Genotoxicity, cytotoxicity and gene expression in patients undergoing elective surgery under isoflurane anaesthesia

There are numerous studies reporting on the effects of inhalation anaesthesia in cells of exposed individuals but not much is known about the ability of isoflurane (ISF) to induce oxidative DNA damage. However, surgery is often associated with a temporary perioperative immunological alteration, and some volatile anaesthetics seem to contribute to a transient lymphocytopenia after surgery. We conducted a study to evaluate a possible genotoxic effect, including oxidative DNA damage, and apoptosis in peripheral lymphocytes of 20 patients American Society of Anaesthesiologists physical status I undergoing minor elective surgery lasting at least 120 min, under anaesthesia with ISF. We also investigated the expression of several genes in blood cells. Blood samples were collected at three time points: before anaesthesia (T(1)), 2 h after the beginning of anaesthesia (T(2)) and on the first post-operative day (T(3)). General DNA damage and oxidised bases (Fpg and endo III-sites) in blood lymphocytes were evaluated using the comet assay. Lymphocytes were phenotyped and apoptosis was evaluated by flow cytometry. In addition, expressions of hOGG1 and XRCC1, genes involved in DNA repair, and BCL2, a gene related to apoptosis, were assessed by quantitative real-time polymerase chain reaction. Results showed no statistically significant difference in the level of DNA damage and oxidised bases among the three sampling times. Anaesthesia with ISF did not increase the percentage of cells in early or late apoptosis in cytotoxic or helper T lymphocytes. Lower hOGG1 and BCL2 expressions were detected at T(3) in comparison to the other two previous time points, and there was significantly lower expression of XRCC1 at T(3) in relation to T(2). In conclusion, the exposure to ISF did not result in genotoxicity and cytotoxicity in lymphocytes and in toxicogenomic effect in leukocytes, although DNA repair and apoptosis-related genes were down-regulated on the first post-operative day.     

Radio-adaptive response,individual radio-sensitivity and correlation of base excision repair gene polymorphism (hOGG1, APE1, XRCC1, and LIGASE1) in human peripheral blood mononuclear cells exposed to gamma radiation

Radio-adaptive response (RAR) is a biological mechanism, where cells primed with a low dose exhibit reduced DNA damage with a high challenging dose. Single nucleotide polymorphisms (SNPs) of DNA repair genes including base excision repair (BER) pathway are known to be associated with radio-sensitivity but involvement in RAR is not yet understood. In the present study, attempt was made to correlate genotype frequencies of four BER SNPs [hOGG1(Ser326Cys), XRCC1(Arg399Gln), APE1(Asp148Glu) and LIGASE1(A/C)] with DNA damage, repair and mRNA expression level among 20 healthy donors (12 adaptive and 8 nonadaptive). Our results revealed that LIGASE1 (p = .002) showed significant correlation with DNA damage and mRNA expression level with increasing dose. hOGG1 (Ser326Cys), XRCC1 (Arg399Gln) and LIGASE1(A/C) polymorphisms showed significant difference with DNA damage (%T) and mRNA expression profile in primed cells among adaptive donors. In conclusion, BER gene polymorphisms play important role in identifying donors with radio-sensitivity and RAR in human cells.     

hOGG1(326), XRCC1(399) and XRCC3(241) polymorphisms influence micronucleus frequencies in human lymphocytes in vivo

