Alternative sampling strategies for the assessment of ...

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Current Opinion in

Toxicology

Alternative sampling strategies for the assessment of biomarkers of exposure Lisa Delahaye1,a, Bram Janssens1,2,a and Christophe Stove1 Abstract

Biomarkers of exposure reflect the extent to which an individual has been exposed to a particular agent. This review focuses on covalently modified endogenous molecules, adducted proteins or DNA, which can more easily –and longer– be quantified than the involved agents or metabolites, which are mostly reactive, short-living electrophiles. More specifically, we address the quantitation of these biomarkers of exposure via alternative sampling strategies, along with the advantages and challenges associated with these strategies. While immunochemical approaches may be used for quantitation, recently, high-end bioanalytical strategies are increasingly being applied. DNA adducts of multiple agents have been determined in saliva, oral fluid, sputum, oral tissue or breast milk. Microsampling strategies have not yet been applied for DNA-adducts, analytical sensitivity still being the limiting factor. In contrast, microsampling strategies (e.g. dried blood spots), as well as other alternative sampling strategies, have been used successfully for the determination of multiple protein adducts. However, also here, absolute quantification poses a real challenge. The many advantages offered by alternative sampling strategies makes them a very promising complement in the analytical toolbox, e.g. for large-scale epidemiological research, where they may be a valid substitute for the traditionally used blood, plasma and serum samples. Addresses 1 Laboratory of Toxicology, Department of Bioanalysis, Faculty of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, Belgium 2 Scientific Institute of Public Health (WIV-ISP), Rue Juliette Wytsman 14, 1050 Ixelles, Belgium Corresponding author: Stove, Christophe (christophe.stove@ugent. be) a Equally contributed.

Current Opinion in Toxicology 2017, 4:43–51 This review comes from a themed issue on Translational ToxicologyBiomarkers (2017) Available online 2 June 2017 For a complete overview see the Issue and the Editorial http://dx.doi.org/10.1016/j.cotox.2017.05.003 2468-2020/© 2017 Elsevier B.V. All rights reserved.

Keywords Alternative sampling strategies, Adduct, DNA, Proteins, Biomarker, Dried blood spots. Abbreviations 4-ABP, 4-aminobiphenyl; AFB1, aflatoxin B1; B[a]P, benzo[a]pyrene; BChE, butyrylcholinesterase; DBS, dried blood spot; dG-C8, N-(deoxyguanosin-8-yl); DPS, dried plasma spot; ESI, electron spray ionization;

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GB, sarin; GC, gas chromatography; HPB, 4-hydroxy-1-(3-pyridyl)-1butanone; LC, liquid chromatography; MS, mass spectrometry; MS/MS, tandem mass spectrometry; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)1-butanone; NNN, N0 -nitrosonornicotine; NSI, nanospray ionization; OF, oral fluid; OPNA, organophosphorus nerve agent; PAH, polycyclic aromatic hydrocarbons; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine; SF-ICP, sector-field inductively coupled plasma; VAMS, volumetric absorptive microsampling/microsamples.

1. Introduction Biomarkers of exposure can be looked at from a wide variety of perspectives, ranging from the actual agent (e.g. a heavy metal), to metabolites (pointing at exposure to e.g. a certain drug), to other short- or long-term indicators. They are typically used to evaluate to what extent there has been external or internal exposure to an (offending) agent. In the context of this review, we refer to ‘biomarkers of exposure’ as endogenous molecules that are covalently altered following exposure to an agent. This agent can be e.g. a therapeutic drug, a noxious substance encountered in the workplace or an environmental pollutant. Not within the scope of this review is the assessment of endogenous molecules whose expression is up- or downregulated following exposure (often referred to as ‘biomarkers of effects’), or the detection of the molecules themselves or their direct metabolites. Yet, some of the discussed sampling strategies may also be used for determination of these substances. In essence, the biomarkers of exposure covered here are large molecules e proteins or DNA e that have undergone a modification following exposure (Fig. 1). These biomarkers of exposure allow a more accurate determination of the internal dose of a substance than environmental measurements per se [1]. Furthermore, chemical carcinogens and other toxic substances can be converted to reactive electrophiles, which are short-lived, rendering direct detection in a target tissue virtually impossible from a practical-point-of-view [2]. Their reactivity results in the formation of adducts with nucleophilic groups of DNA and proteins, allowing long-term detection of the initial exposure (days to months) [3,4]. While in DNA the N7 position of the guanine residue is the most nucleophilic site, in proteins typically sulfhydryl groups of cysteine residues are targeted, as well as lysine, histidine and tyrosine residues [5e7]. More specifically, the focus of this review will lie on how these biomarkers of exposure can be assessed -primarily quantitatively-when using so-called ‘alternative sampling strategies’ (Fig. 1). These encompass the collection of ‘traditional’ samples (blood, plasma, serum or urine) in an

