Clarence A. Broomfield
United States Army Medical Research Institute of Chemical Defense
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Featured researches published by Clarence A. Broomfield.
Biochemical Pharmacology | 1978
Larrel W. Harris; William C. Heyl; David L. Stitcher; Clarence A. Broomfield
Abstract The effects of 1,1′-oxydimethylene bis-(4- tert -butylpyridinium chloride) (SAD-128) and decamethonium on reactivation by oxime of Soman- and Sarin-inhibited erythrocyte acetylcholinesterase (AChE; EC 3.1.1.7) are reported. The inclusion of SAD-128 or decamethonium (10 −3 M) together with either TMB-4 or Toxogonin markedly augmented the reactivation of Soman- or Sarin-inhibited AChE; the recovery of enzyme activity was more than doubled in each instance when compared to the recovery in the presence of oxime alone. No reactivation was observed with SAD-128 or decamethonium in the absence of oxime. The i.v. ld 50 of SAD-128 in rabbits is 11.9 mg/kg. When 5 mg/kg of SAD-128 was given i.v. to rabbits 2 min before poisoning with Soman. 15–20 per cent of the blood cholinesterase was protected from inhibition by Soman. The aging rate in vitro of Soman-inhibited erythrocyte AChE was decreased by a factor of 1.9 with 10 −3 M SAD-128. A concentration of 1.4 × 10 −4 M SAD-128 reduced the rate of acetylcholine hydrolysis by erythrocyte AChE in vitro to 50 per cent of control. We suggest that SAD-128 protects against inhibition by Soman due to its ability to function as a reversible inhibitor and that SAD-128 and decamethonium might be allosteric modifiers of the inhibited enzyme, rendering it more susceptible to reactivation by TMB-4 or Toxogonin.
Biochemical Pharmacology | 1989
James S. Little; Clarence A. Broomfield; Mary K. Fox-Talbot; Lisa Jane Boucher; Brian MacIver; David E. Lenz
The properties of a rat liver enzyme that hydrolyzes organophosphorus (OP) inhibitors of cholinesterases were studied. The rates of hydrolysis of OP inhibitors were determined by continuous titration of released hydrogen ions, using a pH stat method. Centrifugation of homogenates at 205,000 g for 30 min demonstrated that the activity was in the soluble fraction. Hydrolysis of sarin, soman, and diisopropyl phosphorofluoridate (DFP), but not of tabun, was stimulated by the addition of Mn2+ and Mg2+. Hydrolysis of sarin greater than soman greater than tabun greater than DFP. Unlike other OP hydrolases that preferentially hydrolyze the non-toxic isomers of soman, this enzyme hydrolyzed all four soman isomers at approximately the same rate. This result was obtained in vitro by gas chromatographic analysis of enzyme-catalyzed soman hydrolysis and confirmed in vivo by demonstrating reduced toxicity in mice of soman partially hydrolyzed by this enzyme. Km and Vmax were determined by fitting V vs [S] to a hyperbolic function using regression analysis. Km values ranged from 1.1 mM for soman to 8.9 mM for tabun. Vmax values ranged from 54 nmol/min/mg protein for DFP to 2694 for sarin. The enzyme was stable for at least 2 months at -90 degrees but was inactivated by heating at 100 degrees for 5 min. Elution profiles from gel filtration by high pressure liquid chromatography showed that the hydrolytic activity for the OP inhibitors eluted in a single peak, suggesting that a single enzyme was responsible for the observed hydrolysis. Further purification and characterization of this enzyme should prove useful for the development of methods for detection, detoxification, and decontamination of these cholinesterase inhibitors.
