Regulacijski mehanizmi gonadotropnog sustava sisavaca pod djelovanjem organokloriranih i S-triazinskih spojeva : doktorska disertacija - Šimić, Branimir, biokemičar; Kniewald, Jasna; Rendić, Slobodan; Lipovac, Krešo; Čepelak, Ivana (Zagreb : B. Šimić, 1995)
Sažetak: Mehanizam djelovanja androgenih hormona vezan je uz metaboličke procese u neuroendokrinim i ciljnim tkivima, te uz nastajanje specifičnih androgen-receptor kompleksa, kao preduvjetom za daljnji slijed fizioloških procesa i ispoljavanje krajnjih učinaka u organizmu. Testosteron se u androgen-ovisnim tkivima reducira djelovanjem 5[alfa]-reduktaze u 5[alfa]-dihidrotestosteron (DHT), koji posjeduje najizraženiju androgenu aktivnost. Djelovanjem 3[alfa]-hidroksisteroid dehidrogenaze (3[alfa]-HSD) se DHT reducira u 5[alfa]-androstan-3[alfa],17[beta]-diol (3[alfa]-diol). Istovremeno uslijed djelovanja 17[beta]-hidroksisteroid dehidrogenaze (17[beta]-HSD) iz testosterona nastaje androst-4-en-3,17-dion (androstendion), te 5[alfa]-redukcijom 5[alfa]-androstan-3,17-dion (5[alfa]-dion).
DHT se u androgen-ovisnim tkivima veže na specifične receptore u citoplazmi, čime počinje proces translokacije aktiviranog hormon-receptor kompleksa u jezgru, vezanja na kromatin, te se procesom transkripcije omogućava sinteza novih proteina. U ovom radu je ispitivan in vitro i in vivo utjecaj organokloriranog insekticida lindana, s-triazinskih herbicida atrazina i prometrina i organofosfornog insekticida malationa na regulacijske mehanizme reprodukcijskih procesa u hipotalamo-hipofizno-gonadnom sustavu: na metaboličku pretvorbu testosterona i na nastajanje DHT-receptor kompleksa. Također je ispitivan utjecaj atrazina na reprodukcijsku sposobnost štakora. U hipofizi mužjaka štakora in vitro lindan inhibira aktivnost 5[alfa]-reduktaze i 17[beta]-HSD, a u hipotalamusu samo aktivnost 5[alfa]-reduktaze. Inhibicije su potpuno nekompetitivnog tipa.
Smanjeno nastajanje 3[alfa]-diola u oba tkiva in vitro posljedica je inhibirane redukcije testosterona u DHT kao supstrata za 3[alfa]-HSD. U hipotalamusu mužjaka štakora tretman lindanom dnevnom dozom od 60 mg kg-1 tjelesne mase per os, tijekom 7 dana, ne izaziva značajne promjene u metabolizmu testosterona. Tretman nižom dozom (20 mg kg-1) izaziva stimulirajući učinak na aktivnost 5[alfa]-reduktaze i 3[alfa]-HSD. U hipofizi prepubertalnih štakora su neposredno poslije tretmana lindanom (dnevne doze od 60 mg kg-1) stimulirane aktivnosti 5[alfa]-reduktaze, 3[alfa]-HSD i 17 [beta]-HSD. Sedam dana nakon završetka tretmana enzimski su sustavi inhibirani. U hipofizi spolno zrelih mužjaka štakora djelovanje lindana in vivo inhibira aktivnost 5[alfa]-reduktaze i 17[beta]-HSD, dok se aktivnost 3[alfa]-HSD povećava.
