54830aa722b8d56af16fd4c7bf08779b ijms_27_05031.pdf a0df9597a6d6379bb4d4a53cd61695db40f3153a ijms_27_05031.pdf db4b093a8d7192ae413bc75e239ab654dd97bbed0663577cb691094af259d6af ijms_27_05031.pdf Title: Long-Term PET-Nanoplastic Exposure Alters DNA Damage Response Capacity in BEAS-2B Human Bronchial Epithelial Cells Subject: Chronic inhalation exposure to nanoplastics, specifically polyethylene terephthalate (PET) nanoplastics (PET-NPLs) is an emerging health concern, yet the long-term consequences for genomic stability and DNA damage response (DDR) capacity in bronchial epithelial cells remain poorly characterized. For this study, human bronchial epithelial BEAS-2B cells were continuously exposed to PET-NPLs for over 20 weeks, after which elevated basal DNA genotoxic damage was observed, as assessed by the alkaline comet assay. In addition, a broad transcriptional suppression of the DDR, with 27 of 84 profiled genes involved in DDR showing reduced expression relative to passage-matched control was observed. The suppressed genes span ATM/ATR checkpoint signaling, homologous recombination (HR), base excision repair (BER), nucleotide excision repair (NER), and apoptotic pathways. To determine whether chronic PET-NPL exposure altered susceptibility to acute genotoxic challenge in a damage-type-specific manner, cells were treated with methyl methanesulfonate (MMS), ultraviolet-C (UV-C) radiation, or bleomycin. While MMS and UV-C induced comparable levels of DNA damage in control and PET-exposed cells, bleomycin produced significantly greater damage in PET-exposed cells, indicating selective sensitization to doble-strand breaks (DSB)-type and oxidative genotoxic insults. Transcriptional profiling during bleomycin challenge identified 18 DDR genes with relatively higher expression in PET-exposed cells compared to passage-matched controls, encompassing HR, BER, ATM/ATR signaling, the Fanconi anemia pathway, and apoptosis. Furthermore, PET-exposed cells retained significantly higher residual DNA damage after 3 h of bleomycin challenge, indicating a persistent early repair deficit. Together, these findings suggest that chronic PET-NPL exposure specifically compromises the bronchial epithelial DDR, with potential implications for long-term genomic stability in respiratory epithelia subjected to nanoplastic inhalation. Keywords: nanoplastics; PET; long-term exposure; DNA damage response; genotoxicity Author: Michelle Morataya-Reyes, Aliro Villacorta, Raquel Egea, Joan Martín-Pérez, Javier Gutiérrez-García, Susana Pastor, Ricard Marcos and Alba Hernández Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25; modified using OpenPDF 1.4.2 CreationDate: Tue Jun 2 14:20:12 2026 CEST ModDate: Tue Jun 2 14:28:01 2026 CEST Custom Metadata: yes Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 18 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 1962901 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- GYDIEN+URWPalladioL-Roma Type 1 Custom yes yes yes 133 0 ZCNCDW+URWPalladioL-Ital Type 1 Custom yes yes yes 134 0 AIRHKL+VnURWPalladioL Type 1 Custom yes yes yes 135 0 CUDEYE+URWPalladioL-Bold Type 1 Custom yes yes yes 136 0 WUZQWY+EURM10 Type 1 Builtin yes yes yes 178 0 FGLHJM+CMSY10 Type 1 Builtin yes yes yes 223 0 CIDFont+F3 CID TrueType Identity-H yes no yes 217 0 CIDFont+F2 CID TrueType Identity-H yes no yes 218 0 MPUDFL+PazoMath Type 1 Builtin yes yes yes 277 0 CIDFont+F2 CID TrueType Identity-H yes no yes 239 0 CIDFont+F3 CID TrueType Identity-H yes no yes 271 0 CIDFont+F2 CID TrueType Identity-H yes no yes 272 0 CIDFont+F2 CID TrueType Identity-H yes no yes 322 0 CIDFont+F3 CID TrueType Identity-H yes no yes 344 0 CIDFont+F2 CID TrueType Identity-H yes no yes 345 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2026-06-30 04:13:46 CEST RepresentationInformation: ijms_27_05031.