Involvement of environmentally relevant toxic metal mixture in Alzheimer's disease pathway alteration and protective role of berberine: Bioinformatics analysis and toxicogenomic screening

https://doi.org/10.1016/j.fct.2022.112839Get rights and content

Highlights

  • Pb + Cd + MeHg + As - environmentally relevant toxic metal mixture linked to AD

  • Berberine antagonized effects of toxic metal mixture on AD genes

  • Predicted interactions (45.29%) and physical interactions (18.39%) – most prominent

  • Apoptotic signaling pathway was the main enriched biological process

  • Computational analysis showed berberine as a promising agent for AD treatment

Abstract

We aimed to examine the molecular basis of the positive effect of berberine against environmentally relevant toxic metal-linked Alzheimer's disease (AD). The Comparative Toxicogenomic Database (CTD) retrieved a set of genes common to lead, cadmium, methylmercury and arsenic linked to AD development and a set of genes through which berberine exerts a therapeutic mode of action in AD. GeneMania prediction server revealed detailed gene interactions, while Metascape highlighted protein-protein interaction enrichment (PPIE). SwissADME evaluated physicochemical properties of berberine. Berberine had an antagonistic effect for the majority of genes mutual for AD and toxic metal mixture: ACHE, APP, BAX, BCL2, CASP3, HMOX1, IL1B, MAPT, SOD2, TNF. Gene network analysis revealed interactions predicted by the server (45.29%) and physical interactions (18.39%) as the most important. Enriched biological processes analysis showed apoptotic signaling pathway, positive regulation of organelle organization and response to oxidative stress as dominant pathways involved in berberine protective effects against toxic metal mixture, while PPIE analysis showed regulation of apoptotic signaling pathway as the main gene ontology process targeted by berberine. Physicochemical properties and pharmacokinetics of berberine are in concordance with its beneficial properties in AD due to the high gastrointestinal absorption and capability to pass the blood-brain barrier.

Introduction

Alzheimer's disease (AD) is the most common neurodegenerative disease and the most frequent cause of dementia (Checkoway et al., 2011). According to the Global, regional, and national burden of Alzheimer's disease and other dementias (1990–2016), these disorders are considered an increasing global health challenge, currently affecting 40–50 million people, with projections to elevate to 66 million by 2030 and 115 million by 2050 (GBD, 2016 Dementia Collaborators; Honjo et al., 2012). The hallmarks of AD, a progressive neurodegenerative disorder, are extracellular amyloid beta (Aβ) plaques and intracellular neurofibrillary tangles (NFTs) with high synaptic defects (Correia et al., 2012). This disorder is characterized by cognitive, functional, and behavioral alterations (Cummings et al., 2018).

Among the different pathological patterns, environmentally relevant toxic metals are known as agents that affect brain development (Brown et al., 2005). Many studies have suggested a linkage between neurodegenerative disorders and increase in the level of toxic metals in blood of the patients with neurodegenerative disorders (Cavaleri, 2015; HasanImad Lafta, 2021). A study involving a Sicilian population living in an environment potentially contaminated with metals found a stronger association between AD and metals (copper (Cu), selenium (Se), zinc (Zn), lead (Pb), and mercury (Hg)) when compared to the other neurodegenerative disorders, such as multiple sclerosis (Giacoppo et al., 2014). However, Lee et al. (2012) showed that there was no statistical significance between blood/serum toxic metals (Pb, cadmium (Cd) and Hg) level and decrease in cognitive function. According to Park et al. (2014) serum Pb, Cd, Hg, and arsenic (As) levels were not directly related to abnormal cognition in AD, although there were some clinically significant results regarding Pb and Cd. The findings of Pamphlett and Kum Jew (2015) neither suggested that toxic metal uptake (Hg, bismuth (Bi), and silver (Ag)) could cause hyperphosphorylation of tau in locus ceruleus neurons later in life, nor that the numbers of locus ceruleus neurons containing toxic metals correlated with neurons containing hyperphosphorylated tau.

