Regulatory Toxicology and Pharmacology 70 (2014) 673–680
Contents lists available at ScienceDirect
Regulatory Toxicology and Pharmacology journal homepage: www.elsevier.com/locate/yrtph
Thyroid tumor formation in the male mouse induced by fluopyram is mediated by activation of hepatic CAR/PXR nuclear receptors D. Rouquié ⇑, H. Tinwell, O. Blanck, F. Schorsch, D. Geter, S. Wason, R. Bars Bayer SAS, Bayer CropScience, 355 rue Dostoïevski, CS 90153, 06906 Sophia Antipolis, France
a r t i c l e
i n f o
Article history: Received 15 July 2014 Available online 17 October 2014 Keywords: Mouse thyroid carcinogen Mode of action Car/Pxr nuclear receptors Threshold carcinogen Thyroid follicular cell proliferation
a b s t r a c t Fluopyram, a broad spectrum fungicide, caused an increased incidence of thyroid follicular cell (TFC) adenomas in males at the highest dose evaluated (750 ppm equating to 105 mg/kg/day) in the mouse oncogenicity study. A series of short-term mechanistic studies were conducted in the male mouse to characterize the mode of action (MOA) for the thyroid tumor formation and to determine if No Observed Effect Levels (NOELs) exist for each key event identified. The proposed MOA consists of an initial effect on the liver by activating the constitutive androstane (Car) and pregnane X (Pxr) nuclear receptors causing increased elimination of thyroid hormones followed by an increased secretion of thyroid stimulating hormone (TSH). This change in TSH secretion results in an increase of TFC proliferation which leads to hyperplasia and eventually adenomas after chronic exposure. Car/Pxr nuclear receptors were shown to be activated as indicated by increased activity of specific Phase I enzymes (PROD and BROD, respectively). Furthermore, evidence of increased T4 metabolism was provided by the induction of phase II enzymes known to preferentially use T4 as a substrate. Additional support for the proposed MOA was provided by demonstrating increased Tsh b transcripts in the pituitary gland. Finally, increased TFC proliferation was observed after 28 days of treatment. In these dose–response studies, clear NOELs were established for phase 2 liver enzyme activities, TSH changes and TFC proliferation. Furthermore, compelling evidence for Car/Pxr activation being the molecular initiating event for these thyroid tumors was provided by the absence of the sequential key events responsible for the TCF tumors in Car/Pxr KO mice when exposed to fluopyram. In conclusion, fluopyram thyroid toxicity is mediated by activation of hepatic Car/Pxr receptors and shows a threshold dependent MOA. Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction Fluopyram (N-(2-[3-chloro-5-(trifluoromethyl)-2-pyridinyl]ethyl)-2-(trifluoromethyl) benzamide) is a broad spectrum fungicide developed by Bayer CropScience for the control of fungi such as white mold, black dot and botrytis. It belongs to the class of fungicides described as SDHI fungicides due to their ability to inhibit succinate dehydrogenase (Complex II) within the fungal mitochondrial respiratory chain. At the end of the mouse cancer bioassay, fluopyram was shown to induce thyroid follicular cell (TFC) hyperplasia in both males and females. However, fluopyram was only shown to be a inducer of TFC tumors in males. This induction of tumors was weak both in terms of severity (benign adenomas only) and incidence (7/50 in the high dose group vs 1/50 in the control group) (Table 1). In order to identify the mode of action (MOA) of thyroid toxicity and to ⇑ Corresponding author. Fax: +33 (0)4 93 95 84 54. E-mail address: [email protected] (D. Rouquié). http://dx.doi.org/10.1016/j.yrtph.2014.10.003 0273-2300/Ó 2014 Elsevier Inc. All rights reserved.
