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Biomarkers of environmental risk factors for prevention and research
Trends in Analytical Chemistry 52 (2013) 275–281
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Trends in Analytical Chemistry journal homepage: www.elsevier.com/locate/trac
Meeting Report Biomarkers of environmental risk factors for prevention and research
challenges the user to collaborate with colleagues in other fields to achieve the desired reduction of chemical risk and prevention of disease. 2. Background
1. Introduction Human biological monitoring (HBM) is the repeated, controlled measurement of chemical or biochemical markers in body fluids, tissues or other accessible samples from human subjects exposed to chemical, physical or biological risk factors in the workplace and/or the general environment [5]. This applied field of analytical chemistry is different from the application of biomarkers in a clinical setting and is directed towards the reduction of exposure to chemicals and prevention of disease. In the early development of the concept of HBM, occupational exposures and prevention of occupational disease were important foci. As the sensitivity and the specificity of analytical methods improved, HBM was increasingly used to characterize exposures of the general population. Currently, there are large-scale national HBM programs in the USA, Canada, more than 15 European countries and also some Asian countries, such as South Korea. In addition to biomarkers of exposure, biomarkers of susceptibility and biomarkers of early physiological responses were used to understand the mechanisms leading to the onset of disease and to identify sensitive groups. Susceptibility and early effect biomarkers are not exclusive to the field of HBM and are also used in a clinical setting as clinical markers of diagnosis, prognosis or for clinical research. There are some new challenging fields of HBM application, such as: describing the total of all internal exposures to environmental factors in new research programs involving thousands of study subjects; evaluating interventions to mitigate environmental risk factors in developing countries; and, classifying exposures of victims of chemical incidents in the interest of medical support, or treatment and risk communication. The development of new biomarkers relies much on analytical techniques and most researchers in this field have a background in laboratory-based research. Once laboratory methods are validated, there are still many challenges in the application of these new biomarkers, such as the kinetic patterns in different biological media, the flawless collection of samples and the pre-treatment and storage of biological specimens. Particular challenges in the interaction with study participants engaged in HBM campaigns concern ethical implications, informed consent, the potential burden of invasive sample collection and the communication of HBM results to individuals and groups. HBM is therefore much more than the technical performance of a chemical analysis and it doi:http://dx.doi.org/10.1016/j.trac.2013.10.004
The International Symposium on Biological Monitoring in Occupational and Environmental Health (ISBM) series is organized by the Scientific Committee on Occupational Toxicology (SCOT) of the International Commission on Occupational Health (ICOH), in collaboration with the ICOH Scientific Committee on Toxicology of Metals (SCTM) and the ICOH Scientific Committee on Rural Health (SCRH). The mission of SCOT is to promote research in all areas of occupational toxicology. A current focus of SCOT is the use of HBM in the exposure, effect and susceptibility assessment of chemicals in workplaces and the general environment based on sound scientific and ethical principles. With its focus on laboratory-based research, most participants have backgrounds in chemistry with access to analytical laboratories having infrastructures for trace analysis of biological matrices, invariably blood and urine, but also saliva, hair, exhaled air and other human tissues. The 9th ISBM was held on 9–11 September 2013 in the Lowry Centre, Salford Quays, Manchester, historically a hot spot of occupational and environmental hazards due to heavy industry, but now an attractive modern business center annexed to a residential area. The 9th International Symposium was hosted by the Health and Safety Laboratory located in Buxton, 40 km southeast of the meeting venue. Previous meetings were in Kyoto (1992), Parma (1994), Helsinki (1996), Seoul (1998), Banff (2001), Heidelberg (2004), Beijing (2007) and Helsinki (2010). Reports of most previous meetings have been published [10–13]. 3. Biomarkers to support classification of carcinogenic risk factors The contribution of biomarkers to disentangle the complex mechanisms implicated in the process of carcinogenesis was the subject of a keynote contribution from Kurt Straif of the International Agency for Research on Cancer in Lyon, France (e-mail [email protected]). Straif explained that evidence from epidemiology studies often drives the classification of an environmental factor as a Group 1 human carcinogen but, even without ‘‘sufficient’’ evidence from population-based observational studies, it is possible to classify an environmental factor as a (‘Group 1’) human carcinogen. Such supportive evidence could be provided by HBM studies in exposed humans, such as worker populations. Currently, some 950 environmental factors are classified (some repeatedly, see http://monographs.iarc.fr/). Of these evaluated substances, more than 100 are placed in Group 1. The task of hazard classification is described in a series of monographs. The 100th monograph was devoted to a critical update on target sites and mechanisms of carcinogens in Group 1. An increase in the
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contribution of mechanistic data to Group 1 evaluations was noted and biomarker studies have contributed to this development. Substances classified as human carcinogens, for which the evidence in humans was less than ‘‘sufficient’’, include: ethylene oxide, 2,3,7,8-tetrachlorodibenzo-para-dioxin, neutrons, gallium arsenide, benzo[a]pyrene, dyes metabolized to benzidine, 4,40 -methylenebis(2-chloroaniline), 2,3,4,7,8-pentachlorodibenzofurane and dioxin-like polychlorinated biphenyls. To illustrate the role of biomarkers, Straif elaborated the case of benzo[a]pyrene: the evidence from epidemiological studies was evaluated as inadequate, but ‘‘sufficient evidence’’ from cancer bioassays was further supported by strong evidence for involvement of the diol-epoxide in lung and skin cancer, radical cations in skin tumors and the role of benzo[a]pyrene-7,8-quinone and reactive oxygen species generation, the aryl hydrocarbon receptor (AhR) and immunosuppression as potential additional mechanisms. Data in human cells, in vitro, provided evidence for the involvement of benzo[a]pyrene-diolepoxide (BPDE) DNA adducts in lung explants and mammary epithelial cells. Decisive evidence came from HBM studies performed in coke-oven workers and chimney sweeps, confirming a role of elevated levels of BPDE DNA adducts and specific Ki-RAS and TP53 mutations. Straif anticipated that the development and the validation of new biomarkers applied in molecular epidemiology studies would increasingly provide mechanistic evidence to support a causal relationship with carcinogenicity in humans in future IARC evaluations. 4. Biomarkers in characterization of the internal exposome The ‘‘exposome’’ concept was discussed previously during the 2010 ISBM meeting in a keynote paper by Christopher Wild, current Director of IARC, who introduced the term. It refers to the totality of environmental exposure from conception onwards. The external exposome relates to a myriad of environmental factors, whereas the internal exposome is characterized by measurements of biological materials using repeated sampling of biological media, especially during critical life stages, such as early childhood, puberty, and pregnancy. Biomarkers cover a wide range of molecules, ranging from xenobiotics and their metabolites in blood (metabolomics) to covalent complexes with DNA and proteins (adductomics). In a keynote lecture, Paolo Vineis (Imperial College, London, e-mail [email protected]) elaborated on the use of the exposome concept in large population-based studies, such as the EPIC study that relies on bio-banked biological materials of >400,000 human subjects. In these large cohorts, biomarkers are used to help understand the relationships between environmental factors and disease [4,9]. The latency between exposure and the onset of disease often covers many years and, for some diseases, such as cancer, several decades. Therefore, it is not surprising that 65% of cancers cannot be attributed to a specific cause. A possible approach to bridge the gap between cause and disease was introduced by Chadeau-Hyam and co-workers [2,3], who advocated the use of mechanism-based biomarkers in the ‘‘meet-in-the-middle’’ principle. An example was the finding that DNA methylation of the AhR repressor gene (AHRR) was associated with a former smoking status [8]. Whereas cotinine is a poor marker for classification of former smokers, AHRR provides a high score in the receiver operator curve (ROC) of 0.77, suggesting that DNA methylation is a suitable and persistent marker to classify a person as a former smoker. Vineis and his team will apply these, and other novel biomarkers, in a recently funded European Union (EU) project (EXPOsOMICS). Data will be collected on the internal and external exposome within on-going studies that collectively reflect all life stages from conception to old age. The internal exposome will be analyzed using omics-based approaches, such as
