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.
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.
In February–March, 2023, a Working Group of 20 scientists from 10 countries met at the invitation of the International Agency for Research on Cancer (IARC) in Lyon, France, to finalise their ...evaluation of the carcinogenicity of four agents: anthracene, 2-bromopropane, butyl methacrylate (BMA), and dimethyl hydrogen phosphite (DMHP). Multiple studies showed that 2-bromopropane induced DNA damage in human leukocytes, micronucleus formation in rodents, and gene mutations in bacteria. Monograph Working Group Members RC Cattley (USA)–Meeting Chair; H Kromhout (The Netherlands); M Sun (USA); EJ Tokar (USA) –Subgroup Meeting Chairs; MA-E Abdallah (UK); AK Bauer (USA); KR Broadwater (USA); L Campo (Italy); E Corsini (Italy); KA Houck (USA); G Ichihara (Japan); M Matsumoto (Japan); S Morais (Portugal); J Mráz (Czechia); T Nomiyama (Japan); K Ryan (USA); H Shen (People's Republic of China); T Toyoda (Japan); KH Vähäkangas (Finland); MG Yakubovskaya (Russian Federation) Declaration of interests All Working Group members declare no competing interests Invited Specialists IJ Je Yu, Hyundai Calibration & Certification Technologies Co Ltd, Republic of Korea Declaration of interests IJY reports being an employee of HCT Co., Ltd., a former spin-off of Hyundai Electronics, whose work may involve competing interests for the topics covered at this IARC Monographs meeting Representatives C Dellavalle, National Cancer Institute, USA Declaration of interests CD declares no competing interest Observers G Tuschl, Methacrylate REACH Task Force (MRTF), Germany Declaration of interests GT is employed with Rohm GmbH, manufacturer of butyl methacrylate.
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.
•Compartmental model to simulate formation and elimination of adducts with globin.•Toxicokinetics of the adducts in globin and their cleavage products in the urine.•Non-invasive biomonitoring to assess long-term exposures to electrophilic chemicals.
Dihydromyricetin (DHM) is a natural flavonoid showing several health promoting effects such as protective activity during severe alcohol intoxication. The mechanism underlying the effects of DHM on ...alcohol metabolism is virtually unknown. The present paper is focused on clarifying the role of DHM in the liver alcohol elimination at its molecular level. First, impact of DHM on alcohol dehydrogenase (ADH) activity in vitro and the enzyme induction in vivo was examined. Neither the ADH activity nor the enzyme expression were influenced by DHM. Next, the effect of DHM during alcohol intoxication were studied on primary hepatocytes isolated from EtOH-premedicated and untreated rats. The viability of cells exposed to alcohol, estimated based on the released enzymes, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), was slightly affected by DHM. Although the expected hepatoprotective effect of DHM was not fully achieved, DHM (in a concentration manner) proved to reduce the level of ROS/RNS in hepatocytes. However, no change in the rate of alcohol metabolism in vivo was found when rats were administered with a single or repeated dose of ethanol supplemented with DHM. In conclusion, the proposed positive effect of DHM during alcohol intoxication has not been proven. Moreover, there is no effect of DHM on the alcohol metabolism. The "hoped-for" DHM hepatoprotective activity can be attributed to the reduction of ROS/RNS levels in cells.
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.
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.
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-MS
2
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.
A simple environmentally friendly one-step synthetic procedure was developed for S- and N-methylcarbamoylation of amino acids and their derivatives in buffered aqueous solutions. N-Succinimidyl ...N-methylcarbamate (SNMC) and N,S-dimethylthiocarbamate (DMTC) were used as synthetic equivalents to replace highly hazardous methyl isocyanate (MIC). SNMC reacted rapidly in both S- and N-methylcarbamoylations affording nearly quantitative conversions (>97 %) of all tested compounds after 2-3 h at 50 °C at pH 8.2 and 9.5-10 for S- and N-methylcarbamoylations, respectively. Under the same conditions, DMTC reacted more slowly (48 h for N-methylcarbamoylations) but with some polar amino acids it provided products of higher purity than SNMC. Similarly, N-trideuteriomethyl-S-methylthiocarbamate (DMTC-d
3
) was used to synthesize N
α
- and N
ε
-trideuteriomethylcarbamoyl derivatives of valine and lysine, respectively. Prepared compounds will be used in toxicological research as authentic standards in the analyses for protein adducts derived from MIC or its metabolic precursor, N,N-dimethylformamide.
•The ECF mediated derivatization - LLME for the comprehensive biomarker coverage.•The 13C2 and D3 ECF labeling for the definite structural elucidation of products.•The automated GC-MS/MS method ...examined on certified urine materials.•Biomonitoring of combined and cumulative exposures to several industrial solvents.•Simultaneous screening of biomarkers and protic endogenous metabolites in 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.
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