British Joumal of Dermatology (1975) 92, 143
Photochemistry of tetrachlorosalicylanilide and its relevance to the persistent light reactor A.K.DAVIES, N.S.HILAL, J.F.McKELLAR AND G.O.PHILLIPS Department of Chemistry and Applied Chemistry, University of Salford, Salford M5 4WT Accepted for publication 4 April 1974
SUMMARY
The photochemistry of 3,5,3'j4'-tetrachlorosalicylanilidc has been studied in solution under carefully controlled conditions. When irradiated in a buffered solution of pH 7 4 (physiological pH), three atoms of chlorine are liberated from the molecule instead of one as suggested by earlier photochemical work. From this observation a mechanism is proposed to explain the long-term photobiological effect of this compound in skin i.e. that of the persistent light reactor.
The photobiological effect on the skin of 3,5,3',4'-tetrachlorosaIicylanilide (T4CSA), originally employed as an antibacterial agent in soap, has been recognized for over a decade (Wilkinson, 1961). A curious feature, and one that has as yet no satisfactory explanation, is that the skin can remain highly sensitive to sunlight for months or even years after termination of all known contact with the sensitizer (Willis & Kligman, 1968). A patient with this long-term light sensitive condition has been described as a 'persistent light reactor' (Jillson & Baughman, 1963). T4CSA can have both short-term and long-term photobiological effects on the skin and the shortterm efFect appears readily explicable in terms of its reported photochemistry (Jenkins, Welti & Baines, 1964; Coxon, Jenkins & Welti, 1965). In aqueous alcoholic solutions of neutral pH, T4CSA is present in both non-ionic and anionic forms (pK = 5-6) but it is only the anionic form I (see below) that is photochemically active to near ultra-violet radiation.
(I)
The free radical II formed on photolysis has been shown to combine readily with y-globulin and protein in rat serum (Jenkins et al., 1964). It would thus appear likely that II is readily capable of combining with the protein of human skin. However the long-term sensitizing effect, as seen in the persistent light reactor, cannot be satisfactorily explained by this mechanism of a single photolytic Correspondence to: Dr. J. F. McKellar, Department of Chemistry and Applied Chemistry, University of Salford.
c
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step except, of course, by assuming that T4CSA is retained in the skin for very long periods of time (Willis & Kligman, 1968). MATERIALS AND METHODS
T4CSA was obtained from Eastman Kodak Ltd, and purified by repeated recrystallization from hot aqueous-ethanolic solution till a sharp melting point of 150-151 C was obtained (literature 149-151 C). Protamine sulphate was obtained from Sigma Co. Ltd, and used without further purification. The alcohols used (ethanol, propan-2-oI and ethylcne glycol) were purified by refiuxing under acid conditions with 2,4-dinitrophenylhydrazine. All other chemicals used were o f Analar' grade. The light source was a 220 W Hanovia UVS medium pressure lamp. This lamp has a strong emission line at 365 nm which can be isolated with a Chance OXi filter. The filter has an absorbance of less than 05 in the wavelength range 330-390 nm with minimum absorbance at 365 nm. This 365 nm irradiation is strongly absorbed by the photochemically active anionic form I of T4CSA (Coxon et al., 1965). The reaction cell was cylindrical in shape (diameter 35 cms; height 6-5 cms) and constructed of pyrex glass. The volume of solution irradiated in each experiment was 25 ml. During irradiation the solution was continuously stirred by having a small magnetic follower encased in PTFE within the cell and this follower was activated by a rotating magnet beneath the cell. All irradiations were carried out with the reaction cell in a thermostatically controlled bath at 25 C. Although earlier work (Jenkins et al.