examine if the tumors were induced by a threshold dependent MOA, a program of mechanistic studies was conducted in the male mouse. Since the initial adverse finding in the thyroid occurred only after 12 months of exposure and consisted of a slight increase in TFC hyperplasia, it was anticipated that the elucidation of the underlying molecular basis for this chronically observed effect would be challenging. Furthermore, the MOAs responsible for thyroid tumors have been well described in the rat but much less information is published concerning the mouse (Dellarco et al., 2006; Hill et al., 1989). Both genotoxic and non-genotoxic agents have been shown to induce TFC tumors (Hurley, 1998). Fluopyram did not show any effect in the battery of genotoxicity/mutagenicity studies (in vitro assays ± S9: Ames test; chromosome aberration test in V79 cells; HPRT test in V79 cells; in vivo: mouse bone marrow micronucleus assay) excluding genotoxic or mutagenic effects as causes of the thyroid tumors. Among the non-genotoxic agents, direct and indirect thyroid toxicants have been described. Based on the profile of rat hepatic enzyme induction (Tinwell et al., 2014) and thyroid
674
D. Rouquié et al. / Regulatory Toxicology and Pharmacology 70 (2014) 673–680
effects in the rat and mouse obtained in the standard rodent regulatory studies, an MOA involving an indirect liver mediated thyroid effect was proposed for the thyroid tumor formation. In rats, a number of chemical substances (e.g. Acetochlor, Bromacil, Thiazopyr) have been shown to induce TFC hyperplasia and tumors through an MOA that involves perturbation of thyroid hormone homeostasis via a liver mediated reduction of circulating thyroid hormones (Hurley, 1998). Homeostatic responses to reduced thyroid hormone concentrations result in a compensatory increase in the release of thyroid-stimulating hormone (TSH) from the pituitary gland, which in turn stimulates the thyroid gland to increase thyroid hormone synthesis and release. Persistent elevation of TSH levels leads to TFC hypertrophy and hyperplasia, which if maintained (due to continuous exposure to the substance) can eventually lead to neoplasia. In contrast, regarding the mouse there is only limited information available in the literature about compounds known to induce liver-mediated increased T4 clearance causing increased TFC proliferation and thyroid tumors (Blanck et al., 2009; O’Shea and Williams, 2002; Pascussi et al., 2008; Phillips et al., 2009; Qatanani et al., 2005). The objective of this investigation was to evaluate the MOA for thyroid tumor formation in male mice induced by fluopyram based on the conceptual framework proposed by the World Health Organization – International Programme on Chemical Safety (WHO – IPCS) as described by Sonich-Mullin et al. (2001). The MOA for the carcinogenic effect on the mouse thyroid gland was proposed to be secondary to enhanced metabolism of the thyroid hormone thyroxine (T4) triggered by initial activation of hepatic Car and Pxr nuclear receptors. A series of mechanistic studies in male mice were conducted showing the dose and temporal concordance of the specific key events demonstrating the validity of the proposed MOA. Moreover, the use of a Car/Pxr knock out (KO) mouse model confirmed unequivocally activation of Car and Pxr as the initial molecular event and that the thyroid effects were secondary to increased metabolism and elimination of thyroid hormones. Finally, No Observed Effect Levels (NOELs) could be identified for each of the key events, which provided evidence that fluopyram acts through a threshold-dependent MOA. 2. Materials and methods 2.1. Chemicals Fluopyram (International Union of Pure and Applied Chemistry name: (N-(2-[3-chloro-5-(trifluoromethyl)-2-pyridinyl]ethyl)-2(trifluoromethyl)benzamide); CAS 658066-35-4; purity: 94.7% w/ w) was supplied by Bayer CropScience AG (Monheim, Germany). 2.2. Animals and housing Male C57BL/6J mice obtained from Charles River Laboratories (St Germain-sur-l’Arbresle, France) and male Car/Pxr KO mice with a C57BL/6J genetic background obtained from Taconic Farms
(Germantown, New York, 12526, USA; (Scheer et al., 2008)) were housed and maintained as described previously (Ludwig et al., 2012). Animals were acclimatized to laboratory conditions for at least 5 days prior to the start of treatment and were 10 weeks old at the start of treatment.
2.3. Dosing and experimental design A summary of the mechanistic studies conducted is presented in Table 2. In an initial study (Study#1) fluopyram suspended in 0.5% methyl cellulose was administered to wild type C57BL/6J mice (15 male mice per group) orally by gavage at a daily dose of 0 (control), 100, and 300 mg/kg body weight corresponding respectively to 750 and 2000 ppm dietary concentrations, for 3 consecutive days, using a dose volume of 5 ml/kg body weight. Subsequently three 28-day studies were conducted in which groups of 15 male mice were exposed to control diet (A04CP1– 10; Scientific Animal Food and Engineering, Augy, France) or fluopyram incorporated into the diet. In an initial dose–response mechanistic study (Study#2), wild type C57BL/6J mice were exposed at dietary dose levels of 30, 75, 150, 600 or 750 ppm fluopyram; two additional subgroups of control mice and mice exposed to 750 ppm were sacrificed following a 28-day recovery phase during which they were maintained on control diet following the 28-day exposure period. A second dose–response mechanistic study (Study#3) was conducted to specifically investigate the TFC proliferation. The same study design as Study#2 was used except that an additional dose level of 1500 ppm fluopyram was evaluated and additional subgroups of mice were included at this dose level to assess reversibility of the effects instead of the 750 ppm dose level. To quantitatively assess TFC proliferation, animals were exposed to bromodeoxyuridine (BrdU, Sigma–Aldrich, France) at 0.8 g/l in drinking water for one week before sacrifice. In the fourth mechanistic study (Study#4), wild type and Car/ Pxr KO male mice were exposed for 28 days either to control diet or fluopyram at the tumorigenic dose of 750 ppm. Animals were also exposed to 0.8 g/l BrdU in drinking water for one week before sacrifice to assess TFC proliferation. In every study, clinical observations were performed daily and body weights and physical examinations were recorded weekly. All animals were sacrificed by isoflurane (Baxter Maurepas, France) inhalation followed by exsanguination under deep anesthesia. In Study#1, at necropsy, plasma samples were prepared from blood samples from each animal for hormone measurements. The pituitary gland from each mouse was collected. In Study#2, at necropsy, the liver and the pituitary gland were excised from each mouse, trimmed free of fat and connective tissue, and weighed. In Study#3, at necropsy, the thyroid gland was excised from each mouse, trimmed free of fat and connective tissue, and weighed. In Study#4, at necropsy, the liver, the thyroid gland and the pituitary gland were excised from each mouse, trimmed free of fat and connective tissue, and weighed.
