Acute toxicity of VX, soman and sarin in male and female guinea pigs at different ages
To evaluate the effects of age and sex on the sensitivity of guinea pigs to VX, soman, and sarin, the nerve agents were injected subcutaneously between the shoulder blades of male and female animals at different ages: PND5-10 (neonatal), PND35-45 (prepubertal) ad PND120-150 (adult). As indicated in Methods, the published LD50s of VX, soman and sarin for prepubertal male guinea pigs (see ) were used as the starting test doses for the neonatal and adult guinea pigs. A minimum of six doses of each nerve agent were evaluated in each age group.
Median lethal doses (LD50s) for the nerve agents VX, soman and sarin in neonatal, prepubertal and adult guinea pigs of both sexes.
The sc LD50s for VX, soman and sarin were derived from the probit analysis of the 24-h mortality dose-response relationships (). The LD50s of soman did not vary significantly with the age or sex of the guinea pigs (). In contrast, the LD50s of sarin and VX were lower for adult males than for age-matched female guinea pigs as well as male and female guinea pigs of all other ages ( and ). The probit slopes of the dose-response relationships for each of the three nerve agents for male and female guinea pigs were very steep. For neonatal and adult male guinea pigs, the slopes were, respectively: 19.1 and 21.2 for VX, 11.9 and 13.9 for soman, and 27.1 and 26.6 for sarin. For neonatal, prepubertal and adult female guinea pigs the slopes were, respectively: 21.9, 12.8 and 10.9 for VX, 8.4, 27.9, and 14 for soman, and 19.8, 24.9 and 23.9 for sarin. The steep probit slopes indicate that a small increase in the dose of the nerve agents is sufficient to cause a large reduction in the survival of the guinea pigs.
Mortality dose-response relationships for VX, soman and sarin in male and female guinea pigs at PND5-10, PND35-45 and PND120-150
In the present dose-response studies, response variations among animals of differing sizes were taking into account by the use of OPs in doses proportional to the animals’ body weights. The assumption that the amount of OP/unit of animal weight can factor the differences in the subjects’ body weights only holds true if the molecular target for the toxicants increases proportionally to the body weight of the animals. This is not the case for many drugs whose toxicities are significantly influenced by the size of the animals (see Anderson and Weber, 1975
and references therein). As shown here, however, age- and sex-dependent changes in the body weights of the guinea pigs did not parallel the age and sex dependencies of the LD50s of the nerve agents. In each group of animals exposed to a given nerve agent, the mean weights of male and female guinea pigs increased significantly from PND5-10 to PND120-150 (). Further, adult male guinea pigs were significantly heavier than adult female guinea pigs (). If the total amount of OP/total body weight rather than the amount of OP/kg body weight administered to the animals determined their sensitivity to the OPs, the LD50s of all nerve agents should have decreased gradually from neonatal to prepubertal to adult animals of a given sex. In addition, the LD50s of all nerve agents should have been lower in male than in female guinea pigs. Instead, the LD50 of soman remained unchanged regardless of weight, age or sex of animals. In addition, the LD50s of VX and sarin were within the same range regardless of the weights of neonatal and prepubertal guinea pigs of both sexes and adult female guinea pigs. Thus, higher sensitivity of adult male guinea pigs to VX and sarin cannot be explained by the increase in total body weight of these animals.
Weights of male and female guinea pigs at PND5-10, PND35-45 and PND120-150 measured at 2 h prior to their challenge with VX, soman or sarin
There were qualitative differences in the signs of intoxication presented by guinea pigs challenged with VX, soman or sarin. Of the three nerve agents, VX was the slowest acting, with life-threatening effects occurring only at 3–5 h after the challenge with 1xLD50 (for operational definition of life-threatening effects, see paragraph below). Sarin was the fastest acting nerve agent with life-threatening signs developing within 10–45 min after an exposure to 1xLD50. Life-threatening effects occurred within 30–120 min after the challenge of the guinea pigs with 1xLD50 soman.
In each age and sex group, guinea pigs presented clear signs of acute intoxication when challenged with nerve agent doses ≥ 0.8xLD50. Facial twitches, chewing, slight hyperlocomotion and head tremors were common signs observed in guinea pigs challenged with 0.8xLD50 VX, soman or sarin. With increasing doses of soman or sarin, animals presented progressively with forelimb clonus, increased secretions, muscle fasciculations, rearing, strong grinding, gnashing or clenching of the teeth (bruxism), all limb clonus and convulsions, and severe respiratory distress. Between 10 min and 2 h after the challenge with 1xLD50 sarin or soman, the toxic signs progressed from the peripheral nicotinic and muscarinic effects to the CNS effects. The onset of the CNS effects and the time to lethality were shortened with increasing doses of soman or sarin. At doses > 1xLD50 the signs of acute toxicity progressed from hyperlocomotion and tremors to life-threatening convulsions 10–20 min after the challenge.
Tonic-clonic convulsions triggered by ≥ 1xLD50 soman or sarin became life-threatening when they occurred unremittingly and lasted longer than 10 min or when they recurred for longer than 10 min with the animals exhibiting immobility between the recurrences. Animals that underwent recurrent or continuous convulsions when challenged with ≥ 1xLD50 sarin developed severe respiratory distress within 2–3 minutes of the onset of the convulsions. In contrast, gasping was a condition that developed 10–15 min after the onset of soman-induced convulsions. There is evidence from clinical studies that 10 min of continuous convulsions is sufficient to damage neurons and that unremitting or recurrent seizures lasting longer than 10 min are unlikely to self-terminate (García Peñas et al., 2007
). Thus, following the Institutional Animal Care and Use Committee (IACUC) guidelines, the animals were euthanized as soon as life-threatening signs of unremitting or recurrent convulsions developed.
