1ENZYME MARKERS OF ENZYME MARKERS OF TOXICITYTOXICITYWhy do we need enzyme markers?In vivomonitoringSerial samplingEarly detection of metabolic changesDetection of organ-specific effectsEstablishment of “NO EFFECT” levelDetermination of toxic mechanismIs required by regulatory agenciesENZYMES: “highly specialized proteins that facilitate biochemical reactions that otherwise would proceed at a much lower rate”Are usually confined to a specific cellular (membrane, cytosol, mitochondria) and/or organ locationSensitive to membrane integrity, changes in metabolism, excretion, inactivationThe magnitude of response often correlates with the severity of damageWhy “enzyme markers” ?2BIOMARKERS: molecular, biochemical, or cellular alterations that are measurable in biological samples (tissues, cells or fluids)Where do enzyme markers fit?Markers of internal dose: blood & urine levels, fat concentrations, exhaled breath, metabolites in urineMarkers of biologically active dose: DNA & protein adducts (both in cells and in body fluids)Markers of early biological effect: genetic alterations in target and reporter genes, nuclear aberrations, altered enzymatic activitiesMarkers of altered structure/function: enzyme markers, proliferation, cell differentiation, differential expression of genes, cellular/tissue changesWhere do enzyme markers fit?From: Kensler T.W. SOT 1992 (AM#2)Laboratory evaluation of organ-specific toxicityIMPORTANT ISSUES TO REMEMBER:Cell types differ in susceptibility to toxic agentsOne organ – many cell typesCellular injury vs.organ function impairmentOxygen concentration gradientsMetabolizing enzymes (e.g., Cyt. P450) concentration gradients3P.C.K.C.I.C.® LiverCancer.com ® EM Atlas of Mammalian Tissues in the Internet(Universität Mainz, Germany) ® Rashmil SaxenaLocalization of damage:Centrilobular (zone 3):¾ Most hepatotoxicants (CCl4, APAP)¾ Less oxygen + high P450 concentrationPeriportal (zone 1):¾ Phosphorus, aflatoxin, allyl alcohol¾ High oxygen + highest dose at the siteMidzonal (zone 2): berylliumMassive necrosis: iproniazid, MAO inhibitorsLIVER TOXICITY123Portal triadCentral VeinCholestatic InjuryCytotoxic InjuryDisturbances of hepatic function/clearanceLIVER TOXICITY4Serum enzyme testsHepatic excretory tests* Alterations in chemical constituents of the liver* Histological analysis of liver injuryEvaluation of liver toxicity in vivoZimmerman classification of serum enzymes to monitor liver injury:1. Cholestatic Injury (ALP, 5’-NT, GGT)2. Cytotoxic Injury:A. Somewhat non-specific enzymes (AST; LDH)B. Enzymes that are found mainly in liver (ALT)C. Enzymes that are found only in liver (OCT; SDH)3. Enzymes relatively insensitive to hepatic injury (e.g., creatine phosphokinase)4. Enzymes that demonstrate reduced serum activity in liver disease (cholinesterase)LIVER TOXICITYfrom Hayes A.W. Principles and Methods of Toxicology, 4thEdition (Taylor & Francis, 2000)ANIT – α-naphtylisothiocyanateBSP – sulfobromophthalein5LIVER TOXICITYI. Markers of cholestatic injury:A. Enzymatic:Alkaline Phosphatase [AP, ALP] (membrane)Hydrolyzes phosphate esters (e.g. ATP) at pH>7.0Normal circulating levels contributed by: intestine/bone (rat), intestine/bone/liver/placenta (humans)Many isoforms: humans-3, rats-2Affected by diet, age, pregnancy and other factorsNot a very reliable marker in rat studies (diet, strain)LIVER TOXICITYI. Markers of cholestatic injury:A. Enzymatic:5’-Nucleotidase [5'-NT] (membrane)Hydrolyzes nucleoside 5’-monophosphatesNormally present in: kidney, intestinal mucosa, etc.Many isoforms: humans-3, rats-2Is made soluble from membranes by a detergent or bile acids – released during cholestasisLIVER TOXICITYI. Markers of cholestatic injury:A. Enzymatic:γ-Glutamyl Transpeptidase [GGT] (membrane)Participates in the transfer of amino acids across the cellular membrane and in glutathione metabolismHigh concentrations are found in the liver and kidney GGT is measured in combination with other tests: ALP is increased in hepatobiliary disease and bone disease; GGT is elevated in hepatobiliary disease, but not in bone disease6I. Markers of cholestatic injury:B. Non-enzymatic markers:Total Serum Bile AcidsSynthesized in the liver, important for digestion and absorption of lipids and lipid-soluble vitaminsRelatively sensitive, early marker of cholestasisCould be affected by altered enterohepatic circulation and impaired hepatic function LIVER TOXICITYLIVER TOXICITYI. Markers of cholestatic injury:B. Non-enzymatic markers:Plasma Bilirubin (Direct and Total)Heme Æ biliverdin Æ bilirubin Æ conjugated bilirubinCholestasis: direct (conjugated) is > 50% total bilirubinHemolysis: direct (conjugated) is < 50% total bilirubinLIVER TOXICITYII. Markers of hepatocellular injury:A. Somewhat non-specific enzymes:Aspartate aminotransferase [AST] (cytosol/mitochondria)L-aspartate + 2-oxoglutarate ÅÆoxaloacetate + glutamatea.k.a.: serum glutamate-oxaloacetate transaminase (SGOT)Normally present in a wide variety of tissues (skeletal muscle, heart muscle, liver, etc.)AST in serum is stable: RT- 3 days; frozen – 30 daysRed blood cells are loaded with AST: be careful (hemolysis)7II. Markers of hepatocellular injury:A. Somewhat non-specific enzymes:Lactate Dehydrogenase [LDH] (cytosol)pyruvate ÅÆ L-lactateNormally present in a wide variety of tissuesFive isoenzymes, isoenzyme profile may help identify specific tissue origin (LDH-5 Æ liver; LDH-1,-2 Æ kidney)LIVER TOXICITYII. Markers of hepatocellular injury:B. Enzymes found mainly in liver:Alanine aminotransferase [ALT] (cytosol)L-alanine + α-ketoglutarate ÅÆ pyruvate + glutamatea.k.a.: serum glutamate-pyruvate transaminase (SGPT)Greatest activity is found in the liverActivity can be found in serum and CSF, but not in urineStable at RT, frozen and refrigeratedHemolysis has a negligible effect on ALT activityLIVER TOXICITYLIVER TOXICITYII. Markers of hepatocellular injury:C. Enzymes almost exclusively located in liver:Ornithine carbamyl transferase [OCT] (mitoch.)ornithine Æ citrullineIs found in liver (<97%) and sm. intestine (<2%)Activity increases in both acute and chronic liver diseaseDiagnostically useful in all speciesAs sensitive as histopathological examination of the liverIs elevated after acute obstruction of bile flow8LIVER TOXICITYII. Markers of hepatocellular injury:C. Enzymes almost exclusively located in liver:Sorbitol dehydrogenase [SDH] (cytosol)D-sorbitol ÅÆ