Expression of xenobiotic-metabolizing enzymes in propagatable cell cultures and induction of micronuclei by 13 compounds.

Activities of various xenobiotic-metabolizing enzymes were determined in 18 cell lines. Activities of cytochrome P450 reductase, microsomal epoxide hydrolase and glutathione transferase were detectable in all lines. The highest values were similar to the activities found in freshly isolated rat hepatocytes. Catalase activity was also present in all 12 investigated cell lines. Activity of UDP-glucuronosyl transferase was high in some lines, but low or undetectable in others. Activity of cytosolic epoxide hydrolase was not measurable in most lines, and was low in the others. Metabolism of benzo[a]pyrene was observed in eight out of nine examined lines, no activity being found in V79 cells. V79 and three epithelial cell lines were then used as target cells in a genotoxicity assay in which the frequency of micronucleated cells was determined. In V79 cells, 7,12-dimethyl- benz[a]anthracene, benzo[a]pyrene, benzo[a]pyrene-trans-7,8-dihydrodiol, aflatoxin B1, N-nitrosomorpholine and 2-acetylaminofluorene showed negative responses, whereas N-methyl-N'-nitro-N-nitrosoguanidine, 9-hydroxybenzo[a]pyrene, 2-nitrofluorene, dibenz[a,h]anthracene 1,2-catechol, dibenz[a,h]anthracene, 1,2-quinone hydroquinone and p-benzoquinone proved positive in the test. All 13 compounds, however, induced micronuclei in rat intestinal cells (IEC-17 and IEC-18) and in embryonal human liver cells (HuFoe-15). Thus, these epithelial cell lines are capable of activating and detecting a broad spectrum of chemically diverse genotoxic compounds. They may also be useful for the detection of hazardous compounds whose active metabolites are not able to penetrate from the extracellular space into the indicator cell.

Growth factor requirements of childhood acute leukemia: establishment of GM-CSF-dependent cell lines.

Eight permanent cell lines were established from cells of 50 consecutive patients with childhood acute leukemia. Three cell lines required growth factor-containing conditioned media. Analysis using blocking antisera and recombinant granulocytic macrophage (GM) colony-stimulating factor (CSF) identified GM-CSF as a growth factor required to establish the latter three cell lines and necessary for their continuous proliferation in chemically defined medium. Two of the GM-CSF-dependent cell lines were derived from patients with undifferentiated T- and a biphenotypic B-myelomonocytic leukemia, which suggests that GM-CSF might maintain proliferation of leukemias originating from immature progenitor cells. Cytogenetic analysis indicated that all established leukemic cell lines were aneuploid, with six lines containing chromosomal alterations related to those observed in the leukemic cells of the patient. Two patients did not have an abnormal clone identified in the marrow but did yield an aneuploid cell line. These studies indicate that GM-CSF-dependent leukemic cell lines can be established in a fraction of childhood leukemia. These cell lines lend themselves to studies aimed at the evaluation in vitro of the role of growth factors in controlling proliferation and differentiation of leukemic cells.

Establishment and characterization of an undifferentiated human T leukemia cell line which requires granulocyte-macrophage colony stimulatory factor for growth.

A human leukemia cell line (TALL-101) was established from the bone marrow of a patient with an undifferentiated acute T cell leukemia using the conditioned medium (CM) of the human T cell leukemia virus (HTLV) II-transformed human cell line J-LB1. Immunofluorescence analysis on the original leukemic cells indicated the presence of T cell markers (Leu-1, Tdt, and T11); however, the established TALL-101 cell line expressed only antigens commonly present on progenitor cells, thymocytes, and myelomonocytic cells, but not on mature T cells. A high percentage of TALL-101 cells displayed the Tac antigen which was down-regulated upon incubation in the presence of recombinant human (rH) interleukin 2 (IL 2). Interferon (IFN)-gamma induced the appearance of class II histocompatibility leukocyte antigens (HLA) and of a T cell marker (3A1), and enhanced the expression of transferrin receptors on these cells. Further evidence for a T cell lineage of the TALL-101 cell line was provided by both chromosomic and genotypic analysis showing a translocation in chromosome 14 typical of T cell leukemias, and a rearrangement of the T-beta receptor locus. The growth-promoting activity in the J-LB1-CM was identified as granulocyte-macrophage colony stimulatory factor (GM-CSF), a growth factor which stimulates proliferation of normal myelomonocytic cells and other progenitor cells, but not known to have an effect on T cells. Dose response curves of [3H]thymidine incorporation and growth indicated that TALL-101 cells were sensitive to very low concentrations of rHGM-CSF, 5 ng/ml inducing maximal proliferation in chemically defined medium. The TALL-101 cell line is strictly GM-CSF-dependent for growth: upon depletion of GM-CSF from the culture medium, the cells stop proliferating immediately and die within 1 to 2 wk. The overall data, showing that GM-CSF is able to support the growth of a highly undifferentiated T cell leukemia, strongly suggests that this factor might have similar growth promoting effects on other immature T cell leukemias, and possibly, on normal T cell progenitors.

