Feeding of grey squirrels (Sciurus carolinensis) with the contraceptive agent DiazaCon™: effect on cholesterol, hematology, and blood chemistry.

Grey squirrels (Sciurus carolinensis) are an invasive species in Britain and Italy. They have replaced native red squirrels (Sciurus vulgaris) throughout most of Britain, and cause damage to trees. Currently, lethal control is used to manage grey squirrel populations in Britain, but nonlethal methods might be more acceptable to the public. One such method is contraception with 20,25-diazacholesterol dihydrochloride (DiazaCon™). DiazaCon™ inhibits the conversion of desmosterol to cholesterol, resulting in increasing desmosterol concentrations and decreasing cholesterol concentrations. Because cholesterol is needed for the synthesis of steroid reproductive hormones, such as progesterone and testosterone, inhibition of cholesterol synthesis indirectly inhibits reproduction. Desmosterol is used as a marker of efficacy in laboratory studies with species that do not reproduce readily in captivity. Grey squirrels were gavaged with a DiazaCon™ solution for 2 days, and then fed DiazaCon™-coated peanuts for an additional 8 days at target doses of 50 and 100 mg DiazaCon™ per kg body weight. There was a significant difference in cholesterol concentrations in the treatment groups compared to the control group. Cholesterol was reduced by ≥ 40% for 2 months in both treatment groups. There were no differences among groups with respect to blood chemistry and hematology parameters, and mean values are reported. The mean overall dose of DiazaCon™ received was 29.0 ± 1.6 and 55.3 ± 4.3 mg/kg in the low (50 mg/kg) and high dose (100 mg/kg) groups, respectively. DiazaCon™ might provide an effective, acceptable alternative to lethal control.

Effectiveness of twenty, twenty-five diazacholesterol, avian gonadotropin-releasing hormone, and chicken riboflavin carrier protein for inhibiting reproduction in Coturnix quail.

Contraception may provide a useful nonlethal management tool when it is desirable to reduce populations of birds. We tested the efficacy of 20,25 diazacholesterol, and immunization with avian gonadotropin-releasing hormone (AGnRH-I) and chicken riboflavin carrier protein (cRCP) as contraceptives and investigated their modes of action in Coturnix quail (Coturnix coturnix japonica). Females that were paired with males treated with 20,25 diazacholesterol produced lower percentages of eggs that were fertile and hatched. Females treated with 20,25 diazacholesterol and paired with control males laid fewer eggs, and lower percentages of their eggs were fertile and hatched. Treatment with 20,25 diazacholesterol reduced testosterone levels in males and progesterone levels in females. Nonesterified cholesterol levels were reduced, whereas desmosterol levels increased in birds treated with 20,25 diazacholesterol. Treatment with AGnRH-I and cRCP immunocontraceptive vaccines did not decrease average egg production and hatchability or hormone levels, but this failure might have been due to the vaccination protocol. If registered, wildlife managers may be able to use 20,25 diazacholesterol when other methods, such as lethal control, are undesirable for reducing damage caused by specific breeding behaviors such as the building of nests.

Structure-activity relationship of aza-steroids as PI-PLC inhibitors.

A number of aza-steroids were synthesized as potent phosphatidylinositol phospholipase C (PI-PLC) inhibitors. The epimeric mixtures 22,25-diazacholesterol (8a) and 3beta-hydroxy-22,25-diazacholestane (8b) were among the most active of these inhibitors, with IC(50) values of 7.4 and 7.5 microM, respectively. The 20alpha epimer, 8a2 (IC(50)=0.64 microM), whose stereochemistry at C-20 coincides with that of cholesterol, was found 50 times more potent than the 20beta epimer, 8a1 (IC(50)=32.2 microM). In diaza-estrone derivatives, the 3-methoxy group on the aromatic A-ring of 23 exhibited moderate PI-PLC inhibitory activity (IC(50)=19.7 microM), while compound with a free hydroxyl group (21) was inactive. However, in diaza-pregnane derivatives, epimers with a 3-hydroxyl group (8a, IC(50)=7.4 microM) exhibited more potent PI-PLC inhibitory activity than their counterparts with 3-methoxyl group on the non-aromatic A-ring (26, IC(50)=17.4 microM). We have illustrated in our previous publication that 3-hydroxyl-6-aza steroids are potent PI-PLC inhibitors.(3) However, simultaneous presence of the 6-aza and 22,25-diaza moieties in one molecule as in 13, led to loss of activity. Epimeric mixture 8a showed selective growth inhibition effects in the NCI in vitro tumor cell screen with a mean GI(50) value (MG-MID) of 5.75 microM for 54 tumors.

