The effect of co-administration of berberine, resveratrol, and glibenclamide on xenobiotic metabolizing enzyme activities in diabetic rat liver.

It is possible to use plant-derived antioxidant molecules in the form of dietary supplements. However, dietary supplement-drug interaction pattern has not been well defined for most of these products. The aim of this study was to determine the effects of berberine, resveratrol, and glibenclamide on xenobiotic metabolizing enzyme activities in diabetic rats. Streptozotocin was administered to create experimental diabetes. Resveratrol (5 mg/kg) (R), glibenclamide (5 mg/kg) (G), and berberine (10 mg/kg) (B) were administered individually or in combinations in DMSO by intraperitoneal administration route to the diabetic rats. DMSO was also given to non-diabetic control (C) and diabetic control (D) groups. Livers of rats were taken under anesthesia at the end of the treatment period (12 days). Ethoxyresorufin O-deethylase (EROD), pentoxyresorufin O-depentylase (PROD), aniline 4-hydroxylase (A4H), erythromycin N-demethylase (ERND), glutathione S-transferase (GST), catalase (CAT), and glutathione reductase (GR) activities were measured in microsomes and cytosols. In addition, histomorphological studies were also performed in the liver tissues. EROD activity of D+R was significantly higher than C and D+R+B. PROD activity of D+R was significantly higher than C, D, D+R+G, D+R+B, and D+R+B+ G. PROD activity of D+B was significantly higher than C and D+R+B. ERND activity of D+R was significantly higher than D+R+G and D+R+B. GST activity of D+R was significantly higher than D+R+G. CAT activity of D+B was significantly lower than C. It is clear that co-administration of resveratrol, berberine, and glibenclamide modifies some of the important xenobiotic metabolizing enzyme activities. Resveratrol and berberine have the potential to cause dietary supplement-drug interaction.

Decreasing myocardial estrogen receptors and antioxidant activity may be responsible for increasing ischemia- and reperfusion-induced ventricular arrhythmia in older female rats.

AIMS: This study aimed to investigate the relationship between ischemia- and reperfusion-induced arrhythmia and blood serum estrogen levels, myocardial estrogen receptor levels, antioxidant enzyme activities, and the effects of the estrogen receptor blocker, fulvestrant (ICI 182 780). MAIN METHODS: A total of 102 female Sprague-Dawley rats of different ages (2-3, 6-7, 14-15, and 20-21 months) were used in this study. Myocardial ischemia was produced by ligation of the descending branch of the left anterior descending coronary artery, and reperfusion was produced by releasing this artery. An electrocardiogram (ECG) and blood pressure were recorded for 6 min of ischemia and 6 min of reperfusion. The levels of superoxide dismutase (SOD), malondialdehyde (MDA), catalase (CAT), estrogen receptor α (ERα), and estrogen receptor ß (ERß) in myocardial tissue and 17 beta-estradiol (E2) in blood serum were measured via enzyme-linked immunosorbent assay (ELISA). The results were compared using a Mann-Whitney U test, one-way analysis of variance (ANOVA), and a student's t-test. KEY FINDINGS: It is not the changes in serum estrogen levels but the decreasing myocardial estrogen receptors and antioxidant activities that could be responsible for the occurrence of more severe arrhythmia in response to reperfusion in older female rats. SIGNIFICANCE: The death rate due to a heart attack in younger men is higher than in women. However, it equalizes after the menopausal stage in women. In this study, the reason for the increasing sudden post-menopausal death rate in women was investigated experimentally.

Monitoring of pollution in the western Black Sea coast of Turkey by striped red mullet (Mullus surmuletus).

The striped red mullet (Mullus surmuletus) is an economically important demersal fish species. In this study, our aim was to monitor the pollution in the western Black Sea coast of Turkey using striped red mullet as a bioindicator species. Fish samples were caught from four different locations in the western Black Sea coast of Turkey in 2006, 2009-2011, and 2016. Highly elevated cytochrome P4501A (CYP1A)-related 7-ethoxyresorufin O-deethylase (EROD) activities were measured in striped red mullet caught from Zonguldak Harbor in all of the sampling years. The lowest EROD activities were measured in fish samples caught from Kefken. In addition to the EROD activity measurements, glutathione S-transferase (GST), glutathione reductase, and catalase activities were also measured in the striped red mullet samples. Higher GST and catalase activities were measured in the striped red mullet samples caught from Zonguldak Harbor than from Kefken in 2016. These results indicate that the striped red mullet is responsive to CYP1A inducer pollutants. This study covers intermittent measurements of the biomonitoring data from the striped red mullet caught around the western Black Sea coast of Turkey, over a 10-year period.

