Heavy metals are widely used in industrial, agricultural, and technological applications, have become a significant threat to public health [1]. Even at low exposure levels, these metals can damage multiple organ systems [2]. Among commonly encountered heavy metals, arsenic, lead, mercury, and cadmium are classified among the top 10 most toxic substances according to the Substance Priority List of the Agency for Toxic Substances and Disease Registry [3]. These four metals are recognized as priority hazardous substances by the World Health Organization, owing to their high toxicity and persistence in the environment. They can enter the human body through inhalation, ingestion, and dermal absorption [4]. Once absorbed, heavy metals accumulate in various tissues and primarily affect the kidneys, lungs, liver, gastrointestinal tract, hematological system, and both the central and peripheral nervous system [5], and Among these, neurotoxicity has emerged as a major public health concern [4]. The excessive accumulation of heavy metals leads to the overproduction of reactive oxygen species (ROS), causing an imbalance in the redox state between pro-oxidants and antioxidants which is termed oxidative stress [6]. In addition, heavy metals exhibit a strong affinity for thiol groups present in enzymes and proteins essential for cellular defense, further impairing antioxidant capacity. Chronic exposure to these metals may ultimately lead to apoptotic cell death [7].

Importantly, exogenous neurotoxic heavy metals such as Pb²⁺ , Hg²⁺, As⁵⁺, As³⁺, and Cd²⁺, can penetrate the blood-brain barrier (BBB) and potentially interact with endogenous neurotoxic factors, such as glutamate, nitric oxide, and misfolded proteins, thereby exacerbating neurotoxicity [8].

Glutamate is the most abundant excitatory neurotransmitter in both the central and peripheral nervous systems and is also considered an endogenous neurotoxin. It plays an essential role in neuronal development, synaptic plasticity, and memory formation [5]. Glutamate exerts its effects through both synaptic and non-synaptic receptors. Among these, three families of ionotropic glutamate receptors (iGluRs) have been identified: N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and kainite receptors. These receptors are non-selective cation channels that mediate the influx of Na⁺, K⁺, and small amounts of Ca²⁺. Additionally, three groups of metabotropic glutamate receptors (mGluRs) act via G protein-coupled mechanisms to regulate glial and neuronal excitability [9]. However, excessive glutamate release from neurons and glial cells causes sustained neuronal depolarization, triggering a process known as excitotoxicity [10]. This pathological condition involves intracellular Ca²⁺ overload, overexpression of neuronal nitric oxide synthase (nNOS), mitochondrial dysfunction, increased ROS production, endoplasmic reticulum stress, and lysosomal enzyme activation [5].

Furthermore, glutamate accumulation disrupts the cystine/glutamate antiporter (Xc-), leading to reduced uptake of cystine (CySS), an essential precursor for glutathione (GSH) synthesis. This disruption results in GSH depletion, accumulation of free radicals, impaired Ca²⁺ homeostasis, and ultimately neuronal cell death [11]. Notably, Ca²⁺-independent oxidative glutamate toxicity through Xc- dysfunction can occur even in the absence of glutamate receptors [12]. This mechanism has been implicated in various neurological diseases, including Parkinson’s disease, Alzheimer’s disease, epilepsy, and ischemic brain injury [13].

The HT-22 cell line, derived from mouse hippocampal neurons, is widely used as an in vitro model to study oxidative glutamate toxicity independent of ionotropic glutamate receptors [14]. These immortalized cells possess characteristics similar to those of mature hippocampal neurons in vivo, including high responsiveness to glutamate and excitatory properties [15]. Although HT-22 cells lack functional iGluRs, they are highly sensitive to elevated levels of extracellular glutamate.

Exposure to glutamate induces oxidative cell death in HT-22 cells in a dose-and time-dependent manner, involving both necrotic and apoptotic pathways [16].

Drug combination studies involve the evaluation of two or more compounds administered at fixed doses or concentrations within a single formulation, considering differences in pharmacokinetics and physiological responses [17]. The interaction between drugs administered in combination can result in additive, synergistic, antagonistic, or independent effects, each of which can lead to significant biochemical changes in the body [18]. Although various studies have investigated the toxic effects of chemical mixtures on the skin, eyes, and respiratory system, only a limited number have quantitatively evaluated the effects of chemical combinations.

