Tioconazole

Azole compounds play a key role as antifungals in agriculture and in human mycoses and as nonsteroidal antiestrogens in the treatment of estrogen-responsive breast tumors in postmenopausal women. This broad use of azoles is based on their inhibition of certain pathways of steroidogenesis by high-affinity binding to the enzymes sterol 14[alpha]-demethylase and aromatase. Sterol 14[alpha]-demethytase is crucial for the production of meiosis-activating sterols, which recently were shown to modulate germ cell development in both sexes of mammals. Aromatase is responsible for the physiologic balance of androgens and estrogens. At high doses, azole fungicides and other azole compounds affect reproductive organs, fertility, and development in several species. These effects may be explained by inhibition of sterol 14[alpha]-demethylase and/or aromatase. In fact, several azole compounds were shown to inhibit these enzymes in vitro, and there is also strong evidence for inhibiting activity in vivo. Furthermore, the specificity of the enzyme inhibition of several of these compounds is poor, both with respect to fungal versus nonfungal sterol 14[alpha]-demethylases and versus other P450 enzymes including aromatase. To our knowledge, this is the first review on sterol 14[alpha]-demethylase and aromatase as common targets of azole compounds and the consequence for steroidogenesis. We conclude that many azole compounds developed as inhibitors of fungal sterol 14[alpha]-demethylase are inhibitors also of mammalian sterol 14[alpha]-demethylase and mammalian aromatase with unknown potencies. For human health risk assessment, data on comparative potencies of azole fungicides to fungal and human enzymes are needed. Key words: aromatase, GYP19, CYP51, endocrine disruption, follicle fluid meiosis-activating sterol (FF-MAS), lanosterol, meiosis-activating sterols, sterol 14[alpha]-demethylase, testis meiosis-activating sterol (T-MAS).

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Azole fungicides show a broad antifungal activity and are used either to prevent fungal infections or to cure an infection. Therefore, they are important tools in integrated agricultural production. According to their chemical structure, azole compounds are classified into triazoles and imidazoles; however, their antifungal activity is due to the same molecular mechanism. The cell membrane assembly of fungi and yeast is disturbed by blocking the synthesis of the essential membrane component ergosterol. This fundamental biochemical mechanism is the basis for the use of azole fungicides in agriculture and in human and veterinary antimycotic therapies. The enzyme involved is sterol 14[alpha]-demethylase, which is found in several phyla. In mammals, it converts lanosterol into the meiosis-activating sterols (MAS). These precursors of cholesterol have been recently discovered to modulate the development of male and female germ cells.

Aromatase is another target enzyme of azole compounds. In steroidogenesis, it converts androgens into the corresponding estrogens. The importance of androgens and estrogens for the development of reproductive organs, for fertility, and in certain sex steroid-dependent diseases is well known. Therefore, azole compounds can be directed against aromatase to treat estrogen-responsive diseases.

The broad use of biologically active compounds in human therapy as well as in nonhuman applications may involve some risks, as exemplified by emerging antibiotic resistance. In agriculture, fungi and yeast are well known to develop resistance to azoles, and some molecular mechanisms of resistance development have been described (Joseph-Horne and Hollomon 1997). The significance of the agricultural azole resistance for human clinical antimycotic therapies has been recently discussed in Europe, but is not clarified yet (Hof 2001).

The antifungal properties of azoles and the issue of azole resistance in agriculture and medicine, however, are not the topics of this review. Our focus is rather on the increasing evidence for adverse effects of high doses of azole fungicides on the mammalian steroid metabolism.

Use of Azoles

Azole fungicides in agriculture. According to the U.S. Environmental Protection Agency (EPA), in 1997 approximately 244,000 and 37,000 tons of fungicides were sold worldwide and in the United States, respectively (U.S. EPA 1999). Unfortunately, precise data on the proportion of azole fungicides are not available. In Switzerland, nearly 40 tons of azoles are sold per year, which is approximately 5% of active ingredients in fungicides.

Maximum residue limits (MRLs) of pesticides approved in Switzerland are listed in the Swiss Ordinance on Foreign Substances and Toxic Components in Foodstuffs (Federal Authorities of the Swiss Confederation 2000). Within the group of the azole fungicides, currently 16 triazoles (bitertanol, cyproconazole, difenoconazole, epoxiconazole, fluquinconazole, flusilazole, flutriafol, hexaconazole, metconazole, myclobutanil, penconazole, propiconazole, tebuconazole, triadimefon, triadimenol, triticonazole) and three imidazoles (imazalil, prochloraz, and triflumizole) are listed. Additionally, there are triazoles with nonfungicidal applications (e.g., azocyclotin is used as an acaricide, paclobutrazole as a growth regulator, carfentrazone as a herbicide, and isazophos as an insecticide). An imidazole moiety is also found in the chloroacetanilid herbicide metazachlor.