Modulation of xenobiotic metabolizing enzyme activities in rat liver by co-administration of morin, endosulfan, and 7,12-dimethylbenz[a]anthracene.

Morin is a flavonoid which is present in many plants. Endosulfan and 7,12-dimethylbenz[a]anthracene (DMBA) are toxic chemicals that humans are exposed to in their daily lives. In this study, the protective role of morin was investigated in endosulfan and DMBA treated rats. Eight groups, each comprising seven 2.5-month-old adult male Wistar rats (weighing 170-255 g), were used. Endosulfan, morin, and DMBA were administered individually or in combinations, at 5 mg/kg body weight (bw) (three times/week), 25 mg/kg bw (three times/week), and 30 mg/kg bw (once/week for three weeks) via oral gavage, respectively. On day 54 of the administration period, the rats were killed. DMBA + endosulfan co-administration significantly increased CYP1A1-, CYP1A2-, CYP2E-, and GST-associated activities in the rats compared to the control. DMBA + endosulfan + morin significantly increased CYP1A1, CYP1A2, CYP3A, and GST associated activities in the rats relative to the control. Histopathological studies were performed to investigate protective effects of morin on liver damage. The results indicated that DMBA + endosulfan treatment induced liver damage, and morin reduced this damage. These findings suggest that CYP1A, CYP3A, and GST enzyme activities participate in the protective mechanism of morin against endosulfan and DMBA induced toxicity.

Combined use of PAH levels and EROD activities in the determination of PAH pollution in flathead mullet (Mugil cephalus) caught from the West Black Sea coast of Turkey.

The aim of this study was to determine the extent of polycyclic aromatic hydrocarbon (PAH) pollution by measuring PAH levels and 7-ethoxyresorufin-O-deethylase (EROD) activities in flathead mullet (Mugil cephalus) samples caught from the West Black Sea coast of Turkey. The fish samples were caught in August 2008-2011. The levels of 13 PAHs were measured by high-performance liquid chromatography (HPLC) in the liver of fish. Most of the measured PAHs had three rings (low molecular weight). The frequencies of detection of PAHs were higher in fish samples caught from Zonguldak Harbour and Gülüç Stream Mouth than those from Sakarya River Mouth, Amasra and Kefken. EROD activities and cytochrome P4501A (CYP1A) protein level were also measured in the fish liver microsomes. Highly elevated EROD activities and CYP1A levels were measured in the mullet samples caught from Zonguldak Harbour and Gülüç Stream than those from Amasra and Kefken. The detection of PAHs in the liver of fish samples shows recent exposure to PAHs. The chemical analyses of PAHs and EROD activity results together reflected the extent of PAH pollution in the livers of fish caught from the West Black Sea coast of Turkey. The results indicate that Zonguldak Harbour is the most polluted site in the West Black Sea coast of Turkey.

Mechanism of inhibition of CYP1A1 and glutathione S-transferase activities in fish liver by quercetin, resveratrol, naringenin, hesperidin, and rutin.

Quercetin, resveratrol, naringenin, hesperidin, and rutin are phenolic compounds/flavonoids that may have roles in the reduction of cancer susceptibility. In this study, in vitro modulatory effects of them were studied on liver CYP1A1 associated 7-ethoxyresorufin-O-deethylase (EROD) activity and glutathione S-transferase (GST) activity of leaping mullet (Liza saliens). All of the phenolic compounds/flavonoids used exerted an inhibitory effect on both EROD and GST activities of fish. Quercetin, resveratrol, hesperidin, and rutin were found to inhibit EROD activity in a competitive manner; on the other hand, naringenin was found to inhibit EROD activity in a noncompetitive manner. Ki values of quercetin, resveratrol, naringenin, hesperidin, and rutin were calculated from Dixon plots as 0.12 µM, 0.67 µM, 2.63 µM, 18 µM and 0.1 mM, respectively. Resveratrol, quercetin, and hesperidin were found to inhibit GST activity in a competitive manner; on the other hand, rutin and naringenin were found to inhibit GST activity in a mixed-type manner. Ki values of resveratrol, quercetin, hesperidin, naringenin, and rutin were calculated from Dixon plots as 3.2 µM, 12.5 µM, 45 µM, 128 µM, and 150 µM, respectively. The results suggest that quercetin and resveratrol containing foods are effective in the prevention and treatment of cancer.