adductomics, metabonomics, transcriptomics, epigenomics and proteomics. For this purpose, fresh blood and urine specimens will be collected from individuals, who are followed by personal exposure monitoring to collect data on the external exposome. This involves instrumentation, such as compact direct-reading sensors for ultrafine particles and PM, both linked to a smartphone. The key concept of this study is the calibration of land use, and similar conventional models, for thousands of subjects and for a large range of diseases, including asthma, cardiovascular disease, cancer and neurodevelopment-related disease. The internal and external exposome data will be combined in an improved risk assessment for this range of diseases in the second phase of the project (see also http://www.exposomicsproject.eu/). 5. New insights in dermal absorption Two contributions challenged current beliefs as regards dermal absorption of substances used in consumer products and in work places. In an in vitro study of di(2-ethylhexyl) phthalate (DEHP), Nancy Hopf of the Institute for Work and Health (IST, Lausanne, Switzerland, e-mail [email protected]) presented new data suggesting that the dermal uptake of this plasticizer, at room temperature, represents a contribution to overall DEHP exposure. The study was performed using ex vivo, human viable skin, obtained from cosmetic surgery in a flow-through diffusion cell. Where undiluted DEHP was tested, no DEHP was detected in the reservoir fluid until 30 h. However, DEHP emulsified in water (with no further additives) was recovered from the reservoir fluid within 8 h and the primary metabolite mono-(2-ethylhexyl) phthalate (MEHP) penetrated the skin within 6 h. DEHP appears to enhance its own dermal absorption, similarly to precursors that are sometimes used in medical drugs to enhance dermal absorption. In another presentation on dermal absorption of chemicals by consumers, Jacqueline Biesterbos, (Radboud University Medical Center, Nijmegen, The Netherlands, e-mail [email protected]) discussed early results of the dermal absorption of the cyclosiloxane decamethylcyclopentasiloxane (D5), undiluted, or as ingredient in night cream, and deodorant. Healthy volunteers were exposed for 1 h and subsequently followed for another 5 h. Systemic uptake was evaluated by determination of the D5 content in end-exhaled air. In each of these three exposures, the level of D5 in end-exhaled air was similar to the baseline excretion of D5 observed in the same volunteer, following 24 h of not using any personal-care products. The researchers noticed that the experimental design was sensitive to inhalation of D5 released from the skin into the breathing zone, either by emission from residues of the topical application, or by back-diffusion of D5 taken up previously by the skin. Analysis of urine samples will be carried out in order to confirm the results obtained for exhaled air. N-methyl-2-pyrrolidone (NMP) is well known for its wide application as an organic solvent for degreasing and cleaning. It is popular due to its low vapor pressure, but a disadvantage of the use of this substance is a rapid uptake through the skin and its systemic toxicity, in particular neurotoxicity and reproductive toxicity. Due to these limitations, NMP is increasingly substituted by a structural analogue, N-ethyl-2-pyrrolidone (NEP). This new substance is not classified yet, but the toxicological data available suggest that this alternative substance has similar toxicity to NEP. Holger Koch from IPA, Ruhr-University Bochum Germany (email [email protected]), presented a new GC-MS method for the determination of urinary metabolites of NEP [i.e., 5-hydroxy-Nethyl-2-pyrrolidone (5-HNEP) and 2-hydroxy-N-ethylsuccinimide (2-HESI)]. These explain approximately 50% of the oral dose in human volunteers. 5-HNEP has an estimated half-life of 7 h, whereas 2-HESI is excreted at a slower rate with a half-life of 16 h and is still detectable 96 h after administration. In a field
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study presented by Stephan Koslitz, from the same institute, it was shown that, in a group of 23 automotive spray painters, working with a blend of NMP and NEP, the aforementioned metabolites were detected in urine in addition to the two NMP metabolites [5-hydroxy-N-methyl-2-pyrrolidone (5-HNMP) and 2-hydroxy-Nmethyl-succinimide (2-HMSI)]. The uptake of both NMP and NEP was not prevented, despite the use of rubber gloves. As expected, based on the volunteer study presented by Koch, the elimination of 2-HMSI and 2-HESI was slower than that of the respective hydroxyl metabolites. As a consequence, the highest levels of 2-HMSI and 2-HESI were not observed at the end of the work shift but the following morning prior to the next shift. 6. Permethrin HBM For HBM of permethrin, the excretion of urinary metabolites 3phenoxybenzoic acid (3-BPA) and 3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylic acid (DCCA) are used. Thomas Göen of the University of Erlangen-Nurnberg (e-mail thomas.goeen@ ipasum.med.uni-erlangen.de) presented the results of an interlaboratory comparison of the performance of this analysis for 25 urine samples from Germany and 22 samples from Japan. Although the laboratories used different methods of derivatization in pre-treatment, the results of the GC–MS analyses corresponded very well with correlation coefficients of R2 = 0.9437 and R2 = 0.9368 for 3-BPA and DCCA, respectively. Michèle Bouchard and her PhD student, Mylène Ratelle, of the University of Montreal, Quebec, Canada (e-mail [email protected]) studied the time profiles of metabolites 3PBA and DCCA in blood and urine following oral administration in six volunteers. Maximum plasma levels were reached at 2 h post exposure. The average apparent half-life in serum was 10.5 h for cis-DCCA, 11 h for trans-DCCA and 18 h for 3-BPA. The peak rate in urine of the various metabolites was observed within 8 h and the average apparent elimination half-life was less than 14 h, leading to >97% of the dose eliminated after 48 h. Bernd Rossbach of the University Medical Center of the Johannes Gutenberg University of Mainz, Germany (e-mail [email protected]) studied the uptake of permethrin from impregnated clothes used to protect forestry workers from Lyme disease. He compared the excretion of urinary metabolites in three groups of volunteers wearing the impregnated trousers and jackets during a simulated work shift of 8 h under three different conditions: (a) default, (b) increased temperature of 25°C and increased relative humidity of 65%, and (c) the same as (b) but with a simulated, increased workload. Urine was analyzed for the permethrin metabolites cis-DCCA, trans-DCCA, and 3-BPA. The urinary metabolite levels were similar before exposure and increased between 8 h and 16 h after cessation of exposure with median concentrations of the total of the metabolites of 5.62 lg L1, 10.63 lg L1 and 15.95 lg L1 for the aforementioned conditions, respectively. These results suggest that permethrin absorption from impregnated clothes increases at elevated temperature and higher relative humidity and also with increased physical exercise. However, this last increase may also be the result of an increase in the formation of the metabolites due to increased perfusion of the liver. 7. HBM and incidents From The Netherlands, two presentations discussed the implementation of HBM following industrial incidents. Henri Heussen of Arbo Unie (Occupational Health Service) Harderwijk, The Netherlands (e-mail [email protected]) presented the use of HBM following a chemical-plant blaze in Moerdijk’s industrial area on 5 January 2011. The clean-up workers
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had to deal with heavy contamination of soil and surface water with monocyclic and polycyclic aromatic hydrocarbons. The concern of potential dermal uptake of these substances made the use of HBM appropriate. However, the study plan implemented by the cleaning company failed because the kinetics of urinary metabolite excretion was not taken into account when planning the collection of urine samples. This resulted in data that were largely unsuitable for assessing the possible uptake as a result of the clean-up activity. In a second presentation, Paul Scheepers of the Radboud University Medical Centre responded to this problem by referring to the guidance prepared by the Dutch Community Health Services, in collaboration with the National Institute of Public Health and the Environment (RIVM), which offers an algorithm to calculate, for each biomarker of interest, the most appropriate window of time for sample collection, taking into account the elimination half-life and the limit of quantification for each biomarker. The guidance is aimed at public health services and could also be useful for occupational health services, but then adaptations would need to be made. In addition to supporting the decision whether to use HBM (or to not use it) and the preparation of an adequate study plan, a service desk supports an HBM study by supplying appropriate materials for sample collection, pre-treatment, storage and transportation to a laboratory with extensive experience in analysis. A network of 10 laboratories has been established to support rapid analysis of 224 biomarkers for 132 different chemical substances. For more than 50% of the biomarkers, these laboratories have certificates of the German EQUAS Inter-Laboratory Quality Assurance Scheme. That an appropriate HBM response to unintended direct contact with chemicals can be effective was shown by Michael Bader of BASF, Ludwigshafen (e-mail [email protected]), who monitored workers of several industrial production plants. To assess benzene exposure, urine samples were analyzed for the metabolites S-phenylmercapturic acid (SPMA) and t,t-muconic acid (ttMA), as well as for unmetabolized urinary benzene. Plant managers received reports on the outcome of these analyses on a daily basis and could immediately intervene in cases of unacceptable uptake of benzene. Elevated ttMA was observed, for example, during the dismantling of installations and the emptying of pipes and pumps. Bader also presented results from an HBM program after an accident in 2005, involving a blast from a heated barrel resulting in carbonization of pyraclostrobin, which yielded p-chloroaniline (p-CA) as a process emission. In addition to plant workers, fire fighters and paramedics (a total of 145 persons) were involved in an HBM campaign, which demonstrated elevated urine levels of p-CA in several cases. The last presentation of the session covered reports of fume events in airplanes by cabin crews, who also reported a variety of health complaints in relation to those events. Tobias Weiss and his team at IPA, Bochum, Germany (e-mail [email protected]) have developed sensitive methods for the determination of triortho-cresylphosphate (o-TCP) and chemical analogues (organophosphate flame retardants). These substances are added to oils used in jet engines and are supposed to enter the plane interior as bleed air. Similar chemicals are also used as flame retardants in the interior of airplanes. A first attempt to detect elevated exposures in members of cabin crews in Germany using urinary biomarkers did not confirm that those persons were internally exposed to o-TCP due to fume events. Rather, they appeared to be exposed to organophosphate flame retardants slightly above baseline levels. Mechanics who had direct contact with the oils during maintenance of jet engines had somewhat elevated levels of o-TCP urinary metabolites, which were likely to be a consequence of dermal absorption.
