Carboxylesterases and

In addition to ester bonds with P (Section 10.2.1, Figures 10.1 and 10.2), some OPs have other ester bonds not involving P, which are readily broken by esteratic hydrolysis to bring about a loss of toxicity. Examples include the two carboxylester bonds of malathion, and the amido bond of dimethoate (Figure 10.2). The two carboxylester bonds of malathion can be cleaved by B-esterase attack, a conversion that provides the basis for the marked selectivity of this compound. Most insects lack an effective carboxylesterase, and for them malathion is highly toxic. Mammals and certain resistant insects, however, possess forms of carboxylesterase that rapidly hydrolyze these bonds, and are accordingly insensitive to malathion toxicity. 

Ross MK, Crow JA (2007) Human carboxylesterases and their role in xenobiotic and endobi-otic metabolism. J Biochem Mol Toxicol 21 187-196... 

Wheelock CE, Shan G, Ottea J (2005) Overview of carboxylesterases and their role in the metabolism of insecticides. J Pestic Sci 30 75-83... 

Carboxylesterases and amidases catalyze hydrolysis of carboxy esters and carboxy amides to the corresponding carboxylic acids and alcohols or amines. In general those enzymes capable of catalyzing hydrolysis of carboxy esters are also amidases, and vice versa (110). The role of these enzymes in metabolsim of drugs and insecticides has been reviewed (111, 112). In addition to the interest in mammalian metabolism of drugs and environmental chemicals, microbial esterases have been used for enantioselective hydrolyses (113, 114). 

Cross-tolerance between disulfoton and another organophosphate, chlorpyrifos, was observed in mice (Costa and Murphy 1983b). Because of this cross-tolerance, a benefit is derived as a result of this interaction. In the same study, propoxur-tolerant mice were tolerant to disulfoton but not vice versa. Propoxur (a carbamate) is metabolized by carboxylesterases, and these enzymes are inhibited in disulfoton-tolerant animals disulfoton-tolerant animals are more susceptible to propoxur and/or carbamate insecticides than are nonpretreated animals. In another study, disulfoton-tolerant rats were tolerant to the cholinergic effects of octamethyl pyrophosphoramide (OMPA) but not parathion (McPhillips 1969a, 1969b). The authors were unable to explain why the insecticides OMPA and parathion caused different effects. 

Natural (-)-cocaine (7.57, Fig. 7.8), which has the (2/ ,3S)-configuration, is a relatively poor substrate for hepatic carboxylesterases and plasma cholinesterase (EC 3.1.1.8), and also a potent competitive inhibitor of the latter enzyme [116][121], In contrast, the unnatural enantiomer, (+)-(2S,3/ )-cocaine, is a good substrate for carboxylesterases and cholinesterase. Because hydrolysis is a route of detoxification for cocaine and its stereoisomers, such metabolic differences have a major import on their monooxygenase-catalyzed toxification, a reaction of particular effectiveness for (-)-cocaine. 

The transesterification of cocaine to cocaethylene is an enzymatic reaction catalyzed by microsomal carboxylesterases and blocked by inhibitors of serine hydrolases [124][125], In Chapt. 3, we have discussed the mechanism of serine hydrolases, showing how a H20 molecule enters the catalytic cycle to hydrolyze the acylated serine residue in the active site of the enzyme. In the case of cocaine, the acyl group is the benzoylecgoninyl moiety (Fig. 7.9,d ), which undergoes esterification with ethanol according to Steps e and/ (Fig. 7.9). 

Simple alkyl or aryl thioesters are commonly assayed as substrates of hydrolases, witness the hydrolysis of phenyl thioesters by horse serum carbox-ylesterase [150], For most substrates investigated, e.g., phenyl thioacetate, phenyl thiopropionate, and phenyl thiobutyrate (7.66, R = Me, Et, and Bu, respectively), kcat values were found, which were a few times larger than those of corresponding nitrophenyl esters, whereas the affinities were lower by approximately one order of magnitude. Methyl and phenyl esters of various linear thioacids were also found to be good substrates of mammalian liver carboxylesterases and serum cholinesterases [151]. 

