Drug metabolism in cell culture. II. Studies on the metabolic route of DDC analogues

1977 ◽  
Vol 55 (3) ◽  
pp. 552-559 ◽  
Author(s):  
William J. Racz ◽  
Lynette Hunter ◽  
Richard G. F. Wanless ◽  
Miriam V. McDonald ◽  
Joyce A. Moffat
Keyword(s):  
Cell Culture ◽  
Half Life ◽  
Aminolevulinic Acid ◽  
Piperonyl Butoxide ◽  
Metabolic Route ◽  
Nitrophenyl Phosphate ◽  
Intact Embryo ◽  
Embryo Liver ◽  
Ala Synthetase ◽  
A Minor

To elucidate the major metabolic pathways of DDC analogues, the effect of inhibitors of oxidative and hydrolytic microsomal enzymes on the rate of metabolism of these compounds in chick embryo liver cell cultures has been examined. The metabolism of 3,5-diethoxycarbonyl-2,4,6-trimethylpyridine (Ox-DDC) was inhibited threefold by piperonyl butoxide and SKF-525A (β-diethylaminoethyl-diphenyl-n-propylacetate-HCl), but only onefold by bis(p-nitrophenyl)phosphate (BNPP). This indicates that this compound is metabolized predominately by an oxidative route and hydrolysis is a minor pathway. The half-life of 3,5-diethoxycarbonyl-2,6-dimethylpyridine (4-desmethyl-Ox-DDC) was increased 20-fold by BNPP, but no change was observed with piperonyl butoxide, indicating that this compound is metabolized exclusively by hydrolytic mechanisms. Since both piperonyl butoxide and BNPP produced similar changes in the half-life of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC), it appears that this compound is metabolized equally via oxidative and hydrolytic mechanisms. In contrast to the intact embryo where Ox-DDC is rapidly metabolized and the effect of the agent on δ-aminolevulinic acid (δ-ALA) synthetase (δ-aminolaevulinate synthetase; EC 2.3.1.37) activity is brief and hence porphyrin accumulation does not occur, in cell culture Ox-DDC is metabolized at a rate sufficiently slow so that induction of δ-ALA synthetase does occur.

Drug-Induced Porphyrin Biosynthesis. X. Potentiation of Propanidid-Induced Elevation of δ-Aminolevulinic Acid Synthetase and Porphyrins in Chick Embryo Liver by the Carboxylesterase Inhibitor Bis-[p-nitrophenyl] Phosphate

1973 ◽  
Vol 51 (9) ◽  
pp. 700-704 ◽  
Author(s):  
Hillel Taub ◽  
G. S. Marks
Keyword(s):  
Chick Embryo ◽  
Aminolevulinic Acid ◽  
Ester Group ◽  
Synthetase Activity ◽  
Chick Embryos ◽  
Drug Induced ◽  
Duration Of Action ◽  
Nitrophenyl Phosphate ◽  
Embryo Liver ◽  
Ala Synthetase

Propanidid, an ultra short-acting non-barbiturate anesthetic containing an ester group, induces δ-aminolevulinic acid (ALA)-synthetase and porphyrin accumulation in 17-day-old chick embryo liver. The potency and duration of action of propanidid in inducing ALA-synthetase activity and porphyrin accumulation was markedly increased when administered to chick embryos which had been pretreated with bis-[p-nitrophenyl] phosphate, an inhibitor of liver carboxylesterase.

A comparison of the porphyrin-inducing activity of barbiturates and benzodiazepines in chick embryo liver cells

1980 ◽  
Vol 58 (8) ◽  
pp. 991-995 ◽  
Author(s):  
S. Zimmer ◽  
H. Taub ◽  
G. S. Marks
Keyword(s):  
Cell Culture ◽  
Chick Embryo ◽  
Liver Cell ◽  
Aminolevulinic Acid ◽  
Synthetase Activity ◽  
Great Caution ◽  
Test Systems ◽  
Embryo Liver ◽  
Ala Synthetase ◽  
Hypnotic Dose

