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.     

Haemoglobin adducts as biomarkers of exposure to the herbicides propanil and fluometuron

Abstract

Aromatic amine herbicides, including propanil, fluometuron, alachlor, trifluralin, and pendimethalin, were examined for their ability to form haemoglobin adducts in rats as potential biomarkers of exposure. Many aromatic amines are known to form haemoglobin adducts via conversion to the nitroso metabolite and binding of this metabolite to cysteinyl groups on haemoglobin. Since red blood cells are long lived, adducts formed with these cells may be reliable biomarkers of exposure with the potential for showing progressive accumulation. Gas chromatographic-mass spectrometric analyses of haemoglobin revealed that adducts were formed in rats treated with the rice herbicide propanil and the cotton herbicide fluometuron. Adducts were not detected with the herbicides alachlor, trifluralin, or pendamethalin.     

Distribution of biomarkers of human exposure to persistent organic pollutants from the group of organohalogen compounds as a result of the impact of the environment

Abstract

Organohalogen compounds constitute one of the important groups of persistent organic pollutants (POPs). Among them, due to their long-term health effects, one should pay attention on polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs), and perfluoroalkylated substances (PFASs). In case of that anthropogenic group of environmental pollution, the scientific world faces a problem of not only checking their toxic influences on the human organism at different age, from the natal period till late elderly years, but also monitoring the levels of such a numerous group of compounds in various environments, including human tissues and body fluids. This gave birth to a concept of checking the levels of selected biomarkers of exposure in the human organism, calculating body burden and assessing the hazard exposure to human beings. This article is an attempt to answer the question whether testing only biomarkers for different groups of pollutants is enough to determine the threat to different human populations. CB-153 levels represent a significant share in the sum of the six indicator NDL-PCBs (42.96%). In contrary to PCBs, in the case of PBDEs, not only BDE-47 is a biomarker of exposure to the entire PBDEs group, the congener BDE-153 cannot be omitted. Among the compounds belonging to PFASs, only four are detected in the biological material. The PFOS is the dominant representative of this group in the blood samples. It constitutes approximately 75% of the total PFASs.     

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.     

Nickel exposure and plasma levels of biomarkers for assessing oxidative stress in nickel electroplating workers

Context: The mechanism of nickel-induced pathogenesis remains elusive.

Objective: To examine effects of nickel exposure on plasma oxidative and anti-oxidative biomarkers.

Materials and methods: Biomarker data were collected from 154 workers with various levels of nickel exposure and from 73 controls. Correlations between nickel exposure and oxidative and anti-oxidative biomarkers were determined using linear regression models.

Results: Workers with a exposure to high nickel levels had significantly lower levels of anti-oxidants (glutathione and catalase) than those with a lower exposure to nickel; however, only glutathione showed an independent association after multivariable adjustment.

Discussion and conclusion: Exposure to high levels of nickel may reduce serum anti-oxidative capacity.     

De-novo identification of specific exposure biomarkers of the alternative plasticizer di(2-ethylhexyl) terephthalate (DEHTP) after low oral dosage to male volunteers by HPLC-Q-Orbitrap-MS

Context: Human exposure biomonitoring relies on the availability of specific, sensitive biomarkers. For emerging chemicals, the identification (prediction, synthesis, verification) of such biomarkers is time and cost intensive.

Objective: This study aimed to further elucidate the urinary metabolic profile of the plasticizer di(2-ethylhexyl) terephthalate (DEHTP) in search of probably additional biomarkers of exposure.

Materials and methods: Urine samples of an oral low-dose volunteer study were analysed by HPLC-Q-Orbitrap-MS combined with a commercial data mining software. Metabolite identification was based on isotopic pattern, accurate masses of product ions and excretion profiles.

Results: Nine phase I metabolites of DEHTP were tentatively identified by HPLC-Q-Orbitrap-MS. Four previously described, side chain oxidized monoester metabolites were confirmed in all samples. In addition, five previously unknown downstream metabolites were tentatively identified.

