Evidence of altered redox homeostasis in trichothiodystrophy

in Redox Experimental Medicine
Authors:
Valeria Cordone Department of Environmental and Prevention Sciences, University of Ferrara, Ferrara, Italy

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Anna Guiotto Department of Environmental and Prevention Sciences, University of Ferrara, Ferrara, Italy
Department of Animal Science, Plants for Human Health Institute, North Carolina State University, Kannapolis, North Carolina, USA

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Vivian Bucay Bucay Center for Dermatology and Aesthetics, San Antonio, Texas, USA

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Alessandra Pecorelli Department of Environmental and Prevention Sciences, University of Ferrara, Ferrara, Italy
Department of Food, Bioprocessing and Nutrition Sciences, Plants for Human Health Institute, North Carolina State University, Kannapolis, North Carolina, USA

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Giuseppe Valacchi Department of Environmental and Prevention Sciences, University of Ferrara, Ferrara, Italy
Department of Animal Science, Plants for Human Health Institute, North Carolina State University, Kannapolis, North Carolina, USA
Department of Food and Nutrition, Kyung Hee University, Seoul, South Korea

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Correspondence should be addressed to G Valacchi: gvalacc@ncsu.edu

*(V Cordone and A Guiotto contributed equally to this work)

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Objective

Trichothiodystrophy (TTD) is a rare hereditary disease whose prominent feature is brittle hair. Additional clinical signs are physical and neurodevelopmental abnormalities and in about half of the cases hypersensitivity to UV radiation. Although the mutations involved in this condition have been characterized, the correlation between the molecular defects and the plethora of clinical symptoms is not well understood. Recently, the presence of a redox imbalance in TTD has been suggested although no clear evidence has been reported on this aspect.

Methods

In the present study, we evaluated the redox status of fibroblasts isolated from a TTD patient. In addition, to understand the ability of TTD cells to respond to oxidative insults, the cells were challenged with H2O2. Mitochondrial O2 •− and mitochondrial membrane potential were measured in different oxidative conditions. In addition, protein levels of NRF2 and BACH1 were also analyzed in response to H2O2.

Results

The results suggested an aberrant mitochondrial response to oxidative stimuli, an increased baseline oxidative stress status in TTD, and an altered NRF2/BACH1 level.

Conclusions

This study emphasizes the altered redox homeostasis in TTD pathogenesis and mitochondria functionality.

Significance statement

Focusing on mitochondria homeostasis and redox imbalance could represent an alternative therapeutic target for this condition to improve patients’ clinical features.

Abstract

Objective

Trichothiodystrophy (TTD) is a rare hereditary disease whose prominent feature is brittle hair. Additional clinical signs are physical and neurodevelopmental abnormalities and in about half of the cases hypersensitivity to UV radiation. Although the mutations involved in this condition have been characterized, the correlation between the molecular defects and the plethora of clinical symptoms is not well understood. Recently, the presence of a redox imbalance in TTD has been suggested although no clear evidence has been reported on this aspect.

Methods

In the present study, we evaluated the redox status of fibroblasts isolated from a TTD patient. In addition, to understand the ability of TTD cells to respond to oxidative insults, the cells were challenged with H2O2. Mitochondrial O2 •− and mitochondrial membrane potential were measured in different oxidative conditions. In addition, protein levels of NRF2 and BACH1 were also analyzed in response to H2O2.

Results

The results suggested an aberrant mitochondrial response to oxidative stimuli, an increased baseline oxidative stress status in TTD, and an altered NRF2/BACH1 level.

Conclusions

This study emphasizes the altered redox homeostasis in TTD pathogenesis and mitochondria functionality.

Significance statement

Focusing on mitochondria homeostasis and redox imbalance could represent an alternative therapeutic target for this condition to improve patients’ clinical features.

Introduction

Trichothiodystrophy (TTD) is a rare autosomal recessive multisystem disorder, characterized by brittle nails and hair due to keratinocytes lacking sulfur-rich proteins (Itin et al. 2001). Particularly, by observing the hair from TTD subjects under polarising microscopy, it shows an alternating light and darkness banding pattern, termed ‘tiger tail banding’, due to the reduced cysteine amount in the hair (in TTD is less than 50% of the normal content) (Liang et al. 2005). Indeed, the name of the pathology recalls this feature (from Greek ‘tricho’, hair; ‘thio’, sulphur; ‘dys’, faulty; ‘trophe’, nourishment). The incidence rate for TTD is approximately 1 per million live births, and the prevalence rate has been estimated at 1 per 830,000 in Europe (Kleijer et al. 2008).

