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Dec 28, 2016 - Wu, M.S.; Sun, D.S.; Lin, Y.C.; Cheng, C.L.; Hung, S.C.; Chen, P.K.; Yang ... Wong, M.S.; Chen, C.W.; Hsieh, C.C.; Hung, S.C.; Sun, D.S.; Chang, ...
nanomaterials Article

Visible Light-Responsive Platinum-Containing Titania Nanoparticle-Mediated Photocatalysis Induces Nucleotide Insertion, Deletion and Substitution Mutations Der-Shan Sun 1,2 , Yao-Hsuan Tseng 3 , Wen-Shiang Wu 1 , Ming-Show Wong 4 and Hsin-Hou Chang 1,2, * 1 2 3 4

*

Department of Molecular Biology and Human Genetics, Tzu-Chi University, Hualien 97004, Taiwan; [email protected] (D.-S.S.); [email protected] (W.-S.W.) Nanobiomedical Research Center, Tzu-Chi University, Hualien 97004, Taiwan Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei 10607, Taiwan; [email protected] Department of Materials Science and Engineering, National Dong-Hwa University, Hualien 97401, Taiwan; [email protected] Correspondence: [email protected]; Tel.: +886-3-8565301 (ext. 2667)

Academic Editor: Guogang Ren Received: 14 October 2016; Accepted: 22 December 2016; Published: 28 December 2016

Abstract: Conventional photocatalysts are primarily stimulated using ultraviolet (UV) light to elicit reactive oxygen species and have wide applications in environmental and energy fields, including self-cleaning surfaces and sterilization. Because UV illumination is hazardous to humans, visible light-responsive photocatalysts (VLRPs) were discovered and are now applied to increase photocatalysis. However, fundamental questions regarding the ability of VLRPs to trigger DNA mutations and the mutation types it elicits remain elusive. Here, through plasmid transformation and β-galactosidase α-complementation analyses, we observed that visible light-responsive platinum-containing titania (TiO2 ) nanoparticle (NP)-mediated photocatalysis considerably reduces the number of Escherichia coli transformants. This suggests that such photocatalytic reactions cause DNA damage. DNA sequencing results demonstrated that the DNA damage comprises three mutation types, namely nucleotide insertion, deletion and substitution; this is the first study to report the types of mutations occurring after photocatalysis by TiO2 -VLRPs. Our results may facilitate the development and appropriate use of new-generation TiO2 NPs for biomedical applications. Keywords: visible light-responsive photocatalyst; lacZ α-complementation

TiO2 ;

nanoparticle;

DNA mutation;

1. Introduction Antibacterial agents, such as antibiotics and disinfectants, are crucial for personal hygiene, water treatment and food production and in healthcare facilities to control the spread of infectious diseases. The overuse of antibiotics and the emergence of antibiotic-resistant and virulent microbial strains has necessitated the urgent development of alternative sterilization technologies. Despite several advancements in antibiotics research, antibiotic-resistant bacterial infections have become a major clinical challenge worldwide because the frequency of outbreaks and epidemics remains high [1]. Photocatalysts are potentially useful in various settings for reducing pathogen transmission in public environments. Titanium dioxide or titania (TiO2 ) substrates, which are primarily induced using ultraviolet (UV) light, are the most frequently-used photocatalysts for antibacterial applications [2–4]. The photon energy excites electrons from the valence band to the conduction band, generating positive Nanomaterials 2017, 7, 2; doi:10.3390/nano7010002

