Objectives of this lecture are to: Explain the steps involved to exert a toxic effect.

1. General ToxicologyMechanisms of ToxicityLec. 34th Year2017-2018College of Pharmacy/University of MustansiriyahDepartment of Pharmacology & ToxicologyLecturer: Rua Abbas Al-Hamdy

2. Objectives of this lecture are to: • Explain the steps involved to exert a toxic effect. • Determine the steps involved in delivery of the toxic agent from the site of exposure to the target. • Explain absorption versus presystemic elimination. • Determine mechanisms of distribution to & away from the target. • Explain excretion versus reabsorption. • Explain toxication versus detoxication. • Explain how toxicants interact with endogenous target molecules & how some toxicants could alter the biological environment.

3. Mechanisms of toxicity: • The most complex path to toxicity involves certain steps (Fig. 1): • First step: the toxicant is delivered to its target or targets. • Second step: • a. interacting with endogenous target molecules • b. or altering the environment • Third step: triggering perturbations in cell function &/or structure, • Fourth step: repair mechanisms at the molecular, cellular, &/or tissue levels

4. When the perturbations induced by the toxicant exceed repair capacity or when repair becomes malfunctional , toxicity occurs. • Tissue necrosis, cancer, & fibrosis are examples of chemically induced toxicities that follow this four step course.

5. Figure 1. Potential stages in the development of toxicity after chemical exposure.

6. Step 1: Delivery from the site of exposure to the target: • Theoretically, the intensity of a toxic effect depends on: • the concentration & • persistence of the ultimate toxicant at its site of action.

7. Increased concentration is facilitated by: • absorption, • distribution to the site of action, • reabsorption, & • toxication • While: • presystemic elimination, • distribution away from the site of action, • excretion, • & detoxication will decrease the toxicant concentration at its target.

8. Absorption versus presystemic elimination: • Absorption: • Transfer of a chemical from the site of exposure into the systemic circulation is called absorption. • Transporters contribute to gastrointestinal (GI) absorption of some chemicals; however, the vast majority of toxicants traverse epithelial barriers via diffusion.

9. Factors that influence absorption include: • concentration, • surface area of exposure, • characteristics of the epithelial layer through which the toxicant is being absorbed, & • usually most important, lipid solubility because lipid soluble molecules are absorbed most easily into cells.

10. Presystemic elimination: • During transfer from the site of exposure to the systemic circulation, toxicants may be eliminated. • This is common for chemicals absorbed from the gastrointestinal (GI) tract because they must first pass: • through the GI mucosal cells, • into the liver (enterohepatic circulation), & • then lung (pulmonary circulation) before being distributed to the rest of the body (systemic circulation).

11. Presystemic or first pass elimination generally reduces the toxic effects of chemicals that reach their target sites by way of the systemic circulation. • However presystemic elimination may contribute to injury of the digestive mucosa, the liver, & the lungs because these processes necessitate toxicant delivery to those sites.

12. Mechanisms facilitating distribution to a target: • 1. Porosity of the capillary endothelium: • Endothelial cells in the hepatic sinusoids & in the renal peritubular capillaries have large fenestrae (50 to 150 nm in diameter) that permit passage of even proteinbound xenobiotics. • This relatively free filtration promotes the accumulation of chemicals in the liver & kidneys.

13. 2. Specialized transport across the plasma membrane: • Specialized ion channels & membrane transporters can contribute to the intracellular delivery of toxicants, making those cells targets. • Some examples are: • Na+,K+ ATPase, • voltage gated Ca2+ channels, • carrier mediated uptake, & • endocytosis.

14. 3. Accumulation in cell organelles: • Amphipathic xenobiotics with a protonatable amine group & lipophilic character accumulate in lysosomes as well as mitochondria. • Lysosomal accumulation occurs by pH trapping, while mitochondria accumulation takes place electrophoretically.

