Andrew Jenner

Oxidative Damage to Proteins, Lipids, and DNA in Cortical Brain Regions from Patients with Dementia with Lewy Bodies

Abstract: Dementia with Lewy bodies (DLB) forms the second most common pathological subgroup of dementia after Alzheimers disease. The present study compares the levels of oxidative damage to proteins, lipids, and DNA bases in cortical brain areas from patients with DLB with levels in matched control tissues. Overall, there was a trend for protein carbonyl levels to be increased in all areas, but a significant difference was found only in the parietal and temporal lobes. No differences were observed in the levels of lipid peroxidation. Measurement of products of damage to DNA bases showed increased levels of thymine glycol, 8‐hydroxyguanine, 2,6‐diamino‐4‐hydroxy‐5‐formamidopyrimidine, 5‐hydroxycytosine, 5‐hydroxyuracil, 5‐hydroxymethyluracil, and xanthine. Xanthine levels were increased in the DLB group in the parietal, occipital, and temporal lobes, indicating that peroxynitrite or other deaminating species may be involved. The finding of increased protein carbonyls and increased DNA base products in cortical regions from DLB patients indicates that oxidative stress may play a role in DLB.

DNA damage in human respiratory tract epithelial cells: damage by gas phase cigarette smoke apparently involves attack by reactive nitrogen species in addition to oxygen radicals

Treatment of human respiratory tract tracheobronchial epithelial cells with gas‐phase cigarette smoke led to dose‐dependent DNA strand breakage that was highly correlated with multiple chemical modifications of all four DNA bases. The pattern of base damage suggests attack by hydroxyl radicals (OH:). However, by far the most important base damage in quantitative terms was formation of xanthine and hypoxanthine, presumably resulting from deamination of guanine and adenine respectively. Hence, DNA damage by cigarette smoke may involve reactive nitrogen species as well as reactive oxygen species.

DNA strand breakage and base modification induced by hydrogen peroxide treatment of human respiratory tract epithelial cells

Treatment of human respiratory tract epithelial cells with H2O2 led to concentration‐dependent DNA strand breakage that was highly‐correlated with multiple chemical modifications of all four DNA bases, suggesting that damage is due to hydroxyl radical, OH.. However, the major base damage occurred to adenine. Hence, conclusions made about the occurrence and the extent of oxidative DNA damage on the basis only of changes in 8‐hydroxyguanine should be approached with caution.

Vitamin C Protects Against Hypochlorous Acid–Induced Glutathione Depletion and DNA Base and Protein Damage in Human Vascular Smooth Muscle Cells

Hypochlorous acid (HOCl), generated by myeloperoxidase released from activated macrophages, is thought to contribute to vascular dysfunction and oxidation of low density lipoproteins (LDLs) in atherogenesis. We have previously shown that HOCl exposure can cause chlorination and oxidation of isolated DNA and that vitamin C protects human arterial smooth muscle cells against oxidized LDL–mediated damage. We report in the present study that vitamin C attenuates HOCl-induced DNA base and protein damage and depletion of intracellular glutathione (GSH) and ATP in human arterial smooth muscle cells. Cells were pretreated in the absence or presence of 100 &mgr;mol/L vitamin C (24 hours) and then exposed to HOCl (0 to 500 &mgr;mol/L, 0 to 60 minutes) in the absence of vitamin C. Intracellular GSH and ATP levels were depleted by HOCl treatment, and gas chromatography–mass spectroscopy revealed a concentration- and time-dependent increase in DNA base oxidation and protein damage (measured as 3-chlorotyrosine). Pretreatment of smooth muscle cells with vitamin C significantly reduced the extent of HOCl-induced DNA and protein damage and attenuated decreases in intracellular ATP and GSH. Our findings suggest that physiological levels of vitamin C provide an important antioxidant defense against HOCl-mediated injury in atherosclerosis.

