Optimization of ovine bone decalcification for increased cellular detail: a parametric study.

There are many published methods of decalcifying bone for paraffin histology; however, the current literature lacks details regarding the processing of ovine tissue. Ovine bone tissue presents challenges, as samples are often denser and larger than other comparative animal models, thus increasing decalcification times. Trifluoroacetic Acid (TFAA) has previously been used to decalcify ovine bone samples for histological analysis. Unfortunately, TFAA is a strong acid and often results in loss of cellular detail, especially in the connected soft tissue. This is generally manifested as over staining with eosin, and a decrease in cellular features which impacts overall histological interpretation. It is well known that leaving tissue in acid for long periods degrades cellular detail; therefore, minimizing decalcification time is critical to accurately determine cellular morphology. Six decalcification solutions (8% TFAA, 20% TFAA, 8% formic acid, 20% formic acid, Formical-4, and XLCal, and three temperatures (room temperature, 30°C, 37°C), were examined to determine their effects on cellular detail in ovine vertebrae and humeral heads. These data clearly indicate that 20% formic acid at 30°C yielded better decalcification rates (2.6 d ± 0.9 d) and cellular detail (none to mild changes) for the vertebrae samples, and 20% formic acid at RT yielded the best cellular detail (none to minimal loss) for humerus samples with a moderate amount of time (6.5 d ± 1.7). These results should establish the optimal demineralization procedures for ovine bone used in scientific studies resulting in improved cellular detail while minimizing decalcification times.

TNF-alpha expression patterns as potential molecular biomarker for human skin cells exposed to vesicant chemical warfare agents: sulfur mustard (HD) and Lewisite (L).

Studies were conducted to examine the effect of two vesicant chemical warfare agents (VCWA), one of them an arsenical, on cytokine gene expression in normal human epidermal keratinocyte (NHEK) cells. We tested 2,2'-dichlorethylsulfide (sulfur mustard, military designation HD) and 2,chlorovinyldichloroarsine (Lewisite, military designation L), which have significant differences in their chemical, physical, and toxicological properties. Human tumor necrosis factor-alpha (hTNF-alpha) cytokine was detected by using the enzyme-linked immunosorbent assay, a protein multiplex immunoassay, Luminex100, and reverse transcription-polymerase chain reaction (RT-PCR). The messenger RNA expression of hTNF-alpha was determined to provide a semi-quantitative analysis. HD-stimulated NHEK induced secretion of hTNF-alpha in a dose-dependent manner. Dose response effect of Lewisite decreased hTNF-alpha levels. Time-response data indicated that the maximum response for HD occurred at 24 h with an associated cytotoxic concentration of 10(-4) mol/L. NHEK cells stimulated with 10(-4) mol/L HD for 24 h at 37 degrees C increased detectable levels of hTNF-alpha from 5 to 28 ng/ml at an index of cell viability between 85 to 93% as detected by Luminex100. Our results indicated that the increased levels of hTNF-alpha by HD are dependent on the primary cultures, cell densities, and chemical properties of the stimulation. Lewisite under the same conditions as HD caused a reduction of hTNF-alpha from control levels of 1.5 ng/ml to 0.3 ng/ml after stimulation (10(-4) mol/L), with an index of cell viability of reverse similar 34%. We analyzed the transcriptional of hTNF-alpha gene and found that HD (10(-6) to 10(-4) mol/L) activates hTNF-alpha gene in cultured NHEK and that L at 10(-6) to 10(-4) mol/L markedly reduces hTNF-alpha gene. We conclude that the pro-inflammatory mediator, hTNF-alpha, could be a potential biomarker for differentiating between exposure of HD or L.

Putative role of proteolysis and inflammatory response in the toxicity of nerve and blister chemical warfare agents: implications for multi-threat medical countermeasures.