Current Opinion in Toxicology 2017, 4:43–51

44 Translational Toxicology-Biomarkers (2017)

Fig. 1

Schematic diagram of adduct formation and potential sampling strategies.

alternative way, as well as the collection of ‘alternative’ samples in non- or minimally invasive ways [8,9]. A typical example of the former is the collection of dried blood spots (DBS) (i.e. the collection of blood in an unconventional manner), while examples of the latter include sampling of e.g. oral fluid (OF), hair and a wide variety of other matrices. Recent improvements enotably increased sensitivitye in the available instrumentarium have allowed these ‘alternative sampling strategies’ to become more and more implemented as routine sampling techniques, even for the determination of low analyte concentrations in minute amounts of sample. While these alternative sampling strategies may offer distinct advantages over conventional sampling strategies, they are also typically associated with some challenges or limitations. Table 1 provides an overview of the advantages and challenges of the alternative sampling strategies, applied for assessment of biomarkers of exposure, that are discussed in this review. Some alternative sampling strategies offer advantages in storage, transport and analyte stability compared to conventional samples. While there is no general rule as to which sampling methods offer the best analyte stability, it can be expected that dry matrices suffer less from analyte degradation compared to liquid matrices [12,23,24]. Transportation of cell-based matrices (e.g. exfoliated bladder cells, oral buccal cells) will often require cooled transportation and an addition of excipient(s) for storage, making them less suited for home sampling. Not only the biomatrix itself, but also how the sample is collected, can influence the analyte stability and storage conditions [18].

2. DNA adducts Both the 4 base-nucleotides and the deoxyribose phosphate backbone of DNA may be subject to covalent modification by genotoxic compounds and/or their Current Opinion in Toxicology 2017, 4:43–51

metabolites [25]. During the formation of adducts, typically a nucleophilic atom within the DNA molecule reacts with an electrophilic atom in the genotoxic compound [26]. Furthermore, cyclical structures can be formed after rearrangement reactions or reactions with compounds with 2 reactive sites. Inter- and intrastrand DNA linking can also occur through the reaction with bifunctional chemicals. All these types of bonds can influence the molecular shape and hydrogen bonding characteristics on a lower level and distort the whole helix-shape of DNA on a higher level. The damage caused by these changes in both chemical and physical structure can give rise to errors (mutations) during the transcription or may even prevent transcription from taking place. As such, the formation of DNA adducts is typically associated with carcinogenesis, which is the reason why they are useful biomarkers for assessing exposure to carcinogenic compounds. It should be noted however that, as organisms have several levels of detection and repair to prevent these types of DNA damage, DNA adducts are typically only present at rather low concentrations in human (and animal) tissue (0.01e10 adducts per 108 nucleotides). As a consequence, an extensively optimized sample preparation (including DNA extraction and hydrolysis) needs to be combined with ultra-sensitive, accurate and specific analytical techniques for detection and/or quantification [27,28]. (semi-)Quantitative techniques include 32P-postlabeling, immuno- and electrochemical methods, fluorescence and phosphorescence detection, and mass spectrometry. The last few years, tandem and/or high-resolution mass spectrometry (MS), preceded by liquid chromatography (LC), has become the method of choice [26,27,29,30]. MS-based technologies also allow seamless integration of stably labeled internal standards, which may compensate for losses or oxidative processes taking place during sample preparation and/or for matrix effects in the MS source. Typical biological sources for DNA analysis are blood (lymphocytes or whole fraction of nucleated white blood cells) and www.sciencedirect.com

Alternative sampling strategies for the assessment of biomarkers of exposure Delahaye et al.

Table 1

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Advantages and challenges of the different alternative sampling techniques discussed in this review.

