Cyclosporine-induced endoplasmic reticulum stress triggers tubular phenotypic changes and death.

The molecular mechanisms by which cyclosporine induces chronic nephrotoxicity remain poorly understood. A previous transcriptomic study suggested that cyclosporine might induce endoplasmic reticulum (ER) stress in human tubular cells. The aim of the present study was to characterize the features of tubular ER stress induced by cyclosporine and to investigate its effects on cell differentiation and viability. Using primary cultures of human tubular cells, we confirmed that cyclosporine is responsible for ER stress in vitro. This was also confirmed in vivo in the rat. In vitro, cyclosporine and other ER stress inducers were responsible for epithelial phenotypic changes leading to the generation of protomyofibroblasts, independent of transforming growth factor-beta signaling. RNA interference directed against cyclophilin A supported the role of its inhibition in triggering ER stress as well as epithelial phenotypic changes induced by cyclosporine. Salubrinal, which is known to protect cells from ER stress, significantly reduced epithelial phenotypic changes and cytotoxicity induced by cyclosporine in vitro. Salubrinal also reduced cyclosporine nephrotoxicity in rat kidneys. Thus, we describe a novel mechanism that initiates dedifferentiation and tubular cell death upon cyclosporine treatment. These results provide an interesting framework for further nephroprotective therapies by targeting ER stress.

Purification of a new cytochrome P-450 from human liver microsomes.

Using a classical methodology of purification consisting of three chromatographic steps (Octyl-Sepharose, DEAE-cellulose, CM-cellulose) we have purified a new cytochrome P-450 from human liver microsomes. It was called cytochrome P-450(9). It has been proven to be different from all precedingly purified human liver microsomal cytochrome P-450 isozymes by its immunological and electrophoretical properties. It does not cross-react with any rat liver cytochrome P-450 and anti-cytochrome P-450(9) does not recognize rat liver microsomes; thus this cytochrome P-450(9) is specific to humans. This cytochrome P-450 isozyme exists in low amounts in human liver microsomes and exhibits an important quantitative polymorphism. In reconstituted system, cytochrome P-450(9) is able to hydroxylate all substrates tested but is not specific of any; its exact role in xenobiotic metabolism in man remains to be elucidated.

Presence of proteins recognized by mammalian cytochrome P-450 antibodies in Euglena gracilis.

We have attempted to probe three microsomal cytochrome P-450 isozymes in Euglena gracilis using immunochemical methods. They cross-react with anti-rat cytochrome P4502C11, cytochrome P4502E, and cytochrome P4502B. Activities of alkoxyphenoxazone dealkylation have been tested in living cells. In untreated cultures, the amount of proteins recognized by anti-cytochrome P4502C11 or anti-cytochrome P4502E is high. Phenobarbital treatment increased the levels of microsomal proteins recognized by antibody to cytochrome P4502B, as well as dealkylases of pentoxyresorufin, but decreased the level of proteins recognized by anti-cytochrome P450C11 or cytochrome P4502E. These results suggest that these unicellular algae may contain different isozymes of microsomal cytochromes P-450, comparable to those in mammalian liver. They are cytochrome P-450 equivalents of mammalian isoenzymes 2C, 2E and 2B. However, we could not demonstrate ethanol induction of cytochrome P-450 equivalent to isoenzyme 2E. Its role in xeno- or endobiotic metabolism remains to be elucidated.

High yield purification and characterization of engineered human P450 1A2 and generation of immuno-inhibitor antibodies.

