Human Blood Cell DNA Research Articles

Copper(II) generates ROS and RNS, impairs antioxidant system and damages membrane and DNA in human blood cells.

Copper (Cu) is widely used in various industries, and human exposure to this metal results in severe multi-organ toxicity, which is thought to be due to the generation of free radicals by Fenton-like reaction. The generation of reactive oxygen as well as nitrogen species and free radicals results in induction of oxidative stress in the cell. We have studied the effect of different concentrations of Cu(II) on human erythrocytes and lymphocytes. Incubation of erythrocytes with copper chloride, a Cu(II) compound, enhanced the production of reactive oxygen and nitrogen species, decreased glutathione and total sulphydryl content and increased protein oxidation and lipid peroxidation. All changes were in a Cu(II) concentration-dependent manner. This strongly suggests that Cu(II) causes oxidative damage in erythrocytes. The activities of major antioxidant enzymes were altered, and antioxidant power was lowered. Cu(II) treatment also resulted in membrane damage in erythrocytes as seen by electron microscopy and lowered activities of plasma membrane-bound enzymes. Incubation of human lymphocytes with Cu(II) resulted in DNA damage when studied by the sensitive comet assay. These results show that Cu(II) exerts cytotoxic and genotoxic effects on human blood cells probably by enhancing the generation of reactive oxygen and nitrogen species.

Effect of age and sex on the level of DNA strand breaks and oxidatively damaged DNA in human blood cells

DNA damage measured by the comet assay is a well-established biomarker in studies on environmental and occupational exposures, dietary factors and clinical outcomes. Age and sex are typically regarded as confounding factors that are controlled by either selective inclusion criteria of subjects or adjustment in the statistical analysis. This review assesses the influence of age and sex on levels of DNA damage in leukocytes in study populations of healthy subjects. Analysis of unadjusted results in the studies indicates that the level of DNA strand breaks increases by 1% per year (95% CI: 0.9%–1.1%, linear regression analysis weighted for the number of subjects in the individual studies). The studies also show a slightly higher level of DNA strand breaks in men than women (8%, 95% CI: 0.4%–17%) in unadjusted analyses, which is not robust in studies with adjusted analyses. The attenuation of effect in adjusted analyses of DNA strand breaks in leukocytes indicates that the effect of age and sex may be driven by differences in lifestyle factors or other exposures. There do not appear to be differences related to age and sex on basal levels of oxidatively damaged DNA in leukocytes. In summary, the results indicate influences of both age and sex on DNA damage in the comet assay, which may be mediated by lifestyle factors or external exposures rather than direct effects of age and sex.

Countering effects of a combination of podophyllotoxin, podophyllotoxin β-D-glucoside and rutin hydrate in minimizing radiation induced chromosomal damage, ROS and apoptosis in human blood lymphocytes

The present study was conceptualized with the aim of developing a safe radioprotector for human application against radiation induced toxicity. For this study, a formulation (G-002M) prepared by combining three active principles isolated from rhizomes of Podophyllum hexandrum, was evaluated for its potential to protect genomic DNA of human blood cells exposed to different doses of radiation (5,7&10Gy). Blood samples were pretreated (-1hr to exposure) with G-002M. Parameters of Premature Chromosome Condensation (PCC) assay like PCC-index, PCC-rings and PCC-fragments were used to estimate radiation induced chromosomal aberrations. Radiation (7Gy) induce ROS generation and its modulation by G-002M was determined by flow-cytometry and fluorescent microscopy while apoptosis (0,2,24&48 hr) was analyzed using TUNEL assay. Effect on spindle organization in G2/M arrested cells by all the three compounds individually was studied using immunofluorescence microscopy. Irradiation caused dose dependent linear increase in PCC-rings and fragments, while decline in PCC index. G-002M pretreatment significantly decreased these chromosomal aberrations at all the radiation doses and assisted cell survival as indicated by increased PCC index compared with radiation only group. Significant decrease in radiation induced intracellular ROS (45 ± 3%) and apoptosis (49.9%) was also exhibited by the formulation. On podophyllotoxin treatment, most of the cells have shown blocked spindles however, depicted normal arrangement. G-002M also demonstrated a highly significant Dose Modifying Factor or DMF (PCC-rings: 2.27 and PCC-fragments: 1.60). Present study based on many parameters along with DMF study, strongly suggests that G-002M is an effective formulation with a potential to minimize chromosomal damage even at very high radiation doses.

