Inhibition of chick embryo hepatic uroporphyrinogen decarboxylase by components of xenobiotic-treated chick embryo hepatocytes in culture

1989 ◽  
Vol 67 (3) ◽  
pp. 246-249 ◽  
Author(s):  
C. A. James ◽  
G. S. Marks
Keyword(s):  
Chick Embryo ◽  
Protein Concentration ◽  
Enzyme Assay ◽  
Uroporphyrinogen Decarboxylase ◽  
Optimum Ph ◽  
Embryo Liver ◽  
Liver Homogenates ◽  
Previous Demonstration ◽  
Sodium Phenobarbital ◽  
Cytochrome P 450

Uroporphyrinogen decarboxylase (UROG-D) activity in the 10 000 g supernatant of 17-day-old chick embryo liver homogenates was determined by measuring the conversion of pentacarboxylporphyrinogen I to coproporphyrinogen I. The optimum pH of the enzyme was found to be approximately 6.0 and enzyme activity was found to be linear with protein concentrations ranging from 0.3 to 2.0 mg/mL. At a protein concentration of 1.2 mg/mL and pH 6.0, the activity was found to be linear for a reaction time of 50 min and to be approximately 10 pmol/(mg protein∙min). This enzyme assay was used to demonstrate that a UROG-D inhibitor, previously reported to accumulate in rodent liver, also accumulates in 3,3′4,4′-tretrachlorobiphenyl (TCBP) and sodium phenobarbital (PB) treated chick embryo hepatocytes in culture. This result accords with the previous demonstration of a TCBP- and PB-induced decrease in UROG-D activity in this system. Uroporphyrin accumulation in chick embryo hepatocyte culture is interpreted as resulting from a combination of two mechanisms, viz., inhibition of UROG-D activity and uroporphyrinogen oxidation to uroporphyrin catalyzed by a cytochrome P-450 isozyme.Key words: uroporphyrinogen decarboxylase, enzyme inhibition, chick embryo hepatocytes, tetrachlorobiphenyl, porphyria.

Porphyrinogenic effects in chick embryo liver cell culture of chloramphenicol analogues that are mechanism-based inactivators of cytochrome P-450

1991 ◽  
Vol 69 (4) ◽  
pp. 526-530 ◽  
Author(s):  
R. P. Green-Thompson ◽  
D. S. Riddick ◽  
J. E. Mackie ◽  
G. S. Marks ◽  
J. R. Halpert
Keyword(s):  
Cell Culture ◽  
Chick Embryo ◽  
Rat Liver ◽  
Liver Cell ◽  
Prosthetic Group ◽  
Uroporphyrinogen Decarboxylase ◽  
Compound A ◽  
Liver Cell Culture ◽  
Embryo Liver ◽  
Cytochrome P 450

Structural analogues of chloramphenicol (CAP) cause mechanism-based inactivation of rat liver cytochrome P-450 (P450) either via protein acylation or destruction of the heme prosthetic group. The goal of the present work was to determine whether CAP analogues that cause loss of the P450 heme moiety also cause porphyrin accumulation in chick embryo liver cell culture. The porphyrin profiles produced by exposure of cells to CAP analogues (160 μM) were determined by high-performance liquid chromatography with fluorescence detection. Of three CAP analogues that do not cause loss of the heme moiety of rat liver P450IIB1, two dichloroacetamides were not porphyrinogenic. The third compound, a chlorofluoroacetamide, caused porphyrin accumulation. This result may be due to the presence of P450 isozymes in chick embryo hepatocytes, distinct from rat liver P450IIB1, that are susceptible to destruction by this analogue. Of four CAP analogues that inactivate rat liver P450IIB1 with concomitant heme loss, a dichloroacetamide and two chlorofluoroacetamides caused porphyrin accumulation. The remaining compound, a monochloroacetamide, was not porphyrinogenic, perhaps because the P450 apoprotein cannot be reconstituted with fresh heme drawn from the regulatory "free heme pool" following inactivation by this analogue. Alternatively, there may be no P450 isozyme in chick embryo liver cell culture that is susceptible to inactivation by this compound.Key words: cytochrome P-450, chloramphenicol, chick embryo hepatocyte, mechanism-based inactivation, uroporphyrinogen decarboxylase.

