ABSTRACT
Catalytic scavengers of organophosphates (OPs) are considered very promising antidote candidates for preventing the adverse effects of OP intoxication as stand alone treatments. This study aimed at correlating the in-vivo catalytic efficiency ((kcat/KM)[Enzyme]pl), established prior to the OP challenge, with the severity of symptoms and survival rates of intoxicated animals. The major objective was to apply a theoretical approach to estimate a lower limit for (kcat/KM)[Enzyme]pl that will be adequate for establishing the desired kcat/KM value and plasma concentration of efficacious catalytic bioscavengers. Published data sets by our group and others, from in vivo protection experiments executed in the absence of any supportive medicine, were analyzed. The kcat/KM values of eight OP hydrolyzing enzymes and their plasma concentrations in four species exposed to OPs via s.c., i.m. and oral gavage, were analyzed. Our results show that regardless of the OP type and the animal species employed, sign-free animals were observed following bioscavenger treatment provided the theoretically estimated time period required to detoxify 96% of the OP (t96%) in vivo was ≤10 s. This, for example, can be achieved by an enzyme with kcat/KM = 5 × 107 M-1 min-1 and a plasma concentration of 0.4 µM ((kcat/KM)[Enzyme]pl = 20 min-1). Experiments in which animals were intoxicated by i.v. OP injections did not always conform to this rule, and in some cases resulted in high mortality rates. We suggest that in vivo evaluation of catalytic scavengers should avoid the unrealistic bolus i.v. route of OP exposure.
Subject(s)
Aryldialkylphosphatase/blood , Biocatalysis , Neuroprotection , Organophosphate Poisoning/blood , Organophosphate Poisoning/enzymology , Phosphoric Triester Hydrolases/blood , Animals , Buffers , Chemical Warfare Agents/toxicity , Guinea Pigs , Haplorhini , Humans , Reproducibility of Results , SolutionsSubject(s)
Antioxidants/pharmacology , Ascorbic Acid/analogs & derivatives , Lipoproteins, HDL/blood , Oxidants/pharmacology , Sodium Hypochlorite/pharmacology , Aryldialkylphosphatase/blood , Ascorbic Acid/pharmacology , Carboxylic Ester Hydrolases/blood , Chloramines/blood , Humans , Oxidation-Reduction , Phosphoric Triester Hydrolases/bloodABSTRACT
Developing improved medical countermeasures against chemical warfare agents (nerve agents) is urgently needed but time-consuming and costly. Here we introduce a robot-assisted liquid handling system with warming, cooling and incubating facilities to screen the detoxifying properties of biological and chemical materials against nerve agents. Two biological tests were established and plasma from various species, DFPase and three cyclodextrins were used as test materials. In test 1, plasma was mixed with sarin or VX and the inhibitory potency of the incubate was determined with human acetylcholinesterase (AChE) at 0, 30 and 60 min. In test 2, test materials and nerve agents were mixed and incubated. Between 0 and 40 min samples were taken and incubated for 3 min with AChE and the residual AChE inhibition was determined to enable the semi-quantitative evaluation of the detoxification kinetics. The automated assays proved to be highly reproducible. It was possible to pre-select detoxifying reagents with test 1 and to determine more detailed detoxifying kinetics with test 2. In conclusion, the automated assay may be considered as a versatile tool for the high-throughput screening of potential detoxifying materials against different nerve agents. With this two-step assay it is possible to screen effectively for detoxifying materials in a high-throughput system.
Subject(s)
Biological Assay/methods , Biological Warfare Agents , Cholinesterase Reactivators/pharmacology , Drug Evaluation, Preclinical/methods , Animals , Biological Assay/instrumentation , Biomarkers , Cyclodextrins/pharmacology , Drug Evaluation, Preclinical/instrumentation , Erythrocytes/drug effects , Guinea Pigs , Humans , In Vitro Techniques , Macaca mulatta , Organothiophosphorus Compounds/blood , Organothiophosphorus Compounds/toxicity , Phosphoric Triester Hydrolases/blood , Rabbits , Rats , Robotics , Sarin/blood , Sarin/toxicity , Software , Species Specificity , SwineABSTRACT
Paraoxonase 1 (PON1) is a cardioprotective enzyme associated with high-density lipoprotein (HDL). We tested the hypothesis that vitamin C protects HDL and PON1 from deleterious effects of hypochlorous acid, a proinflammatory oxidant. In our experiments, HDL (from human plasma) or diluted human plasma was incubated with hypochlorite in either the absence (control) or presence of vitamin C before measuring chemical modification and PON1 activities. Vitamin C minimized chemical modification of HDL, as assessed by lysine modification and accumulation of chloramines. In the absence of vitamin C, chloramines accumulated to 114 +/- 4 micromol/L in HDL incubated with a 200-fold molar excess of hypochlorite; but addition of vitamin C (200 micromol/L) limited formation to 36 +/- 6 micromol/L (P < .001). In plasma exposed to hypochlorite, IC(50) values of 1.2 +/- 0.1, 9.5 +/- 1.0, and 5.0 +/- 0.6 mmol/L were determined for PON1's phosphotriesterase, arylesterase, and (physiologic) lactonase activities, respectively. Vitamin C lessened this inhibitory effect of hypochlorite on PON1 activities. In plasma supplemented with vitamin C (400 micromol/L), PON1 phosphotriesterase activity was 72% +/- 17% of normal after incubation with hypochlorite (2 mmol/L), compared with 42% +/- 6% for unsupplemented plasma (P < .05). Similar effects were seen for other PON1 activities. In some experiments, vitamin C also appeared to reverse hypochlorite-mediated loss of PON1 phosphotriesterase activity; but this effect was not observed for the other PON1 activities. In conclusion, vitamin C attenuated hypochlorite-mediated loss of PON1 activity in vitro and may, therefore, preserve cardioprotective properties of HDL during inflammation.