Increasing the efficiency of HR (homologous recombination) is important for a successful knock-in. Rad51 is mainly involved in homologous recombination and is associated with strand invasion. The HR-related mismatch repair system maintains HR fidelity by heteroduplex rejection and repair. Therefore, the purpose of this study is to control Rad51, which plays a critical role in HR, through UV-induced DNA damage. It is also to confirm the effect on the expression of MMR related genes (Msh2, Msh3, Msh6, Mlh1, Pms2) and HR-related genes closely related to HR through treatment with the MMR inhibitor CdCl2. The mRNA expression of Rad51 gene was confirmed in both HC11 cells and mouse testes, but the mRNA expression of Dmc1 gene was confirmed only in mouse testes. The protein expression of Rad51 and Dmc1 gene increased in UV-irradiated HC11 cells. After 72 hours of treatment with 1 μm of CdCl2, the mRNA expression level of Msh3, Pms2, and Rad51 decreased, but the mRNA expression level of Msh6 and Mlh1 increased in HC11 cells. There was no significant difference in Msh2 mRNA expression between CdCl2 untreated-group and the 72 hours treated group. In conclusion, HR-related gene (Rad51) was increased by UV-induced DNA damage. Treatment of the MMR inhibitor CdCl2 in HC11 cells decreased the mRNA expression of Rad51.

In this study, we investigated the protective effects of Ulva lactuca methanol extracts against ultraviolet B (UVB)-induced DNA damage in HaCaT cells. First, the contents of general and antioxidative nutrient contents of Ulva lactuca were measured. The moisture, carbohydrate, crude protein, crude fat and ash were 14.01%, 44.80%, 23.19%, 3.10% and 14.90%, respectively. Magnesium that acts as DNA repair enzyme cofactor was the most abundant mineral followed by Ca, P and Fe. The total phenolic and anthocyanoside contents of Ulva lactuca were 2.69 mg/g and 0.13 mg/g, respectively. Cells treated with Ulva lactuca methanol extracts for 24 hours post UVB exposure increased cell viability in a concentration-dependent manner compared to the non-treated control. Also, Ulva lactuca methanol extracts decreased the levels of UVB-induced DNA damage such as cyclobutane pyrimidine dimer and DNA damage response (DDR) proteins such as p-p53 and p21. These results suggest that Ulva lactuca methanol extracts comprising physiological active substances such as Mg, polyphenols and anthocyanosides promote DNA repair by regulating genes related with DDR.

This study was investigated to test whether the zygote recognized the topoisomerase II beta (TOP2B) mediated DNA fragmentation in epididymal spermatozoa or the nuclease degradation in vas deferens spermatozoa by testing for the presence of gammaH2AX (γH2AX). The γH2AX is phosphorylation of histone protein H2AX on serine 139 occurs at sites flanking DNA double-stranded breaks (DSBs). The presence of γH2AX in the pronuclei of mouse zygotes which were injected with DNA broke epididymal spermatozoa was tested by immunohistochemistry at 5 and 9 h post fertilization, respectively. Paternal pronuclei that arose from epididymal spermatozoa treated with divalent cations did not stain for γH2AX at 5 h. On the other hand, in embryos injected with vas deferences spermatozoa that had been treated with divalent cations, γH2AX was only present in paternal pronuclei, and not the maternal pronuclei at 5 h. Interestingly, both pronuclei stained positively for γH2AX for all treatments and controls at 9 h after sperm injection. In conclusion, the embryos recognize DNA that is damaged by nuclease, but not by TOP2B because H2AX in phosphorylated in paternal pronuclei resulting from spermatozoa treated with fragmented DNA from vas deferens spermatozoa treated with divalent cations, but not from epididymal spermatozoa treated the same way.

