Hepatic Peroxisome Proliferation Research Articles

Subchronic oral toxicity of di- n-octyl phthalate and di(2-ethylhexyl) phthalate in the rat

The subchronic oral toxicity of di(2-ethylhexyl) phthalate (DEHP) and di- n-octyl phthalate (DNOP) was studied. Groups of 10 male and 10 female Sprague-Dawley rats were administered DEHP in the diet at 0, 5, 50, 500 or 5000 ppm for 13 wk. In a separate study, groups of 10 male and 10 female Sprague-Dawley rats were given DNOP (5, 50, 500 and 5000 ppm) in the diet while control groups received basal diet containing 4% corn oil and positive control groups were fed a diet containing 5000 ppm DEHP. Growth rate and food consumption were not affected by treatment with either compound. Hepatomegaly was observed in the highest dose groups of both sexes administered DEHP but not in the DNOP-treated animals. At the highest dose, DNOP caused threefold (females) and 12-fold (males) increases in liver ethoxyresorufin- O-deethylase activity while DEHP did not. Mild changes in serum biochemistries were mostly confined to rats in the highest dose group of DEHP, and included increased serum albumin and albumin/globulin ratio in both sexes and decreased cholesterol in female rats. Mild vacuolations in the Sertoli cells were observed in male rats exposed to 500 ppm DEHP. At 5000 ppm DEHP, there was mild to moderate seminiferous tubule atrophy and Sertoli cell vacuolation in males, and rats of both sexes showed hepatic peroxisome proliferation. Both DEHP and DNOP at 5000 ppm caused mild histological changes in the thyroid consisting of reduced follicle size and colloid density, and the liver consisting of endothelial nuclear prominence, nuclear hyperchromicity and anisokaryosis. There was accentuation of zonation of the hepatic lobules and increased perivenous cytoplasmic vacuolation in DNOP-treated rats. Trace quantities (3–5 ppm) of DEHP and DNOP were detected in the liver, and 15–31 ppm were found in adipose tissue of the highest dose groups. The no observed-effect-level was judged to be 50 ppm in the diet or 3.7 mg/kg body weight/day for DEHP, and 500 ppm or 36.8 mg/kg body weight/day for DNOP.

Comparison of the Effects of Cinnamyl Anthranilate on Hepatic Peroxisome Proliferation and Cell Replication in the Rat and Mouse

Comparison of the Effects of Cinnamyl Anthranilate on Hepatic Peroxisome Proliferation and Cell Replication in the Rat and Mouse

Induction of cytochrome P4501 A1 and P4504A1 activities andperoxisomal proliferation by furnonisin B1

The effects of repeated exposure to fumonisin B1 (FB1) on hepatic and renal mixed function oxidase activities and peroxisomal proliferation has been examined in rats following intraperitoneal administration at three dose levels (0.125, 0.25, and 2.5 mg/kg) once a day for 6 days. At the two highest doses, FB1 increased the renal and hepatic N-demethylation of erythromycin (CYP3A1) and the hepatic O-deethylation of ethoxyresorufin (CYP1A1). FB1, at the highest dose of 2.5 mg/kg, also increased the renal O-deethylation of ethoxyresorufin. The liver, but not the kidney, was also susceptible to FB1-dependent induction of the 12- and 11-hydroxylation of lauric acid, suggesting induction of the CYP4A subfamily. Immunoblot studies employing solubilized microsomes from FB1-treated rats revealed that FB1, at the two highest doses, increased the apoprotein levels of CYP1A1 and CYP4A1. The same treatment with FBI increased the β-oxidation of palmitoyl-coenzyme A (CoA) in liver homogenates, and immunoblot analysis showed an increase in the apoprotein levels of the trans-2-enoyl-CoA hydratase trifunctional protein. The possible implications of these findings to the hepatocarcinogenicity of this mycotoxin are discussed.

Induction of cytochrome P4501A1 and P4504A1 activities and peroxisomal proliferation by fumonisin B1.

