ARCHIVED - Toxicology Research

Mission: To protect and improve the health of the people of Canada through toxicology Research conducted to ensure a safe food supply.

Objective: To utilize toxicology research methods to identify and characterize food based human health hazards.

The Toxicology Research Division is responsible for the identification and investigation of potential health hazards associated with chemical contaminants, including those of environmental origin, agricultural chemicals, natural food toxicants and constituents, and food additives. Research is carried out on the carcinogenic, mutagenic, reproductive, neurotoxic and other potentially harmful effects of these chemicals, including alterations to the immune system.

The Toxicology Research Division is located in the Sir Frederick Banting building, Tunney's Pasture, Ottawa, Ontario Canada. KlA OL2 (postal Locator 2202Dl). There is a staff of 37 research scientists, biologists and technicians and it is part of the Bureau of Chemical Safety, Food Directorate, Health Protection Branch.

• General Toxicology Section
• Reproduction and Teratogenicity
• lmmunotoxicity
• Genotoxicity and Carcinogenesis Section
• Carcinogenicity
• Mycotoxins and Biochemical Toxicology
• Cytogenetics
• Molecular Toxicology
• Mutagenicity
• Biochemical Toxicology and Pharmacokinetics
• Pathology Section
• Toxicologic Pathology
• Molecular Pathology

Chief, Toxicology Research Division

Rekha Mehta, Ph.D.
Tel: (613) 957-0987
Rekha_Mehta@hc-sc.gc.ca

Research Interests:

Toxicology and mechanism of action relating to : cyclic halogenated hydrocarbon food contaminants inluding PCB, Dioxins, Furans; natural toxins including shellfish toxins (domoic acid and okadaic acid); mycotoxins such as vomitoxin ; antioxidants, including synthetic phenols such as BHA, antioxidant mechanism of action, biologically based risk assessment, carcinogenicity.

General Toxicology Section

The section undertakes the design, conduct and evaluation of toxicological studies with food additives, food and environmental contaminants, and pesticides which find their way into human food sources. Such studies include a multi-disciplinary team of researchers who employee a variety of laboratory animal species in an attempt to find toxicological endpoints which are relevant to human health (i.e. reproduction, immunological effects, etc). Additionally, such studies must conform with the requirements of Good Laboratory Practices to ensure acceptance by international regulatory agencies. Expertise in the design and conduct of acute, sub-chronic and chronic-carcinogenicity studies using such chemicals as the food additive saccharin, the pesticide/environmental pollutant hexachlorobenzene, the mycotoxin commonly known as vomitoxin, the banned pesticide toxaphene as well as the environmental pollutant PCB (polychlorinated biphenyls).

Reproduction and Development

Reproductive function - with emphasis on male reproduction. Endocrine disruption - effects of exposure to organochlorine food contaminants in utero and during lactation on the development of the male reproductive system. Mechanism of action of food contaminants on the male reproductive system - especially with respect to disruption of androgen biosynthesis and metabolism and also with sperm function. Expertise includes Enzymology:- radiometric assays of steroidogenic enzymes in male and female reproductive tissues and in male and female liver; Animal handling; Tissue subcellular fractionation; Isotope handling; Incubations; Organic extraction; Chromatography; Scintillation spectrometry; Radioautography; Recrystallization; Kinetic analysis

Molecular Biology:- Determination of mechanism of action of food contaminants: RNA extraction. Gel chromatography; RT-PCR Cell Biology:- to examine the effects of food contaminants on androgen receptor activation and inactivation. Luminometry

Immunology/Immunotoxicology

Development, maintenance and implementation of the Immunotoxicology Program within the Food Directorate and the design of experimental protocols and execution of immunotoxicity studies to support regulation. The Immunotoxicology Program includes several internationally validated techniques. These are applied to the study of potential adverse effects of chemicals on the immune system of humans, non-human primates and rodents. The program provides a novel approach for the evaluation of chemical-induced immunotoxicity of food additives as well as environmental chemicals which inadvertetly enter the food chain. Examples of chemicals which have been investigated to date include: environmental contaminants such as PCBs, toxaphene and chlordane; mycotoxins such as deoxynivalenol and fumonisins; and metals such as lead and mercury. The Immunotoxicology Program includes techniques for the quantitative and functional assessment of effects of chemicals on specific humoral and cellular aspects of the immune system as well as on non-specific parameters of the immune system. The program also includes flow cytometry and tissue culture capabilities. Techniques routinely applied to research projects include: quantification of T lymphocytes and their subsets, B lymphocytes, natural killer cells, and the phagocytic mononuclear cells in peripheral blood-, spleen- or thymus- derived mononuclear cell preparations using flow cytometry; evaluation of humoral immunity by immunizing experimental animals with T- and B- dependent antigens and by determining antigen-specific antibodies by the plaque forming cell assay, ELISA, hemagglutination or complement-dependent lysis techniques; evaluation of cell-mediated immunity using the mitogen-induced lymphocyte proliferation technique (tritiated thymidine incorporation) and the one-way mixed lymphocyte culture technique; evaluation of the delayed-type hypersensitivity response using sensitization and challenge with dinitrochlorobenzene (non-human primates), and sensitization and challenge with oxazolone in rats and mice; evaluation of non-specific immunity using flow cytometric analysis of the phagocytic activity of monocytes (peripheral blood) and macrophages (peritoneal exudate cells); evaluation of calcium mobilization using flow cytometric techniques; and evaluation of resistance to bacteria using the Listeria monocytogenes infectivity model. The Immunotoxicology Program is constantly updated and new methods are adapted and/or developed to adequately serve the needs of the Program. Standardization and harmonization of immunotoxicology methods at the National and International levels are two ongoing processes within the Immunotoxicology Program.