Despite the contrasts in chemistry and toxicity, for blister and nerve chemical warfare agents there may be some analogous proteolytic and inflammatory mediators and pathological pathways that can be pharmacological targets for a single-drug multi-threat medical countermeasure. The dermal-epidermal separation caused by proteases and bullous diseases compared with that observed following exposure to the blister agent sulfur mustard (2,2'-dichlorodiethyl sulfide) has fostered the hypothesis that sulfur mustard vesication involves proteolysis and inflammation. In conjunction with the paramount toxicological event of cholinergic crisis that causes acute toxicity and precipitates neuronal degeneration, both anaphylactoid reactions and pathological proteolytic activity have been reported in nerve-agent-intoxicated animals. Two classes of drugs already have demonstrated multi-threat activity for both nerve and blister agents. Serine protease inhibitors can prolong the survival of animals intoxicated with the nerve agent soman and can also protect against vesication caused by the blister agent sulfur mustard. Poly (ADP-ribose) polymerase (PARP) inhibitors can reduce both soman-induced neuronal degeneration and sulfur-mustard-induced epidermal necrosis. Protease and PARP inhibitors, like many of the other countermeasures for blister and nerve agents, have potent primary or secondary anti-inflammatory pharmacology. Accordingly, we hypothesize that drugs with anti-inflammatory actions against either nerve or blister agent might also display multi-threat efficacy for the inflammatory pathogenesis of both classes of chemical warfare agent.

Suppression of sulfur mustard-increased IL-8 in human keratinocyte cell cultures by serine protease inhibitors: implications for toxicity and medical countermeasures.

The toxicity of the chemical warfare blistering agent sulfur mustard (2,2'-dichlorodiethyl sulfide; SM) has been investigated for nearly a century; however, the toxicological mechanisms of SM remain obscure and no antidote exists. The similarity of dermal-epidermal separation caused by SM exposure, proteolysis, and certain bullous diseases has fostered the hypothesis that SM vesication involves proteolysis and/or inflammation. Compound screening conducted by the US Army Medical Research Institute of Chemical Defense established that topical application of three tested serine protease inhibitors could reduce SM toxicity in the mouse ear vesicant model. Although most of the drugs with efficacy for SM toxicity in rodent models are anti-inflammatory compounds, no in vitro assay is in current use for screening of potential anti-inflammatory SM antidotes. IL-8 is a potent neutrophil chemotactic cytokine that is increased in human epidermal keratinocyte (HEK) cell cultures following exposure to SM and has been proposed as a marker for SM-induced inflammation. This study was conducted to establish in vitro screening of IL-8 in SM-exposed HEK as a possible model for evaluating candidate compounds prior to in vivo testing. We chose two protease inhibitors, one from those shown as successful in the MEVM (ethyl p-guanidinobenzoate hydrochloride, ICD 1579) and a prototypic inhibitor of trypsin, N-tosyl-L-lysine chloromethyl ketone (TLCK). TLCK (62.5 to 1000 micromol/L) or ICD 1579 (31.25 to 1000 micromol/L) was added to HEK cell cultures 1 h after SM exposure (200 micromol/L) and dose-dependently suppressed SM-increased IL-8. The suppression of SM-increased IL-8 by a class of drug candidate compounds such as protease inhibitors may provide a mechanistic marker that helps predict future medical countermeasures for SM toxicity and reduces the need for testing in animal models.

Sulfur mustard-stimulated protease: a target for antivesicant drugs.

