Evaluation of aqueous dimethyl trisulfide as an antidote to a highly lethal cyanide poisoning in a large swine model.

BACKGROUND: Cyanide is a rapid acting, lethal, metabolic poison and remains a significant threat. Current FDA-approved antidotes are not amenable or efficient enough for a mass casualty incident. OBJECTIVE: The objective of this study is to evaluate short and long-term efficacy of intramuscular aqueous dimethyl trisulfide (DMTS) on survival and clinical outcomes in a swine model of cyanide exposure. METHODS: Anesthetized swine were instrumented and acclimated until breathing spontaneously. Potassium cyanide infusion was initiated and continued until 5 min after the onset of apnea. Subsequently, animals were treated with intramuscular DMTS (n = 11) or saline control (n = 10). Laboratory values and DMTS blood concentrations were assessed at various time points and physiological parameters were monitored continuously until the end of the experiment unless death occurred. A subset of animals treated with DMTS (n = 5) were survived for 7 days to evaluate muscle integrity by repeat biopsy and neurobehavioral outcomes. RESULTS: Physiological parameters and time to apnea were similar in both groups at baseline and at time of treatment. Survival in the DMTS-treated group was 90% and 30% in saline controls (p = 0.0034). DMTS-treated animals returned to breathing at 12.0 ± 10.4 min (mean ± SD) compared to 22.9 ± 7.0 min (mean ± SD) in the 3 surviving controls. Blood collected prior to euthanasia showed improved blood lactate concentrations in the DMTS treatment group; 5.47 ± 2.65 mmol/L vs. 9.39 ± 4.51 mmol/L (mean ± SD) in controls (p = 0.0310). Low concentrations of DMTS were detected in the blood, gradually increasing over time with no elimination phase observed. There was no mortality, histological evidence of muscle trauma, or observed adverse neurobehavioral outcomes, in DMTS-treated animals survived to 7 days. CONCLUSION: Intramuscular administration of aqueous DMTS improves survival following cyanide poisoning with no observed long-term effects on muscle integrity at the injection site or adverse neurobehavioral outcomes.

Analysis of the Soil Fumigant, Dimethyl Disulfide, in Swine Blood by Dynamic Headspace Gas Chromatography-Mass Spectroscopy.

Plant parasites and soilborne pathogens directly reduce the overall yield of crops, vegetables, and fruits, negatively impacting the market demand for these products and their net profitability. While preplant soil fumigation helps maintain the consistent production quality of high-value cash crops, most soil fumigants are toxic to off-target species, including humans. Dimethyl disulfide (DMDS) has recently been introduced as a relatively low toxicity soil fumigant. Although DMDS exhibits low toxicity compared to other soil fumigants, it is volatile and exposure can cause eye, nasal, and upper respiratory tract irritation, skin irritation, nausea, dizziness, headache, and fatigue. While there is one analysis method available for DMDS from biological matrices, it has significant disadvantages. Hence, in this study, a dynamic headspace gas chromatography-mass spectroscopy (DHS-GC-MS) method was developed for the analysis of DMDS in swine whole blood. This method is highly sensitive and requires only three steps: 1) acid denaturation, 2) addition of internal standard, and 3) DHS-GC-MS analysis. The method produced a wide linear range from 0.1 - 200 µM with an excellent limit of detection of 30 nM. Intra- and interassay accuracy (100±14% and 100±11%, respectively) and precision (<5% and <6% relative standard deviation, respectively) were also excellent. The method worked well to quantify the DMDS levels in the blood of dimethyl trisulfide (DMTS)-treated swine (i.e., DMDS is a byproduct of DMTS treatment) with no interfering substances at or around the retention time of DMDS (i.e., 2.7 min). This simple, rapid, and extremely sensitive method can be used for the quantification of DMDS levels in blood to verify exposure to DMDS or to monitor levels of DMDS following DMTS treatment (e.g., for cyanide poisoning).

Analysis of potential cyanide antidote, dimethyl trisulfide, in whole blood by dynamic headspace gas chromatography-mass spectroscopy.

