toxicology1 pt2

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toxicology1 pt2
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review for tox exam 1 pt 2
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  1. VOLUME OF DISTRIBUTION (VD)
    • total body water may be divided into 3 distinct compartments: plasma water, interstitial water and intracellular water.
    • VD is defined as the proportion of a chem in body that is found in plasma.
    • VD can be determined by dissolving a known amount of a dye in an unknown volume of water and then---VD=amount of dye added/conc in water.
    • in vivo then VD=amount of chem administered/conc in plasma.
    • toxicants stored in tissues have restricted volume of distribution.
  2. VOLUME OF DISTRIBUTION
  3. TISSUE DISTRIBUTION
  4. STORAGE OF TOXICANTS
    • toxicants are often concentrated in tissues: some achieve high conc at the site of action, CO in Hb, paraquat in lungs.
    • storage sites are also called storage depots and are protective mechanisms: conc in storage depot are in equilibrium with the plasma conc.
    • biological half life (t ½) of stored toxicants is very long.
  5. STORAGE DEPOTS
    • plasma proteins.
    • liver and kidney.
    • fat.
    • bone.
  6. PLASMA PROTEINS AS SOTRAGE DEPOT
    • albumins bind to many substances Ca++, Cu++, Zn++, bilirubin, uric acid, penicillin, salicylates, histamines, barbitrurates.
    • transferrin transfers Fe++.
    • lipoproteins bind to lipid soluble compounds.
    • many therapeutic agents remain attached to plasma proteins.
    • the amount of protein bound toxicant is not available for distribution.
    • sometimes another substance can displace a substance previously bound to plasma proteins.
    • examples. strong sulfonamide (an antibiotic) can displace an antidiabetic drug previously bound to plasma proteins. can lead to hypoglycemia.
    • similarly xenobiotics can displace normal physiologic compounds previously bound to plasm proteins. example, dieldrin and acrylonitrile.
  7. LIVER AND KIDNEY AS STORAGE DEPOTS
    • liver and kidney have high capacity to bind and eliminate toxicants.
    • liver biotransforms and kidney eliminates the toxicants.
    • mechanism of binding is not completely understood.
    • a protein ligand in liver cytoplasm has high affinity to organic acids, carcinogens and corticosteroids.
    • a protein metaliothionin in kidney and liver binds to metal ions such as Cd++ and Zn++.
    • liver also stores Pb++.
  8. FAT AND BONE AS STORAGE DEPOTS
    • FAT
    • many xenobiotics are fat soluble (chloroform, DDT, PCB).
    • - a fat soluble xenobiotics enter by simple diffusion.
    • - a substance with high PC will accumulate in fat.
    • - obese people at high risk.
    • - DDT was found in soldiers of WWII.

    • BONE
    • bone is a depot for F, Pb and Sr.
    • - F and OH are readily exchangeable, equilibrium between blood and bone.
    • - case of bone seekers Ra++.
  9. BARRIERS FOR DISTRIBUTION: BLOOD BRAIN BARRIER (BBB)
    a selective barrier to CNS for certain chemicals.

    • four important features of BBB:
    • - tightly bound capillary endothelium with no pores.
    • - endothelial cells with an ATP dependent transporter multidrug-resistant protein that exudes chemicals.
    • - capillaries surrounded by glial cells astrocytes.
    • - protein conc in interstitial fluids of CNS is much lower than those in other body fluids.

