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PubMed1981

Characterization of the alpha-bungarotoxin receptor in chick-embryo retina.

Primary cultures and membranes fractions from chick embryo retina bind iodinated alpha-bungarotoxin, a highly selective ligand for nicotinic acetylcholine receptors from skeletal muscle and fish electric organ. The binding is saturable with an equilibrium dissociation constant (Kd) of 0.75 +/- 0.09 nM. The pseudo-first-order rate constant (k+1) for binding at 37 degrees C is 1.76 +/- 10(5) M-1 s-1, the dissociation rate constant (k-1) at 37 degrees C is 1.15 x 10(-4) s-1. Nicotinic cholinergic ligands and local anaesthetics inhibit alpha-bungarotoxin binding. In the case of carbamoylcholine, the inhibition of binding depends on the time of exposure to this cholinergic agonist. alpha-Bungarotoxin has no effect on the carbamoylcholine-induced stimulation in sodium permeability of cultured retinal neurons. On sucrose density gradients containing Triton X-100, the toxin binding site sediments with an apparent sedimentation coefficient of 10.5-11 S. Detergent-solubilized alpha-bungarotoxin receptor cross reacts with antisera raised against nicotinic acetylcholine receptors from Torpedo marmorata. These results are interpreted as indicating that alpha-bungarotoxin binds to retinal nicotinic acetylcholine receptors without affecting cholinergic receptor function.

Animals↗

PubMed1969

Local stimulation of growth in primary cultures of chick embryo fibroblasts.

Confluent, slowly growing primary cultures of chick embryo fibroblasts were stimulated to grow more rapidly by making discontinuities in the monolayers by a "wounding" procedure. Growth was measured by mitotic index and by the fraction of cells synthesizing DNA as determined by autoradiography. Faster growth was confined to free cells at the edges of the discontinuities. Cells enclosed on all sides were not stimulated to renewed DNA synthesis, even when within 0.2 mm of the discontinuities. The time of appearance of increased rate of growth induced by wounding was measured and found to be similar to the time of a transient growth stimulation among enclosed cells induced by a change of medium. The increased rate of growth among free cells was not transient, however, and persisted as long as it was examined. To rule out possible artifacts incurred in wounding, a free edge was created by seeding trypsinized cells as a confluent sheet in a confined area of the tissue culture plate. Here also the growth rate of cells along the edge was much higher than in the confluent areas. It was concluded that interactions at short range between cells in culture can play an important role in regulating their growth.

Animals↗

PubMed1988

CO2 production of the chick embryo during the first day of post-laying development.

The carbon dioxide production of the chick embryo cultured in vitro has been determined during the first 24 h of post-laying development using a non-invasive conductometric microtechnique. The mean CO2 production of the whole blastoderm (1) increased from 16 nmol/h at laying to 231 nmol/h at early neurulation, (2) became dependent on exogenous glucose and (3) was closely linked to mechanical tension generated in the blastoderm (loosening from vitelline membrane resulted in a decrease of 56%). In our experimental conditions, no significant influence of carbonic anhydrase on the CO2 production has been detected. The value of the respiratory exchange ratio varied from about 3 at pregastrular stages to 1 at neurula stage and CO2 was produced transiently in presence of antimycin A. Such results indicate that the source of CO2 is not exclusively mitochondrial and that the relative proportions of mitochondrial and non-mitochondrial CO2 productions might vary significantly throughout the early development. Our findings confirm that the metabolism of the chick embryo becomes more and more oxidative from laying onwards and suggest that the modifications of metabolism observed during the studied period of development could be associated with functional differentiation.

2,4-Dinitrophenol↗

PubMed1975

Nonspecific nature of the stimulus to DNA synthesis in cultures of chick embryo cells.

