Kelulut honey (Heterotrigona itama) as a multi-target neuroprotective strategy against bisphenol A-induced neurotoxicity

Abstract

Bisphenol A (BPA) is a synthetic chemical widely used in the production of plastics and epoxy resins due to its low cost, durability, and heat resistance.Recognised as an endocrine-disrupting chemical, BPA has raised growing concern regarding its potential effects on brain development, particularly during prenatal and early postnatal life. BPA-induced neurotoxicity involves multiple interconnected mechanisms, including oxidative stress, neuroinflammation, mitochondrial dysfunction, synaptic impairment, and neuroendocrine disruption, which collectively contribute to cognitive and behavioural abnormalities. Kelulut honey, produced by stingless bees (Heterotrigona itama), contains various bioactive compounds such as polyphenols, flavonoids, organic acids, and trehalulose. These compounds possess antioxidant, anti-inflammatory, and neuroprotective properties and may modulate pathways involved in neuronal survival, synaptic plasticity, inflammatory regulation, NMDA receptor signalling, and estrogen-related pathways. This narrative review summarises current findings on the neuroprotective potential of kelulut honey against BPA-induced neurotoxicity, highlights existing research gaps, and discusses future directions for further mechanistic and translational studies.

Graphical Abstract

Highlights

• •BPA exposure occurs through dietary intake, dermal absorption, inhalation, and environmental contamination.
• •BPA-induced neurotoxicity involves oxidative stress, mitochondrial dysfunction, neuroinflammation, and endocrine disruption.
• •BPA disrupts synaptic plasticity, calcium signalling, neurotransmitter balance, and BDNF-associated pathways.
• •Heterotrigona itama honey demonstrates promising neuroprotective and anti-inflammatory effects owing to its abundance of bioactive molecules.
• •H. itama honey may exert multitarget neuroprotective effects against BPA-induced cognitive and neuronal dysfunction.

1. Introduction

Cognitive and neurodevelopmental disorders associated with environmental toxicant exposure have become an increasing global health concern. Among environmental contaminants, bisphenol A (BPA) has garnered considerable attention due to its widespread use in polycarbonate plastics and epoxy resins. BPA is a synthetic endocrine-disrupting chemical (EDC) capable of mimicking endogenous oestradiol and interfering with hormonal signalling pathways [1], [2], [3]. Since BPA exposure is pervasive and difficult to avoid, concerns regarding its long-term effects on neurodevelopment and cognitive function continue to rise. Pregnant women and developing fetuses are particularly vulnerable to BPA exposure as BPA can cross the placental barrier and undergo maternal-fetal transfer [4], [5], [6]. Increasing evidence suggests that prenatal and early-life BPA exposure may contribute to persistent cognitive and behavioural abnormalities. Among the affected brain regions, the hippocampus appears especially vulnerable because of its essential role in learning, memory formation, and synaptic plasticity during neurodevelopment.

Current evidence indicates that BPA-induced neurotoxicity involves multiple interconnected mechanisms rather than oxidative stress alone. BPA exposure has been shown to stimulate excessive reactive oxygen species (ROS) generation, thereby disrupting redox homeostasis and contributing to mitochondrial dysfunction, neuroinflammation, synaptic impairment, and neuronal degeneration [4], [7], [8]. BPA has also been associated with disruption of neurotransmitter homeostasis, calcium signalling, neurotrophic regulation, and synaptic plasticity, ultimately contributing to impairments in learning and memory [9], [10], [11]. Emerging evidence indicates that BPA-induced neurotoxicity is mediated through complex molecular mechanisms involving endocrine disruption, mitochondrial dysfunction, apoptosis, autophagy dysregulation, inflammasome activation, and perturbation of neuronal signalling pathways linked to cognitive regulation and neuronal integrity, including Nrf2, NF-κB, PI3K/Akt, BDNF/CREB, and mTOR pathways [4], [9], [12], [13]. Since these pathways are closely interconnected, therapeutic approaches capable of regulating multiple neurotoxic mechanisms may provide broader neuroprotective benefits during neurodevelopmental injury.

Due to the limitations and potential adverse effects linked with current therapeutic interventions, increasing attention has been directed toward natural bioactive compounds with neuroprotective potential. Among these, Heterotrigona itama honey has attracted growing interest due to its rich composition of polyphenols, flavonoids, phenolic acids, organic acids, and trehalulose [14], [15], [16]. Previous studies demonstrated that H. itama honey possesses antioxidant, anti-inflammatory, cytoprotective, and neuromodulatory properties [15], [17], [18]. Existing studies further suggests that H. itama honey may regulate redox homeostasis, preserve synaptic plasticity, enhance neurotrophic signalling, and modulate pathways associated with neuronal survival, including Nrf2-mediated antioxidant defence, NF-κB inflammatory signalling, ERK/CREB/BDNF signalling, and mTOR-associated pathways [17], [18], [19]. Importantly, these pathways substantially overlap with mechanisms involved in BPA-induced neurotoxicity, suggesting that H. itama honey may exert multitarget neuroprotective effects against environmentally induced neuronal injury.

Although previous studies have separately discussed BPA-induced neurotoxicity and the biological activities of natural products, the mechanistic relationship between BPA-induced cognitive dysfunction and the neuroprotective effects of H. itama honey remains poorly understood. Therefore, this review hypothesises that H. itama honey may attenuate BPA-induced neurotoxicity through coordinated regulation of oxidative stress, neuroinflammation, mitochondrial dysfunction, endocrine disruption, synaptic impairment, and neurotrophic dysregulation. This review aims to critically evaluate the molecular and neurobiological mechanisms underlying BPA-induced cognitive dysfunction and to summarise current evidence regarding the potential neuroprotective effects of H. itama honey against environmentally induced neurodevelopmental and cognitive impairment.

2. Neurotoxicity effects of BPA

2.1. BPA exposure: sources and pathways

Due to its extensive application in polycarbonate plastics and epoxy resin manufacturing, BPA is frequently detected in food and beverages as a result of migration from packaging materials and containers [3], [4]. Dietary intake, therefore, represents the primary route of human exposure. Additional exposure may occur through dermal absorption and inhalation, particularly in occupational settings involving plastics, food packaging, and thermal paper handling [5], [20]. Importantly, BPA can cross both the placental and blood-brain barriers, exposing the developing brain during critical stages of neurodevelopment [21], [22]. Since prenatal and early-life exposure has been associated with persistent cognitive and behavioural abnormalities, BPA exposure remains an important concern in neurodevelopmental toxicology. Fig. 2.1 summarises the major routes of BPA exposure and their potential effects on neurodevelopment and cognitive function.

Fig. 2.1.

Major routes of BPA exposure and their potential effects on neurodevelopment and cognitive function.

2.2. Molecular mechanisms of BPA-induced neurotoxicity

BPA-induced neurotoxicity involves multiple interconnected mechanisms that collectively impair neuronal survival, synaptic plasticity, and cognitive regulation. Current evidence indicates that BPA disrupts endocrine signalling, redox homeostasis, mitochondrial function, neuroinflammatory pathways, synaptic communication, autophagy regulation, and epigenetic stability, ultimately contributing to cognitive and behavioural abnormalities [4], [9], [23].

As a xenoestrogen, BPA structurally mimics oestradiol and interacts with estrogen receptors (ERα and ERβ), androgen receptors, and the G protein-coupled estrogen receptor (GPER30), thereby interfering with hormonal signalling pathways involved in neuronal differentiation, synaptic maturation, and hippocampal development [2], [24], [25]. BPA exposure has also been linked with disruption of the hypothalamic-pituitary-gonadal (HPG) axis and altered neurotransmitter regulation during neurodevelopment [4].

Oxidative stress and mitochondrial dysfunction are recognised as major contributors to BPA-induced neuronal injury. BPA exposure induces ROS production, impairs antioxidant defence systems, disrupts mitochondrial membrane potential, suppresses ATP production, and promotes abnormal autophagic activity, thereby increasing neuronal vulnerability to apoptosis and synaptic dysfunction [12], [13], [26], [27]. BPA has also been associated with suppression of protective signalling pathways, including Nrf2/HO−1 and AKT/mTOR, which further impairs cellular stress adaptation and neuronal survival [12], [13].

In addition, BPA activates neuroinflammatory pathways through microglial activation and amplifies the production of pro-inflammatory mediators, including IL−1β, TNF-α, and COX−2 [28], [29].Emerging evidence further suggests that BPA-induced neurotoxicity involves epigenetic dysregulation, including altered DNA methylation, histone modification, and microRNA expression, which may contribute to persistent abnormalities in neuronal signalling and cognitive function [6], [30], [31].

