Cytochrome P-450 Component Research Articles

The Grapefruit Effect: Interaction between Cytochrome P450 and Coumarin Food Components, Bergamottin, Fraxidin and Osthole. X-ray Crystal Structure and DFT Studies.

Coumarins are plant-derived secondary metabolites. The crystal structure of three coumarins—bergamottin, osthole and fraxidin—are described and we analyze intermolecular interactions and their role in crystal formation. Bergamottin is a furanocoumarin found in citrus plants, which is a strong inhibitor of the principal human metabolizing enzyme, cytochrome P450 3A4 (CYP3A4). The crystal structure determinations of three coumarins give us the geometrical parameters and reveal the parallel-displaced π–π stacking and hydrogen bonding intermolecular interactions used for molecular assembly in the crystal structure. A quite strong (less than 3.4 Å) stacking interaction of bergamottin appears to be a determining feature that distinguishes it from other coumarins studied in this work. Our DFT computational studies on the three natural products of the same coumarin family docked into the active site of CYP3A4 (PDB 4D78) show different behavior for these coumarins at the active site. When the substrate is bergamottin, the importance of π-π stacking and hydrogen bonding, which can anchor the substrate in place, appears fundamental. In contrast, fraxidin and osthole show carbonyl coordination to iron. Our docking calculations show that the bergamottin tendency towards π–π stacking is important and likely influences its interactions with the heme group of CYP3A4.

Biomarker considerations in monitoring petrogenic pollution using the mussel Mytilus galloprovincialis.

Mussels are worldwide bioindicators in pollution monitoring since they fulfil the requirements for being good sentinels. However, some methodological concerns arise in the use of particular biomarkers, particularly those displaying low enzymatic rates and/or limited responsiveness to chemicals and biological-related variability. In the present study, the suitability of oxidative stress and detoxification parameters when using mussels as sentinels of polycyclic aromatic hydrocarbon (PAH) pollution is addressed. Present results show that the S9 subcellular fraction of the digestive gland in mussels is an adequate and convenient matrix where to measure most pollution-related biomarkers. Furthermore, this work constitutes the first evidence of the potential suitability of using particular carboxylesterase (CE) activities in determining PAHs exposure in mussels. This fact could imply the replacement of more controversial cytochrome P450 components (phase I oxidation), which are only measurable in microsomal fractions, by CEs (measured in S9 fractions) as good alternatives for phase I reactions in PAH-exposed mussels. Some methodological considerations, such as the need of including commercial purified proteins in biomarker determinations for quality assurance, are evaluated.

A virus-induced gene-silencing system for functional genetics in a betalainic species, Amaranthus tricolor (Amaranthaceae).

Premise of the StudyResearch in Amaranthaceae could be accelerated by developing methods for targeted gene silencing. Most amaranths, including Amaranthus tricolor, produce betalains. However, the physiological and ecological roles of these pigments are uncertain. We sought to establish a virus‐induced gene‐silencing (VIGS) method for amaranths, using silencing of betalain pigments as a proof‐of‐principle.MethodsWe targeted AtriCYP76AD1, a putative cytochrome P450 component of the betalain biosynthetic pathway, using VIGS, and compared two different methods of introducing the VIGS construct into plants. We measured transcript abundance and concentrations of betalains and their l‐DOPA precursor in VIGS‐treated plants, and compared these to controls.ResultsWe observed that when AtriCYP76AD1 was targeted by VIGS in normally red plants, AtriCYP76AD1 and the related genes AtriCYP76AD6 and AtriCYP76AD5 had diminished transcript abundance. Furthermore, newly emergent petioles and leaves of VIGS‐treated plants appeared green, betacyanin accumulation was strongly reduced, and l‐DOPA accumulation was increased. No betaxanthin could be detected in this variety of A. tricolor, either before or after VIGS treatment.DiscussionThese results help to establish the genetic basis of betalain synthesis in amaranths. Furthermore, this is the first report of VIGS in amaranths and demonstrates the potential of this technique for basic and applied research in these species.