U prostati prepubertalnih štakora lindan potiče metaboličku pretvorbu testosterona in vitro, a u prostati spolno zrelih životinja djeluje kao inhibitor 5[alfa]-reduktaze i 17[beta]-HSD. U testisu štakora promjene u metabolizmu testosterona u prisutnosti lindana su izraženije uz niske koncentracije lindana. Atrazin u hipofizi prepubertalnih mužjaka štakora in vitro inhibira aktivnost 5[alfa]-reduktaze, a izaziva povećanje aktivnosti 3[alfa]-HSD. In vivo (doza atrazina od 120 mg kg-1 tjelesne mase tijekom 7 dana) povećava pretvorbu testosterona u androstendion i 5[alfa]-dion. Promjene su ispoljene i sedmog dana nakon završetka tretmana povećanom aktivnosti 5[alfa]-reduktaze. Neposredno nakon tretmana atrazinom spolno zrelih mužjaka štakora pretvorba u androstendion je inhibirana, a aktivnosti 5[alfa]-reduktaze i 3[alfa]-HSD su povećane.
Redukcija u DHT sedmog je dana nakon završetka tretmana inhibirana. U prostati i atrazin i prometrin inhibiraju 5[alfa]-reduktazu in vitro, dok pretvorbu testosterona u androstendion i 5[alfa]-dion atrazin stimulira i in vitro i in vivo. Lindan i atrazin pokazuju izraziti inhibicijski učinak na nastajanje specifičnog DHT-receptor kompleksa i in vitro i in vivo u prostati štakora. Inhibicija je potpuno nekompetitivnog tipa, a promjene su reverzibilne. Malation također inhibira mahanizam nastajanja DHT-receptor kompleksa u prostati štakora. Lindan i malation u smjesi, kao i smjesa atrazina i prometrina, suprimiraju redukciju testosterona u DHT u prostati štakora in vitro jače nego pojedini herbicid u istim koncentracijama zasebno.
Osim toga, ove smjese pesticida povećanom aktivnosti 3[alfa]-HSD usmjeravaju metabolizam testosterona in vitro na produkte slabijeg androgenog djelovanja. Smjesa lindana i malationa izraženije inhibira proces vezanja DHT na receptore u citosolu prostate, nego svaki insekticid pojedinačno. Učinci lindana i atrazina na reprodukcijski sustav mužjaka štakora su reverzibilni, ali u mladih životinja izraženiji i dugotrajniji. Lindan različito djeluje na neuroendokrine strukture mužjaka štakora ovisno o starosnoj dobi životinja. Izazvane promjene su rezultat kombiniranog djelovanja na više kontrolnih mehanizama u metabolizmu androgenih hormona. Mogle bi biti posljedica neuroloških promjena u središnjem živčanom sustavu, kao i neposrednog djelovanja u samom tkivu. Atrazin ispoljava u mužjaka štakora kombinirana antigonadotropna i antiandrogena svojstva.
Djelovanje slično antiandrogenima pokazuje se kao primarni učinak u fazi izloženosti životinja i neposredno poslije tretmana: promjene u metabolizmu testosterona u hipofizi, u nastajanju DHT-receptor kompleksa u citosolu prostate, te promjene masa sjemenih mjehurića i prostate. Antigonadotropna aktivnost je iskazana i u poslijetretmanskom periodu: promjena aktivnosti 5[alfa]-reduktaze u hipofizi, povećana masa hipofize. Djelovanje antrazina u reprodukcijskom sustavu ženki štakora sličnije je progestagenim nego estrogenim svojstvima. Atrazin izaziva poremećaj ovulacijskog ciklusa ometajući mehanizam proestrusa, te promjene u masi hipofize, uterusa i ovarija. Promjene fizioloških procesa u uterusu, gonadama i hipotalamo-hipofiznom sustavu ženki štakora izazvane djelovanjem atrazina su reverzibilne.