Table 1 Incidence of thyroid follicular cell (TFC) hyperplasia and thyroid tumors observed at the end of carcinogenicity study in mice exposed to fluopyram. Data are presented as the number of animals affected in each group. At the chronic phase sacrifice (12 months), TFC hyperplasia was observed in 2/9 males at 150 ppm and 2/10 males at 750 ppm. Dietary level (ppm) [mg/kg/day]
Group size TFC hyperplasia TFC adenoma * **
p < 0.05. p < 0.01.
Males
Females
0
30 [4.2]
150 [20.9]
750 [105]
0
30 [5.3]
150 [26.8]
750 [129]
50 4 1
50 6 1
50 21** 3
50 32** 7*
48 17 3
50 8* 1
50 19 3
50 33** 1
675
D. Rouquié et al. / Regulatory Toxicology and Pharmacology 70 (2014) 673–680 Table 2 Summary of the studies conducted in the mechanistic program.
a
Study#
Type of mice
Objectives
Duration
Fluopyram doses
Parameters
1 2
WT WT
Hormone measurements Dose–response evaluation of key events and reversibility
3 days 28 days + 28 days recovery
0, 100 and 300 mg/kg/daya 0, 30, 75, 150, 600 and 750 ppm. 0 and 750 ppm for the recovery
3
WT
4
WT and KO
Dose–response evaluation of key event and reversibility Confirmation of the key events
28 days + 28 days recovery 28 days
0, 30, 75, 150, 600, 750 and 1500 ppm. 0 and 1500 ppm for the recovery 0, 750 ppm
Plasma T4 and TSH; Tsh b transcripts Hepatic phase 1 and 2 enzyme activities; Tsh b transcripts TFC proliferation Hepatic phase 1 and 2 enzyme activities; Tsh b transcripts; TFC proliferation
Dose levels corresponding to 750 and 2000 ppm respectively dietary concentrations.
2.4. Hormone measurements
2.8. Thyroid follicular cell proliferation
T4 and TSH levels were determined in individual plasma samples from Study#1 using respectively the Clinical Assays™ GammaCoat™ 125I Total T4 RIA Kit (DIASORIN, France) and the kit rat TSH (Institute of Isotopes Ltd., Hungary).
For each animal, the thyroid gland and a portion of the duodenum (as positive marker of the proliferation) were preserved 48 h in 10% neutral buffered formalin. Six sections of the thyroid gland with the duodenum were processed and embedded in paraffin wax. Paraffin-embedded tissues were prepared, sectioned at 5 lm, and stained with hematoxylin (Sigma, France) and eosin (Merck, France). Immunohistochemical staining demonstrating the incorporation of BrdU and the determination of the labeling index were performed to assess TFC proliferation index on each animal. The immunohistochemical reaction included incubation with a monoclonal antibody (Dako) raised against BrdU, amplification with a secondary biotinylated antibody and a streptavidin– horseradish peroxidase complex, detection of the complex with the chromogen diamino-benzidine (DAB) and nuclear counterstaining with hematoxylin. The proliferation index was calculated on more than 1000 counted follicular cells per thyroid gland.
