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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

UV rays in sunlight are an important factor in the degradation of chemicals. In this study, we investigated the degradation of nonionic surfactants, nonylphenol polyethoxylates (NPEOs) with 10 or 70 ethylene oxide (EO) units using UVA, B and C, and their genotoxic change based on phosphorylation of histone H2AX (γ-H2AX), a marker of DNA damage. NPEOs were degraded dependent on the energy of UV, that is, UVC having the highest energy was most effective, whereas UVA having the lowest energy caused little change. The EO side chain of NPEO(70) was broken near the benzene ring by UV, producing NPEOs with a shortened EO chain (around 10 units). The generation of γ-H2AX reflected the pattern of degradation; shortening of the EO chain changed NPEO(70) into an inducer for γ-H2AX, and degradation of NPEO(10) attenuated the genotoxicity. The γ-H2AX generated by NPEO(10) and UV-degraded NPEO(70) was independent of the cell cycle. The formation of DNA double strand breaks detected by gel electrophoresis was consistent with the results for γ-H2AX. These results suggested that UV rays can make NPEOs harmless or genotoxic according to the degradation of the EO side chain, the effects being dependent on wavelength.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

The use of synthetic detergents is increasing every year. Nonylphenol polyethoxylates (NPEO(n), where n is the number of ethylene oxide units [EO]) are widely used as nonionic surfactants in detergents for industrial and household use. Because of the large scale of their use, organic materials in waste water are contaminated by NPEOs [1-4]. NPEOs released in environments are broken down by microorganisms; first, aerobically into nonylphenol di- and monoethoxylates, then, anaerobically into nonylphenols [4, 5]. The biodegraded products are stable, and have been reported to be toxic to both marine and freshwater species [5, 6].

Natural sunlight can cause the transformation of NPEOs. Ultraviolet (UV) rays in sunlight have proven effective at degrading both NPEOs and biodegraded nonylphenols [7-9]. The UV spectrum can be divided into three based on wavelength: UVA (320–400 nm), UVB (280–320 nm) and UVC (200–280 nm). UVC is absorbed by the ozone layer and does not reach the earth. The shorter wavelength UVB has higher energy than UVA, and consequently, contributes to the photodegradation of chemical compounds in the environment [10, 11]. On exposure to UVB, NPEOs showed characteristic degradation patterns dependent on the lengths of their EO chains [7]. However, UV wavelength-dependent degradation and changes of toxicity have not been examined.

Our recent study found that UVB made nongenotoxic NPEOs genotoxic by shortening of EO units [12]. NPEOs with short degraded EO chains produced by exposure to UVB, induced serious DNA damage, DNA double strand breaks (DSBs). We also have elucidated the genotoxic potential of NPEO(n) having various EO units (= 0–70); the genotoxicity was strongly dependent on the number of EO units, that is, NPEOs having fewer EO units (= 0–15) showed a strong ability to induce DNA damage, whereas NPEOs with longer side chains like NPEO(70) had attenuated genotoxicity [13]. Promotion of carcinogenesis by nonylphenols was reported via a mechanism involving the stimulation of cell proliferation and induction of oxidative DNA damage [14, 15]. Therefore, DNA damage induced by NPEOs degraded by UV is an important target for further risk assessments.

We have been using the phosphorylation of histone H2AX (γ-H2AX) as a marker for DNA damage. The generation of γ-H2AX, originally identified as an early event after the direct formation of DSBs by ionizing radiation [16], is now considered to occur also after the indirect formation of DSBs caused by the collision of the replication forks at sites of DNA damage including oxidative bases, DNA adducts, etc. [17, 18]. We previously reported that γ-H2AX was generated following the exposure of cells to various suspected DNA-damaging agents including several environmental chemicals and pharmacological agents [19-24]. In addition to the advantage that γ-H2AX can be detected in response to many types of DNA damage, we and other researchers are convinced that γ-H2AX provides a considerably more sensitive and convenient measurement of DNA damage than other techniques such as pulse field gel electrophoresis and comet assays [17, 18].

