biomarkers of toxicity

Question 1

what is a biomarker

Answer 1

a characteristic that is objectively measured and evaluated as an indicator of:
normal biological processes, pathological processes, pharmacological responses to a therapeutic intervention

Question 2

what are applications of biomarkers in toxicology

Answer 2

confirmation of exposure

evaluation of biological consequences of exposure

detection of initial and intermediate stages of pathological processes

to enable to basing of decisions regarding interventions before adverse effects appear

to identify susceptible individuals in a population

Question 3

what are groups that biomarkers of toxicology are classified into

Answer 3

exposure

effects/response to the xenobiotic

susceptibility of an organism to toxic effects of a particular toxin

Question 4

how are biomarkers of exposure used

Answer 4

measurement of toxic compound or its metabolite or the product of an interaction between a chemical and a target molecule (or early biochemical change) in biological samples from individuals

shows the organism has been exposed

indicates the level of toxic compound after absorption

they will also reflect the distribution of the chemical or its metabolites throughout the organism

they can also refer to the amount stored in a body compartment or whole body, e.g conc of PCB in blood reflects the amount accumulated in fatty tissues

Question 5

how do toxin level measurements at different sites relate to health effects and external exposure

Answer 5

internal dose measurements:

amount absorbed-amount delivered to tissue- amount delivered to cells- amount delivered to macromolecules- amount delivered to critical site- biologically effective dose

earlier in the list has increased relation to external exposure, later in the list has increased mechanistic linkage to health effects

Question 6

what are the external, internal and effective doses

Answer 6

external dose: amount of a chemical agent in contact with organism

internal dose: the total amount of a chemical agent absorbed by the organism over a period of time e.g blood conc of chemical following absorption

effective dose: true extent of exposure to target molecule/structure/cell

Question 7

what are the most common biomarkers of exposure

Answer 7

xenobiotics: parent xenobiotic or its metabolites

alkylating xenobiotics: adducts between xenobiotic and DNA and proteins

oxidising xenobiotics: damaged macromolecules (DNA, lipid, proteins), increases in oxidised glutathione, induction of superoxide dismutase

Question 8

what are examples of biomarkers of exposure by measuring conc

Answer 8

lead: lead exposure can be quantified in blood, significant effects in children can occur before long term exposure takes place, there is no apparent threshold for neurodevelopmental effects in children
methylmercury: the major source of exposure to methylmercury is from consumption of contaminated fish, it is neurotoxic and levels are detected in hair and blood

polycyclic aromatic hydrocarbons (PAHs): PAHs are a class of compounds found in crude oils, mineral oils, bitumes and tars, many individual PAHs are genotoxic carcinogens, urinary 1-OHP is used as a biomarker of exposure

cadmium: nephrotoxic, urinary cadmium is directly correlated with conc of cadmium in renal cortes (site of injury), can also be measured in blood

Question 9

during which phases of toxin exposure are different biomarkers effective

Answer 9

phase 1: exposure and absorption
phase2: distribution and metabolism
phase 3: interaction with endogenous macromolecules
phase 4: subclinical changes
phase 5: clinical signs

biomarkers of susceptibility are effective over all phases

biomarkers of exposure are effective from phase 2, except for adduct biomarkers which are effective from phase 3

biomarkers of effect are effective from phase 4

Question 10

give examples of types of xenobiotics capable of causing DNA adducts

Answer 10

polycylic aromatic hydrocarbons such as benzopyrenes and anthracenes

Question 11

what are stages of DNA adducts

Answer 11

1. formation of adducts
2. secondary modifications of DNA e.g strand breakage or an increase in the rate of DNA repair
3. structural perturbations in DNA become fixed and the affected cells often show altered function
4. DNA mutation and consequences