Overall, BPA-induced neurotoxicity arises through coordinated disruption of interconnected oxidative, inflammatory, mitochondrial, endocrine, and synaptic pathways, ultimately contributing to neuronal dysfunction and cognitive impairment. Fig. 2.2 summarises the interconnected molecular and cellular mechanisms involved in BPA-induced neurotoxicity and their contribution to neuronal dysfunction and cognitive impairment.

Fig. 2.2.

Interconnected molecular and cellular mechanisms involved in BPA-induced neurotoxicity and cognitive dysfunction. BPA exposure disrupts endocrine signalling, redox homeostasis, mitochondrial function, neuroinflammatory pathways, neuronal signalling, synaptic plasticity, autophagy regulation, and epigenetic stability, ultimately contributing to neuronal damage, cognitive impairment, and behavioural abnormalities.

2.3. Synaptic dysfunction and cognitive impairment following BPA exposure

The developing brain is highly vulnerable to BPA exposure because BPA can cross both the placental and blood-brain barriers during critical stages of neurodevelopment [21], [22]. Prenatal and early-life exposure has been associated with persistent cognitive and behavioural abnormalities, including deficits in learning, memory, attention, and emotional regulation [32], [33]. These neurodevelopmental effects appear to involve disruption of neuronal differentiation, synaptic maturation, and neural circuit formation within the hippocampus and cerebral cortex [4], [21].

Among the affected brain regions, the hippocampus appears particularly susceptible to BPA-induced neurotoxicity due to its essential role in synaptic plasticity and memory formation. Experimental studies demonstrated that developmental BPA exposure alters dendritic morphology, reduces dendritic spine density, impairs long-term potentiation (LTP), and disrupts glutamatergic neurotransmission, ultimately contributing to deficits in spatial learning and memory [34], [35]. At the molecular level, BPA exposure downregulates NMDA receptor subunits, including GluN2A and GluN2B, together with synaptic proteins such as PSD−95 and synapsin I, which are essential for synaptic maturation and neuronal communication [35], [36]. BPA-induced disruption of NMDA receptor signalling may further impair calcium-dependent synaptic plasticity and memory consolidation through altered CaMKII-associated pathways [34], [35].

In addition to synaptic dysfunction, BPA exposure disrupts neurotransmitter homeostasis by increasing glutamate levels while reducing GABA, serotonin, and dopamine signalling within the hippocampus and prefrontal cortex [28], [37]. BPA exposure has also been associated with reduced expression of GABA-related enzymes and transporters, including GAD67, GABA-T, and GAT−1, suggesting impaired inhibitory neurotransmission and increased neuronal excitability [37]. These alterations may contribute to excitotoxicity, synaptic instability, and behavioural dysregulation.

BPA-induced cognitive dysfunction is also closely associated with suppression of neurotrophic signalling pathways involved in neuronal survival and synaptic maintenance. Several studies have demonstrated reduced BDNF expression together with impaired ERK/CREB and BDNF/TrkB signalling following developmental BPA exposure [38], [39]. In addition, altered expression of microRNAs associated with calcium signalling and synaptic plasticity has been reported, suggesting that epigenetic dysregulation may contribute to persistent synaptic abnormalities and long-term cognitive vulnerability [40].

Collectively, current evidence suggests that BPA-induced cognitive impairment arises through coordinated disruption of synaptic signalling, neurotransmitter balance, calcium homeostasis, neurotrophic regulation, and neuronal connectivity. Since these mechanisms are closely interconnected with oxidative stress and mitochondrial dysfunction, therapeutic approaches capable of modulating multiple neurotoxic pathways may provide broader neuroprotective effects during neurodevelopmental injury. In this context, Heterotrigona itama honey has gained increasing interest because its phenolic and flavonoid compounds may regulate antioxidant defence, neuroinflammatory signalling, mitochondrial function, and neurotrophic pathways associated with synaptic plasticity and neuronal survival [41], [42], [43], [44].

Table 2.1 summarises the major interconnected mechanisms underlying BPA-induced neurotoxicity and their associated cognitive and behavioural outcomes.

Table 2.1.

Major molecular and cellular mechanisms involved in BPA-induced neurotoxicity and their associated cognitive and behavioural consequences.

BPA-induced neurotoxic mechanisms	Key molecular alterations	Main affected brain region(s)	Cognitive and behavioural consequences	Key references
Endocrine disruption	Dysregulation of ERα/ERβ, androgen receptor, GPER30, and HPG axis signalling	Developing brain, hippocampus, cerebral cortex	Abnormal neurodevelopment and sex-dependent cognitive dysfunction	Costa, Cairrao [2], [4], [24]
Oxidative stress	Increased ROS production, lipid peroxidation, impaired antioxidant defence, and suppression of Nrf2/HO−1 signalling	Hippocampus, cerebral cortex	Oxidative neuronal injury and memory impairment	Lee et al., [12], [13], [27]
Mitochondrial dysfunction	Disrupted mitochondrial membrane potential, reduced ATP production, accumulation of dysfunctional mitochondria, and abnormal autophagic activity	Hippocampal neurons	Synaptic dysfunction, neuronal apoptosis, and cognitive deficits	Meng et al., [13], [26]
Neuroinflammation	Microglial activation and increased IL−1β, TNF-α, and COX−2 production through NF-κB-associated pathways	Hippocampus, prefrontal cortex	Neuroinflammation, anxiety-like behaviour, and cognitive decline	Al-Shami et al., [28], [29]
Synaptic dysfunction	Reduced GluN2A/GluN2B, PSD−95, and synapsin I expression, impaired NMDA receptor signalling and CaMKII-associated pathways, disrupted LTP	Hippocampus	Impaired synaptic plasticity, learning deficits, and memory impairment	Hu et al., [34], [35], [36]
Neurotransmitter imbalance	Increased glutamate levels with reduced GABA, serotonin, and dopamine signalling; altered GAD67, GABA-T, and GAT−1 expression	Hippocampus, prefrontal cortex	Excitotoxicity, impaired inhibitory neurotransmission, and behavioural dysregulation	Al-Shami et al., [28], [37]
Neurotrophic signalling impairment	Reduced BDNF expression and impaired ERK/CREB and BDNF/TrkB signalling	Hippocampus	Reduced neuroplasticity and impaired memory consolidation	Hyun et al., [38], [39]
Autophagy and mTOR dysregulation	Impaired AKT/mTOR signalling, disrupted autophagic activity, and impaired cellular stress adaptation	Hippocampus, dentate gyrus	Increased neuronal vulnerability and impaired neuronal survival	Lee et al., [12], [13]
Epigenetic dysregulation	Altered DNA methylation, histone modification, and microRNA expression associated with calcium signalling and synaptic plasticity	Developing hippocampus	Persistent synaptic abnormalities and long-term cognitive vulnerability	Engdahl et al., [30], [31], [40]

Abbreviations: AKT, protein kinase B; ATP, adenosine triphosphate; BDNF, brain-derived neurotrophic factor; BPA, bisphenol A; CaMKII, calcium/calmodulin-dependent protein kinase II; CAT, catalase; COX−2, cyclooxygenase−2; ERα, estrogen receptor alpha; ERβ, estrogen receptor beta; ERK, extracellular signal-regulated kinase; GABA, gamma-aminobutyric acid; GABA-T, gamma-aminobutyric acid transaminase; GAD67, glutamate decarboxylase 67; GAT−1, gamma-aminobutyric acid transporter−1; GPER30, G protein-coupled estrogen receptor 30; HPG axis, hypothalamic-pituitary-gonadal axis; IL−1β, interleukin−1 beta; LTP, long-term potentiation; MAPK, mitogen-activated protein kinase; mTOR, mammalian target of rapamycin; NF-κB, nuclear factor kappa B; Nrf2/HO−1, nuclear factor erythroid 2-related factor 2/heme oxygenase−1; NMDA, N-methyl-D-aspartate; PSD−95, postsynaptic density protein−95; ROS, reactive oxygen species; SOD, superoxide dismutase; TNF-α, tumour necrosis factor alpha.

3. Neuroprotective potential of heterotrigona itama honey

3.1. Bioactive composition relevant to neuroprotection

Natural products enriched with bioactive compounds have gained increasing attention as potential neuroprotective agents due to their antioxidant, anti-inflammatory, cytoprotective, and neuromodulatory properties. Among these, stingless bee honey, particularly Heterotrigona itama honey, has emerged as a promising functional food with potential relevance in neurodevelopmental and neurodegenerative disorders [15], [43], [45]. Several preclinical studies demonstrated that H. itama honey may preserve hippocampal neuronal integrity, improve cognitive performance, and regulate pathways associated with neuronal survival and synaptic plasticity [42], [43], [46].

