PARP-1 overexpression does not protect HaCaT cells from DNA damage induced by SiO₂ nanoparticles

Abstract

Nano-SiO₂ is increasingly used in diagnostic and biomedical research because of its ease of production and relatively low cost and which is generally regarded as safe and has been approved for use as a food or animal feed ingredient. Although recent literature reveals that nano-SiO₂ may present toxicity and DNA damage, however, the underlying mechanism remains poorly understood. Since in previous studies, we found that nano-SiO₂ treatment down-regulated the expression of the poly(ADP-ribose) polymerases-1 (PARP-1), a pivotal DNA repair gene, in human HaCaT cells and PAPR-1 knockdown can aggravate DNA damage induced by nano-SiO₂. Therefore, we speculate whether PARP-1 overexpression can protect DNA from damage induced by nano-SiO₂. However, our data demonstrated that overexpression of PARP-1 in HaCaT cells slightly enhanced the cellular proliferation of undamaged cells, when compared with both empty vector control cells and parental cells, but had drastic consequences for cells treated with nano-SiO₂. The PARP-1 overtransfected cells were sensitized to the cytotoxic effects and DNA damage of nano-SiO₂ compared with control parental cells. Meanwhile, flow cytometric analysis of nano-SiO₂ stimulated poly(ADP-ribose) synthesis revealed consistently larger fractions of cells positive for this polymer in the PARP-1 overexpression cells than in control clones. Combining our previous research on PARP-1 knockdown HaCaT cells, we hypothesize that an optimal level of cellular poly(ADP-ribose) accumulation exists for the cellular recovery from DNA damage.

Introduction

With the development of nanotechnology and materials science, engineered nanoparticles have been mass produced and widely applied [1, 2]; however, they pose a threat to large populations who may be exposed at the industrial as well as at the consumer levels [3, 4]. During mining, processing, and industrial usage, workers are exposed to these minerals. Apart from the occupational exposure, environmental exposure to nano-SiO₂ dust is also very common due to its presence in the soil, which becomes airborne under arid, windy conditions or during agricultural, urban, and construction activities. Information about the safety and probable hazards of this mineral are urgently desired. With this aim, the field of nanotoxicology has significantly emerged to deal with the potential risk of ultrafine particles [5–7]. Nano-SiO₂ are one of the most abundant inorganic elements in PM 2.5 and are easier to be brought into the atmosphere via increasing applications in daily life [8]. Yang et al. [7] observed that the intratracheal instillation of nano-SiO₂ activates macrophages in the lung and leads to inflammation in BALB/c mice. Also, the in vitro studies indicate that nano-SiO₂ induce mitochondrial toxicity and cell dysfunction [9]. Owing to their unique nano-scale, nanoparticles are provided with many special physicochemical properties and thereby may yield extraordinary hazards to human health [10, 11].

Nano-SiO₂ particles enter the body through breathing and skin exposure and accumulate in organs such as the lungs, liver, brain, and heart [2]. The skin is the largest organ in the human body, which provides protection against heat, cold, electromagnetic radiation, and chemical damage. Indeed, skin cells are likely to have the highest frequency of exposure to nanoparticles. Hence, a safety evaluation of nanoparticles using dermal cells is essential [12]. Based on this consideration, using the HaCaT human keratinocyte cell line as a model system, we studied the effects of nano-SiO₂ particles on cell function.

PARP-1 is the most important number of poly(ADP-ribose) polymerases (PARP) family, which is responsible for the 80% formation of poly(ADP-ribose). PARP family constitute a family of enzymes involved in the regulation of many cellular processes such as DNA repair, recombination, proliferation, and genomic stability [13]. Since PAPR-1 knockdown in HaCaT cells can aggravate DNA damage induced by nano-SiO₂, we suppose that PARP-1 overexpression can protect HaCaT cells from DNA damage induced by nano-SiO₂. In order to study the influence of increased PARP-1 activity upon cellular resistance toward DNA damage, we established HaCaT cell lines that constitutively overexpression full-length human PARP-1 and speculated that increased poly(ADP-ribosyl)ation capacity could be associated with a protective effect. Here, we report on the characterization of transfected lines and effects upon cellular survival of treatment with nano-SiO₂.

