Blood Protein Adducts Research Articles

Идентификация фосфонилированных пептидов с применением МАЛДИ-мишени, функционализированной стеаратом лантана

As markers of intoxication, adducts of blood proteins with organophosphorus compounds (OPs) allow establishing the fact of poisoning and, furthermore, enable identification of the OPs by the attached residue. This study aimed to develop a method of specific and selective extraction of blood protein adducts carrying OPs on the surface of a matrix-assisted laser desorption/ionization (MALDI) target functionalized with multimolecular structures based on lanthanum stearate using metal affinity chromatography. We have shown the ability of adsorbent to retain both full-size and dealkylated adducts of blood proteins with OPs. The developed method allowed extraction and identification of peptides of human serum albumin and human butyrylcholinesterase modified with various OPs (after incubation of human blood plasma with OPs in concentrations from 1 to 100 ng/mL), which makes this approach applicable for the purposes of OPs identification in the context of investigation of real cases of intoxication.

Literature review and evaluation of biomarkers, matrices and analytical methods for chemicals selected in the research program Human Biomonitoring for the European Union (HBM4EU).

Humans are potentially exposed to a large amount of chemicals present in the environment and in the workplace. In the European Human Biomonitoring initiative (Human Biomonitoring for the European Union = HBM4EU), acrylamide, mycotoxins (aflatoxin B1, deoxynivalenol, fumonisin B1), diisocyanates (4,4'-methylenediphenyl diisocyanate, 2,4- and 2,6-toluene diisocyanate), and pyrethroids were included among the prioritized chemicals of concern for human health. For the present literature review, the analytical methods used in worldwide biomonitoring studies for these compounds were collected and presented in comprehensive tables, including the following parameter: determined biomarker, matrix, sample amount, work-up procedure, available laboratory quality assurance and quality assessment information, analytical techniques, and limit of detection. Based on the data presented in these tables, the most suitable methods were recommended. According to the paradigm of biomonitoring, the information about two different biomarkers of exposure was evaluated: a) internal dose = parent compounds and metabolites in urine and blood; and b) the biologically effective = dose measured as blood protein adducts. Urine was the preferred matrix used for deoxynivalenol, fumonisin B1, and pyrethroids (biomarkers of internal dose). Markers of the biological effective dose were determined as hemoglobin adducts for diisocyanates and acrylamide, and as serum-albumin-adducts of aflatoxin B1 and diisocyanates. The analyses and quantitation of the protein adducts in blood or the metabolites in urine were mostly performed with LC-MS/MS or GC-MS in the presence of isotope-labeled internal standards. This review also addresses the critical aspects of the application, use and selection of biomarkers. For future biomonitoring studies, a more comprehensive approach is discussed to broaden the selection of compounds.

Detection of Benzo[a]pyrene Diol Epoxide Adducts to Histidine and Lysine in Serum Albumin In Vivo by High-Resolution-Tandem Mass Spectrometry.

Electrophilic diol epoxide metabolites are involved in the carcinogenicity of benzo[a]pyrene, one of the widely studied polycyclic aromatic hydrocarbons (PAHs). The exposure of humans to this PAH can be assessed by measuring stable blood protein adducts, such as to histidine and lysine in serum albumin, from their reactive metabolites. In this respect, measurement of the adducts originating from the genotoxic (+)-anti-benzo[a]pyrene diol epoxide is of interest. However, these are difficult to measure at such low levels as are expected in humans generally exposed to benzo[a]pyrene from air pollution and the diet. The analytical methods detecting PAH-biomarkers still suffer from low selectivity and/or detectability to enable generation of data for calculation of in vivo doses of specific stereoisomers, for evaluation of risk factors and assessing risk from exposures to PAH. Here, we suggest an analytical methodology based on high-pressure liquid chromatography (HPLC) coupled to high-resolution tandem mass spectrometry (MS) to lower the detection limits as well as to increase the selectivity with improvements in both chromatographic separation and mass determination. Method development was performed using serum albumin alkylated in vitro by benzo[a]pyrene diol epoxide isomers. The (+)-anti-benzo[a]pyrene diol epoxide adducts could be chromatographically resolved by using an HPLC column with a pentafluorophenyl stationary phase. Interferences were further diminished by the high mass accuracy and resolving power of Orbitrap MS. The achieved method detection limit for the (+)-anti-benzo[a]pyrene diol epoxide adduct to histidine was approximately 4 amol/mg serum albumin. This adduct as well as the adducts to histidine from (−)-anti- and (+/−)-syn-benzo[a]pyrene diol epoxide were quantified in the samples from benzo[a]pyrene-exposed mice. Corresponding adducts to lysine were also quantified. In human serum albumin, the anti-benzo[a]pyrene diol epoxide adducts to histidine were detected in only two out of twelve samples and at a level of approximately 0.1 fmol/mg.

