Dissection and weighing of accessory sex glands after formalin fixation, and a 5-day assay using young mature rats are reliable and feasible in the Hershberger assay

The rodent Hershberger assay has been used predominantly by the pharmaceuti cal industry to evaluate androgenic and antiandrogenic chemicals for potent ial therapeutic use. However, this assay has not yet been formally validate d and standardized for use in toxicology testing. There are many variations in the protocol used for this assay. The weight of androgen-dependent tiss ues is a definitive endpoint in the Hershberger assay. To find out the reli able assay protocol with feasibility. although many possible factors may af fect assay reliability, the present study consist of a series of three sepa rate experiments focused on method of dissection and weighing of accessory sex glands (ASGs: ventral and dorso-lateral prostate, seminal vesicles toge ther with coagulating glands, and Cowper's glands), animal age and number o f doses. Furthermore, male pubertal. assay, an alternative to the Hershberg er assay, was also examined its reliability. Experiment 1 explored whether reliably accurate ASG weights can be obtained after formalin fixation. The ASGs were collected from castrated male rats (11 weeks of age) treated dail y with corn oil, or testosterone propionate (TP, 0.25 mg/kg/day, s.c.) and p,p'-DDE (0 or 100 mg/kg/day, p.o.) for 5 days. One day after the final tre atment, the ASGs were removed carefully and weighed separately, and then fi xed overnight in a 10% neutral-buffered formalin and weighed again. After t hat, the tissues were dried overnight in an oven and weighed again. A high correlation between fresh and fixed tissue weights, and a high correlation between fixed and dried tissue weights were noted. The changes in the tissu e weight due to fixation were marginal and were proportional to the fresh w eights of the individual tissue. Standard deviation of the fixed tissue wei ght in each group and the magnitude of responses to TP or p,p'-DDE in fixed tissues were equivalent to those in fresh or dried tissues. These findings indicate that formalin fixation does not interfere with interpretation of assay results, and this procedure was used in the subsequent experiments. E xperiments 2 and 3 explored whether animal age or treatment duration altere d assay sensitivity. In Experiment 2, antiandrogenic effect of p,p'-DDE (10 0 mg/kg/day) was detected after 5-and 10-day treatment irrespective of anim al age (7 vs 11 weeks). In Experiment 3, antiandrogenic effects of flutamid e (1 and 10 mg/kg/day) and p,p'-DDE (10 and 100 mg/kg/day) were compared be tween two different protocols, the 10-day assay using peripubertal rats and the 5-day assay using young mature rats. Results demonstrated that both pr otocols could significantly detect antiandrogenic effects of flutamide and p,pl-DDE. These findings demonstrate that (1) dissection and weighing of AS Gs after formalin fixation is reliable in the Hershberger assay, (2) when t his procedure is used, the 5-day Hershberger assay using young mature rats, expected to be more feasible assay than the 10-day assay using peripuberta l rats, is also reliable as well as the 10-day assay using peripubertal rat s. Furthermore, we confirmed that male pubertal assay with use of dissectio n and weighing of fixed tissues also reliable. (C) 2001 Elsevier Science Ir eland Ltd. All rights reserved.