European Journal of Pharmacology, 44 (1977) 191--193
191
© Elsevier/North-Holland Biomedical Press Short c o m m u n i c a t i o n THE PREVENTION OF ALLOXAN-INDUCED DIABETES IN MICE BY DIMETHYL SULFOXIDE RICHARD E. HEIKKILA Department of Neurology, Mount Sinai School of Medicine, Fifth Avenue and lOOth Street, New York, New York 10029, U.S.A.
Received 26 April 1977, accepted 27 April 1977
R.E. HEIKILA, The prevention of alloxan-induced diabetes in mice by dimethyl suIfoxide, European J. Pharmacol. 44 (1977) 191--193. Dimethyl sutfoxide (DMSO, 7.3 g/kg) administered to mice prior to alloxan completely protected against the diabetogenic actions of 50 mg/kg alloxan. The same close of DMSO provided a partial protection against 75 mg/kg alloxan. This protection against alloxan-induced diabetes is consistent with the scavenging of the hydroxyl radical by DMSO. Dimethyl sulfoxide
DMSO
Alloxan-induced diabetes
1. Introduction Alloxan is a widely used pharmacological tool which causes an experimental diabetes when injected into laboratory animals such as rats or mice (Rerup, 1970). Alloxan is selectively taken up by the insulin-producing fl cells of the pancreas and subsequently causes their destruction (Hammarstrom and Ullberg, 1966). Two of the characteristics of alloxaninduced diabetes are a degranulation o f the beta cells as determined by histochemical means and an elevated blood glucose (see for example, Rerup, 1970). We have previously shown that ethanol and several other structurally similar alcohols, as well as thiourea, all of which are very good h y d r o x y l radical scavengers, protected mice from the diabetogenic actions of alloxan (Heikkila et al., 1976). These data indicated a role for the h y d r o x y l radical in alloxaninduced diabetes. In the present s t u d y we have tested the capacity of dimethyl sulfoxide (DMSO} to prevent alloxan-induced diabetes. DMSO, a widely used pharmacological agent, was chosen because it is an excellent h y d r o x y l
Hydroxyl radical
radical scavenger (Ashwood-Smith, 1975) and also because it is a relatively non-toxic agent which can be tolerated by animals at rather high doses (Rubin, 1975). The data of the" present study indicate t h a t DMSO can protect against the diabetogenic action of alloxan.
2. Materials and methods Male Swiss-Webster mice (25 -+ 3 g) were used in the experiments. Food and water were withheld from the mice 3--4 h prior to atloxan administration. 30 min prior to alloxan, the mice received an i.p. injection of 0.02 ml/ g of isotonic saline or of DMSO [7.3 g/kg, 33% DMSO in saline (v/v)]. After the half hour wait, the mice were injected into the tail vein with 50 or 75 mg of alloxan/kg in saline (0.2 ml/animal). The alloxan solution was made just prior to injection and kept on ice and used as quickly as possible, since it is felt that alloxan is unstable (Rerup, 1970). 1 h after alloxan, the mice were given free access to food and water. The mice were decapitated 72 h after alloxan, 100 pl blood was col-
192
R.E. HEIKKILA
lected, and blood glucose was d e t e r m i n e d by a glucose oxidase assay. The DMSO was obtained f r om Fisher Chemicals (Fairlawn, New Jersey, U.S.A.), the alloxan m o n o h y d r a t e from Calbiochem (Los Angeles, Cal., U.S.A.) and the glucose assay kit from Worthington Biochemicals (Freehold, New Jersey, U.S.A.).
mals receiving DMSO prior to 75 mg/kg alloxan, 7 had blood glucose levels less than 175 mg%, which can be considered within the normal range. The other 4 mice had blood glucose levels of 3 0 3 , 3 9 8 , 4 1 5 and 485 mg%. This compares with the range of 424--711 mg% in animals receiving 75 mg/kg alloxan alone. DMSO by itself had no effect on 72 h blood glucose levels (table 1).