Sampling strategy

Advantages

Challenges

DBS [9–12]

Ease of sampling, possibility of home or remote sampling Convenient transport and storage of samples Small blood volumes Reduced risk of infection Simplification of sample preparation procedures Amenable to automated sample processing and analysis

Guarantee on sample quality Contamination risk High sensitivity required Chromatographic effect and influence of the site of punching Influence of spotted volume Hematocrit effect Conversion to corresponding plasma values may be needed Correlation between venous and capillary blood concentrations Preparation requires additional centrifugation step or devices for in situ DPS generation (adding a cost) Equivalence of concentrations in blood filtrate and plasma needs to be demonstrated Cfr. DBS, except for chromatographic effect, spotted volume and hematocrit effect

Dried plasma spots (DPS) Cfr. DBS [8,9] No conversion factor needed for comparison with standard plasma (or serum) values Volumetric absorptive microsampling (VAMS) of blood [8,9] Saliva/OF [8,13,14] Skin [15–17] Sputum [8] Oral tissue [18]

Cfr. DBS No/Reduced hematocrit effect

No need for qualified personnel No privacy issues Readily available No privacy issues No infection risk Little infection risk during sampling Alternative for pulmonary tissue samples

Breast milk [19,20]

No need for qualified personnel No privacy issues Readily available Non invasive matrix If applicable, available at a relatively large volume

Urine/Urinary bladder cells (Dried Urine) [21–23]

No need for qualified personnel Readily available DUS: cfr. other dried matrices

organ tissue (biopsy). DNA can also be collected via less invasive sampling techniques, such as via sputum, buccal and cervical mucosa, sperm, urine (exfoliated bladder cells), placenta, hair and breast milk (exfoliated breast epithelial cells) [31,32]. The high sensitivity that is required imposes the need for sufficient sample. This is likely the reason that, as far as we are aware, no real microsampling strategies (e.g. DBS) have been applied for detecting DNA adducts, although ongoing efforts to further improve assay sensitivity may render this possible in the near future. Also further deployment of untargeted strategies, using high-resolution MS, for DNA adductome mapping is increasingly receiving attention [33e36]. As regards alternative sampling strategies, most attention has been directed towards the oral cavity, given its easy accessibility and its obvious exposure to tobacco or dietary carcinogens. In this context, molecular changes (including the presence of DNA adducts) in cells in/from the oral cavity have been demonstrated to be similar to those in bronchial epithelial cells, which is consistent with the “field carcinogenesis concept” of cancers of the lung and upper aerodigestive tract [37]. Hence, although www.sciencedirect.com

Flow rate and composition is influenced by multiple factors Standardization of sample collection method needed Contamination risk Standardization of sample collection method needed Semi-invasive Reduced analyte stability (presence of water) Transportation and storage less convenient Standardization of sample collection method needed Transportation and storage less convenient Only applicable in lactating women Lactation may have an effect on the result Transportation and storage less convenient Not possible to directly generate a microsample

more studies are needed, oral fluid and/or buccal cells may be considered as surrogates of other, less easily accessible cells, which is interesting in the perspective of predicting (risks of) cancer in e.g. smokers. 2.1. Saliva/OF OF is a combination of different kinds of fluids esaliva, secretionse of the accessory glands, gingival fluid and oronasopharyngeal secretions-along with enzymes, other proteins, electrolytes, bacteria, epithelial cells and food debris [38]. It can be collected via suction, swabbing, drooling and expectoration or by using commercial OF collectors. Samples can be collected without stimulation (passive drooling), with chemical stimulation or with mechanical stimulation [39e41]. While several studies on biomarkers of exposure have referred to ‘saliva’, these actually used ‘oral fluid’. Hence, we refer to ‘oral fluid’ further. Analyses typically start from DNA isolated from several (2e4) ml of OF, which is subjected to digestion by multiple enzymes (e.g. DNase, nuclease, alkaline phosphatase and phosphodiesterase) to obtain adducted mononucleosides. Current Opinion in Toxicology 2017, 4:43–51