The rate of DNA synthesis in chick embryo cultures deprived of serum is stimulated 5- to 20-fold by a large variety of substances, including subtoxic concentrations of certain metal ions such as Zn++, Cd++, and Hg++. The stimulatory concentrations of Zn++ and Cd++ have sharp optima, which are just below the concentrations that produce frank morphological damage in each case. A much wider gap exists between stimulatory and morphologically damaging concentrations of Hg++. These metal ions also stimulate RNA synthesis, and the uptake of 2-deoxy-D-glucose. The stimulatory effects of Zn++, but not those of Hg++, are prevented by treatment with EDTA. Although medium from cultures stimulated by Zn++ or Hg++ retains its stimulatory capacity for a new set of cultures, the capacity in the case of Zn++-treated cultures is almost entirely lost upon addition of EDTA. It is also lost upon dialysis of conditioned medium from cultures treated with either Zn++ or Hg++. It is concluded that the stimulatory effect is the direct result of interaction between metal ions and cells, and not to the release of growth-stimulatory materials from the cells. The stimulation is thus seen as a non-specific event resulting in an integrated, metabolic response by the cells.

Animals↗

PubMed1988

Flow cytometric evaluation of cell cycle characteristics during in vitro differentiation of chick embryo chondrocytes.

The cell cycle kinetic characteristics of chick endochondral chondrocytes differentiating in vitro were studied by flow cytometry. In addition, the synthesis of type I and type X collagens of the same cells was evaluated by immunoprecipitation. Dedifferentiated cells, derived from chick embryo tibiae and grown attached to a substratum, were characterized by type I collagen synthesis, a high growth fraction (GF = 0.94), minimal cell loss factor (phi = 0.02), and a total cell cycle time of the proliferating cells of about 17 h (tG1 = 8 h, tS = 5 h, and tG2 + M = 4 h). Transfer of dedifferentiated cells to suspension culture on agarose-coated dishes induced differentiation to hypertrophic chondrocytes. These were characterized by type X collagen synthesis, a low growth fraction (GF = 0.52), maximal cell loss factor (phi = 1.0), and a total cell cycle time of the proliferating cells of about 73 h (tG1 = 53 h, tS = 12 h, and tG2 + M = 8 h). The transition from dedifferentiated chondrocytes to hypertrophic chondrocytes was accompanied by large increases of the duration of all the cell cycle phases and of the number of quiescent and degenerating cells. Associated with these alterations in cell cycle kinetics was a switch from type I to type X collagen synthesis. Further preliminary data suggest that the population of differentiating chondrocytes (a state between dedifferentiated and hypertrophic chondrocytes) comprises a heterogeneous population of fast and slow growing cells.

Animals↗

PubMed1976

Synthesis of elastin. A rapid formation of lysine-derived crosslinks by chick embryo aorta.

Aortas of 13-day-old chick embryo were labeled for 0.5 hr with [14C]lysine and subjected to a serial extraction after chase for 1-24 hr with [12C]lysine. Substantial radioactivity was found in insoluble elastin after 3 hr chase. The effect of beta-amino-propionitrile on labeling with [14C]lysine was also examined. Each fraction was hydrolyzed and applied to a short column on an amino acid analyzer. Radioactivity was found in desmosine and isodesmosine of insoluble elastin as early as 1 hr after the beginning of chase. The radioactivity increased rapidly at 2 hr and very slowly thereafter. A large count, which was separated into five peaks on a long column, was observed in other lysine derivatives at 2 hr and increased steadily up to 24 hr, while the lysine count decreased from 1 : 0.5 to 1 : 6 against lysine derivatives and from 1 : 0.04 to 1 : 0.9 against quarter-desmosine after 24 hr. The oxidation of lysine residues incorporated during the 0.5 hr pulse was almost completed during the first 1 hr of chase, and these oxidized residues were incorporated into crosslinks during the following 1 hr. It is suggested that poorly crosslinked elastin accumulated in the soluble fractions. The presence of crosslinking derived from lysine residues was also indicated in the microfibril fraction.

Amino Acids↗

PubMed1980

Interferon induction by viruses. VI. Reovirus: virion genome dsRNA as the interferon inducer in aged chick embryo cells.