The neuroprotective potential of H. itama honey is largely attributed to its rich phenolic and flavonoid composition. Several bioactive compounds identified in stingless bee honey, including quercetin, kaempferol, apigenin, gallic acid, caffeic acid, and catechin, possess strong antioxidant and anti-inflammatory properties [41], [43], [47].

In addition to direct free radical scavenging activity, these compounds may regulate intracellular pathways involved in oxidative stress responses, mitochondrial homeostasis, neuroinflammation, and neuronal signalling. Previous studies have demonstrated that stingless bee honey enhances endogenous antioxidant defence systems through activation of the KEAP1-NRF2/HO−1 pathway, together with increased expression of antioxidant enzymes such as superoxide dismutase (SOD−1) and heme oxygenase−1 (HO−1) [44]. Since oxidative stress and mitochondrial dysfunction are major contributors to BPA-induced neurotoxicity, modulation of these pathways may help preserve neuronal survival and synaptic integrity during neurodevelopmental stress.

Several molecular pathways associated with BPA-induced neurotoxicity also overlap with pathways potentially regulated by H. itama honey. Current evidence suggests that stingless bee honey may modulate Nrf2-mediated antioxidant defence, NF-κB-associated inflammatory signalling, MAPK/ERK pathways, and BDNF-related neurotrophic regulation [42], [43], [44]. These pathways are closely involved in neuronal resilience, synaptic plasticity, mitochondrial function, and cognitive regulation. Importantly, the biological activity of H. itama honey may extend beyond general antioxidant activity because several of its phenolic compounds have been associated with the regulation of cellular stress adaptation and neuronal signalling pathways under oxidative and inflammatory conditions.

Nevertheless, despite increasingly promising findings, direct evidence evaluating the protective effects of H. itama honey specifically against BPA-induced neurotoxicity remains limited. Most currently available studies primarily investigated general antioxidant, anti-inflammatory, or cognitive effects in non-BPA experimental models. Consequently, the precise molecular mechanisms underlying H. itama-mediated neuroprotection during BPA-induced neurodevelopmental injury remain incompletely understood. Further mechanistic studies integrating behavioural, molecular, synaptic, and neurochemical analyses are therefore required to clarify its therapeutic relevance in BPA-associated neurotoxicity.

3.2. Phenolic compounds and antioxidant regulation

Current evidence suggests that the neuroprotective potential of H. itama honey extends beyond general antioxidant activity and may involve coordinated regulation of multiple pathways associated with BPA-induced neurotoxicity. Since BPA-induced cognitive dysfunction is mediated through interconnected oxidative, mitochondrial, inflammatory, synaptic, and neuroendocrine mechanisms [4], [23], modulation of these pathways may contribute to preservation of neuronal survival and cognitive function during neurodevelopmental injury.

One of the major mechanisms potentially involved in H. itama-mediated neuroprotection is regulation of oxidative stress and mitochondrial homeostasis. Several phenolic and flavonoid compounds identified in stingless bee honey, including quercetin, kaempferol, apigenin, and caffeic acid, have been associated with activation of endogenous antioxidant defence pathways, particularly KEAP1-NRF2/HO−1 signalling [41], [44]. Activation of these pathways may help attenuate excessive ROS accumulation, preserve mitochondrial membrane integrity, and reduce neuronal vulnerability during oxidative stress conditions. Since mitochondrial dysfunction and impaired antioxidant responses are major contributors to BPA-induced neurotoxicity, preservation of mitochondrial homeostasis may represent an important component of the neuroprotective effects of H. itama honey.

In addition to oxidative stress regulation, H. itama honey may also modulate neuroinflammatory pathways associated with BPA-induced neuronal injury. Several studies demonstrated that stingless bee honey and its bioactive compounds suppress NF-κB-associated inflammatory signalling and reduce production of pro-inflammatory mediators under oxidative and inflammatory conditions [43], [44]. Since persistent neuroinflammation contributes to microglial activation, synaptic dysfunction, and neuronal damage following BPA exposure [28], attenuation of inflammatory signalling may help preserve neuronal integrity and synaptic communication.

A growing body of evidence further suggests that stingless bee honey may influence pathways involved in synaptic plasticity and neurotrophic regulation. Previous studies reported increased brain-derived neurotrophic factor (BDNF) expression and improved cognitive performance following stingless bee honey supplementation in experimental models [42], [45], [48]. Since BPA-induced cognitive dysfunction has been associated with dysregulation of the ERK/CREB/BDNF signalling pathway [38], [39], modulation of these pathways may contribute to preservation of synaptic function during neurodevelopmental stress.

Nevertheless, despite increasingly promising findings, direct mechanistic evidence specifically evaluating the effects of H. itama honey in BPA-induced neurotoxicity remains limited. Most available studies primarily investigated general oxidative stress, inflammation, or cognitive dysfunction in broader experimental models rather than direct prenatal BPA exposure paradigms. Therefore, while current findings support the possibility that H. itama honey may exert multitarget neuroprotective effects against BPA-induced neuronal injury, further mechanistic studies integrating behavioural, molecular, synaptic, and neurochemical analyses are still required to clarify its precise therapeutic relevance. Fig. 3.1 summarises the proposed neuroprotective mechanisms of Heterotrigona itama honey against BPA-induced neurotoxicity through coordinated regulation of oxidative stress, mitochondrial dysfunction, neuroinflammation, synaptic impairment, and neuronal survival pathways.

Fig. 3.1.

Proposed neuroprotective mechanisms of Heterotrigona itama honey against BPA-induced neurotoxicity. Bioactive compounds present in H. itama honey may attenuate oxidative stress, mitochondrial dysfunction, neuroinflammation, inflammasome activation, synaptic impairment, and neuronal injury through coordinated regulation of antioxidant, neurotrophic, and neuronal survival pathways, thereby contributing to preservation of cognitive function and hippocampal integrity.

3.3. Experimental evidence of neuroprotective and cognitive benefits

Accumulating evidence suggests that the neuroprotective effects of H. itama honey extend beyond general antioxidant activity and may involve modulation of pathways associated with neuronal survival, synaptic plasticity, neuroinflammation, and cognitive regulation [41], [43]. This is particularly relevant in BPA-induced neurotoxicity, where neuronal injury results from interconnected mechanisms involving oxidative stress, mitochondrial dysfunction, glutamatergic dysregulation, endocrine disruption, and synaptic impairment [4], [23]. Since these pathways collectively contribute to cognitive dysfunction, preservation of neuronal signalling and synaptic integrity may be equally important as antioxidant regulation in limiting neurodevelopmental injury.

Several experimental studies indirectly support the neuroprotective potential of H. itama honey in conditions associated with neuronal dysfunction and cognitive impairment. Mustafa et al. [46] demonstrated that dietary supplementation with H. itama honey promoted better spatial memory performance in mice and concurrently increased the expression levels of BDNF and Itpr1. Similarly, Zulkifli et al. [43] suggested that stingless bee honey may modulate neurotrophic and synaptic signalling pathways involved in learning and memory processes. Given the crucial role of BDNF signalling in neuronal differentiation, synaptic maturation, and hippocampal plasticity [38], [39], these findings indicate the potential of stingless bee honey to enhance neuronal resilience against neurotoxic insults.

Comparable findings have also been reported in pathological models associated with oxidative stress, excitotoxicity, and neurodegeneration. In metabolic syndrome-induced rats, stingless bee honey supplementation improved spatial memory performance, reduced anxiety-like behaviour, and preserved hippocampal neuronal integrity [45], [49]. In addition, Zulkifli et al. [43] demonstrated improved spatial memory and increased hippocampal neuronal viability following stingless bee honey administration in kainic acid-induced seizure models. Since seizure-associated neurotoxicity involves excitotoxicity, mitochondrial dysfunction, and oxidative stress [43], these findings may suggest that stingless bee honey helps preserve neuronal homeostasis and protect against neuronal injury.

Several studies also suggest that stingless bee honey may influence neurochemical and inflammatory pathways associated with behavioural dysfunction. Shaheran et al. [42] reported that stingless bee honey supplementation increased BDNF expression, maintained serotonin and dopamine balance, reduced pro-inflammatory cytokine levels, and preserved hippocampal neuronal morphology under chronic restraint stress conditions. These findings were accompanied by improvements in anxiety- and depression-like behaviours, suggesting possible involvement of neurotrophic and neurochemical regulatory pathways. In addition, findings by Cheng et al. [44] revealed that stingless bee honey and its phenolic-rich extract restored oxidative balance through KEAP1-NRF2 pathway activation and enhanced expression of antioxidant-related proteins, particularly SOD−1 and HO−1.