Materials and Methods

Chemicals and antibodies

Human epidermal keratinocyte cell line HaCaT was purchased from China Center for Type Culture Collection (Wuhan, Hubei, China). MEM culture media were purchased from Hyclone Laboratories, Inc. (Logan, UT, USA). Lipofectamine 2000, fetal bovine serum (FBS), penicillin–streptomycin for cell culture, and trypsin were purchased from Gibco/Invitrogen (Carlsbad, CA, USA). Cell counting kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan). The primary antibodies mouse anti-γH2AX antibody was purchased from Millipore Corporate (Palm Springs, CA, USA), mouse anti-PAR monoclonal antibody was supplied by Trevigen INC (Gaithersburg, MD USA), and FITC-conjugated secondary antibody was purchased from southern biotechnology, Inc (Birmingham, AL, USA).

Particles and their characteristics

The 15-nm nano-SiO₂ was purchased from Wan Jing New Material Co. Ltd (Hangzhou, Zhejiang, China), and the micrometer SiO₂ particle (micro-SiO_2, 1–5 μm) was supplied by Sigma-Aldrich (Sigma, SL, USA). The distribution and zeta potential of the SiO₂ particles was tested by use of Nicomp 380/ZLS submicron particle sizer (Particle Sizing Systems, Santa Barbara, CA, USA). Transmission electron microscopy is also used to characterize the SiO₂ particles. Crystal structure was characterized by Scintag XDS 2000 diffract meter (Scintag, Inc., Cupertino, CA, USA). The purity of the samples was analyzed by use of a Thermo Elemental X7 ICP-MS spectrometer (Thermo Scientific, Waltham, MA, USA).

Plasmids and retroviral infections

The PAPR-1 overexpression vector pBABE-PAPR-1 was constructed by our laboratory. Human PARP-1 cDNA, containing the complete coding region, was isolated by PCR amplification. The human PARP-1 CDS (3042 bp fragment of cDNA) was PCR amplified from chromosomal cDNA of HaCaT with primers 5′-GAAGATCTTC CGCCAC ATGGCGGAGTCTTCGGAT-3′(sense) and 5′-CCGCTCGAGCGGCCACAGGGAGGTCTTAAAATTG-3′ (antisense). The underlined are restriction digest sites. The resulting fragment was cloned into a pBABE-puro vector, which was generously provided gift by Dr. W.C. Hahn doctor (Dana Farber Cancer Institute, Boston, USA). Recombination sequence was digested by snabIand SalI and then sequenced by Shanghai Sangon Service. To create amphotropic retroviruses, pBabe-PAPR-1 was introduced into HaCaT cells using retroviral infection and purified by selection with puromycin (1 μg mL⁻¹). In transfection experiments, 0.5 × 10⁶ 293 T cells were seeded in 100 × 100 mm culture dishes (corning, NY, USA) and transfected with Lipofectamine 2000 (Invitrogen, CA, USA) adopting the manufacturer’s protocol. Assays were performed with 4 μg per dish of purified plasmid DNA of either empty pBABE-puro as control or pBABE–PARP-1 construct together with 2 μg per dish of pCL-ampho vector for puromycin selection of transfected cells. After 24 h, cells were incubated for further 24 or 72 h in culture medium supplemented with 1 μg mL⁻¹ puromycin. Apart from seeding 0.25 × 10⁶ cells/dish and omitting pBabe-puro and puromycin selection, the same procedure was employed in transient transfection assays. To verify the transfection efficiency of the system, we used a retroviral vector with a GFP marker (pMIG) as a reference system.

RT-qPCR assay

Total RNA was extracted from cells using high pure RNA extraction method (Roche, Germany) and treated with DNase I. cDNA was synthesized from 1 μg of total RNA using Super Script III reverse transcriptase (Invitrogen, San Diego, CA, USA) with random hexamers. To perform qPCR, the ranges of linear amplification for the target gene and for the ACTB genes were studied. Quantification of PARP-1 mRNA expression by qPCR using SYBR green I amplification of each target cDNA was performed in a MX 4000 Thermocycler Sequence Detection System (Stragene, CA, USA). qPCR analysis was performed according to the instruction of SYBR^® ExScript ™ RT-PCR Kit (Takara, China). Data were normalized relative to the expression level of ACTB for each sample primers used for qPCR were human PARP-1:5’-CCCAGGGTCTTCGGATAG-3′(sense) and 5’-AGCGTGCTTCAGTTCATAC-3′ (antisense); ACTB: 5’ CCCAGGGTCTTCGGATAG −3′ (sense) and 5’ AGCGTGCTTCAGTTCATAC-3′ (antisense).

