All solvents and chemicals were commercially available (HPLC grade) and used without additional purification. Chlorpromazine hydrochloride (CPZ; CAS 69-09-0), sodium dodecyl sulfate (SDS; CAS 151-21-3), anthracene (ANT; CAS 120-112-7) and (S)-(+)-Ketoprofen (KP; CAS 22161-81-5) were purchased from Sigma-Aldrich (Madrid, Spain). Gefitinib hydrochloride (GFT; CAS 184475-55-6) was provided by MedChemExpress (New Jersey, United States). 4-Defluoro-4-hydroxy gefitinib (DF-GFT; CAS 847949-50-2) and O-Demorpholinopropyl gefitinib (DMOR-GFT; CAS 184475-71-6) were acquired from Santa Cruz Biotechnology (Dallas, United States) and O-Demethyl gefitinib (DMT-GFT; CAS 847949-49-9) was purchased from Toronto Research Chemicals (North York, Canada). Stock solutions were prepared in DMSO as vehicle, whereas GFT, CPZ and SDS were dissolved in ultrapure water (Milli-Q^®). 1,4-dihydro-1,2-dimethylbenzoic acid (DMBA) was obtained from a Birch reduction synthesis. Dulbecco’s Modified Eagle Medium (DMEM, low glucose with pyruvate and glutamine), Dulbecco’s Modified Eagle Medium (DMEM, low glucose with pyruvate and without glutamine and phenol red), fetal bovine serum (FBS), penicillin–streptomycin (1.0 U/mL × 10⁵ U/mL, 1.0 μg/mL × 10⁵ μg/mL) and ethylenediaminetetraacetic acid (EDTA) were supplied by Honeywell Fluka (North Carolina, United States). Trypsin–EDTA (0.25%–0.02%) was provided by Cultek (Madrid, Spain). Phosphate buffered saline solution (PBS, pH 7.4), neutral red dye, human serum albumin fatty acid free (HSA), polyoxyethylenesorbitan monolaurate (TWEEN 20) and sodium hydroxide (NaOH) were purchased from Sigma-Aldrich (Madrid, Spain). Low melting point agarose was provided by Pronadisa (Madrid, Spain). Tris(hydroxymethyl)aminomethane, Linoleic acid (LA), Methyl Linoleate (ML), 1,1,3,3-Tetraethoxypropane (TEP), 2-Thiobarbituric acid (TBA) and 2,6-Di-tert-butyl-4-methylphenol (BHT) were supplied by Sigma-Aldrich (Madrid, Spain). CometAssay^® Lysis Solution was purchased from R&D systems (Minneapolis, United States). 2,4-Dinitrophenylhydrazine Hydrochloride (DNPH) was acquired from Santa Cruz Biotechnology (Dallas, United States). Trichloroacetic acid (TCA) was purchased from Labbox (Barcelona, Spain). SYBR™ Gold DNA, and CellMask™ Orange Plasma membrane stains were acquired from Invitrogen (Madrid, Spain). RedDot™ Far-Red Nuclear was supplied by Biotium (California, United States). Apo-ONE^® Homogeneous Caspase-3/7 assay and CytoTox-ONE™ Homogeneous Membrane Integrity Assay were received from Promega (Madison, United States). Deoxyribonucleic acid sodium salt from calf thymus was purchased from Sigma-Aldrich (Madrid, Spain).

Human keratinocytes cells (HaCaT) were grown in 75 cm² plastic flasks in DMEM supplemented with 10% FBS and penicillin/streptomycin (100 U/mL, and 100 μg/mL) in a humidified incubator (100% relative humidity) at 37°C with 5% CO₂ atmosphere. Cells were routinely passed twice a week for maintenance (1:5 splitting ratio) and a trypan blue exclusion test was used to ensure that the cultures were viable before each experiment.

