Biomarkers for mercury biomonitoring

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					Biomarkers for Mercury Biomonitoring

 Proyecto QSP “Campaña Regional para la Minimización de las Fuentes
 Domésticas de Mercurio con Acciones de Intervención en la comunidad
 para la protección de la salud del niño y la mujer en la Argentina,
 Chile, Paraguay, Uruguay, Bolivia y Perú”. Buenos Aires, 3 de
 Diciembre 2008

                 Marcelo Enrique Conti
            Sapienza, University of Rome, Italy
People are continuously exposed to thousands of
natural and man-made chemicals (i.e. 100.106 –
REACH) through the external environment, food
habits and lifestyles.

Using modern analytical technology it is now
possible to measure a large number of chemicals
and their metabolites present in the human
organism (in blood, tissues, urine, hair, etc.).
The biomonitoring is a procedure well known
since 1927 when the first paper on the use of the
analysis of lead in urine in exposed workers was

Today biomonitoring is largely used to control the
health risk of people occupationally and non-
occupationally exposed.
Programmes on the biomonitoring are currently in
progress in the USA and in Europe.

The biomonitoring evaluates the exposure by
comparison with appropriate reference values and
goes by the knowledge of the relationship between
environmental exposure and deriving degree of
adverse health effects.
When a health risk is revealed, legislators may
decide to ban a product or restrict its usage to
applications with lower risks for human health.

Biomonitoring techniques are becoming, in fact,
common tools for decision-makers in the health
and environmental field.
   Valutazione Rischio Chimico

 Qualunque   strumento operativo o
 processo venga utilizzato per effettuare
 la valutazione del rischio degli agenti
 chimici pericolosi (misure, stime,
 algoritmi) dovrà tenere conto di tutti i
 requisiti minimi della VRC previsti
 dall’art. 223 del Dlgs 81/08.
              Valutazione del Rischio Chimico
                 Requisiti minimi Art. 223
   a) le loro proprietà pericolose;
   b) le informazioni sulla salute e sicurezza comunicate
    dal produttore o dal fornitore tramite la relativa
    scheda di sicurezza predisposta ai sensi dei decreti
    legislativi 3 febbraio 1997, n. 52 e 16 luglio 1998, n.
    285 e successive modifiche;
   c) il livello, il tipo e la durata dell’esposizione;
   d) le circostanze in cui viene svolto il lavoro in
    presenza di tali agenti, compresa la quantità degli
          Valutazione del Rischio Chimico
           Requisiti minimi Art. 223 (II)

   e) i valori limite di esposizione professionale o i valori
    limite biologici; di cui un primo elenco è riportato
    negli allegati XXXVIII e XXXIX;

   f) gli effetti delle misure preventive e protettive
    adottate o da adottare;

   g) se disponibili, le conclusioni tratte da eventuali
    azioni di sorveglianza sanitaria già intraprese.
Valore limite di esposizione professionale: è il limite
  della concentrazione media ponderata nel tempo di
  un agente chimico nell'aria all'interno della zona di
  respirazione di un lavoratore in relazione ad un
  determinato periodo di riferimento;

Valore limite biologico: il limite della concentrazione del
relativo agente, di un suo metabolita, o di un indicatore
  di effetto, nell'appropriato mezzo biologico.
Valori limite esposizione professionale
Valore limite biologico
     Strumenti per la valutazione del rischio
   Stime del rischio (valutazioni di tipo cautelativo
    basate su dati disponibili)

   Algoritmi (valutazioni di tipo semiquantitativo basate
    su parametri indicizzati di calcolo)

   Misure (determinazioni degli inquinanti in ambienti di
Biomarkers suggest the occurrence of toxicological
events much earlier than the emergence of those
effects that can be evaluated.

