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Cytochrome P-450 in inflamed bovine mammary tissue
162
Cytoehrome P450 and Oxidative Stress
17.5 CYTOCHROMEP450 (P450) MEDIATEDHzO2 PRODUCTION AND ITS ROLE IN MUTAGENESIS. H.A. Awney, M.H. Mostafa, H.V. Gelboin and M.J. Myers Laboratory of Molecular Carcinogenesis NCI, NIH, Bethesda, MD 20892 USA
The contribution of oxygen radicals generated by P450s IAI and IA2 towards carcinogenicity was examined. H20z generated by substrate activation was used as a measure of oxy-radical production, with mutagenicity assessed using the oxy-radical sensitive strain TA 102. Benzo(a)pyrene (BaP) metabolism increased HzOz production in methylcholanthrene microsomes 4-fold over baseline. BaP augmented HzOz production was inhibited 50% using the anti-P450 monoclonal antibody (MAb) I - 7 - I . There was no effect on HzOz production during the metabolism of TrpP I, TrpPz, IQ, MelQ, 7-12 DMBA ethoxycoumarin, ethoxyresorufin, acetanilide, or BeP. HzOz production during metabolism of BaP using cDNA expressed P450 IAI, IA2, or combinations of IAIIA2, I A I - I I I A 4 or I A I - I I I A 3 did not increase HzOz production over baseline values. Only BaP and the (-)trans 7-8 diol metabolite demonstrated oxygen radical-mediated mutagenicity. This response was inhibited by MAb I - 7 - I by 50% and 70% for BaP and BaP 7-8 d i o l , respectively. These results demonstrate that P450 is involved in the increased production of HzOz which occurs during metabolism of BaP, but is not the ultimate source of the elevated levels of HzOz. Furthermore, t h i s effect is substrate specific and not a general phenomena for a l l P450 IAI and IA2 substrates.
17.7
INTERACTION OF INCREASED MICROSOMAL OXYGEN RADICALS AND THE STABILITY OF CYTOCHROME P-450 IN DIETARY ZN DEFICIENT RATS Zhaoming Xu and Tammy M. Bray Department of Nutritional Sciences, University of Guelph Guelph, Ontario, Canada, N1G 2W1 It has been proposed that an essential biochemical function of Zn is to maintain the stability of membrane structure. Previous research has found Zn deficiency caused a leakage of I-I202 from the NADPH-dependent mixed function oxidase system (MFO) and a significant increase in total iron concentration of microsomes. Microsomai membranes isolated from Zn deficient rats were more susceptible to lipid peroxidation. The objective of this study is to evaluate if the increased microsomal oxygen radical generation in Zn deficient rats would affect the stability of cytochrome P-450. The total cytochrome P-450, the conversion of eytochrome P-450 to P-420, the NADPH-dependent MFO activity and MDA concentration were used as the criteria for measurement of the stability. When liver microsomes were treated with protein denaturants, acetone and urea, P-420/P-450 was increased from 6- to 14-fold. When microsomes were treated with, llzO2-Fe2+/Fe3÷, xanthine/xanthine oxidase, there was a significant decrease in P-450 concentration which was not converted to 1'-420. Similarly, when rats were challenged with ferrous nitrilotriacetic acid (FeNTA) or CCI4, chemicals known to cause free radicals, the ratio of P-420/P-450 was not changed but P-450 concentration in liver microsomes was depressed by 25% when compared to the control. When rats were fed a Zn deficient diet, the P-450 concentration in liver microsomes was depressed hut not converted to P-420 when compared to the controls. The results suggest that the free radicals generated in microsomes may cause structural destruction of cytochrome P-450 rather than conversion to P-420.
CYCLOSPORINE A METABOLISM IS RELATED TO MICROSOMAL LIPID PEROXIDATION Francesco Serino, Henry W. Strobel, Kimberly L. Napoli, Joachim Grovel, Div. of Clin. Pharmacol., Dept. of Pharmacol., Dept. of Biochem., Div. of Org. Transpl., Dept., of Surg., Univ. of Texas Med. Schl., Houston, Texas 77030, USA.
