CHAPTER - 3

Cellular toxicity of difenoconazole, dimethomorph and pyrimethanil in

Chinese hamster ovary (CHO) and humanliver (HepG2) cells

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3.1. IntroductionTo 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

Cl

hCl

•"O-y \ ^ J

^ > A \H3C— O O— CHa

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

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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. PLHC- 1 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 liver weight and induced

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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 methods3.2.1. Cell cultureThe CHO and HepG2 cells were obtained from NCCS, India and cultured as

described in chapter 2 .

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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 treatmentDue 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 viabilityThe 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 assayThe 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 analysisResults were expressed as meantS.E.M. and data were analyzed using one

way analysis of variance (ANOVA) with Dunnett post hoc test to determine

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significance relative to unexposed control. In all cases, p<0.05 was

considered significant.

3.3. Results

3.3.1. Cytotoxicitya, DifenoconazoleA 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.

I03

01

100

80

60

40

20

4r B

i Be ■j” - -B ■■ m

H m : 1Q mm

E zE 1 =" - -B •I

Control 0.01 0.025 0.05 0.1

□ CHO iiHepG2

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.

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b. DimethomorphIn 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.).

‘>hUSu

*osojro§

100 -

80

40

20 ■

nCHO H HepG2

Control 0.01 0.025 0.05 0.1 0.2 0.4 0.6 0.8 1.0 2.0

I_____________________^ _____________________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.

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c. PyrimethanilIn 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.

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utu«■c■aco

□ CH0QHepG2

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 viabilityBefore 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 (table3.1.).

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Table 3.1. Cells viability in treatment and control groups as assessed by the trypan blue dye exclusion method.

Groups Cell viability (% of 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 ± 0.50 . 1 0 78 ±0.3 95 ± 0.7EMS^ 98 ± 0.4 97.1 ± 1.3

Control 1 0 0 ± 0 . 6 100 ±0.4

Dimethomorph (mM)0 . 0 1 97 ± 0.8 97 + 0.40.025 96 ± 1.2 97.3 ± 1.30.05 98.5 ± 0.7 98 ±0.60 . 1 98.4 ± 0.5 92.3 ± 0.20 . 2 96.2 ± 1.3 93.6 ± 0.7EMS^ 98.2 ±0.9 94 ±0.3Control 1 0 0 ± 0 , 2 100 ± 0.4

Pyrimethanil (mM)

0 . 0 1 97 ±0.2 95.4 + 0.20.025 96.4 ±0.1 92.7 ±0.60.05 97.1 ±0.9 95 ±0.40 . 1 98 ± 0.5 91.5 ±0.60 . 2 97.6 + 0.8 97 + 0.3EMS* 99 ± 0.4 95 ± 0.4

Control 1 0 0 ± 0 . 6 100 ±0.5

Data represents mean ± S.E.M. of three Independent experiments.

EMS, ethyl methanesuifonate (1mM) - positive control.

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3.3.3. DNA damaging potential of fungicidesThe 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 (table3.2. 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.

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Table 3.2. Effect of difenoconazole on the Comet parameters in CHO and HepG2 cells.

A. CHO cellsGroups Olive tail moment

(arbitrary unit)Tail DMA (%)

Control 1.3 ±0.1 12.4 ±0.6

EMS^ 17.9 ± 1.4*** 40 ± 2.8***

Difenoconazole (mM)

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

B. HepG2 cellsGroups Olive tail moment

(arbitrary unit)Tail DNA (%)

Control 1 . 1 ± 0 . 1 8 . 6 ±0,7

EMS^ 19.4 ±3.8*** 42.9 ± 2.1***

Difenoconazole (mM)0 . 0 1 1.1 ±0.04 8.9 ±2.7

0.025 1.3 ±0.2 11.3 10.50.05 1.3±0.1 11.7 ±0.90 . 1 0 1.32 ±0.1 1 1 . 6 ± 0 . 2

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.

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Table 3.3. Dimethomorph induced genotoxicity as evident by the Comet

parameters in CHO and HepG2 cells.

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A. CHO cellsGroups Olive tail moment

(arbitrary unit)Tail DMA (%)

Control 1 . 1 ±0.06 9.7 ± 0.1

EMS* 15.9 ± 0.9*** 60.7 ±1.5***Dimethomorph (mM)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**

B. HepG2 cells

Groups Olive tail moment(arbitrary unit)

Tail DNA (%)

Control 1.3 ± 0.1 10.5 ±1.2EMS^ 18.5 ± 3.2*** 51.2 ± 2.5***Dimethomorph (mM)0 . 0 1 1.3 ± 0.1 1 1 . 6 + 1 . 0

0.025 1.4 ±0.1 11.8 ±0.90.05 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**

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.

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Table 3.4. Pyrimethanil induced DNA damage as evident by the Comet parameters in CHO and HepG2 cells.A. CHO cellsGroups Olive tail moment

(arbitrary unit)Tail DNA (%)

Control 1 . 1 ± 0 . 1 8.5 ±0.7EMS* 15.7 ±1.5*** 59.1 ± 4.8***Pyrimethanil (mM)0 . 0 1 1.2 ±0.3 8 . 6 ± 0 . 8

0.025 1 . 2 ± 0 . 2 9.0 ±0.30.05 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**

B. HepG2 cellsGroups Olive tail moment

(arbitrary unit)Tail DNA (%)

Control 1 .0 ± 0 . 1 7.8 ±0.1

EMS^ 21.5 ±2.6*** 47.1 ±1.9***Pyrimethanil (mM)0 . 0 1 1 . 1 ± 0 . 1 8.3 ±0.70.025 1 , 2 ± 0 . 1 8 . 6 ±0.40.05 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*

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.

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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.

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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 non- genotoxic 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

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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.

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9 0

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; while pyrimethanil and dimethomorph demonstrated the genotoxic potential. However there is still a need for some other confirmatory studies for validating

these results.

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