Neuropeptide Y Expression Confers Benzo[a]pyrene Induced DNA Damage and Microtubule Disruption in Human Neuroblastoma SH-SY5Y Cells

Benzo[a]pyrene (B[a]P), is a family member of polycyclic aromatic hydrocarbons and a widespread environmental pollutant and neurotoxicant that contribute to the development of cancer. Microtubules are polymers of tubulin that form part of the cytoskeleton and target for anticancer drugs. Furthermore, NPY significantly increased the percentage of cells in S and G2/M phases. However, little is known about the specific role of NPY in proliferation and the underlying protective mechanism remains unclear. Hence, the aim of this work was to investigate the effect of B[a]P on SH-SY5Y neuroblastoma cells and to explore the potential mechanism for alteration of tubulin-microtubule equilibrium causing mitotic arrest and NPY expression. The present findings showed B[a]P treatment significantly increase number of SH-SY5Y cells in S and G2/M phase as compared to G1 phase and provokes cell cycle arrest that correlated with significant decrease in G0/G1 cells. Immunofluorescence study showed significantly distorted tubulin arrangement from metaphasic plate in formation of bipolar mitotic spindle apparatus. Further, higher doses of B[a]P treatment lead to chromosomal abnormalities accompanied by DNA damage due ROS causing oxidative stress showing significant decrease in tubulin protein around spindle. The results of present study demonstrated that NPY exerts a proliferative and protective effect on B[a]P-induced oxidative stress in a dose-dependent manner in vitro and importantly, these effects may be mediated via mitotic arrest and involved in spindle arrangement during cell division. Our findings addresses a novel pathological outcomes of B[a]P-induced NPY expression by oxidative stress through spindle abnormalities leading to microtubule disruption.


