Toxicokinetic relationship between the adducts in globin and their cleavage products in the urine: Implications for human biomonitoring.

Globin adducts of various chemicals, persisting in organism over the whole lifetime of erythrocytes, have been used as biomarkers of cumulative exposures to parent compounds. After removal of aged erythrocytes from the bloodstream, cleavage products of these adducts are excreted with urine as alternative, non-invasively accessible biomarkers. In our biomonitoring studies on workers exposed to ethylene oxide, its adduct with globin, N-(2-hydroxyethyl)valine, and the related urinary cleavage product N-(2-hydroxyethyl)-L-valyl-L-leucine have been determined. To describe a toxicokinetic relationship between the above types of biomarkers, a general compartmental model for simulation of formation and removal of globin adducts has been constructed in the form of code in R statistical computing environment. The essential input variables include lifetime of erythrocytes, extent of adduct formation following a single defined exposure, and parameters of exposure scenario, while other possible variables are optional. It was shown that both biomarkers reflect the past exposures differently as the adduct level in globin is a mean value of adduct levels across all compartments (subpopulations of erythrocytes of the same age) while excretion of cleavage products reflects the adduct level in the oldest compartment. Application of the model to various scenarios of continuous exposure demonstrated its usefulness for human biomonitoring data interpretation.

New Aminobiphenylcysteine Derivatives in Globin and Urine of Rats Dosed with 4-Aminobiphenyl, a Tobacco Smoke Carcinogen.

The 4-biphenylnitrenium ion (BPN), a reactive metabolic intermediate of the tobacco smoke carcinogen 4-aminobiphenyl (4-ABP), can react with nucleophilic sulfanyl groups in glutathione (GSH) as well as in proteins. The main site of attack of these S-nucleophiles was predicted using simple orientational rules of aromatic nucleophilic substitution. Thereafter, a series of presumptive 4-ABP metabolites and adducts with cysteine were synthesized, namely, S-(4-amino-3-biphenyl)cysteine (ABPC), N-acetyl-S-(4-amino-3-biphenyl)cysteine (4-amino-3-biphenylmercapturic acid, ABPMA), S-(4-acetamido-3-biphenyl)cysteine (AcABPC), and N-acetyl-S-(4-acetamido-3-biphenyl)cysteine (4-acetamido-3-biphenylmercapturic acid, AcABPMA). Then, globin and urine of rats dosed with a single ip dose of 4-ABP (27 mg/kg b.w.) was analyzed by HPLC-ESI-MS2. ABPC was identified in acid-hydrolyzed globin at levels of 3.52 ± 0.50, 2.74 ± 0.51, and 1.25 ± 0.12 nmol/g globin (mean ± S.D.; n = 6) on days 1, 3, and 8 after dosing, respectively. In the urine collected on day 1 (0-24 h) after dosing, excretion of ABPMA, AcABPMA, and AcABPC amounted to 1.97 ± 0.88, 3.09 ± 0.75, and 3.69 ± 1.49 nmol/kg b.w. (mean ± S.D.; n = 6), respectively. On day 2, excretion of the metabolites decreased by one order of magnitude followed by a slower decrease on day 8. Regarding the possible formation of AcABPC from ABPC, N-acetylation of the amino group at the biphenyl moiety prior to that at cysteine appears to be very unlikely. Thus, the structure of AcABPC indicates the involvement of N-acetyl-4-biphenylnitrenium ion (AcBPN) and/or its reactive ester precursors in in vivo reactions with GSH and protein-bound cysteine. ABPC in globin might become an alternative biomarker of the dose of toxicologically relevant metabolic intermediates of 4-ABP.

HPLC-ESI-HRMS2 Determination of N-(2-Hydroxyethyl)-l-valyl-l-leucine in Human Urine: Method Validation.