The interferon-inducing particle (IFP) activity of avian and human reoviruses in aged chick embryo cells was determined by analyzing dose (multiplicity)-response (interferon yield) curves. These curves fit best a model in which each cell infected with greater than or equal to 1 IFP produces a quantum yield of interferon. Avian reovirus stocks contained as many as 60 times more IFP than plaque-forming particles (PFP). Upon UV-irradiation the ratio of IFP:PFP became 197, suggesting that virtually every physical particle of avian reovirus could function as an interferon-inducing particle. Thus, about one-third of the non-infectious particles were intrinsically IFP and the other two-thirds could be converted to IFP status at an optimal dose of UV radiation, the equivalent of 9.4 lethal hits, i.e., 8000 ergs/mm2. UV-irradiated avian reovirus induced about twice the usual yield of interferon on a per cell basis. Wildtype human reovirus (type 3) and mutants ts201(A,RNA+) and ts447(C,RNA-) were excellent inducers of interferon, but only about 1 in 3 infectious particles functioned as an interferon-inducing particle, meaning that virtually all physical particles failed to function as IFP, UV-irradiation of human reoviruses resulted in a slight loss of IFP activity. Our data support the hypothesis that virion genome dsRNA constitutes the interferon inducer moiety of avian reoviruses and that in its permissive host cell the processing of genome dsRNA from most particles to a putative recognition site in the cytoplasm occurs naturally with a high probability. For human reovirus this is a much rarer event which may be intrinsic only to infectious virus, and may require limited transcription for expression. The sensitivity of the avian reovirus-aged chick embryo cell system recommends it for further study on the mechanism of interferon induction by virions containing pre-existing dsRNA.

Animals↗

PubMed1980

Chick embryo fibroblasts produce two forms of hyaluronidase.

Cultured chick embryo fibroblasts derived from skin and skeletal muscle exhibit hyaluronidase activity both associated with the cell layer and secreted into the medium. Although both forms of the enzyme have a number of similar characteristics (R.W. Orkin and B.P. Toole, 1980, J. Biol. CHem. 255), they differ in thermal stability at neutral pH and in behavior on ion-exchange chromatography. Both forms of the enzyme are equally stable at acidic pH for long intervals, but the cell-associated hyaluronidase is significantly less stable than the secreted froms at neutral pH and at temperatures more than or equal to 30 degrees C. Neither the presence of proteases nor inhibitors of hyaluronidase appear to be involved in the cell-asspcoated enzyme. Chromatography of the two forms of hyaluronidase on carboxymethyl cellulose reveals that most (60-90 percent) of the secreted form of the enzyme elutes at a lower ionic strength than the cell- associated enzyme. Treatment of the secreted form of hyaluronidase with neuraminidase shifts its elution profile on carboxymethyl cellulose toward that of the cell-associated form, and also decreases its thermal stability at neutral pH. In contrast, treatment of the secreted form of hyaluronidase with alkaline phosphatase has no detectable effect. These data suggest that the secreted hyaluronidase differs from the cellular form in possessing additional sialic acid residues which endow the former with increased stability in the extracellular milieu.

Alkaline Phosphatase↗

PubMed2000

Incorporation of ethanolamine into insulin-sensitive glycosylated phosphatidylinositol of chick embryo fibroblasts.

