Kenneth Steven Hirsch

Inhibition of central nervous system aromatase activity: A mechanism for fenarimol-induced infertility in the male rat

Fenarimol (alpha-(2-chlorophenyl)-alpha(4-chlorophenyl)-5-pyrimidine-methanol), a pyrimidine carbinol agricultural fungicide, was previously reported to cause a dose-related decrease in fertility in rats (K. S. Hirsch, E. R. Adams, D. G. Hoffman, J. K. Markham, and N. V. Owen (1986), Toxicol. Appl. Pharmacol. 86, 391-399). Based on the results of a number of reproduction studies (K. S. Hirsch, E. R. Adams, D. G. Hoffman, J. K. Markham, and N. V. Owen (1986), Toxicol. Appl. Pharmacol. 86, 391-399), the infertility appeared to be associated with an impairment of male sexual behavior. When [14C]fenarimol was administered to the dam, high concentrations of radioactivity were observed in the neonatal hypothalamus, which functions in the development and subsequent expression of male sexual behavior. In the present studies fenarimol exhibited neither antiandrogenic nor antiestrogenic activities. The compound did, however, prevent the increase in nuclear estrogen receptors in the brain which normally occurs in the male during the early postnatal period. These results suggested that fenarimol might be acting to inhibit estrogen biosynthesis (via the aromatase enzyme complex) within the central nervous system. [3H]Testosterone was administered to neonatal rats, and the tritiated metabolites were isolated. Testosterone and dihydrotestosterone (17 beta-hydroxy-5 alpha-androstan-3-one) concentrations were similar in all treatment groups. Tritiated estrogens were detected in the brain cell nuclei from control neonates but not in neonates exposed to fenarimol. Fenarimol was also observed to inhibit rat ovarian aromatase activity in vitro. These data indicate that the decrease in male sexual behavior and the infertility associated with exposure to fenarimol were, most likely, due to inhibition of aromatase activity within the central nervous system.

Platelet Derived Growth Factor (Pdgf), Androgens and Inflammation: Possible Etiologic Factors in the Development of Prostatic Hyperplasia

Benign prostatic hyperplasia (BPH) is characterized by varying degrees of epithelial and stromal hyperplasia in association with inflammation. Although androgens are known to be important for the growth and function of the prostate, their role in the development of BPH is unclear. The release of platelet-derived growth factor (PDGF) in response to inflammation suggests that PDGF may participate in the development of BPH. Cultured prostate cells derived from patients with BPH were examined for the presence of functional PDGF and androgen receptors. The cells expressed PDGF receptors and responded to PDGF stimulation by the activation of the PDGF signal transduction pathway and a dose-dependent stimulation of cell proliferation. Even though the cells expressed androgen receptors, dihydrotestosterone failed to elicit a mitogenic response. While the role of androgens in BPH remains unclear, these results suggest that inflammation and, specifically, PDGF may be important etiologic factors in the development of BPH.

Studies to elucidate the mechanism of fenarimol-induced infertility in male rats

Fenarimol (alpha-(2-chlorophenyl)-alpha-(4-chlorophenyl)-5- pyrimidinemethanol a pyrimidine carbinol fungicide, caused a dose-related decrease in male fertility in Wistar rats. The effect was particularly evident in the anatomically normal progeny of dams treated with fenarimol throughout gestation and lactation. Based on the observation that the infertility was associated with the absence of vaginal sperm at the time of mating, the effect appeared to be the result of an absence of male sexual behavior. Fenarimol does not readily cross the placenta but does concentrate in milk, reaching three- to fivefold higher concentrations than those observed in the maternal plasma. These results suggest that fenarimol might be acting to block the perinatal development of male patterns of sexual behavior which involves the action of gonadal steroids within the central nervous system (CNS). To test this hypothesis, [14C]fenarimol was administered to dams and the radioactivity measured in the brains of the neonates. Radioactivity in the hypothalamus was three- to fourfold higher and the half-life four times longer than that observed in the remainder of the brain. Since the hypothalamus is believed to play a key role in the development and expression of male sexual behavior, it appears likely that fenarimol is acting centrally to decrease male sexual behavior, thereby decreasing male fertility.

Discovery and development of a novel class of nonsteroidal aromatase inhibitors

Efforts to develop a novel class of nonsteroidal aromatase inhibitors began with the discovery that the infertility in male rats exposed to high levels of the agricultural fungicide, fenarimol (alpha-(2-chlorophenyl)-alpha-(4-chlorophenyl)-5-pyrimidine-methanol), was attributable to the inhibition of aromatase activity within the central nervous system during the critical neonatal period. Although fenarimol was not particularly potent in inhibiting rat ovarian microsome aromatase activity in vitro (50% inhibition (IC50) = 4.1 microM). Subsequent testing of a number of analogues led to the identification of LY56110 (alpha,alpha-bis(4-chlorophenyl)-5-methylpyrimidine) which exhibited an IC50 of 29 nM. LY56110 was orally active, blocking the testosterone-induced increase of uterine weight and ovarian estrogen biosynthesis in immature female rats. In rats with established DMBA-induced mammary carcinoma, complete tumor regression was observed in 80% of the animals. Development of LY56110 was, however, stopped because of its effects on hepatic microsomal enzymes and an unacceptably long half-life. Structural modifications resulted in the development of the indenopyrimidines. LY113174 (8-chloro-5-(4-chlorophenyl)-5H-indeno less than 1, 2D greater than pyrimidine) was highly effective in vitro (IC50 = 24 nM) and in vivo but was far less potent than LY56110 with respect to induction of hepatic microsomal enzymes. LY113174 exhibited an acceptable biological half-life and had no effect on cholesterol side-chain cleavage. The indenopyrimidines appear to be a novel class of nonsteroidal aromatase inhibitors which may prove useful in the treatment of estrogen-dependent diseases.

