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. Author manuscript; available in PMC: 2021 Sep 1.
Published in final edited form as: Mech Ageing Dev. 2020 Jul 22;190:111318. doi: 10.1016/j.mad.2020.111318

tBHP treatment as a model for cellular senescence and pollution-induced skin aging

Sophia Wedel 1,*, Ines Martic 1, Nina Hrapovic 2, Susanne Fabre 2, Corina T Madreiter-Sokolowski 3, Thomas Haller 4, Gerhard Pierer 5, Christian Ploner 5, Pidder Jansen-Dürr 1, Maria Cavinato 1
PMCID: PMC7610711  EMSID: EMS123020  PMID: 32710895

Abstract

Accumulation of senescent cells promotes the development of age-related pathologies and deterioration. In human skin, senescent cells potentially impair structure and function by secreting a mixture of signaling molecules and proteases that influence neighboring cells and degrade extracellular matrix components, such as elastin and collagen. One of the key underlying mechanisms of senescence and extrinsic skin aging is the increase of intracellular reactive oxygen species and resulting oxidative stress. Tert-butyl hydroperoxide (tBHP) is a known inducer of oxidative stress and cellular damage, acting at least in part by depleting the antioxidant glutathione. Here, we provide a detailed characterization of tBHP-induced senescence in human dermal fibroblasts in monolayer culture. In addition, results obtained with more physiological experimental models revealed that tBHP treated 3D reconstructed skin and ex vivo skin developed signs of chronic tissue damage, displaying reduced epidermal thickness and collagen fiber thinning. We, therefore, propose that tBHP treatment can be used as a model to study the effects of extrinsic skin aging, focusing mainly on the influence of environmental pollution.

Keywords: oxidative stress, cigarette smoke, epidermal thinning, collagen degradation

1. Introduction

Cellular senescence describes a distinct phenotype of cell cycle arrest that is coupled with morphological changes, senescence-associated β-galactosidase (SA-β-galactosidase) activity and the secretion of a mixture of several cytokines, chemokines, proteases, and growth factors, which are collectively known as the senescence-associated secretory phenotype (SASP) (Muñoz-Espín and Serrano, 2014). Acute and short-term appearance of senescent cells has been associated with tumor suppression, wound healing and tissue development, whereas longterm presence of senescent cells has been described as detrimental and a driver of age-related pathologies and deterioration (Baker et al., 2016; van Deursen, 2014). Cellular senescence can be induced by a high number of internal and external factors that have the potential to damage cells with such severity that they are not able to recover and, consequently, cannot reverse the cell cycle arrest (Höhn et al., 2017).

Senescent cells occur in human skin (Ressler et al., 2006) and their presence potentially impairs tissue function and youthful appearance by supporting the formation of visible signs of skin aging, such as wrinkle formation and skin laxity (Demaria et al., 2015). Skin aging is an intricate process, orchestrated by a number of intrinsic and extrinsic factors that potentially induce changes in epidermal and dermal structure (Wang and Dreesen, 2018). Skin aging manifests in the epidermis by thinning of the layer and by an increasing number of pigmentation irregularities. Aged dermis is characterized by a breakdown of extracellular matrix components, such as collagen and elastic fibers (Kim and Park, 2016). Environmental stressors, such as ultravioletradiation, tobacco smoke and air pollution are of main interest in the field of skin aging research, as skin cells are usually exposed to these stressors (Krutmann et al., 2016). The key mechanism by which environmental factors damage the skin is the intracellular accumulation of reactive oxygen species (ROS) and subsequent induction of oxidative stress (Kim and Park, 2016). In the context of skin aging, ROS are known to directly damage DNA, proteins and lipids, to trigger the downregulation of collagen production and to induce the expression of matrix metalloproteases (MMPs), which are enzymes specifically targeting and decomposing collagen and elastin fibers (Cavinato and Jansen-Dürr, 2017).

Tert-butyl hydroperoxide (tBHP) is a known inducer of oxidative stress and has previously been used to induce cellular senescence in different types of cells (Dumont et al., 2000; Kučera et al., 2014; Unterluggauer et al., 2003). The chemical is known to deplete cellular antioxidant defense mechanisms (Crane et al., 1983; Zavodnik et al., 1998), to produce radicals that initiate lipid peroxidation (Davies, 1989) and to reduce mitochondrial membrane potential (ΔΨm) in neuronal cells (Wang et al., 2019). Notably, the plastic industry and several other industries use tBHP for chemical processes, such as polymerizing, deodorizing and bleaching. Direct cutaneous contact and inhalation of vapors are risk factors for workers, but normally do not concern the general public (National Center for Biotechnology Information. PubChem Database. tert-Butyl hydroperoxide, Source=HSDB, https://pubchem.ncbi.nlm.nih.gov/source/hsdb/837#section=Toxicity-Summary (accessed on Mar. 9, 2020)). Due to the chemical’s oxidative stress-inducing properties and the fact that it is used in industrial processes and potentially pollutes working environments, we assume that tBHP treatment of skin cells and tissue can be used as a model to study the effects of environmental pollutants.

Here, we present and characterize a model of tBHP-induced senescence in human dermal fibroblasts. Additionally, we examine the effects of tBHP treatment on 3D reconstructed skin equivalents and ex vivo skin.

