IN VITRO SYSTEMS
Effects of single pesticides and binary pesticide mixtures on estrone production in H295R cells
Wiebke Prutner . Petra Nicken . Eberhard Haunhorst . Gerd Hamscher · Pablo Steinberg
Received: 29 January 2013 / Accepted: 16 May 2013 @ Springer-Verlag Berlin Heidelberg 2013
Abstract The aim of the present study was to deter- mine whether the human adrenocortical carcinoma cell line H295R can be used as an in vitro test system to inves- tigate the effects of binary pesticide combinations on estrone production as biological endpoint. In the first step ten pesticides selected according to a tiered approach were tested individually. The anilinopyrimidines cyprodinil and pyrimethanil as well as the dicarboximides iprodione and procymidone increased estrone concentration, while the triazoles myclobutanil and tebuconazole as well as the strobilurins azoxystrobin and kresoxim-methyl decreased estrone concentration in the supernatant of H295R cells. The N-methylcarbamate methomyl did not show any effects, and the phthalimide captan reduced estrone con- centration unspecifically due to its detrimental impact on cellular viability. When cyprodinil and pyrimethanil, which belong to the same chemical group and increase estrone
production, were combined, in most of the cases the overall effect was solely determined by the most potent compound in the mixture (i.e., cyprodinil). When cyprodinil and pro- cymidone, which belong to different chemical groups but increase estrone production, were combined, in most cases an additive effect was observed. When cyprodinil, which increased estrone production, was combined with either myclobutanil or azoxystrobin, which decreased estrone production, the overall effect of the mixture was in most cases either entirely determined by myclobutanil or at least partially modulated by azoxystrobin. In conclusion, H295R cells appear to be an adequate in vitro test system to study the effect of combining two pesticides affecting estrone production.
Keywords Additivity . Antagonism . Estrone production . H295R cells . Mixture toxicity · Pesticides
W. Prutner . P. Nicken . G. Hamscher . P. Steinberg Institute for Food Toxicology and Analytical Chemistry, University of Veterinary Medicine Hannover, Bischofsholer Damm 15, 30173 Hannover, Germany e-mail: Pablo.Steinberg@tiho-hannover.de
Present Address: W. Prutner Federal Institute for Occupational Safety and Health (BAuA), Friedrich-Henkel-Weg 1-25, 44149 Dortmund, Germany
E. Haunhorst Lower Saxony State Office for Consumer Protection and Food Safety (LAVES), Röverskamp 5, 26203 Wardenburg, Germany
Present Address: G. Hamscher Institute of Food Chemistry and Food Biotechnology, Justus- Liebig-University, Heinrich-Buff-Ring 58, 35392 Giessen, Germany
Introduction
Pesticides are utilized in agriculture to protect plant health, to ensure high crop yields and to preserve the quality of plant products. The active compounds, which are made use of during the vegetation, storage or transport phase, may still be detected in the form of pesticide residues on or in food items reaching the consumer. If residues of more than one pesticide are identified in one and the same food sam- ple, then the burden is due to the so-called multiple pesti- cide residues. Between the years 2002 and 2010, data on the analysis of pesticide residues in food samples in Ger- many revealed that at least one-third of them (32.8-41.6 %) contained multiple pesticide residues (BVL 2012a, b). In some cases, for example, between 2008 and 2010 2 % of all food samples analyzed for pesticide residues in Germany,
the amount of the quantified residues exceeded the legally fixed “Maximum Residue Levels” (MRLs). Only in very few cases, for example, between 2005 and 2010 0.3 % of all food samples analyzed for pesticide residues in Ger- many, the amount of the quantified residues exceeded the corresponding MRLs as well as the toxicologically sub- stantiated “Acceptable Daily Intake” (ADI) and/or “Acute Reference Dose” (ARfD) values, so that they posed a potential health risk for consumers.
In general, the MRL, ADI and ARfD values are only issued for single active compounds and are therefore only applicable when assessing the potential risk associated with a single pesticide residue. However, when assessing the potential risks that may emanate from multiple pesticide residues, it has to be questioned whether the risk assess- ment concept applied up to the present time and based on the adherence to single MRL, ADI and ARfD values is alto- gether adequate. Concretely, there are concerns that multiple pesticide residues taken up with food could lead to additive or synergistic toxic effects once they have been absorbed in the gastrointestinal tract. Hence, in the case of multiple pes- ticide residues, one has to raise the question as to whether it would not be more adequate to apply a cumulative risk assessment concept instead of assessing every single com- pound in a mixture independently of each other. In fact, cumulative assessment concepts for mixtures have been pro- posed by different expert groups and agencies (ILSI 1999; COT 2002; EPA 2002; CVUA 2007; EFSA 2008; VKM 2008). Moreover, concrete approaches have been developed in the last few years for organophosphates, N-methyl-car- bamates, triazines, chloroacetanilides and triazoles (Boon and van Klaveren 2003; Jensen et al. 2003; EPA 2006a, b, c, 2007; Boobis et al. 2008; EFSA 2009). In the aforemen- tioned cases, the individual compounds in a group share the same mechanism/mode of action regarding the toxic effects they elicit in mammals and only differ in their poten- cies. Consequently, their potential cumulative risk can be assessed by assuming dose addition. However, much more difficult to assess is the potential risk of a mixture, in which the individual compounds do show different mechanisms/ modes of action, but whose effects are biologically related to each other, so that interactions in terms of synergism or antagonism may occur. Since no cumulative risk assessment concept has been developed for such interacting mixtures so far, the European Food Safety Agency (EFSA) recommends to conduct an individual case-by-case assessment whenever interactions between pesticide residues are considered plau- sible due to a biological relationship (EFSA 2008).
