Mutagenesis vol. 19 no. 5 pp. 341--347, 2004
doi:10.1093/mutage/geh040
Genotoxicity and cytotoxicity assessment in lake drinking water
produced in a treatment plant
Annamaria Buschini, Pamela Carboni, Silvia Frigerio1,
Mariangela Furlini, Laura Marabini1, Silvano Monarca2,
Paola Poli3, Sonia Radice1 and Carlo Rossi
Dipartimento di Genetica Antropologia Evoluzione, Università di Parma,
Parco Area delle Scienze 11A, 43100 Parma, Italy, 1Dipartimento di
Farmacologia, Chemioterapia e Tossicologia ‘E. Trabucchi’, Università di
Milano, via Vanvitelli 32, 20129 Milano, Italy and 2Dipartimento di Igiene e
Sanità Pubblica, Università di Perugia, Via del Giochetto, 06123 Perugia, Italy
Chemical analyses and short-term mutagenicity bioassays
have revealed the presence of genotoxic disinfection byproducts in drinking water. In this study, the influence of
the different steps of surface water treatment on drinking
water mutagen content was evaluated. Four different samples were collected at a full-scale treatment plant: raw lake
water (A), water after pre-disinfection with chlorine
dioxide and coagulation (B), water after pre-disinfection,
coagulation and granular activated carbon filtration (C)
and tap water after post-disinfection with chlorine dioxide
just before its distribution (D). Water samples, concentrated by solid phase adsorption on silica C18 columns,
were tested in human leukocytes and HepG2 hepatoma
cells using the comet assay and in HepG2 cells in the micronuclei test. A significant increase in DNA migration was
observed in both cell types after 1 h treatment with filtered
and tap water, and, to a lesser extent, chlorine dioxide predisinfected water. Similar findings were observed for the
induction of ‘ghost’ cells. Overloading of the carbon filter,
with a consequent peak release, might explain the high
genotoxicity found in water samples C and D. Cell toxicity
and DNA damage increases were also detected in metabolically competent HepG2 cells treated with a lower concentration of tap water extract for a longer exposure time
(24 h). None of the water extracts significantly increased
micronuclei frequencies. Our monitoring approach
appears to be able to detect contamination related to the
different treatment stages before drinking water consumption and the results suggest the importance of improving
the technologies for drinking water treatment to prevent
human exposure to potential genotoxic compounds.
Introduction
Mutagenicity of drinking water is due not only to industrial,
agricultural and urban pollution but also to disinfection treatments (WHO, 1996; Calderon, 2000; Hofer and Shuker, 2000).
In particular, it has been demonstrated that chlorination, the
most widely used method of disinfecting water, leads to
the formation of numerous mutagenic and/or carcinogenic byproducts (DPBs) (Rook, 1974, 1977; Meier et al., 1986; Meier,
1988; Agarwal and Neton, 1989; Cantor, 1997; Richardson,
1998; Lee et al., 2001; Plewa et al., 2002; Woo et al., 2002;
3
Richardson et al., 2003). Furthermore, epidemiological studies
in Finland, the USA and Taiwan have shown an association
between mutagenicity of chlorinated drinking water and cancer
of the urinary and gastrointestinal tracts (Koivusalo et al.,
1994, 1995; Schenk et al., 1998; Tao et al., 1999).
Chlorine dioxide (ClO2 ) is widely used as an alternative
disinfectant because it does not produce appreciable levels of
trihalomethanes, even though some possible hazardous byproducts, including chlorite and chlorate, are produced (EPA,
1994). Moreover, ClO2 is a strong oxidant that reacts with
organic material to produce a variety of oxidized by-products
(Tan et al., 1987; Kim et al., 1999; Dabrowska et al., 2003).
Considering the wide use of ClO2 as a disinfectant, it is
desirable to find efficient methods to remove its unwanted byproducts and/or decrease the necessary amount of chlorine
dioxide used in the post-disinfection step. A reduction in the
concentration of natural organic matter in treated water is
therefore of great importance in order to decrease ClO2
demand (Swietlik et al., 2002). ClO2 water disinfection is
generally followed by granular activated carbon (GAC) filtration for the removal of oxidized natural organic matter and
inorganic by-products (Paune et al., 1998; Badriyha et al.,
2003). Furthermore, GAC filtration has been reported to efficiently remove chlorites (Swietlik et al., 2003). On the other
hand, relatively rapid saturation of the filter bed with the high
molecular weight portion of natural organic matter sharply
increases the ClO2 demand of GAC effluents (Swietlik et al.,
2003).