Identity, chemical structure and physicochemical and other properties of azole fungicides, as well as their metabolic pathways in animals, plants and soils, are described elsewhere (Roberts and Hutson 1999; Tomlin 1997).

Azoles in treatment of human diseases. In the treatment of systemic and dermal mycoses, azoles play a pivotal role (Bodey 1992; Georgopapadakou 1998). They show significantly fewer side effects in comparison with other antimycotics such as amphotericin B, they can be applied after the emergence of resistance to other antimycotics (Espinel-Ingroff 1997), and they are inexpensive.

Individual azole compounds are found in many different antimycotic formulations. In Switzerland, three triazoles (terconazole, itraconazole, fluconazole) and eight imidazoles (clotrimazole, miconazole, econazole, ketoconazole, tioconazole, isoconazole, oxiconazole, and fenticonazole) are in use (Documed AG 2002).

Another significant application of azoles is the management of advanced estrogen-responsive breast tumors in postmenopausal women (Murray 2001; Santen and Harvey 1999). In these women, the estrogen levels dramatically decrease due to the lack of synthesis in the ovaries. At this time point, the adipose tissue becomes the main site of estrogen production. In patients suffering from breast cancer, the tumor and the surrounding tissues express aromatase and hence produce estrogen as well (Santner et al. 1997). This local production can substantially support the growth of estrogen-responsive tumors, and treatment with azole compounds (letrozole, anastrozole, vorozole, or fadrozole) suppresses estrogen production dramatically in these patients. Consequently, tumor growth can be blocked, and sometimes even remission occurs (Bhatnagar et al. 2001; Bisagni et al. 1996; de Jong et al. 1997; Dixon et al. 2000). In Switzerland, currently the two triazoles letrozole and anastrozole are in use at recommended doses of 2.5 mg/day or 1 mg/day, respectively (Documed AG 2002), partially replacing the classical nonsteroidal antiestrogen tamoxifen.

In a clinical trial, anastrozole was also effective in the treatment of estrogen-dependent endometriosis (Takayama et al. 1998). Furthermore, letrozole was used to treat boys with delayed puberty and short stature to induce growth (Wickman et al. 2001). The rationale behind this treatment is that azoles inhibit estrogen-dependent bone maturation. This leads to a delayed closure of the epiphysial growth plates and thereby allows growth.

These examples demonstrate the increasing importance of azole compounds in sex-steroid hormone-dependent diseases.

Target Enzymes of Azoles in Steroidogenesis

In this section we review the molecular characteristics and the involvement in steroidogenesis of both the sterol 14[alpha]-demethylase (encoded by the CYP51 gene) and the aromatase (encoded by the CYP19 gene), and we summarize the results of inhibition studies with azole compounds.

Sterol 14[alpha]-demethylase. Sterol 14[alpha]-demethylase is a member of the superfamily of heme-containing cytochrome P450 enzymes involved in metabolism of endogenous and xenobiotic substances. The antifungal effect of azoles is due to inhibition of sterol 14[alpha]-demethylase in fungi and yeast, thereby blocking the biosynthesis of ergosterol (Espinel-Ingroff 1997; Georgopapadakou 1998; Joseph-Horne and Hollomon 1997). The subsequent lack of ergosterol is detrimental because ergosterol is an essential sterol component in the membranes of fungi and yeast.

Sterol 14[alpha]-demethylase is not only expressed in fungi and yeast but is also found in many other species ranging from bacteria to mammals. The DNA sequences encoding sterol 14[alpha]-demethylase of many fungi and yeast are known, as well as the sequences of mice, rats, pigs, and humans (Debeljak et al. 2000; Kojima et al. 2000; Nitahara et al. 1999; Stromstedt et al. 1996). On the protein level, the amino acid sequences are highly conserved along the phylogenetic tree. This fact is considered by many authors as an indication of the pivotal role of sterol 14[alpha]-demethylase in all organisms. The homology of the amino acid sequence level between rats and humans is 93% and 40% between fungi and humans (Stromstedt et al. 1996). In humans, the sterol 14[alpha]-demethylase is expressed in many different tissues (Raucy et al. 1991).