2. Material and Methods

2.1. Cell Culture

Human diploid fibroblasts derived from newborn foreskin (HFF-1, ATCC® #SCRC-1041, Manassas, Virginia, USA), HSEK (Human Skin Epidermal Keratinocytes) and HSDF (Human Skin Dermal Fibroblasts) were cultivated at 37 °C and 5% CO2. HFFs from passage 2 - 15 were used for subsequent experiments. The HSEK and HSDF were isolated from abdominal skin derived from plastic surgery. The skin was donated by patients from the Universitätsklinik für Plastische, Rekonstruktive und Ästhetische Chirurgie, Innsbruck, after informed consent according to current guidelines of the Ethikkommission der Medizinischen Universität Innsbruck. The HSEK were cultivated in Dermalife K Medium (LL-000, Lifeline Cell Technology, USA). Fibroblasts were cultivated in Dulbecco’s Modified Eagle’s Medium (DMEM D5546, Sigma, Steinheim Germany) with 10% fetal bovine serum, 4 mM L-Glutamine and 1% Penicillin-Streptomycin.

2.2. Induction of Stress-induced Premature Senescence using tBHP

Cells were treated with 50 μM tBHP by pipetting the respective volume of tBHP (diluted in DMEM) directly into the media. After 1 h, the cells were washed two times with Dulbecco’s Phosphate Buffered Saline (DPBS) and then incubated in DMEM for at least 4 h for recovery. The cells were treated twice a day for four days (D1 – D4) and were kept in culture for at least 9 days.

2.3. Counting Cells and Estimation of Cumulative Population Doublings (cPDL)

To determine the number of cells, a CASY® Cell counter & Analyzer System (OMNI Life Science, Bremen, Germany) was used. For this purpose, cells were washed in DPBS, detached in trypsin-ethylenediaminetetraacetic acid (EDTA) 0.05% solution and counted. The cPDL were calculated as described by Hutter et al. (2004).

2.4. RNA Isolation, cDNA Synthesis and RT-qPCR

RNA was isolated using the RNeasy® Mini Kit by Qiagen according to the manufacturer’s protocol. The RNA concentration was measured with a Nanodrop 2000 (Thermo Scientific) system. cDNA synthesis was performed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems by Thermo Fisher Scientific, Vienna, Austria) according to the manufacturer’s protocol. Following primer pairs were tested in RT-qPCR: p21 (forward, GGG ACA GCA GAG GAA GAC C; reverse, GGC GTT TGG AGT GGT AGA AA), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, housekeeper) (forward, GAG TCA ACG GAT TTG GTC GT; reverse, GAT CTC GCT CCT GGA AGA TG), IL1α (forward, TCA GCA AAG AAG TCA AGA TGG C; reverse, CAT GGA GTG GGC CAT AGC TT), IL6 (forward, AAG CCA GAGC TGT GCA GAT GAG TA; reverse, TGT CCT GCA GCC ACT GGT TC), IL8 (forward, ACC GGA AGG AAC CAT CTC AC; reverse, AAA CTG CAC CTT CAC ACA GAG), MMP1 (CAT CGT GTT GCA GCT CAT GA; reverse, ATG GGC TGG ACA GGA TTT TG); SERPINB2 (forward, TCT CAG AGG AGC ATT GCC CG; reverse, GTG CAA GAA ATG CTG GTT GTT C), TNFRSF10C (forward, CTG CTG CCA GTC CTA GCT TAC; reverse, GGA CAC TCC TCC CCC TTG AA), TNFRSF10D (forward, CGT GTA GAA CAG GGT GTC CC; reverse TGG GGT TTT CCC AGT GGA AC), SERPINE2 (forward, TCA AGG GTC TGT GGA AAT CA; reverse, CGT GGT AGG GCA GTT CAA TG),COL5A3 (forward, AGG TGA TCA GGG GAA ACC CG; reverse, GGG GAC CGG GAA ATC CAA TAG), ACAN (forward, GAC TTC CGC TGG TCA GAT GG; reverse, CGT TTG TAG GTG GTG GCT GTG), PTX3 (forward, TGC CGG CAG GTT GTG AAA C; reverse, GCC TCA TTG GTC TCA CTG GA), PAPPA (forward, ATC ACA GGG CTG TAT GAC AAA TG; reverse, TTG ATG GTG GTC ACT TGC CG), FAM46C (forward, CTT CTA TTG CCC AGT TTC CCC; reverse, CCT GAT CCC AGT TGA GCA CG), SCD (forward, CCC GAC GTG GCT TTT TCT TC; reverse, GCC AGG TTT GTA GTA CCT CCT C), COL1A1 (forward, CAG GCA AAC CTG GTG AAC A; reverse, CTC GCC AGG GAA ACC TCT). The RT-qPCR was carried out by a LightCycler® 480II (Roche, Vienna, Austria).

2.5. Protein Isolation

For protein isolation a modified radioimmunoprecipitation assay (RIPA) buffer was prepared as follows: 50 mM Tris-HCL (pH 7.4), 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, 150 mM NaCl, 2 mM EDTA, 50 mM NaF, 2 μg/ml Aprotinin, 1 mM PMSF and 1mM activated Na-Orthovanadate. The cells were washed three times with DPBS and modified RIPA buffer was added to the dish. Working on ice, the cells were scraped off with a Costar® Cell Lifter 3008 and the resulting cell solution was transferred into a collection tube. Afterwards, the cells were frozen in liquid N2 and thawed repeatedly. After centrifugation at 9500 g for 10 min at 4 °C, the supernatant was transferred into a new tube. The protein concentration was measured by Pierce® BCA Protein Assay Kit according to the manufacturer’s protocol.