A wide range of other clinical characteristics with variable degrees of severity can be seen in TTD individuals, i.e. physical and mental retardation, microcephaly, small stature, unusual facial features, ichthyotic skin, decreased fertility, cataracts, recurrent infections, anemia, and signs of premature aging (Faghri et al. 2008). Skin photosensitivity is also described in about 50% of TTD patients (PS-TTD) and is associated with defects in nucleotide excision repair (NER) of UV-damaged DNA (Orioli & Stefanini 2019). Although the NER deficiency (the most common mutations involve ERCC2 – or XPD – gene) in TTD is indistinguishable from that reported in Xeroderma Pigmentosum type D, TTD patients do not show augmented risks of skin cancers (Wijnhoven et al. 2005, Orioli & Stefanini 2019).

Non-photosensitive TTD (NPS-TTD) is characterized by biallelic mutations in genes not implied in DNA repair processes, like MPLKIP, RNF113A, GTF2E2, CARS1, TARS1, MARS1, and AARS1 (Nakabayashi et al. 2005, Corbett et al. 2015, Kuschal et al. 2016, Kuo et al. 2019, Theil et al. 2019, Botta et al. 2021). For instance, a recent paper highlighted the role of RNF113A in modulating the redox cellular balance, bringing up the idea that possible oxidative stress (OS) might play a role in this condition (Cho et al. 2024).

In fact, despite the known genetic mutations of both PS and NPS-TTD, the correlation between the molecular defects and the plethora of clinical symptoms is poorly understood and the idea that a corrupted redox homeostasis is involved in this pathology has been hypothesized.

The term ‘oxidative stress’ describes a series of events, leading to damage to biological macro-molecules, as a consequence of an imbalance between cellular antioxidant defence and reactive oxygen species (ROS) production. OS is a key player in aging (Maldonado et al. 2023), as well as a large number of human pathologies, including neurological diseases (Pecorelli et al. 2011, Dias et al. 2013, Liu et al. 2022), cancer (Hayes et al. 2020, Arfin et al. 2021), inflammatory bowel disease (Li et al. 2023).

As known, ROS can be produced during cellular metabolism, inflammatory responses or exposure to physical and chemical agents (Markkanen 2017). Recently, Lerner and colleagues showed that XPD-mutated primary fibroblasts from TTD subjects presented high basal levels of intracellular ROS (Lerner et al. 2019). In addition, it has been demonstrated that NER may be implicated in the repair of lesions generated by ROS, like 8-oxo-7,8-dihydroguanine (8-oxoG) (Shafirovich et al. 2016).

In light of this, the present work was designed to characterize and evaluate the redox homeostasis, mitochondrial functionality, and the triggering of an appropriate antioxidant response in an ex-vivo TTD model. For these purposes, skin fibroblasts deriving from one TTD patient (n = 1) were used as they represent a good model to study the molecular mechanisms involved in neurological disorders, as well as multi-system broad-spectrum pathologies (Auburger et al. 2012, Cervellati et al. 2015, Cordone et al. 2019).

Materials and methods

Human skin fibroblast isolation and culture

Primary fibroblasts were isolated from a 3-mm skin biopsy from one healthy and one TTD subject, as previously described (Sticozzi et al. 2013, Cordone et al. 2019). Cells were kept in culture with low glucose-DMEM medium (cat. 11054020, Thermo Fisher Scientific Inc.) supplemented with fetal bovine serum (10% v/v), L-glutamine (2 mM) and antibiotics (100 IU/mL penicillin, 100 µg/mL streptomycin) (cat. 26140079, 25030081 and 15140122, Thermo Fisher Scientific Inc., respectively) and incubated at 37°C in a humidified atmosphere (5% CO2). All the subsequent experiments were performed by using fibroblasts between the sixth and tenth passage in vitro.

Human skin biopsies were obtained from biological waste material during diagnostic procedures of skin cancer and represented normal tissues adjacent to cancer tissues. Tissue collection was carried out in compliance with the Declaration of Helsinki of the World Medical Association and signed written informed consent.

Cell treatments

For the experiments reported in this study, control and TTD cells were starved for 12 h with 1% FBS-supplemented medium. Subsequently, fibroblasts were treated for 1, 6, and 24 h with 100 µM of hydrogen peroxide (H2O2) (cat. H1009, Sigma-Aldrich), as previously reported (Pecorelli et al. 2015, 2020a ). In the experiments with MitoTEMPO (cat. SML0737, Sigma-Aldrich), cells were first pre-treated with 1 µM of MitoTEMPO for 2 h, and then the surnatant was replaced with H2O2- and MitoTEMPO-containing medium for the indicated time-points, as described above.

Oxidants production analysis

The analysis of oxidant production was performed by reactive oxygen species (ROS) Detection Reagents (cat. MP36103, Invitrogen), as previously described (Pasqui et al. 2024). After the overnight starvation, fibroblasts were incubated in darkness with 2′,7′-dichlorofluorescein diacetate (DCFDA) in 1% FBS-containing medium for 30 min at 37°C in a humidified 5% CO2 atmosphere. At the end of the incubation, the medium was removed, and fibroblasts were washed once with D-PBS in order to eliminate the excess of the probe. Then, the cells were incubated for 1, 6, and 24h with new media containing the treatments. At the end of each treatment, DCF fluorescence, as a measure of oxidant production, was determined by SpectraMax iD3 plate reader (Molecular Devices, LLC., San Jose, CA, USA), at 485 nm (excitation filter) and 530 nm (emission filter). Data were normalized against cell density determination, performed by sulforhodamine B (SRB) staining.

