Relationship between the neurotoxicities of Soman, Sarin and Tabun, and acetylcholinesterase inhibition

Acetylcholinesterase (AChE)-induced chewing movements, tremors, convulsions and hind limb abduction at doses of 50-85% LD50 in rats were monitored in order to determine whether the severity of these different signs would correlate with brain AChE levels and the time course of such a relationship. 30 min after subcutaneous (s.c.) injection of Soman, the intensities of toxic signs were significantly correlated with the degree of striatal AChE inhibition. In the case of Sarin, the corresponding r-values were not significant except for tremors. For Tabun-induced chewing, tremor and hind-limb abduction, the r-values were significant. The neurotoxicity was most intense between 15 min to 2 h after treatment, but at 2 or 6 h, the r-values were well below 0.5. The inhibition of brain AChE was maximal by 30 min and was still high at 24 h.     

Acute effects of Soman, Sarin, and Tabun on cyclic nucleotide metabolism in rat striatum

Rats were injected sc with 120 micrograms/kg Soman, 120 micrograms/kg Sarin or 240 micrograms/kg Tabun. At 15 min, 2 h, or 6 h after administration, animals were decapitated along with saline-treated controls, and striatal activities of nucleotide cyclases and phosphodiesterases and striatal cyclic nucleotide levels were determined. All three agents had two similar effects on rat striatal cyclic nucleotide systems: they all increased cyclic GMP levels 15 min after their administration, and they all decreased guanylate cyclase activity 2 h after administration. There were also some different effects elicited by these three organophosphorus compounds. Different effects of Soman and Sarin seem to be mainly due to their different potencies, which in turn influence the time course of their actions. Tabun is quite different from Soman and Sarin in several respects: it rarely causes convulsions at sub-lethal doses, it has no effects on striatal cyclic AMP levels, and it affects enzyme activities 6 h after its administration. These differences may be due to the presence of cyanide instead of fluoride in its structure: i.e., this may be responsible for the different effects of Tabun on striatal cyclic nucleotide systems, and perhaps other biochemical effects. These results also indicate that other neurotransmitter systems, in addition to the cholinergic system, may be involved in organophosphate-induced toxicity.     

Efficacy of Mono- and Bis-Pyridinium Oximes Versus Soman, Sarin and Tabun Poisoning in Mice

Efficacy of Mono- and Bis-Pyridinium Oximes Versus Soman, Sarinand Tabun Poisoning in Mice. Clement, J.G. (1983). Fundam. Appl.Toxicol. 3:533–535. Various oximes (PAM, toxogonin, TMB-4,HS-6, HI-6, HGG-12, HGG-42) combined with atropine were comparedas antidotes of soman, sarin and tabun poisoning in non-fastedCD-1^® male mice. TMB-4 was the most toxic oxime with ani.p. LD₅₀ value of 80 mg/kg and HI-6 was the least toxic oximewith an i.p. LD₅₀ of 588 mg/kg. Upon comparing ED₅₀ values,HGG-42 was the most effective oxime versus soman and tabun poisoningwhereas, HI-6 was the most effective oxime versus sarin poisoning.Further research needs to be done to explain the distinct differencesin efficacy of the oximes versus poisoning by soman, sarin ortabun.     

An assessment of comparative acute toxicity of diisopropyl-fluorophosphate,Tabun, Sarin,and Soman in relation to cholinergic and GABAergic enzyme activities in rats

The sc LD50s (μmol/kg) in rats for diisopropylfluorophosphate (DFP), Tabun, Sarin, and Soman were 14.5, 1.9, 1.4, and 0.88, respectively. The relative potency was as follows: DFP < Tabun < Sarin < Soman (1:7.6:10.4:16.4). The relative potencies correlated with the in vitro acetylcholinesterase (AChE) inhibition (in terms of the IC50) by these compounds, in whole brain homogenates or the purified bovine erythrocyte AChE. There was a dose versus time for mortality relationship for all four compounds; the average time for death decreased with increase in dose. However, there was no correlation between time for death and the extent of AChE inhibition. The striatal as well as other regional (medulla, diencephalon, cortex, and cerebellum) AChE activity was inhibited over 90% of the control, by the lethal doses of these compounds. None of the lethal or sublethal doses had any apparent effect on choline acetyltransferase (CAT) or GABA-transaminase activities. Glutamic acid decarboxylase activity was increased by Soman, Sarin, and Tabun at certain lethal doses but was not affected by DFP even at the lethal dose. The results indicate that (a) the acute toxicity of organophosphate acetylcholinesterase inhibitors is directly related to the inhibition of AChE though there is a wide difference in their potency; (b) a substantial inhibition of AChE activity (over 90% of control) is necessary for lethality to ensue after an acute exposure and the margins between lethal and nonlethal doses are extremely small; and (c) qualitative differences seem to exist among the various organophosphates in affecting noncholinergic neurotransmitter enzymes.     

