Thomas W. Sawyer

Stimulation of Ca2+ influx through ATP receptors on rat brain synaptosomes: identification of functional P2X7 receptor subtypes

ATP receptors of the P2X class have previously been identified on autonomic nerve endings and on a limited population of CNS neurons. In the present study P2X receptors on mammalian cortical synaptosomes have been identified by a variety of functional and biochemical studies. In choline buffer ATP analogues caused concentration/time dependent Ca2+ influx. Relative to the effects caused by ATP, benzoylbenzoyl ATP (BzATP) was about seven times more active than ATP while 2‐me‐S‐ATP and ATPγS were much less active. α,β‐me‐ ATP and β,γ‐me‐ATP were virtually inactive. In sucrose buffer, relative to choline buffer, the activity of BzATP was more than doubled while activity in sodium buffer was reduced. Moreover, the P2X antagonists PPADS or Brilliant Blue G both significantly attenuated influx. These observations suggest the presence of P2X receptors on synaptosomes which subserve Ca2+ influx. This activity profile of the ATP analogues and the response to blocking agents are characteristic of responses of P2X7 receptors. Influx was unaffected by the VSCC inhibitors ω‐CTx‐MVIIC and (−) 202 – 791, indicating that ATP induced Ca2+ influx occurred primarily through P2X receptors. P2X7 receptor protein was identified by Western blotting and immunohistochemical staining. Purified preparations were devoid of significant concentrations of GFAP or the microglial marker OX‐42 but contained greatly enriched amounts of syntaxin and SNAP 25. The various pharmacological and biochemical studies were all consistent with the presence of functional P2X7 receptors.

Characterization of the protective effects of l-nitroarginine methyl ester (l-NAME) against the toxicity of sulphur mustard in vitro

The protective effects of L-nitroarginine methyl ester (L-NAME) against the toxicity of sulphur mustard (HD) was characterized in primary cultures of chick embryo forebrain neurons. These effects were not associated with the known nitric oxide synthase-inhibiting characteristics of this compound. No protection was evident in immature (1 day old) cultures. However, L-NAME pre-treatment resulted in a concentration-dependent protection against the toxicity of HD in mature (5 days and older) cultures, with maximal protection reaching greater than 220% at 5 mM L-NAME in terms of increasing the LC50 of control HD-treated cultures. Maximal protective effects were also achieved when L-NAME treatment was delayed up to 3 h post-HD exposure. These effects were reduced by 5 h and absent when the L-NAME was added to the cultures 8 h after HD exposure. Protection against the toxicity of HD was dependent on the continued presence of L-NAME in the medium and was persistent up to 48 h after HD exposure. This compound is one of the most effective drugs yet identified in protecting against the toxicity of HD and is the only one that exerts its effects therapeutically.

Efficacy of an oximate-based skin decontaminant against organophosphate nerve agents determined in vivo and in vitro

Recent Canadian research efforts have been directed towards the development of a reactive skin decontaminant (RSD) lotion active against classical nerve agents and mustard. The formulation presently under study consists of a 1.25 molal solution of potassium 2,3-butanedione monoximate (KBDO) in polyethylene glycol methylether 550. Although this formulation has shown good efficacy, concern has been expressed as to the potential toxicity of the reaction products of KBDO and organophosphate (OP) nerve agents. This report details the high efficacy of this lotion in inactivating OPs as measured by the systemic toxicity of the OP/RSD mixtures in rats. In addition, primary cultures of chick embryo neurons were also used to test the efficacy of the RSD. By relating the anticholinesterase activity in these cultures of the OP/RSD mixture to that of pure OP standards, a sensitive measure of the value of the RSD in inactivating tabun, sarin, soman and VX was obtained. Experiments with all four nerve agents in this in vitro system provided a good correlation with the in vivo data, and also indicated that the inactivation process was time- and agent-dependent and also related to the ratio of OP to RSD.

Effect of lowered temperature on the toxicity of sulphur mustard in vitro and in vivo

Primary cultures of chick embryo neurons were exposed to sulphur mustard (HD) and L-nitroarginine methyl ester (L-NAME) and then incubated at either 25 or 37 degrees C. Lowering the temperature of the cultures decreased the 24-h toxicity of HD, but did not increase the efficacy of L-NAME protection. However, the length of time post-HD treatment in which L-NAME was maximally effective in protecting against HD toxicity was dramatically enhanced, out to 12 h after HD exposure. In addition, the persistence of L-NAME protection of the cells against HD was significantly lengthened. Tests conducted in human skin keratinocytes also showed that lowering the incubation temperature of actively proliferating, just-confluent or post-confluent cultures significantly and persistently decreased the cytotoxicity of HD. The persistence of L-NAME protection was increased in non-proliferating cells. Finally, cooling of HD-vapour exposed sites on hairless guinea pigs for 4.5 h decreased the severity of the resultant lesions out to 72 h post-exposure.

Toxicity of sulphur mustard in adult rat lung organ culture

The toxicity of the chemical warfare agent sulphur mustard, (bis-(2-chloroethyl)sulphide, HD), was examined in adult rat lung organ cultures. Assessment of HD-induced damage by the MTT cytotoxicity assay indicated that the median lethal concentration (LC50) of HD in these cultures was reproducible, and in the microM range. Damage to the lung slices was expressed only after a latent period of 48 h and did not increase significantly with longer expression times. Histopathological examination of HD-treated lung cultures showed that the structural changes in the lung tissue paralleled the toxicity measured biochemically, and were also similar to the damage found in animals and man exposed to HD in vivo. This in vitro model offers a useful tool with which to study the toxicity and mechanism of action of sulphur mustard.

Parallel development of acetylcholinesterase in vivo and in primary neuron surface culture

The development of acetylcholinesterase (AChE) activity in primary surface cultures of mouse cortical neurons and in mouse brain was examined. The specific activity of AChE in culture increased over 600% during a 3 week period and closely paralleled the development of AChE observed in vivo. The results obtained in this study show that a developmental increase in AChE can be obtained in primary surface neuron cultures, and that the high degree of cellular organization previously deemed necessary for this development in vitro is not as important as previously thought.

Aryl hydrocarbon hydroxylase (AHH) induction by polybrominated biphenyls (PBBs): Enhancement by photolysis

Abstract Irradiation of the commercial polybrominated biphenyl (PBB) mixture, fireMaster BP-6, in cyclohexane solution at 300 nm for 930 min resulted in a marked diminution of the major components of the mixture. Administration of the photolyzed PBB mixture or fireMaster BP-6 to immature mall Wistar rats caused dose-related decreases in thymus weight and increases in hepatic microsomal benzo[a]pyrene hydroxylase (AHH), 4-dimethylaminoantypyrine N-demethylase and NADPH-cytochrome c reductase activities and cytochrome P-450 content. The dose effecting half-maximal AHH induction for the photolyzed PBBs (9 mg · kg−1) was approximately 6 times lower than that of fireMaster Bp-6 (50 mg · kg−1). Futhermore, the concentration of photolyzed PBBs (2 μM) required to displace 50% of the specifically-bound [3H]TCDD from its high-affinity cytosolic Ah receptor was approximately 150 times lower than that required for fireMaster BP-6 (300 μM), as measured by sucrose density gradient centrifugation analysis. The results suggest that the photolysis of the commercial PBB mixture yields products which possess increased biologic activity.