The effects of repeated exposure to fumonisin B1 (FB1) on hepatic and renal mixed function oxidase activities and peroxisomal proliferation has been examined in rats following intraperitoneal administration at three dose levels (0.125, 0.25, and 2.5 mg/kg) once a day for 6 days. At the two highest doses, FB1 increased the renal and hepatic N-demethylation of erythromycin (CYP3A1) and the hepatic O-deethylation of ethoxyresorufin (CYP1A1). FB1, at the highest dose of 2.5 mg/kg, also increased the renal O-deethylation of ethoxyresorufin. The liver, but not the kidney, was also susceptible to FB1-dependent induction of the 12- and 11-hydroxylation of lauric acid, suggesting induction of the CYP4A subfamily. Immunoblot studies employing solubilized microsomes from FB1-treated rats revealed that FB1, at the two highest doses, increased the apoprotein levels of CYP1A1 and CYP4A1. The same treatment with FB1 increased the beta-oxidation of palmitoyl-coenzyme A (CoA) in liver homogenates, and immunoblot analysis showed an increase in the apoprotein levels of the trans-2-enoyl-CoA hydratase trifunctional protein. The possible implications of these findings to the hepatocarcinogenicity of this mycotoxin are discussed.

Comitogenicity of eicosanoids and the peroxisome proliferator ciprofibrate in cultured rat hepatocytes

Several hypolipidemic drugs and environmental contaminants induce hepatic peroxisome proliferation and hepatic tumors when administered to rodents. These chemicals increase the expression of the peroxisomal β-oxidation pathway and the cytochrome P-450 4A family, which metabolize lipids, including eicosanoids and their precursor fatty acids. We previously found that the peroxisome proliferator ciprofibrate decreases the level of eicosanoids in the liver and in cultured hepatocytes. In this study, we examined the effect of prostaglandins E2 and F2α (PGE2 and PGF2α), leukotriene C4 (LTC4) and the peroxisome proliferator ciprofibrate on DNA synthesis in cultured hepatocytes. Primary rat hepatocytes were cultured on collagen gels in serum-free L-15 medium with varying concentrations of eicosanoids and ciprofibrate, and the absence or presence of growth factors. Ciprofibrate lowered hepatocyte eicosanoid concentrations; the addition of eicosanoids restored their levels. After a 48-h exposure with [3H]-thymidine, DNA synthesis was determined by measuring [3H]-thymidine incorporation into DNA. The addition of PGE2, PGF2α, and LTC4 to cultures along with ciprofibrate increased DNA synthesis, whereas treatment with ciprofibrate or eicosanoids alone resulted in a much smaller increase. The addition of epidermal growth factor (EGF) to the eicosanoid-ciprofibrate combination increased DNA synthesis more than EGF or the eicosanoid-ciprofibrate combination alone. The PGF2α-ciprofibrate combination also was comitogenic with transforming growth factor-α and hepatocyte growth factor. The addition of both ciprofibrate and prostaglandins also blocked the growth inhibitory effect of transforming growth factor-β on DNA synthesis induced by EGF. These results show that the eicosanoids PGE2, PGF2α, and LTC4 are comitogenic with the peroxisome proliferator ciprofibrate in cultured rat hepatocytes. © 1996 Wiley-Liss, Inc.

Comitogenicity of eicosanoids and the peroxisome proliferator ciprofibrate in cultured rat hepatocytes.

Several hypolipidemic drugs and environmental contaminants induce hepatic peroxisome proliferation and hepatic tumors when administered to rodents. These chemicals increase the expression of the peroxisomal beta-oxidation pathway and the cytochrome P-450 4A family, which metabolize lipids, including eicosanoids and their precursor fatty acids. We previously found that the peroxisome proliferator ciprofibrate decreases the level of eicosanoids in the liver and in cultured hepatocytes. In this study, we examined the effect of prostaglandins E2 and F2 alpha (PGE2 and PGF2 alpha), leukotriene C4 (LTC4) and the peroxisome proliferator ciprofibrate on DNA synthesis in cultured hepatocytes. Primary rat hepatocytes were cultured on collagen gels in serum-free L-15 medium with varying concentrations of eicosanoids and ciprofibrate, and the absence or presence of growth factors. Ciprofibrate lowered hepatocyte eicosanoid concentrations; the addition of eicosanoids restored their levels. After a 48-h exposure with [3H]-thymidine, DNA synthesis was determined by measuring [3H]-thymidine incorporation into DNA. The addition of PGE2, PGF2 alpha, and LTC4 to cultures along with ciprofibrate increased DNA synthesis, whereas treatment with ciprofibrate or eicosanoids alone resulted in a much smaller increase. The addition of epidermal growth factor (EGF) to the eicosanoid-ciprofibrate combination increased DNA synthesis more than EGF or the eicosanoid-ciprofibrate combination alone. The PGF2 alpha-ciprofibrate combination also was comitogenic with transforming growth factor-alpha and hepatocyte growth factor. The addition of both ciprofibrate and prostaglandins also blocked the growth inhibitory effect of transforming growth factor-beta on DNA synthesis induced by EGF. These results show that the eicosanoids PGE2, PGF2 alpha, and LTC4 are comitogenic with the peroxisome proliferator ciprofibrate in cultured rat hepatocytes.

