Alkylglycidic acids: potential new hypoglycemic agents

A series of alkylglycidic acid analogues and derivatives were synthesized and tested for their ability to inhibit long-chain fatty acid oxidation in vitro and to lower blood sugar in rats. The extent of inhibition of carnitine acyl transferase, the enzyme at the mitochondrial membrane necessary to transport long-chain fatty acids into the mitochondria for subsequent beta-oxidation, was determined for the series. Structure-activity relationships using in vitro inhibition of [1-14C]palmitic acid oxidation in rat hemidiaphragm muscle indicate that potent activity resides mainly in 2-alkyl (C12-C16) glycidates. Replacement of the oxirane ring with cyclopropyl, thiirane, or other rings diminishes activity, as does substitution of the glycidate ring at the 3-position. In vivo potency in the rat glucose tolerance test roughly parallels the hemidiaphragm results. The lead compound, methyl 2-tetradecylglycidate (8), is a potent hypoglycemic agent following oral administration to several animal species. The hypoglycemic analogues interfere with fatty acid oxidation by specific and irreversible inhibition of mitochondrial carnitine palmitoyl transferase-A.     

Alkylthio acetic acids (3-thia fatty acids)--a new group of non-beta-oxidizable peroxisome-inducing fatty acid analogues--II. Dose-response studies on hepatic peroxisomal- and mitochondrial changes and long-chain fatty acid metabolizing enzymes in rats

The activity of key enzymes involved in oxidation and esterification of long-chain fatty acids was investigated after male Wistar rats were treated with different doses of sulfur substituted fatty acid analogues, 1,10-bis(carboxymethylthiodecane) (BCMTD, non-beta-oxidizable and non-omega-oxidizable), 1-mono(carboxymethylthiotetradecane) (CMTTD, trivial name, alkylthio acetic acid, non-beta-oxidizable) and 1-mono(carboxyethylthiotetradecane) (CETTD trivial name, alkylthio propionic acid, beta-oxidizable). The sulfur substituted dicarboxylic acid and the alkylthio acetic acid induced in a dose-dependent manner the mitochondrial, microsomal and especially the peroxisomal palmitoyl-CoA synthetase activity, the mitochondrial and cytosolic palmitoyl-CoA hydrolase activity, the mitochondrial and especially the microsomal glycerophosphate acyltransferase activity and the peroxisomal beta-oxidation, especially revealed in the microsomal fraction. Morphometric analysis of randomly selected hepatocytes revealed that BCMTD and CMTTD treatment increased the number, size and volume fraction of peroxisomes and mitochondria. Thus, the observed changes in the specific activity of fatty acid metabolizing enzymes with multiple subcellular localization can partly be explained as an effect of changes in the s-values of the organelles as proliferation of mitochondria and peroxisomes occurred. The most striking effect of the alkylthio propionic acid was the formation of numerous fat droplets in the liver cells and enhancement of the hepatic triglyceride level. This was in contrast to BCMTD treatment which decreased the hepatic triglyceride content. In conclusion, the results provide evidence that administration of non-beta-oxidizable fatty acid analogues had much higher in vivo potency in inducing hepatomegaly and key enzymes involved in fatty acid metabolism, including proliferation of peroxisomes and mitochondria than is exhibited in the beta-oxidizable, alkylthio propionic acid. Moreover, the dicarboxylic acid was apparently three to six times more potent than the alkylthio acetic acid in inducing peroxisomal beta-oxidation and peroxisome proliferation when considered on a mumol/day basis. As palmitic acid and hexadecanedioic acid only marginally affected these hepatic responses, it is conceivable that the potency of the selected compounds as proliferators of peroxisomes and inducers of the associated enzymes depends on their accessibility for beta-oxidation.     

Effects of sodium 2-[5-(4-chlorophenyl)pentyl]-oxirane-2-carboxylate (POCA) on intermediary metabolism in isolated rat-liver cells

In hepatocytes isolated from meal-fed rats, sodium 2-[5-(4-chlorophenyl)pentyl]oxirane-2-carboxylate (POCA) decreased the rate of lipogenesis measured as incorporation of ³H from ³H₂O into glycerolipids and cholesterol. Moreover, POCA inhibited the oxidation of added oleate, whereas oleate esterification was stimulated. In hepatocytes from 24-hr-starved rats, inhibition of gluconeogenesis by POCA was observed only with gluconeogenic precursors which require pyruvate carboxylation. This inhibition was secondary to impaired oxidation of long-chain fatty acids by POCA. It is concluded that, in addition to its inhibition of long-chain fatty acid oxidation, POCA interferes with de novo synthesis of cholesterol and fatty acids. On the other hand, neither fatty acid esterification nor the conversion of oxaloacetate into glucose are affected by POCA.     

