The cytochrome P-450 isoenzyme CYP2E1 in the biological processing of industrial chemicals : consequences for occupational and environmental medicine

The importance of the isoform CYP2E1 of the human cytochrome P-450 superfamily of enzymes for occupational and environmental medicine is derived from its unique substrate spectrum that includes a number of highly important high-production chemicals, such as aliphatic and aromatic hydrocarbons, solvents and industrial monomers (i.a. alkanes, alkenes, aromatic and halogenated hydrocarbons). Many polymorphic genes, such as CYP2E1, show considerable differences in allelic distribution between different human populations. The polymorphic nature of the human CYP2E1 gene is significant for inter-individual differences in toxicity of its substrates. Since the substrate spectrum of CYP2E1 includes many compounds of basic relevance to industrial toxicology, a rationale for metabolic interactions of different CYP2E1 substrates is provided. In-depth research into the inter-individual phenotypic differences of human CYP2E1 enzyme activities was enabled by the recognition that the 6-hydroxylation of the drug chlorzoxazone is mediated by CYP2E1. Studies on CYP2E1 phenotyping have pointed to inter-individual variations in enzyme activities. There are consistent ethnic differences in CYP2E1 enzyme expression, mostly demonstrated between European and Japanese populations, which point to a major impact of genetic factors. The most frequently studied genetic polymorphisms are the restriction fragment length polymorphisms PstI/RsaI (mutant allele: CYP2E1*5B) located in the 5′-flanking region of the gene, as well as the DraI polymorphism (mutant allele: CYP2E1*6) located in intron 6. These polymorphisms are partly related, as they form the common allele designated CYP2E1*5A. Striking inter-ethnic differences between Europeans and Asians appear with respect to the frequencies of the CYP2E1*5A allele (only approximately 5% of Europeans are heterozygous, but 37% of Asians are, whilst 6% of Asians are homozygous). Available studies indicate a wide variation in human CYP2E1 expression, which are very likely based on complex gene-environment interactions. Major inter-ethnic differences are apparent on the genotyping and the phenotyping levels. Selected cases are presented where inter-ethnic variations of CYP2E1 may provide likely explanations for unexplained findings concerning industrial chemicals that are CYP2E1 substrates. Possible consequences of differential inter-individual and inter-ethnic susceptibilities are related to individual expressions of clinical symptoms of chemical toxicity, to results of biological monitoring of exposed workers, and to the interpretation of results of epidemiological or molecular-epidemiological studies.

A new Salmonella typhimurium NM5004 strain expressing rat glutathione S-transferase 5-5 : use in detection of genotoxicity of dihaloalkanes using an SOS/umu test system

The Escherichia coli mu operon was subcloned into a pKK233-2 vector containing rat glutathione S-transferase (GST) 5-5 cDNA and the plasmid thus obtained was introduced into Salmonella typhimurium TA1535. The newly developed strain S.typhimurium NM5004, was found to have 52-fold greater GST activity than the original umu strain S.typhimurium TA1535/pSK1002. We compared sensitivities of these two tester strains, NM5004 and TA1535/ pSK1002, for induction of umuC gene expression with several dihaloalkanes which are activated or inactivated by GST 5-5 activity. The induction of umuC gene expression by these chemicals was monitored by measuring the cellular P-galactosidase activity produced by umuC'lacZ fusion gene in these two tester strains. Ethylene dibromide, 1-bromo-2-chloroethane, 1,2-dichloroethane, and methylene dichloride induced umuC gene expression more strongly in the NM5004 strain than the original strain, 4-Nitroquinoline 1-oxide and N-methyl-N'-nitro-N-nitrosoguanidine were found to induce umuC gene expression to similar extents in both strains. In the case of 1-nitropyrene and 2-nitrofluorene, however, NM5004 strain showed weaker umuC gene expression responses than the original TA1535/ pSK1002 strain, 1,2-Epoxy-3-(4'-nitrophenoxy)propane, a known substrate for GST 5-5, was found to inhibit umuC induction caused by 1-bromo-2-chloroethane. These results indicate that this new tester NM5004 strain expressing a mammalian GST theta class enzyme may be useful for studies of environmental chemicals proposed to be activated or inactivated by GST activity.

