Biomarkers for Mercury Biomonitoring

1
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 E-mail marcelo.conti_at_uniroma1.it
2
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.).
3
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
published. Today biomonitoring is largely used
to control the health risk of people
occupationally and non-occupationally exposed.
4
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.
5
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.
6
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 dallart. 223
  del Dlgs 81/08.

7
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
  dellesposizione
• d) le circostanze in cui viene svolto il lavoro
  in presenza di tali agenti, compresa la quantità
  degli stessi

8
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.

9

• 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.

10
Valori limite esposizione professionale
11
Valore limite biologico
12
Strumenti per la valutazione del rischio chimico

• 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 lavoro)

13
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.
14
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.
15
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.
16
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.
17
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 infant.
18
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.
19
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).
20
Biomarkers can be classified in i) biomarkers
of exposure ii) biomarkers of effect iii)
biomarkers of susceptibility.
21
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.
22
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 body.
They comprise measurements of the compounds in
appropriate samples, such as blood, serum or
urine.
23
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.
24
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.
25
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 chemicals.
26
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)
27
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 ß2-microglobulin, protein HC
(1-Microglobulin) and the enzyme
N-acetylglucosaminidase can be determined.
28
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. DNA adducts may be detected in white blood
cells or tissue biopsies, and specific DNA
fragments may be excreted in the urine.
29
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.
30
The most frequently used biomarker of effect is
probably the inhibition of cholinesterase caused
by certain insecticides, namely, organophosphates
and carbamates. 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.
31
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
plasma. Other exposures may induce the synthesis
of a protective protein in the body.
32
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 biomarkers. Among the candidates for
possible use as biomarkers are the so-called
stress proteins, previously known as heat shock
proteins.
33
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.
34
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)
35
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.
36
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.
37
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
biomonitoring).
38
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).
39
If an individual has become sensitized to a
particular exposure, then specific antibodies can
be detected in serum.
40
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
susceptibility. 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.
41
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).
42
Tossicologia del mercurio
Hg(0) essendo volatile si deposita nei polmoni
attraverso i quali si incorpora nellorganismo
(reni e cervello). Hg(II) viene incorporato
attraverso il tratto gastrointestinale e
attraverso la pelle. Gli effetti negativi
dellintossicazione acuta si mitigano rapidamente
dato che la vita media del catione nelluomo 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
neurotossici.
43
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.
44
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.
45
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).
46
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.
47
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.
48
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.
49
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.
50
Methylmercury  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 fish.
51
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.
52
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.
53
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 values. 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.
54
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.
55
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.
56
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 population.
57
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.
58
Methylmercury
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)
59
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.
60
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)
61
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.
62
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.
63
Biomarkers for decreased kidneys function include
increasing in urinary proteins and elevation of
serum creatinine or ß2-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.
64
Tubular changes in workers include an increasing
urinary excretion of NAG, ß-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 cant be assessed as biomarker
of effect still now.
65
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.
66
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).
67
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 ?-glutamyl
transpeptidase are involved in the excretion of
methyl-Hg.
68
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).
69
Conclusions 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)
70
More attention should be paid to the development
of the truly health-based biomarkers of exposure
based on the dose-effect and dose-response
relationships. 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 activity.
71
References 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 978-1-84564-002-6.