A biomarker is defines as: „...a change, produced
by a contaminant, at biochemical or cellular level
of a process, a structure or a function that can be
measured in a biological system.
This change provides information (qualitative,
semi-quantitative or quantitative) about the
chemical source, and on the correlation between
the biological effects and the environmental
contamination levels.
A contaminant can cause
...primary toxicity at biochemical and molecular
levels (alterations in enzymatic activity, DNA level
secondly, through cascade events can cause toxicity
at cellular, tissue or organism levels.
It is well-known that lifestyle is implicated in
determining the risks for development of cancers,
circulatory diseases, neurodegenerations,

other chronic diseases.

Similarly, life events and periods, such as
childhood, reproduction and senescence, may
affect the distribution of chemicals within the body.
During pregnancy, as an example, many chemicals
may pass the placental barrier causing exposure of
the foetus.

Lactation may result in excretion of lipid-soluble
chemicals, thus leading to a decreased retention in
the mother along with an increased uptake by the
During weight loss or development of osteoporosis,
stored chemicals may be released, which can then
result in a renewed and protracted “endogenous”
exposure of target organs.

Other factors may affect individual absorption,
metabolism, retention and distribution of chemical
compounds and they have to be taken in account
when a biomarker has to be measured.
The most important features of a biomarker are: i),
stability (to allow the biological sample
preservation); ii), sensitivity (i.e., low probability
of false negative); and iii), specificity (i.e., low
probability of false positive).
Moreover a biomarker must reflect the interaction
(qualitative or quantitative) of the host biological
system with the compound of interest and it has to
be reproducible qualitatively and quantitatively
with respect to time (short- and long-term).
Biomarkers can be classified in:

i) biomarkers of exposure;
ii) biomarkers of effect;
iii) biomarkers of susceptibility.
       Biomarkers of exposure
Biomarker    of   exposure,   the   first   kind   of
biomarkers used in human biomonitoring studies,
may be an exogenous compound or its metabolite
(i.e., a metal or a metal compound) inside the body,
an interactive product between the compound (or
metabolite) and an endogenous component, or
another event related to the exposure.
Often, there is not a clear distinction between
exposure and effect biomarkers. For example,
adducts formation could reflect an effect rather
than the exposure.
However, biomarkers of exposure usually indicate
changes in the functions of the cell, tissue or total
They comprise measurements of the compounds in
appropriate samples, such as blood, serum or
Volatile chemicals concentration may be assessed
in   exhaled    breath,    after    inhalation    of
contamination-free air.

Biomarkers of exposure may be used to identify
exposed individuals or groups, quantify their
exposure, assess their health risks, or to assist in
diagnosis of diseases with environmental or
occupational etiology.
For example, exposure to a particular solvent may
be   evaluated    from    data   on      the   actual
concentration of the solvent in the blood at a
particular time following the exposure.

This measurement will reflect the amount of the
solvent that has been absorbed into the body.

Some of the absorbed amount will be exhaled due
to the vapour pressure of the solvent.
Biomarkers of exposure alone do not give
information on the sources or levels of exposure;
when, where, how, or how many times the exposure
occurred; or any relationships between exposure and
health effects.
Recent technological advances in genomics, proteomics,
and metabolomics are providing new tools for investigating
endogenous chemicals that can be used to characterize an
individual's exposure to a single chemical or a mixture of
 Biomarkers of effect

Biomarkers of effect are referred to reversible
biochemical and functional alterations than can be
measured in a target tissue of the organism.
A BEF may be an endogenous component, or a
measure of the functional capacity, or a marker of the
state or balance of the body or organ system, as
affected by the exposure.
It is usually a pre-clinical marker of pathology and can
be specific or non-specific. (Alimonti and Mattei, 2008)
Both types of biomarkers of effect are useful biomarkers of
early (critical) effects.

For example the detection of early damage to the kidney
tubules caused by exposure to Cd using urinary levels of
low molecular weight proteins such as β2-microglobulin,
protein    HC    (1-Microglobulin)   and   the   enzyme   N-
acetylglucosaminidase can be determined.
Technical developments have been occurred with
biomarkers of effect to mutagenic chemicals.

