Lead induces mouse skin fibroblast apoptosis by disrupting intracellular homeostasis

Lead (Pb) is a critical industrial and environmental contaminant that can cause pathophysiological changes in several cellular and organ systems and their processes, including cell proliferation, differentiation, apoptosis, and survival. The skin is readily exposed to and damaged by Pb, but the mechanisms through which Pb damages cells are not fully understood. We examined the apoptotic properties of Pb in mouse skin fibroblast (MSF) in vitro. Treatment of fibroblasts with 40, 80, and 160 μM Pb for 24 h revealed morphological alterations, DNA damage, enhanced caspase-3, -8, and -9 activities, and apoptotic cell population. Furthermore, apoptosis was dosage (0–160 μM) and time (12–48 h) dependent. Concentrations of intracellular calcium (Ca2+) and reactive oxygen species were increased, and the mitochondrial membrane potential was decreased in exposed cells. Cell cycle arrest was evident at the G0/G1 phase. The Bax, Fas, caspase-3 and -8, and p53 transcript levels were increased, whereas Bcl-2 gene expression was decreased. Based on our analysis, Pb triggers MSF apoptosis bydisrupting intracellular homeostasis. Our findings enrich the knowledge about the mechanistic function of Pb-induced cytotoxicity on human skin fibroblasts and could potentially guide future Pb health risk assessments.

Hoechst 33258 staining. Briefly, coverslips were sterilized and added to culture plates. Cells were allowed to grow on coverslips, treated with Pb 2+ for 24 h, and subsequently entered the logarithmic growth phase. The control group was established simultaneously. After discarding the culture medium, a fixative was introduced prior to rinsing and staining with Hoechst 33258. Anti-fluorescence quenching sealing solution was dripped onto the glass slide, and coverslips with cells were covered on the glass slide, ensuring that cells were fully in contact with the sealing solution while avoiding bubble formation. Cells were observed and photographed under a confocal microscope.
Comet assay. Upon cell harvest, cells were rinsed in cold phosphate-buffered saline (PBS), and 1 × 10 5 cells/ mL was suspended in PBS prior to the comet assay as described in the Alkaline Comet Assay kit protocol (Trevigen Inc., Gaithersburg, MD, USA). The cell suspension was combined with low-melting-point agarose, and the samples were transferred with a pipette onto comet slides prior to storage in ice-cold lysis buffer (Trevigen Inc.) at 4 °C for 35 min. The comet slides were electrophoresed with alkaline electrophoresis buffer and treated with www.nature.com/scientificreports/ 70% ethanol for 6 min before drying. DNA was stained with SYBR green I (Sigma) and photographed under a confocal microscope.
Transmission electron microscopy (TEM). The cells were collected, rinsed twice with PBS, fixed in 2.5% glutaraldehyde and then in 1% osmium tetroxide, and rinsed using 0.1 mol/L of PBS. An ethanol series (30%, 50%, 70%, 80%, 90%, and 100%) was used for cell dehydration, followed by cell embedding in an epoxy resin using acetone, which was left to solidify. Ultrathin sections (50 nm) of the resin were cut using an ultrathin microtome (LEICAUC6i) and stained using uranyl acetate and lead citrate prior to imaging using a transmission electron microscope (JEM-2000Ex; JEOL Ltd., Tokyo, Japan).
Annexin V-fluorescein isothiocyanate (FITC)/PI double-labeling. Apoptosis was assessed based on the Annexin V-FITC Apoptosis Detection kit directions. Upon harvest, cells were diluted to 5 × 10 5 cells/mL with binding buffer. Next, 5 μL each of PI and FITC were introduced to each 100 μL cell suspension sample, followed by incubation in 400 μL binding buffer for 10 min. Cells were analyzed via flow cytometry (FC500, Beckman Coulter, Brea, CA). A minimum of 10,000 cells were assessed per sample.

Statistical analysis.
All experiments were performed in triplicate to verify reproducibility. Data are presented as mean ± standard deviation (SD), and analysis was carried out using the Statistical Analysis System (SAS) software (SAS Institute Inc., Cary, NC, USA) and comparison via a multiple comparison test (DUNCAN). P < 0.05 was set as the significance threshold.

