Catechins Variously Affect Activities of Conjugation Enzymes in Proliferating and Differentiated Caco-2 Cells.

The knowledge of processes in intestinal cells is essential, as most xenobiotics come into contact with the small intestine first. Caco-2 cells are human colorectal adenocarcinoma that once differentiated, exhibit enterocyte-like characteristics. Our study compares activities and expressions of important conjugation enzymes and their modulation by green tea extract (GTE) and epigallocatechin gallate (EGCG) using both proliferating (P) and differentiated (D) caco-2 cells. The mRNA levels of the main conjugation enzymes were significantly elevated after the differentiation of Caco-2 cells. However, no increase in conjugation enzymes' activities in differentiated cells was detected in comparison to proliferating ones. GTE/EGCG treatment did not affect the mRNA levels of any of the conjugation enzymes tested in either type of cells. Concerning conjugation enzymes activities, GTE/EGCG treatment elevated glutathione S-transferase (GST) activity by approx. 30% and inhibited catechol-O-methyltransferase (COMT) activity by approx. 20% in differentiated cells. On the other hand, GTE as well as EGCG treatment did not significantly affect the activities of conjugation enzymes in proliferating cells. Administration of GTE/EGCG mediated only mild changes of GST and COMT activities in enterocyte-like cells, indicating a low risk of GTE/EGCG interactions with concomitantly administered drugs. However, a considerable chemo-protective effect of GTE via the pronounced induction of detoxifying enzymes cannot be expected as well.

Hyperoxia-induced changes in estradiol metabolism in postnatal airway smooth muscle.

Supplemental oxygen, used to treat hypoxia in preterm and term neonates, increases the risk of neonatal lung diseases, such as bronchopulmonary dysplasia (BPD) and asthma. There is a known sex predilection for BPD, but the underlying mechanisms are not clear. We tested the hypothesis that altered, local estradiol following hyperoxia contributes to pathophysiological changes observed in immature lung. In human fetal airway smooth muscle (fASM) cells exposed to normoxia or hyperoxia, we measured the expression of proteins involved in estrogen metabolism and cell proliferation responses to estradiol. In fASM cells, CYP1a1 expression was increased by hyperoxia, whereas hyperoxia-induced enhancement of cell proliferation was blunted by estradiol. Pharmacological studies indicated that these effects were attributable to upregulation of CYP1a1 and subsequent increased metabolism of estradiol to a downstream intermediate 2-methoxyestradiol. Microarray analysis of mouse lung exposed to 14 days of hyperoxia showed the most significant alteration in CYP1a1 expression, with minimal changes in expression of five other genes related to estrogen receptors, synthesis, and metabolism. Our novel results on estradiol metabolism in fetal and early postnatal lung in the context of hyperoxia indicate CYP1a1 as a potential mechanism for the protective effect of estradiol in hyperoxia-exposed immature lung, which may help explain the sex difference in neonatal lung diseases.

Myelophil ameliorates brain oxidative stress in mice subjected to restraint stress.

We evaluated the pharmacological effects of Myelophil, a 30% ethanol extract of a mix of Astragali Radix and Salviae Radix, on oxidative stress-induced brain damage in mice caused by restraint stress. C57BL/6 male mice (eight weeks old) underwent daily oral administration of distilled water, Myelophil (25, 50, or 100mg/kg), or ascorbic acid (100mg/kg) 1h before induction of restraint stress, which involved 3h of immobilization per day for 21days. Nitric oxide levels, lipid peroxidation, activities of antioxidant enzymes (superoxide dismutase, catalase, and glutathione redox system enzymes), and concentrations of adrenaline, corticosterone, and interferon-γ, were measured in brain tissues and/or sera. Restraint stress-induced increases in nitric oxide levels (serum and brain tissues) and lipid peroxidation (brain tissues) were significantly attenuated by Myelophil treatment. Restraint stress moderately lowered total antioxidant capacity, catalase activity, glutathione content, and the activities of glutathione reductase, glutathione peroxidase, and glutathione S-transferase; all these responses were reversed by Myelophil. Myelophil significantly attenuated the elevated serum concentrations of adrenaline and corticosterone and restored serum and brain interferon-γ levels. Moreover, Myelophil normalized expression of the genes encoding monoamine oxidase A, catechol-O-methyltransferase, and phenylethanolamine N-methyltransferase, which was up-regulated by restraint stress in brain tissues. These results suggest that Myelophil has pharmacological properties protects brain tissues against stress-associated oxidative stress damage, perhaps in part through regulation of stress hormones.