One of the mechanisms of the skin blistering effect (vesication) of sulfur mustard (bis-(2-chloroethyl)sulfide, HD) is believed to be via the stimulation of specific protease(s) at the dermal-epidermal junction. Cultured normal human epidermal keratinocytes (NHEK) were used as a model to study and characterize protease stimulated by the mustards 2-chloroethyl ethyl sulfide (CEES), 2-chloro-N-(2-chloroethyl)-N-methylethanamine hydrochloride (nitrogen mustard, HN(2)) and HD. The results obtained using a chromozym (TRY) peptide substrate protease assay revealed the optimum mustard concentrations and time for protease stimulation to be about 200 microM (CEES), 100 microM (HN(2)) and 100 microM (HD) and 16 h. The mustard-stimulated protease was membrane bound and was inhibited by adding a Ca(2+) chelator (either 2 mM EGTA (ethylene glycol-bis(amino ethyl ether) N,N,N',N' tetraacetic acid) or 50 microM BAPTA AM (1,2-bis(z-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, tetraacetoxy methyl ester) alone or in combination), a serine protease inhibitor diisopropyl fluoro-phosphate (DFP, 1 mM), or a protein synthesis inhibitor cycloheximide (35 microM) in the extracellular medium. These results suggest that mustard toxicity may involve the stimulation of trypsin/chymotrypsin-like serine protease, dependent on Ca(2+) and new protein synthesis. Protein purification by gel exclusion and hydrophobic chromatography produced a 70-80 kDa protease, which had an amino acid sequence homologous with a mammalian-type bacterial serine endopeptidase. Based on this information, research is in progress to identify the protease stimulated by HD in NHEK and to determine whether its inhibitors are useful as prospective antivesicant drugs.

Evidence for nonacetylcholinesterase targets of organophosphorus nerve agent: supersensitivity of acetylcholinesterase knockout mouse to VX lethality.

The possibility that organophosphate toxicity is due to inhibition of targets other than acetylcholinesterase (AChE, EC 3.1.1.7) was examined in AChE knockout mice. Mice (34-55 days old) were grouped for this study, after it was determined that AChE, butyrylcholinesterase (BChE), and carboxylesterase activities had reached stable values by this age. Mice with 0, 50, or 100% AChE activity were treated subcutaneously with the nerve agent VX. The LD50 for VX was 10 to 12 microg/kg in AChE-/-, 17 microg/kg in AChE+/-, and 24 microg/kg in AChE+/+ mice. The same cholinergic signs of toxicity were present in AChE-/- mice as in wild-type mice, even though AChE-/- mice have no AChE whose inhibition could lead to cholinergic signs. Wild-type mice, but not AChE-/- mice, were protected by pretreatment with atropine. Tissues were extracted from VX-treated and untreated animals and tested for AChE, BChE, and acylpeptide hydrolase activity. VX treatment inhibited 50% of the AChE activity in brain and muscle of AChE+/+ and +/- mice, 50% of the BChE activity in all three AChE genotypes, but did not significantly inhibit acylpeptide hydrolase activity. It was concluded that the toxicity of VX must be attributed to inhibition of nonacetylcholinesterase targets in the AChE-/- mouse. Organophosphorus ester toxicity in wild-type mice is probably due to inhibition or binding to several proteins, only one of which is AChE.

Protective action of the serine protease inhibitor N-tosyl-L-lysine chloromethyl ketone (TLCK) against acute soman poisoning.

Soman-poisoned rats display cholinergic crisis, a systemic mast cell degranulation characteristic of anaphylactic reactions and an excitotoxin-like sequential seizure and neuronal degeneration. The protection of guinea pigs from soman lethality by prophylactic administration of the serine protease inhibitor suramin suggests a possible proteolytic component in soman poisoning. The present study tested the effect of N-tosyl-L-lysine chloromethyl ketone (TLCK), an inhibitor of trypsin-like serine proteases, on soman-induced toxic signs (convulsions, righting reflex) and survival time. Nine control guinea pigs receiving 2 x LD(50) (56 microg kg(-1), s.c.) of soman immediately followed by a therapeutic dose of atropine sulfate (17.4 mg kg(-1) i.m.) experienced severe convulsions, and 8/9 lost the righting reflex. Six of these nine animals expired within 65 min; the three remaining animals survived 24 h to termination of the experiment. When a second group of animals were given TLCK (12 mg kg(-1), i.p.) 30 min prior to a 2 x LD(50) soman challenge and atropine-sulfate therapy, 5/9 experienced convulsions and only 3/9 lost the righting reflex. All nine animals survived beyond 4 h, with six surviving to 24 h. Compared with soman controls, prophylaxis with TLCK significantly prevented the loss of righting reflex (P = 0.05) and enhanced 4-h survival (P = 0.005). Although, convulsions were reduced and 24-h survival was improved in TLCK-treated animals, these results were not statistically significant. The protection from soman toxicity by chemically distinct protease inhibitors such as suramin and TLCK suggests a role for pathological proteolytic pathways in soman intoxication.

