X-irradiated Human Lymphocytes Research Articles

Protective effects of citrus and rosemary extracts on UV-induced damage in skin cell model and human volunteers

Ultraviolet radiation absorbed by the epidermis is the major cause of various cutaneous disorders, including photoaging and skin cancers. Although topical sunscreens may offer proper skin protection, dietary plant compounds may significantly contribute to lifelong protection of skin health, especially when unconsciously sun UV exposed. A combination of rosemary and citrus bioflavonoids extracts was used to inhibit UV harmful effects on human HaCaT keratinocytes and in human volunteers after oral intake. Survival of HaCaT cells after UVB radiation was higher in treatments using the combination of extracts than in those performed with individual extracts, indicating potential synergic effects. The combination of extracts also decreased UVB-induced intracellular radical oxygen species (ROS) and prevented DNA damage in HaCaT cells by comet assay and decreased chromosomal aberrations in X-irradiated human lymphocytes. The oral daily consumption of 250mg of the combination by human volunteers revealed a significant minimal erythema dose (MED) increase after eight weeks (34%, p<0.05). Stronger protection was achieved after 12weeks (56%, p<0.01). The combination of citrus flavonoids and rosemary polyphenols and diterpenes may be considered as an ingredient for oral photoprotection. Their mechanism of action may deserve further attention.

Discrimination between complete and incomplete chromosome exchanges in X-irradiated human lymphocytes using FISH with pan-centromeric and chromosome specific DNA probes in combination with telomeric PNA probe

Purpose : To discriminate precisely between radiation-induced complete and incomplete chromosome exchanges using chromosome painting together with the detection of the centromeres and telomeres in one FISH assay. Materials and methods : Human lymphocytes were exposed in vitro to X-rays at a dose of 4 Gy. Chromosome aberrations were analysed using the FISH technique in combination with a whole chromosome-specific DNA probe for chromosome 8, human pan-centromeric DNA and telomeric PNA probes. Results : The combined FISH assay has improved the resolution of detecting chromosomal exchanges in human lymphocytes. Results indicate that the frequency of observed incomplete exchange patterns was 21% when telomeric signals were ignored during the analysis. When the telomeric signals were included in the analysis a large proportion of apparently incomplete exchange patterns appeared complete and should be re-classified. The percentage of true incomplete exchanges was found to be less than 5%. Conclusion : The combination of chromosome painting and the detection of centromeres and telomeres enable unequivocal discrimination between incomplete and complete exchanges. The fraction of true incomplete exchanges observed in X-irradiated human lymphocytes was found to be low in comparison with previous reports in the literature.

Kinetics of the formation of chromosome aberrations in X-irradiated human lymphocytes, using PCC and FISH

In order to study the initial frequencies and define kinetics of the formation of chromosomal exchanges in X-irradiated human lymphocytes, the premature chromosome condensation (PCC) technique was employed in combination with fluorescence in situ hybridization (FISH) with a composite probe for human chromosome 8 and a pan-centromeric probe for the whole genome. Human lymphocytes were X-irradiated (0.5, 1, 2, 3, 4 and 6 Gy), fused with mitotic Chinese hamster ovary (CHO) cells immediately or 1, 3, 6, 12 and 18 h after irradiation. Immediately after irradiation chromosomal breaks, dicentrics and translocations showed a linear dose-response. Unrejoined chromosome breaks were the most frequent types of aberrations (about 85%) observed. About 15% of total aberrations were chromosome exchanges of 65% of these were translocations and 35% were dicentrics. The chromosomal exchanges initially observed were mostly incomplete, with no complex exchanges at doses of 1 and 2 Gy, at higher doses (3–6 Gy) complex exchanges were observed and their frequencies increased with increasing post incubation time. Following different recovery times, repair kinetics of breaks for different doses of irradiation was studied. The shapes of the curves obtained for breaks as well as chromosome exchanges were linear-quadratic. The linear yield component, α, is formed entirely in the fast process that can be manifested in the early plateau, while component β developed slowly in the subsequent hours. The kinetics of breaks rejoining was exponential, almost 50% of breaks rejoined after 1 h and at 18 h about 20% of breaks remained. At low doses of 1 and 2 Gy most of the exchanges were formed immediately and at higher doses, the frequency of exchanges increased with kinetics similar to that observed for the rejoining of breaks. However, the kinetics was different for different doses of irradiation. The frequency of dicentrics increased at doses above 2 Gy following 3 h recovery time, but for the translocations effect was pronounced even at 1 h recovery time. The frequency of incomplete exchanges (i.e., terminal translocations) decreased with post irradiation time and at 18 h was 30–40% less than the frequency obtained immediately after irradiation. The increase in the total translocations as a function of time between irradiation and fusion was due to a rapid increase in complete exchanges (i.e., reciprocal translocations). The frequency of ring chromosomes immediately after irradiation, also increased linearly, however, it was 3–5 times lower than dicentrics and remained almost constant in number for different doses and at different post-irradiation times.