In this study, we analyzed the degradation of NPEO(10) and (70) after exposure to different wavelengths of UV (UVA, B and C), and the correlation with their genotoxic change. The genotoxicity of degraded NPEOs was examined based on the generation of γ-H2AX.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

NPEOs and UV irradiation

NPEO(n) (= 10 and 70) kindly provided by NOF Co., Japan, were dissolved in water at a concentration of 10 mm, and exposed to UV in a glass dish 15 mm in diameter and 10 mm in height (1 mL per dish). To avoid water evaporation, the glass dish was sealed with UV-transmittable film (Dura Seal: Diversified Biotech, Dedham, MA). A UVA lamp (HP-30LM; Atto Co., Japan) with an emission wavelength of 320–380 nm and maximum peak of 365 nm, a UVB lamp (HP-30LM; Atto) with a 280–320 nm emission and maximum peak of 312 nm and a UVC lamp (HP-30C; Atto) with a 180–280 nm emission and maximum peak of 254 nm were used without cutoff filter. The spectra were shown in our previous study [25]. During the exposure, fluences were simultaneously measured and integrated using a radiometer (ATV-3W; Atto) with 365, 312 and 254 nm detectors placed at the same distance as the glass dish from the UV source. The approximate irradiances of UVA, UVB and UVC at the sample level were 4.6, 8.5 and 8.2 J cm−2 h−1, respectively.

Analysis of NPEOs degraded by UV irradiation

The degraded NPEOs were detected using high performance liquid chromatography (HPLC). The conditions for the HPLC analysis were given in a previous study [7]. In brief, NPEOs and UV-degraded products were separated on a silica column (TSKgel Silica-150 [4.6 mmID × 25 cm]). The mobile phase solvent A was 30% acetonitrile and solvent B was 80% acetonitrile. Elution was carried out with a liner gradient from 100% A to 100% B over 30 min.

Cells and culture conditions

The human breast adenocarcinoma cell line MCF-7 (provided by Japanese Collection of Research Bioresources, Japan) was maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 100 U mL−1 of penicillin/streptomycin at 37°C in an atmosphere of 5% CO2. All experiments were performed with exponentially growing cells.

Immunofluorescence staining for detection of γ-H2AX

The cells treated with UV-irradiated NPEOs in Lab-Tek chamber slides (Nalge Nunc, IL) were immediately fixed in 2% paraformaldehyde for 30 min at room temperature and then in 100% methanol for 20 min at −20°C. Fixed cells were immersed in buffer containing 100 mm Tris-HCl, 50 mm EDTA and 0.5% Triton X-100 for 5 min at room temperature for better permealization, and blocked with 1% bovine serum albumin (BSA) for 30 min at 37°C. Cells were incubated with a primary antibody against phospho-H2AX (mouse monoclonal) (Millipore Bedford, MA, 1:200) for 2 h, then with a secondary antibody conjugated with fluorescein isothiocyanate (FITC) (Jackson Immuno Research Laboratories, PA). To confirm the distribution of foci, the nucleus was stained with propidium iodide (PI) (20 μg mL−1). Images were acquired on a fluorescence microscope (BX51, Olympus, Japan).

Western blot analysis of γ-H2AX

The cells treated with UV-irradiated NPEOs were lysed in lysis buffer (50 mm Tris [pH 8.0], 5 mm EDTA, 150 mm NaCl, 0.5% Nonidet P-40 and 1 mm phenylmethylsulfonyl fluoride [PMSF]). The samples were separated by 12.5% SDS-PAGE, and blotted onto polyvinylidine fluoride (PVDF) membranes. After blocking with 1% nonfat milk, the membranes were incubated overnight at 4°C with a primary antibody against phospho-H2AX (rabbit polyclonal) (1:1000) or actin (Santa Cruz Biotechnol. CA, 1:1000), then with a secondary antibody conjugated with horseradish peroxidase (Jackson Immuno Research Laboratories, PA) for 1 h. The bands of γ-H2AX were visualized with an enhanced chemiluminescence detection kit (GE Healthcare Ltd. UK).

