Evaluacija citotoksičnih i genotoksičnih efekata permetrina upotrebom in vitro mikronukleus testa

The objectives of this investigation were to evaluate the effects of the synthetic pyrethroid insecticide permethrin on mitotic activity and appearance of micronuclei in cultured human lymphocytes. In the standard cytokinesis-blocked micronucleus test in vitro protocol, four experimental concentrations were used: 1.2, 3, 6 and 12 mg/ml. The concentration of 6 mg/ml was calculated to correspond to the LD50 dose obtained in rats. The cytokinesis block proliferation index (CBPI), as a parameter of cell cycle kinetics, was decreased at all concentrations of permethrin except the lowest one. The analysis of micronucleus frequencies, however, point to the absence of genotoxic effects.Cilj ovih istraživanj bio je da se izvrši evaluacija efekata sintetičkog piretroidnog insekticida permetrina analizom mitotske aktivnosti i pojave mikronukleusa u kulturama humanih limfocita. U standardnom in vitro mikronukleus testu upotrebljene su četiri eksperimentalne koncentracije permetrina: 1.2, 3, 6 i 12 mg/ml. Koncentracija od 6 mg/ml proračunata je da odgovara LD50 dozi kod pacova. Vrednosti proliferacionog indeksa pri blokiranoj citokinezi (CBPI) kao pokazatelja ćelijske kinetike, smanjene su pri svim koncentracijama permetrina osim najniže. Analize frekvenci mikronukleusa, međutim, ukazuju na odsustvo genotoksičnih efekata

Mechanisms of genotoxic effects of hormones

A concept that compounds commonly present in biological systems lack genotoxic and mutagenic activities is generally in use, hence a low number of endogenous substances have ever been tested to mutagenicity. Epidemiological and experimental analyses indicated, however, that sexual steroids could contribute to initiation and/or continuation of malign diseases. Detailed studies using methods of biochemistry, molecular biology, cytogenetics and other branches, showed that not only epigenetic mechanisms, such as a stimulation of cell proliferation, but also certain hormones, that can express genotoxic effects, such as covalent DNA modification, then chromosomal lesions and chromosomal aberrations, are in the background of malign transformation under activities of hormones. In the case of oestrogens, it was shown that excessive hormonal stimulation led to a metabolic conversion of these hormones to reactive intermediates with formation of reactive oxygenic derivates, so that cells were virtually under conditions of oxidative stress. Individual and tissue susceptibility to occurrence of deterioration of DNA and other cell components generally results from the differences in efficiency of enzymic and non-enzymic mechanisms of resistance against oxidative stress. Besides, steroid thyeroid hormones and catecholamine (dopamine, noradrenaline/norepinephrine and adrenaline) can express genotoxic effects in some test-systems. It is interesting that all above mentioned hormones have a phenolic group. Data on possible genotoxic effects of peptide and protein hormones are very scarce, but based on the available literature it is considered that this group of hormones probably lacks mutagenic activities. The possibility that hormones, as endogenous substances, express mutagenic activities results from the fact that DNA is, regardless of chemical and metabolic stability susceptible, to a certain extent, to changeability compatible with the processes of the biological evolution

