Janice Rice Okita

Prostaglandin-Metabolizing Enzymes During Pregnancy: Characterization of NAD+-Dependent Prostaglandin Dehydrogenase, Carbonyl Reductase, and Cytochrome P450-Dependent Prostaglandin Omega-Hydroxylase

Prostaglandins E2 and F2 alpha regulate a number of physiological functions in reproductive tissues, and concentrations of these bioactive modulators increase during pregnancy. Corresponding to the increase in circulating levels of prostaglandins during pregnancy is an increase in enzymes that metabolize these agents. Three prostaglandin-metabolizing enzymes induced during pregnancy are NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH), NADPH-dependent carbonyl reductase, and cytochrome P450-dependent prostaglandin omega- or 20-hydroxylase. This review discusses the biochemical properties, regulation, and possible functions of these three enzymes.

Differential effects of phthalates on the testis and the liver.

Abstract Phthalates have been shown to elicit contrasting effects on the testis and the liver, causing testicular degeneration and promoting abnormal hepatocyte proliferation and carcinogenesis. In the present study, we compared the effects of phthalates on testicular and liver cells to better understand the mechanisms by which phthalates cause testicular degeneration. In vivo treatment of rats with di-(2-ethylhexyl) phthalate (DEHP) caused a threefold increase of germ cell apoptosis in the testis, whereas apoptosis was not changed significantly in livers from the same animals. Western blot analyses revealed that peroxisome proliferator-activated receptor (PPAR) α is equally abundant in the liver and the testis, whereas PPARγ and retinoic acid receptor (RAR) α are expressed more in the testis. To determine whether the principal metabolite of DEHP, mono-(2-ethylhexyl) phthalate (MEHP), or a strong peroxisome proliferator, 4-chloro-6(2,3-xylindino)-2-pyrimidinylthioacetic acid (Wy-14,643), have a differential effect in Sertoli and liver cells by altering the function of RARα and PPARs, their nuclear trafficking patterns were compared in Sertoli and liver cells after treatment. Both MEHP and Wy-14,643 increased the nuclear localization of PPARα and PPARγ in Sertoli cells, but they decreased the nuclear localization of RARα, as previously shown. Both PPARα and PPARγ were in the nucleus and cytoplasm of liver cells, but RARα was predominant in the cytoplasm, regardless of the treatment. At the molecular level, MEHP and Wy-14,643 reduced the amount of phosphorylated mitogen-activated protein kinase (activated MAPK) in Sertoli cells. In comparison, both MEHP and Wy-14,643 increased phosphorylated MAPK in liver cells. These results suggest that phthalates may cause contrasting effects on the testis and the liver by differential activation of the MAPK pathway, RARα, PPARα, and PPARγ in these organs.

Characterization of a cytochrome P450 from di(2-ethylhexyl) phthalate-treated rats which hydroxylates fatty acids

A cytochrome P450 was purified from liver microsomes of rats treated with di(2-ethylhexyl) phthalate (DEHP). DEHP is a member of a group of structurally diverse compounds which have been classified as peroxisome proliferators and are inducers of cytochromes P450 which hydroxylate lauric acid and other fatty acids. The P450 isolated from DEHP-treated rats (P450DEHP) was observed to have a Mr value of 51 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a maximum absorbance of 452 nm in its reduced carbon monoxide bound state. The amino terminal residue for P450DEHP was alanine and an 18-amino acid segment at the N-terminal region was identified. The N-terminal amino acid for the P450 4A1 from clofibrate-treated rats is methionine and alignment of the N-terminal segment of the P450DEHP with P450 4A1 indicated that the first four amino acids were absent. There were two amino acid differences between the two P450s in this 18-amino acid segment; in P450DEHP an alanine and a phenylalanine were substituted for serines in P450 4A1. The P450DEHP was found to catalyze the hydroxylation of several saturated fatty acids, having the highest turnover activity with laurate (82.1 nmol 12-OH-laurate formed/min/nmol P450). Myristate, palmitate, and stearate were also metabolized but at decreasing rates. Cytochrome b5 stimulated laurate 12-hydroxylation 10-fold in a reconstituted system. Laurate was not metabolized at its 11-carbon atom; however, the longer chain length fatty acids were metabolized at the (omega-1)-carbon atom in addition to the omega-carbon atom. A polyclonal antibody to the P450DEHP recognized three protein bands in liver microsomes from control and DEHP-treated rats on Western blot analysis, but only two protein bands from phenobarbital-treated rats.

