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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