Biomarkers as biological indicators of xenobiotic exposure

JOURNAL OF APPLIED TOXICOLOGYJ. Appl. Toxicol. 21, 245–255 (2001)DOI:10.1002/jat.769

REVIEW ARTICLE

Biomarkers as Biological Indicators ofXenobiotic Exposure

Fernando Gil* and Antonio PlaDepartment of Legal Medicine and Toxicology. University of Granada, Madrid, Spain

Key words: biomarkers; human and environmental toxicity; classification; risk assessment; Biochemical and molecular markers.

The presence of a xenobiotic in the environment always represents a risk for living organisms. However, totalk about impregnation there is a need to detect toxicity in the organism, and the concept of intoxicationis related to specific organ alterations and clinical symptoms. Moreover, the relationship between the toxiclevels within the organism and the toxic response is rather complex and has a difficult forecast because itdepends on several factors, namely toxicokinetic and genetic factors. One of the methods to quantify theinteraction with xenobiotics and its potential impact on living organisms, including the human being, ismonitoring by the use of the so-called biomarkers. They can provide measures of the exposure, toxic effectand individual susceptibility to environmental chemical compounds and may be very useful to assess andcontrol the risk of long-term outcomes associated with exposure to xenobiotic (i.e. heavy metals, halogenatedhydrocarbons, pesticides). Copyright 2001 John Wiley & Sons, Ltd.

INTRODUCTION

Definition

The National Academy of Sciences defines a biomarkeror biological marker as a xenobiotically induced alterationin cellular or biochemical components or processes, struc-tures or functions that is measurable in a biological systemor sample.1 Silbergeld et al.2 defines biological markers asphysiological signals that reflect exposure, early cellularresponse or inherent or acquired susceptibilities, whichprovide a new strategy for resolving some toxicologicalproblems.

Sensu stricto, we define a biomarker as a biologicalresponse to a chemical or a group of chemical agents butnot the presence of the agent or its metabolites within thebody (internal dose).3 However, there is no doubt that themeasurement of a xenobiotic in a biological system orsample is a bioindicator of exposure, and thus it could beconsidered a biomarker.

Biological monitoring has advantages over environmen-tal monitoring because it measures the internal dose ofa compound. Inter-individual differences in absorption,bioavailability, excretion and DNA repair should be takeninto account. Moreover, intra-individual differences, as aconsequence of particular physiopathological alterationsoccurring in a specific period of time, also should be con-sidered. This involves an individualized biological controlto evaluate the exposure to a particular xenobiotic.4 Theorganism acts as an integrator of exposure and several

* Correspondence to: F. Gil, Departamento de Medicina Legal y Tox-icologia, Facultad de Medicina, Universidad de Granada, C/Avda.Madrid 11, 18071 Granada, SPAIN. E-mail: [email protected]

physiological factors, which modulate the uptake of toxic.Thus, we may state that a collective cannot be assimilatedas a homogeneous group of individuals exposed to a xeno-biotic of physicochemical properties under reproducibleand standard conditions.

The use of biological markers in the evaluation ofdisease risk has increased markedly in the last decade.Biomarkers are observable end points that indicate eventsin the processes leading to disease. They are particu-larly useful in the evaluation of progressive diseases thatmanifest their symptoms long after exposure to initiatingfactors. In such cases, traditional early warning symptomsof developing disease may be lacking. At the same time,the disease, once clinically apparent, may be essentiallyirreversible.5

The two main research fields in the use of biomarkersin toxicology are enviromental toxicology and industrialtoxicology, the latter being one of the most relevant andimportant branches of medical toxicology.

Conditions and limitations of biological monitoring

A rational biological monitoring is possible only whensufficient toxicological information has been gathered onthe mechanism of action and on the toxicokinetic of xeno-biotics, including absorption, distribution, metabolism andexcretion.6 Biological monitoring cannot be applied forassessing exposure to substances that exhibit their toxiceffects at the sites of first contact, i.e. primary lung irritantsor those that are poorly absorbed.

For other xenobiotics that are absorbed significantlyand exert a systemic toxic action, a biological monitor-ing test may provide different information, depending onour current knowledge of the relationships between exter-nal exposure, internal exposure and the risk of adverse

Copyright 2001 John Wiley & Sons, Ltd. Received 7 January 2001

246 F. GIL AND A. PLA

effects. If only the relationship between external exposureand the internal dose is known, the biological parametercan be used as an index of exposure but it provides lit-tle information on the health risk. But if a quantitativerelationship has been established between internal doseand adverse effects, and the internal dose effects and theinternal dose response relationships are known, biologicalmonitoring allows for a direct health risk assessment andthus for effective prevention of the adverse effects.7

Specificity of biomarkers

The ideal biomarker should have the following characteris-tics:8

(i) Sample collection and analysis is simple and reliable.(ii) The biomarker is specific for a particular type of

exposure.(iii) The biomarker only reflects a subclinical and rever-

sible change.(iv) Relevant intervention or other preventive effort can

be considered.(v) Use of the biomarker is regarded as ethically accept-

able.