P450 S12, an engineered human P450 1A2 containing the 88-first amino-acids of the P450 1A1, demonstrates particularly high expression level in yeast while exhibiting catalytic properties very similar to the moderately expressed natural human P450 1A2. To facilitate P450 purification by nickel chelate chromatography, C-terminal extensions including histidine tags were tested. The -G(H)4 extension was found to be particularly efficient for permitting high expression levels without any catalytic alteration. This engineered P450 was purified to electrophoretic homogeneity (18 nmol/mg of protein) at a very high yield (87%) without any detectable formation of P420. P450 S12 activities were reconstituted in the presence of yeast and Arabidopsis thaliana (ATR1) NADPH-P450 reductases. The plant reductase supported better ethoxyresorufin-, methoxyresorufin- and phenacetin-O-dealkylase activities than the yeast reductase in reconstituted systems. Interestingly, polyclonal antibodies raised against purified P450 S12 selectively recognized in Western blot and fully immuno-inhibited the natural or recombinant P450 1A2 with very limited or no cross-reaction with P450 1A1 and other isoenzymes.

Identification of the major human hepatic cytochrome P450 involved in activation and N-dechloroethylation of ifosfamide.

Two NADPH-dependent metabolic routes for the anticancer drug ifosfamide, 4-hydroxylation (activation) and N-dechloroethylation (a detoxication pathway), were studied in human liver microsomes to identify the cytochrome P450 enzymes involved. Naringenin, a grapefruit aglycone and an inhibitor of cytochrome P450 3A4 (CYP3A4)-catalysed reactions, was found to inhibit ifosfamide activation and N-dechloroethylation by human liver microsomes. IC50 for both reactions was of the order of 70 microM. The CYP3A4-specific inhibitor triacetyloleandomycin inhibited ifosfamide N-dechloroethylation by human liver microsomes with an IC50 of approximately 10 microM. Furthermore, anti-human CYP3A4 antiserum inhibited by about 80% N-dechloroethylation of ifosfamide by human liver microsomes. The relative levels of cytochromes P450 1A, 2C, 2E and 3A4 in 12 human livers were determined by western blotting analysis. A strong correlation (P < 0.001) was observed between CYP3A4 expression and both activation and N-dechloroethylation of ifosfamide. A role for human CYP3A4 in both pathways of ifosfamide metabolism was thus demonstrated. This was substantiated by the observation that the nifedipine oxidase activities of the 12 samples of human liver microsomes correlated with ifosfamide activation (P < 0.009) and N-dechloroethylation (P < 0.001). These findings have important clinical implications. The involvement of the same key cytochrome P450 enzyme in both reactions prohibits selective inhibition of the N-dechloroethylation pathway, as might be desirable to reduce toxic side effects. They also demonstrate the need to consider interaction with co-administered drugs that are CYP3A4 substrates.

Pregnenolone-7beta-hydroxylating activity of human cytochrome P450-1A1.

In many human and murine tissues, both pregnenolone and dehydroepiandrosterone are hydroxylated at the 7alpha and 7beta positions by a cytochrome P450-containing microsomal complex. The 7alpha- and 7beta-hydroxysteroids produced were shown to activate an immune response in mice. Based upon identification by crystallization to constant specific activity and gas chromatography-mass spectrometry analysis, we ascertained that a yeast-expressed human cytochrome P450-1A1 was able to 7beta-hydroxylate pregnenolone (K(M) from 3.2 +/- 0.5 to 4.1 +/- 0.4 microM, turnover number from 117 +/- 15 to 135 +/- 13 pmol/min/nmol of cytochrome P450-1A1). The other human cytochromes P450 tested did not produce identifiable quantities of 7alpha- or 7beta-hydroxylated derivatives of pregnenolone or dehydroepiandrosterone. These findings indicate that cytochrome P450-1A1 involvement in the 7beta-hydroxylation of pregnenolone may contribute to the production of the 7-hydroxylated steroids necessary for activation of the immune defences.

Human cytochromes P450 expressed in Escherichia coli: production of specific antibodies.

Cytochromes P450 (CYP) constitute a superfamily of enzymes involved in the metabolism of xenobiotics. Within the same subfamily, the isoforms present strong similarities, making them difficult to characterize and differentiate. Using heterologous expression in bacteria, five pure human CYP (1A1, 1A2, 2C9, 2E1, 3A4) were easily obtained and used as antigens to raise specific antibodies. These antibodies were characterized for their specificity and sensitivity by immunoblots; anti-CYP3A4 was immunoinhibitor. These antibodies could be used in association with other means to identify the CYPs responsible for production of a given metabolite. The use of our human recombinant CYP1A2 as antigen and the corresponding specific antibody enabled us to quantify the CYP1A2 content in 43 human livers. The average level was 69 pmol of CYP1A2/mg of microsomal proteins. Finally, these antibodies were also used to evaluate the level of heme incorporation in human microsomal CYP expressed in yeasts.