These inconsistent results require further in silico, in vitro, in vivo and observational studies, although there are multiple hypotheses about the relation between AD and metals. Several studies suggested that homeostasis of essential biometals (calcium (Ca), magnesium (Mg), manganese (Mn),Cu, Zn, and iron (Fe)) is impaired in case of AD, while some studies pointed out the pathophysiological impact of metal imbalance in brain (Kabir et al., 2020). Harmful effects of toxic metals are well-known and their omnipresence is particularly important due to their long lasting effects on the brain (Kabir et al., 2020; Karri et al., 2016). Toxic metals are usually present as mixtures, and Pb, Cd, Hg and As are among the major toxic agents found in the environment (Anushruti et al., 2015). There is growing interest in neurotoxic effects of mixtures of these metals, especially considering that the literature indicates various potential mechanisms of toxicity when exposed to a single metal (Karri et al., 2016; Anushruti et al., 2015.

It has been suggested that Pb causes oxidative DNA damage and APP gene hypomethylation, which results in overexpression of the APP gene, increased APP production, and increased activity of transcription factor Sp1 which regulates proteins associated with AD. Consequently, Pb promoted Aβ aggregation and caused plaque formation in brain (Lee et al., 2018). Cadmium induced the aggregation of Aβ oligomers or fibrils by blocking the Aβ1–42 ion channel. Furthermore, Cd blocks M1 receptor that causes overexpression of AChE-S and downregulation of AChE-R and, finally, increases the expression of GSK3β that leads to hyperphosphorylation of tau (Kabir et al., 2020). Mercury binds to tubulin causing its dysfunction and inhibits the polymerization of tubulin to micro-tubulin. These changes result in formation of neurofibrillary tangles and senile plaques. Mercury also induced oxidative stress damage resulting in the increased phosphorylation state of tau protein (Lee et al., 2018). Arsenic exposure is connected to the loss of spatial memory, increased expression of APP and sAPPβ, inflammation and oxidative stress in the brain, which is consistent with the previous research on AD (Rahman et al., 2020).

Environmentally relevant toxic metals also affect genes, while changes on the gene level are deemed as one of the crucial etiological factors for development of neurodegenerative disorders (Cicero et al., 2017; Karri et al., 2016). Expression levels of certain genes, such as APP, PSEN1, PSEN2, APOE4 were found up-regulated in AD patients (Yadav et al., 2021). The authors concluded that there was no significant correlation between metals dyshomeostasis (As, Pb, Cd, Hg, Al, Zn, Fe, Cu) and gene expression level. However, they suggested that a large sample size of AD patients and further examination were required (Yadav et al., 2021).

On the other side, current therapy for AD (e.g. rivastigmine, galantamine, and donepezil) relies on increasing the availability of acetylcholine at cholinergic synapses (Hong-Qi et al., 2012). However, poor pharmacokinetics and pharmacodynamics of medicines are recognized as common cause of therapy failure (Tiwari et al., 2019). Non-selectivity and side effects of the current therapy have led to the increasing efforts for development of natural treatment strategies in AD (curcumin, resveratrol, ginsenosides, flavonoids, terpenoids, phytocannabinoids, berberine, etc.) (Costa et al., 2017; Ghareeb et al., 2010; Sagud et al., 2021).

Berberine, an isoquinoline alkaloid isolated from traditional Chinese medicinal herbs, and its derivatives demonstrated multiple pharmacological benefits for treatment of diabetes, hyperlipidemia, osteoporosis, inflammation, bacterial, protozoal and viral infections, and cancer (Zhang et al., 2015, 2016). Also, there is evidence suggesting that berberine may act as a promising neuroprotective agent against many neurological disorders such as depression, anxiety, cerebral ischemia, and AD (Kulkarni and Dhir, 2010; Simões Pires et al., 2014). Berberine has been shown to exert physiological effects similar to reference AD drugs (Cai et al., 2016; Habtemariam, 2016; He et al., 2017; Ji and Shen, 2012; Kumar et al., 2015).

