Numerical data for systemic effects of photoactivated 5,10,15,20-tetrakis(N-methylpyridinium-3-yl) porphyrin on healthy Drosophila melanogaster

Porphyrins are commonly used as photosensitizing agents (PA) in photodynamic therapy (PDT), a non-invasive method predominantly used for the treatment of subcutaneous tumors. Development of novel PA with increased tissue selectivity and effectiveness is important for the wider use of PDT in the treatment of various types of cancers.  We used Drosophila melanogaster as a model organism to study the systemic effects of 5,10,15,20-tetrakis(N-methylpyridinium-3-yl)-porphyrin (TMPyP3). First, we defined the optimal feeding protocol and showed that TMPyP3 was absorbed and retained longer in the neuronal than the non-neuronal extracts. Hydrogen peroxide (H2O2) concentration increased 24 hours after the photoactivation of orally delivered TMPyP3 but only in the head extracts. After 7 days, regardless of the photoactivation, TMPyP3 led to lower concentration of H2O2, which correlated with the decreased climbing ability measured as negative geotaxis. The results suggest that systemic treatment with TMPyP3 may interfere with redox regulation and thus impair cellular signaling and behavioral output. Further studies are needed to establish the disruptive effect that porphyrins have on redox homeostasis, how long it persists, and mechanistic differences of retention between different tissues.

Methods
Stability of TMPyP3 in nutrient media - figure 3.A

We sampled 25 mg of M1, M2 and M3, and dissolved them in distilled water in duplicates to a final volume of 1 mL. Samples were heated for 30 minutes at 77 °C and centrifuged for 10 minutes at 15 000 rpm. 200 µL of each media supernatant in triplicate (with and without TMPyP3) was added to a 96-well plate, and fluorescence emission spectra of TMPyP3 was recorded using excitation wavelength of 420 nm and emission in 550-800 nm range.

TMPyP3 concentration in the tissue after the oral administration - figure 3.B, 4A,B and 5A,B

To determine the amount of TMPyP3 that was present in the whole body, we prepared a whole-body homogenate immediately after feeding. Using lysis buffer consisting of 1 x phosphate-buffered saline (1 x PBS) at pH 7.4 in the presence of 0.1% (v/v) Triton X-100 (Sigma Aldrich). The volume of extraction buffer was determined as the proportion of 300 µL of buffer and 5 mg tissue sample. NaCl, KCl, Na2HPO4, and KH2PO4, were of analytical grade. After mechanical homogenization, samples were incubated in an ice bath for 20 min and centrifuged at 14.000 rpm for 30 minutes at 4 °C. The concentration of TMPyP3 in the tissue extracts was determined using a calibration curve for TMPyP3 in PBS. The fluorescence emission of the samples was obtained using a Tecan Infinity m1000Pro microplate reader, with excitation wavelength of 420 nm, and an emission of 710 nm. To determine the absorbed concentration of TMPyP3 after one and seven days, in the head or body extract, we applied the same approach of extraction and measurement of TMPyP3. In this test, the flies were starved for 16 hours and fed 40 µM TMPyP3 for 180 minutes on M3 in the dark (Figure 2.). After feeding, the flies were either exposed to red-light emitting diodes for 10 minutes and then transferred to the fresh M3 media, or directly transferred to fresh M3 and left on it for one, or seven days in constant darkness before biochemical and behavioral measurements.

Quantification of the hydrogen peroxide (H2O2) - figure 4.C,D and 5.C,D

For body homogenates five headless bodies were used, and for head homogenates 32 heads. Samples were prepared using the same protocol as for the quantification of TMPyP3. H2O2 concentrations in the body and head homogenates were determined using the calibration curve for dihydroethidium (DHE, ≥95%, Sigma Aldrich) with known H2O2 concentration [21]. To eliminate DHE oxidation induced by the environment, each of the measured sample relative fluorescence units (RFU) were corrected for dilution, and RFU of DHE was subtracted from the RFU of the sample. The reaction mixture contained 1 x PBS (pH 7.4), 10 μM DHE and 5 μL of homogenate with a final volume of 200 µL. The microplate with samples was incubated for 30 minutes at 37°C in the dark. The amount of H2O2 in the homogenates was measured using the microplate reader Tecan Infinite Pro200 with an excitation wavelength of 480 nm and emission at 625 nm.

 Negative geotaxis - figure 6

We measured negative geotaxis as the percent of flies that climbed over the half point of the vertical wall of the vial, at the height of 10 cm, five seconds after being knocked to the bottom of the vial. The flies were first transferred to the test vials and left for 30 minutes to adapt to the new environment. Testing involved tapping vials three times on a hard surface and taking photos after five seconds. From the image analysis, we calculated the percentage of flies that crossed the midline of 10 cm vial in five second intervals. Measurements were repeated five times, with the interval of one minute between measurements. We tested flies that were exposed either for 180 minutes or 24 hours to 40 μg of TMPyP3 in M3 medium and illuminated with 40 mW/cm2 for 10 or 20 minutes, then aged seven days on M3 in constant darkness.