Toxicology.pdf

VETS2007 Introductory Toxicology Prof Rachel Allavena (contributions to slide material from Drs Ian Wilkie and Ross McKenzie) Course structure (2 lectures) 1. What is toxicology 2. Why are we studying toxicology 3. Defining toxicity 4. The ‘relativeness’ of the property of toxicity 5. Poisons and...

VETS2007 Introductory Toxicology Prof Rachel Allavena (contributions to slide material from Drs Ian Wilkie and Ross McKenzie) Course structure (2 lectures) 1. What is toxicology 2. Why are we studying toxicology 3. Defining toxicity 4. The ‘relativeness’ of the property of toxicity 5. Poisons and the body (ADME- in brief) 6. The pathophysiological mechanisms of poisoning 7. Tolerance, resistance, chronic toxicity & threshold dose 8. Basic principles of diagnosis & treatment Learning objectives Define toxicology specific terminology Explain the concepts/ definitions of relativeness of toxicity, hazard, risk, resistance and tolerance, biotransformation, bioactivation and induction Apply understanding of resistance, tolerance and species differences in toxin metabolism to clinical scenarios Explain the basic concepts of the pathophysiology of toxins, especially a cellular level Apply the basic principles of diagnosis and treatment of toxicity to clinical scenarios Instant feedback Male Rats get bladder cancer from saccharin artificial sweetener because:A.They drink lots of artificially sweetened cappuccinos and caffeine is a promoter B. Rats are smaller than humans, and are therefore affected at smaller doses of saccharin C. Rats have a high urine pH, protein and calcium phosphate levels that interact with the saccharin causing microcrystals which promote bladder cancer D.Humans develop a tolerance to saccharin because of increased levels of alcohol dehydrogenase induced by ethyl ethanol consumption Instant feedback Western grey kangaroos can eat Gastrolobium spp containing fluoroacetate and survive, Eastern grey kangaroos die. This is an example of:A. Tolerance B. Resistance C. Induction D. Exogenous biotransformation What is toxicology- definitions Toxicology: – the study of the adverse effects of xenobiotics (xeno = stranger/ foreigner; bios = life) Definitions Xenobiotic- chemical found in an organism that is not expected to be present in it. Examples include toxins, drugs, pollutants. Xenobiotics may not necessarily be harmful! Poison – a xenobiotic substance that after being contacted or entering the body by inhalation, ingestion, dermal contact or injection in sufficient quantity will cause a detrimental effect in an organism. Toxins or toxicants strictly defined as a poison of biological origin. Often the terms toxin and poison are used interchangeably What is toxicology- definitions Venom- a xenobiotic of biological origin that is injected via bite, sting or sharp body part to exert detrimental effects Toxicity is a measure of the harmfulness of a xenobiotic. Materials may have low toxicity, mild toxicity, extreme toxicity. Toxicosis / Intoxication / Poisoning is the syndrome or condition of adverse health effects caused by exposure to a toxicant Toxicology Toxicon (Greek- poison) logos (scientific study) Toxicology deals with the identification & characterisation of poisons, their physical and chemical properties, fate in the body (absorption, distribution, metabolism, excretion), their biological effects and treatment (Gupta 2007) The effects of ionising radiation and pharmaceuticals at toxic doses are also considered within toxicology There are many sources of xenobiotics 1. Accidental exposures to household chemicals, plants, food stuffs, pharmaceuticals 2. Accidental or deliberate misuse of therapeutic or illegal drugs 3. Native & introduced plants in environment 1. Grazing animals- $100 million/ year in economic loss; many pasture species have toxic potential 2. Garden plants like Oleander & Brunfelsia 4. Byproducts of industry and society 1. Chemicals & other materials improperly disposed of, e.g. old lead-acid batteries, or chemicals leaching from old dump sites 2. Accidental spills and deliberate releases 1. Humans, domestic species and wildlife affected 5. Malicious poisoning (forensic toxicology) 1. less common now than a generation ago, but occasional cases still occur Minamata disease “Minamata disease was first discovered in Minamata city in Japan in 1956. It was caused by the release of methylmercury in the industrial wastewater from the Chisso Corporation's chemical factory, which continued from 1932 to 1968. This highly toxic chemical bioaccumulated in shellfish and fish in Minamata Bay and the Shiranui Sea, which when eaten by the local populace resulted in mercury poisoning. While cat, dog, pig, and human deaths continued over more than 30 years, the government and company did little to prevent the pollution.” (source Wikipedia) ‘Tomoko Uemura in her bath’ by W. Eugene Smith Minamata disease ‘During its investigations, the committee uncovered anecdotal evidence of the strange behaviour of cats and other wildlife in the areas surrounding patients' homes. From around 1950 onward, cats had been seen to have convulsions, go mad and die. Locals called it the "cat dancing disease" (猫踊り病 neko odori byō), owing to their erratic movement. Crows had fallen from the sky, seaweed no longer grew on the seabed and fish floated dead on the surface of the sea.’ (source Wikipedia) Why do vets study toxicology As vets we need to recognise, diagnose and treat poisonings 1. Animal welfare- prevention (client education) and relief of suffering (treatment) in poisoned animals 2. Economics- prevention or reduction in economic losses especially in livestock (plant poisonings), residues in meat (Endosulfan residues) 3. Ecosystem health- environmental pollutants (DDT), disturbance of ecological balance 4. Implications in human health- toxicosis in domestic animals or wildlife may have public health implications (methyl mercury), safety of chemicals (paraquat) Can you think of other examples, especially veterinary? DDT and raptors DDT or Dichloro-diphenyl-trichloroethane widely used insecticide from 1945. Highly successful eradication of malaria in North America and Europe with DDT vector control It is very soluble in fats and most organic solvents and practically insoluble in water. (source world of molecules website) Bioconcentrate up the food chain due to fat solubility Associated with thin shelled eggs and infertility in raptors- major declines in raptor populations Feminisation of alligators in Florida Rachel Carson’s 1962 book “The Silent Spring” leads to public outcry and the banning of DDT in many jurisdictions DDT scientific evidence now in dispute about whether DDT really to blame. Toxicity is a relative property "All things are poison and nothing is without poison, solely the dose determines that a thing is not a poison." OR the dose makes the poison Auroleus Phillipus Theostratus Bombastus Von Hohenheim 1493-1541 (referred to himself as ‘Paracelcus’- wonder why?) Ethyl alcohol: Small dose – fun; Large dose – potentially fatal Toxicity is a relative property  Even substances vital for life can act as poisons at extreme doses  Oxygen  Water: hypotonic blood> osmotic redistribution causes cell swelling> fatal cerebral oedema  Human fatalities “Wee for a Wii contest” https://www.nbcchicago.com/n ews/local/indiana-mom-diesof-water-toxicity-afterdrinking-too-much-water-onvacation-family-says/3203387/  LD50 90g/kg in rats  Table salt (NaCl) fatal cerebral edema with excess salt or normal salt levels with inadequate water  pigs on salty rations or livestock reintroduced to water after a period of deprivation  Zinc, selenium, iron, fluoride, copper- are all essential for adequate function of biological enzymes and processes but are poisonous in excess Wee for a Wii On January 12, 2007, Jennifer Strange, a 28-year-old woman and a mother of 3, from Rancho Cordova California, was found dead in her home by her mother, hours after trying to win one of Nintendo's Wii game consoles. KDND 107.9 "The End" radio station's "Hold Your Wee for a Wii" contest, involved drinking large quantities of water without urinating. A nurse called the radio station to warn them about the danger in which they were putting people, but the disc jockeys were less than impressed. Lucy Davidson, the winner of the contest, was severely sickened while picking up her prize. Civil charges against the radio station were filed by Jennifer's family,[20] and the family was eventually awarded $16.5 million in the ensuing wrongful death lawsuit.[21] The FCC launched its own investigation to determine if the station violated the terms of its operating license. (Wikipedia) Defining toxicity: Is everything poisonous? Potentially, yes, but practically, no First, we should distinguish between toxicity and hazard To be hazardous, material must be toxic AND it must: 1. Be likely to be encountered & in a form that can be assimilated 2. Be likely to be eaten, inhaled, or come in contact with the integument in sufficient amounts to cause harm 3. Reach a site within the body where it can do harm which often requires uptake & transport 4. Interact with crucial metabolic pathways and these may vary from species to species Defining toxicity: Is everything poisonous? THUS Materials which are: 1. Unlikely to be encountered by animals in any form, 2. Exist in a form which cannot be assimilated, 3. Need large volumes, are highly unpalatable, or are emetic (cause vomiting) at sub lethal doses, Are not likely to cause poisoning even though they may be highly toxic These materials can be considered non-hazardous, or of low hazard... Hazard= the potential of an agent under defined conditions of exposure to cause an adverse effect Thus hazard is a Risk: a situation that increases the probability of injury or