An analysis of OF for the presence of adducts of 4 heterocyclic aromatic amines, which are formed during smoking and the cooking of meat, was done by Bessette et al. [42]. Detection of the N-(deoxyguanosin-8-yl) (dGC8)-adduct of 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP), via LC-MS3, was deemed to be especially interesting, as it was found in 45% of the “eversmokers”-group. Several other smoking-related DNA adducts have been determined in OF by LC-MS/MS, amongst which adducts originating from the a,b-unsaturated aldehydes acrolein and crotonaldehyde, three etheno adducts, as well as O2-, N3- and O4-ethylthymidine and N3- and N7-ethylguanine adducts [43e 45]. A feasibility study on the use of OF as a source for Pt-DNA adducts utilized sector-field inductively coupled plasma mass spectrometry (SF-ICP-MS) for adduct quantification [46]. This study found that there were challenges in obtaining OF samples of good quality and that there was a low correlation between the OF and the leukocyte Pt-DNA levels. Hence, the conclusion was that more research is required before embarking upon detection of these adducts in OF as an alternative to blood. 2.2. Oral tissue The harvesting of oral buccal cells offers another alternative matrix for the detection of DNA adducts. Given the rapid turnover of the mucosal surface cells (5e6 days), DNA adducts observed in this matrix likely reflect recent exposure to carcinogens [47]. Blommaert et al. [48] collected oral buccal cells by means of swiping with a cotton swab for the analysis of DNA adducts by carboplatin and cisplatin, two anticancer drugs. Semi-quantitative analysis was done via microdensitometrical measurement of the staining intensity of the nuclei. The study concluded that immunocytochemical analysis of Pt-DNA adducts in sampled oral buccal cells was feasible and gave reproducible results. Stepanov et al. [49] sampled oral buccal cells by means of a cytobrush to detect pyridyloxobutylated DNA adducts, via LC-MS/MS analysis of 4-hydroxy-1(3-pyridyl)-1-butanone (HPB), which can in vitro be released from these adducts. Two possible sources of these adducts are the tobacco-specific carcinogens N0 nitrosonornicotine (NNN) and 4-(methylnitrosamino)1-(3-pyridyl)-1-butanone (NNK), making these adducts candidates as biomarkers for tobacco-specific DNAdamage. HPB levels were on average 52-fold higher in the oral buccal cells from smokers compared to nonsmokers, many times higher than in previous studies in lung or other tissues. While these findings suggest that the HPB-releasing DNA adducts in oral buccal cells could be potential biomarkers for NNN and NNK, further research is required to strengthen this hypothesis. Using DNA extracted from buccal cells that were collected from mouthwash, which had been gargled for 1 min, Bessette et al. could identify 6- and 8-hydroxyPdG DNA adducts in smokers, using data-dependent Current Opinion in Toxicology 2017, 4:43–51

constant neutral loss MS3 scanning [50]. Also acroleinderived 1,N2-propanodeoxyguanosines, exocyclic DNA adducts formed after exposure to cigarette smoke or by lipid peroxidation, were quantified in oral buccal cells collected via the cytobrush technique, albeit by antibody-based techniques [51,52]. Using a mouthwash procedure, Balbo et al. [53] collected buccal cell samples for LC-MS/MS-based analysis of the presence of N2-ethylidenedeoxyguanosine, which is formed through the reaction of DNA with acetaldehyde, the major ethanol metabolite. The study, applied on healthy volunteers consuming limited amounts of ethanol, found clear dose-dependent increases of this adduct shortly after ethanol consumption, although large interindividual differences were observed. The authors hypothesized that oral cell DNA could be a valuable target for further investigation in head and neck carcinogenesis. 2.3. Sputum Sputum is the mucus obtained from the lower respiratory tract and can be used as a matrix for alternative sampling. The collection of sputum is considered a semi-invasive sampling technique. Both induced and non-induced collection is possible, the latter occurring after inhalation of a hypertonic saline aerosol. During this treatment, sputum is periodically collected until at least 5 mL is obtained. Sputum contains, just as urine, exfoliated tissue cells, from which DNA can be extracted. Besarati Nia et al. [54] analyzed 4-aminobiphenyl (4ABP) and polycyclic aromatic hydrocarbons (PAH), tobacco smoke constituents with carcinogenic effects, in induced sputum of smokers and non-smokers. An immunohistochemical peroxidase assay was used for semi-quantitative analysis. The study found that smokers had significantly higher 4-ABP-adducts than non-smokers. 2.4. Breast epithelial cells/breast milk Breast cancer is one of the most prevalent forms of cancer. Besides genetic factors, the exposure to environmental chemicals, such as PAHs and aromatic and heterocyclic amines, is considered a potential risk factor [55]. In this context, the analysis of DNA adducts in exfoliated luminal epithelial cells, which can easily be isolated from breast milk, may be promising as these constitute the cells from which most breast cancers arise. It should be noted however, that ductal cells may be different in lactating and nonlactating women, hormonal profiles may affect cell processes, and factors related to milk production may affect the metabolism of chemicals of interest [20]. Gorlewska-Roberts et al. [56] collected milk samples from nonsmoking mothers for the analysis of 3 categories of adducts, originating from PhIP, 4-ABP and benzo[a] pyrene (B[a]P). In 48% of the cases one or more of the www.sciencedirect.com