An Assessment of Comparative Acute Toxicity of Diisopropylfluorophosphate, Tabun, Sarin, and Soman in Relation to Cholinergic and GABAergic Enzyme Activities in Rats

An Assessment of Comparative Acute Toxicity of Diisopropylfluorophosphate,Tabun, Sarin, and Soman in Relation to Cholinergic and GABAergicEnzyme Activities in Rats. SIVAM, S. P., HOSKJNS, B., AND HO,I. K. (1984). Fundam. Appl. Toxicol. 4, 531–538. The scLD50s (µmol/kg) in rats for diisopropylfluorophosphate(DFP), Tabun, Sarin, and Soman were 14.5, 1.9, 1.4, and 0.88,respectively. The relative potency was as follows: DFP <Tabun < Sarin < Soman (1:7.6:10.4:16.4). The relativepotencies correlated with the in Vitro acetylcholinesterase(AChE) inhibition (in terms of the IC50) by these compounds,in whole brain homogenates or the purified bovine erythrocyteAChE. There was a dose versus time for mortality relationshipfor all four compounds; the average time for death decreasedwith increase in dose. However, there was no correlation betweentime for death and the extent of AChE inhibition. The striatalas well as other regional (medulla, diencephalon, cortex, andcerebellum) AChE activity was inhibited over 90% of the control,by the lethal doses of these compounds. None of the lethal orsublethal doses had any apparent effect on choline acetyhransferase(CAT) or GABA-trans-aminase activities. Glutamic acid decarboxylaseactivity was increased by Soman, Sarin, and Tabun at certainlethal doses but was not affected by DFP even at the lethaldose. The results indicate that (a) the acute toxicity of organophosphateacetylcholinesterase inhibitors is directly related to the inhibitionof AChE though there is a wide difference in their potency,(b) a substantial inhibition of AChE activity (over 90% of control)is necessary for lethality to ensue after an acute exposureand the margins between lethal and nonlethal doses are extremelysmall; and (c) qualitative differences seem to exist among thevarious organophosphates in affecting noncholinergic neurotransmitterenzymes.     

Continuous Administration of Low Dose Rates of Physostigmine and Hyoscine to Guinea-pigs Prevents the Toxicity and Reduces the Incapacitation Produced by Soman Poisoning

Abstract— A regime was developed, using mini-osmotic pumps, for the continuous subcutaneous administration of low doses of physostigmine (12.1, 9.7, 4.85 and 2.43 μgh^?1), in combination with hyoscine (1.94 or 0.39 μg h^?1), to guinea-pigs for up to 13 days. Physostigmine, in combination with hyoscine, inhibited plasma Cholinesterase, and red blood cell and brain acetylcholinesterase, in a concentration-dependent manner, did not affect the normal growth rate of guinea-pigs, and produced no obvious signs of poisoning. A dose rate of 4.85 μg h^?1 physostigmine and 1.94 μg h^?1 hyoscine was required to inhibit red cell acetylcholinesterase by 30% and brain acetylcholinesterase by 5–15%, with an accompanying plasma hyoscine concentration of 700–850 pg mL^?1. There was an apparent decline in red cell acetylcholinesterase activity during the 13 days. Hyoscine levels were higher in the cholinergic-rich areas of the brain than in the plasma. Continuous pretreatment (1 or 6 days) with physostigmine (4.84 μg h^?1) and hyoscine (1.94 μg h^?1) provided complete protection against the lethal effects, and minimized the incapacitation and weight loss produced by soman at a dose equivalent to the LD99 value. Following soman challenge, guinea-pigs exhibited early signs of soman poisoning, but generally these signs of poisoning were minimal by 1–2 h. Extending the pretreatment time to 13 days protected 75% of the guinea-pigs against the lethal effects of soman poisoning. Red cell acetylcholinesterase activity, 24 h after soman poisoning, was higher following continuous pretreatment with physostigmine and hyoscine than after acute treatment with atropine.     

