DOI: 10.5061/dryad.bk3j9kdm0
title: Numerical data for systemic effects of photoactivated 5,10,15,20-tetrakis(N-methylpyridinium-3-yl) porphyrin on healthy Drosophila melanogaster

description: 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.
description: <em>Stability of TMPyP3 in nutrient media - figure
 3.A</em> 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.
 <em>TMPyP3 concentration in the tissue after the
 oral administration - figure 3.B, 4A,B and
 5A,B</em> 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, Na<sub>2</sub>HPO<sub>4</sub>, and
 KH<sub>2</sub>PO<sub>4</sub>, 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.
 <em>Quantification of the hydrogen peroxide
 (</em>H<sub>2</sub>O<sub>2</sub>)
 - 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.
 H<sub>2</sub>O<sub>2</sub> concentrations in the
 body and head homogenates were determined using the calibration curve for
 dihydroethidium (DHE, ≥95%, Sigma Aldrich) with known
 H<sub>2</sub>O<sub>2</sub> 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
 H<sub>2</sub>O<sub>2</sub> in the homogenates was
 measured using the microplate reader Tecan Infinite Pro200 with an
 excitation wavelength of 480 nm and emission at 625 nm.
  <em>Negative geotaxis - figure
 6</em> 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/cm<sup>2</sup> for 10 or 20 minutes, then aged seven
 days on M3 in constant darkness.
description: # Numerical data for systemic effects of photoactivated
 5,10,15,20-tetrakis(N-methylpyridinium-3-yl) porphyrin on healthy
 Drosophila melanogaster #### The first sheet in Excel called PDT is the
 results generated for figure three in the publication (F3). **Data
 generated under part F3.A** refer to measurements of **fluorescence**
 **emission spectra of TMPyP3** was recorded using excitation wavelength of
 420 nm and emission in 550-800 nm range. Medium 1 (**M1**) was a standard
 nutrient medium (73 mL tap water, 4.5 mL molasses, 1.1 g table sugar, 2.6
 g inactivated yeast, 0.9 g type II agar, 0.75 mL propionic acid (Sigma
 Aldrich, 99%) and 0.75 mL of 10 % p-hydroxybenzoic acid methyl ester
 (NIPAGINE, Roth, 99%) dissolved in 95% ethanol (VWR). Medium 2 (**M2**)
 was a combination of agar, sugar, and yeasts (75 mL tap water, 1.5 g type
 II agar, 3 g table sugar and 6.9 g inactivated yeast).Medium 3 (**M3**)
 was agar and sugar without yeasts (75 mL tap water, 1.5 g type II agar, 3
 g table sugar). 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. **Data generated under part F3.B**
 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.
 **After 16 hours of starvation**, flies were transferred to **M3** with
 the addition of **40 µM TMPyP3 for 30, 90 or 180 minutes** and kept in the
 incubator in the dark. The **non-starved** group of flies was transferred
 to M3 supplemented with **40 µM TMPyP3 for 24 hours** and kept under the
 same conditions as the starved group. To determine the amount of TMPyP3
 that was present in the whole body, we prepared a whole-body homogenate
 immediately after feeding. We used whole body for this measurements and
 prepare two samples measured in triplicates. 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 second sheet
 in Excel called PDT is the results generated for figure four in the
 publication (F4). **Data generated under part F4.A,B** 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. **After 16
 hours of starvation**, flies were transferred to **M3** with the addition
 of **40 µM TMPyP3 for 180 minutes. **After feeding, the flies were either
 exposed to red-light emitting diodes for **10 minutes 40 mW/cm2** and then
 transferred to the fresh M3 media (**LIGHT**), or directly transferred to
 fresh M3 (**NO LIGHT**) and left on it for **one (F4.A), or seven (F4.B)
 days** in constant darkness before measurements. We collect separately **5
 headless bodies per sample**, in total 3 biological replicas measured in
 triplicate (technical replicas). 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. **Data generated under
 part F4.C,D** flies **starved for 16 hours and then fed with** 40 µM of
 TMPyP3 for 180 minutes on M3 in the dark. After feeding, flies were
 separated in two groups, one group was illuminated with **40 mW/cm2 for 10
 minutes** (**Light, L**), and the other was the ‘no light control’ (**No
 Light, NL**). Flies were then transferred to the fresh M3 medium and left
 for **one (F.4C)** or **seven days (F.4D)** in darkness to avoid potential
 effect of illumination on TMPyP3 activation. For body homogenates five
 headless bodies were used. In total 3 biological replicas measured in
 triplicate of technical replicas. 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.
 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. The third
 sheet in Excel called PDT is the results generated for figure four in the
 publication (F5). **Data generated under part F5.A,B** 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. **After 16
 hours of starvation**, flies were transferred to **M3** with the addition
 of **40 µM TMPyP3 for 180 minutes. **After feeding, the flies were either
 exposed to red-light emitting diodes for 10 minutes **40 mW/cm2** and then
 transferred to the fresh M3 media (**LIGHT**), or directly transferred to
 fresh M3 (**NO LIGHT**) and left on it for one (**F5.A**), or seven (F5.B)
 days in constant darkness before measurements. We collect separately **32
 heads per sample**. In total 3 biological replicas measured in triplicate
 of technical replicas. 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. Data generated under
 part F5.C,D flies **starved for 16 hours** and then fed with **40 µM of
 TMPyP3 for 180 minutes on M3 in the dark**. After feeding, flies were
 separated in two groups, one group was illuminated with 40 mW/cm2 for 10
 minutes (**Light, L**), and the other was the ‘no light control’ (**No
 Light, NL**). Flies were then transferred to the fresh M3 medium and left
 for **one (F.4C**) or **seven days (F.4D)** in darkness to avoid potential
 effect of illumination on TMPyP3 activation. We collect separately 32
 heads per sample. In total 3 biological replicas measured in triplicate of
 technical replicas. Samples were prepared using the same protocol as for
 the quantification of TMPyP3. H2O2 concentrations in the homogenates were
 determined using the calibration curve for dihydroethidium (DHE, ≥95%,
 Sigma Aldrich) with known H2O2 concentration. 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. The forth sheet in Excel called PDT is the
 results generated for figure four in the publication (F6). Negative
 geotaxis measures the speed of vertical climbing after knocking flies to
 the bottom of the vial. 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.