Xenobiotic Carboxylic Acids Research Articles

Interactions between the in vitro metabolism of xenobiotics and fatty acids. The case of ibuprofen and other chiral profens.

Xenobiotic carboxylic acids can act as substrates for the pathways of fatty acid biotransformation. Thus, they can form abnormal fatty acids by addition of 2-carbon units, or be conjugated with cholesterol, bile acids, carnitine and other amino acids. Finally, xenobiotic carboxylic acids can substitute endogeneous fatty acids in glycerolipid synthesis producing hybrid triacylglycerols and phospholipids (Caldwell, 1985; Caldwell and Marsh, 1983; Huston, 1982). In order to enter the routes of lipid biochemistry, xenobiotic acids like natural fatty acids must be activated by coenzyme A. The acyl-CoA thioesters thus formed are pivotal in the metabolism of xenobiotic acids (Fig. 1).

Overview of coenzyme A metabolism and its role in cellular toxicity

Coenzyme A (CoASH) has a clearly defined role as a cofactor for a number of oxidative and biosynthetic reactions in intermediary metabolism. Formation of acyl-CoA thioesters from organic carboxylic acids activates the acid for further biotransformation reactions and facilitates enzyme recognition. Xenobiotic carboxylic acids can also form CoA-thioesters, and the resulting acyl-CoA may contribute to the compound's toxicity. Generation of an unusual or poorly-metabolized acyl-CoA from a xenobiotic may lead to cellular metabolic dysfunction through several types of mechanisms including: (1) inhibition of key metabolic enzymes by the acyl-CoA; (2) sequestration of the total cellular CoA pool as the unusual acyl-CoA; (3) physical-chemical effects of the acyl-CoA; and (4) sequestration and depletion of carnitine as the acyl group is transformed from the acyl-CoA to form the corresponding acylcarnitine. Many of these toxicities are similar to sequelae observed in the inherited organic acidurias in which endogenously-generated acyl-CoAs accumulate secondary to an enzymopathy. Insights into the cellular mechanisms of xenobiotic acyl-CoA accumulation have been derived from model systems developed to understand organic acidemias, such as the methylmalonyl-CoA accumulation of the methylmalonic acidurias. The relevance of acyl-CoA accretion to human pathophysiology has now been well established, and identification of the relevant mechanism of toxicity can allow implementation of strategies to minimize the metabolic injury. Additionally, recognition of the potential for acyl-CoA mediated xenobiotic injury should result in improved rational drug design and earlier recognition of such toxicity when it develops.

Xenobiotic acyl-CoA formation: evidence of kinetically distinct hepatic microsomal long-chain fatty acid and nafenopin-CoA ligases

Multiplicity of hepatic microsomal coenzyme A ligases catalyzing acyl-CoA thioester formation is an important factor for consideration in relation to the metabolism of xenobiotic carboxylic acids. In this study the kinetic characteristics of rat hepatic microsomal nafenopin-CoA ligase were studied and compared with those of long-chain fatty acid (palmitoyl) CoA ligase. The high affinity component of palmitoyl-CoA formation was inhibited by nafenopin ( K i 53 μM) and ciprofibrate ( K i 1000 μM). Analagous to palmitoyl-CoA, nafenopin-CoA formation was catalyzed by an apparent high affinity low capacity isoform ( K m 6 ± 2.5 μM, (V max 0.33 ± 0.12 nmol/mg per min) which was inhibited competitively by palmitic acid (mean K i 1.7 μM, n = 5) and R-ibuprofen (mean K i 10.8 μM, n = 5) whilst ciprofibrate and clofibric acid were ineffective as inhibitors. The intrinsic metabolic clearance of nafenopin to nafenopin-CoA (V max/ K m 0.057 ± 0.011 nmol/mg/min ± M) was similar to that reported recently for the formation of ibuprofenyl-CoA by rat liver microsomes. Evidence of both a substantial difference between the K m and K i for nafenopin and lack of commonality with regard to xenobiotic inhibitors suggests that the high affinity microsomal nafenopin-CoA and long-chain fatty acid-CoA ligases are kinetically distinct. Thus until the current ‘long-chain like’ xenobiotic-CoA ligases are fully characterised in terms of substrate specificity, inhibitor profile, etc, it will be impossible to rationalize (and possibly predict) the metabolism and hence toxicity of xenobiotic carboxylic acids forming acyl-CoA thioester intermediates.

