Showing papers in "Xenobiotica in 1998"
375 citations
TL;DR: The absence and presence of m-HPPA and hippuric acid is suggested to be due to a combination of differences in dietary precursors of substrates for glycine conjugation and a dietary dependent redistribution of the intestinal microflora responsible for breakdown of plant phenolics and aromatic amino acids.
Abstract: 1. A combined in vivo and in vitro study has been devised to investigate an observation, obtained by 1H NMR of urine, that Alp:AprSD (Wistar derived) rats kept under standard husbandry conditions did not excrete urinary hippuric acid (HA). meta-(hydroxyphenyl)-propionic acid ?m-HPPA? was identified as the major aromatic component in urine samples lacking HA. 2. Examination of urine from Alp:APrSD and Zucker (obese negative) rats fed various diets showed that the lack of HA/presence of m-HPPA was due to diet and not to the strain of animal. This observation was reinforced by the demonstration that the administration of benzoic acid (BA) to rats not previously excreting urinary HA resulted in the return of this component to the urinary excretion profile. Thus rats receiving the standard diet were still capable of glycine conjugation. 3. Changing the diet of rats excreting m-HPPA led to the cessation of m-HPPA excretion and the return of HA urine excretion. Interestingly, switching back to the original diet did not cause the loss of HA and the re-emergence of m-HPPA. 4. In vitro studies on the two enzyme systems responsible for glycine conjugation (benzoyl CoA:synthetase and benzoyl CoA:glycine N-acyltransferase) in isolated liver mitochondria showed that m-HPPA did not inhibit either enzyme. However, m-HPPA was not found to be a substrate for the first reaction step explaining why it was found in the urine as the free acid and not as a glycine conjugate. 5. The absence and presence of m-HPPA and hippuric acid is suggested to be due to a combination of differences in dietary precursors of substrates for glycine conjugation and a dietary dependent redistribution of the intestinal microflora responsible for breakdown of plant phenolics and aromatic amino acids. Taken collectively this study emphasises how a simple diet change can cause a profound change in metabolism.
189 citations
TL;DR: Sixteen naturally occurring flavonoids were investigated as substrates for cytochrome P450 in uninduced and Aroclor 1254-induced rat liver microsomes and results indicate that demethylation only occurs in the B-ring when the methoxy group is positioned at C4', and not at the C3'-position.
Abstract: 1. Sixteen naturally occurring flavonoids were investigated as substrates for cytochrome P450 in uninduced and Aroclor 1254-induced rat liver microsomes. Naringenin, hesperetin, chrysin, apigenin, tangeretin, kaempferol, galangin and tamarixetin were all metabolized extensively by induced rat liver microsomes but only to a minor extent by uninduced microsomes. No metabolites were detected from eriodictyol, taxifolin, luteolin, quercetin, myricetin, fisetin, morin or isorhamnetin. 2. The identity of the metabolites was elucidated using lc-ms and 1H-nmr, and was consistent with a general metabolic pathway leading to the corresponding 3',4'-dihydroxylated flavonoids either by hydroxylation or demethylation. Structural requirements for microsomal hydroxylation appeared to be a single or no hydroxy group on the B-ring of the flavan nucleus. The presence of two or more hydroxy groups on the B-ring seemed to prevent further hydroxylation. The results indicate that demethylation only occurs in the B-ring when the methoxy group is positioned at C4', and not at the C3'-position. 3. The CYP1A isozymes were found to be the main enzymes involved in flavonoid hydroxylation, whereas other cytochrome P450 isozymes seem to be involved in flavonoid demethylation.
183 citations
TL;DR: The distribution and excretion of orally administered 14C-labelled 2,2',4,4'-tetrabromodiphenyl ether (TBDE) have been studied in rat and mouse and only a few of the hydroxylated metabolites were present, indicating selective retention of these metabolites.
Abstract: The distribution and excretion of orally administered 14C-labelled 2,2',4,4'- etrabromodiphenyl ether (TBDE) have been studied in rat and mouse.TBDE was efficiently absorbed and stored in adipose tissue where high concen rations were observed in both species.In the rat, 86 % of the dose was retained after 5 days, while 14 % was excreted via the aeces and < 0-5 % via the urine.The mouse excreted 20 % of the dose via the faeces and 33 % via the urine, the latter s a hydrophilic and labile metabolite.Metabolites covalently bound to macromolecules and lipids were noted in tissues nd faeces from both species.The major individual compound was parent TBDE in the faeces and tissues lthough small amounts of five hydroxylated metabolites were indicated by GC-MS.In plasma from both rat and mouse only a few of the hydroxylated metabolites were resent, indicating selective retention of these metabolites.
165 citations
TL;DR: The molecular genetics of the human cytochrome P450 monooxygenase superfamily was studied in this paper. But the results were limited to the human P450 superfamily.
