Oxidative DNA damage and 8-hydroxy-2-deoxyguanosine DNA glycosylase/apurinic lyase in human breast cancer

To test the hypothesis that oxidative stress is involved in breast cancer, we compared the levels of 8-hydroxy-2-deoxyguanosine (8-oxo-dG), an oxidized DNA base common in cells undergoing oxidative stress, in normal breast tissues from women with or without breast cancer. We found that breast cancer patients (N = 76) had a significantly higher level of 8-oxo-dG than control subjects (N = 49). The mean ( +/- SD) values of 8-oxo-dG/10(5) dG, as measured by high-performance liquid chromatography electrochemical detection, were 10.7 +/- 15.5 and 6.3 +/- 6.8 for cases and controls, respectively (P = 0.035). This difference also was found by immunohistochemistry with double-fluorescence labeling and laser-scanning cytometry. The average ratios (x10(6)) of the signal intensity of antibody staining to that of DNA content were 3.9 +/- 7.2 and 1.1 +/- 1.4 for cases (N = 57) and controls (N = 34), respectively (P = 0.008). There was no correlation between the ages of the study subjects and the levels of 8-oxo-dG. Cases also had a significantly higher level of 8-hydroxy-2-deoxyguanosine DNA glycosylase/apurinic lyase (hOGG1) protein expression in normal breast tissues than controls (P = 0.008). There was no significant correlation between hOGG1 expression and 8-oxo-dG. Polymorphism of the hOGG1 gene was very rare in this study population. The previously reported exon 1 polymorphism and two novel mutations of the hOGG1 gene were found in three of 168 cases and two of 55 controls. In conclusion, normal breast tissues from cancer patients had a significantly higher level of oxidative DNA damage. The elevated level of 8-oxo-dG in cancer patients was not related to age or to deficiency of the hOGG1 repair gene.     

Accumulation of 8-oxo-2'-deoxyguanosine and increased expression of hMTH1 protein in brain tumors

Oxidative DNA damage generated by an attack of reactive oxygen species causes mutation or cell death that may lead to various diseases and may be related to initiation or progression of carcinogenesis. 8-Oxo-2'-deoxyguanosine (8-oxo-dG) is a major oxidative DNA damage product that can result in mutation, and hMTH1, human MutT homolog protein 1, has been identified as an enzyme that hydrolyzes 8-oxo-dGTP to the monophosphate, thus preventing accumulation of 8-oxo-dG in DNA. With immunohistochemical approaches, we investigated accumulation of 8-oxo-dG and expression of hMTH1 in brain tumor tissues obtained from surgical and autopsy cases, including 42 neuroepithelial tumors, 5 meningiomas, 2 metastatic brain tumors, and 1 schwannoma. 8-Oxo-dG accumulation and hMTH1 expression were increased in various brain tumors. Nuclei of brain tumor cells were immunoreactive for 8-oxo-dG in all cases. In most cases, both nuclei and cytoplasm of the tumor cells were immunoreactive for hMTH1. Both 8-oxo-dG accumulation and hMTH1 expression were most evident in high-grade gliomas, indicating that oxidative stress was high in these gliomas. Thus, the defense mechanism against such oxidative stress may be enhanced as well. These results suggest that oxidative stress may play a role in tumor progression.     

Impairment of antioxidant enzymes, lipid peroxidation and 8-oxo-2'-deoxyguanosine in advanced epithelial ovarian carcinoma of a Spanish community

In the present study, we describe the changes of antioxidant enzyme activities and other oxidative stress-related parameters in a mediterranean cohort of women affected with epithelial ovarian carcinoma (EOC). For that purpose, the most representative enzymatic activities, such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) and the oxidized/reduced glutathione (GSSG/GSH) ratio have been analyzed in tumor tissue biopsies and compared with the normal tissue of the same patient. As oxidation products, the levels of malondialdehyde (MDA) as an indication of lipid peroxidation, and the DNA damaged base 8-oxo-2'-deoxyguanosine (8-oxo-dG) have been also measured. Advanced EOC show reduced levels of SOD and CAT, while that of GPx is increased when compared with non-neoplastic tissue. The levels of GSH are increased giving as a result a reduction of the oxidative stress marker GSSG/GSH ratio comparing normal ovarian tissue with tumor tissue. In addition, the oxidation products MDA and 8-oxo-dG are significantly increased in tumor tissue, suggesting a shift of oxidative metabolisms towards a pro-oxidation state and potential gene instability in malignant ovary cells. The possible implication of the redox changes and DNA damage in tumor development is discussed.     

