Ethoxycoumarin Deethylase Activity Research Articles

Metabolism of 7-ethoxycoumarin, flavanone and steroids by cytochrome P450 2C9 variants.

CYP2C9 is a human microsomal cytochrome P450c (CYP). Much of the variation in CYP2C9 levels and activity can be attributed to polymorphisms of this gene. Wild-type CYP2C9 and mutants were coexpressed with NADPH-cytochrome P450 reductase in Escherichia coli. The hydroxylase activities toward 7-ethoxycoumarin, flavanone and steroids were examined. Six CYP2C9 variants showed Soret peaks (450nm) typical of P450 in reduced CO-difference spectra. CYP2C9.38 had the highest 7-ethoxycoumarin de-ethylase activity. All the CYP2C9 variants showed lower flavanone 6-hydroxylation activities than CYP2C9.1 (the wild-type). CYP2C9.38 showed higher activities in testosterone 6β-hydroxylation, progesterone 6β-/16α-hydroxylation, estrone 11α-hydroxylation and estradiol 6α-hydroxylation than CYP2C9.1. CYP2C9.40 showed higher testosterone 17-oxidase activity than CYP2C9.1; CYP2C9.8 showed higher estrone 16α-hydroxylase activity and CYP2C9.12 showed higher estrone 11α-hydroxylase activity. CYP2C9.9 and CYP2C9.10 showed similar activities to CYP2C9.1. These results indicate that the substrate specificity of CYP2C9.9 and CYP2C9.10 was not changed, but CYP2C9.8, CYP2C9.12 and CYP2C9.40 showed different substrate specificity toward steroids compared with CYP2C9.1; and especially CYP2C9.38 displayed diverse substrate specificities towards 7-ethoxycoumarin and steroids.

Omeprazole inhibits oxidative drug metabolism. Studies with diazepam and phenytoin in vivo and 7-ethoxycoumarin in vitro.

The effect of treatment with omeprazole, a substituted benzimidazole, on the elimination of diazepam and phenytoin was studied in two groups of 8 subjects. Omeprazole given orally in a daily dose of 40 mg over 7 days decreased diazepam plasma clearance from 22.4 +/- 2.8 to 10.1 +/- 1.5 ml/kg X h and prolonged the half-life of diazepam from 36.9 +/- 4.1 to 85.0 +/- 14.7 h. Plasma concentrations of desmethyldiazepam, a diazepam metabolite, were reduced after omeprazole treatment. Omeprazole also reduced the plasma clearance of phenytoin from 25.1 +/- 2.0 to 21.4 +/- 1.8 ml/kg X h and prolonged its half-life from 20.7 +/- 1.9 to 26.3 +/- 2.7 h. Renal excretion of the major metabolite of phenytoin (p-hydroxyphenyl-phenyl-hydantoin) was not changed. Omeprazole did not affect the volume of distribution and the plasma protein binding of either diazepam or phenytoin. In vitro studies with human liver microsomes showed that omeprazole in equimolar concentrations (0.5 mM) was a stronger inhibitor than cimetidine of 7-ethoxycoumarin deethylase activity. These data confirm that omeprazole interferes with the elimination of other drugs by an inhibition of the drug-metabolizing monooxygenase system of the human liver.

Mechanism-Based Inactivation of Lung-Selective Cytochrome P450 CYP2F Enzymes

3-Methylindole (3MI) is a pneumotoxin that requires P450-catalyzed metabolic activation (dehydrogenation), to an electrophilic methylene imine to elicit toxicity. Previous studies have shown that the human pulmonary cytochrome P450 enzyme, CYP2F1, and its goat analog, CYP2F3, catalyzed the dehydrogenation of 3MI. However, it was not known whether the dehydrogenation product could bind to active site nucleophilic residues to inactivate these enzymes. Therefore, the purpose of this study was to determine whether 3MI is a mechanism-based inhibitor of CYP2F3 and CYP2F1. The results showed that both enzymes were highly susceptible to 3MI-mediated suicide inactivation. The k(inact) and the K(I) for CYP2F3 were 0.09/min and 160 microM, respectively, and the approximate partition ratio was 220. Although CYP2F3 lost approximately 80% of its activity in 30 min, a concurrent loss of its reduced carbon monoxide complex was not observed, suggesting that the heme was not destroyed/modified during the inactivation. The exogenous nucleophile, glutathione, did not protect CYP2F1 from 3MI-mediated inactivation, suggesting that the reactive intermediate did not diffuse from the active site before inactivation events. Dialysis of 3MI-inactivated CYP2F3 did not restore activity, and alternate substrates protected CYP2F3. In addition, 3MI inhibited the 7-ethoxycoumarin deethylase activity of human CYP2F1 in a time- and concentration-dependent manner; the k(inact) and K(I) were 0.025/min and 49 microM, respectively. In conclusion, this study presents evidence that 3MI is a mechanism-based inhibitor of both CYP2F3 and CYP2F1, which are important enzymes in the bioactivation of pneumotoxicants such as 3MI or 1,1-dichloroethylene or carcinogens such as naphthalene, benzene, and styrene.

