Hydrogen peroxide and coffee induce G:C-->T:A transversions in the lacI gene of catalase-defective Escherichia coli.
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The mutagenicity of hydrogen peroxide (H2O2) was compared with that of coffee, a complex mixture which generates H2O2. An Escherichia coli strain defective in catalase activity (katG katE double mutant) and carrying a single copy mucAB (pRW144) plasmid was constructed to enhance the mutagenic response to oxidants. The ability of the mucAB genes to influence the type, frequency and distribution of H2O2-induced mutations was also investigated in isogenic bacteria lacking pRW144. Induced mutational spectra were characterized and compared with that of spontaneous mutagenesis. A total of 444 independent forward mutations affecting the first 210 bp of the lacI gene were identified by DNA sequence analysis. The spontaneous mutation spectrum showed no bias (P = 0.52) for substitutions at G:C base pairs. In contrast, in the H2O2-induced spectrum substitutions occurred preferentially at G:C base pairs (P < 0.0001) with a preponderance of G:C-->T:A transversions (43.4% of H2O2-induced mutants versus 17.3% of spontaneous mutants). These data support the view that 7,8-dihydro-8-oxoguanine is the main premutagenic lesion induced by H2O2 and that catalase-defective bacteria have elevated levels of 8-oxoguanine in chromosome DNA after H2O2 exposure. Coffee produced a similar distribution of mutational events as H2O2 (P > 0.05), suggesting that this compound may be the main cause of the coffee-induced mutagenesis. The presence of plasmid pRW144 did not affect the frequency of H2O2-induced G:C-->T:A transversions, but caused an increase in A:T-->T:A transversions and a decrease in -1 base frameshifts. Although the frequencies of G:C-->T:A transversions were similar in all three induced spectra (H2O2 and coffee +/- pRW144), differences were observed in location of mutations throughout the target gene. (+info)
Tissue and organ expression of catalase in acatalasemic beagle dogs.
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Acatalasemic Beagle dogs which were maintained in our laboratories showed no sign of catalase activity at all in the erythrocytes, and glutathione peroxidase and superoxide dismutase were at normal levels. Immunoblotting analysis demonstrated that no catalase protein is detectable in their erythrocytes. On the other hand, catalase activity was detected in other tissues and organs, albeit at varying, lower levels than in normal dogs. Quantitative immunoblotting analysis consistently demonstrated that the catalase protein is expressed in the liver and kidneys of acatalasemic dogs in proportion to the activity in these organs. The catalase mRNA expressions in the blood, liver and kidneys in acatalasemic dogs were almost the same as those in normal dogs. These results suggested that catalytically normal catalase protein is translated from mRNA in the tissues and organs including erythrocytes, but in erythrocytes this enzyme protein is disposed of by an unknown mechanism. (+info)
cDNA cloning and expression of mutant catalase from the hypocatalasemic mouse: comparison with the acatalasemic mutant.
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Mutant catalase cDNAs from the hypocatalasemic and acatalasemic mice were cloned and expressed in bacteria. A novel missense mutation, Asp (AAT) to Ser (AGT), was identified at amino acid position 439 of the hypocatalasemic catalase. Analysis of recombinant catalase mutants revealed that the mutation is responsible for the reduced activity of hypocatalasemic catalase and the unstable tetrameric structure of acatalasemic catalase was also suggested. (+info)
Characterization of hydrogen peroxide removal reaction by hemoglobin in the presence of reduced pyridine nucleotides.
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Hydrogen peroxide removal rates by hemoglobin were enhanced in the presence of reduced pyridine nucleotides. The species which had the activity to oxidize pyridine nucleotides was purified from human blood and identified as hemoglobin A. Hydrogen peroxide removal rates by hemoglobin A without reduced pyridine nucleotides at 0.2 mM hydrogen peroxide were 0.87+/-0.11 micromol/s/g hemoglobin, and the removal rates using 0.2 mM NADH and NADPH were 2.02+/-0.20 and 1.96+/-0.31 micromol/s/g hemoglobin, respectively. We deduced that the removal reaction by hemoglobin included formations of methemoglobin and the ferryl radical and reduction of the latter with pyridine nucleotides. The hydrogen peroxide removal ability by hemoglobin was less than that by catalase but was larger than that by glutathione peroxidase-glutathione reductase system at 0.2 mM hydrogen peroxide. Under acatalasemic conditions, it was suggested that NAD(P)H were important factors to prevent the oxidative degradation of hemoglobin. (+info)
Properties of acatalasic cells growing in vitro.
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Acatalasia, a disease due to homozygosity for a Mendelian gene, is characterized by the absence of the enzyme catalase from the tissues of the human body. Red cells from heterozygotes have enzyme activities about one-half normal. In this paper, the development of cell lines from skin biopsies on an affected homozygote, a heterozygote, and eight control patients is described. The cell type is the euploid "fibroblast." It was found that acatalasic cells lacked the enzyme, even after growing for many months in a medium rich in catalase. The control lines all had mean catalase activities double or more that of the heterozygous line. Selection experiments, in which the growth of cells exposed for 20 minutes to varying concentrations of hydrogen peroxide was measured, did not provide a system for preferentially eliminating acatalasic cells. Certain other experiments bearing on the enzymatic defect in this disease were performed. (+info)
Acatalasemia sensitizes renal tubular epithelial cells to apoptosis and exacerbates renal fibrosis after unilateral ureteral obstruction.