Genotoxicity and Carcinogenesis Section

The development of cellular and molecular markers for the early detection of toxicological, genotoxic and carcinogenic effects of natural and synthetic food additives/contaminants, and the modulation of such adverse effects by nutrients. Using animal models, some of these biomarkers are currently being applied for cancer risk assessment of mycotoxins and pesticide residues at various organ sites such as the liver, kidney, mammary gland, esophagus and colon. The ultimate application of these and other appropriate biomarkers in molecular epidemiology studies in order to enhance human cancer risk assessment and primary prevention strategies for food contaminants. Methodology and techniques employed in the lab include: Subcellular and macromolecular (DNA, RNA, protein) tissue fractionation (liver, esophagus, kidney, stomach, colon, intestine); Hepatocyte isolation; Surgical procedures (hepatectomy, organ perfusion); HPLC ,Thin layer chromatography, Gas chromatography; isotope analysis, enzyme kinetics, fluorimetry spectrophotometry; serum enzymes Histochemistry - (gamma-glutamyl transpeptidase, Silver-staining nucleolar organiser regions)Immunohistochemistry -(glutathione S-transferase alpha, µ and pi,_Oncogene proteins, Cyclins, Proliferating cell nuclear antigen, Apoptosis) Flow cytometry -(cell cycle analysis Glutathione S-transferase alpha, µ and pi_,Oncogene proteins) Autoradiography -[³ H]- thymidine to determine cell proliferation; DNA adduct analysis - (Aflatoxin B₁ -DNA adducts, 8-hydroxydeoxyguanosine DNA methylation - (Restriction enzyme analysis of 5-methylcytosine.) Tissue levels of S-adenosyl-L-methionine, 5'-methylthioadenosine and S-adenosylhomocysteine ELISA - Oncogene proteins in serum

Natural Toxins (mycotoxins, shell-fish toxins)

Design and implementation of short and long term toxicology studies using rats or mice to investigate the toxicity and mechanism of action of natural toxins and food contaminants. expertise in protocol development in the following subdisciplines: immunotoxicology and immunoassays, hematology, clinical chemistry, tissue culture and in vitro toxicity assays. Methodology includes development and implementation of enzyme-linked immunosorbant assays (ELISAs) and standard enzyme assays for clinical endpoints measurement of platelet function and biochemistry; preparation and culture of lymphocytes for immunotoxicological endpoints; maintenance and manipulation of continuous cell lines for in vitro studies; design, validation and application of in vitro toxicity assays for a range of endpoints, including cell death, enzyme induction, dye uptake and membrane transport

Molecular Toxicology

Areas of expertise are molecular biology and protein chemistry. Areas of interest are molecular toxicology and molecular properties of antigens. Typically, projects involve the monitoring of transcription and protein expression of target genes in isolated tissues and organs as a result of a potential toxicological event or challenge. Some of the markers of interest are cytochrome P450 1A1, 1A2, 1B1, 2B1/2, 2E1, 2C11, 3A1, and 4A1; GSTa, GSTb, GSTp, PKC's, cyclin dependent kinases, cell cycle inhibitors p21 and p27; and cyclin D and cyclin E. The lab is also involved in protein isolation and characterization of antibodies and proteins of interest. Commonly used techniques are: DNA and RNA isolation; Cloning Of DNA and PCR fragments; PCR , RT-PCR and differential display PCR; DNA sequencing.; Northern blotting ;Southern blotting ; Western blotting ;Relative quantitation of target mRNA and proteins via densitometry of RT-PCR products or western blots; Agarose and acrylamide gel electrophoresis of DNA and RNA. 1D and 2D SDS-poly acrylamide gel electrophoresis of proteins; FPLC purification of proteins

