Evaluation of P19 cells for studying mechanisms of developmental toxicity: application to four direct-acting alkylating agents.
P19 cells are pluripotent murine embryonic carcinoma (EC) cells that can be induced by all-trans-retinoic acid (RA) to differentiate into cells that are biochemically and morphologically similar to cells of the central nervous system. We have established these cells as a reproducible cell system to evaluate potential effects of agents disrupting neuronal differentiation. The viability of P19 cells was assessed using a neutral red assay. Uptake of [3H]-gamma-amino butyric acid ([3H]GABA) was assessed as a marker of neuronal differentiation. We observed significant increases in [3H]GABA over time, corresponding with the appearance of cells with neuronal morphologies. 2,4-Diaminobutyric acid, a specific inhibitor of high affinity neuronal GABA uptake, reduced [3H]GABA uptake by approximately 75%. Additionally, [3H]GABA uptake in cells treated with dimethylsulfoxide (DMSO), which differentiate into mesodermal derivatives, was approximately 25% of uptake observed in RA-exposed, neuronally differentiated P19 cells. The morphology of P19 cell cultures correlated with [3H]GABA uptake: high [3H]GABA uptake was only observed in cultures with distinct neuronal morphologies. These results suggest that [3H]GABA uptake is a good indicator of neuronal differentiation in P19 cells. The responsiveness of P19 cells to developmental toxicants was assessed by comparing effects in P19 cells with effects observed previously in primary cultures of differentiating rat embryo midbrain (CNS) cells. Alkylating agents chosen for this investigation include methylnitrosourea (MNU), ethylnitrosourea (ENU), methyl methanesulfonate (MMS), and ethyl methanesulfonate (EMS). The rank order of potency of these alkylating agents in the CNS cells was MMS > MNU > ENU > EMS. With the exception of ENU, concentrations that caused effects on growth and differentiation in the P19 cells were very comparable to those causing similar effects in CNS cell cultures. Our results with P19 cells suggest that this EC cell line may also be a useful in vitro cell system for studying mechanisms of developmental toxicity, with the advantages of being an established cell line.
Seeley
M R
MR
Department of Environmental Health, University of Washington, Seattle 98105-6099, USA.
Faustman
E M
EM
D000477:Alkylating Agents / Q000633:toxicity*; D000613:Aminobutyrates / Q000494:pharmacology; D000818:Animals; D018906:Antineoplastic Agents, Alkylating / Q000633:toxicity; D018236:Carcinoma, Embryonal; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D005020:Ethyl Methanesulfonate / Q000633:toxicity; D005038:Ethylnitrosourea / Q000633:toxicity; D008297:Male; D008636:Mesencephalon / Q000187:drug effects / Q000196:embryology; D008741:Methyl Methanesulfonate / Q000633:toxicity; D008770:Methylnitrosourea / Q000633:toxicity; D051379:Mice; D009474:Neurons / Q000187:drug effects*; D014212:Tretinoin; D014407:Tumor Cells, Cultured / Q000187:drug effects; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
D000477:Alkylating Agents; D000613:Aminobutyrates; D018906:Antineoplastic Agents, Alkylating; C005959:2,4-diaminobutyric acid; D005680:gamma-Aminobutyric Acid; D014212:Tretinoin; D008770:Methylnitrosourea; D005020:Ethyl Methanesulfonate; D008741:Methyl Methanesulfonate; D005038:Ethylnitrosourea; D004121:Dimethyl Sulfoxide