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Studies on Neuroendocrine Effects of Atrazine in Male Rats

Figure 1 presents the introduction sentences to the lecture. The sentence given by McGeer in Figure 2 probably is the crucial point to express the importance of the brain, its function and understanding all the mechanisms that are involved in the brain activities. Figures 3-10 very briefly present the main neuroendocrine and gonadal functions in the male reproductive processes. Feedback regulation of testicular function is given in Figure 3. Testicular function is regulated by LH and FSH. LH stimulates steroidogenesis and testosterone production by binding to receptor on the plasma membrane of the Leydig cells and activating adenylate cyclase thus increasing intracellular cAMP. This feedback appears to be accomplished at the hypothalamus through inhibition of GnRH release, GnRH production, or both. GnRH is normally released in a pulsatile fashion, and maximal production of the gonadotropins (LH and FSH) occurs only when this pulsatile pattern of GnRH release is stimulated. Prolonged exposure to constantly elevated levels of GnRH appears to result in target-cell desensitization and a profound suppression of LH and FSH release ; thus long-acting GnRH analogs are being tested as contraceptive agents. Feedback regulation of FSH secretion includes FSH binding to the Sertoli cells and promotes the synthesis of androgen-binding protein (ABP). ABP is glycoprotein that binds testosterone, but is distinctive from intracellular androgen receptor. Figure 4 illustrated the general features of the lipophilic steroid hormone. These lipophilic molecules diffuse through the plasma membrane of all cells, but only encounter their specific, high-affinity receptor in target cells. The hormone-receptor complex next undergoes a temperature and salt-dependent activation reaction that results in size, conformation and surface charge changes that render it able to bind to chromatin. The "activation" process is starting at cytoplasmic level and the complex is translocated to nucleus. Hormone-receptor complex binds to specific regions of DNA and activates or inactivates specific genes. By selectively effecting gene transcription and the production of the respective mRNAs, the amounts of specific proteins are changed and metabolic processes are influenced. The effect of each steroid hormone is quite specific. A multicellular organism is useful to devide into three compartments as is presented on Figure 5. The producing cell features a production machinery, i.e. the ensamble of molecules and organelles needed in the case of neuronal cells to reuptake a signal. A target cell features two properties, receptivity and responsivity. Any exogenous signal may change these properties. Figure 6 shows the known action of steroid-receptor complexes. Receptors for several classes of steroid hormones have been cloned and their primary structures - aminoacid sequences delineated from cDNA. Steroid receptor was divided into domains A-E. More conserved is domain C, proposed to interact with DNA. Domain E contains the steroid binding site. Domain D contains the nuclear localization signal. As is shown on the intercept picture, only a small part of domain C directly contacts DNA, i.e. a double loop structure at which Zn atoms acts as structural organizers thus forming what is called a "zinc finger". The physiological action of testicular hormone is presented in Figure 7 in which are involved androgens, testosterone and DHT. The classic target cells for DHT are prostate, seminal vesicles, external genitalia and genital skin. Targets for testosterone include embryonic structures, spermatogonia, muscles, bone, kidney and brain. Free testosterone enters cells through the plasma membrane by either passive or facilated diffusion. Target cells retain testosterone, presumably because the hormone associates with a specific intracellular receptor. Target cell contains 5a-reductase, that converts testosterone to DHT. The affinity with the receptor for DHT exceeds that for testosterone. Nuclear localization of testosterone/DHT-receptor complex is a prerequisite for androgen action. Binding of the receptor-steroid complex to chromatin may involve a subsequent activation step. T/DHT-receptor presumably activates specific genes. Shematic presentation of androgen biosynthesis in the testicular microsomal membrane is shown in Figure 8. The five enzymes are localized in the microsomal fraction in rat testis. Various substrates for testosterone biosynthesis can enter the microsomal compartment and might proceed via the D4 -pathway from one reaction to the next. The suggested characteristics for the steroid hormone binding site, i..e. for progesterone is shown in Figure 9. Progesterone binding globulin as a folded polypeptide chain is surrounding in some fashion the steroid molecule. A hydrogen donor forms a hydrogen bond with 3-oxo group, which is essencial for strong interaction. Other bindings are tight hydrophobic. All together creates conformational changes of protein receptor to fit with steroid hormone. Any inhibition changes the conformation, an inhibitor binding on the receptor but not on the active site disturhed the process of conversion. Pesticides act by this way. Figure 10 presents shematically 5a-reductase deficiency or receptor disorders. In both cases in which either testosterone or DHT receptor is detected abnormal in some manner, there are a number of cases in which all measureble entities, including receptor, are normal, but the patients (always genetic males) have variable degree of feminization. The extent of the abnormality of differentiation is related to the severity of the deficit. Genetic males who completely lack functioning receptors have testis and produce testosterone, but have complete feminization of the external genitalia (so called "testicular feminization syndrom"). It is interesting that no comparable deficiences in estrogen synthesis or action have been identified. In consideration to these specific action of steroid hormones presented by the above very short explanations, the aim of our reserach about possible atrazine effects in the different reproductive responsible tissues are presented on Figure 11. Figures 12 to 16 show briefly the different protocols used in the research: 14C-testosterone for detection of testosterone metabolites