Cyanide is a rapidly acting and highly toxic chemical. It inhibits cytochrome c oxidase in the mitochondrial electron transport chain, resulting in cellular hypoxia, cytotoxic anoxia and potentially death. In order to overcome challenges associated with current cyanide antidotes, dimethyl trisulfide (DMTS), which converts cyanide to less toxic thiocyanate in vivo, has gained much attention recently as a promising next-generation cyanide antidote. While there are three analysis methods available for DMTS, they each have significant disadvantages. Hence, in this study, a dynamic headspace (DHS) gas chromatography-mass spectroscopy method was developed for the analysis of DMTS from rabbit whole blood. The method is extremely simple, involving only acidification of a blood sample, addition of an internal standard (DMTS-d6) and DHS-GC-MS analysis. The method produced a limit of detection of 0.04 µM for DMTS with dynamic range from 0.2 to 50 µM. Inter- and intraassay accuracy (100 ± 15% and 100 ± 9%, respectively), and precision (<10% and <9% relative standard deviation, respectively) were good. The validated method performed well during pharmacokinetic analysis of DMTS from the blood of rats treated with DMTS, producing excellent pharmacokinetic parameters for the treatment of cyanide exposure. The method produced significant advantages over current methods for analysis of DMTS and should be considered as a "gold standard" method for further development of DMTS as a potential next-generation cyanide countermeasure.

Caspase-2 deficiency protects mice from diabetes-induced marrow adiposity.

Type I (T1) diabetes is an autoimmune and metabolic disease associated with bone loss. Bone formation and density are decreased in T1-diabetic mice. Correspondingly, the number of TUNEL positive, dying osteoblasts increases in bones of T1-diabetic mice. Moreover, two known mediators of osteoblast death, TNFα and ROS, are increased in T1-diabetic bone. TNFα and oxidative stress are known to activate caspase-2, a factor involved in the extrinsic apoptotic pathway. Therefore, we investigated the requirement of caspase-2 for diabetes-induced osteoblast death and bone loss. Diabetes was induced in 16-week old C57BL/6 caspase-2 deficient mice and their wild type littermates and markers of osteoblast death, bone formation and resorption, and marrow adiposity were examined. Despite its involvement in extrinsic cell death, deficiency of caspase-2 did not prevent or reduce diabetes-induced osteoblast death as evidenced by a twofold increase in TUNEL positive osteoblasts in both mouse genotypes. Similarly, deficiency of caspase-2 did not prevent T1-diabetes induced bone loss in trabecular bone (BV/TV decreased by 30 and 50%, respectively) and cortical bone (decreased cortical thickness and area with increased marrow area). Interestingly, at this age, differences in bone parameters were not seen between genotypes. However, caspase-2 deficiency attenuated diabetes-induced bone marrow adiposity and adipocyte gene expression. Taken together, our data suggest that caspase-2 deficiency may play a role in promoting marrow adiposity under stress or disease conditions, but it is not required for T1-diabetes induced bone loss.

The bone marrow microenvironment contributes to type I diabetes induced osteoblast death.

Type I diabetes increases an individual's risk for bone loss and fracture, predominantly through suppression of osteoblast activity (bone formation). During diabetes onset, levels of blood glucose and pro-inflammatory cytokines (including tumor necrosis factor α (TNFα)) increased. At the same time, levels of osteoblast markers are rapidly decreased and stay decreased chronically (i.e., 40 days later) at which point bone loss is clearly evident. We hypothesized that early bone marrow inflammation can promote osteoblast death and hence reduced osteoblast markers. Indeed, examination of type I diabetic mouse bones demonstrates a greater than twofold increase in osteoblast TUNEL staining and increased expression of pro-apoptotic factors. Osteoblast death was amplified in both pharmacologic and spontaneous diabetic mouse models. Given the known signaling and inter-relationships between marrow cells and osteoblasts, we examined the role of diabetic marrow in causing the osteoblast death. Co-culture studies demonstrate that compared to control marrow cells, diabetic bone marrow cells increase osteoblast (MC3T3 and bone marrow derived) caspase 3 activity and the ratio of Bax/Bcl-2 expression. Mouse blood glucose levels positively correlated with bone marrow induced osteoblast death and negatively correlated with osteocalcin expression in bone, suggesting a relationship between type I diabetes, bone marrow and osteoblast death. TNF expression was elevated in diabetic marrow (but not co-cultured osteoblasts); therefore, we treated co-cultures with TNFα neutralizing antibodies. The antibody protected osteoblasts from bone marrow induced death. Taken together, our findings implicate the bone marrow microenvironment and TNFα in mediating osteoblast death and contributing to type I diabetic bone loss.