Recurrent or unremitting convulsions were not triggered even by the highest doses of VX tested in this study, i.e., approximately 1.2xLD50. VX-induced convulsions were brief and showed spontaneous termination. In general, peripheral muscarinic and nicotinic signs of intoxication were more prominent with VX than with sarin or soman, and gasping was the most severe sign of VX poisoning. Animals that were challenged with ≥ 1xLD50 VX and developed life-threatening respiratory distress were euthanized. Animals survived the challenge with any given dose of VX, soman or sarin if they showed: (i) no clear signs of intoxication, (ii) only mild peripheral muscarinic and nicotinic signs, or (iii) mild peripheral effects accompanied by short-lasting CNS effects.
Apparent potencies of VX, soman and sarin to inhibit in vitro brain, whole blood, RBC, and plasma AChE from neonatal, prepubertal and adult guinea pigs of both sexes
In whole blood, RBC, plasma and brain extracts from prepubertal male guinea pigs, a linear relationship was observed between the change of absorbance units/mg protein and the reaction time (). The Pearson’s regression coefficient was 1 in all cases. The same results were obtained using tissue samples from male and female guinea pigs of different ages (data not shown). The AChE activity defined by the rate of hydrolysis of acetylthiocholine normalized to the amount of protein was highest in the brain extracts compared to the plasma, RBC or whole blood in all groups (). In addition, as reported in a previous study (Meinecke and Oettel, 1967
), levels of AChE activity were not significantly different between guinea pig plasma and RBC (). There was no age- or sex-related difference in the AChE activity in any of the tissues analyzed ().
Rate of AChE hydrolysis of acetylthiocholine in different tissues from male and female guinea pigs at PND5-10 and PND35-45
The concentration-response relationships for VX, soman, and sarin to inhibit brain, RBC, plasma and whole blood AChE from the different groups of guinea pigs were fitted by the Hill equation ( and ). The slopes of all curves were very similar and close to unity, indicating no cooperativity in the interactions between a given nerve agent and the enzyme. The IC50s, on the other hand, differed depending on the tissue. The curves for each VX, soman, and sarin to inhibit AChE in brain extracts from neonatal and prepubertal guinea pigs of both sexes were always displaced to the right compared to the other tissues ( and ). Accordingly, the estimated IC50s for VX, soman, and sarin to inhibit AChE in brain extracts were significantly larger than their respective IC50s to inhibit RBC, plasma or whole blood AChE (). The rank order of potency for the nerve agents to inhibit AChE in RBC and plasma from neonatal and prepubertal guinea pigs of both sexes was: soman > VX > sarin.
Concentration-response relationships for in vitro inhibition of AChE in different tissues from male and female guinea pigs at PND5-10
Concentration-response relationships for in vitro inhibition of AChE in different tissues from male and female guinea pigs at PND35-45
Apparent potencies (IC50s) for nerve agents to inhibit in vitro AChE activity in brain extracts, RBCs, plasma and whole blood of male and female guinea pigs of different ages.
Correlation between LD50s and IC50s for AChE inhibition in neonatal and prepubertal guinea pigs of both sexes
To determine whether the lethal potencies of the nerve agents correlated with their potencies to inhibit AChE in the brain, whole blood, plasma and RBCs from neonatal and prepubertal guinea pigs of both sexes, we analyzed the plots of the LD50s vs. IC50s for all three nerve agents in each tissue from all four animal groups. Given the sex differences identified for VX-induced inhibition of AChE in the various tissues of prepubertal male and female guinea pigs and between this and the younger age groups, the data for VX on prepubertal animals was not included in the plots. The Pearson’s regression coefficient was highest for the correlation between lethal potencies and potencies to inhibit brain AChE (). No positive correlations existed between the lethal potencies and the potencies of the nerve agents to inhibit AChE in the plasma, RBC or whole blood.
Correlational analysis of the relationship between LD50s and IC50s for in vitro inhibition of AChE in various tissues from neonatal and prepubertal guinea pigs of both sexes
AChE inhibition by VX, soman and sarin in brain extracts from adult male guinea pigs
If AChE inhibition was the sole mechanism underlying the toxicity of the nerve agents, the increased lethal potencies of VX and sarin in adult male guinea pigs should be paralleled by an increased apparent potency of each nerve agent to inhibit in vitro AChE activities in brain extracts from those animals. Thus, AChE activity was measured in the absence and in the presence of known concentrations of VX, soman or sarin applied to brain extracts from adult male guinea pigs. The IC50s for each nerve agent to inhibit AChE activity in the brain extracts were estimated by fitting the concentration-response relationships with the Hill equation (). The estimated IC50 values were 3.38 ± 0.10 nM, 2.90 ± 0.10 nM, and 7.27 ± 0.12 nM for VX, soman and sarin, respectively. The IC50s for VX and sarin to inhibit AChE activity in brain extracts from adult male brains were significantly higher than those needed to inhibit AChE activity in brain extracts from neonatal and prepubertal male guinea pigs (p < 0.01 according to ANOVA followed by Tukey’s post hock test). The positive correlation observed between the nerve agents’ LD50s and IC50s for inhibition of AChE in brain extracts (see ) was disrupted by the inclusion of the data obtained from adult male guinea pigs (data not shown). The linear regression of all data points, including data from adult male guinea pigs, had a Pearson’s coefficient (r2) of 0.43.
Concentration-response relationships for in vitro inhibition of AChE in brain extracts from male guinea pigs at PND120-150