Fluorescencijska hibridizacija in situ (FISH) u detekciji kromosomskih oštećenja nastalih kao posljedica profesionalne izloženosti ionizirajućem zračenju

For more than two decades, chromosome aberration analysis has been used in biomonitoring of occupational and environmental exposure to ionising radiation. Chromosome aberration analysis is a method used to detect unstable aberrations in the lymphocytes of irradiated personnel. In turn, stable chromosome aberrations that arise some time after exposure are detected using the multicolour fluorescence in situ hybridisation. This is a technique which dyes each pair of chromosomes with different colour. Due to the dynamics of unstable aberration formation, chromosome aberration analysis is more suitable for genome damage assessment of recent exposures. On the other hand, fluorescence in situ hybridisation gives information on chromosome instability caused by long-time occupational exposure to ionising radiation. Considering the high cost of fluorescence in situ hybridisation and the uncertainty of the result, it should be used in biodosimetry only when it is absolutely necessary.Posljednjih dvadesetak godina analiza kromosomskih aberacija rabi se kao glavna citogenetička tehnika u biodozimetriji osoba profesionalno izloženih zračenju. Metoda omogućava utvrđivanje prisutnosti nestabilnih tipova oštećenja u limfocitima ozračenih osoba. Međutim, njome nije moguće istodobno utvrditi i prisutnost stabilnih tipova oštećenja kao što su translokacije. U tu svrhu potrebno se koristiti višebojnom fluorescencijskom hibridizacijom in situ (mFISH) kojom je moguće svaki par kromosoma obojiti drugom bojom. Zbog same dinamike nastanka nestabilnih kromosomskih oštećenja analiza kromosomskih aberacija, kao citogenetička tehnika, prikladnija je za procjenu genomskih oštećenja u kratkom vremenu nakon ozračivanja, dok bi FISH mogao naći primjenu u detekciji stabilnih oštećenja genoma koja nastaju kao posljedica dugotrajne profesionalne izloženosti zračenju. Međutim, zbog visokih troškova same tehnike i zahtjevnosti njezine izvedbe, FISH kao tehnika u biodozimetriji rabila bi se samo u slučajevima kada podatke o stupnju oštećenja genoma nije moguće dobiti primjenom ni jedne druge citogenetičke tehnike

Efficacy of HUMN criteria for scoring the micronucleus assay in human lymphocytes exposed to a low concentration of p, p, -DDT

The cytokinesis-block micronucleus (CBMN) assay is one of the standard cytogenetic tools employed to assess chromosomal damage subsequent to exposure to genotoxic/cytotoxic agents, and is widely applicable to plant, animal and human cells. In the present study, the CBMN assay was used to assess the baseline damage in binuclear human peripheral blood lymphocytes exposed to 25 microg/L p, p'-DDT for 1, 2, 24, and 48 h by measuring the frequency of micronuclei, nucleoplasmic bridges and nuclear buds. These new scoring criteria facilitated the detection of different types of clastogenic and aneugenic effects induced by this type of pollutant. With these criteria, CBMN can also be used to measure nucleoplasmic bridges which are considered to be consequences of chromosome rearrangements and nuclear buds which are biomarkers of altered gene amplification and gene dosage. The total number of micronuclei observed in binuclear human peripheral blood lymphocytes of the exposed samples (ranging from 32 to 47) was significantly greater (P < 0.05) than that detected in the unexposed (0 time) control sample, where the total number of micronuclei was 7. The number of nucleoplasmic bridges and nuclear buds obtained after 24 and 48 h was also significantly (P < 0.05) greater in the samples treated with p, p'-DDT than in the unexposed control samples. Thus, our results confirmed the usefulness of the new criteria applicable for the CBMN assay employed in measuring the DNA damage and its role of a sensitive cytogenetic biomarker

Evaluation of DNA Damage in Radiotherapy-Treated Cancer Patients Using the Alkaline Comet Assay