A pooled analysis of five biomonitoring studies was performed to assess the influence of hOGG1(326), XRCC1(399) and XRCC3(241) gene polymorphisms on micronuclei (MN) frequency in human peripheral blood lymphocytes, as measured by the ex vivo/in vitro cytokinesis-block micronucleus (CBMN) assay. Each study addressed a type of occupational exposure potentially able to induce DNA strand breakage (styrene, ionising radiation, cobalt/hard metal, welding fumes and inorganic arsenite compounds), and therefore MN, as a result of base excision repair and double-strand break repair deficiencies. The effect of genotype, age, exposure to genotoxic agents and smoking habit on MN induction was determined using Poisson regression analysis in 171 occupationally exposed male workers and in 132 non-exposed male referents. The analysis of genotype-genotype, genotype-smoking and genotype-exposure interactions by linear combinations of parameters showed significantly higher MN frequencies in the following subsets: (i) occupationally exposed workers carrying either the Thr/Thr or the Thr/Met XRCC3(241) genotypes compared to their referent counterparts (P < 0.001) and (ii) carriers of the Met/Met XRCC3(241) genotype compared to Thr/Thr XRCC3(241) carriers, as far as they are non-exposed and bear the variant (Ser/Cys or Cys/Cys) hOGG1(326) genotype (P < 0.01). Significantly lower MN frequencies were observed in carriers of the variant hOGG1(326) genotype compared to Ser/Ser hOGG1(326) carriers in the subgroup of non-smokers with Thr/Thr XRCC3(241) genotype (P < 0.01). Stratified analysis by occupational exposure showed a significant MN increase with smoking in occupationally exposed carriers of the Arg/Gln XRCC1(399)genotype (P < 0.001). In contrast, a significant MN decrease with smoking was observed in referents carrying the Ser/Ser hOGG1(326) genotype (P < 0.01). These findings provide evidence that the combination of different DNA repair genes and their interaction with environmental genotoxic agents may modulate MN induction. Understanding the complexity of the relationships between exposure, DNA repair and MN frequencies require larger scale studies and complementary biomarkers.     

Transient Effects of Anesthesia on Leukocyte Apoptosis and Monocyte Cytokine Stimulation: A Clinical Study

The effects of anesthetics on immune cell apoptosis and cytokine stimulation were studied in a prospective study. American Society of Anesthesiologists I/II patients underwent elective inguinal hernia repair or varicose veins stripping surgery and were randomized to either epidural anesthesia (n = 14) or general anesthesia with sevoflurane (n = 19) or propofol (n = 15). Blood was sampled before anesthesia induction (T0), at the end of surgery (T1), and 6 h later (T2). Apoptosis was determined by ANNEXIN-V staining of white blood cells; monocytes were isolated and stimulated for cytokine production. Results were compared with 10 healthy volunteers well-matched for age and gender. Apoptosis of lymphocytes and monocytes was increased in the epidural and sevoflurane groups at T2. Propofol group had increased production of interleukin-6 at T1 and sevoflurane and epidural groups had decreased production of tumor necrosis factor-alpha at T2. Results emphasize the modulation of immune function by epidural and sevoflurane but not propofol anesthesia in a clinical setting.     

Cadmium Induced Cell Apoptosis,DNA Damage,Decreased DNA Repair Capacity,and Genomic Instability during Malignant Transformation of Human Bronchial Epithelial Cells

Cadmium and its compounds are well-known human carcinogens, but the mechanisms underlying the carcinogenesis are not entirely understood. Our study was designed to elucidate the mechanisms of DNA damage in cadmium-induced malignant transformation of human bronchial epithelial cells. We analyzed cell cycle, apoptosis, DNA damage, gene expression, genomic instability, and the sequence of exons in DNA repair genes in several kinds of cells. These cells consisted of untreated control cells, cells in the fifth, 15th, and 35th passage of cadmium-treated cells, and tumorigenic cells from nude mice using flow cytometry, Hoechst 33258 staining, comet assay, quantitative real-time polymerase chain reaction (PCR), Western blot analysis, random amplified polymorphic DNA (RAPD)-PCR, and sequence analysis. We observed a progressive increase in cell population of the G0/G1 phase of the cell cycle and the rate of apoptosis, DNA damage, and cadmium-induced apoptotic morphological changes in cerebral cortical neurons during malignant transformation. Gene expression analysis revealed increased expression of cell proliferation (PCNA), cell cycle (CyclinD1), pro-apoptotic activity (Bax), and DNA damage of the checkpoint genes ATM, ATR, Chk1, Chk2, Cdc25A. Decreased expression of the anti-apoptotic gene Bcl-2 and the DNA repair genes hMSH2, hMLH1, ERCC1, ERCC2, and hOGG1 was observed. RAPD-PCR revealed genomic instability in cadmium-exposed cells, and sequence analysis showed mutation of exons in hMSH2, ERCC1, XRCC1, and hOGG1 in tumorigenic cells. This study suggests that Cadmium can increase cell apoptosis and DNA damage, decrease DNA repair capacity, and cause mutations, and genomic instability leading to malignant transformation. This process could be a viable mechanism for cadmium-induced cancers.     