The Effects of Maternally–administered Morphine on Rat Foetal Development and Resultant Tolerance to the Analgesic Effect of Morphine

Groups of 6–9 pregnant (sperm = Day 1) Sprague-Dawley (Simonsen) rats were injected with morphine sulphate, 20 mg/kg subcutaneously, on gestational days 2–5, 7–9 or 11–13. Pups delivered by Caesarian section on gestational day 22 were essentially free of any observable teratogenic changes including bone defects. However, offspring of morphine-treated rats (20 mg/kg/day subcutaneously on gestational days 17, 18, 19 and 20) which were allowed to litter normally exhibited on day 12–13 a significantly (P < 0.05) decreased response to a nocioceptive (55° surface) stimulus 30 min. following an injection of morphine sulphate 0.45 mg/kg subcutaneously (offspring of morphine-treated mothers, 16.8 sec. ± 2.3 S.E.M. versus control offspring, 25.2 sec. ± 1.7 S.E.M.). On days 21–22 of age larger doses of morphine were required to elicit an analgesic response in the young. Following an injection of morphine sulphate, 10 mg/kg subcutaneously, the maternal animals also showed significant reduction in reaction time 30 minutes later (treated, 7.6 sec. ± 0.6 S.E.M. versus control, 16.1 sec. ± 0.6 S.E.M.) and 60 minutes later (treated, 7.5 sec. ± 0.9 S.E.M. versus control, 20.8 sec. ± 2.9 S.E.M.) as compared to paired waterinjected control pregnant animals.     

Genetic Variation of Drug–Metabolizing Enzymes in the Wistar Rat

Abstract Rats of the Wistar/Af/Han/Mol/(Han 67) strain were preselected for the inducibility of the hepatic soluble aldehyde dehydrogenase, after treatment with phenobarbital (80 mg/kg daily, intraperitoneally, for 4 days) and open liver biopsy. Animals with highly induced aldehyde dehydrogenase also had a 50 and 40 % difference in the induction of D–glucuronolactone dehydrogenase and NADPH–cytochrome c reductase respectively. Other enzymes of the hepatic drug hydrox–ylation and glucuronidation did not show significant variation in the induction pattern between the two genetic groups. After inbreeding, the second filial generation was used in order to determine enzyme activities without any pretreatment. The basal activities of aldehyde and D–glucuronolactone dehydrogenase, but not of NADPH–cytochrome c reductase, were also different in the untreated animals. Urinary excretion of D–glucaric acid was the same in the two groups, both in the basal and the induced state.     

Age–Dependent Effects of Ethanol on Central Monoamine Synthesis in the Male Rat

Abstract: In the present study, the effect of ethanol on central monoamine synthesis in developing and adult male rats was studied by measuring the accumulation of DOPA and 5–hydrotryptophan after inhibition of aromatic amino acid decarboxylase. Before adolescence, ethanol caused a decrease of DOPA accumulation in the whole rat brain, while after adolescence ethanol increased the DOPA synthesis. Ethanol had no effect on the serotonin synthesis at any of the ages studied. It is suggested that hormonal events occuring during puberty may be of importance for the stimulatory properties of ethanol observed at adult age     

Long-term Effects of the Anticholinesterases Sarin and Soman on Latencies of Muscle Action Potentials in Mouse Diaphragm Muscle

Abstract— In-vivo administration of the irreversible anticholinesterases sarin and soman has been shown to produce long-term effects on latency and variability of latency of muscle action potentials in in-vitro mouse diaphragm muscle preparations. The maximum observed effects occurred three days post-soman administration and seven days post-sarin administration, and were no longer detectable 28 days later. With both anticholinesterases the increase in latency, and variability of latency, was reduced by pyridostigmine pretreatment. Therapeutic administration of pralidoxime mesylate effectively prevented the sarin-induced effects when given after a delay of 24 h. In contrast, the effectiveness of pralidoxime mesylate declined rapidly when its administration was delayed following soman. These findings are consistent with this action of soman and sarin being a product of acetylcholinesterase inhibition. The results obtained with sarin suggest that a period of acetylcholinesterase inhibition in excess of 24 h is required to trigger the events leading to the production of this long-term effect.     