Oncogenicity Testing of 2-Ethylhexanol in Fischer 344 Rats and B6C3F1 Mice

2-Ethylhexanol (2EH) is a weak nongenotoxic hepatic peroxisome proliferator in the rat. It is a high-volume chemical intermediate in the preparation of the plasticizers bis-(2-ethylhexyl) adipate (DEHA), bis-(2-ethylhexyl) phthalate (DEHP), and tris-(2-ethylhexyl) phosphate (TEHP), which are weak hepatocellular tumorigens in female mice. In consequence, the oncogenic potential of 2EH was evaluated in male (M) and female (F) rats and mice (50 animals/sex/group). Oral gavage doses of 2EH in 0.005% aqueous Cremophor EL (polyoxyl-35 castor oil) were given five times a week to rats: 0 (water), 0 (vehicle), 50, 150, and 500 mg/kg for 24 months, and to mice: 0 (water), 0 (vehicle), 50, 200, and 750 mg/kg for 18 months. Statistical comparisons of data were made between vehicle controls and treatment groups. There were no differences of biological significance between data from vehicle and water control groups. In rats, there were no dose-related changes at 50 mg/kg. There was reduced body weight gain at 150 mg/kg (M, 16; F, 12%) and 500 mg/kg (M, 33; F, 31%) and an increased incidence of lethargy and unkemptness. There were dose-related increases in relative liver, stomach, brain, kidney, and testis weights at sacrifice. Female rat mortality was markedly increased at 500 mg/kg. There was marked aspiration-induced bronchopneumonia in rats at 500 mg/kg; hematologic, gross, and microscopic changes, including tumors, were otherwise comparable among all rat groups. In mice at 50 and 200 mg/kg there were no dose-related changes and essentially no time-dependent or time-independent adverse trends in liver tumor incidence at the 5% significance level. At 750 mg/kg mouse body weight gain was reduced (M, 26; F, 24%), and mortality increased (M and F, 30%) versus vehicle controls. At 750 mg/kg there was a slight increase in nonneoplastic focal hyperplasia in the forestomach of mice (M 5/50, F 4/50) versus vehicle controls (M 1/50, F 1/50). There were increases in mouse relative liver (F, 21%) and stomach (M, 13%; F, 19%) weights at 750 mg/kg. There was a 12% incidence of hepatic basophilic foci and an 18% incidence of hepatocellular carcinomas in male mice at 750 mg/kg, not statistically significant compared with either control by Fisher's exact test. There was a 12% incidence of hepatic basophilic foci and a 10% incidence of hepatocellular carcinomas in female mice at 750 mg/kg, statistically significant (p< 0.05) compared with vehicle but not with water controls by Fisher's exact test. There were no metastases. Time-dependent and -independent statistical analyses showed an adverse trend in the incidence of hepatocellular carcinomas in male and female mice, correlated with toxicity (expressed as mortality) at 750 mg/kg. The time-adjusted incidence of hepatocellular carcinomas in male mice (18.8%) was within the historical normal range at the testing facility (0–22%), but that in females (13.1%) lay outside the normal range (0–2%). Under the conditions of these studies 2EH was not oncogenic in rats, but there were weak adverse trends in hepatocellular carcinoma incidence in mice at high dose levels which may have been associated with toxicity. The major effects of chronic dosing were mortality in female rats at 500 mg/kg and in male and female mice at 750 mg/kg, accompanied by reductions in body weight gain in rats at 150 and 500 mg/kg and in mice at 750 mg/kg. Direct comparison of any tumorogenic effects of 2EH given alone to female mice with those due to 2EH formedin vivofrom DEHA, DEHP, or TEHP is limited by the high mortality caused by 2EH in female mice at equivalent doses of 2EH. While 2EH may be a contributing factor in the hepatocellular carcinogenesis in female mice associated with the chronic administration of DEHA and DEHP, it is unlikely to be the entire proximate carcinogen.

