Ricin is a toxalbumin derived from the castor bean plant, Ricinus communis. Ricinine is an alkaloid (3-cyano-4-methoxy-N-methyl-2-pyridone) that shares a common plant source with ricin, and its presence in urine infers ricin exposure. A new quantification method for ricinine was developed that uses solid-phase extraction to prepare 1-mL urine samples (81% recovery) for a 5-min, isocratic high-performance liquid chromatography method, followed by electrospray ionization tandem mass spectrometry. Protonated molecular ions were selected in the multiple reaction monitoring mode and quantified by isotope dilution with (13)C(6)-labelled ricinine as the internal reference. Urine pools enriched with ricinine at two concentrations were characterized as quality control materials and then used to validate the method. The method limit of quantification was 0.083 ng/mL, even with a confirmation ion of low relative abundance. Ricinine was stable in human urine when heated at 90 degrees C for 1 h, and during storage at 25 degrees C and 5 degrees C for 3 weeks. The method was applied to an animal exposure study, a crude ricin preparation scheme, and a forensic analysis. These studies show that ricinine can be measured in rat urine at least 48 h after exposure. Ricinine is present in crude preparations of ricin, and it can be found in human urine after a lethal exposure to ricin.

The nitrogen mustards bis(2-chloroethyl)ethylamine (HN1), bis(2-chloroethyl)methylamine (HN2), and tris(2-chloroethyl)amine (HN3) have the potential to be used as chemical terrorism agents because of their extreme vesicant properties. We modified a previously reported method to incorporate automated solid-phase extraction, improve chromatography, and include the urinary metabolite for HN3. The improved method was used to measure levels of the urinary metabolites N-ethyldiethanolamine (EDEA), N-methyldiethanolamine (MDEA), and triethanolamine (TEA) in rats dosed with HN1, HN2, and HN3, respectively, and to establish background levels of EDEA, MDEA, and TEA in human urine samples from a population with no known exposure to nitrogen mustards. Rat dosing experiments confirmed that EDEA, MDEA, and TEA could be detected in urine for at least 48 h after exposure to HN1, HN2, and HN3, respectively. Substantial amounts of EDEA (89 ng/mL), MDEA (170 ng/mL), and TEA (1105 ng/mL) were measured in the urine of rats exposed to 10 mg HN1, HN2, and HN3, respectively, 48 h after exposure. The background concentrations for TEA in the human population ranged from below the limit of detection (LOD 3 ng/mL) to approximately 6500 ng/mL. Neither EDEA (LOD 0.4 ng/mL) nor MDEA (LOD 0.8 ng/mL) was detected above the LOD in the human samples.

: Surgical production of the isolated, perfused porcine skin flap (IPPSF) model involved resection and suturing of an area of inguinal skin to form a tubular flap. Two days after production, the flap was cannulated and perfused in an environmentally controlled chamber. The objective of this work was to establish the IPPSF technology at Battelle for examining the dermatotoxicity of topically applied sulfur mustard (HD) and for assessing candidate countermeasures. Data obtained from an initial set of untreated flaps indicated that the technology transfer was successful. However, subsequent flaps exhibited a slow deadline in metabolism regardless of treatment (untreated, ethanol, or HD in ethanol). The effects of HD in ethanol were not observed as previously reported. Flaps perfused with media made with a different bovine serum albumin exhibited some of the anticipated effects, but these changes were independent of topical flap treatment Battelle personnel were not able to consistently duplicate the effects of HD applied on flaps as previously reported. Flap performance appeared to be highly sensitive to variations in vascular anatomy, harvesting procedures, and perfusion media composition and pH. The results indicated that the IPPSF model was unsuitable for assessing the efficacy of candidate countermeasures against topically applied HD.

Abstract : The mouse ear model has been used at the U.S. Army Medical Institute of chemical Defense (USAMRICD) to assess dermal injury caused by sulfur mustard (HD) and to evaluate potential pre- and posttreatment compounds for efficacy against percutaneous HD injury. In this model, the effects of HD with or without treatment compounds are evaluated macroscopically by edema and microscopically by histopathological changes associated with HD injury. The objectives of Task 9543 were first to transition this technique to the Battelle Medical Research and Evaluation Facility (MREF), and then to evaluate candidate antivesicant drugs for their efficacy at eliminating or lessening the effects of HD-induced tissue damage. By June 1997 the mouse ear model had been transitioned and validated at the MREF. The MREF has evaluated 334 candidate HD treatment compounds provided by USAMRICD, and 73 passed the initial screening. Of these, 23 were anti-inflammatory agents, 21 were protease inhibitors, 15 were inhibitors of poly-ADP-ribose polymerase, 12 were HD scavengers, and 2 were other classes of compounds. Several of the successful HD scavengers and anti-inflammatory agents were also tested as posttreatments (applied 10 minutes after HD) and showed some efficacy.