The common linear precursor squalene is converted to the basic sterol structures in fungi, yeast, plants, and animals by complex reactions, including cyclizations. These sterols are the substrates of the sterol 14[alpha]-demethylase (Figure 1). In all species, the sterol 14[alpha]-demethylase oxidatively demethylates C-14 of these basic sterols. The reaction has been studied in detail and it was shown to closely resemble the demethylation reaction at C-10 catalyzed by aromatase (Shyadehi et al. 1996). In fungi, the sterol 14[alpha]-demethylase reaction leads to an important precursor of ergosterol. In plants, the sterol 14[alpha]-demethylase reaction metabolizes obtusifoliol and provides precursors for biosynthesis of phytosterols. In animals, the sterol 14[alpha]-demethylase reaction is part of the metabolic pathway leading to biosynthesis of cholesterol. Cholesterol in turn is the substrate for the production of many other sterols (e.g., the sex steroid hormones). In more detail, C-14 demethylation of lanosterol in animals produces the follicle fluid meiosis-activating sterol (FF-MAS), which is further metabolized to the testis meiosis-activating sterol (T-MAS). Otherwise, if lanosterol is reduced by the sterol [DELTA]24-reductase to 24,25-dihydrolanosterol before demethylation, the resulting metabolites are MAS-412 and MAS-414, respectively (Figure 2; Byskov et al. 1995).

[FIGURE 1 OMITTED]

Although the specific inhibition of fungal sterol 14[alpha]-demethylase by competitive, reversible binding to the heme moiety of the enzyme is the intended effect of azoles in agriculture and clinical antifungal treatments (Ji et al. 2000; Podust et al. 2001; Yoshida and Aoyama 1987), some azole compounds are not very specific in this respect. Clotrimazole, miconazole, sulconazole, tioconazole, and ketoconazole were shown to inhibit many different P450 enzymes (Zhang et al. 2002).

The inhibition of sterol 14[alpha]-demethylase by azoles, expressed as the half maximal inhibitory concentration (I[C.sub.50]), depends on the azole derivative and to a lesser extent on the source of sterol 14[alpha]-demethylase (Van den Bossche et al. 1987). For example, the I[C.sub.50] values for triadimenol and tebuconazole on the sterol 14[alpha]-demethylase prepared from the phytopathogen fungus Ustilago maydis are 0.07 and 0.05 nM, respectively, whereas for sterol 14[alpha]-demethylase prepared from the plant Sorghum bicolor, the I[C.sub.50] values are only slightly higher (0.24 and 0.16 nM, respectively; Lamb et al. 2001). Ketoconazole and itraconazole, respectively, exhibited I[C.sub.50] values of 60 and 80 nM on Candida albicans sterol 14[alpha]-demethylase and of 580 and 610 nM on human sterol 14[alpha]-demethylases, both enzymes heterologously expressed in yeast (Lamb et al. 1999). For ketoconazole, itraconazole, and fluconazole, I[C.sub.50] values of 180 nM, 330 nM, and 170 [micro]M, respectively, were reported for rat sterol 14[alpha]-demethylase expressed in Escherichia coli (Nitahara et al. 1999). In crude extracts of yeast, the sterol 14[alpha]-demethylase is inhibited by flusilazole approximately 100 times more efficiently than in crude cell-free extracts from rat liver (Trzaskos and Henry 1989). This difference was nearly abolished upon further purification of the enzymes. According to the authors, the differences in the crude extracts are due to different contents in other azole-susceptible P450 enzymes that may scavenge the azoles.

In conclusion, the direct comparison of the I[C.sub.50] values from different studies is hampered by considerable variations in the study designs and test materials used, such as enzyme purity.

Aromatase. Another important P450 enzyme involved in the steroidogenesis is aromatase. Like sterol 14[alpha]-demethylase, aromatase catalyzes the oxidative demethylation of sterols. In contrast to sterol 14[alpha]-demethylase, which has several substrates in different phyla, aromatase demethylates C10 and specifically converts androstenedione and testosterone, resulting in estrone and estradiol, respectively (Figure 1). However, other nonsteroid substrates of the aromatase, such as 7-ethoxycoumarin are also known (Toma et al. 1996). The aromatase coding DNA sequences of several animals (Hickey et al. 1990; McPhaul et al. 1988; Tanaka et al. 1992; Trant 1994) and humans (Evans et al. 1986; Means et al. 1989; Toda and Shizuta 1993) have been published. On the protein level, the amino acid sequence homology between aromatase from fish and humans is about 50% (Trant 1994) and between rats and humans is about 78% (Hickey et al. 1990). in mammals, aromatase is mainly expressed in the brain and the gonads, but it is also found in placental, adipose, and bone tissue (Conley and Hinshelwood 2001).

