CHAPTER - 3
Cellular toxicity of difenoconazole,
dimethomorph and pyrimethanil in
Chinese hamster ovary (CHO) and human
liver (HepG2) cells
Chapter-3
3.1. Introduction
To combat fungal infections on crops, farmers use various types of fungicides
like pyrimethanil, difenoconazole, dimethomorph etc. which have good
efficacy against target species. However, the indiscriminate use of fungicides
poses grave environmental and health consequences (Boers et al. 2008;
Cockburn et al. 2011; Oliver et al. 2011). Since fungicides are extensively
sprayed on vegetables and fruits (Chen et al. 2011; Erasmus et al. 2011)
there are chances that their residues may persist in improperly washed
vegetables and fruits. Therefore, human population may unintentionally get
exposed to these fungicides.
in the present study an attempt was made to assess the cellular toxicity of
difenoconazole, dimethomorph and pyrimethanil fungicides in mammalian
cells (figure 3.1.). The fungicides studied in the present work were scheduled
for toxicity evaluation at the joint FAOMHO meeting on pesticide residues
(JMPR 2007).
Cl
h
Cl
Cl
-y
\^ J
•"O
^ >
A \O— CHa
H3C— O
Figure 3.1. Chemical structures of the fungicides (a) difenoconazole
(b) dimethomorph and (c) pyrimethanil.
Difenoconazole is an azole-based fungicide that is widely used to control
fungal growth on fruits, vegetables and cereals. The mode of action of
difenoconazole is through the inhibition of cytochrome P450 enzyme- sterol
14a-demethyiase which catalyzes the biosynthesis of ergosterol in fungi
(Vanden Bossche et al. 1988). Besides sterol 14a-demethy[ase, azoles also
76
Chapter-3
non-specifically modulate the expression of various other cytochrome P450s
in different animals (Sun et al. 2005; Hinfray et al. 2006; Sun et al. 2006).
Difenoconazole has been known to induce hepatocellular adenomas in
rodents (Pest
Management
Regulatory Agency
1999).
It has
been
demonstrated that difenoconazole induces toxicity in fish liver cell line. PLHC1 and mortality in juvenile rainbow trout (Knauer et al. 2007). Although
difenoconazole has been reported to be negative in majority of the short term
genotoxicity tests (JIVIPR 2005a), however in vivo studies demonstrated that
azole fungicides induce mutations in C57BL/6 Big Blue mice (Ross et al.
2009; 2010).
Dimethomorph, a cinnamic acid derivative, is a member of the morpholine
group of fungicides and consists of a mixture of the E and Z isomers in
approximately equal proportions. Dimethomorph is used to control downy
mildews, late blights, crown and root rots in grapes, potatoes, tomatoes and
other vegetables. Morpholines disrupt fungal cell wall by inhibiting the A®, A^~
sterol isomerase and the A® ^'‘-sterol
reductase steps of the sterol (I.e.
ergosterol) biosynthesis in fungi. The metabolism of dimethomorph in
mammals is through demethylation of one of the methoxy groups, A large
number of 0 -conjugated products and various degradation products of
morpholine are also formed (EPA 1998). Tiil date no attempt has been
undertaken to elucidate the detailed toxicity evaluation of dimethomorph.
Previous study has reported that dimethomorph induces chromosomal
aberrations in human lymphocytes and V79 cells (CEPA 2001).
Pyrimethanil is an anilinopyrimidine type of fungicide that has a wide range of
activity against powdery mildews and botrytis in cereals, apples and grapes.
The anilinopyrimidine class of fungicides inhibit the synthesis of fungal
hydrolytic enzymes.
Studies have shown that pyrimethanil induced thyroid tumors in rodents
(Hurley 1998). Various short-term toxicity studies in rats and mice showed
pyrimethanil
administration
increased
77
liver
weight
and
induced
Chapter-3
histopathological changes in liver and thyroid (JMPR 2005c).
