Cultured Chick Embryo Liver Cells Research Articles

A direct immunofluorescence test for the rapid detection of avian influenza virus antigen in tissue impression smears

A direct immunofluorescent test on tissue impression smears for the simple, rapid, cheap and sensitive detection of avian influenza virus antigen is described. Cross immunofluorescence tests carried out on four avian influenza conjugates demonstrated that a conjugated anti-serum prepared against one influenza virus subtype would stain heterologous subtypes. Direct immunofluorescence on tissue impression smears from broilers infected with either avirulent or highly pathogenic avian influenza viruses was compared with virus isolation in embryonated hens' eggs, chick embryo liver and chick embryo fibroblast cell cultures. Immunofluorescence was as sensitive as virus isolation in eggs. In some cases antigen was detected by immunofluorescence where virus isolation failed due to the presence of antibody or the onset of autolysis. Virus isolation in chick embryo liver was more successful than chick embryo fibroblasts but not as sensitive as embryonated eggs.

Toxicity of polychlorinated biphenyl with special reference to porphyrin metabolism.

Oral administration of a commercial PCB mixture to chickens caused a hepatic-type porphyria characterized by hepatic accumulation and urinary excretion of uroporphyrin. To clarify the mechanism of the porphyrinogenic activity of these PCBs, we studied the structural requirement of synthetic PCB for porphyrinogenic activities by using the cultured chick embryo liver cells and examined the relationship between induction of delta-aminolevulinic acid (ALA) synthetase and inhibition of uroporphyrinogen decarboxylase. We established that the porphyrinogenic effect of PCBs exhibits a sharply defined structure-activity relationship in that only 3,4,3',4'-tetrachlorobiphenyl and 3,4,5,3',4',5'-hexachlorobiphenyl produced a marked accumulation of uroporphyrin. We also demonstrated that in ALA-supplemented cultures, these same compounds lead to accumulation of a large amount of uroporphyrin III, whereas with other PCBs, which were weak inducers of porphyrin synthesis, the accumulated porphyrin was mostly protoporphyrin. was mostly protoporphyrin. Kinetic studies of the sequential decarboxylation of uroporphyrinogen with purified uroporphyrinogen decarboxylase were performed. The 3,4,3',4'-tetrachlorobiphenyl and 3,4,5,3',4',5'-hexachlorobiphenyl strongly inhibit uroporphyrinogen decarboxylase directly at two steps, i.e. first in the formation of hexacarboxylic porphyrinogen III from heptacarboxylic porphyrinogen III and second in the formation of heptacarboxylic porphyrinogen III from uroporphyrinogen III. The results confirmed that porphyrinogenic PCBs primarily inhibit uroporphyrinogen decarboxylase, leading to a depletion of heme as a result of which synthesis of ALA synthetase increased.

Toxicity of Polychlorinated Biphenyl with Special Reference to Porphyrin Metabolism

Oral administration of a commercial PCB mixture to chickens caused a hepatic-type porphyria characterized by hepatic accumulation and urinary excretion of uroporphyrin. To clarify the mechanism of the porphyrinogenic activity of these PCBs, we studied the structural requirement of synthetic PCB for porphyrinogenic activities by using the cultured chick embryo liver cells and examined the relationship between induction of delta-aminolevulinic acid (ALA) synthetase and inhibition of uroporphyrinogen decarboxylase. We established that the porphyrinogenic effect of PCBs exhibits a sharply defined structure-activity relationship in that only 3,4,3',4'-tetrachlorobiphenyl and 3,4,5,3',4',5'-hexachlorobiphenyl produced a marked accumulation of uroporphyrin. We also demonstrated that in ALA-supplemented cultures, these same compounds lead to accumulation of a large amount of uroporphyrin III, whereas with other PCBs, which were weak inducers of porphyrin synthesis, the accumulated porphyrin was mostly protoporphyrin. was mostly protoporphyrin. Kinetic studies of the sequential decarboxylation of uroporphyrinogen with purified uroporphyrinogen decarboxylase were performed. The 3,4,3',4'-tetrachlorobiphenyl and 3,4,5,3',4',5'-hexachlorobiphenyl strongly inhibit uroporphyrinogen decarboxylase directly at two steps, i.e. first in the formation of hexacarboxylic porphyrinogen III from heptacarboxylic porphyrinogen III and second in the formation of heptacarboxylic porphyrinogen III from uroporphyrinogen III. The results confirmed that porphyrinogenic PCBs primarily inhibit uroporphyrinogen decarboxylase, leading to a depletion of heme as a result of which synthesis of ALA synthetase increased.

