DOGS DOSED WITH DEET

Responsible & Performing Organization: Armed Forces Radiobiology Research Institute, Bethesda, MD

Objective: The objectives of this research are: (1) to develop radioprotective drugs that significantly protect radiation-exposed individuals against injuries that manifest either early as acute radiation illness, or as late-arising disease; (2) to develop effective post-exposure therapies that enhance recovery and repair of radiation-injured tissues and organ systems; (3) to assess and to modify, if needed, the putatively radioprotective agent in order to minimize any residual toxicity and performance decrementing effects; and (4) to engineer simple, easy to apply, and long-lasting drug delivery system(s) for these protective and therapeutic agents.

Approach: The approach of this research involves the use of both small, short-lived rodents and large, long-lived animals (canines) to test the efficacy and toxicity of radioprotectants and therapeutics given as single agents or in combination. Measures of blood-forming tissue (hematopoietic tissue/bone marrow) structure and functional capacity, survival protection, and drug toxicity against specific tissues and tissue injuries under various radiation exposure conditions are being sought for several classes of radioprotectants and therapeutics. These protective and therapeutic agents include aminothiols and related analogs, synthetic and natural immunomodulators, recombinant growth factors and cytokines, vitamins, androstene steroids, and several simple organic compounds with recognized gene-modifying functions. These agents are assessed initially for toxicity in unirradiated animals using both clinical and behavioral response indicators. Subsequently, tests for protective efficacy are conducted in animals that have been stressed by whole-body radiation exposures. For these tests, principal endpoints include measures of blood-forming system (hematopoietic system) sparing and determinations of the capacity to maintain and/or restore critical levels of circulating blood cells. From these tests, several of these agents have been identified as being nontoxic and moderately radioprotective in terms of early- and late-arising radiation injuries. In all these studies, a sufficient number of animals are used in order to provide sufficient statistical power to the tests that allow results to be appropriately and accurately interpreted and reported.

Erythrocyte and plasma cholinesterase activity in male and female rhesus monkeys before and after exposure to sarin.

Woodard CL, Calamaio CA, Kaminskis A, Anderson DR, Harris LW, Martin DG.

United States Army Medical Research & Development Command, Fort Detrick, Frederick, Maryland 21702-5012.

The rhesus monkey (Macaca mulatta) has a menstrual cycle similar to the human. Differences in hormone levels have been demonstrated between the sexes and in females during the menstrual cycle but these differences in terms of organophosphorus toxicity have not been explored. Plasma cholinesterase (ChE/BuChE) and erythrocyte (RBC) acetylcholinesterase (AChE) activity were measured before and after exposure to the organophosphorus compound sarin (11 micrograms/kg, i.v.; 0.75 LD50) in six male and six female rhesus monkeys. After baseline measurements were obtained, sarin was administered to atropinized monkeys to determine in vivo differences between the sexes in their response to sarin. With the baseline values, the intraanimal and intragroup BuChE/AChE variations were found to be minimal. Following sarin intoxication and 2-PAM treatment no significant differences were seen between the sexes in the rate of reactivation of BuChE or AChE by 2-PAM. The rate of aging of sarin phosphonylated RBC AChE between the sexes was also similar. De novo regeneration of RBC AChE and plasma BuChE after sarin intoxication was different between the male and female monkeys. The female plasma BuChE recovery rate was 48% slower than the male recovery rate, while the early (first 63 days) RBC AChE recovery rate was 24.5% faster in the females. In conclusion, there probably are not any clinically significant differences between male and female rhesus monkeys acutely intoxicated with sarin. However, on subsequent exposure clinical differences may be observed due to substantial differences in the rate of de novo synthesis of both plasma BuChE and RBC AChE.

Acute toxicity of cyclohexylmethylphosphonofluoridate (CMPF) in rhesus monkeys: serum biochemical and hematologic changes.

Young GD, Koplovitz I.

Arch Toxicol 1995;69(6):379-83
U.S. Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD 21010-5425, USA.

