Semester

Summer

Date of Graduation

2001

Document Type

Dissertation

Degree Type

PhD

College

Davis College of Agriculture, Natural Resources and Design

Department

Biochemistry

Committee Chair

Gregory W. Konat.

Abstract

This study addresses the genotoxicity of oxidative stress. We induced oxidative stress in cultured rat oligodendrocytes by exposure to hydrogen peroxide (H2O2). Intact genomic DNA was isolated by agarose embedding and its fragmentation was assessed by field inversion gel electrophoresis (FIGE). To detect single strand fragmentations, the DNA was digested with S1 nuclease before FIGE. We found that the exposure of oligodendrocytes to pathological concentrations of H2O2, induces a rapid digestion of nuclear chromatin in a pattern consistent with higher order chromatin degradation (HOCD). HOCD, the hallmark of programmed cell death (PCD), proceeds through single strand scissions that accumulate at matrix attachment regions (MAR). Within 10 min of the addition of 1mM H2O2, a discrete pool representing approximately 45% of oligodendrocyte chromatin underwent single strand digestion into ≥400 kb fragments. Subsequently, chromatin was digested into 50--200kb fragments. Detectable chromatin digestion could be elicited with H2O2 concentrations as low as 50 muM. Chelating intracellular calcium with 1,2-bis( o-aminophenoxy)ethane-N,N,N',N' -tetraacetic acid tetra(acetoxymethyl) ester (BAPTA/AM) prior to H 2O2 exposure inhibited HOCD in a dose dependent manner. However, neither removing extracellular calcium, nor blocking calcium release from mitochondria and endoplasmic reticulum with 8-(N,N-diethylamino)-ocyl-3,4,5-trimethoxybenzoate (TMB-8) affected the digestion. Moreover, increasing intracellular calcium with A23187 calcium ionophore did not induce HOCD. These results indicate that HOCD is not directly mediated by calcium, but resting level of calcium is required for the process. Subsequent study in nuclei purified from C6 glioma cells revealed that the endonuclease responsible for digestion is calcium-independent, but magnesium-dependent. Magnesium-induced HOCD was not affected by the removal of calcium from nuclei with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N ',N'-tetraacetic acid (EGTA), but was practically inhibited in nuclei prepared from BABTA/AM-pretreated cells. In conclusion, although H2O2-induced HOCD is not directly mediated by the increase of intracellular calcium concentration, normal resting levels of intracellular calcium are required for the maintenance of MAR-associated, magnesium-dependent endonuclease in an active form. HOCD induced by oxidative stress may be an important mutagenic factor leading to cellular transformation via somatic mutations.

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