Differential induction and regulation of peroxisomal enzymes: predictive value of peroxisome proliferation in identifying certain nonmutagenic carcinogens

Hypolipidemic drugs and certain plasticizers markedly increase the number of peroxisomes in liver parenchymal cells. Continued exposure to peroxisome proliferators has been shown to produce essentially similar pleiotropic responses leading eventually to the development of liver tumors in rats and mice. These agents are not mutagenic in short-term test systems and do not appear to interact with or damage DNA. Accordingly, the events leading to or associated with the induction of peroxisome proliferation have been postulated to play a role in the development of liver tumors. Recent evidence indicates that persistent peroxisome proliferation leads to the formation of 8-hydroxyguanosine in rat liver DNA, which supports the role for oxidative stress. The mRNAs of the three peroxisomal beta-oxidation genes are induced over 20-fold in the livers of rats treated with nafenopin, Wy-14643, BR-931, and other structurally diverse peroxisome proliferators. This increase in beta-oxidation mRNAs is evident within 30 min to 1 hr and was maximal 8 to 16 hr after the administration of a single dose of these agents by gavage. The peroxisomal catalase and urate oxidase mRNAs increase about 2-fold in the livers of rats treated chronically with peroxisome proliferators. These results indicate that peroxisome proliferators differentially regulate different peroxisomal enzymes. The tissue specificity of peroxisomal beta-oxidation gene regulation by xenobiotics supports the contention that the development of liver tumors following exposure to peroxisome proliferators correlates well with the inducibility of peroxisome proliferation and the beta-oxidation genes. Although these agents are known to exert mitogenic response in liver, it is unlikely that stimulation of DNA synthesis alone is responsible for tumor development. Cell proliferation may, however, play a secondary role. The morphological phenomenon of peroxisome proliferation should serve as a simple, sensitive, and valuable biological indicator for the identification of nongenotoxic or nonmutagenic chemicals that may be carcinogenic. An understanding of the cellular and molecular basis of peroxisome proliferation is a prerequisite for the evaluation of toxicological implications of this phenomenon.     

Lack of induction of hepatic DNA damage on long-term administration of peroxisome proliferators in male F-344 rats

In order to evaluate the relationship between hydrogen peroxide (H2O2) generation and subsequent DNA damage caused by peroxisome proliferation, we examined DNA damage and changes in peroxisomal beta-oxidation activity in rat liver. Male F-344 rats were given orally clofibrate, bezafibrate or di(2-ethylhexyl)phthalate (DEHP) for up to 78 weeks. In rats fed DEHP for 52 or 78 weeks hepatocarcinomas or neoplastic nodules were found. In rats treated for 2 weeks with peroxisome proliferators, peroxisomal beta-oxidation activity was increased 10-17 times over control levels. After long-term treatment (20-78 weeks), the level of peroxisomal beta-oxidation activity remained 3-13-times higher in each group. When single strand DNA breaks were measured by a DNA-alkaline elution technique, no increase in DNA damage was observed in livers from rats fed peroxisome proliferators for 2, 40 or 78 weeks. In rats bearing hepatocarcinomas induced by DEHP, the hepatic DNA showed significant breaks; the rate of DNA-alkaline elution was found to increase approximately 5-fold. No significant increase in hepatic lipid peroxide level was observed in each group. These results show that although prolonged treatment with peroxisome proliferators induces markedly peroxisomal beta-oxidation activity, the active oxygen species from peroxisomal beta-oxidation are not enough to give rise to significant DNA damage. Moreover, the change in the activity of peroxisomal beta-oxidation may not relate to hepatocarcinogenesis induced by peroxisome proliferators.     

Haloacetate-induced oxidative damage to DNA in the liver of male B6C3F1 mice

Brominated and chlorinated haloacetates (HAs) are by-products of drinking water disinfection. Dichloroacetate (DCA) and trichloroacetate (TCA) are hepatocarcinogenic in rodents, but the brominated analogs have received little study. Prior work has indicated that acute doses of the brominated derivatives are more potent inducers of oxidative stress and increase the 8-hydroxydeoxyguanosine (8-OH-dG) content of the nuclear DNA in the liver. Since, DCA and TCA are also known as weak peroxisome proliferators, the present study was intended to determine whether this activity might be exacerbated by peroxisomal proliferation. Classical responses to peroxisome proliferators, cyanide-insensitive acyl-CoA oxidase activity and increased 12-hydroxylation of lauric acid, were elevated in a dose-related manner in mice maintained on TCA and clofibric acid (positive control), but not with DCA, dibromoacetate (DBA) or bromochloroacetate (BCA). Administration of the HAs in drinking water to male B6C3F1 mice for periods from 3 to 10 weeks resulted in dose-related increases in 8-OH-dG in nuclear DNA of the liver with DBA and BCA, but not with TCA or DCA. These findings indicate that oxidative damage induced by the haloacetates is, at least in part, independent of peroxisome proliferation. In addition, these data suggest that oxidative damage to DNA may play a more important role in the chronic toxicology of brominated compared to the chlorinated haloacetates.     