3. Results 4. Discussion Alloxan administration caused the e x p e c t e d increase in blood glucose characteristic o f experimental diabetes at bot h 50 and 75 mg/kg (table 1). Note the higher mean blood glucose level in the mice receiving 75 mg/kg. Animals injected with DMSO prior to 50 mg/kg alloxan were com pl e t e l y p r o t e c t e d against the diabetogenic action of alloxan and exhibited blood glucose levels in the normal range at 72 h. Most animals treated with DMSO prior to 75 mg/kg of alloxan were prot e c t e d against th e diabetogenic actions of alloxan (table 1). For example, of the 11 ani-
TABLE 1 Blood glucose levels in Swiss-Webster mice 72 h after alloxan administration (50 mg/kg in Experiment A, 75 mg/kg in Experiment B). Some mice received DMSO (7.3 g/kg) 0.5 h prior to alloxan. Experiments were run as described in Materials and methods. The number of animals in each group is in parentheses. Group
Blood glucose (mg% +_S.E.M.)
Experiment A Control DMSO AUoxan DMSO + alloxan
120 ± 7 114 _+10 411± 30 131 ± 11
(10) (9) (17) (13) 1
Experiment B Control DMSO Alloxan DMSO + alloxan
115+- 15 131 -+ 11 573 -+ 26 266 ~ 40
(8) (9) (13) (11) 1
1 p < 0.001 compared to alloxan alone.
Alloxan can be reduced in vitro to dialuric acid by agents like ascorbic acid (Deamer et al., 1971). Dialuric acid can then spontaneously react in vitro with oxygen (autoxidize) to form h y d r o g e n peroxide (H202) the superoxide radical (O2 ;) and the h y d r o x y l radical (.OH) (Cohen and Heikkila, 1974). H202, the two-electron reduction p r o d u c t of oxygen, has been det ect ed in vivo in mice after the administration of alloxan (Heikkila et al., 1974). It is likely that 02% the one electron reduction p r o d u c t of oxygen, and .OH, which can form by a reaction bet w een H202 and O2 ~ (Haber and Weiss, 1934), also form in vivo after alloxan administration. All three of these species (H202, 02% and .OH) are very reactive with k n o w n cytotoxicities and might be the causative species in alloxan-induced diabetes. In particular .OH is known to be very toxic and is t h o u g h t to be the primary species responsible for the damaging effects of ionizing radiation (Ashwood-Smith, 1975). In a previous study we d e m o n s t r a t e d how several .OH trapping agents p r o t e c t e d against alloxan-induced diabetes (Heikkila et al., 1976). There was a good correlation between the capacity of the agents tested to react with the h y d r o x y l radical and their capacity to prevent alloxan-induced diabetes when t hey were administered to mice at relatively high doses. This suggested that .OH might be responsible for alloxan-induced diabetes. If this were so, ot her chemicals can be administered in relatively high doses to experimental animals and which have a high reactivity with .OH should
DMSO AND ALLOXAN DIABETES also p r o t e c t against a l l o x a n - i n d u c e d d i a b e t e s . D M S O can be given t o e x p e r i m e n t a l anim a l s in high doses. F o r e x a m p l e , it has b e e n r e p o r t e d t h a t t h e LDs0 o f D M S O i n j e c t e d i.p. in rats is 23 g/kg a n d in m i c e is b e t w e e n 14.7 a n d 17.7 g / k g ( R u b i n , 1 9 7 5 ) . M o r e o v e r , doses as high as 1 g / k g h a v e b e e n a d m i n i s t e r e d cutan e o u s l y to h u m a n s o v e r e x t e n d e d (14 d a y ) p e r i o d s ( R u b i n , 1 9 7 5 ) . D M S O is also highly r e a c t i v e w i t h t h e h y d r o x y l radical, w h i c h as s t a t e d a b o v e is g e n e r a t e d b y ionizing radiation. The well-known