Characterization on the toxic mechanism of two fluoroquinolones to trypsin by spectroscopic and computational methods

Abstract

Ciprofloxacin (CPFX) and enrofloxacin (ENFX), two of the most widely used fluoroquinolones (FQs), pose a great threat to humans and the ecosystem. In this study, the toxic mechanisms between the two FQs and trypsin were evaluated by means of multiple spectroscopic methods, as well as molecular docking. During the fluorescence investigations, both FQs quenched the intrinsic fluorescence of trypsin effectively, which was due to the formation of moderately strong complexes (mainly through van der Waals forces and hydrogen bonds). The binding of two FQs not only caused the conformational and micro-environmental changes of trypsin, but also changed its molecular activity; shown by the UV–Visible absorption spectroscopy, synchronous fluorescence spectroscopy, and functional tests. The established methods in this work can help to comprehensively understand the transport of FQs in the human body.     

Investigation on the interaction between triclosan and bovine serum albumin by spectroscopic methods

Abstract

Multi-spectroscopic and molecular docking methods were used to study the interaction between triclosan (TCS) and bovine serum albumin (BSA). The results indicated that the fluorescence quenching of BSA by TCS was due to the formation of TCS–BSA complex through static quenching. This result was also demonstrated by time-resolved fluorescence experiment. The binding constants and number of binding sites between TCS and BSA were 1.30?×?10⁵ M^?1 and 1.17 at 298?K, respectively. The thermodynamic parameters were studied in detail which suggested that hydrophobic forces and hydrogen bond played major roles in the TCS–BSA interaction. Moreover, the site marker competitive experiments and docking studies revealed that TCS could bind BSA into site I in subdomain IIA. All the results of UV–vis spectrophotometry, circular dichroism spectroscopy and synchronous fluorescence spectroscopy showed that interaction between TCS and BSA induced conformation changes of BSA.     

Interaction of tebuconazole with bovine serum albumin: determination of the binding mechanism and binding site by spectroscopic methods

Abstract

This study investigates the interaction between tebuconazole and bovine serum albumin (BSA) in a physiological buffer (pH = 7.4) using the fluorescence quenching method to obtain the apparent binding constants (K) and number of binding sites (n) in the interaction between tebuconazole and BSA. The results revealed that tebuconazole can quench the intrinsic fluorescence of BSA through a static quenching procedure. It also shows that the thermodynamic parameters of enthalpy change (ΔH) and entropy change (ΔS) are negative, indicating that the interaction of tebuconazole with BSA is mainly driven by van der Waals forces and hydrogen bonds. The process of binding was a spontaneous process in which Gibbs free energy change was negative. The distance of r between the donor (BSA) and acceptor (tebuconazole) was calculated to be 0.68?nm based on Forster’s non-radiative energy transfer theory. Analysis of synchronous fluorescence, three-dimensional fluorescence and circular dichroism (CD) spectra demonstrates that tebuconazole can induce conformational changes of BSA.     

Binding of the veterinary drug tetracycline to bovine hemoglobin and toxicological implications

Tetracycline (TC) is a widely used veterinary drug in animal breeding and fishery. Because of its low bioavailability, the TC residue extensively exists in the environment (e.g. soils, lakes and rivers), which can enter the human body, being potentially harmful. Hemoglobin (Hb) is a protein responsible for oxygen carrying in the vascular system of animals. The aim of this study was to investigate the interaction of bovine hemoglobin (BHb) with TC through spectroscopic and molecular modeling methods. The experimental results revealed that TC can interact with BHb with one binding site to form a TC-BHb complex, mainly through van der Waals interactions and hydrogen bonds. The UV-visible absorption, synchronous fluorescence, and circular dichroism (CD) results revealed that the binding of TC can cause conformational and some microenvironmental changes of BHb, which may affect BHb physiological functions. The synchronous fluorescence experiment disclosed that TC binds into BHb central cavity, which was verified by molecular modeling study. The work contributes to clarify the molecular mechanism of TC toxicity in vivo.     

