Pesticides Research No. 111 2007 – Effects of azole fungicides on the function of sex and thyroid hormones

Effects of azole fungicides on the function of sex and thyroid hormones

Summary

Background

There is a growing concern of permanent damage to the endocrine and nervous systems after exposure to even low levels of pesticides under development. Fetuses (i.e., pregnant women) and children are therefore considered as particularly vulnerable groups in relation to pesticide exposure.

The main objective of this project was to investigate the effects of three frequently used triazole fungicides, tebuconazole, propiconazole, and epoxiconazole on the endocrine system with special focus on sex and thyroid hormones. In addition, we wanted to compare the results with our previous findings for the imidazole fungicide, prochloraz, which was shown to possess endocrine disrupting effects in a number of in vitro and in vivo studies. If the triazole compounds possess similar effects as prochloraz these effects are likely to be general for the group of azole fungicides.

Azole fungicides are used in large amounts for control of fungi in grain production and to a lesser extent in the flower, vegetable and fruit production. Thus, in 2005 a total amount of 100 t of azole fungicides was used in Denmark. The fungicides are relatively fat-soluble and readily absorbed in the gastrointestinal tract. Therefore, the public is exposed to the fungicides when residues exist in food products. Residues of azole fungicides are monitored in vegetable and fruit, but only few azole fungicides are monitored in grain and grain products. For instance, the most widely used azole, epoxiconazole, is not monitored, making it impossible to do a complete dietary exposure assessment of the Danish population. In general, the azole fungicides have a low acute toxicity but the knowledge about their potential health risks at low chronic exposures is very limited. We have recently shown that the azole fungicide, prochloraz, can interact with several receptors, including the estrogen receptor (ER), androgen receptors (AR), and the aryl hydrocarbon receptor (AhR), in cell-based in vitro assays. Besides, prochloraz was anti-androgenic in adult male rats and affected the serum concentration of thyroid hormones. After exposure during pregnancy and lactation it caused feminization of male and virilization of female offspring.

The AhR is a nuclear transcription factor that, after binding of ligand, causes increased gene transcription of AhR responsive genes such as the CYP1 gene family. The classical AhR ligand is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) but also other planar aromatic polycyclic molecules activates the receptor. No natural endogenous ligand has yet been identified. AhR mediated induction of CYP1A1 and 1A2 will enhance the metabolism of estradiol to 2- and 4-hydroxy estradiol (Rifkind, 2006) and could therefore affect the hormone concentration in the organism.

In this project we have investigated whether the three actually most widely used triazole fungicides in Denmark: epoxiconazole, propiconazole, and tebuconazole, possess similar properties as the imidazole fungicide prochloraz regarding interactions with the ER, AR, and AhR, as well as effects on steroid hormone synthesis and the activity of the enzyme aromatase that converts testosterone to estrogen. The effects were investigated by established in vitro assays. Furthermore, we have established a bioassay (T-screen) for detecting binding and activation of the thyroid receptor (TR) and used the assay to investigate the ability of prochloraz, epoxiconazole, propiconazole, and tebuconazole to be thyroid hormone agonists or antagonists. Additionally, we have investigated anti-androgenic effects of propiconazole and tebuconazole in vivo in young adult rats using the Hershberger assay. Finally, developmental effects of epoxiconazole and tebuconazole were investigated in rat offspring after exposure during pregnancy and lactation.

The reason why propiconazole was replaced by epoxiconazole in the developmental study was that we, after the project was initiated, were informed that the use of epoxiconazole had increased markedly during 2004 and now was the most widely used azole fungicide within Danish agricultural production.

In vitro effects

The three azole fungicides: propiconazole, tebuconazole, and epoxiconazole all had the potential, like prochloraz, to act as endocrine disruptors via several different mechanisms although the potencies were less than for prochloraz in some of the assays.

Hence, the three triazoles were less potent than prochloraz to act as ER antagonists (prochloraz > tebuconazole » epoxiconazole » propiconazole) and AhR agonists (prochloraz >>> tebuconazole » epoxiconazole » propiconazole) while the potency as AR antagonists was similar. Like prochloraz, the triazoles were aromatase inhibitors, although again, with lower potency than prochloraz (prochloraz >epoxiconazole » tebuconazole > propiconazole).

The ability to interfere with steroid hormone biosynthesis was investigated in the human adrenocortical carcinoma cell line H295R. Like prochloraz, all three triazole fungicides caused enhanced production of progesterone and reduced production of testosterone and estradiol. This indicates that enzymes involved in the conversion of progesterone to testosterone are inhibited.

Convincing interactions with the TR were not found for either prochloraz or any of the triazole fungicides.

