Pholochnnicrri. awl Phorohiolouv. Vol. 28. pp. 59 1-594. 0 Pergarnon Press Ltd.. 1978. Printed in Great Britain
PHOTOCHEMICAL REACTIONS OF CHLORPROMAZINE; CHEMICAL AND BIOCHEMICAL IMPLICATIONS 1. ROSENTHAL*.E. BEN-HUR,A. PRAGER and E. RIKLIS Israel Atomic Energy Commission. Nuclear Research Center-Negev. Beer-Sheva. Israel (Received 7 March 1978; accepted 13 March 1978)
Abstract-The photoexcited chlorpromazine reacts with methanol to yield promazine and 2-methoxypromazine by two different reaction pathways: hydrogen atom abstraction and nucleophilic attack. respectively. When the photoexcitation of chlorpromazine is performed in the presence of protein or nucleic acids, chlorpromazine binds to the biopolymer. This binding is drastically pH-dependent and correlates to the phototoxic effect exhibited in chlorpromazine-photosensitization of E. coli. No photodynamic damage of E. coli attributed to CPZ-sensitization of molecular oxygen could be detected.
INTRODUCTION
Negev, Israel. The deuterated methanol was purchasea from Roth OHG. Chlorpromazine(CPZ.1a) is the major representative The irradiations of CPZ in methanol were carried out of phenothiazine-type drugs employed in psycho- at room temperature in cylindrical Pyrex cells provided therapy (Forrest, 1974). The abnormal photoreactions with greaseless stopcocks. set in a "merry-go-round" apparatus. Hanovia 450 W high pressure mercury vapour which accompany CPZ treatment such as exaggerated lamp, cooled internally with running water and jacketed sensitivity t o sunburn, hyperpigmentation of the skin in a Pyrex filter ( I 1 300nm) was employed as an irrddiaand ocular opacity have justified investigations of its tion source. In a typical experiment, a solution of 20mg photochemical properties. In oxygenated aqueous CPZ. HCI dissolved in 4 m/ solvent was thoroughly degassed by several freeze-thaw cycles. The solution was exc = 254 nm) under- irradiated for 2 h followed by removal of the solvent under solutions, photoexcited CPZ (i goes photooxidation via its cation radical t o yield vacuum. The reaction products were separated by preparaCPZ-5-oxide (Iwaoka and Kondo. 1974; Felmeister tive thin layer chromatography (Merck PSC-Fertig-platten and Discher, 1964) in addition t o several other oxi- Kieselgel 60F254. eluted by a mixture of benzene, methdation products (Felmeister and Discher, 1964; anol, ammonium hydroxide, 6 : 14:O.I). For biological irradiations, the samples were exposed Huang and Sands, 1964). When the photooxidation to two tubular fluorescent black-light lamps (Lamp No. was dye-sensitized (i. exc > 530 nm), the primary 50058, U.V. Products, Inc.) which emit in a wide band chemical modification was the cleavage of the N-side ( 3 W 0 0 n m ) with a maximum at about 360nm. The incichain (Rosenthal et a/.. 1977). In the absence of dent flux on the sample was 2OJ/m*/s, as measured by a black light meter (model J-221. UV Products. Inc.). oxygen, the UV-irradiation of CPZ provoked the Binding of CPZ ro hiopolyrners. An irradiated aqueous cleavage of the chlorine function at position 2. This solution ( I m/) of bovine serum albumin (IO'Z,) and process resulted in the formation of promazine, 2-sub- 3H-CPZ (1%) was gel filtered through a Sephadex G-25 stituted promazines (Huang and Sands, 1967; Grant column (1.4 x 15cm). The elution was done with water and Greene, 1972; Davies et ul., 1976) and polymeric and 2 m / fractions were collected. The radioactivity was determined in I m/ samples in a dioxane-based cocktail material (Huang and Sands, 1967). Although earlier and counted in a Packard liquid scintillation counter. work suggested an ionic mechanism for this reaction E. coli cells exposed in the presence of ('H)-CPZ were (Grant and Greene. 