Mechanisms of Toxicity

1. Mechanisms of Toxicity To understand how a toxicant enters an organism  how it interacts with target molecules  how the organism deal with the insult

2. To provide a rational basis for  interpreting descriptive toxicity data estimating the probability that a chemical will cause harmful effects  establishing procedures toprevent or antagonize the toxic effects  designing drugs and industrial chemicals that are less hazardous  developing pesticides that are more selectively toxic for their target organisms

3. Example for better understanding of fundamental physiologic and biochemical process  Cancer and carcinogen  Parkinson’s disease and MPTP

4. Step 1-Delivery:from the site of exposure to the target Step 2a-Reaction of the ultimate toxicant with the target molecule Step 2b- Alteration of biological environment Step 3-Cellular dysfunction, injury Step 4-Inappropriate repair or adaptation

7. Ultimate toxicant is the chemical species that reacts with the endogenous target molecule or critically alter the biological environment, initiating structural and /or functional alteration that result in toxicity. Parent compounds  Metabolites of parent compounds  Reactive oxygen or nitrogen species Endogenous molecules

9. Absorption vs. presystemic elimination Influencing factors for absorption  concentration of the chemical at the absorbing surface  the area of the exposed site  the characteristics of the epithelial layer  the intensity of the subepithelial microcirculation  physicochemical properties of the toxicant-lipid solubility Presystemic elimination  Usually for chemicals absorbed from GI tract first pass through GI mucosal cells, liver, and lung

10. in the hepatic sinusoids in the renal peritubular capillaries ion channels protein transporters endocytosis-toxicant-protein complex membrane recycling amphipathic xenobiotics with a protonable amino group and lipophilic character organic and inorganic cations and PAH bind /release to melanin (polyanionic aromatic polymer) Mechanisms facilitating distribution to a target Porosity of the capillary endothelium Specialized transport across the plasma membrane Accumulation in cell organelles (lysosomes and mitochondria) Reversible intracellular binding

11. Homework p48 1. Explain the mechanism of cardiac toxicity of lipophilic local anethetics ( e.g. tetracaine, bupivacaine). 2. Why amine ( e.g. amiodarone) can cause phospholipidosis? 3. Why melanin-containing cells are more sensitive to cations and polycyclic aromatics?

12. Mechanisms opposing distribution to a target Binding to plasma protein DDT and TCDD are bound to high M.W. protein or lipoprotein Specialized barriers (for hydrophilic toxicants) blood-brain barrier reproductive cells Distribution to storage sites (where they do not exert effects) Association with intracellular binding proteins metallothionein Export from cells by ATP dependent transports multidrug-resistance protein (P-glycoprotein) in brain cappilary endothelial cell, oocyte stem cell, and tumor cell

14. Excretion Hydrophilic, ionized chemicals Renal glomeruli-hydrostatically filter Proximal renal tubular cells-active transport Hepatocyte Nonvolatile, highly lipophilic chemicals Excretion by the mammary gland Excretion in bile in association with biliary micelles and /or phospholipid vesicles Intestinal excretion Volatile, nonreactive toxicant Pulmonary capillaries into the alveoli

15. Reabsorption • Renal tubule • diffusion-lipid solubility, ionization (pH) • carriers and transporters- • peptide transporter sulfate transporter (chromate & molybdate), phosphate transporter (arsenate) • Intestinal mucosa • Biliary, gastric, and intestinal excretion • secretion by salivary glands and exocrine pancreas • lipid solubility

16. Toxication (metabolic activation) • Formation of electrophilic Metabolites (table3-2) • molecules containing an electron-deficient atom with • partial or full positive charge • insertion of an oxygen atom • conjugated double bonds are formed • Heterolytic bond cleavage, C-O • Free radials • accepting an electron from reductases (fig.3.3) • losing an electron and form free radical by peroxidase • homolytic fission of a covalent bond • (CCl4CCl3., HO., Fenton reaction) • Nucleophiles (relatively uncommon) • HCN from amygdalin, CO • Redox-active reactants

20. Detoxication No functional groups add a functional group (OH,COO) by cytP450 then endogenous acid (glucuronic acid, sulfuric acid) by transferase Nucleophiles Conjugation at the nucleophilic functional group (OH, SH) Electrophiles (Metal ion, etc) conjugated with the SH of glutathione specific mechanism: epoxide hydrolase-epoxidediols, arene dihydrodiols carboxylesterase DT-diaphorase alcohol dehydrogenase

22. Free radicals O2. --.superoxide dismutase HOOH-glutathione peroxidase, catalase peroxyl radical-glutathione, -tocopherol, ascorbic acid ONOO--selenocysteine-containing glutathione peroxidase, selenoprotein P, oxyhemoglobin, heme-containig peroxidase, albumin peroxidase-generated free radical-electron transfer from glutathione Protein toxin-extra- and intracellular protease toxins with disulfide bond are inactivated by thioredoxin