Ferrochelatase and N-alkylated porphyrins

The final step in heme synthesis is catalyzed by the mitochondrial enzyme, ferrochelatase. Characterization of this enzyme has been complicated by a number of factors including the dependence of enzyme activity on lipids. Purification of ferrochelatase from rat and bovine sources has been achieved only relatively recently using blue Sepharose CL-6B chromatography. When 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC) is given to animals, it produces a hepatic porphyria resembling human variegate porphyria thus providing an experimental system in which to study this disease. DDC has been found to cause the accumulation of a green pigment, identified as N-methyl protoporphyrin IX (N-MePP), which is a potent inhibitor of ferrochelatase. The source of the N-methyl substituent of N-MePP was found to be the 4-methyl group of DDC. Considerable evidence indicates that the protoporphyrin IX moiety of N-MePP originates from the heme moiety of cytochrome P-450 and that DDC is a suicide substrate for this hemoprotein. Some studies suggest that cytochrome P-450 isozymes differ in their susceptibility to destruction by DDC and its 4-alkyl analogues. Griseofulvin has also been reported to inhibit hepatic ferrochelatase in rodents but not in the 17-day old chick embryo nor in hepatocyte culture systems. Thus, the mechanism by which griseofulvin produces an experimental porphyria in chick embryo liver cell culture is different from that for rodents.

Effect of stanozolol on delta-aminolaevulinic acid synthase and hepatic monooxygenase activity in man and rat.

Stanozolol is an anabolic steroid which is used in the treatment of aplastic anaemia and has been recently advocated for the prophylaxis of vascular thrombosis. Similar steroid substances stimulate the activity of delta-aminolaevulinic acid synthase (ALA S), the rate limiting enzyme of haem biosynthesis, in rat hepatocytes and chick embryo liver cell cultures and activate acute hepatic porphyria. In the present study stanozolol (10 mg daily for 14 days) has been shown to increase significantly leucocyte ALA S activity in 9 healthy male subjects. There was a concomitant rise in urinary ALA and total porphyrin excretion but no change in antipyrine kinetics or urinary 6 B hydroxycortisol excretion. In a complementary study in male Sprague Dawley rats, stanozolol administered intraperitoneally, produced a dose-dependent increase in hepatic ALA S activity without changing hepatic cytochrome P 450 content. Stanozolol has been clearly shown to elevate ALA S activity, probably directly, and thereby, porphyrin production without affecting hepatic monooxygenase activity. This porphyrinogenic effect may be relevant to the successful treatment of aplastic anaemia with anabolic steroids. Leucocyte ALA S activity may provide a human system for the study of drug porphyrinogenicity in vivo.

Inhibition of uroporphyrinogen decarboxylase by 3,3',4,4'-tetrachlorobiphenyl in chick embryo liver cell culture.

An assay was developed to measure uroporphyrinogen decarboxylase activity in 17-day-old chick embryo liver cells in culture. Decarboxylation products arising from the action of uroporphyrinogen decarboxylase upon uroporphyrinogen III were quantitated using high-pressure liquid chromatography. 3,3',4,4'-Tetrachlorobiphenyl (TCBP) was added to chick embryo liver cells and 24 h later the uroporphyrinogen decarboxylase activity in the cells was measured; a significant inhibition was found. On the other hand, when TCBP was added directly to the crude uroporphyrinogen decarboxylase enzyme no inhibition was noted. The inhibition of uroporphyrinogen decarboxylase observed in this study explains the accumulation of uroporphyrin and heptacarboxylic porphyrin in chick embryo liver cell cultures treated with TCBP.

