Biological activity of alkylthio and arylthio derivatives of 2-tert-butyl-1,4-benzoquinone

U ovoj disertaciji su sintetisana jedinjenja sa istom farmakoforom kao i biološki aktivna jedinjenja poreklom iz prirodnih proizvoda, alkiltio i ariltio derivati 2-terc-butil-1,4-benzohinona – TBQ i ispitana je njihova biološka aktivnost. Jedinjenja pokazuju dobar antioksidativni potencijal i dovode do produkcije slobodnih radikala, pri čemu se najviše ističe 2-terc-butil-5,6-(etileneditio)-1,4-benzohinon (derivat 4), koji zajedno sa 2-terc-butil-5-(propiltio)-1,4-benzohinonom (derivat 3) pokazuje najjači antineurodegenerativni potencijal. Mikrodilucionim testom uočena je jača antimikrobna aktivnost prema gram-pozitivnim bakterijama, naročito prema Staphylococcus aureus i Bacillus subtilis. Svi derivati, osim 2-terc-butil-6-(feniltio)-1,4-benzohinona (derivat 6), pokazuju manju toksičnost od TBQ prema Artemia salina. MTT i MTS testom je uočeno da jedinjenja, generalno, pokazuju umeren citotoksični efekat prema humanim ćelijskim linijama dok je derivat 4 pokazao najjači efekat. Jedinjenja su pokazala jači efekat na inhibiciju migracije tumorske A549 ćelijske linije u odnosu na zdravu MRC-5 ćelijsku liniju. Genotoksična aktivnost jedinjenja primenom SOS/umuC testa na prokariotskom model sistemu nije uočena, niti je primećena interakcija sa plazmidnom DNK. Komet testom, na eukariotskim model sistemima, pokazano je da izabrane hemijske modifikacije pojačavaju genotoksični potencijal dok je derivat 4 pokazao najjači efekat, uključujući indukciju dvolančanih oštećenja DNK γH2AX testom i značajno veće zaustavljanje ćelijskog ciklusa HepG2 ćelija u G2/M fazi u odnosu na TBQ. Analizom svih dobijenih rezultata, derivat 4 je pokazao najjaču biološku aktivnost.In this dissertation, compounds with the same pharmacophore as well as biologically active compounds originating from natural products, alkylthio and arylthio derivatives of 2-tert-butyl-1,4-benzoquinone - TBQ, were synthesized, and their biological activity was examined. The compounds have good antioxidant potential and lead to the production of free radicals, with 2-tert-butyl-5,6-(ethylenedithio)-1,4-benzoquinone (derivative 4) standing out the most, along with 2-tert-butyl-5-(propylthio)-1,4-benzoquinone (derivative 3), showing the strongest antineurodegenerative potential. A stronger antimicrobial activity against gram-positive bacteria, particularly Staphylococcus aureus and Bacillus subtilis, was observed through a microdilution test. All derivatives, except 2-tert-butyl-6-(phenylthiol)-1,4-benzoquinone (derivative 6), exhibit lower toxicity than TBQ to Artemia salina. Using MTT and MTS assays, compounds generally have moderate cytotoxic effects on human cell lines, with derivative 4 showing the strongest effect. The compounds have demonstrated a stronger effect on inhibiting the migration of the tumor A549 cell line compared to the healthy MRC-5 cell line. No genotoxic activity of the compounds was observed using the SOS/umuC test on the prokaryotic model system, nor was there any interaction with plasmid DNA. On eukaryotic model systems, using the comet assay, selected chemical modifications enhance the genotoxic potential, with derivative 4 showing the strongest effect, including induction of double-strand DNA damage as assessed by the γH2AX test, and significantly greater cell cycle arrest in the G2/M phase in HepG2 cells compared to TBQ. All obtained results reveal that derivative 4 exhibits the strongest biological activity

