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Toxicity Profiles in Rats Treated with Tumorigenic and Nontumorigenic Triazole Conazole Fungicides: Propiconazole, Triadimefon, and Myclobutanil

¹ Environmental Carcinogenesis Division, National Health and Environmental Effects Research Laboratory, ORD, USEPA, Research Triangle Park, North Carolina 27711, USA
² Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, ORD, USEPA, Research Triangle Park, North Carolina 27711, USA

Correspondence: Address correspondence to: Douglas C. Wolf, Environmental Carcinogenesis Division, U.S. EPA, MD-B143-06, 109 TW Alexander Dr., Research Triangle Park, NC 27711, USA; e-mail:wolf.doug{at}epa.gov

	Abstract

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Conazoles are a class of azole based fungicides used in agriculture and as pharmaceutical products. They have a common mode of antifungal action through inhibition of ergosterol biosynthesis. Some members of this class have been shown to be hepatotoxic and will induce mouse hepatocellular tumors and/or rat thyroid follicular cell tumors. The particular mode of toxic and tumorigenic action for these compounds is not known, however it has been proposed that triadimefon-induced rat thyroid tumors arise through the specific mechanism of increased TSH. The present study was designed to identify commonalities of effects across the different conazoles and to determine unique features of the tissue responses that suggest a toxicity pathway and a mode of action for the observed thyroid response for triadimefon. Male Wistar/Han rats were treated with triadimefon (100, 500, 1800 ppm), propiconazole (100, 500, 2500 ppm), or myclobutanil (100, 500, 2000 ppm) in feed for 4, 30, or 90 days. The rats were evaluated for clinical signs, body and liver weight, histopathology of thyroid and liver, hepatic metabolizing enzyme activity, and serum T3, T4, TSH, and cholesterol levels. There was a dose-dependent increase in liver weight but not body weight for all treatments. The indication of cytochrome induction, pentoxyresorufin O-dealkylation (PROD) activity, had a dose-related increase at all time points for all conazoles. Uridine diphopho-glucuronosyl transferase (UDPGT), the T4 metabolizing enzyme measured as glucuronidation of 1-naphthol, was induced to the same extent after 30 and 90 days for all three conazoles. Livers from all high dose treated rats had centrilobular hepatocyte hypertrophy after 4 days, while only triadimefon and propiconazole treated rats had hepatocyte hypertrophy after 30 days, and only triadimefon treated rats had hepatocyte hypertrophy after 90 days. Thyroid follicular cell hypertrophy, increased follicular cell proliferation, and colloid depletion were present only after 30 days in rats treated with the high dose of triadimefon. A dose-dependent decrease in T4 was present after 4 days with all 3 compounds but only the high doses of propiconazole and triadimefon produced decreased T4 after 30 days. T3 was decreased after high-dose triadimefon after 4 days and in a dose-dependent manner for all compounds after 30 days. Thyroid hormone levels did not differ from control values after 90 days and TSH was not increased in any exposure group. A unique pattern of toxic responses was not identified for each conazole and the hypothesized mode of action for triadimefon-induced thyroid gland tumors was not supported by the data.

Key Words: Thyroid • pesticide • endocrine disruptor • UDPGT • enzyme induction

	Introduction

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Triazole-containing azole fungicides (conazoles) have a broad antifungal activity and can prevent as well as treat fungal infections. Their antifungal characteristic is due to their ability to block the synthesis of ergosterol, which is an essential component of the fungal cell membrane. It is this general feature that makes this class of chemicals suitable for use in agriculture as crop protection products as well as veterinary and human medicine as antifungal drugs. Myclobutanil is used for grape fungus and triadimefon and propiconazole are used on fruits, grains, and grasses such as golf courses (Cabras and Angioni, 2000; Haith and Rossi, 2003). Besides applicator exposure, humans can become exposed through runoff from treated fields or golf courses and through the air after aerosol application (Egaas et al., 1999; Haith and Rossi, 2003; Kim et al., 2003; Nag and Dureja, 2003). Environmental exposure to conazoles tends to be low, 0.001 to 0.087 mg/L in surface waters and 0.01 to 0.22 mg/kg in winemaking residues (Cabras and Angioni, 2000; Haith and Rossi, 2003).