2.5. Hepatic enzyme activity The activity of cytochrome P450 enzymes was determined in mice exposed to fluopyram for 28 days in Study #2 and Study #4. Portions of the liver from all animals per treatment group were weighed and homogenized for microsomal preparations in order to determine specific inducible cytochrome P450 and uridine diphosphate glucuronyltransferase enzymes using T4 as substrate (UGTT4). Microsomal pentoxyresorufin-O-depentylation (PROD) was used as a marker for Cyp2b activity, and was measured according to Burke et al. (1985). Cyp3a activity was measured as the O-debenzylation of benzyloxyquinoline (BQ) according to Burke et al. (1985). Microsomal UGT activity was measured using T4 as substrate. Briefly, individual mouse liver microsomes were incubated with 125I-thyroxine for 60 min at 37 °C and T4 and T4-glucuronide formation was determined by HPLC with radioflow detector.
2.6. Total RNA isolation Total RNA was isolated from pituitary samples from individual control and treated animals using RNeasy Mini kits (Qiagen). Quality controls were performed based on the ribosomal RNA electrophoretic profiles using a Bioanalyser (Agilent Technologies). Only samples with an RNA integrity number (RIN) >7.0 (Agilent software) were used for further analyses.
2.7. Quantitative PCR analysis Ten microgram of individual total RNA from pituitary glands of control and treated animals were used for reverse transcription (RT) using the High Capacity cDNA Archive kit (Applied Biosystems, France). The quantitative PCR assays were performed in duplicate using Taqman probes, (Assay on demand, Applied Biosystems, France), 1/50 diluted first strand cDNA, AmpliTaq GoldÒ PCR Master Mix on an ABI prism 7900 HT machine (Applied Biosystems, France) (Rouquie et al., 2009). The relative quantity (RQ) value of b subunit Tsh transcript (Tsh b) was calculated using bactin as reference gene for the quantitative calculations. A negative control condition was included in which H2O MQ was used as template instead of first strand cDNA.
2.9. Statistical analyses Statistical analyses were performed as described previously (Kennel et al., 2004) for body and organ weights, gene transcript measurements, total cytochrome P450 content and hepatic enzyme activities. TFC proliferation data were analyzed by first using the Levene test (Sokal and Rohlf, 1981) to compare the homogeneity of group variances. If the Levene test was not significant (p > 0.05), the means of the exposed groups were compared to the mean of the control group using the Dunnett test (1-sided; (Dunnett, 1955)). If the Levene test was significant (p 6 0.05), the data were transformed using the log transformation. If the Levene test on log transformed data was not significant (p > 0.05), the means of the exposed groups were compared to the mean of the control group using the Dunnett test (1-sided) on log transformed data. If the Levene test was significant (p 6 0.05) even after log transformation, the means of the exposed groups were compared to the mean of the control group using the Dunn test (1-sided; (Dunnett, 1955)). 3. Results 3.1. Key event #1: Liver Car/Pxr activation with induction of Phase I metabolic enzymes As evidence of Car/Pxr nuclear receptor activation, Cyp2b and Cyp3a corresponding enzyme activities as measured by pentoxyresorufin-O-depentylation (PROD) and benzylquinoline debenzylation (BQ), respectively, were found to be clearly induced at the tumorigenic dose level of 750 ppm in wild type C57BL/6J (WT) mice (Figs. 1A and 2A). These activities returned to essentially control levels when treatment was followed by a 28-day recovery
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D. Rouquié et al. / Regulatory Toxicology and Pharmacology 70 (2014) 673–680
C57BL/6J
A Dosing
Recovery
Dosing
150 100 50
**
0 750
0
750
0
Recovery
Dosing
40 30 20
**
10
750
Fluopyram (ppm)
B
Car/Pxr KO
**
50
200
0
Dosing
60
**
250
C57BL/6J
A
BQ Activity nmol /min/mg protein
PROD Activity pmol /min/mg protein
300
Car/Pxr KO
0 0
C57BL/6J
750
0 750 Fluopyram (ppm)
0
750
300
** **
200
**
B
**
C57BL/6J 60
**
50 150 100
**
50 0 0
30
75
150
600
750
Fluopyram (ppm)
Fig. 1. PROD enzyme activities in male mice liver following 28 days fluopyram treatment. Data are presented as the mean of PROD activity ± SD (pmol/min/mg protein). (A) In C57BL/6J mice following dietary exposure for 28 days to 0 or 750 ppm fluopyram; following the same dietary exposure supplemented by a 28 day recovery phase and in Car/Pxr KO mice following dietary exposure to 0 or 750 ppm fluopyram for 28 days. (B) In C57BL/6J mice following a dietary exposure for 28 days to 0, 30, 75, 150, 600 and 750 ppm fluopyram. ⁄p 6 0.05; ⁄⁄p 6 0.01.