HaCaT cells were seeded on sterile round glass coverslips and incubated in 24-well plates (2.5 × 10⁴ cells/well). After 24 h of incubation, DMEM medium was replaced with 1 mL of fresh medium containing drugs (GFT, DMT-GFT or DMOR-GFT) at 15 μM followed by staining with CellMask™ Orange Plasma membrane and RedDot™ Far-Red nuclear stains (1:10,000 and 1:200 dilutions, respectively). Cells treated with the compounds were incubated for 1 h whereas CellMask™ Orange Plasma membrane and RedDot™ Far-Red nuclear stains were incubated for 30 and 10 min respectively at 37°C. Then, coverslips were washed three times for 5 min with PBS and finally mounted in glass slides using a solution of mowiol. Through sequential mode, a Leica SP5 confocal microscope was used for cell imaging. The excitation wavelengths were 405 nm for GFT, DMT-GFT and DMOR-GFT. For CellMask™ Orange Plasma membrane and RedDot™ Far-Red nuclear stains, the excitation wavelengths were 543 nm and 662 nm, respectively. The maxima emission wavelengths were 450, 567, and 694 nm for the drug and its metabolites, plasma membrane and nuclear stains, respectively.

Irradiations under UVA light conditions were performed with an LCZ-4 photoreactor equipped with six top and eight sides Hitachi lamps (λ _max = 350 nm, Gaussian distribution; Luzchem, Canada), which emit 94% UVA and 2% UVB radiation, respectively. The samples for in vitro HaCaT NRU phototoxicity assay were irradiated in 96-well transparent plates, for comet assay 24-well transparent plates were used while in both protein and lipid photooxidation assay irradiations were performed in 6-well transparent wells. All irradiations were carried out through the lid of the plates which does not absorb beyond 310 nm for the purpose of reducing the direct effect of UVB radiation over the cell cultures. Since cell viability after irradiation was higher than 90%, the UV dose selected was suitable for the photogenotoxicity experiments, therefore, false-positive results triggered by DNA fragmentation as a result of cell death were avoided. Moreover, in order to prevent overheating, plates were kept on ice during the irradiation process and the temperature was controlled by ventilation.

Neutral red uptake phototoxicity test (NRU) was assessed according to the OECD Guideline 432 (OECD, 2019) in keratinocyte cells (HaCaT) with additional minor modifications (Garcia-Lainez et al., 2018). Chlorpromazine (CPZ) and sodium dodecyl sulfate (SDS) served as the positive phototoxic and negative non-phototoxic controls, respectively. CPZ is a widely used typical antipsychotic drug with well-known phototoxic properties as already reported (Palumbo et al., 2016). In our recent study, GFT has shown a very significant phototoxic potential in HaCaT cells (Tamarit et al., 2021). Therefore, the phototoxicity behavior of the metabolites was studied in parallel with the parent drug.

Concisely, for each compound, two 96-well plates were seeded at a density of 2.0 × 10⁴ cells/well. Next day, HaCaT cells in a fresh DMEM medium without phenol red were treated with the compounds (GFT, DF-GFT, DMT-GFT and DMOR-GFT) at eight concentrations ranging from 2.5 to 500 μM except for DMOR-GFT which was added in a concentration range from 125 to 0.625 μM. Additional plates were processed with CPZ (from 1.57 to 500 µM) and SDS (from 3.13 to 500 µM). All plates were incubated for 1 h in dark conditions. Then, for each sample, in the presence of ice, one plate was irradiated with a non-cytotoxic dose of 5 J/cm² UVA (UVA Light), whereas the other was kept in a dark box (Dark). Next, drug solutions were replaced with fresh DMEM medium, and plates were further incubated overnight. Next day, neutral red solution (50 μg/mL) was added into the wells and incubated for 2 h at 37°C. Later, cells were washed once with PBS and neutral red was recovered from lysosomes in 100 μL of the extraction buffer [distilled water 50% (v/v), ethanol 49.5% (v/v) and acetic acid 0.5% (v/v)]. Lastly, the absorbance of the plates was read at 550 nm on a Synergy H1 microplate reader. For each compound, dose–response curves were performed to establish the concentration causing a reduction of 50% of neutral red uptake (IC₅₀) in dark and UVA light conditions. Afterwards, photoirritation factor (PIF) values were calculated using the following equation: P I F = I C 50   D a r k   /   I C 50   U V A   L i g h t

Conforming to OECD Guideline 432 (OECD, 2019), a substance is labelled as “non-phototoxic” when PIF is <2, “probably phototoxic” if PIF is between 2 and 5 and “phototoxic” if PIF is >5.