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Tissue homeostasis is determined by the balance between oxidants and antioxidants. Catalase is an important antioxidant enzyme regulating the level of intracellular hydrogen peroxide and hydroxyl radicals. The effect of catalase deficiency on renal tubulointerstitial injury induced by unilateral ureteral obstruction (UUO) has been studied in homozygous acatalasemic mutant mice (C3H/AnLCs(b)Cs(b)) compared with wild-type mice (C3H/AnLCs(a)Cs(a)). Complete UUO caused interstitial cell infiltration, tubular dilation and atrophy, and interstitial fibrosis with accumulation of type IV collagen in obstructed kidneys (OBK) of both mouse groups. However, the degree of injury showed a significant increase in OBK of acatalasemic mice compared with that of wild-type mice until day 7. The deposition of lipid peroxidation products including 4-hydroxy-2-hexenal, malondialdehyde, and 4-hydroxy-2-nonenal was severer in dilated tubules of acatalasemic OBK. Apoptosis in tubular epithelial cells significantly increased in acatalasemic OBK at day 4. Expression of caspase-9, a marker of mitochondrial pathway-derived apoptosis, increased in dilated tubules of acatalasemic mice. The level of catalase activity remained low in acatalasemic OBK until day 7 without compensatory upregulation of glutathione peroxidase activity. The data indicate that acatalasemia exacerbated oxidation of renal tissue and sensitized tubular epithelial cells to apoptosis in OBK of UUO. This study demonstrates that catalase deficiency enhanced tubulointerstitial injury and fibrosis in a murine model of UUO and thus supports the protective role of catalase in this model. (+info)
Inhibitory effects of prior low-dose X-ray irradiation on carbon tetrachloride-induced hepatopathy in acatalasemic mice.
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The catalase activities in blood and organs of the acatalasemic (C3H/AnLCs(b)Cs(b)) mouse of C3H strain are lower than those of the normal (C3H/AnLCs (a)Cs(a)) mouse. We examined the effects of prior low-dose (0.5 Gy) X-ray irradiation, which reduced the oxidative damage under carbon tetrachloride-induced hepatopathy in the acatalasemic or normal mice. The acatalasemic mice showed a significantly lower catalase activity and a significantly higher glutathione peroxidase activity compared with those in the normal mice. Moreover, low-dose irradiation increased the catalase activity in the acatalasemic mouse liver to a level similar to that of the normal mouse liver. Pathological examinations and analyses of blood glutamic oxaloacetic and glutamic pyruvic transaminase activity and lipid peroxide levels showed that carbon tetrachloride induced hepatopathy was inhibited by low-dose irradiation. These findings may indicate that the free radical reaction induced by the lack of catalase and the administration of carbon tetrachloride is more properly neutralized by high glutathione peroxidase activity and low-dose irradiation in the acatalasemic mouse liver. (+info)
Telmisartan inhibits both oxidative stress and renal fibrosis after unilateral ureteral obstruction in acatalasemic mice.
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BACKGROUND: Reactive oxygen species are involved in many of the angiotensin II signalling pathways. We have thus investigated whether the angiotensin II type 1 (AT1) receptor antagonist, telmisartan, can inhibit the accelerated renal fibrosis and excess oxidative stress, which occurs after unilateral ureteral obstruction (UUO) in acatalasemic mice. METHODS: The effect of daily intraperitoneal injection of telmisartan (0.1-0.3 mg/kg body weight) on the renal tubulointerstitial injury induced by UUO has been studied in homozygous acatalasemic mutant mice (C3H/AnLCs b Cs b) and wild-type mice (C3H/AnLCs a Cs a). We evaluated the systemic blood pressure of the mice on the seventh day. In addition, the tubulointerstitial expression of collagens type I and type IV, the p22-, p47- and p67-phox subunits of NADPH oxidase, 4-hydroxy-2-nonenal, and 4-hydroxy-2-hexenal lipid peroxidation products were assessed by immunohistochemistry. The level of apoptosis was determined by terminal deoxynucleotidyl transferase nick end-labelling analysis, while the mRNA level of the p22-, p47- and p67-phox subunits was quantified by real-time PCR. The renal content of each of the antioxidant enzymes catalase, glutathione peroxidase and superoxide dismutase was determined by specific assay. RESULTS: Obstructed kidneys from acatalasemic mice exhibited increased tubulointerstitial deposition in dilated tubules of collagens type I and IV, lipid peroxidation products, and the p22/p47/p67-phox subunits of NADPH oxidase. The level of the p22/p47/p67-phox subunit mRNA, and of apoptosis in tubular epithelial cells, was also increased compared with those from wild-type kidneys. Treatment with telmisartan attenuated all of the changes and prevented renal fibrosis in a dose-dependent manner; despite the low dose (0.1 mg/kg). The treatment did not lower the systemic blood pressure. The catalase activity remained low in acatalasemic obstructed kidneys without compensatory upregulation of glutathione peroxidase or superoxide dismutase activity; the level of neither anti-oxidant enzymes in obstructed kidneys was affected by telmisartan. CONCLUSIONS: The AT1 receptor antagonist telmisartan ameliorated renal fibrosis after UUO by inhibition of oxidative stress, even under acatalasemic conditions. (+info)