Five benzodiazepines, flurazepam, nitrazepam, diazepam, oxazepam, and chlordiazepoxide, have been tested for porphyrin-inducing activity in chick embryo liver cell culture and for δ-aminolevulinic acid (ALA) synthetase inducing activity in the 17-day-old chick embryo. Flurazepam and nitrazepam were found to have considerably lower potency than secobarbital whereas diazepam, oxazepam, and chlordiazepoxide were less potent than phenobarbital in these test systems. The following conclusions were arrived at. (1) A hypnotic dose of flurazepam or nitrazepam would be less likely than a comparable dose of secobarbital to increase ALA synthetase activity in a patient with hereditary hepatic porphyria. (2) A sedative dose of diazepam, oxazepam, or chlordiazepoxide would be less likely than a comparable dose of phenobarbital to increase ALA synthetase activity in a patient with hereditary hepatic porphyria. (3) The benzodiazepines, although apparently less likely to precipitate an attack than barbiturates, should be used with great caution in porphyria patients.

Inhibition of ferrochelatase by N-methylprotoporphyrin IX is not accompanied by δ-aminolevulinic acid synthetase induction in chick embryo liver cell culture

1982 ◽  
Vol 60 (2) ◽  
pp. 212-215 ◽  
Author(s):  
Susan P. C. Cole ◽  
Gerald S. Marks ◽  
Paul R. Oritz de Montellano ◽  
Kent L. Kunze
Keyword(s):  
Cell Culture ◽  
Chick Embryo ◽  
Liver Cell ◽  
Aminolevulinic Acid ◽  
Synthetase Activity ◽  
Liver Cell Culture ◽  
Embryo Liver ◽  
Ala Synthetase

N-Methylprotoporphyrin has been shown to markedly inhibit ferrochelatase activity in chick embryo liver cell culture without inducing δ-aminoievulinic acid (ALA) synthetase activity. This result supports the idea that the effects of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC) on ALA synthetase activity and ferrochelatase activity are dissociated and that inhibition of ferrochelatase alone is not sufficient to cause induction of ALA synthetase. We conclude that the porphyrinogenic activity of DDC can be explained only in part by the actions of N-methylprotoporphyrin.

Drug-Induced Porphyrin Biosynthesis—VIII. Investigation of the Importance of an Allyl Group for Activity in Substituted Acetamides

1973 ◽  
Vol 51 (11) ◽  
pp. 863-868 ◽  
Author(s):  
G. S. Marks ◽  
V. Krupa ◽  
M. W. Roomi
Keyword(s):  
Cell Culture ◽  
Chick Embryo ◽  
Liver Cell ◽  
Aminolevulinic Acid ◽  
Developmental Differences ◽  
Synthetase Activity ◽  
Drug Induced ◽  
Liver Cell Culture ◽  
Embryo Liver ◽  
Ala Synthetase

Allylisopropylacetamide (AIA) was found to be considerably more potent than propylisopropylacetamide (PIA) in inducing hepatic δ-aminolevulinic acid (ALA) synthetase activity and hepatic porphyrin accumulation in the 17-day-old chick embryo, chickens, and mice of the CBA/J strain. AIA and PIA were shown to be approximately equipotent in inducing ALA synthetase activity and porphyrin accumulation in chick embryo liver cell culture. It is likely that the unusual responsiveness of chick embryo liver cell culture to PIA is not due to species or developmental differences but to the large amount of PIA available to the cultured liver cells.

scholarly journals Porphyrin Synthesis and Heme Synthetase Activity in Pyridoxine-Responsive Anemia

Blood ◽  
1968 ◽  
Vol 32 (6) ◽  
pp. 979-988 ◽  
Author(s):  
WILLAM RALPH VOGLER ◽  
ELIZABETH S. MINGIOLI
Keyword(s):  
Aminolevulinic Acid ◽  
Intermediate Compound ◽  
Normal Rate ◽  
Synthetase Activity ◽  
Iron Stores ◽  
Excess Iron ◽  
Porphyrin Synthesis ◽  
Ala Synthetase ◽  
A Minor