Mitochondrial ROS measurement

Mitochondrial ROS production was evaluated by confocal microscopy using the specific probe MitoSOX™ Red (cat. M36008, Thermo Fisher Scientific Inc.). According to the manufacturer’s instructions, fibroblasts were seeded on coverslips, and after treatments, they were incubated for 10 min at 37°C in HBSS buffer containing a final concentration of 5 µM of MitoSOX™ Red probe. Cells were then washed with D-PBS three times and fixed for 10 min with 10% neutral buffered formalin at room temperature. The washes with D-PBS were repeated two times, and the nuclei were counterstained with 4′,6- diamidino-2-phenylindole (DAPI) (cat. D1306, Thermo Fisher Scientific Inc.) (1:50,000 dilution in D-PBS) for 3 min. The coverslips were then mounted on microscope slides using the Fluoromount-G mounting medium (cat. 5018788, Thermo Fisher Scientific Inc.). Images were acquired by Zeiss LSM 710 confocal microscope (Carl Zeiss) with a 40× objective and analyzed using Java-based Fiji-ImageJ software and reported as mean fluorescence intensity (Cordone et al. 2022).

Mitochondrial membrane potential assay

An evaluation of mitochondrial membrane potential (MMP, ΔΨm) was performed by the specific probe MitoTracker® Orange CMTMRos (cat. M7510, Thermo Fisher Scientific Inc.). Cells were seeded and grown on coverslips inside a 24-well plate filled with the appropriate culture medium. At the end of the treatments, a new medium containing a final concentration of 100 nM of the probe was added to the cells, and incubated for 45 min at 37°C (5% CO2). The cells were then quickly washed with PBS and fixed for 10 min at room temperature with 10% neutral buffered formalin. After fixation, the cells were washed three times with D-PBS, the nuclei were stained with DAPI, and the coverslips were mounted onto glass, as reported above. Images at 40× magnification were collected immediately by Zeiss LSM 710 confocal microscope (Carl Zeiss). Results were analyzed by Java-based Fiji-ImageJ software and given as mean fluorescence intensity (Kholmukhamedov et al. 2013, Neikirk et al. 2023).

C11-BODIPYTM Lipid Peroxidation (LPO) Assay

The detection of lipid peroxidation in TTD and CTRL live fibroblasts was performed by using BODIPYTM 581/591 C11, a sensitive fluorescent probe able to localize into live cell membranes (cat. D3861, Thermo Fisher Scientific Inc.) (Vadarevu et al. 2021). Briefly, 40,000 cells were plated on 12-mm coverslips placed in a 6-well plate and incubated overnight in 1% FBS-supplemented medium, then treated with hydrogen peroxide, as described in the ‘Cell treatments sub-section. At the end of the treatments, the medium was replaced with a new medium containing a final concentration of 1 µM of BODIPY™ 581/591 C11 and incubated for 30 min in the incubator. Subsequently, three washes with D-PBS were performed and the fibroblasts were fixed with 10% neutral buffered formalin at room temperature for 10 min. Fibroblasts were then washed with D-PBS, and incubated with DAPI solution, as previously described. Finally, the cells were mounted onto microscope slides. 40×-magnification images were immediately acquired by Zeiss LSM 710 confocal microscope (Carl Zeiss). The levels of lipid peroxidation were determined by monitoring the increase of the green fluorescence intensity (emission at 510 nm). Digital images were analyzed by using Java-based Fiji-ImageJ software. Results were shown as mean fluorescence intensity.

Immunofluorescence analysis

Cells were seeded in a 6-well plate containing coverslips. After the overnight starvation and the H2O2 treatments, cells were washed twice with D-PBS, then fixed in 10% neutral buffered formalin for 10 min at room temperature, and permeabilized with D-PBS containing 0.25% (v/v) Triton X-100 for 10 min at 4°C, as previously described (Pecorelli et al. 2020b ). Non-specific binding sites were blocked with 1% (w/v) bovine serum albumin (BSA, cat. A7906, Sigma-Aldrich) in D-PBS for 45 min. Cells were then incubated overnight with primary antibodies diluted in 0.25% (w/v) BSA-containing D-PBS at 4°C. The following primary antibodies were used: anti-BACH1 (cat. HPA034949, Sigma-Aldrich, dilution 1:250); anti-NRF2 (cat. sc-365949, Santa Cruz Biotechnology, dilution 1:100); anti-4-hydroxynonenal (4-HNE) (cat. AB5605, Merk Millipore, dilution 1:400); and anti-p-H2AX (cat. sc-517348, Santa Cruz Biotechnology, dilution 1:100). The day after, cells were washed with D-PBS, and incubated with solutions of fluorescent dye-cross adsorbed secondary antibodies (cat. A-11008, Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488; cat. A-11004, Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 568; cat. A-11057, Donkey anti-Goat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 568, Thermo Fisher Scientific Inc., dilution 1:1,000) in 0.25% BSA-containing D-PBS for 1 h at room temperature. After further washes with D-PBS and the counterstaining with DAPI, the coverslips were mounted onto microscope slides and photographed by Zeiss LSM 710 confocal microscope (Carl Zeiss) at 40× magnification. The analysis was performed using Java-based Fiji-ImageJ software. Results were given as mean fluorescence intensity.