1972). a more recent report washed by centrifugation, and precipitated with 5% cold explained the formation of these products by radical TCA(trich1oroaceticacid). The precipitate was dissolved in 0.1 N NaOH and heated to 100°C for 30min to hydrolyze recombinations (Davies et al.. 1976). RNA. DNA and proteins were precipitated with cold TCA, It was the purpose of this study to clarify the and DNA extracted by heating the precipitate in 5% TCA mechanistic aspects of the photochemistry of C P Z to 100'C for 10 min. The remaining protein pellet was dissolved in 0.1 N NaOH. The labeled CPZ bound to the and to extend its reaction t o biological systems. various macromolecular fractions was determined by liquid scintillation spectrometry. Control experiments indiMATERIALS AND METHODS cate that this procedure does not induce significant release CPZ. HCI, promazine. HCI. 2-methoxypromazine and 2- of bound CPZ. Bacterial cell growrh and suruiual. Bacteria, E . coli CS chlorphenothiazinewere kindly donated by Rhone-Poulenc 4033 (wild type) and a repair deficient double mutant E. Co.. and were used without further purification. (3H)-CPZ coli CS 4034 uur A6B5 (both endo I ) were grown to a was a commercial product from Nuclear Research Center- density of about 1 x lo9 cells/m/ in minimal medium, harvested by centrifugation and resuspended in phosphate buffered saline pH = 7 containing 100pg/m/ CPZ. After *Also at the Department of Chemistry, The Weizmann about IOmin at room temperature, which was sufficient for maximal sensitization, the cells were irradiated with Institute of Science, Rehovot, Israel. 59 1
1. ROSENTHAL pr a/.
592
near UV light. serially diluted. and plated on nutrient agar. Viability counts were made after 16 h incubation at 37°C. RESULTS AND DISCUSSION
Chernical reactions of photoexcited CPZ Initially, we investigated the photochemical reaction of CPZ in dry methanol in the absence of oxygen. ,CH3 ICH,),
N,
(a)
R =CL
(b) The isotopic composition of the methoxy group in Ic is identical with that of the original methyl group in the methyl alcohol. Thus, no hydrogen scrambling occurs along the reaction pathway between methyl and hydroxyl hydrogen as could be expected for the suggested all-radical mechanism (Davies et al., 1976). An ionic, photonucleophilic substitution of the chlorine by methoxy group is in agreement with this observation
PZ - C1* Ia
+ CH30--*
PZ - OCH, Ic
+ CI-
Alternatively, heterolytic photofission of CPZ to give PZ' and C1- followed by ionic recombination of promazinyl cation and methoxy anion might explain this I resu It. This mechanistic scheme is similar to the reported The direct photolysis of CPZ(1a) (1 > 300nm) resulted in almost total conversion to promazine (Ib) photoreduction and nucleophilic photosubstitution of and 2-methoxypromazine (Ic), in addition to minor halides in haloaromatic compounds (Pinhey and amounts of decomposition products. Ib and Ic were Rigby, 1969). separated by chromatography and identified unBiochernical reactions of photoexcited CPZ ambiguously by spectroscopic methods (IR, UV. MS, The photoinduced reaction of CPZ with biomoleNMR) and comparison with authentic samples (Charpentier. 1951. 1952). Control experiments showed that cules was first tested in oifro with bovine serum albuunder identical irradiation conditions, Ib and Ic were min (BSA). The elution profile of BSA after near UV photostable. that is both of them originated indepen- irradiation in the presence of 3H-labeled C P Z is dently from CPZ. It was. however. noted that the shown in Fig. I. BSA was eluted in the first peak presence of N-alkyl side chain is not a critical require- and evidently binds the drug as a result of irradiation. ment for this reaction. Thus, 2-chlorphenothiazine In our experimental conditions, up to seven CPZ under similar reaction conditions was converted to molecules could be bound to one molecule of BSA. phenothiazine (9004) and 2-methoxyphenothiazine (ca Two additional radioactive peaks of intermediate mol wt between BSA and C P Z are photoproducts of 2%). Valuable information regarding the mechanism involved could be obtained by performing the reaction in selectively labeled methanol, under otherwise identical conditions, followed by mass-spectroscopic analysis of the reaction products. Thus. CHIOH. CH30D. C D 3 0 H and C D 3 0 D were separately employed as solvents. The results are summarized in Table 1. These results show that: (a) The methyl group of the solvent is the exclusive source for the hydrogen atom which replaced chlorine. This observation suggests a homolytical cleavage of the carbonxhlorine bond in the photoexcited C P Z followed by the hydrogen atom abstraction from the solvent by the promazinyl radical: P Z - Cl* + PZ. Ia