Question 12

how are adducts between xenobiotics and proteins usually monitored

Answer 12

using haemoglobin and albumin

e.g haemoglobin adducts are formed after exposure to several compounds including ethylene oxide and aniline

albumin adducts in blood can be a biomarker of exposure to PAHs

Question 13

what do DNA and protein adducts cause

Answer 13

protein: loss of function, enzyme inhibiton, trigger immune response, deplete cell defense, trigger cell death pathway

DNA: carcinogensis, teratogenesis

Question 14

describe biomarkers of effect/response

Answer 14

measurement of an alteration/physiological effect in an organism indicating exposure has had an effect

changes in molecular, biochemical, tissue or functional levels

ideal if they can be detected early and before adverse effects are irreversible

the requirement to be labelled as a biomarker of effect is that the state of the exposed organism has to change e.g modifcations in some parameters in blood, alterations of specific enzyme activities, increased protein expression

often biomarkers of effect are not specific for a single causative agent

includes biomarkers of: genotoxicity, pathological damage, monitoring of disease progression and prognosis

Question 15

how might biomarkers of effect be used

Answer 15

they may be used directly in hazard identification and dose response assessment

Question 16

give examples of biomarkers of response/effect

Answer 16

oxidising compounds: induction of antioxidant enzymes such as SOD

organophosphorous and carbamate insecticides: inhibition of acetylcholinesterase in red blood cells

heavy metals: induction of metallothioneins

Question 17

describe biomarkers of susceptibility

Answer 17

these biomarkers identify individuals who are particular susceptible to xenobiotics

e.g an individuals response to a drug may differ due to polymorphisms in the genome

Question 18

what classes may susceptibility biomarkers associated with ADRs be classified into

Answer 18

drug metabolising enzymes
drug transporters

^these 2; individual genetic variations influence pharmacokinetics and dynamics leading to changes in exposure conc

human lymphocyte antigens (HLAs): polymorphisms in HLAs are considered factors for increased susceptibility to hypersensitivity reactions

Question 19

what properties would an ideal toxicology biomarker have

Answer 19

it would be:

1. Specific
2. Sensitive
3. Known baseline values – narrow intra-individual variation is desirable
4. Reflect early stages of toxicity
5. Relationship to injury progression and prognosis
6. Translatable between species
7. Easily collected – i.e. invasively
8. Reliably measured – simple to perform, rapid turnaround time
9. Found in a substantial fraction of population – to allows validation
10. Predictive
11. Half-life long enough to be measured but not too long
12. Allow measurement of parent compound over metabolites allowing direct
    measure of exposure

Question 20

give examples of drug induced hepatotoxicity

Answer 20

liver tumours, cytotoxic injury, fatty liver, cirrhosis,

Question 21

what are AST and ALT

Answer 21

alanine aminotransferase (ALT) and aspartate aminotransferase (AST)

are transaminases that catalyse the transfer of alpha amino groups from amino acids to keto acids

they are released into the extracellular space when hepatocytes are damaged

ALT is mostly found in cytosol and detected first and more often in mild injury

AST is located in cytosol and mitochondria

neither are specific for liver: AST is found in liver, heart, skeletal muscle and kidney,
ALT has highest activity in liver but also found in kidney, myocardium and skeletal muscle

ALT is more sensitive and specific biomarker than AST

Question 22

how are ALT and AST used as biomarkers of hepatic injury

Answer 22

the ratio of ALT to AST is useful for differentiating drug induced liver injury from other organ injury

usually AST

Question 23

how is glutamate dehydrogenase used as a biomarker for toxicity

Answer 23

glutamate dehydrogenase (GLDH), is a mitochondrial enzyme; performs oxadative deamination of glutamate as part of urea cycle

used as a marker of mitochondrial injury

more liver specific than ALT and AST

highest concentration in liver (centrilobular regions) with smaller amounts found in kidney, small intestine, heart and muscle

levels of GLDH are normally low in most species, and increased serum GLDH is indicative of damaged or necrotic hepatocytes

