Binding of Sudan II and IV to lecithin liposomes and E. coli membranes: insights into the toxicity of hydrophobic azo dyes
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Sudan red compounds are hydrophobic azo dyes, still used as food additives in some countries. However, they have been shown to be unsafe, causing tumors in the liver and urinary bladder in rats. They have been classified as category 3 human carcinogens by the International Agency for Research on Cancer. A number of hypotheses that could explain the mechanism of carcinogenesis have been proposed for dyes similar to the Sudan red compounds. Traditionally, investigations of the membrane toxicity of organic substances have focused on hydrocarbons, e.g. polycyclic aromatic hydrocarbons (PAHs), and DDT. In contrast to hydrocarbons, Sudan red compounds contain azo and hydroxy groups, which can form hydrogen bonds with the polar head groups of membrane phospholipids. Thus, entry may be impeded. They could have different toxicities from other lipophilic hydrocarbons. The available data show that because these compounds are lipophilic, interactions with hydrophobic parts of the cell are important for their toxicity. Lipophilic compounds accumulate in the membrane, causing expansion of the membrane surface area, inhibition of primary ion pumps and increased proton permeability.
This work investigated the interactions of the amphiphilic compounds Sudan II and IV with lecithin liposomes and live Escherichia coli (E. coli). Sudan II and IV binding to lecithin liposomes and live E. coli corresponds to the Langmuir adsorption isotherm. In the Sudan red compounds – lecithin liposome solutions, the binding ratio of Sudan II to lecithin is 1/31 and that of Sudan IV to 1/314. The binding constant of the Sudan II-lecithin complex is 1.75 × 104 and that of the Sudan IV-lecithin complex 2.92 × 105. Besides, the influences of pH, electrolyte and temperature were investigated and analyzed quantitatively. In the Sudan red compounds – E.coli mixture, the binding ratios of Sudan II and Sudan IV to E.coli membrane phospholipid are 1/29 and 1/114. The binding constants of the Sudan II – and Sudan IV- E.coli membrane phospholipid complexes are 1.86 × 104 and 6.02 × 104. Over 60% of Sudan II and 75% of Sudan IV penetrated into E.coli, in which 90% of them remained in the E.coli membrane.
Experiments of Sudan II and IV binding to lecithin liposomes and live E. coli indicates that amphiphilic compounds may besequestered in thelecithin liposomes and membrane phospholipid bilayer according to the Langmuir adsorption law. Penetration into the cytosol was impeded and inhibited for Sudan red compounds. It is possible for such compounds themselves (excluding their metabolites and by-products)not result directly in terminal toxicity. Therefore, membrane toxicity could be manifested as membrane blocking and membrane expansion. The method established here may be useful for evaluating the interaction of toxins with membranes.
KeywordsLecithin Membrane Phospholipid Langmuir Adsorption Isotherm Amphiphilic Compound Liposome Solution
Many toxic chemicals, e.g. drugs and harmful additives that are formed or utilized in daily life, accumulate persistently in biological systems. Sudan red compounds I – IV, which are hydrophobic azo dyes, are still used as food additives in some countries because of their low cost and bright color  but they have been shown to be unsafe, causing tumors in the liver or urinary bladder  in rats. Sudan I – IV have been classified as category 3 carcinogens to humans by International Agency for Research on Cancer , and the European Union does not allow Sudan dyes as food additives [4, 5]. A number of hypotheses that could explain the mechanism of carcinogenesis have been proposed for dyes similar to the Sudan red compounds .
Traditionally, investigations of lipid peroxidation damage by Sudan red compounds have been performed only on mouse and rabbit sera. Recently, Stiborova  compared experimental animals with humans and determined that cytochromes P450 and live microsomes are essential for extrapolating animal carcinogenicity data to human health risk assessment. Although Sudan dyes are indirect carcinogens, they generate metabolites that are converted to several active mutagens and carcinogens in humans [8, 9], such as aniline or 1-amido-2-naphthol, which can be metabolized by hepatic microsomes into benzene and naphthol. These end products can then combine with DNA and RNA to destroy cells .
To cause terminal toxicity, a chemical must penetrate the cell so as to affect the function of a target biomolecule. However, the cell membrane, which consists of a lipid bilayer and membrane proteins along with sugar polymers as a support layer, acts as a natural barrier and often plays a protective role in normal cellular activity. Cell membranes perform a number of essential functions such as transport of nutrients, ion conduction, signal transduction, cell migration, etc. The plasma membrane represents the primary permeability barrier of a cell and therefore regulates the inflow of substrates and the outflow of products. Normal cell activity will be seriously compromised if the cell is persistently exposed to a toxic medium. The partitioning of hydrocarbons in membrane buffer systems has been studied [11, 12]. The available data show that because of their lipophilic character, these compounds interact with hydrophobic parts of the cell, and this is important in the mechanism of toxicity. Lipophilic compounds accumulate in the membrane, causing "expansion" of the membrane surface area, inhibition of primary ion pumps and increased proton permeability .