loss. Toxic vs Hazardous: an example Table salt (NaCl) is essential to life- sodium and chloride are major blood electrolytes, roles in osmotic balance, biological membrane function etc. ~1 gram/Kg bodyweight NaCl lethal (land) animals Amounts sufficient to cause acute toxicity not only difficult to consume, but highly emetic Animal vomits or drinks adequate fresh water So NaCl is toxic, but not hazardous under normal circumstances The toxicity of NaCl is constant, but some conditions make it significantly more hazardous: Drinking seawater if no fresh water available. For most land mammals, this is a very dangerous situation, because they cannot excrete excess Na+ without exacerbating dehydration. Normal or excess salt in feed (pigs) with prolonged water deprivation and then free access to water Toxic vs Hazardous: an example Acute to sub-acute salt intoxication Most common: housed pigs deprived of H2O for 8-12 hours Pathogenesis: Pig rations high in salt (palatability). Dehydration causes haemoconcentration> in the brain astrocytes gradually adapt to the high osmolality of blood> cytoplasmic osmotic pressure increases When water restored: pig drinks copious quantities, blood osmolality falls sharply Astrocyte membrane pumps can’t cope with difference between internal & external osmotic pressure so cell gains water, → cerebral oedema → brain swelling in an inflexible skull compresses blood vessels> ischaemia of cortex> death or clinical signs of brain injury Acute swelling of the brain may be severe enough to force the caudal cerebellum out of the foramen magnum (called cerebellar coning) If the pig survives for 2-3 days, there will be laminar necrosis of the cortex, and large numbers of eosinophils clustered around adjacent blood vessels Images: Noah’s arkive Defining toxicity: Is everything poisonous? We don’t label everything in the world as a poison. A working definition of ‘toxicity’ is therefore required. When consideration is given to: Route of exposure, That the material has to get to active site, Dosage being crucial Thus: A poison is any substance, which, when assimilated into an animal’s body by any route, in sufficient amounts, has a deleterious effect on one or more physiological functions. How toxic is toxic? We also need quantitative ranking of toxicity, to compare & help us assign risk to the myriad of potential poisons in our world New xenobiotics continuously enter our world - among others: • Therapeutic/ Pharmaceutical agents • Food additives • Household & industrial chemicals (e.g. PFAS) OLD measure was the lethal dose 50 or LD50 (now considered unacceptable from animal welfare standpoint but you still see many poisons reported as LD50) Example: How toxic is toxic? Monensin Toxicity of most substances varies for individuals can vary by very large amounts between species A lethal dose of Monensin (a coccidiostat & growth promotant) is: Horse: 2mg/Kg Cow: 26mg/Kg Chicken: 145mg/Kg Low LD50 = more toxic! Horses are >70x more susceptible to monensin than chickens, and >10x than cattle Accidental poisonings of horses from eating ruminant feed If you look at ruminant feed pellets they will have warnings not to feed to horses Horses die of acute cardiac failure, myocardial necrosis Defining toxicity: Ranking There are several different systems for ranking toxicity, each of which has slightly different figures, but they are all similar. European Commission specifies substances as: • Very toxic: - if ingested, aspirated or injected at doses of 25 mg/kg. • Approx. one tenth of a teaspoonful of this type of material would kill an average person Many toxins (botulinum toxin) are much more potent than this! Toxic: substances which kill at doses of 25-200mg / Kg. Approx. 0.5 to 5 teaspoonfuls Harmful: materials which are toxic in doses of between 500 and 1500mg/Kg. table salt! 1000mg/kg Check out https://www.visualcapitalist.com/visually-ranking-biotoxins-in-nature/ Paraquat (source Wikipedia) N,N′-dimethyl-4,4′-bipyridinium dichloride, one of the most widely used herbicides in the world Highly toxic animals and people- uncoupled redox cycling of the molecule generates free radicals damaging lung membranes. A single ‘swig’ immediately spat out is fatal 18 people have died from paraquat in Australia since 2000 It is a suicide method of choice in the developing world because of ready availability Ingestion causes death from pulmonary failure via ARDS or pulmonary fibrosis if victim survives acute phase Chronic low level exposure is linked to Parkinson’s disease Paraquat Toxic ranking varies with route of exposure In acute toxicity studies using laboratory animals, paraquat has been shown to be highly toxic by the inhalation route and has been placed in Toxicity Category I (the highest of four levels) for acute inhalation effects. However, the EPA has determined that particles used in agricultural practices (400 to 800 μm) are well beyond