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Photocatalysts are potentially useful in various settings for reducing pathogen transmission in public environments. Titanium dioxide or titania (TiO2) substrates, which are primarily induced holesusing (electron vacancy) in the valence band. The excited electrons and holes are trapped on the TiO2 ultraviolet (UV) light, are the most frequently-used photocatalysts for antibacterial surfaces. These holes mayexcites then recombine and light or heat,band, resulting applicationselectrons [2–4]. Theand photon energy electrons from therelease valenceenergy band toas the conduction in inefficient photocatalysis. theyinmay react with atmospheric and oxygen to yield generating positive holes Alternatively, (electron vacancy) the valence band. The excited water electrons and holes are a reactive oxygen (ROS),These such electrons as hydrogen peroxide, hydroxyl radicals ·OH) or superoxide trapped on thespecies TiO2 surfaces. and holes may then recombine and (release energy as − )heat, light resulting inare inefficient photocatalysis. they may microorganisms. react with atmospheric anions (O2or [5]. These ROS powerful biocides thatAlternatively, eliminate pathogenic However, water and oxygen yieldatabactericidal reactive oxygen species (ROS), such asdamage hydrogen peroxide, human exposure to UVtolight levels can considerably skin and eyehydroxyl tissues [6,7], radicals (· OH) or superoxide anions (O 2−) [5]. These ROS are powerful biocides that eliminate which limits the use of conventional UV light-induced TiO2 substrates in environments where human pathogenic microorganisms. However, human exposure to UV light at bactericidal levels can exposure may occur. This problem can be resolved by impurity doping TiO2 with different elements, considerably damage skin and eye tissues [6,7], which limits the use of conventional UV such as carbon, sulfur, nitrogen and silver, which shifts the excitation wavelength from the UV region to light-induced TiO2 substrates in environments where human exposure may occur. This problem can the visible light region [2,8–19]. Simultaneously, this process may also recombination be resolved by impurity doping TiO2 with different elements, such as reduce carbon, the sulfur, nitrogen and rates of thesilver, electron andshifts holethe pairs. Visiblewavelength light-responsive antibacterial photocatalysts (which a higher which excitation from the UV region to the visible light regionhave [2,8–19]. quantum efficiency under sunlight than do UV light-responsive photocatalysts) can be safely used in Simultaneously, this process may also reduce the recombination rates of the electron and hole pairs. indoor settings to prevent human exposure to UV light [2,8–11,13–16]. Visible light-responsive antibacterial photocatalysts (which have a higher quantum efficiency under sunlight than dotargets UV light-responsive photocatalysts) can be safely used in indoor settings to prevent The molecular of photocatalysis in bacteria (e.g., DNA, RNA, protein and cell membrane) human exposure to UV these light [2,8–11,13–16]. and the intensity at which are affected remain unclear. Because photocatalytic reactions involve The molecular targets of[20,21], photocatalysis in bacteria (e.g., RNA, protein and cell membrane) both oxidation and reduction damage observed inDNA, the target microorganisms differs from and the intensity at which these are affected remain unclear. Because photocatalytic reactions involve that observed with traditional disinfectants, which involve either oxidation or reduction. This is both oxidation and reduction [20,21], damage observed in the target microorganisms differs from probably the reason that we previously observed a unique pattern of photocatalysis-induced bacterial that observed with traditional disinfectants, which involve either oxidation or reduction. This is destruction [2,10]. UV that light-responsive TiO2 induces DNA damage without specified bacterial temperature probably the reason we previously observed a unique pattern of photocatalysis-induced control [22,23]; however, UV light alone can also induce DNA mutation and damage [24,25]. destruction [2,10]. UV light-responsive TiO2 induces DNA damage without specified temperature ◦ In addition, atUV room temperature (25 induce C) induces DNA damage than that In at 4 ◦ C control photocatalysis [22,23]; however, light alone can also DNA more mutation and damage [24,25]. (Figure 1). Thephotocatalysis absorption of light (25 energy can produce heat;damage thus, illumination-induced addition, at illuminated room temperature °C) induces more DNA than that at 4 °C (Figure 1). The absorption of illuminated light energy candamage. produce heat; thus, illumination-induced heat also potentially has a major role in triggering DNA However, visible light-responsive heat also potentially has a major in triggering damage. However, visible light-responsive photocatalyst (VLRP)-induced DNArole mutations haveDNA not been clearly characterized thus far. Therefore, photocatalyst (VLRP)-induced DNA mutations have not been clearly characterized the exact photocatalysis-induced DNA damage, without perturbations of the effects of thus heat far. and UV Therefore, the exact photocatalysis-induced DNA damage, without perturbations of the effects of light, warrants further investigation. In the present study, we used a previously-reported visible heat and UV light, warrants further investigation. In the present study, we used a previouslylight-responsive platinum-containing titania (TiO2 -Pt) photocatalytic nanoparticle (NP) [11,16] to reported visible light-responsive platinum-containing titania (TiO2-Pt) photocatalytic nanoparticle address this question. Ourthis data revealed that VLRPs canthat induce DNA mutations. (NP) [11,16] to address question. Our data revealed VLRPs can induce DNA mutations.