Use of Cytochrome P450 Enzyme Isolated From <italic>Bacillus Stratosphericus sp.</italic> as Recognition Element in Designing Schottky-Based ISFET Biosensor for Hydrocarbon Detection

Cytochrome P450 enzyme isolated from an extremophile Bacillus Stratosphericus sp. was used as the recognition element for an ion-sensitive field effect transistor (ISFET)-based biosensor. The fabricated ISFET is Schottky-based and has silicon dioxide as a sensing layer. The cytochrome P450 component in its present purified form (pH-7.2) in 5% agarose was laid over the SiO2 gate to examine its capability of generating electronic response in the presence of n-hexadecane and reduced nicotinamide adenine dinucleotide phosphate. The partially purified preparation was obtained from the lysozyme treated spheroplast via ion exchange chromatography followed by the gel filtration chromatography. The variation of potential difference between the gate and the source with respect to pH for the bare ISFET and the variation of gate and source potential difference with respect to the concentration of the measurand at constant current have been recorded for analyses. The observed correlation showed good repeatability of the sensor output suggesting a potential functional sensing device for hydrocarbon detection. The sensitivity parameters for the device have been determined to be 50.65 mV/pH and 54.34 mV/molar for the bare ISFET and enzyme field-effect transistor, respectively.

Effect of endoplasmic reticulum stress on metabolic and stress signaling and kidney-specific functions in Madin-Darby bovine kidney cells.

Recent studies demonstrated induction of endoplasmic reticulum (ER) stress in tissues of cows after parturition, but knowledge about the effect of ER stress on important cellular processes, such as critical signaling and metabolic pathways, in cattle is scarce. Thus, the present study aimed to investigate the effect of ER stress induction on nuclear factor-κB (NF-κB), nuclear factor E2-related factor 2 (Nrf2), and sterol regulatory element-binding protein (SREBF1) pathway in Madin-Darby bovine kidney (MDBK) cells, a widely used in vitro model in ruminant research. To consider the kidney origin of MDBK cells, the effect on renal distal tubular cell-specific functions, such as transport processes and regulation of 1,25(OH)2D3 levels, was also studied. Treatment of MDBK cells with 2 different ER stress inducers, thapsigargin (TG) and tunicamycin (TM), strongly induced ER stress as evident from induction of ER stress target genes, increased phosphorylation of PKR-like ER kinase, and enhanced splicing of X-box binding protein 1. The TM decreased the protein concentration of NF-κB p50 and the mRNA levels of the NF-κB target genes. Likewise, TG decreased the mRNA concentration of tumor necrosis factor and tended to decrease NF-κB p50 protein and mRNA levels of NF-κB target genes. The mRNA levels of most of the Nrf2 target genes investigated were reduced by TG and TM in MDBK cells. Both ER stress inducers reduced the mRNA levels of SREBF1 and its target genes in MDBK cells. Interestingly, TG decreased, but TM increased the mRNA level of the Ca2+ binding protein calbindin 1, whereas the mRNA level of the plasma membrane Ca2+-transporting ATPase 1 remained unchained. The mRNA level of the cytochrome P450 component 24A1 involved in 1α-hydroxylation of 25(OH)D3 was strongly elevated, whereas the mRNA level of the cytochrome P450 component 27A1 catalyzing the breakdown of 1,25(OH)2D3 was markedly reduced by both ER stress inducers. The concentration of 1,25(OH)2D3 in the supernatant of MDBK cells was increased by approximately 15% by both TG and TM. The present study indicates that under conditions of ER stress, critical signaling pathways, such as NF-κB, Nrf2, and SREBF1, are inhibited, whereas the formation of 1,25(OH)2D3 is stimulated in bovine MDBK cells. Future studies are necessary to clarify the physiological relevance of these findings.