Since
pyrimethanil is extensively used in vineyards, therefore there are chances that
it's residues can percolate into nearby water-bodies and contaminate them,
thus pose a threat to the aquatic plants. In this regard studies were conducted
to assess the toxicity of pyrimethanil in aquatic plants and it was observed
that pyrimethanil was highly toxic to vascular plants like Lemna minor and
green alga Scenedesmus acutus (Verdisson et a!. 2001).
In light of the wide exposure pattern and relatively scarce toxicological data on
difenoconazole, pyrimethanil and dimethomorph, it is imperative to study the
toxicological effects of these fungicides under in vitro conditions. The findings
of the study shall also pave the way for further detailed mechanistic studies of
these fungicides.
Cell lines of different origins have widely been used as in vitro model for
general toxicity studies because they are well characterized and more
homogenous than primary cultures. They have been used to predict potential
toxic effects as well as the mechanism of new toxicants on human body. Even
the regulatory authorities like OECD approve the use of cell lines in toxicity
testing (OECD 1997). In the present study we have used Chinese hamster
ovary (CHO) and human hepatoma (HepG2) cell lines for predicting the
cytotoxic and genotoxic potential of the selected fungicides. Both CHO and
HepG2 cell lines are well established in vitro toxicity models being used for
toxicological assessment of a variety of xenobiotics (Fussell et at. 2 0 1 1 ; Kim
2011; Raza et al. 2011; Yang et al. 2011).
3.2. Materials and methods
3.2.1. Cell culture
The CHO and HepG2 cells were obtained from NCCS, India and cultured as
described in chapter 2 .
78
____________ _____________________________ ;________ Chapter-3
For MTT assay the cells were seeded In 96 well plates (1X10'^ cells/well) and
for the Comet assay cells were cultured for 24 h in 24 well plates (5x10'^
cells/well),
3.2.2. Preparation of fungicide treatment
Due to the Insolubility of the fungicides in water, they were first dissolved in
minimum amount of DMSO (concentration should not be more than 0.1% in
culture medium). For each fungicide, stock of concentration 2.0 mM was
prepared, which was serially diluted to subsequent working concentrations of
0.01, 0.025, 0.05, 0.1 and 0.2, 0.4, 0.6, 0.8, and 1.0 mM.
3.2.3. MTT assay
CHO and HepG2 cells were exposed to various concentrations (0,01-2.0 mM)
of the fungicides and incubated for 6 h at 37°C. The MTT assay was carried
out according to the method of Mossman et al (1983) with slight modifications
as described in Chapter 2 .
3.2.4. Cell viability
The cells were incubated with fungicides for 6 h at different concentrations
and assayed for viability using trypan dye exclusion (Phillips 1973).
3.2.5. Comet assay
The cells were exposed to different concentrations of fungicides for 6 h in a 24
well cell culture plate and for each concentration, duplicate wells were used.
After the treatment, cells were harvested by using 0.06% trypsin-EDTA. The
cell pellet was re-suspended in 100 |jl of PBS and mixed with 100 nl of 1%
LMA and slides were prepared and processed as described in Chapter 2.
3.2.6. Statistical analysis
Results were expressed as meantS.E.M. and data were analyzed using one
way analysis of variance (ANOVA) with Dunnett post hoc test to determine
79
Chapter-3
significance relative to unexposed control. In all cases,
p<0.05 was
considered significant.
3.3. Results
3.3.1. Cytotoxicity
a, Difenoconazole
A concentration-dependent cytotoxicity was observed in CHO cells after 6 h of
difenoconazole exposure (figure 3.2.). At the concentrations 0.01-0.05 mM no
significant cytotoxicity was observed as evident by MTT results. However at
0.1 mM and higher concentrations there was a significant (p<0.05) reduction
in nnitochondrial activity.
In HepG2 cells also difenoconazole induced a concentration-dependent
cytotoxicity as evident by the MTT results (figure 3.2.). A comparative analysis
revealed that difenoconazole induced aggravated cytotoxic response in CHO
cells as compared to HepG2 cells.