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Table 1 New and improved biomarkers of exposure Biomarker
Biological material
Method
Type of study
Affiliation of contributor and references
Acrylonitrile
Urine
LC–MS/MS
Smokers
Thomas Schettgen ([email protected])
Urine
lLC-ICP-MS
Worker survey
Elizabeth Leese ([email protected])
Benzene
N-acetyl-S-(2-cyanoethyl)cysteine (CEMA), N-acetyl-S-(1-cyano-2hydroxyethyl)cysteine (CHEMA) Arsenobetaine (AB), arsenite (As3+), Arsenate (As5+), dimethylarsinate (DMA), monomethylarsonate (MMA) S-phenylmercapturic acid
Urine
ELISA
Worker survey
Beryllium
Beryllium
Urine
Quadrupole ICP-MS
Unexposed reference values
Bis(2-propylheptyl)phthalate (DPHP)
Mono-2-(propyl-6hydroxyheptyl)phthalate (OHMPHP), Mono-2-(propyl-6oxoheptyl)phthalate (oxo-MPHP), mono-2-(propyl-6-carboxyhexyl)phthalate (cx-MPHxP) Cotinine (COT), trans-3’hydroxycotinine (HCOT) Decamethylcyclopentasiloxane (D5)
Urine
LC–MS/MS
Volunteer study
Lathan Ball ([email protected]) Jacky Morton ([email protected]) Gabriele Leng ([email protected])
Urine
Online-SPE-UPLC-MS/MS
General population
Exhaled air
TD-GC-MS
Volunteers study
Exhaled air, blood
GC-FID, ATD-GC-FID
Volunteer study
Blood, Urine
GC-MS (MIH), LC–MS/MS (AMCC)
Worker’s survey
Thomas Göen ([email protected])
Urine
UHPLC–ESI–MS/MS
General population
Urine Urine
GC–MS GC–MS
Volunteer study Worker’s study
Cristina Sottani ([email protected]) Jaroslav Mráz ([email protected]) H.M. Koch ([email protected])
Urine
LC–MS/MS
Volunteer study
Arsenic species
Nicotine Decamethylcyclopenta-siloxane (D5)
Methamidophos
1,1-Difluoroethane 1,1,1-Trifluororethane 1,1,1,2-Tetrafluoroethane 1,1,1,3,3-Pentafluoropropane 3-Methyl-5-isoproylhydantoin (MIH, released from N-terminal Hb adduct), N-acetyl-S-(Nmethylcarbamoyl)cysteine (AMCC) Ethylenethiourea (ETU), propylenethiourea (PTU) 2-Ethoxyacetic acid (EEA) 5-Hydroxy-N-ethyl-2-pyrrolidone (5HNEP); 2-hydroxy-Nethylsuccinimide (2-HESI) Methamidophos
Octamethylcyclotetrasiloxane (D4)
Octamethylcyclotetrasiloxane (D4)
Exhaled air
TD–GC–MS
Volunteer study
(1S,6R)-(+)-3-carene
Urine
GC–PCI–MS/MS
Volunteer study
Urine
GC–PCI–MS/MS
Volunteer study
Urine
GC–PCI–MS/MS
Volunteer study
Blood
GC–HRMS–NICI
General population
Isocyanurate
3-Caren-10-ol, 3-caren-10-carboxylic acid (1R,2R,5R)-cis-verbenol, (1R,2S,5R)trans-verbenol, (1S,5R)-(+)-myrtenol (1S, 5S)-trans-carveol, (1R, 5S)-ciscarveol, (1S, 2S, 4R)-limonene-1,2diol, perillyl alcohol, perillic acid, limonene-8,9-diol 5-Isopropyl-3[4-(4aminobenzyl)phenyl]hydantoin Isotriamine
Urine
UPLC-MS/MS
Worker survey
Indium
Indium
Serum
ICP–MS
Worker survey
2- and 3-Nitrobenzanthrone
2-Aminobenzanthron-3ylmercapturic acid (2-ABA-MA), 3aminobenzanthron-3-ylmercapturic acid (3-ABA-MA)
Urine
LC-ESI-MS/MS
Rat study
1,1-Difluoroethane 1,1,1-Trifluoroethane 1,1,1,2-Tetrafluoroethane 1,1,1,3,3-Pentafluoropropane N,N-dimethylformamide (DMF)
Dithiocarbamates (DTC) 2-Ethoxyethanol N-ethyl-2-pyrrolidone (NEP)
(1R,5S)-(+)-a-pinene, (1S, 5S)-(-)-a-pinene (R)-(+)-limonene
4,4’-Methylenediphenyl diisocyanate (MDI)
Ilse van Overmeire ([email protected]) Gwendolyn Beckmann ([email protected]) Lena Ernstgård ([email protected])
F. Garner (fi[email protected]) G. Beckmann ([email protected]) L. Schmidt ([email protected]) [6] L. Schmidt ([email protected]) [6] L. Schmidt ([email protected]) [6]
Gabriele Leng ([email protected]) L.A. Nylander-French ([email protected]) Mi-young Lee ([email protected]) Igor Linhart ([email protected])
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Chemical substance
o-Tricresyl phosphate Bifenthrin/cyhalothrin
ELISA, Enzyme-linked immunosorbent assays; GC-HRMS-NICI, Gas chromatography high-resolution mass spectrometry negative-ion chemical ionization; GC-NCI-MS, Gas chromatography negative chemical ionization mass spectrometry; GC-PCI-MS, Gas chromatography positive chemical ionization mass spectrometry; ICP–MS, Inductively-coupled plasma mass spectrometry; SHS–GC–MS, Static headspace gas chromatography mass spectrometry; SPME, Solid-phase microextraction; UHPLC–ESI–MS/MS, Ultra-high performance liquid chromatography electrospray ionisation tandem mass spectrometry; UPLC–MS/MS, ultra-performance liquid chromatography tandem mass spectrometry; lLC, Micro liquid chromatography; lLC–ICP-MS, Micro liquid chromatography inductively-coupled plasma mass spectrometry.
Urine Urine
GC–MS/MS GC–MS/MS
Worker survey General population
Silvia Fustinoni (silvia.fustinoni@ unimi.it) Tobias Weiss ([email protected]) [7] Thomas Schettgen ([email protected]) Worker survey/general population LC–MS/MS Urine, hair Terbuthylazine
Tebuconazole
t-Butylhydroxy-tebuconazole (TEBOH), t-butylcarboxy-tebuconazole (TEB-COOH) Terbutylazine (TBA), desethylterbutylazine (DET) o,o-Dicresylphosphate 3-(2-Chloro-3,3,3-trifluoroprop-1enyl)-2,2dimethylcyclopropanecarboxylic acid (TFP-acid);cis- and trans-3-(2,2dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic acid (cis- and trans DCCA); cis-3-(2,2dibromovinyl)-2,2dimethylcyclopropanecarboxylic acid (DBCA); 4-chloro-isopropyl benzeneacetic acid (CPBA); 3phenoxybenzoic acid (3-PBA); 4fluoro-3-phenoxybenzoic acid (FPBA); 2-methyl-3-phenylbenzoic acid (2-MPA)
Urine
LC–MS/MS
Worker survey
Silvia Fustinoni (silvia.fustinoni@ unimi.it)
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8. Other promising new developments Some new methods were introduced to tackle old and contemporary chemical exposures. Most new analytical methods are highly specific, sensitive, mass spectrometry (MS)-based and involve highly sophisticated pre-treatment (see Table 1). Other methods rely on the use of immunoassays for pre-screening, such as the ELISA-based dip-stick test for on-site testing of benzene exposures for urinary S-phenylmercapturic introduced by Lathan Ball of Biomark, Cardiff, United Kingdom (e-mail lball@ abbiomonitoring.co.uk). Urine samples with positive scores need to be confirmed by a more laborious, costly analysis using gas chromatography (GC)–MS/MS (Tables 2 and 3). Investigation of epigenetic modification of biomarker results has also entered into the field of HBM. An interesting example of this is the determination of DNA methylation on urinary biomarker results in workers exposed to 1,6-hexamethylene diisocyanate (HDI) in a pilot study presented by Leena Nylander-French of the University of North Carolina, Chapel Hill, NC, USA (e-mail [email protected]). In a group of 20 automotive spray-painters, blood DNA was analyzed for 5-methyl-CpG marks associated with urinary 1,6-hexamethylene diamine (HDA) levels using linear mixed modeling accounting for exposure, ethnicity, age and smoking status. Significant DNA methylation marks were associated with individual differences in urinary HDA levels. Bioinformatics analysis revealed two significant, predicted, networks associated with this phenotype in HDI exposed workers. The identified genes are involved in antigen processing and presentation, as well as immune responses, which appear to be related to plausible working mechanisms for HDI toxicity. These preliminary findings demonstrate the high potential of using DNA methylation marks associated with individual difference in urinary biomarker levels that may be instrumental in identification of susceptible subpopulations in future studies (Table 2). A new approach to the use of covalent binding of alkylating agents to proteins was proposed by Jaroslav Mráz of the National Institute of Public Health, Prague, Czech Republic (e-mail [email protected]). It was hypothesized that adducted blood proteins, such as globin, will undergo proteolysis and that amino-acid adducts will appear in urine. Unlike adducts to DNA, there are no known repair mechanisms for protein adducts. Structural modifications will therefore persist until complete recycling at the end of the life span of the protein. Native amino acids are reused, but it is likely that modified amino acids may follow a different fate and may be excreted with feces or urine. It is supposed that these adducted protein fragments in urine could be used as biomarkers of exposure. What is interesting about this proposal is that these biomarkers will still carry the structural information, which may be used to identify the specific chemical substances involved in the exposure. To study this hypothesis, amino-acid adducts derived from the common alkylating substances methyl isocyanate and ethylene oxide were synthesized and injected intraperitoneally in rats. Analysis of urine collected over 48 h showed that the expected amino-acid adducts for methyl isocyanate [N-(methylcarbamoyl)valine and Ne-(methylcarbamoyl)lysine)] and ethylene oxide [N-(2-hydroxyethyl)valine and S-(2-hydroxyethyl)cysteine)] could be detected by liquid chromatography electrospray ionization (LC-ESI)–MS/MS. More than 90% of the excreted adducts were retrieved (unchanged or further metabolized) in the first 24 h. This is a promising first step in exploring the full potential of this novel approach. Future research will demonstrate whether these adducts can be detected following administration of the parent compounds. For application in humans, these species will have to be detected in the presence of a myriad of endogenous, native amino acids, of which many more may also have
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Table 2 Biomarkers of susceptibility Chemical substance
Biomarker of susceptibility
Adverse health effect
Study population
Affiliation of contributor and references