Acute effects of a sarin-like organophosphorus agent,bis(isopropyl methyl)phosphonate,on cardiovascular parameters in anaesthetized,artificially ventilated rats

The organophosphorus compound sarin irreversibly inhibits acetylcholinesterase. We examined the acute cardiovascular effects of a sarin-like organophosphorus agent, bis(isopropyl methyl)phosphonate (BIMP), in anaesthetized, artificially ventilated rats. Intravenous administration of BIMP (0.8 mg/kg; the LD50 value) induced a long-lasting increase in blood pressure and tended to increase heart rate. In rats pretreated with the non-selective muscarinic-receptor antagonist atropine, BIMP significantly increased both heart rate and blood pressure. In atropine-treated rats, hexamethonium (antagonist of ganglionic nicotinic receptors) greatly attenuated the BIMP-induced increase in blood pressure without changing the BIMP-induced increase in heart rate. In rats treated with atropine plus hexamethonium, intravenous phentolamine (non-selective α-adrenergic receptor antagonist) plus propranolol (non-selective β-adrenergic receptor antagonist) completely blocked the BIMP-induced increases in blood pressure and heart rate. In atropine-treated rats, the reversible acetylcholinesterase inhibitor neostigmine (1 mg/kg) induced a transient increase in blood pressure, but had no effect on heart rate. These results suggest that in anaesthetized rats, BIMP induces powerful stimulation of sympathetic as well as parasympathetic nerves and thereby modulates heart rate and blood pressure. They may also indicate that an action independent of acetylcholinesterase inhibition contributes to the acute cardiovascular responses induced by BIMP.     

Effect of Age and Route of Administration on LD50 of Lithium Chloride in the Rat *

Abstract: Male inbred rats were used to determine the lethal dose of lithium chloride. The rats were 3 weeks, 6 weeks, 3 months and 6 months old, and lithium chloride was given intraperitoneally, subcutaneously and orally. In relation to age, LD50 was significantly higher in rats of 6 weeks after oral administration than LD50 in rats of 3 and 6 months. No differences were found following intraperitoneal and subcutaneous administration. In relation to route of administration, LD50 was significantly higher in rats of 3 and 6 weeks after oral administration than LD50 after intraperitoneal and subcutaneous administration. This difference was not found in rats of 3 and 6 months. It is concluded that age and route of administration are of significance for LD50 of lithium chloride in the rat.     

Studies on the Effects of Orally Administered Dicyclohexyl Phthalate in the Rat

Abstract: The oral administration of 500–2500 mg/kg/day dicyclohexyl phthalate (DCHP) to young male Sprague-Dawley rats for 7 days resulted in liver enlargement and induction of some parameters of hepatic xenobiotic metabolism. Additional studies indicated that the hepatic enzyme induction resembled that of sodium phenobarbitone rather than that of polycyclic hydrocarbons. Morphological examination of the livers of DCHP treated rats revealed centrilobular cell hypertrophy and ultrastructural examination demonstrated marked proliferation of the smooth endoplasmic reticulum. Mitochondrial structure and numbers of peroxisomes (microbodies) were not affected. DCHP treatment did not affect kidney and testes weights but some histological evidence of testicular damage was obtained with 2500 mg/kg/day of DCHP. The metabolites of DCHP, namely monocyclohexyl phthalate (MCHP) and cyclohexanol, also induced certain parameters of hepatic xenobiotic metabolism. MCHP, but not cyclohexanol also produced marked testicular atrophy. It is concluded that DCHP is a weak drug-type inducer of hepatic xenobiotic metabolism in the rat and the hepatic effects of this phthalate diester are different from those of di-(2-ethylhexyl) phthalate.     

The Effects of Carbidopa Dose and Time and Route of Administration on Systemic L-Dopa Levels in Rats

The effects of carbidopa dose and time and route of administration on systemic plasma levels of parenterally and nonparenterally administered L-dopa were examined in rats. Intravenous coadministration of L-dopa + carbidopa resulted in significant (P < 0.05) carbidopa-dependent increases in both the area under the plasma L-dopa concentration versus time profile (AUC; +27%) and the plasma L-dopa half-life ( . Simultaneous duodenal or rectal carbidopa administration did not alter the L-dopa i.v. pharmacokinetic profile. Carbidopa pretreatment significantly increased the i.v. L-dopa AUC ( + 38 and +82% for i.v. and duodenal pretreatments, respectively) compared to simultaneous administration. Both i.v. and duodenal carbidopa increased duodenal L-dopa AUC to a similar extent ( + 282 and +239% for i.v. and duodenal administration, respectively). Rectal studies indicated poor absorption of both L-dopa and carbidopa, with no demonstrable effect on plasma L-dopa. The results indicate that the timing and route of carbidopa and L-dopa administration are important in determining the extent of i.v. or duodenal L-dopa systemic availability. The rat model affords results similar to those reported in human studies and may be useful for more extensive evaluation of L-dopa and carbidopa interactions.     