Western blot analysis

Cells (1 × 10⁶) were washed twice with PBS, and proteins were extracted with lysis buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100, and 100 mg mL⁻¹ PMSF) for 15 min on ice. Equal amounts of protein were separated by 10% sodium dodecyl sulphatepolyacrylamide gel electrophoresis and transferred to PVDF membrane using a standard protocol. Membranes were then blocked with TBST (TBS with 0.05% Tween20) containing 5% fat-free milk for 1 h and incubated with human monoclonal antibody against PARP-1 (Santa cruz, CA, USA) at a 1:500 dilution for 12 h at 4°C. Membranes were then washed with TBST and incubated with horseradish peroxidase conjugated goat anti-mouse immunoglobulin G for 1 h. The blots were developed by using the ECL system (Pierce, Chicago, USA).

Cell proliferation assay

Cells were cultured in 96-well plates at a concentration of 1000 cells/well for 1, 2, 3, 4, 5, and 6 days, respectively. The cells were ultimately treated with MTT solution and incubated for 4 h, followed by the addition of 150 μL DMSO and incubation for 15 min. An ELISA microplate reader (BioTek, Winooski, VT, USA) was used to measure the absorbance at 490 nm [optical density (OD) value].

Cell cycle analysis

Cell cycles were analyzed as follows: after the chemical treatment, the cells were collected by trypsin and washed with ice-cold PBS (pH 7.4) and fixed in 70% ethanol overnight at 4°C. Fixed cells were washed with PBS before incubation with 50 μg mL⁻¹ DNasefree RNase A (Invitrogen, CA, USA) at 37°C for 30 min, then 10 μg mL⁻¹ propidium iodide (PI; Sigma-Aldrich, MO, USA) at 4°C for 15 min. Fluorescence was measured on a Beckman-coulter (Beckman Coulter, CA, USA) flow cytometer. At least 10 000 cells were measured for each sample. ModFit LT software was used to analyze the cell cycle distribution. Samples were analyzed in triplicate. Three independent experiments were performed for each group.

Analysis of chromosome aberrations

A total of 1 × 10⁵ cells were seeded into a 10 cm² plate. Four hours before the end of the experiment, colchicine (BBI, Grand Island, NY) was administered at 0.3 μg mL⁻¹ and metaphase chromosomes were prepared. The cells were collected and treated with 0.075 mol L⁻¹ potassium chloride, fixed in Carnoy’s solution (methanol:acetic acid, 3:1), and spread on glass slides by the air drying method. The specimens were stained with a 3% Giemsa solution in 0.075 mol L⁻¹ phosphate buffer (pH 6.8) for 25 min. The aberrations scored were gaps, breaks, exchanges, dicentrics, O-rings, and fragmentations were observed by microscope (Olympus CX31, Olympus, Japan). Over 100 cells from each experimental group were scored blindly for the number of mitotic cells by a well-trained observer, and all of the observations were double-checked by a second observer. Three replicates are required to ensure credibility of the results, and at the same time, the standard deviation data of the replicates within the group can be calculated. Cytogenetic toxicity experiments needed to meet strict data analysis standards. If the CV value is greater than 10%, it should be removed.

Micronucleus scoring via microscopy

To see whether PARP-1 overexpression affects basal or DNA damage induced MN formation, HaCaT, HaCaT-V, and HaCaT-T cells were treated 1 μg mL⁻¹ mitocycin C for 12 and 24 h, and recovered for 12, 24, 36, and 48 h followed by scoring of MN. The cells were collected and treated with 0.075 mol L⁻¹ potassium chloride, fixed in Carnoy’s solution (methanol:acetic acid, 3:1), and spread on glass slides by the air drying method. Staining was accomplished by submerging slides for 20 min in 3% Giemsa solution in 0.075 mol L⁻¹ phosphate buffer (pH 6.8). An Olympus CX31 microscope was used for MN measurements at 400× magnification. For each culture, 1000 mononucleotide, non-apoptotic, non-necrotic cells were analyzed for the presence of MN (1000 cells per culture × duplicate cultures = 2000 cells per time point). For a cell to be scored as MN containing, the MN event(s) had to be approximately round in shape, exhibit similar staining characteristics as the main nucleus, be less than one-third the size of the main nucleus, and could touch but not overlap the main nucleus (HUMN criteria) [14]. Each experimental group was scored blindly for the number MN by a well-trained observer, and all of the observations were double-checked by a second observer. Three replicates are required to ensure credibility of the results, and at the same time, the standard deviation data of the replicates within the group can be calculated. Cytogenetic toxicity experiments need to meet strict data analysis standards. If the CV value is greater than 10%, it should be removed.