ALT, AST and GLDH usually give complementary data

Question 24

how is sorbitol dehydrogenase used as a biomarker for toxicity

Answer 24

catalyses the reversible oxidation/reduction reaction between sorbitol and fructose

fairly specific to liver but is also found in small quantities in kidney and testes

has a short half life and limited stability needs to be analysed as soon as possible

used as biomarker for hepatotoxicity

Question 25

what is cholestasis

Answer 25

an impairment of bile flow from the liver, either due to functional defect in bile formation in hepatocytes or from impairment in bile secretion and flow in the bile ducts

Question 26

how is alkaline phosphatase used as a biomarker

Answer 26

they are a group of isoenzymes that dephosphorylate a variety of molecules in body tissue non specific ALP forms the majority of circulating ALP, located in liver, bone and kidney hepatic ALP is present on the surface of bile duct epithelia levels increase in cholestasis; increased synthesis and release of ALP, accumulating bile salts also increase its release from cell surface a 2 fold isolated elevation of serum ALP or an ALT:ALP ratio of no more than 2 is considered as key biomarker of cholestatic drug induce liver failure serum ALP levels vary w age

Question 27

how is gamma glutamyltransferase used as a biomarker

Answer 27

present in hepatocytes and biliary epithelial cells, renal tubules, pancreas and intestine GGT is not specific to liver, but predominant source of increased serum GGT activity is the liver GGT cleaves C-terminal glutamyl groups from amino acids and transfers them to another peptide or to an amino acid, important in glutathione metabolism increases occur earlier and persist longer than ALP in cholestatic disorders has high sensitivity for liver disease/injury but lack specificity for type of injury in alcoholic liver disease GGT serum levels can be markedly altered (>10 times the upper reference value) where ALP levels may be normal or only slightly altered due to high sensitivity for liver disease, GGT can be useful for identifying causes of altered ALP levels, ALP is usually first test for cholestasis

Question 28

what are bilirubin and bile acids

Answer 28

2 major components of bile total bilirubin (TBL) has 2 forms: unconjugated (indirect): bound to albumin and is the dominant form of total bilirubin in blood conjugated (direct): very small amounts in blood because it is normally excreted into bile

Question 29

what might altered bilirubin levels mean

Answer 29

sometimes hydrophobic drugs or fatty acids can cause elevation of unconjugated bilirubin due to displacement from albumin serum bilirubin levels reflect the ability of the hepatocyte to take up unconjugated bilirubin in blood, conjugate it with glucuronic acid and secreting it into bile where it is broken down in the intestine by bacteria an obstruction to bile flow will increase conjugated bilirubin within hepatocytes which will then be refluxed back into blood and spill into urine, resulting in an increase in total bilirubin, that is usually due to mostly to conjugated or direct bilirubin increased total bilirubin causes jaundice the conc of bile acids in serum can be elevated due to the inability of the liver to extract bile acids

Question 30

what is the difference in serum level and renal clearance of bile acids in impaired enterohepatic circulation compared to normal

Answer 30

normal levels: serum: 2-10 umol/L renal clearance: urine 8umol/L impaired enterohepatic circulation: serum 12-300umol/L renal clearance: urine 120umol/L

Question 31

how are plasma proteins used as a biomarker

Answer 31

albumin, clotting factors and globulins (but not gamma globulin) are synthesised in liver albumin is most abundant protein in blood plasma and is major determinant of plasma oncotic pressure and transports numerous substances normally serum total protein conc is in direct proportion to serum albumin conc plasma protein alterations are generally associated w decreased production (liver) or increased loss (kidney) or acute inflammatory conditions

Question 32

what is a classic biomarker for the diagnosis of acute kidney injury

Answer 32

decrease in creatinine clearance detected via a rise in serum creatinine and an increase in blood urea nitrogen

Question 33

what is creatinine

Answer 33

produced spontaneously from creatine and phosphocreatine creatinine is freely filtered and not reabsorbed so reflects filtration power of the kidney (glomerular filtration rate) creatinine is also secreted by the tubular cells in the tubular lumen especially if renal function is impaired and is altered by muscle mass creatinine clearance is more reliable and compares amount of creatinine in blood with that in urine in a 24 hour period glomerular filtration rate=[ Urinary creatinine x volume]/ Serum creatinine

