Melatonin inhibits Benzo(a)pyrene-Induced apoptosis through activation of the Mir-34a/Sirt1/autophagy pathway in mouse liver
Graphical abstract
Introduction
Benzo(a)pyrene (BaP), a polycyclic aromatic hydrocarbon, is formed from the incomplete combustion of organic matter at temperatures between 300° and 600 °C. The ubiquitous compound is found in coal tar, tobacco smoke and many foods, especially grilled meats. The dietary intake of BaP per day by the general population is different in various communities. For example, the daily intake of BaP in the USA and Korea is estimated to be 2.2 μg/day and 124.55 ng/day, respectively, whereas an inhabitant of East Germany could approximately consume 1.0–3.3 μg/day of Bap (Fritz, 1972; Hattemer-Frey and Travis, 1991; Lee and Shim, 2007). The epoxide metabolites (BPDE) of BaP can bind to DNA, form DNA adduct and finally, caused to mutation (Shiizaki et al., 2013). For this reason, it is listed as a group 1 carcinogen by the International Agency for Research on Cancer (IARC) (Baird et al., 2005; Phillips and Venitt, 2012). BaP can be absorbed through oral, inhalation, and/or dermal exposure (Ramesh et al., 2001). After absorption, BaP is distributed primarily into the liver, kidneys, and the urinary bladder (Yamazaki et al., 1987). The primary site of BaP toxicity and metabolism is the liver which contains the majority of the enzymes required for bioactivation of BaP (Delgado-Roche et al., 2019).
As part of the BaP metabolism process, reactive oxygen species (ROS) are generated resulting in oxidative damage of intracellular macromolecules such as DNA and proteins, alterations in cellular structures and cell cycle progression (Guo et al., 2015). Oxidative stress and mitochondrial dysfunction are important mechanisms involved in BaP-induced hepatotoxicity (Ji et al., 2016), ultimately leading to cytotoxicity and ROS-mediated apoptotic cell death (Guo et al., 2015). BaP also is capable of inducing apoptosis via other mechanisms including DNA damage-related p53 pathway (Gao et al., 2011) and/or covalently metabolic intermediates-nuclear DNA binding (Stolpmann et al., 2012). Oral exposure to BaP has been reported to active a p53 DNA-damage response, which involves angiogenesis, induction of apoptosis, and growth signals (Labib et al., 2012).
Apoptosis or programmed cell death is a highly regulated process that results in cell death with energy consumption (Elmore, 2007). Caspases and the Bcl-2 family are important proteins, which are essential to activate apoptosis pathway (Cory and Adams, 2002; Pop and Salvesen, 2009). In addition, Bnip3 (BCL2/adenovirus E1B 19 kDa protein-interacting protein 3) is an apoptotic Bcl-2 protein (Zhang and Ney, 2009), which can modulate the permeability state of the outer mitochondrial membrane resulting in increased ROS production, mitochondrial depolarization and cytochrome c release. Bnip3 is a pro-apoptotic protein which can induce cell death and apoptosis (Burton and Gibson, 2009). Previous studies have shown BaP-induced apoptosis in different organs such as liver, lung and brain (He et al., 2016; Qin et al., 2015; Sakthivel et al., 2019). It has been demonstrated that BaP-induced apoptosis could regulate by several natural compounds including catechin and resveratrol (Banerjee et al., 2016; Shahid et al., 2016).