1-Methoxy-3-indolylmethyl DNA adducts in six tissues, and blood protein adducts, in mice under pak choi diet: time course and persistence.

We previously showed that purified 1-methoxy-3-indolylmethyl (1-MIM) glucosinolate, a secondary plant metabolite in Brassica species, is mutagenic in various in vitro systems and forms DNA and protein adducts in mouse models. In the present study, we administered 1-MIM glucosinolate in a natural matrix to mice, by feeding a diet containing pak choi powder and extract. Groups of animals were killed after 1, 2, 4 and 8 days of pak choi diet, directly or, in the case of the 8-day treatment, after 0, 8 and 16 days of recovery with pak choi-free diet. DNA adducts [N2-(1-MIM)-dG, N6-(1-MIM)-dA] in six tissues, as well as protein adducts [τN-(1-MIM)-His] in serum albumin (SA) and hemoglobin (Hb) were determined using UPLC-MS/MS with isotopically labeled internal standards. None of the samples from the 12 control animals under standard diet contained any 1-MIM adducts. All groups receiving pak choi diet showed DNA adducts in all six tissues (exception: lung of mice treated for a single day) as well as SA and Hb adducts. During the feeding period, all adduct levels continuously increased until day 8 (in the jejunum until day 4). During the 14-day recovery period, N2-(1-MIM)-dG in liver, kidney, lung, jejunum, cecum and colon decreased to 52, 41, 59, 11, 7 and 2%, respectively, of the peak level. The time course of N6-(1-MIM)-dA was similar. Immunohistochemical analyses indicated that cell turnover is a major mechanism of DNA adduct elimination in the intestine. In the same recovery period, protein adducts decreased more rapidly in SA than in Hb, to 0.7 and 37%, respectively, of the peak level, consistent with the differential turnover of these proteins. In conclusion, the pak choi diet lead to the formation of high levels of adducts in mice. Cell and protein turnover was a major mechanism of adduct elimination, at least in gut and blood.

Hemoglobin Adducts and Urinary Metabolites of Arylamines and Nitroarenes.

Arylamines and nitroarenes are intermediates in the production of pharmaceuticals, dyes, pesticides, and plastics and are important environmental and occupational pollutants. N-Hydroxyarylamines are the toxic common intermediates of arylamines and nitroarenes. N-Hydroxyarylamines and their derivatives can form adducts with hemoglobin (Hb-adducts), albumin, DNA, and tissue proteins in a dose-dependent manner. Most of the arylamine Hb-adducts are labile and undergo hydrolysis in vitro, by mild acid or base, to form the arylamines. According to current knowledge of arylamine adduct-formation, the hydrolyzable fraction is derived from the reaction products of the arylnitroso derivatives that yield arylsulfinamide adducts with cysteine. Hb-adducts are markers for the bioavailability of N-hydroxyarylamines. Hb-adducts of arylamines and nitroarenes have been used for many biomonitoring studies for over 30 years. Hb-adducts reflect the exposure history of the last four months. Biomonitoring of urinary metabolites is a less invasive process than biomonitoring blood protein adducts, and urinary metabolites have served as short-lived biomarkers of exposure to these hazardous chemicals. However, in case of intermittent exposure, urinary metabolites may not be detected, and subjects may be misclassified as nonexposed. Arylamines and nitroarenes and/or their metabolites have been measured in urine, especially to monitor the exposure of workers. This review summarizes the results of human biomonitoring studies involving urinary metabolites and Hb-adducts of arylamines and nitroarenes. In addition, studies about the relationship between Hb-adducts and diseases are summarized.

Simplified Method for Quantifying Sulfur Mustard Adducts to Blood Proteins by Ultrahigh Pressure Liquid Chromatography−Isotope Dilution Tandem Mass Spectrometry.

