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480 Session 2: Measurement of Oxidative Stress in viva 2~5 EVIDENCE FOR IN VIVO OXIDATIVE DAMAGE ASSOCIATED WITH KETOACIDOSIS: HEINZ BODY FORMATION IN DIABETIC CATS Mary M. Christopher, John Broussard and Mark E. Peterson Department of Physiological Sciences, University of Florida, Gainesville, FL 32610 USA; Animal Medical Center, New York, NY 10021 USA. DETECTION OF C8-METHYLGUANINE IN DNA FROM RATS 2:6 ADMINISTERED THE INORGANIC COMPOUND HYDRAZINE L.F. Gomes; S.S. Zavan and 0. August0 School of Pharmaceutical Sciences and Institute of Chemistry, Univeraidade de Sao Paulo, Sao Paulo, S.P., CP 20780,01498-970 Brasil Feline hemoglobin is uniquely susceptible to oxidative denaturation such that Heinz body formation is a highly sensitive indicator of in vivo oxidative stress in this species. Oxidative damage is considered to play an important role both in the pathogenesis of diabetes and in the development of diabetic complications. We investigated a number of biochemical changes in 30 cats with spontaneous diabetes mellitus (and 15 control cats) and evaluated the relationship of these changes to erythrocyte oxidative damage. Diabetic cats had significantly more Heinz bodies (11.62 i 2.90%) than control cats (0.57 f 0.15%), and ketotic cats had significantly more Heinz bodies (23.07 f 9.55%) than non-ketotic cats. Percent Heinz bodies directly correlated with plasma p- hydroxybutyrate concentration (r2 = 0.386; p = 0.0002), as well as with hypochloridemia and increased anion gap. Only cats with >30% Heinz bodies had decreased erythrocyte CSH. There was not a significant association between Heinz bodies and degree of anemia, hyperglycemia, or glycosylated hemoglobin. Indeed, in vitro experiments indicated that feline RBC were relatively impermeable to external glucose. Ketotic cats also had significantly elevated plasma levels of D-lactate (372.4 k 86.7 pmol/L) compared to non-ketotic (143.6 + 56.2 pmol/L) and control (39.4 + 15.6 pmol/L) cats. These data indicate that ketone metabolism, rather than hyper- glycemia, is associated with oxidative hemoglobin damage in cats, and implicate cytochrome I’450 2El as a potential source of oxygen radical generation in animals with ketosis. Baaed on in vitro studies, we have recently suggested that metabolic activation of formaldehyde hydrazone to methyl radicals may be of importance in regard to hydrazine-mediated DNA alkylation. Previously, it has been demonstrated that hydrazine (NH2NH2) methylates DNA with formation of N7- MeGua and O%leGua, products expected from methyldiazonium ion attack on DNA (Becker et al., Carcinogenesis, 2: 1181-1188, 1981). We now report the identification of C8-MeGua, the product expected from methyl radical attack on DNA in hydrolysates of liver DNA from rats treated with hydrazine. First, an improved procedure for detecting and identifying C8-MeGua was developed based on HPLC with electrochemical detection (El/2 = 0.93V). The levels of methylated adducts (pmol/mol Gua) obtained in the liver of treated animals (single LD and 20 for C8-MeGua, 7 6 dose, 6h exposure) were: 520. 5000 N -MeGua and ObMeGua respectively. Parallel in viw spin-trapping experiments with N-tert-butyla- phenylnitrone (PBN) demonstrate biliary excretion of a PBN- radical adduct whose splitting constants (aN = 162mT; aH = 0.36mT) are similar to those expected for the PBN-methyl radical adduct. Taken together these results indicate that methyl radicals may be important in hydrazine mediated genotoxicity. 2:7 CELLULAR ELECTROPHYSIOLOGY AS A MEASURE OF IN VW0 ESR SPIN TRAPPING EVIDENCE FOR HYDROXYL 2:8 OXIDATIVE DAMAGE IN CARDIAC MYOCYTES. RADICAL MEDIATED TOXICITY OF PARAQUAT AND COPPER Peggy L. Barrington, IN RATS. Department of Pharmacology, Northwestern University, Chicago, Illinois, 6061 1 USA. A variety of pathological conditions including several degenerative diseases associated with aging may involve oxidative damage. Damage to cell membranes is thought to be part of oxidative pathology, therefore monitoring membrane function should be an assay for oxidative damage in intact cells. Isolated ventricular myocytes from either cat or dog hearts were exposed to free radical generating systems: dihydroxyfumaric acid, xanthine/xanthine oxidase or H,O,. Cellular membrane potentials were recorded with either high resistance microelectrodes or lower resistance patch-pipettes. The action potential configuration was altered in a progressive, reproducible, sequence. The effects were qualitatively the same and independent of the generating system or animal species used. The reproducible sequence of changes in the action potential implies that oxidative damage occurs at specific site(s) within the cell. Interactions occurred between the patch- pipette recording method and the appearance of oxidative damage. These experimental artifacts could be accounted for with detailed experimental design and use of multiple regressions and analyses of variance. Cells exposed via the patch-pipette to intracellular EGTA I5 mM) and extracellular H,O, were monitored during voltage clamp protocols to determine if H,O, altered cellular action potentials by direct, modification of specific ion channels. The results suggest that H,O, does not act directly on channel proteins. The changes in membrane function do not result from direct damage to ion channel proteins but more likely reflect damage to regulatory or metabolic functions of the cell. The exact site(s) of oxidative damage and the underlying electrophysiological mechanism of the altered action potentials remain to be determined. Ronald P. Mason, Phillip M. Hanna and Maria B. Kadiiska National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC 27709 The electron spin resonance (ESR) spin trapping technique was employed to study the role of Cu(ll) in the generation of hydroxyl radical during paraquat (Pa”) intoxication in rats. A secondary radical trapping technique was used for the detection of hydroxyl radical generated in viva during copper- mediated PQ” toxicity. According to this technique (Burkitt and Mason (1991) Proc. Nat. Acad. Sci. USA 88: 8440-8444), the hydroxyl radical generated reacts with dimethyl sulfoxide (DMSO) in viva to form the methyl radical, which is then spin trapped by phenyl A!-terf-butylnitrone (PBN). The relatively stable methyl radical adduct of PBN was detected in the bile of rats 2 hours after treatment with simultaneous doses of CuSO,. PQ*‘, PBN, and DMSO. Bile samples were collected into Cu(l)- and Fe(ll)-stabilizing agents in order to prevent the occurrence of radical adducts generated exvivo in bile during its collection. The analysis of radical adducts excreted via the biliary route provide strong ESR evidence for the generation of the hydroxyl radical as a result of the known futile enzymatic redox cycling of PQ*‘, with copper playing an essential mediatory role. No radical adducts were detected when either CuSO, or PQ” was excluded. From a different perspective, in viva copper- dependent hydroxyl radical generation could be said to be promoted by PQ”. This is the first report of ESR evidence for this synergetic hydroxyl radical generation by copper and PQ” in a whole animal.