In the present study pesticide residues were screened for active compounds supposed to affect the same biological pathway in mammals. The data referred to fruit and veg- etable samples, which were analyzed for pesticide residues by the Lower Saxony State Office for Consumer Protection
and Food Safety (LAVES, Oldenburg, Germany) in 2006. The data screening demonstrated that fungicides were the most frequently observed pesticide group among the multi- ple pesticide residues. As shown in Fig. 1, a variety of pesti- cides and particularly fungicides are known or suspected to influence the mammalian biosynthesis of sexual hormones and/or to be sexual hormone receptor agonists or antago- nists in vitro and/or in vivo (Mason et al. 1985; Ostby et al. 1999; Vinggaard et al. 2000; Andersen et al. 2002; Sander- son et al. 2002; Lu et al. 2004; Pfeil 2007; Kjærstad et al. 2010a). Moreover, fungicides such as the phthalimide cap- tan and the strobilurins azoxystrobin, kresoxim-methyl and trifloxystrobin are known to inhibit mitochondrial respira- tion by blocking the electron transport chain in the inner mitochondrial membrane, thereby leading to decreased ATP biosynthesis (Budimir et al. 1976; Becker et al. 1981; Pfeil 2007). This decrease in ATP generation may indi- rectly lead to a dysregulation of steroidogenesis (Allen et al. 2006; Midzak et al. 2011). Furthermore, it is known that the inhibition of cholesterol transfer from the outer to the inner mitochondrial membrane may also impair the production of sexual steroids (Rone et al. 2009).
Despite the diverse mechanisms, which may lead to the disruption of the endocrine system, the present study only focused on pesticides that are known or presumed to modu- late the biosynthesis of sexual hormones. The H295R cell line was chosen as an in vitro test system for the experi- ments. These cells were subcloned from the human pluri- potent adrenocortical carcinoma cell line NCI-H295 estab- lished by Gazdar et al. (1990) and contain a high number of mitochondria as well as all enzymes needed for sexual hormone biosynthesis. The aim of the present study was to determine whether the H295R cells can be used as an in vitro test system to investigate the effects of binary pes- ticide mixtures on estrone production as biological end- point. In the first step, ten pesticides were selected based on a tiered approach (see “Materials and Methods” section, “Pesticide selection”) and individually tested in H295R cells. Thereafter, four binary pesticide combinations were selected, and their overall effects on estrone production in H295R cells were tested. Each combination consisted of two pesticides that (i) enhance estrone production or (ii) modulate estrone production in opposing manners.
Materials and methods
Pesticide selection
The selection of the pesticides to be tested individually was based on residue data provided by LAVES, Germany. In a food survey performed by LAVES in 2006, more than 160 different pesticides in about 1,250 fruit and vegetable
cholesterol
CYP11A
azoxystrobin captan kresoxim-methyl trifloxystrobin
cholesterol
pregnenolone
mitochondrion
iprodione
pregnenolone
iprodione methomyl pirimicarb procymidone ??? propamocarb
inhibition
3ßHSD I + II
CYP17
stimulation
???
controversial findings
progesterone
dehydroepiandrosterone
transport
CYP17
36HSD I + II
conversion
androstenedione
difenoconazole fenarimol imazalil penconazole prochloraz
17ßHSD3
CYP19 (aromatase)
testosterone
estrone
5a-reductase 1 + 2
CYP19
17@HSD1 + 7
tebuconazole ??? triadimenol
dihydrotestosterone
estradiol
estrogen receptors
prochloraz
dimethoate ??? fenarimol prochloraz procymidone
androgen receptors
chlorpyriphos fenarimol pirimicarb (+ 17ß-estradiol) propamocarb (+ 17ß-estradiol)
carbendazim
samples were detected. Starting from this broad range of substances, ten pesticides were selected based on a four- step procedure: (1) Any pesticide detected in less than 10 food samples was excluded from further considerations. By doing so, the number of substances was reduced from more than 160 to 66. (2) Those pesticides included in plant protection products whose placing on the market and use were prohibited according to the European Council Direc- tive 79/117/EEC or whose use was restricted by no later than December 31, 2007, according to Commission Regu- lation (EC) No. 2076/2002 were excluded. Thereby, the number of pesticides was reduced from 66 to 57. (3) Only those pesticides known to modulate aromatase (cytochrome P450 19) and/or mitochondrial activity were further con- sidered. In this case, compounds belonging to the same pesticide class or being structurally related to substances with known mode of action were also taken into account. In this way, the number of pesticides was reduced from 57 to 22. (4) From the remaining 22 pesticides, ten active sub- stances were finally selected for the present study, based on their occurrence as residues in the same sorts of fruits and vegetables. These were azoxystrobin, captan, cyprodinil, iprodione, kresoxim-methyl, methomyl, myclobutanil, pro- cymidone, pyrimethanil and tebuconazole (Fig. 2). All sub- stances are used as fungicides, except for methomyl, which is an insecticide and acaricide.
