Chick Embryo Liver Cells Research Articles

Heat Inactivation of a Duck Adenovirus Serologically Indistinguishable from Adenovirus 127

Egg-drop syndrome 1976 (EDS-76), first reported in the Netherlands in late 1976 (10) and seen since in Ireland (5,6) and Japan (12,13), is a disease characterized by depressed egg production in chickens, loss of shell pigmentation, and soft-shelled or shell-less eggs (5,6,10,12,13). Several hemagglutinating adenovirus isolates have been associated with EDS-76, including isolate 127 (6), BC-14 (3), and JPA-1 (13). In the United States, hemagglutinating adenoviruses have been isolated from clinically normal ducks (2,11). These isolates have been shown to be serologically indistinguishable from the agents known to cause EDS (2,11). It has been shown in our laboratory that the Cornell isolate of duck adenovirus (DAV) does not replicate and produce hemagglutinating (HA) activity in chicken embryo liver cell cultures (4), which is in contrast to the European isolates (6,9,13) and one other domestic isolate (11). It was of interest, therefore, to investigate other possible differences of the Cornell DAV.

Metal ion-mediated regulation of heme oxygenase induction in cultured avian liver cells.

The induction of heme oxygenase (EC 1.14.99.3) in response to various metal treatments was investigated in monolayer cultures of chick embryo liver cells maintained in a chemically defined serum-free medium. The most potent heme oxygenase-inducing action was exhibited by CO2+, Cd2+, Sb3+, As3+, and Au1+ followed by lesser induction observed with Cu2+, Fe2+, and Fe3+. Mn2+, Ni2+, Se4+, Sn2+, and Zn2+ were without effect. In contrast to the marked inducing effect of Co2+ on heme oxygenase, Co-protoporphyrin IX decreased the enzyme activity in a dose-dependent manner. Addition of Zn2+ (20 microM) to Co2+-treated liver cell cultures revealed a striking ability of Zn2+ to block completely Co2+-induced heme oxygenase. Simultaneous addition of Mn2+ (50 microM) to Co2+-treated cells also blocked Co2+-induced heme oxygenase (approximately 50%). These findings in tissue culture confirm those made earlier in whole animals (Drummond, G. S., and Kappas, A. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 5331-5335) and indicate that these effects of Zn2+ and Mn2+ are exerted directly in liver cells. Addition of cysteine (400 microM) to the cultures also inhibited heme oxygenase induction by Co2+ substantially. Cycloheximide and actinomycin D blocked the induction of heme oxygenase, indicating that increased heme oxygenase activity by metal treatment is dependent on fresh RNA and protein synthesis. The half-life of the enzyme was calculated to be approximately 15 h after treatment with cycloheximide. These findings provide further evidence that metal ions can regulate heme oxygenase synthesis directly in isolated liver cells and that the metal-metal interactions which lead to blockade of the enzyme induction do not involve extrahepatic tissues.

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.

The Effect of Incubation Temperature on the Propagation of Duck Adenovirus (Virus 127-Like) in Duck and Chicken Cells

Duck adenovirus (Cornell strain) was propagated in duck and chicken embryo cells at 37.5 C and at 40 C. In duck cells, virus levels, as indicated by HA titers, peaked earlier at 40 C than at 37.5 C. High titers were eventually observed in duck cells at both temperatures. In chicken embryo fibroblasts, no titers were observed at 37.5 C, whereas low titers were observed at 40 C. Evidence of virus propagation was not detected in chicken embryo liver and kidney cells.