The neuroprotective relevance of stingless bee honey has also been explored in neurodegenerative disease models. Shaikh et al. [50] demonstrated that kelulut honey reduced amyloid plaque accumulation, phosphorylated tau expression, neuronal apoptosis, and hippocampal histopathological damage in amyloid-β-induced rats. Similarly, Shajahan et al. [51] reported improvements in cognition, neurotransmitter balance, locomotor behaviour, and neuronal preservation following stingless bee honey administration in aluminium chloride and D-galactose-induced neurodegeneration models. Interestingly, several studies observed histological and behavioural improvement despite only modest changes in oxidative stress or inflammatory biomarkers, suggesting that the neuroprotective effects of stingless bee honey may involve selective modulation of interconnected neuronal pathways rather than simple antioxidant activity alone.

Nevertheless, despite increasingly promising findings, direct mechanistic evidence specifically investigating the effects of H. itama honey against BPA-induced neurotoxicity remains limited. Most available evidence is derived from broader neurological or metabolic disease models rather than from direct developmental BPA exposure paradigms. Therefore, although current findings support the possibility that H. itama honey exerts multitarget neuroprotective effects involving antioxidant regulation, neuroinflammatory suppression, neurotrophic signalling, and preservation of neuronal integrity, further mechanistic studies integrating behavioural, molecular, neurochemical, and synaptic analyses are still required to clarify its therapeutic relevance in BPA-associated neurodevelopmental dysfunction. Table 3.1 summarises the major bioactive compounds and proposed neuroprotective mechanisms of H. itama honey relevant to pathways involved in BPA-induced neurotoxicity.

Table 3.1.

Bioactive compounds and proposed neuroprotective mechanisms of Heterotrigona itama honey relevant to BPA-induced neurotoxicity.

Bioactive compounds/components	Proposed molecular targets and pathways	Reported neuroprotective effects	Key References
Quercetin	Activation of KEAP1-NRF2/HO−1 signalling, regulation of oxidative stress and mitochondrial homeostasis	Reduces oxidative stress, preserves mitochondrial integrity, and supports neuronal survival	Cheng et al., [41], [44]
Kaempferol	Modulation of antioxidant defence and inflammatory signalling pathways	Attenuates oxidative and inflammatory neuronal injury	Fadzil et al., [41], [43]
Apigenin	Regulation of antioxidant and neurotrophic signalling pathways	Supports synaptic plasticity and neuronal resilience	Fadzil et al., [41], [42]
Caffeic acid	Regulation of ROS accumulation and inflammatory responses	Protects against oxidative stress-associated neuronal dysfunction	Cheng et al., [44], [47]
Catechin	Antioxidant and cytoprotective activity	Reduces oxidative neuronal damage and supports cellular homeostasis	Shamsudin et al., [43], [47]
Gallic acid	Modulation of oxidative stress and inflammatory pathways	Protects neuronal integrity and reduces cellular stress	Fadzil et al., [41], [47]
Phenolic-rich extract	Activation of KEAP1-NRF2/HO−1 signalling and increased antioxidant enzyme expression (SOD−1, HO−1)	Improves oxidant-antioxidant balance and neuronal stress adaptation	Cheng et al., [44]
Neurotrophic pathway modulation	Regulation of ERK/CREB/BDNF-associated pathways	Enhances synaptic plasticity, neuronal survival, learning, and memory	Muhammad Abdul Kadar et al., [42], [45], [48]
Anti-inflammatory activity	Suppression of NF-κB-associated inflammatory signalling and pro-inflammatory mediators	Reduces neuroinflammation and preserves synaptic communication	Cheng et al., [43], [44]
Neuroprotective and cognitive effects in experimental models	Preservation of hippocampal neuronal integrity and neurotransmitter balance	Improves cognitive performance, spatial memory, and behavioural outcomes	Arshad et al., [46], [49], [51]

Abbreviations: BDNF, brain-derived neurotrophic factor; CREB, cyclic AMP response element-binding protein; ERK, extracellular signal-regulated kinase; HO−1, heme oxygenase−1; KEAP1, Kelch-like ECH-associated protein 1; NF-κB, nuclear factor kappa B; NRF2, nuclear factor erythroid 2-related factor 2; ROS, reactive oxygen species; SOD−1, superoxide dismutase−1.

4. Mechanisms of Heterotrigona itama-mediated protection against-mediated protection against BPA neurotoxicity

4.1. Regulation of oxidative stress and mitochondrial dysfunction

Oxidative stress is recognised as a major contributor to BPA-induced neurotoxicity because excessive ROS production disrupts multiple pathways involved in neuronal survival, synaptic regulation, and mitochondrial homeostasis [4], [13]. Rather than functioning as an isolated mechanism, oxidative stress interacts closely with mitochondrial dysfunction, neuroinflammation, calcium dyshomeostasis, and impaired neurotrophic signalling, thereby contributing to progressive neuronal injury during neurodevelopment. Experimental studies demonstrated that BPA exposure suppresses endogenous antioxidant defence systems, including glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), together with increased lipid peroxidation and mitochondrial impairment [27], [52], [53]. Persistent disruption of redox homeostasis may subsequently impair ATP production, weaken synaptic signalling, and increase neuronal vulnerability to apoptotic and pyroptotic injury pathways.

Within this context, current evidence suggests that the neuroprotective effects of H. itama honey may involve coordinated regulation of oxidative and mitochondrial stress pathways rather than simple free radical scavenging alone. Several phenolic and flavonoid compounds present in stingless bee honey, including quercetin, kaempferol, apigenin, and caffeic acid, have been associated with activation of KEAP1-NRF2/HO−1 signalling and preservation of mitochondrial integrity under oxidative stress conditions [41], [43], [44]. Activation of these pathways may help restore antioxidant defence capacity, attenuate excessive ROS accumulation, and improve cellular stress adaptation during BPA-induced neuronal injury. In addition, maintenance of mitochondrial homeostasis may help preserve synaptic function and neuronal survival, as mitochondrial dysfunction is closely linked to calcium imbalance, excitotoxicity, and neuroinflammatory responses.

Current findings also suggest that oxidative stress interacts closely with inflammatory and neurotrophic pathways involved in cognitive regulation. Excessive ROS accumulation may suppress ERK/CREB/BDNF signalling, disrupt NMDA receptor-associated calcium homeostasis, and activate NF-κB-dependent inflammatory responses, thereby aggravating synaptic dysfunction and neuronal injury [10], [27], [38]. Since several bioactive compounds present in H. itama honey have been associated with modulation of antioxidant, inflammatory, and neurotrophic pathways, its neuroprotective effects may involve broader regulation of interconnected cellular stress responses rather than antioxidant activity alone.

Nevertheless, despite increasingly promising evidence, direct mechanistic studies specifically evaluating the effects of H. itama honey on oxidative and mitochondrial pathways during BPA-induced neurodevelopmental toxicity remain limited. Most available findings are derived from broader oxidative stress and neurodegenerative disease models rather than direct prenatal BPA exposure paradigms. Therefore, further studies integrating mitochondrial function, synaptic signalling, neuroinflammatory responses, and neurotrophic regulation are still required to clarify the mechanistic relevance of H. itama honey in BPA-associated neurodevelopmental injury.

4.2. Modulation of neuroinflammation and cell death pathways

Neuroinflammation is increasingly recognised as a central mechanism underlying BPA-induced neurotoxicity rather than merely a secondary consequence of oxidative injury [4]. Current evidence suggests that BPA-induced inflammatory signalling interacts closely with mitochondrial dysfunction, calcium dyshomeostasis, excitotoxicity, and impaired synaptic plasticity, thereby contributing to progressive neuronal dysfunction during neurodevelopment. Accumulating evidence indicates that BPA exposure promotes neuroinflammatory responses through microglial activation and increased production of cytokines such as IL−1β, TNF-α, IL−6, and COX−2 in cognition-related brain regions [4], [28], [29]. Since prolonged microglial activation may disrupt synaptic remodelling, neuronal communication, and hippocampal plasticity, persistent neuroinflammation likely contributes directly to BPA-associated cognitive impairment rather than functioning solely as a downstream response to cellular stress.