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P-postlabeled adducts could be identified using HPLC analysis with UV-VIS detection, corroborating earlier findings by Thompson et al. [39]. A follow-up study suggested an association between 4-ABP adduct levels in cells isolated from breast milk and the use of hair coloring products, as well as a role for the metabolic genotype [57]. With respect to the latter, it is unclear to what extent an ‘overall’ metabolic phenotype is reflected locally, in the breast tissue e and in the milk derived thereof.

2.5. Urine/urinary bladder cells Urinary bladder cells, which can be obtained from urine, can be a source of DNA adducts. Furthermore, urine on its own contains waste products, including DNA fragments. The analysis of urinary N7-(2-hydroxyethyl)guanine, an adduct of the carcinogen ethylene oxide, was performed by Huang et al. [22], utilizing an on-line solid-phase extraction isotope-dilution LC-MS/MS method, starting from only 140 mL of urine. The on-line set-up drastically enhanced the throughput capacity of the system, even for a matrix as complex as urine. Also following solidphase extraction, two 1,N2-propano-20 -deoxyguanosine adducts could be quantified, starting from only 200 mL of urine [21]. The requirement of merely 140 or 200 mL of urine might be a stepping stone for the application of dried urine spots for these and other applications.

3. Protein adducts The protein adduct that is chosen as a biomarker of exposure is greatly determined by the matrix from which a sample is being taken. Typically, the biomarker of choice will be an adduct with the most abundant protein in a given matrix, e.g. albumin or hemoglobin in blood or keratin in skin. Protein adducts offer the advantage over DNA adducts that they are not actively repaired and are typically chemically stable. This results in a long lifetime, essentially the same as that of the original protein (e.g. for hemoglobin this is 2e3 months). While immunological assays (e.g. ELISA) targeting the specific modification have been marketed for a selected number of adducts, LC-MS/MS, as well as gas chromatography coupled to tandem mass spectrometry (GC-MS/MS) are the prime techniques being applied [27,58]. Sample preparation may include an immunoaffinity-based step, which may help to achieve exquisite sensitivity, either by depleting unwanted proteins, or by allowing enrichment of the protein or peptide of interest. Sample preparation also typically includes a step wherein an adducted single amino acid or small peptide is released. This can either be via modified Edman degradation (as applied to release adducted N-terminal valine from hemoglobin), via other chemical reactions, or via conventional proteolysis using enzymes [59]. A true challenge for the absolute quantification of analytes via this procedure is the non-availability of suitable standards www.sciencedirect.com