Potential toxicity of amphenicol antibiotic: binding of chloramphenicol to human serum albumin

Antibiotics are widely used in daily life but their abuse has posed a potential threat to human health. To evaluate the toxicity of chloramphenicol (CAP) at the protein level, the interaction between CAP and human serum albumin (HSA) was investigated by fluorescence, Ultraviolet–visible (UV–Vis) absorption, Fourier transform infrared (FT-IR) spectroscopy and molecular docking methods. Fluorescence data revealed that the fluorescence quenching of HSA by CAP was the result of the formation of CAP–HSA complex, and the binding constant was determined to be 3.196?×?10⁴ L mol^?1 at 310 K. The thermodynamic determination indicated that the interaction was driven by enthalpy change and entropy change together, where the multiple hydrogen bonds (CAP and the residues Arg 222 and His 242 of HSA) and van der Waals forces were the dominant binding force. The site marker competition revealed that CAP bound into sub-domain IIA of HSA. The binding of CAP induced the drastic reduction in α-helix conformation and the significant enhancement in β-sheet conformation of HSA. Molecular docking study further confirmed the binding mode obtained by experimental study. This work provides a new quantitative evaluation method for antibiotics to cause the protein damage.     

New insights into the characterization of the binding of tetracycline analogues with lysozyme: a biophysical study

Tetracycline (TC), chlortetracycline (CTC) and oxytetracycline (OTC) are the most common members of the widely used veterinary drug tetracyclines, the residue of which in the environment can enter human body, being potentially harmful. Lysozyme is a monomeric protein widely distributed in the nature including human beings, having many physiological and pharmaceutical functions. The aim of this study was to examine the interaction of lysozyme with the three tetracyclines (TC, CTC and OTC) through spectroscopic and molecular modeling methods. The experimental results revealed that all the three tetracyclines (TCs) can interact with lysozyme with one binding site to form TCs-lysozyme complex, mainly through electrostatic forces with the affinity order: CTC > TC > OTC. The binding of TCs can cause conformational and some microenvironmental changes of lysozyme. Furthermore, molecular docking was applied to define the specific binding sites, the results of which show that all the three TCs can bind into lysozyme cleft and interact with the key active-site residues Glu 35 or Asp 52, resulting in competitive inhibition of lysozyme activity. The accurate and full basic data in the work is beneficial to clarifying the binding mechanism of TCs with lysozyme in vivo.     

Sunlight-induced degradation of soil-adsorbed veterinary antimicrobials Marbofloxacin and Enrofloxacin

Marbofloxacin (MAR) and Enrofloxacin (ENR), two largely employed veterinary Fluoroquinolones (FQs), were found to be present at the micrograms per kilogram level in agricultural soils of South Lombardy (Italy) several months after manuring. Distribution coefficients (K_d) from sorption experiments indicated a strong binding to the soil. Soil samples fortified with environmentally significant FQs amounts (0.5 mg kg⁻¹) were exposed to solar light that promoted extensive degradation (80%) of both drugs in 60-150 h. Thus, photochemistry could be considered a significant depollution path in the soil, although it was two orders of magnitudes slower than in aqueous solution and a fraction of the drug (ca. 20%) remained unaffected. For MAR the photoprocess was the same as in solution, and involved cleavage of the tetrahydrooxadiazine ring. On the contrary, with ENR only some of the photoproducts determined in water (those arising from a stepwise oxidation of the piperazine side chain) were observed. Substitution of the 6-fluoro by a hydroxyl group and reduction did not occur in the soil, supporting the previous contention that such processes required polar solvation of FQs. Consistently with this rationalization, the irradiation of thin layers of solid drugs led to essentially the same products distribution as in the soil. From the environmental point of view it is important to notice that photodegradation mainly affects the side-chains, while the fluoroquinolone ring, to which the biological effect is associated, is conserved up to the later stages of the degradation.     

