Genotoxicity in the hepatocyte/DNA repair test and toxicity to liver mitochondria of 1-hydroxyanthraquinone and several dihydroxyanthraquinones

1-Hydroxyanthraquinone and dihydroxyanthraquinones (alizarin, quinizarin, anthrarufin and chrysazin) were examined for genotoxicity in the hepatocyte/DNA repair test and for effects on oxidative phosphorylation in isolated rat liver mitochondria. Of the anthraquinone compounds tested, 1-hydroxyanthraquinone and 1,8-dihydroxyanthraquinone (chrysazin) induced DNA repair synthesis in rat hepatocytes, indicating their genotoxic activity. Only 1,2-dihydroxyanthraquinone (alizarin) exerted an uncoupling and inhibitory effect on mitochondrial respiration. The possible relationships of the genotoxic potencies and the uncoupling activities of these anthraquinones to their chemical structures are discussed.Abbreviations ADP adenosine-5-diphosphate - ETP electron transport particles - RC respiratory control - TdR thymidine deoxyribonucleotide - UDS unscheduled DNA synthesis     

Effects of pretreatment with inducers of hepatic mixed function oxidases on DNA repair elicited by various compounds in hepatocytes from adult and neonatal rats

Studies were conducted to assess the effects of inducers of hepatic mixed function oxidases on DNA repair responses to 13 different genotoxic agents in hepatocytes from adult male mice. Phenobarbital pretreatment increased DNA repair elicited by diethylnitrosamine but had no effect on responses to the other compounds. Pretreatment with p,p-dichlorodiphenyltrichloroethane, 3-methylcholanthrene or -naphthoflavone induced the DNA repair responses to a variety of activation-dependent carcinogens. DNA repair responses to the direct-acting alkylating agents methyl methanesulfonate and N-methyl-N-nitro-N-nitrosoguanidine were not increased by any of the pretreatments, which indicated that the pretreatment-related enhancement of responses to the other compounds was due to induction of their metabolic activation. Taken together, the findings suggest that Aroclor, or other pretreatments, may increase the sensitivity of the hepatocyte DNA repair assay for detecting the genotoxicity of certain compounds; however, the potential benefit may be limited due to specific features of the assay. In contrast, Aroclor pretreatment did not produce any enhancement of in vivo DNA repair elicited by dimethylnitrosamine, diethylnitrosamine, o-aminoazotoluene, 2-acetylaminofluorene, 3-methylcholanthrene or aflatoxin B₁, and thus does not appear to be useful for improving the sensitivity of the in vivo/in vitro assay.Whereas the amount of DNA repair produced by dimethylnitrosamine was not increased by classical inducers of liver microsomal enzymes, pretreatment with pyrazole greatly augmented in vitro and in vivo DNA repair responses to dimethylnitrosamine; responses to diethylnitrosamine were increased to a lesser degree by pyrazole pretreatment. The effects of lactational exposure to enzyme inducing agents on DNA repair in neonatal hepatocytes was also investigated.Abbreviations 2-AAF 2-acetylaminofluorene - 4-AB 4-aminobiphenyl - 6-AC 6-aminochrysene - AFB aflatoxin B₁ - ARO Aroclor 1254 - o-AT o-aminoazotoluene - B(a)P benzo[a]pyrene - B-NF beta-naphthoflavone - BZ benzidine - DDT p,p-dichlorodiphenyltrichloroethane - DDE p,p-dichlorodiphenyldichloroethylene - DEN diethylnitrosamine - DMBA 7,12-dimethylbenzanthracene - DMN dimethylnitrosamine - 3-MC 3-methylcholanthrene - MMS methyl methanesulfonate - MNNG N-methyl-N-nitro-N-nitrosoguanidine - 2-NA 2-naphthylamine - NNG net nuclear grains - PB phenobarbital - PYR pyrazole     