Cell viability

HaCaT cells were cultured in MEM media containing 10% FBS and 5% carbon dioxide (CO₂) at 37°C. Nano-SiO₂ particles of different concentrations were administered when the cell confluence reached up to 80%, and the cells were treated for 24 h. The final concentrations of nano-SiO₂ were 100, 80, 60, 40, 20, 10, 5, and 0 μg mL⁻¹. After the treatment, the cells were incubated with CCK-8 for 2 h. After thoroughly mixing, the plate was read at 450 nm for optical density that is directly correlated with the cell quantity. The inhibitory rate of cell growth was calculated from the relative absorbance at 450 nm. The absorbance was measured at 450 nm using a microplate reader (BioTek, Winooski, VT, USA). The reference wavelength is 630 nm. The inhibitory rate (IR) of the cell growth was figured out by the formula provided by the kit: Viability (%) = [(OD0 − ODdose)/OD0] × 100%.

Comet assay

After the chemical treatment, the cells were collected by trypsin and re-suspended in PBS at a density of 1.0 × 10⁶ mL⁻¹. The alkaline comet assay was performed according to TAO,S [15]. Comet were imaged using the IX51 fluorescence microscope (excitation filter 549 nm, barrier filter 590 nm, OLYMPUS). Individual comets’ analysis was performed using Comet Assay Software Project (CASP)-1.2.2 (University of Wroclaw, Poland). We chose two commonly used parameters to do the following analysis: tail DNA percentage (%) and tail length (DNA migration, μm). Values were all expressed as means ± standard deviation (SD).

The flow cytometry assay and immunofluorescence assay of γ-H2AX

Flow cytometry assay of γ-H2AX was measured according to the manufacturer’s protocol. Phosphorylated histone H2AX was immunostained according to the method of Xu et al. [16]. Briefly, 70% ethanol-fixed cells were permeabilized with 0.5% Triton X-100 in PBS. After centrifugation, the cell pellet was incubated in 100 μL PBS containing 1% bovine serum albumin (BSA) and a monoclonal antibody specifically recognizing phospho-Ser10 on histone H2A at room temperature. After being washed with PBS containing 1% BSA, cells were incubated with FITC-conjugated goat anti-mouse IgG antibody diluted at a ratio of 1:400 in the dark. After being washed again, the cells were incubated with PBS before the fluorescence was measured by flow cytometry.

Quantitation of cellular poly(ADP-ribose) levels

To analyze cellular poly(ADP-ribose) formation, exponentially growing cells that had been treated or not with nano-SiO₂ for 24 h. Cells were fixed according to modified protocol of previous publication [17]. Cells were washed with PBS, trypsinized, and fixed as described below. The cells were fixed with 1% paraformaldehyde in PBS for 15 min on ice. After fixation, the cells were immunostained according to the procedure of Shin et al. [18].

Statistical analysis

The experiments were replicated three independent times, and data were expressed as mean ± standard deviation. Statistical analysis of the data was carried out using ANOVA, followed by Tukey’s HSD post hoc test (equal variances) or Dunnett’s T3 post hoc test (unequal variances). Otherwise, the nonparametric Kruskal–Wallis test was used. In the study of DNA damage by the comet assay, Student’s t-test for independent samples was also used. These tests were performed using SPSS software, version 16.0 for windows. Significance was declared when P-value was less than 0.05. The χ2 analysis and Fisher’s exact tests were performed to compare data for incidences.

Results

Stably PARP-1 overexpression HaCaT cells were constructed successfully

A retroviral vector with a GFP marker (pMIG) as a reference system for transfection efficiency showed that 293 T cells were successfully transfected, GFP expression was detected almost in all the cells (Fig. 1). As expected, there is high transfection efficiency in retroviral transcription system and HaCaT cells transfected with pBABE-PAPR-1 resulted in higher expression of PARP-1 mRNA and protein than those with empty vector or the parental cells. Results showed that in transiently cells PARP-1 mRNA were almost 4-fold increase and in stably cells that were more than 5-fold increase higher than the controls (Figs 2A and C and S1). Protein expression level was consistent with the mRNA result (Fig. 2B and D). PAPR-1 overexpression HaCaT cells were established successfully.

Figure 1 .

The efficiency of transfection in 293T and HaCaT cells. (A) The GFP expression in 293T after 24h transfection; (B) The GFP expression in 293T after 48h transfection; (C) The GFP expression in HaCaT cell 24h after first transfection; (D) The GFP expression in HaCaT cell 48h after first transfection.