Serum paraoxonase (PON1) isozymes: the quantitative analysis of isozymes affecting individual sensitivity to environmental chemicals.

In a recent study on Gulf War veterans who developed delayed neurotoxicity symptoms, we found their levels of serum paraoxonase (PON1) isozyme type Q to be significantly lower than in the control, unaffected veteran group. These results were obtained in 25 ill veterans and 20 well control subjects, of which 10 were deployed and 10 were nondeployed battalion members who remained in the United States during the Gulf War. The blood samples were also assayed for serum butyrylcholinesterase in our laboratory, and more recently in Dr. C. Broomfield's laboratory for somanase and sarinase activities. The cholinesterase activities showed no significant correlation with the PON1 isozyme levels or the severity of the clinical symptoms, but the somanase and sarinase levels ran parallel to the PON1 type Q isozyme concentrations. Although there is no direct evidence that these Gulf War veterans were directly exposed to or encountered either of these nerve gases, they may have been exposed to some environmental or chemical toxin with a similar preference for hydrolysis by the PON1 type Q isozyme. The number of subjects is relatively small, but the results should encourage other investigators to examine both the individual phenotypes and the levels of PON1 isozymes in other groups exhibiting neurological symptoms.

The role of interleukin-6 (IL-6) in human sulfur mustard (HD) toxicology.

The authors applied in vitro models of controlled damage to human epidermal keratinocytes (HEKs), human skin fibroblasts (HSFs), and human breast skin tissue (HBST) to examine the mechanism responsible for sulfur mustard (HD)-induced interleukin-6 (IL-6) alterations. Treatment with 100 microM HD for 24 hours resulted in a significant increased amount of IL-6 being secreted by HEKs (HD-exposed to control ratio [E/C] = 4.15 +/- 0.07) and by HSFs (E/C = 7.66 +/- 0.04). Furthermore, the HD-induced secretion of IL-6 in HEKs was neutralized with monoclonal human IL-6 antibodies. The secretion of IL-6 in HBST supernatant exposed to HD produced conflicting results. Although an increase of IL-6 was observed in control superfusion media from HBST, IL-6 levels were observed to decrease as the concentration of HD increased. Time course of IL-6 mRNA levels were performed using a competitive polymerase chain reaction (PCR) and human IL-6 mRNA assay detection kit in control and HD (100 microM)-treated HEKs cells. IL-6 mRNA transcripts in HD-exposed HEKs were first observed within 2 hours, dropped at 5 to 6 hours, and increased by approximately 2.2-fold and 8.5-fold at 24 to 48 hours after HD exposure, respectively, as detected by the Xplore mRNA Quantification System. Surface-enhanced laser desorption ionization (SELDI) mass spectrometry was also applied to study the secretion pattern of IL-6 on lysate preparations of HBST. A peak in the area of 23,194 to 23,226 Da was detected using antibody coupled to the chip. This peak was assigned to correspond to the mass of the IL-6 glycoprotein. Recombinant human IL-6 (rhIL-6) exposed to HD lacked the second disulfide bridge and was partially unfolded, as determined by nuclear magnetic resonance-nuclear Overhauser enhancement and exchange spectroscopy (NMR-NOESY). The disappearance of the resonance peak at 3.54 ppm and the appearance of a new chemical shift at 1.85 ppm suggested that a change in structure had occurred in the presence of HD. From the data, the possibility cannot be excluded that IL-6 might be involved in the early event of structural changes of the signal transducer glycoprotein that indirectly initiates the cascade of events such as skin irritation and blister formation observed in the pathophysiology of HD injury.