Determination of organochlorine pesticide concentrations in flathead mullet (Mugil cephalus) caught from the western Black Sea coast of Turkey.

The objective of this study was to determine the levels of 14 organochlorine pesticides (OCPs) in flathead mullet (Mugil cephalus) caught from the western Black Sea coast of Turkey. The fish samples were caught from five different locations of the western Black Sea coast of Turkey in August 2009. Organochlorine pesticides were extracted from the liver tissues, and then the levels of OCPs were measured using gas chromatography with an electron capture detector. Organochlorine pesticides were detected in all locations. The levels of total OCPs in fish samples ranged between 0.224 and 1.103 µg g(-1) dry weight in the western Black Sea coast of Turkey. DDT, beta-HCH, and endosulfan I were the dominant OCPs in the fish samples. The levels of DDT in fish samples ranged between 0.081 and 0.186 µg g(-1) dry weight. The levels of total HCH in fish samples ranged between 0.007 and 0.376 µg g(-1) dry weight in the western Black Sea coast of Turkey. Although the usage of OCPs was banned in Turkey, the results of this study clearly indicated the presence of OCPs in the western Black Sea coast of Turkey and exposure of living organisms to these chemicals.

Aldrin epoxidation in flathead mullet (Mugil cephalus): possible involvement of CYP1A and CYP3A.

The primary objective of this study was to determine specific cytochrome P450 isozyme(s) involved in the metabolism of aldrin to its toxic metabolite dieldrin in flathead mullet (Mugil cephalus) liver microsomes. To identify the cytochrome P450 isozyme responsible for the aldrin metabolism in mullet liver, the effects of mammalian-specific cytochrome P450 inhibitors and substrates were determined in the epoxidation reaction of aldrin. CYP3A-related inhibitors, ketoconazole, SKF-525A, and cimetidine, inhibited the metabolism of aldrin. The contribution of CYP1A to the aldrin metabolism was shown by the inhibition of 7-ethoxyresorufin-O-deethylase activity in the presence of aldrin. The results indicate that CY1A and CYP3A are the cytochrome P450s involved in aldrin epoxidase activity in mullet. In addition, the suitability of aldrin epoxidase activity for monitoring of environmental pollution was also assessed in the fish samples caught from four different locations of the West Black Sea coast of Turkey.

Assessment of pollution in the West Black Sea Coast of Turkey using biomarker responses in fish.

Aim of this study was to determine the extent of pollution in the West Black Sea Coast of Turkey by measuring CYP1A associated EROD activity, phase II enzyme, glutathione S-transferase and antioxidant enzymes, catalase and glutathione reductase activities and immunochemical detection of CYP1A protein level in the liver of mullet. The fish samples were caught from six locations having a varying degree of pollution in the West Black Sea Region of Turkey in August 2005, 2006 and 2007. Mullets caught from Zonguldak Harbour, Eregli Harbour and Gülüç Stream's Mouth displayed 6-9-fold higher EROD, 2-4-fold higher glutathione S-transferase and 2-3-fold higher catalase activities than the reference site, Amasra. Total polyaromatic hydrocarbon levels in mullets caught from these locations were also significantly higher (2-4-fold) than Amasra. The results of this study indicate that Zonguldak Harbour, Eregli Harbour and Gülüç Stream are highly polluted by polycyclic aromatic hydrocarbons and related contaminants.

Purification of CYP2B-like protein from feral leaping mullet (Liza saliens) liver microsomes and its biocatalytic, molecular, and immunological characterization.

In this study, CYP2B-immunoreactive protein was purified to electrophoretic homogeneity from the liver microsomes of leaping mullet. The purified cytochrome P450 (CYP) gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis having a M(r) of 49,300 Da. Absolute absorption spectrum of the purified CYP showed a maximum at 417 nm and CO-difference spectrum of dithionite-reduced cytochrome P450 gave a peak at 450 nm. The purified CYP was found to be active in N-demethylation of benzphetamine, erythromycin, and ethylmorphine, and O-dealkylation of pentoxyresorufin in the reconstituted system. However, it was unable to catalyze O-dealkylation of ethoxyresorufin, methoxyresorufin, benzyloxyresorufin, and hydroxylation of lauric acid and aniline. The purified CYP showed strong cross-reactivity with anti-sheep lung CYP2B, a homologue of CYP2B4. N-terminal amino acid sequence of the mullet P450 had the highest degree of homology with CYP2Bs among the known CYPs. Spectral, electrophoretic, immunochemical, N-terminal amino acid sequence, and biocatalytic properties of the purified CYP are most similar to those of mammalian cytochrome P4502B. All these data indicate that the purified CYP is certainly 2B-like. In this study, we not only purified biocatalytically active CYP2B-like protein from fish, but also demonstrated detailed functional properties of CYP2B-like protein for the first time.