the respirable range and therefore inhalation toxicity is not a toxicological endpoint of concern. Paraquat is toxic (Category II) by the oral route and moderately toxic (Category III) by the dermal route. Paraquat will cause moderate to severe eye irritation and minimal dermal irritation, and has been placed in Toxicity Categories II and IV (slightly toxic) respectively for these effects.US EPA Other considerations 1. Unknown toxicities New chemicals - not enough data/experience to know if any effects of chronic exposure, especially carcinogenesis. These compounds may be suspect on basis of chemical structure Have not been fully tested in at-risk species 2. A substance may have low acute toxicity for adults, but could be teratogenic (cross placenta & affect foetus) Vitamin A, thalidomide 3. Persistent (fat soluble) compounds – may have very low acute toxicity, because encountered in very small doses, but can accumulate in fat stores. If acute nutritional stress organs may be exposed to high doses when fat mobilized INTERACTION OF THE BODY AND THE XENOBIOTIC Important concepts: Active sites, disposition, biotransformation, bioactivation, induction. Poisons and active sites To cause adverse effects a toxicant: 1. Must be able to reach the effector site in concentrations and amounts sufficient to cause significant damage Completely insoluble (in fat or water), or non-volatile compounds usually have little or no toxicity Exception: radioactive substances 2. The ‘victim’ must have cell structures or biochemical/ metabolic pathways that will be adversely affected by the compound or its metabolites. These vary from species to species. 3. Poisons reach the active site by: Ingestion, injection, skin exposure or inhalation Then via body fluids, or direct diffusion to reach targets within cells Toxin disposition Dose response is mediated through toxin disposition Disposition = absorption (A), distribution (D), metabolism/biotransformation (M) and excretion (E) mechanisms acting on the poison Toxicokinetics- kinetic processes of ADME at toxic doses/ preclinical safety testing dose response curves many shapes Pharmacokinetics- kinetics of ADME at pharmacologically relevant doses- dose response curve is usually linear Toxicodynamics – the effects elicited by the xenobiotic and active metabolites at systemic exposures used in toxicity testing This is what we focus on in 2007 and VETS3050: What does the poison do to the animal’s body? Disposition- metabolism Many xenobiotics are potentially toxic, so animals have evolved mechanisms to neutralise & excrete them Most common alteration of the xenobiotic is to convert it from a lipophilic to a hydrophilic form, suitable for excretion This process called biotransformation i.e metabolism Liver responsible for most biotransformation, but also significant activity in lungs & kidneys (renal tubules): major injured target organs! Site of biotransformation varies with species and xenobiotic Biotransformation is a 2 phase process Phase I. Degradative reaction involving oxidation, reduction or hydrolysis: Occurs in SER, requires enzymes called the Cytochrome P450 (CYPs)/Mixed Function Oxidases (MFO) Metabolites may be more or less toxic than parent compound. However for toxic compounds phase I metabolites are often highly reactive molecules. Phase II. Synthetic reaction involving conjugation with a hydrophilic molecule ready for excretion, e.g. via bile, urine, or mucus enzymes in cytosol, e.g. glucuronyl transferase A small number of compounds have low, or no toxicity as ingested, but after metabolic alteration (usually in phase I) become toxic = bioactivation Phase I reactions Cytochrome p450 (CYP) Most body tissues but liver has high concentrations of CYPs with promiscuous specificity A single xenobiotic may be metabolised by multiple CYPs each with different kinetics Some reactions produce high energy state molecules facilitating Phase II conjugation Some products may be highly toxic unstable molecules Other non-CYP phase I enzymes exist eg alcohol dehydrogenase, epoxide hydrase et al Phase II reactions Products of phase II reactions are more water soluble, more easily excreted, less toxic than parents or phase I metabolites. Phase II enzymes include glucuronosyl and glutathione transferases Glutathione (GSH) conjugates electrophiles Scavenges free radicals Depletion of GSH stores to <20% increases susceptibility to free radical damage> DNA, lipid membranes, proteins> toxicity Excreted products out in urine or bile then faeces. Principal function of biotransformation is to reduce toxicity by altering compound & preparing for elimination However, bioactivation is opposite effect: e.g. Crofton Weed Ageratina adenophora If eaten by horses, causes severe respiratory dysfunction because some component, possibly 9-oxo-10,11 dehydroagerophorone (DHA), which is not cytotoxic in its native state, is bioactivated in