One of the major oxysterols in the human circulation is 4␤-hydroxycholesterol formed from cholesterol by the drug-metabolizing enzyme cytochrome P450 3A4. Deuterium-labeled 4␤-hydroxycholesterol was injected into two healthy volunteers, and the apparent half-life was found to be 64 and 60 h, respectively. We have determined earlier the half-lives for 7␣-, 27-, and 24-hydroxycholesterol to be ϳ0.5, 0.75, and 14 h, respectively. Patients treated with certain antiepileptic drugs have up to 20-fold increased plasma concentrations of 4␤-hydroxycholesterol. The apparent half-life of deuteriumlabeled 4␤-hydroxycholesterol in such a patient was found to be 52 h, suggesting that the high plasma concentration was because of increased synthesis rather than impaired clearance. 4␤-Hydroxycholesterol was converted into acidic products at a much slower rate than 7␣-hydroxycholesterol in primary human hepatocytes, and 4␤-hydroxycholesterol was 7␣-hydroxylated at a slower rate than cholesterol by recombinant human CYP7A1. CYP7B1 and CYP39A1 had no activity toward 4␤-hydroxycholesterol. These results suggest that the high plasma concentration of 4␤-hydroxycholesterol is because of its exceptionally slow elimination, probably in part because of the low rate of 7␣-hydroxylation of the steroid. The findings are discussed in relation to a potential role of 4␤-hydroxycholesterol as a ligand for the nuclear receptor LXR.4␤-Hydroxycholesterol is one of the quantitatively most important oxysterols in human circulation (1). We have recently shown that it is formed by the drug-metabolizing enzyme cytochrome P450 3A4 (CYP3A4) 1 (1). Preliminary experiments showed that the formation of this oxysterol by human liver microsomes was relatively slow. The high plasma levels of the oxysterol are therefore surprising, and we hypothesized that this may be a consequence of slow metabolism. Therefore, in this work, we determined the rate of elimination of deuteriumlabeled 4␤-hydroxycholesterol from plasma. Oxysterols are generally degraded to bile acids, and the rate-limiting step in this conversion is the introduction of a hydroxyl group in the 7␣-position of the steroid. Alternative pathways for bile acid biosynthesis start with oxidation of the steroid side chain by CYP27A1 and CYP46. Therefore, we have studied the possibility that these cytochromes are active toward 4␤-hydroxycholesterol. The metabolism of 4␤-hydroxycholesterol was studied in human primary hepatocytes, control, and transfected cells and by incubations with recombinant enzymes. In addition, fecal samples from three untreated subjects and one subject treated with carbamazepine were analyzed for 4␤-hydroxylated bile acids. Based on these experiments, we present evidence that 4␤-hydroxycholesterol has an unusually long halflife in plasma and that this is the result of slow elimination, particularly slow 7␣-hydroxylation that is the rate-limiting step for further conversion into bile acids.

This article aims to give an overview on the characterization, properties and regulation of enzymes, particularly the cytochrome (CYP) P450 enzymes, in the formation of bile acids from cholesterol. Bile acids are biologically active molecules that promote absorption of dietary lipids in the intestine and stimulate biliary excretion of cholesterol. Bile acids and oxysterols, formed from cholesterol, act as ligands to nuclear receptors regulating the expression of important genes in cholesterol homeostasis. Thus, the bioactivation of cholesterol into bile acids is crucial for regulation of cholesterol homeostasis. The primary human bile acids, cholic acid and chenodeoxycholic acid, are formed from cholesterol via several pathways involving many different enzymes. Many of these enzymes are cytochrome P450 (CYP) enzymes, introducing a hydroxyl group in the molecule. The "classic" pathway of bile acid formation starts with a 7alpha-hydroxylation of cholesterol by CYP7A1 in the liver. The "acidic" pathway starts with a hepatic or extrahepatic 27-hydroxylation by CYP27A1. There also exist some quantitatively minor pathways which may be of importance under certain conditions. Formation of cholic acid requires insertion of a 12alpha-hydroxyl group performed by CYP8B1. Oxysterols are precursors to bile acids, participate in cholesterol transport and are known to affect the expression of several genes in cholesterol homeostasis. Enzymes with capacity to form and metabolize oxysterols are present in liver and extrahepatic tissues. The enzymes, nuclear receptors and transcription factors involved in bile acid biosynthesis are potential pharmaceutical targets for the development of new drugs to control hypercholesterolemia and to prevent atherosclerosis and other diseases related to disturbed cholesterol homeostasis. The review will also discuss some inborn errors of bile acid biosynthesis and the recently acquired knowledge on the genetic defects underlying these diseases.

