Sigrun H. Sterri

Factors modifying the toxicity of organophosphorous compounds including Soman and Sarin.

I N T R O D U C T I O N The primary target for the organophosphorous insecticides and nerve agents, such as Satin and Soman, is the enzyme acetylcholinesterase (ACHE). The inhibit ion of AChE is followed by an increase in the level of ACh, in the brain up to about 100% (Fonnum and Guttormsen, 1969). The symptoms accompanying poisoning by the organophosphorous compounds: increased secretion, gastrointestinal disturbance, miosis, tremors and at last death through anoxia may be explained by a larg~ excess of ACh at the cholinergic receptors and thereby an ove:rstimulation of the cholinergic system. The cause of death is anoxia due to a combination of factors such as central respiratory paralysis, severe bronchoconstriction and weakness or paralysis of the accessory muscles of respirat ion(Sire, 1975). : In the simple and well acknowledged scheme there are several factors which are often neglected, but which may be important in discussing therapy or prophylaxis against organophosphorous poisoning. In this paper we wi l l discuss the possible contribution of enzymes other than AChE to the toxicity of the organophosphorous compounds. In particular we wish to focus the attention to aliesterases, a group of esterases, which are inhibited by Satin and Soman to almost the same extent as ACHE. These enzymes could act almost as scavengers by reacting i r revers ib lywi th the organophosph0rous compounds and thereby removing them before they react wi th the ACHE. There are also other enzymes wi th a high aff inity for organophosphorous compounds and which inactivate these compounds by hydrolysis. In the present paper we wi l l demonstrate the efficiency of the latter in a perfused liver preparation.

Three related brain nuclear receptors, NGFI-B, Nurr1, and NOR-1, as transcriptional activators

Three related orphan nuclear receptors that are expressed in the brain, NGFI-B, Nurr1, and NOR-1, were studied to compare their function as transcriptional activators. NGFI-B was able to activate (in the absence of added hormone) in CV1 cells both an NGFI-B-responsive luciferase reporter gene (containing eight copies of a response element for NGFI-B upstream of a basal prolactin promoter driving the luciferase gene, NBRE8-LUC), a similar thyroid hormone-receptor-responsive reporter gene (TRE3-LUC), and a reporter gene with an authentic promoter from aXenopus vitellogenin gene containing two binding sites for the estrogen receptor (vit-LUC). NGFI-B activated NBRE8-LUC and TRE3-LUC (but not the vit-LUC) with an amino-terminal activation domain. Nurr1 was less promiscuous as a transcriptional activator, activating the NBRE8-LUC better than NGFI-B, but less than NGFI-B at the other reporter genes. NOR-1 activated only the NBRE8-LUC reporter gene. These results indicate that closely related nuclear receptors may differentiate between response elements or promoters and that different activation mechanisms exist depending on the promoter. This may contribute to regulation of specificity of target gene expression in the brain.

A radiochemical assay method for carboxylesterase, and comparison of enzyme activity towards the substrates methyl [1-14C] butyrate and 4-nitrophenyl butyrate

A radiochemical assay for carboxylesterase based on the substrate methyl[1-14C]butyrate is described. The blank value corresponds to 1.04 micrograms (liver)-1.44 mg (plasma) of tissues with the highest and lowest activity respectively, which constitute the sensitivity of the method. The hydrolysis of methyl butyrate and 4-nitrophenyl butyrate by plasma, liver, lung, heart, diaphragm, cerebrum, kidney and duodenum of rat have been compared. The results showed that the two substrates were hydrolysed preferentially by two different groups of the enzyme. The effect of selective esterase inhibitors showed that both groups can be characterized as carboxylesterase, because bis-4-nitrophenyl phosphate inhibits the hydrolysis of both substrates, physostigmine has only a slight effect and EDTA is no inhibitor. The exception is the enzyme in the duodenum which is inhibited by all three inhibitors. The effect of phenobarbital induction and soman treatment on enzyme activity towards the two substrates were similar. Sex difference in the plasma activity towards methyl butyrate, but not 4-nitrophenyl butyrate, indicates that the group of carboxylesterase preferentially hydrolyzing 4-nitrophenyl butyrate may be the most important for the detoxification of soman.