Theoretically, with the ten aforementioned pesticides, one could form 45 binary pesticide mixtures, but in the present study the number was limited to four combinations based on exposure-related and toxicology-related consid- erations. Briefly, the frequency with which each potential binary pesticide mixture was detected as a real residue combination in single food samples was taken into account. For this purpose, pesticide residue data from 2,945 fruit and vegetable samples analyzed by LAVES between 2005 and 2008 were considered. Moreover, the outcome of the experiments, in which the ten compounds were tested indi- vidually in H295R cells, was taken into account, thereby putting special emphasis on the potency of the individual substances to modulate estrone production. In the end, the following four binary pesticide mixtures were selected: cyprodinil + pyrimethanil, cyprodinil + procymidone, cyprodinil + myclobutanil and cyprodinil + azoxystrobin. A detailed rationale for the selection of these four pesticide combinations is given in the “Results” section.
Chemicals
The pesticides azoxystrobin (CAS No. 131860-33-8), cyprodinil (CAS No. 121552-61-2), iprodione (CAS No. 36734-19-7), kresoxim-methyl (CAS No. 143390-89-0), methomyl (CAS No. 16752-77-5), myclobutanil (CAS
H
H
N
N
N
N
CH3
cyprodinil
pyrimethanil
N
N
CH3
CH3
H3C
CN
HO
CH3
C l
C
(CH2)3CH3
tebuconazole
CH3
CH2
myclobutanil
N
Cl
N
N
N
N
N
CH3
N
N
O
azoxystrobin
kresoxim-methyl
O
O
O
CN
H3C
OCH3
H3C
O
N
CO2CH3
O
C l
O
Cl
O
CH3
iprodione
N
N
procymidone
N
CONHCH(CH3)2
CH3
C l
O
Cl
O
O
O
S
N
methomyl
CH3
O
N
CH3
N
SCCl3
captan
H
CH3
O
No. 88671-89-0), procymidone (CAS No. 32809-16-8), pyrimethanil (CAS No. 53112-28-0) and tebuconazole (CAS No. 107534-96-3) were purchased from Dr. Ehren- storfer (Augsburg, Germany) and captan (CAS No. 133- 06-2) from Sigma-Aldrich (Schnelldorf, Germany). Their purities ranged from 97.5 to 99.5 % as specified by the manufacturer. 4-Androsten-4-ol-3,17-dione (CAS No. 566-48-3) was used as a positive control for the reduction in estrone production and 8-bromoadenosine 3’,5’-cyclic monophosphate sodium salt (CAS No. 76939-46-3) as a positive control for the increase in estrone production; both were purchased from Sigma-Aldrich. Estrone (CAS No. 53-16-7, purity ≥99 %) obtained from Sigma-Aldrich was used for the matrix calibration in cell culture medium. Stock solutions of the aforementioned compounds were
prepared in dimethyl sulfoxide (DMSO, purity ≥99.5 %, purchased from Carl Roth, Karlsruhe, Germany). Aliquots of the stock solutions were stored at -20 ℃ until use.
Cells and cell culture media
The cell line H295R was obtained from ATCC (Manas- sas, VA, USA). NuSerumTM as well as ITS+TM Premix Universal Culture Supplement was purchased from BD Biosciences (Heidelberg, Germany) and penicillin/strep- tomycin (10,000 units/ml and 10,000 µg/ml), DMEM/ Ham’s F-12 powder medium, 1 M HEPES buffer solution and 7.5 % w/v sodium bicarbonate solution from Bio- chrom (Berlin, Germany). The basal DMEM/Ham’s F-12 medium consisted of 12.12 g DMEM/Ham’s F-12 powder
medium, 20 ml 1 M HEPES buffer solution, 20 ml 7.5 % w/v sodium bicarbonate solution and 1 liter distilled water. H295R cells were cultured in 75-cm2 cell culture flasks (Nunc, Langenselbold, Germany) using the “maintenance” cell culture medium (500 ml basal DMEM/Ham’s F-12 medium, 12 ml NuSerum™M, 5 ml penicillin/streptomycin and 5 ml ITS + TM Premix Universal Culture Supplement). When H295R cells were incubated with single pesticides or binary mixtures of them, a “treatment” cell culture medium consisting of the basal DMEM/Ham’s F-12 medium, 2 % Ultroser® SF (steroid-free) and maximally 0.1 % (v/v) DMSO was used.