Question 34

how is blood urea nitrogen used as a biomarker

Answer 34

urea is the primary end product of protein catabolism in most species and BUN is freely filtered by the glomerulus however urea is reabsorbed to varying degrees in the nephron back into the bloodstream which comprises its value as a biomarker also the rate of urea production is related to protein in diet, however usually the production of urea is sufficiently constant to provide a reasonable index of renal function with serum BUN being inversely proportional to glomerular filtration rate while serum creatinine and BUN levels are considered important biomarkers significant and potentially irreversible damage may have already occurred by the time changes are observed

Question 35

how is kidney injury molecule 1 used as a biomarker

Answer 35

KIM-1 is a type 1 transmembrane protein containing a immunoglobulin-like domain and a mucin domain in response to acute kidney injury the N terminal immunoglobulin and mucin ectodomain are cleaved by metalloproteinase from the apical surface of the proximal tubule and the ectodomain appears in urine soon after injury it is not detectable in normal kidney tissue or urine but expression is upregulated on the apical surface of proximal tubule cells in response to injury KIM-1 is sensitive and specific, has no interferences w pathologies unrelated to kidney and is highly stable in urine KIM-1 is virtually absent in normal kidneys and is predominantly expressed in tubular cells that also express dedifferentiation and proliferation markers, it is unknown if it actively regulates the inflammation process or its expression is just a response to damage, but it is believed to play a role in renal regeneration processes patients within the highest KIM-1 quartile have a 3.2 fold higher odds ratio for dailysis or hospital death compared to patients within the lowest quartile

Question 36

how is NGAL used as a biomarker

Answer 36

neutrophil gelatinase associated lipocalin NGAL is a protein of the lipocalin family lipocalin proteins bind and transport small molecules NGAL was orginally identified in neutrophils but is also expressed in kidney, liver and epithelial cells increased in response to inflammation, infection, intoxication, ischemia, acute kidney injury and neoplastic transformation it has been shown to have protective functions against infection and ischemic kidney injury thought to be through bacteriostatic and anti apoptotic effects only low levels of NGAL are detectable in urine normally immediately following acute kidney injury NGAL is upregulated in the distal part of the nephron, this leads to increased urinary and plasma NGAL levels, presumably due to secretion from nephron epithelia impaired proximal tubular reabsorption in the setting of proximal tubular injury may further potentiate increased NGAL levels in urine NGAL is a good biomarker for acute kidney injury and its outcomes NGAL levels are dependent on gender, age, liver function and correlate w inflammatory parameters

Question 37

how is cystatin C used as a biomarker

Answer 37

cystatin C is a protein produced by all nucleated cells at a constant rate it is freely filtered by glomerulus, completely reabsorbed by the proximal tubulues and is not secreted by the renal tubules, considered a marker for glomerulus serum cystatin C level has no association w age, sex and muscle mass and is therefore thought to be a superior marker for glomerular filtration rate than serum creatinine level, using cystatin C based equations or combined equations including serum creatinine and cystatin C cystatin C is thought to be a predictor of outcome in patients with kidney failure and for staging chronic kidney disease however cystatin C levels can be affected by inflammation, hyperthyroidism, high dose steroids and triglyceride levels can also be used as a biomarker of cardiovascular events

Question 38

how are various biomarkers of acute kidney injury effective with respect to time from injury

Answer 38

NGAL is quickest; effective almost immediately from injury up until 24 hours after KIM-1 is effective for 24 hours from 3-6 hours after injury cystatin C is effective from just under 24 hours to just over 48 hours after injury creatinine is effective from just under 48 hours after injury onwards

