Lauric Acid Hydroxylase Activity Research Articles

Determination of CYP4A11-catalyzed lauric acid 12-hydroxylation by high-performance liquid chromatography with radiometric detection.

The human CYP4.411 gene encodes a P450 protein that has been isolated from human kidney (, ) and liver () and purified to apparent homogeneity. CYP4All mRNA is present in greater abundance in kidney than in liver, whereas it is absent in lung (). Various fatty acids, including lauric acid (, , ), are substrates for recombinant CYP4Al l. Experiments with a panel of individual cDNA-expressed P450 enzymes indicate that CYP4Al l is a major lauric acid 12-hydroxylase (). Immunoinhibition experiments with heterologous and antihuman CYP4Al l antibodies have further established that CYP4Al l contributes a major fraction of the lauric acid 12-hydroxylase activity in human liver microsomes (, , ). Accordingly, microsomal lauric acid 12-hydroxylase activity is used as a marker for human hepatic CYP4A11. Although CYP4A11 is a major lauric acid 12-hydroxylase in human liver, lauric acid 11 -hydroxylation in this tissue is catalyzed by CYP2E1 and not CYP4A11 (, ). Several analytical methods are available for the quantification of 12-hydroxylauric acid, including the use of nonradioactive lauric acid as the substrate by a highperformance liquid chromatographic (HPLC) assay with fluorescence detection (, ). This chapter describes a radiometric HPLC assay for the determination of lauric acid 12-hydroxylase activity.

Efficient Assembly of Functional Cytochrome P450 2C2 Requires a Spacer Sequence between the N-terminal Signal Anchor and Catalytic Domains

Cytochromes P450 (P450) are anchored to the endoplasmic reticulum membrane by an N-terminal transmembrane sequence with the catalytic domain facing the cytoplasmic side. Within the peptide sequence linking these two domains in P450 2C2 is a glycine-rich region from residues 22 to 28. To examine the role of this region, deletion and substitution mutations were constructed, and the activities and spectral properties were determined for the mutant proteins expressed in COS-1 cells, insect cells, and bacteria. Deletion of residues 22 to 28 or substitution of 7 valines for this region inactivated the proteins in COS-1 cells, and no P450 species was detected for these mutations in bacteria or insect cells. Substitution of the three glycine residues with alanine or proline or the entire sequence from 22 to 28 with 7 alanines did not reduce lauric acid hydroxylase activity of the proteins expressed in COS-1 cells. Reducing the number of alanines substituted to 4, 3, and 2 progressively decreased activity in COS-1 cells to undetectable levels when 2 alanines were substituted. The loss of activity in COS-1 cells correlated with decreased expression of hemoprotein with a reduced difference spectrum of 450 nm (P450 species) and a corresponding increase in the inactive P420 species in insect cells and bacteria. The activities expressed per nanomole of P450 in insect microsomes were similar for P450 2C2 and the alanine substitution mutants, including the mutant with 2 alanines which was inactive in COS-1 cells. The rates of conversion of P450 to P420 resulting from incubation at 48 degrees C in vitro were not changed sufficiently to explain the increase in expressed P420 observed for the mutants with 3 or 7 alanines substituted. These data are consistent with a role for the residue 22-28 region as a linker that facilitates the folding of P450; however, once the protein is properly folded into the functional P450 species, this region has little influence on the stability and activity of the enzyme.

Induction of anti-trout lauric acid hydroxylase immunoreactive proteins by peroxisome proliferators in bluegill and catfish

Various chemicals such as phthalate esters, hypolipidemic drugs, solvents like trichloroethylene, and certain herbicides have been identified as peroxisome proliferating agents (PPAs). In many vertebrates, species- and even gender-specific sensitivities to peroxisome proliferation (PP) and toxicity, have been described and may correlate with inducibility of lauric acid hydroxylase activity. Little is known about PP in fish. The rainbow trout does not appear to be inducible for lauric acid hydroxylases (LMC1 and LMC2; CYP2M1 and CYP2K1, respectively) in response to the hypolipidemic drug, clofibrate. Fish other than trout, such as bluegill and catfish, are more acutely sensitive to phthalate ester toxicity. In this study, bluegill and catfish were examined for inducibility of anti-trout LMC1 and LMC2 immunoreactive proteins, 48 h after intra-pentoneal (i.p.) administration of olive oil vehicle, 200 mg/kg clofibrate, or 100 mg/kg ciprofibrate. In control animals, immunoreactive proteins were recognized in a sex-specific manner in both bluegill and catfish liver, and in catfish kidney. In treated animals, male immunoreactive proteins were induced to a greater extent than female, and ciprofibrate was the more potent inducer. The response of bluegill and catfish, therefore, is similar to the response of PPA-sensitive species.

