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One of the major diseases observed in recent decades in sea turtles is fibropapillomatosis (Baptistotte et al. 2004; Brito et al. 2004; Herbst et al. 1998). Initially described during the 1930s in Florida, fibropapillomatosis is characterized by the presence of one or multiple external cutaneous tumors of varying size, which may be found on various parts of the body; occasionally there are visceral fibromas (Herbst 1994). Fibroepithelial tumors are commonly found on the turtle's eyes, including the conjunctivae and skin obstruct vision, and may interfere with feeding and locomotion, while visceral nodules may fatally disrupt normal organ function (Herbst 1994, Herbst et al. 1998). In advanced disease stages, affected animals may become weak, anemic, and in some cases even blind if the cornea is significantly involved.

The prevalence of this disease is associated with highly populated coastal areas polluted by large amounts of agricultural, domestic and industrial wastes or marine biotoxins (Adnyana et al. 1997; Aguirre & Lutz 2004; Foley et al. 2005; Herbst 1994). Some studies also show a correlation with tumor-associated viruses, and there is no effective treatment for this disease other than surgical removal (Ene et al. 2005; Herbst et al. 1998; Matushima et al. 2001; Quackenbush et al. 1998).

The histological characteristics of fibropapillomatosis include stromal and epidermal hyperplasic proliferation. Epithelial cells may have large nuclear pleomorphism and it is possible to observe cytoplasmic vacuolization, skin cell degeneration and fibroblast proliferation. The presence of eosinophilic intranuclear inclusions in epidermal cells may be observed microscopically (Matushima et al. 2001; Schumacher 1996). Dense connective tissue tumors in internal organs associated with proliferation and purulent inflammatory process was seen in a single fibropapilloma-afflicted turtle in Brazil (Brito et al. 2004). Clinical diagnosis may be based on observations of obvious external tumors, which may be classified as circumscribed, infiltrating or disseminated (Knobl et al. 2011). Although tumors are benign, their physical presence may cause difficulty in swimming and consequently locomotion, making food capture and breathing inefficient and leading to increased risk of predation (Baptistotte et al. 2001; Herbst 1994).

Photodynamic therapy (PDT) has been studied and applied to cancer and infectious disease treatments in multiple areas of human medicine (Dai et al. 2009; Dolmans et al. 2003). Currently, its use is considered one of the most investigated treatments for the control, prevention and cure of non-melanoma skin cancers (Braathen 2007; Brown 2004). PDT has also been used in humans to treat skin lesions caused by many viral infections. Examples can include lesions caused by human papilloma virus (HPV), cutaneous warts known as Verrucae vulgaris or Verrucae plana (also caused by HPV), Molluscum contagiosum (MC), which is a DNA poxvirus and Herpes simplex (Dai et al. 2009).

In the PDT procedure, the human or animal patient generally receives a systemic or local administration of a non-toxic photosensitizer (PS), which is selectively retained in tumor tissue via a few possible mechanisms. The selective drug uptake may take 10 minutes to over 24 hours according to the PS chemical structure and the manner of drug delivery. During the drug uptake time, also referred as pre-irradiation time (PIT), the photosensitized area must be kept out of direct illumination. If the PS is administered systemically, the patient should be kept in a low-luminosity room until sufficient PS elimination. Locally administered PS may require only a light-absorbing bandage to cover the photosensitive area and prevent damage to healthy tissue. After PIT, the photosensitized target-tissue is irradiated by visible or near-infrared low-intensity light at a specific wavelength and time to “activate” the PS molecules. The light absorption by the photosensitizing agent in the presence of molecular oxygen results in photochemical processes that form a variety of reactive oxygen species (ROS) and cause severe cellular damage leading to necrosis and/or apoptosis (Ribeiro 2005). Apart from cell injury by ROS, PDT may induce a local inflammatory process, reducing the ability of the tumor to evade immune system recognition and consequently enhancing the antitumor immune response (Davids 2008).

The first reports of PDT in animals were described in the early 1980s when hematoporphyrin derivative PSs were administered intravenously in dogs and cats prior to laser irradiation. Although these studies included a wide variety of tumor types, most tumors were considered responsive, demonstrating the clinical potential of PDT in the treatment of solid tumors (Lucroy 2002).

Methylene blue is a low-cost drug approved by the FDA and it has been widely used in medical practice and biomedical research involving PDT. It is a phenothiazinum salt characterized by a tricyclic heteroaromatic structure, with irrelevant toxicity in low concentrations (Lim et al. 2013). Its selectivity to cellular organelles such as mitochondria, lysosomes and nuclei is mainly linked to its positive charge and lipophilic nature that facilitates the crossing of cell membranes (Gabrielli et al. 2004; Tardivo et al. 2005). The photodynamic reactions, and consequently the local ROS formation, may be caused by their electronic excitation that is induced by red light (600-670 nm) absorption provided by any light source set at the proper irradiation parameters (e.g., laser, LED, filtered broad spectrum light, etc.). In addition, the associated electromagnetic radiation at wavelengths between 600 and 1300 nm (red and near-infrared light) exhibits privileged penetration into biological tissues, allowing effective photodynamic treatments at greater depth (Sternberg et al. 1998; Sternberg & Dolphin 1996). Although MB employment in oncology is relatively recent, investigations have shown positive in vivo activity against a wide range of tumor types including bladder cancer, inoperable esophageal tumors, adenocarcinomas and other non-melanoma skin cancers (Perussi 2007).

