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Noninvasive monitoring of oxidative stress via DNA biomarkers
174
Cancer and Oxidative Stress
18.29 RADIATION DEATH AND ACUTE RADIATION TOXICITY: MODIFICATION BY DIETARY SQUALENE Suk Y. Oh, Heidi Storm, Stata Norton, Bruce Kimler Department of Dietetics and Nutrition, Pharmacology, Toxicology and Therapeutics, Radiation Oncology, University of Kansas Medical Center, Kansas City, KS 66103 It is hypothesized that dietary squalene protects against
radiation injury by scavenging reactive oxygen species. Squalene, a water insoluble intern~ediate in the biosynthesis of cholesterol, is a polyunsaturated isoprenojd containing six isoprene units. The purpose of the present study was to compare the effects of a squalene-supplemented diet (2% by weight) and a regular diet on body weights, leukocyte counts and survival rates of C3H mice following exposure to ionizing radiation. After a feeding period of 2 weeks, animals were exposed to one of three doses of ionizing radiation, 6 Gy, 7 Gy or 8 Gy. Survivors were held for 30 days post irradiation. Mice did not lose weight at 6 Gy. At 7 Gy, surviving mice (squalene-treated) began to lose weight 16d post irradiation. Less leukopenia was observed in squalene (SQ)-fed mice than in control (C) mice at all radiation doses. Percent lymphocytes was significantly higher in SQ-fed mice (p= <0.02), as indicated by WBC differential. At 6 and 7 Gy, squalene-supplemented animals survived significantly longer than animals fed the control diet (6 Gy: SQ, no deaths, C, ST50=21.4; 7 Gy: SQ, ST50 = 15.7, C, ST50-12.2, where ST50-probit calculation of day at which 50% of animals died). Survival rates were not statistically different at 8 Gy. It is concluded that squalene reduces the toxicity of ionizing radiation, possibly by interfering with free radical reactions.
18.31
N O N I N V A S I V E M O N I T O R I N G OF O X I D A T I V E STRESS V I A DNA BIOMARKERS M i c h a e l G. Simic and D a v i d S. B e r g t o l d N a t i o n a l I n s t i t u t e of S t a n d a r d s and Technology, Gaithersburg, MD 20899, U.S.A. D e s p i t e n u m e r o u s complexities, the u l t i m a t e goal of m o n i t o r i n g free r a d i c a l p r o c e s s e s in vivo has been s u c c e s s f u l in a limited n u m b e r of cases. T h y m i d i n e glycol and 8 - h y d r o x y d e o x y g u a n e s i n e , two p r o d u c t s of ON r a d i c a l r e a c t i o n s w i t h DNA, have b e e n t e s t e d for s p e c i f i c i t y of OH r a d i c a l reactions in vivo. In i r r a d i a t e d m i c e and humans, the i n c r e a s e of t h e s e p r o d u c t s in the urine is p r o p o r t i o n a l to the total e n e r g y input, i.___~e.,the n u m b e r of O H r a d i c a l s g e n e r a t e d in soft tissue b y i o n i z i n g radiations. This finding supports the v a l u e of t h e s e p r o d u c t s as true b i o m a r k e r s of OH r a 4 i c a l r e a c t i o n s in vivo. In the a b s e n c e of radiation, w e find that d i e t a r y caloric intake in humans p l a y s a critical role in the levels of t h e s e b i o m a r k e r s in the urine. The c o r r e l a t i o n b e t w e e n r a d i a t i o n - i n d u c e d and e n d o g e n o u s g e n e r a t i o n of t h e s e O H radical b i o m a r k e r s in v i v o s t r o n g l y indicates c o n t i n u o u s g e n e r a t i o n of O H r a d i c a l s by m e t a b o l i c processes. This is further c o r r o b o r a t e d by the new f i n d i n g that d i e t a r y c o m p o s i t i o n p l a y s a critical role in the u r i n a r y levels of t h e s e biomarkers. The a p p a r e n t c o r r e l a t i o n b e t w e e n b i o m a r k e r levels, d i e t a r y calories, type of foods, l o n g e v i t y , and cancer will be p r e s e n t e d and p o s s i b l e m e c h a n i s m s of o x i d a t i v e stress discussed.