Cytochrome P450 expression and activities in human tongue cells and their modulation by green tea extract

The expression, inducibility, and activities of several cytochrome P450 (CYP) enzymes were investigated in a human tongue carcinoma cell model, CAL 27, and compared with the human liver model HepG2 cells. The modulation effects of green tea on various CYP isoforms in both cell lines were also examined. RT-PCR analysis of CAL 27 cells demonstrated constitutive expression of mRNA for CYPs 1A1, 1A2, 2C, 2E1, 2D6, and 4F3. The results were negative for CYP2A6, 2B6/7, 3A3/4, and 3A7. Both cell lines displayed identical expression and induction profiles for all of the isoforms examined in this study except 3A7 and 2B6/7, which were produced constitutively in HepG2 but not Cal-27 cells. CYP1A1 and 1A2 were both induced by treatment with β-napthoflavone as indicated by RT-PCR and Western blotting, while CYP2C mRNA was upregulated by all-trans retinoic acid and farnesol. RT-PCR and Western blot analysis showed that the expressions of CYP1A1 and 1A2 were induced by green tea extract (GTE), which also caused an increase in mRNA for CYP2E1, CYP2D6, and CYP2C isoforms. The four tea catechins, EGC, EC, EGCG and ECG, applied to either HepG2 or Cal-27 cells at the concentration found in GTE failed to induce CYP1A1 or CYP1A2, as determined by RT-PCR. Of the isoforms that were apparently induced by GTE, only 7-ethoxycoumarin deethylase (ECOD) activity could be detected in CAL 27 or HepG2 cells. Interestingly, mRNA and protein for CYP1A1 and CYP1A2 were detected in both cell lines, and although protein and mRNA levels of CYP1A1 and CYP1A2 were increased by GTE, the observed ECOD activity in both cell lines was decreased.

Establishment of a Novel Host, High-Red Yeast That Stably Expresses Hamster NADPH–Cytochrome P450 Oxidoreductase: Usefulness for Examination of the Function of Mammalian Cytochrome P450

A novel strain ofSaccharomyces cerevisiaeuseful for expression studies of mammalian microsomal cytochrome P450s was established and named High-red yeast. Hamster NADPH–cytochrome P450 oxidoreductase (P450 reductase) cDNA to be introduced into yeast was isolated from a hamster liver cDNA library. The cDNA was 2421 bp long and contained an entire coding region for 667 amino acids. The NH2-terminal amino acid sequence deduced from the hamster P450 reductase cDNA was identical with that of the enzyme purified from hamster livers except for deletion of the initial methionine. A δ-sequence derived from yeast retrotransposon Ty was cloned and used as a sequence for homologous recombination in a yeast genome.S. cerevisiaeYPH500 was transformed with a multi-integration cassette containing the expression unit of the hamster P450 reductase and the δ-sequence. The transformant showing the highest activity of the P450 reductase was named High-red yeast. High-red yeast carried more than six copies of the multi-integration cassettes in a single chromosome and retained the multi-integration cassettes over a period of 100 generations under nonselective culture conditions, indicating that this yeast was a mitotically stable transformant. The microsomes prepared from High-red yeast had 20 times the P450 reductase activity of the microsomes prepared from the parental yeast. Due to the high activity of the hamster P450 reductase, the 7-ethoxycoumarin deethylase activity of mouse CYP1A1 expressed in High-red yeast was 250 times higher than the activity of mouse CYP1A1 expressed in the parental yeast.