Effect of the Peroxisome Proliferator, Ammonium Perfluorooctanoate (C8), on Hepatic Aromatase Activity in Adult Male Crl:CD BR (CD) Rats

The incidence of Leydig cell adenomas increases in CD rats fed for 2 years with the hepatic peroxisome proliferator, ammonium perfluorooctanoate (C8). Treatment with C8 increased the serum concentration of estradiol in 2-week gavage studies, and feeding studies at various time points up to 2 years, and was also accompanied by increases in liver weight and hepatic β-oxidation activity. Since peroxisome proliferators induce both hepatic β-oxidation and specific cytochrome P450 enzymes, C8 may also induce aromatase (cytochrome P450-19A1), the cytochrome P450 monooxygenase which converts androgens to estrogens. This hypothesis was investigated in the present study. Adult male CD rats were dosed daily by gavage for 14 days with 0, 0.2, 2, 20, or 40 mg C8/kg body wt. An additional group, the pair-fed control, was fed at a rate matched to the daily consumption by the 40 mg C8/kg group. Treatment with C8 produced a dose-dependent decrease in body weight, and increases in absolute and relative liver weights, and in the protein yield of hepatic microsomes. These C8-induced changes were associated with a 2-fold increase in the serum concentration of estradiol and up to a 16-fold increase in total hepatic aromatase activity. A significant linear correlation was established between serum estradiol and total hepatic aromatase activity. The absolute weights and the aromatase activity of the testes were not affected by C8. Hepatic peroxisomal β-oxidation activity and the microsomal concentration of total cytochrome P450 were also increased by C8. A comparison of estimated EC50 values suggested that these parameters may be less sensitive to induction by C8 than hepatic aromatase activity. Co-incubation of control liver microsomes with C8 in the aromatase assay for 2 hr dose dependently reduced the apparent aromatase activity. This inhibition of aromatasein vitrobut increasein vivowas further investigated using cultured rat hepatocytes. Decreases in aromatase activity were found after up to 42 hr of treatment with C8, but the enzyme activity was increased almost 2-fold after 66 hr. The results of this study suggest that the increased serum concentration of estradiol produced by C8 in rats is at least partly due to a direct effect on the liver to increase synthesis of estradiol through induction of aromatase cytochrome P450 in the endoplasmic reticulum.

The Direct Effect of Hepatic Peroxisome Proliferators on Rat Leydig Cell Functionin Vitro

A review of the literature indicates that some compounds which produce hepatic peroxisome proliferation in rats also appear to produce Leydig cell adenomas, and some also affect the serum concentrations of testosterone and estradiol. Previous studies with the peroxisome proliferator ammonium perfluorooctanoate showed a direct effect on Leydig cells to alter steroidogenesis. It was therefore proposed that peroxisome proliferators in general may directly affect Leydig cell function to produce Leydig cell tumors by some undetermined mechanism. The present study investigated whether the following peroxisome proliferators directly affect Leydig cell functionin vitro:2,4-dichlorophenoxyacetic acid, ammonium perfluorooctanoate, acetylsalicylic acid, clofibric acid, ciprofibrate, gemfibrozil, tiadenol, tibric acid, trichloroacetic acid, trichloroethylene, and Wyeth 14,643. Leydig cells, isolated from adult Crl:CDBR rats (12–16 weeks old), were treated with peroxisome proliferator for 21 hr and the medium was assayed for estradiol. The function of the treated Leydig cell was evaluated by measuring the release of testosterone in response to human chorionic gonadotropin (hCG). In general, the peroxisome proliferators reduced the hCG-stimulated release of testosterone and either reduced or had no effect on the baseline release of testosterone. Of the 11 peroxisome proliferators, 8 increased the release of estradiol from Leydig cells treated for 1 day. Two more compounds were found to increase estradiol production when the treatment period was extended to 2 days. These effects were seen at noncytotoxic doses and at concentrations similar to those achieved in rat serum in dietary studies. The results suggest that peroxisome proliferators, as a class of compounds, directly modify the steroidogenic function of Leydig cellsin vitro.Some of these compounds are known to produce Leydig cell tumors in rats, but this association has yet to be established for other peroxisome proliferators. This suggests that compounds which directly affect Leydig cell functionin vitromay also induce Leydig cell tumorsin vivo.Further investigations are necessary to address the mechanism for thein vitroeffects on Leydig cells and to clarify the apparent relationship between peroxisome proliferator-induced changes in Leydig cell function and the development of Leydig cell tumors.

Hepatic peroxisome proliferation in vitamin A-deficient mice without a simultaneous increase in peroxisomal Acyl-CoA oxidase activity

Vitamin A-adequate and vitamin A-deficient C57B1/6 mice were treated for ten days with 0.02% (w/w) perfluorooctanoic acid (PFOA) in their diet. Treated vitamin A-adequate and -deficient mice demonstrated approximately the same increases in liver somatic index (g liver/g body weight) (somewhat more than 2-fold) and mitochondrial protein content (5-fold). PFOA treatment resulted in a 26-fold increase in hepatic peroxisomal lauroyl-CoA oxidase activity in vitamin A-adequate mice, whereas the same activity was unchanged in vitamin A-deficient mice. Vitamin A deficiency itself caused a 3- to 4-fold increase in cytosolic catalase activity and a smaller increase in the activity of microsomal cytochrome P-450 IVA (lauric acid ω- and ω-1 hydroxylase) in this same organ. The induction of the activities of these enzymes was less prominent in vitamin A-deficient mice compared with the effect caused by PFOA in vitamin A-adequate mice, resulting in approximately the same maximal values for these parameters in both groups (i.e. approx. 21 mmol/g liver · min and 350 nmol/g liver · min, respectively). A 70 kDa protein, presumably the multifunctional protein, was shown by Commassie blue staining of SDS-polyacrylamide gels and by immunoblotting (with antibodies towards the multifunctional protein) to be induced to approximately the same degree in vitamin A-adequate and -deficient mice. A morphometric study revealed that PFOA causes the same extent of hepatic peroxisome proliferation in vitamin A-deficient as in vitamin A-adequate mice. The possibility that PFOA exerts its effect in vivo through at least two different mechanisms is discussed.

Effect of the Peroxisome Proliferator, Ammonium Perfluorooctanoate (C8), on Hepatic Aromatase Activity in Adult Male CrI:CD BR (CD) Rats

The incidence of Leydig cell adenomas increases in CD rats fed for 2 years with the hepatic peroxisome proliferator, ammonium perfluorooctanoate (C8). Treatment with C8 increased the serum concentration of estradiol in 2-week gavage studies, and feeding studies at various time points up to 2 years, and was also accompanied by increases in liver weight and hepatic β-oxidation activity. Since peroxisome proliferators induce both hepatic β-oxidation and specific cytochrome P450 enzymes, C8 may also induce aromatase (cytochrome P450-19A1), the cytochrome P450 mono oxygenase which converts androgens to estrogens. This hypothesis was investigated in the present study. Adult male CD rats were dosed daily by gavage for 14 days with 0, 0.2, 2, 20, or 40 mg C8/kg body wt. An additional group, the pair-fed control, was fed at a rate matched to the daily consumption by the 40 mg C8/kg group. Treatment with C8 produced a dose-dependent decrease in body weight, and increases in absolute and relative liver weights, and in the protein yield of hepatic microsomes. These C8-induced changes were associated with a 2-fold increase in the serum concentration of estradiol and up to a 16-fold increase in total hepatic aromatase activity. A significant linear correlation was established between serum estradiol and total hepatic aromatase activity. The absolute weights and the aromatase activity of the testes were not affected by C8. Hepatic peroxisomal β-oxidation activity and the microsomal concentration of total cytochrome P450 were also increased by C8. A comparison of estimated EC50 values suggested that these parameters may be less sensitive to induction by C8 than hepatic aromatase activity. Co-incubation of control liver microsomes with C8 in the aromatase assay for 2 hr dose dependently reduced the apparent aromatase activity. This inhibition of aromatase in vitro but increase in vivo was further investigated using cultured rat hepatocytes. Decreases in aromatase activity were found after up to 42 hr of treatment with C8, but the enzyme activity was increased almost 2-fold after 66 hr. The results of this study suggest that the increased serum concentration of estradiol produced by C8 in rats is at least partly due to a direct effect on the liver to increase synthesis of estradiol through induction of aromatase cytochrome P450 in the endoplasmic reticulum.

Induction of peroxisomal beta-oxidation and P-450 4A-dependent activities by pivalic and trichloroacetic acid in rat liver and kidney.

The influence of pivalic acid (PIV), a compound often used to make pro-drugs, and of the structurally related trichloroacetic acid (TCA), on several hepatic and renal enzymes was investigated in Sprague-Dawley rats, following a 4-day treatment period. The PIV and TCA treatments resulted in a similar and selective induction (2-3 times) of peroxisomal palmitoyl-CoA oxidase and the cytochrome P-450 4A dependent microsomal (omega)- and (omega-1)-lauric acid activities, both in liver and kidney. Western blot analysis of liver and kidney microsomes from PIV- and TCA-treated rats, using antibody to the P-450 4A1, revealed induction of members of the P-450 4A subfamily. These results suggest that PIV, like TCA, is a renal and hepatic peroxisome proliferator in rats, and further support the previously indicated close association between the peroxisomal fatty acid beta-oxidation enzymes and microsomal P-450 4A sub-family enzymes.

Gemfibrozil-induced peroxisome proliferation and hepatomegaly in male F344 rats.

Gemfibrozil is a widely used hypolipidemic drug in humans that causes peroxisome proliferation and hepatocarcinogenesis in rodents. The induction of hepatomegaly and hepatic peroxisome proliferation (measured as peroxisomal acyl CoA oxidase activity), was determined and compared to another peroxisome proliferator, WY-14,643 (0.1% in the diet) in male F344 rats. In a 21-day study, dietary no-observable-effect and lowest-observable-effect levels of gemfibrozil for both hepatomegaly and peroxisome proliferation were 0.002% and 0.005%, respectively. In a 42-day study, dietary concentrations of 0.9-2.0% gemfibrozil induced a similar magnitude of hepatomegaly to WY-14,643 (2.3-fold) but a higher level of peroxisome proliferation (16-18-fold) than the maximum induction for WY-14,643 (13-fold). The plateau in magnitude of gemfibrozil-induced peroxisome proliferation across the 0.9-2.0% dietary concentrations was associated with a plateau in serum concentration of gemfibrozil (approximately 20 micrograms/ml), similar to concentrations reported in human subjects receiving oral gemfibrozil. These results indicate that maximal induction of peroxisome proliferation by gemfibrozil can exceed that of a more potent compound such as WY-14,643, and further suggest that maximal induction of peroxisome proliferation can be limited by steady-state serum concentrations. Moreover, the reported lack of hepatic responses to gemfibrozil in humans is unlikely to be the result of inefficacy or unavailability of this drug, compared to other peroxisome proliferators, in rodents.

The Herbicide Dicamba (2-Methoxy-3,6-dichlorobenzoic Acid) Is a Peroxisome Proliferator in Rats

The Herbicide Dicamba (2-Methoxy-3,6-dichlorobenzoic Acid) Is a Peroxisome Proliferator in Rats. Espandiari, P., Thomas, V, A., Glauert, H. P., O'Brien, M., Noonan, D., and Robertson, L. W. (1995). Fundam. Appl. Toxicol. 26, 85-90.The widely used broad leaf herbicide, dicamba, or Banvel, is similar in structure to xenobiotics which induce hepatic drug metabolism or proliferation of hepatic peroxisomes in rodents. The ability of xenobiotics to effect these hepatic changes often portends their positive outcomes in chronic bioassays. Dicamba's ability to induce hepatomegaly and peroxisome proliferation was studied in male and female Sprague-Dawley rats. Rats were placed on feed containing 0, 0.001, 0.01, 0.1, or 1% dicamba or 0.01% ciprofibrate for 3 weeks. Dicamba had no effect on relative liver weights or feed efficiency in either female or male rats at all doses tested. Dicamba, however, caused a statistically significant increase in peroxisomal β-oxidation activity in liver homogenates from rats of both sexes fed 1% dicamba. Fatty acyl CoA-oxidase activity was increased in male rats fed 1% dicamba. A protein of Mr 80 kDa was visible when liver homogenates of female or male rats fed 1% dicamba were subjected to SDS-PAGE. Lauric acid hydroxylase activity and CYP4A-reactive protein were increased in microsomes from male rats fed the highest level of dicamba. Moreover, dicamba was observed to transcriptionally upregulate the peroxisome proliferator-activated receptor (PPAR), a peroxisome prolifexator sensitive receptor previously shown to be linked to the transcriptional regulation of a variety of peroxisome-specific enzymes. These studies show that dicamba is a peroxisome proliferator in rats. Although dicamba was not an efficacious inducer of peroxisomal enzymes in these rats, dicamba's ability to transcriptionally activate the PPAR and induce peroxisomal and related enzymes must be considered in the safety evaluation of this herbicide.

