Biomedical subjects
Publications and source records attributed to J R Lakowicz.
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In recognition of the co-carcinogenic effects of particulate matter and chemical carcinogens, we investigated the effect of particulate silica on the rates of membrane uptake of 1,2-benzanthracene. The fluorescence emission spectra and the apparent quantum yields of benzanthracene and dependent upon adsorption to silica and upon the surface density of benzanthracene on the silica. The fluorescence spectral shifts which occur upon transfer of benzanthracene from the silica surface to phospholipid vesicles provided a convenient means to quantitate the membrane uptake of benzanthracene from particulates. The rate of benzanthracene uptake by dipalmitoyl-L-alpha-phosphatidylcholine vesicles was independent of the concentration of lipid, indicating that the rate-limiting step may involve its solubilization in the aqueous phase. These uptake rates were also independent of the surface density of benzanthracene on the silica, indicating that the benzathracene molecules are dispersed uniformly on the silica surface. Rates of membrane uptake of benzanthracene from the crystalline, microcrystalline, and the silica-absorbed states were compared, and are greatly enhanced by a reduction in crystal size. Silica-adsorbed benzanthracene had the most rapid rate of membrane uptake. Silica did not cause disruption of the lipid vesicles. These results indicate that particulates can enhance the cellular availability of the chemical carcinogens.
Diffusional motions of 1,6-diphenyl-1, 3, 5-hexatriene (DPH) were observed by differential polarized phase fluorometry. The measurements indicated that the depolarizing rotations of DPH in propylene glycol are isotropic. The results in vesicles of dimyristoyl-l-alpha-phosphatidylcholine indicated that diffusional rotations of DPH are dominated by hindered torsional motions. Combined use of both differential phase and steady-state anisotropy measurements showed that the average rotational angle of DPH, at times long compared to the fluorescence lifetime, is limited to about 23 degrees at temperatures below the transition temperature of the lipid and that these rotations become less hindered above the transition temperature. The evidence that the depolarizing rotations of DPH in a lipid bilayer are different from those in an isotropic solvent calls into question the meaning of membrane microviscosity as determined by fluorescence anisotropy.
The fluorescence emissions of chrysene, N-ethylcarbazole, and 1,6-diphenylhexatriene undergo large spectral shifts or changes in quantum yield, or both, upon their uptake from particulates by phospholipid vesicles. This membrane uptake of carcinogen and carcinogen-like molecules by model membranes does not result in any disruption of the lipid bilayers. The fluorescence emission of chrysene, when bound to silica, was found to be sensitive to the surface density of chrysene on the silica. These observations demonstrate the feasibility of using fluorescence spectroscopy to measure the rates of exchange of carcinogens from particulate matter to cell membranes and to characterize the surface distribution of chemical carcinogens on particulate matter. Comparison of the uptake rate of chrysene from the unperturbed crystal state, sonicated crystals, and the silica-adsorbed state demonstrated that the last condition results in the most rapid transport of chrysene into model membranes. This information should prove valuable in understanding the cocarcinogenic effects of particulates and polynuclear aromatic hydrocarbons.
Differential polarized phase fluorometry has been used to investigate the depolarizing motions of 1,6-diphenyl-1,3,5-hexatriene (DPH) in the isotropic solvent propylene glycol and in lipid bilayers of dimyristoyl-L-alpha-phosphatidylcholine (DMPC), dipalmitoyl-L-alpha-phosphatidylcholine (DPPC), and other phosphatidylcholines. Differential phase fluorometry is the measurement of differences in the phase angles between the parallel and perpendicular components of the fluorescence emission of a sample excited with sinusoidally modulated light. The maximum value of the tangent of the phase angle (tan Delta(max)) is known to be a function of the isotropy of the depolarizing motions. For DPH in propylene glycol the maximum tangent is observed at 18 degrees C, and this tangent value corresponds precisely with the value expected for an isotropic rotator. Additionally, the rotational rates determined by steady-state polarization measurements are in precise agreement with the differential phase measurements. These results indicate that differential phase fluorometry provides a reliable measure of the probe's rotational rate under conditions where these rotations are isotropic and unhindered.Rotational rates of DPH obtained from steady-state polarization and differential phase measurements do not agree when this probe is placed in lipid bilayers. The temperature profile of the tan Delta measurements of DPH in DMPC and DPPC bilayers is characterized by a rapid increase of tan Delta at the transition temperature (T(c)), followed by a gradual decline in tan Delta at temperatures above T(c). The observed tanDelta(max) values are only 62 and 43% of the theoretical maximum. This defect in tanDelta(max) is too large to be explained by any degree of rotational anisotropy. However, these defects are explicable by a new theory that describes the tan Delta values under conditions where the probe's rotational motions are restricted to a limiting anisotropy value, r(infinity). Theoretical calculations using this new theory indicate that the temperature dependence of the depolarizing motions of DPH in these saturated bilayers could be explained by a rapid increase in its rotational rate (R) at the transition temperature, coupled with a simultaneous decrease in r(infinity) at this same temperature. The sensitivity of the tan Delta values to both R and r(infinity) indicates that differential phase fluorometry will provide a method to describe more completely the depolarizing motion of probes in lipid bilayers.
