ASSESSING HUMAN EXPOSURES WITH BIOLOGICAL MARKERS

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ASSESSING HUMAN EXPOSURES WITH BIOLOGICAL MARKERS

• Conrad D. Volz, DrPH, MPH
• Department of Environmental and Occupational
  Health, Graduate School of Public Health,
  University of Pittsburgh
• Co-Director Exposure Assessment, Center for
  Environmental Oncology, University of Pittsburgh
  Cancer Institute
• Scientific Director, Center for Healthy
  Environments and Communities
• Lecture 20

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General

• Biological markers represent events or changes in
  human biological systems as a result of exposure
  or disease (US NRC, 1991b).
• They are classified as markers of exposure,
  effect, and susceptibility and are considered to
  represent events along a theoretical continuum
  from causal exposure to resulting health outcome
  (US NRC, 1987 Schulte, 1989). See Hatch papers
  for theoretical description.

Major Text WHO Exposure Assessment
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General

• Biological markers represent a means to monitor
  environmental exposure by characterizing an
  individual's total dose of a contaminant from all
  sources of exposure. Remember that dose is mass
  of contaminant over some time.
• The main advantage of this strategy is in
  evaluation of an individual's total exposure
  using a measure which integrates over all
  exposure sources and is influenced by human
  behavior.
• Biological markers are believed to be more
  predictive of health effects than external
  measures of exposure.
• Biological markers address important exposure
  assessment needs
• characterizing an individual's or a
  population's exposure
• generating population distributions of dose
• identifying the environmental and demographic
  determinants of exposure.

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Disadvantages of Biological Monitoring

• The main disadvantage of biological markers is
  the difficulty in characterizing the individual
  sources which contribute to the subject's total
  exposure.
• When developing and utilizing biological markers,
  understanding the toxicokinetics of the
  contaminant in the system is crucial to
  characterize the biological variability and to
  determine whether the biological marker is valid
  for exposure assessment purposes at the
  concentration of interest.
• There is an ethical concern in relying on
  biomonitoring especially in known occupational
  exposure assessment.

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What is a Biological Marker of Exposure? A
biological marker of exposure is defined as a
xenobiotic substance or its metabolite(s) or the
product of an interaction between a xenobiotic
agent and some target molecule(s) or cell(s) that
is measured within a compartment of an organism
(US NRC, 1989 IPCS, 1993)-after exposure to the
xenobiotic contaminant or a physical exposure,
such as ionizing radiation.
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Biological Markers of Effect and Susceptibility

• Biological markers of effect are measurable
  biochemical, physiological, behavioral or other
  alterations within an organism that, depending
  upon the magnitude, can be recognized as
  associated with an established or possible health
  impairment or disease (IPCS, 1993).
• Biological markers of susceptibility are
  indicators of inherent or acquired abilities of
  an organism to respond to the challenge of
  exposure to a specific xenobiotic substance
  (IPCS, 1993).
• Example-Different genotypes of BCHE-K, PON-192,
  and PON-55 may be related to the severity of
  adverse health effects of organophosphorus
  pesticide exposure. (Carboxylic esterase and its
  associations with long-term effects of
  organophosphorus pesticides. Biomed Environ Sci.
  2007 Aug20(4)284-90. )

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Situations which are bestsuited for biological
monitoring.

• Ideally, a biological marker of exposure should
  be
• 1. Chemical-specific.
• 2. Detectable in trace quantities.
• 3. Available by non-invasive techniques.
• Inexpensive to assay.
• Relate consistently and quantitatively to the
  extent of exposure and ideally also integrate the
  exposure over time (Bond et al., 1992).
• Currently there are very few biological markers
  that possess all these characteristics.

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Sampling of Blood

• Blood is frequently used for biological
  monitoring, especially in clinical settings such
  as occupational medicine.
• Blood can integrate all sources of exposure,
  including internal sources, and provide an
  indication of current internal dose.
• Since blood transports all agents throughout the
  organism, it represents an opportunity to sample
  all types of contaminants, such as gases,
  solvents, metals and fat-soluble compounds.

