CYP

Oxidative metabolism is the most commonly encountered metabolic clearance pathway of xenobiotics by mammalian systems. Similar pathways are often encountered in microorganisms and have been used to metabolise xenobiotics. This paper reviews the background to these observations and the potential uses they imply. It proposes that although microbial metabolism can have a useful role in preparative biocatalysis, any predictive value to man is negated by the lack of correlation with mammalian systems and may potentially be misleading. q

Cumulative evidence demonstrated effective downstream metabolism of pregnenolone in renal tissue. The aim of this study was to evaluate the expression and functional activity of cytochrome P450 side chain cleavage enzyme (CYP11A1), which converts cholesterol into pregnenolone, in adult rat kidney. Immunohistochemical labeling for CYP11A1 was observed in renal cortex and medulla, on structures identified as distal convoluted tubule and thick ascending limb of Henle's loop, respectively. Immunoblotting analysis corroborated the renal expression of the protein in inner mitochondrial membrane fractions. The incubation of isolated mitochondria with the membrane-permeant cholesterol analogue 22Rhydroxycholesterol resulted in efficient formation of pregnenolone, the immediate precursor for the synthesis of all the steroid hormones. The low progesterone production rate observed in these experiments suggested a poor activity of 3␤-hydroxysteroid dehydrogenase enzyme in renal mitochondria. The steroidogenic acute regulatory protein (StAR), involved in the mitochondrial import of cholesterol, was detected in renal tissue at both mRNA and protein level. Immunostaining for StAR showed similar distribution to that observed for CYP11A1. The expression of StAR and CYP11A1 was found to be higher in medulla than in cortex. This enhanced expression of steroidogenesis-related proteins correlated with a greater pregnenolone synthesis rate and higher steroid hormones tissular content measured in medulla. In conclusion, we have established the expression and localization of StAR and CYP11A1 protein, the ability of synthesizing pregnenolone and a region-specific content of sex hormones in the adult rat kidney. These data clearly show that the kidney is a steroid hormones synthesizing organ. It is proposed that the existence in the kidney of complete steroidogenic machinery would respond to a physiological significance.

Polymorphisms that occur in the CYP1A1, CYP17 and TNF-α genes affects hyperkeratinzation process, sebum production and inflammation in acne vulgaris. Polymorphisms of CYP1A1, CYP17 and TNF-α genes can be identified by using PCR and sequencing techniques. The aim of this study is to demonstrate the role of polymorphisms of CYP1A1, CYP17, and TNF-α genes and the interaction polimorphisms of CYP1A1, CYP17 and TNF-α genes to severe acne vulgaris. This study was conducted as an observational study with case-control method, in 64 patients with severe acne, and as controls 73 patients with mild acne and healthy people. Criteria based on Combined Acne Severity Classification. Biospesimen blood 1 ml taken from vena mediana cubiti then performed PCR and sequencing to determine the sequence of nucleotides in DNA fragments. The conclution from the study shows that polymorphisms of CYP1A1, CYP17, TNF-α genes and the interaction polimorphisms of CYP1A1, CYP 17 and TNF-α genes is not a risk factor for severe acne vulgaris, but polymorphism of CYP1A1 gene is a risk factor for acne vulgaris.

Human cytochrome P45017␣ (CYP17), present in mammalian adrenal and gonadal tissues, catalyses both steroid 17-hydroxylation and C17,20 lyase reactions, producing intermediates for the glucocorticoid and androgenic pathways, respectively. The characterisation of this complex enzyme was initially hampered due to low level in vivo expression of CYP17. Heterologous expression systems have contributed greatly to our current knowledge of CYP17's dual catalytic activity. However, due to the hydrophobic nature of this membrane-bound protein, primarily truncated and modified forms of CYP17 are currently being expressed heterologously. Although the N-terminally modified enzyme has been well characterised, protein structure and function studies still necessitate the expression of unmodified, wild-type CYP17. We report here the expression of a catalytically active, unmodified human CYP17 in the industrial methylotrophic yeast, Pichia pastoris. A typical P450 carbon monoxide difference spectrum, with an absorption maximum at 448 nm and a substrate-induced type I spectrum were recorded using a detergent-solubilised cellular fraction containing CYP17. The expressed enzyme catalysed the conversion of progesterone to 17-hydroxyprogesterone as well as 16-hydroxyprogesterone, a product unique to human and chimpanzee CYP17. This is the first report showing the heterologous expression of a fully functional human steroidogenic cytochrome P450 enzyme in P. pastoris.

