m6A-modified RIPK4 facilitates proliferation and cisplatin resistance in epithelial ovarian cancer.

BACKGROUND: Cisplatin (DDP)-based chemotherapy is a common chemotherapeutic regimen for the treatment of advanced epithelial ovarian cancer (EOC). However, most patients rapidly develop chemoresistance. N6-methyladenosine (m6A) is a pervasive RNA modification, and its specific role and potential mechanism in the regulation of chemosensitivity in EOC remain unclear. METHODS: The expression of RIPK4 and its clinicopathological impact were evaluated in EOC cohorts. The biological effects of RIPK4 were investigated using in vitro and in vivo models. RNA m6A quantification was used to measure total m6A levels in epithelial ovarian cancer cells. Luciferase reporter, MeRIP-qPCR, RIP-qPCR and actinomycin-D assays were used to investigate RNA/RNA interactions and m6A modification of RIPK4 mRNA. RESULTS: We demonstrated that RIPK4, an upregulated mRNA in EOC, acts as an oncogene in EOC cells by promoting tumor cell proliferation and DDP resistance at the clinical, database, cellular, and animal model levels. Mechanistically, METTL3 facilitates m6A modification, and YTHDF1 recognizes the specific m6A-modified site to prevent RIPK4 RNA degradation and upregulate RIPK4 expression. This induces NF-κB activation, resulting in tumor growth and DDP resistance in vitro and in vivo. CONCLUSIONS: Collectively, the present findings reveal a novel mechanism underlying the induction of DDP resistance by m6A-modified RIPK4, that may contribute to overcoming chemoresistance in EOC.

P(V)-Promoted Rh-Catalyzed Highly Regioselective Hydroformylation of Styrenes under Mild Conditions.

Hydroformylation of olefins is widely used in the chemical industry due to its versatility and the ability to produce valuable aldehydes with 100% atom economy. Herein, a hybrid phosphate promoter was found to efficiently promote rhodium-catalyzed hydroformylation of styrenes under remarkably mild conditions with high regioselectivities. Preliminary mechanistic studies revealed that the weak coordination between the Rhodium and the P=O double bond of this pentavalent phosphate likely induced exceptional reactivity and high ratios of branched aldehydes to linear products.

Divergent Chemo- and Biocatalytic Route to 16ß-Methylcorticoids: Asymmetric Synthesis of Betamethasone Dipropionate, Clobetasol Propionate, and Beclomethasone Dipropionate.

16ß-Methylcorticoids are among the most important glucocorticoid steroids for the treatment of various dermatological disorders, respiratory infections, and other allergic reactions elicited during inflammatory responses of the human body. Betamethasone dipropionate, clobetasol propionate, and beclomethasone dipropionate are particularly noteworthy for their synthetic intractability. Despite five decades of research, these 16ß-methylcorticoids have remained challenging synthetic targets owing to insurmountable issues of reactivity, selectivity, and cost efficiency associated with all previously explored strategies. We herein report our practicability-oriented strategy toward the unified stereoselective synthesis of 16ß-methylcorticoids in 12.6-14.0 % overall yield from commercially available 9α-hydroxyandrost-4-ene-3,17-dione (9α-OH-AD). In this approach, the chiral C16ß-Me and C17α-OH groups of the corticosteroid D ring were installed via a substrate-controlled diastereo- and enantioselective Mn-catalyzed oxidation-reduction hydration of Δ4,9(11),16 -triene-3,20-dione. The C1-C2 double bond of the corticosteroid A ring was constructed using an unprecedented engineered 3-ketosteroid-Δ1 -dehydrogenase (MK4-KstD)-catalyzed regioselective Δ1 -dehydrogenation of Δ4,9(11) -diene-3,21-dione. This strategy provides a general method and a key precursor for the divergent synthesis of a variety of glucocorticoids and related steroidal drugs.

Urinary Profile of Alkylated DNA Adducts and DNA Oxidative Damage in Sulfur Mustard-Exposed Rats Revealed by Mass Spectrometry Quantification.