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Components of Blood Available for Sampling

• Whole blood consists of all the blood
  components and is preferable when the
  distribution of the analyte between plasma and
  cellular elements is unknown (Que Hee, 1993).
• Red blood cells make up a large portion of
  blood and their primary role is to transport
  oxygen via hemoglobin throughout the body. Mature
  red blood cells contain no nucleus and therefore
  no DNA, and have a 120-day lifetime. Chemicals
  that interact with hemoglobin, such as carbon
  monoxide, are found in red blood cells.

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Components of Blood Available for Sampling
Continued

• Numerous types of white blood cells are present
  in blood. These cells have a half-life ranging
  from 18-20 days to decades (Carrano Natarajan,
  1988).
• White blood cells are circulating cells that have
• DNA can itself be altered or its expression can
  be changed as a result of exposure to a genotoxic
  agent, so white blood cell DNA may be used for
  biomarkers of exposure to genotoxic agents
  (Carrano Natarajan, 1988 Kelsey, 1990).
  Interpretation of genotoxic response is
  complicated because DNA damage can result in
  either cell death or removal of the marker by DNA
  repair, or may alter cell functions (Perera,
  1987). Regardless of this, correlations have been
  seen between environmental exposures and DNA
  adducts.
• A DNA adduct is an abnormal section of DNA which
  is bonded to a contaminant. DNA adducts are used
  as biological markers of exposure. Acetaldehyde
  DNA adducts are common in cigarette smokers.

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Components of Blood Available for Sampling
Continued

• Plasma and serum represent the non-cellular
  component of blood.
• Plasma is a straw-coloured aqueous solution of
  electrolytes, non-electrolytes and macromolecules
  (including clotting factors).
• Serum is plasma without the clotting factors
  (Que Hee, 1993).
• Plasma represents a component of whole blood
  (approximately 60), and it may contain the most
  biologically active fraction of blood borne
  contaminants, since plasma is in more immediate
  contact with tissues (Silbergeld, 1993). Plasma
  can be used for analysis of lipophilic chemicals,
  thereby avoiding the need for fat sampling.

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Components of Blood Available for Sampling

• Blood proteins can be sensitive monitoring tools
  for chemicals that bind to macromolecules
  including DNA (Osterman-Golkar et al., 1976 Bond
  et al., 1992).
• Protein adducts, unlike DNA adducts, are not
  repaired and may prove to be a useful dosimeter
  of mutagen exposure (Grassman Haas, 1993 Que
  Hee, 1993).
• Hemoglobin and albumin are two proteins available
  for use in exposure assessment. Hemoglobin is
  located in red blood cells in high concentration
  and has the half-life of red blood cells
  (120days) albumin is present in serum and has a
  half-life of 21 days.
• Because of their differing biological half-lives,
  these proteins can be used to investigate the
  timing of exposure.

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Sampling of Urine

• The concentrations of compounds found in urine
  usually reflect time-weighted averages in plasma
  during collection and storage in the bladder (Que
  Hee, 1993).
• The presence of a contaminant or its metabolite
  in urine generally represents recent exposure,
  though in some cases it may represent release
  from storage within the body (Lauwerys, 1983).
  (Release of lipopylic chemicals from adipose
  tissue during weight reduction is an example of
  release from body storage.)
• Urine can be analysed for metabolites of organic
  chemicals (e.g., benzene and styrene), metals
  (e.g., arsenic and mercury) and pesticides as
  well as for mutagenic potential (Lauwerys, 1983
  Baselt, 1988 Que Hee, 1993).
• Since collection of urine samples is
  non-invasive, some investigators feel that, when
  validated, urine may be a better sampling medium
  than blood for monitoring (Smith Suk, 1994).

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Types of Urine Sampling, Advantages and
Disadvantages

• Spot urine samples are relatively easy to
  collect but there may be significant variability
  with respect to exposure prediction as a result
  of metabolism, liquid consumption and kidney
  function.
• First morning void samples have less
  variability since they are more concentrated than
  spot samples, but require motivated subjects to
  collect the samples.
• Twenty-four hour urine samples control much of
  the intraindividual variability but require
  highly motivated subjects in order to collect
  useful samples (Baselt, 1988).
• To make the results of urine monitoring
  comparable between individuals, analytical
  results are frequently standardized to creatinine
  concentration or specific weight. Standardization
  reduces some of the variability of body size and
  urinary output (Lauwerys).

