Cytochrome cell culture

Imidazole antimycotics, ketoconazole, clotrimazole, and miconazole are potent inhibitors of various cytochrome P450-isoenzymes that also affect the metabolism of retinoids. They were fust shown to inhibit the metabolism of RA in F9 embryonal carcinoma cells. When tested in vitm liarazole, a potent CYP-inhibitor, suppressed neoplastic transformation and upregulated gap junctional communication in murine and human fibroblasts, which appeared to be due to the presence of retinoids in the serum component of the cell culture medium. Furthermore, liarazole magnified the cancer chemopreventive activity of RA and (3-carotene in these experiments by inhibiting RA-catabolism as demonstrated by absence of a decrease in RA-levels in the culture medium in the presence of liarazole over 48 h, whereas without liarazole 99% of RA was catabolized. In vivo, treatment with liarazole and ketoconazole reduced the accelerated catabolism of retinoids and increased the mean plasma all-irans-RA-concentration in patients with acute promyelocytic leukemia and other cancels. 

STADLER, R., ZENK, M.H., The purification and characterization of a unique cytochrome P-450 enzyme from Berberis stolonifera plant cell cultures, J. Biol. Chem., 1993,268, 823-831. 

Paine, A. J. and Hockin, L. J. (1980). Nutrient imbalance causes the loss of cytochrome P-450 in liver cell culture formulation of culture media which maintain cytoch rome P-450 at in vivo concentrations. Biochem. Pharmacol. 29 3215-3218. 

Flavanone 3 -hydroxylase (F3 H ECl.14.13.21 CYP75B) activity was initially identified in microsomal preparations of golden weed (Haplopappus gracilis) [110]. E3 H from irradiated parsley cell cultures was later biochemically analyzed and characterized as a cytochrome P450 having an absolute requirement for NADPH and molecular oxygen as cofactors [111]. The enzyme has been shown to have activity with flavanones, flavones, dihydroflavonols, and flavonols, but does not appear to have activity with anthocyanidins [111]. The first cDNA clone for E3 H was isolated from Petunia [112]. It has been suggested that E3 H may serve as an anchor for the proposed flavonoid multi-enzyme complex on the cytosolic surface of the endoplasmic reticulum [44]. 

F3 H activity from the microsomal fraction of cell cultures of C. sinensis (L.) Osbeck cv. Hamlin (Hamlin) has been biochemically characterized and verified to be a cytochrome P450 [113]. The enzyme was shown to use naringenin, dihy-drokaempferol, and kaempferol, but not apigenin as substrates. E3 H activity was also demonstrated from young leaves of Hamlin orange as well as from the flavedo of Hamlin orange, Marsh grapefruit, and Lisbon lemon fruits [113]. The fact... 

Doostdar H, Shapiro JP, Niedz R, Burke MD, McCollum TG, McDonald RE, Mayer RT (1995) A cytochrome P450 mediated naiingenin 3 -hydroxylase from sweet orange cell cultures. Plant Cell Physiol 36(l) 69-77... 

Cytochrome P450 2D6 and 3A4 activities. Saw palmetto extract, administered to healthy volunteers (six men and six women) for 14 days at generally recommended doses, did not alter the disposition of coadministered dextromethorphan and alprazolam primarily dependent on the CYP2D6 or CYP3A4 pathways for elimination b Cytotoxic activity. Extract from saw palmetto, in LNCaP cell culture, produced cell... 

Limited data were available to evaluate the toxic effects of ethylbenzene in humans. Liver and kidney weights were increased in rats following exposure to ethylbenzene with no signs of hepatic necrosis. Cytochrome P450 enzymes were induced in both liver and kidney of ethylbenzene-exposed rats. Ethylbenzene caused changes in dopamine levels in brain and prolactin secretion in rats exposed for three to seven days. In rat brain cell cultures, ethylbenzene decreased the activity of several integral membrane enz5unes. 

CYP3A4 has been expressed in human lymphoblasts (Crespi et al., 1991a). The expression level in this report was quite low. Recent modifications to the promoter for cDNA expression and coexpression of OR have led to a 40-fold increase in catalytic activity. The mean testosterone 6j8-hydroxylase activity in microsomes from h3A4/OR cells (cultured in the presence of dexamethasone to induce cytochrome P450 reductase activity) [1000 pmol/(mg min)] is comparable to the mean values observed in human liver microsomes [1070 pmol/(mg min) Yamazaki et al., 1993]. The turnover number for testosterone and CYP3A4 in the human lymphoblasts was 15/min for endogenous OR levels and increased to 44/min with OR coexpression. 

Inhibition by 7-hydroxyflavone was competitive with respect to the substrate androstenedione. Flavonoids of the 5,7-dihydroxyflavone series could bind to the active site human cytochrome P-450 aromatase with affinity. The flavonoid kaempferol inhibited aromatase enzyme activity competitively in a human Glyoxalase cell culture system. Such results suggest that diets rich in these compounds could contribute to the control of estrogen-dependent conditions, such as breast cancer. 

Figure 13 Dose- and time-dependent induction of CYP3A4 (A), CYP3A5 (B), and CYP3A7 (C) mRNAs in human cultured hepatocytes incubated with rifampicin. Cells cultured on Matrigel-coated 96-well plates were incubated with increasing doses of rifampicin, and RNA was harvested at times indicated. Specific CYP mRNAs were determined by TaqMan RT-PCR. Abbreviations. CYP, cytochrome P450 RT-PCR, realtime reverse transcriptase-polymerase chain reaction.

Owens, I.S., and D.W. Nebert. Aryl hydrocarbon hydroxylase induction in mammalian liver-derived cell cultures. Stimulation of "cytochrome P1450-ass ocia ted" enzyme activity by many inducing compounds. Mol. Pharmacol. 11 94-104, 1975. 

Currently, there are many methods available for determining cell death by apoptosis in cell cultures and tissues. These methods are based essentially on changes that occur in apoptotic cells. During apoptosis, several phenomena can be observed, such as DNA fragmentation, chromatin condensation, nuclear fragmentation, cytoplasm acidification, cytochrome c release from the mitochondria, exposure of intracellular phospholipids and the activation and breakdown of proteins. These apoptotic phenomena can be detected by direct or indirect methods, on cell populations or on individual cells that are representative of a population. The main principles used by the different detection methods are ... 

A feature of some pterocarpan phytoalexins (e.g., pisatin and glyceollin of pea and soybean, respectively) is their hydroxylation at position 6a, a reaction catalyzed by a microsomal cytochrome P450 monooxygenase.49 50 A cDNA encoding this enzyme was recently characterized from elicited soybean cell cultures.51 The microsomal protein, expressed in yeast cells, catalyzed the stereoselective hydroxylation of (6a/ , lla/ )-3,9-dihydroxypterocarpan to its 6a-hydroxy derivative. It was also demonstrated that the enzyme expression is regulated at the transcriptional level.51... 

FALKENHAGEN, H., STOCKIGT, J., Enzymatic biosynthesis of vomilenine, a key intermediate of the ajmaline pathway, catalyzed by a novel cytochrome P450-dependent enzyme from plant cell cultures of Rauwoljia serpentina. Z. Naturforsch., 1995, 50C, 45-53. 

MEIJER, A.H., DE WAAL, A., VERPOORTE, R., Purification of cytochrome P450 enzyme geraniol 10 hydroxylase from cell cultures of Catharanthus roseus., J. Chromatog., 1993,35,237-249. 

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