Assessment of human exposure to di-isodecyl phthalate using oxidative metabolites as biomarkers

Di-isodecyl phthalate (DiDP), primarily used as a plasticiser, is a mixture of isomers with predominantly ten-carbon branched side chains. Assessment of DiDP exposure has not been conducted before because adequate biomarkers were lacking. In 129 adult volunteers with no known exposure to DiDP, the urinary concentrations of three oxidative metabolites of DiDP: monocarboxyisononyl phthalate (MCiNP), monooxoisodecyl phthalate (MOiDP) and monohydroxyisodecyl phthalate (MHiDP), previously identified in DiDP-dosed rats, were estimated by solid-phase extraction coupled to high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) using the respective oxidative metabolites of di(2-ethylhexyl)phthalate since authentic standards of the DiDP oxidative metabolites were unavailable. Interestingly, the hydrolytic monoester of DiDP, monoisodecyl phthalate (MiDP), was not detected in any of the samples, while MCiNP, MHiDP and MOiDP were detected in 98%, 96% and 85%, respectively, of the samples tested. MCiNP was excreted predominantly in its free form, whereas MOiDP was excreted as its glucuronide. MCiNP, MHiDP and MOiDP eluted as clusters of multiple peaks from the HPLC column probably due to the presence of numerous structurally similar isomers present in commercial DiDP formulations. The urinary concentrations of these oxidative metabolites correlated significantly (p<0.0001) with each other, thus confirming a common precursor. The urinary concentrations of these DiDP oxidative metabolites also correlated significantly (p<0.0001) with oxidative metabolites of di-isononyl phthalate (DiNP) suggesting the potential presence of DiNP isomers in commercial DiDP or simultaneous use of DiDP and DiNP in consumer products. The concentrations presented are semiquantitative estimates and should be interpreted cautiously. Nevertheless, the higher frequency of detection and higher urinary concentrations of MCiNP, MHiDP and MOiDP than of MiDP suggest that these oxidative metabolites are better biomarkers for DiDP exposure assessment than MiDP. These data also suggest that unless oxidative metabolites are measured, the prevalence of exposure to DiDP will probably be underestimated.     

Measurement of eight urinary metabolites of di(2-ethylhexyl) phthalate as biomarkers for human exposure assessment.

Human metabolism of di(2-ethylhexyl) phthalate (DEHP) is complex and yields mono(2-ethylhexyl) phthalate (MEHP) and numerous oxidative metabolites. The oxidative metabolites, mono(2-ethyl-5-oxohexyl) phthalate (MEOHP), mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), mono(2-ethyl-5-carboxypentyl) phthalate (MECPP) and mono(2-carboxymethylhexyl) phthalate (MCMHP), have been considered to be better biomarkers for DEHP exposure assessment than MEHP because urinary levels of these metabolites are generally higher than MEHP, and their measurements are not subject to contamination. The urinary levels of the above metabolites, and of three other recently identified DEHP oxidative metabolites, mono(2-ethyl-3-carboxypropyl) phthalate (MECPrP), mono-2-(1-oxoethylhexyl) phthalate (MOEHP), and mono(2-ethyl-4-carboxybutyl) phthalate (MECBP), were measured in 129 adults. MECPP, MCMHP and MEHHP were present in all the samples analysed. MEHP and the other oxidative metabolites were detected less frequently: MEOHP (99%), MECBP (88%), MECPrP (84%), MEHP (83%) and MOEHP (77%). The levels of all DEHP metabolites were highly correlated (p<0.0001) with each other, confirming a common parent. The ? and ?-1 oxidative metabolites (MECPP, MCMHP, MEHHP and MEOHP) comprised 87.1% of all metabolites measured, and thus are most likely the best biomarkers for DEHP exposure assessment. The percentage of the unglucuronidated free form excreted in urine was higher for the ester linkage carboxylated DEHP metabolites compared with alcoholic and ketonic DEHP metabolites. The percentage of the unglucuronidated free form excreted in urine was higher for the DEHP metabolites with a carboxylated ester side-chain compared with alcoholic and ketonic metabolites. Further, differences were found between the DEHP metabolite profile between this adult population and that of six neonates exposed to high doses of DEHP through extensive medical treatment. In the neonates, MEHP represented 0.6% and MECPP 65.5% of the eight DEHP metabolites measured compared to 6.6% (MEHP) and 31.8% (MECPP) in the adults. Whether the observed differences reflect differences in route/duration of the exposure, age and/or health status of the individuals is presently unknown.     