Biološka aktivnost alkiltio i ariltio derivata 2-terc-butil-1,4-benzohinona

U ovoj disertaciji su sintetisana jedinjenja sa istom farmakoforom kao i biološki aktivna jedinjenja poreklom iz prirodnih proizvoda, alkiltio i ariltio derivati 2-terc-butil-1,4-benzohinona – TBQ i ispitana je njihova biološka aktivnost. Jedinjenja pokazuju dobar antioksidativni potencijal i dovode do produkcije slobodnih radikala, pri čemu se najviše ističe 2-terc-butil-5,6-(etileneditio)-1,4-benzohinon (derivat 4), koji zajedno sa 2-terc-butil-5-(propiltio)-1,4-benzohinonom (derivat 3) pokazuje najjači antineurodegenerativni potencijal. Mikrodilucionim testom uočena je jača antimikrobna aktivnost prema gram-pozitivnim bakterijama, naročito prema Staphylococcus aureus i Bacillus subtilis. Svi derivati, osim 2-terc-butil-6-(feniltio)-1,4-benzohinona (derivat 6), pokazuju manju toksičnost od TBQ prema Artemia salina. MTT i MTS testom je uočeno da jedinjenja, generalno, pokazuju umeren citotoksični efekat prema humanim ćelijskim linijama dok je derivat 4 pokazao najjači efekat. Jedinjenja su pokazala jači efekat na inhibiciju migracije tumorske A549 ćelijske linije u odnosu na zdravu MRC-5 ćelijsku liniju. Genotoksična aktivnost jedinjenja primenom SOS/umuC testa na prokariotskom model sistemu nije uočena, niti je primećena interakcija sa plazmidnom DNK. Komet testom, na eukariotskim model sistemima, pokazano je da izabrane hemijske modifikacije pojačavaju genotoksični potencijal dok je derivat 4 pokazao najjači efekat, uključujući indukciju dvolančanih oštećenja DNK γH2AX testom i značajno veće zaustavljanje ćelijskog ciklusa HepG2 ćelija u G2/M fazi u odnosu na TBQ. Analizom svih dobijenih rezultata, derivat 4 je pokazao najjaču biološku aktivnost.Nisam sigurna koji tip licence se stavlja za doktorsku disertaciju kao i to da li ide otvoreni ili zatvoreni pristup (pretpostavljam da je otvoreni). Takođe valjda sam ja nosilac autorskih prava pa sam stavila svoje ime. Možda sam i tu poglešila

APPLICATION OF IN VITRO AND IN SITU BIOASSAYS FOR EVALUATION OF WATER GENOTOXIC POTENTIAL

Human activity has the great negative impact on water quality. As a consequence, deterioration of freshwater ecosystems has the effect on whole wildlife. Some of the pollutants which are realised into water could interact with DNA molecule and lead to DNA damage. The Velika Morava River is one of the larger tributaries of the Danube River. The major ecological problem of the Velika Morava River basin is discharging of municipal and industrial wastewaters. The aim of our study was the evaluation of genotoxic potential of the Velika Morava River basin. For this purpose, we employed battery of in vitro and in situ bioassays. From 9 selected sites the water samples and blood samples of Alburnus alburnus were collected. Within in vitro approach, the genotoxicity of water samples was tested using the SOS/umuC test on Salmonella thyphimurium TA1535/pSK1002 and alkaline comet assay on the HepG2 cell line. On the other hand, in situ approach included the alkaline and Fpg modified comet assay and the micronucleus test on bleak (A. alburnus) erythrocytes. The obtained results indicated differences of in vitro and in situ tests to evaluate genotoxic potential. The effects of genotoxicity were evident only in tests performed in situ, which is not surprising considering constant exposure of collected organisms to environmental stress. Differential sensitivity was found also within in situ tests where the alkaline comet assay showed the highest potential in distinguishing between sites. So, in accordance with results, we could conclude that application of the battery of in vitro and in situ bioassays is appropriate for assessment of genotoxic potential

Ispitivanje mutagenog i genotoksičnog potencijala derivata tert-butilhinona SOS/umuC testom i alkalnim komet testom na MRC-5 i HepG2 ćelijskim linijama