Acetyl-CoA Synthesizing Enzymes in Cholinergic Nerve Terminals

Abstract: The activities of five enzymes involved in acetyl‐CoA synthesis, pyruvate dehydrogenase complex, ATP citrate lyase, carnitine acetyl‐transferase, acetyl‐CoA synthetase, and citrate synthase, were determined in normal nucleus interpeduncularis and nucleus interpeduncularis in which cholinergic terminals were removed following lesion of the habenulo‐interpeduncular tract. The activities of aspartate transaminase, fumarase, and GABA transaminase also were determined to compare the effect of lesion on other mitochondrial enzymes which are not linked to the biosynthesis of ACh. In normal nucleus interpeduncularis the activities of carnitine acetyltransferase and pyruvate dehydrogenase complex were higher than the activity of ChAT (choline acetyltransferase), whereas the activities of acetyl‐CoA synthetase and citrate synthase were considerably lower than that of ChAT. The effect of the lesion separated the enzymes into two groups: the activities of pyruvate dehydrogenase complex, carnitine acetyltransferase, fumarase and aspartate transaminase decreased by 30–409%, whereas the activities of the other enzymes decreased 5–15%. ChAT activity was in all cases less than 159% of normal. It could be concluded that none of the acetyl‐CoA synthesizing enzymes decreased to the degree that ChAT did. Only pyruvate dehydrogenase complex and carnitine acetyltransferase seem to be localized in cholinergic terminals to a significant degree. ATP citrate lyase as well as acetyl‐CoA synthetase seem to have less significance in supporting acetyl‐CoA formation in cholinergic nerve terminals.

Carboxylesterases, importance for detoxification of organophosphorus anticholinesterases and trichothecenes

Several different types of experiments, including the use of inhibitors, have shown that carboxylesterases are a major factor in the metabolism and therefore detoxification of organophosphorus compounds such as soman and trichothecene toxins. The development of a new assay method for the enzyme has allowed us to separate the carboxylesterases into two major groups. The carboxylesterases can, however, be further separated by gel filtration, affinity chromatography, isoelectric focusing, and chromatofocusing into several isoenzymes. Liver microsomal carboxylesterases can be separated into five or six isoenzymes whereas guinea-pig plasma contains two isoenzymes. The isoenzymes differ in molecular weights, isoelectric points, substrate specificities, and affinity for inhibitors. Intravenous administration of a carboxylesterase preparation lowered the toxicity of soman in young rats. Carboxylesterases from rat and guinea-pig plasma inhibited by soman could be reactivated by DAM, whereas enzymes from porcine liver were not reactivated. Only one of the isoenzymes from rat liver microsomal preparation was responsible for the metabolism of T-2 toxin to HT-2. The further metabolism of HT-2 was performed by esterases from rat liver cytoplasma. Long-term exposure of the bronchial muscle to low concentration of soman modulate the bronchial contraction.

Carboxylesterases in guinea-pig plasma and liver: Tissue specific reactivation by diacetylmonoxime after soman inhibition in vitro

The carboxylesterase activity in both plasma and liver of guinea-pig were separated into three main peaks by chromatofocusing. Two of the three plasma enzymes were retained by affinity chromatography on Affi-Gel Blue (100-200 mesh). Isoelectric points determined by chromatofocusing or isoelectrofocusing were pI 6.1, pI 5.2 and pI 4.0 for the plasma enzymes, and pI 5.7, pI 5.2 and pI 4.5 for the liver enzymes. The effect of selective esterase inhibitors, soman, physostigmine (cholinesterase inhibitor) and bis-4-nitrophenyl phosphate (carboxylesterase inhibitor), suggested that the three enzymes in both tissues may be regarded as carboxylesterases. However, the pI 5.7 carboxylesterase was partially inhibited by physostigmine, and the pI 4.5 carboxylesterase was almost not affected by bis-4-nitrophenyl phosphate. The ratio between the activities towards 4-nitrophenyl butyrate and methyl butyrate differed among the carboxylesterases in both tissues. All three carboxylesterases in plasma were partially reactivated by diacetylmonoxime after soman inhibition in vitro, but to a different extent. The soman inhibited liver carboxylesterases were not reactivated by diacetylmonoxime.