Introduction
Rising pollution load in the life sustaining ambient environment are primarily due to anthropogenic activities that might bring its adverse consequences on living organisms. The compounds like polycyclic aromatic hydrocarbons (PAHs) are reported to add a significant load of pollutants through incomplete combustion of fossil fuels [1,2]. Benzo[a]pyrene (B[a]P), a prototype of PAH is well known for its carcinogenic or mutagenic role [3,4], however in past decades, its neurotoxic potential is also well documented [5,6,7]. The neurotoxic potential of B[a]P is mainly achieved through neuromorphological alteration, cell cycle arrest, oxidative DNA damage, etc [8,9,10].
The environmental neurotoxicant like B[a]P undergoes metabolic breakdown into more potent toxicants by cytochrome P450 (CYP) enzymes and thus involves in formation of DNA adducts [11,12]. Metabolic activation of B[a]P and CYP induction are responsible to enhance oxidative stress conditions increasing physiological generation of reactive oxygen species (ROS) [13,14]. This anomalous condition in cell milieu induces changes in the cell cycle, DNA damage, chromosomal and mitotic aberrations.
Several studies have highlighted B[a]P-induced ROS production as a consequence of the ability of its metabolites to enter redox cycles via direct and indirect mechanisms [15]. Because of the critical role of free radicals and ROS in the pathogenesis of cancer [16], the enhancement of oxidative stress resulting in the interaction between B[a]P metabolites and antioxidant enzyme activity [7]. The potential role of B[a]P inducing cell cycle arrest through cytoskeletal deformation like microtubule disruption is well reported [17,18]. Microtubules are arranged in a bipolar array with less dynamic minus-ends embedded at the pole and more dynamic plus-ends extending towards the spindle equator and cell cortex [19]. During early mitosis, the nuclear envelope breaks down; microtubules invade the nuclear space and attach to chromosomes at the kinetochore [20]. B[a]P induces microtubule disruption, but the cause behind this manifestation is limiting in literature. Microtubules are highly dynamic structures, which consist of α-and β-tubulin heterodimers and are involved in cell movement, intracellular trafficking, and mitosis with regulation of determination of cell morphology and differentiation.
The SH-SY5Y cells can be successfully differentiated to post-mitotic and more mature neuronal phenotypes. During several decades, the SH-SY5Y cell line has been used as an in vitro model system, detecting the impact of toxicants on mature and developing neurons [21]. Human neuroblastoma SH-SY5Y cells are immature neuroblasts characterized by their low differentiation, pyramidal shape, and obvious axons [21]. SH-SY5Y is a reliable model for studying the neurotoxic effect of B[a]P and for elucidating the mechanisms of NPY expression and as functional markers of neurons [22]. SH-SY5Y cells share some physiological functions with normal neurons [23]. Moreover, an increase in NPY production in the SH-SY5Y cell line can be associated with neurological diseases [24]. B[a]P alters the cytoskeleton, which is made up of three kinds of protein filaments: actin filaments, intermediate filaments and microtubules, and other associated proteins (tubulin) interfere with the tubulinstabilizing process [25]. During mitosis B[a]P metabolites hinders the formation of the mitotic spindle and causes DNA damage and it may also modulate proteins involved in the intracellular transduction pathways. B[a]P also interferes with the assembly of cell microtubule and, in dividing cells, arrests the cells in the G2-M phase. An abnormal spindle structure can be a consequence of B[a]P-induced DNA damage or directly originated by spindle poisons leading to cell cycle arrest [25]. B[a]P-induced multipolar spindle and mitotic arrest coupled to reduced cell proliferation and also increased apoptotic death through mitosis. Alterations in the mitotic spindle apparatus play a major role in the generation of genomic instability through promoting chromosome mis-segregation and aneuploidy.
NPY is the most abundant neuropeptide in the mammalian central nervous system and plays important roles in different pathological conditions such as obesity, anxiety, depression, cognition, epilepsy, chronic pain and other neurodegenerative disorders [26][27][28]. It is usually found that in the peripheral nervous system, NPY is coexists with norepinephrine in the large dense-core vesicles at nerve endings of the sympathetic system. A significant body of literature argues that the arcuate nucleus NPY neurons are involved in energy homeostasis [29]. Whereas, limited information exists regarding the relationship between metabolic activation of B[a]P and NPY over expression causing oxidative stress leading to cell cycle arrest, microtubule distortions and spindle arrangement during cell division. Elevated neuronal activity results in the altered expression of NPY and tubulin protein molecules that may ultimately lead to alterations in the morphology and physiology of hippocampal neurons [30]. These changes may reflect adaptive responses to physiological activation of neurons. NPY has been shown to be present within the neurons of the hippocampus [31,32]. We previously demonstrated that endogenous increase in NPY over expression in hypothalamus and hippocampus was associated with increase body weight oxidative stress following B[a]P administration (i.v.) due its orexigenic effects in rats [33,34].
Hence, the present study was conducted to know whether B[a]P induced DNA damage and cell cycle arrest causing microtubule disruption plays a role in NPY expression in neuroblastoma cells. SH-SY5Y cell lines were differentiated by 10 µM retinoic acid, maintained and treated with different doses of B[a]P (2 and 4µM) for 24 hour. It has been hypothesized that B[a]P-induced oxidative stress and DNA damage leading to cell cycle alterations could be due to different mechanisms that involve the dynamic nature of tubulin protein resulting NPY expression in SHSY5Y cells.

SY5Y cells
After the Undifferentiated (UD) and differentiated (D) SH-SY5Y cells were

Effect of B[a]P on DNA damage using the comet assay in SH-SY5Y cells
The DNA damaging effect of B[a]P on SH-SY5Y cells was evaluated by single cell gel electrophoresis; which compares the percentage of DNA in comet tail with tail moment, a measurement of DNA damage ( Fig. 2A). In separate sets of experiment, the

SHSY5Y cells
The organization of the mitotic spindle was examined by indirect immunofluorescence using an antibody to α-tubulin and DAPI that stains chromatin.
Immunofluorescence staining results revealed that NPY expression significantly

Chemicals
The chemicals used in this experimentation were procured from Sigma-Aldrich Chemicals (St. Louis, MO, USA), unless otherwise mentioned. The quantification of NPY was carried by peptide enzyme immunoassay kit (Bachem, Peninsula Laboratories Inc., San Carlos, CA).