Separation of impurities in diazinon preparations and their effect on porphyrin biosynthesis in tissue culture

The impurities present in commercial diazinon preparations have been examined by high performance liquid chromatography with particular reference to the ability of these compounds to cause porphyrin accumulation in cultures of chicken embryo liver cells. Diazinon and its impurities are readily separated on 10 micron Partisil using cyclohexane-dioxan mixtures. The main impurities are tetraethylpyrophosphate, sulphotetraethylpyrophosphate, 2-isopropyl-6-methylpyrimid-4-one, 2-isopropyl-6-methylpyrimidin-4-thione, and 2-isopropyl-4-ethylthio-6-methylpyrimidine. A previously unreported impurity, 2-isopropyl-6-methyl-4- S-pyrimidinyl diethylthiophosphate (isodiazinon), was also detected. Both diazinon and isodiazinon cause accumulation of coproporphyrin in cultures of chicken embryo liver cells. Isodiazinon has a greater effect on porphyrin biosynthesis in the cultures than has diazinon. It is suggested that the point of interference with porphyrin biosynthesis is towards the end of the pathway.

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.

The influence of propranolol on the concentration of heme and on the activity of δ-aminolevulinate synthase in monolayers of chick embryo liver cells

The addition of propranolol to monolayers of chick embryo liver cells caused a rapid increase in cellular heme, followed by an equally rapid decrease. Subsequently the concentration of heme rose at a relatively slower rate. About 10 hr after addition of propranolol to the medium a plateau level was reached at ± 35% above control values. Changes in the activity of δ-aminolevulinate synthase (ALAS) were negatively correlated with those of cellular heme. Cycloheximide prevented the above phenomenon. ALAS activity was not clearly correlated with the rapid, partial inhibition of protein synthesis, caused by propranolol. These observations are related to the beneficial influence of administration of hemin or of propranolol to patients with acute attacks of hepatic porphyria.

Some biological and physico-chemical properties of egg drop syndrome (EDS) avian adenovirus strain B8/78.

The EDS adenovirus produced either in the allantoic cavity of embryonated duck eggs (A0 virus) or in chicken embryo liver cell cultures (TC0 virus) were comparatively studied by gradient ultracentrifugation in CsCl. The A0 viral particles banded at densities of 1.36 and 1.31 g/ml, whereas, the TC0 viral particle were found at densities of 1.33 and 1.31 g/ml, respectively. The heavy populations contained infectious and hemagglutinating particles if they were not pelleted and sonicated before gradient ultracentrifugation. Pelleting or sonicating the viral particles resulted in loss of hemagglutinating activity of the heavy populations. The light particles were hemagglutinating but not infectious regardless of the previous treatment. The pH- and heat-sensitivity of the B8/78 virus strain was similar to those described for CEL0 (FAV-1) adenovirus (5). The B8/78 virus strain replicated well in tissue cultures of chicken and goose origin causing the formation of intranuclear inclusion bodies in the infected cells.

Growth hormone stimulates drug-induced porphyrin formation in liver cells maintained in a serum-free medium.

Human growth hormone and a few other related polypeptide hormones enhanced porphyrin formation induced by 2-allyl-2-isopropylacetamide in chick embryo liver cells maintained in a serum-free medium. The stimulatory effect of human growth hormone on drug-induced porphyrin formation was significant at 10 ng/ml and increased in a dose-dependent manner. Human growth hormone did not stimulate porphyrin formation in otherwise untreated cultures. These findings thus demonstrate a potentiating effect of growth hormones on hepatic porphyrin formation.

Isolation of viruses from outbreaks of suspected tenosynovitis (viral arthritis) in chickens

Specimens from the legs of chickens from 84 outbreaks of suspected tenosynovitis were examined for the presence of viruses by culture in chick embryo lung or liver cell monolayers. All samples were from broilers or broiler breeders, ranging in age from dead-in-shell embryos to 36 weeks old. Twenty-five outbreaks (29.8 per cent) yielded viruses of which 12 were reoviruses alone, 12 adenoviruses alone, and one, a mixture of both types of virus. Rupture of the gastrocnemius tendon was seen in 12 outbreaks and viruses were isolated from six of these: three were reoviruses and three were adenoviruses. Approximately half the affected flocks from which specimens were received were in the six to 14 week age range. With one exception, all the reovirus isolations were made from chickens of 11 weeks or under, while adenovirus isolations showed more scatter with regard to age.