15. Mechanisms opposing distribution to a target: • 1. Binding to plasma proteins: • Hydrophobic xenobiotics generally bind proteins or lipoproteins in the plasma. • In order to leave the blood & enter cells, these xenobiotics must dissociate from these proteins. • Therefore, strong binding to plasma proteins delays xenobiotics movement across membranes & prolongs their effects & elimination.

16. 2. Specialized barriers: • Brain capillaries lack fenestrae & are joined by extremely tight junctions. • Thus preventing the access of hydrophilic chemicals to the brain except by active transport.

17. 3. Distribution to storage sites: • Some chemicals accumulate in tissues (i.e., storage sites) where they do not exert significant effects. • Such storage decreases toxicant availability for their target sites. • For example, highly lipophilic substances such as chlorinated hydrocarbon insecticides concentrate in adipocyte.

18. 4. Association with intracellular binding proteins: • Binding to nontarget intracellular sites, such as metalothionein, temporarily reduces the concentration of toxicants at the target site. • For example, binding to metalothionein serves such a function in acute cadmium intoxication

19. Excretion versus reabsorption: • Excretion: • Excretion is the removal of xenobiotics from blood & their return to the external environment. • Excretion is a physical mechanism, whereas biotransformation is a chemical mechanism for eliminating the toxicant. • The route & speed of excretion depend largely on the physicochemical properties of the toxicant.

20. The major excretory organs—the kidney & the liver—efficiently remove highly hydrophilic chemicals such as organic acids & bases. • Three rather inefficient processes are available for the elimination of nonvolatile , highly lipophilic chemicals: • excretion from the mammary gland in breast milk, • excretion in bile excretion in bile in association with biliary micelles and/or phospholipid vesicles;& • excretion into the intestinal lumen from blood.

21. Volatile, nonreactive toxicants such as gases & volatile liquids diffuse from pulmonary capillaries into the alveoli & are exhaled.

22. Reabsorption: • Toxicants in the blood are filtered at the glomerulus into the renal tubules. • These filtered toxicants may reenter the blood by diffusing through peritubular capillaries. • Reabsorption by diffusion is dependent on the lipid solubility of the chemical & inversely related to the extent of ionization, because the nonionized molecule is more lipid soluble.

23. Therefore, pH of the tubular fluid affects reabsorption such that acidification favors excretion of weak organic bases & alkalinization favors the elimination of weak organic acids.

24. Toxication versus detoxication: • Toxication: • A number of xenobiotics are directly toxic, whereas other xenobiotics exert a toxic effect through their metabolites. • The increased reactivity by biotransformation may be due to conversion into: • electrophiles, • free radicals, • nucleophiles, or • redoxactive reactants.

25. Detoxication: • Biotransformations that eliminate the ultimate toxicant or prevent its formation are called detoxications. • Examples are: • Detoxication of toxicants with no functional groups. • Detoxication of free radicals:

26. Detoxication of toxicants with no functional groups: • In general , chemicals without functional groups, such as benzene & toluene, are detoxicated in two phases: • Initially, a functional group such as hydroxyl or carboxyl is introduced into the molecule, most often by cytochrome P450 enzymes. • Next, an endogenous acid, such as glucuronic acid, sulfuric acid, or an amino acid, is added to the functional group by a transferase.

27. With some exceptions, the final products are inactive, highly hydrophilic organic acids that are readily excreted.

28. Detoxication of free radicals: • Detoxication & elimination of O2•− is important because it can be converted into much more reactive compounds such as: • the hydroxyl radical (HO•), • nitrogen dioxide (•NO2), & • the carbonate anion radical (CO3•−)

29. Superoxide dismutases (SODs), located in the cytosol & the mitochondria , convert O2•− to hydrogen peroxide (HOOH). • Subsequently, HOOH is reduced to water by cytosolic glutathione peroxidase or peroxisomal catalase.

30. Step 2: Reaction of the ultimate toxicant with the target molecule: • Practically all endogenous compounds are potential targets for toxicants. • The most prevalent & toxicologically relevant targets are nucleic acids (especially DNA), proteins, & membranes.