Alkylation reagents, represented by sulfur mustard (SM), can damage DNA molecules directly as well as lead to oxidative stress, causing DNA lesions indirectly. Correspondingly, two types of biomarkers including alkylated DNA adducts and oxidative DNA adducts are commonly involved in the research of DNA damage evaluation caused by these agents. However, the correlations and differences of the occurrence, duration, severity, and traceability between alkylation and oxidation lesions on the DNA molecular level reflected by these two types of biomarkers have not been systematically studied. A simultaneous determination method for four alkylated DNA adducts, i.e., N7-(2-hydroxyethylthioethyl)2'-guanine (N7-HETEG), O6-(2-hydroxyethylthioethyl)-2'-guanine (O6-HETEG), N3-(2-hydroxyethylthioethyl)-2'-adenine (N3-HETEA), and bis(2-ethyl-N7-guanine)thioether (Bis-G), and the oxidative adduct 8-hydroxy-2'-deoxyguanosine (8-OH-dG) in urine samples by isotope-dilution high-performance liquid chromatography-tandem mass spectrometry (ID-HPLC-MS/MS) was built with a lower limit of detection of 0.02 ng/mL (except Bis-G, 0.05 ng/mL) and a recovery of 79-111%. The profile of these adducts was simultaneously monitored in urine samples after SD rats' dermal exposure to SM in three dose levels (1, 3, and 10 mg/kg). The time-effect and dose-effect experiments revealed that when exposed to SM, DNA alkylation lesions would happen earlier than those of oxidation. For the two types of biomarkers, alkylated DNA adducts showed an obvious dose-effect relationship and could be used as internal exposure dose and effect biomarkers, while 8-OH-dG did not show a correlation with exposure dose, demonstrating that it was more suitable as a biomarker for DNA oxidative lesions but not an indicator for the extent of cytotoxicity and internal exposure.

The value of HPV genotypes combined with clinical indicators in the classification of cervical squamous cell carcinoma and adenocarcinoma.

BACKGROUND: To investigate the differences in HPV genotypes and clinical indicators between cervical squamous cell carcinoma and adenocarcinoma and to identify independent predictors for differentiating cervical squamous cell carcinoma and adenocarcinoma. METHODS: A total of 319 patients with cervical cancer, including 238 patients with squamous cell carcinoma and 81 patients with adenocarcinoma, were retrospectively analysed. The clinical characteristics and laboratory indicators, including HPV genotypes, SCCAg, CA125, CA19-9, CYFRA 21-1 and parity, were analysed by univariate and multivariate analyses, and a classification model for cervical squamous cell carcinoma and adenocarcinoma was established. The model was validated in 96 patients with cervical cancer. RESULTS: There were significant differences in SCCAg, CA125, CA19-9, CYFRA 21-1, HPV genotypes and clinical symptoms between cervical squamous cell carcinoma and adenocarcinoma (P < 0.05). Logistic regression analysis showed that SCCAg and HPV genotypes (high risk) were independent predictors for differentiating cervical squamous cell carcinoma from adenocarcinoma. The AUC value of the established classification model was 0.854 (95% CI: 0.804-0.904). The accuracy, sensitivity and specificity of the model were 0.846, 0.691 and 0.899, respectively. The classification accuracy was 0.823 when the model was verified. CONCLUSION: The histological type of cervical cancer patients with persistent infection of high-risk HPV subtypes and low serum SCCAg levels was more prone to being adenocarcinoma. When the above independent predictors occur, the occurrence and development of cervical adenocarcinoma should be anticipated, and early active intervention treatment should be used to improve the prognosis and survival of patients.

Red Fluorescence of Eu3+-Doped ZnAl-LDH Response to Intercalation and Release of Ibuprofen.

A drug delivery system with identification function is attractive and important. For this reason, the red fluorescence of Eu3+-doped ZnAl-LDH response to intercalation and release of ibuprofen (IBU) has been studied. X-ray diffraction(XRD) results showed that the basal spacing of the Eu3+-doped ZnAl-LDH varied from 8.85 to 12.04 Å after the intercalation of IBU. The release of the IBU from the Eu3+-doped ZnAl-LDH was carried out in simulated intestinal medium (phosphate buffer solutions with pH 7.4 and 37 °C), and the releasing behavior of IBU exhibited an initial rapid release followed by a slow release. Moreover, the present delivery system has slower release of drug than those of other LDH-based delivery systems. Interestingly, the intercalation of IBU into the Eu3+-doped ZnAl-LDH obviously reduced the red fluorescence of the Eu3+-doped ZnAl-LDH, whereas the red fluorescence was recovered after the release of IBU. This fluorescent responsiveness may be a favorable signal for detecting the delivery and release of IBU. Therefore, the Eu3+-doped ZnAl-LDH with red fluorescence would be potential application as drug delivery system with identification function because of its cheapness, non-toxicity, good biocompatibility, and little damage to biological tissue.

