[Determination of 35 antibiotics and four ß-receptor agonists in teenagers and analysis of exposure status].

Antibiotics are mainly used for disease treatment and prevention, and ß-receptor agonists are mainly used in the clinical treatment of respiratory diseases. Both types of drugs are also widely used in animal husbandry and aquaculture to promote animal growth and prevent disease. These drugs enter the human body through many routes and cause harm to human health. Teenagers are in a critical period of growth and development, and long-term antibiotic exposure may have adverse effects on their bodies. In this study, 442 teenagers aged 11-15 years were recruited from a middle school to investigate the body burden of various antibiotics and ß-receptor agonists. The seven categories of antibiotics, including five macrolides, four tetracyclines, 10 quinolones, 11 sulfonamides, three ß-lactams, one quinoxaline, and one lincosamide, and four ß-receptor agonists were determined by isotope dilution and solid phase extraction coupled with ultra performance liquid chromatography-tandem mass spectrometry. Analyte levels were corrected using urine creatinine, and detection rates were used for data analysis. Pearson's chi-squared test was used to analyze the correlations between detection rate and gender, age, or body mass index (BMI). Logistic regression was used to evaluate the correlation between detection rate and different groups after adjusting for confounding factors. The results showed that 397 teenagers had at least one antibiotic or ß-receptor agonist in their urine, with a total detection rate of 89.8%. A total of 29 antibiotics and ß-receptor agonists were detected, and the detection rate of each compound ranged from 0.2% to 59.0%. Doxycycline, oxytetracycline, and azithromycin were the top three drugs with the highest detection rates (59.0%, 56.1%, and 34.6%, respectively). Tetracyclines and macrolides were the two antibiotic categories detected most often, with detection rates of 81.9% and 42.3%, respectively. Among the antibiotics investigated, preferred veterinary antibiotics (PVAs) had the highest detection rate (85.1%), followed by human antibiotics (HAs) (41.0%). The overall detection rate of ß-receptor agonists was 2.7%. Statistical analysis showed that the male was prone to be exposed to tetracycline antibiotics (odds ratio (OR)=2.17). The detection rates of macrolides differed among the different age groups and were higher in those aged 12-13 years than in those aged 11 years. As the BMI of the teenagers increased, the detection rate of macrolides gradually increased. After adjusting for age and gender, teenagers with obesity were found to be 2.35 times more likely to be exposed to macrolides than those with a normal weight. The findings suggest that teenagers are generally exposed to low levels of antibiotics, that food and the environment may be the main sources of antibiotic exposure in teenagers, and that macrolide exposure may be associated with adolescent obesity.

The association between per-/polyfluoroalkyl substances in serum and thyroid function parameters: A cross-sectional study on teenagers living near a Chinese fluorochemical industrial plant.

Thyroid hormones (THs) play an important role in a wide range of crucial biological functions related to growth and development, and thyroid antibodies (TAs) can influence the biosynthesis of THs. Epidemiological studies have indicated that per- and polyfluoroalkyl substances (PFAS) could induce thyroid disruption, but studies on teenagers living in areas with high PFAS exposure are limited. This cross-sectional study focused on 836 teenagers (11- 15 years) living near a Chinese fluorochemical industrial plant. Decreased levels of free thyroxine (FT4, ﹤9.6 pmol/L, abnormal rate = 19.0 %) and elevated levels of free triiodothyronine (FT3, ï¹¥6.15 pmol/L, abnormal rate = 29.8 %) were observed. Correlations of serum PFAS concentrations and TAs/THs were analyzed. Increased PFOA was identified as a risk factor of decreased FT4 by using unadjusted (OR: 11.346; 95 % CI: 6.029, 21.352, p < 0.001) and adjusted (OR: 12.566; 95 % CI: 6.549, 24.115, p < 0.001) logistic regression models. In addition, significantly negative correlations were found between log10 transformed PFOA and FT4 levels using linear (unadjusted: ß = -1.543, 95 % CI: -1.937, -1.148, p < 0.001; adjusted: ß = -1.534, 95 % CI: -1.930, -1.137, p < 0.001) and BKMR models. For abnormal FT3, a significantly positive association between PFHxS and FT3 levels was observed in a regression model (unadjusted: ß = -0.903, 95 % CI: -1.212, -0.595, p < 0.001; adjusted: ß = -0.894, 95 % CI: -1.204, -0.583, p < 0.001), and PFHxS was identified as a risk factor (unadjusted: OR: 4.387; 95 % CI: 2.619, 7.346, p < 0.001; adjusted: OR: 4.527; 95 % CI: 2.665, 7.688, p < 0.001). Sensitivity analyses confirmed the robustness of the above results. This study reported the elevated PFAS exposure and thyroid function of teenagers living near a fluorochemical industrial plant from China.

