SVIBOR - Project code: 4-07-059

MINISTRY OF SCIENCE AND TECHNOLOGY

Strossmayerov trg 4, HR - 10000 ZAGREB
tel.: +385 1 459 44 44, fax: +385 1 459 44 69
E-mail: ured@znanost.hr

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Project code: 4-07-059


BIOLOGICAL MARKERS IN REPRODUCTIVE TOXICOLOGY


Main researcher: KNIEWALD, JASNA (21481)


Assistants
Type of research: basic
Duration from: 02/01/91. to 02/28/96.
Papers on project (total): 52
Papers on project quoted in Current Contents: 8
Institution name: Prehrambeno-biotehnološki fakultet, Zagreb (58)
Department/Institute: Dept. for chemistry and biochemistry, Laboratory for toxicology Dept. for biochemical engineering, Laboratory for technology of vitamins and hormones
Address: 41000 ZAGREB, Pierottijeva ul.6
City: 10000 - Zagreb, Croatia
Communication
Phone: 385 (0)1 440-202
Fax: 385 (0)1 418-230
E-mail: knic@mapbf.pbfrng.hr
Summary: The influence of the selected pesticides, organochlorine insecticides, lindane and malathion, and s-triazine herbicides, atrazine and prometryne, on the biochemical processes responsible for androgen and estrogen activities in the process of reproduction, was studied. Results demonstrate the strong inhibitory effects of pesticides on the biological markers selected as three groups of enzymes responsible for testosterone metabolism - 5 alpha-reductase, 3 alpha- and 17 beta - hydroxysteroid dehydrogenase in rat and porcine anterior pituitary and prostate, either in vivo or in vitro. Binding of dihydrotestosterone (DHT) to its specific receptors was followed by measuring the number of specific binding sites on the receptor molecule, under in vitro and in vivo influence of the selected pesticides. The significant impact of lindane and atrazine on that process, detected in rat prostate as a model system, was shown as a decrease in the number of the specific binding sites in the cytosol. Also in rat uterus significant decrease of the formation of oestradiol-receptor complex was detect under the influence of atrazine and prometryne, whereas Kd values remain the same order of magnitude. The reproductive performance in rats after in vivo exposure to atrazine was demonstrated on ovarian cycling, male sexual activity, on litter size and on the rate of conception during the mating of Fo and F1 generations. The possible prolonged effect of premating atrazine on rat females and their offsprings indicate a longlasting effect of premating atrazine treatment on the treated females and on the behaviour of their adult offsprings. Changes in the content of polypeptides Mr 25000 and 28000 in rat and porcine anterior pituitary were found after either in vivo or in vitro atrazine and lindane influence. After cytosol protein purification it was possible to detect that changes correspond to prolactin and growth hormone (GH). Development and introduction of cell culture technology of pituitary, ovarian and Leydig cells in monolayer, as well as plant cells are followed by electron microscopy and in our study is providing faster testing of endogenous effects of pesticides and other biologically active compounds or their metabolites.

Keywords: neuroregulation, steroid hormones, pesticides, receptors, 5 - alpha reductase, pituitary, hypothalamus, prostate, ovary, reproduction, in vivo, in vitro, toxicology, environment, feedback mechanism, 17 beta - hydroxysteroid dehydrogenase, 3 alpha - hydroxysteroid dehydrogenase, rat, sheep, pig