Statistical analysis

Statistical analyses were performed by using Statsoft Statistica10 and GraphPad Prism 8 software. Data were expressed as means ± standard error of the mean (s.e.m.) and are the results of three different cultures from cells of n = 1 control subject and n = 1 TTD patient. Factorial analysis of variance (ANOVA), with post-hoc Tukey’s tests was applied. A P value of <0.05 was regarded as statistically significant for all the tests.

Results

TTD fibroblasts oxidative response to H2O2 challenges

In order to investigate the possible impairment of redox homeostasis in TTD primary fibroblasts, we first stimulated cells with H2O2 (100 µM) and then evaluated both oxidants production (Fig. 1A) and superoxide anion levels (Fig. 1B and C).

Figure 1
Figure 1

Overall oxidant production and mitochondrial ROS levels in TTD fibroblasts. (A) Levels of total oxidants in control and TTD fibroblasts in basal condition or exposed to hydrogen peroxide challenge (100 µM) for 1h, 6h and 24h, assessed by DCFDA assay. (B) Representative images and (C) analysis of MitoSOX™ Red fluorescence staining in control and TTD fibroblasts, pre-treated with MitoTEMPO (1 µM for 2 h) and then co-incubated or not with hydrogen peroxide (100 µM) for 1 h, 6 h, and 24 h. Nuclei are counterstained with DAPI. Objective 40×. Data were expressed as means ± S.E.M., and are the result of three different cultures of cells from n = 1 control subject and n = 1 TTD patient. *indicates significant differences between CTRL and TTD groups at the same treatment conditions (*P < 0.05; **P < 0.01; ***P < 0.001). #indicates significant differences of H2O2/MitoTEMPO/H2O2+MitoTEMPO-exposed groups versus the corresponding basal conditions (##P < 0.01; ###P < 0.001); +indicates significant differences between CTRL and TTD groups treated with H2O2+MitoTEMPO vs only H2O2 at the same time point (+P < 0.05; +++P < 0.001). Results were analyzed by factorial ANOVA, with post-hoc Tukey’s multiple comparisons test. CTRL, control subject; TTD, trichothiodystrophy.

Citation: Redox Experimental Medicine 2024, 1; 10.1530/REM-24-0010

In particular, it was observed that TTD cells presented higher levels of oxidants already in basal condition, as compared to basal CTRL fibroblasts (Fig. 1A). As expected, the H2O2-challenge was able to trigger an increased oxidants generation in CTRL cells, starting after 1h of exposure (as compared to baseline CTRL). In TTD fibroblasts the treatment with H2O2 was able to further significantly augment the signal related to oxidants, with respect to the CTRL cells at all the considered time-points (1h, 6h, and 24h of H2O2 treatment) (Fig. 1A).

We then analyzed the mechanistic role of mitochondrial ROS (mtROS) in primary TTD fibroblast response, challenged with H2O2, by using MitoTEMPO, a mitochondrial-targeted antioxidant and specific scavenger of mitochondrial superoxide (Peoples et al. 2019). The generation of mtROS, mainly superoxide anion, measured by MitoSOX-related fluorescence levels (Fig. 1B) was enhanced in basal TTD cells, as compared to CTRL, and raised after 1h-, 6h- and 24h-exposure to H2O2 in both CTRL and TTD cells, with a higher fluorescence signal in treated TTD cells (Fig. 1C). The treatment with MitoTEMPO for 6 and 24h in TTD cells led to a decrease of the mtROS production, to the levels of CTRL cells. In addition, a significant reduction of mtROS upon H2O2-treatment was achieved by 1h-MitoTEMPO exposure in CTRL cells (+++P < 0.01 vs CTRL+H2O2 1 h), while only after 6h-MitoTEMPO stimulation in TTD fibroblasts (Fig. 1B and C).

Taken together, these results showed an unbalanced redox homeostasis in TTD primary cells, with a greater response in terms of oxidants generation to the pro-oxidant challenge, as compared to control fibroblasts. Furthermore, the mitochondria seemed to play a key role in this redox impairment, since MitoTEMPO differently affected H2O2-induced mtROS generation in CTRL and TTD cells.

