Cellular and Molecular Toxicology Core
The Cellular and Molecular Toxicology Core (CMTC) is established on the foundation of the DNA Damage and Toxicology Core. The core provides state-of-the-art research facilities and services in a cost-effective manner with the necessary expertise in the areas of cellular and molecular analysis of tissue injury. The core also provides training to COBRE Project Leaders to gain new knowledge and skills that are essential for them to establish their independent careers. CMTC is directed by Alexei G. Basnakian.
Core Director: Alexei G. Basnakian, MD PhD DSc received his MD from the Sechenov Moscow Medical School, and PhD and DSc degrees from the Russian Academy of Medical Science, both in the field of Biochemistry. He had postdoctoral trainings in molecular biology at the Harvard Medical School and in toxicology/cancer research at the National Center for Toxicological Research (NCTR), Food and Drug Administration (FDA). Dr. Basnakian is a tenured Professor in the Department of Pharmacology and Toxicology and Research Career Scientist at the Veteran’s Hospital in Little Rock, Arkansas. He is an author of more than 85 peer-reviewed papers and 15 reviews or book chapters. Dr. Basnakian is an Editorial Board member of three biomedical journals, and a member of NIH, AHA and VA grant study sections. His research interests are in the field of enzymatic DNA damage associated with toxicity, tissue injury and cell death.
General. The Core provides in vitro and in vivo toxicity assessments and measurements of toxicants. In vitro toxicity assessment is based on a variety of cell-, ELISA-, FACS- and colorimetry-based assays. Examples of such assays are LDH release, MTT, PI, TUNEL, and Comet assays. In vivo toxicity assessment includes testing toxic compounds in rodents, blood collections, blood assays, and assessment of tissue injury (DNA/RNA damage-safe tissue collection, nuclei Comet assay, DNA fragmentation assays, 8OHdG ELISA, and quantitative TUNEL assay, combined, if necessary, with quantitative immunocytochemistry (qIHC) for oxidative, DNA damage, apoptotic, autophagy or inflammation markers). The Core also provides measurements of toxicants in biological fluids. These include: catalytic iron (CatFe), thiocyanate, and modified lipoproteins.
Toxicity assessment in vitro. The Core performs the following in vitro assays, some of which subcontract Flow Cytometry Core services:
- Assessment of cell death mechanism in suspension cell populations
- Analysis of cellular senescence
- Analysis of intracellular production of reactive oxygen species (ROS)
- Analysis of radiation-, hypoxia- or drug-induced oxidative DNA damage
- TUNEL assay
- Analysis of cell cycle
- Cobblestone area–forming cell (CAFC) assay
- Competitive repopulation assay (CRA)
Upon the completion of analyses, resulting in data are entered in Excel spreadsheets, and statistical analyses performed by ANOVA and Student’s t test or by nonparametric statistical tests as appropriate. Description of the assays and representative images for publications are provided upon request.
Measurement of systemic functional toxicity in vivo. For the assessment of systemic (organ) functional toxicity, mice will be administered the compound of interest, and blood is collected pre-dose and once a month until euthanasia. Measurement of 14 blood parameters indicative of organ function (toxicities) is performed using VetScan VS2 instrument equipped with the Comprehensive Diagnosis Kit (Abaxis, Union City, CA). The measurements include: alanine aminotransferase (ALT), albumin (ALB), alkaline phosphatase (ALP), amylase (AMY), blood urea nitrogen (BUN), calcium (CA), creatinine (CRE), globulin (GLOB), glucose (GLU), phosphorus (PHOS), potassium (K+), sodium (NA+), total bilirubin (TBIL), and total protein (TP). These markers are indicators of tissue injury as follows:
- liver (ALT, ALB, ALP, GLU, BUN, TBIL, TP)
- kidney (ALB, AMY, ALB, BUN, CRE, CA, TP)
- heart (ALT, NA+)
- pancreas (AMY, GLU)
- bone (ALP)
- parathyroid (ALP, CA, PHOS)
- intestine (ALP, GLOB, NA+, PHOS, TP), or
- immune system (GLOB)
In addition, blood cell count can be analyzed using the VetScan.
Measurement of systemic structural toxicity in vivo. To measure acute and delayed (long-term) structural toxicities, tissues and organs are collected after euthanasia and fixed. Tissue blocks in paraffin will be prepared and stored. Acute tissue injury will be measured using TUNEL assay, and quantification of fibrosis is used to assess a delayed injury.
The TUNEL assay has a unique advantage over other tissue injury assays because it is highly sensitive, quantitative, and universal for in vitro and in vivo.
For the TUNEL staining, 4-mm thick tissue sections are cut, dewaxed, rehydrated in phosphate-buffered saline (PBS), and processed using the In Situ Cell Death Detection Kit (Roche Diagnostics, Indianapolis, IN). Images are taken using an Olympus IX-81 microscope (Olympus America Inc., Center Valley, PA) equipped with a digital camera Hamamatsu ORCA-ER (Hamamatsu Photonics K.K., Hamamatsu City, Japan). Slidebook 6 software (SciTech Pty Ltd., Australia) is used for the image capturing and analysis. The areas of the total nuclear DNA (DAPI-positive) and fragmented nuclear DNA (TUNEL-positive) are masked and mean color density of the area is measured within each tissue compartment. The results are presented as the percentage of TUNEL-positive nuclear DNA area in the total nuclear DNA area calculated for individual cells.
Quantitative immunohistochemistry with or without TUNEL can be used to assess mechanisms of cell death in vivo and in vitro. The core can perform new immunohistochemical stainings and/or analyze previously stained, fluorescent or color slides. Slides can be used for measurements of area, and overlap between areas (colocalization), or mean intensity (level of protein expression).
Molecular Cytogenetics Service: This facility offers an array of conventional as well as cutting-edge molecular cytogenetic services including G-banded karyotyping, analysis of stable and unstable chromosomal aberrations by solid staining or G-banding, whole chromosome or locus-specific multiple fluorescence in situ hybridization (mFISH), telomere painting, and Spectral karyotyping (SKY) in cells from human and research animal sources. Cytogenetic studies are performed on fresh cells or tissues including peripheral blood, bone marrow, skin, and solid tumors.
Use of our instruments by customers. Some customers prefer to use our microscopes with the software for image analyses. The use of image analysis software alone is also available (after brief training provided by the core) and is free of change.
Training, consulting and writing. The core also provides training to faculty, postdocs, and graduate and undergraduate students, as well as consultation for customers. The Core Director helps customers to write sections related to the core services in grant proposals and research papers.
Cost. The core services are free for the Center for Studies of Host Response to Cancer Therapy Project Leaders and Pilot Project Awardees. All other investigators are welcome to use our services for a fee. The price-list can be found at the site DNA Damage and Toxicology Core site.