Since toxicogenomics combines bioinformatics and conventional toxicology to explore the interaction between genes and the environment, it provides better understanding of gene functions in specific biological pathways and enables identifying potential genomic targets. Also, the use of toxicogenomic databases enables screening of toxicants and potential protective agents, finding overlapping pathways and matching them to a specific disease that is also affected by the changes in the same pathways (Boverhof and Zacharewski, 2006; Breda et al., 2014; Liu et al., 2020; Tung et al., 2020). Additionally, computational chemistry tools can facilitate rapid prediction of pharmacokinetic and physicochemical key parameters for examined molecules to elucidate their potential future application (Daina et al., 2017).

Therefore, the aim of this investigation was to: (i) examine the molecular mechanisms of altered signaling pathways in AD linked to the environmentally relevant toxic metals (Pb, Cd, MeHg as an organic/neurotoxic form of mercury and As) and possible beneficial effect of berberine, by applying in silico toxicogenomics analysis, including pathway and process enrichment analysis; (ii) predict cheminformatic key parameters for the targeted molecule, berberine, to evaluate previously obtained results; and (iii) demonstrate the potential of in silico tools to obtain preliminary screening results that could serve as a good reference point for further research.

Section snippets

CTD analysis

Based on our previous results (Živančević et al., 2021), further exploration of linkage between AD development and exposure to environmentally relevant toxic metals (Pb, Cd, MeHg and As), as well examination of the potential beneficial role of berberine in combating AD, was performed by Comparative Toxicogenomics Database (CTD; http://CTD.mdibl.org). The reported chemical–gene/protein interactions analysis presents the data downloaded in September 2021. CTD is a publicly available,

Results

CTD data curation has revealed the genes related to each of the examined toxic metals and linked to AD development (Table 1). Targeted gene sets were retrieved from CTD “Disease” data-tabs for each investigated toxic metal. Gene set for berberine was related to its therapeutic properties (annotated in the “Direct Evidence” tab). Thereafter, the mutual genes for toxic metal mixture and berberine were obtained via MyVenn CTD tool. Data-mining revealed that Pb, Cd, MeHg and As affected 39, 51, 22,

Discussion

Despite the extensive research, the etiopathology of AD remains largely unknown, while no effective treatment is currently available. Alzheimer's Disease is characterized by degenerative process of damaging cholinergic neurons across the brain regions and, therefore, has detrimental effects on conscious awareness, attention, and memory function (Krauskopf et al., 2020). In the development of AD, extracellular aggregates of Aβ peptides result in disrupted cell functioning (Krauskopf et al., 2020

Conclusion

This investigation has suggested that berberine exhibits its potential protective effect in AD induced by the toxic metal mixture via following genes: ACHE, APP, BAX, BCL2, CASP3, HMOX1, IL1B, MAPT, SOD2 and TNF. Individual toxic metals mainly exhibited expression and activity opposite of the regulation by berberine in the case of APP, IL1B MAPT, SOD2 and TNF. Surprisingly, individual toxic metals exhibited similar expression and activity as berberine in the case of ACHE, BAX, BCL2, CASP3 and

Funding

This work was partially supported by The Ministry of Education, Science and Technological Development of the Republic of Serbia (451-03-68/2020-14/200161).

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

CRediT authorship contribution statement

Katarina Živančević: Conceptualization, Methodology, Formal analysis, Investigation, Data curation, Software, Writing – original draft, Visualization. Katarina Baralić: Methodology, Supervision. Dragica Bozic: Methodology, Supervision. Evica Antonijević Miljaković: Supervision. Aleksandra Buha Djordjevic: Supervision. Marijana Ćurčić: Supervision. Zorica Bulat: Supervision. Biljana Antonijević: Supervision, Project administration, Funding acquisition. Petar Bulat: Supervision. Danijela

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.

Acknowledgement

The authors gratefully acknowledge the support from The Malaysian Society of Toxicology (MySOT) and the International Union of Toxicology (IUTOX) in the form of the CTDC11 Fellowship Award for attending the 11th Congress of Toxicology in Developing Countries where the abstract of this investigation was presented. The authors gratefully acknowledge the support from the Serbian Society of Toxicology (SETOX).

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