A quantitative genetic analysis of tissue-specific catalase activity inMus musculus

Tissue-specific catalase activity in 3-week-old animals from inbred mouse strains 129/ReJ, BALB/c, C3H/HeAnl/Cas-1^b, C3H/HeSnJ, C3H/S, C57BL/6J, and Swiss-Webster was found to be highly variable by analysis of variance (P=0.01). Appropriate crosses were made among strains which were classified as normal (BALB/c, C3H/HeSnJ, C3H/S), hypocatalasemic (129/ReJ, C57BL/6J), and acatalasemic (C3H/HeAnl/Cas-1^b) with respect to blood catalase activity to study the inheritance of the blood, kidney, liver, and lung catalase activity levels in a number of generations (reciprocal F₁'s, F₂, two backcrosses —BC₁ and BC₂— and some RI lines). Segregation analysis and statistical methods which tested different models of inheritance as well as calculations of heritability were used in an effort to assess and evaluate genetic parameters that affect catalase activity. Results indicate that the inheritance of blood catalase activity in the cross involving acatalasemic and normal (BALB/c, C3H/HeSnJ) strains is compatible with the single-locus difference between the parental strains; however, the difference between the acatalasemic and the hypocatalasemic strain (C57BL/6J) would require additional genetic interaction for a satisfactory explanation. A similar pattern of generalization also applies to the inheritance of kidney catalase activity. The segregation pattern for the liver and lung catalase activity in most crosses is significantly different from the expectations of the single locus model. These results are compatible with the concept that a number of genes must affect tissue-specific catalase activity in mice. These may include previously described (e.g., Ce-1 and Ce-2) or novel genetic regulators/modifiers which interact with a single structural gene (Cas-1) or its product to produce the catalase phenotype characteristic of specific tissues in each strain.This investigation was supported by a Natural Sciences and Engineering Research Council of Canada operating grant to S.M.S.     

Modulation of selenium-dependent glutathione peroxidase (Se-GSH-Px) activity in mice

Selenium-dependent glutathione peroxidase activity was assessed in the liver, kidney, lung and blood of mice from seven strains (129/ReJ, BALB/c, C3H/HeSnJ, C3H/S, C57BL/6J, Csb, and S.W.) at five ages (newborn, 21, 70, 175 and +500 days old). Activity was highest in the liver (0.25 U/mg protein) followed by blood hemolysate (0.16 U/mg protein) with kidney and lung displaying similar, comparatively lower levels of activity (0.14 and 0.12 U/mg protein respectively). Although activity was shown by statistical analysis to be not significantly different among the strains (p = 0.05), age-associated, strain-specific changes in enzyme activity were noted to be highly significant (p = 0.001). Also, ethanol administered in drinking water resulted in a marked reduction in selenium-dependent glutathione peroxidase activity during both short- (1-2 weeks) and long- (5-6 weeks) term treatment periods. Changes in this enzyme due to aging and after exposure to xenobiotics such as ethanol may have serious ramifications given the importance of this enzyme in the detoxification of reactive oxygen metabolites.     

Effect of ethanol in vivo on enzymes which detoxify oxygen free radicals

The effects of ethanol administered as a 15% solution in drinking fluid on weight gain, soluble liver protein and the activity of the three enzymes of oxygen radical metabolism (i.e., superoxide dismutase, catalase, and glutathione peroxidase) were studied in five inbred strains of mice (129/ReJ, BALB/c, C3H/HeSnJ, C57BL/6J, Csb) and Sprague Dawley rats, relative to age, sex, and genotype matched controls. Animals maintained on ethanol exhibited lower weight gains and elevation of soluble liver protein than controls. Total superoxide dismutase, catalase and glutathione peroxidase activity in ethanol-treated animals were in general reduced in comparison to that of their matched controls, with each strain showing genotype specific enzyme activity. Such ethanol feeding results are attributed to the direct and indirect effects of this treatment protocol and raise the possibility that ethanol-fed animals may be susceptible to free radical damage and at least some of the cellular damages observed following ethanol challenges could be attributed to the reduced level of these protective enzymes.     