8-oxo-dG elevated in children during leukemia treatment

Changes in oxidative stress in children undergoing chemotherapy for acute lymphoblastic leukemia (ALL) have not been well documented. To determine whether the measurement of the DNA oxidized base 8-oxodeoxyguanosine (8-oxo-dG) may be a useful biomarker in this population, the authors conducted an observational study on 103 children with ALL. Blood samples were collected at diagnosis, during interim maintenance (IM), and during delayed intensification (DI). Blood mononuclear cell 8-oxo-dG, measured with an immunohistochemical method, decreased from diagnosis to IM (P = .01) and increased between IM and DI (P < .01). In a pilot study, bone marrow was also collected from 16 patients at diagnosis and after 28 days of treatment, but 8-oxo-dG remained the same. The relationship between plasma and dietary intake of antioxidants and the level of 8-oxo-dG was also explored. There was a direct relationship between the intake of vitamin E at diagnosis and bone marrow 8-oxo-dG (P = .03) and an inverse relationship between beta-carotene intake and blood 8-oxo-dG at IM (P = .03) and vitamin A in-take and blood 8-oxo-dG at DI (P = .003). Plasma vitamin C (P = .02) and total carotenoids (P = .01) were inversely related to blood 8-oxo-dG at IM. In contrast, higher plasma E/total lipid levels were associated with higher 8-oxo-dG at IM and DI (P < .01). At IM, patients with higher 8-oxo-dG had an increased risk of chemotherapy dose reduction (P = .04). In conclusion, the level of 8-oxo-dG in blood mononuclear cells decreases after the start of chemotherapy and increases during aggressive chemotherapy in children with ALL.     

A multi-biomarker approach to study the effects of smoking on oxidative DNA damage and repair and antioxidative defense mechanisms

We investigated the effects of smoking-induced oxidative stress in healthy volunteers (21 smokers versus 24 non-smokers) by quantifying various markers of oxidative DNA damage and repair, and antioxidative defense mechanisms. Lymphocytic 7-hydroxy-8-oxo-2'-deoxyguanosine (8-oxo-dG) levels measured by high performance liquid chromatography with electrochemical detection, were significantly lower in smokers as compared with non-smokers (38.6 +/- 5.2 versus 50.9 +/- 4.6/10(6) dG, P = 0.05). The levels of oxidized pyrimidine bases in lymphocytes of smokers quantified by the endonuclease III-modified comet assay were non-significantly lower than those of non-smokers (% DNA in tail: 13 +/- 3 versus 14 +/- 2; tail length: 69 +/- 13 versus 96 +/- 10; tail moment: 6416 +/- 1220 versus 7545 +/- 1234). Urinary excretion levels of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) assessed by enzyme-linked immunosorbent assay did not differ significantly between smokers and non-smokers (197 +/- 31 versus 240 +/- 33 ng/body mass index, P = 0.3). Overall DNA repair activity expressed as unscheduled DNA synthesis in blood leukocytes, was not significantly different between smokers and non-smokers (2.9 +/- 0.3 versus 3.3 +/- 0.3, P = 0.4). Plasma antioxidative capacity measured by the Trolox equivalent antioxidant capacity assay was slightly higher in smokers as compared with non-smokers (440 +/- 16 versus 400 +/- 15 microM Trolox equivalent, P = 0.09), and it was significantly related to lymphocytic 8-oxo-dG levels (r = 0.4, P = 0.001). Genotyping of human 8-OH-dG glycosylase/apurinic lyase and glutathione S-transferase M1 showed that a polymorphism in either or both of the two genes does not affect any of the quantified biomarkers. We conclude that oxidative stress imposed by cigarette smoking has a low impact upon certain pathways involved in DNA damage and the antioxidative defense system.     