Sulfur mustard binds to reactive cysteine residues, forming a stable sulfur-hydroxyethylthioethyl [SHETE]adduct that can be used as a long-term biomarker of sulfur mustard exposure in humans. The digestion of sulfur mustard-exposed blood samples with proteinase K following total protein precipitation with acetone produces the tripeptide biomarker [S-HETE]-Cys-Pro-Phe. The adducted tripeptide is purified by solid phase extraction, separated by ultra high pressure liquid chromatography, and detected by isotope dilution tandem mass spectrometry. This approach was thoroughly validated and characterized in our laboratory. The average interday relative standard deviation was ≤ 9.49%, and the range of accuracy was between 96.1 and 109% over a concentration range of 3.00 to 250. ng/mL with a calculated limit of detection of1.74 ng/mL. A full 96-well plate can be processed and analyzed in 8 h, which is 5 times faster than our previous 96-well plate method and only requires 50 μL of serum, plasma, or whole blood. Extensive ruggedness and stability studies and matrix comparisons were conducted to create a robust, easily transferrable method. As a result, a simple and high-throughput method has been developed and validated for the quantitation of sulfur mustard blood protein adducts in low volume blood specimens which should be readily adaptable for quantifying human exposures to other alkylating agents.

A 10-minute point-of-care assay for detection of blood protein adducts resulting from low level exposure to organophosphate nerve agents

The OrganoTox test is a rapid, point-of-care assay capable of detecting clinically relevant organophosphate (OP) poisoning after low-level exposure to sarin, soman, tabun, or VX chemical nerve agents. The test utilizes either a finger stick peripheral blood sample or plasma specimen. While high-level nerve agent exposure can quickly lead to death, low-level exposure produces vague, nondescript signs and symptoms that are not easily clinically differentiated from other conditions.In initial testing, the OrganoTox test was used to detect the presence of blood protein-nerve agent adducts in exposed blood samples. In order to mimic the in vivo exposure as closely as possible, nerve agents stored in organic solvents were spiked in minute quantities into whole blood samples. For performance testing, 40 plasma samples were spiked with sarin, soman, tabun, or VX and 10 normal plasma samples were used as the negative control. The 40 nerve agent-spiked plasma samples included 10 replicates of each agent. At the clinically relevant low-level exposure of 10ng/ml, the OrganoTox test demonstrated 100% sensitivity for soman, tabun, and VX and 80% sensitivity for sarin. The OrganoTox test demonstrated greater than 97% specificity with 150 blood samples obtained from healthy adults. No cross-reactivity or interference from pesticide precursor compounds was found.A rapid test for nerve agent exposure will help identify affected patients earlier in the clinical course and trigger more appropriate medical management in a more timely manner.

Synthetic Approaches To Obtain Amino Acid Adducts of 4,4′-Methylenediphenyl Diisocyanate

4,4'-Methylenediphenyl diisocyanate (MDI) is the most important isocyanate used in the chemical industry. Lung sensitization and asthma are the main types of damage after exposure to MDI. Albumin adducts of MDI might be involved in the etiology of sensitization reactions. It is therefore necessary to have sensitive and specific biomarkers such as blood protein adducts to monitor people exposed to isocyanates. For the discovery of new isocyanate adducts with blood proteins present in vivo, new synthetic standards are needed. To achieve this, we developed five methods to obtain amino acid adducts of MDI. We synthesized and isolated MDI adducts of aspartic acid, glutamic acid, cysteine, and valine. The new adducts were characterized by LC-MS/MS and NMR. We synthesized the corresponding isotope-labeled MDI adducts to develop analytical methods using LC-MS/MS. Glutathione adducts of isocyanates are an important way of transportation of the reactive isocyanates to distant sites from the original site of exposure. Therefore, we used N-acetyl-cysteine adducts of MDI as reactants: N-acetyl-S-[[4-(4-aminobenzyl)phenyl]carbamoyl]-cysteine (MDI-AcCys) and N-acetyl-S-[[4-(4-acetylaminobenzyl)phenyl]carbamoyl]-cysteine (AcMDI-AcCys). MDI-AcCys or AcMDI-AcCys formed adducts with albumin, N(α)-acetyl lysine, and valine. Isotope-labeled albumin adducts (= d(4)-MDI-albumin) were synthesized from d(4)-MDI-AcCys and albumin. d(4)-MDI-albumin can be used as an internal standard to analyze biological samples. Such an internal standard will not correct only for the extraction recovery of the adducts but also for the potential variation of the enzymatic digestions used in the procedure to analyze albumin adducts of MDI. The synthetic procedures described in this manuscript will be applicable to the synthesis of amino acid adducts from other isocyanates.