The relationship between mitochondrial activation and toxicity of some substituted carboxylic acids

The activation of 4-bromocrotonic acid, 4-bromo-2-octenoic acid, valproic acid, and 3-methylglycidic acid by conversion to their CoA thioesters and the effects of these carboxylic acids on palmitoylcarnitine-supported respiration were studied with rat liver and rat heart mitochondria. 4-Bromocrotonic acid was activated by both liver and heart mitochondria, whereas 4-bromo-2-octenoic acid and valproic acid were only activated by liver mitochondria. 3-Methylglycidic acid was not a substrate of mitochondrial activation. All of the carboxylic acids that were activated also inhibited palmitoylcarnitine-supported respiration. 3-Methylglycidoyl-CoA was found to irreversibly inhibit 3-ketoacyl-CoA thiolase in a concentration-dependent and time-dependent manner. Together, these results lead to the conclusion that substituted medium-chain carboxylic acids, which enter mitochondria directly, may inhibit β-oxidation as long as they are activated and perhaps further metabolized in the mitochondrial matrix to compounds that sequester CoA and/or inhibit β-oxidation enzymes. Liver is more susceptible to inhibition by such xenobiotic carboxylic acids due to the broader substrate specificity of its mitochondrial medium-chain acyl-CoA synthetase (EC 6.2.1.2).

Inhibition of rat peroxisomal palmitoyl-CoA ligase by xenobiotic carboxylic acids

ATP-dependent coenzyme A (CoA) ligases catalyse the formation of the acyl-CoA thioesters of xenobiotic carboxylic acids and the formation of xenobiotic-CoAs has been implicated as being a causative factor in peroxisomal proliferation. In this study we have demonstrated using rat liver peroxisomes that the formation of palmitoyl-CoA is inhibited by a variety of xenobiotic carboxylic acids. Palmitoyl-CoA formation exhibited biphasic kinetics indicative of two isoforms, a high affinity ( K ml 2.3 μM) low capacity form and a low affinity ( K m2 831 μM ) high capacity form. These forms were differentially inhibited by a range of xenobiotics. However, it would appear that the low affinity component may not contribute to any major extent to the formation of xenobiotic-CoAs in vivo. At a concentration of 1 mM, greater than 20% inhibition of the high affinity form was observed with the 2-arylpropionates, ibuprofen, naproxen, benoxaprofen, fenoprofen, indoprofen, ketoprofen, tiaprofenic acid and cicloprofen, the hypolipidaemics, nafenopin and ciprofibrate, and the herbicides, silvex and 2,4,5-trichlorophenoxyacetate. Valproic acid, clofibric acid, salicylic acid and 2,4-dichlorophenoxy-acetate were non-inhibitory at all concentrations studied (0.1–2.5 mM). Analysis of the type of inhibition established that only nafenopin ( K i, 430 μM ) and ciprofibrate ( K i, 97 μM ) were competitive inhibitors of palmitoyl-CoA formation suggesting that they bind at the active site and thus potentially function as alternative substrates for the peroxisomal ligase. Notably, clofibric acid which has previously been shown to form clofibroyl-CoA in peroxisomes did not interact with the palmitoyl-CoA ligase thereby suggesting that activation is mediated via an alternative peroxisomal CoA ligase. In addition, the xenobiotic inhibitors of the peroxisomal palmitoyl-CoA ligase differed from those previously reported for the equivalent microsomal enzyme suggesting that the organellar forms may be functionally distinct. This study establishes that numerous xenobiotic carboxylic acids interact with the peroxisomal palmitoyl-CoA ligase; however, it would appear that relatively few function as alternative substrates. The toxicological ramifications of peroxisomally mediated xenobiotic-CoA formation and the identification of other peroxisomal xenobiotic-CoA ligase(s) remain to be elucidated.