"Nonpolarity paving" in substrate tunnel of a Limnobacter sp. Phenylacetone monooxygenase for efficient single whole-cell synthesis of esomeprazole.

Baeyer-Villiger monooxygenase (BVMO) mediated sulfoxidation is a sustainable approach for the synthesis of esomeprazole. In this work, a novel phenylacetone monooxygenase from Limnobacter sp. (LnPAMO) was found to have trace activity for synthesis of enantiopure esomeprazole. Through engineering in the substrate tunnel using a mutagenesis strategy called "nonpolarity paving" and some modifications in cofactor binding domains, a mutant harboring 15 mutations (LnPAMO Mu15) was obtained with 6.6 × 103-fold higher activity to convert omeprazole sulfide into esomeprazole. The activities of the mutant for synthesis of (S)-methyl phenyl sulfoxide and (S)-pantoprazole also increased much, indicating the versatility of the mutant for sulfoxide synthesis. Importantly, no over-oxidation byproduct omeprazole sulfone was detected in the sulfoxidation products by both mass spectrometry and HPLC analysis. Then NADP-dependent Burkholderia stabili formate dehydrogenase was ligated behind Mu15 along with a ribosome binding site sequence in pET-28a for co-expression. By single whole-cell of recombinant Escherichia coli BL21 coexpressing Mu15 and formate dehydrogenase, omeprazole sulfide was efficiently converted into esomeprazole without production of sulfone (16 g/L substrate, enantiomeric excess > 99.9% (S) and > 99% conversion) and the space-time-yield reached 1.67 g product/L/h.

Dynamically monitoring cellular γ-H2AX reveals the potential of carcinogenicity evaluation for genotoxic compounds.

Amongst all toxicological endpoints, carcinogenicity might pose the greatest concern. Genetic damage has been considered an important underlying mechanism for the carcinogenicity of chemical substances. The demand for in vitro genotoxic tests as alternative approaches is growing rapidly with the implementation of new regulations for compounds. However, currently available in vitro genotoxicity tests are often limited by relatively high false positive rates. Moreover, few studies have explored carcinogenicity potential by in vitro genotoxicity testing due to the shortage of suitable toxicological biomarkers to link gene damage with cancer risk. γ-H2AX is a recently acknowledged attractive endpoint (biomarker) for evaluating DNA damage and can simultaneously reflect the DNA damage response and repair of cells. We previously reported an ultrasensitive and reliable method, namely stable-isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS), for detecting cellular γ-H2AX and evaluating genotoxic chemicals. More importantly, our method can dynamically monitor the specific processes of genotoxic compounds affecting DNA damage and repair reflected by the amount of γ-H2AX. To clarify the possibility of using this method to assess the potential carcinogenicity of genotoxic chemicals, we applied it to a set of 69 model compounds recommended by the European Center for the Validation of Alternative Methods (ECVAM), with already-characterized genotoxic potential. Compared to conventional in vitro genotoxicity assays, including the Ames test, the γ-H2AX assay by MS has high accuracy (94-96%) due to high sensitivity and specificity (88% and 100%, respectively). The dynamic profiles of model compounds after exposure in HepG2 cells were explored, and a mathematical approach was employed to simulate and quantitatively model the DNA repair kinetics of genotoxic carcinogens (GCs) based on γ-H2AX time-effect curves up to 8 h. Two crucial parameters, i.e., k (rate of γ-H2AX decay) and t50 (time required for γ-H2AX from maximum decrease to half) estimated by the least squares method, were achieved. An open web server to help researchers calculate these two key parameters and profile simulated curves of the tested compound is available online ( http://ccb1.bmi.ac.cn:81/shiny-server/sample-apps/prediction1/ ). We detected a positive association between carcinogenic levels and k and t50 values of γ-H2AX in tested GCs, validating the potential of using this MS-based γ-H2AX in vitro assay to help preliminarily evaluate carcinogenicity and assess genotoxicity. This approach may be used alone or integrated into an existing battery of in vitro genetic toxicity tests.