Human keratinocytes convert 25(OH)D 3 to hormonally active 1␣,25(OH) 2 D 3 and respond to its antiproliferative/prodifferentiating action in vitro and in vivo. Levels and activity of 1␣,25(OH) 2 D 3 are short-lived. 1␣,25(OH) 2 D 3 induces 24-hydroxylase (CYP24) that rapidly metabolizes the hormone, yielding a cascade of side-chain oxidized products and this eventually results in the loss of activity. Aiming at stabilizing the levels of active hormone, we have searched for potent, selective inhibitors of CYP24. Selective inhibition was crucial in order to avoid impairment of 1␣,25(OH) 2 D 3 synthesis, catalyzed by 1␣-hydroxylase -a related member of cytochrome P-450 (CYP) superfamily. We describe here the testing protocol, using primary human keratinocyte cultures as an appropriate source of CYP24 and 1␣-hydroxylase, 3 H-25(OH)D 3 (at physiological concentrations) as substrate and sensitive HPLC techniques to analyze the complex metabolite profiles. Four hundred potential inhibitors were screened by this method; most of them were synthesized in our laboratory. These compounds (entitled azoles) were capable of direct binding to the heme iron and of additional interactions with other parts of the enzyme. In this paper, we present VID400 and SDZ 89 -443, as first examples of powerful selective CYP24 inhibitors. As anticipated, these compounds increased the levels of 1␣-hydroxylated products generated from 3 H-25(OH)D 3 and extended their lifetime. Importantly, blocking of 24-hydroxylation led to a switch in metabolism, namely to preferential conversion of 1␣,25(OH) 2 D 3 to 1␣,25(OH) 2 -3epi-D 3 . As spin-off from our program, selective inhibitors of 1␣-hydroxylase were also found (e.g. SDZ 88 -357). Using 3 H-25(OH)D 3 as substrate in the absence of SDZ 88 -357, CYP24 showed high preference for freshly generated 1␣-hydroxylated metabolites over abundant 25(OH)D 3 . In the presence of SDZ 88 -357, we noticed a great increase in 24-hydroxylation of 3 H-25(OH)D 3 . Besides their use as valuable tools in elucidating regulatory mechanisms, inhibitors of VD hydroxylases may give rise to novel therapeutic strategies, especially in defects of cell growth and differentiation.

<p>Motherisk safety guidelines for codeine use in breastfeeding pamphlet given to all expectant mothers with planned caesarean sections at St. Michaels Hospital in Toronto, Ontario, Canada (14). Women were advised to take codeine for as short a period as possible (3–4 days postpartum) and were advised to seek the care of a physician if they required pain medication beyond this point. Mothers were also advised to breastfeed before taking codeine to maximize the time to eliminate codeine in between feeds. ©Motherisk Program and The Hospital for Sick Children. Reprinted with permission from the Canadian Family Physician.</p

The human adrenal cortex produces aldosterone, cortisol and the so-called adrenal androgens, dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEAS). Within the adult adrenal, the zona glomerulosa produces aldosterone, the zona fasciculata cortisol and the zona reticularis both DHEA and DHEAS. The processes regulating aldosterone and cortisol synthesis are well defined; however, the mechanisms regulating the production of DHEA(S) remain elusive. The emphasis of this review is based on increasing evidence that cytochrome b(5), DHEA sulfotransferase and 3 beta-hydroxysteroid dehydrogenase play crucial roles in regulating production of DHEA(S). Insight into the mechanisms that regulate the synthesis of these key components of DHEA(S) synthesis should provide important clues to the regulation of adrenal androgen biosynthesis.

Several factors influencing the carbon isotope ratios (CIR) of endogenous urinary steroids have been identified in recent years. One of these should be the metabolism of steroids inside the body involving numerous different enzymes. A detailed look at this metabolism taking into account differences found between steroids excreted as glucuronides or as sulphates and hydrogen isotope ratios of different steroids pointed out possibility of unequal CIR at the main production sites inside the male body -the testes and the adrenal glands.