Tissue-specific developmental regulation of superoxide dismutase (SOD-1 and SOD-2) activities in genetic strains of mice

The activity levels of CuZn superoxide dismutase (SOD) (SOD-1) and Mn SOD (SOD-2) in liver, kidney, and lung were assessed in newborn and 3-, 10-, 25-, and 70-week-old females from seven genetic strains (BALB/c, Cs^b, C3H/HeSnJ, C3H/S, C57BL/6J, Swiss-Webster, and 129/ReJ) of mice. Total SOD enzyme activity was high at birth and declined somewhat with old age (70 weeks) in the liver and increased in both kidney and lung from newborn to 25 weeks. The activity level of SOD-1 was found to be highly variable among strains at different ages in liver, with little change associated with aging in the kidney, and showed a strain-specific increase during aging in the lung. In general, SOD-2 activity was lower than SOD-1 activity in liver and lung but levels of the two forms of this enzyme were similar in the kidney. The SOD-2 activity increased with age with little variation among strains in kidney. The increase in this form of the enzyme with age was relatively small and strain specific in lung and highly variable among strains in the liver. The Cs^b genotype (acatalasemic) at age 70 weeks showed an exceptionally high SOD-1 activity associated with an exceptionally low SOD-2 activity in the liver. Changes in enzyme activity with age in different tissues associated with differences in activity level among genotypes (of the type reported here for SOD-1 and SOD-2) may be indicative of a complex system of enzyme regulation. Further studies are needed to explain fully the genetic/molecular mechanism(s) for SOD regulation.This study was financially supported by an NSERC operating grant to S.M.S. N.J.S. was the recipient of a postgraduate scholarship from the NSERC during this period.     

Genetic considerations in the effects of ethanol in mice. I. Genotype-dependent alterations in alcohol dehydrogenase activity

Most genetic studies on individual and racial differences in sensitivity to alcohol intoxication have concentrated on genetic variations associated with structural genes for the enzymes involved in alcohol metabolism, including alcohol dehydrogenase (ADH; E.C. 1.1.1.1). We studied the ethanol-induced regulation of ADH following chronic administration of ethanol in mice. Newly weaned males from six inbred strains (BALB/c, C3H/HeSnJ, C3H/S, C57BL/6J, S.W., and 129/ReJ) were subjected to ethanol administration. Alterations in the level of liver ADH activity, relative to matched littermate controls, were evaluated. The change in ADH activity was found to be strain (genotype) specific, which may explain the contradictory results in the literature. Strains which showed induction of ADH activity, in general, reflected a strain-specific time-dependent profile. Strains which showed repression, however, were independent in the degree of repression to the duration of ethanol exposure. Such variable, ethanol-induced regulatory responses (induction/repression) in ADH activity of different genotypes may account for individual and population variations in response to alcohol. Additional work, however, is needed to establish the molecular bases of ADH inducibility and its specific role in relative susceptibility to alcohols.     

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.     

Effects of post low-dose X-ray irradiation on carbon tetrachloride-induced acatalasemic mice liver damage

The catalase activities in the blood and organs of the acatalasemic (C3H/AnLCsb-Csb) mouse of the C3H strain are lower than those of the normal (C3H/AnLCSa-Csa) mouse. We examined the effects of post low-dose (0.5 Gy) X-ray irradiation which reduced the oxidative damage under carbon tetrachloride-induced hepatopathy in acatalasemic or normal mice. As a result, the 0.5 Gy irradiation after carbon tetrachloride administration decreased the glutamic oxaloacetic and glutamic pyruvic transaminase activity in the acatalasemic mouse blood to a level similar to that of the acatalasemic mouse blood not treated with carbon tetrachloride; this is in contrast to a high-dose (15 Gy) irradiation. In the same manner, pathological disorder was improved by 0.5 Gy irradiation. The fat degeneration in normal mice was quickly reduced, in contrast to acatalasemic mice. These findings suggest that low-dose irradiation after carbon tetrachloride administration accelerates the rate of recovery and that catalase plays an important role in the recovery from hepatopathy induced by carbon tetrachloride, in contrast to high-dose irradiation.     

Molecular analysis of an acatalasemic mouse mutant

The Csb acatalasemia mouse mutant differentially expresses reduced levels of catalase activity in a tissue specific manner. In order to pinpoint the molecular lesion that imparts the acatalasemia phenotype in Csb mice we have utilized the polymerase chain reaction technique to isolate catalase cDNA clones from control and Csb mouse strains. Sequence analyses of these cDNA clones have revealed a single nucleotide difference within the coding region of catalase between control and Csb mice. This nucleotide transversion (G----T) is located in the third position of amino acid 11 in the catalase monomer. In control mouse strains glutamine (CAG) is encoded at amino acid 11, while in Csb mice this codon (CAT) encodes histidine. This amino acid is located within a region that forms the first major alpha-helix in the amino-terminal arm of the catalase subunit and, as such, may render the catalase molecule unstable under certain physiological conditions.     

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.     

Characterization of acatalasemia detected in two Hungarian sisters.

Acatalasemia was detected in 2 sisters of a Hungarian family. The pedigree of the family showed hypocatalasemia in the children of the patients and in 1 of their brothers, while the other members of the family had normal blood catalase activity. The biochemical characterization (catalase activity, electrophoretic migration, isoelectric point and enzyme stability) of the blood as well as tissue catalase of the acatalasemic patients yielded a catalase form which did not differ from normal.     