Changes in serum biochemical and hematological parameters were studied in 20 male rhesus monkeys following acute poisoning by the organophosphate nerve agent cyclohexylmethylphosphonofluoridate (CMPF or GF). Animals were challenged with 5 x LD50 GF (233 micrograms/kg, IM) following pretreatment with pyridostigmine (0.3-0.7 mg/kg per 24 h) and treated with atropine (0.4 mg/kg, IM) and either 2-PAM (25.7 mg/kg, IM) or H16 (37.8 mg/kg, IM) at the onset of clinical signs or at 1 min after exposure. Muscle fasciculations, tremors, or convulsions occurred in 19 of 20 animals. Serum biochemical and hematologic parameters were analyzed 2 days and 7 days after exposure and compared to pre-exposure baseline values. Significant increases in creatine kinase (CK), lactate dehydrogenase (LD), aspartate transaminase (AST), alanine transaminase (ALT) and potassium ion (K+), associated with damage to striated muscle and metabolic acidosis, occurred in both oxime-treated groups 2 days after exposure. Total protein, albumin, red blood cell (RBC) count, hemoglobin concentration (Hb) and hematocrit (Hct), were decreased in both oxime-treated groups at 7 days. The results demonstrate that animals exposed to a single high dose of GF and treated with standard therapy exhibit changes in serum biochemical and hematological indices directly and indirectly associated with their clinical presentations.

PMID: 7495375 [PubMed - indexed for MEDLINE]

Histopathologic changes in the brain, heart, and skeletal muscle of rhesus macaques, ten days after exposure to soman (an organophosphorus nerve agent).

Britt JO Jr, Martin JL, Okerberg CV, Dick EJ Jr.

US Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD, USA.

BACKGROUND AND PURPOSE: Soman, an organophosphorus, anticholinergic, chemical warfare nerve agent, is studied at few research facilities, and there have been few pathologic studies of soman-exposed primates. We describe the brain, heart, and skeletal muscle lesions, review lesions described in literature, and discuss possible pharmacologic mechanisms for soman-induced neuron necrosis. METHODS: In this retrospective, histopathologic study, records were obtained for 36 rhesus macaques (Macaca mulatta) that were euthanized 10 days after soman exposure, from a larger group of 103 monkeys that were exposed to soman and used for pharmacologic and lethality studies. RESULTS: Brain lesions were seen in 9 of 15 animals that convulsed and in only 1 of 21 that did not convulse. The brain lesions in our primates were limited to the hippocampus, amygdala, and thalamus (of one animal), and consisted of neuron necrosis and dropout, spongiosis, gliosis, astrocytosis, and vascularization. Heart lesions consisted of myocardial degeneration and necrosis. Three animals had brain and heart lesions, 7 had brain lesions only, and 3 had heart lesions only. Skeletal muscle lesions, although minimal to mild, were in most of the animals, whether they had convulsed, but most had muscular tremors. These lesions were in the biceps brachii (11 of 22 monkeys), anterior tibialis (8/22), biceps femoris (7/22), flexor carpi radialis (5/22), gastrocnemius (3/22), and diaphragm (1/22). The limited literature on soman lesions in primate brain and heart, and the limited information on skeletal muscle lesions, is reviewed. CONCLUSIONS: Brain lesions were not as widespread as reported in other studies of primates and rodents, and were significantly associated with convulsions. Unlike other studies using rodents, we observed poor correlation between heart and brain lesions; thus, a single hypothesis to explain the pathogenesis for the brain and heart lesions may be difficult to establish.

PMID: 10857003 [PubMed - indexed for MEDLINE]

Neurophysiologic and Neuropathologic Effects in Monkeys of Low Level Exposures to Sarin, Pyridostigmine, Pesticides and Botulinumtoxoid