Another therapeutic class to be briefly discussed is that of the lipid-lowering agents known as fibrates, e.g., clofibrate and fenofibrate (8.5). Here also, the acidic metabolite is the active form clofibrate (an ethyl ester) is rapidly hydrolyzed to clofibric acid by liver carboxylesterases and blood esterases [11], Human metabolic studies of fenofibrate (8.5), the isopropyl ester of fenofibric acid, showed incomplete absorption after oral administration, while hydrolysis of the absorbed fraction was quantitative [12], This was followed by other reactions of biotransformation, mainly glucuronidation of the carboxylic acid group. 

Two examples of aryl esters are given in Table 8.5, namely the 4-chloro-phenyl and 4-nitrophenyl esters of nicotinic acid (8.33). Under physiological conditions of pH and temperature, these two compounds were clearly much more susceptible to chemical hydrolysis than the alkyl and arylalkyl esters in Table 8.5. Their affinity for carboxylesterase and human plasma hydrolases, as assessed by the Michaelis constant Km, was generally higher, while nothing can be said regarding Vmax values. 

Brondeau MT, Coulais C, de Ceaurriz J. 1991. Difference in liver and serum malathion carboxylesterase and glucose-6-phosphatase in detecting carbon tetrachloride-induced liver damage in rats. J AppI Toxicol 11 433-435. 

Slatter JG, Su P, Sams JP et al. Bioactivation of the anticancer agent CPT-11 to SN-38 by human hepatic microsomal carboxylesterases and the in vitro assessment of potential drug interactions. Drug Metab Dispos 1997 25 1157-1164. 

Esters, amides, hydrazides, and carbamates can all be metabolized by hydrolysis. The enzymes, which catalyze these hydrolytic reactions, carboxylesterases and amidases, are usually found in the cytosol, but microsomal esterases and amidases have been described and some are also found in the plasma. The various enzymes have different substrate specificities, but carboxylesterases have amidase activity and amidases have esterase activity. The two apparently different activities may therefore be part of the same overall activity. 

Enzymes with carboxylesterase and amidases activity are widely distributed in the body, occurring in many tissues and in both microsomal and soluble fractions. They catalyze the following general reactions ... 

Although carboxylesterases and amidases were thought to be different, no purified carboxylesterase has been found that does not have amidase activity toward the corresponding amide. Similarly enzymes purified on the basis of their amidase activity have been found to have esterase activity. Thus these two activities are now regarded as different manifestations of the same activity, specificity depending on the nature of R, R and R groups and, to a lesser extent, on the atom (O, S, or N) adjacent to the carboxyl group. 

The well-known selectivities of some organophosphates may be explained by the balance of enzymatic events. The reduced toxicity of the insecticide malathion to mammals is largely the result of rapid activation by desulfuration in the insect and the more rapid detoxificaton by carboxylesterases and glutathione transferases in the mammal (3). Design of new pest bioregulators should exploit enhanced activation and decreased detoxification capabilities in the targeted pests. 

Technical malathion (-95% pure) contains several impurities. Of the impurities isolated, four compounds have been shown to be important in insecticide toxicology (Figure 4.5). Umetsu et al. (1977) demonstrated that all of these impurities potentiated the toxicity of purified malathion in rats, with compounds C and D being more active. Further studies showed that these impurities inhibited serum malathion carboxylesterase and liver malathion carboxylesterase in vitro and in vivo in rats (Talcott et al., 1979), which would explain the potentiating activity observed with these impurities because these carboxylesterases... 

There are two types of esterases that are important in metabolizing insecticides, namely, carboxylesterases and phosphatases (also called phosphorotriester hydrolases or phosphotriesterases). Carboxylesterases, which are B-esterases, play significant roles in degrading organophosphates, carbamates, pyrethroids, and some juvenoids in insects. The best example is malathion hydrolysis, which yields both a- and (i-monoacids and ethanol (Figure 8.10). 