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2026-06-29 13:30:10 CEST Size: 1962901 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 552 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: ViewerPreferences: HideToolbar: false HideMenubar: false HideWindowUI: false FitWindow: true CenterWindow: false DisplayDocTitle: false NonFullScreenPageMode: UseNone Direction: L2R ViewArea: CropBox ViewClip: CropBox PrintArea: CropBox PageClip: CropBox PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Item: Title: Results and Discussion Children: Item: Title: Characterization of PET-NPLs and Internalization in BEAS-2B Cells Item: Title: Effects of Long-Term Exposure to PET-NPLs in Basal Genomic Integrity and DDR Gene Expression Item: Title: Damage-Type-Specific Sensitivity of PET-Exposed BEAS-2B Item: Title: Transcriptional Response and DDR of PET-Exposed BEAS-2B After Bleomycin Challenge Item: Title: Environmental Health Relevance of the Chronic Exposure Model Item: Title: Limitations and Future Research Directions Item: Title: Materials and Methods Children: Item: Title: Particle Obtention, Labeling and Characterization Item: Title: Cell Culture and Long-Term Exposure to PET-NPLs Item: Title: Particle Internalization Using Confocal Microscopy Item: Title: DNA Damage Induction Item: Title: The Comet Assay Item: Title: RNA Extraction After DNA Damage Induction Item: Title: Gene Expression Using the RT2 Profiler PCR Array Human DNA Damage Signaling Pathway Item: Title: Data Analysis of the RT2 Profiler PCR Array Human DNA Damage Signaling Pathway Item: Title: Conclusions Item: Title: References Info: Title: Long-Term PET-Nanoplastic Exposure Alters DNA Damage Response Capacity in BEAS-2B Human Bronchial Epithelial Cells Author: Michelle Morataya-Reyes, Aliro Villacorta, Raquel Egea, Joan Martín-Pérez, Javier Gutiérrez-García, Susana Pastor, Ricard Marcos and Alba Hernández Subject: Chronic inhalation exposure to nanoplastics, specifically polyethylene terephthalate (PET) nanoplastics (PET-NPLs) is an emerging health concern, yet the long-term consequences for genomic stability and DNA damage response (DDR) capacity in bronchial epithelial cells remain poorly characterized. For this study, human bronchial epithelial BEAS-2B cells were continuously exposed to PET-NPLs for over 20 weeks, after which elevated basal DNA genotoxic damage was observed, as assessed by the alkaline comet assay. In addition, a broad transcriptional suppression of the DDR, with 27 of 84 profiled genes involved in DDR showing reduced expression relative to passage-matched control was observed. The suppressed genes span ATM/ATR checkpoint signaling, homologous recombination (HR), base excision repair (BER), nucleotide excision repair (NER), and apoptotic pathways. To determine whether chronic PET-NPL exposure altered susceptibility