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Mutagenicity and genotoxicity of water treated for human consumption induced by chlorination by-products.

Introduction

Most of the routine analyses performed for drinking water quality are focused on physicochemical and microbiological tests. Based on these analyses, some criteria for water used for human consumption have been established. In this way, with the introduction of water disinfection, the population can be ensured that their drinking water is likely to be free of waterborne infectious diseases (Boorman et al., 1999). Alternatives methodologies exist, but none of them offers continuous protection against pathogens through the distribution network.

Since the 1970s, a new risk for human health appeared in drinking water, as findings showed that it can contain mutagenic and carcinogenic compounds known as disinfection by-products (DBPs) (Hemming, Holmbom, Reunanen, & Kronberg, 1986; Kusamran et al., 1994; Meier, Blazak, & Knohl, 1987). When chlorine reacts with humic and fulvic acids present naturally in the water, it can produce several compounds such as trihalomethanes, halofuranes, haloacetic acids, halophenols, halopropanones, and others that are well known for their mutagenic and carcinogenic properties (Langvik & Holmbom, 1994; Richardson, Although most of the information presented in the Journal refers to situations within the United States, environmental health and protection know no boundaries. The Journal periodically runs International Perspectives to ensure that issues relevant to our international membership, representing over 20 countries worldwide, are addressed. Our goal is to raise diverse issues of interest to all our readers, irrespective of origin. Plewa, Wagner, Schoeny, & DeMarini, 2007; Richardson, Simmons, & Rice, 2002; Shi et al., 2009). A large number of those compounds have been isolated from chlorinated waters (McDonald & Komulainen, 2005; Richardson et al., 2007). The trihalomethanes (THMs) include chloroform (CH[Cl.sub.3]), dibromochloromethane (CH[Br.sub.2]Cl), bromodichloromethane (CHBr[Cl.sub.2]), and bromoform (CH[Br.sub.3]); these compounds represent between 5% and 20% of the total DBPs (Fayad, 1993).

Other compounds with similar properties have been identified and quantified in chlorinated water, and it is believed that they are responsible for the rest of the mutagenic activity. Within the compounds detected are the haloacetic acids (HAAs) (Krasner et al., 2006) and chlorohydroxyfuranones (MXs) (Kronberg & Vartiainen, 1988; Smeds, Vartiainen, Maki-Paakkanen, & Kronberg, 1997). Hemming and co-authors (1986) identified and quantified 3-chloro-4-(dichloromethyl)5-hydroxy-2(5H)-furanone (MX), one of the most potent bacterial mutagens that is the product of the reaction of chlorine with the organic material present in water. Figure 1 shows the structure of MX and its open isomeric forms, Z-MX ([Z]-2-chloro-4-[dichloromethyl]-4-oxo-butenoic acid) and E-MX ([E]-2-chloro-3-[dichloromethyl]4-oxobutenoic acid) (Franzen & Kronberg, 1994; Richardson et al., 2007).

MX can be found at low levels (2-310 ng/L) in drinking water (Kronberg, Christman, Singh, & Ball, 1991; McDonald & Komulainen, 2005; Wright et al., 2002). It has been estimated, however, that this compound may contribute about 3% to 67% of the total mutagenicity of chlorinated waters, inducing a wide spectrum of mutations in bacterial and mammalian cells (Hyttinen, Myohanen, & Jansson, 1996; Jansson & Hyttinen, 1994; Maki-Paakkanen & Hakulinen, 2008; Wright et al., 2002). In Salmonella typhimurium strains TA100 and TA102, MX induces damage of the DNA by base-pair substitution (Hemming et al., 1986; Kronberg & Vartiainen, 1988) and in Salmonella typhimurium TA98, a bacterial strain sensitive to frameshift mutations, MX produces loss or gain of a pair of bases (DeMarini, AbuShakra, Felton, Patterson, & Shelton, 1995). TA98 and TA100 have been widely used to test a numerous series of chemical mutagens and carcinogens.