The primary enzyme blocked by the conazoles is sterol 14--sterol demethylase (CYP51, lanosterol 14--demethylase), the only member of the cytochrome family present in animals, plants, fungi, and prokaryotes (Lamb et al., 2001). Inhibition occurs by binding to the heme iron of the enzyme which, in fungi, results in depletion of ergosterol needed for normal fungal membranes (Lamb et al., 2001). This evolutionarily conserved enzyme is important in cholesterol and vitamin D synthesis (Zarn et al., 2003). In vertebrate species, conazoles have complex effects on hepatic and nonhepatic microsomal enzymes (Leslie et al., 1988; Walker, 1998; Egaas et al., 1999). They act as both inducers and inhibitors of cytochrome P450s depending on the tissue and specific conazole.

Conazoles have been shown to affect the activity and expression of a number of P450s in the liver. Propiconazole induces the activities of CYP1A1, CYP1A2, CYP2B1/2, CYP2B6, and CYP3A4 and inhibits CYP2C11 (Ronis et al., 1994; Walker, 1998). Ketoconazole has been shown to induce CYP1A1; CYP2B and CYP3A2 in rat liver and inhibit CYP1A1, CYP1A2, CYP2A6, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 in human liver microsomes (Ronis et al., 1994; Zhang et al., 2002). In addition to altered expression and activity of cytochromes, other metabolizing enzyme activities are also altered including glutathione-S-transferase (GST) and those that metabolize lauric acid and testosterone (Walker, 1998; Egaas et al., 1999).

Many pesticides including the conazoles are hepatotoxic and hepatocarcinogenic in mice and also induce thyroid follicular cell tumors in rats (INCHEM, 1981, 1987, 1992; Hasegawa and Ito, 1992; Federal Register, 1996; Hurley et al., 1998). In a review of 240 pesticides, including fungicides, screened for tumorigenicity, 37 induced thyroid endocrine disruption or other thyroid alterations, and 27 induced thyroid follicular cell tumors (Hurley et al., 1998). Thyroid gland tumors were the second most common tumor, after liver, of pesticide-induced tumors. Of the pesticides that induce thyroid tumors 92% are for rats only, with 33% positive for males only and none for females only. This gender difference is also seen in the consistently greater serum thyroid-stimulating hormone (TSH) level in males. Unlike rodents, in humans, women tend to have a higher incidence of thyroid cancer than men. The majority of thyroid cancers in humans arise from the follicular epithelium and are of 2 main types, papillary and follicular, which develop by 2 different molecular pathways (Williams, 1995). In rodents there is no molecular correlate to a separation of thyroid cancer into morphological entities and the major factors associated with rodent thyroid carcinogenesis are exposure to a mutagenic insult and/or growth stimulation by TSH hypersecretion (Williams, 1995).

The modes of action for thyroid tumorigenesis in rodents have been shown to include iodide deficiency, inhibition of iodide uptake, inhibition of thyroid peroxidase, inhibition of thyroxine (T4) release, follicular cell damage, inhibition of conversion of T4 to tri-iodothyronine (T3), increased T4 and T3 metabolism and excretion, and direct cellular damage including mutagenicity (Hill et al., 1998; Hurley et al., 1998). To aid in the determination of the biological relevance of rodent thyroid tumors for human health risk assessment, it is necessary to show how the chemical in question increases thyroid growth, alters thyroid hormones, and its site of action relative to thyroid function (Hill et al., 1998). In addition to these features information on the dose correlation to effects, their reversibility, and if the thyroid lesion progresses in response to continued treatment help in better describing the potential for antithyroid activity (Hill et al., 1998).