The physiologic balance between different sex steroid hormones is crucial for the development, maintenance, and function of the reproductive system as well as for the differentiation of the sexual phenotype during ontogeny. Estrogens (estrone and estradiol) are products of the androgens (androstenedione and testosterone), and the reaction is catalyzed by aromatase. In mammals, differentiation of the male phenotype depends not only on testosterone but also on estradiol generated from testosterone by neuronal aromatase in central nervous system. Therefore, disturbances in aromatase expression and/or changes in its catalytic activity are expected to exhibit negative effects on reproduction parameters. In fact, in transgenic male mice overexpressing aromatase in the testis, the serum estradiol level is increased (Fowler et al. 2000). Half of these animals were infertile and had larger testes and a significantly increased incidence of Leydig cell tumors in testes. In male and female aromatase knockout mice (ArKO), the estradiol levels are below detection limits in both sexes. In males, the testosterone levels are elevated in the first 12-14 weeks only, and female ArKO mice showed a pronounced increase in testosterone levels (Fisher et al. 1998; Robertson et al. 1999).

Aromatase can be inhibited competitively and reversibly by azole compounds, as seen with sterol 14[alpha]-demethylase. The I[C.sub.50] values of the azole fungicides prochloraz, imazalil, propiconazole, triadimenol, and triadimefon on microsomal aromatase from human placenta were 0.04, 0.34, 6.5, 21, and 32 [micro]M, respectively (Andersen et al. 2002; Vinggaard et al. 2000). In a rat ovary aromatase assay, the therapeutic drugs anastrozole, fadrozole, and letrozole exhibited I[C.sub.50] of 0.025, 0.007 and 0.007 [micro]M, respectively (Odum and Ashby 2002). Not only azoles but also natural plant constituents such as flavonoids are able to significantly inhibit the aromatase (Jeong et al. 1999).

Biological Effects of Azole Compounds

Based on the inhibitory activity of azoles on key enzymes involved in sex steroid hormone synthesis, it is likely that effects on fertility, sexual behavior, and reproductive organ development will occur depending on dose level and duration of treatment of laboratory animals. Therefore, we reviewed the toxicology monographs of the Joint FAO/WHO (Food and Agriculture Organization/World Health Organization) Meeting on Pesticide Residues (JMPR) on some azole fungicides by focusing on effects that might be induced by disturbed steroidogenesis (Appendix).

Role of sterol 14[alpha]-demethylase in germ cell development. As discussed above, in mammals, sterol 14[alpha]-demethylase either directly converts lanosterol into FF-MAS or, after preceding reduction of lanosterol by sterol [DELTA]24-reductase into MAS-412. Sterol [DELTA]14-reductase then converts FF-MAS in T-MAS and MAS-412 in MAS-414 (Figure 2). These metabolites of lanosterol have been regarded only as precursors of cholesterol without any biological function in animals. This view dramatically changed recently with the observation that FF-MAS isolated from human follicle fluid and T-MAS isolated from bull testis as well as the MAS-412 and MAS-414 induced resumption of meiosis in cultivated mouse oocytes (Byskov et al. 1995).

[FIGURE 2 OMITTED]

In rats, expression of sterol 14[alpha]-demethylase in postmeiotic spermatids is significantly higher than in premeiotic spermatids (Stromstedt et al. 1998) and is restricted to discrete stages of the spermatid development (Majdic et al. 2000). In the developing rat testis, high levels of T-MAS were found. These increased T-MAS levels were due to the selective up-regulated expression of cholestogenic genes, namely the pre-MAS genes such as sterol 14[alpha]-demethylase, and down-regulated post-MAS genes (Tacer et al. 2002). In the human hepatoma cell line HepG2 and in rat testes, progestins inhibited the cholesterol biosynthesis, and consequently the levels of FF-MAS and T-MAS were increased (Lindenthal et al. 2001). In mouse oocytes, in vitro resumption of meiosis was induced by the specific blocking of the sterol [DELTA]14-reductase, and hence the FF-MAS level was elevated (Leonardsen et al. 2000). In another study, only denuded oocytes responded to higher FF-MAS levels but not cumulus-cell-enclosed oocytes (Downs et al. 2001). Resumption of meiosis in vitro by increased FF-MAS levels was also observed in human oocytes (Cavilla et al. 2001). The biological significance of the MAS and their implications on fertilization were recently reviewed (Byskov et al. 2002; Rozman et al. 2002). However, other studies failed to show these pronounced effects of MAS on oocytes (Tsafriri et al. 1998, 2002; Vaknin et al. 2001).