Ultrasensitive UPLC–MS/MS method for analysis of etheno-DNA adducts in human white blood cells

Etheno-DNA adducts are generated by interaction of cellular DNA with exogenous environmental carcinogens and end products of lipid peroxidation. It has been determined that 1,N6-etheno-2’-deoxyadenosine (εdA) and 3,N4-etheno-2’-deoxycytidine (εdC) adducts formed in human white blood cells can be used to serve as biomarkers of genetic damage mediated by oxidative stress. In this study, we developed an ultrasensitive ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method used to detect and quantify εdA and εdC adducts in human white blood cells. The percent recoveries of εdA and εdC adducts were found to be 88.9% ± 2.8 and 95.7% ± 3.7, respectively. The detection limits were ∼1.45 fmol for εdA and ∼1.27 fmol for εdC in 20 μg of human white blood cell DNA samples, both εdA and εdC adducts could be detected using only ∼ 5 μg of DNA per sample. For validation of the method, 34 human blood cell DNA samples were assayed and the results revealed a significant difference (P < 0.01) between levels (fmol/μg DNA) of 0.82 ± 0.83 (standard deviation [SD]) (range: 0.15–3.11) for εdA, 3.28 ± 3.15 (SD) (range: 0.05–9.6) for εdC in benzene-exposed workers; and 0.04 ± 0.08 (SD) (range: 0.0–0.27) for εdA and 0.77 ± 1.02 (SD) (range: 0.10–4.11) for εdC in non-benzene-exposed workers. Our method shows a high sensitivity and specificity when applied to small amounts of human white blood cell DNA samples; background levels of εdA and εdC could be reproducibly detected. The ultrasensitive and simple detection method is thus suitable for applications in human biomonitoring and molecular epidemiology studies.

Quality assurance of genotyping array for detection and typing of human papillomavirus

The aim of this study was to evaluate the sensitivity, specificity, reliability and reproducibility of the EasyChip ® HPV blot for human papillomavirus (HPV) genotyping. Type-specific sensitivity and specificity for 39 types of HPV (HPV 6, 11, 16, 18, 26, 31, 32, 33, 35, 37, 39, 42, 43, 44, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 62, 66, 67, 68, 69, 70, 72, 74, 82, CP8061, CP8304, L1AE5, MM4, MM7 and MM8) were examined. The operating environment, reliability, reproducibility and blot interpretation were assessed by a quality assurance system. Each batch experiment contained samples from 89 cervical specimens and 7 extrinsic controls. Caski, HeLa and Jurkat cells, male human blood cell DNA and sterile water were used to assess reliability. Furthermore, pairs of sibling controls were used to assess reproducibility. The overall sensitivity of HPV detection was 1–50 copies of HPV genome equivalent. There was no cross-reactivity with amplicons of other HPV genotypes. One hundred batch experiments demonstrated that the reliability was excellent. The intra-batch and inter-batch reproducibility was 98 and 97%, respectively. It was concluded that the EasyChip ® HPV blot is a highly sensitive, reliable and reproducible tool for detection and identification of HPV genotypes.