2.6. Westernblot

Equal protein amounts, were loaded onto a SDS polyacrylamid-gel, separated by discontinuous electrophoresis in 1x SDS buffer and transferred onto a PVDF membrane by western blotting using a standard protocol (Greussing et al., 2013). The proteins were detected with Millipore Immobilon™ Western Chemiluminescent HRP Substrate and exposed to X-Ray films (FujiFilm Super RX, FujiFilm). Quantification of protein levels was done using ImageJ software and band intensity was normalized to the loading control GAPDH. The following primary antibodies were used: pRB (#9308S Cell Signaling), p53 (#SC-126 (DO-1), Santa Cruz), p21 waf1/cip1 (#2947S Cell Signaling), p-p53 (Serin15) (#92845S Cell Signaling), GAPDH (#SC-25778 Santa Cruz);

2.7. Cytochemistry for SA-β-galactosidase

To evaluate the activity of SA-β-galactosidase, the cells were stained as described (Greussing et al., 2013). The percentage of senescent cells was calculated by dividing the number of blue positive cells by the total cell number in a given area. At least 400 cells were counted per group.

2.8. Determination of Apoptotic and Necrotic Cell Death by flow cytometry Using AnnexinV/PI Staining

Cells were stained with AnnexinV/PI for detection of apoptotic and necrotic cells according to the manufacturer’s protocol (FITC Annexin V Apoptosis Detection Kit I, BD Pharmingen™, Vienna, Austria). The probes were measured using the BD FACS Canto II flow cytometer. Per group, the measurements were performed in triplicates. The percentage of apoptotic cells is presented as the sum of necrotic (Annexin V negative/PI positive), early (Annexin V positive/PI negative) and late stage (Annexin V positive/PI positive) apoptotic cells.

2.9. Measurement of Cell Surface Area

Cell surface area was estimated on using ImageJ Software of at least 80 cells per group.

2.10. γH2AX Immunofluorescence

HFF-1 were seeded on top of glass cover slips for immunofluorescence experiments and fixed in 4% paraformaldehyde. The cells were permeabilized in phosphate buffered saline (PBS) containing 0.1% sodium citrate and 0.3% Triton-X and blocking was performed in 1% bovine serum albumin in PBS. The cells were incubated in γH2AX antibody (#2577S, Cell Signaling) overnight at 4 °C. Alexa Fluor anti-rabbit 488 was used as a secondary antibody. The nuclei were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI) and the cells were analyzed using Cell Voyager CV1000 Yokogawa (Visitron Systems). Quantification of nuclear γH2AX fluorescence intensity was performed using ImageJ software.

2.11. Determination of Intracellular ROS Levels by DHE (Dihydroethidium) Staining

To assess intracellular ROS levels, the cells were stained with DHE (Molecular Probes, Vienna, Austria) and measured with a BD FACS Canto II flow cytometer as described (Cavinato et al., 2016).

2.12. Determination of Mitochondrial ROS Levels by CM-H2XRos Staining

For determination of mitochondrial ROS levels, the cells were trypsinized and stained with 100 nM CM-H2XRos (Molecular Probes, Vienna, Austria) for 30 min at 37 °C. The cells were washed with PBS and fluorescence was measured with a BD FACS Canto II flow cytometer. Treatment with 0.5 μM rotenone was used as a positive control.

2.13. Determination of Mitochondrial Membrane Potential by JC-1 Staining

Mitochondrial membrane potential was analyzed using the fluorescent probe JC-1. The cells were trypsinized, stained using 0.5 μg/ml JC-1 solution for 30 min at 37 °C and washed. Subsequently, fluorescence was measured using a BD FACS Canto II flow cytometer. For a positive control, cells were incubated with 5 μM Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP).

2.14. Relative mitochondrial DNA (mtDNA) / nuclear DNA (nDNA) ratio

Genomic DNA was isolated from three independent replicates per group using the PureLink™ Genomic DNA Mini Kit (Invitrogen). DNA concentration was assessed using a Nanodrop 2000 (Thermo Scientific) system. 100ng DNA were used in each qPCR reaction containing either a set of primers binding mtDNA (MT-ND4 [forward, ACT CTC ACT GCC CAA GAA CT; reverse, GTG TGA GGC GTA TTA TAC CA] or MT-COX1 [forward, TAC GTT GTA GCC CAC TTC CAC T; reverse, AGT AAC GTC GGG GCA TTC CG]) or nDNA (GAPDH; B2M [forward, GAA TTC ACC CCC ACT GAA AA; reverse, CTC CAT GAT GCT GCT TAC A]). All four primer pairs were used in reactions containing DNA from every sample. The qPCR was carried out by a LightCycler® 480II (Roche, Vienna, Austria). Relative mitochondrial copy number from either of the two mitochondrial genes was calculated with either of the two nuclear genes using the ΔΔCt method, resulting in four values for each sample. Presented relative mtDNA/nDNA ratios were given as mean ± SD derived from the resulting four values.

2.15. RNAseq Analysis

Total RNA was isolated and purified using the RNeasy® Mini Kit (Qiagen) and the RNeasy® MinElute Kit (Qiagen). Samples were sent to Eurofins Genomics (Konstanz, Germany) for further processing of RNA sequencing (RNAseq) by Inview Explore. To analyze the provided data from Eurofins, the list of differentially expressed genes, using a cutoff at 1.5 (log2) fold change, was uploaded for a functional annotation analysis by DAVID bioinformatics resources 6.8 (Huang et al., 2009a, 2009b). In total, 63 entries of diseases were displayed with p-value < 0.01. From these 63 entries double entries and entries with very high similarity were excluded. Additionally, we decided to exclude entries that were not relevant for skin, aging or tobacco use. To specifically look for aging related genes, a gene list was extracted from the genetic association database correlated with aging (GAD_Disease ‘Aging’), which was counterchecked with the list of differentially expressed genes from the RNAseq. To highlight the regulated genes with a cutoff of 2 (log2) fold change, a heatmap was created using Morpheus Versatile matrix visualization and analysis software, https://software.broadinstitute.org/morpheus.

