UCTD and SLE patients show increased levels of oxidative and DNA damage together with an altered kinetics of DSB repair

Immunological tolerance is a critical feature of the immune system; its loss might lead to an abnormal response of lymphocytes causing autoimmune diseases. One of the most important groups belonging to autoimmune disorders is the connective tissue diseases (CTD). CTD are classified among systemic rheumatic diseases and include pathologies such as systemic lupus erythematosus (SLE), and undifferentiated CTD (UCTD). In this study, we evaluated oxidative and genome damage in peripheral blood lymphocytes from patients with SLE and UCTD, further classified on the basis of disease activity and the presence/absence of a serological profile. Oxidative damage was evaluated in cell membrane using the fluorescent fatty acid analogue BODIPY 581/591 C11. The percentage of oxidised lymphocytes in both SLE and UCTD patients was higher than in the control group, and the oxidative stress correlated positively with both disease activity and autoantibody profile. The γH2AX focus assay was used to quantify the presence of spontaneous double strand breaks (DSBs), and to assess the abilities of DSBs repair system after T cells were treated with mitomycin C (MMC). Subjects with these autoimmune disorders showed a higher number of γH2AX foci than healthy controls, but no correlation with diseases activity and presence of serological profile was observed. In addition, patients displayed an altered response to MMC-induced DSBs, which led their peripheral cells to greatly increase apoptosis. Taken together our results confirmed an interplay among oxidative stress, DNA damage and impaired DNA repair, which are directly correlated to the aggressiveness and clinical progression of the diseases. We propose the evaluation of these molecular markers to better characterize SLE and UCTD, aiming to improve the treatment plan and the quality of the patients’ life.

Evaluation of the liver and blood micronucleus, and comet assay end points in a 14-day repeated-dose study with methyl carbamate and 1,3-propane sultone

The repeated-dose liver micronucleus (RDLMN) assay is a novel method for detecting genotoxic chemicals. Two carcinogens methyl carbamate (MC) and 1,3-propane sultone (PS) were evaluated for the liver micronucleus in a 14-day repeated-dose study with Crl: CD (SD) IGS rats. Additionally, micronucleated reticulocytes (MN-RET) in peripheral blood and DNA damage (alkaline comet assay) in the liver were also assessed in the same animals. Ten groups of five male Crl: CD (SD) IGS rats were treated once daily with MC (300, 600 or 1200 mg/kg/day), PS (37.5, 75 or 150 mg/kg/day), negative control or three positive controls by oral gavage for 15 days. Blood samples were collected at 3 h after the last administration for determining MN-RET frequencies (%MN-RET), and the livers were sampled for determining the frequency of micronuclei and DNA damage. MC was negative in the comet assay, liver micronucleus assay and reticulocyte micronucleus assay, while PS was positive in all three assays. These results are consistent with the previous genotoxic findings of MC and PS. Therefore, the liver micronucleus assay can be effectively integrated into repeated-dose studies in animals. Moreover, integration of multiple genotoxicity end points into one study can reduce the number of animals, boost the experimental efficiency, and provides a comprehensive evaluation of the genotoxic potential of chemicals.

Primary mesenchymal stromal cells in co-culture with leukaemic HL-60 cells are sensitised to cytarabine-induced genotoxicity, while leukaemic cells are protected

Tumour microenvironments are hallmarked in many cancer types. In haematological malignancies, bone marrow (BM) mesenchymal stromal cells (MSC) protect malignant cells from drug-induced cytotoxicity. However, less is known about malignant impact on supportive stroma. Notably, it is unknown whether these interactions alter long-term genotoxic damage in either direction. The nucleoside analogue cytarabine (ara-C), common in haematological therapies, remains the most effective agent for acute myeloid leukaemia, yet one-third of patients develop resistance. This study aimed to evaluate the bidirectional effect of MSC and malignant cell co-culture on ara-C genotoxicity modulation. Primary MSC, isolated from patient BM aspirates for haematological investigations, and malignant haematopoietic cells (leukaemic HL-60) were co-cultured using trans-well inserts, prior to treatment with physiological dose ara-C. Co-culture genotoxic effects were assessed by micronucleus and alkaline comet assays. Patient BM cells from chemotherapy-treated patients had reduced ex vivo survival (P = 0.0049) and increased genotoxicity (P = 0.3172) than untreated patients. It was shown for the first time that HL-60 were protected by MSC from ara-C-induced genotoxicity, with reduced MN incidence in co-culture as compared to mono-culture (P = 0.0068). Comet tail intensity also significantly increased in ara-C-treated MSC with HL-60 influence (P = 0.0308). MSC sensitisation to ara-C genotoxicity was also demonstrated following co-culture with HL60 (P = 0.0116), which showed significantly greater sensitisation when MSC-HL-60 co-cultures were exposed to ara-C (P = 0.0409). This study shows for the first time that malignant HSC and MSC bidirectionally modulate genotoxicity, providing grounding for future research identifying mechanisms of altered genotoxicity in leukaemic microenvironments. MSC retain long-term genotoxic and functional damage following chemotherapy exposure. Understanding the interactions perpetuating such damage may inform modifications to reduce therapy-related complications, such as secondary malignancies and BM failure.

