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Biomedical subjects

K E Herbert

Publications and source records attributed to K E Herbert.

At least 19 recordsLinked to original sources
PubMed2005

Dietary supplementation with different vitamin C doses: no effect on oxidative DNA damage in healthy people.

BACKGROUND: Antioxidants are believed to prevent many types of disease. Some previous studies suggest that dietary supplementation with vitamin C results in a decrease in the level of one of the markers of oxidative damage-8-oxoguanine in the DNA of peripheral blood mononuclear cells (PBMC). AIM OF TRIAL: To investigate the effect of different dose levels of dietary supplementation with vitamin C on oxidative DNA damage. METHODS: A randomised double-blind placebo-controlled trial was carried out using three different levels (80, 200 and 400 mg) of dietary vitamin C supplementation in a healthy population of 160 volunteers; supplementation was for a period of 15 weeks followed by a 10 week washout period. Peripheral blood samples were obtained every 5 weeks from baseline to 25 weeks. RESULTS: An increase in PBMC vitamin C levels was not observed following supplementation in healthy volunteers. There was no effect found on 8-oxoguanine measured using HPLC with electrochemical detection for any of the three supplemented groups compared to placebo. 8-oxoadenine levels were below the limit of detection of the HPLC system used here. CONCLUSIONS: Supplementation with vitamin C had little effect on cellular levels in this group of healthy individuals, suggesting their diets were replete in vitamin C. The dose range of vitamin C used did not affect oxidative damage in PBMC DNA.

Adolescent↗
PubMed2005

A phase II study of dexamethasone, ifosfamide, cisplatin and etoposide (DICE) as salvage chemotherapy for patients with relapsed and refractory lymphoma.

The 4-day combination of dexamethasone, ifosfamide, cisplatin, and etoposide (DICE) is a salvage regimen for lymphoma. We report a prospective phase II multi-center trial of a modified DICE regimen in relapsed or refractory Hodgkin (HL) or non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL), constituting a single day of intravenous administration followed by 3 days of oral administration, aimed at reducing inpatient days without losing efficacy. Forty patients (median age 56, range 25 - 79) were included: 28 (70%) NHL, 9 (23%) HL and 3 (8%) CLL. Fifty-three per cent had received 2 prior treatment regimens. International Prognostic Index (IPI) was 2 in 75% of NHL patients. Patients aged 55 and those with previous autologous stem cell transplantation (ASCT) started on a lower-dose regimen, with dose escalation possible in 2 patients. Overall response rate was 41%. Thirty-eight per cent of patients had stable disease. With a median of 3.1 years of follow-up, estimated progression-free survival (PFS) and overall survival (OS) rates at 3 years were 15% and 43% respectively. OS was longer in the < 55 compared to the 55 age cohort (P = 0.0091), longer for HL than NHL (P = 0.59 and 0.039 respectively) and longer for Low/Low-Int IPI than High/High-Int IPI (P = 0.0074 and 0.0009 respectively). Median duration of inpatient stay was 3 days. There were no treatment-related deaths. In conclusion, this modification of DICE is an effective and well tolerated salvage regimen, even in this poor prognosis group of patients. Further clinical studies of DICE in first relapse and in older patients, possibly with the addition of rituximab, are warranted.

Adult↗
PubMed2004

Graft-versus-lymphoma effect in refractory cutaneous T-cell lymphoma after reduced-intensity HLA-matched sibling allogeneic stem cell transplantation.

Cutaneous T-cell lymphomas (CTCL) are rare diseases that, in their advanced stages or in transformation, have a poor prognosis. Autologous stem cell transplantation (Au-SCT) after high-dose therapy has yielded disappointing results. Allogeneic transplantation (allo-SCT) provides the potential advantage of an immune-mediated graft-versus-lymphoma (GVL) effect. Reduced-intensity allo-SCT potentially offers a GVL effect, but with diminished toxicity related to the induction regimen; however, published experience with this approach in CTCL is limited. We report a series of three patients (age 35-49) with advanced, refractory (n=2) or transformed (n=1) CTCL who underwent reduced-intensity allo-SCT in the context of active disease. All three survived the peri-transplant period and, despite later having disease relapse, all exhibited evidence of a GVL effect. Relapses of the disease were in the context of immune suppression for graft-versus-host disease (GVHD), and when immune suppression was reduced, responses were regained. A comparison is made of these results to those in a review of the published literature to date. We conclude that while a GVL can be achieved for CTCL with reduced-intensity allogeneic transplantation, the clinical benefits are short lived and novel approaches are required to obtain sustained remissions.

