Addition Of Cytochrome P-450 Inhibitors Research Articles

Multistage defense response of microalgae exposed to pharmaceuticals in wastewater

Recently, the use of microalgae for bioremediation of pharmaceuticals (PhAs) has attracted increasing interest. However, most studies focused more on microalgae removal performance, its defensive response to the PhAs during wastewater treatment remains unexplored. Herein, microalgal three defensive systems have been investigated in synthetic wastewater, with six PhAs as the typical drug. Results show that PhAs could bind to EPS, and this action in turn could help to alleviate the direct toxicity of PhAs to microalgae. Subsequently, the physiological analyses revealed the increase of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) activities, potentially reducing the oxidative stress induced by PhAs. Furthermore, the enzyme activities of cytochrome P450 (CYP450) and glutathione-S-transferase (GST) were significantly upregulated after exposure to SMX, CIP and BPA, followed by a significant decrease in biodegradation rates after the addition of CYP450 inhibitors, suggesting that the biotransformation and detoxification of PhAs occurred. Meanwhile, molecular docking further revealed that CYP450 could bind with PhAs via hydrogen bond and hydrophobic interaction, which proved their abilities to be metabolized and form transformation products in microalgae. These findings provide an advancing understanding of microalgae technologies to improve the treatment of wastewater contaminated with PhAs.

Identification of the cytochrome P450 involved in the degradation of neonicotinoid insecticide acetamiprid in Phanerochaete chrysosporium.

We previously reported that cytochrome P450 s play critical roles in neonicotinoid insecticide biodegradation by white-rot fungi. Here, we investigated the biodegradation of acetamiprid (ACET) by Phanerochaete chrysosporium to identify the cytochrome P450 involved in this degradation process. During a 20-day incubation period, P. chrysosporium degraded 21% and 51% of ACET in ligninolytic and nonligninolytic media, respectively. The degradation rate of ACET was markedly decreased by the addition of cytochrome P450 inhibitors. Recombinant cytochrome P450s in P. chrysosporium (PcCYP) were heterologously expressed in Saccharomyces cerevisiae strain AH22, and the PcCYP involved in ACET degradation was identified. The results showed that CYP5147A3 can degrade ACET, and two ACET metabolites, N'-cyano-N-methyl acetamidine and 6-chloro-3-pyridinemethanol, were identified. To the best of our knowledge, this study provides the first characterization of the fungal cytochrome P450 that is responsible for the degradation and detoxification of ACET.

Bioremediation of the neonicotinoid insecticide clothianidin by the white-rot fungus Phanerochaete sordida

Clothianidin (CLO) is a member of the neonicotinoid pesticides, which have been widely used worldwide over the last two decades. However, its toxicity for bees and neurological toxicity for humans are urgent problems. Here, the degradation of CLO by the white-rot fungus Phanerochaete sordida was examined in nitrogen-limited liquid medium. After incubation for 20days at 30°C, 37% of CLO was degraded in the cultures. High-resolution ESI–MS and NMR analyses of the culture supernatant identified N-(2-chlorothiazol-5-yl-methyl)-N′-methylurea (TZMU) as a metabolite of CLO degradation. The addition of cytochrome P450 inhibitors to the culture medium markedly reduced the degradation of CLO by P. sordida. And manganese peroxidase, a major ligninolytic enzyme secreted by this fungus, were not carried out CLO degradation. The effects of CLO and TZMU on the viability of the neuronal cell line Neuro2a demonstrated that P. sordida effectively degrades CLO into a metabolite that lacks neurotoxicity.

Biodegradation of octyltin compounds by Cochliobolus lunatus and influence of xenobiotics on fungal fatty acid composition

The perturbation in the growth and fatty acid profile of the microscopic fungus Cochliobolus lunatus IM 4417 in the presence of octyltin compounds (trioctyltin – TOT, dioctyltin – DOT and monooctyltin – MOT) was studied. Fungal resistance to the tested organotins decreased with a reduction in the number of octyl groups bonded with a tin atom. Also, the fatty acid unsaturation index decreased according to the mentioned scheme. Among all tested octyltin compounds, TOT was removed with the highest efficiency. The efficiency of MOT removal was correlated with the initial concentration of the compounds and for concentrations 20 and 100mgl−1 reached the value of 75% and 40%, respectively. Elimination of octyltins depended on the metabolic activity of the fungus and was not the result of passive sorption. During bioconversion of TOT the hydroxylated derivative of substrate was detected. Moreover, the addition of cytochrome P-450 inhibitors significantly reduced the metabolism of octyltin compounds. Thus, it is postulated that the process of degradation of octyltin compounds is similar to that described for tributyltin (TBT) and it is mediated by cytochrome P-450.

Biotransformation of the organochlorine pesticide trans-chlordane by wood-rot fungi

There is very limited information on the biotransformation of organochlorine pesticide chlordane by microorganisms, and no systematic study on the metabolic products and pathways for chlordane transformation by wood-rot fungi has been conducted. In this study, trans-chlordane was metabolized with the wood-rot fungi species Phlebia lindtneri, Phlebia brevispora and Phlebia aurea, which are capable of degrading polychlorinated dibenzo-p-dioxin and heptachlor epoxide. At the end of 42 days of incubation, over 50% of trans-chlordane was degraded by the fungal treatments in pure cultures. These fungi transformed trans-chlordane to at least eleven metabolites including a large amount of hydroxylated products such as 3-hydroxychlordane, chlordene chlorohydrin, heptachlor diol, monohydroxychlordene and dihydroxychlordene. P. lindtneri particularly can metabolize oxychlordane, a recalcitrant epoxide product of chlordane, into a hydroxylated product through substitution of chlorine atom by hydroxyl group. The present results suggest that hydroxylation reactions play an important role in the metabolism of trans-chlordane by these Phlebia species. Additionally, transformation of trans-chlordane and production of hydroxylated metabolites were efficiently inhibited by the addition of cytochrome P450 inhibitors, piperonyl butoxide and 1-aminobenzotriazole, demonstrating that fungal cytochrome P450 enzymes are involved in some steps of trans-chlordane metabolism, particularly in the hydroxylation process.