Benzo[a]pyrene Treatment
The B[a]P was solubilised in DMSO and the working solution of 10 mM concentration was prepared in cell culture media. In our study, we used both

Single cell gel electrophoresis (Comet assay)
The comet assay was carried out under alkaline conditions as reported earlier [35] to evaluate DNA strand breaks in individual SHSY5Y cells. Microscope slides were pre-coated with 0.75% normal melting agarose and stored at 4 0 C. The cells were grown for 24hrs in DMEM medium supplemented with 10% FBS, incubated at 5%

Effect of Benzo[a]pyrene on phases of cell cycle by flow cytometry
The effect of B[a]P treatment on cell cycle was evaluated on the basis of cellular DNA content as reported earlier [35]. SH-SY5Y was grown in 60mm plates containing DMEM and 10% FBS at 37ºC in 5% CO 2 for 24hr. After the cells were confluent, they were washed twice with PBS and then treated with B[a]P for 24hrs.
After treatment, cells were trypsinized, rinsed with PBS, and fixed in ice-cold 70% ethanol for one hour at 4ºC. Fixed cells were then centrifuged for 5min at 3000 rpm to obtain cell pellet, which was re-suspended in 1ml DNA staining buffer [50 µg

Effect of Benzo[a]pyrene on spindle morphology by immunofluorescence
Immunofluorescence analysis was performed as previously described [35].

NPY expression in SHSY5Y cells after Benzo[a]pyrene treatment by immunofluorescence analysis
Cultured SHSY5Y cells after B[a]P treatment were subsequently fixed by applying ice-cold methanol and acetone (1:1) for 10 minutes at 20°C, blocked, and incubated with the monoclonal rabbit anti-NPY antibodies (1:500 diluted with 1% BSA in PBS; Millipore, Bedford, MA) at room temperature for 1 hr. After being washed with PBST three times, the cells were incubated with phycoerythrin (PE)-conjugated secondary antibody (1:300 in 1% BSA in PBS) for 1 hr at room temperature. The cells in cover slips were washed once with PBST, followed by PBS; then cells were next counterstained with DAPI. Finally, the cover slip edges were sealed and microscopic images (Carl Zeiss, Jena, Germany) were taken by fluorescence microscope equipped with a charge-coupled digital camera and fluorescence intensity was estimated by using Image J software.

Statistical analysis
Data have been expressed as mean ±SEM and were analyzed for statistical significance by applying one-way analysis of variance (ANOVA) and post hoc analysis was done by Newman-Keul's test (Sigma Stat software, Jandel Scientific USA). The number of replicates for each experiment was three and difference below the probability level p< 0.05 was considered statistically significant.