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

Biotransformation and porphyrinogenic action of hexachlorobenzene and its metabolites in a primary liver cell culture

Hexachlorobenzene (HCB) is metabolized in a primary culture of chick embryo liver cells and causes porphyrin accumulation within 24 h after administration. The HCB-metabolites, pentachlorothiophenol (PCThP), pentachlorobenzene (PeCB) and pentachlorophenol (PCP) identified in liver cell culture are already known from long-term experiments with rats. The pattern of accumulated porphyrins is comparable with the pathological porphyrin pattern observed in oral feeding studies with warm blooded laboratory animals. Protein bound radioactivity was found in cell cultures treated with [ 14C]HCB. Addition of the monooxygenase-inhibitor piperonyl butoxide or ascorbic acid decreased the irreversible binding of 14C-metabolites. The results show that biotransformation of HCB fulfils an essential role in the onset of porphyria. Since none of the main HCB-metabolites could induce a pathological porphyrin pattern, a reactive intermediate capable of reacting with glutathione or thiol-groups of uroporphyrinogen decarboxylase (UROG-D) is believed to be responsible for the inhibition of UROG-D. The chick embryo liver cell system may be considered as a useful and sensitive system for studying the metabolism of xenobiotics in relation to their toxicity.

The potent heme oxygenase inducing action of arsenic and parasiticidal arsenicals.

The administration of trivalent arsenic, either as sodium arsenite or as the trypanocidal drug melarsoprol, to rats produced a profound induction of microsomal heme oxygenase (EC 1.14.99.3) in both liver and kidney and a concomitant decrease in cytochrome P-450 content. In addition, perturbations of delta-aminolevulinate synthase were observed which showed an initial decline followed by a rebound increase in the activity of this enzyme with arsenical treatment. Pentavalent arsenic did not induce hepatic heme oxygenase but did induce the enzyme in kidney, although to a lesser extent (50%) than trivalent arsenic. Treatment of isolated chick embryo liver cells in vitro with sodium arsenite or the parasiticidal drug melarsoprol also showed a potent induction of heme oxygenase. These findings describe a new and potent ability of arsenic and parasiticidal arsenicals to induce heme oxygenase resulting in enhanced degradation of cellular heme.

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.

Evidence for the synthesis of a somatomedin similar to insulin-like growth factor I by chick embryo liver cells.

Chick embryo liver cells were incubated in serum-free and hormone-free medium and shown to produce a mitogen starting after the first day in culture. This mitogen was purified by gel filtration and ion-exchange chromatography and identified by several assay systems as a somatomedin similar to human insulin-like growth factor I (IGF-I). This somatomedin stimulates thymidine incorporation into DNA of chick fibroblasts, it reacts in an IGF-specific protein binding assay and it cross-reacts immunologically with antibodies prepared against human IGF-I, but not with those against IGF-II. Its behaviour on ion-exchange chromatography and on sodium dodecylsulfate/polyacrylamide gel electrophoresis is indistinguishable from that of authentic human IGF-I. Concomitantly with the mitogenic activity, chick liver cells produce IGF-binding protein. Addition of purified human IGF-I to the liver cells inhibits production of the binding protein. Chick liver cells may thus be used to study synthesis and regulation of IGF and its binding protein.

Development of a microtitre fluorescent antibody test for serological detection of adenovirus infection in birds

The development of a microtitre fluorescent antibody test, for detection of adenovirus antibody is described. Chick embryo liver cells, infected with fowl adenovirus were found to be more suitable for the test than chick kidney, since a proportion of the chick kidney cells were found to contain non-viral cytoplasmic inclusions which were easily confused with specific viral fluorescence at the low power magnifications used in the test. It was demonstrated that a single positive serum would stain cultures infected with 11 different serotypes of fowl adenovirus, and two serotypes of turkey adenovirus with only minor differences in titre, thus confirming the presence of a common antigen in fowl and turkey adenoviruses. In a survey for adenovirus antibody a total of 595 avian sera (453 fowl and 142 turkey) were screened by double immuno-diffusion and microtitre fluorescence. A total of 52.6% of the sera was positive by double immuno-diffusion while 70.8% were positive by microtitre fluorescence.

