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Peroxy Radical-Induced DNA Damage: Mutation and Base Damage Pattern

Hemv Rodrieuez’, Michael R. Valentine&d John Termin? *Biotechnology Division, NIST, Gaithersburg, MD, USA ‘Department of Molecular Biology, Beckman Research Institute, CA, USA

Oxidative Stress has been suggested to be a major contributor to aging and the degenerative diseases associated with aging, such as cancer, Alzheimer’s disease, Parkinson’s disease, male infertility, and diabetes. To better understand the molecular mechanisms underlying ROS-induced DNA damage, we investigated the frequencies of DNA base modifications and strand break densities caused by peroxy radical (ROO*) oxidation which has a half-life of 0.5-7 seconds, compared to the short lived hydroxyl radical (OH*) of lo’9 seconds. Transfection of DNA exposed to micmmolsr amounts of peroxy radical resulted in a 30-fold increase in mutation frequency in non-SOS inducible cells. Sequencing analysis of DNA isolated from mutants revealed that 88% consisted of transversions at G, with nearly equal number of G to C and G to T mutants. Electrophoretic analysis of peroxy radical treated DNA exposed to NaOH, Nth, and Fpg proteins demonstrated that abasic sites are not formed, while guanine modifications dominated the spectrum. Using LMPCR technology, we are currently mapping the sites of DNA damage along the ~53 and PGKI gene. Preliminary results reveal that the DNA sequence context determines the local probability of DNA damage as was previously shown with the hydroxyl radical (Rodriguez, H. et. al, 1997, Cancer Res. 57). Interestingly, the base damage signature for the peroxy radical is unique from that of the hydroxyl radical suggesting the involvement of different DNA damaging pathways.

DIASTEREOSELECI-IVE OXIDATION AND REPAIR OF PROTEIN METHIONINE RESIDUES Chnstrnn and Victor Sharm. Depnrtment o Phnrmaceutical Chemistry, University of Kansas, Lawrence, KS 6604 If

The oxidative modification of proteins is a hallmark of biological aging. Recently, we have demonstrated the age-dependent accumulation of methionine sulfoxide, L-MetSO, on the calcium-sensor protein calmodulin, associated with functional changes. An open question is why L-MetSO accumulates despite the presence of the repair enzyme methionine sulfoxide reductase.<BR> In general, the oxidation of L-Met can lead to two diastereomeric products, L-Met-d-SO and L-Met-l-SO. Here, we will show that the repair of protein-bound L-MetSO is diastereoselective to the L-Met-d-SO isoform, i.e. may be incomplete in vivo. Furthermore, the oxidation of protein L-Met by various biologically relevant reactive oxygen species (peroxyl radicals, singlet oxygen, hypochlorous acid and peroxynitrite) shows a preferred formation of the L-Met-l-SO diastereomer, i.e. the diastereomer not repaired by methionine sulfoxide reductase. For calmoduin, which contains 9 L-Met residues, the extent of diastereoselectivity depends on the location of the target L-Met residue in the protein, suggesting an effect of protein structure and sequence on the diastereoselectivity.

Together, these factors may account for the age-dependent accumulation of protein-bound L-MetSO observed in vivo. Support: NIA POlAG12993

PROOXIDANT PROPERTY OF ANTIESTROGEN ICI 182,780: PHYSIOLOGICAL RELEVANCE. Nalini Santanam and Sampath Parthasarathy. Department of Gynecology & Obstetrics, Emory University, Atlanta, GA.

The antioxidant property of estrogens is attributed for its cardioprotective effect. However, our recent studies (N. Santanam et al. J Lipid Res. 1998. 39:2111-2118) indicate that estradiol (E2) has both antioxidant and prooxidant property depending on its concentrations. Apart from its antioxidant property, estrogens also have other hormonal properties, which may be mediated through estrogen receptor. ICI 182,780 has been used extensively as an estrogen receptor antagonist. However, looking at the structure (figure 1) bofh E2 and ICI 182,780 have a phenolic group, which is attributed to its antioxidant property. We therefore, compared the antioxidant (or prooxidant) property of these two compounds. Both the compounds at l- 5 p M concentrations protected Isolated LDL

ICI 182,780

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(100 pg) against in vitro oxidation catalyzed by 5 p M copper. The ICI compound was less potent antioxidant compared to E2. The lag tme for 1 pM of E2 Vs ICI was (120 Vs 90 min). However surprismgly, at physiological concentrations (0.05 - 5 nM) similar to E2, ICI also promoted peroxidase mediated oxidation of LDL in vitro m a dose dependent manner. Again, ICI compound was less potent than E2 (lag time for 1 nm of E2 Vs ICI was 60 Vs 80 min). Since these effects are concentration dependent, we conclude that one has to use caution in interpreting the data when regarding the participation of cellular receptor(s).

L-CARNOSINE @-ALANYL-L-HISTIDINE) AND L- HISTIDINE BUT NOT P-ALANINE PREVENT PROTEIN GLYCATION Norbert W. Seidler, Sharagim S. Shokry, and Justin Nauth University of Health Sciences, Department of Biochemistry, Kansas City, MO 64106

Glyceraldehyde 3-phosphate (Glyc3P) glycates (or non- enzymatically glycosylates) cardiac aspartate aminotransferase (cAAT) and thereby inhibits cAAT activity and causes distinct changes in the cAAT electrophoretic mobility. L-Camosine (L- Car) protects against Glyc3P-induced loss of enzyme activity and prevents Glyc3P-induced changes in protein structure. This study examined the chemical components of L-Car, which is a dipeptide @-alanyl-L-histidine), and determined the ability of these components to prevent Glyc3P-induced protein modification. Purified cAAT was incubated with 1OmM of either L-Car, b-alanine (B-Ala), L-histidine (L-His) or imidazole in the presence or absence of Glyc3P (5OOpM) at 37°C for 4 days and analyzed by SDS-PAGE. L-Car and L-His, but not P-Ala or imidazole, prevented protein modification. When cAAT was incubated with Glyc3P (500p.M) at 37°C for 5 days at various concentrations of L-His, we observed protection against protein glycation at L-His to Glyc3P ratios of 5: 1. These observations suggest that the histidinyl moiety of L-Car may be in part responsible for L-Car’s anti-glycation properties.

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