Effects of various inducers on the expression of cytochromes P-450 IIC8, 9, 10 and IIIA in cultured adult human hepatocytes.

Primary cultures of adult human hepatocytes were used to determine the capability of the human liver to respond to phenobarbital, 3-methylcholanthrene, troleandomycin and rifampicin, four compounds known to be potent inducers of hepatic cytochrome P-450 in various animal species. Both mRNA and corresponding protein of two major isoenzymes, that is, P-450 IIC8, 9, 10 and P-450 IIIA, were measured after daily exposure to the drugs for 3 days. Phenobarbital and rifampicin were found to increase the levels of P-450 IIC8, 9, 10 mRNA and protein while troleandomycin and 3-methylcholanthrene were ineffective. Different effects were obtained for P-450 IIIA. Both mRNA and related protein were markedly increased by troleandomycin and rifampicin and decreased by 3-methylcholanthrene. mRNAs were slightly increased by phenobarbital. The results demonstrate that human hepatocytes retain drug-inducible P-450 isoenzymes in primary culture and represent a unique approach to investigate regulation of human liver drug metabolizing enzymes.

The human colonic microflora influences the alterations of xenobiotic-metabolizing enzymes by catechins in male F344 rats.

As other xenobiotics, polyphenols are metabolized both by the endogenous detoxication system and the gut microflora. We hypothesized that the presence of a gut microflora may account for the effect of catechins on phase I and II xenobiotic-metabolizing enzymes and that the human bacterial metabolites may be different from those of a rodent gut microflora. Therefore, the effects of 2% (+)-catechin or 2% (-)-epicatechin were studied in germ free (GF) rats and rats inoculated with the flora of a human volunteer (HFA). In addition, the catechins were administered in ethanol as a vehicle. In the liver, (+)-catechin or (-)-epicatechin decreased the total amount of CYP450 in both GF and HFA rats while the isoenzyme CYP2E1 decreased. In GF rats only, CYP2C11 increased when compared to the rats treated with the vehicle alone. (+)-catechin increased the specific activity of UGT-chloramphenicol in GF rats only and that of cytosolic glutathion-S-transferase (GST) in HFA rats only. In the intestine, (+)-catechin and (-)-epicatechin increased the specific activity of UGT-4-methylumbelliferone in both GF and HFA rats and that of UGT- chloramphenicol in HFA rats only. In conclusion, the presence of a human flora in rats is able to modify the inducing effect of catechins on the UGT and GST activities suggesting the involvement of bacterial metabolites. The alterations on CYP 450 are independent of the presence of a human gut flora.

Halofantrine metabolism in microsomes in man: major role of CYP 3A4 and CYP 3A5.