It is clear that if we agree with the last definition only afew biomarkers fit well. Biomarkers range from those thatare highly specific, such as an enzyme of the haeme path-way, aminolevulinic acid dehydratase (ALAD), which isinhibited only by lead or the inhibition of acetylcholineesterase (AChE), which is specific to the organophos-phorus and carbamate pesticides, to those that are non-specific, namely the effects on the immune system or DNAthat can be caused by a wide variety of chemical agents(see Table 1).3

An important aspect to be considered is the complemen-tation among biomarkers that results in a higher degreeof specificity. Thus, metallothionein induction may occurby exposure to a variety of metals. However, if a mea-surement of one specific metal is performed in a bio-logical fluid, i.e cadmium in urine, and it is found ele-vated over normal values, the evaluation of the inductionof metallothionein would enhance the specificity of themeasurement, which in turn would increase even moreif a preclinical renal alteration such as renal proteinuria(i.e. β2-microglobulin) is detected. This feature is relevant

Table 1—Biomarkers listed in order of decreasing specificityto xenobiotics3

Biomarker Xenobiotic

Inhibition of ALAD Lead

Inhibition of AChE Organophosphorus

compounds and carbamates

Induction of

metallothionein

Metals (cadmium)

Eggshell thinning DDT, DDE, Dicofol

Porphyrin Organochlorine compounds

(OCs)

Heat shock proteins Metals and OCs

Immune response Metals, OCs and polycyclic

aromatic hydrocarbons

(PAHs)

DNA and haemoglobin

adducts

PAHs, nitrosamine, aromatic

amines, chemotherapeutic

agents

because currently there are a number of biomarkers oftoxic response in different tissues, organs, and systems thatare non-specific. However, the establishment of new rela-tionships between biomarkers may contribute to increasetheir specificity.

CLASSIFICATION OF BIOMARKERS

Biomarkers are generally classified into three groups:exposure, effects and susceptibility.2,3,9,10

Biomarkers of exposure

These allow measurement of the internal dose by chemicalanalysis of the toxic compound or metabolite in bodyfluids or excreta such as blood, urine and exhaled air(Table 2).10,11

Internal dose also may mean the amount of a chem-ical stored in one or several body compartments or inthe whole body. This usually applies to cumulative toxicchemicals. For example, the concentration of polychlo-rinated biphenyl (PCB) in blood is a reflection of theamount accumulated in the main sites of deposition (i.e.fatty tissues). The internal dose reflects the amount ofchemical bound to the critical sites of action.12

Bernard and Lauwerys classified the biomarkers ofexposure into two subgroups–selective and non-selec-tive—according to their selectivity test, which is based onthe direct measurement of unchanged chemicals or theirmetabolites in biological media. The non-selective testsare used as non-specific indicators of exposure to a groupof chemicals. As examples of non-selective exposuretests, the determination of diazo-positive metabolites inurine for monitoring exposure to aromatic amines, theanalysis of thioethers in urine and the determination ofthe mutagenic activity of urine can be cited.

When assessing the usefulness of a particular exposurebiomarker, one must consider two aspects of validity: ana-lytical and toxicokinetic. For optimal analytical qualitystandardization is needed, but the specific requirementsvary considerably between individual toxicants. Majorareas of concern include: preparation of the individual,sampling procedure and sampling handling; and the mea-surement procedure, which encompasses technical factorssuch as calibration and quality assurance procedures. Lifeevents, such as reproduction and senescence, also mayaffect the toxicokinetic of a xenobiotic.8

Biomarkers of susceptibility

These serve as indicators of a particular sensitivity of indi-viduals to the effect of a xenobiotic or to the effectsof a group of such compounds. They can be geneticmarkers that include alterations in chromosomal struc-ture, such as restriction fragment length polymorphisms(RFLPs), polymorphism of enzyme activities, etc.13 Afterthe exposure of an organism to a xenobiotic it suffers abiotransformation process in two phases. In the first phasea primary metabolite, usually oxidized and more or lessactive, originates by the specific action of the microso-mal P-450 cytochrome isoenzymic family. In the secondphase, the primary metabolite is transformed into anothersecondary metabolite, which is usually inactive. Some

Copyright 2001 John Wiley & Sons, Ltd. J. Appl. Toxicol. 21, 245–255 (2001)

BIOMARKERS FOR XENOBIOTIC EXPOSURE 247

Table 2—Examples of selective exposure biomarkers

I. Inorganic compounds

Cadmium Cd Urine <2 µg g−1 creatinine

Cd Blood <0.5 µg 100 ml−1

Mercury Hg Urine <5 µg g−1 creatinine

Hg Blood <1 µg 100 ml−1

Lead Pb Blood <30 µg 100 ml−1

Pb Urine <50 µg g−1 creatinine

Pb (after 1 g EDTA) Urine <600 µg 24 h−1

Zinc Zn Urine <0.7 mg g−1 creatinine

Zn Serum <15 µg l−1

II. Organic compounds

n-Hexane 2-Hexanol Urine 0.2 mg g−1 creatinine

2,5-Hexanodione Urine 2 mg g−1 creatinine

Benzene Phenol Urine <20 mg g−1 creatinine

Benzene Blood <5 µg 100 ml−1

Benzene Exhaled air <0.022 ppm

Styrene Mandelic acid Urine 1 g g−1 creatinine

Phenylglyoxilic acid Urine 350 mg g−1 creatinine

Styrene Blood 0.055 mg 100 ml−1

Styrene Exhaled air 18 ppm

Aniline Aniline Urine 0.75 mg g−1 creatinine

p-Aminophenol Urine 10 mg g−1 creatinine

Methaemeglobin Blood <2%

Ethylene glycol Oxalic acid Urine <50 mg g−1 creatinine

M-n-butylacetone 2,5-Hexanodione Urine 4 mg g−1 creatinine

Acetone Acetone Urine <2 mg g−1 creatinine

Acetone Blood <0.2 mg 100 ml−1

individuals with a low cytochrome P-450 activity will bemore resistant to the generation of primary active metabo-lites, whereas those exhibiting a low activity of enzymesinvolved in the second phase will show a lower formationof phase II inactive metabolites, increasing the toxicity.

