Steady-state Fluorescence Polarization Research Articles

Insights into the Interaction Landscape of the EVH1 Domain of Mena.

The Enabled/VASP homology 1 (EVH1) domain is a small module that interacts with proline-rich stretches in its ligands and is found in various signaling and scaffolding proteins. Mena, the mammalian homologue of Ena, is involved in diverse actin-associated events, such as membrane dynamics, bacterial motility, and tumor intravasation and extravasation. Two-dimensional (2D) 1H-15N HSQC NMR was used to study Mena EVH1 binding properties, defining the amino acids involved in ligand recognition for the physiological ligands ActA and PCARE, and a synthetic polyproline-inspired small molecule (hereafter inhibitor 6c). Chemical shift perturbations indicated that proline-rich segments bind in the conserved EVH1 hydrophobic cleft. The PCARE-derived peptide elicited more perturbations compared to the ActA-derived peptide, consistent with a previous report of a structural alteration in the solvent-exposed β7-β8 loop. Unexpectedly, EVH1 and the proline-rich segment of PTP1B did not exhibit NMR chemical shift perturbations; however, the high-resolution crystal structure implicated the conserved EVH1 hydrophobic cleft in ligand recognition. Intrinsic steady-state fluorescence and fluorescence polarization assays indicate that residues outside the proline-rich segment enhance the ligand affinity for EVH1 (Kd = 3-8 μM). Inhibitor 6c displayed tighter binding (Kd ∼ 0.3 μM) and occupies the same EVH1 cleft as physiological ligands. These studies revealed that the EVH1 domain enhances ligand affinity through recognition of residues flanking the proline-rich segments. Additionally, a synthetic inhibitor binds more tightly to the EVH1 domain than natural ligands, occupying the same hydrophobic cleft.

Mechanistic Insights into Cellular and Molecular Targets of Zinc Oxide Quantum Dots (ZnO QDs) in Fungal Pathogen, Candida albicans: One Drug Multi-Targeted Therapeutic Approach.

Rationally designed multitargeted drugs, known as network therapeutics/multimodal drugs, have emerged as versatile therapeutic solutions to combat drug-resistant microbes. Here, we report novel mechanistic insights into cellular and molecular targets of ZnO quantum dots (QDs) against Candida albicans, a representative of fungal pathogens. Stable, monodispersed 4-6 nm ZnO QDs were synthesized using a wet chemical route, which exhibited dose-dependent inhibition on the growth dynamics of Candida. Treatment with 200 μg/mL ZnO QDs revealed an aberrant morphology and a disrupted cellular ultrastructure in electron microscopy and led to a 23% reduction in ergosterol content and a 53% increase in intracellular reactive oxygen species. Significant increase in steady-state fluorescence polarization and fluorescence lifetime decay of membrane probe 1,6-diphenyl-1,3,5-hexatriene (DPH) in treated cells, respectively, implied reduction in membrane fluidity and enhanced microviscosity. The observed reduction in passive diffusion of fluorescent Rhodamine 6G across the membrane validated the intricate relationship between ergosterol, membrane fluidity, and microviscosity. An inverse relationship existing between ergosterol biosynthetic genes, ERG11 and ERG3 in treated cells, related well with displayed higher susceptibilities. Furthermore, treated cells exhibited impaired functionality and downregulation of ABC drug efflux pumps. Multiple cellular targets of ZnO QDs in Candida were validated by in silico molecular docking. Thus, targeting ERG11, ERG3, and ABC drug efflux pumps might emerge as a versatile, nano-ZnO-based strategy in fungal therapeutics to address the challenges of drug resistance.

