Showing papers in "Chemical Research in Toxicology in 2007"

Mechanistic applicability domain classification of a local lymph node assay dataset for skin sensitization.

Abstract: The goal of eliminating animal testing in the predictive identification of chemicals with the intrinsic ability to cause skin sensitization is an important target, the attainment of which has recently been brought into even sharper relief by the EU Cosmetics Directive and the requirements of the REACH legislation. Development of alternative methods requires that the chemicals used to evaluate and validate novel approaches comprise not only confirmed skin sensitizers and non-sensitizers but also substances that span the full chemical mechanistic spectrum associated with skin sensitization. To this end, a recently published database of more than 200 chemicals tested in the mouse local lymph node assay (LLNA) has been examined in relation to various chemical reaction mechanistic domains known to be associated with sensitization. It is demonstrated here that the dataset does cover the main reaction mechanistic domains. In addition, it is shown that assignment to a reaction mechanistic domain is a critical first step in a strategic approach to understanding, ultimately on a quantitative basis, how chemical properties influence the potency of skin sensitizing chemicals. This understanding is necessary if reliable non-animal approaches, including (quantitative) structure-activity relationships (Q)SARs, read-across, and experimental chemistry based models, are to be developed.

Toxicity of luminescent silica nanoparticles to living cells.

Abstract: Luminescent nanomaterials can provide high-intensity and photostable luminescent signals when used as labeling materials for the determination of trace amounts of analytes. However, a major concern that has arisen is whether the nanomaterials cause toxic effects in living systems. Here, we address this problem through a systematic investigation of the cytotoxicity and genotoxicity of luminescent silica nanoparticles. These nanoparticles are intensely luminescent labeling materials for ultrasensitive determination of biological samples. The investigation of genotoxicity of the nanomaterials was carried out from two perspectives. First, the integrity of the DNA was examined by detecting DNA base modification, strand breaks, and increased DNA repair activity to recover the damage. Second, different sets of cellular DNAs, including nuclear DNA extracts and the whole genomic DNAs, were examined. Furthermore, to fully assess DNA damage by the nanoparticles, isolated genomic DNAs were directly exposed to the nanoparticles. The cytotoxicity of the nanoparticle was detected by measuring the cell proliferation rate, cell death, and death patterns (necrosis and apoptosis) after the nanoparticle treatments. Results show no significant toxic effects due to the luminescent nanoparticles at the molecular and cellular levels below a concentration of 0.1 mg/mL. Our study indicates that the luminescent silica nanoparticle is a promising labeling reagent for various biomedical applications.

Possible Controversy Over Dietary Polyphenols: Benefits vs Risks

Abstract: Dietary phytochemicals including flavonoids, polyunsaturated fatty acids, tocopherols, and others have been the subject of increasing amounts of research for their potential beneficial effects as both disease preventive and therapeutic agents ( 1, 2). For example, the major polyphenolic compounds in green tea (Camellia sinensis , Theaceae), the catechins, have been extensively studied for their cancer preventive activity and have shown activity in a number of animal models of carcinogenesis at organ sites including the lung, prostate, oral cavity, intestine, colon, liver, and skin ( 3). A number of mechanisms have been proposed to account for the disease preventive effects of dietary polyphenols; antioxidative and free radical scavenging activities are often cited ( 4, 5). Indeed, these compounds have strong antioxidant activities in vitro. Such effects, however, have been harder to demonstrate in vivo and to correlate with disease prevention in vivo. Whereas diet-derived compounds are generally regarded as safe based on their long history of use in the diet and/or as traditional medicines, it is becoming increasingly apparent that these compounds could have deleterious effects (i) at pharmacological concentrations, (ii) in certain vulnerable populations, and (iii) in certain disease or polypharmaceutical contexts ( 6). Herein, we will review the available data on the toxic potential of polyphenols as a prototypical class of dietary phytochemicals. As specific examples, we will discuss the prooxidative vs antioxidant potentials of tea catechins, the hepatic and intestinal toxicities of high doses of tea catechins, and the potential DNA damaging effects and leukemiogenic activities of flavonoids. We interpret the results of these studies with a consideration of the factors governing the bioavailability of the test compounds. We feel that the bioavailability will ultimately govern the occurrence of toxic events in vivo. More in-depth studies on the potential adverse effects of dietary phytochemicals are required in order to assess the potential toxicities and to determine their potential usefulness as disease preventive and treatment agents. Ongoing human intervention studies should include protocols to assess potential adverse effects including hepatotoxicity.

Applying mechanisms of chemical toxicity to predict drug safety.

Abstract: Toxicology can no longer be used only as a science that reacts to problems but must be more proactive in predicting potential human safety issues with new drug candidates. Success in this area must be based on an understanding of the mechanisms of toxicity. This review summarizes and extends some of the concepts of an American Chemical Society ProSpectives meeting on the title subject held in June 2006. One important area is the discernment of the exact nature of the most common problems in drug toxicity. Knowledge of chemical structure alerts and relevant biological pathways are important. Biological activation to reactive products and off-target pharmacology are considered to be major contexts of drug toxicity, although defining exactly what the contributions are is not trivial. Some newer approaches to screening for both have been developed. A goal in predictive toxicology is the use of in vitro methods and database development to make predictions concerning potential modes of toxicity and to stratify drug candidates for further development. Such predictions are desirable for several economic and other reasons but are certainly not routine yet. However, progress has been made using several approaches. Some examples of the application of studies of wide-scale biological responses are now available, with incorporation into development paradigms.

