toxicology1 pt2

• 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.

• 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.

• plasma proteins.
• liver and kidney.
• fat.
• bone.

• 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.

• 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++.

• 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++.

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.

• 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.

• 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.

• 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.

• 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.

• most substances including amino acids and glucose are recovered in the proximal tubules (PT), ---75% reabsorption.
• hence PT are prime targets for many toxicants.

involves transport of solvents. organic acids, sulfates, conjugates, strong bases, etc.

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• 

• 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.

• 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!

• 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.

• 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.

• if a subs is filtered but not reabsorbed, its PCR = GFR.
• no real example. inulin, a foreign inert subs, is used to determine 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.

• 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

-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.

• 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.

• 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.

• 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.

• 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).

bacterial metabolism; for example DDT→DDE.

• 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.

• 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.

• 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.

• 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.

• 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.

• 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.

reactions catalyzed by XBE are generally divided into two groups: Phase I and Phase II.

• 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.

• 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.

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• 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.

• 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.

• cytochrome P450.
• alcohol, aldehyde, ketone oxidation-reduction systems.
• alcohol dehydrogenase.
• aldehyde dehydrogenase.
• monoamine oxidase.
• peroxidase.

• 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 O^2-, gained 6(2) e^-, 12 e^- gained (reduced).

• NADP⁺: oxidized.
• NADPH: reduced.
• NAD⁺: oxidized.
• NADH: reduced.
• FAD⁺: oxidized.
• FADH₂: reduced (accepts both H^- and H⁺).

• 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.

• 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.

• 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.

• remove sulfate group, amine group, and halogen.
• 

• alcohol→aldehyde→carboxilic acid
• 
• aldehyde→alcohol ketone→alcohol

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.

• 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.

• 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.

• peroxide dependent co-oxidation.
• added to reduce substances in the body and to produce alcohols.
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• REDUCTION
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• EPOXIDE HYDROLASE
• 

• 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.

• forms mercapturic acid as end product.
• located in cytosol.
• 

• mitochondrial enzyme; its substrate is thiosulfate.
• 

• 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.

• 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.

• makes drugs more water soluble, transports amino acids across cell membranes.
• reduces disulfide bonds.
• most concentrated in the liver.
• depletion leads to cell death.

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

• 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.

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.

• 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.

• 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.

• 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