As diethylnitrosamine (DEN) effect on cell proliferation, DNA damage and stem cell marker(s) expression have been largely unknown in mouse normal hepatocytes (AML-12 cells) cultured over a short-term period, this study was conducted to examine the cell proliferation, Ataxia telangiectasia mutated (ATM) and epithelial cell adhesion molecule (EpCAM) and Neighbor of Punc E 11 (Nope) expression in AML-12 cells treated with DEN for 24 and 48 h. Cells were treated with DEN (25-800 μg/mL) and cell phenotype was determined, and the MTT assay was used to quantify the proliferation of cells treated with DEN. Expression and distribution of ATM in AML-12 cells were determined by indirect immunofluorescence microscopy. And Western blot analysis of EpCAM and Nope was performed. Cell viability was significantly increased in response to all doses of DEN treatment compared to control at 24 h (p<0.05 or p<0.01). However, there was no significant increase at 48 h, even though it showed increased trend. Immunofluorescence staining of ATM showed that there was an increase of ATM expression at doses of 50, 100 and 200 μg/mL of DEN treatment, showing strong nuclear staining. Furthermore, Western blot analysis showed that DEN treatment showed increased trend of EpCAM and Nope expression. Taken together, DEN treatment increased cell proliferation in AML- 12 cells, and it was associated with increased ATM expression.

The acetyltransferase Tip60 (Kat5) is a member of the MYST family of HATs that was initially identified as a cellular protein. TIP60 acetylates histone and non-histone proteins, and is involved in diverse biological processes, including apoptosis, cell cycle, and DNA damage responses. In this study, a specific inhibitor of TIP60, Nu 9056, was used to study the function and its regulatory mechanism of Tip60 in the porcine preimplantation embryonic development. The results showed that inhibition of TIP60 impaired the embryonic development due to induce DNA damage through ATM-p53-p21 pathway, it was evidenced by expression of γH2A in the nuclei of blastocysts. In addition, TIP60 inhibition decreased efficiency of DNA repair by regulating P53 binding protein 1 expression. Furthermore, autophagy was induced following TIP60 inhibition through modulating microtubule-associated protein 1A/1B-light chain 3 expression. In conclusion, the results suggest that TIP60 plays a critical role in early embryonic development via regulation of DNA damage and its repairs.

The cryopreservation of sperm has become the subject of research for successful artificial insemination technologies. Antifreeze proteins (AFPs), one of the factors necessary for effective cryopreservation, are derived from certain Antarctic organisms. These proteins decrease the freezing point of water within these organisms to below the temperature of the surrounding seawater to protect the organism from cold shock. Accordingly, a recent study found that AFPs can increase the motility and viability of spermatozoa during cryopreservation.To evaluate this relationship, we performed cryopreservation of boar sperm with AFPs produced in the Arctic yeast Leucosporidium sp. AFP expression system at four concentrations (0, 0.01, 0.1, and 1 μg/ml) and evaluated motility using computer assisted sperm analysis. DNA damage to boar spermatozoa was measured by the comet assay, and sperm membrane integrity and acrosome integrity were evaluated by flow cytometry. The results showed that motility was positively affected by the addition of AFP at each concentration except 1 μg/ml (p<0.001).Although cryopreservation with AFP decreased the viability of the boar sperm using, the tail DNA analyses showed that there was no significant difference between the control and the addition of 0.1 or 0.01 μg/ml AFP. In addition, the percentage of live sperm with intact acrosomes showed the least significant difference between the control and 0.1 μg/ml AFP (p<0.05), but increased with 1 μg/ml AFP (p<0.001). Our results indicate that the addition of AFP during boar sperm cryopreservation can improve viability and acrosome integrity after thawing.

CDK2 inhibition plays a central role in DNA damage–induced cell cycle arrest and DNA repair. However, whether CDK2 also influences early porcine embryo development is unknown. In this study, we examined whether CDK2 is involved in the regulation of oocyte meiosis and early embryonic development of porcine. We found that disrupting CDK2 activity with RNAi or an inhibitor did not affect meiotic resumption or MII arrest. However, CDK2 inhibitor-treated embryos showed delayed cleavage and ceased development before the blastocyst stage. Disrupting CDK2 activity is able to induce sustained DNA damage as demonstrated by the formation of distinct γH2AX foci in nuclei of day 3- and day 5-embryos. Inhibiting CDK2 triggers a DNA damage checkpoint by activating of the ATM-P53-P21 pathway. However, the mRNA expression of genes involved in non-homologous end-joining (NHEJ) or homologous recombination (HR) pathways for double strand break (DSB) repair reduced after administering CDK2 inhibitor to 5-day-old embryos. Furthermore, CDK2 inhibition caused apoptosis in day 7 blastocysts. Thus, our results indicate that an ATM-P53-P21 DNA damage checkpoint is intact in the absence of CDK2; however, CDK2 is important for proper repair of the damaged DNA by either directly or indirectly influencing DNA repair-related gene expression.