Tea polyphenol (-)-epigallocatechin-3-gallate: a new trapping agent of reactive dicarbonyl species.

Abstract: Previous studies have demonstrated that reactive dicarbonyl compounds [e.g., methylglyoxal (MGO) and glyoxal (GO)] irreversibly and progressively modify proteins over time and yield advanced glycation end products (AGEs), which are thought to contribute to the development of diabetes mellitus and its subsequent complications. Thus, decreasing the levels of MGO and GO will be an effective approach to reduce the formation of AGEs and the development of diabetic complications. In our studies to find nontoxic trapping agents of reactive dicarbonyl species from dietary sources, we found that (-)-epigallocatechin-3-gallate (EGCG), the major bioactive green tea polyphenol, could efficiently trap reactive dicarbonyl compounds (MGO or GO) to form mono- and di-MGO or GO adducts under physiological conditions (pH 7.4, 37 degrees C). The products formed from EGCG and MGO (or GO), combined at different ratios, were analyzed using LC/MS. We also developed a method to purify the two major mono-MGO adducts of EGCG without derivatization, and their structures were identified as stereoisomers of mono-MGO adducts of EGCG based on their 1D and 2D NMR spectra. Our LC/MS and NMR data showed that positions 6 and 8 of the EGCG A-ring were the major active sites for trapping reactive dicarbonyl compounds. We also found that EGCG lost its trapping efficacy under acidic conditions (pH

Aryl hydrocarbon receptor-mediated effects of chlorinated polycyclic aromatic hydrocarbons

Abstract: Chlorinated polycyclic aromatic hydrocarbons (ClPAHs) with 3–5 rings are ubiquitous environmental contaminants. However, toxicities of ClPAHs remain unclear. In this study, aryl hydrocarbon receptor (AhR)-mediated activities of ClPAHs were investigated by using a yeast assay system. All environmentally relevant 18 ClPAHs showed the AhR activities in the test; the activities were elevated with the number of chlorine atoms on the lower molecular weight PAH (∼three-ring and fluoranthene derivatives) but not for higher molecular weight ClPAHs (>four-ring). The similar trends were also observed in certain ClPAHs-induced cytochrome P450 1A1 expression in MCF-7 cells. The structure–activity relationship between the AhR activity and the corresponding solvent accessible surface area of ClPAHs revealed a parabolic relationship, with approximately 350 A2/molecule as the optimal dimensions as the ligand for binding to AhR. These findings indicate that the spatial dimensions of ClPAHs apparently influence their abilit...

Quantitation of acrolein-derived (3-hydroxypropyl)mercapturic acid in human urine by liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry: effects of cigarette smoking.

Abstract: Recently published data suggest that acrolein (1), a toxic but weakly carcinogenic constituent of cigarette smoke, may be involved as a causative factor for the mutations frequently observed in the p53 tumor suppressor gene in lung cancer in smokers. Biomarkers are needed to further assess the possible relationship between acrolein uptake and cancer. In this study, we analyzed (3-hydroxypropyl)mercapturic acid (3-HPMA, 2) in human urine. 3-HPMA is a major metabolite of acrolein in laboratory animals. The method employs [13C3]3-HPMA as an internal standard, with analysis and quantitation by LC-APCI-MS/MS-SRM. Clean, readily quantifiable chromatograms were obtained. The method was accurate and precise and required only 0.1 mL of urine. Median levels of 3-HPMA were significantly higher (2900 pmol/mg of creatinine, N=35) in smokers than in nonsmokers (683 pmol/mg of creatinine, N=21) (P=0.0002). The effect of smoking was further assessed by determining the levels of 3-HPMA before and after a 4 week smoking cessation period. There was a significant 78% decrease in median levels of urinary 3-HPMA after cessation (P<0.0001). The relationship between the levels of urinary 3-HPMA and those of acrolein-derived 1,N2-propanodeoxyguanosine (PdG) adducts in lung was investigated in 14 smokers. There was a significant inverse relationship between urinary 3-HPMA and alpha-hydroxy-PdG (3) but not gamma-hydroxy-PdG (4) or total adduct levels. The results of this study clearly demonstrate that acrolein uptake in smokers is significantly higher than in nonsmokers and underline the need for further investigation of the possible relationship of acrolein uptake to lung cancer.

Electrophilic chemistry related to skin sensitization. Reaction mechanistic applicability domain classification for a published data set of 106 chemicals tested in the mouse local lymph node assay.

Abstract: This article presents an overview of electrophilic reaction mechanisms relevant to skin sensitization, with reference to a published skin sensitization test data set for 106 chemicals. Where appropriate to aid the interpretation, additional data on a small number of further compounds are also discussed. It is shown that there is a close correspondence in the way differences and similarities in skin sensitization potency of chemicals relate to differences and similarities in their physical organic chemistry and electrophilic reaction mechanistic chemistry. The 106 chemicals are classified into their reaction mechanistic applicability domains, and reactivity−sensitization trends are analyzed for each domain: the Michael acceptor and pro-Michael acceptor electrophile domain; the SNAr electrophile domain; the SN2 electrophile domain; the Schiff base electrophile domain; the acyl transfer electrophile domain; and the non-electrophilic non-pro-electrophilic domain. The last of these domains should be populated...

Mercurial-induced hydrogen peroxide generation in mouse brain mitochondria: protective effects of quercetin.