Anti-androgenic effects in vivo

In the Hershberger assay, used for investigating anti-androgenic effects in vivo, propiconazole and tebuconazole (50, 100, or 150 mg/kg bw/day) had no effect on neither reproductive organ weights or on hormone levels, except for the highest dose of propiconazole, for which there was a significant increase in the serum concentration of follicle stimulating hormone (FSH). The only significant effect of the azole fungicides on gene expression was a decrease in of expression of ornithine decarboxylase (ODC) mRNA in prostate at all doses of propiconazole and at the highest dose of tebuconazole. This gene is regulated by androgens but is also regulated via other pathways. Since none of the other anti-androgenic end points (weight of androgen dependent organs, expression of other androgen responsive genes or serum concentration of LH) were affected we believe that the effects observed on ODC mRNA expression are due to a non-androgen-regulated pathway.

In conclusion, none of the triazole fungicides, propiconazole or tebuconazole, had any androgen receptor blocking effect in vivo in the Hershberger test at doses at or below 150 mg/kg bw/day. This is in contrast to prochloraz that induced anti-androgenic effects in this assay at doses between 50 and 150 mg/kg bw/day.

Effects on offspring after perinatal exposure

Pregnant rats were dosed with epoxiconazole (15 or 50 mg/kg bw/day) or tebuconazole (50 or 100 mg/kg bw/day). Some dams were chosen for caesarian section at gestational day 21 (GD21) to evaluate effects on sexual differentiation in the fetuses. Other dams delivered normally and the pups were examined at birth or at post natal day (PND) 13 or 16 after exposure during lactation. The highest dose of epoxiconazole caused marked fetal lethality and problems with normal delivery. Therefore only two litters were born normally and the remaining dams in this group had caesarian sections between GD23 and GD25 and they were included in the group having caesarian sections at GD 21. Interpretation of the results from examination of the offspring from the high dose epoxiconazole group is therefore hampered and no final conclusions can be drawn.

The following effects were observed for dams, fetuses, and offspring:

Both epoxiconazole and tebuconazole caused an increased gestational length as we have also previously seen for prochloraz (30 mg/kg bw/day). This effect is probably caused by a marked increase in plasma concentrations of progesterone in the mothers induced by both fungicides.

The anogenital distance (AGD) was increased in the female pups in the highest tebuconazole dose group and the lowest epoxiconazole dose group indicating a virilizing effect on the females. No effect on AGD was seen in the newborn male pups. The testosterone level in testis from the male fetuses was decreased by tebuconazole, while progesterone and 17a-hydroxyprogesterone levels were increased at both doses. Furthermore, tebuconazole caused a significant increase in the number of nipples in the male pups and a tendency towards decreased plasma testosterone concentration in male pups. Thus, the overall picture is that tebuconazole virilizes the females and feminizes the male pups.

Epoxiconazole had an apparent growth-promoting effect on the male offspring. A higher birth weight was seen which is probably caused by the enhanced testosterone level in the epoxiconazole-dosed mothers. The testosterone may have acted as a growth promoter and may have caused the increased AGD in the fetuses and the insignificant tendency towards an increased weight of androgen dependent reproductive organs in male pups. In summary, the results indicate that the profile of effects induced by epoxiconazol on male offspring is different from the effects induced by prochloraz and tebuconazol. Epoxiconazol has marked fetotoxic effects but the mechanisms behind and the potential role of endocrine disruption for these effects cannot be ruled out from this study.

Conclusion

Overall the results from this study indicate that azole fungicides in general have a similar profile of action in vitro but that the profile of action in vivo is different and varies from chemical to chemical. The four azole fungicides all have the potential to act via several modes of actions. The potencies of the three triazoles included in this study, epoxiconazole, propiconazole, and tebuconazole, were similar or less than for the imidazole fungicide, prochloraz, in the applied in vitro assays. Propiconazole and tebuconazole did not possess androgen receptor blocking effect in vivo in the Hershberger test at doses at or below 150 mg/kg bw/day. Despite this, both epoxiconazole and tebuconazole were capable of inducing effects on reproductive development in the offspring after exposure in utero. Both epoxiconazole and tebuconazole virilized the female offspring, and tebuconazole also caused feminizing effects in male offspring. In addition epoxiconazol had marked fetotoxic properties. The common features for the tested azole fungicides are that they all increase gestational length, they virilize female pups, and they increase progesterone levels.

These effects are likely due to several different mechanisms operating simultaneously and thereby enhancing the integrated biological response. However, the effects on steroid hormone synthesis in vitro, the lack of effect in the Hershberger assay (where effects on steroid synthesis is omitted by using testosterone supplemented castrated male rats) combined with the effects on reproductive developmental after perinatal exposure strongly indicate that one of the main responsible mechanisms is disturbance of key-enzymes involved in the synthesis of steroid hormones.

The observed effects deserve further investigations in order to unveil the doses needed to induce the effects and to obtain more information on the mechanisms involved. The results from the project add new knowledge about the toxic mechanisms of azole fungicides and their potential for inducing health effects on reproduction and the endocrine system and will contribute to a better risk assessment of this widely used group of fungicides.