MX also induced a wide variety of DNA damage in mammalian cells in vitro (Jansson & Hyttinen, 1994; Maki-Paakkanen & Hakulinen, 2008) including human cells (Chang, Daniel, & Deangelo, 1991) such as sister chromatid exchange (SCE), chromosomal aberrations (Hyttinen et al., 1996; Jansson et al., 1993), DNA strand breaks and different kinds of mutations (Hyttinen et al., 1996; Richardson et al., 2007), and other effects (King, Hester, Warren, & DeMarini, 2009). MX has been classified by the International Agency of Research on Cancer (IARC) as a possible carcinogen in humans (type 2B) (International Agency of Research on Cancer [IARC], 2004).

Several studies have concluded that a potential risk of cancer is associated with the consumption of chlorinated water (Cantor, 1997; Tao, Zhu, & Matanoski, 1999). IARC (1995) conducted research that concluded that a positive correlation exists between chlorinated water consumption and the development of kidney and bladder cancer. Nevertheless, for the amount of factors to be considered, IARC considers that not enough evidence exists to classify DBPs as carcinogenic agents in humans. By contrast, the World Health Organization (WHO) recommendation prioritized the latter for authorities wishing to meet the disinfection by-products and the microbiological guidelines (World Health Organization [WHO], 2004).

According to WHO, MX concentrations of 1.8 [micro]g/L are associated with [10.sup.-5] cancer risk for a 60 kg adult drinking 2 L of water per day (WHO, 2004). In 2001, some studies were initiated in Colombia to understand the mutagenic effect of chlorinated and nonchlorinated waters in one of the principal water treatment plants (Villa Hermosa) in the city of Medellin (Melendez, Zuleta, Marin, Calle, & Salazar, 2001). The authors found mutagenicity before and after the chlorination process and concluded that the pollution from the river that supplies water to the plant and DBP formation from the chlorination process were responsible for the mutagenicity.

In our study we evaluated the mutagenic and genotoxic effect of water extracts taken from San Cristobal water treatment plant in Medellin, Colombia, increasing the number of treatment plants previously evaluated for mutagenicity by DBPs in Colombia. The main goal of our study was to identify the potential risk to the population due to the presence of these compounds in drinking water.

Methods

Water Sampling and Sample Workup Procedure

Water samples were obtained from two different areas of the San Cristobal plant: (1) immediately before chlorination and (2) after the chlorination process but before water distribution. Samples were taken manually at a total volume of 80 L. The pH was adjusted to 2 with concentrated HCl. The samples were passed through columns filled with XAD-2 and XAD-7 (1:1) sorbants at a flow rate of 15 mL/min., according to procedure described by Meier and co-authors (Meier, Knohl, et al., 1987) and Stahl (1991), with some modifications. The elution was performed with 300 mL of acetone and 300 mL of methanol. The volume of the eluent was concentrated on a roto-evaporator at 55[degrees]C. The samples were weighed and kept at -20[degrees]C for further mutagenic and genotoxic assays.

Mutagenic Test (Ames Test)

The mutagenic activity of water extracts and MX were determined by means of the Ames test (Maron & Ames, 1983), using two strains (TA98 and TA100) of Salmonella typhimurium, with metabolic activity (with mixture S9, made from a fraction of rat liver homogenate) and without metabolic activity (without mixture S9) to detect indirect mutagenic activity. The 2-aminofluorene (2-AF, 10 [micro]g/plate) was used as a positive control. Sterile distilled water was used as a negative control. The tests were conducted using three doses in duplicate, with a minimum of three independent experiments. The answer is positive when the number of mutations is at least doubled in contrast with the negative control, according to the criteria suggested by WHO (2004).

Genotoxic Test (Comet Assay)

To determine the level of damage of the DNA of human lymphocytes, the technique single cell gel electrophoresis (SCGE) or comet assay was utilized according to the protocol described by Singh and co-authors (1988) with slight modifications. Between 5,000 and 50,000 human lymphocytes were isolated from total blood by Ficoll density gradient centrifugation (Duthie, Ross, & Collins, 1995), which were incubated with the water extracts.

The viability of lymphocytes was determined with the trypan blue (0.2%) exclusion test, before and after the treatment showing values greater than 91%. Hydrogen peroxide (50 uM) was used as a positive control; dimethyl sulphoxide (DMSO) and phosphate buffered saline (PBS) as negative control.