본 연구의 목적은 NIH3T3와 HepG2 세포에서 에탄올유도 세포독성 및 유전독성에 대하여 녹차엑기스(GTE)와 epigallocatechin-3-gallate (EGCG)의 보호작용을 평가하는데 있다. 세포생존율은 MTT assay를 실시하였으며 DNA 손상도는 Comet assay로 실시한 결과 에탄올은 농도의존적인 세포독성과 유전독성을 나타내었다. 한편 GTE와 EGCG는 에탄올 유도 세포독성 및 DNA 손상에 대하여 유의성 있는 억제효과를 나타내었으며 DPPH시험과 LDL oxidation 및 8OH-2``dG 생성시험에서 항산화효과를 나타내었다. 한편 녹차성분 함유 시판 리큐르주도 순수 에탄올에 비하여 세포독성억제 및 DNA 손상억제효과를 나타내었다. 이상의 시험결과 GTE와 함유 EGCG는 항산화성 유전독성억제기전을 통한 에탄올독성저감 물질로 판단된다.

본 연구는 식용 버섯의 조리방법에 따른 항산화 생리활성의 평가를 위해 수행되었으며, 산화적 스트레스에 의한 DNA 손상 감소 효과를 통해 조리방법을 달리한 버섯 추출물의 유전독성학적 방호효과를 살펴보았다. Human lymphocyte에 조리방법을 달리한 3가지 버섯(느타리, 팽이, 표고)의 추출물을 처리하고, hydrogen peroxide(H2O2)로 산화적 손상을 준 후, DNA 감소 효과를 Comet assay로 평가한 결과, 모든 시료군에서 산화적 손상에 의한 DNA 손상 감소 효과를 나타냈다. 3가지 버섯 모두 비조리군이 조리군보다 높은 효과를 나타냈는데, 이는 조리과정에 의한 페놀성 화합물의 감소로 인한 것으로 보이며, 조리군 중에서 볶기와 전이 비교적 낮은 DNA 손상 감소 효과를 나타낸 것은 조리 시 첨가되었던 대두유의 가열 산화에 의한 것으로 사료된다. 결론적으로, 조리된 버섯은 생버섯에 비해 산화적 스트레스에 의한 DNA 손상 감소효과가 낮으나, 양성 대조군과 비교하였을 때 손상을 유의적으로 감소시킨 것으로 나타났다. 또한, 본 연구에서 사용한 네 가지 조리법(굽기, 데치기, 볶기, 전) 중 DNA 손상 감소에 효과적인 조리법은 대두유를 사용하지 않은 굽기와 데치기인 것으로 판단된다.

In the present study, the effects of extracts from Korean plants on the DNA damage response in HaCaT cells exposed to ultraviolet B (UVB) were investigated. The activity of cells treated for 24 hr with ethanol extracts from Vaccinium spp. (VS), and Vitis vinifera L (VV) alone was similar to that of the non-treated control, but gradually decreased at concentrations above 200μg/mL. However, when post-incubation of UVB-exposed cells was carried out for 24 hr in medium containing VS or VV extracts, the cell activity increased in a concentration-dependent manner compared with that in the normal growth medium. The cell viability of UVB-exposed cells also increased when post-incubated in medium containing VS or VV extracts, in a concentration-dependent manner. Nuclear fragmentation analysis showed that post-incubation with VS or VV extracts decreased the UVB-induced apoptosis by about 10 and 13%, respectively, of that in cells post-incubated in growth medium. After 24 hr of post-incubation in medium containing VS or VV extracts, the level of CPD and 8-OHdG decreased in time- and concentration-dependent manners. Overall these results suggest that VS and VV extracts assist the survival of UVB-exposed cells, in accordance with the respective decrease in the levels of UVB-induced DNA damage.