Modulation of the cholinergic activity of bronchial muscle during inhalation of soman

Abstract The main routes for penetration of nerve agents such as soman, sarin, and tabun to the body are through the respiratory tree, gastrointestinal tract, and the skin. The signs and symptoms of poisoning are those of excess acetylcholine. The cause of death is anoxia due to a combination of factors: (1) sudden respiratory paralysis, (2) severe bronchial constriction, (3) excess accumulations of bronchial and salivary gland secretions, (4) weakness of the accessory muscles of respiration. Intoxication through inhalation will rapidly manifest itself through factors (2) and (3) ( Sim, 1975 ). We have previously investigated enzymes, soman hydrolases, aliesterases, and butyrylcholinesterase, which inactivate soman in the body after different types of exposures ( Fonnum and Sterri, 1981 ; Sterri et al. , 1983 ). The aim of the present investigation was to study the barrier these enzymes constitute in the respiratory tract to the entry of soman into the blood stream during inhalation exposure. In addition we have studied the effects of toxic doses of soman on the function of the bronchial smooth muscle. Finally we have focused the attention on the modification of the bronchial muscle contraction by peptides, muscarinic agonists and antagonist, adenosine, and serotonin.

Tolerance Development to Toxicity of Cholinesterase Inhibitors

Publisher Summary Tolerance to AChE inhibitors has been noted using different forms of administration and in several species, such as mice, rats, guinea pigs, and man. Tolerance can develop in several ways. It often occurs because of receptor changes either in the number of receptors or by decreased affinity of the receptor molecule. However, it can also occur because of the presence of other proteins that can bind or inactivate the inhibitor, and thereby make it less readily available. Examples are, binding of the inhibitor to carboxylesterases (CarbEs), buturylcholinesterases (BuChEs), or other binding proteins such as albumin. In addition, tolerance can be achieved through more rapid metabolism of the OP compounds by OP-hydrolyzing enzymes such as paraoxonases (PONs) and somanases. Organophosphates (OPs) inhibit the enzyme AChE, and thereby increase the level of acetylcholine (ACh) in the synaptic gap. The acute toxic effects of OPs are because of accumulation of ACh at the muscarinic and nicotinic receptors. It has been noted by several investigators that during prolonged exposure to an OP, the physiological effects often diminish more than expected from the degree of AChE inhibition, or that repeated additions of OP give lower responses with time. These observations were seen irrespective of inhibition of blood AChE. The decrease in response to repeated administration could not be explained by a reduced inhibitory effect of soman on AChE or by a more rapid de novo synthesis of AChE.

CHAPTER 68 – Role of Carboxylesterases in Therapeutic Intervention of Nerve Gas Poisoning

Publisher Summary Carboxylesterases (CarbEs) constitute an important group of enzymes that play a major role in the hydrolytic biotransformation of a large number of prodrugs. The scavenger function of CarbE in blood plasma is the principal reason for the variation in nerve gas toxicity between the species, in both the presence and absence of carbamate prophylaxis. The scavenger role is primarily a consequence of the different content of CarbE protein in the plasma of different species, and is not necessarily linked to its enzymatic activity. Plasma CarbE has proved to have an important role in nerve gas poisoning both with and without therapeutic intervention, and is natures own example of an effective scavenger of nerve gases being present in the blood. Both CarbE and other B-esterases have proved to be effective as artificially supplied scavengers, but since they are high molecular weight proteins some practical problems may be difficult to overcome for practical use.

The Role of Carboxylesterases in Therapeutic Intervention of Nerve Gases Poisoning

There is a large variation in the toxic dosage and also in the therapeutic effects between different species poisoned with nerve agents such as sarin and soman. These discrepancies can be explained to a large extent by the variation of carboxylesterase (CarbE) in plasma of different species. CarbE in plasma acts as a scavenger and removes the nerve agent before it reaches its target acetylcholinesterase in the nervous tissue. In this chapter, we explain how high toxic doses in rodents and low toxic doses in humans can be explained by plasma CarbE in the former, but not the latter. The low therapeutic index in rodents poisoned with nerve gases and treated prophylactively with carbamates like physostigmine and therapeutically with oximes and the high therapeutic index in humans are explained by the same mechanism.