After the treatment, 10[micro]L of the cellular suspension was mixed with 75[micro]L of low-melting-point agarose (LMA) 0.5% (w/v) at 37[degrees]C and placed onto microscope slides precoated with normal-melting-point agarose (NMA). The cellular suspension was covered with a cover slip and maintained at 4[degrees]C for five minutes. The cover slip was removed and a third layer of agarose was added and cooled up to 4[degrees]C. The slides were immersed in a lysis solution adjusted to pH 10 (NaCl 2.5 molars [M], [Na.sub.2]EDTA 100 millimolar [mM], TRIS 10 mM, Sarcosinate 1%, Triton X-100 1%, and DMSO 10%) at 4[degrees]C for 90 minutes.

The layers were placed in a buffer solution ([Na.sub.2]EDTA 1 mM, NaOH 300 mM, pH 13) for electrophoresis to allow DNA unwinding. The electrophoresis was conducted at 4[degrees]C for 30 minutes at 25 volts and 300 milliamperes. After this procedure, the slides were rinsed with a neutralizing buffer (tris-HCl 0.4 M, pH 7.5) for 15 minutes and dehydrated with methanol. The slides were stained with 40 [micro]L of acridine orange (5[micro]g/mL) and examined with a fluorescence microscope equipped with an excitation filter of 450-490 nm, using a magnification of 10x and 40x.

The slides were ready for image analysis using a fluorescence microscope with a camera. Twenty-five randomly selected comet cells from each slide were analyzed with an ocular-micrometer; two slides were done by doses. The DNA damage was evaluated by measuring the length of the resulting image (nuclei diameter plus migrated DNA comet tail) in microns and an average was calculated. The effect of the doses over the migration of the DNA was analyzed by the Duncan test with [alpha] = .05.

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis of MX and E-MX

Twelve liters of each water sample were concentrated on a column of 12 x 40 cm filled with XAD-2 and XAD-7 (1:1) with a flow rate of 20 mL/min. The adsorbed organics were eluted with 300 mL of ethyl acetate (EtAc). The EtAc extracts were concentrated in a roto-evaporator. The MX and E-MX were determined in the extract after derivatization with methanol at 70[degrees]C for 2 hours in a [H.sub.2]S[O.sub.4] 2% (v/v) (sulfuric acid) solution.

The GC-MS analyses for non-volatile organochlorinated compounds were performed on a gas chromatograph Agilent G1530A (HP 6890A) equipped with a selective mass detector 5973N MSD. The separation of the components in the GC-MS analyses was performed on a HP-5MS fused silica column (30 m x 0.25 mm i.d., film thickness 0.25 [micro]n). The carrier gas was helium at a flow rate of 1 mL/min. The oven temperature was programmed from 80[degrees]C to 230[degrees]C at a rate of 6[degrees]C/min.; the injector temperature was kept at 250[degrees]C. Electron impact ([EI.sup.+]) ionization was at 70 electron volts.

GC/Electron Capture Detector (ECD) Analyses for THMs

The standard solutions for calibration were prepared from a standard calibration mix. They were prepared in iso-octane for chloroform and n-pentane for bromoform, bromodichloromethane and dibromochloromethane.

For the quantification of THMs, 1 mL of fresh water treated from the water treatment plant San Cristobal was extracted with 0.5 mL of n-pentane or iso-octane for chloroform (David, Sandra, & Klee, 1997). Chloroform was extracted in iso-octane because of the co-elution with n-pentane during the chromatographic analysis.

The analyses were preformed in a gas chromatograph Shimadzu 15-A, with an injector SPL-G9 and ECD. The system was equipped with a DB-1 column (30 m, 0.53 mm i.d., film thickness 1.5 um). The carrier gas was helium (5.7 psi) with an isothermal temperature of 45[degrees]C for bromoform, bromodichloromethane, and dibromochloromethane and for chloroform a temperature program of 45[degrees]C for 2.6 minutes with a ramp of 20[degrees]C/ min. up to 125[degrees]C for 3.0 min. The temperature of the injector and the detector were 250[degrees]C and 310[degrees]C, respectively.

