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 pharmaceutical industry to evaluate androgenic and antiandrogenic chemicals for potential therapeutic use. However, this assay has not yet been formally validated and standardized for use in toxicology testing. There are many variations in the protocol used for this assay. The weight of androgen-dependent tissues is a definitive endpoint in the Hershberger assay. To find out the reliable assay protocol with feasibility, although many possible factors may affect assay reliability, the present study consist of a series of three separate experiments focused on method of dissection and weighing of accessory sex glands (ASGs: ventral and dorso-lateral prostate, seminal vesicles together with coagulating glands, and Cowper's glands), animal age and number of doses. Furthermore, male pubertal assay, an alternative to the Hershberger 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 daily 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 treatment, the ASGs were removed carefully and weighed separately, and then fixed overnight in a 10% neutral-buffered formalin and weighed again. After that, 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 tissue weight due to fixation were marginal and were proportional to the fresh weights of the individual tissue. Standard deviation of the fixed tissue weight 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. Experiments 2 and 3 explored whether animal age or treatment duration altered assay sensitivity. In Experiment 2, antiandrogenic effect of p,p'-DDE (100 mg/kg/day) was detected after 5-and 10-day treatment irrespective of animal age (7 vs 11 weeks). In Experiment 3, antiandrogenic effects of flutamide (1 and 10 mg/kg/day) and p,p'-DDE (10 and 100 mg/kg/day) were compared between two different protocols, the 10-day assay using peripubertal rats and the 5-day assay using young mature rats. Results demonstrated that both protocols could significantly detect antiandrogenic effects of flutamide and p,p'-DDE. These findings demonstrate that (1) dissection and weighing of ASGs after formalin fixation is reliable in the Hershberger assay, (2) when this procedure is used, the 5-day Hershberger assay using young mature rats, expected to be more feasible assay than the 10-day assay using peripubertal rats, is also reliable as well as the 10-day assay using peripubertal rats. Furthermore, we confirmed that male pubertal assay with use of dissection and weighing of fixed tissues also reliable.
Yamada
T
T
Environmental Health Science Laboratory, Sumitomo Chemical Company, Ltd, 3-1-98, Kasugade-naka, Konohana-ku, Osaka, Japan. yamadat8@sc.sumitomo-chem.co.jp
Sunami
O
O
Kunimatsu
T
T
Kamita
Y
Y
Okuno
Y
Y
Seki
T
T
Nakatsuka
I
I
Matsuo
M
M
D000728:Androgens / Q000633:toxicity; D000818:Animals; D003633:Dichlorodiphenyl Dichloroethylene / Q000633:toxicity; D004210:Dissection; D005557:Formaldehyde; D006727:Hormone Antagonists / Q000633:toxicity; D008297:Male; D009919:Orchiectomy; D009929:Organ Size / Q000187:drug effects; D011467:Prostate / Q000187:drug effects / Q000473:pathology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D012669:Seminal Vesicles / Q000187:drug effects / Q000473:pathology*; D012680:Sensitivity and Specificity; D012741:Sexual Maturation / Q000187:drug effects; D013739:Testosterone / Q000633:toxicity; D013997:Time Factors; D016707:Tissue Fixation; D018675:Toxicity Tests / Q000379:methods*
D000728:Androgens; D006727:Hormone Antagonists; D005557:Formaldehyde; D013739:Testosterone; D003633:Dichlorodiphenyl Dichloroethylene