Sensitive Untargeted Screening of Nerve Agents and Their Degradation Products Using Liquid Chromatography-High Resolution Mass Spectrometry.

Nerve agents (NAs) are notorious chemical warfare agents that pose a serious threat to national security and public health. The total number of theoretical chemicals of NAs and their degradation products (DPs) exceeds 410 000, according to 1.A.01-1.A.03 in the Schedules of Chemicals of the Chemical Weapons Convention, which poses great challenges for identification and verification. A three-step integrated untargeted screening strategy was developed based on high-resolution mass spectrometry. First, an extensible homemade library for targeted screening of common classical agents was established. Second, a set of in-source collision-induced dissociation mass spectrometry (MS)-alerting ions was extracted and concluded based on fragmentation behavior studies, which included 40 specific alerting ions and 10 types of characteristic structural fragments from total NAs and their DPs. A novel "alerting ion" searching method was developed to rapidly and sensitively screen whether or not nerve agent-related compounds were present and of which type they were. Third, we built a theoretical exact mass database including 202 accurate masses or molecular formulas, which could cover all structural possibilities of the NAs and their DPs. Comprehensively, the elemental composition of pseudomolecular ions, fragment ions, MS/MS spectra, and isotope pattern information were obtained from the full scan MS/data dependent-MS2 experiments and elucidated for identification of the candidates selected in the screening step. This strategy was successfully applied to the identification of unknown chemicals in real samples with good stability and a low limit of detection of 1-10 ng/mL. These procedures are applicable for trace forensic investigations in cases of the alleged use of nerve agents.

Distinct Orchestration and Dynamic Processes on γ-H2AX and p-H3 for Two Major Types of Genotoxic Chemicals Revealed by Mass Spectrometry Analysis.

Genotoxic chemicals act by causing DNA damage, which, if left unrepaired, can have deleterious consequences for cell survival. DNA damage response (DDR) gets activated to repair or mitigate the effects of DNA damage. Histone H2AX and H3 phosphorylation biomarkers (γ-H2AX and p-H3) have attracted great attention as they play pivotal roles in the DDR. Simultaneous quantitation of γ-H2AX and p-H3 in exposed cells may monitor the toxicity of genotoxic chemicals and to some extent reflect the subsequent DDR process. Reported here is the first comprehensive characterization of distinct orchestration and dynamic processes on cellular γ-H2AX and p-H3 for two major types of genotoxic chemicals, clastogens and aneugens, by stable isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS). We find that clastogens significantly induce an increase in γ-H2AX and a decrease in p-H3; aneugens have no obvious effect on γ-H2AX, whereas induce either an increase or a decrease in p-H3. In addition, the specific profiles of clastogens and aneugens affecting DNA damage may be dynamically observed, which in turn provides insights into the processes involving DNA damage repair as well as transcription. Taken together, these results suggest that robust LC-MS/MS analysis of γ-H2AX and p-H3 can not only quantitatively differentiate mechanistic information on clastogens and aneugens but also dynamically present the detail profiles of DNA damage and repair processes.

MRI evaluation of endopelvic fascial swelling and analysis of influencing factors in patients with uterine fibroids after high-intensity focused ultrasound ablation.

