DSC and Raman study on the interaction of DDT [1,1,1-trichloro-2,2-bis(p-chlorophenyl)-ethane] with liposomal phospholipids
Introduction
DDT[1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane; (ClC6H4)2CHCCl3], is an organochlorine insecticide that has found a broad range of agricultural and non-agricultural applications worldwide since 1939. In the late 70, DDT was banned in most parts of the world, except for use in controlling emergencies in public health problems and actually, DDT is seldom used to control vector-borne diseases, such as malaria. Nevertheless, as a consequence of the appearance of insects resistant to other pesticides, a larger use of DDT has been again proposed, rending again of great interest the studies on its toxicity.
Because of its chemical characteristics, DDT can undergo long-range transport through the atmosphere in a process known as “global distillation” where DDT migrates from warmer regions to colder regions through repeated cycles of volatilization from soil and water surfaces followed by deposition of DDT onto surfaces through dry and wet deposition processes. This long-range transport results in the wide dispersion of DDT and its metabolites throughout the world, even into remote areas, such as the Arctic or Antarctic regions [1].
Similarly to many other chlorinated pesticides and chlorinated substances of industrial interest, like, for e.g. the PCBs (polychlorinated biphenyls), DDT accumulated in the environment and considerable amounts of DDT have been detected in soil after more than 20 years subsequently to the last treatment with this product [2].
The high lipophilicity of DDT, associated with a high stability and persistence, favors accumulation in tissues of animals, man, and other members of ecological chains. Toxic concentrations may be reached after long-term exposure to small doses [3].
DDT, like organochlorines, appears to associate preferentially with lipid-rich biological structures as a consequence of the very high partition coefficients of the compound in hydrophobic phases (hexane/water partition coefficient: log P = 4.96) [4], [5], [6]. Therefore, the phospholipids and, in particular, the biomembranes are good candidates as targets of DDT action and blood may serve as a carrier of the insecticide [7]. Up to today the DDT–phospholipid interaction has been studied by different techniques. A NMR study [8] of the interaction between DDT and lecithin suggested that the site of the interaction is localized in the lipid polar head. On the contrary, X-ray studies on oriented films and crystalline powders of DDT–phospholipid mixtures concluded that no interactions occurred [9], whereas fluorescence polarization studies on similar systems evidenced the effect of the interaction above the phase transition temperature of the lipid [10]. Moreover, it has been evidenced in the literature that the modifications induced in the membrane structure can play a role in the development of the DDT toxicity [11], [12].
In this paper, the interactions between DDT and multilamellar vesicles (liposomes) of DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) and DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine) were studied by means of differential scanning calorimetry (DSC) and Raman spectroscopy.
DMPC liposomes are widely used as a model system of biomembranes since lecithins are the major component of most mammalian biomembranes. On the contrary, DMPE liposomes are a useful model for nervous tissue cell membranes because in this kind of tissue cephalins are present in a significant amount and, consequently, their behaviour could be related to the neurotoxicity.
Both DSC and Raman spectroscopy have been proved to be very useful techniques to study the changes induced in model biomembranes by foreign substances. Indeed, liposomes exhibit a characteristic thermal behaviour by heating, showing a sharp endothermic gel to liquid crystal transition whose peak temperature and shape are strongly modified by interactions with other substances, thus reflecting the changes induced in the bilayer structure [13], [14], [15].
By plotting the intensity ratio of some characteristic Raman bands as a function of the temperature, sharp changes are observed in correspondence of the transition temperatures. The slope of the curve as well the difference between the values of the intensity ratios before and after the phase transition is related to the structural changes of the inner hydrophobic core in the liposome structure [14], [16], [17]. Combining DSC and Raman data, the type of the interaction, the depth of the penetration in the bilayer as well as the conformational changes in the hydrophobic chain structure, can be investigated.
Moreover, Raman spectroscopy offers a useful tool to examine also localized interactions, studying the behaviour of the vibrational stretching modes of each group.
Section snippets
Materials and methods
Synthetic DMPC and DMPE were obtained from Sigma Chemical Co. with purity guaranteed greater than 99% (TLC) and thus used without further purification. DDT is a Fluka Chemical Co. product and its purity was checked before the use by GC–MS technique and found greater than 98%. GC analysis was carried out on a Perkin Elmer AutoSystem XL gas chromatograph equipped with a Perkin Elmer TurboMass mass spectrometer and a PTE5 capillary column (30 m length and 0.32 mm i.d.), using He as carrier gas.
DMPC–DDT liposomes
Table 1 reports the values of Tm (main transition temperature), ΔH (enthalpy of transition) and ΔT1/2 (half width of the peak) measured in all the DMPC/DDT systems during the heating cycles, as well as Tmc (main transition temperature in cooling cycles). The DDT content ranges from 0.25% to 30.0% w/w and the corresponding molar ratio DDT/DMPC ranges from 4.8 × 10−3 to 8.2 × 10−1.
The values obtained for Tm, ΔH and ΔT1/2 in pure DMPC liposomes (23.7 °C, 25.7 kJ mol−1 and 0.5 °C, respectively) were in
Discussion
The structure of phospholipids bilayer and its thermal behaviour have been the subject of many theoretical and experimental works and some models have been proposed to explain the experimental results both in pure liposomes and in the presence of foreign substances [20], [21], [22]. It has been shown that in the presence of small, lipophilic substances, able to insert deeply into the hydrophobic bilayer, a simple solution model well agrees with the experimental data [20]. In this it is assumed
Conclusions
The effect of DDT on liposome structures is notable even in the presence of very small amounts of the foreign added substances. The changes observed in both DMPC and DMPE liposomes in presence of DDT denote that it can interact with the phospholipids and that the interaction mainly involves the external part of the hydrophobic core of the bilayer. This behaviour was evidenced particularly in the DMPE liposomes, as the insertion of DDT molecules into the central part of the bilayer seems to be
Acknowledgment
This work was supported by grants of Bologna University (Ricerca fondamentale orientata—ex 60%).
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