radioprotective effects o f D M S O are t h o u g h t t o be d u e t o its reactivit y w i t h t h e h y d r o x y l radical ( A s h w o o d S m i t h , 1 9 7 5 ) . I t has b e e n r e c e n t l y s h o w n t h a t several -OH t r a p p i n g agents including D M S O c o u l d i n h i b i t p r o s t a g l a n d i n b i o s y n t h e s i s (Pang a n a m a l a et al., 1976). This suggested t h a t •O H m i g h t be an i m p o r t a n t i n t e r m e d i a t e in t h e b i o s y n t h e s i s o f p r o s t a g l a n d i n s and m a y perhaps help explain the anti-inflammatory a c t i o n s o f D M S O ( P a n g a n a m a l a et al., 1976). In t h e c u r r e n t s t u d y D M S O c o m p l e t e l y p r o t e c t e d against t h e d i a b e t o g e n i c a c t i o n s o f a l l o x a n at 50 m g / k g a n d p a r t i a l l y p r o t e c t e d against a l l o x a n at 75 m g / k g {table 1, Results section). A l t h o u g h t h e r e are p e r h a p s alternative e x p l a n a t i o n s f o r t h e p r o t e c t i o n against the alloxan-induced diabetes, the protection by DMSO, taken together with our previously p u b l i s h e d d a t a on the p r e v e n t i o n o f alloxani n d u c e d d i a b e t e s b y .OH t r a p p e r s {Heikkila et al., 1 9 7 6 ) s u p p o r t t h e c o n c e p t t h a t .OH is an i m p o r t a n t t o x i c i n t e r m e d i a t e in alloxani n d u c e d d i a b e t e s . It is t h u s i n t e r e s t i n g t h a t a species like .OH m i g h t p e r h a p s be crucial in p r o s t a g l a n d i n s y n t h e s i s b u t w h e n g e n e r a t e d in a relatively u n c o n t r o l l e d f a s h i o n , as f r o m a l l o x a n o r f r o m ionizing r a d i a t i o n , m a y cause cell d e a t h .
193 Acknowledgements This work was supported by the Clinical Research Center for Parkinson's and Allied Diabetes and by Grant NS-05184 from the U.S. Public Health Service. The assistance of Mr. Bradley Winston in preliminary experiments is greatly appreciated. References Ashwood-Smith, M.J., 1975, Current concepts concerning radioprotective and cryoprotective properties of dimethyl sulfoxide in cellular systems, Ann. N.Y. Acad. Sci. 243,246. Cohen, G. and R.E. Heikkila, 1974, The generation of hydrogen peroxide, superoxide radical and hydroxyl radical by 6-hydroxydopamine, dialuric acid and related cytotoxic agents, 249, 2447. Deamer, D.W., R.E. Heikkila, R.V. Panganamala, G. Cohen and D.G. Cornwell, 1971, The alloxan-dialuric acid cycle and the generation of hydrogen peroxide, Physiol. Chem. Physics 3, 26. Haber, F. and J. Weiss, 1934, The catalytic decomposition of hydrogen peroxide by iron salts, Proc. Roy. Soc. Ser. A., 147,332. Hammarstrom, L. and S. Ullberg, 1966, Specific uptake of labelled alloxan in the pancreatic islets, Nature (London) 212,708. Heikkila, R.E., H. Barden and G. Cohen, 1974, Prevention of alloxan-induced diabetes by ethanol administration, J. Pharmacol. Exptl. Therap. 190, 501. Heikkila, R.E., B. Winston, G. Cohen and H. Barden, 1976, Alloxan-induced diabetes: evidence for hydroxyl radical as a cytotoxic intermediate, Biochem. Pharmacol. 25, 1085. Panganamala, R.V., H.V. Sharma, R.E. Heikkila, J.C. Geer and D.G. Cornwell, 1976, Role of hydroxyl radical scavengers dimethyl sulfoxide, alcohols and methional in the inhibition of prostaglandin biosynthesis, Prostaglandins 11, 599. Rerup, C.C., 1970, Drugs producing diabetes through damage of the insulin secreting cells, Pharmacol. Rev. 22,485. Rubin, L.F., 1975, Toxicity of dimethyl sulfoxide alone and in combination, Ann. N.Y. Acad. Sci. 243, 98.