DETECTION OF CONJUGATED DIENES OF DIFFERENT SOURCE IN RAT'S TISSUE LIPIDS
18.30
Sebastiano Banni, Fraucesco P. Corongiu, Maria A. Dessi, Benito LombardiI and Maria G. Salgo. Dipartimento di Biologia Sperimentale, Sezione di Patologia Sperimentale, Universita' di Cagliari, Cagliari, Italy. 1Department of Pathology, School of Medicine, University of Pittsburgh, Pittsburgh, PA. Partially hydrogenated fats (PHF) are widely consumed by humans and are used in the preparation of semi-synthetic diets for biological studies in experimental animals. It is well established that partial hydrogenation of fats generates a wide range of unusual positional and geometric fatty acid isomers that are absorbed and deposited in human and rat tissues. As a byproduct of partial hydrogenation, some (0.3-0.6%) of the fatty acids contain conjugated dienes. Conjugated dienes are also formed in the oxidation of polyunsaturated fatty acids (PUFA's) to hydroperoxides, and because of their strong absorption in the U.V. region around 234 nm they have been used for years to detect lipid peroxidation. Thus, detection of conjugated dienes in tissue lipids of rats fed a diet containing PHF could lead to a misinterpretation of the results. We came across recently one such an example, in trying to confirm the occurrence of liver membrane lipid peroxidation in rats fed a choline-devoid (CD) diet. The purified CD diets used in our and other laboratories for carcinogenesis studies have a high content of fat (15%), 10% of which is Primex, a commercial mixture of partially hydrogenated soybean and palm oil, and 5% corn oil. ~These diets are hepatocarcinogenic, and have been claimed to trigger a peroxidation of liver membrane lipids that would lead to initiation of liver cells. In this communication we describe methodological approaches to differentiate the source of conjugated dienes in animals fed diets containing PHF.
LIPID P E R O X I D E AND SCHIFF R E A C T I V E M A T E R I A L PRODUCED BY G L A - T R E A T E D CANCER CELLS Schumpei Takeda, David F. Horrobin, Mehar Manku, G r e g o r y Ells, Tim Sanford and V a l e r i e Simmons Efamel R e s e a r c h Institute, PO Box 818, Kentville, NS, Canada B4N 4H8 G a m m a - l i n o l e n i c acid (GLA) is an essential fatty acid w h i c h kills cancer cells at doses which do not harm normal cells. Iron p o t e n t i a t e s the lethal effect while vitamin E (VE) inhibits it. We have i n v e s t i g a t e d lipid p e r o x i d a t i o n in human breast cancer cells (ZR-75-1) by: i. Schiff staining of cultured living cancer cells. 2. Detection of t r i p h e n y l p h o s p h i n e oxide (TPP0) formation. 3. Detection of c o n j u g a t e d diene formation at 235-245 nm absorption. ZR-75-1 cells were seeded at 5,000 cells/cm2 with 10% F B S M E M : next day m e d i u m was replaced with 5% F B S M E M c o n t a i n i n g 100~M GLA, 100~M GLA + 10~g FeCI2.4H20 or no additive. Three days after supplementation, cells in the 24-well m u l t i d i s h were stained with S c h i f f ' s r e a g e n t , followed by lipid e x t r a c t i o n for Schiff reactive material (SRM) and assay at 544 n m in m e t h a n o l solution. Tumor cell killing, LDH activity, SRM, the a b s o r b a n c e at 235-245 n m and TPPO formation were highest in the GLA + Fe supplemented cells with the cultures w i t h GLA ~ alone giving intermediate values, and lowest in the control cells. These results indicate that Tumor cell k i l l i n g b y G L A i s a c c o m p a n i e d by increased lipid p e r o x i d a t i o n in cancer cells. Normal cells n e i t h e r undergo lipid p e r o x i d a t i o n nor die when exposed to similar GLA concentrations. These o b s e r v a t i o n s suggest new ways of t r e a t i n g cancer w i t h o u t harming normal cells.