ω- and (ω-1)-hydroxylation of eicosanoids and fatty acids by high-performance liquid chromatography

Publisher Summary This chapter discusses the ω and (ω-1)-hydroxylation of eicosanoids and fatty acids by high-performance liquid chromatography (HPLC). Cytochromes P450 of the IVA family catalyze the hydroxylation of a number of endogenous substrates including prostaglandins (PG), leukotriene B4 (LTB4), and fatty acids at their ω and ω-1 carbon atoms. ω-Oxidation is a major route in prostaglandin metabolism, with a large proportion of prostaglandins being excreted in the urine as ω-oxidized products. Hydroxylated products are formed enzymatically by incubating fatty acids or prostaglandins with lung microsomes from pregnant rabbits, liver microsomes from diethylhexyl phthalate (DEHP)-treated rats, or purified cytochromes P450 from these tissues in reconstituted enzyme systems. The enzymatic rates of formation of products are calculated based on the percentage of radioactivity for the given metabolite relative to the total radioactivity eluting from the HPLC column.

Modification of lipoperoxidative effects of dichloroacetate and trichloroacetate is associated with peroxisome proliferation

Pretreatment of male B6C3F1 mice with clofibric acid (CFA) or trichloroacetic acid (TCA) in the drinking water results in a marked decrease in the lipoperoxidative response as measured by the production of thiobarbituric acid reactive substances (TBARS) in mouse liver homogenates following acute dosing with TCA or dichloroacetic acid (DCA). Pretreatment with TCA or CFA also increased palmitoyl-CoA oxidase activity, microsomal 12-(omega) hydroxylation of lauric acid and expression of P450 4A isoforms. At the doses utilized, DCA-pretreatment did not increase the level of P450 4A protein, or markers of peroxisome proliferation. However, DCA-pretreatment did result in enhanced levels of TBARS, following acute dosing with DCA, compared to controls. Pretreatment with DCA, TCA, or CFA did not alter p-nitrophenol hydroxylation (an assay specific for P450 2E1), and no increases in immunodetectable P450 2E1, 4A, 1A1/2, 2B1/2 or 3A1 protein were observed. Assays from CFA- and TCA-pretreated mice suggest that the reduction in the TBARS response seen in TCA-pretreated animals results from activities associated with peroxisome proliferation. This might result from the induction of systems efficient in scavenging of peroxide intermediates or detoxification of aldehyde by-products of lipid peroxidation.

Peroxisome Proliferators Disrupt Retinoic Acid Receptor Alpha Signaling in the Testis

Abstract Peroxisome proliferators include a diverse group of chemicals, some of which have been demonstrated to be testicular toxicants. However, the mechanism by which peroxisome proliferators, such as phthalates, cause testicular damage is not clear. It is known that retinoic acid receptor alpha (RARα) and its retinoic acid ligand, the acid form of vitamin A, are required for spermatogenesis. It has been demonstrated that the absence of RARα gene or vitamin A in the animal leads to testis degeneration and sterility. Therefore, any compound that disrupts the action of vitamin A in the testis could potentially be damaging to male fertility. The current investigation examined a novel hypothesis that a mechanism of degeneration by peroxisome proliferators in the testis is due, in part, to disruption of the critical RARα signaling pathway. We show that peroxisome proliferators were able to disrupt the retinoic acid-induced nuclear localization of RARα and the retinoic acid-stimulated increase in transcriptional activity of a retinoic acid-responsive reporter gene in Sertoli cells. Concomitantly, peroxisome proliferators increased the nuclear localization of PPARα and the transcriptional activity of a peroxisome proliferator-responsive reporter gene in these cells. These results indicate that peroxisome proliferators can indeed shift the balance of nuclear localization for RARα and PPARα, resulting in deactivation of the critical RARα transcriptional activity in Sertoli cells.

Effects of diethyl phthalate and other plasticizers on laurate hydroxylation in rat liver microsomes

Diethyl phthalate (DEP) is used in pharmaceutical coatings, cosmetics, and plastic films to wrap foods. There is a health concern associated with the exposure to certain phthalate esters because they belong to a class of compounds referred to as peroxisome proliferators which have been shown to increase the incidence of liver tumors when administered to rats. In this study, we have compared DEP to four other commonly used plasticizers, 2-diethylhexyl phthalate (DEHP), dibutyl phthalate (DBP), 2-diethylhexyl adipate (DEHA), and acetyltributyl citrate (ATBC), for their ability to induce the cytochrome P450-mediated fatty acid ω-hydroxylation system, which is one of the initial cellular responses when animals are treated with peroxisome proliferators. The administration of DEHP, DBP, and DEHA to rats increased the specific activity of laurate 12-hydroxylase from 2.8 ± 1.1 in control rats to 30.3 ± 11.6, 14.5 ± 4.1, and 9.7 ± 1.9 nmol 12-hydroxylaurate formed/min/nmol P450, respectively. In contrast, laurate 12-hydroxylase activity in DEP-and ATBC-treated rats were 4.4 ± 1.2 and 4.4 ± 1.0 nmol 12-hydroxylaurate formed/min/nmol P450, respectively. In addition, whereas DEHP increased peroxisomal palmitoyl-CoA oxidation 6-fold, DEP increased this activity only 1.3-fold. Two protein bands, at 51 and 52 kDa, were found to increase 6- to 12-fold in microsomes of DEHP-, DBP-, and DEHA-treated rats, but these bands were increased only 2-fold in DEP- or ATBC-treated rats.