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Example Atrazine Metabolite from UrineChem Res
Toxicol. 1993 Jan-Feb6(1)107-16.

• Enzyme-linked immunosorbent assays (ELISAs) are
  reported useful for the detection of atrazine and
  its principle metabolite in human urine. The
  ELISAs can be used with crude urine or following
  extraction and partial purification.
• GC, MS, and HPLC techniques can be used to
  confirm and complement the ELISA methods for
  qualitative and quantitative detection of urinary
  metabolites.
• A series of samples from workers applying this
  herbicide confirmed a mercapturic acid conjugate
  of atrazine as a major urinary metabolite. The
  mercapturate was found in concentrations at least
  10 times that of any of the N-dealkylated
  products or the parent compound.
• Atrazine mercapturic acid was isolated from urine
  using affinity extraction based upon a polyclonal
  antibody for hydroxy-s-triazines and yielded
  products sufficiently pure for structure
  confirmation by MS/MS.
• In a pilot study monitoring applicators, a
  relationship between cumulative dermal and
  inhalation exposure and total amount of atrazine
  equivalents excreted over a 10-day period was
  observed. On the basis of these data, we propose
  that an ELISA for the mercapturate of atrazine
  could be developed as a useful marker of
  exposure.
• Lucas AD, Jones AD, Goodrow MH, Saiz SG, Blewett
  C, Seiber JN, Hammock BD., Department of
  Entomology, University of California, Davis 95616.

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Exhaled Breath

• Breath analysis is useful for assessing recent
  exposure to gases (e.g., carbon monoxide) and
  organic vapors and solvents (e.g., acetone and
  toluene).

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Types of Exhaled Breath Samples

• Exhaled breath can be a mixture of inhaled and
  exhaled air. If the exhaled biological marker is
  not present in inhaled air, then exhaled breath
  analysis is an effective means to measure
  internal exposure. For example, when alcohol has
  an internal source only (i.e., ingestion) a mixed
  breath sample is appropriate.
• Alveolar air provides a measure of the air that
  is in equilibrium with the blood in the deep lung
  (Bond et al., 1992). For analytes present in
  inhaled air, it is necessary to collect an
  alveolar air sample.

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Saliva Sampling

• Glands at four locations in the mouth produce
  saliva the secretion rate varies at each
  location. Chemicals enter saliva via passive
  diffusion from plasma. Therefore, saliva may
  become a useful tool to non-invasively
  characterize plasma levels of contaminants
  (Silbergeld, 1993).
• Social science research has used saliva sampling
  because of its ease of collection and storage
  (Dabbs, 1991, 1993). Contaminants found in saliva
  include cotinine, drugs, metals, organic
  solvents, pesticides and steroid hormones (Tomita
  Nishimura, 1982 Nigg Wade, 1992 Silbergeld,
  1993).

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Sampling Keratinized Tissues (hair and nails)

• Keratinized tissues, primarily hair and toenails,
  are practical sampling media for evaluation of
  past exposure to metals (Bencko et al., 1986
  Bencko, 1991 Subramanian, 1991 Kemper, 1993,
  Bencko, 1995).
• Toenails are usually the medium of choice because
  these media
• - integrate exposures over a period of
  months.
• -contain relatively larger concentrations of
  trace elements than blood or urine and
• -are easy to collect, store and transport
  (Garland et al., 1993 Kemper, 1993).

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Sampling of Hair

• Hair can be used to study exposure to
  environmental tobacco smoke (ETS).
• The German Environmental Survey (Krause et al.,
  1992) it was concluded that in large population
  studies nicotine and continine in urine as well
  as nicotine in hair are useful indicators of
  exposure for different levels of active and
  passive smoking.
• Continine and nicotine concentrations in hair
  have also been used to study fetal exposure by
  maternal smoking (Klein et al., 1993).
• Hair has also successfully been used in studies
  evaluating exposure to organic mercury (Suzuki et
  al., 1989) or PCB (Que Hee, 1993).
• Hair grows approximately 1 cm/30 days (Que Hee,
  1993) and can be evaluated along the shaft to
  provide a profile of exposure over time. Since
  growth rates of hair differ based on body
  location, standardization of sampling location is
  crucial.

