Embryonic catalase protects against ethanol embryopathies in acatalasemic mice and transgenic human catalase-expressing mice in embryo culture.

Reactive oxygen species (ROS) have been implicated in the mechanism of ethanol (EtOH) teratogenicity, but the protective role of the embryonic antioxidative enzyme catalase is unclear, as embryonic activity is only about 5% of maternal levels. We addressed this question in a whole embryo culture model. C57BL/6 mouse embryos expressing human catalase (hCat) or their wild-type (C57BL/6 WT) controls, and C3Ga.Cg-Cat(b)/J catalase-deficient, acatalasemic (aCat) mouse embryos or their wild-type C3HeB/FeJ (C3H WT) controls, were explanted on gestational day (GD) 9 (plug=GD 1), exposed for 24h to 2 or 4mg/mL EtOH or vehicle, and evaluated for functional and morphological changes. hCat and C57BL/6 WT vehicle-exposed embryos developed normally, while EtOH was embryopathic in C57BL/6 WT embryos, evidenced by decreases in anterior neuropore closure, somites developed, turning and head length, whereas hCat embryos were protected (p<0.001). Maternal pretreatment of C57BL/6 WT dams with 50kU/kg PEG-catalase (PEG-cat) 8h prior to embryo culture, which increases embryonic catalase activity, blocked all EtOH embryopathies (p<0.001). Vehicle-exposed aCat mouse embryos had lower yolk sac diameters compared to WT controls, suggesting that endogenous ROS are embryopathic. EtOH was more embryopathic in aCat embryos than WT controls, evidenced by reduced head length and somite development (p<0.01), and trends for reduced anterior neuropore closure, turning and crown-rump length. Maternal pretreatment of aCat dams with PEG-Cat blocked all EtOH embryopathies (p<0.05). These data suggest that embryonic catalase is a determinant of risk for EtOH embryopathies.

Methanol teratogenicity in mutant mice with deficient catalase activity and transgenic mice expressing human catalase.

The role of catalase in methanol (MeOH) teratogenesis is unclear. In rodents it both detoxifies reactive oxygen species (ROS) and metabolizes MeOH and its formic acid (FA) metabolite. We treated pregnant mice expressing either high (hCat) or low catalase activity (aCat), or their wild-type (WT) controls, with either MeOH (4g/kg ip) or saline. hCat mice and WTs were similarly susceptible to MeOH-initiated ophthalmic abnormalities and cleft palates. aCat and WT mice appeared resistant, precluding assessment of the developmental impact of catalase deficiency. Catalase activity was respectively increased at least 1.5-fold, and decreased by at least 35%, in hCat and aCat embryos and maternal livers. MeOH and FA pharmacokinetic profiles were similar among hCat, aCat and WT strains. Although the hCat results imply no ROS involvement, embryo culture studies suggest this may be confounded by maternal factors and/or a requirement for higher catalase activity in the hCat mice.

Catalase -262C>T polymorphisms in Hungarian vitiligo patients and in controls: further acatalasemia mutations in Hungary.

Catalase is the main regulator of hydrogen peroxide metabolism. In vitiligo patients there are conflicting data on its activity and no data on the effect of -262C>T polymorphism in the catalase gene. Blood catalase activity, -262C>T polymorphism and acatalasemia mutations were examined in 75 vitiligo patients and in 162 controls, in Hungary. We measured blood catalase activity and conducted analyses with PCR-SSCP, polyacrylamide gel electrophoresis and silver staining in combination with RFLP and nucleotide sequencing. Comparison of the wild (CC) genotype and the mutant (TT) genotype in the vitiligo patients revealed a non significant (P > 0.19) increase in blood catalase. Male controls with the CT genotype had significantly (P < 0.04) lower blood catalase activity than CC genotype controls. Female vitiligo patients with CC genotype had lower (P < 0.04) blood catalase than female controls. The frequency of wild genotype (CC) and C alleles is significantly (P < 0.04) decreased in Hungarian controls when compared to controls in Slovenia, Morocco, UK, Greece, Turkey, USA, China. The detection of a novel acatalasemia mutation (37C>T in exon 9) and the 113G>A (exon 9) mutation in Hungary are further proofs of genetic heterogeneity origin of acatalasemia mutations. In conclusion, the -262 C>T polymorphism has a reverse effect on blood catalase in vitiligo patients and in controls. In controls the mutant genotypes and alleles are more frequent in Hungary than in several other populations. The new acatalasemia mutations are further examples of heterogeneity of acatalasemia.

Embryonic catalase protects against endogenous and phenytoin-enhanced DNA oxidation and embryopathies in acatalasemic and human catalase-expressing mice.