2.16. Production of 3D Reconstructed Skin (Skin Equivalents)

For the production of 3D skin equivalents HSDF fibroblasts were used. The production of 3D skin equivalents was performed as described (Cavinato et al., 2016).

2.17. Preparation of Skin Biopsies

Skin tissue from breast and abdominal areas was received from Strandkliniken, Stockholm, Sweden, after plastic surgery with the patients’ consent according to the current guidelines of the ethics committee Regionala Etikprövningsnämnden, Stockholm, Sweden. Biopsies were taken with 8 mm biopsy punches, avoiding sites with visible interferences, such as stretch marks, redness, accumulation of hair follicles and hypo- or hyper-pigmented spots etc. Subcutaneous fat was removed as well as blood vessels, and the biopsies were submerged in RPMI 1640 W/GLUTAMAX-I (#11554516, Fisher) overnight. The following day, the biopsies were submitted to air-liquid interface, submerging dermis in medium, whereas epidermis was in contact with air. From each patient, six biopsies were taken - three of them remained untreated, while three were treated with tBHP.

2.18. Treatment of 3D Skin Equivalents and Skin Biopsies with tBHP

Skin biopsies and reconstructed skin were treated with 40 μM tBHP for 1 h by adding tBHP into the dermis-surrounding medium. The skin biopsies and the reconstructed tissues were incubated for 1 h at 37°C until washed twice with DPBS and recovered in RPMI medium and keratinocyte growth medium, respectively, for at least 4 h. The tissue was treated twice a day for three consecutive days. Two days of recovery followed until the samples were taken and processed for histology and RNA isolation.

2.19. Processing for Histology of 3D Reconstructed Skin and Skin Biopsies

The reconstructed and ex vivo skin tissue were fixed in 4% paraformaldehyde solution and processed for embedding in paraffin blocks. The blocks were cut in 5 μm sections and stained with hematoxylin and eosin (H&E). Skin biopsy cuts were also stained for Fontana Masson using the Sigma-Aldrich Fontana Masson stain kit, Masson’s Trichrome staining, and Picrosirius Red staining for collagen fibers. Pictures were acquired with a LEICA DMLS Microscope (Leica Mikroskopie und Systeme, Wetzlar). Polarization images were recorded with a true color RGB camera (Spot insight, Diagnostic Instruments Inc., USA) on a Zeiss inverted microscope (Axiovert 35) equipped with a Zeiss LD Achroplan 40 × long distance objective, NA 0.6. Crossed polarizers were placed between the condenser (0.55 NA; DIC 0.3-0.4 position) and the rear aperture of the objective. Polychromatic white light (3200 K; 100 W halogen lamp) was used throughout.

2.20. Measurement of Epidermal Thickness

Epidermal thickness was estimated using ImageJ software in 50 spots across one biopsy. Biopsies were taken in triplicates, resulting in 150 measurements per sample. In reconstructed skin, 40 measurements per sample were performed.

2.21. RNA Isolation, cDNA synthesis and RT-qPCR from Skin Biopsies

Skin biopsies were transferred to FastPrep Lysing Matrix D tubes (MP Biomedicals), containing RLT Buffer (RNeasy® Mini Kit (Qiagen)) + β-Mercaptoethanol. To lyse the tissue, the biopsies were shook in a TissueRuptor FastPrep-24 Classic Instrument (MP Biomedicals). The homogenate was transferred from the tubes to RNeasy® Mini Kit (Qiagen) columns and RNA isolation was continued according to the manufacturer’s protocol. cDNA was synthesized using iScript advanced cDNA synthesis kit for RT-qPCR (#170-8843, Biorad) following the supplier’s instructions. RT-qPCR was performed using a CFX96TM Optics Module (Bio-Rad) with the following primers: GAPDH (housekeeper) (# qHsaCED0038674, Biorad), COL1A1 (#qHsaCED0043248, Biorad).

2.22. Collagen Fiber Density Measurement

Collagen fiber density in Masson’s trichrome stained sections was measured as described using picture analysis ImageJ software (Hong et al., 2020). For papillary dermis, per patient 6 pictures were analyzed. For reticular dermis, 9 pictures per patient were analyzed. Picrosirius Red staining was analyzed using Adobe Photoshop color histograms. The ratio between red and green pixels was calculated. Per patient and per dermal layer, 6 pictures were analyzed. Biopsies from two different patients were used.

2.23. Statistical analysis

If not differently stated, results are displayed as mean values ± standard deviation (SD) of n = 3. Statistical analysis was done using Student’s t-test.

3. Results

3.1. tBHP treatment induces stress-induced premature senescence in skin fibroblasts

Human skin fibroblasts were exposed to tBHP twice a day for four consecutive days (D1 - D4) and kept in culture until D14 after seeding (Figure 1A). Untreated skin fibroblasts served as the control. Various tBHP concentrations were tested and cell proliferation was monitored by counting the cells and calculating cumulative population doublings (cPDL; Supplementary Figure 1A). We determined the concentration of 50μM tBHP to be most suitable, as serial exposure to such stresses led to a pronounced decrease in cell divisions (Figure 1B, Supplementary Figure 1B), which was accompanied by an increase in cyclin-dependent-kinase inhibitor p21 mRNA and protein levels on D4 and D9 (Figure 1C and 1D). Treatment with tBHP activated the p53 signaling pathway by significantly enhancing p53 phosphorylation at serin15 on D4 (Figure 1D). The activation of p53 signaling supported cell cycle arrest in combination with decreased pRB phosphorylation on D4 and D9 (Figure 1D). SA-β-galactosidase staining resulted in ~90% of SA-β-galactosidase positive cells on D9 after tBHP treatment (Figure 1E, Supplementary Figure 1C), while the percentage of apoptotic cells was slightly increased compared to control cells (Supplementary Figure 1D). Senescence-associated morphological changes, such as cellenlargement and loss of spindle like shape, were seen on D9 in tBHP treated cells (Figure 1F). Cell surface measurement showed that tBHP treatment led to a three to four-fold increase of cell size compared to control cells (Figure 1F).