Abstract Previous in vitro studies had shown that reticulocytes from a patient with pyridoxine-responsive anemia (PRA) incorporated glycine-2-14C into heme at 11-25 per cent of the normal rate. Incorporation of the intermediate compound, δ-aminolevulinic acid-4-14C (ALA), varied from 46 to 81 per cent of normal. These findings suggested two defects in the synthesis of heme: a major one prior to formation of ALA, probably ALA synthetase, and a minor one somewhere between ALA and heme. This minor defect was investigated in a patient with PRA during a period of repeated phlebotomies resulting in a 30 gram reduction in iron stores. Porphyrin synthesis from ALA was measured in vitro and found to be normal. Heme synthetase activity in particulate fractions of reticulocytes measured by radioiron incorporation into heme was 50 per cent less than normal when the iron stores were high and significantly improved when the iron stores were reduced to normal. Repeat studies showed that incorporation of ALA-4-14C into heme by intact reticulocytes equaled the normal rate. Glycine incorporation was still reduced. Concomitant with the improvement in heme synthetase activity was an improvement in anemia. These results suggest that excess iron is a significant cause of the reduced heme synthetase activity observed in this patient with pyridoxine-responsive anemia.

Drug-Induced Porphyrin Biosynthesis—XII. Levels of Cytochrome P-450 in Chick Embryo Liver following Administration of Allylisopropylacetamide and Propylisopropylacetamide

1974 ◽  
Vol 52 (4) ◽  
pp. 891-895 ◽  
Author(s):  
V. Krupa ◽  
J. C. Creighton ◽  
M. Freeman ◽  
G. S. Marks
Keyword(s):  
Chick Embryo ◽  
Aminolevulinic Acid ◽  
The Other ◽  
Synthetase Activity ◽  
Drug Induced ◽  
Liver Cytochrome ◽  
Time Period ◽  
Embryo Liver ◽  
Ala Synthetase ◽  
Cytochrome P 450

Allylisopropylacetamide caused a decrease in the level of chick embryo liver cytochrome P-450 1 h after administration, followed by an elevation above control levels at a later time period. Propylisopropylacetamide on the other hand did not produce an early decrease in cytochrome P-450 but produced an elevation of cytochrome P-450 at a later time period. Since propylisopropylacetamide is an inducer of δ-aminolevulinic acid synthetase activity and porphyrin accumulation in chick embryo liver, it was concluded that a loss of cytochrome P-450 is not a prerequisite for ALA-synthetase induction as is thought to be the case in rats.

Patterns of porphyrin accumulation in response to chemicals in chick embryo liver cells

1983 ◽  
Vol 61 (6) ◽  
pp. 546-553 ◽  
Author(s):  
G. S. Marks ◽  
S. B. Follows ◽  
D. T. Zelt ◽  
S. P. C. Cole
Keyword(s):  
Chick Embryo ◽  
Biosynthetic Pathway ◽  
Aminolevulinic Acid ◽  
Liver Cells ◽  
Uroporphyrinogen Decarboxylase ◽  
Coproporphyrinogen Oxidase ◽  
Site Of Action ◽  
Endogenous Substrate ◽  
Embryo Liver ◽  
Ala Synthetase

Four patterns of porphyrin accumulation were observed by high-pressure liquid chromatography when chemicals were added to chick embryo liver cells. These patterns provide a guide to the site of action of the chemicals. Protoporphyrin accumulated in response to 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC), a result consistent with its ability to inhibit ferrochelatase. Uroporphyrin and heptacarboxylic acid porphyrin accumulated in response to 3,3′,4,4′-tetrachlorobiphenyl, 2,2′,4,4′,6,6′-hexachlorobiphenyl, and 3,5-diethoxycarhonyl-2,4,6-trimethylpyridine, a result suggesting inhibition of uroporphyrinogen decarboxylase. Coproporphyrin was the major porphyrin to accumulate in response to allylisopropylacetamide, aromatic amides, and steroids, a result suggesting inhibition of coproporphyrinogen oxidase. A mixture of uroporphyrin, heptacarboxylic acid porphyrin and coproporphyrin accumulated in response to aromatic di- and mono-esters, aliphatic diesters, and aliphatic amides. The pattern observed after addition of excess δ-aminolevulinic acid (ALA) the endogenous substrate of the pathway to the cells was proto- > copro- > uro-porphyrin. This pattern resembled that produced by DDC but by none of the other chemicals. It was concluded that porphyrin accumulation can not be attributed solely to the induction of ALA-synthetase. It appears that porphyrin-inducing chemicals exert an additional effect on one or other of the enzymes of the heme biosynthetic pathway.