Results and Discussion

Mutagenicity of the Water Extracts Before and After Chlorination

Figure 2 shows the mutagenic activity of the water extracts before and after chlorination in Salmonella typhimurium, in strains TA98 and TA100, in presence and absence of the mixture S9. The water extracts for human consumption in the San Cristobal plant exhibited mutagenicity to the TA98 and TA100 strains of Salmonella typhimurium in absence of the mixture S9 (Table 1, Figure 2).

Table 1 shows the mutagenic activity of water extracts of Salmonella TA98 and TA100. The water extracts with chlorine and without mixture S9 exhibited a significant mutagenic activity in both strains. For the TA98 strain, the three concentrations tested (1, 2, 3 mg/plate) produced four-, six-, and five-fold increases, respectively, in mutant frequency relative to the negative control without mixture S9. Regarding the same control, the TA100 strain, the three concentrations tested (1, 2, 3 mg/plate) produced four-, six- and seven-fold increases, respectively. Those results indicate that the extracts contain a strong mutagenic activity, according to the criteria established by WHO (Coulston & Dunne, 1980). Furthermore, due to the fact that the extract presented mutagenic activity to both strains suggests that the mechanism to damage the DNA involves frameshift mutations (loss or gain of a pair of bases) as substitution of a pair of bases (Benigni, 2005; King et al., 2009).

The mutagenic activity for both extracts of chlorinated water for both strains (TA98 and TA100) was reproducible with a relatively low standard deviation (10.3% and 3.8% coefficient of variation, respectively) over six assays developed in a period of four months.

The TA98 strain presented a linear mutagenic response until 2 mg of extract (Figure 2), where it reaches the maximal number of revertants/plate. The highest dose used (3 mg/ plate) decreased by about 16% the number of revertants/plate regarding the previous dose, which can be explained by a toxic effect to this level of dose. For the TA100 strain (Figure 2) a linear effect was observed for all doses tested.

In contrast, the water extract without chlorination did not present mutagenic activity for any of the strains used in condition of presence or absence of S9 (Table 1 and Figure 2); this indicates that the substances responsible for the mutagenic activity of the extracts are formed during the chlorination process. Similar results were found in water extracts collected from Finland and Russia (Smeds et al., 1997).

Table 1 shows that the addition of the mixture S9 that contains microsomal enzymes responsible for the metabolic activity, i.e., cytochrome P450, lessens almost completely the mutagenicity of the extracts of chlorinated waters. These results are consistent with those of other researchers that report a reduction of the mutagenic activity in chlorinated waters in the presence of S9 in Salmonella typhimurium, strain TA100 (Backlund, Kronberg, Pensar, & Tikkanen, 1985). These results indicate that the presence of S9 reduces markedly the activity of the mutagenic substances that are present in the extracts; the average reduction of the mutagenicity was 51% for the strain TA98 and 68% for the strain TA100, suggesting that the mutagenic substances present in the extracts have a direct-acting mutagenicity. Previous work demonstrated that MX is a directly acting mutagen and its mutagenic affects in vitro are greatly reduced in the presence of liver enzymes-S9 mix (Franzen, Goto, Tanabe, & Morita, 1998).

According to the mutagenicity obtained for the extracts, it is possible to infer that a good recovery of the mutagenic compounds occurred using the procedure of absorption by XAD resins and acidifying previously the water samples at pH 2. This is in agreement with the fact that the resins are made of relative nonpolar materials (styrene-divinylbenzene copolymers) and at this pH the nonionic form of the MX is favored (Rezemini, Vaz, & Carvalho, 2008). That apparently is one of the compounds that is contributing to the main part of the mutagenic activity of the extracts.

Genotoxic Effect of the Water Extracts of the DNA of Human Lymphocytes

Figure 3 shows that the extracts of chlorinated water produced a significant migration of the DNA (tail of the comet) compared with the nonchlorinated extracts. Figures 4 and 5 show photomicrographs obtained during the comet assay for different doses used with the extracts of chlorinated water 0.1, 0.4, 0.7, and 1.0 mg; the negative and positive controls and a representative dose of the water extracts without chlorination (0.4 mg). The remaining doses of these extracts are not shown as they are very similar to those presented. The photomicrographs show how the extracts of chlorinated water produce migration of the DNA in the electrophoresis gel in a progressive way according to the dose, indicating that the extracts of chlorinated water contain compounds that are able to induce strand breaks and labile sites in the DNA of human lymphocytes. The damage is dependent on the dose, with the higher concentration of the extract, the greater the DNA migration, which is equivalent to greater damage. In contrast, the same concentrations 0.1, 0.4, 0.5, and 0.7 mg of the water extract without chlorination (only the photomicrograph of the dose 0.4 mg is shown) did not produce visible genotoxic damage (Figure 5C).

