Nutrient removal in a sequencing batch biofilm reactor (SBBR) using a vertically moving biofilm system.

In this paper, the performance of a sequencing batch biofilm reactor (SBBR) using an anoxic/anaerobic phase followed by an aerobic phase for nutrient removal from wastewater was investigated. In the laboratory SBBR unit, biofilm was grown on a plastic biofilm media module, which was vertically moved up into the air and down into the bulk fluid during the aerobic phase. The vertical movement of the biofilm module supplied oxygen to the microorganisms. The module was submerged in the wastewater during the anoxic/anaerobic phase. The percentage removals of total chemical oxygen demand (COD), total nitrogen (TN) and soluble orthophosphate (PO4-P) were 95.4%, 68.3% and 89.5% respectively at influent concentrations of COD 773 mg l(-1), TN 49.9 mg l(-1) and PO4-P 16.5 mg l(-1). The effluent COD was 35 mg l(-1), NH4-N 10.2 mg l(-1), NO3-N 5.5 mg l(-1), soluble PO4-P 1.7 mg l(-1) and suspended solids (SS) 19 mg l(-1).

The cancer preventative agent resveratrol is converted to the anticancer agent piceatannol by the cytochrome P450 enzyme CYP1B1.

Resveratrol is a cancer preventative agent that is found in red wine. Piceatannol is a closely related stilbene that has antileukaemic activity and is also a tyrosine kinase inhibitor. Piceatannol differs from resveratrol by having an additional aromatic hydroxy group. The enzyme CYP1B1 is overexpressed in a wide variety of human tumours and catalyses aromatic hydroxylation reactions. We report here that the cancer preventative agent resveratrol undergoes metabolism by the cytochrome P450 enzyme CYP1B1 to give a metabolite which has been identified as the known antileukaemic agent piceatannol. The metabolite was identified by high performance liquid chromatography analysis using fluorescence detection and the identity of the metabolite was further confirmed by derivatisation followed by gas chromatography-mass spectrometry studies using authentic piceatannol for comparison. This observation provides a novel explanation for the cancer preventative properties of resveratrol. It demonstrates that a natural dietary cancer preventative agent can be converted to a compound with known anticancer activity by an enzyme that is found in human tumours. Importantly this result gives insight into the functional role of CYP1B1 and provides evidence for the concept that CYP1B1 in tumours may be functioning as a growth suppressor enzyme.

Conformationally restricted hybrid analogues of the hormone 1 alpha,25-dihydroxyvitamin D(3): design, synthesis, and biological evaluation.

Four new conformationally restricted hybrid analogues of the hormone 1 alpha-25-dihydroxyvitamin D(3) (1,25D3) have been synthesized in a convergent manner by combining enantiomerically pure C,D-ring ketones (-)-15 and (-)-17 with racemic 1-hydroxymethyl A-ring phosphine oxide (+/-)-18. Parent hybrid analogue 6, which combines the calcemia-inactivating 1 beta-hydroxymethyl A-ring modification with the antiproliferation- activating 20-epi-22-oxa-25-hydroxydiethyl C,D-ring side chain modification, is comparable in potency to 1,25D3 at the low nM level in inhibiting proliferation in a wide assortment of malignant cell lines in vitro with extremely low calcemic activity in vivo. Surprisingly, both conformationally restricted analogues of 6 (8b and 9b), which incorporate rigidifying units at their 25-hydroxyl side chain termini, retained the desirable antiproliferative, transcriptional, and calcemic activities of the parent compound.

Regulation, function, and tissue-specific expression of cytochrome P450 CYP1B1.

Cytochrome P450 CYP1B1 is a relatively recently identified member of the CYP1 gene family. The purpose of this commentary is to review the regulatory mechanisms, metabolic specificity, and tissue-specific expression of this cytochrome P450 and to highlight its unique properties. The regulation of CYP1B1 involves a variety of both transcriptional and post-transcriptional mechanisms. CYP1B1 can metabolize a range of toxic and carcinogenic chemicals in vitro but in some cases with a unique stereoselectivity. Estradiol 4-hydroxylation appears to be a characteristic reaction catalyzed by human CYP1B1. However, there are considerable species differences regarding the regulation, metabolic specificity, and tissue-specific expression of this P450. In humans CYP1B1 is overexpressed in tumor cells, and this has important implications for tumor development and progression and the development of anticancer drugs specifically activated by CYP1B1.

Maternal cigarette smoking and oxygen diffusion across the placenta.