Linoleic acid photosensitized oxidation assay was performed as described previously (Seto et al., 2013) with minor adjustments exposed in prior studies (Zeb and Ullah, 2016). For this, a solution of linoleic acid (1 mM) in 20 mM PBS (pH 7.4) containing 0.05% Tween 20 was prepared and irradiated in the presence of GFT or its metabolites (DMT-GFT or DMOR-GFT) in a concentration of 100 µM. KP 200 µM was taken as a positive control (Seto et al., 2013). Lipid peroxidation was monitored with TBA-reactive substances assay (TBARS) (Zeb and Ullah, 2016) adding 4 mM TBA and 10 µL BHT solution in glacial acetic acid to the irradiated samples (500 µL). Then, samples were heated at 95 °C for 60 min. After 10 min of cooling, the absorbance of the samples was measured at 532 nm for the determination of TBARS. A standard curve of TEP was used to determine the total of malondialdehyde (MDA) produced. The data was analyzed statistically using a Two-way ANOVA (Analysis of Variance) technique.

Protein photooxidation assay was assessed according to Colombo et al. (2016) with minor modifications as explained below. The photooxidative activity of GFT has already been evaluated in our previous study (Tamarit et al., 2021), which has proved its protein photooxidation capability. Briefly, a solution of HSA (5 mg/mL, 1 mg protein/sample) was prepared in PBS and irradiated alone or in the presence of 100 μM of GFT, DMT-GFT or DMOR-GFT with a UVA dose of 15 J/cm². The amount of HSA oxidation in each sample was determined immediately after irradiation by incubating the samples for 60 min at room temperature with 200 µL of 2,4-dinitrophenylhydrazine (DNPH) 10 mM to create stable protein-DNP hydrazone adducts. After incubation, proteins were precipitated with 20% TCA solution and incubated on ice for 15 min. Next, samples were washed twice with ethanol/ethyl acetate 1:1 (v/v) containing 20% TCA, in order to reduce the protein loss, followed by its resolubilization in 100 μL guanidine buffer (6 M). Finally, absorbance at 375 nm was registered using the Synergy H1 microplate reader and the HSA oxidation degree was expressed as nmol of carbonyl per mg protein as displayed in the next equation: C a r b o n y l   c o n t e n t   n m o l / m g   p r o t e i n = A 375 / ε m M P × 100

Where A₃₇₅ is the absorbance of the sample at 375 nm, ε^mM is the corrected millimolar extinction coefficient (6.364) and P the amount of protein from standard well.

The statistical analysis of the data was conducted using a Two-way ANOVA (Analysis of Variance) method.

The comet assay (single-cell gel electrophoresis) was performed as already detailed in Garcia-Lainez et al. (2018) in order to detect both single and double strand breaks and alkaline labile sites on nuclear DNA. Briefly, HaCaT cells in exponential growth were harvested by trypsinization and kept in ice-cold PBS for 2 h to neutralize any DNA damage produced during trypsinization step, as has been previously demonstrated in FSK cells (Agúndez et al., 2020). Then, 24-wells plates (1.0 × 10⁴ cells/well) were seeded and treated with 100 μM of GFT or its metabolites (DMT-GFT or DMOR-GFT) for 30 min at 4°C in darkness to minimize cell aggregation and inhibit DNA repair. The reference photogenotoxic control of this assay was CPZ (10 μM). Afterwards, one plate was irradiated under 2 J/cm² UVA light dose (4 J/cm² for DMT-GFT and DMOR-GFT) while the other one was kept in darkness as the negative control. Later, both irradiated and non-irradiated cells were detached from the plates and 100 µL of cell suspension was mixed with 100 µL 1% low melting point agarose solution and loaded onto FLARE^® slides. Then, slides were incubated on ice to allow drop jellification. Finally, slides were embedded in a box with lysis buffer to initiate the lysis of cells and incubated overnight at 4°C. Next day, slides were placed in electrophoresis tank filled with 1 L of cold alkaline electrophoresis buffer (0.2 M NaOH, 1 mM EDTA in distilled water and pH ≥ 13). The electrophoresis was run at 21 V (1 V/cm) for 30 min and kept at 4°C. Then, slides were washed twice with MilliQ water and DNA fixation accomplished by a serial dehydration with 70% ethanol and 100% ethanol solutions during 5 min and followed by drying for 2 h at 37°C. Nuclear DNA was stained with a SYBR Gold^® bath (1:10,000 in Tris-EDTA buffer) for 30 min at 4°C and slides were kept in darkness until its visualization. Comets (nucleoids and tails) were visualized using a fluorescence microscope (Leica DMI 4000B). DNA damage was quantified by counting and analyzing at least 100 DNA comets. Finally, total comet score (TCS) was obtained with the classification of six DNA damage types (Møller, 2006) applying the formula: C l .   0 × 0 + C l .   1 × 1 + C l .   2 × 2 + C l .   3 × 3 + [ C l .   4 × 4 + C l .   5 × 5 + C l .   6 × 6 ] / 6

