Polycyclic Aromatic Compounds Chlorinated

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Chlorinated Polycyclic Aromatic Hydrocarbons Associated with Drinking Water Disinfection: Synthesis, Formation under Aqueous Chlorination Conditions and Genotoxic Effects a

Miguel Pinto , Marlene Rebola b

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, Henriqueta Louro , Alexandra M. c

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M. Antunes , Silvia S. José , Maria Rocha , Maria João Silva & Ana Sofia Cardoso

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Instituto Nacional de Saúde, Dr. Ricardo Jorge, I.P., Departamento de Genética Humana, Lisbon, Portugal b

Instituto Superior Técnico, Universidade de Lisboa, Centro de Química Estrutural, Lisbon, Portugal c

Instituto Nacional de Saúde, Dr. Ricardo Jorge, I.P., Departamento Saúde Ambiental, Lisbon, Portugal Published online: 13 Jun 2014.

To cite this article: Miguel Pinto, Marlene Rebola, Henriqueta Louro, Alexandra M. M. Antunes, Silvia S. José, Maria Rocha, Maria João Silva & Ana Sofia Cardoso (2014) Chlorinated Polycyclic Aromatic Hydrocarbons Associated with Drinking Water Disinfection: Synthesis, Formation under Aqueous Chlorination Conditions and Genotoxic Effects, Polycyclic Aromatic Compounds, 34:4, 356-371, DOI: 10.1080/10406638.2014.891143 To link to this article: http://dx.doi.org/10.1080/10406638.2014.891143

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Polycyclic Aromatic Compounds, 34:356–371, 2014 C Taylor & Francis Group, LLC Copyright ISSN: 1040-6638 print / 1563-5333 online DOI: 10.1080/10406638.2014.891143

Chlorinated Polycyclic Aromatic Hydrocarbons Associated with Drinking Water Disinfection: Synthesis, Formation under Aqueous Chlorination Conditions and Genotoxic Effects Miguel Pinto,1 Marlene Rebola,1,2,3 Henriqueta Louro,1 Alexandra M. M. Antunes,2 Silvia S. Jose, ´ 3 Maria Rocha,3 Maria Joao ˜ Silva,1 and Ana Sofia Cardoso3 1

´ Instituto Nacional de Saude, Dr. Ricardo Jorge, I.P., Departamento de Gen´etica Humana, Lisbon, Portugal 2 Instituto Superior T´ecnico, Universidade de Lisboa, Centro de Qu´ımica Estrutural, Lisbon, Portugal 3 ´ ´ Ambiental, Instituto Nacional de Saude, Dr. Ricardo Jorge, I.P., Departamento Saude Lisbon, Portugal Polycyclic aromatic hydrocarbons (PAHs) are among the most persistent and toxic organic micropollutants present in water and several of them are mutagenic and carcinogenic. Although it has been shown that chlorinated derivatives of PAHs (Cl-PAHs) may be formed during the water chlorination procedure, little is known about their potential genotoxic and carcinogenic effects. The objectives of the present work were to prepare and characterize the major chlorinated derivatives of benzo[a]pyrene (BaP) and fluoranthene (Fluo), to develop an analytical methodology for their quantification in water samples and to analyse their potential genotoxicity. Chlorinated standards were prepared by a newly developed two phase method (water/n-hexane) using sodium

Received 1 August 2013; accepted 1 February 2014. ´ Address correspondence to Ana Sofia Cardoso, Departamento Saude Ambiental, ´ Instituto Nacional de Saude, Dr. Ricardo Jorge, Av. Padre Cruz, 1649-016 Lisbon, Portugal. E-mail: [email protected] Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/gpol.

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Synthesis, Formation, and Genotoxicity of Chlorinated PAH in Drinking Water hypochlorite. 6-Chloro-benzo[a]pyrene was selectively obtained from BaP, while 1,3dichloro-fluoranthene and 3-chloro-fluoranthene were obtained from Fluo. All products were isolated and characterized by nuclear magnetic resonance and mass spectrometry. The formation of BaP- and Fluo-chlorinated derivatives under aqueous chlorination conditions was observed using a SPE-HPLC-FLD methodology. In addition, the cytotoxic and genotoxic activities of the three chlorinated derivatives were analyzed in comparison to their parent compounds, in a human-derived hepatoma cell line using the neutral red uptake and comet assays, respectively. The results showed that, at the equimolar doses of 100 and 125 μM, 6-Cl-BaP was able to induce a significantly higher level of DNA damage than BaP, suggesting a more potent genotoxic effect. In contrast, neither Fluo nor its chlorinated derivatives were genotoxic in the same cell line. The identification of new and possibly hazardous water chlorination by-product from PAHs emphasizes the need to minimize total organic carbon content of raw water and the implementation of safer water disinfection methods. Key Words: benzo[a]pyrene, chlorinated polycyclic aromatic hydrocarbons, chromatographic methodology, fluoranthene, genotoxicity, water disinfection by-products