Progress on the road to new nerve agent treatments.

In the 50 years since nerve agents were developed a great deal has been learned about their acute toxicity, treatment and prophylactic strategies. However, the currently fielded treatments are not significantly different from those available at the end of World War II. Reasons for this lack of progress and strategies to circumvent those intrinsic problems that have impeded progress are discussed, with emphasis on the development of scavengers to be introduced as prophylactics that will significantly reduce the effective dose and thus protect against multiple times the normal LD50.

Expression of biologically active human butyrylcholinesterase in the cabbage looper (Trichoplusia ni).

This investigation examined the utility of three recombinant protein-expression systems (COS cells, insect cells and insect larvae) to cost-effectively produce biologically active human butyrylcholinesterase (BuChE). It was determined that baculovirus-infected insect cells (Sf9 and High 5) expressed 3.5- and 8.2-fold, respectively, more active enzyme than COS-7 cells. Baculovirus-infected cabbage looper (Trichoplusia ni) insect larvae produced over 26 times more than High 5 cells; in fact, one baculovirus-infected insect larva provided more active protein than 100 ml of insect cell culture. Analysis of the larvally expressed proteins revealed that the vast majority of BuChE expressed was inactive due to extensive degradation that occurred in vivo. However, the active form of BuChE does have enzyme kinetics similar to those of its human serum counterpart. Cabbage looper larvae were also examined for their ability to serve as an in vivo animal model to study protection against anti-cholinesterase toxicity. This was unsuccessful due to their high tolerance to the very toxic organophosphorus compounds tested. This tolerance may be attributed at least in part to a novel endogenous organophosphorus acid anhydride hydrolase activity that is capable of hydrolysing the chemical-warfare nerve agents sarin (isopropyl methylphosphonofluoridate) and soman (pinacolyl methylphosphonofluoridate). These results show that cabbage looper larvae can serve as an inexpensive recombinant protein-expression system for human BuChE.

Response of normal human keratinocytes to sulfur mustard: cytokine release.

Cytokines play a major role in both acute and chronic inflammatory processes, including those produced by sulfur mustard (2,2'-dichlorodiethyl sulfide, HD). This study describes responses of normal human epidermal keratinocytes (NHEK) to HD, defined by interleukin-1beta (IL-1beta), IL-6, IL-8 and tumor necrosis factor alpha (TNF-alpha) release. Commercially available enzyme-linked immunosorbent assay (ELISA) kits were used to measure the cytokine release in NHEK during exposure to 100 and 300 microM of HD. Exposure to 100 microM HD increased the release of cytokines. The amounts of IL-8 and TNF-alpha present in cell suspensions increased up to 59-fold and 4-fold, respectively, above control levels when NHEK were exposed to 300 microM HD. Exposure of NHEK to 300 microM HD had a highly variable effect on the release of IL-1beta, where sometimes the secretion of IL-1beta increased above baseline level and at other times it decreased in cell suspensions. Supernatants were collected from cell culture flasks 24 h after exposure of 100 and 300 microM HD and significantly increased levels of IL-6 were observed. Interleukin-6 was released in a concentration-dependent manner, 3.6-fold up to 8.4-fold, respectively, in supernatant. These pro-inflammatory mediators IL-1beta, IL-8, TNF-alpha and IL-6 may play an important role in HD injury. The present findings suggest that the cytokine changes detected could be used as potential biomarkers of cutaneous vesicant injury.

Exposure of human epidermal keratinocyte cell cultures to sulfur mustard promotes binding of complement C1q: implications for toxicity and medical countermeasures.