Where Cl. is the class of DNA damage according to the visual scoring.

The results are expressed in 1–100 arbitrary units, where class 0 comets are comets with no DNA damage while class 6 comets represent comets with maximum DNA damage. Two-way ANOVA (Analysis of Variance) method was used for the statistical analysis of the data.

Results are presented as mean ± standard deviation retrieved from the results of at least three independent experiments. Data were analyzed and regression methods were developed using either GraphPad or OriginLab software. Statistical significance was obtained from the Student’s t-test and p values lower than 0.05 were considered significant (*p < 0.05; **p < 0.01; ***p < 0.001).

In vitro phototoxicity testing enables the early detection and screening of substances or compounds that may exhibit phototoxic properties. Thus, in a first stage, in vitro neutral red uptake (NRU) phototoxicity studies were performed with HaCaT cells to determine the phototoxic potential of GFT and its metabolites upon exposure to UVA light (5 J/cm²).

Having established the phototoxic potential of GFT (Tamarit et al., 2021), DMT-GFT and DMOR-GFT (Table 1), the fluorescence spectral characteristics of these compounds were analyzed in order to investigate the photophysical differences between GFT and its metabolites in different biological media. Thus, the emission spectra (λ _exc = 320 nm) were recorded in PBS solution and in the presence of different biomolecules (lipids, protein and DNA) or inside keratinocyte cells. As displayed in Figure 5 and Table 2, an evident red shift in the emission maximum occurred for the metabolites in all models, probably due to emission from the phenolate form. Expectedly, in terms of fluorescence intensity, the protein environment increased fluorescence emission in all cases, especially for the DMOR-GFT metabolite, which was markedly enhanced. On this basis, it can be foreseen that this effect is also observed once the compounds are inside keratinocyte cells (Figure 5).

In parallel, fluorescence quantum yield (Φ_F) of the internalized compounds were also determined, by comparison with anthracene as standard (Φ _F = 0.27 in ethanol) [26]. Hence, both GFT and DMT-GFT showed similar values, Φ _F = 0.07 and Φ _F = 0.04, respectively. In contrast, DMOR-GFT fluorescence inside the cells revealed a measurable enhancement of the quantum yield (Φ _F = 0.1) (Supplementary Figure S3).

Considering the intrinsic fluorescence properties stated above, confocal microscopy was used to define the intracellular colocalization of the compounds. Keratinocytes were seeded in coverslips and treated with GFT 15 μM, DMT-GFT 25 µM and DMOR-GFT 15 µM and further labeled with both RedDot™ Far-Red Nuclear (far red fluorescence) and CellMask™ Orange Plasma membrane stains (red fluorescence). After 1 h incubation, the uptake was efficiently observed in all compounds. A cytoplasmic distribution was shown in all cases without a predominant specific localization in any organelle (Figure 6).

Single-cell gel electrophoresis (comet assay) under alkaline conditions was carried out to disclose DNA damage as a result of single strand and double strand breaks as well as alkali-labile sites on chromosomic DNA of individual human keratinocytes. Hence, HaCaT cells were embedded in low melting point agarose on a slide and incubated with the compounds for 1 h. Then, samples were exposed to UVA light for 5 min (10 min for DMT-GFT and DMOR-GFT) and alkaline electrophoresis was performed after cell lysis. During electrophoresis, damaged DNA migrates from the nucleus yielding to formation of comet nucleoids and tails, which were visualized by fluorescence after SYBR Gold staining. Percentage of DNA damage was calculated following the classification of the images in six different classes. As shown in Figure 7, GFT displayed a significant damage (around 72%) after 5 min of UVA light irradiation. Likewise, DMT-GFT generated high percentage of DNA damage (around 54%). In contrast, irradiation of DMOR-GFT for 5 min did not promote DNA damage given that the nucleoids remained intact compared to control cells. Further irradiation of DMOR-GFT up to 10 min led to a significant degree of DNA damage (around 45%).