[Determination of sixteen antibiotics and four ß-agonists in human urine samples using ultra-performance liquid chromatography-tandem mass spectrometry based on high-throughput automatic solid-phase extraction].

An analytical method combining high-throughput automatic solid-phase extraction with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed to determine 16 antibiotics (macrolides, tetracyclines, quinolones, and sulfonamides) and 4 ß-agonists (terbutaline, salbutamol, ractopamine, and clenbuterol) in human urine samples. After thawing at room temperature, 1 mL of urine was sampled and the internal standard was added, followed by the addition of 200 µL ammonium acetate buffer and 20 µL ß-glucuronidase, and the mixture was incubated at 37 ℃ overnight. Automatic solid-phase extraction was used to extract the target compounds from the urine samples, and the recoveries were compared using different solid-phase extraction 96-well plates (PRiME MCX, Sep-Pak C18, PRiME HLB), types and volumes of rinse solutions and eluents. Satisfactory recoveries of the 20 target compounds were obtained using the Oasis PRiME HLB 96-well plate, with 1.5 mL 10% (v/v) methanol aqueous solution and 2.0 mL methanol as the rinse solution and eluent, respectively. The eluent was concentrated under nitrogen gas at 45 ℃, and the recoveries of the target compounds were compared under different conditions (completely or almost dry, drying to 1 mL, and adding water as a protective agent), and the recovery rate was optimal when water was added as a protective agent. In this study, two types of analytical columns (ACQUITY BEH C18 and ACQUITY HSS T3) and different gradient elution procedures and mobile phases were compared. The optimal chromatographic effect was realized using an HSS T3 column (100 mm×3.0 mm, 1.8 µm) and 0.1% (v/v) formic acid aqueous solution-0.1% (v/v) formic acid in acetonitrile as the mobile phase in gradient elution at a flow rate of 0.3 mL/min. Comparing the peaks observed using different proportions of methanol aqueous solution and the initial mobile phase as the injection solvent revealed that 30% (v/v) methanol aqueous solution was the optimal solution in terms of peak shape and signal-to-noise ratio. MS was conducted using positive electrospray ionization (ESI+) in multiple reaction monitoring (MRM) mode, and the MS parameters were optimized, including the curtain (CUR) and collision gases (CAD). The standard curve obtained using this method exhibited a good linearity (correlation coefficient>0.997), and the respective limits of detection and quantification were 0.02-0.12 ng/mL and 0.06-0.41 ng/mL. At spiked levels of 0.25, 2.5, and 12.5 ng/mL, the recoveries were in the range of 81.7%-120.0% (except that of tetracycline), the intra- and inter-day RSDs (n=6) were 1.1%-11.0% and 1.2%-13.0%, respectively. Azithromycin, trimethoprim, terbutaline, salbutamol, ractopamine, and clenbuterol displayed moderate matrix effects, but all targets exhibited weak matrix effects after correction using the isotope internal standard. To evaluate the accuracy of this method, BCR-503 (containing salbutamol and clenbuterol) and internal quality control samples were used and the concentrations of salbutamol and clenbuterol were within the reference ranges. Additionally, the mean concentrations of the 20 target compounds of two different internal quality control samples after 7 measurements were in the ranges of 0.44-0.59 ng/mL (0.5 ng/mL) and 1.72-2.16 ng/mL (2.0 ng/mL), respectively, which were satisfactory. In this study, the analytical method employed automatic sample pretreatment with a 96-well solid-phase extraction plate, and the detection efficiency was considerably improved. This method displays the advantages of simple operation, ideal recovery, a high sensitivity and weak matrix effect, which satisfies the requirements for the simultaneous determination of 16 antibiotics and 4 ß-agonists in human urine samples. This study provides a crucial method for use in monitoring antibiotics and ß-agonists in human urine and studying their exposure characteristics and health risks.

Molecular mechanism for the activation of the potent hepatotoxin acetylhydrazine: Identification of the initial N-centered radical and the secondary C-centered radical intermediates.