OBJECTIVETo use a large population-based case-control study to test the association between the common genetic variant rs743572 (−34 T to C), prostate cancer risk and circulating levels of several hormones.To use a large population-based case-control study to test the association between the common genetic variant rs743572 (−34 T to C), prostate cancer risk and circulating levels of several hormones.SUBJECTS AND METHODSA previous meta-analysis concluded that reported associations between rs743572 in the promoter of CYP17A1 and prostate cancer risk might reflect publication bias, but a few recent studies reported associations with prostate cancer risk and data suggesting that rs743572 is functional. We genotyped 824 prostate cancer cases and 737 population-based controls, and applied unconditional logistic regression to estimate the association between rs743572 and prostate cancer risk. We also used linear regression of transformed testosterone, androstanediol glucuronide, dehydroepiandrosterone sulphate, androstenedione, sex hormone-binding globulin and oestradiol (circulating levels) measured for controls, to estimate the association between these levels and rs743572. The linear models were adjusted for age and laboratory batch.A previous meta-analysis concluded that reported associations between rs743572 in the promoter of CYP17A1 and prostate cancer risk might reflect publication bias, but a few recent studies reported associations with prostate cancer risk and data suggesting that rs743572 is functional. We genotyped 824 prostate cancer cases and 737 population-based controls, and applied unconditional logistic regression to estimate the association between rs743572 and prostate cancer risk. We also used linear regression of transformed testosterone, androstanediol glucuronide, dehydroepiandrosterone sulphate, androstenedione, sex hormone-binding globulin and oestradiol (circulating levels) measured for controls, to estimate the association between these levels and rs743572. The linear models were adjusted for age and laboratory batch.RESULTSMen with different genotypes had similar circulating levels of all the hormones measured (all P < 0.05). In the case-control comparison using unconditional unadjusted logistic regression, the odds ratios (95% confidence interval) for prostate cancer were 1.07 (0.87–1.32) and 0.94 (0.71–1.25) for the dominant and recessive models, respectively, and for the co-dominant model, 1.10  (0.88–1.36) and 0.99 (0.73–1.35) for carriers of one or two copies of the C allele, respectively. There was no evidence of heterogeneity in the odds ratios by tumour stage (all P > 0.3) and grade (all P > 0.3).Men with different genotypes had similar circulating levels of all the hormones measured (all P < 0.05). In the case-control comparison using unconditional unadjusted logistic regression, the odds ratios (95% confidence interval) for prostate cancer were 1.07 (0.87–1.32) and 0.94 (0.71–1.25) for the dominant and recessive models, respectively, and for the co-dominant model, 1.10  (0.88–1.36) and 0.99 (0.73–1.35) for carriers of one or two copies of the C allele, respectively. There was no evidence of heterogeneity in the odds ratios by tumour stage (all P > 0.3) and grade (all P > 0.3).CONCLUSIONThe results of the present study are consistent with the conclusions of the previous meta-analysis, and suggest that rs743572 has no role in the risk of prostate cancer for men of Caucasian origin.The results of the present study are consistent with the conclusions of the previous meta-analysis, and suggest that rs743572 has no role in the risk of prostate cancer for men of Caucasian origin.

The steroidogenic cytochrome P450 17 (CYP17) enzyme produces dehydroepiandrosterone (DHEA), which is the most abundant circulating endogenous sex steroid precursor. DHEA plays a key role in e.g. sexual functioning and development. To date, no rapid screening assay for effects on CYP17 is available. In this study, a novel assay using porcine adrenal cortex microsomes (PACMs) was described. Effects of twenty-eight suggested endocrine disrupting compounds (EDCs) on CYP17 activity were compared with effects in the US EPA validated H295R (human adrenocorticocarcinoma cell line) steroidogenesis assay. In the PACM assay DHEA production was higher compared with the H295R assay (4.4 versus 2.2 nmol/h/mg protein). To determine the additional value of a CYP17 assay, all compounds were also tested for interaction with CYP19 (aromatase) using human placental microsomes (HPMs) and H295R cells. 62.5% of the compounds showed enzyme inhibition in at least one of the microsomal assays. Only the cAMP inducer forskolin induced CYP17 activity, while CYP19 was induced by four test compounds in the H295R assay. These effects remained unnoticed in the PACM and HPM assays. Diethylstilbestrol and tetrabromobisphenol A inhibited CYP17 but not CYP19 activity, indicating different mechanisms for the inhibition of these enzymes. From our results it becomes apparent that CYP17 can be a target for EDCs and that this interaction differs from interactions with CYP19. Our data strongly suggest that research attention should focus on validating a specific assay for CYP17 activity, such as the PACM assay, that can be included in the EDC screening battery.

Dietary foods and fruits possess an array of flavonoids with unique chemical structure and diverse bioactivities relevant to cancer. Numerous epidemiological studies have validated the inverse relation between the consumption of flavonoids and the risk of cancer. Flavonoids possess cancer blocking and suppressing effects. Flavonoids modulate various CYPs involved in carcinogen activation and scavenging reactive species formed from carcinogens by CYP-mediated reactions. They induce biosynthesis of several CYPs. They are involved in the regulation of enzymes of phase-II responsible for xenobiotic biotransformation and colon microflora. Since cytochromes P450, P-gp and phase-II enzymes are involved in the metabolism of drugs and in the processes of chemical carcinogenesis, interactions of flavonoids with these systems hold great promise for their therapeutic potential. The role of flavonoids also includes the inhibition of activation of pro-carcinogens, inhibition of proliferation of cancer cells, selective death of cancer cells by apoptosis, inhibition of metastasis and angiogenesis, activation of immune response against cancer cells, modulation of the inflammatory cascade and the modulation of drug resistance. This has greatly extended the goal of cancer therapy from eradicating the affected cells to control of the cancer phenotype. Phytotherapy is being used in combination with other therapies as phytonutrients have been shown to work by nutrient synergy.