Figure 2 .

Overexpression of PARP-1 in transfected cells. (A) mRNA levels of PARP-1 in HaCaT cells, HaCaT cells transfected with empty pBABE-puro vector (HaCaT-V) and pBABE/PARP-1 vector (HaCaT-PAPR-1) cells after transient transfection; (B) Western blot analysis of PARP-1 in different clones after transient transfection; (C) mRNA levels of PARP-1 in different clones after stable transfection. (A) Western blot analysis of PARP-1 in different clones after stable transfection. ^*P < 0.05 compared HaCaT group; #P < 0.05 compared with HaCaT-V group.

Biologic characteristics of transfected cells

Cell morphology observation, cell cycle analysis, growth curve analysis, micronucleus formation induced by mitomycin C, and chromosome stability aberration assay of HaCaT, PAPR-1 overexpression HaCaT-PARP-1 cells transfected with empty vector HaCaT-V cells were examined to be the characteristics of the role of PAPR-1. As shown in Fig. 3C, a striking variation in the growth pattern was observed among stable transfectants from the third day to the sixth day. The HaCaT-PARP-1 cells showed higher growth rate compared with the controls (P < 0.05). Result showed that cells overexpression PAPR-1 has a little change in its morphology (Fig. 3A), increasing its S phase in cell cycle distribution (Fig. 3B). The chromosome was stable when no agent treated. PARP-1 overexpression can cause it hypersensitive to DNA damage agent, but PARP-1 overexpression cells recovered from damage more quickly. Expression of PARP-1 failed to increase significantly the frequency of chromosome aberrations (Fig. 3D). When we analyzed the chromosomal aberrations of those cell lines, we found that the majority (about 90%) of the metaphases had a diploid number (2n = 46) of chromosomes and about 10% were aneuploid. In addition, structural aberrations including single chromosomal breaks, abnormal centromeres, and ring chromosomes were also observed in 4% of HaCaT cells and 3% in HaCaT-PARP-1 cells (Table 1). This observation showed that PARP-1 overexpression can proceed without wide spread genomic instability.

Figure 3 .

The biologic characteristics of PARP-1 overexpression cells. (A) Morphological changes of HaCaT, HaCaT-V, HaCaT-PAPR-1 cells; (B) Cell cycle profiles of HaCaT, HaCaT-V, HaCaT-PAPR-1 cells; (C) Cell growth curves of HaCaT, HaCaT-V, HaCaT-PAPR-1 cells; (D) The rate of micronucleus induced by mitocycin C of different times. ^*P < 0.05 compared with HaCaT group; ^#P < 0.05 compared with HaCaT-V group.

Table 1.

Percentage of chromosomal abnormalities in different groups (%, means ± SD)

Cells	Abnormality in number				Abnormality in structure
	2n	<2n	>2n	Rate of abnormality (%)	Normal	Breakage	Others	Rate of abnormality(%)
HaCaT	90 ± 1.2	2 ± 0.4	8 ± 0.6	10.0	96 ± 1.8	3 ± 0.4	1 ± 0.1	4.0
HaCaT-V	90 ± 2.6	1 ± 0.4	9 ± 0.5	10.0	93 ± 0.6	5 ± 0.4	2 ± 0.4	7.0
HaCaT-PARP-1	93 ± 0.6	1 ± 0.4	6 ± 0.5	7.0	97 ± 0.6	2 ± 0.4	1 ± 0.4	3.0

Particle characterization

The characterization results of 15-nm SiO₂ nanoparticles and standard SiO₂ (micro-sized SiO₂) were summarized in Table 2. The hydrodynamic sizes of the 15-nm SiO₂ nanoparticles in MEM suspension after 24 h was 14.6 ± 0.3 nm, indicating that little aggregation occurred and solution was uniform. The TEM images were showed in Fig. 4. The zeta potential was −59.70 mV for 15-nm SiO₂ nanoparticles and −13.23 mV for standard SiO₂. The XRD analysis clearly showed the structure of SiO₂ nanoparticles and standard SiO₂ was all amorphous. No metal is detected in 15-nm SiO₂ nanoparticles. The metal impurity data of standard silicon dioxide were provided by U.S. Silica Company.

Table 2.