and involved enzyme activities ; Scatchard analysis of specific binding sites on receptors for 3H-DHT ; detection by SDS-PAGE low molecular proteins bands in pituitary tissue ; histochemical and immunohistochemical studies of ultrastructural changes in male rat pituitary, testis, prostate and seminal vesicles. Figure 17 presents the summary of in vitro and Figures 18 and 19 in vivo possible influence of atrazine and its metabolite deethylatrazine on testosterone conversion into DHT (5a-reductase activity). Details can be found in the published papers: Babić-Gojmerac, T., Kniewald, Z., Kniewald, J.: Testosterone metabolism in neuroendocrine organs in male rats under atrazine and deethylatrazine influence, Journal of steroid Biochemistry 33 (1989) 141-146 ; Kniewald, J., Osredečki, V., Gojmerac, T., Zechner, V., Kniewald, Z.: Effects of s-triazine compounds on testosterone metabolism in the rat prostate, Journal of Applied Toxicology 15 (1995) 215-218. Figure 20 presents testosterone conversion into metabolites in anterior pituitary of 21 days male rats from mother treated with atrazine or deethylatrazine (1.66 mg/kg b.w./ daily) s.c. from the first day of pregnancy. Details can be find in the published paper: Kniewald, J., Peruzović, M., Gojmerac, T., Milković, K., Kniewald, Z.: Indirect influence of s-triazines on rat gonadotropic mechanism at early postnatal period, Journal of steroid Biochemistry 27 (1987) 1095-1000. The reversibility of atrazine influence (120 mg/kg b.w./7 days/daily p.o.) on DHT-receptor complex formation in the rat prostate cytosol of 28-day-old and adult (90 days) Fisher rats, is shown on Figure 21. Details can be find in the published paper: Šimić, B., Kniewald, Z., Davies, J., Kniewald, J.: Reversibility of the inhibitory effect of atrazine and lindane on cytosol 5a-dihydrotestosterone receptor complex formation in rat prostate. Journal of Environmental Contamination and Toxicology 46 (1991) 92 - 99. Sucrose density gradient pattern of DHT-receptor complex in 28-day-old male rat hypothalamus is presented in Figure 22. Atrazine in vitro decreases 8S fraction vs. control value. Details are in the paper: Kniewald, J., Mildner, P., Kniewald, Z.: Effects of s-triazine herbicides on 5a-dihydrotestosterone receptor complex formation in hypothalamus and ventral prostate, in Pharmacological Modulations of Steroid Action (Genazzani et al, eds.), Raven Press, New York (1980) pp.159-169. Detection of possible in vivo effects of atrazine (120 mg/kg b.w./7 days/daily p.o.) on low molecular weight proteins in male rat pituitary is presented in Figures 23-25. After the purification of protein bands by gel filtration the decrease of 18% in protein band 24 kD was found to correspond to LH. Possible influences of atrazine treatment (120 mg/kg b.w./60 days/twice a week) on ultractructural changes in male rat hypothalamus, pituitary, testis, prostate and seminal vesicles are shown on Figures 26-30. There were not any histochemical differences between control and atrazine treated male and female (120 mg/kg b.w./10 days/daily) at the basal hypothalamus. Immunohistochemical test on LH and FSH did not show any significant presence of LH or FSH in hypothalamic tissue. The possible presence of LH or FSH in this area necessary for maintenance of feedback mechanism, is probably under the dectability of this method. Therefore in the future the sensitivity of the method should be increased by application of immunofluorescency and electron microscopy. Figure 27 presents immunohistochemical and histochemical treated slices of male rat pituitary. By colouring with hemalaum-eosin, basophilic pituitary cells are colored. By application of immunohistochemical test it was possible to differentiate very clearly anterior lobe (pars distalis) with high LH and FSH activity as well as posterior lobe. The FSH is positive in anterior lobe but not on the other parts of pituitary. Atrazine stimulates synthesis of FSH in males, while in females FSH was reduced and LH increased after atrazine treatment. Cross-sectionated seminiferus tubule from rat testis is shown in Figures 28 and 29. Between the seminiferus tubules the interstitial tissue with red colored Leydig cells are seen. On the periphery of seminiferus tubules from control rat the primary and secondary spermatocytes and spermatogonia are found. The tails of spermatozoa are present towards the periphery of seminiferous tubules. In atrazine treated testes many structural changes could be detected. An irregularity in the center of semiferous tubules is present, primary and secondary spermatocytes and spermatogonia are present, and the tails of spermatozoa are distributed irregularly in the center and in periphery of the lumen. Until now just a small part of samples were examined by electron microscopy and in the lumen were found macrophages, that usually are not present in this tissue. In testes by approving immunohistochemical test for FSH (Figure 29), in control semiferus tubules, FSH is dominantly present on the periphery and is concentrated within the spermatocytes or spermatogonia. In atrazine treated rats, FSH presence is within the whole irregular organized seminiferus tubules. Further research on LH detection and electron microscopy will try to explain why the higher presence of FSH at anterior pituitary level in atrazine treated male rats has the higher content also in testes. The androgen synthesis, testosterone and its conversion to active DHT are significantly reduced. Histochemistry of prostate tissue (Figure 30) shows the tubular alveolar gland with large irregular cavities. It was not possible to detect any significant difference between control and atrazine treated animal. Our previous research on DHT-receptors in prostate, showed the reduction of DHT specific binding sites on receptor molecules. This is in the direct correlation with activity of LH in androgen synthesis and metabolism. At seminal vesicles evidently mucosal folds are more tighty packed in atrazine treated vs. control. The weight of seminal vesicles in atrazine treated rat was much lower and this picture shows a typical atrophy of the gland (Figure 30). Figure 31 shows the summary of our presented results. Further experiments are in progress and will be discussed in some future occasions.

atrazine; male rat; reproduction; neuroendocrine tissues

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