Analysis of human urine for fifteen phthalate metabolites using automated solid-phase extraction

We improved our previous analytical method to measure phthalate metabolites in urine as biomarkers for phthalate exposure by automating the solid-phase extraction (SPE) procedure and expanding the analytical capability to quantify four additional metabolites: phthalic acid, mono-3-carboxypropyl phthalate, mono-isobutyl phthalate (miBP), and monomethyl isophthalate. The method, which involves automated SPE followed by isotope dilution-high performance liquid chromatography (HPLC)-electrospray ionization (ESI)-tandem mass spectrometry (MS), allows for the quantitative measurement of 15 phthalate metabolites in urine with detection limits in the low ng/ml range. SPE automation allowed for the unattended sequential extraction of up to 100 samples at a time, and resulted in an increased sample throughput, lower solvent use, and better reproducibility than the manual SPE. Furthermore, the modified method permitted for the first time, the separation and quantification of mono-n-butyl phthalate (mBP) and its structural isomer miBP. The method was validated on spiked pooled urine samples and on pooled urine samples from persons with no known exposure to phthalates.     

Studies using specific biomarkers for human exposure assessment to exogenous and endogenous chemical agents.

The extent of formation of carcinogen adducts with DNA and protein may be used to assess the biologically effective dose of these carcinogens in the tissue under study. In normal human tissues, such carcinogen adducts arise in part from exposures to exogenous genotoxic compounds, although it has been shown that endogenously formed carcinogens also make a significant contribution to the observed DNA and protein damage. Although, highly sensitive analytical methods, such as immunoassay, 32P-postlabelling and mass spectrometry have been developed and successfully applied to measure carcinogen adducts, further methodological advances are making these methods more amenable to molecular epidemiological studies. Thus, the use of immunoslot blot assays allows a higher sample throughput for adduct quantification. Liquid chromatographic separations of adducts, either for their radiochemical detection following 32P-postlabelling or for their determination by mass spectrometry, improves the specificity and applicability of these techniques. In this review, the sensitivities and specificities of the analytical methods used for adduct detection are compared and the limitations of these methods described.     

Assessment of Anopheles salivary antigens as individual exposure biomarkers to species-specific malaria vector bites

The development of parasitological immunity against malaria affects the ability to detect infection, the efficiency of the local human parasite reservoir at infecting mosquitoes, and the response to reintroduction of parasites to previously cleared areas. Observations of similar age-trends in detected prevalence and mean parasitaemia across more than an order-of-magnitude of variation in baseline transmission complicate simple exposure-driven explanations. Mathematical models often employ age-dependent immune factors to match the observed trends, while the present model uses a new detailed mechanistic model of parasite transmission dynamics to explain age-trends through the mechanism of parasite diversity. Illustrative simulations are performed for multiple field sites in Tanzania and Nigeria, and observed age-trends and seasonality in parasite prevalence are recreated in silico, proffering possible mechanistic explanations of the observational data. Observed temporal dynamics in measured parasitaemia are recreated for each location and age-prevalence outputs are studied. Increasing population-level diversity in malaria surface antigens delays development of broad parasitological immunity. A local parasite population with high diversity can recreate the observed trends in age-prevalence across more than an order of magnitude of variation in transmission intensities. Mechanistic models of human immunity and parasite antigen diversity can recreate the observed temporal patterns for the development of parasitological immunity across a wide range of transmission intensities. This has implications for the distribution of disease burden across the population, the human transmission reservoir, design of elimination campaigns, and development and roll-out of potential vaccines.     

