PXR nuclear receptors

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

676

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 <30 ppm. These results, particularly those from the KO mice, clearly show the link between Car/Pxr activation and subsequent induction of Phase I enzymes following fluopyram exposure. 3.2. Key event #2: Phase II liver enzyme induction leading to increased serum T4 clearance and consequently decreased circulating T4

BQ Activity nmol /min/mg protein

PROD Activity pmol /min/mg protein

250

**

40 30

**

20

**

**

10 0 0

30

75 150 Fluopyram (ppm)

600

750

Fig. 2. BQ enzyme activities in male mice liver following 28 days fluopyram treatment. Data are presented as the mean of BQ 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 28day 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.

hepatic Phase II enzyme activity data provides evidence that fluopyram induces the activity of the UGT enzyme responsible for the conjugation of T4 at P150 ppm and that Car/Pxr activation is obligatory for the induction of this enzyme activity (Fig. 3B). A decrease of T4 plasma levels was recorded following treatment of WT mice with fluopyram for 3 days at 100 and 300 mg/ kg/day corresponding to 750 and 2000 ppm dietary concentrations (Fig. 4A). Overall the data provide evidence that the increase in UGT activities induced by fluopyram results in an increased elimination of thyroid hormones. 3.3. Key event #3: Increased thyroid stimulating hormone (TSH)

An increase in the activity of uridine diphosphate glucuronyltransferase that conjugates T4 (UGT-T4) and therefore allows for increased metabolism and clearance of thyroid hormones was recorded following treatment of WT mice with fluopyram for 28 days at the tumorigenic dose of 750 ppm (Fig. 3A). This change was fully reversed following a 28-day recovery period (Fig. 3A). In contrast, the activity measured in Car/Pxr KO mice exposed to fluopyram was essentially comparable to the untreated control group (Fig. 3A). In the dose–response characterization, although there was a clear statistical increase in UGT-T4 activity at 600 ppm, the absence of a statistically significant response for UGT-T4 at 750 ppm is likely due to assay variability (Fig. 3B). Overall, this

Due to difficulties in detecting clear changes in TSH plasma levels (Fig. 4B), pituitary ‘‘thyroid-stimulating hormone, beta’’ (Tsh b) transcript levels were measured by qPCR analysis (Fig. 4C). Tsh b is a gene that provides instructions for making the specific protein sub-unit of TSH. Thus, an increase in Tsh b transcripts can serve as a surrogate marker for increased TSH levels. A statistically significant induction of Tsh b transcripts (+49%) was recorded in the 3-day oral gavage study at 300 mg/kg/day. After a 28-day exposure in the diet, a significant induction of this transcript was observed in the pituitary gland at 750 ppm fluopyram which was fully reversed following a 28-day recovery

677

D. Rouquié et al. / Regulatory Toxicology and Pharmacology 70 (2014) 673–680

C57BL/6J Dosing

Recovery

A 50.0

Dosing

**

1.0

40.0

0

750

0

750

0

750

Fluopyram (ppm)

B

C57BL/6J

10.0

5.0 4.0

1.0

0.5

100

300

0

100

300

2.0 1.0

C

2.0

Tsh transcript Relative quantitiy

1.5

0

3.0

0.0

**

**

20.0

B

2.0

**

30.0

0.0

0.5

0.0

T4-glucuronidation (pmol/min/mg)

C57BL/6J

Plasma TSH (ng/mL)

T4-glucuronidation (pmol/min/mg)

1.5

Car/Pxr KO

Plasma T4 (nmol/L)

A

1.5

**

1.0 0.5

0.0 0

30

75

150

600

750

0.0 0

Fluopyram (ppm)

100

300

Fluopyram ( mg/kg/day )

Fig. 3. Uridine diphosphate glucuronyltransferases (Ugt) enzyme activities using T4 as substrate in male mice liver following 28 days fluopyram treatment. Data are presented as the mean of Ugt-T4 activity (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.

Fig. 4. Hormone measurements following 3 day fluopyram treatment at 0, 100 and 300 mg/kg/day. Data are presented as mean values ± SD. (A) Plasma T4 levels (nmol/l). (B) Plasma TSH levels (ng/ml). (C) Relative quantity Tsh b transcript levels. ⁄ p 6 0.05; ⁄⁄p 6 0.01.