Flow cytometric analysis of γ-H2AX and cell cycle distribution

The cells treated with UV-irradiated NPEOs were fixed in ice-cold 70% ethanol and kept at −20°C for at least 2 h. The fixed cells were centrifuged at 400 g rpm for 5 min and washed twice with PBS. They were resuspended in PBS containing 0.2% triton X-100 and 1% BSA (BSA-T-PBS) and kept at room temperature for 15 min. Cells were incubated with phospho-H2AX (mouse monoclonal) (1:200) for 1 h, then with a secondary antibody conjugated with FITC (1:200) (Jackson Immuno Research Laboratories, PA) for 1 h in BSA-T-PBS. After the immunoreaction, the cells were resuspended in BSA-T-PBS containing 1 μg mL−1 RNase A. PI (10 μg mL−1) was added prior to the measurement for the cell cycle analysis. The fluorescence intensity of FITC and PI was determined using flow cytometry (FCM) (FACS CANTTM II; Becton Dickinson, Franklin Lakes, NJ). At least 10 000 cells per sample were analyzed.

Detection of DNA double strand breaks

DSBs were detected with a biased sinusoidal field gel electrophoresis (BSFGE) system (Atto, Japan). The cells treated with UV-irradiated NPEOs were solidified in 1% low-melting agarose. The agarose plugs were treated with proteinase K (0.5 mg mL−1) and ribonuclease A (1 mg mL−1), and electrophoresed in a 0.8% agarose gel. The gel was visualized by staining with ethidium bromide.

All experiments above were repeated two or three times.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Degradation of NPEOs by UV having different wavelengths

The degradation of NPEO(10) and NPEO(70) after UVA, B and C irradiation was analyzed using HPLC. Each degraded product of NPEO(10) and NPEO(70) was separated on the same HPLC column using a mobile phase buffer of acetonitrile/water as described in 'Materials and methods' (Fig. 1). The retention times of NPEO(10) and NPEO(70) were 4 min and 20–25 min, respectively. In Fig. 1A, the peak of NPEO(10) became smaller with exposure to UVB and UVC, whereas the degradation induced by UVA was slight.

image

Figure 1. HPLC analysis of UV-irradiated NEPOs. UV (~1000 J cm−2)-irradiated NPEO(10) (A) and NPEO(70) (B) were analyzed using HPLC.

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In Fig. 1B, the peak of NPEO(70) decreased after UV irradiation and the peaks of NPEOs having short side chains (around 10 units long) appeared, which were remarkable after UVB and UVC irradiation. UVA caused some degradation of NPEO(70), which was observed as a decrease in the peak of NPEO(70) and the appearance of a peak of NPEO (around 10). Intermediate peaks between NPEO(10) and (70), for example NPEO(40), did not appear under any UV irradiation. With UVC irradiation at 1000 J cm−2, the peak of NPEO(70) completely disappeared and the peaks of NPEOs having short side chains (around 10 units) also became negligibly small.

Generation of γ-H2AX after treatment with UV-irradiated NPEOs

We recently showed NPEO(10), not NPEO(70), to be genotoxic, using γ-H2AX, a marker of DNA damage [13]. The toxicity changed with the degradation of EO units by UVB irradiation [12]. As NPEOs showed different degradation rates dependent on UV wavelengths, a corresponding change in genotoxic ability was expected. Figure 2A shows images of the γ-H2AX generated by NPEO(10). NPEO(10) produced a remarkable number of γ-H2AX foci in the nucleus. UVB and UVC, but not UVA, decreased the number (Fig. 2B). Conversely, NPEO(70) generated no foci of γ-H2AX until after UV irradiation (Fig. 2C). NPEO(70) exposed to UVB (500 J cm−2) produced γ-H2AX, but that exposed to an excess dose (1000 J cm−2) did not. UVC (250 J cm−2) caused similar levels of γ-H2AX (data not shown) and excess exposure (500 and 1000 J cm−2) gradually decreased the amount. For UVA, 1000 J cm−2 was needed for the formation of γ-H2AX foci.

image

Figure 2. Images of formation of γ-H2AX foci after treatment with UV-irradiated NPEOs. (A) Magnified images of γ-H2AX foci produced by treatment with UV-irradiated NPEO(10) (300 μm) for 4 h. Nuclei were stained with PI. Left panels: γ-H2AX, center panels: nuclei stained by PI, right panels: merged images. (B) γ-H2AX produced by NPEO(10) exposed to UVA, B and C (500 and 1000 J cm−2). (C) γ-H2AX produced by NPEO(70) exposed to UVA, B and C (500 and 1000 J cm−2).

