Elsevier

Toxicology Letters

Volume 148, Issues 1–2, 14 March 2004, Pages 91-94
Toxicology Letters

Primary cultures of microglial cells for testing toxicity of anticancer drugs

https://doi.org/10.1016/j.toxlet.2003.12.058Get rights and content

Abstract

Toxicity of anticancer agents on normal neural cells during chemotherapy of primary or secondary brain tumors is a clinical problem of increasing relevance and concern. In this perspective, here we used primary cultures of rat cortical microglia as an in vitro paradigm of normal glia to investigate the neurotoxicity of anticancer agents. The effects of two compounds frequently used for treatment of brain tumors, methotrexate (MTX) and temozolomide (TMZ), were compared to those of a known microglial activator, bacterial lipopolysaccharide (LPS); cell viability and metabolism was assessed by the MTS assay.

We found that LPS, in the low-intermediate range of concentrations, strongly activates microglia cells, but a highly significant decrease in viability was observed from 100 ng/ml onward. TMZ has no effect at concentrations of clinical interest, whereas MTX significantly increases cell metabolism at 30 μM, a phenomenon possibly reflecting MTX neurotoxicity observed in patients.

Introduction

The pharmacological treatment of primary or metastatic brain tumors is a cutting-edge field in modern medicine. The therapeutic approach to these disorders is biased by serious prognosis in the short period: criteria of drug choice are essentially limited to the consideration of pharmacokinetics parameters that may facilitate penetration and accumulation within the central nervous system (CNS). Drugs that easily cross the blood brain barrier by virtue of their liposolubility are more suitable for use. Among them are nitrosoureas, the first compounds shown to be active on brain tumors (Edwards et al., 1980), and temozolomide (TMZ), a novel methylating agent recently approved for the treatment of recurrent high grade gliomas (Yung et al., 1999). Moreover, TMZ has shown activity against CNS metastases from solid tumors and brain lymphoma (Biasco et al., 2001, Reni et al., 2000, Tentori et al., 2002). In addition, if high doses are adopted or the drug is given via intracerebroventricular (ICV) infusion, other less lipophylic compounds can be used, i.e. methotrexate (MTX).

When an antineoplastic agent is chosen, generally the sensitivity of cerebral tissue to its actions is poorly considered, nor is the fact that cytotoxic properties of the drug may affect not only neoplastic cells but also normal tissues surrounding the neoplasm. Neurotoxicity induced by antineoplastic agents during chemotherapy of brain tumors (Shapiro and Young, 1984) has been rarely considered as a problem in clinical research; however, it can be foreseen that this problem might become more relevant in the future, as a consequence of increased cytotoxic activity of the antineoplastic drugs and the awaited prognostic improvements of these patients.

In this light, we have used a previously validated model, i.e. primary cultures of rat cortical microglia (Vairano et al., 2002), as an in vitro paradigm of normal glial cell, and investigated the effects of treatments with anticancer agents frequently used in brain tumors, namely TMZ and MTX. Cell viability was assessed by the MTS [3-(4,5-dimethylthiazol-2-yl)-5-(4-sulfophenyl)2H-tetrazolium] test; MTS is a tetrazolium salt which is reduced to a colored formazan product by reducing enzymes present only in metabolically active, viable cells (Berridge and Tan, 1993). The effects of the above drugs in this paradigm were compared to that of bacterial lipopolysaccharide (LPS), a tool commonly used to activate microglia (Rivest, 2003).

Section snippets

Reagents

Lipopolysaccharide from Escherichia coli, serotype 026:B6, and methotrexate were obtained from Sigma Chemicals Co. (St. Louis, MO, USA); temozolomide was a kind gift from Schering-Plough Research Institute (Kenilworth, NY, USA).

Cell cultures

Cultures of purified cortical rat microglial cells were prepared from the cerebral cortex of 1–2 days old Wistar rats as previously described. The use of animals for this experimental work has been approved by the Italian Ministry of Health (licensed authorization to P.

Results and conclusions

Fig. 1A shows that LPS activates microglial cells with a unique dose-response pattern: a marked metabolic activity is associated to relatively low concentrations, whereas the metabolic activity of cells is reduced at higher concentrations, and is significantly lower than controls at 100 and 1000 ng/ml LPS. These variations do not appear to be associated to changes in cell proliferation, as the total protein content (Vairano et al., 2002) is not modified by LPS treatments. Moreover, the

Acknowledgements

Supported by a ‘Giovane Ricercatore’ grant to Cinzia Dello Russo on a project entitled: “Citotossicità gliale da antiblastici: valutazione del danno e meccanismi di neuroprotezione.”

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