Oxidative stress and reactive oxygen species (ROS) such as hydrogen peroxide (H(2)O(2)), which is detoxified by catalase, are implicated in fetal death and birth defects. However, embryonic levels of catalase are only ∼ 5% of adult activity, and its protective role is not understood completely. Herein, we used mutant catalase-deficient mice [acatalasemic (aCat)] and transgenic mice expressing human catalase (hCat), which, respectively, exhibited 40-50% reductions and 2-fold elevations in the activities of embryonic and fetal brain catalase, to show that embryonic catalase protects the embryo from both physiological oxidative stress and the ROS-initiating antiepileptic drug phenytoin. Compared to wild-type (WT) catalase-normal controls, both untreated and phenytoin-exposed aCat mice exhibited a 30% increase in embryonic DNA oxidation and a >2-fold increase in embryopathies, both of which were completely blocked by protein therapy with exogenous catalase. Conversely, compared to WT controls, untreated and, to a lesser extent, phenytoin-exposed hCat mice were protected, with untreated hCat embryos exhibiting a 40% decrease in embryonic DNA oxidation and up to a 67% decrease in embryopathies. Embryonic catalase accordingly plays an important protective role, and both physiological and phenytoin-enhanced oxidative stress can be embryopathic.

Altered methanol embryopathies in embryo culture with mutant catalase-deficient mice and transgenic mice expressing human catalase.

The mechanisms underlying the teratogenicity of methanol (MeOH) in rodents, unlike its acute toxicity in humans, are unclear, but may involve reactive oxygen species (ROS). Embryonic catalase, although expressed at about 5% of maternal activity, may protect the embryo by detoxifying ROS. This hypothesis was investigated in whole embryo culture to remove confounding maternal factors, including metabolism of MeOH by maternal catalase. C57BL/6 (C57) mouse embryos expressing human catalase (hCat) or their wild-type (C57 WT) controls, and C3Ga.Cg-Catb/J acatalasemic (aCat) mouse embryos or their wild-type C3HeB/FeJ (C3H WT) controls, were explanted on gestational day (GD) 9 (plug=GD 1), exposed for 24 h to 4 mg/ml MeOH or vehicle, and evaluated for functional and morphological changes. hCat and C57 WT vehicle-exposed embryos developed normally. MeOH was embryopathic in C57 WT embryos, evidenced by decreases in anterior neuropore closure, somites developed and turning, whereas hCat embryos were protected. Vehicle-exposed aCat mouse embryos had lower yolk sac diameters compared to C3H WT controls, suggesting that endogenous ROS are embryopathic. MeOH was more embryopathic in aCat embryos than WT controls, with reduced anterior neuropore closure and head length only in catalase-deficient embryos. These data suggest that ROS may be involved in the embryopathic mechanism of methanol, and that embryonic catalase activity may be a determinant of teratological risk.

Low catalase activity in blood is associated with the diabetes caused by alloxan.

BACKGROUND: Hydrogen peroxide is enzymatically processed by catalase, and catalase deficiency in blood is known as acatalasemia. We examined whether low catalase activity is a risk factor for diabetes mellitus. METHODS: Blood glucose, insulin and glucose tolerance test were examined in acatalasemic and normal mice under non-stress and oxidative stress conditions. Alloxan administration was used as oxidative stress. RESULTS: Alloxan, which was a drug that caused diabetes mellitus, mostly generated hydrogen peroxide by the reaction of alloxan and reduced glutathione, in vitro. Incidence of hyperglycemia in alloxan-untreated acatalasemic mice was as low as that in the normal mice. However, the incidence of acatalasemia mice treated with alloxan was higher than that in normal mice, and the number of pancreatic beta-cells in the acatalasemic mice was less than that in normal mice. CONCLUSION: These results indicate that low catalase activity in the blood is associated with the diabetes mellitus caused by alloxan administration.

Peroxisomes and aging.

Peroxisomes are indispensable for proper functioning of human cells. They efficiently compartmentalize enzymes responsible for a number of metabolic processes, including the absolutely essential beta-oxidation of specific fatty acid chains. These and other oxidative reactions produce hydrogen peroxide, which is, in most instances, immediately processed in situ to water and oxygen. The responsible peroxidase is the heme-containing tetrameric enzyme, catalase. What has emerged in recent years is that there are circumstances in which the tightly regulated balance of hydrogen peroxide producing and degrading activities in peroxisomes is upset-leading to the net production and accumulation of hydrogen peroxide and downstream reactive oxygen species. The factor most essentially involved is catalase, which is missorted in aging, missing or present at reduced levels in certain disease states, and inactivated in response to exposure to specific xenobiotics. The overall goal of this review is to summarize the molecular events associated with the development and advancement of peroxisomal hypocatalasemia and to describe its effects on cells. In addition, results of recent efforts to increase levels of peroxisomal catalase and restore oxidative balance in cells will be discussed.

Catalase enzyme mutations and their association with diseases.