The DNA strand break evaluated with the comet assay could be produced for an adduct reparation mechanism possibly formed by the reaction of MX with DNA bases. According to Lindahl & Andersson (1972) the DNA adducts are repaired by N-glycosylases originating at an apurinic/apyrimidinic site (AP site) that can be repaired by AP-endonucleases or hydrolyzed by alkalis producing a DNA break, that is visualized in the length of the "tail of the comet."

Analytical Detection of THMs and MX/E-MX

Using the GC-MS technique, MX, and E-MX were identified in extracts of chlorinated water. Using scan mode the compounds of interest coeluted with some other compounds, however using single ion monitoring (SIM) the compounds were clearly identified by looking at the fragments (m/z) 198.9, 200.9, and 202.9 for MX, corresponding to the isotopic group OC[H.sub.3] in the MX; and the fragments 241.0 and 243.0 for the loss of chlorine in the E-MX molecule and the resulting molecule due to the loss of the group E-MX with the fragments 244.9 and 246.9. These fragments were used by other authors to identify these molecules in chlorinated waters (Kronberg, Holmbom, Reunanen, & Tikkanen, 1988; Smeds, Vartiainen, Maki-Paakkanen, & Kronberg, 1997). In Table 2 it is possible to observe a correspondence in the abundances for both the standards and the water extracts that confirms the presence of MX and E-MX in the samples. Quantification of these compounds was not possible at the time of the analysis due to unavailability of the standard when the analysis was performed.

The GC-ECD analysis was used to quantify four trihalomethanes: chloroform, bromoform, bromodichloromethane, and dibromochloromethane. Table 3 reports the quantity detected for each. The average concentration in the samples were 1.34 [micro]g/L for CHBr[Cl.sub.2], 0.24 [micro]g/L for CH[Br.sub.2]Cl, 0.30 [micro]g/L for CH[Br.sub.3] and 5.77 [micro]g/L for CHC[L.sub.3]. The sum of the four THMs studied was 7.65 [micro]g/L, an amount that is very small compared to the amount permitted by international regulations of 80 [micro]g/L (National Primary Drinking Water Regulations, 2006). Nevertheless, depending on the period of sampling and some physicochemical conditions, the concentration found in drinking water may vary (Loyola-Sepulveda, Lopez-Leal, Munoz, Bravo-Linares, & Mudge, 2009).

Conclusion

The purpose of this research was to evaluate the mutagenic as well genotoxic effects of water treated with chlorine in the San Cristobal plant. The results show the following:

a) The concentrated water extracts after the chlorination process were mutagenic to bacteria and genotoxic for mammal cells (human lymphocytes), evaluated through the Ames test and comet assay, respectively. The use of both protocols in the same study allows a correlation between mutagenic and genotoxic events that are complementary, constituting a good tool for the evaluation of the sequential steps that can lead to a carcinogenic process.

b) The mutagens formed during the chlorination process are of direct action and are inhibited by enzymes of metabolic activation as the one contained in the mixture S9.

c) The detection by GC-MS of MX and its isomer E-MX in the extracts of chlorinated water suggests that part of the mutagenicity and carcinogenicity of these extracts might be attributed to the presence of these compounds classified by WHO (Coulston & Dunne, 1980) as potent mutagens of direct action.

d) The quantiication of the THMs in the water extracts indicates that these compounds are present in a minimal amount, less than the permitted by the international regulations, and consequently their contribution to the mutagenicity and genotoxicity of the water extracts may not be significant.

Further studies should be conducted to evaluate the mechanisms for what these compounds such as the MX and its isomers produce damage to the DNA. Also the data could be of interest for future study to specifically understand the mechanism involved in the genotoxicity and it could also be helpful to implement regulation to set criteria for acceptable limits of these compounds in water treated by chlorination. It will also open room to introduce mandatory testing for the presence of these compounds in drinking water.

