Male fertility: core chemical structure in pharmacological research.

Research in male fertility is of interest in connection with both contraception and treatment of sterility. Several compounds have been shown to inhibit fertility by various mechanisms. The most significant of them are reviewed, including some recently described indazole carboxylic acids. Concerning sterility treatment, no effective treatment is so far available, with the exception of hormonal dysfunctions. The hypothesized role of an excess of free radicals in male sterility suggests a potential therapeutic value of free radicals scavengers. In this connection, indazole derivatives appear of special interest, as some of them display an anti-denaturant activity resulting in a peculiar scavenger-like activity. Some synthetic approaches are proposed, leading to new compounds of potential interest either as male contraceptives or in the treatment of male sterility.

Two new male contraceptives exert their effects by depleting germ cells prematurely from the testis.

The three currently available male contraceptive approaches are 1) the barrier method such as the condom, 2) hormonal methods by disrupting the pituitary-testicular axis so as to impair spermatogenesis, and 3) immunological methods by preparing vaccines against male-specific antigens. We hereby describe an alternative approach in which attachments of developing germ cells onto the seminiferous epithelium are disrupted, thereby inducing their premature release into the tubular lumen. This in turn leads to infertility. A panel of analogues based on the core structure of 1-(2,4-dichlorobenzyl)-indazole-3-carboxylic acid was synthesized. These compounds were subjected to an in vivo screening assay assessing their effects in inducing the expression of testin, a testicular marker whose expression correlates with the integrity of Sertoli-germ cell junctions. An induction of testin expression in the testis signifies a disruption of Sertoli-germ cell junctions that is followed by depletion of germ cells from the seminiferous epithelium. Two compounds, namely 1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide (AF-2364) and 1-(2,4-dichlorobenzyl)-indazole-3-acrylic acid (AF-2785), were identified that caused detachment of germ cells, in particular round and elongated spermatids, from the epithelium inducing their premature release into the tubular lumen as confirmed by histological analysis. Adult rats receiving several oral doses of either one of these compounds became infertile within 3-7 wk after the epididymal sperm reserve was exhausted. Depending on the dosing of the administered compound, rats became infertile for 4-14 wk before their fertility gradually bounced back, illustrating the reversibility and efficacy of these new compounds. Also, these compounds did not appear to impair the hypothalamus-pituitary-testicular axis because the serum levels of LH, FSH, and testosterone of the treated animals did not change significantly when compared to control rats. In addition, results of serum microchemistry illustrate that liver and kidney function was not affected in animals treated with both compounds.

Inhibition of heat-induced denaturation of albumin by nonsteroidal antiinflammatory drugs (NSAIDs): pharmacological implications.

The activity of nonsteroidal antiinflammatory drugs (NSAIDs) in rheumatoid arthritis is not only due to the inhibition of the production of prostaglandins, which can even have beneficial immunosuppressive effects in chronic inflammatory processes. Since we speculated that these drugs could also act by protecting endogenous proteins against denaturation, we evaluated their effect on heat-induced denaturation human serum albumin (HSA) in comparison with several fatty acids which are known to be potent stabilizers of this protein. By the Mizushimas assay and a recently developed HPLC assay, we observed that NSAIDs were slightly less active [EC50 to approximately 10(-5)-10(-4) M] than FA and that the HPLC method was less sensitive but more selective than the turbidimetric assay, i.e. it was capable of distinguishing true antiaggregant agents like FA and NSAIDs from substances capable of inhibiting the precipitation of denatured protein aggregates. In conclusion, this survey could be useful for the development of more effective agents in protein condensation diseases like rheumatic disorders, cataract and Alzheimers disease.

Reversible inhibition of spermatogenesis in rats using a new male contraceptive, 1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide.

