Cellular Dysfunction and Resultant Toxicities in General Toxicology

1. General ToxicologyMechanisms of ToxicityLec. 44thYear2017-2018College of Pharmacy/University of MustansiriyahDepartment of Pharmacology & ToxicologyLecturer: Rua Abbas Al-Hamdy

2. Objectives of this lecture are to: • Explain cellular dysfunction & resultant toxicities. • Explain toxic alterations of internal & external cellular maintenance. • Define necrosis & apoptosis, & explain the steps involved in each one. • Define repairing & adaptation mechanisms. • Explain how could inappropriate repair & adaptation result in toxicity.

3. Step 3: Cellular dysfunction & resultant toxicities: Reaction of toxicants with a target molecule may result in impaired cellular function as the third step in the development of toxicity (Figure 1).

4. Figure 1. The third step in the development of toxicity: alteration of the regulatory or maintenance function of the cell.

5. Step 3 involves: • Toxicant-induced cellular dysregulation, which includes: • Dysregulation of gene expression • Dysregulation of ongoing cellular activity • Or, • Toxic alterations of cellular maintenance, which includes: • Impairment of internal cellular maintenance • Impairment of external cellular maintenance

6. Dysregulation of gene expression: • Dysregulation of gene expression may occur at: • elements that are directly responsible for transcription, • at components of the intracellular signal transduction pathway, • & at the synthesis, storage, or release of the extracellular signaling molecules.

7. Dysregulation of ongoing cellular activity: Toxicants can adversely affect ongoing cellular activity of electrically excitable cells, while dysregulation of signaling mechanisms in non excitable cells, such as exocrine secretory cells, Kupffer cells, & pancreatic β-cells is usually less consequential.

8. Dysregulation of electrically excitable cells: Many xenobiotics cause perturbation of ongoing cellular activity in excitable cells, such as neurons, skeletal , cardiac, & smooth muscle cells due to an alteration in: a. the concentration of neurotransmitters, b. receptor function, c. intracellular signal transduction, or d. the signal terminating processes.

9. Alteration in neurotransmitter levels: • Chemicals may alter synaptic levels of neurotransmitters by interfering with their synthesis, storage, release, or removal from the vicinity of the receptor. • For example: • Inhibition of acetylcholinesterase by organophosphate or carbamate insecticides prevents the hydrolysis of acetylcholine, resulting in massive stimulation of cholinergic receptors & a cholinergic crisis.

10. Toxicant–neurotransmitter receptor interactions: Some chemicals interact directly with neurotransmitter receptors, including: agonists that associate with the ligand-binding site on the receptor & mimic the natural ligand, antagonists that occupy the ligand-binding site, activators, & inhibitors that bind to a site on the receptor that is not directly involved in ligand binding.

11. Toxicant–signal transducer interactions: • Many chemicals alter neuronal and/or muscle activity by acting on signal transduction processes. • For example: • Dichlorodiphenyltrichloroethane (DDT) causes activation of voltage-gated Na+ channels, located in neurons & muscle cells, resulting in over excitation. • In contrast, chemicals that block voltage-gated Na+ channels (such as tetrodotoxin) cause paralysis.

12. Toxicant–signal terminator interactions: • The cellular signal generated by cation influx is terminated by removal of the cations through cation exporters. • Inhibition of cation export may prolong excitation, as occurs with the blockade of Ca2+- activated K+ channels by Ba2+, which is accompanied by potentially lethal neuroexcitatory & spasmogenic effects.

13. Toxic alteration of cellular maintenance: • Impairment of internal cellular maintenance: Mechanisms of toxic cell death: • There are three critical biochemical disorders that chemicals inflicting cell death may initiate, namely,: • Adenosine triphosphate (ATP) depletion, • sustained rise in intracellular Ca2+, & • overproduction of reactive oxygen species (ROS) & reactive nitrogen species (RNS).

14. Depletion of ATP: • The role of ATP: • ATP plays a central role in cellular maintenance both as a chemical for biosynthesis and as the major source of energy. • Chemical energy is released by hydrolysis of ATP to  adenosine diphosphate (ADP) or adenosine monophosphate (AMP). • The ADP is rephosphorylated in the mitochondria by ATP synthase via a process termed oxidative phosphorylation.