17.6
The clinical use of cyclosporine A (CsA) is impeded by its renal, hepatic, and neurologic toxicity whose molecular mechanism is yet unexplained. Tissues subject to toxicity contain cytochrome P-450 and participate in the metabolism of CsA. Phenobarbital-induced rat liver microsomes metabolized CSA and caused lipid peroxidation which was detected as malondialdehyde (MDA). Both processes were time(0-360 min) and concentration- (0-i mMol CsA) dependent. P-450 competitors (e.g., aminopyrine) and inhibitors (e.g. ketoconazole) reduced both the concentration of monohydroxylated CsA-metabolites (detection by high-pressure liquid chromatography) and the formation of MDA. At 40 #Mol CsA, the production of MDA started after i0 min of incubation. Maximal MDA formation reached 70% of MDA produced by equimolar concentrations of carbon tetrachloride. In the same system aminopyrine did not cause MDA formation but the production of formaldehyde. The involvement of oxygen radicals was demonstrated by the inhibition caused by superoxide dismutase (500 U/ml) and catalase (500 U/ml), while EDTA (2 mMol) and eeruloplasmin (20 U/ml) were ineffective.
CYTOCHROME P-450 IN INFLAMED BOVINE MAMMARY TISSUE F. Atroshi, P. Kaipainen, T. Westermarck, J. Parantainen and H. Saloniemi Department of Animal Hygiene, College of Veterinary Medicine, Box 6, Helsinki 55; Helsinki Central Institution, 02400 Kirkkonummi; Research laboratories, Leiras Co Ltd, Helsinki, Finland Cytochromes P-450 are responsible for the oxidative metabolism of vartious endogenous substances. These enzymes can also be inducede by a wide variety of xenobiotics, drugs, and hormones. Cytochromes P-450 exist in several tissues. During the oxidation functional hydroxyl groups are generated to the substances to be eliminated, which allows the final metabolism by conjugation. Jarasch e t a l . (1977) have described the presence of a b-type cytochrome in endoplasmic reticulum of lactating mammary gland epithelial cells from various species, and a similar pigment complex has also been found in the milk fat globule membranes.The content of cytochrome P-450 in mastitic and healthy lactating bovine mammary gland was analyzed. Evidence for the existence of cytochrome P-450 in mastitic tissues was found. The amount of cytochrome P-450 formed in the assay ranged from 0.017 to 0.031 nmole/mg protein.The highest levels were found in severely inflamed samples, whereas and tracers or no cytochrome P-450 were found in healthy tissues. The significance of cytochrome P-450 in mastitic mammary gland is discussed in relation to host defence involving oxidative killing of pathogens.
17.8
Cytoehrome P450 and Oxidative Stress
17.5 CYTOCHROMEP450 (P450) MEDIATEDHzO2 PRODUCTION AND ITS ROLE IN MUTAGENESIS. H.A. Awney, M.H. Mostafa, H.V. Gelboin and M.J. Myers Laboratory of Molecular Carcinogenesis NCI, NIH, Bethesda, MD 20892 USA
The contribution of oxygen radicals generated by P450s IAI and IA2 towards carcinogenicity was examined. H20z generated by substrate activation was used as a measure of oxy-radical production, with mutagenicity assessed using the oxy-radical sensitive strain TA 102. Benzo(a)pyrene (BaP) metabolism increased HzOz production in methylcholanthrene microsomes 4-fold over baseline. BaP augmented HzOz production was inhibited 50% using the anti-P450 monoclonal antibody (MAb) I - 7 - I . There was no effect on HzOz production during the metabolism of TrpP I, TrpPz, IQ, MelQ, 7-12 DMBA ethoxycoumarin, ethoxyresorufin, acetanilide, or BeP. HzOz production during metabolism of BaP using cDNA expressed P450 IAI, IA2, or combinations of IAIIA2, I A I - I I I A 4 or I A I - I I I A 3 did not increase HzOz production over baseline values. Only BaP and the (-)trans 7-8 diol metabolite demonstrated oxygen radical-mediated mutagenicity. This response was inhibited by MAb I - 7 - I by 50% and 70% for BaP and BaP 7-8 d i o l , respectively. These results demonstrate that P450 is involved in the increased production of HzOz which occurs during metabolism of BaP, but is not the ultimate source of the elevated levels of HzOz. Furthermore, t h i s effect is substrate specific and not a general phenomena for a l l P450 IAI and IA2 substrates.