These compounds are reactive and may form
adducts with macromolecules, such as proteins or

DNA adducts may be detected in white blood cells
or tissue biopsies, and specific DNA fragments
may be excreted in the urine.
Other macromolecules may also be changed by adduct
formation or oxidation.
In particular, such reactive compounds may generate
haemoglobin adducts that can be determined as
biomarkers of effect to these compounds.
For the purpose of occupational health, these
biomarkers should be restricted to those that indicate
subclinical or reversible biochemical changes, such as
inhibition of enzymes.
The most frequently used biomarker of effect is
probably the inhibition of cholinesterase caused by
certain insecticides, namely, organophosphates and
In most cases, this effect is entirely reversible, and the
enzyme inhibition reflects the total exposure to this
particular group of insecticides.
Some exposures do not result in enzyme inhibition but
in increased activity of an enzyme. This is the case of
several enzymes belonging to the P450 family.
They may be induced by exposures to certain
solvents and polyaromatic hydrocarbons.
Generally, the enzyme activity is determined
indirectly in vivo by managing a compound that is
metabolized by that particular enzyme, and then
the breakdown product is measured in urine or
Other exposures may induce the synthesis of a
protective protein in the body.
The best example is probably metallothionein, which
binds Cd and promotes its excretion; Cd exposure is
one of the factors that result in increased expression of
the metallothionein gene.
Similar protective proteins may exist but have not yet
been explored sufficiently to become accepted as
Among the candidates for possible use as biomarkers
are the so-called stress proteins, previously known as
heat shock proteins.
These proteins are generated by a range of
different organisms in response to a variety of
adverse exposures.
The urinary excretion of proteins with a small
molecular weight, such as albumin, may be used as
a biomarker of early kidney damage.
Relating to genotoxic effects, chromosomal
aberrations or formation of micronuclei can be
detected by microscope observation. Damage may
also be revealed by adding a dye to the cells during
cell division.
Exposure to a genotoxic agent can then be visualized
as an increased exchange of the dye between the two
chromatids of each chromosome (sister chromatid
exchange, SCE).
Chromosomal aberrations are related            to   an
increased risk of developing cancer.
More sophisticated assessment of genotoxicity is
based on particular point mutations in somatic cells,
that is, white blood cells or epithelial cells obtained
from the oral mucosa (Alimonti and Mattei, 2008)
Biomarkers of susceptibility

Biomarkers of susceptibility are indices of the
individual predisposition (hereditary or acquired)
to suffer xenobiotic effects, that is, to be
particularly sensitive to the effects of a single
compound or of a group of such chemicals.
People working under identical conditions may show
interindividual variation in the intensity of the
effects of a particular degree of exposure.
Therefore differences are seen in health impairment
in workers similary exposed to the same substance.
Genetic screening and genetic monitoring.
A clear distinction must be drown between genetic
tests which are intended to detect inherited
characteristics, which may point to greater
susceptibility to certain conditions (genome
screening) and genetic tests which aim to find
changes in the hereditary material, which are the
result of exposure to harmful agents (genetic
Genetic biomonitoring can form part of the
periodic medical examination of employees and is
specially designed to asses the effects of exposure
to carcinogenic or mutagenic agents in the
workplace    (somatic   mutations,       chromosome
aberrations, micronuclei, aneuploidy).
If an individual has become sensitized to a
particular exposure, then specific antibodies can
be detected in serum.
A major problem is to determine the joint effect of
mixed exposures at work. In addition, personal
habits and drug use may result in an increased
For example, tobacco smoke usually contains a
considerable amount of Cd.
...a heavy smoker who has accumulated substantial
amounts of Cd in the body will be at increased risk
of developing cadmium-related kidney disease.
In the environment Hg occurs in metallic form, as
inorganic Hg and organic Hg.
Generally, environmental levels of Hg are quite low
between 10 and 20 ng/m3 of Hg have been measured in
urban outdoor air (i.e., hundreds of times lower than
safe levels to breathe) or less than 5 ng/L in surface
waters (i.e. about a thousand times lower than safe
drinking water).
    Tossicologia del mercurio
Hg(0): essendo volatile si deposita nei polmoni attraverso i quali si incorpora nell’organismo (reni e

Hg(II): viene incorporato attraverso il tratto gastrointestinale e attraverso la pelle. Gli effetti
negativi dell’intossicazione acuta si mitigano rapidamente dato che la vita media del catione
nell’uomo è di circa 60 giorni.