Human glutathione S-transferase T1-1 enhances mutagenicity of 1,2-dibromoethane, dibromomethane and 1,2,3,4-diepoxybutane in Salmonella typhimurium

The rat theta class glutathione S-transferase (GST) 5-5 has been shown to affect the mutagenicity of halogenated alkanes and epoxides. In Salmonella typhimurium TA1535 expressing the rat GST5-5 the number of revertants was increased compared to the control strain by CH2Br2, ethylene dibromide (EDB) and 1,2,3,4-diepoxybutane (BDE); in contrast, mutagenicity of 1,2-epoxy-3-(4'-nitrophenoxy)propane (EPNP) was reduced. S.typhimurium TA1535 cells were transformed with an expression plasmid carrying the cDNA of the human theta ortholog GST1-1 either in sense or antisense orientation, the latter being the control. These transformed bacteria were utilized for mutagenicity assays. Mutagenicity of EDB, BDE, CH2Br2, epibromohydrin and 1,3-dichloroacetone was higher in the S.typhimurium TA1535 expressing GSTT1-1 than in the control strain. The expression of active enzyme did not affect the mutagenicity of 1,2-epoxy-3-butene or propylene oxide, GSTT1-1 expression reduced the mutagenicity of EPNP. Glutathione S-transferase 5-5 and GSTT1-1 modulate genotoxicity of several industrially important chemicals in the same way. Polymorphism of the GSTT1 locus in humans may therefore cause differences in cancer susceptibility between the two phenotypes.

Nanocrystalline Janus films of inorganic materials prepared at the liquid-liquid interface

The interface between toluene and water has been employed to prepare ultrathin Janus nanocrystalline films of metal oxides, metal chalcogenides and gold, wherein the surface on the organic-side is hydrophobic and the aqueous-side is hydrophilic. We have changed the nature of the metal precursor or capping agent in the organic layer to increase the hydrophobicity. The strategy employed for this purpose is to increase the length of the alkane chain in the precursor or use a perfluroalkane derivative as precursor or as a capping agent. The hydrophobicity and hydrophilicity of the Janus films have been determined by contact angle measurements. The morphology of hydrophobic and hydrophilic sides of the film have been examined by field emission scanning electron microscopy.

Hybrid Materials Combining Photoactive 2,3-Didecyloxy Anthracene Physical Gels and Gold Nanoparticles

Organic/inorganic hybrid gels have been developed in order to control the three-dimensional structure of photoactive nanofibers and metallic nanoparticles (NPs). These materials are prepared by simultaneous self-assembly of the 2,3-didecyloxyanthracene (DDOA) gelator and of thiol-capped gold nanoparticles (AuNPs). TEM and fluorescence measurements show that alkane-thiol capped AuNPs are homogeneously dispersed and tightly attached to the thermoreversible fibrillar network formed by the organogelator in n-butanol or n-decanol. Rheology and thermal stability measurements reveal moreover that the mechanical and thermal stabilities of the DDOA organogels are not significantly altered and that they remain strong, viscoelastic materials. The hybrid materials display a variable absorbance in the visible range because of the AuNPs, whereas the strong luminescence of the DDOA nanofibers is efficiently quenched by micromolar amounts of AuNPs. Besides, we obtained hybrid aerogels using supercritical CO2. These arc very low-density porous materials showing fibrillar networks oil which fluorinated gold NPs arc dispersed. These hybrid materials are of high interest because of their tunable optical properties and are under investigation for efficient light scattering.

