Kjell Wikvall

Human cytochromes P450 in health and disease

There are 18 mammalian cytochrome P450 (CYP) families, which encode 57 genes in the human genome. CYP2, CYP3 and CYP4 families contain far more genes than the other 15 families; these three families are also the ones that are dramatically larger in rodent genomes. Most (if not all) genes in the CYP1, CYP2, CYP3 and CYP4 families encode enzymes involved in eicosanoid metabolism and are inducible by various environmental stimuli (i.e. diet, chemical inducers, drugs, pheromones, etc.), whereas the other 14 gene families often have only a single member, and are rarely if ever inducible or redundant. Although the CYP2 and CYP3 families can be regarded as largely redundant and promiscuous, mutations or other defects in one or more genes of the remaining 16 gene families are primarily the ones responsible for P450-specific diseases—confirming these genes are not superfluous or promiscuous but rather are more directly involved in critical life functions. P450-mediated diseases comprise those caused by: aberrant steroidogenesis; defects in fatty acid, cholesterol and bile acid pathways; vitamin D dysregulation and retinoid (as well as putative eicosanoid) dysregulation during fertilization, implantation, embryogenesis, foetogenesis and neonatal development.

6α-Hydroxylation of taurochenodeoxycholic acid and lithocholic acid by CYP3A4 in human liver microsomes

The aim of the present study was to identify the enzymes in human liver catalyzing hydroxylations of bile acids. Fourteen recombinant expressed cytochrome P450 (CYP) enzymes, human liver microsomes ...

Porcine microsomal vitamin D(3) 25-hydroxylase (CYP2D25). Catalytic properties, tissue distribution, and comparison with human CYP2D6.

The metabolic activation of the prohormone vitamin D3 requires a 25-hydroxylation that has been reported to be catalyzed by both mitochondrial CYP27A and a microsomal vitamin D3 25-hydroxylase in the liver. CYP27A has been extensively studied, but its role as a physiologically important vitamin D3 25-hydroxylase has been questioned. The present paper reports that the microsomal vitamin D325-hydroxylase, purified from pig liver, converted vitamin D3 into 25-hydroxyvitamin D3 in substrate concentrations which are within the physiological range (apparentK m = 0.1 μm). The enzyme 25-hydroxylated vitamin D3, 1α-hydroxyvitamin D3 and vitamin D2 and also converted tolterodine, a substrate for human CYP2D6, into its 5-hydroxymethyl metabolite. Tolterodine inhibited the microsomal 25-hydroxylation, whereas quinidine, an inhibitor of CYP2D6, did not markedly inhibit the reaction. The primary structure of the microsomal vitamin D3 25-hydroxylase, designated CYP2D25, shows 77% identity with that of human CYP2D6. Northern blot and reverse transcription-polymerase chain reaction experiments revealed that CYP2D25 mRNA is expressed in higher levels in liver than in kidney and in small amounts in adrenals, brain, heart, intestine, lung, muscle, spleen, and thymus. Experiments with human liver microsomes and recombinantly expressed CYP2D6 strongly indicate that the microsomal 25-hydroxylation of vitamin D3 in human liver is catalyzed by an enzyme different from CYP2D6.

24-, 25- and 27-hydroxylation of cholesterol by a purified preparation of 27-hydroxylase from pig liver

Pig liver mitochondria were found to catalyze 27-, 25- and 24-hydroxylation of cholesterol at relative rates of about 1:0.2:0.04. An apparently homogeneous preparation of pig liver mitochondrial cytochrome P-450-27 was found to catalyze the same three hydroxylations at about the same relative rates when reconstituted with adrenodoxin and adrenodoxin reductase. The 24-hydroxycholesterol formed was shown to consist of one of the two possible stereoisomers. When using specifically deuterium-labeled substrates a significant isotope effect was observed in the case of 24-hydroxylation (KH/KD > 10), but not 25-hydroxylation (KH/KD = 1.1), or 27-hydroxylation (KH/KD = 1.1). The difference between the 24-hydroxylation and the other two hydroxylations may be due to different interactions between cholesterol and the same enzyme, with a resulting difference with respect to the rate-limiting step in the reaction. The physiological significance of the mitochondrial 24-hydroxylation is discussed.

Regulation of human vitamin D3 25-hydroxylases in dermal fibroblasts and prostate cancer LNCaP cells

In this study, we examined whether 1α,25-dihydroxyvitamin D3 (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 D3 25-hydroxylase activity in fibroblasts was suppressed by both calcitriol and efavirenz. In LNCaP cells, consumption of substrate (1α-hydroxyvitamin D3) was used as indicator of metabolism because no 1α,25-dihydroxyvitamin D3 product could be determined. The amount of 1α-hydroxyvitamin D3 remaining in cells treated with calcitriol was significantly increased. Taken together, 25-hydroxylation of vitamin D3 was suppressed by calcitriol and drugs. The present study provides new information indicating that 25-hydroxylation of vitamin D3 may be regulated. In addition, the current results may offer a possible explanation for the impaired bone health after treatment with certain drugs.

Oxysterol 7 alpha-hydroxylase activity by cholesterol 7 alpha-hydroxylase (CYP7A)

A 7α-hydroxylation is necessary for conversion of both cholesterol and 27-hydroxycholesterol into bile acids. According to current theories, cholesterol 7α-hydroxylase (CYP7A) is responsible for the former and oxysterol 7α-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, 7α-hydroxylation of various oxysterols in liver and kidney was investigated. Surprisingly, human cholesterol 7α-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 7α-hydroxylase enzyme fraction from pig liver showed 7α-hydroxylase activity toward the same oxysterols as metabolized by expressed recombinant human and rat CYP7A. The 7α-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 7α-hydroxylase, in addition to the previously known human oxysterol 7α-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.