Acknowledgements: The authors would like to thank Dr. Bruce Ames of the University of California, Berkeley, for his donation of the strains of Salmonella typhimurium. Also thanks to the water treatment plant San Cristobal for facilitating the collection of the water samples.

Corresponding Author: Elizabeth RinconBedoya, Assistant Professor, Universidad Austral de Chile, Facultad de Ciencias, Institute de Quimica, Las encinas 220, ex hotel isla teja, Valdivia, Chile. E-mail: elizabethrincon@uach.cl.

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Elizabeth Rincon-Bedoya, PhD

Nelly Velasquez

Jairo Quijano, PhD

Claudio Bravo-Linares, PhD

Did You Know?

The Davis Calvin Wagner Award was first presented in 1981 to Richard L. Roberts. This award was not presented to anyone in 2012. To view of list of past notable recipients of this honor, go to www.sanitarians.org/aas-awards/.

TABLE 1

Mutagenicity of the Water Extracts With and Without Chlorine
in the Strains TA98 and TA100

Doses       Mixture         Average Mutagenicity
(mg/Plate)    S9           (Rev/Plate) [+ or -] SD
                                     TA98

                         Chlorine +         Chlorine -

1.0            +       38 [+ or -] 7.3    31 [+ or -] 2.9
1.0            -       69 [+ or -] 7.4    21 [+ or -] 2.1
2.0            +       39 [+ or -] 6.8    32 [+ or -] 9
2.0            -      111 [+ or -] 10.8   24 [+ or -] 4.3
3.0            +       51 [+ or -] 12     26 [+ or -] 6.6
3.0            -       93 [+ or -] 9.7    23 [+ or -] 1.2
C- *           +       31 [+ or -] 4.0    30 [+ or -] 8.6
C- *           -       18 [+ or -] 4.9    16 [+ or -] 2.9

Doses              Average Mutagenicity
(mg/Plate)       (Rev/Plate) [+ or -] SD
                          TA100

               Chlorine +         Chlorine -

1.0         151 [+ or -] 10    135 [+ or -] 0.5
1.0         434 [+ or -] 5     135 [+ or -] 13
2.0         193 [+ or -] 6     156 [+ or -] 17
2.0         660 [+ or -] 44    138 [+ or -] 4
3.0         248 [+ or -] 9.9   159 [+ or -] 10
3.0         781 [+ or -] 29    137 [+ or -] 12
C- *        129 [+ or -] 11    137 [+ or -] 14
C- *        114 [+ or -] 12    110 [+ or -] 12

* Negative control: distilled water.

TABLE 2

Selected Ions for the Identification of MX and E-MX and
Comparison of Their Relative Abundances With the Standards

Compound     Fragment       m/z    Relative Abundance

                                   Standard    Sample

MX         M-OC[H.sub.3]   198.9     0.54       0.53
                           200.9     1.00       1.00
                           202.9     0.64       0.68

E-MX           M-Cl        241.0     0.62       0.70
                           243.0     0.43       0.48

           M-OC[H.sub.3]   244.9     1.00       1.00
                           246.9     0.90       0.94

TABLE 3

Height and Concentrations (Mg/L) of Four Trihalomethanes
in the Chlorinated Water Samples From the San Cristobal
Treatment Plant

Sample     CHBr[Cl.sub.2]      CH[Br.sub.2]Cl

          H     [micro]g/L    H     [micro]g/L

1        7833      1.29      914       0.24
2        8055      1.33      900       0.24
3        8435      1.39      917       0.24
Average   -        1.34       -        0.24

Sample       CH[Br.sub.3]         CHC[L.sub.3]

           H     [micro]g/L     H      [micro]g/L

1         250       0.27      14,029      5.86
2         300       0.32      12,484      6.34
3         287       0.31       5796       5.11
Average    -        0.30        -         5.77
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Title Annotation:INTERNATIONAL PERSPECTIVES
Author:Rincon-Bedoya, Elizabeth; Velasquez, Nelly; Quijano, Jairo; Bravo-Linares, Claudio
Publication:Journal of Environmental Health
Article Type:Report
Geographic Code:3COLO
Date:Jan 1, 2013
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