Hepatic Peroxisomal and Drug Metabolizing Activity in CD-1 Mice after Oral Treatment with a Novel 5-Lipoxygenase Inhibitor

The effects of zileuton, a 5-lipoxygenase inhibitor, on hepatic peroxisomal enzyme activity as well as hepatic drug metabolizing activity in male and female CD-1 mice were assessed after oral administration of the drug (50, 150, or 450 mg/kg/day) for 14 days. The effects were compared to those in mice receiving clofibrate (CLOF; 462 mg/kg/day, po) or sodium phenobarbital (PB; 50 mg/kg/day, po). Zileuton pretreatment caused hepatomegaly and elevated liver peroxisomal KCN-insensitive palmitoyl CoA oxidase activity in a dose-dependent manner. However, these changes were marginal (≤121% increase), when compared to those elicited by CLOF (∼370% increase). In both sexes, zileuton pretreatment also caused a dose-dependent increase in the levels of liver microsomal cytochrome P450 2B and cytochrome P450 4A (CYP4A) proteins, and their associated monooxygenase activity. In the case of CYP4A, the induction of lauric acid 12-hydroxylase activity by zileuton was more pronounced in female (maximal 851% increase) than in male mice (maximal 111% increase). Based on the dose normalized response observed in CD-1 mice, zileuton can be considered a relatively weak inducer of peroxisome enzyme activities (cf. CLOF) and a moderate inducer of cytochromes P450. Moreover, zileuton exhibits characteristics of both a PB- and a CLOF-type hepatic enzyme inducer, especially in the female mice.

Sex differences in constitutive level of renal lauric acid hydroxylase activities and CYP4A-related proteins in mice.

The constitutive level of renal fatty acid hydroxylase was examined in ddY mice by measuring the activities of lauric acid omega- and (omega-1)-hydroxylase, (LA12H and LA11H respectively). The activities of both LA12H and LA11H of male mice were significantly higher than those of female mice. This sex difference in renal lauric acid hydroxylase (LAH) activity exists in other strains of mice, including Balb/c, C57BL/6, C3H/HeN and C3H/HeJ. Renal LAH activities are at significantly low levels in both sexes of immature animals, but are sexually differentiated in the mature state. In male mice, orchiectomy caused a drastic decrease in renal LAH activities, and the activities were restored by testosterone treatment to above the level of the intact animal. In female mice, ovariectomy and estradiol treatment had no effect on the activities, but testosterone treatment caused an increase in the activities to the level of the intact male animal. These results suggested that testosterone is a key regulatory factor in the level of LAH activity in mouse kidney. The administration of dexamethasone and clofibrate affected the level of LAH activity. The above data are consistent with the level of CYP4A-related proteins (band H and L protein) measured by using anti-rat CYP4A1 antibodies.antibodies. The antibodies inhibited both LA12H and LA11H activities. The level of band H protein was regulated by testosterone and dexamethasone. On the other hand, the level of band L protein was regulated by clofibrate. These results suggest that distinct modes of regulation exist in the level between band H and L protein in mouse kidney.

Interleukin 1 beta and interleukin 6 repress clofibric acid induction of different P450 isoforms in cultured foetal rat hepatocytes.

1. Expression of various P450 subfamilies (1A, 2A, 2B, 2C, 3A) have been studied in cultured foetal rat hepatocytes after treatment with clofibric acid, a peroxisome proliferator and prototypic CYP4A inducer in vitro. Ethoxyresorufin O-deethylase activity (EROD, a CYP1A-related activity) as well as 7 alpha-, 16 alpha-, 2 alpha- and 6 beta-testosterone hydroxylase activities (CYP2A, 2B, 2C11 and 3A respectively) were determined during culture. Levels of the corresponding P450 apoproteins were measured by Western blotting. 2. Clofibric acid was able to induce all the P450-dependent activities studied. In most cases this induction required the additional presence of dexamethasone, an agent which promotes differentiation and favours long-term maintenance of the hepatocytes. 3. The major pro-inflammatory cytokines, IL-1 beta and IL-6, decrease the levels of the clofibric acid-induced P450 isoforms, except CYP1A, which was insensitive to IL-6, previous studies having shown that IL-1 beta represses lauric acid 12-hydroxylase activity after induction by clofibric acid. The effects of these cytokines were clearly dose- and time-dependent. The decrease in enzyme activity correlated with a decrease in apoprotein content. 4. The ability of clofibric acid to induce P450 isoforms highlights the complexity of P450 regulation by peroxisome proliferators. Our results confirm, moreover, that different P450 subfamilies are differentially affected by IL-1 beta and IL-6.