Poslednjih godina, istraživanja u terapiji kancera usmerena su na korišćenje prirodnih proizvoda, naročito poreklom iz marinskih organizama. Hidrohinon avarol i hinon avaron, izolovani iz Dysidea avara pokazuju dobru antikancerogenu aktivnost, ali njihova proizvodnja nije dovoljna za potencijalnu kliničku primenu. Kao potencijalna zamena koristi se model baziran na tert-butilhinonu (TBQ) koji je sličan po strukturi avarolu i avaronu. Međutim, interakcije TBQ sa molekulom DNK mogu dovesti do neželjenih efekata, zbog čega je važno ispitati genotoksičnu aktivnost. U ovom radu ispitivan je genotoksični potencijal TBQ i njegovih alkilamino i aralkilamino derivata u različitim test sistemima. Interakcija sa molekulom DNK je prvo ispitivana u acelularnom modelu na plazmidnoj DNK. Prokariotski test sistem je bio SOS/umuC test na bakteriji Salmonella typhimurium TA1535/pSK1002, a eukariotski komet test na ćelijskim linijama HepG2 (humani karcinom jetre) i MRC-5 (zdravi fibroblasti pluća fetusa). U acelularnom modelu, rezultati su pokazali da TBQ i njegovi derivati ne interaguju direktno sa plazmidom pUC19. U SOS/umuC testu jedino TBQ pokazuje slab genotoksični potencijal i to pri najvećim testiranim koncentracijama. Genotoksični potencijal uočen je primenom komet testa, uglavnom pri najvećim koncentracijama testiranih jedinjenja, ali je on znatno niži u odnosu na benzo(a)piren i etopozid koji su u eksperimentu korišćeni kao pozitivne kontrole

Defensive secretions of millipedes Megaphyllum unilineatum (C. L. Koch, 1838), Pachyiulus hungaricus (Karsch, 1881) and Cylindroiulus boleti (C. L. Koch, 1847) (Diplopoda, Julida) as antimicrobial agents in the inhibition of biofilms of Pseudomonas aeruginosa PAO1 and Staphylococcus aureus

INTRODUCTION: In recent years, the emphasis of the scientific community has been placed on the invention of new antimicrobial agents due to the increasing resistance of bacteria to antibiotics. However, serious global health concern is focused on bacterial biofilms, a complex structure of a microbiome made up of colonies of bacteria or individual bacterial cells in a group, attached to a surface. Bacterial biofilms are highly resistant to antimicrobial agents and grow on the surfaces of medical implants such as sutures, catheters, and dental implants. Given that plants and animals are a valuable source of natural biologically active products, they are a good basis for finding new antimicrobial and antibiofilm agents. Bacterial strains of Pseudomonas aerug- inosa PAO1 and Staphylococcus aureus are known for biofilm production and cause opportunistic and chronic infections in humans, some of which are due to biofilm production. Due to their characteristic way of life, millipedes (Diplopoda) are char- acterized by a diverse and complex defense against predators, which includes the secretion of various chemical compounds that are toxic, repellent, or tasteless to predators. Analyzes have shown that millipedes produce chemical compounds such as phenols, alkaloids, quinones, terpenoids, cyanogenic compounds, and fatty acid esters, which showed antimicrobial ac- tivity, among other. Representatives of the order Julida, which are frequent in Republic of Serbia, produce defense secretions that are chemically very complex (the most complex within Diplopoda) and exhibit antimicrobial, antioxidant, and neurode- generative potential, so they represent a good basis for the invention of new antibiofilm agents. OBJECTIVES: Objectives are to determine the inhibition of biofilm formation and degradation of the formed biofilm of P. aeruginosa PAO1 and S. aureus by defense secretions of selected millipede species from the family Julidae as well as to determine their anti- microbial activity. METHOD / DESIGN: Biofilm formation was quantified by the crystal violet staining method, while antimicrobial activity was examined using the broth dilution minimum inhibitory concentration (MIC) test. RESULTS: Defensive secretions of Megaphyllum unilineatum (MUN), Pachyiulus hungaricus (PHU), and Cylindroiulus boleti (CBO) showedantimicrobial activity against S. aureus with MIC values of 0.03, 0.06, and 0.06 mg/mL, respectively. On P. aeruginosa PAO1, defense secretions did not show antimicrobial activity even at the highest tested concentration of 1 mg/mL for MUN while for PHU and CBO the MIC was 1 mg/mL, which is most likely due to the high resistance of this bacterial strain. The antibiofilm effect was observed in all tested defense secretions and was more pronounced against S. aureus than against P. aeruginosa PAO1. The strongest biofilm inhibition of S. aureus was at the highest tested concentrations (2 × MIC) with percentages of inhibition of CBO: 88.6%, PHU: 73.7%, and MUN: 67.2%. Degradation of already formed S. aureus biofilm was shown at lower tested defensive secretions concentrations (MIC/4), about 40% of biofilm degradation for MUN and PHU and about 30% for CBO. The strongest inhibition of P. aeruginosa PAO1 biofilm formation was observed at the highest tested concentrations of defensive secretions, 1 and 0.5 mg/mL for PHU (82 and 54%), and CBO (64.3 and 38.5%) while MUN had the strongest activity at the lowest tested concentration of 0.06 mg/mL (34.3%). All examined defense secretions had similar degradation activity of P. aeruginosa PAO1 biofilm with stronger activity at lower tested concentrations (about 30%). Defensive secretions of MUN and PHU extracted in DMSO solvent showed a stronger antibiofilm effect compared to the same ethanol extracts. CONCLUSIONS: The defense secretions of MBO, PHU, and CBO show a good basis for further investigations of their use as antimicrobial agents, especially against S. aureus