The oral male contraceptive agent 1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide (AF2364) is a new analogue of indazole-carboxylic acid. AF2364 was orally administered to rats at 50 mg/kg body weight once weekly for five consecutive weeks. The effects on fertility efficacy, hormonal profile, organ weights, tissue morphology, and serum microchemistry were examined. Complete infertility was noted in rats 29 days after the initial dose of AF2364 and continued until 90 days. Fertility resumed in 25% of the group after 104 days and had resumed in 75% of the rats by the last mating at 197 days. Morphological examination of the testis showed rapid exfoliation of elongated spermatids and the generation of large multinucleated cells 6 days after the first treatment, with depletion of most germ cells after 40 days. Normal spermatogenesis was noted in 95% of the tubules in the animals that were fertile at 210 days. Morphological analysis of the epididymal compartments revealed reduced lumen size, whereas the prostate exhibited an increase in the glandular lumen with a reduction in epithelium height. No morphological changes were detected in the kidney, liver, and cerebrum by light microscopy. Kidney and liver function, as evaluated by serum chemistry, were not affected by the drug treatment. AF2364 did not alter the levels of FSH, and only minimal changes were noted for LH and testosterone, suggesting that the hypothalamic-pituitary-testicular axis was not affected. These results illustrate the potential of AF2364 as a male contraceptive.

Inhibition of calcium oxalate precipitation by bile salts.

BACKGROUND: Both urinary and biliary stones can contain calcium. Bile salts (BA), which are known to bind Ca2+, are commonly used to dissolve the latter but not the former. METHODS: The effect of physiologic BA on calcium oxalate (CaOx) precipitation was evaluated by a recently developed method. RESULTS: The Ca2+ binding properties of BA were confirmed by small but significant decreases in pH observed following addition of CaCl2 to bile acids solutions. More importantly, BA inhibited CaOx precipitation with effective concentrations of approximately 10-3 mol/L. The clinical relevance of the latter observation is presently unknown but it is of note that in the same in vitro assay, the activity of BA appeared comparable to that of citric acid, the most common drug for urolithiasis. Although BA do not reach mmol/L levels in urine, they are known to change the physicochemical properties of this fluid, possibly slowing down the crystal growth process. However, the hypothetical therapeutic use of BA in former stone patients would present at least two major problems: (i) hepatotoxicity and (ii) lithogenic activity, due to hyperoxaluria subsequent to increased intestinal absorption of oxalate. CONCLUSION: The ability of BA in effectively binding calcium ions and in inhibiting the precipitation of CaOx appears of interest from both a physiopathologic and a pharmacologic point of view, even if it does not currently seem exploitable for prophylactic or therapeutic purposes.

Immunohistologic markers of immune activation and changes of glycosylation of serum proteins in primary Sjögren's syndrome.

OBJECTIVE: [corrected] To assess the possible correlations between the immune activation of certain surface antigens at the lip salivary gland (LSG) level, and changes in glycosylation of serum proteins in primary Sjögren's syndrome (SS). METHODS: LSG biopsy samples were obtained from 22 SS patients (mean age 56.3 years; mean disease duration 70.8 months) and prepared for immunohistochemical analysis using murine monoclonal antibodies for interleukin-2 receptor (IL-2R) (CD25) and for the class II major histocompatibility antigen HLA-DR. The glycosylation of serum proteins was evaluated in all patients by an enzyme-linked lectin assay (ELLA) using concanavalin A (Con A). RESULTS: In LSG specimens the presence of IL-2R was observed at the infiltrating level, mainly periductally, in 13 (59%) cases and on the epithelial cells of 14 (64%) patients. In 13 out of 22 SS patients (59%) a marked positivity both of the infiltrates and of the epithelium was found for anti-HLA-DR monoclonal antibody. The degree of expression of different antigens on LSG samples was correlated with their histologic class according to Tarpley evaluation. The positivity for IL-2R and HLA-DR molecules on glandular tissues was correlated. A significant increase in the total Con A reactivity of serum proteins was found in those patients expressing IL-2R and HLA-DR antigens on LSG specimens. CONCLUSIONS: The co-expression of IL-2R and HLA-DR antigens on both the epithelium and infiltrates of LSG is consistent with a participation of these cells in the immune process of SS. Moreover, changes in the glycosylation of serum proteins seem to be related to the presence of these immunoactivation markers of the disease at the LSG level, suggesting that the control of protein glycosylation could be mediated by the same mechanisms involved in the tissue damage of SS.