Mechanism of Resistance to Deltamethrin inCydia pomonella(L.) (Lepidoptera: Tortricidae)

The pathways for deltamethrin detoxification were studied in fifth instar larvae of two resistant strains ofCydia pomonellaobtained by mass selection (with deltamethrin (Rv) or diflubenzuron (Rt)) of resistant field populations from southeastern France. Compared to a laboratory susceptible strain (S), these strains exhibited respectively a 3170- and 1600-fold resistance ratio to topically applied deltamethrin. The esterase inhibitorS,S,S-tributyl phosphorotrithioate synergized deltamethrin in both resistant and susceptible strains, whereas the mixed-function oxidase (mfo) inhibitor piperonyl butoxide (PBO) synergized deltamethrin only in R strains (Rv and Rt). The two inhibitors together totally overcame the deltamethrin resistance of Rv and Rt strains. While ethoxycoumarin deethylase activities, measured at individual level in microtitration plates, were higher in R strains than with S, those of glutathioneS-transferases showed little difference and those of esterase showed no difference. Moreover, the involvement of mfo in the resistance to deltamethrin was confirmed byin vivometabolization of [14C] deltamethrin by guts of fifth instar larvae. Metabolites resulting from the ester bond hydrolysis, such as phenocyanol and PBald, were common to S and R strains. The mfo-dependent hydroxylated derivatives (torc-CHO-t-deltamethrin, 4′ or 5-HO-deltamethrin and 4′ or 5-HO-PB-acid) were generated only in Rv and Rt strains and were inhibited by PBO. These results may provide the basis for further studies in genetic of resistance inC. pomonella.

Expression of CYP71B7, a Cytochrome P450 Expressed Sequence Tag fromArabidopsis thaliana

The systematic sequencing of anonymous cDNA clones (expressed sequence tags or ESTs) from the plantArabidopsis thalianahas identified a number of cDNAs with similarity to known cytochrome P450 sequences. The partial sequence of one of these cDNAs, 5G6, indicated that it was likely to encode a full-length cytochrome P450 monooxygenase (cyt P450) sequence. In this paper we describe the complete sequence of this clone, which has been designatedCYP71B7in accordance with the nomenclature for the cyt P450 gene superfamily. The cDNA was used to determine the pattern of expression of the corresponding gene inA. thaliana.Northern hybridization analysis indicated that maximal expression ofCYP71B7occurred in rosette leaves. Weaker hybridizing bands were also detected by Northern analysis of RNA from roots, leaves, flowers, and siliques. No expression could be detected in stem tissue. Southern analysis indicated that theCYP71B7gene was likely to exist as a single copy in the genome ofA. thaliana. CYP71B7was expressed episomally in yeast, and microsomes prepared from transgenic yeast exhibited a carbon monoxide difference spectrum characteristic of cyt P450. Microsomes from yeast expressingCYP71B7were assayed for enzymatic activity with synthetic model cyt P450 substrates. Microsomes from yeast cells expressingCYP71B7or those from control cells exhibited no detectable NADPH-supported 7-ethoxycoumarin or 7-ethoxyresorufin deethylase activities. However, in the presence of cumene hydroperoxide, activity was observed with microsomes from cells expressing CYP71B7 with 7-ethoxycoumarin as substrate. Organic hydroperoxides are well known to support cyt P450 catalysis in the absence of electrons from NADPH. The yeast microsomes contained high levels of endogenous NADPH–ferricytochrome P450 reductase (CPR) activity. The data suggest that thisA. thalianacyt P450, although expressed in an active form, is incapable of accepting electrons from the endogenous yeast CPR protein.

Investigations on the biotransformation capacity of organophosphates in the mollusc Dreissena polymorpha P

Microsomes of Dreissena polymorpha were able to biotransform the organophosphates thiometon and disulfoton significantly after 1 h incubation at 10 °C. Contrarily to vertebrates, biotransformation with mussel microsomes occurred without the addition of reduction equivalents (NADPH), at a considerably lower extent however. Metabolites of thiometon and disulfoton were detected with gas chromatography/mass spectrometry (GC/MS) analysis. The P450 content of whole mussel tissue was 8 pmol mg −1 protein being two orders of magnitude lower than the amounts measured in mammal liver. Ethoxycoumarin deethylase (ECOD) activity could be measured at 10 °C in microsomes of Dreissena (22 pmol min −1 mg −1 protein) but not at 37 °C. ECOD activity was inhibited in the presence of NADPH (measured activity 3 pmol min −1 mg −1).