Biomonitoring of human exposure to reactive electrophilic chemicals such as ethylene oxide (EO) has been commonly based on the determination of adducts with N-terminal valine in blood protein globin, but a systematic search has also been undertaken to find surrogate markers enabling non-invasive sampling. Recently, N-(2-hydroxyethyl)-L-valyl-L-leucine (HEVL) has been identified as an ultimate cleavage product of EO-adducted globin in the urine of occupationally exposed workers. Herein, full validation of the analytical procedure consisting of solid-phase extraction of HEVL from urine samples (2 mL) followed by high-performance liquid chromatography-electrospray ionization-high-resolution mass spectrometry determination using deuterium-labeled HEVL as an internal standard (IS) is described. Method limit of quantitation is 0.25 ng/mL, and its selectivity is excellent as demonstrated by the invariable ratio of the qualifier and quantifier ion intensities across diverse urine samples and synthetic standard. The linear calibration model was applicable over the whole concentration range tested (0.25-10 ng/mL). The method accuracy assessed as a recovery of HEVL using a spiking experiment was 98-100%. Within-day precision of the method ranged from 1.8% to 3.0%, while the results from consecutive analytical runs conducted within 1 week or within 10-150 weeks differed in the range of 2.2-9.7%. The stability study on urine samples (-20°C up to 3 years, freeze-and-thaw up to 10 cycles) as well as on aqueous solutions (5°C up to 4 months) indicated no relevant changes in HEVL concentration (≤4%) over the time tested. Analytical responses of both HEVL and IS correlated with urinary creatinine as an index of matrix composition, but this matrix effect was mostly eliminated using the HEVL/IS peak area ratio, attaining the IS-normalized relative matrix effect <3%. In conclusion, the method complied successfully with the bioanalytical method validation criteria, making it a reliable tool for HEVL determination in human biomonitoring.

Health effects of exposure to isocyanates in a car factory.

OBJECTIVES: Isocyanates are known to induce occupational diseases. The aim of this work was to assess the health effects of exposure to isocyanates and to test the sensitivity of selected parameters for early detection of isocyanate-related allergic diseases. METHODS: In total, 35 employees from one factory were tested: 26 workers exposed to isocyanates (exposed group) and nine office workers (control group). All subjects filled in a questionnaire regarding possible health problems. Fractional exhaled nitric oxide (FeNO) and spirometry were measured for each subject at the same time during two consecutive working days. A urine sample was taken for a biological exposure test (BET). RESULTS: No significant difference was found between the exposed and control groups for spirometry parameters and FeNO. However, in the exposed group, FeNO was highly elevated (> 50 ppb) in five subjects (all reporting health problems at the workplace, all with normal spirometry and non-smokers). The BET revealed a significant difference (p < 0.001) between the exposed and control groups for 4,4´-methylenediphenyl diamine (MDA) in the urine. CONCLUSIONS: Our examination showed the usefulness of the BET in monitoring of workplace exposure to isocyanates and the importance of FeNO in monitoring of allergic inflammation of airways in non-smoking employees with normal spirometry.

Co-Exposure to Aristolochic Acids I and II Increases DNA Adduct Formation Responsible for Aristolochic Acid I-Mediated Carcinogenicity in Rats.

The plant extract aristolochic acid (AA), containing aristolochic acids I (AAI) and II (AAII) as major components, causes aristolochic acid nephropathy (AAN) and Balkan endemic nephropathy (BEN), unique renal diseases associated with upper urothelial cancer. Recently (Chemical Research in Toxicology 33(11), 2804-2818, 2020), we showed that the in vivo metabolism of AAI and AAII in Wistar rats is influenced by their co-exposure (i.e., AAI/AAII mixture). Using the same rat model, we investigated how exposure to the AAI/AAII mixture can influence AAI and AAII DNA adduct formation (i.e., AA-mediated genotoxicity). Using 32P-postlabelling, we found that AA-DNA adduct formation was increased in the livers and kidneys of rats treated with AAI/AAII mixture compared to rats treated with AAI or AAII alone. Measuring the activity of enzymes involved in AA metabolism, we showed that enhanced AA-DNA adduct formation might be caused partially by both decreased AAI detoxification as a result of hepatic CYP2C11 inhibition during treatment with AAI/AAII mixture and by hepatic or renal NQO1 induction, the key enzyme predominantly activating AA to DNA adducts. Moreover, our results indicate that AAII might act as an inhibitor of AAI detoxification in vivo. Consequently, higher amounts of AAI might remain in liver and kidney tissues, which can be reductively activated, resulting in enhanced AAI DNA adduct formation. Collectively, these results indicate that AAII present in the plant extract AA enhances the genotoxic properties of AAI (i.e., AAI DNA adduct formation). As patients suffering from AAN and BEN are always exposed to the plant extract (i.e., AAI/AAII mixture), our findings are crucial to better understanding host factors critical for AAN- and BEN-associated urothelial malignancy.