Two-dimensional fluid viscosity measurement in microchannel flow using fluorescence polarization imaging

This study describes the development of a noncontact and two-dimensional fluid viscosity measurement technique based on fluorescence polarization microscopy. This technique exploits fluorescence depolarization due to rotational Brownian motion of fluorophores and determines fluid viscosity in microchannel flow by measuring steady-state fluorescence polarization. The main advantage of the technique is that planar distributions of fluid viscosity can be visualized by noncontact optical measurement, while commonly-used mechanical viscometers measure the viscosity of bulk liquids. Moreover, steady-state polarization measurements are realized using a simpler experimental setup compared to other noncontact techniques such as time-resolved fluorescence lifetime/polarization measurements. The relationship between the fluid viscosity (μ) and the fluorescence polarization degree (P) was experimentally obtained using casein molecules labeled with fluorescein isothiocyanate as a fluorescent probe. The fluid viscosity was controlled within the range of 0.7–3.0 mPa s, which is the range often encountered in biological materials, by mixing sucrose or glucose with the solution. The fluid temperature was maintained uniform at 30 °C during the measurement. The calibration result showed that 1/P linearly increased with 1/μ which qualitatively agreed well with the theoretical prediction. The measurement uncertainty was 7.5%–9.5% based on the slope of the calibration curve. The viscosity gradient generated by the mass diffusion between the two solutions co-flowing in the Y-shaped microchannel was clearly visualized under uniform temperature conditions by applying the calibration curve. Finally, the influence of the temperature change on P was experimentally evaluated. The results supported the applicability of the present technique for visualization of the viscosity distribution induced by temperature change. These results confirmed the feasibility of the present technique for analyzing microscale viscosity fields associated with mass transport or temperature change.

Kinetics of Membrane Protein-Detergent Interactions Depend on Protein Electrostatics.

Interactions of a membrane protein with a detergent micelle represent a fundamental process with practical implications in structural and chemical biology. Quantitative assessment of the kinetics of protein-detergent complex (PDC) interactions has always been challenged by complicated behavior of both membrane proteins and solubilizing detergents in aqueous phase. Here, we show the kinetic reads of the desorption of maltoside-containing detergents from β-barrel membrane proteins. Using steady-state fluorescence polarization (FP) anisotropy measurements, we recorded real-time, specific signatures of the PDC interactions. The results of these measurements were used to infer the model-dependent rate constants of association and dissociation of the proteomicelles. Remarkably, the kinetics of the PDC interactions depend on the overall protein charge despite the nonionic nature of the detergent monomers. In the future, this approach might be employed for high-throughput screening of kinetic fingerprints of different membrane proteins stabilized in micelles that contain mixtures of various detergents.

Detergent Desorption of Membrane Proteins Exhibits Two Kinetic Phases.

Gradual dissociation of detergent molecules from water-insoluble membrane proteins culminates in protein aggregation. However, the time-dependent trajectory of this process remains ambiguous because the signal-to-noise ratio of most spectroscopic and calorimetric techniques is drastically declined by the presence of protein aggregates in solution. We show that by using steady-state fluorescence polarization (FP) spectroscopy the dissociation of the protein-detergent complex (PDC) can be inspected in real time at detergent concentrations below the critical micelle concentration. This article provides experimental evidence of the coexistence of two distinct phases of the dissociations of detergent monomers from membrane proteins. We first noted a slow detergent predesolvation process, which was accompanied by a relatively modest change in the FP anisotropy, suggesting a small number of dissociated detergent monomers from the proteomicelles. This predesolvation phase was followed by a fast detergent desolvation process, which was highlighted by a major alteration in the FP anisotropy. The durations and rates of these phases were dependent on both the detergent concentration and the interfacial PDC interactions. Further development of this approach might lead to the creation of a new semiquantitative method for the assessment of the kinetics of association and dissociation of proteomicelles.

Quantification of Membrane Protein-Detergent Complex Interactions.