Figure 1. tBHP treatment induces stress-induced premature senescence in HFF-1.

Figure 1

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A) Schematic overview of experimental setup and treatment protocol. B) HFF-1 cells were treated with 50μM tBHP and cumulative population doublings (cPDL) were calculated. Data represents mean values ± SD, n=3; C) RT-qPCR was performed to check for p21 mRNA levels; Data represents mean values ± SD, n=3; D) Protein expression of phosphorylated pRB, p53, p-p53 (Serin15) and p21 was measured by Westernblot on D4 and D9 of the experiment. Quantification of protein expression was performed using ImageJ software; Data represents mean values ± SD, n=3; E) tBHP treated and control cells were stained for SA-β-galactosidase activity on D9 of the experiment; Representative pictures of stained cells are shown. Data represents mean values ± SD, n=3; F) Cell surface measurement was performed using ImageJ software. Pictures were taken on D9 of the experiment, representative pictures are shown, Data represents mean values ± SEM, n=80 (randomly chosen from 3 independent samples); Statistical analysis was calculated using t-test (*P < 0.05, **P < 0.01, ***P < 0.001);

3.2. Oxidative stress-induced DNA and mitochondrial damage are underlying factors of tBHP-induced senescence

To assess whether DNA damage was a possible cause of p53 signaling pathway activation and cell cycle arrest, we measured DNA damage accumulation by immunofluorescence staining of the histone variant γH2AX. An increase in γH2AX foci accumulation in the nuclei of tBHP treated cells was clearly visible (Figure 2A, Supplementary Figure 2). γH2AX positive spots accumulated already after two tBHP treatments (D1). The number of spots and fluorescence intensity peaked on D4 and decreased slightly until D7. tBHP treated samples showed increased fluorescence intensity compared to control samples at all measurement points. Intracellular ROS levels, as measured by the fluorescent probe DHE, were also significantly induced by tBHP treatment (Figure 2B and 2C). Similar to the γH2AX-associated DNA damage pattern, DHE mean fluorescence was changed in a transient manner. tBHP treatment induced mitochondrial ROS formation (i.e., increased staining of mitochondria with the redox-sensitive probe CM-H2XRos) on D4, D7 and D9, but not on D1 (Figure 2C and 2D). Mitochondrial ROS levels stayed constantly elevated after tBHP treatment and were accompanied by a drastic loss of mitochondrial membrane potential (ΔΨm), as semi quantitatively reflected by JC-1 fluorescence measurement (Figure 2E). In the presence of high ΔΨm, JC-1 aggregates are formed, emitting red fluorescence, whereas in the presence of low ΔΨm, monomeric JC-1 emits green fluorescence (Sivandzade et al., 2019). Substantial changes in JC-1 fluorescence composition were measurable on D4, D7 and D9 after tBHP treatment, indicating severe mitochondrial damage. Furthermore, tBHP treatment led to an increase in relative mtDNA/nDNA ratio at all measurement points (Figure 2F). On D4, D7 and D9 the mtDNA/nDNA ratio doubled the ratio of control cells (Figure 2F), suggesting an increase in mitochondrial content with senescence induction.

Figure 2. Oxidative stress-induced DNA and mitochondrial damage are underlying factors of tBHP-induced senescence.

Figure 2

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A) γH2AX was stained using Immunofluorescence; nuclei were counterstained using DAPI; Representative pictures are shown; Green nuclear fluorescence intensity was measured using ImageJ software, scale bar = 20 μm; Data represents mean values ± SD, n = 90 (randomly chosen from 3 independent samples); B) DHE staining was performed to measure intracellular ROS levels using flow cytometry; Data represents mean values ± SD, n = 3; C) Representative histogram of DHE fluorescence flow cytometry measurements on D9; D) CM-H2XRos staining was performed to measure mitochondrial ROS using flow cytometry; Data represents mean values ± SD, n = 3; E) JC-1 staining was performed and used as a proxy to measure mitochondrial membrane potential using flow cytometry. Data represents mean values ± SD, n=3; F) mtDNA/nDNA ratio was measured by qPCR; Data represents mean values ± SD, n=4; Statistical analysis was calculated using t-test (*P < 0.05, **P < 0.01, ***P < 0.001)

3.3. tBHP-induced senescence is connected to tobacco smoke induced pathologies and aging

