Mutation Research 534 (2003) 145–154

Effect of dithiocarbamate pesticide zineb and its
commercial formulation, azzurro

IV. DNA damage and repair kinetics assessed by single cell
gel electrophoresis (SCGE) assay on Chinese

hamster ovary (CHO) cells

Marina González, Sonia Soloneski, Miguel A. Reigosa, Marcelo L. Larramendy∗
Laboratorio de Citogenética, Cátedra de Citologı́a, Facultad de Ciencias Naturales y Museo, Universidad Nacional de La Plata,

Calle 37 Nro. 668 7 Mo “B”, 1900 La Plata, Argentina

Received 20 February 2002; received in revised form 17 September 2002; accepted 9 October 2002

Abstract

Single cell gel electrophoresis (SCGE) was used to analyse dithiocarbamate zineb- and the zineb-containing technical
formulation azzurro-induced DNA damage and repair in CHO cells. Cells were treated with zineb (50.0�g/ml) or azzurro
(100.0�g/ml) for 80 min, washed and reincubated in pesticide-free medium for 0–12 h until SCGE. Viability of treated cells
(0 h) did not differ from control remaining unchanged up to 6 h of incubation. After 12 h, viability decreased up to 70 and 54%
in zineb- and azzurro-treated cultures, respectively. SCGE revealed at 0 h the absence of undamaged cells and an increase of
slightly damaged and damaged cells in zineb-treated cultures or by an increase in damaged cells in azzurro-treated cultures. For
both chemicals, a time-dependent repair of pesticide-induced DNA damage within a 0–12 h post-treatment incubation period
was observed. Overall, damaged cells decreased as a function of the repair time for both pesticides while the slightly damaged
cells decreased as a function of the repair time of zineb-induced DNA damage. Concomitantly, a time-dependent increase
of undamaged cells was observed within the 0.5–12 h repair time for both pesticides. At 12 h after treatment, no differences
in the frequencies of undamaged, slightly damaged and damaged cells were found between both zineb- or azzurro-treated
cultures and control values as well as between zineb- and azzurro-treated cells. Immediately after exposure, nuclear DNA
from zineb and azzurro-treated cells were larger and wider than nuclear DNA from untreated cells. When damaged cells were
allowed to repair, a time-dependent decrease of the amount of free DNA migrating fragments was observed committed only
to damaged cells but not in slightly or undamaged cells. On the other hand, no time-dependent alteration on nuclear DNA
width within the 0–12 h repair period was observed.
© 2002 Elsevier Science B.V. All rights reserved.

Keywords:Comet assay; Chinese hamster ovary (CHO) cells; Zineb; Azzurro; Dithiocarbamates; Repair kinetics

∗ Corresponding author. Tel.:+54-221-489-0860;
fax: +54-221-423-3340.
E-mail address:m larramendy@hotmail.com (M.L. Larramendy).

1. Introduction

Among ethylene bis(dithiocarbamate) pesticides,
the ethylene bis(dithiocarbamate)zinc (zineb) is a
widely used foliar fungicide and is registered for

1383-5718/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved.
PII: S1383-5718(02)00257-7



146 M. Gonźalez et al. / Mutation Research 534 (2003) 145–154

use on a large number of fruits, vegetable and field
crops, as well as on a large number of ornamental
plants and for the treatment of several seeds. Zineb
is also registered for use as fungicide in paints and
for mould control on fabrics, leather, linen, painted
surfaces, surfaces to be painted and paper, plastic and
wood surfaces[1]. Though carcinogenic and terato-
genic properties were reported for ethylenethiourea,
the main metabolic and degradation product of the
ethylene bis(dithiocarbamate) pesticides is well docu-
mented[2], very little is known about the deleterious
effect of zineb[1,2].