Assessment of reference values for DNA damage detected by the comet assay in human blood cell DNA

Genotoxicity measured by the comet assay is expressed by different researchers using parameters that are not easy to conceptualize, except for percent tail DNA (%T) or visual score (arbitrary units). A total of 125 publications have reported genotoxicity as DNA damage (representing strand breaks, alkaline labile sites, and transient repair sites), endonuclease III (ENDOIII), or formamidopyrimidine DNA glycosylase (FPG) sensitive sites. I have recalculated the visual score so that it is expressed in the range of 0-100, similar to that of %T. Similar values were obtained for DNA damage and ENDOIII sites, regardless of whether of the data were reported as %T or visual score. Thus, these endpoints can be used interchangeably, assuming that the visual score is expressed in the 0-100 range. Pooled analysis of %T and visual score data showed that the median (25-75%) values of DNA damage, ENDOIII, and FPG sites were 8.6 (4.4-14.5), 11.0 (4.2-19.5), 7.6 (3.2-14.2), respectively. The duration of alkaline treatment and electrophoresis had no significant effect on the level of DNA damage. There was a positive correlation between age and the level of DNA damage. A sub-analysis of DNA damage obtained from European countries showed a negative correlation with latitude. In conclusion, reference values for DNA lesions measured by the comet assay are around 7-11 %T or arbitrary units.

Quantification of mitochondrial DNA in human blood cells using an automated detection system

The 4977 bp deletion of mitochondrial DNA (mtDNA) accumulates in postmitotic tissues with advancing age. The purpose of our study was to detect and quantify these deletion even in blood cells with a high turnover activity. Whole venous blood, isolated human platelets and peripheral blood mononuclear cells (PBMCs) were collected from 10 unrelated donors aged 20–71 years and total DNA was extracted. PCR was performed for total and mutated mtDNA using two different primer pairs and two fluorogenic probes labeled with the fluorescent dyes FAM and VIC. Specific PCR products were generated, detected and quantified in a real-time PCR. The amplification products of total and deleted mtDNA could be detected in each sample and did not exhibit any differences in the amount of the deleted mtDNA in whole blood, human platelets or PBMCs. Our data did not show any accumulation of the 4977 bp deletion with increasing age as it was observed for several other tissues.

Incorporation of zidovudine into leukocyte DNA from HIV-1-positive adults and pregnant women, and cord blood from infants exposed in utero.

The nucleoside analog 3'-azido-3'-deoxythymidine (ZDV) has widespread clinical use but also is carcinogenic in newborn mice exposed to the drug in utero and becomes incorporated into newborn mouse DNA. This pilot study was designed to determine ZDV incorporation into human blood cell DNA from adults and newborn infants. In this prospective cohort study, peripheral blood mononuclear cells (PBMC) were obtained from 28 non-pregnant adults and 12 pregnant women given ZDV therapy, six non-pregnant adults with no exposure to ZDV, and six non-pregnant adults who last received ZDV > or = 6 months previously. In addition, cord blood leukocytes were obtained from 22 infants of HIV-1-positive, ZDV-exposed women and from 12 infants unexposed to ZDV. There were 11 mother-infant pairs involving HIV-1 -positive women. DNA was extracted from PBMC obtained from non-pregnant HIV-1-positive adults taking ZDV, pregnant HIV-1-positive women given ZDV during pregnancy, and from adults not taking ZDV. Cord blood leukocytes were examined from infants exposed to ZDV in utero and from unexposed controls. DNA samples were assayed for ZDV incorporation by anti-ZDV radioimmunoassay (RIA). The majority (76%) of samples from ZDV-exposed individuals, pregnant women (8 of 12), non-pregnant adults (24 of 28), or infants at delivery (15 of 22), had detectable ZDV-DNA levels. The range of positive values for ZDV-treated adults and infants was 25-544 and 22-452 molecules ZDV/10(6) nucleotides, respectively. Analysis of 11 mother-infant pairs showed variable ZDV-DNA incorporation in both, with no correlation by pair or by duration of drug treatment during pregnancy. Two of the 24 samples from individuals designated as controls were positive by anti-ZDV RIA. The 20-fold range for ZDV-DNA values in both adults and infants suggested large interindividual differences in ZDV phosphorylation. Incorporation of ZDV into DNA was detected in most of the samples from ZDV-exposed adults and infants. Therefore, the biologic significance of ZDV-DNA damage and potential subsequent events, such as mutagenicity, should be

DNA adducts as exposure biomarkers and indicators of cancer risk.