Acetylhydrazine (AcHZ), a major human metabolite of the widely-used anti-tuberculosis drug isoniazid (INH), was considered to be responsible for its serious hepatotoxicity and potentially fatal liver injury. It has been proposed that reactive radical species produced from further metabolic activation of AcHZ might be responsible for its hepatotoxicity. However, the exact nature of such radical species remains not clear. Through complementary applications of ESR spin-trapping and HPLC/MS methods, here we show that the initial N-centered radical intermediate can be detected and identified from AcHZ activated by transition metal ions (Mn(III)Acetate and Mn(III) pyrophosphate) and myeloperoxidase. The exact location of the radical was found to be at the distal-nitrogen of the hydrazine group by 15N-isotope-labeling techniques via using 15N-labeled AcHZ we synthesized. Additionally, the secondary C-centered radical was identified unequivocally as the reactive acetyl radical by complementary applications of ESR spin-trapping and persistent radical TEMPO trapping coupled with HPLC/MS analysis. This study represents the first detection and unequivocal identification of the initial N-centered radical and its exact location, as well as the reactive secondary acetyl radical. These findings should provide new perspectives on the molecular mechanism of AcHZ activation, which may have potential biomedical and toxicological significance for future research on the mechanism of INH-induced hepatotoxicity.

Serum concentrations of per-/polyfluoroalkyl substances and its association with renal function parameters among teenagers near a Chinese fluorochemical industrial plant: A cross-sectional study.

Currently, studies on the association between per-/polyfluoroalkyl substances (PFAS) concentrations and the renal function of residents, especially teenagers, living near fluorochemical industrial plants, are relatively rare, and not all these studies suggested associations. In this cross-sectional study, 775 local teenagers (11-15 years old) were included, and serum concentrations of 18 PFAS were measured. Perfluorooctanoic acid (PFOA) was found to be the dominant PFAS with a concentration of 22.3-3310 ng/mL (mean = 191 ng/mL), accounting for 71.5-99.1% of ΣPFAS. Statistical analyses demonstrated that internal exposure of perfluoroalkyl carboxylic acids (PFCA, C8-C10) was related to the plant. In addition, the prevalence rate of chronic kidney disease (CKD) (35.0%) in the participants was relatively high. A significantly positive association was observed between the increase in PFOA concentration and increasing risk of CKD (OR = 1.741; 95% CI: 1.004, 3.088; p = 0.048) by adjusting for gender, age, body mass index (BMI), and household income. Similar positive correlation was also observed in PFHpA with CKD (OR = 1.628, 95% CI: 1.031, 2.572; p = 0.037). However, no significant correlation was observed for concentrations of other PFAS and CKD (p > 0.05). Furthermore, linear regression analyses demonstrated that none of the PFAS concentrations were significantly correlated with estimated glomerular filtration rate (eGFR) or urine albumin/urine creatinine ratio (ACR) (p > 0.05). However, a significantly negative correlation was observed between PFOA concentration and abnormal ACR (ß = -0.141, 95% CI: -0.283, 0.001; p = 0.048) after stratifying by CKD. Sensitivity analyses further confirmed these results. This cross-sectional study is the first, to our knowledge, to investigate the association between PFAS concentrations and renal function in teenagers living near a Chinese industrial plant. Further prospective and metabonomic studies are needed to interpret the results and clarify the biological mechanisms underlying this association.

Structure-Activity Relationship Investigation on Reaction Mechanism between Chlorinated Quinoid Carcinogens and Clinically-Used Aldoxime Nerve-Agent Antidote under Physiological Condition.

Pyridinium aldoximes are best-known therapeutic antidotes used for clinical treatment of poisonings by organophosphorus nerve-agents and pesticides. Recently, we found that pralidoxime (2-PAM, a currently clinically used nerve-agent antidote) could also detoxify tetrachloro-1,4-benzoquinone (TCBQ), which is a carcinogenic quinoid metabolite of the widely used wood preservative pentachlorophenol under normal physiological conditions, via an unusually mild and facile Beckmann fragmentation mechanism accompanied by radical homolysis. However, it is not clear whether the less-chlorinated benzoquinones (CnBQs, n ≤ 3) act similarly; if so, what is the structure-activity relationship? In this study, we found that (1) The stability of reaction intermediates produced by different CnBQs and 2-PAM was dependent not only on the position but also the degree of Cl-substitution on CnBQs, which can be divided into TCBQ- and DCBQ (dichloro-1,4-benzoquinone)-subgroup; (2) The pKa value of hydroxlated quinones (Cn-1BQ-OHs, the hydrolysis products of CnBQs), determined the stability of corresponding intermediates, that is, the decomposition rate of the intermediates depended on the acidity of Cn-1BQ-OHs; (3) The pKa value of the corresponding Cn-1BQ-OHs could also determine the reaction ratio of Beckmann fragmentation to radical homolysis in CnBQs/2-PAM. These new findings on the structure-activity relationship of the halogenated quinoid carcinogens detoxified by pyridinium aldoxime therapeutic agents via Beckmann fragmentation and radical homolysis reaction may have broad implications on future biomedical and environmental research.