Abstract 5701: Determination of new biomarkers to monitor the dietary consumption of isothiocyanates

Abstract Background. Isothiocyanates (ITCs) found in cruciferous vegetables have demonstrated cancer preventive activity in animals. Increased dietary intake of ITCs has been shown to be associated with a reduced cancer risk in humans. ITCs exert their cancer chemopreventive action by multiple mechanisms, for example, by modulating the activities of phase I and phase II drug metabolism enzymes, by inhibiting the cell cycle and histone deacetylase, and by causing apoptotic cell death. In cells, protein adducts account for most of total cellular ITC-uptake at 4h after treatment. The time course of this protein binding correlates well with the inhibition of proliferation and the induction of apoptosis. In animal studies, glutathione conjugates are the major products of ITCs. The major urinary excretion products of ITCs in human are N-acetyl cysteine conjugates. Urinary metabolites might provide the exposure history of the last 24h, if the urine of the full next day is collected. Therefore, stable biomarkers such as reaction products with blood proteins are needed which reflect a larger time span of the ITC-exposure history. Results. We developed a method to determine serum albumin adducts of allyl isothiocyanate (AITC), benzylisothiocyanate (BITC), phenylethyl isothiocyanate (PEITC), and sulforaphane (SFN) in humans. The feasibility of the method was tested on biological samples collected previously for a diet-study. The volunteers were randomly assigned to three groups: Group A (n=29): the diet was rich in flavonoids, but not supplemented. Group B (n=29), the diet was a normal iso-caloric diet with an adequate administration of fruit and vegetables. Group C (n=27): the diet was based on supplementation of the normal diet with additional flavonoids in the form of green tea and soy products. All subjects were male smokers. Blood samples were collected at the beginning and after 4 weeks. After enzymatic digestion of albumin we determined the adducts of the ITCs with lysine (Lys) using LC-MS/MS and isotope dilution method. At the beginning of the study (and after 4 weeks) 5.9% (2.4%), 83.5% (96.5%), 11.8% (15.3%), and 31.8% (32.9%) samples were found positive for AITC-Lys, BITC-Lys, PEITC-Lys, and SFN-Lys respectively. Significant differences among the groups were found at the beginning of the study. SFN-Lys levels in group C were significantly higher than in group A and B, and BITC-levels in group C were significantly lower than in group A and B. These differences disappear after 4 weeks. Conclusions. This new method enables to quantify ITC adducts in blood proteins from large prospective studies about diet and cancer. Protein adducts are involved in the chemopreventive effects of ITCs. Therefore, blood protein adducts are a potential surrogate marker for the effects of ITCs at the cellular level. This new technique will improve the assessment of ITC exposure and the power of studies on the relationship between ITC intake and cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5701.

Benzene exposure: An overview of monitoring methods and their findings

Benzene has been measured throughout the environment and is commonly emitted in several industrial and transportation settings leading to widespread environmental and occupational exposures. Inhalation is the most common exposure route but benzene rapidly penetrates the skin and can contaminant water and food resulting in dermal and ingestion exposures. While less toxic solvents have been substituted for benzene, it still is a component of petroleum products, including gasoline, and is a trace impurity in industrial products resulting in continued sub to low ppm occupational exposures, though higher exposures exist in small, uncontrolled workshops in developing countries. Emissions from gasoline/petrochemical industry are its main sources to the ambient air, but a person's total inhalation exposure can be elevated from emissions from cigarettes, consumer products and gasoline powered engines/tools stored in garages attached to homes. Air samples are collected in canisters or on adsorbent with subsequent quantification by gas chromatography. Ambient air concentrations vary from sub-ppb range, low ppb, and tens of ppb in rural/suburban, urban, and source impacted areas, respectively. Short-term environmental exposures of ppm occur during vehicle fueling. Indoor air concentrations of tens of ppb occur in microenvironments containing indoor sources. Occupational and environmental exposures have declined where regulations limit benzene in gasoline (<1%) and cigarette smoking has been banned from public and work places. Similar controls should be implemented worldwide to reduce benzene exposure. Biomarkers of benzene used to estimate exposure and risk include: benzene in breath, blood and urine; its urinary metabolites: phenol, t, t-muconic acid ( t, tMA) and S-phenylmercapturic acid ( sPMA); and blood protein adducts. The biomarker studies suggest benzene environmental exposures are in the sub to low ppb range though non-benzene sources for urinary metabolites, differences in metabolic rates compared to occupational or animal doses, and the presence of polymorphisms need to be considered when evaluating risks from environmental exposures to individuals or potentially susceptible populations.

