoglobin content, urea, creatinine & brain condition of treated rats. It was concluded that methanolic extract of phoenix dactylifera clearly ameliorated these damaging effects of linezolid.
Biography:
Dr. Ben Valdez obtained his PhD in Biochemistry at Louisiana State Univ. He did his post-doctoral training at Baylor College of Medicine in Houston, TX and became an Assistant Professor. His laboratory cloned the genes and corresponding cDNAs for RNA helicase II/Gu α and β and discovered their functions. His lab identified the functions of treacle, encoded by the TCOF1 gene, in the expression and methylation of pre-ribosomal RNA. In 2005, he transferred to the Dept of Stem Cell Transplantation and Cellular Therapy, UT MD Anderson Cancer Center where he is now an Associate Professor. His current research focuses on the identification of safe and efficacious conditioning regimen for hematopoietic stem cell transplantation for patients with hematological disorders. He conceptualized and proved the efficacy of combined DNA alkylators, nucleoside analogs, and epigenetic modifiers in leukemia, lymphoma and multiple myeloma cells, and proposed a model called the “loop of death” to explain the cytotoxic synergism of these drugs. He developed an assay for cellular efflux of chemotherapeutic drugs which is relevant to understanding drug interactions. Using this assay, Dr. Valdez discovered the differential effects of HDAC inhibitors on cellular drug transporters which have tremendous implications for using epigenetic modifiers in combination chemotherapy. The results of his pre-clinical studies have been used as bases for several clinical trials at UT MD Anderson Cancer Center
Abstract:
Statement of the Problem: Hematopoietic stem cell transplantation is an effective treatment for a variety of hematological disorders. Its success partly depends on the optimization of the pre-transplant conditioning regimen.
Methodology & Theoretical Orientation: To identify an efficacious regimen, we exposed cells to different drug combinations, analyzed their cytotoxicity and identified their molecular mechanisms of interaction using various techniques.
Findings: We have shown the synergistic cytotoxicity of DNA alkylating agents (AA) and nucleoside analogs (NA) in leukemia and lymphoma cells and proposed a mechanistic model called the “loop of death”. Exposure of cells to a nucleoside analog initiates DNA damage resulting in chromatin remodeling and makes genomic DNA more susceptible to DNA alkylation. DNA damage response is then activated and the loop of DNA damage, chromatin remodeling, and DNA alkylation continues until the tumor cells commit to apoptosis. Using this model and the [AA+NA] combination as a backbone to identify drugs that may further enhance its anti-tumor activity, we hypothesized that epigenetic modifiers would amplify the loop of death. Indeed, inhibitors of histone deacetylases (HDACi) and DNA methyl transferases (DNMTi), which facilitate relaxation of chromatin, were found to be synergistic with [AA+NA]. Since active DNA repair may contribute to decreased efficacy of these drug combinations, we also examined the inclusion of DNA repair inhibitors such as olaparib. Addition of olaparib to [AA+NA] caused significant apoptosis by activation of the DNA-damage response, inhibition of PARP activity and DNA repair, production of reactive oxygen species and depolarization of the mitochondrial membranes.
Conclusion & Significance: Our pre-clinical studies have been translated to the clinic and results from some of our clinical trials will be presented. Overall, our pre-clinical and clinical results suggest that the conditioning regimen for HSCT may be optimized by combining drugs that provide synergistic cytotoxicity based on their molecular mechanisms of action.
Biography:
Katarzyna Miranowicz-Dzierżawska works in Laboratory of Toxicology, a part of the Department of Chemical, Aerosol and Biological Hazards in the Central Institute for Labour Protection – National Research Institute. She was graduated from the Faculty of Pharmacy (specialization: pharmaceutical analysis) of the Medical University of Warsaw and she prepared her dissertation (Ph.D.): “The evaluation of interaction between chosen organic solvents” at the Collegium Medicum of Jagiellonian University, Cracow. The main area of her professional interest are problems of human in the working environment and the toxicity of chemical substances as well as methods of estimating the toxicity of substances in vitro. She was also engaged in preparation of documentation of maximum allowable levels of occupational exposure and characteristics of hazardous substances in the work of the Interdepartmental Committee mandated with updating and verification of the Threshold Limit Values (TLVs) for agents harmful to human health.
Abstract:
Statement of the Problem: Cell culture system could be a useful model for aging-related changes. The aim of the study was to assess whether there are differences between the results of determination of preservatives cytotoxicity obtained on senescent cells in different age.
Methodology & Theoretical Orientation: Experiments were conducted to determine the cytotoxicity of four preservatives: methylparaben, propylparaben, 2-phenoxyethanol and benzalkonium chloride on subsequent passages of senescent human lung CCD-8Lu (ATCC CCL-201TM) fibroblasts. The tests were carried out in passages no. 10 / 18. Xenobiotics cytotoxicity was evaluated using two cell viability assays: MTT assay, determining metabolic activity of cells, and NRU assay, assessing the integrity of cell membranes. The IC50 values were used as the main measure for comparing the cytotoxicity of tested compounds.