Residual disinfection is used to provide a partial safeguard
against low level contamination and regrowth within the distribution system. Consequently, disinfectants are present in tap
water together with the disinfection by-products. Even if the
exposure level is low, the presence of these compounds in
drinking water must be taken into account because of lifelong
human exposure.
This study is part of a research project, supported by the
Italian Ministry for Universities (MURST), on potential genotoxic risk from the treatment of surface water for human
consumption. We propose a methodological approach to evaluate the influence of the different steps of drinking water treatment (pre-disinfection, GAC filtration and post-disinfection) at
the beginning of the distribution system on water mutagenicity.
Water genotoxicity was evaluated by the single cell gel
electrophoresis (SCGE or comet assay) on fresh human leukocytes and human HepG2 hepatoma cells and by the micronuclei
(MN) test on HepG2 cells. The sensitivity of the comet assay
allows rapid detection of the genotoxic potential of compounds
and has been shown to be useful for in vivo and in vitro
biomonitoring of environmental pollutants, as reported in
many reviews (Anderson et al., 1998; Cotelle and Ferard,
1999; Olive, 1999; Kassie et al., 2000; Møller et al., 2000;
Sasaki et al., 2000; Shen and Ong, 2000; Collins and
To whom correspondence should be addressed. Tel: +39 0521 905608; Fax: +39 0521 905604; Email: [email protected]
Mutagenesis vol. 19 no. 5 ß UK Environmental Mutagen Society 2004; all rights reserved.
341
A.Buschini et al.
Horvathova, 2001). The MN test, a validated method for
detecting both clastogenic and aneugenic activity (Knasmuller
et al., 1998), could be a useful tool for the evaluation of
drinking water genotoxic load.
Materials and methods
Chemicals
Reagents for electrophoresis, normal melting point and low melting point
agarose, dimethyl sulfoxide (DMSO), ethidium bromide (EtBr), fluorescein
diacetate (FDA), ethylmethane sulfonate (EMS), cell culture medium, foetal
bovine serum, penicillin-streptomycin and general laboratory chemicals were
from Sigma (Sigma-Aldrich Co. Ltd, Milan, Italy).
Water sampling and concentration
The water samplings were carried out at a full-scale treatment plant producing
drinking water from Lake Trasimeno (Central Italy) serving about 30 000
inhabitants. The lake water analysis (Table I) indicated high concentrations
of total organic carbon (TOC), with significant seasonal variation. Furthermore,
high amounts of bromide (0.6--0.7 mg/l), a potential precursor of disinfection
by-products, were found in the lake water.
The stages of drinking water treatment were as follows: 1, pre-disinfection
with ClO2 and coagulation; 2, rapid sand filtration; 3, GAC-filtration; 4, postdisinfection with ClO2 and distribution in the mains. The samples were
collected during different water treatment steps (Figure 1): raw lake water
(A), water after pre-disinfection with ClO2 and coagulation (B), water after
pre-disinfection, coagulation and GAC filtration (C) and tap water after postdisinfection with ClO2 , just before its distribution (D).
The water samples (100 l) were acidified with sulphuric acid (pH 2),
concentrated using solid phase adsorption with 10 g trifunctional silica gel
C18 cartridges (Sep-Pak Plus C18 Environmental Cartridges; Waters Chromatography, Milford, MA). Cartridge activation was made in sequence with ethyl
acetate, dichloromethane, methanol and distilled water (40 ml of each solvent)
(Monarca et al., 2002). The water was sucked through the cartridge (20 l/
cartridge) with a pump in a multisample concentration system (VAC ELUT
Table I. Physico-chemical characteristics of raw lake water in different
seasonal sampling periods
Water temperature ( C)
pH
TOC (mg/l)
COD (mg/l)
BOD (mg/l)
Nitrates (mg/l NO3 )
Autumn
Winter
Summer
17
8.5
6.4
19
1.3
0.11
8.5
8.5
9.4
35
2.1
0.16
22
8.6
5.8
17
0.9
50.1
SPS 24; Varian, Leini, Italy). The extracts, eluted from cartridges with ethyl
acetate, dichloromethane and methanol (40 ml of each solvent), were first dried
with a rotary evaporator, then redissolved in DMSO (1 leq /40 ml) and later used
for in vitro assays.