Effects of silver nanoparticles on oxidative DNA damage–repair as a function of p38 MAPK status: A comparative approach using human Jurkat T cells and the nematode Caenorhabditis elegans

The large‐scale use of silver nanoparticles (AgNPs) has raised concerns over potential impacts on the environment and human health. We previously reported that AgNP exposure causes an increase in reactive oxygen species, DNA damage, and induction of p38 MAPK and PMK‐1 in Jurkat T cells and in Caenorhabditis elegans. To elucidate the underlying mechanisms of AgNP toxicity, here we evaluate the effects of AgNPs on oxidative DNA damage–repair (in human and C. elegans DNA glycosylases hOGG1, hNTH1, NTH‐1, and 8‐oxo‐GTPases—hMTH1, NDX‐4) and explore the role of p38 MAPK and PMK‐1 in this process. Our comparative approach examined viability, gene expression, and enzyme activities in wild type (WT) and p38 MAPK knock‐down (KD) Jurkat T cells (in vitro) and in WT and pmk‐1 loss‐of‐function mutant strains of C. elegans (in vivo). The results suggest that p38 MAPK/PMK‐1 plays protective role against AgNP‐mediated toxicity, reduced viability and greater accumulation of 8OHdG was observed in AgNP‐treated KD cells, and in pmk‐1 mutant worms compared with their WT counterparts, respectively. Furthermore, dose‐dependent alterations in hOGG1, hMTH1, and NDX‐4 expression and enzyme activity, and survival in ndx‐4 mutant worms occurred following AgNP exposure. Interestingly, the absence or depletion of p38 MAPK/PMK‐1 caused impaired and additive effects in AgNP‐induced ndx‐4(ok1003); pmk‐1(RNAi) mutant survival, and hOGG1 and NDX‐4 expression and enzyme activity, which may lead to higher accumulation of 8OHdG. Together, the results indicate that p38 MAPK/PMK‐1 plays an important protective role in AgNP‐induced oxidative DNA damage–repair which is conserved from C. elegans to humans. Environ. Mol. Mutagen. 55:122–133, 2014. © 2013 Wiley Periodicals, Inc.     

BER修复基因多态性与肺癌易感性的研究进展

碱基切除修复(base excision repair,BER)通路是DNA损伤修复的关键通路,通路中的8-羟基鸟嘌呤DNA糖苷酶基因(human 8-oxoguanine glycosylase,hOGG1),人类X线交叉互补修复基因(X-ray repair cross-complementing group 1,XRCC1),MutY homolog(MUTYH)基因的单核苷酸多态性影响BER通路中重要的酶和蛋白质的功能,导致修复障碍,最终引起癌症发生.DNA损伤修复基因单核苷酸多态性和肺癌易感性的研究结果尚存在争议,本文对近年来BER通路基因hOGG1Ser326Cys,XRCC1 Arg194Trp,XRCC1 Arg280His,XRCC1 Arg399Gln,XRCC1-77T>C和MUTYHHis324Gln多态性与肺癌易感性的关系的研究进行汇总,并探讨了多项研究对BER基因多态性与不同肺癌亚型的关系以及与吸烟之间关系.基因多态性与肺癌易感性关系受多因素影响,其相关性尚待进一步探索.     

BER修复基因多态性与肺癌易感性的研究进展

碱基切除修复(base excision repair,BER)通路是DNA损伤修复的关键通路,通路中的8-羟基鸟嘌呤DNA糖苷酶基因(human 8-oxoguanine glycosylase,hOGG1),人类X线交叉互补修复基因(X-ray repair cross-complementing group 1,XRCC1),MutY homolog(MUTYH)基因的单核苷酸多态性影响BER通路中重要的酶和蛋白质的功能,导致修复障碍,最终引起癌症发生.DNA损伤修复基因单核苷酸多态性和肺癌易感性的研究结果尚存在争议,本文对近年来BER通路基因hOGG1Ser326Cys,XRCC1 Arg194Trp,XRCC1 Arg280His,XRCC1 Arg399Gln,XRCC1-77T>C和MUTYHHis324Gln多态性与肺癌易感性的关系的研究进行汇总,并探讨了多项研究对BER基因多态性与不同肺癌亚型的关系以及与吸烟之间关系.基因多态性与肺癌易感性关系受多因素影响,其相关性尚待进一步探索.     