Mutageni efekti hormona

There is a widely accepted view that the endogenous substances, including hormones, do not have any mutagenous effect when present in the usual physiological concentrations. However, beside relative stability and permanence of the genetic material, its changeability is also needed to provide for the biological evolution. Thus, it is possible to expect that certain reactions, due to the complexity of the mechanism of the signal transduction under the effect of hormones, still lead to the creation of reactive derivatives able to inter-react with the DNA molecules thus encouraging the emergence of mutations. This paper gives a survey of the exploration of the hormones' genotoxic effects in various test-systems, namely, from the bacteric through the cell cultures and experiments in vivo upon laboratory guinea-pigs, up to the determination of the mutagenous effects in the people that were treated by hormones. The steroid hormones' effects are described in detail in the literature while, on the other hand, there is not sufficient knowledge yet about possible changes of the genetic material under the influence of the non-steroid hormones. The examinations of the steroid hormones geonotoxicity in the bacterial systems have mainly given negative results. In more complex eukaryotic systems in vitro and in vivo most of the steroid hormones manifest mutagenous effects, though the result may vary depending one the type of cell or the biological species used in the experiment. As for the non-steroid hormones, though they have been studied less, it seems that they do not mostly express mutagenous effects. Today it is clear that the steroid hormones (especially estrogen) are completely cancerigenic and that they are capable of encouraging the process of cancerogenesis both by inducing covalent simulating the cell division (tumor promoters).Široko je prihvaćeno gledište da endogene supstance, uključujući hormone, nemaju mutagene efekte kada su prisutni u uobičajenim fiziološkim koncentracijama. Međutim, pored relativne stabilnosti i stalnosti genetičkog materijala, potrebna je i njegova promenljivost da bi postojala biološka evolucija. Stoga je moguće očekivati da izvesne reakcije, usled kompleksnosti mehanizama transdukcije signala pod dejstvom hormona, ipak dovode do stvaranja reaktivnih derivata sposobnih da interreaguju sa molekulima DNK i potpomognu nastanak mutacija. U ovom radu dat je pregled istraživanja genotoksičnih efekata hormona u različitim test-sistemima: od bakterijskih, preko kultura ćelija, in vivo eksperimenata na laboratorijskim životinjama, do utvrđivanja mutagenih efekata kod ljudi tretiranih hormonima. Efekti steroidnih hormona detaljno su opisani u literaturi, dok se nasuprot tome, još uvek nedovoljno zna o mogućim promenama naslednog materijala pod uticajem nesteroidnih hormona. Ispitivanja genotoksičnosti steroidnih hormona u bakterijskim sistemima uglavnom su dala negativne rezultate. U kompleksnijim eukariotskim sistemima in vitro i in vivo većina steroidnih hormona ispoljava mutagene efekte, mada rezultat može da varira zavisno od tipa ćelije ili biološke vrste upotrebljene u eksperimentu. Sto se tiče nesteroidnih hormona, mada su znatno manje proučavani, stiče se utisak da uglavnom ne ispoljavaju mutagene efekte. Danas je jasno da su steroidni hormoni (naročito estrogeni) kompletni kancerogeni sposobni da podstaknu proces kancerogeneze, kako indukcijom kovalentnih oštećenja DNK koja se mogu prevesti u mutacije (tumor inicijatori) tako i putem stimulacije deobe ćelija (tumor promotori)

Epigenetički mehanizmi kancerogeneze

The biological activity of many cancerogenic agents is manifested in genotoxic mechanisms by changing the information encoded in the DNA molecules. However, it becomes much clearer that there are non-genotoxic (epigenetic) cancerogens whose action is manifested through changes of the gene's activity without causing mutations. The transformation of the normal cell into a malign one requires a greater number of genetic and/or epigenetic changes that undergo at lead three phases, namely, initiation, promotion and progression. The epigenetic cancerogenes realize their effect primarity in the promotion phase, primarily, by stimulating the cell division (mitogenesis). Unlike the genotoxic agents affecting the DNA molecule, the non-genotoxic cancerogenes can affect a considerably greater number of cell constituents and products, that is, the components of the signal transduction system, the protein factors involved in the regulation of replication, reparation and transcription of the DNA. Besides, the non-genotoxic cancerogenes can change a degree of methylation of thenucleus DNA and chromatin conformation. Despite prominent differences between the genotoxic and the non-genotoxic cancerogenes, it is necessary to stress such a way of division is somewhat imprecise regarding the fact that the chemical cancerogenes often exert their effect through a combination of mechanisms while the primary mechanisms of effect can vary depending on the type of the cell.Biološka aktivnost mnogih kancerogenih agenasa ostvaruju se genotoksičnim mehanizmima, promenom informacije kodirane u molekulima DNK. Sve više jasno je, međutim, da postoje negenotoksični (epigenetički) kancerogeni koji deluju preko promena aktivnosti gena bez izazivanja mutacija. Za transformaciju normalne ćelije u malignu potreban je veći broj genetičkih i/ili epigenetičkih promena, pri čemu se razlikuju najmanje tri faze: inicijacija, promocija i progresija. Epigenetički kancerogeni svoje efekte ostvaruju prevashodno u fazi promocije, pre svega stimulacijom deobe ćelija (mitogeneza). Za razliku od genotoksičnih agenasa koji deluju na molekul DNK, negenotoksični kancerogeni mogu uticati na znatno veći broj ćelijskih sastojaka i produkata: komponenti sistema za transdukciju signala, proteinskih faktora uključenih u regulaciju replikacije, reparacije i transkripcije DNK. Pored toga, negenotoksični kancerogeni mogu da promene stepen metilacije jedarne DNK i konformacije hromatina. Uprkos markantnim razlikama između genotoksičnih i negenotoksičnih kancerogena, potrebno je istaći da je ovakav način podele donekle neprecizan, s obzirom da hemijski kancerogeni često deluju kombinacijom mehanizama, dok primarni mehanizam dejstva može da varira zavisno od tipa ćelije