Benzene
GSTT1, GSTM1 GSTA1, GSTP1, mEHex3, mEHex4, NQ01, CYP2E1, CYP1A, MPO, Circulating mitochondrial DNA
Cancer
301 benzene-exposed subjects
N. Mazitova ([email protected])
Neurological and respiratory dysfunction Cancer
86 persons with confirmed exposure to off-gassing haloalkane pesticides in storage facilities or residential environments 255 workers exposed to styrene in the marble, plastics and boat industry and 49 non-exposed workers 129 workers in the footwear industry and 13 controls
Lygia Therese Budnik ([email protected]) [1] Maria José Prieto Castelló ([email protected]) Maria José Prieto Castelló ([email protected])
Halo-alkane-based pesticides Styrene
GSTM1, EPHX1
Toluene
CYP2E1
Cytogenetic damage
Table 3 Biomarkers linking physiological responses to potential adverse effects Chemical substance
Biomarker
Related adverse health effect
Aim of the study
Affiliation of contributor and references
Arsenic (inorganic)
MircoRNAs
Cellular pathology
Claudio Minoia ([email protected])
Benzene
Chromosome aberration and micronuclei
Cancer
Cadmium
a1-microglobulin (a1-MG), b2microglobulin (b2-MG) and Nacetyl-b-D-glucosaminidase (NAG) DNA methylation marks
Tubular dysfunction
Role of microRNAs, negative regulators of gene expression in pathological cellular processes Influence of low exposures of benzene on biomarkers of cytogenetic damage Association of previous disease with cadmium exposure parameters Identify individual differences in DNA methylation associated with biomarker levels Study of association of manganese and problematic behavior in school children in Bahia, Brazil Relationships between the measured biomarkers of effect and other health-outcome measures and exposure estimates Identify biomarkers of exposure and early health indicators of low-level VOCs from building materials and consumer products Assess the feasibility of using cell-proliferation ratio of lymphocytes in umbilical cord blood as an indicator of subclinical pathology in fetus
1,6-Hexamethylene diisocyanate (HDI)
Occupational asthma
Manganese
Manganese in hair
Neurobehavioral effects
Silica
Clara cell protein (CC16), lung surfactant proteins (A&D) Kidney injury molecule-1 (KIIM-1), neutrophil gelatinase-associated lipocalin (NGAL) and cytin C VOC and 8’-oxo-7,8-dihydro-2’deoxyguanosine (8-OH-dG) in urine
Lung and kidney
Volatile organic compounds
Reproduction toxic substances
MTT cell-proliferation ratio
DNA damage and/or DNA oxidation
Sub-clinical pathology in fetus
been structurally modified (e.g., oxidative protein damage). This will require highly-selective, effective enrichment of the analytes of interest. 9. Environmental exposures and developing countries Due to the increased sensitivity and the specificity of analytical techniques applied in contemporary HBM campaigns, many of the recent research projects are related to environmental exposures and consumer safety. As an example, Roel Smolders of VITO, Belgium (e-mail [email protected]) presented data from the EUfunded HBM survey, carried out in 2011 and 2012 in 17 European countries as part of the COPHES-DEMOCOPHES project involving 1800 mother-child pairs. Aggregated fish consumption could be related to mercury and cadmium in hair, and dietary levels related to urinary cadmium and urinary cotinine were associated with the effectiveness of anti-smoking programs. A limitation of this study was the availability and quality of contextual information.
Piero Lovreglio ([email protected]) Masayuki Ikeda ([email protected]) Leena Nylander-French ([email protected]) José A Menezes-FIlho ([email protected])
Howard Mason ([email protected])
Massimiliano Mascelloni ([email protected])
Isabella Karakis ([email protected])
In a keynote lecture, Stephan Böse-O’Reilly of the University of München (e-mail [email protected]) explained the health effects resulting from the use of mercury in small-scale gold mining. Approximately 15% of gold is mined in an artisanal manner in 70 different countries across the globe and is responsible for the largest environmental burden of mercury (1100 tons of mercury per annum). This type of mining has great environmental implications, but local and regional authorities do not give much priority to its impact on health. Smelting mercury from ore is associated with highest exposures and leads to serious effects on the brain. Most effects are related to the toxicity of metallic mercury and are characterized by dysfunction of the cerebellum leading to ataxia and coordination problems. Children suffer similar exposures to parents, as most of the gold-related activities are not separated from the residential environment. Urinary mercury levels in children were sometimes even higher than the values in mothers, who were in direct contact with the mercury. Böse-O‘Reilly conducted his research in many countries, such
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as Indonesia (Kalimantan and Sulawesi), Tanzania, Zimbabwe and Mongolia, and is promoting mercury-free alternative gold mining (e.g., using borax). HBM could be a valuable tool to demonstrate the effect of such interventions and to help address the problems with metal toxicity in the developing parts of the world. 10. Conclusions The potential of HBM is driven by the increased technical performance of laboratory-based techniques. In addition to sensitivity and specificity, the capacity for high-throughput analysis of biological specimen appears to be critical to the future growth of HBM in large-scale population-based studies. The applications of HBM evolved from high single exposure in subgroups (e.g., workers) to low aggregated exposures in the general populations. HBM now faces the challenge of contributing to characterizing the totality of the internal exposome, not once in an individual’s lifetime, but frequently, and at different critical life stages, in cohorts of thousands of study subjects. An important role remains for targeted approaches that can be used in growing fields of application, such as dermal absorption of chemical substances in workers and consumers, and exposure characterization following chemical incidents and disasters and interventions in developing countries. Acknowledgements The authors thanks the cited contributors for their consent to publish this report and for suggestions made to improve its quality. The author would also like to express their gratitude to Paul Aston of AB Biomonitoring for some final editing. References [1] L.T. Budnik, S. Kloth, X. Baur, A.M. Preisser, H. Schwarzenbach, Circulating mitochondrial DNA as biomarker linking environmental chemical exposure to early preclinical lesions elevation of mtDNA in human serum after exposure to carcinogenic halo-alkane-based pesticides, PLoS One 8 (5) (2013 May 31) e64413, http://dx.doi.org/10.1371/journal.pone.0064413. Print 2013. [2] M. Chadeau-Hyam, T.J. Athersuch, H.C. Keun, M. De Iorio, T.M. Ebbels, M. Jenab, C. Sacerdote, S.J. Bruce, E. Holmes, P. Vineis, Meeting-in-the-middle using metabolic profiling – a strategy for the identification of intermediate biomarkers in cohort studies, Biomarkers 16 (1) (2011 Feb) 83–88, http:// dx.doi.org/10.3109/1354750X.2010.533285. Epub 2010 Nov 29.
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[3] M. Chadeau-Hyam et al, Deciphering the complex: methodological overview of statistical models to derive OMICS-based biomarkers, Environ. Mol. Mutagen. 54 (7) (2013 Aug) 542–557. [4] D.G. Hebels, P. Georgiadis, H.C. Keun, T.J. Athersuch, P. Vineis, R. Vermeulen, L. Portengen, I.A. Bergdahl, G. Hallmans, D. Palli, B. Bendinelli, V. Krogh, R. Tumino, C. Sacerdote, S. Panico, J.C. Kleinjans, T.M. de Kok, M.T. Smith, S.A. Kyrtopoulos, EnviroGenomarkers Project Consortium, Performance in omics analyses of blood samples in long-term storage: opportunities for the exploitation of existing biobanks in environmental health research, Environ. Health Perspect 121 (4) (2013 Apr) 480–487. [5] M. Manno, C. Viau, in collaboration with, J. Cocker, C. Colosio, L. Lowry, A. Mutti, M. Nordberg, S. Wang, Biomonitoring for occupational health risk assessment (BOHRA), Toxicol. Lett. 192 (1 (January)) (2010) 3–16, http:// dx.doi.org/10.1016/j.toxlet.2009.05.001. [6] L. Schmidt, V.N. Belov, Th. Göen, Sensitive monitoring of monoterpene metabolites in human urine using two-step derivatisation and positive chemical ionisation–tandem mass spectrometry, Anal. Chim. Acta 793 (2013) 26–36, http://dx.doi.org/10.1016/j.aca.2013.07.046. [7] B.K. Schindler, T. Weiss, A. Schütze, S. Koslitz, H.C. Broding, J. Bünger, T. Brüning, Occupational exposure of air crews to tricresyl phosphate isomers and organophosphate flame retardants after fume events, Arch. Toxicol. 87 (4 (April)) (2013) 645–648, http://dx.doi.org/10.1007/s00204-012-0978-0. [8] N.S. Shenker, P.M. Ueland, S. Polidoro, K. van Veldhoven, F. Ricceri, R. Brown, J.M. Flanagan, P. Vineis, DNA methylation as a long-term biomarker of exposure to tobacco smoke, Epidemiology 24 (5 (September)) (2013) 712–716, http://dx.doi.org/10.1097/EDE.0b013e31829d5cb3. [9] P. Vineis et al, Advancing the application of omics-based biomarkers in environmental epidemiology, Environ. Mol. Mutagen. 54 (7 (August)) (2013) 461–467. [10] P.T.J. Scheepers, G.A.H. Heussen, Trends Anal. Chem. 21 (2002) 11. [11] P.T.J. Scheepers, G.A.H. Heussen, Biomarkers 10 (2005) 80. [12] P.T.J. Scheepers, G.A.H. Heussen, Biomarkers 13 (2008) 133. [13] P.T.J. Scheepers, Trends Anal. Chem. 30 (2011) 415.