Kinetics and Mechanism of the Reaction of Coherently Synchronized Oxidation and Dehydrogenation of Cyclohexane by Hydrogen Peroxide

Inducing effect of hydrogen peroxide on synchronous oxidation reaction is accompanied by the occurrence of two interconnected and interacting reactions. The reaction of Н 2 O 2 decomposition (primary) generates the leading active • OH and HO • 2 free radicals in a system. During the interaction of active and free radicals with the substrate, the conversion of the substrate occurs in the secondary reaction, coherently-synchronized with the primary one. The mechanism of such coherently synchronized reactions is being examined in the process of cyclohexane oxidation with hydrogen peroxide in homogeneous and heterogeneous systems.The process of coupling dehydrogenation of cyclohexane to cyclohexene and cyclohexadiene with hydrogen peroxide was carried out without catalyst. This process was also carried out at a fairly low temperature in a heterogeneous catalytic system.Biomimetic catalyst, which simulates the basic functions of the enzyme group of oxidoreductase - catalase and monooxygenase, was used as the catalyst here. Characterised by its highly active and selective action these biomimetic catalysts are synthesized based on iron-porphyrin complexes simulating the active component of cytochrome P-450.Based on this experimental researches, the complex reaction, consisting of parallel-sequential oxidation and dehydrogenation reactions, which are coherently synchronized, proceeds during the process of cyclohexane oxidation with biomimetic catalyst. Depending on the reaction parameters it is possible to deliberately adjust the direction of oxidation reaction and reaction rate.

Kinetics and Mechanism of the Reaction of Coherently Synchronized Oxidation and Dehydrogenation of Cyclohexane by Hydrogen Peroxide

Inducing effect of hydrogen peroxide on synchronous oxidation reaction is accompanied by the occurrence of two interconnected and interacting reactions. The reaction of Н 2 O 2 decomposition (primary) generates the leading active • OH and HO • 2 free radicals in a system. During the interaction of active and free radicals with the substrate, the conversion of the substrate occurs in the secondary reaction, coherently-synchronized with the primary one. The mechanism of such coherently synchronized reactions is being examined in the process of cyclohexane oxidation with hydrogen peroxide in homogeneous and heterogeneous systems.The process of coupling dehydrogenation of cyclohexane to cyclohexene and cyclohexadiene with hydrogen peroxide was carried out without catalyst. This process was also carried out at a fairly low temperature in a heterogeneous catalytic system.Biomimetic catalyst, which simulates the basic functions of the enzyme group of oxidoreductase - catalase and monooxygenase, was used as the catalyst here. Characterised by its highly active and selective action these biomimetic catalysts are synthesized based on iron-porphyrin complexes simulating the active component of cytochrome P-450.Based on this experimental researches, the complex reaction, consisting of parallel-sequential oxidation and dehydrogenation reactions, which are coherently synchronized, proceeds during the process of cyclohexane oxidation with biomimetic catalyst. Depending on the reaction parameters it is possible to deliberately adjust the direction of oxidation reaction and reaction rate.

CYP24A1 inhibition facilitates the anti-tumor effect of vitamin D3 on colorectal cancer cells

The effects of vitamin D3 have been investigated on various tumors, including colorectal cancer (CRC). 25-hydroxyvitamin-D3-24-hydroxylase (CYP24A1), the enzyme that inactivates the active vitamin D3 metabolite 1,25-dihydroxyvitamin D3 (1,25-D3), is considered to be the main enzyme determining the biological half-life of 1,25-D3. During colorectal carcinogenesis, the expression and concentration of CYP24A1 increases significantly, suggesting that this phenomenon could be responsible for the proposed efficacy of 1,25-D3 in the treatment of CRC. The aim of this study was to investigate the anti-tumor effects of vitamin D3 on the human CRC cell line Caco-2 after inhibition of the cytochrome P450 component of CYP24A1 activity. We examined the expression of CYP24A1 mRNA and the effects of 1,25-D3 on the cell line Caco-2 after inhibition of CYP24A1. Cell viability and proliferation were determined by means of sulforhodamine-B staining and bromodeoxyuridine incorporation, respectively, while cytotoxicity was estimated via the lactate dehydrogenase content of the cell culture supernatant. CYP24A1 expression was measured by real-time reverse transcription polymerase chain reaction. A number of tetralone compounds were synthesized to investigate their CP24A1 inhibitory activity. In response to 1,25-D3, CYP24A1 mRNA expression was enhanced significantly, in a time- and dose-dependent manner. Caco-2 cell viability and proliferation were not influenced by the administration of 1,25-D3 alone, but were markedly reduced by co-administration of 1,25-D3 and KD-35, a CYP24A1-inhibiting tetralone. Our data suggest that the mechanism of action of co-administered KD-35 and 1,25-D3 does not involve a direct cytotoxic effect, but rather the inhibition of cell proliferation. These findings demonstrate that the selective inhibition of CYP24A1 by compounds such as KD-35 may be a new approach for enhancement of the anti-tumor effect of 1,25-D3 on CRC.