Effect of dibromoacetic acid on DNA methylation, glycogen accumulation, and peroxisome proliferation in mouse and rat liver.

Dibromoacetic acid (DBA) is a drinking water disinfection by-product. Its analogs, dichloroacetic acid (DCA) and trichloroacetic acid (TCA), are liver carcinogens in rodents. We evaluated the ability of DBA to cause DNA hypomethylation, glycogen accumulation, and peroxisome proliferation that are activities previously reported for the two other haloacetic acids. Female B6C3F1 mice and male Fischer 344 rats were administered 0, 1,000, and 2,000 mg/l DBA in drinking water. The animals were euthanized after 2, 4, 7, and 28 days of exposure. Dibromoacetic acid caused a dose-dependent and time-dependent decrease of 20%-46% in the 5-methylcytosine content of DNA. Hypomethylation of the c-myc gene was observed in mice after 7 days of DBA exposure. Methylation of 24 CpG sites in the insulin-like growth factor 2 (IGF-II) gene was reduced from 80.2% +/- 9.2% to 18.8% +/- 12.9% by 2,000 mg/l DBA for 28 days. mRNA expression of the c-myc and IGF-II genes in mouse liver was increased by DBA. A dose-dependent increase in the mRNA expression of the c-myc gene was also observed in rats. In both mice and rats, DBA caused dose-dependent accumulation of glycogen and an increase of peroxisomal lauroyl-CoA oxidase activity. Hence, DBA, like DCA and TCA, induced hypomethylation of DNA and of the c-myc and IGF-II genes, increased mRNA expression of both genes, and caused peroxisome proliferation. Again like DCA, DBA also induced glycogen accumulation. These results indicate that DBA shares biochemical and molecular activities in common with DCA and/or TCA, suggesting that it might also be a liver carcinogen.     

Metabolism and lipoperoxidative activity of trichloroacetate and dichloroacetate in rats and mice.

Trichloroacetate (TCA) and dichloroacetate (DCA) have been shown to be hepatocarcinogenic in mice when administered in drinking water. However, DCA produces pathological effects in the liver that are much more severe than those observed following TCA treatment in both rats and mice. To identify potential mechanisms involved in the liver pathology, the biotransformation of TCA and DCA was investigated in male Fischer 344 rats and B6C3F1 mice. Rodents were administered 5, 20, or 100 mg/kg [14C]TCA or [14C]DCA as a single oral dose in water. Elimination was examined by counting radioactivity in urine, feces, exhaled air, and carcass. Blood concentration over time curves were constructed for both TCA and DCA at the 20 and 100 mg/kg doses. Analysis of the data reveals two significant differences in the systemic clearance of TCA relative to DCA. First, DCA was much more extensively metabolized than TCA. More than 50% of any single dose of TCA was excreted unchanged in the urine of both rats and mice. In contrast, less than 2% of any dose of DCA was recovered in the urine as the parent compound. Second, while the blood concentration over time curves for TCA were similar in rats and mice, the blood concentrations of DCA were markedly greater in rats compared to those in mice, both when DCA was administered and when DCA resulted from metabolism of TCA. DCA was detected in the urine of TCA-treated animals and chloroacetate was found in the urine of DCA-treated animals. These metabolic products would be expected to arise from a free radical-generating, reductive dechlorination pathway. To evaluate the ability of acute doses of TCA and DCA to elicit a lipoperoxidative response, additional groups of mice were administered 0, 100, 300, 1000, and 2000 mg/kg TCA or DCA and thiobarbituric acid-reactive substances (TBARS) measured in liver homogenates. Both TCA and DCA enhanced the formation of TBARS in a dose-dependent manner, thereby providing further evidence of a reductive metabolic pathway. DCA was found to be the more potent of the chlorinated acetates in increasing TBARS formation in the livers of both rats and mice. In view of these data, it appears that the more extensive metabolism and rapid rate of elimination of DCA relative to TCA and the more potent lipoperoxidative activity of DCA may be important factors in the pathological effects associated with DCA treatment.     

