Identification, Quantification and In-Vitro Genotoxicity of Major Polyaromatic Hydrocarbons Produced by Sugarcane Fly Ash Emitted from Sugarmill

Sugarcane burning during harvest and non-harvest season emits various pollutants like volatile organic compounds (VOCs), alkanes, and PAHs (Polya-romatic hydrocarbons) in the surrounding environment. Among these pollutants, PAHs are of uttermost concern due to their high level of toxicity. Burning of sugarcane bagase in sugar mill results in the production of fly ash. Fly ash is produced as a result of sugarcane bagasse burning in sugar mills. In present study, fly ash that comes out from the sugar mill chimney was collected from Western Uttar Pradesh, India and used for further analysis. High temperature and incomplete combustion inside chimney lead to the formation of PAHs. Extraction of PAHs present in fly ash samples was done by ultrasonication method and was identified with GC-FID (gas chromatogra-phy-flame ionization detector). Results exhibit the presence of eight PAHs in fly ash samples where the Benzo(a)pyrene and Naphthalene were found to be in high concentration. Furthermore, we have evaluated toxic effects of fly ash and Polyaromatic hydrocarbons (Standard of BaP & Nap) through different methods i.e. MTT, ROS and comet assay. Significant reduction (p < 0.001) in cell viability was noted in cells treated with fly ash as compared to control. Fly ash samples were also found to induce significant oxidative stress in HeLa cells, which ultimately causes DNA damage. Therefore, it may be concluded that the fly ash samples are toxic to the environment due to the presence of PAHs. Hence, the present study plays an important role in determining the harmful effects of PAHs and their source of occurrence.


Introduction
India is the second largest producer of sugarcane (341,200 TMT) after Brazil (739,267 TMT harvest) [1]. In India, Maharashtra and Uttar Pradesh are the leading states in terms of sugarcane production. Sugar industries in Uttar Pradesh are one of the largest sugar industries in the Indian economy. Currently, there are 28 sugar mills in Uttar Pradesh, out of which 6 are located in the western zone, 8 are in eastern zone and rest 14 are in the central zone. Thus, there are 23 sugar factories which are functional in Uttar Pradesh in the co-operative sector having a total crushing capacity of 60,000 TCD [2].
During harvesting season sugarcane is used in the sugar making process. Bagasse is the remaining crushed sugarcane stalk after the juice extraction and is used in sugar mills as fuel to generate electricity and eventually emitted from chimney in the form of fly ash [3]. Burning of bagasse also leads to the emission of many gaseous pollutants and particles into the environment which has an adverse impact on the plantation and health of neighbouring residents [4].
The particles released from sugar mills have similar properties to the particles emitted from combustion and fuel burning. Organic carbon aerosols discharged during the combustion process consist of various compounds, out of which those having three or more fused aromatic rings are polycyclic aromatic hydrocarbons (PAHs) [5]. There are basically two sources of PAHs emission in the atmosphere: natural and anthropogenic. As estimated, the total global atmospheric emission of PAHs is 520 Gg/year comprising of biofuel (58%), wildfire (17.0%), consumer product usage (7%), traffic oil (5%) and domestic coal (4%) as the major sources in 2004 [6].
In general, the information is limited regarding pollution caused by the burning of sugarcane and its impact on flora and fauna; even if available most of it relates to plantations of sugarcane in Brazil. Previous research has reported the existence of PAHs in various sugarcane products. Sugarcane juice is the main source of benzo(b)fluoranthene, benzo(a)pyrene, Benzo(a)anthracene, and benzo(k) fluoranthene. Burning of sugarcane during the harvest season contributes towards increased levels of PAHs. Different types of PAHs such as dibenz[a,h]anthracene, benz[a]anthracene, benzo[k]fluoranthene benzo [b]fluoranthene and benzo[a]-pyrene have been reported in Cachaca, an alcoholic beverage common in Brazil.
Studies suggest that PAHs contamination in cane sugar is the result of the burning of sugarcane during harvest season and residues of PAHs in final products depend on the processing and refining processes [7]. Sugarcane burning leads to the formation of fly soot which itself is a source of a large number of PAHs [8]. Along with the PAHs, sugarcane fly ash is also the major source of various elements and Elemental analysis can be done by EDXRF technique [9].
PAHs have various health effects on human beings; chronic effect includes decreased immune function, kidney and liver damage (e.g., jaundice), cataracts, breathing problems while acute health effects include nausea, diarrhea, vomiting, and eye irritation. Naphthalene, BaP, and benzo (a) anthracene exposed experimental animals show embryotoxic effects [6]. Previous studies have shown that PAHs induce intracellular ROS through intracellular aldo-keto reductase activity which in turn converts PAH-metabolites to PAH-o-quinones which are strong electron acceptors [10].
In this study, we measured the concentration of PAHs in fly ash of sugarcane bagasse and investigated their effects on the cell line (HeLa). Cell viability, generation of reactive oxygen species and DNA damage at various concentrations were studied. Studies done by Mackay et al. show that two and three-ring PAHs have the highest water solubility and are cytotoxic in nature [11]. In a risk assessment conducted for PAHs, naphthalene was found to be a harmful compound due to its direct cytotoxic action [12]. Therefore, among all PAHs Benzo (a)pyrene (BaP) and Naphthalene (Nap) were used as positive control.