Quantification of 22 phthalate metabolites in human urine

Phthalates are ubiquitous industrial chemicals with high potential for human exposure. Validated analytical methods to measure trace concentrations of phthalate metabolites in humans are essential for assessing exposure to phthalates. Previously, we developed a sensitive and accurate automated analytical method for measuring up to 16 phthalate metabolites in human urine by using on-line solid phase extraction coupled with isotope dilution-high performance liquid chromatography (HPLC)-electrospray ionization-tandem mass spectrometry. To include the measurement of seven additional analytes, including oxidative metabolites of diisononyl and diisodecyl phthalates, two chemicals used extensively in numerous consumer products, we used a novel nontraditional HPLC solvent gradient program. With this approach, we achieved adequate resolution and sensitivity for all 22 analytes with limits of detection in the low ng/mL range, without increasing the analytical run time. The method also has high accuracy with automatic recovery correction, high precision, and excellent sample throughput with minimal matrix effects. Although it is possible to measure these 22 phthalate metabolites with adequate precision and accuracy at sub-parts-per-billion levels, additional information, including toxicokinetic data, is needed to demonstrate the usefulness of these phthalate metabolites for exposure assessment purposes.     

Urinary hydroxylated metabolites of polycyclic aromatic hydrocarbons as biomarkers of exposure in asphalt workers

Background. Fumes and vapours released during laying of hot asphalt mix have been recognised as a major source of exposure for asphalt workers. Objectives. We investigated the relationships between inhalation exposure to asphalt emissions and urinary biomarkers of polycyclic aromatic hydrocarbons (PAHs) in asphalt workers (AW, n=75) and in ground construction workers (CW, n=37). Methods. Total polyaromatic compounds (PAC) and 15 priority PAHs in inhaled air were measured by personal sampling. Hydroxylated PAH metabolites (OH-PAHs) (2-naphthol, 2-hydroxyfluorene, 3-hydroxyphenanthrene, 1-hydroxypyrene, 6-hydroxychrysene and 3-hydroxybenzo[a]pyrene) were determined in urine spot samples collected in three different times during the work week. Results. Median vapour-phase PAC (5.5 µg m^-3), PAHs (≤50 ng m^-3) and OH-PAHs (0.08-1.11 µg l^-1) were significantly higher in AW than in CW, except in the cases of air naphthalene and 2-naphthol. Airborne levels of particle-phase contaminants were similar in the two groups and much lower than vapour-phase levels; metabolites of particulate PAHs were never found in quantifiable amounts. An appreciable increase in OH-PAH levels during the work day and work week was found in AW; median levels for 2-hydroxyfluorene, 3-hydroxyphenanthrene and 1-hydroxypyrene were, respectively, 0.29, 0.08 and 0.18 at baseline; 0.50, 0.18 and 0.29, pre-shift; 1.11, 0.44 and 0.44 µg l^-1, post-shift. Each OH-PAH exhibited a characteristic profile of increase, reflecting differences in half-lives of the parent compounds. In non-smoking subjects, positive correlations were found between vapour-phase PAC or PAHs and OH-PAHs both in pre- and post-shift samples (0.34 ≤ r≤69). Smokers exhibited 2-5-fold higher OH-PAHs than non-smokers, at any time and at both workplaces. Conclusions. Our results suggest that OH-PAHs are useful biomarkers for monitoring exposure to asphalt emissions. The work-related exposure to PAC and PAHs was low in all AW, but urinary metabolites reflected exposure satisfactorily.     

Peroxiredoxins as biomarkers of oxidative stress

Background

Peroxiredoxins (Prxs) are a class of abundant thiol peroxidases that degrade hydroperoxides to water. Prxs are sensitive to oxidation, and it is hypothesized that they also act as redox sensors. The accumulation of oxidized Prxs may indicate disruption of cellular redox homeostasis.

Scope of review

This review discusses the biochemical properties of the Prxs that make them suitable as endogenous biomarkers of oxidative stress, and describes the methodology available for measuring Prx oxidation in biological systems.