horse lung tissue. DHA is toxic to mice, but causes liver injury, because it is bioactivated in liver in that species, (a good demonstration of how species may react very differently to drugs & chemicals). IE bioactivation with species specific differences Early Crofton-weed poisoning, horse lung: Multiple firm nodules throughout lung tissue Histology of above: Nodules are due to foci of fibrosis & metaplastic change of alveolar epithelium (right) caused by metabolites of a compound in the weed. Toxicity is a complex process Rumen microflora are often involved in destroying or modifying toxins L-tryptophan in lush pasture converted to 3-methylindole but rumen bacteria> Blood to lung> metabolised by Clara cell CYP metabolism = free radicals> interstitial damage to lung> ARDS Young animals don’t have the rumen microflora- resistant Alteration in rumen microflora can modulate toxicity in adult cattle Learn more: https://www.fas.scot/article/fog-fever/ Lecture 2 Have a break Induction CYPs have capacity for up-regulation if increased amount of substrate encountered = induction Organisms adapt and become resistant to xenobiotic load Once demand falls off, the CYP that has increased drops back to ‘normal’ levels SER hypertrophy and glassy hypertrophied cytoplasm can be seen with EM or light microscopy of affected liver cells Exposure to the same or chemically similar compound may cause induction in a single CYP e.g. valium and alcohol! Alcohol and testosterone! Induction is usually a good thing – means liver can safely handle more of the material causing it. However….. Induction may lower toxic threshold Induction may be detrimental if a xenobiotic is so rapidly metabolised that it produces an excess of toxic metabolite in higher local concentrations. Sometimes CYP induction to one compound impacts the toxicity of an incidental/ unrelated compound. Carbon tetrachloride CCl4 was used as a flukicide for sheep (Unknown) compounds in pasture plants = CYP induction> The regular therapeutic dose of CCl4 became 100 times more toxic! Induction continued Induction increasing the metabolism of hormones or therapeutic agents may have detrimental effects e.g. Chronic intake of alcohol causes induction of Alc. dehydrogenase & the Microsomal Ethanol Oxidising System (a CYP/MFO) These enzymes ^ testosterone metabolism in males = feminising effect + ^ metabolism of barbiturates & diazepam = ^ dose required for sedation in alcoholics Summary: Induction usually decreases potential toxicity of target compounds, but may increase toxicity, depending on the metabolic product(s) of the induced enzyme(s) What are the pathophysiological mechanisms of toxicity? Limited answers – don’t know the molecular mode of action of many toxins. Further examples will be discussed in VETS3050/ VETS3017 Don’t even know what the toxic compound is in some cases! If function of vital cells is significantly compromised, or large number of cells affected, severe clinical effects or death of animal occurs. CSx and lesions manifest based on the ‘target organ(s)’ Pathophysiological mechanisms of toxicity? Toxins function at a cellular level, i.e. the compound interferes with any or all of: 1. Energy supply 1. e.g. Fluoroacetate blocks Kreb’s citric acid cycle 2. Membrane integrity or function 1. Free radical lipid peroxidation of biological membranes What organelles does this affect? e.g. Paraquat- uncoupled redox cycling produces free radicals 3. Inhibition of an enzyme/ metabolic pathway- bind a co-factor/ competition for substrate/ irreversibly binding active site etc 1. Antimetabolites: successfully replace normal enzyme substrate. E.g. anticoagulant rodenticide competitive inhibition vitamin K epoxide reductase affects clotting factors 2,7,9,10. 4. DNA damage – cell death, mitoses prevented, cancer Pathophysiology: DNA Toxins which affect DNA may cause subacute to chronic intoxication in two ways: 1. Compounds or their metabolites react with DNA = epigenetic effects or mutations = death or dysfunction of the cell Eg Pyrrolizidine alkaloids (plant-derived compounds) metabolised in liver> metabolites form adducts with nucleic acids> kill cells sub-acutely, prevent mitoses or may initiate neoplasia 2. May initiate and/or promote the development of neoplasia. Toxins which initiate neoplasia are called carcinogens Because exposure & neoplasia maybe separated by many years, identification of carcinogens is often very difficult Why particular clinical signs associated with a toxin? • Effect of toxin usually depends on which organ most affected i.e. the most vulnerable metabolic pathways • Characteristics of cells which make them uniquely susceptible (muscle & ionophores; brain and heart- energy and electrical sensitivities; high levels of CYP in liver & renal cells) • Some toxins affect all organs, but clinical signs due to dysfunction of most sensitive organ e.g. cyanide blocks Kreb’s