The aim of this study was to test whether or not adaptations in partial, total and specific oxygen diffusive conductances occur in the placentae of women who smoke cigarettes during pregnancy and help to compensate for intrauterine fetal hypoxic stress. Tissue sections were randomly sampled from human term placentae divided into two groups (non-smokers and smokers) according to maternal smoking status. In smokers, status was expressed as either declared smoking rate or level of plasma cotinine (the major metabolite of nicotine). Sections were analysed stereologically to estimate key structural quantities (vascular volumes, exchange surface areas, tissue diffusion distances). These were combined with previously-published physicochemical quantities (oxygen-haemoglobin reaction rates and tissue oxygen diffusion coefficients) in order to estimate the partial conductances of six tissue compartments of the oxygen pathway: maternal erythrocytes and plasma, villous trophoblast, villous stroma (including fetal capillary wall), fetal plasma and erythrocytes. From partial conductances and birthweights, total and specific conductances were calculated for each placenta. Results were assessed statistically by analyses of variance and t -tests. Despite apparent improvements in the partial conductances of the maternal erythrocytes and plasma, total and specific conductances did not alter significantly in smoking groups. However, the relative biases affecting these estimates may be different in smokers and non-smokers. We conclude that total conductance does not increase in placentae associated with maternal smoking. However, given that the fetus suffers chronic hypoxic stress as a consequence of smoking (evidenced here by elevated haematocrits), even a constant diffusive conductance implies a reduced transplacental partial pressure gradient. This could be a contributory factor to the reduced birthweight.

Bioflavonoids: selective substrates and inhibitors for cytochrome P450 CYP1A and CYP1B1.

Interactions of six naturally occurring flavonoids (acacetin, diosmetin, eriodictyol, hesperetin, homoeriodictyol, and naringenin) with human cytochrome P450 (CYP1) enzymes were studied. The flavones acacetin and diosmetin were potent inhibitors of ethoxyresorufin O-dealkylase (EROD) activity of CYP1A and CYP1B1. Hydroxy and/or methoxy substitutions at the 3' and 4' positions in the flavonoid structures were the major factors involved in conveying selectivity for the different cytochrome P450 enzymes. Eriodictyol, homoeriodictyol and naringenin were very poor inhibitors of human CYP1A EROD activity (IC(50)4 microM). Hesperetin and homoeriodictyol selectively inhibited human CYP1A1 and CYP1B1. Only homoeriodictyol selectively inhibited human CYP1B1 (IC(50) 0.24 microM). Hesperetin was O-demethylated by both human CYP1A1 and 1B1 to eriodictyol, which was then further metabolized by the same enzymes. Hesperetin was not metabolized by human CYP1A2 or CYP3A4.

A quantitative study on the effects of maternal smoking on placental morphology and cadmium concentration.

The aim of this study was to quantify the effects of maternal cigarette smoking on placental morphology, paying particular attention to variables known to be influential in facilitating oxygen diffusion. Structural quantities were estimated by stereological analyses of placental samples drawn from non-smoking and smoking women whose smoking habits were assessed both subjectively (from volunteered cigarette consumption) and objectively (by determining levels of plasma cotinine, a major metabolite of nicotine). Concentrations of placental cadmium were also measured. In the smoking group, maternal and fetal haematocrits were elevated and mean birthweight was reduced. Within placentae, the most significant alterations were increases in cadmium levels, the relative volumes of maternal intervillous space, the relative surface areas of fetal capillaries and decreases in the relative and absolute volumes of fetal capillaries. Findings indicate that changes in capillary volume are the result of a decrease in mean capillary diameter rather than total length. The mean thickness of the trophoblast component of the villous membrane was also increased in the smoking group. Although increased haematocrits suggest that fetuses of smoking mothers suffer hypoxic stress, these morphological changes are likely to compromise, rather than assist, transplacental oxygen transfer. This is in marked contrast to the adaptive changes seen in pregnancies associated with preplacental hypoxia and suggests that other factors might be compromising the fetoplacental unit. Finally, although the morphological changes associated with maternal smoking seem to be the result of an all-or-none, rather than dose-dependent, effect, the available evidence is not conclusive.

In vitro evaluation of sedative drug losses during extracorporeal membrane oxygenation.