Although the detailed function of the MAS is not fully understood yet, the existing studies indicate an important role of these sterols in the development of testis and spermatids and possibly also an impact on the oocyte development.

Ejects induced by aromatase inhibition. Azole fungicides described in the JMPR monographs (see Appendix) and azole compounds used in the management of breast cancer share some common effects in vitro and in vivo.

The potential of azoles to lower estradiol levels and to delay ovulation in rats has been known for several years as is exemplified by an isomer of flutriafol (Middleton et al. 1986; Milne et al. 1987).

Although on the molecular level (e.g., I[C.sub.50] values) no profound species differences are seen, distinct effects are observed in laboratory animals after azole treatment. In dogs, exposure to letrozole induced histopathologic changes in the testis, whereas in rats no such effects were observed (Walker and Nogues 1994). In female rats, letrozole (Sinha et al. 1998) and vorozole (De Coster et al. 1990) considerably lowered estrogen levels and reduced uterus weights. In a mouse MCF-7 xenograft model, tumor growth was effectively inhibited by letrozole (Lee et al. 1995). The same effect on rat mammary tumor growth was seen with letrozole and a derivative thereof, in addition to a prominent reduction of estradiol levels and uterus weights (Schieweck et al. 1993). In male cynomolgus monkeys, vorozole lowered the estradiol levels (Tuman et al. 1991) and in quails (Foidart et al. 1994) and in female musk shrews (Rissman et al. 1996) it changed sexual behavior. The treatment of growing male rats with vorozole impaired skeletal development and the mineralization of bones (Vanderschueren et al. 1997).

In European pond turtles (Belaid et al. 2001) and in whiptail lizards (Wennstrom and Crews 1995), letrozole affected sex determination, as did fadrozole in chickens and turkeys (Burke and Henry 1999). Juvenile sexually undifferentiated fathead minnows were shown to masculinize when exposed to fadrozole in combination with methyltestosterone (Zerulla et al. 2002). In another study on fathead minnows exposed to fadrozole, reduced brain aromatase activity was accompanied by reduced estradiol and vitellogenin levels, and a decrease in mature oocytes and an increase in sperms in testes was observed (Ankley et al. 2002). In nonhuman primates, it was shown that fadrozole, letrozole, and a derivative of letrozole inhibited the aromatase (Shetty et al. 1997, 1998). Although in female animals the estrogen levels were significantly decreased, the development of follicles seemed normal but the animals did not become pregnant. In the males, the treatment resulted in elevated testosterone levels, and the production of spermatocytes was significantly reduced within the first 30 days of treatment. In contrast, within the first 90 or 120 days of treatment, in ArKO mice the spermatogenesis seemed normal and the spermatozoa in vitro were as fertile as the wild-type spermatozoa (Fisher et al. 1998; Robertson et al. 1999). Yet there were fewer litters with these males. The authors suggest alterations in mounting behavior as being the reason for this discrepancy (Robertson et al. 2001). Additionally, elevated prostate weights without malignant changes were observed in ArKO male mice (McPherson et al. 2001).

Many of the effects described can be seen as the result of a disturbed balance of androgens and estrogens, provoked either by chemically blocking aromatase or by the lack of a functional aromatase in the case of knockout animals. In animal studies to investigate the chemical blockage of the aromatase by azoles, it cannot be excluded that the sterol 14[alpha]-demethylase also was blocked. Interpretations of these results therefore should include not only changed androgen and estrogen levels but also a disturbed balance of MAS levels.

Summary

Several azole compounds were shown to inhibit the aromatase and to disturb the balance of androgens and estrogens in vivo. In fact, the clinical use of azole compounds in estrogen-dependent diseases is based on this effect. Additionally, azole antifungals developed to inhibit the sterol 14[alpha]-demethylase of fungi and yeast in agriculture and medicine are also inhibiting aromatase. Therefore, these antifungals may unintentionally disturb the balance of androgens and estrogens. Until now, it is not clear whether this effect is compensated by an increased expression of aromatase or by other unknown mechanisms.