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(i.e. adducted proteins) and internal standards (labeled analogue of adducted proteins). While the former are ideally applied for setting up calibration curves, labeled internal standards are essential to correct for all eor as many as possiblee variations of the sample preparation and analysis, including ewhen applicablee the enzyme digestion process. Labeled adducted peptides, which are also subject to enzymatic digestion, thereby yielding the labeled adducted analyte, can be used as the best available alternative. Yet, there is no guarantee that these peptides will be affected by the sample preparation in the same way as the protein. An alternative approach to tackle (part) of this issue has been suggested by Grigoryan et al. [60], who used a ”housekeeping peptide” in combination with a conventional internal standard. This housekeeping peptide also originates from the adducted protein and should be generated with a similar efficiency as the peptide of interest. Quantitation of adduct levels can then be performed after correction for the amount of measured housekeeping peptide and relative to the signal of the internal standard. 3.1. Blood-based matrices The emergence of new-generation analytical instrumentation has made it possible to use microsamples such as DBS, dried plasma spots (DPS) and so-called volumetric absorptive microsamples (VAMS) instead of conventionally sampled blood or plasma. VAMS are obtained using handheld devices consisting of a plastic handle and a hydrophilic polymer tip that absorbs a fixed volume of blood, irrespective of the hematocrit [61,62]. The resulting microsamples can be used for a wide variety of toxicological applications, amongst which is the measurement of protein adducts [10,63]. It should be kept in mind, though, that also some drawbacks are associated with these microsampling techniques, as outlined in Table 1. Many attempts are being made to make remote sampling more easy and to cope with DBS-associated issues, such as those imposed by the (unknown) hematocrit [64e66]. Below we summarize applications that were successful in determining protein adducts, starting from microsamples. Undoubtedly, many more applications will follow in the (near) future. The first report about the detection of protein adducts in DBS dates back to 2008, when Funk et al. determined a benzene oxide-hemoglobin adduct in adult and newborn DBS via GC-MS [67]. Complete DBS, generated by volumetric deposition of 50 mL of blood onto filter paper, were analyzed and yielded similar results for the benzene oxide-hemoglobin adduct as corresponding blood samples. If this method were to be applied on partial punches from non-volumetrically applied samples, the impact of DBS-related parameters like hematocrit effect, spotted volume and site of punching would need to be evaluated as well, even though the Current Opinion in Toxicology 2017, 4:43–51


applied procedure included a normalization based upon hemoglobin measurements.

Their findings showed that VAMS are a viable alternative to DBS for the analysis of OPNA-BChE adducts.

Several albumin adducts have been determined in dried microsamples. Using LC-MS/MS, Xue et al. determined an adduct of albumin with aflatoxin B1 (AFB1), a type of mycotoxin produced mainly by Aspergillus species, in DBS [68]. While in samples from exposed animals an excellent correlation between DBS and serum data (both normalized to albumin content) was observed, this correlation was only moderate between DBS and corresponding serum samples from exposed humans. It is not clear to what extent this is owing to DBS-related parameters. The determination of sulfur mustardalbumin adducts via mLC-MS/MS has recently been reported by John et al. [69]. These authors analyzed dried plasma samples, using 10-mL VAMS, 100 mL DPS and Noviplex DUO Cards (generating a quantitative DPS from a drop of blood). All three sampling procedures were reported to give satisfactory results with respect to the investigated validation parameters.

3.2. Human skin Also skin proteins offer the possibility of adduct monitoring. Obvious candidates there are the different keratins, which are abundant in human skin and have multiple nucleophilic sites. Adducts can be formed via direct reaction with reactive compounds or following CYP-mediated metabolism (bio-activation), taking place in the suprabasal layer of the epidermis. The latter holds true for e.g. naphthalene, which is metabolized to the reactive naphthalene-1,2-oxide, which can react with nearby keratin proteins (primarily keratin 1 and 10). The resulting adducts allow monitoring over a longer time period, since the migration from the basal layer to the stratum corneum takes up about 15e30 days [74]. As a consequence, periodic non-invasive sampling, via tape stripping, may allow longer-term follow-up. Application of an ELISA-based procedure has been described in a series of reports by Kang-Sickel et al. [16,17] and Jiang et al. [75] for quantitative assessment of naphthyl-keratin adducts in tape-strip samples from air force fuel-cell maintenance workers, occupationally exposed to jet fuel. The results were normalized using the total amount of protein (considered to be primarily the target keratins) recovered from the tape-strips. Naphthyl-keratin adducts were found not to correlate with dermal naphthalene levels and to correlate only moderately with urinary naphthalene levels. The authors provide two possible explanations for this: first, the adducts reflect both short-term and longer-term exposure, while the presence of naphthalene itself only reflects recent exposure; second, the presence of adducts may greatly depend on the metabolic activity in the skin, which may show substantial interindividual variability [16]. It is anticipated that also for the detection of keratin adducts, LC-MS/MS methods will soon emerge as tools to assess dermal exposure (distinguishable from other routes of exposure).