The interaction of lipophilic compounds with the phospholipid bilayer causes dramatic changes in cell membrane structure. For example, accumulation of such compounds in the hydrophobic part of the membrane will disturb the interactions between the phospholipid acyl chains, modifying membrane fluidity and eventually leading to swelling of the bilayer. Furthermore, the lipid annuli that surround membrane-embedded proteins will also change, possibly altering protein conformations . Because Sudan red compounds have groups capable of forming hydrogen bonds, their toxic effects on the membrane should differ from those of apolar hydrocarbons.
In this study, we have investigated the interactions of the amphiphilic compounds Sudan II and IV with both lecithin liposomes and live E. coli membrane phospholipids. Polar compounds with hydrogen-bond forming groups e.g. -OH, = O, -NH2, -N = N- have been shown to accumulate on the external surfaces of membranes according to the Langmuir adsorption isotherm . Entry into the liposome center and cytosol is impeded for amphiphilic compounds, so it is possible for them not to cause the terminal toxicity. However, their metabolites and by-products could be terminally toxic, possibly formed by exo-enzyme or photocatalysis [15, 16, 17]. Membrane toxicity could therefore be manifested as membrane blocking and membrane expansion. The method established in the present work could be valuable for evaluating the interaction of toxins with membranes and will be applicable to research in the environmental and life sciences, and medicine.
Results and discussion
Absorption spectra and structural analysis
In order to investigate the interaction of these compounds with lecithin liposomes, the mixtures must be centrifuged. However, the centrifugal pellet appears light yellow in ethanol solution, so the background interference was estimated by a dual-wavelength correction  as follows:
and λ1 and λ2 are the measurement wavelengths. λ1 was selected at 400 nm and λ2 at 495 nm for Sudan II and 515 nm for Sudan IV. The symbol AS/Lλ 2is the real absorbance of a Sudan compound adsorbed by the lecithin liposomes; Aλ 1and Aλ 2are the absorbances of the above solutions at λ1 and λ2. Both a and b are correction constants. A L λ 1and A L λ 2are the absorbances of a lecithin-ethanol solution at λ1 and λ2 without any Sudan compound and A s λ 1and A S λ 2are those of a Sudan compound in ethanol solution without liposomes. For Sudan II, a = 1.66 and b = 0.642; for Sudan IV, a = 2.73 and b = 0.621. These values were calculated from curves 1 – 3 in Figure 1.
Interaction of liposomes with the Sudan red compounds
Standard curves for determination of Sudan red compounds in ethanol and pH 7.0 phosphate buffer solution (PBS)
Upper limit, nM
Wave- length, nm
Linear correlation coefficient
A = 0.0155CL (2)
A = 0.0121CL - 0.001 (3)
A = 0.0318CL - 0.0018 (4)
A = 0.0196CL- 0.0007 (5)
The phospholipid micelle will bind a polar hydrophobic organic compound via polar bonds, e.g. ion pairing, hydrogen bonds and dispersion forces, corresponding to the molecular monolayer adsorption model. The units of various non-covalent bonds determine the binding type, binding position and interaction strength of an organic compound . With respect to biological activity, non-covalent interactions are common because they are non-specific. A more complicated molecule with more active polar groups, e.g. Sudan IV, often forms a more stable complex. Thus, Sudan IV binding to lecithin liposomes cannot be reversed easily. The binding of Sudan II and IV by liposomes indicates thatentry into the liposome center may be impeded for them, as further argued on the basis of the following experiments.
Effect of ionic strength, pH and temperature
Analysis of E. coli growth state
Distribution of Sudan red compounds in E. coli
Interaction of Sudan red compounds with cells
Penetration into the cytosol is impeded for Sudan red compounds, and it is possible for them not to cause the terminal toxicity. This work provides a useful experimental strategy for the quantitative assessment of potential membrane toxicity of hydrophobic compounds with polar groups and recognition of the terminally toxic substances. This technique may be useful in a variety of research fields including environmental and life sciences, and medicine.