Intracellular signal transduction pathways as targets for neurotoxicants.

The multiple cascades of signal transduction pathways that lead from receptors on the cell membrane to the nucleus, thus translating extracellular signals into changes in gene expression, may represent important targets for neurotoxic compounds. Among the biochemical steps and pathways that have been investigated are the metabolism of cyclic nucleotides, the formation of nitric oxide, the metabolism of membrane phospholipids, the activation of a multitude of protein kinases and the induction of transcription factors. This brief review will focus on the interactions of three known neurotoxicants, lead, ethanol and polychlorinated biphenyls, with signal transduction pathways, particularly the family of protein kinase C isozymes, and discusses how such effects may be involved in their neurotoxicity.

Analysis of lonidamine in rat serum and testis by high performance liquid chromatography.

A HPLC method for the determination of lonidamine in serum and testis, suitable for pharmacological studies in the rat and other mammals, has been developed. Briefly, 0.5 mL of serum or about 0.2 g of testicular tissue were extracted with ethyl acetate and evaporated to dryness under nitrogen. The residue was redissolved in methanol and an aliquot was injected onto a C18 column eluted with a mobile phase consisting of acetonitrile:water (51:49, v/v), containing 0.1% trifluoroacetic acid. The eluate was monitored at 230 nm with a sensitivity of 0.05 AUFS. By this method, the pharmacokinetics and the serum and testicular levels of the drug up to 120 h after the administration of one single dose (100 mg/kg body weight) of lonidamine to Sprague-Dawley rats have been studied. Results were highly variable, as previously reported, but a very good linear correlation was found between the serum and the testicular levels, suggesting that, in the rat, and possibly in the human, testicular levels could be estimated based on the serum concentrations.

Antidenaturant drugs for cataract and other condensation diseases.

'Condensation diseases' are heterogeneous pathological conditions in which the primary pathogenetic step is the loss of solubility of specific substances, resulting in the formation of a condensed phase. Typical examples are cataract, nephrolithiasis, gallstone disease and certain rheumatic conditions in which protein denaturation, aggregation and precipitation may occur. Since the condensing molecules are often proteins, antidenaturant agents should be considered rational drugs for the treatment of these diseases. Surprisingly, however, only a few molecules with these properties are currently available for therapeutic use, including bendazac for cataract.

Effects of harmine on dopamine output and metabolism in rat striatum: role of monoamine oxidase-A inhibition.

In vivo microdialysis was used to investigate the effects of acute injections of harmine on extracellular concentrations of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxindoleacetic acid (5-HIAA) in the striatum of awake rats. Administration of harmine in doses of 0.5, 2.5, and 10 mg/kg (i.p.) elicited a dose-dependent increase of the dopamine efflux to 152, 173, and 243% and a decrease in DOPAC to 52, 36, and 10%, and HVA to 67, 45, and 20% throughout, respectively; 5-HIAA concentrations were decreased to 81, 74, and 72% only. In contrast to D-amphetamine, which also increases dopamine release and decreases its metabolites, the stimulatory action of harmine on dopamine release in the striatum was totally abolished in the presence of tetrodotoxin (1 microM). Similar to monoamine oxidase (MAO)-A inhibitors, harmine potentiated the stimulatory effect of D-amphetamine (10 microM), infused by reverse microdialysis in the striatum, on dopamine release. Pre-treatment with the benzodiazepine receptor antagonist flumazenil (5 mg/kg, i.p.) did not modulate the effect of harmine on striatal dopamine release and metabolism. Administration of the reversible MAO-A inhibitor, moclobemide (20 mg/kg, i.p.), induced an increase in dopamine to 256% and a decrease in DOPAC, HVA, and 5-HIAA to 30, 24, and 62%, respectively, reproducing a pattern similar to that of harmine. Taken together, these results indicate that harmine affects the brain dopamine system probably by acting as a MAO-A inhibitor and not as an inverse agonist for the benzodiazepine receptors.