18.32
Cancer and Oxidative Stress
18.29 RADIATION DEATH AND ACUTE RADIATION TOXICITY: MODIFICATION BY DIETARY SQUALENE Suk Y. Oh, Heidi Storm, Stata Norton, Bruce Kimler Department of Dietetics and Nutrition, Pharmacology, Toxicology and Therapeutics, Radiation Oncology, University of Kansas Medical Center, Kansas City, KS 66103 It is hypothesized that dietary squalene protects against
radiation injury by scavenging reactive oxygen species. Squalene, a water insoluble intern~ediate in the biosynthesis of cholesterol, is a polyunsaturated isoprenojd containing six isoprene units. The purpose of the present study was to compare the effects of a squalene-supplemented diet (2% by weight) and a regular diet on body weights, leukocyte counts and survival rates of C3H mice following exposure to ionizing radiation. After a feeding period of 2 weeks, animals were exposed to one of three doses of ionizing radiation, 6 Gy, 7 Gy or 8 Gy. Survivors were held for 30 days post irradiation. Mice did not lose weight at 6 Gy. At 7 Gy, surviving mice (squalene-treated) began to lose weight 16d post irradiation. Less leukopenia was observed in squalene (SQ)-fed mice than in control (C) mice at all radiation doses. Percent lymphocytes was significantly higher in SQ-fed mice (p= <0.02), as indicated by WBC differential. At 6 and 7 Gy, squalene-supplemented animals survived significantly longer than animals fed the control diet (6 Gy: SQ, no deaths, C, ST50=21.4; 7 Gy: SQ, ST50 = 15.7, C, ST50-12.2, where ST50-probit calculation of day at which 50% of animals died). Survival rates were not statistically different at 8 Gy. It is concluded that squalene reduces the toxicity of ionizing radiation, possibly by interfering with free radical reactions.
18.31
N O N I N V A S I V E M O N I T O R I N G OF O X I D A T I V E STRESS V I A DNA BIOMARKERS M i c h a e l G. Simic and D a v i d S. B e r g t o l d N a t i o n a l I n s t i t u t e of S t a n d a r d s and Technology, Gaithersburg, MD 20899, U.S.A. D e s p i t e n u m e r o u s complexities, the u l t i m a t e goal of m o n i t o r i n g free r a d i c a l p r o c e s s e s in vivo has been s u c c e s s f u l in a limited n u m b e r of cases. T h y m i d i n e glycol and 8 - h y d r o x y d e o x y g u a n e s i n e , two p r o d u c t s of ON r a d i c a l r e a c t i o n s w i t h DNA, have b e e n t e s t e d for s p e c i f i c i t y of OH r a d i c a l reactions in vivo. In i r r a d i a t e d m i c e and humans, the i n c r e a s e of t h e s e p r o d u c t s in the urine is p r o p o r t i o n a l to the total e n e r g y input, i.___~e.,the n u m b e r of O H r a d i c a l s g e n e r a t e d in soft tissue b y i o n i z i n g radiations. This finding supports the v a l u e of t h e s e p r o d u c t s as true b i o m a r k e r s of OH r a 4 i c a l r e a c t i o n s in vivo. In the a b s e n c e of radiation, w e find that d i e t a r y caloric intake in humans p l a y s a critical role in the levels of t h e s e b i o m a r k e r s in the urine. The c o r r e l a t i o n b e t w e e n r a d i a t i o n - i n d u c e d and e n d o g e n o u s g e n e r a t i o n of t h e s e O H radical b i o m a r k e r s in v i v o s t r o n g l y indicates c o n t i n u o u s g e n e r a t i o n of O H r a d i c a l s by m e t a b o l i c processes. This is further c o r r o b o r a t e d by the new f i n d i n g that d i e t a r y c o m p o s i t i o n p l a y s a critical role in the u r i n a r y levels of t h e s e biomarkers. The a p p a r e n t c o r r e l a t i o n b e t w e e n b i o m a r k e r levels, d i e t a r y calories, type of foods, l o n g e v i t y , and cancer will be p r e s e n t e d and p o s s i b l e m e c h a n i s m s of o x i d a t i v e stress discussed.