Paul T.J. Scheepers Gwendolyn Beckmann Jacqueline W.H. Biesterbos Research Lab Molecular Epidemiology, Department for Health Evidence, Radboud University Medical Center, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands Tel.: +31 24 3616878; Fax: +31 24 3613505. E-mail address: [email protected] (P.T.J. Scheepers)
Contents lists available at ScienceDirect
Trends in Analytical Chemistry journal homepage: www.elsevier.com/locate/trac
Meeting Report Biomarkers of environmental risk factors for prevention and research
challenges the user to collaborate with colleagues in other fields to achieve the desired reduction of chemical risk and prevention of disease. 2. Background
1. Introduction Human biological monitoring (HBM) is the repeated, controlled measurement of chemical or biochemical markers in body fluids, tissues or other accessible samples from human subjects exposed to chemical, physical or biological risk factors in the workplace and/or the general environment [5]. This applied field of analytical chemistry is different from the application of biomarkers in a clinical setting and is directed towards the reduction of exposure to chemicals and prevention of disease. In the early development of the concept of HBM, occupational exposures and prevention of occupational disease were important foci. As the sensitivity and the specificity of analytical methods improved, HBM was increasingly used to characterize exposures of the general population. Currently, there are large-scale national HBM programs in the USA, Canada, more than 15 European countries and also some Asian countries, such as South Korea. In addition to biomarkers of exposure, biomarkers of susceptibility and biomarkers of early physiological responses were used to understand the mechanisms leading to the onset of disease and to identify sensitive groups. Susceptibility and early effect biomarkers are not exclusive to the field of HBM and are also used in a clinical setting as clinical markers of diagnosis, prognosis or for clinical research. There are some new challenging fields of HBM application, such as: describing the total of all internal exposures to environmental factors in new research programs involving thousands of study subjects; evaluating interventions to mitigate environmental risk factors in developing countries; and, classifying exposures of victims of chemical incidents in the interest of medical support, or treatment and risk communication. The development of new biomarkers relies much on analytical techniques and most researchers in this field have a background in laboratory-based research. Once laboratory methods are validated, there are still many challenges in the application of these new biomarkers, such as the kinetic patterns in different biological media, the flawless collection of samples and the pre-treatment and storage of biological specimens. Particular challenges in the interaction with study participants engaged in HBM campaigns concern ethical implications, informed consent, the potential burden of invasive sample collection and the communication of HBM results to individuals and groups. HBM is therefore much more than the technical performance of a chemical analysis and it doi:http://dx.doi.org/10.1016/j.trac.2013.10.004
The International Symposium on Biological Monitoring in Occupational and Environmental Health (ISBM) series is organized by the Scientific Committee on Occupational Toxicology (SCOT) of the International Commission on Occupational Health (ICOH), in collaboration with the ICOH Scientific Committee on Toxicology of Metals (SCTM) and the ICOH Scientific Committee on Rural Health (SCRH). The mission of SCOT is to promote research in all areas of occupational toxicology. A current focus of SCOT is the use of HBM in the exposure, effect and susceptibility assessment of chemicals in workplaces and the general environment based on sound scientific and ethical principles. With its focus on laboratory-based research, most participants have backgrounds in chemistry with access to analytical laboratories having infrastructures for trace analysis of biological matrices, invariably blood and urine, but also saliva, hair, exhaled air and other human tissues. The 9th ISBM was held on 9–11 September 2013 in the Lowry Centre, Salford Quays, Manchester, historically a hot spot of occupational and environmental hazards due to heavy industry, but now an attractive modern business center annexed to a residential area. The 9th International Symposium was hosted by the Health and Safety Laboratory located in Buxton, 40 km southeast of the meeting venue. Previous meetings were in Kyoto (1992), Parma (1994), Helsinki (1996), Seoul (1998), Banff (2001), Heidelberg (2004), Beijing (2007) and Helsinki (2010). Reports of most previous meetings have been published [10–13]. 3. Biomarkers to support classification of carcinogenic risk factors The contribution of biomarkers to disentangle the complex mechanisms implicated in the process of carcinogenesis was the subject of a keynote contribution from Kurt Straif of the International Agency for Research on Cancer in Lyon, France (e-mail [email protected]). Straif explained that evidence from epidemiology studies often drives the classification of an environmental factor as a Group 1 human carcinogen but, even without ‘‘sufficient’’ evidence from population-based observational studies, it is possible to classify an environmental factor as a (‘Group 1’) human carcinogen. Such supportive evidence could be provided by HBM studies in exposed humans, such as worker populations. Currently, some 950 environmental factors are classified (some repeatedly, see http://monographs.iarc.fr/). Of these evaluated substances, more than 100 are placed in Group 1. The task of hazard classification is described in a series of monographs. The 100th monograph was devoted to a critical update on target sites and mechanisms of carcinogens in Group 1. An increase in the
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contribution of mechanistic data to Group 1 evaluations was noted and biomarker studies have contributed to this development. Substances classified as human carcinogens, for which the evidence in humans was less than ‘‘sufficient’’, include: ethylene oxide, 2,3,7,8-tetrachlorodibenzo-para-dioxin, neutrons, gallium arsenide, benzo[a]pyrene, dyes metabolized to benzidine, 4,40 -methylenebis(2-chloroaniline), 2,3,4,7,8-pentachlorodibenzofurane and dioxin-like polychlorinated biphenyls. To illustrate the role of biomarkers, Straif elaborated the case of benzo[a]pyrene: the evidence from epidemiological studies was evaluated as inadequate, but ‘‘sufficient evidence’’ from cancer bioassays was further supported by strong evidence for involvement of the diol-epoxide in lung and skin cancer, radical cations in skin tumors and the role of benzo[a]pyrene-7,8-quinone and reactive oxygen species generation, the aryl hydrocarbon receptor (AhR) and immunosuppression as potential additional mechanisms. Data in human cells, in vitro, provided evidence for the involvement of benzo[a]pyrene-diolepoxide (BPDE) DNA adducts in lung explants and mammary epithelial cells. Decisive evidence came from HBM studies performed in coke-oven workers and chimney sweeps, confirming a role of elevated levels of BPDE DNA adducts and specific Ki-RAS and TP53 mutations. Straif anticipated that the development and the validation of new biomarkers applied in molecular epidemiology studies would increasingly provide mechanistic evidence to support a causal relationship with carcinogenicity in humans in future IARC evaluations. 4. Biomarkers in characterization of the internal exposome The ‘‘exposome’’ concept was discussed previously during the 2010 ISBM meeting in a keynote paper by Christopher Wild, current Director of IARC, who introduced the term. It refers to the totality of environmental exposure from conception onwards. The external exposome relates to a myriad of environmental factors, whereas the internal exposome is characterized by measurements of biological materials using repeated sampling of biological media, especially during critical life stages, such as early childhood, puberty, and pregnancy. Biomarkers cover a wide range of molecules, ranging from xenobiotics and their metabolites in blood (metabolomics) to covalent complexes with DNA and proteins (adductomics). In a keynote lecture, Paolo Vineis (Imperial College, London, e-mail [email protected]) elaborated on the use of the exposome concept in large population-based studies, such as the EPIC study that relies on bio-banked biological materials of >400,000 human subjects. In these large cohorts, biomarkers are used to help understand the relationships between environmental factors and disease [4,9]. The latency between exposure and the onset of disease often covers many years and, for some diseases, such as cancer, several decades. Therefore, it is not surprising that 65% of cancers cannot be attributed to a specific cause. A possible approach to bridge the gap between cause and disease was introduced by Chadeau-Hyam and co-workers [2,3], who advocated the use of mechanism-based biomarkers in the ‘‘meet-in-the-middle’’ principle. An example was the finding that DNA methylation of the AhR repressor gene (AHRR) was associated with a former smoking status [8]. Whereas cotinine is a poor marker for classification of former smokers, AHRR provides a high score in the receiver operator curve (ROC) of 0.77, suggesting that DNA methylation is a suitable and persistent marker to classify a person as a former smoker. Vineis and his team will apply these, and other novel biomarkers, in a recently funded European Union (EU) project (EXPOsOMICS). Data will be collected on the internal and external exposome within on-going studies that collectively reflect all life stages from conception to old age. The internal exposome will be analyzed using omics-based approaches, such as