Although fundamentally significant in structural, chemical, and membrane biology, the interfacial protein-detergent complex (PDC) interactions have been modestly examined because of the complicated behavior of both detergents and membrane proteins in aqueous phase. Membrane proteins are prone to unproductive aggregation resulting from poor detergent solvation, but the participating forces in this phenomenon remain ambiguous. Here, we show that using rational membrane protein design, targeted chemical modification, and steady-state fluorescence polarization spectroscopy, the detergent desolvation of membrane proteins can be quantitatively evaluated. We demonstrate that depleting the detergent in the sample well produced a two-state transition of membrane proteins between a fully detergent-solvated state and a detergent-desolvated state, the nature of which depended on the interfacial PDC interactions. Using a panel of six membrane proteins of varying hydrophobic topography, structural fingerprint, and charge distribution on the solvent-accessible surface, we provide direct experimental evidence for the contributions of the electrostatic and hydrophobic interactions to the protein solvation properties. Moreover, all-atom molecular dynamics simulations report the major contribution of the hydrophobic forces exerted at the PDC interface. This semiquantitative approach might be extended in the future to include studies of the interfacial PDC interactions of other challenging membrane protein systems of unknown structure. This would have practical importance in protein extraction, solubilization, stabilization, and crystallization.

Interrogating Detergent Desolvation of Nanopore-Forming Proteins by Fluorescence Polarization Spectroscopy.

Understanding how membrane proteins interact with detergents is of fundamental and practical significance in structural and chemical biology as well as in nanobiotechnology. Current methods for inspecting protein-detergent complex (PDC) interfaces require high concentrations of protein and are of low throughput. Here, we describe a scalable, spectroscopic approach that uses nanomolar protein concentrations in native solutions. This approach, which is based on steady-state fluorescence polarization (FP) spectroscopy, kinetically resolves the dissociation of detergents from membrane proteins and protein unfolding. For satisfactorily solubilizing detergents, at concentrations much greater than the critical micelle concentration (CMC), the fluorescence anisotropy was independent of detergent concentration. In contrast, at detergent concentrations comparable with or below the CMC, the anisotropy readout underwent a time-dependent decrease, showing a specific and sensitive protein unfolding signature. Functionally reconstituted membrane proteins into a bilayer membrane confirmed predictions made by these FP-based determinations with respect to varying refolding conditions. From a practical point of view, this 96-well analytical approach will facilitate a massively parallel assessment of the PDC interfacial interactions under a fairly broad range of micellar and environmental conditions. We expect that these studies will potentially accelerate research in membrane proteins pertaining to their extraction, solubilization, stabilization, and crystallization, as well as reconstitution into bilayer membranes.

Fluorescence and Phosphorescence Anisotropy from Oriented Films of Thermally Activated Delayed Fluorescence Emitters.

Anisotropy within three TADF materials has been observed using steady-state fluorescence polarization. This technique has allowed for the observation of differences in polarization within dilute solution, and both unstretched and stretched films; the latter producing highly aligned molecules within the sample. Using these aligned films differences in anisotropy can be observed between the emission from the 1LE and 1CT states and upon exciting different absorption bands. Furthermore, polarization observed from time-resolved measurements highlights the strong vibronic coupling between charge-transfer and local triplet states.

Molecular Simulation and Experimental Characterization of Ionic-Liquid-Based Cosolvent Extraction Solvents

The use of ionic liquid (IL)/polar covalent molecule (PCM) cosolvent mixtures is a significant new pathway to extract and separate oils and other high-value components from algae and other bio-oil-bearing biomasses. In this work, light-scattering and steady-state fluorescence polarization techniques were utilized to characterize cosolvent behavior at the micro- and macroscales, and their measurements correlated to lipid extraction yields. In addition, molecular dynamics simulations were employed to probe the fundamental interactions between IL and PCMs in solution. Both experimental and computational (radial distribution functions and clustering analysis) data confirmed the increased aggregation between IL cations and anions with increasing methanol concentration. For the 1-ethyl-3-methylimidazolium methyl sulfate/methanol (MeOH) system, this ion aggregation appears to correlate with experimentally observed decreases in the maximum lipid extraction yields for MeOH concentrations above 80 vol %. At these concentrations, MeOH appears to completely surround the aggregates, thereby removing the effect of the IL and diminishing the extraction yield. The successful simulation of experimental observations provides new knowledge on not only the relationship between a molecular-level cosolvent structure and its impact upon the solubility of the extracted material, which in this case are lipids, but also the ability to improve future designs of IL-based cosolvent mixtures for a broad range of extractable materials.