Defective expression of cytochrome P450 proteins in the liver of the genetically obese Zucker rat

The hepatic expression of xenobiotic-metabolising cytochrome P450 isoforms in the genetically obese Zucker rat, a model of obesity, was compared to that of its lean littermate. Cytochrome P450 (CYP) levels were determined using diagnostic substrates and/or immunologically in Western blot analyses. When compared with the lean Zucker rat, the obese animal exhibited hyperglycaemia, hypercholesterolaemia, marked hyperinsulinaemia and hypertriglyceridaemia but was normoketonaemic. CYP3A and CYP1A2 levels were higher in the obese Zucker rat when compared with the lean littermate but, in contrast, a protein recognised by human CYP2D6 and, to a lesser extent, CYP2C11 levels were lower. Pretreatment with acetone, dexamethasone and clofibrate resulted in enhanced p -nitrophenol hydroxylase (CYP2E), erythromycin N -demethylase (CYP3A) and lauric acid hydroxylase (CYP4A) activities respectively in the liver of the lean Zucker rat but, in contrast, the obese Zucker rat was refractive to such treatment; similarly, hepatic apoprotein levels of the CYP2E and CYP4A subfamilies were increased markedly only in the lean Zucker rat. It is concluded that CYP2E, CYP3A and CYP4A subfamilies are poorly expressed in the obese Zucker rat, and this rat strain may serve as a good model for elucidating the molecular mechanisms of induction of these cytochrome P450 proteins.

Defective expression of cytochrome P450 proteins in the liver of the genetically obese Zucker rat

The hepatic expression of xenobiotic-metabolising cytochrome P450 isoforms in the genetically obese Zucker rat, a model of obesity, was compared to that of its lean littermate. Cytochrome P450 (CYP) levels were determined using diagnostic substrates and/or immunologically in Western blot analyses. When compared with the lean Zucker rat, the obese animal exhibited hyperglycaemia, hypercholesterolaemia, marked hyperinsulinaemia and hypertriglyceridaemia but was normoketonaemic. CYP3A and CYP1A2 levels were higher in the obese Zucker rat when compared with the lean littermate but, in contrast, a protein recognised by human CYP2D6 and, to a lesser extent, CYP2C11 levels were lower. Pretreatment with acetone, dexamethasone and clofibrate resulted in enhanced p-nitrophenol hydroxylase (CYP2E), erythromycin N-demethylase (CYP3A) and lauric acid hydroxylase (CYP4A) activities respectively in the liver of the lean Zucker rat but, in contrast, the obese Zucker rat was refractive to such treatment; similarly, hepatic apoprotein levels of the CYP2E and CYP4A subfamilies were increased markedly only in the lean Zucker rat. It is concluded that CYP2E, CYP3A and CYP4A subfamilies are poorly expressed in the obese Zucker rat, and this rat strain may serve as a good model for elucidating the molecular mechanisms of induction of these cytochrome P450 proteins.

72 Mechanistic studies of tamoxifen hepatocarcinogenesis: Peroxisomal proliferation

The antiestrogenic drug tamoxifen (TAM) is a strong liver carcinogen in the rat. As peroxisome proliferators induce liver cancer probably by an epigenetic mechanism the effect of TAM on peroxisomes, peroxisomal enzymes and peroxisome proliferator inducible CYP4AI activity was studied in the rat. Comparisons were made with the new noncarcinogenic antiestrogen, toremifene (TOR), and with clofibrate (CLO). TAM, TOR (0.10 mmol/kg) or CLO (0.10 or 1.03 mmol/kg) were administered daily to female SD-rats p.o. for 14 days. Morphometric analysis of hepatocytes was performed. Peroxisomal β-oxidation (β-OX) activity and hepatic CYP4AI-dependent lauric acid 12-hydroxylase (LAH) and catalase activities were measured. The volume density (VD) of peroxisomes increased 2.4-fold in the high-dose CLO rats. In the TAM rats the VD of peroxisomes or mitochondria increased 1.8-or 1.3-fold, respectively. In the TOR rats the VD of mitochondria increased l.4-fold. Either TAM or TOR had no effect on peroxisomal β-OX or on microsomal LAH activities. The high-dose CLO increased the β-OX rate 3.4-fold and LAH activity over 10-fold (P