Acetazolamide teratology and its association with carbonic anhydrase inhibition in the mouse

Acetazolamide, an inhibitor of the enzyme carbonic anhydrase (E.C. 4.2.1.1.), causes a unique congenital anomaly characterized by postaxial reduction of the distal portion of the right forelimb. To gain an understanding of the mechanism of teratogenesis, the activity of carbonic anhydrase in sensitive and resistant mouse strains, and its inhibition by acetazolamide, were examined. Differences in teratologic sensitivity were found not to be attributable to differences in maternal or embryonic drug levels. Enzyme inhibition at acetazolamide concentrations ranging between 10(-11) and 10(-5) M did not differ between the mouse strains when adult erythrocytes or day 12 embryos were assayed. However, in day 10 embryos, the period of maximum teratologic susceptibility, a small strain difference was found which suggested that the form of carbonic anhydrase in the susceptible CBA/J strain at this time is somewhat more sensitive to inhibition by acetazolamide than the form found in the resistant SWV strain. The results suggest further that more than one isozyme of carbonic anhydrase may be present in all three samples.

Endocrine effects of a new histamine H2-receptor antagonist, nizatidine (LY139037), in the male rat

A new orally active histamine H2-receptor antagonist, nizatidine (LY139037), was evaluated in male rats for effects on mechanisms regulating accessory sex organ growth and function. Cimetidine antagonized androgen binding to cytosolic receptors in vitro while nizatidine had no effect. Nizatidine and cimetidine were administered at the ED50, 5 X ED50, or 10 X ED50 doses for inhibition of gastric acid secretion previously determined using in vivo dog and rat models. The relative potencies of both agents to antagonize histamine H2-receptor-mediated gastric acid secretory responses have been confirmed in human clinical trials. Neither nizatidine nor cimetidine antagonized the in vivo uptake or nuclear translocation of radiolabeled androgen into the hypothalamic-preoptic-amygdala, pituitary, or ventral prostate. Nizatidine, given at doses equal to and 10 X the ED50 gastric acid secretion inhibitory values, and cimetidine (10 X ED50 value) had no effect on the response of male accessory sex organs to a submaximally stimulating dose of androgen in castrated rats. High doses of dietary nizatidine (greater than 500 mg/kg-day) administered for 6 months did not alter intact rat male accessory sex organ weights or circulating androgen levels relative to untreated controls. Acute administration of either nizatidine or cimetidine produced transient elevations in plasma prolactin (PRL) levels. Cimetidine was more potent and consistent than nizatidine in producing these increases in circulating PRL. The data described herein support the contention that unlike cimetidine, nizatidine is not a pharmacological antagonist of androgen action and has less of a stimulatory effect upon plasma prolactin. Taken together, these studies indicate that in the male rat, nizatidine exhibits a large therapeutic index between its gastric antisecretory activity and potential endocrinological effects.

Carbonic anhydrase distribution in rodent embryos and its relationship to acetazolamide teratogenesis.

The carbonic anhydrase inhibitor, acetazolamide, leads to a unique distal postaxial right forelimb deformity in rats and CBA/J mice, but SWV mice are completely resistant. Using Hanssons histochemical method, the distribution of carbonic anhydrase and its inhibition by acetazolamide in rat, CBA/J mouse, and SWV mouse embryos were compared. Carbonic anhydrase activity was demonstrable in many tissues of sensitive rat and CBA/J mouse embryos and in resistant SWV mouse embryos. The forelimb buds of resistant and sensitive embryos possess carbonic anhydrase activity in the area between the ectoderm and adjacent mesenchyma with no localization of enzyme activity corresponding to the malformation seen in acetazolamide teratogenesis. This suggests that carbonic anhydrase in the forelimbs is not the primary site of action for acetazolamide. A distinctive staining pattern of nucleated erythrocytes in resistant embryos indicated the presence of a low activity form of carbonic anhydrase in nearly half of the erythrocytes. A five-to tenfold greater amount of acetazolamide was needed to completely inhibit carbonic anhydrase activity in nucleated erythrocytes from resistant embryos than in those from sensitive embryos. The existence of a low activity form of carbonic anhydrase in SWV embryo erythrocytes may be the basis of resistance to acetazolamide teratogenesis.

Translocatable estrogen receptors in rat splenocytes

Estradiol has previously been shown to suppress the response of the cellular immune system of the rat while enhancing the production of IgM antibodies. Analysis of the cytosol from rat splenocytes showed saturation of specific binding sites at concentrations of between 80 and 160 nM [3H]-estradiol with an approximate Kd of 12 nM. Competitive binding studies showed a dose-dependent decrease in the binding of [3H]-estradiol to the receptor in the presence of increasing concentrations of unlabeled estradiol. Dexamethasone, progesterone and R1881 (synthetic androgen) had no effect on the binding of [3H]-estradiol. The in vivo administration of estradiol resulted in increased nuclear binding of [3H]-estradiol as compared to vehicle treated controls. These results indicate that rat splenocytes possess specific, translocatable estrogen receptors which may be responsible for the observed modulation of the immune system.