Concentration, spatial distribution, and health risk assessment of PFASs in serum of teenagers, tap water and soil near a Chinese fluorochemical industrial plant.

Discharges released from fluorochemical industrial plants lead to severe contamination of the environment with per- and polyfluoroalkyl substances (PFASs), which may pose risks to human health. In this study, 187 serum samples from teenagers (age = 14 years), 22 tap water samples and 40 soil samples were collected in areas within 0-11 km of a fluorochemical industrial plant in Huantai County, Shandong Province, and concentrations of 18 PFASs were quantified by UPLC-MS/MS. Perfluorooctanoic acid (PFOA) was found to be predominant, concentrations of which ranged from 40.4 to 845 ng/mL in serum, from 2.88 to 19.3 ng/L in tap water, from 4.40 to 189 ng/g in soil, and accounting for 84.1-98.6%, 15.9-79.8%, and 73.8-96.7% of the total PFASs, respectively. Statistical analysis demonstrated that concentrations of perfluorinated carboxylic acids (PFCAs) in soil (C5-C9) and serum (C8-C10) were associated with the industrial plant. And PFOA concentrations in tap water were not relevant to the industrial plant, which were comparable with the non-contaminated area and lower than the threshold value recommended by U.S. EPA (70 ng/mL), indicating that the contribution to the high concentration of serum PFOA of local teenagers by drinking water was limited. Moreover, PFCAs in soil only made a limited contribution to the serum PFCAs of local residents by direct inhalation and dermal exposure, but the potential health risk by the soil via food chain should be paid attention to. Furthermore, health risk assessment demonstrated that high concentrations of PFOA in serum could pose potential health risk to local teenagers. Therefore, effective measures should be taken to attenuate the health risks caused by the industrial plant to local residents, and further epidemiological studies should be carried out in the future.

Potent Oxidation of DNA by Haloquinoid Disinfection Byproducts to the More Mutagenic Imidazolone dIz via an Unprecedented Haloquinone-Enoxy Radical-Mediated Mechanism.

Halogenated quinones are a class of carcinogenic intermediates and newly identified chlorination disinfection byproducts in drinking water. We found recently that halogenated quinones could enhance the decomposition of hydroperoxides independent of transition-metal ions and formation of the novel quinone enoxy/ketoxy radicals. Here, we show that the major oxidation product was 2-amino-5-[(2-deoxy-ß-d-erythro-pentofuranosyl)amino]-4H-imidazol-4-one (dIz) when the nucleoside 2'-deoxyguanosine (dG) was treated with tetrachloro-1,4-benzoquinone (TCBQ) and t-butyl hydroperoxide (t-BuOOH). The formation of dIz was markedly inhibited by typical radical spin-trapping agents. Interestingly and unexpectedly, we found that the generated quinone enoxy radical played a critical role in dIz formation. Using [15N5]-8-oxodG, dIz was found to be produced either directly from dG or through the transient formation of 8-oxodG. Based on these data, we proposed that the production of dIz might be through an unusual haloquinone-enoxy radical-mediated mechanism. Analogous results were observed in the oxidation of ctDNA by TCBQ/t-BuOOH and when t-BuOOH was substituted by the endogenously generated physiologically relevant hydroperoxide 13S-hydroperoxy-9Z,11E-octadecadienoic acid. This is the first report that halogenated quinoid carcinogens and hydroperoxides can induce potent oxidation of dG to the more mutagenic product dIz via an unprecedented quinone-enoxy radical-mediated mechanism, which may partly explain their potential carcinogenicity.

An unexpected new pathway for nitroxide radical production via more reactve nitrogen-centered amidyl radical intermediate during detoxification of the carcinogenic halogenated quinones by N-alkyl hydroxamic acids.