Cell types
Leukocytes were isolated from heparin-treated peripheral blood obtained
by venipuncture from healthy non-smoking donors using the procedure outlined in a previous report (Poli et al., 1999). The human HepG2 hepatoma
cells were obtained from the American Type Cell Culture Collection (Istituto
Zooprofilattico, Brescia, Italy). The cells were grown in RPMI 1640 medium
supplemented with 10% foetal bovine serum, 0.03% glutamine, 0.11 g/l sodium
pyruvate, 100 mg/ml streptomycin and 100 U/ml penicilin in culture dishes at
37 C, 96% relative humidity and 5% CO2 .
Cytotoxicity assays
Short-term exposure (1 h). Toxicity was checked immediately after exposure
on cells treated for 1 h at 37 C. Leukocyte survival was determined by the
FDA/EtBr assay (Merk and Speit, 1999). Toxicity in HepG2 cells was
measured by the Trypan blue exclusion method.
Long-term exposure (24 hours). Two different tests, neutral red uptake (NRU)
and lactate dehydrogenase (LDH) release, were carried out on HepG2 cells in
order to exclude highly toxic concentrations (cell survival 560% in the NRU
assay was considered toxic).
The NRU assay was performed according to the method of Zhang et al.
(1990). The cells were distributed over a 96-well microplate 24 h before
treatment. The medium was removed from the microplates and 200 ml of
neutral red solution (50 mg/ml in RPMI 1640) was added to each well. After
2 h incubation at 37 C, the medium was removed and the cells rapidly washed
with 200 ml of fixative (1% formaldehyde, 1% CaCl2 ). A solution of 1% acetic
acid and 50% ethanol was added (200 ml) to each well to extract the dye. After
1 h at room temperature, the plates were read at 540 nm.
The LDH release assay was performed using a CytoTox96 non-radioactive
cytotoxicity assay (Promega). The CytoTox 96 Assay quantitatively measures
LDH, a stable cytosolic enzyme that is released upon cell lysis. LDH released
into the culture medium and total LDH of lysed cells were evaluated. The ratio
released LDH:total LDH, expressed as a percentage with respect to the control
group, was a measure of the level of cell membrane damage. Briefly, the cells
were seeded on a 96-well microplate 24 h before treatment, which was performed with serum-free medium to avoid interaction with the substrate. After
the treatment time, 25 ml of medium were moved to a new multiwell plate to
evaluate the LDH released from cells into the medium, while the remaining
medium was replaced with fresh serum-free medium. The cells were lysed by
adding 15 ml of a lysis solution (9% Triton X-100 in water) and incubated for
60 min at 37 C. After incubation, the plate was centrifuged (250 g for 4 min)
and then 25 ml of the supernatant were transferred to another plate. Thereafter,
25 ml of Substrate Mix solution was added to each well (both supernatant and
lysed cells) and incubated at room temperature for 30 min. The reaction was
stopped with 25 ml of Stop solution (1 M acetic acid) and the plates read at
490 nm (Victor2 Wallac spectrophotometer).
Comet assay
The assay was performed according to Singh et al. (1988): cell lysis at 4 C
overnight (2.5 M NaCl, 10 mM Na2 EDTA, 10 mM Tris--HCl, 1% Triton X-100
and 10% DMSO, pH 10), DNA unwinding for 20 min in an alkaline electrophoretic buffer (1 mM Na2 EDTA, 300 mM NaOH, pH > 13, 0 C), electrophoresis for 20 min (0.78 V/cm, 300 mA) at 0 C in the same buffer,
neutralization (0.4 M Tris--HCl, pH 7.5). The DNA was stained with 100 ml
EtBr (2 mg/ml) before examination at 4003 magnification under a fluorescent
microscope equipped with a BP 515--560 nm excitation filter and an LP 580 nm
barrier filter. The samples were coded and evaluated blind (50 cells for each of
two replicate slides per data point). The assay was only performed with at least
70% cell survival (Tice et al., 2000). All tests were repeated (three independent
replicates per data point).
