**1. Introduction**

As reported carbendazim (methyl-2-benzimidazole carbamate) is used to be a systemic fungicide [1]. Both carbendazim and its parent benomyl [methyl 1-(butylcarbamoyl)-2-benzimidazole carbamate] are exhibiting low acute toxicity [2]. In contrast, carbendazim and benzimidazole chemicals induced severe reproductive and developmental toxicity in rodents [3–6]. Reports showed that carbendazim and benomyl exhibited testicular toxicity such as sloughing of immature spermatids [7, 8], inhibition of microtubule assembly [9], seminiferous tubular atrophy [10], and testicular atrophy and infertility [11] in male rats. Both carbendazim and benomyl induced developmental toxicity in rodents. Prenatal treatment of carbendazim to rats during pregnancy exhibited embryonic death, growth retardation, and developmental abnormalities including exencephaly, microphthalmia and hydronephrosis in offspring [3, 12]. Treatment of benomyl to pregnant rats induced craniocerebral and systemic malformations such as cleft palate, hydrocephalus, and exencephaly in offspring of male and female rats [13]. In contrary to the more reports available on the reproductive and developmental toxicity of carbendazim and benomyl, studies for endocrine-disrupting activity or mode of action of the these two fungicides remains unclear. The earlier report on endocrine activity for carbendazim might be Rehnberg et al. (1989). They showed that administration of male rats with carbendazim raised testosterone concentration and the levels of androgen binding protein in the interstitial and seminiferous tubule fluid, meaning an association between endocrine disruption activity and carbendazim toxicity [14]. Recently Rama et al. (2014) reviewed and reported that carbendazim induced reproductive toxicity and possible hormonal effects in rats [15]. They reviewed the previous reports and generalized that carbendazim have androgenic effects acting directly in the androgen receptors and/or increasing the expression of androgen receptors. Some chemicals were reported to increase or decrease AR expression. Bisphenol A was reported to increase AR expression [16] while di-n-butyl phthalate (DBP) [17, 18] and sodium valproate [19] decrease it. We found out that it is common for estrogen receptor agonist and androgen receptor antagonist but not for androgen agonist in pesticides. Based on the chemical structure it seems to determine the AR agonist or antagonist. The degree of increase or decrease of AR expression might be depended on the chemical structure, which shared with nature ligand dihydrotestosterone. This manuscript would like to combine our unpublished data and previous studies to infer that androgen receptor plays an important role in benomyl- and carbendazim-induced reproductive and developmental toxicity and endocrine-disrupting activity in rats.

animal facility in Taiwan Agricultural Chemicals and Toxic Substances Research Institute, Taichung, Taiwan. The animal rooms were kept on a 12-h light and dark cycle, 23 ± 2°C, and 50 ± 10% relative humidity. When they were transported to animal room, the rats were quarantined for at least 1 week and opened on the bases of enough body weight gain and without clinical signs of disease of injury. Both carbendazim and benomyl with 99% pure were a gift from Sinon Co., Taichung, Taiwan. These two pesticides were suspended in polyethylene glycol 200 and treated to animals orally by gavage in a volume of 2 ml/kg body weight, once daily. In reproductive toxicity studies, male rats were administered with these two pesticides and/or flutamide for 28 days. In developmental toxicity studies, both male and female rats were administered with 200 mg/kg carbendazim or 100 mg/kg benomyl for 28 days. Then the female rats were mated with male within each treatment group for 14 days. No treatment was carried out during the mating period. Pregnant dams with plug detected were kept to deliver the offspring at term and conception rates were calculated. All rat offspring were weaned at

Androgen Receptor Plays a Vital Role in Benomyl- or Carbendazim-Induced Reproductive…

http://dx.doi.org/10.5772/intechopen.78276

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21 days postnatal and then fed up to 6 week old [20].

to Oakberg [22] and Hess [23].

(3) In vitro androgen receptor binding assay

(2) Organ and tissue weight, morphology, and histopathological examination

Both testis and epididymis were weighed by right and left sides. The incidence of abnormal morphology was recorded. The half of testis or epididymis each was fixed in 10% neutral phosphate-buffered formalin solution for subsequent histopathological examinations. Tissues of testis and epididymis were processed by standard histopathological processes and stained using haematoxylin and eosin for light-microscopic examinations. Both testis and epididymis sections were stained with the Giemsa staining periodic acid-Schiff methods and then counterstained using haematoxylin as reported in Simoes and Schoning [21]. Both testis and epididymis histology were evaluated and histopathological findings were scored according

The ligand binding assay was processed to determine the concentration of androgen receptor in rat tissue according to Nonneman et al. [24]. The ligand [1, 2, 3, 5, 6, 7-3H(N)]-5α-androstan-17β-ol-3-one (dihydrotestosterone, 5α-DHT) (110–150 Ci/mmol) was obtained from NEN Life Science Products, Inc., Boston. Nonlabelled 5α-DHT was obtained from Sigma Chemical Company, St. Louis, MO, and recrystallized from ethanol prior to use. Both rat testis and epididymis were homogenized in ice-cold low-salt TEDG buffer, pH 7.4, consisting of 10 mM Tris, 1.5 mM ethylenediaminetetraacetic acid (EDTA), 10% glycerol, and 1 mM each of dithiothreitol, phenylmethylsulfonyl fluoride, and sodium molybdate as described by Hardy et al. [25]. Tissue homogenates were centrifuged at 30,000 × g for 1 h and the supernatant were processed to use as the low-salt extract. Before analysis, the endogenous steroids were removed from the low-salt extract by incubation with dextran-coated charcoal. The binding of [3H]-5α-DHT to androgen receptor of testis and epididymis extract each was determined by competitive inhibition binding using nonlabelled 5α-DHT. Charcoal-treated testicular and epididymal extract each was incubated with 1 nM [3H]-5α-DHT at 4°C for 24 h. The nonspecific binding was carried out with incubating the extract with 100-fold excess nonlabelled 5α-DHT. Unbinding [3H]-5α-DHT was isolated from the binding steroid by adding the extract to packed hydroxyapatite in the low-salt TEDG buffer. Mixture were incubated for 30 min with several mixings and then centrifuged at 600 × g for 3 min at 4°C. After that the supernatant was aspirated. An aliquot of the packed HAP was washed 4 times with ice-cold 50 mM Tris buffer, pH 7.3. In determination of total binding,