^ 1964; Coxon et a/., 1965) had shown that chloride ions are released on photolysis of the anionic form of T4CSA in aqueous alcoholic solution, it was not specified whether the irradiated solutions were either aerobic or anaerobic. Our preliminary experiments showed that chloride ions are released under both conditions but more rapidly so when the solution is anaerobic. Before each irradiation, therefore, virtually all the oxygen was removed by presaturating the solution with nitrogen containing less than 5 p.p.m. of oxygen. Also, for those experiments carried out with the solution buffered at physiological pH (pH = 74) the citric acid: disodium hydrogen phosphate mixture recommended by Hale (1965) was used. The fluorescence measurements were made with a Baird-Atomic 'Fluorispec' spectrofluorimeter. RESULTS AND DISCUSSION
Initially the photochemical experiments of Coxon et al. (1965) were repeated by Williams (1971) in these laboratories. 0-2% aqueous-alcoholic solutions of T4CSA were photolysed with 365 nm irradiation and the course of the reaction monitored by potentiometric titration of the chloride ions produced by the reaction (R^alkyl group); (2)
In agreement with Coxon et al. (1965) it was found that with a solution of pH 11, for example, one chloride ion was liberated per molecule of T4CSA photolysed. Thus there is no reason to doubt the validity of reaction (i) as a primary step in the photochemistry of T4CSA and, in our view, as a possible process for initiating the short-term photobiological effect. When, however, T4CSA was irradiated under the same experimental conditions but at a lower concentration (0-02" „) it was found that one molecule of T4CSA is not only capable of yielding one chloride ion but can, in fact, yield up to three chloride ions depending upon the initial pH of the solution. It should be noted that the experiments of Coxon et al. (1965), Williams (1971) and the experimental data shown in Fig. i (a) were carried out with unbuffered solutions, the pH of the solution thus decreasing during the course of the reaction due to the liberation of protons by reaction (2).
Tetrachlorosalicylanilide and the persistent light reactor
145
Of more significance with regard to the mechanism of the long-term photobiological effect of T4CSA, we believe, are the results shown in Fig. i (b). In this experiment the conditions were the same as in Fig. i (a) but the solution was buffered at physiological pH. It is seen that on irradiation three chloride ions were liberated per molecule of T4CSA photolysed.
20
40
60
20
60
Time of IrrodiatJon [min)
FIGURE I. Photolysis of T4CSA in nitrogen-saturated 2:3 propan-2-ol,water solutions, (a) unbuffered solutions, initial p H = O ) 4-0; • , 7 5 ; • , i i - 6 and n , 130. (b) bufFered solution, pH = A, 7 4.
This interesting observation suggests the following mechanism for the long-term effect of T4CSA. If, following reaction (i), the radical II attaches itself to the protein moiety by means of a direct chemical bond then this could obviously increase the retention time of the compound in the protein. Also, as we have shown here, if the protein-attached trichloro-dcrivative is still potentially photochemically active then the long term photochemical sensitivity receives a logical explanation. To test this theory the following experiment was carried out. T4CSA at a concentration of 2 x 10 ^* moI/1 was photolysed under neutral pH conditions in the presence of a simple protein-type compound, protamine sulphate (West et al.y 1966}. The concentration of the protamine sulphate was 0 4 % (by
146
A.K.Davies et al.
weight) and the solvent was a mixture of water and ethylene glycol, the protamine sulphate not being sufficiently soluble for the simpler alcohols to be used. The progress of the reaction was followed by spectrofluorimetry. This method was chosen because the photochemically active anionic form of T4CSA is quite strongly fluorescent with an emission maximum at about 450 nm. The corresponding T3CSA and T2CSA also fluoresce in the same wavelength region but more weakly so; TiCSA and salicylanilide itself are virtually non-fluorescent.
400
500 Wavelength (nm)
600
FIGURE 2. (a) Fluorescence from protamine-bound T4CSA formed after 15 min irradiation, (b) Absence of fluorescence after a further 30 min irradiation.