Effects of Benomyl on Cytochrome P450 Monooxygenase Systems in the Hep G2 Cell Line

Changes in the cytochrome P450 monooxygenase system were investigated in Hep G2 cells treated for 24 hours with various concentrations of benomyl. Decreases in both benzo[a]pyrene hydroxylase (AHH) and ethoxyresorufin deethylase (EROD), markers of the P4501A1 isoenzyme, were noted. Ethoxycoumarin deethylase (ECOD), a marker of the P4502B1 isoenzyme, showed a dose-dependent increase. Characterisation by SDS-polyacryl-amide gel electrophoresis of Hep G2 cell microsomal proteins revealed a decrease in the polypeptide bands at 55.5kD (P4501A1) and 48kD (P4501A1 and P4502B1) and an increase in the polypeptide band at 52kD (P4502B1). Benomyl induced a decrease in cytochrome P4501A1 and an increase in cytochrome P4502B1 in Hep G2 cells, as indicated by variations in AHH, EROD and ECOD activity, and by characterisation of microsomal proteins by SDS-polyacryl-amide gel electrophoresis.

Microsomal function in biliary obstructed rats: effects of S-adenosylmethionine

Backgrounds/Aims: S-adenosylmethionine has been reported to have beneficial effects in the treatment of different chronic liver diseases and to protect against different hepatotoxic agents. The aim of this study was to investigate whether S-adenosylmethionine treatment might contribute to improved microsomal function in chronically biliary obstructed rats. Methods: Secondary biliary cirrhosis was induced by 28 days of bile duct obstruction. Groups of control and Cirrhotic animals received S-adenosylmethionine (10 mg/kg per day) through the experimental period. Results: Bile duct obstruction resulted in a marked incrase in lipid peroxidation levels and decreases in glutathione concentration, microsomal membrane fluidityy, microsomal cytochrome P-450 content, NADPH-cytochrome P-450 reductase activity and the activities of the aniline hydroxylase, aminopyrine demethylase and ethoxycoumrin deethylase. Reductions in glutathione and cytochrome P-450 concentration were not corrected by S-adenosylmethionine, but lipid peroxidation, the decrease in the activities of the various microsomal monooxygenases and the reduction in microsomal membrane fluidity were partially prevented. A significant relationship was found between membrane fluidity and aniline hydroxylase, aminopyrine demethylase or ethoxycoumarin deethylase activities. Conclusions: S-adenosylmethionine administration partially preserves microsomal function. This effect could be associated to the protection of membrane function by restoring transmethylation reactions.

Cryopreservation of Porcine Hepatocyte Cultures

Cryopreservation of freshly isolated hepatocytes is regarded the standard procedure for long term storage of liver cells. However, a dramatic loss in cell number, viability, and differentiated cell function is usually inevitable, and, upon thawing, hepatocytes do not attach well in culture. Freezing of hepatocyte cultures has already been shown to reduce these hazardous effects, probably due to the beneficial impact of a repair phase that allows the cells to restore their membranes during the first days of culture prior to and after freezing. We have compared the performance of porcine hepatocytes from slaughterhouse organs that were frozen at day 3 of immobilizing gel culture and stored at −80°C for increasing time intervals up to 30 days (three groups;n= 3 each). Control groups consisted of batch identical hepatocyte cultures, either frozen at a fixed cooling rate (−1°C/min;n= 3), subjected to rapid freezing (−10°C/min;n= 3) or left without any freezing (n= 4). Upon thawing, light and electron microscopic studies revealed a cell survival rate of 70.3 ± 10.5% (mean ± SD;n= 9) in hepatocyte cultures after adjusted rate-controlled freezing. In these groups, the length of storage time did not influence results. Functional parameters, like albumin secretion values and ethoxycoumarin deethylase activity (cytochrome P450-IA1) were comparable to non–frozen cultures after a recovery time of 48 h post-thaw. Fixed cooling rates at −1°C and −10°C, respectively, showed a greater reduced viability (35.4 ± 7.4%; 9.87 ± 2.8%) and a severely impaired post-thaw functional performance of cultures. Cryopreservation of hepatocyte cultures could provide a practical means of establishing a bank of hepatocyte cultures. This could be extremely useful for the development of a hybrid artificial liver device, and it might support repeated investigations on batch-controlled hepatocyte cultures, especially with respect to human cells.