Major conclusions

Two Prx oxidation products accumulate in cells under increased oxidative stress: an intermolecular disulfide and a hyperoxidized form. Methodologies are available for measuring both of these redox states, and oxidation has been reported in cells and tissues under oxidative stress from external or internal sources.

General significance

Monitoring the oxidation state of Prxs provides insight into disturbances of cellular redox homeostasis, and complements the use of exogenous probes of oxidative stress. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.     

Haemoglobin adducts as biomarkers of exposure to tobacco-related nitrosamines

A sensitive gas chromatography/mass spectrometry (GC/MS) method was developed to measure nitrosamine-haemoglobin adducts (HPB-Hb) (4-hydroxy-3-pyridinyl-1-butanone) at trace levels in red blood cells of smoking and non-smoking mothers and their newborn babies. GC/MS methods with chemical ionization (CI) of methane reagent gas in both positive and negative ion mode as well as electron ionization (EI) were studied to determine differences in sensitivity among the various ionization methods. Detection limits using both positive and negative chemical ionization modes were found to be 30 fmol HPB, whereas detection using electron impact modes yielded a detection limit of 80 fmol HBP. In order to apply the various methods of detection to tobacco-exposed samples from human populations, we characterized adduct levels in maternal as well as paired fetal samples obtained from mothers exposed to tobacco smoke during pregnancy. Maternal samples were characterized using serum cotinine levels and were classified as non-smokers, passively smoke-exposed women, less than one pack per day smokers and greater than one pack per day smokers. Paired maternal and fetal blood samples were obtained at delivery for qualitative and qualitative analysis of nitrosamine adducts. Comparative derivatization of HPB released under alkaline hydrolysis conditions was performed using O-bis(trimethylsilyl)-trifluoroacetamide (BSTFA) and 2,3,4,5,6-pentafluorobenzoylchloride (PFBC). Both negative CI and positive CI modes of analysis were compared to the more widely accepted EI modes of mass spectrometric analysis. These results suggest that both NICI and PICI modes of detection offer a greater sensitivity of adduct characterization when compared with EI ionization techniques and that either NICI or PICI modes are preferably applicable towards the detection of human biomarker assessment of tobacco-related nitrosamines.     

Determination of three phthalate metabolites in human urine using on-line solid-phase extraction-liquid chromatography-tandem mass spectrometry

An on-line solid-phase extraction-liquid chromatography-tandem mass spectrometry (on-line SPE-HPLC-MS/MS) method was developed for the analysis of metabolites of three phthalate esters in human urine at the low nanogram per milliliter level. The recoveries were above 84.3% and relative standard deviations varied from 0.8 to 4.8%. The compounds along with their deuterated internal standards were detected in the negative ion mode by selective reaction monitoring and the accuracy of the method was improved by isotope dilution. Monobutyl phthalate was detected with median level of 22.5 ng/ml. The median levels for monobenzyl phthalate and monoethylhexyl phthalate were less than the limit of quantitation (LOQ). The on-line SPE-HPLC-MS/MS method allowed the possibility of determining these metabolites within a short time, with increased sensitivity and by using decreased amounts of sample and solvent.     

Formaldehyde-induced DNA adducts as biomarkers of in vitro human nasal epithelial cell exposure to formaldehyde