Abstract: (1998). Molecular genetics of the human cytochrome P450 monooxygenase superfamily. Xenobiotica: Vol. 28, No. 12, pp. 1129-1165.
138 citations
TL;DR: No clear structure-activity or selectivity relationship was observed in mouse liver microsomes from induced animals and analysis of the kinetics of inhibition revealed that the inhibition in most cases was non-competitive in nature.
Abstract: 1. The effect of the phenolic compounds protocatechuic acid, chlorogenic acid, tannic acid,gallates and silybinon ethoxyresorufin O-dealkylase(CYP1A1),methoxyresorufin O-dealkylase (CYP1A2) and pentoxy-O-dealkylase (CYP2B) was examined in mouse liver microsomes from induced animals. 2. All compounds tested could inhibit cytochrome P450-mediated enzyme activities, but to different extents. Tannic acid was the most potent inhibitor, especially toward EROD activity with an IC50 = 2.6 μM. Synthetic dodecyl gallate was also relatively selective toward this enzyme activity with an IC50 = 120 μM. 3. Protocatechuic acid,chlorogenicandsilybin were moreselectivetowards PRODand MROD activities. Their relative inhibitory potency for PROD activity was as follows: chlorogenic acid > protocatechuic acid > silybin > dodecyl gallate > propyl gallate. Protocatechuic acid was a more effective inhibitor of MROD activity than chlorogenic acid, and propyl gallate more effective than dodecyl gallate. Thus, no clear structure-ac...
129 citations
TL;DR: In vitro assessment of human cytochrome P450 is described in this paper, where it is shown that P450 can be classified into two classes: 1) in vitro and 2) in vivo.
Abstract: (1998). In vitro assessment of human cytochrome P450. Xenobiotica: Vol. 28, No. 12, pp. 1167-1202.
121 citations
TL;DR: CYP2C9 and, to a much lesser extent, CYP3A4 were found to convert ME to M7, and kinetics of the formation of M7 by the individual enzymes resulted in a Km = 9.6 microM and Vmax = 8.4 pmol/min/mg protein for 2C9, while human liver microsomes formed mostly only the 5-hydroxymethylderivative.
Abstract: 1. The metabolism of Meloxicam (ME) and the cytochrome(s) P450 (CYPs) involved were analysed by using primary human hepatocytes, human liver microsomes and microsomes from recombinant human B-lymphoblastoid cell lines. 2. While human hepatocytes were capable of converting ME to a 5-hydroxymethyl metabolite (M7) and then to a 5-carboxyderivative (M5), human liver microsomes formed mostly only the 5-hydroxymethylderivative. The kinetics of the formation of M7 by human liver microsomes were biphasic with Km = 13.6 +/- 9.5 and 381 +/- 55.2 microM respectively. The corresponding Vmax were 33.7 +/- 24.2 and 143 +/- 83.9 pmol/min/mg protein respectively. 3. CYP2C9 and, to a much lesser extent, CYP3A4 were found to convert ME to M7. The involvement of 2C9 was demonstrated by inhibition of tolbutamide hydroxylase activity in the presence of ME, inhibition of ME metabolism by sulphaphenazole, correlation between ME metabolism and tolbutamide hydroxylase activity and active metabolism of ME by recombinant 2C9. The involvement of 3A4 was shown by inhibition of ME metabolism by ketoconazole, correlation between ME metabolism and nifedipine oxidase activity and metabolism of ME by recombinant 3A4. Kinetics of the formation of M7 by the individual enzymes resulted in a Km = 9.6 microM and Vmax = 8.4 pmol/min/mg protein for 2C9 and a Km = 475 microM and Vmax = 23 pmol/min/mg protein for 3A4.
120 citations
TL;DR: This review considers the human cytochrome P450 (CYP) enzymes responsible for the metabolism of drugs and investigates why a compound is metabolized by a particular isoform (substrate± structure activity relationships, SSAR) and how to assess the expression of that isoform in vivo.
Abstract: (1998). Human cytochrome P450s: selectivity and measurement in vivo. Xenobiotica: Vol. 28, No. 12, pp. 1095-1128.
115 citations
TL;DR: To determine which human cytochrome P450 isoform(s) is responsible for the metabolism of ivermectin, chemical inhibitors including sulphaphenazole, quinidine, furafylline, troleandomycin (TAO) and diethyldithiocarbamate (DDC) were used to evaluate their effect on iverMectin metabolism.