Purpose: To evaluate endopelvic fascial swelling in patients with uterine fibroids after high-intensity focused ultrasound (HIFU) ablation on magnetic resonance imaging (MRI) and investigate the factors that influence endopelvic fascial swelling.Methods: MRI and clinical data from 188 patients with uterine fibroids who were treated with HIFU were analyzed retrospectively. The patients were divided into a fascial swelling group and a non-swelling group, and the degree of swelling was graded. Fascial swelling was set as the dependent variable, and factors such as baseline characteristics and HIFU parameters, were set as the independent variables. The relationship between these variables and fascial swelling was analyzed by univariate and multivariate analyses. Correlations between the factors and the degree of fascial swelling were evaluated by Kruskal-Wallis test.Results: The univariate analysis revealed that the fibroid location, distance from the fibroid to the sacrum, sonication time, treatment time, treatment intensity, therapeutic dose (TD), and energy efficiency (EEF) all affected the endopelvic fascial swelling (p < 0.05). Subsequently, multivariate analysis showed that the distance from the fibroid to the sacrum was significantly correlated with fascial swelling (p < 0.05). Moreover, TD and sonication time were significantly positively correlated with the degree of fascial swelling (p < 0.05). The incidence of sacrococcygeal pain was significantly correlated with fascial swelling (p < 0.05).Conclusion: The distance from the fibroid to the sacrum was a protective factor for fascial swelling. TD and sonication time were significantly positively correlated with the degree of fascial swelling.

New near-infrared emissions and energy transfer in Er3+ -doped MgAl layered double hydroxides.

A series of Er3+ -doped magnesium aluminium layered double hydroxides (Er3+ -doped, MgAl-LDHs) with different Mg2+ /(Al3+ +Er3+ ) molar ratios were synthesized using the hydrothermal method. Compositional and structural analyses suggest that the Er3+ -doped MgAl-LDHs kept a hexagonal structure while the Mg2+ /(Al3+ +Er3+ ) molar ratio was at 1.0-4.1. The downconverted emission spectra of the Er3+ -doped MgAl-LDHs showed a red emission at 650 nm and strong infrared emissions at 720, 780, and 850 nm. These infrared emissions were hardly observed in previous downconverted emission spectra of Er3+ -doped materials. In the analysis of the Er3+ energy levels and in relevant published literature, the energy transfer diagram for Er3+ -doped in MgAl-LDHs is described, and infrared emissions at 720, 780, and 850 nm may be attributed to 4 F7/2 →4 I13/2 , 2 H11/2 →4 I13/2 , and 4 S3/2 →4 I13/2 transitions of Er3+ , respectively. Er3+ -doped MgAl-LDHs could have potential application as marking and targeting agents in the processes for drug delivery in consideration of the strong near-infrared Er3+ emissions, as well as the special layered structure of MgAl-LDH.

Synthesis, Characterization, and Identification of New in Vitro Covalent DNA Adducts of Divinyl Sulfone, an Oxidative Metabolite of Sulfur Mustard.

Divinyl sulfone (DVS) is an important oxidative metabolic product of sulfur mustard (SM) in vitro and in vivo. Although DVS is not a classical blister agent, its high reactivity and toxicity induced by vinyl groups can also cause blisters like SM upon contact with the skin, eyes, and respiratory organs. The purpose of this paper was to identify whether DVS could covalently bind to DNA bases to form new DNA adducts in cells in vitro. A series of adducts were synthesized and characterized using purine, nucleoside, or DNA, separately, as starting materials. The covalent site, pattern, and relative reactivity of adduct formation were identified and discussed in detail. The results showed that five high abundance site-specific DNA adducts, including two monoadducts and three cross-linked adducts, were obtained when DNA was used as a substrate. When HaCaT cells were exposed to 30 µM of DVS, four new DNA adducts containing monoadducts and cross-linked adducts were found and identified in cells, including N3-A monoadduct, N7-G monoadduct, N7G-N7G bis-adduct, and N3A-N7G cross-linked adduct. Among them, the abundance of N3-A monoadduct was 10 times higher than that of the other three adducts. DNA adduct formation with DVS showed significant differences from that observed with SM. The observation of these new DNA adduct in vitro cells revealed that DNA damage could be also induced by DVS.

Accumulation of intact sulfur mustard in adipose tissue and toxicokinetics by chemical conversion and isotope-dilution liquid chromatography-tandem mass spectrometry.

