scholarly journals Immunochemical detection of different isoenzymes of cytochrome P-450 induced in chick hepatocyte cultures

1989 ◽  
Vol 258 (1) ◽  
pp. 237-245 ◽  
Author(s):  
P Sinclair ◽  
J Frezza ◽  
J Sinclair ◽  
W Bement ◽  
S Haugen ◽  
...  
Keyword(s):  
Chick Embryo ◽  
Rabbit Antiserum ◽  
Enzyme Assays ◽  
Chick Embryos ◽  
Chemical Inducers ◽  
Hepatic Microsomes ◽  
Immunochemical Detection ◽  
Demethylase Activity ◽  
Cytochrome P 450

This study investigated whether the same cytochrome P-450 (P-450) isoenzymes were inducible in cultures of chick-embryo hepatocytes as in the liver of chicken embryos. We purified two isoenzymes of cytochrome P-450 from the livers of 17-day-old-chick embryos: one of molecular mass approx. 50 kDa induced in vivo by the phenobarbital-like inducer glutethimide, and the second of approx. 57 kDa induced by 3-methylcholanthrene. Rabbit antiserum against the 50 kDa protein inhibited benzphetamine demethylase activity in hepatic microsomes (microsomal fractions) from glutethimide-treated chick embryo. Antiserum to the 57 kDa protein inhibited ethoxyresorufin de-ethylase activity in hepatic microsomes from methylcholanthrene-treated chick embryo. Cultured chick hepatocytes were treated with chemicals known to induce isoenzymes of P-450 in rodent liver. The induced P-450s were quantified spectrophotometrically and characterized by immunoblotting and enzyme assays. From these studies, chemical inducers were classified into three groups: (i) chemicals that induced a P-450 isoenzyme of 50 kDa and increased benzphetamine demethylase activity: glutethimide, phenobarbital, metyrapone, mephenytoin, ethanol, isopentanol, isobutanol, lindane, lysodren; (ii) chemicals that induced a P-450 isoenzyme of 57 kDa and increased ethoxyresorufin de-ethylase activity: 3-methylcholanthrene and 3,3',4,4'-tetrachlorobiphenyl; and (iii) the mono-alpha-substituted 2,3',4,4',5-pentabromobiphenyl, which induced both proteins and both activities. The immunochemical data showed that chick-embryo hepatocytes in culture retain the inducibility of glutethimide- and methylcholanthrene-induced isoenzymes of P-450 that are inducible in the liver of the chicken embryo.

Cytochrome P-450 and hepatic drug biotransformation during progressive cardiac disease in cardiomyopathic hamsters

1979 ◽  
Vol 57 (3) ◽  
pp. 302-304 ◽  
Author(s):  
Kenneth W. Renton ◽  
James C. Baker ◽  
Leslie E. Bailey
Keyword(s):  
Heart Failure ◽  
Cardiac Disease ◽  
Disease Process ◽  
Microsomal Protein ◽  
Hepatic Drug ◽  
Drug Biotransformation ◽  
Hepatic Microsomes ◽  
Cardiomyopathic Hamsters ◽  
Demethylase Activity ◽  
Cytochrome P 450

Reductions in cytochrome P-450 levels and aminopyrine N-demethylase activity of hepatic microsomes obtained from cardiomyopathic hamsters (BIO 14.6) occurred at all stages of the disease before the development of congestive heart failure (CHF). Cytochrome b5 levels were reduced only in animals with CHF when compared with age-matched controls (BIO.RB). Total microsomal protein and p-nitrophenol glucuronidation were not affected by the disease process. We conclude that the reduction in cytochrome P-450 levels and N-demethylase activity in cardiomyopathic hamsters is not a consequence of CHF, but is one of the manifestations of the disease process.

Nonadditive effects of combined in vivo hepatic microsomal enzyme inducers in the Wistar rat

1983 ◽  
Vol 61 (9) ◽  
pp. 983-988 ◽  
Author(s):  
Larry S. Gontovnick ◽  
Geoffrey Sunahara ◽  
Gail D. Bellward
Keyword(s):  
Adult Female ◽  
Wistar Rat ◽  
Female Rats ◽  
Male Rats ◽  
Hepatic Microsomes ◽  
Trans Stilbene ◽  
Microsomal Enzyme Inducers ◽  
Nonadditive Effects ◽  
Cytochrome P 450