Generally, cells have evolved a number of repair mechanisms to reduce DNA damage; however, if the repair is faulty, DNA lesions can result in long-term mutations that can ultimately result in carcinogenic effects. For this reason, a complementary set of experiments were performed for the purpose of studying the capability of HaCaT cells to repair the nuclear DNA photodamage produced by GFT and its metabolites after UVA light irradiation. In brief, HaCaT cells containing GFT, DMT-GFT or DMOR-GFT were irradiated and processed for comet assay with two different incubation time (6 h and 18 h) before cell lysis was performed. Finally, DNA damage was monitored as mentioned earlier. Apparently, cells treated with GFT recovered considerably from the initial DNA damage although a residual damage was still remanent even when the recovery time reached 18 h (41%) (Figure 7). This trend was not found either with DMT-GFT or with DMOR-GFT treated cells, which maintained the initial damage intact (Figure 7). More details are provided in the supplementary information (Supplementary Figure S4).

Apoptosis, frequently referred to as programmed cell death, plays a crucial role in the regulation of the cellular lifecycle (Kumar, 2007), however; excessive activation of this process can lead to critical diseases (Kam and Ferch, 2000). The apoptosis event involves, in most cases, the activation of the so-called zymogens, which are evidenced to be the precursors of the well-known caspase enzymes (Kumar, 2007). Upon apoptotic signals, caspases are activated and, through their proteolytic activity, initiate protein digest inducing cell death (Morgan and Thorburn, 2001). The key effector caspases in mammals are caspase-3, -6 and -7, being caspase-3 the most frequently involved in apoptosis (Kumar, 2007). Based on these facts, the contribution of GFT or its metabolites to apoptosis upon UVA light exposure was investigated in HaCaT cells using Apo-ONE homogeneous caspase-3/7 assay. The kit contains a profluorescent Rhodamine 110 (Z-DEVD-R110), which serves as a substrate for both caspase-3 and caspase-7. Consequent to the cleavage and removal of the DEVD peptide by caspase-3/7, Rhodamine 110 (the leaving group) becomes intensely fluorescent. Considering this, caspase-3/7 activity was monitored by fluorescence and represented as a relative change, indicating the level of apoptosis activation inside the cells. Thus, both GFT and DMOR-GFT induce a concentration dependent activation of caspase-3/7 after UVA light exposure, as shown in Figures 8A, C, specially at the concentration close to the IC₅₀ value (5 µM and 3 μM, respectively). In the case of DMT-GFT (Figure 8B), caspase-3/7 activity was similar between the concentration of 2.5 µM and the concentration corresponding to IC₅₀ (5 µM).

L-Lactate dehydrogenase (LDH) is a stable cytoplasmic enzyme that catalyzes the conversion of lactate to pyruvate, as it converts NAD⁺ to NADH during glycolysis (Kaja et al., 2015). LDH-release assay is used to assess the level of plasma membrane damage since the permeabilization of the membrane cause the leakage of this enzyme out of the cells (Chan et al., 2013). Bearing in mind that the key factor for necrotic cells is the permeabilization of the plasma membrane, the measurement of LDH-release can be considered an indicator for necrosis. However, it is interesting to highlight that the leakage of LDH may also be involved in apoptotic events in late stages (Parhamifar et al., 2013). As shown in Figure 8D, a small but significant percent of LDH-release was displayed for both GFT at a concentration near the IC₅₀, respectively. Contrary, DMT-GFT (Figure 8E) did not show any effect on the LDH release. Additionally, in Figure 8F, a significant percentage of LDH-release is also observed.

In summary, gefitinib represents an important targeted therapy for certain types of cancer, offering personalized treatment options and potentially improved outcomes. However, it is crucial to carefully consider the photo(geno)toxic potential of both the parent drug and its metabolites.