扩展细胞色素P4502A6对大位阻底物的适应性

细胞色素P450酶(P450)的分子改造及其催化的生物转化是当前的两个研究热点.本研究以扩展P450 2A6的底物适应性为目标,通过尝试对P450 2A6酶进行N-端修饰提高表达量、筛选随机突变库以及点突变改变活性位点重要氨基酸残基等手段,在两个随机突变体中未筛选到功能突变体,但通过点突变重要氨基酸残基获得了新的突变体P450 2A6 (N297Q/I300A).该突变体具有比野生型和已报道的其它突变体更广泛的底物适应性,能够催化氧化大位阻底物6-苄氧基吲哚(6-OBzl-indole)生成蓝绿色物质.该物质经质谱测定为靛蓝类二聚体化合物.实验表明,其中两个氨基酸残基的变化都是关键突变,此结果与最新的P450 2A6突变体晶体结构研究相符.计算机模拟显示,I300A突变导致活性口袋在铁卟啉环的垂直和水平方向都增大,使其能接纳在两个方向都有位阻的底物;而297位的天冬酰胺是一个功能保守的残基,N297Q的变化可能与氢键等弱作用有关.图4表1参29     

Cytochrome P-450 monooxygenase system and benzo(a)pyrene metabolism in echinoderms

A comparative study on mixed-function oxygenase (MFO) systems was carried out on four echinoderm species: the asteroidsAsterias rubens andMarthasterias glacialis, a holothurianHolothuria forskali and an echinoidEchinus esculentus. Cytochromes P-450 andb ₅ and the MFO system-associated NADH-ferricyanide reductases NADH-cytochromec reductase, NADPH-cytochromec reductase and benzo(a)pyrene (BP) hydroxylase activities were present in microsomal fractions of pyloric caeca ofA rubens andM. glacialis and in the haemal plexus ofH. forskali. In contrast, cytochrome P-450 and BP hydroxylase activity were not detectable in the gonads ofE. esculentus. The tissue and subcellular distribution of the MFO system was studied inA. rubens. MFO system components were found in the stomachs and gonads, although detection of cytochrome P-450 in the latter tissue was difficult. Sex-related differences were not significant. The contents of the MFO system components and BP hydroxylase activities were highest in the microsomal (100 000 ×g) fractions, but MFO system components were also found in the mitochondrial (12 000 ×g) and cytosolic fractions. The BP hydroxylase activity in pyloric caeca microsomes ofA. rubens was NADPH-dependent and was inhibited by several agents known to be inhibitors of vertebrate cytochromes P-450. In the former respect, the characteristics of the MFO system were more like those of vertebrates and crustaceans than that of molluscs.     

双酚AF对雄性斑马鱼卵黄蛋白原水平与芳香化酶基因表达的影响

双酚AF(4,4'-六氟-2-二酚,BPAF)对生物有机体具有内分泌干扰作用。为研究低剂量BPAF对水生生物的效应,本研究选择成年雄性斑马鱼为研究对象,考察了0.005、0.05和0.5 mg·L~(-1)3种浓度BAPF暴露30 d对血浆中卵黄蛋白原(VTG)含量、2种卵黄蛋白原基因(vtg~(-1)和vtg-3)表达和2种芳香酶基因(cyp 19a与cyp 19b)表达的影响。结果表明:在0.005 mg·L~(-1)浓度暴露30 d后,血浆中VTG含量显著升高,随着暴露浓度的升高,促进作用不显著;BPAF暴露对不同组织中的4种基因存在不同的影响,0.005 mg·L~(-1)BPAF暴露可诱导脑部cyp19b、肝脏中cyp19a和性腺中vtg~(-1)、vtg-3和cyp19b基因表达;0.5 mg·L~(-1)BPAF暴露可导致肝脏中vtg~(-1)、vtg-3、性腺中cyp19a等基因显著上调。实验结果表明,BPAF具有雌性激素样效应,可诱导雄性斑马鱼体内部分组织卵黄蛋白原基因和芳香酶基因的表达。BPAF可引起斑马鱼血浆中的VTG含量的上升,从而干扰由VTG所参与的下丘脑-垂体-性腺轴与免疫系统的正常生理过程。     

Cytochrome P450 monooxygenase specific activity reduction in wheat <Emphasis Type="Italic">Triticum aestivum</Emphasis> induced by soil roxithromycin stress

