Müllerian inhibiting substance inhibits testosterone synthesis in adult rats.

Müllerian inhibiting substance (MIS) is a gonadal hormone that causes regression of the Müllerian ducts during male sexual differentiation. Postnatally, MIS inhibits the proliferation and differentiation of immature Leydig cells, and transgenic mice that overexpress MIS have decreased serum testosterone concentrations. To elucidate the effects of MIS on androgen regulation in the postnatal testis, we examined testosterone synthesis in adult Sprague-Dawley rats following intratesticular and intraperitoneal injections of MIS. Intratesticular MIS injection achieved high local concentrations of MIS (574.0 +/- 60.0 ng/mL) at 4 hours, with a corresponding decline in serum testosterone concentrations to 0.7 +/- 0.1 ng/mL, compared to 1.1 +/- 0.2 ng/mL with intraperitoneal MIS and 1.6 +/- 0.1 ng/mL with intratesticular vehicle (IT-Veh) (P < .001). Intratesticular administration of MIS (IT-MIS) resulted in much higher serum and testicular interstitial fluid MIS concentrations than the intraperitoneal route. To directly examine the testosterone production rate in MIS-treated animals, we isolated Leydig cells from MIS and vehicle-injected testes. Primary Leydig cells exposed to MIS had a lower testosterone production rate and decreased expression of p450c17 (hydroxylase/lyase) and luteinizing hormone (LH) receptor mRNAs than that of vehicle-injected controls or the noninjected contralateral testis. In conclusion, intratesticular administration of MIS caused a decline in serum testosterone concentrations by decreasing the rate of testosterone biosynthesis, confirming that MIS can regulate adult Leydig cell androgen production. The ability of MIS to down-regulate mRNA expression of the p450c17 and LH receptor genes suggests that this effect is mediated transcriptionally. These data indicate that, in addition to its role in embryonic differentiation of the male reproductive tract, MIS has a regulatory function in the postnatal testis. We conclude that one such function is for MIS to directly inhibit adult Leydig cell steroidogenesis.

Local inhibition of sebaceous gland growth by topically applied RU 58841.

The biological activity of a series of nonsteroidal, pure androgen receptor inhibitors was compared using the Syrian hamster ear skin sebaceous gland model. RU 58841, RU 56187, RU 38882 and cyproterone acetate were applied topically for 4 weeks on the ventral ear pinna of sexually mature male Syrian hamsters. Their order of efficacy was as follows: RU 58841 > RU 56187 > RU 38882 > cyproterone acetate. Maximal reduction of 60% in the size of the sebaceous glands was observed in hamsters treated with RU 58841 at a dose of 10 micrograms per day. This degree of inhibition occurred without any systemic side effects as shown by the absence of inhibition on the contralateral untreated ear pinna. Longer treatment did not produce greater inhibition since extending the treatment period from 4 weeks to 12 weeks showed similar data. The effect of RU 58841 was reversible since the inhibited sebaceous glands returned to normal size within 4 weeks after the cessation of the topical applications. The potent localized inhibition of sebaceous glands by RU 58841 demonstrates the excellent potential of this compound as a topical drug for the treatment of acne and other androgen-mediated disorders.

Low methionine ingestion by rats extends life span.

Dietary energy restriction has been a widely used means of experimentally extending mammalian life span. We report here that lifelong reduction in the concentration of a single dietary component, the essential amino acid L-methionine, from 0.86 to 0.17% of the diet results in a 30% longer life span of male Fischer 344 rats. Methionine restriction completely abolished growth, although food intake was actually greater on a body weight basis. Studies of energy consumption in early life indicated that the energy intake of 0.17% methionine-fed animals was near normal for animals of their size, although consumption per animal was below that of the much larger 0.86% methionine-fed rats. Increasing the energy intake of rats fed 0.17% methionine failed to increase their rate of growth, whereas restricting 0.85% methionine-fed rats to the food intake of 0.17% methionine-fed animals did not materially reduce growth, indicating that food restriction was not a factor in life span extension in these experiments. The biochemically well-defined pathways of methionine metabolism and utilization offer the potential for uncovering the precise mechanism(s) underlying this specific dietary restriction-related extension of life span.

The possibility of lidocaine ion pair absorption through excised hairless mouse skin.

The purpose of the present research was to test the ion pair absorption hypothesis with respect to the topical route of drug delivery. The experiment consisted of preparing various lidocaine-n-alkanoate ion pairs, then characterizing them by proton magnetic resonance spectroscopy, elemental analysis and conductivity. Percutaneous absorption studies through excised hairless mouse skin were carried out using ethanolic solution of radiolabeled 14C-lidocaine-octanoate, 14C-lidocaine-decanoate and 14C-lidocaine-dodecanoate. Studies were conducted under steady-state conditions using Bronaugh's flow-through apparatus and normal saline as the receptor fluid. Ethanolic solution of a lidocaine base served as a control. The apparent differences in flux between lidocaine and the various ion pairs were statistically significant (p less than 0.05). The differences among the fluxes of the various ion pairs were not statistically significant (p greater than 0.05), nor were the differences in lag times (p greater than 0.05). The difference between the flux values of lidocaine-1-14C-dodecanoate and 14C-lidocaine-dodecanoate infers that lidocaine-dodecanoate did not cross the excised, full-thickness, hairless mouse skin as an intact 1:1 ion pair. The formation of weakly associated ion pairs was suggested by the apparent low-association constants (Ka = 15-17 liters/mol) obtained at 25 degrees C in methanol by conductometric analysis.