Results
Growth dynamics. The MSF growth curve presented a strong "S" outline, and the PDT was approximately 32 h. The cells entered a latent phase from days 1-2, entering the logarithmic phase from days 2-5. The density peaked on day 5, reached a plateau on day 6, and subsequently declined (Fig. 1a).
Cell survival assay. The MTT assay was utilized to determine the mitochondrial succinate dehydrogenase activity and detect mitochondrial function, which is widely used to evaluate cell viability. Pb 2+ reduces the viability of MSFs. Cell survival decreased considerably with increased Pb 2+ concentration and duration of exposure; in other words, cell survival reduced in a dose-and time-dependent manner (Fig. 1b). www.nature.com/scientificreports/ AO/EB double staining. In normal cells, the nuclear chromatin is pyknotic or beaded. The 40 µM treatment group showed numerous early apoptotic cells with pale yellow nuclei. In the 80 µM treatment group, some cells exhibited uneven morphology and late apoptosis. The 160 µM treatment group exhibited a considerable number of late apoptotic cells, with orange-red nucleosomes, concentrated chromatin, variable sizes, and relatively thick staining. Some late apoptotic cells were close to death (Fig. 1c).
Hoechst 33258 staining. The nuclear chromatin of control cells was uniformly stained, displaying round or ovoid nuclei, and emitting strong fluorescence. The treatment group showed cell chromatin concentration, uneven coloring, nuclear pyknosis, and bright spots, with nuclear pyknosis worsening with increasing Pb 2+ concentration ( Fig. 2a).
Comet assay. To analyze DNA damage, 100 fibroblasts were randomly selected from each group. The DNA of control cells was confined to the nucleus, while DNA strands of Pb 2+ -treated cells were broken, with DNA fragments traveling along the electric field, forming a bright fluorescent trail (Fig. 2b). The trail size indicated injury severity, which increased with increasing Pb 2+ doses (Fig. 2d), thereby indicating that Pb 2+ could disrupt the DNA integrity of MSFs.
TEM. The control cells exhibited abundant cell surface microvilli, uniform cytoplasm, intact nuclear membrane structure, clear and complete nucleoli, and uniform staining. Considering Pb 2+ -treated cells, cell surface microvilli disappeared, vacuoles appeared in the cytoplasm, and the nuclear membrane structure was damaged. The chromatin of apoptotic cells exhibited chromatin was condensed, marginalized, and segmented into clumps (Fig. 2c).

Cell cycle analysis.
With increasing Pb 2+ concentration, the proportion of G0/G1 cells increased, and the proportion of S and G2 phase cells decreased accordingly. Based on these results, there was a dose-dependent association between Pb 2+ levels and cell cycle dysregulation, indicating that Pb 2+ inhibited DNA synthesis in the www.nature.com/scientificreports/ G0/G1 phase (Fig. 4a). In the diploid DNA peak (sub-G1 population), the proportion of apoptotic cells increased in a dose-dependent fashion (Fig. 4b).
MMP. JC-1 is a lipophilic cationic stain that exclusively enters mitochondria and reversibly changes it from a green to a red color with increasing MMP. In normal cells, JC-1 accumulates in the mitochondria and shows red fluorescence. In apoptotic cells, JC-1 exists as a monomer with green cytoplasm, indicating a low MMP. The proportion of C-gated cells indicates the alterations in the MMP; a larger quantity represents a decrease in the MMP (Fig. 5a). Accordingly, the MMP of treated cells was significantly reduced when compared with that of control cells (P < 0.01) (Fig. 5b).
Intracellular Ca 2+ homeostasis. Upon treatment with higher Pb 2+ concentrations, apoptotic cells showed a higher proportion and intensity of green fluorescence, indicating an increase in free Ca 2+ levels. The green fluorescence observed in 160 μM Pb 2+ -treated cells was evenly distributed, suggesting a severe disruption in intracellular Ca 2+ homeostasis (Fig. 6a).

ROS analysis.
The higher the Pb 2+ concentration, the greater the number of apoptotic cells and the stronger the green fluorescence (Fig. 6b). The green fluorescence intensity increased, suggesting that ROS concentration also increased. The green fluorescence was distributed throughout the cells in the 80 and 160 μM Pb2 + treatment groups.  Bax and Bcl-2 are expressed in the outer mitochondrial membrane, and the Bax/Bcl-2 ratio strongly regulates apoptosis. Based on the qPCR analysis, treatment with 160 µM Pb 2+ significantly increased Bax, Fas, caspase-3, caspase-8, and p53 levels in mouse fibroblasts (P < 0.01) but significantly decreased Bcl-2 expression (P < 0.01, Fig. 7b). these results indicate that Pb 2+ treatment increased the Bax/Bcl-2 ratio in mouse fibroblasts.

