Henry L. Meier

Molecular basis for mustard-induced vesication☆☆☆

A biochemical hypothesis explaining the generation of pathology in human skin by mustard gas (HD) is presented which links the initiation of DNA damages to local alterations of metabolism and subsequent development of blisters. The proposed sequence involves HD alkylation of purines in DNA which are processed to form apurinic sites. Apurinic endonucleases act at these sites to produce backbone breaks in DNA which cause activation of the chromosomal enzyme poly(ADP-ribose)polymerase. This enzyme utilizes NAD+ as a substrate and, at vesicating doses of HD, would deplete the cells of their NAD+ content. The depletion in NAD+ would cause inhibition of glycolysis, and the resulting accumulation of common intermediates would stimulate the NADP+-dependent hexosemonophosphate shunt (HMS). Such stimulation of the HMS has been associated with DNA damage and enhancement of protease synthesis and release. These proteases could be responsible for development of subepidermal blisters which result from fluid accumulation in the cavity created by separation of the moribund basal cell layer from the basement membrane--a characteristic feature of HD-exposed human skin. Partial validation of this biochemical hypothesis has been achieved. DNA alkylated with either monofunctional or bifunctional sulfur mustards, followed by spontaneous or enzymatic depurination, was shown to be sensitized to degradation by apurinic endonuclease. Studies on the effect of HD on human skin grafted to athymic nude mice demonstrated dose- and time-related decreases in NAD+ levels. These decreases in NAD+ levels preceded and correlated to the predicted severity of pathology. The participation of poly(ADP-ribose)polymerase activity in the HD-induced NAD+ loss was substantiated by prevention of this loss in the presence of inhibitors of the enzyme. Additional supporting evidence for the proposed mechanism was obtained at the cellular level by studies which utilized human leukocytes. The subsequent involvement of the HMS and proteases in HD-induced vesication is discussed.

Nasal challenge with ragweed pollen in hay fever patients. Effect of immunotherapy.

Challenge of the nasal mucosa of allergic subjects with specific allergen induces not only the expected sneezing and rhinorrhea, but also the appearance in nasal secretions of mediators commonly associated with activation of mast cells or basophils: histamine, leukotrienes, prostaglandin D2 (PGD2), kinins, and TAME ([3H]-N-alpha-tosyl-L-arginine methyl ester)-esterase. To determine whether specific immunotherapy alters mediator release in vivo, nasal pollen challenge was used to compare 27 untreated highly sensitive ragweed (RW)-allergic subjects with 12 similarly sensitive patients receiving long-term immunotherapy (3-5 yr) with RW extract (median dose, 6 micrograms RW antigen E). The two groups were equally sensitive based on skin tests and basophil histamine release. The immunized group had a diminished response as demonstrated by (a) the treated group required higher pollen doses to excite sneezing or mediator release; (b) significantly fewer subjects in the treated group released mediators at any dose (TAME-esterase [P = 0.005], PGD2 [P = 0.04]), and (c) the treated group released 3-5-fold less mediator (TAME-esterase [P = 0.01], and histamine [P = 0.02]).

Biochemical manipulation of intracellular glutathione levels influences cytotoxicity to isolated human lymphocytes by sulfur mustard

Glutathione (GSH) is the major nonprotein thiol that can protect cells from damage due to electrophilic alkylating agents by forming conjugates with the agent. Sulfur mustard (HD) is an electrophilic alkylating agent that has potent mutagenic, carcinogenic, cytotoxic, and vesicant properties. Compounds that elevate or reduce intracellular levels of GSH may produce changes in cytotoxicity induced by sulfur mustard. Pretreatment of human peripheral blood lymphocytes (PBL) for 72 hr with 1 mM buthionine sulfoximine (BSO), which reduces intracellular GSH content to approximately 26% of control, appears to sensitize these in vitro cells to the cytotoxic effects of 10 μM HD but not to higher HD concentrations. Pretreatment of PBL for 48 hr with 10 mM N-acetyl cysteine (NAC), which elevates intracellular glutathione levels to 122% of control, appears to partially protect these in vitro cells from the cytotoxic effects of 10 μM HD but not to higher HD concentrations. Augmentation of intracellular levels of glutathione may provide partial protection against cytotoxicity of sulfur mustard.

The use of human epidermal keratinocytes in culture as a model for studying the biochemical mechanisms of sulfur mustard toxicity.

Human epidermal keratinocytes in culture were studied to evaluate their usefulness in demonstrating toxic events following exposure to sulfur mustard. Exposure of keratinocytes to sulfur mustard over a concentration range of 1–1000 μM HD, reduced NAD+ levels from 96% to 32% of control levels. When keratinocytes were exposed to a concentration of 300 μM HD, NAD+ levels began to fall at 1 hour and reached a plateau of 47% of control levels at 4 hours. Niacinamide, an inhibitor of the enzyme poly(ADP-ribose) polymerase, partially protected mustard-exposed cells against NAD+ depletion. It also protected cellular viability as assessed by vital staining 24 hours after exposure. This protection was not seen in long-term (72 hr) cultures. These studies suggest that human epidermal keratinocytes in culture can serve as a usefulin vitro model for research into the biochemical mechanisms of sulfur mustard-induced cutaneous injury.