The 1986 Upjohn Award Lecture. Interaction of chemicals with hemoproteins: implications for the mechanism of action of porphyrinogenic drugs and nitroglycerin

1987 ◽  
Vol 65 (6) ◽  
pp. 1111-1119 ◽  
Author(s):  
Gerald S. Marks
Keyword(s):  
Chick Embryo ◽  
Guanylate Cyclase ◽  
Inhibitory Activity ◽  
Liver Cells ◽  
Enzymatic Reactions ◽  
Uroporphyrinogen Decarboxylase ◽  
Nitrite Ion ◽  
Catalytic Activation ◽  
Embryo Liver ◽  
Cytochrome P 450

The ferrochelatase inhibitory activity of a variety of analogues of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC) was studied in chick embryo liver cells. The ferrochelatase inhibitory activity of the 4-butyl, 4-pentyl, and 4-hexyl analogues was considered to be due to catalytic activation by cytochrome P-450 leading to heme alkylation and formation of the corresponding N-alkylporphyrins. The relative ferrochelatase inhibitory activity of the DDC analogues has implications for a postulated model of the binding of porphyrins in the ferrochelatase active site. 3-[2-(2,4,6-Trimethylphenyl)thioethyl]-4-methylsydnone (TTMS) was shown to be a potent porphyrinogenic agent and to inhibit ferrochelatase in chick embryo liver cells. A related sydnone, 3-benzyl-4-phenylsydnone did not inhibit ferrochelatase activity. These results supported the idea that the porphyrinogenicity of TTMS was due to catalytic activation by cytochrome P-450 leading to heme alkylation and formation of N-vinylprotoporphyrin which inhibits ferrochelatase. Polychlorinated biphenyls, phenobarbital, nifedipine, and a large number of structurally different chemicals which are porphyrinogenic in chick embryo liver cells inhibit uroporphyrinogen decarboxylase by an unknown mechanism. Thus drug-induced porphyrin biosynthesis in chick embryo liver cell culture appears to be caused by inhibition of either ferrochelatase or uroporphyrinogen decarboxylase. The biotransformation of nitroglycerin by human red blood cells is due to a combination of a sulfhydryl-dependent enzymatic process and an interaction with reduced hemoglobin. Biotransformation of nitroglycerin was shown to occur only with the deoxy form of hemoglobin and to involve a two-electron denization, resulting in the oxidation of two molecules of heme iron (II) per mole of nitroglycerin biotransformed to glyceryl dinitrate and nitrite anion. Since nitroglycerin biotransformation appears to be involved in the mechanism of nitroglycerin-induced vasodilation, we have suggested the following hypothesis: biotransformation of nitroglycerin in vascular smooth muscle might occur by interaction of nitroglycerin with the iron (ferrous) of guanylate cyclase-bound heme. The nitrite ion formed may be converted via nitrous acid to nitric oxide. This in turn would combine with the heme moiety of guanylate cyclase to activate the enzyme and through a series of enzymatic reactions cause vasodilation.

scholarly journals Uroporphyrin accumulation produced by halogenated biphenyls in chick-embryo hepatocytes. Reversal of the accumulation by piperonyl butoxide

1986 ◽  
Vol 237 (1) ◽  
pp. 63-71 ◽  
Author(s):  
P R Sinclair ◽  
W J Bement ◽  
H L Bonkovsky ◽  
R W Lambrecht ◽  
J E Frezza ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Chick Embryo ◽  
De Novo ◽  
Covalent Binding ◽  
Piperonyl Butoxide ◽  
Uroporphyrinogen Decarboxylase ◽  
Drug Induced ◽  
Cytochrome P 450 ◽  
Inhibition Of Metabolism ◽  
Methylenedioxyphenyl Compounds

Cultures of chick-embryo hepatocytes were used to study the mechanism by which 3,4,3′,4′-tetrachlorobiphenyl and 2,4,5,3′,4′-pentabromobiphenyl cause accumulation of uroporphyrin. In a previous paper, an isoenzyme of cytochrome P-450 induced by 3-methylcholanthrene had been implicated in this process [Sinclair, Bement, Bonkovsky & Sinclair (1984) Biochem. J. 222, 737-748]. Cells treated with 3,4,3′,4′-tetrachlorobiphenyl and 5-aminolaevulinate accumulated uroporphyrin and heptacarboxyporphyrin, whereas similarly treated cells accumulated protoporphyrin immediately after piperonyl butoxide was added. Piperonyl butoxide also restored haem synthesis as detected by incorporation of radioactive 5-aminolaevulinate into haem, and decrease in drug-induced 5-aminolaevulinate synthase activity. The restoration of synthesis of protoporphyrin and haem by piperonyl butoxide was not affected by addition of cycloheximide, indicating recovery was probably not due to protein synthesis de novo. Piperonyl butoxide also reversed uroporphyrin accumulation caused by 3,4,5,3′,4′,5′-hexachlorobiphenyl, mixtures of other halogenated biphenyls, lindane, parathion, nifedipine and verapamil. The effect of piperonyl butoxide was probably not due to inhibition of metabolism of these compounds, since the hexachlorobiphenyl was scarcely metabolized. Other methylenedioxyphenyl compounds, as well as ellipticine and acetylaminofluorene, also reversed the uroporphyrin accumulation caused by 3,4,3′,4′-tetrachlorobiphenyl. SKF-525A (2-dimethylaminoethyl-2,2-diphenyl valerate) did not reverse the uroporphyrin accumulation caused by the halogenated biphenyls, but did reverse that caused by phenobarbital and propylisopropylacetamide. We conclude that the mechanism of the uroporphyrin accumulation cannot be due to covalent binding of activated metabolites of halogenated compounds to uroporphyrinogen decarboxylase.

