Protective effect of an inhibitor of nitric oxide synthase on sulphur mustard toxicity in vitro.

Although sulphur mustard is one of the oldest chemical warfare agents, its mechanism of toxic action is still not understood and as a consequence, no antidotes exist that are effective against this agent. Pretreatment of chick embryo neuron cultures with the well-known nitric oxide synthase (NOS) inhibitor L-nitroarginine methyl ester (L-NAME) was found to confer significant protection against sulphur mustard-induced cell death. However, these protective effects were not mediated through the inhibition of NOS. These findings may provide clues to the eventual understanding to sulphur mustard toxicity, and also suggest that L-NAME has significant novel pharmacological effects other than the inhibition of NOS.

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.

Cytotoxicity of the MEIC test chemicals in primary neurone cultures.

50 Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) test chemicals were assayed for cytotoxicity in primary cultures of chick embryo forebrain neurones using the MTT and neutral red assays. The neutral red assay was consistently more sensitive to chemical toxicity; however, both assays were equally predictive when compared with in vivo toxicity data obtained from the Registry of Toxic Effects of Chemical Substances. High correlations were obtained when comparing the in vitro data with ip rodent toxicity data, but these correlations decreased significantly when oral toxicity data were used. The predictive value of the in vitro data for oral human toxicity was generally poor, but comparable with its value in predicting oral rodent toxicity. In a limited study with 10 of the MEIC test chemicals, the cytotoxicity of some compounds was dependent on the degree of differentiation of the neurone cultures, suggesting that this culture system may not only be sensitive to the basal cytotoxicity of chemicals, but also to toxic effects specific to the specialized differentiated functions of the central nervous system.

Bioassay of organophosphate nerve agents in soil using neuronal tissue cultures.

A soil sample originating from an area of suspected chemical warfare activity was subjected to chemical analysis and bioassay. Sarin and several related compounds were confirmed in the soil by capillary column gas chromatography-mass spectrometry (GC-MS); however, the binding of these compounds to the soil hindered quantitation. The chemical results were then compared to those obtained by bioassay in primary cultures of chick embryo forebrain neurons. By comparing the sample's anticholinesterase activity against those of purified standards in chick embryo neuron cultures, a reasonable agreement was found between the chemical and bioassay semi-quantitative estimates of sarin content in the soil extract. Furthermore, the in vitro system appears to offer a sensitive technique for the estimation of sarin remaining bound to the soil following solvent extraction as well as for an assessment of the potential toxicity of the contaminated soil in vivo.

Anticholinesterase activity of organophosphate nerve agents in neuronal tissue culture.

Primary chick embryo forebrain culture was examined with respect to its usefulness as a model to study organophosphate (OP) anticholinesterases. The acetylcholinesterase (AChE) activity of these cultures increased with time in culture and paralleled the development in ovo of this enzyme. The neuronal cells were extremely sensitive to the anticholinesterase effects of OP nerve agents, and experiments with soman indicated that enzyme inhibition was rapid and persistent. The potencies of several OP nerve agents as inhibitors of AChE activity in vitro paralleled their literature-reported toxicities in vivo.

Effect of metabolism on the anticholinesterase activity of parathion and paraoxon in primary neuron cultures.

The anticholinesterase activity of parathion and paraoxon, and the effects of metabolism on this activity, were examined in primary cultures of chick embryo forebrain neurons. Paraoxon was an extremely potent inhibitor of acetylcholinesterase activity, with a median inhibitory concentration (IC(50)) more than 800 times more potent than parathion. Incubation of these two compounds with rat hepatic S-9 fractions, before neuron treatment, dramatically altered their activity, though with opposite effects. Parathion, when preincubated with control S-9 (from saline-treated rats), exhibited increased anticholinesterase activity, whereas paraoxon was much decreased in potency. The relative anticholinesterase activity of these compounds after incubation with the control S-9 fractions was much more predictive of the relative toxicites of these compounds in vivo. Preincubation of these compounds with hepatic S-9 fractions that had been obtained from rats induced with either phenobarbitone or 3-methylcholanthrene, resulted in a decreased activation of parathion and an increased deactivation of paraoxon compared with control S-9.