Fig. 1. Schematic diagram of sampling points in a plant for drinking water
treatment. A, raw lake water; B, water after pre-disinfection with chlorine
dioxide and coagulation; C, sand- and GAC-filtered water; D, tap water after
post-disinfection with chlorine dioxide.
342
Leukocytes. The cells were treated for 1 h at 37 C with different concentrations
of the test compound. The highest concentration (1.25 leq /ml corresponding to
50 ml extract/ml) was determined on the basis of DMSO toxicity (unpublished
data). A positive control (2 mM EMS) was performed. The comet parameters
were determined using an image analysis system (Cometa ReleaseÒ 2.1; Sarin,
Florence, Italy) and the tail moment (TM) was chosen to represent the data on
genotoxic effects. Cells with a complete disintegration of the head region,
‘ghost cells’, were also registered. An SPSS 11 statistical package was applied.
Statistical differences between controls and treated samples were first determined with the non-parametric Wilcoxon rank sum test for each experiment
(Anderson et al., 1994). The mean values from the repeated experiments were
used in a one-way analysis of variance test. If a significant F value (P 5 0.05)
was obtained, a Dunnett’s C multiple comparison analysis was conducted.
Genotoxicity and cytotoxicity of lake water
Results
Cytotoxicity assays
In short-term (1 h) treated cells, the survival rate was 493% in
all samples, with no significant relation to the concentration,
from 0.25 to 1.25 leq /ml (results not shown).
The cytotoxicity data in HepG2 cells, evaluated by two
different methods (the NRU and LDH assays) at different
concentrations (0.125--0.500 leq /ml) of water extracts after
24 h treatment, are reported in Figure 2 (results expressed as a
percentage versus control). In both assays the tap water (D) significantly reduced the cell survival rate (0.125 and 0.250 leq /ml
in the LDH and NRU assays, respectively).
Comet asssay in human leukocytes
A wide seasonal variability in genotoxicity in human
leukocytes (Table II) was detected by the comet assay in previous samplings (autumn and winter) of raw and ClO2 predisinfected water. The disinfection process was able to reduce
(autumn) or increase (winter) the genotoxic activity of raw
water in relation to the seasonal chemical characteristics of
lake water (Table I). In summer, sand- and GAC-filtered
water (C) and tap water (D) were sampled together with raw
(A) and ClO2 pre-disinfected (B) water. The results relating to
the summer sampling are presented in Figure 3.
Raw lake water (A). The water extract did not significantly
induce DNA damage or ‘ghost’ cells in human leukocytes,
confirming the high seasonal variability in lake water
genotoxicity (see Table II).
ClO2 pre-disinfected water (B). The water sampled after
pre-disinfection with ClO2 and coagulation showed very
weak effects on DNA: a significant increase in DNA migration with respect to the control was only observed at the
highest concentration (P 5 0.05, 13.6 ± 1.0 versus 10.5 ±
0.7 mm). The number of ‘ghost’ cells was not affected by
the treatment. As reported (Table II), the disinfection process
can alter, either increasing (winter) or reducing (autumn),
raw water genotoxicity according to the lake water characteristics (Table I).
100
Cell survival (%)
Micronuclei assay
HepG2 cells were grown on microscope slides seeded in Quadriperm plates
(Heraeus Instruments, Hanau, Germany) for 24 h before treatment. After 24 h
treatment, the medium was removed and cytocalasin B (final concentration
3 mg/ml) was added. For fixation, the slides were treated with a cold
hypotonic solution (5.6 mg/l KCl). Conventional air-dried preparations
were made: the cells on the slides were fixed with a 3:1 methanol/acetic
acid solution for 15 min and the slides dried overnight. To detect MN in
binucleated cells, the slides were stained with Giemsa Stain Modified
(Sigma) for 30 min. A total of 1000 binucleated cells/slide (three replicates
per data point) were scored with a light microscope for the evaluation of MN
frequencies. The response was considered positive when the mean MN
frequency was at least more than twice the spontaneous frequency (Hartmann et al., 2001). A statistical analysis was performed with a x2 test using
the PRISM statistical package.
