Cytochrome P450IVA1 Research Articles

Conjugated linoleic acid is a potent naturally occurring ligand and activator of PPARα

We have previously shown that a mixture of dietary conjugated derivatives of linoleic acid (conjugated linoleic acid, CLA) induces peroxisome proliferator-responsive enzymes and modulates hepatic lipid metabolism in vivo. The present studies demonstrate that CLA is a high affinity ligand and activator of peroxisome proliferator-activated receptor α (PPARα) and induces accumulation of PPAR-responsive mRNAs in a rat hepatoma cell line. Using a scintillation proximity assay (SPA), CLA isomers were shown to be ligands for human PPARα with a rank order of potency of (9Z,11E)>(10E,12Z)>(9E,11E)> furan-CLA (IC50 values from 140 nm to 400 nm). Levels of acyl-CoA oxidase (ACO), liver fatty acid-binding protein (L-FABP), and cytochrome P450IVA1 (CYP4A1) mRNA were induced by CLA in FaO hepatoma cells. Even though linoleate and CLA were incorporated into lipids of hepatoma cells to the same extent, linoleate had little or no effect on ACO, CYP4A1, or L-FABP mRNA. In agreement with its binding potency, (9Z,11E)-CLA was the most efficacious PPARα activator in the mouse PPARα-GAL4(UAS)5-CAT reporter system.▪These data indicate that CLA is a ligand and activator of PPARα and its effects on lipid metabolism may be attributed to transcriptional events associated with this nuclear receptor. Also, (9Z,11E)-CLA is one of the most avid fatty acids yet described as a PPARα ligand.—Moya-Camarena, S. Y., J. P. Vanden Heuvel, S. G. Blanchard, L. A. Leesnitzer, and M. A. Belury. Conjugated linoleic acid is a potent naturally occurring ligand and activator of PPARα. J. Lipid Res. 1999. 40: 1426–1433.

Comparison of Dose–Response Relationships for Induction of Lipid Metabolizing and Growth Regulatory Genes by Peroxisome Proliferators in Rat Liver

The regulation of gene expression via the peroxisome proliferator-activated receptor (PPAR) is believed to be critical in the effects of peroxisome proliferators on lipid metabolism and possibly in hepatocarcinogenesis. The involvement of PPAR in the peroxisome proliferator-mediated induction of fatty acid metabolizing genes such as acyl-CoA oxidase (ACO), fatty acid-binding protein (FABP), and cytochrome P450IVA1 (CYP4A1) has been clearly demonstrated. However, the induction by peroxisome proliferators of important growth regulatory genes such asc-mychas not been investigated extensively. In these studies we examined the dose–response relationships for the induction of mRNA for the PPAR-regulated and lipid metabolizing genesACO, FABP,andCYP4A1and compared them to the immediate early genec-myc.Liver mRNA from rats fed various amounts of the peroxisome proliferator Wy14,643 for 13 weeks was utilized. The lipid metabolism and growth regulatory genes were induced by subchronic administration of Wy14,643 but to varying degrees and with different sensitivities. The lowest dose that resulted in a significant change in ACO and FABP expression was 10 ppm. The mRNA for CYP4A1 and c-myc was significantly affected at the lowest dose examined (5 ppm). Also, the maximal induction ranged from 105-fold (CYP4A1) to less than 10-fold (FABP) relative to vehicle-treated animals. The accumulation of mRNA for ACO, FABP, and CYP4A1, but not c-myc, showed typical receptor-mediated dose–response relationships. The effects on gene expression were compared to rates of hepatic cell proliferation, a pertinent marker of tumor promotion and hepatocarcinogenesis. Surprisingly, ACO mRNA showed an excellent correlation (r2= 0.9) while c-myc mRNA exhibited a poor correlation (r2= 0.3) with cell proliferation in rat liver. Although the differences between the dose–response relationships of ACO and c-myc mRNA accumulation may suggest immediate early genes are not controlled by PPAR, evidence from PPARα null mice support this receptor in both lipid metabolism and growth regulatory genes. This study shows the complexity of responses mediated by peroxisome proliferators, with ACO being a good marker of PPAR-mediated events as well as cell proliferation, while c-myc, a known growth regulatory gene, was induced by Wy14,643 partially via PPAR but did not correlate well with cell proliferation.

Carcinogenic and anticarcinogenic effects of dehydroepiandrosterone in the liver of male and female rats

Dehydroepiandrosterone (DHEA), an adrenal steroid in humans, shows a certain anticarcinogenic effect in rat liver when it is administered simultaneously with strong carcinogens such as N-nitrosomorpholine (NNM). On the other hand, DHEA is an hepatocarcinogen in the rat when administered at high doses for more than 18 months. The hepatocellular tumors arise from amphophilic cell foci and exhibit a well-differentiated pheno-type. Wlien administered subsequent to NNM, DHEA acts as a tumor promoter in rat liver. Preneoplastic lesions of the glycogenotic/basophilic cell lineage induced by NNM are modulated to amphophilic lesions by DHEA. Female animals are more sensitive than males in respect of the carcinogenic and tumor-enhancing effect of DHEA. The higher sensitivity in females may be due to higher blood levels of DHEA andlor different metabolism and elimination of the drug, as compared to males. DHEA markedly induces cytochrome P450IVA, particularly in the liver of male rats. This effect may explain the rapid metabolism and elimination of the substance and the lower carcinogenicity in males. Both preneoplastic amphophilic foci and neoplasms are characterized by a strong proliferation of mitochondria. DHEA also induces peroxisome proliferation and susceptibility to lipid peroxidation in both genders. Furthermore, DHEA causes a reduction in activity and expression of key enzymes of carbohydrate metabolism and of glycogen content in the liver, which may be related to modulation of hepatocarcinogenesis.

Hepatic zonation of the induction of cytochrome P450 IVA, peroxisomal lipid beta-oxidation enzymes and peroxisome proliferation in rats treated with dehydroepiandrosterone (DHEA). Evidence of distinct zonal and sex-specific differences.

Dehydroepiandrosterone (DHEA) is an intermediate product in the synthesis of male and female sex hormones in the adrenal cortex of man. In livers of rats and mice DHEA increases the levels of cytochrome P450 IVA and peroxisomal beta-oxidation enzymes associated with peroxisome proliferation. Prolonged treatment of rats with DHEA induces liver tumors that are more frequent in females arising mainly in the periportal regions of the liver lobule (Metzger et al., Toxicol. Pathol. 23, 591-605, 1995). Because of paucity of information on hepatic zonation of peroxisomal response to DHEA and controversial reports on gender-specific differences of its effects the present study was undertaken using qualitative immunohistochemical and quantitative immunoelectron microscopical techniques in addition to Western blotting. Rats were treated for 24 weeks with 0.6% DHEA supplied with diet. Immunoblot analysis revealed marked induction of peroxisomal beta-oxidation enzymes, which by quantitative analysis was equally strong in male and female animals, whilst catalase and urate-oxidase were not increased. Cytochrome P450 IVA, in contrast, was induced significantly stronger in male than in female rats. Immunohistochemistry confirmed the induction of cytochrome P450 IVA showing a marked lobular gradient in female animals with strong induction in pericentral and almost no induction in periportal regions of the liver lobule. In male animals cytochrome P450 IVA was expressed more uniformly across the liver lobule. A similar sex specific zone-dependent response was observed for peroxisomes. DHEA induced in females a significant zonal gradient with marked peroxisome proliferation and a strong induction of peroxisomal hydratase/dehydrogenase in pericentral hepatocytes and a much smaller response in periportal regions. Livers of male animals, in contrast, showed a uniform peroxisomal proliferation to DHEA with only slight zonal differences. The striking homologies of the induction patterns of cytochrome P450 IVA and the peroxisome proliferation in both sexes support the notion of a functional relationship. In view of the almost exclusive periportal localization of DHEA-induced tumors in female rats in contrast to the pericentral localization of the peroxisomal proliferation shown by this study, it seems likely that other factors in addition to peroxisome proliferation may contribute to the hepatocarcinogenic effect of DHEA.

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.

Synergistic induction of acyl-CoA oxidase activity, an indicator of peroxisome proliferation, by arachidonic acid and retinoic acid in Morris hepatoma 7800C1 cells

Morris hepatoma 7800C1 cells (a Wistar rat cell line) were exposed to 100 μM arachidonic acid in the medium for seven days. This treatment resulted in 150% and 60% increases (above control activities) in acyl-CoA oxidase (which catalyzes the first step in peroxisomal β-oxidation) and catalase activities, respectively. Arachidonic acid (C20:4) can be metabolized to 20- and 19-hydroxyarachidonic acid by cytochrome P-450IVA and it was shown that our cells are capable of forming 20-hydroxyarachidonic acid. However, 20-hydroxyarachidonic acid (0.l–0.8 μM, 4 days) had no effects on lauroyl-CoA oxidase and catalase activities in Morris hepatoma cells. Treatment of 7800C1 cells with 100 μM all- trans-retinoic acid resulted in inductions of catalase (160% above the control activity) and carnitine acetyltransferase (140% above the control activity) activities. The activity of lauroyl-CoA oxidase was often, but not always, slightly induced by treatment with all- trans-retinoic acid. When all- trans-retinoic acid was administered together with arachidonic acid, these two compounds had a synergistic effect on the induction of acyl-CoA oxidase activity (almost 700% above the control activity). However, treatment of Morris hepatoma cells with the man-made peroxisome proliferator, perfluorooctanoic acid, together with all- trans-retinoic acid did not result in any synergistic effect on this same enzyme activity. In summary, this study (1) corroborates findings from transfection experiments indicating that the heterodimer PPAR-RXR α activates transcription of the acyl-CoA oxidase gene using the Morris hepatoma cell line; (2) shows that arachidonic acid induces the activity of lauroyl-CoA oxidase; (3) suggests that transcription of the catalase gene is not regulated by a PPAR-RXR α heterodimer in this system; and (4) demonstrates that peroxisome proliferation in Morris hepatoma cells by perfluorooctanoic acid is not as dependent on the level of retinoic caused by arachidonic acid.

Effect of 70% partial hepatectomy on developmental expression of cytochrome P450 IVA1 within intrasplenically transplanted fetal hepatocytes from spontaneously hypertensive rats

Fetal hepatocytes were harvested at 20 days of gestation from spontaneously hypertensive rats (SHR) and then transplanted into recipient adult SHR spleens. Morphological examination of the recipient spleens revealed that after 4 weeks, large masses of hepatocytes were present in the red pulp, as well as cord-like structures. Immunochemical determinations revealed that cytochrome P450 IVA1 (cytochrome P450 LAω) could be detected without any prior induction. Cytochrome P450 LAω was detected in fetal hepatocyte microsomes from the spleens 6 weeks after fetal hepatocyte transplantation. Cytochrome P450 LAω and peroxisomal acyl CoA oxidase were expressed 6 weeks after transplantation in a 70% hepatectomized host. These results demonstrate that fetal hepatocytes can be successfully transplanted to the spleens of recipient hosts, and that fetal hepatocytes appear to grow and develop functional cytochrome P450 metabolizing systems.

Determination of phthalate-induced rat liver cytochrome P-450 IVA1 by a fluorimetric enzymatic assay and by chemiluminescence detection on Western blots

An enzymatic assay for cytochrome P-450 IVA1 as determined by the formation of 12-hydroxylauric acid from lauric acid, using liquid chromatography with fluorescence detection has been compared with a commercial Western blot kit for this protein using enzyme enhanced chemiluminescence detection. The comparison was performed with samples of liver homogenates of rats exposed to the plasticizer di(ethylhexyl)phthalate (DEHP). Both methods showed sufficient sensitivity to detect the protein in control animals. The comparison resulted in a good correlation between both methods with a correlation coefficient of 0.96. The resulting no-effect levels for DEHP were also found to be the same. It is concluded that although the enzymatic method has a better reproducibility and dynamic range, both methods can be used for the determination of the cytochrome P-450 IVA1 in rat liver homogenates as an indicator of peroxisome proliferation.

Effects of perfluorooctanoic acid — a potent peroxisome proliferator in rat — on Morris hepatoma 7800C1 cells, a rat cell line

In this study, Morris hepatoma 7800C1 cells (from rat) were exposed to 500 μM perfluorooctanoic acid (PFOA) in the culture medium for 7 days. This treatment resulted in inductions of catalase, lauroyl-CoA oxidase (which catalyzes the first step in peroxisomal β-oxidation) and of cytochrome P-450IVA (specialized for omega-and omega-1 hydroxylation of fatty acids). Northern blot analysis revealed that the level of mRNA for peroxisomal fatty acyl-CoA oxidase was enhanced in cells treated with PFOA. Inductions of the enzymes mentioned above are generally connected with peroxisome proliferation in vivo. This work also includes a comparison between the activities of catalase, lauroyl-CoA oxidase, DT-diaphorase and glutathione transferase in rat liver homogenate and 7800C1 cells in order to investigate to what extent this cell line differs from the situation in vivo. The findings suggest that the cells selectively lost most of their peroxisomes during transformation into a cell line and subsequent propagation. The control activities of catalase and lauroyl-CoA oxidase (marker enzymes for peroxisomes) were only about 2% of the corresponding enzyme activities in rat liver. In addition, a morphological study revealed that the frequency of peroxisomes in 7800C1 cells is very low. The control activity of glutathione transferase in 7800C1 cells was 11% of the corresponding activity in rat liver homogenate, whereas the level of DT-diaphorase was virtually the same in 7800C1 cells as in rat liver. Electron microscopic investigation of the control cultures revealed all signs of viable cells, with well-developed cell organelles. Treatment of 7800C1 cells with 500 μM PFOA has little effect on cellular morphology.

Peroxisome proliferator-activated receptors: finding the orphan a home

The peroxisome proliferator-activated receptor (PPAR) is a member of the steroid hormone receptor superfamily and is activated by a variety of fibrate hypolipidaemic drugs and non-genotoxic rodent hepatocarcinogens that are collectively termed peroxisome proliferators. A key marker of peroxisome proliferator action is the peroxisomal enzyme acyl CoA oxidase, which is elevated about ten fold in the livers of treated rodents. Additional peroxisome proliferator responsive genes include other peroxisomal β-oxidation enzymes and members of the cytochrome P450 IVA family. A peroxisome proliferator response element (PPRE), consisting of an almost perfect direct repeat of the sequence TGACCT spaced by a single base pair, has been identified in the upstream regulatory sequences of each of these genes. The retinoid X receptor (RXR) forms a heterodimer with PPAR and binds to the PPRE. Furthermore, the RXR ligand, 9-cis retinoic acid, enhances PPAR action. Retinoids may therefore modulate the action of peroxisome proliferators and PPAR may interfere with retinoid action, perhaps providing one mechanism to explain the toxicity of peroxisome proliferators. Interestingly, a variety of fatty acids can activate PPAR supporting the suggestion that fatty acids, or their acyl CoA derivatives, may be the natural ligands of PPAR and that the physiological role of PPAR is to regulate fatty acid homeostasis. Taken together, the discovery of PPAR has opened up new opportunities in understanding how lipid homeostasis is regulated, how the fibrate hypolipidaemic drugs may act and should lead to improvements in the assessment of human risk from peroxisome proliferators based upon a better understanding of their mechanism of action.

Induction of peroxisomal fatty acid beta-oxidation and liver fatty acid-binding protein by peroxisome proliferators. Mediation via the cytochrome P-450IVA1 omega-hydroxylase pathway

Both the enzymes of peroxisomal fatty acid beta-oxidation and the liver fatty acid-binding protein (L-FABP) are induced in the liver by peroxisome proliferators, such as clofibrate (CF), as well as high fat diets. One proposed mechanism for this induction is that it represents an adaptive response to altered intracellular fatty acid fluxes, mediated by dicarboxylic fatty acids formed via the cytochrome P-450IVA1 omega-oxidation pathway. The studies presented in this paper were designed to investigate the role of the products of P-450IVA1 omega-oxidation in the regulation of peroxisomal beta-oxidation and L-FABP. In primary hepatocyte cultures exposed to CF, the increase in P-450IVA1 activity preceded the induction of peroxisomal beta-oxidation and L-FABP. The CF-mediated increases in peroxisomal beta-oxidation and L-FABP, but not P-450IVA1, could be significantly inhibited pretranslationally by concurrent exposure of cultured hepatocytes to inactivators of cytochromes P-450, such as 1-aminobenzotriazole and 10-undecynoic acid. Hexadecanedioic acid, a 16-carbon dicarboxylic fatty acid, that is poorly metabolized in hepatocytes, induced peroxisomal beta-oxidation and L-FABP, but not P-450IVA1, via a pretranslational mechanism that was not inhibited by 1-aminobenzotriazole. Long-chain monocarboxylic acids were without such inducing effect. In further studies, non-beta-oxidizable dicarboxylic acid analogs were found to display greater potency as inducers of peroxisomal beta-oxidation when compared to hexadecanedioic acid. The inducing effects of the dicarboxylic acid analogs were also independent of the P-450 omega-oxidation pathway. The results of these studies suggest that the regulation of peroxisomal beta-oxidation enzymes and L-FABP is mediated, to a significant extent, by poorly metabolized long-chain dicarboxylic acids formed via the P-450IVA1 pathway.

Non-genotoxic hepatocarcinogenesis in vitro: the FaO hepatoma line responds to peroxisome proliferators and retains the ability to undergo apoptosis

A range of hepatoma cell lines (RH1, HTC, FaO, 7800C1 and MH1C1), has been studied with the aim of establishing an in vitro model to investigate the molecular mechanisms of hepatocarcinogenicity induced by the peroxisome proliferator class of non-genotoxic carcinogens. In view of speculation that peroxisome proliferators suppress hepatocyte apoptosis in vivo, we have placed particular emphasis on evaluating whether hepatoma cell lines retain the ability to undergo apoptotic cell death. Expression of the liver-specific differentiation marker albumin and the peroxisome proliferator-activated receptor (PPAR) was highest in the Reuber hepatoma cell line, FaO. This cell line also demonstrated the most marked response to the peroxisome proliferator nafenopin with a 2.2-fold induction of the microsomal enzyme cytochrome p450IVA1. This response was found to display intercellular heterogeneity by immunocytochemistry. Thus, the FaO cell line maintained characteristics of hepatocytes, both in vivo and in vitro, in terms of expression of constitutive and inducible markers. However, none of the cell lines tested mirrored the hyperplastic response of hepatocytes to nafenopin, since no increase in cell growth kinetics was observed on addition of nafenopin to the growth medium. The mode of cell death in confluent FaO cultures was characterised as apoptosis, by fluorescence microscopy and agarose gel electrophoresis of extracted DNA. Cells detaching from confluent FaO cultures exhibited chromatin condensation and DNA fragmentation patterns characteristic of cels undergoing apoptotic death.Interestingly, no apoptosis was seen in monolayer cells, suggesting that apoptosis in vitro is associated with cell shrinkage and detachment similar to that documented for the liver in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

An in vitro model of rodent nongenotoxic hepatocarcinogenesis

An in vitro model of liver in which rat hepatocytes are maintained as cocultures with nonparenchymal epithelial cells (NPC) derived from liver has been developed and characterized with respect to maintenance of hepatocyte viability and differentiated function. The system was then evaluated as a model for studying peroxisome proliferator-induced rodent liver nongenotoxic carcinogenesis. Within the coculture model, hepatocyte viability and morphology were maintained for 1 month or more within a system that is both easily accessible for microscopic examination and is free of any additives that may lead to artifacts. Even after 1 month or more, hepatocyte cocultures retained expression of the constitutive liver marker albumin. In addition, they maintained the ability to show induction of the peroxisome proliferator-inducible enzymes peroxisomal bifunctional enzyme (PBE) and cytochrome P450IVA1 in response to the peroxisome proliferator nafenopin. After 4 weeks, NPC cocultures showed a six- and a fourfold induction of PBE and cytochrome P450IVA1 expression, respectively, which compared well with the three-and fivefold induction seen in freshly isolated cells. This was paralleled by an increase in the cytoplasmic volume fraction of peroxisomes averaging eightfold. Interestingly, great heterogeneity was exhibited between adjacent hepatocytes in terms of the degree of peroxisome proliferation, a finding reflected by immunocytochemical staining which indicated heterogeneity in the level of expression of the peroxisome proliferator-inducible enzymes. Other cell lines representing different tissue types, morphologies, and species were also examined for their ability to support hepatocyte survival but were found to be ineffective, with the exception of a bovine corneal endothelial cell line. This line supported hepatocyte survival and maintenance of differentiated function but to a lesser extent than that observed with NPC. Ultrastructural examination of NPC cocultures revealed extensive interhepatocyte junctional complexes and interdigitation of adjacent membranes together with the presence of bile canalicular structures. There were no junctional complexes between the hepatocytes and the supporting feeder cells with any contact being limited to a close association of the hepatocytes with the extracellular matrix presumably produced by the NPC. The data demonstrate that hepatocytes maintained in vitro within an NPC coculture system retain differentiated function and the ability to respond to the peroxisome proliferator class of nongenotoxic carcinogens. Cocultures will provide us with a model system for the study of changes in hepatocyte growth regulation during rodent liver nongenotoxic carcinogenesis.

Differential tissue‐specific expression and induction of cytochrome P450IVA1 and acyl‐CoA oxidase

We have examined the tissue-specific expression and inducibility of acyl-CoA oxidase and cytochrome P450IVA1 (P450IVA1) RNA in rats. Groups of three rats were dosed daily by gavage with methylclofenapate at 25 mg/kg in 5 ml/kg corn oil for nine weeks, or were administered a vehicle control. P450IVA1 and acyl-CoA oxidase RNA were detected using an RNase protection assay. Similar levels of acyl-CoA oxidase RNA were present in control liver and kidney, but the level of this RNA in lung, muscle and testis was 6-11%, and in pancreas was 0.13%, of that in liver. Treatment of rats with methylclofenapate led to an 11-fold induction of acyl-CoA oxidase RNA in liver and also produced a significant induction of this RNA in kidney, lung, muscle and testis of 1.7-fold, 1.3-fold, 2-fold and 1.7-fold, respectively. Acyl-CoA oxidase RNA was not induced in pancreas. P450IVA1 RNA was present in control liver and also in kidney of control rats at 28% of the level in liver. In contrast to acyl-CoA oxidase RNA, P450IVA1 RNA was not detected in lung, pancreas or testis. Methylclofenapate treatment of rats led to an 18-fold induction of P450IVA1 RNA in liver, and a sevenfold induction in kidney. Induction of P450IVA1 was not detected in any of the other tissues examined. Quantification of the relative amounts of acyl-CoA oxidase and P450IVA1 RNA in control liver revealed that acyl-CoA oxidase RNA was present in a 17.5-fold molar excess over P450IVA1 RNA. Western blotting with an anti-P450IVA IgG revealed two bands of similar apparent molecular mass in liver and kidney microsomes, but not in microsomes from the testis of control rats. Methylclofenapate treatment of rats caused an increase in the intensity of these bands in microsomes from liver, but no induction was obvious in kidney. Immunocytochemical staining for both the microsomal P450IVA and peroxisomal acyl-CoA oxidase proteins was restricted to the proximal convoluted tubule in the kidney cortex, with staining being most intense in the S3 region.

Induction of acyl-CoA oxidase and cytochrome P450IVA1 RNA in rat primary hepatocyte culture by peroxisome proliferators

We have characterized the induction of acyl-CoA oxidase and cytochrome P450IVA1 RNAs in a primary hepatocyte culture system in vitro, using a sensitive and specific RNAse protection assay. Hepatocytes were cultured with a maximal inducing dose of the peroxisome proliferator clofibric acid (1 mM), or vehicle control, for 4 days, and the level of RNAs compared with the level in rats which had been treated with corn oil or clofibric acid (300 mg/kg) for 4 days. The level of acyl-CoA oxidase and P450IVA1 RNAs in 4-day-old control hepatocytes was less than 2% of that in control liver. However, the level of these RNAs in RNA from treated hepatocytes was 61% of that in liver RNA from treated rats. Hepatocytes were treated with the potent peroxisome proliferator methylclofenapate (100 microM), and the induction of RNAs determined at various times after exposure. P450IVA1 RNA was significantly induced 1 h after dosing, rising to 34-fold above control after 8 h, whereas acyl-CoA oxidase RNA was not significantly induced until 4 h, increasing to 5.2-fold above control after 8 h. A similar time course of induction was seen after treatment of hepatocytes with 100 microM-nafenopin, 100 microM-methylclofenapate, 1 mM-clofibric acid or 1 mM-mono(ethylhexyl) phthalate, suggesting that the differential time course of induction of P450IVA1 and acyl-CoA oxidase RNAs is not related to the esterification, structure or potency of the peroxisome proliferator, but is intrinsic to the process of peroxisome proliferation. Hepatocytes were treated with methylclofenapate in the presence and absence of cycloheximide. P450IVA1 RNA was significantly induced by methylclofenapate in the presence of cycloheximide, rising to 17-fold above control after 8 h. However, no induction of acyl-CoA oxidase RNA was detected in the presence of cycloheximide. Therefore we characterize the induction of acyl-CoA oxidase and P450IVA1 RNAs in primary hepatocyte culture in vitro as a faithful model of the induction response in rat liver, and suggest that induction of P450IVA1 RNA is a primary event in the process of peroxisome proliferation.