Ethyl chloroformate mediated gas chromatographic-mass spectrometric biomonitoring of acidic biomarkers of occupational exposure and endogenous metabolites in human urine.

Numerous industrial organic pollutants such as aromates, alkoxyalcohols, other organic solvents and monomers are absorbed, metabolized, and finally excreted in urine mostly as carboxylic acids that are determined as biomarkers of exposure. For a number of these xenometabolites, biological limits (levels of biomarkers in biological material) have been established to prevent damage to human health. Till now, most of the analytical procedures used have been optimized for one or a few analytes. Here, we report a more comprehensive approach enabling rapid GC-MS screening of sixteen acidic biomarkers in urine that are metabolized in the human body from several important industrial chemicals; benzene, toluene, styrene, xylenes, alkoxyalcohols, carbon disulfide, furfural and N,N-dimethylformamide. The new method involves immediate in situ derivatization - liquid liquid microextraction of urine by an ethyl chloroformate-ethanol-chloroform-pyridine medium and GC-MS analysis of the derivatized analytes in the lower organic phase. The xenometabolite set represents diverse chemical structures and some of hippuric and mercapturic acids also provided unusual derivatives that were unambiguously elucidated by means of new ethyl chloroformates labeled with stable isotopes and by synthesis of the missing reference standards. In the next step, an automated routine was developed for GC-MS/MS analysis using a MetaboAuto® sample preparation workstation and the new method was validated for fourteen metabolites over the relevant concentration range of each analyte in the spiked pooled human urine. It shows good linearity (R2 ≥ 0.982), accuracy (from 85% to 120%), precision (from 0.7% to 20%) and recovery (from 89% to 120%). The method performance was further successfully proved by GC-MS/MS analysis of the certified IP45 and RM6009 reference urines. Moreover, we show that the new method opens up the possibility for biomonitoring of combined and cumulative occupational exposures as well as for urinary metabolite profiling of persons exposed to harmful industrial chemicals.

Novel aminoarylcysteine adducts in globin of rats dosed with naphthylamine and nitronaphthalene isomers.

Novel aminonaphthylcysteine (ANC) adducts, formed via naphthylnitrenium ions and/or their metabolic precursors in the biotransformation of naphthylamines (NA) and nitronaphthalenes (NN), were identified and quantified in globin of rats dosed intraperitoneally with 0.16 mmol/kg b.w. of 1-NA, 1-NN, 2-NA and 2-NN. Using HPLC-ESI-MS2 analysis of the globin hydrolysates, S-(1-amino-2-naphthyl)cysteine (1A2NC) together with S-(4-amino-1-naphthyl)cysteine (4A1NC) were found in rats given 1-NA or 1-NN, and S-(2-amino-1-naphthyl)cysteine (2A1NC) in those given 2-NA or 2-NN. The highest level of ANC was produced by the most mutagenic and carcinogenic isomer 2-NA (35.8 ± 5.4 nmol/g globin). The ratio of ANC adduct levels for 1-NA, 1-NN, 2-NA and 2-NN was 1:2:100:3, respectively. Notably, the ratio of 1A2NC:4A1NC in globin of rats dosed with 1-NA and 1-NN differed significantly (2:98 versus 16:84 respectively), indicating differences in mechanism of the adduct formation. Moreover, aminonaphthylmercapturic acids, formed via conjugation of naphthylnitrenium ions and/or their metabolic precursors with glutathione, were identified in the rat urine. Their amounts excreted after dosing rats with 1-NA, 1-NN, 2-NA and 2-NN were in the ratio 1:100:40:2, respectively. For all four compounds tested, haemoglobin binding index for ANC was several-fold higher than that for the sulphinamide adducts, generated via nitrosoarene metabolites. Due to involvement of electrophilic intermediates in their formation, ANC adducts in globin may become toxicologically more relevant biomarkers of cumulative exposure to carcinogenic or non-carcinogenic arylamines and nitroarenes than the currently used sulphinamide adducts.