Abstract: 1. Ivermectin was extensively metabolized by human liver microsomes to at least 10 metabolites. The structure of many of them (mostly hydroxylated and demethylated) was determined by 1H-NMR and LC/MS. 2. To determine which human cytochrome P450 isoform(s) is responsible for the metabolism of ivermectin, chemical inhibitors including sulphaphenazole, quinidine, furafylline, troleandomycin (TAO) and diethyldithiocarbamate (DDC) were used to evaluate their effect on ivermectin metabolism. TAO, a specific inhibitor of cytochrome P4503A4, was the most potent inhibitor, inhibiting the total metabolism as well as formation of each metabolite. Metabolism was also inhibited by an anti-human cytochrome 3A4 antibody by 90%. 3. When ivermectin was incubated with microsomes from cells expressing CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1 or 3A4 at 4 mg/ml protein concentrations, metabolic activity was only detected with the microsomes containing CYP3A4. The metabolic profile from cDNA-expressed CYP3A4 microsomes was qualitatively similar to that from human liver microsomes. 4. Thus, cytochrome P4503A4 is the predominant isoform responsible for the metabolism of ivermectin by human liver microsomes.
105 citations
TL;DR: The homology models of CYP2 family enzymes appear to show self-consistency with the currently accumulated information from site-directed mutagenesis and chemical modification of amino acid residues known to affect redox partner interactions.
Abstract: 1. The construction of three-dimensional models of mammalian cytochromes P450 from the CYP2 family is reported based on protein sequence alignment with CYP102, a bacterial P450 of known crystal structure. 2. The homology models of CYP2 family enzymes appear to show self-consistency with the currently accumulated information from site-directed mutagenesis and chemical modification of amino acid residues known to affect redox partner interactions. 3. The generation of these models from the recently reported crystal structure of substrate-bound CYP102 enables the exploration of likely active site contacts with specific substrates of CYP2 family isozymes.
TL;DR: The results suggest that CYP3A4 and CYP1A1 are responsible for the hydroxylation of these endogenous steroids, as well as xenobiotics, in human liver.
Abstract: 1. Hydroxylation activities toward steroid hormones were determined for eleven forms of human hepatic cytochrome P450s expressed in yeast Saccharomyces cerevisiae cells. Microsomes were prepared from the yeast cells and assayed for their regioselectivity of hydroxylation toward progesterone, pregnenolone, dehydroepiandrosterone (DHEA) and oestrone. 2. 6 beta-Hydroxylation of progesterone was catalysed most efficiently by CYP3A4, followed by CYP2D6. CYP3A4 showed the highest progesterone 16 alpha-hydroxylation activity, followed by CYP1A1 and CYP2D6. 16 alpha-Hydroxylation of pregnenolone was catalysed efficiently by CYP1A1 and CYP3A4. Only CYP3A4 exhibited 16 alpha-hydroxylase activities toward DHEA and oestrone. 3. Addition of nifedipine, a typical substrate of CYP3A4, inhibited the 6 beta- and 16 alpha-hydroxylation of progesterone by CYP3A4. 4. These results suggest that CYP3A4 and CYP1A1 are responsible for the hydroxylation of these endogenous steroids, as well as xenobiotics, in human liver.
TL;DR: The mouse and rat exhibit substantial differences in the gender expression of flavin-containing monooxygenase (FMO) forms, and Methimazole, imipramine and thiobenzamide are much better substrates for FMO1 than for F MO3 or FMO5.
Abstract: 1. The mouse and rat exhibit substantial differences in the gender expression of flavin-containing monooxygenase (FMO) forms. Hepatic FMO1 is gender-dependent in both species, selective to the male in rat, female in mouse. Human FMO1 is nearly undetectable. FMO3 in mouse is gender-specific to the female, but gender-independent in rat and man. FMO5 is gender-independent for mouse, rat and man. 2. Gender differences in substrate metabolism do not reflect overall FMO or isoform differences. Methimazole, imipramine and thiobenzamide are much better substrates for FMO1 than for FMO3 or FMO5. 3. Activities of microsomal samples toward these substrates reflect the relative abundance of FMO1. Hepatic samples show a 3-fold greater activity toward methimazole in the female mouse and male rat. Human microsomal samples show minimal activity. 4. Developmentally, FMO1 and FMO5 are expressed in foetuses as early as gestation days 15 and 17 and equally between genders until puberty. FMO3 is not found until 2 weeks post-partum and is found equally in the male and female until 6 weeks post-partum when it becomes undetectable in the male. 5. An event takes place after birth but before puberty that confers the ability to produce FMO3. The developmental pattern observed for mouse FMO3 is similar to human FMO3.
TL;DR: Only CaCo-2 cells were able to produce metabolites similar to those observed in in vivo metabolism studies, whereas all other cell lines were metabolically incompetent and may be used in studies of intestinal biotransformation.
