Analytical Toxicology & Toxicological Tests

General Toxicology: Analytical Toxicology and Toxicological Tests

• Presentation for teaching purposes only.

Analytical Toxicology

• Involves using chemistry and chemical properties to identify (qualitative) and/or quantify (quantitative) xenobiotics in a sample.

Sample Preparation

• Sample collection and preparation is crucial for accurate lab results.
• Factors influencing collection: Case animal, distinguishing between alive or dead animals for ante-mortem or post-mortem exams.
• Factors influencing collection: Purpose; Clinical, forensic (crime solving), or environmental research to quantify xenobiotics.
• Collect samples without contamination using clean, disinfected materials.
• Use resistant and inert containers like plastic or glass to avoid sample contamination.
• Refrigerate (4°C) or freeze samples (ideally -20°C) until analysis, especially if lab analysis is delayed.
• Avoid using ethanol, formalin or other preservative liquids.
• Identify samples correctly with animal ID, date, and place.

Ante-Mortem Sample Collection

• Whole Blood: 5-10 ml, use EDTA, heparin
• Serum: 5-10 ml
• Urine: 25-50 ml, stored in a plastic screw-capped tube
• GI contents: As much as possible (100g or more) of each sample. Representative samples are stomach or rumen contents and feces
• Hair: 1-2g
• CSF: 1-2ml
• Milk: 30ml

Post-Mortem Sample Collection

• Whole blood/serum/urine: Collect as for ante-mortem samples.
• Liver: approximately 100-250g. Use plastic containers.
• Kidney: approximately 100-250g. Use plastic containers.
• Brain: Take one-half brain, sagittal cut.
• Fat: approximately 100g, wrap in foil inside plastic
• GI contents: Collect as much as possible (100g or more) of each sample. Include representative samples of stomach/rumen contents, intestinal contents and feces, package separately.
• Ocular fluid or whole eyeball: Collect as much as possible, or the whole eyeball, aqueous preferred
• Bone: Take 100g or more.
• Spleen: Collect 100g, treat as for liver samples.
• Lung: Collect 100g, treat as for liver samples.
• Hair/skin: Collect as for ante-mortem samples. If dermal exposure is suspected

Environmental Sample collection

• Bait/Source: Collect 200 ml or g. Use a clean jar, plastic container, or whirl-pak.
• Feed: Collect 500 g or more. Take composite of representative samples for each lot or batch.
• Plants: Collect entire plant or representative portion, including root, fresh, pressed/dry, or frozen.
• Water: Collect 1 liter or more, in a clean jar with a plastic lid for metals.

Analytical Toxicology: Limits of Detection

• Limit of Detection (LOD): The lowest amount of a substance identifiable by a technique or distinguished from zero, using both qualitative and quantitative methods.
• Limit of Quantification (LOQ): The lowest amount of a substance quantifiable by a technique, using quantitative methods.

Thin-Layer Chromatography (TLC)

• Stationary Phase: A thin layer of silica gel spread on a glass surface.
• Mobile Phase: A solvent that moves up the plate.
• Process: A spot of a mixture of substances is applied on a TLC plate. Substances migrate based on interactions with the stationary phase, separating into single compounds. The pattern is revealed using natural light, colorimetric substances, or fluorescence.
• Retention Factor (Rf): Indicates how far a compound travels relative to the solvent front. Rf= distance travelled by the component / distance travelled by the solvent.
• Determining compounds: The compound with a larger Rf value is less polar. Less polar compounds do not stick to the stationary phase as easily as compounds with a lower Rf value.
• Example: TLC can determine the substances in a dog's urine.

Gas Chromatography (GC)

• An analytical separation technique used to analyze volatile substances in the gas phase.
• Sample Prep: Dissolve the sample in a solvent, vaporize, and inject into a column containing the stationary phase.
• Mobile Phase: A chemically inert gas carries the molecules of the analyte through the heated column.
• Measurement: Retention time (Rt), the time it takes to move a certain distance, is measured.

High-Performance Liquid Chromatography (HPLC)

• An analytical technique used to separate compounds soluble in a liquid solvent (mobile phase).
• Stationary Phase: is inside a small steel column
• Measurement uses Retention time (Rt) instead of Rf.

Complementary Techniques

• TLC, GC, and HPLC separate substances from a mixture, and they are called separation techniques
• GC, LC and HPLC techniques are not always enough for substance identification.
• To precisely identify and quantify substances in complex mixtures a molecular and atomic level analysis is needed

Mass Spectrometry (MS)

• Measures the mass of molecules in a substance.
• Ionizer: Converts the substance into smaller, positively charged molecules.
• Positively Charged Molecules under a Magnetic Field: The molecules deflect based on their mass.
• Detector: Measures the deflection.
• Works with isomers by identifying different patterns that originate from the different molecular conformations
• The goal is to identify the different patterns of fragments that originated. It is the weight of each fragment that created a separate peak pattern.
• The relative abundance pattern helps quantify and identify the complex mixture.
• Example: Can be used for distinguishing between isomers due to their distinct fragmentation patterns and mass-to-charge ratios.

Atomic Absorption Spectroscopy (AAS)

• A spectrometer atomizes a sample in a flame or small carbon tube inside a furnace.
• Determines how the sample interacts with light. Uses an atomization source, a lamp producing light at a wavelength that is specific to each element, a monochromator, and a detector.