Insulin sensitive glycosylated phosphatidylinositol (GPI) from chick embryo fibroblasts was isolated and partially characterized. [(3)H]Ethanolamine was incorporated into lipids different from phosphatidylethanolamine, as shown by two sequential thin layer chromatographies (TLC) using an acidic solvent system followed by a basic solvent system. Other isotopes, myo-[(3)H]inositol, [(3)H]glucosamine, [(3)H]galactose, and [(3)H]palmitic acid were also incorporated into these lipids. These lipids were separated into two peaks on the second basic TLC, designated as peaks I and II from the origin. Insulin stimulation of cells caused a rapid breakdown of these two lipids. These two lipids were treated by nitrous acid and phosphatidylinositol-specific phospholipase C (PI-PLC). The radioactivity of peak I lipid was decreased by both treatments, and that of peak II lipid was also decreased by PI-PLC treatment but not significantly by nitrous acid treatment. Peak II lipid did not fulfill the criteria for GPI. Tritium released by the treatment of PI-PLC of peak I lipid was recovered in the aqueous phase. [(3)H]Ethanolamine-labeled peak I lipid was hydrolyzed by acid treatment and the hydrolysis products were analyzed by TLC and high performance liquid chromatography (HPLC). Tritium label was recovered as native label at the rate of 95%. [(3)H]Ethanolamine of peak I lipid was reductively methylated completely with formaldehyde and cyanoborohydride, as shown by HPLC analysis. The results indicate that peak I lipid contains primary ethanolamine as a glycan component and is insulin-sensitive free GPI.

Animals↗

PubMed1975

Nerve growth factor-induced rapid activation of RNA labeling in dorsal root ganglionic dissociates from chick embryos.

Dorsal root ganglionic cells, from 8-day chick embryo, undergo anabolic declines when incubated in vitro without Nerve Growth Factor (NGF) or other supportive agents. To determine whether the decline could be opposed by delayed administration of the NGF, cells were incubated without the factor for varying times, then supplies with it and tested periodically with pulses of radiouridine or radioleucine. The decline in RNA labeling was actually reversed by a delayed addition of NGF, and the effect was fully elicited within less than 10 min from the treatment. With delays up to 6 hr, this rapid activation by NGF fully restored the incorporation rate exhibited by fresh cells or by cells continuously incubated with NGF. From the hour 8 on, the NGF-induced activation of RNA labeling fell progressively shorter of restoring maximal performance until, by 18 hr, it was no longer significant. The residual (irreversible) decline in RNA labeling, starting after 6 hr, developed with a time pattern coincidental with that of the irreversible decline in protein labeling also displayed by those untreated cells, and similar to the appearance of RNA degradation and the acceleration of protein degradation. All four such "degenerative" events were fully prevented by NGF when administered with delays shorter than 6 hr, and only interrupted (or delayed) by NGF delivered at later times. Additional experiments revealed that, over the first 6 hr, NGF also prevented or reversed a decline in TCA-soluble radioactivity of the cells, an effect which was not blocked by actinomycin D. This rapid increase of soluble radioactivity could be responsible for the rapid activation of RNA labeling and may also be involved in the prevention by NGF of the later developing degenerative events.

Animals↗

PubMed1986

Studies of the influence of chloro-substituent sites and conformational energy in polychlorinated biphenyls on uroporphyrin formation in chick-embryo liver cell cultures.

Treatment of cultured chick-embryo liver cells with polychlorinated biphenyls (PCB) results in decreased uroporphyrinogen decarboxylase activity and increased uroporphyrin accumulation. In the present study we examined the effect of the chloro- or bromo-substituent sites in biphenyls (BP) on uroporphyrin accumulation in cultured hepatocytes and the three-dimensional structure of these congeners determined by molecular orbital calculations using a MNDO ('modified neglect of diatomic overlap') method. Among 20 congeners examined, those which were effective in stimulating porphyrin accumulation contained at least two Cl or Br atoms at the lateral adjacent positions in each phenyl ring, e.g. 3,4,3',4'-tetrachloro-, 2,4,3',4'-tetrachloro-, 3,4,5,3',4',5'-hexachloro- and 3,4,5,3',4',5'-hexabromobiphenyl, whereas those which contained less than two halogen atoms or more than three halogen atoms in each phenyl ring or those which contained halogen atoms at 2,2'-positions were not effective. On the basis of the conformational energy (delta E, difference from the most stable conformational energy), which is calculated as a function of the dihedral angle (theta) between the two phenyl rings, biphenyl congeners can be classified into four groups with different conformations. The conformation of active PCB was relatively flexible, whereas inactive species had a rigidly angulated conformation. Furthermore, the calculated probability of the conformation distribution for each congener indicated that the probability of co-planarity was higher for active biphenyls than for inactive congeners. These structural characteristics suggest the significance of both the chloro-substituent sites and the conformational energy reflecting the phenyl-ring twist angles in determining the inhibitory effect of PCB on uroporphyrinogen decarboxylase activity.