Variations on the standard protocol design of the hepatocyte DNA repair assay

Several variations on the standard primary rat hepatocyte DNA/repair assay were evaluated for their ability to enhance the sensitivity of this genotoxicity test system. The use of hamster hepatocytes proved to be a much more sensitive system than rat hepatocytes for detecting the DNA repair inducing ability of the nitrosamines, dimethylnitrosamine and diethylnitrosamine, and the aromatic amines, 2-acetylaminofuorene, 9-aminoacridine, 1-naphthylamine and benzidine. In addition, hamster hepatocytes were a more sensitive indicator of the genotoxicity of the azo dyes, o-aminoazotoluene, Congo Red and Evans Blue. However, the azo reduction product of the azo dyes Congo Red, Trypan Blue and Evans Blue, benzidine and o-tolidine, respectively, were active in both rat and hamster hepatocytes at concentrations that were 10–100 fold lower than the parent dyes. This suggests that little or no azo reduction of the dyes occurred in the in vitro assay systems. The in vivo-in vitro variation of the rat hepatocytes DNA/repair assay exhibited a positive DNA repair response with the azo dye solvent Yellow S, which was negative in the standard in vitro assay. The in vivo-in vitro hepatocyte DNA repair assay was also more sensitive for detecting the genotoxic activity of Evans Blue, which was positive in the in vivo-in vitro assay and equivocal in the standard in vitro assay. Also, Solvent Yellow 14 was negative in the in vitro assay, but induced an equivocal DNA repair response in the in vivo-in vitro assay system. A treatment/³H-thymidine labeling period of approximately 18 hours, compared to 4 hours, was demonstrated to be superior for detecting the DNA repair elicited by the mutagens 4-nitroquinoline-1-oxide, mitomycin C, dimethylnitrosamine and methyl methanesulfonate in the in vitro rat hepatocyte assay. There was little or no difference observed between the 4 hour and 18 hour treatment/ labeling incubation periods for the detection of DNA repair induced by 2-acetylaminofluorene, aflatoxin B₁, and benzidine. The data suggest that these several variations on the standard rat hepatocyte DNA/ repair assay should be considered when evaluating the genotoxicity of chemicals for safety purposes.Abbreviations 2-AAF 2-acetylaminofluorene - o-AT o-aminoazotoluene - DMN dimethylnitrosamine - DMSO dimethylsulfoxide - FMN flavin mononucleotide - MMS methyl methanesulfonate - 4-NQO 4-nitroquinoline-1-oxide - PRI Pharmakon Research International - RTI Research Triangle Institute     

Effect of varying the exposure and 3H-thymidine labeling period upon the outcome of the primary hepatocyte DNA repair assay

The results presented in this report demonstrate that an 18–20 hour exposure/³H-thymidine DNA labeling period is superior to a 4 hour incubation interval for general genotoxicity screening studies in the rat primary hepatocyte DNA repair assay. When DNA damaging agents which give rise to bulky-type DNA base adducts such as 2-acetylaminofluorene, aflatoxin Bi and benzidine were evaluated, little or no difference was observed between the 4 hour or an 18–20 hour exposure/labeling period. Similar results were also noted for the DNA ethylating agent diethylnitrosamine. However, when DNA damaging chemicals which produce a broader spectrum of DNA lesions were studied, differences in the amount of DNA repair as determined by autoradiographic analysis did occur. Methyl methanesulfonate and dimethylnitrosamine induced repairable DNA damage that was detected at lower dose levels with the 18–20 hour exposure/labeling period. Similar results were also observed for the DNA cross-linking agents, mitomycin C and nitrogen mustard. Ethyl methanesulfonate produced only a marginal amount of DNA repair in primary hepatocytes up to a dose level of 10^–3M during the 4 hour incubation period, whereas a substantial amount of DNA repair was detectable at a dose level of 2.5 × 10^–4M when the 18–20 hour exposure/labeling period was employed. The DNA alkylating agent 4-nitroquinoline-1-oxide, which creates DNA base adducts that are slowly removed from mammalian cell DNA, induced no detectable DNA repair in hepatocytes up to a toxic dose level of 2 × 10^–5M with the 4 hour exposure period, whereas a marked DNA repair response was observed at 10^–5M when the 18–20 hour exposure/labeling period was used.Abbreviations 2AAF 2-acetylaminofluorene - AB₁ aflatoxin B₁ - BENZ benzidine - DEB diepoxybutane - DEN diethylnitrosamine - DMN dimethylnitrosamine - EMS ethyl methanesulfonate - MITC mitomycin C - MMS methyl methanesulfonate - NG mean net nuclear grain counts - NM nitrogen mustard - 4NQO 4-nitroquinoline-N-oxide     

No detectable DNA excision repair in UV-exposed hepatocytes from two catfish species