The aim of this study was to evaluate primary DNA damage and the dynamics of the repair of radiotherapy-induced DNA lesions in non-target cells of cancer patients. This study included patients diagnosed with different solid tumors who received radiotherapy. The levels of DNA damage were evaluated using the alkaline comet assay on peripheral blood leukocytes. Altogether four blood samples per patient were collected: before and after receiving the first dose of radiotherapy, in the middle of radiotherapy cycle, and after the last dose of radiotherapy. The results indicate that after the first radiation dose significantly increased levels of DNA damage were recorded in almost all cancer patients compared to their baseline values. Specific patterns of DNA damage were recorded in samples analyzed in the middle of radiotherapy and after receiving the last dose, indicating the possibility of adaptive response in some patients. Our results indicate that persistence of post-irradiation damage in peripheral blood leukocytes (and possibly in other non-target cells) of cancer patients that are strong determinants for the secondary cancer risk. Moreover, the alkaline comet assay was confirmed as a rapid and sensitive assay for the assessment of genome damage after in vivo irradiation

Procjena citogenetičkih oštećenja u medicinskog osoblja profesionalno izloženog niskim dozama ionizirajućeg zračenja na odjelima nuklearne medicine

The aim of this study was to provide data on genetic hazards associated with occupational exposure to low doses of ionising radiation in nuclear medicine departments. The DNA damage in peripheral blood lymphocytes of medical staff was assessed using the chromosome aberration test. Altogether 120 subjects (60 exposed and 60 controls) participated in the study. The exposed subjects showed significantly higher frequencies of chromosome aberrations than controls. Significant inter-individual differences in DNA damage within the exposed population indicate different genome sensitivity. Age and sex were not confounding factors, while smoking increased DNA damage only in control subjects. This study suggests that chronic exposure to low doses of ionising radiation in nuclear medicine departments causes cytogenetic damage. For this reason, exposed medical personnel should minimise radiation exposure wherever possible. Our results also point to the significance of biological indicators, which provide information about the actual risk for the radiation-exposed individuals.Ionizirajuće zračenje izaziva različita oštećenja u živim stanicama, čija je pojava ovisna o dozi kojoj su stanice izložene, apsorbiranoj dozi, trajanju izloženosti te osjetljivosti tkiva. Osoblje profesionalno izloženo zračenju pod povećanim je rizikom od štetnih učinaka ionizirajućeg zračenja samo u slučajevima nepoštivanja propisa sigurnosti pri radu. Dugogodišnja profesionalna izloženost zračenju za posljedicu može imati zloćudne bolesti (rak) i genske mutacije. Unatoč intenzivnim istraživanjima, genetska oštećenja proizašla iz izloženosti osoblja ionizirajućem zračenju još nisu potpuno razjašnjena, a posebice ona proizašla iz izloženosti niskim dozama. Cilj ovog istraživanja bio je proučiti genetska oštećenja nastala pod utjecajem profesionalne izloženosti niskim dozama ionizirajućeg zračenja u osoblja zaposlenog na odjelima nuklearne medicine. Procjena oštećenja DNA u limfocitima periferne krvi izloženoga medicinskog osoblja provedena je s pomoću testa analize kromosomskih aberacija. Istraživanje je obuhvatilo ukupno 120 ispitanika (60 izloženih i 60 kontrolnih). U izloženih ispitanika utvrđena je statistički značajno povišena učestalost kromosomskih aberacija u usporedbi s kontrolnom skupinom. Unutar izložene skupine uočene su i značajne interindividualne razlike u razini oštećenja DNA, koje upućuju na različitu genetsku osjetljivost. Dob i spol ispitanika nisu značajnije utjecali na razinu oštećenja genoma, a navika pušenja utjecala je na porast razine oštećenja DNA samo u ispitanika kontrolne skupine. Na osnovi dobivenih rezultata vidljivo je da kronična profesionalna izloženost niskim dozama ionizirajućeg zračenja na odjelima nuklearne medicine izaziva genotoksična oštećenja u limfocitima periferne krvi medicinskog osoblja. Kako bi se izbjegli potencijalni genotoksični učinci, izloženost medicinskog osoblja, kad god je to moguće, treba smanjiti na najmanju moguću razinu. Dobiveni rezultati također naglašavaju važnost primjene bioloških indikatora koji pružaju niz informacija o stvarnom i trenutačnom riziku za izložene ispitanike koji se ne mogu dobiti iz fizikalne dozimetrije