We found previously that nitroxide radical of desferrioxamine (DFO•) could be produced from the interaction between the classic iron chelating agent desferrioxamine (DFO, an N-alkyl trihydroxamic acid) and tetrachlorohydroquinone (TCHQ), one of the carconogenic quinoind metabolites of the widely used wood preservative pentachlorophenol. However, the underlying molecular mechanism remains unclear. Here N-methylacetohydroxamic acid (N-MeAHA) was synthesized and used as a simple model compound of DFO for further mechanistic study. As expected, direct ESR studies showed that nitroxide radical of N-MeAHA (Ac-(CH3)NO•) can be produced from N-MeAHA/TCHQ. Interestingly and unexpectedly, when TCHQ was substituted by its oxidation product tetrachloro-1,4-benzoquinone (TCBQ), although Ac-(CH3)NO• could also be produced, no concurrent formation of tetrachlorosemiquinone radical (TCSQ•) and TCHQ was detected, suggesting that Ac-(CH3)NO• did not result from direct oxidation of N-MeAHA by TCSQ• or TCBQ as proposed previously. To our surprise, a new nitrogen-centered amidyl radical was found to be generated from N-MeAHA/TCBQ, which was observed by ESR with the spin-trapping agents and further unequivacally identified as Ac-(CH3)N• by HPLC-MS. The final product of amidyl radical was isolated and identified as its corresponding amine. Analogous radical homolysis mechanism was observed with other halogenated quinoid compounds and N-alkyl hydroxamic acids including DFO. Interestingly, amidyl radicals were found to induce both DNA strand breaks and DNA adduct formation, suggesting that N-alkyl hydroxamic acids may exert their potential side-toxic effects via forming the reactive amidyl radical species. This study represents the first report of an unexpected new pathway for nitroxide radical production via hydrogen abstration reaction of a more reactive amidyl radical intermediate during the detoxification of the carcinogenic polyhalogenated quinones by N-alkyl hydroxamic acids, which provides more direct experimental evidence to better explain not only our previous finding that excess DFO can provide effective but only partial protection against TCHQ (or TCBQ)-induced biological damage, and also the potential side-toxic effects induced by DFO and other N-alkyl hydroxamic acid drugs.

Unexpected activation of N-alkyl hydroxamic acids to produce reactive N-centered free radicals and DNA damage by carcinogenic chlorinated quinones under normal physiological conditions.

We found recently that benzohydroxamic acid (BHA) could detoxify the chlorinated quinoid carcinogens via an unusual Lossen rearrangement reaction. However, it is not clear what would happen when the nitrogen hydrogen of BHA was substituted with methyl and other alkyl groups. Here we show that N-methyl benzohydroxamic acid (N-MeBHA, a simple model compound for the classic iron-chelator deferoxamine, which is a typical N-alkyl trihydroxamic acid) could react with 2,5-dichloro-1,4-benzoquinone (DCBQ) to form a relatively stable initial carbon-oxygen bonding conjugation intermediate CBQ-O-N-MeBHA. However, the major final product was identified, unexpectedly, as a carbon-nitrogen bonding conjugate CBQ(OH)-N(CH3)-COAr, which is the rearranged isomer of CBQ-O-N-MeBHA. Interestingly, a new 18-line nitrogen-centered radical and a carbon-centered quinone ketoxy radical were observed by the ESR spin-trapping method, which was further confirmed by HPLC-MS and 15N-isotope labeling methods. We further found that both new DNA adducts and DNA strand breaks could be produced by the reactive nitrogen-centered radical. Taken together, we propose that the reaction between DCBQ and N-MeBHA was not via the Lossen rearrangement, but rather through a novel radical homolysis and recoupling pathway. Analogous results were observed for other chlorinated quinones and N-alkyl hydroxamic acids including the widely-used trihydroxamate iron-chelating drug deferoxamine. This represents the first report of unexpected radical pathway for the reaction between chlorinated quinones and N-alkyl hydroxamic acids under normal physiological conditions, which may have broad biological and environmental significance for future study of carcinogenic chloroquinones and hydroxamic acid drugs.

Molecular mechanism for the activation of the anti-tuberculosis drug isoniazid by Mn(III): First detection and unequivocal identification of the critical N-centered isoniazidyl radical and its exact location.