Mechanism of inhibition of purified leaping mullet (Liza saliens) NADPH-cytochrome P450 reductase by toxic metals: aluminum and thallium.

Aluminum and thallium may reach life-threatening levels in aquatic systems in the near future because of their extensive use in various industrial fields. It is therefore important to study the mechanism of toxicity of aluminum and thallium on fish enzymes. To this aim, the effects of aluminum and thallium on the activity of purified leaping mullet (Liza saliens) cytochrome P450 reductase, an essential component of the important cytochrome P450 system, have been studied. Results indicated that both metal ions strongly inhibited the NADPH-cytochrome P450 reductase. The IC50 values of AlCl3 and TlCl3 were estimated to be 34 microM and 3 microM, respectively. The Lineweaver-Burk plot and Dixon plot revealed that both metal ions noncompetitively inhibited the purified mullet cytochrome P450 reductase. The K(i) values of Al3+ and Tl3+ were calculated from Dixon plots as 8.9 and 5.6 microM, respectively. The inhibitory effects of Al3+ and Tl3+ on purified cytochrome P450 reductase were partially recovered by 1 mM EDTA. Additionally, tin and magnesium were shown to have no apparent effect on purified mullet cytochrome P450 reductase.

Effects of diabetes on rabbit kidney and lung CYP2E1 and CYP2B4 expression and drug metabolism and potentiation of carcinogenic activity of N-nitrosodimethylamine in kidney and lung.

There are limited number of studies regarding the influence of diabetes on the regulation of cytochrome P450s and associated drug metabolizing enzyme activities especially in extrahepatic tissues such as kidney. However, there is almost no such study in lung. Alloxan-induced diabetes did not change CYP2B4 expression as measured with immunoblot analysis and associated enzyme, benzphetamine N-demethylase, activity in rabbit kidney and lung. Induction of cytochrome P4502E1 by diabetes was identified by immunochemical detection on Western blots in the lung and kidney microsomes of rabbits. In parallel to CYP2E1 induction, aniline 4-hydroxylase and p-nitrophenol hydroxylase activities were markedly increased in diabetic rabbit lung and kidney. CYP2B4 and CYP2E1 dependent drug metabolism did not show any tissue variation in diabetic rabbit. These findings are in contrast to those of rats, mice and hamster. The results of the present work, in combination with those of the previous work [Arinç, E., Arslan, S., Adali, O., 2005. Differential effects of diabetes on CYP2E1 and CYP2B4 proteins and associated drug metabolizing enzyme activities in rabbit liver. Arch. Toxicol. 79, 427-433], indicate the existence of species-dependent response of CYP-dependent drug metabolizing enzymes to diabetes. A procarcinogen and food contaminant, N-nitrosodimethylamine (NDMA), is converted to its carcinogenic form after it is activated with NDMA N-demethylase. In the current study, a statistically significant increase of liver, kidney and lung NDMA N-demethylase activity associated with CYP2E1 was shown in diabetic rabbit. Thus, it is expected that, the risk of nitrosamine induced carcinogenesis will be greater in liver, kidney and lung of the diabetic subjects.

Effect of mercury, cadmium, nickel, chromium and zinc on kinetic properties of NADPH-cytochrome P450 reductase purified from leaping mullet (Liza saliens).