Discussion and conclusion: The excretion profiles obtained by HPLC-Q-Orbitrap-MS were in good agreement with quantitative HPLC-QqQ-MS data. For the newly discovered metabolites, plausible excretion profiles, similar to the ones of the known metabolites, were obtained. The presented approach proved to be successful for metabolite screening in urine samples after low-dose exposure and will be applied in future human metabolism studies for a fast, reliable and cost effective identification of specific biomarkers of exposure.     

The oxidative products of methionine as site and content biomarkers for peptide oxidation

Biomarkers for peptide/protein oxidation under oxidative stress (OS) hold both incredible application potential as well as significant challenges. In this article, liquid chromatography and mass spectrometry were applied to establish a new method for evaluating the oxidation site and degree of peptide oxidized, with its oxidative product serving as biomarker. In the three model peptides, peptide FMRF (containing a methionine) was prone to undergo oxygen addition under UV/H₂O₂ oxidization, forming a sulfoxide (FM(O)RF) with a stable chromatographic peak separate from the model peptides. The oxidation content of FMRF, expressed as S_FM(O)RF/(S_FM(O)RF + S_FMRF), is positively correlated with oxidation time. Based on sequence analysis of FM(O)RF, the oxidation mechanism (site and extent) of FMRF under UV/H₂O₂ oxidization was explicitly clarified. By comparing the specific injury to each model peptide, we found that the oxidative products of Met‐containing peptides are good biomarkers for OS. This research not only expands the range of biomarkers for OS, but also provides an efficient and accurate method for evaluating oxidation damage to peptides and even proteins. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Critical window of male reproductive tract development in rats following gestational exposure to di-n-butyl phthalate

BACKGROUND: Gestational exposure to di-n-butyl phthalate (DBP), a ubiquitous environmental contaminant, has been shown to interfere with the development of the male reproductive tract by acting as an antiandrogen. This study was conducted to identify the critical days for the abnormal development of the male reproductive tract, specifically the testis and epididymis. METHODS: Timed-pregnant Sprague-Dawley rats were dosed with DBP at 500 mg/kg/day on gestation day (GD) 14 and 15, 15 and 16, 16 and 17, 17 and 18, 18 and 19, or 19 and 20 (GD 0=plug day). Anogenital distance (AGD) was measured on postnatal day (PND) 1 and 13, while areloa number was recorded on PND 13 only. After weaning, males were allowed to mature to PND 90 at which time they were necropsied. Areloa number and AGD were recorded and testes, epididymides, seminal vesicles, prostate gland, kidneys, and liver weighed. Blood serum was collected and assayed for total testosterone concentration. RESULTS: There were no observable effects on litter size, sex ratio, serum testosterone concentration, or mortality of pups. Statistically significant permanent reductions in AGD were seen in males exposed prenatally to DBP on GD 15 and 16 or GD 18 and 19. On PND 13, areola were present in males exposed to DBP on GD 15 and 16, 16 and 17, 17 and 18, and 19 and 20. However, permanent retention occurred only in males after DBP exposure on GD 16 and 17. Exposure to DBP on only GD 17 and 18 elicited a reduction in epididymal weights; while exposure on only GD 16 and 17 caused a significant increase in the weights of the testes due to edema. In this study, epididymal and testicular malformations were most prevalent after exposure to DBP on any gestational day. Epididymal malformations, characterized by agenesis of various regions and small or flaccid testes were significantly increased in DBP-exposed males only on GD 16 and 17. CONCLUSIONS: These findings suggest that 2-day DBP exposure is highly detrimental to the developing reproductive tract of the male fetus and the critical window for abnormal development is GD 16-18.     

Duration of exposure to environmental carcinogens affects DNA-adduct level in human lymphocytes

Background and objective: An important issue in human biomonitoring is determining how exposure duration affects the kinetics of molecular biomarkers. In this study we compare the influence of exposure variables on DNA adducts.

Methods: DNA adducts were analysed by 32P-postlabelling in lympho/monocytes of 677 Caucasian subjects.

Results: After correction for other variables, DNA adducts increased depending on the length of occupational and smoke exposures. Higher DNA adducts were detected in workers with more than 14 years of exposure than in workers with shorter exposures (RR?=?1.19, p?=?0.049) and in smokers with more than 10 years of exposure than in smokers with shorter exposure (RR?=?1.21, p <0.001).