Analysis of aromatic DNA adducts and 7,8-dihydro-8-oxo- 2'-deoxyguanosine in lymphocyte DNA from a case-control study of lung cancer involving minority populations

The purpose of this study was to examine the level of smoking-related aromatic DNA adducts and oxidative DNA damage in current smokers from a lung cancer case-control study in African Americans and Mexican Americans. In addition, mutagen sensitivity (bleomycin-induced chromatid breaks), a marker of genetic susceptibility, was assessed in these patients and correlated with the level of DNA damage. Lymphocyte DNA from cases and age-, sex-, and ethnicity-matched controls was analyzed for aromatic DNA adducts (43 cases and 47 controls) and the level of 7, 8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG) was determined in 46 cases and 48 controls using (32)P-postlabeling. Overall, lung cancer cases had significantly (P < 0.05) higher levels of aromatic DNA adducts and 8-oxo-dG (mean+/-SEM; 6.03+/-1.16/10(8) nucleotides and 5.82+/-0.77/10(5) nucleotides, respectively) compared to the controls (2.80+/-0.36/10(8) nucleotides and 3.65+/-0.56/10(5) nucleotides, respectively). The case-control differences for these two biomarkers were especially evident in current smokers. Both male and female lung cancer cases had higher levels of aromatic DNA adducts compared to the corresponding controls but only in men was the difference statistically significant (P=0.002). Cases who started smoking at earliest age had highest levels of aromatic DNA adducts and 8-oxo-dG. The level of aromatic DNA adducts in lung cancer cases, but not controls, was positively correlated with bleomycin-induced chromatid breaks (P=0.011). In contrast, the level of 8-oxo-dG was not correlated with mutagen sensitivity in either cases or controls or with the level of aromatic DNA adducts. The data suggest that levels of both aromatic DNA adducts and 8-oxo-dG may be useful in predicting risk of lung cancer in these minority populations. The correlation between aromatic DNA adducts and mutagen sensitivity in lung cancer cases and the trend for higher levels of DNA damage in cancer cases who started smoking earliest are particularly interesting and merit further study.     

Transition metals in personal samples of PM2.5 and oxidative stress in human volunteers.

Ambient particulate matter (PM) has been associated with increased risk of lung cancer. One proposed mechanism is that PM induces oxidative stress mediated by transition metals contained within this mixture. We examined the relationship between the personal exposure to water-soluble transition metals in PM(2.5) and oxidative DNA damage. In 49 students from central Copenhagen, we determined PM(2.5) exposure by personal sampling twice in 1 year, and measured in these PM(2.5) samples the concentration of the soluble transition metals vanadium, chromium, iron, nickel, copper, and platinum. Collected lymphocytes and 24-hour urine samples were analyzed for DNA damage in terms of 7-hydro-8-oxo-2'-deoxyguanosine (8-oxodG). We found that the 8-oxodG concentration in lymphocytes was significantly associated with the vanadium and chromium concentrations with a 1.9% increase in 8-oxodG per 1 microg/L increase in the vanadium concentration and a 2.2% increase in 8-oxodG per 1 microg/L increase in the chromium concentration. We have previously reported that in this study population the personal exposure to PM(2.5) was associated with an increase in 8-oxodG in lymphocytes. However, vanadium and chromium were associated with the 8-oxodG concentration in lymphocytes independently of the PM(2.5) mass concentration. The four other transition metals were not associated with 8-oxodG in lymphocytes and none of the transition metals was significantly associated with 8-oxodG in urine. Our results could indicate that vanadium and chromium present in PM(2.5) have an effect on oxidative DNA damage that is independent of particle mass and/or other possible toxic compounds contained within this particulate mixture.     

Androgen manipulation alters oxidative DNA adduct levels in androgen-sensitive prostate cancer cells grown in vitro and in vivo

