Study of the biological effects of polyamine putrescine in different test organisms

Author(s)

Franco Dani Campos Pereira

• Document type: Doctoral Thesis
• Press: Rio Claro. 2017-07-18.
• Institution: Universidade Estadual Paulista (Unesp). Instituto de Biociências. Rio Claro
• Defense date: 2017-05-19
• Advisor: Maria Aparecida Marin Morales; Grasiela Dias de Campos Severi Aguiar

Abstract

The quality of surface water and groundwater has been threatened by different sources of environmental contamination. The cemeteries have stood out in this question, mainly for making available to the environment a liquid produced by the cadaveric decomposition, denominated of necroslurry. Necroslurry is rich in chemicals, among them a polyamine called putrescine, a substance that plays important physiological functions in organisms. However, if this substance is present in higher concentrations than the physiological ones, it can cause problems for the exposed organisms, because, due to its high bioactivity, it becomes a contaminant dangerous to living beings. As this type of contamination has been little studied ecotoxicologically, this work is pioneering in this approach. For the evaluation of the biological effects triggered by the exposure to different concentrations of putrescine, several tests with different bioindicators were carried out. Biological assays, in vitro and in vivo, were developed with the test organisms Allium cepa, Salmonella typhimurium, culture of human cells (HepG2) and Wistar rats. The genotoxic potential were evaluted in meristematic cells of A. cepa by the chromosomal aberration test and the mutagenic potencial by the micronucleus analysis. The meristematic cells exposed to the concentration of 23 mg/kg of putrescine showed a significant increase in the index of mutagenicity and increase in the frequency of chromosomal adherence, indicating aneugenic action of the substance. The mutagenic potential of putrescine was evaluated by the Ames test, with S. typhimurium TA 98 and TA 100 strains (with and without metabolizing fraction). A positive response was observed for the two strains used after metabolic activation, showing that the metabolic process of putrescine increases its potential to cause mutations in the DNA. Cytotoxicity (MTT and resazurin), genotoxicity (comet) and, mutagencicity test (micronucleus with block of cytokinesis) were developed with HepG2 cells. We also evaluated the gene expression patterns of genes involved in the xenobiotic metabolism pathway in response to damage to DNA repair and oxidative stress. Cytotoxicity tests allowed the selection of non-cytotoxic concentrations, which were used in the other experiments with HepG2. Concentrations of 46.3, 138.9, 231.5 and 324.1 mg/kg, corresponding respectively to 10, 30, 50 and 70 % of the LD50 for rats (463 mg/kg) were established. From the comet assay it was observed that all the concentrations tested were significant for the tail intensity criterion, whereas for the tail length and tail moment criteria only the concentrations of 10 and 50 % were significant. There was an increase in the frequency of MN at concentrations of 10, 30 and 50 %, while the frequency of nuclear changes (buds and chromosomal bridges) were significant only at the highest concentration tested (70 %). The expression of genes related to oxidative stress increased in relation to the control group. There was also increased expression of DNA repair genes, especially at concentrations of 10 and 50 %. Genes related to xenobiotic metabolism had greater expression in the cells exposed to higher concentrations. These results indicate a genotoxic and mutagenic action of putrescine for HepG2 cells and show that the substance was able to modulate the levels of gene expression of these studied genes. Wistar rats were exposed by gavage to three different concentrations of putrescine (10, 30 and 50 % of LD50) for 56 consecutive days. Biometric, hematological and biochemical parameters were evaluated, which provided systemic information about the toxic potential of the substance. The exposed animals presented altered parameters for organ weight such as liver, kidney, lung; changes in hematological indices (increase of red blood cells, platelets and cells of the immune system); and alterations in biochemical parameters, such as disturbances in glycemic and cholesterolemic indexes. The genotoxic potential of putrescine was evaluated in the blood cells of the rats by the comet assay and the mutagenic potential by the micronucleus test in bone marrow. In these organisms, putrescine was genotoxic for all exposed groups, but more intense in the group exposed to the lowest concentration (46.3 mg/kg). This same concentration induced a significant increase of Micronucleated Polychromatic Erythrocytes (PCEMN), confirming its genotoxic potential. Reproductive parameters such as sperm counts in the testicles and epididymides, protein expression of enzymes involved in testosterone biosynthesis, morphological, morphometric and stereological alterations in the testicles and morphological aspects of the epididymides, as well as evaluation of oxidative stress in the testicles and epididymides were also evaluated. The results showed a strong action of putrescine on the reproductive indices, such as decrease in sperm production, decrease in the expression of enzymes important for testosterone biosynthesis (CYP11A1, 17β-HSD and StAR) and induction of oxidative stress and morphological changes. In view of all these evaluations, it was possible to aggregate information on the biological effects promoted by the exposure of different organisms to the different concentrations of putrescine evaluated. These results contribute to information concerning the toxicity of this substance and alert to its potential as an environmental contaminant. (AU)

• FAPESP's process: 13/08279-4 - Study of the biological effects of the diamine putrescine in different test organisms
• Grantee: Franco Dani Campos Pereira
• Support Opportunities: Scholarships in Brazil - Doctorate