Sulfur mustard (SM) is a powerful vesicant and one of the most harmful chemical warfare agents. Although having been studied for a long time, it is still difficult to fully elucidate the mechanisms of SM poisoning, and there is no effective antidote or specific treatment for SM injury. The investigations on toxicokinetics and tissue distribution of SM will help to understand its toxicity and provide a theoretical basis for pretreatment and therapy of SM poisoning. But the metabolic trajectory or fate of intact SM in vivo remains unclear, and there are insufficient experimental data to elucidate, due to its high reactivity and difficulty in biomedical sample analysis. In this paper, a sensitive method for the detection and quantification of intact SM in blood or tissues using isotope-dilution LC-MS/MS coupled with chemical conversion was developed. By transforming highly reactive SM into stable derivative product, the real concentration of intact SM in biological samples was obtained accurately. The toxicokinetics and tissue distribution studies of intact SM in rats were successfully profiled by the novel method after intravenous (10 mg/kg) or cutaneous administration (1, 3 and 10 mg/kg). The SM level in blood with peak time at 30-60 min determined in cutaneous exposure experiment was found much higher than previously reported, and the mean residence time in blood extended to 1-1.5 h. A significant accumulation of intact SM was observed in adipose tissues, including the perirenal fat, epididymal fat, subcutaneous fat and brown fat, in which the concentrations of SM were at least 15 times greater than those in non-adipose tissues in cutaneous exposed rats. The recovery of SM in body fat was calculated as 3.3 % of bioavailable SM (the bioavailability after cutaneous exposure was evaluated as 16 %). Thus, the adipose tissue was important for SM distribution and toxicity, which may pioneer a new model for both the prevention and treatment of SM exposure.

Reversible Fluorescent Probe for Selective Detection and Cell Imaging of Oxidative Stress Indicator Bisulfite.

In this paper, we report a benzothiazole-functionalized cyanine fluorescence probe and demonstrate that it is selectively reactive to bisulfite, an intermediate indicator for oxidative stress. The selective reaction can be monitored by distinct ratiometric fluorescence variation favorable for cell imaging and visualization. The original probe can be regenerated in high yield through the elimination of bisulfite from the product by peroxides such as hydrogen peroxide, accompanied by fluorescence turning on at 590 nm, showing a potential application for the detection of peroxides. We successfully applied this probe for fluorescence imaging of bisulfite in cancer cells (MCF-7) treated with bisulfite and hydrogen peroxide as well as a selective detection limit of 0.34 µM bisulfite in aqueous solution.

Analysis of different fates of DNA adducts in adipocytes post-sulfur mustard exposure in vitro and in vivo using a simultaneous UPLC-MS/MS quantification method.

Sulfur mustard (SM) is a powerful alkylating vesicant that can rapidly penetrate skin, ocular, and lung bronchus mucous membranes and react with numerous nucleophiles in vivo. Although the lesion mechanisms of SM remain unclear, DNA damage is believed to be the most crucial factor in initiating SM-induced toxicity. Four major DNA adducts were identified for retrospective detection and DNA lesion evaluation, namely, N(7)-[2-[(2-hydroxyethyl)thio]-ethyl]guanine (N(7)-HETEG), bis(2-ethyl-N(7)-guanine)thioether (Bis-G), N(3)-(2-hydroxyethylthioethyl)-2'-adenine (N(3)-HETEA), and O(6)-[2-[(2-hydroxyethyl)thio]-ethyl]guanine (O(6)-HETEG). Because of previous observations that the levels of SM-DNA adducts were relatively higher in adipose-rich organs, such as the brain, we focused on the in vitro and in vivo fates of the DNA adducts in exposed adipocytes. A UPLC-MS/MS method developed in our laboratory was used to profile the N(7)-HETEG, Bis-G, and N(3)-HETEA levels in human mature adipocytes (HA-s) that had differentiated from human subcutaneous preadipocytes (HPA-s). This method was also used to profile three other cell lines related to the targeting of major tissues, including human keratinocytes (HaCaT), human hepatocytes (L-02), and human lung fibroblasts (HLF). Long-lasting adduct persistence and a high proportion of Bis-G were found in exposed adipocytes in vitro. The survival properties of exposed adipocytes were also tested. At the same time, the fate of SM-DNA adducts in vivo was characterized using a rat model exposed to 1 and 10 mg/kg doses of SM. The level of DNA adducts in the exposed adipose tissue (AT) was much lower than those in other organs studied in our previous work. The adduct persistence behavior was observed in AT with an extremely high proportion of Bis-G, which was higher than N(7)-HETEG. In light of these results, we suggest that an adipose-rich environment may promote the formation of Bis-G and that adipocyte-specific DNA repair mechanisms may result in adduct persistence and the survival of adipocytes after SM exposure. These conclusions should be further investigated.