Objective and Approach: EVALUATE POSSIBLE ADDITIVE OR POTENTIATING EFFECTS OF SARIN, BOTULINUM TOXOID, DEET, PB, AND CHLORPYRIFOS EXPOSURES IN PRODUCING NEUROPATHIES, TESTING USING PERIODIC ENZYME ANALYSES AND NEUROPHYSIOLOGIC EVALUATION, AND NEUROMUSCULAR HISTOPATHOLOGY AT THE TERMINATION OF THE STUDY WILL YIELD VALUABLE INFORMATION ON THE INTERACTIONS OF SARIN BOTULINUM TOXOID DEET, PB AND CHLORPYRIFOS EXPOSURES. BECAUSE OF THE GENOTYPIC AND PHENOTYPIC RESEMBLANCE TO HUMANS AND THE PHYLOGENETIC AND PHYSIOLOGIC SIMILARITIES AND THE PHYLOGENETIC AND PHYSIOLOGIC SIMILARITIES, THE MONKEY (MACACA MULATTA) IS THE ANIMAL MODEL OF CHOICE FOR EVALUATING POTENTIAL TOXICITY OF COMPOUNDS IN HUMANS. THE PROPOSED STUDY WILL ADDRESS TWO HYPOTHESES 1) LOW-LEVEL SARIN EXPOSURES IN COMBINATION WITH VACCINATION AGAINST BOTULINUM TOXIN DO NOT PRODUCE NEUROPHYSIOLOGIC AND NEUROPATHOLOGIC EFFECTS IN MONEKYS. 2) NEUROPHYSIOLOGIC AND NEUROPATHOLOGIC EFFECTS OBSERVED IN MONKEYS VACCINATED AGAINST BOTULINUM TOXIN, TREATED WITH PB, DEET AND CPF, AND EXPOSED TO LOW LEVELS OF SARIN ARE NO GREATER THAN THOSE OBSERVED IN ANIMALS TREATED WITH PB, DEET AND/OR CPF.

Lesions of acute inhaled lethal ricin intoxication in rhesus monkeys.

Wilhelmsen CL, Pitt ML.

Pathology Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD, USA.

Five unimmunized adult rhesus monkeys weighing 5.9-6.3 kg were challenged with a precalculated, inhaled dose of 20.95-41.8 micrograms/kg of aerosolized ricin. Two males and three females either died or were killed at the onset of respiratory distress between 36 and 48 hours post-ricin inhalation and were necropsied. Consistent gross and microscopic lesions were confined to the thoracic cavity. All monkeys had multifocal to coalescing fibrinopurulent pneumonia, diffuse necrosis, and acute inflammation of airways, and nearly diffuse alveolar flooding, with peribronchovascular edema. All monkeys also had purulent tracheitis, fibrinopurulent pleuritis, and purulent mediastinal lymphadenitis. One male monkey and one female monkey had bilateral adrenocortical necrosis. We attributed the cause of death to asphyxiation following massive pulmonary alveolar flooding. The lesions of acute inhaled ricin intoxication in rhesus monkeys closely resembled those lesions reported in rats with acute inhaled ricin intoxication.

PMID: 8740703 [PubMed - indexed for MEDLINE]

Key Words: LABORATORY ANIMALS RA IV MUSTARD GAS ANTIOXIDANTS LIPOSOMES INFLAMMATION FREE RADICALS ADULT RESPIRATORY DISTRESS SYNDROME

Objective: THIS PROJECT WILL (A) INVESTIGATE THE PATHOPHYSIOLOGY OF MUSTARD INJURY AND THE EFFICACY OF LIPOSOMES AS A COUNTERMEASURE; AND (B) ATTEMPT TO IMPROVE ANTIOXIDANT LIPOSOMES AS A COUNTERMEASURE FOR MUSTARD INJURY. SULFUR MUSTARD IS A VESICATING AGENT THAT CAN BE DISPERSED BY AN AEROSOL OR AN EXPLOSIVE BLAST. IT IS PRIMARILY AN "INCAPACITATING" WEAPON THAT PRODUCES BOTH ACUTE AND CHRONIC EFFECTS ON EXPOSED MUCOSAL AND CUTANEOUS AREAS. IN LARGE DOSES IT CAN HAVE SYSTEMIC TOXICITY. ALTHOUGH MUSTARD GAS HAS BEEN STUDIED SINCE WORLD WAR I, NEITHER ITS MECHANISM OF ACTION NOR AN ANTIDOTE IS KNOWN. IT IS CLEAR, HOWEVER, THAT MUSTARD GAS INITIATES AN INFLAMMATORY PROCESS, AND REASONABLE TO ASSUME THAT TREATMENTS WHICH MINIMIZE THIS PROCESS HOLD THERAPEUTIC PROMISE