Sampling
Sugarcane fly ash sampling was done from Kisan Sahkari Chini Mill of Bisalpur ( Figure 1), located in Bareilly, western Uttar Pradesh, India in the month of February 2015. It was established in 1977-78 and its capacity is 2750 TCD (Tones of Cane per day). Its total Cane Area is 9549 hectare [2]. Bisalpur is located at 28.3˚N 79.8˚E. It has an average altitude of 156 meters (512 ft). Fly ash samples were collected randomly from the soil surface within a distance of 15 -20 m from the sugar mill chimney which was further mixed to form a composite sample. After that, the sample was wrapped in an aluminum foil then sealed in pre-coded zip-loc polyethylene bags for carrying to the laboratory. Before processing, undesirable fractions of coarser particles were removed from the samples and then samples were kept in desiccators to avoid moisture.

Sample Preparation and Clean up Procedure
Sample preparation was done as per the method described by Sun et al., 1998  [13]. Extraction was done by ultrasonication method followed by solid phase extraction (SPE) clean-up. In the ultrasonication method, 10 ml dichloroform (DCM) containing 1 g of fly ash sample was sonicated for 30 minutes. The sample was centrifuged at 12,000 rpm for 5 min and the supernatant was collected.
The same procedure was repeated again by addition of 10 ml of fresh DCM. Finally, the sample was filtered through 90 mm GF/C glass microfiber filter paper (Whatman International, UK). Clean up the procedure of the sample was done by the protocol described by Zamperlini et al., 2000 [14]. Silica column was loaded by 5 ml of fly ash sample. Additional 5 ml methylene chloride was loaded and an aliquot was collected and concentrated to 1 ml under N 2 stream and stored in GC vials for further analysis by GC-FID.

Sample Preparation for EDXRF
Fly ash samples were grinded in mortar and pestle to make a homogeneous fine powder. Fly ash sample was mixed with boric acid (as a supporter base) and then by applying 10 tons pressure on the sample, pressed powder samples were prepared. The elemental concentration was evaluated in 1 g sample using energy dispersive X-ray fluorescence (EDXRF) spectroscopy (Epsilon5 PANalytical) [15].

Cell Line and Culture Conditions
HeLa cells were obtained from National Centre for Cell Science (NCCS), Pune.

Comet Assay
After treatment of cells with BaP, Nap and fly ash samples for 24 h, harvesting and fixation of cells using 1:3 glacial acetic acid/methanol was done and then finally the cells were washed with PBS. Comet assay (single cell gel electrophoresis) was employed to ascertain the damage in DNA. Single cell gel electrophoresis was carried out according to the protocol described by Meena, R. & Paulraj, R. [15]. In brief, microscopic slides were coated with 0.5% agarose and 10 µl single cell suspensions of treated cells were implanted in the coated layer of agarose. The prepared slides were dipped in a jar filled with cold lysis solution (2.5M NaCl, 100 mM EDTA disodium salt, 1% Triton X, 10 mM Tris HCl, pH-10) and after that were incubated at 4˚C for two hours. In order to enable unwinding of DNA, the slides were immersed in electrophoresis buffer (75 mM NaOH, 1 mM EDTA, pH > 12) and left for 20 minutes. After that, electrophoresis was carried out at 25 V, 300 mA for 20 min. DNA present in each cell was neutralized with 0.4 M Tris buffer (pH 7.5) and then stained using ethidium bromide. Cellular DNA damage in cells was observed under the fluorescence microscope (Carl Zeiss, Germany). Slides were prepared for different groups and overall 50 cells from each slide were chosen in a non-specific way for further analysis. Evaluation of results was done using Comet IV software (Perceptive Instruments Ltd, UK).

Reactive Oxygen Species (ROS)
2',7'Dichlorofluorescein-diacetate (DCFHDA) dye was used to measure the generation of intracellular ROS (reactive oxygen species) generated in HeLa cells. To measure the intracellular ROS being generated in separate cells, 5 × 10 5 cells were seeded over coverslip in 6-plated well and incubated overnight for allowing the attachment. Following day, cells were treated with fresh media containing 100 ppb of BaP, Nap and fly ash. Cells were left for incubation for 4 hrs at 37˚C. On completion of the incubation period, the coverslip was removed from the culture plate and was stained with 40 μM with DCFHDA for about 30 min. Surplus dye was cleansed-off using 1X PBS. Coverslip was fixed on a glass slide and was observed under a fluorescence microscope (Nikon ECLIPSE TiE, Tokyo, Japan) [16].

Statistical Analysis
Statistical analysis was done using Graph pad prism 5.03. Data were expressed as mean ± SD, statistical analysis was done using two-way ANOVA and Bonferroni post tests. The p values < 0.01 and p < 0.001 were considered significant with respect to their control counterpart. All in vitro experimental assays were performed in triplicates.

Identification and Quantification of PAHs
PAHs are semi-volatile compounds having two or more aromatic rings. Burning of bagasse in the boiler leads to fly ash formation. Further identification and quantification of fly ash samples were done by GC-FID in split mode. Figure  2(a) and Figure 2 Table 1 and Table 2

ED XRF
The Inorganic compounds present in sugarcane fly ash were analyzed by EDXRF and the result shows that SiO 2 is having the maximum concentration (79%) (  [20].  [21]. Previous study has shown that fly ash generated from the coal-fired power station is a source of highly toxic metals like As, Cd, Pb, etc. in addition to the metals that are crucial for health but are present in trace amount. A similar finding has been found in this study also which shows the presence of As, Pb, Rb, Cu, etc and other elements that are toxic in nature [22].
A. Verma et al.

Cell Viability
The cytotoxicity of sugarcane fly ash sample, Benzo(a)pyrene (BaP) and Naphthalene (Nap) samples was evaluated by measuring the cell viability of HeLa cells using MTT assay. Cell viability was found to be decreasing in a dose-dependent manner in pure standards of BaP, Nap, and Fly ash samples. BaP-standard was found to be most cytotoxic as it induces significant (p < 0.001) toxicity in all doses except 5 ppb conc. while Nap was found to significantly reduce (p < 0.001) cell viability in high doses i.e. 50 and 100 ppb (Figure 3). Fly ash samples were found to induce significant (P < 0.001) toxicity in 50 and 100 ppb doses as com- through apoptosis [23]. In contrast to this, Schirmer et al. have shown that naphthalene causes the highest direct cytotoxicity among the 16 studied PAHs due to its increased water solubility and lipophilic properties [12].