Optimization of fermentation conditions for the production of human soluble catechol-O-methyltransferase by Escherichia coli using artificial neural network.

The aim of this work was to optimize the temperature, pH and stirring rate of the production of human soluble catechol-O-methyltransferase (hSCOMT) in a batch Escherichia coli culture process. A central composite design (CCD) was firstly employed to design the experimental assays used in the evaluation of these operational parameters on the hSCOMT activity for a semi-defined and complex medium. Predictive artificial neural network (ANN) models of the hSCOMT activity as function of the combined effects of these variables was proposed based on this exploratory experiments performed for the two culture media. The regression coefficients (R(2)) for the final models were 0.980 and 0.983 for the semi-defined and complex medium, respectively. The ANN models predicted a maximum hSCOMT activity of 183.73 nmol/h, at 40 °C, pH 6.5 and stirring rate of 351 rpm, and 132.90 nmol/h, at 35 °C, pH 6.2 and stirring rate of 351 rpm, for semi-defined and complex medium, respectively. These results represent a 4-fold increase in total hSCOMT activity by comparison to the standard operational conditions used for this bioprocess at slight scale.

Berries and ellagic acid prevent estrogen-induced mammary tumorigenesis by modulating enzymes of estrogen metabolism.

To determine whether dietary berries and ellagic acid prevent 17beta-estradiol (E(2))-induced mammary tumors by altering estrogen metabolism, we randomized August-Copenhagen Irish rats (n = 6 per group) into five groups: sham implant + control diet, E(2) implant + control diet (E(2)-CD), E(2) + 2.5% black raspberry (E(2)-BRB), E(2) + 2.5% blueberry (E(2)-BB), and E(2) + 400 ppm ellagic acid (E(2)-EA). Animals were euthanized at early (6 wk), intermediate (18 wk), and late (24 wk) phases of E(2) carcinogenesis, and the mammary tissue was analyzed for gene expression changes using quantitative real-time PCR. At 6 weeks, E(2) treatment caused a 48-fold increase in cytochrome P450 1A1 (CYP1A1; P < 0.0001), which was attenuated by both BRB and BB diets to 12- and 21-fold, respectively (P < 0.001). E(2) did not alter CYP1B1 levels, but both berry and EA diets significantly suppressed it by 11- and 3.5-fold, respectively, from baseline (P < 0.05). There was a 5-fold increase in 17beta-hydroxysteroid dehydrogenase 7 (17betaHSD7), and this was moderately abrogated to approximately 2-fold by all supplementation (P < 0.05). At 18 weeks, CYP1A1 was elevated by 15-fold in E(2)-CD and only E(2)-BB reduced this increase to 7-fold (P < 0.05). Catechol-O-methyltransferase expression was elevated 2-fold by E(2) treatment (P < 0.05), and all supplementation reversed this. At 24 weeks, CYP1A1 expression was less pronounced but still high (8-fold) in E(2)-treated rats. This increase was reduced to 3.2- and 4.6-fold by E(2)-BRB and E(2)-EA, respectively (P < 0.05), but not by E(2)-BB. Supplementation did not alter the effect of E(2) on steroid receptors. The diets also significantly suppressed mammary tumor incidence (10-30%), volume (41-67%), and multiplicity (38-51%; P < 0.05). Berries may prevent mammary tumors by suppressing the levels of E(2)-metabolizing enzymes during the early phase of E(2) carcinogenesis.