Peroxisome proliferating sulphur- and oxy-substituted fatty acid analogues are activated to acyl coenzyme A thioesters.

In liver homogenates from untreated rats the sulphur-substituted fatty acid analogues tetradecylthioacetic acid (CMTTD) was activated to its acyl-coenzyme A thioester. The activation was found to take place in the mitochondrial, microsomal and peroxisomal fractions. The activity of CMTTD-CoA synthetase was 50% compared to palmitoyl-CoA synthetase in all cellular fractions. When rats were treated with the peroxisome proliferating sulphur-substituted fatty acid analogues CMTTD and 3-dithiahexadecanedioic acid (BCMTD), the CMTTD-CoA synthetase activity was induced in mitochondrial, peroxisomal and microsomal fractions. Palmitoyl-CoA synthetase was induced proportionally. In rats treated with tetradecylthiopropionic acid (CETTD) of low peroxisome proliferating potency, the activities of CMTTD-CoA synthetase and palmitoyl-CoA synthetase were inhibited in mitochondrial and microsomal fractions. In contrast, all three sulphur-substituted acids induced the activity of palmitoyl-CoA synthetase and CMTTD-CoA synthetase in peroxisomes. Both the CMTTD-CoA and palmitoyl-CoA synthetase activities were induced by CMTTD and BCMTD, in close correlation to the induction of peroxisomal beta-oxidation. During the three treatment regimes, CMTTD-CoA synthetase activity ran parallel to the palmitoyl-CoA synthetase activity at a rate of 50% in all cellular fractions. Thus, CMTTD is assumed to be activated by the long-chain acyl-CoA synthetase enzyme. Rats were treated for 5 days with sulphur- and oxy-substituted fatty acid analogues, clofibric acid and fenofibric acid. All compounds which induced peroxisomal beta-oxidation activity in vivo could be activated to their respective CoA thioesters in liver homogenate. CETTD which induced peroxisomal beta-oxidation only two-fold, was activated at a rate of 50% compared to palmitate. Fenofibric acid induced peroxisomal beta-oxidation 9.6-fold, while it was activated at a rate of only 10% compared to palmitate. Thus, no correlation was found between rate of activation in vitro and induction of peroxisomal activity in vivo. On the other hand, tetradecylsulfoxyacetic acid (TSOA) and tetradecylsulfonacetic acid (TSA) (sulphuroxygenated metabolites of CMTTD) with no inductive effects, were not activated to their respective CoA derivatives. Altogether the data suggest that the enzymatic activation of the peroxisome proliferating compounds is essential for their proliferating activity, but the rate of activation does not determine the potency of the proliferators. The role of the xenobiotic-CoA pool in relation to the whole coenzyme A profile during peroxisome proliferation is discussed.     

Early induction of reparative hyperplasia in the liver of B6C3F1 mice treated with dichloroacetate and trichloroacetate

Trichloroacetate (TCA) and dichloroacetate (DCA) were administered at concentrations of 0, 300, 1000 or 2000 mg/l in the drinking water of male B6C3F1 and male and female Swiss-Webster mice for up to 14 days. At 2, 5 or 14 days of treatment, mice were injected with [3H]thymidine 2 h prior to sacrifice. The livers were examined histologically and autoradiographically and DNA was isolated and counted. As observed in chronic studies dichloroacetate induced a marked increase in liver weights, but only after 14 days of treatment and local necrosis in both B6C3F1 and Swiss-Webster mice. A significant increase in the labeling index of hepatocytes was observed in animals treated with DCA, but only at 14 days of treatment. No such increases were observed in animals treated with TCA. In contrast, significant increases in [3H]thymidine were observed in the livers of both DCA- and TCA-treated animals after 5 days of treatment. This effect remained apparent with TCA after 14 days of treatment. These data support the hypothesis that the tumorigenic effect of DCA is strongly influenced by necrosis and reparative hyperplasia. On the other hand, the carcinogenic effects of TCA appear to be more closely associated with [3H]thymidine incorporation that can be separated from cell division, suggesting an elevated rate of repair synthesis of DNA. Thus the carcinogenic effects of TCA (and perhaps lower doses of DCA) may involve damage to DNA.     