Available reported data of the deleterious effect
of the zineb is scarce and even contradictory. It has
been demonstrated that zineb can be considered as a
non-mutagenic agent when using plate incorporation
assay withSalmonella typhymuriumwhereas gene
conversion and point mutation assays withSaccha-
romyces cerevisaeandBacillus subtillisgave positive
results[3–5]. Zineb exerted a high dose-related cy-
totoxicity in BALB/c 3T3 mouse cells in vitro but
only in the absence of an exogenous metabolising
system[6]. Trigathy et al.[7] reported zineb as pos-
itive genotoxic agent to somatic and germ cells in
Drosophila. While Chernov and Khitsenko[8] ob-
served an increased incidence of lung tumours after its
oral administration to C57BL mice, negative results
have been also reported in other mouse strains[8,9]
and in rats[10,11]. Moreover, after its subcutaneous
administration, systematic reticulum-cell carcinoma
and a variety of sarcomas were observed in mice
[12] and rats[10], respectively. In humans, develop-
ment of sulphaemoglobinaemia, haemolytic anaemia
and Heinz body formation has been reported in one
person suffering hypocatalasemia after contact with
zineb[13]. Even though zineb has been considered a
non-mutagenic agent in bacterial systems by the Inter-
national Agency for Research on Cancer[1], this does
not necessarily imply that the dithiocarbamate cannot
directly damage genetic material. In favour with this
assumption, are reports of the induction of point muta-
tions [3–5], chromosomal aberrations in lymphocytes
from crop workers occupationally exposed to zineb
[14], and our recent report of its ability to induce
chromosomal aberrations, in human lymphocytes in
vitro [15]. We have also demonstrated that zineb as
well as the zineb-containing formulation azzurro are
not only able to induce micronuclei in human lympho-

cytes in vitro, but also that such induction is limited
to B CD20+ and T suppressor/cytotoxic CD8+ cells
among the different lymphocyte subsets[16].

In a previous study, Soloneski et al.[17] reported
that both zineb and azzurro induced a significant
increase in SCEs and a delay in cell-cycle progres-
sion of Chinese hamster ovary (CHO) cells. Further-
more, we analysed the capacity of the single cell
gel electrophoresis (SCGE) methodology in detect-
ing lesions introduced in the DNA of CHO cells
exposed to a pulse treatment of either zineb and the
zineb-containing technical formulation azzurro within
a 1.0–100.0�g/ml dose range. However, no attempt
to analyse the repair kinetics of the induced lesions
have been assessed[17]. In the present study we
analysed whether CHO cells are able to repair the
zineb-induced DNA strand breaks and to provide fur-
ther knowledge of the real risk of the genetic damage
of the dithiocarbamate pesticide.

2. Materials and methods

2.1. Chemicals

Ethylene bis(dithiocarbamate)zinc (zineb; CAS
no. 12122-67-7) was obtained from Riedel-de Haën
(Pestanal®, Hanover, Germany). Azzurro (zineb 70%)
was kindly provided by Chemiplant (Buenos Aires,
Argentina). Dimethyl sulphoxide (DMSO) was pur-
chased from Sigma.

2.2. Cell cultures and pesticide treatment

CHO cells were grown in Ham’s F10 medium
(Gibco, Grand Island, NY) supplemented with 10%
foetal calf serum (Gibco), 100 units/ml of penicillin
(Gibco) and 10�g/ml of streptomycin (Gibco) at
37◦C in a 5% CO2 atmosphere. Prior to drug treat-
ment, exponentially growing CHO cells were de-
tached with a rubber-policeman, collected by centrifu-
gation and resuspended in complete culture medium
and then counted. Afterwards, aliquots containing
3.5 × 105 cells/ml were incubated during 80 min at
37◦C in a 5% CO2 atmosphere in culture medium
containing the test compounds. Both zineb and az-
zurro were dissolved in DMSO prior to use and then
were diluted in culture medium such the addition of



M. Gonźalez et al. / Mutation Research 534 (2003) 145–154 147

100�l to cultures allowed them to reach the required
concentration. Zineb and azzurro were used at the
final concentration of 50.0 and 100.0�g/ml, respec-
tively. The final solvent concentration was lower than
1% for all the treatments. Negative control (untreated
cell and solvent-vehicle-treated cells) were performed
and run simultaneously with pesticide-treated cul-
tures. None of the treatments produced significant
pH changes in the culture medium even the highest
concentration of zineb and azzurro. Thereafter, the
cells were washed twice with pesticide-free complete
culture medium, collected by centrifugation, resus-
pended in 1.0 ml of pesticide-free complete culture
medium. The SCGE and cell viability assays were
performed immediately after the 80 min treatment or
after a post-treatment incubation period of 0–12 h at
37◦C in a 5% CO2 atmosphere with harvesting times
at 0, 0.5, 1, 2, 3, 6 and 12 h. Cultures were duplicated
for each experimental point, during at least three in-
dependent experiments. The same batches of culture
medium, sera and reagents were used throughout
the study.