adductomics, metabonomics, transcriptomics, epigenomics and proteomics. For this purpose, fresh blood and urine specimens will be collected from individuals, who are followed by personal exposure monitoring to collect data on the external exposome. This involves instrumentation, such as compact direct-reading sensors for ultrafine particles and PM, both linked to a smartphone. The key concept of this study is the calibration of land use, and similar conventional models, for thousands of subjects and for a large range of diseases, including asthma, cardiovascular disease, cancer and neurodevelopment-related disease. The internal and external exposome data will be combined in an improved risk assessment for this range of diseases in the second phase of the project (see also http://www.exposomicsproject.eu/). 5. New insights in dermal absorption Two contributions challenged current beliefs as regards dermal absorption of substances used in consumer products and in work places. In an in vitro study of di(2-ethylhexyl) phthalate (DEHP), Nancy Hopf of the Institute for Work and Health (IST, Lausanne, Switzerland, e-mail [email protected]) presented new data suggesting that the dermal uptake of this plasticizer, at room temperature, represents a contribution to overall DEHP exposure. The study was performed using ex vivo, human viable skin, obtained from cosmetic surgery in a flow-through diffusion cell. Where undiluted DEHP was tested, no DEHP was detected in the reservoir fluid until 30 h. However, DEHP emulsified in water (with no further additives) was recovered from the reservoir fluid within 8 h and the primary metabolite mono-(2-ethylhexyl) phthalate (MEHP) penetrated the skin within 6 h. DEHP appears to enhance its own dermal absorption, similarly to precursors that are sometimes used in medical drugs to enhance dermal absorption. In another presentation on dermal absorption of chemicals by consumers, Jacqueline Biesterbos, (Radboud University Medical Center, Nijmegen, The Netherlands, e-mail [email protected]) discussed early results of the dermal absorption of the cyclosiloxane decamethylcyclopentasiloxane (D5), undiluted, or as ingredient in night cream, and deodorant. Healthy volunteers were exposed for 1 h and subsequently followed for another 5 h. Systemic uptake was evaluated by determination of the D5 content in end-exhaled air. In each of these three exposures, the level of D5 in end-exhaled air was similar to the baseline excretion of D5 observed in the same volunteer, following 24 h of not using any personal-care products. The researchers noticed that the experimental design was sensitive to inhalation of D5 released from the skin into the breathing zone, either by emission from residues of the topical application, or by back-diffusion of D5 taken up previously by the skin. Analysis of urine samples will be carried out in order to confirm the results obtained for exhaled air. N-methyl-2-pyrrolidone (NMP) is well known for its wide application as an organic solvent for degreasing and cleaning. It is popular due to its low vapor pressure, but a disadvantage of the use of this substance is a rapid uptake through the skin and its systemic toxicity, in particular neurotoxicity and reproductive toxicity. Due to these limitations, NMP is increasingly substituted by a structural analogue, N-ethyl-2-pyrrolidone (NEP). This new substance is not classified yet, but the toxicological data available suggest that this alternative substance has similar toxicity to NEP. Holger Koch from IPA, Ruhr-University Bochum Germany (email [email protected]), presented a new GC-MS method for the determination of urinary metabolites of NEP [i.e., 5-hydroxy-Nethyl-2-pyrrolidone (5-HNEP) and 2-hydroxy-N-ethylsuccinimide (2-HESI)]. These explain approximately 50% of the oral dose in human volunteers. 5-HNEP has an estimated half-life of 7 h, whereas 2-HESI is excreted at a slower rate with a half-life of 16 h and is still detectable 96 h after administration. In a field
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study presented by Stephan Koslitz, from the same institute, it was shown that, in a group of 23 automotive spray painters, working with a blend of NMP and NEP, the aforementioned metabolites were detected in urine in addition to the two NMP metabolites [5-hydroxy-N-methyl-2-pyrrolidone (5-HNMP) and 2-hydroxy-Nmethyl-succinimide (2-HMSI)]. The uptake of both NMP and NEP was not prevented, despite the use of rubber gloves. As expected, based on the volunteer study presented by Koch, the elimination of 2-HMSI and 2-HESI was slower than that of the respective hydroxyl metabolites. As a consequence, the highest levels of 2-HMSI and 2-HESI were not observed at the end of the work shift but the following morning prior to the next shift. 6. Permethrin HBM For HBM of permethrin, the excretion of urinary metabolites 3phenoxybenzoic acid (3-BPA) and 3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylic acid (DCCA) are used. Thomas Göen of the University of Erlangen-Nurnberg (e-mail thomas.goeen@ ipasum.med.uni-erlangen.de) presented the results of an interlaboratory comparison of the performance of this analysis for 25 urine samples from Germany and 22 samples from Japan. Although the laboratories used different methods of derivatization in pre-treatment, the results of the GC–MS analyses corresponded very well with correlation coefficients of R2 = 0.9437 and R2 = 0.9368 for 3-BPA and DCCA, respectively. Michèle Bouchard and her PhD student, Mylène Ratelle, of the University of Montreal, Quebec, Canada (e-mail [email protected]) studied the time profiles of metabolites 3PBA and DCCA in blood and urine following oral administration in six volunteers. Maximum plasma levels were reached at 2 h post exposure. The average apparent half-life in serum was 10.5 h for cis-DCCA, 11 h for trans-DCCA and 18 h for 3-BPA. The peak rate in urine of the various metabolites was observed within 8 h and the average apparent elimination half-life was less than 14 h, leading to >97% of the dose eliminated after 48 h. Bernd Rossbach of the University Medical Center of the Johannes Gutenberg University of Mainz, Germany (e-mail [email protected]) studied the uptake of permethrin from impregnated clothes used to protect forestry workers from Lyme disease. He compared the excretion of urinary metabolites in three groups of volunteers wearing the impregnated trousers and jackets during a simulated work shift of 8 h under three different conditions: (a) default, (b) increased temperature of 25°C and increased relative humidity of 65%, and (c) the same as (b) but with a simulated, increased workload. Urine was analyzed for the permethrin metabolites cis-DCCA, trans-DCCA, and 3-BPA. The urinary metabolite levels were similar before exposure and increased between 8 h and 16 h after cessation of exposure with median concentrations of the total of the metabolites of 5.62 lg L1, 10.63 lg L1 and 15.95 lg L1 for the aforementioned conditions, respectively. These results suggest that permethrin absorption from impregnated clothes increases at elevated temperature and higher relative humidity and also with increased physical exercise. However, this last increase may also be the result of an increase in the formation of the metabolites due to increased perfusion of the liver. 7. HBM and incidents From The Netherlands, two presentations discussed the implementation of HBM following industrial incidents. Henri Heussen of Arbo Unie (Occupational Health Service) Harderwijk, The Netherlands (e-mail [email protected]) presented the use of HBM following a chemical-plant blaze in Moerdijk’s industrial area on 5 January 2011. The clean-up workers
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had to deal with heavy contamination of soil and surface water with monocyclic and polycyclic aromatic hydrocarbons. The concern of potential dermal uptake of these substances made the use of HBM appropriate. However, the study plan implemented by the cleaning company failed because the kinetics of urinary metabolite excretion was not taken into account when planning the collection of urine samples. This resulted in data that were largely unsuitable for assessing the possible uptake as a result of the clean-up activity. In a second presentation, Paul Scheepers of the Radboud University Medical Centre responded to this problem by referring to the guidance prepared by the Dutch Community Health Services, in collaboration with the National Institute of Public Health and the Environment (RIVM), which offers an algorithm to calculate, for each biomarker of interest, the most appropriate window of time for sample collection, taking into account the elimination half-life and the limit of quantification for each biomarker. The guidance is aimed at public health services and could also be useful for occupational health services, but then adaptations would need to be made. In addition to supporting the decision whether to use HBM (or to not use it) and the preparation of an adequate study plan, a service desk supports an HBM study by supplying appropriate materials for sample collection, pre-treatment, storage and transportation to a laboratory with extensive experience in analysis. A network of 10 laboratories has been established to support rapid analysis of 224 biomarkers for 132 different chemical substances. For more than 50% of the biomarkers, these laboratories have certificates of the German EQUAS Inter-Laboratory Quality Assurance Scheme. That an appropriate HBM response to unintended direct contact with chemicals can be effective was shown by Michael Bader of BASF, Ludwigshafen (e-mail [email protected]), who monitored workers of several industrial production plants. To assess benzene exposure, urine samples were analyzed for the metabolites S-phenylmercapturic acid (SPMA) and t,t-muconic acid (ttMA), as well as for unmetabolized urinary benzene. Plant managers received reports on the outcome of these analyses on a daily basis and could immediately intervene in cases of unacceptable uptake of benzene. Elevated ttMA was observed, for example, during the dismantling of installations and the emptying of pipes and pumps. Bader also presented results from an HBM program after an accident in 2005, involving a blast from a heated barrel resulting in carbonization of pyraclostrobin, which yielded p-chloroaniline (p-CA) as a process emission. In addition to plant workers, fire fighters and paramedics (a total of 145 persons) were involved in an HBM campaign, which demonstrated elevated urine levels of p-CA in several cases. The last presentation of the session covered reports of fume events in airplanes by cabin crews, who also reported a variety of health complaints in relation to those events. Tobias Weiss and his team at IPA, Bochum, Germany (e-mail [email protected]) have developed sensitive methods for the determination of triortho-cresylphosphate (o-TCP) and chemical analogues (organophosphate flame retardants). These substances are added to oils used in jet engines and are supposed to enter the plane interior as bleed air. Similar chemicals are also used as flame retardants in the interior of airplanes. A first attempt to detect elevated exposures in members of cabin crews in Germany using urinary biomarkers did not confirm that those persons were internally exposed to o-TCP due to fume events. Rather, they appeared to be exposed to organophosphate flame retardants slightly above baseline levels. Mechanics who had direct contact with the oils during maintenance of jet engines had somewhat elevated levels of o-TCP urinary metabolites, which were likely to be a consequence of dermal absorption.