100
□ CHO iiHepG2
4r B
80
I03
60
”
0
-
-B
i
40
1
20
"
Be ■j
:1
■■ m
H
m
Q
mm
1=
E zE
-B
•I
-
Control
0.01
0.025
0.05
0.1
Concentration of difenoconazole (mWI)
Figure 3,2. Cytotoxicity of difenoconazole in CHO and HepG2 cells.
Data represents mean ± S.E.M. of three independent experiments.
*p<0.05, ” p<0.01, *” p<0-001, significant with respect to control.
80
Chapter-3
b. Dimethomorph
In CHO cells, dimethomorph Induced significant (p<0.05) cytotoxicity at 0.4
mM concentration (59.3%), the viability further decreased to 31.1% at 0.6 mM
(figure 3.3.). While in HepG2 cells, the cytotoxicity was observed at 0.6 mM
and higher concentrations (figure 3.3.).
100
-
nCHO H HepG2
80
‘>
hU
uS
*o
s
jroo
§
40
20
■
Control 0.01
0.025
0.05
0.1
0.2
I_____________________^
0.4
0.6
0.8
1.0
_____________________
Concentration of dimethomorph (mlW)
Figure 3.3. Dimethomorph induced cytotoxicity In CHO and HepG2 cells.
Data represents mean ± S.E.M. of three independent experiments.
‘p<0.05, **p<0.01, ***p<0.001, significant with respect to control.
81
2.0
_________________________________________________ Chapter-3
c. Pyrimethanil
In CHO cells pyrimethanil significantly (p<0.05) induced cytotoxicity at 1.0 nnM
and higher concentrations (figure 3.4.).
The MTT results also demonstrated that in HepG2 cells the cytotoxic
response was evident only at 2.0 mM concentration (figure 3.4.).
The MTT results showed that the cytotoxicity of fungicides studied was in the
order, difenoconazole>dimethomorph>pyrimethanil in both CHO and HepG2
cells.
100
□ C H 0Q H epG 2
u
tu
«
■
c
■a
co
Control ,0.01
0.02S
0.05
0.1
0.2
0.4
0.6
0.8
1.0
2.0
Concentration of pyrimetharjil (mM)
Figure 3.4. Pyrimethani! induced cytotoxicity in CHO and HepG2 cells.
Data represents mean ± S.E.M. of three independent experiments.
p<0.05, p<0.01,
p<0.001, significant with respect to control.
3.3.2. Cell viability
Before conducting the Comet assay experiments, the cell viability in control
and treatment groups were assessed by Trypan blue dye exclusion method.
The following concentrations of fungicides were used for the Comet
experiments; the cell viability exceeded 90% in each of these groups (table
3.1.).
82
Chapter-3
Table 3.1. Cells viability in treatment and control groups as assessed by the
trypan blue dye exclusion method.
Groups
Cell viability (% o f respective control)
CHO cells
HepG2 cells
Difenoconazole (mM)
0.0 1
98.2 ± 0.5
97 ± 0.1
0.025
98 ±0.3
98 ± 0.6
0.05
97.6 ± 0.1
94.5 ± 0.5
0 .10
78 ±0.3
95 ± 0.7
EMS^
98 ± 0.4
97.1 ± 1.3
Control
1 0 0 ± 0.6
100 ±0.4
0 .0 1
97 ± 0.8
97 + 0.4
0.025
96 ± 1.2
97.3 ± 1.3
0.05
98.5 ± 0.7
98 ±0.6
0 .1
98.4 ± 0.5
92.3 ± 0.2
0.2
96.2 ± 1.3
93.6 ± 0.7
EMS^
98.2 ±0.9
94 ±0.3
Control
1 0 0 ± 0,2
100 ± 0.4
0.0 1
97 ±0.2
95.4 + 0.2
0.025
96.4 ±0.1
92.7 ±0.6
0.05
97.1 ±0.9
95 ±0.4
0 .1
98 ± 0.5
91.5 ±0.6
0 .2
97.6 + 0.8
97 + 0.3
EMS*
99 ± 0.4
95 ± 0.4
Control
1 0 0 ± 0.6
100 ±0.5
Dimethomorph (mM)
Pyrimethanil (mM)
Data represents mean ± S.E.M. of three Independent experiments.