Evaluacija genotoksičnih efekata tiroksina primenom in vivo citogenetičkog testa na Swiss albino miševima

Thyroid hormones enhance aerobic metabolism favoring oxidative stress which may lead to covalent damage of various molecules including DNA. Previous investigations revealed that thyroid hormones induce DNA damage on human lymphocytes and sperm in the in vitro Comet assay. However, cytogenetic evaluation of genotoxic effects of thyroxine gave equivocal results: increase of sister chromatid exchanges, and no incerase of micronuclei in cultured human lymphocytes. Therefore, the aim of the present study was to further evaluate the possible genotoxic effects of thyroxine using in vivo cytogenetic test on Swiss albino mice. Three experimental concentrations of thyroxine were used (0.1 mg/kg, 0.5 mg/kg and 2.5 mg/kg). The mice were divided into several groups depending on the duration of the treatment with thyroxine. Thus, we treated mice for 1, 3, 7 and 10 days. Positive (Nmethyl- N'-nitro-N-nitrosoguanidine) and negative controls were also formed for the same time periods. Cytogenetic endpoinds (numerical and structural aberrations, chormosome gaps and breaks) were analysed in bone marrow cells from femures. The results obtained in this investigation showed that thyroxine has not induced chromosome damage or aberrations. This is in agreement with our previous analysis of micronuclei in human peripheral blood lymophocytes treated with thyroxine. On the other hand, we observed a decrease of mitotic index especially in animals treated for a longer period of time with the highest dose of thyroxine. Therefore, it can be concluded that thyroxine does not induce genotoxic effects which could be detected by cytogenetic analysis.Tireoidni hormoni podstiču aerobni metabolizam favorizujući oksidativni stres koji može da dovede do kovalentnih oštećenja različitih molekula uključujući i DNK. U prethodnim istraživanjima otkriveno je da tireoidni hormoni indukuju oštećenja molekula DNK u humanim limfocitima i spermi u in vitro Komet testu. Međutim, citogenetička evaluacija genotoksičnih efekata tiroksina dala je kontradiktorne rezultate: povećanje razmena sestrinskih hromatida bez porasta učesalosti mikronukleusa u kulturama humanih limfocita. Stoga je cilj istraživanja u ovom radu bio da dodatno ispitamo moguće genotoksične efekte tiroksina koristeć i in vitro citogenetički test na Swiss albino miševima. Upotrebljene su tri eksperimentalne koncentracije tiroksina (0,1 mg/kg, 0,5 mg/kg and 2.5 mg/kg). Miševi su podeljeni u nekoliko grupa zavisno od dužine tretmana tiroksinom: 1, 3, 7 i 10 dana. U istim vremenskim periodima miševi su tretirani pozitivnom (N-metil-N'-nitro-N-nitrozogvanidin) i negativnom kontrolom. Analizirani su citogenetički parametri (numeričke i strukturne aberacije hromozoma, gapovi i prekidi na hromozomima) u ćelijama kostne srži izolovanim iz femura. Rezultati dobijeni u ovom istraživanju ukazuju da tiroksin ne indukuje hromozomske prekide i aberacije, što je u saglasnosti sa našim prethodnim zapažanjima na humanim limfocitima u kulturi. Istovremeno, primetili smo smanjenje mitotskog indeksa, naročito kod životinja tretiranih u dužem vremenskom periodu sa visokim dozama tiroksina. Prema tome, može se zaključiti da tiroksin ne indukuje genotoksične efekte koji mogu da se detektuju citogenetičkim analizama

