biomarkers of toxicity Flashcards
what is a biomarker
a characteristic that is objectively measured and evaluated as an indicator of:
normal biological processes, pathological processes, pharmacological responses to a therapeutic intervention
what are applications of biomarkers in toxicology
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
what are groups that biomarkers of toxicology are classified into
exposure
effects/response to the xenobiotic
susceptibility of an organism to toxic effects of a particular toxin
how are biomarkers of exposure used
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
how do toxin level measurements at different sites relate to health effects and external exposure
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
what are the external, internal and effective doses
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
what are the most common biomarkers of exposure
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
what are examples of biomarkers of exposure by measuring conc
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
during which phases of toxin exposure are different biomarkers effective
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
give examples of types of xenobiotics capable of causing DNA adducts
polycylic aromatic hydrocarbons such as benzopyrenes and anthracenes
what are stages of DNA adducts
- formation of adducts
- secondary modifications of DNA e.g strand breakage or an increase in the rate of DNA repair
- structural perturbations in DNA become fixed and the affected cells often show altered function
- DNA mutation and consequences
how are adducts between xenobiotics and proteins usually monitored
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
what do DNA and protein adducts cause
protein: loss of function, enzyme inhibiton, trigger immune response, deplete cell defense, trigger cell death pathway
DNA: carcinogensis, teratogenesis
describe biomarkers of effect/response
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
how might biomarkers of effect be used
they may be used directly in hazard identification and dose response assessment
give examples of biomarkers of response/effect
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
describe biomarkers of susceptibility
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
what classes may susceptibility biomarkers associated with ADRs be classified into
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
what properties would an ideal toxicology biomarker have
it would be:
- Specific
- Sensitive
- Known baseline values – narrow intra-individual variation is desirable
- Reflect early stages of toxicity
- Relationship to injury progression and prognosis
- Translatable between species
- Easily collected – i.e. invasively
- Reliably measured – simple to perform, rapid turnaround time
- Found in a substantial fraction of population – to allows validation
- Predictive
- Half-life long enough to be measured but not too long
- Allow measurement of parent compound over metabolites allowing direct
measure of exposure
give examples of drug induced hepatotoxicity
liver tumours, cytotoxic injury, fatty liver, cirrhosis,
what are AST and ALT
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
how are ALT and AST used as biomarkers of hepatic injury
the ratio of ALT to AST is useful for differentiating drug induced liver injury from other organ injury
usually AST
how is glutamate dehydrogenase used as a biomarker for toxicity
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
how is sorbitol dehydrogenase used as a biomarker for toxicity
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
what is cholestasis
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
how is alkaline phosphatase used as a biomarker
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
how is gamma glutamyltransferase used as a biomarker
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
what are bilirubin and bile acids
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
what might altered bilirubin levels mean
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
what is the difference in serum level and renal clearance of bile acids in impaired enterohepatic circulation compared to normal
normal levels:
serum: 2-10 umol/L
renal clearance: urine 8umol/L
impaired enterohepatic circulation:
serum 12-300umol/L
renal clearance: urine 120umol/L
how are plasma proteins used as a biomarker
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
what is a classic biomarker for the diagnosis of acute kidney injury
decrease in creatinine clearance detected via a rise in serum creatinine and an increase in blood urea nitrogen
what is creatinine
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
how is blood urea nitrogen used as a biomarker
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
how is kidney injury molecule 1 used as a biomarker
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
how is NGAL used as a biomarker
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
how is cystatin C used as a biomarker
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
how are various biomarkers of acute kidney injury effective with respect to time from injury
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
how can an ECG be used as a biomarker
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
what are biomarkers of necrosis
troponins and myoglobin
how is troponins used as a biomarker
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
how is myoglobin used as a biomarker
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
how is C reactive protein used as a biomarker
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
how are natriuretic peptides used as biomarkers
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