    • BBB is not completely developed at birth, hence:
    • - morphine is 3-10X effective in new born rats.
    • - phenobarbital produces encephalomyelopathy in new born rats.
  10. BBB FORMATION:
  11. BARRIERS FOR DISTRIBUTION: PLACENTA
    • anatomically placenta is several layers of cells.
    • placenta: protects fetus, provide nutrients, eliminates wastes, maintains hormonal concentrations and activities.
    • many drugs cross placental barrier.
    • diffusion is the most common mechanism for transport.
    • it also has capacity for biotransformation.
  12. EXCRETION OF TOXICANTS
    • simple forms of life excrete into surrounding medium, usually water.
    • during evolution when environment changed from marine water to fresh water to land, excretion became complex.
    • most common routes for excretion in terrestrial environment include renal and hepatic, other minor routes include milk, expired air, etc.
  13. URINARY EXCRETION
    • kidneys primarily are excretory organs for polar and hydrophilic substances.
    • kidney is designed for this activity, nephrons, renal tubules, pelvis, ureter, bladder and urethra.
    • formation of urine involves 3 processes: glomerular filtration, tubular reabsorption and tubular secretion.
  14. GLOMERULAR FILTRATION
    • plasma is primarily filtered under pressure generated by heart.
    • glomerular filtration rate (GFR) is 180L/d, 125ml/min.
    • entire plasma is filtered except blood cells and large proteins.
    • any factor that affects hydrostatic pressure affects GFR.
  15. TUBULAR REABSORPTION
    • most substances including amino acids and glucose are recovered in the proximal tubules (PT), ---75% reabsorption.
    • hence PT are prime targets for many toxicants.
  16. TUBULAR SECRETION
    involves transport of solvents. organic acids, sulfates, conjugates, strong bases, etc.
  17. URINARY EXCRETION PROCESS
  18. URINARY EXCRETION
    • toxicants pass through same stages as other substances.
    • polar or ionic subs are excreted not reabsorbed.
    • basic toxicants are excreted in an acidic urine.
    • acidic toxicants are excreted in a basic urine.
    • phenobarbital (PB) is a weak organic acid (pKa 7.2) administration of Na2CO3 alkalinizes the urine and helps eliminate PB.
    • salicylates (aspirin--acetyl salicylic acid) are also eliminated by administration of Na2CO3.
  19. URINARY EXCRETION: TUBULAR ACTIVE SECRETION
    • one for organic acids such as para amino hippuric acid.
    • another for organic bases such as N-methyl nicotinamide.
    • active transport system is located in the basolateral walls of PT, many subs compete for this system.
    • case of penicillin in short supply during WWII: penicillin actively secreted by organic acid transport system. patients were given probenecid along with penicillin which increased the t ½ of penicillin.
    • if an acid competes with the uric acid (UA) transport system, UA accumulates resulting in gout!
  20. URINARY EXCRETION
    • average 1 ml urine/min.
    • for every 125ml of plasma filtered 124ml is reabsorbed producing only 1 ml urine--1.5L/d out of possible 180L.
    • urine has high concentration of wastes and other substance that are regulated by kidney.
    • a small change in filtration can cause a large increase in urine production.
  21. PLASMA CLEARANCE (PCR)
    • volume of plasma cleared of a subs/min---index of kidney function.
    • PCR=mg of secreted subs in the urine per minute/mg of that subs in each ml plasma.
    • example: PCR of urea = 12mg/0.2mg = 60ml; means that 60 ml of plasma is cleared of plasma is cleared of urea.
    • GFR was determined by this concept using an inert subs inulin (intern carbohydrate).
    • GFR = 0.125 mg inulin in urine/0.001 mg/ml plasma = 125 ml/min.
  22. PCR = GFR
    • if a subs is filtered but not reabsorbed, its PCR = GFR.
    • no real example. inulin, a foreign inert subs, is used to determine GFR.
  23. PCR < GFR
    • if a subs is filtered and reabsorbed but not secreted, its PCR < GFR.
    • example, glucose: its PCR is zero; all of glucose is reabsorbed.
    • urea 50% is cleared its PCR is 60 ml.
  24. PCR > GFR
    • if a subs is filtered and secreted but not reabsorbed, its PCR > GFR. example H+.
    • TS allows kidney to effectively clear plasma of H+.
    • only 20% plasma is filtered/ min and 80% continues to flow into the capillaries.
    • only way to clear that plasma of H+ is thru active TS.
    • if quantity of H+↑, it is secreted actively. example; if 25 ml more of plasma is cleared--then its PCR is 150 ml which is more than 125 ml, hence PCR > GFR
  25. FECAL EXCRETION
    -non-absorbed ingesta: non-absorbed portion of xenobiotics contributes to fecal excretion (not very significant).