Disposition Kinetic of Amoxicillin in Healthy and Nephropathic Goats with Immunological and Residual Level in Blood and Tissues

In the present investigation, we evaluated disposition kinetics of amoxicillin in healthy as well as nephropathy black-Bengal goats of either sex through single intravenous (IV) route along with metabolism aspects in all vital organs and urine after repeated therapeutic (TP, 10 mg/kg) and one half of therapeutic dose (HTP, 5 mg/kg) consecutively for 60 days through intramuscular administration (IM), evaluation of blood biochemistry and liver cytochrome p450 component. Higher ClB, AUC and lower MRT values in nephropathic goats compared to healthy goats indicates rapid distribution and elimination of drug from plasma when amoxicillin was administered to goats with acute renal failure. Continuous administration through intramuscular route up to 60 days revealed biochemical, hematological, histopathological changes in therapeutics as well as below therapeutic dosage level in blackBengal goats. Present study also revealed immunosuppressive effect during 60 days continuous intramuscular administration and confirmed stimulatory effect on Cytochrome p 450 enzyme system.

Severe ovarian hyperstimulation syndrome after letrozole-gonadotropin stimulation: a case report

Ovarian hyperstimulation syndrome (OHSS) results from an exaggerated response to ovulation induction, characterized by a fluid shift from the intravascular space to third space compartments. The incidence of moderate OHSS is approximately 3–6% and severe OHSS occurs in 0.1–3% of treatment cycles [1, 2]. A number of causal mechanisms have been investigated, involving estradiol (E2), human chorionic gonadotropin (hCG), and vascular endothelial growth factor (VEGF). A strong association between E2 concentrations and risk for developing OHSS has been observed consistently [3]. A growing awareness and interest in treatments aimed at fertility preservation (FP) has led many women to pursue urgent oocyte or embryo cryopreservation. Those with estrogen-sensitive cancers often receive adjunctive treatment with letrozole, in efforts to limit exposure to elevated estrogen concentrations that typically result from exogenous gonadotropin stimulation; letrozole inhibits estrogen production by binding competitively to the cytochrome P450 component of the aromatase enzyme. Combination stimulation regimens yield results comparable to those achieved with standard gonadotropin stimulation protocols, but are associated with significantly lower E2 levels [4]. The risk of OHSS for women receiving adjunctive treatment with letrozole generally is considered low, because E2 levels are markedly lower than in cycles stimulated with gonadotropins alone. In fact, no cases of severe OHSS have been reported in a patient receiving combined treatment with letrozole and gonadotropins. Here, we report a case of severe OHSS arising in a woman with breast cancer after stimulation with exogenous gonadotropins, despite treatment with letrozole and having only moderately elevated E2 concentrations.