Abstract: Plants of the genus Polygala have been shown to possess protective effects against neuronal death and cognitive impairments in neurodegenerative disorders related to excitotoxicity. Moreover, previous reports from our group have shown the neuroprotective effects of the plant Polygala paniculata against methylmercury (MeHg)-induced neurotoxicity. In this work, we have examined the potential protective effects of three compounds (7-prenyloxy-6-methoxycoumarin, quercetin, and 1,5-dihidroxi-2,3-dimethoxy xanthone) from Polygala species against MeHg- and mercuric chloride (HgCl2)-induced disruption of mitochondrial function under in vitro conditions using mitochondrial-enriched fractions from mouse brain. MeHg and HgCl2 (10–100 µM) significantly decreased mitochondrial viability; this phenomenon was positively correlated to mercurial-induced glutathione oxidation. Among the isolated compounds, only quercetin (100–300 µM) prevented mercurial-induced disruption of mitochondrial viability. Moreover, quercetin, wh...

Fragrance compound geraniol forms contact allergens on air exposure. Identification and quantification of oxidation products and effect on skin sensitization.

Abstract: Fragrances are common causes of contact allergy Geraniol (trans-3,7-dimethyl-2,6-octadiene-1-ol) is an important fragrance terpene It is considered a weak contact allergen and is used for fragrance allergy screening among consecutive dermatitis patients Analogous to other monoterpenes studied, such as limonene and linalool, geraniol has the potential to autoxidize on air exposure and form highly allergenic compounds The aim of the present study was to investigate and propose a mechanism for the autoxidation of geraniol at room temperature To investigate whether allergenic compounds are formed, the sensitizing potency of geraniol itself, air-exposed geraniol, and its oxidation products was determined using the local lymph node assay in mice The results obtained show that the allylic alcohol geraniol follows an oxidation pattern different from those of linalool and limonene, which autoxidize forming hydroperoxides as the only primary oxidation products The autoxidation of geraniol follows two paths, originating from allylic hydrogen abstraction near the two double bonds From geraniol, hydrogen peroxide is primarily formed together with aldehydes geranial and neral from a hydroxyhydroperoxide In addition, small amounts of a hydroperoxide are formed, analogous to the formation of the major linalool hydroperoxide The autoxidation of geraniol greatly influenced the sensitizing effect of geraniol The oxidized samples had moderate sensitizing capacity, quite different from that of pure geraniol The hydroperoxide formed is believed to be the major contributor to allergenic activity, together with the aldehydes geranial and neral On the basis of the present study and previous experience, we recommend that the possibility of autoxidation and the subsequent formation of contact allergenic oxidation products are considered in risk assessments performed on fragrance terpenes

Failure of MTT as a Toxicity Testing Agent for Mesoporous Silicon Microparticles

Abstract: In this work, it is shown that the common toxicity indicator, MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide), will fail to predict the toxicity of porous silicon (PSi) microparticles. This is due to the spontaneous redox reactions where the MTT is reduced and the PSi particle surfaces are oxidized simultaneously. MTT was shown to even react with thermally oxidized and carbonized forms of PSi particles, although the treatment did give an enhanced protection against the unwanted reactions as compared to as-anodized PSi particles. The observed levels of cellular viability with the MTT assay were much higher than expected in the presence of Caco-2 cells, even considering the spontaneous reduction of MTT at PSi surfaces. The results indicate that the redox reaction is further enhanced inside living cells. Thus, we recommend that MTT should not be used to test the cytotoxicity of drug formulations containing PSi microparticles. The study also shows that since PSi particles are capable of reducing the MTT, they will also be able to reduce other species as well. This should be taken into account when considering future applications for the porous silicon particles. The completely oxidized SiO2 particles (MCM-41 and SBA-15) were shown to work as expected with the MTT assay and showed no inherent oxidation/reduction.

Prediction of in vivo potential for metabolic activation of drugs into chemically reactive intermediate: correlation of in vitro and in vivo generation of reactive intermediates and in vitro glutathione conjugate formation in rats and humans.

Abstract: The covalent binding of reactive intermediates to macromolecules might have potential involvement in severe adverse drug reactions. Thus, quantification of reactive metabolites is necessary during the early stage of drug discovery to avoid serious toxicity. In this study, the relationship between covalent binding and glutathione (GSH) conjugate formation in rat and human liver microsomes were investigated using 10 representative radioactive compounds that have been reported as hepatotoxic or having other toxicity derived from their reactive intermediates: acetaminophen, amodiaquine, carbamazepine, clozapine, diclofenac, furosemide, imipramine, indomethacin, isoniazid, and tienilic acid, all at a concentration of 10 microM. The GSH conjugate formation rate correlates well with the covalent binding of radioactivity (both rat and human, r2 = 0.93), which suggests that quantification of the GSH conjugate can be used to estimate covalent binding. To quantify the GSH-conjugation rate with non-radiolabeled compounds in vitro, the validation study for the determination of GSH conjugate formation using 35S-GSH by radio-HPLC was useful to predict metabolic activation. Following oral administration of 20 mg/kg of the radiolabeled compounds to rats, radioactivity that covalently bound to plasma and liver proteins was determined. The in vivo maximum covalent binding level in liver based on the free fraction of plasma area under the concentration curve (AUC) and in vitro covalent binding rate was found to correlate well (r2 = 0.79). Therefore, this model for in vitro covalent binding studies in human and rat and in vivo rat studies might be useful in predicting human metabolic activation of compounds.