Materials and instruments
Lecithin (Sinopharm Cheam), 20 mg/ml, was suspended in deionized water and then dispersed ultrasonically at maximal amplitude at 4°C for 5 cycles of 15 s interspersed with 45 s periods of rest . Stock solutions of Sudan II (0.80 mM) and Sudan IV (0.10 mM) (Shanghai Chemical Reagents Center of National Medicine Group of China) were prepared in absolute ethanol. Britton-Robinson (B-R) buffers at pH 2.18, 3.13, 4.16, 5.72, 7.16, 8.69, 9.62 and 10.38 were prepared and were used to examine the effect of solution pH on the interactions of the Sudan red compounds with liposomes. A pH 7.0 PBS was prepared by mixing 0.8% NaCl, 0.02% KCl, 0.0144% Na2HPO4 and 0.024% KH2PO4 and was used to re-suspend bacterial pellets. Sterile L-B nutrient medium containing 1.0% peptone, 0.5% yeast extract and 1.0% NaCl was prepared and used for culturing E. coli. The E. coli strain used was provided by the Environmental Biological Laboratory of Tongji University.
A Model Lambda-25 spectrometer (Perkin-Elmer Instruments, USA) with version-2.85.04 UV-WinLab software was used to record the absorption spectra of the Sudan solutions and to measure turbidities and absorptions. A Model JY92-II Ultrasonic Cell Disruptor (Ningbo Xinzhi Instruments, China) was used to disrupt the bacterial cells. A Model Anke TGL-16C High-speed Centrifuge (Shanghai Anting Sci. Instruments, China) with 7.0 ml plastic tubes was used to separate the liposomes and cell membranes. Solution pHs were measured with a Model pHS-25 Acidity Meter (Shanghai Precise Instruments., China). A Model TS-030 Thermostat Cycling Bath (Shanghai Yiheng S&T, China) was used to maintain the experimental temperature between room temperature and 70°C. A Model SK3300H Ultrasonic Cleaning Device (Shanghai Ultrasonic Cleaning Instruments, China) was used to accelerate the dissolution of solutes and to disperse the phospholipids. A Model CHA-2 Thermostat Gas Bath Vibrator (Jintan E-tong Electrons, Jiangsu, China) and a Model SPX-80BS-II Biochemical Incubator (Shanghai Cimo Medical instruments, China) were used to culture the bacteria. A Model Optima 2100 DV ICP- OES (PerkinElmer Instruments, USA) was used to measure the phosphorus content of the membrane phospholipids. A Model GM-100 diaphragm vacuum pump (Tengda Filtration Devices of Tianjin, China) was used to volatilize the organic reagent in the membrane extracts to yield the E. coli membrane residue.
In-vitro interaction of Sudan red compounds with liposomes
A series of mixtures containing 50 μl of liposome suspension and aliquots of 0.8 mM Sudan II (0 to 120 μl) were diluted to 5.00 ml with deionized water. After mixing for 10 s in the ultrasonic cleaning device, they were centrifuged for 20 min at 7000 × g. Each pellet was dissolved in 5.00 ml of ethanol. A dual-wavelength spectral correction method  was used to eliminate background interference. The solutions were measured at 400 and 495 nm, and a standard curve of Sudan II in the liposomes was generated. Applying the same procedures, a standard curve prepared from aliquots of the 0.1 mM stock solution of Sudan IV was used to measure the interaction of Sudan IV with the liposomes, except that data were collected at 400 and 515 nm.
Culture of E. coli and determination of membrane phospholipids
E. coli was selected as the model microorganism for further experiments with the Sudan dyes. Several cycles of the E. coli strain were grown in 250 ml of L-B nutrient medium in culture bottles in the thermostated gas bath vibrator at 37°C. Bacterial growth was monitored by turbidimetric measurement at 600 nm. The E. coli cultures were also treated with MTT  and the absorbance measured at 555 nm to determine the number of living cells.
After the E. coli growth reached stationary phase, 5.00 ml of the zymotic fluid from the culture bottle was placed in a 7-ml centrifuge tube. The sample was centrifuged for 5 min in 3500 × g to sediment the bacteria, and the yellow supernatant was removed. The pellet was resuspended with PBS (2.5 ml), mixed well, and centrifuged again to obtain a pellet. The pellet was suspended and dispersed in PBS (2.5 ml) by ultrasonication for 10 × 5 s at 200 w interspersed by 10 s intervals of rest. After centrifugation, the supernatant was removed, the pellet was dissolved in 2.50 ml dichloromethane and the organic phase was volatilized under vacuum. The dried residue was treated  with 4 ml H2SO4, 4 ml HCl and 5 ml water to prepare a clear solution. The concentration of phosphorus was determined by ICP-OES . From this, the molarity of the membrane phospholipid species in the E. coli culture medium was calculated.
Interactions of Sudan red compounds with E. coli
The authors sincerely thank the National Natural Science Foundation of China (No. 20477030) and the Shanghai Fundamental Research Projects (No. 04JC14072 and 05JC14059) for financially supporting this work.
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