Distribution of DNA adducts and corresponding tissue damage of Sprague-Dawley rats with percutaneous exposure to sulfur mustard.

Sulfur mustard (SM) is a highly reactive alkylation vesicant and cytotoxic agent that has been recognized as an animal and human carcinogen. Although the exact mechanism of toxicology is vague, DNA alkylation seems to be responsible for the triggering of apoptosis. In this study, after male adult Sprague-Dawley rats were cutaneous exposed to a low concentration of SM at parts-per-million levels, their lungs, livers, pancreases, spleens, marrow, and brains were collected at 11 different time points and analyzed. N7-[2-[(2-hydroxyethyl)thio]-ethyl]guanine (N7-HETEG), N3-[2-[(2-hydroxyethyl)thio]-ethyl]adenine (N3-HETEA), and bis[2-(guanin-7-yl)ethyl]sulfide (Bis-G) as the biomarkers for DNA damage were measured in the vital tissues by isotope dilution ultraperformance liquid chromatography tandem mass spectrometry (ID-UPLC-MS/MS). At the same time, general variations and pathological changes were monitored and detected to evaluate the tissue damage. Time- and dose-dependent data showed that SM had strong permeability and reactivity and that three SM-DNA adducts were detected in all investigated tissues only after 10 min after exposure. Obvious dose-dependency was observed except in the brain and pancreas. Most times to peak (tmax) of all three adducts were less than 3 h, while half-lifetimes (t1/2) were less than 24 h. We also suggested that the lipophilic SM can easily pass through the blood-brain barrier and can be stored in the fatty organs. To the best of our knowledge, the abundant adducts in marrow were found and reported for the first time. The surveillance of N7-HETEG in vivo, which was the most abundant adduct, may be the most efficient indicator to validate SM exposure even without any symptoms. Bis-G can be regarded as a biomarker of effect, which is directly related to the extent of damage. The most abundant Bis-G was found in the most sensitive tissues, marrow, spleen, and lung, which is in good accordance with histopathologic results. General variations and pathological changes were evaluated as well. After cutaneous exposure to SM, the body weights of rats heavily decreased in the first 4 days and were inversely proportional to the applied doses, and then recovered at the last experimental day except for those of the rats at the highest dosing level, in which the relative weights of rat spleens were obviously lost. Moreover, we found remarkable histological changes of the lung and skin, such as encephalemia, at the very beginning of the sampling procedure, and plentiful mononuclear cells in marrow appeared 6 h after exposure. The micronucleus test of marrow cells showed that the micronucleus rate had a positively dose-dependent effect.

Determination of gaseous sulfur dioxide and its derivatives via fluorescence enhancement based on cyanine dye functionalized carbon nanodots.

The development of convenient methods for sulfur dioxide and its derivatives analysis is critically important because SO2 causes worldwide serious environmental problems and human diseases. In this work, we show an unprecedented example of an energy-transfer-based fluorescence nanoprobe for selective and quantitative detection of SO2, through molecular engineering of the fluorescent carbon nanodots by a cyanine dye which have a unique reactivity to bisulfite, achieving a detection limit of 1.8 µM with a linear relationship (R(2) = 0.9987). The specific detection was not interfered with other potential coexisted species. In addition, the probe is demonstrated for the determination of SO2 gas in aqueous solution as well as for visually monitoring of SO2 gas in air. This nanomaterial based probe is easily prepared, fast responding, and thus potentially attractive for extensive application for the determination of SO2 and other similar air pollutants.

Abundance of four sulfur mustard-DNA adducts ex vivo and in vivo revealed by simultaneous quantification in stable isotope dilution-ultrahigh performance liquid chromatography-tandem mass spectrometry.