to acute genotoxic challenge in a damage-type-specific manner, cells were treated with methyl methanesulfonate (MMS), ultraviolet-C (UV-C) radiation, or bleomycin. While MMS and UV-C induced comparable levels of DNA damage in control and PET-exposed cells, bleomycin produced significantly greater damage in PET-exposed cells, indicating selective sensitization to doble-strand breaks (DSB)-type and oxidative genotoxic insults. Transcriptional profiling during bleomycin challenge identified 18 DDR genes with relatively higher expression in PET-exposed cells compared to passage-matched controls, encompassing HR, BER, ATM/ATR signaling, the Fanconi anemia pathway, and apoptosis. Furthermore, PET-exposed cells retained significantly higher residual DNA damage after 3 h of bleomycin challenge, indicating a persistent early repair deficit. Together, these findings suggest that chronic PET-NPL exposure specifically compromises the bronchial epithelial DDR, with potential implications for long-term genomic stability in respiratory epithelia subjected to nanoplastic inhalation. Keywords: nanoplastics; PET; long-term exposure; DNA damage response; genotoxicity Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25; modified using OpenPDF 1.4.2 CreationDate: Tue Jun 02 14:20:12 CEST 2026 ModDate: Tue Jun 02 14:28:01 CEST 2026 ID: 0x61302e898f97c75ece340014a3af4d9e, 0x17b101a93d33a6d2edc910d46ef2875a Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: WUZQWY+EURM10 FontSubset: true FirstChar: 13 LastChar: 22 FontDescriptor: FontName: WUZQWY+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true ToUnicode: true Font: BaseFont: GYDIEN+URWPalladioL-Roma FontSubset: true FirstChar: 1 LastChar: 248 FontDescriptor: FontName: GYDIEN+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: MPUDFL+PazoMath FontSubset: true FirstChar: 68 LastChar: 68 FontDescriptor: FontName: MPUDFL+PazoMath Flags: Symbolic FontBBox: -40, -283, 878, 946 FontFile: true ToUnicode: true Font: BaseFont: ZCNCDW+URWPalladioL-Ital FontSubset: true FirstChar: 2 LastChar: 150 FontDescriptor: FontName: ZCNCDW+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: AIRHKL+VnURWPalladioL FontSubset: true FirstChar: 160 LastChar: 225 FontDescriptor: FontName: AIRHKL+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: CUDEYE+URWPalladioL-Bold FontSubset: true FirstChar: 3 LastChar: 237 FontDescriptor: FontName: CUDEYE+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: FGLHJM+CMSY10 FontSubset: true FirstChar: 0 LastChar: 25 FontDescriptor: FontName: FGLHJM+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true ToUnicode: true Pages: Page: Sequence: 1 Annotations: Annotation: Subtype: Link Rect: 34, 204, 124, 223 Annotation: Subtype: Link Rect: 88, 77, 152, 84 Annotation: Subtype: Link Rect: 34, 64, 119, 72 Annotation: Subtype: Link Rect: 34, 773, 180, 809 Annotation: Subtype: Link Rect: 515, 773, 560, 809 Annotation: Subtype: Link Rect: 157, 643, 169, 656 Annotation: Subtype: Link Rect: 324, 643, 335, 656 Annotation: Subtype: Link Rect: 545, 643, 556, 656 Annotation: Subtype: Link Rect: 104, 630, 115, 642 Annotation: Subtype: Link Rect: 198, 630, 209, 642 Annotation: Subtype: Link Rect: 317, 630, 328, 642 Annotation: Subtype: Link Rect: 421, 27, 560, 37 Page: Sequence: 2 Annotations: Annotation: Subtype: Link Rect: 214, 717, 221, 726 