The effect of beclobric acid and clofibric acid on peroxisomal beta-oxidation and peroxisome proliferation in primary cultures of rat, monkey and human hepatocytes

The peroxisome-proliferating effects of clofibric acid and beclobric acid were studied in primary cultures of hepatocytes derived from rat, monkey (Macaca fascicularis) and human liver. Determination of peroxisomal fatty acid beta-oxidation and morphometrical analysis of the peroxisomal compartment were performed after incubation of 1-day-old hepatocyte cultures for 3 days with either compound. In rat liver cell cultures both compounds gave a 10-fold increase in peroxisomal beta-oxidation, a 3-fold increase in the relative number of peroxisomes and a 1.5-fold increase in the mean size of peroxisomes. Beclobric acid gave its maximal effect at a concentration of 10 microM, which is at least one order of magnitude lower than the maximum-effect concentration of clofibric acid. At concentrations greater than 300 microM beclobric acid was cytotoxic. No stimulation of peroxisomal fatty acid beta-oxidation was found in either monkey or human hepatocyte cultures. Morphometrical analysis also showed no increase in the peroxisomal compartment in cultures derived from these species, as indicated by the lack of increase in both relative number and size of peroxisomes. In all three species tested beclobric acid was equally cytotoxic for hepatocytes in vitro. These results are of relevance for the interpretation of the peroxisome-proliferating effects of clofibrate and similar compounds in rats. Since peroxisome proliferation may be correlated to increased hepatic tumour incidences in the rat, the absence of peroxisome proliferation in primates suggests the absence of tumourogenic activity by hypolipidemic compounds in these species.     

Hypomethylation of DNA and the insulin-like growth factor-II gene in dichloroacetic and trichloroacetic acid-promoted mouse liver tumors

Dichloroacetic acid (DCA) and trichloroacetic acid (TCA) are mouse liver carcinogens. DNA hypomethylation is a common molecular event in cancer that is induced by DCA and TCA. Hypomethylation of DNA and the insulin-like growth factor-II (IGF-II) gene was determined in DCA- and TCA-promoted liver tumors. Mouse liver tumors were initiated by N-methyl-N-nitrosourea and promoted by either DCA or TCA. By dot-blot analysis using an antibody for 5-methylcytosine, the DNA in DCA- and TCA-promoted tumors was demonstrated to be hypomethylated. The methylation status of 28 CpG sites in the differentially methylated region-2 (DMR-2) of mouse IGF-II gene was determined. In liver, 79.3 +/- 1.7% of the sites were methylated, while in DCA- and TCA-treated mice, only 46.4 +/- 2.1% and 58.0 +/- 1.7% of them were methylated and 8.7 +/- 2.6% and 10.7 +/- 7.4% were methylated in tumors. The decreased methylation found in liver from mice exposed to DCA or TCA occurred only in the upstream region of DMR-2, while in tumors it occurred throughout the probed region. mRNA expression of the IGF-II gene was increased in DCA- and TCA-promoted liver tumors but not in non-involved liver from DCA- and TCA-exposed mice. The results support the hypothesis that DNA hypomethylation is involved in the mechanism for the tumorigenicity of DCA and TCA.     

Subchronic 90 day toxicity of dichloroacetic and trichloroacetic acid in rats

Male Sprague-Dawley rats were treated with either dichloroacetic acid (DCA) or trichloroacetic acid (TCA) in the drinking water at levels of 0, 50, 500 and 5000 ppm for a period of 90 days to determine the toxicities associated with subchronic exposure. All animals were sacrificed and examined for gross and histopathologic lesions, serochemical changes, immune dysfunction, hepatic peroxisomal and mixed function oxidase enzyme induction and organ-body weight changes. Animals treated with DCA had decreased body weight gains (500 and 5000 ppm) and decreased total serum protein (all doses). Rats given either TCA (5000 ppm) or DCA (500 or 5000 ppm) had increased liver and kidney organ to body weight ratios. Rats offered DCA had significantly elevated alkaline phosphatase (500 and 5000 ppm) and alanine-amino transferase (5000 ppm). No consistent immunotoxicity was observed in animals exposed to either compound. Rats treated with 5000 ppm TCA or DCA had significantly increased hepatic peroxisomal beta-oxidation activity. These data, along with histopathologic changes, suggest that TCA and DCA produce substantial systemic organ toxicity to the liver and kidney during a 90-day subchronic exposure, although only at doses greater than those expected to occur in the environment.     