Abstract: 1. Certain chemicals and drugs in addition to metabolically activated carcinogens are substrates for intestinal cytochrome P450s (CYPs) and a number of cell lines are available which could be used in metabolism studies. These include the rat duodenal cell line IEC 6, rat ileal IEC 18, foetal human HuTu 80, foetal human small intestinal FHS 74, human duodenal HCT 8 and human colon CaCo-2 cells, but they lack thorough biochemical characterization. 2. The aim of the present study was therefore to investigate the mRNA and protein expression of CYP1A1, CYP1A2, CYP2C9/10, CYP2E1 and CYP3A. In addition, the metabolism of the immunosuppressant drug tacrolimus and of the procarcinogen 7,12- dimethyl-benz[a]anthracene (DMBA) was studied to obtain information on the functional activity on these cell lines. 3. Of all the cell lines tested only CaCo-2 cells expressed CYP1A1 at the protein and mRNA level, but the CYP2E1 and CYP3A protein was also detected in CaCo-2 and FHS 74 cells. It is of considerable interest that ...
TL;DR: The three-dimensional structures of the CYP2C enzymes are consistent with known experimental evidence from site-directed mutagenesis, antibody recognition and regiospecificity of substrate metabolism, and variations in substrate specificity can be rationalized in terms of single amino acid residue changes within the putative active site region.
Abstract: 1. The results of molecular modelling of human CYP2C isozymes, CYP2C9 and CYP2C19, are reported based on an alignment with a bacterial form of the enzyme, CYP102. 2. The three-dimensional structures of the CYP2C enzymes are consistent with known experimental evidence from site-directed mutagenesis, antibody recognition and regiospecificity of substrate metabolism. 3. The variations in substrate specificity between CYP2C9 and CYP2C19 can be rationalized in terms of single amino acid residue changes within the putative active site region, of which I99H appears to be the most significant.
TL;DR: Directly coupled HPLC-NMR-MS and 19F-N MR spectroscopy proved to be efficient techniques for the unequivocal and rapid determination of the urinary metabolic fate and excretion balance of fluorinated xenobiotics without the need for radiolabelling.
Abstract: 1. The metabolic fate and urinary excretion of 2-bromo-4-trifluoromethylaniline has been studied in rat using 19F-NMR spectroscopic and directly coupled HPLC-NMRMS methods. The compound was dosed to Sprague-Dawley rats (50 mg kg−, i.p.) and urine collected over 0-8, 8-24 and 24-48 h post-dosing. 2. A total urinary recovery of 53.5 ± 7.0% of the dose was achieved up to 48 h after dosing. The major metabolite in the urine was identified as 2-amino-3-bromo-5-trifluoromethylphenylsulphate accounting for a total of 35.7 ± 6.2% of the dose. 3. Further metabolites detected were 2-bromo-4-trifluoromethylphenylhydroxylamine-N-glucuronide (9.7 ± 0.2% of the dose), 2-bromo-4-trifluoromethylaniline-N-glucuronide (3.0 ± 0.3%) and 2-amino-3-bromo-5-trifluoromethylphenylglucuronide (2.8 ± 0.4). Minor metabolites, including 2-bromo-4-trifluoromethylphenylhydroxylamine-O-glucuronide, 2-amino-3-bromo-5-trifluoromethylphenol and 2-bromo-4- trifluoromethylphenylsulphamate, in total accounted for 2.3 ± 0.9% of the dose. 4. Di...
TL;DR: Results suggest that humans with Leu allele of CYP2C9 have lower Vmax's for S-warfarin 7-hydroxylation and tolbutamide methyl hydroxylations than those with wild-type and Cys allele CYP 2C9, although the K(m)'s are not very different in liver microsomes of these three groups of humans.
Abstract: 1. Tolbutamide methyl hydroxylation and racemic warfarin 7-hydroxylation activities were determined in liver microsomes of 39 Japanese and 45 Caucasians genotyped for the cytochrome P450 (P450 or CYP) 2C9 gene into three groups, namely the wild-type (Arg144·Ile359), and two heterozygous Cys allele (Cys144·Ile359) and Leu allele (Arg144·Leu359) variants. 2. Good correlations were found between tolbutamide methyl hydroxylation and racemic warfarin 7-hydroxylation activities in liver microsomes of Japanese and Caucasians. Humans with the Cys allele CYP2C9 variant, which was detected in 22% of Caucasians, were found to have similar catalytic rates to those of the wild-type in the oxidations of tolbutamide and racemic warfarin, whereas humans with the Leu allele, which was detected in 8% Japanese and 7% Caucasian samples, had lower catalytic rates than those of other two groups. 3. The rates of 6- and 7-hydroxylation of racemic warfarin were correlated well with those of S-warfarin, but not R-warfarin, in huma...
TL;DR: This cryopreservation method is suitable for storing liver slices to be used for comparing drug metabolism patterns, at least qualitatively, between species, and retained the measured drug metabolism activities.