+ CH30H
--*
PZ - H Ib
+ CHIOH
Table 1. Mass spectroscopic analysis of products Product ratio Reaction solvent CHjOH CH3OD CD3OH CD30D
1b:Ic 1 :0.5 1 :0.5
I :0.8 I :0.8
Molecular weight 2-MethoxyPromazine promazine 284 284 285 285
314 314 317 317
Figure 1. Elution profile of BSA irradiated in the presence of 'H-CPZ. Free jH-CPZ was eluted in fractions 25-30 (not shown). NUV is near UV light (300-400nm).
Photochemistry of chlorpromazine
PH
1.0
1.5
.
DNA
A
RNA
?ROTEIN
D
* /
I
I0
/
10 IRRADIAIION
/
. so 1lME
40
(mil)
593
quently repair it. Obviously, there is an additional possibility that endonuclease itself is photoinactivated by CPZ, thus explaining the large shoulder region in the survival curve. Results obtained with human adenovirus 5 (Day and Dimattina, 1977) indicate that CPZ-induced damage in DNA is repairable, therefore, the latter possibility seems most likely. In any event, the biological significance of CPZ-induced damage in DNA was evidenced by its mutagenic effect as established separately by the Ames' test (Ames et al., 1975). These results will be published separately (Green et al., in preparation). Identical phototoxic effects of CPZ as reflected in the survival curves were obtained by exposure in the absence or presence of oxygen (Fig. 3). Since CPZsensitized photoprcduction of singlet molecular oxygen has been documented (Davies et al., 1976), this observation excludes a possible photodynamic destruction due to excited oxygen. Additional support for this conclusion was provided by control experiments performed in the presence of oxygen and competitive quenchers of '0,such as histidine or DABCO. In concentrations up to mM, these compounds had no effect on the photosensitization of E. coli by CPZ (Fig. 3). In this regard, it is noted that photohemolysis of erythrocytes sensitized by CPZ
Figure 2. Binding of 'H-CPZto macromolecules in E. coli. DMF(P) is the dose modifying factor for light-induced binding of CPZ to proteins.
CPZ. BSA-free. Similar results were obtained when DNA served as substrate. Thus, 3 h exposure resulted in binding of 8"/, of free CPZ to DNA, compared with 8% binding following 1.5 h exposure with BSA (Fig. 1). Photoinduced binding of CPZ to DNA, RNA and proteins in uiuo was established by using E. coh cells. The results (Fig. 2.) show that the major amount of drug binds to proteins as calculated on a per cell basis. The biological effect of this photochemical binding is reflected in the survival of E. coli cells (CS-4033, wild type). Near UV light or CPZ, when applied separately, did not affect the survival of the bacterial cells. However, the simultaneous exposure to both factors led to an exponential decrease in survival after an initial shoulder region (Fig. 3). Control experiments showed that preirradiated CPZ added to a suspension of E. coli cells did not affect their survival. Since part of the photoinduced binding of the drug is to DNA, it was of interest to compare the sensitivity of the wild type bacteria to isogenic mutant cells deficient in UV-specific endonuclease responsible for repair of DNA damage (E. coli CS-4034). It seems that the deficiency in this endonuclease increases the sensitivity of the mutant cells to photoexcited CPZ only slightly. Thus, the contribution to cell lethality of CPZ-binding to DNA is either of minor importance, or the endonuclease enzyme is unable to recognize the CPZ-induced damage in DNA and conse-
\
bO3b
\'
Figure 3. Survival curves of irradiated E . coli cells in the presence of CPZ at pH 6.5 and 7.0. CS-4033 -0-, -4under nitrogen; -0-, +under air; -Xin the presence of histidine and air. CS-4034 -A-, -A- under air. Irradiation without oxygen was done by bubbling nitrogen through the cell suspension for 30min prior to' and during near UV exposure.