2.3. Cell viability

Cell viability was determined using the ethidium
bromide/acridine orange assay described elsewhere
[18]. Briefly, one aliquot of 5�l of a 1:1 freshly
prepared mixture of ethidium bromide (100�g/ml,
Sigma) and acridine orange (100�g/ml, Sigma) was
mixture with 50�l of the cell suspension. After-
wards, cells were analysed using an Olympus BX50
fluorescence photomicroscope equipped with ap-
propriated filter combination. Viable cells appear
green-fluorescent whereas orange-stained nuclei indi-
cate dead cells. At least, 500 cells were counted per
experimental point, and results expressed as percent-
age of viable cells among all cells. Cell viability was
expressed as proportion of living cells.

2.4. Single cell gel electrophoresis (SCGE)
assay

The remaining cell culture (950�l) was used for
microgel electrophoresis. The comet assay was per-
formed following the alkaline procedure described
by Singh[19] with minor modifications. Slides were
cleaned with 100% ethanol and air-dried. Two so-

lutions containing 0.5% normal melting agarose
(NMA), and 0.5% low melting agarose (LMA) so-
lution in Ca2+–Mg2+-free PBS were performed.
Briefly, 75�l of 0.5% NMA was transferred onto
a pre-cleaned slide, spread evenly, and placed at
37◦C to solidify the agarose. Afterwards, 95�l of
0.5% LMA together with 7× 103 cells (20�l cell
suspension+ 75�l of 0.5% LMA) was applied, cov-
ered with a coverslip, and placed at 4◦C for 15 min.
After this layer had solidified, a third layer of 75�l of
0.5% LMA was added, and slides placed at 4◦C for
15 min. Immediately after, slides were immersed in
ice-cold freshly prepared lysis solution (1% sodium
sarcocinate, 2.5 M NaCl, 100 mM Na2EDTA, 10 mM
Tris, pH 10.0, 1% Triton X-100, 10% DMSO) and
then lysed in the dark at 4◦C for an overnight period.
After the overnight period, slides were placed in a
horizontal electrophoresis device filled with freshly
prepared electrophoresis buffer (1 mM Na2EDTA,
300 mM NaOH) for 20 min at 4◦C to allow the cel-
lular DNA to unwind, followed by electrophoresis
in the same buffer at 4◦C for 20 min at 25 V and
250 mA. Afterwards, slides were neutralised with a
solution comprising 0.4 M Tris–HCl (pH 7.5) and
stained with 4′,6-diamidino-2-phenylindole (DAPI;
Vectashield mounting medium H1200; Vector Labo-
ratories, Burlingame, CA, USA). Slides were coded
and scored blind by one cytogeneticist. Analysis of
the slides was performed in a Olympus BX50 fluores-
cence photomicroscope equipped with appropriated
filter combination with a X63 fluorescence objec-
tive. Cellular images were acquired with the Leica
IM50 Image Manager (Imagic Bildverarbeitung AG),
based on an integrated high-sensitivity monochrome
charge-coupled device (CCD) camera and automated
capture image software. The cellular nucleus diame-
ter and the comet length, determined as the diameter
of the nucleus plus migrated DNA, were measured
by CarioFISH 1.2 software. Width of the nucleus
and comet length (expressed in�m) were determined
from 50 randomly captured cells per experimental
point of each experiment. Two parallel slides were
performed for each experimental point. Cells were
visually graded into four categories as previously
suggested by us[17]. Depending on DNA damage
level the categories were as follows: undamaged
(no tail comet, diameter≤30�m), slightly damaged
(diameter 31–45�m), damaged (tail comet length



148 M. Gonźalez et al. / Mutation Research 534 (2003) 145–154

Fig. 1. Microphotographs of CHO cells treated with zineb show-
ing various degrees of DNA damage: (A) undamaged cell (diam-
eter≤30�m); (B) slightly damaged cell (no tail comet, diameter
31–45�m); (C) damaged cell (tail comet length >45�m).