A Novel FRET Technique to Characterize the Oligomerization State of Protein-Protein Interactions

Protein-protein interactions are the fundamental driving force of numerous cellular processes and cell signaling pathways. Characterizing whether proteins interact as dimers, trimers, or higher oligomers is essential to understanding these interactions. Several microscopy and advanced imaging techniques relying on Forster resonance energy transfer (FRET) between identical fluorophores (homo-FRET) have been developed to estimate protein stoichiometry. The increased FRET in oligomers is detected by measuring depolarization or emission time. Homo-FRET methods have a strong advantage in requiring only a single fluorophore, greatly simplifying sample preparation in comparison to conventional hetero-FRET methods. However, most homo-FRET methods require sophisticated imaging equipment, and both theoretical models and applications have been restricted to the study of membrane-bound proteins. Using a simple bulk homo-FRET and laser photobleaching approach, we demonstrate the feasibility of characterizing the oligomerization state of an interacting protein in-vitro. To simulate oligomers in a proof of concept, we constructed an extensive repertoire of fusion proteins with 1-6 consecutive green fluorescent protein (GFP) domains. We show how the resulting homo-FRET (measurable via steady-state anisotropy or fluorescence polarization) is proportional to the oligomerization state of proximal GFP domains. For the first time, this is demonstrated with soluble proteins. In both membrane and soluble proteins, oligomerization increases FRET and therefore anisotropy. However for soluble proteins oligomerization also slows fluorophore rotation, leading to a size-dependent decrease in anisotropy. Through gradual photobleaching of fluorophores these two effects can be distinguished, and the oligomerization state of a labeled protein of interest can be estimated. We therefore show how the theoretical framework developed for membrane proteins needs to be adjusted to account for this additional degree of freedom in soluble proteins. Overall, bulk homo-FRET and laser photobleaching is a promising method to determine the oligomerization state of a protein of interest, which can have a low concentration (0.1-0.5 µM) and needs only a single fluorescent label. The method requires only a photometer or microplate reader capable of measuring steady-state anisotropy.

Azadioxatriangulenium: exploring the effect of a 20 ns fluorescence lifetime in fluorescence anisotropy measurements

Azaoxatriangulenium (ADOTA) has been shown to be highly emissive despite a moderate molar absorption coefficient of the primary electronic transition. As a result, the fluorescence lifetime is ~20 ns, longer than all commonly used red fluorescent organic probes. The electronic transitions in ADOTA are highly polarised (r0 = 0.38), which in combination with the long fluorescence lifetime extents the size-range of biomolecular weights that can be detected in fluorescence polarisation-based experiments. Here, the rotational dynamics of bovine serum albumin (BSA) are monitored with three different ADOTA derivatives, differing only in constitution of the reactive linker. A detailed study of the degree of labelling, the steady-state anisotropy, and the time-resolved anisotropy of the three different ADOTA-BSA conjugates are reported. The fluorescence quantum yields (ϕfl) of the free dyes in PBS solution are determined to be ~55%, which is reduced to ~20% in the ADOTA-BSA conjugates. Despite the reduction in ϕfl, a ~20 ns intensity averaged lifetime is maintained, allowing for the rotational dynamics of BSA to be monitored for up to 100 ns. Thus, ADOTA can be used in fluorescence polarisation assays to fill the gap between commonly used organic dyes and the long luminescence lifetime transition metal complexes. This allows for efficient steady-state fluorescence polarisation assays for detecting binding of analytes with molecular weights of up to 100 kDa.

Fluorescence life-time imaging and steady state polarization for examining binding of fluorophores to gold nanoparticles.