Atorvastatin therapy safety from the aspect of genotoxicity

Primjena statina danas predstavlja jedan od osnovnih pristupa u liječenju bolesti koje su vezane uz povišenu razinu kolesterola u krvi. Osnovni cilj djelovanja ove skupine lijekova usmjeren je prema snižavanju ukupne koncentracije kolesterola u krvi inhibicijom djelovanja 3-hidroksi-3-metilglutaril koenzim A (HMG-CoA) reduktaze. Da bi se postigao što bolji terapijski učinak u što kraćem vremenskom razdoblju, primjenjuju se različite koncentracije ovih lijekova u rasponu od 10 mg/dan do 80 mg/dan. Rezultati dosadašnjih epidemioloških istraživanja pokazali su da određene koncentracije ovih lijekova mogu ostaviti posljedice na okolne stanice i tkiva nakon dulje izloženosti, od kojih su najčešće bolesti skeletnih mišića, jetrene bolesti, povećane vrijednosti serumskih transaminaza, te miopatija koja može voditi u rabdomiolizu i bubrežno zatajenje. Istraživanja lijekova koji se koriste za snižavanje razine kolesterola u krvi provode se od njihovog otkrića u ranim sedamdesetim godinama prošloga stoljeća. Iako su se pristupi istraživanjima mijenjali tijekom godina, cilj im je uvijek bio isti – pronaći one najpogodnije te one doze u kojima bi njihova djelotvornost bila najučinkovitija, a rizik od mogućih posljedica najmanji. Ovaj rad stoga donosi pregled dosadašnjih istraživanja vezanih uz sigurnost atorvastatina u terapijske svrhe i smjernice za provođenje protokola za tesiranje lijekova.The application of statin today represents one of the basic approaches to the treatment of the disease connected to high levels of cholesterol in the blood. Statins are a widely used group today found in different generic names of cholesterol-lowering agents that act by inhibiting 3-hydroxy 3-methylglutaryl CoA (HMG CoA) reductase, an enzyme which catalyses the rate-limiting step in cholesterol biosynthesis. In order to achieve the best therapeutical effects in the shortest period of time various concentrations of these drugs are administered in the range of containing 10 mg/day to 80 mg/day atorvastatin. The results of recent epidemiological researches have shown that determined concentrations of these drugs administered over a longer period can have serious side effects. The most important adverse effects in clinical practice are disease of the skeletal muscles, asymptomatic increases in liver transaminases, progressive liver disease and myopathy which can lead to rhabdomyolysis and cause acute renal failure. Researches regarding this group of drugs have been conducted since the early seventies of the last century with the same effort; to find suitable dosage that would be effective in reducing the cholesterol level. Even though the research approaches have changed through the years, the aim has always been the same; to find the most adequate and most effective dosage with the lowest side effect risk. This review paper brings therefore an overview of recent researches regarding atorvastatin therapy safety with guidelines for protocols used in drug research

Presence of Dichlorodiphenyltrichloroethane (DDT) in Croatia and Evaluation of Its Genotoxicity

The DDT was one of the most commonly used pesticide in mid 20th century and even though its use is banned it is detectible in water, soil, fish, meat and milk products. Using comet assay and micronucleus test we have managed to detect genotoxic properties of this pesticide on human peripheral blood lymphocytes. Results obtained in this research indicate the need for further environmental and food monitoring, and cytogenetic research using sensitive methods in detection of primary genome damage after exposure to DDT to establish the impact of such chemicals on human genome and health