DNA repair is a critical process in protecting cellular genetic information from mutation. Nucleotide excision repair (NER) is a mechanism by which cells correct DNA damage caused by agents that form bulky covalent adducts and UV photoproducts such as thymine dimers and 6-4 photoproduct. NER, sometimes called dark repair, is generally accepted as being low in fish compared to mammals. This study was designed to quantitate NER in two related catfish species that have known differential sensitivities to liver carcinomas. The original hypothesis was that the more cancer resistant species, channel catfish (Ictalurus punctatus), would have more efficient DNA repair compared to the more sensitive brown bullhead (Ameriurus nebulosus). In order to measure NER, primary cultured hepatocytes of both species were exposed to UV light (10-40 J/m2) and collected at 0, 24, 48 and 72 h after exposure. Total DNA was extracted from the cells and incubated with T4 endonuclease V. Using alkaline gel electrophoresis, endonuclease sensitive sites (ESS) were quantified. Results from the ESS assay indicated there was a UV dose-response increase in thymine dimers from 0 to 40 J/m2. However, no repair (decrease in number of ESS) occurred in either fish species over a 72-h time period. When cells were exposed to photoreactivating fluorescent light, repair was detected. These studies highlight the difficulty of measuring NER in fish and are consistent with the low levels of NER reported by other researchers in fish.     

Genotoxicity of epoxy resin hardeners in the hepatocyte primary culture/DNA repair test

The genotoxicity of 9 chemicals used as epoxy resin hardeners was examined in the DNA repair test with rat hepatocytes. DNA repair synthesis was elicited by 7 chemicals, i.e., 4-aminodiphenyl ether, 4,4-diaminodiphenyl ether, 3,4,4′-triaminodiphenyl ether, 3,3′-dichloro-4,4′-diaminodiphenyl ether, 1,3-phenylenedi-4-aminophenyl ether, 4,4′-diaminodiphenyl methane and 4,4′-methylene-bis(2-chloroaniline).The positive results obtained with 4 epoxy resin hardeners of unknown carcinogenicity, i.e., 4-aminodiphenyl ether, 3,4,4′-triaminodiphenyl ether, 3,3′-dichloro-4,4′-diaminodiphenyl ether and 1,3-phenylene-di-4-aminophenyl ether suggest that they may be carcinogens. The genotoxicity of 1,4-phenylene-di-4-aminophenyl ether, of unknown carcinogenicity, and 4,4′-diaminodiphenyl sulfone, for which there is no sound proof of carcinogenicity, was not confirmed in the DNA repair test. The result with 4,4′-diaminodiphenyl sulfone was in agreement with its lack of mutagenicity in Salmonella typhimurium.     

Efficient lipid-mediated transfection of DNA into primary rat hepatocytes

Cationic lipids are an effective means for transfecting nucleic acids into a variety of cell types. Very few of these lipids, however, have been reported to be effective with primary cells. We report on the efficacy of several commercially available cationic lipid reagents to transfect plasmid DNA into primary rat hepatocytes in culture. The reagents tested in this study include TransfectAce, LipofectAmine, Lipofectin, N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammoniumchloride (DOTMA), (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methylsulfate (DOTAP), and cetyltrimethyl-ammonium bromide/dioleoylphosphatidylethanol-amine (CTAB/DOPE). Electron micrographic (EM) studies indicate that similar size Lipofectin and DOTAP vesicles contain DNA-like material internally and that these vesicles attach to the cell membrane. DOTAP vesicles are multilamellar, appear as clusters, and have a high DNA-to-lipid ratio. Lipofectin vesicles appear to attach to the cell surface as individual vesicles. The EM observations are consistent with current theories on the mechanism of transfection by cationic lipids. While Lipofectin has proven to be effective in transfection studies of primary cells in culture, we have found DOTAP to be a viable alternative. DOTAP yields transfection rates in hepatocytes comparable to DOTMA and Lipofectin, however, at lower concentrations of reagent and at considerably less cost. Optimal conditions for transfecting 5 μg of plasmid DNA with DOTAP were achieved by utilizing multilamellar (vortexed) vesicles at a concentration of 15 μg DOTAP per 2 ml media in 60-mm plates for 2 h transfection time. In this study, DOTAP has proven to be economical, easy to prepare, and very effective in transfecting DNA into primary rat hepatocytes.     