Animals↗

PubMed1984

Properties of chick embryo chondrocytes grown in serum-free medium.

Chick embryo tibial chondrocyte growth and activities were compared in serum-free and serum-supplemented media. A basal salts medium containing equal volumes of Ham's F-12 and Dulbecco's modified Eagle's medium was supplemented with 10% fetal calf serum or with a mixture of bovine insulin, transferrin, fibroblast growth factor, dexamethasone, a prostaglandin E1 supplement, and a liposome supplement. Chondrocytes grew at identical rates in both media. Insulin, liposomes, and fibroblast growth factor were required for optimum growth in the serum-free medium, but removal of transferrin, dexamethasone, or prostaglandin E1 had little effect on the growth rate. In the serum-supplemented medium, the chondrocytes synthesized Type II collagen, Mr = 59,000 collagen, and both the large, cartilage-specific and the small ubiquitous proteochondroitin SO4 species typically produced by cultured chondrocytes. In the serum-free medium there was a shift toward synthesis of Type I collagen and a loss of the capacity to synthesize Mr = 59,000 collagen and the cartilage-specific proteochondroitin SO4. The loss of capacity for cartilage-specific proteochondroitin SO4 synthesis began immediately after replacement of the serum with the mixture of defined growth factors and the rate of loss was retarded but not reversed when serum was added back in place of the growth factors. When the serum and the mixture of growth factors were added together to the basal medium at the time of cell plating, the chondrocytes grew rapidly and retained their normal phenotype observed in serum-supplemented cultures. Thus, the serum appears to contain factors which are required for retention of the chondrocyte phenotype in culture over and above those factors necessary for cell growth.

Animals↗

PubMed1986

Immunocytochemical study of the development of vasotocin/mesotocin in the hypothalamo-hypophysial system of the chick embryo.

The hypothalamo-hypophysial system of the chick embryo has been studied with a monoclonal antibody which cross-reacts with arginine vasotocin and mesotocin, using thick (100 micron) sections in conjunction with a peroxidase-conjugated rabbit anti-mouse antibody. Although weakly stained perikarya occur occasionally in the tuberal region on embryonic days 6 and 7, the most consistent immunostaining of perikarya is found in the periventricular region of the caudal midhypothalamus at the level of the optic chiasm after embryonic day 8 1/2. Synthesis of peptides, therefore, takes place while the cells are close to their site of origin. Between embryonic days 9 and 10, beaded axons run along the anterior median eminence closely apposed to the adenohypophysis, thereby forming the anlage of the zona externa. The axons of the hypothalamo-neurohypophysial tract surround the neural lobe between embryonic days 11 and 12. The caudal to rostral wave of neuronal maturation that occurs during development appears to be due to a progressive differentiation of the periventricular zone, as well as the migration of perikarya. The early periventricular perikarya at embryonic day 8 1/2 send processes rostrally in a wing-shaped formation that extends both dorso- and ventrolaterally. From embryonic days 10 to 12, perikarya can be observed in the wing-like extensions, apparently migrating to rostral levels. The dorsolateral pathway gives rise at its midportion to the lateral cell group, whereas those perikarya migrating more laterally form the anlage of the external supraoptic nucleus. The ventrolateral wing-shaped extension of perikarya appears to be directed toward the ventral group and those lateral perikarya continuous with it. The location of mature neuronal cell groups is well established by embryonic day 17.

Animals↗

PubMed1980

Stereospecific D-glucose transport in mixed membrane and plasma membrane vesicles derived from cultured chick embryo fibroblasts.

