Adult Rat Liver Epithelial Cells Research Articles

Inhibition of repair of O6-methyldeoxyguanosine and enhanced mutagenesis in rat-liver epithelial cells

The pro-mutagenicity of chemically-induced methylation of DNA at the O 6 position of dexoyguanosine was studied in cultured adult rat liver epithelial cells. To modify the level of O 6-methyldeoxyguanosine (O 6-medGuo) resulting from exposure to an alkylating agent, partial depletion of the O 6-alkylguanine-DNA alkyltransferase (AGT) repair system was produced by pretreatment of ARL 18 cells with a non-toxic dose of exogenous O 6-methylguanine (O 6-meG). Exposure of cells to 0.6 mM O 6-meG for 4 h depleted AGT activity by about 40%. Intact and pretreated cells were exposed to a range of doses of N-methyl- N′-nitro- N-nitrosoguanidine (MNNG), and mutagenesis at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus was quantified by measurement of 6-thioguanine-resistant mutants. The mutagenicity of MNNG was dose dependent and was greater in O 6-meG pretreated cultures than in intact cultures. Immunoslot blot measurement of O 6-medGuo employing a mouse monoclonal antibody demonstrated that MNNG produced O[su6-medGuo and that the intact liver cells were efficient in eliminating this lesion from their DNA. Since depletion of AGT would be expected to affect the rate of elimination of only O 6-medGuo, it is concluded that this lesion is highly pro-mutagenic.

Enrichment and characterization of clonogenic epithelial cells from adult rat liver and initiation of epithelial cell strains.

A highly efficient method is described for obtaining proliferative epithelial cells from adult rat livers for the reproducible establishment of liver epithelial cell strains. When cells were isolated from livers of 10- to 15-wk-old male Fischer 344 rats by a collagenase-perfusion method, collected by centrifugation at 50 X g for 5 min, and cultured in Williams' medium E containing fetal bovine serum and dexamethasone, colonies of epithelial cells different in size and morphology from hepatocytes were obtained. Sequential perfusion with collagenase and dispase yielded numerous epithelial cell colonies. When isolated cells were fractionated by differential centrifugation, the great majority of hepatocytes were sedimented at 50 X g for 1 min, whereas many non-hepatocytic cells remained in the supernatant and could be sedimented by a second centrifugation at 50 X g for 5 min. Culture of the two fractions revealed that almost all the epithelial cell colonies were derived from cells in the non-hepatocytic cell fraction. The epithelial cells were cytochemically negative for gamma-glutamyl transpeptidase activity, whereas an increase in the activity was detected in hepatocytes with duration in culture. Ultrastructural characteristics of hepatocytes were not found in the cells of newly established cell strains. These results suggest that adult rat liver epithelial cells propagable in culture were derived from a cell type other than the hepatocyte.

The response of an established line of rat liver cells to thyroid hormone

The response of an established line of non-transformed adult rat liver epithelial cells (ARL 15) to thyroid hormone (T 3) (3,5,3′-triiodothyronine) was characterized. Exposure of confluent monolayers to 1·10 −8 M T 3 for 3 days increased O 2 consumption ( QO 2) between 14–58%, ouabain-sensitive Rb + uptake 26%, (Na + + K +)-ATPase activity 32%, α-glycerophosphate dehydrogenase activity 103% and cytochrome oxidase activity 208%. The ARL 15 cells, maintained in continuous culture, therefore, exhibit the hallmarks of an authentic physiological response to thyroid hormone.

Amino acid transport in established adult rat liver epithelial cell lines.

The capacities of Na+-dependent transport of alpha-aminoisobutyrate, glutamine and glutamate in four established and three transformed rat liver epithelial cell lines were found to be considerably higher than those of isolated and cultured hepatocytes. At least for transport systems A and G- this seemed to be due to elevated values of Vmax, whereas the values for Km were quite comparable to those of hepatocytes. In contrast to hepatocytes, however, no significant hormonal stimulation of amino acid uptake could be detected in the cell lines. Each normal cell line expressed a distinct pattern of transport capacities with respect to the three systems measured and this was not altered by chemical transformation of the lines. The individual patterns of the lines showed no similarity to presumptive patterns of subpopulations of liver parenchymal cells. In particular, there was no evidence for a direct relationship of one of the cell lines with a small subpopulation of parenchymal cells located adjacent to hepatic venules as revealed by additional measurements of glutamine synthetase, a marker enzyme for this particular subpopulation. It is concluded that established rat liver epithelial cell lines express features characteristic of normal hepatocytes with respect to amino acid transport, but have developed a distinct phenotype adapted to a rapid, hormone-independent growth in vitro. Alteration of their phenotype by transformation is not coupled with a further increase in amino acid transport capacity.