Establishment and cellular characteristics of a hepatocyte cell line (oums-31) derived from an acatalasemic mouse

Summary Liver cell lines with very low catalase activity were established from an acatalasemic mouse. Hepatocytes isolated by a collagenase-liver-perfusion technique were cultured in Williams’ E medium supplemented with 10% fetal bovine serum. The acatalasemic liver cell line showed approximately 20% of the catalase activity of a normal mouse liver cell line, whereas its glutathione peroxidase activity was approximately equal to that of the normal liver cell line. DNA sequence analysis of this cell line showed the same mutation in the catalase gene as is seen in the acatalasemic mouse. Our observation of intracellular content of hydrogen peroxide (H₂O₂) radical and increased susceptibility of the cells to H₂O₂ were compatible with the existence of low catalase activity in the acatalasemic mouse. This hepatocyte cell line should be useful for studying effects of oxidative radical stress at the cellular level.     

Polyacrylamide gradient gel electrophoretic studies of residual catalase in acatalasemia

Erythrocyte catalse in a Japanese-type acatalasemia and a normal control subject was separated by chromatofocusing with or without prior partial purification with DEAE-cellulose. Fractions were analyzed by polyacrylamide gradient gel electrophoresis for catalse activity and protein stain. Chromatofocusing revealed no marked difference in pI values between normal and acatalasemic catalases with or without partial purification. In the gel electrophoresis, molecular weights were also similar; two bands of catalase activity with molecular weights of 290,000 and 350,000 for the acatalasemia and of 280,000 and 360,000 for the normal control were found in the partially purified preparations. The molecular weight of normal catalase in untreated hemolysate was 250,000. Normal catalse was identified as protein bands on polyacrylamide gradient gel after fractionation of hemolysate by chromatofocusing. A more sensitive method for protein stain is still required for demonstration of residual catalse protein on the gel.     

Adjustment function among antioxidant substances in acatalasemic mouse brain and its enhancement by low-dose X-ray irradiation

The catalase activities in blood and organs of the acatalasemic (C3H/AnLCsbCsb) mouse of the C3H strain are lower than those of the normal (C3H/AnLCsaCsa) mouse. We conducted a study to examine changes in the activities of antioxidant enzymes, such as catalase, superoxide dismutase (SOD) and glutathione peroxidase (GPX), the total gluathione content, and the lipid peroxide level in the brain, which is more sensitive to oxidative stress than other organs, at 3, 6, or 24 hr following X-ray irradiation at doses of 0.25, 0.5, or 5.0 Gy to the acatalasemic and the normal mice. No significant change in the lipid peroxide level in the acatalasemic mouse brain was seen under non-irradiation conditions. However, the acatalasemic mouse brain was more damaged than the normal mouse brain by excessive oxygen stress, such as a high-dose (5.0 Gy) X-ray. On the other hand, we found that, unlike 5.0 Gy X-ray, a relatively low-dose (0.5 Gy) irradiation specifically increased the activities of both catalase and GPX in the acatalasemic mouse brain making the activities closer to those in the normal mouse brain. These findings may indicate that the free radical reaction induced by the lack of catalase is more properly neutralized by low dose irradiation.     

Methanol metabolism in acatalasemic mice

The methanol metabolism in acatalasemic mice was studied by administering [14C]methanol and [14C]formic acid to acatalasemic and normal mice and determining the radioactivity of exhaled carbon dioxide. Methanol metabolism was also studied in acatalasemic and normal mice treated with 3-amino-1,2,4-triazole (AT), which is known to be an inhibitor of catalase (EC 1.11.1.6). The metabolism of methanol and formic acid was inhibited in acatalasemic mice as seen by reduced [14C]CO2 production. Similar results were obtained when AT was given prior to the methanol injection into the normal and acatalasemic mice. The results indicate the peroxidative activity of catalase plays the major role in the methanol metabolism in mice. On the other hand similar studies with [1-14C] ethanol showed that the metabolism of ethanol was not inhibited in acatalasemic mice.     