In this study, we examined whether 1␣,25-dihydroxyvitamin D 3 (calcitriol), phenobarbital, and the antiretroviral drug efavirenz, drugs used by patient groups with high incidence of low bone mineral density, could affect the 25-hydroxylase activity or expression of human 25-hydroxylases in dermal fibroblasts and prostate cancer LNCaP cells. Fibroblasts express the 25-hydroxylating enzymes CYP2R1 and CYP27A1. LNCaP cells were found to express two potential vitamin D 25-hydroxylases-CYP2R1 and CYP2J2. The presence in different cells of nuclear receptors vitamin D receptor (VDR), pregnane X receptor (PXR), and constitutive androstane receptor (CAR) was also determined. Phenobarbital suppressed the expression of CYP2R1 in fibroblasts and CYP2J2 in LNCaP cells. Efavirenz suppressed the expression of CYP2R1 in fibroblasts but not in LNCaP cells. CYP2J2 was slightly suppressed by efavirenz, whereas CYP27A1 was not affected by any of the two drugs. Calcitriol suppressed the expression of CYP2R1 in both fibroblasts and LNCaP cells but had no clear effect on the expression of either CYP2J2 or CYP27A1. The vitamin D 3 25-hydroxylase activity in fibroblasts was suppressed by both calcitriol and efavirenz. In LNCaP cells, consumption of substrate (1␣-hydroxyvitamin D 3 ) was used as indicator of metabolism because no 1␣,25-dihydroxyvitamin D 3 product could be determined. The amount of 1␣-hydroxyvitamin D 3 remaining in cells treated with calcitriol was significantly increased. Taken together, 25-hydroxylation of vitamin D 3 was suppressed by calcitriol and drugs. The present study provides new information indicating that 25-hydroxylation of vitamin D 3 may be regulated. In addition, the current results may offer a possible explanation for the impaired bone health after treatment with certain drugs.The metabolic activation of vitamin D is initiated by 25-hydroxylation of the side chain followed by a 1␣-hydroxylation. 1␣,25-Dihydroxyvitamin D 3 (calcitriol), the biologically most active form of vitamin D 3 , is known as a calciumregulating hormone but is involved also in other processes such as modulation of the immune system and cell proliferation and differentiation. At the moment, at least four enzymes capable of 25-hydroxylation of vitamin D 3 and/or vitamin D 2 have been described in humans, including the mitochondrial CYP27A1 and the microsomal CYP2R1, CYP2J2, and CYP3A4 (Ohyama and Yamasaki, 2004;Prosser and Jones, 2004). CYP3A4 is reported to prefer the nonphysiological form vitamin D 2 over vitamin D 3 (Gupta et al., 2004). Thus, several possible candidates for a vitamin D 3 25-hydroxylase have been suggested; from a regulatory perspective, however, the physiological roles of these proposed 25-hydroxylases remain poorly defined. In this context, it is interesting that vitamin D 3 25-hydroxylation occurs also in certain extrahepatic tissues (e.g., the prostate). Regulation of human vitamin D 3 25-hydroxylation may be particularly important in extrahepatic tissues and might be a means of controlling cellular le...