Cellular electrophysiology as a measure of oxidative damage in cardiac myocytes

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480 Session 2: Measurement of Oxidative Stress in viva

2~5 EVIDENCE FOR IN VIVO OXIDATIVE DAMAGE ASSOCIATED WITH KETOACIDOSIS: HEINZ BODY FORMATION IN DIABETIC CATS Mary M. Christopher, John Broussard and Mark E. Peterson Department of Physiological Sciences, University of Florida, Gainesville, FL 32610 USA; Animal Medical Center, New York, NY 10021 USA.

DETECTION OF C8-METHYLGUANINE IN DNA FROM RATS 2:6

ADMINISTERED THE INORGANIC COMPOUND HYDRAZINE L.F. Gomes; S.S. Zavan and 0. August0

School of Pharmaceutical Sciences and Institute of Chemistry,

Univeraidade de Sao Paulo, Sao Paulo, S.P.,

CP 20780,01498-970 Brasil

Feline hemoglobin is uniquely susceptible to oxidative denaturation such that Heinz body formation is a highly sensitive indicator of in vivo oxidative stress in this species. Oxidative damage is considered to play an important role both in the pathogenesis of diabetes and in the development of diabetic complications. We investigated a number of biochemical changes in 30 cats with spontaneous diabetes mellitus (and 15 control cats) and evaluated the relationship of these changes to erythrocyte oxidative damage. Diabetic cats had significantly more Heinz bodies (11.62 i 2.90%) than control cats (0.57 f 0.15%), and ketotic cats had significantly more Heinz bodies (23.07 f 9.55%) than non-ketotic cats. Percent Heinz bodies directly correlated with plasma p- hydroxybutyrate concentration (r2 = 0.386; p = 0.0002), as well as with hypochloridemia and increased anion gap. Only cats with >30% Heinz bodies had decreased erythrocyte CSH. There was not a significant association between Heinz bodies and degree of anemia, hyperglycemia, or glycosylated hemoglobin. Indeed, in vitro experiments indicated that feline RBC were relatively impermeable to external glucose. Ketotic cats also had significantly elevated plasma levels of D-lactate (372.4 k 86.7 pmol/L) compared to non-ketotic (143.6 + 56.2 pmol/L) and control (39.4 + 15.6 pmol/L) cats. These data indicate that ketone metabolism, rather than hyper- glycemia, is associated with oxidative hemoglobin damage in cats, and implicate cytochrome I’450 2El as a potential source of oxygen radical generation in animals with ketosis.