Intracellular reactive oxygen species (ROS) may cause oxidative DNA damage, resulting in the formation of adducts such as 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) and the cyclic pyrimidopurinone N-1, N(2) malondialdehyde-2'-deoxyguanosine (M(1)dG). These adducts have been associated with carcinogenesis, genomic instability and clonal evolution. We tested two hypotheses in human prostate cancer cells grown in vitro and in a xenograft model: (1) treatment of androgen-sensitive cells with DHT increases levels of oxidative DNA adduct levels; (2) flutamide, a competitive androgen receptor antagonist, prevents DHT-induced changes. Levels of M(1)dG and 8-oxo-dG adducts were determined by immunoslot blot and liquid chromatography-tandem mass spectrometry. M(1)dG and 8-oxo-dG levels were significantly higher than control levels in LNCaP cells exposed to supra-physiological concentrations (25-100 nM) of DHT (both P<0.05 by ANOVA). Flutamide pre-treatment completely prevented this increase. In the xenograft model, tumour levels of M(1)dG were decreased by 46% (P=0.001 by Mann-Whitney Test) in flutamide-treated animals compared to controls. The changes demonstrated suggest that oxidative DNA adducts may serve as biomarkers of the efficacy of androgen manipulation in chemoprevention trials.     

PARP1 expression,activity and ex vivo sensitivity to the PARP inhibitor,talazoparib (BMN 673), in chronic lymphocytic leukaemia

In chronic lymphocytic leukemia (CLL), mutation and loss of p53 and ATM abrogate DNA damage signalling and predict poorer response and shorter survival. We hypothesised that poly (ADP-ribose) polymerase (PARP) activity, which is crucial for repair of DNA breaks induced by oxidative stress or chemotherapy, may be an additional predictive biomarker and a target for therapy with PARP inhibitors.We measured PARP activity in 109 patient-derived CLL samples, which varied widely (192 – 190052 pmol PAR/10⁶ cells) compared to that seen in healthy volunteer lymphocytes (2451 – 7519 pmol PAR/10⁶ cells). PARP activity was associated with PARP1 protein expression and endogenous PAR levels. PARP activity was not associated with p53 or ATM loss, Binet stage, IGHV mutational status or survival, but correlated with Bcl-2 and Rel A (an NF-kB subunit). Levels of 8-hydroxy-2′-deoxyguanosine in DNA (a marker of oxidative damage) were not associated with PAR levels or PARP activity. The potent PARP inhibitor, talazoparib (BMN 673), inhibited CD40L-stimulated proliferation of CLL cells at nM concentrations, independently of Binet stage or p53/ATM function.PARP activity is highly variable in CLL and correlates with stress-induced proteins. Proliferating CLL cells (including those with p53 or ATM loss) are highly sensitive to the PARP inhibitor talazoparib.     

Multidimensional scaling analysis identifies pathological and prognostically relevant profiles of circulating T‐cells in chronic lymphocytic leukemia

Antitumor immunity in chronic lymphocytic leukemia (CLL) is hampered by highly dysfunctional T‐cells. Although certain T‐cell subsets have been reported to be of prognostic significance in this disease, their interplay is complex and it remains incompletely understood which of these subsets significantly drive CLL progression. Here, we determined immunological profiles of 24 circulating T‐cell subsets from 79 untreated individuals by multiparametric flow cytometry. This screening cohort included healthy donors, patients with monoclonal B‐cell lymphocytosis (MBL), Rai 0 CLL and advanced CLL. We applied multidimensional scaling analysis as rigorous and unbiased statistical tool to globally assess the composition of the circulating T‐cell environment and to generate T‐cell scores reflecting its integrity. These scores allowed clear distinction between advanced CLL and healthy controls, whereas both MBL and Rai 0 CLL showed intermediate scores mirroring the biological continuum of CLL and its precursor stages. T‐cell stimulation and suppression assays as well as longitudinal T‐cell profiling showed an increasingly suppressive regulatory function initiating at the MBL stage. Effector function was impaired only after transition to CLL and partially recovered after chemoimmunotherapy. In an independent validation cohort of 52 untreated CLL cases, aberrant T‐cell profiles were significantly associated with shorter time to treatment independently of other prognostic parameters. Random forest modeling predicted regulatory T‐cell, gamma/delta and NKT‐cells, as well as exhaustion of the CD8+ subset as potential drivers of progression. Our data illustrate a pathological T‐cell environment in MBL that evolves toward a more and more suppressive and prognostically relevant profile across the disease stages.     