Species and strain sensitivity to the induction of peroxisome proliferation by chloroacetic acids

B6C3F1 mice and Sprague-Dawley rats were provided drinking water containing 6-31 mM (1-5 g/liter) trichloroacetic acid (TCA), 8-39 mM (1-5 g/liter) dichloroacetic acid (DCA), or 11-32 mM (1-3 g/liter) monochloroacetic acid (MCA) for 14 days. TCA and DCA, but not MCA, increased the mouse relative liver weight in a dose-dependent manner. Rat liver weights were not altered by TCA or DCA treatment, but were depressed by MCA. Hepatic peroxisome proliferation was demonstrated by (1) increased palmitoyl-CoA oxidase and carnitine acetyl transferase activities, (2) appearance of a peroxisome proliferation-associated protein, and (3) morphometric analysis of electron micrographs. Mouse peroxisome proliferation was enhanced in a dose-dependent manner by both TCA and DCA, but only the high DCA concentration (39 mM) increased rat liver peroxisome proliferation. MCA was ineffective in both species. Three other mouse strains (Swiss-Webster, C3H, and C57BL/6) and two strains of rat (F344 and Osborne-Mendel) were examined for sensitivity to TCA. TCA (12 and 31 mM) effectively enhanced peroxisome proliferation in all mouse strains, especially the C57BL/6. A more modest enhancement in the Osborne-Mendel (288%) and F344 rat (167%) was seen. Dosing F344 rats with 200 mg/kg TCA in water or corn oil for 10 days increased peroxisome proliferation 179 and 278%, respectively, above the vehicle controls. These studies demonstrate that the mouse is more sensitive than the rat with respect to the enhancement of liver peroxisome proliferation by TCA and DCA and suggest that if peroxisome proliferation is critical for the induction of hepatic cancer by TCA and DCA, then the rat should be less sensitive or refractory to tumor induction.     

Accumulation of medium chain acyl-CoAs during beta-oxidation of long chain fatty acid by isolated peroxisomes from rat liver.

We have reported fatty alcohol synthesis accompanied by chain elongation in liver peroxisomes (Biochim. Biophys. Acta, 1346, 38 (1997)). In the present experiment, we studied what kind of acyl-CoA(s) destined to be utilized as primer for fatty alcohol synthesis accumulate(s) during peroxisomal beta-oxidation. Peroxisomes were prepared from rat liver treated with clofibrate, a peroxisome proliferator, and incubated with [U-14C]palmitate, in order to investigate acyl-CoAs after beta-oxidation. At 1 mM concentration, MgATP activated beta-oxidation, but inhibited beta-oxidation at concentrations higher than 1 mM. After incubation of peroxisomes with palmitate, various acyl-CoAs were formed. Among medium-chain labelled acyl-CoAs, octanoyl-CoA was mainly detected. These results suggest that octanoyl-CoA accumulates during beta-oxidation of palmitate. When peroxisomes were incubated with [9,10-(3)H]palmitate and [9,10-(3)H]stearate, among medium-chain acyl-CoAs, octanoyl-CoA and decanoyl-CoA were primarily detected, respectively, suggesting the occurrence of at least 4 cycles of beta-oxidation of both fatty acids by peroxisomes.     

Induction of fatty acid beta-oxidation and peroxisome proliferation in the liver of rhesus monkeys by DL-040, a new hypolipidemic agent

Many structurally unrelated hypolipidemic agents and certain phthalate-ester plasticizers induce hepatomegaly and proliferation of peroxisomes in liver parenchymal cells of rodents, but there is relatively limited evidence regarding the ability of such compounds to induce peroxisome proliferation in the livers of nonrodent species including man. The present study was designed to determine if DL-040 (4-(((1,3-benzodioxol)-5-yl)methyl)amino-benzoic acid), a newly developed hypolipidemic agent, induces peroxisome proliferation in the liver of adult rhesus monkeys. Feeding of DL-040 (300 mg/kg body wt for 1 week; and 400 mg/kg body wt for 10 weeks) caused a significant increase in peroxisome population as determined by ultrastructural and morphometric analyses. The DL-040-induced peroxisome proliferation was accompanied by increases in the levels of catalase, carnitine acetyltransferase and the peroxisomal fatty acid beta-oxidation system. As expected, DL-040 caused a significant reduction of serum cholesterol and low density lipoprotein content. These data suggest that hepatic peroxisome proliferation is inducible in nonhuman primates at dose levels that exceed therapeutic levels.     