Triadimefon-induced rat thyroid tumors are hypothesized to be a result of increased hepatic metabolism and biliary excretion of T4 leading to increased TSH and overstimulation of the thyroid leading to neoplasia (Capen, 1997). Generally, microsomal enzyme inducers can also nonspecifically induce UDPGT activity, which would result in increased excretion of T4 by general induction of UDPGTs. Up-regulation of UDPGT yields T4 and T3 glucuronic acid and biliary excretion (Barter and Klaassen, 1992a, 1992b, 1994; Liu et al., 1995; Saito et al., 1991). A proposed mechanism for thyroid tumor promotion is induction of UDPGT resulting in increased elimination of T4 followed by decreased serum T4 and increased serum TSH, which causes increased follicular cell proliferation (Capen, 1997). With persistent follicular cell stimulation, thyroid tumors form in the rat (Klaassen and Hood, 2001). Triadimefon does not directly damage DNA (Kevekordes et al., 1996), suggesting that a nongenotoxic mode of action is driving the development of triadimefon-induced thyroid gland tumors. The present study was designed to describe a mode of action for the thyroid tumorigen, triadimefon in the rat, characterize the time- and dose-dependent alterations induced by myclobutanil, propiconazole, and triadimefon, and to determine if traditional toxicology methods could differentiate these conazoles.

	Materials and Methods

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One hundred and thirty, 13/exposure group, male Wistar/Han rats approximately 7 weeks old were treated with myclobutanil, propiconazole, or triadimefon at 3 concentrations in the feed for 4, 30, or 90 days (Table 1). The highest concentration had been a tumorigenic dose in previous pesticide registration studies or, in the case of myclobutanil, the MTD was negative for a tumorigenic response. These conazoles were selected based on their tumor response in rats and mice. Triadimefon induces both rat thyroid gland and mouse liver tumors; propiconazole induces only mouse liver tumors and myclobutanil is negative for a tumor response in any tissue. Animals were examined daily for morbidity and weighed weekly. In addition feed consumption was assessed weekly.

Feed Preparation and Analysis
All feed was prepared and analyzed by Bayer Crop-Sciences at Stilwell, KS, following GLP. Every 2 weeks the feeds were prepared and shipped to the US EPA in Research Triangle Park, NC. Briefly, triadimefon (96.1% purity), propiconazole (94.2% purity), and myclobutanil (95.8% purity) were dissolved in acetone and added dropwise to Purina Mills Certified Rodent Diet 5002 Meal in a Hobart mixer for blending. The mixed feed, as powdered meal, was stored at room temperature in covered plastic containers until the feed was shipped to the US EPA, at which time it was stored at 4°C. Feeds were sampled at least twice from different locations within the container. Quantitative analyses of the levels of conazoles in the feed were performed by Bayer CropSciences using LC-MS/MS with deuterated internal standards and methanol extracts of the feed samples. The target concentrations and measured mean concentrations (± SD) of each conazole determined over the 21-week period are presented in Table 1.

Histology
Quantitative assessment was made of thyroid and liver histopathology. All slides were read without knowledge of treatment or time. The liver alterations were scored based on severity of hepatocyte hypertrophy, which was the only alteration present except in a few slides which also had hepatocyte vacuolation in addition to the hypertrophy. The lesion scores were: 0 – no lesion present, 1 – centrilobular hypertrophy, 2 – centrilobular and midzonal hypertrophy, 3 – pan lobular hypertrophy, 4 – pan lobular hypertrophy with cytoplasmic vacuolation. The thyroid gland was scored for colloid depletion, follicular cell hypertrophy, and/or follicular cell hyperplasia as previously described (Hooth et al, 2001; Khan et al., 2005).

In addition to lesion scores, cell proliferation indices were quantitated for hepatocytes and thyroid follicular cells. The tissues were stained for the presence of proliferating cell nuclear antigen (PCNA) as previously described (McDorman et al., 2003). The cells were counted using counting software, Cytology Histology Recognition Information System (CHRIS, Sverdrup, Fort Walton Beach, FL) as previously reported (Medinsky et al., 1999). Briefly, 20 random images at 200X magnification were collected for the liver and thyroid gland. Labeled and unlabeled nuclei were identified within the program. The number of labeled nuclei per approximately 1,000 total nuclei were identified for each organ to calculate a labeling index.