Notably, more than 95% of toxicological studies have focused on the effects of individual chemicals, often neglecting the potential interactions between compounds [19]. However, humans are frequently exposed to multiple chemicals, either simultaneously or sequentially, highlighting the importance of mixture-based approaches for exposure assessment, hazard identification, and risk evaluation [20]. Various analytical methods have been developed to assess chemical interactions. Among these, isobologram analysis and combination index (CI) method are two of the most widely recognized and reliable approaches for quantitative evaluation of synergistic, additive, or antagonistic effect of drug or chemical combinations [21, 22].

Although several studies have demonstrated the individual neurotoxic effects of heavy metals and glutamate, quantitative data assessing their combined interactions are lacking [23, 24].

Therefore, to elucidate the interaction mechanisms between heavy metals and glutamate, we aimed to (1) evaluate the individual and combined neurotoxic effects of heavy metals and glutamate in vitro, (2) quantitatively analyze their interaction patterns using combination analysis, and (3) assess their combined effects on reduced intracellular glutathione (GSH) levels.

In this study, we quantitatively evaluated the neurotoxic effects of combination of heavy metals and glutamate in HT-22 cells, a mouse hippocampal neuronal cell line.

Heavy metals are major environmental pollutants because of their high toxicity, widespread industrial use, and potential for bioaccumulation [28]. Natural sources, such as volcanic eruptions and Earth’s crust, along with anthropogenic activities, including mining, industrial operations, and agricultural practices, have significantly accelerated human exposure to heavy metals [29, 30].

The central nervous system is particularly vulnerable to metal-induced toxicity [28]. Among various heavy metals, Pb²⁺, Hg²⁺, As⁵⁺, As³⁺, and Cd²⁺ are the most commonly identified elements associated with adverse health effects. These metals can trigger a range of critical intracellular events, including oxidative stress, mitochondrial dysfunction, DNA fragmentation, endoplasmic reticulum stress, and activation of apoptotic pathways. Such abnormalities can disrupt normal neurophysiological functions and contribute to the development of neurodegenerative diseases, including Alzheimer’s disease, amyotrophic lateral sclerosis, autism spectrum disorder, and Parkinson’s disease [31]. Once these metals enter the human body through inhalation, ingestion, or dermal contact, they are capable of crossing the BBB and interact with endogenous neurotoxic molecules [32].

Despite being the most abundant excitatory amino acid involved in various essential metabolic and neurotransmission pathways, glutamate is one of the major endogenous neurotoxins in the brain. However, excessive release of glutamate from both neurons and glial cells can initiate a form of cell death known as excitotoxicity [31]. Furthermore, elevated extracellular glutamate levels can reverse the function of the cystine/glutamate antiporter system Xc-, thereby inhibiting cystine uptake. This leads to a reduction in intracellular GSH levels and induces a form of cell injury known as oxidative glutamate toxicity [33].

Additionally, a recent study has reported that monosodium L-glutamate (MSG), commonly used as a food additive to enhance the umami flavor, may induce neurodegeneration in certain brain regions and trigger neurochemical and behavioral alterations [34]. Although numerous studies have investigated the neurotoxic effects of individual heavy metals and glutamate, quantitative evaluation of their combined interactions, particularly in hippocampal neuronal cell lines, remains limited. Most contemporary toxicological research and health risk assessments continue to focus on individual chemical effects and often overlook the potential risks posed by combined or interactive exposures [35].

Pb²⁺, Hg²⁺, As⁵⁺, As³⁺, and Cd²⁺ are systemic toxicants that can damage multiple organs even at low levels of exposure [36].