Toxicity testing in environmental monitoring: the role of enzymatic biosensors.

Biological toxicity testing is a rapidly expanding field involving numerous bioanalytical techniques. The enzymatic biosensors are valuable screening tools to identify pollutants and/or toxic agents in the environment and/or in food matrices, thus representing a valid alternative to animal testing in analytical toxicology. Inhibition based biosensors here presented have been proved to represent alternative assays for the toxicity evaluation of warfare agents and endocrine disrupting chemicals as well as algal toxins (phycotoxins) in the contamined sea foods (mainly clams and other mollusks). Results obtained by inhibition studies performed by means of several enzymatic biosensors indicate the reliability of the proposed method and the possibility to extend such an experimental approach to other toxicants as a simple, rapid and cheap biotest, to be used easily also "on the spot".

Changes of lipocalin type prostaglandin D-synthase in the seminal plasma of subfertile man.

It was proposed that lipocalin type prostaglandin D synthase (L-PGD-S), a bifunctional protein both synthesizing PGD2 and transporting retinoids and other lipophilic ligands, could be involved in the development and the maturation of sperm. In the present study, the seminal plasma (SP) of 59 adult males was analyzed by standard WHO methods and immunoblotting, using a monospecific polyclonal antibody directed against L-PGD-S. Briefly, aliquots of SP (2.5 microl), were fractionated by polyacrylamide electrophoresis in the presence of sodium dodecyl sulfate, the blots were stained and densitometrically analyzed. To obtain quantitative data, the aliquot of SP was selected within the linear part of the dose/band intensity curve and a proper quality control was analyzed in all blots to normalize the intensity of the bands of different experiments. A significant reduction (p<0.05) of the L-PGD-S levels was observed in severe oligozoospermic patients compared to normozoospermic subjects and a significant correlation between L-PGD-S levels and sperm concentration was found, as reported by other authors. Further studies are warranted to evaluate the possible diagnostic and pharmacological applications of these observations.

Relaxant effects of Hydrastis canadensis L. and its major alkaloids on guinea pig isolated trachea.

Hydrastis or goldenseal, one of the most popular medicinal herbs in the U.S.A., is used in mild pathological conditions like cold and flu, based on the pharmacological properties of its active components, berberine (anticholinergic, antisecretory, and antimicrobial) and beta-hydrastine (astringent). We previously reported the relaxant effect of a total ethanolic extract of hydrastis on carbachol precontracted isolated guinea pig trachea, and with the present study, using the same experimental model, we aimed at evaluating the contribution of its major alkaloids, berberine, beta-hydrastine, canadine and canadaline to the total effect. Furthermore, using specific pharmacological tools, like timolol and xanthine amine congener, we attempted to elucidate its mechanism of action. The EC50 of berberine, beta-hydrastine, canadine and canadaline, were 34.2+/-0.6, 72.8+/-0.6, 11.9+/-1.2 and 2.4+/-0.8 microg/ml, respectively. Timolol effectively antagonized the effect of canadine (EC50 = 19.7+/-3.0 microg/ml) and canadaline (EC50 = 17.1+/-1.2 microg/ml) but not that of berberine and beta-hydrastine, while xanthine amine congener antagonized the effect of beta-hydrastine (EC50 = 149.9+/-35.3 microg/ml) and canadaline (EC50 = 26.1+/-3.0 microg/ml) but not that of berberine and canadine. Besides, the hydrastis extract, at concentrations between 0.01 and 0.1 microg/ml, potentiated the relaxant effect of isoprenaline on carbachol-precontracted isolated guinea pig trachea. These data, which are insufficient to draw definite mechanistic conclusions, indicate that the aforementioned alkaloids may act by interacting with adrenergic and adenosinic receptors.

In vitro evaluation of antioxidant activity by electrophoresis and high performance liquid chromatography.