Figure 1. Influence of heat on photocatalyst-induced DNA damage. Plasmid pBlueScript II SK DNA Figure 1. Influence of heat on photocatalyst-induced DNA damage. Plasmid pBlueScript II SK+ DNA was transformed to Escherichia coli (E. coli) competent cells after photocatalysis environments set to ◦ was transformed to Escherichia coli (E. coli) competent cells after photocatalysis environments set to 4 C 4 °C and 25 °C. The level of DNA damage involving ultraviolet (UV)- and heat-induced nanoscaleand 25 ◦ C. The level of DNA damage involving ultraviolet (UV)- and heat-induced nanoscale-TiO2 TiO2 film-mediated photocatalysis was indicated by the reduction of transformants. * p < 0.05 vs. 4 °C film-mediated photocatalysis was indicated by the reduction of transformants. * p < 0.05 vs. 4 ◦ C group. group. n = 6, three experiments with two replicates. n = 6, three experiments with two replicates. +

2. Results

2. Results 2.1. Involvement of Heat in VLRP-Induced Plasmid DNA Damage Photocatalysts can absorb light energy and produce heat [26]; simultaneously, heat can also induce ROS production and DNA damage [27]. Herein, we observed that the temperature of photocatalytic

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2.1. Involvement of Heat in VLRP-Induced Plasmid DNA Damage

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Photocatalysts can absorb light energy and produce heat [26]; simultaneously, heat can also ◦ C. To investigate whether films underROS UV production irradiation rapidly increased, than 200 that induce and DNA damageeasily [27].reaching Herein, more we observed the temperature of temperature is involved in photocatalytic damage, weeasily used reaching UV-irradiated single-layer TiO2 photocatalytic films under UV irradiation DNA rapidly increased, more than 200 °C. To + ◦ thin films [13]whether to catalyze plasmid (pBlueScript II SK DNA ) in environments at 25 C and 4 ◦ C. investigate temperature is DNA involved in photocatalytic damage, we used UV-irradiated After transforming plasmid DNA into Escherichia (E. coli) cells,atwe single-layer TiO2 the thinphotocatalyzed films [13] to catalyze plasmid DNAcompetent (pBlueScript II SK+) incoli environments ◦ C contained considerably more transformants observed that the experimental samples catalyzed at 4 25 °C and 4 °C. After transforming the photocatalyzed plasmid DNA into competent Escherichia coli ◦ C (Figure 1). These results suggest that illumination-induced heat also than at 25that (E.did coli)those cells,catalyzed we observed the experimental samples catalyzed at 4 °C contained considerably plays a major role in inducing DNA damage. more transformants than did those catalyzed at 25 °C (Figure 1). These results suggest that illumination-induced heat also plays a major role in inducing DNA damage. 2.2. VLRP Induces Plasmid DNA Damage 2.2. VLRP Induces Plasmid DNA Damage Both UV light and heat contributed to the DNA damage noted herein; thus, to analyze the DNA UV lightinduced and heatthrough contributed to the DNA damage noted herein; thus, to analyze the DNA damageBoth specifically photocatalysis, we performed a photocatalysis of plasmid DNA damage specifically induced through photocatalysis, we performed a photocatalysis of plasmid DNA using visible light-responsive TiO2 -Pt NPs as compared to UV-responsive pure-anatase TiO2 NPs at 2-Pt NPs as compared UV-responsiveconsiderably pure-anatasedecreased TiO2 NPs with at 4 ◦using C for 1visible h. Thelight-responsive results revealedTiO that the number of E. colitotransformants °C for 1 h. results revealed that the number(Figure of E. coli2A; transformants considerably decreased the4increase inThe visible light illumination intensity TiO2 -Pt groups), indicating that thewith DNA the increase in visible light illumination intensity (Figure 2A;intensity TiO2-Pt groups), indicating that the damage is induced in a dose-dependent manner. Because light of 104 lux is an effective dose 4 lux is an effective DNA damage is induced in a dose-dependent manner. Because light intensity of 10 to reduce the E. coli transformants (Figure 2A), we used that as the constant illumination intensity dose to reduce the E. coli time transformants we Here, used we thatnoted as thea decrease constant illumination with increasing illumination to obtain (Figure a kinetic2A), result. in the number intensity with increasing illumination time to obtain a kinetic result. Here, we noted a decrease in the of transformants, associated with the increasing illumination time (Figure 2B). The DNA samples of number of transformants, associated with the increasing illumination time (Figure 2B). The DNA those dark groups were covered with aluminum foil to prevent photocatalysis, and thus, no particular samples of those dark groups were covered with aluminum foil to prevent photocatalysis, and thus, response occurred. Because the pure anatase TiO2 NPs are UV-responsive, it is reasonable that no DNA no particular response occurred. Because the pure anatase TiO2 NPs are UV-responsive, it is damage was observed in the “TiO2 light” groups (Figure 2A,B). These results confirm that VLRPs can reasonable that no DNA damage was observed in the “TiO2 light” groups (Figure 2A,B). These results elicit DNAthat damage. confirm VLRPs can elicit DNA damage.