Mixed membrane vesicles prepared from cultured chick embryo fibroblasts possess a stereospecific D-glucose transport system, the properties of which are identical to those of the system in intact cells. Uptake of D-glucose proceeds without chemical alteration. The rate of stereospecific uptake of D-glucose into the mixed vesicles is 70% greater than that of the homogenate and uptake is directly proportional to membrane protein concentration. Stereospecific D-glucose uptake appears linear for 0.3 min, reaches a maximum at 2--5 min, and declines to zero by 5 h as L-glucose enters the vesicles. Uptake is osmotically sensitive and inhibited by cytochalasin B (Ki = 0.13 microM) and the structural analogues of D-glucose : D-mannose, 2-deoxy-D-glucose, 3-O-methyl-D-glucose, D-galactose and maltose, but not by sucrose of L-glucose. Uphill counterflow can be demonstrated and the apparent activation energy displays a transition from 47.7 kcal/mol below 11 degrees C to 18.1 kcal/mol above 11 degrees C. Stereospecific uptake rates of mixed vesicles prepared from Rous sarcoma virus-transformed cells are increased 30% over control values, and are increased 66% in vesicles derived from cells incubated for 24 h in glucose-free medium. Plasma membrane vesicles prepared from these cells by a dextran cushion centrifugation procedure display a 9-fold increase in the specific activity of stereospecific D-glucose uptake relative to the homogenate. Extraction of these membranes with dimethylmaleic anhydride (5 mg/mg protein) results in substantial or complete removal of major polypeptides of molecular weight 40 000, 55 000, 75 000, 78 000 and 200 000 with no loss in total uptake activity. Following extraction, major polypeptides of molecular weight 28 000, 33 000 and 68 000 remain in the membrane residue.

Animals↗

PubMed1975

Reversible alterations in the mitotic cycle of chick embryo cells in various states of growth regulation.

Chick embryo cells which have been kept overnight at pH 6.8 in the absence of serum multiply very slowly. Only a small fraction of cells is in the S period at any given time, and the rate of uptake of 2-deoxy-D-glucose is very low. Upon raising the pH to 7.4 and adding serum ("turn-on") the uptake of 2-deoxy-D-glucose increases immediately; the rate of DNA synthesis increases after a lag of about 4 hours, and represents an increase in the fraction of cells synthesizing DNA. the uptake of 2-deoxy-D-glucose is rapidly returned to its original low rate at any time by again lowering the pH and removing serum ("turn-off"). The synthesis of DNA in the culture remains constant or continues to rise at a markedly reduced rate following the same treatment. Lowering pH or removing serum independently of each other is less efficient at inhibiting the increase in DNA synthesis than the combined treatment but each accomplishes a similar result. Cultures which have been "turned-off" during the early stages of the rapid increase in DNA synthesis, resume their prior rate of increase immediately if "turned-on" again within 2.5 hours. If the cultures have been "turned-off" for 5.5 hours before restoring the "turn-on", there is a 2 hour delay before they resume an increased rate of DNA synthesis. The results until shortly before, or at the time of the onset of the S period. Up to 96% of the cells in post-confluent cultures growing in conventional medium become labeled upon continuous, prolonged exposure to 3H-thymidine. Seventy-eight percent of the cells in serum-deprived cultures growing at a very low rate become labeled. These and other considerations suggest that the inhibition of cell multiplication by high population density or serum deprivation is caused by a lengthening of the time cells remain in the prereplicative G1 period rather than by shifting cells into a qualitatively distinct G0 period. There may, however, be a period common to all cells regardless of growth rate, in which cells are not progressing toward the S period. The length of this variable period would then determine the growth rate of a population of cells.

Animals↗

PubMed1977

A technique for introducing localized long-lasting implants in the chick embryo.