Quantitation of DNA adducts in human tissues has been achieved with highly sensitive techniques based on adduct radiolabeling, antisera specific for DNA adducts or modified DNA, and/or adduct structural characterization using chemical instrumentation. Combinations of these approaches now promise to elucidate specific adduct structures and provide detection limits in the range of 1 adduct/10(9) nucleotides. Documentation of human exposure and biologically effective dose (i.e., chemical bound to DNA) has been achieved for a wide variety of chemical carcinogens, including polycyclic aromatic hydrocarbons (PAHs), aromatic amines, heterocyclic amines, aflatoxins, nitrosamines, cancer chemotherapeutic agents, styrene, and malondialdehyde. Due to difficulties in exposure documentation, dosimetry has not been precise with most environmental and occupational exposures, even though increases in human blood cell DNA adduct levels may correlate approximately with dose. Perhaps more significant are observations that lowering exposure results in decreasing DNA adduct levels. DNA adduct dosimetry for environmental agents has been achieved with dietary contaminants. For example, blood cell polycyclic aromatic hydrocarbon-DNA adduct levels were shown to correlate with frequency of charbroiled meat consumption in California firefighters. In addition, in China urinary excretion of the aflatoxin B1-N7-guanine (AFB1-N7-G) adduct was shown to increase linearly with the aflatoxin content of ingested food. Assessment of DNA adduct formation as an indicator of human cancer risk requires a prospective nested case-control study design. This has been achieved in one investigation of hepatocellular carcinoma and urinary aflatoxin adducts using subjects followed by a Shanghai liver cancer registry. Individuals who excreted the AFB1-N7-G adduct had a 9.1-fold adjusted increased relative risk of hepatocellular carcinoma compared to individuals with no adducts. Future advances in this field will be dependent on chemical characterization of specific DNA adducts formed in human tissues, more-precise molecular dosimetry, efforts to correlate DNA adducts with cancer risk, and elucidation of opportunities to reduce human DNA adduct levels.

Human DNA adduct measurements: state of the art.

Human DNA adduct formation (covalent modification of DNA with chemical carcinogens) is a promising biomarker for elucidating the molecular epidemiology of cancer. Classes of compounds for which human DNA adducts have been observed include polycyclic aromatic hydrocarbons (PAHs), nitrosamines, mycotoxins, aromatic amines, heterocyclic amines, ultraviolet light, and alkylating cancer chemotherapeutic agents. Most human DNA adduct exposure monitoring has been performed with either 32P-postlabeling or immunoassays, neither of which is able to chemically characterize specific DNA adducts. Recently developed combinations of methods with chemical and physical end points have allowed identification of specific adducts in human tissues. Studies are presented that demonstrate that high ambient levels of benzo[a]pyrene are associated with high levels of DNA adducts in human blood cell DNA and that the same DNA adduct levels drop when the ambient PAH levels decrease significantly. DNA adduct dosimetry, which has been achieved with some dietary carcinogens and cancer chemotherapeutic agents, is described, as well as studies correlating DNA adducts with other biomarkers. It is likely that some toxic, noncarcinogenic compounds may have genotoxic effects, including oxidative damage, and that adverse health outcomes other than cancer may be correlated with DNA adduct formation. The studies presented here may serve as useful prototypes for exploration of other toxicological end points.