Characterization of 15-nm SiO₂ nanoparticles and micro-SiO₂

Solution	Concentration	Average sizec	Size in solution d	Shape	Zeta potentiald	Purity c
Nano-SiO2a	2%	15 nm	14.6 ± 0.3 nm	Amorphous	−59.70 mV	99.99%
Standard SiO2  b	2%	1 ~ 5 μm	1 ~ 5 μm	Amorphous	−13.23 mV	99.99%

^aSupplied by Hangzhou Wanjing new material limited company.

^bSupplied by Sigma Aldrich American.

^cAccording to the manufacture.

^dUsing NICOMP 380ZLS submicron particle sizer.

^eICP-MS Thermo Elemental X7.

Figure 4 .

Characterization of nano-SiO₂. (A) Transmission electron microscopic image of nano-SiO₂. (B) Size distribution of nano-SiO₂ (14.6 ± 0.3 nm).

PAPR-1 gene overexpression decreased viability of HaCaT and HaCaT-PARP-1 cells after treated by nano-SiO₂ and micro-SiO₂ particles

A cytotoxicity screen is done in order to determine a proper dose of nano-SiO₂ used to treatment HaCaT cells. We showed that parameter IC₅₀ determined by cell viability detection 24 h after treatment might potentially be used as a reference concentration before conducting cytotoxicity assay. Treatment of HaCaT cells and HaCaT-PARP-1 cells by 15-nm SiO₂ particles resulted in significantly decreased cell viability in a dose-dependent manner. The relationship of inhibitory rate of the cell growth and the dosages was analyzed by SPSS 15.0 software, and the IC₅₀ (50% concentration of inhibition) value was therefore figured out. A dose-dependent cytotoxicity of nano-SiO₂ toward HaCaT cells and HaCaT-PARP-1 cells were found: 50% inhibitory concentrations IC₅₀ value after 24 h treatment was 27.27 ± 1.5 and 22.18 ± 1.4 μg mL⁻¹, respectively (Fig. 5). The dosage of 10 μg mL⁻¹ (about 1/3 ~ 1/2 IC₅₀ for HaCaT-PARP-1) was used as the maximum dosage in this study, and 2.5 and 5 μg mL⁻¹ were selected as the final dosages used in the subsequent experiments. After HaCaT cells and HaCaT-PARP-1 cells were treated by nano-SiO₂ or standard silicon dioxide at 2.5, 5, and 10 μg mL⁻¹ for 24 h, the CCK-8 absorbance decreased as a function of dosage levels. The viability of HaCaT-PARP-1 cells treated by nano-SiO₂ was 90.04, 82.00, and 69.00%, respectively, compared to the control group and standard silicon dioxide group (P < 0.05), the cell viability of HaCaT cells treated by nanoparticles was 97.5, 89.4, and 84.1%, respectively. There was statistical difference compared to the control group and standard group (P < 0.05). HaCaT-PARP-1 cells showed tendency more sensitive to nano-SiO₂.

Figure 5 .

Viability of HaCaT and HaCaT-PARP-1 cells after 24-h exposure to 15-nm SiO₂ particles Values were mean ± SD from three independent experiments. ^*P < 0.05 vs control cells; & P < 0.05 vs HaCaT cells of the same dose of nano-SiO₂.

PAPR-1 gene overexpression increased DNA damage in HaCaT cells and HaCaT-PARP-1 cells induced by nano-SiO₂ particles

Comet assay, also called single cell gel electrophoresis (SCGE), determines a combination of single-strand breaks, double-strand breaks, and alkaline labile sites [19]. Comet assay showed that there were significant increases in tail length, percentage of DNA in tail, after HaCaT cells and HaCaT-PARP-1 were treated with nano-SiO₂ at all the examined concentration (Fig. 6A and B). At 5 and 10 μg mL⁻¹ dose, nano-SiO₂ caused more DNA damage and more percentage of DNA in the tail in HaCaT-PARP-1 cells than HaCaT cells (P < 0.05); however, take tail DNA% as example, 34.72, 27.14, 21.84, and 10.90% for the group of 10, 5, and 2.5 μg mL⁻¹ and micro-SiO₂, respectively. γH2AX is a phosphorylated histone H2AX that can act as a marker of DNA double-strand breaks (DSBs). The effect of nano-SiO₂ on γH2AX formation was detected by flow cytometry assay. HaCaT cells exposed to nano-SiO₂ of different concentrations for 24 h dramatically increase γH2AX formation compared to HaCaT cells at 10 μg mL⁻¹ (P < 0.05), suggesting that nano-SiO₂ induced the serious DNA damage and at 10 μg mL⁻¹ group, HaCaT-PARP-1 group was more serious than HaCaT group (Fig. 6C).