Isolation and culture of hepatocytes from human liver of immediate autopsy

Summary Human livers were removed at immediate autopsy (IA) from brain death patients within 1 h after cessation of cardiac function. Viable hepatocytes were isolated successfully from these IA livers by perfusion of an intack lobe with collagenase or by digestion of a small tissue wedge with collagenase-dispase. The yields of hepatocytes ranged from 1 to 3 × 10⁶ cells/g liver in the five cases studied. Approximately 70 to 90% of the cells excluded trypan blue dye. In the isolated hepatocytes, 632 pmol/mg protein of cytochromep ₄₅₀ and 536. pmol/mg protein cytochromeb ₅ were measured. The cells attached to the dishes in 4 h and produced monolayer cultures with a high success rate. The cells maintained in primary cultures for several days and developed ultrastructural features characteristic of human hepatocytes in vivo. The cultured hepatocytes can hydroxylate benzo[a]pyrene, conjugate the metabolites, and have a benzo[a]pyrene hydroxylase activity of 48.7 pmol/mg DNA per h, which is comparable to that of rat hepatocytes. The liver cells repaired DNA damage caused by exposures to aminofluorene and acetylaminofluorene in culture. This work was supported by EPA Grants R-809835-01-1, R-809599010 and DOE Contract DE-A505-83ER60158. Cobtribution no. 1762 from the Cellular Pathobiology Laboratory, University of Maryland School of Medicine.     

The comet assay for DNA damage and repair

The comet assay (single-cell gel electrophoresis) is a simple method for measuring deoxyribonucleic acid (DNA) strand breaks in eukaryotic cells. Cells embedded in agarose on a microscope slide are lysed with detergent and high salt to form nucleoids containing supercoiled loops of DNA linked to the nuclear matrix. Electrophoresis at high pH results in structures resembling comets, observed by fluorescence microscopy; the intensity of the comet tail relative to the head reflects the number of DNA breaks. The likely basis for this is that loops containing a break lose their supercoiling and become free to extend toward the anode. The assay has applications in testing novel chemicals for genotoxicity, monitoring environmental contamination with genotoxins, human biomonitoring and molecular epidemiology, and fundamental research in DNA damage and repair. The sensitivity and specificity of the assay are greatly enhanced if the nucleoids are incubated with bacterial repair endonucleases that recognize specific kinds of damage in the DNA and convert lesions to DNA breaks, increasing the amount of DNA in the comet tail. DNA repair can be monitored by incubating cells after treatment with damaging agent and measuring the damage remaining at intervals. Alternatively, the repair activity in a cell extract can be measured by incubating it with nucleoids containing specific damage.     

Genotoxicity of pyrene oxide and 1-nitropyrene oxides in hepatocyte primary culture/DNA repair test

The genotoxicity of a pyrene oxide, 1-nitropyrene (NP) oxides and other related compounds was examined in the hepatocyte primary culture (HPC)/DNA repair test. Pyrene 4,5-oxide and both 1-NP-4,5-oxide and 1-NP-9,10-oxide elicited clearly positive responses of DNA repair. In this assay, 1-NP itself was weakly positive. However, other related chemicals such as pyrene, 1-nitro-3-hydroxypyrene, 1-nitro-6-hydroxypyrene, and 1-nitro-8-hydroxypyrene did not generate positive responses.     

Reliability of the hepatocyte primary culture/DNA repair test in testing of coded carcinogens and noncarcinogens

The hepatocyte primary culture/DNA repair test was evaluated for its reliability using a series of coded samples. Among the 30 chemicals tested, 15 were general reference compounds and 15 were chemicals that had been tested for carcinogenicity in the U.S. National Cancer Institute Bioassay Program. The latter group were from the same lot that had been used for the in vivo testing and had also been tested for mutagenicity in the Ames test. From the group of 15 reference compounds, 5 were positive for DNA repair and all 5 were carcinogens. Of the 10 samples scored as negative, 4 were noncarcinogens and 6 were carcinogens. Among the 6 carcinogens were 3 compounds whose carcinogenicity probably does not involve the production of DNA damage. From the 15 coded chemicals that were tested for carcinogenicity by the NCI in long-term animal studies, 7 were scored as positive. 5 of these were judged carcinogenic in the in vivo bioassays and the other 2, which were also mutagenic in Salmonella, showed some indication of carcinogenicity. Of the 8 compounds that were scored as negative, 5 were noncarcinogenic. Among the 3 carcinogens that were not detected, there was at least one whose carcinogenicity probably does not involve DNA damage. Thus, the results of this study indicate that positive results in the hepatocyte primary culture/DNA repair test are highly specific for carcinogens and that the test is also highly sensitive in the detection of DNA-damaging genotoxic carcinogens.     