Formaldehyde (FA) is a mutagen that, at high concentrations and long durations, has been reported to cause nasal cancer in rats and in some humans. The level of FA-induced modified DNA in nasal cells should serve as a biomarker of FA exposure and effect. In the present study, a high-performance liquid chromatography (HPLC)-ultraviolet (UV) method at 254 nm was developed and optimized to detect and quantify hydroxymethyldeoxynucleosides after the isolated DNA in exposed human nasal epithelial cells (HNEC) was enzymically digested. Normal and modified deoxynucleosides were successfully resolved from one another and from tissue and enzyme blank interferences. The viability of HNEC exposed to FA in solution for 24 h decreased, and there was a linear dose response between % nonviability and FA dose from 10 to 500 microg/mL. Amounts of 18.0 +/- 1.5 pmol N6-dA and 12.0 +/- 1.2 pmol N2-dG derivatives were determined in a 10 microL injection after 1.4 x 10(7) HNEC (106 microg DNA) were exposed to 500 microg/mL in solution. The respective tissue concentrations in pmol hydroxymethyldeoxynucleoside/mg DNA were 170 +/- 14 and 113 +/- 11. The lower quantifiable limits were about 97 and 88 pmol/mg DNA, respectively. Diffusive exposure of HNEC to air FA up to 100 ppm (v/v) for 24 h did not produce quantifiable hydroxymethylnucleosides. FA-modified deoxynucleosides may be useful biomarkers for FA exposure in biological monitoring samples taken by nasal lavage or brush biopsy.     

Prenatal exposure to pesticides: analysis of human placental acetylcholinesterase, glutathione S-transferase and catalase as biomarkers of effect.

Pre- and perinatal exposure to pesticides is deleterious on foetal and neonatal development, but information regarding possible effects on environmental low-dose exposure to pesticides is scarce. Most epidemiological studies of the health effect of pesticides have been based on self-reported information. However, detailed information on past pesticide use is difficult to reconstruct. This is a current study conducted among pregnant mothers attending a delivery care and perinatal programme at a public hospital. The study investigates biomarkers of early effects in placentas from women living in an area with an intensive use of pesticides in the northern part of Patagonia, province of Río Negro, Argentina, and it assesses the consistency of the information provided by self-reports. The study confirms that placental acetylcholinesterase and catalase activities are significantly associated with periods of organophosphorus pesticides application, while glutathione S-transferase is not affected. We found a positive correlation between environmental exposure to organophosphorus pesticides and carbamate insecticides and newborn head circumference. The findings provide a further indication of a link between placenta acetylcholinesterase and catalase activity and prenatal exposure to pesticides in population studies. Both placenta enzymes may be used as biomarkers in health surveillance programmes for early diagnosis of exposure related alterations produced by organophosphorus pesticides and carbamate pesticides.     

Design issues in cross-sectional biomarkers studies: urinary biomarkers of PAH exposure and oxidative stress

Cross-sectional biomarker studies can provide a snapshot of the frequency and characteristics of exposure/disease in a population at a particular point in time and, as a result, valuable insights for delineating the multi-step association between exposure and disease occurrence. Three major issues should be considered when designing biomarker studies: selection of appropriate biomarkers, the assay (laboratory validity), and the population validity of the selected biomarkers. Factors related to biomarker selection include biological relevance, specificity, sensitivity, biological half-life, stability, and so on. The assay attributes include limit of detection, reproducibility/reliability, inter-laboratory variation, specificity, time, and cost. Factors related to the population validity include the frequency or prevalence of markers, greater inter-individual variation than intra-individual variation, intra-class correlation coefficients (ICC), association with potential confounders, invasiveness of specimen collection, and subject selection. Three studies are selected to demonstrate different features of cross-sectional biomarker studies: (1) characterizing the determinants of the biomarkers (study I: urinary PAH metabolites and environmental particulate exposure), (2) relationship of multiple biomarkers of exposure and effect (study II: relationship between urinary PAH metabolites and oxidative stress), and (3) evaluating gene-environmental interaction (study III: effect of genetic polymorphisms of GSTM1 on the association of green tea consumption and urinary 1-OHPG levels in shipbuilding workers).     