191
© Elsevier/North-Holland Biomedical Press Short c o m m u n i c a t i o n THE PREVENTION OF ALLOXAN-INDUCED DIABETES IN MICE BY DIMETHYL SULFOXIDE RICHARD E. HEIKKILA Department of Neurology, Mount Sinai School of Medicine, Fifth Avenue and lOOth Street, New York, New York 10029, U.S.A.
Received 26 April 1977, accepted 27 April 1977
R.E. HEIKILA, The prevention of alloxan-induced diabetes in mice by dimethyl suIfoxide, European J. Pharmacol. 44 (1977) 191--193. Dimethyl sutfoxide (DMSO, 7.3 g/kg) administered to mice prior to alloxan completely protected against the diabetogenic actions of 50 mg/kg alloxan. The same close of DMSO provided a partial protection against 75 mg/kg alloxan. This protection against alloxan-induced diabetes is consistent with the scavenging of the hydroxyl radical by DMSO. Dimethyl sulfoxide
DMSO
Alloxan-induced diabetes
1. Introduction Alloxan is a widely used pharmacological tool which causes an experimental diabetes when injected into laboratory animals such as rats or mice (Rerup, 1970). Alloxan is selectively taken up by the insulin-producing fl cells of the pancreas and subsequently causes their destruction (Hammarstrom and Ullberg, 1966). Two of the characteristics of alloxaninduced diabetes are a degranulation o f the beta cells as determined by histochemical means and an elevated blood glucose (see for example, Rerup, 1970). We have previously shown that ethanol and several other structurally similar alcohols, as well as thiourea, all of which are very good h y d r o x y l radical scavengers, protected mice from the diabetogenic actions of alloxan (Heikkila et al., 1976). These data indicated a role for the h y d r o x y l radical in alloxaninduced diabetes. In the present s t u d y we have tested the capacity of dimethyl sulfoxide (DMSO} to prevent alloxan-induced diabetes. DMSO, a widely used pharmacological agent, was chosen because it is an excellent h y d r o x y l
Hydroxyl radical
radical scavenger (Ashwood-Smith, 1975) and also because it is a relatively non-toxic agent which can be tolerated by animals at rather high doses (Rubin, 1975). The data of the" present study indicate t h a t DMSO can protect against the diabetogenic action of alloxan.
2. Materials and methods Male Swiss-Webster mice (25 -+ 3 g) were used in the experiments. Food and water were withheld from the mice 3--4 h prior to atloxan administration. 30 min prior to alloxan, the mice received an i.p. injection of 0.02 ml/ g of isotonic saline or of DMSO [7.3 g/kg, 33% DMSO in saline (v/v)]. After the half hour wait, the mice were injected into the tail vein with 50 or 75 mg of alloxan/kg in saline (0.2 ml/animal). The alloxan solution was made just prior to injection and kept on ice and used as quickly as possible, since it is felt that alloxan is unstable (Rerup, 1970). 1 h after alloxan, the mice were given free access to food and water. The mice were decapitated 72 h after alloxan, 100 pl blood was col-
192
R.E. HEIKKILA
lected, and blood glucose was d e t e r m i n e d by a glucose oxidase assay. The DMSO was obtained f r om Fisher Chemicals (Fairlawn, New Jersey, U.S.A.), the alloxan m o n o h y d r a t e from Calbiochem (Los Angeles, Cal., U.S.A.) and the glucose assay kit from Worthington Biochemicals (Freehold, New Jersey, U.S.A.).
mals receiving DMSO prior to 75 mg/kg alloxan, 7 had blood glucose levels less than 175 mg%, which can be considered within the normal range. The other 4 mice had blood glucose levels of 3 0 3 , 3 9 8 , 4 1 5 and 485 mg%. This compares with the range of 424--711 mg% in animals receiving 75 mg/kg alloxan alone. DMSO by itself had no effect on 72 h blood glucose levels (table 1).