Hemoglobin Adducts and Mercapturic Acid Excretion of Acrylamide and Glycidamide in One Study Population

The aim of this study was to determine the relationship between the oxidative and reductive metabolic pathways of acrylamide (AA) in the nonsmoking general population. For the first time both the blood protein adducts and the urinary metabolites of AA and glycidamide (GA) were quantified in an especially designed study group with even distribution of age and gender. The hemoglobin adducts N-carbamoylethylvaline (AAVal) and N-( R, S)-2-hydroxy-2-carbamoylethylvaline (GAVal) were detected by GC-MS/MS in all blood samples with median levels of 30 and 34 pmol/g of globin, respectively. Concentrations ranged from 15 to 71 pmol/g of globin for AAVal and from 14 to 66 pmol/g of globin for GAVal. The ratio GAVal/AAVal was 0.4-2.7 (median = 1.1). The urinary metabolites were determined by LC-MS/MS. Of all urine samples examined 99% of N-acetyl- S-(2-carbamoylethyl)- l-cysteine (AAMA) levels and 73% of N-( R/ S)-acetyl- S-(2-carbamoyl-2-hydroxyethyl)- l-cysteine (GAMA) levels were above the LOD (1.5 microg/L). Concentrations ranged from <LOD to 229 microg/L (median = 29 microg/L) for AAMA and from <LOD to 85 microg/L (median = 7 microg/L) for GAMA. The ratio of GAMA/AAMA varied from 0.004 to 1.4 (median = 0.3). Using hemoglobin adduct levels in blood and mercapturic acid excretion in urine for calculation of daily AA intake gave practically identical values. The median daily intakes were 0.43 (0.21-1.04) microg/kg of body weight(bw)/day using Hb adducts and 0.51 (<LOD-2.32) microg/kg of bw/day using mercapturic acids for calculations. Children take up approximately 1.3-1.5 times more AA per kilogram of body weight than adults. The ratio GAMA/AAMA is significantly higher in the group of young children (6-10 years) with a median level of 0.5. A gender-related difference in internal exposure and metabolism was not observed.

ANALYSIS OF POLYCYCLIC AROMATIC HYDROCARBONS IN AMNIOTIC FLUID SAMPLES FROM SMOKERS AND NONSMOKERS

Previous studies from this laboratory have focused on the characterization of blood protein adducts formed in utero as a result of maternal smoking during pregnancy. These biological samples, obtained during the third trimester of pregnancy, at delivery, have clearly shown a correlation between maternal smoking histories and exposure of the fetus to tobacco smoke carcinogens, including 4-aminobiphenyl and benzo(a)pyrene. In the present study, we examined exposure of the fetus during the first trimester of pregnancy to various environmental carcinogens, particularly those found in tobacco smoke. Amniotic fluid samples were obtained from women undergoing routine amniocentesis between 16 and 20 weeks gestational age. Amniotic fluid, produced by the fetal lungs and kidneys, is an important part of pregnancy and fetal development and this fluid surrounds the fetus throughout pregnancy. In these studies, samples of amniotic fluid were obtained from nonsmokers as well as 0.5 pack per day smokers, 1.0 pack per day smokers, and greater than 2.0 pack per day smokers. Maternal smoking status was determined by questionnaire as well as assessment of amniotic fluid for cotinine via immunoassay. Amniotic fluid samples were extracted and analyzed for the presence of polycyclic aromatic hydrocarbons (PAHs). A clear correlation was found between levels of maternal smoking and PAHs in the amniotic fluid. Amniotic fluid 1-hydroxypyrene levels ranged from 1.54 ± 0.12 μ g/L in nonsmokers to 11.72 ± 0.67 μ g/L in women smoking greater than 2 pks/da, indicating approximately a 10X increase over nonsmokers. Similar results were found 3-hydroxybenzo(a)pyrene, 6-hydroxybenzo(a)pyrene, and 3,6-dihydroxybenzo(a)pyrene, metabolites of the carcinogen benzo(a)pyrene as well as with the 9-hydroxy and 9,10-dihydroxy metabolites of anthracene. The 5-hydroxymethyl metabolite of 5-methylchrysene was found to range in concentrations from 1.65 ± 0.11 μ g/L in nonsmokers to 12.67 ± 0.79 μ g/L in greater than 2 packs per day smokers. These results demonstrate that amniotic fluid can serve as a biological marker of exposure to tobacco related polycyclic aromatic hydrocarbons. Identification of potentially harmful compounds detected at an early stage of pregnancy may prevent subsequent exposures to the fetus and as a result decrease the risk of potential genotoxic as well as teratogenic events.