Recent studies have further demonstrated that BPA-induced neuroinflammation is associated with activation of inflammasome-related pathways, particularly the nucleotide-binding oligomerisation domain-like receptor pyrin domain-containing 3 (NLRP3) signalling pathway. Activation of the NLRP3 inflammasome subsequently promotes caspase−1 activation and maturation of IL−1β and IL−18, thereby exacerbating inflammatory damage and neuronal stress responses [13]. Importantly, increasing evidence suggests that BPA-induced inflammasome activation is closely associated with mitochondrial dysfunction and excessive ROS accumulation, indicating that oxidative and inflammatory pathways are mechanistically interconnected rather than independently activated processes [26], [28], [54]. This interaction appears particularly relevant during neurodevelopment because immature neuronal systems exhibit increased susceptibility to mitochondrial stress, inflammatory injury, and altered calcium signalling.

Next, research to date also suggests that BPA exposure may induce pyroptotic neuronal death through NLRP3/caspase−1/gasdermin D (GSDMD)-dependent pathways. Al-Shami et al. (2024) demonstrated increased NLRP3 expression, caspase−1-positive neurons, gliosis, microglial activation, and dentate gyrus injury in the hippocampus and prefrontal cortex following developmental BPA exposure. Similarly, Wang et al. [27] reported increased NLRP3, ASC, caspase−1, GSDMD, IL−1β, and IL−18 expression in human neuroblastoma cells exposed to BPA, supporting involvement of inflammasome-associated pyroptotic signalling. Notably, inhibition of caspase−1, estrogen receptor signalling, and oxidative stress partially attenuated these effects [27], suggesting that BPA-induced pyroptosis may involve coordinated interaction between ROS-dependent, endocrine-associated, and inflammasome-mediated pathways rather than a single isolated mechanism. Since pyroptosis is characterised by membrane rupture, inflammatory cytokine release, and propagation of inflammatory injury, persistent activation of these pathways may further aggravate synaptic dysfunction and neurobehavioural abnormalities during neurodevelopment.

Within this context, the neuroprotective potential of H. itama honey may extend beyond antioxidant activity by modulating inflammatory and cellular stress-related signalling pathways. Bioactive phenolic and flavonoid compounds found in stingless bee honey, including quercetin, kaempferol, and caffeic acid, have been reported to suppress NF-κB-mediated inflammatory signalling and reduce the production of pro-inflammatory cytokines under oxidative and inflammatory conditions [42], [43], [44]. Experimental studies also reported reductions in TNF-α, IL−1β, and apoptotic activity following stingless bee honey supplementation in stress-associated and neurodegenerative models [42], [45]. Nevertheless, whether these anti-inflammatory effects directly involve modulation of inflammasome-associated signalling pathways remains unclear. Since inflammatory activation, mitochondrial dysfunction, and pyroptotic signalling are highly interconnected during BPA-induced neurotoxicity, it is plausible that the neuroprotective effects of H. itama honey involve coordinated regulation of multiple cellular stress pathways rather than selective inhibition of individual inflammatory mediators.

Despite increasingly promising findings, several mechanistic limitations remain unresolved. Most currently available studies primarily evaluate broad inflammatory biomarkers without directly investigating upstream inflammasome regulators, pyroptotic signalling dynamics, or synaptic-inflammatory interactions during developmental BPA exposure. In addition, direct evidence specifically linking H. itama honey with modulation of NLRP3/caspase−1/GSDMD pathways remains limited. Therefore, further mechanistic studies integrating inflammasome signalling, mitochondrial regulation, synaptic plasticity, and neurobehavioural outcomes are still required to clarify whether H. itama honey can genuinely attenuate inflammasome-associated neurodevelopmental injury following BPA exposure.

4.3. Synaptic protection and neurotrophic regulation

Synaptic dysfunction is increasingly recognised as a major mechanism underlying BPA-induced cognitive impairment, particularly within the hippocampus, where neuronal signalling and synaptic plasticity are essential for learning and memory. BPA exposure disrupts glutamatergic neurotransmission, alters NMDA receptor subunit expression, impairs calcium homeostasis, and suppresses signalling pathways involved in neuronal survival and synaptic maturation [34], [38], [55], [56]. Since synaptic development during prenatal and early postnatal stages depends heavily on tightly regulated calcium-dependent signalling, disruption of these pathways may contribute directly to long-term abnormalities in hippocampal connectivity and cognitive function [34], [38].

Among the NMDA receptor subunits, GluN2A and GluN2B are particularly important for calcium-dependent synaptic plasticity and memory formation [34], [38]. Several studies demonstrated that BPA exposure disrupts the normal GluN2A/GluN2B balance, leading to impaired synaptic transmission and destabilisation of hippocampal circuitry [34], [35], [40]. Dysregulated NMDA receptor activity may further promote excessive calcium influx, mitochondrial dysfunction, oxidative stress, and excitotoxic neuronal injury [55], [56]. In addition, BPA exposure reduced expression of synaptic proteins, including PSD−95, synapsin I, and GluR1, all of which are important for maintenance of synaptic structure and neuronal communication [23], [34], [35]. These findings suggest that BPA-induced cognitive dysfunction involves coordinated disruption of synaptic signalling, neuronal plasticity, and hippocampal network stability rather than isolated oxidative injury alone.

BPA exposure also suppresses pathways involved in neurotrophic regulation and neuronal survival. Several studies reported reduced PKC/ERK/CREB and ERK-BDNF signalling together with impaired BDNF expression following developmental BPA exposure [10], [38]. Since BDNF signalling is closely linked to NMDA receptor activity, dendritic remodelling, synaptic maturation, and long-term potentiation (LTP), suppression of these pathways may contribute directly to impaired learning and memory processes [38]. In addition, altered expression of microRNAs associated with calcium signalling and synaptic plasticity has been reported following BPA exposure, suggesting that epigenetic dysregulation may contribute to persistent synaptic abnormalities and long-term cognitive vulnerability [40].

Importantly, BPA appears to exert dose- and exposure-dependent effects on NMDA-mediated calcium signalling. Acute low-dose exposure has been reported to transiently enhance glutamate-induced calcium influx and NMDA receptor activity, whereas chronic or developmental exposure consistently disrupts calcium homeostasis, reduces NMDA receptor subunit expression, and impairs synaptic plasticity [34], [35], [57]. Persistent calcium dysregulation appears to play a greater role in long-term hippocampal dysfunction than transient receptor activation alone. Since calcium signalling interacts closely with mitochondrial homeostasis, oxidative stress, and neuroinflammatory pathways, disruption of these interconnected mechanisms may progressively impair neuronal communication and synaptic resilience during neurodevelopment.

Within this context, H. itama honey has attracted increasing interest because several of its phenolic and flavonoid compounds, including quercetin, apigenin, kaempferol, and caffeic acid, may regulate pathways associated with synaptic plasticity and neuronal survival [43], [46]. Experimental studies demonstrated that stingless bee honey supplementation increased BDNF expression and improved cognitive performance in several experimental models [42], [45], [46]. Current evidence also suggests possible modulation of MAPK/ERK, CREB, and BDNF-associated signalling pathways following stingless bee honey supplementation [42], [43], [46]. Since these pathways are closely involved in synaptic maturation, neuronal communication, and hippocampal plasticity, preservation of neurotrophic signalling may represent an important component of the neuroprotective effects of H. itama honey.

Nevertheless, several mechanistic limitations remain unresolved. Most currently available studies primarily evaluated behavioural outcomes, antioxidant activity, or BDNF expression without directly examining NMDA receptor-associated signalling, calcium homeostasis, synaptic protein regulation, or electrophysiological changes following BPA exposure. In addition, direct evidence evaluating the effects of H. itama honey on GluN2A/GluN2B balance, PSD−95, synapsin I, GluR1 expression, and calcium-dependent synaptic plasticity during BPA-induced neurodevelopmental injury remains limited. Therefore, further mechanistic studies integrating synaptic signalling, mitochondrial regulation, calcium homeostasis, neurotrophic pathways, and neurobehavioural analyses are still required to clarify the molecular basis of its neuroprotective effects during developmental neurotoxicity.

4.4. Neuroendocrine and estrogen receptor regulation

As an established endocrine-disrupting chemical, BPA disrupts hormonal signalling pathways essential for neurodevelopment and cognitive regulation. Its structural similarity to endogenous estrogen enables interaction with estrogen receptors, especially ERα and ERβ, consequently affecting neuronal differentiation, hippocampal maturation, and synaptic plasticity [58], [59]. Experimental evidence further suggests that BPA alters receptor phosphorylation and downstream transcriptional activity, indicating that its neurotoxic effects involve dysregulation of estrogen receptor signalling rather than simple estrogen mimicry alone [58], [60]. Since estrogen receptor signalling is closely involved in hippocampal connectivity and memory formation, prolonged BPA exposure during prenatal and early postnatal development may increase susceptibility to persistent cognitive and behavioural abnormalities.

