antarelix and Body-Weight

The Hershberger assay for antiandrogens and modifiers of steroid biosynthesis uses surgically-castrated rats. We described an adaptation of the assay using the GnRH inhibitor Antarelix in place of surgical castration [Ashby J, Lefevre PA, Deghenghi R, Wallis N. Regulatory Toxicology and Pharmacology 34:188-203, 2001], and concomitantly described changes in expression of the androgen-dependent prostatic genes PBP C3, TRPM-2, and ODC as a possible complement to gravimetric analysis of the sex accessory tissues (SAT) [Nellemann C, Vinggaard AM, Dalgaard M, Hossaini A, Larsen J-J. Toxicology 163:29-38, 2001.. The present study describes the results of combining these two modifications into a single assay. During the course of these experiments it was shown that SD rats gave similar results to AP rats and that the higher stimulatory dose of testosterone propionate (TP) used in our experiments gave stronger assay responses to FLU than the lower dose of TP used by some earlier investigators. The potent antiandrogen flutamide (FLU) and the weak antiandrogen DDE were used to evaluate this modified assay.. For all parameters studied (SAT weights and changes in expression of the 3 prostatic genes) FLU gave the expected positive results. The weak antiandrogen DDE gave variable and mainly non-reproducible responses. Use of DDE as a weak antiandrogen accelerated assessment of the new assay.. Possible reasons for this failure to detect DDE are discussed, and it is concluded that the modified assay is unsuitable for use in its present form. The use of gene expression analyses together with evaluation of SAT weights is a promising tool as an early and sensitive marker of antiandrogen action, but more work is needed on the choice of time frame as well as the selection of genes to monitor.

Topics: Androgen Antagonists; Androgen-Binding Protein; Animals; Biomarkers; Body Weight; Clusterin; Dichlorodiphenyl Dichloroethylene; Flutamide; Gene Expression; Glycoproteins; Gonadotropin-Releasing Hormone; Male; Molecular Chaperones; Oligopeptides; Orchiectomy; Organ Size; Ornithine Decarboxylase; Prostate; Prostatein; Rats; Rats, Sprague-Dawley; Secretoglobins; Testosterone Propionate; Uteroglobin

Five rodent diets have been evaluated for their possible effect on the sexual development of the rat. Groups of 12 pregnant Alpk rats were fed one of the following combinations of diets during pregnancy and postnatally: RM3/RM1, AIN-76A/AIN-76A, RM3/AIN-76A, Teklad Global 2016 (Global)/Global and Purina 5001/Purina 5001. AIN-76A is phytoestrogen-free while the other diets contained varying amounts of phytoestrogens. The phytoestrogens genistein and daidzein were determined in the diets studied, and the concentrations found agreed with earlier estimates. RM3/RM1 was selected as the control group, as this has been used routinely in this laboratory for the past decade. Determinations were made in offspring of the times of vaginal opening and first estrus among the females, and of prepuce separation and testes descent among the males. At postnatal day (PND) 26 the females from 6 of the 12 litters were terminated and tissue weights measured. Males from 6 of the 12 litters were similarly studied at PND 68. Animals from the remaining litters were transferred to RM1 diet at PND 70. Termination of the study was at PND 128 (males) and PND 140 (females) when body weights and tissue weights were determined. Marked differences in body weight, sexual development, and reproductive tissue weights were observed for rats maintained on AIN-76A or Purina 5001, with only minimal effects among rats maintained on the Global diet. These comparisons were against RM3/RM1 as the reference diet. However, using Purina 5001 as the reference diet reversed the direction of the differences seen when using RM3/RM1 as the reference diet. The differences observed when using RM3/RM1 as reference diet occurred mainly postnatally. In addition, the fact that similar differences were seen for the phytoestrogen-free diet, AIN-76A, and the phytoestrogen-rich diet, Purina 5001, indicate that these effects are more likely to be caused by nutritional differences between the diets that then have centrally mediated effects on rodent sexual development, rather than individual dietary components affecting peripheral estrogen receptors (ER). This proposal is supported by abolition of the uterotrophic activity of AIN-76A and Purina 5001 (relative to RM3/RM1) in the immature rat by coadministration of the gonadotrophin-releasing hormone (GnRH) antagonist ANTARELIX: The present data indicate that choice of diet may influence the timing of sexual development in the rat, and consequently, that when evaluating

Topics: Age Factors; Animal Feed; Animals; Animals, Newborn; Biological Assay; Body Weight; Diet; Estrogens, Non-Steroidal; Female; Genistein; Isoflavones; Male; Oligopeptides; Organ Size; Phytoestrogens; Plant Preparations; Pregnancy; Rats; Rats, Inbred Strains; Rats, Wistar; Sexual Maturation; Weaning