A Microfluorometric Method for Measuring Ethoxycoumarin-O-deethylase Activity on Individual Drosophila melanogaster Abdomens: Interest for Screening Resistance in Insect Populations

We developed a method for measuring ethoxycoumarin deethylase (ECOD) activity using a single Drosophila abdomen. The activities obtained were well correlated with the classic method from microsomes (r = 0.902). This new method, performed in microtitration plates, was at least six times more sensitive compared to the conventional cuvet fluorometric one. Moreover, it was possible among a large number of insects to differentiate those with low or high ECOD activities. This improved procedure has been checked upon crosses between resistant strain (with high ECOD activity) and susceptible strain (with low ECOD activity). The results demonstrate the possible separation of resistant phenotypes and emphasize the importance of this approach in assessing the spreading of insecticide resistance in natural populations of insects.

Piperine effects on the expression of P4502E1, P4502B and P4501A in rat.

1. Treatment of rat with piperine (PIP) (1.4 mmol/kg, 3 days ip injections) resulted in an approximate two-fold increase in total liver microsomal P450 content relative to that in uninduced animals. 2. 4-Nitrophenol and aniline hyroxylase activities in the hepatic microsomes prepared from rat treated with PIP decreased by 30 and 28% respectively as compared with control. Immunoblot analyses also revealed decreased P4502E1 levels in hepatic microsomes from PIP-treated animals. 3. In contrast with P4502E1 suppression, hepatic 2B1 and 2B2 levels were significantly increased in PIP-induced animals, as evidence by both metabolic activity and immunoblot analysis of the liver microsomal fractions. The rate of hexobarbital hydroxylase activity in microsomes from PIP-treated animals was markedly elevated and was inhibited by approximately 62% in the presence of monoclonal anti-P4502B IgG. Immunoblot analyses demonstrated that P4502B1 and 2B2 levels in hepatic microsomes from PIP-treated animals were comparable with those from phenobarbital-treated animals. 4. 7-Ethoxycoumarin deethylase activity was elevated approximately two-fold in PIP-induced animals and was 17% of that derived from 3-methylcholanthrene-induced animals. 7-ethoxycoumarin deethylase activity in PIP-induced hepatic microsomes was inhibited 63% in the presence of monoclonal anti-P4501A antibody. Immunoblot analysis confirmed the increase in P4501A levels by PIP, which was 15% of that in hepatic microsomes from 3-methylcholanthrene-induced animals. 5. PIP treatment failed to affect microsomal epoxide hydrolase (mEH) and glutathione S-transferases (GST) expression, as indicated by immunoblot analyses using polyclonal antibodies toward mEH and GST subunits Ya, Yb1, Yb2 and Yc. 6. These results demonstrate that PIP treatment suppressed P4502E1 expression and enhanced 2B and 1A expression, whereas this agent failed to affect hepatic mEH and GST expression.

Antimutagenicity of Maillard reaction products from amino acid/sugar model systems against 2-amino-3-methylimidazo-[4,5-f]quinoline: the role of pyrazines.

The antimutagenicity of dichloromethane extracts from eight amino acid/sugar model systems was determined using Salmonella typhimurium TA98 against 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ) in the presence of Aroclor 1254-induced rat hepatic S9. The Maillard reaction products in the dichloromethane extracts were then quantified and qualified by capillary gas chromatography and gas chromatography-mass spectrometry, respectively. Pyrazines and furans were found to be the major Maillard reaction products yielded in the extracts. Moreover, the antimutagenicity of dichloromethane extracts correlated positively with the total amounts of pyrazines and furans. To elucidate the mechanism of antimutagenicity of dichloromethane extracts, the inhibitory effect of pyrazines on ethoxycoumarin deethylase activity in Aroclor 1254-induced hepatic microsomes was examined. We also studied the effects of pyrazines on IQ metabolism by Aroclor 1254-induced microsomes using high-performance liquid chromatography. The antimutagenicity of pyrazines correlated positively with both the inhibition of cytochrome P-450 IA2-linked ethoxycoumarin deethylase in hepatic microsomes and the inhibition of N-hydroxy-IQ formation from IQ metabolism by hepatic microsomes. Thus we concluded that pyrazines in dichloromethane extracts from eight amino acid/sugar model systems play an important role in the antimutagenicity of IQ. Moreover, we concluded that the modifying effect of pyrazines on the mutagenicity of IQ is mediated through interaction with microsomal activating enzymes to inhibit the major active metabolite in N-hydroxy-IQ formation.