The effects of X-ray irradiation on development and reproduction of Spodoptera litura were examined. Eggs, larvae, pupae, and adults were irradiated at target doses of 10 - 250 Gy. When eggs were irradiated with 30 Gy, egg hatching was completely inhibited. When irradiated to the larvae, pupation was inhibited at 100 Gy and larval period was delayed. When irradiated to the pupae, emergence was inhibited at over 100 Gy. When irradiated to the adults, longevity and fecundity did not show any differences. However, egg hatching was significantly decreased at 70 Gy and above. Also, X-ray irradiation was not induced the rapid death of S. litura. Reciprocal crosses between irradiated and unirradiated moths demonstrated that males were more radiotolerant than females. The levels of DNA damage in S. litura adults were evaluated using the alkaline comet assay. Our results indicate that X-ray irradiation increased levels of DNA damage. The recovery of DNA damage in S. litura adults increased as time passed. But DNA damage hasn't recovered fully. These results indicate that X-ray irradiation induced abnormal development and reproduction by DNA damage in S. litura.

The effect of electron beam irradiation on development and reproduction of susceptible strain (S) and imidacloprid-resistance strain (IMI-R) of Aphis gossypii were compared. Nymphs and adults of S and IMI-R strain were irradiated at target doses of 50, 100, 150, and 200 Gy. When nymphs were irradiated, emergence rate was not affected at all target dose, but number of F1 nymphs was perfectly inhibited at 150 Gy in both strains when irradiated to the adults, longevity slightly decrease at 150 Gy and above. Fecundity was strongly decreased at 100 Gy, but was not completely inhibited even at 200 Gy. Emergence rate of F1 nymph was decreased at 100 Gy and completely inhibited at 200 Gy. However, there was no significant differences on development and reproduction of S and IMI-R strain. We also conducted the comet assay immediately after irradiation and over the following 7 day periods. In addition, we performed quantitative real-time PCR on several genes.

The effect of electron beam irradiation on development and reproduction of imidacloprid-resistant (R strain) and -susceptible (S strain) Aphis gossypii were compared. Nymphs and adults of R and S strain were irradiated at target doses of 50, 100, 150, and 200 Gy. When nymphs were irradiated, emergence was not affected at all target dose, but number of F1 nymph was perfectly inhibited at 150 Gy in both strains. When irradiated to the adults, longevity slightly decrease at 150 Gy and above. Fecundity was strongly decreased at 100 Gy, but was not completely inhibited even at 200 Gy. Emergence of F1 nymph was decreased at 100 Gy and completely inhibited at 200 Gy. However, there were no significant differences on development and reproduction of R and S strain. We also conducted the comet assay immediately after irradiation and over the following 10 day period. Severe DNA fragmentation in A. gossypii cells was observed just after irradiation and the damage was repaired during the post-irradiation period in a time-dependent manner. These results suggest that electron beam irradiation induced abnormal development, reproduction, and DNA damage in A. gossypii, but there were no significant differences between R and S strain.

This study was undertaken to determine free radical scavenging capacity and oxidative DNA damage protecting activity of methanol extract of red tea stem. The extract was subjected to assess their antioxidant potential using various in vitro systems such as DPPH•, ABTS•+ , super oxide and nitric oxide free radicals and it exhibited IC50 values of 68.88 ± 1.1, 12.08 ± 0.65, 404.38 ± 1.6, 93.6 ± 2.7, µg/mL respectively. Red tea extract also showed ferric reducing ability (FRAP) with 2606.85 mmol Fe (II)/g of extract. Furthermore, Methanol extract of red tea stem showed significant DNA damage protecting activity in concentration dependent manner against H2O2+UV induced photolysis on pUC19 plasmid DNA. Results of this study showed that the methanol extract of Red Tea stem has strong antioxidant potential along oxidative DNA damage protecting capacity that would be the significant sources of natural antioxidants, which might be helpful in preventing the progress of various oxidative stress generated diseases. Further study is necessary for isolation and characterization of the active antioxidants, which may serve as a potential source of natural antioxidant.