After irradiating for 15 min unbound T4CSA was removed by precipitating the protamine sulphate from solution with addition of propan-2-ol at - 7 8 C to avoid denaturation. The mixture was then centrifuged. The precipitate was retained and the supernatant tested for fluorescence emission. This procedure was repeated several times until the supernatant showed nofluorescenceemission indicating that all the chemically unbound T4CSA had been removed. The precipitated protamine sulphate containing the bound T4CSA was then redissolved in aqueous solution and its fluorescence emission is shown in Fig. 2. This result clearly indicates that radical-protein binding takes place on irradiation of T4CSA; a conclusion that is supported by the earlier experiments of Jenkins et al. (1964). When the solution was further irradiated for a period of 30 min it was found that all the fluorescence emission of the T4CSA bound complex had disappeared indicating photochemical conversion to the nonflourescent derivatives in agreement with the proposed mechanism.
Tetrachlorosalicylanilide and the persistent light reactor
147
ACKNOWLEDGMENTS
We thank Dr J.S.Gardiner, Medical Officer of ICI Ltd, (Pharmaceuticals Division) for his valuable advice during the coiirse ofthe work. REFERENCES CoxoN, J.A., JENKINS, P.P. & WELTI, D . (1965) The efFect of light on balogenated salicylanilides. Photochemistry and Phoxobiology, 4, 713.
HALE, L.J. (1965) Biological Laboratory Data. Chapman and Hall, London. JENKINS, F.P., WELTI, D . & BAINES, D . (1964) Photocbemical reactions of tetrachlorosalicylanilides. Nature, 201,
827. JiLLSON, O.F. & BAUGHMAN, R . D . (1963) Contact pbotodermatitis from bithionol. Archives of Dermatology, 88, 409. WEST, E.S., TODD, W.R., MASON, H . S . & VAN BRUGGEN, J . T . (1966) Textbook of Biochemistry, p. 299- Macmillan,
New York. WILKINSON, D.S. (1961) Patcbtest reactions of certain balogenated salicylanilides. British Journal of Dermatology, 73»2I3WILLIAMS, F.A. (1971) Photochemistry of tetrachlorosalicylanilide. M.Sc. Thesis, University of Salford. WILLIS, I. & KLIGMAN, A.M. (1968) Mechanism of the persistent light veactoi. Journal of Investigative Dermatology, 5I) 385-
Photochemistry of tetrachlorosalicylanilide and its relevance to the persistent light reactor A.K.DAVIES, N.S.HILAL, J.F.McKELLAR AND G.O.PHILLIPS Department of Chemistry and Applied Chemistry, University of Salford, Salford M5 4WT Accepted for publication 4 April 1974
SUMMARY
The photochemistry of 3,5,3'j4'-tetrachlorosalicylanilidc has been studied in solution under carefully controlled conditions. When irradiated in a buffered solution of pH 7 4 (physiological pH), three atoms of chlorine are liberated from the molecule instead of one as suggested by earlier photochemical work. From this observation a mechanism is proposed to explain the long-term photobiological effect of this compound in skin i.e. that of the persistent light reactor.
The photobiological effect on the skin of 3,5,3',4'-tetrachlorosaIicylanilide (T4CSA), originally employed as an antibacterial agent in soap, has been recognized for over a decade (Wilkinson, 1961). A curious feature, and one that has as yet no satisfactory explanation, is that the skin can remain highly sensitive to sunlight for months or even years after termination of all known contact with the sensitizer (Willis & Kligman, 1968). A patient with this long-term light sensitive condition has been described as a 'persistent light reactor' (Jillson & Baughman, 1963). T4CSA can have both short-term and long-term photobiological effects on the skin and the shortterm efFect appears readily explicable in terms of its reported photochemistry (Jenkins, Welti & Baines, 1964; Coxon, Jenkins & Welti, 1965). In aqueous alcoholic solutions of neutral pH, T4CSA is present in both non-ionic and anionic forms (pK = 5-6) but it is only the anionic form I (see below) that is photochemically active to near ultra-violet radiation.