MeHg(II): i composti contenenti il catione MeHg+ sono fra le sostanze più tossiche, tanto per i loro
effetti che per la loro incidenza osservata su intere popolazioni. Questo catione forma composti
liposolubili che possono dare fenomeni di bioaccumulazione (catena alimentare). Inoltre questi
composti possono attraversare facilmente la membrana emato-encefalica agendo come potenti
A potential source of exposure is Hg released from
dental amalgam fillings, which can contain
approximately 50 % of metallic Hg.
Some people may be exposed to high levels of
methyl Hg if they eat often fish, shellfish, or
marine mammals.
Hg is not essential to living cells; its absorption
distribution and biotransformation are influenced
significantly by its valence state.
Chronic exposure to Hg vapor results in toxicity of
the central nervous system including tremors,
increased excitability and delirium.
Elemental Hg is eventually oxidized to Hg (II) in
the body by the hydrogen peroxidase-catalase
pathway and is primarily excreted via the kidneys.
However, a small portion may be exhaled.
Ingestion of inorganic, oxidized Hg can result in
abdominal cramping, ulceration and renal toxicity.
Inhalation of Hg° vapor is associated with an acute,
corrosive bronchitis or pnaeumonitis.
Hg has a strong affinity for sulfur, and Hg primary
mode of toxic action in living organisms is thought to
be the interference of enzyme function and protein
synthesis by binding to sulfhydryl or thiol groups.
Excretion by kidneys is the primary route of
elimination of oxidized Hg, and because of its strong
affinity for protein, proteinuria is a symptom
associated with exposure to Hg(II).
Organic Hg is highly lipophilic and exposure occurs
primarily via consumption of contaminated fish.

Both methyl-Hg and Hg° cross the placental
(inducing   teratogenic   effects)   and blood-brain
barrier where they can be oxidized and accumulated

Methyl Hg can react directly with important
receptors in the nervous system, such as the
acetycholine receptors in the peripheral nerves.
Carcinogenicity   and   mutagenicity    are   not
commonly associated with Hg exposure.

Instead, the IARC have not classified Hg as
human carcinogenicity, whilst EPA has determined
that Hg chloride and methyl-Hg are possible
human carcinogens.
Biomarkers of Hg exposure

Blood and urine Hg concentrations are commonly
used as biomarkers of exposure. Urine Hg is a
biomarker used for detecting elemental and
inorganic forms of Hg.
The reference values, such as the reference dose
(RfD) published by US EPA, or the Provisionally
Tolerated Weekly Intake (PTWI) (WHO-FAO),
reflect the levels of exposure that should prevent
humans    from   suffering   adverse   effects   of
environmental exposure.
Provisional tolerable weekly intake (PTWI) of 1.6
µg/kg bw. The Committee considered this PTWI to be
sufficient to protect the developing fetus, the most
sensitive subgroup of the population. The Committee
also reaffirmed its position that fish are an important
part of a balanced nutritious diet and that this has to be
appropriately considered in public health decisions
when setting limits for methylmercury concentrations in
The International Commission on Occupational
Health (ICOH) and the International Union of Pure
and Applied Chemistry (IUPAC) Commission on
Toxicology determined that a mean value of 2 μg/L
was the background blood level in persons who do
not eat fish.
Reference values
Human Biomonitoring Commission of the German Federal
Environmental Agency (Wilhelm et al., 2004). Reference values
indicate the upper margin of the current background exposure
of the general population.