In Vivo Metabolism of Aristolochic Acid I and II in Rats Is Influenced by Their Coexposure.

The plant extract aristolochic acid (AA), containing aristolochic acid I (AAI) and II (AAII) as major components, causes aristolochic acid nephropathy and Balkan endemic nephropathy, unique renal diseases associated with upper urothelial cancer. Differences in the metabolic activation and detoxification of AAI and AAII and their effects on the metabolism of AAI/AAII mixture in the plant extract might be of great importance for an individual's susceptibility in the development of AA-mediated nephropathies and malignancies. Here, we investigated in vivo metabolism of AAI and AAII after ip administration to Wistar rats as individual compounds and as AAI/AAII mixture using high performance liquid chromatography/electrospray ionization mass spectrometry. Experimental findings were supported by theoretical calculations using density functional theory. We found that exposure to AAI/AAII mixture affected the generation of their oxidative and reductive metabolites formed during Phase I biotransformation and excreted in rat urine. Several Phase II metabolites of AAI and AAII found in the urine of exposed rats were also analyzed. Our results indicate that AAI is more efficiently metabolized in rats in vivo than AAII. Whereas AAI is predominantly oxidized during in vivo metabolism, its reduction is the minor metabolic pathway. In contrast, AAII is mainly metabolized by reduction. The oxidative reaction only occurs if aristolactam II, the major reductive metabolite of AAII, is enzymatically hydroxylated, forming aristolactam Ia. In AAI/AAII mixture, the metabolism of AAI and AAII is influenced by the presence of both AAs. For instance, the reductive metabolism of AAI is increased in the presence of AAII while the presence of AAI decreased the reductive metabolism of AAII. These results suggest that increased bioactivation of AAI in the presence of AAII also leads to increased AAI genotoxicity, which may critically impact AAI-mediated carcinogenesis. Future studies are needed to explain the underlying mechanism(s) for this phenomenon.

N-(2-Hydroxyethyl)-l-valyl-l-leucine: a novel urinary biomarker of ethylene oxide exposure in humans.

Ethylene oxide (EO), a carcinogenic chemical used as an industrial intermediate and sterilant, forms covalent adducts with DNA and proteins. The adduct with N-terminal valine [N-(2-hydroxyethyl)-l-valine, HEV] in blood protein globin has been employed as a principal biomarker of cumulative exposures to EO. However, as sampling of blood is inconvenient in routine occupational health practice, a non-invasive alternative to globin analysis has been investigated. Following identification of N-(2-hydroxyethyl)-l-valyl-l-leucine (HEVL) as ultimate cleavage product of EO-adducted globin excreted in the rat urine, here we report for the first time on the presence of HEVL in the urine of humans. In 18 sterilization workers, urinary HEVL ranged from 0.67 to 11.98 µg/g creatinine (mean ± SD: 5.04 ± 3.14 µg/g creat) and correlated with HEV: HEVL (µg/g creat) = 0.833 HEV (nmol/g globin) + 1.19 (R2 = 0.45). As unexpectedly high levels of urinary HEVL were found also in controls (mean ± SD: 0.97 ± 0.37 µg/g creat, n = 32), HEVL is not proposed for the accurate assessment of sub-ppm exposures to EO. On the other hand, non-invasive sampling and facile work-up procedure predetermine HEVL for screening purposes to identify subjects approaching to or exceeding occupational exposure limit for EO (1.8 mg/m3) to be re-examined by the more sensitive reference analysis for HEV.