Citogenetička analiza dejstva adrenalina na humane limfocite u kulturi

Epinephrine can modulate mitotic activity of normal and malignant cells and exhibit genotoxic potential in some test-systems. It is assumed that metabolic conversion of phenolic groups in the catechol ring of epinephrine leads to the formation of reactive derivatives and superoxide anions capable of damaging cellular molecules including DNA. The aim of the present study was to evaluate the cytotoxic and genotoxic effects of epinephrine on human peripheral blood lymphocytes in vitro. The lowest concentration of epinephrine used in these experiments (5x10-10 M) was calculated to be in the range of the physiological blood level of epinephrine in humans. Three experimental concentrations corresponded to minimal (2x10-7 M), average (10-6 M) and maximal (5x10-6 M) therapeutic doses in human medicine. In addition, the highest concentrations exceeded the maximal therapeutic dose 10-fold (5x10-5 M) and 30-fold (1.5x10-4 M), respectively. On the basis of the results obtained it can be concluded that epinephrine had no influence on the appearance of chromosome aberrations under the described experimental conditions. However, mitotic index was significantly lower in cultures treated with the three highest concentrations of epinephrine used in this investigation.Adrenalin može da promeni mitotsku aktivnost normalnih i malignih ćelija Pored toga, adrenalin ispoljava genotoksične efekte u nekim test-sistemima. Smatra se da metabolička konverzija fenolnih grupa u kateholnom prstenu adrenalina dovodi do stvaranja reaktivnih derivata i superoksidnih anjona sposobnih da oštete molekule u ćeliji, uključujući DNK. Cilj ovog istraživanja bio je da se izvrši evaluacija citotoksičnih i genotoksičnih efakata adrenalina na limfocite periferne krvi čoveka in vitro. Najniža eksperimentalna koncentracija adrenalina korišćena u ovom eksperimentu (5x10-10 M) proračunata je tako da bude uporedljiva sa fiziološkom koncentracijom adrenalina u krvi čoveka. Tri eksperimentalne koncentracije odgovaraju minimalnim (2x10-7 M), srednjim (10-6 M) i maksimalnim (5x10-6 M) terapijskim dozama u humanoj medicini. Pored toga, dve najviše koncentracije veće su od maksimalne terapijske doze deset (5x10-5 M) i trideset puta (1.5x10-4 M). Na osnovu dobijenih rezultata može se zaključiti da adrenalin nije značajno uticao na pojavu hromozomskih aberacija pod opisanim eksperimentalnim uslovima. Međutim, tri najviše koncentracije adrenalina upotrebljene u ovom eksperimentu dovode do značajnog smanjenja mitotske aktivnosti

Analiza DNK oštećenja izazvanog tiazofurinom u humanim ćelijama pune krvi primenom in vitro komet testa

Objective. Inosine 5’-monophosphate dehydrogenase (IMPDH) activity in cancer cells is increased. Tiazofurin selectively inhibits the activity of IMPDH, and it has been granted for the treatment of different cancers and new viral diseases. Its widespread use was limited because exposure to tiazofurin under certain circumstances was found to have a higher frequency of severe non-hematologic toxicity. Therefore, the objective of this study was to examine genotoxic action and inducement of DNA damage of tiazofurin using the comet assay. Methods. The ability of tiazofurin to induce DNA damage was evaluated using single-cell gel electrophoresis (SCGE) technique/comet assay. Human whole blood cells were exposed to three final concentrations of tiazofurin (1 µM/mL, 2 µM/mL, and 5 µM/mL) for 30 min in vitro. Results. Our results indicate that tiazofurin produced a significant level of DNA damage on whole blood cells after 30 min of exposure vs. control. All tested concentrations were significantly comet-forming, in a concentration-dependent manner. Conclusion. Our investigation on the tiazofurin-treated cells and their relationship to the formation of DNA damage demonstrated that the genotoxic effect was induced after exposure to tiazofurin under described conditions.Cilj. Aktivnost inozin 5'-monofosfat dehidrogenaze (IMPDH) povec'ana je u c'elijama karcinoma. Tiazofurin selektivno inhibira aktivnost IMPDH i odobren je za lečenje različitih karcinoma i novih virusnih bolesti. Njegova široko rasprostranjena upotreba bila je ograničena jer je utvrđeno da je izloženost tiazofurinu pod određenim okolnostima imala vec'u incidencu ozbiljne nehematološke toksičnosti. Stoga je cilj ove studije bio da se pomoc'u komet testa ispita genotoksično delovanje i izazivanje DNK oštec'enja tiazofurinom. Metode. Sposobnost tiazofurina da izazove DNK oštec'enje procenjena je primenom elektroforeze DNK pojedinačnih ćelija (SCGE) / komet testa. Ćelije pune krvi su bile izložene trima konačnim koncentracijama tiazofurina (1 µM/mL, 2 µM/mL, and 5 µM/mL) tokom 30 minuta in vitro. Rezultati. Naši rezultati ukazuju na to da je tiazofurin proizveo značajan nivo DNK oštec'enja na c'elijama pune krvi nakon 30 minuta izlaganja u odnosu na kontrolu. Sve ispitivane koncentracije su dovele do značajnog nastanka kometa, pri čemu je nivo oštećenja rastao s koncentracijom. Zaključak. Naše istraživanje c'elija tretiranih tiazofurinom i njihova reakcija na izazivanje DNK oštec'enja pokazalo je da je tiazofurin ispoljio genotoksični efekat pod opisanim uslovima