Sedative agents are routinely administered to critically ill patients, both on and off extracorporeal membrane oxygenation (ECMO), to enable patients to be comfortable and facilitate patient management. It has been observed empirically in our paediatric intensive care unit that doses of sedative drugs required to achieve desired levels of sedation in ECMO patients are far greater than those used in non-ECMO patients. These differences could not simply be accounted for by differences in patient types, clinical status or sedation levels. We therefore undertook an in vitro evaluation of drug binding in ECMO circuits. This study investigated how the polyvinyl chloride (PVC) and silicone rubber components of neonatal ECMO circuits affect drug delivery in patients through drug sorption. Phase 1 investigated drug uptake by the two polymers in static solutions of known concentrations of four commonly used sedative drugs: lorazepam, midazolam, diazepam and propofol. Phase 2 involved the setting up of a complete neonatal ECMO circuit, injecting the drug solutions pre reservoir at a flow rate of 350 ml/min and collecting samples post-oxygenator for analysis. Phase 1 results revealed significant uptake of drugs with losses in the range 40-98% and in the order propofol > diazepam > midazolam > orazepam. Phase 2 results were similar and in the first 40 min of running an ECMO circuit only 10% of propofol passed through the circuit. These results may help to explain observed clinical phenomena and raise important issues regarding drug dosing in ECMO patients.

Triggered enzymatic degradation of a water-soluble polymer solution using a novel inhibitor.

Controlled enzymatic degradation of solutions of guar galactomannan, a naturally occurring polysaccharide, offers a powerful tool to enhance its usage in a variety of applications. In this study, we use viscometry to investigate the use of tris(hydroxymethyl)aminomethane (Tris) as a reversible, pH-dependent inhibitor to control the degradation of guar galactomannan by Aspergillus niger beta-mannanase. Viscosity measurements of pH 9 guar solutions containing 25 mM Tris show no reduction in the viscosity when treated with beta-mannanase. In contrast, samples containing no Tris show significant reduction in viscosity within an hour. Samples maintained at a pH of 9 in the presence of Tris for several hours show no change in viscosity; however, reduction of the pH of the sample to 4 initiates enzymatic degradation of the guar solution resulting in a rapid reduction in solution viscosity. This reversible, pH-dependent inhibition is possibly a result of the protonation state of Tris as well as key amino acids in the active site.

Rat cytochromes P450 (CYP) specifically contribute to the reductive bioactivation of AQ4N, an alkylaminoanthraquinone-di-N-oxide anticancer prodrug.

1. The bioreductive activation of the alkylaminoanthraquinone di-N-oxide prodrug AQ4N has been characterized in rat hepatic tissue using HPLC. 2. AQ4N was shown to be metabolized to two products, namely AQM, the two electron reduced mono-N-oxide, and AQ4, the four electron reduced active cytotoxic agent. 3. Metabolism was shown to occur in microsomes with an apparent Km = 30.29 microM and Vmax = 1.05 nmol/mg/min. 4. Bioreduction was dependent on anaerobic conditions and the presence of the reduced cofactor NADPH. Ketoconazole (100 microM) and carbon monoxide both inhibited AQ4N metabolism inferring a role for cytochrome P450 (CYP). 5. Microsomes from phenobarbitone and isoniazid-pretreated animals significantly (p < 0.05) enhanced the formation of AQ4 from AQ4N indicating a role for CYP2B and 2E respectively. The involvement of both CYP2B and 2E was confirmed by the use of CYP-specific inhibitors. 6. In conclusion, the involvement of rat hepatic CYP in the reductive bioactivation of the novel antitumour prodrug AQ4N has been established in detail for the first time. These findings highlight an important interspecies difference between the metabolism of AQ4N in rat and man which was shown earlier to be mediated by CYP3A enzymes. The pharmacological significance of this is discussed.

A comparison of basal and induced hepatic microsomal cytochrome P450 monooxygenase activities in the cynomolgus monkey (Macaca fascicularis) and man.