Treatment of the H295R cells with single pesticides or binary pesticide mixtures
After thawing, H295R cells were passaged at least five times and maximally used until passage 16. To test the individual compounds, 35,000 cells in 150 ul “mainte- nance” cell culture medium per well were seeded into a 96-well plate (Greiner Bio-One, Frickenhausen, Ger- many). Thereafter, the 96-well plate was placed in a cell incubator for 24 h at 37 ℃ and 5 % CO2. The “mainte- nance” cell culture medium was then substituted by the “treatment” cell culture medium including the compounds to be tested individually at a final concentration of 0.01, 0.1, 0.3, 1, 3, 10, 30 and 100 uM. The test substance solutions were prepared immediately before adding them to the cell culture medium by diluting one aliquot of the corresponding stock solution in “treatment” cell culture medium. The different pesticide concentrations were prepared in such a way that the final concentration of DMSO in each well never exceeded 0.1 % (v/v). Due to its limited solubility in the treatment cell culture medium, azoxystrobin could only be tested up to a concentration of 30 µM. It has to be pointed out that iprodione, procy- midone and kresoxim-methyl crystallized out in the cell culture medium at a concentration of 100 uM each dur- ing the 24-h incubation period. After cell treatment, 70 ul of the supernatant was immediately taken from each well and frozen at -20 ℃. At a later stage, these samples were used to measure estrone concentration by means of an ELISA kit (DRG Instruments, Marburg, Germany). The remaining supernatant in each well was used to deter- mine the degree of cytotoxicity with the CytoTox-ONE™M Homogenous Membrane Integrity Assay (Promega, Man- nheim, Germany).
The procedure to test the binary pesticide mixtures was identical to the one for the individual compounds with one exception: Only 25,000 cells per well were seeded. This modification was considered necessary because preliminary experiments with single substances had shown that pesti- cides that increased estrone production and were applied
in high concentrations to H295R cells occasionally led to estrone concentrations exceeding the linear estrone concen- tration range measured with the ELISA kit. Since samples were generally not diluted for estrone measurement, these experiments had to be repeated. In order to avoid potential repetitions in case of binary combinations of compounds that increase estrone production, the cell number was pre- cautionally lowered to 25,000 cells per well. Three concen- trations in the 0.1-100 µM range were chosen individually for each compound of the binary combination. In general, these were the highest concentration not leading to a signif- icant change in the estrone concentration (i.e., a no-effect concentration), the concentration leading to an approxi- mately half-maximal change and the concentration leading to a maximal change in the estrone concentration in the cell culture supernatant when compared to the solvent control. Each of the three concentrations of the one compound in the binary mixture was tested with each of the three con- centrations of the other compound in the binary mixture, so that nine concentration combinations per binary pesticide mixture were actually tested.
Each 96-well plate included a blank (i.e., no cells), a solvent control with 0.1 % (v/v) DMSO and two positive controls (1 µM 4-androsten-4-ol-3,17-dione and 100 µM 8-bromoadenosine 3’,5’-cyclic monophosphate sodium salt). Each concentration of the individual compounds and each concentration combination of the binary pesticide mixtures were tested in triplicate within each experiment. In the case of the individual compounds and the binary pes- ticide mixtures, three and five independent experiments, respectively, were performed.
Estrone measurement
The samples consisting of 70 ul supernatant of each well were thawed at room temperature. Fifty microliters per sample was then pipetted into a 96-well estrone ELISA plate. The sample processing and estrone quantification were conducted according to the instructions of the man- ufacturer. But instead of using the manufacturer’s estrone standard solutions dissolved in serum, a matrix calibra- tion was performed by diluting estrone in fresh cell cul- ture medium used for treatment. This modification was considered necessary because the extinction values for the estrone standard curve dissolved in cell culture medium were shifted upward in a parallel manner when compared to those for the estrone standard curve dissolved in serum. The matrix calibration was always freshly prepared on the day when the estrone ELISA was conducted by using one aliquot of the estrone stock solution stored at -20 ℃. The coefficient of determination regarding the linearity of the standard curve was always above 0.99. A preliminary con- trol experiment demonstrated that even at 100 M none of
Estrone concentration change in the cell culture medium
120
100
(% of control)
Estrone concentration change in the cell culture medium (% of control)
120
100
80
80
60
60
40
40
20
20
0
₹
₹
0
-20
-20
0.01
0.1
1
10
100
0.01
0.1
1
10
100
Cyprodinil (UM)
Pyrimethanil (uM)
Estrone concentration change (% of control) in the cell culture medium
120
Estrone concentration change in the cell culture medium (% of control)
120
100
100
80
80
60
60
40
40
20
20
0
1
0
-20
-20
0.01
0.1
1
10
100
0.01
0.1
1
10
100
Iprodione (UM)
Procymidone (uM)
Estrone concentration change in the cell culture medium (% of control)
20
Estrone concentration change in the cell culture medium (% of control)
20
0
0
I
-20
-20
-40
-40
-60
-60
-80
-80
-100
.
-100
0.01
0.1
1
10
100
0.01
0.1
1
10
100
Myclobutanil (uM)
Estrone concentration change in the cell culture medium (% of control)
20
Estrone concentration change in the cell culture medium (% of control)
Tebuconazole (UM)
20
0
1
*
0
-20
-20
-40
-40
-60
-60
-80
-80
-100
-100
0.01
0.1
1
10
0.01
0.1
1
10
100
Azoxystrobin (uM)
Kresoxim-methyl (uM)
Estrone concentration change in the cell culture medium
120
100
(% of control)
80
60
40
20
0
-20
0.01
0.1
1
10
100
Methomyl (μM)
Springer
the pesticides tested cross-reacted >0.001 % with the anti- bodies of the ELISA kit.
Cytotoxicity assay
Forty microliters of each supernatant of the H295R cells treated for 24 h with single compounds or binary mixtures of them was pipetted into a 96-well plate. Cytotoxicity was determined with the CytoTox-One™M Homogeneous Membrane Integrity Assay from Promega (Mannheim, Ger- many) according to the instructions of the manufacturer.