Estrogen receptor signalling also interacts closely with pathways involved in glutamatergic neurotransmission and synaptic plasticity. Activation of ERα, ERβ, and G-protein coupled estrogen receptor (GPER) regulates dendritic spine formation, long-term potentiation (LTP), and expression of synaptic proteins required for neuronal communication and memory consolidation [61], [62]. In addition, estrogen receptor-associated signalling pathways converge with ERK/MAPK, PI3K/Akt, CREB, and NMDA receptor-associated calcium signalling cascades that support neuronal survival and synaptic stability [63], [64]. Consequently, disruption of these interconnected pathways during neurodevelopment may impair hippocampal maturation and weaken synaptic resilience.

Several studies demonstrated that developmental BPA exposure suppresses estrogen receptor-associated synaptic signalling pathways within the hippocampus. Perinatal BPA exposure impaired ERα-mediated PKC/ERK/CREB and BDNF signalling, together with reductions in PSD−95, synapsin I, GluR1, and NMDA receptor subunits [58]. BPA exposure has also been associated with altered dendritic spine formation, impaired glutamatergic neurotransmission, and abnormal synaptic ultrastructure, suggesting disruption of both neuronal connectivity and synaptic organisation during neurodevelopment [55], [56], [57]. Since BDNF signalling interacts closely with NMDA receptor activity and calcium-dependent synaptic plasticity, suppression of these pathways may contribute directly to impaired learning and memory following developmental BPA exposure.

Importantly, BPA-induced neurotoxicity does not appear to occur exclusively through classical ERα/ERβ pathways. Increasing evidence suggests that BPA may also act through non-classical mechanisms involving estrogen-related receptors (ERRs), GPER, oxidative stress pathways, and NMDA receptor-associated calcium signalling [57], [60]. In hippocampal neurons, BPA rapidly enhanced glutamate-induced calcium influx through ERK- and p38-associated NMDA receptor signalling pathways [57]. Persistent calcium dysregulation may subsequently promote mitochondrial dysfunction, ROS accumulation, excitotoxicity, and neuronal injury, thereby linking endocrine disruption with synaptic impairment and neurodegenerative processes [55], [56]. These findings suggest that oxidative stress and calcium dyshomeostasis function as major downstream mediators of BPA-associated neuroendocrine toxicity rather than isolated pathological events.

Prenatal and perinatal developmental stages appear particularly vulnerable to BPA exposure because these periods involve rapid synaptogenesis, neuronal differentiation, and maturation of estrogen-sensitive hippocampal circuitry. Recent transcriptomic studies demonstrated that developmental BPA exposure altered expression of genes associated with neurogenesis, synaptic development, and neuronal maturation in a sex-dependent manner [6], [65]. BPA exposure has also been associated with impaired postnatal neurogenesis and persistent dysregulation of hippocampal developmental pathways, suggesting that BPA-induced neuroendocrine toxicity may involve sustained molecular reprogramming rather than transient hormonal disruption alone [66].

Within this context, H. itama honey has attracted increasing attention because several of its phenolic and flavonoid compounds, including quercetin, kaempferol, apigenin, and caffeic acid, possess antioxidant and neuroprotective properties associated with neuronal survival and synaptic regulation [43], [45]. Experimental studies demonstrated that stingless bee honey supplementation increased BDNF expression, improved cognitive performance, and preserved neuronal function in models of oxidative and neurobehavioural stress [42], [46]. Current evidence also suggests possible modulation of MAPK/ERK, CREB, and BDNF-associated pathways following stingless bee honey supplementation [42], [43]. Since estrogen receptor signalling is functionally interconnected with these neurotrophic and synaptic pathways, preservation of these signalling networks may indirectly support neuronal resilience during BPA-induced neurodevelopmental stress.

Nevertheless, direct evidence linking H. itama honey with estrogen receptor regulation during BPA-induced neurotoxicity remains limited. Most currently available studies primarily evaluated antioxidant activity, behavioural outcomes, or neurotrophic markers without directly examining ERα/ERβ signalling, GPER-associated pathways, or estrogen-dependent synaptic regulation. In addition, several proposed mechanisms are currently inferred from broader flavonoid literature rather than direct mechanistic studies involving H. itama honey itself. Therefore, although stingless bee honey demonstrates promising neuroprotective potential, its specific role in modulating estrogen receptor-associated pathways during BPA-induced neurodevelopmental toxicity remains insufficiently characterised.

Overall, current evidence suggests that BPA-induced neuroendocrine disruption contributes significantly to hippocampal dysfunction through coordinated impairment of estrogen receptor signalling, NMDA receptor-associated calcium regulation, neurotrophic signalling, and synaptic plasticity. Emerging findings indicate that H. itama honey may provide indirect neuroprotective effects through modulation of interconnected antioxidant, neurotrophic, and synaptic pathways. However, direct mechanistic investigations examining the influence of H. itama honey on ERα/ERβ-associated signalling during developmental BPA exposure are still lacking. Therefore, further investigations are needed to elucidate the molecular mechanisms underlying the potential neuroprotective effects of H. itama honey against BPA-induced neurodevelopmental damage.

4.5. Integrated neuroprotective mechanisms of Heterotrigona itama against BPA-induced neurotoxicity

Current evidence suggests that BPA-induced neurotoxicity arises through coordinated dysregulation of multiple interconnected pathways involving oxidative stress, mitochondrial dysfunction, neuroinflammation, calcium dyshomeostasis, synaptic impairment, neurotrophic suppression, and endocrine disruption rather than through a single isolated mechanism. These pathological processes collectively contribute to hippocampal dysfunction, impaired neuronal connectivity, and cognitive decline during neurodevelopment. Importantly, oxidative stress appears to function as a major upstream mediator capable of simultaneously disrupting mitochondrial integrity, activating inflammatory signalling, impairing calcium homeostasis, suppressing neurotrophic pathways, and destabilising synaptic communication. Consequently, therapeutic approaches capable of modulating several interconnected pathways may provide broader neuroprotective effects than interventions targeting individual downstream mechanisms alone. Within this context, H. itama honey has emerged as a promising neuroprotective candidate because of its rich phenolic and flavonoid composition together with its multitarget biological activities.

The neuroprotective potential of H. itama honey is largely attributed to bioactive compounds, including quercetin, kaempferol, apigenin, catechin, gallic acid, caffeic acid, and p-coumaric acid, which possess antioxidant, anti-inflammatory, neurotrophic, and cytoprotective properties [41], [43], [44], [47]. However, current evidence suggests that these compounds do not function solely through direct free radical scavenging. Instead, stingless bee honey appears to regulate intracellular pathways involved in cellular adaptation to oxidative and inflammatory stress, including Nrf2-Keap1-ARE, NF-κB, MAPK/ERK, CREB, and BDNF-associated signalling pathways [43], [46]. This mechanistic complexity is particularly relevant because BPA-induced neurotoxicity involves simultaneous disruption of oxidative, inflammatory, mitochondrial, synaptic, and neuroendocrine pathways.

Among these mechanisms, preservation of redox and mitochondrial homeostasis appears to represent a central component of H. itama-mediated neuroprotection. BPA exposure disrupts endogenous antioxidant defence systems, impairs mitochondrial membrane potential, suppresses ATP production, and promotes excessive ROS accumulation, thereby increasing neuronal vulnerability to oxidative and mitochondrial injury [13], [26], [54]. Since mitochondrial dysfunction contributes directly to calcium dyshomeostasis, inflammatory activation, synaptic impairment, and neuronal death signalling, maintenance of mitochondrial integrity may indirectly stabilise several downstream neurotoxic pathways simultaneously. Several studies demonstrated that stingless bee honey supplementation increased antioxidant enzyme activity, reduced oxidative damage biomarkers, and enhanced Nrf2-associated antioxidant signalling, suggesting activation of endogenous cellular defence mechanisms rather than transient antioxidant activity alone [44], [48].

Regulation of mammalian target of rapamycin (mTOR)-associated pathways may further contribute to these integrative protective effects. The PI3K/Akt/mTOR signalling pathway plays an important role in neuronal survival, mitochondrial quality control, autophagy regulation, synaptic plasticity, and cellular energy homeostasis. Increasing evidence suggests that BPA exposure disrupts autophagic flux, impairs mitochondrial stress adaptation, and promotes excessive inflammatory and oxidative signalling during neurodevelopmental injury [13], [26]. Since oxidative stress, impaired mitophagy, mitochondrial dysfunction, and neuroinflammation are highly interconnected during BPA-induced neurotoxicity, dysregulation of mTOR-associated pathways may further aggravate neuronal vulnerability and synaptic dysfunction. Several flavonoids present in H. itama honey, including quercetin and kaempferol, have also been associated with modulation of intracellular stress adaptation and survival pathways under oxidative and inflammatory conditions [67], [68]. Nevertheless, direct mechanistic evidence linking H. itama honey with mTOR regulation during BPA-induced neurodevelopmental toxicity remains limited.