Graft vs Tumor Effect↗
PubMed2001

Insulin resistance as a major determinant of increased coronary heart disease risk in postmenopausal women with Type 2 diabetes mellitus.

AIM: To investigate the risk factors associated with clinically defined coronary heart disease (CHD) in women with Type 2 diabetes mellitus (DM). METHODS: CHD status was assessed via standard history and resting electrocardiogram in 41 postmenopausal diabetic and 41 age- and body mass index-matched normoglycaemic women recruited from a community-based cohort. The following parameters were assessed: body composition by dual energy X-ray absorptiometry, blood pressure, metabolic and lipoprotein profile and haemostatic factors. RESULTS: Diabetic women with CHD (n = 14) had greater insulin resistance, calculated by homeostasis model assessment (10.2 (7.0-14.8) vs. 6.5 (5.5-7.7), P = 0.010), and higher plasminogen activator inhibitor-1 (PAI-1) levels (45 (29-69) vs. 24 (19-32) ng/ml, P = 0.013), than those without CHD. They also had higher triglycerides (2.9 (2.2-3.8) vs. 2.1 (1.8-2.4) mmol/l, P = 0.016) and a trend towards reduced low-density lipoprotein particle size (25.5 +/- 0.6 vs. 25.8 +/- 0.5 nm, P = 0.097). In a logistic regression model, insulin resistance was a significant independent predictor of CHD status (odds ratio = 1.33, 95% confidence interval = 1.06-1.68, P = 0.015). In contrast, in normoglycaemic women the major risk factors for CHD were elevated cholesterol, apolipoprotein(a), apolipoprotein B and systolic blood pressure (P = 0.018, P = 0.016, P = 0.006 and P = 0.049, respectively). CONCLUSIONS: Increased insulin resistance in association with elevated PAI-1 and dyslipidaemia appears to underpin the increased risk of CHD in women with Type 2 DM. Therapeutic approaches that increase insulin sensitivity may serve to reduce CHD risk in this vulnerable group. Diabet. Med. 18, 476-482 (2001)

Absorptiometry, Photon↗
PubMed2000

Urinary 8-oxo-2'-deoxyguanosine--source, significance and supplements.

Oxidative damage to cellular biomolecules, in particular DNA, has been proposed to play an important role in a number of pathological conditions, including carcinogenesis. A much studied consequence of oxygen-centred radical damage to DNA is 8-oxo-2'-deoxyguanosine (8-oxodG). Using numerous techniques, this lesion has been quantified in various biological matrices, most notably DNA and urine. Until recently, it was understood that urinary 8-oxodG derives solely from DNA repair, although the processes which may yield the modified deoxynucleoside have never been thoroughly discussed. This review suggests that nucleotide excision repair and the action of a specific endonuclease may, in addition to the nucleotide pool, contribute significantly to levels of 8-oxodG in the urine. On this basis, urinary 8-oxodG represents an important biomarker of generalised, cellular oxidative stress. Current data from antioxidant supplementation trials are examined and the potential for such compounds to modulate DNA repair is considered. It is stressed that further work is required to link DNA, serum and urinary levels of 8-oxodG such that the kinetics of formation and clearance may be elucidated, facilitating greater understanding of the role played by oxidative stress in disease.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2000

Immunochemical quantitation of UV-induced oxidative and dimeric DNA damage to human keratinocytes.

There is growing evidence to suggest that solar radiation-induced, oxidative DNA damage may play an important role in skin carcinogenesis. Numerous methods have been developed to sensitively quantitate 8-oxo-2'deoxyguanosine (8-oxodG), a recognised biomarker of oxidative DNA damage. Immunoassays may represent a means by which the limitations of many techniques, principally derived from DNA extraction and sample workup, may be overcome. We report the evaluation of probes to thymine dimers and oxidative damage in UV-irradiated cells and the DNA derived therefrom. Thymine dimers were most readily recognised, irrespective of whether in situ in cells or in extracted DNA. However, using antibody-based detection the more subtle oxidative modifications required extraction and, in the case of 8-oxodG, denaturation of the DNA prior to successful recognition. In contrast, a recently described novel probe for 8-oxodG detection showed strong recognition in cells, although appearing unsuitable for use with extracted DNA. The probes were subsequently applied to examine the relative induction of lesions in cells following UV irradiation. Guanine-glyoxal lesions predominated over thymine dimers subsequent to UVB irradiation, whereas whilst oxidative lesions increased significantly following UVA irradiation, no induction of thymine dimers was seen. These data support the emerging importance of oxidative DNA damage in UV-induced carcinogenesis.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2000

Development of a quality control material for the measurement of 8-oxo-7,8-dihydro-2'-deoxyguanosine, an in vivo marker of oxidative stress, and comparison of results from different laboratories.