Molecular changes in erythrocyte membranes induced by long-term administration of clofibrate.

It has been reported that an enantiomer ((S)-(-)-4) of clofibric acid, and the racemate, can block the chloride conductance of skeletal muscle membrane. It has also been reported that several analogs of clofibric acid inhibit the HCO3(-)-Cl- exchange of erythrocytes. Since the two effects are probably similar biophysical membrane phenomena, the possibility of a common molecular mechanism arises. We exposed Sprague-Dawley male rats to long-term administration of clofibrate and 20,25-diazacholesterol (20,25-D) for comparison, at equipotent doses. Clofibrate (but not 20,25-diazacholesterol) produced a significant increase in density of the 220,000 Da band (beta-spectrin) and a decrease, also significant, in density of bands 2.1, 2.2, 2.3, 2.6 (syndeins or ankyrins) and of bands 4.1 and 6. Thus, clofibrate exhibits a manifold effect on the protein profile of the erythrocyte membrane cytoskeleton which, due to the lack of effect of 20-25-D, does not seem to be produced by the hypolipidemic effect per se, and thus deserves further study.

Effect of chemical carcinogens on cholesterol biosynthetic pathways in the skin of mice.

The metabolism of zymosterol and mevalonic acid was studied in vitro using the skin homogenates of 3-methylcholanthrene (3MC), diazacholesterol-treated and untreated mice. In normal mouse skin only lathosterol was a major metabolite and the other metabolites, cholesta-5,7,24-trien-3 beta-ol, desmosterol, cholesterol and 7-dehydrocholesterol were much less abundant. However, in the skin homogenate of mice treated with 3MC lathosterol production was depressed, while the production of cholesta-5,7,24-trien-3 beta-ol and desmosterol was significantly increased, the cholesterol level being normal or a little higher. In contrast, in the skin homogenate of mice administered diazacholesterol the production of both lathosterol and cholesterol was almost completely blocked with the slightly increased production of cholesta-5,7,24-trien-3 beta-ol and desmosterol. The metabolism in vitro of [2-14C]-mevalonic acid was quite similar to that of zymosterol, and no additional product which might possibly have been produced by the Kandutsch-Russell pathway was observed. Two pathways for cholesterol biosynthesis, therefore, may exist in mouse skin; the first is lanosterol----zymosterol----5 alpha-cholesta-7,24-dien-3 beta-ol----lathosterol----7-dehydrocholesterol----cholesterol, and the second by Bloch: lanosterol----zymosterol----5 alpha-cholesta-7,24-dien-3 beta-ol----cholesta-5,7,24-trien-3 beta-ol----desmosterol----cholesterol. When mouse skin is treated with 3-MC the former pathway is virtually blocked and the latter is stimulated, keeping the level of cholesterol in the tissue constant or a little higher than normal, which seems to be a significant change in the early stage of chemical carcinogenesis.

Alteration of cholesterol biosynthetic pathways in the skin of mice administered polycyclic aromatic hydrocarbons.

When polycyclic aromatic hydrocarbons were applied solely or together with a tumor promoter (12-O-tetradecanoylphorbol-13-acetate) to the skin of mice, a marked decrease in the level of lathosterol was observed, reflecting a significant change in the metabolism of sterols. Yet the total amount of cholesterol was not changed. When diazacholesterol (a metabolic inhibitor) was administered to mice, both desmosterol and 5 alpha-cholesta-7,24-dien-3 beta-ol accumulated in the skin, whereas the level of lathosterol decreased. These results seem to suggest that a significant portion of lathosterol is formed via 5 alpha-cholesta-7,24-dien-3 beta-ol in addition to the pathway through methostenol. When polycyclic aromatic hydrocarbon was applied to the skin of the mouse treated with diazacholesterol, a significant increase of desmosterol and a marked drop of the level of 5 alpha-cholesta-7,24-dien-3 beta-ol were observed. These results strongly suggest that polycyclic aromatic hydrocarbons perturb the metabolism of sterol in the skin of mice while keeping the total amount of cholesterol unchanged. A similar metabolism also seems to be operating in tumor tissue itself.