citric acid cycle but most energy depended organs are brain and heart. Foxgloves and digitaliscardiac glycosides affect heart Monensins affect ion channels in muscle Why are particular clinical signs associated with a given toxin? Summary Organ vulnerability can be explained by:- 1. First port of call (intestinal mucosa, liver) 2. Site of concentration (kidney & oxalates) 3. Site of bioactivation (liver, lung, kidney) Unique organ susceptibilities Example: Cardiovascular System: Electrical conducting system + sensitive to anoxia Muscle contractility (Digitalis ↑ Ca++ flux, small amount increases strength of contraction, too much lethal) Ca++ ion channels – disrupted by ionophores (monensin, etc.) What organ? Toxin may have primary effect in one organ, but clinical signs are due to a secondary effects in another organ. Or there are species differences. e.g. hepatic encephalopathy This liver is from a dog which ate horse meat contaminated with a toxin from a plant (Indigofera sp.) which the horse ate. Horses show CNS signs before signs of general liver failure, but dogs show severe liver failure before CNS signs. Learn more at: https://agriculture.vic.gov.au/biosecurity/animaldiseases/important-animal-diseases/indospicine-toxicity-in-dogs Paracetamol and cats Cats lack the glucuronyl transferase enzyme pathways to metabolise paracetamol/ acetaminophen and excrete it Metabolism is shunted to oxidation pathways Phase I metabolism by CYP2E1 bioactivates the paracetamol to a highly reactive quinone imine> GSH depletion> Free radical damage to cell membranes Toxicity effects the liver and blood Cats die from methaemoglobinaemia and Heinz body formation in their red blood cells> haemolytic anaemia (blood) + liver necrosis (centrolobular) Asphyxiate with decreased oxygen carrying capacity Cat: Paracetamol toxicosis Cats present with: Methaemoglobinaemia with Heinz body formation  Subcutaneous oedema  Cyanosis  Haemoglobinuria In addition to acute hepatotoxicity Normal Methaemoglobinaemia Heinz bodies in erythrocytes Haemoglobinuria Resistance & tolerance Some animals have an unusual capacity to withstand normally toxic doses of certain xenobiotics This property is the result of either: Resistance an innate property, developed by selection over many generations, & possessed by all members of the species. OR Tolerance which is an acquired trait of an individual Resistance Resistance to some toxins acquired through natural selection over long periods of time. i.e. Species level. Survival advantage for utilization of food sources that would otherwise be unavailable. Potential mechanisms include: 1. Enhanced hepatic excretion (endogenous biotransformation) 2. Modification of compounds by gut microflora (exogenous biotransformation) 3. Modification or circumvention of biochemical pathways that are vulnerable to the toxin Resistance Koalas survive on a diet consisting entirely of Eucalyptus leaves containing large amounts of toxic essential oils These compounds are toxic to most other animals, but through enhanced hepatic (endogenous) biotransformation, and the action of gut flora transforming some of the oils into non-toxic metabolites (exogenous biotransformation), they manage quite well… Resistance Gastrolobium spp. contain high fluoracetate in West. Aust. - local marsupials can utilize this high protein food source because they have evolved high fluoracetate-specific defluorinase activity Western Grey kangaroos are resistant; Eastern Grey’s will die Resistance Humans Tolerance and tolerance mechanisms Tolerance is an individual’s acquired or ‘conditioned’ resistance to a normally toxic dose of a xenobiotic Potential mechanisms for tolerance: 1. Ingestion of sublethal amounts altering absorption, storage, or elimination rate Example: Arsenic & strychnine ‘tonics’ (archaic therapeutic agents) if given over long periods induce tolerance to lethal doses Possibly induction of metallothioneins, sulphydryl-rich proteins involved in storage of heavy metals ^ metallothion production enables sequestration of normally lethal Arsenic dose Tolerance mechanisms Up-regulation (induction) of metabolic pathways (or alternative pathways) This happens with many therapeutic agents eg. barbiturates & benzodiazepines Therapeutic, or even smaller doses of some compounds up-regulate certain hepatic CYPs with broad substrate specificity Many drugs need increased doses for same effect if administered for a long time to achieve the correct therapeutic effect. This can also be seen with illegal drugs like prescription opiods and heroin. Princess bride Chronic verse Acute toxicity Many toxins have acute onset & clinical course – exposure causes clinical effects &/or death within minutes to hours Chronic toxicity usually means long exposure period -. NB onset of clinical signs is often acute e.g. Copper poisoning of sheep However, onset & clinical course may both be chronic Chronic