Inhibition of steroid-mediated induction of δ-aminolevulinic acid synthase by 2-diethylaminoethyl-2,2-diphenylvalerate HCl (SKF 525-A)

The inhibition of the steroid-mediated induction of δ-aminolevulinate synthase, the rate-limiting enzyme in hepatic porphyrin-heme biosynthesis, by 2-diethylaminoethyl-2,2-diphenylvalerate HCl (SKF 525-A) as studied in cultured chick embryo liver cells. The formation of porphyrins in response to cyproterone, a synthetic steroid, was inhibited in a time-dependent manner by SKF 525-A, an inhibitor of several drug metabolizing enzyme systems. This action is a result of an inhibitory effect of SKF 525-A on the cyproterone-mediated induction of δ-aminolevulinate synthase; SKF 525-A laso inhibited the induction of the enzyme by the naturally occurring 5β-H steroids, etiocholanolone and pregnanolone. Employing [ 3H]etiocholanolone, we provide evidence that this inhibition is not associated with either decreased uptake or an altered metabolism of the steroid. Moreover, approx. 4–6-fold more radioactivity was associated with [ 3H]etiocholanolone-treated cells cultured in the presence of SKF 525-A. Alternative mechanisms for the induction of δ-aminolevulinate synthase by steroids are proposed which do not require the interaction of steroid-receptor complex with the genome.

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

N-Methylprotoporphyrin has been shown to markedly inhibit ferrochelatase activity in chick embryo liver cell culture without inducing delta-aminolevulinic 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.

Polychlorobiphenyls that induce δ-aminolevulinic acid synthetase inhibit uroporphyrinogen decarboxylase in cultured chick embryo liver cells

Polychlorobiphenyls that induce δ-aminolevulinic acid synthetase inhibit uroporphyrinogen decarboxylase in cultured chick embryo liver cells

Prevention by estradiol of aflatoxin-induced cytotoxicity in cultured chick embryo liver cells.

Aflatoxin B1 at a final concentration of 10(-5) M depressed the sytheses of DNA, RNA and protein in cultured chick embryo liver cells. The same dose produced ultrastructural changes in the nucleus, such as nucleolar compactness or segregation of fibrillar and granular components and in the cytoplasmic area, such as vesiculation, dilatation or degranulation of endoplasmic meticulum. These acute toxic effects of aflatoxin B1 were partially decreased by an addition of 4 X 10(-5) M estradiol-17 beta. Namely estradiol-17 beta significantly reduced the nucleolar compactness and segregation of fibrillo-granular components but did not improve the vesiculation, dilatation and degranulation of endoplasmic reticulum. Estradiol-17 beta also protected the liver cells from the aflatoxin B1-induced inhibition of nucleic acid and protein synthesis. These results suggest that the protective effect of estradiol-17 beta against the acute hepatotoxicity of aflatoxin B1 is mainly due to an antagonistic interaction of both compounds on the synthesis of nucleic acid in nucleolus.

Inhibition of hepatic ferrochelatase by the four isomers of N-methylprotoporphyrin IX

The four isomers of N-methylprotoporphyrin IX, obtained by chemical synthesis and purified by high pressure liquid chromatography, have been shown to be equally potent as inhibitors of the ferrochelatase enzymes from rat liver and from cultured chick embryo liver cells.

Isolation of rotaviruses from turkeys and chickens: Demonstration of distinct serotypes and RNA electropherotypes

Six isolates of rotavirus were made from turkeys and two from chickens. Three of these required trypsin treatment for isolation and serial passage in cell cultures. The remainder were isolated without trypsin treatment. Most of these viruses were isolated in chick embryo liver cell cultures from the faeces of birds aged under 1 week. In six of the eight instances, rotavirus isolation was associated with enteric disturbance, characterised by signs such as diarrhoea, poor or abnormal appetite, abnormally fluid or gaseous intestinal contents or increased mortality. Cross immunofluorescence tests showed that while avian and mammalian rotaviruses shared a common group antigen, the avian viruses were more closely related to each other than to the Nebraska calf rotavirus isolate. On the basis of serum neutralisation tests seven of the eight avian rotaviruses were grouped into three serotypes, with two turkey isolates (Ty1 and Ty3) and a chicken (Ch1) virus being the prototype strains. The remaining virus, Ty2, was intermediate in type between Ty1 and Ch1. Analysis of the RNA of these viruses by polyacrylamide gel electrophoresis showed that they could also be grouped into a number of electropherotypes. The isolates which were serologically distinct were also electrophoretically distinct. Similarly the five isolates which belonged to the Ty3 sero-type were electrophoretically identical. Analysis of the serological and electrophoretic differences suggested that RNA segment 5 may code for a type-specific antigen.