Discussion
Environmental pollution attributed to heavy metals caused by human activities is a global concern. Pb, a heavy metal responsible for environmental pollution, is a non-biodegradable inorganic pollutant widely distributed in www.nature.com/scientificreports/ soil-water-air ecosystems owing to its easy migration and transformation 26 . Pb can cause a series of behavioral, biochemical, and physiological dysfunctions in organisms 27 . Toxicological and epidemiological studies have documented Pb-induced toxicity in several cells, organs, and organ systems 28 . Pb accumulation in living organisms has deleterious long-term effects, given that Pb induces cytotoxicity in various cell types through distinct cytotoxic pathways 29 . For instance, Pb suppresses the proliferation of human leukemia cells by inducing cell cycle arrest, DNA damage, and accelerating apoptosis via caspase-3 activation 30 . Pb mediates mitochondrial permeability transition pore (MPTP) opening, releases mitochondrial cytochrome c, and induces apoptosis of proximal tubule cells. Furthermore, Pb can induce apoptosis of rat proximal tubular cells through endoplasmic reticulum stress 31 . The skin is one of the major target organs for Pb poisoning. However, the Pb toxicity pathway  www.nature.com/scientificreports/ in the skin remains elusive. Herein, we present evidence to suggest that Pb exposure suppresses MSF cell survival and promotes cell apoptosis. Pb has been shown to significantly reduce TM3 cell proliferation in a concentration-and time/dependent manner 29 . Following treatment of HK-2 cells with increasing concentrations of lead nitrate (0-50 µM) for 24 h, intracellular ROS levels increased, whereas glutathione (GSH) levels decreased substantially in a dose-dependent manner 32 . Our findings are consistent with prior toxicological investigations involving other cell lines, showing a dose-dependent decrease in MSF survival, as evidenced by the MTT assay and Annexin V-FITC/PI double labeling. Annexin V-FITC/PI double labeling showed that Pb strongly promoted apoptosis, and the apoptoticpromoting effect was more notable with increasing Pb concentrations. AO/EB staining, Hoechst 33258 staining, and comet assay were used to demonstrate that Pb could induce MSF apoptosis. The membrane permeability of early apoptotic cells was relatively low, and ethidium bromide did not easily enter the cell interior; the cells were light yellow, with pyknotic or bead-like nuclear chromatin. The membrane permeability of late apoptotic cells was higher than that of early-stage apoptotic cells, but differed in terms of membrane rupture, with cells appearing orange-red and relatively more stained; some late apoptotic cells were close to death to a certain extent (Fig. 1c). Our assay results indicated that Pb could induce DNA damage, which is a unique change in apoptotic cells. The comet assay is a sensitive method for assessing individual cell nucleosomal DNA fragmentation. Ultrastructure analysis by electron microscopy is considered the optimal approach for identifying cell apoptosis. Based on our TEM analysis, Pb could induce typical apoptotic morphological changes, including cell shrinking, mitochondrial depolarization, subcellular structure destruction, and chromatin condensation in MSFs. In addition, flow cytometric analysis revealed that Pb treatment considerably increased the proportion of apoptotic cells in a concentration-dependent manner. Our results are consistent with those of previous studies, in which Pb accelerated apoptosis in TM3 cells 33 , cardiomyocytes 34 , and human monocyte-macrophage (THP-1) cells 35 .