Future of toxicology--iron chelators and differing modes of action and toxicity: the changing face of iron chelation therapy.

Abstract: Iron (Fe) chelation therapy was initially designed to alleviate the toxic effects of excess Fe evident in Fe-overload diseases. However, the novel toxicological properties of some Fe chelator-metal complexes have shifted appreciable focus to their application in cancer chemotherapy. Redox-inactive Fe chelator complexes are well suited for the treatment of Fe-overload diseases, whereas Fe chelator complexes with high redox activity have shown promising results as chemotherapeutics against cancer. Within this perspective, we discuss the different modes of action and toxicological profiles of Fe chelators, including analogues of 2-pyridylcarboxaldehyde isonicotinoyl hydrazone, di-2-pyridylketone isonicotinoyl hydrazone, di-2-pyridylketone thiosemicarbazone, and the clinically trialed chelator 3-aminopyridine-2-carboxaldehyde thiosemicarbazone. The potential application of these agents in the changing face of Fe chelation therapy is discussed.

Development and evaluation of an electrochemical method for studying reactive phase-I metabolites: correlation to in vitro drug metabolism.

Abstract: A reactive metabolite may react covalently with proteins or DNA to form adducts that ultimately may lead to a toxic response Reactive metabolites can be formed via, for example, cytochrome P450-mediated phase 1 reactions, and in this study, we report the development and evaluation of an electrochemical method for generating reactive metabolites Paracetamol was used as a test compound to develop the method The stability of the electrochemically generated N-acetyl-p-benzoquinoneimine (NAPQI) from paracetamol was investigated at 37 °C at pH 50, 74, and 90 The highest stability of NAPQI was observed at pH 74 The reaction rate between NAPQI and glutathione (GSH) was studied with cyclic voltammetry NAPQI reacted quantitatively with GSH within 130 ms The reactivity of NAPQI toward other nucleophiles was investigated, and for the reaction with N-acetyltyrosine, a time-dependent formation of a conjugate with N-acetyltyrosine was observed from 0 to 4 min The applicability of the method was evaluated wit

Arsenic metabolism and thioarsenicals in hamsters and rats.

Abstract: The tissue distribution and chemical forms of arsenic were compared in two animal species with different metabolic capacity and toxicity to arsenic. Hamsters and rats were given a single oral dose of arsenite (iAsIII) at 5.0 mg As/kg body weight, and then the concentrations of arsenic were determined; more than 75% of the dose accumulated in rat red blood cells (RBCs) in the form of dimethylarsinous acid (DMAIII), whereas less than 0.8% of the dose accumulated in hamster RBCs, mostly in the form of monomethylarsonous acid (MMAIII). Reflecting the low accumulation in RBCs, more than 63% of the dose was recovered in hamster urine within one week (7.8-fold higher than that in rat urine). The quantity of arsenic distributed in the liver and kidneys was significantly higher in hamsters than in rats, and arsenic in livers stayed much longer in hamsters than in rats. Arsenic accumulated more and was retained longer in the kidneys than in the livers in both animals, and in hamster kidneys, it accumulated at levels higher than those in rat kidneys in the form of MMAIII bound to proteins. In the first 24 h urine, dimethylmonothioarsinic (DMMTAV) and dimethyldithioarsinic (DMDTAV) acids were detected in hamsters, but only DMMTAV was found in rats, together with an unknown arsenic metabolite in both animals. The unknown urinary arsenic metabolite was identified as monomethylmonothioarsonic acid (MMMTAV; CH3As(=S)(OH)2). The present results indicate that in hamsters, arsenic does not accumulate in RBCs, and therefore, hamsters exhibit a more uniform tissue distribution and faster urinary excretion of arsenic than rats. In addition, arsenic was thiolated more in hamsters than in rats excreting mono and dimethylated thioarsenicals in urine.

Enniatin exerts p53-dependent cytostatic and p53-independent cytotoxic activities against human cancer cells.

Abstract: Worldwide, multiple Fusarium mycotoxins occur as contaminants of cereals with important impacts on human and animal health. The aim of this study was to investigate the effects of the widespread Fusarium secondary metabolite enniatin (ENN), a cyclic hexadepsipeptide, on human cell growth and survival. While short-term exposure (up to 8 h) to ENN at nanomolar concentrations slightly but significantly stimulated cell proliferation, it showed profound apoptosis-inducing effects especially against various human cancer cell types at low micromolar concentrations (already after 24 h of treatment). Several cellular changes indicative for programmed cell death such as cell shrinkage, chromatin condensation, DNA fragmentation, and apoptotic body formation were observed. Correspondingly, the cleavage of poly(ADP-ribosyl)polymerase and the activation of multiple caspases accompanied a distinct loss of mitochondrial membrane potential. To investigate the impact of apoptosis- and cell cycle-regulating proteins on ENN activity, HCT116 cells with homozygously disrupted p53, p21, or bax genes were analyzed. In vitality assays, no significant influences of these proteins on the anticancer activity of ENN were detectable. In contrast, 3H-thymidine incorporation revealed a significantly more efficient block of DNA synthesis in p53 wild-type as compared to knock-out cells. Accordingly, fluorescence-activated cell sorting analysis demonstrated a stronger ENN-induced cell cycle arrest in the G0/G1 phase. Profound ENN-mediated induction of p53 and the p53-downstream cell cycle inhibitor p21 were detectable in p53 wild-type cells by Western blotting. P53-independent p21 induction was also detectable at higher ENN concentrations in p53 (-/-) cells. In contrast, bax activation by ENN was independent of the cellular p53 status. In summary, our results suggest that short-term exposure to very low ENN concentrations, for example, via food intake, might have tumor-promoting functions based on growth stimulation. In contrast, elevated ENN concentrations exert profound p53-dependent cytostatic and p53-independent cytotoxic activities especially against human cancer cells, suggesting a potential quality of ENN as an anticancer drug.