Acute toxicity assessment of defense secretions of Megaphyllum bosniense (Verhoeff, 1897) and M. unilineatum (C. L. Koch, 1838) (Diplopoda, Julida) on Artemia salina.

INTRODUCTION: Different orders within the class Diplopoda possess a variety of chemical compounds in their defense secretions: quinones, phenolics, alcohols, aldehydes, ketones, esters, alkaloids, cyanogenic compounds. Defensive secretions of species from the order Julida are regarded as the most complex within Diplopoda, and they are blends of several classes of chemical com- pounds: quinones, esters and ketones. Numerous biological activities of these secretions have been reported: antimicrobial, antioxidative, antineurodegenerative, cytotoxic and embryotoxic on zebrafish. Besides zebrafish embryos, Artemia salina is one of the common model organisms in toxicity assessment which has not been used for screening of toxicity of millipedes’ defensive secretions. OBJECTIVES: The main goal of this study was to examine the toxic effects of defensive secretions of two species from the order Julida [Megaphyllum bosniense (MBO) and M. unilineatum (MUN)] using Artemia salina (ARC test). METHOD / DESIGN: Adult individuals of M. bosniense were collected during April and May of 2021 on Mt. Avala, near Belgrade, while adults of M. unilineatum were collected during the same period in the Krnjača, suburb of Belgrade. After the capture, millipedes were kept in plastic boxes containing ground cover from the collecting site. The boxes were regularly sprayed with water to maintain high humidity. Due to the fact that the sample was female-biased, defensive secretions of female specimens were used for further analyses. Excretion of defensive secretions was elicited from glands of mentioned species via mechanical stress in closed glass vials. Secretions collected from both species were dissolved in 10 ml of hexane, concentrated under reduced pressure in a rotary evaporator (Rotavapor R-210, Buchi) at 40°C to a dry residue, and redissolved in 50% dimethylsulphoxide (DMSO). The stock concentration of extracts used in ARC test was 20 mg/mL. Before treatments, eggs of A. salina were incubated for 72h with constant lighting and aeration. For the purposes of the experiment, stage II and III larvae were used (separated by phototaxis in 300 ml of seawater). In a plate with 24 wells, 900 μl of seawater with larvae (10-15 per well) was placed and then 100 μl of tested extracts (range of concentrations 0,1 mg/mL - 0,003125 mg/mL) was added. Potassium dichromate (K2Cr2O7) was used as a positive control and DMSO was used as solvent control. The total number of individuals per well was counted after 24h and 48h, as well as the number of living and dead individuals. These data were used for esti- mation of survival rate and determination of LC50 value. The experiment was done in triplicate. RESULTS: Our results show that secretions from both species exhibit a toxic effect on the survival of the chosen model organism, with the MBO extract showing weaker activity in comparison with MUN extract. The LC50 value after 24h was about the same for both species (LC50=73,23 μg/mL for MBO and LC50=68,56 μg/mL for MUN). The LC50 value for MBO after 48h was 47,18 μg/ mL, while LC50 value in the same period for MUN was 29,12 μg/mL. Positive control (LC50= 13,5 μg/mL) showed three times stronger effects in relation to MBO and twice as strong when compared to MUN extract. It has also been shown that the num- ber of surviving individuals decreases with increasing concentration of tested extracts and the increasing incubation time. CONCLUSIONS: The defense secretions of both tested millipede species show toxic effects in the ARC test. It is shown that MBO extract has a weaker toxic effect than the MUN extract. This result can be linked with the fact that esters of long-chain fatty acids are dominant compounds in MBO, while MUN is almost exclusively benzoquinone-based. Esters detected in MBO are generally regarded as low-toxic compounds, but with the potential to interact with compounds from other chemical classes. However, as MBO achieved toxic effects and many esters that are detected in MBO are new natural products and their biological potential is unknown, further extensive studies are needed to determine their toxicological potential