INTRODUCTION Chlorine is the most widely used drinking-water disinfectant in water treatment plants but has the disadvantage of being highly reactive with organic matter present in the source water, producing a high variety of disinfection byproducts (DBPs). Among DBPs, the most prominent and well characterized are trihalomethanes (THMs), especially chloroform, bromodichloromethane, dibromochloromethane, and bromoform, the latter three being genotoxic and all four being carcinogenic to rodents (1). In addition, several epidemiologic studies suggested an association between human exposure to DBPs and an increased incidence of bladder and colorectal cancers (2, 3). Besides THMs, other DBPs (e.g., halogenated acetic acids, halogenated acetonitriles, chloral hydrate, and chlorinated phenols) have already been identified as potential carcinogens. It has been estimated that these DBPs globally account for approximately half of the total by-products formed in chlorinated water (1, 4), raising the need for more studies to identify new halogenated DBPs and to characterize their potential hazards. Polycyclic aromatic hydrocarbons (PAHs) are a ubiquitous class of compounds that are formed during incomplete combustion of organic substances in natural, urban, and industrial environments. Surface water, used as raw water, can be contaminated with PAHs due to discharges of untreated industrial effluents, municipal wastewater, forest fires, and solubilization of organic material from contaminated soils. Thus, the formation of chlorinated-PAHs (Cl-PAHs) derivatives seems possible following chlorination of PAH-containing water, remaining the most stable ones in the treated water (5–8). This assumption is supported by the previous detection of some Cl-PAHs in environmental samples including urban air, automobile exhausts, emissions from municipal waste

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incineration, coal combustion (9), river sediments and, more importantly, in chlorinated tap water (8, 9). Considering that humans are continuously exposed to very low quantities of water contaminants (ingestion of water and inhalation/dermal absorption while showering/bathing/swimming) through lifetime, the potential long term adverse health effect of this kind of unregulated DBPs deserves closer consideration and, if possible, quantification. The adverse effects of some PAHs (especially those containing four or more aromatic rings) have been essentially associated to their ability to alter genome integrity, i.e., to their genotoxic properties that can mediate their carcinogenicity (10, 11). There are also strong epidemiological evidences demonstrating a relationship between PAHs exposure and several forms of human bladder, lung, and gastrointestinal cancer (12, 13). In contrast, data on genotoxicity of halogenated PAHs are scarce and the few studies that have addressed this issue showed a higher genotoxic potency of chlorinated derivatives as compared to their parent compounds. This is illustrated by the stronger mutagenicity of the chlorinated derivatives of phenanthrene, pyrene, and benzo[e]pyrene as assessed by the Ames test (14, 15). Among PAHs, benzo[a]pyrene (BaP) and fluoranthene (Fluo) present different genotoxic activities as BaP is a potent mutagen and is classified by the International Agency for Research on Cancer (16) as carcinogenic to humans (group 1), while the mutagenic and carcinogenic properties of Fluo remain to be demonstrated (17, 18). The present article had three objectives: (1) to synthesise and characterize the major chlorinated derivatives of BaP and Fluo; (2) to develop an analytical methodology for their quantification in treated water samples; and (3) to analyze their genotoxic potential in a human cell line. The human-derived hepatoma (HepG2) cell line was used given that it presents the double advantage of efficiently metabolizing PAHs through phase I and phase II pathways and of retaining many of the morphologic characteristics of liver cells. In addition, this cell line has been considered as a sensitive cell line for genotoxicity assessment (19) using, among others, the simple, fast and cost-effective comet assay (20).

MATERIALS AND METHODS Chemicals R BaP and Aliquat 336 (N-methyl-N,N-dioctyloctan-1-ammonium chloride) were purchased from Sigma-Aldrich Qu´ımica, S.A. (Madrid, Spain) and used as received. Ultra-pure water was produced by Milli-Q device from Millipore Corporation (Billerica, MA, USA). All reagents used in the neutral red (NR) uptake and comet assays were obtained from Merck (Darmstadt, Germany) and Sigma-Aldrich (St. Louis, MO). All cell culture medium and supplements were obtained from Invitrogen (Carlsbad, CA).

Synthesis, Formation, and Genotoxicity of Chlorinated PAH in Drinking Water


Instrumentation Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance II 400 spectrometer (Bruker Biospin, Billerica, USA). Semipreparative liquid chromatography (HPLC) separations were conducted on a Dionex system (Dionex Corporation, Sunnyvale CA, USA), using a Luna C18 column (250 × 10 mm, 5 μm; Phenomenex) with an isocratic acetonitrile (ACN) elution at a flow rate of 3 mL/min. The fluorescence spectra were recorded in a Perkin Elmer LS 50B Luminescence Spectrometer (PerkinElmer, Waltham, Massachusetts, USA) using a window from 288–900 nm with an excitation wavelength of 288 nm, a scan speed of 350, an excitation slit and emission slit of 7.5. Spectra were recorded at 37◦ C, every 15 min during 4 h. Identification and quantification of Cl-PAHs was performed using an Agilent 1100 series HPLC instrument, equipped with a fluorescence detector (FLD) from Agilent Technologies Inc. (Santa Clara, USA), using a C18 ChromSep column SS 250 × 4.6 mm from Varian (Varian BV, Herculesweg, Middelburg, The Netherlands) with an isocratic ACN elution using the following wavelenghts 295/440 nm (excitation/emission wavelenghts). A solid phase extraction (SPE) Tracer-Teknokroma twelve positions vacuum manifold (Tracer-Teknokroma, Barcelona, Spain) was used. The cartridges (C18 - 500 mg/6 mL - supplied by Varian) were previously conditioned with 7 mL of acetonitrile and 14 mL of ultra-pure water. Aqueous samples were passed through the cartridges at maximum rate allowed. The glass material was then washed with 20 mL of acetonitrile/water (30/100) and subsequently passed through the cartridges. These were then dried by blowing N2 for 20 min and the adsorbed Cl-PAHs were eluted with 10 mL n-hexane; the cartridges were washed with 2 mL n-hexane that were added to the elutriate. The solutions were concentrated to 1.0 mL, with N2 blowing, after the addition of 2 mL acetonitrile. Gas chromatography with mass spectrometric detection (GC-MS) Varian 4000 (Varian BV, Herculesweg, Middelburg, The Netherlands) was used for the identification of PAHs and their chlorinated derivatives, with a VF-5ms column (30 m × 0.25 mm × 0.25 μm) from Varian. The ion trap mass spectrometer was operated in full scan mode.