>45�m) and highly damaged (dying or dead cells;
Fig. 1).

2.5. Statistical analysis

The non-parametric Kruskal–Wallis test was used
to compare the global effect of a pesticide over con-
trol cells while individual comparisons between pairs
of data were performed using the Mann–Whitney test
employing the SPSS 9.0 software. The level of signif-
icance chosen was 0.05 unless indicate otherwise.

3. Results

Since no differences of cell viability were ob-
served between negative and positive controls (un-
treated and DMSO-treated cells, respectively), pooled
data are presented for control cultures. Cell viabil-
ity after 80 min treatment with 50.0�g/ml of zineb
or 100.0�g/ml of azzurro showed no significant
changes until 12 h of incubation when a significant
decrease reaching values of 70 and 54% in zineb- and
azzurro-treated cultures, respectively, was observed
(P < 0.05; Fig. 2). Statistical analysis revealed that
at this harvesting time, cell viability was lower in
azzurro-treated cultures than in those cells treated
with zineb (P < 0.05; Fig. 2).

Equivalent frequencies of undamaged/damaged
cells revealed by SCGE assay from both negative
and positive controls (untreated and DMSO-treated
cells) were observed, then pooled data are presented

Fig. 2. Effect of in vitro treatment with zineb (50.0�g/ml; black
squares) and the zineb-containing technical formulation azzurro
(100.0�g/ml; empty circles) on Chinese hamster ovary (CHO)
cells viability. Cell viability was determined using the ethidium
bromide/acridine orange assay and expressed as the proportion
of living cells at different harvesting times after a 80 min pulse
treatment. For each harvesting time, pooled data from three inde-
pendent experiments are reported as mean values± S.E. (y-axis)
and plotted against incubation time (x-axis). ∗P < 0.05.



M. Gonźalez et al. / Mutation Research 534 (2003) 145–154 149

for control cultures. Moreover, a clear increase in
DNA damage after fungicide 80 min pulse treatment
(0 h) was revealed by the total absence of undamaged
cells and by either an increase over control values of
the proportion of slightly damaged (P < 0.01) and

Fig. 3. Time course of the repair of in vitro DNA damage induced in Chinese hamster ovary (CHO) cells by a 80 min pulse treatment
(0 h) treatment with zineb (50.0�g/ml; A) and the zineb-containing technical formulation azzurro (100.0�g/ml; B) detected by SCGE.
Electrophoresis was performed at 4◦C for 20 min at 25 V and 250 mA, and cells were stained with DAPI. For each harvesting time, pooled
data from three independent experiments are reported, a total 150 randomly selected cells were visualised for DNA damage. Cells classified
as undamaged (empty bars), slightly damaged (grey bars) and damaged (black bars) and their frequencies plotted against harvesting times.

damaged cells (P < 0.01) in zineb-treated cultures
(Figs. 1 and 3A) or by an increase in the frequency of
damaged cells (P < 0.01) in azzurro-treated cultures
(Fig. 3B). No highly damaged cells were observed
either in zineb- (Fig. 3A) or azzurro-treated cells



150 M. Gonźalez et al. / Mutation Research 534 (2003) 145–154

(Fig. 3B). For both chemicals, a time-dependent repair
of zineb- and azzurro-induced DNA damage was ob-
served by a progressive decrease of slightly damaged
and damaged cells simultaneously with an increase in
the frequency of undamaged cells within the 12 h re-
pair period. A regression test showed that the number
of damaged cells decreased as a function of the repair
time of both zineb- (r = −0.73, P < 0.01; Fig. 3A)
or azzurro-induced DNA damage (r = −0.86, P <

Fig. 4. Zineb-induced DNA damage and repair in CHO cells
measured by SCGE. Electrophoresis was performed at 4◦C for
20 min at 25 V and 250 mA, and cells were stained with DAPI.
For each harvesting time, pooled DNA damage relative to 150
zineb-treated individual cells from three independent experiments
are reported. The nuclear DNA length (A) and the nuclear DNA
width (B) expressed in�m are plotted against the different repair
times. The data are displayed as box plots, where they-axis shows
the range data. Each box encloses 50% of the data, with the median
value of the variable displayed as a line. The top and the bottom
of the box mark the limits±25% of the variable population. The
lines extending from the top and the bottom of each box mark
the minimum and maximum values that fall within an acceptable
range. Any value outside this range is displayed as an individual
point (empty circles).

