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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.
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.
- plasma proteins.
- liver and kidney.
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.
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++.
FAT AND BONE AS STORAGE DEPOTS
- 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 is a depot for F, Pb and Sr.
- - F and OH are readily exchangeable, equilibrium between blood and bone.
- - case of bone seekers Ra++.
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.
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.
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.
- 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.
URINARY EXCRETION PROCESS
- 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.
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!
- 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.
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.
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.
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.
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
-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.
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.
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.
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.
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).
FECAL EXCRETION: INTESTINAL FLORA
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.
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.
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.
- 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 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.
PHASE I AND PHASE II REACTIONS
reactions catalyzed by XBE are generally divided into two groups: Phase I and Phase II.
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.
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.
PHASE I AND PHASE II REACTIONS
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.
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.
- cytochrome P450.
- alcohol, aldehyde, ketone oxidation-reduction systems.
- alcohol dehydrogenase.
- aldehyde dehydrogenase.
- monoamine oxidase.
- 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).
- 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.
ISOLATION BY DIFFERENTIAL CENTRIFUGATION
- 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.
- -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.
HYDROXYLATION OF ALIPHATIC OR AROMATIC CARBON. ALIFATIC/AROMATIC→ALCOHOL
EPOXIDATION OF DOUBLE BOND (ALKENE). DOUBLE BOND→EPOXIDE
N-, O-, AND S-DEALKYLATION; METAL GROUPS→FORMALDEHYDE
S-OXIDATION AND N-HYDROXYLATION.
DEAMINATION, DESULFURATION AND DEHALOGENATION.
- remove sulfate group, amine group, and halogen.
ALCOHOL, ALDEHYEDE, KETONE OXIDATION-REDUCTION SYSTEMS
- alcohol→aldehyde→carboxilic acid
- aldehyde→alcohol ketone→alcohol
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.
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.
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.
- peroxide dependent co-oxidation.
- added to reduce substances in the body and to produce alcohols.
REDUCTION AND HYDROLYSIS
- EPOXIDE HYDROLASE
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.
SULFATE CONJUGATION (SULFOTRANSFERASE)
- forms mercapturic acid as end product.
- located in cytosol.
- mitochondrial enzyme; its substrate is thiosulfate.
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.
- 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.
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.
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
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.
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.
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.
MODIFICATION OF BIOTRANSFORMATION: GENDER AND AGE DIFFERENCES
- -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.
- -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.
MODIFICATION OF BIOTRANSFORMATION: EFFECT OF DIET, CIRCADIAN RHYTHM, HORMONES AND PREGNANCY
- -mineral and vitamin deficiency and starvation decrease biotransformation.
- CIRCADIAN RHYTHM
- biotransformation is related to time of day because of endocrine changes, example GSH.
- -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.
- -many enzymes decrease during pregnancy, related to progesterone which is an inhibitor of some enzymes
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