Investigations on the interaction of the phototoxic alkaloid coralyne with serum albumins

The interaction of the phototoxic alkaloid coralyne with bovine and human serum albumins (BSA, HSA) was investigated. Absorbance and fluorescence quenching experiments revealed the formation of strong complexes. Based on the binding parameters calculated from Stern-Volmer quenching method, coralyne has higher affinity to BSA (∼10⁵ M⁻¹) compared to HSA (∼10⁴ M⁻¹). Forster resonance energy transfer studies showed that the specific binding distances between Trp (donor) of the proteins and coralyne (acceptor) were 2.95 and 3.10 nm, respectively. The bindings were favored by negative enthalpy and a stronger favorable entropy contribution. The heat capacity values for binding to BSA and HSA were similar, indicating the involvement of similar molecular forces in the complexation. Competitive binding experiments using site markers demonstrated that coralyne binds to site I (subdomain IIA) of both proteins. The secondary structure of the proteins was altered, suggesting a small but definitive partial unfolding on complexation.     

Adsorption and oxidation of fluoroquinolone antibacterial agents and structurally related amines with goethite

Seven members (ciprofloxacin, enrofloxacin, norfloxacin, ofloxacin, lomefloxacin, pipemidic acid, and flumequine) of the popular fluoroquinolone antibacterial agents (FQs) were found to adsorb strongly to goethite with 50-76% of the added FQ adsorbed under the experimental conditions. The adsorption isotherms fitted well to the Langmuir model. Adsorption was accompanied by slow oxidation of the FQs (except for flumequine) by goethite yielding a range of hydroxylated and dealkylated products. The oxidation kinetics showed different stages in reaction rate, mostly likely caused by accumulation of Fe(II) species on the oxide surface that slowed the reaction. Structurally related amines 1-phenylpiperazine, N-phenylmorpholine, aniline, and N,N-dimethylaniline were found to be oxidized by goethite without significant adsorption. The results strongly indicate that the carboxylic group of FQs is critical for adsorption while the piperazine ring is susceptible to oxidation. A radical mechanism is proposed for the oxidation of FQs by goethite which involves formation of a surface complex between the FQ and surface-bound Fe(III) through adsorption, and initial oxidation at the piperazinyl N1 atom to form radical intermediates that ultimately lead to the final products. This study indicates that Fe oxides in aquatic sediments may well play an important role in the natural attenuation of fluoroquinolone antibacterial agents.     

Detection of antibacterial-like activity on a silica surface: fluoroquinolones and their environmental metabolites