0.01; Fig. 3B) as well as the number of slightly dam-
aged cells of zineb-induced DNA damage (r = −0.69,
P < 0.01;Fig. 3A). Concomitantly, a time-dependent
increase in the number of undamaged cells was ob-
served within the 0.5–12 h repair time either in zineb-
(r = 0.78, P < 0.01) or in azzurro-treated cultures
(r = 0.85, P < 0.01; Fig. 3). At 12 h after treat-
ment, no differences in the frequencies of undamaged,
slightly damaged and damaged cells were found be-
tween both zineb- or azzurro-treated cultures and con-
trol values (P > 0.05) as well as between zineb- and
azzurro-treated cells (P > 0.05; Fig. 3).

Mann–Whitney test demonstrates that both the
length and width of nuclear DNA from zineb-treated

Fig. 5. Zineb-induced DNA damage and repair in CHO cells
measured by SCGE. Electrophoresis was performed at 4◦C for
20 min at 25 V and 250 mA, and cells were stained with DAPI.
For each harvesting time, pooled DNA damage relative to 150
zineb-treated individual cells from three independent experiments
are reported. The nuclear DNA length (A) and the nuclear DNA
width (B) expressed in�m for undamaged (empty bars), slightly
damaged (grey bars) and damaged cells (black bars) are plotted
against are plotted against the different repair times. The data are
displayed as box plots (for details see the caption ofFig. 4).



M. Gonźalez et al. / Mutation Research 534 (2003) 145–154 151

cells immediately after exposure (0 h) are higher and
wider than untreated cells, respectively (P < 0.01;
Fig. 4). When damage cells were allowed to repair, a
time-dependent decrease of the amount of free DNA
fragments that could migrate (i.e. a strong reduction
in the average of nuclear DNA length) was observed
(r = −0.60, P < 0.05; Fig. 4A). Statistical analysis
reveals that such strong reduction of nuclear DNA
length was committed to damaged cells (P < 0.01)
among the treated cell population (Fig. 5A). On the
other hand, no time-dependent alteration on nuclear
DNA width within the 0–12 h repair period was ob-
served (r = −0.2, P > 0.05; Fig. 4B), though an
increase in nuclear DNA width in regard to control

Fig. 6. Azzurro-induced DNA damage and repair in CHO cells
measured by SCGE. Electrophoresis was performed at 4◦C for
20 min at 25 V and 250 mA, and cells were stained with DAPI.
For each harvesting time, pooled DNA damage relative to 150
azzurro-treated individual cells from three independent experiments
are reported. The nuclear DNA length (A) and the nuclear DNA
width (B) expressed in�m are plotted against the different repair
times. The data are displayed as box plots. The data are displayed
as box plots (for details see the caption ofFig. 4).

values was observed either for damaged cells during
the 12 h repair time (P < 0.01) or for slightly dam-
aged cells found at 6 h (P < 0.01; Fig. 5B). Overall,
the zineb-damaged cell population did not recover
completely the features of the control cells even after
12 h repair being the nuclear DNA from treated cells
longer (Figs. 4A and 5A) and wider (Figs. 4B and
5B) than control cells, respectively (P < 0.01).

The length and width of nuclear DNA from
azzurro-treated cells are larger and wider than un-
treated cells immediately after exposure (0 h,P <

0.01; Fig. 6). During the 0–12 h repair time, a
time-dependent decrease in the average of nuclear

Fig. 7. Azzurro-induced DNA damage and repair in CHO cells
measured by SCGE. Electrophoresis was performed at 4◦C for
20 min at 25 V and 250 mA, and cells were stained with DAPI.
For each harvesting time, pooled DNA damage relative to 150
azzurro-treated individual cells from three independent experiments
are reported. The nuclear DNA length (A) and the nuclear DNA
width (B) expressed in�m for undamaged (empty bars) and
damaged cells (black bars) are plotted against are plotted against
the different repair times. The data are displayed as box plots (for
details see the caption ofFig. 4).



