LAPORAN PENELITIAN HIBAH PENELITIAN STRATEGIS NASIONAL TAHUN ANGGARAN 2009
Potensi Formalin Dalam Meningkatkan Kerentanan Mutasi Gen Parp-1 dan Suseptibilitas Terhadap Bahan Karsinogenik
Peneliti Utama Dr. Dra. Sri Widyarti, M.Si. Anggota Wibi Riawan, SSi.
Dibiayai oleh Direktorat Jenderal Pendidikan Tinggi, Departemen Pendidikan Nasional, Sesuai Dengan Surat Perjanjian Pelaksanaan Hibah Penelitian Strategis Nasional Nomor 0174.0/023-04.2/XV/2009 tanggal 31 Desember 2008, dan berdasarkan SK Rektor nomor: 160/SK/2009, tanggal 7 Mei 2009
UNIVERSITAS BRAWIJAYA NOVEMBER 2009
RINGKASAN
SRI WIDYARTI. Jurusan Biologi Fakultas MIPA Universitas Brawijaya, Malang. Potensi Formalin Dalam Meningkatkan Kerentanan Mutasi Gen Parp-1 dan Suseptibilitas Terhadap Bahan Karsinogenik. Meskipun dapat menimbulkan berbagai gangguan kesehatan, formalin telah disalahgunakan sebagai pengawet makanan. Sejauh ini belum ada keluhan dari masyarakat tentang dampak makanan berformalin. Sebagai bahan genotoksik, formalin berpotensi menyebabkan kerusakan DNA sehingga berdampak pada peningkatan resiko kanker. Namun sejauh ini belum diketahui apakah paparan formalin dapat berdampak langsung pada DNA, sehingga menigkatkan kerentanan terhadap resiko kanker. Gen Parp-1 sebagai penyandi protein PARP yang berperan dalam mekanisme perbaikan kerusakan DNA, mempunyai fungsi luas antara lain sebagai faktor trakskripsi untuk enzim MnSOD. Untuk mendapatkan penjelasan ilmiah tentang dampak formalin pada level transkriptom, maka penelitian ini disusun untuk membuktikan ada tidaknya perubahan level mRNA PARP-1 dan mRNA MnSOD keterkaitannya dengan kerentanan hewan coba Mus musculus terhadap bahan karsinogenik benzapiren. Mencit dibagi menjadi enam kelompok, yaitu: (1) kelompok kontrol tanpa paparan formalin dan benzapiren (F0B0); (2) kelompok formalin 5 mg/kg BB (F5B0); (3) kelompok formalin 25 mg/kg BB (F25B0); (4) kelompok benzapiren (F0B); (5) kelompok formalin 5 mg/kg BB + benzapiren (F5B); (6) kelompok formalin 25 mg/kg BB + benzapiren (F25B). Sediaan formalin dalam bentuk cairan dimasukkan langsung ke lambung mencit lewat kerongkongan menggunakan sonde. Pemberian paparan dilakukan setiap hari selama 60 hari (paparan sub kronis). Sediaan benzapiren 200 mg/kg BB dilarutkan dalam minyak jagung diberikan secara injeksi intraperitoneal pada hari ke 30 paparan formalin. Setelah perlakuan, mencit dibunuh kemudian dibedah secara aseptis, dan diambil organ hepar dan paru untuk dilakukan isolasi RNA, isolasi protein dan preparasi histoteknik untuk analisis imunohistokimia. Isolasi RNA total dilakukan menggunakan NucleoSpin RNA-II Kit. Kadar RNA diukur menggunakan spektrofotometer uv-vis. Probe untuk analisis level mRNA MnSOD berupa cDNA yang dibuat dari hasil RT-PCR menggunakan primer forward MnSOD 5’GACCTGCCTTACGACTATGG-3’ dan primer reverse MnSOD 5’GAGCTTGCTCCTTATTGAAGG-3’. Probe cDNA selanjutnya dilabel menggunakan Biotin-Nick Translation Mix. Probe untuk analisis level mRNA PARP-1 berupa antisense mRNA PARP-1 exon 21 terlabel biotin 5’CTTGGGTAACTTGCTGATATGTGAAGC-3’ (1st Base). Pengujian level mRNA pada sampel menggunakan teknik hibridisasi ex situ menggunakan dot blot pada membran Nylon. Level mRNA dihitung berdasarkan densitas warna dot yang dianalisis menggunakan software Quantity One 4.8.6 (Biorad). Isolasi protein total dilakukan dalam buffer lisis (50 mM Tris pH 7.4, 5 mM EDTA pH 8, 1% Triton X-100, 0,1 mM PMSF). Protein yang diperoleh diukur kadarnya menggunakan metode UV Absorption pada λ 280 nm. Analisis ekspresi protein PARP-1 dilakukan dengan metode Western