Compounds that are known to increase the hepatic microsomal cytochrome P-450 dependent monooxygenases were adminstered to adult female rats, alone or in combination, to determine whether their effects on certain substrate oxidations were additive. 3-Methyleholanthrene (3-MC) and pregnenolone-16α-carbonitrile (PCN), known to induce different forms of cytochrome P-450, when administered together increased benzo[a]pyrene oxidation to the same level as observed following 3-MC treatment alone. Phenobarbital (Pb) and PCN when administered concomitantly increased benzo[a]pyrene, aminopyrine, and ethylmorphine metabolism to the same extent as seen following PCN administration alone. Both compounds are known to induce different forms of cytochrome P-450. Nonadditive effects were also observed with Pb and spironolactone, as well as with Pb and trans-stilbene oxide. Treatment of adult male rats with either PCN or 3-MC resulted in significantly smaller increases in benzo[a]pyrene oxidation than observed in adult female rats. These results suggest that oxidative metabolism in hepatic microsomes is not the sum of activities of a number of cytochrome P-450s, but may represent the activity of a single predominant hemeprotein. In addition, it appears that the oxidation of substrate by a particular cytochrome P-450, in intact microsomes, is greatly influenced by the presence of another form.

scholarly journals Mechanistic studies of the inhibition of hepatic uroporphyrinogen decarboxylase in C57BL/10 mice by iron-hexachlorobenzene synergism

1986 ◽  
Vol 238 (3) ◽  
pp. 871-878 ◽  
Author(s):  
A G Smith ◽  
J E Francis ◽  
S J E Kay ◽  
J B Greig ◽  
F P Stewart
Keyword(s):  
Additional Treatment ◽  
Mechanistic Studies ◽  
Iron Species ◽  
Uroporphyrinogen Decarboxylase ◽  
Microsomal Cytochrome ◽  
Hepatic Microsomes ◽  
Iron Treatment ◽  
Maximum Inhibition ◽  
Cytochrome P 450

Porphyria was induced in C57BL/10 mice with iron overload by a single oral dose (100 mg/kg) of hexachlorobenzene (HCB). Within 2 weeks hepatic uroporphyrinogen decarboxylase (EC 4.1.1.37) was inhibited, reaching a maximum (greater than 95%) at 6-8 weeks. There was no recovery by 14 weeks, despite a fall in liver HCB concentrations to only 6% of the day-3 value. The major rise in hepatic porphyrin levels occurred after 4 weeks and secondary inhibition of uroporphyrinogen synthase (EC 4.2.1.75) was inferred from the progressively greater proportion of uroporphyrin I present relative to the III isomer. Plasma alanine aminotransferase (EC 2.6.1.2) activity was also elevated. Although, in further studies, total microsomal cytochrome P-450 content and ethoxyphenoxazone de-ethylase activity reached a peak a few days after dosing and had declined significantly at the time of maximum inhibition of the decarboxylase, additional treatment of HCB-dosed mice with a cytochrome P1-450 inducer, beta-naphthoflavone, enhanced the inhibition, whereas piperonyl butoxide, an inhibitor of cytochrome P-450, partially protected. Uroporphyrinogen decarboxylase was not radiolabelled in vivo by [14C]HCB. There was no major difference in the ability to hydroxylate HCB between hepatic microsomes from induced C57BL/10 mice and those from the insensitive DBA/2 strain. By contrast, lipid peroxidation, in the presence of NADPH, was 8-fold greater in control C57BL/10 microsomes than in DBA/2 microsomes and was stimulated by iron treatment (although not by HCB). The results suggest that the inhibition of hepatic uroporphyrinogen decarboxylase is unlikely to be due to a direct effect of a metabolite of HCB but to another process requiring a specific cytochrome P-450 isoenzyme and an unknown iron species.

scholarly journals Inhibition of uroporphyrinogen decarboxylase activity. The role of cytochrome P-450-mediated uroporphyrinogen oxidation

1990 ◽  
Vol 269 (2) ◽  
pp. 437-441 ◽  
Author(s):  
R W Lambrecht ◽  
J M Jacobs ◽  
P R Sinclair ◽  
J F Sinclair
Keyword(s):  
Chick Embryo ◽  
Fold Increase ◽  
Treated Mouse ◽  
Decarboxylase Activity ◽  
Chick Embryos ◽  
Uroporphyrinogen Decarboxylase ◽  
Chemically Induced ◽  
Cytochrome P 450 ◽  
Generating System