Sulfur mustard (HD)-increased proteolytic activity, HD-enhanced expression of Fc receptor (FcR) on human epidermal keratinocytes (HEK) and associated inflammatory responses may contribute to HD pathology. Like the FcR, the first component of the classical complement (C') cascade, C1q, binds to the Fc region of antibody to mediate inflammatory responses. Complement C1q binds specifically to the C1q receptor (C1qR) on the blebs of apoptotic human keratinocytes and is proposed as a cell surface marker for apoptosis. Assays by fluorescent antibodies demonstrated significantly enhanced binding of C1q to HEK cell cultures exposed to HD. The cell populations of HEK that showed enhanced C1q binding also demonstrated an intermediate uptake of propidium iodide that was greater than in viable unexposed cells but less than in dead cells. The HD-enhanced C1q binding was concentration-dependent, negative by flow cytometry or weakly positive by digital scanning microscopy at 100 microM and positive by both methods at 300 microM. Binding of C1q was also time-dependent, weakly positive at 8 h, and positive at 16 and 24 h after HD exposure. The HD-increased C1qR that binds C1q to the surface of HEK might be a contributing mechanism or a marker for the inflammation and vesication associated with HD exposure.

Characterization of a soluble mouse liver enzyme capable of hydrolyzing diisopropyl phosphorofluoridate.

A novel mouse liver soluble fraction DFPase which has organophosphatase activities with sarin, soman and tabun, was purified and characterized. However, it lacks paraoxonase and arylesterase activities with paraoxon and phenyl acetate, respectively. This DFPase closely resembles and may be identical with the one purified by Little et al. in 1989 from the soluble fraction of rat liver, based on its substrate specificity, size (approximately 39 kDa) and its stimulation by several metal ions, namely magnesium, manganese and cobalt. Sequencing of our purified mouse liver DFPase showed it to be identical in its amino acid sequence with the recently identified senescence marker protein-30 (SMP-30) by Fujita et al. in 1996. Other senescence marker proteins possessing high structural homology with the mouse SMP-30 have also been found and sequenced from human and rat livers. There is no structural homology between the senescence marker protein family and the group of mammalian paraoxonases. Thus, it is clear that there are at least two distinct, unrelated families of mammalian liver enzymes that share DFPase activity.

Protein engineering of a human enzyme that hydrolyzes V and G nerve agents: design, construction and characterization.

Because of deficiencies in the present treatments for organophosphorus anticholinesterase poisoning, we are attempting to develop a catalytic scavenger that can be administered as prophylactic protection. Currently known enzymes are inadequate for this purpose because they have weak binding and slow turnover, so we are trying to make an appropriate enzyme by protein engineering techniques. One butyrylcholinesterase mutant, G117H, has the desired type of activity but reacts much too slowly. This communication describes an attempt to determine the reason for the slow reaction so that a more efficient enzyme might be designed. The results indicate that the mutation at residue 117 has resulted in a distortion of the transition state of the reaction of organophosphorus compounds with the active site serine. This information will be used to develop other mutants that avoid transition state stabilization sites.

Response of normal human keratinocytes to sulfur mustard (HD): cytokine release using a non-enzymatic detachment procedure.