A multibiomarker approach in Mytilus galloprovincialis to assess environmental quality

A multibiomarker approach was carried out for the first time in the South Portuguese Coast using Mytilus galloprovincialis, to assess environmental quality, establish if there are adverse biological responses associated to different sources of anthropogenic contamination and to determine spatial and seasonal trends. For this purpose the battery of biomarkers selected was: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidases (GPx total and Se dependent), Cytochrome P450 component system, Glutathione-S-transferase (GST) and acetylcholinesterase (AChE), metallothionein (MT) and lead-delta-aminolevulinic acid dehydratase (ALAD), lipid peroxidation (LPO) and Condition Index (CI) along with the determination of PAHs and metals (Cd, Cu, Ni, Pb and Zn). Results show that despite the levels of both organic and metallic contaminants in these eight spots in the South Coast of Portugal not being particularly high compared with other contaminated/polluted sites worldwide, the selected battery of biomarkers responded efficiently to the environmental changes and allowed an environmental assessment between seasons and sites. Different spatial and seasonal responses were evident along the South Coast of Portugal, meaning that the contamination is not homogeneous. This does not only reflect different competition, origin and intensity of contamination, but also different environmental conditions (e.g. temperature, salinity). Along the South Portuguese Coast site 8 was the most contaminated, while site 2 was considered the least contaminated. Despite environmental factors possibly causing difficulties in the general interpretation of biomarker data, those that better responded to environmental contamination were CYP450, SOD-mit and T-GPx for the summation SigmaPAHs, MT (digestive gland) for metals (especially Cu), ALAD for Pb and LPO for both organic and metallic contamination. These biomarkers were also positively correlated with temperature in summer, revealing this as a more stressful/critical season. In future environmental contamination assessments there is no need to analyse the components b5, P418, NADH and NADPH of phase I MFO system, and MT in the gills, since their responses are not evident.

Immunochemically detected nuclear envelope-associated cytochrome P-450 component(s) in rat hepatocyte culture lines.

The presence and location of cytochrome P-450 in Donryu rat hepatocyte culture lines, Ac2F cells and 3 other cell lines were assessed by indirect immunofluorescence examination using anti-cytochrome P-450 monoclonal antibodies. Ac2F cells and other hepatocyte cell lines were selectively stained at their nuclear envelope, but not the cytoplasm, with a monoclonal antibody selective to a high-spin form of cytochrome P-448 (P-448H), although this monoclonal antibody stained primary cultured normal rat hepatocytes at both cellular components and did not stain hepatoma cells of 2 transplantation lines. The results of unscheduled DNA synthesis assay with Ac2F cells using several carcinogenic aromatic amines (4-aminoazobenzene derivatives and amino acid pyrolysis products) suggested that this nuclear envelope-associated cytochrome P-450 activates a restricted portion of these aromatic amines, i.e., a tryptophan pyrolysis component and a glutamic acid pyrolysis component. These results indicate that rat hepatocyte culture lines lack (or contain a reduced amount of) the cytoplasmic cytochrome P-450 but maintain a characteristic type of cytochrome P-450, probably a kind of cytochrome P-448H in their nuclear envelope, and this may be involved in oxidative metabolism of a restricted portion of aromatic amines.

Subcellular/tissue distribution and responses to oil exposure of the cytochrome P450-dependent monooxygenase system and glutathione S-transferase in freshwater prawns (Macrobrachium malcolmsonii, M. lamarrei lamarrei)