It was previously shown that uroporphyrinogen oxidation is catalysed by a form of cytochrome P-450 induced by 3-methylcholanthrene [Sinclair, Lambrecht & Sinclair (1987) Biochem. Biophys. Res. Commun. 146, 1324-1329]. We have now measured uroporphyrinogen oxidation and uroporphyrinogen decarboxylation simultaneously in 10,000 g supernatants from the livers of methylcholanthrene-treated mice and chick embryos incubated with an NADPH-generating system. We found that uroporphyrinogen oxidation is associated with inhibition of uroporphyrinogen decarboxylase activity. The decreased uroporphyrinogen decarboxylase activity was not due to depletion of substrate, since decarboxylase activity was not increased by a 2.6-fold increase in uroporphyrinogen. Uroporphyrinogen oxidation and the associated inhibition of decarboxylase activity were also observed with liver supernatant from methylcholanthrene-treated chick embryo; both actions required the addition of 3,3′,4,4′-tetrachlorobiphenyl. Uroporphyrinogen oxidation catalysed by microsomes from a methylcholanthrene-treated mouse inhibited the uroporphyrinogen decarboxylase activity in the 100,000 g supernatant. Ketoconazole, an inhibitor of cytochrome P-450, prevented both uroporphyrinogen oxidation and the inhibition of uroporphyrinogen decarboxylation. The addition of ketoconazole to mouse supernatant actively oxidizing uroporphyrinogen inhibited the oxidation and restored decarboxylation. The latter finding suggested that a labile inhibitor was formed during the oxidation. These results suggest uroporphyrinogen oxidation may be important in the mechanism of chemically induced uroporphyria.

Growth hormone depresses ethylmorphine demethylase activity: correlation with decreased levels of fast-turnover cytochrome P-450 in hypophysectomized female rats

1988 ◽  
Vol 66 (7) ◽  
pp. 868-872
Author(s):  
Birgit M. Vockentanz ◽  
Bruce B. Virgo
Keyword(s):  
Growth Hormone ◽  
Cytochrome C ◽  
Female Rats ◽  
Monooxygenase System ◽  
Demethylase Activity ◽  
Hepatic Monooxygenase ◽  
Cytochrome P 450 ◽  
Slow Turnover ◽  
Nadph Cytochrome C Reductase

The hepatic monooxygenase system was studied in hypophysectomized female rats infused for 5 days with ovine growth hormone (GH). At 7.5 μg∙h−1 GH decreased the total cytochrome P-450 by 16%; at 10 μg∙h−1 it reduced both cytochrome P-450 (31%) and the activity of ethylmorphine demethylase (31%). GH did not alter the activities of NADPH cytochrome c reductase or aniline hydroxylase. The lower GH dose decreased the amount of fast- and slow-turnover P-450 by 11 and 38%, respectively, while the higher dose decreased both by 49%. The loss of demethylase activity therefore correlates with the loss of fast-tumover P-450. This component is relatively more abundant in the female (fast: slow turnover of 4.3) than the male (fast: slow turnover of 2.5). GH did not affect the half-lives of the P-450 components, suggesting that it decreases their synthesis. The P-450 concentration in microsomes from GH-treated animals did not increase after incubation with hemin, suggesting that in vivo the hormone does not lower P-450 synthesis via depression of heme. Puromycin mimicked the effect of GH and when given with the hormone their effects on the P-450 levels were multiplicative (p < 0.05), suggesting different modes of action and that GH does not decrease P-450 by acting at translation.

scholarly journals Role of haem in the induction of cytochrome P-450 by phenobarbitone. Studies in chick embryos in ovo and in cultured chick embryo hepatocytes

1981 ◽  
Vol 198 (2) ◽  
pp. 321-329 ◽  
Author(s):  
U Giger ◽  
U A Meyer
Keyword(s):  
Chick Embryo ◽  
Inhibitory Effect ◽  
Chick Embryos ◽  
In Ovo ◽  
Haem Biosynthesis ◽  
Rate Limiting ◽  
Cytochrome P 450 ◽  
Hepatic Cytochrome

The role of haem synthesis during induction of hepatic cytochrome P-450 haemoproteins was studied in chick embryo in ovo and in chick embryos hepatocytes cultured under chemically defined conditions. 1. Phenobarbitone caused a prompt increase in the activity of 5-aminolaevulinate synthase, the rate-limiting enzyme of haem biosynthesis, and in the concentration of cytochrome P-450. This induction response occurred without measurable initial destruction of the haem moiety of cytochrome P-450. 2. When intracellular haem availability was enhanced by exogenous haem or 5-aminolaevulinate, phenobarbitone-medicated induction of cytochrome P-450 was not affected in spite of the well known repression of 5-aminolaevulinate synthase by haem. These data are consistent with the concept that haem does not regulate the synthesis of cytochrome P-450 haemoproteins. 3. Acetate inhibited haem biosynthesis at the level of 5-aminolaevulinate formation. When intracellular haem availability was diminished by treatment with acetate, phenobarbitone-medicated induction was decreased. 4. This inhibitory effect of acetate on cytochrome P-450 induction was reversed by exogenous haem or its precursor 5-aminolaevulinate. These data suggest that inhibition of haem biosynthesis does not decrease synthesis of apo-cytochrome P-450. Moreover, they indicate that exogenous haem can be incorporated into newly formed aop-cytochrome P-450.