: The objective of this study was to investigate the pharmacologic modulation of biomarkers for evaluating therapeutics against HD. The molecular biomarkers investigated, using a ribonuclease protection assay (RPA) to determine messenger ribonucleic acid (mRNA) levels, were mediators of inflammation, including several cytokines and chemokines, tenascin, and ornithine decarboxylase. Biochemical biomarkers investigated were serum amyloid P (SAP), interleukin-6 (IL-6), and interleukin-1 alpha (IL-1alpha) using ELISAs, and activity of myeloperoxidase (MPX) using a spectrophotometric method. Other endpoints included histopathology and edema measurements. Exposure to HE) resulted in a time-dependent increase in mRNA levels of monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-2 (MiP-2), macrophage inflammatory protein-1 alpha (MIP-1alpha) and interleukin- 1 beta (IL-1Beta). Exposure to HD also resulted in edema and apparent increases in the protein levels of SAP and IL-6, and in the activity of MPX. Four drug treatments, olvanil, dexamethasone, hydrocortisone, and indometbacin, were shown to modulate HD-induced inflammation.

Vesication and skin irritation studies were conducted in hairless guinea-pigs to determine the vesicant and skin irritation potential of chemically-neutralized Chemical Agent Identification Sets (CAIS), The CAIS are training items that contain chemical warfare-related material-sulfur mustard (HD), nitrogen mustard (HN) or lewisite (L)-and were declared obsolete in 1971, Animals were dosed topically with 'test article'-neat HD, 10% agent/chloroform solutions or product solutions (waste-streams) from neutralized CAIS-and evaluated for skin-damaging effects (gross and microscopic). Product solutions from the chemical neutralization of neat sulfur mustard resulted in microvesicle formation, All agent-dosed (HD or agent/chloroform solutions) sites manifested microblisters as well as other histopathological lesions of the skin. Waste-streams from the neutralization of agent (agent/chloroform or agent/charcoal) were devoid of vesicant activity. Cutaneous effects (erythema and edema) were consistent with the skin-injurious activity associated with the neutralizing reagent 1,3-dichloro-5,5-dimethylhydantoin (DCDMH), Chemical neutralization of CAIS was effective in eliminating/reducing the vesicant property of CAIS containing agent in chloroform or agent on charcoal but was inefficient in reducing the vesicant potential of CAIS containing neat sulfur mustard, (C) 1998 John Wiley & Sons, Ltd.

A study is being initiated to investigate subtle neurobehavioral effects and neuropathology in rats due to exposure to combinations of low levels of Sarin (GB), N,N-diethyl-m-toluamide (DEET), chlorpyrifos (CPF), and pyridostigmine bromide (PB). A similar study is being initiated in rhesus monkeys to investigate neurophysiologic effects and neuromuscular pathology due to exposure to a combination of GB, DEET, CPF, and PB, along with vaccination with botulinum toxoid. A description of these studies is presented.

Abstract : The objective of this work was to determine the dose-response relationship between the duration of a topical exposure of sulfur mustard vapors to hairless guinea pigs (HGPs) and the incidence of microblisters observed histologically. The dose-response curve was compared to results of similar work performed at the U.S. Army Medical Research Institute of Chemical Defense (USAMRICD). The effects of ketamine hydrochloride anesthesia on HGP skin reflectance were also examined. The exposure time needed to produce a 50 percent incidence of microblisters in HGP skin exposed to saturated HD vapors was approximately 7.5 min. Comparison with USAMRICD study results indicated that the HGPs used in the present study were more tolerant of HD vapors by approximately 2 min. However, the probit analysis slopes exhibited the same rate of increase in microblisters as a function of HD vapor exposure time. The model was thus validated for use in screening prophylactic and therapeutic treatments against mustard injury. Ketamine hydrochloride anesthesia significantly reduced the baseline (pre-dose) level of red chromaticity on HGP skin test sites.