The effect of 3-methylcholanthrene on postlanosterol cholesterol biosynthetic pathways in mouse skin.

Repeated topical application of 3-methylcholanthrene to the backs of BALB/c mice lowered the tissue levels of lathosterol and provitamin D3, intermediates in one of the cholesterol biosynthetic pathways (the delta 7 pathway), though the activities of lathosterol 5-desaturase and provitamin D3 7-reductase were similar to those of control animals. These results seemed to indicate that carcinogen treatment exerted a depressive effect at some earlier step(s) than lathosterol synthesis. However, the content of cholesterol in mouse skin was not lowered in these animals, suggesting that another biosynthetic pathway might be activated. When diazacholesterol, which is known to inhibit the conversion of desmosterol to cholesterol, was administered together with the carcinogen, a marked accumulation of desmosterol was observed compared to animals given only diazacholesterol. Since desmosterol is an intermediate in the pathway in which the delta 24 double bond is reduced at the final step (the delta 24 pathway), this seemed to suggest that the delta 24 pathway was activated by carcinogen treatment.

Effect of 22,25-diazacholesterol dihydrochloride on the spermatogenesis of a wild rat, Bandicota bengalensis.

The effects of 22,25-diazacholesterol dihydrochloride (SC-12937) on the spermatogenesis of the bandicoot rat (Bandicota bengalenesis), heretofore unreported on any mammalian species, were studied. Rats were given a single subcutaneous injection of SC-12937 at a dosage of 100 or 200 mg/kg body weight and were killed on days 2 or 10 post-injection. Quantitative analysis of the germinal epithelium was performed on testicular sections (5 micron) stained with periodic acid-Schiff-hematoxylin to assess spermatogenesis. Treatment with SC-12937 at both dosages was found to interfere with the spermatogenic process of this pest rat. In addition to its antispermatogenic action, at higher dosage (200 mg/kg), this compound produced a marked suppression in accessory organ function. The results indicate that a single administration of SC-12937 in low dosage can cause spermatogenic inhibition without affecting accessory organ function in the bandicoot rat.

Effects of diazacholesterol dihydrochloride (SC-12937), an avian antifertility agent, on rat testis.

The present study was undertaken to evaluate the effectiveness of an avian chemosterilant, 20, 25-diazacholesterol dihydrochloride (SC-12937), on the rat testis. Adult male rats were injected intraperitoneally with 10 mg (Group 1) or 30 mg (Group 2) of SC-12937/kg/d or with vehicle alone (Group 3) for 10 days, and were killed 24 hours after the last injection. A wide range of variation in the appearance of affected seminiferous tubules was observed in the testis of SC-12937-treated rats at both dose levels. This ranged from apparently normal-looking seminiferous tubules to almost completely atrophied tubules with no cells. Affected tubules exhibited intraepithelial vacuoles of varying size, multinucleated giant cells, germ cell exfoliation, and tubular atrophy. The presence of severely damaged and entirely normal seminiferous tubules adjacent to one another in the same section was noteworthy. The changes appeared to be dose-related. A greater number (34.6%) of affected tubules were observed in rats receiving 30 mg of SC-12937 compared with the ones receiving 10 mg of this compound (19.6%). The Sertoli cells also were affected by this drug and exhibited cytoplasmic vacuolation, a marked increase in the accumulation of lipid droplets and myeloid bodies. Necrotic Sertoli cells also were observed in the severely affected tubules. The possible mechanism of antispermatogenic action of SC-12937 in rats has been discussed briefly.

Sterol composition and biosynthesis in mouse salivary glands.