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.

Haem control in experimental porphyria. The effect of haemin on the induction of delta-aminolaevulinate synthase in isolated chick-embryo liver cells.

2-Allyl-2-isopropylacetamide-mediated induction of hepatic porphyria was studied in isolated chick-embryo liver cells. Increased delta-aminolaevulinate synthase activity occurred within 1h of induction and continued to increase for 8h. Protoporphyrins synthesized during this time accumulated to a concentration 10-fold greater than that in the control. Removal of 2-allyl-2-isopropylacetamide from the cells by washing at 3h immediately inhibited further increases in delta-aminolaevulinate synthase synthesis. However substitution of 2-allyl-2-isopropylacetamide at 3h by deferoxamine methane-sulphonate, an inhibitor of haem synthesis, allowed continued delta-aminolaevulinate synthase induction at an unaltered rate, even though this agent did not, by itself, induce enzyme synthesis. Exogenously added haemin was shown completely to inhibit 2-allyl-2-isopropylacetamide-mediated delta-aminolaevulinate synthase induction at concentrations as low as 20nm, a value that is less than the reported physiological one. The duration of inhibition was dependent on the concentration of added haemin and was followed by a period of delta-aminolaevulinate synthase synthesis at a rate similar to that of the control. These data are consistent with the hypothesis that delta-aminolaevulinate synthase synthesis is regulated by the concentration of intracellular haem and that induction is initiated by 2-allyl-2-isopropylacetamide-mediated destruction of haem. Induction of delta-aminolaevulinate synthase was shown to be dependent on both RNA and protein synthesis, and a study of the comparative effects of cordycepin, cycloheximide and haem has shown that, at haemin concentrations up to 50nm, the inhibition of delta-aminolaevulinate synthase synthesis followed kinetics similar to the effect of cordycepin, with no synergism between cordycepin and 50nm-haemin. However, at a haemin concentration of 2mum, the inhibition of delta-aminolaevulinate synthase synthesis followed similar kinetics to the effect of cycloheximide. These data demonstrate the control of delta-aminolaevulinate synthase synthesis by low concentrations of haemin and suggests that the primary effect of haemin is at the level of transcription.

Interaction of epithelial cells and fibroblasts in culture: Effect on glycosaminoglycan levels

Twelve- to fifteen-day chick embryo liver cells (epithelial) were cultured on top of confluent chick embryo fibroblasts to produce an in vitro model of an epithelial-mesenchymal interacting system. This cocultivation resulted in a marked increase in hyaluronic acid (HA) levels and a decrease in chondroitin sulfate (CS) levels, either in total or in proportion to HA, compared with the two cell types cultured separately. The liver cells cultured alone produced little or no detectable glycosaminoglycans (GAGs). Cocultivation of increasing numbers of liver cells with fibroblasts resulted in a progressive increase and decrease of HA and CS levels, respectively, and the combined effect of these changes was a progressive increase in the HA/CS ratio. Fibroblasts cultured in liver-cell-conditioned growth medium also showed increased levels of HA, but in contrast to cocultivation, an increase in CS and no change in the HA/CS ratio. Liver cells cultured in fibroblast-conditioned growth medium showed no changes in GAG level. This suggests that under conditions of cocultivation the fibroblasts alone could be responsible for the increased HA levels and that the decreased CS levels are a result of conditions produced by the close proximity of the two cell types. In vivo most connective tissues immediately adjacent to epithelial tissues are also characterized by a matrix rich in HA and these results support the concept that some epithelial tissues are able to modulate the GAG composition of adjacent connective tissues and thereby affect their immediate extracellular environment.