3. Results 4. Discussion Alloxan administration caused the e x p e c t e d increase in blood glucose characteristic o f experimental diabetes at bot h 50 and 75 mg/kg (table 1). Note the higher mean blood glucose level in the mice receiving 75 mg/kg. Animals injected with DMSO prior to 50 mg/kg alloxan were com pl e t e l y p r o t e c t e d against the diabetogenic action of alloxan and exhibited blood glucose levels in the normal range at 72 h. Most animals treated with DMSO prior to 75 mg/kg of alloxan were prot e c t e d against th e diabetogenic actions of alloxan (table 1). For example, of the 11 ani-
TABLE 1 Blood glucose levels in Swiss-Webster mice 72 h after alloxan administration (50 mg/kg in Experiment A, 75 mg/kg in Experiment B). Some mice received DMSO (7.3 g/kg) 0.5 h prior to alloxan. Experiments were run as described in Materials and methods. The number of animals in each group is in parentheses. Group
Blood glucose (mg% +_S.E.M.)
Experiment A Control DMSO AUoxan DMSO + alloxan
120 ± 7 114 _+10 411± 30 131 ± 11
(10) (9) (17) (13) 1
Experiment B Control DMSO Alloxan DMSO + alloxan
115+- 15 131 -+ 11 573 -+ 26 266 ~ 40
(8) (9) (13) (11) 1
1 p < 0.001 compared to alloxan alone.
Alloxan can be reduced in vitro to dialuric acid by agents like ascorbic acid (Deamer et al., 1971). Dialuric acid can then spontaneously react in vitro with oxygen (autoxidize) to form h y d r o g e n peroxide (H202) the superoxide radical (O2 ;) and the h y d r o x y l radical (.OH) (Cohen and Heikkila, 1974). H202, the two-electron reduction p r o d u c t of oxygen, has been det ect ed in vivo in mice after the administration of alloxan (Heikkila et al., 1974). It is likely that 02% the one electron reduction p r o d u c t of oxygen, and .OH, which can form by a reaction bet w een H202 and O2 ~ (Haber and Weiss, 1934), also form in vivo after alloxan administration. All three of these species (H202, 02% and .OH) are very reactive with k n o w n cytotoxicities and might be the causative species in alloxan-induced diabetes. In particular .OH is known to be very toxic and is t h o u g h t to be the primary species responsible for the damaging effects of ionizing radiation (Ashwood-Smith, 1975). In a previous study we d e m o n s t r a t e d how several .OH trapping agents p r o t e c t e d against alloxan-induced diabetes (Heikkila et al., 1976). There was a good correlation between the capacity of the agents tested to react with the h y d r o x y l radical and their capacity to prevent alloxan-induced diabetes when t hey were administered to mice at relatively high doses. This suggested that .OH might be responsible for alloxan-induced diabetes. If this were so, ot her chemicals can be administered in relatively high doses to experimental animals and which have a high reactivity with .OH should
DMSO AND ALLOXAN DIABETES also p r o t e c t against a l l o x a n - i n d u c e d d i a b e t e s . D M S O can be given t o e x p e r i m e n t a l anim a l s in high doses. F o r e x a m p l e , it has b e e n r e p o r t e d t h a t t h e LDs0 o f D M S O i n j e c t e d i.p. in rats is 23 g/kg a n d in m i c e is b e t w e e n 14.7 a n d 17.7 g / k g ( R u b i n , 1 9 7 5 ) . M o r e o v e r , doses as high as 1 g / k g h a v e b e e n a d m i n i s t e r e d cutan e o u s l y to h u m a n s o v e r e x t e n d e d (14 d a y ) p e r i o d s ( R u b i n , 1 9 7 5 ) . D M S O is also highly r e a c t i v e w i t h t h e h y d r o x y l radical, w h i c h as s t a t e d a b o v e is g e n e r a t e d b y ionizing radiation. The well-known