Concepts and Terminology

1. Concepts and Terminology Toxicology Is the study of poisons, including their chemical properties and biological effects. Toxicant an alternative term for poison. Toxin: A poison that originates from biological processes also called a biotoxin. Examples; Mycotoxins (fungal toxins) and zootoxins (animal toxins). Many plants are also known to be toxic when consumed by specific types of animals. Toxicity:The quantity or amount of a poison that causes a toxic effect. Toxicosis:A disease state that results from exposure to a poison.

2. Toxicology versus pharmacology Pharmacology is the study of chemicals (drugs) used at doses to achieve therapeutic (beneficial) effects on an organism. Toxicology is the study of chemicals (toxicants) that produce a harmful (detrimental) effect on an organism. Dose: The amount of toxicant that is received per animal. Dosage: The amount of toxicant per unit of animal mass or weight. It can also be expressed as the amount of toxicant per unit of mass or weight per unit of time. For examples, a dog could receive a dosage of chemical at the rate of 2 mg/kg/day. When conducting traditional acute, subacute, subchronic, or chronic studies, the length and frequency of exposure are also noted. For examples, rats may receive a chemical dosage of 2.5 mg/kg/day for 2 years.

3. Route of exposure: The most common routes of exposure are inhalation, oral, and dermal, with some variations for each. Less frequently used routes of exposure include rectal, sublingual, subcutaneous, and intramuscular. Threshold dose: The highest dose of a toxicant at which toxic effects are not observed. Lethal dose (LD) or median lethal dose (MLD) (LD50): The dose that will kill 50% of a group of animals during some period of observation in acute toxicity study. Lethal concentration (LC) or minimal toxic dose: is the lowest concentration of a chemical or drug in a matrix (usually feed or water) that causes death. Effective dose (ED).The dose of drug or toxicant or therapeutic agent that produces some desired effect in 50% of a population.

4. Therapeutic index (TI). Defined by the equation : TI = LD50 ED50 the TI is a unitless estimate that characterizes the relative safety of a drug or chemical. The larger the TI, the more “safe” a chemical is relative to another with a smaller TI. For example, if chemical X has an LD50 of 1000 mg/kg and an ED50 of 10 mg/kg, the TI would be 100 (the mg/kg units cancel). Compare this to chemical Y, which has an LD50 of 50 mg/kg and an ED50 of 40 mg/kg. The TI of chemical Y is 1.25, a much less safe chemical when compared with chemical X.

5. Standard safety margin (SSM) or margin of safety (MoS). Defined by the equation: LD1 SSM = ED99 the SSM, like the TI, is a unitless estimate that characterizes the relative safety of a drug or chemical, but much more conservative data are used. The larger the SSM, the more safe the chemical tends to be relative to other chemicals with smaller SSMs.

6. Exposure duration. The length of time an animal is exposed to a drug or chemical. In general, there are four subgroups: - Acute: Exposure to a single or multiple doses during a 24-hour period. The LD50 is often determined during acute exposure studies. - Subacute: Exposure to multiple doses of a toxicant for greater than 24 hours but for as long as 30 days. - Subchronic: Exposure lasting from 1 to 3 months. - Chronic: Exposure for 3 months or longer.

7. Hazard (risk): a chemical or drug will cause harm under certain conditions. Toxic effect : damage effect to certain biological system or process caused by poison or drug in high dose. Side effect : secondary predicted undesired effect that accompanied the therapeutic effect. Adverse effect: unpredicted undesired effect caused by drug used at recommended dose ex. Allergy of penicillin.

8. The dose-response relationship • The result of exposure to the dose is any measurable quantifiable, or observable indicator. The response depends on the quantity and route of chemical exposure or administration within a given period. Two types of dose–response relationships exist, depending on the numbers of subjects and doses tested. Graded Dose–Response The graded dose–response describes the relationship of an individual test subject or system to increasing and/or continuous doses of a chemical.