Studies on the efficacy of methyl esters of n-alkyl fatty acids as penetration enhancers.

The efficacy of the methyl esters of medium chain n-alkyl fatty acids as penetration enhancers was evaluated in vitro using various animal and human skins with minoxidil as the test drug. Both methyl nonanoate and methyl caprate at a 10% concentration were found to be effective penetration enhancers for a 2% solution of minoxidil in alcohol USP. The percent of the applied radioactive dose of minoxidil penetrated after 17 h was 5-8 times greater for methyl non-anoate and methyl caprate enhanced solutions than for a 2% solution of minoxidil in alcohol USP alone or with the addition of 10% Azone, dimethylsulfoxide (DMSO) or N,N-diethyl-m-toluamide (DEET). The penetration enhancing activity of methyl caprate was effective for human, mouse, and hamster skins. Methyl caprate also enhanced the penetration of vitamin D3, erythromycin, triamcinolone acetonide, testosterone, and hydrocortisone.

Animal models of androgen-dependent disorders of the pilosebaceous apparatus. 1. The androchronogenetic alopecia (AGA) mouse as a model for male-pattern baldness.

The androchronogenetic alopecia (AGA) mouse if a mutant strain which expresses androgen-dependent baldness. Daily s.c. injection of testosterone (T) induced thinning of the hair coat along the upper dorsum after 4 weeks of treatment. After 12 to 14 weeks this diffuse alopecia eventually eveloped into a bald area which extended to the middorsum. Dihydrotestosterone was more effective than T in stimulating the onset of AGA. In this model, T produced the alopecia by decreasing the rate of hair growth, decreasing the duration of anagen, and markedly prolonging the duration of telogen. When applied topically at a concentration of 5%, cyproterone acetate delayed the progression of the T-mediated hair loss. However, this inhibitory effect occurred through systemic means as evidenced by decrease in the size of the submaxillary gland. Chronic feeding of androgen-treated female AGA mice with a diet containing 0.01% minoxidil also inhibited the development of alopecia. Skin and core temperatures were found to be higher in minoxidil-treated animals than in the placebo-treated controls. Minoxidil at a topical dose of 1% did not produce any effect. Increasing the dose to 2% caused a slight retardation of the development of alopecia. However, a 60% inhibition was observed at a topical dose of 5% minoxidil after 12 weeks of treatment (p less than 0.03). The data demonstrate that hair loss in the AGA mouse is androgen dependent and that this mutant strain can serve as a suitable model for the screening of compounds, such as antiandrogens and vasodilators, which may influence the balding process.

Synergistic antiandrogenic effects of topical combinations of 5 alpha-reductase and androgen receptor inhibitors in the hamster sebaceous glands.

The androgenic action of dihydrotestosterone (DHT) is antagonized by agents that compete with testosterone for the 5 alpha-reductase enzyme and by agents that block the binding of DHT to its receptor. The topical synergistic effect of 5 alpha-reductase (5 alpha RI) and androgen receptor inhibitors (ARI) was determined by measurement of the sebaceous gland size (SGS) of the ventral ear skin of the intact, sexually mature male Syrian hamsters. Progesterone (P), a 5 alpha RI, and spironolactone (SL), an ARI, produced a dose responsive decrease in SGS at topical concentrations of 0.01% to 5.0%. At concentrations of 1, 3, and 5%, P and SL combinations produced neither an additive nor synergistic inhibition of SGS. At very low concentrations of up to 0.10%, neither P nor SL alone produced any effect on SGS. When combinations of these two steroids were applied at low concentrations, SGS decreased unilaterally to approximately 50%. This synergy occurred best at a P:SL ratio of 1:2. The lower effective concentrations of P may be explained by its greater percutaneous absorption. Synergy was also demonstrated at low concentrations with other antiandrogens: cyproterone acetate, canrenone, hydroxyflutamide, and N-N-diethyl-4-methyl-3-oxo-4-aza-5 alpha-androstane- 17 beta-carboxamide. The use of anti-androgen combinations at low concentrations is of value because of the decreased risk of systemic side effects while maintaining potent topical efficacy.

Effects of toxic shock syndrome Staphylococcus aureus, endotoxin and tampons in a mouse model.