Subcellular fractions (mitochondrial, cytosolic and microsomal) prepared from the tissues (hepatopancreas, muscle and gill) of freshwater prawns Macrobrachium malcolmsonii and Macrobrachium lamarrei lamarrei were scrutinized to investigate the presence of mixed function oxygenase (MFO) and conjugating enzymes (glutathione-S-transferase, GST). Cytochrome P450 (CYP) and other components (cytochrome b(5); NADPH-cytochrome c (CYP) reductase and NADH-cytochrome c-reductase activities) of the MFO system were predominantly present in the hepatic microsomal fraction of M. malcolmsonii and M. lamarrei lamarrei. The results are in agreement with the notion that monooxygenase system is mainly membrane bound in the endoplasmic reticulum, and that the hepatopancreas is the major metabolic tissue for production of biotransformation enzymes in crustaceans. Further, the prawns were exposed to two sublethal (0.9 ppt (parts per thousand) and 2.3 ppt) concentrations of oil effluent. At the end of 30th day, hydrocarbons and detoxifying enzymes were analysed in the hepatopancreas. The accumulations of hydrocarbon in the tissues gradually increased when exposed to sublethal concentrations of oil effluent and were associated with significantly enhanced levels of cytochrome P450 (180.6+/-6.34 pmol mg(-1) protein (P<0.05 versus control, 136.5+/-7.1 pmol mg(-1) protein) for 2.3 ppt and 305.6+/-8.5 pmol mg(-1) protein (P<0.001 versus control, 132.3+/-6.8 pmol mg(-1) protein] for 0.9 ppt of oil exposed M. malcolmsonii; 150+/-6.5 pmol mg(-1 )protein (P<0.01 versus control, 84.6+/-5.2 pmol mg(-1) protein) for 2.3 ppt and 175+/-5.5 pmol mg(-1) protein (P<0.01 versus control, 87.6+/-5.4 pmol mg(-1) protein) for 0.9 ppt of oil exposed M. lamarrei lamarrei), NADPH cytochrome c-reductase activity (14.7+/-0.6 nmol min(-1 )mg(-1) protein (P<0.05 versus control, 6.8+/-0.55 nmol min(-1 )mg(-1) protein) for 2.3 ppt and 12.1+/-0.45 nmol min(-1 )mg(-1) protein (P<0.01 versus control, 6.9+/-0.42 nmol min(-1 )mg(-1) protein) for 0.9 ppt of oil exposed M. malcolmsonii; 12.5+/-0.31 nmol min(-1 )mg(-1) protein (P<0.001 versus control, 4.6+/-0.45 nmol min(-1 )mg(-1) protein) for 2.3 ppt and 9.6+/-0.32 nmol min(-1 )mg(-1) protein (P<0.01 versus control, 4.9+/-0.41 nmol min(-1 )mg(-1) protein) for 0.9 ppt of oil exposed M. lamarrei lamarrei) and cytochrome b(5 )(124.8+/-3.73 pmol mg(-1) protein (P<0.01 versus control, 76.8+/-4.2 pmol mg(-1) protein) for 2.3 ppt and 115.3+/-3.86 pmol mg(-1) protein (P<0.01 versus control, 76.4+/-4.25 pmol mg(-1 )protein) for 0.9 ppt of oil exposed M. malcolmsonii and 110+/-3.11 pmol mg(-1) protein (P<0.01 versus control, 63.7+/-3.24 pmol mg(-1 )protein) for 2.3 ppt and 95.3+/-2.63 pmol mg(-1) protein (P<0.01 versus control, 61.4+/-2.82 pmol mg(-1) protein) for 0.9 ppt of oil exposed M. lamarrei lamarrei). The enhanced levels of biotransformation enzymes in oil-exposed prawns demonstrate a well-established detoxifying mechanism in crustaceans, and the response offers the possibility of use as a biomarker for the early detection of oil pollution.

PTH increases renal 25(OH)D3-1alpha -hydroxylase (CYP1alpha) mRNA but not renal 1,25(OH)2D3 production in adult rats.

The capacity of parathyroid hormone (PTH) to stimulate renal 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] production declines with age in the rat. The purpose of these studies was to determine whether this decline is due to a decreased capacity of PTH to increase the mRNA levels of CYP1alpha, the cytochrome P-450 component of the 25(OH)D(3)-1alpha-hydroxylase. Young (2 mo) and adult (12 mo) male Fischer 344 rats were parathyroidectomized (PTX). After 72 h, PTX rats were injected with PTH or vehicle at 24, 6, and 3 h before death, and renal CYP1alpha mRNA levels were measured by ribonuclease protection assay. In young rats, PTH markedly increased plasma 1,25(OH)(2)D(3) and renal 1,25(OH)(2)D(3) production. However, in adult rats, the response to PTH was less than 30% of that seen in young rats. Renal CYP1alpha mRNA levels, on the other hand, were increased over fivefold by PTH in both young and adult rats. In in vitro studies, PTH/forskolin increased CYP1alpha mRNA levels over twofold in renal slices from both young and adult PTX rats. These studies demonstrate that the decreased capacity of PTH to increase 1,25(OH)(2)D(3) production in adult rats is not due to decreased induction of CYP1alpha mRNA.