I
A
B
80
C
60
D
40
20
0
0
0.125
0.250
0.500
Leq/ml
II
160
% LDH vs. control
HepG2 cells. The exposure times were 1 or 24 h at 37 C with different
concentrations of test compounds. After treatment, the cells were trypsinized
(0.1% trypsin solution, 5 min), centrifuged (80 g for 3 min), resuspended in
medium and pressed through a syringe (21 G) twice to obtain individual cells.
The comet parameters were determined using an image analysis system
(MetamorphÒ ; Crisel Instruments, Rome, Italy). TM was chosen to represent
the data on genotoxic effects. Ghost cells were also registered. Statistically
significant (P 5 0.05) differences between treatment groups were determined
with one-way ANOVA using a PRISM statistical package.
Control
120
0.062 Leq/ml
80
0.125 Leq/ml
0.250 Leq/ml
40
0
sample
D
C
B
A
Fig. 2. Cytotoxicity to HepG2 cells of different concentrations of water
concentrates after 24 h treatment evaluated using the NRU (I) and LDH (II)
assays. The results (means ± SD of three independent experiments) are
expressed as percent versus control. P 5 0.05. A, raw lake water; B, water
after pre-disinfection with chlorine dioxide and coagulation; C, sand- and
GAC-filtered water; D, tap water after post-disinfection with chlorine
dioxide.
Table II. Comet assay in human leukocytes treated with raw and chlorine
dioxide (ClO2 ) disinfected water extracts (leq /ml) sampled during two seasonal
periods (autumn and winter) at the drinking water treatment plant
Concentration
(leq /ml)
0
0.25
0.50
0.75
1.00
1.25
Autumn
Winter
Raw water
ClO2
10.2
16.6
15.9
20.3
36.9
42.5
10.2
9.9
10.8
12.6
59.7
52.9
±
±
±
±
±
±
1.1
1.3
1.6
2.6
2.7
3.7
±
±
±
±
±
±
1.1
1.0
1.1
1.7
4.6
4.9
Raw water
ClO2
10.8
11.0
13.3
24.7
73.3
98.2
10.8
14.7
68.8
84.0
93.6
84.6
±
±
±
±
±
±
0.9
1.2
0.8
2.6
5.8
3.7
±
±
±
±
±
±
0.9
1.1
6.3
7.1
4.6
4.9
Median tail moment values are reported (means ± SD of three independent
experiments). The lowest effective extract concentrations (P 5 0.05)
are in bold.
Sand- and GAC-filtered water (C). The sand and GAC filtering processes increased the genotoxic potency of water
in human leukocytes. Both DNA migration and ‘ghost’ cell
number were linearly related to extract concentrations (r2 5
0.74 and 0.82 for TM and ghost cells, respectively). The lowest
effective concentrations were 0.25 leq /ml for TM and 0.75
leq /ml for ‘ghost’ cells.
Tap water (D). The extract of water sampled at the end of
the drinking water treatment increased, with a dose--response
343
A.Buschini et al.
Raw water (A)
Chlorinated water (B)
100
60
100
40
60
40
Median TM (µm)
80
Ghost Cells (%)
Median TM (µm)
80
40
60
40
Ghost Cells (%)
60
20
20
*
20
20
0
0
0
0
0
0.25 0.50 0.75 1.00 1.25
Leq/ml
0.25 0.50 0.75 1.00 1.25
Leq/ml
Filtered water (C)
60
Tap water (D)
100
60
100
80
40
40
˚˚˚
˚˚˚
0
20
0
0
60
20
***
20
Ghost Cells (%)
60
40
***
Median TM (µm)
40
Median TM (µm)
80
Ghost Cells (%)
0
0.25 0.50 0.75 1.00 1.25
Leq/ml
0
20
0
0
0.25 0.50 0.75 1.00 1.25
Leq/ml
Fig. 3. Comet assay on human leukocytes. Migration of DNA as median tail moment (TM) (bars) and percentage ghost cells (line) are reported (50 cells for
each of two replicate slides per data point of each experiment; means ± SD of three
independent experiments). Positive control (2 mM EMS): TM 5 51.06 ± 5.92
mm; 2.43 ± 0.98% ghost cells. TM, P 5 0.05, P 5 0.001; ‘Ghost’ cells, P 5 0.001 (Dunnett’s C).