Approach: THE PROJECT WILL BE CARRIED OUT BY A MULTIDISCIPLINARY GROUP COMPOSED OF CHEMISTS, PHYSICIANS AND PHYSIOLOGISTS FROM A CONSORTIUM OF SIX UNIVERSITY, MILITARY AND PRIVATE ORGANIZATIONS, THE MUSTARD CONSORTIUM. ONGOING TESTING OF THE LIPOSOMAL FORMULATIONS WILL OCCUR IN EACH LABORATORY, AND MEMBERS OF THE GROUP WILL INTERACT AND COLLABORATE AS DISCOVERIES ARE MADE. IT IS ANTICIPATED THAT THIS APPROACH WILL GREATLY ACCELERATE DEVELOPMENT OF AN ANTIDOTE BY BRINGING THE INSIGHTS AND METHODS OF A VARIETY OF DISCIPLINES TO BEAR ON MUSTARD-RELATED INJURY. THIS PROJECT INVESTIGATES THE PATHOPHYSIOLOGY OF INJURY FROM SULFUR MUSTARD AT THE CELLULAR AND MOLECULAR LEVEL. IT WILL ALSO PURSUE RECENT EVIDENCE THAT SURVIVAL AFTER EXPOSURE TO MUSTARD GAS IS ENHANCED BY ANTIOXIDANTS AND ANTI-INFLAMMATORY AGENTS; THIS WILL BE DONE IN EXPERIMENTS THAT EXPLORE THE EFFICACY OF LIPID- AND WATER-SOLUBLE ANTIOXIDANTS ENCAPSULATED IN LIPOSOMES.

Responsible Organization: U.S. Army Medical Research and Materiel Command, Ft. Detrick, MD

Objective: Sulfur mustard (HD) is a severely cytotoxic vesicant used as a chemical warfare agent in various conflicts recently. The Principal Investigator (PI) has found that post-exposure treatment with the widely used antiseptic, povidone iodine (PI) Ointment, protected guinea pig skin against HD toxicity even at a time interval of 20 minutes between exposure and treatment. These preliminary studies need further validation before approval for use in humans. The objective of the research is to determine the relationship between PI and (1) SM dose; (2) the time interval between exposure and treatment; and (3) the period of time that PI has to be left on the skin to obtain maximal potection. The research also will investigate the mechanism of PI-induced protection. Further studies will verify the association between HD skin toxicity and other neurogenic inflammatory factors such as calcitonin gene-related peptide, vasoactive intestinal peptide, and nitric oxide.

Title: Nuclear, Biological, and Chemical (NBC) Interactions and Countermeasures

Responsible Organization: Armed Forces Radiobiology Research Institute, Bethesda, MD

Keywords: Laboratory Animals, rat, mouse, guinea pig, Cell tissue culture, ionizing radiation, BW/CW agents, mustard gas, nerve agent, Animals. . . .

Objective: This research program has three major objectives. The first objective is to determine the major health effects that result from exposure to ionizing radiation in combination with exposure to biological and/or chemical warfare (BWCW) agents, endemic diseases, and their vaccines and treatments. The second objective is to determine the effectiveness of ionizing radiation in neutralizing BW and CW agents. The third objective is to develop computer models to predict incapacitation and mortality from exposure to ionizing radiation in combination with BWCW agents.

Approach: The first objective is approached by three experimental strategies. First, the interaction of radiation in combination with BW agents on antibody production, on hematopoietic endpoints, cytokine production, cytotoxicity, and gastrointestinal motility is being assessed. Second, the effect of radiation in combination with mustard agents on DNA mutagenesis and cell transformation is being examined. Third, interactions of radiation with nerve agents and pyridostigmine on seizure mechanisms, neuromuscular junction, behavior, and thermoregulation is being assessed. Research methodologies utilized in these studies involve the use of rats, mice, guinea pigs, as well as cell tissue culture techniques. The second objective is approached by determining radiation kill curves for inactivation of BW agent simulants. The third objective is approached by integrating new experimental data on combined radiation and BW exposure with existing computer models that predict downwind collateral effects. This research will ultimately generate new protocols, based on the results of our Nuclear, Biological, and Chemical (NBC) bioeffects experiments, which will enable U.S. Armed Forces to accomplish military operations in an NBC environment while optimizing both survival and military performance. Preserving combatants health in a combined NBC environment will improve warfighting operations and optimize mission capabilities.