Reduced mitochondrial membrane potential in fibroblasts isolated from a TTD patient

In homeostatic conditions, between the inner and outer mitochondrial membranes, an electrochemical gradient ensures the ATP production and the maintenance of the mitochondrial membrane potential (MMP, ΔΨm), which is in turn essential for the correct mitochondrial activities (e.g. preservation of the electrochemical potential of hydrogen ions, as well as the process of energy storage during oxidative phosphorylation) (Zorova et al. 2018).

Since an over-generation of mtROS may often be the result of mitochondrial impairments/dysfunctions (Guo et al. 2013, Palma et al. 2024), we wanted to evaluate MMP in CTRL and TTD cell models, as an indicator of mitochondrial integrity/homeostasis.

In particular, we found that H2O2 was able to induce in CTRL cells a strong decrease in the MMP after 1 and 6 h of exposure, while upon 24h-treatment this response seemed to return to the basal condition (Fig. 2A and B). In TTD fibroblasts, ΔΨm was constitutively lower than non-treated CTRL cells (P < 0.001), with levels of MMP comparable to those observed in CTRL cells challenged with H2O2 (for 1 and 6 h) (Fig. 2B). In addition, we did not notice any substantial response in terms of ΔΨm in TTD cells, following H2O2 exposure at all the considered time-points (vs basal TTD). Indeed, also after 6 and 24h-H2O2 challenge TTD cells presented a reduced MMP, as compared to CTRL cells stimulated for the same time (Fig. 2B).

Figure 2
Figure 2

H2O2 affects mitochondrial membrane potential differently in CTRL and TTD fibroblasts. (A) Representative images and (B) analysis of MitoTracker Orange fluorescence signal in control and TTD fibroblasts exposed to hydrogen peroxide challenge (100 µM) for 1 h, 6 h and 24 h. Nuclei are counterstained with DAPI. Objective 40×. Bar = 100 µm. Data were expressed as means ± s.e.m., and are the result of three different cultures of cells from n = 1 control subject and n = 1 TTD patient. *indicates significant differences between CTRL and TTD groups at the same treatment conditions (*P < 0.05; ***P < 0.001). #indicates significant differences of H2O2-exposed groups versus the corresponding basal conditions (###P < 0.001). $indicates significant differences of H2O2-exposed groups versus the previous time-points ($$$P < 0.001). Results were analyzed by factorial ANOVA (with 2 × 2 × 3 design), with post-hoc Tukey’s multiple comparisons test. CTRL, control subject; TTD, trichothiodystrophy.

Citation: Redox Experimental Medicine 2024, 1; 10.1530/REM-24-0010

Altogether, our data seemed to indicate an alteration in mitochondrial function of TTD cells, both in basal and after a pro-oxidant stimuli. This impaired mitochondrial response was consistent with the augmented mtROS generation in TTD fibroblasts (as reported in Fig. 1B and C), suggesting a possible vicious circle in mitochondrial dysfunction.

Increased lipid peroxidation levels and DNA damage in TTD fibroblasts

After detecting unbalanced oxidants and mtROS production in TTD primary cells, we next explored the effect of H2O2-treatment on the levels of lipid peroxidation (LPO) and DNA damage in CTRL and TTD cells. LPO is considered to be involved in the pathogenesis of several disorders such as cancer, atherosclerosis, inflammatory conditions, and neurological diseases (Pecorelli et al. 2011, Zhong & Yin 2015, Di Domenico et al. 2017, Nègre-Salvayre et al. 2017). By using the oxidation-sensitive BODIPY C11 LPO sensor, we could detect the presence of ROS in the cell membranes (green fluorescence). In particular, the levels of the oxidized BODIPY C11 signal raised only after 24h-H2O2 exposure in CTRL fibroblasts (Fig. 3A and B). Conversely, TTD cells exhibited constitutive high levels of oxidized BODIPY C11 signal, while upon H2O2 treatment the green fluorescence levels were first reduced (1h-treatment vs basal TTD) and then strongly augmented at a longer time-point (24h-treatment vs basal TTD and vs CTRL+H2O2 24h) (Fig. 3A and B).