Zinc Concentrations in Human Plasma and in Rat Plasma and Tissues during Lithium Administration

Abstract: Zinc concentrations, determined with atomic absorption spectrophotometry, did not differ significantly in plasma from manic-depressive patients about to start lithium treatment and in patients having been in lithium treatment for six months or 1-2 years. Plasma zinc concentrations and zinc concentrations in liver, muscle, kidney, bone, and skin did not differ in control rats and rats given lithium with the food for four weeks.     

Excretion of Zinc in Rat Bile – A Role of Glutathione

Abstract: Fractionation of the bile from rats injected with ⁶⁵ZnCl₂ (5 μmol/kg) showed that zinc was mainly bound to low molecular weight compounds eluted corresponding to the zinc-glutathione complexes. Diethylmaleate (3.9 mmol/kg), cyclohexene oxide (4.9 mmol/kg) and acrylamide (3.5 mmol/kg) administered intraperitoneally to rats caused a rapid decrease in the endogenous excretion of both zinc and reduced glutathione into bile. This depression probably reflects the conjugation of the aforementioned substances to glutathione in the liver cells. These results indicate that zinc is transferred from liver to bile by glutathione dependent process and most likely as zinc-glutathione complexes.     

Enterohepatic Circulation,Urinary Excretion and Laxative Action of Some Bisacodyl Derivatives after Intragastric Administration in the Rat

Abstract: Bisacodyl (BIS), the parent diphenol (DES) and its sulphuric acid di-ester (picosulphate = PICO) were given by stomach tube to fasted rats at a dose of 3.1 μmol/100 g rat. Bile was sampled in the periods 0–6, 6–12 and 12–18 hrs after drug administration, and assayed for total diphenol (= free + conjugated) by HPLC. Mean fractions (% of dose±S.E.M.) excreted in 5 rats per compound and period were: BIS 74.0±4.7, 51.9±7.9 and 30.8±2.5; DES 41.2±4.3, 46.8±4.7 and 25.1±2.5; PICO 9.0±0.9, 26.0±5.4 and 19.6±3.1. Only minor amounts were excreted as free diphenol. Urine samples taken by bladder puncture and assayed as above furthermore showed that the renal excretion of total diphenol was insignificant compared to the amounts excreted in bile. Practically no diphenol was present in urine 0–6 hrs after the administration of PICO. In experiments with BIS and DES at 0.85 μmol/100 g, total diphenol excreted in bile during 0–6 hrs was: BIS 67.1±2.6 (n = 5); DES: 55.4±3.0 (5). - The latency time for laxative effect was studied in groups of 10 unfasted rats per compound. Cumulative time response curves showed that PICO caused diarrhoea more promptly at 0.85 μmol/100 g than either BIS or DES. In most rats, this delayed action of BIS and DES persisted also at 1.7 μmol/100 g. At 3.1 μmol/100 g, however, the majority of the rats reacted as promptly to these two compounds as to PICO. These results are discussed in relation to the biliary excretion experiments, and interpreted in terms of the relative importance at the different dose levels of: 1. The enterohepatic recirculated fraction, and 2. The non-absorbed fraction, which passes directly to the large intestine. For PICO, the latter fraction is the single determinant of the effect, which is triggered when the di-ester is being hydrolyzed to active diphenol in this part of the GI-tract.     

Initial sensitivity and acute tolerance to ethanol in the P and NP lines of rats

We recently reported that selectively bred, alcohol-preferring (P) and alcohol-nonpreferring (NP) rats differ in sensitivity to a single sedative-hypnotic dose of ethanol, as measured by performance in the jump test. The present study examines the contributions of initial sensitivity and acute tolerance development to this difference. Initial sensitivity, assessed by brain alcohol content upon loss of the aerial righting reflex, was not significantly different between P and NP groups given 3 g ethanol/kg body weight intraperitoneally. Acute tolerance was indexed from blood alcohol concentrations (BAC) upon recovery of jumping performance following two successive ethanol doses. Practiced P and NP rats were required to jump 35 cm to a descending platform following the IP injection of 2.0 g ethanol/kg. The NP group took signiificantly longer (74 min) than the P (33 min) group whereupon BAC₁ of NP rats (234 mg%) was significantly lower than that of P rats (250 mg%). A second injection (1.0 g/kg) was given immediately after the animals reached the 35 cm criterion. Again, NP rats took significantly longer (124 min) than P rats (52 min) to jump 35 cm and BAC₂ of NP animals was lower (295 mg%) than that of P rats (343 mg%). The difference between BAC₂ and BAC₁, the measure of tolerance development, was significantly larger for P rats (90 mg%) than for NP rats (61 mg%). No significant differences in blood ethanol elimination weree observed between the groups. The data indicate no difference in initial sensitivity between P and NP animals but that P rats develop acute tolerance more rapidly and/or to a greater degree than do NP rats. The results are consistent with a relationship in these selectively bred lines of rats between alcohol preference and the development of acute tolerance.     