Cd (non smokers): blood - 1.0 μ/l ; urine 0.8 μg/l.
Pb : blood – 70 μg/l (female) ; 90μg/l ( male).
Hg : (consumption of fish ≤ 3 x a month) blood – 2 μg/l (no
amalgam fillings) urine – 1.0 μ/l.
As : ( no fish consumption) urine – 15.0 μg/l.
Pt : (no dental inlays, crowns, bridges) urine –0.01μg/l.
The American Conference of Governmental Industrial
Hygienists      (ACGIH)        and      the    Deutsche
Forschungsgemeinschaft (DFG), the two main
organizations involved in the setting of BM reference
values differ their approach to and definitions of these
BEIs are understood as advisory levels that may be
exceeded by individuals in the observed group.

ACGIH has already published BEI values for 37
substances or groups of substances.
The DFG BAT values are defined as "the maximum
permissible quantity of a chemical substance or its
metabolites, or the maximum possible deviation from the
norm for biological parameters induced by these
substances in exposed humans. The BAT values are
considered the ceiling values for healthy individuals".
They are intended to protect the workers from work-
related health impairments. DFG has so far determined
BAT values for 50 substances or groups of substances.
Occupational limits are a ACGIH-BEI of 15 µg/l and
a BAT of 25 µg/l.
But blood Hg levels peak quickly soon after short-
term exposures, so measurements should be made
soon after exposure.
Human Biological Monitoring Values (HBM) recommended by
 the German Commission on Human Biological Monitoring
         (March 1999) (Jakubowski and Trzcinka-Ochocka, 2005)
                                         HBMI                 HBMII

Mercury in urine   Children and adults   5μg/g creat.   20 μg/g creat.
Mercury in blood   Children and adults   5μg/l          15 μg/l

HBMI- The concentration of an environmental toxin in human
biological material, below which there is no risk of advance
health effects.
HBM II- The concentration above which there is increased risk
of adverse health effects in susceptible individuals in the general
A strong correlation has been found among the
amount of fish swallowed, the Hg fish level and the
Hg hair level.

Expired air samples have been considered as
possible biomarkers of exposure for Hg, but results
showed that expired air can only be used soon after
short-term exposure to Hg vapours.

The effects of methylmercury on the adult differ both in
quantitative and qualitative terms from the effects
observed after prenatal or postnatal exposure. The critical
organ is the nervous System and the critical effects
include developmental neurologic abnormalities in
human infants, and paraesthesia in adults. The foetus is at
particular risk. Prenatal exposure leads to psychomotor
retardation in infants. Developmental neurologic
abnormalities are considered the critical effects in the
infant population
(Jakubowski and Trzcinka-Ochocka, 2005)
Hair is a biomarker of long-term exposure to
methylmercury. Once mercury is incorporated into hair, it
remains unchanged. The level of mercury in hair (Hg-
H) is dependent on fish consumption
The dose-response relationship between maternal hair
concentration and the frequency of health effects in
children was used by the IPCS for the purpose of risk
assessment. At peak mercury levels in maternal hair at
above 70 μg/g, there is a high risk (more than 30%) of
neurological disorder in the children, and a 5% risk may
be associated with a peak mercury level of 10-20 μg/g in
maternal hair.
The present background level of Hg-H, associated with
no or low fish consumption or a low fìsh methylmercury
concentration, amounts to from 0.25 to 0.8 μg/g.