Human & Experimental Toxicology | 1999
Carmen M. Arroyo; Robert J. Schafer; E M Kurt; Clarence A. Broomfield; Alasdair J. Carmichael
Cytokines play a major role in both acute and chronic inflammatory processes, including those produced by sulfur mustard (HD). This study describes responses of normal human epidermal keratinocyte (NHEK) cells to 2,2′-dichlorodiethyl sulfide, sulfur mustard (HD), defined by interleukin-1 beta (IL-1β), interleukin-6 (IL-6), inter-leukin-8 (IL-8), and tumor necrosis factor-alpha (TNF-α) release. A new method for detaching cell to cell adhesion between keratinocytes has been applied. This method permits the characterization of endogenous fluid from cellular content that could be applied for the development of therapeutic intervention. NHEK (typical average cell density 4.4×106 cells/mL) were exposed to HD (100 and 300 μM) in keratinocyte growth medium (KGM) for 24 h at 37°C in humidified air. Commercially available enzyme-linked immunosorbent assay (ELISA) kits were used to measure the cytokine release in NHEK during exposure to 100 and 300 μM of HD. Exposure to 100 μM HD increased release of cytokines. IL-1β (exposed: 1.41×10-5 pg/ cell±1.60×10-6 pg/cell: control 7.10×10-6 pg/ cell±1.20× 10-6 pg/cell), TNF-α (exposed: 1.06× 10-5 pg/cell±7.3× 10-7 pg/cell; control: 4.04×10-6±2.80×10-7 pg/cell) and IL-8 (exposed: 3.71×10-5 pg/ cell±3.26×10-6 pg/cell; control: 2.99×10-6 pg/cell±8.80×10-7 pg/cell) were significantly enhanced when NHEK cells were detached from culture flasks by non-enzymatic procedures. Cell suspensions of NHEK released low amounts of IL-6 when exposed to 100 μM for 24 h (exposed: 1.47×10-6±1.60×10-7 pg/cell; control: 1.28×10-6± 8.40×10-8 pg/cell). However, cell suspensions of NHEK increased levels of IL-6 after exposure to 300 μM HD (4.67×10-5 pg/cell±3.90×10-6 pg/cell; control: 3.99× 10-6 pg/cell±5.50×10-7 pg/cell). The amount of IL-8 and TNF-α present in cell suspensions increased up to 59-fold and fourfold, respectively, above control levels when NHEK cells were exposed to 300 μM HD. Exposure of NHEK to 300 μM HD had a highly variable effect on the release of IL-1β, where sometimes the secretion of IL-1β increased above baseline level and other times decreased in cell suspensions. Supernatants were collected from cell culture flasks 24 h after exposure of 100 and 300 μM and significantly increased levels of IL-6 were observed. IL-6 was released in a concentration-dependent manner, 3.6- fold up to 8.4-fold, respectively, in supernatant. These proinflammatory mediators IL-1β, IL-8, TNF-α and IL-6 may play an important role in HD injury. The present findings suggest that cytokine changes detected could be used as potential biomarkers of cutaneous vesicant injury.
Journal of Applied Toxicology | 1996
F. M. Cowan; T.‐M. Shih; D. E. Lenz; J. M. Madsen; Clarence A. Broomfield
The neurotoxicity of organophosphorus (OP) compounds involves the inhibition of acetylcholinesterase (AChE), causing accumulation of acetylcholine (ACh) at synapses. However, cholinergic crisis may not be the sole mechanism of OP toxicity. Adverse drug reactions caused by synergistic toxicity between drugs with distinct pharmacological mechanisms are a common problem. Likewise, the multiple pharmacological activities of a single molecule might also contribute to either toxicity or efficacy. For example, certain OP compounds (e.g. soman) exhibit anti‐AChE activity and also act as secretagogues by inducing mast cell degranulation with associated autacoid release and anaphylactoid reactions. Anaphylactoid shock can produce a lethal syndrome with symptoms of respiratory failure and circulatory collapse similar to the physiological sequelae observed for OP poisoning. Moreover, the major classes of drugs used as antidotes for OP intoxication can affect anaphylaxis. Acetylcholine can act as an agonist of autacoid release, and autacoids such as histamine can augment soman‐induced bronchial spasm. In concert with the demonstrably critical role of cholinergic crisis in OP toxicity, the precepts of neuroimmunology indicate that secondary adverse reactions encompassing anaphylactoid reactions may complicate OP toxicity.
