a pH Dependent Switch in DHP Oxidation Mechanism

Undergraduate Basi

Inhibition of Liver Cytochrome P450 Isoforms 1A1 and 1A2 by Brazilian Acai Berry Extracts

Undergraduate Basi

Characterization of the Cytochrome P450 CYP234: Expression in Rat Small Intestine and Role in Retinoic Acid Biotransformation from Retinal

The sites of expression in the small intestine and the function of CYP2J4, a recently identified rat cytochrome (P450) isoform found to be predominantly expressed in the small intestine, were characterized. Immunoblot analysis with a polyclonal antibody to heterologously expressed CYP2J4 revealed that expression of CYP2J4 was at the highest level in the distal duodenum and jejunum and decreased toward the ileum. Villous cells expressed higher levels of CYP2J4 than crypt cells. Isoform-specific RNA polymerase chain reaction indicated that a related P450 isoform, CYP2J3, was only a minor form in rat small intestine. Since the intestinal mucosa is exposed to high levels of dietary nutrients, we hypothesized that CYP2J4 may be active toward diet-derived factors. We determined that purified, heterologously expressed CYP2J4 is active toward all-trans- and 9-cis-retinal in reconstituted systems, producing the corresponding retinoic acids as the major products. Apparent Km values for the formation of retinoic acids were 54 and 49 µM, respectively, and apparent Vmax values were 20 and 21 nmol/min/nmol P450, respectively. These activities were readily inhibited by a polyclonal anti-CYP2J4 antibody. Rat enterocyte microsomes were also active with all-trans-retinal to produce all-trans-retinoic acid in the presence of NADPH, and the majority of retinoic acid synthesis activity was inhibited by the polyclonal anti-CYP2J4 antibody. These findings suggest that CYP2J4 plays a major role in intestinal microsomal metabolism of retinal to retinoic acid and may be involved in the maintenance of retinoid homeostasis in the small intestine in vivo

Peroxo-iron and Oxenoid-iron Species as Alternative Oxygenating Agents in Cytochrome P450-catalyzed Reactions: Switching by Threonine-302 to Alanine Mutagenesis of Cytochrome P450 2B4.

ABSTRACT Among biological catalysts, cytochrome P450 is unmatchedi n its multiplicityo f isoforms,i nducers, substrates,a nd typeso f chemical reactionsc atalyzed.I n the presents tudy,e videncei s givent hatt hisv ersatilityex tendst o the nature of the active oxidant. Althoughm echanistice vidence froms everal laboratoriesp ointst o a hypervalentir onoxenoid species in P450-catalyzed oxygenation reactions, Akhtar and colleagues [Akhtar,M ., Calder, M. R., Corina, D. L. & WrightJ, . N. (1982) BiochemJ. . 201,5 69-580] proposed that in steroidd eformylatioenf fectedb y P450 aromatase an iron-peroxos pecies is involvedW. e have shownm orer ecently that purifiedl iver microsomalP 450 cytochromesi,n cluding phenobarbital-inducedP 450 2B4, catalyze the analogous deformylationo f a series of xenobiotica ldehydes with olefin formationT. he investigationp resentedh ere on the effecto f site-directedm utagenesiso f threonine-302t o alanine on the activitieso f recombinantP 450 2B4 with N-terminala mino acids 2-27 deleted [2B4 (A2-27)] makes use of evidencef rom otherl aboratoriest hat the correspondingm utationi n bacterial P450s interferesw ith the activationo f dioxygent o the oxenoid species by blockingp rotond eliveryt o the active site. The rates of NADPH oxidation,h ydrogenp eroxide production, a nd productf ormationf romf ours ubstrates,i ncluding formaldehydef rom benzphetamineN -demethylationa,c etophenone from1 -phenylethanoolx idation,c yclohexanolf rom cyclohexaneh ydroxylationan, d cyclohexenef romc yclohexane carboxaldehyded eformylationw, ere determinedw ith P450s 2B4, 2B4 (A2-27), and 2B4 (A2-27) T302A. Replacement of the threoniner esidue in the truncatedc ytochromge ave a 1.6- to 2.5-foldi ncrease in peroxidef ormationin the presenceo fa substrate,b ut resultedi n decreased productf ormationf rom benzphetamine (9-fold), cyclohexane (4-fold), and 1-phenylethanol (2-fold).I n sharp contrast,t hed eformylatioonf c yclohexane carboxaldehydeb y the T302A mutantw as increased about 10-foldO. n the basis oft hesef indingsa nd our previous evidencet hat aldehyded eformylationis supportedb y added H202, but not by artificial oxidants, we conclude that the iron-peroxys pecies is the directo xygend onor. It remains to be established which of the many other oxidative reactions involvingP 450 utilize this species and the extentt o which peroxo-irona nd oxenoid-ironf unctiona s alternativeo xygenating agents with the numerous isoforms of this versatile catalyst