Effect of training and anabolic-androgenic steroids on drug metabolism in rat liver.

The effects of anabolic-androgenic steroid administration and exercise training on various aspects of hepatic function were investigated in sedentary and trained (treadmill for 12 wk) male and female rats treated orally with fluoxymesterone or methylandrostanolone (2 mg.kg-1 body weight, 5 d.wk-1 for 8 wk). The mean values of serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total and direct bilirubin, and total- and high-density lipoprotein-cholesterol remained within normal range in all groups of male animals. The same is true for female rats, except for an increase in alkaline phosphatase activity in the steroid-treated groups. Hepatic microsomal aniline p-hydroxylase activity was reduced in male and increased in female rats by either steroid, whereas no significant effect was detected on 7-ethoxycoumarin deethylase activity. The levels of cytochrome P-450 and cytochrome b5 were markedly decreased by the anabolic-androgenic steroid treatment in male rat microsomes, but neither the steroid administration nor exercise training induced significant changes in the cytochrome levels of female rat livers. Taking into account the significant increase in microsomal protein yield elicited by fluoxymesterone or methylandrostanolone treatment both in males and females, it is noteworthy that the total monooxygenase activities and cytochrome P-450 content, expressed on a per gram liver basis, were significantly increased in female whereas they were apparently unchanged in male rats. In conclusion, the present data show that the prolonged ingestion of high doses of anabolic-androgenic steroids, either with or without concurrent exercise training, can modify in a sex-dependent manner the capacity of rat liver to metabolize drugs without affecting classical serum indicators of hepatic function.

Cytochrome P450 in trypanosomatids

Post-mitochondrial supernatant extracts prepared from bloodstream forms of Trypanosoma brucei brucei, T. cruzi epimastigotes, Leishmania donovani promastigotes and Crithidia fasciculata have been found to catalyse cytochrome P450-dependent reactions. Appreciable ethoxycoumarin deethylase and ethoxyresorufin deethylase activities were found in all of the above trypanosomatids, with T. cruzi epimastigotes having the highest activity (57.1 and 10.7 pmol/min/mg protein, respectively). In all four species these reactions were inhibited by the cytochrome P450 inhibitors carbon monoxide, proadifen and metyrapone. In contrast to rat liver microsomes, the trypanosomatid extracts showed no detectable pentoxyresorufin depentylase or pentamidine hydroxylase activity. Both C. fasciculata and T. b. brucei post-mitochondrial supernatants showed carbon monoxide difference spectra consistent with the presence of cytochrome P450 (9.6 and 6.3 pmol/mg protein, respectively). An additional hemoprotein which gave a carbon monoxide difference peak at 420 nm was also detected in C. fasciculata and T. b. brucei microsomes and C. fasciculata mitochondria. Subcellular fractionation of both early and late log C. fasciculata showed that the ethoxycoumarin deethylase activity was enriched in the microsomal fraction.

Molecular basis for a functionally unique cytochrome P450IIB1 variant.

Liver microsomes from phenobarbital-treated rats of four inbred strains expressing distinct allelic variants of cytochrome P450IIB1 were analyzed. The Wistar Munich (WM) strain exhibited 5- to 10-fold lower androstenedione 16 beta-hydroxylase activity (a specific P450IIB1 marker) than the Lewis, Wistar Kyoto, and Wistar Furth strains. The androstenedione 16 beta-hydroxylase in the WM liver microsomes was refractory to inactivation by N-(2-p-nitrophenethyl)chlorofluoroacetamide, a selective P450IIB1 inactivator in the other three strains. Purified P450IIB1-WM was insensitive to the inactivator and exhibited 5-fold lower androstenedione 16 beta-hydroxylase, testosterone 16-hydroxylase, and 7-ethoxycoumarin deethylase activities but the same benzphetamine demethylase activity and slightly higher androstenedione 16 alpha-hydroxylase activity than a P450IIB1 purified from outbred Sprague-Dawley rats, which appears to correspond to the form in Lewis rats. The stereoselectivity of androstenedione 16-hydroxylation catalyzed by P450IIB1-WM (16 beta-OH:16 alpha-OH = 1.4) is thus distinct from that (16 beta-OH:16 alpha-OH = 12-15) of other P450IIB1 preparations described. A cDNA encoding P450IIB1-WM was cloned and sequenced, revealing a single amino acid substitution (Gly-478----Ala) compared with the published sequence (Fujii-Kuriyama, Y., Mizukami, Y., Kawajiri, K., Sogawa, K., and Muramatsu, M. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 2793-2797). Heterologous expression of P450IIB1 and P450IIB1-WM confirmed the striking difference in androstenedione metabolite profiles, strongly implicating the involvement of Ala-478 in defining the distinctive catalytic properties of P450IIB1-WM.