Rezultatima ovog rada pokazano je da izloženost organokloriranim, s-triazinskim i organofosfornim pesticidima može znatno utjecati na fiziološke procese uključene u sustav reprodukcije. Lindan, atrazin, prometrin i malation interferiraju s biokemijskim procesima u neuroendokrinim organima i androgen-ovisnim tkivima, te djeluju na gonadotropni mehanizam u mužjaka štakora. U ženki štakora atrazin blokira estrogen-ovisne funkcije hipofize i uterusa, te djelujući antiestrogeno izaziva poremećaj neuroendokrine kontrole funkcija ovarija. Stoga zagađenost okoliša pesticidima predstavlja opasnost, kako za sadašnju populaciju, tako i za buduće generacije, zbog njihove izrazite postojanosti u prirodi i mutagenih promjena koje oni mogu izazvati.
Summary: The enzymatic conversions of androgen hormones, as well as the formation of specific androgen-receptor complex in neuroendocrine structures and target tissues, are the prerequisite for androgen action in the physiological processes and for the exertion of a final androgenic effect. Through the influence of 5[alfa]-reductase androgen-dependent tissues metabolize testosterone into 5[alfa]-dihydrotestosterone (DHT), which is known to possess the highest androgenic activity. Subsequently, 3[alfa]-hydroxysteroid dehydrogenase (3[alfa]-HSD) converts DHT into 5[alfa]-androstane-3[alfa],17[beta]-diol (3[alfa]-diol).
Another metabolic pathway exists in the central and peripheral tissues, through which testosterone is converted into androst-4-ene-3,17-dione (androstenedione) under the influence of 17[beta]-hydroxysteroid dehydrogenase (17[beta]-HSD), and further through the 5[alfa]-reduction into 5[alfa]-androstane-3,17-dione (5[alfa]-dione). In androgen-dependent tissues DHT forms a complex with its specific cytoplasmic receptor. This is followed by translocation of activated hormone-receptor complex into the nucleus, binding to chromatin, induction of transcription and initiation of protein synthesis.
In this study, the in vitro and in vivo effects of an organochlorine insecticide lindane, of s-triazine herbicides atrazine and prometryne, and of an organophosphorus insecticide malathion on the mechanisms which regulate the reproductive processes in hypothalamo-pituitary-gonadal axis (the conversion of testosterone into its metabolites, and the formation of DHT-receptor complex) were investigated. Also, the reproductive performance in the rat under the influence of atrazine was studied. Lindane inhibited in vitro the activities of 5[alfa]-reductase and 17[beta]-HSD in male rat pituitary, and of 5[alfa]-reductase in hypothalamus. The inhibition of both enzymes was characterized as a fully noncompetitive.
In the presence of lindane the suppressed conversion of testosterone into DHT, which is the substrate for 3[alfa]-HSD, resulted in the lower formation of 3[alfa]-diol. A lindane oral dose of 60 mg kg-1 bw, given daily for 7 days, did not cause any significant changes of testosterone metabolism in male rat hypothalamus. The treatment with a lower lindane dose (20 mg kg-1) stimulated the activities of 5[alfa]-reductase and of 3[alfa]-HSD. On the first day after the termination of lindane treatment (daily dose of 60 mg kg-1 bw) the activities of 5[alfa]-reductase, of 3[alfa]-HSD, and of 17[beta]-HSD were stimulated in the pituitary of prepubertal rat. Seven days following the termination of treatment these enzymic systems were inhibited.
In sexually mature rats lindane enhanced the activity of pituitary 3[alfa]-HSD, but inhibited the activities of 5[alfa]-reductase and of 17[beta]-HSD. In the prepubertal rat prostate lindane stimulated the in vitro metabolism of testosterone, while in the prostate of sexually mature rat lindane acted as an inhibitor of the activities of 5[alfa]-reductase and of 17[beta]-HSD. In rat testis, the changes of testosterone metabolism in vitro were evident in the presence of low lindane concentrations only. Atrazine inhibited in vitro the activity of 5[alfa]-reductase, and it caused an augmentation of the activity of 3[alfa]-HSD in the pituitary of prepubertal rat. In vivo, atrazine in a daily dose of 120 mg kg-1 bw for 7 days augmented the conversion of testosterone into androstenedione, and further into 5[alfa]-dione.