Cytogenetic Effects of Met-enkephalin (Peptid-M) on Human Lymphocytes

The structure, complementary structure and cytogenetic/proliferative effects of the Met-enkephalin on human peripheral blood lymphocytes were analyzed. Met-enkephalin, i.e. Peptid-M (LUPEX®), is a low molecular weight synthetic pentapeptide that corresponds to thymus Met-enkephalin. Its structure was examined by means of NMR spectroscopy. The influence of Met-enkephalin on in vitro normalization of chromosomally aberrant lymphocytes of patients suffering from different immune-mediated diseases, was analyzed by the sensitive cytogenetic tests for screening and detection of genome damages in human lymphocytes. The tests showed that in vitro stimulation of human lymphocytes with the Met-enkephalin led to dissapearance of different types of chromosome aberrations, reduction in the number of micronuclei, decrease in the frequency of sister chromatid exchange (SCE) and apoptosis as well as a cytostatic effect on mitosis cycles. They have also confirmed normalization of chromosomally aberrant cell findings in patients suffering from different immune-mediated diseases. These results suggest a possible role of Met-enkephalin (Peptid-M) in immunotherapy of different diseases which involve chromosomal aberrations as well as abnormal cell proliferation and offer new approaches to immunotherapy by the use of Peptid-M. Based on the molecular recognition theory and the SCA method, peptide complementary to Peptid-M was designed, synthesized and denoted Peptide-D. Peptide-D is a calpastatin fragment. Predicted ligand-receptor interaction between both peptides is confirmed by the results showing that Peptide-D blocked the Peptid-M induced lymphocyte proliferation in a dosedependent manner

Cytogenetic Effects of Met-enkephalin (Peptid-M) on Human Lymphocytes

The structure, complementary structure and cytogenetic/proliferative effects of the Met-enkephalin on human peripheral blood lymphocytes were analyzed. Met-enkephalin, i.e. Peptid-M (LUPEX®), is a low molecular weight synthetic pentapeptide that corresponds to thymus Met-enkephalin. Its structure was examined by means of NMR spectroscopy. The influence of Met-enkephalin on in vitro normalization of chromosomally aberrant lymphocytes of patients suffering from different immune-mediated diseases, was analyzed by the sensitive cytogenetic tests for screening and detection of genome damages in human lymphocytes. The tests showed that in vitro stimulation of human lymphocytes with the Met-enkephalin led to dissapearance of different types of chromosome aberrations, reduction in the number of micronuclei, decrease in the frequency of sister chromatid exchange (SCE) and apoptosis as well as a cytostatic effect on mitosis cycles. They have also confirmed normalization of chromosomally aberrant cell findings in patients suffering from different immune-mediated diseases. These results suggest a possible role of Met-enkephalin (Peptid-M) in immunotherapy of different diseases which involve chromosomal aberrations as well as abnormal cell proliferation and offer new approaches to immunotherapy by the use of Peptid-M. Based on the molecular recognition theory and the SCA method, peptide complementary to Peptid-M was designed, synthesized and denoted Peptide-D. Peptide-D is a calpastatin fragment. Predicted ligand-receptor interaction between both peptides is confirmed by the results showing that Peptide-D blocked the Peptid-M induced lymphocyte proliferation in a dosedependent manner

The biological effects of diagnostic cardiac imaging on chronically exposed physicians: the importance of being non-ionizing

Ultrasounds and ionizing radiation are extensively used for diagnostic applications in the cardiology clinical practice. This paper reviewed the available information on occupational risk of the cardiologists who perform, every day, cardiac imaging procedures. At the moment, there are no consistent evidence that exposure to medical ultrasound is capable of inducing genetic effects, and representing a serious health hazard for clinical staff. In contrast, exposure to ionizing radiation may result in adverse health effect on clinical cardiologists. Although the current risk estimates are clouded by approximations and extrapolations, most data from cytogenetic studies have reported a detrimental effect on somatic DNA of professionally exposed personnel to chronic low doses of ionizing radiation. Since interventional cardiologists and electro-physiologists have the highest radiation exposure among health professionals, a major awareness is crucial for improving occupational protection. Furthermore, the use of a biological dosimeter could be a reliable tool for the risk quantification on an individual basis