I. R ~ E N T H AerL ul.
594
also pi.oceeds by a non-singlet oxygen pathway (Nilsson cr af.. 1975). We found that the phototoxic effect of CPZ on E. coli is drastically controlled by the pH of the sohtion. Thus, a decrease in pH from 7.0 to 6.5 enhances the photosensitivity of the cells to a very large extent (Fig. 3) and causes a suppression of any photochemical linkage to nucleic acids in favour of that to protein (Fig, 2). Since E. coli is hardly affected by this change in pH, a pH-dependent photochemistry of C P Z must be responsible for this effect. It is interesting that the same result obtained in bufferedH 2 0 at pH = 6.5 could be duplicated in unbuffered D2° Cmeasured pH which corresponds to a pD value of 6.5 (Thomson, 1963)]. An increased reaction rate in D 2 0 as compared to H 2 0 medium has been-_
'"*
suggested as a test for singlet oxygen (Merkel a;Td Kearns. 1973). The drastic pH effect observed in the CPZ-photosensitization warrants against overlooking this parameter in using this test for singlet oxygen diagnosis. These results clearly indicate that the photoexcited C P Z exhibit strong toxic effects in uiuo due to its binding to biomacromolecular components. Of particular significance is the large extent of binding to proteins, which might thus generate a new antigen. Such a process can provide the explanation for CPZinduced photoallergic syndrome. Acknowledgements-We thank Rhone-Poulenc Company for supplying chlorpromazine. HCI, promazine. HCI. 2methoxypromazine and 2-chlorphenothiazine. We also thank Dr. C . Van Sluis for the bacterial cells.
REFERENCES
Ames, B. N.. J. McCann and E. Yamasaki (1975) Mutar. Res. 31. 347-364. Charpentier. P. (1951) U.S. Patent 2. 519.886 (C.A. 45, 673c). Charpentier, P., P. Gaillot, R. Jacob. J. Gaudechon and P. Buisson (1952) C. r. hebd. Shanc. Acad. Sci. Paris 235. 59-60. Davies. A. K.. S. Navaratnam and G . 0. Phillips (1976) J . Cherii. Soc.. Perkin I1 2529. Day. R. S . and M. Dimattina (1977) Cheiri.-Biol. Inreracr. 17. 89-97. Felmeister. A. and C. A. Discher (1964) J . Pharinac. Sci. 53. 756762. Forrest. 1. S.. C . J. Carr and E. Usdin (1974) Advances in Biochernical Psychopharrrracology. Vol. 9. Raven Press, New York. Grant. F. W. and J. Greene (1972) Toxicology and Applied Pharmacology 23. 71-74. Huang. C. L. and F. L. Sands (1964) J . Chrornatogr. 13. 246249. Huang. C. L. and F. L. Sands (1967) J . Pharrrrac. Sci. 56. 259-264. Iwaoka. T. and M. Kondo (1974) Bull. Cherrr. SOC. (Jpn) 47. 980-986. Merkel. P. B. and D. R. Kearns (1973) J. Am. Chsii. Soc. 95. 58865890. Nilsson. R.. G. Swanbeck and G. Wennersten (1975) Photochern. Phorobiol. 22. 183-186. Pinhey. J. T. and R. D. G. Rigby (1969) Tetrahedron Leu.. 1271-1274. Rosenthal. I.. T. Bercovici and E. Frimer (1977) J. Herercycl. Chern. 14. 355-357. Thomson. J. F. (1963) Biological E'icrs of Deuterium. p. 4. Pergamon Press, Oxford.