'Metabonomics': understanding the metabolic responses of living systems to pathophysiological stimuli via multivariate statistical analysis of biological NMR spectroscopic data

1. In this study we explored the relationship between specific acyl-CoA esters and induction of acyl-CoA binding protein (ACBP) and enzymes related to the proliferation of peroxisomes. Male Wistar rats were administered a single dose (150mg/day/kg) of sulphur-substituted fatty acid analogues, and the effects of tetradecylthioacetic acid and 3-thiadicarboxylic acid, which both act as peroxisome proliferators, were compared with the effects of tetradecylthiopropionic acid and palmitic acid which do not induce peroxisome proliferation.

2. The hepatic level of total long-chain acyl-CoA was significantly increased within 12?h of feeding these fatty acids, except in rat fed tetradecylthioacetic acid. Hplc chromatograms of liver extracts prepared from rat fed tetradecylthioacetic acid showed that tetradecyl-thioacetyl-CoA ester accumulated in the liver 4h after feeding and had disappeared after 24h. In liver extracts of the tetradecylthiopropionic acid-treated rat tetradecylthiopropionyl-CoA was not observed, but the appearance of a new long-chain acyl-CoA ester, probably a metabolite of tetradecylthiopropionic acid, was detected. This new peak reached a maximum 4?h after feeding. In rat fed tetradecylthioacetic acid and 3-thiadicarboxylic acid the hepatic level of fatty acyl-CoA oxidase mRNA increased 8h after feeding, while the acyl-CoA oxidase activity had increased after 12h.

3. The early accumulation of specific tetradecylthioacetyl-CoA suggests that this ester may be a possible mediator of the induction of fatty acyl-CoA oxidase. The level of hepatic acyl-CoA binding protein, long-chain acyl-CoA hydrolase activity and long-chain acyl-CoA synthetase activity did not change after a single dose of all four fatty acids. Prolonged administration of 3-thia fatty acids resulted, however, in a dose- and time-dependent increase in hepatic ACBP content and ACBP mRNA level. The amount of ACBP increased in parallel to the long-chain acyl-CoA hydrolase activity. The correlated induction of fatty acyl-CoA binding protein and long-chain acyl-CoA hydrolase seems to be dependent on a sustained accumulation of total long-chain acyl-CoA esters.     

Effects of clofibric and beclobric acid in rat and monkey hepatocyte primary culture: influence on peroxisomal and mitochondrial β-oxidation and the activity of catalase,glutathione S-transferase and glutathione peroxidase

The effect of hypolipidaemic compounds on peroxisomal fatty acid β-oxidation and on peroxisome morphology in the liver differs widely between rodent and primate species. We studied the relative importance of peroxisomal and mitochondrial β-oxidation of palmitate in primary cultures of hepatocytes isolated from rat and monkey liver in the absence or presence of clofibric acid or beclobric acid. It was demonstrated that it is possible to differentiate between peroxisomal and mitochondrial β-oxidation activities in intact cells. Overall β-oxidation of palmitate was ca. 30% higher in rat hepatocytes than in monkey liver cells. In both monkey and rat cell cultures the mitochondrial component was over 90% of the total palmitate β-oxidation. In rat hepatocyte culture clofibric acid and beclobric acid caused a 5- to 8-fold stimulation of peroxisomal β-oxidation, while in monkey cells this activity was not significantly increased. However, in cells derived from both species mitochondrial palmitate β-oxidation was increased (rat 2.5-fold; monkey 1.5-fold). These results indicate that the species differences in the increase in peroxisomal fatty acid oxidation are not a result of an inability to metabolize fatty acids in rat liver cell mitochondria. A comparison of the activity of enzymes involved in the detoxification of hydrogen peroxide showed that catalase and glutathione-S-transferase activity is 2.9-fold higher in monkey hepatocytes than in rat liver cells, while glutathione peroxidase activity was 1.6-fold higher in rat cells. When a comparison between both species is made for the ratio of hydrogen peroxide production over catalase activity, it can be concluded that this peroxide will have much smaller possibilities to escape from the peroxisomal compartment in monkey hepatocytes. These findings suggest that species differences in these enzyme activities can contribute to differences in susceptibility for peroxisome proliferator-induced carcinogenicity between rodents and primates. Received: 3 January 1994/Accepted: 11 April 1994     

The inhibition of long-chain fatty acyl-CoA synthetase by enoximone in rat heart mitochondria.