Lymphocytes from hepatic inflammatory infiltrate kill rat hepatocytes in primary culture

In the last few years it has become possible in the liver to isolate lymphocytes from inflammatory infiltrates and to culture them in vitro. Most of the lymphocyte clones obtained are CD 8 + cytotoxic cells, but interactions between these lymphocytes and hepatocytes in primary culture have not been analysed previously. In this study, cloned human T lymphocytes from liver biopsies and from the peripheral blood of patients with chronic hepatitis B or primary biliary cirrhosis, after phenotypical and functional characterization into CD 8+ or CD 4+ cytotoxic lymphocytes, were activated in an antigen-independent fashion by adding either anti CD 3 or anti CD 2/R-3 monoclonal antibodies to the cell suspension. The activated cells were then coincubated with rat hepatocytes in primary culture. The killing capacity of the activated lymphocytes was monitored by light and electron microscopy and by measurement of lactic dehydrogenase (LDH)-release into the culture medium. It was found that cytotoxic CD 8 +, but not CD 4 + helper lymphocytes very effectively killed hepatocytes. The killing effect was dependent on the time of cocultivation and on effector-target (E/T) ratio. Total breakdown of the hepatocyte monolayer was achieved after 10–20 h coculture and at an E/T ratio of 10 to 1. As LDH-release in the culture medium reached about 80% of the total LDH-content, most of the hepatocytes were lysed by activated lymphocytes. Cytotoxic activity of clones obtained from different biopsies was comparable with that of clones from peripheral blood. Hepatocytes in primary culture seem to be very sensitive to the killing capacity of activated cytotoxic lymphocytes. Supported by DFG grants Ra 362/5-2 and SFB 311 A7 (G.R.) and A5 (H.P.D.)     

Isolation of 8-methoxypsoralen accessible DNA domains from chromatin of intact cells

The chromatin organization of living mammalian cells was probed using 8-methoxypsoralen (MOP). In intact cells, MOP intercalates into DNA domains which are also preferentially accessible to micrococcal nuclease. After UV_{365 nm} irradiation of MOP-treated cells, this chemical forms bifunctional adducts crosslinking the two strands of DNA. Following extraction of cellular DNA, heat denaturation and renaturation at low temperature, the fraction of crosslinked DNA is obtained following enzymatic hydrolysis of unhybridized, non-crosslinked DNA ny nuclease S1 treatment. An application of this procedure in the isolation of 8-methoxypsoralen-accessible DNA domains during DNA excision repair is shown.Abbreviations MOP 8-methoxypsoralen - UV ultraviolet light     

Cell density dependent morphological changes in adult rat hepatocytes during primary culture

In order to gain morphological insights about the cell density dependency, hepatocytes cultured at a low cell density (less than about 0.1 X 10(5) nuclei (cm2)-1) and at a high cell density (greater than about 1 X 10(5) nuclei (cm2)-1) were examined ultrastructurally 24 h after plating (just prior to the beginning of DNA synthesis). The results were as follows: (i) glycogen rosettes disappeared completely in low density culture as compared with sections from an intact liver. In contrast, glycogen rosettes were still present in high density culture. (ii) Polysomes seemed increased in low density culture in comparison with those seen in sections from an intact liver and from the high density culture. (iii) In low density culture, the shape of mitochondria deviated from that of hepatocytes in an intact liver and the mitochondria often lost a characteristic close contact with rough endoplasmic reticulum (rough ER). (iv) In low density culture, bundles of filamentous structure were detected, which were not found in an intact liver or high density culture. The following features were found only in high density culture; (v) numerous villous cytoplasmic protrusions developed along the area facing adjacent cells, and seemed to intertwine with each other, and (vi) between the hepatocytes, only abortive junctions were found. These results indicate that the hepatocytes cultured at a low density express most of the characteristics of the hepatocytes in a regenerating liver and the features of the cells cultured at a high density are very similar to those of the hepatocytes in sections from an intact liver.     

Long-term culture of primary rat hepatocytes with high albumin secretion using membrane-supported collagen sandwich

Primary cultured rat hepatocytes in a membrane-supported collagen sandwich maintained their normal cell morphology and high level of albumin secretion for over 56 days. It was found that the existence of an upper layer of collagen gel is crucial for long-term culture and that the transference of cellular nutrients between the culture media and hepatocytes from both the upper and the lower sides of gel layers promotes albumin secretion. These facts suggest that the membrane-supported collagen sandwich mimics well thein vivo environment of hepatocytes. This method has great potential for the long-term culture of primary cells.     