The Influence of Bilayer Composition on the Gel to Liquid Crystalline Transition

We report molecular dynamics simulations of bilayers using a united atom model with explicit solvent molecules. The bilayer consists of the single tail cationic surfactant behenyl trimethyl ammonium chloride (BTMAC) with stearyl alcohol (SA) as the cosurfactant. We study the gel to liquid crystalline transitions in the bilayer by varying the amount of water at fixed BTMAC to SA ratio as well as by varying the BTMAC to SA ratio at fixed water content. The bilayer is found to exist in the tilted, Lβ′ phase at low temperatures, and for the compositions investigated in this study, the Lβ′ to Lα melting transition occurred in the temperature range 330−338 K. For the highest BTMAC to SA composition (2:3 molar ratio), a diffuse headgroup−water interface is observed at lower temperatures, and an increase in the d-spacing occurs prior to the melting transition. This pretransition swelling is accompanied by a sharpening in the water density variation across the headgroup region of the bilayer. Signatures of this swelling effect which can be observed in the alkane density distributions, area per headgroup, and membrane thickness are attributed to the hydrophobic effect. At a fixed bilayer composition, the transition temperature (>338 K) from the Lβ′ to Lα transition obtained for the high water content bilayer (80 wt %) is similar to that obtained with low water content (54.3 wt %), confirming that the melting transition at these water contents is dominated by chain melting.

Influence of Oxide Particle Network Morphology on Ion Solvation and Transport in ``Soggy Sand''Electrolytes

The role of oxide surface chemical composition and solvent on ion solvation and ion transport of ``soggy sand'' electrolytes are discussed here. A ``soggy sand'' electrolyte system comprising dispersions of hydrophilic/hydrophobic functionalized aerosil silica in lithium perchlorate methoxy polyethylene glycol solution was employed for the study. Static and dynamic rheology measurements show formation of an attractive particle network in the case of the composite with unmodified aerosil silica (i.e., with surface silanol groups) as well as composites with hydrophobic alkane groups. While particle network in the composite with hydrophilic aerosil silica (unmodified) were due to hydrogen bonding, hydrophobic aerosil silica particles were held together via van der Waals forces. The network strength in the latter case (i.e., for hydrophobic composites) were weaker compared with the composite with unmodified aerosil silica. Both unmodified silica as well as hydrophobic silica composites displayed solid-like mechanical strength. No enhancement in ionic conductivity compared to the liquid electrolyte was observed in the case of the unmodified silica. This was attributed to the existence of a very strong particle network, which led to the ``expulsion'' of all conducting entities from the interfacial region between adjacent particles. The ionic conductivity for composites with hydrophobic aerosil particles displayed ionic conductivity dependent on the size of the hydrophobic chemical moiety. No spanning attractive particle network was observed for aerosil particles with surfaces modified with stronger hydrophilic groups (than silanol). The composite resembled a sol, and no percolation in ionic conductivity was observed.

Temperature dependent free energy surface of polymer folding from equilibrium and quench studies

Langevin dynamics simulation studies have been employed to calculate the temperature dependent free energy surface and folding characteristics of a 500 monomer long linear alkane (polyethylene) chain with a realistic interaction potential. Both equilibrium and temperature quench simulation studies have been carried out. Using the shape anisotropy parameter (S) of the folded molecule as the order parameter, we find a weakly first order phase transition between the high-temperature molten globule and low-temperature rodlike crystalline states separated by a small barrier of the order of k(B)T. Near the melting temperature (580 K), we observe an intriguing intermittent fluctuation with pronounced ``1/f noise characteristics'' between these two states with large difference in shape and structure. We have also studied the possibilities of different pathways of folding to states much below the melting point. At 300 K starting from the all-trans linear configuration, the chain folds stepwise into a very regular fourfold crystallite with very high shape anisotropy. Whereas, when quenched from a high temperature (900 K) random coil regime, we identify a two step transition from the random coiled state to a molten globulelike state and, further, to a anisotropic rodlike state. The trajectory reveals an interesting coupling between the two order parameters, namely, radius of gyration (R-g) and the shape anisotropy parameter (S). The rodlike final state of the quench trajectory is characterized by lower shape anisotropy parameter and significantly larger number of gauche defects as compared to the final state obtained through equilibrium simulation starting from all-trans linear chain. The quench study shows indication of a nucleationlike pathway from the molten globule to the rodlike state involving an underlying rugged energy landscape. (C) 2010 American Institute of Physics. doi:10.1063/1.3509398]

