Pyruvate affords protection from zinc toxic insults in the murine HT-22 hippocampal neuronal cell line Elizabeth V. Berry, Nicola L. Darvill and Nick J. Toms. School of Biomedical and Life Sciences, University of Surrey, Guildford, GU2 7XH.

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The putative neuromodulator Zn²⁺ is reported to be sequestered in glutamatergic hippocampal synaptic vesicles. During acute CNS injury (e.g. ischaemia) excessive glutamate receptor activation is known to induce neurodegeneration. However, increased extracellular Zn²⁺ levels may also participate in inducing neuronal cell death (Koh et al., 1996). The present study employed mouse hippocampal HT-22 cells to examine the mechanisms of Zn²⁺-induced hippocampal neurotoxicity.

HT-22 cells were cultured (5% CO₂, 37ºC) in 96 well plates (5 x 10³ cells well^-1) in DMEM / 10% FCS and exposed to either ZnSO₄ or staurosporine for up to 24 hours, together with the appropriate test compound of interest. Cell viability was then determined via monitoring methylthiazoletetrazolium bromide (MTT) reduction (Hansen et al., 1989). Additionally, lactate dehydrogenase (LDH) release was also employed to assess cell viability, using a commercially available kit (Roche). For the LDH release assay, cells were seeded in 24 well plates (4 x 10³ cells/cm²) and maintained in DMEM / 2% FCS. MTT reduction data are expressed as percentage mean (± s.e.m.) of untreated cellular MTT reduction. LDH release cell viability data are expressed as percentage mean (± s.e.m.) of total cellular LDH content as determined by 0.1% v/v Triton X-100 addition. All experiments were accompanied with a HEPES buffer (10 mM, pH 7.4) control. Statistical analyses were performed using either a one-way ANOVA (Dunnett's post test) or paired Student's t-test, where appropriate.

When tested over 24 h, Zn²⁺ induced widespread cell death (pEC₅₀ = -3.9 ± 0.1, n=6). Significant cell death occurred after 1 hr exposure to 200 µM Zn²⁺ (57.8% ± 3.7% MTT reduction, n=4, P<0.01). Neither the antioxidants, trolox (100 µM) nor N,N'-diphenyl-1,4-phenylenediamine (DPPD, 1 µM) afforded significant protection versus 200 µM Zn²⁺ insults (24 h, n=4). The broad-spectrum kinase inhibitor staurosporine (1 µM, 24 h) induced cell death (control = 2.3% ± 0.3% and staurosporine = 30.0% ± 5.8% total LDH released, n=4, P<0.01), an effect inhibited by the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone (z-VAD-FMK, 100 µM) (3.5% ± 1.8% total LDH released, n=4, P<0.05). In contrast, 100 µM z-VAD-FMK failed to afford significant protection against 200 µM Zn²⁺ insults (n=4). When examined over 24 hrs, either 5 mM or 10 mM pyruvate limited significantly 200 µM Zn²⁺-induced cytotoxicity (81.5% ± 13.2% MTT reduction, n=4, P<0.01 and 100.4% ± 11.6% MTT reduction, n=4, P<0.01 respectively). However, only 10 mM pyruvate (103.6% ± 15.6% MTT reduction, n=5, P<0.05) significantly limited a 6 h 200 µM Zn²⁺ insult (47.2% ± 7.7% MTT reduction, n=5).

We conclude that HT-22 cells are vulnerable to Zn²⁺ via a mechanism independent of caspase activation and cellular oxidative stress, but which may involve glycolytic inhibition.

Hansen, M.B. et al. (1989) J. Immunol. Meth. 119, 203-210.
Koh, J.Y. et al. (1996) Science 272, 1013-1016.

We wish to thank Dr P. Maher (The Scripps Research Institute, La Jolla, California) for donating the HT-22 cell line.