Results
The ten pesticides shown in Fig. 2 were first tested indi- vidually regarding their ability to modulate estrone con- centration in the supernatant of H295R cells (Figs. 3 and 4; Table 1). Cyprodinil and to a lesser extent pyrimethanil as well as iprodione and procymidone increased the estrone concentration in the supernatant of H295R cells, while myclobutanil and tebuconazole as well as azoxystrobin and kresoxim-methyl (to a lesser extent) reduced it (Fig. 3). Methomyl exerted no influence on the aforementioned parameter (Fig. 3). Iprodione only increased the amount of estrone at a concentration of 100 µM, but at this concen- tration it also crystallized out in the cell culture medium. Crystals were also observed in the cell culture medium in the case of kresoxim-methyl and procymidone at a con- centration of 100 µM. None of the nine aforementioned compounds was cytotoxic in the concentration range tested (0.01-100 µM; data not shown). However, in the case of captan, a significant cytotoxicity was observed at the con- centrations of 30 µM (26 %) and 100 µM (74 %) (Fig. 4). Since at a concentration of 100 µM captan all H295R cells were morphologically altered, it was only tested up to a concentration of 30 p.M. As shown in Fig. 4, 30 p.M captan decreased estrone concentration by 66 %, this effect most probably being due to its strong cytotoxicity. The maximal alterations (increase or decrease) in estrone concentration
(compared to the corresponding solvent control and expressed in percentage) mediated by the ten individual pesticides are listed in Table 1.
In the second step, the binary pesticide mixtures to be tested in H295R cells were selected. To do so, the fre- quency with which each binary pesticide mixture composed of two of the ten compounds previously tested was detected in food samples of the state of Lower Saxony, Germany, between 2005 and 2008 (the 15 most often detected binary mixtures are shown in Table 2) and the potency of each of these compounds to enhance or inhibit estrone biosynthesis were taken into account. Based on the aforementioned cri- teria, the following four combinations were chosen: cypro- dinil + pyrimethanil, cyprodinil + procymidone, cyprod- inil + myclobutanil and cyprodinil + azoxystrobin.
The cyprodinil + pyrimethanil combination was selected because: (1) this pesticide mixture was the sec- ond most frequently detected pesticide residue combi- nation in fruits and vegetables (Table 2); (2) cyprodinil and pyrimethanil were the two compounds that enhanced estrone biosynthesis to the greatest extent (Fig. 3, Table 1); and (3) the combination of two compounds, which belong to the same pesticide class and enhance estrone biosynthesis, was adequate to test whether the binary mixture would lead to an additive or synergistic effect or whether the compound enhancing estrone bio- synthesis to the greatest extent (in this case cyprodinil) would determine the overall effect of the mixture. If both compounds were added in the lowest concentration, no increase in estrone biosynthesis due to the mixture was observed (Fig. 5, combination A1). If only one of the two compounds in the mixture was present at its no-effect concentration, the estrone concentration in the cell culture medium increased depending on the concentration of the second compound (for cyprodinil see Fig. 5, combinations B1 and C1; for pyrimethanil see Fig. 5, combinations A2 and A3). The extent of the estrone increase was similar to that observed when the H295R cells were treated with the single compounds in the corresponding higher concentra- tions. If cyprodinil was present at a concentration in the
Estrone concentration change in the cell culture medium (% of control)
20
100
0
I
Cytotoxicity (%)
80
-20
60
-40
40
-60
-80
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0
£
I
I
0.01
0.1
1
10
0
0.01
0.1
0.3
1
3
10
30
100
Captan (UM)
Captan (uM)
| Pesticide class | Compound | Maximal alteration in the estrone amount in the supernatant (%)ª | Concentration of the pesticide that leads to the maximal alteration (p.M) |
|---|---|---|---|
| Anilinopyrimidines | Cyprodinil | +84 ± 28 | 30 |
| Pyrimethanil | +47 ± 20 | 100 | |
| Dicarboximides | Iprodione | +27 ± 17 | 100 |
| Procymidone | +40 ± 10 | 30 | |
| Triazoles | Myclobutanil | -95±1 | 100 |
| Tebuconazole | -96±1 | ≥30 | |
| Strobilurins | Azoxystrobin | -19 ± 10 | ≥10 |
| Kresoxim-methyl | -37 ±13 | 100 | |
| N-Methylcarbamate | Methomyl | – | – |
| Phthalimide | Captan | -66 ± 10 | 30b |
ª The results are expressed as percentage increase (+) or decrease (-) when compared to the corresponding solvent control. Shown is the mean ± standard deviation of three independent experiments
b ” Estrone in the supernatant was not quantified at 100 uM captan due to clearly recognizable cytotoxicity
| Binary pesticide combination | Number of food samples in which the binary pesticide mixture was detected |
|---|---|
| Cyprodinil + azoxystrobin | 208 |
| Cyprodinil + pyrimethanil | 80 |
| Iprodione + azoxystrobin | 73 |
| Cyprodinil + iprodione | 65 |
| Cyprodinil + procymidone | 49 |
| Cyprodinil + myclobutanil | 43 |
| Iprodione + procymidone | 41 |
| Tebuconazole + cyprodinil | 38 |
| Pyrimethanil + azoxystrobin | 37 |
| Procymidone + azoxystrobin | 29 |
| Cyprodinil + kresoxim-methyl | 27 |
| Pyrimethanil + procymidone | 25 |
| Myclobutanil + captan | 24 |
| Iprodione + captan | 22 |
| Tebuconazole + iprodione | 20 |
mixture that led to a half-maximal increase in estrone bio- synthesis and pyrimethanil at the two higher concentra- tions, the effect of the combination was stronger than that of the individual compounds (Fig. 5, combinations B2 and B3). If cyprodinil was present in its highest concentration in the mixture, it determined the increase in the estrone amount in the cell culture medium by itself, indepen- dently of the pyrimethanil concentration in the mixture (Fig. 5, combinations C1, C2 and C3).