bloting menggunakan mouse monoklonal anti-PARP sebagai antibodi primer, dan goat anti-mouse IgG phosphatase-labeled sebagai antibodi sekunder. Analisis proliferasi dilakukan dengan metode imunohistokimia menggunakan mouse monoclonal anti-PCNA sebagai antibodi primer, dan goat anti-mouse IgG biotin-labeled sebagai antibodi sekunder. Analisis apoptosis dilakukan dengan metode imunohistokimia menggunakan APO-BrdU-IHC In Situ DNA Fragmentation Assay Kit. Histopatologi dilakukan menggunakan pewarnaan HE. Hasil dari penelitian dapat disimpulkan sebagai berikut. Level mRNA MnSOD hepar pada perlakuan formalin 2 mg/kg BB lebih tinggi dari pada perlakuan benzapiren. Hal ini mengindikasikan bahwa stres oksidatif yang disebabkan oleh formalin lebih tinggi dibandingkan dengan stres oksidatif yang disebabkan oleh benzapiren. Pada hepar, peningkatan level mRNA MnSOD semakin meningkat secara signifikan pada perlakuan formalin 2 mg/kg BB tanpa atau dengan benzapiren. Perlakuan yang serupa pada paru justru menyebabkan menurunnya level mRNA MnSOD. Hal tersebut mengindikasikan bahwa stres oksidatif yang lebih tinggi, justru menyebabkan hambatan atau penekanan transkripsi mRNA MnSOD pada paru, namun tidak terjadi pada hepar. Paru lebih rentan terhadap stres oksidatif dibandingkan dengan hepar. Kata Kunci: benzapiren, formalin, level mRNA, MnSOD, PARP-1
SUMMARY
SRI WIDYARTI. Biology Department, Faculty of Sciences, Brawijaya University, Malang. The Potency of Formalin on Increasing Suceptibilty to Parp-1 mutation and Carcinogenic Agent Benzapyrene. Formaldehyde (FA), which leads to irritation of mucous membranes, has also been reported to have toxic effects on the respiratory tract (the lungs, nose), gastrointestinal system, skin, eye, testicles and menstrual functions. FA has also a strong tendency to combine with protein, DNA, RNA which leads to allergic reactions, cytotoxicity, genotoxicity, mutagenic and cancerogenic actions. Benzo(a)pyrene is widespread environmental carcinogenesis derived from incomplete combustion of organic materials such as gasoline, diesel fuel, coal and oil. FA or benzo(a)pyrene are also stress oksidatif agent which can induce increasing antioxidant enzymatic such as MnSOD. The objective of this research is to observe the level of mRNA MnSOD and mRNA PARP-1 after exposure of FA with or without benzo(a)pyrene. Two-month olds of male mice (Mus musculus) divided into 5 groups: (1) control: (2) formalin 5 mg/kg BW; (3) formalin 25 mg/kg BW; (4) kelompok benzapiren (5) formalin 5 mg/kg BW + benzapiren; (6) formalin 25 mg/kg BW + benzapiren. Total RNA was isolated from organ with NucleoSpin RNA-II Kit. Probe cDNA MnSOD was synthezed using RT-PCR with primer forward MnSOD 5’-GACCTGCCTTACGACTATGG-3’ dan primer reverse MnSOD 5’-GAGCTTGCTCCTTATTGAAGG-3’. Labeling cDNA with
Biotin-Nick Translation Mix. Probe for mRNA PARP-1 is antisense mRNA PARP-1 exon 21 5’- CTTGGGTAACTTGCTGATATGTGAAGC-3’ labeled biotin. Analysis of mRNA level using ex situ hybridisation with dot blot on Nylon membrane. Level of mRNA was analyzed using software Quantity One 4.8.6. The result shown that level of mRNA MnSOD hepar on treatment formalin 2 mg/kg BW more higher than benzo(a)pyrene treatment. It conclude that oxidative stress because of formalin more higher than benzo(a)pyrene. On hepar, increasing of mRNA MnSOD level more increase significantly on treatment of formalin 2 mg/kg BB with or without benzo(a)pyrene. This same treatment cause depleted on lung level mRNA MnSOD. Keywords: benzo(a)pyrene, formalin, level mRNA, MnSOD, PARP-1
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