Interplay between Helicobacter pylori and host gene polymorphisms in inducing oxidative DNA damage in the gastric mucosa

Infection by Helicobacter pylori is the most important risk factor for gastric cancer. However, only a small fraction of colonized individuals, representing at least half of the world's population, develop this malignancy. In order to shed light on host-microbial interactions, gastric mucosa biopsies were collected from 119 patients suffering from dyspeptic symptoms. 8-Hydroxy-2'-deoxyguanosine (8-oxo-dG) levels in the gastric mucosa were increased in carriers of H.pylori, detected either by cultural method or by polymerase chain reaction, and were further increased in subjects infected with strains positive for the cagA gene, encoding the cytotoxin-associated protein, cagA. Oxidative DNA damage was more pronounced in males, in older subjects, and in H.pylori-positive subjects suffering from gastric dysplasia. Moreover, 8-oxo-dG levels were significantly higher in a small subset of subjects having a homozygous variant allele of the 8-oxoguanosine-glycosylase 1 (OGG1) gene, encoding the enzyme removing 8-oxo-dG from DNA. Conversely, they were not significantly elevated in glutathione S-transferase M1 (GSTM1)-null subjects. Thus, both bacterial and host gene polymorphisms affect oxidative stress and DNA damage, which is believed to represent a key mechanism in the pathogenesis of gastric cancer. The interplay between bacterial and host gene polymorphisms may explain why gastric cancer only occurs in a small fraction of H.pylori-infected individuals.     

吸烟引起人体氧化损伤的可能机制

背景与目的： 通过多项生物标志物研究吸烟对DNA氧化损伤、脂质过氧化和氧化防御机制的影响。 材料与方法： 选取年龄在18～25岁的男性吸烟志愿者60名和非吸烟者30名,分为吸烟组和非吸烟组。分别通过高效液相色谱-电化学检测法(HPLC-ECD)测定24 h尿样中8-羟基脱氧鸟苷(8-OHdG)水平;应用彗星实验测定外周血淋巴细胞DNA链的断裂情况;采用高效液相色谱-紫外线检测法(HPLC-UV)测定24 h尿样中丙二醛(MDA)、丙酮(ACON)和戊醛(PP)水平;采静脉血测定血浆超氧化物歧化酶(SOD)、谷胱苷肽过氧化氢酶(GPX)和过氧化氢酶含量。 结果： 吸烟组尿8-OHdG和外周血淋巴细胞DNA的断裂分别比非吸烟组高185％和97 ％(P均<0.01),尿MDA、ACON和PP较非吸烟组显著增高(P均<0.01),SOD、GPX和过氧化氢酶分别较非吸烟组低15％,10％和9％(P≤0.01)。 结论： 吸烟可以引起机体的氧负荷,造成DNA氧化损伤、脂质过氧化和抗氧化酶的改变。     

Can 8-oxo-dG be used as a predictor for individual radiosensitivity?

PURPOSE: To develop predictive tests for individual radiosensitivity of tumor patients. METHODS AND MATERIALS: Acute skin reactions were clinically scored among 40 women after 46 Gy, given with 2 Gy fractions to breast and regional lymph nodes, adjuvant after surgery. The acute skin reactions were compared to the excretion of 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG) in urine, determined by high-performance liquid chromatography (HPLC) with electrochemical detector. Specimens of urine were collected before and during postoperative radiation treatment at given intervals. We compared a group of 9 patients with the most pronounced skin reactions with another group of 8 patients with almost no skin reactions at 46 Gy. RESULTS: The level of 8-oxo-dG excreted in urine during 8 h was measured. After normalizing the excretion to irradiated volumes, dose per volume and excretion before irradiation, the 8-oxo-dG level in urine was significantly (p < 0.001) lower for the patients with pronounced skin reactions as compared to patients with minor skin reactions, at an accumulated dose of 12 Gy. In addition, the background level of 8-oxo-dG excreted before treatment started, was significantly (p = 0.043) lower for patients with minor skin reactions as compared to patients with pronounced skin reactions. The background level of 8-oxo-dG was corrected for body weight and normalized to BMI. CONCLUSION: We suggest that the excretion of 8-oxo-dG into urine of breast cancer patients is a possible marker for acute radiosensitivity.     