Neuroinflammatory signalling also appears to represent an important mechanistic target of H. itama honey-mediated neuroprotection. BPA exposure activates NF-κB-associated inflammatory pathways, promotes microglial activation, and increases production of pro-inflammatory cytokines, including TNF-α, IL−1β, and IL−6, thereby amplifying neuronal injury and synaptic dysfunction [4], [69]. Increasing evidence further suggests that BPA-induced neuroinflammation involves inflammasome-associated and pyroptotic signalling pathways closely linked with mitochondrial dysfunction and oxidative stress. Several studies demonstrated that stingless bee honey supplementation reduced inflammatory cytokine levels together with improvements in neuronal integrity and hippocampal morphology [45], [50]. Experimental findings also suggest suppression of NF-κB and p38 MAPK signalling together with enhancement of Nrf2-associated antioxidant pathways following stingless bee honey supplementation [43], [44], [48]. Since oxidative stress and neuroinflammation reinforce each other during neurodevelopmental injury, simultaneous regulation of these pathways may represent an important therapeutic advantage of H. itama honey.

Apart from oxidative and inflammatory regulation, H. itama honey may also indirectly support synaptic plasticity and neurotrophic signalling pathways disrupted during BPA exposure. BPA-induced suppression of NMDA receptor-associated signalling, calcium homeostasis, PSD−95, synapsin I, GluR1 expression, and ERK/CREB/BDNF pathways contributes substantially to impaired neuronal communication and hippocampal dysfunction during neurodevelopment [10], [23], [38]. Several studies demonstrated that stingless bee honey supplementation increased BDNF expression, preserved neurotransmitter balance, reduced corticosterone levels, and improved behavioural performance in stress-associated experimental models [42], [43], [46]. Current evidence also suggests modulation of MAPK/ERK, CREB, and BDNF-associated pathways involved in neuronal survival and synaptic plasticity [42], [43], [46]. Since BDNF signalling is functionally interconnected with NMDA receptor activity and calcium-dependent synaptic regulation, preservation of neurotrophic signalling may indirectly stabilise neuronal communication during oxidative and inflammatory stress conditions.

The neuroprotective effects of H. itama honey may additionally involve modulation of neuroendocrine and estrogen-associated pathways disrupted by BPA exposure. BPA interferes with ERα/ERβ-mediated signalling pathways involved in hippocampal plasticity, neuronal survival, and synaptic maturation through dysregulation of ERK/CREB/BDNF and NMDA receptor-associated pathways [58], [61], [63]. Polyphenolic compounds such as quercetin, kaempferol, and apigenin may contribute to neuroprotection through interactions with oestrogen-related signalling pathways. However, direct experimental evidence specifically linking H. itama honey with ERα/ERβ regulation during BPA-induced neurotoxicity remains insufficient.

Importantly, the neuroprotective effects of H. itama honey appear to involve coordinated regulation of interconnected cellular stress pathways rather than selective modulation of a single molecular target. Since multiple pathological pathways involved in BPA-induced neurotoxicity are closely interconnected, modulation of several mechanisms may provide more effective neuroprotection than targeting a single pathway alone. This multitarget mechanism may represent a significant therapeutic advantage in neurodevelopmental toxicology, where neuronal injury typically involves dysregulation of multiple signalling networks rather than a single pathological process.

Nevertheless, several important mechanistic limitations remain unresolved. Most currently available evidence evaluating the neuroprotective effects of H. itama honey is derived from preclinical or indirect disease models, whereas direct mechanistic studies involving prenatal BPA-induced neurotoxicity remain scarce. Considerable variability also exists in honey composition, botanical source, geographical origin, dosage, and extraction methods, all of which may influence biological activity and experimental reproducibility. In addition, current evidence remains limited regarding the precise molecular interactions between H. itama honey and pathways associated with mitophagy, inflammasome activation, pyroptosis, estrogen receptor signalling, and NMDA receptor-associated calcium regulation. Therefore, although H. itama honey demonstrates promising multitarget neuroprotective potential, further mechanistic and translational investigations are still required to clarify its molecular targets and determine its therapeutic relevance in BPA-associated neurodevelopmental toxicity. Table 4.1 summarises the integrated molecular mechanisms involved in BPA-induced neurotoxicity and the proposed neuroprotective relevance of Heterotrigona itama honey.

Table 4.1.

Integrated molecular mechanisms involved in BPA-induced neurotoxicity and proposed neuroprotective relevance of Heterotrigona itama honey.

Pathological effects of BPA	Major mechanisms involved	Proposed neuroprotective relevance ofH. itamahoney	Key references
Oxidative stress	Excessive ROS generation, lipid peroxidation, glutathione depletion, and impaired endogenous antioxidant defence	Enhances SOD, CAT, and GPx activity, reduces oxidative damage, and activates Nrf2-Keap1-ARE-associated antioxidant signalling	Cheng et al., [15], [44], [48]
Mitochondrial dysfunction	Mitochondrial membrane depolarisation, ATP depletion, mitochondrial ROS accumulation, and apoptotic activation	Preserves mitochondrial integrity, attenuates mitochondrial oxidative stress, and supports cellular stress adaptation pathways	Han et al., [67], [68], [70]
Neuroinflammation	NF-κB activation, microglial activation, increased TNF-α, IL−1β, and IL−6 production	Suppresses inflammatory signalling and reduces pro-inflammatory cytokine expression	Kobayashi et al., [15], [50], [69]
Inflammasome activation and pyroptosis	NLRP3/caspase−1/GSDMD activation, IL−1β maturation, and inflammatory neuronal injury	May attenuate inflammasome-associated inflammatory signalling through regulation of oxidative and inflammatory stress pathways	Al-Shami et al., [13], [26], [28]
Calcium dyshomeostasis and excitotoxicity	Excessive NMDA receptor-mediated calcium influx, disrupted calcium signalling, and excitotoxic neuronal injury	May stabilise calcium-dependent neuronal signalling indirectly through antioxidant and neurotrophic pathway regulation	Wang et al., [55], [56], [57]
Synaptic dysfunction	Reduced PSD−95, synapsin I, GluR1, and NMDA receptor subunit expression; and impaired glutamatergic neurotransmission	Supports synaptic stability and neuronal communication through ERK/CREB/BDNF-associated signalling pathways	Flores et al., [23], [38], [42]
Neurotrophic dysregulation	Suppression of ERK/CREB/BDNF signalling and impaired synaptic plasticity	Increases BDNF expression and supports neuronal survival, synaptic plasticity, and hippocampal function	Mustafa et al., [42], [43], [46]
Neuroendocrine disruption	Dysregulation of ERα/ERβ-associated signalling, impaired hippocampal maturation and neuronal plasticity	Flavonoid-associated modulation of estrogen-sensitive neuroprotective pathways may indirectly support synaptic resilience	Brann et al., [58], [61], [63]
Autophagy and mitophagy dysregulation	Impaired autophagic flux, disrupted mitochondrial quality control, and excessive cellular stress signalling	May support cellular stress adaptation and mitochondrial homeostasis through modulation of intracellular survival pathways	Han et al., [13], [26], [67]
Cognitive and behavioural impairment	Hippocampal dysfunction, impaired learning and memory, and disrupted neuronal connectivity	Improves cognitive performance, preserves hippocampal structure, and reduces stress-associated behavioural abnormalities	Muhammad Abdul Kadar et al., [42], [45]

Abbreviations: ARE, antioxidant response element; ATP, adenosine triphosphate; BDNF, brain-derived neurotrophic factor; BPA, bisphenol A; CAT, catalase; CREB, cAMP response element-binding protein; ERα, estrogen receptor alpha; ERβ, estrogen receptor beta; ERK, extracellular signal-regulated kinase; GPER, G-protein coupled estrogen receptor; GPx, glutathione peroxidase; GSH, glutathione; HO−1, heme oxygenase−1; IL−1β, interleukin−1 beta; IL−6, interleukin−6; Keap1, Kelch-like ECH-associated protein 1; MAPK, mitogen-activated protein kinase; MDA, malondialdehyde; NF-κB, nuclear factor kappa B; NLRP3, NOD-like receptor family pyrin domain containing 3; NMDA, N-methyl-D-aspartate; Nrf2, nuclear factor erythroid 2-related factor 2; PI3K, phosphoinositide 3-kinase; PSD−95, postsynaptic density protein 95; ROS, reactive oxygen species; SOD, superoxide dismutase; TNF-α, tumour necrosis factor alpha.