31. Types of reactions: • Noncovalent binding • Covalent binding • Electron transfer • Hydrogen abstraction • Enzymatic reactions

32. Noncovalent binding: • Hydrophobic interactions, hydrogen bonding, & ionic bonding are forms of noncovalent binding. • in noncovalent binding, a toxicant can interact with targets such as membrane receptors, intracellular receptors, ion channels, & certain enzymes. • For example, such interactions are responsible for the binding of strychnine to the glycine receptor on motor neurons in the spinal cord, & binding of warfarin to vitamin K 2,3-epoxide reductase.

33. Noncovalent binding usually is reversible because of the comparatively low bonding energy.

34. Covalent binding: • Being practically irreversible, covalent binding permanently alters endogenous molecules. • Covalent adduct formation is common with electrophilic toxicants such as nonionic & cationic electrophiles & radical cations. • These toxicants react with nucleophilic atoms that are abundant in biological macromolecules, such as proteins & nucleic acids.

35. Nucleophilic toxicants are, in principle, reactive toward electrophilic endogenous compounds. • However, such reactions are infrequent due to the rarity of electrophilic biomolecules. • Carbon monoxide, cyanide & hydrogen sulfide are examples of nucleophiles that form coordinate covalent bonds with iron in various heme proteins.

36. Electron transfer: • Electron transfer chemicals can exchange electrons to oxidize or reduce other molecules, leading to formation of harmful byproducts. • For example, chemicals can oxidize Fe(II) in hemoglobin to Fe(III), producing methemoglobinemia.

37. Hydrogen abstraction: Neutral free radicals can readily abstract H atoms from endogenous compounds, subsequently converting those compounds into radicals.

38. Enzymatic reactions: • A few toxins act enzymatically on specific target proteins. • For example, botulinum toxin hydrolyzes the fusion proteins that assist in exocytosis of the neurotransmitter acetylcholine in cholinergic neurons. • This causes inhibition of acetylcholine release, most importantly from motor neurons, resulting in paralysis.

39. Effects of toxicants on target molecules: • Dysfunction of target molecules • Destruction of target molecules • Neoantigen formation

40. Dysfunction of target molecules: • Chemicals can inhibit the function of target molecules by blocking neurotransmitter receptors or ion channels & inhibiting enzymes. • Toxicants may also interfere with the template function of DNA through the covalent binding of chemicals to DNA.

41. Destruction of target molecules: • Toxicants may alter the primary structure of endogenous molecules by means of cross linking & fragmentation. • Examples: Lipid peroxidation & DNA fragmentation. • Lipid peroxidation: Free radicals such as HO• can initiate peroxidative degradation of lipids by hydrogen abstraction from fatty acids.

42. Lipid peroxidation not only destroys lipids in cellular membranes, but also generates free radicals, & electrophiles which can go on to harm adjacent molecules (e.g., membrane proteins) or more distant molecules (e.g., DNA). • DNA fragmentation: Examples of DNA fragmentation caused by toxicants are: • single strand breaks (SSBs), & • doubles strand breaks (DSBs).

43. Neoantigen formation: • Covalent binding of xenobiotics or their metabolites to proteins may evoke an immune response. • Some chemicals (e.g., nickel) bind to proteins spontaneously. • Other chemicals may obtain reactivity by autooxidation (e.g., the allergens in poison ivy).

44. Toxicity not initiated by reaction with target molecules: • Some xenobiotics not only interact with a specific endogenous target molecule to induce toxicity but also, instead, alter the biological microenvironment (step 2b in Fig.1 ) • Included here are : • chemicals that alter H+ ion concentrations in the aqueous biophase, such as acids. • 2. solvents & detergents that physicochemically alter the lipid phase of cell membranes.

45. 3. other xenobiotics that cause harm merely by occupying a site or space. For example, by occupying bilirubin binding sites on albumin, compounds such as the sulfonamides induce bilirubin toxicity (kernicterus) in neonates.