Thirty-five novel substituted imidazolyl methylene biphenyls have been synthesized as CYP17 inhibitors for the potential treatment of prostate cancer. Their activities have been tested with recombinant human CYP17 expressed in Escherichia coli. Promising compounds were tested for selectivity against CYP11B1, CYP11B2, and hepatic CYP enzymes 3A4, 1A2, 2B6 and 2D6. The core rigidified compounds (30–35) were the most active ones,

of the 3 genes. Our analysis reveals that GMC gene expression levels of cyp19 alone can be used as a robust predictor of phenotypic sex in L. sylvaticus tadpoles. In addition, we validated this method measuring cyp19 mRNA levels in S. tropicalis GMCs. We propose measuring cyp19 as a tool to study the effects of chemical contaminants (including endocrine disrupting compounds) on amphibian gonadal development and sex ratios in the future.

is the key enzyme in the biosynthesis of proinflammatory leukotrienes. The 5-LO gene is a primary target of 1α,25dihydroxyvitamin D 3 (1α,25(OH) 2 D 3 ) and its expression is prominently increased during myeloid cell differentiation. Since no functional vitamin D response element (VDRE) has been reported for this gene so far, we performed in silico screening of the whole 5-LO gene area (84 kb, including 10 kb promoter region) and identified 22 putative VDREs. Both gelshift and reporter gene assays identified four of these candidates as functional VDREs. Their approximate positions are −2,250 (promoter), + 21,400 (intron 2), + 42,000 (intron 4) and + 50,600 (intron 5) in relation to the transcription start site (TSS). Remarkably, the VDRE at position +42,000 is one of the strongest known VDREs of the human genome. Chromatin immunoprecipitation (ChIP) assays demonstrated simultaneous association of vitamin D receptor (VDR), retinoid X receptor (RXR) and RNA polymerase II (Pol II) to the 5-LO gene regions containing two of these four putative VDREs. This indicates DNA looping of the TSS to even very distant gene regions. In summary, we suggest that the upregulation of the primary 1α,25(OH) 2 D 3 target 5-LO is mediated in vivo by a prominent VDRE in intron 4.

Two cytochrome P450 17␣-hydroxylase/17,20-lyase (CYP17) isoforms have been identified in the South African Angora goat (Capra hircus) and have been implicated as the primary cause of hypocortisolism in this subspecies. These goats are the most efficient fibre producing, but least hardy, small stock breed in Southern Africa. Their inability to cope with prolonged exposure to cold and the resulting stock loss which occurs during winter have been the subject of numerous studies. The two isoforms are encoded for by two separate genes, a novel finding for a mammalian species. The enzymes have unique catalytic properties and differ with respect to their 17,20-lyase activities towards 17-hydroxypregnenolone and subsequent androgen production. In vivo assays confirmed that the three resulting genotypes differed in their ability to produce cortisol in response to intravenous insulin injection implicating CYP17 as the primary cause of the observed hypocortisolism.

The cytochrome P450 proteins are monooxygenases which catalyze many reactions involved in drug metabolism and synthesis of cholesterol, steroids and other lipids. CYP17A1 and CYP19A1 proteins involved in steroidogenesis. Majority o polymorphisms have yet not been evaluated for their possible damaging effects on protein structure and function of both the genes. In present analysis, we tried to find out the effect of damage associated nsSNPs of both CYP17A1 and CYP19A1 gene by using various computational algorithms. Out of 205 and 246 nsSNPs reported for CYP17A1 and CYP19A1 gene, total 13 and 7 were predicted to affect the structure and function respectively. R125Q, R416H and F453S substitutions of CYP17A1 gene were not reported previously hence provide the new dataset of unexplored SNPs for their validation. By our results we also found that substitution of mainly Arginine or Phenyl alanine within conserved domain of redox binding, heam binging and redox partner site would cause partial or complete loss of function of both CYP17 and CYP19 gene.

In order to understand the activity specificity of the hamster cytochrome P450 17␣-hydroxylase/17,20-lyase (P450c17), we have studied its structure/activity using three hamster P450c17 recombinant mutants (T202N/D240N/D407H). In transiently transfected COS-1 cells, the mutation T202N reduced 17␣-hydroxylation of pregnenolone and progesterone to 24 and 44% of wild type (WT), respectively, followed by reduced 17,20-cleavage to 71 and 67%, respectively. On the other hand, the mutation D240N decreased specifically 17,20-lyase activity to 61% of WT when incubated with pregnenolone while the mutation D407H only decreased 17␣-hydroxylation to 46% when incubated with progesterone.

AIM OF THE STUDY: The aim of this study was to investigate whether Sutherlandia frutescens, subsp. microphylla (family: Fabaceae/Leguminosa), which is traditionally used to treat symptoms of chronic stress generally associated with increased circulating ...