scholarly journals Conversion of 5-aminolaevulinate into haem by liver homogenates. Comparison of rat and chick embryo

1981 ◽  
Vol 198 (3) ◽  
pp. 595-604 ◽  
Author(s):  
J F Healey ◽  
H L Bonkowsky ◽  
P R Sinclair ◽  
J F Sinclair
Keyword(s):  
Chick Embryo ◽  
Rat Liver ◽  
Biosynthetic Pathway ◽  
Frozen Storage ◽  
Relevant Information ◽  
Porphobilinogen Deaminase ◽  
Embryo Liver ◽  
Liver Homogenates ◽  
Rate Limiting ◽  
Kinetics Of

1. We have studied the kinetics of the conversion of 5-aminolaevulinate into haem and haem precursors in homogenates of livers of rats and chick embryos. Homogenates of fresh liver from both species efficiently convert 5-aminolaevulinate into haem. After frozen storage for 1 year, homogenates of rat, but not chick, liver have decreased rates of formation of haem with accumulation of more protoporphyrin. The rate of haem formation after storage is restored by addition of Fe2+ and menadione. 2. At all initial concentrations of 5-aminolaevulinate tested (2 microM-1 mM), homogenates of rat liver accumulate less protoporphyrin than haem. In contrast, homogenates of chick embryo liver accumulate more protoporphyrin than haem at concentration of 5-aminolaevulinate greater than 10 microM. Conversion of protoporphyrin into haem by homogenates of fresh or frozen chick embryo liver is not increased by addition of Fe2+. 3. Homogenates of liver from both species accumulate porphobilinogen; the kinetic parameters for this process reflect those of 5-aminolaevulinate dehydratase. 4. The results show that the rate-limiting enzyme for the hepatic conversion of 5-aminolaevulinate into protoporphyrin is porphobilinogen deaminase. In addition, chick liver, compared with rat liver, has only about one-fifth the activity of ferrochelatase, the final enzyme of the haem biosynthetic pathway, which inserts Fe2+ into protoporphyrin to form haem. 5. Comparison of these results with previous studies indicates that the homogenate system described here provides physiologically and clinically relevant information for study of hepatic haem synthesis and its control.

Inhibition of chick embryo hepatic uroporphyrinogen decarboxylase by components of xenobiotic-treated chick embryo hepatocytes in culture. II.

1992 ◽  
Vol 70 (7) ◽  
pp. 939-942 ◽  
Author(s):  
R. P. Green-Thompson ◽  
S. M. Kimmett ◽  
G. S. Marks
Keyword(s):  
Chick Embryo ◽  
Enzyme Inhibition ◽  
Culture System ◽  
Hepatocyte Culture ◽  
Uroporphyrinogen Decarboxylase ◽  
Soluble Component ◽  
Sodium Phenobarbital

A variety of xenobiotics, viz., 3,3′,4,4′-tetrachlorobiphenyl (TCBP), sodium phenobarbital (PB), 3,5-diethoxycarbonyl-2,4,6-trimethylpyridine (OX-DDC), and nifedipine, cause a decrease in uroporphyrinogen decarboxylase (UROG-D) activity, accompanied by uroporphyrin accumulation, in chick embryo hepatocytes in culture. In this study the activity of 17-day-old chick embryo hepatic UROG-D was determined by measuring the conversion of pentacarboxylporphyrinogen I to coproporphyrinogen I, and it was shown that a UROG-D inhibitor, previously reported to accumulate in TCBP-treated and PB-treated chick embryo hepatocytes in culture, also accumulates in OX-DDC-treated and nifedipine-treated chick embryo hepatocytes in culture. It was concluded that the accumulation of a UROG-D inhibitor provides an explanation for the UROG-D inhibition observed in this culture system with xenobiotics that cause uroporphyrin accumulation. Studies of the UROG-D inhibitory fraction isolated from the 10 000 × g, 40 000 × g, and 100 000 × g supernatant fractions of cultured chick embryo hepatocyte homogenate led to the conclusion that the UROG-D inhibitor is derived from a soluble component of the homogenate.Key words: uroporphyrinogen decarboxylase, chick embryo hepatocyte culture, nifedipine, 3,5-diethoxycarbonyl-2,4,6-trimethylpyridine, enzyme inhibition.

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