Synthesis 6-Chloro-benzo[a]pyrene. R A solution containing Aliquat 336 (3.5 mg, 8.66 μmol), sodium hypochlorite (NaOCl) (800 μL), and HCl 0.1 M solution (800 μL) in water (4 mL) was added to a solution of BaP (21.6 mg, 85.6 μmol) in n-hexane (10 mL). Following 10 min of stirring, the organic phase was extracted and purified by semipreparative HPLC. 6-Chloro-benzo[a]pyrene (6-Cl-BaP) was obtained in 96%

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yield (21.6 mg). 1H-NMR (acetone-d6 ): δ (ppm) 9.32–9.29 (1H, m, H10), 9.26 (1H, d, J = 9.2, H11), 8.86–8.84 (1H, m, H7), 8.55 (1H, d, J = 9.5, H5), 8.50 (1H, d, J = 9.2, H12), 8.42 (1H, d, J = 8.3, H1), 8.30 (1H, d, J = 7.5, H3), 8,23 (1H, d, J = 9.5, H4), 8.13–8.10 (1H, m, H2), 8.02–7.99 (2H, m, H9 + H8); GCMS (EI): m/z (%) 288 (33) [M + 2]+, 286 (100) [M]+, 250 (35) [M-HCl]+; FExS (λmax , nm): 242, 266, 288, 306, 323, 340, 359, 381; FEmS (λmax , nm): 343, 359, 417, 441, 481. 3-Chloro-fluoranthene and 1,3-dichloro-fluoranthene. R A solution containing Aliquat 336 (11.3 mg, 27.6 μmol), NaOCl (140.7 μL), and HCl 0.1 M solution (800 μL) in water (2 mL) was added to a solution of Fluo (50.01 mg, 247.3 μmol) in n-hexane (3 mL). Following 30 min of stirring the organic phase was extracted and purified by preparative thin layer chromatography (TLC) (silica, n-hexane). Although 57.7% Fluo remained unreacted, two chlorinated products were obtained: 3-chloro-fluoranthene (3-ClFluo), obtained in 4.1% yield (2.4 mg)—1H-NMR similar to that reported by Van Haeringen et al. (21), GC-MS (EI): m/z (%): 238 (33) [M + 2]+, 236 (100) [M]+; 1,3-dichloro-fluoranthene (1,3-Cl2 -Fluo), obtained in 4.2% yield (2.8 mg). 1 H-NMR (400 MHz, acetone-d6 ) δ (ppm): 8.33–8.31 (1H, m, H10/H7), 8.24 (1H, d, J = 7.2, H6/4), 8.13–8.09 (2H, m, H10/7 + H6/4), 7.85 (1H, dd, J = 8.4 and 7.2, H5), 7.75 (1H, s, H2), 7.55–7.51 (2H, m, H9 + H8), GC-MS (EI): m/z (%): 274 (10) [M + 4]+, 272 (64) [M + 2]+, 270 (100) [M]+.

Identification of BaP- and Fluo-chlorinated Derivatives Formation under Aqueous Chlorination Conditions A solution containing BaP (100 mg/L, 250 μL) and NaOCl (7.5%, 0.5 μL) was added to ultra-pure water (1 L). Following acidification to pH 3 (5, 22), the resulting mixture was stirred for 30 min, 4 h and 24 h. This procedure was repeated for 4 h and 24 h and all experiments were performed in duplicates. At the end of each experiment, samples were analyzed by SPE-HPLC-FLD. The chlorinated product 6-Cl-BaP was consistently identified in all solutions analysed (based on the co-elution with the synthetic standard). For the chlorination of Fluo, similar conditions were used, and 3-Cl-Fluo and 1,3-Cl2 -Fluo were identified in all solutions analyzed, on basis of equal retention times upon co-injection with the corresponding synthetic standards.

Analysis of Cytotoxic and Genotoxic Effects Cell Culture. The human-derived hepatoma (HepG2) cell line was obtained from the American Type Culture Collection (ATCC, Rockville, MD). Cells were cultured