Baaed on in vitro studies, we have recently suggested that

metabolic activation of formaldehyde hydrazone to methyl

radicals may be of importance in regard to hydrazine-mediated

DNA alkylation. Previously, it has been demonstrated that

hydrazine (NH2NH2) methylates DNA with formation of N7-

MeGua and O%leGua, products expected from methyldiazonium ion attack on DNA (Becker et al.,

Carcinogenesis, 2: 1181-1188, 1981). We now report the

identification of C8-MeGua, the product expected from methyl

radical attack on DNA in hydrolysates of liver DNA from rats

treated with hydrazine. First, an improved procedure for detecting

and identifying C8-MeGua was developed based on HPLC with

electrochemical detection (El/2 = 0.93V). The levels of

methylated adducts (pmol/mol Gua) obtained in the liver of

treated animals (single LD

and 20 for C8-MeGua, 7 6 dose, 6h exposure) were: 520. 5000

N -MeGua and ObMeGua respectively.

Parallel in viw spin-trapping experiments with N-tert-butyla-

phenylnitrone (PBN) demonstrate biliary excretion of a PBN-

radical adduct whose splitting constants (aN = 162mT; aH =

0.36mT) are similar to those expected for the PBN-methyl radical

adduct. Taken together these results indicate that methyl radicals

may be important in hydrazine mediated genotoxicity.

2:7 CELLULAR ELECTROPHYSIOLOGY AS A MEASURE OF IN VW0 ESR SPIN TRAPPING EVIDENCE FOR HYDROXYL 2:8 OXIDATIVE DAMAGE IN CARDIAC MYOCYTES. RADICAL MEDIATED TOXICITY OF PARAQUAT AND COPPER Peggy L. Barrington, IN RATS. Department of Pharmacology, Northwestern University, Chicago, Illinois, 6061 1 USA.

A variety of pathological conditions including several degenerative diseases associated with aging may involve oxidative damage. Damage to cell membranes is thought to be part of oxidative pathology, therefore monitoring membrane function should be an assay for oxidative damage in intact cells. Isolated ventricular myocytes from either cat or dog hearts were exposed to free radical generating systems: dihydroxyfumaric acid, xanthine/xanthine oxidase or H,O,. Cellular membrane potentials were recorded with either high resistance microelectrodes or lower resistance patch-pipettes. The action potential configuration was altered in a progressive, reproducible, sequence. The effects were qualitatively the same and independent of the generating system or animal species used. The reproducible sequence of changes in the action potential implies that oxidative damage occurs at specific site(s) within the cell. Interactions occurred between the patch- pipette recording method and the appearance of oxidative damage. These experimental artifacts could be accounted for with detailed experimental design and use of multiple regressions and analyses of variance. Cells exposed via the patch-pipette to intracellular EGTA I5 mM) and extracellular H,O, were monitored during voltage clamp protocols to determine if H,O, altered cellular action potentials by direct, modification of specific ion channels. The results suggest that H,O, does not act directly on channel proteins. The changes in membrane function do not result from direct damage to ion channel proteins but more likely reflect damage to regulatory or metabolic functions of the cell. The exact site(s) of oxidative damage and the underlying electrophysiological mechanism of the altered action potentials remain to be determined.

Ronald P. Mason, Phillip M. Hanna and Maria B. Kadiiska National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC 27709

The electron spin resonance (ESR) spin trapping technique was employed to study the role of Cu(ll) in the generation of hydroxyl radical during paraquat (Pa”) intoxication in rats. A secondary radical trapping technique was used for the detection of hydroxyl radical generated in viva during copper- mediated PQ” toxicity. According to this technique (Burkitt and Mason (1991) Proc. Nat. Acad. Sci. USA 88: 8440-8444), the hydroxyl radical generated reacts with dimethyl sulfoxide (DMSO) in viva to form the methyl radical, which is then spin trapped by phenyl A!-terf-butylnitrone (PBN). The relatively stable methyl radical adduct of PBN was detected in the bile of rats 2 hours after treatment with simultaneous doses of CuSO,. PQ*‘, PBN, and DMSO. Bile samples were collected into Cu(l)- and Fe(ll)-stabilizing agents in order to prevent the occurrence of radical adducts generated exvivo in bile during its collection. The analysis of radical adducts excreted via the biliary route provide strong ESR evidence for the generation of the hydroxyl radical as a result of the known futile enzymatic redox cycling of PQ*‘, with copper playing an essential mediatory role. No radical adducts were detected when either CuSO, or PQ” was excluded. From a different perspective, in viva copper- dependent hydroxyl radical generation could be said to be promoted by PQ”. This is the first report of ESR evidence for this synergetic hydroxyl radical generation by copper and PQ” in a whole animal.