9. Graded dose–response curve for caffeine HCl • chloramphenicolHCl(●) atropine sulfate , and phenol

10. Quantal Dose–Response The quantal dose–response is determined by the distribution of responses to increasing doses in animals of test subjects or systems. This relationship is generally classified as an “all-or-none effect” in which the test system or organisms are quantified as either responders or non-responders. A typical quantal dose–response curve is illustrated in Figure by the LD50 (lethal dose 50%) distribution.

11. Quantal dose–response curve showing experimental derivation and graphic estimation of LD50

12. Three general assumptions must be considered when evaluating the dose-response relationship: 1. The chemical interacts with a molecular or receptor site to produce a response. 2. The production of the response, or the degree of response, is correlated to the concentration of the chemical at that receptor site. 3. The concentration of the chemical at the receptor site is related to the dose of chemical received.

13. Toxicokinetics Toxicant Exposure Entrance to Body Ingestion Skin Inhalation Absorption into Blood Stream and Distribution to Body Tissues and Organs Toxicity Storage Excretion Metabolism

14. xenobiotic(foreign compound): they are substance which not enter any biological process or used as a source of energy or nutrition such as heavy mental Absorption Defines how much of a chemical passes into the body over a period of time. Different routes of exposure produce different absorption patterns, which can vary both within a species (intraspecies variation) and between different species (interspecies variation). For a xenobiotic to exert a toxic effect, it must reach its site of actions. It must reach to the body by crossing any number of body membranes (e.g., skin, lung, gastrointestinal tract, and red blood cell membranes). Composition of these membranes varies, resulting in various levels of resistance to penetration. For example, the skin more resistance to penetration than the lung alveolar surface.

15. Absorption can be described in terms of bioavailability (F), which is the quantity or percentage portion of the total chemical that is absorbed and available to be processed (DME) by the animal. In the case of intravenous administration, F = 100% because all of the xenobiotic enters the animal. Inhalation, oral, and dermal are the three usual routes of exposure to xenobiotics. Inhalation (pulmonary) Inhalation exposure to chemicals occurs when the chemical is dissolved in the ambient air inhaled by the animal. The chemical first reaches the nasal passages, when some absorption can take place before it enters the trachea, bronchi, and finally the alveoli, the chemical can cross the very thin alveolar wall and enter the blood

16. Oral (gastrointestinal) Chemicals can enter the gastrointestinal tract in either contaminated food or water sources. Depending on the physicochemical properties of the chemical. For example, some chemicals are unstable in the stomach’s acidic environment and can be destroyed to varying degrees, resulting in decreased absorption. On the other chemicals are readily absorbed from the stomach and enter the small intestine, absorption through the intestinal mucosa and then into the blood. Portal circulation delivers them to the liver, a major metabolic organ of the body. Dermal (percutaneous) Three key events must occur for percutaneous absorption to take place. • First, the chemical must be soluble in the vehicle (solvent) that is applied to the skin. • Second, it must be able to penetrate the thick keratin layer of the epidermis. • Third, it must make its way through the lower cells of the epidermis and into a blood vessel.

17. Xenobiotics can pass through body membranes by either passive transport or active transport. 1. Passive transport. Passive transport requires no energy expenditure on the body’s part to transport the xenobiotic across a cell membrane. Passive transport occurs via two mechanisms: simple diffusion and filtration. a. Simple diffusion. Simple diffusion depends on both the lipid solubility and the size of the molecule. In biological matrices, most xenobiotics exist in a solution as either an ionized or un-ionized form. Un-ionized (uncharged) molecules have greater lipid solubility than the ionized forms. Xenobiotic will penetrate a body membrane, three facts must be known: (1) whether the xenobiotic is a weak acid or a weak base (2) the pH of the biological matrix (3) the association constant of the xenobiotic (or pKa, the pH at which 50% of the xenobiotic is ionized and 50% is un-ionized).