Two types of susceptibility biomarkers can be distin-guished: polymorphisms activating system markers andpolymorphisms of detoxicating systems. Polymorphismsof activating systems are measurements of the activity ofcytochrome P-450 isoenzymes. The family of cytochromeP-450 enzymes is involved in the toxicity of several xeno-biotics; associated with the P-450 cytochromes there are awide range of enzyme activities, referred to as monooxy-genase activities. A number of studies have suggested thatthe various cytochrome P-450 enzymes differ substan-tially in their amino acid sequences and thus are likelyto be encoded by distinct genes. This has been confirmedby comparisons of the complete amino acid sequences ofover 71 forms of cytochrome P-450 and of the nucleotidesequences of their corresponding cDNAs and of severalgenes.11

A roman numeral corresponding to its specific classdesignates each form of cytochrome P-450. The mostimportant classes that constitute the different forms ofcytochrome P-450 are I–IV. These cytochrome classescomprise several subclasses that are designated by a com-bination of a letter (A,B,C,D,..) and an identifying ara-bic numeral, i.e. IA1, IIC8, etc. The most important areIA1 (representing Aryl Hidrocarbon Hydroxylase (AHH)activity), IIC8, IID6 and IIE1. There have been a num-ber of studies trying to establish a relationship betweenspecific cytochrome P-450 activities and some diseasesdue to environmental toxic exposure, especially cancer.However, there are no definitive conclusions.14,15

Markers of polymorphisms of detoxicating systems aremeasurements of the activity of conjugating enzymessuch as glutathione-S-transferases, acetyltransferases, sul-fotransferases, glucuronyltransferases and paraoxonase.For instance, predisposition to cancer has been corre-lated with genetic polymorphisms of N -acetyltransferases.N -Acetyltransferase is an enzyme involved in the deac-tivation of aromatic amines. After acetylation there isenhanced excretion in urine. In a group of arylamine-exposed workers, the slow acetylators are at increased riskof bladder cancer versus rapid acetylators. Another exam-ple is glutathione-S-transferase µ, an enzyme involved inthe detoxification of reactive metabolites. Half of the pop-ulation has no functional allele for this enzyme and no orlow enzyme activity. These persons are at increased riskof squamous cell carcinoma of the lung.11

Finally, several organophosphates can be inactivated(hydrolysed) by paraoxonase (PON1). Human paraox-onase exhibits an important polymorphism and in humansthree genotypes have been detected: individuals homozy-gous for the low activity allele; individuals homozy-gous for the high activity allele; and individuals whoare heterozygous. Thus paraoxonase activity can be usedas a biomarker of susceptibility to organophosphoruscompounds.16 The polymorphism also is observed withthe oxons of methyl parathion, chlorthion and ethyl 4-nitrophenyl phenylphosphonate (EPN). However, it is notobserved with the oxon of chlorpyrifos.

Several pieces of evidence suggest that high levelsof serum paraoxonase are protective against poisoningby organophosphorus pesticides whose active metabolitesare substrates of this enzyme.17 Birds, which have verylow levels of serum paraoxonase, are very sensitive toparathion, diazinon-oxon and pirimiphos-oxon compared



with mammals who have higher levels of this enzyme.18

After injection of partially purified rabbit paraoxonaseinto rats, an increased resistance to the toxic effectsof paraoxon was observed. Recent studies indicate thatadministration of paraoxonase might have therapeuticvalue in the case of organophosphate intoxication.19

Response or effect biomarkers

Response or effect biomarkers are indicative of biochem-ical changes within an organism as a result of xenobi-otic exposure. The ideal biomarkers should be detectedearly and be able to show adverse effects before they areirreversible. Those are the most studied biomarkers andthey include modifications in some parameters of bloodcomposition, alterations of specific enzyme activities, theappearance of DNA adducts, localized mRNA and pro-tein increases and the appearance of specific antibodies(autoantiboides) against a xenobiotic or a particular cellu-lar fraction.10

It is noticeable that it is not always easy to distinguishbetween an exposure and a response biomarker. Perhapsthe most typical example is the formation of a DNAadduct—an exposure biomarker whose formation resultsfrom the reaction of a xenobiotic with the DNA, whichin turn constitutes the cellular response. Moreover, itis evident that a particular response requires a previousexposure to the xenobiotic. Below, we consider somesignificant examples of response biomarkers.

Respiratory system. Several studies have suggestedthat low-molecular weight proteins (LMWP) specific forthe lung might serve as peripheral biomarkers of lungtoxicity.20 A lung biomarker, measurable in serum, bron-choalveolar fluid (BAL) and sputum, has been identifiedrecently. This biomarker is a microprotein initially iso-lated in 1974 from urine (Urine Protein 1) of patients withrenal tubular dysfunction and subsequently identified asthe major secretory product of the lung Clara cells, whichare non-ciliated cells localized predominantly in terminalbronchioles. This protein, called Clara cell protein (CC16),is a homodimer of 15.8 kDa. Clara cells are particularlysensitive to toxic lung injury and they contain most of thelung cytochrome P-450 activity, which gives them highxenobiotic metabolizing activity.21,22 Several lines of evi-dence indicate that CC16 is a natural immunoregulatorprotecting the respiratory tract from unwanted inflam-matory reactions. The CC16 has been shown to inhibitthe activity of cytosolic phospholipase A2, a key enzymein inflammatory processes. Phospholipase A2 is the rate-limiting enzyme in the production of arachidonic acid, thesubstrate for the synthesis of prostaglandin and leukotrienemediators of inflammation.23 By inhibiting phospholipaseA2, CC16 also could prevent the degradation of lung sur-factant phospholipids.24

The CC16 secreted in the respiratory tract diffuses pas-sively by transudation into plasma, where it is eliminatedrapidly by glomerular filtration before being taken up andcatabolized in proximal tubule cells. Studies reviewed byBernard suggest that CC16 in BAL fluid, sputum or serumis a sensitive and relatively specific indicator of acute orchronic bronchial epithelium injury.