Amino acid and protein targets of 1,2-diacetylbenzene,a potent aromatic gamma-diketone that induces proximal neurofilamentous axonopathy

Determining the key events in the induction of liver cancer in mice by trichloroethylene (TRI) is important in the determination of how risks from this chemical should be treated at low doses. At least two metabolites can contribute to liver cancer in mice, dichloroacetate (DCA) and trichloroacetate (TCA). TCA is produced from metabolism of TRI at systemic concentrations that can clearly contribute to this response. As a peroxisome proliferator and a species-specific carcinogen, TCA may not be important in the induction of liver cancer in humans at the low doses of TRI encountered in the environment. Because DCA is metabolized much more rapidly than TCA, it has not been possible to directly determine whether it is produced at carcinogenic levels. Unlike TCA, DCA is active as a carcinogen in both mice and rats. Its low-dose effects are not associated with peroxisome proliferation. The present study examines whether biomarkers for DCA and TCA can be used to determine if the liver tumor response to TRI seen in mice is completely attributable to TCA or if other metabolites, such as DCA, are involved. Previous work had shown that DCA produces tumors in mice that display a diffuse immunoreactivity to a c-Jun antibody (Santa Cruz Biotechnology, SC-45), whereas TCA-induced tumors do not stain with this antibody. In the present study, we compared the c-Jun phenotype of tumors induced by DCA or TCA alone to those induced when they are given together in various combinations and to those induced by TRI given in an aqueous vehicle. When given in various combinations, DCA and TCA produced a few tumors that were c-Jun+, many that were c-Jun-, but a number with a mixed phenotype that increased with the relative dose of DCA. Sixteen TRI-induced tumors were c-Jun+, 13 were c-Jun-, and 9 had a mixed phenotype. Mutations of the H-ras protooncogene were also examined in DCA-, TCA-, and TRI-induced tumors. The mutation frequency detected in tumors induced by TCA was significantly different from that observed in TRI-induced tumors (0.44 vs 0.21, p < 0.05), whereas that observed in DCA-induced tumors (0.33) was intermediate between values obtained with TCA and TRI, but not significantly different from TRI. No significant differences were found in the mutation spectra of tumors produced by the three compounds. The presence of mutations in H-ras codon 61 appeared to be a late event, but ras-dependent signaling pathways were activated in all tumors. These data are not consistent with the hypothesis that all liver tumors induced by TRI were produced by TCA.     

Trichloroacetate stimulation of liver DNA synthesis in male and female mice

Male and female B6C3F1 mice were given trichloroacetate (TCA) by gavage for 11 days. Livers from untreated male and female mice were unremarkable by histopathologic examination. In livers from mice receiving 1000 mg/kg body weight, the centrolobular hepatic cords showed slight changes, which included increased eosinophilic staining and rare apoptosis. Areas in the intermediate zone were noted where the architecture of the liver hepatic cords was subtly changed. The changes in cord architecture seemed to define nodular areas where cellular proliferation in animals treated with TCA had occurred. No histopathologic differences were noted between the livers of treated or control, male and female animals. Mitosis and DNA synthesis were examined using incorporation of ³H]thymidine into liver cells. ³H]Thymidine incorporation into extracted liver DNA of animals receiving TCA was significantly increased over controls in all treatment groups. Autoradiographic examination of liver sections showed that the incorporation of label in control animals was predominantly in peri-sinusoidal cells, whereas the majority of radiolabel incorporation in TCA-treated animals was found in intermediate zone cells that appeared to be mature hepatocytes. No outstanding differences in the distribution of radiolabel in the liver sections from male or female mice were noted. When incorporation of ³H]thymidine was quantified by enumeration of labeled liver cells following autoradiography, incorporation of the radiolabel into hepatocytes increased with the dose of TCA given but there was no increase in radiolabel in peri-sinusoidal cells. Increased mitotic figures in intermediate zone cells resembling mature hepatocytes were noted in all mice treated with TCA. These results suggest that increased DNA synthesis and mitosis may contribute tumorigenesis by TCA.     

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.     