One possible mechanism by which cocarcinogenesis occurs was investigated. The effect of a particulate, silica, upon the rate of membrane uptake of a polynuclear aromatic hydrocarbon, benz[a]anthracene (BA), was studied. The fluorescence emission spectrum and quantum yield of BA, when adsorbed to silica, underwent spectral shifts upon addition of phospholipid vesicles. These spectral changes appeared to result from the transfer of BA from the silica surface into the lipid bilayer. We used these spectral changes to measure the rates of membrane uptake of BA from the silica-adsorbed state and from the crystalline and microcrystalline states of BA. Our studies demonstrated that adsorption of BA to silica resulted in an increased rate of uptake of BA into dipalmitoyl-L-alpha-phosphatidylcholine vesicles compared to the uptake rate into these vesicles from crystalling states. Such particle-enhanced membrane uptake of chemical carcinogens may be of importance in explaining the enhanced carcinogenicity of the polynuclear aromatic hydrocarbons in the presence of particulate matter.
The case of methylmercury pollution has demonstrated the profound importance of understanding biologically mediated transformation reactions that yield organometallic compounds with a high potential for bioaccumulation and toxicity. Toxic elements that form organometallic compounds, especially the metal-alkyls (e.g., methylmercury), deserve special concern. Most metal-alkyls are poisonous to the central nervous systems of higher organisms, and these compounds do accumulate in cells. Metal-alkyls that are stable in water, and that have been reported to be synthesized biologically, can be formed from the following toxic elements: Hg, Sn, As, Se, Te, Pd, Au, Tl and Pb. In this report we present details of the mechanisms for biological methylation of certain metals and metalloids with special emphasis on those elements that are widely dispersed in the biosphere. In addition we present preliminary results on the use of flourescence quenching techniques to determine cellular diffusion rates and partition coefficients for methylmercuric chloride.
Chlorinated hydrocarbons, such as the pesticide lindane (gamma-hexachlorocyclohexane), quench the fluorescence of carbazole. The observed quenching is a result of the molecular contacts which occur upon diffusional collisions. Because the amount of quenching depends upon the collisional frequency between carbazole and pesticide, this phenomenon provides a measure of both the diffusional rate of lindane and its local concentration. The carbazole fluorophore is localized within phosphatidylcholine bilayers by cosonicating the lipid with a newly synthesized phospholipid, beta-(11-(9-carbazole)-undecanoyl)-L-alpha-phosphatidylcholine. Using this probe in dimyristoyl-L-alpha-phosphatidylcholine vesicles, and the above mentioned quenching phenomena, we determined the lindane diffusion rate within the bilayer to be 5.7.10-7 cm2/s at 37 degrees C. Measurement of the apparent quenching constant at various dimyristoyl phosphatidylcholine concentrations yielded a lipid-water partition coefficient for lindane of 9500, which is in agreement with the value of 8980 obtained by our equilibrium dialysis experiments. Vesicles of dimyristoyl-L-alpha-phosphatidylcholine become saturated with lindane at a pesticide to lipid molar ratio of approx. 0.28. These results demonstrate the possibility of using the quenching of carbazole fluorescence to investigate the transport and partitioning of pesticides within biological membranes. This ability should prove useful in studies of the interactions of chlorinated hydrocarbons with cell membranes.