The mechanism by which enoximone, a reported phosphodiesterase inhibitor, inhibits the oxidation of long-chain fatty acids was studied in isolated rat heart mitochondria using a series of 14C-labeled substrates. Enoximone decreased palmitate oxidation in a time- and concentration-dependent manner. Fifty percent inhibition of palmitate oxidation was achieved with 250 microM of enoximone. In contrast to its effect on palmitate, enoximone (250 microM) increased octanoate oxidation by 30%, whereas pyruvate oxidation was unaffected by enoximone. At that dose there was no effect on the oxidation of palmitoyl-CoA and palmitoyl carnitine. The degree of palmitate oxidation inhibited by enoximone was parallel to the inhibition of acyl-CoA synthetase in both rat heart mitochondria and microsomes. These results suggest that enoximone is a reversible inhibitor of long-chain fatty acyl-CoA synthetase. Moreover, the reaction, which is catalyzed by this enzyme, is a rate-limiting step in the pathway of fatty acid oxidation in rat heart mitochondria.     

Stimulation of fatty acid utilization by sodium clofibrate in rat and monkey hepatocytes

The acute effects of sodium clofibrate (NaCPIB) on the metabolism of [1-14C]palmitate, [1-14C]octanoate, [1-14C]butyrate, and [2-3H]glycerol by freshly isolated hepatocytes were tested to explore its mechanism of action. Labeled long-, medium-, and short-chain fatty acids were incorporated into all the major lipid classes and were oxidized to 14CO2 by the liver cells. The partitioning of labeled fatty acids from lipogenic towards oxidative pathways was inversely related to fatty acid chain length. [1-14C]Palmitate was incorporated mainly into cellular triglycerides and phospholipids; [1-14C]octanoate, mainly into triglycerides and free cholesterol; and [1-14C]butyrate, mainly into free cholesterol and phospholipids of the cells. NaCPIB (1-3 mM) rapidly stimulated the esterification of labeled palmitate or glycerol to triglycerides, but drug levels greater than 5 mM were inhibitory to esterification. NaCPIB (1 mM) increased the oxidation of [1-14C]palmitate to 14CO2 by either rat or monkey hepatocytes and enhanced the release of labeled lipids from [2-3H]glycerol-prelabeled cells into the extracellular medium. Accelerated [1-14C]octanoate incorporation into glycerolipids and sterols and increased [1-14C]octanoate conversion to 14CO2 were observed in rat liver cells incubated with 1 mM NaCPIB. In contrast, the same drug level stimulated the oxidation of [1-14C]butyrate to 14CO2 but greatly diminished its incorporation into hepatocellular sterols or glycerolipids. These results indicate that (a) NaCPIB acutely alters hepatic ultilization of fatty acids by actions at diverse loci; (b) these metabolic alterations vary with fatty acid chain length; and (c) these effects are probably due to rapid changes in biochemical regulatory mechanism and/or in substrate channelling within the cells. These data further suggest that the early hypolipidemic effect of the drug in rats and primates may be related to an enhanced hepatic oxidation of long-chain fatty acids, but cannot be attributed simply to a reduction in their esterification to complex lipids.     

Propionyl-L-carnitine as potential protective agent against adriamycin-induced impairment of fatty acid beta-oxidation in isolated heart mitochondria.

