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Articles by R. W. L Godschalk
Total Records ( 6 ) for R. W. L Godschalk
  N Gungor , A Haegens , A. M Knaapen , R. W. L Godschalk , R. K Chiu , E. F. M Wouters and F. J. van Schooten
 

Chronic pulmonary inflammation is associated with increased lung cancer risk, but the underlying process remains unknown. Recently, we showed that activated neutrophils inhibit nucleotide excision repair (NER) in pulmonary epithelial cells in vitro via the release of myeloperoxidase (MPO). To evaluate the effect of neutrophils on NER in vivo, mice were intratracheally instilled with lipopolysaccharide (LPS) (20 µg), causing acute lung inflammation and associated neutrophil influx into the airways. Three days post-exposure, phenotypical NER capacity was assessed in lung tissue homogenate. LPS exposure inhibited pulmonary NER by ~50%. This finding was corroborated by down-regulation of the NER-associated genes Xpa and Xpf. To further elicit the role of neutrophils and MPO in this process, we utilized MPO-deficient mice as well as mice in which circulating neutrophils were depleted by antibody treatment. LPS-induced inhibition of pulmonary NER was not affected by either Mpo–/– or by depletion of circulating neutrophils. This contrasts with our previous in vitro observations, suggesting that inhibition of pulmonary NER following acute dosing with LPS is not fully mediated by neutrophils and/or MPO. In conclusion, these data show that LPS-induced pulmonary inflammation is associated with a reduction of NER function in the mouse lung.

  P Moller , L Moller , R. W. L Godschalk and G. D. D. Jones
 

The alkaline single cell gel electrophoresis (comet) assay has become a widely used method for the detection of DNA damage and repair in cells and tissues. Still, it has been difficult to compare results from different investigators because of differences in assay conditions and because the data are reported in different units. The European Comet Assay Validation Group (ECVAG) was established for the purpose of validation of the comet assay with respect to measures of DNA damage formation and its repair. The results from this inter-laboratory validation trail showed a large variation in measured level of DNA damage and formamidopyrimidine DNA glycosylase-sensitive sites but the laboratories could detect concentration-dependent relationships in coded samples. Standardization of the results with reference standards decreased the inter-laboratory variation. The ECVAG trail indicates substantial reliability for the measurement of DNA damage by the comet assay but there is still a need for further validation to reduce both assay and inter-laboratory variation.

  L Forchhammer , C Johansson , S Loft , L Moller , R. W. L Godschalk , S. A. S Langie , G. D. D Jones , R. W. L Kwok , A. R Collins , A Azqueta , D. H Phillips , O Sozeri , M Stepnik , J Palus , U Vogel , H Wallin , M. N Routledge , C Handforth , A Allione , G Matullo , J. P Teixeira , S Costa , P Riso , M Porrini and P. Moller
 

The comet assay has become a popular method for the assessment of DNA damage in biomonitoring studies and genetic toxicology. However, few studies have addressed the issue of the noted inter-laboratory variability of DNA damage measured by the comet assay. In this study, 12 laboratories analysed the level of DNA damage in monocyte-derived THP-1 cells by either visual classification or computer-aided image analysis of pre-made slides, coded cryopreserved samples of cells and reference standard cells (calibration curve samples). The reference standard samples were irradiated with ionizing radiation (0–10 Gy) and used to construct a calibration curve to calculate the number of lesions per 106 base pair. All laboratories detected dose–response relationships in the coded samples irradiated with ionizing radiation (1.5–7 Gy), but there were overt differences in the level of DNA damage reported by the different laboratories as evidenced by an inter-laboratory coefficient of variation (CV) of 47%. Adjustment of the primary comet assay end points by a calibration curve prepared in each laboratory reduced the CV to 28%, a statistically significant reduction (P < 0.05, Levene's test). A large fraction of the inter-laboratory variation originated from differences in image analysis, whereas the intra-laboratory variation was considerably smaller than the variation between laboratories. In summary, adjustment of primary comet assay results by reference standards reduces inter-laboratory variation in the level of DNA damage measured by the alkaline version of the comet assay.

  C Johansson , P Moller , L Forchhammer , S Loft , R. W. L Godschalk , S. A. S Langie , S Lumeij , G. D. D Jones , R. W. L Kwok , A Azqueta , D. H Phillips , O Sozeri , M. N Routledge , A. J Charlton , P Riso , M Porrini , A Allione , G Matullo , J Palus , M Stepnik , A. R Collins and L. Moller
 