Detection of incomplete exchanges and interstitial fragments in X-irradiated human lymphocytes using a telomeric PNA probe

Purpose : The detection of incomplete exchanges and interstitial fragments by fluorescence in situ hybridization using a telomeric peptide nucleic acid (PNA) probe. Materials and methods : Isolated human lymphocytes were exposed in vitro to X-rays at a dose of 3 Gy. Aberrations were analysed in the first mitosis after irradiation using a telomeric PNA probe. Results : After an acute dose of 3 Gy, only about 16% of the cells contained a pair of incomplete chromosome elements with telomeric signals at only one terminal end. Acentric interstitial fragments, lacking telomeric signals, were observed with a frequency of 0.56 per cell, which was very similar to the dicentric frequency (0.61 per cell). Conclusions : The low frequency of incompleteness suggests that most of the non-reciprocal interchanges observed using chromosome painting must originate from terminal exchanges rather than from incomplete exchanges. Furthermore, it was estimated that about 78% of excess acentric fragments originate from interstitial fragments and only 22% from terminal fragments.

Kinetics of the Formation of Chromosome Aberrations in X-Irradiated Human Lymphocytes: Analysis by Premature Chromosome Condensation with Delayed Fusion

Human lymphocytes irradiated with graded doses of up to 5 Gy of 150 kV X rays were fused with mitotic CHO cells after delay times ranging from 0 to 14 h after irradiation. The yields of dicentrics seen under PCC conditions, using C-banding for centromere detection, and of excess acentric fragments observed in the PCC experiment were determined by image analysis. At 4 Gy the time course of the yield of dicentrics shows an early plateau for delay times up to 2 h, then an S-shaped rise and a final plateau which is reached after a delay time of about 8 to 10 h. Whereas the dose-yield curve measured at zero delay time is strictly linear, the shape of the curve obtained for 8 h delay time is linear-quadratic. The linear yield component, alpha D, is formed entirely in the fast process manifested in the early plateau, while component beta D2 is developed slowly in the subsequent hours. Analysis of the kinetics of the rise of the S-shaped curve for yield as a function of time leads to the postulate of an "intermediate product" of pairwise DNA lesion interaction, still fragile when subjected to the stress of PCC, but gradually processed into a stable dicentric chromosome. It is concluded that the observed difference in the kinetics of the alpha and beta components explains a number of earlier results, especially the disappearance of the beta component at high LET, and opens possibilities for chemical and physical modification of the beta component during the extended formation process after irradiation observed here.

Micronuclei and 3AB index in X-irradiated human lymphocytes in G1 and G1 phases

We applied the cytokinesis-block micronucleus assay to observe the radiobiological response of human lymphocytes after X-ray treatment in the G0 and G1 phases. In addition, we used 3-aminobenzamide (3AB) to measure the 3AB index in the two phases. The experimental results show that the at 2 Gy the MN yield and the 3AB index are dependent on the cell phase and show considerable inter-individual variability. The radiation-induced MN frequency obtained for 33 subjects is 0.470 ± 0.063 for the G0 phase and 0.689 ± 0.139 for the G1 phase; the 3AB index values are 0.326 ± 0.144 and 0.067 ± 0.058 for G0 and G1 phases, respectively. At the individual level, the 3AB index for the G1 phase correlations inversely with the cytogenetic effects observed in that phase. We discuss the possibility of applying the MN test combined with the 3AB index to lymphocytes at different phases to study the individual response to radiation (individual radiosensitivity).