The cytotoxicity of Bleomycin A 2 on adult rat liver epithelial cell lines at different stages of spontaneous cell transformation

Cytotoxic effects of Bleomycin A 2 on adult rat liver epithelial cell lines were evaluated by three methods: the incorporation rate of ( 3H)thymidine for DNA biosynthesis, the incorporation rate of L-( 3H)leucine for protein biosynthesis and Giemsa dye staining of surviving cells. Chromosome investigations at successive passages of cell lines have shown that spontaneous chromosome abnormalities in distribution and structure after 15–20 passages, i.e. 50 to 60 cell generations, were the earliest morphological sign of spontaneous transformation. In this study a highly spontaneously transformed cell line was very sensitive to the drug. Another cell line at the beginning of spontaneous transformation appeared to be insensitive although on further passage it became more sensitive. The use of microtitration plates made it easier for us to undertake a comparative study of the different parameters. Following Bleomycin A 2 exposure, Giemsa staining gave the best evaluation of cell killing whereas thymidine incorporation allowed the estimation of cell recovery. The antineoplastic effect of Bleomycin A 2 can probably be used to evaluate the malignant potential of different rat liver epithelial cell lines.

Differences in responses of 4 adult rat-liver epithelial cell lines to a spectrum of chemical mutagens

An assay for mutagenesis at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in adult rat-liver epithelial cell cultures (ARL) has been developed to take advantage of the capacity of this cell type to metabolically activate promutagens/procarcinogens. A survey of the effect of 5 types of activation-dependent mutagens/carcinogens on 4 ARL lines indicates that the ARL/HGPRT mutagenesis assay with the 4 target cell lines is able to detect a spectrum of activation-dependent carcinogens. Individual ARL lines, however, responded quite differently to a given carcinogen. The ARL/HGPRT mutagenesis assay system thus offers distinct possibilities for the study of the control of chemical biotransformation processes. However, in light of the specificity of the various cell lines to respond to a particular class of mutagens under the current assay condition, this particular assay system cannot be readily applied to routine screening of suspected environmental mutagens of unknown requirements for metabolic activation. Nevertheless, for agents with a structure related to those activated by a specific line, this system can be used to study mutagenesis resulting from intact cellular metabolism.

Sister-chromatid exchange induction by polycyclic aromatic hydrocarbons in an intact cell system of adult rat-liver epithelial cells

Adult rat-liver epithelial cell lines possess intrinsic metabolic capability for the biotransformation of xenobiotics and thus, are sensitive to a broad spectrum of mutagens/carcinogens in a mutagenesis assay at the hypoxanthine-guanine phosphoribosyl transferase locus. To provide another end-point of biological significance in these lines, we have investigated the application of adult rat-liver epithelial cell line 18 in a sister-chromatid exchange assay. Significant dose-dependent increases in the sister-chromatid exchange frequency occurred when liver cells were exposed to benzo[ a]pyrene and 7,12-dimethylbenz[ a]anthracene. A weak but positive response was elicited by benz[ a]anthracene. The present observations thus confirm the capacity of these cells to generate genotoxic metabolites from activation-dependent mutagens/carcinogens and indicate a relationship between the production of mutations and sister-chromatid exchanges by polycyclic aromatic hydrocarbons.

Inhibition of intercellular communication between liver cells by the liver tumor promoter 1,1,1-trichloro-2,2-bis( p-chlorophenyl)ethane

A dose dependent inhibition of intercellular communication (metabolic cooperation) between primary cultures of rat liver hepatocytes and an established adult rat liver epithelial cell 6-thioguanine resistant strain by the liver tumor promoter 1,1,1-trichloro-2,2-bis ( p-chlorophenyl)ethane (DDT) is demonstrated. This in vitro assay is proposed to evaluate the tumor promoting activity of oncogenic agents shown to be non-genotoxic in the liver culture systems.