Pesticides that contain chlorine have been shown to quench the fluorescence of carbazole-labeled phospholipids. Incorporation of these carbazole-labeled phospholipds into model membranes provides a system that allows the rapid determination of the uptake rates of chlorinated hydrocarbons into model membranes. This technique can be used in the determination of diffusion rates and partition coefficients of chlorine-containing organic compounds in model membrane systems, and hence may provide a method by which the bioaccumulation potential of synthetic chlorine-containing compounds can be estimated.
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Fluorescence spectroscopy can reveal the dynamic properties of proteins, membranes and nucleic acids on the nanosecond timescale. Dynamic processes which can affect the fluorescence spectral characteristics of biopolymer-bound fluorophores include dipolar relaxation around excited state dipoles, rotational diffusion of fluorophores, and permeation of bipolymers of fluorescence quenchers. The occurrence of these processes is revealed by the time-dependence of the Stokes's shifts, the time dependence of fluorescence anisotropies and the quenching of fluorescence, respectively. We will describe each of these processes in detail using examples of data obtained for membrane-bound fluorophores. In addition, we will review the fluorescence spectral evidence for nanosecond structural fluctuations in proteins and nucleic acids. In total, fluorescence spectroscopy indicates that both proteins and membranes fluctuate rapidly on the nanosecond and subnanosecond timescale. In contrast, base pairs in double-helical DNA appear to be immobile on this timescale.
We describe the synthesis and spectral characterization of a rhenium metal-ligand complex. This complex reacts with sulfhydryl groups via an iodoacetamide side chain on the phenanthroline ligand and displays a high limiting anisotropy near 0.35 when excited at 442 nm. When covalently linked to human serum albumin, this complex displayed a mean decay time of about 1 micros. This decay time is appropriate for measuring rotational correlation times on the microsecond time scale as may occur for large macromolecular complexes.
An autoclavable sensing film was developed for monitoring dissolved CO2. The sensing film, based on fluorescence resonance energy transfer (FRET), consisted of a fluorescent donor, an acceptor, and a quaternary ammonium hydroxide, which were doped in a two-component silicone film. As no aqueous solution was used in the sensing film matrix, the sensing film was unaffected by osmotic pressure. Fluorescence lifetime was selected as the sensing parameter, and measured in frequency domain using phase fluorometry. Upon exposure to 20% CO2-saturated water, a 43 degrees increase in phase angle was observed at 100 MHz. The process was fully reversible when the sensing film was exposed to nitrogen-saturated water. The estimated response and recovery times for 90% signal change were 1 min (for a step change from 0 to 6.7% CO2-saturated water) and 1.5 min (for a step change from 6.7 to 3.3% CO2-saturated water). When used for on-line monitoring of dissolved CO2 produced by a culture of Escherichia coli, the sensing film showed a similar trend to that obtained from off-line measurements using a wet chemistry analyzer.
Investigations of the mechanism of particle-enhanced uptake of benzo [a] pyrene into model membranes and microsomes have been extended by studying the effects of particulates on the rates of uptake of four additional polynuclear aromatic hydrocarbons (PAH) into model membranes. Adsorption of dibenzo [a, h] anthracene, benzo [g,h,i] perylene, and 3-methylcholanthrene to the surface of particles resulted in enhanced rates of membrane uptake of these PAH. Adsorption to the fibrous asbestos minerals (chrysotile and anthophyllite) produced the greatest enhancement in membrane uptake rates compared to uptake from microcrystalline dispersions of the PAH. Adsorption of these PAH to nonfibrous minerals resulted in uptake rates intermediate between those of the asbestos-adsorbed and microcrystalline states. In contrast, adsorption of dibenzo-[c,g] carbazole to particles did not result in enhanced uptake. In this case, the rates of uptake in the absence and presence of particles were all extremely high, presumably as a result of the high water solubility of this PAH. The methods described in this paper permit characterization of particles-PAH mixtures in terms of the rate of PAH uptake into membranes.
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