Determination of oxidative DNA base damage by gas chromatography-mass spectrometry. Effect of derivatization conditions on artifactual formation of certain base oxidation products

GC-MS is a widely used tool to measure oxidative DNA damage because of its ability to identify a wide range of base modification products. However, it has been suggested that the derivatization procedures required to form volatile products prior to GC-MS analysis can sometimes produce artifactual formation of certain base oxidation products, although these studies did not replicate previously-used reaction conditions, e.g. they failed to remove air from the derivatization vials. A systematic examination of this problem revealed that levels of 8-hydroxyguanine, 8-hydroxyadenine, 5-hydroxycytosine and 5-(hydroxymethyluracil) in commercial calf thymus DNA determined by GC-MS are elevated by increasing the temperature at which derivatization is performed in our laboratory. In particular, 8-hydroxyguanine levels after silylation at 140 degrees C were raised 8-fold compared to derivatization at 23 degrees C. Experiments on the derivatization of each undamaged base revealed that the artifactual oxidation of guanine, adenine, cytosine and thymine respectively was responsible. Formation of the above products was potentiated by not purging with nitrogen prior to derivatization. Increasing the temperature to 140 degrees C or allowing air to be present during derivatization did not significantly increase levels of the other oxidized bases measured. This work suggests that artifactual oxidation during derivatization is restricted to certain products (8-hydroxyguanine, 8-hydroxyadenine, 5-hydroxycytosine and 5-[hydroxymethyluracil]) and can be decreased by reducing the temperature of the derivatization reaction to 23 degrees C and excluding as much air possible. Despite some recent reports, we were easily able to detect formamidopyrimidines in acid-hydrolyzed DNA. Artifacts of derivatization are less marked than has been claimed in some papers and may vary between laboratories, depending on the experimental procedures used, in particular the efficiency of exclusion of O2 during the derivatization process.

Influence of inducers and inhibitors of cytochrome P450 on the hepatotoxicity of hydrazine in vivo

Hydrazine hepatotoxicity in vivo, as manifested by triglyceride accumulation, depletion of ATP and reduced glutathione (GSH) was shown to be dose related. The effect of pretreatment of rats with various inhibitors and inducers of cytochrome P450 on these dose-response relationships was investigated. Pretreatment with the inhibitor piperonyl butoxide increased triglyceride accumulation whereas pretreatment with the inducers phenobarbital and β-naphthoflavone (BNF) resulted in reduced triglyceride accumulation. Pretreatment with the inducers acetone and isoniazid also enhanced triglyceride accumulation. Only phenobarbital pretreatment also significantly reduced GSH and ATP depletion. A linear correlation was found between hepatic glutathione and ATP levels in non-pretreated animals given various doses of hydrazine. However, exponential relationships were found between hepatic triglycerides and both hepatic ATP and glutathione. The results suggest that i) the hepatotoxicity of hydrazine can be modulated by inducing or inhibiting particular isoenzymes of cytochrome P450, ii) ATP and GSH depletion may not be directly involved in the development of fatty liver.

Effect of acute and repeated exposure to low doses of hydrazine on hepatic microsomal enzymes and biochemical parameters in vivo

A single dose of hydrazine (3 mg·kg−1 i.p.) caused hepatic accumulation of triglycerides and depletion of ATP in rats after 9 h. Repeated exposure of rats to hydrazine (≊2.5 mg·kg−1 per day) for 10 days resulted in depletion of hepatic reduced glutathione (GSH) and triglycerides. Repeated exposure to hydrazine also caused a significant (time dependent) induction ofp-nitrophenol hydroxylase (NPH) activity together with changes in other hepatic microsomal enzymes. These included 7-pentoxyresorufinO-deethylase (PROD) and 7-ethoxyresorufin O-de ethylase (EROD) activity, total cytochrome P450, cytochrome b5 and cytochrome P450 reductase activity. Repeated exposure to lower levels of hydrazine (≊0.250 mg·kg−1 per day) caused no significant hepatic biochemical or microsomal changes after 5 or 10 days except for an increase in NPH activity (17%) and liver ATP (15%) after 5 days.