period on a control diet (Figs. 1A and 2A). Definitive confirmation of Car/Pxr activation was established by comparing WT and Car/ Pxr KO mice exposed to 750 ppm fluopyram; PROD was increased 1.4-fold in KO mice compared to 69.8-fold in WT mice, whereas BQ activity was reduced 1.5-fold in KO mice compared to an induction of 5.5-fold in WT mice following 28 days of exposure (Figs. 1A and 2A). In a 28-day dose–response study in WT mice, these specific enzyme inductions were observed in a dose-related manner from the lowest dose tested of 30 ppm (Figs. 1B and 2B) and therefore the NOEL was defined as
Contents lists available at ScienceDirect
Regulatory Toxicology and Pharmacology journal homepage: www.elsevier.com/locate/yrtph
Thyroid tumor formation in the male mouse induced by fluopyram is mediated by activation of hepatic CAR/PXR nuclear receptors D. Rouquié ⇑, H. Tinwell, O. Blanck, F. Schorsch, D. Geter, S. Wason, R. Bars Bayer SAS, Bayer CropScience, 355 rue Dostoïevski, CS 90153, 06906 Sophia Antipolis, France
a r t i c l e
i n f o
Article history: Received 15 July 2014 Available online 17 October 2014 Keywords: Mouse thyroid carcinogen Mode of action Car/Pxr nuclear receptors Threshold carcinogen Thyroid follicular cell proliferation
a b s t r a c t Fluopyram, a broad spectrum fungicide, caused an increased incidence of thyroid follicular cell (TFC) adenomas in males at the highest dose evaluated (750 ppm equating to 105 mg/kg/day) in the mouse oncogenicity study. A series of short-term mechanistic studies were conducted in the male mouse to characterize the mode of action (MOA) for the thyroid tumor formation and to determine if No Observed Effect Levels (NOELs) exist for each key event identified. The proposed MOA consists of an initial effect on the liver by activating the constitutive androstane (Car) and pregnane X (Pxr) nuclear receptors causing increased elimination of thyroid hormones followed by an increased secretion of thyroid stimulating hormone (TSH). This change in TSH secretion results in an increase of TFC proliferation which leads to hyperplasia and eventually adenomas after chronic exposure. Car/Pxr nuclear receptors were shown to be activated as indicated by increased activity of specific Phase I enzymes (PROD and BROD, respectively). Furthermore, evidence of increased T4 metabolism was provided by the induction of phase II enzymes known to preferentially use T4 as a substrate. Additional support for the proposed MOA was provided by demonstrating increased Tsh b transcripts in the pituitary gland. Finally, increased TFC proliferation was observed after 28 days of treatment. In these dose–response studies, clear NOELs were established for phase 2 liver enzyme activities, TSH changes and TFC proliferation. Furthermore, compelling evidence for Car/Pxr activation being the molecular initiating event for these thyroid tumors was provided by the absence of the sequential key events responsible for the TCF tumors in Car/Pxr KO mice when exposed to fluopyram. In conclusion, fluopyram thyroid toxicity is mediated by activation of hepatic Car/Pxr receptors and shows a threshold dependent MOA. Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction Fluopyram (N-(2-[3-chloro-5-(trifluoromethyl)-2-pyridinyl]ethyl)-2-(trifluoromethyl) benzamide) is a broad spectrum fungicide developed by Bayer CropScience for the control of fungi such as white mold, black dot and botrytis. It belongs to the class of fungicides described as SDHI fungicides due to their ability to inhibit succinate dehydrogenase (Complex II) within the fungal mitochondrial respiratory chain. At the end of the mouse cancer bioassay, fluopyram was shown to induce thyroid follicular cell (TFC) hyperplasia in both males and females. However, fluopyram was only shown to be a inducer of TFC tumors in males. This induction of tumors was weak both in terms of severity (benign adenomas only) and incidence (7/50 in the high dose group vs 1/50 in the control group) (Table 1). In order to identify the mode of action (MOA) of thyroid toxicity and to ⇑ Corresponding author. Fax: +33 (0)4 93 95 84 54. E-mail address: [email protected] (D. Rouquié). http://dx.doi.org/10.1016/j.yrtph.2014.10.003 0273-2300/Ó 2014 Elsevier Inc. All rights reserved.