Much higher Hg-H levels result from the consumption of
large amounts of fish or sea mammals. The mean Hg-H
levels in the Faroe Island population amounted from 1.6
μg/g (one fish meal per week) to 5.2 μg/g (four fish
meals per week). In the Madeira fishermen and their
families, it amounted to 38.9 μg/g in men and 10.4 μg/g
in women (Jakubowski and Trzcinka-Ochocka, 2005)
Despite the numerous long-term studies and
considerable efforts of the researchers, the so-called
'health-based' reference values have been proposed
and validated only for several chemical substances or
groups of substances.
These recommendations are of great value to health
professionals because the health effect of exposure can
be predicted directly from the determination of a
biomarker of exposure. It is possible to predict early
direct health effects of lead based substances on blood
lead levels.
Such measurements can be interpreted without knowing
the results of environmental monitoring.
 Biomarkers of effect

A number of possible biomarkers of effect have
been investigated, especially for neurological and
renal dysfunctions.
For example, the toxic effects observed at kidney
level have been well correlated with blood and
urine levels.
Biomarkers for decreased kidneys function include
increasing in urinary proteins and elevation of
serum creatinine or β2-microglobulin.

Biomarkers for biochemical changes include
eicosanoids, fibronectin, kallikrein activity, and
glycosaminoglycans in urine.

Glomerular changes have been reported as
increased high-molecular weight proteinuria.
Tubular changes in workers include an increasing
urinary excretion of NAG, β-galactosidase, and
retinol binding protein. (Alimonti and Mattei, 2008)

But toxic kidneys parameters are not specific
markers for Hg exposure and may be a
consequence        of    other     concurrent          chemical
exposures      and      they     can‟t   be    assessed      as
biomarker of effect still now.
The neurophysiological and neuropsychological health
effects of Hg have been extensively studied in
occupationally exposed individuals.

Neurological changes induced by Hg may look like
exposure to other chemicals that can cause damage to
the brain. Some studies have examined the relationship
between nerve function and Hg levels in blood, urine, and
Tissue levels of Hg have also been found to
correlate with impaired nerve function. But also in
this case no biomarkers of effect are established.

Potential biomarkers for the autoimmune effects of
mercury have been examined and they include
measurement          of     antiglomerular        basement
membrane antibodies, anti-DNA antibodies, serum
IgE complexes, and total IgE (Alimonti and Mattei, 2008).
Biomarkers of susceptibility

Various factors affect the absorption, distribution,
biotransformation, excretion and, consequently,
toxicity of Hg. In the case of methyl-Hg, reduced
glutathione and γ-glutamyl transpeptidase are
involved in the excretion of methyl-Hg.
The glutathione S-transferase (GST) gene family is
involved in the detoxification of electrophilic
compounds by conjugation and         (a study conducted by

Brambila et al.)   showed that various GST genes are
activated in rats exposed to Hg, indicating that
individuals with specific genotypes could be better
protected against the cytotoxicity of Hg.      (Alimonti and
Mattei, 2008).
BM of exposure and early health effects of exposure
should be considered as the prophylactic activity. BM
has an important role to play in both health
surveillance and exposure assessment in occupational
settings and in identifying hot spots and developments
in trends of exposure in the general environment.
There are important discrepancies in the approach
towards the role of BM between Europe and the USA
(occupational medicine or occupational hygiene).
(Jakubowski and M. Trzcinka-Ochocka, 2005)
More attention should be paid to the development
of the truly health-based biomarkers of exposure
based on the dose-effect and dose-response
The practical implementation of BM as well as the
ethical problems can be solved in enterprises
where a close cooperation between the health
service, management and employees is a routine
Biological monitoring of exposure. Trends and Key Developments
M. Jakubowski and M. Trzcinka-Ochocka. J. Occup. Health, 2005, 47, 22 – 48.

Revised and new reference values for some trace elements in blood and urine for human
biomonitoring in environmental medicine
M. Wilhelm, U. Ewers, C. Schulz, Int J of Hyg and Env Health, 2004, 207, 69-73.

Large-scale biological monitoring in Japan.
M. Ogata, T. Numano, M. Hosokawa, H. Michitsuji, Sci Total Env,1997,199, 197-204

Biomarkers for human biomonitoring (Chapter 6)

A. Alimonti, D. Mattei (2008) in: M.E. Conti (Ed.) Biological Monitoring: Theory And
Applications. The Sustainable World, 17, 163-211, WIT press, Southampton, ISBN: 978-1-

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