BER修复基因多态性与肺癌易感性的研究进展

碱基切除修复(base excision repair,BER)通路是DNA损伤修复的关键通路,通路中的8-羟基鸟嘌呤DNA糖苷酶基因(human 8-oxoguanine glycosylase,hOGG1),人类X线交叉互补修复基因(X-ray repair cross-complementing group 1,XRCC1),MutY homolog(MUTYH)基因的单核苷酸多态性影响BER通路中重要的酶和蛋白质的功能,导致修复障碍,最终引起癌症发生.DNA损伤修复基因单核苷酸多态性和肺癌易感性的研究结果尚存在争议,本文对近年来BER通路基因hOGG1Ser326Cys,XRCC1 Arg194Trp,XRCC1 Arg280His,XRCC1 Arg399Gln,XRCC1-77T>C和MUTYHHis324Gln多态性与肺癌易感性的关系的研究进行汇总,并探讨了多项研究对BER基因多态性与不同肺癌亚型的关系以及与吸烟之间关系.基因多态性与肺癌易感性关系受多因素影响,其相关性尚待进一步探索.     

Links between DNA-repair gene polymorphisms and chromosomal aberrations in lung-cancer patients

This work presents the results of research into the links between DNA-repair gene polymorphisms and chromosomal aberrations in lung-cancer patients. Significant positive links have been found between lung cancer and the hOGG1 G/G and XPD G/G genotypes. A significant negative association has been found between the hOGG1 C/C genotypes and lung cancer. It has been shown that metaphases with chromosomal aberrations were significantly more frequent in lung-cancer patients than in the control group. Levels of chromosomal aberrations in the carriers of all APE1 and XPD genotypes, XRCC1 G/G genotype, ADPRT T/T genotype, and hOGG1 C/C genotypes reliably differed between the study and control groups. Statistically significant differences have been found between the lung-cancer patients carrying the XPD T/T genotype and those with G/G genotype of this gene.     

Differential susceptibility to monomeric HIV gp120-mediated apoptosis in antigen-activated CD4+ T cell populations

To support the hypothesis that indirect mechanisms mediated by viral products like the HIV envelope glycoprotein gp120 could be responsible for T lymphocyte depletion in HIV infection, we developed a system in which the impairment of T cell functions could be investigated in vitro. In particular, we characterized the conditions that allow T lymphocytes repeatedly stimulated with an antigen to be sensitive or resistant to gp120-mediated apoptotic signals. To achieve this goal, a panel of antigen-specific CD4⁺ T cell clones and primary CD4⁺ T lymphocytes were treated for 2 and 18 h with saturating amounts of monomeric gp120 (without cross-linking with specific antibodies) and antigen-driven T cell proliferation and apoptosis were analyzed. We show that monomeric gp120 induces apoptosis only in T lymphocytes repeatedly stimulated with the antigen, that primary T lymphocytes are resistant to programmed cell death mediated by monomeric gp120, but are sensitive to anti-CD4 antibodies, and that gp120-mediated apoptosis is dependent on the period of time between the binding of gp120 to CD4 and the encounter with antigen. To investigate the different susceptibility to gp120 induced apoptosis of primary CD4⁺ and T cell clones further, the number of membrane CD4 molecules and their affinity for gp120, together with Bcl-2 and Fas expression, were studied. Our data suggest that a down-modulation of membrane CD4 together with high expression of the Bcl-2 gene and protein characterizes the susceptibility to apoptosis of gp120-treated cells. In conclusion, our results define the phenotypic features of T cells susceptible to HIV gp120-induced apoptosis and demonstrate that the same clonotype, depending on the activation state, may present a differential sensitivity to apoptosis induction.     