To further investigate the changes caused by tBHP treatment in human skin fibroblasts, RNAseq datasets were generated from samples of control and tBHP treated HFF-1 on D9. Differentially expressed genes were sorted for log2 fold change and the top upregulated and downregulated genes (cutoff 1.5) were submitted to a functional annotation analysis using DAVID. ‘Tobacco use disorder’ was the top entry when looking for gene association database diseases (GAD_Disease, Figure 3A). Other smoking related pathologies, like mouth neoplasms, blood pressure, cholesterol (LDL), cardiovascular disease, type 2 diabetes and chronic obstructive pulmonary disease were found as well, implying similarities of gene expression patterns between tBHP treated cells and tobacco smoke exposure. Because SASP is understood as the main component by which senescent cells influence aging and the aging phenotype, we used the DAVID functional annotation tool to search for aging and SASP related KEGG pathways influenced by tBHP treatment. DAVID provided a list that consisted of KEGG pathways involving signal transduction of secreted proteins, indicating that SASP plays an important role in tBHP-induced senescence (Figure 3B). To further test the regulation of SASP genes, we counterchecked the list of differentially expressed genes (cutoff log2 fold change 2) with the aging-associated genes provided by GAD_Disease ‘aging’ (Figure 3C). Many well-described SASP genes, like MMP1, MMP3, SERPINB2, IL1A, EGF and IL8 were found to be upregulated in the tBHP-induced senescence system. To validate the RNAseq datasets, RT-qPCR was performed for MMP1, MMP3, IL1A, IL6, IL8, SERPINB2 (Figure 3D) and others (Supplementary Figure 3). To reveal more about their expression pattern over time, we measured mRNA levels on D1, D4, D7 and D9. All except for MMP3 showed an immediate response to tBHP and were induced already on D1. Interestingly, IL6 expression was induced on D1 and then downregulated on the following days.

Figure 3. tBHP-induced senescence is connected to tobacco smoke induced pathologies and aging.

Figure 3

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A) DAVID functional annotation analysis was performed using RNAseq datasets. GAD_DISEASE results are displayed according to –log10 (p-value); B) Results for KEGG pathways are displayed according to –log10 (p-value); C) Differentially expressed genes (cut-off log2 fold change ±2) were counterchecked with GAD_DISEASE_Aging genes and reoccurring genes are displayed in a heatmap Color code: red – upregulated; blue – downregulated; D) Gene expression of RNAseq was validated by RT-qPCR for several SASP genes. Data represents mean values ± SD, n = 3; Statistical analysis was calculated using t-test (*P < 0.05, **P < 0.01, ***P < 0.001)

3.4. tBHP treatment of 3D reconstructed skin equivalents reduces epidermal thickness and increases epidermal thickness variance in ex vivo skin

To investigate whether tBHP treatment would affect skin in a more complex context than a fibroblast monolayer, we produced 3D skin equivalents and added tBHP to the media that submerged the dermis, but did not touch the epidermis. 3D skin equivalents that were not exposed to tBHP were used as controls. One characteristic of aged and damaged skin is loss of epidermal thickness. Therefore, we measured epidermal thickness of the reconstructed skin epidermal layer from the basal layer to the top of the stratum granulosum. Epidermal thickness of the tBHP treated skin equivalents was decreased, as could be demonstrated by H&E stained sections (Figure 4A). Image analysis confirmed that the epidermal height of tBHP treated skin equivalents decreased to almost half compared to control skin equivalents (Figure 4B). Skin equivalents are a well-established model to study effects of skin treatments, but do not reach the complexity level of skin tissue. We, therefore, took biopsies from skin tissue that derived from plastic surgery patients. Tissues from the breast area as well as abdominal sites were obtained. Epidermal areas were, in contrast to dermal areas, not submerged in media and did not have direct contact with the chemical, neither did control biopsies. When measuring epidermal thickness in the skin biopsies, only a slight decrease could be detected (Figure 4C and 4D), but tBHP treatment increased the variance of measurements within a sample (Figure 4D and 4E). Whereas the epidermis of control biopsies showed an overall consistent thickness, tBHP treated biopsies were marked by very thin sites that neighbored much thicker parts.

Figure 4. tBHP treatment of 3D reconstructed skin equivalents reduces epidermal thickness and increases epidermal thickness variance in ex vivo skin.

Figure 4

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A) H&E staining was performed for 3D skin equivalents paraffin embedded sections. Dashed yellow lines indicate epidermal-dermal junction. Representative pictures are shown; B) Epidermal thickness measured in 3D skin equivalents using ImageJ software at 40 sites per group; Data represents mean values ± SD, n = 3; C) H&E staining was performed for ex vivo skin. Representative pictures are shown; D) Epidermal thickness was measured in ex vivo skin using ImageJ software at 150 sites in each group; Data represents mean values ± SD, n = 2; E) Variance of epidermal thickness measurements is displayed; Data represents mean values ± SD, n = 2. Statistical analysis was calculated using t-test (*P < 0.05, **P < 0.01, ***P < 0.001)

3.5. tBHP treatment of ex vivo skin induces collagen-associated changes in the dermis, including fiber thinning

Because disorders in skin pigmentation are well-known characteristics of aged skin, we aimed to understand whether tBHP treatment of skin biopsies influenced melanin content. The biopsies were stained using Fontana Masson stain and melanin content was analyzed. However, the data remained inconclusive (data not shown). Interestingly, we were able to detect darker staining in the dermis of tBHP treated biopsies (Figure 5A). These differences in collagen fiber color between tBHP treated and control biopsies could be seen in breast and abdominal tissue to the same extent. Decreased synthesis of collagen is another feature of aging skin. To investigate collagen production, we performed RT-qPCR for COL1A1 mRNA levels with HFF-1 monolayer samples in the tBHP-induced senescence model (Figure 5B). COL1A1 was clearly downregulated at all four measurement points examined. This indicated that two tBHP stresses were sufficient to decrease COL1A1 mRNA levels in cells. Additionally, we isolated RNA from the biopsies that were treated with tBHP and controls, and observed that COL1A1 mRNA levels were decreased in the skin tissue as well (Figure 5C). To further investigate dermal composition, skin sections were stained with Masson’s trichrome (Figure 5D, Supplementary Figure 4). This technique stains collagen fibers blue, nuclei black and muscle and cytoplasm red. In tBHP treated samples the blue color was generally lighter and more red structures could be seen, especially in the reticular dermis (Figure 5D, Supplementary Figure 4). Collagen fibers were packed densely in control samples, whereas in tBHP treated samples, we measured less compact structures (Figure 5E and 5F). These results were valid for papillary dermis as well as for reticular dermis (Figure 5E and 5F, respectively). As the Masson’s trichrome staining results suggested differences of collagen fiber thickness between treated and untreated samples, Picrosirius red staining was used to confirm these findings. Picrosirius red stains collagen fibers to appear red under normal light microscopy. Under polarized light microscopy, it highlights the birefringence of collagen fibers, making the thicker fibers appear red and the thinner fibers green. We detected significantly more green structures in the tBHP treated samples compared to control (Figure 5G). Image analysis affirmed clear differences between tBHP treated and control samples. The red/green ratio was decreased in the tBHP treated group (Figure 5H and 5I). This also applied for both parts of the dermis, papillary and reticular dermis (Figure 5H and 5I, respectively).