Research goals: The direct action of pesticides on target tissues, responsiblefor biochemical processes in the reproductive system, and on thecentral nervous system have not been studied enough. Pesticidesin micro and submicro amounts are able to influence thebiochemical processes responsible for the reproductive systemwith the hereditary effects. Aim of our work was to study theinfluence of some representative pesticides commercially used inCroatia, organochlorine insecticides, lindane and malathion, ands-triazine herbicides, atrazine and prometryne, to thebiochemical processes in the gonadotrophic feedback mechanisms ofmammals, based on our previously published results. Our studyincluded the influence of these pesticides on the binding ofsexual hormones to the receptors, on the activities of the enzymes responsible for the normal function of androgen hormones in the gonadotropic feedback mechanism and to introduce cellculture technology in providing faster testing of endogenouseffect of pesticides. Both in vitro and in vivo, neuroimpact ofselected pesticides was measured at the anterior pituitary andhypothalamic levels, by the study of inhibitory effects on thebiological markers selected as three groups of enzymes,responsible for testosterone metabolism - 5 alpha-reductase, 3alpha- and 17 beta - hydroxysteroid dehydrogenase. Throughout our research the obtained results demonstrate the inhibitory effects of lindane, malathion, atrazine and prometryne, on all tested enzymes. In our study binding of DHT to its specific receptorswas followed by measuring the number of specific binding sites onthe receptor molecules at the cytosol level, under in vitro andin vivo influence of the selected pesticides. At the same time Kd values remained unchanged; type of binding remain the same, but the degree of free specific binding sites was lower. The kinetic studies of inhibition confirm a fully non-competitive type of inhibition. The reproductive performance in rats after in vivo exposure to atrazine demonstrated the loss of body weight in Fo females, in Fo males a decrease in prostate and seminal vesicle weights. In Fo females atrazine induces transient prolongation of the estrous cycle. The results indicate a longlasting effectof premating atrazine treatment on the treated females and on the behaviour of their adult offsprings. Changes in the low molecular weight protein content in rat and porcine pituitary Mr 25000 and28000 were found after either in vivo or in vitro atrazine orlindane influence. Purification of porcine cytosol anteriorpituitary proteins enabled us to detect that the changescorrespond in the decrease of prolactin and a lower increase in GH. The cell culture technology of pituitary cells, ovariancells and Leydig cells was introduced to provide faster testing of endogenous effects of pesticides. Leydig cells isolated from rat testis were used as the biological marker for testosterone biological activity, and the study of progesterone metabolism by 17 beta-hydroxysteroid dehydrogenase and the influence of antiandrogens and atrazine.

COOPERATION - PROJECTS


  1. Name of project: PN 873 Hormones, reproduction and environmental influence; Principal Investigator Kniewald, Jasna
    Name of institution: USDA Animal Science Institute, Germplasm and Gamete Physiology Laboratory, Livestock and Poultry Sciences Institute, Beltsville Agricultural Research Center
    City: 20705 - Beltsville, Maryland, USA

  2. Name of project: Usvajanje i razvijanje tehnologije kulture stanica sisavaca za proizvodnju proteina terapeutske primjene; glavni istraživač Kniewald, Zlatko
    Name of institution: PLIVA, farmaceutska, kemijska, prehrambena i kozmetička industrija
    City: 10000 - Zagreb, Croatia

COOPERATION - INSTITUTIONS


  1. Name of institution: USDA, Beltsville Agricultural Research Center, Germplasm and Gamete Physiology Laboratory, Livestock and Poultry Sciences Institute
    Type of institution: State institute
    Type of cooperation: Joint project
    City: 20705 - Beltsville, Maryland, USA

  2. Name of institution: PLIVA d.d.
    Type of institution: Economical/Production
    Type of cooperation: Joint project
    City: 10000 - Zagreb, Croatia

OTHER ACHIEVEMENTS


  1. Name: Biotechnology organization in Croatia in relation to EFB, publ. EFB Newsletter, June 1993
    Type of achievement: Other
    Authors: Marić Vladimir, Kniewald Zlatko

  2. Name: Biotechnology in Croatia: Development and Future Perspectives, predavanje u plenarnoj sekciji 'Biotechnology and Business' Central/Eastern European Conference,Prag, Češka Republika, 1993
    Type of achievement: Other
    Authors: Kniewald Zlatko

  3. Name: Usvajanje i razvijanje tehnologije i kulture stanica sisavaca za proizvodnju proteina terapeutske namjene
    Type of achievement: Technology
    Authors: Kniewald Zlatko, Čajavec Stanko,

  4. Name: II stručni seminar i prezentiranje proizvoda - predavanje 'Hormoni i primjena hormon aktivnih preparata u kozmetici', 1992
    Type of achievement: Other
    Authors: Kniewald Zlatko
Other information about the project.
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