Background, scope, and aims

Antibacterial fluoroquinolones (FQs) are third-generation antibiotics that are commonly used as therapeutic treatments of respiratory and urinary tract infections. They are used far less in intensively farmed animal production systems, though their use may be permitted in the veterinary treatments of flocks or in medicated feeds. When used, only a fraction of ingested parent FQ actually reaches the in vivo target site of infection, while the remainder is excreted as the parent FQ and its metabolized products. In many species?? metabolism, enrofloxacin (EF) is converted into ciprofloxacin (CF) while both FQs are classified as parent FQs in human treatments. It is therefore likely that both FQs and their metabolic products will contribute to a common pool of metabolites in biological wastes. Wastes from intensive farming practices are either directly applied to agricultural land without treatment or may be temporarily stored prior to disposal. However, human waste is treated in sewage treatment plants (STPs) where it is converted into biosolids. In the storage or treatment process of STPs, FQs and their in vivo metabolites are further converted into other environmental metabolites (FQEMs) by ex vivo physicochemical processes that act and interact to produce complex mixtures of FQEMs, some of which have antibacterial-like activities. Biosolids are then often applied to agricultural land as a fertilizer amendment where FQs and FQEMs can be further converted into additional FQEMs by soil processes. It is therefore likely that FQ-contaminated biowaste-treated soils will contain complex mixtures of FQEMs, some of which may have antibacterial-like activities that may be expressed on bacteria endemic to the receiving agricultural soil environment. Concern has arisen in the scientific and in the general community that repeated use of FQ-contaminated biowaste as fertilizer amendments of nutrient-impoverished agricultural land may create a selective environment in which FQ-resistant bacteria might grow. The likelihood of this happening will depend, to some extent, on whether bioactive FQEMs are first synthesized from the parent FQs by the action and interaction of in vivo and ex vivo processes producing bioactive FQEMs in biowastes and biosolids. The postulated creation of a selective environment will also depend, in part, on whether such bioactive FQEMs are biologically available to bacteria, which may, in turn, be influenced by soil type, amendment regime, and the persistence of the bioactive FQEMs. Additionally, soil bacteria and soil processes may be affected in different ways or extents by bioactive FQEMs that could possibly act additively or synergistically at ecological targets in these non-target bacteria. This is an important consideration, since, while parent FQs have well-defined ecological targets (DNA gyrase and topoisomerase IV) and modes of bactericidal action, the FQEMs and their possible modes of action on the many different species of soil bacteria is less well studied. It is therefore understandable that there is a lack of conclusive evidence directly attributing biosolid usage to any increase in FQ-resistant bacteria detected in biowaste-amended agricultural soil. However, a lack of evidence may simply imply that a causal relationship between biosolid usage programs and any detection of low levels of FQ-resistant bacteria in soils has yet to be established, rather than an assumption of no relationship whatsoever. Based on results presented in this paper, the precautionary principle should be applied in the usage of FQ-contaminated biosolids as fertilizer amendments of agricultural land. The aim of this research was to test whether any bioactive FQEMs of EF could be synthesized by aerobic fermentation processes using Mycobacterium gilvum (American Tissue Culture Collection) and a mixed culture of microorganisms derived from an agricultural soil. High-performance thin-layer chromatography (HPTLC) and bioautography were tested as screening techniques in the detection and analysis of bioactive FQEMs.

Materials and methods

FQEMs derived from M. gilvum and mixed (soil) culture aerobic ferments were fractionated using preparative HPTLC. A standard strain of Escherichia coli was then used as the reporter organism in a bioautography assay in the detection of bioactive-FQEMs on a mid-section of the HPTLC plate. Plate sections were reassembled, and a photograph was taken under low-intensity ultraviolet (UV) light to reveal regions that contained analytes that had UV chromophores and antibacterial-like activities.

Results and discussion

Many fractionated FQEMs displayed antibacterial-like activity while bound to silica gel HPTLC plates. These results also provide evidence that sufficient quantities of biologically active FQEMs were biologically available from a silica gel surface to prevent the adherent growth of E. coli. Six to seven FQEMs derived from EF using aerobic fermentation processes had antibacterial-like activities, while two FQEMs were also detectable using UV light. Furthermore, similar banding patterns of antibacterial-like activity were observed in both the monoculture (M. gilvum) and mixed culture bioautography assays, indicating that similar processes operated in both aerobic fermentations, either producing similar biologically active FQEMs or biologically active FQEMs that had similar physicochemical properties in both ferments. The simplest explanation for these findings is that the tested agricultural soil also contained mycobacteria that metabolized EF in a similar way to the purchased standard monoculture M. gilvum. Additionally, the marked contrast between the bioautography results and the UV results indicated that the presence of UV chromophores is not a prerequisite for the detection of antibacterial-like activity.

Conclusions

A reliance on spectrophotometric techniques in the detection of bioactive FQEMs in the environment may underestimate component antibacterial-like activity and, possibly, total antibacterial-like activity expressed by EF and its FQEMs. The described bioautography method provides a screening technique with which antibacterial-like activities derived from EF and possibly other FQs can be detected directly on silica gel HPTLC plates.