Spectroscopic investigations of phase transitions in complex solids

Spectroscopic methods have provided information of seminal importance in understanding phase transitions in solids. After briefly examining some fundamental concept related to phase transitions, we shall discuss several case studies particularly involving the use of vibrational (IR and Raman) spectroscopy. Examples will include both order-disorder and displacive transitions. Under the former are included transitions in nitrates, ammonium halides, alkylammonium salts, plastic state of C60 and superionic conductors (specially CsHSO4). In addition, we shall discuss some aspects of incommensurate phase transitions, the glass transition and electronic phase transitions. Transitions of phosphonitrilic halide tetramers and alkane dicarboxylic acids are also examined

Solubilization of n-decane into n-decyl-polyoxyethylene surfactants: A cavity forming free energy based model

The present paper reports the results of a theoretical study of the forces and factors driving the solubilization of n-alkane solubilizates into the micellar core of some non-ionic surfactants, based on a micellar model which includes the cavity forming free energy as a component of micellization. The solubilizate is n-decane and the non-ionic surfactants considered are n-decyl-polyoxyethylene surfactants. The extent of solubilization, i.e. the mole fraction of the solubilizate within the core has been calculated. The results indicate that the incorporated solubilizate has more translational and rotational degrees of freedom as compared to those of the tail parts of the surfactants present in the core. This drives the total free energy of aggregation after solubilization into a more favourable direction. The results are in fair agreement with the experimental results.

Vesicle formation from dimeric ion-paired amphiphiles. Control over vesicular thermotropic and ion-transport properties as a function of intra-amphiphilic headgroup separation

A novel series of vesicle-forming ion-paired amphiphiles, bis(hexadecyldimethylammonium)alkane dipalmitate (1a-1h), containing four chains were synthesized with two isolated headgroups. In each of these amphiphiles, the two headgroup charges are separated by a flexible polymethylene spacer chain -[(CH2)(m)]- of varying lengths (m) such that the length and the conformation of the spacer chain determine the intra-"monomer" headgroup separation. Transmission electron microscopy indicated that each of these forms bilayer membranes upon dispersion in aqueous media. The vesicular properties of these aggregates have been examined by differential scanning calorimetry and temperature-dependent fluorescence anisotropy measurements. Interestingly, their T-m values decreased with the increase in the m value. Thus while the apparent T-m of the lipid with m = 2 (1a) is 74.1 degrees C, the corresponding value observed for the lipid with m = 12 (1h) is 38.9 degrees C. The fluorescence anisotropy values (r) for 1b-1g were quite high (r similar to 0.3) compared to that of 1h (r similar to 0.23) at 20-30 degrees C in their gel states. On the other hand, the r value for vesicular 1b beyond melting was higher (0.1) compared to any of those for 1c-1h (similar to 0.04-0.06). X-ray diffraction of the cast films was performed to understand the nature and the thickness of these membrane organizations. The membrane widths ranged from 30 to 51 A as the m values varied. The entrapment of a small water-soluble solute, riboflavin, by the individual vesicular aggregates, and their sustenance: under an imposed transmembrane pH gradient have also been examined. These results show that all lipid vesicles entrap riboflavin and that generally the resistance to OH- permeation decreases with the increase in m value. Finally,all the above observations were comparatively analyzed, and on the basis of the calculated structures of these lipids, it was possible to conclude that membrane propel-ties can be modulated by spacer chain length variation of the ion-paired amphiphiles.

Ferromagnetism Exhibited by Nanoparticles of Noble Metals

Gold nanoparticles with average diameters in the range 2.515 nm, prepared at the organic/aqueous interface by using tetrakis( hydroxymethyl) phosphonium chloride (THPC) as reducing agent, exhibit ferromagnetism whereby the saturation magnetization M(S) increases with decreasing diameter and varies linearly with the fraction of surface atoms. The value of M(S) is higher when the particles are present as a film instead of as a sol. Capping with strongly interacting ligands such as alkane thiols results in a higher M(S) value, which varies with the strength of the metal-sulfur bond. Ferromagnetism is also found in Pt and Ag nanoparticles prepared as sols, and the M(S) values vary as Pt > Au > Ag. A careful study of the temperature variation of the magnetization of Au nanoparticles, along with certain other observations, suggests that small bare nanoparticles of noble metals could indeed possess ferromagnetism, albeit weak, which is accentuated in the presence of capping agents, specially alkane thiols which form strong metal-sulfur bonds.