In this study, we investigated the neurotoxic effects of individual and combined exposures to heavy metals and glutamate in a hippocampal neuronal cell line. Our results demonstrated that cell viability decreased in a concentration-dependent manner for both glutamate and each heavy metal when applied individually. Notably, co-exposure to glutamate and heavy metals at both low concentration ratio [LCR; Glu: HM = 1:0.0025] and high concentration ratio [HCR; Glu: HM = 1:0.025] concentration ratios resulted in a more pronounced reduction in cell viability than that of individual treatments. We selected the Glu: HM concentration ratios of 1:0.0025 and 1:0.025 based on IC values (inhibitory concentration) of cell viability data from our MTT assay for glutamate and each of the heavy metals. We used the combined concentrations within the IC10 to IC30 range, which is essential to evaluate any potential synergistic or additive interactions while preventing excessive cytotoxicity that could obscure mild toxicological effects. Specifically, the low concentration ratio refers to the IC10 of glutamate and each heavy metals, allowing assessment under subthreshold ranges, whereas the 1:0.025 ratio investigates interactions at higher concentration of heavy metals which fall in a steep range in dose-response curves.

In the environment, chemical toxicities rarely occur in isolation but typically exist as complex mixtures. Certain chemicals can enhance the toxic potential of others, resulting in greater toxicity than expected from individual exposure alone [37]. Therefore, it is essential to conduct quantitative analyses of these chemical mixtures to better understand their combined toxicological effects.

Accordingly, we employed a mathematically validated and widely used pharmacodynamic/ toxicodynamic method to evaluate the chemical interactions, known as the median-effect and CI model, based on the isobologram equation, a methodology that has been extensively applied in many studies to evaluate chemical-chemical interaction [38-41]. This approach allows for the quantitative assessment of interaction types, where CI values less than 1 indicate synergism, equal to 1 indicate additivity, and greater than 1 indicate antagonism [26].

Median-effect and CI analyses revealed that at Fa levels of 0.25, 0.5, and 0.75, the combination of Pb²⁺ and glutamate exhibited effects ranging from near-additive to synergistic under LCR condition, and clear synergistic effects under HCR condition. Consistently, isobologram analysis at Fa = 0.5 level also demonstrated synergistic effects between Pb²⁺ and glutamate under both LCR and HCR conditions. Therefore, we conclude that Pb²⁺ exhibits a consistent synergistic effect with glutamate.

Pb²⁺ is a well-known systemic toxicant that poses serious health risks to both adults and children [42]. In particular, Pb²⁺ exerts significant neurotoxic effects owing to its strong ability to cross the BBB, which is facilitated by its capacity to substitute for calcium ions. Once in the brain, Pb²⁺ accumulates in regions such as the prefrontal cortex, hippocampus, and cerebellum, leading to neuronal injury that can contribute to a range of neurological disorders, including behavioral impairments, neurodevelopmental delays, and brain atrophy associated with Alzheimer’s and Parkinson’s diseases [43].

Naturally present in the Earth’s crust, lead contamination arises from both natural and anthropogenic sources such as mining, smelting, manufacturing, and recycling processes. Pb²⁺ is still widely used in consumer products such as jewelry, toys, paints, stained glass, and certain forms of traditional medicine. According to the World Health Organization, there is no known safe blood lead concentration, and levels as low as 3.5 μg/dL have been associated with reduced intelligence, behavioral problems, and learning deficits in children [44].

Reduced GSH, a ubiquitous tripeptide thiol, is one of the key antioxidant involved in both intracellular and extracellular defense mechanisms, as well as in maintaining redox homeostasis in neurons [35]. Decreased GSH levels in the brain have been implicated in the pathogenesis of several neurodegenerative diseases [45, 46].

Based on these findings, we investigated the effects of individual and combined exposure to Pb²⁺ and glutamate on GSH levels. Our results showed that co-treatment with Pb²⁺ and glutamate at a low concentration ratio significantly decreased GSH levels in hippocampal neuronal cells.

Co-exposure to heavy metals (Pb²⁺, Hg²⁺, As⁵⁺, As³⁺, and Cd²⁺) and glutamate resulted in greater neurotoxicity than individual exposures. Among the tested metals, Pb²⁺ exhibited consistent synergistic effects with glutamate under both LCR and HCR. In contrast, Hg²⁺ showed antagonistic effects under LCR and synergistic effects under HCR, while As⁵⁺ exhibited antagonism at LCR and additivity at HCR. As³⁺ and Cd²⁺ demonstrated antagonistic effects with glutamate under both conditions. Notably, co-treatment with Pb²⁺ and glutamate significantly reduced intracellular GSH levels in the hippocampal neuronal cell line, suggesting enhanced oxidative stress under combined exposure.