The use of adult rat liver cultures in the detection of the genotoxicity of various polycyclic aromatic hydrocarbons.

The hepatocyte primary culture (HPC)--DNA repair test and the adult rat liver epithelial cell (ARL)--hypoxanthine-guanine phosphoribosyl transferase (HGPRT) mutagenesis assay are two in vitro short-term tests that possess intrinsic capability for xenobiotic biotransformation. Both assays detected the genotoxicity of a variety of carcinogenic polycyclic aromatic hydrocarbons. Thus, these two tests, which embody intact cellular metabolism, are useful for the evaluation of this class of carcinogens and provide results that strengthen those obtained in tests dependent upon subcellular metabolism.

Cell cycle-specific mutagenesis at the hypoxanthine phosphoribosyltransferase locus in adult rat liver epithelial cells.

The cell cycle specificity of chemical mutagenesis was studied by use of two cell synchronization techniques, one a nontoxic technique involving serum deprivation and the other a double thymidine block, to obtain rat liver epithelial cells in different phases of the cell cycle to be exposed to chemical mutagens. For both methyl methanesulfonate and N-methyl-N'-nitro-N-nitrosoguanidine, there was a cell cycle specificity of chemical mutagenesis, with the most sensitive phase being the period of DNA synthesis.

Characterization of analog resistance and purine metabolism of adult rat-liver epithelial cell 8-azaguanine-resistant mutants

Adult rat-liver epithelial cultures were sensitive to the lethal effects of 8-azaguanine (AG), but lines contained variants resistant to AG. The frequency of retrievable AG-resistant colonies varied with both the concentration of AG used and the seeding density of the population under selection. Cells resistant to AG were also cross-resistant to 6-thioguanine and unable to grow in medium containing hypoxanthine, aminopterin and thymidine. Resistance was stable. AG resistance was due to a deficiency of hypoxanthine-guanine phosphoribosyl transferase (HGPRTase) activity which was not caused by an inhibitor. In the assay for HGPRTase, a substantial amount of product appeared as inosine (In) in addition to inosine monophosphate (IMP). Purine nucleoside phosphorylase will generate In from hypoxanthine and, indeed, the cells did possess this activity. However, several findings indicated that the In was derived from IMP by catabolism by 5′-nucleotidase (NTase): (1) IMP decreased as In increased and (2) the inhibitors of NTase, adenosine monophosphate and thymidine triphosphate, reduced the generation of In by over 90% without inhibiting purine nucleoside phosphorylase. The cells possessed substantial NTase activity, 35% of which was located in the cytosol along with 69% of HGPRTase. Several lines of evidence suggested that the NTase activity limited the amount of 8-azaguanylic acid presented to the cells by catabolising the nucleotide and, thereby, reducing the toxicity of available AG.

Rat Hepatocyte Primary Cell Culture-Mediated Mutagenesis of Adult Rat Liver Epithelial Cells by Procarcinogens

SummaryRat hepatocyte primary cell (HPC) cultures which were unable to be subcultured were used as a metabolizing feeder system for established lines of adult rat liver (ARL) cultures in studies of procar-cinogen-induced mutagenesis. Treatment of a mixed culture of HPC and ARL cells with 7,12-dimethylbenz(a)anthracene, dimethyl-nitrosamine, or 2-acetylaminofluorene for 24 hr resulted in an increase in the incidence of 8-azaguanine-resistant mutants that was not observed with treatment of ARL cells alone. Thus, HPC cultures can be used as a feeder system for enhancing mutagenesis in established lines of ARL cells by procarcin-ogens requiring metabolic activation.

Adult rat liver epithelial cells for studies of carcinogenic activity of safrole

Adult rat liver epithelial cells for studies of carcinogenic activity of safrole

Long-term cell culture of adult rat liver epithelial cells

The livers of adult rats were dissociated after collagenase-hyaluronidase perfusion. A high yield of viable cells was obtained and these regularly gave rise to continuous lines of polygonal epithelial cell types.