EVALUATION OF THE CONDENSED CHROMATIN INDEX IN MITOGEN-STIMULATED LYMPHOCYTES PUVA TREATED PATIENTS

Öz

UV-A ve psoralenlerle birlikte kullanılan fotokemoterapi (PUVA) vitiligo ve psoriasis gibi bazı deri hastalıklarında yaygın olarak kullanılan bir tedavidir. PUVA tedavisi aynı zamanda çeşitli hücre tiplerinde apoptosis ve kromatin yoğunlaşmasına neden olmaktadır. Bu çalışmanın amacı PUVA ile tedavi edilen hastaların mitojenle uyarılmış lenfositlerinde yoğunlaşmış kromatin indeksinin (YKi) , kardeş kromatid değişim (KKD) ve mikronukleus (MN) metodları gibi DNA hasarlarının belirlenmesinde bir test olarak k u l l a n ı l ı p k u l l a n ı l m a y a c a ğ ı n ı t e s p i t etmektir.Çekirdekteki kromatin yoğunluğunu ölçmek için özel bir bilgisayar programı yapıldı. Çalışmaya PUVA tedavisi alan 20 hastanın mitojenle uyarılmış lenfositleri ile başlandı. Her hastanın sıfırıncı saatte (n=20) ve tedavinin 20’ci (n=16), 40’ıncı (n=12) ve 60’ıncı (n=10) seans sonrası, mitojenle uyarılmış 100 interfaz çekirdeği YKi ortalaması değerlendirildi. Bu seanslar sırasıyla 57.81 ± 23.85, 181.43 ± 46.42, 299.56 ± 61.03 jul/cm2 UVA and 0.6x20, 0.6x40, 0.6x60 mg/kg methoxypsoralen (8-MOP)’e karşılık gelmektedir. Tedavi öncesi (sıfirıncı saat) ile 20. ve 40. seans sonrası elde edilen YK değerleri arasında bir fark bulunamamıştır ( p> 0.05 ). Bununla birlikte , tedavi öncesi ile 60. seans sonrası arasında istatistiksel olarak önemli bir fark bulunmuştur ( p<0.05 ). YKi ‘de duyarlılık, yaklaşık 300 jul/cm2 UVA ve 0.6x20, 0.6x40, 0.6x60 mg/kg 8-MOP’a karşılık gelen PUVA tedavisinin ortalarından sonra başlamaktadır

Anahtar Kelimeler

• PUVA, Yoğunlaşmış kromatin, bilgisayarla analiz

PUVA Tedavisi Gören Hastaların Mitojenle Uyarılmış Lenfositlerindeki Yoğunlaşmış Kromatin İndeksinin Değerlendirilmesi

Öz

Photochemotherapy with psoralens and UV-A (PUVA) is a treatment used widely in some skin disease like as vitiligo and psoriasis. PUVA treatment induces also chromatin condensation and apoptosis in several cell types. The aim of this study is to assay if the condensed chromatin index (CCi) in stimulated lymphocytes of PUVA treated patients may be used as a test method for the detection of DNA damage like the micronucleus (MN) and the sister chromatid exchange (SCE) methods. A special image analysis procedure has been designed to evaluate CCi in the nucleus. The work was initiated with the Mitogen- stimulated lymphocytes of 20 PUVA treated patients. The mean CCi (CCmi) have been evaluated at zero time (n=20) and after 20 (n=16), 40 (n=12), 60 (n=10) sessions of PUVA treatment in 100 mitogen- stimulated interphase nuclei for each patient. These sessions correspond reciprocally to 57.67 ± 23.74, 181.43 ± 46.42 and 299.56 ± 61.03 joules/cm2 of UVA and 0.6x20, 0.6x40, 0.6x60 mg/kg of methoxypsoralen (8-MOP). There was no difference between CCmi obtained before treatment (zero time) and after 20, 40 sessions of PUVA treatment (p> 0.05). However, statistically significant difference was found between untreated and 60 sessions treated individuals (p<0.05). CCi sensitivity begins after the moderate term of PUVA treatment corresponding to nearly 300 Joules/cm2 of UVA and 0.6x60 mg/kg of 8MOP