A significant reduction of CC16 in serum is an indica-tor of Clara cell number and integrity. After adjustment

for age, a linear dose–response relationship was appar-ent between smoking history and serum CC16, the latterdecreasing on average by ca. 15% for each 10 pack-year smoking history.25 Serum CC16 also was found tobe decreased in several occupational groups chronicallyexposed to silica, dust and welding fumes and lung dis-eases (cancer, asthma and patients with chronic obstruc-tive pulmonary disease).26

The increased concentration of CC16 in serum also canbe used to detect an acute or chronic disruption of thebronchoalveolar/blood barrier integrity. Increased serumlevels of CC16 have been observed in sarcoidosis andadult respiratory distress syndrome (ARDS). This con-firm that in pathological conditions the barrier betweenthe surface of respiratory epithelium and the vascularcompartment may be disrupted, upsetting the diffusionalequilibrium between CC16 in serum and in the respiratorytract. The existence of an enhanced passage of proteinsacross the blood/bronchoalveolar space barriers, e.g. inacute exposure in animals by inhalation or systemic routeswith pneumotoxic agents (4-ipomeanol, 3-methylfuran,naphthalene, trichloroethylene, 1,2-dichloroethylene, etc.),is supported by the significant elevation of albumin,β2-microglobulin and other plasma proteins in BAL flu-ids. These findings open new perspectives in the assess-ment of lung toxicity by suggesting that readily diffusiblelung-specific proteins may serve as peripheral markers ofpneumotoxicity.

Blood system. The most studied biomarkers of effectare those related to the alterations of haeme synthesis.The enzyme ALAD is involved in the haeme biosyntheticpathway and the assay is highly specific for lead exposureand effect. The inhibition of ALAD has been shown tobe a reliable indicator of the effect of lead in studieson humans and animals (especially several species offish and birds—eagles, starlings, ducks and geese). Oneof the most important advantage of this biomarker inecotoxicology is that animal sacrifice is not required; theeffect is slowly reversed, with ALAD values returning tonormal only after ca. 4 months.3,27 – 29

Haeme biosynthesis normally is closely regulated andlevels of porphyrins are ordinarily very low. Some organo-chlorines (OCs) cause the formation of excess amountsof hepatic, highly carboxylated porphyrins. The twoOCs that are most involved in inducing porphyria inmammals and birds are hexacholorobenzene (HCB) andthe PCBs.3,30

Haemoglobin adducts are formed from exposure of sev-eral compounds (ethylene oxide, acrylamide, 3-amino-1,4-dimethyl-5OH-pyrido-indole, 4-aminobiphenyl-2,6-dim-ethylaniline,etc.). Acrylamide is an important neurotoxicagent causing a peripheral neuropathy to experimental ani-mals as well as to humans, and it has been shown to be apotential carcinogen. The conversion rate of acrylamideto glycidamide (reactive metabolite epoxide responsi-ble for neurotoxicity) is significantly correlated with thehaemoglobin adducts of acrylamide. These adducts areuseful as biomarkers of acrylamide-induced peripheralneuropathy.31 Because of the relatively long lifespan ofthe red blood cells (4 months in humans), haemoglobinadducts have been used advantageously for integratingconcentrations of genotoxic substances in the blood.



Nervous system. Despite its obvious importance withintoxicology, the area of neurotoxicity seems to beprogressing more slowly than other fields with regardto biological monitoring. The complexity of the nervoussystem and its distinctive peculiarities, together with theproblems associated with determination of precise targetsfor neurotoxic action, are certainly responsible for thislimited advancement. Neurochemical measurements fordetecting neurotoxicity in humans are limited by theinaccessibility of target tissue. Thus, a necessary approachfor identifying and characterizing neurotoxicity is thesearch for neurochemical parameters in peripheral tissuesthat are obtained easily and ethically in humans and couldrepresent a marker for the same parameters in nervetissue.32

Perhaps the most significant and useful example ofa specific biomarker of neurotoxicity is the inhibitionof AChE caused by organophosphorus compounds orcarbamate pesticides. The enzyme activity is present inseveral tissues although inhibition generally is determinedfrom blood samples (whole blood or plasma) and thebrain. This biomarker has been used in human toxicologyand is studied widely in ecotoxicology (birds, mammalsand aquatic species). For example, inhibition of AChE inbrain can be taken as proof of mortality in birds, whereasin other animals, such as fish, there is a wider variabilityof lethal inhibition, in the range of 40–80%.33 – 35

The decrease in AChE activity in brain may remainfor several weeks after the toxic exposure, which isadequately correlated with the effect, in contrast to thatoccurring in blood with a lower lifespan. Nevertheless,measuring the blood AchE activity has the advantageof easy sampling because there is no need for animalsacrifice.36

Butyrylcholinesterase (BuChE)—an example of a non-specific biomarker of neurotoxicity—sometimes is stud-ied in parallel with AChE but its physiological role isunknown and its degree of inhibition is not related sim-ply to toxic effect. Other parameters involved in neuro-transmission are the target for a variety of neurotoxicantxenobiotics. These parameters are measured in red bloodcells, lymphocytes and fibroblasts.32,37

Several active bioamines are liberated from the nerveending by exocytosis, a process that is triggered by aninflux of Ca2+, and are inactivated by re-uptake andmethylation mediated by catechol-O-methyltransferase(COMT). Because of its intracellular localization, mono-amine oxidase (MAO) plays a strategic role in inactivatingcatecholamines that are free within the nerve terminal andnot protected by storage vesicles. Isoenzymes of MAOhave been characterized with differential substrate speci-ficities; MAO-A preferentially deaminates norepinephrineand serotonin, whereas MAO-B acts on a broad spec-trum of phenylethylamines. The MAO-B is a microsomalenzyme and its amino acid sequences from human cerebralcortex and consequently platelets were shown to be iden-tical. Platelet MAO-B activity appears to reflect reliablythe enzyme activity in the nervous system.