Biochemical and Biophysical Research Communications | 1992
Charles B. Millard; Clarence A. Broomfield
The three-dimensional structure of human serum butyrylcholinesterase (BuChE) was modeled using a computer-based amino acid replacement strategy and the known coordinates of crystallized acetylcholinesterase (AChE) from Torpedo californica. The BuChE model was then energetically minimized with dynamic iterations of an adopted basis Newton-Raphson algorithm and the program CHARMM. Hypothetical glycosylation of this structure based upon the known carbohydrate composition of the enzyme was also performed. The glycosylated, minimized model predicts that the tertiary structure of BuChE could be very similar to AChE but that the entrance of the narrow channel leading toward its active site triad probably differs. All nine of the known N-linked oligosaccharides of BuChE are predicted to occur away from the putative active site channel and most are located on one face of the monomer.
Human & Experimental Toxicology | 1995
Carmen M. Arroyo; Robert L. von Tersch; Clarence A. Broomfield
Tumour necrosis factor (TNF) is a monokine produced by monocytes and macrophages in response to different stim uli. To determine whether vesicant agents such as half- mustard gas (H-MG; chemical structure: ClCH2CH 2SCH2CH3) may induce the release of TNF-α in human monocytes (THP-1), ELISA experiments were con ducted at different post exposure times. The results indi cate that: (1) Significant increases in the TNF-α (pg mL-1) concentration were observed as a function of time when THP-1 cells were exposed to 100 μL of 2 M H-MG. A spe cific serine-type protease inhibitor, Nα-p-tosyl-L-lysine chloromethyl ketone (TLCK), led to partial but significant inhibition of TNF activation. (2) Furthermore, this labora tory detected the generation of spin adducts of 2-methyl-2- nitrosopropane (MNP) having a resemblance to MNP adducts generated from hydrogen atom abstraction of pro tein constituents.1 The EPR/Spin Trapping data indicate the trapping of by-products of protein degradation after exposure to H-MG. TNF-α may play a role as a biochemi cal marker for pathophysiological changes induced by H- MG or related agents.
Toxicology and Applied Pharmacology | 1979
Larrel W. Harris; David L. Stitcher; William C. Heyl; Claire N. Lieske; John R. Lowe; James H. Clark; Clarence A. Broomfield
Abstract p-Nitrophenyl di-n-butylphosphinate (DBP) is a strong inhibitor of cholinesterase in vitro and in vivo. The half-times for regeneration of DBP-inhibited brain and blood enzyme activity were found to be 103.2 and 47.2 hr, respectively. These values are similar to those found for mice given soman, an irreversible inhibitor of cholinesterase. The absence of spontaneous reactivation of enzyme activity in vivo is supported by in vitro studies on DBP-inhibited eel acetylcholinesterase; no increase in enzyme activity was observed in 48 hr. After maximum inhibition with DBP, male rats were treated im with 44 μmol/kg of TMB-4, 2-PAM, or HS-6. TMB-4 treatment, but not 2-PAM or HS-6, resulted in significant reactivation of inhibited blood enzyme activity. 2-PAM was also markedly less effective than TMB-4 in restoring DBP-inhibited eel enzyme activity. Treatment with atropine alone and with 44 μmol/kg TMB-4, 2-PAM, or HS-6 im at 20 min after poisoning with 2.5 × LD50 DBP resulted in complete protection with TMB-4, 20% protection with 2-PAM or HS-6, and no protection with atropine alone. The data indicate that the response to therapy of animals receiving DBP depends upon the particular oxime used for treatment in conjuction with atropine; 2-PAM may not be the oxime of choice in the treatment of DBP intoxication.