PHOTOCHEMICAL REACTIONS OF CHLORPROMAZINE; CHEMICAL AND BIOCHEMICAL IMPLICATIONS 1. ROSENTHAL*.E. BEN-HUR,A. PRAGER and E. RIKLIS Israel Atomic Energy Commission. Nuclear Research Center-Negev. Beer-Sheva. Israel (Received 7 March 1978; accepted 13 March 1978)
Abstract-The photoexcited chlorpromazine reacts with methanol to yield promazine and 2-methoxypromazine by two different reaction pathways: hydrogen atom abstraction and nucleophilic attack. respectively. When the photoexcitation of chlorpromazine is performed in the presence of protein or nucleic acids, chlorpromazine binds to the biopolymer. This binding is drastically pH-dependent and correlates to the phototoxic effect exhibited in chlorpromazine-photosensitization of E. coli. No photodynamic damage of E. coli attributed to CPZ-sensitization of molecular oxygen could be detected.
INTRODUCTION
Negev, Israel. The deuterated methanol was purchasea from Roth OHG. Chlorpromazine(CPZ.1a) is the major representative The irradiations of CPZ in methanol were carried out of phenothiazine-type drugs employed in psycho- at room temperature in cylindrical Pyrex cells provided therapy (Forrest, 1974). The abnormal photoreactions with greaseless stopcocks. set in a "merry-go-round" apparatus. Hanovia 450 W high pressure mercury vapour which accompany CPZ treatment such as exaggerated lamp, cooled internally with running water and jacketed sensitivity t o sunburn, hyperpigmentation of the skin in a Pyrex filter ( I 1 300nm) was employed as an irrddiaand ocular opacity have justified investigations of its tion source. In a typical experiment, a solution of 20mg photochemical properties. In oxygenated aqueous CPZ. HCI dissolved in 4 m/ solvent was thoroughly degassed by several freeze-thaw cycles. The solution was exc = 254 nm) under- irradiated for 2 h followed by removal of the solvent under solutions, photoexcited CPZ (i goes photooxidation via its cation radical t o yield vacuum. The reaction products were separated by preparaCPZ-5-oxide (Iwaoka and Kondo. 1974; Felmeister tive thin layer chromatography (Merck PSC-Fertig-platten and Discher, 1964) in addition t o several other oxi- Kieselgel 60F254. eluted by a mixture of benzene, methdation products (Felmeister and Discher, 1964; anol, ammonium hydroxide, 6 : 14:O.I). For biological irradiations, the samples were exposed Huang and Sands, 1964). When the photooxidation to two tubular fluorescent black-light lamps (Lamp No. was dye-sensitized (i. exc > 530 nm), the primary 50058, U.V. Products, Inc.) which emit in a wide band chemical modification was the cleavage of the N-side ( 3 W 0 0 n m ) with a maximum at about 360nm. The incichain (Rosenthal et a/.. 1977). In the absence of dent flux on the sample was 2OJ/m*/s, as measured by a black light meter (model J-221. UV Products. Inc.). oxygen, the UV-irradiation of CPZ provoked the Binding of CPZ ro hiopolyrners. An irradiated aqueous cleavage of the chlorine function at position 2. This solution ( I m/) of bovine serum albumin (IO'Z,) and process resulted in the formation of promazine, 2-sub- 3H-CPZ (1%) was gel filtered through a Sephadex G-25 stituted promazines (Huang and Sands, 1967; Grant column (1.4 x 15cm). The elution was done with water and Greene, 1972; Davies et ul., 1976) and polymeric and 2 m / fractions were collected. The radioactivity was determined in I m/ samples in a dioxane-based cocktail material (Huang and Sands, 1967). Although earlier and counted in a Packard liquid scintillation counter. work suggested an ionic mechanism for this reaction E. coli cells exposed in the presence of ('H)-CPZ were (Grant and Greene. 1972). a more recent report washed by centrifugation, and precipitated with 5% cold explained the formation of these products by radical TCA(trich1oroaceticacid). The precipitate was dissolved in 0.1 N NaOH and heated to 100°C for 30min to hydrolyze recombinations (Davies et al.. 1976). RNA. DNA and proteins were precipitated with cold TCA, It was the purpose of this study to clarify the and DNA extracted by heating the precipitate in 5% TCA mechanistic aspects of the photochemistry of C P Z to 100'C for 10 min. The remaining protein pellet was dissolved in 0.1 N NaOH. The labeled CPZ bound to the and to extend its reaction t o biological systems. various macromolecular fractions was determined by liquid scintillation spectrometry. Control experiments