Induction of carboxylesterases and epoxide hydroloases would also affect the toxicity of insecticides. For example, host plant induction of 1-naphthyl acetate esterase would decrease the toxicity of certain insecticides containing an ester linkage such as organo-phosphates, pyrethroids, and some juvenile hormone analogs and, possibly, carbamates. 

Sterri, S.H., Johnsen, B.A., Fonnum, F. (1985b). A radiochemical assay method for carboxylesterase, and comparison of enzyme activity towards the substrates methyl[l- C]butyrate and 4-nitrophenyl butyrate. Biochem. Pharmacol. 34 2779-85. 

We have used the growth effects and pathologies associated with L-ascorbic acid deficiency as a basis for the determination of the biological potency of related compounds (Table I). At a dietary concentration of 0.5 mM, L-ascorbic acid and dehydroascorbic acid were fully active, as well as some ester derivatives including the 6-myristate and 2-phosphate compounds. The insect may be metabolically like the guinea pig because both were able to utilize those esters (17), Carboxylesterases and phosphatases probably converted those derivatives to the free vitamin (18). The 6-bromo compound was less active and apparently cannot be metabolized to L-ascorbic acid or only poorly so. 

Hemolymph JHE from Day 2 of the fifth instar larvae of T. ni was used (L3D2), diluted 1 500 with 0.08M phosphate buffer (pH-7.4 with 0.1% phenylthiourea to inhibit tyrosinases). The main reason for choosing this insect was that a great deal of effort has been put into the characterization of larval carboxylesterases and JHE in T. ni (39,40). In L3D2 larvae, the JHE titer is near its maximum (19). CIO 3H labeled JH III (New England Nuclear) and unlabeled JH III (Calbiochem) were used as substrate solubilized in abs.ethanol. 

Malathion (chemathion, mala-spray) requires conversion to malaoxon (replacement of a sulfur atom with oxygen in vivo, conferring resistance to mammalian species). Malathion can be detoxified by hydrolysis of the carboxyl ester linkage by plasma carboxylesterases, and plasma car-boxylesterase activity dictates species resistance to malathion. The detoxification reaction is much more rapid in mammals and birds than in insects. Malathion has been employed in aerial spraying of relatively populous areas for control of Mediterranean fruit flies and mosquitoes that harbor and transmit viruses harmful to human beings (e.g.. West Nile encephalitis virus). Evidence of acute toxicity from malathion arises only with suicide attempts or deliberate poisoning. 

Karanth, S.. and Pope. C. (2000). Carboxylesterase and A-eslerase activities during maturation and aging Relationship to the toxicity of chlorpyrifos and parathion in rats. To.xicol Set. 58, 282-289. 

Age-related differences in kinetic parameters of detoxification can partially explain the increased sensitivity of the young to acute exposure to chlorpyrifos and other OPs (Mortensen et ai, 1996 Moser et ai, 1998 Padilla et ai, 2000, 2004). Thus, in vitro assays show that differences in vivo are not due to intrinsic differences in sensitivity of the target enzyme (Mortensen el ai, 1998). Furthermore, differences in liver microsomal metabolism, which mediates activation and/or inactivation of some pesticides, do not adequately explain the increased sensitivity (Benke and Murphy, 1975 Brodeur and DuBoks, 1%7). Differences in detoxification pathways correlate better with age sensitivity. B-esterases (e.g., carboxylesterases) and A-esterase.s bind to and/or hydrolyze, and thus detoxify, some cholinesterase-inhibiting pesticides (Jokanovic et al., 1996 Maxwell, 1992). These pathways are much less well developed in the young, and maturation of these systems tracks the decreasing sensitivity to acute exposure to chlorpyrifos and other OPs (Attcrberry el ai, 1997 Benke and Murphy, 1975 Brodeur and DuBois, 1967 Chanda et ai, 1997, 2002 Mendoza, 1976 Mortensen et ai, 1996, 1998 ... 

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