Improvements in the Methodology of Monitoring Sulfur Mustard Exposure by Gas Chromatography-Mass Spectrometry Analysis of Cleaved and Derivatized Blood Protein Adducts

An analytical method for determining exposure to 2,2'-dichlorodiethyl sulfide (sulfur mustard, HD) has been enhanced. The method is based on the cleavage of adducted HD (protein-hydroxyethylthioethyl esters) to produce thiodiglycol. Following cleavage, a deuterated internal standard is added, and the analytes are extracted, derivatized, and analyzed by gas chromatography-negative ion chemical ionization-mass spectrometry. Inclusion of a concentration step, addition of solid sodium bicarbonate to neutralize excess derivatization reagent, and optimization of method and instrument conditions provided dramatic increases in signal-to-noise ratio. A five-day precision and accuracy study was conducted, including interday and intraday unknown analysis. Linearity was verified by a R(2) > 0.9995 for all five curves evaluated. The precision and accuracy of the assay were demonstrated to be excellent by evaluation of the interday and intraday unknown samples (< 10% relative standard deviation and relative error in most cases). Statistical treatment of the method blanks and calibration results demonstrated a reduction in the limit of quantitation from 25 nM (HD, human plasma, in vitro) to 1.56 nM. Sample and calibration stability through the analytical sequence was established by the inclusion of continuing calibration verification standards (< 5% error). Short-term sample stability was verified by reinjection of a calibration set after 18 days (R(2) = 0.9997). Quantitative agreement with the previous method was supported by the analysis of a 50 nM standard protein sample (HD, rat plasma) with both methodologies (< 1% error).

Monitoring sulfur mustard exposure by gas chromatography-mass spectrometry analysis of thiodiglycol cleaved from blood proteins.

A gas chromatography-mass spectrometry method for determining exposure to the chemical warfare agent 2,2'-dichlorodiethyl sulfide (sulfur mustard; HD) has been developed. The technique is based upon quantitating thiodiglycol (TDG) released from blood protein adducts that are formed upon exposure to HD. Protein was precipitated from plasma, whole blood, or packed red blood cells (RBCs) and then treated with sodium hydroxide to liberate protein-bound TDG. The TDG was derivatized with pentafluorobenzoyl chloride that enabled sensitive detection by negative-ion chemical ionization. Octadeuterothiodiglycol was used as an internal standard. Exposure of human plasma to HD (25 nM to 400 nM) resulted in a linear relationship (r2 = 0.9995) between HD concentration and released TDG levels with means ranging from 2.0 to 38 pg/mg protein. The coefficients of variation expressed as a percentage for the data points ranged from 2 to 11.5%. The application of this procedure was demonstrated in two HD animal exposure models. African green monkeys (Chlorocebus aethiops) were exposed intravenously to 1 mg/kg HD, and TDG levels in blood samples were analyzed out to 45 days post-exposure. Mean TDG levels were determined to be 220 pg/mg protein on day 1 and declined to 10 pg/mg protein on day 45. Yorkshire cross pigs (Sus scrofa) were cutaneously exposed to neat liquid HD, and TDG levels in plasma were determined out to 21 days following exposure. Mean TDG levels were found to be 60 pg/mg protein on day one and decreased to an average of 4 pg/mg protein on day 21. The data from this study indicate that the assay is sensitive and provide a relatively simple approach to assay TDG cleaved from blood proteins at relatively long time frames (21-45 days) after HD exposure. The utility of the method has been demonstrated in vivo in a non-human primate and pig HD exposure model.

Carcinogen derived biomarkers: applications in studies of human exposure to secondhand tobacco smoke

Objective: To review the literature on carcinogen derived biomarkers of exposure to secondhand tobacco smoke (SHS). These biomarkers are specifically related to known carcinogens in tobacco smoke and include urinary...