Production of methemoglobin (met Hb) is effective in treating or preventing cyanide intoxication due to the affinity of cyanide for met Hb. This paper describes an automated method for measuring met Hb in mouse blood utilizing the Roche COB AS FARA centrifugal analyzer and a modified Evelyn–Malloy procedure. Blood samples were spiked with various quantities of potassium ferricyanide to produce met Hb, and automated and manual analyses of these samples were compared. Following validation of the COB AS method, two known met Hb inducers, sodium nitrite and p-aminopropiophenone (PAPP), and two sulfur donor compounds, sodium thiosulfate and ICD #1021, not known to produce met Hb were tested in vivo. Five mice per compound were injected IP and blood samples were analyzed periodically over a time period of approximately 2 h. The automated technique requires only a drop of blood, which allows repeated sampling from the same animal, and can be adapted for blood from different species. A nalyses are rapid and sensitive, and results are reproducible.

: Two human skin culture models, also known as human skin equivalents (HSEs), and dermatomed (split thickness) natural human skin (NHS) were assessed for use in studies investigating the dermatotoxic effects of sulfur mustard BIS(2-CHLOROETHYL) SULFIDE; HD. The HSEs examined were model ZK1300 from Advanced Tissue Sciences (ATS, La Jolla, CA) and Epiderm from MatTek Corporation (Ashland, MA). NHS specimens were obtained either fresh from the Ohio State University branch of the Cooperative Human Tissue Network (Columbus, OH), or cryopreserved from the Ohio Valley Tissue and Skin Center (OVTSC, Cincinnati, OH). The utility of each skin model was assessed by histopathology and by several viability indices. Tissue samples were processed for both light and electron microscopy and examined for consistency among HSE lots and among NHS patients in exhibiting normal ultrastructural details. In particular, the samples were examined for the presence of a continuous, intact basement membrane and an epidermal-dermal interface resembling that found in normal, living human skin.

The efficacies of atropine (ATR)/ 2-PAM and ATRl HI-6 in treating male rhesus monkeys injected with the neurotoxic organophosphonate (OP) agents GA, GB, GD, GF, or VX were compared. Experiments were conducted using no more than 8 monkeys per OP and treatment regimen. Doses were selected using a modified up-and-down experimental design, challenging one monkey per day per OP and treatment. Results were used to approximate the median lethal dose (MLD) for groups of treated monkeys or monkeys given only a vehicle following challenge with an OP. Mortality and signs of intoxication with each treatment were statistically compared. Doses of 2-PAM (25.7 mgl kg) or HI-6 (50 mgl kg) and atropine (0.4 mg free base! kg) were given in a single intramuscular (IM) injection 1 min following challenge with an OP. Strong and well-defined relationships between agent dose and 10-h lethality were observed in untreated animals. The 10-h IM OP MLDs for untreated monkeys were estimated to be 80, 43, 8.0, 22, and 7.4 μg/kg for GA, GB, GD, GF, and VX, respectively. No statistical differences w ere found between AT Rl 2-PAM and AT Rl HI-6 treatment efficacies in preventing lethality for any of the OPs. Both oxime combinations appear to provide protection against a 2 × 10-h MLD of GA, GF, or VX; only

: Vesication and skin irritation studies were conducted in hairless guinea pigs to ascertain the vesicant and skin irritation potential of chemically-neutralized Chemical Agent Identification Sets (CAIS). CAIS are training items that contain agent (HD, HN, or L) and were declared obsolete in 1971. Animals were dosed with 'test article' EITHER NEAT HD, 10% agent/chloroform solutions or product solutions (wastestreams) and evaluated for skin-damaging effects (gross and light microscopic). Product solutions from the chemical neutralization of CAIS (either agent/chloroform or agent/charcoal) produced no microblisters. Dermal application of product solutions from the neutralization of neat HD resulted in microvesicle formation (vesication). All agent-dosed (either agent/chloroform solutions or HD) sites exhibited micro- blisters as well as other histopathologic lesions. Skin irritant effects were consistent with the skin-injurious activity (erythema and edema) associated with the neutralizing reagent 1 ,3-DICHLORO-5,5-DIMETHYLHYDANTOIN (DCDMH).

Studies were conducted under this task to Assess the time and concentration dependent nature of niacinamide (NM) protection against HD-induced NAD+ depletion and cytotoxicity. The HD concentrations used assess the time dependent nature of cytotoxicity and NAD+ depletion, and the impact of NM and niacin (NI) treatment were 13,62, 101, and 171 mu-M HD. Three concentrations of NM and NI were (0.01,0.1 and 1 mM) selected by USAMRICD for evaluation at 2, 4,8, 12, 16,20,24,48, and 72 hours after exposure to HD. Cytotoxicity and total culture NAD+ content were assessed. NAD+ concentrations following the addition of 1 mM NM frequently were significantly greater than those observed for the HD-exposed controls especially at 171mu-M HD. Multiple addition of NM had little protective effect relative to that by provided by pretreatment alone. At the 171mu-M HD concentration, the single addition of NM provided marginal but statistically significant (p<=0.05) protection. Comparisons of the different NM addition modes yielded mixed results, but usually the number of viable cells was greater with multiple additions of NM. NI did not provide protection against HD-induced NAD+ depletion or cytotoxicity.