Figure 5. tBHP treatment of ex vivo skin leads to collagen-associated changes in the dermis.

Figure 5

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A) Fontana Masson staining was performed on ex vivo skin sections. Arrowheads indicate stained collagen fibers; Dashed yellow line indicates epidermal-dermal junction; Representative pictures are shown; B) HFF-1 were treated with 50 μM tBHP and RT-qPCR measurements were performed for COL1A1 mRNA expression. Data represents mean values ± SD, n = 3; C) RNA was isolated from ex vivo skin tissue and COL1A1 mRNA expression was measured using RT-qPCR. Data represents mean values ± SD, n = 3; D) Ex vivo skin sections were stained with Masson’s trichrome. Representative pictures of collagen fibers of reticular dermis are shown; E) Collagen fiber density was measured in the papillary dermis; Data represents mean values ± SD, n = 2; F) Collagen fiber density was measured in the reticular dermis; Data represents mean values ± SD, n = 2; G) Picrosirius red staining was performed on ex vivo skin sections, pictures of same areas were taken in brightfield and under polarized light. Dashed yellow line indicates epidermal-dermal junction. Representative pictures are shown; H) Measurement of red to green ratio using color histograms for Picrosirius red polarized light pictures of papillary dermis. Data represents mean values ± SD, n = 2; I) Measurement of red to green ratio using color histogram for picrosirius red polarized light pictures of reticular dermis; Data represents mean values ± SD, n = 2. Statistical analysis was calculated using t-test (*P < 0.05, **P < 0.01, ***P < 0.001)

4. Discussion

Tert-butyl hydroperoxide, an organic peroxide, is a known inducer of oxidative stress and has been used to induce senescence in different cell types (Dumont et al., 2000; Kučera et al., 2014; Unterluggauer, 2003). tBHP is widely used in a variety of industrial processes and workers are possibly exposed to this chemical, while its potential dermal toxicity is not completely understood. In the current study, we have demonstrated that tBHP treatment induced aging-associated changes, including cellular senescence, epidermal thinning and collagen fiber thinning, in skin fibroblasts, 3D reconstructed skin and ex vivo skin, respectively. Aged skin tissue is characterized by an accumulation of senescent cells that eventually impairs tissue health and composition (Demaria et al., 2015). DNA damage and consequent activation of the p53 signaling pathway are major triggers of cellular senescence (López-Otín et al., 2013). Although, p53 signaling is one of the key mechanisms to initiate cell cycle inhibition and early-state senescence, the SASP and mitochondrial dysfunction are important for full senescence development and maintaining cell-cycle arrest (van Deursen, 2014; von Zglinicki et al., 2020). We showed that DNA and mitochondrial damage were underlying mechanisms of tBHP-induced senescence and that DNA damage accumulated during the acute stress phase (D1-D4) but decreased afterwards (D7). This transient change was not visible in mitochondrial ROS and membrane potential measurements, indicating that DNA damage could be, at least in part, repaired in tBHP treated fibroblasts, but mitochondrial damage burden could not be reduced. As previously reported, elevated mitochondrial ROS production and loss of membrane potential often are accompanied with increased mitochondrial mass in senescent cells (Korolchuk et al., 2017). In accordance, we observed that tBHP treated cells depicted clear accumulation of mtDNA with senescence induction compared to proliferating control cells. Increased mitochondrial biogenesis as well as decreased clearance efficiency of damaged mitochondria by mitophagy could be underlying mechanisms of such increased mtDNA/nDNA ratio, but remain to be elucidated. We suggest that tBHP-induced senescence led to a vicious cycle of mitochondrial ROS production and damaged mitochondria accumulation. This vicious cycle has already been implicated as one of the primarily responsible mechanisms of the aging process (Harman, 1992) and has been associated with extrinsic skin aging (Naidoo and Birch-Machin, 2017).