N-(2-Hydroxyethyl)-L-valyl-L-leucine in rat urine as a hydrolytic cleavage product of ethylene oxide adduct with globin.

Ethylene oxide (EO), a genotoxic industrial chemical and sterilant, forms covalent adducts with DNA and also with nucleophilic amino acids in proteins. The adduct with N-terminal valine in globin [N-(2-hydroxyethyl)valine (HEV)] has been used in biomonitoring of cumulative exposures to EO. Here we studied in rats the fate of EO-adducted N-termini of globin after life termination of the erythrocytes. Rat erythrocytes were incubated with EO to produce the HEV levels in globin at 0.4-13.2 µmol/g as determined after acidic hydrolysis. Alternative hydrolysis of the isolated globin with enzyme pronase afforded N-(2-hydroxyethyl)-L-valyl-L-leucine (HEVL) and N-(2-hydroxyethyl)-L-valyl-L-histidine (HEVH), the EO-adducted N-terminal dipeptides of rat globin α- and ß-chains, respectively. The ratio of HEVL/HEVH (1:3) reflected higher reactivity of EO with the ß-chain. The EO-modified erythrocytes were then given intravenously to the recipient rats. HEVL and HEVH were found to be the ultimate cleavage products excreted in the rat urine. Finally, rats were dosed intraperitoneally with EO, 50 mg/kg. Herein, the initial level of globin-bound HEVL (11.7 ± 1.3 nmol/g) decreased almost linearly over 60 days corresponding to the life span of rat erythrocytes. Daily urinary excretion of HEVL was almost constant for 30-40 days, decreasing faster in the subsequent phase of elimination. Recoveries of the total urinary HEVL from its globin-bound form were 84 ± 6% and 101 ± 17% after administrations of EO and the EO-modified erythrocytes, respectively. In conclusion, urinary HEVL appears to be a promising novel non-invasive biomarker of human exposures to EO.

Determination of N-(2-hydroxyethyl)valine in globin of ethylene oxide-exposed workers using total acidic hydrolysis and HPLC-ESI-MS2.

Ethylene oxide (EO), an industrial intermediate and gaseous sterilant for medical devices, is carcinogenic to humans, which warrants minimization of exposure in the workplaces. The principal analytical strategy currently used in biomonitoring of exposure to EO consists in the conversion of N-(2-hydroxyethyl) adduct at the N-terminal valine (HEV) in globin to a specific thiohydantoin derivative accessible to GC-MS analysis (modified Edman degradation, MED). Though highly sensitive, the method is laborious and, at least in our hands, not sufficiently robust. Here we developed an alternative strategy of HEV determination based on acidic hydrolysis (AH) of globin followed directly by HPLC-ESI-MS2 analysis. Limit of quantitation is ca. 25 pmol HEV/g globin. Comparative analyses of globin samples from EO-exposed workers by both the AH-based and MED-based methods provided results that correlated well with each other (R2 > 0.95) but those obtained with AH were significantly more accurate (according to external quality control programme G-EQUAS) and repeatible (5% and 6% for intra-day and between-day analyses, respectively). In conclusion, the new AH-based method surpassed MED being similarly sensitive, much less laborious and more reliable, thus applicable as an effective tool for biomonitoring of EO in exposure control and risk assessment.

Urinary Cadmium and Cotinine Levels and Hair Mercury Levels in Czech Children and Their Mothers Within the Framework of the COPHES/DEMOCOPHES Projects.