Pyruvate inhibits clofibrate-induced hepatic peroxisomal proliferation and free radical production in rats

In an effort to identify the effects of the 3-carbon compound pyruvate on free radical production, we measured hepatic total peroxisomal beta-oxidation and catalase activity and the production of lipofuscin-like products in male Sprague-Dawley rats consuming an adequate diet supplemented with pyruvate, vitamin E, or the peroxisome proliferator and free radical enhancer clofibrate for 22 days (n = 5 in each group). Clofibrate feeding induced hepatomegaly, a fivefold increase in total peroxisomal beta-oxidation activity, and a threefold increase in hepatic lipofuscin-like products (P < .05). Pyruvate but not vitamin E inhibited the increase in liver size by 70% (P < .05). Both pyruvate and vitamin E completely inhibited clofibrate-induced increases in lipofuscin-like products (P < .05). Pyruvate but not clofibrate or vitamin E increased plasma concentrations of the nitric oxide metabolites nitrite and nitrate (P < .05). We conclude that with clofibrate-induced peroxisomal proliferation and free radical production, pyruvate will inhibit peroxisomal proliferation and free radical production, inhibit free radical-induced lipid peroxidation, and enhance metabolism of nitric oxide.

Histochemistry of Peroxisomes: Overview.

Peroxisomes are present in virtually all eukaryotic cells and known to contain more than fifty enzymes. Their size, number and composition of enzymes vary considerably between different species and between different cell types within the same species. During the past two decades, there has been an explosion in the amount of information about their biogenesis, peroxisomal targeting signals, the role of these organelles in lipid metabolism, in the synthesis of cholesterol, bile acids and glycerolipids, and in the degradation of uric acid. The importance of peroxisomes in mammalian metabolism is underscored by the discovery of carcinogenic peroxisomes proliferators and by the occurrence of peroxisomal genetic diseases. Of considerable importance is the implication of induction of hepatic peroxisome proliferation and the peroxisomal fatty acid β-oxidation enzyme system in the development of hepatocellular carcinomas in rats and mice by structurally diverse peroxisome proliferators. The immunocytochemical and in situ hybridization procedures are extremely valuable in identifying qualitative and quantitative changes of specific enzymes in animals exposed to peroxisome proliferators. Peroxisome proliferators appear to exert their pleiotropic responses by activating a receptor known as peroxisome proliferatoractivated receptor. An understanding of the ontogeny of this receptor in relation to the expression of peroxisomal genes is an important consideration in assessing the implications of peroxisome proliferatorinduced toxicity and the role of peroxisomes in genetic diseases.

Evaluation of the Genetic Toxicity of the Peroxisome Proliferator and Carcinogen Methyl Clofenapate, Including Assays Usin Muta TM Mouse and Big Blue™ Transgenic Mice

The rodent liver carcinogen and hepatic peroxisome proliferator methylclofenapate (MCP) has been evaluated for genetic toxicity in a range of in vitro and rodent genotoxicity assays. It gave a negative response in each of the following assays: mutagenicity to S. typhimurium and E. coli (+/- S9 mix, plate and pre-incubation assays), clastogenicity to cultured human lymphocytes and CHO cells (+/- S9 mix), a mouse bone marrow micronucleus assay (24h and 48h sampling), a rat liver assay for UDS in vivo (12h sampling), assays for lac I (Big Blue) and lac Z (Muta Mouse) mutations in the liver of transgenic mice, and an assay of the ability of MCP to modify the mutagenicity to the liver of dimethylnitrosamine in both transgenic mutation assays. The micronucleus and UDS assays were conducted using a single administration of MCP at its maximum tolerated dose, while the transgenic assays were conducted using nine daily administrations of MCP at its cancer bioassay dose level. These nine daily administrations were shown to double the weight of the liver of non-transgenic, Big Blue and Muta Mice, as well as leading to a dramatic proliferation of peroxisomes (electron microscopy) in the livers of each strain. These changed parameters had returned to control levels when the mutation analyses were conducted (10 days after the final dose of MCP). Despite the liver enlargement observed following MCP administration, no evidence of mitotic activity was observed in treated livers, although an increased number of cells were undergoing replicative DNA synthesis during the final 3 days of the 9 days of administration (BUdR assessment of S-phase). Liver biochemistry parameters (ALT, AST, AP, CK, GGT and albumin) were unaffected by the chronic (9 day) administration of MCP indicating an absence of hepatic toxicity. These combined observations favour a non-genotoxic mechanism of action for the hepatic carcinogenicity of MCP. The clastogenicity in vitro of the perixisome proliferator Wyeth 14,643 has been confirmed in CHO cells, but it is noted that this chemical is more soluble than is MCP. In particular, at the highest dose level at which MCP could be tested, Wy 14,643 was also non-clastogenic.