Induction of hepatic CYP1A2 by the oral administration of caffeine to rats: lack of association with the Ah locus

Caffeine was administered to male Wistar albino rats for two weeks at three concentrations, namely 0.1, 0.2 and 0.3%, and hepatic cytochrome P450-dependent mixed-function oxidases determined. Caffeine administration gave rise to a marked, dose-dependent increase in the O-deethylation of ethoxyresorufin and, to a lesser extent, in the O-depentylation of pentoxyresorufin. Erythromycin N-demethylase, p-nitrophenol hydroxylase and lauric acid hydroxylase activities, as well as total cytochrome P450 content were unaffected by this treatment. Immunoblot analysis revealed that caffeine gave rise to a dose-dependent increase in the hepatic CYP1A2, and at the highest dose only, CYP2B apoprotein levels. Apoprotein levels of CYP3A and CYP2E1 were not modulated by the treatment with caffeine at all dose levels studied. Caffeine could not displace [ 3H]TCDD from the rat hepatic cytosolic Ah receptor. Computer analysis showed that caffeine is essentially a planar molecule with an area/depth 2 ratio 4.8, characteristic of CYP1A substrates/inducers. Molecular modelling revealed that the caffeine molecule could orientate itself within the putative CYP1A2 active site so as to facilitate demethylation of the N-1, N-3 and N-7 positions. However, at physiological pH, the N-9 nitrogen atom is likely to be partially protonated, allowing it to participate in an electrostatic interaction with the negatively-charged glutamate 318-residue, favouring N-3 demethylation, the major pathway of metabolism in both humans and animals. In conclusion caffeine, being essentially planar, is an inducer of CYP1A2 in rat liver.

The Effects of Diquat and Ciprofibrate on mRNA Expression and Catalytic Activities of Hepatic Xenobiotic Metabolizing and Antioxidant Enzymes in Rat Liver

Although the mechanisms responsible for chemically induced oxidative stress are under intense investigation, little is known about the effects of prooxidant chemicals on the expression of drug-metabolizing enzymes. We examined the effects of diquat (0.1 mmol/kg, ip) and ciprofibrate (0.025% w/w, diet), chemicals which induce oxidative stress via different biochemical mechanisms, on the steady-state messenger RNA (mRNA) levels of six cytochrome P450 enzymes, seven glutathione S-transferase (GST) isoenzymes, UDP-glucuronosyl transferase 1-06 (UGT1*06), γ-glutamylcysteine synthetase (γGCS), NADP(H):quinone oxidoreductase (quinone reductase), Cu/Zn superoxide dismutase (SOD), catalase, and 18S ribosomal RNA in the livers of male Sprague-Dawley rats. Effects of chemical treatments on mRNA levels were compared to changes in catalytic activities for selected enzymes. Ciprofibrate treatment selectively decreased CYP1A2 mRNA expression, whereas both chemicals suppressed CYP3A2 mRNA expression. CYP4A1 mRNA expression and lauric acid hydroxylase activities were induced by ciprofibrate treatment, whereas diquat treatment moderately increased CYP4A1 mRNA levels without affecting lauric acid hydroxylase activities. The steady-state mRNA levels encoding constitutively expressed GST isozymes (Ya1, Ya2, Yb1, Yb2, and Yc1) were decreased by diquat exposure, and the mRNA encoding four of the five constitutively expressed GSTs (Ya1, Ya2, Yb1, and Yc1) were also decreased by ciprofibrate treatment. Nonconstitutively expressed or low constitutively expressed genes (CYP1A1, CYP2B1, CYP2B2, GST Yc2, GST Yf, and UGT1*06) were not induced by exposure to the prooxidants. Changes in isozyme-specific catalytic activities were more consistent with the observed changes in mRNA expression for the GSTs than for the P450s. Both treatments had inhibitory effects on hepatic GSH biosynthesis by decreasing γGCS large-subunit mRNA expression, γGCS catalytic activities, and hepatic GSH concentrations. Cu/Zn SOD and quinone reductase mRNA levels were increased after ciprofibrate exposure, whereas Cu/Zn SOD mRNA expression was decreased in the diquat-treated animals. The results of this study indicate that diquat and ciprofibrate can decrease the expression profile of a number of phase I, phase II, and antioxidant enzymes and inhibit GSH biosynthesis. These effects may involve the pre-translational loss of hepatic mRNAs, possibly due to accelerated production of reactive oxygen species.