The MAO-B activity is used clinically as a marker ofthe pharmacological effects of MAO inhibitors, such as inthe treatment of Parkinson’s disease. The MAO-B activityin platelets has been used as a biomarker of the effects ofstyrene and perchloroethylene occupational exposures,38

which are known to cause dopamine depletion. Changes inMAO-B could represent an adaptive response to dopamine

depletion and, alternatively, styrene or its metabolite(s)might exert a direct inhibitory effect on the enzyme.37,39

Another example of a neurotoxic biomarker involvedin delayed toxicity is the inhibition of neuropathy targetesterase (NTE). Several organophosphorus compounds(Mipafox, Methamidofos, etc.), after a single dose,induce delayed neuropathy characterized by symmetricalaxonal degeneration, which implicates NTE inhibition andnot AChE. Organophosphate-induced delayed neuropathy(OPIDN) is characterized by a lag period of ca.1–3 weeks, from the moment of intoxication to theappearance of clinical symptoms. The first intoxicationwas described with TOCP (tri-ortho-cresylphosphate).40,41

In experimental assays the measurement of NTE inlymphocytes has been used as a biomarker of effectand there is a good correlation between NTE activity inbrain and lymphocytes after 24 h of an acute exposure toneurotoxic organophosphorus compounds.42

In conclusion, the complexity of the nervous systemdoes not allow a rapid and easy development of sensi-tive, specific and reliable biomarkers for neurotoxicity,although the biomarkers presented in this review appearpromising.

Urinary biomarkers. Long-term exposure to cer-tain nephrotoxic compounds—heavy metals (lead, mer-cury, cadmium and chromium), halogenated hydro-carbons (chloroform), organic solvents (toluene), ther-apeutic agents (aminoglucosides, amphotericine B,acetaminophen)—may cause progressive degenerativechanges in the kidney. However, because of its largereserve capacity, the clinical signs of renal damage arenot apparent until the injury is extensive and consequentlyirreversible. The prevention of renal disease requires theuse of more sensitive tests capable of detecting renaleffects at a stage when they are still reversible or atleast not so advanced as to trigger a progressive renaldisease.43 – 47

In practice, one usually recommends the determinationof at least two plasma-derived proteins in the urine: ahigh-molecular-weight protein (HMWP) such us albuminfor the early detection of a glomerular barrier defectand a low-molecular-weight protein (LMWP) such asretinol-binding protein for the early screening of proximaldamage (Table 3).48,49

Injury to the kidney can be detected by measuring theurinary activity of kidney-derived enzymes. The lysoso-mal enzyme β-N -acetyl-D-glucosaminidase (NAG) hasbeen proposed as an index of nephrotoxicity. Advantagesof this enzyme include its stability in urine and its highactivity in the kidney. The diagnostic value of NAG can beimproved further by measuring the B isoenzyme (lesionalform released with fragments of cell membranes).50

Destruction of renal tissue also can be detected by mea-suring kidney components in urine that, when they arequantified by immunochemical methods, are referred toas renal antigens. These have been proposed as urinarymarkers of nephrotoxicity and include: carbonic anhy-drase, alanine aminopeptidase and adenosine deaminase-binding protein for the proximal tubule; fibronectin forthe glomerulus; and Tamm–Horsfall glycoprotein for thethick ascending limb of the loop of Henle.50 – 55

It is important to realize that this battery of tests doesnot permit the detection of effects on all areas or segments



Table 3—Biomarkers of renal effects44

Serum

Markers of glomerular

filtration

Creatinine, β2-microglobulin

Markers of the

glomerular basal

membrane (GBM)

integrity

Laminin and anti-GBM

antibodies

Urine

Plasma-derived proteins

High molecular weight Albumin, transferrin

Low molecular weight β2-Microglobulin,

retinol-binding protein,

α1-microglobulin, Clara cell

protein, α-amylase

Kidney-derived

components

Enzymes Gluathione-S-transferase,

β-N-acetylglucosaminidase

Antigens

Glomerulus Fibronectin, laminin

Proximal tubule Brush border antigens (alkaline

phosphatase)

Loop of Henle Tamm–Horsfall glycoprotein

Others Glycosaminoglycans,

prostanoids

of the kidney or nephron. No sensitive biomarker isavailable to detect effects on the deep medulla, the papillaor the distal tubule. Also, there is no biomarker to detectand follow the progression of active fibrotic processes thatmay insidiously and irreversibly reduce the renal function(i.e. interstitial fibrosis).

Immune system. Direct effects of xenobiotics canaffect the immune system and lead to decreased resistanceto infections or tumours, alter the course of autoimmu-nity or induce hypersensitivity reactions. Data of severalimmunotoxic agents (dioxin, polychlorinated biphenyls,immunotherapeutic drugs, etc.) are derived mainly fromanimal research (mouse and rat), although a few biomark-ers exist that provide specific information on immunotox-icity in humans.56,57

The biomarkers proposed for assessing immunotoxi-city in humans are listed in Table 458 and include fullblood count, antibody-mediated immunity (immunoglob-ulin concentrations in serum), phenotypic analysis of lym-phocytes by flow cytometry, cellular immunity study,measurement of antibodies and markers of inflamma-tory response and, finally, examination of non-specificimmunity.

A variety of factors may modify the immune function,including drugs (non-steroidal anti-inflammatory, vitamincomplexes, etc.), biological parameters (gender, age, preg-nancy) and other factors (diet, alcohol consumption, circa-dian rhythms, stress, nutritional state, sleep disturbances,etc.).

Within the field of ecotoxicology, the resistance toinfection in ducks exposed to organochloride pesticideshas been studied by measuring the cellular activitiesinvolved in the immune response, particularly the in vitrophagocytic capacity from kidney-isolated macrophages inan number of species.