Recommendations

It is recommended that both bioassay and instrumental analytical techniques be used in any measurement of hazard and risk relating to antibacterial-like activities in the environment that are derived from fluoroquinolone antibiotics and their environmental metabolites.     

Environmental metabolites of fluoroquinolones: synthesis, fractionation and toxicological assessment of some biologically active metabolites of ciprofloxacin

Background, aim, and scope

Biowastes produced by humans and animals are routinely disposed of on land, and concern is now growing that such practices provide a pathway for fluoroquinolone (FQs) antibacterial agents and their environmental metabolites (FQEMs) to contaminate the terrestrial environment. The focus of concern is that FQs and FQEMs may accumulate in amended soils to then adversely impact on the terrestrial environment. One postulated impact is the development of a selective environment in which FQ-resistant bacteria may grow. To find evidence in support of an accumulation of antibacterial-like activity, it was first necessary to establish whether any biologically active FQEMs could be synthesized by physicochemical factors that are normally present in the environment. However, many FQEMs are not commercially available to be used as standards in such studies. FQEMs were therefore synthesized using well-defined processes. They were subsequently analyzed using spectroscopy (UV-vis) and high performance liquid chromatography with mass spectral detection. The antibacterial-like activities of fractionated FQEMs were then assessed in novel bacterial growth inhibition bioassays, and results were compared to those obtained from instrumental analyses.

Materials and Methods

Parent FQs were either exposed to sunlight or were synthesized using defined aerobic microbial (Mycobacterium gilvum or a mixed culture derived from an agricultural soil) fermentation processes. Mixtures of FQEMs derived from photo- and (intracellular) microbial processes were isolated by preparative chromatography and centrifugation techniques, respectively. Mixtures were subsequently fractionated using analytical high-performance thin layer chromatography (HPTLC), and excised analytes were tested in bioautography assays for their antibacterial-like activities. Two bacteria, Escherichia coli (E. coli) and Azospirillum brasilense (A. brasilense) were used as reporter organisms in testing FQ standards and any subtle differences between biologically active FQEMs of ciprofloxacin (CF).

Results and discussion

FQEMs produced in the photo-synthetic process had UV-vis profiles that were indistinguishable from the parent FQs, and yet mass spectral data revealed the presence of N-formylciprofloxacin (FCF). In contrast, the UV-vis profiles of FQEMs synthesized by M. gilvum and a mixed culture of microorganisms had UV-vis profiles that were similar to one another and markedly different to the parent fluoroquinolones. Mass spectral studies confirmed the presence of FCF and N-acetylciprofloxacin in both microbial ferments. In addition, a photo-FQEM (Cp 6), three M. gilvum FQEMs (Cm 5, Cm 8, and Cm 10) and a mixed culture FQEM (Cs 6) of CF and many other FQEMs of CF, norfloxacin (NF), and enrofloxacin (EF) were fractionated using HPTLC, although their identities have yet to be confirmed. Differences between bioautography results were obtained when E. coli or A. brasilense were used as reporter organisms. Parent FQs (CF and EF) and the FQEMs of CF (Cp 6, Cm 8, and Cs 6) displayed antibacterial-like activity when using E. coli as the reporter organism. In contrast, A. brasilense was insensitive to parent CF and sensitive to EF and all tested FQEMs of CF. Results are consistent with photo- and microbial processes modifying CF in different ways, with the latter changing the UV-vis chromophores. It can be inferred that a lack of detection of analytes (especially photo-FQEMs) when using UV-vis does not necessarily indicate an absence of analyte. Additionally, similarities between the UV-vis profiles of FQEMs extracted from the (monoculture) M. gilvum and the mixed culture microbial aerobic ferments are consistent with similar processes operating in both ferments. Results of HPTLC and bioautography studies revealed that mixtures of (photo- and microbial) FQEMs could be fractionated into individual components.

