Abstract
Protoporphyrin IX (PpIX) is a precursor of heme synthesis and is known to be an active photosensitizer and precursor of photosensitizers applied in photodynamic therapy (PDT) and photodynamic diagnostics (PDD). On irradiation with visible light, PpIX undergoes phototransformation, producing photoproducts which may also be phototoxic and increase its efficacy. The mechanism of PpIX phototransformation depends on environmental characteristics and can be different in vitro and in vivo. In this paper, we present a comparative study of the photoactivity of synthetic PpIX and PpIX extracted from the Harderian gland of ssp Rattus novergicus albinus rats, along with their photoproducts toward murine B16F-10 melanoma cells. It was observed that when irradiated with visible light the endogenous PpIX demonstrates photocytotoxicity ten times higher than the synthetic PpIX. The photoproduct of endogenous PpIX also possesses phototoxicity, though slightly lower than that of PpIX itself. The rate of cell internalization for both endogenous PpIX and its photoproduct was eightfold greater than that obtained for the synthetic porphyrin. This difference might result from a complexation of the native PpIX with some amphiphilic compounds during its synthesis within the Harderian glands, which facilitates the cell uptake of PpIX. Fluorescence microscopy images show that both endogenous and synthetic porphyrins are localized after uptake predominantly in the mitochondrial region of cells.
Similar content being viewed by others
References
Ben-Hur E, Horowits B (1995) Advances in photochemical approaches for blood sterilization. Photochem Photobiol 62(3):383–388
Bonnett R (1995) Photosensitizers of the porphyrin and phthalocyanine series for photodynamic therapy. Chem Soc Rev Camb 24(1):19–33
Bonnett R, Martinez G (2001) Photobleaching of sensitisers used in photodynamic therapy. Tetrahedron 54:9513–9547
Brancaleon L, Moseley H (2002) Effects of photoproducts on the binding properties of protoporphyrin IX to proteins. Biophys Chem 96:77–87
Brown SB, Brown EA, Walker I (2004) The present and future role of photodynamic therapy in cancer treatment. Lancet Oncol 5:497–508
Chieffi G, Chieffi BG, Di Matteo L, D’Istria M, Minucci S, Varriale B (1997) Cell biology of the harderian gland. Int Rev Cytol 168:1–80
Clennan EL (2000) New mechanistic and synthetic aspects of singlet oxygen chemistry. Tetrahedron 56:9151–9179
Dickson EFG, Pottier RH (1995) On the role of the protoporphyrin IX photoproducts in photodynamic therapy. J Photochem Photobiol, B 29:91–93
Djeridane Y (1994) The harderian gland and its excretory duct in the WISTAR rat. A histological and ultrastructural study. J Anat 184:553–566
Dysart JS, Patterson MS (2005) Characterization of photofrin photobleaching for singlet oxygen dose estimation during photodynamic therapy of MLL cells in vitro. Phys Med Biol 50:2597–2616
Dysart JS, Patterson M (2006) Photobleaching kinetics, photoproduct formation, and dose estimation during ALA induced PpIX PDT of MLL cells under well oxygenated and hypoxic conditions. Photochem Photobiol Sci 5:73–81
Ericson MB, Grapengiesser S, Gudmundson F, Wennbrg AM, Larkö O, Moan J, Rosén A (2003) A spectroscopic study of the photobleaching of protoporphyrin IX in solution. Lasers Med Sci 18:56–62
Finlay JC, Mitra S, Patterson MS, Foster TH (2004) Photobleaching kinetics of photofrin in vivo and in multicell tumour spheroids indicates two simultaneous bleaching mechanisms. Phys Med Biol 49:4837–4860
Gallegos EE, De Leon Rodrigues I, Martinez GL, Perez ZAJ (1999) In vitro study of protoporphyrin IX induced by delta—aminolevulinic acid in normal and cancerous cells of the human cervix. Arch Med Res 30(3):163–170
Georgakoudi I, Foster TH (1998) Singlet oxygen–versus non singlet oxygen-mediated mechanisms of sensitizer photobleaching and their effects on photodynamic dosimetry. Photochem Photobiol 67(6):612–625
Gibson SL, Nguyen ML, Havens JJ, Barbarin A, Hilf R (1999) Relationship of delta–aminolevulinc acid induced protoporphyrin IX levels to mitochondrial content in neoplastic cells in vitro. Biochem Biophys Res Commun 265(2):315–321