in DMEM-F12 medium containing L-Glutamax and HEPES buffer (25 mM), supplemented with 15% heat-inactivated fetal bovine serum, 1% penicillin/streptomycin, and 1.5% amphotericin B and maintained at 37◦ C, in 5% CO2 humidified atmosphere. Cytotoxicity. Cytotoxicity was measured by the NR uptake assay, carried out according to Repetto et al. (23) with modifications. For each experiment, 3 replicate cultures of HepG2 cells (104 cells/well) were initiated and, after 24 h, were exposed to several concentrations of BaP and 6-Cl-BaP (0.1–125 μM) and to several concentrations of Fluo and its chlorinated derivatives (25–125 μM), during 24 h. Solvent controls (dimethylsulfoxide (DMSO) or acetonitrile) were included in all experiments. After exposure of cells, the NR solution was added at a final concentration of 50 μg/mL. Cells underwent a 3 h incubation period and were then rinsed with phosphate buffer saline; the NR was extracted with ethanol:water:acetic acid (50:49:1) for 20 min and its absorbance was measured at 540 nm using a Multiskan Ascent spectrophotometer (Thermo Labsystems). The relative cell viability (%) of treated cells was calculated as the ratio between the mean absorbance of treated and control cells. The results were expressed as the mean value (±SE) of 3 independent experiments per treatment condition. Genotoxicity. The alkaline comet assay was performed as described by Tice et al. (20), with minor modifications. HepG2 cells (5 × 104 cells/well) were exposed to concentrations of BaP and 6-Cl-BaP, ranging from 50–125 μM, and to Fluo and its derivatives at final concentrations of 75 and 100 μM, for 24 h. Solvents (DMSO or acetonitrile) and a positive control (ethyl methanesulfonate, EMS) were included. After exposure, cells were rinsed, detached by trypsinization and embedded in low melting point agarose (0.7%); two microgels were prepared for each treatment condition by dropping 40 μL of cell suspension onto microscope slides pre-coated with normal melting point agarose. After solidification, cells were submitted to lysis (2.5 M NaCl, 100 mM Na2 EDTA.2H2 O, 10 mM Tris-HCl, 1% N-laurylsarcosine, 10% DMSO, 1% Triton X-100, pH 10) for 1 h, at 4◦ C. DNA was then allowed to unwind for 20 min, in cold alkaline electrophoresis buffer (300 mM NaOH, 1 mM Na2 EDTA.2H2 O, pH > 13) and electrophoresis was run for 20 min, at 25 V and 300 mA, at 4◦ C. Following neutralization (0.4 M Tris, 4 M HCl, pH 7.5), slides were stained with ethidium bromide (0.125 μg/μL). One hundred randomly selected nucleoids were analysed in replicate slides under a fluorescence microscope (Zeiss, Axioplan 2) using the Comet Imager 2.2. software (MetaSystems, GmbH). For each slide the mean percentage of DNA in tail, which is linearly related to breaks frequency, was registered as a measure of DNA damage level. The results

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were expressed as the mean value (±SE) of 3 independent experiments per treatment condition.

Statistical Analysis Statistical analysis of data was performed using SPSS Statistics 17.0 software. NR assay data were analyzed either by the 2-tailed Student’s t-test, or by the Mann-Whitney U-test, depending on data normality (tested through the Shapiro–Wilk’s test). ANOVA was applied to analyze the comet assay results obtained for each chemical, following logarithmic transformation of data in order to obtain a normal distribution of means; the 2-tailed Student’s t-test was used to compare the level of DNA breaks produced by each chemical concentration with that of the respective solvent control. Statistical significance, for all assays, was assumed for p ≤ 0.05.

RESULTS AND DISCUSSION Some studies have shown that brominated, iodinated and chlorinated derivatives of organic compounds (e.g., chloro-pyrene, halonitromethanes, chloro-, and bromo-benzoquinones) persist in chlorinated water and can be genotoxic (14, 24), raising concern about their impact on human health, especially regarding cancer risk. In this study, in addition to their preparation and analysis, we also provide data on the cytotoxicity and genotoxicity of 6-Cl-BaP, 3-Cl-Fluo, and 1,3-Cl2 -Fluo (Figure 1), in comparison to their parent compounds.

Preparation and Characterization of Chlorinated PAHs Standards A key difficulty preventing the quantification of Cl-PAHs in water is the lack of commercial standards to be used in the development of analytical methods for their detection. The synthetic methods commonly used for the synthesis of chlorinated derivatives of BaP and Fluo have the disadvantages of being either time consuming and/or using highly toxic reagents and/or solvents (21, 25), and thus more expedite and safer methods are needed to prepare Cl-PAHs. Sodium hypochlorite (NaOCl) in acid medium is a non toxic chlorine source that has already been used for the chlorination of PAHs in aqueous medium. However, due to the hydrophobic character of PAHs, this procedure is not suitable for synthetic purposes. This drawback could be easily overcome knowing that NaOCl can be transferred into organic phase (where the PAHs are soluble) in the presence of a phase transfer catalyst (26). Effectively, in this work, we have developed a two-phase (water/n-hexane), easy, and fast synthetic procedure that, using hypochlorite in presence of a catalytic amount of the quaterR 336 (used as phase transfer reagent), succeeded nary ammonium salt Aliquat on the chlorination of both BaP and Fluo. Whereas chlorination of BaP gave



Figure 1: Structures of benzo[a]pyrene (BaP), 6-chloro-benzo[a]pyrene (6-Cl-BaP), fluoranthene (Fluo), 3-chloro-fluoranthene (3-Cl-Fluo), and 1,3-dichloro-fluoranthene (1,3Cl2 -Fluo).