The cyprodinil + procymidone combination was selected because: (1) this pesticide mixture was a fre- quently detected pesticide residue combination in fruits and vegetables (Table 2), and (2) even though iprodione, which belongs to the same pesticide class as procymidone, was more frequently detected as part of a residue combination than procymidone, its combination with cyprodinil or azox- ystrobin was not selected for the present study for two rea- sons: (1) Iprodione was a less potent stimulator of estrone biosynthesis than procymidone; (2) iprodione increased the amount of estrone in the supernatant of H295R cells only at a concentration that also resulted in crystal formation; and (3) in contrast to the first combination tested, the two com- pounds enhance estrone biosynthesis but belong to differ- ent pesticide classes.
If both compounds were added in the lowest concentra- tion, no increase in estrone biosynthesis due to the mixture was observed (Fig. 6, combination A1). If only one of the two compounds in the mixture was present at its no-effect concentration, the estrone concentration in the cell culture medium increased depending on the effective concentration of the second compound (for cyprodinil see Fig. 6, com- binations B1 and C1; for procymidone see Fig. 6, com- binations A2 and A3). The extent of the estrone increase was similar to that observed when the H295R cells were incubated with the single compounds in the correspond- ing higher concentrations. If cyprodinil was present at a concentration in the mixture that led to a half-maximal increase in estrone biosynthesis and procymidone at the two higher concentrations, an additive effect was observed (Fig. 6, combinations B2 and B3). If cyprodinil was present in its highest concentration in the mixture, it determined the increase in the estrone amount in the cell culture medium
140
Estrone concentration change in the cell culture medium (%ofcontrol)
120
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60
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-20
☒ Cyprodinil (uM)
0.1
0.1
0.1
10
10
10
30
30
30
☐ Pyrimethanil (uM)
3
30
100
3
30
100
3
30
100
☐ Combination
A1
A2
A3
B1
B2
B3
C1
C2
C3
140
Estrone concentration change in the cell culture medium (%ofcontrol)
120
100
80
60
T
T
40
20
0
-20
☒ Cyprodinil (uM)
0.1
0.1
0.1
10
10
10
30
30
30
☐ Procymidone (uM)
3
10
30
3
10
30
3
10
30
☐ Combination
A1
A2
A3
B1
B2
B3
C1
C2
C3
by itself, independently of the procymidone concentration in the mixture (Fig. 6, combinations C1, C2 and C3).
The cyprodinil + myclobutanil combination was selected because: (1) this pesticide mixture was often detected as pesticide residue combination in fruits and vegetables (Table 2), and (2) cyprodinil and myclobutanil exerted very strong but contrasting effects on estrone bio- synthesis (Fig. 3, Table 1). Hence, the combination was adequate to test whether the two compounds would neutral- ize themselves regarding the effects on estrone biosynthe- sis or whether one of the compounds would determine the overall effect of the mixture.
If both compounds were added in the lowest concen- tration, no increase or decrease in estrone biosynthesis due to the mixture was observed (Fig. 7, combination A1). Only in the case that myclobutanil was present at its no-effect concentration (0.01 µM) in the mixture, did
cyprodinil at concentrations of 10 or 30 pM increase the estrone amount in the cell culture medium (Fig. 7, com- binations B1 and C1). At a concentration of 1 or 30 uM, myclobutanil determined the overall effect of the mix- ture, that is, it led to a decrease in the estrone levels in the cell culture medium, independently of the amount of cyprodinil added (Fig. 7, combinations A2, A3, B2, B3, C2 and C3).
The cyprodinil + azoxystrobin combination was selected because: (1) this pesticide mixture was by far the most often detected residue combination in fruits and vegetables (Table 2), and (2) cyprodinil exerted a very strong enhancing effect on estrone biosynthesis, whereas azoxystrobin reduced estrone biosynthesis to a very lim- ited extent (about 20 %) when compared to myclobutanil (Fig. 3, Table 1). Hence, the combination was adequate to test whether the two compounds would neutralize
120
Estrone concentration change in the cell culture medium (% ofcontrol)
100
80
60
40
20
0
-20
-40
-60
-80
I
-100
Cyprodinil (uM)
0.1
0.1
0.1
10
10
10
30
30
30
Myclobutanil (uM) 0.01
1
30
0.01
1
30
0.01
1
30
Combination
A1
A2
A3
B1
B2
B3
C1
C2
C3
120
Estrone concentration change in the cell culture medium (%ofcontrol)
100
80
60
40
20
0
-20
-40
Cyprodinil (uM)
0.1
0.1
0.1
10
10
10
30
30
30
Azoxystrobin (uM) 3
10
30
3
10
30
3
10
30
Combination
A1
A2
A3
B1
B2
B3
C1
C2
C3
themselves regarding the effects on estrone biosynthesis or whether cyprodinil would determine the overall effect of the mixture in the presence of a weak inhibitor of estrone biosynthesis. If cyprodinil was present at a no- effect concentration, azoxystrobin determined the over- all effect of the mixture, a decrease in the amounts of estrone in the cell culture medium, at all three concentra- tions tested (Fig. 8, combinations A1-A3). In the case that cyprodinil was present at a concentration of 10 or 30 µM in the mixture, azoxystrobin counteracted the effect of cyprodinil in a concentration-dependent manner (Fig. 8, combinations B1-B3 and C1-C3) up to the extent that at a concentration of 30 µM azoxystrobin in the mixture (almost), no net increase in the concentration of estrone in the cell culture medium was measured (Fig. 8, combina- tions B3 and C3).