Figure 2. (A) Dose-dependent and (B) kinetic responses, with increasing illumination density and Figure 2. (A) Dose-dependent and (B) kinetic responses, with increasing illumination density and with increasing time, respectively. The visible light stimulated TiO2-Pt photocatalysis-mediated DNA with increasing time, respectively. The visible light stimulated TiO2 -Pt photocatalysis-mediated DNA damage was determined by the reduction of transformants. The DNA samples of those dark groups damage was determined by the reduction of transformants. The DNA samples of those dark groups were covered with aluminum foil to prevent the photocatalysis. UV-responsive TiO2 NPs were used were covered with aluminum foil to prevent the photocatalysis. UV-responsive TiO2 NPs were used as control materials. ** p < 0.01 vs. respective TiO2-Pt dark groups. n = 6, three experiments with two as control materials. ** p < 0.01 vs. respective TiO2 -Pt dark groups. n = 6, three experiments with replicates. two replicates.

2.3. Application of VLRP-Induced DNA to Different Plasmids 2.3. Application of VLRP-Induced DNA to Different Plasmids We subsequently investigated whether our noted visible light-responsive TiO2-Pt NP-mediated We subsequently investigated whether ourapplicable noted visible light-responsive NP-mediated photocatalysis-induced DNA damage is also to different plasmids.TiO We2 -Pt employed two photocatalysis-induced DNA damage also applicable different plasmids. We employed two additional plasmids, pGEM-2KS and is pET21, which are to used primarily in bacterial recombinant additional plasmids, pGEM-2KS and pET21, which are used primarily in bacterial recombinant protein protein expression [28–38], and observed that VLRPs induced considerable damage in all three + (Figure plasmids,[28–38], including theobserved pBlueScript II SK 3). expression and that VLRPs induced considerable damage in all three plasmids, + including the pBlueScript II SK (Figure 3).

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Figure 3. Significant visible light stimulated TiO2 -Pt-mediated photocatalysis-induced DNA damage occurred in the pGEX-2KS,visible pET16 and pBlueScript SK+ plasmids. Notably, these plasmids all -Pt-mediated photocatalysis-induced DNA Figure 3. Significant light stimulated TiO2II Figure 3. Significant visible light stimulated TiO 2-Pt-mediated photocatalysis-induced DNA damage + vs. respective displayed similar reductions after the photocatalysis. ** p < 0.01; * p < 0.05 dark groups. damage occurred in the pGEX-2KS, pET16 and pBlueScript II SK plasmids. Notably, these in the pGEX-2KS, pET16 and pBlueScript II SK+ plasmids. Notably, these plasmids all n = 6,occurred three experiments with two replicates. plasmids all displayed similar reductions after the photocatalysis. ** p < 0.01; * p < 0.05 vs. displayed similar reductions after the photocatalysis. ** p < 0.01; * p < 0.05 vs. respective dark groups. respective dark n = 6, three experiments with two replicates. n = 6, three experiments with twogroups. replicates.