Propionyl-L-carnitine (PLC), a natural short-chain derivative of L-carnitine, has been tested in this study as a potential protective agent against adriamycin (ADR)-induced cardiotoxicity in isolated rat heart myocytes and mitochondria. In cardiac myocytes, ADR (0.5 mM) caused a significant (70%) inhibition of palmitate oxidation, whereas, PLC (5 mM) induced a significant (49%) stimulation. Addition of PLC to ADR-incubated myocytes induced 79% reversal of ADR-induced inhibition of palmitate oxidation. In isolated rat heart mitochondria, ADR produced concentration-dependent inhibition of both palmitoyl-CoA and palmitoyl-carnitine oxidation, while PLC caused a more than 2.5-fold increase in both substrates. Preincubation of mitochondria with 5 mM PLC caused complete reversal of ADR-induced inhibition in the oxidation of both substrates. Also ADR induced concentration-dependent inhibition of CPT I which is parallel to the inhibition of its substrate palmitoyl-CoA. In rat heart slices, ADR induced a significant (65%) decrease in adenosine triphosphate (ATP) and this effect is reduced to 17% only by PLC. Results of this study revealed that ADR induced its cardiotoxicity by inhibition of CPT I and beta-oxidation of long-chain fatty acids with the consequent depletion of ATP in cardiac tissues, and that PLC can be used as a protective agent against ADR-induced cardiotoxicity.     

Differences in the induction of carboxylesterase isozymes in rat liver microsomes by perfluorinated fatty acids

1. Differences in the ability of metabolically-inert peroxisome proliferators (perfluoro-n-decanoic acid (PFDA, C₁₀), perfluoro-n-octanoic acid (PFOA, C₈), perfluorooctane sulphonic acid (PFOS, C₈) and 1-H,1-H-pentadecafluoro-n-octanol (PFOL, C₈)) to induce three forms of hepatic microsomal carboxylesterase, namely RL1, RL2 and RH1, in the male rat were studied by measuring changes in hydrolytic activities towards p-nitrophenyl acetate (PNPA), isocarboxazid (ISOC) and butanilicaine (BUTA), which are thought to be specific substrates for RL1, RL2 and RH1, respectively, and by evaluating changes in the contents of the three isozymes by radial immunodiffusion assay with specific antibodies.

2. The administration of PFDA rather specifically decreases PNPA hydrolase activity and RL1 content. On the other hand, PFOA, PFOS and PFOL markedly increase all three hydrolase activities and the content of all three isozymes (except RH1 in the case of PFOA, where the increase was not statistically significant).

3. The correlations between hydrolase activities and isozyme contents supported specificity of the three substrates, with the exception that the content of the predominant isozyme, RL2, showed a higher correlation with BUTA hydrolase activity than with ISOC hydrolase activity.

4. In conclusion, we have demonstrated that metabolically-inert perfluorinated fatty acids induce hepatic microsomal carboxylesterase isozymes, as determined by radial immunodiffusion analysis using specific antibodies. This is the first report that perfluorinated fatty acid affect carboxylesterase isozymes in rat liver microsomes, and is indicative of the importance of peroxisome proliferators in hepatic metabolism of xenobiotics. Further work is needed to determine the regulatory mechanisms involved.     

Mitochondrial 3-hydroxy-3-methylglutaryl coenzyme A synthase and carnitine palmitoyltransferase II as potential control sites for ketogenesis during mitochondrion and peroxisome proliferation

3-Thia fatty acids are potent hypolipidemic fatty acid derivatives and mitochondrion and peroxisome proliferators. Administration of 3-thia fatty acids to rats was followed by significantly increased levels of plasma ketone bodies, whereas the levels of plasma non-esterified fatty acids decreased. The hepatic mRNA levels of fatty acid binding protein and formation of acid-soluble products, using both palmitoyl-CoA and palmitoyl-L-carnitine as substrates, were increased. Hepatic mitochondrial carnitine palmitoyltransferase (CPT) -II and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase activities, immunodetectable proteins, and mRNA levels increased in parallel. In contrast, the mitochondrial CPT-I mRNA levels were unchanged and CPT-I enzyme activity was slightly reduced in the liver. The CoA ester of the monocarboxylic 3-thia fatty acid, tetradecylthioacetic acid, which accumulates in the liver after administration, inhibited the CPT-I activity in vitro, but not that of CPT-II. Acetoacetyl-CoA thiolase and HMG-CoA lyase activities involved in ketogenesis were increased, whereas the citrate synthase activity was decreased. The present data suggest that 3-thia fatty acids increase both the transport of fatty acids into the mitochondria and the capacity of the beta-oxidation process. Under these conditions, the regulation of ketogenesis may be shifted to step(s) beyond CPT-I. This opens the possibility that mitochondrial HMG-CoA synthase and CPT-II retain some control of ketone body formation.     