Focus on anastrozole and breast cancer

SUMMARYThis commentary article provides an overview of recent clinical research trials involving anastrozole and its evolving role in the management of breast cancer.Anti-aromatase agents inhibit the cytochrome P-450 component of the aromatase enzyme complex responsible for the final step of estrogen biosynthesis in peripheral tissues which are the main source of estrogen in postmenopausal women. Anastrozole is a third-generation non-steroidal aromatase inhibitor. It has been shown to be superior to megestrol acetate, in terms of survival and adverse effects, as a second-line therapy in postmenopausal women with estrogen receptor (ER)- and/or progesterone receptor (PgR)-positive advanced breast cancer. Phase III clinical trials have also demonstrated that anastrozole significantly prolongs the time to tumour progression compared with tamoxifen as a first-line therapy for ER- and/or PgR-positive advanced breast cancer in postmenopausal women. Furthermore, the preliminary results of the Arimidex, Tamoxifen, Alone and in Combination (ATAC) study have shown that adjuvant anastrozole is superior to tamoxifen in terms of disease-free survival (DFS), non-musculoskeletal adverse effects and prevention of contralateral breast cancer in postmenopausal women with early, ER-positive breast cancer. Although longer follow-up is required to assess the long-term effects of anastrozole on bone mineral density, cognitive function and overall survival, the drug has been recently approved for adjuvant use in postmenopausal women with early, ER-positive breast cancer who are unable to tolerate tamoxifen or at an increased risk of developing thromboembolism or endometrial cancer.* Arimidex (anastrozole) is a registered tradename of AstraZeneca Pharmaceuticals, Concord Pike, Wilmington DE, USAThe potential role of anastrozole in the neoadjuvant setting, the management of DCIS, premenopausal breast cancer and breast cancer prevention is currently being investigated.

The evolving role of aromatase inhibitors in breast cancer.

Anti-aromatase agents inhibit the cytochrome p-450 component of the aromatase enzyme complex responsible for the final step of estrogen biosynthesis in peripheral tissues. These drugs can be classified into first-generation (e.g., aminoglutethimide), second-generation (e.g., formestane and fadrazole), and third-generation (e.g., anastrozole, letrozole, and exemestane) agents. Anti-aromatase agents can also be divided into type I and type II inhibitors. Type I inhibitors have a steroidal structure similar to androgens and inactivate the enzyme irreversibly by blocking the substrate-binding site, and are therefore known as aromatase inactivators. Type II inhibitors are nonsteroidal and their action is reversible. This article reviews the recent evidence regarding the role of third-generation aromatase inhibitors in the management of breast cancer. Relevant PubMed listed articles and presentations at recent international symposia were reviewed. There is a growing body of evidence supporting the role of third-generation aromatase inhibitors (anastrozole, letrozole, and exemestane) as first-line and second-line therapy for estrogen receptor (ER)- and/or progesterone receptor (PgR)-positive advanced breast cancer in postmenopausal women, and as a neoadjuvant therapy in postmenopausal women with hormone receptor-positive invasive breast cancer unsuitable for breast-conserving surgery. Furthermore, the preliminary results of the ATAC (Arimidex, Tamoxifen, Alone and in Combination) study have shown that adjuvant anastrozole is superior to tamoxifen in terms of disease-free survival (DFS), adverse effects, and prevention of contralateral breast cancer in postmenopausal women with early, ER-positive breast cancer. However, longer follow-up is required to assess the long-term effects of these agents on bone mineral density, cognitive function, and overall survival prior to considering their routine use in the adjuvant setting instead of tamoxifen. The potential role of these drugs in the management of ductal carcinoma in situ (DCIS), premenopausal breast cancer, and breast cancer prevention is worth investigating.

