1. Basics of general toxicology Lecture No. 2 Copyright © Prof. MVDr. Zdeňka Svobodová, DrSc., Mgr. Zuzana Široká, PhD.

2. Definition of toxin: Toxin is any substance that even in small quantities or low concentrations, after a single administration or repeated exposure, cause severe damage to, or the death of, the organism To quote Professor Švagr, "any substance qualitatively or quantitatively foreign to the organism that causes chemical or physical damage to that organism should be classified as a toxin" Factors affecting toxicity: Playe's principle of CCC (DCC) can be applied: - Concentration (Doses) -Complexation -Competition

3. Concentration: The dose (concentration) issue has been mentioned above (it is generally true that the higher the dose, the stronger the effects) Some toxins also exhibit a phenomenon called hormesis. Such substances have the opposite effect in small doses than in large doses: - We most frequently come across the so-called radiation hormesis – small dose of radiation has protective effects - Methylene blue used as a reduction agent in cases of NO 2 - poisoning causes methaemoglobinemia at higher concentrations

4. Complexation: A general principle of toxicology says that what is soluble is toxic Eg. LC 50 of CuSO 4.5 H 2 O for carp is 10 mg.l -1 in pond water or 1 mg.l -1 in ground water the explanation of the difference is that pond water contains much more organic substances, which form with copper less soluble and absorbable organocopper complexes, therefore less toxic Complexation characteristics are used in treating metal poisoning cases. A proven home remedy is milk with whisked egg whites. The proteins and the metal salts form insoluble complexes and these cannot be absorbed The so-called chelate-forming substances administered in the case of metal poisoning also prevent the absorption of metals because of formation of insoluble complexes

5. They include: - ethylenediaminetetraacetic acid (EDTA) - mainly in lead poisoning - compounds containing SH groups, e.g. dimercaprol (BAL) – no longer used in clinical practice today - SH ligands – macromolecular sorbents – used to block the absorption or to interrupt the enterohepatic circulation of metals - D – penicilamin – used for chelation of practically all metals EDTA Penicilamin

6. Competition: Substances compete for the receptor, enzyme or site of absorption The competition between Cl - and NO 2 - on fish gills may serve as an example. Both Cl - and NO 2 - are absorbed to the body through eosinophilic cells, the so-called chloride cells in the gills. Chloride ions always have primacy. It means that in water with abundance of chloride ions, the chloride cells are all occupied by chlorides, which block the absorption of NO 2 - to the bodies of fish and negative effects related with it Another example is the competition between methyl alcohol and ethyl alcohol for the alcohol dehydrogenase enzyme, whose affinity to the ethyl alcohol is 100 times higher. This characteristic is taken advantage of in the treatment of methyl alcohol and ethylene glycol poisonings Other examples are competition between toxic metals (Cd, Pb, Hg) and Zn and Cu for SH groups contained in amino acids or competition of anticoagulation rodenticides and vitamin K in formation of active clotting factors

7. Synergism and antagonism Synergism Interaction of 2 or more agents so that their effects are combined There are two types of synergism, the additive and the potentiation synergism Additive synergism means that effects of two types of agents are added up. An example is the interaction between organophosphates and carbamates. The mechanism of action of the two agents is the same – they inhibit the acetylcholinesterase enzyme, which results in an accumulation of acetylcholine at nerve synapses In the case of potentiation synergism, effects of the interacting agents are multiplied. Pyrethroids and piperonylbutoxid are an example of potentiation synergism. Piperonylbutoxid markedly potentiates the effect of pyrethroids, and in practise pyrethroids are used as insecticides potentiated by piperonylbutoxid

8. Antagonism interaction of two or more agents that in combination have an overall effect which is less than the sum of their individual effects We distinguish between two types of antagonism, i.e. the chemical and the functional type - Chemical antagonism is exemplified by neutralization occurring when an acid combines with a base, or a combination of cation-active and anion-active tensides in water. The same principle of chemical antagonism is also used in chelate treatment of metal poisoning