We have clarified the contribution of the different enzymes involved in the N-debutylation of halofantrine in liver microsomes in man. The effect of ketoconazole and cytochrome P450 (CYP) 3A substrates on halofantrine metabolism has also been studied. The antimalarial drug halofantrine is metabolized into one major metabolite, N-debutylhalofantrine. In microsomes from nine livers from man, N-debutylation of halofantrine was highly variable with apparent Michaelis-Menten constant V(max) and K(m) values of 215+/-172 pmol min(-1) mg(-1) and 48+/-26 micromol L(-1), respectively, (mean+/-standard deviation). Formation of N-debutylhalofantrine was cytochrome P450 (CYP)-mediated. Studies using selective inhibitors of individual CYPs revealed the role of CYP 3As in the formation of N-debutylhalofantrine. alpha-Naphthoflavone, a CYP 3A activator, increased metabolite formation. In microsomes from 12 livers from man the rate of N-debutylation of halofantrine correlated strongly with CYP 3A4 relative levels (P = 0.002) and less strongly, but significantly, with CYP 2C8 levels (P = 0.025). To characterize CYP-mediated metabolism of halofantrine further, incubations were performed with yeast microsomes expressing specific CYP 3A4, CYP 3A5, CYP 2D6, CYP 2C8 and CYP 2C19 from man. The rate of formation of N-debutylhalofantrine was six- and twelvefold with CYP 3A4 than with CYP 3A5 and CYP 2C8, respectively. CYP 2D6 and CYP 2C19 did not mediate the N-debutylation of halofantrine, but, because in-vivo CYP 2C8 is present at lower concentrations than CYP 3A in the liver in man, the involvement of CYP 3As would be predominant. Diltiazem, erythromycin, nifedipine and cyclosporin (CYP 3A substrates) inhibited halofantrine metabolism. Similarly, ketoconazole inhibited, non-competitively, formation of N-debutylhalofantrine with an inhibition constant, K(i), of 0.05 microM. The theoretical percentage inhibition of halofantrine metabolism in-vivo by ketoconazole was estimated to be 99%. These results indicate that both CYP 3A4 and CYP 3A5 metabolize halofantrine, with major involvement of CYP 3A4. In-vivo, the other CYPs have a minor role only. Moreover, strong inhibition, and consequently increased halofantrine cardiotoxicity, might occur with the association of ketoconazole or other CYP 3A4 substrates.

Cytochrome P4502C9 is the principal catalyst of racemic acenocoumarol hydroxylation reactions in human liver microsomes.

The oral anticoagulant acenocoumarol is given as a racemic mixture. The (S)-enantiomer is rapidly cleared and is the reason why only (R)-acenocoumarol contributes to the pharmacological effect. The objective of the study was to establish the cytochrome P450 (CYP) enzymes catalyzing the hydroxylations of the acenocoumarol enantiomers. Of various cDNA-expressed human CYPs, only CYP2C9 hydroxylated (S)-acenocoumarol. Hydroxylation occurred at the 6-, 7-, and 8-position with equal K(m) values and a ratio of 0.9:1:0.1 for V(max). CYP2C9 also mediated the 6-, 7-, and 8-hydroxylations of (R)-acenocoumarol with K(m) values three to four times and V(max) values one-sixth times those of (S)-acenocoumarol. (R)-Acenocoumarol was also metabolized by CYP1A2 (6-hydroxylation) and CYP2C19 (6-, 7-, and 8-hydroxylation). In human liver microsomes one enzyme only catalyzed (S)-acenocoumarol hydroxylations with K(m) values < 1 microM. In most of the samples tested the 7-hydroxylation of (R)-acenocoumarol was also catalyzed by one enzyme only. The 6-hydroxylation was catalyzed by at least two enzymes. Sulfaphenazole could completely inhibit in a competitive way the hydroxylations of (S)-acenocoumarol and the 7-hydroxylation of (R)-acenocoumarol. The 6-hydroxylation of (R)-acenocoumarol could be partially inhibited by sulfaphenazole, 40 to 50%, and by furafylline, 20 to 30%. Significant mutual correlations were obtained between the hydroxylations of (S)-acenocoumarol, the 7-hydroxylation of (R)-acenocoumarol, the 7-hydroxylation of (S)-warfarin, and the methylhydroxylation of tolbutamide. The results demonstrate that (S)-acenocoumarol is hydroxylated by a single enzyme, namely CYP2C9. CYP2C9 is also the main enzyme in the 7-hydroxylation of (R)-acenocoumarol. Other enzymes involved in (R)-acenocoumarol hydroxylation reactions are CYP1A2 and CYP2C19. Drug interactions must be expected, particularly for drugs interfering with CYP2C9. Also, drugs interfering with CYP1A2 and CYP2C19 may potentiate acenocoumarol anticoagulant therapy.

Intralobular distribution and quantitation of cytochrome P-450 enzymes in human liver as a function of age.