On the substrate specificity of human CYP27A1 implications for bile acid and cholestanol formation

The mitochondrial sterol 27-hydroxylase (CYP27A1) is required for degradation of the C27-sterol side chain in bile acid biosynthesis. CYP27A1 seems, however, to have roles beyond this, as illustrated by patients with a deficient sterol 27-hydroxylase due to mutations of the CYP27A1 gene [cerebrotendinous xanthomatosis (CTX)]. These subjects have symptoms ranging from accumulation of bile alcohols and cholestanol to accelerated atherosclerosis and progressive neurologic impairment. The present work describes a detailed investigation on the substrate specificity of recombinant human CYP27A1. In accordance with some previous work with rat liver mitochondria, the activity in general increased with the polarity of the substrate. An obvious example was the finding that cholesterol was 27-hydroxylated more efficiently than cholesterol oleate but less efficiently than cholesterol sulfate. The oxysterols 24S-hydroxycholesterol and 25-hydroxycholesterol were 27-hydroxylated less efficiently than cholesterol, possibly due to steric hindrance. Surprisingly, sterols with a 3-oxo-Δ4 structure were found to be hydroxylated at a much higher rate than the corresponding sterols with a 3β-hydroxy-Δ5 structure. The rates of hydroxylation of the sterols were: 7α-hydroxy-4-cholesten-3-one>4-cholesten-3-one>7α-hydroxycholesterol>24-hydroxy-4-cholesten-3-one> cholesterol>25-hydroxy-4-cholesten-3-one>24-hydroxycholesterol⩾25-hydroxycholesterol. The possibility is discussed that the findings may have implications for oxysterol-mediated regulation of gene expression. The very high activity of CYP27A1 towards the cholestanol precursor 4-cholesten-3-one may be of importance in connection with the accumulation of cholestanol in patients with CTX.

Identification of CYP3A4 as the major enzyme responsible for 25-hydroxylation of 5β-cholestane-3α,7α,12α-triol in human liver microsomes

Human liver microsomes catalyze an efficient 25-hydroxylation of 5 beta-cholestane-3 alpha,7 alpha, 12 alpha-triol. The hydroxylation is involved in a minor, alternative pathway for side-chain degr ...

7α-Hydroxylation of 26-hydroxycholesterol, 3β-hydroxy-5-cholestenoic acid and 3β-hydroxy-5-cholenoic acid by cytochrome P-450 in pig liver microsomes

Pig liver microsomes were found to catalyze the 7α‐hydroxylation of several potential bile acid precursors besides cholesterol. 26‐Hydroxycholesterol, 3β‐hydroxy‐5‐cholestenoic acid and 3β‐hydroxy‐5‐cholenoic acid were all efficiently converted into the 7α‐hydroxylated products. Two cytochrome P‐450 fractions showing 7α‐hydroxylase activity could be isolated. One fraction catalyzed 7α‐hydroxylation of 26‐hydroxycholesterol. 3β‐hydroxy‐5‐cholestenoic acid and 3β‐hydroxy‐5‐cholenoic acid but was inactive towards cholesterol. The other fraction catalyzed 7α‐hydroxylation of cholesterol in addition to the other substrates. 26‐Hydroxycholesterol in equimolar concentration did not inhibit the cholesterol 7α‐hydroxylase activity of this fraction. It is concluded that liver microsomes contain a cytochrome P‐450 catalyzing 7α‐hydroxylation of 26‐hydroxycholesterol, 3β‐hydroxy‐5‐cholestenoic acid and 3β‐hydroxy‐5‐cholenoic acid. The results indicate that this cytochrome P‐450 is different from that catalyzing 7α‐hydroxylation of cholesterol.

1α,25-Dihydroxyvitamin D3 exerts tissue-specific effects on estrogen and androgen metabolism.

It is well-known that 1α,25-dihydroxyvitamin D(3) and analogs exert anti-proliferative and pro-differentiating effects and these compounds have therefore been proposed to be of potential use as anti-cancer agents. Due to its effects on aromatase gene expression and enzyme activity, 1α,25-dihydroxyvitamin D(3) has been proposed as an interesting substance in breast cancer treatment and prevention. In the present study, we have examined the effects of 1α,25-dihydroxyvitamin D(3) on estrogen and androgen metabolism in adrenocortical NCI-H295R cells, breast cancer MCF-7 cells and prostate cancer LNCaP cells. The NCI-H295R cell line has been proposed as a screening tool to study endocrine disruptors. We therefore studied whether this cell line reacted to 1α,25-dihydroxyvitamin D(3) treatment in the same way as cells from important endocrine target tissues. 1α,25-Dihydroxyvitamin D(3) exerted cell line-specific effects on estrogen and androgen metabolism. In breast cancer MCF-7 cells, aromatase gene expression and estradiol production were decreased, while production of androgens was markedly increased. In NCI-H295R cells, 1α,25-dihydroxyvitamin D(3) stimulated aromatase expression and decreased dihydrotestosterone production. In prostate cancer LNCaP cells, aromatase expression increased after the same treatment, as did production of testosterone and dihydrotestosterone. In summary, our data show that 1α,25-dihydroxyvitamin D(3) exerts tissue-specific effects on estrogen and androgen production and metabolism. This is important knowledge about 1α,25-dihydroxyvitamin D(3) as an interesting substance for further research in the field of breast cancer prevention and treatment. Furthermore, the observed cell line-specific effects are of importance in the discussion about NCI-H295R cells as a model for effects on estrogen and androgen metabolism.