Serum Hormone and Lipid Analyses
Total circulating serum thyroid hormone levels of T3, T4, and TSH were analyzed using previously established protocols (Khan et al., 2005). Serum levels for cholesterol, high-density lipoproteins, and triglycerides were measured using the Roche Hitachi 717 Chemistry Analyzer (LabCorp, Research Triangle Park, NC).

Hepatic Enzyme Activity
Liver microsomes were prepared in accordance with procedures of Matsuura et al.(1991), with some modifications. Briefly, fresh livers were washed with cold 1.15% KCl and 0.25 M sucrose and minced and then transferred to a cold Beckman centrifuge tube, to which 15 ml of cold 0.25 M sucrose was added. Liver samples were homogenized and centrifuged at 9,000 x g for 20 minutes at 4°C in a Beckman L8 70 ultracentrifuge. The supernatant was then centrifuged at 105,000 x g for 60 minutes at 4°C. The pellet was homogenized in cold storage buffer (pH 7.5, K₂HPO₄: 10 mM, DTT: 0.1 mM, EDTA: 1 mM, glycerol: 20% (v/v)), diluted 1:1 with cold 0.25 M sucrose. Pelleted sample material was resuspended and the microsomal suspensions were aliquoted into Nunc vials (Nunc-Nalgene, Rochester, NY). The samples were stored in liquid nitrogen until assays were performed. Microsomal protein levels were determined by the Lowry assay (Lowry et al., 1951) using bovine serum albumin as the protein standard.

Cytochrome P450 enzyme activities were assessed with alkoxyresorufin O-dealkylation (AROD) assays, as described by Burke et al. (1994), with some modifications. These assays were based on activity measures of ethoxyresorufin O-dealkylation (EROD), pentoxyresorufin O-dealkylation (PROD), and methoxyresorufin O-dealkylation (MROD). Reaction mixtures (3 ml) were prepared in 4-sided, clear methacrylate cuvettes containing phosphate buffer (Na salt, 0.1 M, pH, 7.4), MgCl₂ (3.3 mM), alkoxyresorufin (4.9 µM), NADP (78 mM), G-6-P (198 mM), and G-6-P DH (24 U/ml) and were incubated at 37°C for 2 minutes. Microsomes were added to the mixtures to initiate the reactions. The final concentration range of microsomes was 0.1 mg protein/ml. The fluorescence of the mixtures was measured at 37°C on a Perkin-Elmer LS-50 fluorometer with an excitation wavelength of 550 nm and an emission wavelength of 585 nm. Data were collected every 3 sec. for 5 minutes. AROD activities were expressed as rates of resorufin formation, and were calculated based on the fluorescence of a standard curve of resorufin. Values are expressed as the mean ± SD in pmol resorufin formed/min/mg microsomal protein. Microsomes from each animal were assayed in duplicate with variances in the duplicates less than 10%

The assay procedure for UDP-glucuronosyltransferase used was that described by Mackenzie and Hanninen (Mackenzie and Hanninen, 1980) using 1-naphthol as substrate. Briefly, a 3-ml reaction solution consisting of 50 µM 1-naphthol and microsomes (30–300 µg protein) in 0.05 M sodium phosphate buffer pH, 7.4 containing 4 mM magnesium chloride was placed in a 4-sided clear plastic cuvette and incubated for 3 minutes. Uridine 5'-diphosphoglucuronic acid (1.5 mM) was then added to start the reaction. The formation of 1-naphthyl-β-D-glucuronide was continuously monitored for 5 minutes at 37°C at an excitation wavelength of 293 nm and an emission wavelength at 335 nm using a Perkin-Elmer LS-50 Fluorometer with the slits set at 5 nm. The UGT activity was calculated using a standard curve based on the fluorescence of a series of 1-naphthol-β-glucuronide standard concentrations. The UDP-glucuronosyl transferase activity was expressed as nmol 1-naphthyl-β-D-glucurconide formed/mg microsomal protein/min. Assays were performed in duplicate.