Sulfur mustard lowers nicotinamide adenine dinucleotide concentrations in human skin grafted to athymic nude mice

Human skin grafted to athymic nude mice shows a decrease in nicotinamide adenine dinucleotide (NAD+) concentrations when exposed to sulfur mustard (HD). The lowering of NAD+ is dependent on both dose of HD and time after exposure. When HD is applied to grafted skin at 127 micrograms/cm2, the decrease in NAD+ begins immediately after exposure, approaches minimal values by 4 hr. NAD+ returned to normal within 18 hr. With higher concentrations of HD, NAD+ concentrations fall precipitously within 4 hr, reach a minimum value at 18 hr, and seem to remain at this depressed value for at least 72 hr. NAD+ loss appears to precede and be proportional to tissue injury. Pretreatment of mice with 3-aminobenzamide, a specific inhibitor of poly(ADP-ribose) polymerase, seems to lessen the effect of HD. At higher concentrations of HD, human grafts from mice pretreated with 3-aminobenzamide show significant protection from loss of NAD+ levels after 4 hr.

Anaphylactic release of a prekallikrein activator from human lung in vitro.

We have demonstrated the in vitro IgE-mediated release of a prekallikrein activator from human lung. The lung prekallikrein activator was partially purified by sequential chromatography on sulfopropyl-Sephadex, DEAE-Sephacel, and Sepharose 6B. Purified human prekallikrein was converted to its active form (kallikrein) by the lung protease. The generated kallikrein was shown to be biologically active; that is, it generates bradykinin from purified human high-molecular weight kininogen and also cleaves benzoyl-propyl-phenyl-arginyl-p-nitroanilide, a known synthetic substrate of kallikrein. The lung prekallikrein activator differs from the known physiologic activators of prekallikrein (the activated forms of Hageman factor) with respect to: (a) size (it has a mol wt of approximately 175,000); (b) synthetic substrate specificity (D-propyl/phenyl/arginyl-p-nitroanilide is a substrate for the activated forms of Hageman factor, but not the lung protease); (c) antigenic specificity (an anti-Hageman factor immunoadsorbent column did not remove significant amounts of the lung protease, while it removed most of the activity of activated Hageman factor fragments); and (d) inhibition profile (the lung proteases was not inhibited by corn trypsin inhibitor). This prekallikrein activator provides a physiologic mechanism by which prekallikrein can be directly activated during IgE-mediated reactions of the lung. While the role of this lung prekallikrein activator in immediate hypersensitivity reactions and in other inflammatory processes is not clear, it does represent a first and important interface between IgE-mediated reactions and the Hageman factor-dependent pathways of the inflammatory response.

Release of elastase from purified human lung mast cells and basophils - Identification as a Hageman factor cleaving enzyme

Elastase, a serine protease, is capable of inducing severe lung destruction in experimental animal models. We now report that this proteinase exists preformed in neutrophil-free sonicates of purified human lung mast cells (>98% purity) and in circulating peripheral blood basophils (>97% purity). The elastase levels in both cell types (41–174 ng/106 cells) represents approximately 3–20% of those found in human neutrophils; both cell types released their elastase following anti-IgE and ionophore A23187 challenge. The apparent molecular size of the mast cell enzyme on Sephadex G-100 gel filtration, as well as its inhibition profile, was identical to that of purified human neutrophil elastase. This mast cell elastase is identical to our previously reported mast cell-derived Hageman factor cleaving activity. Mast cell-, basophil-, and neutrophil-derived elastases cleave Hageman factor into fragments of 52,000 and 28.000 Da; cleavage by all three enzymes is inhibited by preincubation with polyclonal antibodies directed against human neutrophil elastase.

Ultrastructural Correlates of the Protection Afforded by Niacinamide against Sulfur Mustard-Induced Cytotoxicity of Human Lymphocytes in Vitro

Sulfur mustard (HD) has been shown to cause a concentration-dependent decrease in viability of human lymphocytes in vitro as measured by dye exclusion; this decrease is preventable by inhibitors of poly(adenosine diphosphatase ribose) polymerase such as niacinamide. The present study investigates the morphologic correlates of the protection afforded by niacinamide through scanning and transmission electron microscopic analysis of human lymphocytes incubated in the presence or absence of 10(3) M niacinamide for 24 h at 37 degrees C and exposed in vitro to 10(-3) M HD. Lymphocytes exposed to HD alone demonstrated 30% to 40% viability and loss of microvilli, large cytoplasmic vacuoles, extensive blebbing of the perinuclear envelope, loss of cytoplasmic organelles, condensation of nuclear chromatin, and multiple perforations of the plasmalemma. In the presence of niacinamide HD-treated lymphocytes had a viability of 87% and, except for blunting of the microvilli, essentially normal ultrastructure. Although the sequence of observed ultrastructural changes was not established, results of this morphologic study suggest that, in addition to the prevention of plasmalemmal defects and dye infusion, the mechanism of niacinamide protection appears to include preservation of the morphologic and functional integrity of cellular organelles.

Histamine Release by Esterase Inhibitors

The serine esterase inhibitor diisopropyl fluorophosphate (DFP) has been shown to inhibit IgE-dependent histamine release. Recently, it has been demonstrated that lower concentrations of DFP enhanced

Regulation by serine esterase of histamine release from human leukocytes—I: Direct release of histamine by the serine esterase inhibitors diisopropyl fluorophosphate (DFP) and soman (GD)

The serine esterase inhibitor diisopropyl fluorophosphate (DFP) had been reported previously to inhibit IgE-dependent histamine release. Recently, it has been demonstrated that lower concentrations of DFP enhance IgE-dependent histamine release and inhibit desensitization. This manuscript describes the abilities of several esterase inhibitors to cause release of histamine from human leukocytes (basophils), by a process that is IgE-independent. This esterase inhibitor-induced histamine release appears to be by a non-cytotoxic mechanism that requires calcium and is temperature dependent. These histamine release processes occurred over a longer period of time than IgE-dependent release. Direct release of histamine by these small molecular weight inhibitors and inhibition of desensitization both suggest that one or more serine esterases are involved in the regulation of histamine release from human basophils.