König K, Schneckenburger H, Kück A, Steiner R (1993) In vivo photoproduct formation during PDT with ALA–induced endogenous porphyrins. J Photochem Photobiol B 18:287–290
Lim CK, Razzaque MA, Luo J, Farmer PB (2000) Isolation and characterization of protoporphyrin glycoconjugates from rat harderian gland by HPLC, capillary electrophoresis and HPL/electrospray ionization MS. Biochem J 347:757–761
Menezes PFC (2006). Estudos espectroscópicos e citotóxicos do Photogem® fotodegradado e dos fotoprodutos formados pela irradiação com laser. 2006. 182f. (Tese Doutorado em Ciências: Química Analítica) - Instituto de Química de São Carlos da Universidade de São Paulo. São Carlos
Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63
Nicola JH, Reis ER, Nicola EMD, Metze K (2001) Efeito fotodinâmico em glândula Harderiana de ratos WISTAR. Revista da Sociedade Brasileira de Laser 1(4):21–23
Ochsner M (1997) Photophysical and photobiological processes in the photodynamic therapy of tumors. J Photochem Photobiol B 39:1–18
Oleinick NK, Morris LR, Belichenko I (2002) The role of apoptosis in response to photodynamic therapy: what, where, why, and how. Photochem Photobiol Sci 1:1–21
Reis ER, Nicola EMD, Nicola JH (2005) Harderian gland of WISTAR rats revised as a protoporphyrin IX producer. Brazil J Morphol Sci 22(1):43–51
Reis ER, Metze K, Nicola EMD, Nicola JH, Borissevitch IE (2013) Photodynamic activity of protoporphyrin IX in Harderian glands of WISTAR rats: monitoring by gland fluorescence. J Lumin 137:32–36
Reis ER, Nicola EMD, Metze K and Nicola JH (2000) Photodynamic effect produced by HeNe radiation in Harderian glands of WISTAR rats: an experimental model for PDT studies. Laser-Tissue Interaction XI: Photochemical, Photothermal and Photomechanical, United States of America 1(8): 40–45
Rotomskis R, Vaiacaitis V, Piskarskas A (1993) Time-resolved absorption spectroscopy of hematoporphyrin and its photoproducts. Chem Phys Lett 202(34):233–236
Rotomskis R, Streckyte Gand Bagdonas S (1997) Phototransformations of sensitizers photoproducts formed in aqueous solutions of porphyrins. J Photochem Photobiol B 39:172–175
Sakai T, Yohro T (1981) A histological study of the Harderian gland of Mongolian gerbils, Meriones meridianus. Anat Rec 100:259–270
Sobolev AS, Jans DA, Rosenkranz AA (2000) Targeted intracellular delivery of photosensitizers. Prog Biophys Mol Biol 73:51–90
Spike RC, Payne AP, Moore MR (1992) Porphyrins and their possible significance in Harderian glandsin. In: Weeb SM, Hoffman RA, Puig-Domingo ML, Reiter RJ (eds) Harderian glands porphyrin: metabolism, behavioral and endocrine effects. Springer, Berlin, pp 125–193
Spike RC, Stewart SL, Murray K, Kelly FM, Maharaj JA, Payne PA, Moore MR (1990) Porphyrin synthesis in the Harderian gland and other tissues of golden hamster during pregnancy and lactation. Mol Aspects Med 11:151–152
Tanielian C, Mechin R, Seghrouchni R, Schweitzer C (2000) Mechanistic and kinetic aspects of photosensitization in the presence of oxygen. Photochem Photobiol 71:12–19
Theodossiou T, MacRobert AJ (2002) Comparison of the photodynamic effect of exogenous photoprotoporphyrin and protoporphyrin IX on PAM 212 murine keratinocytes. Photochem Photobiol 76(5):530–537
Weinstein GD, Mc Cullough JL, Nelson JS, Berns MWJ (1991) Low dose photofrin II photodynamic therapy of psoriasis. J Invers Dermatol 96(4):573
Zeng H, Korbelik M, McLean DI, MacAulay C, Lui H (2002) Monitoring photoproduct formation and photobleaching by fluorescence spectroscopy has the potential to improve PDT dosimetry with a verteroporfin-like photosensitizer. Photochem Photobiol 75(4):398–405
Acknowledgements
The authors thank to the Brazilian Agencies CNPq (research Grant no 305303/2013-9) and Medical Sciences College (UNICAMP) for supporting this work.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Reis, E.R., Ferreira, L.P., Nicola, E.M.D. et al. Comparative study of phototoxicity of protoporphyrin IX synthetic and extracted from ssp Rattus novergicus albinus rats toward murine melanoma cells. Eur Biophys J 47, 601–609 (2018). https://doi.org/10.1007/s00249-018-1283-5
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00249-018-1283-5