Enzyme catalase seems to be the main regulator of hydrogen peroxide metabolism. Hydrogen peroxide at high concentrations is a toxic agent, while at low concentrations it appears to modulate some physiological processes such as signaling in cell proliferation, apoptosis, carbohydrate metabolism, and platelet activation. Benign catalase gene mutations of 5' noncoding region (15) and intron 1 (4) have no effect on catalase activity and are not associated with disease. Catalase gene mutations have been detected in association with diabetes mellitus, hypertension, and vitiligo. Decreases in catalase activity in patients with tumors is more likely to be due to decreased enzyme synthesis rather than to catalase mutations.Acatalasemia, the inherited deficiency of catalase has been detected in 11 countries. Its clinical features might be oral gangrene, altered lipid, carbohydrate, homocysteine metabolism and the increased risk of diabetes mellitus. The Japanese, Swiss, and Hungarian types of acatalasemia display differences in biochemical and genetic aspects. However, there are only limited reports on the syndrome causing these mutations. These data show that acatalasemia may be a syndrome with clinical, biochemical, genetic characteristics rather than just a simple enzyme deficiency.

A novel catalase mutation detected by polymerase chain reaction-single strand conformation polymorphism, nucleotide sequencing, and western blot analyses is responsible for the type C of Hungarian acatalasemia.

Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) screening was used for searching mutations of the catalase gene in two Hungarian hypocatalasemic families. A syndrome-causing mutation was found in a PCR product containing exon 7 and its boundaries. Nucleotide sequence analyses detected a G to T substitution at position 5 of intron 7. The effect of this splice site mutation was confirmed by Western blot analyses demonstrating a decreased catalase protein level in these patients. These findings represent a novel type (C) of catalase mutations in the Hungarian acatalasemic/hypocatalasemic patients.

cDNA cloning and expression of mutant catalase from the hypocatalasemic mouse: comparison with the acatalasemic mutant.

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.

Purification and characterization of liver catalase in acatalasemic beagle dog: comparison with normal dog liver catalase.

Catalase from acatalasemic dog liver was purified to homogeneity and its properties were compared with those of normal dog liver catalase. The purified acatalasemic and normal dog liver catalases were found to have the same molecular weight (230,000 Da) and isoelectric point (pI: 6.0-6.2) and both enzymes contained four hematins per molecule. The catalytic activity of catalase from acatalasemic dog was normal. Furthermore, there was no difference between the acatalasemic and normal dog catalases in the binding affinity to NADPH (apparent Kd: 0.11-0.12 microM) and in the sensitivity to oxidative stress by hydrogen peroxide, the normal substrate of catalase. The acatalasemic dog enzyme was stable only in a narrow pH range (pH 6-9) although the normal enzyme was stable in a wide pH range (pH 4-10). Acatalasemic dog liver catalase also showed a slight low thermal stability at 37 degrees C and the heat-lability was remarkable at 45 degrees C, compared to the normal dog enzyme. These results indicated that the acatalasemic dog catalase is catalytically normal although it is associated with an unstable molecular structure.

cDNA cloning of mutant catalase in acatalasemic beagle dog: single nucleotide substitution leading to thermal-instability and enhanced proteolysis of mutant enzyme.

The mutant catalase purified previously from acatalasemic dog liver was heat-labile but possessed normal activity, suggesting a mutation within the coding region distal from the catalytic site. The nucleotide and deduced amino acid sequences of acatalasemic beagle dog catalase were determined by analysis of cDNA obtained by 5'- and 3'-RACE and reverse transcriptase-polymerase chain reaction (RT-PCR) methods. Comparative analysis of cDNA sequences of normal and acatalasemic dog catalases indicated a single nucleotide difference where alanine(327) (G macro CT) was substituted with threonine (ACT). The mutant catalase, which was overexpressed in COS-1 cells, was heat-labile as previously observed with the purified enzyme from acatalasemic dog liver, indicating that this amino acid substitution can lead to structural instability. No catalase protein and activity were detected by immunoblotting and spectrophotomeric assay in acatalasemic dog reticulocytes although almost the same level of mRNA expression as that in the normal reticulocytes was observed. Pulse-labeling and immunoprecipitation examination indicated that the level of catalase synthesis in the acatalasemic dog reticulocytes was almost the same (approximately 80%) as that in the normal reticulocytes. On the other hand, the synthesized mutant catalase in reticulocytes was rapidly degraded (t(1/2): 1.8 h) compared with the normal catalase (t(1/2): 14.0 h) and this degradation was almost completely inhibited by lactacystin (LC). These results suggested that the proteolytic degradation mediated most likely by proteasome might be involved in disposing of the mutant catalase in acatalasemic erythroid cells.

Tissue and organ expression of catalase in acatalasemic beagle dogs.

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.