Nanocomposites as multifunctional agents are capable of combing imaging and cell biology technologies. The conventional methods used for validation of the conjugation process of nanoparticles (NPs) to fluorescent molecules such as spectroscopy analysis and surface potential measurements, are not sufficient. In this paper we present a new and highly sensitive procedure that uses the combination of (1) fluorescence spectrum, (2) fluorescence lifetime, and (3) steady state fluorescence polarization measurements. We characterize and analyze gold NPs with Lucifer yellow (LY) surface coating as a model. We demonstrate the ability to differentiate between LY-GNP (the conjugated complex) and a mixture of coated NP and free dyes. We suggest the approach for neuroscience applications where LY is used for detecting and labeling cells, studying morphology and intracellular communications. Histograms of Fluorescence lifetime imaging (FLIM) of free LY dye (Left) in comparison to the conjugated dye to gold nanoparticles, LY-GNP (Middle) enable the differentiation between LY-GNP (the conjugated complex) and a mixture of coated NP and free dyes (Right).

Biophysical alterations in lipid rafts from human cerebral cortex associate with increased BACE1/AβPP interaction in early stages of Alzheimer's disease.

In the present study, we have assessed the biophysical properties of lipid rafts from different brain areas in subjects exhibiting early neuropathological stages of Alzheimer's disease (AD). By means of steady-state fluorescence polarization analyses using two environment-sensitive fluorescent probes, we demonstrate that lipid rafts from cerebellum, and frontal and entorhinal cortices, exhibit different biophysical behaviors depending on the stage of the disease. Thus, while membrane anisotropies were similar in the cerebellum along stages, lipid rafts from frontal and entorhinal cortices at AD stages I/II and AD III were significantly more liquid-ordered than in control subjects, both at the aqueous interface and hydrophobic core of the raft membrane. Thermotropic analyses demonstrated the presence of Arrhenius breakpoints between 28.3-32.0 °C, which were not influenced by the disease stage. However, analyses of membrane microviscosity (ηapp) demonstrate that frontal and entorhinal lipid rafts are notably more viscous and liquid-ordered all across the membrane from early stages of the disease. These physicochemical alterations in lipid rafts do not correlate with changes in cholesterol or sphingomyelin levels, but to reduced unsaturation index and increased saturate/polyunsaturated ratios in phospholipid acyl chains. Moreover, we demonstrate that β-secretase/AβPP (amyloid-β protein precursor) interaction and lipid raft microviscosity are strongly, and positively, correlated in AD frontal and entorhinal cortices. These observations strengthens the hypothesis that physical properties of these microdomains modulate the convergence of amyloidogenic machinery toward lipid rafts, and also points to a critical role of polyunsaturated fatty acids in amyloidogenic processing of AβPP.

Fluorescence anisotropy measurements in solution: Methods and reference materials (IUPAC Technical Report)

After recalling the basic relations relevant to both steady-state and time-resolved fluorescence polarization, it is shown how the values of steady-state polarized intensities recorded experimentally usually need to be corrected for systematic effects and errors, caused by instrumentation and sample properties. A list of selected reference values of steady-state fluorescence anisotropy and polarization is given. Attention is also paid to analysis of time-resolved fluorescence anisotropy data obtained by pulse fluorometry or phase and modulation fluorometry techniques. Recommendations for checking the accuracy of measurements are provided together with a list of selected time-resolved fluorescence anisotropy data as reported in the literature.