^ EMS, ethyl methanesuifonate (1mM) - positive control.
83
_____________ Chapter-^
3.3.3. DNA damaging potential of fungicides
The DNA damaging potential of different fungicides showed varied response
as described below.
Difenoconazole did not induce significant DNA damage either in CHO or
HepG2 cells as shown by the Comet assay data at all concentrations (table
3.2. A, B).
However, there was a significant (p<0.05) concentration dependent Induction
in the DNA damage in CHO cells after 6 h of exposure to dimethomorph at
0.025 mM and higher concentrations (table 3.3. A). Also, in HepG2 cells,
dimethomorph induced a significant (p<0.G5) DNA damage at 0.05 and higher
concentrations as evident by various Comet assay parameters (table 3.3. B).
Pyrimethanil also induced significant (p<0.05) DNA damage at 0.05 mM and
higher concentrations in CHO and HepG2 cells exposed for 6 h (table 3.4. A;
B).
The DNA damaging potential of the three fungicides was found to be in the
order dimethomorph>pyrlmethanil>difenoconazole in both the cell lines.
84
Chapter-3
Table 3.2. Effect of difenoconazole on the Comet parameters in CHO and
HepG2 cells.
A. CHO cells
Tail DMA (%)
Control
Olive tail moment
(arbitrary unit)
1.3 ±0.1
EMS^
17.9 ± 1.4***
40 ± 2.8***
0 .0 1
1.2± 0.04
1 1 . 8 ± 0 .6
0.025
1 .2 ± 0.1
11.7 ± 0.7
0.05
1 . 1 + 0.1
11.5 ±0.9
Groups
12.4 ±0.6
Difenoconazole (mM)
B. HepG2 cells
Groups
Olive tail moment
Tail DNA (%)
(arbitrary unit)
Control
1 . 1 ± 0.1
8 . 6 ±0,7
EMS^
19.4 ±3.8***
42.9 ± 2.1***
0 .0 1
1.1 ±0.04
8.9 ±2.7
0.025
1.3 ±0.2
11.3 10.5
0.05
1.3±0.1
11.7 ±0.9
0 .10
1.32 ±0.1
1 1 . 6 ± 0 .2
Difenoconazole (mM)
Values represent mean of three experiments ± S.E.M.
* EMS, ethyl methanesulfonate (1 mM) - positive control.
***p<0 . 0 0 1 when compared to control.
85
__________________________________________________ Chapter-3
Table 3.3. Dimethomorph induced genotoxicity as evident by the Comet
parameters in CHO and HepG2 cells.
A. CHO cells
Groups
Olive tail moment
Tail DMA (%)
(arbitrary unit)
Control
1 . 1 ±0.06
9.7 ± 0.1
EMS*^
15.9 ± 0.9***
60.7 ±1.5***
0.0 1
1.2 ± 0 .1
11.5 + 0.9
0.025
1.3 ±0.2*
1 1 . 6 ± 0 .2
0.05
1 . 3 ± 0 .1 *
11.9 ±0.2**
0 .1
1.5 ±0.1**
12.1 ±0.5**
0.2
1 . 5 ± 0 .2 **
13.2 ± 1.3**
Olive tail moment
Tail DNA (%)
Dimethomorph (mM)
B. HepG2 cells
Groups
(arbitrary unit)
Control
1.3 ± 0.1
10.5 ±1.2
EMS^
18.5 ± 3.2***
51.2 ± 2.5***
0 .0 1
1.3 ± 0.1
1 1 .6 + 1.0
0.025
1.4 ±0.1
11.8 ±0.9
0.05
1.9 ± 0.1**
12.7 ±1.6*
0.1
2 . 0 + 0 .1 **
13.1 +0.3**
0.2
2 . 1 ± 0 .1 **
^ 13.7± 1.1**
Dimethomorph (mM)
Values represent mean of three experiments ± S.E.M.