Sterol components of mouse submandibular, sublingual and parotid glands were studied by thin-layer and gas-liquid chromatography. The major sterol was cholesterol (5-cholesten-3 beta-ol; 26.7, 28.0, 18.8 micrograms/mg protein respectively), with minor amounts of squalene, lathosterol (5 alpha-cholest-7-en-3 beta-ol), desmosterol (cholesta-5,24-dien-3 beta-ol), lanosterol (4,4',14-trimethyl-5 alpha-cholesta-8,24-dien-3 beta-ol), dihydrolanosterol (4,4',14-trimethyl-5 alpha-cholest-8-en-3 beta-ol) and methylstenol. Chromatograms of salivary sterols were similar to those of liver, and different from those of skin in which the amount of lathosterol was much higher. Administration of the anticholesterolaemic agent, 20,25-diazacholesterol, resulted in accumulation of desmosterol in all tissues tested, and additional sterols also accumulated in skin. The gas-liquid chromatographic profiles of salivary sterols from the treated animals were similar to those of liver, but different from those of skin. Thus sterols were synthesized in salivary glands and the biosynthetic pathway was via C24-unsaturated side-chain intermediates, as in liver. This was verified by showing that [2-14C]-mevalonate was incorporated into sterols in vitro when it was incubated with homogenates of these glands.

3-Alkyl-3-hydroxyglutaric acids: a new class of hypocholesterolemic HMG CoA reductase inhibitors. 1.

Derivatives of 3-hydroxy-3-methylglutaric acid (HMG), a portion of the substrate for HMG CoA reductase, were prepared and tested for their inhibitory action against rat liver HMG CoA reductase and for their hypocholesterolemic activity. Structure-dependent competitive inhibition was observed. Optimal structures had a free dicarboxylic acid with an alkyl group of 13-16 carbons at position 3. 3-n-Pentadecyl-3-hydroxyglutaric acid (3j) (IC50 = 50 microM) reduced serum cholesterol in the Triton-treated rat and HMG CoA reductase activity in the 20,25-diazacholesterol-treated rat.

Cholesterol synthesis and nerve regeneration.

In this report, we examine the requirement of cholesterol biosynthesis and its axonal transport for goldfish optic nerve regeneration. Cholesterol, labeled by intraocular injection of [3H]mevalonolactone, exhibited a delayed appearance in the optic tectum. Squalene and other minor components were labeled but not transported. Following optic nerve crush, the amount of labeled cholesterol transport was elevated, while retinal labeling was not altered relative to control fish. A requirement for cholesterol biosynthesis is inferred from the inhibition of neurite outgrowth in retinal explants caused by the cholesterol synthesis inhibitor, 20,25-diazacholesterol. The inhibition of growth could be overcome by addition of mevalonolactone, but not cholesterol, to the medium. Intraperitoneal administration of 200 nmol of diazacholesterol resulted in 92-98% inhibition of retinal cholesterol synthesis and accumulation of labeled desmosterol and other lipids in fish retina and brain which persisted for 2 weeks. Diazacholesterol-treated fish showed no reduction in the amount of lipid-soluble radioactivity transported following intraocular injection of [3H]mevalonolactone, but there were alterations in the chromatographic pattern of the transported labeled lipids. In contrast to its effects on neurite outgrowth in vitro, diazacholesterol did not inhibit optic nerve regeneration in vivo, as measured both by arrival of labeled rapidly transported protein at the tectum and by time required for the return of visual function.

Effects of 20,25-diazacholesterol on cholesterol synthesis in cultured chick muscle cells: a radiogas chromatographic and mass spectrometric study of the post-squalene sector.

Radiogas chromatography, used in conjunction with mass spectrometry, has been used to analyze the sterol content of cultured chick muscle cells. Seven sterols, plus lanosterol, were detected. These sterols conformed to a linear biosynthetic pathway linking lanosterol and cholesterol. The reaction sequence is: C-14 demethylation, C-4 demethylation, delta 8 leads to delta 5 double bond rearrangement, delta 24 double bond reduction. When chick cells were treated with increasing concentrations of 20,25-diazacholesterol, components of this pathway and aberrant products accumulated. These accumulations suggest that diazacholesterol affects reductases, double bond isomerases and the C-14 demethylation enzymes of sterol biosynthesis.

Lymphocyte abnormality in human myotonic dystrophy and experimental drug-induced myotonia.