Toxicity of Dimethylmonothioarsinic Acid toward Human Epidermoid Carcinoma A431 Cells

Abstract: Chronic ingestion of arsenic-contaminated drinking water induces skin lesions and urinary bladder cancer in humans. It is now recognized that thioarsenicals such as dimethylmonothioarsinic acid (DMMTA (V)) are commonly excreted in the urine of humans and animals and that the production of DMMTA (V) may be a risk factor for the development of the diseases caused by arsenic. The toxicity of DMMTA (V) was compared with that of related nonthiolated arsenicals with respect to cell viability, uptake ability, generation of reactive oxygen species (ROS), and cell cycle progression of human epidermoid carcinoma A431 cells, arsenate (iAs (V)), arsenite (iAs (III)), dimethylarsinic acid (DMA (V)), and dimethylarsinous acid (DMA (III)) being used as reference nonthiolated arsenicals. DMMTA (V) (LC 50 = 10.7 microM) was shown to be much more cytotoxic than iAs (V) (LC 50 = 571 microM) and DMA (V) (LC 50 = 843 microM), and its potency was shown to be close to that of trivalent arsenicals iAs (III) (LC 50 = 5.49 microM) and DMA (III) (LC 50 = 2.16 microM). The greater cytotoxicity of DMMTA (V) was associated with greater cellular uptake and distribution, and the level of intracellular ROS remarkably increased in A431 cells upon exposure to DMMTA (V) compared to that after exposure to other trivalent arsenicals at the respective LC 50. Exposure of DMMTA (V) to cells for 24 h induced cell cycle perturbation. Namely, the percentage of cells residing in S and G2/M phases increased from 10.2 and 15.6% to 46.5 and 20.8%, respectively. These results suggest that although DMMTA (V) is a pentavalent arsenical, it is taken up efficiently by cells and causes various levels of toxicity, in a manner different from that of nonthiolated pentavalent arsenicals, demonstrating that DMMTA (V) is one of the most toxic arsenic metabolites. The high cytotoxicity of DMMTA (V) was explained and/or proposed by (1) efficient uptake by cells followed by (2) its transformation to DMA (V), (3) producing ROS in the redox equilibrium between DMA (V) and DMA (III) in the presence of glutathione.

Screening and identification of GSH-trapped reactive metabolites using hybrid triple quadruple linear ion trap mass spectrometry.

Abstract: The present study describes a new analytical approach for the detection and characterization of GSH-trapped reactive metabolites using multiple reaction monitoring (MRM) as the survey scan to trigger the acquisition of enhanced product ion (EPI) spectra on a triple quadrupole linear ion mass spectrometer. The MRM scan step was carried out following up to 114 MRM transitions from the protonated molecules of potential GSH adducts to their product ions derived from a neutral loss of 129 or 307 Da. MRM transition protocols were constructed on the basis of common bioactivation reactions predicted to occur in human liver microsomes (HLM). The effectiveness and reliability of the approach were evaluated using acetaminophen, diclofenac, and carbamazepine as model compounds. The total ion chromatograms of the MRM for the HLM incubations with these compounds and GSH clearly displayed a number of GSH adducts, including acetaminophen-GSH adducts and carbamazepine-GSH adducts that were not previously observed in HLM i...

Detection and quantitation of acrolein-derived 1,N2-propanodeoxyguanosine adducts in human lung by liquid chromatography-electrospray ionization-tandem mass spectrometry.

Abstract: Acrolein, a widely distributed environmental pollutant, reacts with dGuo in DNA to form two pairs of 1,N2-propano-dGuo adducts: (6R/S)-3-(2'-deoxyribos-1'-yl)-5,6,7,8-tetrahydro-6-hydroxypyrimido[1,2-a]purine-10(3H)one (alpha-OH-Acr-dGuo) and (8R/S)-3-(2'-deoxyribos-1'-yl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2-a]purine-10(3H)one (gamma-OH-Acr-dGuo). alpha-OH-Acr-dGuo is more mutagenic and mainly induces G --> T transversions. A recent study demonstrated that acrolein-DNA adducts are preferentially formed in p53 mutational hotspots in human lung cancer, but there are no reports on the presence of these adducts in the human lung. To directly investigate this question, we have developed a sensitive and specific liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method for the quantitative analysis of Acr-dGuo adducts in DNA. Our method is based on the enzymatic hydrolysis of DNA isolated from the human lung in the presence of [13C10,15N5]Acr-dGuo as internal standards. Acr-dGuo adducts are enriched from the hydrolysates by solid-phase extraction and analyzed by LC-ESI-MS/MS using selected reaction monitoring. The method is accurate and precise, and the identity of the adducts was confirmed by monitoring different transitions from the same parent ion and by carrying out reactions with NaOH and NaBH4, which produced N2-(3-hydroxypropyl)-dGuo or 1,N2-(1,3-propano)-dGuo from gamma-OH-Acr-dGuo and alpha-OH-Acr-dGuo, respectively. Thirty DNA samples from lung tissue were analyzed, and Acr-dGuo adducts were detected in all samples. Both alpha-OH- and gamma-OH-Acr-dGuo were observed in most of the samples; total adduct concentrations ranged from 16-209 adducts/109 nucleotides. These results demonstrate for the first time that both types of Acr-dGuo adducts are present in human lung DNA. There was no difference in adduct levels between current and ex-smokers. Collectively, the results support a plausible role for acrolein as one cause of p53 mutations in the human lung.