indiMATERIALS AND METHODS cate that this procedure does not induce significant release CPZ. HCI, promazine. HCI. 2-methoxypromazine and 2- of bound CPZ. Bacterial cell growrh and suruiual. Bacteria, E . coli CS chlorphenothiazinewere kindly donated by Rhone-Poulenc 4033 (wild type) and a repair deficient double mutant E. Co.. and were used without further purification. (3H)-CPZ coli CS 4034 uur A6B5 (both endo I ) were grown to a was a commercial product from Nuclear Research Center- density of about 1 x lo9 cells/m/ in minimal medium, harvested by centrifugation and resuspended in phosphate buffered saline pH = 7 containing 100pg/m/ CPZ. After *Also at the Department of Chemistry, The Weizmann about IOmin at room temperature, which was sufficient for maximal sensitization, the cells were irradiated with Institute of Science, Rehovot, Israel. 59 1
1. ROSENTHAL pr a/.
592
near UV light. serially diluted. and plated on nutrient agar. Viability counts were made after 16 h incubation at 37°C. RESULTS AND DISCUSSION
Chernical reactions of photoexcited CPZ Initially, we investigated the photochemical reaction of CPZ in dry methanol in the absence of oxygen. ,CH3 ICH,),
N,
(a)
R =CL
(b) The isotopic composition of the methoxy group in Ic is identical with that of the original methyl group in the methyl alcohol. Thus, no hydrogen scrambling occurs along the reaction pathway between methyl and hydroxyl hydrogen as could be expected for the suggested all-radical mechanism (Davies et al., 1976). An ionic, photonucleophilic substitution of the chlorine by methoxy group is in agreement with this observation
PZ - C1* Ia
+ CH30--*
PZ - OCH, Ic
+ CI-
Alternatively, heterolytic photofission of CPZ to give PZ' and C1- followed by ionic recombination of promazinyl cation and methoxy anion might explain this I resu It. This mechanistic scheme is similar to the reported The direct photolysis of CPZ(1a) (1 > 300nm) resulted in almost total conversion to promazine (Ib) photoreduction and nucleophilic photosubstitution of and 2-methoxypromazine (Ic), in addition to minor halides in haloaromatic compounds (Pinhey and amounts of decomposition products. Ib and Ic were Rigby, 1969). separated by chromatography and identified unBiochernical reactions of photoexcited CPZ ambiguously by spectroscopic methods (IR, UV. MS, The photoinduced reaction of CPZ with biomoleNMR) and comparison with authentic samples (Charpentier. 1951. 1952). Control experiments showed that cules was first tested in oifro with bovine serum albuunder identical irradiation conditions, Ib and Ic were min (BSA). The elution profile of BSA after near UV photostable. that is both of them originated indepen- irradiation in the presence of 3H-labeled C P Z is dently from CPZ. It was. however. noted that the shown in Fig. I. BSA was eluted in the first peak presence of N-alkyl side chain is not a critical require- and evidently binds the drug as a result of irradiation. ment for this reaction. Thus, 2-chlorphenothiazine In our experimental conditions, up to seven CPZ under similar reaction conditions was converted to molecules could be bound to one molecule of BSA. phenothiazine (9004) and 2-methoxyphenothiazine (ca Two additional radioactive peaks of intermediate mol wt between BSA and C P Z are photoproducts of 2%). Valuable information regarding the mechanism involved could be obtained by performing the reaction in selectively labeled methanol, under otherwise identical conditions, followed by mass-spectroscopic analysis of the reaction products. Thus. CHIOH. CH30D. C D 3 0 H and C D 3 0 D were separately employed as solvents. The results are summarized in Table 1. These results show that: (a) The methyl group of the solvent is the exclusive source for the hydrogen atom which replaced chlorine. This observation suggests a homolytical cleavage of the carbonxhlorine bond in the photoexcited C P Z followed by the hydrogen atom abstraction from the solvent by the promazinyl radical: P Z - Cl* + PZ. Ia
+ CH30H
--*
PZ - H Ib
+ CHIOH
Table 1. Mass spectroscopic analysis of products Product ratio Reaction solvent CHjOH CH3OD CD3OH CD30D
1b:Ic 1 :0.5 1 :0.5
I :0.8 I :0.8
Molecular weight 2-MethoxyPromazine promazine 284 284 285 285
314 314 317 317
Figure 1. Elution profile of BSA irradiated in the presence of 'H-CPZ. Free jH-CPZ was eluted in fractions 25-30 (not shown). NUV is near UV light (300-400nm).