Statistical Analysis
Numerical data were evaluated for statistical significance at the p < 0.05 level. Both individual comparison test statistics to control, Student’s t-test, and a multiple comparison statistic to control, Dunnett’s test, were used (JMP, SAS, Cary, NC).

	Results

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All rats survived to the end of the study or their appointed termination. There were no significant differences in bodyweight gain across the treatments (data not shown). Livers tended to be larger after treatment with the high doses of the 3 conazoles than controls, although not statistically significant (Table 2). There were no macroscopic alterations present at necropsy. The major histologic alteration in the liver was hepatocyte hypertrophy (Table 2). The hypertrophy was more prominent after triadimefon treatment where there was both dose- and time-dependence to the incidence and severity of treatment. Consistent with the hepatocyte hypertrophy and greater hepatic size, liver from rats treated with the high doses of the conazoles also had increased hepatocyte cell proliferation, which was back to normal rates after 30 days in triadimefon and propiconazole treated rats and by 90 days after any of the treatments (Table 3).

View this table: [in this window] [in a new window]   Table 4 Time course of the effects of myclobutanil, propiconazole and triadimefon on AROD activities in rat hepatic microsomes.

View larger version (24K): [in this window] [in a new window]   Figure 1 Time-dependent increase of cytochrome P450 enzyme metabolizing activity induction after treatment with the high dose of each conazole as measured by the pentoxyresorufin O-dealkylation (PROD) assay.

View larger version (54K): [in this window] [in a new window]   Figure 2 Time- and treatment-dependent increased uridine diphosphoglucuronosyltransferase (UDPGT) metabolizing enzyme activity in the liver.

	Discussion

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The liver response and induction of metabolizing enzymes in rats from the present study has been reported for propiconazole in rats and quail (Ronis and Badger, 1995). Propiconazole increased liver/body weight ratios in rats and quail along with a 3–4-fold increase in P450 content and increased EROD, PROD, MROD, BROD activities, and lauric acid and testosterone metabolism in quail (Ronis et al., 1994; Ronis and Badger, 1995; Walker, 1998). In addition to the P450 enzyme induction, UDPGT was uniformly induced. Various classes of UDPGT may have overlapping substrate specificity for T4 such that a large number of microsomal enzyme inducers can also induce UDPGT and therefore potentially increase the excretion of T4 (Saito et al., 1991; Barter and Klaassen, 1992a, 1992b; Barter and Klaassen, 1994).

In general, there is good correlation between hepatic UDPGT activity and reduction of serum T4 levels suggesting that increased hepatic UDPGT activity is responsible for T4 reduction (Liu et al., 1995). T4 conjugation with glucuronic acid is the rate-limiting step in T4 biliary excretion. However, T4 is not a specific substrate for a particular class of UDPGT, and various classes of UDPGT may have overlapping substrate specificity for T4 such that many microsomal enzyme inducers would increase the excretion of T4 (Saito et al., 1991; Barter and Klaassen, 1992a, 1992b; Barter and Klaassen, 1994). Although T4 metabolism is not specific to a particular UDPGT, some UDPGT isoforms tend to preferentially metabolize T3 or T4. It is reported that T3 and T4 are glucuronidated by different UGT enzymes (Beetstra et al., 1991), T4 by 1A and T3 by 2B. Bilirubin UGT (UGT1A1) and phenol UGT (UGT1A6) have been shown to glucuronidate T4 while androsterone UGT (UGT2B2) glucuronidates T3 (Vansell and Klaassen, 2002). Therefore, induction of UDPGT could account for the general decrease in circulating T4 and T3 in the present study.