scholarly journals Metal ion interactions in the control of haem oxygenase induction in liver and kidney

1980 ◽  
Vol 192 (2) ◽  
pp. 637-648 ◽  
Author(s):  
G S Drummond ◽  
A Kappas
Keyword(s):  
Metal Ions ◽  
Metal Ion ◽  
Simultaneous Administration ◽  
Ion Interactions ◽  
Liver And Kidney ◽  
Demethylase Activity ◽  
Haem Oxygenase ◽  
Metal Ion Interactions ◽  
Cytochrome P 450

Mn2+ and Zn2+ exhibit a striking ability to block the induction by Sn2+ and Ni2+ of haem oxygenase (EC 1.14.99.3) in kidney. The blocking effects of Mn2+ and Zn2+ were found to be greatest on simultaneous administration, time-dependent when administered up to 8 h before the inducing metal ions, and ineffective when administered as little as 10 min after the inducing metal ions. The decreases in cytochrome P-450 and haem contents and the sequential changes in delta-aminolaevulinate synthase (EC 2.3.1.37) activity that occur concomitant with haem oxygenase induction were largely eliminated with simultaneous or prior treatment with Mn2+ or Zn2+, but not when Mn2+ or Zn2+ was administered after Sn2+ or Ni2+. Mn2+ and Zn2+ did not increase the catabolism of the enzyme in vivo. Zn2+ on simultaneous administration was also able substantially to block the induction of haem oxygenase by Co2+, Cd2+ and Ni2+ in liver. The Zn2+ blockade of Cd2+ induction was examined in detail, and prior or simultaneous administration of Zn2+ was found to be effective in blocking the induction of haem oxygenase and the concomitant decreases in cytochrome P-450 and haem contents, ethylmorphine demethylase activity and the sequential changes in delta-aminolaevulinate synthase activity. Zn2+ administration 10 min or more after Cd2+ was ineffective in preventing the occurrence of these perturbations in haem metabolism. These findings describe a new and striking biological property of Mn2+ and Zn2+, and indicate the existence of significant metal ion interactions in the control of haem metabolism.

scholarly journals Evidence that in chick embryos destruction of hepatic microsomal cytochrome P-450 haem is a general mechanism of induction of δ-aminolaevulinate synthase by porphyria-causing drugs

1980 ◽  
Vol 190 (3) ◽  
pp. 519-526 ◽  
Author(s):  
L K Lim ◽  
G Srivastava ◽  
J D Brooker ◽  
B K May ◽  
W H Elliott
Keyword(s):  
Chick Embryo ◽  
Enzyme Induction ◽  
General Mechanism ◽  
Chick Embryos ◽  
In Ovo ◽  
Microsomal Cytochrome ◽  
Primary Mechanism ◽  
Embryo Liver ◽  
The Relationship ◽  
Cytochrome P 450

A variety of prophyrinogenic compounds were tested for their effect in ovo on chick-embryo liver microsomal cytochrome P-450 haem concentration and mitochondrial delta-aminolaevulinate synthase activity. With all drugs tested, there was a 30—50% decrease in cytochrome P-450 haem concentration within 1 h of treatment, and this was closely followed by an increase in delta-aminolaevulinate synthase activity. The relationship was independent of the extent of enzyme induction and is consistent with the proposal that drug-mediated destruction of cytochrome P-450 haem is the primary mechanism of induction of delta-aminolaevulinate synthase. After induction, synthesis of delta-aminolaevulinate synthase could be maintained by inhibiting further haem synthesis. These studies suggest that induction of porphyria is a combination of two distinct processes: (a) induction of delta-aminolaevulinate synthase synthesis by destruction of cytochrome P-450 haem and consequent depletion of cellular free haem; (b) maintenance of continued delta-aminolaevulinate synthase synthesis by preventing replenishment of cellular haem either by inhibiting haem synthesis and/or by promoting continuous removal of newly synthesized haem.

Sign in / Sign up

Export Citation Format

Share Document