3.5. Key event #5: Increased thyroid cell hyperplasia

period (Fig. 5A). In contrast, the level of pituitary Tsh b transcripts measured in Car/Pxr KO mice exposed to fluopyram was comparable to the untreated control group (Fig. 5A). Finally, a dose-related increase in Tsh b transcript levels starting at 600 ppm was recorded in the dose–response evaluation resulting in a NOEL of 150 ppm (Fig. 5B).

Sustained cell proliferation resulted in a low incidence (20–22%) of TFC hyperplasia in male mice at 150 and 750 ppm after 12 months of treatment (Table 1). After 18 months, the incidence was 42% (21/50 animals) and 64% (32/50 animals) in these respective treatment groups compared to the 12 month interval (Table 1).

3.4. Key event #4: Increased thyroid follicular cell proliferation (TFC)

4. Discussion

A dose-related increase in TFC proliferation index measured after incorporation of BrdU was recorded following treatment with fluopyram for 28 days at the tumorigenic dose of 750 ppm, and also at 1500 ppm (Fig. 6A). The evaluation of a dose level of 1500 ppm in addition to the tumorigenic dose level (750 ppm) was performed due to the anticipated weak thyroid effects of fluopyram after an exposure period of 28 days. This effect was reversible after treatment at 1500 ppm followed by 28 days on a control diet (Fig. 6A). In WT and Car/Pxr KO mice treated at 750 ppm, a significant increase was observed in the TFC proliferation index for WT mice, whereas the level of cell proliferation of the fluopyram treated KO mouse group was similar to the KO control value (Fig. 6A). The absence of increased cellular proliferation in Car/ Pxr KO mice demonstrates that the activation of the Car/Pxr receptor is obligatory for the induction of the TFC alterations in mice exposed to fluopyram. In a dose–response study in WT mice, a dose-related increase in TFC proliferation was observed starting from 150 ppm resulting in a NOEL of 75 ppm (Fig. 6B).

Following a lifetime exposure to 750 ppm fluopyram an increased incidence of TFC adenomas was observed in male mice. In order to elucidate the MOA responsible for the thyroid effects and to determine if the overall MOA was threshold dependent, a series of short-term mechanistic dose–response studies were conducted in the male mouse. Since thyroid effects were postulated to be the consequence of indirect effects in the liver and the pituitary, the mechanistic studies focused on identification and characterization of key events in these target tissues in addition to the later effects on the thyroid. The TFC is one of the most common target sites for tumorigenesis in long-term toxicity studies in rodents (IARC, 1999, 2001). Both genotoxic and non-genotoxic agents have been shown to induce TFC tumors (Hurley, 1998). Fluopyram did not show any genotoxic or mutagenic potential in the battery of genotoxicity/ mutagenicity studies (data not shown); therefore, a genotoxic mechanism causing the thyroid effects was ruled out. Non-genotoxic agents share the capacity to induce thyroid effects in rodents

678

D. Rouquié et al. / Regulatory Toxicology and Pharmacology 70 (2014) 673–680

C57BL/6J

A Dosing

Recovery

A

C57BL/6J

Dosing

Dosing 70.0

3.0

**

2.0 1.5 1.0 0.5

Recovery

Dosing

50.0

**

40.0 30.0 20.0 10.0 0.0

0.0 0

750

0

750

0

0

750

750

1500

3.0

**

2.5

*

2.0 1.5 1.0

1500

0

750

C57BL/6J

B

C57BL/6J

60.0 TFC Prolliferation index

B

0

Fluopyram (ppm)

Fluopyram (ppm)

Tsh transcript Relative quantitiy

Car/Pxr KO

**

60.0 TFC Prolliferation index

2.5 Tsh transcript Relative quantitiy

Car/Pxr KO

**

50.0

**

40.0

**

30.0 20.0 10.0

0.5

0.0

0.0 0

30

75

150

600

750

Fluopyram (ppm)

0

30

75

150

600

750

1500

Fluopyram (ppm)

Fig. 5. Tsh b transcript levels in male mice liver following 28 days fluopyram treatment. Data are presented as the means ± SD (Relative quantity). (A) In C57BL/6J mice following a 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 for 28 days to 0 or 750 ppm fluopyram for 28 days. (B) In C57BL/6J mice following dietary exposure for 28 days to 0, 30, 75, 150, 600 and 750 ppm fluopyram. ⁄p 6 0.05; ⁄⁄p 6 0.01.