The measurement of 8-oxo-7,8-dihydro-2'-deoxyguanosine is an increasingly popular marker of in vivo oxidative damage to DNA. A random-sequence 21-mer oligonucleotide 5'-TCA GXC GTA CGT GAT CTC AGT-3' in which X was 8-oxo-guanine (8-oxo-G) was purified and accurate determination of the oxidised base was confirmed by a 32P-end labelling strategy. The lyophilised material was analysed for its absolute content of 8-oxo-dG by several major laboratories in Europe and one in Japan. Most laboratories using HPLC-ECD underestimated, while GC-MS-SIM overestimated the level of the lesion. HPLC-ECD measured the target value with greatest accuracy. The results also suggest that none of the procedures can accurately quantitate levels of 1 in 10(6) 8-oxo-(d)G in DNA.

Biomarkers↗
PubMed1999

Discrepancies in the measurement of UVC-induced 8-oxo-2'-deoxyguanosine: implications for the analysis of oxidative DNA damage.

Ultraviolet (UV) light-induced indirect, oxidative damage to DNA has received increasing attention with respect to the mutagenic and carcinogenic effects of solar radiation. An oxidative lesion that has raised particular interest because of its qualitative and quantitative importance is 8-oxo-2'-deoxyguanosine. This deoxynucleoside lesion is most frequently measured by high performance liquid chromatography with electrochemical detection (HPLC-EC) following enzymatic hydrolysis of DNA or as the base equivalent, 8-oxoguanine, by gas chromatography-mass spectrometry (GC-MS) following acid hydrolysis of DNA. We have noted a discrepancy in the literature whereby the levels of 8-oxo-2'-deoxyguanosine measured by HPLC-EC in UVC-irradiated DNA are significantly higher than when 8-oxoguanine is measured by GC-MS. By making use of the availability of both HPLC-EC and stable-isotope dilution GC-MS methodologies in our laboratory we have confirmed the discrepancy noted in the literature by parallel analysis of the same UVC-irradiated calf thymus DNA samples. Furthermore, analysis of the UVC-induced product by UV-visible spectrophotometry, voltammetry and its detection by a monoclonal antibody which recognises 8-oxo-2'-deoxyguanosine strongly suggests that the product is indeed 8-oxo-2'-deoxyguanosine. Partial explanation for this discrepancy could be an inordinate resistance of UVC-irradiated DNA to formic acid hydrolysis. However, we cannot completely exclude the possibility that there is a formic acid-labile species which co-elutes with 8-oxo-2'-deoxyguanosine in enzymatically digested UVC-irradiated DNA. Whether this phenomenon is unique to UV-irradiation damage or occurs with other systems that cause oxidative damage to DNA awaits further investigation. Irrespective of the exact mechanism, there will be significant implications for the analysis of oxidative DNA damage.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1999

Immunochemical detection of glyoxal DNA damage.

The relevance of reactive oxygen species (ROS) in the pathogenesis of inflammatory diseases is widely documented. Immunochemical detection of ROS DNA adducts has been developed, however, recognition of glyoxal-DNA adducts has not previously been described. We have generated a polyclonal antibody that has shown increased antibody binding to ROS-modified DNA in comparison to native DNA. In addition, dose-dependent antibody binding to DNA modified with ascorbate alone was shown, with significant inhibition by desferrioxamine, catalase, and ethanol. Minimal inhibition was observed with uric acid, 1,10-phenanthroline and DMSO. However, antibody binding in the presence of EDTA increased 3500-fold. The involvement of hydrogen peroxide and hydroxyl radical in ascorbate-mediated DNA damage is consistent with ascorbate acting as a reducing agent for DNA-bound metal ions. Glyoxal is known to be formed during oxidation of ascorbate. Glyoxylated DNA, that previously had been proposed as a marker of oxidative damage, was recognised in a dose dependent manner using the antibody. We describe the potential use of our anti-ROS DNA antibody, that detects predominantly Fenton-type mediated damage to DNA and report on its specificity for the recognition of glyoxal-DNA adducts.

Animals↗
PubMed1998

Novel repair action of vitamin C upon in vivo oxidative DNA damage.