Immunoassays

• A type of ELISA test is performed in a multi-well plate, with each well containing immobilized antibodies specific for an analyte.
• When a sample is introduced, present analyte becomes immobilized by binding to the antibody.
• A solution containing the enzyme's substrate is added.
• The reaction between the enzyme and substrate produces a signal, usually in the form of a color change.
• ELISA used for screening purposes using selective techniques to confirm

Measurement of Acetylcholinesterase Activity (AChE)

• This enzyme is reduced by exposure to substances such as carbamate and organophosphate pesticides.
• Labs test AChE level to check the exposure and poisoning
• The Ellman method is used involving adding a reagent to a blood or brain sample. This is enzyme kinetic assay in which acetylthiocholine reacts with a reagent producing a product with a yellow color. The rate of color change is measured with a spectrophotometer.
• Comparison with normal levels to determine poisoning.

Toxicological Tests- Main Goals

• General Toxicity: Evaluation of a substance's toxicity in acute, subacute, or chronic exposure.
• Specific Effects: Evaluate reproductive/carcinogenic effects of a substance.

Evaluating Toxicity

• Recent years have seen alternatives to animal testing emerging; however, animal testing is still crucial for assessing toxicity.
• 3R's Concept: Replace the use of animals with other types of studies and reduce the number of animals used.

Animal Testing

• Animal tests have historically predicted toxicity in humans.
• Test methods include controlling chemical exposure, controlling environmental conditions and the study of toxicity mechanisms
• Animal tests are in parallel with human testing but are not always able to be conducted

Animal Usage Factors

• The route of exposure, age of test animals, test animal sex, and dose level administered correlate as close as possible to humans

Acute Toxicity Tests

• Historically the first toxicity tests conducted.
• Acute toxicity tests can provide data on the relative toxicity that may arise from a single limited period of time.
• Standardized Tests are available for tests on the oral, inhalation, ,dermal exposures

Subchronic Toxicity Tests

• Determine toxicity likely to arise from repeated exposures over weeks to months.
• Conducted through standardized oral, inhalation and dermal exposures.

Chronic Toxicity Tests

• Determine toxicity from exposure for a substantial portion of a subject's life.
• Similar to Subchronic tests, but conducted over a longer period of time and involve larger groups of animals.

Carcinogenicity Tests

• Similar to Chronic Tests but extend over a longer period, and use larger groups of animals
• Provide an assessment of cancer potential

Reproductive Toxicity Testing

• Determine the effects of substances on gonadal function, conception, birth, and offspring development.
• Measured by defects caused by toxic agents in both males and females using species such as Rats and mice .

Developmental Toxicity Tests

• Used primarily to determine hazard regarding potential effects of prenatal exposure on the developing foetus.
• Most important developmental phase is the organogenesis period.
• Compounds categorized as teratogenic/fetotoxic by recording structural malformations, developmental retardation/mortality in rodent models.

Alternative methods of Toxicology Testing

• Teratogenic effects cause structural malformations.
• Fetotoxic effects lead to growth retardation, the death of a foetus

Cutaneous/Dermal Toxicity Tests

• Animals are monitored for skin reactions/dermal effects and findings may show clinical, or microscopic pathological evidence

Dermal Irritation and Sensitization

• Alibino rabbits provide high permiabilty and senitivity to toxic agents
• Immunoassay tests done on guinea pig and test substance

Ocular Toxicity Tests:

• Historical method test substance applied to eye of test subject and monitored over time, typically Albino Rabbit
• A reaction on cornea, conjunctiva, or iris, can occur with only simple reversible irritation

Toxicity Tests Using Cell Cultures for Modeling

• In-vitro testing system should be sensitive and yield low levels of false-neg/positives
• Measurements include objective endpoint reproducibility and control

Emerging Approaches and Methods: In Silco Methods

• Virtual Tissue Models are developed by the U.S. EPA"s National Center for Computational Toxicology (NCCT).
• Incorporate chemical simulation capable of estimating effects of chemical impact on human development and will help minimize reliance on animal trials.

Emerging Approaches and Methods: Chip Models

• Tissue Chip and Organs-on-chips test using Models include human cell cultures that are placed on a computer chip and studied there such as lungs on a chip

Emerging Approaches and Methods: Induced Pluripotent Stem Cells

• hiPSCs using in vitro cultures can create mammalian cells as cellular platforms in investigations studying disease pathophysiology
• The cells can function as cellular platforms that assist in target identification for drug treatments.

Measuring Oxidative Stress

• The imbalance of ROS production and antioxidant defence can damage lipids, proteins, and DNA, which may lead to cellular dysfunction. Certain chemicals, pollutants, and the drugs can all factor in oxidative stress resulting in the body's damaged defence mechanism.
• Lipid peroxidation from lipids, Protein oxidative modification and DNA Oxidation can all indicate oxidative damage.

Oxidative Stress Enzymes

• These enzymes help our cells leading with ROS
• These enzymes include Superoxide Dismutase, Catalase, Glutathione Peroxidase , Gluthatione Reductase , Peroxi redoxins

Toxicology testing (at point of Contact)

• Point of Contact testing can carried close to subjects in order to obtain immediate results, typically to detect for substance, therapeutic drug use or abuse

OECD

• Used in risk analysis, regulatory safety testing and subsequent chemical notification and registration, used in Toxicology

OECD

• OECD guidelines cover a wide range of toxicological tests that provide standardized protocols for conducting studies and data interpretation

Presentation on analytical toxicology, which uses chemistry to identify and quantify xenobiotics in samples. Proper sample collection and preparation are crucial, considering factors like animal status (alive/dead) and purpose (clinical, forensic, environmental). Samples should be collected without contamination, stored in inert containers, and refrigerated or frozen.