Environmental pollution, including contamination in biological, physical or chemical form, is a potent contributor to the skin aging process (Krutmann et al., 2016). Air pollution is a complex mixture of different types of molecules and its composition depends on geography and time, including seasonal changes or even daily events, such as rush hours (Péter et al., 2015). Different types of pollutants induce oxidative stress and thereby potentially drive extrinsic skin aging (Kim and Park, 2016). tBHP, known to induce oxidative stress, can be recognized as such a molecule and pollutant, possibly contaminating numerous working environments in industrial production. We were able to show that tBHP treatment induced oxidative stress in skin fibroblasts and cellular senescence in a similar manner as cigarette smoke extract exposure (Yang et al., 2013). By analyzing gene expression of tBHP treated fibroblasts, we found common associations with tobacco smoke exposure, indicating parallels in molecular pathway activation and response to the stresses. MMP upregulation, especially of MMP1 and MMP3, is a major contributor to tobacco smoke induced skin aging (Drakaki et al., 2014). In our tBHP senescence model, MMP1 and MMP3, among other SASP factors, were increased on mRNA level and varied throughout the senescence induction, supporting the idea of the SASP as a very complex and dynamic network. Epidermal thinning, as a result of slowed keratinocyte proliferation and regeneration, has been pointed out as a marker of aged skin (Wang and Dreesen, 2018). In tBHP treated 3D reconstructed skin, we found a reduction in epidermal thickness. Interestingly, a similar phenotype was reported for 3D reconstructed skin, where senescent fibroblasts were included into the matrix (Weinmüllner et al., 2020). This could imply that the fibroblasts responded to the stressor similarly, independent of being embedded in a skin equivalent or not. Even though they were immersed in a collagen matrix, the cells seemed to be affected by the tBHP treatment. In our experimental setups of 3D reconstructed and ex vivo skin, the epidermal keratinocytes did not have direct contact to the stressor in contrast to the subjacent fibroblasts, implying that crosstalk between dermal and epidermal cell types via signaling molecules caused the epidermal thickness variation. We suggest that in tBHP treated 3D reconstructed skin, the fibroblasts released signaling molecules, possibly SASP factors, which altered keratinocyte differentiation and caused epidermal thinning. The transmitters responsible for this communication still need to be determined.

In extrinsically aged skin, collagen deposition can be decreased by the interplay of two mechanisms – impaired production or accelerated degradation of collagen (Kim et al., 2016). Both mechanisms could be responsible for the reduced collagen deposition observed in tBHP treated ex vivo skin. COL1A1 mRNA downregulation in tBHP treated samples implied a reduction in collagen synthesis. Collagen degradation, for example by MMP1, was likely, as tBHP treatment of skin fibroblasts resulted in a clear induction of MMP1 in cell monolayer. Several reports have demonstrated that thinning of collagen bundles is a characteristic feature of aged skin, specifically related to exposition of skin to environmental damages (Abd El-Aal, N.H. et al., 2012; El-Domyati et al., 2002). In Masson’s trichrome stained tBHP treated skin biopsies, red structures seemed to be more abundant than in control samples. We assume that tBHP treatment reduced the thickness of dermal collagen fibers uncovering these structures. The accumulation of thinner fibers in tBHP treated biopsies, as evidenced by Picrosirius Red staining, supported this idea.

The skin is the outermost organ of the body and its surface is challenged by environmental pollution (Drakaki et al., 2014). Even though healthy skin shields the body from molecules via barrier function, it has been reported that chemicals can be absorbed by the organ and released into the blood stream, for example nicotine from cigarette smoke (Torres et al., 2018). Also inhalation and oral intake of toxins lead to their distribution throughout the body, eventually reaching the skin “from the inside” (Soeur et al., 2017). By adding tBHP to the culture media, we mimicked exposure of dermal cells and skin to toxins via this systemic route.

As previously mentioned, the composition of environmental pollution is highly divergent and depends on a multitude of factors (Péter et al., 2015). Given these variations, the simplicity of our model, which exposes cells and skin tissue to a single chemical, can be understood as a limiting factor. However, tBHP treatment induced similar pathways and responses as exposure to environmental pollution, especially tobacco smoke, in skin cells and in intact skin. In conclusion, our findings suggest that repetitive tBHP treatment is a suitable model to study extrinsic skin aging in different experimental setups with varying complexity, namely dermal fibroblasts, 3D reconstructed skin and ex vivo skin. The presented model can be applied to answer research questions about basic molecular mechanisms as well as support the development of anti-skin-aging strategies.

Supplementary Material

Supplementary Figures

Funding

This work was supported by an award from Aktion D. Swarovski KG 2018, [Project Nr. 281886, University of Innsbruck], by an Erasmus+ fellowship for traineeship, by FWF [P 31582-B26] and by the Doctoral Programme Ageing and Regeneration (DP AGE_REG), University of Innsbruck. C.T.M-S. is currently funded by an Erwin Schroedinger Abroad Fellowship [J4205-B27].

Abbreviations

cPDL

cumulative Population Doublings

D1

Day One

DAPI, 4’

6-diamidino-2-phenylindole

ΔΨm

Mitochondrial Membrane Potential

DHE

Dihydroethidium

DMEM

Dulbecco’s Modified Eagle’s Medium

DPBS

Dulbecco’s Phosphate Buffered Saline

EDTA

Ethylenediaminetetraacetic acid

FCCP

Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone

GAD_Disease ‘Aging’

Genetic Association Database Correlated with Aging

GAPDH

Glyceraldehyde 3-phosphate dehydrogenase

H&E

Hematoxylin and Eosin Staining

HFF-1

Human Foreskin Fibroblasts

HSDF

Human Skin Dermal Fibroblasts

HSEK

Human Skin Epidermal Keratinocytes

MMPs

Matrix metalloproteases

mtDNA

mitochondrial DNA

nDNA

nuclear DNA

PBS

Phosphate Buffered Saline

RIPA

Radioimmunoprecipitation Assay

RNAseq

RNA Sequencing

ROS

Reactive Oxygen Species

SASP

Senescence-associated Secretory Phenotype

SA-β-galactosidase

Senescence-associated β-galactosidase

SD

Standard Deviation

tBHP

tert-butyl hydroperoxide

Footnotes

Declarations of interest: None

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