relationship, both DNA migration (r2 5 0.75) and ‘ghost’ cell
number (r2 5 0.90). At 0.25 leq /ml, the median DNA migration
was significantly higher than after treatment with the C sample
at the same concentration (P 5 0.001), even if the slopes of the
curves were similar for tap water and filtered water (25 versus
28). The lowest effective concentration for ghost cell induction
was 0.50 leq /ml (0.75 leq /ml for the ‘filtered water’); the slope
of the curve was 35, versus 22 for the ‘filtered water’.
344
Comet and micronuclei assays in HepG2 cells
The comet assay was also performed in metabolically competent HepG2 cells following the 1 h exposure time previously
used in fresh human leukocytes and a longer exposure (24 h),
as used for the MN assay (Figure 4). For the long exposure
assay, a 0.125 leq /ml extract concentration was chosen to avoid
high cell mortality (Figure 2I), as recommended by the comet
assay guidelines (Tice et al., 2000); the short exposure assay
Genotoxicity and cytotoxicity of lake water
1 hour
80
*
30
60
20
40
10
20
0
0
CTR
A
B
C
D
Micronuclei/ 1000 cells
**
Gho st cells (%)
Median TM (µm)
100
**
40
12
10
8
6
4
2
0
sample
A
B
C
D
1 Leq/ml
Control
24 ho urs
*
100
60
40
40
*
20
20
Gho st cells (%)
80
60
*
Median TM (µm)
80
0
0
A
Control
B
0.0625 Leq/ml
C
D
sample
0.125 Leq/ml
Fig. 4. Comet assay on HepG2 cells after 1 and 24 h treatment. Migration of
DNA as median tail moment (TM) (bars) and percentage ‘ghost’ cells
(open diamonds) are reported (50 cells for each of two replicate slides per
data point of each experiment; means ± SD of three independent
experiments). P 5 0.05, P 5 0.01. A, raw lake water; B, water after
pre-disinfection with chlorine dioxide and coagulation; C, sand- and
GAC-filtered water; D, tap water after post-disinfection with chlorine
dioxide.
was performed at a higher concentration (1 leq /ml) due to the
low mortality rate.
Raw lake water (A). The sample did not appear able to significantly induce DNA damage or increase ‘ghost’ cell number
in human HepG2 cells after either 1 or 24 h treatment at all
concentrations.
ClO2 pre-disinfected water (B). The short-term (1 h) exposure, at a relatively high concentration (1 leq /ml), was able to
detect the genotoxicity induced in HepG2 cells by the extract
of pre-disinfected water. The number of ‘ghost’ cells also
increased with respect to the control group due to the treatment
(P 5 0.05). No DNA migration increase was observed after
the 24 h exposure to lower concentrations.
Sand- and GAC-filtered water (C). The GAC filtering process
also increased the effects of the water extract in HepG2 cells.
Both DNA migration and number of ‘ghost’ cells were
increased after a 1 h treatment at 1 leq /ml concentration. No
significant effects were observed after the 24 h treatment.
Tap water (D). Both DNA migration and number of ‘ghost’
cells were increased after a 1 h treatment with tap water extract
0.062 Leq/ml
sample
0.125 Leq/ml
Fig. 5. Induction of micronuclei (MN) in HepG2 cells by different
concentrations of water extracts after 24 h treatment (1000 binucleated cells/
slide scored; means ± SD of three independent cultures). A, raw lake water;
B, water after pre-disinfection with chlorine dioxide and coagulation; C,
sand- and GAC-filtered water; D, tap water after post-disinfection with
chlorine dioxide.
at 1 leq /ml. A similar effect was observed after 24 h treatment
at 0.125 leq /ml, with a TM value and ‘ghost’ cell number
significantly higher than the control.
The concentrations used for the MN assay were 0.062 and
0.125 leq /ml, with a cell survival percentage 460% for all the
water extract samples (Figure 2I). The frequency of MN in
HepG2 cells after 24 h treatment with the different sampled
water extracts was not significantly different from the control
(Figure 5) when considering a doubling of the spontaneous
frequency a positive effect (Hartmann et al., 2001).