selectively 6-Cl-BaP in 96% yield, chlorination of Fluo revealed to be less efficient, providing two chlorination products, 3-Cl-Fluo and 1,3-Cl2 -Fluo, in app. 4% yield each, using similar reaction conditions. However, under extended reaction time (72 h), higher 3-Cl-Fluo and 1,3-Cl2 -Fluo yields were obtained as a clear indication that Fluo chlorination is slower when compared with that of BaP. Noteworthy is the fact that with increased reaction times other nonidentified products were formed. 6-Cl-BaP was identified on the basis of indistinguishable 1H-NMR and mass spectra when recorded under similar conditions, as well as equal retention times (HPLC-DAD, HPLC-FLD) upon co-injection with the corresponding synthetic standard pre-prepared by adaptation of Mulder et al. (25) method. Identification of 3-Cl-Fluo was based upon indistinguishable 1H-NMR spectrum when compared with literature data (21) and the mass spectrometry (MS) data (see experimental section) was consistent with the structure ascribed. The first evidence for the formation of the dichlorinated Fluo product, 1,3-Cl2 -Fluo, was provided by mass spectrometry, where the mass spectra (EI) exhibited the ions [M]+ at m/z 270, [M + 2]+ at m/z 272 and [M + 4]+ at m/z 274, characteristic of dichloro-derivatives. 1H-NMR spectrum was consistent with the MS data presenting signals corresponding to 8 aromatic protons, as a clear evidence for the existence of two substituents in the Fluo ring system (see experimental

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Figure 2: Expanded region of the 1H-13C HMBC spectrum of 1,3- Cl2 -Fluo, displaying the connectivities between the proton H2 and carbons C3a and C10b. The connectivity of proton H5 to the carbon C3a was also decisive in ascribing the positions 1 and 3 as the chlorinated positions.

section and supplementary data). It should be noted that because 1,3-Cl2 -Fluo was isolated in very small amounts, as such, it was not possible to obtain a 13 C-NMR spectrum with good signal/noise ratios. Nonetheless, carbon resonance assignments were performed on the basis of the correlations observed in the more sensitive inverse bidimensional experiments, HMQC and HMBC. The positions of substitution were established based on the 1H-13C-three bond correlations observed in the HMBC spectrum (Figure 2): proton H2 (singlet at 7.75 ppm) presented a three-bond correlation with a quaternary carbon at 127.4 ppm, compatible with C3a; the fact that proton H5 (double doublet, 7.85 ppm) also presented 1H-13C-three bond correlation with this quaternary carbon is entirely consistent with 1 and 3 as the positions of chlorination.

Identification of BaP- and Fluo-chlorinated Derivatives Formation under Aqueous Chlorination Conditions The procedure used for aqueous chlorination of BaP and Fluo involved the use of NaOCl under acidic conditions. Acidification to pH 3 was previously



Figure 3: Viability of HepG2 cells following 24 h exposure to different concentrations of (A) BaP and 6-Cl-BaP; (B) Fluo, 3-Cl-Fluo and 1,3-Cl2 -Fluo. Results are expressed as the mean percentage of viable cells (±SE) of 3 independent experiments.

used by other authors for the aqueous chlorination of PAHs. Namely, Oyler et al. (5, 22) demonstrated that reactions performed at pH > 6 tended to produce mainly oxygenated products while reactions at pH < 6, specifically pH = 3, tended to produce both chlorinated and oxygenated products. Acidification to pH = 3 was used in the current work, since the objective of these experiments was mainly to evaluate the formation of chlorinated PAHs under aqueous chlorination conditions, and rather than to reproduce the procedures used in water treatment plants (WTPs). At these conditions, 6-Cl-BaP was consistently identified by SPE-HPCL-FLD methodology in aqueous solutions of BaP treated with NaOCl, under acidic conditions, after 30 min, 4 h, and 24 h of contact time. This identification was based on equal retention times upon co-injection with the corresponding synthetic standards. Likewise, the chlorinated derivatives 3-Cl-Fluo and 1,3-Cl2 -Fluo were consistently identified in Fluo solutions, under the same experimental conditions. These results unequivocally show that when water contaminated with PAHs is subjected to aqueous chlorination conditions, the chlorinated derivatives of BaP and Fluo can be promptly formed and persist for at least 24 h. These findings suggest that formation of these and other PAHs chlorinated by-products might occur in WTPs during chlorinebased water disinfection methods.

Analysis of Cytotoxic and Genotoxic Effects Treatment of HepG2 cells with BaP and 6-Cl-BaP during 24 h resulted in dose-related decreases in cell survival evaluated by the NR assay (Figure 3A).