Table 1. Animal ID and anatomical site, size, pigmentation and outcomes of treated tumors. Lesions treated by PDT received 0.5 mL of MB at 300 μM and were irradiated by a low-intensity red diode laser emitting 100 mW at 660 nm. Each square centimeter of the lesion received 16 J of light energy, for 160 seconds. Control groups received either only light irradiation (light + MB -) or MB inoculation (light - MB +). Lesions were classified as responsive when macroscopic aspects indicating severe tumor necrosis were observed up to 30 days after the first treatment session.

In this study, we attempted to treat well-developed fibropapilloma tumors employing PDT mediated by methylene blue as the PS in association with a red laser low-intensity light. We treated five green turtles (Chelonia mydas) that had multiple skin tumor lesions compatible with cutaneous fibropapillomatosis (Fig. 1A) in the Municipal Aquarium of Santos/SP. The lesions presented different characteristics regarding size, location, and pigmentation (Table 1). Fibropapillomatosis was diagnosed in each turtle through a clinical examination. Turtles were isolated in individual tanks for better evaluation.

Lesions were treated by two PDT applications within a 15-day interval and consisted of two injections of 0.5 ml intralesional methylene blue (MB - concentration of 300 μM - Sigma Aldrich) at the base of all tumors, followed by 5 min of pre-irradiation time in the dark to allow for cellular uptake, and the administration of a continuous wave red diode laser irradiation operating at 100 mW of optical power, wavelength (λ) of 660 nm, for 160 seconds per point, resulting in 16 J of energy per point and an energy density of 560 J/cm² per point (Fig. 1B). These irradiation parameters do not generate any relevant temperature increase and all biological effects are expected to be due to products formed by MB-mediated photochemical reactions. Each square centimeter of the lesion surface was illuminated according to the parameters described above. Consequently, the number of irradiation points per lesion varied according to the lesion size. To evaluate the response to isolated MB or light interventions, two control lesions were randomly treated exclusively by intralesional MB inoculation and other two separate lesions received low-intensity laser irradiation at the same previously described parameters but free of MB inoculation (Table 1). All treated lesions were evaluated weekly by simple descriptive analysis for a time period of 30 days (Table 1).

Figure 1. Aspects of one PDT-treated tumor before any intervention (A) and at the first irradiation procedure (B), after MB inoculation. One week after the first PDT session (C), tumor is highly swollen due to intense inflammatory process. Two (D) and (E) three weeks after first PDT session, macroscopic aspects indicating severe tumor necrosis can be observed. At the fourth week (F) the tumor has spontaneously detached from the turtle’s tissue.

At the seventh day post-treatment, we observed that all PDT-treated lesions were dark blue in color and swollen with a firm consistency (Fig. 1C). Lesions that were only treated with a laser did not show any noticeable macroscopic alterations. The lesions where MB was administered alone also presented with a dark blue color - similar to those treated with PDT. However, no further macroscopic alterations were observed.

At the fourteenth day, PDT-treated lesions began to take on a soft consistency, having a light blue color and with classical macroscopic characteristics of tissue necrosis (Fig. 1D). Lesions treated with laser or MB alone did not show any changes in appearance.

Twenty-one days after the first PDT session, lesions showed a partial loss of adhesion to the skin, were light brown in color, had a soft consistency and obvious tissue necrosis characteristics (Fig. 1E). On the thirtieth day, the treated lesions were totally or partially detached from the epidermis (Fig. 1F), and were easily removed using tweezers. After two PDT treatments all lesions showed local macroscopic changes consistent with cellular death. The regression time for all PDT-treated lesions was approximately the same, regardless of the tumor initial size, pigmentation, or location.

Some turtles with multiple lesions had one of the tumors randomly selected to be exclusively treated by MB or low-intensity red laser alone. Tumors not treated by the whole PDT scheme (i.e., MB associated to irradiation) did not show any noticeable macroscopic changes, such as signs of inflammation or regression, during the experimental time and were classified as non-responsive. This observation strongly suggests that relevant cellular and tissue damage may only be achieved when PS was excited by light at a specific wavelength, time and intensity (Moor 2000). In addition, it indicates that PDT does not trigger relevant systemic antitumor immunity. This conclusion agrees with the literature concerning the selectivity of PS to tumor cells and its phototoxic action being observed only in the irradiated site (Machado 2000; Luksiene 2003).

Our results indicate that MB-mediated photodynamic therapy is a potential new treatment for sea turtle fibropapillomatosis tumors. It is a low-cost ambulatory procedure that can be contemplated as a minimally invasive alternative treatment when the surgical procedure is not available. The evaluation of all mechanisms involved in the tumor regression provided by this treatment, such as the aspects of the histopathology, cellular death, molecular signaling and gene expression, and the possibility of employing PDT as postsurgical treatment to avoid recurrence deserves further investigation.

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