Binding of Dimethylarsinous Acid to Cys-13α of Rat Hemoglobin Is Responsible for the Retention of Arsenic in Rat Blood

Abstract: The metabolism, disposition, and carcinogenicity of arsenic differ dramatically between humans and rats. To understand the molecular basis of these differences, we have characterized arsenic species in rats that were treated with inorganic arsenate (iAsV), monomethylarsonic acid (MMAV), or dimethylarsinic acid (DMAV) for up to 15 weeks. Arsenic significantly accumulated in the red blood cells (RBCs) of rats in the form of hemoglobin (Hb) complexed with dimethylarsinous acid (DMAIII), regardless of whether the rats were treated with iAsV, MMAV, or DMAV, suggesting rapid methylation of arsenic species followed by strong binding of DMAIII to rat Hb. The binding site for DMAIII was identified to be cysteine 13 in the α-chain of rat Hb with a stoichiometry of 1:1. Over 99% of the total arsenic (maximum 2.5−3.5 mM) in rat RBCs was bound to Hb for all rats examined (n = 138). In contrast, only 40−49% of the total arsenic (maximum ∼10 μM) in rat plasma was bound to proteins. The ratios of the total arsenic in RBC...

AHR- and DNA-damage-mediated gene expression responses induced by benzo(a)pyrene in human cell lines.

Abstract: Carcinogens induce complex transcriptional responses in cells that may hold key mechanistic information. Benzo(a)pyrene (BaP) modulation of transcription may occur through the activation of the aryl hydrocarbon receptor (AHR) or through responses to DNA damage. To characterize further the expression profiles induced by BaP in HepG2 and MCF-7 cells obtained in our previous study, they were compared to those induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which activates AHR but does not bind to DNA, and anti-benzo(a)pyrene- trans-7,8-dihydrodiol-9,10-epoxide (BPDE), which binds directly to DNA but does not activate AHR. A total of 22 genes had altered expression in MCF-7 cells after both BaP and TCDD exposure, and a total of 29 genes had altered expression in HepG2 cells. In both cell lines, xenobiotic metabolism was upregulated through induction of NQO1, MGST1, and CYP1B1. A total of 78 expression changes were induced by both BaP and BPDE in MCF-7 cells, and a total of 29 expression changes were induced by both BaP and BPDE in HepG2 cells. These genes were predominantly involved in cell cycle regulation, apoptosis, and DNA repair. BaP and BPDE caused the repression of histone genes in both cell lines, suggesting that regulation of these genes is an important component of the DNA damage response. Interestingly, overlap of the BPDE and TCDD gene expression profiles was also observed. Furthermore, some genes were modulated by BaP but not by TCDD or BPDE, including induction of CRY1 and MAK, which may represent novel signaling pathways that are independent of both AHR activation and DNA damage. Promoter analysis identified candidate genes for direct transcriptional regulation by either AHR or p53. These analyses have further dissected and characterized the complex cellular response to BaP.

Protein targets of reactive electrophiles in human liver microsomes

Abstract: Liver microsomes are widely used to study xenobiotic metabolism in vitro, and covalent binding to microsomal proteins serves as a surrogate marker for toxicity mediated by reactive metabolites. We have applied liquid chromatography-tandem mass spectrometry (LC-MS-MS) to identify protein targets of the biotin-tagged model electrophiles 1-biotinamido-4-(4'-[maleimidoethylcyclohexane]-carboxamido)butane (BMCC) and N-iodoacetyl-N-biotinylhexylenediamine (IAB) in human liver microsomes. The biotin-tagged peptides resulting from in-gel tryptic digestion were enriched by biotin-avidin chromatography and LC-MS-MS was used to identify 376 microsomal cysteine thiol targets of BMCC and IAB in 263 proteins. Protein adduction was selective and reproducible, and only 90 specific cysteine sites in 70 proteins (approximately 25% of the total) were adducted by both electrophiles. Differences in adduction selectivity correlated with different biological effects of the compounds, as IAB- but not BMCC-induced ER stress in HEK293 cells. Targeted LC-MS-MS analysis of microsomal glutathione-S-transferase cysteine 50, a target of both IAB and BMCC, detected time-dependent adduction by the reactive acetaminophen metabolite N-acetyl-p-benzoquinoneimine during microsomal incubations. The results indicate that electrophiles selectively adduct microsomal proteins, but display differing target selectivities that correlate with differences in toxicity. Analysis of selected microsomal protein adduction reactions thus could provide a more specific indication of potential toxicity than bulk covalent binding of radiolabeled compounds.

Stabilization of C60 nanoparticles by protein adsorption and its implications for toxicity studies.