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No significant solvent cytotoxicity was observed within the tested concentration range. Comparison of survival data obtained at the lowest concentrations tested (10 and 50 μM) indicated that BaP was approximately 2-fold more cytotoxic than 6-Cl-BaP. This was also supported by the observation that a reduction of cells viability to about 50% was achieved after exposure of cells to 5 μM of BaP (44.49 ± 7.42%, p = 0.043) and to 100 μM of 6-Cl-BaP (60.58 ± 15.61%, p = 0.024). Interestingly, at the two highest concentrations tested (100 and 125 μM), a similar significant reduction of viability to app. 40% cell survival was induced by both compounds (e.g., p = 0.044 and 0.048, for 125 μM of BaP and 6-Cl-BaP, respectively). Cells exposure to different concentrations of Fluo, 3-Cl-Fluo, and 1,3-Cl2 -Fluo allowed survival of 60–80% of cells, i.e., did not induce a significant cytotoxic effect (Figure 3B). Taken together, the cell lethality data of BaP and Fluo agree with those reported by Audebert et al. (18) based on the quantification of ATP concentrations, showing that HepG2 cells incubation with 100 μM BaP reduced viability to 70% while Fluo did not show any toxicity up to 100 μM. The toxic effect of these PAHs was also compared by the MTT test in a human breast cancer cell line, MCF-7 cells, showing major mechanistic differences, given that BaP (app. 50 μM) caused cell cycle delay and apoptosis while Fluo (app. 40 μM) demonstrated no significant effects on cell proliferation (27). In addition, the present results show that the introduction of chlorine atoms in BaP or Fluo molecules did not increase their cytotoxic effect in HepG2 cells. Genotoxicity was assessed by the comet assay and data, expressed as the percentage of DNA in the comet tail are presented in Figure 4. A marginally significant increase in the level of DNA strand breaks was observed at the highest BaP concentration tested (125 μM) as compared to the solvent control (p = 0.057). 6-Cl-BaP treatment produced a significant increase in DNA damage (p = 0.001) in HepG2 cells, being able to raise significantly the percentage of DNA in tail at 100 μM and 125 μM (p = 0.012 and 0.017, respectively), as compared to the solvent control (Figure 4A). Furthermore, a statistical comparison of the mean percentage of DNA in tail induced by equimolar and equitoxic concentrations of both compounds (100 and 125 μM), confirmed that 6-Cl-BaP exerted a significantly higher DNA damaging effect than its parent compound (p = 0.041 and 0.038, respectively). Although the comet assay allows the quantification of primary DNA lesions, i.e., lesions that are not fixed and can still be reversed through DNA repair, a positive result has been closely associated to irreversible genotoxic effects, such as chromosome breaks (28). The present finding that 6-Cl-BaP might be a stronger genotoxicant than BaP in a human cell line is in line with previous studies that showed a higher mutagenic potential of chlorinated derivatives of phenanthrene, benzo[e]pyrene (15), and pyrene (14) through the Ames test, comparatively to the parent compounds. In contrast, neither Fluo nor its chlorinated derivatives were able to induce DNA strand breaks (Figure 4B). This differential genotoxic effect of BaP and Fluo



Figure 4: DNA damaging effect of: (A) BaP, 6-Cl-BaP, (B) Fluo, 3-Cl-Fluo and 1,3-Cl2 -Fluo; in HepG2 cells, assessed by the comet assay. 0 μM corresponds to solvent controls. Results are expressed as the mean percentage of DNA in tail (±SE) from 3 independent experiments. ∗ Statistical significant difference when compared to the respective solvent control; statistical significant difference between equimolar concentrations of BaP and 6-Cl-BaP.

in HepG2 cells is in agreement with the recent findings of Audebert et al. (18) using the γH2AX assay in the same cell line. The same authors also confirmed that HepG2 cells efficiently metabolize BaP and Fluo, through Phase I and Phase II pathways that are highly expressed in HepG2 cells (19), giving rise to several intermediate metabolites, the most mutagenic being BaP-diol epoxide that forms guanine and, to a lesser extent, adenine and cytosine adducts (29), resulting in DNA strand breaks. It must be pointed out that the concentrations of both PAHs and its derivatives tested in our study were far higher than the concentrations found in water resources to which human exposure can occur. Several recent studies addressing the status of PAHs contamination in drinking source water in different regions of China, described the occurrence of PAHs in the range of ng/L-μg/L (30–32), arising from several anthropogenic and industrial sources (33, 34). Although in vitro genotoxicity assays have the advantage of providing expedite results, the concentrations of chemicals tested are hardly comparable to the human exposure levels. However, for hazard identification purposes and assuming that 6-Cl-BaP could have a mechanism of action similar to that of BaP, the positive findings obtained in this study suggest that its formation in water might also imply a relevant concern for human health. Moreover, the biopersistence of PAHs and their the capacity to accumulate in the human organism should be also taken into account, given that it raises the levels of internal exposure along the lifetime. Finally, the coexistence of several PAHs and halogenated PAHs have been identified in the same environmental matrices (32, 34, 35) and thereby humans will be exposed to a mixture of these organic compounds rather to an individual compound,

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possibly giving rise to interactions whose toxic effects and risk for humans are extremely difficult to predict.

CONCLUSIONS A new, cost-efficient, simple, and fast synthetic methodology was developed for the preparation of BaP and Fluo chlorinated products. Although it resulted more efficiently for BaP chlorination, affording selectively 6-Cl-BaP in 96% yield, than for Fluo, where both 3-Cl-Fluo and 1,3-Cl2 -Fluo were obtained in lower yield, the usefulness of this methodology for the chlorination of other aromatic compounds is anticipated. The SPE-HPLC-FLD methodology developed enabled the identification of 6-Cl-BaP, 3-Cl-Fluo, and 1,3-Cl2 -Fluo in water samples upon hypochlorite treatment in acidic conditions. These results support the possible occurrence of these Cl-PAHs in water, following treatment with chlorine disinfectants. The synthesized chlorinated derivatives 3-Cl-Fluo and 1,3-Cl2 -Fluo were not genotoxic in HepG2 cells, but high concentrations of 6-Cl-BaP were able to induce higher levels of DNA damage than BaP. While it is undeniable that disinfection of drinking-water is essential for public health protection from outbreaks of waterborne infectious and parasitic diseases, disinfection of drinking-water by chlorination (and other oxidative treatments) should ideally be performed only on raw waters with minimal or minimized content of total organic carbon and of suspended particles, to reduce formation of potentially toxic by-products and to decrease the risk from long-term human exposure to those hazardous products.