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.

Extrahepatic expression of P450 proteins in insulin-dependent diabetes mellitus.

1. Male Wistar rats were rendered diabetic by the administration of a single intraperitoneal dose of streptozotocin; the levels of the xenobiotic-inducible P450 proteins were determined in the lung and kidney using diagnostic substrates and immunoblotting employing polyclonal antibodies. The glutathione conjugation system in the cytosol of these tissues was also investigated. 2. The onset of insulin-dependent diabetes did not influence the O-dealkylations of methoxyresorufin, ethoxyresorufin and pentoxyresorufin in either kidney or lung. 3. Lauric acid hydroxylase activity, however, was induced in the kidney but no activity was detectable in the lung. Immunoblot analysis of kidney microsomes using antibodies to P4504A1 revealed the presence of two bands, both of which were clearly inducible in diabetes. In pulmonary microsomes a single faint band was detected which also appeared to be higher in the diabetic rats. 4. Aniline p-hydroxylase activity was not detectable in the kidney, but activity was measurable in the lung and was suppressed in diabetes. Immunoblot analysis of pulmonary microsomes using antibodies to P4502E1 immunodetected a single band which was suppressed in diabetes. In the kidney microsomes a single band was also detected which was, however, markedly elevated in diabetes. 5. Glutathione S-transferase activity was modestly higher in the kidney, but not lung, of the diabetic animals. Glutathione reductase and total glutathione levels were not influenced by the presence of diabetes. 6. It is concluded that streptozotocin-induced insulin-dependent diabetes modulates extrahepatic P450 proteins, the effect being both tissue- and isoform-specific.

Identification by in Vitro Mutagenesis of the Interaction of Two Segments of C2MstC1, a Chimera of Cytochromes P450 2C2 and P450 2C1

A hybrid cytochrome P450, C2MstC1, with 306 N-terminal amino acids derived from cytochrome P450 2C2 sequence and 184 C-terminal amino acids from cytochrome P450 2C1 acquires a novel progesterone 21-hydroxylase activity which is absent in the parent enzymes. Extension of the cytochrome P450 2C2 sequence to residue 382 reduced progesterone hydroxylase activity to 5% of that of C2MstC1, while further extension to residue 411 or 462 increased activity back to about 30 or 40%, respectively. In the chimera with cytochrome P450 2C2 sequence to residue 382, substitution of cytochrome P450 2C1 amino acids at positions 368, 369, and 374 increased progesterone hydroxylase activity to a level equivalent to that of C2MstC1. In the chimera with cytochrome P450 2C2 sequence extending to residue 411, substitutions of P450 2C1 amino acids at positions 386 and 388, in addition those at 368, 369, and 374, were required to obtain activities equivalent to that of C2MstC1, which suggests an interaction between these two regions. The lauric acid hydroxylase activities of all chimeras and mutant cytochromes P450 differed by 2-fold or less, demonstrating that the changes in progesterone hydroxylase activity reflected altered interactions with the substrate. Alignment of cytochrome P450 2C1 sequence with cytochromes P450cam, P450BM-3, and P450terp predicts that residues 368/369 and 386/388 are in adjacent antiparallel strands of the same beta-sheet, in agreement with the experimental data suggesting an interaction between these two regions.

Modulation of the rat hepatic cytochrome P450 composition by long-term streptozotocin-induced insulin-dependent diabetes.