scholarly journals Half-life modeling of basic fibroblast growth factor released from growth factor-eluting polyelectrolyte multilayers

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ivan Ding ◽  
Amy M. Peterson
Keyword(s):  
Cell Culture ◽  
Growth Factor ◽  
Fibroblast Growth Factor ◽  
Basic Fibroblast Growth Factor ◽  
Half Life ◽  
Cell Types ◽  
Polyelectrolyte Multilayers ◽  
Enzyme Linked Immunosorbent Assay ◽  
Fibroblast Growth ◽  
Cell Culture Study

AbstractGrowth factor-eluting polymer systems have been widely reported to improve cell and tissue outcomes; however, measurements of actual growth factor concentration in cell culture conditions are limited. The problem is compounded by a lack of knowledge of growth factor half-lives, which impedes efforts to determine real-time growth factor concentrations. In this work, the half-life of basic fibroblast growth factor (FGF2) was determined using enzyme linked immunosorbent assay (ELISA). FGF2 release from polyelectrolyte multilayers (PEMs) was measured and the data was fit to a simple degradation model, allowing for the determination of FGF2 concentrations between 2 and 4 days of culture time. After the first hour, the FGF2 concentration for PEMs assembled at pH = 4 ranged from 2.67 ng/mL to 5.76 ng/mL, while for PEMs assembled at pH = 5, the concentration ranged from 0.62 ng/mL to 2.12 ng/mL. CRL-2352 fibroblasts were cultured on PEMs assembled at pH = 4 and pH = 5. After 2 days, the FGF2-eluting PEM conditions showed improved cell count and spreading. After 4 days, only the pH = 4 assembly condition had higher cells counts, while the PEM assembled at pH = 5 and PEM with no FGF2 showed increased spreading. Overall, the half-life model and cell culture study provide optimal concentration ranges for fibroblast proliferation and a framework for understanding how temporal FGF2 concentration may affect other cell types.

scholarly journals Ultrastructural and Biophysical Characterization of Hepatitis C Virus Particles Produced in Cell Culture

2010 ◽  
Vol 84 (21) ◽  
pp. 10999-11009 ◽  
Author(s):  
Pablo Gastaminza ◽  
Kelly A. Dryden ◽  
Bryan Boyd ◽  
Malcolm R. Wood ◽  
Mansun Law ◽  
...  
Keyword(s):  
Cell Culture ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Structural Characteristics ◽  
Buoyant Density ◽  
Hcv Rna ◽  
Membrane Bilayer ◽  
Biophysical Characterization ◽  
Negative Stain ◽  
A Minor

ABSTRACT We analyzed the biochemical and ultrastructural properties of hepatitis C virus (HCV) particles produced in cell culture. Negative-stain electron microscopy revealed that the particles were spherical (∼40- to 75-nm diameter) and pleomorphic and that some of them contain HCV E2 protein and apolipoprotein E on their surfaces. Electron cryomicroscopy revealed two major particle populations of ∼60 and ∼45 nm in diameter. The ∼60-nm particles were characterized by a membrane bilayer (presumably an envelope) that is spatially separated from an internal structure (presumably a capsid), and they were enriched in fractions that displayed a high infectivity-to-HCV RNA ratio. The ∼45-nm particles lacked a membrane bilayer and displayed a higher buoyant density and a lower infectivity-to-HCV RNA ratio. We also observed a minor population of very-low-density, >100-nm-diameter vesicular particles that resemble exosomes. This study provides low-resolution ultrastructural information of particle populations displaying differential biophysical properties and specific infectivity. Correlative analysis of the abundance of the different particle populations with infectivity, HCV RNA, and viral antigens suggests that infectious particles are likely to be present in the large ∼60-nm HCV particle populations displaying a visible bilayer. Our study constitutes an initial approach toward understanding the structural characteristics of infectious HCV particles.

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