152 M. Gonźalez et al. / Mutation Research 534 (2003) 145–154

DNA length was achieved (r = −0.68, P < 0.01;
Fig. 6A). On the other hand, no time-dependent al-
teration on nuclear DNA width within the 0–12 h
repair time was observed (r = −0.31, P > 0.05;
Fig. 6B), though an increase in its width from dam-
aged cells was observed in regard to control values
during the whole repair time period (P < 0.01) but
not in the undamaged cells (Fig. 7B). Overall, the
azzurro-damaged cell population did not completely
achieve the nuclear DNA length (Figs. 6A and 7A)
and the width of the control cells (Figs. 6B and 7B)
even after a 12 h post-treatment repair time appear-
ing the DNA migration longer and wider than the
untreated cell population (P < 0.01).

4. Discussion

We employed the alkaline SCGE assay to anal-
yse the repair kinetics of lesions introduced in the
DNA of CHO cells after an 80 min pulse treatment of
50.0�g/ml zineb and 100.0�g/ml azzurro within the
first 12 h after exposure. We have recently reported
equivalent levels of DNA damage in CHO cells in-
duced by these different pesticide doses[17]. The
present results are in total accord with our previous
observations[17]. Accordingly, we could assume that
the effect induced by azzurro can be accounted for the
zineb component of the mixture alone, and suggest
that a non-clastogenic agent(s) other than zineb might
be present in the technical formulation containing
70% zineb. However, the identity of the components
of the commercial product was not made available
to us.

The reports concerning the use of the SCGE assay
for assessing damage and/or repair of pesticides are
scarce, specially among carbamates. Quantification of
DNA single-strand breaks induction has been reported
in mouse splenocytes and peripheral lymphocytes af-
ter oral administration of thiram as well as in human
lymphocytes in vitro[20]. Garaj-Vrhovac and Zeljezic
[21,22]have evaluated the risk assessment of workers
occupationally exposed to a mixture of different agro-
chemicals. They found that after pesticide exposure,
increased DNA damage in circulating lymphocytes,
namely tail length and tail moment were observed
which decreased significantly compared with the first
sampling point after workers spend 6–8 months out of

the pesticide exposure zone[21,22]. Blasiak et al.[23]
analysed the DNA damaging effect of malathion and
both its major metabolites malaoxon and isomalathion
in human lymphocytes in vitro. They observed that
while malathion did not cause any significant changes
in the comet length of the lymphocytes, its metabo-
lites introduced damage to DNA in a dose-dependent
manner, and that the effect of malaoxon was more pro-
nounced than that caused by isomalathion[23]. Saleha
et al. [24] studied damage and repair of DNA strand
breaks induced by monocrotophos in the nucleated
erythrocytes ofTilapia mossambicain vivo. Their re-
sults revealed that the DNA damage induced within
a 0.313–4.375 ppm dose range of monocrotophos re-
turned to control levels after a 96 h repair period[24].

Our SCGE results demonstrated that zineb and az-
zurro induced a significant increase not only in the
number of CHO damaged cells but also in the comet
tail length. In the genotoxicity, measured by the SCGE,
the effects on cell viability could be ruled out since cell
viability after 80 min treatment with both test com-
pounds showed non-significant changes until 12 h of
repair time when it significantly decreased reaching
values of 70 and 54% in zineb- and azzurro-treated
cultures. It should be pointed out that at the concen-
trations employed, no evidence of a relationship be-
tween cytotoxic effect and comet length was observed,
though equivalent doses of 50.0�g/ml of zineb and
100.0�g/ml of azzurro, respectively, induced a com-
plete inhibition of cellular growth of CHO cells after
a continuous 36 h treatment[17].