Isoniazid (INH), the most-widely used anti-tuberculosis drug, has been shown to be activated by Mn(III) to produce the reactive carbon-centered isonicotinic acyl radical, which was considered to be responsible for its anti-tuberculosis activity. However, it is still not clear whether the previously-proposed N-centered isoniazidyl radical intermediate can be initially produced or not; and if so, what is its exact location on the hydrazine group, distal- or proximal-nitrogen? Through complementary applications of ESR spin-trapping and HPLC/MS methods, here we show that the characteristic and transient N-centered isoniazidyl radical intermediate can be detected and identified from INH activation uniquely by Mn(III)Acetate not by Mn(III) pyrophosphate. The exact location of the radical was found to be at the distal-nitrogen of the hydrazine group by 15N-isotope-labeling techniques via using 15N-labeled INH. Diisonicotinyl hydrazine was identified as a new reaction product from INH/Mn(III). Analogous results were observed with other hydrazides. This study represents the first detection and unequivocal identification of the initial N-centered isoniazidyl radical and its exact location. These findings should provide a new perspective on the molecular mechanism of INH activation, which may have broad biomedical and toxicological significance for future research for more efficient hydrazide anti-tuberculosis drugs.

Mechanism of unprecedented hydroxyl radical production and site-specific oxidative DNA damage by photoactivation of the classic arylhydroxamic acid carcinogens.

The carcinogenicity of N-hydroxy-2-acetamidofluorene (N-OHAAF), the major genotoxic metabolite of the classic model aromatic amine (AA) carcinogen 2-acetylaminofluorene, has been attributed mainly to the formation of DNA adducts via arylnitrenium upon enzymatic activation. Here, we show, unexpectedly, that exposure of N-OHAAF to UV or sunlight irradiation can not only induce the formation of the well-known covalent DNA adducts, but, more interestingly, simultaneous generation of oxidative DNA damage was also observed as measured by the formation of DNA single-/double-strand breaks (SSBs/DSBs) and 8-oxo-2'-deoxyguanosine (8-oxodG), which were partly inhibited by the typical hydroxyl radical (•OH) scavengers. Electron spin resonance spin-trapping and fluorescent studies unequivocally confirmed that the highly reactive •OH was generated from photolysis of N-OHAAF. Further DNA sequencing investigations suggest that photoactivation of N-OHAAF caused preferential cleavage at guanine, thymine and cytosine sites. More importantly, the formation of 8-oxodG and DSBs were also observed when fibroblast Balb/c-3T3 cells were co-exposed to N-OHAAF/UV irradiation as measured by double immunofluorescence staining. Taken together, we propose that both •OH and amidyl radicals can be readily produced via N-OH homolysis in N-OHAAF by photoirradiation, which can induce both oxidative and covalent DNA damage. This represents the first report of •OH production and site-specific DNA damage via photoactivation of the genotoxic hydroxamic acid intermediate, which provides a new free radical perspective to better understand the molecular mechanism for the carcinogenicity of AAs.

An unusual double radical homolysis mechanism for the unexpected activation of the aldoxime nerve-agent antidotes by polyhalogenated quinoid carcinogens under normal physiological conditions.

We have recently shown that the pyridinium aldoximes, best-known as therapeutic antidotes for chemical warfare nerve-agents, could markedly detoxify the carcinogenic tetrachloro-1,4-benzoquinone (TCBQ) via an unusual double Beckmann fragmentation mechanism. However, it is still not clear why pralidoxime (2-PAM) cannot provide full protection against TCBQ-induced biological damages even when 2-PAM was in excess. Here we show, unexpectedly, that TCBQ can also activate pralidoxime to generate a reactive iminyl radical intermediate in two-consecutive steps, which was detected and unequivocally characterized by the complementary application of ESR spin-trapping, HPLC/MS and nitrogen-15 isotope-labeling studies. The same iminyl radical was observed when TCBQ was substituted by other halogenated quinones. The end product of iminyl radical was isolated and identified as its corresponding reactive and toxic aldehyde. Based on these data, we proposed that the reaction of 2-PAM and TCBQ might be through the following two competing pathways: a nucleophilic attack of 2-PAM on TCBQ forms an unstable transient intermediate, which can decompose not only heterolytically to form 2-CMP via double Beckmann fragmentation, but also homolytically leading to the formation of a reactive iminyl radical in double-steps, which then via H abstraction and further hydrolyzation to form its corresponding more toxic aldehyde. Analogous radical homolysis mechanism was observed with other halogenated quinones and pyridinium aldoximes. This study represents the first detection and identification of reactive iminyl radical intermediates produced under normal physiological conditions, which provides direct experimental evidence to explain only the partial protection by 2-PAM against TCBQ-induced biological damages, and also the potential side-toxic effects induced by 2-PAM and other pyridinium aldoxime nerve-agent antidotes.

Unusual Double Beckmann Fragmentation Reaction under Physiological Conditions.