    • BILIARY EXCRETION
    • -most important component of fecal excretion.
    • -liver is at strategic location for removal of toxicants and is also the major site for metabolism.
    • -toxicants excreted into the bile are often classified based upon their conc in bile/conc in plasma.
  26. TOXICANTS EXCRETED INTO BILE CLASSIFICATION
    • class A: ratio 1; Na, K, glucose, Hg, Th, Cs, Co, etc.
    • class B: ratio > 1 (usually 10-1000) bile acids, bilirubin, Pb, As, Mg, xenobiotics (toxicants).
    • class C: ratio < 1; inulin, albumin, Zn, Fe, Au, Cr, etc.
    • little is known about the mechanism of excretion of classes A and C in the bile.
    • substance most likely to be excreted in the bile are class B substances.
  27. FECAL EXCRETION: BILIARY EXCRETION
    • subs with enterohepatic recirculation exhibit a longer t ½ and the subs in organisms with injured liver exhibit a shorter t ½.
    • ↑bile secretion→ ↑xenobiotic excretion→↓toxicity.
    • hepatic secretion is not well developed in new borns, hence xenobiotics are more toxic to them.
    • hepatic parenchymal cellular elimination must be efficient to avoid any accumulation of xenobiotics.
  28. HEPATIC PARENCHYMAL CELLULAR EXCRETION
    • cells convert toxicants into polar metabolites.
    • these metabolites cannot cross plasma membranes and need an active transport mechanism for elimination.
    • hepatocyte plasma membranes have a variety of transport systems.
    • multidrug resistance protein one (mdr 1) and multiresistant drug protein two (mrp2) are for transport into bile.
    • mrp3 and mrp6 transport xenobiotics into blood.
  29. FECAL EXCRETION: INTESTINAL EXCRETION
    • some toxicant appear in feces due to:
    • -incomplete absorption.
    • -being a biliary product.
    • -secretion into saliva or gastric, intestinal or pancreatic juices.
    • -secretion by respiratory tract and then swallowed into GI.

    intestinal secretion is the major route of excretion for dioxin and PCBs.

    • subs in GI are not toxic until absorbed.
    • -for this reason there is a clinical significance of emesis (vomiting).
  30. FECAL EXCRETION: INTESTINAL FLORA
    bacterial metabolism; for example DDT→DDE.
  31. EXHALATION
    • substances for this kind of secretion are in gaseous phase.
    • volatile substances are in equilibrium with gas and liquid phase for example ethanol, ether, etc.there is no specific mechanism for excretion, simply diffusion.
  32. OTHER ROUTES OF ELIMINATION
    • cerebrospinal fluid (CSF):
    • -very specific route for lipid soluble subs such as CNS metabolites.
    • -active transport from CSF to blood.

    • sweat and saliva:
    • -simple diffusion of lipid soluble subs, may cause dermatitis.