Characterization of an epithelial, nearly diploid liver cell strain, from Chinese hamster, able to activate promutagens.

Epithelial liver cells of the Chinese hamster (CHEL cells) were propagated in culture for 35 passages. At favourable cell densities, the population doubling time in normal medium, was 20 h. L-Tyrosine amino transferase activity was retained at a measurable level, but its enhancement by dexamethasone was detected solely in cells of early passages. Pyruvate kinase was strongly activated by fructose-1,6-biphosphate at low substrate concentrations. These enzymatic properties suggest that the CHEL cells are derived from a sub-population of parenchymal hepatocytes or from cells closely related to parenchymal hepatocytes. With a lag period of a few hours, CHEL cultures metabolized benzo[a]pyrene. In cell homogenates the various monooxygenase activities investigated were below the detection limits. However, other xenobiotic-metabolizing activities, such as cytochrome P-450 reductase, glutathione transferase and UDP-glucuronosyl-transferase were high, with levels comparable to those observed in freshly isolated rat parenchymal cells. Epoxide hydrolase activity was also detected, but was lower than in the liver. The CHEL cells were able to activate benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene and aflatoxin B1 to mutagens, as shown in a co-culture assay with V79 cells, in which acquisition of resistance to 6-thioguanine was studied. At early passages, the CHEL cells had a near diploid set of chromosomes. Then, gradually the frequency of cells with slight changes in the number of chromosomes and the frequency of tetraploids were increased. During the observation period (up to passage 20) the modal number of chromosomes shifted from 22 to 23. No gross morphological changes in the cultures were noticed during the 20 passages.(ABSTRACT TRUNCATED AT 250 WORDS)

Search for cell culture systems with diverse xenobiotic-metabolizing activities and their use in toxicological studies.

Many toxic effects are not caused by the administered compound itself, but are due to metabolites. All cell types express some xenobiotic-metabolizing enzymes, but levels and patterns are very variable. Critical metabolic steps may occur within the target cell and/or at other sites. This complex situation is difficult to mimic in vitro. The further problem is that cells that are taken into culture tend to rapidly cease the expression of important xenobiotic-metabolizing enzymes. Part of the problem may be solved by the addition of exogenous metabolizing systems, for example, in the form of freshly isolated hepatocytes, crude subcellular preparations, or purified enzymes. In these systems, the plasma membrane of the target cell may act as a barrier for the active metabolite and thereby lead to false negative results. The alternative is the use of metabolically active target cells. We therefore screened 18 cell lines for monooxygenase, cytochrome P-450 reductase, epoxide hydrolase, glutathione transferase, and UDP-glucuronosyl transferase activities. In further studies, IEC-17, IEC-18, and HuFoe-15 cells showed their capabilities of activating a broad spectrum of structurally heterogenous promutagens, as indicated by the induction of micronuclei. These cells, however, were not suited for the study of a more relevant genetic end point, the induction of hereditary functional changes (gene mutations), implying that a compromise had to be made on the level of the toxicodynamics. In the second approach, cDNAs encoding the rat cytochromes P-450IA1 and P-450IIB1, set under the control of a constitutive promoter, were transfected into V79 Chinese hamster cells, which do not express cytochromes P-450 but are ideal target cells for gene mutation assays. The resulting substrains (XEM1, XEM2, XEM3; SD1) stably expressed cytochromes P-450IA1 and P-450IIB1, respectively, and showed the corresponding monooxygenase activities. Aflatoxin B1, cyclophosphamide, dibutylnitrosamine, and benzo[a]pyrene mutated SD1 and/or XEM1 and XEM2 cells, but were inactive in parental V79 cells. The mutagenicity of benzo[a]pyrene 7,8-trans-dihydrodiol was about 1000 times more potent in XEM1 and XEM2 cells than in SD1 and V79 cells. Other promutagens were inactive in V79 as well as in the genetically engineered daughter lines. This system therefore is not yet optimal in general screening for the detection of new mutagens, but appears ideal in the identification of critical xenobiotic-metabolizing enzymes for a given mutagen.