Discussion
Among the in vitro eukaryotic test systems most commonly used to study the effects of single pesticides and pesticide mixtures on sexual hormone receptors are MCF-7 cells transiently transfected with a reporter construct contain- ing an estrogen-responsive element (Bonefeld Jørgensen et al. 1997; Vinggaard et al. 1999a; Andersen et al. 2002), CHO K1 cells transfected with the cDNA for the human androgen receptor (Vinggaard et al. 1999a; Andersen et al. 2002) and U-2 OS cells transfected with the cDNA for the human androgen and estrogen receptors (Sonneveld et al. 2005; van der Burg et al. 2010a, b). Moreover, H295R cells are also used to study the biological effects of pesticide mixtures, whereby in this case the biological endpoint ana- lyzed is steroid biosynthesis (Kjærstad et al. 2010b). In the
present study H295R cells have successfully been used to investigate the effects of single pesticides as well as binary mixtures of them on estrone biosynthesis. The advantage of using H295R cells is that they contain a high number of mitochondria as well as all enzymes needed for sexual hor- mone biosynthesis found in the human adult adrenal cortex and the gonads (Gazdar et al. 1990). Thus, pesticides inter- fering with sexual hormone biosynthesis either at the level of mitochondria or at the level of the enzymes involved in steroidogenesis (e.g., aromatase) can be studied individu- ally or as mixtures.
When tested individually, the anilinopyrimidines cypro- dinil and pyrimethanil enhanced estrone biosynthesis, whereby cyprodinil was the most potent compound. More- over, the maximal concentration of estrone measured in the cell culture medium after cyprodinil treatment of the cells was almost twice as high as in the case of pyrimeth- anil. The fact that the two anilinopyrimidines enhanced estrone biosynthesis is in line with the data on fenarimol, a pyrimidine fungicide known to exert estrogenic effects in vitro as well as in vivo (Vinggaard et al. 1999b, 2000, 2005; Andersen et al. 2002, 2006; Sanderson et al. 2002). Whereas in the case of fenarimol it has been shown that it activates estrogen receptors (ER) and inhibits aromatase activity (Andersen et al. 2002), the mechanism(s) by which cyprodinil and pyrimethanil enhance estrone biosynthesis remains presently unknown. The decrease in estrone pro- duction observed when increasing the concentration of cyprodinil from 30 to 100 µM is not due to cytotoxicity, as monitored by a lactate dehydrogenase leakage assay.
The triazole fungicides myclobutanil and tebuconazole led to a strong inhibition of estrone production in H295R cells. In the present study estrone biosynthesis was inhib- ited by tebuconazole at the concentrations between 0.3 and 30 µM, and no cytotoxicity was observed in this concentra- tion range. In contrast, Sanderson et al. (2002) reported that aromatase activity in H295R cells was inhibited at the con- centrations of tebuconazole ≥30 µM and argued that the aromatase inhibition was most probably due to an unspe- cific cytotoxic effect (Sanderson et al. 2002). Moreover, it has been documented that tebuconazole (Taxvig et al. 2007) and myclobutanil (Goetz et al. 2009) also inhibit cytochrome P450 17, which is involved in the conversion of pregnenolone to androstenedione, a direct precursor of estrone. Taken together, the strong inhibitory effect of the two aforementioned triazole fungicides on estrone biosyn- thesis most probably is the consequence of the inhibition of the aromatase as well as cytochrome P450 17.
The strobilurins azoxystrobin and kresoxim-methyl inhibit the mitochondrial electron transport chain, which is needed for the conversion of cholesterol to pregnenolone (Payne and Hales 2004). Therefore, it was speculated that disruption of the mitochondrial respiratory chain could
influence steroidogenesis. In fact, the two strobilurins led to a 20-40 % inhibition of estrone biosynthesis, which was modest if compared to the inhibition induced by myclobu- tanil and tebuconazole (about 95 %). At a kresoxim-methyl concentration of 100 µM, crystals were observed in the cell culture medium after the 24-h treatment period and cyto- toxicity was about 6 % (data not shown), so that one cannot discard the possibility that part of the 40 % inhibition of estrone biosynthesis mediated by kresoxim-methyl could be the result of a cytotoxic effect elicited by the crystals.