    • milk, eggs and placenta (fetus):
    • -important because subs may go from mothers to babies.
    • -since milk pH is 6.5 and that of blood is 7.4 only basic subs are excreted in the milk.
    • -also lipid soluble subs such as xenobiotics (DDT, PCBs, PBBs, Pb, etc) appear in the milk.
    • -eggs have polar metabolites; this is good for mother but not for developing embryo.
    • -placenta is not a good barrier; case of DES in mothers to vaginal cancer in daughters.
  33. BIOTRANSFORMATION OF XENOBIOTICS
    • lipophilic compounds can enter, accumulate and get excreted unchanged in urine, feces, bile, expired air or sweat.
    • except for perspiration chemicals depend on water solubility for excretion.
    • water soluble toxicants are readily excreted and lipophilic ones are stored.
    • end results of metabolism is to form hydrophilic metabolites.
    • the hydrophilicity makes a chemical ionic and hence reduces its absorption.
  34. BIOTRANSFORMING ENZYMES
    • xenobiotic transformation (XB) is the principal mechanism for homeostasis.
    • XB is accomplished by a limited number of enzymes (D) with broad substrate specificities.
    • synthesis of some of these E is triggered by xenobiotics themselves.
    • XBE play an important role in synthesizing the same molecule they biotransform, example steroids.
  35. METABOLISM
    • metabolites: conversion products of substances, often mediated by enzyme reactions.
    • bioactivation (activation): production of metabolites that are more toxic than the parent substance.
    • detoxication: production of metabolites that are less toxic than the parent substance.
  36. BIOTRANSFORMATION VS METABOLISM
    • biotransformation: transformation of an endogenous or xenobiotic substance.
    • metabolism: total fate of a xenobiotic including absorption, distribution, biotransformation and elimination.
    • products of biotransformation are called metabolites.
  37. PHASE I AND PHASE II REACTIONS
    reactions catalyzed by XBE are generally divided into two groups: Phase I and Phase II.
  38. PHASE I REACTIONS
    • involve hydrolysis, oxidation and reduction.
    • these reactions expose or introduce a functional group (-OH, -NH2, -SH, -COOH).
    • usually result in a small increase in hydrophilicity.
  39. PHASE II REACTIONS
    • include glucuronidation, sulfation, acetylation, methylation, conjugations with amino acids or glutathione.
    • cofactors for these reactions react with functional groups already present or those introduced by phase I reactions.
    • usually a result in a large increase in hydrophilicity and promote excretion.
    • phase II reactions may or may not be preceded by phase I reactions.
  40. PHASE I AND PHASE II REACTIONS
  41. NOMENCLATURE OF XBE
    • XBE have broad overlapping substrate specificities.
    • this makes it difficult to name these enzymes after the reactions they catalyze.
    • many XBE have been cloned and sequenced.
    • for many of them a nomenclature system has been developed.
  42. DISTRIBUTION OF XBE
    • organ and cellular distribution:
    • -liver is the richest source of XBE.
    • -also found in skin, lung, nasal mucosa, eye and GI tract.
    • -also kidney, adrenal, pancreas, spleen, heart, brain, testis, ovary, placenta, plasma, erythrocytes, platelets, lymphocytes and aorta.

    • subcellular distribution:
    • -primarily in smooth endoplasmic reticulum (microsomes).
    • -soluble fractions of cytoplasm (cytosol).
    • -smaller amounts in mitochondria, nuclei and lysosomes.
  43. OXIDATION
    • cytochrome P450.
    • alcohol, aldehyde, ketone oxidation-reduction systems.
    • alcohol dehydrogenase.
    • aldehyde dehydrogenase.
    • monoamine oxidase.
    • peroxidase.
  44. OXIDATION-REDUCTION
    • oxidation: addition of H+. loss of e-.
    • reduction: removal of H+. gain of e-.

    • 4 Fe (s) + 3 O2 (g) → 2 Fe2O3 (s)
    • 4 Fe → 4 Fe3+, lost 4(3) e-, 12 e- lost (oxidized).
    • 3 O2 → 6 O2-, gained 6(2) e-, 12 e- gained (reduced).
  45. COMMON COFACTORS
    • NADP+: oxidized.
    • NADPH: reduced.
    • NAD+: oxidized.
    • NADH: reduced.
    • FAD+: oxidized.
    • FADH2: reduced (accepts both H- and H+).
  46. CYTOCHROME P450
    • most important phase I enzyme system.
    • works in conjunction with NDPH-cytochrome P450 reductase.
    • both are embedded in the phospholipid matrix of smooth endoplasmic reticulum (microsomes).
    • NADPH transfers electrons to cytochrome P450.
    • in the reduced form it can bind to ligands such as O2 and carbon monoxide (CO).
    • the complex between reduced cytochrome P450 and CO absorbs light maximally at 450 nm; hence the name cytochrome P450.
  47. ISOLATION BY DIFFERENTIAL CENTRIFUGATION
  48. MONOOXYGENASES
    • give one atom of O2 to substrate (xenobiotic) and one atom to water.
    • R + O2 + NAD(P)H → ROH + H2O + NAD(P)+.
    • Phe + O2 + NADH + H+ → Tyr + NAD+ + H2O.
  49. CYTOCHROME P450
    • monooxygenase:
    • -gives one atom of molecular O2 to the substrate and one atom to water.