Abstract: 1. Precision-cut liver slices represent a suitable and convenient in vitro preparation for studying metabolism and toxicity mechanisms of drugs and toxic chemicals.Particularly in the case of human liver slices, cryopreservation would enable more efficient utilization of this scarce and irregularly available tissue. 2. Liver slices from consecutive human livers were cryopreserved using a method previously developed for rat and monkey liver slices. This procedure involves incubation in 12% dimethyl sulphoxide for 30 min on ice and direct immersion into liquid nitrogen. 3. Functional integrity of cryopreserved human liver slices, as compared with that of fresh liver slices, was maintained at 66±8% (alanine aminotransferase activity retained in the slices), 78±7% (urea synthesis), 88±14% (testosterone hydroxylation), 84±7% (N-deethylation of lidocaine) and 88±10% (total O-deethylation of 7-ethoxycoumarin). The ratios of testosterone metabolites did not change on cryopreservation. 4. These results show that t...
TL;DR: By correlation analysis, chemical inhibition studies and the use of cDNA-expressed CYPs, ZAL N-deethylation to DZAL in human liver appears to be catalysed by CYP3A isoforms.
Abstract: 1. The metabolism of Zaleplon (CL-284,846; ZAL) has been studied in human liver microsomal preparations and in cDNA-expressed human cytochrome P450 (CYP) isoforms. 2. Human liver microsomes catalysed the NADPH-dependent N-deethylation of ZAL to DZAL (CL-284,859), but not to two other known in vivo metabolites, namely M1 (CL-345,644) and M2 (CL-345,905). Sigmoidal kinetic plots were observed for ZAL deethylation indicating positive cooperativity. 3. The metabolism of ZAL to DZAL was determined in a characterized bank of 24 human liver microsomal preparations. Good correlations (r2 = 0.734-0.937) were observed with caffeine 8-hydroxylase, diazepam 3-hydroxylase, dextromethorphan N-demethylase and testosterone 2 beta-, 6 beta- and 15 beta-hydroxylase activities, which are all catalysed by CYP3A isoforms. In contrast, poor correlations (r2 = 0.152-0.428) were observed for enzymatic markers for CYP1A2, CYP2A6, CYP2C9/10, CYP2D6, CYP2E1 and CYP4A9/11. 4. The metabolism of ZAL to DZAL in human liver microsomes was inhibited to 6-15% of control by 5-50 microM of the mechanism-based CYP3A inhibitor troleandomycin. Whereas some inhibition of DZAL formation was observed in the presence of 200 microM diethyldithiocarbamate, 5-50 microM furafylline, 2-20 microM sulphaphenazole, 50-500 microM S-mephenytoin and 1-10 microM quinidine had little effect. 5. Using human B-lymphoblastoid cell microsomes containing cDNA-expressed CYP isoforms, ZAL was metabolized to DZAL by CYP3A4, hut not to any great extent by CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1. 6. In contrast with ZAL, the NADPH-dependent N-deethylation of M2 to M1 proceeded at only a very low rate with both human liver microsomes and cDNA-expressed CYP3A4. 7. In summary, by correlation analysis, chemical inhibition studies and the use of cDNA-expressed CYPs, ZAL N-deethylation to DZAL in human liver appears to be catalysed by CYP3A isoforms.
TL;DR: The results suggest that both tissue slicers can readily produce precision-cut liver slices for studies of xenobiotic metabolism and toxicity, however, the data suggest that for any given application of precision- cut tissue slices it is desirable to establish optimal culture conditions.
Abstract: 1. In this study we have compared freshly cut and cultured precision-cut rat liver slices produced by the Krumdieck and Brendel-Vitron tissue slicers. 2. No significant differences were observed in levels of protein, potassium, total glutathione (i.e. GSH and GSSG), reduced glutathione (GSH) and cytochrome P450 and activities of 7-ethoxyresorufin O-deethylase and 7-benzoxyresorufin O-debenzylase in freshly cut rat liver slices produced by the two tissue slicers. However, levels of oxidized glutathione (GSSG) were significantly greater in liver slices produced with the BrendelVitron tissue slicer. 3. Precision-cut rat liver slices produced with both tissue slicers were cultured for 0 (i.e. a 1-h preincubation), 24 and 72 h in a dynamic organ culture system in an atmosphere of either 95% O2/5% CO2 or 95% air/5% CO2. 4. Apart from small differences in glutathione levels in 0 and 24 h cultured liver slices, no significant differences were observed in the parameters measured between liver slices prepared with ...
TL;DR: The design and function of a modular, small-scale-type bioartificial liver cell culture model is described lending itself for drug metabolism studies but maintaining also typical hepatospecific properties.