Table 4—Biological markers of immunotoxicity in humans58

Full blood count (includes lymphocyte count)

Study of antibody-mediated immunity

Immunoglobulin concentrations in serum (IgM, IgG,

IgA, IgE)

Phenotypic analysis of lymphocytes by flow cytometry

Surface markers (CD3, CD4, CD8, CD20, CD23, etc.)

Study of cellular immunity

Delayed-type hypersensitivity on skin

Natural immunity to blood group antigens (anti-A,

anti-B)

Autoantibodies and markers of inflammatory response

C-Reactive protein

Autoantibodies to nuclei, DNA and mitochondria

Measure of non-specific immunity

Interleukine analysis (ELISA or reserve transcription

polymerase chain reaction)

Natural killer cell activity (CD56 or CD60)

Phagocytosis (chemiluminescence)

Measurement of complement components

Biomarkers of DNA damage. At present, many tech-nological approaches permit the detection of covalentinteractions of xenobiotics with proteins and other macro-molecules. For example, several biomolecules (haemoglo-bin, serum albumin, etc.) have carboxyl, amino or sulf-hydryl reactive groups that can interact with electrophiliccompounds. Human DNA adduct formation (covalentmodification of DNA with chemical carcinogens) has beenshown to correlate with the incidence of a carcinogenicprocess and is a promising biomarker for elucidating themolecular epidemiology of cancer.59

There is a sequence of events between the first interac-tion of a xenobiotic with DNA and consequent mutation:the first stage is the formation of adducts; the next stagemay be secondary modifications of DNA, such as strandbreakage or an increase in the rate of DNA repair; andthe third stage is reached when the structural perturba-tions in the DNA become fixed and the affected cells oftenshow altered function. One of the most widely used assaysto measure chromosomal aberrations is sister chromatidexchange (SCE). Finally, when the cells divide, damagecaused by xenobiotics can lead to DNA mutation and con-sequent alterations in the descent.3,60

Some examples of toxics compounds capable of form-ing human DNA adducts are given in Table 5, includ-ing polycyclic aromatic hydrocarbons, aromatic amines,heterocyclic amines, micotoxins and chemotherapeuticagents.61,62 Biological monitoring to detect human and ani-mal DNA adducts includes 32P post-labelling and recentlyimmunoassays using adduct-specific antibodies.63,64 Theycan be detected in blood (lymphocytes), urine or tissuehomogenates from biopsy (gastric mucosa, liver, etc.),although the study of DNA adducts is not feasible in rou-tine analysis. Damage of DNA has been studied in thefield of Ecotoxicology for several marine species (fresh-water fish, snapping turtle, etc.) that can be exposed tobenzo[a]pyrene.3,65 – 67

Future investigations will focus on the implementa-tion and design of studies to assess the associationbetween DNA adduct formation and cancer risk fromtoxic compounds. Although this association is strongly



Table 5—Xenobiotics capables of human DNA-adductsformation

N-Nitrosamines 4-(N-Nitrosomethylamino-1-(3-

pyridyl)-1-butanone

(NNK)

N-Nitrosodimethylamine (NDMA)

Diethylnitrosamine (DEN)

Polycyclic aromatic

hydrocarbons

Benzo[a]pyrene (BaP)

7,12-Dimethylbenzo[a]anthracene

(DMBA)

Aromatic amines 2-Acetylaminofluorane (2-AAF)

4-Aminobiphenyl (4-ABP)

4-Iminobiphenyl (4-IBP)

Heterocyclic amines 2-Amino-3,8-dimethylimidazo-

quinoxaline

Mycotoxins Aflatoxin B1

Ochratoxin A

Chemotherapeutic

agents

Cisplatin

Mitomycin C

Procarbazine

Dacarbazine

8-Methoxypsoralen

Others Ulltraviolet light

Oxidative damage

Malondialdehyde (endogenous)

supported by animal studies, it remains to be ascer-tained whether adducts also are a necessary compo-nent of carcinogenesis in humans. Many studies arebeing designed now to correlate metabolic polymor-phisms, urinary metabolites, chromosomal aberrationsand protein and DNA adducts, and it is possible inthe future to obtain promising results from the com-bined use of these biomarkers in the evaluation of can-cer risk.

Biomarkers of gene expression. The development ofmany tumours related to xenobiotics is associated with theaberrant expression of genes that encode proteins involvedin cellular growth. This aberrant expression can involvea quantitative difference (overexpression of the protein)and a qualitative difference (expression of a mutant formof the protein). Although these biomarkers are affectednot only by toxic compounds, it is very important toestablish potential confounding factors and to assess thesensitivity, specificity and predictive value of these tests.Table 6 shows the biomarkers of gene expression, whichinclude:63,68,69

(i) Growth factors.(ii) Oncoproteins:

(a) growth factor receptors;(b) other oncogene proteins;(c) tumour-suppressor gene proteins.

These biomarkers have been studied in easily obtainablebiological fluids such as serum, plasma, urine and BAL byenzyme-linked immunoadsorbent assay (ELISA), radioim-munoassay (RIA) or immunoblotting.