Pyridinium aldoximes, which are best-known as therapeutic antidotes for organophosphorus chemical warfare nerve-agents and pesticides, have been found to markedly detoxify polyhalogenated quinones, which are a class of carcinogenic intermediates and recently identified disinfection byproducts in drinking water. However, the exact chemical mechanism underlying this detoxication remains unclear. Here we demonstrate that pralidoxime can remarkably facilitate the dechlorination/hydroxylation of the highly toxic tetrachloro-1,4-benzoquinone in two-consecutive steps to generate the much less toxic 2,5-dichloro-3,6-dihydroxy-1,4-benzoquonine, with rate enhancements of up to 180 000-times. On the contrary, no accelerating effect was noticed with O-methylated pralidoxime. The major reaction product from pralidoxime was identified as its corresponding nitrile (2-cyano-1-methylpyridinium chloride). Along with oxygen-18 isotope-labeling studies, a reaction mechanism was proposed in which nucleophilic substitution coupled with an unprecedented double Beckmann fragmentation reaction was responsible for the dramatic enhancement in the detoxification process. This represents the first report of an unusually mild and facile Beckmann-type fragmentation that can occur under normal physiological conditions in two-consecutive steps. The study may have broad biomedical and environmental significance for future investigations of aldoxime therapeutic agents and carcinogenic polyhalogenated quinones.

Mechanism of Intrinsic Chemiluminescence Production from the Degradation of Persistent Chlorinated Phenols by the Fenton System: A Structure-Activity Relationship Study and the Critical Role of Quinoid and Semiquinone Radical Intermediates.

We found recently that intrinsic chemiluminescence (CL) could be produced by all 19 chlorophenolic persistent organic pollutants during environmentally friendly advanced oxidation processes. However, the underlying mechanism for the structure-activity relationship (SAR, i.e., the chemical structures and the CL generation) remains unclear. In this study, we found that, for all 19 chlorophenol congeners tested, the CL increased with an increasing number of chlorine atoms in general; and for chlorophenol isomers (such as the 6 trichlorophenols), the CL decreased in the order of meta- > ortho-/para-Cl-substituents with respect to the -OH group of chlorophenols. Further studies showed that not only chlorinated quinoid intermediates but also, more interestingly, chlorinated semiquinone radicals were produced during the degradation of trichlorophenols by the Fenton reagent; and the type and yield of which were determined by the directing effects, hydrogen bonding, and steric hindrance effect of the OH- and/or Cl-substitution groups. More importantly, a good correlation was observed between the formation of these quinoid intermediates and CL generation, which could fully explain the above SAR findings. This represents the first report on the structure-activity relationship study and the critical role of quinoid and semiquinone radical intermediates, which may have broad chemical and environmental implications for future studies on remediation of other halogenated persistent organic pollutants by advanced oxidation processes.

Mechanism of synergistic DNA damage induced by the hydroquinone metabolite of brominated phenolic environmental pollutants and Cu(II): Formation of DNA-Cu complex and site-specific production of hydroxyl radicals.

2,6-Dibromohydroquinone (2,6-DBrHQ) has been identified as an reactive metabolite of many brominated phenolic environmental pollutants such as tetrabromobisphenol-A (TBBPA), bromoxynil and 2,4,6-tribromophenol, and was also found as one of disinfection byproducts in drinking water. In this study, we found that the combination of 2,6-DBrHQ and Cu(II) together could induce synergistic DNA damage as measured by double strand breakage in plasmid DNA and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formation, while either of them alone has no effect. 2,6-DBrHQ/Cu(II)-induced DNA damage could be inhibited by the Cu(I)-specific chelating agent bathocuproine disulfonate and catalase, but not by superoxide dismutase, nor by the typical hydroxyl radical (•OH) scavengers such as DMSO and mannitol. Interestingly, we found that Cu(II)/Cu(I) could be combined with DNA to form DNA-Cu(II)/Cu(I) complex by complementary application of low temperature direct ESR, circular dichroism, cyclic voltammetry and oxygen consumption methods; and the highly reactive •OH were produced synergistically by DNA-bound-Cu(I) with H2O2 produced by the redox reactions between 2,6-DBrHQ and Cu(II), which then immediately attack DNA in a site-specific manner as demonstrated by both fluorescent method and by ESR spin-trapping studies. Further DNA sequencing investigations provided more direct evidence that 2,6-DBrHQ/Cu(II) caused preferential cleavage at guanine, thymine and cytosine residues. Based on these data, we proposed that the synergistic DNA damage induced by 2,6-DBrHQ/Cu(II) might be due to the synergistic and site-specific production of •OH near the binding site of copper and DNA. Our findings may have broad biological and environmental implications for future research on the carcinogenic polyhalogenated phenolic compounds.