: The U.S. Army Project Manager for Non-stockpile Chemical Materiel (PMNSCM) has a requirement to develop and field a transportable Rapid Response System (RRS) to chemically treat Chemical Agent Identification Sets (CAIS) containing vesicant chemical warfare agents (i.e., HE), HN, and L). The proposed operation consists of removing ampules from CAIS and crushing them in a decontamination solution under engineering controls. After chemical neutralization of the agents, the wastestreams are to be sent to a commercial hazardous waste disposal facility. The proposed chemical neutralization process for the detoxification of CMS is based on the oxidizing agent 1,3-dichloro-5,5-dimethylhydantoin (DCDMlI). The oxidant/solvent system selected for the neutralization process consisted of DCDMH in 50:50 chloroform/t-butanol with about 3 percent water. Neat RD, synthesized CAIS components (10 percent agent in chloroform), and wastestream samples from chemically-neutralized CMS were evaluated for skin action (vesicancy potential). The study was conducted in accordance with Good Laboratory Practices (GLP) regulations of the U. S. Environmental Protection Agency. Male hairless guinea-pigs were used as the animal model. The research consisted of three phases: Phase I consisted of the evaluation of the analytical technique for RD, HN and L in wastestreams and waste stream sample analyses, Phase II consisted of dose-ranging and vesication optimization studies, and Phase III was comprised of vesicancy testing of wastestreams resulting from the chemical neutralization of CAIS.

A prophylactic treatment is needed to circumvent constraints of the current therapy for cyanide (CN-) intoxication: intravenous administration of sodium nitrite and sodium thiosulfate. Experiments were conducted to compare the anticyanide effects of three candidate pretreatment methemoglobin (MHb)-forming compounds in an anesthetized animal model. The experiment was conducted in 5 periods, with each of 9 animals receiving the vehicle control in period 1, 0.2 mg/kg p-aminopropiophenone (PAPP) in period 2, and 2.5 mg/kg WR242511 in period 3. Four of the animals received the vehicle control in period 4 followed by 7 mg/kg p-aminoheptanophenone (PAHP) in period 5, and 5 animals received 7 mg/kg PAHP in period 4 followed by the vehicle control in period 5. Sodium cyanide (NaCN) infusions for PAPP, PAHP, and WR242511 experiments were initiated when the predicted MHb level was approximately 5%. Infusions were stopped 10 s after no functional breaths (respiratory arrest) were observed. Blood samples were collected for hemoglobin (Hb), MHb, and total blood CN- levels at scheduled time points. Time to respiratory arrest, percent MHb, and NaCN dose were the primary response parameters. All pretreatment regimens effectively mitigated the effects of NaCN poisoning compared to the vehicle control (p < .05). No discernable differences in protection were provided by the three compounds. The results indicated that the protective effect is related to the MHb level rather than the specific pretreatment drug.

: Task 92-28 was conducted to determine the protective efficacy provided by low methemoglobin (MHb) levels against a continuous sodium cyanide (NaCN) infusion, and to compare and quantitate the efficacy of long-acting and short-acting MHb-forming compounds in anesthetized canines. The time to respiratory arrest (TRA) was measured, after which the animals were revived with 10 mg/kg of hydroxylamine iv. The average estimated protective percent MHb was 5.4 percent with a 95 percent upper tolerance bound of 6.4 percent. Three compounds that induce the formation of MHb, p-aminopropiophenone (PAPP), p-aminoheptanophenone (PAHP), and an 8-aminoquinoline (WR242511), were effective in mitigating the effects of NaCN poisoning when compared to the controls. No statistically significant differences in efficacy were observed among the three compounds. Doses of two times the average concentration required to induce respiratory arrest (2xAvTRA) were tested in animals pretreated with PAPP. All of the PAPP pretreated anesthetized animals achieved approximately 6.5 percent MHb, did not cease breathing, and did not require any other medical intervention to recover from the NaCN challenge.

: This task was conducted to determine the minimum dose of pyridostigmine (PYR), and the associated level of erythrocyte acetycholinesterase inhibition (AChE-I), that provides protection from 5 X 48-br GD LD50 of untreated monkeys. Monkeys were injected im with GD and treated with 0.4 mg atropine (ATR) free base and 25.7 mg pralidoxime (2-PAM) per kg BW.