Figure 3
Figure 3

Increased Lipid peroxidation and DNA damage levels in TTD fibroblasts. (A)Representative images and (B)analysis of the immunocytochemical assay with the BODIPYTM 581/591 C11 marker. Peroxidized lipids are shown in green on cells treated with or without hydrogen peroxide (100 µM) for 1h, 6h and 24h. (C and E)Representative images and (D and F)analysis of 4-hydroxynonenal-protein adducts (4-HNE-PAs) and p-H2AX fluorescence levels, detected by IF, in control and TTD fibroblasts, treated as reported above. Objective 40×. Bar = 100 µm. Data were expressed as means ± S.E.M., and are the result of three different cultures of cells from n = 1 control subject and n = 1 TTD patient. *indicates significant differences between CTRL and TTD groups at the same treatment conditions (**P < 0.01; ***P < 0.001). #indicates significant differences of H2O2-exposed groups versus the corresponding basal conditions (#P < 0.05; ##P < 0.01; ###P < 0.001). $indicates significant differences of H2O2-exposed groups versus the previous time-points ($P < 0.05; $$P < 0.01; $$$P < 0.001). Results were analyzed by factorial ANOVA (with 2 × 2 × 3 design), with post-hoc Tukey’s multiple comparisons test. CTRL, control subject; TTD, trichothiodystrophy.

Citation: Redox Experimental Medicine 2024, 1; 10.1530/REM-24-0010

We then analyzed the levels of 4-hydroxynonenal-protein adducts (4-HNE-PAs) in our ex-vivo model, since 4-HNE is a highly reactive aldehyde end-product and a biomarker of oxidative stress-induced LPO. It is generated by peroxidation of polyunsaturated fatty acids (PUFAs) in membrane lipid bilayers and its toxicity is mainly due to alterations of cellular functions by the formation of covalent adducts with proteins (Di Domenico et al. 2017). As shown in Fig. 3C and D, the levels of 4-HNE-PAs were enhanced in basal TTD cells, as compared to CTRL fibroblasts, which was consistent with BODIPY C11 data (Fig. 3A and B). The H2O2 challenge was able to elicit an augment of 4-HNE-PAs levels only in CTRL, but not in TTD cells. Of note, the 4-HNE-PAs signal in TTD fibroblasts, both basal and H2O2-stimulated, were similar to those detected in H2O2-treated CTRL cells (Fig. 3C and D), suggesting that TTD 4-HNE-PAs were already at maximum levels at baseline.

Then, since ROS and H2O2 exposure can also induce DNA double-strand breaks (DSBs) (Li et al. 2006), by using fluorescent microscopy, we evaluated the levels of H2AX phosphorylation (Ser 139), as a crucial check-point in the recognition and repair of DSBs (Löbrich et al. 2010). In CTRL cells, H2O2 induced an augment of H2AX phosphorylation after 1h of exposure, followed by a cellular response able to reduce this up-regulation (although not at baseline) (CTRL+H2O2 6h and 24h vs basal CTRL). While, H2AX phosphorylation levels were enhanced in TTD cells already in basal condition, showing a further increase upon 1h of H2O2 challenge, which remained significantly higher after 6h of exposure (vs basal TTD and vs CTRL+H2O2 6h), and decreased only at 24h of treatment. These data suggest that TTD cells have a high steady-state level of DSBs (the enhancement of p-H2AX can be due to other cellular pathways, besides OS (Ivashkevich et al. 2012)) and that their DNA repair machinery responds at a slower rate than CTRL cells.

Altered NRF2-BACH1 cross-talk in TTD fibroblasts

In order to elucidate the molecular mechanisms underlying the oxidative damage and the pro-oxidant milieu observed in the TTD ex-vivo model, as a first attempt, we investigated the possible involvement of the master regulator of the antioxidant response, nuclear factor erythroid 2-related factor 2 (NRF2). In addition, we also analyzed the role played by another oxidative stress-responsive transcription factor, BACH1, which by binding to antioxidant response elements (AREs) like NRF2, can prevent the NRF2 binding to the same promoter region, and the expression of genes encoding for antioxidant and detoxification enzymes, as well as cytoprotective proteins (Ahuja et al. 2021, Nishizawa et al. 2023).

The nuclear translocation of the two transcription factors was evaluated after 1 h of H2O2 -/+ MitoTEMPO challenge. As shown in Fig. 4A and C, TTD fibroblasts were characterized by a constitutive decrease of BACH1 nuclear levels, as compared to basal CTRL cells (***P < 0.001). Moreover, CTRL cells exhibited a reduction of BACH1 levels into the nucleus after all the treatments (vs basal CTRL fibroblasts, as indicated by #), while in TTD cells BACH1 nuclear signal remained approximately unchanged.

Figure 4
Figure 4

Impaired NRF2–BACH1 interplay in TTD fibroblasts. (A)Representative images and (B and C) analysis of nuclear NRF2 and BACH1 levels, by immunofluorescence, in control and TTD fibroblasts, pre-treated with MitoTEMPO (1 µM for 2 h) and then co-incubated or not with hydrogen peroxide (100 µM) for 1 h. Nuclei are counterstained with DAPI. Objective 40×. Data were expressed as means ± s.e.m., and are the result of three different cultures of cells from n = 1 control subject and n = 1 TTD patient. *indicates significant differences between CTRL and TTD groups at the same treatment conditions (*P < 0.05; ***P < 0.001). #indicates significant differences of H2O2/MitoTEMPO/H2O2+MitoTEMPO-exposed groups versus the corresponding basal conditions (#P < 0.05; ###P < 0.001); +indicates significant differences between CTRL and TTD groups treated with H2O2+MitoTEMPO vs only H2O2 (+P < 0.05; ++P < 0.01). Results were analyzed by factorial ANOVA, with post-hoc Tukey’s multiple comparisons test. CTRL, control subject; TTD, trichothiodystrophy.