Mutagenicity

A focus on in vitro approaches to studying the toxicology of food associated chemicals and examining the biological mechanisms involved. The laboratory has been primarily involved in assessing test chemicals for mutagenic activity and effects on DNA damage and repair mechanisms. Ongoing work includes the development of continuous cell line based methods to aid in minimizing the use of animal models. The activities of the laboratory have recently expanded to include a variety of in vitro assays for evaluating the neurotoxic and endocrine disruptor activities of test chemicals. Techniques include cell culture; Chinese hamster V79/HGPRT mutagenesis assays; Ames Salmonella mutagenesis assays; assays for DNA adduct formation, damage removal, strand breakage and DNA repair; replication; DNA and protein quantification; colony forming assays; colourimetric and fluorescent viability assays; radiolabelling uptake assays; molecular biology, construction of DNA vectors and gene expression techniques; DNA transfection and reporter gene assays; immunohistochemistry

Biochemical Toxicology, Pharmacokinetics

Expertise in the field of renal toxicology with an emphasis on the development and standardization of a program to evaluate renal function and renal damage following exposure to nephrotoxicants. In addition, the toxicokinetics of contaminants may also be evaluated. Interests also include the study of xenobiotic metabolism with emphasis on a family of metabolizing enzymes including the cytochrome P450's. Methodology and techniques include; Determinants of renal function and/or damage; Urinalysis parameters including measurement of enzymuria, proteinuria, urinary creatinine and osmolality; Organic ion transport measurements in renal cortical slices; Toxicokinetic model of renal clearance; Cytochrome P450 analysis including assays for specific isozymes (CYP1A1, CYP2B, CYP3A); Xenobiotic metabolism, toxicology of chemical mixtures.

Pathology Section

Evaluation of tissue and cellular injury induced by chemical compounds, natural and/or man made, environmental pollutants, which enter the food chain or utilized as therapeutic products. Pathology encompasses the laboratories of histopathology, neurotoxicologic pathology, haematology and molecular pathology. A diversified approach is used to identify the target sites, the nature and extent of tissue/cellular damage induced by potentially toxic compounds, their safety margins and potential impact on human health. The techniques used, are among others: immunohistochemistry, morphometry, transmission electron microscopy and molecular biology methodologies. Current research projects include: sea food toxins, mycotoxins, dioxins, excitatory amino acids, effects of prenatal exposure to polyhalogenated aromatic hydrocarbons on brain development, markers of neural injury and degeneration, glutamate receptors, application of novel methodology to toxicologic pathology, circumventricular organs/ autonomic nervous system and the cardiac conducting system as target sites for xenobiotic toxicity and cardiotoxicity.

Molecular Pathology

Molecular Toxicology with focus on the heart and brain as vital target organs for the effect/action of xenobiotics. Application of molecular biology methodologies to toxicologic pathology and safety assessment of chemical compounds. Glutamate receptors in brain and peripheral tissues i.e. heart, kidney, and testes, as potential mediators of excitotoxicity. These receptors are differentially localized to specific cellular types suggesting that they have a pivotal role in the homeotasis of the organism. Neurotoxicity of environmental/food toxins using molecular markers for neural injury. Glutamate, GABA and dopamine receptors in food safety and toxicity. Developmental neurotoxicology.

Expertise: bacterial genetics, plasmids, DNA sequencing, construction of genomics banks and species specific probes for the PGPRs ( plant growth promoting rhizobacteria), Southerns, extraction of total and mRNA, RT-PCR, semi-quantitative PCR, Westerns, Northerns, differentially display of mRNA using RT-PCR, cell culture, immunochemistry, and in situ hybridization.

Toxicologic Pathology

General Pathology for most animal species, (including Zoo Animal Diseases, Wildlife- Fish-Diseases.) Comparative Pathology via collaboration with the Medical School of Ottawa University; the Ottawa Heart Institute research on development of ventricular assist devices (artificial Heart Program); research in conjunction with the University is carried out in the field of Drug Side Effects, and Drug Toxicity with emphasis on pathology related to longterm treatment / exposure. Consulting to the regulatory groups in the Food Directorate, and in the Therapeutic Products Program as it pertains to preclinical, and clinical studies.

Current research involves morphometry, histochemistry, scanning- and transmission electronmicroscopy in a variety of species of laboratory animals including primates.

Seafood Toxins : Pathologic effects of short, and longterm exposure, with special emphasis on the study of receptors and target sites in the nervous- and circulatory system of rats and monkeys. Light-, and electronmicroscopic studies of peroxisome proliferation, and phospholipidosis produced by various classes of chemicals.