15. Impairment of oxidative phosphorylation is detrimental to cells. • For example: • Arsenite acts as inhibitors of hydrogen delivery to the electron transport chain acting on pyruvate dehydrogenase. • Phosgene acts inhibitor of oxygen delivery to the electron transport chain, causing impairment of pulmonary gas exchange.

16. Sustained rise of intracellular Ca2+: • Toxicants induce elevation of cytoplasmic Ca2+ levels by : • promoting Ca2+ influx into the cytoplasm, • inhibiting Ca2+ efflux from the cytoplasm, or • by inducing its leakage from the mitochondria or the endoplasmic reticulum

17. Movement of Ca2+ down its concentration gradient from extracellular fluid to the cytoplasm could occur through: • opening of the ligand gated Ca2+ channels [e.g., by glutamate & capsaicin acting on ligand-gated channels in neurons],or • opening of voltage gated Ca2+channels,or • damage to the plasma membrane by chemicals [e.g., exogenous detergents, phospholipases in snake venoms, & carbon tetrachloride (CCl4).

18. Toxicants also may increase cytosolic Ca2+ through: decreasing Ca2+ efflux by inhibition of Ca2+ transporters (e.g., acetaminophen, bromobenzene, CCl4 & chloroform are covalent binders that inhibit Ca2+-ATPase in cell membrane).

19. Sustained elevation of intracellular Ca2+ is harmful because it can result in: • depletion of energy reserves by inhibiting the ATPase used in oxidative phosphorylation, • dysfunction of microfilaments, • activation of hydrolytic enzymes, & • generation of ROS & RNS.

20. Overproduction of ROS & RNS: • A number of xenobiotics can direct y generate ROS and RNS, such as the redox cyclers and transition metals. • Overproduction of ROS & RNS can be secondary to intracellular hypercalcemia.

21. Mitochondrial permeability transition (MPT) & the worst outcome: Necrosis: • An abrupt increase in the mitochondrial inner-membrane permeability, termed MPT. • Causative factors of MPT are: • Mitochondrial Ca2+ uptake, • decreased mitochondrial membrane potential, • generation of ROS &RNS, • depletion of ATP, &

22. MPT is believed to be caused by the opening of a proteinaceous pore that spans both mitochondria membranes & is permeable to solutes of 1500 Da. • This opening permits free influx of protons into the matrix space, causing rapid & complete dissipation of the membrane potential, cessation of ATP synthesis, & the osmotic influx of water causing mitochondrial swelling.

23. An alternative outcome of MPT: Apoptosis: • Chemicals that ultimately cause necrosis may also induce apoptosis. • The necrotic cell swells & lyses, with a consequent inflammatory response, while the apoptotic cells shrinks; its nuclear & cytoplasmic materials condense, & then it breaks into membrane bound fragments (apoptotic bodies) that are phagocytosed without inflammation.

24. In contrast to the random sequence of multiple metabolic defects that a cell suffers on its way to necrosis, the routes to apoptosis are ordered involving cascade like activation of catabolic processes that finally disassemble the cell. • Most, if not all , chemical induced cell deaths will involve the mitochondria, and that MPT is a crucial event.

25. Another related event is release into the cytoplasm of cytochrome c (cyt c). • cyt c is a small hemeprotein that normally resides in the mitochondrial intermembrane space attached to the surface of inner membrane.

26. The significance of cyt c release is two-fold: • The loss of cyt c will block ATP synthesis, & potentially thrust the cell toward necrosis. • Simultaneously, the unleashed cyt c represents an initial link in the chain of events directing the cell to the apoptotic path……Why? • On binding, together with ATP, to an adapter protein, cyt c can induce activation of proteins called caspases or cysteine proteases that cleave cytoplasmic proteins into fragments, beginning apoptosis.

27. What determines the form of cell death (i.e. necrosis or apoptosis)? • Toxicants tend to induce apoptosis at low exposure levels or early after exposure at high levels, whereas they cause necrosis later at high exposure levels. • Recent research suggests a larger toxic insult causes necrosis rather than apoptosis….why? • because necrosis incapacitates the cell such that it is unable to undergo apoptosis.