Microcarrier-attached rat hepatocytes as a xenobiotic-metabolizing system in cocultures

A method for the primary culture of rat liver cells on collagen-coated dextran microcarriers is described. Ethoxycoumarin deethylase (EOD) activity 24 hr after inoculation was comparable for liver cells cultured on microcarriers and on collagen-coated dishes. Cells were cultured on microcarriers for up to 48 hr and retained 25% of the initial EOD-activity that was seen in freshly isolated liver cells. Microcarrier-attached hepatocytes were cocultured with BALB/c 3T3 cells to study the metabolism-mediated cytotoxicity of cyclophosphamide (CPA). In the absence of hepatocytes, growth of 3T3 cells was not affected by CPA at concentrations up to 3600 microM. In coculture with hepatocytes, cytotoxicity of CPA was expressed in a time- and concentration-dependent manner. At high concentrations, CPA slightly depressed the EOD-activity of hepatocytes. Our results indicate that cocultivation of microcarrier-attached rat liver cells with target cells represents a valuable approach to the study of the metabolism-mediated toxicity of xenobiotics in vitro.

A Sensitive and Accurate Assay for 7-Ethoxycoumarin Deethylase Activity Determination Using Column-Switching High-Performance Liquid Chromatography

To meet the need for the determination of the low activities of cytochrome P450-catalyzed 7-ethoxycoumarin deethylation (ECDE) in most extra-hepatic tissues and cultured cells, a reliable, sensitive, and accurate assay, which uses a modified column-switching high-performance liquid chromatographic system and is essentially free of sample pretreatment, has been developed. With this system, the recovery of the 7-hydroxycoumarin product added to the biological tissue-containing incubation medium was 87%. With ECDE activity as low as 1 pmol/mg of protein/min, replicate measurements with variation coefficients lower than 5% were easily obtainable. ECDE activities in several extrahepatic tissues and cultured cells were determined with the technique to illustrate its usefulness.

Dietary selenium protects against acute toxicity of T‐2 toxin in rats

The efficacy of dietary selenium (Se) supplementation on acute toxicity of T-2 toxin was investigated. Wistar male rats were divided into six groups with 15 rats in each and fed for 6 weeks ad libitum a semi-synthetic diet containing either 0.03 (groups 1 and 2), 0.5 (groups 3 and 4) or 2.5 mg Se/kg (groups 5 and 6). By the end of the experiment the rats in groups 2, 4 and 6 were administered once per os 3.8 mg/kg body weight T-2 toxin, while the animals in groups 1, 3 and 5 received equal doses of the solvent. Twenty-four hours after administration of the toxin the surviving rats were sacrificed and the liver microsomes isolated and determined for activities of enzymes relating to xenobiotics metabolism and Se. The results showed that feeding the rats 2.5 mg Se/kg diet increased the deethylation rate of 7-ethoxycoumarin by 42% and slightly decreased (20%) glutathione-S-transferase activity. Twenty-four hours after the administration of T-2 toxin the lethality percentages in groups 2, 4 and 6 were 47%, 27% and 20%, respectively. Furthermore, administration of T-2 toxin to group 6 rats resulted in a significant decrease in the level of cytochrome P-450 and 7-ethoxycoumarin deethylase activity (to 78% and 51%, respectively) compared to the control group. At the same time a 72% increase in the UDP-glucuronosyltransferase activity and of 61% in epoxide hydrolase activity compared to the control group was found. Similarly, although somewhat smaller, changes were seen in the group 4 rats receiving 0.5 mg Se/kg diet.(ABSTRACT TRUNCATED AT 250 WORDS)