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-1beta), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-alpha (TNF-alpha) 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 x 10(6) cells/mL) were exposed to HD (100 and 300 microM) 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 microM of HD. Exposure to 100 microM HD increased release of cytokines. IL-1beta (exposed: 1.41 x 10(-5) pg/ cell+/-1.60 x 10(-6) pg/cell: control 7.10 x 10(-6) pg/ cell+/-1.20 x 10(-6) pg/cell), TNF-alpha (exposed: 1.06 x 10(5) pg/cell+/-7.3 x 10(-7)pg/cell; control: 4.04 x 10(-6)+/-2.80 x 10(-7) pg/cell) and IL-8 (exposed: 3.71 x 10(-5) pg/ cell+/- 3.26 x 10(-6) pg/cell; control: 2.99 x 10(-6) pg/cell+/-8.80 x 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 microM for 24 h (exposed: 1.47 x 10(-6)+/-1.60 x 10(-7) pg/cell; control: 1.28 x 10(-6)+/-8.40 x 10(-8) pg/cell). However, cell suspensions of NHEK increased levels of IL-6 after exposure to 300 microM HD (4.67 x 10(-5) pg/cell+/-3.90 x 10(-6) pg/cell; control: 3.99 x 10(-6) pg/cell+/-5.50 x 10(-7) pg/cell). The amount of IL-8 and TNF-alpha present in cell suspensions increased up to 59-fold and fourfold, respectively, above control levels when NHEK cells were exposed to 300 microM HD. Exposure of NHEK to 300 microM HD had a highly variable effect on the release of IL-1beta, where sometimes the secretion of IL-1beta 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 microM 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 pro-inflammatory mediators IL-1beta, IL-8, TNF-alpha 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.

Purification and characterization of protease activated by sulfur mustard in normal human epidermal keratinocytes.

A membrane-bound protease induced by sulfur mustard in cultured normal human epidermal keratinocytes (NHEK) was purified and partially characterized. Maximum enzyme stimulation occurred at 16 hr after normal human epidermal keratinocytes were exposed to 300 microM sulfur mustard. Purification to homogeneity of the protease was accomplished by Triton X-100 solubilization, ultracentrifugation, and dialysis, followed by ion-exchange chromatography through DEAE-cellulose and finally hydrophobic column chromatography through phenyl Sepharose. Analysis of the purified enzyme by SDS-PAGE revealed a single polypeptide at the 80 kDa region. Further investigation of biochemical properties showed that a synthetic serine-specific Chromozym TRY peptide and the physiological protein laminin were good substrates for this enzyme. Moreover, this enzyme was inhibited mostly by the serine-protease inhibitors leupeptin and di-isopropyl fluorophosphate and not by the cysteine protease inhibitor E-64 or the metalloprotease inhibitor 1,10-phenanthroline (Component H, CH), indicating the serine protease nature of this enzyme. This enzyme had a pH optimum in the range of 7.0 to 8.0. Amino acid sequencing of the purified enzyme revealed that this enzyme belongs to the endopeptidase family (serine protease), and is homologous with a mammalian-type bacterial serine endopeptidase that can preferentially cleave K-X, including K-P. These results suggest that serine-protease stimulation may be one of the mechanisms of mustard-induced skin blister formation, and that some specific serine-protease inhibitors may be useful for the treatment of this sulfur mustard toxicity.

Sulfur mustard exposure enhances Fc receptor expression on human epidermal keratinocytes in cell culture: implications for toxicity and medical countermeasures.

Sulfur mustard (HD) is a chemical warfare blister agent. The biochemical basis of HD-induced vesication is unknown, and no antidote currently exists. Basal epidermal cells are a major site of HD toxicity in vivo, with inflammation and HD-increased proteolytic activity implicated as factors that contribute to HD pathology. Fc receptors (FcR) bind to the Fc region of antibody to mediate many effector and regulatory functions that can influence inflammatory responses. FcR are found on all types of immune cells and are also expressed on the surface of human keratinocytes. Assay by fluorescent antibodies demonstrated significantly enhanced CD32 (FcRII) and CD16 (FcRIII) on human epidermal keratinocyte (HEK) cell cultures at 8 to 24 h after exposure to HD (50, 100 and 200 micromol/L). The enhanced CD32 was time- and concentration-dependent and agreed well with the time course of increased proteolysis and cutaneous pathology observed during HD vesication. HD-increased FcR on the surface of HEK might be a mechanism of vesication.

Organophosphorus acid anhydride hydrolase activity in human butyrylcholinesterase: synergy results in a somanase.