Conclusions

Bioactive FQEMs of ciprofloxacin, as a representative FQ, can be synthesized by photo- and microbial processes, and their detection required the use of both instrumental and bioautography analytical techniques. It is likely that such FQEMs will also be present on agricultural land that has been repeatedly amended with FQ-contaminated biosolids.

Recommendations and perspectives

The use of instrumental analytical techniques alone and especially photometric detection techniques will underestimate antibacterial-like activities of FQEMs. Moreover, the extraction technique(s) and the selected toxicological endpoint(s) require careful consideration when assessing bioactivity. It is therefore recommended that instrumental analytical techniques and several bioautography assays be performed concurrently, and bioautography assays should use a variety of reporter organisms. Two types of bacterial growth bioassays are recommended in any assessment of antibacterial-like activity derived from CF (and possibly from other FQs). A standardized E. coli bioassay should be used as a general screening procedure to facilitate intra- and inter-laboratory exchange of data. Additionally, soil-specific (region-specific) growth inhibition bioassays should be undertaken using several species of endemic soil bacteria. It is likely that the two sets of data will be useful in future risk assessment processes.     

Study on the mechanism of action between dimethyl phthalate and herring sperm DNA at molecular level

Dimethyl phthalate (DMP), a typical phthalic acid ester, is widespread in the environment and causes extensive concern due to its adverse effects on human health. To understand the genotoxicity of DMP at molecular level, the toxic interaction of DMP with herring sperm (hs) deoxyribonucleic acid (DNA; hs-DNA) was investigated in vitro under simulated physiological conditions using multi-spectroscopic techniques and a molecular modeling method. The results of Ultraviolet-Visible absorption spectroscopy, fluorescence emission spectroscopy, and circular dichroism spectra indicated that DMP interacts with hs-DNA in a groove-binding mode that changes the double helical structure of DNA. The binding constant and the number of binding sites calculated from the fluorescence quenching data were 565.718 L mol^?1 and 0.7872, respectively. A molecular modeling study revealed that DMP tends to bind with DNA in the A-T-rich regions of minor groove and that hydrogen bonding and van der Waals forces play main roles in the interaction. This research can help to elucidate the mechanism of DMP toxicity in vivo.     

Study on the binding interaction between perfluoroalkyl acids and DNA

Perfluoroalkyl acids (PFAAs) are carcinogens, and elucidating their DNA binding properties is crucial for understanding PFAA genotoxicity. We have investigated the binding mode and affinity of five PFAAs to seven DNA molecules using fluorescence displacement and molecular docking analysis. DNA conformational changes upon PFAA binding were also examined by circular dichroism (CD). The data revealed that DNA intercalation was the dominant interaction mode of the PFAAs; however, these molecules also bound to grooves. The dissociation constants for the PFAAs ranged between 0.11 and 1,217.14 μM, and between 3.46 and 2,141.21 μM for DNA intercalation and groove binding, respectively. PFAAs that contain longer carbon chains had stronger DNA intercalation affinities. Binding to DNA was stronger for perfluoroalkyl sulfonates than for perfluorcarboxyl acids that contain the same number of carbons. This observation is postulated to arise from the presence of more fluorine and oxygen atoms in perfluoroalkyl sulfonates acting as hydrogen bond donors that facilitate stronger DNA intercalation. The binding of the PFAAs to DNA showed some CT-DNA sequence selectivity. Molecular docking analysis confirmed the DNA binding mode and affinities of the PFAAs. CD analysis revealed that the PFAAs weakened DNA base stacking and loosened DNA helicity. The present study has improved our understanding of the formation of PFAA–DNA adducts.     