    • family of enzymes:
    • -many different forms of P450 have been identified but only one form of reductase.
    • -P450 forms are designated as CYP1, 2, 3 and subforms as CYP2A, B etc.
    • -absorption maximum at 450 nm.
    • -present primarily in liver microsomes.
    • -involved in: detoxication of xenobiotics, biosynthesis of steroid hormones, synthesis of bile salts.
  50. CYTOCHROME P450
  51. HYDROXYLATION OF ALIPHATIC OR AROMATIC CARBON. ALIFATIC/AROMATIC→ALCOHOL
  52. EPOXIDATION OF DOUBLE BOND (ALKENE). DOUBLE BOND→EPOXIDE
  53. N-, O-, AND S-DEALKYLATION; METAL GROUPS→FORMALDEHYDE
  54. S-OXIDATION AND N-HYDROXYLATION.
    S-R'→S=O N-H→N-OH
  55. DEAMINATION, DESULFURATION AND DEHALOGENATION.
    • remove sulfate group, amine group, and halogen.
  56. ALCOHOL, ALDEHYEDE, KETONE OXIDATION-REDUCTION SYSTEMS
    • alcohol→aldehyde→carboxilic acid
    • aldehyde→alcohol ketone→alcohol
  57. ACTIVATION OF XENOBIOTICS BY CYTOCHROME P450
    biotransformation by cytochrome P450 does not always led to detoxication.

    • many reaction results in activation of parent compounds such as:
    • -benz[a]pyrene by CYP1A1.
    • -acetaminophen by CYP1A2, CYP2E1.
    • -acrylonitrile, styrene, vinyl chloride by CYP2E1.
    • -aflatoxin B1 by CYP3A4.

    many of the compounds activated by cytochrome P450 can also be detoxified by P450.
  58. P450 KNOCKOUT MICE
    • several strategies are developed to explore role of P450 in activation of xenobiotics.
    • P450 levels in rodents can be increased by a variety of inducers which in turn can result in increase in xenobiotic toxicity.
    • P450 activity can be decreased by a variety of inhibitors which in turn can result in decrease in xenobiotic toxicity.
    • transgenic mice that lack one or more P450 enzymes are commonly called as knockout or null mice.
    • these mice have provided a new strategy to evaluate role of specific P450 enzymes in xenobiotic activation.
    • the studies in these mice are relevant to humans.
  59. MONOAMINE OXIDASE (MAO)
    • MAO catalyzes oxidative deamination.
    • a reaction similar to that by P450 which catalyze oxidative deamination producing an aldehyde and ammonia gas from a primary amine.
  60. PEROXIDASE
    • peroxide dependent co-oxidation.
    • added to reduce substances in the body and to produce alcohols.
  61. REDUCTION AND HYDROLYSIS
    • REDUCTION
    • EPOXIDE HYDROLASE
  62. PHASE II ENZYME REACTIONS
    • are biosynthetic and require energy to drive reactions.
    • are accomplished by activating cofactors or substrates to high energy intermediates.
    • examples: sulfate conjugation (sulfotransferase), n-acetyl transferase, glutathione S-transferase (GST), rhodanese.
  63. SULFATE CONJUGATION (SULFOTRANSFERASE)
  64. N-ACETYL TRANSFERASE
  65. GLUTATHIONE S-TRANSFERASE
    • forms mercapturic acid as end product.
    • located in cytosol.
  66. RHODANESE
    • mitochondrial enzyme; its substrate is thiosulfate.
  67. PHASE II REACTIONS
    • preferred routes of excretion of conjugates of xenobiotics.
    • - glucuronides < 250 mw by kidney and > 250 mw by bile.
    • - sulfates, mercapturic acids and thiocyanates by kidney and acetylated conjugates by bile.

    • extrahepatic biotransformation:
    • -lungs, kidney, skin and GI mucosa, intestinal microbes.
  68. GLUTATHIONE (GSH)
    • reduced glutathione (GSH) is a tripeptide consisting of 3 amino acids glutamine, cysteine and glycine.
    • it is widely found in all forms of life.
    • plays an essential role in the health of organisms.
    • GSH is the predominant non-protein thiol and functions as redox buffer.
  69. FUNCTIONS OF GLUTATHIONE (GSH)
    • makes drugs more water soluble, transports amino acids across cell membranes.
    • reduces disulfide bonds.
    • most concentrated in the liver.
    • depletion leads to cell death.
  70. MODIFICATION OF BIOTRANSFORMATION: INHIBITION OF XBE
    agents that inhibit protein synthesis: cobalt, aminotriazole.

    agents that affect tissue levels of necessary cofactors: sulfoximine inhibits GSH synthesis, diethyl maleate depletes GSH.