ActionDest: 90 Annotation: Subtype: Link Rect: 419, 703, 426, 712 ActionDest: 90 Annotation: Subtype: Link Rect: 427, 703, 434, 712 ActionDest: 90 Annotation: Subtype: Link Rect: 372, 661, 379, 670 ActionDest: 90 Annotation: Subtype: Link Rect: 205, 633, 212, 642 ActionDest: 90 Annotation: Subtype: Link Rect: 212, 633, 219, 642 ActionDest: 90 Annotation: Subtype: Link Rect: 519, 619, 526, 628 ActionDest: 90 Annotation: Subtype: Link Rect: 526, 619, 533, 628 ActionDest: 90 Annotation: Subtype: Link Rect: 300, 577, 307, 586 ActionDest: 90 Annotation: Subtype: Link Rect: 307, 577, 319, 586 ActionDest: 90 Annotation: Subtype: Link Rect: 508, 536, 520, 545 ActionDest: 90 Annotation: Subtype: Link Rect: 521, 536, 533, 545 ActionDest: 90 Annotation: Subtype: Link Rect: 428, 508, 440, 517 ActionDest: 90 Annotation: Subtype: Link Rect: 441, 508, 453, 517 ActionDest: 90 Annotation: Subtype: Link Rect: 285, 410, 297, 419 ActionDest: 90 Annotation: Subtype: Link Rect: 213, 382, 225, 391 ActionDest: 90 Annotation: Subtype: Link Rect: 226, 382, 238, 391 ActionDest: 90 Annotation: Subtype: Link Rect: 259, 341, 270, 350 ActionDest: 90 Annotation: Subtype: Link Rect: 271, 341, 283, 350 ActionDest: 90 Annotation: Subtype: Link Rect: 510, 299, 522, 308 ActionDest: 204 Annotation: Subtype: Link Rect: 544, 271, 556, 280 ActionDest: 204 Annotation: Subtype: Link Rect: 421, 27, 560, 37 Page: Sequence: 3 Annotations: Annotation: Subtype: Link Rect: 392, 432, 399, 444 ActionDest: 24 Annotation: Subtype: Link Rect: 448, 379, 460, 388 ActionDest: 90 Annotation: Subtype: Link Rect: 461, 379, 473, 388 ActionDest: 204 Annotation: Subtype: Link Rect: 333, 348, 340, 360 ActionDest: 24 Annotation: Subtype: Link Rect: 391, 240, 402, 249 ActionDest: 204 Annotation: Subtype: Link Rect: 199, 153, 206, 165 ActionDest: 24 Annotation: Subtype: Link Rect: 200, 126, 207, 138 ActionDest: 24 Annotation: Subtype: Link Rect: 218, 101, 230, 109 ActionDest: 204 Annotation: Subtype: Link Rect: 421, 27, 560, 37 Page: Sequence: 4 Annotations: Annotation: Subtype: Link Rect: 282, 703, 294, 712 ActionDest: 90 Annotation: Subtype: Link Rect: 295, 703, 307, 712 ActionDest: 204 Annotation: Subtype: Link Rect: 547, 235, 554, 247 ActionDest: 154 Annotation: Subtype: Link Rect: 312, 154, 324, 163 ActionDest: 90 Annotation: Subtype: Link Rect: 324, 154, 336, 163 ActionDest: 90 Annotation: Subtype: Link Rect: 337, 154, 349, 163 ActionDest: 204 Annotation: Subtype: Link Rect: 349, 154, 361, 163 ActionDest: 204 Annotation: Subtype: Link Rect: 421, 27, 560, 37 Page: Sequence: 5 Annotations: Annotation: Subtype: Link Rect: 421, 27, 560, 37 Page: Sequence: 6 Annotations: Annotation: Subtype: Link Rect: 536, 742, 543, 754 ActionDest: 154 Annotation: Subtype: Link Rect: 214, 675, 226, 684 ActionDest: 204 Annotation: Subtype: Link Rect: 226, 675, 238, 684 ActionDest: 204 Annotation: Subtype: Link Rect: 340, 589, 347, 601 ActionDest: 154 Annotation: Subtype: Link Rect: 544, 547, 551, 559 ActionDest: 155 Annotation: Subtype: Link Rect: 421, 27, 560, 37 Page: Sequence: 7 Annotations: Annotation: Subtype: Link Rect: 254, 703, 266, 712 ActionDest: 90 Annotation: Subtype: Link Rect: 267, 703, 279, 712 ActionDest: 90 Annotation: Subtype: Link Rect: 369, 591, 381, 600 ActionDest: 204 Annotation: Subtype: Link Rect: 384, 591, 396, 600 ActionDest: 204 Annotation: Subtype: Link Rect: 455, 370, 467, 379 ActionDest: 204 Annotation: Subtype: Link Rect: 198, 342, 210, 351 ActionDest: 204 Annotation: Subtype: Link Rect: 292, 314, 304, 323 ActionDest: 204 Annotation: Subtype: Link Rect: 286, 270, 293, 282 ActionDest: 34 Annotation: Subtype: Link Rect: 546, 214, 553, 226 ActionDest: 34 Annotation: Subtype: Link Rect: 405, 130, 412, 142 ActionDest: 34 Annotation: Subtype: Link Rect: 421, 27, 560, 37 Page: Sequence: 8 Annotations: Annotation: Subtype: Link Rect: 401, 353, 408, 365 ActionDest: 154 Annotation: Subtype: Link Rect: 225, 189, 237, 198 ActionDest: 204 Annotation: Subtype: Link Rect: 238, 189, 250, 198 ActionDest: 204 Annotation: Subtype: Link Rect: 492, 62, 499, 74 ActionDest: 330 Annotation: Subtype: Link Rect: 421, 27, 560, 37 Page: Sequence: 9 Annotations: Annotation: Subtype: Link Rect: 405, 742, 412, 754 ActionDest: 330 Annotation: Subtype: Link Rect: 295, 630, 302, 642 ActionDest: 154 Annotation: Subtype: Link Rect: 459, 118, 466, 130 ActionDest: 330 Annotation: Subtype: Link Rect: 421, 27, 560, 37 Page: Sequence: 10 Annotations: Annotation: Subtype: Link Rect: 409, 703, 421, 712 ActionDest: 204 Annotation: Subtype: Link Rect: 421, 703, 433, 712 ActionDest: 204 Annotation: Subtype: Link Rect: 368, 563, 380, 572 ActionDest: 204 Annotation: Subtype: Link Rect: 380, 563, 392, 572 ActionDest: 204 Annotation: Subtype: Link Rect: 502, 161, 514, 170 ActionDest: 204 Annotation: Subtype: Link Rect: 426, 147, 438, 156 ActionDest: 204 Annotation: Subtype: Link Rect: 494, 119, 506, 128 ActionDest: 204 Annotation: Subtype: Link Rect: 544, 105, 556, 114 ActionDest: 400 Annotation: Subtype: Link Rect: 421, 27, 560, 37 Page: Sequence: 11 Annotations: Annotation: Subtype: Link Rect: 204, 453, 216, 462 ActionDest: 90 Annotation: Subtype: Link Rect: 216, 453, 228, 462 ActionDest: 400 Annotation: Subtype: Link Rect: 455, 439, 467, 448 ActionDest: 400 Annotation: Subtype: Link Rect: 468, 440, 480, 448 ActionDest: 400 Annotation: Subtype: Link Rect: 317, 102, 329, 111 ActionDest: 204 Annotation: Subtype: Link Rect: 330, 102, 342, 111 ActionDest: 204 Annotation: Subtype: Link Rect: 342, 102, 354, 111 ActionDest: 400 Annotation: Subtype: Link Rect: 421, 27, 560, 37 Page: Sequence: 12 Annotations: Annotation: Subtype: Link Rect: 289, 661, 301, 670 ActionDest: 400 Annotation: Subtype: Link Rect: 301, 661, 313, 670 ActionDest: 400 Annotation: Subtype: Link Rect: 421, 27, 560, 37 Page: Sequence: 13 Annotations: Annotation: Subtype: Link Rect: 255, 468, 267, 478 ActionDest: 400 Annotation: Subtype: Link Rect: 258, 91, 273, 103 ActionDest: 34 Annotation: Subtype: Link Rect: 421, 27, 560, 37 Page: Sequence: 14 Annotations: Annotation: Subtype: Link Rect: 223, 631, 238, 643 ActionDest: 43 Annotation: Subtype: Link Rect: 421, 27, 560, 37 Page: Sequence: 15 Annotations: Annotation: Subtype: Link Rect: 536, 591, 560, 602 Annotation: Subtype: Link Rect: 165, 578, 377, 589 Annotation: Subtype: Link Rect: 421, 27, 560, 37 Page: Sequence: 16 Annotations: Annotation: Subtype: Link Rect: 408, 607, 446, 618 Annotation: Subtype: Link Rect: 131, 569, 168, 579 Annotation: Subtype: Link Rect: 344, 543, 382, 554 Annotation: Subtype: Link Rect: 388, 543, 424, 554 Annotation: Subtype: Link Rect: 318, 517, 356, 528 Annotation: Subtype: Link Rect: 321, 492, 560, 503 Annotation: Subtype: Link Rect: 104, 