Toxicity of organophosphate nerve agents and related phosphonylated oximes compared to their anticholinesterase activity in neuron cultures.

Oximes, such as pralidoxime and toxogonin, are important therapeutic agents for the treatment of organophosphate (OP) nerve agent poisoning. Oximes can react with these nerve agents to give intermediates, phosphonylated oximes, which may be equally toxic to the parent OP. The sc LD50s of a series of phosphonylated 2-butanone and 2,3-butanedione monoximes were compared to the sc LD50s of their parent OPs (tabun, sarin, and VX) in CD-1 mice. In every case the derivatives were significantly less toxic than their parent nerve agents. Times to death, and to signs of poisoning, were inversely proportional to the dose of test compound, and in all mortalities, blood serum acetylcholinesterase (AChE) was severely inhibited. The relative potencies of these compounds, as well as soman, cyclohexyl methylphosphonofluoridate, and diisopropyl fluorophosphate, as inhibitors of AChE in primary cultures of mouse embryo neurons, correlated with their in vivo toxicities. The results indicate that mouse embryo neuron cultures may be a useful model with which to study this class of compounds.

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.

The determination of urinary 3-trans-hydroxyproline (3 OHP). II. Normal values in neonates, infants and preschool children.

3-trans-hydroxyproline is a hydroxyproline isomer present in collagens. It is more abundant in basement membrane collagen (collagen type IV) where it can be found in a relation of 12 per 1000 amino acid residues. This implicates its probable use as a marker substance for collagen type IV metabolism. Up to now only hydrolyzed urine samples were examined for the presence of this amino acid, thus indicating total urinary excretion. We are reporting the free urinary 3 OHP content of neonates, infants and preschool children. Neonates (n = 23) showed mean excretion of 0.363 micrograms/mg creatinine (SD +/- 0.127), SEM 0.026, min. 0.197, max. 0.598. Infants (n = 11) showed mean excretion of 1.501 micrograms/mg creatinine (SD +/- 0.468), SEM 0.141, min. 0.908, max. 2.553. Preschool children (n = 16) had a mean excretion of 1.442 micrograms/mg creatinine (SD +/- 0.637), SEM 0.159, min. 0.318, max. 2.342. As determined by statistical calculations, neonates differed from infants and preschool children significantly (p less than 0.0005). Infants did not differ from preschool children, however (p less than 0.4). The reason for the low urinary excretion of 3 OHP in the neonatal period could be explained by the low enzyme activities which are physiologically observed in this developmental period in general and in the special case of a presumably low activity of the 3-prolyl-hydroxylase. Infants and preschool children presented levels 5 times higher than their neonatal mates, probably reflecting the increased 3-prolyl-hydroxylase activity which in turn is a marker of collagen synthesis.

The determination of urinary 3-trans-hydroxyproline (3 OHP). I. Normal values in school children and adults.

3-trans-hydroxyproline is a hydroxyproline isomer present in collagens. It is more abundant in basement membrane collagen (collagen type IV) where it can be found in a relation of 12 residues per 1000 amino acid residues. This implicates its probable use as a marker substance for collagen type IV metabolism. Up to now only hydrolyzed urine samples were examined for the presence of this amino acid, thus indicating total urinary excretion. We are reporting the free urinary 3 OHP content of adults and school children. School children (age group 8-18 years) showed mean excretion of 1.066 micrograms/mg creatinine (SD +/- 0.376), SEM 0.054, min. 0.516; max. 2.397, range: 1.881. Adults (age 19-29 years) showed mean excretion of 0.618 micrograms/ml creatinine (SD +/- 0.465), SEM 0.134, min. 0.055; max. 1.634, range: 1.579. As given by the statistical calculation of unpaired t-test, adults showed significantly lower urinary 3 OHP levels: t = 3.518 and p = less than 0.0005. This can be explained by increased collagen metabolism of the growing organism and is in congruence with data reported in literature for total urinary 3 OHP excretion.