A technique is described for making chronic, localized implants of drug-impregnated Silastic in early chick embryos. This silicone material has been shown to gradually and uniformly release its impregnated contents under such conditions. Following implantation of pieces of Silastic treated with the metaphase-blocking drugs colchicine and vinblastine into the wing-buds of 41/2-5 day chick embryos, 53-75% survived 4-5 days, at which time they were sacrificed. Non-impregnated (control) Silastic was also implanted in a group of embryos. In this group, 90% survived 4-5 days. The antimitotic effects of the drugs were very well localized to implanted wings, which were reduced in size by approximately 55% at the time of sacrifice (day 8-9 of incubation). In control embryos, the implanted wings were normal in size and general morphology. It is suggested that the technique could have numerous applications for the study of neuroembryology or other aspects of embryogenesis.

Animals↗

PubMed1981

Inhibition of in vitro keratinization of chick embryo skin cultured with hepatectomized rat serum.

In the present study organ cultures of 6 day-old chick embryo matatarsal skin were used to determine the effect of hepatectomized rat serum in modulating epithelial differentiation. Six day-old chick embryo skin, maintained in organ culture for 6-7 days with a medium containing adult rat serum, was seem to undergo differentiation and keratinization faster then in vivo. Epidermis cultured for 6-7 days with a medium containing partially hepatectomized rat serum does not differentiate and the squamous layer is absent. Studies with 3H-cystine show a sharp decrease in the labelled amino-acids incorporation in the cultures added with serum obtained after partial hepatectomy.

Animals↗

PubMed1994

Two pathways for chromium(VI)-induced DNA damage in 14 day chick embryos: Cr-DNA binding in liver and 8-oxo-2'-deoxyguanosine in red blood cells.

Previous studies have shown that chromium(VI) induced cell-specific types of DNA damage, i.e. DNA cross-links in liver and DNA strand breaks in red blood cells, in 14 day chick embryos. Direct and indirect pathways for chromium(VI)-mediated DNA damage, in the form of Cr-DNA binding and 8-oxo-2'-deoxyguanosine (8-oxo-dG) respectively, were examined in liver and red blood cells of 14 day chick embryos. Levels of hepatic Cr-DNA binding increased in a Cr(VI) dose-dependent manner. Cr-DNA binding in red blood cells was 10-fold lower than in liver, although the Cr-uptake in red blood cells was only 2-fold lower than in liver. The level of 8-oxo-dG formation in red blood cells increased at all Cr(VI) doses tested but peaked at 0.10 mmol Cr(VI)/kg, whereas no increase in 8-oxo-dG levels over background levels was observed in liver of Cr(VI)-treated embryos. The possible role of glutathione in modulating Cr(VI)-induced DNA damage was examined by using L-buthionine-R,S-sulfoximine (BSO) to deplete glutathione. No changes in glutathione levels were observed in either liver or red blood cells of embryos treated with Cr(VI) in the presence or absence of BSO pretreatment. Ascorbate levels in liver and red blood cells were not affected by treatment of embryos with chromium(VI), BSO or Cr(VI) and BSO. Depletion of glutathione by BSO resulted in a small increase of chromium uptake in liver of embryos treated with 0.050 and 0.10 mmol Cr(VI)/kg, but had no effect on hepatic chromium uptake at 0.20 mmol Cr(VI)/kg. BSO had no effect on chromium uptake in red blood cells. Depletion of glutathione had no effect on hepatic or red blood cells Cr-DNA binding in embryos treated with Cr(VI). However, depletion of glutathione significantly decreased the 8-oxo-dG levels in red blood cells at all Cr(VI) doses tested. Levels of 8-oxo-dG in liver of Cr(VI)-treated embryos remained at background in the presence or absence of BSO pretreatment. These results indicate that Cr(VI)-induced DNA damage in 14 day chick embryos is through a direct interaction of chromium with DNA in liver, but is through an indirect oxidative pathway in red blood cells. It appears that glutathione plays an important role in chromium(VI)-induced formation of 8-oxo-dG in red blood cells.

8-Hydroxy-2'-Deoxyguanosine↗