Single turnover studies of oxidative halophenol dehalogenation by horseradish peroxidase reveal a mechanism involving two consecutive one electron steps: toward a functional halophenol bioremediation catalyst.

Horseradish peroxidase (HRP) catalyzes the oxidative para-dechlorination of the environmental pollutant/carcinogen 2,4,6-trichlorophenol (2,4,6-TCP). A possible mechanism for this reaction is a direct oxygen atom transfer from HRP compound I (HRP I) to trichlorophenol to generate 2,6-dichloro 1,4-benzoquinone, a two-electron transfer process. An alternative mechanism involves two consecutive one-electron transfer steps in which HRP I is reduced to compound II (HRP II) and then to the ferric enzyme as first proposed by Wiese et al. [F.W. Wiese, H.C. Chang, R.V. Lloyd, J.P. Freeman, V.M. Samokyszyn, Arch. Environ. Contam. Toxicol. 34 (1998) 217–222]. To probe the mechanism of oxidative halophenol dehalogenation, the reactions between 2,4,6-TCP and HRP compounds I or II have been investigated under single turnover conditions (i.e., without excess H2O2) using rapid scan stopped-flow spectroscopy. Addition of 2,4,6-TCP to HRP I leads rapidly to HRP II and then more slowly to the ferric resting state, consistent with a mechanism involving two consecutive one-electron oxidations of the substrate via a phenoxy radical intermediate. HRP II can also directly dechlorinate 2,4,6-TCP as judged by rapid scan stopped-flow and mass spectrometry. This observation is particularly significant since HRP II can only carry out one-electron oxidations. A more detailed understanding of the mechanism of oxidative halophenol dehalogenation will facilitate the use of HRP as a halophenol bioremediation catalyst

Cytochrome P450 expression and activities in rat, rabbit and bovine tongue

Xenobiotic metabolism in the tongue has received little attention in the literature. In the present study, we report a comparative analysis of constitutive cytochrome P450 (CYP) expression and activities in the tongue. First we compared catalytic activities of rabbit, rat and bovine tongue samples using the probe substrates 4-nitrophenol, 1-phenylethanol, caffeine and 7-ethoxycoumarin. Rabbit tongue samples showed the highest activities for all substrates. We then compared the activities in rat and rabbit tongue with those in the rabbit liver, along with the effects of P450 inhibitors on specific activities. Combined, the activity studies indicate that CYP1A1 is active in rabbit tongue cells, but CYP1A2, CYP3A6 and CYP2E1 are below limits of detection. RT-PCR was also used to compare mRNA levels of 11 different rabbit and six different rat P450 isoforms in the tongue to those in the liver of these two species. Only CYP2E1, CYP1A1 and CYP4A4 were detected at significant levels in the rabbit tongue. None of the six rat isoforms probed were observed in the tongue. Although 4-nitrophenol activity was observed in the rabbit tongue samples, the kinetic parameter Km was inconsistent with the involvement of CYP2E1. We suggest that although CYP2E1 is expressed in the tongue, it is rapidly degraded in this organ, and the nitrophenol hydroxylation and caffeine hydroxylation we observe is the result of activity of CYP1A1