radioprotective effects o f D M S O are t h o u g h t t o be d u e t o its reactivit y w i t h t h e h y d r o x y l radical ( A s h w o o d S m i t h , 1 9 7 5 ) . I t has b e e n r e c e n t l y s h o w n t h a t several -OH t r a p p i n g agents including D M S O c o u l d i n h i b i t p r o s t a g l a n d i n b i o s y n t h e s i s (Pang a n a m a l a et al., 1976). This suggested t h a t •O H m i g h t be an i m p o r t a n t i n t e r m e d i a t e in t h e b i o s y n t h e s i s o f p r o s t a g l a n d i n s and m a y perhaps help explain the anti-inflammatory a c t i o n s o f D M S O ( P a n g a n a m a l a et al., 1976). In t h e c u r r e n t s t u d y D M S O c o m p l e t e l y p r o t e c t e d against t h e d i a b e t o g e n i c a c t i o n s o f a l l o x a n at 50 m g / k g a n d p a r t i a l l y p r o t e c t e d against a l l o x a n at 75 m g / k g {table 1, Results section). A l t h o u g h t h e r e are p e r h a p s alternative e x p l a n a t i o n s f o r t h e p r o t e c t i o n against the alloxan-induced diabetes, the protection by DMSO, taken together with our previously p u b l i s h e d d a t a on the p r e v e n t i o n o f alloxani n d u c e d d i a b e t e s b y .OH t r a p p e r s {Heikkila et al., 1 9 7 6 ) s u p p o r t t h e c o n c e p t t h a t .OH is an i m p o r t a n t t o x i c i n t e r m e d i a t e in alloxani n d u c e d d i a b e t e s . It is t h u s i n t e r e s t i n g t h a t a species like .OH m i g h t p e r h a p s be crucial in p r o s t a g l a n d i n s y n t h e s i s b u t w h e n g e n e r a t e d in a relatively u n c o n t r o l l e d f a s h i o n , as f r o m a l l o x a n o r f r o m ionizing r a d i a t i o n , m a y cause cell d e a t h .
193 Acknowledgements This work was supported by the Clinical Research Center for Parkinson's and Allied Diabetes and by Grant NS-05184 from the U.S. Public Health Service. The assistance of Mr. Bradley Winston in preliminary experiments is greatly appreciated. References Ashwood-Smith, M.J., 1975, Current concepts concerning radioprotective and cryoprotective properties of dimethyl sulfoxide in cellular systems, Ann. N.Y. Acad. Sci. 243,246. Cohen, G. and R.E. Heikkila, 1974, The generation of hydrogen peroxide, superoxide radical and hydroxyl radical by 6-hydroxydopamine, dialuric acid and related cytotoxic agents, 249, 2447. Deamer, D.W., R.E. Heikkila, R.V. Panganamala, G. Cohen and D.G. Cornwell, 1971, The alloxan-dialuric acid cycle and the generation of hydrogen peroxide, Physiol. Chem. Physics 3, 26. Haber, F. and J. Weiss, 1934, The catalytic decomposition of hydrogen peroxide by iron salts, Proc. Roy. Soc. Ser. A., 147,332. Hammarstrom, L. and S. Ullberg, 1966, Specific uptake of labelled alloxan in the pancreatic islets, Nature (London) 212,708. Heikkila, R.E., H. Barden and G. Cohen, 1974, Prevention of alloxan-induced diabetes by ethanol administration, J. Pharmacol. Exptl. Therap. 190, 501. Heikkila, R.E., B. Winston, G. Cohen and H. Barden, 1976, Alloxan-induced diabetes: evidence for hydroxyl radical as a cytotoxic intermediate, Biochem. Pharmacol. 25, 1085. Panganamala, R.V., H.V. Sharma, R.E. Heikkila, J.C. Geer and D.G. Cornwell, 1976, Role of hydroxyl radical scavengers dimethyl sulfoxide, alcohols and methional in the inhibition of prostaglandin biosynthesis, Prostaglandins 11, 599. Rerup, C.C., 1970, Drugs producing diabetes through damage of the insulin secreting cells, Pharmacol. Rev. 22,485. Rubin, L.F., 1975, Toxicity of dimethyl sulfoxide alone and in combination, Ann. N.Y. Acad. Sci. 243, 98.