Fig. 6. Thyroid follicular cell (TFC) proliferation index in male mice liver following 28 day fluopyram treatment. Data are presented as the mean labeling index ± SD based on the evaluation of at least 1000 TFC for BrdU staining. (A) In C57BL/6J mice following dietary exposure for 28 days to 0, 750 or 1500 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 dietary exposure for 28 days to 0, 30, 75, 150, 600, 750 and 1500 ppm fluopyram. ⁄p 6 0.05; ⁄⁄p 6 0.01.

via a perturbation of the hypothalamus–pituitary–thyroid feedback mechanism leading to an increase in circulating TSH, the hormone that stimulates TFCs to proliferate and to produce T4. Growth factors (e.g. insulin/insulin-like growth factor I, epidermal growth factor, basic fibroblast growth factor) are also known to be involved but to a lesser extent (Hard, 1998; Kondo et al., 2006). A prolonged increase in plasma TSH levels in rodents is known to induce a continuum of thyroid proliferative effects, starting with follicular cell proliferation, hypertrophy/hyperplasia and ultimately tumors. Chemicals can induce a sustained TSH increase by many ways: (1) a direct effect on the thyroid causing a decrease of de novo biosynthesis of thyroxine (e.g. inhibition of thyroid peroxidase) or an inhibition of the transport of inorganic iodide into the follicular cell (iodide pump); (2) damage to follicular cells; and (3) inhibition of thyroid hormone release into the blood. Outside the thyroid, chemicals can cause (4) inhibition of the conversion of T4 to T3 by 50 -monodeiodinase at various sites in the body; and (5) enhancement of the metabolism and excretion of thyroid hormone by the liver, largely through the action of UGT enzymes (Hill et al., 1989; Hood et al., 1999; Hurley, 1998; McClain, 1992). This work describes a series of converging experimental results showing that the TFC adenomas induced by fluopyram in the male mouse are due to increased elimination of thyroxine produced by the induction of specific hepatic enzyme activities leading to pituitary–thyroid axis imbalance. In the liver, activation of Car/Pxr as observed by increases in the Phase I enzymes PROD (specific for Car) and BQ (specific for Pxr)

was demonstrated (Figs. 1A and 2B). Specific induction of Phase 2 UGTs activity was also observed at the tumorigenic dose (Fig. 3A). More specifically, UGT activities were measured using T4 as substrate. The glucuronidation of T4 facilitates the elimination of thyroid hormones via urinary and biliary clearance resulting in decreased circulating thyroid hormone levels (Hood and Klaassen, 2000; Klaassen and Hood, 2001). In accordance with the proposed MOA, decreased plasma T4 levels were recorded at the tumorigenic dose level of fluopyram after 3- (Fig. 4A), 14and 28-day treatment periods (data not shown). These data are supported by limited T4 clearance studies which were conducted in male mice intravenously injected with radioactive T4. The results showed that pre-treatment with 2000 ppm fluopyram for 4 days resulted in a clear increased clearance of T4 (+69%) at the earliest time point measured (40 min), when compared to control mice (data not shown). As a consequence of increased T4 clearance, a compensatory mechanism in the pituitary results in increased plasma TSH levels. However, no clear plasma TSH alterations were observed in our studies (Fig. 4B). The difficulty in precisely evaluating plasma hormone levels is likely a reflection of the weak ability of fluopyram to perturb the pituitary–thyroid axis (illustrated by the necessity to treat for 12 months before detecting thyroid histopathological changes and the observation of only benign tumors in just a few animals (7/50) at the end of the cancer bioassay). To get around the difficulty of directly assessing TSH plasma levels, the more sensitive technique of measuring pituitary Tsh b transcript levels was employed as a biomarker that reflects the

679

D. Rouquié et al. / Regulatory Toxicology and Pharmacology 70 (2014) 673–680 Table 3 Summary of NOELs for each liver, pituitary and thyroid parameter measured in the mechanistic and standard toxicity studies.