Factors influencing variable oxidative hemolysis of inbred mouse erythrocytes

The hemolysis of erythrocytes from certain inbred mouse strains (e.g., BALB/c) in response to hydrogen peroxide stress has been shown to be correlated with the type of hemoglobin beta chain (Kruckeberg, W.C., et al. (1987) Blood 70, 909-914). The characteristic hemolytic response of BALB/c red cells to oxidative stress resembles that of human red cells in that carbon monoxide and iron chelators inhibit hemolysis of both. Gross hemoglobin oxidation rates were similar in hemolytic (BALB/c) and nonhemolytic (C57BL/6) strains. The rate and degree of in vitro catalase inhibition by sodium azide was also the same for the two strains. Even in the presence of this catalase inhibitor the assayable hydrogen peroxide disappeared within seconds of its addition, yet hemolysis was not observed for about 15 min. The mechanism underlying this delay between hydrogen peroxide addition and disappearance and subsequent hemolysis is under investigation.     

Tests of genetic allelism between four inbred mouse strains with absent corpus callosum.

Inbred mouse strains that lack the corpus callosum connecting the cerebral hemispheres in the adult differ from the C57BL/6J strain at several relevant but unknown loci. To identify at least one major locus that influences axon guidance, different strains showing phenotypically similar defects were crossed to test for allelism. If the F1 hybrid between two strains with the same brain defect is phenotypically normal, it is much more likely that the two strains will differ at fewer loci than will an acallosal strain and C57BL/6J. This approach proved to be very informative. Five reasonable models of inheritance involving two or three loci were assessed, and the data justified rejection of all but one hypothesis. A total of 479 mice were obtained from four inbred strains prone to absence of the corpus callosum (BALB/cWah1, BALB/cWah2, I/LnJ, and 129/ReJ), one normal strain (C57BL/6J), and 11 F1 hybrids among them. Because the size of forebrain axon bundles is generally greater in mice with larger brains, and because whole brain size is certainly polygenic, the phenotypically normal groups were used to derive a standard index of the degree of corpus callosum deficiency relative to brain size. Results demonstrated clearly that the hybrid between BALB/cWah1 and 129/ReJ is normal, whereas the crosses among the BALB/c substrains and I/LnJ yielded many mice with deficient corpus callosum. I/LnJ crossed with 129/ReJ also produced some animals with callosal defects. The data were consistent with a model in which the difference between BALB/c and 129/ReJ involves two loci, whereas the defect in I/LnJ involves homozygosity at three loci, which impairs development more severely.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunotitration of the catalase in the blood of Japanese subjects and mice suffering from acatalasemia and hypocatalasemia.

Catalase in hemolysates of normal, heterozygous hypocatalasemic and acatalasemic Japanese was immunotitrated with an anti-human blood catalase rabbit serum. Equivalence points were calculated from the regression lines between catalase activity added and catalase activity remaining in the supernatant. Catalase activities at the equivalence points of Japanese normal, hypocatalasemia and acatalasemia were similar. The results indicate that the specific activities of catalase in the normal and of the variant bloods are identical. Catalase in hemolysates of normal and variant mice was immunotitrated with an anti-mouse liver catalase rabbit serum. In contrast to Japanese acatalasemic subject, the equivalence points of catalase in heterozygous hypocatalasemic, homozygous hypocatalasemic, acatalasemic and normal hemolysates were different, and the ratios of specific activity in these variant mice to that in normal were 0.72, 0.46 and 0.21, respectively. The differences in catalase activities at equivalence points were also supported by the statistical analysis on parameters of regression lines of catalase activities remaining in the supernatant on catalase activities added in the immunotitration. These findings suggest that the molecular properties of residual catalase of Japanese acatalasemia and those of mouse acatalasemia are entirely different.     

Relationship of hydrogen peroxide production by Mycoplasma pulmonis to virulence for catalase-deficient mice

Mycoplasma pulmonis, an etiological agent of murine pneumonia, produced about 0.065 mumoles of hydrogen peroxide (H(2)O(2)) per hr per 10(10) colony-forming units. When glucose was present at a concentration of 0.01 m, H(2)O(2) production was increased by 50%. To determine if H(2)O(2) production by M. pulmonis could be correlated with virulence, normal, acatalasemic, and acatalatic mice were infected with the organism. Three days after infection with M. pulmonis significantly more acatalatic mice had pneumonia than did normal or acatalasemic mice. The pneumonia in acatalatic mice was also more severe than in the other two groups. Five days after infection, pneumonia in the acatalatic mice was resolved, whereas normal mice were severely affected. The presence of pneumonia and the severity were correlated with the recovery of M. pulmonis from the lesions. In vitro studies of the effect of catalase on M. pulmonis showed that exogenously supplied catalase stimulated the growth of M. pulmonis at 37 C and prolonged its survival at 25 C. Hemolysis of sheep blood, guinea pig blood, rabbit blood, and normal and acatalasemic mouse blood by M. pulmonis was inversely related to the catalase activity of the erythrocytes. These findings suggest that H(2)O(2) secretion contributes to the virulence of M. pulmonis and to the death of the microorganism in the absence of host catalase.