Tissue‐resident memory T cells are epigenetically cytotoxic with signs of exhaustion in human urinary bladder cancer

Tissue‐resident memory T (T_RM) cells are CD8⁺ T lymphocytes that reside in the tissues, including tumours. This T cell subset possesses a magnitude of cytotoxicity, but its epigenetic regulation has not been studied. Here, we investigate the impact of perforin DNA methylation in T_RM cells and correlate it with their functional potential. Fifty‐three urothelial urinary bladder cancer (UBC) patients were recruited prospectively. The DNA methylation status of the perforin gene (PRF1) locus in T_RM cells was investigated by pyrosequencing. Flow cytometry with ViSNE analysis and in‐vitro stimulation were used to evaluate T_RM cell phenotypes. We discovered that tumour T_RM cells have low DNA methylation in the PRF1 locus (32·9% methylation), which corresponds to increased numbers of perforin‐expressing T_RM cells. Surprisingly, programmed cell death 1 (PD‐1) expression is high in tumour T_RM cells, suggesting exhaustion. Following interleukin‐15 and T cell receptor stimulation, perforin and T‐bet expressions are enhanced, indicating that T_RM cells from tumours are not terminally exhausted. Moreover, a high number of T_RM cells infiltrating the tumours corresponds to lower tumour stage in patients. In conclusion, T_RM cells from UBC tumours are epigenetically cytotoxic with signs of exhaustion. This finding identifies T_RM cells as potential new targets for cancer immunotherapy.     

The influence of genetic polymorphisms in XRCC3 and ADH5 genes on the frequency of genotoxicity biomarkers in workers exposed to formaldehyde

The International Agency for Research on Cancer classified formaldehyde as carcinogenic to humans because there is “sufficient epidemiological evidence that it causes nasopharyngeal cancer in humans”. Genes involved in DNA repair and maintenance of genome integrity are critically involved in protecting against mutations that lead to cancer and/or inherited genetic disease. Association studies have recently provided evidence for a link between DNA repair polymorphisms and micronucleus (MN) induction. We used the cytokinesis‐block micronucleus (CBMN assay) in peripheral lymphocytes and MN test in buccal cells to investigate the effects of XRCC3 Thr241Met, ADH5 Val309Ile, and Asp353Glu polymorphisms on the frequency of genotoxicity biomarkers in individuals occupationally exposed to formaldehyde (n = 54) and unexposed workers (n = 82). XRCC3 participates in DNA double‐strand break/recombination repair, while ADH5 is an important component of cellular metabolism for the elimination of formaldehyde. Exposed workers had significantly higher frequencies (P < 0.01) than controls for all genotoxicity biomarkers evaluated in this study. Moreover, there were significant associations between XRCC3 genotypes and nuclear buds, namely XRCC3 Met/Met (OR = 3.975, CI 1.053–14.998, P = 0.042) and XRCC3 Thr/Met (OR = 5.632, CI 1.673–18.961, P = 0.005) in comparison with XRCC3 Thr/Thr. ADH5 polymorphisms did not show significant effects. This study highlights the importance of integrating genotoxicity biomarkers and genetic polymorphisms in human biomonitoring studies. Environ. Mol. Mutagen. 54:213–221, 2013. © 2013 Wiley Periodicals, Inc.     

Transforming growth factor-β2 induces apoptosis of murine T cell clones without down-regulating bcl-2 mRNA expression

