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 β-chains, respectively. The ratio of HEVL/HEVH (1:3) reflected higher reactivity of EO with the β-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.
- MeSH
- biologické markery moč MeSH
- dipeptidy metabolismus moč MeSH
- erytrocyty MeSH
- ethylenoxid toxicita MeSH
- globiny metabolismus MeSH
- hydrolýza MeSH
- krysa rodu rattus MeSH
- leucin MeSH
- monitorování životního prostředí MeSH
- nebezpečné látky toxicita MeSH
- valin chemie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Publikační typ
- abstrakt z konference MeSH
- Publikační typ
- abstrakt z konference MeSH
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.
- MeSH
- biologické markery metabolismus moč MeSH
- epoxidové sloučeniny chemie metabolismus MeSH
- globiny metabolismus moč MeSH
- hydrolýza MeSH
- krysa rodu rattus MeSH
- látky znečišťující životní prostředí chemie metabolismus moč MeSH
- monitorování životního prostředí MeSH
- potkani Wistar MeSH
- xenobiotika MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
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.
- MeSH
- biologické markery metabolismus moč MeSH
- epoxidové sloučeniny metabolismus moč MeSH
- erytrocyty účinky léků metabolismus MeSH
- globiny metabolismus moč MeSH
- hydrolýza MeSH
- krysa rodu rattus MeSH
- potkani Wistar MeSH
- tandemová hmotnostní spektrometrie MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- xenobiotika metabolismus moč MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
- MeSH
- antioxidancia metabolismus MeSH
- chromatografie kapalinová MeSH
- glutathionreduktasa genetika MeSH
- hypoxie metabolismus MeSH
- krysa rodu rattus MeSH
- myokard metabolismus MeSH
- oxidační stres fyziologie MeSH
- peroxiredoxiny genetika MeSH
- reperfuzní poškození myokardu metabolismus MeSH
- superoxiddismutasa genetika MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Publikační typ
- abstrakt z konference MeSH
- Publikační typ
- abstrakt z konference MeSH
- Publikační typ
- abstrakt z konference MeSH