DETECTION OF CONJUGATED DIENES OF DIFFERENT SOURCE IN RAT'S TISSUE LIPIDS
18.30
Sebastiano Banni, Fraucesco P. Corongiu, Maria A. Dessi, Benito LombardiI and Maria G. Salgo. Dipartimento di Biologia Sperimentale, Sezione di Patologia Sperimentale, Universita' di Cagliari, Cagliari, Italy. 1Department of Pathology, School of Medicine, University of Pittsburgh, Pittsburgh, PA. Partially hydrogenated fats (PHF) are widely consumed by humans and are used in the preparation of semi-synthetic diets for biological studies in experimental animals. It is well established that partial hydrogenation of fats generates a wide range of unusual positional and geometric fatty acid isomers that are absorbed and deposited in human and rat tissues. As a byproduct of partial hydrogenation, some (0.3-0.6%) of the fatty acids contain conjugated dienes. Conjugated dienes are also formed in the oxidation of polyunsaturated fatty acids (PUFA's) to hydroperoxides, and because of their strong absorption in the U.V. region around 234 nm they have been used for years to detect lipid peroxidation. Thus, detection of conjugated dienes in tissue lipids of rats fed a diet containing PHF could lead to a misinterpretation of the results. We came across recently one such an example, in trying to confirm the occurrence of liver membrane lipid peroxidation in rats fed a choline-devoid (CD) diet. The purified CD diets used in our and other laboratories for carcinogenesis studies have a high content of fat (15%), 10% of which is Primex, a commercial mixture of partially hydrogenated soybean and palm oil, and 5% corn oil. ~These diets are hepatocarcinogenic, and have been claimed to trigger a peroxidation of liver membrane lipids that would lead to initiation of liver cells. In this communication we describe methodological approaches to differentiate the source of conjugated dienes in animals fed diets containing PHF.
LIPID P E R O X I D E AND SCHIFF R E A C T I V E M A T E R I A L PRODUCED BY G L A - T R E A T E D CANCER CELLS Schumpei Takeda, David F. Horrobin, Mehar Manku, G r e g o r y Ells, Tim Sanford and V a l e r i e Simmons Efamel R e s e a r c h Institute, PO Box 818, Kentville, NS, Canada B4N 4H8 G a m m a - l i n o l e n i c acid (GLA) is an essential fatty acid w h i c h kills cancer cells at doses which do not harm normal cells. Iron p o t e n t i a t e s the lethal effect while vitamin E (VE) inhibits it. We have i n v e s t i g a t e d lipid p e r o x i d a t i o n in human breast cancer cells (ZR-75-1) by: i. Schiff staining of cultured living cancer cells. 2. Detection of t r i p h e n y l p h o s p h i n e oxide (TPP0) formation. 3. Detection of c o n j u g a t e d diene formation at 235-245 nm absorption. ZR-75-1 cells were seeded at 5,000 cells/cm2 with 10% F B S M E M : next day m e d i u m was replaced with 5% F B S M E M c o n t a i n i n g 100~M GLA, 100~M GLA + 10~g FeCI2.4H20 or no additive. Three days after supplementation, cells in the 24-well m u l t i d i s h were stained with S c h i f f ' s r e a g e n t , followed by lipid e x t r a c t i o n for Schiff reactive material (SRM) and assay at 544 n m in m e t h a n o l solution. Tumor cell killing, LDH activity, SRM, the a b s o r b a n c e at 235-245 n m and TPPO formation were highest in the GLA + Fe supplemented cells with the cultures w i t h GLA ~ alone giving intermediate values, and lowest in the control cells. These results indicate that Tumor cell k i l l i n g b y G L A i s a c c o m p a n i e d by increased lipid p e r o x i d a t i o n in cancer cells. Normal cells n e i t h e r undergo lipid p e r o x i d a t i o n nor die when exposed to similar GLA concentrations. These o b s e r v a t i o n s suggest new ways of t r e a t i n g cancer w i t h o u t harming normal cells.
18.32