Melatonin (5 methoxy-N-acetyltryptamine) is an endogenous hormone synthesized from the amino acid tryptophan and circulating melatonin is produced primarily by the pineal gland at night (Tan et al., 2015). The melatonin synthesis and secretion is enhanced by darkness while inhibited by light. Melatonin is involved in the regulation of circadian rhythms such as the neuroendocrine rhythm, the sleep-wake rhythm, and the body temperature cycle (Karasek and Winczyk, 2006; Tordjman et al., 2017). The effects of melatonin on blood pressure (Scheer et al., 2004), autonomic cardiovascular regulation (Nishiyama et al., 2001), retinal function (Iuvone et al., 2005), and regulation of the immune system (Carrillo-Vico et al., 2005) have been reported. In addition, melatonin has several pharmacological activities such as hepatoprotective (Oleshchuk et al., 2019; Sheen et al., 2016; Tunon et al., 2011), neuroprotective (Alghamdi, 2018), gastroprotective (Brzozowski et al., 2005), and anti-cancer effects (Asghari et al., 2017). Furthermore, melatonin has been shown to exhibit a potent antioxidant activity through its ability to scavenge free radical oxygen species, an indirect effect that results in reduction of nitric oxide formation or induction of antioxidant enzymes, thereby protecting cells and tissues against oxidative stress damage (Galano et al., 2013; Reiter et al., 2016). Other studies have proposed that melatonin has a protective role through modulation of inflammation (Mauriz et al., 2013), apoptosis (Bizzarri et al., 2013; Tunon et al., 2011), and autophagy (Roohbakhsh et al., 2018; San-Miguel et al., 2015; Stacchiotti et al., 2019).
Autophagy, a highly conserved catabolic process, allows the orderly degradation and recycling of cellular components. As part of the autophagy process of supporting cellular homeostasis, the process responds to different conditions including starvation, hypoxia, and oxidative stress (Hashemzaei et al., 2017). Autophagy contributes to degradation of cellular content by the lysosomes which involves removal of cytosolic components, misfolded protein aggregates and damaged or ectopic organelles (e.g., mitochondria and endoplasmic reticulum) (Barangi et al., 2019). Generally, autophagy is considered a protector and survival response to starvation or a toxic environment insult and is critical to normal cell physiology. However, sometimes, dysregulated autophagy switches to autophagic (non-apoptotic) programmed cell death (Kroemer and Levine, 2008). Melatonin has been reported to attenuate neuronal apoptosis markers or to increase basal autophagy proteins as a protective pathway in peripheral sciatic nerves and dorsal root ganglion in oxaliplatin-administered rats (Areti et al., 2017). In contrast, pre-ischemia melatonin treatment has showed anti-fibrogenic effects against CCl4-induced fibrosis through inhibition of autophagy (San-Miguel et al., 2015).
The autophagy pathway is mediated by the autophagy-related (Atg) genes. Microtubule associated protein 1 light chain 3 (LC3; mammalian homologues of yeast Atg8) and Beclin-1 (mammalian homologues of yeast Atg6) are two such autophagic proteins that have been reported to contribute to the induction and regulation of autophagy and autophagosome formation (Petibone et al., 2017). Moreover, Beclin-1 interacts with the anti-apoptotic Bcl-2 family and thus regulates autophagy. It has been demonstrated that the Bcl-2/Beclin-1 complex plays a key role in the crosstalk between autophagy and apoptosis (Wirawan et al., 2010). On the other hand, autophagy can be regulated by other factors such as transcription factor activity like NF-κB and Sirtuin activity (Wang et al., 2019).
Sirtuins, a nicotinamide adenine dinucleotide (NAD+)-dependent protein, contains either mono-ADP-ribosyltransferase activity or deacylase activity, including deacetylase, desuccinylase, demalonylase, demyristoylase, and/or depalmitoylase activity (Mayo et al., 2017). Sirtuin 1 (silent information regulator 1; Sirt1), one of seven isoforms, occurs both in the cytosol and nucleus of mammals (Tanno et al., 2007). Sirt1 plays a pivotal role in antioxidant defense (Hariharan et al., 2010), inflammation (Carloni et al., 2016), autophagy (Jang et al., 2012), and apoptosis (Zhang et al., 2016). It was reported that melatonin enhanced Sirt1 and autophagy proteins, but reduced inflammation and apoptosis markers in brain injury (Carloni et al., 2016). Recently, Ren et al. illustrated that melatonin could ameliorate hepatocellular damage induced by chronic intermittent hypoxia via activating Sirt1-mediated autophagy pathway (Ren et al., 2019).