Abstract: 1. The aim of the study was the development of a small-scale liver cell bioreactor maintaining tissue monoxygenase activity and hepatospecific activities over at least 2 weeks. 2. For characterization the antihypertensive drug urapidil was used as a model compound to study maintenance of metabolic activity. Tissue-specific parameters assessed included urea and albumin secretion as well as cellular integrity. The problem of the use of serum in bioreactor cultures is addressed. 3. Bioreactor runs could be performed in serum- and lactate-free cultures with a joint recovery of oxidative biotransformation capacity for urapidil as well as tissue-specific markers. LDH release was reduced with older cultures. Fibronectin was shown as a contributing factor for cell attachment. 4. In the present study the design and function of a modular, small-scale-type bioartificial liver cell culture model is thus described lending itself for drug metabolism studies but maintaining also typical hepatospecific properties.
TL;DR: In vitro-in vivo extrapolation indicates that the degree of inhibition of CYP1A2, 2C9 and 3A4 activity which could theoretically be produced in vivo by propofol is relatively low (40-51%); this is considered unlikely to have any pronounced clinical significance.
Abstract: 1. The potential of propofol to inhibit the activity of major human cytochrome P450 enzymes has been examined in vitro using human liver microsomes. Propofol produced inhibition of CYP1A2 (phenacetin O -deethylation), CYP2C9 (tolbutamide 4-hydroxylation), CYP2D6 (dextromethorphan O -demethylation) and CYP3A4 (testosterone 6 beta hydroxylation) activities with IC = 40, 49, 213 and 32 mu M respectively. K for propofol against all of these enzymes with the exception of CYP2D6, where propofolishowed little inhibitory activity, was 30, 30 and 19 mu M respectively for CYPs 1A2, 2C9 and 3A4. 2. Furafylline, sulphaphenazole, quinidine and ketoconazole, known selective inhibitors of CYPs 1A2, 2C9, 2D6 and 3A4 respectively, were much more potent than propofol having IC = 0.8, 0.5, 0.2 and 0.1 mu M; furafylline and sulphaphenazole yielded K = 0.6 and 0.7 mu M respectively. i 3. The therapeutic blood concentration of propofol (20 mu M; 3-4 mu g ml) together with the in vitro K estimates for each of the major human P4...
TL;DR: The metabolic profile was totally modified by beta-glucuronidase hydrolysis, showing that most of the metabolites were glucuronic acid conjugates.
Abstract: 1. [R-2,6-3H]-4-n-nonylphenol was synthesized and a single dose (5 mg, 1850 KBq) orally administered to rainbow trout. After 48 h, the radioactivity present in the bile amounted 5.5%. More than ten biliary metabolites were separated by hplc and collected for subsequent mass spectrometry analysis. The metabolic profile was totally modified by β-glucuronidase hydrolysis, showing that most of the metabolites were glucuronic acid conjugates. 2. Conjugated metabolites were identified by lc-ms analysis and their aglycones were analysed by gc-ms analysis as TMS and acetyl derivatives. 3. The major metabolite accounted for 52 ± 11% of the biliary radioactivity and was identified as nonylphenol-glucuronide. 4. Nonylphenol was hydroxylated at both ω and ω-1 positions of the alkyl chain, giving 9-hydroxynonylphenol and 8-hydroxynonylphenol. 5. 9-Hydroxynonylphenolwasoxidized to the corresponding acid,and subsequently β-oxidized, yielding 7-(4-hydroxyphenyl)heptanoic acid, 5-(4-hydroxyphenyl)pentanoic acid, 3-(4-hydr...
TL;DR: It is concluded that formation of tamsulosin metabolites, AM-1 and M-1, is catalysed by CYP3A4 whereas that of M-3 andM-4 isCatalysed by CyP2D6, however, minor contributions from other CYPs cannot be excluded.
Abstract: 1. The in vitro human liver metabolism of the alpha1-adrenoceptor blocker tamsulosin was investigated. When 14C-tamsulosin was incubated with human liver microsomes, it was converted to five known urinary metabolites and at least three unknown metabolites. Of the former group, the predominant metabolite was the O-deethylated metabolite (M-1), followed by the o-ethoxyphenoxy acetic acid (AM-1) and the m-hydroxylated metabolite (M-3). 2. There was a good linear relationship between AM-1 formation and testosterone 6beta-hydroxylase activity in microsomes from each of 10 individual donors. The rate of M-1 formation also correlated with the same activity, albeit the correlation curve did not pass through the origin. By contrast, the rates of M-3 and the O-demethylated metabolite (M-4) formation correlated with dextromethorphan O-demethylase activity. 3. Ketoconazole strongly inhibited AM-1 formation and reduced that of M-1 by c. 60%. Immunoinhibition studies using anti-rat antibodies supported these results. The formation of M-3 and M-4 was inhibited by quinidine and sparteine. 4. It is concluded that formation of tamsulosin metabolites, AM-1 and M-1, is catalysed by CYP3A4 whereas that of M-3 and M-4 is catalysed by CYP2D6. However, minor contributions from other CYPs cannot be excluded.