We have used immunohistochemical, immunoblotting and messenger RNA blotting approaches to study the distribution and quantitation of three cytochrome P-450 enzymes, namely P-450 IA2, P-450 IIC and P-450 IIIA and, for comparison, epoxide hydrolase and NADPH-cytochrome P-450 reductase in human liver. Age-related changes in both the amounts and the intralobular distributions of these enzymes were demonstrated, and the enzymes differ in these regards: In fetal liver, P-450 IA2 and P-450 IIC were very low, when present at all, whereas P-450 IIIA, epoxide hydrolase and the reductase were already abundant and found in all the hepatocytes. During the postnatal period, P-450 IIC dramatically increased and was observed in all hepatocytes, the centrilobular ones being more intensely stained. P-450 IIIA was restricted to centrilobular and midzonal hepatocytes in normal adult liver. P-450 IA2 showed this same intralobular distribution; however, its presence was detected only several weeks or months after birth as judged both by immunohistochemical and immunoblotting techniques. Epoxide hydrolase and NADPH-cytochrome P-450 reductase were easily visualized in all hepatocytes regardless of the age of the donor; in child and adult livers, centrilobular hepatocytes were more intensely stained. Immunoreactive protein contents and corresponding messenger RNA levels correlated well with immunohistochemical observations. No major modification was seen in fibrotic liver, whereas both positive and negative cells were observed in cirrhotic liver nodules for all enzymes studied.

Overexpression of glutamine synthetase in human primary liver cancer.

BACKGROUND/AIMS: We have identified several clones specifically expressed during malignant cell proliferation by screening a complementary DNA library constructed from a human primary liver cancer with subtractive probes. One clone was identified as the glutamine synthetase (GS) transcript. Its expression is tightly regulated during development, especially in the hepatic lobule. Because this enzyme is involved in nitrogen homeostasis, it might contribute to tumor development/progression in primary liver cancer. METHODS: We compared the expression of GS messenger RNA (mRNA) and protein in tumorous and nontumorous liver from 34 patients with primary liver cancers, using a combination of Northern blot, dot blot, western blot, and determination of GS enzyme activity. RESULTS: GS mRNA was higher in tumors versus nontumors in 23 of 34 primary liver cancer samples. GS activity was higher in 6 of 8 selected primary liver cancer samples with high RNA levels. GS protein levels were proportional to enzyme activity. A major GS transcript of 2.8 kilobase was detected by Northern blotting and sequencing. This comprised the minor 1.8-kb transcript and a long 3' untranslated region; the latter contained an AT-rich zone, fully conserved in the chicken, mouse, and rat, which might be important for stability. CONCLUSIONS: Our results show an overexpression of GS in human primary liver cancers and, thus, point to its potential involvement in hepatocyte transformation.

Hepatic and intestinal cytochrome P-450, glutathione-S-transferase and UDP-glucuronosyl transferase are affected by six types of dietary fiber in rats inoculated with human whole fecal flora.

The effects of six different sources of dietary fiber (inulin, wheat brain, carrot, cocoa, pea and oat fiber) on hepatic and intestinal cytochrome P-450 (EC 1.14.14.1), glutathione-S-transferase (GSH-T, EC 2.5.1.18) and UDP-glucuronosyl transferase (UDPG-T, EC 2.4.1.17) were studied using germ-free F344 rats subsequently inoculated with a human whole fecal flora. In the liver, the total concentration of P-450 was significantly lower in the wheat bran-fed group than in the carrot-fed group. The 2E1 form of P-450, involved in nitrosamine metabolism, was enhanced in the carrot-fed group compared with those fed most other types of fiber. Compared with the pea-fed group, rats fed cocoa had a lower constitutive 2C11 form and a higher 1A2 form. A very high concentration of small intestinal 1A1 form--involved in "toxication" reactions--was observed in rats fed cocoa. The specific activity of hepatic GSH-T was significantly higher in rats fed inulin than in all other groups, except the carrot-fed group. In the colon, GSH-T specific activity was twice as high in the oat-fed group as in the wheat bran-fed counterpart. Small intestinal GSH-T activity and hepatic and intestinal UDPG-T activities were unaffected by diet. Results are discussed in relation to potential health benefits.

