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

2. People are continuously exposed to thousands of natural and man-made chemicals (i.e – 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 dall’art. 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 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 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 β2-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 nell’organismo (reni e cervello).
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 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 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
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
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 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 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.

64. 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.

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 γ-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,
Large-scale biological monitoring in Japan.
M. Ogata, T. Numano, M. Hosokawa, H. Michitsuji, Sci Total Env,1997,199,
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, , WIT press, Southampton, ISBN: