Science of the Total Environment 904 (2023) 166556 Contents lists available at ScienceDirect Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv Morphologically different hydroxyapatite nanoparticles exert differential genotoxic effects in Drosophila Merve Güneş a, Burçin Yalçın a, Ayşen Yağmur Burgazlı a, Ghada Tagorti a, Emre Yavuz b, Esin Akarsu b, Nuray Kaya a, Ricard Marcos c, *, Bülent Kaya a, * a Department of Biology, Faculty of Sciences, Akdeniz University, Antalya, Turkey b Department of Chemistry, Faculty of Sciences, Akdeniz University, Antalya, Turkey c Department of Genetics and Microbiology, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Spain H I G H L I G H T S G R A P H I C A L A B S T R A C T • Shape and size of hydroxyapatite nano­ particles (HAP-NPs) modulate hazard­ ous effects. • The toxic effects of spherical HAP-NPs were higher than those of the rod form. • HAP-NPs may have potential apoptotic effects. • DNA repair pathways were affected by exposure to HAP-NPs. A R T I C L E I N F O A B S T R A C T Editor: Damia Barcelo Hydroxyapatite (HAP) occurs naturally in sedimentary and metamorphic rocks and constitutes the hard struc­ tures in many organisms. Since synthetic nano-sized HAP (HAP-NPs) are used in orthopedic applications and for Keywords: heavy metal remediation in aquatic and terrestrial media, both environment and humans are exposed to them. Caspase Due to the concerns about their potential hazards, the genotoxic effects that round/rod forms of HAP-NPs were Drosophila melanogaster investigated in Drosophila using the wing-spot and the comet assays. Furthermore, caspase activities were Single-cell gel electrophoresis evaluated to examine the activation of cell death pathways. As a novelty, the expression of 36 genes involved in Somatic mutation and recombination DNA repair DNA repair was investigated, as a tool to indirectly determine DNA damage induction. Obtained sizes were Gene expression 35–60 nm (roundHAP-NPs) and 45–90 nm (rodHAP-NPs) with a low Zeta-potential (− 1.65 and 0.37 mV, respectively). Genotoxicity was detected in the wing-spot (round form), and in the comet assay (round and rod- like HA-NPs). In addition, increased expression of Caspases 3/7, 8, and 9 activities were observed. For both HAP forms, increased changes in the expression were observed for mismatch repair genes, while decreased expression was observed for genes involved in ATM, ATR, and cell cycle pathways. The observed changes in the repair pathways would reinforce the view that HAP-NPs have genotoxic potential, although more markedly in the round form. Thus, the environmental presence of engineered nanoparticles, including HAPs, raises concerns * Corresponding authors. E-mail addresses: ricard.marcos@uab.es (R. Marcos), bkaya@aldeniz.edu.tr (B. Kaya). https://doi.org/10.1016/j.scitotenv.2023.166556 Received 16 June 2023; Received in revised form 3 August 2023; Accepted 23 August 2023 Available online 24 August 2023 0048-9697/© 2023 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). M. Güneş et al. Science of the Total Environment 904 (2023) 166556 about potential effects on human health. It is essential that the effects of their use are carefully assessed and monitored to ensure safety and to mitigate any potential adverse effects. 1. Introduction approaches to detect genotoxicity, such as the detection of somatic mutation and recombination and the detection of DNA breaks (comet Biomaterials are compounds used to increase body functions and/or assay), toxicogenomic tools have also been used. This approach, using to replace damaged tissues which have played a relevant role in trans­ high-performance omics technologies, analyze and interpret changes in forming medicine over the last few decades (Montoya et al., 2021). In gene expression resulting from exposure to genotoxicants. Molecular this context, calcium phosphate-based biomaterials are widely used for profiling methods, including bioinformatics techniques, permit the repairing bone defects, due to their similarities to the inorganic com­ simultaneous analysis of a multitude of gene variants to search for genes ponents of human bones. Among them, hydroxyapatite (HAP) stands out prone to damage, detecting patterns and mechanisms of genotoxicity, (Filip et al., 2022). Besides its therapeutic uses, HAP has many other and identifying specific gene expression profiles that can provide bio­ ones such as removing heavy metal pollution from aquatic and terres­ markers of exposure and risk (Portugal et al., 2022). (Portugal et al., trial systems and wastewater (Brazdis et al., 2021). Composites created 2022). The used toxicogenomic tools will help to understand the geno­ with HAP are used as environmentally friendly cleaners for paint toxicity data obtained in standard assays, showing the way of how future removal from wastewater (Pai et al., 2021). Thus, among the potential genotoxicity studies should be carried out. Our final goal is to compre­ materials for fluoride removal, HAP and HAP-based composites stand hensively investigate the genotoxic effects of HAP-NPs in Drosophila, as a out with excellent performance at neutral and near-neutral pH (Rath­ suitable in vivo model, to assess the safety of these materials. nayake et al., 2022). Furthermore, Since HAP is soluble in the acidic environment of the stomach, the dissolved ions reach the intestine 2. Materials and methods similarly to other calcium sources and for this reason, they are proposed to be used as food additives (Enax et al., 2022). 2.1. Chemicals It is important to remark that HAPs are naturally found either in mammals (bone structures), aquatic organisms (spines and shells), Calcium nitrate tetrahydrate [Ca(NO3)2.4H2O], ammonium dihy­ plants, and algae, as well as in limestone minerals (Mohd Pu’ad et al., drogen phosphate [NH4H2PO4], diammonium dihydrogen phosphate 2019; Kalpana and Nagalakshmi, 2023). Since the production of HAP in [(NH4)2HPO4], ortho-phosphoric acid, propionic acid, Arabic gum, nano-size is synthetically feasible, their applications in various fields, chloral hydrate, glycerol, sodium hydroxide (NaOH), ethylenediamine particularly medicine, is steadily growing. Nevertheless, there is limited tetra acetic acid (Na2EDTA), sodium chloride (NaCl), Trizma Base Bio­ data on the potential toxicity of HAP (Righi et al., 2023). Ultra, sodium hydroxide (NaOH), ethidium bromide (EtBr), phosphate Nanotechnology permits the molecular design and production of buffer (PBS), phenylthiourea, Triton-X, sarcosine, Tris HCl, Na2H2P2O7, materials in the nano-range controlling size, morphology, and other β-glycerophosphate, and Coomassie Blue were purchased from Sigma physicochemical properties (Ignjatovic et al., 2020). Specifically, Aldrich (Saint Louis, MO, USA). Low melting agar, and low melting agar nanotechnology permits to obtain biomaterials of increasing biocom­ (DNA grade) were purchased from AppliChem (Darmstadt, Germany). patibility by controlling their structural properties, such as pore volume, All chemicals were used in their analytical grade, without further shape, diameter, and solubility (Bonilla-Represa et al., 2020). Thus, purification. compared to conventional ceramic formulations, the nanoparticles of apatite (HAP-NPs) present characteristics such as surface particle size 2.2. Synthesis of nano hydroxyapatite and pore size more efficient in their interactions with other molecules. As for the other nanomaterials, the potential hazards associated to Various approaches such as sol-gel, hydrothermal, co-precipitation, the use of HAP-NPs have been pointed out. A recent review did not find wet/dry and mechanical-chemical reactions can be used to synthesize associated hazards (Zaffarin et al., 2021), confirming the previous re­ HAP (Bonilla-Represa et al., 2020; Osuchukwu et al., 2021; Venkatesan ported for nanoscopic calcium phosphate (Epple, 2018). Nevertheless, in and Anil, 2021). Considering the advantages of each one of the methods, exposed Wistar rats (300 mg/kg bw for 45 days) HAP-NPs reduced the it is also possible to combine processes for HAP synthesis. Among them, levels of stomach antioxidant enzymes as well as glutathione (Mosa the ionic mixing of calcium and phosphorus in the sol-gel method has et al., 2020). In addition, round and rod-shaped HAP-NPs delayed em­ provided a specific microstructure and superior physical and chemical bryo hatching in zebrafish (3–100 μg/mL for 7 2 h) (Zhao et al., 2013), homogeneity (Panda et al., 2021); accordingly, this synthesis approach which would agree with data reporting that some nanomaterials prop­ was used in our study. erties, such as shape and size, influence the toxicological profile of nanomaterials (Dong et al., 2019). Accordingly, further studies on the 2.3. Round form of nano hydroxyapatite hazardous effects of HAP-NPs, considering shape characteristics are required. Round-like hydroxyapatite nanoparticles (roundHAP-NPs) were The aim of this study was to evaluate the potential toxicity of two synthesized according to our previous study (Yavuz et al., 2021). The different forms of HAP-NPs in D. melanogaster using different toxicity reaction was carried out by keeping the atomic ratio 1.67 for Ca/P. assays. For such purpose, two different HAP-NPs shapes were synthe­ Firstly, 78.88 g of Ca(NO3)2.4H2O and 129.6 mL of ethanol were stirred sized, namely round and nanorods HAP-NPs. To determine their po­ until they became a homogeneous solution. Then, the pH of the solution tential hazardous effects, Drosophila was used as a model organism, was set at 10 by adding NH3. Afterwards, 23.2 g of NH4H2PO4 and 186.5 which was reported to present many advantages when testing nano­ mL of distilled water were stirred to gain a homogenous phosphate so­ materials (Alaraby et al., 2016). Furthermore, considering the metabolic lution. Following this, the pH of the solution was set at 10 by adding similarities of Drosophila with mammals (dietary inputs including NH3. Next, the calcium solution was added (2.4 g/min) over a stirring nutrient intake-digestion-absorption-storage, xenobiotic metabolism phosphate solution. The resulting solution was kept for 22 h at room system, antioxidant enzymes and DNA replication and repair systems), it temperature and centrifuged at 5000 rpm for 5 min. Finally, the ob­ seems that this model organism can be successfully used to investigate tained HAP particles were washed six times with distilled water and the genotoxic potential of different agents (Mishra et al., 2017; Furla­ dried under vacuum at 70 ◦ C for 24 h. After that, HAP-NPs particles were netto et al., 2018; Naves et al., 2018). In addition to the classical calcined at 750 ◦ C for 4 h. 2 M. Güneş et al. Science of the Total Environment 904 (2023) 166556 2.4. Rod form of nano hydroxyapatite at which the radiation is absorbed and form the fingerprint regions of the measured substance. These regions provide essential information Rod-like hydroxyapatite particles (rodHAP-NPs) were produced about the synthesis or processing conditions used to obtain synthetic using a modified chemical precipitation technique setting the atomic HAP-NPs and help to distinguish it from other types of calcium phos­ ratio to 1.67 for Ca/P (Nagyne-Kovacs et al., 2018). Briefly, 17.712 g of phate. Thus, roundHAP-NPs and rodHAP-NPs were investigated by FTIR Ca(NO3)2.4H2O and 5.940 g of (NH4)2HPO4 were dissolved in 32.288 spectroscopy using a Bruker-Tensor 27 (Billerica, MA, USA) device at 4 mL and in 44.06 mL of distilled water, respectively, to prepare homo­ cm− 1 resolution in transmission mode ranging from 400 to 4000 cm− 1. geneous calcium and phosphate solutions. Then, the pH of the solutions was adjusted to 10 by adding NH3. Afterwards, the calcium solution was 2.6. Drosophila strains and exposure added (2.4 g/min) over a stirring phosphate solution and stirred for 1 h. The final solution was centrifuged at 5000 rpm for 5 min and, finally, the The Oregon strain of Drosophila melanogaster was used for all the obtained HAP-NPs were calcined at 500 ◦ C for 5 h. experiments except for the wing-spot assay where both, the multiple wing hair (mwh) and flare (flr) strains were used (Fig. 1). Initially, a wide range of doses (from 10 to 1000 ppm) was chosen to determine those to 2.5. Nanoparticle characterization be used in the study. Since intense precipitation was observed in the applications of HAP-NPs at 400, 800, and 1000 ppm doses, the highest 2.5.1. Transmission electron microscopy and zeta potential analysis dose applicable was determined to be 200 ppm. Accordingly, we decide Transmission electron microscopy (TEM) was used to visualize HAP- to use four different doses (25, 50,100, and 200 ppm). Distilled water NPs and determine their dispersion and aggregation status, size, and was used in the reconstitution of the HAP-NPs suspensions and, conse­ shape. In this study, for morphological examinations of roundHAP-NPs quently, it was used as the negative control. and rodHAP-NPs a ZEISS brand LEO906 TEM model (Oberkochen, Germany) was used. The zeta potential of nanoparticles was measured to understand their stability and potential interactions in colloidal systems 2.7. The Drosophila wing somatic mutation and recombination test (Rasmussen et al., 2020). Stability measurements of HAP-NPs were performed in a Malvern Zetasizer Nano-ZS. The wing somatic mutation and recombination test (SMART) in Drosophila was performed according to Graf et al. (1984). To proceed, 2.5.2. Fourier transform infrared spectroscopy analysis mwh/mwh males were crossed with flr3/ ln (3LR) TM3, BdS females, and Atomic groups have unique fundamental vibrational modes and their eggs from this cross were collected during 8-h periods. The resulting peaks in a Fourier Transform Infrared Spectroscopy (FTIR) spectrum three-day-old larvae were transferred to test tubes where 4.5 g represent specific chemical bonds and functional groups (Dee et al., Drosophila Instant Medium were hydrated with 9 mL of 25, 50, 100, and 2002). When molecules are exposed to infrared light, radiation at fre­ 200 ppm of roundHAP-NPs and rodHAP-NPs dispersions. Larvae were quencies matching their fundamental vibrational modes is absorbed. kept in this environment until they became adults. Ethyl­ The FTIR spectra show the peak values corresponding to the frequencies methanesulfonate (EMS, 1 mM) was used as the positive control. Wings Fig. 1. Scheme of the study. Three different in vivo experiments and one in vitro experiment were conducted in D. melanogaster. Three databases were used for bioinformatic analyses. 3 M. Güneş et al. Science of the Total Environment 904 (2023) 166556 were removed from the emerged adults and mounted on microscopy 2.11. Gene ontology and pathway enrichment analysis slides with Faure’s solution. The wing preparations were analyzed under a light microscope at 40× magnification to detect mutant clones. The Gene Ontology Consortium was created by combining infor­ mation from three model organism databases, FlyBase, Mouse Genome 2.8. The Drosophila single cell gel electrophoresis/comet test Informatics (MGI), and Saccharomyces Genome Database (SGD) (Ash­ burner et al., 2000). This system, which provides a basis for the The comet (single cell gel electrophoresis) assay, detecting DNA computational analysis of large-scale molecular biology and genetics breaks in larvae hemocytes, was performed according to the Carmona experiments, has data on >43,000 GO terms, approximately 1,500,000 et al. (2011) method. Eight-hour-old eggs were collected from the gene products and >5000 species by 2022. The Comparative Tox­ Drosophila Oregon strain to get third instar larvae, which were exposed icogenomic Database (CTD; http://ctdbase.org/) is a digital ecosystem to 25, 50, 100, and 200 ppm roundHAP-NPs and rodHAP-NPs for 24 h. (that holds and can link toxicology data for chemicals, genes, pheno­ Distilled water was used as negative control and 4 mM EMS as a positive types, exposures, and related diseases (Davis et al., 2021). The molecular control. For each dose, 50 cells were counted on a fluorescence micro­ functions of the 36 genes whose expression was measured were inves­ scope (Nikon Eclipse E200, Tokio, Japan) at 40× magnification. The tigated using the gene ontology and pathway enrichment approach. percentage of DNA in the tail was used as the evaluation parameter. All measurements were made with a computer-connected fluorescent mi­ 2.12. Gene-interactions network construction croscope and automatic measurement with the COMET Assay-IV (Version 4.11, Instem, UK) software. The GeneMANIA tool finds the genes most closely linked between the networks and selected traits. Thus, GeneMANIA (http://genemania.org) is a user-friendly website established to identify the relationships be­ 2.9. Caspase activity as indicator of apoptosis tween gene lists and their functions and to generate hypotheses about them (Warde-Farley et al., 2010). This site has >2000 networks with To measure caspase activity, 8-h-old eggs were collected from the approximately 164,000 genes and 600 million interactions. Twenty-two Drosophila Oregon strain. Haemocytes were isolated from 96 h larvae genes showing increase and decrease in the RT2-PCR system were added and exposure to HAP-NPs was performed in vitro. The Promega Caspase- to this database and an estimate was made about the relationship size Glo 3/7, Caspase-Glo 8, and Caspase-Glo 9 Assay (Madison, WI, USA) between them (Franz et al., 2018). kits were used to determine caspase activities in Drosophila larvae he­ mocytes. The bicinchoninic acid (BCA) assay was used to determine the 2.13. The gene pathway Visualizer tool amount of protein in the isolated hemocytes. Cell lysates and a caspase kit were added to 96 well plates in a 1:1 ratio (100 μL:100 μL). The plates The flymine.org is the genomic and proteomic database for were mixed for 30 s at 300–500 rpm with the help of a plate-shaker. Drosophila and other insects. This website enables complex queries to be Afterwards, the plates were incubated for 1 h at room temperature. created from a simple scan initiated with one or more data (Lyne et al., The amount of luminescence at the end of the time was measured with a 2007). Over time, this database has been gathered under the umbrella of multi-module plate reader (Promega Multi Detection System, Madison, intermine.org by combining data from many model organisms, such as USA). Arabidopsis, yeast, maggot, mouse, and humans (Lyne et al., 2022). This gene pathway visualizer has been used to determine the associations 2.10. The gene expression analysis array involving the selected genes. The RT2 Profiling PCR Array (Quiagen, Hilden, Germany) enables a 2.14. Data analysis fast and reliable, allowing to perform faster and more tests than tradi­ tional PCR methods. The arrays are pathway-driven panels of the In the Drosophila wing SMART test, the statistical analysis of the data manufacturer’s lab-validated qPCR assays with integrated patented was made using the binomial conditional test in the MICROSTA package controls to ensure a successful experiment every time. Each array in this program. Data from the Drosophila comet assay and caspase activities system can be individually designed and modified to suit unique assays were evaluated with the Dunnett’ multiple comparison test using experimental needs. The human gene homology ratio of the genes whose analysis of variance (ANOVA) in the IBM SPSS 20 package program. The expression was investigated within the scope of the study was verified by results of DNA damage signaling pathway PCR array were analyzed flybase.org. The RT profiles prepared for Drosophila gene expressions using the RT2 Profiles PCR Array Data Analysis Software version 3.5. used the DNA Damage Signaling Pathway PCR Array (Qiagen) in 96-well This program calculates fold change/regulation in gene expressions PCR array plates. It is designed to run two doses in 96-well plates in a using the delta-delta CT (cycle threshold) method. Delta CT is calculated panel format. The lowest and highest dose tested in the experiment were between the gene of interest (to be measured) and housekeeping genes. used to detect differences in gene activity. Then, Delta-delta CT calculations, Delta CT (Experiment), and Delta CT The RT2-PCR study was made as recently published (Yalcin et al., (control) calculations are made. Fold changes are calculated using the 2022). Thirty individuals from the treated larvae were collected in formula 2-ΔΔCT. The data analysis report is obtained from Qiagen’s microcentrifuge tubes. Total mRNA isolation from larvae was performed GeneGlobe web portal. In bioinformatic analysis, the significance of using the RNeasy Mini Isolation Kit (Qiagen). After the RNAs were iso­ enrichment in molecular functions is calculated by the hypergeometric lated, the amount and quality of RNA was determined using the Nano­ distribution and adjusted for multiple testing using the Bonferroni drop (Thermo Fisher Scientific, USA) device. After sufficient RNA was method. isolated, cDNA synthesis was performed with the RT2 First Strand Kit. The cDNAs were tested with the RT2-PCR instrument (Applied Bio­ 3. Results systems, USA) to determine whether the expression of genes in the DNA damage pathway was altered. A vital advantage of the RT2 Profiler PCR 3.1. Synthesis and characterization Array is the control tests PCR (3 wells), reverse-transcription control (3 wells), and genomic DNA control (1 well). Five wells contain house­ As shown in the TEM images, the synthesized HAP-NPs were round keeping genes (Act42A, Gapdh1, RpL32, SdhA, and Tbp), which are used and rod-like (Fig. 2a, c). The roundHAP-NPs and rodHAP-NPs lengths to calculate normalization in the analysis system. The remaining 36 range from 35 to 60 nm and 45–90 nm, respectively. The Zeta potential wells contain genes involved in DNA repair pathways. demonstrated that the surface charge of roundHAP-NPs was − 1.65 mV 4 M. Güneş et al. Science of the Total Environment 904 (2023) 166556 Fig. 2. Characterization of HAP-NPs. TEM images of synthesized HAP-NP and Zeta potential show roundHAP-NPs (a, b) and rodHAP-NPs (c, d). Fig. 3. The FTIR spectra of roundHAP-NPs and rodHAP-NPs. 5 M. Güneş et al. Science of the Total Environment 904 (2023) 166556 and rodHAP-NPs 0.37 mV (Fig. 2b, d). spots, as well as the twin spots, were recorded. The obtained results are The FTIR spectra for roundHAP-NPs and rodHAP-NPs (Fig. 3) show indicated in Fig. 4, where distilled water and EMS (4 mM) were used as a the functional groups of the obtained HAPs. Typical PO3− 4 bands appear negative and positive controls, respectively, at 472, 569, 602, 962, 1036, and 1093 cm− 1. The absorbed water (H2O) According to the results, roundHAP-NPs caused a statistically sig­ is observed in bands at 1634 and 3443 cm− 1. The hydroxyl groups (OH-) nificant genotoxic effect at 50, 100, and 200 ppm when the frequency of in the HAP crystal structure are observed at 632 and 3571 cm− 1. The total clones was determined. It should be noted that these effects did not weak carbonate bands (CO2− − 1 3 ) are observed at 875 and 1456 cm . As a follow a dose-response relationship. As observed, rodHAP-NPs were summary, the obtained results confirm that both forms of HAP-NPs were unable to cause statistically significant genotoxic effects at the studied effectively synthesized. doses. Representative clone images of the types of mutant spot observed in the wing blade are shown in Fig. 4 (b-e). 3.2. The induction of somatic mutation and recombination 3.3. The induction of DNA strand breaks Adult flies were collected from the exposure tubes and kept in tubes containing 70 % ethanol. The analyses were performed by scanning the As observed in Fig. 5, the positive control (4 mM EMS), caused sta­ wings of Drosophila individuals to detect changes in the normal tistically significant DNA damage levels when compared to the negative morphology of wing hairs. The observed mutant flr3 and mwh single control group (distilled water), supporting the validity of the reported Fig. 4. Genotoxic effects of roundHAP-NPs and rodHAP-NPs on the wings of transheterozygous (mwh/flr3) individuals (a). Images of small mwh single spot (b), large mwh single spot (c), flr3 spot (d), and twin spots (e). Distilled water was used as negative control and 1 mM EMS was used as positive control. *P ≤ 0.05 (binominal conditional test). 6 M. Güneş et al. Science of the Total Environment 904 (2023) 166556 Fig. 5. DNA damage analysis in Drosophila hemocytes by using the comet assay. DNA damage induced by roundHAP-NPs and rodHAP-NPs (a). Representative comet images from the negative control (b), the positive control (c), and apoptotic cells (d). *P ≤ 0.05, ** P ≤ 0.01, P ≤ 0.001 (ANOVA, Tukey test). data. When the effects of roundHAP-NPs were analyzed, a statistically much more present in roundHAP-NPs exposed individuals, have been significant DNA damage induction was observed but only at the applied considered as consequence of apoptotic processes. Undamaged and 100 ppm dose (Fig. 5a) (P < 0.01). Similar effects were observed when damaged comet images are given in Fig. 5b-d. rodHAP-NPs were evaluated since only the dose of 100 ppm induce significant effects (P < 0.01). It should be indicated that at the highest 3.4. Caspase activity dose (200 ppm) many comets with a small and pale head and with almost all the DNA in their tail were observed. These comets, denoted as The potential induction of apoptosis by roundHAP-NPs and rodHAP- hedgehog comets are not scored and, consequently, this is the main NPs was measured, and the obtained results are indicated in Fig. 6. As reason of the lack of significance of the reported data. These figures, observed, roundHAP-NPs at the dose of 100 ppm only induced 7 M. Güneş et al. Science of the Total Environment 904 (2023) 166556 Fig. 6. Results of Caspase-3/7, Caspase-8, and Caspase-9 activities. RoundHAP-NPs (a), rodHAP-NPs (b). *P ≤ 0.05, ** P ≤ 0.01, P ≤ 0.001 (ANOVA, Tukey test). statistically significant increases in the activity of caspase-9. At the Table 1 concentration of 200 ppm the activity of the three caspases (caspase-3/ Analysis of changes in gene expression levels. Those genes showing increases or 7, caspase-8, and caspase-9) shows statistically significant increases decreases with fold changes over 2 are indicated. (relatively P < 0.01 and P < 0.001) (Fig. 6a). These results show that rodHAP-NPs induced higher effects, as 25 ppm 200 ppm compared with those induced by roundHAP-NPs. Thus, the lowest ▴ FC ▾ FC ▴ FC ▾ FC exposure (25 ppm) increased statistically the activity of caspase-3/7 and roundHAP-NPs caspase-9 and, at the highest concentrations (100 and 200 ppm), Mlh1 3.86 spel1 3.89 Rad51C 4.49 caspase-8 and caspase-9 activities increased markedly (P < 0.001) Rad51C 4.15 Mlh1 3.32 MCPH1 5.92 Brca2 3.13 MCPH1 8.04 (Fig. 6.b). Rad51D 4.08 Rad51D 4.32 3.5. Results from the gene expression analysis rodHAP-NPs Mlh1 5.34 PNKP 18.03 spel1 29.79 Brca2 2.97 Rev1 17.42 dATM 12.88 The effects of roundHAP-NPs on the expression of the selected genes dap 10.94 punt 7.95 involved in DNA repair signaling pathways are shown in Table 1. As mu2 10.92 Dxpa 7.25 observed, the Mlh1 gene is overexpressed in both 25 and 200 ppm RpLP2 6.67 Rev1 6.20 treatments, and these effects were statistically significant (P < 0.05). mus101 2.24 Pms2 6.30 Rad9 6.07 spel1 4.73 Rad51D 5.41 Similarly, three other genes (MCPH1, Rad51C, and Rad51D) showed stg 4.89 statistically significant decreases after exposures to 25 and 200 ppm of Sir2 4.22 Hus1-like 4.56 roundHAP-NPs. ▴: Increased gene expression Rad17 4.53 Interestingly, the exposure to rodHAP-NPs induced higher levels of ▾: Decreased gene expression mu2 4.24 FC: Fold change Cdk7 4.00 changes than those observed after roundHAP-NPs exposure, and most of them involved under-expression. Thus, although no genes reached the stablished significance levels (fold >2) at the exposure of 200 ppm, three and Sir2) and twelve after exposures to 200 ppm (spel1, dATM, punt, genes (mus101, Mlh1, and Brca) were overexpressed at 25 ppm exposure. Dxpa, Rev1, Rad9, Rad51D, stg, Hus1-like, Rad17, mu2, and Cdk7). Regarding the genes showing under-expression, eight were deregulated Comparing both types of exposures, only three genes were altered at after exposures to 25 ppm (PNKP, Rev1, dap, mu2, RpLP2, Pms2, spel1, 8 M. Güneş et al. Science of the Total Environment 904 (2023) 166556 both doses (Rev1, mu2, and spel1). importance of the interactions between genes in the management of DNA repair functions. It must be pointed out that >10 % of the genes of 3.6. Gene ontology and pathway enrichment the entire genome are involved in the different functions of the DNA repair system, either in the process of recognizing the damage, in cutting The Gene Ontology (GO) is an extensive bioinformatics database the damaged region, or repairing the gaps in the DNA, with the com­ showing species’ gene and gene product characteristics. When the mo­ plexes formed by the genes coming together. lecular functions of the genes involved in the study (Table 2) are examined, it is observed that those involved in catalytic activities and 3.7. Gene-interaction network construction binding to DNA are specially highlighted. The molecular function of each one of the selected genes can arise The interactions in the molecular network of the 22 genes showing a from their proteins or RNAs and can consist of a single gene product or a statistically significant increase or decrease in the RT2-PCR were combination of multiple gene products. The table highlights the determined by using Genemania. Accordingly, 20 genes associated with Table 2 Molecular functions of the genes involved in this study. A molecular process that can be carried out by the action of a single macromolecular machine, usually via direct physical interactions with other molecular entities. Function in this sense denotes an action, or activity, that a gene product (or a complex) performs (CTD Set Analyzer, https://ctdbase.org/tools/analyzer.go, threshold = 0.01). Highest GO term name GO term ID P- Corrected P- Annotated Annotated genes Genome GO value value genes quantity frequency level CDK7|ERCC1|FEN1|MLH1|OGG1|PCNA| 5.34E- 213/45141 3 Catalytic activity, acting on DNA GO:0140097 7.29E-19 13 PMS2|POLH|RAD1|RAD17|RAD50| 22 genes: 0.47 % RAD51D|XRCC1 1.02E- ERCC1|FEN1|OGG1|PCNA|PNKP|POLH| 65/45141 5 Damaged DNA binding GO:0003684 1.40E-15 9 18 RAD1|REV1|XRCC1 genes: 0.14 % CDK7|ERCC1|FEN1|MLH1|OGG1|PCNA| Catalytic activity, acting on a nucleic 5.47E- 514/45141 2 GO:0140640 7.45E-14 13 PMS2|POLH|RAD1|RAD17|RAD50| acid 17 genes: 1.14 % RAD51D|XRCC1 1.12E- ERCC1|FEN1|OGG1|PMS2|PNKP|RAD1| 179/45141 4 Nuclease activity GO:0004518 1.53E-09 8 12 RAD50|XRCC1 genes: 0.40 % 2.05E- ERCC1|FEN1|OGG1|PMS2|PNKP|RAD50| 106/45141 5 Endonuclease activity GO:0004519 2.80E-09 7 12 XRCC1 genes: 0.23 % BRCA2|ERCC1|FEN1|MLH1|OGG1|PCNA| 2.56E- 1799/45141 4 DNA binding GO:0003677 3.49E-08 14 PMS2|PNKP|POLH|RAD1|RAD50| 11 genes: 3.99 % RAD51D|REV1|XRCC1 2.79E- 46/45141 4 Deoxyribonuclease activity GO:0004536 3.80E-07 5 ERCC1|FEN1|RAD1|RAD50|XRCC1 10 genes: 0.10 % ATP-dependent activity, acting on 2.79E- CDK7|MLH1|PMS2|RAD17|RAD50| 110/45141 2 GO:0008094 3.81E-07 6 DNA 10 RAD51D genes: 0.24 % ATP-dependent DNA damage sensor 1.79E- 21/45141 3 GO:0140664 2.45E-06 4 MLH1|PMS2|RAD50|RAD51D activity 09 genes: 0.05 % 3.18E- 24/45141 2 DNA damage sensor activity GO:0140612 4.34E-06 4 MLH1|PMS2|RAD50|RAD51D 09 genes: 0.05 % 6.12E- 28/45141 5 Endodeoxyribonuclease activity GO:0004520 8.34E-06 4 ERCC1|FEN1|RAD50|XRCC1 09 genes: 0.06 % BRCA2|ERCC1|FEN1|MLH1|OGG1|PCNA| 9.49E- 2835/45141 3 Nucleic acid binding GO:0003676 1.29E-05 14 PMS2|PNKP|POLH|RAD1|RAD50| 09 genes: 6.28 % RAD51D|REV1|XRCC1 BRCA2|CDK7|ERCC1|FEN1|MLH1|OGG1| 1.64E- 4755/45141 2 Heterocyclic compound binding GO:1901363 2.24E-05 17 PCNA|PMS2|PNKP|POLH|RAD1|RAD17| 08 genes: 10.53 % RAD50|RAD51D|REV1|SIRT2|XRCC1 1.72E- 103/45141 5 Single-stranded DNA binding GO:0003697 2.35E-05 5 BRCA2|ERCC1|MLH1|PMS2|RAD51D 08 genes: 0.23 % BRCA2|CDK7|ERCC1|FEN1|MLH1|OGG1| 2.00E- 4817/45141 2 Organic cyclic compound binding GO:0097159 2.72E-05 17 PCNA|PMS2|PNKP|POLH|RAD1|RAD17| 08 genes: 10.67 % RAD50|RAD51D|REV1|SIRT2|XRCC1 BRCA2|CDK7|ERCC1|FEN1|MLH1|OGG1| 2.09E- 4832/45141 1 Catalytic activity GO:0003824 2.85E-05 17 PCNA|PMS2|PNKP|POLH|RAD1|RAD17| 08 genes: 10.70 % RAD50|RAD51D|REV1|SIRT2|XRCC1 Single-stranded DNA 2.38E- 8/45141 6 GO:0000014 3.25E-05 3 ERCC1|RAD50|XRCC1 endodeoxyribonuclease activity 08 genes: 0.02 % 2.44E- 39/45141 1 Small molecule sensor activity GO:0140299 3.33E-05 4 MLH1|PMS2|RAD50|RAD51D 08 genes: 0.09 % Hydrolase activity, acting on ester 2.99E- ERCC1|FEN1|OGG1|PMS2|PNKP|RAD1| 651/45141 3 GO:0016788 4.08E-05 8 bonds 08 RAD50|XRCC1 genes: 1.44 % 7.01E- 11/45141 3 Mismatch repair complex binding GO:0032404 9.57E-05 3 MLH1|PCNA|PMS2 08 genes: 0.02 % 9.35E- 12/45141 6 Mismatched DNA binding GO:0030983 1.28E-04 3 MLH1|PCNA|PMS2 08 genes: 0.03 % 3′ Overhang single-stranded DNA 1.75E- 3/45141 7 GO:1990599 0.00239 2 ERCC1|XRCC1 endodeoxyribonuclease activity 06 genes: 0.01 % 1.98E- ERCC1|FEN1|OGG1|PCNA|PMS2|PNKP| 2010/45141 2 Hydrolase activity GO:0016787 0.0027 10 06 RAD1|RAD50|SIRT2|XRCC1 genes: 4.45 % 2.51E- CDK7|MLH1|PMS2|RAD17|RAD50| 511/45141 1 ATP-dependent activity GO:0140657 0.00342 6 06 RAD51D genes: 1.13 % 9 M. Güneş et al. Science of the Total Environment 904 (2023) 166556 the 3 genes (Mlh1, Brca2, mus101) showing an increased expression, 3.8. Gene pathway visualizer emerged. These genes show a 39.37 % of physical interaction, a 21.05 % of co-expression, and a 3.49 % of genetic interaction (Fig. 7). In the In Fig. 8, squares represent the genes studied and the circles repre­ analysis performed in GeneMANIA, 20 genes were identified as showing sent the pathways in which the genes are connected. Pie slices in the associations with the 18 genes which expression was significantly circle represent pathways that contain more than one gene. Genes decreased. These genes show 31.82 % physical interaction, 37.73 % co- involved in DNA repair are shown in groups. Cell cycle control genes, expression, and 0.56 % genetic interaction. mismatch repair system genes, nucleotide excision damage detection, Fig. 7. Genes associated with over- expressed (a) and downregulation (b) in RT2-PCR analysis. Physical Interaction: Data of protein-protein interactions. Two or more gene products are interrelated if they found to interact protein-protein. Data collected from BioGRID and Path­ wayCommons. Predicted: Represents the relationship between genes and proteins. Data collected from ortology databases such as Human2Mouse, yeast2worm, YeastNet. Co-expression: Two or more gene connected if their expressions are similar in gene expression analysis. Data collected from Gene Expression Omnibus (GEO). Co-localization: Genes expressed same tissue and gene products identified in the same cellular localization. Shared protein domains: Gene products linked in the same protein domain. Data collected from InterPro, SMART and Pfam. Genetic interaction: Genes are functionally asso­ ciated. Data are collected from BioGRID. 10 M. Güneş et al. Science of the Total Environment 904 (2023) 166556 Fig. 8. Gene Pathway Visualizer. Square nodes represent genes. Circular nodes represent pathway annotations for the genes. Edges connect genes and pathways. By default, the most closely related genes and pathways are shown. All genes are involved in DNA repair processes, and some of them are in specialized groups. Thus, tefu and dap are involved in tp53-related path­ ways, and tefu, dap, and stg are involved in G1/S DNA damage checkpoints. Spel1, mlh1, and pms2 are involved in mismatch repair and in the MutSalpha complex-associated mismatch repair. The xpac and cdk7 are included in the NER system together. The Rad17, Rad9, mus101, Hus1-like, and tefu are involved in double strand repair system. and apoptosis genes are included in these groupings. Some of the genes behavior were observed after rodHAP-NPs exposure (Dan et al., 2019). are specifically involved in a particular pathway (Xpac, Mlh1, Pms2 and Only one study focuses on the potential genotoxicity of HAP-NPs, spel1); but other are involved in multiple pathways, including the cell showing that resveratrol was able to reduce the genotoxic effects cycle, repair systems, and apoptotic processes. Thus, Xpac and Cdk7 are detected by the comet assay; pointing out that oxidatively DNA damage involved in the nucleotide excision repair system and work together in was caused (Turna Demir, 2022). In this context, our study tries to the NER system. In addition, cdk has a role in the cell cycle. Genes such emphasize the potential mechanisms underlying HAP-NPs genotoxicity. as spell1, Mlh1 and Pms2, involved in the recognition of damage in A complete physiochemical characterization of the synthesized HAP- mismatch repair, work together and have been grouped separately. The NPs was carried out including Zeta potential, which is a key parameter genes dap and tefu/dATM are involved in several steps of DNA repair, that indicates the surface charge of nanoparticles, which in turn affects particularly in the mechanisms of senescence and apoptosis. MCPH1, their dispersion and aggregation behavior (Rasmussen et al., 2020). The dap, stg and Cdk7 are involved in cell cycle control, while the Rad family, aggregation observed in the TEM results is correlated with the zeta mus101 and Hus1-like, are involved in the detection of DNA damage and potential values of the roundHAP-NPs and rodHAP-NPs. In fact, for bone in the activation of the repair systems. tissue applications it is desired that HAP have a negative or near nega­ tive Zeta potential (Doostmohammadi et al., 2012). Furthermore, FTIR 4. Discussion results confirmed the efficient synthesis of both rod-like and round-like forms of HAP-NPs (Panda et al., 2003; van der Houwen et al., 2003;, Sun In this study, the genotoxic effects of HAP-NPs are discussed at the et al., 2006; Wang et al., 2006; Lin et al., 2007; Salarian et al., 2009; Ren gene level based in data supported by genotoxicity tests and bio­ et al., 2014). informatic analyses. Thus, the toxicity of HAP-NPs was comprehensively Our results in the wing-spot assay show that exposure to roundHAP- investigated by in vitro, in vivo, and in silico analyses. Bio-ceramic NPs NPs caused significant genotoxic effects in Drosophila somatic cells, such as HAP have been used in medicine for a long time, mainly in bone which not was observed after rodHAP-NPs exposure. It is thought that tissue regeneration (Bahadar et al., 2016). Thus, factors such as the genotoxic effects induced by roundHAP-NPs result from their easier biocompatibility, interactions with surrounding tissues, and potential cell uptake due to their shape. In addition, this shape has shown to in­ toxicity using in vivo models have been largely evaluated (Deveci et al., crease oxidative damage in different models (Zhao et al., 2011; Xu et al., 2020; Ferreira-Ermita et al., 2020). Regarding the few studies carried 2012). When the comet assay was applied, a significant increase of DNA out using Drosophila, roundHAP-NPs exposure was reported to cause breaks was observed in the hemocytes of larvae exposed to 100 ppm. behavioral phenotypic disorders in both larvae and adults, as well as Nevertheless, this increase was not observed at the dose of 200 ppm. oxidative and cell damages (Pappus et al., 2017; Sahu and Mishra, Interestingly, at this high dose, comets with a small and pale head and 2020). Nevertheless, no adverse effects such as ROS and lipid peroxi­ with almost all the DNA in their tail were observed. These comets are dation induction, or alterations in adult climbing and larval crawling called “hedgehog” (Collins et al., 2008) and, although initially these 11 M. Güneş et al. Science of the Total Environment 904 (2023) 166556 images were associated with apoptosis, further studies proposed that at both doses of the roundHAP-NPs, and at the low dose of the rodHAP- they correspond to one level on a continuum of genotoxic damage, NPs, revealing the relevance of that repair pathway. The Mlh1 gene consequently they do not represent a diagnostic of apoptosis (Lorenzo works in the mismatch repair system to repair point mutations (Sia et al., et al., 2013). Independently of the content of the previous discussion 2001). As the bioinformatic analysis indicates, Mlh1 works together with about the meaning of the hedgehog comets, the induction of apoptosis Pms2 and spel1 in the mismatch repair pathway. The decrease in these can result in a DNA fragmentation with the consequent loss of such two genes may indicate that this specific repair system does not work fragments during the electrophoresis step. In this way we would explain properly. On the other hand, the decreased expression of Rad9 and Hus1 the non-significant effects observed at the dose of 200 ppm. To deter­ genes after rodHAP-NPs exposure can be considered as an indicator of mine the potential role played by apoptosis in the observed effects more DNA damage induction. The complex formed by Rad9, Rad1, and Hus1 specific biomarkers must be used. genes is called “9-1-1” and it is a complex regulating checkpoint Caspases are proteins involved in the programed cell death. In signaling pathways after DNA damage (Parrilla-Castellar et al., 2004). addition, they can regulate various pathological conditions such as Furthermore, it was also determined that the expression of dap, sir2, and neurodegeneration, autoimmune diseases, and cancer (Wu et al., tefu, which are involved in apoptotic pathways, decreased after 2016a). Their essential biological role supposes a high conservation over roundHAP-NPs exposure. Caspase results indicate that both intrinsic and the evolution. Thus, caspases are approximately 50 % conserved be­ extrinsic apoptotic pathways are activated after high exposure doses. tween humans and Drosophila (Crawford et al., 2012). In Drosophila, the Decreased expression of genes suppressing apoptosis and increased initiator caspase DRONC activity, a Caspase-2 and Caspase-9 homo­ caspase activities may point out that roundHAP-NPs exposure induces logue, is required for developmental and stress-induced apoptosis apoptosis. After exposures to the rodHAP-NPs, the expression of DNA (Dorstyn and Kumar, 2008). DICER, the Drosophila homologue of repair genes did not increase statistically, but caspase activities caspase-3, which acts as an effector caspase, has been associated with increased. Accordingly, it can be considered that rodHAP-NPs (with a developmental apoptosis (Fan and Bergmann, 2010). The high conser­ lower potential ability to be internalized), induce apoptosis by causing vation in those genes linked to intrinsic apoptotic pathways suppose that caspase activities, both in the internal and external pathways. Brca2, Drosophila can be considered as a good model organism for studying the whose expression is increased in the repair of strand breaks, was over- roles of caspases operating during the initiation and implementation of expressed after low dose exposures. Moreover, Ca ions released from apoptotic cell death. (Fogarty and Bergmann, 2014). The caspase levels HAP-NPs into the cell could be internalized by the mitochondria causing in Drosophila were investigated after exposures to different agents such oxidative stress and apoptosis (Liu et al., 2021; Xia et al., 2022). All this as the antibiotic cefotaxime, and cerium and zinc nanoparticles, togethers would indicate that apoptosis can be induced through DNA showing increases in Caspase-3 and Caspase-9 activities (Filipiak et al., strand breaks caused by HAP-NP exposures. 2012; Rahul et al., 2015). In our study, it has been determined that As our results show, it is relevant to investigate the toxic potential of roundHAP-NPs caused an increase in Caspase-3/7 and Caspase-9 ac­ different forms of HAP-NPs since the effects can be modulated by their tivity, and a decrease in Caspase-8 activity. On the other hand, rodHAP- size/shape. This was demonstrated in one study using four different NPs caused a decrease in Caspase-3/7 and Caspase-9 activity, but types of HAP-NPs (needle, round, thin rod, and long rod) where a dose- increased Caspase-8 activity. All this would confirm the induction of dependent increased in cytotoxicity and apoptosis was observed in rat intrinsic and extrinsic apoptosis pathways by HAP-NPs exposure (Wu osteoblast cell lines. In addition, it was observed that the toxic effect et al., 2016b). According to our results, exposures to low doses of occurred mostly in needle-shaped particle exposure, with mail effects in roundHAP-NPs (with high potential to internalize into the cells) can long rod HAP-NP exposures (Xu et al., 2012). This was confirmed by activate intracellular ROS-induced death pathways, while extrinsic other studies showing that HAP-NPs in short rod and microsphere forms apoptotic pathway initiators would be activated at high doses of cause increased ROS and DNA damage (comet) in osteoblast primary rodHAP-NPs. The roundHAP-NPs and rodHAP-NPs have similar zeta cells (Dai et al., 2014) In addition, cytotoxicity, ROS, and LDH levels potential, suggesting that the rate of aggregation is similar in both. were induced in kidney epithelial cells exposed to different HAP-NPs These results were also supported by TEM images. The results show that forms such as spheres, needles, rods, and plates (Rao et al., 2014). the morphological structure is one of the important factors affecting the It is assumed that the repair of DNA damage is not carried out by the toxicity mechanism. These differential roles of the different shapes decrease/increase of a single gene, but by the activation of complex would agree with the observed by exposing different NPs with different structures that arise with the expression of many genes (Evans et al., shapes, sizes, and chemical composition (Sohaebuddin et al., 2010). 2004; Mannuss et al., 2012). The genes interact with each other in In eukaryotes, genomic stability is maintained under stress condi­ complex pathways and can be controlled by the same transcriptional tions induced by endogenous and exogenous sources. Thus, genomic factors in the same biological pathway, and expected to be functionally stability is maintained by DNA repair and cycle checkpoints genes. similar (Allocco et al., 2004). Although pathways can be studied and Major DNA repair pathways are functionally conserved among pro­ analyzed independently, they depend on the expression of interrelated karyotes, primitive and higher eukaryotes, and the proteins involved in genes. Changes such as mutations in the functions of genes in the hier­ these repair processes show a high degree of amino acid identity. With archical system may cause changes in the functions of the pathways (Oti the genome studies, it has been determined that Drosophila is one of the and Brunner, 2007; Grimes et al., 2019). The fact that genes responsible model organisms with high similarities (Klovstad et al., 2008). Thus, for similar functions share features such as interaction with each other approximately 77 % of the genes identified in human diseases show and expression patterns has highlighted the term “guilt by association” homology with Drosophila genes (Reiter et al., 2001). In the present (GBA) (Gillis and Pavlidis, 2012). GBA has become a phenomenon used study the expression of genes working in nucleotide excision repair, base to predict genes in certain products or pathways (van Dam et al., 2018), excision repair, mismatch repair, repair of double-strand breaks, ATM/ and gene ontology explains the relationships of gene groups in networks ATR signaling pathways, and cell cycle checkpoints were investigated in with each other by statistically making sense (Gillis and Pavlidis, 2012). Drosophila, as a suitable model organism for investigating DNA damage In the post-genomic era, data sets in the literature and gene expression repair in multicellular organisms. In fact, DNA repair research has its analyses by researchers continue defining the interactions between roots in the harmful effects of Muller’s X-rays on the Drosophila genome, genes and their relationships in the network and constitutes the basis for nearly 100 years ago when that pioneering study was published. In our determining candidate genes in the pathways (Handl et al., 2005; Chiu study, thirty-six genes belonging to NER and BER, MMR, double chain et al., 2013). Accordingly, to identify the molecular pathways involved breaks repair system, ATM-ATR signaling pathways, cell cycle check­ is of capital relevance. In such way, bioinformatics analyses permit to points, and apoptosis were analyzed in response to HAP-NPs exposure. identify which genes are working together to repair DNA damage Our results show that the expression of the Mlh1 gene was increased induced, in our case, by HAP-NPs. In this context, the genotoxicity data 12 M. Güneş et al. Science of the Total Environment 904 (2023) 166556 obtained in the wing-spot and the comet assays can be better explained Sherlock, G., 2000. Gene ontology: tool for the unification of biology. Nat. Genet. 25 (1), 25–29. https://doi.org/10.1038/75556. when the involved pathways are identified. Augustine, R., Hasan, A., Primavera, R., Wilson, R.J., Thakor, A.S., Kevadiya, B.D., 2020. Compared to micro-sized HAPs, HAP-NPs have a larger surface area, Cellular uptake and retention of nanoparticles: insights on particle properties and a higher mechanical strength, and a more bioactive structure. In addi­ interaction with cellular components. Mater. Today Commun. 25, 101692 https:// tion, it binds to the target tissue more effectively both structurally and doi.org/10.1016/j.mtcomm.2020.101692. Bahadar, H., Maqbool, F., Niaz, K., Abdollahi, M., 2016. Toxicity of nanoparticles and an functionally. HAP-NPs, which releases more Ca, stands out regarding overview of current experimental models. Iran. Biomed. J. 20 (1), 1–11. https://doi. bioactivation in the target tissue (Webster et al., 2000). The Ca release, org/10.7508/ibj.2016.01.001. which is an important feature for biocompatibility, brings along intra­ Bonilla-Represa, V., Abalos-Labruzzi, C., Herrera-Martinez, M., Guerrero-Perez, M.O., 2020. Nanomaterials in dentistry: state of the art and future challenges. cellular toxicity problems (Ewence et al., 2008). Size differences is an Nanomaterials-Basel. 10, (9) https://doi.org/10.3390/nano10091770. important factor in assessing the toxicity of nanoparticles (Park et al., Brazdis, R.I., Fierascu, I., Avramescu, S.M., Fierascu, R.C., 2021. Recent progress in the 2011; Kaygisiz and Cigerci, 2017; Sukhanova et al., 2018). Although the application of hydroxyapatite for the adsorption of heavy metals from water matrices. Materials (Basel) 14 (22), 6898. https://doi.org/10.3390/ma14226898. entry of NPs into the cell varies depending on the cell and particle Carmona, E.R., Guecheva, T.N., Creus, A., Marcos, R., 2011. Proposal of an in vivo comet structure, small-sized particles have a higher potential to cause toxicity assay using haemocytes of Drosophila melanogaster. Environ. Mol. Mutagen. 52 (2), because their facility internalize (Augustine et al., 2020). As proved in 165–169. https://doi.org/10.1002/em.20604. Chiu, W.A., Euling, S.Y., Scott, C.S., Subramaniam, R.P., 2013. Approaches to advancing this study, HAP properties like size, shape, and synthesis time are factor quantitative human health risk assessment of environmental chemicals in the post- affecting the biological response. genomic era. Toxicol. Appl. Pharmacol. 271 (3), 309–323. https://doi.org/10.1016/ j.taap.2010.03.019. Collins, A.R., Oscoz, A.A., Brunborg, G., Gaivao, I., Giovannelli, L., Kruszewski, M., 5. Conclusions Smith, C.C., Stetina, R., 2008. The comet assay: topical issues. Mutagenesis. 23 (3), 143–151. https://doi.org/10.1093/mutage/gem051. In summary, our findings highlight the potential hazardous effects of Crawford, E.D., Seaman, J.E., Barber 2nd, A.E., David, D.C., Babbitt, P.C., Burlingame, A. L., Wells, J.A., 2012. Conservation of caspase substrates across metazoans suggests HAP-NPs in Drosophila. This study showed that sol-gel-produced HAP- hierarchical importance of signaling pathways over specific targets and cleavage site NPs cause mutations and DNA strand breaks in Drosophila. Changes in motifs in apoptosis. Cell Death Differ. 19 (12), 2040–2048. https://doi.org/10.1038/ the expression of genes involved in different DNA repair pathways have cdd.2012.99. been correlated with results in genotoxicity assays. Furthermore, cas­ Dai, C., Duan, J., Zhang, L., Jia, G., Zhang, C., Zhang, J., 2014. Biocompatibility of defect-related luminescent nanostructured and microstructured hydroxyapatite. pase activity increased when apoptosis-suppressed genes decreased. The Biol. Trace Elem. Res. 162 (1–3), 158–167. https://doi.org/10.1007/s12011-014- data obtained in this study are consistent with the literature, as the 0151-0. dimensional and shape differences of nanoparticles may affect the Dan, P., Sundararajan, V., Ganeshkumar, H., Gnanabarathi, B., Subramanian, A.K., Venkatasubu, G.D., Ichihara, S., Ichihara, G., Mohideen, S.S., 2019. Evaluation of genotoxic potential. In line with the data obtained, the development of hydroxyapatite nanoparticles - induced in vivo toxicity in Drosophila melanogaster. new strategies to produce safer HAP-NPs seems to be necessary to Appl. Surf. Sci. 484, 568–577. https://doi.org/10.1016/j.apsusc.2019.04.120. improve their clinical applications. Davis, A.P., Grondin, C.J., Johnson, R.J., Sciaky, D., Wiegers, J., Wiegers, T.C., Mattingly, C.J., 2021. Comparative toxicogenomics database (CTD): update 2021. Nucleic Acids Res. 49 (D1), D1138–D1143. https://doi.org/10.1093/nar/gkaa891. CRediT authorship contribution statement Dee, K.C., Puleo, D.A., Bizios, R., 2002. An Introduction to Tissue-biomaterial Interactions: Tissue-Biomaterial. Wiley-Liss, Inc. Deveci, M.Z.Y., Gonenci, R., Canpolat, I., Kanat, O., 2020. In vivo biocompatibility and Bülent Kaya, Nuray Kaya, and Merve Güneş planned the experi­ fracture healing of hydroxyapatite-hexagonal boron nitride-chitosan-collagen ments. Esin Akarsu and Emre Yavuz carried out the HA characterization. biocomposite coating in rats. Turk. J. Vet. Anim. Sci. 44 (1), 76–88. https://doi.org/ Merve Güneş, Burçin Yalçın, Ayşen Yağmur Burgazlı, and Ghada Tagorti 10.3906/vet-1906-21. Dong, L., Tang, S., Deng, F., Gong, Y., Zhao, K., Zhou, J., Liang, D., Fang, J., Hecker, M., carried out the rest of the experiments, analyzed the data, carried out the Giesy, J.P., Bai, X., Zhang, H., 2019. Shape-dependent toxicity of alumina statistical analysis, and prepared tables/figures. Merve Güneş, Bülent nanoparticles in rat astrocytes. Sci. Total Environ. 690, 158–166. https://doi.org/ Kaya, Nuray Kaya, and Ricard Marcos wrote the final manuscript. 10.1016/j.scitotenv.2019.06.532. Doostmohammadi, A., Monshi, A., Salehi, R., Fathi, M.H., Karbasi, S., Pieles, U., Daniels, A.U., 2012. Preparation, chemistry and physical properties of bone-derived Declaration of competing interest hydroxyapatite particles having a negative zeta potential. Mater. Chem. Phys. 132 (2–3), 446–452. Dorstyn, L., Kumar, S., 2008. A biochemical analysis of the activation of the Drosophila The authors declare that they have no known competing financial caspase DRONC. Cell Death Differ. 15 (3), 461–470. https://doi.org/10.1038/sj. interests or personal relationships that could have appeared to influence cdd.4402288. the work reported in this paper. Enax, J., Meyer, F., Schulze Zur Wiesche, E., Epple, M., 2022. On the application of calcium phosphate micro- and nanoparticles as food additive. Nanomaterials (Basel) 12 (22), 4075. https://doi.org/10.3390/nano12224075. Data availability Epple, M., 2018. Review of potential health risks associated with nanoscopic calcium phosphate. Acta Biomater. 77, 1–14. https://doi.org/10.1016/j.actbio.2018.07.036. Evans, M.D., Dizdaroglu, M., Cooke, M.S., 2004. Oxidative DNA damage and disease: Data will be made available on request. induction, repair and significance. Mutat. Res. Rev. Mutat. Res. 567 (1), 1–61. https://doi.org/10.1016/j.mrrev.2003.11.001. Acknowledgments Ewence, A.E., Bootman, M., Roderick, H.L., Skepper, J.N., McCarthy, G., Epple, M., Neumann, M., Shanahan, C.M., Proudfoot, D., 2008. Calcium phosphate crystals induce cell death in human vascular smooth muscle cells a potential mechanism in This work was supported by the Akdeniz University Scientific atherosclerotic plaque destabilization. Circ. Res. 103 (5), E28–E34. https://doi.org/ Research Projects Coordination Unit [Grant numbers FDK-2019-4457, 10.1161/Circresaha.108.181305. Fan, Y., Bergmann, A., 2010. The cleaved-Caspase-3 antibody is a marker of Caspase-9- FBA-2018-3285]. like DRONC activity in Drosophila. Cell Death Differ. 17 (3), 534–539. https://doi. org/10.1038/cdd.2009.185. References Ferreira-Ermita, D.A.C., Valente, F.L., Carlo-Reis, E.C., Araújo, F.R., Ribeiro, I.M., Cintra, C.C.V., Borges, A.P.B., 2020. Characterization and in vivo biocompatibility analysis of synthetic hydroxyapatite compounds associated with magnetite Alaraby, M., Annangi, B., Marcos, R., Hernandez, A., 2016. Drosophila melanogaster as a nanoparticles for a drug delivery system in osteomyelitis treatment. Results Mater. 5, suitable in vivo model to determine potential side effects of nanomaterials: a review. 10063. https://doi.org/10.1016/j.rinma.2020.100063. J. Toxicol. Environ. Health B 19 (2), 65–104. https://doi.org/10.1080/ Filip, D.G., Surdu, V.A., Paduraru, A.V., Andronescu, E., 2022. Current development in 10937404.2016.1166466. biomaterials-hydroxyapatite and bioglass for applications in biomedical field: a Allocco, D.J., Kohane, I.S., Butte, A.J., 2004. Quantifying the relationship between co- review. J. Funct. Biomater. 13 (4), 248. https://doi.org/10.3390/jfb13040248. expression, co-regulation and gene function. BMC Bioinformatics 5, (18). https:// Filipiak, M., Tylko, G., Pyza, E., 2012. Zinc induces caspase-dependent mitochondrial doi.org/10.1186/1471-2105-5-18. pathway of the programmed cell death in haemocytes of Drosophila melanogaster. Ashburner, M., Ball, C.A., Blake, J.A., Botstein, D., Butler, H., Cherry, J.M., Davis, A.P., Biometals. 25 (3), 507–516. https://doi.org/10.1007/s10534-012-9530-1. Dolinski, K., Dwight, S.S., Eppig, J.T., Harris, M.A., Hill, D.P., Issel-Tarver, L., Kasarskis, A., Lewis, S., Matese, J.C., Richardson, J.E., Ringwald, M., Rubin, G.M., 13 M. Güneş et al. Science of the Total Environment 904 (2023) 166556 Fogarty, C.E., Bergmann, A., 2014. Detecting Caspase Activity in Drosophila Larval Pai, S.D., Kini, M.S., Selvaraj, R., 2021. A review on adsorptive removal of dyes from Imaginal Discs, 1133, pp. 109–117. https://doi.org/10.1007/978-1-4939-0357-3_7. wastewater by hydroxyapatite nanocomposites. Environ. Sci. Pollut. Res. 28 (10), Franz, M., Rodriguez, H., Lopes, C., Zuberi, K., Montojo, J., Bader, G.D., Morris, Q., 2018. 11835–11849. https://doi.org/10.1007/s11356-019-07319-9. GeneMANIA update 2018. Nucleic Acids Res. 46 (W1), W60–W64. https://doi.org/ Panda, R.N., Hsieh, M.F., Chung, R.J., Chin, T.S., 2003. FTIR, XRD, SEM and solid state 10.1093/nar/gky311. NMR investigations of carbonate-containing hydroxyapatite nano-particles Furlanetto, M.P., Grivicich, I., Dihl, R.R., Lehmann, M., de Souza, D.S., Plentz, R.D.M., synthesized by hydroxide-gel technique. J. Phys. Chem. Solids 64 (2), 193–199. 2018. In vivo analysis of photobiomodulation genotoxicity using the somatic https://doi.org/10.1016/S0022-3697(02)00257-3. mutation and recombination test. Photomed. Laser Surg. 36 (10), 536–540. https:// Panda, S., Biswas, C.K., Paul, S., 2021. A comprehensive review on the preparation and doi.org/10.1089/pho.2018.4468. application of calcium hydroxyapatite: a special focus on atomic doping methods for Gillis, J., Pavlidis, P., 2012. “Guilt by association” is the exception rather than the rule in bone tissue engineering. Ceram. Int. 47 (20), 28122–28144. https://doi.org/ gene networks. PLoS Comput. Biol. 8 (3), e1002444 https://doi.org/10.1371/ 10.1016/j.ceramint.2021.07.100. journal.pcbi.1002444. Pappus, S.A., Ekka, B., Sahu, S., Sabat, D., Dash, P., Mishra, M., 2017. A toxicity Graf, U., Wurgler, F.E., Katz, A.J., Frei, H., Juon, H., Hall, C.B., Kale, P.G., 1984. Somatic assessment of hydroxyapatite nanoparticles on development and behaviour of mutation and recombination test in Drosophila melanogaster. Environ. Mutagen. 6 (2), Drosophila melanogaster. J. Nanopart. Res. 19, (4) https://doi.org/10.1007/s11051- 153–188. https://doi.org/10.1002/em.2860060206. 017-3824-8. Grimes, T., Potter, S.S., Datta, S., 2019. Integrating gene regulatory pathways into Park, M.V.D.Z., Neigh, A.M., Vermeulen, J.P., de la Fonteyne, L.J.J., Verharen, H.W., differential network analysis of gene expression data. Sci. Rep. 9 (1), 5479. https:// Briede, J.J., van Loveren, H., de Jong, W.H., 2011. The effect of particle size on the doi.org/10.1038/s41598-019-41918-3. cytotoxicity, inflammation, developmental toxicity and genotoxicity of silver Handl, J., Knowles, J., Kell, D.B., 2005. Computational cluster validation in post-genomic nanoparticles. Biomaterials. 32 (36), 9810–9817. https://doi.org/10.1016/j. data analysis. Bioinformatics. 21 (15), 3201–3212. https://doi.org/10.1093/ biomaterials.2011.08.085. bioinformatics/bti517. Parrilla-Castellar, E.R., Arlander, S.J., Karnitz, L., 2004. Dial 9-1-1 for DNA damage: the Ignjatovic, N.L., Markovic, S., Jugovic, D., Uskokovic, V., Uskokovic, D.P., 2020. From Rad9-Hus1-Rad1 (9-1-1) clamp complex. DNA Repair (Amst) 3 (8–9), 1009–1014. molecules to nanoparticles to functional materials. J. Serb Chem. Soc. 85 (11), https://doi.org/10.1016/j.dnarep.2004.03.032. 1383–1403. https://doi.org/10.2298/JSC200426035I. Portugal, J., Mansilla, S., Pina, B., 2022. Perspectives on the use of Toxicogenomics to Kalpana, M., Nagalakshmi, R., 2023. Nano hydroxyapatite for biomedical applications assess environmental risk. Front. Biosci. 27 (10), 294. https://doi.org/10.31083/j. derived from chemical and natural sources by simple precipitation method. Appl. fbl2710294. Biochem. Biotechnol. 195 (6), 3994–4010. https://doi.org/10.1007/s12010-022- Rahul, S.J., Naz, F., Siddique, Y.H., 2015. Evaluation of the toxic potential of cefotaxime 03968-8. in the third instar larvae of transgenic Drosophila melanogaster. Chem. Biol. Interact. Kaygisiz, S.Y., Cigerci, I.H., 2017. Genotoxic evaluation of different sizes of iron oxide 233, 71–80. https://doi.org/10.1016/j.cbi.2015.03.004. nanoparticles and ionic form by SMART, Allium and comet assay. Toxicol. Ind. Rao, P.J., Pelletier, M.H., Walsh, W.R., Mobbs, R.J., 2014. Spine interbody implants: Health 33 (10), 802–809. https://doi.org/10.1177/0748233717722907. material selection and modification, functionalization and bioactivation of surfaces Klovstad, M., Abdu, U., Schupbach, T., 2008. Drosophila brca2 is required for mitotic and to improve osseointegration. Optop. Surg. 6 (2), 81–89. https://doi.org/10.1111/ meiotic DNA repair and efficient activation of the meiotic recombination checkpoint. os.12098. PLoS Genet. 4 (2), e31 https://doi.org/10.1371/journal.pgen.0040031. Rasmussen, M.K., Pedersen, J.N., Marie, R., 2020. Size and surface charge Lin, K.L., Chang, J., Cheng, R.M., Ruan, M.L., 2007. Hydrothermal microemulsion characterization of nanoparticles with a salt gradient. Nat. Commun. 11 (1), 2337. synthesis of stoichiometric single crystal hydroxyapatite nanorods with mono- https://doi.org/10.1038/s41467-020-15889-3. dispersion and narrow-size distribution. Mater. Lett. 61 (8–9), 1683–1687. https:// Rathnayake, A., Hettithanthri, O., Sandanayake, S., Mahatantila, K., Rajapaksha, A.U., doi.org/10.1016/j.matlet.2006.07.099. Vithanage, M., 2022. Essence of hydroxyapatite in defluoridation of drinking water: Liu, Q., Xiang, P.P., Chen, M.Y., Luol, Y., Zhao, Y., Zhu, J.Y., Jing, W.W., Yu, H., 2021. a review. Environ. Pollut. 311, 119882 https://doi.org/10.1016/j. Nano-sized hydroxyapatite induces apoptosis and osteogenic differentiation of envpol.2022.119882. vascular smooth muscle cells via JNK/c-JUN pathway. Int. J. Nanomedicine 16, Reiter, L.T., Potocki, L., Chien, S., Gribskov, M., Bier, E., 2001. A systematic analysis of 3633–3648. https://doi.org/10.2147/Ijn.S303714. human disease-associated gene sequences in Drosophila melanogaster. Genome Res. Lorenzo, Y., Costa, S., Collins, A.R., Azqueta, A., 2013. The comet assay, DNA damage, 11 (6), 1114–1125. https://doi.org/10.1101/gr.169101. DNA repair and cytotoxicity: hedgehogs are not always dead. Mutagenesis. 28 (4), Ren, F.Z., Ding, Y.H., Leng, Y., 2014. Infrared spectroscopic characterization of 427–432. https://doi.org/10.1093/mutage/get018. carbonated apatite: a combined experimental and computational study. J. Biomed. Lyne, R., Smith, R., Rutherford, K., Wakeling, M., Varley, A., Guillier, F., Janssens, H., Mater. Res. A 102 (2), 496–505. https://doi.org/10.1002/jbm.a.34720. Ji, W., McLaren, P., North, P., Rana, D., Riley, T., Sullivan, J., Watkins, X., Righi, S., Prato, E., Magnani, G., Lama, V., Biandolino, F., Parlapiano, I., Carella, F., Woodbridge, M., Lilley, K., Russell, S., Ashburner, M., Mizuguchi, K., Micklem, G., Iafisco, M., Adamiano, A., 2023. Calcium phosphates from fish bones in sunscreen: 2007. FlyMine: an integrated database for Drosophila and Anopheles genomics. an LCA and toxicity study of an emerging material for circular economy. Sci. Total Genome Biol. 8 (7), R129. https://doi.org/10.1186/gb-2007-8-7-r129. Environ. 862, 160751 https://doi.org/10.1016/j.scitotenv.2022.160751. Lyne, R., Bazaga, A., Butano, D., Contrino, S., Heimbach, J., Hu, F.Y., Kalderimis, A., Sahu, S., Mishra, M., 2020. Hydroxyapatite nanoparticle causes sensory organ defects by Lyne, M., Reierskog, K., Stepan, R., Sullivan, J., Wise, A., Yehudi, Y., Micklem, G., targeting the retromer complex in Drosophila melanogaster. Nanoimpact 19, 100237. 2022. HumanMine: Advanced Data Searching, Analysis and Cross-species https://doi.org/10.1016/j.impact.2020.100237. Comparison, 2022. Database-Oxford. https://doi.org/10.1093/database/baac054. Salarian, M., Solati-Hashjin, M., Shafiei, S.S., Salarian, R., Nemati, Z.A., 2009. Template- Mannuss, A., Trapp, O., Puchta, H., 2012. Gene regulation in response to DNA damage. directed hydrothermal synthesis of dandelion-like hydroxyapatite in the presence of Biochim. Biophys. Acta, Gene Regul. Mech. 1819 (2), 154–165. https://doi.org/ cetyltrimethylammonium bromide and polyethylene glycol. Ceram. Int. 35 (7), 10.1016/j.bbagrm.2011.08.003. 2563–2569. https://doi.org/10.1016/j.ceramint.2009.02.031. Mishra, N., Srivastav, R., Agrawal, U.R., Tewari, R.R., 2017. An insight into the Sia, E.A., Dominska, M., Stefanovic, L., Petes, T.D., 2001. Isolation and characterization genotoxicity assessment studies in dipterans. Mutat. Res. Rev. Mutat. Res. 773, of point mutations in mismatch repair genes that destabilize microsatellites in yeast. 220–229. https://doi.org/10.1016/j.mrrev.2016.10.001. Mol. Cell. Biol. 21 (23), 8157–8167. https://doi.org/10.1128/Mcb.21.23.8157- Mohd Pu’ad, N.A.S., Koshy, P., Abdullah, H.Z., Idris, M.I., Lee, T.C., 2019. Syntheses of 8167.2001. hydroxyapatite from natural sources. Heliyon. 5 (5), e01588 https://doi.org/ Sohaebuddin, S.K., Thevenot, P.T., Baker, D., Eaton, J.W., Tang, L., 2010. Nanomaterial 10.1016/j.heliyon.2019.e01588. cytotoxicity is composition, size, and cell type dependent. Part. Fibre Toxicol. 7, 22. Montoya, C., Du, Y., Gianforcaro, A.L., Orrego, S., Yang, M.B., Lelkes, P.I., 2021. On the https://doi.org/10.1186/1743-8977-7-22. road to smart biomaterials for bone research: definitions, concepts, advances, and Sukhanova, A., Bozrova, S., Sokolov, P., Berestovoy, M., Karaulov, A., Nabiev, I., 2018. outlook. Bone Res. 9 (1) https://doi.org/10.1038/s41413-020-00131-z. Dependence of nanoparticle toxicity on their physical and chemical properties. Mosa, I.F., Abd, H.H., Abuzreda, A., Assaf, N., Yousif, A.B., 2020. Bio-evaluation of the Nanoscale Res. Lett. 13 https://doi.org/10.1186/s11671-018-2457-x. role of chitosan and curcumin nanoparticles in ameliorating genotoxicity and Sun, Y.X., Guo, G.S., Wang, Z.H., Guo, H.Y., 2006. Synthesis of single-crystal HAP inflammatory responses in rats’ gastric tissue followed hydroxyapatite nanorods. Ceram. Int. 32 (8), 951–954. https://doi.org/10.1016/j. nanoparticles’ oral uptake. Toxicol. Res. 9 (4), 493–508. https://doi.org/10.1093/ ceramint.2005.07.023. toxres/tfaa054. Turna Demir, F., 2022. Protective effects of resveratrol against genotoxicity induced by Nagyne-Kovacs, T., Studnicka, L., Kincses, A., Spengler, G., Molnar, M., Tolner, M., nano and bulk hydroxyapatite in Drosophila melanogaster. J. Toxicol. Environ. Lukacs, I.E., Szilagyi, I.M., Pokol, G., 2018. Synthesis and characterization of Sr and Health, Part A 85 (20), 850–865. https://doi.org/10.1080/ mg-doped hydroxyapatite by a simple precipitation method. Ceram. Int. 44 (18), 15287394.2022.2101568. 22976–22982. https://doi.org/10.1016/j.ceramint.2018.09.096. van Dam, S., Vosa, U., van der Graaf, A., Franke, L., de Magalhaes, J.P., 2018. Gene co- Naves, M.P.C., de Morais, C.R., Silva, A.C.A., Dantas, N.O., Spano, M.A., de Rezende, A. expression analysis for functional classification and gene-disease predictions. Brief. A.A., 2018. Assessment of mutagenic, recombinogenic and carcinogenic potential of Bioinform. 19 (4), 575–592. https://doi.org/10.1093/bib/bbw139. titanium dioxide nanocristals in somatic cells of Drosophila melanogaster. Food Chem. van der Houwen, J.A.M., Cressey, G., Cressey, B.A., Valsami-Jones, E., 2003. The effect of Toxicol. 112, 273–281. https://doi.org/10.1016/j.fct.2017.12.040. organic ligands on the crystallinity of calcium phosphate. J. Cryst. Growth 249 Osuchukwu, O.A., Salihi, A., Abdullahi, I., Abdulkareem, B., Nwannenna, C.S., 2021. (3–4), 572–583. https://doi.org/10.1016/S0022-0248(02)02227-3. Synthesis techniques, characterization and mechanical properties of natural derived Venkatesan, J., Anil, S., 2021. Hydroxyapatite derived from marine resources and their hydroxyapatite scaffolds for bone implants: a review. SN. Appl. Sci. 3, (10) https:// potential biomedical applications. Biotechnol. Bioprocess Eng. 26 (3), 312–324. doi.org/10.1007/s42452-021-04795-y. https://doi.org/10.1007/s12257-020-0359-0. Oti, M., Brunner, H.G., 2007. The modular nature of genetic diseases. Clin. Genet. 71 (1), Wang, Y.J., Chen, J.D., Wei, K., Zhang, S.H., Wang, X.D., 2006. Surfactant-assisted 1–11. https://doi.org/10.1111/j.1399-0004.2006.00708.x. synthesis of hydroxyapatite particles. Mater. Lett. 60 (27), 3227–3231. https://doi. org/10.1016/j.matlet.2006.02.077. 14 M. Güneş et al. Science of the Total Environment 904 (2023) 166556 Warde-Farley, D., Donaldson, S.L., Comes, O., Zuberi, K., Badrawi, R., Chao, P., Yalcin, B., Gunes, M., Kursun, A.Y., Kaya, N., Marcos, R., Kaya, B., 2022. Genotoxic Franz, M., Grouios, C., Kazi, F., Lopes, C.T., Maitland, A., Mostafavi, S., Montojo, J., hazard assessment of cerium oxide and magnesium oxide nanoparticles in Shao, Q., Wright, G., Bader, G.D., Morris, Q., 2010. The GeneMANIA prediction Drosophila. Nanotoxicol. 16 (3), 393–407. https://doi.org/10.1080/ server: biological network integration for gene prioritization and predicting gene 17435390.2022.2098072. function. Nucleic Acids Res. 38, W214–W220. https://doi.org/10.1093/nar/gkq537. Yavuz, E., Erdem, R., Kucuksayan, E., Akarsu, E., Akarsu, M., 2021. Preparation and Webster, T.J., Ergun, C., Doremus, R.H., Siegel, R.W., Bizios, R., 2000. Enhanced characterization of polyethylene glycol functional hydroxyapatite/polycaprolactone functions of osteoblasts on nanophase ceramics. Biomaterials. 21 (17), 1803–1810. electrospun biomembranes for bone tissue engineering applications. Fiber Polym. 22 https://doi.org/10.1016/S0142-9612(00)00075-2. (5), 1274–1284. https://doi.org/10.1007/s12221-021-0560-6. Wu, Y., Zhao, D., Zhuang, J., Zhang, F., Xu, C., 2016b. Caspase-8 and Caspase-9 Zaffarin, A.S.M., Ng, S.F., Ng, M.H., Hassan, H., Alias, E., 2021. Nano-hydroxyapatite as a functioned differently at different stages of the cyclic stretch-induced apoptosis in delivery system for promoting bone regeneration in vivo: a systematic review. human periodontal ligament cells. PLoS One 11 (12), e0168268. https://doi.org/ Nanomaterials-Basel. 11, (10) https://doi.org/10.3390/nano11102569. 10.1371/journal.pone.0168268. Zhao, X., Heng, B.C., Xiong, S., Guo, J., Tan, T.T., Boey, F.Y., Ng, K.W., Loo, J.S., 2011. In Wu, Z.H., Yang, S.L., Wu, W., 2016a. Shape control of inorganic nanoparticles from vitro assessment of cellular responses to rod-shaped hydroxyapatite nanoparticles of solution. Nanoscale. 8 (3), 1237–1259. https://doi.org/10.1039/c5nr07681a. varying lengths and surface areas. Nanotoxicol. 5 (2), 182–194. https://doi.org/ Xia, Y., Li, B., Zhang, F., Wu, Q., Wen, S., Jiang, N., Liu, D., Huang, C., Liu, S., 2022. 10.3109/17435390.2010.503943. Hydroxyapatite nanoparticles promote mitochondrial-based pyroptosis via Zhao, X., Ong, K.J., Ede, J.D., Stafford, J.L., Ng, K.W., Goss, G.G., Loo, S.C., 2013. activating calcium homeostasis and redox imbalance in vascular smooth muscle Evaluating the toxicity of hydroxyapatite nanoparticles in catfish cells and zebrafish cells. Nanotechnol. 33, (27) https://doi.org/10.1088/1361-6528/ac61ca. embryos. Small 9 (9–10), 1734–1741. https://doi.org/10.1002/smll.201200639. Xu, Z.L., Liu, C.S., Wei, J., Sun, J., 2012. Effects of four types of hydroxyapatite nanoparticles with different nanocrystal morphologies and sizes on apoptosis in rat osteoblasts. J. Appl. Toxicol. 32 (6), 429–435. https://doi.org/10.1002/jat.1745. 15