The effects of catechol-O-methyltransferase inhibition on estrogen metabolite and oxidative DNA damage levels in estradiol-treated MCF-7 cells

Many of the major identified risk factors for breast cancer are associated with exposure to endogenous estrogen. In addition to the effects of estrogen as a growth factor, experimental and epidemiological evidence suggest that catechol metabolites of estrogen also contribute to estrogen carcinogenesis by both direct and indirect genotoxic mechanisms. O-Methylation catalyzed by catechol-O-methyltransferase (COMT) is a Phase II metabolic inactivation pathway for catechol estrogens. We and others have found that a polymorphism in the COMT gene, which codes for a low activity variant of the COMT enzyme, is associated with an increased risk of developing breast cancer; therefore, the goal of the current study was to investigate the role of decreased COMT activity on estrogen catechol levels and on oxidative DNA damage, as measured by 8-hydroxy-2'-deoxyguanosine (8-oxo-dG) levels. MCF-7 cells were pretreated with dioxin as a means to increase estrogen metabolism to catechol estrogens, then treated with estradiol (E2) +/- Ro 41-0960, a COMT-specific inhibitor. After extraction from culture medium, estrogen metabolites were separated using an high-performance liquid chromatography-electrochemical detection method. As expected, dioxin dramatically increased E2 oxidative metabolism, primarily to its 2-OH and 2-methoxy metabolites. The COMT inhibitor blocked 2-methoxy E2 formation. This was associated with increased 2-hydroxy E2 (2-OH E2) and 8-oxo-dG levels. In the presence of COMT inhibition, increased oxidative DNA damage was detected in MCF-7 cells exposed to as low as 0.1 microM E2, whereas in the absence of COMT inhibition, no increase in 8-oxo-dG was detected at E2 concentrations < or =10 microM. This study is the first to show that O-methylation of 2-OH E2 by COMT is protective against oxidative DNA damage caused by 2-OH E2, a major oxidative metabolite of E2.     

Nuclear oxidative damage correlates with poor survival in colorectal cancer

Oxidative DNA damage results from DNA adducts such as 8-oxo-7, 8 dihydro-2′-deoxyguanosine (8-oxo-dG), which is a pro-mutagenic lesion. No known association between 8-oxo-dG, disease progression and survival exists in colorectal cancer (CRC). We examined levels of 8-oxo-dG in sporadic CRC to determine its relationship with pathological stage and outcome. A total of 143 CRC patients and 105 non-cancer patients were studied. Nuclear and cytoplasmic 8-oxo-dG was assessed using immunohistochemistry. Double immunofluorescence using 8-oxo-dG and manganese superoxide dismutase (MnSOD) antibodies localised cytoplasmic 8-oxo-dG. Apoptosis was detected using TUNEL. Nuclear staining levels were similar in tumour tissue and matched normal mucosa in both epithelial (P=0.22) and stromal (P=0.85) cells. Epithelial cytoplasmic staining was greater in tumour tissue (P<0.001). Double immunofluorescence localised cytoplasmic 8-oxo-dG to mitochondria. Epithelial and stromal nuclear 8-oxo-dG decreased with local disease spread, but highest levels were found in distant disease (P<0.01). Survival was related to epithelial nuclear and stromal staining in normal mucosa (P<0.001) and tumour (P<0.01) but was unrelated to cytoplasmic staining. Normal control cells in tissue from cancer patients with high levels of 8-oxo-dG failed to undergo cell death. 8-oxo-dG may be an important biomarker of disease risk, progression and survival for CRC patients.     

Comparative analysis of 8-oxo-2' -deoxyguanosine in DNA by 32P- and 33P- postlabeling and electrochemical detection