Abstract: Dispersion stability of nanoparticles of C60 under a model condition simulating a physiological environment was studied by dynamic light scattering. Although the C60 nanoparticles at a concentration of 9.3 x 10(-6) M (6.7 microg/mL) coagulated and precipitated out rapidly in phosphate buffered saline, coagulation was suppressed completely when HSA was present at concentrations above 1 mg/mL. DLS results show that the HSA molecules adsorb onto the surfaces of the C60 nanoparticles, thereby forming a protective layer, and prevent salt-induced coagulation. DLS results also indicate that the HSA molecules take an expanded conformation on the surface. Our findings suggest that C60 nanoparticles can be stabilized in the physiological environment even if they are not deliberately stabilized by using stabilizers and are of significant implications for the on-going efforts to evaluate the cytotoxicity of C60 nanoparticles in which no such effect has been considered.

Metabolism of Benzo[a]pyrene in Human Bronchoalveolar H358 Cells Using Liquid Chromatography–Mass Spectrometry

Abstract: Benzo[a]pyrene (B[a]P), a representative polycyclic aromatic hydrocarbon (PAH), is metabolically activated by three enzymatic pathways: by peroxidases (e.g., cytochrome P450 peroxidase) to yield radical cations, by P4501A1/1B1 monooxygenation and epoxide hydrolase to yield diol epoxides, and by P4501A1/1B1 monooxygenation, epoxide hydrolase, and aldo-keto reductases (AKRs) to yield o-quinones. In humans, a major exposure site for environmental and tobacco smoke PAH is the lung; however, the profile of B[a]P metabolites formed at this site has not been well characterized. In this study, human bronchoalveolar H358 cells were exposed to B[a]P, and metabolites generated by peroxidase (B[a]P-1,6- and B[a]P-3,6-diones), from cytochrome P4501A1/1B1 monooxygenation [3-hydroxy-B[a]P, B[a]P-7,8- and 9,10-trans-dihydrodiols, and B[a]P-r-7,t-8,t-9,c-10-tetrahydrotetrol (B[a]P-tetraol-1)], and from AKRs (B[a]P-7,8-dione) were detected and quantified by RP-HPLC, with in-line photo-diode array and radiometric detection,...

3,3'-diselenodipropionic acid, an efficient peroxyl radical scavenger and a GPx mimic, protects erythrocytes (RBCs) from AAPH-induced hemolysis.

Abstract: 3,3'-diselenodipropionic acid (DSePA), a derivative of selenocystine, has been synthesized and examined for antioxidant activity, glutathione peroxidase (GPx) activity, and cytotoxicity. The effect of DSePA on membrane lipid peroxidation, release of hemoglobin, and intracellular K+ ion as a consequence of erythrocyte (red blood cells or RBCs) oxidation by free radicals generated by 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH) were used to evaluate the antioxidant ability. Lipid peroxidation, hemolysis, and K+ ion loss in RBCs were assessed, respectively, by formation of thiobarbituric acid reactive substances (TBARS), absorbance of hemoglobin at 532 nm and flame photometry. The IC50 values for lipid peroxidation, hemolysis, and K+ ion leakage were 45+/-5, 20+/-2, and 75+/-8 microM, respectively. DSePA treatment prevented the depletion of glutathione (GSH) levels in RBCs from free-radical-induced stress. DSePA is a good peroxyl radical scavenger and the bimolecular rate constant for the reaction of DSePA with a model peroxyl radical, trichloromethyl peroxyl radical (CCl 3O2*), was determined to be 2.7x10(8) M(-1) s(-1) using a pulse radiolysis technique. DSePA shows GPx activity with higher substrate specificity towards peroxides than thiols. The cytotoxicity of DSePA was studied in lymphocytes and EL4 tumor cells and the results showed that DSePA is nontoxic to these cells at the concentrations employed. These results when compared with two well-known selenium compounds, sodium selenite and ebselen, indicated that DSePA, although it shows lesser GPx activity, has higher free radical scavenging ability and lesser toxicity.

Verification of the structural alerts for Michael acceptors

Abstract: A diverse series of polarized alpha,beta-unsaturated and related compounds were evaluated for reactivity with a spectrophotometric assay using the sulfhydryl group in the form of the cysteine residue of the tripeptide GSH as a model nucleophile. The reactive end point (RC 50) calculations were compared to previously described structural alerts based on conventional organic chemistry. This comparison focused on polarized alpha,beta-unsaturates, including ones containing an aldehyde, ketone, ester, sulfoxide, sulfone, sulfonate, nitro, or cyano moiety as well as ortho- and para-pyridino compounds and ortho- and para-quinones. The alerts were coded by substructure and are available in open-source software ( http://sourceforge.net/projects/chemeval). Comparisons of reactivity between selected analogues revealed that only the polarized alpha,beta-unsaturates were reactive. These results verified the coded structural alerts that define the applicability domain for Michael acceptor electrophiles.

Biological effects of particles from the paris subway system.

Abstract: Particulate matter (PM) from atmospheric pollution can easily deposit in the lungs and induce recruitment of inflammatory cells, a source of inflammatory cytokines, oxidants, and matrix metalloproteases (MMPs), which are important players in lung structural homeostasis. In many large cities, the subway system is a potent source of PM emission, but little is known about the biological effects of PM from this source. We performed a comprehensive study to evaluate the biological effects of PM sampled at two sites (RER and Metro) in the Paris subway system. Murine macrophages (RAW 264.7) and C57Bl/6 mice, respectively, were exposed to 0.01–10 µg/cm2 and 5–100 µg/mouse subway PM or reference materials [carbon black (CB), titanium dioxide (TiO2), or diesel exhaust particles (DEPs)]. We analyzed cell viability, production of cellular and lung proinflammatory cytokines [tumor necrosis factor α (TNFα), macrophage inflammatory protein (MIP-2), KC (the murin analog of interleukin-8), and granulocyte macrophage-colon...