TL;DR: The findings show that nicotine is a potent, rapid but transient inducer of CYP1A1 in the rat lung and suggest that the alkaloid is a likely contributor to CYP 1A1 induction by cigarette smoke.
Abstract: 1. We have examined the catalytic activities (7-ethoxyresorufin O -deethylase \[EROD] and 7-methoxyresorufin O -demethylase \[MROD]), protein levels (Western blot analysis) andmRNA levels (Northern blot analysis) of cytochrome P4501A (CYP1A1 and CYP1A2) in the lung, liver and kidney following a single 2.5 mg kg (15.4 mu mol kg) subcutaneous dose of nicotine to the female Sprague-Dawley rat. 2. Only in lung microsomes was EROD activity significantly induced by nicotine treatment.The activity increased 4.4-fold at 6 h after treatment relative to controls, peaked at 12 h at 14.7-fold the control activity and returned to near control level at 24 h. 3. In parallel with EROD activity, CYP1A1 immunoreactive protein abundance was altered significantly by nicotine treatment only in the lung, peaking at 12 h and decreasing towards control levels thereafter. 4. Following subcutaneous nicotine treatment, CYP1A1 mRNA was detectable in the lung at 6 and 12 h but not at 24 h, was slightly elevated in the kidney at 12 h ...
TL;DR: There was an extensive accumulation of radioactivity in the maternal liver after 14C-4-OH-TCB administration and the investigated compounds resulted in a small or no effect on EROD/MROD activity in maternal liver and these enzyme activities were not detectable in either exposed or control foetal liver.
Abstract: 1. At day 17 of pregnancy, 1 day after maternal intravenous administration (5-50 mumol/kg body wt) of 4-OH-3,5,3',4'-tetrachlorobiphenyl (4-OH-TCB; a CB-77 metabolite), a limited dose-dependent decrease was found both in foetal and maternal total thyroxine (T4) levels (76-81% of control at 50 mumol/kg). Similarly, a 50 mumol/kg dose of a 4-OH-3,5,2',3',4'-pentachlorobiphenyl (4-OH-PeCB1) decreased total T4 levels, whereas 4-OH-2,3,5,3',4'-pentachlorobiphenyl (4-OH-PeCB2) showed no clear effect (both 4-OH-pentaCBs are CB-105 metabolites). Earlier administration (gestation day 10 or 13) of the 4-OH-PCBs had no effect on total T4 at day 17. 2. Placental transfer of 14C-4-OH-TCB to the foetal compartment was dose-related and accumulated mainly in foetal plasma at levels 2-fold those in the maternal plasma at the dose interval 0.5-5.0 mumol/kg body wt, whereas at higher doses (20 and 50 mumol/kg body wt) the foetal and maternal plasma levels were similar. A break-point in the foetal dose/plasma concentration curve at 5.0 mumol/kg indicates saturation of a high-affinity ligand binding above this dose. 3. There was an extensive accumulation of radioactivity in the maternal liver after 14C-4-OH-TCB administration (20-30% of the administered dose). In spite of this the investigated compounds resulted in a small or no effect on EROD/MROD activity in maternal liver and these enzyme activities were not detectable in either exposed or control foetal liver.
TL;DR: C cultured human liver slices can be used to evaluate the effect of chemicals derived from cruciferous and other vegetables on CYP isoforms and it is demonstrated that DIM induces CYP1A isoforms in culturedhuman liver slices.
Abstract: 1. The effect of 3,3′-diindolylmethane (DIM), an indole derivative derived from cruciferous vegetables, on cytochrome P450 (CYP) isoforms in the CYP1A and CYP3A subfamilies has been studied in 72-h cultured human liver slices. 2. In cultured human liver slices 50 μM DIM induced 7-ethoxyresorufin O-deethylase and to a lesser extent 7-methoxyresorufin O-demethylase activities. 3. Western immunoblotting of liver slice microsomes was performed with antibodies to ratCYP1A2 and human CYP3A4. Compared with control liver slice microsomes (dimethyl sulphoxide-only treated), DIM induced levels of CYP1A2 but had little effect on levels of CYP3A4. The treatment of human liver slices with 2 μg ml of the polycholorinated biphenyl mixture Aroclor 1254 also resulted in an induction of levels of CYP1A2, but had no effect on CYP3A4. 4. These results demonstrate that DIM induces CYP1A isoforms in cultured human liver slices. Some variability in the magnitude of induction of enzyme activities by DIM was observed in four huma...