The actual target enzyme of azole antifungals, the fungal sterol 14[alpha]-demethylase, is expressed in many species including humans, and it is highly conserved through evolution. Hence, it seems reasonable to assume that most of the azole antifungals used in agriculture and medicine as well as azoles used in management of breast cancer also act as inhibitors on human sterol 14[alpha]-demethylase to an unknown extent. Unfortunately, there are few studies on the inhibition of mammalian sterol 14[alpha]-demethylases by azole compounds.

The impact of inhibition of the mamalian sterol 14[alpha]-demethylase on the cholesterol pool is not clear. Because dietary cholesterol may compensate disturbances in cholesterol levels, the cholesterol pool might be not a very sensitive parameter for sterol 14[alpha]-demethylase inhibition. However, spermatogenesis and oogenesis are in part controlled by MAS, and the conversion of lanosterol to MAS exclusively relies on sterol 14[alpha]-demethylase activity. The data available provide good evidence that azoles directed against the ergosterol biosynthesis of fungi and yeast have the potential to influence the levels of FF-MAS, T-MAS, androgens, and estrogens in animals and humans.

To our knowledge, structure-activity relationships of azoles with respect to their ability to discriminate between sterol 14[alpha]-demethylase and aromatase have hardly been investigated. There is also a lack of data on enzyme regulation of sterol 14[alpha]-demethylase and aromatase after azole exposure and on the putative impact of dietary cholesterol. Inhibition of other P450 enzymes by azoles is even less investigated.

The toxicologic profiles of individual azole fungicides, as summarized in the Appendix, provide evidence for endocrine effects. In fact, many of these fungicides have effects on prostate, testis, uterus, and ovaries as well as on fertility, development, and sexual behavior. The current database does not allow us to establish causal relationships of these effects with inhibition of sterol 14[alpha]-demethylase and/or aromatase, but the overall view strongly suggests a connection with disturbed steroidogenesis.

Based on the clear effects on steroidogenic enzymes in vitro, animal studies should be performed with concomitant analysis of androgen, estrogen and MAS levels and investigations on inhibition of the fungal sterol 14[alpha]-demethylase, mammalian sterol 14[alpha]-demethylases, and aromatase are clearly necessary.

The Endocrine Disrupter Screening and Testing Advisory Committee (EDSTAC) of the U.S. EPA has recommended the aromatase inhibition assay as one of the possible testing system to identify endocrine disrupters (EDSTAC 1998). Since the publication of the EDSTAC report in 1998, recent studies on the impact of MAS on spermatogenesis and oogenesis have been published and, as a consequence, sterol 14[alpha]-demethylase should also be considered as a candidate testing system for identifying endocrine disrupters.

Exposure estimates based on residue values from supervised field trials are performed by JMPR. These estimates indicate that the average daily intake of individual azole fungicides is approaching at most a very low percentage of the acceptable daily intake (ADI) established by the JMPR. According to residue data from food random sampling, the MRLs are hardly ever exceeded. Nevertheless, several azole fungicides are found in the candidate list of putative endocrine disrupters within the European Union Community strategy for endocrine disrupters (European Commission DG ENV 2002).

In conclusion, azole fungicides interact with several P450 enzymes in different species and have the potential to affect the endocrine system by interacting with steroidogenesis. Due to the knowledge of the common mechanism on steroidogenesis in target organisms and mammalians, we suggest that azole fungicides should be considered as a group when MRLs are allocated. It will be the challenge to design more specific inhibitors of fungal sterol 14[alpha]-demethylases that do not affect animal sterol 14[alpha]-demethylases and aromatases.

Appendix

Usually, the hazard assessments of pesticides by the JMPR are based on data of in vitro and animal studies providing no observable effect levels (NOEL) for delineation of individual ADIs in humans. Based on the ADI and residue levels in plants after application of recommended use levels, MRLs are allocated to individual foods by the authorities.

In vivo studies of some azole fungicides are summarized below with special focus on effects putatively connected to disturbed steroidogenesis. In the allocation procedure for an ADI by the JMPR, these effects are included.

