Cultured Rat Liver Epithelial Cells Research Articles

Spectrophotometric determination of intracellular pH with cultured rat liver epithelial cells.

A spectrophotometric method using 6-carboxyfluorescein (CF) was developed to determine intracellular pH in anchorage-dependent monolayers of control cells of rat hepatic origin. Until now, such studies have been carried out with ascites cells in suspension, which lack specific controls for comparative studies. The rat cell line is grown on plastic Leighton tube slides which fit directly into 3 cm spectrophotometer cuvettes. One sample, without CF, serves as a control for the light-scattering properties of the cell monolayers. Steady-state determinations show a decline in intracellular pH from 7.3 to 6.8 ten minutes after the addition of glucose and quercetin. Kinetic determinations show that with the addition of glucose to substrate-free cells the rate of acid formation is -0.02 pH units/min; the addition of quercetin results in a further acceleration of the kinetic rate to -0.10 pH units/min. In both types of analyses, the change in intracellular pH is standardized with nigericin and external buffers, based on the decrease in the maximum absorption of CF at 492 nm. The results demonstrate that even with anchorage-dependent monolayers of a control hepatocyte line which produces very little acid, this spectrophotometric method permits determinations sufficiently sensitive for analysis of intracellular pH.

The effects of epidermal growth factor and the state of confluence on enzymatic activities of cultured rat liver epithelial cells.

In cultured normal rat liver epithelial cells, the specific activity and/or isozyme expression of NADH-diaphorase (NADH-D), pyruvate kinase (PK), glucose-6 phosphate dehydrogenase (G6PD), gamma-glutamyl transpeptidase (GGT), and alkaline phosphatase (AP) were markedly dependent on the growth state of the cultures. Proliferating, preconfluent cells had higher specific activities of PK, NADH-D, and G6PD but lower activities of GGT and AP than did the more stationary confluent cells. Addition of epidermal growth factor [EGF] to the media of proliferating cells enhanced the specific activities of PK, NADH-D, G6PD, GGT, and lactate dehydrogenase (LDH) of these cells, but the specific activity of AP was markedly depressed. The increase in activity of PK and GGT by EGF appeared to involve new protein synthesis, whereas the effect of EGF on AP appeared to involve the EGF-directed suppression of the synthesis of a form of AP that is produced exclusively by cells in confluent cultures. Furthermore, the preconfluent cells were more responsive to the action of EGF on AP than were confluent cells, i.e., the EGF-mediated decrease in AP activity was seen at lower concentration in preconfluent than in confluent cells. Paradoxically, confluent cells exhibited a two-to threefold higher capacity to bind [125 I]EGF because of an increase in surface receptor number. The results of this study indicate that enzymatic or other biochemical studies performed on cultured cells must take into account the growth-state of the cultures. EGF can modulate enzyme activity in growing and nongrowing cells; one effect of EGF is to maintain higher activity of glycolytic enzymes, suggesting that EGF or EGF-like factors may contribute to the high rate of glycolysis in certain neoplasms.

Induction of sister chromatid exchanges by carcinogens mediated through cultured rat liver epithelial cells.

A cloned, untransformed rat liver cell-line, LNRL (with the normal diploid karyotype and epithelial morphology) was tested for use as the metabolizing component in a co-cultivation system for carcinogen-screening using sister chromatid exchanges (SCE) as the criterion. The well characterized early cultures of LNRL cells were co-cultivated with Chinese hamster ovary (CHO) cells, the target commonly used for the induction of SCE, and exposed to different concentrations of various chemicals for 24 h. Two polycyclic hydrocarbons, 7,12-dimethylbenz[a]anthracene and benzo[a]pyrene did not produce any SCE in CHO cells cultured alone, but produced a significant number of SCE in CHO co-cultivated with LNRL. The aromatic amine, 2-acetylaminofluorene, and the potent carcinogenic mycotoxin, aflatoxin B1, induced SCE to a limited extent even in CHO cells cultivated alone, but in the presence of liver cells the SCE frequency was greatly increased. Aflatoxin G2, the least potent of the aflatoxins, also produced a response in the co-cultivation system. These results indicate that cultured liver cells can be used as the metabolizing cells in co-cultivation systems for carcinogen-screening. The advantages of this assay over those employing liver microsomal fractions are discussed.

Enhancement of mutagenesis during cell replication of cultured liver epithelial cells

The susceptibility to mutagenesis of proliferating and non-proliferating mammalian cells was studied in cultured rat liver epithelial cells. Cells brought to growth quiescence by a non-toxic means were stimulated to proliferate and both types of cultures were exposed to methyl methanesulfonate (MMS). Cultures enriched in proliferating cells were more susceptible to both the toxic and mutagenic action of the mutagen than were quiescent cultures with a low level of proliferation.

Test for malignant transformation of rat liver cells in culture: cytology, growth in soft agar, and production of plasminogen activator.

Three parameters were evaluated as diagnostic of the malignant potential of cultured rat liver epithelial cells: cytology, growth in soft agar, and production of extracellular plasminogen activator. A total of 22 tumorigenic and nontumorigenic cultures from 15 cell lines were sent coded from their originators to two different laboratories for the evaluation of these three parameters. Cytologic diagnosis and growth in soft agar were reliable means of determining the malignant potential of the cultured cells. However, the production of extracellular plasminogen activator showed little correlation with tumorigenicity. Of cytologic properties evaluated, the two that correlated best with malignant potential were increased cytoplasmic basophilia and and increased nuclear:cytoplasmic ratio.