2.4. VLRP Induces Mutations in Plasmid DNA

VLRP Induces Mutations in Plasmid DNA 2.4.2.4. VLRP Induces Mutations in Plasmid DNA To further investigate whether photocatalysis induces mutated DNA damage, we performed an α-complementation analysis of the β-galactosidase gene lacZ using pBlueScript II SK+ and To further investigate whether photocatalysis induces mutated DNA damage, we performed To further investigate whether photocatalysis induces mutated DNA damage, we performed an α- an of the β-galactosidase gene lacZ using pBlueScript II SK+expressed and E. coliα-complementation XL1-blue (Figureanalysis S1). Notably, E. coli XL1-blue cells with wild-type plasmids complementation analysis of the β-galactosidase gene lacZ using pBlueScript II SK+ and E. coli E. coliα-peptide, XL1-blue (Figure S1). Notably, E.the coli XL1-bluelacZ cellstowith wild-type plasmidsinexpressed functional which complements defective produce blue colonies the presence XL1-blue (Figurewhich S1). Notably, E. coli XL1-blue cellslacZ withtowild-type expressed α-peptide, complements the defective produce plasmids blue colonies in the(Figure presence4A,B). of thefunctional chromogenic substrate 5-bromo-4-chloro-3-indolyl-βD-galactopyranoside (X-gal) functional α-peptide, which complements the defective lacZ to produce blue colonies in the (Figure of the chromogenic substrate 5-bromo-4-chloro-3-indolyl-βD-galactopyranoside (X-gal) By contrast, the mutant transformant cells containing damaged DNA in the lacZα region did not of thethe chromogenic substrate 5-bromo-4-chloro-3-indolyl-β-galactopyranoside (X- did 4A,B).presence By contrast, mutant transformant cells containing damaged D DNA in the lacZα region produce blue colonies and instead remained white. Thus, using this method, we can differentiate lacZα not produce blue 4A,B). colonies instead white. Thus, using this method, we can differentiate gal) (Figure Byand contrast, theremained mutant transformant cells containing damaged DNA in the mutation-type (white) and wild-type (blue) clones. Even at an extremely low frequency (14/2000; lacZαlacZα mutation-type (white) andblue wild-type (blue) clones. Even white. at an Thus, extremely lowmethod, frequency region did not produce colonies and instead remained using this 0.7%),(14/2000; visible light-responsive TiO2 -Pt NP-mediated photocatalysis can induce white colony formation 0.7%), visible light-responsive TiO 2-Pt NP-mediated photocatalysis can induce white colony we can differentiate lacZα mutation-type (white) and wild-type (blue) clones. Even at an (Figure 4C), indicating thatindicating mutations generated the lacZα region. formation (Figure 4C), thatwere mutations were in generated in the lacZα region. extremely low frequency (14/2000; 0.7%), visible light-responsive TiO2-Pt NP-mediated photocatalysis can induce white colony formation (Figure 4C), indicating that mutations were generated in the lacZα region.

Figure 4. Detection of mutated clones using lacZ α-peptide complementation. (A,B) After being

Figure 4. Detection of mutated clones using lacZ α-peptide complementation. (A,B) After being complemented with lacZ α-peptide expression, the transformants are displayed as blue colonies on complemented with lacZ α-peptide expression, the transformants are displayed as blue colonies on the the agar plates with 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside. The VLRP TiO2-Pt agar plates with 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside. The VLRP TiO2 -Pt NP-mediated NP-mediated photocatalysis markedly reduces the number of transformants, compared with the photocatalysis markedly reduces the TiO number of transformants, compared with the control groups control groups using UV-responsive 2 NPs, under visible light illumination. (C) Quantified results usingshow UV-responsive illumination. (C)colonies. Quantified 2 NPs, under that TiO2-Pt TiO photocatalysis can visible induce light the formation of white This results indicatesshow that that TiO2 -Pt photocatalysis can induce formation of white colonies. This indicates that mutations hit the mutations hit the lacZα region the because of a loss-of-function (loss-of-complementation) phenotype, with theofwild-type plasmid-transformed blue colonies. ND: no detected colony. * p < 0.05 lacZαcompared region because a loss-of-function (loss-of-complementation) phenotype, compared with the vs. both blue groups of TiO2-visible light and ND: TiO2-Pt-dark; ** p