The COPHES/DEMOCOPHES twin project was performed in 2011-2012 in 17 European countries to harmonize all steps of the human biomonitoring survey. Urinary cadmium, cotinine, phthalate metabolites, and hair mercury were measured in children (N = 120, 6-11 years) and their mothers of reproductive age, living in urban or rural areas. Cadmium in mothers' and children's urine was detected at a geometric mean (GM) concentration 0.227 and 0.109 µg/L, respectively; 95th percentile (P95) was 0.655 and 0.280 µg/L in mothers and children, respectively. No age-related, education-related, or urban versus rural differences were observed within the frame of each population group. Cadmium urinary level in mothers was about twofold compared with children. Higher levels were obtained in all smoking mothers but not in occasionally smoking or mothers and children exposed to environmental tobacco smoke (ETS). Mercury values in mothers were significantly higher in urban than in rural populations but not in children. GM and P95 for mercury in children's hair were 0.098 and 0.439 µg/g and in mothers' hair were 0.155 and 0.570 µg/g. Concentrations for mercury in the Czech samples were lower than European average. Hair mercury increased significantly with consumption of fish or seafood and with number of amalgam tooth fillings (in children). A positive association was found with family educational level. No influence of age was observed. Urinary cadmium and hair mercury levels were lower than health-based guidelines with one exception. High levels of urinary cotinine were found in the 12 smoking mothers (GM approximately 500 µg/L); lower levels in occasionally smoking mothers, N = 11 (34.5 µg/L). The mean cotinine levels in nonsmoking mothers who reported daily exposure to ETS was 10.7 µg/L. A similar mean value (10.8 µg/L) was obtained in six children who had daily exposure to ETS. In children without exposure to ETS, the mean cotinine level was 1.39 µg/L urine. Cotinine in the urine of children demonstrates limited protection of the Czech children against exposure to ETS.

S-(3-Aminobenzanthron-2-yl)cysteine in the globin of rats as a novel type of adduct and possible biomarker of exposure to 3-nitrobenzanthrone, a potent environmental carcinogen.

3-Nitrobenzanthrone (3-NBA), a potent environmental mutagen and carcinogen, is known to be activated in vivo to 3-benzanthronylnitrenium ion which forms both NH and C2-bound adducts with DNA and also reacts with glutathione giving rise to urinary 3-aminobenzanthron-2-ylmercapturic acid. In this study, acid hydrolysate of globin from rats dosed intraperitoneally with 3-NBA was analysed by HPLC/MS to identify a novel type of cysteine adduct, 3-aminobenzanthron-2-ylcysteine (3-ABA-Cys), confirmed using a synthesised standard. The 3-ABA-Cys levels in globin peaked after single 3-NBA doses of 1 and 2 mg/kg on day 2 to attain 0.25 and 0.49 nmol/g globin, respectively, thereafter declining slowly to 70-80% of their maximum values during 15 days. After dosing rats for three consecutive days with 1 mg 3-NBA/kg a significant cumulation of 3-ABA-Cys in globin was observed. 3-ABA-Cys was also found in the plasma hydrolysate. Herein, after dosing with 1 and 2 mg 3-NBA/kg the adduct levels peaked on day 1 at 0.15 and 0.51 nmol/ml plasma, respectively, thereafter declining rapidly to undetectable levels on day 15. In addition, sulphinamide adducts were also found in the exposed rats, measured indirectly as 3-aminobenzanthrone (3-ABA) split off from globin by mild acid hydrolysis. Levels of both types of adducts in the globin samples parallelled very well with 3-ABA/3-ABA-Cys ratio being around 1:8. In conclusion, 3-ABA-Cys is the first example of arylnitrenium-cysteine adduct in globin representing a new promising class of biomarkers to assess cumulative exposures to aromatic amines, nitroaromatics and heteroaromatic amines.

Biological fate of styrene oxide adducts with globin: Elimination of cleavage products in the rat urine.