Photochemistry of chlorpromazine
PH
1.0
1.5
.
DNA
A
RNA
?ROTEIN
D
* /
I
I0
/
10 IRRADIAIION
/
. so 1lME
40
(mil)
593
quently repair it. Obviously, there is an additional possibility that endonuclease itself is photoinactivated by CPZ, thus explaining the large shoulder region in the survival curve. Results obtained with human adenovirus 5 (Day and Dimattina, 1977) indicate that CPZ-induced damage in DNA is repairable, therefore, the latter possibility seems most likely. In any event, the biological significance of CPZ-induced damage in DNA was evidenced by its mutagenic effect as established separately by the Ames' test (Ames et al., 1975). These results will be published separately (Green et al., in preparation). Identical phototoxic effects of CPZ as reflected in the survival curves were obtained by exposure in the absence or presence of oxygen (Fig. 3). Since CPZsensitized photoprcduction of singlet molecular oxygen has been documented (Davies et al., 1976), this observation excludes a possible photodynamic destruction due to excited oxygen. Additional support for this conclusion was provided by control experiments performed in the presence of oxygen and competitive quenchers of '0,such as histidine or DABCO. In concentrations up to mM, these compounds had no effect on the photosensitization of E. coli by CPZ (Fig. 3). In this regard, it is noted that photohemolysis of erythrocytes sensitized by CPZ
Figure 2. Binding of 'H-CPZto macromolecules in E. coli. DMF(P) is the dose modifying factor for light-induced binding of CPZ to proteins.
CPZ. BSA-free. Similar results were obtained when DNA served as substrate. Thus, 3 h exposure resulted in binding of 8"/, of free CPZ to DNA, compared with 8% binding following 1.5 h exposure with BSA (Fig. 1). Photoinduced binding of CPZ to DNA, RNA and proteins in uiuo was established by using E. coh cells. The results (Fig. 2.) show that the major amount of drug binds to proteins as calculated on a per cell basis. The biological effect of this photochemical binding is reflected in the survival of E. coli cells (CS-4033, wild type). Near UV light or CPZ, when applied separately, did not affect the survival of the bacterial cells. However, the simultaneous exposure to both factors led to an exponential decrease in survival after an initial shoulder region (Fig. 3). Control experiments showed that preirradiated CPZ added to a suspension of E. coli cells did not affect their survival. Since part of the photoinduced binding of the drug is to DNA, it was of interest to compare the sensitivity of the wild type bacteria to isogenic mutant cells deficient in UV-specific endonuclease responsible for repair of DNA damage (E. coli CS-4034). It seems that the deficiency in this endonuclease increases the sensitivity of the mutant cells to photoexcited CPZ only slightly. Thus, the contribution to cell lethality of CPZ-binding to DNA is either of minor importance, or the endonuclease enzyme is unable to recognize the CPZ-induced damage in DNA and conse-
\
bO3b
\'
Figure 3. Survival curves of irradiated E . coli cells in the presence of CPZ at pH 6.5 and 7.0. CS-4033 -0-, -4under nitrogen; -0-, +under air; -Xin the presence of histidine and air. CS-4034 -A-, -A- under air. Irradiation without oxygen was done by bubbling nitrogen through the cell suspension for 30min prior to' and during near UV exposure.