examine if the tumors were induced by a threshold dependent MOA, a program of mechanistic studies was conducted in the male mouse. Since the initial adverse finding in the thyroid occurred only after 12 months of exposure and consisted of a slight increase in TFC hyperplasia, it was anticipated that the elucidation of the underlying molecular basis for this chronically observed effect would be challenging. Furthermore, the MOAs responsible for thyroid tumors have been well described in the rat but much less information is published concerning the mouse (Dellarco et al., 2006; Hill et al., 1989). Both genotoxic and non-genotoxic agents have been shown to induce TFC tumors (Hurley, 1998). Fluopyram did not show any effect in the battery of genotoxicity/mutagenicity studies (in vitro assays ± S9: Ames test; chromosome aberration test in V79 cells; HPRT test in V79 cells; in vivo: mouse bone marrow micronucleus assay) excluding genotoxic or mutagenic effects as causes of the thyroid tumors. Among the non-genotoxic agents, direct and indirect thyroid toxicants have been described. Based on the profile of rat hepatic enzyme induction (Tinwell et al., 2014) and thyroid
674
D. Rouquié et al. / Regulatory Toxicology and Pharmacology 70 (2014) 673–680
effects in the rat and mouse obtained in the standard rodent regulatory studies, an MOA involving an indirect liver mediated thyroid effect was proposed for the thyroid tumor formation. In rats, a number of chemical substances (e.g. Acetochlor, Bromacil, Thiazopyr) have been shown to induce TFC hyperplasia and tumors through an MOA that involves perturbation of thyroid hormone homeostasis via a liver mediated reduction of circulating thyroid hormones (Hurley, 1998). Homeostatic responses to reduced thyroid hormone concentrations result in a compensatory increase in the release of thyroid-stimulating hormone (TSH) from the pituitary gland, which in turn stimulates the thyroid gland to increase thyroid hormone synthesis and release. Persistent elevation of TSH levels leads to TFC hypertrophy and hyperplasia, which if maintained (due to continuous exposure to the substance) can eventually lead to neoplasia. In contrast, regarding the mouse there is only limited information available in the literature about compounds known to induce liver-mediated increased T4 clearance causing increased TFC proliferation and thyroid tumors (Blanck et al., 2009; O’Shea and Williams, 2002; Pascussi et al., 2008; Phillips et al., 2009; Qatanani et al., 2005). The objective of this investigation was to evaluate the MOA for thyroid tumor formation in male mice induced by fluopyram based on the conceptual framework proposed by the World Health Organization – International Programme on Chemical Safety (WHO – IPCS) as described by Sonich-Mullin et al. (2001). The MOA for the carcinogenic effect on the mouse thyroid gland was proposed to be secondary to enhanced metabolism of the thyroid hormone thyroxine (T4) triggered by initial activation of hepatic Car and Pxr nuclear receptors. A series of mechanistic studies in male mice were conducted showing the dose and temporal concordance of the specific key events demonstrating the validity of the proposed MOA. Moreover, the use of a Car/Pxr knock out (KO) mouse model confirmed unequivocally activation of Car and Pxr as the initial molecular event and that the thyroid effects were secondary to increased metabolism and elimination of thyroid hormones. Finally, No Observed Effect Levels (NOELs) could be identified for each of the key events, which provided evidence that fluopyram acts through a threshold-dependent MOA. 2. Materials and methods 2.1. Chemicals Fluopyram (International Union of Pure and Applied Chemistry name: (N-(2-[3-chloro-5-(trifluoromethyl)-2-pyridinyl]ethyl)-2(trifluoromethyl)benzamide); CAS 658066-35-4; purity: 94.7% w/ w) was supplied by Bayer CropScience AG (Monheim, Germany). 2.2. Animals and housing Male C57BL/6J mice obtained from Charles River Laboratories (St Germain-sur-l’Arbresle, France) and male Car/Pxr KO mice with a C57BL/6J genetic background obtained from Taconic Farms
(Germantown, New York, 12526, USA; (Scheer et al., 2008)) were housed and maintained as described previously (Ludwig et al., 2012). Animals were acclimatized to laboratory conditions for at least 5 days prior to the start of treatment and were 10 weeks old at the start of treatment.