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These changes were analyzed in detail by Western blotting (Fig. 3). UVA irradiation only slightly decreased the amount ofγ-H2AX generated by NPEO(10) (Fig. 3A). Moreover, 500 J cm−2 of UVB and 250 J cm−2 of UVC markedly suppressed γ-H2AX production by NPEO(10). With NPEO(70) (Fig. 3B), high doses (750–1000 J cm−2) of UVA and doses of UVB above 250 J cm−2 resulted in γ-H2AX production. γ-H2AX was also generated with small doses of UVC, but excess exposure reversed the process. These results were consistent with the results of formation of γ-H2AX foci in Fig. 2.

image

Figure 3. Generation of γ-H2AX after treatment with UV-irradiated NPEOs. γ-H2AX after treatment with UV-irradiated NPEO(10) (A) and NPEO(70) (B) (100 μm) for 4 h was detected using Western blotting. Actin was used as a standard for the equal loading of proteins for SDS-PAGE.

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Cell cycle-dependent generation of γ-H2AX was examined using FCM (Fig. 4). After treatment with NPEO(10), γ-H2AX was formed independently of the cell cycle. The γ-H2AX produced by NPEO(10) disappeared after exposure to UVB and UVC. Conversely, NPEO(70) produced no γ-H2AX in any phase of the cell cycle. UV irradiation caused NPEO(70) to generate γ-H2AX. The generation of γ-H2AX by NPEO(70) exposed to UVA, B and C was independent of the cell cycle.

image

Figure 4. Flow cytometric analysis of γ-H2AX and cell cycle phases after treatment with UV-irradiated NPEOs. Cells treated with NPEOs for 4 h were fixed and immune stained with γ-H2AX antibody and PI as described in 'Materials and methods'. The cells were analyzed using FCM and cell cycle-dependent formation of γ-H2AX was revealed as dotted blots. (A): NPEO(10), (B): NPEO(70).

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Formation of DSBs after treatment with UV-irradiated NPEOs

As the generation of γ-H2AX is attributed to the formation of DSBs, DSBs induced by treatment with UV-irradiated NPEO(10) and NPEO(70) for 4 h were detected by BSFGE (Fig. 5). NPEO(10) caused DNA migration, but NPEO(70) did not. UVA irradiation did not influence the migration, whereas UVB and UVC irradiation attenuated it in a dose-dependent manner. Conversely, irradiation caused NPEO(70) to damage DNA, with UVC most effective, then UVB and UVA. These results were consistent with the data for γ-H2AX in Figs. 2-4.

image

Figure 5. Formation of DSBs after treatment with UV-irradiated NPEOs. Cells treated with NPEOs for 4 h were solidified in 1% low-melting agarose and treated as described in 'Materials and methods'. The gel stacks containing the cells were loaded onto a 0.8% agarose gel, and BSFGE was carried out. (A): NPEO(10), (B): NPEO(70).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

UV has been used to degrade several environmental contaminants [26-28]. For some chemicals, the degradation process is advanced in the presence of ozone and photocatalysts [28-30]. The UV spectrum can be divided into UVA (320–400 nm), UVB (280–320 nm) and UVC (200–280 nm), with the energy ranking in decreasing order: UVC > UVB > UVA. In this study, we found that NPEOs were degraded dependent on the energy of UV, that is, UVC was most effective and UVA caused little change. As the benzene ring in NPEOs absorbs shorter wavebands of UV having high energy, the degradation by UVB and UVC is more effective than that by UVA.

The changes in HPLC patterns following exposure to UVB or UVC suggested the EO side chain to be degraded with the benzene ring. The photolysis of the EO side chain differed between NPEO(10) and (70). This had been found in our previous study using UVB [7], that is, the EO side chain of NPEO(10) was gradually degraded from the end of the EO unit, but that of NPEO(70) was degraded from near the benzene ring. As shown in Supplementary Fig. 1, NPEO(10) lost its side chain, depending on the dose of UVB and C. NPEO(70) was degraded by UVA, B and C, resulting in the production of NPEOs having around 10 EO units, not NPEOs having more EO units (Fig. 1). Ahel et al. [31] reported that photochemical degradation of both NPEOs and NP was mainly due to sensitized photolysis and that direct photolysis was slow, in which singlet oxygen was not important. Some reports showed that hydroxyl radicals can react with polyethoxylated chains of alcohol ethoxylates and octylphenol ethoxylates as well as aromatic rings [32, 33]. We suspected that the reactive oxygen species (ROS) produced following energy absorption of UVB and UVC by benzene rings would attack the EO side chains of NPEOs; however, the exact mechanism was not clarified.