An approach based on liquid chromatography/electrospray ionization-mass spectrometry to detect diol metabolites as biomarkers of exposure to styrene and 1,3-butadiene

Styrene and 1,3-butadiene are important intermediates used extensively in the plastics industry. They are metabolized mainly through cytochrome P450-mediated oxidation to the corresponding epoxides, which are subsequently converted to diols by epoxide hydrolase or through spontaneous hydration. The resulting styrene glycol and 3-butene-1,2-diol have been suggested as biomarkers of exposure to styrene and 1,3-butadiene, respectively. Unfortunately, poor ionization of the diols within electrospray mass spectrometers becomes an obstacle to the detection of the two diols by liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS). We developed an LC/ESI-MS approach to analyze styrene glycol and 3-butene-1,2-diol by means of derivatization with 2-bromopyridine-5-boronic acid (BPBA), which not only dramatically increases the sensitivity of diol detection but also facilitates the identification of the diols. The analytical approach developed was simple, quick, and convincing without the need for complicated chemical derivatization. To evaluate the feasibility of BPBA as a derivatizing reagent of diols, we investigated the impact of diol configuration on the affinity of a selection of diols to BPBA using the established LC/ESI-MS approach. We found that both cis and trans diols can be derivatized by BPBA. In conclusion, BPBA may be used as a general derivatizing reagent for the detection of vicinal diols by LC/MS.     

Quantitation of five nevirapine oxidative metabolites in human plasma using liquid chromatography-tandem mass spectrometry

A multiple-reaction-monitoring LC/MS/MS method for the analysis of nevirapine oxidative metabolites, 2-hydroxynevirapine, 3-hydroxynevirapine, 8-hydroxynevirapine, 12-hydroxynevirapine, and 4-carboxynevirapine, in human plasma was developed and validated. The metabolites were isolated from 50 microL heparinized plasma by enzymatic hydrolysis of the glucuronide conjugates to the free metabolite followed by protein precipitation with acetonitrile. Peaks were quantitated at 3.03 min for the 4-carboxynevirapine metabolite, at 3.72, 4.27, 5.27, and 5.73 min for the positional 2-hydroxynevirapine, 12-hydroxynevirapine, 3-hydroxynevirapine, and 8-hydroxynevirapine metabolites, respectively, and 2.30 min for the internal standard, pirenzepine. The assay was accurate and precise based on assay validation controls over the nominal range of 0.010-1.0 mg/L. The average accuracy at the lowest concentration quality control (QC) sample was 16% (difference from theoretical value) for 8-hydroxynevirapine, all others were closer to their known respective standards. Within- and between-day precisions were within 12% for quality control samples for all five metabolites. Repetitive thawing and freezing did not have an effect on any metabolite through a minimum of three cycles. Thawed samples, remaining in plasma for 4 h before extraction, were within 5% of theoretical value. Stability of the extracted samples on the autosampler at room temperature was evaluated for 48 h and was observed to be within 12% of a fresh analytical sample for 2-hydroxynevirapine and 3-hydroxynevirapine; other metabolites were within 6% of theoretical value. The utility of the analytical method was demonstrated using trough steady-state plasma samples collected from 48 patients in a hepatic impairment study.     

Cholesterol ozonation products as biomarkers for ozone exposure in rats.

Cholesterol, 1, which is present in both the lung lining fluid and cell membranes of lung tissue, reacts with ozone in aqueous systems to give 3 beta-hydroxy-5-oxo-5,6-secocholestan-6-al (2) as the major product. Reaction of 2 with 2,4-dinitrophenyl hydrazine (DNPH) in aqueous solutions, liposomes or lung extracts affords the anti and syn DNPH derivatives of 2 (3b and 3c) and of the rearrangement product 3,5-dihydroxy-B-norcholestane-6-carboxaldehyde (3a). These derivatives also are detected in lung tissue extracts from rats exposed to 1.3 ppm ozone for 12 hr.     