(I)
The free radical II formed on photolysis has been shown to combine readily with y-globulin and protein in rat serum (Jenkins et al., 1964). It would thus appear likely that II is readily capable of combining with the protein of human skin. However the long-term sensitizing effect, as seen in the persistent light reactor, cannot be satisfactorily explained by this mechanism of a single photolytic Correspondence to: Dr. J. F. McKellar, Department of Chemistry and Applied Chemistry, University of Salford.
c
143
144
A.K.Davies et al.
step except, of course, by assuming that T4CSA is retained in the skin for very long periods of time (Willis & Kligman, 1968). MATERIALS AND METHODS
T4CSA was obtained from Eastman Kodak Ltd, and purified by repeated recrystallization from hot aqueous-ethanolic solution till a sharp melting point of 150-151 C was obtained (literature 149-151 C). Protamine sulphate was obtained from Sigma Co. Ltd, and used without further purification. The alcohols used (ethanol, propan-2-oI and ethylcne glycol) were purified by refiuxing under acid conditions with 2,4-dinitrophenylhydrazine. All other chemicals used were o f Analar' grade. The light source was a 220 W Hanovia UVS medium pressure lamp. This lamp has a strong emission line at 365 nm which can be isolated with a Chance OXi filter. The filter has an absorbance of less than 05 in the wavelength range 330-390 nm with minimum absorbance at 365 nm. This 365 nm irradiation is strongly absorbed by the photochemically active anionic form I of T4CSA (Coxon et al., 1965). The reaction cell was cylindrical in shape (diameter 35 cms; height 6-5 cms) and constructed of pyrex glass. The volume of solution irradiated in each experiment was 25 ml. During irradiation the solution was continuously stirred by having a small magnetic follower encased in PTFE within the cell and this follower was activated by a rotating magnet beneath the cell. All irradiations were carried out with the reaction cell in a thermostatically controlled bath at 25 C. Although earlier work (Jenkins et al.^ 1964; Coxon et a/., 1965) had shown that chloride ions are released on photolysis of the anionic form of T4CSA in aqueous alcoholic solution, it was not specified whether the irradiated solutions were either aerobic or anaerobic. Our preliminary experiments showed that chloride ions are released under both conditions but more rapidly so when the solution is anaerobic. Before each irradiation, therefore, virtually all the oxygen was removed by presaturating the solution with nitrogen containing less than 5 p.p.m. of oxygen. Also, for those experiments carried out with the solution buffered at physiological pH (pH = 74) the citric acid: disodium hydrogen phosphate mixture recommended by Hale (1965) was used. The fluorescence measurements were made with a Baird-Atomic 'Fluorispec' spectrofluorimeter. RESULTS AND DISCUSSION
Initially the photochemical experiments of Coxon et al. (1965) were repeated by Williams (1971) in these laboratories. 0-2% aqueous-alcoholic solutions of T4CSA were photolysed with 365 nm irradiation and the course of the reaction monitored by potentiometric titration of the chloride ions produced by the reaction (R^alkyl group); (2)
In agreement with Coxon et al. (1965) it was found that with a solution of pH 11, for example, one chloride ion was liberated per molecule of T4CSA photolysed. Thus there is no reason to doubt the validity of reaction (i) as a primary step in the photochemistry of T4CSA and, in our view, as a possible process for initiating the short-term photobiological effect. When, however, T4CSA was irradiated under the same experimental conditions but at a lower concentration (0-02" „) it was found that one molecule of T4CSA is not only capable of yielding one chloride ion but can, in fact, yield up to three chloride ions depending upon the initial pH of the solution. It should be noted that the experiments of Coxon et al. (1965), Williams (1971) and the experimental data shown in Fig. i (a) were carried out with unbuffered solutions, the pH of the solution thus decreasing during the course of the reaction due to the liberation of protons by reaction (2).
Tetrachlorosalicylanilide and the persistent light reactor
145
Of more significance with regard to the mechanism of the long-term photobiological effect of T4CSA, we believe, are the results shown in Fig. i (b). In this experiment the conditions were the same as in Fig. i (a) but the solution was buffered at physiological pH. It is seen that on irradiation three chloride ions were liberated per molecule of T4CSA photolysed.