Effects of green tea extracts on gene expression in HepG2 and Cal-27 cells

Green tea extract is known to contain compounds that are able to produce antioxidant effects in many types of living cells. Treatment of cultured human hepatoma (HepG2) cells with green tea extract resulted in dramatically increased expression of at least 15 genes that are present on a commercial human drug metabolism gene array. RT-PCR was used to confirm the microarray results, and analysis of the 5'-flanking region of each of these genes revealed potential electrophile/antioxidant response elements. Members of the acetyl transferase, epoxide hydrolase, sulfotransferase and glutathione transferase gene families were strongly induced. In addition, the human tongue carcinoma cell line Cal-27 did not respond to green tea extract in the same way, as none of the induced genes in the HepG2 cells were induced in the Cal-27 cells. The lack of induction of detoxification enzymes in the Cal-27 cell line may help to explain the previously observed increased cytotoxicity of green tea catechins on this cell line

Defluorination of 4-fluorophenol by Cytochrome P450BM3-F87G: Activation by long Chain Fatty Aldehydes.

Cytochrome P450BM3-F87G catalyzed the oxidative defluorination of 4-fluorophenol, followed by reduction of the resulting benzoquinone to hydroquinone via the NADPH P450-reductase activity of the enzyme. The k catand K m for this reaction were 71 ± 5 min-1 and 9.5 ± 1.3 mM, respectively. Co-incubation of the reaction mixture with long chain aldehydes stimulated the defluorination reaction, with the 2,3-unsaturated aldehyde, 2-decenal producing a 12-fold increase in catalytic efficiency. At 150 µM aldehyde, k cat increased to 158 ± 4, while K m decreased to 1.8 ± 0.2. The effects of catalase, glutathione and ascorbate on the reaction were all consistent with a direct oxygen insertion mechanism, as opposed to a radical mechanism. The study demonstrates the potential use of P450BM3 mutants in oxidative defluorination reactions, and characterizes the novel stimulatory action of straight chain aldehydes on this activity

Oxymyohemerythrin: discriminating between O2 release and autoxidation

Myohemerythrin (Mhr) is a non-heme iron O2 carrier (with two irons in the active site) that is typically found in the retractor muscle of marine ‘peanut’ worms. OxyMhr may either release O2, or undergo an autoxidation reaction in which hydrogen peroxide is released and diferric metMhr is produced. The autoxidation reaction can also be promoted by the addition of certain anions to Mhr solutions. This work, using recombinant Themiste zostericola Mhrs, contrasts the results of environmental effects on these reactions. For the O2 release reaction, &#x0394V‡(21.5°C)=+28±3 cm3 mol-1, &#x0394H‡(1 atm)=+22±1 kcal mol-1, and &#x0394S‡(1 atm)=+ 28±4 eu. The autoxidation reaction (pH 8.0, 21.5°C, 1 atm) displays different kinetic parameters: &#x0394V‡=-8±2 cm3 mol-1, &#x0394H‡=+24.1±0.7 kcal mol-1, and &#x0394S‡=+1±1 eu. Autoxidation in the presence of sodium azide is orders of magnitude faster than solvolytic autoxidation. The &#x0394V‡ parameters for azide anation and azide-assisted autoxidation reaction are +15±2 and +59±2 cm3 mol-1, respectively, indicating that the rate-limiting steps for the Mhr autoxidation and anation reactions (including O2 uptake) are not associated with ligand binding to the Fe2 center. The L103V and L103N oxyMhr mutants autoxidize &#x2248 103–105 times faster than the wild-type protein, emphasizing the importance of leucine-103, which may function as a protein ‘gate’ in stabilizing bound dioxygen

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 ß-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