Binding of peptides corresponding to the carboxy-terminal region of human-β-defensins-1–3 with model membranes investigated by isothermal titration calorimetry

Human-β-defensins HBD-1–3 are important components of the innate immune system. Synthetic peptides Phd-1–3 with a single disulphide bond, spanning the cationic C-terminal region of HBD-1–3, have antimicrobial activity. The interaction of Phd-1–3 with model membranes was investigated using isothermal titration calorimetry (ITC) and steady-state fluorescence polarization to understand the biophysical basis for the mechanism of antimicrobial action. Calorimetric titration of POPE:POPG (7:3) vesicles with peptides at 25°C and 37°C showed complex profiles with two distinct regions of heat changes. The data indicate binding of Phd-1–3 at 37°C to both negative and zwitterionic lipid vesicles is exothermic with low enthalpy values (ΔH~−1.3 to −2.8kcal/mol) as compared to amphipathic helical antibacterial peptides. The adsorption of peptides to negatively charged lipid membranes is modulated by electrostatic interactions that are described by surface partition equilibrium model using Gouy–Chapman theory. However, this model could not explain the isotherms of peptide binding to zwitterionic lipid vesicles. Fluorescence polarization of TMA-DPH (1-[4-(trimethylammonio) phenyl]-6-phenyl-1,3,5-hexatriene) and DPH (1,6-diphenyl-1,3,5-hexatriene) located in the head group and acyl chain region respectively, indicates that the peptides interact with interfacial region of negatively charged membranes. Based on the results obtained, we conclude that adsorption of cationic peptides Phd-1–3 on lipid surface do not result in conformational change or pore formation. It is proposed that interaction of Phd-1–3 with the negatively charged lipid head group causes membrane destabilization, which in turn affects the efficient functioning of cytoplasmic membrane proteins in bacteria, resulting in cell death.

Altered membrane lipid dynamics and chemoprevention by non-steroidal anti inflammatory drugs during colon carcinogenesis.

The present work focuses on the anti-neoplastic role of non steroidal anti-inflammatory drugs (NSAIDs) in modulating the biophysical parameters of the colonic membranes in 1,2-dimethylhydrazine dihydrochloride (DMH) induced carcinogenesis. The steady-state fluorescence polarization technique was applied to assess membrane fluidity, membrane polarity and lipid phase states. The decline in cholesterol content, biosynthesis and cholesterol: phospholipids ratio with DMH treatment indicates more fluidity associated with carcinogenesis. The DMH group had shown lower order parameter indicating more fluidity whereas NSAIDs resulted in increasing the membrane lipid order. The converging effects of these changes were more in membrane phase separations and membrane phase state. In DMH treatment membrane shows lesser phase separation or high polarity, and more liquid crystalline state while for NSAID groups membranes have higher phase separations or low polarity, and more of the gel phase. Further, NSAIDs induced anti-proliferative effects were evidently observed by apoptosis in the colonocytes by using acridine orange-ethidium bromide fluorescent staining and Terminal de-oxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The results suggest that NSAIDs induced alteration in the membrane biophysical parameters may be an important initiating event for the chemopreventive action.

Effect of Dimensionality in Dendrimeric and Polymeric Fluorescent Materials for Detecting Explosives

Steady-state Stern−Volmer analysis is uniformly used to assess in solution the efficiency of a sensing molecule for a particular analyte. We use a combination of steady-state Stern−Volmer analysis and time-resolved photoluminescence (TRPL) to determine the underlying mechanisms by which fluorescent sensing materials comprised of fluorene-based chromophores sense nitro-based explosive analytes. The ability of two first-generation dendrimers comprised of bifluorene-containing chromophores to sense explosive analytes was compared with the chemically related polymer poly(9,9-di-n-octylfluoren-2,7-diyl). One dendrimer was planar with a single chromophore with the second having four chromophores tetrahedrally arranged around an adamantyl center. All the materials had high photoluminescence quantum yields of around 90% and were able to sense explosive analytes via quenching of their fluorescence. The three-dimensional dendrimer based upon the adamantyl core was found to have the highest Stern−Volmer constants for all the analytes tested with the planar dendrimer also proving to be on average superior to the polymer. The TRPL measurements showed that sensing occurred by a combination of collisional and static quenching with the proportion of collisional quenching being based on the number of aromatic units in the analyte. Steady-state fluorescence polarization anisotropy measurements of the three materials revealed that for the three-dimensional dendrimer an exciton can migrate between all of the chromophores, meaning that an exciton formed on one chromophore of the dendrimer can be quenched by an analyte interacting with a second chromophore. This gives rise to the potential for sensing response amplification and explains its superior performance to the planar dendrimer and polymer.