An obstacle to the widespread adoption of the Hershberger antiandrogen assay is the surgical castration procedure required to produce androgen deficiency in the test animals. Here we describe two chemical treatments that produce similar effects to surgical castration. The first is use of ethane dimethane sulphonate (EDS), a specific toxin to the testosterone-producing Leydig cells of the mature testes. The second class of compound is the decapeptide inhibitors of the gonadotrophin-releasing hormone (GnRH), compounds such as Antarelix and Antide. Administration of either EDS or the GnRH inhibitors results in loss of weight of the testes, epididymides, and sex-associated tissues. Co-administration of testosterone to these animals leads to reversal of the induced effects. The basic test protocol for both of these assay modifications is described. Flutamide was used as a representative potent antiandrogen, and DDE as an example of a weakly active antiandrogen. The 5alpha-reductase inhibitor finasteride was used to inhibit the transformation of testosterone to dihydrotestosterone. It is shown that the EDS assay is sensitive to the antiandrogen flutamide, but that it fails to detect the weaker antiandrogen DDE. In contrast, the Antarelix assay performs as well as does the classical castration assay, leading to the detection as antiandrogens of flutamide, DDE, and finasteride. It is concluded that the GnRH inhibition Hershberger assay is more convenient to conduct than the original surgical castration assay, and it involves less stress to the test animals.

Topics: 5-alpha Reductase Inhibitors; Androgen Antagonists; Animals; Body Weight; Dichlorodiphenyl Dichloroethylene; Dose-Response Relationship, Drug; Enzyme Inhibitors; Finasteride; Flutamide; Gonadotropin-Releasing Hormone; Hormone Antagonists; Hormone Replacement Therapy; Insecticides; Leydig Cells; Male; Mesylates; Oligopeptides; Orchiectomy; Organ Size; Rats; Rats, Inbred Strains

Diethylstilbestrol (DES) was administered neonatally (Days 2-12; 10 microg on alternate days) to rats, and developmental changes in Sertoli cell function were evaluated at 18, 25, and 35 days of age and compared to those observed in rats administered a GnRH antagonist (GnRHa; Days 2 and 5; 10 mg/kg) or a vehicle (controls). DES and GnRHa treatments resulted in similar reductions in both Sertoli cell numbers (40% for DES, 48% for GnRHa) and suppression of testicular growth at 18 and 25 days, though by 35 days the suppression was more pronounced (p < 0.001) in DES-treated animals. Plasma FSH levels were suppressed markedly at 18 and 25 days, but not at 35 days, in GnRHa-treated rats, whereas in DES-treated rats the FSH levels were suppressed significantly only at 35 days. Both treatments suppressed plasma levels of inhibin B, though this was more pronounced (p < 0.05) in DES- than in GnRHa-treated rats. In controls, Sertoli cell immunoexpression of inhibin alpha, sulfated glycoprotein-1 (SGP-1), and androgen receptor (AR) increased in intensity and changed to an adult, stage-dependent pattern by 25 days. In GnRHa-treated rats these changes were reduced in intensity but were similar to those in controls at 35 days. In DES-treated rats, the increase in intensity and stage-dependent pattern of immunoexpression of inhibin alpha, SGP-1, and AR were virtually absent at 25 days but were present by 35 days. Germ cell volume per Sertoli cell was reduced in GnRHa- and DES-treated rats compared with controls at 18 and 25 days but was significantly greater (p < 0. 001) in DES- than in GnRHa-treated rats at 35 days. The proportion of apoptotic to viable germ cells was increased (p < 0.01) in GnRHa- and DES-treated rats compared with controls at 18 and 25 days; but at 35 days, values in GnRHa-treated rats had declined to control values whereas those for DES-treated rats remained 10-fold elevated (p < 0.001). In adulthood, testis weight and daily sperm production were reduced by 43% and 44%, respectively, in GnRHa-treated rats, but spermatogenesis was grossly normal. Comparable changes were observed in approximately 25% of DES-treated rats, but the majority exhibited > 60% reduction in testis weight with many Sertoli cell-only tubules and very low daily sperm production. Taken together, these data are interpreted as providing evidence for direct modulation of Sertoli cell (maturational) development by DES.

Topics: Aging; Animals; Animals, Newborn; Body Weight; Cell Count; Diethylstilbestrol; Estrogens; Follicle Stimulating Hormone; Male; Oligopeptides; Organ Size; Rats; Rats, Wistar; Sertoli Cells; Spermatogenesis; Testis