Effect of methylglyoxal bis(guanylhydrazone) on hepatic, heart and skeletal muscle mitochondrial carnitine palmitoyltransferase and beta-oxidation of fatty acids

Methylglyoxal bis(guanylhydrazone) (MGBG) is an antileukemic agent and a structural polyamine analogue which inhibits S-adenosyl methionine decarboxylase. However, MGBG also produces profound mitochondrial structural damage and inhibition of fatty acid oxidation. Carnitine palmitoyltransferase-A (CPT-A) is located on the outer surface of the inner mitochondrial membrane and is the putative rate-controlling enzyme for mitochondrial long-chain fatty acid oxidation. The present experiments were designed to determine if MGBG inhibits CPT-A. Liver, heart and skeletal muscle mitochondria were isolated from rats following 24 hr of starvation. Measuring the reaction in the direction of palmitoylcarnitine plus CoA formation from palmitoyl-CoA plus carnitine ("forward reaction"), MGBG was competitive with l-carnitine. The MGBG CPT-A Ki values were (mM): liver, 5.0 +/- 0.6 (N = 15); heart 3.2 +/- 1.2 (N = 3); and skeletal muscle, 2.8 +/- 1.0 (N = 3). Lysis of hepatic mitochondria with Triton X-100 yielded a Ki of 4.0 +/- 2.0, which was not significantly different from intact mitochondria or inverted vesicles (4.9 mM). Purified hepatic CPT had a Ki of 4.2 mM. MGBG did not inhibit purified CPT in the "reverse reaction" (palmitoyl-CoA plus carnitine formation from palmitoylcarnitine plus CoA). Spermine and spermidine, which are structurally similar to MGBG, did not inhibit either CPT activity or acid-soluble product formation from 1-[14C]palmitoyl-CoA. MGBG inhibited mitochondrial state 3 oxidation rates of palmitoyl-CoA and palmitoylcarnitine, as well as of glutamate. However, the fatty acid substrates were considerably more sensitive than glutamate to MGBG inhibition. MGBG also increased hepatic mitochondrial aggregation which was reversed by l-carnitine. Fluorescence polarization, using 1,6-diphenyl-1,3,5-hexatriene (DPH) as a probe, indicated that MGBG increased membrane rigidity in a dose-dependent manner. This effect was not altered by l-carnitine. MGBG also inhibited purified pigeon breast carnitine acetyltransferase (CAT; Ki = 1.6 mM). While MGBG appeared to be competitive with l-carnitine for both CPT and CAT, MGBG also exhibits a number of effects which may be mediated through membrane interaction and which are not reversed by carnitine.     

Carnitine palmitoyltransferase: a viable target for the treatment of NIDDM?

Inhibition of fatty acid oxidation is well recognized as a potentially effective mechanism for controlling glycemia in non-insulin-dependent diabetes mellitus (NIDDM). However, a direct targeting of inhibition of the intramitochondrial beta-oxidation pathway or an indirect modulation of fatty acid oxidation by inhibition of substrate release from adipose stores has been fraught with lack of efficacy, unacceptable side-effects or both. Focus has therefore recently been directed towards the carnitine palmitoyltransferase (CPT) system, a three-component system necessary for the transfer of long-chain fatty acids into the intramitochondrial matrix. This article will briefly review the background for fatty acid oxidation inhibition in NIDDM and then focus on the progress in the biological understanding and drug discovery targeting of the CPT system for the treatment of NIDDM. Based upon the review, it is concluded that mechanism-based hepatic and myocardial toxicities in normal animals and a potential for a lack of human efficacy may pose insurmountable hurdles for the development of CPT inhibitors for the treatment of NIDDM.     

Inhibition of hepatic glutathione-S-transferases by fatty acids and fatty acid esters

Micromolar concentrations of polyunsaturated fatty acids and ascorbate esters of saturated fatty acids were found to cause a marked inhibition of rat and mouse hepatic glutathione-S-transferase (GST) activity towards 1-chloro-2,4-dinitrobenzene. Arachidonic acid was approximately 25 times more potent in inhibiting rat GST than palmitic acid which was the least effective. Both linoleic and arachidonic acids did not inhibit rat liver GST when ethacrynic acid was used as substrate while the reverse was true with 1,2-dichoro-4-nitrobenzene. In contrast, all the chemicals tested inhibited rat liver GST activity towards 4-nitropyridine N-oxide, indicating isozyme specificity.     