The in vivo fate of globin adducts with styrene 7,8-oxide (SO), an electrophilic metabolic intermediate of styrene, was studied in male Wistar rats dosed intraperitoneally with racemic SO, 100mg/kg b.w. Regioisomeric hydroxy(phenyl)ethyl (HPE) adducts at Cys, N-terminal Val, Lys and His in globin were determined and their elimination from blood was followed during 60days, corresponding to life span of rat erythrocytes. In the rat urine, Nα-acetylated products of hydrolytic cleavage of the HPE adducts with Cys, Lys and His were determined. On the first day post-exposure, abundant Nα-acetyl-HPE-Cys adducts (mercapturic acids) formed via direct conjugation of SO with hepatic glutathione were excreted rapidly, but then a much slower phase of elimination reflecting formation of Nα-acetyl-HPE-Cys via cleavage of the adducted globin was observed. A two-phase elimination occurred also in urinary Nα-acetyl-HPE adducts with His and Lys. While a decline by 75-85% during the first 7days post-exposure most likely reflected elimination of adducted albumin, the subsequent slow decline until day 60 corresponded to elimination kinetics of the adducted globin. Thus, the study not only provided original data on the fate of SO-globin adducts but also allowed to reveal general toxicokinetics properties of the urinary cleavage products as a novel type of chemical exposure biomarkers.

Hydrolytic Cleavage Products of Globin Adducts in Urine as Possible Biomarkers of Cumulative Dose: Proof of Concept Using Styrene Oxide as a Model Adduct-Forming Compound.

A new experimental model was designed to study the fate of globin adducts with styrene 7,8-oxide (SO), a metabolic intermediate of styrene and a model electrophilic compound. Rat erythrocytes were incubated with SO at 7 or 22 °C. Levels of specific amino acid adducts in globin were determined by LC/MS analysis of the globin hydrolysate, and erythrocytes with known adduct content were administered intravenously to recipient rats. The course of adduct elimination from the rat blood was measured over the following 50 days. In the erythrocytes incubated at 22 °C, a rapid decline in the adduct levels on the first day post-transfusion followed by a slow phase of elimination was observed. In contrast, the adduct elimination in erythrocytes incubated at 7 °C was nearly linear, copying elimination of intact erythrocytes. In the urine of recipient rats, regioisomeric SO adducts at cysteine, valine, lysine, and histidine in the form of amino acid adducts and/or their acetylated metabolites as well as SO-dipeptide adducts were identified by LC/MS supported by synthesized reference standards. S-(2-Hydroxy-1-phenylethyl)cysteine and S-(2-hydroxy-2-phenylethyl)cysteine, the most abundant globin adducts, were excreted predominantly in the form of the corresponding urinary mercapturic acids (HPEMAs). Massive elimination of HPEMAs via urine occurred within the first day from the erythrocytes incubated at both 7 and 22 °C. However, erythrocytes incubated at 7 °C also showed a slow second phase of elimination such that HPEMAs were detected in urine up to 50 days post-transfusion. These results indicate for the first time that globin adducts can be cleaved in vivo to modified amino acids and dipeptides. The cleavage products and/or their predictable metabolites are excreted in urine over the whole life span of erythrocytes. Some of the urinary adducts may represent a new type of noninvasive biomarker for exposure to adduct-forming chemicals.

Cardioprotective and nonprotective regimens of chronic hypoxia diversely affect the myocardial antioxidant systems.

It has been documented that adaptation to hypoxia increases myocardial tolerance to ischemia-reperfusion (I/R) injury depending on the regimen of adaptation. Reactive oxygen species (ROS) formed during hypoxia play an important role in the induction of protective cardiac phenotype. On the other hand, the excess of ROS can contribute to tissue damage caused by I/R. Here we investigated the relationship between myocardial tolerance to I/R injury and transcription activity of major antioxidant genes, transcription factors, and oxidative stress in three different regimens of chronic hypoxia. Adult male Wistar rats were exposed to continuous normobaric hypoxia (FiO2 0.1) either continuously (CNH) or intermittently for 8 h/day (INH8) or 23 h/day (INH23) for 3 wk period. A control group was kept in room air. Myocardial infarct size was assessed in anesthetized open-chest animals subjected to 20 min coronary artery occlusion and 3 h reperfusion. Levels of mRNA transcripts and the ratio of reduced and oxidized glutathione (GSH/GSSG) were analyzed by real-time RT-PCR and by liquid chromatography, respectively. Whereas CNH as well as INH8 decreased infarct size, 1 h daily reoxygenation (INH23) abolished the cardioprotective effect and decreased GSH/GSSG ratio. The majority of mRNAs of antioxidant genes related to mitochondrial antioxidant defense (manganese superoxide dismutase, glutathione reductase, thioredoxin/thioredoxin reductase, and peroxiredoxin 2) were upregulated in both cardioprotective regimens (CNH, INH8). In contrast, INH23 increased only PRX5, which was not sufficient to induce the cardioprotective phenotype. Our results suggest that the increased mitochondrial antioxidant defense plays an important role in cardioprotection afforded by chronic hypoxia.