18. Once this information is known, the Henderson-Hasselbalch equation for either a weak acid or a weak base can be applied. For a weak acid: [un-ionized] pKa – pH = log [ionized] For a weak base: [ionized] pKa – pH = log [un-ionized] The higher the ratio of un-ionized:ionized, the greater the potential for absorption across a lipid membrane.

19. b. Filtration. Filtration relies on the potency of a membrane. When water flows in bulk across a porous membrane, any solute that is small enough to pass through the pores flows with it. 2. Active transport. Active transport mechanisms usually require an energy expenditure on the body’s part to transport the xenobiotic across a cell membrane. a. Active transport active transport moves xenobiotics against concentration gradients. b. Facilitated transportexpends energy on xenobiotic transport, but it does not occur against a concentration gradient. c. Pinocytosisis another type of active transport mechanism that involves the ability of cells to engulf small masses of xenobiotic and carry it through the cell membrane.

20. Distribution Once absorbed across one of the body’s barriers, the chemical enters the blood so that it can be distributed to the body’s organs and tissues. The chemical leaves the blood and enters the tissues at varying rates, depending on a number of factors: (1) rate of blood flow (generally, the higher the blood flow, the more potential distribution to the organ) (2) the ability of the chemical to traverse the capillary endothelial wall (3) the physiochemical properties of the chemical, such as lipid solubility. The extent of distribution within an animal can be described by the volume of distribution (Vd), which is a proportion between the amount of a chemical found in the blood to the total amount of drug in the body at any given time. The equation is

21. Vd = AC(t) / CB AC(t) is the total amount of the chemical in the body at time CB is the concentration of the chemical in the blood . Examining the Vd equation, the higher the numerator or the lower the denominator, the higher the Vd. Restated, the higher the total chemical in the body or the lower the concentration in the blood, the higher the Vd. Therefore, the higher the Vd, the higher the distribution from the blood to the tissues.

22. Metabolism(biotransformation) Metabolism of chemicals varies, ranging from simple hydrolysis, to glutathione conjugation, to no metabolism at all. The liver possesses the most metabolism capacity, regardless of species, other organs such as the kidneys, gastrointestinal tract, skin, heart, and brain also have considerable metabolic capabilities. The metabolism of a xenobiotic usually occurs in several steps. As stated earlier, a key component of metabolism is to convert the xenobiotic into a water-soluble form. so it can be excreted from the body. metabolic conversion can be categorized into two steps or phases. Phase I metabolism converts a polar, lipophilicxenobiotics into more polar and more hydrophilic metabolites via liberation of functional groups that can be used during phase II. Phase I metabolism uses a wide assortment of reactions that processes the xenobiotic via hydrolysis, oxidation, or reduction pathways.

23. Phase II conjugates either the xenobiotic itself or its metabolite formed during phase I metabolism with a functional group that results in a multifold increase in water solubility.   Excretion usually occurs via the kidney (urine), gastrointestinal tract (feces), or lungs (exhalation of volatile chemicals); however, other excretory mechanisms do exist (e.g., tears, sweat, skin exfoliation). Not all xenobiotics are completely absorbed, particularly via oralexposure. If absorption is less than 100%, the xenobiotic can continue down the gastrointestinal tract and either be metabolized by gut microbes or be passed unmetabolized out of the body via feces, some xenobiotics may be metabolized, excreted via the bile.

24. Other non renal routes of excretion include milk, cerebrospinal fluid, sweat, and saliva. Determining the sum of clearance pathways can be defined by the following equation: CLB = CLR + CLNR CLB is the total amount of xenobiotic and its metabolites CLR is the amount cleared via the urine CLNR is the sum of all non renal pathways. Treatment Antidotes Antidotes are therapeutic agents that have a specific action against the activity or effect of a toxicant. Antidotes can be broadly classified as chemical or pharmacologic antidotes.