Neurosteroids are defined as steroids de novo synthesized in the central nervous system. While the production of neurosteroids is well documented in mammals and amphibians, there is less information about teleosts, the largest group of fish. Teleosts have long been known for their high brain aromatase and 5α-reductase activities, but recent data now document the capacity of the fish brain to produce a large variety of sex steroids. This article aims at reviewing the available information regarding expression and/or activity of the main steroidogenic enzymes in the brain of fish. In addition, the distribution of estrogen, androgen, and progesterone nuclear receptors is documented in relation with the potential sites of production of neurosteroids. Interestingly, radial glial cells acting as neuronal progenitors, appear to be a potential source of neurosteroids, but also a target for centrally and/or peripherally produced steroids.

As considerable inter-species differences exist in xenobiotic metabolism, developing new pharmaceutical therapies for use in different species is fraught with difficulties. For this reason, very few medicines have been registered for use in rabbits, despite their importance in inter alia meat and fur production. We have developed a rapid and sensitive screening system for drug safety in rabbits based on cytochrome P450 enzyme assays, specifically CYP1A1, CYP1A2 and CYP3A6, employing an adaptation of the luciferin-based clinical assay currently used in human drug screening. Short-term (4-h) cultured rabbit primary hepatocytes were treated with a cytochrome inducer (phenobarbital) and 2 inhibitors (alpha-naphthoflavone and ketoconazole). In parallel, and to provide verification, New Zealand white rabbits were dosed with 80 mg/kg phenobarbital or 40 mg/kg ketoconazole for 3 d. Ketoconazole significantly increased CYP3A6 gene expression and decreased CYP3A6 activity both in vitro and in...

CYP2B is a drug-metabolizing enzyme expressed in the liver and brain that metabolizes a variety of centrally acting drugs (e.g. propofol, bupropion and nicotine), endogenous neurochemicals (e.g. serotonin and testosterone) and toxins (e.g. chlorpyrifos). Human CYP2B6 is found at higher levels in the brains of smokers, and 7-day nicotine treatment induces rat brain CYP2B while not altering hepatic CYP2B. We characterized the time course of rat brain CYP2B induction by nicotine and determined if nicotinic acetylcholine receptors (nAChRs) mediated this induction. Rats were treated once daily with 1 mg/kg nicotine base or saline s.c. for 1 or 7 days and sacrificed from 30 minutes to 7 days after the last injection. Oneday nicotine treatment did not induce brain CYP2B, whereas 7-day nicotine treatment significantly increased CYP2B expression for up to 24 hours in the frontal cortex and brainstem; these levels returned to baseline by 7 days post-treatment. CYP2B activity was also higher at 24 hours in these regions. No change was seen in the cerebellar CYP2B levels or in vivo activity following nicotine treatment. Time of day of treatment and sacrifice altered the magnitude of brain CYP2B induction while chlorisondamine, an irreversible nAChR blocker, pre-treatment did not block CYP2B induction. Seven-day nicotine treatment resulted in an induction of rat brain CYP2B protein and in vivo activity for up to 24 hours, which would suggest greater local drug metabolism by brain CYP2B. Humans or animals exposed to nicotine may have altered therapeutic drug response, brain levels of neurotransmitters and/or neurotoxicity.

Early age at menarche is a risk factor for breast cancer. A previous study reported a significant positive association between the CYP3A4*1B variant allele and early puberty. We investigated whether polymorphisms of the CYP3A4, CYP17, CYP1B1, and CYP1A2 genes predict the age at onset of menarche. Five hundred eighty-three nulliparous women between ages 17 and 35, of various ethnic backgrounds, completed a questionnaire that included information about menstrual history. Samples of DNA were provided and used to genotype these women for polymorphic variants in the four genes. There was no significant difference in mean age at menarche between women who carried two variant CYP17 A2 alleles (12.5 years) and women who carried one or no variant allele (12.5 years) (P ‫؍‬ 0.8, adjusted for ethnic group and year of birth). Similar results were found for the CYP1B1*3 variant allele and for the CYP1A2*1F variant allele. Women who carried two variant CYP3A4*1B alleles had an earlier mean age at menarche (12.0 years) than women who carried one or no variant allele (12.6 years) (P ‫؍‬ 0.02). However, after adjusting for ethnic group and year of birth, no significant differences in mean age at menarche were found. The polymorphic variants of the CYP3A4, CYP17, CYP1B1, and CYP1A2 genes are unlikely to influence age of menarche.