Assessing the quality of marine coastal environments: comparison of scope for growth and Microtox bioassay results of pollution gradient areas in eastern Mediterranean (Greece)

Methods for assessing the quality of marine coastal environments are numerous and rapidly evolving. The integration of physiological parameters termed as Scope for Growth (SfG) and the luminescent bioassay Microtox were applied in several sites of Saronikos and Amvrakikos Gulfs (Greece) to assess the environmental quality and compare the results of the two methods. Each site in the two areas undergoes different types and levels of pollution. Both methods could identify a pollution gradient reflecting the quality of each site. A very good linear correlation was noticed between the two methods (r = 0.98). The stress order, which was similar in both methods, exhibited the expected pollution gradient according to the pressure that each site undergoes. Results suggest that for screening and fast isolation of polluted sites before further analysis, the rapid and simple Microtox assay could operate as an alternative to the more complex SfG method.     

氯消毒对城市污水中DOM的三维荧光特性影响

在城市污水二级出水氯消毒过程中,通过对溶解性有机物(DOM)的三维荧光光谱(3DEEM)分析及反应过程中三卤甲烷(THMs)生成量的连续测定,分析各类荧光物质随加氯反应时间的变化规律,探讨其与THMs生成量之间的关系,以此来推测THMs的主要前驱物质。结果表明,加氯后0～6 h内,各反应时间点三维荧光光谱图的等高线的密集程度较原二级出水明显降低,荧光峰的荧光强度减少40%～70%,说明DOM与氯发生反应,芳香构造化程度及不饱程度降低,从而失去荧光特性。其中,简单芳香族蛋白质(酪氨酸类)、腐殖酸类及富里酸类物质在加氯前后荧光强度变化较大,是生成THMs的主要前驱物质。THMs的生成量随反应时间的增加呈现明显的上升趋势,15 min内各类荧光特性有机物的荧光强度减少约50%左右,同时生成了50.17%的THMs。     

二级出水微滤过程中对醋酸纤维素酯膜的有机污染特征

低压膜过滤技术(包括微滤和超滤)在再生水生产领域正引起越来越广泛的关注。然而如何解决低压膜过滤过程中的膜有机污染问题始终是膜技术所面临的技术挑战。本研究采用醋酸纤维素酯微孔滤膜对二级出水溶解性有机物(EfOM)及其不同亲疏水性组分、蛋白模拟溶液、腐殖酸(HA)等进行恒流过滤实验。对不同有机物污染后的膜表面使用全反射傅立叶红外光谱(ATR-FTIR)和X射线电子能谱(XPS)进行表征。结果表明,相对于蛋白质和EfOM等,HA所造成的膜污染最少。ATR-FTIR的结果同时显示,以官能团而言,更多的氨化物(1535cm-1)、脂肪族物质(2860～2970cm-1)和氢氧根(3400cm-1)存在于膜表面。TMP/V数据比较结果表明,在EfOM各亲疏水性组分、蛋白质和HA的对比中,EfOM中的疏水碱性物质(HPO-B)对膜污染的贡献最大,而HA的膜污染贡献最小。UVA和荧光激发-发射光谱(FEEM)结果表明,HPO-B和蛋白质对醋酸纤维素酯膜的污染贡献较大。综合不同分析手段可以对不同有机物造成膜污染的潜能大小得出如下排序:HPO-B>蛋白质>HPO-A、HPI>HA。     

Humic acid complexation of basic and neutral polycyclic aromatic compounds

Complexation by humic acid (HA) of basic (quinoline) and neutral (naphthalene) polycyclic aromatic compounds (PACs) was compared using fluorescence spectroscopy and equilibrium dialysis (ED). These compounds sorb to HA via cation exchange and hydrophobic interactions, respectively. Ionization of quinoline strongly affects its sorption to HA; maximum sorption is observed at pH close to logKb (4.92), and competition with H+ and electrolyte cation (Li+) is evident. Spectroscopic experiments indicate that quinolinium (QH+) cation fluorescence is quenched via a static mechanism (i.e., a dark complex is formed) when the protonated form is adsorbed via ion exchange to HA. The extent of sorption, calculated from fluorescence data using the Stern-Volmer equation, was compared to independent ED measurements. Although both methods indicated the same trends with solution chemistry, fluorescence quenching data suggested more extensive complexation than that measured using ED. In contrast to ionizable PACs, studied here and previously, interaction of naphthalene with HA is unaffected by changes in solution conditions (pH, ionic strength).     