The exact mechanism(s) of zineb-induced DNA
damage is not known. It is well documented that
the repair kinetic profile of the single-strand breaks
induced by active oxygen species when analysed
by SCGE is totally different from the repair profile
of those zineb-induced lesions. In fact, the in vitro
repair of the active oxygen species in mammalian
cells is very rapid, being the majority of the breaks
resealed within 10 min[25,26]. Accordingly, the pos-
sibility that zineb-introduced lesions into the DNA
through the formation of active oxygen species, pre-
viously suggested by us when using other cytogenetic
end-points [15–17], could be ruled out since the
present results clearly show the necessity of a 12 h
repair period for a complete resealing of the lesions
analysed with the SCGE under our experimental
conditions. Another possible explanation could be



M. Gonźalez et al. / Mutation Research 534 (2003) 145–154 153

alkylation of the DNA molecule by this ethylene
bis(dithiocarbamate) pesticide. It has been previously
demonstrated that single-strand breaks introduced in
the DNA molecule of CHO cells after treatments with
different ethylating agents, e.g.N-ethyl-N-nitrosurea
and N-ethyl-N′-nitro-N-nitrosoguanidine, are long
lasting, being repaired in a period of up to 24 h after
pulse treatment[26].

Interestingly, our results demonstrated that when
damaged cells were allowed to completely repair
zineb- and azzurro-induced DNA damage, the re-
paired cell population did not completely recover
the morphological features of the control cells, i.e.
increased nuclear diameter, even when no free DNA
migrating fragments were observed. The same phe-
nomenon has been observed by Fortini et al.[26]
in mammalian cells after a complete repair of either
X-ray and alkylation-induced DNA damage. Accord-
ingly, these authors suggested that such morpholog-
ical differences could be committed to differences
in the packaging of DNA in those cells undergoing
repair when compared to untreated cells[26].

To date, only 14 fungicides have been evaluated for
their carcinogenicity to humans by the International
Agency for Research in Cancer[1,27–29]. Among
those analysed, zineb has been overall evaluated and
ranked into category 3 (i.e. insufficient available data
to evaluate carcinogenicity to humans) since the de-
gree of evidence for its carcinogenicity properties have
been determined as not adequate data and/or inade-
quate evidence in humans and other animal systems,
respectively[30]. We know that either an in vitro pulse
treatment of zineb as well as one of its currently for-
mulation, azzurro, produce DNA lesions that can be
repaired within a 12 h period suggesting that the risk
of genetic damage is, then, reduced, at least for some
mammalian cells in culture. On the other hand, a com-
pletely different behaviour of zineb-induced lesions
is found after a continuous cellular exposure during
longer time periods. The presence of chromosomal
aberrations in lymphocytes from crop workers occu-
pationally exposed to zineb found by Pilinskaya[14]
and our previous studies demonstrating the ability of
a continuous in vitro treatment in inducing chromoso-
mal aberrations, sister chromatid exchanges and a de-
lay in cell-cycle kinetics in human lymphocytes[15]
could indicate the inability of this cell type to repair, at
least not totally, the lesions during the G0 period. Fur-

thermore, they could also suggest the persistence of
such lesions in the genetic material, potentially com-
prising DNA breakage at sites of oncogenes or tumour
suppressor genes which may play a crucial role in the
induction of malignancies.

Acknowledgements

Marina González and Sonia Soloneski contributed
equally to this work. This study was supported by the
National Agency of Scientific and Technological Pro-
motion, the National Council of Scientific and Tech-
nological Research (CONICET), the Commission of
Scientific Research of Buenos Aires Province (CIC),
and the National University of La Plata (Grant number
11/N325) from Argentina. The authors thank Marı́a
Apeztegúıa, M.Sc. for performing statistical analysis
and to Prof. Marcelo Vilches for language revision.

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	Effect of dithiocarbamate pesticide zineb and its commercial formulation, azzurroIV. DNA damage and repair kinetics assessed by single cell gel electrophoresis (SCGE) assay on Chinese hamster ovary (CHO) cells
	Introduction
	Materials and methods
	Chemicals
	Cell cultures and pesticide treatment
	Cell viability
	Single cell gel electrophoresis (SCGE) assay
	Statistical analysis

	Results
	Discussion
	Acknowledgements
	References