Roxithromycin, as widely used medicine and livestock growth promoter, arouses concern because its occurrence and persistence in soil environments. However, effects of roxithromycin in higher plants are still vague. Accordingly, we hypothesized that roxithromycin-contaminated soil may exhibits ecotoxicological effects in wheat (Triticum aestivum). In this study, effects induced by a gradient concentration of roxithromycin stress (0.01, 0.1, 1, 10, and 100 mg·kg^–1) was investigated in a 7-d soil test in T. aestivum. Results indicated that the specific activity of cytochrome P450 (CYP450) monooxygenase was decreased dramatically with the concentration of roxithromycin in soil. The IC₅₀ value was 8.78 mg·kg^–1 of roxithromycin. On the contrary, the growth related endpoints (i.e., the germination percentage, the biomass and the height), the content related endpoints (i.e., soluble protein content and CYP450 content), and the superoxide dismutase (SOD) activity failed to reveal the roxithromycin-induced effects. Further analysis revealed that the CYP450 monooxygenase specific activity reduction was enzymatic mechanism mediated, other than oxidative stress induced. We conclude that the soil roxithromycin declined the CYP450 monooxygenase activity in T. aestivum by the inhibition of the enzymatic mechanism. Further efforts can include, but are not limited to, investigation of joint effects induced by combined exposure of roxithromycin and the pesticides and evaluation of the similar effects in other higher plants.     

Mutagenicity of polyaromatic hydrocarbons by chemical models for cytochrome P450 in Ames assay

DNA damage is an important step in carcinogenesis. The Ames assay is a short-term screening of carcinogens that induce DNA damage. Most carcinogens require enzymatic activation through oxidation by cytochrome P450 (CYP450) in the presence of S9 mix. A combination of iron (Fe)(III) porphyrin and an oxidant is also able to oxidize compounds as an alternative metabolic pathway to CYP450. Previously it was reported that a chemical model containing a water-soluble 5,10,15,20-tetrakis(1-methylpyridinium4-yl)porphyrinatoiron(III) chloride (4-MPy) and tert-butyl hydroperoxide (t-BuOOH) activated aromatic amines and amides. In this study, a chemical model composed of an Fe porphyrin, water-insoluble 5,10,15,20-tetrakis(pentafluorophenyl)porphyrinatoiron(III) chloride (F₅P) or water-soluble 4-MPy was optimized with an oxidant – t-BuOOH, magnesium monoperoxyphthalate (MPPT), or iodosylbenzene (PhIO). Subsequently the mutagenicity of benzo[a]pyrene (B[a]P) and chrysene in Salmonella typhimurium TA strains was compared. B[a]P was activated by a combination of F₅P or 4-MPy plus MPPT or PhIO in S. typhimurium TA1538. The B[a]P-induced mutagenicity with F₅P plus oxidant was higher than 4-MPy plus oxidant. Mutagenicity of chrysene, a tetracyclic aromatic hydrocarbon, was not detected in the presence of F₅P/PhIO in S. typhimurium TA98, but was activated in the presence of F₅P/MPPT. The F₅P/MPPT activated other polyaromatic hydrocarbons (PAH) in the S. typhimurium TA98 assay including dibenz[a,c]anthracene, dibenz[a,h]anthracene, 3-methylcholanthrene, and benzo[a]anthracene. The results indicated that the F₅P/MPPT was the most efficient model for detecting PAH-induced mutagenicity in the Ames assay.     

Insight into fluorescence properties of 14 selected toxic single-ring aromatic compounds in water: Experimental and DFT study

• The fluorescence peak location of 14 compounds interpreted at protein-like region. • The p-electron system inside aromatic ring contributes to the fluorophore region. • Functional group variation effects the emission spectra. • Decrease in quantum yield and increase in DE is due to atomic weight F>Cl>Br>I. • Theoretically results are in line with experimental ones. Various single-ring aromatic compounds in water sources are of great concern due to its hazardous impact on the environment and human health. The fluorescence excitation-emission matrix (EEMs) spectrophotometry is a useful method to identify organic pollutants in water. This study provides a detailed insight into the fluorescence properties of the 14 selected toxic single-ring aromatic compounds by experimental and theoretical analysis. The theoretical analysis were done with Time-Dependent Density Functional Theory (TD-DFT) and B3LYP/6-31G (d,p) basis set, whereas, Polarizable Continuum Model (PCM) was used to consider water as solvent. The selected compounds displayed their own specific excitation-emission (Ex/Em) wavelengths region, at Ex<280 nm and Em<340 nm, respectively. Whereas the theoretical Ex/Em was observed as, Ex at 240 nm–260 nm and Em at 255 nm–300 nm. Aniline as a strong aromatic base has longer Em (340 nm) than alkyl, carbonyl, and halogens substituted benzenes. The lone pair of electrons at amide substituent serves as a p-electron contributor into the aromatic ring, hence increasing the stability and transition energy, which results in longer emission and low quantum yield for the aniline. The fluorescence of halogenated benzenes illustrates an increase in the HOMO-LUMO energy gap and a decrease in quantum yield associated with atomic size (F>Cl>Br>I). In this study the theoretical results are in line with experimental ones. The understanding of fluorescence and photophysical properties are of great importance in the identification of these compounds in the water.     