Two methods for the analysis of antioxidants, based on polyacrylamide gel electrophoresis (PAGE) and gel permeation high performance liquid chromatography (HPLC) were developed. Both of them exploit the variations of the signal (band or peak) given by human serum albumin (0.2% w/v in 100 mM sodium phosphate pH 7) upon oxidation with hypochlorite (1% of a solution containing 4% active Cl), quantitatively determined by densitometric analysis or peak integration. Based on such changes, two formulas were defined which allowed the determination of the antioxidant activity of ascorbic acid (EC(50,PAGE)=4.8x10(-4) M, EC(50,HPLC)=3.6x10(-4) M), glutathione (EC(50,PAGE)=1.5x10(-4) M, EC(50,HPLC)=2.0x10(-4) M) and melatonin (EC(50,PAGE)=5.2x10(-4) M, EC(50,HPLC)=3.2x10(-4) M), chosen as reference compounds. A good correlation was found between the activities of these substances in the two assays, which are also in good agreement with literature data, indicating that the two methods are essentially equivalent. These assays could be useful for the screening of new antioxidant drugs for pathological conditions such as cataract, rheumatic diseases, atherosclerosis and Alzheimer's disease.

Changes of acute-phase proteins in streptozotocin-induced diabetic rats.

Quantitative and qualitative changes of serum proteins, apart from glycation, have not been sufficiently studied in streptozotocin-induced diabetic rats (D), the most common experimental model for diabetes. Thus, we decided to analyze the serum of diabetic rats by concanavalin A-blotting in comparison with rats with acute inflammation induced by fermented yeast (Y), in which characteristic alterations of serum proteins have been described. Two months after the streptozotocin treatment, the blood glucose levels were highly elevated (456+/-24 vs. 124+/-10 mg/dl, p<0.001, n=12), the body weight was significantly lower than normal (279+/-10 vs. 392+/-6 g, p<0.001, n=12), and serum proteins appeared to be highly glycated (p<0.001) when analyzed by the fructosamine assay, without any significant change in the total serum protein concentration. Analysis by concanavalin A-blotting, revealed a significant decrease of alpha1-inhibitor-3 (alpha1-I3, p<0.05) and an increase of the beta chain of haptoglobin (beta-Hp, p<0.05) in both D and Y rats (n=3) compared with control animals. However, acute inflammation caused a marked rise of two prominent acute phase proteins, alpha2-macroglobulin and hemopexin, which did not change appreciably in diabetic rats. Further work will be necessary to evaluate the physiopathological significance of these phenomena which could result from changes of both concentration and glycosylation of the aforementioned proteins.

Rat testicular myotubularin, a protein tyrosine phosphatase expressed by Sertoli and germ cells, is a potential marker for studying cell-cell interactions in the rat testis.

The full-length cDNA encoding the entire open reading frame (ORF) of rat myotubularin (rMTM) was isolated from a rat testis expression library by PCR. Among the three approximately 2.9-kb cDNAs that were sequenced, one clone was different from the other two clones. It contained seven extra amino acids of FVVLNLQ; this short stretch of extra sequence was found between Gln(421) and Phe(422) within the SET (Suvar3-9, Enhancer-of-zeste, Trithorax) interacting domain (SID) of rMTM. The rMTM ORF had 1,713 bp encoding for a 571 amino acid polypeptide and a calculated molecular weight of 65.8 kDa. A comparison between its deduced amino acid sequence and the GenBank database using BLAST revealed a 53.1% identity with human myotubularin protein (hMTM1), which is a member of the protein tyrosine phosphatase (PTP) family associated with X-linked myotubular myopathy. A 22 amino acid peptide NH(2)-TKVNERYELCDTYPALLAVPAN was synthesized based on the deduced amino acid sequence of rMTM and used for antibody production. By using immunoblot analysis, a 66-kDa protein was indeed detected in both Sertoli and germ-cell cytosols. rMTM mRNA was found in various tissues but was predominantly expressed in the testis, ovary, and skeletal muscle. Sertoli cell rMTM expression was stimulated by germ cells and enhanced when inter-Sertoli junctions were being assembled in vitro. A drastic reduction in testicular rMTM steady-state mRNA level correlated with the depletion of germ cells from the testis in vivo following either glycerol or lonidamine treatment. These results indicate that rMTM is a rat homologue of hMTM1 that may be a useful marker in monitoring the events of cell-cell interactions in the testis.