^ EMS, ethyl methanesulfonate (1 mM) - positive control.
*p<0.05, **p<0.01, ***p<0.001 when compared to control.
86
Chapter-3
Table 3.4. Pyrimethanil induced DNA damage as evident by the Comet
parameters in CHO and HepG2 cells.
A. CHO cells
Groups
Olive tail moment
Tail DNA (%)
(arbitrary unit)
Control
1.1 ± 0 .1
8.5 ±0.7
EMS*
15.7 ±1.5***
59.1 ± 4.8***
0.0 1
1.2 ±0.3
8 .6 ± 0.8
0.025
1.2 ± 0 .2
9.0 ±0.3
0.05
1.3±0.1*
9.5 ± 0.6*
0.1
1.4±0.1*
10.2 ± 0.7*
0.2
1 . 6 ± 0 .1 **
12.2 ± 0.7**
Olive tail moment
Tail DNA (%)
Pyrimethanil (mM)
B. HepG2 cells
Groups
(arbitrary unit)
Control
1 .0 ± 0 . 1
7.8 ±0.1
EMS^
21.5 ±2.6***
47.1 ±1.9***
0.0 1
1 . 1 ± 0 .1
8.3 ±0.7
0.025
1 ,2 ± 0.1
8 . 6 ±0.4
0.05
1.3 ±0.2*
8 . 8 ± 0.9*
0 .1
1,3 ±0.3*
8.9 ±0.8*
0 .2
1.4 ±0.1**
9.9 ± 0.7*
Pyrimethanil (mM)
Values represent mean of three experiments + S.E.M,
* EMS, ethyl methanesulfonate (1 mM) - positive control.
* p<0,05, ** p<0.01, ***p<0.001 when compared to control.
87
_____________ ___________ _________________________ Chapter-3
3.4. Discussion
The
data
of the
present
study
demonstrated
that
difenoconazole,
dimethonnorph and pyrimethanil exhibited cytotoxicity in CHO and HepG2
cells, although to different extents.
The cytotoxicity data demonstrated that in comparison to other fungicides,
difenoconazole exerted highest cytotoxic response (in both CHO and HepG2
ceils). Analysis of the cytotoxic response of difenoconazole in the two ceil
types indicated that CHO cells were more sensitive than HepG2 cells to
difenoconazole. For example, at 0.2 mM concentration difenoconazole
decreased the mitochondrial activity to 37% in CHO cells; however at the
same concentration the fungicide reduced the mitochondrial activity to only
81.8% in HepG2 cells. In other words it can be expected that difenoconazole
perse is more cytotoxic in comparison to its metabolites. It has been reported
that difenoconazole is quickly metabolized in liver and yields CGA 205374
and CGA 189138 that are then conjugated to glucuronic, sulfate and glycine
moieties (JMPR 2005a). These conjugated products are hydrophilic in nature
and therefore are rapidly excreted out of the body. Since in comparison to
CHO,
HepG2
cells
are
richer
in xenobiotic
metabolizing
enzymes,
difenoconazole may be detoxified to water-soluble and less toxic metabolites.
This could also help in explaining the aggravated cytotoxic response of
difenoconazole in CHO cells by comparison with HepG2 cells. Our results are
in accordance to the previous studies which have reported the cytotoxic
potential of the triazole-based fungicides (Daniel et al. 2007; Chen et at.
2008).
On the basis of our results and previous investigations it may be postulated
that difenoconazole can induce cytotoxicity by three mechanisms: viz. ( 1 )
Induction of oxidative stress, (2) Disruption of plasma membrane and (3)
Inhibition of mitochondrial activity.
88
________________________ ___ _____________________ Chapter-3
There are known relationships between oxidative stress and toxicity,
increased cellular oxidant levels can alter various biomolecules (proteins,
lipids and DNA) as well as alter many signalling pathways inside cell.