Question 39

how can an ECG be used as a biomarker

Answer 39

xenobiotics primarly interfere w metabolic processes or alter ionic processes causing necrosis and inflammation of the myocardium cardiotoxicity can be detect by changes in ionic flow of the heart; detected by an ECG; a change in QRS, increase in amplitude of T wave and ST elevation/ depression, Q wave evolution are indicators of injury

Question 40

what are biomarkers of necrosis

Answer 40

troponins and myoglobin

Question 41

how is troponins used as a biomarker

Answer 41

they are regulatory proteins found in skeletal and cardiac muscle contains three subunits: troponin I, troponin T and Troponin C troponin C binds calcium, I binds actin and inhibits actin-myosin interaction and T binds tropomyosin when calcium binds to troponin C the myosin binding site on actin is exposed both T and I subunits have distinct isoforms for each muscle type; there are specific cardiac isoforms (cardiac troponin T and I) trace amount of both are present in cytosol cardiac specific troponin T and I are the most specific biomarkers related to cardiac damage during injury cardiac troponins are released into bloodstream troponin levels begin to rise 2-8 hours after the onset of myocardial injury such as myocardial infarction, peaks 24-48 hours after injury there is direct relationship between degree of elevation of troponin levels and prognosis

Question 42

how is myoglobin used as a biomarker

Answer 42

low molecular weight haem protein found in cytoplasm of both skeletal and cardiac muscle rapidly released into circulation upon tissue necrosis and injury to muscle tissue including myocardium, myoglobin is generally accepted as one of the earliest markers to appear serum levels increase within 1 to 3 hours of muscle damage and peak at 6-9 hours, return to normal by 24-36 hours since myoglobin is also present in skeletal muscle elevated levels are not specific for heart it is a negative predictor of myocardial injury in setting o mycardial infarction; if myoglobin levels remain within reference range 8 hours after onset of chest pain MI can be ruled out peak size gives an indication of injury with adoption of troponin testing as standard for acute injury the value of myoglobin as a marker has significantly decreased

Question 43

how is C reactive protein used as a biomarker

Answer 43

it is an acute phase protein produced by liver in response to cytokine production during tissue injury, inflammation or infection the main biologic function of C reactive protein is its ability to recognise pathogens and damaged cells of the host and to mediate their elimination by recruiting the complement system and phagocytic cells CRP is also produced in smooth muscle cells within coronary arteries and expressed preferentially in disease or injured vessels CRP has been shown to affect expression of adhesion molecules, impact fibrinolysis and alter endothelial dysfunction and mediate atherogenesis CRP is a non specific marker of inflammation, levels are increased in acute infections, inflammatory diseases such as rheumatoid arthritis, increased BMI, advanced age, and hypertension CRP measurement serves as an overall indicator of inflammation in a given individual regardless of the source of the inflammation, but is considered one of the strongest predictors of risk levels of traditional CRP vary significantly in inflammatory disease, however high sensitivity CRP immunoassays detect low range differences and this assay is the focus for cardiovascular disease

Question 44

how are natriuretic peptides used as biomarkers

Answer 44

the natriuretic peptide family play a major role in the regulation of cardiovascular, endocrine and renal homeostasis the synthesis and secretion of natriuretic peptides (ANP and BNP) from cardiac atria and ventricles is predominantly due to wall stretch, ventricular dilation and ischaemia ANP and BNP derive from precursors, the prohormones which contain a single peptide sequence at the N terminal end these hormones are natural antagonists for sympathetic nervous system and the renin-angiotensin-aldosterone axis; they protect the CV and other systems from the effects of volume overload plasma conc of these peptides is increased in diseases or injury characterised by an increased fluid volume such as renal failure and congestive heart failure BNP and proNT-BNP (produced by cleavage of proBNP, is the inactive part), are approved biomarkers for heart failure, used for diagnosis and monitoring of patients with CHF concentrations are related to the severity of symptoms, the degree of left ventricular dysfunction and cardiac filling pressures however elevated BNP conc are also found in diseases such as unstable angina, hypertension, low BMI and pulmonary disease females have higher levels than men, levels also increase w age