Key events

a b

CAR/PXR activation: (Indirectly measured) P450 enzyme activity: PROD BQ Ugt-T4 Decreased plasma T4 Tsh b transcript TFC increased proliferation TFC hyperplasia TFC adenoma

NOEL (ppm)

Treatment duration

Reversiblea

Key event detected in Car/Pxr KO mice at 750 ppm

<30 <30 75 ND 150 75 30 150

3, 14, 28 days 3, 14, 28 days 28 days 3, 14, 28 days 3, 28 days 28 days 12 and 18 months 18 months

Yes following 28 days at 750 ppmb

No No No Not determined No No Not determined

Not determined

Reversibility determined after cessation of fluopyram for 28 days. Reversibility for TFC increased proliferation assessed at 1500 ppm.

activity of the pituitary gland to secrete this particular hormone (Hill et al., 1998; DeVito et al., 1999; Elcombe et al., 2002). Quantitative PCR assessments revealed an induction of Tsh b transcript levels at the tumorigenic dose level. Finally, the increased TFC proliferative effect induced by fluopyram exposure provides further evidence of the validity of the proposed MOA. This key event is of particular relevance since it represents the first step of the continuum of effects leading to adenomas. Importantly, data generated for fluopyram demonstrated that the series of causallylinked key events induced following 28 days of treatment at the tumorigenic dose were reversible upon exposure cessation. In the spirit of a weight of evidence approach as recommended in the WHO – IPCS framework, the data generated showed converging evidences supporting the proposed MOA. However the difficulties in detecting clear hormone effects led us to characterize the effects of fluopyram in a Car/Pxr KO mouse model to unequivocally confirm the proposed MOA. The results obtained with the Car/Pxr KO mouse model confirmed the proposed MOA by showing the necessity of functional hepatic Car and Pxr nuclear receptors for the detection of fluopyram treatment effects in the liver, pituitary gland and thyroid gland. Specifically, in KO mice treated with fluopyram at the tumorigenic dose only marginal changes in PROD and BQ activities were observed, and there was no induction of UGT-associated Phase II enzyme. No increase in TFC proliferation was observed, thus demonstrating that the activation of these nuclear receptors is necessary to trigger the key events causally linked with the thyroid adverse effects. In addition, these data in KO mice demonstrate that fluopyram is not directly interfering with thyroid hormone biosynthesis since if this was the case, an increase in Tsh b transcripts and an increased TFC proliferation would have been observed in both wild type and KO mice after fluopyram exposure at the tumorigenic dose level (Figs. 5A and 6A). Dose–response concordance is also critical in demonstrating both the MOA and threshold effects induced by a chemical. In addition to the adenomas detected only at 750 ppm at the end of the cancer bioassay, TFC hyperplasia was detected from 150 ppm in male mice (Table 1). The molecular basis of TFC hyperplasia and adenoma are essentially the same, therefore one could have anticipated detecting key events related to the proposed MOA from 150 ppm. In the present mechanistic studies, NOELs have been identified for the key events causally involved in the tumor formation, namely UGT-T4 activity, Tsh b transcripts induction and TFC proliferation. The induction of Cyp2B and Cyp3A enzyme activities were considered as associated events and not causally linked with the initiation and the progression of the thyroid effects but they did nevertheless provide a means of demonstrating Car/Pxr activation. For these associated events, at the dose levels investigated NOELs were not identified. However, for Key Event 2 (UGT-T4