Levels of polycyclic aromatic hydrocarbons in amniotic fluid samples from smokers and nonsmokers

Previous studies from this laboratory have focused on the characterization of blood protein adducts formed in utero as a result of maternal smoking during pregnancy. These biological samples, obtai...

Biomarkers of exposure to heterocyclic amines: approaches to improve the exposure assessment

Various methods of exposure assessment, such as questionnaires, sometimes combined with pictures of cooked meat, have been employed in investigations on the relationship between heterocyclic amines (HA) and health effects. However, as the content of heterocyclic amines vary greatly with cooking conditions, it is difficult to obtain an accurate estimate of the exposure. To improve the exposure assessment, the use of biomarkers has been investigated. The metabolism of 2-amino-3,8-dimethylimidazo[4,5- f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP) is well characterised. In humans, the major part of the dose is excreted in urine within 24–48 h following a meal. A few percent is excreted as parent compounds, whereas the major part is metabolites. Urinary level of parent HA reflects only recent exposure. However, the pattern of excreted metabolites might indicate the capacity to activate or detoxify HAs. The excretion of glucuronide conjugates of N-hydroxy-PhIP and N-hydroxy-MeIQx could be a marker for the N-hydroxylation capacity and the dose of the proximate metabolites. Recently, we proposed 5-OH-PhIP as a marker for the ultimate reactive metabolite of PhIP, since it is formed from this compound as a by-product along with the formation of PhIP–DNA adducts. In a search for biomarkers reflecting exposure over some time, blood protein adducts with a longer lifespan have been investigated, and PhIP adducts of serum albumin and haemoglobin from meat-consuming humans were recently reported. Many compounds, like drugs, nicotine and narcotics, bind to melanin in hair and give information on exposure for longer time periods. In mice, PhIP is irreversibly incorporated in a dose-dependent manner into hair, and in humans exposed to an ordinary diet, it was found to vary from <50 to 5000 pg PhIP/g hair. The incorporation is also dependent on the content of eumelanin. The use of PhIP in hair as a biomarker of exposure is promising, but needs validation, using other methods of exposure assessment.

Synthesis of adducts with amino acids as potential dosimeters for the biomonitoring of humans exposed to toluenediisocyanate.

Toluenediisocyanates (TDI) are important intermediates in the chemical industry. Among the main damages after low levels of TDI exposure are lung sensitization and asthma. Protein adducts of TDI might be involved in the etiology of sensitization reactions. Blood protein adducts are used as dosimeters for modifications of macromolecules in the target organs where the disease develops. The functional groups of cysteine, tyrosine, serine, lysine, tryptophan, histidine, and N-terminal amino acids are potential reaction sites for isocyanates. Especially the N-terminal amino acids, valine, and aspartic acid of hemoglobin and albumin, respectively, are reactive toward electrophilic xenobiotics. To develop methods for the quantitation of protein adducts of 2,4- and 2,6-TDI, we reacted 3-nitro-4-methylphenyl isocyanate (1a) with single amino acids and reduced the nitro group using catalytic hydrogenation or ammonium formate with palladium on carbon yielding N-[(3-amino-4-methylphenyl)carbamoyl]valine (2a), N-[(3-amino-4-methylphenyl)carbamoyl]aspartic acid (8a), N(alpha)-acetyl-N(epsilon)-[(3-amino-4-methylphenyl)carbamoyl]lysine (12a), and N(alpha)-acetyl-O-[(3-amino-4-methylphenyl)carbamoyl]serine (15a). The same reactions were performed with 5-nitro-2-methylphenyl isocyanate (1b) and 3-nitro-2-methylphenyl isocyanate (1c). The valine adducts were boiled in acid to obtain the corresponding hydantoins: 3-(3-amino-4-methylphenyl)-5-isopropylimidazoline-2,4-dione (5a), 3-(5-amino-2-methylphenyl)-5-isopropylimidazoline-2,4-dione (5b), and 3-(3-amino-2-methylphenyl)-5-isopropylimidazoline-2,4-dione (5c). A method for the detection of N-terminal adducts with valine in biological samples was developed. The tripeptide adduct N-[(3-amino-4-methylphenyl)carbamoyl]valyl-glycyl-glycine (19a) was hydrolyzed with acid in the presence of globin and the internal standard N-[(3-amino-4-methylphenyl-d(6))carbamoyl]valyl-glycyl-glycine (19d). The released hydantoins were determined by LC/MS/MS and after derivatization with pentafluoropropionic anhydride by GC/MS. The determination limit was 0.16 pmol/sample. The same N-terminal adduct with valine was found in globin of a TDI-worker and in two women with polyurethane covered breast implants.