Alterations of protein profile in zebrafish liver cells exposed to methyl parathion: a membrane proteomics approach

Methyl parathion (MP) is an extensively used organophosphorus pesticide, which has been associated with a wide spectrum of toxic effects on environmental organisms. The aim of this study is to investigate the alterations of membrane protein profiles in zebrafish liver (ZFL) cell line exposed to MP for 24 h using proteomic approaches. Two-dimensional gel electrophoresis revealed a total of 13 protein spots, whose expression levels were significantly altered by MP. These differential proteins were subjected to matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry analysis, and nine proteins were identified to be membrane proteins, among which seven were up-regulated, while two were down-regulated. In addition, the mRNA levels corresponding to these differential membrane proteins were further analyzed by quantitative real-time PCR. And the differential expression of arginase-2 was specially validated via Western blotting. Regarding the physiological functions, these proteins are involved in molecular chaperon, cytoskeleton system, cell metabolism, signal transduction, transport and hormone receptor respectively, suggesting the complexity of MP-mediated toxicity to ZFL cell. These data could provide useful insights for better understanding the hepatotoxic mechanisms of MP and develop novel protein biomarkers for effectively monitoring MP contamination level in aquatic environment.     

Behaviour and biodegradation of sulfonamides (p-TSA, o-TSA, BSA) during drinking water treatment

Three sulfonamides -para-toluenesulfonamide (p-TSA), ortho-toluenesulfonamide (o-TSA) and benzenesulfonamide (BSA) - have recently been detected in groundwater within a catchment area of one drinking water treatment plant (DWTP), which is located downstream of a former sewage farm. The degradation pathways of p-TSA, o-TSA and BSA were investigated during drinking water treatment with incubation experiments and an experimental filter. Incubation experiments showed that p-TSA is removed during the treatment by microbiological processes. Removal of p-TSA is performed by adapted microorganisms only present in polluted groundwater. The elimination in an experimental filter of 1.6m length applying filtration velocities from 2 to 6 m h(-1) was approximately 93% of p-TSA. The microbial degradation rates in the incubation experiment were approximately 0.029 microg l(-1) h(-1) (zero order reaction). In the experimental filter, the reaction rate constants were around 0.0063 s(-1) for all filtration velocities (1st order reaction). Drinking water treatment does not reduce the concentration of o-TSA and BSA under conditions encountered in Berlin. p-TSA, o-TSA and BSA were only measured in the low microg l(-1) concentrations range in the purified water.     

Investigating interactions of phenanthrene with dissolved organic matter: limitations of Stern-Volmer plot

Although linear binding isotherms of hydrophobic organic chemicals (HOCs) with dissolved organic matter (DOM) are widely reported, several studies showed nonlinear HOC-DOM interactions. This study pointed out that fluorescence static quenching modeling (FSQM), which often uses a Stern-Volmer type plot to process the data from fluorescence quenching experiments, is conceptually different from the classic Stern-Volmer equation. We also emphasized that although linear Stern-Volmer plots are generally observed in literature, it does not necessarily indicate a linear HOC-DOM interaction. According to both mathematical simulation and laboratory sorption experiments in this study, nonlinear interactions could be concealed by the use of Stern-Volmer plot. Moreover, this study tested the two assumptions for applying FSQM to process binding data. Our results showed that binding coefficient (K(DOC)) for phenanthrene is neither independent of free solute concentration, nor DOM concentration, which is a critical limitation for using FSQM in a form of Stern-Volmer equation to examine HOC-DOM interactions. Therefore, the true characteristics of HOC-DOM interactions need to be examined using different ways of experimental design and data processing.