3-(3,4-Dihydroxyphenyl)adenine, a urinary DNA adduct formed in mice exposed to high concentrations of benzene.

Metabolism of benzene, an important environmental and industrial carcinogen, produces three electrophilic intermediates, namely, benzene oxide and 1,2- and 1,4-benzoquinone, capable of reacting with the DNA. Numerous DNA adducts formed by these metabolites in vitro have been reported in the literature, but only one of them was hitherto identified in vivo. In a search for urinary DNA adducts, specific LC-ESI-MS methods have been developed for the determination in urine of six nucleobase adducts, namely, 7-phenylguanine, 3-phenyladenine, 3-hydroxy-3,N(4) -benzethenocytosine, N(2) -(4-hydroxyphenyl)guanine, 7-(3,4-dihydroxyphenyl)guanine and 3-(3,4-dihydroxyphenyl)-adenine (DHPA), with detection limits of 200, 10, 260, 50, 400 and 200 pg ml(-1) , respectively. Mice were exposed to benzene vapors at concentrations of 900 and 1800 mg m(-3) , 6 h per day for 15 consecutive days. The only adduct detected in their urine was DHPA. It was found in eight out of 30 urine samples from the high-exposure group at concentrations of 352 ± 146 pg ml(-1) (mean ± SD; n = 8), whereas urines from the low-exposure group were negative. Assuming the DHPA concentration in the negative samples to be half of the detection limit, conversion of benzene to DHPA was estimated to 2.2 × 10(-6) % of the absorbed dose. Thus, despite the known high mutagenic and carcinogenic potential of benzene, only traces of a single DNA adduct in urine were detected. In conclusion, DHPA is an easily depurinating adduct, thus allowing indication of only high recent exposure to benzene, but not long-term damage to DNA in tissues.

Protective effects of dexrazoxane against acute ischaemia/reperfusion injury of rat hearts.

Dexrazoxane (DEX), an inhibitor of topoisomerase II and intracellular iron chelator, is believed to reduce the formation of reactive oxygen species (ROS) and protects the heart from the toxicity of anthracycline antineoplastics. As ROS also play a role in the pathogenesis of cardiac ischaemia/reperfusion (I/R) injury, the aim was to find out whether DEX can improve cardiac ischaemic tolerance. DEX in a dose of 50, 150, or 450 mg·(kg body mass)(-1) was administered intravenously to rats 60 min before ischaemia. Myocardial infarct size and ventricular arrhythmias were assessed in anaesthetized open-chest animals subjected to 20 min coronary artery occlusion and 3 h reperfusion. Arrhythmias induced by I/R were also assessed in isolated perfused hearts. Only the highest dose of DEX significantly reduced infarct size from 53.9% ± 4.7% of the area at risk in controls to 37.5% ± 4.3% without affecting the myocardial markers of oxidative stress. On the other hand, the significant protective effect against reperfusion arrhythmias occurred only in perfused hearts with the dose of DEX of 150 mg·kg(-1), which also tended to limit the incidence of ischaemic arrhythmias. It is concluded that DEX in a narrow dose range can suppress arrhythmias in isolated hearts subjected to I/R, while a higher dose is needed to limit myocardial infarct size in open-chest rats.