Citation: Redox Experimental Medicine 2024, 1; 10.1530/REM-24-0010

About NRF2, we observed an augmented nuclear localization of this transcription factor in TTD cells after H2O2 challenge (vs basal TTD and vs CTRL+H2O2) (Fig. 4A and B), maybe due to the combination of exogenous pro-oxidants and endogenous high levels of oxidants, as shown in Fig. 1. While, by scavenging mtROS with MitoTEMPO, we found a significant decrease of nuclear NRF2 signal in both CTRL and TTD fibroblasts exposed to H2O2, as compared to the pro-oxidant stimuli alone (Fig. 4A and B), although the NRF2 levels in TTD cells remained higher than CTRL ones (* P <0.05; CTRL+H2O2+MitoTEMPO vs TTD+H2O2+MitoTEMPO).

Taken together, these results seemed to reveal an altered NRF2–BACH1 interplay in TTD fibroblasts, with an augmented translocation of NRF2 into the nuclear compartment of TTD cells upon pro-oxidant challenge, as a likely consequence of a higher OS, as compared to CTRL cells; whereas presumably, the antioxidant defense system of CTRL fibroblasts were sufficient to cope with the H2O2 acute stimulus, without resorting to NRF2 response.

Discussion

Our findings demonstrate, for the first time, the presence of a redox imbalance, associated with an impaired response to pro-oxidant stimuli, in primary skin fibroblasts obtained from a patient with TTD. In particular, our reported results of a constitutive increase of intracellular oxidants and mtROS levels, together with augmented LPO damage at the plasma membrane and proteins (as shown by BODIPY C11 and 4-HNE-PAs levels) seem to indicate a reduced antioxidant defense in TTD cells and suggest that oxidative events are likely to be persistent. In addition, upon a 1 h-pro-oxidant challenge only TTD cells, and not the CTRL ones, induced the translocation of NRF2 into the nuclear compartment, thus likely needing a further antioxidant response to cope with the stress. In this regard, Cho and colleagues found that RNF113A deficiency in HeLa cells was able to trigger the NRF2 pathway, upon an acute treatment (30 min) with H2O2 (Cho et al. 2024).

Although it is still not clarified whether OS is the cause or the consequence of the phenotypic manifestations typical of TTD, these data add new evidence to the concept of an OS as a key player in both multi-system and genetic pathologies (Perluigi & Butterfield 2012, Cervellati et al. 2015, Ohl et al. 2016, Vona et al. 2021).

We also found that mitochondria are involved in the pro-oxidant milieu of TTD, as shown by the reduced membrane potential and the enhanced levels of mtROS. These latter were reduced to the levels of control cells, by the treatment with MitoTEMPO, a specific scavenger of mitochondrial superoxide anion. This may corroborate the hypothesis of a reduced/malfunctioning antioxidant defense system even at the mitochondrial level. Defective antioxidant response and mitochondrial dysfunctions, known as ‘mitochondriopathies’, are central hallmarks of several neurologic disorders, genetic pathologies, inflammatory and skin problems (Shulyakova et al. 2017, Missiroli et al. 2020, Sreedhar et al. 2020, Liskova et al. 2021).

However, due to the rarity of the pathology, the effects of TTD-related mutations on mitochondrial dysfunctions and OS are poorly studied. Besides the already mentioned paper by Cho and colleagues (Cho et al. 2024), another work linked OS to one of the genes involved in TTD – MARS1 – can be phosphorylated at Ser209 and Ser825 by an extracellular signal-related kinase (ERK1/2) in response to OS, resulting in a reduced specificity for tRNAMet and in more frequent methionine incorporation into newly synthetized proteins, thus increasing cellular reductive capacity (Lee et al. 2014, Sung et al. 2022). Hence, cells harboring MARS1 mutations may be more sensitive to oxidative stress, suggesting that these cells respond to OS by promiscuously charging Met to different tRNAs.

It should be anyway mentioned that this study was performed by isolating cells from only one patient as TTD is a very rare disease and it is very difficult to obtain samples. Therefore, before extrapolating these findings to TTD patients more investigations need to be conducted with the aim to confirm our results.

Declaration of interest

G Valacchi is an editorial board member of Redox Experimental Medicine. He was not involved in the peer review of this manuscript. The authors declare no competing interests.

Funding

This work did not receive any specific grant from any funding agency in the public, commercial, or not-for-profit sector.

