Developmental toxicity of alkylating agents in differentiating cell cultures

Abstract

Alkylating agents are well known carcinogens and teratogens. Highest exposures occur in occupational settings, although other lifestyle factors also contribute to exposure. The four alkylating agents used in this study were methylmethanesulfonate (MMS), methylnitrosourea (MNU), ethylmethanesulfonate (EMS), and ethylnitrosourea (ENU). In the micromass rat whole embryo cell culture system, significant concentration-dependent decreases in viability and differentiation were observed for both midbrain (CNS) and limb bud (LB) cells. CNS cells were more sensitive than LB cells, and effects on differentiation occurred at lower concentrations than effects on viability. The order of potency was MMS $>$ MNU $>$ ENU $>$ EMS. Next we adapted the P19 embryonal carcinoma cell line as a system for studying mechanisms of developmental toxicity. Using all-trans retinoic acid to induce neuronal differentiation, we confirmed that uptake of ($\sp3$H) -GABA is a good indicator of neuronal differentiation in P19 cells. As in the micromass cells, significant concentration-dependent decreases in both viability and differentiation were observed in alkylating agent-treated P19 cells. Furthermore, effects in the P19 cells occurred at concentrations which were very comparable to concentrations causing similar effects in the CNS cells, for all agents except ENU, which was much less toxic in P19 cells as compared with CNS cells. We also used O$\sp6$-benzylguanine (O$\sp6$-Bg) to inhibit the activity of the O$\sp6$-alkylguanine-DNA-alkyltransferase protein which repairs adducts at the O$\sp6$ position of guanine. O$\sp6$-Bg caused substantial, significant decreases in both viability and differentiation of cells treated with the methylating agents, MMS and MNU. Although O$\sp6$-Bg did reduce viability and differentiation in cells treated with EMS, the effects were not as great as with the methylating agents, and they were not significant. ENU O$\sp6$-Bg only slightly decreased viability and differentiation. The last aspect of our study was to investigate the mode of cell death in neuronally differentiating P19 cells treated with alkylating agents, using both an ELISA to quantify cytosolic oligonucleosomes, and transmission electron microscopy to observe ultrastructural morphology. We observed significant concentration-dependent increases in cytosolic oligonucleosomes only for MNU and ENU. Additionally, with all four alkylating agents, increases in lethality were greater than increases in cytosolic oligonucleosomes. The ultrastructural morphology of treated cells showed morphologies characteristic of both apoptosis and necrosis, including condensation of chromatin and cytoplasm, apoptotic bodies, phagocytosis, increased numbers of lysosomes, and myelin bodies.