Crow et al. [70] determined tyrosine adducts with organophosphorus nerve agents (OPNAs; tabun, GB, soman, cyclosarin, VR, VX and VM) in 50 mL of plasma, serum or lysed whole blood. Considering the small sampling volume, it can be assumed that the applied method can also be used for DBS, DPS or VAMS, as these require sample volumes in the same order of magnitude. However, again, this requires evaluation of several additional aspects (cfr. Table 1). As a matter of fact, for many applications for which albumin is a target for protein adduction (as recently reviewed by Ref. [71]), DBS or DPS might be applied, as also suggested by our own preliminary data on e.g. detectability of a paracetamol-albumin adduct in DBS. Apart from hemoglobin and albumin, covalent adduct formation with enzymes has also been monitored to assess exposure. The best known example here is butyrylcholinesterase (BChE), which loses its activity following covalent binding of OPNAs. Perez et al. [24] demonstrated that DBS, DPS and dried serum spots can be used for BChE enzyme activity measurements via a modified Ellman assay, as well as for OPNA-BChE adduct measurements, via isotope-dilution LC-MS/ MS. The use of dried samples was shown to offer the advantage of increased stability, both in terms of BChE activity and intactness of the BChE adduct. While normalization to total protein allowed to cope with intraspot variability caused by unknown volume and punch location, the effect of hematocrit as a variable was not investigated. Hamelin et al. [72] investigated the determination of different OPNA-BChE adducts in DBS and VAMS. The latter allow an even more convenient sampling [73], eliminate or drastically reduce the influence of the hematocrit [61] and do not suffer from inhomogeneity issues, which are observed in DBS [72]. Current Opinion in Toxicology 2017, 4:43–51

4. Conclusion In this review we have discussed the alternative sampling strategies for the assessment of biomarkers of exposure that are currently being used. Alternative matrices for detection of DNA adducts encompass OF, oral tissue, sputum, breast milk and breast tissue cells and urinary bladder cells. Since protein adducts are generally present at higher concentrations, more work has been done on the exploration of the suitability of microsampling strategies (e.g. DBS, DPS, VAMS) to allow their detection. Apart from skin tissue and bloodbased microsamples, protein adducts as biomarkers of exposure have not been determined in hair, dried urine spots (or small volume urine samples) or any other matrix that was collected via an alternative sampling technique. While some sampling strategies can offer an advantage in storage and analyte stability, these www.sciencedirect.com


parameters should always be evaluated for new analytee biomatrix combinations. The ease of sampling and nonor minimal invasiveness are clearly advantages offered by alternative sampling strategies that may result in their use in e.g. large-scale epidemiological surveys. In this context, we believe that the use of alternative sampling strategies for adduct detection eeither DNA or protein adductse as biomarkers of exposure is promising and will continue to grow in future.

Acknowledgment The authors would like to thank Phebe De Coene for her help with the selection of articles for this review paper and Michiel Janssens for help with illustration. This work was supported by the Research Foundation e Flanders (grant n G0E0916N) and by the Belgian Science Policy Office (BELSPO, Ministerial Decree 23/12/2015 & 1/12/2016).

Conflict of interest

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Elsevier; 2016:279–336. http://dx.doi.org/10.1016/B978-0-12802025-8.00013-1. 10. Stove CP, et al.: Dried blood spots in toxicology: from the cradle to the grave? Crit Rev Toxicol 2012, 42:230–243. http:// dx.doi.org/10.3109/10408444.2011.650790. 11. De Kesel PM, et al.: Hemato-critical issues in quantitative analysis of dried blood spots: challenges and solutions. Bioanalysis 2013, 5:2023–2041. http://dx.doi.org/10.4155/ bio.13.156. 12. McDade TW, Williams S, Snodgrass JJ: What a drop can do: dried blood spots as a minimally invasive method for integrating biomarkers into population-based research. Demography 2007, 44:899–925. 13. Alves A, et al.: Human biomonitoring of emerging pollutants through non-invasive matrices: state of the art and future potential. Anal Bioanal Chem 2014, 406:4063–4088. http:// dx.doi.org/10.1007/s00216-014-7748-1. 14. Gallardo E, Queiroz JA: The role of alternative specimens in toxicological analysis. Biomed Chromatogr 2008, 22:795–821. http://dx.doi.org/10.1002/bmc.1009.

The authors declare no conflict of interest regarding this work.

15. Escobar-Chavez JJ, et al.: The tape-stripping technique as a method for drug quantification in skin. J Pharm Pharm Sci 2008, 11:104–130.

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