Discussion
In the present study we have evaluated in vitro the cytotoxic
and genotoxic effects of complex mixtures present in extracts
of water sampled in a drinking water treatment plant during
different potabilization steps (raw water, ClO2 disinfected
water, sand- and GAC-filtered water and finished tap water).
With a short exposure time (1 h), concentrations ranging
from 0.25 to 1.25 leq /ml of all the extracts apparently did not
reduce the survival rate of either HepG2 cells or fresh human
leukocytes immediately after treatment. Significant increases
in DNA migration, detected by the comet assay, were observed
in both cell types after 1 h treatment with filtered and tap water
and, to a lesser extent, in ClO2 pre-disinfected water. Similar
findings were observed for the induction of ‘ghost’ cells, which
represent toxic cellular events, i.e. apoptotic and/or necrotic
cells (Meintieres et al., 2003). The close matching of results
on the two cell types shows that the mixtures tested were
biologically active, irrespective of the cell characteristics.
After extended (24 h) exposure to a low concentration (0.125
leq /ml), the tap water extract induced significant cytotoxic
(Figure 2-II) and genotoxic effects, detected by the comet
assay (Figure 4), in metabolically competent HepG2 cells. A
correlation between primary damage and MN induction was
not found as reported elsewhere in human biomonitoring
studies (Hartmann et al., 1998). The discrepancy between the
results in the two assays could be explained by the difference in
the end-points (Lu et al., 2000). MN are the result of chromosome breaks or mitotic anomalies, which require a passage
345
A.Buschini et al.
through mitosis. On the other hand, DNA effects detected in
the comet assay represent a mitosis-independent end-point,
sensitive in assessing the synergistic DNA damaging potential
of mixtures containing compounds producing repairable DNA
breakage or alkali-labile sites (Van Goethem et al., 1997).
Since ClO2 is a strong oxidant, free radicals could be involved
in the DNA damage.
In conclusion, high cytotoxic and genotoxic potency was
observed in water sampled after GAC filtration and in the
finished tap water. These effects are probably due to saturation
of the GAC filters, followed by uncontrolled releases of DBPs
previously adsorbed because of the competitive effects of a
background of organic compounds (Oxenford and Lykins,
1991; Sorial et al., 1994a,b; Cerminara et al., 1995). Paune
et al. (1998) observed the phenomenon of inverse adsorption of
bromoform with respect to the total contaminant charge.
Furthermore, cytotoxic (Figure 2) and genotoxic (Figure 4,
24 h exposure) compounds can be formed after ClO2 postdisinfection since DPBs may be continuously produced by
the reaction of residual disinfectant with organics, as also
reported in other studies (Schwartz et al., 1979; Monarca
and Meier, 1984; Meier, 1988; Koivusalo et al., 1995;
Tao et al., 1999).
The comet assay appeared to be able to detect genotoxicity
during the different stages of water treatment before drinking
water consumption. In this way, ClO2 was found to be able to
produce genotoxic compounds in surface water rich in DBP
precursors, while GAC filtration was found to be unable to
adsorb these contaminants. However, to verify the hypothesis
of filter saturation as responsible for high cytotoxicity and
genotoxicity, specific investigations to check the functionality
and the effects of saturated and new filters need to be performed using surface water with different chemical characteristics. The present methodological approach seems to give
additional data useful for monitoring drinking water quality
and the results suggest the importance of improving the analytical methods and technologies for drinking water treatment to
prevent human exposure to potential genotoxic compounds.
Acknowledgements
This research was funded by the Italian Ministry of Universities and Research
(MURST 1999). We would like to thank Gillian Mansfield (Head of the
Language Centre, University of Parma) for the English revision of the manuscript. We are grateful to the MURST Research Group: A.Alberti, C.Bolognesi,
G.Cantelli-Forti, E.Chiesara, M.D€orr, C.Elia, D.Feretti, B.Gustavino,
L.Guzzella, P.Hrelia, F.Maffei, L.Mantilacci, M.Monfrinotti, M.Paolini,
S.Richardson, M.Rizzoni, M.Santoro, M.I.Taticchi, A.Thurston, C.Zani,
I.Zerbini.
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Received on December 22, 2003; revised and
accepted on May 28, 2004
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