The increasing use of single cell gel electrophoresis (the comet assay) highlights its popularity as a method for detecting DNA damage, including the use of enzymes for assessment of oxidatively damaged DNA. However, comparison of DNA damage levels between laboratories can be difficult due to differences in assay protocols (e.g. lysis conditions, enzyme treatment, the duration of the alkaline treatment and electrophoresis) and in the end points used for reporting results (e.g. %DNA in tail, arbitrary units, tail moment and tail length). One way to facilitate comparisons is to convert primary comet assay end points to number of lesions/106 bp by calibration with ionizing radiation. The aim of this study was to investigate the inter-laboratory variation in assessment of oxidatively damaged DNA by the comet assay in terms of oxidized purines converted to strand breaks with formamidopyrimidine DNA glycosylase (FPG). Coded samples with DNA oxidation damage induced by treatment with different concentrations of photosensitizer (Ro 19-8022) plus light and calibration samples irradiated with ionizing radiation were distributed to the 10 participating laboratories to measure DNA damage using their own comet assay protocols. Nine of 10 laboratories reported the same ranking of the level of damage in the coded samples. The variation in assessment of oxidatively damaged DNA was largely due to differences in protocols. After conversion of the data to lesions/106 bp using laboratory-specific calibration curves, the variation between the laboratories was reduced. The contribution of the concentration of photosensitizer to the variation in net FPG-sensitive sites increased from 49 to 73%, whereas the inter-laboratory variation decreased. The participating laboratories were successful in finding a dose–response of oxidatively damaged DNA in coded samples, but there remains a need to standardize the protocols to enable direct comparisons between laboratories.

  N Gungor , A. M Knaapen , A Munnia , M Peluso , G. R Haenen , R. K Chiu , R. W. L Godschalk and F. J. van Schooten
 

Chronic inflammation has been recognized as a contributing factor in the pathogenesis of lung cancer. In this process, reactive oxygen species released by neutrophils may play an important role. The aim of the present study was to investigate the capacity of the major neutrophilic oxidant hypochlorous acid (HOCl), which is formed by myeloperoxidase (MPO), to induce DNA damage and mutagenicity in lung cells. HOCl was mutagenic in lung epithelial A549 cells in vitro, showing at physiological concentrations a significant induction of mutations in the HPRT gene. We studied three major types of DNA lesions that could be relevant for this HOCl-induced mutagenicity. Single strand DNA breakage and 8-oxo-7,8-dihydro-2'-deoxyguanosine were not found to be increased following HOCl treatment. On the other hand, HOCl caused a significant increase in the formation of 3-(2-deoxy-β-D-erythro-pentofuranosyl)pyrimido[1,2-]purin-10(3H)-one (M1dG), which can be formed by either malondialdehyde (MDA) or base propenals. We observed an increased MDA formation upon exposure of A549 cells to HOCl, but a role of base propenals cannot be excluded. In line with this, we observed 4-fold increased M1dG adduct levels in mice that were intratracheally instilled with lipopolysaccharide to induce a pulmonary inflammation with neutrophil influx. Depletion of circulating neutrophils significantly reduced pulmonary MPO activity as well as M1dG adducts levels, thereby providing a causal link between neutrophils/HOCl and pulmonary genotoxicity in vivo. Taken together, these data indicate that MPO catalysed formation of HOCl during lung inflammation should be considered as a significant source of neutrophil-induced genotoxicity.

  V Sipinen , J Laubenthal , A Baumgartner , E Cemeli , J. O Linschooten , R. W. L Godschalk , F. J Van Schooten , D Anderson and G. Brunborg
 

Exposure to genotoxins may compromise DNA integrity in male reproductive cells, putting future progeny at risk for developmental defects and diseases. To study the usefulness of sperm DNA damage as a biomarker for genotoxic exposure, we have investigated cellular and molecular changes induced by benzo[a]pyrene (B[a]P) in human sperm in vitro, and results have been compared for smokers and non-smokers. Sperm DNA obtained from five smokers was indeed more fragmented than sperm of six non-smokers (mean % Tail DNA 26.5 and 48.8, respectively), as assessed by the alkaline comet assay (P < 0.05). B[a]P-related DNA adducts were detected at increased levels in smokers as determined by immunostaining. Direct exposure of mature sperm cells to B[a]P (10 or 25 µM) caused moderate increases in DNA fragmentation which was independent of addition of human liver S9 mix for enzymatic activation of B[a]P, suggesting some unknown metabolism of B[a]P in ejaculates. In vitro exposure of samples to various doses of B[a]P (with or without S9) did not reveal any significant differences in sensitivity to DNA fragmentation between smokers and non-smokers. Incubations with the proximate metabolite benzo[a]pyrene-r-7,t-8-dihydrodiol-t9,10-epoxide (BPDE) produced DNA fragmentation in a dose-dependent manner (20 or 50 µM), but only when formamidopyrimidine DNA glycosylase treatment was included in the comet assay. These levels of DNA fragmentation were, however, low in relation to very high amounts of BPDE–DNA adducts as measured with 32P postlabelling. We conclude that sperm DNA damage may be useful as a biomarker of direct exposure of sperm using the comet assay adapted to sperm, and as such the method may be applicable to cohort studies. Although the sensitivity is relatively low, DNA damage induced in earlier stages of spermatogenesis may be detected with higher efficiencies.

 
 
 
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