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Table 1 New and improved biomarkers of exposure Biomarker
Biological material
Method
Type of study
Affiliation of contributor and references
Acrylonitrile
Urine
LC–MS/MS
Smokers
Thomas Schettgen ([email protected])
Urine
lLC-ICP-MS
Worker survey
Elizabeth Leese ([email protected])
Benzene
N-acetyl-S-(2-cyanoethyl)cysteine (CEMA), N-acetyl-S-(1-cyano-2hydroxyethyl)cysteine (CHEMA) Arsenobetaine (AB), arsenite (As3+), Arsenate (As5+), dimethylarsinate (DMA), monomethylarsonate (MMA) S-phenylmercapturic acid
Urine
ELISA
Worker survey
Beryllium
Beryllium
Urine
Quadrupole ICP-MS
Unexposed reference values
Bis(2-propylheptyl)phthalate (DPHP)
Mono-2-(propyl-6hydroxyheptyl)phthalate (OHMPHP), Mono-2-(propyl-6oxoheptyl)phthalate (oxo-MPHP), mono-2-(propyl-6-carboxyhexyl)phthalate (cx-MPHxP) Cotinine (COT), trans-3’hydroxycotinine (HCOT) Decamethylcyclopentasiloxane (D5)
Urine
LC–MS/MS
Volunteer study
Lathan Ball ([email protected]) Jacky Morton ([email protected]) Gabriele Leng ([email protected])
Urine
Online-SPE-UPLC-MS/MS
General population
Exhaled air
TD-GC-MS
Volunteers study
Exhaled air, blood
GC-FID, ATD-GC-FID
Volunteer study
Blood, Urine
GC-MS (MIH), LC–MS/MS (AMCC)
Worker’s survey
Thomas Göen ([email protected])
Urine
UHPLC–ESI–MS/MS
General population
Urine Urine
GC–MS GC–MS
Volunteer study Worker’s study
Cristina Sottani ([email protected]) Jaroslav Mráz ([email protected]) H.M. Koch ([email protected])
Urine
LC–MS/MS
Volunteer study
Arsenic species
Nicotine Decamethylcyclopenta-siloxane (D5)
Methamidophos
1,1-Difluoroethane 1,1,1-Trifluororethane 1,1,1,2-Tetrafluoroethane 1,1,1,3,3-Pentafluoropropane 3-Methyl-5-isoproylhydantoin (MIH, released from N-terminal Hb adduct), N-acetyl-S-(Nmethylcarbamoyl)cysteine (AMCC) Ethylenethiourea (ETU), propylenethiourea (PTU) 2-Ethoxyacetic acid (EEA) 5-Hydroxy-N-ethyl-2-pyrrolidone (5HNEP); 2-hydroxy-Nethylsuccinimide (2-HESI) Methamidophos
Octamethylcyclotetrasiloxane (D4)
Octamethylcyclotetrasiloxane (D4)
Exhaled air
TD–GC–MS
Volunteer study
(1S,6R)-(+)-3-carene
Urine
GC–PCI–MS/MS
Volunteer study
Urine
GC–PCI–MS/MS
Volunteer study
Urine
GC–PCI–MS/MS
Volunteer study
Blood
GC–HRMS–NICI
General population
Isocyanurate
3-Caren-10-ol, 3-caren-10-carboxylic acid (1R,2R,5R)-cis-verbenol, (1R,2S,5R)trans-verbenol, (1S,5R)-(+)-myrtenol (1S, 5S)-trans-carveol, (1R, 5S)-ciscarveol, (1S, 2S, 4R)-limonene-1,2diol, perillyl alcohol, perillic acid, limonene-8,9-diol 5-Isopropyl-3[4-(4aminobenzyl)phenyl]hydantoin Isotriamine
Urine
UPLC-MS/MS
Worker survey
Indium
Indium
Serum
ICP–MS
Worker survey
2- and 3-Nitrobenzanthrone
2-Aminobenzanthron-3ylmercapturic acid (2-ABA-MA), 3aminobenzanthron-3-ylmercapturic acid (3-ABA-MA)
Urine
LC-ESI-MS/MS
Rat study
1,1-Difluoroethane 1,1,1-Trifluoroethane 1,1,1,2-Tetrafluoroethane 1,1,1,3,3-Pentafluoropropane N,N-dimethylformamide (DMF)
Dithiocarbamates (DTC) 2-Ethoxyethanol N-ethyl-2-pyrrolidone (NEP)
(1R,5S)-(+)-a-pinene, (1S, 5S)-(-)-a-pinene (R)-(+)-limonene
4,4’-Methylenediphenyl diisocyanate (MDI)
Ilse van Overmeire ([email protected]) Gwendolyn Beckmann ([email protected]) Lena Ernstgård ([email protected])
F. Garner (fi[email protected]) G. Beckmann ([email protected]) L. Schmidt ([email protected]) [6] L. Schmidt ([email protected]) [6] L. Schmidt ([email protected]) [6]
Gabriele Leng ([email protected]) L.A. Nylander-French ([email protected]) Mi-young Lee ([email protected]) Igor Linhart ([email protected])
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Chemical substance
o-Tricresyl phosphate Bifenthrin/cyhalothrin
ELISA, Enzyme-linked immunosorbent assays; GC-HRMS-NICI, Gas chromatography high-resolution mass spectrometry negative-ion chemical ionization; GC-NCI-MS, Gas chromatography negative chemical ionization mass spectrometry; GC-PCI-MS, Gas chromatography positive chemical ionization mass spectrometry; ICP–MS, Inductively-coupled plasma mass spectrometry; SHS–GC–MS, Static headspace gas chromatography mass spectrometry; SPME, Solid-phase microextraction; UHPLC–ESI–MS/MS, Ultra-high performance liquid chromatography electrospray ionisation tandem mass spectrometry; UPLC–MS/MS, ultra-performance liquid chromatography tandem mass spectrometry; lLC, Micro liquid chromatography; lLC–ICP-MS, Micro liquid chromatography inductively-coupled plasma mass spectrometry.
Urine Urine
GC–MS/MS GC–MS/MS
Worker survey General population
Silvia Fustinoni (silvia.fustinoni@ unimi.it) Tobias Weiss ([email protected]) [7] Thomas Schettgen ([email protected]) Worker survey/general population LC–MS/MS Urine, hair Terbuthylazine
Tebuconazole
t-Butylhydroxy-tebuconazole (TEBOH), t-butylcarboxy-tebuconazole (TEB-COOH) Terbutylazine (TBA), desethylterbutylazine (DET) o,o-Dicresylphosphate 3-(2-Chloro-3,3,3-trifluoroprop-1enyl)-2,2dimethylcyclopropanecarboxylic acid (TFP-acid);cis- and trans-3-(2,2dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic acid (cis- and trans DCCA); cis-3-(2,2dibromovinyl)-2,2dimethylcyclopropanecarboxylic acid (DBCA); 4-chloro-isopropyl benzeneacetic acid (CPBA); 3phenoxybenzoic acid (3-PBA); 4fluoro-3-phenoxybenzoic acid (FPBA); 2-methyl-3-phenylbenzoic acid (2-MPA)
Urine
LC–MS/MS
Worker survey
Silvia Fustinoni (silvia.fustinoni@ unimi.it)
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8. Other promising new developments Some new methods were introduced to tackle old and contemporary chemical exposures. Most new analytical methods are highly specific, sensitive, mass spectrometry (MS)-based and involve highly sophisticated pre-treatment (see Table 1). Other methods rely on the use of immunoassays for pre-screening, such as the ELISA-based dip-stick test for on-site testing of benzene exposures for urinary S-phenylmercapturic introduced by Lathan Ball of Biomark, Cardiff, United Kingdom (e-mail lball@ abbiomonitoring.co.uk). Urine samples with positive scores need to be confirmed by a more laborious, costly analysis using gas chromatography (GC)–MS/MS (Tables 2 and 3). Investigation of epigenetic modification of biomarker results has also entered into the field of HBM. An interesting example of this is the determination of DNA methylation on urinary biomarker results in workers exposed to 1,6-hexamethylene diisocyanate (HDI) in a pilot study presented by Leena Nylander-French of the University of North Carolina, Chapel Hill, NC, USA (e-mail [email protected]). In a group of 20 automotive spray-painters, blood DNA was analyzed for 5-methyl-CpG marks associated with urinary 1,6-hexamethylene diamine (HDA) levels using linear mixed modeling accounting for exposure, ethnicity, age and smoking status. Significant DNA methylation marks were associated with individual differences in urinary HDA levels. Bioinformatics analysis revealed two significant, predicted, networks associated with this phenotype in HDI exposed workers. The identified genes are involved in antigen processing and presentation, as well as immune responses, which appear to be related to plausible working mechanisms for HDI toxicity. These preliminary findings demonstrate the high potential of using DNA methylation marks associated with individual difference in urinary biomarker levels that may be instrumental in identification of susceptible subpopulations in future studies (Table 2). A new approach to the use of covalent binding of alkylating agents to proteins was proposed by Jaroslav Mráz of the National Institute of Public Health, Prague, Czech Republic (e-mail [email protected]). It was hypothesized that adducted blood proteins, such as globin, will undergo proteolysis and that amino-acid adducts will appear in urine. Unlike adducts to DNA, there are no known repair mechanisms for protein adducts. Structural modifications will therefore persist until complete recycling at the end of the life span of the protein. Native amino acids are reused, but it is likely that modified amino acids may follow a different fate and may be excreted with feces or urine. It is supposed that these adducted protein fragments in urine could be used as biomarkers of exposure. What is interesting about this proposal is that these biomarkers will still carry the structural information, which may be used to identify the specific chemical substances involved in the exposure. To study this hypothesis, amino-acid adducts derived from the common alkylating substances methyl isocyanate and ethylene oxide were synthesized and injected intraperitoneally in rats. Analysis of urine collected over 48 h showed that the expected amino-acid adducts for methyl isocyanate [N-(methylcarbamoyl)valine and Ne-(methylcarbamoyl)lysine)] and ethylene oxide [N-(2-hydroxyethyl)valine and S-(2-hydroxyethyl)cysteine)] could be detected by liquid chromatography electrospray ionization (LC-ESI)–MS/MS. More than 90% of the excreted adducts were retrieved (unchanged or further metabolized) in the first 24 h. This is a promising first step in exploring the full potential of this novel approach. Future research will demonstrate whether these adducts can be detected following administration of the parent compounds. For application in humans, these species will have to be detected in the presence of a myriad of endogenous, native amino acids, of which many more may also have
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Table 2 Biomarkers of susceptibility Chemical substance
Biomarker of susceptibility
Adverse health effect
Study population
Affiliation of contributor and references
Benzene
GSTT1, GSTM1 GSTA1, GSTP1, mEHex3, mEHex4, NQ01, CYP2E1, CYP1A, MPO, Circulating mitochondrial DNA
Cancer
301 benzene-exposed subjects