Safety evaluations of food chemicals by “compact” 1. A study of some acyclic terpenes

A group of 19 acyclic terpenes have been evaluated for potential toxicity/carcinogenicity by molecular orbital determinations of their spatial and electronic parameters, and hence prediction of their metabolic activation or detoxication by the cytochrome P-450 (CYP) superfamily of mixed-function oxidase enzymes. Previous studies have characterized the spatial dimensions of the CYP1A1, 1A2 and 2E1 enzymes, which are known to activate mutagens and carcinogens and to be involved in other mechanisms of toxicity. None of the terpenes was found to have shape or electronic parameters appropriate for metabolic activation by CYP1A1 or 1A2, and hence they are unlikely to be carcinogenic or mutagenic. Furthermore, none of these chemicals had spatial parameters critical for substrates of CYP2E, and they are therefore unlikely to induce the formation of reactive oxygen species (ROS) or to initiate or promote malignancy or toxicity by mechanisms involving ROS. However, citral, and others of these terpenes, are known to undergo metabolism to carboxylic acids that may induce CYP4, and are therefore possible inducers of hepatic peroxisomal proliferation at high dosage, which may have implications for possible hepatotoxicity.

The Relevance of Hepatic Peroxisome Proliferation in Rats to Assessment of Human Carcinogenic Risk for Pharmaceuticals

There exists a strong association between the ability of drugs and other chemicals to induce hepatic peroxisomes in rodents and to increase the frequency of liver tumors in these same species. Despite several years of intensive investigation, a plausible mechanism causally relating peroxisome induction to tumor formation has not been found. Two major theories of how such compounds produce tumors - prolonged oxidative stress and cellular growth dysregulation - have limited experimental support. The oxidative stress demonstrated in rodents as a consequence of peroxisomal activity may be irrelevant to man since primates appear to be much less susceptible to peroxisome proliferation than are rats or mice. Cellular growth dysregulation and other effects of test materials that often accompany - but are not directly attributable to - peroxisome proliferation in rodents can be assessed in other, more relevant species if a causal relationship between their presence and tumor development is ultimately shown in rodents. Currently, there is no reason to specifically avoid the clinical assessment and commercial development of new therapeutic drugs that induce peroxisomes in rodents if other measurements indicate the absence of DNA damage or growth dysregulation at reasonable exposures in relevant species.

Modification of Hepatic Immunoglobulin Heavy Chain Binding Protein (BiP/Grp78) Following Exposure to Structurally Diverse Peroxisome Proliferators

Modification of Hepatic Immunoglobulin Heavy Chain Binding Protein (BiP/Grp78) Following Exposure to Structurally Diverse Peroxisome Proliferators. Witzmann, F. A., Parker, D. N., Jarnot, B. M., and Clack, J. W. (1994). Fundam. Appl. Toxicol. 23, 1-8.This investigation was conducted to determine the comparative effect of structurally diverse peroxisome proliferators (PP) on the two-dimensional protein pattern of rat liver whole homogenates. Perfluoro-n-decanoic acid (PFDA), perfluoro-n-octanoic acid (PFOA), clofibrate, and di(2-ethylhexyl)phthalate (DEHP) are all known to cause the proliferation of hepatic peroxisomes and the induction of peroxisomal β-oxidative and microsomal ω-oxidative enzymes. To clarify the mechanistic differences between these compounds with regard to the liver, we examined the unique patterns of protein alteration produced by in vivo exposure to them. Following exposure to various doses, whole liver homogenates were prepared and separated by two-dimensional gel electrophoresis (2DE) using the ISO-DALT system. Stained gels were digitized and protein patterns analyzed using the Kepler 2D gel analysis system. Immunoglobulin heavy chain binding protein (BiP), also known as 78-kDa glucose-regulated protein (Grp78), was identified immunologically and by comigration of recombinant Grp78. BiP is a luminal endoplasmic reticular protein that functions in the assembly and folding of nascent proteins as they enter the ER. The present results suggest a selective posttranslational modification of BiP following PFDA exposure. Single-dose exposure to PFDA was associated with a notable charge modification of BiP that persists up to 30 days. PFOA, clofibrate, and DEHP had less effect in this regard. The identity of BiP/Grp78 as the halothane hepatitis-associated trifluoroacetylated protein was also demonstrated. The nature of this PFDA-associated protein modification (reactive metabolite conjugation, abnormal ribosylation, or phosphorylation) is currently under investigation. These results document PFDA's unique toxicity as a PP and support the utility of 2D gel analysis in toxicity testing.