Subpicogram determination of (+)- anti-benzo[ a]pyrene diol-epoxide adducts in fish albumin and globin by high-performance liquid chromatography with fluorescence detection

A simple and highly sensitive high-performance liquid chromatography/fluorescence method was developed for the determination of benzo[ a]pyrene (BaP)-tetrol I-1 released after acid hydrolysis of (+)- anti-BaP diol-epoxide (BPDE)-blood protein adducts. This method does not require extensive tetrol isolation such as solvent extraction, multiple solid-phase extraction columns and/or immunoaffinity chromatography. Instead, a single cleanup step using an inexpensive, disposable C 18 solid-phase extraction cartridge is performed, which yields 97% recovery of BaP-tetrol I-1. The method included a number of quality assurance measures and was validated using fish albumin (Alb) and globin (Gb) samples fortified with BaP-tetrol I-1. The absolute detection limit ( S/ N=3) was 0.3 pg (0.9 fmol) per injection, corresponding to 450 fg (1.4 fmol) mg −1 Alb and 90 fg (0.3 fmol) mg −1 Gb for 1 ml of blood. The operating linear range was at least 1–100 pg ( r 2=0.99). Coefficients of variation for within-run and between-run assays were <5.5 and <9.3%, respectively. The method was applied to the determination of (+)- anti-BPDE-Alb and Gb adducts in fish intraperitoneally injected with a single (10 mg kg −1) or two doses (25 mg kg −1 each) of BaP.

Biological fate of [ 14C]-1-nitropyrene in rats following intragastric administration

1-Nitropyrene (1-NP), a weak carcinogen associated with diesel exhaust particles, has previously been detected in workplace atmospheres with in-use diesel engines and in the general environment. In order to gain insight in its biological fate, a single dose of [ 14C]-1-NP (27.6 μCi, 750 mg/kg body weight, b.w.) was administered intragastrically to rats and the presence of metabolites in blood and tissue homogenates, and radioactivity associated with blood proteins and tissue DNA, were studied. Early peak levels of radioactivity observed in blood and tissue homogenates indicated a rapid absorption of [ 14C]-1-NP from the gastrointestinal tract. Metabolite patterns observed in plasma, liver and kidney homogenates strongly suggested an important role of the intestinal microflora in the enterohepatic recirculation, but not in nitroreduction of 1-NP prior to absorption from the gastrointestinal tract. This might explain the low levels of radioactivity associated with blood proteins, since 1-nitrosopyrene, a product of nitroreduction of 1-NP, is likely to be involved in protein binding. Levels of radioactivity associated with plasma proteins were approximately four times higher than the levels of radioactivity associated with hemoglobin (401.0 and 84.1 pmol/g protein per μmol 1-NP kg b.w., respectively, at 24 h). Maximal 25% of the associated radioactivity was released following mild alkaline hydrolysis of either hemoglobin or plasma proteins. 1-Aminopyrene was the only released compound after hydrolysis of hemoglobin. In addition to 1-aminopyrene, two more polar unidentified metabolites were detected following hydrolysis of plasma proteins. Association of radioactivity with DNA was highest in the liver at the first moments of observation (7.4 pmol 14C Eq./mg DNA per μmol 1-NP kg b.w.), but decreased rapidly to levels lower than observed for kidney DNA (max. 3.0 pmol 14C Eq./mg DNA per μmol 1-NP kg b.w. at 24 h). In lungs 8–50 times less radioactivity was associated with DNA than observed in the liver and kidneys. The results of this study show, that 1-NP undergoes an extensive and complex biotransformation in vivo, resulting in a variety of metabolites present in blood and tissue homogenates and a diversity of blood protein adducts. Concentrations of plasma metabolites, blood protein adducts and DNA adducts were rather low. In addition, previous studies also showed relatively low concentrations of metabolites present in urine. Therefore, sensitive and selective methods will be needed in order to evaluate the biological fate of 1-NP, associated with diesel exhaust particles, in humans.