A 7␣-hydroxylation is necessary for conversion of both cholesterol and 27-hydroxycholesterol into bile acids. According to current theories, cholesterol 7␣-hydroxylase (CYP7A) is responsible for the former and oxysterol 7␣-hydroxylase (CYP7B) for the latter reaction. CYP7A is believed to have a very high substrate specificity whereas CYP7B is active toward oxysterols, dehydroepiandrosterone, and pregnenolone. In the present study, 7␣-hydroxylation of various oxysterols in liver and kidney was investigated. Surprisingly, human cholesterol 7␣-hydroxylase, CYP7A, expressed as a recombinant in Escherichia coli and COS cells, was active toward 20(S)-hydroxycholesterol, 25-hydroxycholesterol, and 27-hydroxycholesterol. This enzyme has previously been thought to be specific for cholesterol and cholestanol. A partially purified and reconstituted cholesterol 7␣-hydroxylase enzyme fraction from pig liver showed 7␣-hydroxylase activity toward the same oxysterols as metabolized by expressed recombinant human and rat CYP7A. The 7␣-hydroxylase activity toward 20(S)-hydroxycholesterol, 25-hydroxycholesterol, and 27-hydroxycholesterol in rat liver was significantly increased by treatment with cholestyramine, an inducer of CYP7A. From the present results it may be concluded that CYP7A is able to function as an oxysterol 7␣-hydroxylase, in addition to the previously known human oxysterol 7␣-hydroxylase, CYP7B. These findings may have implications for oxysterol-mediated regulation of gene expression and for pathways of bile acid biosynthesis. A possible use of 20(S)-hydroxycholesterol as a marker substrate for CYP7A is proposed.Oxysterols are important degradation products of cholesterol and are intermediates in biosynthesis of steroid hormones and bile acids. These compounds have a broad spectrum of biological effects including modulation of the activity of enzymes involved in cholesterol homeostasis (1-9). Primary bile acids are formed from cholesterol in the liver, either through the "neutral pathway" or the "acidic pathway," involving several cytochrome P450 enzymes. The first and rate-limiting reaction in the neutral pathway is a 7␣-hydroxylation by the cholesterol 7␣-hydroxylase (CYP7A), 1 an enzyme believed to be specific for cholesterol and cholestanol (10, 11). The first step of the acidic pathway is a 27-hydroxylation. 27-Hydroxycholesterol is further 7␣-hydroxylated by an oxysterol 7␣-hydroxylase (27-hydroxycholesterol 7␣-hydroxylase), which does not 7␣-hydroxylate cholesterol (12-17). A cDNA has been isolated encoding an oxysterol 7␣-hydroxylase (CYP7B), which catalyzes 7␣-hydroxylation of 25-hydroxycholesterol, 27-hydroxycholesterol, dehydroepiandrosterone, and pregnenolone (18 -21). In contrast to CYP7A, which is found only in the liver, CYP7B is present also in extrahepatic tissues and organs (21). It has recently been reported that several oxysterols including 20(S)-, 22(R)-, 25-, and 27-hydroxycholesterol are ligands of the liver X receptor (LXR), which functions as a ligand-dependent transcription factor in a het...

ABSTRACT:A new metabolic scheme of flutamide is proposed in this article. Some patients treated with flutamide, a nonsteroidal antiandrogen, have developed severe hepatic dysfunction. Toxic metabolites have been proposed to be responsible for these negative effects. In this study, the qualitative aspects of the in vitro metabolism of flutamide in liver microsomes from human, dog, pig, and rat were evaluated. A direct comparison of the flutamide metabolism in liver and prostate microsomes from pig was made, and the in vivo metabolism of flutamide was investigated in urine from orally treated prostate cancer patients. Liquid chromatography/tandem mass spectrometry was used for analysis. The mass spectrometer was equipped with an electrospray interface and operated in the negative ion mode. In liver microsomes from pig, dog, and rat, extensive hydroxylation of flutamide occurred. One, two, or three hydroxy groups were attached, and isomeric forms were detected for both monohydroxylated and trihydroxylated drug. In pig liver microsomes, isomers of a third metabolite, hydroxylated 4-nitro-3-(trifluoromethyl)-aniline, were also found after incubation with either flutamide or 2-hydroxyflutamide. In human liver microsomes, the pharmacologically active 2-hydroxyflutamide was the only metabolite detected. Several phase I metabolites as well as four intact phase II metabolites could be recovered from the urine samples. For the first time in humans, glucuronic acid conjugates of hydroxylated 4-nitro-3-(trifluoromethyl)-aniline, and mono-and dihydroxylated flutamide were identified, together with hydroxylated 4-nitro-3-(trifluoromethyl)-aniline conjugated with sulfate. In addition, one mercapturic acid conjugate of hydroxylated flutamide, probably formed from flutamide via a reactive intermediate, was detected.

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