Cyanuric acid increases generation of hippocampal reactive oxygen species to induce apoptosis and synaptic impairments via inhibiting activation of the CaMKII/CREB pathway.

Cyanuric acid (CA) has attracted widespread toxicological concern due to reported cases of infant renal injury, with accumulating evidence confirming that CA induces oxidative stress and multi-organ toxicity. While the nephrotoxicity of CA has been extensively investigated, its detailed molecular mechanisms underlying central nervous system toxicity remain incompletely understood, particularly in comparison with its well-documented peripheral organ damage. Primary rat hippocampal neurons were treated with 25 or 50 μg/mL CA for 24 h, and only the 50 μg/mL CA treatment significantly increased intracellular ROS accumulation, reduced neuronal viability, and promoted late apoptosis. Mechanistically, CA triggered mitochondrial caspase-dependent apoptosis by upregulating Bax and cleaved caspase-3 while downregulating Bcl-2, and ROS scavenging with NAC effectively reversed these pro-apoptotic effects. CA selectively suppressed the CaMKII/CREB signaling cascade by decreasing total CaMKII and phosphorylated CaMKII (Thr286) as well as total CREB and phosphorylated CREB (Ser133), without altering total or phosphorylated PKA (Thr197), indicating pathway-specific inhibition mediated by excessive ROS. Pharmacological intervention further confirmed the core mechanism. CaMKII inhibition with KN-93 (10 μM) combined with CA did not exacerbate CA-induced injury beyond CA alone, supporting that CA and KN-93 act through the same pathway, whereas CREB activator forskolin (10 μM) rescued CA-mediated cell injury and apoptosis. In hippocampal slices, CA did not alter basal synaptic transmission or presynaptic release probability but significantly impaired Schaffer collateral-CA1 long-term potentiation (LTP), and both NAC and forskolin markedly restored CA-disrupted synaptic plasticity. In vivo, repeated CA exposure (40 mg/kg/day, i.p., 14 days) induced spatial learning and memory deficits in rats, which were substantially rescued by forskolin co-administration (15 mg/kg, i.p.). Collectively, these results demonstrate that CA triggers hippocampal neuronal apoptosis, synaptic dysfunction, and cognitive impairment through a ROS-mediated CaMKII/CREB suppression axis, revealing a previously unrecognized molecular mechanism underlying CA neurotoxicity and highlighting CREB activation as a potential protective strategy.