Metabolic activation of sulfur mustard leads to oxygen free radical formation.

We recently published electron paramagnetic resonance (EPR) spin trapping results that demonstrated the enzymatic reduction of sulfur mustard sulfonium ions to carbon-based free radicals using an in vitro system containing sulfur mustard, cytochrome P450 reductase, NADPH, and the spin trap α-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) in buffer (A.A. Brimfield et al., 2009, Toxicol. Appl. Pharmacol. 234:128-134). Carbon-based radicals have been shown to reduce molecular oxygen to form superoxide and, subsequently, peroxyl and hydroxyl radicals. In some cases, such as with the herbicide paraquat, a cyclic redox system results, leading to magnified oxygen free radical concentration and sustained tissue damage. Low mustard carbon radical concentrations recorded by EPR in our in vitro system, despite a robust (4.0mM) sulfur mustard starting concentration, led us to believe a similar oxygen reduction and redox cycling process might be involved with sulfur mustard. A comparison of the rate of mustard radical-POBN adduct formation in our in vitro system by EPR at atmospheric and reduced oxygen levels indicated a sixfold increase in 4-POBN adduct formation (0.5 to 3.0 µM) at the reduced oxygen concentration. That result suggested competition between oxygen and POBN for the available carbon-based mustard radicals. In parallel experiments we found that the oxygen radical-specific spin trap 5-tert-butoxycarbonyl-5-methylpyrroline-N-oxide (BMPO) detected peroxyl and hydroxyl radicals directly when it was used in place of POBN in the in vitro system. Presumably these radicals originated from O(2) reduced by carbon-based mustard radicals. We also showed that reactive oxygen species (ROS)-BMPO EPR signals were reduced or eliminated when mustard carbon radical production was impeded by systematically removing system components, indicating that carbon radicals were a necessary precursor to ROS production. ROS EPR signals were completely eliminated when superoxide dismutase and catalase were included in the complete in vitro enzymatic system, providing additional proof of oxygen radical participation. The redox cycling hypothesis was supported by density functional theory calculations and frontier molecular orbital analysis.

Toxicodynamic modeling of highly toxic organophosphorus compounds.

Although the in vitro effect of organophosphorus (OP) compounds on acetylcholine-esterase (AChE) has been studied extensively, the hypothesis that OP inhibition of AChE is the primary mechanism of acute in vivo OP toxicity has been controversial. For example, a recent review (Pope and Liu, 2004) suggested that OP compounds have direct toxic effects on other enzymes, ACh receptors, and receptor/ channel complexes that are independent of AChE inhibition. The purpose of this report is to examine the hypothesis that AChE inhibition is the mechanism of acute toxicity of OP compounds by mathematically modeling the in vivo lethal effects of highly toxic OP compounds and determining the amount of variation in OP toxicity that is explained by AChE inhibition.

A theoretical model of the competition between hydrolase and carboxylesterase in protection against organophosphorus poisoning.

A system of coupled non-linear differential equations describing interactions between organophosphorus compounds (OPs), OP hydrolase, acetylcholinesterase (AChE), and carboxylesterase (CaE) in a single compartment was derived incorporating irreversible combination of OP with AChE, hydrolytic breakdown of OP, and irreversible combination of OP with CaE. The equations were then uncoupled, providing non-linear differential equations on AChE, CaE and OP concentrations. One steady state solution of the AChE equation provided theoretical expressions for the amounts of OP hydrolyzed, bound with CaE, and bound with AChE. Assuming that the LD50 of an OP reflects the dose that depletes AChE to a 'minimal essential' level and that a single compartment model is applicable in vivo, the steady state solution becomes an equation predicting the LD50 from rate constants, initial enzyme levels, and the allowable AChE depletion. Normalization by initial AChE concentration produced a dimensionless relationship describing an 'OP toxicity surface' that clearly demonstrates regions where hydrolysis and CaE offer protection against OP poisoning. The surface can be used to theoretically predict an LD50 given only kinetic rate constants and effective whole-body AChE and CaE levels. Predictions of LD50s of seven OPs in rats were compared with published data. The relationship was found to adequately predict published LD50s spanning 5 orders of magnitude. The OP toxicity surface relationship provides a conceptual tool for use in OP toxicity research but should be particularly useful in predicting the relative protective effects of catalytic and stoichiometric scavenger mechanisms for an OP.

Comparison of hematologic consequences and efficacy of p-aminophenones in mice.