23. Prx(SH)2 PrxS2 2HOH

24. chlopromazine peroxidase

25. Homework p54 • Describe at least 3 ways to prevent peroxynitrite (ONOO-) buildup.

26. When detoxication fails Toxicants may overwhelm detoxication process  exhaustion of the detoxication enzymes  consumption of the cosubstrates  depletion of cellular antioxidants Toxicant inactivates a detoxicating enzyme ONOO-incapacitates Mn-SOD Some conjugation reactions reversed Sometimes detoxication generates potentially harmful byproducts ex. glutathione thiyl radical (GS.) glutathione disulfide (GSSG)

28. Attributes of target molecules DNA, protein, membrane lipids, cofactor  Appropriate reactivity and/or configuration  Accessibility-endogenous molecules that are in the vicinity of reactive chemicals or are adjacent to sites where they are formed ex. enzyme responsible for production of reactive metabolites or the adjacent intracellular structures  Critical function-not all targets for chemicals contribute to the harmful effects ex. CO for Hb but not cytP450

29. Types of reactions Noncovalent binding Covalent binding covalent adduct formation Hydrogen abstraction R-SH, RSOH Electron transfer enzymatic reactions Hydrogen bond, ionic bond ex. Interaction of toxicants with receptors, ion channels, and some enzymes Fe(II)Fe(III) ADP ribosylation-diphthera toxin, cholera toxin

31. Hydrogen abstraction

35. Dysfunction of target molecules Activation- agonist, activator Inhibition- antagonist Alteration in conformation or structure of protein- thiol group Interference with template with the function of DNA aflatoxinbind to G: GCGA HO. 8-hydroxyguanine and 8-hydroxyadenine  mispairing

36. Destruction of target molecules Cross-linking Fragmentation spontaneous degradation after chemical attack hydrolytic degradation Neoantigen formation Covalent binding altered protein evoke immune response drug-protein adduct

38. Toxicity not initiated by reaction with • target molecules • Chemicals that alter H ion concentrations • Acids and substance biotransformed to acids • protonophoric uncoupler • 2. Solvents and detergents alter the lipid phase of cell • membrane and destroy transmembrane solute gradients • 3. Occupying a site or a space • ethylene glycol form water insoluble precipitates in the • renal tubules • sulfomides occupy bilirubin binding sites of albumin

41. Dysregulation of gene expression •  Dysregulation of transcription • Promoter region of the gene • Transcription factors (TFs) • ligand-activated (Table 3-4) • altering the regulatory region of the genes • direct chemical interaction –thalidomide/GCbox • methylation of cytosine • Dysregulation of signal transduction •  Dysregulation of the synthesis, storage, or release of the extracellular signaling molecules

42. Systemic lupus erythemathosus Induced by Procainamide Hydrolazine Inhibit DNA methylation in CD4+T lymphocyte Overexpression of protein for inflammation TCDD hypermethylation in Insulin-like growth factor-2 gene

43. Dysregulation of signal transduction *sinaling molecules to activate TFs ( c-FOS, c-JUN, c-Myc) that control transcriptional activity of genes that influence cell cycle  Altering protein phosphorylation  by kinases,  by phosphatases  Interfering with the GTPase activity of G protein  Disrupting normal protein-protein interaction  Altering the synthesis or degradation of the signaling proteins

44. Extracellulr Signaling molecules

45. Chemically altered signal transduction with proliferative effect Chemically altered signal transduction with antiproliferative effect

46. Dysregulation of ongoing cellular activity dysregulation of electrically excitable cells (Table 3-5) due to an alteration in  the concentration of neurotransmitters  receptor function  intracellular signal transduction  the signal terminating process dysregulation of the activity of other cells ex.liver cells possess -1 adrenergic receptors exocrine secretory cells controlled by Ach receptor

48. Toxic alteration of cellular maintenance • Impairment of internal cellular maintenance: • mechanism of cell death •  ATP depletion (Table 3-6) • Ca accumulation (Table 3-7) • ROS/RNS generation • .

50. Sustained elevation of intracellular Ca2+ • can result in : • Depletion of energy reserve •  mitochondria Ca2+ uptake dissipate membrane • potential •  continuous Ca2+ uptake and export causing • oxidative injury to inner membrane •  impair ATP synthesis •  ATP consumption by the Ca2+ -ATPase (eliminate • the excess Ca2+ • 2.Dysfunction of microfilaments • dissociation of actin filaments from -actinin • and fodrin (anchor proteins)  membrane blebbing