I. R ~ E N T H AerL ul.
594
also pi.oceeds by a non-singlet oxygen pathway (Nilsson cr af.. 1975). We found that the phototoxic effect of CPZ on E. coli is drastically controlled by the pH of the sohtion. Thus, a decrease in pH from 7.0 to 6.5 enhances the photosensitivity of the cells to a very large extent (Fig. 3) and causes a suppression of any photochemical linkage to nucleic acids in favour of that to protein (Fig, 2). Since E. coli is hardly affected by this change in pH, a pH-dependent photochemistry of C P Z must be responsible for this effect. It is interesting that the same result obtained in bufferedH 2 0 at pH = 6.5 could be duplicated in unbuffered D2° Cmeasured pH which corresponds to a pD value of 6.5 (Thomson, 1963)]. An increased reaction rate in D 2 0 as compared to H 2 0 medium has been-_
'"*
suggested as a test for singlet oxygen (Merkel a;Td Kearns. 1973). The drastic pH effect observed in the CPZ-photosensitization warrants against overlooking this parameter in using this test for singlet oxygen diagnosis. These results clearly indicate that the photoexcited C P Z exhibit strong toxic effects in uiuo due to its binding to biomacromolecular components. Of particular significance is the large extent of binding to proteins, which might thus generate a new antigen. Such a process can provide the explanation for CPZinduced photoallergic syndrome. Acknowledgements-We thank Rhone-Poulenc Company for supplying chlorpromazine. HCI, promazine. HCI. 2methoxypromazine and 2-chlorphenothiazine. We also thank Dr. C . Van Sluis for the bacterial cells.
REFERENCES
Ames, B. N.. J. McCann and E. Yamasaki (1975) Mutar. Res. 31. 347-364. Charpentier. P. (1951) U.S. Patent 2. 519.886 (C.A. 45, 673c). Charpentier, P., P. Gaillot, R. Jacob. J. Gaudechon and P. Buisson (1952) C. r. hebd. Shanc. Acad. Sci. Paris 235. 59-60. Davies. A. K.. S. Navaratnam and G . 0. Phillips (1976) J . Cherii. Soc.. Perkin I1 2529. Day. R. S . and M. Dimattina (1977) Cheiri.-Biol. Inreracr. 17. 89-97. Felmeister. A. and C. A. Discher (1964) J . Pharinac. Sci. 53. 756762. Forrest. 1. S.. C . J. Carr and E. Usdin (1974) Advances in Biochernical Psychopharrrracology. Vol. 9. Raven Press, New York. Grant. F. W. and J. Greene (1972) Toxicology and Applied Pharmacology 23. 71-74. Huang. C. L. and F. L. Sands (1964) J . Chrornatogr. 13. 246249. Huang. C. L. and F. L. Sands (1967) J . Pharrrrac. Sci. 56. 259-264. Iwaoka. T. and M. Kondo (1974) Bull. Cherrr. SOC. (Jpn) 47. 980-986. Merkel. P. B. and D. R. Kearns (1973) J. Am. Chsii. Soc. 95. 58865890. Nilsson. R.. G. Swanbeck and G. Wennersten (1975) Photochern. Phorobiol. 22. 183-186. Pinhey. J. T. and R. D. G. Rigby (1969) Tetrahedron Leu.. 1271-1274. Rosenthal. I.. T. Bercovici and E. Frimer (1977) J. Herercycl. Chern. 14. 355-357. Thomson. J. F. (1963) Biological E'icrs of Deuterium. p. 4. Pergamon Press, Oxford.