Bitertanol was evaluated by the JMPR in 1998 (FAO/WHO 1999). An ADI of 0-0.01 rog/kg body weight (bw) was allocated based on a NOEL of 1 mg/kg bw in a three-generation rat study and reduced pup survival rates at 5 mg/kg bw. Histopathologic changes in the adrenal glands of dogs and rats were seen at 1.2 and [greater than or equal to] 81 mg/kg bw, respectively. In dogs, at [greater than or equal to] 5 mg/kg bw, cataracts and reduced prostate weight with histopathologic changes were seen. In male rats, at [greater than or equal to] 300 mg/kg bw, the relative testis weights were increased, and in females, the absolute ovary and adrenal weights were decreased with histopathologic changes. At the maternotoxic level of 100 mg/kg bw, rat fetuses showed effects such as cleft palate and hydrocephalus.

Cyproconazole induced cleft palate, hydrocephalus, and hydronephroses in rat embryos after treatment of the dams with 20 mg/kg bw (the lowest dose tested); [greater than or equal to] 50 mg/kg bw increased incidences of resorptions and dead fetuses were observed (Machera 1995).

Flusilazole was evaluated by the JMPR in 1995 (FAO/WHO 1996). An ADI of 0-0.001 mg/kg bw was allocated based on a NOEL of 0.14 mg/kg bw in a 1-year dog study. At [greater than or equal to] 0.7 mg/kg bw, effects on the liver of the dogs were observed. In a 14-day rat study, the levels of testosterone and estradiol were reduced at [greater than or equal to] 20 mg/kg bw. In isolated Leydig cells, the I[C.sub.50] for testosterone production was 3.5 [micro]M for flusilazole and 1 [micro]M for ketoconazole. In a 2-year rat study, 31 mg/kg bw flusilazole induced Leydig cell tumors of the testis; flusilazole was fetotoxic and embryotoxic at [greater than or equal to] 9 mg/kg bw in a developmental study. Toxicity to the dams was observed at [greater than or equal to] 27 mg/kg bw. In in vitro teratogenicity assays with rat embryos, changes at the branchial apparatus were seen at 6.25 [micro]M (Menegola et al. 2001).

Hexaconazole was evaluated by the JMPR in 1990 (FAO/WHO 1991). An ADI of 0-0.005 mg/kg bw was allocated based on a NOEL of 0.47 mg/kg bw in a 2-year rat study. At 4.7 [greater than or equal to] mg/kg bw an increased incidence of Leydig cell tumors in testis was observed. In a 90-day rat study, histopathologic changes were observed in the adrenal glands at [greater than or equal to] 2.5 mg/kg bw, and reduced testis and adrenal weights were observed at 250 mg/kg bw. In rats, hexaconazole was fetotoxic at [greater than or equal to] 25 mg/kg bw. Isolated rat Leydig cells showed reduced testosterone and increased progesterone production on exposure to hexaconazole (0.1-30 [micro]M).

In a 29-day mouse study, lack of corpora lutea and smaller ovaries were observed in females and histopathologic changes in the testis and epididymis and enlarged adrenals were observed in males at [greater than or equal to] 14 mg/kg bw. In a 90-day dog study, weights of ovaries and testes were reduced at [greater than or equal to] 125 mg/kg bw.

Imazalil was evaluated by the JMPR in 2000 (FAO/WHO 2001). In a 1-year dog study, an ADI of 0-0.03 mg/kg bw was allocated based on a NOEL of 2.5 mg/kg bw and effects on body weight, the liver, and clinical symptoms at 20 mg/kg bw. At a dose range of 5-20 mg/kg bw imazalil was fetotoxic in rats, mice, and rabbits.

Myclobutanil was evaluated by the JMPR in 1992 (FAO/WHO 1993a). An ADI of 0-0.03 mg/kg bw was allocated based on a NOEL of 2.5 mg/kg bw in a 2-year rat study and many effects on the male reproductive system at [greater than or equal to] 10 mg/kg bw. Reduced testis weight, testis atrophy, reduced or absent spermatid production, necrotic epididymis, and atrophy of the prostate were observed with increasing doses.

Penconazole was evaluated by the JMPR in 1992 (FAO/WHO 1993b). An ADI of 0-0.03 mg/kg bw was allocated based on a NOEL of 3 mg/kg bw and reduced testis weight with atrophic changes at [greater than or equal to] 17 mg/kg bw in a 1-year dog study. In long-term studies, increased prostate weights were observed in mice at [greater than or equal to] 9.8 mg/kg bw and in rat testis at 202 mg/kg bw. In a two-generation rat study, relative ovary weights were increased at [greater than or equal to] 125 mg/kg bw. At the maternotoxic level of 500 mg/kg bw, penconazole was also fetotoxic.