8-Oxo-2'-deoxyguanosine (8-oxo-dG) is emerging as a useful marker for oxidative DNA damage. Reported basal levels determined by 32P- postlabeling (PPL) method were 10-fold or more higher than those obtained with HPLC/electrochemical detection (ECD). This discrepancy was investigated. In commercial calf thymus DNA, levels of 4 +/- 1 and 64 +/- 14 8-oxo-dG per 10(6) 2'-deoxynucleosides (dN) were measured by the standard HPLC/ECD and PPL methods, respectively. DNA digestion by micrococcal nuclease/spleen phosphodiesterase and nuclease P1 (as used in the standard PPL method), followed by ECD analysis resulted in a level of 8 +/- 3. In calf thymus DNA spiked with chemically synthesized 8-oxo-dGp to give an increment of 9 8-oxo-dG/10(6) dN, the added standard produced a significant increase with HPLC/ECD but not PPL. After spiking the DNA with 90 8-oxo-dG/10(6) dN, the added 8-oxo-dGp was detectable also with PPL, with a labeling efficiency of 65%. In order to investigate the role of ionizing radiation from 32P for the higher 8-oxo-dG levels in PPL, incubation times and amounts of radioactivity in the phosphorylation reaction with commercial dGp were increased, and external irradiation of commercial dG with 32P was investigated. All modifications resulted in higher values of 8-oxo-dG measured, but the effect was not large enough to fully explain the discrepancy between PPL and HPLC/ECD. Using [gamma-33P]ATP instead of [gamma-32P]ATP or adding [33P]phosphate to a 32P-PPL assay resulted in even higher levels of 8-oxo-dG measured. The increase in 8-oxo-dG levels during the PPL workup is attributed to the presence and oxidation of unmodified dGp in the reaction mixture. For a determination of true basal levels, the PPL method will have to be modified, including the removal of dGp prior to the phosphorylation reaction.      

Monoclonal B Cell Lymphocytosis—What Does It Really Mean?

Monoclonal B cell Lymphocytosis (MBL) or similar terms have been used for decades to describe the presence of light-chain restricted B lymphocytes with uncertain clinical significance, usually having a phenotype consistent with chronic lymphocytic leukemia (CLL). As diagnostic technology improved, ever smaller monoclonal B cell populations were identifiable in the population, and approximately half of people over 90 years old have a minimal (<1 cell/μL) circulating CLL-like B cell population. These minimal CLL-like B cell populations share some molecular characteristics with CLL, but have no clinical significance. In contrast, CLL-like MBL cases detected through hospital investigations are biologically indistinguishable from early stage CLL, but the neoplastic B cell levels are usually stable over time and the risk of progressive disease requiring treatment is much lower than for early stage CLL. However, there is usually partial or complete depletion of normal B cells, with an increased relative risk of severe infection, comparable to early stage CLL, which may impair overall survival.     

Clinical features and outcome of Chinese patients with monoclonal B-cell lymphocytosis

B-cell chronic lymphocytic leukemia (CLL) is the most common type of adult leukemias in the Western countries, however, infrequent in the Eastern. A diagnosis of CLL requires a count of B-lymphocytes ≥5.0 × 10⁹/L. Asymptomatic person with <5.0 × 10⁹/L B-lymphocytes is defined as monoclonal B-cell lymphocytosis (MBL). To compare the clinical characteristics, prognostic factors, and outcome of Chinese patients with MBL and CLL, we present a study from our single centre of 20 patients with MBL and 136 patients with CLL. The factors included: age at diagnosis, gender, direct antiglobulin test (DAT), immunoglobulin heavy chain variable gene (IgHV) mutational status, ZAP-70 protein, CD38 expression level, and molecular cytogenetic aberrations were analyzed in MBL and CLL subgroups. The Kaplan–Meier method was used to construct survival curves, and results were compared using the log-rank test. Patients in the MBL category were slightly older than in the CLL category. There was no significant difference of these clinical and biological characteristics between patients in MBL subgroup and early stage CLL (Binet A). The incidence of positive DAT was significantly increased in CLL patients at Binet B and C, compared with MBL (P = 0.036). IgHV gene mutation in MBL is skewed, with more than 92.3% of subjects harbored mutated IgVH genes (P = 0.025). The proportion of MBL patients with a 13q14 deletion or trisomy 12 was similar to that of CLL patients. Moreover, markers associated with poor prognosis (deletion of 11q22 or 17p13) in these MBL populations were less than those in Binet B and C CLL patients (P = 0.025). No statistically significant differences in ZAP-70 and CD38 status were observed between the MBL and CLL subgroups. During a median follow-up period of 45.5 months, MBL patients had a low probability of progression, with no patients transformed to aggressive non-Hodgkin's lymphoma or dying of CLL-related causes. The overall survival of MBL was very similar to Binet A CLL, but longer than that of CLL patients at advanced stages (Binet B and C) (P = 0.024). Our study demonstrated that a more indolent clinical course and superior clinical outcome for patients with MBL compared to CLL.     