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  • Figure 1

    Overall oxidant production and mitochondrial ROS levels in TTD fibroblasts. (A) Levels of total oxidants in control and TTD fibroblasts in basal condition or exposed to hydrogen peroxide challenge (100 µM) for 1h, 6h and 24h, assessed by DCFDA assay. (B) Representative images and (C) analysis of MitoSOX™ Red fluorescence staining in control and TTD fibroblasts, pre-treated with MitoTEMPO (1 µM for 2 h) and then co-incubated or not with hydrogen peroxide (100 µM) for 1 h, 6 h, and 24 h. Nuclei are counterstained with DAPI. Objective 40×. Data were expressed as means ± S.E.M., and are the result of three different cultures of cells from n = 1 control subject and n = 1 TTD patient. *indicates significant differences between CTRL and TTD groups at the same treatment conditions (*P < 0.05; **P < 0.01; ***P < 0.001). #indicates significant differences of H2O2/MitoTEMPO/H2O2+MitoTEMPO-exposed groups versus the corresponding basal conditions (##P < 0.01; ###P < 0.001); +indicates significant differences between CTRL and TTD groups treated with H2O2+MitoTEMPO vs only H2O2 at the same time point (+P < 0.05; +++P < 0.001). Results were analyzed by factorial ANOVA, with post-hoc Tukey’s multiple comparisons test. CTRL, control subject; TTD, trichothiodystrophy.

  • Figure 2

    H2O2 affects mitochondrial membrane potential differently in CTRL and TTD fibroblasts. (A) Representative images and (B) analysis of MitoTracker Orange fluorescence signal in control and TTD fibroblasts exposed to hydrogen peroxide challenge (100 µM) for 1 h, 6 h and 24 h. Nuclei are counterstained with DAPI. Objective 40×. Bar = 100 µm. Data were expressed as means ± s.e.m., and are the result of three different cultures of cells from n = 1 control subject and n = 1 TTD patient. *indicates significant differences between CTRL and TTD groups at the same treatment conditions (*P < 0.05; ***P < 0.001). #indicates significant differences of H2O2-exposed groups versus the corresponding basal conditions (###P < 0.001). $indicates significant differences of H2O2-exposed groups versus the previous time-points ($$$P < 0.001). Results were analyzed by factorial ANOVA (with 2 × 2 × 3 design), with post-hoc Tukey’s multiple comparisons test. CTRL, control subject; TTD, trichothiodystrophy.

  • Figure 3

    Increased Lipid peroxidation and DNA damage levels in TTD fibroblasts. (A)Representative images and (B)analysis of the immunocytochemical assay with the BODIPYTM 581/591 C11 marker. Peroxidized lipids are shown in green on cells treated with or without hydrogen peroxide (100 µM) for 1h, 6h and 24h. (C and E)Representative images and (D and F)analysis of 4-hydroxynonenal-protein adducts (4-HNE-PAs) and p-H2AX fluorescence levels, detected by IF, in control and TTD fibroblasts, treated as reported above. Objective 40×. Bar = 100 µm. Data were expressed as means ± S.E.M., and are the result of three different cultures of cells from n = 1 control subject and n = 1 TTD patient. *indicates significant differences between CTRL and TTD groups at the same treatment conditions (**P < 0.01; ***P < 0.001). #indicates significant differences of H2O2-exposed groups versus the corresponding basal conditions (#P < 0.05; ##P < 0.01; ###P < 0.001). $indicates significant differences of H2O2-exposed groups versus the previous time-points ($P < 0.05; $$P < 0.01; $$$P < 0.001). Results were analyzed by factorial ANOVA (with 2 × 2 × 3 design), with post-hoc Tukey’s multiple comparisons test. CTRL, control subject; TTD, trichothiodystrophy.

  • Figure 4

    Impaired NRF2–BACH1 interplay in TTD fibroblasts. (A)Representative images and (B and C) analysis of nuclear NRF2 and BACH1 levels, by immunofluorescence, in control and TTD fibroblasts, pre-treated with MitoTEMPO (1 µM for 2 h) and then co-incubated or not with hydrogen peroxide (100 µM) for 1 h. Nuclei are counterstained with DAPI. Objective 40×. Data were expressed as means ± s.e.m., and are the result of three different cultures of cells from n = 1 control subject and n = 1 TTD patient. *indicates significant differences between CTRL and TTD groups at the same treatment conditions (*P < 0.05; ***P < 0.001). #indicates significant differences of H2O2/MitoTEMPO/H2O2+MitoTEMPO-exposed groups versus the corresponding basal conditions (#P < 0.05; ###P < 0.001); +indicates significant differences between CTRL and TTD groups treated with H2O2+MitoTEMPO vs only H2O2 (+P < 0.05; ++P < 0.01). Results were analyzed by factorial ANOVA, with post-hoc Tukey’s multiple comparisons test. CTRL, control subject; TTD, trichothiodystrophy.

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