Liver tumor induction in B6C3F1 mice by dichloroacetate and trichloroacetate

Male and female B6C3F1 mice were administered dichloroacetate (DCA) and trichloroacetate (TCA) in their drinking water at concentrations of 1 or 2 g/l for up to 52 weeks. Both compounds induced hepatoproliferative lesions (HPL) in male mice, including hepatocellular nodules, adenomas and hepatocellular carcinomas within 12 months. The induction of HPL by TCA was linear with dose. In contrast, the response to DCA increased sharply with the increase in concentration from 1 to 2 g/l. Suspension of DCA treatment at 37 weeks resulted in the same number of HPL at 52 weeks that would have been predicted on the basis of the total dose administered. However, none of the lesions in this treatment group progressed to hepatocellular carcinomas. Conversely, the yield of HPL at 52 weeks when TCA treatment was suspended at 37 weeks was significantly below that which would have been predicted by the total dose administered. In this case, 3 of 5 remaining lesions were hepatocellular carcinomas. Throughout active treatment DCA-treated mice displayed greatly enlarged livers characterized by a marked cytomegaly and massive accumulations of glycogen in hepatocytes throughout the liver. Areas of focal necrosis were seen throughout the liver. TCA produced small increases in cell size and much a more modest accumulation of glycogen. Focal necrotic damage did not occur in TCA-treated animals. TCA produced marked accumulations of lipofuscin in the liver. Lipofuscin accumulation was less marked with DCA. These data confirm earlier observations that DCA and TCA are capable of inducing hepatic tumors in B6C3F1 mice and argue that the mechanisms involved in tumor induction differ substantially between these two similar compounds. Tumorigenesis by DCA may depend largely on stimulation of cell division secondary to hepatotoxic damage. On the other hand, TCA appears to increase lipid peroxidation, suggesting that production of radicals may be responsible for its effects.     

Contribution of peroxisomal beta-oxidation system to the chain-shortening of N-(alpha-methylbenzyl)azelaamic acid in rat liver

Hepaptic peroxisomal and mitochondrial beta-oxidation of N-(alpha-methylbenzyl)azelaamic acid (C9), which is a possible metabolic intermediate of Melinamide, a potent hypocholesterolemic drug, were investigated. Isolated hepatocytes generated H2O2 when incubated with C9, indicating that C9 served as the substrate for peroxisomal beta-oxidation. Also with isolated peroxisomes a significant activity of peroxisomal beta-oxidation for C9-CoA measured by following cyanide-insensitive NAD reduction was observed, when the chain-shortened products such as C7 and C5 were detected from the incubation mixture of C9-CoA, and so NADH, acetyl-CoA and C2 units split off from C9-CoA were produced in stoichiometric amounts. In contrast, the mitochondrial beta-oxidation for C9 measured by following ketone body production and antimycin A-sensitive O2 consumption was not detectable, indicating that C9 is not metabolized by mitochondrial beta-oxidation. Comparative study of beta-oxidation capacities in peroxisomes and mitochondria indicate that the beta-oxidation of C9 occurs exclusively in peroxisomes. Also, the formation activity of C2 units liberated from C9 in intact hepatocytes reflects the peroxisomal beta-oxidation activity of liver homogenates with a highly close correlation. Therefore, it is concluded that C9 can be an excellent substrate for estimating peroxisomal beta-oxidation activity in intact cells.     

Influence of chronic administration of valproate on ultrastructure and enzyme content of peroxisomes in rat liver and kidney. Oxidation of valproate by liver peroxisomes

Chronic administration to rats of the anticonvulsant drug, valproate, induced proliferation of liver peroxisomes and selectively increased the activity of the enzymes involved in beta-oxidation in these organelles. In kidney cortex, only a moderate increase in enzyme activity could be recorded. Valproate (1% w/w in the diet for 25 to 100 days) caused the appearance on electron micrographs of unusual tubular inclusions in the matrix of liver peroxisomes. SDS-PAGE analysis of purified peroxisomal fractions from treated rats demonstrated an increase in the content of five polypeptides; four of which most likely correspond to enzymes of the peroxisomal beta-oxidation. It is suggested that the peroxisomal inclusions correspond to the accumulation of these polypeptides in the matrix of the organelle. An in vivo evaluation of the peroxisomal hydrogen peroxide production suggested that valproate itself or one of its metabolites is substrate for peroxisomal beta-oxidation. This was confirmed by in vitro studies. Activation of valproate or its metabolites by liver acyl-CoA synthetase could be demonstrated, although it was 50 times slower than that of octanoate. This reaction further led to a small, but significant production of H2O2 by the action of peroxisomal acyl-CoA oxidase.