Conclusions: Exposure length is the primary factor affecting DNA-adduct level in lympho/monocytes both in smokers and in occupationally exposed subjects.     

Psychological stress increases bilirubin metabolites in human urine

Some authors have suggested that psychological stress induces the production of reactive oxygen species (ROS). Some studies have supported that bilirubin exerts anti-oxidative effects in vivo. However, it is not known whether ROS induced by psychological stress provoke bilirubin oxidation in vivo. We investigated if the concentration of bilirubin oxidative metabolite (BOM), a bilirubin oxidative metabolite, increased in urine from subjects exposed to psychological stress. Sixty healthy male volunteers working in a pharmaceutical company were divided into a Group I which did not attend a conference, a Group II which attended a conference but did not deliver a speech, and a Group III which attended a conference and delivered speeches in the presence of the company executives. Subjective stress was scored (self-rating score) after subjects in Group III delivered their speeches at the conference. Urine was collected on the next day. The BOM concentrations, as measured by enzyme-linked immunosorbent assay, were normalized to the urinary concentration of creatinine. The concentration of BOM in Group III was significantly higher compared to that in Groups I and II (p<0.01 and p<0.05, respectively). Furthermore, in Group III, the concentration of BOM correlated with the self-rating stress score (r=0.53, p<0.01). These findings suggest that emotional stimuli are associated with an increase in the oxidative metabolites of bilirubin in human urine, and that BOMs could be useful markers of psychological stress.     

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.     

Short review of selected fish biomarkers of xenobiotic exposure with an example using fish hepatic mixed-function oxidase

Abstract One group of biological tools that are useful for monitoring exposure to xenobiotics (and hence water quality) have been collectively referred to as biomarkers and are denned in this paper as any biochemical, histological and/or physiological alterations or manifestations of stress. Biomarkers within an aquatic toxicological context generally represent biological responses of individual organisms to xenobiotic exposure (i. e. responses at the whole organism level of biological organization). These include among others, enzyme alterations, bile metabolites, RNA/DNA ratio, adenylate energy charge, skeletal abnormalities, immune dysfunction, behavioural changes and histopathological lesions. Biomarkers can act as effective early warning sentinels to ensure the protection of the integrity of whole ecosystems, including freshwater and marine ecosystems. This paper briefly reviews a selection of fish biomarkers of xenobiotic chemical exposure and discusses their respective strengths and limitations for use in biomonitoring. An example of the application of fish mixed-function oxidase (MFO) and cytochrome P-450 as biomarkers of chemical exposure in Port Phillip Bay is provided. It is concluded that judicious application of biomarkers such as MFO in association with an understanding of the underlying causal mechanisms of induction and toxicity, will contribute to the successful prediction of biological effects of xenobiotic exposure on fish population health.     

Endocannabinoids effect on oxidative status of human platelets

Reactive oxygen species (ROS) are known to regulate platelet activation. Since endocannabinoids behave as platelet agonists, we investigated the effect of two endocannabinoids, 2-arachidonoylglycerol (2AG) and anandamide (AEA) on the oxidative status of human platelets. We have demonstrated that 2AG and AEA stimulate ROS production, superoxide anion formation and lipid peroxidation. The effect is dose and time dependent and mainly occurs through the involvement of cannabinoid receptor 1 (CB1) since all tested parameters are greatly reduced by SR141716, the CB1 specific inhibitor. The specific inhibitor of cannabinoid receptor 2 (CB2) SR144528 produces a very small inhibition. The involvement of syk/PI3K/AKT/mTor pathway in oxidative stress induced by endocannabinoids is shown. Nicotinamide adenine dinucleotide phosphate oxidase seems to be poorly involved in the endocannabinoids effect. Concerning the aerobic metabolism, it has been demonstrated that endocannabinoids reduce the oxygen consumption and adenosine triphosphate synthesis, both in the presence of pyruvate + malate or succinate. In addition, endocannabinoids inhibit the activity of respiratory complexes II, III and IV and increase the activity of respiratory complex I. The endocannabinoids effect on aerobic metabolism seems to be also a CB1 mediated mechanism. Thus, in human platelets oxidative stress induced by endocannabinoids, mainly generated in the respiratory chain through the activation of complex I and the inhibition of complex II, III and IV, may lead to thrombotic events, contributing to cardiovascular diseases.     