The effects of electron beam irradiation on life stage and reproduction of Spodoptera litura were examined. Eggs, larvae, pupae, and adults were irradiated at target doses of 30, 50, 100, 150, 200, and 250 Gy. When eggs were irradiated with 100 Gy, egg hatching was completely inhibited. When irradiated to the larvae, pupation was inhibited at 100 Gy and larval period was delayed. When irradiated to the pupae, emergence was inhibited at over 100 Gy. When irradiated to the adults, longevity and fecundity did not show any differences. However, egg hatching was significantly decreased at 100 Gy and above. Also, electron beam irradiation was not induced the rapid death of S. litura. Reciprocal crosses between irradiated and unirradiated moths demonstrated that males were more radiotolerant than females. Adult longevity was not affected in all stages. The levels of DNA damage in S. litura adults were evaluated using the alkaline comet assay. Our results indicate that electron beam irradiation increased levels of DNA damage. The recovery of DNA damage in S. litura adults increased as time passed. but DNA damage hasn’t recovered fully. These results indicate that electron beam irradiation induced abnormal development and reproduction by DNA damage in S. litura. and as time goes by, the DNA damage was decresed.

The influence of electron beam irradiation on each developmental stage and reproduction of Spodoptera litura were examined. Eggs, larvae, pupae, and adults were irradiated at target doses of 30, 50, 100, 150, 200, and 250 Gy. When eggs were irradiated with 100 Gy, egg hatching was perfectly inhibited. When irradiated to the larvae, pupation was inhibited at 100 Gy and larval period was delayed. When irradiated to the pupae, emergence was inhibited at 100 Gy and above. When irradiated to the adults, longevity and fecundity did not show any differences. However, egg hatching was strongly decreased at 100 Gy and above. Also, electron beam irradiation was not induced the instantaneous death of S. litura. Reciprocal crosses between irradiated and unirradiated moths demonstrated that males were more radiotolerant than females. Adult longevity was not affected in all stages. The levels of DNA damage in S. litura adults were evaluated using the alkaline comet assay. Our results indicate that electron beam irradiation increased levels of DNA damage. These results suggest that electron beam irradiation induced abnormal development and reproduction by DNA damage in S. litura.

수은이 DNA 수복에 미치는 영향을 알아보기 위해 E. fetida를 염화수은(II)과 이온화 방사선에 순차적으로 노출시킨 후, 단세포 겔 전기영동 기법을 이용하여 DNA의 손상 수준과 방사선 조사 후 시간 경과에 따른 수복 양상을 관찰하였다. 염화수은(II)의 농도를 40 mg kg-1으로 하여 48시간 동안 in vivo 노출 시험을 수행한 뒤 20Gy의 감마선을 조사한 결과, 시간이 지날수록 대체로 DNA 손상의 수준이 감소했다. 이온화

E. fetida를 방사선과 수은에 각각 노출시킨 후, 체강세포를 추출하고 단세포 겔 전기영동 기법을 이용하여 DNA의 손상정도와 시간의 경과에 따른 수복 양상을 평가해 보았다. 그 결과, 방사선 조사 후의 시간이 경과할수록 대체로 DNA 손상정도가 감소했으며, 12시간 내에 모든 실험군의 DNA가 완전히 수복되었다. 정확한 수복 완료 시간을 알아보기 위해 OTM 값을 대조군과 비교해 보면 2.5와 5Gy는 방사선 조사 후 약 2시간, 10과 20 Gy

각종 유전독성학적 물질로 인한 생물체내의 영향을 평가해보기 위해 E. fetida를 대상으로 본 연구를 수행하였다. 염화수은에 대한 DNA 손상을 알아보는 실험에서는 노출 시간에 상관없이 노출 농도에 비례한 유전자의 손상이 나타났다. 방사선이 지렁이의 DNA 손상에 미치는 영향을 알아본 실험에서도 역시 방사선 총 선량의 증가에 따라 DNA 손상이 증가하는 경향을 보였다. 염화수은에 48시간 동안 노출시키고 방사선을 조사한 지렁이의 세포를 comet as