Second derivative-high-performance liquid chromatographic-fluorometric detection of porphyrins in chick embryo liver cell culture medium

A high-performance liquid chromatographic system is described which is suitable for the separation and quantitative determination of a mixture of porphyrin esters in nanogram quantities from the culture medium used for maintenance of a monolayer culture of chick embryo liver cells. The desired sensitivity was obtained by coupling a high-performance liquid chromatograph with a second derivative-fluorometric detection system. The most effective method for preparation of the porphyrin methyl esters prior to chromatography was found to be lyophilization of the culture medium prior to esterification with 5% sulphuric acid in methanol.

Metabolism as a prerequisite for the porphyrinogenic action of polyhalogenated aromatics, with special reference to hexachlorobenzene and polybrominated biphenyls (Firemaster BP-6)

1. 1. Pretreatment of chick-embryo liver cell cultures with inducers of the drug-metabolizing enzyme system markedly stimulates the accumulation of porphyrins after exposure to polyhalogenated hydrocarbons. 2. 2. Inhibition of hepatic drug metabolism or addition of compounds which are known to trap electrophiles or radicals protects against the porphyrinogenic action of polyhalogenated hydrocarbons in vitro. A reactive intermediate formed by metabolism of the polyhalogenated hydrocarbon is suggested to react with the catalytic SH-containing part of the uroporphyrinogen decarboxylating enzyme in the liver cytosol. 3. 3. The consequent inhibition of uroporphyrinogen decarboxylase, which leads to porphyrin accumulation, may be a very first indication of cellular damage caused by toxic metabolites of these compounds. 4. 4. The results of this work with the chick embryo liver cell system are in good agreement with the observations in animal experiments.

Porphyrin-inducing Activity of Alfaxolone and Alfadolone Acetate in Chick Embryo Liver Cells

The two steroid components of Alfathesin, alfaxolone and alfadolone acetate, have been tested for porphyrin-inducing activity in chick embryo liver cell culture and for hepatic ALA-synthetase-inducing activity in the 17-day-old chick embryo. In cell culture alfoxolone was shown to have potency comparable to that of thiopental, while alfadolone acetate had low potency. In the 17-day-old chick embryo alfaxolone has a third the potency of thiopental; alfadolone acetate showed low potency. The authors conclude that an induction dose of Alfathesin would be less likely than a comparable dose of thiopental to increase ALA-synthetase activity in a patient with hereditary hepatic porphyria.

Induction of porphyrin synthesis in chick embryo liver cell culture by synthetic polychlorobiphenyl isomers

The effects of a series of synthetic di-tetra- and hexachlorobiphenyl isomers and commercial polychlorinated biphenyls on the porphyrin biosynthesis in chick embryo liver cells in culture were examined. It was found that 3,4,3′,4′-tetra- and 3,4,5,3′,4′,5′-hexachlorobiphenyl isomers were the most active inducers, which were approximately 20 times as active as 1,4-dihydro-3,5-dicarbethoxy-2,4,6-trimethylpyridine (DDC) in porphyrin production. 3,5,3′,5′-Tetra- and 2,3,4,2′,3′,4′-hexachlorobiphenyl isomers were moderate inducers, which were approximately 2.0 to 2.5 times as active as DDC. 2,4,6,2′,4′,6′-Hexachlorobiphenyl showed the same activity as DCC. Compounds such as 4,4′-di-, 2,3,2′,3′-, 2,4,2′,4′- and 2,6,2′,6′-tetrachlorobiphenyl were weak inducers and 2,5,2′,5′-tetrachloro- and decachlorobiphenyl isomers were found to be inactive. Kanechlor-400 was the strongest inducer among the commercial polychlorinated biphenyls investigated. The structural requirements for potent porphyrin-inducing activity of chlorobiphenyl isomers were found to be the para and meta substituted structure causing a more highly conjugated and nearly coplanar conformation. It was found that induction caused by some chlorobiphenyls was subject to feed-back repression by end-product heme. In addition, the metabolism of chlorobiphenyls in mice was influenced by the unsubstituted pairs of carbon atoms in the molecule. These results lead us to postulate the following hypothesis, namely, that strong inducers may displace heme directly and incorporate into a hydrophobic pocket of the apo-represor protein, thus causing an induction of δ-aminolevulinic acid synthetase.