Discussion
In this study, we show the temporal expression of the neuronal peptide NPY in SH-SY5Y human neuroblastoma cell lines, advancing previous studies on NPY expression in wistar rats (6,27). This study was conducted on SH-SY5Y human neuroblastoma cell lines to ascertain the possible link between the B[a]P induced oxidative stress and cycle arrest causing NPY expression with microtubule disruption due to ROS production. NPY is known to play an important role in neuroprotection.
The advantages of using a clonal cell culture, such as SH-SY5Y cells, are that cultures will be consistent, easy to maintain, possess a human phenotype. Hence, it was hypothesized that B[a]P affect tubulin filaments via different mechanisms (e.g. by inducing oxidative stress) with the sequestration of the damaged tubulin monomers and part of the filaments in the granules in SH-SY5Y cells [36]. The use of two different concentration of B[a]P (2 µM and 4 µM) in the present study are higher than the actual concentrations (0.03-0.09 ng/g wet tissue) reported for healthy humans [37,38]. These concentrations were chosen because of their relevance to environmental levels of these PAHs in hazardous waste sites, former industrial sites, and exposure concentrations for 'at risk' populations such as smokers and occupationally exposed individuals [39,40].
The phase contrast images of SHSY5Y cultures at control cells showed no particular neurite outgrowth and the cells have a round shape, whereas B[a]P-treated cells showed significant number of short neurites. The result also showed differentiated SH-SY5Y cells respond differently to toxicants like B[a]P in higher and lower doses with different cell morphology. The present findings addresses the augmented expression of NPY in rat brain following exposure to B[a]P and its correlation with the orexigenic effects depending on the antioxidant activity [15,8,37].
Previous reports suggested that B[a]P exposure leads to cell cycle arrest with significant increase in the number of cells in S-phase of cell cycle [41,42]. Importantly, from late S phase to mitotic onset, the two fully duplicated centrosomes exhibit enhanced recruitment of pericentriolar material components that are essential for microtubule nucleation, a process termed centrosome maturation [41]. When accumulation of pericentriolar material becomes inaccurate at the centrosome, results in a functionally compromised mitotic centrosome with multipolar or disorganized spindles that may further promote mitotic arrest and cell death [41]. The result showed Previous studies showed the central NPY exerts protective effects under stress and is known to play an essential role in the basic mechanisms of stress tolerance by reducing anxiety and depressive-like behaviours [45,46]. B[a]P significantly increases NPY levels in the hypothalamus, and the hippocampus, which may leads to alteration in behavior [47,48]. Hence, B[a]P-induced neurotoxicity might provides new insight into the induction of serious neurodegenerative diseases following exposure to chemical pollutants that produced due to incomplete combustion of fossil fuel causing increasing load of anthropogenic neurotoxicants [49]. The present study also provide for the first time an experimental evidence that B[a]P exposure may stimulates the neuroblastoma SH-SY5Y cell lines to release functionally active NPY causing cell cycle arrest in Sphase by microtubule disruption and spindle formation that might be achieved through oxidative stress and ROS induced spindle abnormalities during cellular metabolism through intracellular calcium [50,51]. These findings identified a novel mechanism by which ROS regulate NPY expression and indicated a potential molecular target for the treatment of oxidative stress-related neurodegenerative diseases.

Conclusion
In conclusion the endogenous NPY release into the hippocampus may induce a compensatory mechanism for neuronal survival after the oxidative damage induced by exposure to B[a]P in SH-SY5Y cell. The possible mechanism of this protection is that NPY may prevents human neuroblastoma SH-SY5Y cells from achieving mitosis and microtubule assembly inhibiting SH-SY5Y cell proliferation by a novel mechanism.
However, NPY may not just be a mere marker of microtubule disruption during development and in metabolic stress. The present findings suggest that NPY may serve (n=3) (Fig 1B). Data were shown as mean ± SEM, * indicates significant difference (*p < 0.05) respect to comparison between groups.

Figure 2. Effect of B[a]P-induced DNA damage in SH-SY5Y cells
Representative photomicrograph of PI fluorescence of SH-SY5Y cells showing DNA damage with comet head and tail (Fig 2A). DNA damage was shown after 24hrs of different doses of B[a]P treatment with comet tail having fragmented DNA and extensive DNA migration indicating B[a]P induced DNA strand breaks where as control cells showed intact very bright nuclei with slight or no DNA migration in SH-SY5Y ( Fig 2B) and extent of DNA migration was quantified in tail moment length.
Histogram for tail moment length in SH-SY5Y cells under control and different doses of B[a]P treated conditions were also shown ( Fig 2C). The tail moment was calculated as a product of tail length and fraction of the DNA in the tail using the image analysis system of Comet Score™ v1.5 (TriTek Corp., USA). Fifty randomly chosen comets were analysed in each group and data are expressed as the mean ± SEM from three independent experiments (n=3). Significant difference (*p<0.05); Scale bar: 40 μm.