2.3. Dosing and experimental design A summary of the mechanistic studies conducted is presented in Table 2. In an initial study (Study#1) fluopyram suspended in 0.5% methyl cellulose was administered to wild type C57BL/6J mice (15 male mice per group) orally by gavage at a daily dose of 0 (control), 100, and 300 mg/kg body weight corresponding respectively to 750 and 2000 ppm dietary concentrations, for 3 consecutive days, using a dose volume of 5 ml/kg body weight. Subsequently three 28-day studies were conducted in which groups of 15 male mice were exposed to control diet (A04CP1– 10; Scientific Animal Food and Engineering, Augy, France) or fluopyram incorporated into the diet. In an initial dose–response mechanistic study (Study#2), wild type C57BL/6J mice were exposed at dietary dose levels of 30, 75, 150, 600 or 750 ppm fluopyram; two additional subgroups of control mice and mice exposed to 750 ppm were sacrificed following a 28-day recovery phase during which they were maintained on control diet following the 28-day exposure period. A second dose–response mechanistic study (Study#3) was conducted to specifically investigate the TFC proliferation. The same study design as Study#2 was used except that an additional dose level of 1500 ppm fluopyram was evaluated and additional subgroups of mice were included at this dose level to assess reversibility of the effects instead of the 750 ppm dose level. To quantitatively assess TFC proliferation, animals were exposed to bromodeoxyuridine (BrdU, Sigma–Aldrich, France) at 0.8 g/l in drinking water for one week before sacrifice. In the fourth mechanistic study (Study#4), wild type and Car/ Pxr KO male mice were exposed for 28 days either to control diet or fluopyram at the tumorigenic dose of 750 ppm. Animals were also exposed to 0.8 g/l BrdU in drinking water for one week before sacrifice to assess TFC proliferation. In every study, clinical observations were performed daily and body weights and physical examinations were recorded weekly. All animals were sacrificed by isoflurane (Baxter Maurepas, France) inhalation followed by exsanguination under deep anesthesia. In Study#1, at necropsy, plasma samples were prepared from blood samples from each animal for hormone measurements. The pituitary gland from each mouse was collected. In Study#2, at necropsy, the liver and the pituitary gland were excised from each mouse, trimmed free of fat and connective tissue, and weighed. In Study#3, at necropsy, the thyroid gland was excised from each mouse, trimmed free of fat and connective tissue, and weighed. In Study#4, at necropsy, the liver, the thyroid gland and the pituitary gland were excised from each mouse, trimmed free of fat and connective tissue, and weighed.
Table 1 Incidence of thyroid follicular cell (TFC) hyperplasia and thyroid tumors observed at the end of carcinogenicity study in mice exposed to fluopyram. Data are presented as the number of animals affected in each group. At the chronic phase sacrifice (12 months), TFC hyperplasia was observed in 2/9 males at 150 ppm and 2/10 males at 750 ppm. Dietary level (ppm) [mg/kg/day]
Group size TFC hyperplasia TFC adenoma * **
p < 0.05. p < 0.01.
Males
Females
0
30 [4.2]
150 [20.9]
750 [105]
0
30 [5.3]
150 [26.8]
750 [129]
50 4 1
50 6 1
50 21** 3
50 32** 7*
48 17 3
50 8* 1
50 19 3
50 33** 1
675
D. Rouquié et al. / Regulatory Toxicology and Pharmacology 70 (2014) 673–680 Table 2 Summary of the studies conducted in the mechanistic program.
a
Study#
Type of mice
Objectives
Duration
Fluopyram doses
Parameters
1 2
WT WT
Hormone measurements Dose–response evaluation of key events and reversibility
3 days 28 days + 28 days recovery
0, 100 and 300 mg/kg/daya 0, 30, 75, 150, 600 and 750 ppm. 0 and 750 ppm for the recovery
3
WT
4
WT and KO
Dose–response evaluation of key event and reversibility Confirmation of the key events
28 days + 28 days recovery 28 days
0, 30, 75, 150, 600, 750 and 1500 ppm. 0 and 1500 ppm for the recovery 0, 750 ppm
Plasma T4 and TSH; Tsh b transcripts Hepatic phase 1 and 2 enzyme activities; Tsh b transcripts TFC proliferation Hepatic phase 1 and 2 enzyme activities; Tsh b transcripts; TFC proliferation
Dose levels corresponding to 750 and 2000 ppm respectively dietary concentrations.
2.4. Hormone measurements
2.8. Thyroid follicular cell proliferation
T4 and TSH levels were determined in individual plasma samples from Study#1 using respectively the Clinical Assays™ GammaCoat™ 125I Total T4 RIA Kit (DIASORIN, France) and the kit rat TSH (Institute of Isotopes Ltd., Hungary).
For each animal, the thyroid gland and a portion of the duodenum (as positive marker of the proliferation) were preserved 48 h in 10% neutral buffered formalin. Six sections of the thyroid gland with the duodenum were processed and embedded in paraffin wax. Paraffin-embedded tissues were prepared, sectioned at 5 lm, and stained with hematoxylin (Sigma, France) and eosin (Merck, France). Immunohistochemical staining demonstrating the incorporation of BrdU and the determination of the labeling index were performed to assess TFC proliferation index on each animal. The immunohistochemical reaction included incubation with a monoclonal antibody (Dako) raised against BrdU, amplification with a secondary biotinylated antibody and a streptavidin– horseradish peroxidase complex, detection of the complex with the chromogen diamino-benzidine (DAB) and nuclear counterstaining with hematoxylin. The proliferation index was calculated on more than 1000 counted follicular cells per thyroid gland.