9. - Functional antagonism - the principle is used for symptomatic treatment in poisoning cases - barbiturates with strongly anticonvulsive effects are administered to victims of strychnine poisoning (strychnine has convulsive effects). Methyl alcohol poisoning is treated by administration of ethyl alcohol. The alcohol dehydrogenase enzyme in the liver "prioritizes" ethyl alcohol oxidation, and the conversion rate of methyl alcohol to formaldehyde and formic acid is thus slowed down. A similarly antagonistic relation exists between ethylene glycol and ethyl alcohol

10. Toxicokinetics – the fate of toxin in the organism Includes absorption, transport and distribution biotransformation and excretion Routes of absorption: - gastrointestinal tract - respiratory apparatus - skin and external mucous membranes - parenterally (s. c., i. m. or i. v. injections), or an injection of snake venom or some other zootoxin

11. Transport and distribution: - Poisons are transported in the blood, where they are either free or bound - The free form is mostly dissolved in the aqueous component of blood plasma - The bound form is fixed to some other blood component, mainly blood plasma proteins (albumin – binds most toxic substances; transferrin and ceruloplasmin – bind metals) and to cellular blood elements and their components (e.g. erythrocytes, haemoglobin) - With regard to distribution, the greatest concentrations of the majority of toxic agents are in the liver and kidneys (e.g. toxic metals bind there to polypeptide metallothionein) - Many toxic substances show specific affinity to specific organs – we speak about tropism of poisons (e.g. hepatotropic poisons – CCl 4, aflatoxin B 1, microcystin, etc.; nephrotropic – ochratoxin, ethylene glycol; neurotropic – organophosphates, carbamates, pyrethroids; hematotropic – CO, NO 2 - )

12. Biotransformation: - Some chemical substances are eliminated from the organism in an unchanged form (e.g. organic compounds of arsenic – arsenobetaine, arsenocholine – pass through the mammalian digestive tract without any changes) - A large majority of toxic substances are altered by biotransformation, while their toxicity is either activated or reduced (detoxification)  One type of biotransformation takes place in the pre-absorption phase: - phosphane (PH 3 ) is released from zinc phosphide (Zn 3 P 2 ) by stomach HCl - HCN is released from cyanogenic glycosides by hydrolytic enzymes activated by stomach HCl - NO 3 - is reduced to NO 2 - by the gastrointestinal tract microflora - NH 3 is released from urea by urease in the rumen - decomposition of digitalised glycosides in the proventriculi of ruminant (reduction of toxicity)

13.  But, the main organ of biotranformation is the liver! Most of organic biotoxicants are biotransformed in two stages: stage 1stage 2 XH X – OH X – O – conjugate The XH xenobiotic is transformed by oxidation (or hydrolytic or reduction) enzymes to a more polar metabolite X – OH, which becomes a substrate for conjugating enzymes

14. stage 1 – monooxygenation (by cytochrome P450) XH → X – OH more polar, but hazardous and potentially mutagenic and carcinogenic substances may be produced stage 2 – conjugation by conjugating enzymes, e.g. glutathion-S- transferase – GST or glucuronyl transferase bind glutathion or glucuronic acid to X – OH and the resulting conjugate is excreted from the body in bile or urine C P450 GST e.g. benzo(a)pyren epoxydiol l-hydroxypyrene - In the process of metal detoxication, metals are being bound to SH groups of metallothioneins. In the case of metal contamination of an organism, its liver increases the production of methallothioneins. Metallothioneins are polypeptides, every third amino acid of is cystein, and they therefore contains a large quantity of SH groups

15. Routes of excretion: - kidney (urine) - liver (bile, and then by faeces) - gastrointestinal tract (faeces) - lungs - mammary gland - salivary glands - sweat, lacrimal and sebaceous glands and their products