Various antithyroid effects have been shown to stimulate tumor development in the rat thyroid gland including inhibition of iodide transport, inhibition of thyroid peroxidase, direct cellular toxicity, inhibition of T4 release, inhibition of conversion of T3, and enhanced metabolism of T4 (Hurley et al., 1998). A downstream effect of all these alterations is increased circulating TSH. In a study comparing pyrethrins and Phenobarbital, both compounds induce a time- and dose-dependent increase of thyroid gland weight, follicular cell hypertrophy, decrease of T3 and T4 and increase of hepatic UDPGT beginning after 7 days of treatment and increase follicular cell proliferation and TSH after 14 days of treatment (Finch et al., 2006).

In the present study the evidence suggests that these potential alterations do not result in sufficient thyroid disruption to result in an increase in TSH. The thyroid gland histopathology is mild and transient and not consistent with what has been reported for iodine uptake inhibitors nor pyrethrin or phenobarbital (Hooth et al., 2001; Khan et al., 2005; Finch et al., 2006). There is no histologic evidence of direct thyroid follicular toxicity. Adequate colloid after 90 days suggests that there is no substantial inhibition of thyroid peroxidase. The data from the present study suggest that an alternate pathway, not requiring persistently elevated TSH, is active in thyroid gland follicular tumor development associated with triadimefon exposure. This suggests a pathway very different from what has been proposed and what has been demonstrated for phenobarbital (Finch et al., 2006).

Although TSH is considered to be the main growth factor for thyroid cells, other growth regulators that can influence thyroid follicular cell proliferation have been identified. These additional growth regulators include insulin like growth factor I (IGF1), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and transforming growth factor-β (TGFβ) (Hard, 1998). Typically high circulating TSH and prolonged cAMP stimulation within the follicular epithelium are necessary to induce cell proliferation (McClain, 1995; Williams, 1995; Hard, 1998; Hill et al., 1998). However, follicular cell growth and proliferation can also be induced through the hormone-receptor by phosphorylation of a tyrosine on the receptor via a tyrosine protein kinase pathway. Receptors for EGF and IGF-1 have tyrosine kinase activity and IGF-1 is necessary for TSH stimulation of follicular proliferation (Hard, 1998, Hill et al., 1998). EGF is synthesized in the thyroid gland and can induce follicular cell proliferation.

Thyroid tumors in humans have been shown to have increased levels of IGF1 and bFGF expression increases during thyroid hyperplasia in the rat (Hard, 1998). Along with induction of these potential positive regulators of growth inhibition of the negative growth regulator of follicular cell proliferation, decreased TGF-β expression could also result in follicular cell proliferation and ultimately proliferative lesions (Hard, 1998). Since, in the present study, there is no increase in circulating TSH in triadimefon exposed rats even though there is an increase in follicular cell proliferation, it is likely that one or more of these alternative mechanisms could be operative in the induction of thyroid follicular cell tumors.

The present study showed that altered metabolism in the liver is a common response to all the conazoles studied and related to the development of thyroid hormone disruption. These data suggest that thyroid tumors induced by triadimefon likely develop by a mode of action that is not consistent with excess circulating TSH. An alternative toxicity pathway likely contributes to thyroid tumor development in rats treated with triadimefon that was not definable using traditional toxicology approaches. In order to identify potential initiating key events driving the toxic responses from exposure to these conazoles, an alternative approach of transcriptional profiling was utilized (Hester et al., 2006). The examination of transcriptional profiles of tissues after exposure to these pesticides should enhance our ability to differentiate between treatments and describe the toxicity pathways that result in the adverse health effect of concern.

	Acknowledgments

 
The authors would like to thank Drs. Kevin Crofton and Suzanne Fenton for helpful review. We would also like to thank the U. S. Triazole Task Force for providing and analyzing the treated feeds. This manuscript does not necessarily reflect opinions or policy of the U.S. EPA nor does mention of trade names constitute endorsement.

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Toxicologic Pathology, Vol. 34, No. 7, 895-902 (2006)
DOI: 10.1080/01926230601047808


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