Cytochrome P450 differences in normal and imposex-affected female whelk Buccinum undatum from the open North Sea

Normal and imposex-affected female Buccinum undatum were sampled from the open North Sea at three locations, one with low, and two with high shipping densities. Cytochrome P450 components and P450 aromatase activity were determined in the microsomal fractions isolated from pooled digestive gland/gonads. Cytochrome P450 aromatase activity was significantly higher (P<0.05) in normal females collected in the low shipping density area (1325±295 fmol/h/mg protein) than levels from imposex animals from a high shipping density area (620±287 fmol/h/mg protein). A negative correlation was found between aromatase activity and organotin body burden (r=−0.99). Levels of CYP450, cytochrome b5 and NADPH cytochrome c reductase activity did not show differences among groups. This is the first field evidence of depressed aromatase activity in imposex affected females, although additional research under laboratory controlled conditions is required to fully understand the mechanisms underlying the development of imposex in this species.

Differential responsiveness of intestinal epithelial cells to 1,25-dihydroxyvitamin D3--role of protein kinase C.

Non-transformed rat intestinal epithelial cell (IEC) lines were used to study the action of 1,25-dihydroxyvitamin D(3) (1,25(OH)2D) in the intestine. The capacity of 1,25(OH)2D to increase the expression of the cytochrome P450 component of the vitamin D 24-hydroxylase (CYP24) was determined in IEC-6 and IEC-18 cell lines. In IEC-6 cells, which are derived from crypt cells isolated from the whole small intestine, 1,25(OH)2D markedly increased expression of CYP24 protein and mRNA within 12 h. In contrast, in IEC-18 cells, which are derived from crypt cells from the ileum only, 1,25(OH)2D did not increase expression of CYP24 until 24-48 h. The maximal levels of CYP24 mRNA seen in the IEC-18 cells were only 31% of the maximal levels seen in the IEC-6 cells. In the presence of 1,25(OH)2D, phorbol esters rapidly increased CYP24 mRNA levels in IEC-18 cells from almost undetectable to levels seen in IEC-6 cells. Protein kinase inhibitors abolished the stimulation by 1,25(OH)2D and by phorbol esters in both cell lines. Stimulation of mRNA levels by phorbol esters required new protein synthesis but stimulation by 1,25(OH)2D did not. These studies demonstrated that the rapid action of 1,25(OH)2D in IEC-6 cells is related to the activation of protein kinase C, an event which is missing in the IEC-18 cells. This differential response to 1,25(OH)2D probably takes place at a post-receptor site, since the number of vitamin D receptors in each cell line was found to be similar.

Purification of cytochrome P450 from filamentous fungus Rhyzopus nigricans

It has been reported in our previous studies that steroid hydroxylation system of Rhizopus nigricans involves cytochrome P450 and NADPH-cytochrome P450 reductase in the electron transport system. Both enzymes are membrane bound and are located in the microsomal preparations of progesterone induced fungal mycelia. In order to identify and characterize the cytochrome P450 component of fungal monoxygenase system, microsomal proteins from induced mycelia were subjected to HIGH Q anion exchange and MONO P (FPLC) anion exchange chromatography. Four fractions containing cytochrome P450 have been resolved on MONO P column. They exhibit CO difference spectra and type II difference spectra with ketoconazole.

Purification of cytochrome P450 from filamentous fungus Rhyzopus nigricans.

It has been reported in our previous studies that steroid hydroxylation system of Rhizopus nigricans involves cytochrome P450 and NADPH-cytochrome P450 reductase in the electron transport system. Both enzymes are membrane bound and are located in the microsomal preparations of progesterone induced fungal mycelia. In order to identify and characterize the cytochrome P450 component of fungal monoxygenase system, microsomal proteins from induced mycelia were subjected to HIGH Q anion exchange and MONO P (FPLC) anion exchange chromatography. Four fractions containing cytochrome P450 have been resolved on MONO P column. They exhibit CO difference spectra and type II difference spectra with ketoconazole.