Effects of water chemistry and concentrations of dissolved organic matter on its fluorescence characteristics and molecular conformation

Previous studies showed that water chemistry and concentrations of dissolved organic matter (DOM) could affect its molecular conformation and binding characteristics with hydrophobic organic contaminants (HOCs). However, the conformational change of DOM resultant from water chemistry and concentrations of DOM was not extensively investigated; therefore, the contradictory reports regarding the binding property with HOCs were available in literature. In this study, the effects of ionic strength, pH and DOM concentrations on the fluorescence properties of two humic acids (HA), namely Fluka HA and Amherst HA, were investigated by three-dimensional excitation-emission matrix fluorescence spectroscopy (3DEEM) and steady-state fluorescence polarization (FP) techniques. The results not only corroborated previous observations obtained by other investigators, but revealed some new information about the fluorescence properties and molecular conformation of the humic acids under different water chemistry and DOM concentration conditions, which could shed light on its binding mechanisms and binding properties with HOCs.

Effects of pyrethroids and endosulfan on fluidity of mitochondria membrane in Chilo suppressalis (Walker)

The effects of pyrethroids and endosulfan on fluidity of mitochondrial membranes from Chilo suppressalis were investigated at different temperatures by steady-state fluorescence polarization using 1,6-diphenyl-1,3,5-hexatriene (DPH) molecule as probe. The results showed that changes in DPH polarization caused by the pyrethroids tested were more at lower temperature than at higher temperature, which showed a negative temperature coefficient of the pyrethroids. On the contrary, change in DPH polarization caused by endosulfan was more at higher temperature than at lower temperature. Endosulfan displayed a positive temperature coefficient. Moreover, these effects of pyrethroids and endosulfan on changes of DPH polarization were dose-dependent in mitochondrial membranes of C. suppressalis. DPH polarization value increased in the mitochondrial membranes treated by all concentrations of pyrethroids and decreased by endosulfan, and changes caused by pyrethroids were more pronounced than by endosulfan.

Membrane Fluidity and Surface Changes During Initiation of 1,2 Dimethylhydrazine-Induced Colon Carcinogenesis: Protection by Zinc

The present study evaluated the modulatory effects of zinc on colonic membrane fluidity and surface abnormalities following 1,2 dimethylhydrazine (DMH)-induced colon carcinogenesis. Rats were segregated into four groups: normal control, DMH treated, zinc treated, DMH + zinc treated. Colon carcinogenesis was initiated through weekly subcutaneous injections of DMH (30 mg/kg body weight) for 8 weeks. Zinc (in the form of zinc sulphate) was supplemented to rats at a dose level of 227 mg/L in drinking water, ad libitum, for the entire duration of the study. Brush border membranes (BBM) were isolated from the colon of rats and the fluidity parameters were assessed by steady-state fluorescence polarization technique using the membrane extrinsic fluorophore 1,6-diphenyl-1,3,5-hexatriene (DPH). The translational diffusion was measured by using the excimer formation of pyrene incorporated in the membrane. The results demonstrated a significant increase in the polarization and anisotropy, accompanied by an increase in order parameter in the membrane preparations from the colon of DMH-injected rats. Further, studies with pyrene fluorophore indicated a marked decrease in membrane microviscosity following DMH treatment. However, the alterations in membrane fluorescence polarization and the fluidity parameters were completely restored following zinc treatment. Drastic alterations in colon surface were noticed after 8 weeks of DMH treatment. However, zinc treatment to DMH-treated rats greatly restored normalcy in the colonic surface. The study concludes that zinc has a strong membrane stabilizing effect and thus has a positive beneficial effect against chemically induced colonic preneoplastic progression in rats.