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.

Absorption, distribution and pathological injury in mice due to ricin poisoning via the alimentary pathway.

The aim of this work was to investigate the potential interactions between intestinal absorbance and ricin poisoning. The Caco-2 cell monolayer and everted intestinal sac (VEIS) models were used. The distribution of ricin in CD-1 mice intoxicated with 0.1 mg/kg of ricin intragastrically was determined by immunohistochemistry. The results showed that ricin could not transfer across the healthy Caco-2 cell monolayer within three hours after poisoning. However, it could pass through the everted rat intestinal wall after 0.5 h of incubation. The toxin in the liver, spleen, lungs and kidneys of mice could be detected as early as 1 h after intoxication. The pathological results were in accordance with the cytotoxicities of ricin in Caco-2, HepG 2, H1299 and MDCK cells, indicating that though no significant symptom in mice could be observed within 3 h after ricin intoxication, important tissues, especially the kidneys, were being injured by the toxin and that the injuries were progressing.

Facile and sensitive measurement of GSH/GSSG in cells by surface-enhanced Raman spectroscopy.

Reduced glutathione (GSH) and the oxidized glutathione (GSSG) are well-known biomolecules in the main constituents of intracellular redox homeostasis system. A rapid, accurate measurement of cellular GSH and GSSG is quite needed in investigating important biochemical events. In this work, we present a novel and sensitive method to monitor intracellular GSH and GSSG concentrations by a portable surface-enhanced Raman spectroscopy (SERS) technique. We introduced a reduction-sensitive reaction-type Raman probe, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) to initiate GSH reduction, itself concomitantly converts to 2-nitro-5-thiobenzoic acid (TNB) to release a strong SERS signal. In a convenient way of inorganic salt MgSO4 induced aggregation of silver nanoparticles substrate, we easily implemented a good discrimination between DTNB and TNB, and a quantitative measurement of GSH and GSSG with a high sensitivity of 10 nM. This SERS method proved its feasible applicability in rapidly and sensitively monitoring GSH depletion behaviors of some notorious alkylating agents, i.e., sulfur mustard and nitrogen mustards in ex vitro or in vitro (cellular response). This SERS method may be very worthwhile in cellular detoxication event via the GSH approach and other GSH involved biomedical researches.

Biological effects of adipocytes in sulfur mustard induced toxicity.

Sulphur mustard (2,2'-dichloroethyl sulfide; SM) is a vesicant chemical warfare agent whose mechanism of acute or chronic action is not known with any certainty and to date there is no effective antidote. SM accumulation in adipose tissue (AT) has been originally verified in our previous study. To evaluate the biological effect caused by the presence of abundant SM in adipocyte and assess the biological role of AT in SM poisoning, in vitro and in vivo experiments were performed. High content analysis revealed multi-cytotoxicity in SM exposed cells in a time and dose dependent manner, and adipocytes showed a relative moderate damage compared with non-adipocytes. Cell co-culture model was established and revealed the adverse effect of SM-exposed adipocyte supernatant on the growth of co-cultured cells. The pathological changes in AT from 10mg/kg SM percutaneously exposed rats were checked and inflammation phenomena were observed. The mRNA and protein levels of inflammation-related adipokines secreted from AT in rats exposed to 1, 3 and 10mg/kg doses of SM were determined by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assays. The expressions of proinflammatory and anti-inflammatory adipokines together promoted the inflammation development in the body. The positive correlations between AT and serum adipokine levels were explored, which demonstrated a substantial role of AT in systemic inflammation responding to SM exposure. Thus, AT is not only a target of SM but also a modulator in the SM toxicity.

Monoclonal antibody, mAb 4C13, an effective detoxicant antibody against ricin poisoning.