It has been reported that, in subjects developing cancer,during the first stages of the disease a significant increase

Table 6—Biomarkers of gene expression69

I. Growth factors

Platelet-derived (PDGF): breast cancer, various carcinomas,

sarcomas, lymphomas, lung fibrosis, pneumoconiosis,

atherosclerosis

Transforming α (TGF-α): breast cancer, various carcinomas

and pneumoconiosis

Transforming β (TGF-β): liver cancer, bladder cancer, breast

cancer, leukaemia, liver and lung fibrosis and

pneumoconiosis

Fibroblast (bFGF): kidney cancer, bladder cancer and other

carcinomas

Epidermal (EGF): stomach and ovarian cancer

Insulin-like (IGF): bladder cancer, ovarian cancer, hepatitis

and cirrhosis

Hepatocytes (HGF): liver cancer, hepatitis and cirrhosis

II. Oncoproteins

IIa. Growth factor receptors

Transmembrane growth factor receptors (encoded by the

erbB-2 oncogene): bladder cancer, ovarian cancer, liver

cancer, lung cancer and others

Epidermal growth factor receptor, EGFR (encoded by the

c-erB-1 oncogene): lung cancer and other carcinomas

IIb. Oncogene proteins

Membrane-associated G proteins or p21 (21 kDa) (encoded

by the ras oncogene): lung cancer, colon cancer, liver

angiosarcoma and others

Nuclear DNA-binding protein (64 kDa) or p54 (54 kDa)

(encoded by the myc oncogene): lung cancer, colon

cancerard, bladder cancer

IIc. Tumour-suppressor gene proteins

Nuclear phosphoprotein p53 (53 kDa) (encoded by the

tumour-suppressor gene p53): liver cancer, breast cancer,

lung cancer, colon cancer and lymphoma

in gene-expression biomarkers related to the specific can-cer is seen.

Biomarkers of oxidative damage. Contaminants suchas polycyclic aromatic hydrocarbons (PAHs), halogenatedaromatic hydrocarbons, heavy metals, selenium, pesticidesand industrial solvents are capable of causing oxidativedamage, especially by free radicals. In response tooxidative stress, there may be adaptive responsesof the antioxidant systems, modification of cellularmacromolecules and tissue damage.

Changes in the antioxidant systems and modifiedmacromolecules can serve as biomarkers for a varietyof xenobiotics. The protective systems included oxidizedglutathione/reduced glutathione, glutathione reductase,catalase, superoxide dismutase and peroxidase activities,ascorbate and α-tocopherol. Macromolecules that may beaffected by free-radical damage include lipids, proteinsand nucleic acids.70 – 72

Metallothioneins. Metallothioneins are small proteinswith low molecular weight (ca. 61 amino acids in mostmammals), cysteine-rich and capable of binding metals(Cd, Cu, Hg, Zn, Co, Bi, Ni and Ag). The cellular roleof metallothioneins is complex and partially unknown;it is believed to protect cells against free heavy metalion-induced damage. These proteins are implicated in themetabolic regulation of Zn and Cu, making these ionsavailable to the cells as necessary and possibly acting as



sulfhydryl-rich scavengers to prevent damage from stress-induced free radicals.

Metallothioneins have been proposed as biomarkers forexposure to metal ions because they are induced by theown metals. Nevertheless, recent studies indicate that theyare inducible also by a variety of non-metallic toxic agents(glucocorticoids) and physiological conditions (nutritionalchanges and pregnancy). Another limitation concerns ana-lytical measurements. Often, methods of evaluation areslower and more expensive than analysis of the metalsthemselves. The recent development of antibody-basedmethods and of messenger RNA assays probably canmake metallothionein a more valid bioindicator.58,73

Heat stress proteins (HSP). Heat stress proteins are animportant group of ATP-dependent proteins that facilitatethe folding of nascent proteins by preventing their aggre-gation and then chaperoning them to sites of membranetranslocation. They were referred to previously as heatshock proteins because of their rapid appearance followingheat stress, although they increased in response to a vari-ety of xenobiotics, including metals and metalloids (espe-cially, arsenite), heavy metals and oxidizing agents.74 – 77

Such biomarkers will be important in the future.Heat shock proteins include four types: hsp 90 (ca.

90 kDa), hsp 70 (ca. 70 kDa), hsp 60 (ca. 60 kDa) andhsps of low molecular weight (ca. 15–30 kDa):

(i) hsp 90 (stress 90). There are two forms: hsp 83is located in the eukaryotic cytosol and grp 94 islocalized in the endoplasmic reticulum of mammals.

(ii) hsp 70 (stress 70). This is the most widely studiedand is located in the cytosol of mammals. It belongsto a multigene family with at least one form that isexpressed constitutively (hsc 70) in unstressed cellsand one or more isoforms (hsp 70) that are onlystress inducible. Both hsp 70 and hsc 70 help torefold denatured proteins that occur in cells followingheat shock or exposure to other proteotoxic stresses.Because these proteins show a very strong responseto protein damage, they are ideal candidates forbiomarkers of sublethal damage.

(iii) hsp 60. This belongs to the family of chaperonins(cpn 60) involved in protein folding. They are local-ized in the mitochondria and may be useful as anorganelle-specific biomarker.

(iv) hsps of low molecular weight. These show substantialhomology with α-crystallins of vertebrates, may beinvolved in actin binding to stabilize microfilamentsand have good potential as biomarkers of effect onsome targets such as the cytoskeleton.

With the development of antibodies to some heat stressproteins, Western blotting is the best method for eval-uating heat stress proteins. Nevertheless, the heat stressprotein responses have been studied only in experimentalassays on laboratory species, culture systems or inverte-brates.

Eggshell thinning. Severe eggshell thinning maylead to breakage of eggs. Such thinning has beenproposed as a biomarker of reproductive damage.Dichlorodiphenylethane (DDE), a metabolite of DDT, isthe most widely studied in many species of birds (pelican,

eagle, etc.). The pathogenic mechanism is not known butcould be related to hormone alterations that interfere incalcium metabolism, which is essential in the formationof the eggshell.