Fluctuation quench and hydrophobic collapse of polymers and biopolymers in water-DMSO binary mixture at low DMSO concentration

Binary mixtures have strong influence on activities of polymers and biopolymers even at low cosolvent concentration. Among the several aqueous binary mixtures studied, water-DMSO especially stands out for its unusual behavior at certain specific concentrations of DMSO. In the present work, we study the effect of water-DMSO binary mixture on polymers and biopolymers by taking a simple linear hydrocarbon chain of intermediate length (n = 30) and the protein lysozyme, respectively. We find that at a mole fraction of 0.05 of DMSO (x(DMSO) = 0.05) in aqueous solution, the hydrocarbon chain adopts the collapsed conformation as the most stable and rigid state. In this case of 0.05 mole fraction of DMSO in bulk, the DMSO concentration in the first hydration layer around the polymer is found to be as large as 17%. Formation of such hydrophobic environment around the polymer is the reason for the collapsed state gaining so much stability. Interestingly, similar quench of conformational fluctuation is also observed for the protein investigated. It is observed that in the case of alkane polymer chains, long wavelength fluctuation gets easily quenched, the polymer being purely hydrophobic. However, in case of the protein, quench of fluctuation is prominent only at the hydrophobic surface, and quench of long wavelength fluctuation becomes insignificant for the full protein. As protein contains both hydrophobic and hydrophilic moieties, the extent of quench of conformational fluctuation with respect to that in pure water is almost half for the biopolymer complex (16.83%) than the same for pure hydrophobic polymer chain (32.43%).

Effects of a Delocalizable Cation on the Headgroup of Gemini Lipids on the Lipoplex-Type Nanoaggregates Directly Formed from Plasmid DNA

Lipoplex-type nanoaggregates prepared from pEGFP-C3 plasmid DNA (pDNA) and mixed liposomes, with a gemini cationic lipid (CL) 1,2-bis(hexadecyl imidazolium) alkanes], referred as (C(16)Im)(2)C-n (where C-n is the alkane spacer length, n = 2, 3, 5, or 12, between the imidazolium heads) and DOPE zwitterionic lipid, have been analyzed by zeta potential, gel electrophoresis, SAXS, cryo-TEM, fluorescence anisotropy, transfection efficiency, fluorescence confocal microscopy, and cell viability/cytotoxicity experiments to establish a structure-biological activity relationship. The study, carried out at several mixed liposome compositions, alpha, and effective charge ratios, rho(eff), of the lipoplex, demonstrates that the transfection of pDNA using CLs initially requires the determination of the effective charge of both. The electrochemical study confirms that CLs with a delocalizable positive charge in their headgroups yield an effective positive charge that is 90% of their expected nominal one, while pDNA is compacted yielding an effective negative charge which is only 10-25% than that of the linear DNA. SAXS diffractograms show that lipoplexes formed by CLs with shorter spacer (n = 2, 3, or 5) present three lamellar structures, two of them in coexistence, while those formed by CL with longest spacer (n = 12) present two additional inverted hexagonal structures. Cryo-TEM micrographs show nanoaggregates with two multilamellar structures, a cluster-type (at low alpha value) and a fingerprint-type, that coexist with the cluster-type at moderate alpha composition. The optimized transfection efficiency (TE) of pDNA, in HEK293T, HeLa, and H1299 cells was higher using lipoplexes containing gemini CLs with shorter spacers at low a value. Each lipid formulation did not show any significant levels of toxicity, the reported lipoplexes being adequate DNA vectors for gene therapy and considerably better than both Lipofectamine 2000 and CLs of the 1,2-bis(hexadecyl ammnoniun) alkane series, recently reported.