Participation of the peroxisomal beta-oxidation system in the chain-shortening of PCA16, a metabolite of the cytosine arabinoside prodrug, YNKO1, in rat liver

When PCA16, a metabolite of the cytosine arabinoside prodrug YNKO1, was incubated with isolated rat hepatocytes, time-dependent H2O2 generation was found. When the hepatocytes obtained from clofibrate-treated rat liver were used as an enzyme source, PCA16-dependent production of H2O2 was increased by around 6-fold. The activity of peroxisomal beta-oxidation for PCA16 assayed by H2O2 generation was 3-fold higher than that for palmitic acid, whereas the activity of mitochondrial beta-oxidation for PCA16 assayed by ketone body production was much less than that for palmitic acid. A subcellular distribution study revealed that the distribution of the activities of beta-oxidation and fatty acyl-CoA oxidase for PCA16-CoA coincided with those of cyanide-insensitive palmitoyl-CoA-dependent beta-oxidation and catalase, a marker enzyme of peroxisomes. The profile of the cofactor requirement for beta-oxidation of PCA16-CoA in isolated peroxisomes was similar to that for palmitoyl-CoA oxidation, and the reaction was not inhibited by KCN. The formation of CoA derivative prior to beta-oxidation reaction was essential. HPLC analysis of metabolites after incubation of PCA16-CoA with isolated peroxisomes demonstrated the production of four metabolites, two of which were identified as PCA14 and PCA12 by fast atomic bombardment-mass spectrometry. These results indicate that peroxisomal beta-oxidation participates in the shortening of the alkyl-side chain of PCA16 and plays an important role in the formation of antileukemic cytosine arabinoside from YNKO1.     

Effect of Rodent Hepatocarcinogenic Peroxisome Proliferators on Fatty Acyl-CoA Oxidase, DNA Synthesis, and Apoptosis in Cultured Human and Rat Hepatocytes

The effects of the rodent hepatocarcinogens clofibric acid and ciprofibrate on the activity of the peroxisomal fatty acyl-CoA oxidase, DNA synthesis, and apoptosis were compared in cultured rat and human hepatocytes. Rat hepatocytes expressed a 10-fold greater level of the peroxisomal fatty acyl-CoA oxidase compared to human hepatocytes. At the highest concentration (1.0 mM), both drugs induced a two- to threefold increase in this enzyme activity in both rat and human hepatocytes. Ciprofibrate (0.1 and 0.2 mM) caused a twofold increase in DNA synthesis in rat hepatocytes, whereas clofibric acid had no effect on DNA synthesis in these cells. In contrast, increasing concentrations of both clofibric acid and ciprofibrate produced inhibition of DNA synthesis in human hepatocytes. By using the terminal transferase dUTP–biotin nick end labeling technique, it was observed that 0.1 and 0.2 mM clofibric acid and ciprofibrate suppressed transforming growth factor-β (TGFβ)-induced apoptosis by 50% in rat hepatocytes, but they had no effect on TGFβ-induced apoptosis in human hepatocytes. Although clofibric acid and ciprofibrate diminished TGFβ-induced apoptosis, they had no effect on the basal apoptotic levels in the rat hepatocyte cultures. However, both drugs significantly increased the percent of apoptotic cells in the human hepatocyte cultures. It is concluded that primary rat and human hepatocyte cultures respond differently to peroxisome proliferators. The differences in effects on DNA synthesis and apoptosis support the hypothesis that human liver cells are refractory to peroxisome proliferator-induced hepatocarcinogenesis.     