Organophosphorus acid anhydride (OP) "nerve agents" are rapid, stoichiometric, and essentially irreversible inhibitors of serine hydrolases. By placing a His near the oxyanion hole of human butyrylcholinesterase (BChE), we made an esterase (G117H) that catalyzed the hydrolysis of several OP, including sarin and VX [Millard et al. (1995) Biochemistry 34, 15925-15930]. G117H was limited, however, because it was irreversibly inhibited by pinacolyl methylphosphonofluoridate (soman); soman is among the most toxic synthetic poisons known. This limitation of G117H has been overcome by a new BChE (G117H/E197Q) that combines two engineered features: spontaneous dephosphonylation and slow aging (dealkylation). G117H/E197Q was compared with the single mutants BChE G117H and E197Q. Each retained cholinesterase activity with butyrylthiocholine as substrate, although kcat/Km decreased 11-, 11- or 110-fold for purified G117H, E197Q, or G117H/E197Q, respectively, as compared with wild-type BChE. Only G117H/E197Q catalyzed soman hydrolysis; all four soman stereoisomers as well as sarin and VX were substrates. Phosphonylation and dephosphonylation reactions were stereospecific. Double mutant thermodynamic cycles suggested that the effects of the His and Gln substitutions on phosphonylation were additive for PSCR or PRCR soman, but were cooperative for the PSCS stereoisomer. Dephosphonylation limited overall OP hydrolysis with apparent rate constants of 0.006, 0.077, and 0.128 min-1 for the PR/SCR, PSCS, and PRCS soman stereoisomers, respectively, at pH 7.5, 25 degrees C. We conclude that synergistic protein design converted an archetypal "irreversible inhibitor" into a slow substrate for the target enzyme.

Unique push-pull mechanism of dealkylation in soman-inhibited cholinesterases.

The pH-dependence and solvent isotope effects of dealkylation in diastereomeric adducts of Electric eel (Ee) and fetal bovine serum (FBS) acetylcholinesterase (AChE) inactivated with P(-)C(+) and P(-)C(-) 2-(3,3-dimethylbutyl) methylphosphonofluoridate (soman) were studied at 4.0 +/- 0.2 degrees C. The rate constant versus pH profiles were fit to a bell-shaped curve for all adducts. Best fit parameters are pK1 4.4-4.6 and pK2 6.3-6.5 for Ee AChE and pK1 4.8-5. 0 and pK2 5.8 for FBS AChE. The pKs are consistent with catalytic participation of the Glu199 anion and HisH+440. Maximal rate constants (kmax) are 13-16 x 10(-3) s-1 for Ee AChE and 8 x 10(-3) s-1 for FBS AChE. The solvent isotope effects at the pH maxima are 1.1-1.3, indicating unlikely proton transfer at the enzymic transition states for the dealkylation reaction. Slopes of log rate constant versus pH plots are near 1 at 25.0 +/- 0.2 degrees C between pH 7.0 and 10.0. In stark contrast, the corresponding adducts of trypsin are very stable even at 37.0 +/- 0.2 degrees C. The rate constants for diastereomers of soman-inhibited trypsin at 37.0 +/- 0.2 degrees C are pH independent and approximately 10(4) times smaller than kmax for analogous adducts with AChE. Dealkylation in soman-inhibited AChEs is estimated to occur at >10(10) times faster than a plausible nonenzymic reaction. Up to 40% of the catalytic acceleration can be attributed to an electrostatic push, and an electrostatic pull provides much of the balance. The results of this work together with results of a product analysis by Michel et al. (1969) can be explained by an initial and rate-determining methyl migration from Cbeta to Calpha. This is driven by the high electron density of residues (Glu199 and Trp84) at a crowded active site and may be concerted with C-O bond breaking. The positive charge at the rate-determining transition state is distributed between Cbeta and His440. A tertiary carbocation may have a fleeting existence before it is trapped by water or neighboring electrons which is likely to be promoted by Glu199 as the proton acceptor.