In the cytoplasm of peripheral blood lymphocytes in 3 of 13 patients with myotonic dystrophy, myelin-like structures were observed electronmicroscopically. Some were connected to the cytoplasmic membranes, and some were surrounded by a limiting membrane possessing acid phosphatase activity. These findings suggest an aberration of cytoplasmic membranes in lymphocytes. Similar structures had appeared in lymphocytes of normal rats that became myotonic with 20, 25-diazacholesterol. These findings suggest that a primary genetic defect in human myotonic dystrophy, which participates in the formation of myelin-like structures in lymphocytes, might be also responsible for the occurrence of a myotonic phenomenon.

Biochemical and morphological effects of 20,25-diazacholesterol on cultured muscle cells.

Effects of 20,25-diazacholesterol (DAC), a myotonia-inducing drug, were evaluated on certain biochemical and morphological properties of embryonic rat muscle cells grown in tissue culture. During DAC treatment, muscle fibers exhibited spontaneous contractions that changed from coarse twitches to finer fibrillation movements, The ultrastructural alterations produced by DAC were smeared Z-lines, disorganized myofibrils, occasional honeycomb appearance of membranes and large vacuoles connected to zipper-like structures. Biochemically, a microsomal fraction prepared from DAC-treated cells (compared to that of normal cells) showed a 30-45 per cent decrease in the isoproterenol-enhanced and the NaF-enhanced adenylate cyclase activity. however, the beta-adrenergic receptors, through which isoproterenol activates the enzyme, showed no change in density or affinity as judged by the binding of [125I]iodohydroxybenzylpindolol. That indicated that DAC treatment caused an uncoupling of beta-receptor-adenylate cyclase interaction. Guanylate cyclase and cyclic GMP-phosphodiesterase were both markedly increased in DAC-treated cells, indicating a greater turnover of cyclic GMP. Binding of [3H]concanavalin A to DAC-treated muscle membranes was decreased 20-40 per cent. The data indicate that DAC exert a direct influence on muscle fibers, affecting their functional, biochemical and morphological properties.

Effects of steroid blockers on LH-induced ovulation in the domestic fowl, Gallus domesticus.

The spontaneous ovulation of hens was suppressed by daily injection of PMSG. The LH injected to overcome the block was accompanied by one of 4 compounds known to inhibit steroidogenesis at different sites in the biosynthetic pathway. A dose of 300 mg aminoglutethimide phosphate, which inhibits the conversion of cholesterol to 20 alpha-hydroxycholesterol, blocked the LH-induced ovulation and prevented the normal rise in plasma progesterone. Metyrapone, an inhibitor of 11 beta-hydroxylase, and SC 12937 and AY 9944, inhibitors of cholesterol synthesis, did not prevent ovulation or the progesterone rise induced by exogenous LH. Administration of progesterone overcame the inhibitory effect of aminoglutethimide, and it is suggested that progesterone is involved in the ovulatory process of the fowl.

Dependence of membrane potential on extracellular ionic concentrations in myotonic goats and rats.

Intracellular potassium concentration([K]i) and the resting sodium-to-potassium permeability ratio (alpha = PNa/PK) were determined by recording resting potentials at 25 degrees C in six solutions with variable potassium concentrations, constant [K+] + [Na+], and constant [K+] X [Cl-] product and fitting the results by nonlinear least squares to the Goldman-Hodgkin-Katz equation. Fits to models including chloride permeability terms suggest that chloride is in Donnan equilibrium in the muscles studied. External intercostal muscle was biopsied from anesthetized normal goats and goats with hereditary myotonia. In normal goat muscle, alpha was 0.012 +/- 0.001 (mean +/- SE, n = 11), and [K]i was 133 +/- 6 mM (n = 9); myotonic goat muscle did not differ from this. Extensor digitorum longus (EDL) muscle was removed from anesthetized male Wistar rats and from like rats pretreated with a single large dose of 20,25-diazacholesterol (DAC). In normal rat EDL, alpha was 0.019 +/- 0.002 (n = 12), and [K]i was 142 +/- 4 mM (n = 12). In the 16-day DAC-treated rats, alpha was significantly reduced (P < 0.002) to 0.010 +/- 0.002 (n = 8), and [K]i was slightly reduced by DAC. It is concluded that DAC reduced PNa in rat fibers. Tetrodotoxin at 5 microM did not affect either parameter in the rats.