Evaluation of the acute toxicity of perfluorinated carboxylic acids using eukaryotic cell lines, bacteria and enzymatic assays

The acute biological activity of a homologous series of perfluorinated carboxylic acids – perfluorohexanoic acid (PFHxA), perfluoroheptanoic acid (PFHpA), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA) and perfluorodecanoic acid (PFDA) – was studied. To analyze the potential risk of the perfluorinated acids to humans and the environment, different in vitro toxicity test systems were employed. The cytotoxicity of the chemicals towards two different types of mammalian cell lines and one marine bacteria was investigated. The viability of cells from the promyelocytic leukemia rat cell line (IPC-81) and the rat glioma cell line (C6) was assayed calorimetrically with WST-1 reagent. The evaluation was combined with the Vibrio fischeri acute bioluminescence inhibition assay. The biological activity of the compounds was also determined at the molecular level with acetylcholinesterase and glutathione reductase inhibition assays. This is the first report of the effects of perfluorinated acids on the activity of purified enzymes. The results show these compounds have a very low acute biological activity. The observed effective concentrations lie in the millimole range, which is well above probable intracellular concentrations. A relationship was found between the toxicity of the perfluorinated carboxylic acids and the perfluorocarbon chain length: in every test system applied, the longer the perfluorocarbon chain, the more toxic was the acid. The lowest effective concentrations were thus recorded for perfluorononanoic and perfluorodecanoic acids.     

Differential activation of nuclear receptors by perfluorinated fatty acid analogs and natural fatty acids: a comparison of human, mouse, and rat peroxisome proliferator-activated receptor-alpha, -beta, and -gamma, liver X receptor-beta, and retinoid X receptor-alpha.

Administration of ammonium salts of perfluorooctanoate (PFOA) to rats results in peroxisome proliferation and benign liver tumors, events associated with activation of the nuclear receptor (NR) peroxisome proliferator-activated receptor-alpha (PPARalpha). Due to its fatty acid structure, PFOA may activate other NRs, such as PPARbeta, PPARgamma, liver X receptor (LXR), or retinoid X receptor (RXR). In this study, the activation of human, mouse, and rat PPARalpha, PPARbeta, PPARgamma, LXRbeta, and RXRalpha by PFOA (including its linear and branched isomers) and perfluorooctane sulfonate (PFOS) was investigated and compared to several structural classes of natural fatty acids and appropriate positive control ligands. An NR ligand-binding domain/Gal4 DNA-binding domain chimeric reporter system was used. Human, mouse, and rat PPARalpha were activated by PFOA isomers and PFOS. PPARbeta was less sensitive to the agents tested, with only PFOA affecting the mouse receptor. PFOA and PFOS also activated human, mouse, and rat PPARgamma, although the maximum induction of PPARgamma was much less than that seen with rosiglitazone, suggesting that PFOA and PFOS are partial agonists of this receptor. Neither LXRbeta nor the common heterodimerization partner RXRalpha was activated by PFOA in any species examined. Taken together, these data show that of the NRs studied, PPARalpha is the most likely target of PFOA and PFOS, although PPARgamma is also activated to some extent. Compared to naturally occurring long-chain fatty acids, e.g. linoleic and alpha-linolenic acids, these perfluorinated fatty acid analogs were more selective and less potent in their activation of the NRs.     

Effect of the peroxisome proliferator perfluorodecanoic acid on growth and lipid metabolism in Sprague Dawley rats fed three dietary levels of selenium

The possible interrelationships between the effects of dietary selenium and perfluorodecanoic acid (PFDA) on growth and lipid metabolism were studied in the male Sprague Dawley rat. Rats were divided into groups and placed on diets containing three levels of selenium (0.04, 0.2, and 1.0 ppm as sodium selenite). Two weeks later, half the rats in each group received a single 35 mg/kg IP injection of PFDA in corn oil, while their pair-fed companion received only vehicle. Rats injected with PFDA stopped gaining weight, and weighed less than pair-fed controls, despite equal food intakes. Two weeks following PFDA administration the rats were killed and plasma cholesterol and triglycerides, and liver peroxisomal enzyme activities were quantified. In contrast to other peroxisome proliferators, PFDA increased plasma triglycerides while decreasing plasma cholesterol. The rate of peroxisomal fatty acid -oxidation was decreased, even though the activity of fatty acyl-CoA oxidase, the first enzyme in the peroxisomal fatty acid -oxidation pathway, was increased. Dietary selenium, other than increasing the liver to body weight ratio, did not alter growth or lipid metabolism. This study demonstrates, for the first time, the existence of a non-hypotriglyceridemic peroxisome proliferator-PFDA.