The dicarboximides iprodione and procymidone led to a stimulation of estrone biosynthesis of 27-40 %, which could have been even stronger if one takes into account that at a concentration of 100 µM, crystals of the two com- pounds were observed in the cell culture medium (data not shown). In contrast, it has been shown that aromatase activity was weakly stimulated (Andersen et al. 2002) or not stimulated at all (Vinggaard et al. 2000) by iprodione in human placental microsomes. When analyzing the discrep- ancy between the results of this study (increase in estrone biosynthesis) and those of Andersen et al. (2002) and Ving- gaard et al. (2000) (slight or no increase in the aromatase activity), a basic difference between H295R cells and human placental microsomes one should take into account is the presence of ER in H295R cells (Somjen et al. 2003; Montanaro et al. 2005). Although it is not clear at the pre- sent time whether ER are involved in the modulation of steroidogenesis in H295R cells (Villeneuve et al. 2007), it has been shown that procymidone enhances vitellogenin production in rainbow trout hepatocytes via a pathway involving the activation of ER (Radice et al. 2004, 2006). Whether this also applies to estrone production in H295R cells remains to be demonstrated.
In the present study captan led to a concentration- dependent inhibition of estrone biosynthesis, but this effect is due to an unspecific cytotoxicity. This observation is in line with a previous study by Suzuki et al. (2004), in which it was shown that captan exerts a strong cytotoxic effect in isolated rat hepatocytes. In contrast, methomyl did not affect estrone biosynthesis at any of the concentra- tions tested in H295R cells, while Andersen et al. (2002) reported that methomyl is a weak inducer of aromatase activity in human placental microsomes. It could be that (1) methomyl is not able to penetrate the plasma cell mem- brane and cannot enhance estrone biosynthesis or (2) the induction of aromatase activity in H295R cells is so weak that estrone levels are not enhanced.
If two fungicides enhancing estrone biosynthesis and belonging to the same chemical group (cyprodinil and pyrimethanil) were combined, in most of the cases the overall effect was solely determined by the more potent compound in the mixture (i.e., cyprodinil). In a few cases a partial additive effect (e.g., when combining 10 uM
cyprodinil with 30 or 100 µM pyrimethanil) was observed, but if the highest concentration of cyprodinil (30 µM) was combined with the two highest concentrations of pyrimeth- anil (30 or 100 µM), the overall effect was determined by cyprodinil alone and no further additive effect was observed. This could be due to the fact that: (1) the increase in estrone biosynthesis leads to the activation of a negative feedback mechanism; (2) the estrone biosynthesis capacity is enhanced to a maximum and cannot be further increased; and (3) the estrone precursor pool is exhausted.
If two fungicides enhancing estrone biosynthesis and belonging to different chemical groups (cyprodinil and procymidone) were combined, in most cases a dose addi- tivity was observed. It could be that the two compounds act at different sites or on different targets in the H295R cells, so that no competitive reactions at a certain site/on a certain target molecule take place. If the highest concen- tration of cyprodinil (30 µM) was combined with 3, 10 or 30 µM procymidone, the overall effect was mostly deter- mined by cyprodinil alone and no significant additive effect was observed. As in the case of combining cyprodinil and pyrimethanil, this could be due to the three possibilities mentioned at the end of the previous paragraph.
If cyprodinil and myclobutanil, two fungicides having opposing effects on estrone biosynthesis, were combined, cyprodinil was only able to enhance estrone biosynthesis in the presence of the lowest concentration of myclobutanil tested (0.01 µM), whereas at the two higher concentrations of myclobutanil tested (1 and 30 µM), the overall effect of the mixture was only determined by this compound. The potent inhibitory effect of myclobutanil is due to the strong inhibition of cytochrome P450 17 as well as aromatase, so that no precursor molecules for estrone biosynthesis are available. If cyprodinil was combined with azoxystrobin, another compound disrupting steroidogenesis, the overall effect of the mixture was again determined by the com- pound inhibiting estrone biosynthesis.
In conclusion, H295R cells have proven to be an ade- quate in vitro test system to study the interaction between two pesticides modulating estrone biosynthesis. Whereas additive and antagonistic effects were documented, in no case a synergistic effect was observed. Future experiments will show whether H295R cells are suited to test more com- plex mixtures of pesticides (i.e., those comprising more than two of them) influencing estrone biosynthesis. In vitro test systems such as the one described in this study offer the advantage that pesticide mixtures, in which the relative concentration of the individual pesticides to each other has to be varied over a large concentration range to determine whether an effect is additive, synergistic or antagonistic, can be tested in a very short period of time and at a low cost when compared to animal experiments. The obvious limitation is that in the case of H295R cells, as they have
been used in the present study, one only tests whether a mixture of potentially hormonally active compounds is able to modulate a single biological endpoint (i.e., estrone levels). From a regulatory point of view, a combination of in vitro test systems, in which the effects of the mixtures on estrone levels, testosterone levels and sexual hormone receptors are analyzed in parallel, is highly recommended. A prerequisite for the acceptance of such a combination of in vitro test systems by regulatory agencies will be that the results obtained in vitro agree with those obtained in ani- mal experiments.
Acknowledgments We thank Dr. Iris Suckrau (LAVES, Oldenburg, Germany) for providing the data on residue contents of fruits and vegetables.
Conflict of interest The authors state that they have no conflict of interest.
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