There appears to be a paucity of data examining the effect of dietary antioxidants on levels of oxidative DNA damage in vivo, limiting evidence-based assessment of antioxidant efficacy, mechanisms and recommendation for optimal intake. We have examined levels of 8-oxo-2'-deoxyguanosine (8-oxodG) in mononuclear cell DNA, serum and urine from subjects undergoing supplementation with 500 mg/day vitamin C. Significant decreases in DNA levels of 8-oxodG were seen, correlating strongly with increases in plasma vitamin C concentration. Furthermore we established a timecourse for sequential, significant increases in serum and urinary 8-oxodG levels. These results illustrate, for the first time in humans, the kinetics of 8-oxodG removal and processing in vivo, suggesting a role for vitamin C in the regulation of DNA repair enzymes and thereby demonstrating a non-scavenging antioxidant effect.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1998

Molecular and cellular effects of ultraviolet light-induced genotoxicity.

Exposure to the solar ultraviolet spectrum that penetrates the Earth's stratosphere (UVA and UVB) causes cellular DNA damage within skin cells. This damage is elicited directly through absorption of energy (UVB), and indirectly through intermediates such as sensitizer radicals and reactive oxygen species (UVA). DNA damage is detected as strand breaks or as base lesions, the most common lesions being 8-hydroxydeoxyguanosine (8OHdG) from UVA exposure and cyclobutane pyrimidine dimers from UVB exposure. The presence of these products in the genome may cause misreading and misreplication. Cells are protected by free radical scavengers that remove potentially mutagenic radical intermediates. In addition, the glutathione-S-transferase family can catalyze the removal of epoxides and peroxides. An extensive repair capacity exists for removing (1) strand breaks, (2) small base modifications (8OHdG), and (3) bulky lesions (cyclobutane pyrimidine dimers). UV also stimulates the cell to produce early response genes that activate a cascade of signaling molecules (e.g., protein kinases) and protective enzymes (e.g., haem oxygenase). The cell cycle is restricted via p53-dependent and -independent pathways to facilitate repair processes prior to replication and division. Failure to rescue the cell from replication block will ultimately lead to cell death, and apoptosis may be induced. The implications for UV-induced genotoxicity in disease are considered.

Animals↗
PubMed1998

Further evidence for a possible role of conformation in the immunogenicity and antigenicity of the oxidative DNA lesion, 8-oxo-2'deoxyguanosine.

Damage to DNA by reactive oxygen species is acknowledged to be an important factor in a number of pathological conditions, including ageing and carcinogenesis. As a consequence, the development of methods for the sensitive detection and quantitation of oxidative DNA lesions has been of paramount importance. The oxidatively modified base product which has achieved most attention is 8-oxodeoxyguanosine (8-oxodG) and is a recognised marker of oxidative DNA damage. Although both polyclonal and monoclonal antibodies have previously been raised to 8-oxodG these have, for the most part failed to recognise this lesion within the DNA polymer. We have, through dilution cloning, produced a monoclonal antibody which appears to preferentially recognise 8-oxodG over deoxyguanosine (dG) in single-stranded oxidatively modified DNA. Such discrimination was not apparent when the DNA was double-stranded. Previous work has shown that 8-oxodG favours the syn glycosidic conformation due to steric repulsion, whereas dG assumes the anti. We present initial data that appear to support the postulate that it is these differences in conformation, in addition to structural recognition of the lesion itself, which are responsible for the discrimination, by our antibody of 8-oxodG over dG in single-stranded DNA.

Animals↗
PubMed1997

32P-postlabelling approaches for the detection of 8-oxo-2'-deoxyguanosine-3'-monophosphate in DNA.

32P-Postlabelling methods have been investigated for the analysis of the oxidative DNA damage lesion 8-oxoguanine. The extent of digestion of commercially available calf thymus DNA and an 8-oxo-2'-deoxyguanosine-3'-monophosphate (8oxodGp) containing oligonucleotide to 2'-deoxynucleotide-3'-monophosphates, using calf spleen phosphodiesterase and micrococcal nuclease, was determined by HPLC. The extent of unmodified nucleotide release from DNA, and the extent of 8oxodGp released from the oligomer did not increase between 1 and 16 h of incubation at 37 degrees C. Normal nucleotide release from DNA was found to be quantitative under these conditions, and 8oxodGp release from the oligomer was in the range of 84-91%. RNA contamination in DNA prepared for 32P-postlabelling severely compromised 8oxodGp analysis. Guanosine-3'-monophosphate (Gp) was found to exhibit similar chromatographic and electrophoretic properties to 8oxodGp and as such compromised both 8oxodGp isolation in enrichment steps and subsequent resolution of the 32P-labelled bisnucleotides by TLC. The effect of ribonuclease A, T1 and T2 was investigated and a combination of A + T1 was found to reduce Gp contamination in DNA samples to levels which no longer interfered with 8oxodGp analysis. We have successfully applied an HPLC enrichment protocol to the analysis of 8oxodGp in calf thymus DNA. Since determination of damage levels in human samples is often restricted by the amount of DNA available for analysis, a novel capillary electrophoresis (CE) technique for the enrichment of 8oxodGp has been developed. The advantage of CE is that it can achieve resolution of 8oxodGp and unmodified deoxynucleotides from much smaller samples and minimises the amount of [gamma-32P]ATP necessary for the analysis.