Ochratoxin A-induced mutagenesis in mammalian cells is consistent with the production of oxidative stress.

Abstract: Ochratoxin A (OTA) is a widespread mycotoxin in food and a powerful nephrocarcinogen in rats. The mutagenicity of OTA has been extensively investigated but with conflicting results, thus leaving open the mechanistic question for OTA carcinogenicity. Here, we examined the mutagenicity of OTA by using well-standardized mutation assays such as the hypoxanthine-guanine phosphoribosyltransferase (HPRT) assay in Chinese hamster V79 cells and the thymidine kinase assay in mouse lymphoma LY5178 cells. OTA-induced HPRT mutations were characterized at the molecular level. In V79 cells, OTA produced a dose- and time-related decrease in cell number as a consequence of the transitory cytostatic effect mediated by G2/M cell cycle arrest. In both mutation assays, OTA was weakly mutagenic and this effect was independent of biotransformation. OTA-induced mutations were characterized by point mutations (48%) and a lack of a detectable reverse-transcription polymerase chain reaction product (52%). The pattern of OTA-induced point mutations was similar to that of spontaneous mutants, suggesting that OTA induced an increase of the endogenous oxidative metabolism but not covalent DNA adducts. Our data support a model where OTA is mutagenic via oxidative DNA damage induction.

Glutathione S-transferase conjugation of organophosphorus pesticides yields S-phospho-, S-aryl-, and S-alkylglutathione derivatives.

Abstract: Pesticide detoxification is a central feature of selective toxicity and safety evaluation. Two of the principal enzymes involved are GSH S-transferases (GSTs) and cytochrome P450s acting alone and together. More than 100 pesticides are organophosphorus (OP) compounds, but with few exceptions, their GSH conjugates have not been directly observed in vitro or in vivo. The major insecticides chlorpyrifos (CP) and diazinon are of particular interest as multifunctional substrates with diverse metabolites, while ClP(S)(OEt) 2 and the cotton defoliant tribufos are possible precursors of phosphorylated GSH conjugates. Formation of GSH conjugates by GST with GSH was studied in vitro with and without metabolic activation by human liver microsomes or P450 3A4 with NADPH. Metabolites were analyzed by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). Five GSH conjugates were identified from CP and chlorpyrifos oxon (CPO), i.e., GSCP and GSCPO in which the 6-chloro substituent of CP and CPO, respectively, is displaced by GSH; S-(3,5,6-trichloropyridin-2-yl)glutathione; S-(3,5-dichloro-6-hydroxypyridin-2-yl)glutathione; and S-ethylglutathione. GST of a human liver microsomal preparation but not P450 3A4 with GSH metabolized CP to GSCP. With GST and GSH, diazinon and diazoxon gave S-(2-isopropyl-4-methylpyrimidin-6-yl)glutathione and ClP(S)(OEt) 2 yielded GSP(S)(OEt) 2. With microsomes, NADPH, GST, and GSH tribufos gave GSP(O)(SBu) 2. The liver of intraperitoneally treated mice contained GSCP from CP, GSP(S)(OEt) 2 from ClP(S)(OEt) 2, and GSP(O)(SBu) 2 from tribufos. GSP(S)(OEt) 2 and GSP(O)(SBu) 2 are the first S-phosphoglutathione metabolites observed in vitro and in vivo directly by LC-ESI-MS. Nine other OP pesticides gave only O-dealkylation in the GST/GSH system. GST-catalyzed metabolism joins P450s and hydrolases as important contributors to OP detoxification.

Iron-loaded synthetic chrysotile: a new model solid for studying the role of iron in asbestos toxicity.

Abstract: The generation of reactive oxygen species and other radicals, catalyzed by iron ions at the fiber surface, is thought to play an important role in asbestos-induced cytotoxicity and genotoxicity, but a direct confirmation of this statement needs the availability of asbestos samples differing only for their iron content, without the interference of other physicochemical features. Synthetic stoichiometric chrysotile nanofibers, devoid of iron or any other contaminant, did not exert genotoxic and cytotoxic effects nor elicited oxidative stress in a murine alveolar macrophage cell line; on the contrary, the same nanofibers, loaded with 0.57% and 0.94% (w/w) iron, induced DNA strand breaks, lipoperoxidation, inhibition of redox metabolism and alterations of cell integrity, similarly to natural chrysotile. On the other hand, the incubation with ferric nitrilotriacetate, a cell-permeating iron complex, even if it caused an intracellular overloading of iron very similar to that elicited by iron-loaded synthetic chrysotile and by natural chrysotile, did not exert any of these effects. This suggests that chrysotile is not toxic by acting simply as a carrier of iron into the cell, but rather that the redox activity of iron is potentiated when organized at the fibers surface into specific crystallographic sites having coordination states able to activate free radical generation. Synthetic chrysotile fibers may be proposed as a standard reference sample and model solids for experimental studies on asbestos fibers aiming to clarify the mechanisms of its toxicity and to synthesize new fibers devoid of pathogenic effects.