Evaluation of morpholine, 3-morpholinone, and N-substituted morpholines in the rat hepatocyte primary culture/DNA repair test

Although mature mammalian sperm are incapable of DNA repair, repair of damaged sperm DNA can occur after fertilization, as the sperm head decondenses and forms the male pronucleus. To quantify the cytogenetic effects of damage to sperm DNA we adapted the sister-chromatid exchange (SCE) test for use in early mouse embryos. After ultraviolet (UV) irradiation of sperm, eggs were fertilized in vitro and cultured for 2 cell cycles in medium containing fluorodeoxyuridine and bromodeoxyuridine; chromosomes were then prepared for SCE analysis. We found that UV-induced SCEs could be detected at the second cleavage division, and that eggs of different strains showed different frequencies of SCEs when fertilized by damaged sperm of a single strain. These results may indicate strain-specific differences in DNA repair of UV-induced DNA lesions by the early mouse embryo.     

The repair of G-quadruplex-induced DNA damage

G4 DNA motifs, which can form stable secondary structures called G-quadruplexes, are ubiquitous in eukaryotic genomes, and have been shown to cause genomic instability. Specialized helicases that unwind G-quadruplexes in vitro have been identified, and they have been shown to prevent genetic instability in vivo. In the absence of these helicases, G-quadruplexes can persist and cause replication fork stalling and collapse. Translesion synthesis (TLS) and homologous recombination (HR) have been proposed to play a role in the repair of this damage, but recently it was found in the nematode Caenorhabditis elegans that G4-induced genome alterations are generated by an error-prone repair mechanism that is dependent on the A-family polymerase Theta (Pol θ). Current data point towards a scenario where DNA replication blocked at G-quadruplexes causes DNA double strand breaks (DSBs), and where the choice of repair pathway that can act on these breaks dictates the nature of genomic alterations that are observed in various organisms.     

Identifying Rattus species using mitochondrial DNA

In recent years, research has shown that geographical variation in mitochondrial DNA of commensal rats provides a strong signal of human dispersal and migration. However, interpretation of genetic variation is complicated by the presence of multiple species of Rattus especially in Island Southeast Asia, by the occurrence of some of these Rattus sp. as subfossils in archaeological and natural sites, and by the difficulty of osteological identification of these remains. Amplification of DNA from ancient sources usually yields only small fragments (～200 bp). We assessed whether we could identify Rattus sp. reliably with DNA barcoding using cytochrome oxidase I (COI) sequences, or tree‐based methods using D‐loop, cytochrome b and COI sequences. Species forming well‐differentiated clades in a molecular phylogeny were accurately identified by both methods, even when we used short DNA fragments. Identification was less accurate for paraphyletic and polyphyletic species. We suggest that taxonomic revisions that recognize cryptic or polytypic species will lead to even greater accuracy of DNA‐based identification methods.     

The effects of dexamethasone on metabolic activity of hepatocytes in primary monolayer culture

Summary The effects of dexamethasone on multiple metabolic functions of adult rat hepatocytes in monolayer culture were studied. Adult rat liver parenchymal cells were isolated by collagenase perfusion and cultured as a primary monolayer in HI/WO/BA, a serum free, completely defined, synthetic culture medium. Cells inoculated into the culture medium formed a monolayer within 24 hr. Electron microscopy showed that the cells in primary culture had a fine structure identical to liver parenchymal cells in vivo, including the observation of desmosomes and bile canaliculi in intercellular space. There was significant gluconeogenesis by the cell 24 hr postinoculation but it had decreased markedly by 48 hr. There was a marked induction of tyrosine aminotransferase (TAT) by dexamethasone, which was maintained for up to 72 hr postinoculation of cells. The transport of α-aminoisobutyric acid into the cells in monolayer culture was stimulated by dexamethasone and was dependent on the concentration of dexamethasone. Albumin synthesis and secretion by the cells was measured by a quantitative electroimmunoassay. Albumin production was shown to increase linearly over an incubation period of 24 to 48 hr postinoculation. Dexamethasone depressed the albumin synthesis. The effects of dexamethasone are slow, and at times require more than 6 hr to show variation from the control, indicating that dexamethasone is not a single controlling hormone. Possibly it functions in a cooperative and coordinating role in the regulation of cell metabolism. This work was performed during Dr. Yamada's tenure as a Postdoctoral Research Fellow of the American Heart Association, Oklahoma Affiliate, and was supported in part by NIH Research Grant HL 18178 awarded to Thomas F. Whayne, Jr., M.D., Ph.D.