Findings: The results showed that the preservatives can be ranked according to the increasing cytotoxic potency towards the tested human diploid lung fibroblasts: 2-phenoxyethanol methylparaben propylparaben benzalkonium chloride. Older cells became less susceptible then the younger ones with cytotoxic effects of the xenobiotics tested in the majority of cases.
Conclusion & Significance: The passage number of diploid human lung fibroblasts had an important impact on the susceptibility of cells to preservatives. The test of the integrity of cell membranes (NRU) seems to be more appropriate to assess the cytotoxic effect of the investigated preservatives on diploid fibroblasts in different ages, which may be related to the mechanism of action of these compounds.
Funding: This paper has been based on the results of a research task I.N.13 carried out within the scope of the fourth stage of the National Programme Improvement of safety and working conditions partly supported in 2017–2019 — within the scope of research and development — by the Ministry of Science and Higher Education/National Centre for Research and Development.
- Toxicology
Location: Radisson Hotel Narita
Session Introduction
Shikha Singh
University of Allahabad, India
Title: Studies on the assessment of pesticide induced stress and apoptosis in fresh water teleost Channa punctatus
Biography:
Shikha Singh is currently working as a Doctoral Fellow at University of Allahabad, India. She has completed her Masters of Science with cytogenetic specialization from University of Allahabad, India. She has presented ten oral posters in different international/national conferences organized in India. She has been awarded with Best Oral Presentation award for her work in Indian Science Congress Association. She also assists as resource person in various academic courses.
Abstract:
The application of chemical pesticides to agricultural land very often contaminate aquatic habitat which in turn causes detrimental effects to the aquatic biota particularly to the economically important non-target organisms like fish. The aim of the present study was to explore the impact of pesticides (triazophos, deltamethrin and their combination) on oxidative stress level in fish, Channa punctatus. The fishes were procured from local animal supplier of Allahabad, India and acclimatized in laboratory condition. The fishes were exposed to different concentrations of pesticides for 96 hours. The LC50 (Lethal Concentration) value for triazophos, deltamethrin and their combination were found to be 0.069 mg/l, 7.17 µg/l and 0.032 mg/l respectively. For the sub-lethal study, fishes were exposed to 5% and 10% of LC50 of pesticides for 96 hours. During the exposure period, the behavioral changes (i.e. opercular movement, surfacing) were observed. Immediately after exposure, the animals were sacrificed and blood and the key organs (brain, liver, kidney, gills and muscles) were collected for biochemical/stress enzymes assay and for apoptotic studies. In the blood parameters, TLC and DLC showed significant change in counts as compared to control with increased rate of apoptosis. In the stress related enzyme activity such as SOD, CAT, GST and levels of GSH and LPO (Lipid Peroxidation) significant changes were recorded with increase in concentration of pesticides. From our study conclude that, the dose dependent exposure of pesticides may impose detrimental threat to the fish population.
Rishikesh K. Tiwari
University of Allahabad, India
Title: Toxicological evaluation of chlorpyrifos, cypermethrin and their combination in earthworm and their impact on Acetylcholinesterase
Biography:
Rishikesh Kumar Tiwari (Doctoral Fellow): Presently working as a Doctoral fellow with Prof. Ravi S. Pandey, Biochemistry Laboratory, Department of Zoology, University of Allahabad, Mr. Rishikesh K. Tiwari has completed his M.Sc. with cytogenetics specialization from University of Allahabad, India. He has presented ten oral/poster in different International/National Conferences organized in India besides ten research/review articles in National/International journal repute and one book chapter in USA based publishing house. He has attended one International workshop and assisted as resource person in various academic course(s).
Abstract:
The increasing applications of pesticides in the agricultural fields have adverse impact on flora and fauna of the soil ecosystem. The role of earthworms in the agricultural practices is well known as they immensely contribute in increasing the quality and fertility of soil. So, it acts as a bioindicator for the ecotoxicological analysis of pesticide induced soil pollution. Therefore, the present study was aimed to explore the impact of chlorpyrifos (an organophosphate; OP), cypermethrin (a pyrethroid) and their combination (chlorpyrifos + cypermethrin) on earthworm, Eudrilus eugeniae. E. eugeniae were exposed to different concentrations of pesticides for 48 h by paper contact toxicity method. The LC50 for commercial grade chlorpyrifos, cypermethrin and their combination were determined as 0.165, 0.066 and 0.020 μg/cm2 respectively. To assess the sub-lethal effect of these pesticides, E. eugeniae were exposed to 5% and 10% of LC50 pesticides for 48 h. Alterations in morpho-behavioural patterns such as coiling, clitellar swelling, mucus release, and bleeding followed by fragmentation of body in earthworms were observed following exposure. Acetylcholinesterase (AChE) activity was assayed in different regions of body segment which exhibits significant (p < 0.05) decrease in AChE activity particularly in pre-clitellar region followed by clitellar and post clitellar regions and in comparison, to whole body. The decreased AChE activity with increasing concentration of pesticides indicates the effect at neuronal level which apparent from the behavioural changes. Therefore, from the present findings it can be concluded that long term exposure to these pesticides could lead to severe and irreparable effects on biochemical mechanisms of earthworms.