Autoradiography↗
PubMed1997

Immunogenicity of DNA damaged by reactive oxygen species--implications for anti-DNA antibodies in lupus.

Reactive oxygen species (ROS) are implicated in the inflammatory, autoimmune, connective tissue disease, systemic lupus erythematosus (SLE), particularly in respect of processes leading to the formation of pathological anti-DNA antibodies. Exposure to ROS increases the antigenicity of DNA for SLE antibodies, but data on the immunogenicity of ROS-DNA are not conclusive. In this study, we have examined the immunogenicity in rabbits, of DNA modified by three hydroxyl radical generating systems. Additionally, we investigated the antigenicity of UVA, UVB, and UVC irradiated DNA for lupus anti-DNA antibodies. Modification of DNA by both ROS and far UV dramatically increased its immunogenicity; the Fe2+ and H2O2 system resulted in antibodies that recognized both native and modified DNA. In our ELISA system, none of the UV antigens showed any antigenicity above native DNA for SLE sera. The data suggested that different profiles of antigenicity and immunogenicity arise dependent on the method of ROS production, but also that ROS-DNA may be a factor in antigen-driven immune complex formation in SLE.

Adult↗
PubMed1997

Analysis of urinary pseudouridine by micellar electrokinetic capillary chromatography.

We describe a capillary electrophoresis procedure, using uridine as an internal standard, for the analysis of urinary pseudouridine following solid-phase extraction. This method retains the advantages of existing chromatographic techniques but has superior resolving power and is technically less demanding. The standard curves were linear and reproducible with a detection limit of 60 fmol; chromatographic analysis was complete in under 10 min. Injection variability was < 5% and multiple independent analyses of the same urine sample for pseudouridine concentration gave coefficients of variation of < 10%. The mean (SD) urinary pseudouridine level in 18 healthy subjects was 16.1 (2.1) nmol/mumol creatinine. For a limited group of subjects where samples were taken more frequently, intra-individual variation averaged 27.5% reflecting variable excretion.

Adolescent↗
PubMed1996

Evidence for sensitisation of DNA to oxidative damage during isolation.

The oxidative base lesion 8-oxo-deoxyguanosine (8-oxo-dG) has been identified in DNA isolated from normal tissue and may occur at elevated levels during disease. However, the use of phenol during DNA extraction may artificially elevate the detected levels of this lesion. Herein, we have performed a comparative methodological study using both pronase E and phenol extraction techniques; native or oxidatively stressed DNA was isolated to determine the validity of each extraction technique for the subsequent determination of 8-oxo-dG. Whilst the yields of DNA were comparable, after pronase E extraction there was no detectable induction of 8-oxo-dG in reextracted naked DNA or peripheral blood mononuclear cell DNA that had been oxidatively stressed. However, phenol extraction enhanced the basal levels of 8-oxo-dG detected, and also induced a significant increase in levels of the modified base after exposure to oxidative stress. The latter was dependent on the presence of foetal calf serum in the extracellular medium. We have confirmed that phenol extraction sensitises native DNA to subsequent oxidative damage. In addition, this work shows that the extent of sensitisation occurring during phenol extraction varies with the degree of oxidative damage already incurred and infers that labile guanine sites generated during oxidative stress may be detected as 8-oxo-dG residues after phenol extraction.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1996

A novel HPLC procedure for the analysis of 8-oxoguanine in DNA.

The chromatographic quantitation of 8-oxoguanine adducts in DNA is widespread in the literature, although results obtained by HPLC of 8-oxodeoxyguanosine do not always agree with levels determined by GC-MS. To help explain this discrepancy, here we describe a novel procedure for the analysis of 8-oxoguanine adducts in DNA. Although it proved difficult to directly quantitate 8-oxoguanine in the presence of high levels of endogenous guanine using conventional reversed-phase HPLC, a simple preincubation of DNA acid hydrolysates with guanase allowed such analyses. The assay relied on our observation that 8-oxoguanine was not a substrate for guanase, and on sensitive electrochemical detection. The limit of detection for 8-oxoguanine was 5 nM or 250 fmol on column. Using this procedure, the background level of 8-oxoguanine in commercially available calf thymus DNA was 0.4 nmol/mg DNA or 3.2 mol/10(5) mol guanine.

Animals↗