Age and Dose Dependency of the Pharmacokinetics and Metabolism of Bisphenol A in Neonatal Sprague-Dawley Rats Following Oral Administration

Previous studies demonstrated the rapid clearance of bisphenolA (BPA) from blood following oral administration to adult ratswith the principal metabolite being BPA-monoglucuronide (BPA-glucuronide).Since the ontogeny of glucuronyl transferases (GT) differs withage, the pharmacokinetics of BPA were studied in neonatal animals.¹⁴C-BPA was administered via gavage at 1 or 10 mg/kg body weightto rats at postnatal day (pnd) 4, pnd 7, pnd 21, or to 11 weekold adult rats (10 mg/kg dose only). Blood (neonates and adults)and selected tissues (neonates) were collected at 0.25, 0.75,1.5, 3, 6, 12, 18, and 24 h postdosing. BPA and BPA-glucuronidein the plasma were quantified by high-performance liquid chromatography;radioactivity in the plasma and tissues was quantified by liquidscintillation spectrometry. The data indicate that neonatalrats at all three ages metabolized BPA to BPA-glucuronide, althoughan age dependency in the number and concentration of plasmametabolites was observed, consistent with the ontogeny of GT.BPA-glucuronide and BPA concentrations in the plasma were greaterin neonates than in adults, except at 24 h postdosing, suggestingan immaturity in the development of hepatic excretory functionin neonatal rats. Nevertheless, the half-lives for the eliminationof BPA-glucuronide in plasma were more rapid in neonatal animalsthan in adults, likely due to reduced microflora ß-glucuronidaseactivity and an absence of enterohepatic recirculation. A dosedependency in the metabolism and pharmacokinetics of BPA administeredto neonates was also observed with nearly complete metabolismof BPA to BPA-glucuronide (94–100% of the plasma radioactivity)at a dose of 1 mg/kg. This was in contrast to finding up to13 different plasma metabolites observed at the 10 mg/kg dose.These data indicate that, from early in neonatal life throughpnd 21, there is sufficient GT activity in rats to efficientlymetabolize BPA to its nonestrogenic metabolite at low doses.     

Studies in the Rat on the Absorption,Biliary Excretion,Laxative Action and Interference with Intestinal Transport of some Oxyphenisatin Derivatives

Abstract The free diphenol oxyphenisatin (I) and its sulphuric acid (II) and acetic acid (III) di-esters are compared. I disappered rapidly from the lumen of tied jejunal loops in vivo; its disapperance was associated with an inhibition of normal fluid absorption and an increase in loop tissue wet weight. II disappeared slowly, and did neither inhibit fluid absorption nor cause an increase in wet weight. Large fractions of I conjugates were excreted in the bile 0-6, 6-12 and 12-18 hrs following intragastric administration of I; after II and III much smaller fractions were excreted, and their time pattern differed from that of I. The absorption and biliary excretion experiments when combined indicate that following intragastric administration in the intact rat, the bulk of I will undergo enterohepatic circulation during its small intestinal transit, while II and III pass largely unabsorbed to the colon. In spite of their direct transit, however, the laxative action of both II and III in concious rats was slower in onset than of I. In this respect, II differed strikingly from the sulphuric acid di-ester of desacetyl-bisacodyl (IV), which acted even more promptly than I. As in the jejunum, II was inactive per se in tied colonic loops; liberation of free I being required to elicite inhibition of colonic electrolyte and fluid absorption. The reason for the long laxative latency period of II and III appears then mainly to be slow colonic hydrolysis of these prodrugs. Supplementary experiments supported this view as far as the water soluble II is concerned, and slow hydrolysis is very likely for such a sparingly soluble drug as III.