Figure 6 .

DNA dmage of HaCaT and HaCaT-PARP-1 cells after 24-h exposure to 15-nm SiO₂ particles. (A) shows tail length of comet; (B) tail DNA percent of comet, (C) shows the results of the quantitative γH2AX fluorescence Values were mean ± SD from three independent experiments. ^*P < 0.05 vs control cells; ^#P < 0.05 vs cells treated by micro-sized SiO₂ particles; & P < 0.05 vs HaCaT cells of the same dose of nano-SiO₂.

PAR formation of HaCaT cells and HaCaT-PARP-1 cells induced by nano-SiO₂ particles

By means of flow cytometry analysis, overexpression of human PARP-1 was detected in almost 100% of the HaCaT-PAPR-1 cell population, while resident PARP in HaCaT cells was not detected under these conditions (data not shown). To quantitate the poly(ADP-ribose) levels, cells growing on 10 × 10 cm dishes were treated with nano-SiO₂ and microsized particles. Poly(ADP-ribose) was quantified as described in the part of materials and methods. In HaCaT-PAPR-1 cells, the basal levels of poly(ADP-ribose) are 2.6-fold higher than in the control HaCaT. Likewise, treatment of the cells with nano-SiO₂ resulted in 2.3-fold higher peak levels of poly(ADP-ribose) in HaCaT-PAPR-1 cells than in HaCaT cells (Figs 7 and S2).

Figure 7 .

Quantitative determination of poly(ADP-ribose) formation in HaCaT cells and HaCaT-PARP-1 cells induced by SiO₂ nanoparticles. Values were mean ± SD from three independent ^*P < 0.05 vs control cells; ^#P < 0.05 vs cells treated by micro-sized SiO₂ particles; &P < 0.05 vs HaCaT cells of the same dose of nano-SiO₂.

Discussion

There are numerous research on the toxicity of nano-SiO₂, but the biological and cellular responses of the nano-SiO₂ are poorly understood. Our findings confirmed that the direct exposure of nano-SiO₂ to PARP-1 overexpressed HaCaT cells aggravates the DNA damage and lead to initiation of cell death pathways. PARPs are defined as cell signaling enzymes that catalyze the transfer of ADP-ribose units from NAD⁺ to a number of acceptor proteins. PARP-1, a pivotal DNA repair gene and the best characterized member of the PARP family, is essential to the repair of DNA single-strand breaks via the base excision repair pathway. We stably transfected a human-PARP-cDNA expression construct into the HaCaT cells. After two subcloning procedures, we established transfectants, called HaCaT-PARP-1, that displayed constitutive overexpression of human PARP-1 in almost 100% of the cells. Puro-resistant clones transfected with empty pBABE-puro vector without detectable expression of human PARP-1 were chosen as vector controls (HaCaT-V). Characteristic results showed that PAPR-1 overexpression had a little change in morphology, increase its S phase in cell cycle distribution, and showed higher growth rate compared with the controls the third day to the sixth day (P < 0.05). Cell cycle is intimately connected with cell replication, division, and proliferation and can be affected by some adverse conditions, including DNA damage, abnormal DNA replication, and nutrient depletion [20]. Cell cycle distribution is usually used to determine and repair DNA damage, thus maintaining the genomic stability of cells [21]. In order to provide extra time for repairing DNA damage, the cells are prevented from activating next stage mitosis at the G2/M phase. At present, our data demonstrate that overexpression of PARP-1 cell cycle increased the number of cells in the S phase while reducing the number of cells in the G0/G1 phase and G2/M phase. Cell cycle is one of the important elements to affect cell proliferation. Our cell growth curve results show that PARP-1 overexpression HaCaT-PARP-1 cells grow faster than its parental cell. These data are proved in the opposite direction of the previous findings on other U2OS cells that knockdown of PARP-1 increased the number of cells in the G0/G1 phase while reducing the number of cells in the S phase and G2/M phase [22].

