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High dietary levels of the non-genotoxic synthetic pyrethroid momfluorothrin increased the incidence of hepatocellular tumors in male and female Wistar rats. Mechanistic studies have demonstrated that the mode of action (MOA) for momfluorothrin-induced hepatocellular tumors is constitutive androstane receptor (CAR)-mediated. In the present study, to evaluate the potential human carcinogenic risk of momfluorothrin, the effects of momfluorothrin (1-1,000 µM) and a major metabolite Z-CMCA (5-1,000 µM) on hepatocyte replicative DNA synthesis and CYP2B mRNA expression were examined in cultured rat and human hepatocyte preparations. The effect of sodium phenobarbital (NaPB), a prototypic rodent hepatocarcinogen with a CAR-mediated MOA, was also investigated. Human hepatocyte growth factor (hHGF) produced a concentration-dependent increase in replicative DNA synthesis in rat and human hepatocytes. However, while NaPB and momfluorothrin increased replicative DNA synthesis in rat hepatocytes, NaPB, momfluorothrin and Z-CMCA did not increase replicative DNA synthesis in human hepatocytes. NaPB, momfluorothrin and Z-CMCA increased CYP2B1/2 mRNA levels in rat hepatocytes. NaPB and momfluorothrin also increased CYP2B6 mRNA levels in human hepatocytes. Overall, while momfluorothrin and NaPB activated CAR in cultured human hepatocytes, neither chemical increased replicative DNA synthesis. Furthermore, to confirm whether the findings observed in vitro were also observed in vivo, a humanized chimeric mouse study was conducted. Replicative DNA synthesis was not increased in human hepatocytes of chimeric mice treated with momfluorothrin or its close structural analogue metofluthrin. As human hepatocytes are refractory to the mitogenic effects of momfluorothrin, in contrast to rat hepatocytes, the data support the hypothesis that the MOA for momfluorothrin-induced rat liver tumor formation is not relevant for humans.

A 5-day Hershberger assay using young mature male rats to detect compounds interfering with androgen receptor (AR)-mediated mechanisms was evaluated for ability to identify p,p'-DDE (a weak AR antagonist) and methyltestosterone (MT, an AR agonist). Fenitrothion, an organophosphate pesticide, was also evaluated in this validated assay. Castrated male Crj:CD(SD)IGS rats (1 week after castration, 11 weeks of age) were subjected to experiments. To determine a suitable value of testosterone propionate (TP) as a reference androgen for detection of antiandrogenic chemicals, castrated male rats were treated daily with TP (0, 0.06, 0.25, 1, 4, or 16 mg/kg/day, s.c.). TP produced increases in weights of ventral prostate, seminal vesicles and levator ani plus bulbocavernosus muscles. Serum androgen level measured by RIA kit (mostly TP) were elevated in a dose-related manner, while the weights of organs with 1 mg/kg/day of TP were nearly equivalent to the maximum responses (i.e., sub-maximal). One hundred mg/kg/day of p,p'-DDE significantly attenuated TP 0.1 mg/kg-induced increases in weights of seminal vesicles and muscles, and TP 1 mg/kg-induced increases in weights of ventral prostate, seminal vesicles and muscles, but did not affect the weight of these organs in either TP 16 mg/kg-treated or intact rats, demonstrating that the dose range of 0.1-1 mg/kg TP is suitable for reference androgen. Oral treatment with 100 mg/kg of MT increased the weights of ventral prostate, seminal vesicles and muscles as strongly as did subcutaneous injection of 1 mg/kg of TP. These findings demonstrate that the 5-day Hershberger assay using young mature as well as immature male rats is a sensitive and valid short-term screening method for the detection of chemicals interfering with AR-mediated mechanisms. To determine whether fenitrothion interferes with AR-mediated mechanisms in vivo, fenitrothion (0, 0.75, 1.5 or 3 mg/kg/day) was administered by gavage for 5 days to castrated rats for androgenicity, or to castrated rats treated with 1 mg/kg TP for antiandrogenicity. Treatment with fenitrothion had no adverse effects on clinical signs, body weight, or liver or kidney weights, but cholinesterase activities in the brain and erythrocytes were significantly suppressed by fenitrothion to, respectively, 77-81% and 66-67% of control levels. In the antiandrogenicity experiment, serum androgen levels of TP-treated, castrated rats did not differ among groups. Treatment with 100 mg/kg of p,p'-DDE as a positive control again significantly attenuated TP-induced increases in weights of the ventral prostate and seminal vesicles, while fenitrothion had no effect on the weights of any organs. In the androgenicity experiment, treatment with 100 mg/kg of MT significantly increased weights of ventral prostate, seminal vesicles and muscles, but fenitrothion had no effects on the weights of any of these organs. These findings yield no evidence that fenitrothion interferes with AR-mediated mechanisms in vivo, consistent with the result of several toxicological bioassays.

To examine the mechanism and toxicological significance of thyroidal tumor observed slightly in a long-term rat study with diethofencarb (isopropyl 3, 4-diethoxycarbanilate), male Sprague-Dawley rats were fed diethofencarb in diets at concentrations of 0, 5, 000 or 20, 000 ppm for 3 months. Examinations mainly for thyroid functions including thyroid uptake of &lt125&gtI, serum thyroid hormone and thyroid stimulating hormone (TSH) level, hepatic UDP-glucuronyltransferase (UDP-GT) activity and histopathological examination in thyroid were performed at week 13. Decreases of body weights and food consumptions were observed at and above 5, 000 ppm. Under these conditions, decrease of serum free T4 and increase of serum TSH level were observed only at 20, 000 ppm, concurrently with liver weight increase at and above 5, 000 ppm and increase of hepatic UDP-GT activity at 20, 000 ppm. However, no compound related effects were noted in thyroid weight, thyroid uptake of &lt125&gtI and gross or histopathological examination in thyroid. These results indicate that the administration of diethofencarb leads to an increase in UDP-GT activity and acceleration of thyroid hormone excretion from the liver. The acceleration causes a decrease in serum free T4 level, triggering the feedback mechanism of the pituitary gland, promotion of TSH release and consequently an increase in serum TSH level. Thus, the slightly higher incidence of thyroid follicular cell tumors observed in the chronic and oncogenicity study with non-genotoxic diethofencarb is considered to be caused by these weak pituitary-thyroid hormonal imbalances. The toxicological significance in humans is extremely low according to the well established facts that the chronic TSH stimulatin would not induce thyroid tumors in humans and humans may be less sensitive than rats in regard to the response to goitrogenic stimuli.

