39
Mutation Research, 247 (1991) 39-44
© 1991 ElsevierSciencePublishersB.V.0027-5107/91/$03.50 ADONIS 002751079100062H MUT 04939
Pro-oxidative interactions of dithranol with human phagocytes promote oxidative damage to DNA of bystander leucocytes D.M.T. Forrester, C.E.J. Van Rensburg and R. Anderson Medical Research Council Unit for the Study of Phagocyte Function, Department of Immunology, Institute for Pathology, University of Pretoria (Republic of South Africa)
(Received23 April 1990) (Revisionreceived27 July 1990) (Accepted27 August1990) Keywords: Dithranol;DNA strand breaks; Neutrophils; Lymphocytes;Oxidants
Summary Dithranol at therapeutic concentrations (5-40 ~tg m1-1) induced strand breaks in human leucocyte D N A in vitro in a dose-related manner. Leucocytes from individuals with chronic granulomatous disease
(CGD) incurred substantially less DNA strand breaks than did normal leucocytes during exposure to dithranol indicating that activated phagocytes are involved. H-7, 4-fl-bromophenacyl bromide (BPB) and staurosporine, all inhibitors of protein kinase C, decreased both dithranol-mediated activation of the phagocyte respiratory burst and induction of DNA strand breaks. Similar effects were observed with the hydrogen peroxide scavenger catalase. These results suggest that dithranol induces DNA strand breaks, mainly as a result of pro-oxidative interactions with phagocytes.
The anti-psoriatic drug dithranol (anthralin) is a tumour promoter for mouse skin (Bock and Bums, 1983) and has been shown to induce strand breaks in human leucocyte DNA in vitro by an unknown mechanism (Birnboim, 1983). Dithranol has also been shown to stimulate the oxidative metabolism of phagocytes, probably via the activation of protein kinase C (PKC) (Anderson, 1989). Activated phagocytes generate superoxide anion (02) from which a complex mixture of
reactive oxidants including hydrogen peroxide (H202) , hypochlorous acid (HOC1), hydroxyl radical ('OH) and lipid hydroperoxides is generated (Babior et al., 1988; Ochi and Cerutti, 1987; Jackson et al., 1989; Samuni et al., 1988). As H202, "OH and lipid hydroperoxides have been implicated in the induction of DNA strand breaks (Ochi and Cerutti, 1987; Meneghini, 1988; Schraufstatter et al., 1988; Jackson et al., 1989), the possibility of a relationship between phagocyte activation and DNA strand break induction by dithranol has been investigated.
Abbreviations: BPB, 4-fl-bromophenacyl bromide; CGD,
Methods
chronic granulomatous disease; LECL, luminol-enhanced chemiluminescence; PMNL, polymorphonuclearleucocytes; SOD, superoxidedismutase. Correspondence: Dr. C.E.J. Van Rensburg, Department of Immunology, Institute for Pathology,Universityof Pretoria, P.O. Box 2034, Pretoria0001 (Republicof South Africa).
Dithranol (1,8-dihydroxy-9-anthrone) was dissolved in dimethyl sulphoxide (DMSO) immediately before required and used at concentrations of 1-100 /~g m1-1. Solvent controls were always included in the various assays.