Prochloraz was evaluated by the JMPR in 2001 (FAO/WHO 2002). An ADI of 0-0.01 mg/kg bw was allocated based on a NOEL of 1.3 mg/kg bw and liver effects at [greater than or equal to] 5 mg/kg bw in a 2-year rat study. In a 90-day rat study, ovary and thyroid weights were increased in females at [greater than or equal to] 6 mg/kg bw. In a 90-day dog study, reduced testis and prostate weights were observed at [greater than or equal to] 7 mg/kg bw. In a rat reproduction study, at [greater than or equal to] 27 mg/kg bw a tendency to prolonged gestation, increased total litter losses, smaller litter sizes, and increased pup mortality was observed. In trout, 25 nM (9.4 [micro]g/L) prochloraz impaired the spermatogenesis (Le Gac et al. 2001).

Propiconazole was evaluated by the JMPR in 1987 (FAO/WHO 1988). An ADI of 0-0.04 mg/kg bw was allocated based on a NOEL of 4 mg/kg bw slight effects on the liver and hematology parameters at [greater than or equal to] 20 mg/kg bw in a 2-year rat study. In a rat reproduction study, reduced testis and epididymis weights in pups were observed at [greater than or equal to] 21 mg/kg bw. However, testis weights of rats were increased at 256 mg/kg bw in a short-term study.

Tebuconazole was evaluated by the JMPR in 1994 (FAO/WHO 1995). An ADI of 0-0.03 mg/kg bw was allocated based on a NOEL of 3 mg/kg bw and histopathologic changes in the adrenal glands and questionable cataracts at 4.5 mg/kg bw in a 1-year dog study. In a 90-day rat study, histopathologic changes in the adrenals were found at [greater than or equal to] 36 mg/kg bw. In rat reproduction studies, reduced litter sizes and reduced survival indices were observed at [greater than or equal to] 73 mg/kg bw. In a mouse teratogenicity study, increased incidences of runts and malformations such as cleft palate were observed at [greater than or equal to] 30 mg/kg bw. In rats and rabbits, malformations and embryotoxicity occurred at [greater than or equal to] 100 mg/kg bw.

Triadimefon was evaluated by the JMPR in 1981/1985 (FAO/WHO 1982, 1986). An ADI of 0-0.03 mg/kg bw was allocated based on a NOEL of 2.5 mg/kg bw and body weight effects and reduced hematopoesis at [greater than or equal to] 25 mg/kg bw in a 2-year rat study. In a rat reproduction study, the male/female sex ratio in [F.sub.2] was reduced at 77 mg/kg bw and the female fertility index was one-third of control. In a supplementing study, treated males (77 mg/kg bw) were mated with untreated females. The pregnancy rate was significantly lower, but the ratio of pregnant to inseminated females was not affected. It was concluded that triadimefon impaired the sexual behavior of the males. The testosterone level of these male rats was doubled. In in vitro teratogenicity studies with rat embryos, changes at the branchial apparatus were seen at 25 [micro]M for both triadimefon and triadimenol (Menegola et al. 2000).

Triadimenol was evaluated by the JMPR in 1989 (FAO/WHO 1990). An ADI of 0-0.05 mg/kg bw was allocated based on a NOEL of 5 mg/kg bw in a rat two-generation study and on retarded development of pups at 20 mg/kg bw. In a mouse short-term study and a dog long-term study, changes in the cholesterol levels were seen at 170 mg/kg bw and 45 mg/kg bw, respectively, and in a 2-year rat study, ovary weights were reduced at 144 mg/kg bw. In rat and rabbit teratogenicity studies, increased incidences of resorptions were observed at [greater than or equal to] 120 mg/kg bw.

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Address correspondence to J.A. Zarn, Food Toxicology Section, Swiss Federal Office of Public Health, Stauffacherstrasse 101, CH-8004 Zurich, Switzerland. Telephone: 41 43 322 21 93. Fax: 41 43 322 21 99. E-mail: juerg.zarn@bag.admin.ch

The contents of this article are solely the responsibility of the authors and do not necessarily represent the official views of the Swiss Federal Office of Public Health.

Received 22 May 2002; accepted 10 September 2002.

Jurg A. Zarn, Beat J. Bruschweiler, and Josef R. Schlatter

Food Toxicology Section, Swiss Federal Office of Public Health, Zurich, Switzerland

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