Information on the mechanism of metal ion inhibition of NADPH-cytochrome P450 reductase is limited. The purpose of the present paper was to elucidate in vitro effect of Hg(+2), Cd(+2), Ni(+2), Cr(+3) and Zn(+2) ions on the purified mullet NADPH-cytochrome P450 reductase. NADPH-cytochrome P450 reductase was purified from detergent-solubilized liver microsomes from leaping mullet (Liza saliens). All of the metal ions caused inhibition of the enzyme activity except Zn(+2). At 50 microM metal concentration, Hg(+2) inhibited the cytochrome P450 reductase activity completely (100%), while, at the same concentrations, Cd(+2), Cr(+3) and Ni(+2) caused 66%, 65% and 37% inhibition, respectively. At 50 microM metal concentration, Zn(+2) had no apparent effect on cytochrome P450 reductase activity. The IC(50) values of HgCl(2), CrCl(3), CdCl(2) and NiCl(2) were estimated to be 0.07 microM, 24 microM, 33 microM and 143 microM, respectively. Of the metal ions tested, Hg(+2) exhibited much higher inhibitory effect at lower concentrations, so it was evidently a more potent inhibitor than the others. All four metal ions displayed noncompetitive type of inhibition mechanism for the purified reductase as analyzed by Dixon plot. K(i) values of Hg(+2), Cr(+3), Cd(+2), and Ni(+2) were calculated from Dixon plots as 0.048 microM, 18 microM, 73 microM and 329 microM, respectively.

Inhibitory effects of divalent metal ions on liver microsomal 7-ethoxyresorufin O-deethylase (EROD) activity of leaping mullet.

The purpose of the present study was to elucidate in vitro effects of Hg(2+), Zn(2+), Ni(2+) and Cd(2+) on cytochrome P4501A1 (CYP1A1) dependent EROD activities in leaping mullet liver microsomes. Fish captured from the most polluted part of Izmir Bay, had highly elevated EROD activities, and induced CYP1A1 protein levels as determined by Western blotting. Although all of the metal ions caused inhibition of the initial velocity of the reaction, Hg(2+) and Cd(2+) exhibited much higher inhibitory effect at lower concentrations and they were evidently more potent inhibitors than others. The inhibitor concentration giving 50% inhibition (IC(50) values) of Zn(2+), Ni(2+), Cd(2+) and Hg(2+) of initial EROD activity were 107, 16, 1.3 and 0.15 micromolar, respectively. Glutathione (GSH) at 0.5 mM final concentration, completely reversed Ni(2+) and Cd(2+) inhibition of EROD activity indicating the protective action of GSH.

Catalyzation of cocaine N-demethylation by cytochromes P4502B, P4503A, and P4502D in fish liver.

Cocaine N-demethylation by microsomal cytochrome P450s is the principal pathway in cocaine bioactivation and hepatotoxicity. P450 isozymes involved in N-demethylation of cocaine have not been elucidated yet and they differ from species to species. In humans and mice, P4503A contributes to cocaine N-demethylase activity, whereas in rats, both P4503A and P4502B participate. In the present study, contribution of different P450 isozymes to cocaine N-demethylase activity was studied in vitro with fish liver microsomes. The specific cocaine N-demethylase activity was found to be 0.672 +/- 0.22 nmol formaldehyde formed/min/mg protein (mean +/- SD, n = 6). Cocaine N-demethylase exhibited biphasic kinetics, and from the Lineweaver-Burk plot, two K(m) values were calculated as 0.085 and 0.205 mM for the high- and low-affinity enzyme. These results indicate that N-demethylation of cocaine in mullet liver microsomes is catalyzed by at least two cytochrome P450 isozymes. Inhibitory effects of cytochrome P450 isozyme-selective chemical inhibitors, ketoconazole, cimetidine, SKF-525A, and quinidine, on cocaine N-demethylase activity were studied at 50, 100, and 500 micro M concentrations of these inhibitors. At 100 micro M final concentrations, ketoconazole (P4503A inhibitor), SKF-525A (inhibitor of both P4502B and P4503A), and cimetidine (P4503A inhibitor) inhibited N-demethylation activity by 73, 69, and 63%, respectively. Quinidine, P4502D-specific inhibitor, at 100 micro M final concentration, reduced N-demethylation activity down to 64%. Aniline, a model substrate for P4502E1, did not alter N-demethylase activity in the final concentration of 100 micro M. IC(50) values were calculated to be 20 micro M for ketoconazole, 48 micro M for cimetidine (both specific P4503A inhibitors), 164 micro M for quinidine (P4502D inhibitor), and 59 micro M for SKF-525A (inhibitor of both P4503A and P4502B). The contribution of P4502B to cocaine N-demethylase activity in mullet liver microsomes was further explored by the use of purified mullet cytochrome P4502B in the reconstituted system containing purified mullet P450 reductase and lipid. The turnover number was calculated as 4.2 nmol HCOH/(min nmol P450). Overall, these results show that P4503A and P4502B are the major P450s responsible for N-demethylation of cocaine, whereas contribution of P4502D is a minor one, and P4502E1 is not involved in the N-demethylation of cocaine in mullet liver microsomes.