In silico, in vitro and in vivo investigation of the toxicity of the combined application of the immunomodulator sulforaphane and inactivated bacteria Pseudomonas aeruginosa

[D. S. Božić]

180 str.

Farmacija - Toksikologija / Pharmacy - Toxicology

U ovoj doktorskoj disertaciji sveobuhvatno je ispitivana toksičnost imunomodulatora sa antitumorskim potencijalom, hemijski dobijenog sulforafana (SFN) i soja Pseudomonas aeruginosa sa hemaglutininskim fimbrijama osetljivim na manozu (PA-MSHA). In silico analiza je pokazala da SFN može da izazove oštećenje hromozoma i senzibilizaciju kože, dejstvom na gene uključene u procese imunskog odgovora, oksidativnog statusa i apoptoze, dok PA-MSHA intereaguje sa genima uključenim u proces regulacije imunskog sistema i apoptoze. U in vitro uslovima, SFN ostvaruje citotoksičan efekat na ćelije karcinoma kolona i to dominantno na HCT-116 u odnosu na HT-29 ćelije, zaustavljanjem ćelijske deobe u G2/M fazi ćelijskog ciklusa. Citotoksičan potencijal kombinovane primene imunomodulatora dovodi do smrti manjeg broja ćelija karcinoma kolona u odnosu na efekat samog SFN. U in vivo ispitivanju na modelu zebrica utvrđene su letalne koncentracije za SFN i PA-MSHA, kao i granični nivoi toksičnosti za praćene efekte na rast i razvoj embriona zebrica. Kombinovana primena SFN i PA-MSHA indukuje štetne efekte na embrionima zebrice u koncentracijama u kojima pojedinačne supstance nisu ispoljile štetan efekat. U in vivo istraživanju na modelu pacova praćene su promene hematoloških i biohemijskih parametara, nivoa hormona, produkcije citokina i drugih proteina kao i parametara oksidativnog statusa i patohistološke promene, među kojima se pro- i antiinflamatorni citokini, kao i parametri oksidativnog statusa izdvajaju kao najosetljiviji na dejstvo imunomodulatora. Dodatno, uočene su dozno-zavisne promene većeg broja praćenih efekata nakon primene pojedinačnih imunomodulatora i utvrđeni su granični nivoi toksičnosti. Kritičan toksični efekat SFN je smanjenje procenta neutrofila, a PA-MSHA povećanje koncentracije transformišućeg faktora beta kod mužjaka pacova. Rezultati ispitivanja pokazuju da kombinacija SFN i PA-MSHA uglavnom izaziva efekte kao i pojedinačna primena PA-MSHA. Rezultati ove doktorske disertacije razjašnjavaju mehanizme toksičnosti pojedinačne i kombinovane primene ispitivanih imunomodulatora, dok utvrđene vrednosti graničnih nivoa toksičnosti za pojedinačne imunomodulatore mogu biti osnova za dalji odabir doza pri njihovoj kombinovanoj primeni.

In this dissertation, the toxicity of immunomodulators with antitumor potential, chemically derived sulforaphane (SFN) and a Pseudomonas aeruginosa strain with mannose-sensitive hemagglutinin fimbriae (PA-MSHA) was comprehensively investigated. In silico analysis showed that SFN can cause chromosomal damage and skin sensitization by affecting genes involved in the processes of immune response, oxidative status and apoptosis, while PA-MSHA interacts with genes involved in the process of immune system regulation and apoptosis. Under in vitro conditions, SFN exerts a cytotoxic effect on colon cancer cells, especially on HCT-116 compared to HT-29 cells, by killing cells in the G2/M phase of the cell cycle. A similar cytotoxic potential of the combined use of immunomodulators was observed, but it leads to the death of fewer colon cancer cells than SFN alone. In an in vivo test in a zebrafish model, lethal concentrations for SFN and PA-MSHA and thresholds for toxicity were determined to monitor the effects on growth and development of zebrafish embryos. The combined administration of SFN and PA-MSHA results in adverse effects on zebrafish embryos at concentrations at which the individual substances showed no adverse effects. In in vivo studies in a rat model, changes in hematological and biochemical parameters, hormone levels, production of cytokines and other proteins, oxidative status parameters and pathohistological changes were observed, with pro- and anti-inflammatory cytokines and oxidative stress parameters being the most sensitive to the effects of immunomodulators. In addition, dose-dependent changes in a number of monitored effects were observed following the administration of individual immunomodulators and thresholds for toxicity were determined. A critical toxic effect of SFN is a decrease in the percentage of neutrophils and of PA-MSHA an increase in the concentration of transforming factor beta in male rats. The test results show that the combination of SFN and PA-MSHA generally has the same effects as the use of PA-MSHA alone. The results of this dissertation clarify the mechanisms of toxicity of the single and combined application of the tested immunomodulators, while the determined thresholds of toxicity of the single immunomodulators can be the basis for further selection of doses for their combined application.

imunomodulatori, sulforafan, Pseudomonas aeruginosa sa hemaglutininskim fimbrijama osetljivim na manozu, in silico, in vitro, pacov, zebrica, kombinovana primena

immunomodulator, sulforaphane, inactivated bacteria Pseudomonas aeruginosa, in silico, in vivo, rat, zebrafish, combined application

615.9:615.277.3(043.3)

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