Expression pattern and prognostic value of N6-methyladenosine RNA methylation key regulators in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is still one of the most common malignancies worldwide. The accuracy of biomarkers for predicting the prognosis of HCC and the therapeutic effect is not satisfactory. N6-methyladenosine (m6A) methylation regulators play a crucial role in various tumours. Our research aims further to determine the predictive value of m6A methylation regulators and establish a prognostic model for HCC. In this study, the data of HCC from The Cancer Genome Atlas (TCGA) database was obtained, and the expression level of 15 genes and survival was examined. Then we identified two clusters of HCC with different clinical factors, constructed prognostic markers and analysed gene set enrichment, proteins’ interaction and gene co-expression. Three subgroups by consensus clustering according to the expression of the 13 genes were identified. The risk score generated by five genes divided HCC patients into high-risk and low-risk groups. In addition, we developed a prognostic marker that can identify high-risk HCC. Finally, a novel prognostic nomogram was developed to accurately predict HCC patients’ prognosis. The expression levels of 13 m6A RNA methylation regulators were significantly upregulated in HCC samples. The prognosis of cluster 1 and cluster 3 was worse. Patients in the high-risk group show a poor prognosis. Moreover, the risk score was an independent prognostic factor for HCC patients. In conclusion, we reveal the critical role of m6A RNA methylation modification in HCC and develop a predictive model based on the m6A RNA methylation regulators, which can accurately predict HCC patients’ prognosis and provide meaningful guidance for clinical treatment.

Assessment of Mutations Induced by Cold Atmospheric Plasma Jet Treatment Relative to Known Mutagens in Escherichia coli

The main bactericidal components of cold atmospheric plasma (CAP) are thought to be reactive oxygen and nitrogen species (RONS) and UV radiation, both of which have the capacity to cause DNA damage and mutations. Here, the mutagenic effects of CAP on Escherichia coli were assessed in comparison to X- and UV-irradiation. DNA damage and mutagenesis were screened for using a diffusion-based DNA fragmentation assay and modified Ames test respectively. Mutant colonies obtained from the latter were quantitated and sequenced. CAP was found to elicit a similar mutation spectrum to X-irradiation, that did not resemble that for UV implying that CAP produced RONS are more likely the mutagenic component of CAP. CAP treatment was also shown to promote resistance to the antibiotic ciprofloxacin. Our data suggest that CAP treatment has mutagenic effects that may have important phenotypic consequences.

Towards an advanced testing strategy for genotoxicity using image-based 2D and 3D HepG2 DNA damage response fluorescent protein reporters

In vitro assessment of mutagenicity is an essential component in the chemical risk assessment. Given the diverse modes of action by which chemicals can induce DNA damage, it is essential that these in vitro assays are carefully evaluated for their possibilities and limitations. In this study, we used a fluorescent protein HepG2 reporter test system in combination with high content imaging. To measure induction of the DNA damage response (DDR), we used three different green fluorescent protein (GFP) reporters for p53 pathway activation. These allowed for accurate quantification of p53, p21 and BTG2 (BTG anti-proliferation factor 2) protein expression and cell viability parameters at a single cell or spheroid resolution. The reporter lines were cultured as 2D monolayers and as 3D spheroids. Furthermore, liver maturity and cytochrome P450 enzyme expression were increased by culturing in an amino acid rich (AAGLY) medium. We found that culture conditions that support a sustained proliferative state (2D culturing with DMEM medium) give superior sensitivity when genotoxic compounds are tested that do not require metabolization and of which the mutagenic mode of action is dependent on replication. For compounds, which are metabolically converted to mutagenic metabolites, more differentiated HepG2 DDR reporters (e.g., 3D cultures) showed a higher sensitivity. This study stratifies how different culture methods of HepG2 DDR reporter cells can influence the sensitivity towards diverse genotoxicants and how this provides opportunities for a tiered genotoxicity testing strategy.