To clarify the mechanism of gonadotropin imbalances and the differential response of rat and mouse testicular interstitial cells (Leydig cells) to procymidone, N-(3,5-dichlorophenyl)-1,2-dimethylcyclopropane-1,2-dicarboximi de, male Sprague-Dawley rats and ICR mice were fed procymidone in diet for 2 weeks at 0, 700, 2,000 and 6,000 ppm for rats, or 0, 1,000, 5,000 and 10,000 ppm for mice. Testosterone and luteinizing hormone (LH) levels in serum, testis or pituitary and the in vitro binding affinities of procymidone, flutamide and related compounds to the androgen receptor in prostate cytosol of rats and mice were examined. Hypergonadotropism in rats and mice was clearly observed in the same order two weeks after the initiation of treatment with procymidone. Increased levels of testosterone and LH in serum at 6,000 ppm and LH in pituitary at and above 700 ppm in rats were observed. In mice, testosterone levels in serum and testis elevated at 10,000 ppm. LH levels in serum and pituitary elevated significantly as well at around 5,000 to 10,000 ppm. In the competitive binding assay, procymidone showed a significant but lower binding affinity comparing to that of cyproterone acetate, the steroidal androgen receptor antagonist, for the androgen receptor in both rats and mice under the condition that unlabeled dihydrotestosterone (DHT) effectively inhibited the binding of [3H]-DHT to the androgen receptor in both species. The relative binding affinity (RBA) of procymidone was of the same order as that of flutamide, a synthetic non-steroidal antiandrogen structurally similar to procymidone. These results indicate that procymidone is an active antiandrogen and the androgen receptor antagonism is the likely mechanism of action.

To investigate the mechanism and toxicological significance of testicular interstitial cell tumors (ICT) observed in a long-term rat study with procymidone, N-(3, 5-dichlorophenyl)-1, 2-dimethylcyclopropane-1, 2-dicarboximide, male Sprague-Dawley rats were fed procymidone in diets for up to 6 months with a positive control group receiving a single subcutaneous injection of cadmium chloride. Examinations mainly for gonadal functions such as serum testosterone and luteinizing hormone (LH), reproductive organ weight and histopathology presented evidence of the indirect involvement of gonadotropins in the production of ICT in rats. A significant increase in both serum testosterone and LH was observed in the early stage at high dietary concentrations of procymidone without any lesion in gonadal systems in histopathology, whereas administration of cadmium chloride produced the expected substantial increase in serum LH and a concomitant decrease in serum testosterone with a marked damaging effect on gonadal systems. Increases in serum testosterone and LH levels in animals receiving procymidone were reversible. The no-effect level for procymidone on serum testosterone and LH was 300 ppm over six months of treatment. The possible mechanism of ICT production in rats by non-genotoxic procymidone, structurally similar to flutamide, a synthetic non-steroidal antiandrogen, is likely to be derived from its induction of a hypergonadotropism due to the competitive binding to the androgen receptor, preventing the normal effect of testosterone to control the circulating level of LH.

To assess the toxicological significance of thyroidal tumor observed slightly in a long-term rat study with diniconazole, (E)-1-(2, 4-dichlorophenyl)-4, 4-dimethyl-2-(1, 2, 4-triazol-1-yl)-1-penten-3-ol, a 3-month subacute feeding study was conducted in male Crj: CD (SD) rats by administering diniconazole in diet at concentrations of 0, 100, 1, 000, or 2, 000 ppm. Examinations mainly for thyroid functions were performed at Weeks 2, 4 and 13. Measurement of serum hormone levels revealed continuous decreases in serum thyroxine (T4) and free T4 levels at and above 1, 00 ppm and increase in serum thyroid stimulating hormone (TSH) level at 2, 000 ppm concurrently with liver weight and hepatic UDP-glucuronyltransferase (UDP-GT) increases at and above 1, 000 ppm. No changes were observed in serum triiodothyronine (T3) and free T3 levels. Increase in thyroid uptake of &lt125&gtI and organification of &lt125&gtI in the thyroid at 2, 000 ppm and thyroid follicular cell hyperplasia at and above 1, 000 ppm were also observed. However, no compound-related changes were observed in autopsy and organ weight in the thyroid. Based on the above results, diniconazole induces increases in the hepatic UDP-GT activity and the thyroid hormone excretion from the liver. The increased excretion of thyroid hormones causes decrease in serum T4 and free T4 levels, triggering the feedback mechanism of the pituitary gland, promotion of TSH release from the pituitary gland and increase in serum TSH level. The increased serum TSH level probably leads to increased &lt125&gtI uptake of thyroid and thyroid follicular cell hyperplasia. Thus, the thyroid tumorigenesis in rats treated with diniconazole is due to the secondary overstimulant effect on the thyroid by increased serum TSH level. The toxicological significance in humans is extremely low and it is unlikely that diniconazole would increase thyroid tumor in humans even if diniconazole were to alter normal thyroid hormone level in humans.