Development and application of oxidative stress biomarkers

Abstract

Oxidative stress may cause a wide variety of free radical reactions to produce deleterious modifications in membranes, proteins, enzymes, and DNA. Reactive Oxygen Species (ROS) generated by myeloperoxidase (MPO) can induce lipid peroxidation and also play an important role in the generation of reactive chlorinating and brominating species. As the universal biomarkers, chemical, and immunochemical approach on oxidatively modified and halogenated tyrosines has been carried out. As amido-type adduct biomarkers, chemical, and immunochemical evaluation of hexanoyl- and propanoyl-lysines, hexanoyl- and propanoyl-dopamines and phospholipids were prepared and developed for application of evaluation of novel antioxidative functional food factors. We have also involved in application of oxidatively modified DNAs such as 8-hydroxy- and 8-halogenated deoxyguanosines as the useful biomarkers for age-related diseases using both in vitro and in vivo systems. Application of these oxidative stress biomarkers for novel type of functional food development and recent approach for development of novel evaluation systems are also discussed.     

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.     

Mitochondrial oxidative stress elicits chromosomal instability after exposure to isocyanates in human kidney epithelial cells

The role of oxidative stress is often attributed in environmental renal diseases. Isocyanates, a ubiquitous chemical group with diverse industrial applications, are known to undergo bio-transformation reactions upon accidental and occupational exposure. This study delineates the role of isocyanate-mediated mitochondrial oxidative stress in eliciting chromosomal instability in cultured human kidney epithelial cells. Cells treated with 0.005 µM concentration of methyl isocyanate displayed morphological transformation and stress-induced senescence. Along the time course, an increase in DCF fluorescence indicative of oxidative stress, depletion of superoxide dismutase (SOD) and glutathione reductase (GR) and consistent accumulation of 8-oxo-dG were noticed. Thus, endogenous oxidative stress resulted in aberrant expression of p53, p21, cyclin E and CDK2 proteins, suggestive of deregulated cell cycle, chromosomal aberrations, centromeric amplification, aneuploidy and genomic instability.     

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.     

Immunochemical detection of oxidative DNA damage in cancer and aging using anti-reactive oxygen species modified DNA monoclonal antibody

The formation of reactive oxygen species (ROS), although a normal cellular activity, is considerably enhanced under chronic inflammatory conditions and ischemia. These species have been implicated in various disorders, mutagenesis, carcinogenesis and aging. Of many macromolecules, DNA is the most susceptible to hydroxyl radical, the most reactive of the ROS. The present study is designed to detect oxidative DNA damage in cancer patients and healthy aged humans using an anti-ROS-DNA monoclonal antibody (mAb). Purified calf thymus DNA fragments (approximate size 400 bp) were modified with ·OH, generated by UV-irradiation (254 nm) of hydrogen peroxide. ROS-modified DNA was characterized by UV-spectroscopy, melting temperature, alkaline sucrose density gradient ultracentrifugation and ion-exchange chromatography. ROS-DNA showed single strand breaks, decrease in T_m, modification of thymine (58.3%) and guanine (20%). The mAb generated against ROS-DNA was characterized for antigen binding specificity by competition ELISA. Monoclonal antibody showed strong binding to ROS-modified DNA, its modified fragments, polynucleotides and bases. With the exception of native DNA, binding of unmodified polynucleotides and bases was much lower. The mAb distinctly recognized DNA samples from lymphocytes of healthy aged humans and gave maximum inhibitions of 49, 53, 64 and 70%, while not reacting with DNA from young population. Similarly, oxidative lesions in DNA from cancer patients were also efficiently recognized by the mAb. DNA from healthy controls served as negative control. The studies demonstrate that the mAb, although cross-reactive, preferentially binds ROS-modified epitopes on DNA. High reactivity of mAb to DNA samples from cancer patients and healthy aged humans indicates increased oxidative stress leading to DNA damage.     

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.