Mapping genes encoding drug-metabolizing enzymes in recombinant inbred mice

Probes for cytochrome P450IVA (P450IVA), α- and π-class glutathione S-transferases (GST), and phenol-metabolizing UDP-glucuronyltransferase (UDPGT-K39) detected restriction fragment length variants (RFLVs) between C57BL 6J and DBA 2J mice. These variants were used to map the P450IVA genes ( Cyp4a) to chromosome 4, close to Mtv-13 and Pmv-19, midway between brown ( b) and Gpd-1; GSTα genes were mapped to chromosome 9, with a cross-hybridizing sequence mapping to another chromosome; the GSTπ genes were mapped to the distal end of chromosome 1 near Pmv-21; one UDPGT-K39 variant to chromosome 1, between Acrg and Emv-17, and another showed linkage to Odc-10 on an unidentified chromosome. No RFLVs were detected with probes for P450IID, P450 reductase, androsterone-metabolizing UDPGT, GSTμ, or microsomal GST.

Localization and differential induction of cytochrome P450IVA and acyl-CoA oxidase in rat liver

The peroxisome proliferators are structurally diverse chemicals which induce hyperplasia, hypertrophy and the proliferation of peroxisomes in the rodent liver. Cytochrome P450IVA1 and peroxisomal enzymes, such as acyl-CoA oxidase, are induced and are early markers of treatment with peroxisome proliferators. In this study, rats were dosed intraperitoneally with the potent peroxisome proliferator methylclofenapate and the hepatic induction response was studied. There was no significant change in the enzyme activities of laurate hydroxylase (cytochrome P450IVA1) or acyl-CoA oxidase in the first 8 h after treatment, but the activities had doubled at 24 h, suggesting that these enzymes are not involved in the mediation of early events in peroxisome proliferation. Hepatic cytochrome P450IVA1 mRNA was significantly increased at 6 and 8 h after treatment, rising to 15-fold above control values at 30 h. In contrast, acyl-CoA oxidase mRNA showed no significant change in the first 8 h, but increased to 13-fold above control values at 24 and 30 h, thereby demonstrating different kinetics of induction of the two mRNAs. In order to determine whether cytochrome P450IVA1 and peroxisomal enzymes were included in the same cells, rats were treated daily with sub-maximal (2 or 5 mg/kg) and maximal (25 mg/kg) inducing doses of methylclofenapate for 4 days. The lobular distribution of induced proteins was determined immunocytochemically with antibodies raised against P450IVA1 and acyl-CoA oxidase. Livers from control animals showed minimal staining for both proteins. However, in the livers of animals treated with 2 or 5 mg of methylclofenapate/kg, both acyl-CoA and P450IVA immunostaining was increased, mainly in the centrilobular area. Immunostaining of serial sections revealed that these proteins were induced in the same region of the lobule. A maximal inducing dose of methylclofenapate (25 mg/kg) caused panlobular induction of both proteins. The results demonstrate that these proteins are induced in a dose-dependent manner in the same, spatially distinct, sensitive region of the liver lobule.