Two pathways for chromium(VI)-induced DNA damage in 14 day chick embryos: Cr--DNA binding in liver and 8-0X0-2'-deoxyguanosine in red blood cells

Previous studies have shown that chromium(VI) induced cell-specifictypes of DNA damage, i.e. DNA cross-links in liver and DNA strandbreaks in red blood cells, in 14 day chick embryos. Direct andindirect pathways for chromium(VI)-mediated DNA damage, in theform of Cr-DNA binding and 8-oxo-2’-deoxyguanosine (8-oxo-dG)respectively, were examined in liver and red blood cells of14 day chick embryos. Levels of hepatic Cr-DNA binding increasedin a Cr(VI) dose-dependent manner. Cr-DNA binding in red bloodcells was 10-fold lower than in liver, although the Cr-uptakein red blood cells was only 2-fold lower than in liver. Thelevel of 8-oxo-dG formation in red blood cells increased atall Cr(VI) doses tested but peaked at 0.10 mmol Cr(VI)/kg, whereasno increase hi 8- oxo-dG levels over background levels was observedin liver of Cr(VI)-treated embryos. The possible role of glutathionehi modulating Cr(VI)-induced DNA damage was examined by usingL-buthionine-R,S-sulfoximine (BSO) to deplete glutathione. Nochanges in glutathione levels were observed in either liveror red blood cells of embryos treated with Cr(VI) in the presenceor absence of BSO pretreatment Ascorbate levels in liver andred blood cells were not affected by treatment of embryos withchromium(VI), BSO or Cr(VI) and BSO. Depletion of glutathioneby BSO resulted in a small increase of chromium uptake in liverof embryos treated with 0.050 and 0.10 mmol Cr(VI)/kg, but hadno effect on hepatic chromium uptake at 0.20 mmol Cr(VI)/kg.BSO had no effect on chromium uptake in red blood cells. Depletionof glutathione had no effect on hepatic or red blood cells Cr-DNAbinding in embryos treated with Cr(VT). However, depletion ofglutathione significantly decreased the 8-oxo-dG levels in redblood cells at all Cr(VI) doses tested. Levels of 8-oxo-dG inliver of Cr(VI)-treated embryos remained at background in thepresence or absence of BSO pretreatment These results indicatethat Cr(VI)-induced DNA damage in 14 day chick embryos is througha direct interaction of chromium with DNA in liver, but is throughan indirect oxidative pathway in red blood cells. It appearsthat glutathione plays an important role in chromium(VI)-inducedformation of 8-oxo-dG in red blood cells.     

Clinical progression and outcome of patients with monoclonal B-cell lymphocytosis

Monoclonal B-cell lymphocytosis (MBL) is a clonal lymphoproliferation with the immunophenotype of chronic lymphocytic leukemia (CLL) but a B-lymphocyte count of less than 5 x 10(9)/l and no lymphadenopathy, organomegaly, cytopenias or symptoms. We performed a retrospective analysis of patients with MBL (n = 46), Rai stage 0 CLL (n = 112) and Rai stage > or =1 CLL (n = 54). Median follow-up and range was 30 (0.1-120) months for MBL, 60 (0.1-309) months for stage 0 CLL and 54 (0.1-309) months for stage > or =1 CLL. None of the MBL patients required treatment compared with 24 of 112 (21%) stage 0 CLL and 28 of 54 (52%) stage > or =1 CLL patients (p < 0.0003). No MBL underwent aggressive transformation compared with 1 of 112 (0.8%) stage 0 CLL and 6 of 54 (11%) stage > or =1 CLL patients (p < 0.0003). Progression-free survival (PFS) appeared improved in MBL compared to stage 0 CLL, although this did not reach statistical significant (p = 0.07) due to the relatively short follow-up in the MBL group; two year PFS was 97.2% for MBL, 93.1% for stage 0 CLL, and 68% for stage > or =1 CLL patients (p < 0.0001 for stage > or =1 CLL compared with MBL and stage 0 CLL). This is the first study of outcome in MBL which demonstrates that patients have an improved disease course compared to stage 0 CLL patients. Over a median 2.5 years of follow-up, no MBL patients required treatment or died of CLL-related causes.