Anahtar Kelimeler

• PUVA, Condensed chromatin, image analysis

Kaynakça

1. Knobler RM. Hönigsmann H, Edelson RL. Psoralen phototherapies. In: Gasparro FP (ed), Psoralen DNA Photobiology , Vol. Il, CRC Press, Boca Raton, FL, 1988, pp 117- 133. 2. Hashimoto
2. K, Kumakiri M. Colloid-amyloid bodies in PUVA-treated human psoriatic patients. J Invest Dermatol 1979, 72:70-80.
3. Enomoto DN. Schellekens PT, Yong SL, Ten Berge IJ, Mekkes JR, Bos JD. Extracorporeal photochemotherapy (photopheresis) induces apoptosis in lymphocytes: a possible mechanism of action of PUVA therapy. Photochem Photobiol 1997, 65:177-180.
4. Coven TR, Walters IB, Cardinale I, Krueger JG. PUVA-induced lymphocyte apoptosis: mechanism of action in psoriasis. Photodermatol, Photoimmunol, Photomed 1999, 15:22-27.
5. Laporte M, Galand P, Fokan D, de Graef C, Heenen M. Apoptosis in established and healing psoriasis. Dermatology 2000, 200 :314-316.
6. Ibuki Y, Goto R. Phototoxicity of benzo [a] pyrene by ultraviolet A irradiation: induction of apoptosis in Jurkat cells. Envıron Toxıcol Phar 2002 ,11: 101-109.
7. Hamurcu Z, Demirtas H, Aşçıoğlu Ö, Dönmez-Altuntaş H, Aktaş E. Micronucleus evaluation in mitogen-stimulated lymphocytes of PUVA treated patients. Tohoku J Exp Med 2002, 198:11-21.
8. Jirsova K Mondys V. Induction of micronuclei and granular chromatin condensation in human skin fibroblasts influenced by cisplatin (cis-DDP) in vitro. Mutat Res 1994, 310: 37- 44.

9. Demirtas H, Cücer N, Boztosun N, Öztürk M. Condensed chromatin enhancement in the phytohemagglutinin-stimulated lymphocyte interphase nuclei of two xeroderma pigmentosum patients and artificial chromatin condensation of healthy cell nuclei by UV irradiation (Letter). Clin Genet 1997, 51:137- 138.
10. Demirtas H, Imamoglu N, Dönmez H, Cücer N, Yılmaz A, Candemir Z Condensed chromatin surface and NORs surface enhancement in mitogen-stimulated lymphocytes of Down syndrome patients. Ann Génét 2001, 44:77-82.
11. Pathak MA, Nghiem P, Fitzpatric TB. Acute and chronic effects of the sun. In : Fitzpatric TB (ed) Dermatology in general medicine, McGraw-Hill, New York 1999, pp 1598-1607.
12. Demirtas H, Candemir Z, Cücer N, Imamoglu N, Dönmez H, Bökesoy I.Essay on the nucleoli survey by the a- and b-satellite DNA probes of the acrocentric chromosomes in mutagen- stimulated human lymphocytes. Ann Génét 2000, 43: 61-68.
13. Zarebska Z, Waszkowska E, Caffieri S, Dall Acqua F. PUVA (psoralen + UVA) photochemotherapy: processes triggered in the cells. Il Farmaco 2000, 55:515-520.
14. Dobrucki J and Darzynkiewicz Z. Chromatin condensation and sensitivity of DNA in stu to denaturation during cell cycle and apoptosis- confocal microscopy study. Micron 2001, 32:645-652.
15. Heng AE, Sauvezie B, Genestier L, Demeoeq F, Dosgilbert A, Deteix P. PUVA aopototic response in activated and resting human lymphocytes. Transfus Apher Sci 2003,28:43- 50.
16. Kleinau O, Böhm F, Lanto B. Different DNA repair time courses in human lymphoid cells after UVA, UVA1, UVB and PUVA in vitro. J Photochem Photobiol B 1997, 41: 103-108.
17. Fenech M. The advantages and disadvantages of the cytokinesis-block micronucleus method. Mutat Res, 1997, 392: 11-18.