Toxicogenomic studies and several other investigations have reported the
possible involvement of ROS in triazole toxicity (Amin and Hamza 2005;
Martin et al. 2007). Moreover, our mechanistic studies have also revealed that
difenoconazole exposure induces ROS generation leading to cytotoxicity
(chapter 3).
Rodriguez and Acosta (1995) proposed that the cytotoxic effects of azoles
may be due to their ability to inhibit cholesterol biosynthesis through the
inhibition of lanosterol 14 a-demethylase enzyme activity. Since cholesterol is
one of the major components of cellular membranes in animals, its depletion
would result in the loss of plasma membrane integrity eventually leading to
cell death. Our studies have shown that difenoconazole exposure increased
the release of intracellular LDH enzyme which indicates the disruption of
plasma membrane integrity (Chapter 3).
MTT assay showed that difenoconazole decreased the mitochondrial activity
in OHO and HepG2 cells. Our results are in concordance with the previous
studies (Rodriguez and Acosta Jr 1996) which has demonstrated that azoles
induce mitochondrial toxicity by inhibiting the mitochondria! succinate
dehydrogenase and NADPH oxidase activity.
The Comet assay results demonstrated that difenoconazole was nongenotoxic in nature. Our results are in concordance with previous studies
(JMPR 2005a) that have demonstrated that neither difenoconazole nor its
metabolites are genotoxic.
The present study is probably the first to demonstrate the in vitro toxicity of
dimethomorph in CHO and human liver cells. Our result demonstrated that
dimethomorph induced cytotoxicity in CHO and HepG2 cells. Morpholines are
89
Chapter-3
known to inhibit the synthesis of sterols in fungal cell membrane therefore it is
possible that dimethomorph non-specifically inhibited the synthesis of sterols
in animal cells which resulted into the fragile cell membrane. The fragile cell
membrane may lead to cell disruption and finally cell death. Comparative
analysis revealed a more pronounced cytotoxic response of dimethomorph in
CHO cells in comparison to HepG2 cells. This may be due to the reason that
dimethomorph might have metabolized to less toxic and water-soluble
metabolites in HepG2 cells.
The present study also revealed that dimethomorph induced DNA damage in
CHO and HepG2 ceils, which indicated its genotoxic potential in these cell
types. Our results are in accordance to the previous study which showed that
dimethomorph
induced chromosomal aberrations
in peripheral human
lymphocytes and Chinese hamster cell line V79 (California environmental
protection agency 2 0 0 1 ).
The cytotoxicity results demonstrated that pyrimethanil was the least cytotoxic
compound amongst the three fungicides. A comparative analysis revealed
that pyrimethanil induced greater cytotoxicity in CHO cells compared to
HepG2 cells. It is hypothesized that since pyrimethanil can be metabolized to
hydrophilic and less toxic glucuronate and sulfate conjugated metabolites
therefore, the cytotoxic response of pyrimethanil was diminished in HepG2
cells. Pyrimethanil induced DNA damage in both CHO and HepG2 cells, in a
dose-dependent manner as evident from the Comet assay. Our results are in
concordance with the earlier study by Lebailly et al (1998) that showed DNA
damaging potential using the Comet assay in mononuclear leukocytes of
farmers exposed to pyrimethanil during the field spray. A comparative
genotoxicity
analysis
demonstrated
that
the
genotoxic
response
of
pyrimethanil was more prominent in CHO in comparison to HepG2 cells;
which indicates that after getting metabolized the genotoxicity of pyrimethanil
diminished, in other words pyrimethanil is per se more genotoxic in
comparison to its metabolites.
90
__________________________________________________ Chapter-3
In
summary,
we
conclude
that
difenoconazole,
dimethomorph
and
pyrimethanil are cytotoxic in both CHO and HepG2 cells. A comparative
cytotoxicity analysis revealed that metabolism is essentially a detoxification
process in case of these fungicides. The study also showed that neither
difenoconazole
nor its metabolites were
genotoxic
in
nature;
pyrimethanil and dimethomorph demonstrated the genotoxic
while
potential.
However there is still a need for some other confirmatory studies for validating
these results.
91