Generation of Intracellular ROS Levels
The (P < 0.001) increase in oxidative stress in HeLa cells as compared to its control.
ROS level was found to increase in higher doses i.e. 50 and 100 ppb. Our positive control samples i.e. Nap and BaP also showed similar results as florescence intensity was higher in positive groups as compared to fly ash sample which suggests that the underlying reason may be attributed to the presence of PAHs. Significant dose dependent increase in oxidative stress in higher doses was reported which indicates that the higher conc. of PAHs was responsible for inducing these changes. Our results are similar to Wilk et al. [24] who reported that PAHs activate the intracellular ROS accumulation which further leads to oxidative DNA damage. In our study, PAHs were found to induce oxidative stress but the intensity was significantly (p < 0.001) higher in pure PAHs standards (BaP and Nap) as compared to fly ash at same concentration. This may be due to decreased concentration of PAHs in fly ash in comparison to pure standards.

DNA Damage
The DNA Damage induced by fly ash was measured through comet assay. The length in fly ash sample was found to be 3 fold higher than control. Fly ash sample which has the lower PAHs concentration recorded the lowest value (77.08 ± 9.2) at 100 ppb concentration as compared to BaP and Nap treated group. However, a lower concentration (5 ppb These results illustrate that the intensity of DNA damage was highest in BaP followed by Nap and Fly ash sample. Our results are in line with Tarantini et al. who performed comet assay of pure B(a)P, Nap and samples made by the extraction of particulate matter from air samples collected in an urban peri-industrial site and concluded that toxicity of Nap is less as compared to B(a)P [25]. Our findings are in concurrence with the previous results which reported approximately 3-times enlargement of tail length as compared to control at higher doses of BaP. The intensity of DNA damage was highest in BaP as compared with other PAHs like fluoranthene, anthracene, pyrene, and phenanthrene [26]. In general, PAHs do not directly induce DNA damage, but are transformed to PAHs metabolites that cause DNA damage through metabolic enzymes and there are 3 major pathways known: the CYP1A1/1B1 and epoxide hydrolase pathway (CYP/EH pathway), CYP peroxidase pathway, and aldo-keto reductases pathway (AKR pathway). Generally, CYPs and other metabolic enzymes mobilized into phenols and catechols metabolize PAHs and quinones resulting in the formation of diol-epoxides, radical cations, or reactive and redox-active o-quinones, all of which may interact with DNA to forms DNA adducts. The reactive metabolites of PAHs may also trigger the development of protein adducts in cells and modify the normal functioning of these proteins [27]. In addition, the metabolites produced by PAHs may initiate increased ROS production which in turn can result in DNA damage, lipid peroxidation or protein denaturation [28] [29]. Results of comet assay showed that DNA damage was maximum in cells treated with B(a)P followed by other PAHs like fluoranthene, anthracene, pyrene, and phenanthrene [26]. Our results show that Fly ash induces the generation of reactive oxygen species which might cause damage to the DNA leading to cell death. In our study, it was reported that the fly ash sample also contains a significant amount of SiO 2 and PAHs which are known to induce DNA damage. Moreover, fly ash was found to induce DNA damage via the oxidative stress in V79 cells in-vitro. The report says that nanosize fly ash and PAHs were found to induce DNA damage which was evaluated through comet assay. Nonetheless, our study is in line with other research which states that the coal and its by-product were observed to induce oxidative stress-dependent DNA damage [30].

Conclusion
From our findings, it may be concluded that fly ash is an important source of PAHs that are cytotoxic and genotoxic in nature. However, their level of toxicity was less as compared to pure individual standards of PAHs (BaP and Nap) but it suggests that higher concentration of fly ash is deleterious to the environment. Possibly, emission of fly ash from mills may leach down in groundwater and may also get deposited in agricultural soil through which it may get biomagnified in the food chain. Therefore, it may cause deleterious effects to both abiotic and biotic factors of the environment. It will not only affect the surrounding flora and fauna but also workers engaged in Sugarmill. Sugarcane bagasse fly ash causes severe environmental pollution which calls for urgent ways of handling the waste.