SUPPLEMENTAL MATERIAL Supplemental data for this article can be accessed on the publisher’s website.

REFERENCES 1. Richardson, S. D., M. J. Plewa, E. D. Wagner, R. Schoeny, and D. M. Demarini. “Occurrence, Genotoxicity, and Carcinogenicity of Regulated and Emerging Disinfection by-Products in Drinking Water: A Review and Roadmap for Research.” Mutation Research 636 (2007): 178–242. 2. IARC. “Some Drinking-Water Disinfectants and Contaminants, Including Arsenic.” IARC Monographs on the Evaluation of Carcinogenic Risks 84 (2004): 1–477. 3. King, W. D., L. D. Marrett, and C. G. Woolcott. “Case-Control Study of Colon and Rectal Cancers and Chlorination By-Products in Treated Water.” Cancer Epidemiology, Biomarkers & Prevention 9 (2000): 813–8. 4. Nikolaou, A. D., S. K. Golfinopoulos, T. D. Lekkas, and M. N. Kostopoulou. “DBP Levels in Chlorinated Drinking Water: Effect of Humic Substances.” Environmental Monitoring Assesment. 93 (2004): 301–19.


Synthesis, Formation, and Genotoxicity of Chlorinated PAH in Drinking Water 5. Oyler, A. R., R. J. Liukkonen, M. K. Lukasewycz, D. A. Cox, D. A. Peake, and R. M. Carlson. “Implications of Treating Water Containing Polynuclear AromaticHydrocarbons with Chlorine—A Gas Chromatographic-Mass Spectrometric Study.” Environmental Health Perspectives 46 (1982): 73–86. 6. Mori, Y., S. Goto, S. Onodera, S. Naito, and H. Matsushita. “Aqueous Chlorination of Tetracyclic Aromatic-Hydrocarbons – Reactivity And Product Distribution.” Chemosphere 22 (1991): 495–501. 7. Onodera, S., K. Igarashi, A. Fukuda, J. Ouchi, and S. Suzuki. “Chemical Changes of Organic Compounds in Chlorinated Water. XVI. Gas ChromatographicMass Spectrometric Studies of Reactions of Tricyclic Aromatic Hydrocarbons with Hypochlorite in Dilute Aqueous Solution.” Journal of Chromatography 466 (1989): 233–49. 8. Shiraishi, H., N. H. Pilkington, A. Otsuki, and K. Fuwa. “Occurrence of Chlorinated Polynuclear Aromatic-Hydrocarbons in Tap Water.” Environmental Science and Technology 19 (1985): 585–90. 9. Ohura, T., K.-I. Sawada, T. Amagai, and M. Shinomiya. “Discovery of Novel Halogenated Polycyclic Aromatic Hydrocarbons in Urban Particulate Matters: Occurrence, Photostability, and AhR Activity.” Environmental Science and Technology 43 (2009): 2269–75. 10. Majer, B. J., V. Mersch-Sundermann, F. Darroudi, B. Laky, K. de Wit, and S. Knasmuller. “Genotoxic Effects of Dietary and Lifestyle Related Carcinogens in Human Derived Hepatoma (HepG2, Hep3B) Cells.” Mutation Research—Fundamental and Molecular Mechanisms of Mutagenesis 551 (2004): 153–66. 11. Lin, T. and M. S. Yang. “Benzo[a]pyrene-induced Necrosis in the HepG(2) Cells via PARP-1 Activation and NAD(+) Depletion.” Toxicology 245 (2008): 147–53. 12. Spink, D. C., S. J. Wu, B. C. Spink, M. M. Hussain, D. D. Vakhania, B. T. Pentecost, and L. S. Kaminsky. “Induction of CYP1A1 and CYP1B1 by benzo[k]fluoranthene and benzo[a]pyrene in T-47D Human Breast Cancer Cells: Roles of PAH Interactions and PAH Metabolites.” Toxicological and Applied Pharmacology 226 (2008): 213–24. 13. Roth, M. J., W.-Q. Wei, J. Baer, C.C. Abnet, G.-Q. Wang, L. R. Sternberg, A. C. Warner, L.L. Johnson, N. Lu, C. A. Giffen, S. M. Dawsey, Y.-L. Qiao, and J. Cherry. “Aryl Hydrocarbon Receptor Expression Is Associated with a Family History of Upper Gastrointestinal Tract Cancer in a High-Risk Population Exposed to Aromatic Hydrocarbons.” Cancer Epidemiology, Biomarkers & Prevention 18 (2009): 2391–6. 14. Colmsjo, A., A. Rannug, and U. Rannug. “Some Chloro Derivatives of Polynuclear Aromatic-Hydrocarbons are Potent Mutagens in Salmonella-Typhimurium.” Mutation Research 135 (1984): 21–9. 15. Lofroth, G., L. Nilsson, E. Agurell, and T. Sugiyama. “Salmonella Microsome Mutagenicity of Monochloro Derivatives of Some Di-tetracyclic, Tri-tetracyclic and Tetracyclic Aromatic-Hydrocarbons.” Mutation Research 155 (1985): 91–4. 16. IARC. “A Review of Human Carcinogens – Chemical Agents and Related Occupations.” IARC Monographs on the Evaluation of Carcinogenic Risks 100, F (2012): 111–44. 17. Stocker, K. J., W. R. Howard, J. Statham, and R. J. Proudlock. “Assessment of the Potential in vivo Genotoxicity of Fluoranthene.” Mutagenesis 11 (1996): 493–6. 18. Audebert, M., A. Riu, C. Jacques, A. Hillenweck, E. L. Jamin, D. Zalko, and J. P. Cravedi. “Use of the Gamma H2AX Assay for Assessing the Genotoxicity of Polycyclic Aromatic Hydrocarbons in Human Cell Lines.” Toxicology Letters 199 (2010): 182–92.