Platinum Drug-DNA Interactions in Human Tissues Measured by Cisplatin-DNA Enzyme-Linked Immunosorbent Assay and Atomic Absorbance Spectroscopy

Studies of platinum drug-DNA adduct formation in tissues of cancer patients have involved both atomic absorbance spectroscopy (AAS), which measures total DNA-bound platinum, and anti-cisplatin-DNA enzyme-linked immunosorbent assay (ELISA), which detects a fraction of the AAS-measurable adduct. These studies were designed to explore mechanisms of drug-DNA interactions, to make correlations with clinical outcome, and possibly to validate DNA adduct measurements for use in occupational and environmental biomonitoring. The results, determined by both ELISA and AAS, demonstrate that cisplatin and its analog carboplatin bind to DNA in many human organs, including kidney, brain, peripheral nerve, and bone marrow, which are sites for drug toxicity. Platinum was also observed bound to ovarian tumor DNA. The adducts were highly persistent, being measurable in tissues obtained at autopsy up to 15 months after the last administration of platinum chemotherapy. A comparison of blood cell DNA adduct levels, determined by ELISA, and the clinical response of 139 patients with ovarian, testicular, colon, or breast cancer demonstrated a strong correlation between failure to form DNA adducts and failure of therapy. Conversely, patients who formed high levels of DNA adduct were most likely to respond favorably. A similar correlation was not observed for adducts determined by AAS; that is, the average total DNA-bound platinum levels were the same for patients who did not respond to therapy and for patients who had any kind of response. Thus, in this study, human blood cell DNA adducts measured by ELISA correlate with tumor remission, while those measured by AAS do not.

Characterization of the DNA damage recognized by the antiserum elicited against cis-diamminedichloroplatinum (II)-modified DNA

A series of in vitro and in vivo studies were performed to characterize DNA damage recognized by an antiserum elicited against DNA modified with cis-diamminedichloroplatinum(II) (cisplatin). Adducts determined by the cisplatin-DNA enzyme-linked immunosorbent assay (ELISA) in human blood cell DNA have been shown to correlate well with positive clinical outcome in testicular and ovarian cancer patients receiving platinum drug-based chemotherapy (Reed et al. (1990) Proc. Natl. Acad. Sci., 84; 5024, and Reed et al. (1988) Carcinogenesis, 9, 1909). DNAs from calf thymus, salmon sperm, pBR322 and synthetic oligonucleotides were modified with cisplatin in vitro before or after specific DNA digestion steps to yield adducted samples of known size and/or chemical composition. These cisplatin modified DNAs were assayed by atomic absorption spectrometry (AAS) to assess absolute platinum content, and by ELISA to determine the antiserum specificity. The antiserum recognizes native cisplatin-modified calf thymus DNA, and native oligonucleotides containing intrastrand cis-Pt (NH3)2-d(pGpG) adducts (Pt-GG) and intrastrand cis-Pt (NH3)2-d(pApG) adducts (Pt-AG). Modified plasmid DNA fragments of varying sizes (down to 309 base pairs) are recognized similarly to cisplatin-modified calf thymus DNA. The antiserum does not cross-react with individual Pt-GG or Pt-AG adducts not bound to DNA. In experiments designed to assess the relationship between adduct measured by ELISA and total platinum bound to DNA as measured by AAS, male and female Sprague-Dawley rats were injected i.p. with cisplatin and a dose response for adduct formation was determined in kidney DNA samples. Values obtained by ELISA were substantially lower than those measured by AAS, and the two were directly related in DNA from kidney tissues of rodents but not in DNA from human nucleated blood cells. In rodent samples the ELISA measured a consistent 0.2% of the total DNA-bound platinum determined by AAS, with a correlation coefficient of 0.91. Among 54 blood cell DNA samples from human patients, which gave measurable adduct values in both ELISA and AAS, the ELISA measured a variable fraction (0.2-33.0%) of the total DNA-bound platinum measured by AAS. We conclude that the cisplatin-DNA ELISA measures a three dimensional lesion in DNA that is formed in direct proportion to total DNA-bound platinum in rat kidney, but that in human biological samples, interindividual variability precludes a relationship that conforms to simple mathematical algorithms.