25. Chemical antidotes specifically interact with or neutralize toxicants. For example, metal chelators such as calcium disodium edetate (CaNa2EDTA) or succimer combine with metals to form soluble metal-chelator complexes that are subsequently eliminated via the kidneys. Pharmacologic antidotes neutralize or antagonize toxicant effects. Such antidotes can prevent formation of toxic metabolites (fomepizole), compete with or block the action of a toxicant at a receptor site (naloxone), or help restore normal function (N-acetylcysteine). The use of some antidotes in food animals can result in food safety concerns. Because of these concerns, extended withdrawal times have been established for ammonium salts for treatment of copper intoxicated sheep (30 days) and for methylene blue for treatment of nitrate/nitrite intoxication in ruminants (180 days).

26. Calcitonin Calcitoninis widely recommended for the treatment of cholecalciferol (vitamin D)-induced hypercalcemia. The recommended protocol is to administer 4 to 6 IU every 6 hours for up to 3 weeks, the side effects such as anorexia, anaphylaxis, and emesis. Pamidronate It is efficacious for the treatment of hypercalcemia associated with several human diseases and in dogs, needs to be administered less frequently than calcitonin. Fomepizole Fomepizolehas replaced ethanol as the antidote of choice for treating ethylene glycol–intoxicated dogs. It is a better inhibitor of alcohol dehydrogenase than ethanol, and its without side effects. Don’t used in cats because of the less effective inhibition of alcohol dehydrogenase in cats.

27. Succimer Succimer is the chelator of choice for the treatment of lead intoxicated small companion animals. N-Acetylcysteine(NAC) It is antidotal for acetaminophen intoxication in humans, which are not readily available for guiding veterinary therapy. the first dose of NAC should be administered within 8 hours of exposure. It can be administered either orally or intravenously. Oral administration has not been associated with adverse effects in humans, whereas intravenous administration has resulted in urticaria, anaphylactoid reactions, and, rarely, death.

28. Enhanced elimination Various methods of increasing the elimination of absorbed toxicants have been advocated. Active charcoal AC, may provide a gastrointestinal “dialysis” effect. In studies using dogs, multiple doses of AC given orally enhanced the elimination of IV administered theophylline. It was hypothesized that theophylline, passing from the circulation into the lumen of the gastrointestinal tract, was adsorbed, thus preventing its reabsorption. Forced diuresis Forced diuresis via the IV administration of sodium-containing solutions is often recommended to hasten the elimination of many toxicants via the kidneys.

29. Ion-trapping Facilitating the removal of absorbed toxicants via the urine by ion-trapping may be indicated in several specific situations. For example, alkalinization of the urine to a pH of 7 or greater with sodium bicarbonate has been shown to enhance the urinary elimination of weak acids such as ethylene glycol, salicylates and Phenobarbital. The administration of ammonium chloride to acidify the urine (pH of 5.5 to 6.5) may enhance the elimination of weak bases such as amphetamine and strychnine Peritoneal dialysis Peritoneal dialysis has also been advocated to enhance the elimination of water-soluble, low-molecular-weight, lithium, salicylate, and theophylline. Other methods for hastening elimination of an absorbed toxicant, such as charcoal hemoperfusion and hemodialysis, are less practical and less available in veterinary medicine.

30. Diagnostic Toxicology Diagnosis depends heavily on a systematic approach that includes sample collection and handling. Such cases requirepiecing together a “diagnostic puzzle” that includes acomplete case history, clinical signs, clinicopathologicfindings, postmortem findings, results from chemicalanalyses, and occasionally bioassay findings. History The primary goal of the history is to identify possible sources of a toxicant. Sources are found by examining the environment, reviewing management practices, and recording the movement and fate of animals, feed, water, and bedding. Recent medication history should be recorded, including information about the substances given, time of administration, amount used, reason for use, and individuals treating the animal.

31. Sample collection and storage Samples for toxicology testing fall into three general categories: environmental, antemortem, and postmortem. Toxicology Samples are often best collected and held until other testing can be completed because results from other tests (e.g., histology, bacteriology) can give information about the organ that is affected and perhaps the poison itself. All samples should be labeled regarding the date, case, source, description, and the clinician taking the sample. Fresh samples should be frozen, which should be kept cool and dry Environmental samples Many toxicants are not detectable in tissues. Thus, environmental samples (e.g., source materials, feeds) are critical to diagnosis in a poisoning case as well as being important for identifying and removing the source of a poison.