1. The specific activities of hepatic microsomal cortisol 6beta-hydroxylase, coumarin 7-hydroxylase, S-mephenytoin 4'-hydroxylase and phenoxazone hydroxylase and the O-dealkylations of seven homologous alkoxyresorufins were < 3-fold different between the untreated (UT) cynomolgus monkey and man. 2. Heptoxy- and octoxyresorufin O-dealkylase, S-mephenytoin N-demethylase and dextromethorphan O-demethylase specific activities were > 6-fold higher, whereas tolbutamide hydroxylase was almost 5-fold lower in the UT monkey than in man. 3. Phenobarbitone induced (2-6-fold) coumarin 7-hydroxylase, cortisol 6beta-hydroxylase, S-mephenytoin N-demethylase, phenoxazone hydroxylase and benzyloxyresorufin O-dealkylase activities, but not the O-dealkylations of pentoxyresorufin or other alkoxyresorufins, in monkey. 4. Rifampicin induced (2-3-fold) cortisol 6beta-hydroxylase, S-mephenytoin 4'-hydroxylase, S-mephenytoin N-demethylase and tolbutamide hydroxylase activities, the O-dealkylations of methoxy-, ethoxy- and propoxyresorufin and CYP2C- and CYP3A-immunorelated proteins in monkey. 5. Dextromethorphan O-demethylase was significantly reduced by both phenobarbitone and rifampicin treatment in monkey. 6. Beta-naphthoflavone induced (8-39-fold) the O-dealkylations of several alkoxyresorufins, the greatest effect being on propoxyresorufin, but had no effect on the other activities measured in monkey. 7. Constitutive hepatic microsomal CYP2D6-immunorelated proteins were expressed at apparently much higher levels in monkey than in man.

Involvement of human cytochromes P450 (CYP) in the reductive metabolism of AQ4N, a hypoxia activated anthraquinone di-N-oxide prodrug.

PURPOSE: To establish the role of the human cytochromes P450 (CYPs) in the reductive metabolism of the novel anthraquinone di-N-oxide prodrug AQ4N. METHODS AND MATERIALS: Metabolism of AQ4N was conducted in a panel of 17 human phenotyped liver microsomes. AQ4N and metabolites were detected by reverse phase isocratic HPLC. CYP inhibitors and Spearman rank correlation were used to determine the significance of AQ4N metabolism versus specific CYP activity and/or expression. RESULTS: Anaerobic metabolism of AQ4N to the 2-electron reduction product, AQM, and the 4-electron reduced tertiary amine, AQ4, occurred in all 17 human liver microsome preparations. The range (+/- SE) for total AQ4N turnover was 14.26 +/- 1.43 nmol/incubate (highest) to 3.65 +/- 1.05 nmol/incubate (lowest). Metabolism was not detected in the absence of NADPH or microsomes. In aerobic incubates, AQM was less than 4% of anaerobic values whereas AQ4 was undetectable. CYP-mediated metabolism of AQ4N was inhibited completely by ketoconazole (KET) and carbon monoxide (CO), two global inhibitors of CYP-mediated metabolism. AQ4N metabolism correlated significantly with probes for CYP 3A, specifically benzoxylresorufin O-dealkylation [r(s) = 0.70,p <0.01] and tamoxifen N-demethylation (r(s) = 0.85, p < 0.01), but not with probes for CYPs 2C, 2D, and 1A. CYP 3A involvement was confirmed by the use of the CYP 3A specific inhibitor, triacetyloleandomycin (TAO), which repressed the formation of AQM to 13% of the uninhibited value and abolished completely the formation of AQ4. Alpha-naphthoflavone (ANF), an inhibitor of CYP 2C and 1A, had no significant effect on AQ4N metabolism. CONCLUSIONS: These data suggest that the human CYP 3A enzymes can contribute to the reductive metabolism of AQ4N. CYP 3A enzymes are highly expressed in a broad spectrum of human cancers. The results show that AQ4N requires anaerobic conditions for CYP 3A-mediated reduction and hence this subfamily of enzymes is likely to selectively activate AQ4N in hypoxic tumors.

Enhanced expression of cytochrome P450 in stomach cancer.

The cytochromes P450 have a central role in the oxidative activation and detoxification of a wide range of xenobiotics, including many carcinogens and several anti-cancer drugs. Thus the cytochrome P450 enzyme system has important roles in both tumour development and influencing the response of tumours to chemotherapy. Stomach cancer is one of the commonest tumours of the alimentary tract and environmental factors, including dietary factors, have been implicated in the development of this tumour. This type of tumour has a poor prognosis and responds poorly to current therapies. In this study, the presence and cellular localization of several major forms of P450, CYP1A, CYP2E1 and CYP3A have been investigated in stomach cancer and compared with their expression in normal stomach. There was enhanced expression of CYP1A and CYP3A in stomach cancer with CYP1A present in 51% and CYP3A present in 28% of cases. In contrast, no P450 was identified in normal stomach. The presence of CYP1A and CYP3A in stomach cancer provides further evidence for the enhanced expression of specific forms of cytochrome P450 in tumours and may be important therapeutically for the development of anti-cancer drugs that are activated by these forms of P450.

Ketoconazole and fluconazole inhibition of the metabolism of cyclosporin A by human liver in vitro.