The effect of injectable platelet-rich fibrin use in the initial treatment of chronic periodontitis

Introduction/Objective. The objective of the study was to investigate whether there are differences in therapeutic effect between initial treatments of chronic periodontitis [scaling and root planning (SRP)] alone and SRP in conjunction with injectable platelet-rich fibrin (I-PRF) application, comparing clinical parameters after three months. Methods. Twenty-four patients with chronic periodontitis who had at least two sites with probing pocket depth (PPD) ≥ 5 mm on contralateral side participated in the study. Using a split-mouth design, the patients were treated with SRP + I-PRF (study group) or SRP only (control group). The clinical parameters, clinical attachment level (CAL), gingival margin level (GML), PPD, bleeding on probing, and plaque index, were recorded on both sides. Results. Compared to baseline, both treatment modalities demonstrated an improvement in investigated clinical parameters. The mean value of CAL was reduced from 1.97 ± 0.75 (0.25–3.31) to 1.07 ± 0.44 (0.12–1.78) in the study group, whereas it decreased from 1.81 ± 0.66 (0.42–2.96) to 1.48 ± 0.55 (0.22–2.30) in the control group. Similarly, the corresponding values for GML and PPD showed statistically significant difference between the groups (p = 0.040 and p = 0.006, respectively). Conclusion. Regardless the limited number of patients in the study, initial periodontal therapy in conjunction with injectable platelet-rich fibrin proved to display significant improvement in all clinical parameters compared to initial periodontal therapy alone

Detection of double-strand breaks in DNA molecules by the γH2AX assay and analysis of the cell cycle after treatment with potential antitumor agents TBQ and its alkylthio and arylthio derivatives.

2-tert-butyl-1,4-benzoquinone (TBQ) and its alkylthio and arylthio derivatives: 2-tert-butyl-5-(isopropylthio)-1,4-ben- zoquinone, 2-tert-butyl-5-(propylthio)-1,4-benzoquinone, 2-tert-butyl-5,6-(ethylenedithio)-1,4-benzoquinone, 2-tert-butyl-5-(phenylthio)-1,4-benzoquinone and 2-tert-butyl-6-(phenylthio)-1,4-benzoquinone were synthe- sized as analogs of biologically active compounds of natural origin with antitumor activity: quinone avarol/avarone. The γH2AX test was used to detect the potential genotoxic effect of TBQ and its derivatives in the HepG2 cell line. In addition, the effect of these substances on the cell cycle was monitored on the same HepG2 model system. Since the phosphorylation of histone H2AX is an early event in the cellular response to DNA double-strand breaks (DSBs), an assay based on the detection of phosphorylated histone H2AX (γH2AX) can be used as a biomarker of genotoxicity and genomic instability. In this work, the γH2AX test and flow cytometry were used to analyze the genotoxic potential and the effect on the cell cycle of TBQ and its derivatives. Double-strand breaks (DSBs) in DNA are detected with γH2AX-specific antibodies, and the cell cycle in the HepG2 cell line is analyzed by flow cytometry. Based on the obtained results, only 2-tert-butyl-5,6- (ethylenedithio)-1,4-benzoquinone induced increased formation of DSBs. Also, the same derivative caused a significantly greater arrest of cells in the G2/M phase of the cell cycle compared to TBQ from about 27% (TBQ) to 34% of the total population with a decrease in the S phase cell population. By forming DSBs, 2-tert-butyl-5,6-(ethylenedithio)-1,4-benzoquinone leads to genomic instability of the HepG2 cell line, which results in cell cycle arrest in the G2/M phase