International Journal of Toxicology | 1996
Ellen M. Kurt; Robert J. Schafer; Clarence A. Broomfield; David W. Kahler; Carmen M. Arroyo
Human keratinocytes (HK) participate in immunologic events influencing the behavior of resident cells and inflammatory cells in skin. It is clear that cytokines, immunologically active protein molecules that bind to specific receptors on target cells, are produced by keratinocytes. Specifically, keratinocytes have recently been recognized to exhibit the capacity to release interleukin (IL) 1β, IL-6, IL-8, and tumor necrosis factor-α (TNF-α), which influence the growth, function, and differentiation of dermal and epidermal cells. Since HK have been demonstrated to be a potent source of various ILs, we investigated the effect of bis-(2-chloroethyl)sulfide [sulfur mustard, HD] in saline upon the expression of these cytokines. Using enzyme-linked immunosorbent assay kits, we found that after a 3-h exposure to 1 mM HD, adult and neonatal normal HK cell cultures show a significant decrease in the expression of all cytokines listed previously, with the exception of TNF-α in adult HK, which is only slightly decreased. These results indicate that IL-1β, IL-6, and IL-8 levels in HK cell cultures 3 h after HD exposure may provide a means for measuring the immunologic response.
Biochemical Pharmacology | 1987
Clarence A. Broomfield; Ignatius J. Dembure; John Cuculis
It has been reported that several bis-quaternary compounds not necessarily having an oxime function can be used to treat soman poisoning in mice. The mechanism for this protection is not clear, but it has been proposed that such compounds may act by blocking muscarinic or nicotinic acetylcholine receptors. We have tested thirty-four compounds for muscarinic binding activity, using displacement of tritiated 3-quinuclidinyl benzilate (QNB) as a criterion, and thirty-two compounds for nicotinic binding based on inhibition of alpha-bungarotoxin (BGT) binding. Only sixteen of these compounds were able to displace QNB from rat brain membranes, and only ten of them affected BGT binding. With one exception, all of the effective compounds belonged to a series of bis-pyridinium compounds that are similar in structure to SAD-128. The binding affinities to muscarinic receptors of all these compounds were low compared to atropine. Some of the compounds bound to nicotinic receptors with affinities approaching that of d-tubocurarine. However, there was not a direct correlation between binding affinity and their reported efficacy against soman.
Journal of Applied Toxicology | 1996
Claire N. Lieske; Connie R. Clark; Lamar Dean Zoeffel; Robert L. von Tersch; John R. Lowe; C.Dahlem Smith; Clarence A. Broomfield; Steven I. Baskin; Donald M. Maxwell
As part of our studies directed at new treatments for cyanide poisoning we examined the effect of temperature on both the non‐catalyzed and the albumin‐catalyzed reactions of cyanide with a colloidal suspension of elemental sulfur (CSES). Using saturated sulfur solutions prepared in two solvents, pyridine (PY) and methyl cellosolve (MC), the reactions were studied at 15.0, 25.0, 30.0 and 37.5°C. For all the cyanolysis reactions (non‐catalyzed and albumin‐catalyzed) there is an enhancement of reaction rate when the organic solvent for the sulfur is MC. Irrespective of the solvent for the CSES, the non‐catalyzed reactions gave linear Arrhenius plots (PY, correlation coefficient = 0.998; MC, correlation coefficient = 0.997). In each case the entropy of activation was positive (14.1 cal K−1 mol−1 for PY and 56.4 cal K−1 mol−1 for MC). In contrast with these results the albumin‐catalyzed reactions generated non‐linear Arrhenius plots and negative entropies of activation. Non‐linear plots were observed with the three albumins studied: human serum albumin, heat‐shock bovine serum albumin and fatty acid‐free bovine serum albumin. The non‐linear plots are the result of a more complex reaction sequence than a simple cyanolysis reaction.
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United States Army Medical Research Institute of Chemical Defense
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