Synthetic tampons and toxic shock syndrome toxin-one (TSST-1)-producing strains of Staphylococcus aureus have been linked to an increased incidence of toxic shock syndrome (TSS). While recent reports attempt to define the tampon connection as the creation of an optimal environment for the production of TSST-1, the role of other factors in disease expression in an animal model remain under investigation. To understand the role of tampons and bacteria, pools of Swiss mice were inoculated with permutations of effluents from TSS strains of S. aureus and Escherichia coli grown inside tampons. Depending on tampon brand, when all 3 factors were combined mortality ranged from 20-100%. In controls inoculated with single effluents, or effluents from growth in the presence of cotton, no deaths were observed. Likewise, when hairless mice were inoculated with exotoxin, endotoxin, and tampon leachables, mortality was 100%. In the absence of any 1 component, mortality ranged from 0-40%. Lethal toxicity can be the result of enhancement, since animal death in apparent shock was observed in all pools containing the 3 components, and in all pools containing effluents of TSS S. aureus and E. coli grown in the presence of synthetic tampons, but not in their absence. A retrospective analysis of fatal vs non-fatal TSS in humans supports the hypothesis of enhancement.

The stage-dependent resistance of the chorion to external chemical damage and its relationship to embryonic diapause in the annual fish, Nothobranchius guentheri.

The resistance of the embryonic chorion of the annual fish Nothobranchius guentheri to chemical damage in vivo was investigated by the exposure of the embryos to protease. Embryos at stages 20, 33, and 43 were the most resistant to enzymatic action. These stages of development correspond respectively to the stages at which diapause I, diapause II, and 'delayed hatching' may occur. The magnitude of the resistance was further enhanced when diapause was induced prior to the treatment.

The local antiandrogenic effect of the intracutaneous injection of progesterone in the flank organ of sexually mature male Syrian golden hamster.

The local antiandrogenic action of progesterone was investigated using the androgen-responsive flank organs of adult, sexually mature male Syrian golden hamsters. The effect of the unilateral injections of a micronized suspension of progesterone into the flank organs was analyzed by the measurement of the weight, area of surface pigmentation, and the cross-sectional area of the sebaceous glands. The weekly injections of 5 mg of progesterone for a period of 3 weeks produced approximately 50% reduction in all three parameters in comparison to the controls. The minimal effective dose of 1 mg per week was determined by the injection of progesterone at doses ranging from 0.1 to 5 mg. These effects were localized only to the treated flank organs since the values for the contralateral side were not significantly different from the controls. The local action of progesterone was further demonstrated by the absence of effect on the weight of seminal vesicles and testes of the treated animals in comparison to the controls.

The hamster ear sebaceous glands. I. Examination of the regional variation by stripped skin planimetry.

A stripped skin planimetric method was developed to measure the ear sebaceous gland areas using a Numonics Graphics Calculator. The ear skin was manually separated from the cartilage and the area of the sebaceous gland units observed from the underside was determined. This procedure is less time-consuming than standard histologic techniques. Using stripped skin planimetry, it was demonstrated that the sebaceous gland size was greatest at the basal region of the ear and decreased toward the periphery. Regional variations in the density of the sebaceous gland units were also observed. In using the ear sebaceous model system it is important to standardize the site of biopsy in order to avoid sampling errors. The increase in ear sebaceous gland area from weaning to sexual maturity in the male hamster parallels the increase in flank organ area. This observation suggests that the ear and the flank organ sebaceous glands are comparable sebaceous models since they show a similarity in their response to the changing hormone levels during sexual maturation.

Stimulation of hamster sebaceous glands by epidermal growth factor.

Subcutaneous injection of epidermal growth factor (EGF) into the pinna of adult female and castrated male Syrian hamsters resulted in an increase in the number of cells per sebaceous gland unit. The effect of EGF on the sebaceous cell number was localized to the treated ear and accompanied by epidermal hyperplasia. The injection of testosterone into the ear also produced an increase in the cell number. When testosterone and EGF were injected together, the two hormones exerted an additive effect on the sebaceous glands of female hamsters. This is the first demonstration of a sebotrophic action of this polypeptide hormone.

The effects of temperature and season of collection on the onset and duration of diapause in embryos of the annual fish Nothobranchius guentheri.

Annual fish are found in temporary bodies of water in habitats of alternating rainy and dry seasons. The populations survive the dry seasons in the form of embryos encased in the bottom mud. Several genera of annual fish have been reported to exhibit diapause (developmental arrest) at three specific stages of development and it has been proposed that through this adaptation the populations are able to survive the variable durations of the dry seasons. The effects of incubation ans spawning temperatures, and season of collection on the onset and duration of diapause in laboratory populations of the annual fish, Nothobranchius guentheri, were studied. At low incubation and spawning temperatures there was a prolongation of Diapause I and Diapause II. In addition, fish spawned at 25 degrees C during the short days of winter produced embryos that entered a prolonged duration of Diapause II, whereas embryos produced during the long days of summer bypassed Diapause II.