Transforming growth factor-β (TGFβ) is a potent immunosuppressive cytokine which inhibits the antigen (Ag)-dependent expansion of T cells both in vitro and in vivo by mechanisms not well defined yet. Here we report that exposure of interleukin (IL)-2-dependent T cell lines to TGFβ₂ results in apoptosis defined by morphology, nucleosomal size DNA fragmentation and in situ DNA end labeling. TGFβ₂-induced T cell apoptosis showed the following characteristics: (1) in contrast to the rapid evolution of apoptosis following IL-2 deprivation, apoptosis of T cells triggered by TGFβ₂ was delayed; (2) cycloheximide prevented TGFβ₂-induced apoptosis of CTLL-2 but not of OVA-7 T helper cells; (3) in contrast to apoptosis following IL-2 deprivation, TGFβ₂-mediated T cell apoptosis was not associated with decreased expression of the proto-oncogenes, bcl-2 or c-myc; (4) TGFβ₂-induced apoptosis was not restricted to IL-2-dependent T cell lines since the IL-4-dependent T cell line, CT.4S, as well as EL4 lymphoma cells, which grow independently of exogenous IL-2, were also susceptible to TGFβ₂-mediated apoptosis. Taken together, these data may present a novel mechanism of TGFβ₂-mediated suppression of T cell expansion in response to Ag and IL-2, the activation of the endogenous death program of apoptosis, which appears to operate independently of direct interactions of TGFβ₂ with the IL-2/IL-2 receptor system.     

Phosphodiesterase inhibitor pentoxifylline,a selective suppressor of T helper type 1- but not type 2-associated lymphokine production,prevents induction of experimental autoimmune encephalomyelitis in Lewis rats

The phosphodiesterase inhibitor pentoxifylline (POX), which is known to have pharmacological effects in animal models of multiorgan failure and endotoxin-mediated shock, was tested for its immunosuppressive potential on T lymphocyte activation in vitro and in vivo. POX was found to have a profound inhibitory effect on both mitogen- and antigen-induced proliferation of CD4⁺ T cells in vitro. This inhibitory activity of the drug could be reproduced by treating T lymphocytes with cAMP analogues during stimulation. Responses of repeatedly in vitro stimulated cells were much more strongly inhibited by the drug and by cAMP analogues than responses of fresh resting lymphocytes. Furthermore, POX could drastically down-regulate tumor necrosis factor regulate production and to a lesser extent interleukin (IL)-2 secretion in activated T cells, but an excess of exogenous IL-2 did not override the antiproliferative effect of the drug. In contrast, the same doses of POX had no inhibitory effect on spontaneous or induced IL-4 and IL-6 production by short-term cultured T lymphocytes, indicating a selective sparing of T helper type 2 (T_h2)-associated lymphokine functions by the drug. To test a potential use of POX as an antiinflammatory agent in T cell-mediated autoimmune disease, the influence of POX on myelin basic protein (MBP)-induced experimental autoimmune encephalomyelitis (EAE) was assessed. The onset of EAE in Lewis rats could almost completely be abrogated by oral administration of POX during the induction phase of disease. Lack of clinical symptoms in POX-treated animals coincided with a marked suppression of MBP-specific T cell reactivity in vitro, without any evidence for a generalized impairment of T cell activity. Collectively, our data suggest the potential use of xanthine derivatives of the POX type as a supporting antiinflammatory therapeutic agent in T_h1 CD4⁺ T cell-mediated autoimmune diseases in animal models and possibly in man.     

Prednisone increases apoptosis in in vitro activated human peripheral blood T lymphocytes

Glucocorticoid hormones (GCH) regulate, through the apoptotic process, the negative selection of immature T cells in the thymus. Because apoptosis seems to occur also in the maintenance of peripheral tolerance, we have investigated whether GCH may induce apoptosis in human mature lymphocytes. Peripheral blood lymphocytes (PBL) or peripheral CD4⁺ and CD8⁺ T cell subsets were cultured in the presence of phytohaemaglutinin (PHA) or PHA and prednisone (PDN) at 10⁻³-10⁻¹²M concentrations for 72, 96 and 120h. Cell cycle and membrane antigen expression were evaluated by flow cytometry and DNA degradation was detected by agarose gel electrophoresis. PDN blocks PBL growth in the G₁ phase of cell cycle and inhibits both IL-2 receptor (IL-2R) expression and IL-2 secretion. Apoptosis is clearly increased by PDN in PHA-activated human PBL, and the apoptotic effect of PDN is stronger on CD8⁺ than on CD4⁺ T lymphocytes. All these effects are dose- and time-dependent. The addition of exogenous IL-2 did not rescue lymphocytes from PDN-increased apoptosis. These results show that PDN increases apoptosis in mature activated human peripheral blood lymphocytes, suggesting a possible role of GCH in the maintenance of immune tolerance at post-thymic level.