369


370

M. Pinto et al. ¨ 19. Knasmuller, S., V. Mersch–Sundermann, S. Kevekordes, F. Darroudi, W. W. Huber, C. Hoelzl, J. Bichler, and B. J. Majer. “Use of Human–Derived Liver Cell Lines for the Detection of Environmental and Dietary Genotoxicants; Current State of Knowledge.” Toxicology 198 (2004): 315–28. 20. Tice, R. R., E. Agurell, D. Anderson, B. Burlinson, A. Hartmann, H. Kobayashi, Y. Miyamae, E. Rojas, J. C. Ryu, and Y. F. Sasaki. “Single Cell Gel/Comet Assay: Guidelines for in vitro and in vivo genetic toxicology testing.” Environmental and Molecular Mutagenesis 35 (200): 206–21. 21. Van Haeringen, C. J., J. Lugtenburg, and J. Cornelisse. “Synthesis and Characterization of Chloro-, Bromo-, Cyano-, Formyl-, and Methylfluoranthenes.” Polycyclic Aromatic Compounds 8 (1996): 9–22. 22. Oyler, A. R., R. J. Liukkonen, M. T. Lukasewycz, K. E. Helkklla, D. A. Cox, and R. M. Carlson. “Chorine “Disinfection” Chemistry of Aromatic Compouns. Polynuclear Aromatic Hydrocarbons: Rates, Products, and Mechanisms.” Environmental Science and Technology 17 (1983): 334–42. 23. Repetto, G., A. del Peso, and J. L. Zurita. “Neutral Red Uptake Assay for the Estimation of Cell Viability/Cytotoxicity.” Nature Protocols 3 (2008): 1125–31. 24. Liviac, D., A. Creus, and R. Marcos. “Genotoxicity Analysis of Two Halonitromethanes, a Novel Group of Disinfection By-products (DBPs), in Human Cells Treated in vitro.” Environmental Research 109 (2009): 232–8. 25. Mulder, P. P. J., N. V. S. Ramakrishna, P. Cremonesi, E. G. Rogan, and E. L. Cavalieri. “Synthesis of 1- and 3-fluorobenzo[a]pyrene.” Chemical Research in Toxicology 6 (1993): 657–61. 26. Mirafzal, G. A. and A. M. Lozeva. “Phase Transfer Catalyzed Oxidation of Alcohols with Sodium Hypochlorite.” Tetrahedron Letters 39 (1998): 7263–6. 27. Ogba, N., C. M. Wang, and S. Raychoudhury. “Differential Effects of Fluoranthene and benzo[a]pyrene in MCF-7 cells.” Journal of Environmental Science and Health A 40 (2005): 927–36. 28. Kirsch-Volders, M., G. Plas, A. Elhajouji, M. Lukamowicz, L. Gonzalez, K. Vande Loock, and I. Decordier. “The in vitro MN Assay in 2011: Origin and Fate, Biological Significance, Protocols, High Throughput Methodologies and Toxicological Relevance.” Archives of Toxicology 85 (2011): 873–99. 29. Dipple, A. “Reactions of Polycyclic Aromatic Hydrocarbons with DNA.” IARC Scientific Publications (1994): 107–29. 30. Chen, Y. Y., L. Z. Zhu, and R. B. Zhou. “Characterization and Distribution of Polycyclic Aromatic Hydrocarbon in Surface Water and Sediment from Qiantang River, China”. Journal of Hazardous Materials 141 (2007): 148–55. 31. Deng, H. M., P. A. Peng, W. L. Huang, and H. Z. Song. “Distribution and Loadings of Polycyclic Aromatic Hydrocarbons in the Xijiang River in Guangdong, South China.” Chemosphere 64 (2006): 1401–11. 32. An, T., M. Qiao, G. Li, H. Sun, X. Zeng, and J. Fu. “Distribution, Sources, and Potential Toxicological Significance of PAHs in Drinking Water Sources within the Pearl River Delta.” Journal of Environmental Monitoring 13 (2011): 1457–63. 33. Ma, Y. G., J. P. Cheng, F. Jiao, K. X. Duo, Z. Rong, M. Li, and W. H. Wang. “Distribution, Sources, and Potential Risk of Polycyclic Aromatic Hydrocarbons (PAHs) in Drinking Water Resources from Henan Province in Middle of China.” Environmental Monitoring and Assessment 146 (2008): 127–38.


Synthesis, Formation, and Genotoxicity of Chlorinated PAH in Drinking Water 34. Zhang, D., T. An, M. Qiao, B.G. Loganathan, X. Zeng, G. Sheng, and J. Fu. “Source Identification and Health Risk of Polycyclic Aromatic Hydrocarbons Associated with Electronic Dismantling in Guiyu town, South China.” Journal of Hazardous Materials 192 (2011):1–7. 35. Sun, J. L., H. Zeng, and H. G. Ni. “Halogenated Polycyclic Aromatic Hydrocarbons in the Environment.”.Chemosphere 90 (2013): 1751–9.

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