40 Leucocyte preparation A mixed leucocyte preparation was isolated as previously described (Birnboim and KanabusKaminska, 1985). Briefly, heparinized venous blood from adult human volunteers was mixed with a solution of NH4C1/Tris. HC1 for 15 min to lyse erythrocytes. Remaining contaminating erythrocytes were removed by hypotonic lysis. The leucocyte preparation was washed twice in indicator-free Hanks balanced salt solution (HBSS, Highveld Biological Supplies, Kelvin, R.S.A.), buffered with 1 mg m1-1 Hepes (N-2-hydroxyethyl piperazine-H-2 ethane sulphonic acid) and 0.28 mg m1-1 CaCIE, pH 7.4. Leucocytes were resuspended to a concentration of 107 ml -l in HBSS and generally consisted of about 70% polymorphonuclear leucocytes (PMNL). Most of the remaining cells were lymphocytes. Purified PMNL were prepared as previously described (Anderson et al., 1987) and resuspended to a concentration of 107 ml-1 in HBSS. Measurement of DNA strand breaks These were measured by alkaline unwinding and determination of ethidium bromide fluorescence using a Hitachi (Tokyo, Japan) model 650-10 fluorescence spectrophotometer with excitation at 520 nm and emission at 590 nm as described previously (Birnboim and Kanabus-Kaminska, 1985). Leucocytes were distributed to siliconized glass vacutainer tubes (106 cells ml - l ) containing prewarmed HBSS (37°C, 10 ml final volume) and preincubated for 15 min. Dithranol (1-40 lag ml-1) was added and incubation continued for a further 20 min when the reaction was stopped by chilling to 0°C and centrifugation for 10 min at 600 g. Treated cells were washed in cold HBSS and resuspended in 1.8 ml HBSS. If catalase (80 lag m1-1) or superoxide dismutase (SOD, 33 and 200 lag m1-1) were used, they were added immediately prior to the addition of dithranol. The respiratory burst inhibitors H-7 (1-(5-isoquinolinesulphonyl)-2-methylpiperazine, 100 laM), BPB (4fl-bromophenacyl bromide, 5 laM) (both obtained from Sigma Chemical Co., St. Louis, MO, U.S.A.), and staurosporine (50 ng m1-1) (Calbiochem) when used, were added before the addition of the cells, and so were present throughout preincubation. Preincubation with BPB was extended to 30
min to allow cellular uptake of the inhibitor whereas preincubation with H-7 was reduced to 5 min to ensure the stability of this inhibitor. The effect of dithranol (20 lag ml -l) on the DNA of leucocytes from two individuals with CGD was also tested. 200 #1 of cell suspension was added to each of a set of 9 tubes labelled 'B', 'P' and 'T' in triplicate and treated as previously described (Birnboim and Kanabus-Kaminska, 1985) except that incubation at 15°C was for 30 min. Untreated (solvent) controls were always included and the percentage of DNA developing strand breaks is calculated according to the formula: % DNA developing strand breaks = 100 - [(% doublestranded DNA remaining after treatment/% double-stranded DNA present in the untreated control sample) × 100]. Luminol-enhanced chemiluminescence (LECL) of mixed leucocytes or pure PMNL 10 6 mixed leucocytes (or PMNL) were added to polystyrene chemiluminescence vials (50 mm x 10 mm), which contained prewarmed (37°C) HBSS and 18 lag ml 1 luminol (5-amino-2,3-dihydro1,4-phthalazine dione; Sigma Chem. Co.; St. Louis; U.S.A.) and preincubated for 15 min at 37°C. 10 lal of the appropriate dilution of dithranol (1-40 lag ml -l) were then added and LECL recorded with an LKB Wallac 1251 chemiluminometer with results expressed in millivolts sec-1 (mVs- 1). Catalase (80 lag ml-1), when used, was added immediately prior to the addition of dithranol whereas if H-7 (100 laM), BPB (5 laM) or staurosporine (50 ng ml -~) were used, they were added before the addition of the cells and so were present throughout the preincubation period. Preincubation periods for BPB and H-7 were adjusted as previously described. The final reaction volume was 1 ml. Tests were also performed using CGD leucocytes. This condition is associated with a congenital deficiency of membrane-associated oxidative metabolism in phagocytes. Appropriate leucocyte-free control systems were included (e.g. luminol + dithranol) to monitor for possible nonspecific interactions of dithranol with the assay system. Oxygen consumption of mixed leucocytes or PMNL A 3-channel Clark-type oxygen electrode (Hansatech, Kings Lynn, Norfolk, U.K.) was used
41 TABLE 1 DITHRANOL INDUCED STRAND BREAKS IN LEUCOCYTE DNA Treatment Leucocytes + 1/~g ml- 1dithranol
% DNA developing strand breaks 5.2+6.6
Leucocytes + 5 #g ml- 1dithranol
20.6+6.0 **
Leucocytes + 10 #g ml- 1dithranol
19.4+7.6 *
Leucocytes + 20 p.g ml- 1dithranol
35.7+8.4 **
Leucocytes + 40/Lg ml- 1dithranol
45.9+6.5 ***
Results are presented as the mean + SEM of 4 different experiments using leucocytes from different donors. * p < 0.05; * * p < 0.025; * * * p < 0.005 with respect to the solvent control sample.
to m e a s u r e c e l l u l a r o x y g e n c o n s u m p t i o n . T h e elect r o d e was r e c a l i b r a t e d e a c h d a y b e f o r e use a n d t h e results e x p r e s s e d as n m o l e s 0 2 u t i l i z e d / 5 m i n / 5 × 10 5 l e u c o c y t e s . M i x e d l e u c o c y t e s o r p u r i f i e d n e u t r o p h i l s (5 × 10 5) w e r e a d d e d to t h e w e l l h o u s ing the oxygen electrode which contained HBSS p r e w a r m e d to 3 7 ° C . Cells w e r e p r e i n c u b a t e d , w i t h stirring, for 15 m i n at 3 7 ° C . A d d i t i o n o f H - 7 (100 # M ) , B P B ( 5 / ~ M ) o r s t a u r o s p o r i n e (50 n g m l - t ) was as d e s c r i b e d p r e v i o u s l y . O n c o m p l e t i o n of p r e i n c u b a t i o n , 10 /xl d i t h r a n o l was a d d e d a n d i n c u b a t i o n c o n t i n u e d for a f u r t h e r 1 0 - 2 0 m i n as r e q u i r e d . T h e final v o l u m e o f t h e r e a c t i o n m i x was lml.
Viability studies N o n e o f t h e c o n c e n t r a t i o n s o f D M S O , dithranol, catalase, SOD, H-7, BPB or staurosporine u s e d h a v e a n y e f f e c t o n cell v i a b i l i t y as m e a s u r e d b y e o s i n (0.1%) e x c l u s i o n a n d l a c t a t e d e h y d r o g e n a s e release.
Statistical analysis R e s u l t s a r e e x p r e s s e d as m e a n v a l u e s + S E M . R e s u l t s o b t a i n e d i n d i t h r a n o l + c a t a l a s e o r respiratory burst-inhibitor-treated systems were compared with the corresponding untreated controis for e a c h e x p e r i m e n t u s i n g S t u d e n t ' s p a i r e d t test.
TABLE 2 EFFECTS OF ANTIOXIDANTS AND RESPIRATORY BURST INHIBITORS ON THE INDUCTION OF DNA STRAND BREAKS BY DITHRANOL Treatment
% DNA developing strand breaks
Leucocytes + 20 #g ml-1 dithranol
35.7 + 8.4
Leucocytes + 80/~g ml- 1catalase + 20/zg ml- 1dithranol
12.2+3.4 ***
Leucocytes + 33 #g ml- 1 SOD + 20/xg ml- 1dithranol
35.3+7.1
Leucocytes + 200 #g ml- ~ SOD + 20/~g ml- 1dithranol
35.5+5.2
Leucocytes+ 100/xM H-7 + 20 #g ml -~ dithranol
19.0+4.0 *
Leucocytes + 50 ng ml- 1 staurosporine + 20 #g ml- 1dithranol
11.6+7.9 *
Leucocytes + 5/~M BPB + 20 #g ml- ~dithranol
2.0+5.5 **
Results are expressed as the mean:l: SEM of 2-7 different experiments using leucocytes from different donors. * p < 0.05; * * p < 0.025; *** P < 0.005 with respect to the sample treated with dithranol alone.
42
Results
TABLE 4
Effect of dithranol on leucocyte DNA
EFFECT OF H-7, STAUROSPORINE, BPB AND CATALASE ON DITHRANOL-INDUCED LECL
D i t h r a n o l c o n c e n t r a t i o n s of 5 /~g m1-1 a n d greater i n d u c e d s t r a n d breaks in leucocyte D N A as shown i n T a b l e 1. W h e n C G D leucocytes were treated with 2 0 / t g ml -~ d i t h r a n o l they developed strand breaks in 10.26 _+ 1.72% of their D N A . While appreciable, this a m o u n t of d a m a g e is m u c h less t h a n that which occurs in n o r m a l leucocytes treated with this d i t h r a n o l c o n c e n t r a t i o n (35.7 + 8.4%), b u t indicates that d i t h r a n o l m a y also directly d a m a g e D N A . U s i n g these d a t a the direct i n j u r y b y dit h r a n o l accounts for a b o u t 28% of total d a m a g e to DNA. The a d d i t i o n of catalase, H-7, s t a u r o s p o r i n e or BPB to the m e d i u m significantly protected leucocyte D N A from d i t h r a n o l - i n d u c e d d a m a g e while SOD was w i t h o u t effect as shown in T a b l e 2.
Treatment Leucocytes (solvent control)
LECL 33.1+
8.2
Leucocytes + 20 # g ml - 1dithranol
770.4 + 146.2
Leucocytes+ 100/~g H-7+20 #g ml 1 dithranol
426.3 + 52.7 **
Leucocytes + 50 ng ml- 1 staurosporine + 20 ~agml- 1dithranol Leucocytes+5/tM BPB+20/tg ml 1 dithranol Leucocytes + 80/.tg ml 1catalase + 20/.tg ml- 1dithranol
20.5 _+ 8.2 * * * 9.0 +
0.8 *
38.8 +
4.1 *
Results are expressed as the mean peak LECL values in mVs- 1+ the SEMs of 3-4 different experiments using leucocytes from different donors. * p
Mutation Research, 247 (1991) 39-44
© 1991 ElsevierSciencePublishersB.V.0027-5107/91/$03.50 ADONIS 002751079100062H MUT 04939
Pro-oxidative interactions of dithranol with human phagocytes promote oxidative damage to DNA of bystander leucocytes D.M.T. Forrester, C.E.J. Van Rensburg and R. Anderson Medical Research Council Unit for the Study of Phagocyte Function, Department of Immunology, Institute for Pathology, University of Pretoria (Republic of South Africa)
(Received23 April 1990) (Revisionreceived27 July 1990) (Accepted27 August1990) Keywords: Dithranol;DNA strand breaks; Neutrophils; Lymphocytes;Oxidants
Summary Dithranol at therapeutic concentrations (5-40 ~tg m1-1) induced strand breaks in human leucocyte D N A in vitro in a dose-related manner. Leucocytes from individuals with chronic granulomatous disease
(CGD) incurred substantially less DNA strand breaks than did normal leucocytes during exposure to dithranol indicating that activated phagocytes are involved. H-7, 4-fl-bromophenacyl bromide (BPB) and staurosporine, all inhibitors of protein kinase C, decreased both dithranol-mediated activation of the phagocyte respiratory burst and induction of DNA strand breaks. Similar effects were observed with the hydrogen peroxide scavenger catalase. These results suggest that dithranol induces DNA strand breaks, mainly as a result of pro-oxidative interactions with phagocytes.
The anti-psoriatic drug dithranol (anthralin) is a tumour promoter for mouse skin (Bock and Bums, 1983) and has been shown to induce strand breaks in human leucocyte DNA in vitro by an unknown mechanism (Birnboim, 1983). Dithranol has also been shown to stimulate the oxidative metabolism of phagocytes, probably via the activation of protein kinase C (PKC) (Anderson, 1989). Activated phagocytes generate superoxide anion (02) from which a complex mixture of
reactive oxidants including hydrogen peroxide (H202) , hypochlorous acid (HOC1), hydroxyl radical ('OH) and lipid hydroperoxides is generated (Babior et al., 1988; Ochi and Cerutti, 1987; Jackson et al., 1989; Samuni et al., 1988). As H202, "OH and lipid hydroperoxides have been implicated in the induction of DNA strand breaks (Ochi and Cerutti, 1987; Meneghini, 1988; Schraufstatter et al., 1988; Jackson et al., 1989), the possibility of a relationship between phagocyte activation and DNA strand break induction by dithranol has been investigated.
Abbreviations: BPB, 4-fl-bromophenacyl bromide; CGD,
Methods
chronic granulomatous disease; LECL, luminol-enhanced chemiluminescence; PMNL, polymorphonuclearleucocytes; SOD, superoxidedismutase. Correspondence: Dr. C.E.J. Van Rensburg, Department of Immunology, Institute for Pathology,Universityof Pretoria, P.O. Box 2034, Pretoria0001 (Republicof South Africa).
Dithranol (1,8-dihydroxy-9-anthrone) was dissolved in dimethyl sulphoxide (DMSO) immediately before required and used at concentrations of 1-100 /~g m1-1. Solvent controls were always included in the various assays.
40 Leucocyte preparation A mixed leucocyte preparation was isolated as previously described (Birnboim and KanabusKaminska, 1985). Briefly, heparinized venous blood from adult human volunteers was mixed with a solution of NH4C1/Tris. HC1 for 15 min to lyse erythrocytes. Remaining contaminating erythrocytes were removed by hypotonic lysis. The leucocyte preparation was washed twice in indicator-free Hanks balanced salt solution (HBSS, Highveld Biological Supplies, Kelvin, R.S.A.), buffered with 1 mg m1-1 Hepes (N-2-hydroxyethyl piperazine-H-2 ethane sulphonic acid) and 0.28 mg m1-1 CaCIE, pH 7.4. Leucocytes were resuspended to a concentration of 107 ml -l in HBSS and generally consisted of about 70% polymorphonuclear leucocytes (PMNL). Most of the remaining cells were lymphocytes. Purified PMNL were prepared as previously described (Anderson et al., 1987) and resuspended to a concentration of 107 ml-1 in HBSS. Measurement of DNA strand breaks These were measured by alkaline unwinding and determination of ethidium bromide fluorescence using a Hitachi (Tokyo, Japan) model 650-10 fluorescence spectrophotometer with excitation at 520 nm and emission at 590 nm as described previously (Birnboim and Kanabus-Kaminska, 1985). Leucocytes were distributed to siliconized glass vacutainer tubes (106 cells ml - l ) containing prewarmed HBSS (37°C, 10 ml final volume) and preincubated for 15 min. Dithranol (1-40 lag ml-1) was added and incubation continued for a further 20 min when the reaction was stopped by chilling to 0°C and centrifugation for 10 min at 600 g. Treated cells were washed in cold HBSS and resuspended in 1.8 ml HBSS. If catalase (80 lag m1-1) or superoxide dismutase (SOD, 33 and 200 lag m1-1) were used, they were added immediately prior to the addition of dithranol. The respiratory burst inhibitors H-7 (1-(5-isoquinolinesulphonyl)-2-methylpiperazine, 100 laM), BPB (4fl-bromophenacyl bromide, 5 laM) (both obtained from Sigma Chemical Co., St. Louis, MO, U.S.A.), and staurosporine (50 ng m1-1) (Calbiochem) when used, were added before the addition of the cells, and so were present throughout preincubation. Preincubation with BPB was extended to 30
min to allow cellular uptake of the inhibitor whereas preincubation with H-7 was reduced to 5 min to ensure the stability of this inhibitor. The effect of dithranol (20 lag ml -l) on the DNA of leucocytes from two individuals with CGD was also tested. 200 #1 of cell suspension was added to each of a set of 9 tubes labelled 'B', 'P' and 'T' in triplicate and treated as previously described (Birnboim and Kanabus-Kaminska, 1985) except that incubation at 15°C was for 30 min. Untreated (solvent) controls were always included and the percentage of DNA developing strand breaks is calculated according to the formula: % DNA developing strand breaks = 100 - [(% doublestranded DNA remaining after treatment/% double-stranded DNA present in the untreated control sample) × 100]. Luminol-enhanced chemiluminescence (LECL) of mixed leucocytes or pure PMNL 10 6 mixed leucocytes (or PMNL) were added to polystyrene chemiluminescence vials (50 mm x 10 mm), which contained prewarmed (37°C) HBSS and 18 lag ml 1 luminol (5-amino-2,3-dihydro1,4-phthalazine dione; Sigma Chem. Co.; St. Louis; U.S.A.) and preincubated for 15 min at 37°C. 10 lal of the appropriate dilution of dithranol (1-40 lag ml -l) were then added and LECL recorded with an LKB Wallac 1251 chemiluminometer with results expressed in millivolts sec-1 (mVs- 1). Catalase (80 lag ml-1), when used, was added immediately prior to the addition of dithranol whereas if H-7 (100 laM), BPB (5 laM) or staurosporine (50 ng ml -~) were used, they were added before the addition of the cells and so were present throughout the preincubation period. Preincubation periods for BPB and H-7 were adjusted as previously described. The final reaction volume was 1 ml. Tests were also performed using CGD leucocytes. This condition is associated with a congenital deficiency of membrane-associated oxidative metabolism in phagocytes. Appropriate leucocyte-free control systems were included (e.g. luminol + dithranol) to monitor for possible nonspecific interactions of dithranol with the assay system. Oxygen consumption of mixed leucocytes or PMNL A 3-channel Clark-type oxygen electrode (Hansatech, Kings Lynn, Norfolk, U.K.) was used
41 TABLE 1 DITHRANOL INDUCED STRAND BREAKS IN LEUCOCYTE DNA Treatment Leucocytes + 1/~g ml- 1dithranol
% DNA developing strand breaks 5.2+6.6
Leucocytes + 5 #g ml- 1dithranol
20.6+6.0 **
Leucocytes + 10 #g ml- 1dithranol
19.4+7.6 *
Leucocytes + 20 p.g ml- 1dithranol
35.7+8.4 **
Leucocytes + 40/Lg ml- 1dithranol
45.9+6.5 ***
Results are presented as the mean + SEM of 4 different experiments using leucocytes from different donors. * p < 0.05; * * p < 0.025; * * * p < 0.005 with respect to the solvent control sample.
to m e a s u r e c e l l u l a r o x y g e n c o n s u m p t i o n . T h e elect r o d e was r e c a l i b r a t e d e a c h d a y b e f o r e use a n d t h e results e x p r e s s e d as n m o l e s 0 2 u t i l i z e d / 5 m i n / 5 × 10 5 l e u c o c y t e s . M i x e d l e u c o c y t e s o r p u r i f i e d n e u t r o p h i l s (5 × 10 5) w e r e a d d e d to t h e w e l l h o u s ing the oxygen electrode which contained HBSS p r e w a r m e d to 3 7 ° C . Cells w e r e p r e i n c u b a t e d , w i t h stirring, for 15 m i n at 3 7 ° C . A d d i t i o n o f H - 7 (100 # M ) , B P B ( 5 / ~ M ) o r s t a u r o s p o r i n e (50 n g m l - t ) was as d e s c r i b e d p r e v i o u s l y . O n c o m p l e t i o n of p r e i n c u b a t i o n , 10 /xl d i t h r a n o l was a d d e d a n d i n c u b a t i o n c o n t i n u e d for a f u r t h e r 1 0 - 2 0 m i n as r e q u i r e d . T h e final v o l u m e o f t h e r e a c t i o n m i x was lml.
Viability studies N o n e o f t h e c o n c e n t r a t i o n s o f D M S O , dithranol, catalase, SOD, H-7, BPB or staurosporine u s e d h a v e a n y e f f e c t o n cell v i a b i l i t y as m e a s u r e d b y e o s i n (0.1%) e x c l u s i o n a n d l a c t a t e d e h y d r o g e n a s e release.
Statistical analysis R e s u l t s a r e e x p r e s s e d as m e a n v a l u e s + S E M . R e s u l t s o b t a i n e d i n d i t h r a n o l + c a t a l a s e o r respiratory burst-inhibitor-treated systems were compared with the corresponding untreated controis for e a c h e x p e r i m e n t u s i n g S t u d e n t ' s p a i r e d t test.
TABLE 2 EFFECTS OF ANTIOXIDANTS AND RESPIRATORY BURST INHIBITORS ON THE INDUCTION OF DNA STRAND BREAKS BY DITHRANOL Treatment
% DNA developing strand breaks
Leucocytes + 20 #g ml-1 dithranol
35.7 + 8.4
Leucocytes + 80/~g ml- 1catalase + 20/zg ml- 1dithranol
12.2+3.4 ***
Leucocytes + 33 #g ml- 1 SOD + 20/xg ml- 1dithranol
35.3+7.1
Leucocytes + 200 #g ml- ~ SOD + 20/~g ml- 1dithranol
35.5+5.2
Leucocytes+ 100/xM H-7 + 20 #g ml -~ dithranol
19.0+4.0 *
Leucocytes + 50 ng ml- 1 staurosporine + 20 #g ml- 1dithranol
11.6+7.9 *
Leucocytes + 5/~M BPB + 20 #g ml- ~dithranol
2.0+5.5 **
Results are expressed as the mean:l: SEM of 2-7 different experiments using leucocytes from different donors. * p < 0.05; * * p < 0.025; *** P < 0.005 with respect to the sample treated with dithranol alone.
42
Results
TABLE 4
Effect of dithranol on leucocyte DNA
EFFECT OF H-7, STAUROSPORINE, BPB AND CATALASE ON DITHRANOL-INDUCED LECL
D i t h r a n o l c o n c e n t r a t i o n s of 5 /~g m1-1 a n d greater i n d u c e d s t r a n d breaks in leucocyte D N A as shown i n T a b l e 1. W h e n C G D leucocytes were treated with 2 0 / t g ml -~ d i t h r a n o l they developed strand breaks in 10.26 _+ 1.72% of their D N A . While appreciable, this a m o u n t of d a m a g e is m u c h less t h a n that which occurs in n o r m a l leucocytes treated with this d i t h r a n o l c o n c e n t r a t i o n (35.7 + 8.4%), b u t indicates that d i t h r a n o l m a y also directly d a m a g e D N A . U s i n g these d a t a the direct i n j u r y b y dit h r a n o l accounts for a b o u t 28% of total d a m a g e to DNA. The a d d i t i o n of catalase, H-7, s t a u r o s p o r i n e or BPB to the m e d i u m significantly protected leucocyte D N A from d i t h r a n o l - i n d u c e d d a m a g e while SOD was w i t h o u t effect as shown in T a b l e 2.
Treatment Leucocytes (solvent control)
LECL 33.1+
8.2
Leucocytes + 20 # g ml - 1dithranol
770.4 + 146.2
Leucocytes+ 100/~g H-7+20 #g ml 1 dithranol
426.3 + 52.7 **
Leucocytes + 50 ng ml- 1 staurosporine + 20 ~agml- 1dithranol Leucocytes+5/tM BPB+20/tg ml 1 dithranol Leucocytes + 80/.tg ml 1catalase + 20/.tg ml- 1dithranol
20.5 _+ 8.2 * * * 9.0 +
0.8 *
38.8 +
4.1 *
Results are expressed as the mean peak LECL values in mVs- 1+ the SEMs of 3-4 different experiments using leucocytes from different donors. * p