Human cytochrome P450 17a-hydroxylase (CYP17) catalyses not only the 17a-hydroxlation of pregnenolone and progesterone and the C17,20-side chain cleavage (lyase) of 17a-hydroxypregnenolone, necessary for the biosynthesis of C 21 -glucocorticoids and C 19 -androgens, but also catalyses the 16a-hydroxylation of progesterone. In efforts to understand the complex enzymology of CYP17, structure/function relationships have been reported previously after expressing recombinant DNAs, encoding CYP17 from various species, in nonsteroidogenic mammalian or yeast cells. A major difference between species resides in the lyase activity towards the hydroxylated intermediates and in the fact that the secretion of C 19 -steroids take place, in some species, principally in the gonads. Because human and higher primate adrenals secrete steroids, CYP17 has been characterized in the Cape baboon, a species more closely related to humans, in an effort to gain a further understanding of the reactions catalysed by CYP17. Baboon and human CYP17 cDNA share 96% homology. Baboon CYP17 has apparent K m and V values for pregnenolone and progesterone of 0.9 lM and 0.4 nmolAEh )1 AEmg protein )1 and 6.5 lM and 3.9 nmolAEh )1 AEmg protein )1 , respectively. Baboon CYP17 had a significantly higher activity for progesterone hydroxylation relative to pregnenolone. No 16a-hydroxylase and no lyase activity for 17a-hydroxyprogesterone. Sequence analyses showed that there are 28 different amino acid residues between human and baboon CYP17, primarily in helices F and G and the F-G loop.

Treatment of rats and adrenocortical cells with ginkgolide B (GKB), a purified component of Ginkgo biloba leaf extracts, reduces the mRNA, protein, and ligand-binding levels of the adrenal peripheral-type benzodiazepine receptor (PBR), a mitochondrial cholesterol-binding protein, leading to decreased corticosteroid synthesis. In the Y1 adrenocortical cell line, GKB reduced both PBR levels and cyclic AMP-induced steroid formation. In these cells, GKB, but not various steroids and vitamins, reduced the expression of a reporter gene driven by the DNA sequence -624/-513 relative to the transcription start site of the PBR encoding gene. GKB treatment did not affect the SV40 promoter and increased the cytochrome P450 17alpha-hydroxylase gene promoter driven expression of the reporter gene. Electrophoretic mobility shift assays (EMSAs) indicated the presence of a functional transcriptional element bound to the -624/-513 DNA fragment. This GKB-induced inhibition of PBR was mediated by an interaction with a transcription factor that binds to the -636/-616 PBR-promoter region. Deletion or mutation of this sequence eliminated the DNA-protein interaction and the inhibitory effect of GKB on PBR gene transcription. This DNA-binding protein could be detected in nuclear extracts of rat brain, liver, and testis, but not kidney. It is also present in the human adrenal glands. However, the inhibitory effect following GKB treatment could be seen only in the adrenal glands. These results demonstrate that the GKB-activated inhibition of glucocorticoid production is due to a specific transcriptional suppression of the adrenal PBR gene and suggest that GKB might serve as a pharmacological tool to control excess glucocorticoid formation.

~ iv ~ "Die Schlange, welche sich nicht häuten kann, geht zugrunde. Ebenso die Geister, welche man verhindert, ihre Meinungen zu wechseln; sie hören auf, Geist zu sein." Friedrich Wilhelm Nietzsche "Il serpente che non può cambiar pelle muore. Lo stesso accade agli spiriti ai quali s'impedisce di cambiare opinione: cessano di essere spiriti.

The present study evaluates the worldwide frequency distribution of CYP2C19 alleles and CYP2C19 metabolic phenotypes (&#39;predicted&#39; from genotypes and &#39;measured&#39; with a probe drug) among healthy volunteers from different ethnic groups and geographic regions, as well as the relationship between the &#39;predicted&#39; and &#39;measured&#39; CYP2C19 metabolic phenotypes. A total of 52 181 healthy volunteers were studied within 138 selected original research papers. CYP2C19*17 was 42- and 24-fold more frequent in Mediterranean-South Europeans and Middle Easterns than in East Asians (P&lt;0.001, in both cases). Contrarily, CYP2C19*2 and CYP2C19*3 alleles were more frequent in East Asians (30.26% and 6.89%, respectively), and even a twofold higher frequency of these alleles was found in Native…

Studies of the regulation of androgen synthesis in steroidogenic cells have focused on both transcriptional and post-translational regulation of the proteins that catalyze these reactions: the P450c17 that catalyzes the production of DHEA or androstenedione in consecutive hydroxylase and lyase activities, and the 17f3-hydroxysteroid dehydrogenase (17~3-HSD) that catalyzes the conversion of androstenedione to testosterone. Our studies of the regulation of the CYP17 lyase activity at the molecular level have utilized species-and tissue-specific differences to identify target regulatory sequences. Adenovirus infection of rat CYP17 promoter/luciferase reporter gene constructs in primary cultures of rat adrenal and rat Leydig cells revealed a rat-specific domain between-1 and-108 bp that cause inhibition of both basal and cAMP-induced CYP17 transcription in the adrenal, but not the Leydig cell. In contrast, similar promoter constructs from other species exhibited substantial cAMP-induced transcriptional activity in the rat adrenal. Mutagenesis of the conserved region of the rat and human proteins reveals significant differences in the amino acid domains required for hydroxylase and lyase activities within and between the two species, consistent with their differential regulation of lyase activity. The 17~-hydroxysteroid dehydrogenase (17[5-HSD) reaction requires a viable glucose transporter system for optimal activity, and a high-energy phosphate was discovered to be the requisite product of glucose metabolism in 17fA-HSD activation. These studies have provided insight into potential mechanisms of control of androgen synthesis in the late steroidogenic pathway, at the transcriptional and post-translational levels.