Long-term dermal exposure to diisononyl phthalate exacerbates atopic dermatitis through oxidative stress in an FITC-induced mouse model

Diisononyl phthalate (DINP), considered to be an environmentally friendly plasticizer, is now widely used. However, the toxic effects of DINP need to be examined, particularly the effects of long-term dermal DINP exposure. Research into the mechanisms underlying these effects is urgently needed. In this study we examined the exacerbation effect of long-term dermal exposure to DINP in fluorescein isothiocyanate (FITC)-induced contact hypersensitivity (CHS) in mice, and sought the potential molecular mechanisms. Forty-nine male Balb/c mice were subjected to a 40-day dermal exposure to saline or one of three concentrations of DINP and then three rounds of sensitization with vehicle or 0.5% FITC. The results of a histopathological examination and measurement of ear swelling as well as immunological and inflammatory biomarkers (total-immunoglobulin (Ig)E and Th cytokines) supported the notion that high doses of DINP may aggravate atopic dermatitis. We also showed that melatonin, an antioxidant, could decrease the levels of oxidative stress and alleviate FITC-induced CHS suggesting that oxidative stress may be one of the molecular mechanisms to explain the exacerbation effect induced by DINP.     

Long-term stability of oxidative stress biomarkers in human serum

The storage time and storage temperature might affect stability of oxidative stress biomarkers, therefore, they have to be analyzed after long-term storage of serum samples. The stability of three biomarkers reflecting oxidative stress: reactive oxygen metabolites (ROM) for hydroperoxides, total thiol levels (TTL) for the redox status and biological antioxidant potency (BAP) for the antioxidant status, was investigated at several time points during 60 months of storage at ?20 and ?80?°C. Biomarkers ROM and BAP showed a very good stability during storage for 60 months at both temperatures. In addition, the correlation of the data after 60 months of storage compared with the starting data was very good with correlation coefficients >0.9. The TTL assay showed good results in serum samples stored at ?80?°C, but not in samples stored at ?20?°C. Serum samples for analysis of the set of oxidative stress biomarkers ROM, BAP and TTL can be stored up to 60 months at ?80?°C. ROM and BAP can also be stored at ?20?°C during this period. The present results are very important for the biomarker-related epidemiological studies that make use of biobanks with samples stored for many years and for new project planning, including sample storage conditions.     

Selected volatile organic compounds as biomarkers for exposure to tobacco smoke

Cutoff levels on the scales for benzene, ethylbenzene, toluene, styrene, o-xylene and m/p-xylene in blood were developed to classify smokers from non-smokers. Self-reported smoking during the last 5 d was used as the true smoking status. Receiver operating characteristics methods that minimized the difference between specificity and sensitivity were used to develop these cutoffs. Data from National Health and Nutrition Examination Survey for the cycle 2005–2006 were used for this purpose. For the total population, a cutoff of 0.038?ng/ml for benzene was able to classify smokers from non-smokers with a sensitivity of 83.6% and specificity of 83.7%.     

Polycyclic nitroarenes (nitro-PAHs) as biomarkers of exposure to diesel exhaust

Diesel exhaust contains numerous genotoxic carcinogens. It is essentially unknown to which extent this source contributes to the total load of these chemicals in humans. One possible approach to the problem is to find suitable biomarkers. To this end five polycyclic mononitroarenes (nitro-PAH) were selected and methods developed to determine the sulfinic acid-type hemoglobin adducts they form in vivo. The nitro-PAHs are: 1-nitropyrene, 2-nitrofluorene, 3-nitrofluoranthene, 9-nitrophenanthrene, and 6-nitrochrysene. Hydrolysis of the hemoglobin adducts yields the respective arylamines which were analyzed by gas chromatography/mass spectrometry. The detection limit was 0.01-0.08 pmol/g Hb. Blood samples were analyzed from 29 bus garage workers, occupationally exposed to diesel exhaust, and from 20 urban hospital workers and 14 rural council workers as controls. Hb adducts above the detection limit were found in most blood samples. The most abundant cleavage products were 1-aminopyrene and 2-aminofluorene with levels ranging from 0.01 to 0.68 pmol/g Hb. However, there was no significant difference between the groups for 1-nitropyrene and 2-nitrofluorene supporting the conclusion that both are widespread environmental contaminants resulting in significant background exposures. A significant difference on a group from individuals from urban and rural areas was found only if all five adducts were added, this may indicate an additional exposure from traffic. The new specific nitro-PAH Hb adducts are proposed to be used as biomarkers to trace the sources and to identify above-background exposures.