Apoptosis, gene-controlled programmed cell death, is crucial for maintaining the internal environmental balance and normal body development. The cell cycle is a highly regulated process that modulates cell development and differentiation 36 . Given that apoptosis stimulation affects the cell cycle, alterations in cell cycle distribution may be related to apoptosis and differentiation. Hence, cell cycle analysis is among the most informative assessments for determining apoptosis. Pb was found to strongly trigger cell cycle arrest in the G0/G1 phase in the HL-60 cell lines 30 . To verify whether the Pb-mediated MSF suppression is related to cell cycle arrest, we examined cell cycle progression using flow cytometry. Based on our analysis, Pb caused G0/G1 phase cycle arrest in MSFs; the proportion of G1 cells and the level of hypodiploid DNA (sub-G1 population) were increased, the amount of S-phase cells was decreased, and cell proliferation was inhibited. Therefore, we hypothesized that Pb interferes with DNA replication and cell cycle distribution, resulting in MSF apoptosis. Mitochondria are necessary for apoptosis and are considered central regulators of apoptosis in vertebrates. One early event of apoptosis involves a reduction in MMP past a particular point. Pb reportedly binds to mitochondria at ion sites, resulting in a decrease in the membrane potential, shifting mitochondrial channels to an active open state 37 . Therefore, cell apoptosis is initiated when mitochondrial polarization, swelling, and membrane rupture occur along with the release of cytochrome C and other cytokines into the cytoplasm 38 . Pb treatment can stimulate the mitochondria-based apoptotic network 39 . Pb-driven mitochondria-based primary cultured rat proximal tubular cell apoptosis was shown to depend on the opening of the MPTP, and the three regulatory components of MPTP (CYPD, ANT, and VDAC) play roles in Pb-linked MPTP opening 34 . We demonstrated that the MMP of MSFs decreased dose-dependently with increased Pb exposure, indicating that Pb-driven MSF apoptosis is associated with mitochondria-based apoptosis. The Pb-based toxic sites in the mitochondria and the relative sensitivity of multiple mitochondria-mediated pathways remain poorly elucidated.
Under normal conditions, the body has a certain amount of ROS. As the messenger of various metabolic and signaling pathways within the body, ROS participates in the transcription of various genes and the expression of related functional proteins by activating and regulating various transcription factors, participating in cell proliferation, differentiation and maintenance of cells and tissues, and body metabolism 40 . ROS has strong reactive activity and oxidation. High ROS concentrations can directly or indirectly cause oxidative damage to lipid substances such as DNA, protein and cell membranes and induce cell apoptosis, senescence, and death 41 . In mouse renal proximal tubular cells as target cells, Pb exposure caused cell apoptosis, necrosis, mitochondrial damage and other cell damage, accompanied by increased intracellular ROS content 42 . Long-term low-dose Pb exposure can impact the structure and function of mouse renal proximal tubule cells. In addition, Pb exposure was found to alter cell morphology and hinder the ability of cells to take up calcium ions. These physiological changes are related to the content of intracellular ROS and occur in a dose-dependent manner 43 . Low concentration, acute Pb exposure could activate the activity of protein kinase C (PKC) in cells, which, in turn, regulated the calcium transport related N-methyl-d-aspartic acid receptor (NMDA) receptor channel, thereby increasing intracellular Ca 2+ content. However, NMDA receptor activation will promote the release of excitatory amino acid glutamate, subsequently triggering an increase in intracellular ROS content, resulting in oxidative stress 44 . Oxidative stress and cell apoptosis are the two main pathways through which Pb exerts its toxic effects. Pb exposure-induced oxidative stress can accelerate the process of cell apoptosis, as well as impact ROS production, consequently affecting the process of oxidative stress 45 .
Ca 2+ , a secondary messenger, is necessary for cell metabolism; an imbalance in Ca 2+ homeostasis can induce apoptosis through various pathways. The initial increase in Ca 2+ may be attributed to the influx of Ca 2+ in the extracellular space or redistribution of intracellular storage, resulting in irreversible or reversible changes in several major pathways 46 . Increased intracellular free Ca 2+ activates multiple signals and events involved in almost all aspects of cellular activity, ultimately inducing apoptosis 47 . Based on our analysis, Pb treatment increased cytoplasmic free Ca 2+ concentration, disrupting intracellular Ca 2+ homeostasis. In workers exposed to Pb, eryptosis associated with oxidative damage seems to be characterized by a rise in free intracellular Ca 2+ , owing to enhanced Ca 2+ channel permeability and diminished Ca 2+ Mg 2+ (ATPase) activity 48  www.nature.com/scientificreports/ Apoptosis may be related to an imbalance in calcium homeostasis, oxidative damage, and mitochondrial damage 49 . Under physiological conditions, the production and clearance of oxygen free radicals are in a dynamic equilibrium. Several factors may reduce the ability of the antioxidant system to scavenge oxygen free radicals, thereby upregulating oxygen free radicals. Pb exposure can induce the generation of oxygen free radicals, and oxidative stress directly damages cell membranes, leading to increased cell membrane permeability 50 . Herein, Pb treatment resulted in increased ROS levels in MSFs. Our results corroborate the findings of a previous study 50 and suggest that Pb can induce oxidative stress in MSFs in vitro. Excessive ROS production has consistently been shown to induce apoptosis.