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Ossified TissueSampling-Teeth and Bones

• Teeth constitute a unique medium for assessment
  of past exposure. Depending on the tooth type and
  part of the tooth, one can reconstruct early
  childhood exposures to bone-seeking elements,
  such as lead (Rabinowitz MB, Leviton A,
  Bellinger DC (1989) Blood lead tooth lead
  relationship among Boston children. Bull Environ
  Contam Toxicol,43 485-492.).
• Electron Paramagnetic Resonance (EPR) tooth
  dosimetry has been used to validate dose models
  of acute and chronic radiation exposure.
  (Retrospective assessment of radiation exposure
  using biological dosimetry chromosome painting,
  electron paramagnetic resonance and the
  glycophorin a mutation assay. Kleinerman RA,
  Romanyukha AA, Schauer DA, Tucker JD. Radiat Res.
  2006 Jul166(1 Pt 2)287-302. )

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Bone Sampling

• Bone represents both past exposure to
  bone-seeking elements and is a source for future
  internal exposure to these elements. The
  concentrations of elements in bone represent
  long-term exposure and storage of contaminants.
  For example, the half-life of lead in bone is
  approximately 10-40 years (Rabinowitz, 1991).
• Although numerous elements can be detected in
  bone tissue using destructive analyses such as
  atomic absorption spectroscopy (AAS), in vivo
  measurement of environmental contaminants in bone
  has been limited to lead (e.g., Somervaille et
  al., 1988 Hoppin et al., 1995).
• Lead concentration in bone can be analyzed
  non-invasively using a technique known as X-ray
  fluorescence (XRF) (Hu et al., 1995).
• Epidemiological studies have established that
  bone-seeking a-particle-emitting radionuclides
  are effective sarcomagenic agents, increasing
  tumor incidence by up to 1000-fold in exposed
  individuals (H. S. Martland and R. E. Humphries,
  Osteogenic sarcoma in dial painters using
  luminous paint. Arch. Pathol. 7, 406 (1929) and
  C. Mays and H. Spiess, Bone sarcomas in patients
  given 224Ra. In Radiation Carcinogenesis
  Epidemiology and Biological Significance (J. B.
  Fraumeni, Ed.), pp 241252. Raven Press, New
  York, 1982.)

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Breast Milk Sampling

• Breast milk sampling represents a non-invasive
  method to estimate body burden of contaminants in
  adipose tissue. The correlation between
  contaminant concentrations in the lipid phase of
  milk and adipose tissue is good (Sim McNeil,
  1992).
• Environmental studies have used breast milk to
  evaluate past exposure to lipophilic chemicals
  (e.g., pesticides and PCBs) and metals (WHO,
  1996b) and to examine potential exposures for
  breast-feeding infants (Niessen et al., 1984
  Davies Mes, 1987 Sikorski et al., 1990 Sim
  McNeil, 1992).
• Organic chemicals found in breast milk have high
  lipid solubility, resistance to physical
  degradation or biological metabolism and slow or
  absent excretion rates (Rogan et al., 1980).
  Breast milk represents a major route of excretion
  of lipophilic chemicals for lactating women
  (Rogan et al., 1980 Sim McNeil, 1992).
• Concentrations of chemicals in breast milk are a
  function of parity, age, body mass, time of
  sampling, nutritional status, lactation period
  and fat content of milk (Rogan et al., 1986 Sim
  McNeil, 1992). Breast milk results are
  generally standardized to milk fat levels.

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Sampling Adipose Tissue

• Exposure assessment studies using adipose tissue
  have been limited primarily to ecological studies
  comparing fat from cadavers or surgical specimens
  to general pollution levels.
• Adipose tissue represents a long-term reservoir
  of lipophilic compounds that the body slowly
  metabolizes and may release into the bloodstream.
  
• Unfortunately there is no non-invasive manner to
  sample fat stores directly, and many subjects see
  fat sampling as exceedingly invasive.
• WHO Human Exposure Assessment

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Sampling Feces

• Most often used for bacteriological exposure
  sampling.
• Feces are a highly fat-soluble medium that
  provides information on compounds of
  high-molecular weight that exit the body via
  biliary excretion (metabolism by liver and
  excretion via bile) and on unabsorbed chemicals
  that enter the body via ingestion.

Main Text for Biomonitoring Material
Extraction-WHO Exposure Assessment