Ricin is a glycoprotein produced in castor seeds and consists of two polypeptide chains named Ricin Toxin A Chain (RTA) and Ricin Toxin B Chain (RTB), linked via a disulfide bridge. Due to its high toxicity, ricin is regarded as a high terrorist risk for the public. However, antibodies can play a pivotal role in neutralizing the toxin. In this research, the anti-toxicant effect of mAb 4C13, a monoclonal antibody (mAb) established using detoxicated ricin as the immunized antigen, was evaluated. Compared with mAb 4F2 and mAb 5G6, the effective mechanism of mAb 4C13 was analyzed by experiments relating to its cytotoxicity, epitope on ricin, binding kinetics with the toxin, its blockage on the protein synthesis inhibition induced by ricin and the intracelluar tracing of its complex with ricin. Our result indicated that mAb 4C13 could recognize and bind to RTA, RTB and exert its high affinity to the holotoxin. Both cytotoxicity and animal toxicity of ricin were well blocked by pre-incubating the toxin with mAb 4C13. By intravenous injection, mAb 4C13 could rescue the mouse intraperitoneally (ip) injected with a lethal dose of ricin (20µg/kg) even at 6h after the intoxication and its efficacy was dependent on its dosage. This research indicated that mAb 4C13 could be an excellent candidate for therapeutic antibodies. Its potent antitoxic efficiency was related to its recognition on the specific epitope with very high affinity and its blockage of protein synthesis inhibition in cytoplasm followed by cellular internalization with ricin.

Systematic safety evaluation on photoluminescent carbon dots.

Photoluminescent carbon dots (C-dots) were prepared using the improved nitric acid oxidation method. The C-dots were characterized by tapping-mode atomic force microscopy, and UV-vis absorption spectroscopy. The C-dots were subjected to systematic safety evaluation via acute toxicity, subacute toxicity, and genotoxicity experiments (including mouse bone marrow micronuclear test and Salmonella typhimurium mutagenicity test). The results showed that the C-dots were successfully prepared with good stability, high dispersibility, and water solubility. At all studied C-dot dosages, no significant toxic effect, i.e., no abnormality or lesion, was observed in the organs of the animals. Therefore, the C-dots are non-toxic to mice under any dose and have potential use in fluorescence imaging in vivo, tumor cell tracking, and others.

Carbon dots: a safe nanoscale substance for the immunologic system of mice.

We aimed at investigating the effect of carbon dots on the BALB/c mice immune system. Mice were respectively treated with different doses of carbon dots and saline. At 1 and 9 days after intravenous administration of carbon dots, splenocyte proliferation, subpopulation of the peripheral lymphocytes, and induction of primary immune responses in mice were investigated. The results showed that high dose of carbon dots could promote the percentages of CD3+ and interferon-γ (IFN-γ) secretion and decrease the proportions of CD4+/CD8+ on the first day after administration. At 9 days post exposure, the proliferation of splenocytes had a significant increase. IFN-γ secretion and proportions of CD3+/CD19+ were also found to have an obvious promotion, and both the percentages of CD4+ and CD8+ T lymphocytes were raised, whereas the expression of cytokines made little change in the treated groups, except for IL-12 which had a slight increase in the 50-mg/kg group. The weight coefficients and histological analysis of the spleen and thymus of the treated mice exerted fewer differences compared with those from the control mice. It suggests that carbon dots could influence the immune functions of normal BALB/c mice by inducing Th1 and Tc responses and that these effects were not enough to induce the morphological change of the immune organs.

Establishment of a method to determine the magnetic particles in mouse tissues.

This work is aimed to evaluate a method to detect the residual magnetic nanoparticles (MNPs) in animal tissues. Ferric ions released from MNPs through acidification with hydrochloric acid can be measured by complexation with potassium thiocyanate. MNPs in saline could be well detected by this chemical colorimetric method, whereas the detected sensitivity decreased significantly when MNPs were mixed with mouse tissue homogenates. In order to check the MNPs in animal tissues accurately, three improvements have been made. Firstly, proteinase K was used to digest the proteins that might bind with iron, and secondly, ferrosoferric oxide (Fe3O4) was collected by a magnetic field which could capture MNPs and leave the bio-iron in the supernatant. Finally, the collected MNPs were carbonized in the muffle furnace at 420°C before acidification to ruin the groups that might bind with ferric ions such as porphyrin. Using this method, MNPs in animal tissues could be well measured while avoiding the disturbance of endogenous iron and iron-binding groups.