The effects of the important antifungal agents, ketoconazole (Ket) and fluconazole (Flu), on the microsomal metabolism of cyclosporin A (CsA) by seven human livers was measured in vitro. A total of eight CsA metabolites were identified by high-performance liquid chromatography, with metabolites AM9 and AM1 predominating. Ket was a stronger inhibitor than Flu for the formation of each of the 8 metabolites; the mean IC50 for the inhibition of total CsA metabolism was 0.26 +/- 0.08 microM and 85.7 +/- 23.9 microM for Ket and Flu, respectively. Inhibition by Ket and Flu was noncompetitive, with Ki = 0.13 microM and 25.1 microM, respectively. There was considerable interindividual variation in the sensitivity of CsA metabolism to inhibition by Ket or Flu and the degree of inhibition was not uniform across the range of individual CsA metabolites. In six of the seven livers tested, Ket and Flu inhibited the aggregate formation of secondary metabolites (AM19, AM49, AM4N9, and AM1c) more than the aggregate formation of primary metabolites (AM9, AM1, and AM4N) and inhibited the formation of AM9 more than AM1. Although the degree of inhibition of total CsA metabolism by Flu correlated directly with the control (uninhibited) rate of total CsA metabolism (r = 0.95), no similar correlation for inhibition by Ket was noted, nor was the magnitude of inhibition by Ket and Flu related. The results are discussed in relation to the inhibition of CsA metabolism by Ket and Flu in patients in vivo and to the possibility of changes in the efficacy and toxicity of CsA as a result of alterations in its metabolite profile.

Tumor-specific expression of cytochrome P450 CYP1B1.

Cytochrome P450 CYP1B1 is a recently cloned dioxin-inducible form of the cytochrome P450 family of xenobiotic metabolizing enzymes. An antibody raised against a peptide specific for CYP1B1 was found to recognize CYP1B1 expressed in human lymphoblastoid cells but not to recognize other forms of cytochrome P450, particularly CYP1A1 and CYP1A2. Using this antibody, the cellular distribution and localization of CYP1B1 were investigated by immunohistochemistry in a range of malignant tumors and corresponding normal tissues. CYP1B1 was found to be expressed at a high frequency in a wide range of human cancers of different histogenetic types, including cancers of the breast, colon, lung, esophagus, skin, lymph node, brain, and testis. There was no detectable immunostaining for CYP1B1 in normal tissues. These results provide the basis for the development of novel methods of cancer diagnosis based on the identification of CYP1B1 in tumor cells and the development of anticancer drugs that are selectively activated in tumors by CYP1B1.

Xenobiotic metabolism in rat, dog, and human precision-cut liver slices, freshly isolated hepatocytes, and vitrified precision-cut liver slices.

Human, rat, and dog phase I and phase II xenobiotic metabolism in precision-cut liver slices and freshly isolated hepatocytes was compared using a range of substrates. Carbamazepine (50 microM) and styrene (2 mM) were used as probes to study the maintenance of cytochrome P450 and epoxide hydrolase-mediated metabolism in male Sprague-Dawley rat, precision-cut liver slices and hepatocytes. Carbamazepine metabolism in both models resulted in the formation of the bioactive 10,11-epoxide (KM = 766 microM and Vmax = 2.5 pmol/min/mg protein in precision-cut slices). Epoxide formation was higher (2.4-fold) in hepatocytes than slices. Styrene was deactivated to styrene diol at a higher rate in hepatocytes (9.7-fold) than slices. The lower rate of metabolism in slices compared with hepatocytes confirms our previous observations using testosterone, 7-ethoxycoumarin, 1-chloro-2,4-dinitrobenzene and 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone in the rat. Testosterone 6 beta-hydroxylation in human liver slices was similar to cultured hepatocytes, but lower than in freshly isolated hepatocytes. 7-Ethoxycoumarin O-deethylation was higher in freshly isolated human hepatocytes, as was the ratio of glucuronide to 7-hydroxycoumarin. Testosterone hydroxylations, 7-ethoxycoumarin O-deethylation, and 1-chloro-2,4-dinitrobenzene conjugation were also lower in male beagle dog slices, compared with freshly isolated hepatocytes. Attempts at long-term preservation of dog liver slices using vitrification and storage for up to 9 days at -196 degrees C resulted in the retention of phase I and phase II metabolism, although conjugation was lower than in freshly prepared slices. Xenobiotic metabolism in short-term incubations is consistently lower in dog and rat precision-cut slices than in freshly isolated hepatocytes; whereas, in humans, this quantitative difference is partly hidden by the large interindividual variation.