Food, Drug, and Cosmetic Dyes: Biological Effects Related to Lipid
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Proc. Natl. Acad. Sd. USA Vol. 74, No. 7, pp. 2914-2918, July 1977 Cell Biology Food, drug, and cosmetic dyes: Biological effects related to lipid solubility (molluscan neurons/toxicity/uncouplers/anticoagulants/brain uptake) HERBERT LEVITAN Department of Zoology, University of Maryland, College Park, Maryland 20742 Communicated by Harry Grundfest, April 29,1977 ABSTRACT Food, drug, and cosmetic dyes of the xanthane system was used previously to provide insights into the cellular type (analogs of fluorescein) were applied to isolated molluscan mechanism of action of nonnarcotic analgesics in vertebrates ganglia and changes in the electrophysiological properties of (10, 11) and it was hoped it might serve a similar function for identified neurons were monitored. The synthetic coloring agents increased the resting membrane potential and conduc- the current problem. The ganglion was isolated from the ani- tance of the neurons in a dose-dependent manner by increasing mal, pinned to Sylgard (Dow Corning, Midland, MI) in the the potassium permeability of the membrane relative to that bottom of a small plastic dish (5 ml) and usually bathed in a of other ions. The relative activity of these anionic dyes was low-potassium physiological saline consisting of 500 mM NaCl, highly correlated with their lipid solubility. The structure-ac- 1 mM KCI, 10 mM CaC12, 50 mM MgCl2, and 10 mM Tris-HCl tivity study of the effects of the dyes on molluscan neuro- at pH 7.8. The potassium concentration was increased by iso- physiology provides a basis for estimating the toxicity and brain tonic substitution for sodium. The capsule enveloping the uptake of the dyes in vertebrates, and predicting their effects ganglion was cut, exposing large, previously identified neurons on metabolism and blood clotting. (12). These neurons were impaled with double-barreled mi- Synthetic dyes have been a constituent of foods, drugs, and cropipettes with tip diameters of 2 Asm or less, and filled with cosmetics for more than 100 years, and the safety of such ad- 3 M KC1. The neuronal membrane potential was monitored ditives has been of concern for almost as long (1). The intended with one barrel using standard electrophysiological technique function of these additives has been to enhance the physical (12). The second barrel was connected to a source of constant appearance of a product, and some proof of their safety, which current pulses so that total membrane conductance could be has in the past been either assumed or ignored, is now required monitored. in many countries before they can be incorporated into items The compounds selected for testing belong to the xanthene intended for human consumption (1-5). Despite such precau- group of dyes and are derivatives of fluorescein (Table 1). tions several coloring agents were used for decades before they Fluorescein itself is frequently used in angiography to visualize were ruled unsafe, and the use of other dyes is currently under the blood vessels of the eye (17) and some of its derivatives are review because deleterious effects have been discovered or are used to color foods and drugs (4). The dye to be tested in the suspected (1, 5). Interest in the biological activity of artificial current system was dissolved in the physiological saline just prior colorants has recently been renewed following the claims that to use, and was applied to the impaled neurons by exchanging a red food dye (FD&C Red No. 2) is carcinogenic (6) and that the normal physiological medium for one containing the some food additives, and colorants in particular, may be re- dye. sponsible for producing behavioral changes and learning disabilities in children (e.g., hyperkinesis, minimal brain dis- RESULTS function) (7-9). The application of the fluorescein dyes to the molluscan ganglia The present study was undertaken in order to determine (i) produced a rapid increase in the membrane potential and whether some types of synthetic dyes, which are added to foods conductance of the impaled neuron (Fig. 1). The amplitude of and drugs purely for cosmetic purposes, are in fact biologically the response to a particular dye was variable and dependent active compounds, and (ii) the physicochemical properties of upon several factors, including the identity of the neuron im- the compounds responsible for their activity. It was found that paled, the animal, the time of year the experiment was per- xanthane type dyes alter the physiological characteristics of formed, and the number of previous exposures to that dye or invertebrate neurons and that their biological activity