Androgen production in the testis is carried out by the Leydig cells, which convert cholesterol into androgens. Previously, isoflavones have been shown to affect serum androgen levels and steroidogenic enzyme activities. In this study, the effects of lifelong exposure to dietary soy isoflavones on testicular microsomal steroidogenic enzyme activities were examined in the rat. F1 male rats were obtained from a multi-generational study where the parental generation was fed diets containing alcohol-washed soy protein supplemented with increasing amounts of Novasoy, a commercially available isoflavone supplement. A control group was maintained on a soy-free casein protein-based diet (AIN93G). The diets were designed to approximate human consumption levels and ranged from 0 to 1046.6 mg isoflavones/kg pelleted feed, encompassing exposures representative of North American and Asian diets as well as infant fed soy-based formula. Activities of testicular 3␤-hydroxysteroid dehydrogenase (3␤-HSD), P450c17 (CYP17), 17␤-hydroxysteroid dehydrogenase (17␤-HSD) were assayed on post natal day (PND) 28, 70, 120, 240 and 360 while 5␣-reducatase was assayed on PND 28. At PND 28, 3␤-HSD activity was elevated by approximately 50% in rats receiving 1046.6 mg total isoflavones/kg feed compared to those on the casein only diet. A similar increase in activity was observed for CYP17 in rats receiving 235.6 mg total isoflavones/kg feed, a level representative of infant exposure through formula, compared to those receiving 0 mg isoflavones from the casein diet. These results demonstrate that rats fed a mixture of dietary soy isoflavones showed significantly altered enzyme activity profiles during development at PND 28 as a result of early exposure to isoflavones at levels obtainable by humans.

17␣-Hydroxylase deficiency is a rare disease caused by mutation of the CYP17 gene, resulting in hypertension, hypokalemia, female sexual infantilism or male pseudohermaphroditism, low blood cortisol and low plasma renin activity. Herein, we report a female Taiwanese with 17␣hydroxylase deficiency. The CYP17 genes of this patient and five members of her family were analyzed by PCR-direct sequencing. One allele of the patient contains a 9-bp (c. 1459-1467 GACTCTTTC: D487, S488, F489) deletion, which is prevalent in Southeast Asia. The other allele has a 6-bp (c. 1480-1485 AAGGTG: K494, V495) deletion and an R496L (c. 1487 G>T) missense mutation, which is a novel mutation. Site-directed mutagenesis, in vitro expression and functional analysis in HEK-293T cells showed that this novel mutation [K494 V495 Del; R496L] resulted in complete loss of 17␣-hydroxylase and 17,20-lyase activity. Thus this novel mutation in the extreme C-terminus abolishes enzyme activity, and when accompanied by a 9-bp deletion at codons 487-489 in the other allele, results in 17␣-hydroxylase/17,20-lyase deficiency in this patient.

In order to understand the activity specificity of the hamster cytochrome P450 17␣-hydroxylase/17,20-lyase (P450c17), we have studied its structure/activity using three hamster P450c17 recombinant mutants (T202N/D240N/D407H). In transiently transfected COS-1 cells, the mutation T202N reduced 17␣-hydroxylation of pregnenolone and progesterone to 24 and 44% of wild type (WT), respectively, followed by reduced 17,20-cleavage to 71 and 67%, respectively. On the other hand, the mutation D240N decreased specifically 17,20-lyase activity to 61% of WT when incubated with pregnenolone while the mutation D407H only decreased 17␣-hydroxylation to 46% when incubated with progesterone. To comprehend the altered activity profiles of these hamster P450c17 mutants, we have elaborated a 3D model of the hamster P450c17 and compared it to our preceding model of the human P450c17. Analysis of the mutants with this model showed that, without direct contact to the substrates, these mutations transmit structural changes to the active site. By analogy, these results support the concept that any cellular changes modifying the external structure of P450c17, such as phosphorylation, could have influence on its active site and enzymatic activities.