Effects of petroleum hydrocarbons on the cytochrome P450 content of the mollusc bivalveMytilus galloprovincialis

The cytochrome P₄₅₀, identified by the characteristic 450 nm spectral peak of its carbon monoxide complex, was detected in the hepatopancreatic microsomes ofMytilus galloprovincialis, collected from Ponteau (Bay of Fos, France) in 1982 and 1983. When mussels were exposed to a variety of petroleum hydrocarbons, the cytochrome P₄₅₀ content increased. It is shown that aromatic hydrocarbons are more effective than alkanes in inducing a high level of cytochrome P₄₅₀. The possible use of this haemoprotein in hydrocarbon pollutant detection is discussed.     

Cytochrome P450 1A activity in liver and fixed wavelength fluorescence detection of polycyclic aromatic hydrocarbons in the bile of tongue-fish (Cynoglossus acrolepidotus, Bleeker) in relation to petroleum hydrocarbons in the eastern Gulf of Thailand.

This investigation was conducted in an area of oil spill along the east coast of Thailand to examine the relations among cytochrome P450 1A activity in liver and PAHs in the bile of the tonguefish and petroleum hydrocarbons in the sediments. PAH sediment concentrations in the reference and oil spill areas were 5.03 +/- 0.42 and 0.21 +/- 0.043 microg(-1) dry weight respectively Cytochrome activity in fish liver from oil spill area was 45.40 +/- 3.50 pmoles/ min/mg protein, almost threefold higher than that from the reference sites. Flourescense detection in bile metabolites at the oil spill area, 69.8 +/- 9.9 flourescense unit was significantly higher than that at the reference sites, 22.9 +/- 5.5 and 22.2 +/- 3.5 fluorescence unit. A strong correlation was found among cytochrome P450 1A activity in liver, PAH of bile metabolites and petroleum hydrocarbons. Both cytochrome and bile metabolites activity decreased seaward varying to the distance from the oil polluted area. We concluded that both detections in tonguefish can be regarded as a complementary biomarkers for the exposure of PAHs in tropical marine environments.     

Studies on cytochrome P450 in Mytilus galloprovincialis: induction by Na-phenobarbital and ability to biotransform xenobiotics

Mytilus galloprovincialis has a hepatopancreatic monooxygenase activity cytochrome P₄₅₀-dependent. The present paper studies the induction of this activity in the hepatopancreatic microsomes after treatment of M. galloprovincialis with Na-phenobarbital. Mussels were collected from the Gulf of La Spezia in 1985. We measured the increase in levels of cytochrome P₄₅₀, b₅, and P₄₂₀ and the 7-ethoxycoumarin deethylase activity. Furthermore, the fraction of mouse liver was replaced by the correspondent mussels' hepatopancreatic fraction in the yeast genotoxicity test to determine the ability of mussels to biotransform cyclophosphamide and styrene oxide. The results suggest that edible mussels are capable of detoxicating styrene oxide but not of bioactivating cyclophosphamide.     

Mechanistic insights into the selective photocatalytic degradation of dyes over TiO2/ZSM-11

● TiO₂/ZSM-11 was prepared by a facile solid state dispersion method. ● Mechanism for photocatalytic degradation of dyes was investigated. ● Both experimental and MD simulations were conducted. ● Chemisorption instead of electrostatic interaction played a critical role. Photocatalytic degradation is a promising way to eliminate dye contaminants. In this work, a series of TiO₂/ZSM-11 (TZ) nanocomposites were prepared using a facile solid state dispersion method. Methyl orange (MO), methylene blue (MB), and rhodamine B (RhB) were intentionally chosen as target substrates in the photocatalytic degradation reactions. Compared to pristine TiO₂, negative effect was observed on MO degradation while promoted kinetics were collected on MB and RhB over TZ composites. Moreover, a much higher photocatalytic rate was interestingly achieved on RhB than MB, which indicated that a new factor has to be included other than the widely accepted electrostatic interaction mechanism to fully understand the selective photodegradation reactions. Systematic characterizations showed that TiO₂ and ZSM-11 physically mixed and maintained both the whole framework and local structure without chemical interaction. The different trends observed in surface area and the photo-absorption ability of TZ composites with reaction performance further excluded both as the promotion mechanism. Instead, adsorption energies predicted by molecular dynamics simulations suggested that differences in the adsorption strength played a critical role. This work provided a deep mechanistic understanding of the selective photocatalytic degradation of dyes reactions, which helps to rationally design highly efficient photocatalysts.