intoxications very hazardous because we may not know animal has been exposed until too late e.g. pyrrolizidine alkaloid poisoning of grazing livestock or copper toxicity in sheep. Chronic/ cumulative toxicants Exposure to small amounts of a toxin over long time before clinical effects. Gradual or sudden onset of clinical signs Occurs with some heavy metal intoxications such as Fe, Cu, which have high storage capacity Example: Cu poisoning of sheep: Sheep very sensitive to Cu levels Accumulate excess in livers – if reach >600ppm (dry weight)> stressor> sudden release from hepatocytes> free radicals damage RBCs> massive haemolysis Right: kidney from sheep with haemoglobin nephrosis due to Cu-induced haemolysis = diffuse dark red colour Mechanisms of chronic intoxication 1. Functional reserve of organs – until significant amount of damage, or high demand on organ, no clinical effect observed eg liver lose >70% Pyrrolizidine alkaloids 2. Intake vs. utilization rate 1. e.g. Vit. D poisoning- adults acutely poisoned, young animals tolerate higher doses longer, because they are using large amounts to grow bones 3. Elimination rate exceeded by uptake; ‘more in than out’. 1. Some compounds metabolised & excreted, but very slowly, exposure above elimination level causes signs of intoxication e.g. copper in sheep. 4. Uptake rates enhanced &/or Storage capacity exceeded 0 Threshold dose Effect→ Threshold dose Why flat here? Dose→ For most toxins, typical shape of dose vs. effect curve is sigmoid – effects start at some minimum dose determined by the toxicity of the compound The more toxic the substance, the closer the threshold dose to zero The dose below the threshold is called the no observable effect level (NOEL) This is sometimes modified to NOAEL ‘no observable adverse effect level’ in drug toxicity testing Contrasts morphologic or physiologic change with damage to the animal Threshold doses As long as the substance does not accumulate, sub-threshold doses may be given repeatedly, with no effect. Cumulative poisons = small amounts accumulate until they reach the threshold at which signs begin to appear (one mechanism of chronic intoxication) Calculating a NOEL for carcinogens is much more difficult than for other types of toxins. Carcinogenic doses vary widely with individual multi-step nature of carcinogenesis (see neoplasia lectures) effects of other xenobiotics or hormones (promoters) may be required Threshold values for carcinogens are therefore very hard to determine accurately, so decisions on allowable exposure tend to be very conservative. Diagnosis of toxicities General principles – VETS3050/VETS3017 and medicine courses have details Diagnosis is very challenging! Peracute conditions - sometimes no lesions, no clinical signs observed Residues of toxins may be small, & some toxins rapidly metabolised - difficult or impossible to detect Toxin assays are very expensive! Poor availability. Minimum screen is $1000-1500. Single assay $700+ Diagnosis of toxicities Analysis of food/vomitus/ stomach content/ urine – not always available, takes time Also organs of excretion and metabolism (liver and kidneys) Interpretation of analytical results often difficult – presence of toxin alone not enough – was amount sufficient to cause intoxication?? Must know species & their susceptibilities (e.g. Cu –toxic level in sheep tissues is way below clinically significant level for cattle) Diagnosis of toxicities “Negative” analysis - what does it mean? Wrong compound asked for? Toxin is labile & decomposed or metabolised to other compounds? (protein toxins) Residues present, but below detection limits of assay? (OPs) Not a toxicosis at all? Antidotes or binders produce a false negative. Treatment of toxicities Often required immediately – animal in extremis 1. Most treatments - ‘symptomatic’ or palliative e.g. Atropine used to prevent muscarinic effects of Carbamate & Organophosphate insecticides – is not an antidote, & does not reverse effects of toxin. Oximes are a true antidote! Barbiturates control spasms of strychnine poisoning, make respiration easier, but do not neutralise or eliminate toxin 2. Reduce exposure! removing any remaining toxin from GIT or skin (emetics, washing) N.B. – emetics are inadvisable for volatile compounds as danger of inhalation Binders- activated charcoal (pool or aquarium charcoal and bentonite clay) 3. Supportive care whatever appropriate to situation • Therapies to help cardiac function or respiration; • Anticonvulsants; • Analgesics Adsorbants and purgatives Rationale for use is to minimize or prevent uptake from GIT & remove from GIT as quickly as possible. 1. Activated charcoal relatively inexpensive for small animals Adsorbs many (polar) toxic compounds = prevents uptake from gut. Fatty material reduces effectiveness Very safe & easy to use (but messy!) 