Cadmium-induced toxicity in rat primary mid-brain neuroglia cultures: role of oxidative stress from microglia

Abstract

This study examined the role of oxidative stress in neurotoxic effects of cadmium chloride (Cd) in rat primary mid-brain neuron-glia cultures. Cd accumulated in neuron-glia cultures and produced cytotoxicity in a dose-dependent manner, with IC(50) of 2.5microM 24 h after exposure. (3)H-dopamine uptake into neuron-glia cultures was decreased 7 days after Cd exposure, with IC(50) of 0.9microM, indicative of the sensitivity of dopaminergic neurons to Cd toxicity. To investigate the role of microglia in Cd-induced toxicity to neurons, microglia-enriched cultures were prepared. Cd significantly increased intracellular reactive oxygen species production in microglia-enriched cultures, as evidenced by threefold increases in 2',7'-dichlorofluorescein signals. Using 5,5-dimethyl-1-pyrroline N-oxide as a spin-trapping agent, Cd increased electron spin resonance signals by 3.5-fold in microglia-enriched cultures. Cd-induced oxidative stress to microglia-enriched cultures was further evidenced by activation of redox-sensitive transcription factor nuclear factor kappa B and activator protein-1 (AP-1), and the increased expression of oxidative stress-related genes, such as metallothionein, heme oxygenase-1, glutathione S-transferase pi, and metal transport protein-1, as determined by gel-shift assays and real-time reverse transcription-PCR, respectively, in microglia-enriched cultures. In conclusion, Cd is toxic to neuron-glia cultures, and the oxidative stress from microglia may play important roles in Cd-induced damage to dopaminergic neurons.

FIG. 1

Cytotoxicity by MTT assay (top) and cellular accumulation of Cd (bottom) in rat primary neuron-glia cultures 24-h posttreatment. The data are mean ± SEM from three independent experiments. *p < 0.05, compared to control.

FIG. 2

Toxicity of Cd to dopaminergic neurons in rat primary neuron-glia cultures 7-day posttreatment using ³H-dopamine uptake assay. The data are mean ± SEM from three independent experiments. *p < 0.05, compared to control.

FIG. 3

Effect of Cd on intracellular ROS generation in rat primary microglia-enriched cultures 2-h posttreatment. The data are mean ± SEM from three independent experiments. *p < 0.05, compared to control.

FIG. 4

Effect of Cd on DMPO–radical adduct formation in rat primary microglia-enriched cultures 1-h posttreatment by ESR analysis. (Top) Representative ESR spectra; (bottom) ESR signal intensity quantification from data of three independent experiments as mean ± SEM. *p < 0.05, compared to control.

FIG. 5

Effect of Cd on NF-κB (left) and AP-1 (right) activation in rat primary microglia-enriched cultures 6-h posttreatment by gel-shift assay. Lane 1, control, lane 2, LPS 1 ng/ml, lane 3, Cd 0.625μM, lane 4, Cd 2.5μM. The data represent one of three independent experiments.

FIG. 6

Effect of Cd on the expression of HO-1, MT-1, GST-pi, and MTP-1 in rat primary microglia-enriched cultures 24-h posttreatment using real-time RT-PCR. The data are expressed as mean ± SEM of three independent experiments. *p < 0.05, compared to control.