γ-H2AX is currently attracting attention as a new biomarker for detecting genotoxic insults [17, 18]. Based on γ-H2AX, we recently showed that the genotoxic effect of NPEOs was strongly dependent on the number of EO units and that UVB irradiation drastically changed the genotoxic potential of NPEO(15) and NPEO(70) [12]. NPEO(15) showed a great ability to form γ-H2AX, which was reduced by UVB irradiation. Conversely, nongenotoxic NPEO(70) was able to generate γ-H2AX after UVB irradiation. In this study, γ-H2AX production reflected the degradation patterns of NPEO(10) and NPEO(70) according to wavelengths of UV as shown in Fig. 1. Dependent on the energy of UV, the generation of γ-H2AX by NPEO(10) was attenuated, whereas NPEO(70) which has no genotoxicity became able to generate γ-H2AX and excess irradiation made it nongenotoxic again. The patterns of DNA migration detected by BSFGE were almost the same. As γ-H2AX has been considered to be caused by DSBs [16], we confirmed the formation of DSBs by NPEO(10) and degraded NPEO(70). From our previous study, the generation of γ-H2AX by NPEO(15) and NPEO(70) degraded by UVB was independent of the cell cycle [12]. γ-H2AX generated by NPEO(10) and NPEO(70) degraded by different wavelengths of UV was also observed throughout the cell cycle (Fig. 4). DNA lesions would later be converted into DSBs as a consequence of the collision of the replication forks. Therefore, if DNA lesions like oxidative bases formed, γ-H2AX would be detected mainly in the S phase [34]. This means that NPEO(10) and degraded NPEO(70) did not form γ-H2AX via replication stress.

DSBs are a serious form of damage which can lead to cell death and mutation. Cell death patterns were similar with the generation of γ-H2AX and DSBs in Figs. 3-5 (Supplementary Fig. 2). Mistakes in the repair of DSBs may be an important factor in the development of genomic instability [35]. At least two mechanisms, homologous recombination (HR) and nonhomologous end joining (NHEJ), are known for the repair of DSBs. Although HR is an error-free repair pathway, NHEJ is an error-prone one. Increased NHEJ misrepair in response to excess DSBs formed by NPEOs and degraded NPEOs could lead to increased genomic instability. Therefore, appearance of genotoxicity in NPEOs with long EO chains after exposure to several wavelengths of UV is important for risk assessment of NPEOs.

In environments, several contaminants with NPEOs would affect degradation. In the absence of contaminants, UVB might be effective for the degradation of NPEOs, whereas in the presence of contaminants, the more permeable UVA might be effective because short wavelengths of UV are easily masked by contaminants; however, selective production of NPEO having short EO chains would be a problem. The intermediates in the degradation of NPEOs by sunlight should receive attention because they could be detrimental to living organisms.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

This work was supported in part by Grants-in-aid for Young Scientific Research (B) (#22710065) and for Scientific Research (C) (#24510084) from the Ministry of Education, Culture, Sports, Science and Technology, and for Research on Risk of Chemical Substances from the Ministry of Health, Labor, and Welfare of Japan.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
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php12002-sup-0001-FigS1-S2.docxWord document232K

Figure S1. HPLC analysis of UV-irradiated NEPO(10). UV (~1000 J cm−2)-irradiated NPEO (10) was analyzed using HPLC (mobile phase: n-hexane, isopropanol and water). NP and NPEO(6) as standards separated as a single peak, whereas NPEO(10) showed multiple peaks because it was produced for industry and contained NPEOs with slightly different side chain lengths. NP and NPEOs with shorter side chains (~10) appeared after UVB and UVC irradiation, depending on the irradiation dose. The gradual degradation and disappearance of side chains were more remarkable after UVC than UVB irradiation.

Figure S2. Cytotoxicity of NPEOs irradiated with UV. Human fibroblasts, ASF4-1 cells, were treated with NPEO(10) (A) and NPEO(70) (B) irradiated (~1000 J cm−2) at a concentration of 100 μm. Cell survival 24 h after the treatment was determined by alamer BlueTM assay (Diagnostic systems Inc., USA). Values are the mean ± SD (= 5). *< 0.05, ***< 0.001

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