5. Research gaps and future perspectives

Although many studies have reported the neurotoxic effects of BPA and the neuroprotective properties of H. itama honey, the relationship between these two areas is still not fully understood. Most studies investigate BPA toxicity and stingless bee honey separately rather than directly examining whether H. itama honey can protect against BPA-induced brain injury. Therefore, the main hypothesis of this review, that H. itama honey may reduce BPA-induced neurodevelopmental damage through regulation of multiple interconnected pathways, still requires stronger experimental evidence.

One major limitation is that most studies involving stingless bee honey focus mainly on antioxidant activity. However, BPA-induced neurotoxicity involves more than oxidative stress alone. Increasing evidence shows that BPA also disrupts NMDA receptor signalling, calcium homeostasis, ERK/CREB/BDNF pathways, synaptic proteins such as PSD−95 and synapsin I, and estrogen receptor-associated signalling involved in learning, memory, and hippocampal development. Despite this, only a limited number of studies have examined whether H. itama honey can directly regulate these neuronal signalling pathways. As a result, it is still unclear whether the neuroprotective effects of H. itama honey occur mainly through antioxidant activity or through broader regulation of synaptic and neurotrophic pathways.

Another important gap is the lack of prenatal and early-life exposure models. Prenatal and early postnatal developmental stages are highly sensitive to BPA because these periods involve rapid brain development, synapse formation, and maturation of hippocampal circuits. However, most current stingless bee honey studies focus on oxidative stress, metabolic disorders, chronic stress, or neurodegenerative conditions rather than developmental neurotoxicity. Therefore, it remains uncertain whether H. itama honey can preserve brain development, neuronal connectivity, and long-term cognitive function following prenatal BPA exposure.

Mitochondrial quality control and inflammatory cell death pathways also remain poorly studied. Increasing evidence suggests that BPA disrupts mitophagy, autophagy, mTOR signalling, NLRP3 inflammasome activation, and pyroptotic pathways through excessive mitochondrial ROS production and impaired cellular stress responses. These mechanisms are now recognised as important contributors to neuronal injury and cognitive dysfunction. However, direct studies examining whether H. itama honey regulates mTOR signalling, PINK1/parkin-mediated mitophagy, caspase−1 activation, gasdermin D-mediated pyroptosis, or inflammasome pathways are still limited. Without investigation of these pathways, the proposed multitarget neuroprotective effects of stingless bee honey remain incomplete.

Another unresolved issue involves the neuroendocrine effects of BPA exposure. BPA is known to disrupt hypothalamic-pituitary-gonadal (HPG) axis regulation and estrogen receptor-associated pathways involved in neuronal maturation and synaptic plasticity. Although several flavonoids found in H. itama honey possess phytoestrogen-like and neuroprotective properties, direct evidence linking stingless bee honey with ERα/ERβ or GPER-associated signalling is still lacking. Future studies should therefore determine whether H. itama honey directly regulates neuroendocrine pathways or whether its effects occur indirectly through antioxidant and anti-inflammatory actions.

Methodological limitations also remain a major concern. Variations in bee species, botanical source, geographical origin, harvesting conditions, storage conditions, extraction methods, dosage, and treatment duration may influence the phenolic composition and biological activity of honey samples. Although H. itama honey consistently demonstrates high phenolic and flavonoid content, the specific compounds responsible for its neuroprotective effects are still not fully identified. Lack of standardisation, therefore, limits reproducibility and makes comparison between studies more difficult.

In addition, the translational potential of H. itama honey remains uncertain because most available evidence comes from preclinical animal studies. The bioavailability, metabolism, blood-brain barrier permeability, and pharmacokinetic behaviour of many bioactive compounds present in stingless bee honey are still poorly understood. Long-term safety, optimal dosage, and clinical applicability also remain unclear.

Future studies should therefore use integrated approaches that evaluate behavioural, molecular, neurochemical, histological, and transcriptomic changes following BPA exposure and H. itama honey supplementation. Particular attention should be given to pathways involved in synaptic plasticity, mitochondrial quality control, mTOR regulation, inflammasome activation, pyroptosis, calcium signalling, and neuroendocrine regulation. Integrative multi-omics approaches, including transcriptomics, proteomics, metabolomics, and epigenetic analyses, may provide deeper insight into the molecular interactions underlying these effects. In addition, future studies should carefully evaluate sex-dependent responses because BPA-induced neurodevelopmental effects may differ between male and female offspring.

Overall, current evidence suggests that H. itama honey may exert multitarget neuroprotective effects through coordinated regulation of oxidative stress, mitochondrial dysfunction, neuroinflammation, synaptic plasticity, neurotrophic signalling, and neuroendocrine pathways disrupted during BPA-induced neurotoxicity. However, direct mechanistic evidence specifically involving prenatal BPA-induced neurodevelopmental injury remains limited. Addressing these research gaps is important not only for understanding the molecular basis of H. itama-mediated neuroprotection, but also for determining whether stingless bee honey has real translational potential as an evidence-based intervention against environmentally induced neurodevelopmental dysfunction.

6. Conclusion

BPA is an environmental neurotoxicant that disrupts brain development and cognitive function through interconnected mechanisms involving oxidative stress, mitochondrial dysfunction, neuroinflammation, calcium dyshomeostasis, synaptic impairment, neurotrophic dysregulation, inflammasome activation, and endocrine disruption. Current evidence indicates that BPA-induced neurotoxicity cannot be explained by oxidative stress alone, as BPA also interferes with NMDA receptor signalling, ERK/CREB/BDNF pathways, mitochondrial quality control, pyroptotic signalling, and estrogen receptor-associated pathways involved in hippocampal maturation and synaptic plasticity. These molecular disturbances collectively contribute to neuronal injury, impaired synaptic communication, and long-term cognitive dysfunction during vulnerable developmental stages.

Within this context, Heterotrigona itama honey has emerged as a promising natural neuroprotective candidate because of its rich phenolic and flavonoid composition together with its antioxidant, anti-inflammatory, neurotrophic, and cytoprotective properties. Increasing evidence suggests that its neuroprotective effects may extend beyond simple antioxidant activity and involve coordinated regulation of interconnected pathways associated with oxidative stress, mitochondrial homeostasis, neuroinflammation, synaptic plasticity, neurotrophic signalling, and neuroendocrine regulation.

Importantly, the main novelty of this review lies in the integration of BPA-induced neurotoxicity mechanisms with the proposed multitarget neuroprotective actions of H. itama honey. Rather than considering BPA-induced neurotoxicity and stingless bee honey as separate areas of investigation, this review integrates current evidence to highlight how H. itama honey may modulate interconnected signalling pathways disrupted by BPA exposure.These pathways include Keap1-Nrf2-ARE, NF-κB, ERK/CREB/BDNF, mTOR signalling, inflammasome signalling, calcium homeostasis, and estrogen receptor-mediated pathways. This integrated perspective provides a clearer mechanistic understanding of how natural bioactive compounds may influence developmental neurotoxicity beyond general antioxidant effects alone.

Nevertheless, direct mechanistic evidence evaluating the effects of H. itama honey on prenatal BPA-induced neurodevelopmental toxicity remains limited. Most available findings are derived from indirect oxidative stress, neurodegenerative, or metabolic disease models rather than dedicated developmental BPA exposure studies. Therefore, further studies integrating behavioural, molecular, neurochemical, histological, electrophysiological, and multi-omics approaches are required to clarify its molecular targets and therapeutic relevance.

Overall, current evidence suggests that H. itama honey possesses promising multitarget neuroprotective potential against BPA-induced neuronal injury and cognitive dysfunction. Clarifying the molecular interactions underlying these protective effects may facilitate to the future development of evidence-based natural interventions targeting environmentally induced neurodevelopmental disorders.

CRediT authorship contribution statement

Siti Khadijah Azmi: Writing – review & editing, Writing – original draft. Nur Faizah Ali: Writing – review & editing, Supervision, Project administration, Conceptualization. Siran Rosfaizah: Writing – review & editing, Supervision, Investigation, Conceptualization.

Funding

This work was supported by the Fundamental Research Grant Scheme (FRGS), Ministry of Higher Education Malaysia (Grant No. 600-RMC/FRGS 5/3 (015/2022)).

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper

Handling Editor Procf. L.H. Lash

Data availability

No data was used for the research described in the article.