The effects of long-term insulin-dependent diabetes on the enzymatic activities of hepatic cytochrome P450 isozymes were determined in rats rendered diabetic by the administration of streptozotocin and killed 4, 8, and 12 weeks following treatment. The O-dealkylations of ethoxyresorufin and pentoxyresorufin were elevated in the diabetic animals throughout the study, the extent of increase being similar at all three time points. p-Nitrophenol hydroxylase activity was induced in the diabetic animals 4 weeks following treatment with streptozotocin, but the extent of increase became less pronounced with the progress of the disease. A modest increase in ethylmorphine N-demethylase activity was also observed but only in the diabetic animals killed 4 weeks after the induction of diabetes. Finally, lauric acid hydroxylase activity was elevated in the diabetic animals 4 weeks following streptozotocin administration but then declined rapidly with the duration of the disease. It is concluded that the duration of diabetes modulates the hepatic cytochrome P450 profile, with the effect being isoenzyme specific. Mechanisms that may account for these changes are discussed.

Induction of the CYP4A subfamily by perfluorodecanoic acid: the rat and the guinea pig as susceptible and non-susceptible species

Male Wistar rats and male Duncan Hartley guinea pigs were treated with one i.p. dose of perfluorodecanoic acid (PFDA) resulting in pronounced hepatomegaly in the rat but not the guinea pig. PFDA treatment also resulted in a 4-fold induction of lauric acid 12-hydroxylase activity in the rat but not the guinea pig, indicating induction of the CYP4A subfamily of isoenzymes. Consistent with this latter conclusion, Western blot analysis of rat liver microsomes using an antibody to CYP4Al and Northern blot analysis of RNA extracts using a CYP4Al cDNA probe, revealed PFDA-dependent induction of the CYP4A subfamily in the rat but not the guinea pig. Taken collectively, our data has demonstrated that PFDA, like other peroxisome proliferators, is also a CYP4A inducer and conforms to the well-documented species specificity in induction for this class of compound.

Fluorimetric Detection of Microsomal Lauric Acid Hydroxylations Using High-Performance Liquid Chromatography After Selective Solvent Partitioning and Esterification with 1-Pyrenyldiazomethane

Abstract A novel and simple method for determining microsomal lauric acid hydroxylase activity is presented. Lauric acid and hydroxy-metabolites are separated using differential acid/base solubilities coupled to solvent partitioning. After esterification with 1-pyrenyldiazomethane, metabolites are quantitated using isocratic high-performance iiquid chromatography with fluorimetric detection. Column washing and equilibration between samples is not required. The method was verified by measuring the induction, in rats, of microsomal lauric acid hydroxylase activity by clofibrate. The method has clear advantages over published radiochemical procedures for measuring the formation of hydroxylated metabolites of lauric acid.

Coordinate induction of hepatic fatty acyl-CoA oxidase and P4504A1 in rat after activation of the peroxisome proliferator-activated receptor (PPAR) by sulphur-substituted fatty acid analogues.

1. In the liver of rat fed a single dose of 3-thia fatty acids, 3-dithiahexadecanedioic acid (3-thiadicarboxylic acid) and tetradecylthioacetic acid, steady-state levels of P4504A1 and fatty acyl-CoA oxidase mRNAs increased in parallel. The increases were significant 8 h after administration, reaching a maximum after 12 h and decreased from 12 to 24 h after administration. 2. The corresponding enzyme activities of P4504A1 and fatty acyl-CoA oxidase were also induced in a parallel manner by the 3-thia fatty acids. The enzyme activities were significantly increased 12 h after administration and increased further after 24 h. This may reflect a possible effect of the 3-thia fatty acids not only on mRNA levels, but also on the translation and degradation rate of the two enzymes. 3. Repeated administration of 3-thia fatty acids resulted in an increase of the specific P4504A1 protein accompanied with an increased lauric acid hydroxylase activity. The correlation between induction of P4504A1 and fatty acyl-CoA oxidase mRNAs and their enzyme activities may reflect a coordinated rather than a causative induction mechanism, and that these genes respond to a common signal. This suggests that the increased P450 activity may not be responsible or be a prerequisite for fatty acyl-CoA oxidase induction. 4. Since the peroxisome proliferator-activated receptor (PPAR) plays a role in mediating the induction of fatty acyl-CoA oxidase, we analysed the activation of PPAR by fatty acids and sulphur-substituted analogues utilizing a chimera between the N-terminal and DNA-binding domain of the glucocorticoid receptor and the putative ligand-binding domain of PPAR. Arachidonic acid activated this chimeric receptor in Chinese hamster ovary cells. Inhibitors of P450 did not affect the activation of PPAR by arachidonic acid. Furthermore, dicarboxylic acids including 1,12-dodecanedioic acid or 1,16-hexadecanedioic acid only weakly activated the chimera. 3-Thidicarboxylic acid, however, was a much more effective activator than the non-sulphur-substituted analogues. In conclusion, the data suggest that the most likely mechanism of the induction process is fatty acid-induced activation of PPAR, which then leads to a coordinated induction of P4504A1 and fatty acyl-CoA oxidase.