An increase of 5[alfa]-reductase activity was found seven days following cessation of the treatment. In sexually mature rat the conversion of testosterone into androstenedione was inhibited, but the activities of 5[alfa]-reductase and of 3[alfa]-HSD were augmented on the first day after the termination of atrazine treatment. On the seventh post-treatment day the conversion of testosterone into DHT was inhibited. In the prostate, both atrazine and prometryne inhibited the activity of 5[alfa]-reductase in vitro. The conversion of testosterone into androstenedione and further into 5[alfa]-dione was stimulated under the in vitro and in vivo influence of atrazine. Both lindane and atrazine exerted in vitro and in vivo the strong inhibitory effects on the formation of specific DHT-receptor complex in rat prostate cytosol.
The inhibition was characterized as a fully noncompetitive, and the reversibility of changes in the complex formation was established. Also, malathion inhibited the mechanism of DHT-receptor complex formation. The mixture of lindane and malathion, as well as the mixture of atrazine and prometryne, brought about a higher suppression of the in vitro conversion of testosterone into DHT in rat prostate, than each pesticide alone in the same concentrations. In the addition, these mixtures of pesticides directed the in vitro testosterone metabolism in prostate towards the metabolites of lower androgenic potency through the stimulation of 3[alfa]-HSD activity. Also, the mixture of lindane and malathion inhibited the process of DHT binding to its specific receptors in rat prostate cytosol more pronounced than each of the pesticide separately.
The changes caused by either lindane or atrazine in the reproductive system of male rat are reversible, but more expressed and of a longer duration in young than in sexually mature animals. Depending on the age of rat, the influence of lindane on the neuroendocrine structures is different and it is the result of combined action on several control mechanisms responsible for the metabolism of androgen hormones. This could be the consequence of neurological changes in the central nervous system caused by lindane, as well as of its direct action in target tissue cells. Atrazine exerted the combined antigonadotropic and antiandrogenic properties in male rat.
The antiandrogen-like action has been expressed as the primary effect of the treatment: the changes of testosterone metabolism in the pituitary and of DHT-receptor complex formation in rat prostate cytosol; the changes in the weight of seminal vesicles and of prostate. The antigonadotropic activity has been shown during the post-treatment period: the changes of pituitary 5[alfa]-reductase activity and the augmentation of the pituitary weight. In the female rat reproductive system atrazine exerted more progestational activity than estrogen-like effects. By interfering with the mechanism of proestrus atrazine disturbed the ovarian cyclicity, and caused the changes of pituitary, uterus and of ovarian weight. The changes of the physiological processes in uterus, ovary and hypothalamo-pituitary system are reversible.
The present results show that the exposure to organochlorine, s-triazine or organophosphorus pesticides may considerably affect the physiological processes involved in the reproductive system. Lindane, atrazine, prometryne and malathion interfere with the biochemical processes in neuroendocrine organs and androgen-dependent tissues, and they exert the influence on the gonadotropic mechanism in male rat. In female rat atrazine disrupts the estrogen-dependent functions of pituitary and uterus. Through its antiestrogenic activity atrazine disturbs the neuroendocrine control of the ovarian function. In consideration of their persistence in the environment, and their possible mutagenic effects, the contamination with pesticide residues indicates the risk for the present population, as well as for the future generations.
Doktor medicinskih znanosti - medicina
mentor: Jasna Kniewald; Komisija za ocjenu: Slobodan Rendić, Jasna Kniewald, Krešo Lipovac, Ivana Čepelak; Komisija za obranu: Slobodan Rendić, Jasna Kniewald, Krešo Lipovac, Ivana Čepelak; datum obrane 22.12.1995.; datum prormocije 10.05.1996.
Sveučilište u Zagrebu, Farmaceutsko-biokemijski fakultet, Zagreb, 1995
Bibliografija: str. 108-122
Summary
Keywords: Humana reprodukcija -- Poremećaji -- Kemijski onečišćivači