Controlled methemoglobin (MHb) formation is one strategy employed to counter cyanide (CN) toxicity. Currently available MHb formers present certain drawbacks and limitations. The purpose of this study was to characterize, in mice, the hematologic effects of the MHb-forming compound p-aminopropiophenone (PAPP), and two structurally-related p-aminophenones, p-aminoheptanoylphenone (PAHP) and p-aminooctanoylphenone (PAOP). Although these three p-aminophenones have been shown previously to be efficacious as pretreatments against CN, a more complete understanding of their hematologic effects is lacking. In addition, because the active form of PAPP has been shown to be its N-hydroxy metabolite, the N-hydroxy metabolites of PAPP, PAHP and PAOP were also tested. Using a hemoximeter, blood samples obtained -2 to +180 min relative to intramuscular (i.m.) or intraperitoneal (i.p.) drug injections were evaluated. Sodium nitrite (NaNO(2)) and the appropriate solvents served as the positive and negative controls, respectively. Dose-, time-, route-, and compound-related effects were observed. MHb and sulfhemoglobin levels increased, whereas levels of those parameters related to oxygen-carrying capacity of the blood, such as, oxygen saturation and oxyhemoglobin decreased. In general, the effects of PAHP and PAOP were longer lasting than those of PAPP and NaNO(2). Furthermore, PAPP and NaNO(2) were equally effective with either route of administration. Conversely, PAHP and PAOP showed larger effects when administered i.p. versus i.m. The animals treated with N-hydroxy metabolites of the p-aminophenones also showed similar changes in the hematological parameters measured. N-hydroxy PAPP was shown to be the most rapidly acting MHb-forming compound examined in this series. It could achieve therapeutic concentrations of MHb within 2 min and thus may be considered as a treatment for CN intoxication. Although additional work is needed, these data provide information that will be useful for the successful development of improved anti-CN MHb formers.

Development of a physiologically based model for the toxicokinetics of C(+/-)P(+/-)-soman in the atropinized guinea pig.

A physiologically based model was developed which describes the in vivo toxicokinetics of the highly reactive nerve agent C(+/-)P(+/-)-soman at doses corresponding to 0.8-6 LD50 in the atropinized guinea pig. The model differentiates between the summated highly toxic C(+/-)P(-)-soman stereoisomers at supralethal doses and the individual nontoxic C(+/-)P(+)-isomers. Several toxicant-specific parameters for the soman stereoisomers were measured in guinea pig tissue homogenates. Cardiac output and blood flow distribution were measured in the atropinized, anesthetized, and artificially ventilated guinea pig. The model was validated by comparison of the time courses for the blood concentrations of the two pairs of stereoisomers in the guinea pig after i.v. bolus administration with the blood concentrations predicted by the model. The predictions put forward for the summated C(+/-)P(-)-isomers are in reasonable agreement with the experimental data obtained after doses corresponding to 2 and 6 LD50. In view of large differences in the rates of hydrolysis of the C(+/-)P(+)-isomers, these two isomers had to be differentiated for satisfactory modeling of both isomers. In order to model the toxicokinetics of C(+/-)P(-)-soman at a dose of 0.8 LD50, the almost instantaneous elimination of the C(+)P(-)-isomer at that dose had to be taken into account. The sensitivity of the predictions of the model to variations in the parameters has been studied with incremental sensitivity analysis. The results of this analysis indicate that extension to a model involving four individual stereoisomers is desirable in view of large differences in the biochemical characteristics of the two C(+/-)P(-)- and C(+/-)P(+)-isomers.

Product lines in a hospital emergency room.

A hospital's emergency department is not a single product, but is a product system made up of multiple product lines. Although the same department staff deals with all the product lines, the ROI for time and resources varies widely among the lines. Each line draws upon a different expertise in the hospital staff and services a different market. Identification of market niches within the broad system of emergency services will enable a hospital to narrow the focus of its marketing efforts on those lines for which a current need can be shown.

Pricing health care services: applications to the health maintenance organization.

This article illustrates how management in one type of service industry, the health maintenance organization (HMO), have attempted to formalize pricing. This effort is complicated by both the intangibility of the service delivered and the relatively greater influence in service industries of non-cost price factors such as accessibility, psychology, and delays. The presentation describes a simple computerized approach that allows the marketing manager to formally estimate the effect of incremental changes in rates on the firm's projected patterns of enrollment growth and net revenues. The changes in turn reflect underlying variations in the mix of pricing influences including psychological and other factors. Enrollment projections are crucial to the firm's financial planning and staffing. In the past, most HMO enrollment and revenue projections of this kind were notoriously unreliable. The approach described here makes it possible for HMOs to fine-tune their pricing policies. It also provides a formal and easily understood mechanism by which management can evaluate and reach consensus on alternative scenarios for enrollment growth, staff recruitment and capacity expansion.

Nutrient pathways of the cruciate ligaments. An experimental study using the hydrogen wash-out technique.

We used the hydrogen wash-out technique to determine the rate of wash-out of an inert tracer from the posterior cruciate ligament in dogs and rabbits after we had detached the ligament from bone and synovial structures, and after we had isolated it from the synovial tissue with aluminum foil. These rates were compared with those from controls and no differences could be found between them. The uptake of tritiated proline administered intravenously was also shown not to be significantly different in the detached ligament as compared with an intact ligament. Isolation of the ligament from contact with the synovial lining prevented the uptake of hydrogen. Cruciate ligaments removed from the knee and placed in the suprapatellar pouch appeared viable after eight to ten days. These findings suggest that diffusion can serve as a major pathway for delivery of nutrients to the cruciate ligaments.