Hydrophobic Side Chain Requirements for Lauric Acid and Progesterone Hydroxylation at Amino Acid 113 in Cytochrome P450 2C2, a Potential Determinant of Substrate Specificity

To determine the requirements for hydrophobic amino acids at position 113 in cytochrome P450 2C2, a series of hydrophobic and uncharged polar amino acids was substituted for isoleucine in P450 2C2 and in C2MstC1, a chimera of P450 2C2 and P450 2C1. Lauric acid bydroxylase activity was determined in COS 1 cells transfected with P450 2C2 mutants and both lauric acid and progesterone hydroxylase activities were determined for C2MstC1 variants. In P450 2C2, 40 to 120% of the wild type (ω-1) lauric acid hydroxylase activity was retained in all hydrophobic mutants, but activity was reduced to near background by substitutions of the polar amino acids, tyrosine and cysteine. Likewise, in C2MstC1 mutants, hydrophobic substitutions were 20 to 50% as active as wild type for lauric acid hydroxylation, and polar amino acids again resulted in strong reductions of activity. In contrast, a different pattern of activity for progesterone C21-hydroxylase was observed for C2MstC1 mutants. A valine substitution had a modest effect on activity but substitutions of leucine and alanine reduced progesterone C21-hydroxylase activity 5- to 7-fold, respectively, and the large hydrophobic amino acid, phenylalanine, reduced activity about 30-fold. No changes in the regiospecificity of progesterone hydroxylation were observed for any of the mutants. Similar steady-state levels of immunoprecipitated, radiolabeled protein were observed for each mutant except for the glycine substitution which resulted in degradation of the protein. The different patterns of the effects of the mutations on progesterone and lauric acid hydroxylase activity provide additional support for the critical role of residue 113 in substrate recognition. The low activities in mutant proteins with hydrophilic amino acid substitutions indicate that the hydrophobic nature of this residue is important. The hydrophobic requirements aren more stringent for a larger, more rigid steroid substrate than for a saturated fatty acid with a flexible hydrocarbon tail.

Cassette mutagenesis of a potential substrate recognition region of cytochrome P450 2C2.

Cassette mutagenesis was used to analyze the effects of mutations at amino acid positions 107-120 in cytochrome P450 2C2 which are part of a predicted substrate recognition site, SRS-1 (Gotoh, O. (1992) J. Biol. Chem. 267, 83-90), and terminate with a highly conserved tryptophan. All the mutant enzymes were expressed in COS1 cells at approximately the same level as wild type as determined by immunoprecipitation of radiolabeled products. Substitutions of amino acids from 107 to 110 and from 116 to 119 resulted in mutant enzymes that retained substantial lauric acid hydroxylase activity suggesting that these residues are not critical determinants of substrate specificity. In contrast, amino acids 112-115 were characterized by strong decreases in activity in at least one mutation tested. A substitution of phenylalanine for valine 112 reduced activity about 3-fold. At position 113, substitution of leucine for isoleucine had little effect, but activity was reduced 15-fold by substitution with cysteine. A conservative mutation at position 114 reduced activity 5-fold, a nonconservative mutation at position 115 resulted in a 30-fold reduction of lauric acid hydroxylation. Substitution of glutamic acid for glycine at position 109 had little effect on activity, while more conservative substitutions of valine for glycines at positions 111 and 117 resulted in about 50- and 7-fold reductions of activity, respectively. Substitution of leucine for tryptophan 120 resulted in a 7-fold reduction of activity indicating that tryptophan at this position contributes to, but is not essential for, enzymatic activity in spite of its presence in nearly all eukaryotic cytochromes P450. The results are consistent with a model in which amino acids 112-114, and possibly 115, align with a loop of bacterial P450cam sequence containing substrate contacting amino acids, tyrosine 96 and phenylalanine 98. Glycines at either end of the loop in cytochrome P450 2C2 appear to be critical for activity.