Effects of adenine arabinoside and coformycin on the kinetics of G2 chromatid aberrations in X-irradiated human lymphocytes.

The kinetics of chromatid aberrations have been studied in human lymphocytes exposed to X-rays in the G2 phase of the cell cycle and incubated with or without the nucleoside analogue 9-beta-D-arabinofuranosyladenine (ara A), known to inhibit the repair of DNA double-strand breaks. In the absence of ara A an exponential decrease in frequencies of chromatid breaks occurred which we interpret as repair. Few breaks were observed if samples were harvested immediately following irradiation. The frequency of chromatid breaks at 1 h after X-irradiation (442 per 100 cells/Gy) was similar to that previously observed in Chinese hamster ovary (CHO) K1 cells. However, the exponential decrease of chromatid breaks between X-irradiation and sampling occurred with a t1/2 of 0.87 h, a faster rate than we have previously observed in CHO K1 cells and was not inhibited by 200 microM ara A alone, in contrast with our previous findings in a human fibroblast line. However, in the presence of the ADA inhibitor coformycin, inhibition of break repair was already observed at an ara A concentration of 100 microM indicating that the apparent unresponsiveness to ara A of lymphocyte chromatid break rejoining results from the deamination of this nucleoside analogue. This deamination effect was confirmed by measurements of DNA synthesis which showed stable inhibition of synthesis by ara A only when coformycin was present. Frequencies of chromatid exchanges in irradiated cells remained constant except at the sampling time directly after irradiation, consistent with the view that chromosomal radiosensitivity remained constant throughout the G2 phase, except for the period immediately prior to mitosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Micronuclei in X-irradiated human lymphocytes

The dose-effect relationship of the frequency of micronuclei in cytokinesis-blocked human lymphocytes after in vitro irradiation of whole blood in the range from 0.2 to 4 Gy was studied. The linear-quadratic response obtained offers a useful technique for dose assessments in cases of radiation injuries above approx. 0.1 Gy whole body dose. The distribution in size of micronuclei in cytochalasin B treated cells ranges to larger diameters than in mononuclear lymphoblasts.

Micronuclei in X-irradiated human lymphocytes

The dose-effect relationship of the frequency of micronuclei in cytokinesis-blocked human lymphocytes after in vitro irradiation of whole blood in the range from 0.2 to 4 Gy was studied. The linear-quadratic response obtained offers a useful technique for dose assessments in cases of radiation injuries above approx. 0.1 Gy whole body dose. The distribution in size of micronuclei in cytochalasin B treated cells ranges to larger diameters than in mononuclear lymphoblasts.

Effects of aphidicolin on repair replication and induced chromosomal aberrations in mammalian cells

The influence of aphidicolin, an inhibitor of polymerase α, on UV-induced repair replication in human skin fibroblasts, as well as in HeLa cells, was determined. In growing fibroblasts and in HeLa cells, aphidicolin had a potentiating effect on UV-induced repair replication, whereas in fibroblasts grown to confluency, aphidocolin had an inhibitory effect. This inhibitory effect was stronger when measured in the presence of hydroxyurea. In HeLa cells the presence of both aphidicolin and hydroxyurea also had an inhibitory effect, but in the presence of hydroxyurea alone, UV-induced repair replication was enhanced. The results of these studies can be explained on the basis of differences in deoxyribonucleotide triphosphate pool sizes in growing and confluent cells. Post-treatment of X-irradiated human lymphocytes in the G 0 and G 1 stages with aphidicolin increased the frequencies of X-ray-induced chromosomal aberrations. Such an increase was not observed in G 1 cells of CHO after similar treatment with X-rays and aphidicolin. However, treatment with aphidicolin, in the G 2 stage, of CHO cells that had been exposed to UV or alkylating agents in the G 1 stage increased the frequencies of induced chromatid breaks. The significance of these results is discussed.