TL;DR: It is concluded that all the flavonoids studied inhibit ECOD activity by interfering with the binding of substrate to the active site and other site(s) of the enzyme and that their structural differences lead to different binding affinities at the active sites and possibly to binding at other sites of the enzymes for the Flavonoids.
Abstract: 1. The inhibitory effects of several naturally occurring flavonoids and related compounds on cytochrome P450-dependent 7-ethoxycoumarin O-deethylase (ECOD) and the structure-activity relationships were studied in liver microsomes from rats treated with 3-methylcholanthrene (MC). 2. All the flavonoids (flavone, apigenin, chrysin, flavonol, fisetin, kaempferol, morin, myrisetin, quercetin, flavanone, hesperetin and naringenin) studied inhibited microsomal ECOD activity in the following order: flavones > flavonols > flavanones, were mixed type inhibitors and had Ki in the range of 0.17-4.5 microM. (+/-)-Catechin had no effect. 3. The double bond between C2 and C3 of the C ring, the keto group and hydroxyl group of this ring in the flavonoids seem to play major roles in inhibiting the ECOD activity. 4. The hydroxyl groups in the C5 and C7 positions of A ring in the flavone and the hydroxyl group in the C3 position of C ring in the flavonol classes, respectively, were important factors for the inhibition of the enzyme. 5. In a series of 3, 5, 7-trihydroxyflavones, the hydroxyl group at the C4 in the B ring was also an important factor for the inhibition of ECOD activity, but hydroxyl groups in other positions of the B ring had little effect on the inhibition. 6. We conclude that all the flavonoids studied inhibit ECOD activity by interfering with the binding of substrate to the active site and other site(s) of the enzyme and that their structural differences lead to different binding affinities at the active site and possibly to binding at other site(s) of the enzyme for the flavonoids.
TL;DR: To avoid misinterpretation and overinterpretation of data, it is essential to have a good understanding of the pharmacokinetic basis of indirect measures of in vivo drugmetabolizing activity, and of appropriate statistical procedures for the analysis of frequency distributions.
Abstract: ethnic dia erences in the in vivo metabolism of drugs and other xenobiotics. The key issues in assessing drug-metabolizing enzyme activity in vivo include separation (with the aid of genotyping) of risk and exposure; choice of the best experimental index to use, the selection and number of subjects for study, and the use of objective criteria for assessing multimodality in frequency distributions of in vivo data. To avoid misinterpretation and overinterpretation of data, it is essential to have a good understanding of the pharmacokinetic basis of indirect measures of in vivo drugmetabolizing activity, and of appropriate statistical procedures for the analysis of frequency distributions. Pharmacokinetic basis for using indirect induces of drug metabolism Partial intrinsic clearance In theory, the closest in vivo measure of the activity of the gene product, that is the enzyme, ea ecting a particular metabolic pathway, i, of a drug is its intrinsic clearance down that route. This partial intrinsic clearance is de® ned in terms of unbound drug concentration in plasma ( CLu int, mi ), and can be viewed as the quotient of the apparent V max and K m of the drug with respect to the enzyme (Wilkinson and Shand 1975). Calculation of partial intrinsic clearance after oral drug administration requires measurement of the urinary recovery of the metabolite [Ae(mi) po ], the AUC (total area under the plasma drug concentration± time curve), plasma binding (indicated by fu, the free fraction) and renal clearance ( CL R ) of the
TL;DR: The results indicate that CP dog hepatocytes are a suitable in vitro system for xenobiotic metabolism since enzyme functions in CP hepatocytes were stabilized and cofactors in freshly thawedCP hepatocytes should be measured and controlled for optimal use.
Abstract: 1. Dog hepatocytes were cryopreserved at 6 × 106 viable cells/ml in a suspension buffer containing 10% DMSO and were stored in liquid nitrogen. 2. The exclusion of trypan blue dye was 96± 2 and 85± 9% in fresh and cryopreserved (CP) hepatocytes, respectively. Albumin synthesis was unaffected by freezing. 3. Ethoxycoumarin and ethoxyresorufin O-deethylase activities were equivalent in fresh and CP hepatocytes. 4. The profile of testosterone metabolism was unaffected by freezing. Total hydroxylase activities were 815± 33 pmol/min/106 CP cells in freshly isolated whole hepatocytes and 463± 24 pmol/min/106 CP whole hepatocytes, but they were equivalent in fresh and CP hepatocyte homogenates supplemented with 250 μM NADPH. 5. Phase 2 enzymes were functional in freshly thawed CP hepatocytes but they required exogenous addition of cofactors (20 μM UDPGA and 1.7 μM PAPS). 6. When placed in suspension for longer times, fresh and CP cell viabilities were 88± 6 and 64± 2% after 4 h. ECOD and EROD activities were equ...