Effects of the bacterial status of rats on the changes in some liver cytochrome P450 (EC 1.14.14.1) apoproteins consequent to a glucosinolate-rich diet.

The aim of the present work was to investigate the influence of the intestinal microflora on the changes in hepatic cytochrome P450 apoproteins induced by dietary glucosinolates. Ten rats harbouring a conventional digestive microflora were offered either a diet containing 390 g myrosinase-free rapeseed meal/kg (n 5) or a control diet devoid of glucosinolates (n 5). A similar trial was performed using germ-free rats. After 4 weeks of exposure to the dietary regimens, animals were slaughtered and their livers removed for preparation of microsomes and analysis of cytochrome P450 (EC 1.14.14.1). The glucosinolate-rich diet decreased the concentration of total cytochrome P450 in conventional rats only (-34%). The bacterial status did not modify the concentration of apoproteins CYP1A2 and CYP2B1/B2, but greatly decreased the concentration of the male constitutive isoform CYP2C11 (-53 and -45% respectively in conventional and germ-free rats). Germ-free rats fed on the glucosinolate-rich diet had a greater concentration of CYP3A (+139%) and a lower concentration of CYP2E1 (-32%) than their counterparts fed on the control diet. However, these differences were absent in conventional animals. On the whole, the influence of the intestinal microflora on the changes in hepatic cytochrome P450 due to the consumption of cruciferous vegetables is very complex and obviously involves different mechanisms according to the apoprotein.

In vitro metabolism of quinidine: the (3S)-3-hydroxylation of quinidine is a specific marker reaction for cytochrome P-4503A4 activity in human liver microsomes.

The aim of this study was to evaluate the (3S)-3-hydroxylation and the N-oxidation of quinidine as biomarkers for cytochrome P-450 (CYP)3A4 activity in human liver microsome preparations. An HPLC method was developed to assay the metabolites (3S)-3-hydroxyquinidine (3-OH-Q) and quinidine N-oxide (Q-N-OX) formed during incubation with microsomes from human liver and from Saccharomyces cerevisiae strains expressing 10 human CYPs. 3-OH-Q formation complied with Michaelis-Menten kinetics (mean values of Vmax and Km: 74.4 nmol/mg/h and 74.2 microM, respectively). Q-N-OX formation followed two-site kinetics with mean values of Vmax, Km and Vmax/Km for the low affinity isozyme of 15.9 nmol/mg/h, 76.1 microM and 0.03 ml/mg/h, respectively. 3-OH-Q and Q-N-OX formations were potently inhibited by ketoconazole, itraconazole, and triacetyloleandomycin. Isozyme specific inhibitors of CYP1A2, -2C9, -2C19, -2D6, and -2E1 did not inhibit 3-OH-Q or Q-N-OX formation, with Ki values comparable with previously reported values. Statistically significant correlations were observed between CYP3A4 content and formations of 3-OH-Q and Q-N-OX in 12 human liver microsome preparations. Studies with yeast-expressed isozymes revealed that only CYP3A4 actively catalyzed the (3S)-3-hydroxylation. CYP3A4 was the most active enzyme in Q-N-OX formation, but CYP2C9 and 2E1 also catalyzed minor proportions of the N-oxidation. In conclusion, our studies demonstrate that only CYP3A4 is actively involved in the formation of 3-OH-Q. Hence, the (3S)-3-hydroxylation of quinidine is a specific probe for CYP3A4 activity in human liver microsome preparations, whereas the N-oxidation of quinidine is a somewhat less specific marker reaction for CYP3A4 activity, because the presence of a low affinity enzyme is demonstrated by different approaches.

The biotransformation of clomipramine in vitro, identification of the cytochrome P450s responsible for the separate metabolic pathways.