2.5. Hepatic enzyme activity The activity of cytochrome P450 enzymes was determined in mice exposed to fluopyram for 28 days in Study #2 and Study #4. Portions of the liver from all animals per treatment group were weighed and homogenized for microsomal preparations in order to determine specific inducible cytochrome P450 and uridine diphosphate glucuronyltransferase enzymes using T4 as substrate (UGTT4). Microsomal pentoxyresorufin-O-depentylation (PROD) was used as a marker for Cyp2b activity, and was measured according to Burke et al. (1985). Cyp3a activity was measured as the O-debenzylation of benzyloxyquinoline (BQ) according to Burke et al. (1985). Microsomal UGT activity was measured using T4 as substrate. Briefly, individual mouse liver microsomes were incubated with 125I-thyroxine for 60 min at 37 °C and T4 and T4-glucuronide formation was determined by HPLC with radioflow detector.
2.6. Total RNA isolation Total RNA was isolated from pituitary samples from individual control and treated animals using RNeasy Mini kits (Qiagen). Quality controls were performed based on the ribosomal RNA electrophoretic profiles using a Bioanalyser (Agilent Technologies). Only samples with an RNA integrity number (RIN) >7.0 (Agilent software) were used for further analyses.
2.7. Quantitative PCR analysis Ten microgram of individual total RNA from pituitary glands of control and treated animals were used for reverse transcription (RT) using the High Capacity cDNA Archive kit (Applied Biosystems, France). The quantitative PCR assays were performed in duplicate using Taqman probes, (Assay on demand, Applied Biosystems, France), 1/50 diluted first strand cDNA, AmpliTaq GoldÒ PCR Master Mix on an ABI prism 7900 HT machine (Applied Biosystems, France) (Rouquie et al., 2009). The relative quantity (RQ) value of b subunit Tsh transcript (Tsh b) was calculated using bactin as reference gene for the quantitative calculations. A negative control condition was included in which H2O MQ was used as template instead of first strand cDNA.
2.9. Statistical analyses Statistical analyses were performed as described previously (Kennel et al., 2004) for body and organ weights, gene transcript measurements, total cytochrome P450 content and hepatic enzyme activities. TFC proliferation data were analyzed by first using the Levene test (Sokal and Rohlf, 1981) to compare the homogeneity of group variances. If the Levene test was not significant (p > 0.05), the means of the exposed groups were compared to the mean of the control group using the Dunnett test (1-sided; (Dunnett, 1955)). If the Levene test was significant (p 6 0.05), the data were transformed using the log transformation. If the Levene test on log transformed data was not significant (p > 0.05), the means of the exposed groups were compared to the mean of the control group using the Dunnett test (1-sided) on log transformed data. If the Levene test was significant (p 6 0.05) even after log transformation, the means of the exposed groups were compared to the mean of the control group using the Dunn test (1-sided; (Dunnett, 1955)). 3. Results 3.1. Key event #1: Liver Car/Pxr activation with induction of Phase I metabolic enzymes As evidence of Car/Pxr nuclear receptor activation, Cyp2b and Cyp3a corresponding enzyme activities as measured by pentoxyresorufin-O-depentylation (PROD) and benzylquinoline debenzylation (BQ), respectively, were found to be clearly induced at the tumorigenic dose level of 750 ppm in wild type C57BL/6J (WT) mice (Figs. 1A and 2A). These activities returned to essentially control levels when treatment was followed by a 28-day recovery
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Fig. 1. PROD enzyme activities in male mice liver following 28 days fluopyram treatment. Data are presented as the mean of PROD activity ± SD (pmol/min/mg protein). (A) In C57BL/6J mice following dietary exposure for 28 days to 0 or 750 ppm fluopyram; following the same dietary exposure supplemented by a 28 day recovery phase and in Car/Pxr KO mice following dietary exposure to 0 or 750 ppm fluopyram for 28 days. (B) In C57BL/6J mice following a dietary exposure for 28 days to 0, 30, 75, 150, 600 and 750 ppm fluopyram. ⁄p 6 0.05; ⁄⁄p 6 0.01.
period on a control diet (Figs. 1A and 2A). Definitive confirmation of Car/Pxr activation was established by comparing WT and Car/ Pxr KO mice exposed to 750 ppm fluopyram; PROD was increased 1.4-fold in KO mice compared to 69.8-fold in WT mice, whereas BQ activity was reduced 1.5-fold in KO mice compared to an induction of 5.5-fold in WT mice following 28 days of exposure (Figs. 1A and 2A). In a 28-day dose–response study in WT mice, these specific enzyme inductions were observed in a dose-related manner from the lowest dose tested of 30 ppm (Figs. 1B and 2B) and therefore the NOEL was defined as