Micronucleus formation induced by mitomycin C of HaCaT, HaCaT-PARP-1 cells, and HaCaT-V cells shows that PARP-1 overexpression can cause it hypersensitive to DNA damage agent, but PARP-1 overexpressed cells recovered from damage more quickly maybe because PARP-1 plays an expansive and multifaceted role in the cellular response to DNA damage, with growing evidence for participation in multiple pathways of DNA damage repair and genome maintenance [23, 24]. Detection of DNA damage is one of the currently appreciated roles within repair pathways. Another possible reason for this may be that the level of poly(ADP-ribose) accumulating upon DNA damage is strictly controlled, and any manipulation of polymer steady-state levels interferes with the cellular recovery from DNA damage. According to the protein-shuttle model proposed by Althaus, poly(ADP-ribose) has a role in the transient removal of histones from DNA to facilitate DNA repair [25]. PARP-1 plays a critical role in the maintenance of chromosome stability. Importantly, PARP-1 is involved in the diverse molecular and cellular functions including transcription, DNA repair and recombination, chromatin remodeling, and genome stability [22]. Chromosome stability aberration assay shows that when there is no external stimulus, PARP-1 overexpression does not affect the stability of cell chromosomes.

Consistent with previous studies, we found that nano-SiO₂ treatment significantly reduced cell viability [12]. No matter in HaCaT cells or PARP-1 overexpressing HaCaT cells, cell viability decreased with the increase of nano-SiO₂ dosage. Surprisingly, when the concentration was greater than 10 μg mL⁻¹, the PARP-1 overexpression group had lower cell viability under the same dosage condition. But, a protective effect of PARP-1 overexpression against DNA damage was never observed. Direct evidence for SiO₂-induced DNA damage was obtained by comet analysis and phosphorylation of histone H2AX at serine 139. Comet assay showed that at 5 and 10 μg mL⁻¹ dose, nano-SiO₂ caused more DNA damage and more percentage of DNA in the tail in HaCaT-PARP-1 cells than HaCaT cells (P < 0.05) and the γH2A formation had the similar result. Histone H2AX serves a critical role in the regulation of DNA damage [26]. Our results are consistent with previous studies, which demonstrated that nano-SiO₂ has great potential for inducing DNA damage [27–29]. Klien and Godnić-Cvar’s result [30] showed that 1–2-day exposure is sufficient to cause DNA strand breaks. However, differences in exposure methods must be considered. The molecular mechanism of HaCaT-PARP-1 cell DNA damage caused by nano-SiO₂ is currently unknown, and multiple mechanisms may underlie the detrimental effect of nano-SiO₂ on HaCaT-PARP-1 cell genomic integrity. Chen and von Mikecz [31] reported that SiO₂ nanoparticles, due to their small size, are capable of reaching the nucleus and interacting with DNA. Martinez et al [32] stated that ROS are involved in DNA damage of purine and pyrimidine bases. What is the role of PARP-1 in the process of nano-SiO₂ induced DNA damage in HaCaT cells? To gain a closer insight into the mechanism of nano-SiO₂ induced toxicity and DNA damage in PARP-1 overexpression HaCaT cells, PAR formation of HaCaT cells and HaCaT-PARP-1 cells induced by nano-SiO₂ were determined. Whether it is HaCaT cells or PARP-1 overexpressing HaCaT cells, treatment of the cells with nano-SiO₂ resulted in 2.3-fold higher peak levels of poly(ADP-ribose) in HaCaT-PAPR-1 cells than in HaCaT cells, the PAR formation ability is higher in PARP-1 overexpressed cells. PARP-1 is an abundantly expressed nuclear enzyme, which uses NAD+ as a substrate to poly(ADPribose)ylate (PARylate) nuclear proteins, including automodification of PARP-1 itself [33, 34]. Cell death and DNA damage might be mediated by varying and/or excessive consumption of NAD⁺ by poly(ADP-ribosyl)ation in response to treatment with DNA damaging agents [35, 36], so we concluded that PARP activation was the reason for the increased sensitivity in HaCaT-PARP-1 cells. PARP-1 overexpression in HaCaT cells leads to increased poly(ADP-ribosyl)ation capacity and does not promote survival after nano-SiO₂ treatment.

Conclusion

In summary, expression of PARP-1 failed to protect the cells from cell cytotoxicity and DNA damage induced by nano-SiO₂, but HaCaT-PARP-1 cells has 2.3-fold higher peak levels of poly(ADP-ribose). Combining with our previous studies, PARP-1 knockdown aggravates the DNA damage induced by nano-SiO₂, we guess higher or lower than normal poly(ADP-ribosyl)ation capacity does not protect cells against the cytotoxic effects of nano-SiO₂. An optimal level of PARP-1 activity to successfully cope with cell cytotoxicity and DNA damage.

Funding

This work was supported by the National Natural Science Foundation of China (81202239); Healthcare Research Projects (SZGW2017015) and Guangdong Medical Research Fund Project (2017096).

Conflicts of interest statement

The authors do not have any potential conflict of interest or financial interests to disclose.