    • agents that inhibit P450:
    • - CO: competes with oxygen for heme in cytochrome.
    • - SKF 525-A, piperonyl butoxide: are noncompetitive inhibitors.
    • -suicide inhibition, activated metabolites bind to heme. examples, carbon tetrachloride and vinyl chloride.

    simple or complex inhibition: some agents inhibit both phase I and II reactions
  71. MODIFICATION OF BIOTRANSFORMATION: INDUCTION OF XBE
    • induction requires de novo protein synthesis.
    • many enzymes including P450 are inducible.
    • most inducing agents studied are phenobarbital, benz[a]pyrene, and 3-methylcholanthrene.
    • others include DDT, aldrin, lindane, chlordane, PCB, PBB, steroids, testosterone, TCDD, etc.
    • mechanism of induction is at the transcriptional level.
    • time course of induction is highly variable: 3-5 days for phenobarbital and few hours for 3-methycholanthrene.
    • induction is reversible, withdrawal of agent resumes basal enzyme activity.
  72. MODIFICATION OF BIOTRANSFORMATION: INDUCTION OF XBE
    enzymes can be induced by many chemical agents: drugs, pesticides, industrial chemicals, natural products and ethanol.

    • inducible enzymes other than P450:
    • - UDP-glucuronosyltransferase by phenobarbital (PB) and 3-methycholanthrene (3-MC).
    • - epoxide hydrolase by PB, 3-MC, butylated hydroxy anisole (BHA) and beta hydroxy toluene (BHT).

    • induction of extrahepatic enzymes:
    • -extrahepatic P450 is not inducible by PB but polycyclic hydrocarbons can induce P450 in lung, kidney, GI and skin.

    • induction of cytosolic enzymes:
    • - except glutathione S-transferase (GST) all other enzymes are not inducible.
    • - GST is inducible by 3-MC, PB and BHA.
  73. ENZYMES
  74. MODIFICATION OF BIOTRANSFORMATION: SPECIES, STRAIN AND GENETIC VARIATION
    • species difference:
    • -qualitative: some species can metabolize a xenobiotic and others cannot.
    • -quantitative: some species can metabolize a xenobiotic more readily than others.
    • -examples: Phase I--rats hydroxylate acetaminophen and rabbits deaminate.
    • Phase II--cats cannot do glucuronic acid conjugation.

    • strain difference:
    • -is under genetic control.
    • -duration of sleep due to hexobarbital is different in Sprague-Dawley and Wistar rats.

    • genetic difference:
    • -different inducing abilitiy of P450 in different species of mice.
  75. MODIFICATION OF BIOTRANSFORMATION: GENDER AND AGE DIFFERENCES
    • GENDER
    • -hexobarbital: males sleep longer than females.
    • -parathion: twice as toxic to females than males.
    • -chloroform: more nephrotoxic in males than in females.
    • -gender difference more common in rats than in mice.

    • AGE
    • -fetus and new born most susceptible.
    • -human P450: 20%-50% activity of adult by 2nd trimester.
    • -in elderly decreased biotransformation is due to: decreased P450, renal and hepatic blood flow, liver size, ability of biliary and urinary excretion and increased fat mass.
  76. MODIFICATION OF BIOTRANSFORMATION: EFFECT OF DIET, CIRCADIAN RHYTHM, HORMONES AND PREGNANCY
    • DIET
    • -mineral and vitamin deficiency and starvation decrease biotransformation.

    • CIRCADIAN RHYTHM
    • biotransformation is related to time of day because of endocrine changes, example GSH.

    • HORMONES
    • -thyroid hormone decreases P450 and monoamine oxidase.
    • -removal of adrenal gland decreases hepatic microsomal activity.
    • -induction of P450 in diabetic patients can be reversed by insulin.

    • PREGNANCY
    • -many enzymes decrease during pregnancy, related to progesterone which is an inhibitor of some enzymes

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