28. Three causatively related cellular events may lead to this incapacitation, namely: • increasing number of mitochondria undergoing MPT , • depletion of ATP, & • failed activation of caspases.

29. Impairment of external cellular maintenance: • Toxicants may also interfere with cells that are specialized to provide support to other cells, tissues, or the whole organism. • Chemicals acting on the liver illustrate this type of toxicity. For example: • Inhibition of hepatic synthesis of coagulation factors by coumarins does not harm the liver, but may cause death by hemorrhage. This is the mechanism of rodenticidal action of warfarin

30. Step 4: Inappropriate repair & adaptation: • The fourth step in the development of toxicity is inappropriate repair. • Many toxicants alter macromolecules, which, if not repaired, cause damage at higher levels.

31. Molecular repair: • Repair of proteins • Repair of lipids • Repair of DNA

32. Repair of proteins: • Thiol groups are essential for the function of numerous proteins. • Oxidation of protein thiols can be reversed by enzymatic reduction that is catalyzed by thioredoxin & glutaredoxin. • Physical or chemical insults may lead to protein denaturation or its aggregation.

33. Repair of lipids: • Peroxidized lipids are repaired by a complex process involving a series of reductants, glutathione peroxidase, & glutathione reductase. • NADPH is needed to recycle the reductants that are oxidized in the process.

34. Repair of DNA: • Despite its high reactivity with electrophiles & free radicals, nuclear DNA is remarkably stable, because: • it is packaged in a condensed form, called chromatin,& • several repair mechanisms are available to correct alterations. • Mitochondrial DNA, however, is not condensed & lacks efficient repair mechanisms.

35. Cellular repair: • Autophagic removal of damaged cell organelles may be viewed as a universal mechanism of cellular repair, • whereas clearance & regeneration of damaged axons is a mechanism specific for peripheral neurons.

36. In autophagy, the cytoplasmic material is engulfed & then encapsulated in a double membrane vesicle, called an autophagosome. • This vesicle fuses with the lysosome to form an autolysosome. • Contents of the autolysosome are degraded into amino acids, lipids, nucleosides, & carbohydrates, which are then transported to the cytosol for further metabolism.

37. Tissue repair: • Repair of injured tissues involves both regeneration of lost cells & the extracellular matrix & reintegration of the newly formed elements into tissues & organs. • The regenerative process is probably initiated by the release of chemical mediators from damaged cells.

38. The extracellular matrix is composed of proteins, glycosaminoglycans, & the glycoprotein & proteoglycans glycoconjugates. These molecules are synthesized in the liver. • Activation of resting stellate cells in liver is mediated chiefly by two growth factors, platelet derived growth factor (PDGF) & transforming growth factor beta (TGF-β).

39. Mechanisms of adaptation: • Adaptation may be defined as a harm induced capability of the organism or increased tolerance to the harm itself. • Adaptation of toxicity may result from biological changes causing: • diminished delivery of the toxicant to the target, • decreased susceptibility of the target, • increased capacity of the organism to repair itself, & • strengthened mechanisms to compensate the toxicant inflicted dysfunction.

40. Toxicity resulting from inappropriate repair & adaptation: • Like repair, dysrepair occurs at the molecular, cellular, & tissue levels. • Some toxicities involve dysrepair at an isolated level , such as a specific enzyme or process, or at different levels, such as tissue necrosis, fibrosis, & chemical carcinogenesis.

41. Fibrosis: • Fibrosis is a pathologic condition characterized by excessive deposition of an extracellular matrix of abnormal composition. • It is a specific manifestation of dysrepair of the chronically injured tissue. • Cellular injury initiates a surge in cellular proliferation & extracellular matrix production. • If increased production of extracellular matrix is not halted, fibrosis develops.

42. TGF-β appears to be a major mediator of fibrogenesis. • Normally, TGF-β production ceases when repair is complete. Failure to halt TGF-β overproduction leads to fibrosis. • TGF-β overproduction could be caused by continuous injury or a defect in the regulation of TGF-β.

43. Carcinogenesis: Chemical carcinogenesis involves inappropriate function of various repair mechanisms, including: (1) failure of DNA repair, (2) failure of apoptosis, & (3) failure to terminate cell proliferation.