454, 142, 465 Annotation: Subtype: Link Rect: 398, 428, 436, 439 Annotation: Subtype: Link Rect: 405, 402, 443, 413 Annotation: Subtype: Link Rect: 245, 365, 283, 375 Annotation: Subtype: Link Rect: 197, 327, 235, 337 Annotation: Subtype: Link Rect: 334, 300, 372, 311 Annotation: Subtype: Link Rect: 378, 300, 414, 311 Annotation: Subtype: Link Rect: 325, 275, 363, 286 Annotation: Subtype: Link Rect: 414, 249, 451, 260 Annotation: Subtype: Link Rect: 216, 225, 253, 235 Annotation: Subtype: Link Rect: 287, 198, 324, 209 Annotation: Subtype: Link Rect: 330, 198, 367, 209 Annotation: Subtype: Link Rect: 131, 174, 168, 184 Annotation: Subtype: Link Rect: 175, 174, 211, 184 Annotation: Subtype: Link Rect: 309, 147, 346, 158 Annotation: Subtype: Link Rect: 256, 109, 293, 120 Annotation: Subtype: Link Rect: 259, 84, 297, 94 Annotation: Subtype: Link Rect: 421, 27, 560, 37 Page: Sequence: 17 Annotations: Annotation: Subtype: Link Rect: 354, 743, 391, 754 Annotation: Subtype: Link Rect: 397, 743, 433, 754 Annotation: Subtype: Link Rect: 196, 718, 234, 729 Annotation: Subtype: Link Rect: 413, 679, 451, 690 Annotation: Subtype: Link Rect: 108, 642, 146, 652 Annotation: Subtype: Link Rect: 177, 616, 215, 627 Annotation: Subtype: Link Rect: 366, 590, 404, 601 Annotation: Subtype: Link Rect: 59, 553, 96, 563 Annotation: Subtype: Link Rect: 59, 514, 96, 524 Annotation: Subtype: Link Rect: 102, 514, 138, 524 Annotation: Subtype: Link Rect: 156, 475, 194, 486 Annotation: Subtype: Link Rect: 200, 475, 236, 486 Annotation: Subtype: Link Rect: 452, 450, 489, 461 Annotation: Subtype: Link Rect: 159, 424, 197, 435 Annotation: Subtype: Link Rect: 402, 399, 440, 410 Annotation: Subtype: Link Rect: 388, 373, 425, 384 Annotation: Subtype: Link Rect: 170, 348, 207, 358 Annotation: Subtype: Link Rect: 293, 322, 330, 333 Annotation: Subtype: Link Rect: 336, 322, 372, 333 Annotation: Subtype: Link Rect: 506, 296, 543, 307 Annotation: Subtype: Link Rect: 180, 272, 218, 282 Annotation: Subtype: Link Rect: 224, 272, 260, 282 Annotation: Subtype: Link Rect: 95, 246, 132, 256 Annotation: Subtype: Link Rect: 216, 221, 253, 231 Annotation: Subtype: Link Rect: 127, 195, 164, 205 Annotation: Subtype: Link Rect: 397, 169, 434, 180 Annotation: Subtype: Link Rect: 440, 169, 476, 180 Annotation: Subtype: Link Rect: 519, 156, 557, 167 Annotation: Subtype: Link Rect: 342, 130, 380, 141 Annotation: Subtype: Link Rect: 235, 92, 272, 103 Annotation: Subtype: Link Rect: 421, 27, 560, 37 Page: Sequence: 18 Annotations: Annotation: Subtype: Link Rect: 105, 731, 143, 742 Annotation: Subtype: Link Rect: 82, 693, 119, 703 Annotation: Subtype: Link Rect: 314, 667, 351, 678 Annotation: Subtype: Link Rect: 358, 667, 394, 678 Annotation: Subtype: Link Rect: 139, 642, 176, 652 Annotation: Subtype: Link Rect: 182, 642, 218, 652 Annotation: Subtype: Link Rect: 90, 591, 128, 601 Annotation: Subtype: Link Rect: 301, 552, 338, 563 Annotation: Subtype: Link Rect: 95, 514, 132, 524 Annotation: Subtype: Link Rect: 138, 514, 174, 524 Annotation: Subtype: Link Rect: 95, 476, 133, 486 Annotation: Subtype: Link Rect: 421, 27, 560, 37 Checksum: 4e4dcc0f Type: CRC32 Checksum: 54830aa722b8d56af16fd4c7bf08779b Type: MD5 Checksum: a0df9597a6d6379bb4d4a53cd61695db40f3153a Type: SHA-1 Checksum: db4b093a8d7192ae413bc75e239ab654dd97bbed0663577cb691094af259d6af Type: SHA-256