Journal of Photochemistry
and Photobiology,
B: Biology,
4 (1990)
329 - 333
329
Preliminary Note The Mechanism of Photohaemolysis ERNEST0
FERNkNDEZt
by Photoproducts of Nalidixic Acid
and ANA MARiA CARDENAS
Laboratorio Fotobiologia, Facultad de Medicina, Casilla 92-V Valparaiso, (Chile) (Received April 11, 1989;accepted
Universidad
de Valparaiko,
May 18, 1989)
Keywords: Photosensitization, phototoxicity, dynamic mechanism, nalidixic acid.
photohaemolysis,
photo-
1. Introduction Nalidixic acid (NA) is an antibacterial drug whose clinical use has been associated with photosensitivity reactions [ 11. NA was reported to be both a photosensitizer [2] and a photolabile drug [ 31; these properties appear to have some correlation with photosensitivity in uiuo. Several photosensitizers are known to cause lysis of red blood cells (RBC) [ 41. A photohaemolysis test has proved to be suitable for assaying damage to cell membranes, since RBC have no organelles and the released haemoglobin is easily measured [ 51. Recently we reported a photohaemolytic mechanism induced by NA and mediated by singlet oxygen and hydroxyl radicals [ 6, 71. Although preirradiated NA was not lytic to RBC, the mixtures of photoproducts showed a greater photohaemolytic effect than did NA itself. This observation suggested that photoproducts from NA could be more phototoxic than NA itself [ 71. The present report describes the photohaemolytic activity of two main products of NA, a decarboxylated photoproduct (l-ethyl-l,4 dihydro-7methyl-Coxo-1,Snaphthyridine (I) and a dimer of this product (II) (Fig. 1). The aim of this work was to compare the photohaemolysis potential of photoproducts I and II with that of NA and to study the effects of antioxidants, hydroxyl radical scavengers and singlet oxygen quenchers on the photohaemolysis induced by both photoproducts, to gain an insight into photohaemolytic mechanisms. 2. Materials and methods NA (99.9% purity) was provided by Laboratorio Chile (Santiago, Chile). Phosphate-buffered saline (PBS) was prepared from 0.01 M monoTAuthor to whom correspondence should be addressed. loll-1344/90/$3.50
@ Elsevier Sequoia/Printed in The Netherlands
330
NA
0
C2H5
‘W5
0
Fig. 1. Structure of nalidixic acid (NA), a decarboxylated (II).
photoproduct
(I) and a dimer
basic and dibasic phosphate, and 0.15 M sodium chloride (pH 7.4) and was used throughout these studies. Product I was obtained by photolysis and thermolysis of NA. The photolysis was performed by irradiation of NA (1 g 1-l) solutions in 0.05 M NaOH with a medium pressure Mercury lamp (Hanovia 450 W) for 35 h. Thermolysis was performed as described by Detzer and Huber [ 81. Product II was obtained by thermolysis of NA [8]. Both products were isolated by chromatographic techniques and identified by their spectral properties; the results corresponded exactly with earlier data [ 81. Freshly isolated RBC obtained from volunteer donors were washed three times with PBS and then added to buffered drug solutions. All other chemicals were of reagent grade. RBC suspensions plus NA or product solutions were irradiated with a 450 W medium pressure Mercury lamp (Hanovia) in a merry-go-round turntable reactor (Ace Class). The light was passed through a Pyrex filter (X > 290 nm). The fluence under the conditions used was approximately 1.5 X lo-l6 quanta s-l as determined by ferrioxalate actinometry. The temperature of the irradiated solutions was kept at 37 f 1 “C. Deoxygenation of RBC suspensions was accomplished by gentle bubbling with nitrogen. Control samples incubated in the dark at 37 “C and irradiated solutions were centrifuged at 2000g. The absorbance of the supematant fluid (haemoglobin) was read at 540 nm by a Spectronic 20 spectrophotometer. A 100% haemolysed solution was prepared by adding 2 ml of 2% v/v RBC suspension to 8 ml of a 0.1% NH,OH solution. All experiments were carried out in triplicate under identical experimental conditions. 3. Results and discussion Product I was found to be the main photoproduct of NA photolysis together with carbon dioxide, ethylamine, diketone and dimers [S]. Never-
331
theless, irradiation of solutions of NA is not an adequate method for preparing photoproducts with high efficiency, owing to the formation of a variety of products. However, the thermolytic technique proposed by Detzer and Huber [8] allows products I and II to be prepared with high efficiency. In the present paper the photohaemolytic effect of NA was compared with the effects of products I and II. As shown in Fig. 2(A), irradiation of RBC with NA (25 pg ml-‘) produced 7% and 39% photohaemolysis within 2 h and 3 h respectively, while product I (at the same concentration) induced 88% photohaemolysis within 2 h and 91% within 3 h (Fig. 2(B)). On the other hand, 25 pg ml-’ of product II produced 10% and 18% photohaemolysis after 2 h and 3 h of irradiation respectively. When RBC were irradiated in the presence of 50 erg ml-’ of product II, photohaemolysis was increased to 58% and 75% after 2 h and 3 h respectively (Fig. 2(C)).
!i~ 0 A
1
I_
f 2
3
0
1
2
3
B IRRADIATION
0
1
2
3
C TIME
(h)
Fig. 2. Photohaemolysis by NA, product I and product II: open symbols (0) indicate photohaemolysis induced by 25 pg ml-’ of NA (A), product I (B) and product II (C); closed symbols (0) indicate photohaemolysis induced by 50 l.(g ml-’ of NA (A), product I (B) and product II (C). Each point is the mean of at least three separate experiments.
No lysis was observed, during 3 and 6 h periods, when RBC were incubated in the dark with 200 I.cgml-’ of product I or product II. Hence the photoproducts of NA are not lytic per se, but produce more photohaemolysis than NA; product I showed a greater photohaemolytic potential than product II. The fact that product I induced more photohaemolysis than NA suggests that the carboxylic group in position 3 is not responsible for the photohaemolytic effect. Moreover, the absence of this group increases this effect, as also observed by Moore et al. [ 21. To determine if lysis of RBC by products I and II occurs by the same mechanism as observed for NA photohaemolysis, RBC were irradiated with product I or product II in the absence of oxygen. No haemolysis was detected over 3 h, suggesting that photohaemolysis by both products is an oxygen-dependent process.
332 TABLE 1 Effect of antioxidants, hydroxyl radicals and a singlet oxygen quencher on the photohaemolysis by NA, product I and product II Reagent
0.5 mM ascorbate 0.4 mM histidine 1.3 mM thiourea
Inhibition
ofphotohaemolysisa
(%)
NA
Product
I
90.0 + 1.6 88.5 + 2.2 91.3 + 1.0
99.0 f 1.5 75.7 + 2.3 99.6 + 1.2
Product
II
100fO 0.0 + 0 2.4 f 0.4
The RBC were irradiated with 100 /.fg ml-’ of NA, product I or product II during a 3 h period either in the presence or in the absence of each reagent. Results are the mean of three experiments. aPer cent inhibition of photohaemolysis was calculated with respect to photohaemolysis induced by NA in the absence of each reagent.
As shown in Table 1, 0.5 mM ascorbate, an antioxidant, inhibited the photohaemolysis induced by NA, product I and product II by 90%, 99% and 100% respectively. The influence of ascorbate on photohaemolysis induced by products I and II indicates that a photo-oxidative step is essential in both photohaemolytic mechanisms. Histidine (0.4 mM), a singlet oxygen quencher [9, lo] and hydroxyl radical scavenger [ 111, inhibited respectively the photohaemolysis induced by NA and product I by 88.5% and 75.7%, but the photohaemolysis by product II was not inhibited (Table 1). Thiourea (1.3 mM), another hydroxyl radical scavenger [ 121, also inhibited photohaemolysis by NA and product I (91.3% and 99.6% respectively); however, photohaemolysis by product II was inhibited by only 2.4% (Table 1). These results show that photohaemolysis induced by product I occurs through a mechanism similar to that found with NA; the photolysis of RBC induced by product II, even if it occurs by an oxygen-dependent mechanism, does not, however, involve participation of singlet oxygen and hydroxyl radicals. In conclusion, the results presented in this paper indicate that the photoproducts of NA produce photohaemolysis by oxygen-dependent mechanisms. Product I is more phototoxic than NA with RBC, and the mechanism of this process is mediated by singlet oxygen and hydroxyl radical species. On the other hand, product II is less photohaemolytic than product I, and exerts its action by a mechanism distinct from that seen with NA or product I. This research was supported by a grant from Direction de Investigation Cientifica, Universidad de Valparaiso (Project U.V. 3/86). The authors thank Dr. Christopher Pogson and Dr. Mario Sapag-Hagar for their helpful comments.
333 1 A. Boisvert and G. Barbeau, Nalidixic acid induced photodermatitis after minimal sun exposure, Drug Intell. Clin. Pharm., 15(2) (1981) 126 - 127. 2 D. E. Moore, V. J. Hemmens and H. Yip, Photosensitization of drugs: nalidixic and oxolinic acids, Photochem. Photobiol., 39 (1984) 57 - 61. 3 E. Fernandez, W. Pen”, R. Vinet and M. E. Hidalgo, Kinetics of photodegradation of nalidixic acid, An. Quim., 82 (1986) 96 - 98. 4 A. C. Allison, I. A. Magnus and A. R. Young, Role of lysosomes and of cell membranes in photosensitization, Nature, 209 (1966) 874 - 878. 5 G. Kahn and B. Fleischaker I, Red blood cell hemolysis by photosensitizing compounds, J. Invest. Dermatol., 56 (1971) 85 - 90. 6 A. M. Cardenas and E. Fernandez, Photohemolysis mechanisms induced by oxoiinic and nalidixic acids, Photochem. Photobiol., 41 S (1985) 115. 7 E. Fernandez, A. M. Cardenas and G. Martinez, Phototoxicity from nalidixic acid: oxygen dependent photohemolysis, Zl Farmaco, Ed. Sci., 42 (1987) 681 - 690. 8 N. Detzer and B. Huber, Photochimie Heterocyclischer Enone I, Tetrahedron, 31 (1975) 1937 - 1941. 9 R. Nilsson, P. B. Merkeland D. R. Kearns, An unambiguous evidence for the participation of singlet oxygen in photodynamic oxidation of aminacids, Photochem. Photobiol., 16 (1972) 117 - 124. 10 R. Nilsson, G. Swanbeck and G. Wennersten, Primary mechanisms of erythrocyte photolysis induced by biological sensitizers and phototoxic drugs, Photochem. Photobiol., 22 (1975) 183 - 186. 11 L. Dorfmann and G. Adams, Reactivity of the hydroxyl radical in aqueous solutions, Publication 46, National Bureau of Standards, Washington, DC, 1973. 12 R. J. Trotta, S. G. Sullivan and A. Stern, Lipid peroxidation and haemoglobin degradation in red blood cells exposed to t-butyl hydroperoxide, Biochem. J., 212 (1983) 759 - 772.
and Photobiology,
B: Biology,
4 (1990)
329 - 333
329
Preliminary Note The Mechanism of Photohaemolysis ERNEST0
FERNkNDEZt
by Photoproducts of Nalidixic Acid
and ANA MARiA CARDENAS
Laboratorio Fotobiologia, Facultad de Medicina, Casilla 92-V Valparaiso, (Chile) (Received April 11, 1989;accepted
Universidad
de Valparaiko,
May 18, 1989)
Keywords: Photosensitization, phototoxicity, dynamic mechanism, nalidixic acid.
photohaemolysis,
photo-
1. Introduction Nalidixic acid (NA) is an antibacterial drug whose clinical use has been associated with photosensitivity reactions [ 11. NA was reported to be both a photosensitizer [2] and a photolabile drug [ 31; these properties appear to have some correlation with photosensitivity in uiuo. Several photosensitizers are known to cause lysis of red blood cells (RBC) [ 41. A photohaemolysis test has proved to be suitable for assaying damage to cell membranes, since RBC have no organelles and the released haemoglobin is easily measured [ 51. Recently we reported a photohaemolytic mechanism induced by NA and mediated by singlet oxygen and hydroxyl radicals [ 6, 71. Although preirradiated NA was not lytic to RBC, the mixtures of photoproducts showed a greater photohaemolytic effect than did NA itself. This observation suggested that photoproducts from NA could be more phototoxic than NA itself [ 71. The present report describes the photohaemolytic activity of two main products of NA, a decarboxylated photoproduct (l-ethyl-l,4 dihydro-7methyl-Coxo-1,Snaphthyridine (I) and a dimer of this product (II) (Fig. 1). The aim of this work was to compare the photohaemolysis potential of photoproducts I and II with that of NA and to study the effects of antioxidants, hydroxyl radical scavengers and singlet oxygen quenchers on the photohaemolysis induced by both photoproducts, to gain an insight into photohaemolytic mechanisms. 2. Materials and methods NA (99.9% purity) was provided by Laboratorio Chile (Santiago, Chile). Phosphate-buffered saline (PBS) was prepared from 0.01 M monoTAuthor to whom correspondence should be addressed. loll-1344/90/$3.50
@ Elsevier Sequoia/Printed in The Netherlands
330
NA
0
C2H5
‘W5
0
Fig. 1. Structure of nalidixic acid (NA), a decarboxylated (II).
photoproduct
(I) and a dimer
basic and dibasic phosphate, and 0.15 M sodium chloride (pH 7.4) and was used throughout these studies. Product I was obtained by photolysis and thermolysis of NA. The photolysis was performed by irradiation of NA (1 g 1-l) solutions in 0.05 M NaOH with a medium pressure Mercury lamp (Hanovia 450 W) for 35 h. Thermolysis was performed as described by Detzer and Huber [ 81. Product II was obtained by thermolysis of NA [8]. Both products were isolated by chromatographic techniques and identified by their spectral properties; the results corresponded exactly with earlier data [ 81. Freshly isolated RBC obtained from volunteer donors were washed three times with PBS and then added to buffered drug solutions. All other chemicals were of reagent grade. RBC suspensions plus NA or product solutions were irradiated with a 450 W medium pressure Mercury lamp (Hanovia) in a merry-go-round turntable reactor (Ace Class). The light was passed through a Pyrex filter (X > 290 nm). The fluence under the conditions used was approximately 1.5 X lo-l6 quanta s-l as determined by ferrioxalate actinometry. The temperature of the irradiated solutions was kept at 37 f 1 “C. Deoxygenation of RBC suspensions was accomplished by gentle bubbling with nitrogen. Control samples incubated in the dark at 37 “C and irradiated solutions were centrifuged at 2000g. The absorbance of the supematant fluid (haemoglobin) was read at 540 nm by a Spectronic 20 spectrophotometer. A 100% haemolysed solution was prepared by adding 2 ml of 2% v/v RBC suspension to 8 ml of a 0.1% NH,OH solution. All experiments were carried out in triplicate under identical experimental conditions. 3. Results and discussion Product I was found to be the main photoproduct of NA photolysis together with carbon dioxide, ethylamine, diketone and dimers [S]. Never-
331
theless, irradiation of solutions of NA is not an adequate method for preparing photoproducts with high efficiency, owing to the formation of a variety of products. However, the thermolytic technique proposed by Detzer and Huber [8] allows products I and II to be prepared with high efficiency. In the present paper the photohaemolytic effect of NA was compared with the effects of products I and II. As shown in Fig. 2(A), irradiation of RBC with NA (25 pg ml-‘) produced 7% and 39% photohaemolysis within 2 h and 3 h respectively, while product I (at the same concentration) induced 88% photohaemolysis within 2 h and 91% within 3 h (Fig. 2(B)). On the other hand, 25 pg ml-’ of product II produced 10% and 18% photohaemolysis after 2 h and 3 h of irradiation respectively. When RBC were irradiated in the presence of 50 erg ml-’ of product II, photohaemolysis was increased to 58% and 75% after 2 h and 3 h respectively (Fig. 2(C)).
!i~ 0 A
1
I_
f 2
3
0
1
2
3
B IRRADIATION
0
1
2
3
C TIME
(h)
Fig. 2. Photohaemolysis by NA, product I and product II: open symbols (0) indicate photohaemolysis induced by 25 pg ml-’ of NA (A), product I (B) and product II (C); closed symbols (0) indicate photohaemolysis induced by 50 l.(g ml-’ of NA (A), product I (B) and product II (C). Each point is the mean of at least three separate experiments.
No lysis was observed, during 3 and 6 h periods, when RBC were incubated in the dark with 200 I.cgml-’ of product I or product II. Hence the photoproducts of NA are not lytic per se, but produce more photohaemolysis than NA; product I showed a greater photohaemolytic potential than product II. The fact that product I induced more photohaemolysis than NA suggests that the carboxylic group in position 3 is not responsible for the photohaemolytic effect. Moreover, the absence of this group increases this effect, as also observed by Moore et al. [ 21. To determine if lysis of RBC by products I and II occurs by the same mechanism as observed for NA photohaemolysis, RBC were irradiated with product I or product II in the absence of oxygen. No haemolysis was detected over 3 h, suggesting that photohaemolysis by both products is an oxygen-dependent process.
332 TABLE 1 Effect of antioxidants, hydroxyl radicals and a singlet oxygen quencher on the photohaemolysis by NA, product I and product II Reagent
0.5 mM ascorbate 0.4 mM histidine 1.3 mM thiourea
Inhibition
ofphotohaemolysisa
(%)
NA
Product
I
90.0 + 1.6 88.5 + 2.2 91.3 + 1.0
99.0 f 1.5 75.7 + 2.3 99.6 + 1.2
Product
II
100fO 0.0 + 0 2.4 f 0.4
The RBC were irradiated with 100 /.fg ml-’ of NA, product I or product II during a 3 h period either in the presence or in the absence of each reagent. Results are the mean of three experiments. aPer cent inhibition of photohaemolysis was calculated with respect to photohaemolysis induced by NA in the absence of each reagent.
As shown in Table 1, 0.5 mM ascorbate, an antioxidant, inhibited the photohaemolysis induced by NA, product I and product II by 90%, 99% and 100% respectively. The influence of ascorbate on photohaemolysis induced by products I and II indicates that a photo-oxidative step is essential in both photohaemolytic mechanisms. Histidine (0.4 mM), a singlet oxygen quencher [9, lo] and hydroxyl radical scavenger [ 111, inhibited respectively the photohaemolysis induced by NA and product I by 88.5% and 75.7%, but the photohaemolysis by product II was not inhibited (Table 1). Thiourea (1.3 mM), another hydroxyl radical scavenger [ 121, also inhibited photohaemolysis by NA and product I (91.3% and 99.6% respectively); however, photohaemolysis by product II was inhibited by only 2.4% (Table 1). These results show that photohaemolysis induced by product I occurs through a mechanism similar to that found with NA; the photolysis of RBC induced by product II, even if it occurs by an oxygen-dependent mechanism, does not, however, involve participation of singlet oxygen and hydroxyl radicals. In conclusion, the results presented in this paper indicate that the photoproducts of NA produce photohaemolysis by oxygen-dependent mechanisms. Product I is more phototoxic than NA with RBC, and the mechanism of this process is mediated by singlet oxygen and hydroxyl radical species. On the other hand, product II is less photohaemolytic than product I, and exerts its action by a mechanism distinct from that seen with NA or product I. This research was supported by a grant from Direction de Investigation Cientifica, Universidad de Valparaiso (Project U.V. 3/86). The authors thank Dr. Christopher Pogson and Dr. Mario Sapag-Hagar for their helpful comments.
333 1 A. Boisvert and G. Barbeau, Nalidixic acid induced photodermatitis after minimal sun exposure, Drug Intell. Clin. Pharm., 15(2) (1981) 126 - 127. 2 D. E. Moore, V. J. Hemmens and H. Yip, Photosensitization of drugs: nalidixic and oxolinic acids, Photochem. Photobiol., 39 (1984) 57 - 61. 3 E. Fernandez, W. Pen”, R. Vinet and M. E. Hidalgo, Kinetics of photodegradation of nalidixic acid, An. Quim., 82 (1986) 96 - 98. 4 A. C. Allison, I. A. Magnus and A. R. Young, Role of lysosomes and of cell membranes in photosensitization, Nature, 209 (1966) 874 - 878. 5 G. Kahn and B. Fleischaker I, Red blood cell hemolysis by photosensitizing compounds, J. Invest. Dermatol., 56 (1971) 85 - 90. 6 A. M. Cardenas and E. Fernandez, Photohemolysis mechanisms induced by oxoiinic and nalidixic acids, Photochem. Photobiol., 41 S (1985) 115. 7 E. Fernandez, A. M. Cardenas and G. Martinez, Phototoxicity from nalidixic acid: oxygen dependent photohemolysis, Zl Farmaco, Ed. Sci., 42 (1987) 681 - 690. 8 N. Detzer and B. Huber, Photochimie Heterocyclischer Enone I, Tetrahedron, 31 (1975) 1937 - 1941. 9 R. Nilsson, P. B. Merkeland D. R. Kearns, An unambiguous evidence for the participation of singlet oxygen in photodynamic oxidation of aminacids, Photochem. Photobiol., 16 (1972) 117 - 124. 10 R. Nilsson, G. Swanbeck and G. Wennersten, Primary mechanisms of erythrocyte photolysis induced by biological sensitizers and phototoxic drugs, Photochem. Photobiol., 22 (1975) 183 - 186. 11 L. Dorfmann and G. Adams, Reactivity of the hydroxyl radical in aqueous solutions, Publication 46, National Bureau of Standards, Washington, DC, 1973. 12 R. J. Trotta, S. G. Sullivan and A. Stern, Lipid peroxidation and haemoglobin degradation in red blood cells exposed to t-butyl hydroperoxide, Biochem. J., 212 (1983) 759 - 772.