We have exploited the reaction of 1,1-carbonylbis(2-methylimidazole) (CBMI) with several 17␤-hydroxy androstanes to synthesize a series of novel C17 steroidal carbamates. Structural elucidation features have been provided for the final compounds based on 1D and 2D NMR techniques, IR spectroscopy, and related literature. The new compounds were tested for inhibition of human cytochrome 17␣-hydroxylase-C 17,20-lyase (CYP17) and androgen receptor (AR) binding and function effects. Their inhibitory potential against PC-3 cell proliferation was also evaluated. Compounds 11 and 23 were found to inhibit CYP17 with IC 50 values of 17.1 and 11.5 M, respectively. The carbamate moiety at C17 allowed tight binding of the synthesized compounds to both wild-type (wt−) and mutated AR. When bound to the mutated AR, the compounds were found to have a dual effect, stimulating transcription at low concentrations while almost fully blocking it at the higher concentrations tested, in the presence of the natural androgen dihydrotestosterone (DHT). Compounds 8 and 12 were the most active against PC-3 cell proliferation with EC 50 values of 2.2 and 0.2 M, respectively.

While Vitamin D insufficiency in the US and European population is rising, epidemiological studies suggest an inverse correlation between low serum levels of 25-hydroxyvitamin D 3 (25-OH-D 3 ) and colorectal cancer incidence. The antimitotic, prodifferentiating and proapoptotic active metabolite 1␣,25-dihydroxyvitamin D 3 (1,25-(OH) 2 -D 3 ) is synthesized also by colonocytes, since these possess Vitamin D synthesizing (CYP27B1) and catabolic (CYP24) hydroxylases similar to the kidney. Early during colon tumor progression, expression of CYP27B1 and of the Vitamin D receptor increases, suggesting an autocrine/paracrine growth control in colon tissue as a physiological restriction against tumor progression. However, in human adenocarcinomas expression of the catabolic CYP24 is also enhanced when compared with adjacent normal mucosa. Therefore, to maintain colonic accumulation of 1,25-(OH) 2 -D 3 its catabolism needs to be restricted. Our studies in mice show that low nutritional calcium causes hyperproliferation of colon crypts and significant elevation of CYP24 expression, which can be completely abrogated by soy feeding. We suggest that phytoestrogens in soy, known to be estrogen receptor modulators, are responsible for decreased CYP24 expression. These results and our observation that 17␤-estradiol can elevate CYP27B1 expression in rectal tissue of postmenopausal women, may underlie the observed protective effect of estrogens against colorectal cancer in females.

Cumulative evidence demonstrated effective downstream metabolism of pregnenolone in renal tissue. The aim of this study was to evaluate the expression and functional activity of cytochrome P450 side chain cleavage enzyme (CYP11A1), which converts cholesterol into pregnenolone, in adult rat kidney. Immunohistochemical labeling for CYP11A1 was observed in renal cortex and medulla, on structures identified as distal convoluted tubule and thick ascending limb of Henle's loop, respectively. Immunoblotting analysis corroborated the renal expression of the protein in inner mitochondrial membrane fractions. The incubation of isolated mitochondria with the membrane-permeant cholesterol analogue 22Rhydroxycholesterol resulted in efficient formation of pregnenolone, the immediate precursor for the synthesis of all the steroid hormones. The low progesterone production rate observed in these experiments suggested a poor activity of 3␤-hydroxysteroid dehydrogenase enzyme in renal mitochondria. The steroidogenic acute regulatory protein (StAR), involved in the mitochondrial import of cholesterol, was detected in renal tissue at both mRNA and protein level. Immunostaining for StAR showed similar distribution to that observed for CYP11A1. The expression of StAR and CYP11A1 was found to be higher in medulla than in cortex. This enhanced expression of steroidogenesis-related proteins correlated with a greater pregnenolone synthesis rate and higher steroid hormones tissular content measured in medulla.

17␣-Hydroxylase deficiency is a rare disease caused by mutation of the CYP17 gene, resulting in hypertension, hypokalemia, female sexual infantilism or male pseudohermaphroditism, low blood cortisol and low plasma renin activity. Herein, we report a female Taiwanese with 17␣hydroxylase deficiency. The CYP17 genes of this patient and five members of her family were analyzed by PCR-direct sequencing. One allele of the patient contains a 9-bp (c. 1459-1467 GACTCTTTC: D487, S488, F489) deletion, which is prevalent in Southeast Asia. The other allele has a 6-bp (c. 1480-1485 AAGGTG: K494, V495) deletion and an R496L (c. 1487 G>T) missense mutation, which is a novel mutation. Site-directed mutagenesis, in vitro expression and functional analysis in HEK-293T cells showed that this novel mutation [K494 V495 Del; R496L] resulted in complete loss of 17␣-hydroxylase and 17,20-lyase activity. Thus this novel mutation in the extreme C-terminus abolishes enzyme activity, and when accompanied by a 9-bp deletion at codons 487-489 in the other allele, results in 17␣-hydroxylase/17,20-lyase deficiency in this patient.