Sirt1 expression in various cellular processes is modulated by microRNAs (miRNAs), small endogenous and highly conserved noncoding RNAs (~22 nucleotide in length) (Karbasforooshan et al., 2018). MicroRNAs modulate post-transcriptional gene expression by targeting mRNA and suppressing protein expression (Razavi-Azarkhiavi et al., 2017). Among the various miRNAs, miR-34a is known to inhibit the expression of Sirt1 protein in different tissues under various pathophysiological processes (Carloni et al., 2016; Karbasforooshan and Karimi, 2018). MiR-34a inhibits Sirt1 expression and increases the acetylation of p53; ultimately leading to apoptosis in human colon cancer cells (Yamakuchi et al., 2008). Melatonin has been reported to trigger autophagy through upregulating Sirt1 and downregulating miR-34a in non-alcoholic fatty liver disease (Stacchiotti et al., 2019) and in a neonatal brain inflammation model (Carloni et al., 2016).
Induction of oxidative stress, followed by apoptosis, is an important mechanism in BaP-induced hepatotoxicity. Since no information was found on the protective effect of melatonin as well as autophagy pathway on BaP-induced hepatotoxicity, we undertook a study to investigate the interaction between melatonin and the miR-34a/Sirt1/autophagy pathway as related to BaP-induced hepatotoxicity. The study investigated whether melatonin had a protective effect against liver injury in BaP treated mice and whether the attenuate BaP-induced oxidative stress and apoptosis were mediated through the miR-34a/Sirt1/autophagy pathway.
Section snippets
Chemicals and reagents
Benzo(a)pyrene (C20H12, >96% purity; sc-257,130) and melatonin (C13H16N2O2, >99% purity; sc-207848) were purchased from the Santa Cruze Biotechnology Company, USA; potassium chloride (KCl), phosphoric acid, and thiobarbituric acid (TBA) were purchased from the Merck Company, Germany; 5, 5′-Dithiobis 2-nitrobenzoic acid (DTNB) and trichloroacetic acid (TCA) were purchased from the Sigma Company, Germany. All of used antibodies were purchased from Cell Signaling Company, USA.
Animals
Thirty male Razi mice
BaP and melatonin effect on the liver enzymes activity
The results in Fig. 1 show that after BaP treatment, the serum AST and ALT activity (IU/L) significantly increased compared to controls (P < 0.0001). Melatonin at a dose of 10 mg/kg did not decrease these elevated levels; however, 20 mg/kg of melatonin statistically attenuated the AST and ALT activity comparing to the BaP treated group (P < 0.001). There were no significant changes in the activity of AST and ALT in mice administered only melatonin compared to control group.
BaP and melatonin effect on MDA and GSH content in mouse liver
MDA is a marker of
Discussion
The current study investigated whether melatonin had the potential to reduce hepatotoxicity arising from oxidative injury and apoptosis induced in mice by BaP through the miR34a/Sirt1/autophagy signaling pathway. BaP (75 mg/kg, orally, 28 days) increased apoptosis following enhancement of ROS production and thus, induced hepatotoxicity in mice, which was confirmed by assessment of oxidative stress markers and histopathology. Moreover, BaP could reduce autophagy markers and miR-34a expression
Conclusion
In conclusion, the enhancement of oxidative stress and apoptosis as well as the reduction of autophagy were shown to be involved in the BaP-induced hepatotoxicity in mice. BaP increased the MDA level and induced apoptosis proteins (Bax/Bcl-2 ratio, Bnip3, and caspase-3) and the miR-34a expression, while decreasing the level of autophagy markers (LC3 II/I ratio, Beclin-1, and Sirt1). Most importantly, this study provides evidence for the hepatoprotective effects of melatonin in BaP-related liver
CRediT authorship contribution statement
Samira Barangi: Data curation, Writing - original draft. Soghra Mehri: Conceptualization, Methodology. Zahra Moosavi: Investigation. A. Wallace Hayesd: Writing - review & editing, Validation. Russel J. Reiter: Writing - review & editing, Validation. Daniel P. Cardinali: Writing - review & editing, Validation. Gholamreza Karimi: Conceptualization, Methodology.
Declaration of competing interest
The Authors declare that they have no conflicts of interest to disclose.
Acknowledgements
Authors are grateful to the Vice Chancellor of Research, Mashhad University of Medical Sciences, Mashhad, Iran for financial support.
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