Stereochemical selectivity in the induction of cytochrome P450IVA1 (P452)-dependent fatty acid hydroxylation and peroxisome proliferation

Induction of hepatic microsomal cytochrome P450IVA1 and peroxisomal enzymes of the β-oxidation spiral were observed when male Long Evans hooded rats were administered optically pure enantiomeric forms and a racemic mixture of a clofibrate analogue [2-[4-(4-chlorophenyl)benzyloxy]-2-phenylacetic acid] at a dose level of 80 mg/kg for 3 days. The R(−)-enantiomer was found to be a more potent inducer of microsomal cytochrome P450IVA1 and its associated lauric acid 12-hydroxylase activity than its corresponding S(+)-antipode. This difference in potency was reflected by a eudismic ratio ( R/ S activity ratio) of approximately 3, whereas the racemic mixture exhibited a potency intermediary between the two isomers. An identical enantiomeric selectivity was observed for the phenomenon of peroxisome proliferation as judged by induction of cyanide-insensitive palmitoyl CoA oxidation and the bifunctional protein of the peroxisomal β-oxidation spiral. The highest potency was shown by the R(−)-isomer resulting in approximately a 3–6-fold increase over the control value. These increases were paralleled by an increase in total carnitine acetyl transferase activity with a eudismic ratio of approximately 4. In addition, immunochemical detection by Western blotting analysis for both the microsomal cytochrome P450IVA1 isozyme and the peroxisomal bifunctional protein was in agreement with the above modulation of catalytic activities. These results are therefore not inconsistent with the hypothesis that cytochrome P450IVA1 induction and peroxisome proliferation are intimately linked. Whether the observed stereochemical selectivity resides in xenobiotic recognition or disposition still remains to be determined.

Microsomal oxidation of dodecylthioacetic acid (a 3-thia fatty acid) in rat liver

[1- 14C]odecylthioacetic acid (DTA), a 3-thia fatty acid, is ω (ω-1)-hydroxylated and sulfur oxygenated at about equal rates in rat liver microsomes. In prolonged incubations DTA is converted to (ω-hydroxydodecylsulfoxyacetic acid. ω-Hydroxylation of DTA is catalysed by cytochrome P450IVA1 (or a very closely related isoenzyme in the same gene family), the fatty acid ω- hydroxylating enzyme. It is absolutely dependent on NADPH and inhibited by CO, and lauric acid is a competing substrate. ω-Hydroxylation of DTA is increased by feeding tetradecylthioacetic acid (TTA), a 3-thia fatty acid, for 4 days to rats. ω-Hydroxylation of [1- 14C]lauric acid is also induced by TTA and other 3-thia carboxylic acids. A close relationship was observed between induction of microsomal ω-hydroxylation of fatty acid and palmitoyl-CoA hydrolase activity. DTA is ω-hydroxylated at about the same rate as the physiological substrate lauric acid. The sulfur oxygenation of DTA is catalysed by liver microsomal flavin-containing monooxygenase (FMO) (EC 1.14.13.8). It is dependent on either NADH or NADPH. The K m value for NADH was approx. five times larger than the K m value for NADPH. It is inhibited by methimazole and not affected by CO. It is not induced by TTA.

Determination of the cytochrome P-450 IV marker, ω-hydroxylauric acid, by high-performance liquid chromatography and fluorimetric detection

The formation of omega-hydroxylauric acid from lauric acid is an indicator of the activity of cytochrome P-450 IV family proteins. The two main metabolites of lauric acid, (omega-1)-and omega-hydroxylauric acid, have been completely separated by reversed-phase high-performance liquid chromatography. Measurement of lauric acid hydroxylase activity in microsomal liver samples, based on derivatization of the substrate and metabolites with the fluorescent agent 4-bromomethyl-6,7-dimethoxycoumarin, is a precise method (coefficient of variation = 7.6 and 10% for omega and (omega-1) metabolites, respectively) with good sensitivity (signal-to-noise ratio in microsomal samples of untreated rats greater than 20). In microsomal fractions from livers of rats treated with di-(2-ethylhexyl)phthalate the extent of omega-hydroxylation of lauric acid increased dose-dependently (ca. ten-fold). The (omega-1)-hydroxylase activity was not altered. A strong correlation between immunochemically determined cytochrome P-450 IVA1 and lauric acid omega-hydroxylase activity was found (r = 0.94, n = 30).