enzyme induction) the NOEL was 75 ppm since an increased (not statistically significant) activity of the UGT-T4 was observed at 150 ppm. Concerning the induction of Tsh b transcripts in the pituitary gland, a NOEL at 150 ppm was observed. Unfortunately, the quantitative relationship between Tsh b expression and plasma TSH levels is not known. However, the data on Tsh b transcripts provide indirect evidence that a compensatory mechanism on the pituitary–thyroid axis was induced following fluopyram treatment, in particular at the tumorigenic dose level. Finally, the NOEL of the most critical key event that drives thyroid hyperplasia and tumors (TFC proliferative index) was established at 75 ppm. Overall, taking into account that these NOELs were determined after 28-day treatment period and the limitation of the measurement of TSH, the dose–response concordance between the key events characterized in these mechanistic studies and the TFC adenomas is reasonably well established (Table 3). In conclusion, despite the weak thyroid effects induced by fluopyram at the end of the mouse cancer bioassay, the early key events implicit in the MOA responsible for the thyroid tumor were identified in short-term studies using a combination of molecular and cellular tools. These key events induced by fluopyram in male mice were identified in a dose and temporal-responsive manner. Compelling evidence for Car/Pxr being the molecular initiating event for these tumors is provided by the absence of the key events in Car/Pxr KO mice when exposed to fluopyram. Finally, NOELs could be identified for each of the key events, which provided evidence that fluopyram is a threshold-dependent rodent carcinogen. Conflict of interest None declared. Acknowledgments The authors would like to thank M.P. Come, B. Labory, D. Cespedes, and H. Lormeau for their technical expertise and A. Blacker, C. Langrand-Lerche and B. Stahl for their helpful discussions. References Blanck, O., Fowles, J., Schorsch, F., Pallen, C., Espinasse-Lormeau, H., Schulte-Koerne, E., Totis, M., Banton, M., 2009. Tertiary butyl alcohol in drinking water induces phase I and II liver enzymes with consequent effects on thyroid hormone homeostasis in the B6C3F1 female mouse. J. Appl. Toxicol. 30, 125–132. Burke, M.D., Thompson, S., Elcombe, C.R., Halpert, J., Haaparanta, T., Mayer, R.T., 1985. Ethoxy-, pentoxy- and benzyloxyphenoxazones and homologues: a series of substrates to distinguish between different induced cytochromes P-450. Biochem. Pharmacol. 34, 3337–3345. Dellarco, V.L., McGregor, D., Berry, S.C., Cohen, S.M., Boobis, A.R., 2006. Thiazopyr and thyroid disruption: case study within the context of the 2006 IPCS Human Relevance Framework for analysis of a cancer mode of action. Crit. Rev. Toxicol. 36, 793–801.

680

D. Rouquié et al. / Regulatory Toxicology and Pharmacology 70 (2014) 673–680

DeVito, M., Biegel, L., Brouwer, A., Brown, S., Brucker-Davis, F., Cheek, A.O., Christensen, R., Colborn, T., Cooke, P., Crissman, J., Crofton, K., Doerge, D., Gray, E., Hauser, P., Hurley, P., Kohn, M., Lazar, J., McMaster, S., McClain, M., McConnell, E., Meier, C., Miller, R., Tietge, J., Tyl, R., 1999. Screening methods for thyroid hormone disruptors. Environ. Health Perspect. 107, 407–415. Dunnett, C.W., 1955. A multiple comparison procedure for comparing several treatments with a control. J. Am. Stat. Assoc. 50, 1096–1121. Elcombe, C.R., Odum, J., Foster, J.R., Stone, S., Hasmall, S., Soames, A.R., Kimber, I., Ashby, J., 2002. Prediction of rodent nongenotoxic carcinogenesis: evaluation of biochemical and tissue changes in rodents following exposure to nine nongenotoxic NTP carcinogens. Environ. Health Perspect. 110, 363–375. Hard, G.C., 1998. Recent developments in the investigation of thyroid regulation and thyroid carcinogenesis. Environ. Health Perspect. 106, 427–436. Hill, R.N., Crisp, T.M., Hurley, P.M., Rosenthal, S.L., Singh, D.V., 1998. Risk assessment of thyroid follicular cell tumors. Environ. Health Perspect. 106, 447–457. Hill, R.N., Erdreich, L.S., Paynter, O.E., Roberts, P.A., Rosenthal, S.L., Wilkinson, C.F., 1989. Thyroid follicular cell carcinogenesis. Fundam. Appl. Toxicol. 12, 629– 697. Hood, A., Hashmi, R., Klaassen, C.D., 1999. Effects of microsomal enzyme inducers on thyroid-follicular cell proliferation, hyperplasia, and hypertrophy. Toxicol. Appl. Pharmacol. 160, 163–170. Hood, A., Klaassen, C.D., 2000. Differential effects of microsomal enzyme inducers on in vitro thyroxine (T(4)) and triiodothyronine (T(3)) glucuronidation. Toxicol. Sci. 55, 78–84. Hurley, P.M., 1998. Mode of carcinogenic action of pesticides inducing thyroid follicular cell tumors in rodents. Environ. Health Perspect. 106, 437–445. IARC, 1999. Species differences in thyroid, kidney and urinary bladder carcinogenesis. In: Proceedings of a Consensus Conference. IARC Sci Publ, Lyon, France, pp. 1–225 (3–7 November 1997). IARC, 2001. Some thyrotropic agents. IARC Monogr. Eval. Carcinog. Risks Hum. 79, 1–725 (i-iv). Kennel, P.F., Pallen, C.T., Bars, R.G., 2004. Evaluation of the rodent Hershberger assay using three reference endocrine disrupters (androgen and antiandrogens). Reprod. Toxicol. 18, 63–73. Klaassen, C.D., Hood, A.M., 2001. Effects of microsomal enzyme inducers on thyroid follicular cell proliferation and thyroid hormone metabolism. Toxicol. Pathol. 29, 34–40.