Eggshell thickness may be estimated by two methods:one utilizes direct measurement with a micrometer andthe other calculates the thickness index as the weight ofthe shell (mg) divided by the product of shell length andbreadth (mm2).3

Vitamin A, thyroxine and thyroxine–TBPA com-plex. Normally, thyroxines in the plasma binds totransthyretin or TBPA (thyroxine-binding prealbumin) andRBP (retinol-binding protein)–vitamin A to form thyrox-ine–TBPA–RBP complex. Certain monohydroxymetabo-lites of 3, 3′, 4, 4′-tetrachlorobiphenyl (OH-TCBs) formedby the monooxygenase system (cytochrome P-450IA1)compete strongly with thyroxine for its binding site uponthe transthyretin. The consequence of the presence of thesemetabolites in blood is the quick loss of thyroxine fromthe circulation and the appearance of symptoms associ-ated with hypothyroidism. Also, the binding of OH-TCBsto transthyretin can cause a conformational change thatleads to a reduction in the attachment of the RBP toTBPA. The RBP and attached retinol (vitamin A) then islost from the blood by glomerular filtration (this complexhas a low molecular weight). The consequences of thisfeature are the appearance of hypovitaminosis A (dermaland epithelial lesions78). These finding have been shownin laboratory studies with mice, rats and monkeys.79 Theincrease of thyroxine and vitamin A in urine represents abiomarker of TCB exposition.

Biomarkers for anticoagulant rodenticides. Warfarinand second-generation anticoagulant rodenticides (flo-coumafen) act as inhibitors of the vitamin K cycle thatoperates in the liver of vertebrates. These compounds actby competing with vitamin K or derivatives and the con-sequence of this antagonism is a failure of the vitamin Kcycle to carboxylate the precursors of clotting proteins.This leads to an extension of blood clotting time, haem-orrhaging and death. The effects of anticoagulant rodenti-cides may be detected at two different levels: monitoringchanges in the vitamin K cycle or monitoring increasesin precursors of clotting proteins in blood. The develop-ment of ELISA assays in the latter years has allowed thedetection of forerunners of clotting proteins in mammalsand birds.3

Biomonitoring of environmental contamination.

(i) Plants have been used widely as biomonitors to anal-yse the environmental impact of pollutants (especiallygaseous air pollutants). Specific biomarkers have beenidentified in sensitive plants. In a few cases it is knownthat excess of a specific compound will give rise to theproduction of a metabolite that is different betweentolerant and sensitive plants.For example, in the presence of an excess of selenium,Se-sensitive plants fail to differentiate between S andSe and incorporate Se in sulfur amino acids (essen-tially selenomethionine and selenocysteine), leading



to the synthesis of enzymes of lower activity (seleno-proteins), which can lead to plant death. On the con-trary, Se-tolerant plants biosynthesize and accumulatenon-protein seleno-amino acids (such as selenocys-tathioneine and Se-methylselenocysteine), which donot cause metabolic problems for the plant. Thus, theoccurrence of selenoproteins in plants is an excellentbiomarker, although their use has not been reportedwidely.Another example is the synthesis of fluorocitrate afterexposure to an excess of fluorine. The plants syn-thesize fluoroacetyl-CoA and then convert it, via thetricarboxylic acid cycle, to fluorocitrate. This com-pound is not recognized by aconitase and as a resultthe fluorocitrate is accumulated, being a very reliablebiomarker for fluorine poisoning.Phytochelatins are proteins with plenty of sulfhydrylgroups and are synthesized during exposure to a heavymetal and anions such as SeO4

2−, SeO32− and AsO4

3−.Dose- and time-dependent relationships have beenestablished under laboratory conditions for cadmium,copper and zinc but for biological monitoring moreresearch is needed.Plant biomarkers respond to a wide variety of envi-ronmental compounds and may be useful to indicatea hazard to plant life. For example, the activity ofthe enzyme peroxidase has been used to biomark theexposure of plants to air pollution, especially SO2.Changes of enzyme systems during the developmentof the plant (seasonal and climatic processes) are notknown well enough yet and plant biomarkers are notas well advanced as animal end points although a goodfuture in the ecotoxicology field has been predicted.3

(ii) Lichens have long been considered important indi-cators of air pollution (air quality), and scientificwork has demonstrated their susceptibility to airbornecompounds such as heavy metals, ozone and sul-fur dioxide. Lichens are widespread and capable ofabsorbing elements directly from the atmosphere and

accumulating them in their tissues. The impact ofanthropogenic activities (bioaccumulation) is particu-larly due to steelworks, glassworks, chemical plants,combustion processes related to energy production andvehicle emissions.Concentrations of Pb, Cd, Hg, Zn, Cu, Cr, Ni, As, V,Al and Fe are reported from lichens (Cetraria nivalis,Xanthoria parientina, Ramalina fastigiata, Parmeliasulcata) that are believed to be highly influenced bysoil dust.80 – 84

(iii) Barks of pine trees (Pinus sylvestris) can be usedas biomonitoring tools to indicate and characterizedepositions of airborne organic and inorganic pollu-tants (heavy metals, sulfate, nitrate, benzo[a]pyrene, α-hexachlorocyclohexane and dichlorodiphenyltrichloro-ethane).85

Growth rates of tree rings can influence the con-centrations of elements in wood. It is possible tocollect wood samples of different ages and analysetheir heavy metal contents to obtain a chronologi-cal record of trace element pollution in the tree’senvironment (dendroanalysis). A basic assumption ofdendroanalysis is the stability of the mineral distribu-tion patterns. Some studies present good correlationsbetween radial distributions of heavy metals in treerings and temporal records of pollution from industryor traffic.86

In conclusion, the markers of biological toxicity rep-resent an important tool in toxicology for three mainreasons:

(i) they allow estimation of the biological effect on thetarget tissue;

(ii) they are markers of subclinical alterations and sensi-ble indicators of pathology, so they may be useful indiagnostic and preventive strategies;

(iii) they consider inter- and intra-individual variability inresponse to xenobiotics.

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Biomarkers as biological indicators of xenobiotic exposure

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