Xenobiotic triacylglycerol formation in isolated hepatocytes

The formation of neutral lipophilic metabolites from five xenobiotic carboxylic acids was studied in isolated rat hepatocytes. Oleic acid was used as a positive control. Rates of formation of lipids lay in the order: oleic acid > phytanic acid > ibuprofen > 3-phenoxybenzoic acid > indomethacin and 3-phenylbutanoic acid (rates were undetectable with the last two substrates). The process was saturable with the maximum rates at about 0.5 mM substrate concentration. Supplementation of the hepatocyte system with glycerol enhanced the yields of lipid products. The hepatocytes also effectively modelled the in vivo metabolism of ibuprofen, 3-phenoxybenzoic acid and 3-phenylbutanoic acid with oxidations and classical conjugation reactions predominating over xenobiotic lipid formation.

Incorporation of xenobiotic carboxylic acids into lipids

Over thirty-six different xenobiotic carboxylic acids have been reported to form xenobiotic lipids. The majority form triacylglycerol analogs or cholesterol esters with fewer reports of polar lipids being formed. As yet there is insufficient information to deduce a relationship between the structure of the xenobiotic acid and its activity as a substrate for lipid biosynthesis, although the ability to form a CoA ester appears to be important. The action of monoacylglycerol acyltransferase, diacylglycerol acyltransferase, lecithin cholesterol acyltransferase and a carboxylesterase in synthesizing xenobiotic lipids has been demonstrated. One xenobiotic lipid has been shown to be the cause of granulomatous changes and there are some indications that others may prove to be of toxicological or pharmacological significance. Detailed investigations into several aspects of xenobiotic lipid biochemistry are still required.

Activation of protein kinase C by an aromatic xenobiotic diacylglycerol analogue

A number of xenobiotic carboxylic acids, including 3-phenoxybenzoic acid (3PBA), have been shown to form ‘hybrid” di- and tri-acylglycerols both in vivo and in vitro. Experiments were carried out to test the hypothesis that naturally occurring xenobiotic diacylglycerols may stimulate protein kinase C. The activity of protein kinase C was measured in the presence of different diacylglycerols. 1-Acyl-2-(3PBA)- sn-glycerol but not l,2-di-3PBA- sn-glycerol stimulated protein kinase C, but less effectively than either dioleoylglycerol or phorbol 12-myristate 13-acetate. The xenobiotic diacylglycerols were also more resistant to lipolysis. The possibility exists therefore that some xenobiotic diacylglycerols may behave, like phorbol diesters, as tumour promoters. Protein kinase C; Xenobiotic lipid; 3-Phenoxybenzoic acid; Diacylglycerol; Phorbol ester

Acyl-CoA esters of xenobiotic carboxylic acids as biochemically active intermediates.

Acyl-CoA esters of xenobiotic carboxylic acids as biochemically active intermediates.

Metabolic conjugation of some carboxylic acids in the horse.

1. 14C-Labelled benzoic acid, salicylic acid and 2-naphthylacetic acid were administered orally to horses, and urinary metabolites investigated by chromatographic and mass spectral techniques. 2. [14C]Benzoic acid (5 mg/kg) was eliminated rapidly in the urine, and quantitatively recovered in 24 h. The major urinary metabolite was hippuric acid (95% of dose) with much smaller amounts of benzoic acid, benzoyl glucuronide and 3-hydroxy-3-phenylpropionic acid. Administration of [ring-D5]benzoic acid together with [14C]benzoic acid to a pony permitted the mass spectral determination of metabolites of the exogenous benzoic acid metabolites in the presence of the same endogenous compounds. 3. [14C]Salicylic acid (35 mg/kg) was eliminated rapidly in the urine, 98% of the 14C dose being excreted in 24 h. The major excretion product was unchanged salicylate (94% of dose). Gentisic acid, salicyluric acid and the ester and ether glucuronides of salicylic acid were very minor metabolites. 4. 2-Naphthyl[14C]acetic acid (2 mg/kg) was excreted very slowly in the urine, with 53 and 77% of the 14C dose being recovered in six days. 2-Naphthylacetylglycine was the major metabolite (26 and 38% dose) and in addition, the glucuronic acid and taurine conjugates were excreted together with unchanged 2-naphthylacetic acid. 5. The study has shown that the horse can utilize glycine, taurine and glucuronic acid for conjugation of xenobiotic carboxylic acids, and that the relative extents of these pathways are governed by the structure of the carboxylic acid.