Kondo, T., Ezzat, S., Asa, S.L., 2006. Pathogenetic mechanisms in thyroid follicularcell neoplasia. Nat. Rev. Cancer 6, 292–306. Ludwig, S., Tinwell, H., Rouquie, D., Schorsch, F., Pallardy, M., Bars, R., 2012. Potential new targets involved in 1,3-dinitrobenzene induced testicular toxicity. Toxicol. Lett. 213, 275–284. McClain, R.M., 1992. Thyroid gland neoplasia: non-genotoxic mechanisms. Toxicol. Lett. 64–65 Spec No, 397–408. O’Shea, P.J., Williams, G.R., 2002. Insight into the physiological actions of thyroid hormone receptors from genetically modified mice. J. Endocrinol. 175, 553–570. Pascussi, J.M., Gerbal-Chaloin, S., Duret, C., Daujat-Chavanieu, M., Vilarem, M.J., Maurel, P., 2008. The tangle of nuclear receptors that controls xenobiotic metabolism and transport: crosstalk and consequences. Annu. Rev. Pharmacol. Toxicol. 48, 1–32. Phillips, J.M., Burgoon, L.D., Goodman, J.I., 2009. Phenobarbital elicits unique, early changes in the expression of hepatic genes that affect critical pathways in tumor-prone B6C3F1 mice. Toxicol. Sci. 109, 193–205. Qatanani, M., Zhang, J., Moore, D.D., 2005. Role of the constitutive androstane receptor in xenobiotic-induced thyroid hormone metabolism. Endocrinology 146, 995–1002. Rouquie, D., Friry-Santini, C., Schorsch, F., Tinwell, H., Bars, R., 2009. Standard and molecular NOAELs for rat testicular toxicity induced by flutamide. Toxicol. Sci. 109, 59–65. Scheer, N., Ross, J., Rode, A., Zevnik, B., Niehaves, S., Faust, N., Wolf, C.R., 2008. A novel panel of mouse models to evaluate the role of human pregnane X receptor and constitutive androstane receptor in drug response. J. Clin. Invest. 118, 3228–3239. Sokal, R.R., Rohlf, F.J. 1981. Levene Test. In: Biometry, W. H. Freeman, New York, pp. 403–407. Sonich-Mullin, C., Fielder, R., Wiltse, J., Baetcke, K., Dempsey, J., Fenner-Crisp, P., Grant, D., Hartley, M., Knaap, A., Kroese, D., Mangelsdorf, I., Meek, E., Rice, J.M., Younes, M., 2001. IPCS conceptual framework for evaluating a mode of action for chemical carcinogenesis. Regul. Toxicol. Pharmacol. 34, 146–152. Tinwell, H., Rouquié, D., Schorsch, F., Geter, D., Wason, S., Bars, R., 2014. Liver tumor formation in female rat induced by fluopyram is mediated by Car/Pxr nuclear receptor activation. Regul. Toxicol. Pharmacol. 70, 648–658.