20
40
60
20
60
Time of IrrodiatJon [min)
FIGURE I. Photolysis of T4CSA in nitrogen-saturated 2:3 propan-2-ol,water solutions, (a) unbuffered solutions, initial p H = O ) 4-0; • , 7 5 ; • , i i - 6 and n , 130. (b) bufFered solution, pH = A, 7 4.
This interesting observation suggests the following mechanism for the long-term effect of T4CSA. If, following reaction (i), the radical II attaches itself to the protein moiety by means of a direct chemical bond then this could obviously increase the retention time of the compound in the protein. Also, as we have shown here, if the protein-attached trichloro-dcrivative is still potentially photochemically active then the long term photochemical sensitivity receives a logical explanation. To test this theory the following experiment was carried out. T4CSA at a concentration of 2 x 10 ^* moI/1 was photolysed under neutral pH conditions in the presence of a simple protein-type compound, protamine sulphate (West et al.y 1966}. The concentration of the protamine sulphate was 0 4 % (by
146
A.K.Davies et al.
weight) and the solvent was a mixture of water and ethylene glycol, the protamine sulphate not being sufficiently soluble for the simpler alcohols to be used. The progress of the reaction was followed by spectrofluorimetry. This method was chosen because the photochemically active anionic form of T4CSA is quite strongly fluorescent with an emission maximum at about 450 nm. The corresponding T3CSA and T2CSA also fluoresce in the same wavelength region but more weakly so; TiCSA and salicylanilide itself are virtually non-fluorescent.
400
500 Wavelength (nm)
600
FIGURE 2. (a) Fluorescence from protamine-bound T4CSA formed after 15 min irradiation, (b) Absence of fluorescence after a further 30 min irradiation.
After irradiating for 15 min unbound T4CSA was removed by precipitating the protamine sulphate from solution with addition of propan-2-ol at - 7 8 C to avoid denaturation. The mixture was then centrifuged. The precipitate was retained and the supernatant tested for fluorescence emission. This procedure was repeated several times until the supernatant showed nofluorescenceemission indicating that all the chemically unbound T4CSA had been removed. The precipitated protamine sulphate containing the bound T4CSA was then redissolved in aqueous solution and its fluorescence emission is shown in Fig. 2. This result clearly indicates that radical-protein binding takes place on irradiation of T4CSA; a conclusion that is supported by the earlier experiments of Jenkins et al. (1964). When the solution was further irradiated for a period of 30 min it was found that all the fluorescence emission of the T4CSA bound complex had disappeared indicating photochemical conversion to the nonflourescent derivatives in agreement with the proposed mechanism.
Tetrachlorosalicylanilide and the persistent light reactor
147
ACKNOWLEDGMENTS
We thank Dr J.S.Gardiner, Medical Officer of ICI Ltd, (Pharmaceuticals Division) for his valuable advice during the coiirse ofthe work. REFERENCES CoxoN, J.A., JENKINS, P.P. & WELTI, D . (1965) The efFect of light on balogenated salicylanilides. Photochemistry and Phoxobiology, 4, 713.
HALE, L.J. (1965) Biological Laboratory Data. Chapman and Hall, London. JENKINS, F.P., WELTI, D . & BAINES, D . (1964) Photocbemical reactions of tetrachlorosalicylanilides. Nature, 201,
827. JiLLSON, O.F. & BAUGHMAN, R . D . (1963) Contact pbotodermatitis from bithionol. Archives of Dermatology, 88, 409. WEST, E.S., TODD, W.R., MASON, H . S . & VAN BRUGGEN, J . T . (1966) Textbook of Biochemistry, p. 299- Macmillan,
New York. WILKINSON, D.S. (1961) Patcbtest reactions of certain balogenated salicylanilides. British Journal of Dermatology, 73»2I3WILLIAMS, F.A. (1971) Photochemistry of tetrachlorosalicylanilide. M.Sc. Thesis, University of Salford. WILLIS, I. & KLIGMAN, A.M. (1968) Mechanism of the persistent light veactoi. Journal of Investigative Dermatology, 5I) 385-