An Exceptionally Facile Two-Step Structural Isomerization and Detoxication via a Water-Assisted Double Lossen Rearrangement.

N-hydroxyphthalimide (NHPI), which is best known as an organocatalyst for efficient C-H activation, has been found to be oxidized by quinoid compounds to its corresponding catalytically active nitroxide-radical. Here, we found that NHPI can be isomerized into isatoic anhydride by an unusually facile two-step method using tetrachloro-1,4-benzoquinone (TCBQ, p-chloranil), accompanied by a two-step hydrolytic dechlorination of highly toxic TCBQ into the much less toxic dihydroxylation product, 2,5-dichloro-3,6-dihydroxy-1,4-benzoquinone (chloranilic acid). Interestingly, through the complementary application of oxygen-18 isotope-labeling, HPLC combined with electrospray ionization quadrupole time-of-flight and high resolution Fourier transform ion cyclotron resonance mass spectrometric studies, we determined that water was the source and origin of oxygen for isatoic anhydride. Based on these data, we proposed that nucleophilic attack with a subsequent water-assisted Lossen rearrangement coupled with rapid intramolecular addition and cyclization in two consecutive steps was responsible for this unusual structural isomerization of NHPI and concurrent hydroxylation/detoxication of TCBQ. This is the first report of an exceptionally facile double-isomerization of NHPI via an unprecedented water-assisted double-Lossen rearrangement under normal physiological conditions. Our findings may have broad implications for future research on hydroxamic acids and polyhalogenated quinoid carcinogens, two important classes of compounds of major chemical and biological interest.

Unexpected unrelated umbilical cord blood stem cell engraft in two patients with severe aplastic anemia that received immunosuppressive treatment: A case report and literature review.

Severe aplastic anemia (SAA) is a life-threatening bone marrow disorder. Bone marrow transplantation is the primary therapy for SAA; however, its efficacy is limited by numerous factors, including lack of histocompatible sibling donor, patient age and graft-versus-host-disease (GVHD) following transplantation. Immunosuppressive treatment (IST) is the first procedure developed for patients without a sibling donor. Our previous study reported that patients administered enhanced IST, in addition to a regime of unrelated umbilical cord blood (UCB) transfusion, exhibited higher efficiency and a reduced rate of relapse. Therefore, the present study reported the cases of 2 patients that received enhanced IST plus unrelated UCB transfusion. These patients exhibited complete hematological recovery with an increased rate of mixed chimerism and demonstrated no signs of GVHD or relapse during the 2-year follow-up period. Thus, enhanced immunosuppressive treatment (low-dose cyclophosphamide and antithymocyte globulin) combined with UCB transfusion may be an effective treatment for patients with SAA.

Assessment of the use of immunosuppressants combined with cord blood for severe aplastic anemia.

BACKGROUND: The objective of this study was two-fold: 1) to investigate the changes of cytokines concentration in relation to severe aplastic anemia (SAA) when treated with immunosuppressants combined with cord blood (IS + CBI). and 2) to assess the curative effect of umbilical cord blood chimerism engraftment. METHODS: We selected 43 patients with SAA all treated with IS + CBI (newly diagnosed group). Among them, a total of 33 patients were treated effectively (effective group) while 10 cases were treated invalidly (invalid group). An additional 20 healthy individuals were selected as control (control group). The expression levels of IL-17, IL-22 and other cytokines in each group were detected by ELISA. The engraftment of cord blood stem cells was detected by using short tandem repeat-polymerase chain reaction (STR-PCR). RESULTS: 1. IL-17, IL-22 and other cytokines expressions in the newly diagnosed group were significantly higher than in the control group. 2. After six months, the levels in the effective group were significantly lower than pre-therapy levels (P < 0.05). The levels in the invalid group did not differ to those observed prior treatment. 3. After one and three months of treatment, a small amount of engraftment was found in the effective group. However, after six months, transplant rejection was observed in all patients. No effective engraftment was observed in the invalid group. CONCLUSION: 1) Th17 and Th22 producing cells in SAA patients significantly increased indicating a positive correlation between these biomarkers and the progression of SAA. 2) During the IS + CBI treatment the maintenance of a normal hematopoietic function depended on immunesup-pressants. Early umbilical cord blood chimerism engraftment may promote hematopoietic recovery.