N. Mazitova ([email protected])
Neurological and respiratory dysfunction Cancer
86 persons with confirmed exposure to off-gassing haloalkane pesticides in storage facilities or residential environments 255 workers exposed to styrene in the marble, plastics and boat industry and 49 non-exposed workers 129 workers in the footwear industry and 13 controls
Lygia Therese Budnik ([email protected]) [1] Maria José Prieto Castelló ([email protected]) Maria José Prieto Castelló ([email protected])
Halo-alkane-based pesticides Styrene
GSTM1, EPHX1
Toluene
CYP2E1
Cytogenetic damage
Table 3 Biomarkers linking physiological responses to potential adverse effects Chemical substance
Biomarker
Related adverse health effect
Aim of the study
Affiliation of contributor and references
Arsenic (inorganic)
MircoRNAs
Cellular pathology
Claudio Minoia ([email protected])
Benzene
Chromosome aberration and micronuclei
Cancer
Cadmium
a1-microglobulin (a1-MG), b2microglobulin (b2-MG) and Nacetyl-b-D-glucosaminidase (NAG) DNA methylation marks
Tubular dysfunction
Role of microRNAs, negative regulators of gene expression in pathological cellular processes Influence of low exposures of benzene on biomarkers of cytogenetic damage Association of previous disease with cadmium exposure parameters Identify individual differences in DNA methylation associated with biomarker levels Study of association of manganese and problematic behavior in school children in Bahia, Brazil Relationships between the measured biomarkers of effect and other health-outcome measures and exposure estimates Identify biomarkers of exposure and early health indicators of low-level VOCs from building materials and consumer products Assess the feasibility of using cell-proliferation ratio of lymphocytes in umbilical cord blood as an indicator of subclinical pathology in fetus
1,6-Hexamethylene diisocyanate (HDI)
Occupational asthma
Manganese
Manganese in hair
Neurobehavioral effects
Silica
Clara cell protein (CC16), lung surfactant proteins (A&D) Kidney injury molecule-1 (KIIM-1), neutrophil gelatinase-associated lipocalin (NGAL) and cytin C VOC and 8’-oxo-7,8-dihydro-2’deoxyguanosine (8-OH-dG) in urine
Lung and kidney
Volatile organic compounds
Reproduction toxic substances
MTT cell-proliferation ratio
DNA damage and/or DNA oxidation
Sub-clinical pathology in fetus
been structurally modified (e.g., oxidative protein damage). This will require highly-selective, effective enrichment of the analytes of interest. 9. Environmental exposures and developing countries Due to the increased sensitivity and the specificity of analytical techniques applied in contemporary HBM campaigns, many of the recent research projects are related to environmental exposures and consumer safety. As an example, Roel Smolders of VITO, Belgium (e-mail [email protected]) presented data from the EUfunded HBM survey, carried out in 2011 and 2012 in 17 European countries as part of the COPHES-DEMOCOPHES project involving 1800 mother-child pairs. Aggregated fish consumption could be related to mercury and cadmium in hair, and dietary levels related to urinary cadmium and urinary cotinine were associated with the effectiveness of anti-smoking programs. A limitation of this study was the availability and quality of contextual information.
Piero Lovreglio ([email protected]) Masayuki Ikeda ([email protected]) Leena Nylander-French ([email protected]) José A Menezes-FIlho ([email protected])
Howard Mason ([email protected])
Massimiliano Mascelloni ([email protected])
Isabella Karakis ([email protected])
In a keynote lecture, Stephan Böse-O’Reilly of the University of München (e-mail [email protected]) explained the health effects resulting from the use of mercury in small-scale gold mining. Approximately 15% of gold is mined in an artisanal manner in 70 different countries across the globe and is responsible for the largest environmental burden of mercury (1100 tons of mercury per annum). This type of mining has great environmental implications, but local and regional authorities do not give much priority to its impact on health. Smelting mercury from ore is associated with highest exposures and leads to serious effects on the brain. Most effects are related to the toxicity of metallic mercury and are characterized by dysfunction of the cerebellum leading to ataxia and coordination problems. Children suffer similar exposures to parents, as most of the gold-related activities are not separated from the residential environment. Urinary mercury levels in children were sometimes even higher than the values in mothers, who were in direct contact with the mercury. Böse-O‘Reilly conducted his research in many countries, such
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as Indonesia (Kalimantan and Sulawesi), Tanzania, Zimbabwe and Mongolia, and is promoting mercury-free alternative gold mining (e.g., using borax). HBM could be a valuable tool to demonstrate the effect of such interventions and to help address the problems with metal toxicity in the developing parts of the world. 10. Conclusions The potential of HBM is driven by the increased technical performance of laboratory-based techniques. In addition to sensitivity and specificity, the capacity for high-throughput analysis of biological specimen appears to be critical to the future growth of HBM in large-scale population-based studies. The applications of HBM evolved from high single exposure in subgroups (e.g., workers) to low aggregated exposures in the general populations. HBM now faces the challenge of contributing to characterizing the totality of the internal exposome, not once in an individual’s lifetime, but frequently, and at different critical life stages, in cohorts of thousands of study subjects. An important role remains for targeted approaches that can be used in growing fields of application, such as dermal absorption of chemical substances in workers and consumers, and exposure characterization following chemical incidents and disasters and interventions in developing countries. Acknowledgements The authors thanks the cited contributors for their consent to publish this report and for suggestions made to improve its quality. The author would also like to express their gratitude to Paul Aston of AB Biomonitoring for some final editing. References [1] L.T. Budnik, S. Kloth, X. Baur, A.M. Preisser, H. Schwarzenbach, Circulating mitochondrial DNA as biomarker linking environmental chemical exposure to early preclinical lesions elevation of mtDNA in human serum after exposure to carcinogenic halo-alkane-based pesticides, PLoS One 8 (5) (2013 May 31) e64413, http://dx.doi.org/10.1371/journal.pone.0064413. Print 2013. [2] M. Chadeau-Hyam, T.J. Athersuch, H.C. Keun, M. De Iorio, T.M. Ebbels, M. Jenab, C. Sacerdote, S.J. Bruce, E. Holmes, P. Vineis, Meeting-in-the-middle using metabolic profiling – a strategy for the identification of intermediate biomarkers in cohort studies, Biomarkers 16 (1) (2011 Feb) 83–88, http:// dx.doi.org/10.3109/1354750X.2010.533285. Epub 2010 Nov 29.
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[3] M. Chadeau-Hyam et al, Deciphering the complex: methodological overview of statistical models to derive OMICS-based biomarkers, Environ. Mol. Mutagen. 54 (7) (2013 Aug) 542–557. [4] D.G. Hebels, P. Georgiadis, H.C. Keun, T.J. Athersuch, P. Vineis, R. Vermeulen, L. Portengen, I.A. Bergdahl, G. Hallmans, D. Palli, B. Bendinelli, V. Krogh, R. Tumino, C. Sacerdote, S. Panico, J.C. Kleinjans, T.M. de Kok, M.T. Smith, S.A. Kyrtopoulos, EnviroGenomarkers Project Consortium, Performance in omics analyses of blood samples in long-term storage: opportunities for the exploitation of existing biobanks in environmental health research, Environ. Health Perspect 121 (4) (2013 Apr) 480–487. [5] M. Manno, C. Viau, in collaboration with, J. Cocker, C. Colosio, L. Lowry, A. Mutti, M. Nordberg, S. Wang, Biomonitoring for occupational health risk assessment (BOHRA), Toxicol. Lett. 192 (1 (January)) (2010) 3–16, http:// dx.doi.org/10.1016/j.toxlet.2009.05.001. [6] L. Schmidt, V.N. Belov, Th. Göen, Sensitive monitoring of monoterpene metabolites in human urine using two-step derivatisation and positive chemical ionisation–tandem mass spectrometry, Anal. Chim. Acta 793 (2013) 26–36, http://dx.doi.org/10.1016/j.aca.2013.07.046. [7] B.K. Schindler, T. Weiss, A. Schütze, S. Koslitz, H.C. Broding, J. Bünger, T. Brüning, Occupational exposure of air crews to tricresyl phosphate isomers and organophosphate flame retardants after fume events, Arch. Toxicol. 87 (4 (April)) (2013) 645–648, http://dx.doi.org/10.1007/s00204-012-0978-0. [8] N.S. Shenker, P.M. Ueland, S. Polidoro, K. van Veldhoven, F. Ricceri, R. Brown, J.M. Flanagan, P. Vineis, DNA methylation as a long-term biomarker of exposure to tobacco smoke, Epidemiology 24 (5 (September)) (2013) 712–716, http://dx.doi.org/10.1097/EDE.0b013e31829d5cb3. [9] P. Vineis et al, Advancing the application of omics-based biomarkers in environmental epidemiology, Environ. Mol. Mutagen. 54 (7 (August)) (2013) 461–467. [10] P.T.J. Scheepers, G.A.H. Heussen, Trends Anal. Chem. 21 (2002) 11. [11] P.T.J. Scheepers, G.A.H. Heussen, Biomarkers 10 (2005) 80. [12] P.T.J. Scheepers, G.A.H. Heussen, Biomarkers 13 (2008) 133. [13] P.T.J. Scheepers, Trends Anal. Chem. 30 (2011) 415.
Paul T.J. Scheepers Gwendolyn Beckmann Jacqueline W.H. Biesterbos Research Lab Molecular Epidemiology, Department for Health Evidence, Radboud University Medical Center, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands Tel.: +31 24 3616878; Fax: +31 24 3613505. E-mail address: [email protected] (P.T.J. Scheepers)