Inhibitory effect of membrane active compounds on the uptake of 14C-∝-aminoisobutyric acid (AIB) in cultured chick embryo liver cells

High concentrations of beta-adrenoceptor blocking drugs with membrane active properties and of the membrane active compounds quinidine and lidocaine inhibit the uptake of ∝-aminoisobutyric acid by chick embryo liver cells in culture. Beta-adrenoceptor blockers without membrane active properties were without effect. These results are in accordance with previous findings which showed partial inhibition of incorporation of amino acids into proteins caused by membrane active drugs in this system.

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

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.

Inhibitory effect of some membrane active drugs on RNA and DNA synthesis in cultured chick embryo liver cells

DL-Propranolol and oxprenolol, both beta-adrenergic receptor blocking agents with membrane active properties, were found to inhibit the incorporation of uridine and thymidine into RNA and DNA respectively in cultures of chick embryo liver cells. The membrane active compounds quinidine and lidocaine had similar effects. D-Propranolol, which has a very slight beta-blocking action only but has a membrane activity similar to DL-propranolol, showed inhibition like that of DL-propranolol. Pindolol and practolol, beta-adrenergic blocking agents nearly devoid of membrane activity, had no effect on incorporation. The inhibition is reversible and non-competitive. It seems to be related to the membrane active properties of the drugs examined.

Induction of aminolevulinate synthase and porphyrins in cultured liver cells maintained in chemically defined medium. Permissive effects of hormones on induction process.

Primary liver cells, isolated from 16- 17-day-old chick embryos, were incubated in a serum-free chemically defined medium (Ham's F12) supplemented with hormones for up to 6 days. The culture method also includes the complete removal of contaminating red cells before the initiation of culture. On the 2nd day in cluture, the level of amino-levulinate (ALA) synthase activity in response to allylisopropylacetamide (AIA) was increased 6-fold in cells grown in F12. Insulin, hydrocortisone, and triiodothyronine alone had no appreciable effects on ALA synthase levels. On the other hand, when added with AIA, insulin, insulin plus hydrocortisone, insulin plus hydrocortisone triiodothyronine increased ALA synthase levels 17-, 50-, 110-fold, respectively. The maximally induced levels of ALA synthase activity by AIA in the presence of insulin, hydrocortisone, and triiodothyronine were approximately 15 nmol of ALA/mg of protein/h, 37 degrees or 3 micronmol of ALA/g of tissue/h, 37 degrees, a value similar to that found in ovo or at least 5 times greater than that found in rat liver. The morphology of hepatocytes was maintained for at least 6 days in culture, although the induction of ALA synthase was reduced after the 4th day unless triiodothyronine was present. Dibutyryl adenosine 3':5'-monophosphate (10(8) M) or glucagon (5x10(8) M) had little effect on the induced as well as noninduced levels of ALA synthase or porphyrins. These data demonstrate a "permissive" effect of insulin, hydrocortisone, and triiodothyronine on the induction of ALA synthase and porphyrins by AIA in cultured chick embryo liver cells. In the absence of insulin hydrocortisone, or triiodothyronine, AIA produces only a slight increase in ALA synthase activity or porphyrins (or both); on the other hand, it produces a marked increase in the enzyme activity and porphyrins when these hormones are added to the culture medium. The term "permissive" is applied to these hormone-dependent effects. A sensitive spectrofluorometric method for heme quantitation allowed us to follow changes in the cellular heme content in hemoglobin-free cultured liver cells. Heme content in the cultured liver cells was approximately 250 pmol/mg of protein at the initiation of culture but gradually declined to 175 pmol/mg of protein at the initiation of culture but gradually declined to 175 pmol/mg of protein during 48 h of incubation. The apparent decrease in heme content may be accounted for by the concomitant increase in protein content in these cells.