SEM–EDS probing of morphological and physiological changes produced by a porphyrin photosensitizer in Psammobatis extenta electrocytes

Abstract

Morphological and physiological changes produced by the intracellular localization of an alkyl long-chain tetraphenylporphyrin photosensitizer in Psammobatis extenta electrocytes were studied by means of SEM–EDS. Immediately after photosensitizer penetration, electrocytes swell and their convex faces lose all invaginations. This effect is due to chloride and sodium ion flux into electrocytes, in accordance with energy dispersive X-ray microanalysis.

Introduction

Photosensitizers have a wide spectrum of applications ranging from material sciences to medical and biological sciences. They are used in photocatalysis to improve semiconductor efficiency. For example, ruthenium bipiridyl complexes or phthalocyanine derivatives are used to improve solar energy TiO₂ absorption range (Nogueira et al., 2004). In medicine, their main application is in the photodynamic therapy of cancer (PDT) as well as in several non-malignant conditions such as age related macular degeneration (van den Berg, 2001). PDT takes advantage of the some photosensitizers high affinity (mainly porphyrin and phthalocyanine derivatives) for cancer cells and the absorption of visible light to generate singlet molecular oxygen (Fournier et al., 2004). This reactive oxygen species can chemically modify important biomolecules, including lipids, proteins, and nucleic acids. These damaged biomolecules cannot then accomplish their function and, consequently, cells die (Davies, 2004, Oleinick et al., 2002). In biology, photosensitizers are used to inactivate bacteria and protozoa (Reddi et al., 2002, Malik et al., 1993). A practical application of this knowledge may be water disinfection for human consumption or biofilm formation inhibition.

To understand cell death in PDT or bacteria inactivation, it is important to know where the photosensitizer is attached in the cell. This is usually done by observing tissues under an optical microscope or scanning electron microscope (SEM) to detect morphological changes induced by photosensitizers in cells due to the chemical modification of biomolecules.

Scanning electron microscopy (SEM) coupled with an energy dispersive X-ray analyzer (EDS) were used to study the morphology (shape and size) and elemental composition of material surfaces. Lately, these techniques have been increasingly used in areas other than material sciences. In 1980, Pearl and Brody reported the presence of aluminum in the neurons of patients with Alzheimer's disease by using EDS/SEM. In biology these techniques are applied to monitor human vascular cell calcification (Proudfoot et al., 1998), and the in vivo tissue response to implants (Liao et al., 2000). In the latter case, EDS has helped to demonstrate the formation of a phosphorous-rich zone in the interface between the bone and the implant. It has also been used to measure the elemental concentration of monovalent cations (Na⁺, Cl⁻, and K⁺) during the degradation phase of apoptosis of the U937 human monoblastoid cell line (Zabiti et al., 2001).

In this work we used SEM–EDS to obtain additional information related to physiological and morphological changes observed by the intracellular localization of 5,10,15,20-tetrakis(4-n-dodecylphenyl)porphyrin (C₁₂TPPH₂) in Psammobatis extenta electrocytes. This species belongs to the Rajidae, one of the three groups of weakly electric fish; others are the Gymnotiformes and the Mormyriforms. Electric organs of the Rajidae electric fish derive from myoblasts of the caudal region (Fessard, 1958, Laget, 1972). Electric organs are made-up of macroscopic cells called electrocytes. Electrocytes are found in an alveolar arrangement on the antero-posterior axis (Bennett, 1987). Synaptic terminal degeneration has also been observed in the adult skate electric organs (Fox et al., 1990, and our observations). The few organelles present in electrocytes can be easily defined by light microscopy (Prado Figueroa and Santiago, 2004). Electrocytes are highly polarized multinuclear cells. P. extenta electrocytes represent an interesting area of research because they are modified primitive cup-shaped cells (Jacob et al., 1994, Prado Figueroa et al., 1995) with myoproteins (Vidal et al., 1997).