Lonidamine and analogue AF2785 block the cyclic adenosine 3', 5'-monophosphate-activated chloride current and chloride secretion in the rat epididymis.

The cystic fibrosis transmembrane conductance regulator (CFTR) or the small conductance cAMP-activated chloride channel encoded by the CFTR gene has been shown to play an important role in the formation of the epididymal fluid microenvironment. Mutation of the gene has led to widespread effects on male reproduction. Like other ion channels, CFTR is amenable to pharmacological intervention. Blocking CFTR in the epididymis could in principle lead to disruption of the epididymal fluid environment. We report for the first time two indazole compounds: lonidamine and 1-(2, 4-dichlorobenzyl)-indazole-3-acrylic acid (AF2785) are potent blockers of CFTR in the epididymis. When added to the external solution under whole-cell patch clamp conditions, AF2785 and lonidamine inhibited the cAMP-activated chloride current in rat epididymal cells with apparent IC(50) values of 170.6 and 631.5 microM, respectively; by comparison the IC(50) value for diphenylamine-2-carboxylate, a well-known chloride channel blocker was 1294 microM. In cultured rat epididymal epithelia mounted in a Ussing chamber, AF2785 and lonidamine inhibited the cAMP-stimulated short-circuit current (a measure of chloride secretion) when added to the apical bathing solution with potency greater than any known chloride channel studied. It is proposed that in view of the important role CFTR plays in male reproduction, further study with these and other new indazole compounds for their CFTR blocking actions can provide a new avenue of research into the development of novel male contraceptives.

[Nephrolithiasis]. / La calcolosi renale.

The development of efficacious techniques for stone elimination, and in particular of extracorporeal lithotripsy, slackened, in the last two decades, the pharmacological research on nephrolithiasis, with the result that the currently available efficacious drugs are very few. Instead, the recent elucidation of some relevant etiopathogenetic aspects, with special reference to the quantitative and qualitative changes of specific urinary inhibitors of crystallization, could open new therapeutic avenues for many lithiasic forms, now considered idiopathic.

Effects of lonidamine on testicular and epididymal proteins in the rat.

The mechanism responsible for the antispermatogenic activity of lonidamine (LND) [1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxylic acid], a drug with low systemic toxicity and lack of significant hormonal effects, is still unclear but may be related to alterations of Sertoli cell proteins. Here, we confirmed that a single oral dose of LND (100 mg/kg b.w.) to sexually mature Sprague-Dawley rats causes shrinkage and weight reduction of the testes after 48 h. These macroscopic changes correlated with histologic alterations revealed by light microscopy, consistent with partially reversible inhibition of spermatogenesis. When the testes and the epididymides of animals treated with or without LND were homogenized and analyzed by the Bradford assay, a significant increase of total protein content was observed after 24 and 48 h. When these homogenates were analyzed by concanavalin blotting, specific changes of the major rat macroglobulins, i.e. alpha(1)-inhibitor-3, alpha(2)-macroglobulin, and alpha(1)-macroglobulin, were noted. In particular, LND caused a decrease of testicular alpha(1)-inhibitor-3, but not an increase of testicular alpha(2)-macroglobulin, indicating a mild local inflammatory response to the drug.

Aflatoxins inhibit prolactin secretion by rat pituitary cells in culture.

Acute effects of aflatoxins (AF), and in particular hormonal actions, have not been examined as much as chronic toxicity. Thus, we studied the effects of specific AF on prolactin (PRL) secretion by rat pituitary cells in culture. AFB1 and AFQ1 (1 x 10(-4) M) reduced the stimulating effect of dimethyl sulfoxide on PRL secretion by cultured rat pituitary cells. The mechanism responsible for this action is still unknown, but it did not seem to be a non specific toxic effect, because AFB1, at the same concentration, did not significantly alter cell viability, as indicated by the Trypan blue dye-exclusion test.