32. Feedsamples For example, mold toxins (e.g., aflatoxin in grain or nitrate from weeds) in a lot of hay occur in “hot spots,” or isolated portions of a lot or bag. Samples are individually labeled. Sample sizes vary, but the optimal size in most instances is about 500 g of material. Moist samples should be frozen. Feeds to be kept dry should be stored in a cool, dry environment in containers. Water samples Water samples are obtained at the source (well, canal, pond), in transit (piping, tankers), in storage tanks, and at the site of exposure. Some chemicals are toxic in water even when present in very low concentrations. Glass preserving jars are useful for water samples. Plastic and metal containers should be avoided. • - Metal and salt samples can be covered with plastic wrap and then the lid; • - Organic chemical samples should be covered with foil and then the lid.

33. Plant samples Many plant poisonings still rely on plant identification for diagnosis. Pastures should be examined, with plants being identified as the investigation proceeds. The amount of the weeds in the bale, stack, or lot should be estimated, and then the weeds can be sent to a laboratory for identification if needed. Diagnosis of plant poisoning also requires evidence of consumption of a plant by the animal. Antemortem samples The toxicology case involves sudden onset of disease or death in a number of animals. Other cases may involve onset of signs, or perhaps merely a decrease in production. The next step in investigating a toxicology case is to examine the affected animals. Clinical effects of toxicants vary, depending on the organs involved, route of exposure, species and host characteristics (e.g., age, past history, environment, use).

34. For example, some poisons may lead to acute signs in multiple animals in a short time after a single exposure. Conversely, toxicants that have a longer period from exposure to onset of signs have incidence rates spread over longer periods of time. For example, clinical pyrrolizidine alkaloid poisoning may not appear until after weeks of exposure. - Whole blood and serum samples are used for a variety of tests. Urine is tested for drugs and some plant toxins. - Testing of ingesta and feces is valuable to determine exposure to a variety of toxicants. The value of these matrices lies in the likelihood that toxicants may be present in high enough levels to be detected when compared with other body fluids and substances. For example, many compounds are diluted and metabolized after absorption. Thus, a compound is at its highest concentration in the rumen or stomach content.

35. - Biopsies are invasive and are done only when other diagnostic options are limited. Liver is the most common tissue that is biopsied for toxicology analysis. • Histology may help identify organ specificity of disease Postmortem samples A toxicology case frequently depends on the postmortem examination for a diagnosis. Although practitioners are trained to perform a necropsy to identify disease and traumatic conditions. The diagnostic laboratories usually have board certified pathologists with specialized training in accepted procedures for investigating this type of case. If the case is accepted, a complete necropsy should be done, not a partial, “keyhole” necropsy. - Photographs of the animal and findings may be helpful in assessing the case. - The urine should be sampled using a syringe. The urine is put into a plastic or glass container; it is not left in the syringe.

36. - Samples of tissue should be fixed in formalin for histology testing. • Some toxicants cause nonspecific lesions. For example, hemorrhagic gastroenteritis may result from a variety of toxicants such as arsenic or even salt poisoning • Analytical toxicology • Many poisons can be identified in environmental samples or tissues. Toxicology test methods range from simple visualization (e.g., identification of plant parts) to modern analytical chemistry methods. • - Analyses for metals are done using spectroscopy. Spectroscopy is rapid and accurate, allowing for analysis of lead in liquids (e.g., blood) within a few hours. • - Tissue analysis requires additional time for digestion of the sample to free the metals before analysis. • - Analyses for organic compounds such as some plant poisons, drugs, or pesticides are done using chromatography.

37. - Chromatography is separation of compounds based on characteristic chemical properties in liquid (high performance liquid chromatography), solid (thin layer chromatography), or gas (gas chromatography).