Antimicrobial activity of 2-tert-butyl-1.4-benzoquinone and its selected alkylthio and arylthio derivatives.

Background: Biologically active compounds, originating from a variety of natural sources: plants, animals and microor- ganisms, have great potential for use as antimicrobial agents. Avarol, a compound originating from the Mediterranean sponge Disidea avara, exhibits a number of biological activities including antimicrobial activity. Considering that, avarol was taken as a model for the synthesis of 2-tert-butyl-1,4-benzoquinone (TBQ) derivatives. Objectives: TBQ was chosen because of its similar chemical structure with avarol who showed strong biological activity but is more accessible and economical than avarol. By selecting the thiol group, we aimed at combining the action of two strong functional groups of natural origin, quinones and thiols, which both have antimicrobial activity. Methods: In this work antimicrobial activity of TBQ and its derivatives: 2-tert-butyl-5-(isopropylthio)-1.4-benzoqui- none, 2-tert-butyl-5-(propylthio)-1.4-benzoquinone, 2-tert-butyl-5,6-(ethylendithio)-1.4-benzoquinone, 2-tert-bu- tyl-5-(phenylthio)-1.4-benzoquinone and 2-tert-butyl-6-(phenylthio)-1.4-benzoquinone was evaluated by the MIC mi- crodilution method on 7 different ATCC bacterial strains. Results: All compounds tested showed stronger antimicrobial activity against Gram positive bacterial strains (Entero- coccus faecalis, Staphylococcus aureus, Bacillus spp.). A strong antimicrobial effect (MIC value less than 100μM) was shown by 2-tert-butyl-5,6- (ethylendithio)-1,4-benzoquinone, 2-tert-butyl-5-(phenylthio)-1.4-benzoquinone deriva- tives and 2-tert-butyl-6-(phenylthio)-1,4-benzoquinone against S.aureus. TBQ, 2-tert-butyl-5-(isopropylthio)-1,4-ben- zoquinone, 2-tert-butyl-5, 6-(ethylendithio)-1,4-benzoquinone and 2-tert-butyl-5-(phenylthio)-1.4-benzoquinone showed strong antimicrobial effect against Bacillus spp. According to our results, chemical modifications of TBQ in- crease its antimicrobial activity while derivative 2-tert-butyl-5,6-(ethylendithio)-1.4-benzoquinone is the best candi- date for further testing

Analiza mikrobiološkog kvaliteta površinskih voda na teritoriji Republike srbije

Neprerađene komunalne i industrijske otpadne vode su jedan od glavnih zagađivača površinskih voda u Srbiji. Sa komunalim otpadnim vodama u površinske vode dospevaju hemijski polutanti i fekalno zagađenje koje može sadržati za čoveka patogene mikroorganizme. U okviru istraživanja vršena je analiza mikrobiološkog kvaliteta vode sa 40 lokaliteta na teritoriji Republike Srbije koji nisu obuhvaćeni programom rutinskog monitoringa. Prisustvo i broj Escherichia coli je odabrano kao parametar fekalnog zagađenja. Rezultati su pokazali da voda sa 57,5% lokaliteta pripada III i IV klasi. Najveći broj zagađenih lokaliteta se nalazi nizvodno od naseljenih mesta, što potvrđuje značaj izgradnje postrojenja za preradu otpadnih voda