Cytochrome P-450 inhibitors alter afferent arteriolar responses to elevations in pressure.

The present study evaluated the effects of cytochrome P-450 inhibitors on the response of the renal microvasculature to changes in renal perfusion pressure and on autoregulation of glomerular capillary pressure using the rat juxtamedullary nephron microvascular preparation perfused in vitro with a cell-free perfusate containing 5% albumin. The basal diameters of the proximal and distal afferent arterioles averaged 28 +/- 1 (n = 32) and 18 +/- 1 micron (n = 23), respectively, at a control perfusion pressure of 80 mmHg. The diameters of these vessels decreased by 8% when perfusion pressure was elevated from 80 to 160 mmHg. After addition of cytochrome P-450 inhibitors (either 17-octadecynoic acid, 20 microM; 7-ethoxyresorufin, 10 microM; or miconazole, 20 microM) to the perfusate, the diameters of the proximal and distal afferent arterioles increased by 6% in response to the same elevation in perfusion pressure. Control glomerular capillary pressure averaged 43 +/- 1 mmHg (n = 32) at a renal perfusion pressure of 80 mmHg and increased by only 9 +/- 1 mmHg when perfusion pressure was elevated to 160 mmHg. Autoregulation of glomerular capillary pressure was impaired after addition of the cytochrome P-450 inhibitors, and it increased by 18 +/- 2 mmHg when perfusion pressure was varied over the same range. These results indicate that cytochrome P-450 inhibitors attenuate the vasoconstrictor response of afferent arterioles to elevations in renal perfusion pressure and impair autoregulation of glomerular capillary pressure, suggesting a possible role for cytochrome P-450 metabolites of arachidonic acid in these responses.

Spin trapping of free radical species produced during the microsomal metabolism of ethanol

Liver microsomes incubated with a NADPH regenerating system, ethanol and the spin trapping agent 4-pyridyl-1-oxide- t-butyl nitrone (4-POBN) produced an electron spin resonance (ESR) signal which has been assigned to the hydroxyethyl free radical adduct of 4-POBN by using 13C-labelled ethanol. The free radical formation was dependent upon the activity of the microsomal monoxygenase system and increased following chronic feeding of the rats with ethanol. The production of hydroxyethyl free radicals was stimulated by the addition of azide, while catalase and OH• scavengers decreased it. This suggested that hydroxyl radicals (OH•) produced in a Fenton-type reaction from endogenously formed hydrogen peroxide were involved in the free radical activation of ethanol. Consistently, the supplementation of iron, under various forms, also increased the intensity of the ESR signal which, on the contrary, was inhibited by the iron-chelating agent desferrioxamine. Microsomes washed with a solution containing desferrioxamine and incubated in a medium treated with Chelex X-100 in order to remove contaminating iron still produced hydroxyethyl radicals, although at a reduced rate. Under these conditions the free radical formation was apparently independent from the generation of OH· radicals, whereas addition of cytochrome P-450 inhibitors decreased the hydroxyethyl radical formation, suggesting that a cytochrome P-450-mediated process might also be involved in the activation of ethanol. Reduced glutathione (GSH) was found to effectively scavenge the hydroxyethyl radical, preventing its trapping by 4-POBN. The data presented suggest that ethanol-derived radicals could be generated during the microsomal metabolism of alcohol probably through two different pathways. The detection of ethanol free radicals might be relevant in understanding the pathogenesis of the liver lesions which are a consequence of alcohol abuse.

In vitro metabolism of o-aminoazotoluene and mutagenesis of Salmonella by the metabolites.

Incubation of hepatocarcinogenic aminoazo dye, o-aminoazotoluene (OAT) with rat liver microsomes together with NADPH and NADH yielded N-hydroxy-OAT (I), 4'-hydroxy-OAT (II) and a smaller amount of 2'-hydroxymethyl-3-methyl-4-aminoazobenzene (III). As an artifact 4,4'-bis(o-tolylazo)-2,2' -dimethylazoxybenzene (IV) was also detected in a small quantity. The mutagenicities of these metabolites were assayed by using Salmonella typhimurium TA98 and TA100 together with S-9 prepared from the livers of rats treated with polychlorinated biphenyl mixture (PCB). OAT and III were strongly mutagenic, but only when S-9 was present. In contrast, I was a strong mutagen regardless of the presence or absence of S-9. II and IV were non-mutagenic. The yields of I, II and III from OAT were pronouncedly reduced by addition of cytochrome P-450 inhibitors, especially by a cytochrome P-448 inhibitor 7,8-benzoflavone. Mutagenesis by OAT was also inhibited by addition of 7,8-benzoflavone. Activation of OAT for mutagenesis was enhanced by pretreatment of the donor rats with PCB or 3-methyl-cholanthrene and to a much lesser extent by phenobarbital. These findings suggest that N-hydroxylation of OAT mainly proceeds via catalysis by cytochrome P-448 and that this process is an obligatory step for the activation of OAT. Synthetic methods for the preparation of new azo compounds such as I, IV and 2',3-dimethyl-4-nitrosoazobenzene are described.