17.7
INTERACTION OF INCREASED MICROSOMAL OXYGEN RADICALS AND THE STABILITY OF CYTOCHROME P-450 IN DIETARY ZN DEFICIENT RATS Zhaoming Xu and Tammy M. Bray Department of Nutritional Sciences, University of Guelph Guelph, Ontario, Canada, N1G 2W1 It has been proposed that an essential biochemical function of Zn is to maintain the stability of membrane structure. Previous research has found Zn deficiency caused a leakage of I-I202 from the NADPH-dependent mixed function oxidase system (MFO) and a significant increase in total iron concentration of microsomes. Microsomai membranes isolated from Zn deficient rats were more susceptible to lipid peroxidation. The objective of this study is to evaluate if the increased microsomal oxygen radical generation in Zn deficient rats would affect the stability of cytochrome P-450. The total cytochrome P-450, the conversion of eytochrome P-450 to P-420, the NADPH-dependent MFO activity and MDA concentration were used as the criteria for measurement of the stability. When liver microsomes were treated with protein denaturants, acetone and urea, P-420/P-450 was increased from 6- to 14-fold. When microsomes were treated with, llzO2-Fe2+/Fe3÷, xanthine/xanthine oxidase, there was a significant decrease in P-450 concentration which was not converted to 1'-420. Similarly, when rats were challenged with ferrous nitrilotriacetic acid (FeNTA) or CCI4, chemicals known to cause free radicals, the ratio of P-420/P-450 was not changed but P-450 concentration in liver microsomes was depressed by 25% when compared to the control. When rats were fed a Zn deficient diet, the P-450 concentration in liver microsomes was depressed hut not converted to P-420 when compared to the controls. The results suggest that the free radicals generated in microsomes may cause structural destruction of cytochrome P-450 rather than conversion to P-420.
CYCLOSPORINE A METABOLISM IS RELATED TO MICROSOMAL LIPID PEROXIDATION Francesco Serino, Henry W. Strobel, Kimberly L. Napoli, Joachim Grovel, Div. of Clin. Pharmacol., Dept. of Pharmacol., Dept. of Biochem., Div. of Org. Transpl., Dept., of Surg., Univ. of Texas Med. Schl., Houston, Texas 77030, USA.
17.6
The clinical use of cyclosporine A (CsA) is impeded by its renal, hepatic, and neurologic toxicity whose molecular mechanism is yet unexplained. Tissues subject to toxicity contain cytochrome P-450 and participate in the metabolism of CsA. Phenobarbital-induced rat liver microsomes metabolized CSA and caused lipid peroxidation which was detected as malondialdehyde (MDA). Both processes were time(0-360 min) and concentration- (0-i mMol CsA) dependent. P-450 competitors (e.g., aminopyrine) and inhibitors (e.g. ketoconazole) reduced both the concentration of monohydroxylated CsA-metabolites (detection by high-pressure liquid chromatography) and the formation of MDA. At 40 #Mol CsA, the production of MDA started after i0 min of incubation. Maximal MDA formation reached 70% of MDA produced by equimolar concentrations of carbon tetrachloride. In the same system aminopyrine did not cause MDA formation but the production of formaldehyde. The involvement of oxygen radicals was demonstrated by the inhibition caused by superoxide dismutase (500 U/ml) and catalase (500 U/ml), while EDTA (2 mMol) and eeruloplasmin (20 U/ml) were ineffective.
CYTOCHROME P-450 IN INFLAMED BOVINE MAMMARY TISSUE F. Atroshi, P. Kaipainen, T. Westermarck, J. Parantainen and H. Saloniemi Department of Animal Hygiene, College of Veterinary Medicine, Box 6, Helsinki 55; Helsinki Central Institution, 02400 Kirkkonummi; Research laboratories, Leiras Co Ltd, Helsinki, Finland Cytochromes P-450 are responsible for the oxidative metabolism of vartious endogenous substances. These enzymes can also be inducede by a wide variety of xenobiotics, drugs, and hormones. Cytochromes P-450 exist in several tissues. During the oxidation functional hydroxyl groups are generated to the substances to be eliminated, which allows the final metabolism by conjugation. Jarasch e t a l . (1977) have described the presence of a b-type cytochrome in endoplasmic reticulum of lactating mammary gland epithelial cells from various species, and a similar pigment complex has also been found in the milk fat globule membranes.The content of cytochrome P-450 in mastitic and healthy lactating bovine mammary gland was analyzed. Evidence for the existence of cytochrome P-450 in mastitic tissues was found. The amount of cytochrome P-450 formed in the assay ranged from 0.017 to 0.031 nmole/mg protein.The highest levels were found in severely inflamed samples, whereas and tracers or no cytochrome P-450 were found in healthy tissues. The significance of cytochrome P-450 in mastitic mammary gland is discussed in relation to host defence involving oxidative killing of pathogens.
17.8