2. Bentonite. Finely powdered clay Cheaper alternative to activated charcoal, generally not as effective, but said to be better at adsorbing mycotoxins. Pool charcoal may be more cost effective. Treatment of toxicities- antidotes In veterinary medicine few true antidotes. Some examples: Anti-venoms for snake bite, tick envenomation- antibodies neutralize the protein venom Vitamin K injections to counter anticoagulant rodenticide poisoning -provides excess of normal substrate, out-competing rodenticide Ethyl alcohol used to treat dogs & cats which drink ethylene glycol (antifreeze). Both compounds metabolized by alcohol dehydrogenase but ethyl alcohol out-competes ethylene glycol, producing less toxic metabolites QLD News Vodka saves cat Tipsy’s life Tom Snowdon, The Courier-Mail July 17, 2017 10:40pm Instant feedback Male Rats get bladder cancer from saccharin artificial sweetener because:A. They drink lots of artificially sweetened cappuccinos and caffeine is a promoter B. Rats are smaller than humans, and are therefore affected at smaller doses of saccharin C. Rats have a high urine pH, protein and calcium phosphate levels that interact with the saccharin causing microcrystals which promote bladder cancer D. Humans develop a tolerance to saccharin because of increased levels of alcohol dehydrogenase induced by ethyl ethanol consumption Instant feedback Male Rats get bladder cancer from saccharin artificial sweetener because:A. They drink lots of artificially sweetened cappuccinos and caffeine is a promoter B. Rats are smaller than humans, and are therefore affected at smaller doses of saccharin C. Rats have a high urine pH, protein and calcium phosphate levels that interact with the saccharin causing microcrystals which promote bladder cancer D. Humans develop a tolerance to saccharin because of increased levels of alcohol dehydrogenase induced by ethyl ethanol consumption Instant feedback Western grey kangaroos can eat Gastrolobium spp containing fluoroacetate and survive. This is an example of:A. Tolerance B. Resistance C. Induction D. Exogenous biotransformation 1. McKenzie RA. 2002 Toxicology for Australian Veterinarians 1st ed CD-ROM. 1. Copies available in the library http://library.uq.edu.au/record=b2091214~S7 2. Library Zen-works platform References Also available on all of the library workstations Go to the ‘Zenworks Application Portal’ window (minimised on bottom taskbar)> Under ‘Specialised Applications’, click on the 'T' group> Double click on the 'Toxicology for Australian Veterinarians' icon. Note – This programme can only run for one user at a time. 3. 2. 3. Order form on blackboard. Students can purchase from Ross for $40 + postage. Otherwise $120 after you graduate. Highly recommended! McKenzie RA 2012 Australia’s poisonous plants, fungi and Cyanobacteria: A guide to species of medical and veterinary importance (CSIRO publishing). 1. Available in the library http://library.uq.edu.au/record=b3087956~S7 2. $195/ copy- could look at bulk order for faculty and students. Dowling RM, McKenzie RA 1993 Poisonous plants a field guide. DPI 1. Great field guide but out of print. 4. Gupta, R.C. Ed 2007 Veterinary toxicology: basic clinical principals. Elsevier AP. SF757.5 V5864 5. Haschek WM, Rousseaux CG, Wallig MA 2010 Fundamentals of Toxicologic Pathology 2nd edition. Academic Press 6. New South Wales Department of Primary Industries Veterinary laboratory manual http://www.dpi.nsw.gov.au/agriculture/vetmanual 1. Guidance on available tests and sample submission VETS2007 toxicology review questions These questions are a guide for your study and reflection on the learning objectives of the course. The answers can be found in provided lecture notes and suggested reference materials. Note Exam will be in MCQ format: see past papers! What is the role of the veterinarian in dealing with intoxications and why do we study toxicology? Define xenobiotic, poison, toxin, venom, disposition, toxicokinetics, toxicodynamics, biotransformation, bioactivation, induction, resistance, tolerance, NOEL, NOAEL, Why is toxicity a ‘relative’ property? What is the difference between toxic and hazardous? How do we quantitate or rank toxicity? What chemical and biological factors affect toxicity and why? Explain the role of biotransformation in both normal metabolism and toxicity situations, what are the mechanisms involved? VETS2007 toxicology review questions Why is diagnosing toxicity and identifying a toxic principal a major challenge? What are the basic pathophysiological mechanisms of toxicity? What unique organ susceptibilities make major organs vulnerable to toxic insults? What is resistance, and what mechanisms confer resistance to a toxicant? What is tolerance, and what mechanisms confer tolerance to a toxicant? What factors affect whether a toxicant will work in an acute or chronic fashion? What are the basic principals of toxicity treatment and diagnosis? Why are toxicities such a challenge for vets?