is highly others. Because of desensitization in the response to repeated correlated with the compounds' lipid solubility. The struc- dye applications and/or a slow irreversible decline in the ture-activity study of the dyes' effects on neurons provided a membrane potential produced by some of the dyes, complete basis for estimating their toxicity and brain uptake in verte- dose-response curves were rarely obtained. In order to have a brates, and predicting their effects on metabolism and blood common base with which to compare the relative effectiveness clotting. of the several dyes, the response of a neuron to each dye was compared with its response to a preceding and subsequent METHODS AND MATERIALS application of 12 mM salicylate, an organic anion of related in Experiments were performed primarily on the buccal ganglia structure known to produce similar effects this system of the marine mollusc Navanax inermis (obtained from Pacific (18-21). This concentration of salicylate produces half the Biomarine Supply Co., Venice, CA). This invertebrate nervous maximum change in membrane potential and conductance and its effects are reversible. The concentration of a particular dye Abbreviations: FD&C denotes approval by the United States Food and that produced an increase in membrane potential equal to that Drug Administration for use in food, drugs, and cosmetics; D&C, ap- produced by 12 mM salicylate was considered the concentration proved for drugs and cosmetics. that would produce half the maximum change in membrane 2914 Downloaded by guest on September 29, 2021 CdYBiology:BgUvitin Proc. Nati. Acad. Sci. USA 74 (1977) 2915 Table 1. Biological activity and partition coefficient of fluorescein analogs R2 NaOm Ri1 cOONa Predicted values Colour Index R <Rl Cso, mM LD5,i (1971) R, Ob- Pre- I50,g F,h mmol/ BUI, Compounda no.b R1 R2 R3 R4 R5 log Pc servede dictedf mM mM kg % 1. Fluorescein (D&C Yellow No. 8) 45350 H H H H H -4.77 200 300 50* 3200* >10* * 2. D&C Orange No. 9 45365 H Cl H Cl H -3.77 38 11* 1000* >10* 2 3. Eosin 2J (D&C Orange No. 6) 45370 H Br H Br H -3.02 22 4 400* >10* 4 4. Erythrosin Y (D&C Orange No. 11) 45425 H I H I' H -2.46 10 2 200* >10* 10 5. Eosin B 45400 H Br NO2 Br NO2 -2.18d 8 6 1 135* >10* 14 6. D&C Red No. 25 45366 H Cl Cl Cl Cl -1.97 - 5 0.9 105* >10* 19 7. Eosin Y (D&C Red No. 23) 45380 H Br Br Br Br -1.27 4 2 0.3 47 >10* 40 8. Erythrosin B (FD&C Red No. 3) 45430 H I II I -0.15d 0.2 0.3 0.06 10 8 82 9. Ext. D&C Red No.5 45435 di-Cl I I I I 1.44 0.03 0.006 2 2 86* 10. Phloxine B (D&C Red No. 28) 45410 . Cl Br Br Br Br 2.02 0.01 0.01 0.003* 1 1 65* 11. Rose Bengal 45440 Cl I I I I 3.14 0.002 0.0005* 0.2* 0.6 25* a FD&C denotes approval by the United States Food and Drug Administration for use in foods, drugs, and cosmetics; D&C, drugs and cosmetics; Ext., external. Fluorescein, Eosin B, Eosin Y, Phloxine B, and Rose Bengal are certified dyes obtained from Matheson, Coleman, and Bell, Norwood, OH; Erythrosin B was a gift from M. Chin, Stange, Co., Chicago IL. b The Colour Index number provides unique identification of the compounds (4). C With the exception of compounds 5 and 8, the octanol/water partition coefficients, P, of the ionized form of the molecules were calculated by applying the additive-constitutive rules established by Hansch et al. (13). d Octanol/water partition coefficients of ionized form of these molecules determined experimentally by Bio- and Pharmaceutical Analysis Section, Midwest Research Institute, Kansas City, MO. Sodium was the co-ion in all cases. The pKa values of Eosin and Errthrosin B were found to be 4.95 and 5.04, respectively. e C5o = concentration, estimated from experimental observations, that would produce halfthe maximal increase in membrane potential ofidentified molluscan neurons. f C50 predicted from least squares fit of data points (Eq. 1). g Iso = concentration that should inhibit oxidative phosphorylation ofisolated rat liver mitochondria by 50%, predicted from an equation describing similar activity of salicylate analogs (see text). 'h F = concentration that should dissolve a standard clot of human plasma in 24 hr, predicted from an equation describing similar activity of salicylate analogs (14). LD5o = concentration that should kill 50% ofthe mice that receive an intravenous administration, predicted from an equation describing similar activity of benzoate analogs (15). BUI = Brain uptake index or-ratio of concentration of organic anion to tritiated water which should be found in rat brain to that injected into the internal carotid artery 15 sec earlier (16). * The partition coefficient for these compounds lies outside the range of data from which the equation was originally calculated.