The aim of the study was to identify the cytochrome P450s (CYPs) that catalyze the biotransformation of clomipramine in vitro. A high-performance liquid chromatography method was developed to assay N-desmethylclomipramine, 8-hydroxyclomipramine, 2-hydroxyclomipramine, 8-hydroxydesmethhylclomipramine, didesmethylclomipramine and 2-hydroxydesmethylclomipramine formed by microsomes prepared from human liver and yeast expressing human CYP1A1, 1A2, 2C8, 2C9, 2C18, 2C19, 2D6 and 3A4. There was a statistically significant correlation between the formation rate of desmethylclomipramine and the immunoquantified concentration of CYP3A4 in 12 human liver microsome preparations (rs = 0.664, P = .028). Ketoconazole was a very potent inhibitor of desmethylclomipramine formation (Ki = 0.054 microM) and microsomes from yeast expressing CYP3A4 were also active in forming the metabolite (formation rate: 25.6 nmol/nmol of CYP per hr). Thus, the results are consistent with the assumption that the N-demethylation of clomipramine is catalyzed by CYP3A4. As expected from in vivo panel studies, CYP2C19 in yeast was also very active in the N-demethylation (formation rate, 145 nmol/nmol of CYP per hr). Fluvoxamine was a potent inhibitor of desmethylclomipramine formation (Ki, 0.15 microM), suggesting that CYP1A2 is a third CYP involved in the N-demethylation. CYP2D6 in yeast microsomes catalyzed the 8-hydroxylation of clomipramine and desmethylclomipramine (formation rates, 65 and 75 nmol/nmol of CYP per hr) and quinidine was a very potent inhibitor (Ki, 0.10 and 0.16 microM). Both results confirm that CYP2D6 catalyzes the 8-hydroxylation in agreement with the results obtained in previous in vivo studies. Besides quinidine, paroxetine, fluoxetine and norfluoxetine, all were potent inhibitors of the 8-hydroxylations (Ki, 0.24-1.5 microM) and sertraline was a less potent inhibitor (Ki, 16 and 27 microM, respectively).

Hepatic cytochrome P450 and UDP-glucuronosyl transferase are affected by five sources of dietary fiber in germ-free rats.

The influence of dietary fiber on xenobiotic-metabolizing enzymes (XME) was assessed using germ-free rats fed inulin and other sources of fiber (wheat bran, carrot, cocoa and oat). The consumption of cocoa fiber greatly modified the hepatic cytochrome P450 isoenzymatic profile, causing a strong enhancement of 1A2 and 2B1/B2 forms, concomitant with a significant decrease of the constitutive form 2C11, compared with all of the other types of fiber. Moreover, rats fed the cocoa fiber diet had a higher specific activity of hepatic UDP-glucuronosyl transferase than their carrot fiber- and wheat bran-fed counterparts. Intestinal UDP-glucuronosyl transferase was unaffected by the type of ingested fiber. Diet composition also did not alter the specific activity of glutathione-S-transferase in the liver, small intestine, or colon. Using earlier results obtained in heteroxenic rats, we show that intestinal microflora plays a key role in some of the effects of fiber on XME, although this is not a necessary prerequisite for all of the liver alterations.

Induction of drug-metabolizing enzymes by tricyclic antidepressants in human liver: characterization and partial resolution of cytochromes P-450.

Drug-metabolizing enzyme activities were determined in liver microsomes from six kidney-transplant donors, one tricyclic antidepressant-treated and five untreated donors. The tricyclic antidepressant treatment modifies neither the overall cytochrome P-450 content of the liver, nor enzymatic activities of 4-nitroanisole demethylase, aniline hydroxylase, epoxide hydrolase and glutathione S-transferase. Only benzphetamine and ketotifen demethylation and conjugation of bilirubin with UDP-glucuronic acid are markedly augmented (more than two-fold). Separation of the different cytochrome P-450 fractions on a DEAE cellulose column indicates a modification of the elution pattern: the fraction increased by tricyclic antidepressants is responsible for the enhanced monooxygenase activity towards benzopyrene and benzphetamine.