Cytogenetic alterations in peripheral cells of Alzheimer’s disease patients

Alzheimer’s disease (AD) is the most frequent progressive neurodegenerative disorder in elderly associated with irreversible cognitive impairment and dementia. The vast majority of AD patients are sporadic (SAD) in which the disease develops after age of 65. Despite of century of research, we lack understanding of the SAD etiology and pathogenesis. Several hypotheses try to explain the main causes of brain degeneration in SAD, one of them assuming that genomic instability and the reentry of certain neurons into the incomplete cell cycle may be the pathogenic basis of the disease. Although the brain is the most affected organ in AD, numerous studies showed structural and functional alterations in peripheral tissues, suggesting that AD is a generalized systemic disorder. Diverse changes in peripheral cells from AD patients are described in literature including cell cycle aberration and chromosome instability, alterations in cell viability, proliferation and apoptosis, oxidative metabolism, amyloid precursor protein and amyloid β protein metabolism, and other cellular processes. The aim of this paper was to summarize and review the results of our investigations and the growing literature data concerning the multiple chromosomal alterations in peripheral cells of AD patients and to consider their possible role in the disease pathogenesis as well as the importance of such investigations. [Projekat Ministarstva nauke Republike Srbije, br. 173034

Influence of cadmium chloride on the frequency of micronuclei in da and AO rats

Heavy metal cadmium (Cd), a well-known environmental hazard, exerts a number of toxic and genotoxic effects. Experimental animals, Albino Oxford (AO) and Dark August (DA) rat strains, were treated intraperitoneally with three different concentrations of cadmium chloride (CdCl2): 0.5, 1, and 2 mg CdCl2 per kg of body weight, while the control animals received equal volume of sterile phosphate buffered saline. In this investigation individuals of both sexes aged 3, 6 and 12 month were used. Frequency of micronuclei formation was evaluated in polychromatic erythrocytes (PCEs), 24h hours after treatment. The results showed that that cadmium-cloride (CdCl2) exibits the genotoxic effects causing an increase of the frequency of micronuclei depending on concentration, sex, age and strain

Kinetika proliferacije i citogenetičke promene u humanim limfocitima pod dejstvom estradiola in vitro

Metabolic conversion of oestrogen phenolic groups may create conditions of oxidative stress accompanied by damage of cellular macromolecules including DNA. The aim of this investigation was to evaluate the cell cycle kinetics and possible cytogenetic changes in cultured human peripheral blood lymphocytes exposed to seven experimental concentrations of 17β-oestradiol (range 10-10 M to 10-4 M). Cell cycle kinetics was analyzed on metaphase spreads prepared for a standard analysis of sister-chromatid exchanges (SCEs) stained by fluorescent-plus-Giemsa (FPG) technique. Cytogenetic changes were monitored by analysis of chromosome damage (gaps and breaks), structural and numerical aberrations. On the basis of the obtained results it can be concluded that oestradiol has no significant influence on cell cycle kinetics and mitotic index of cultured human lymphocytes. However, at estradiol concentration of 7×10-6 M, and at higher concentrations used in this experiment, there was a significant increase of gaps, breaks and aneuploidies. On the other hand, oestradiol treatment has not changed the frequency of polyploid cells. Therefore, it can be concluded that high concentrations of oestradiol pose some genetic risk detectable at cytogenetic level.Metabolička konverzija fenolnih grupa estrogenih hormona može da dovede do oksidativnog stresa praćenog oštećenjima različitih makromolekula u eliji, uključujući DNK. Cilj ovog istraživanja je evaluacija kinetike proliferacije i mogućih citogenetičkih promena u kulturama humanih limfocita pod dejstvom sedam eksperimentalnih koncentracija 17β-estradiola (opseg od 10-10M do10-4 M). Kinetika proliferacije limfocita analizirana je na metafaznim figurama obojenim tehnikom FPG za standardne analize razmena sestrinskih hromatida (SCE). Citogenetičke promene praćene su analizama hromozomskih oštećenja (gapovi i prekidi), strukturnih i numeričkih aberacija hromozoma. Na osnovu dobijenih rezultata može se zaključiti da estradiol ne utiče značajno na mitotsku aktivnost i kinetiku proliferacije limfocita u kulturi. Međutim, pri koncentraciji od 7×10-6 M, kao i pri višim eksperimentalnim koncentracijama korišćenim u ovim eksperimentima, zapažen je porast gapova, prekida i aneuploidija. S druge strane, tretman estradiolom ne menja učestalost poliploidnih ćelija. Prema tome, može se zaključiti da visoke koncentracije estradiola izazivaju izvestan genetički rizik koji se može detektovati na citogenetičkom nivou