Caspases are a family of proteases, and the caspase-induced cascade is critical for the stimuli-induced apoptotic process. Caspase-3 is primarily activated by cytotoxic drugs 51 , and it strongly regulates apoptotic DNA damage. In human leukemia cells, Pb was shown to modulate apoptosis via caspase-3 activation 30 . Herein, Pb-exposed MSFs showed increased caspase-3, -8, and -9 activities. Our results corroborate those of a previous study, which demonstrated that Pb exposure could promote renal cell apoptosis and impair cell function by activating caspase-3 52 .
Bcl-2 is a major contributor to cell death signaling, and the protein family has both anti-and pro-apoptotic members. Bcl-2 regulates the balance between pro-apoptotic members like Bax and pro-survival Bcl-2-like proteins and functions as an anti-apoptotic protein that modulates multiple apoptotic networks. The Bax protein is considered to be oligomeric and can enhance mitochondrial outer membrane permeability 53 . A decrease in the Bax/Bcl-2 ratio suppresses apoptosis, whereas an increase in the Bcl-2 ratio enhances apoptosis 54 . Therefore, the Bax to Bcl-2 ratio strongly modulates cell survival and apoptosis. In addition, p53 inhibits the anti-apoptotic protein Bcl-2 and promotes the pro-apoptotic protein Bax 55 . Bax and Bcl-2 regulate the mitochondrial outer membrane permeability, stimulate cytochrome C release, activate the caspase cascade, and induce apoptosis 56 . The Fas gene is also important in mediating apoptosis. Based on our findings, increasing Pb concentrations enhanced expression levels of Fas, Bax, p53, caspase-3, and caspase-8 and reduced those of Bcl-2. In addition, the Bax to Bcl-2 ratio was increased. Therefore, Pb can interfere with the levels of multiple apoptotic genes in MSFs. In PC12 cells, Pb triggers apoptosis, accompanied by increased caspase-3 and Bax levels and an associated increase in p53 expression, elevated Bax expression, and reduced Bcl-2 levels 57 . Furthermore, Pb can stimulate Fas gene expression and promote testicular germ cell apoptosis in a dose-dependent manner 58 .
Pb affects the levels of oxidants in MSFs and enhances ROS production; in turn, increased ROS concentration leads to the dysregulation of the cellular lipid bilayer and disrupts Ca 2+ transport. Pb leads to instability in calcium homeostasis and destroys membrane ion channels by disrupting electron transport and reducing ATP concentration, ultimately leading to cell apoptosis 59 . Pb-induced apoptosis of MSFs also appears to depend on the mitochondria, given that Pb can affect the MMP. Damaged cell membranes are characterized by a decrease in MMP and interference in the balance of molecules such as Bax and Bcl-2 60 . Alterations in the Bax/Bcl-2 ratio can trigger cytochrome C release and caspase activation and, ultimately, cell apoptosis 61 .
However, given that the present study was an in vitro assessment, limitations are inevitable. Considering one such limitation, it should be noted that the results do not necessarily represent real-world effects of Pb on MSFs in vivo, as various other factors affect cell proliferation and apoptosis in vivo. Another limitation of the study is that protein levels of apoptotic markers were not quantified, which can be considered a future research direction.

Conclusions
In conclusion, Pb induces considerable toxic effects on MSFs, such as decreasing cell survival, accelerating cell morphological alterations, and apoptosis. Treatment with Pb treatment arrested the cell cycle, decreased the MMP, increased the Bax/Bcl-2 ratio, and enhanced caspase-3, -8, and -9 activation, suggesting that Pb can induce MSF apoptosis. These intracellular processes were related to Ca 2+ overload, suppression of DNA formation, oxidative stress, mitochondrial dysfunction, and altered gene expression. Our findings provide useful regarding the etiology of Pb toxicity in the skin and indicate the undesirable outcomes of Pb exposure in humans. This evidence offers a basis for further studies on the Pb pollutant-based toxicity mechanisms to improve health risk assessments.