Sulfur mustard (SM) is a highly reactive alkylating vesicant and causes blisters upon contact with skin, eyes, and respiratory organs. It covalently links with DNAs by forming four mono- or cross-link adducts. In this article, the reference standards of SM-DNA adducts and deuterated analogues were first synthesized with simplified procedures containing only one or two steps and using less toxic chemical 2-(2-chloroethylthio)ethanol or nontoxic chemical thiodiglycol as starting materials. A sensitive and high-throughput simultaneous quantification method of N(7)-[2-[(2-hydroxyethyl)thio]-ethyl]guanine (N(7)-HETEG), O(6)-[2-[(2-hydroxyethyl)thio]-ethyl]guanine (O(6)-HETEG), N(3)-[2-[(2-hydroxyethyl)thio]-ethyl]adenine (N(3)-HETEA), and bis[2-(guanin-7-yl)ethyl]sulfide (Bis-G) in the Sprague-Dawley rat derma samples was developed by stable isotope dilution-ultrahigh performance liquid chromatography-tandem mass spectrometry (ID-UPLC-MS/MS) with the aim of revealing the real metabolic behaviors of four adducts. The method was validated, the limit of detection (S/N ratio greater than 10) was 0.01, 0.002, 0.04, and 0.11 fmol on column for N(7)-HETEG, O(6)-HETEG, Bis-G, and N(3)-HETEA, respectively, and the lower limit of quantification (S/N ratio greater than 20) was 0.04, 0.01, 0.12, and 0.33 fmol on column for N(7)-HETEG, O(6)-HETEG, Bis-G, and N(3)-HETEA, respectively. The accuracy of this method was determined to be 76% to 129% (n = 3), and both the interday (n = 6) and intraday (n = 7) precisions were less than 10%. The method was further applied for the quantifications of four adducts in the derma of adult male Sprague-Dawley rats exposed to SM ex vivo and in vivo, and all adducts had time- and dose-effect relationships. To the best of our knowledge, this is the first time that the real presented status of four DNA adducts was simultaneously revealed by the MS-based method, in which Bis-G showed much higher abundance than the result previously reported and N(3)-HETEA showed much less. It should be noted that since the interstrand cross-linked adduct is believed to stall DNA replication and finally induce a double-strand break, the higher abundance of Bis-G is a great indication of a more serious DNA lesion by SM alkylation.

Monitoring urinary metabolites resulting from sulfur mustard exposure in rabbits, using highly sensitive isotope-dilution gas chromatography-mass spectrometry.

A highly sensitive method for the determination of sulfur mustard (SM) metabolites thiodiglycol (TDG) and thiodiglycol sulfoxide (TDGO) in urine was established and validated using isotope-dilution negative-ion chemical ionization (NICI) gas chromatography-mass spectrometry (GC-MS). TDGO in the samples was reduced with TiCl3, and then determined together with TDG as a single analyte. The sample preparation procedures, including two solid-phase-extraction (SPE) clean-up steps, were optimized to improve the sensitivity of the method. The limits of detection (LOD) for both TDG and TDG plus TDGO (TDG + TDGO) were 0.1 ng mL(-1), and the limits of quantitation (LOQ) for both were 0.3 ng mL(-1). The method was used in a rabbit cutaneous SM exposure model. Domestic rabbits were exposed to neat liquid SM at three dosage levels (0.02, 0.05, and 0.15 LD50), and the urinary excretion of four species of hydrolysis metabolites, namely free TDG, free plus conjugated TDG (total TDG), free TDG + TDGO, and free plus conjugated TDG + TDGO (total TDG + TDGO), was evaluated to investigate the metabolic processes. The total urinary excretion profiles of the metabolites, including the peak time, time window, and dose-response and time-response relationships, were clarified. The results revealed that the concentrations of TDG and TDG + TDGO in the urine increased quickly and then decreased rapidly in the first two days after SM exposure. The cumulative amount of total TDG + TDGO excreted in urine during the first five days accounted for 0.5-1% of the applied dose of SM. It is also concluded that TDG and TDGO in urine existed mainly in free form, the levels of glucuronide and of sulfate conjugates of TDG or TDGO were very low, and most hydrolysis metabolites were present in the oxidized form (TDGO). The study indicates that the abnormal increase of TDG and TDGO excretion levels can be used as a diagnostic indicator and establishes a reference time-window for retrospective analysis and sampling after SM exposure.