Genotoxicity screening via the γH2AX by flow assay.

The measurement of serine139-phosphorylated histone H2AX (γH2AX) provides a biomarker of DNA double-strand breaks (DSBs) and may identify potential genotoxic activity. In order to evaluate a flow cytometry assay for γH2AX detection (hereafter termed the γH2AX by flow assay), 6 prototypical (3 pro- and 3 proximate) genotoxins, i.e. dimethylbenz[a]anthracene (DMBA), 2-acetylaminofluorene (2-AAF), benzo[a]pyrene (B[a]P), methyl methane sulphonate (MMS), methyl nitrosourea (MNU) and 4-nitroquinoline oxide (4NQO), were selected to define assay evaluation criteria. In addition, 3 non-genotoxic cytotoxins (phthalic anhydride, n-butyl chloride and hexachloroethane) were included to investigate the influence of cytotoxicity on assay performance. At similar cytotoxicity levels (relative cell counts; RCC 75-40%) all prototypical genotoxins induced marked concentration-dependent increases in γH2AX compared with the non-genotoxins. As a result, assay evaluation criteria for a positive effect were defined as >1.5-fold γH2AX @ RCC >25%. Twenty five additional chemicals with diverse structures and genotoxic activity were selected to evaluate the γH2AX by flow assay. Results were compared with Ames bacterial and in vitro mammalian genotoxicity tests (mouse lymphoma assay and/or chromosome aberration assay). γH2AX by flow assay results were highly predictive of Ames (sensitivity 100%; specificity 67%; concordance 82%) and in vitro mammalian genotoxicity tests (sensitivity 91%; specificity 89%; concordance 91%) and provide additional evidence that γH2AX is a biomarker of potential genotoxic activity, underpinned mechanistically by the cellular response to DSBs. Discordant findings were predominately attributed to differences in specificity for some mammalian cell genotoxins that are Ames non-mutagens or for "biologically-irrelevant" positives in the mammalian tests. Simple anilines were classified as genotoxic following rat liver S9-mediated bioactivation, however, effects on γH2AX were atypical and limited to a small sub-population of S-phase nuclei. Nevertheless, the γH2AX by flow assay represents a novel genotoxicity assay with the potential to flag both pro- and proximate genotoxins.

An analysis of results from 305 compounds tested with the yeast RAD54-GFP genotoxicity assay (GreenScreen GC)-including relative predictivity of regulatory tests and rodent carcinogenesis and performance with autofluorescent and coloured compounds.

Data from 305 non-proprietary compounds tested using the yeast RAD54-GFP (Green Fluorescent Protein) assay, GreenScreen GC, are presented, together with a detailed comparison with results from in vitro and in vivo genotoxicity tests and rodent carcinogenesis. In addition, observations on reproducibility and the performance of the test with autofluorescent and coloured compounds are described. Like the Ames test, the GreenScreen assay is shown to exhibit high specificity (82%), meaning that compounds with positive results are very likely to be genotoxic carcinogens. This is in contrast to mammalian cell tests established for use in regulatory testing that provide disappointingly low specificity and the inevitable generation of confounding false positive data. The analysis confirmed the observations of earlier studies, showing that a combination of an Ames test (or surrogate) with the yeast test provides high specificity as well as high sensitivity in the identification of rodent carcinogens.

Thoracofemoral bypass using spliced femoral vein with removal of an infected axillobifemoral bypass graft.

A 63-year-old male underwent emergency repair of a ruptured juxtarenal aortic aneurysm via a transabdominal approach using an aorto-bi-iliac Dacron graft. This became infected. A right axillobifemoral bypass was placed and the infected graft was removed with oversewing of the aorta. The patient was re-admitted 8 months later with an infected axillobifemoral prosthesis. We harvested both femoral veins (FV) and spliced them to perform a left thoracobifemoral bypass with simultaneous explantation of the infected graft. The patient remains well with a patent graft 20 months post-operatively.

RNA destabilization by the granulocyte colony-stimulating factor stem-loop destabilizing element involves a single stem-loop that promotes deadenylation.

Granulocyte colony-stimulating factor (G-CSF) mRNA contains two distinct types of cis-acting mRNA destabilizing elements in the 3'-untranslated region. In addition to several copies of the AU-rich element the G-CSF mRNA also contains a destabilizing region that includes several predicted stem-loop structures. We report here that the destabilizing activity resides in a single stem-loop structure within this region. A consensus sequence for the active structure has been derived by site-directed mutagenesis, revealing that a three-base loop of sequence YAU and unpaired bases either side of the stem contribute to the activity. The helical nature of the stem is essential and the stem must be less than 11 bp in length, but the destabilizing activity is relatively insensitive to the sequence within the helix. The stem-loop increases the rate of mRNA deadenylation, most likely by enhancing the processivity of the deadenylation reaction. A protein that binds the stem-loop, but not an inactive mutant form, has been detected in cytoplasmic lysates.

Spontaneous mutation spectra in supF: comparative analysis of mammalian cell line base substitution spectra.

The last decade has seen a dramatic accumulation of mutation data from reporter genes utilized in mutagenesis experiments involving DNA reactive agents allowing comparisons for the mutagenic potential between many different mutagens. When analysing chemically induced mutation spectra it is important to establish the potential spontaneous background before drawing conclusions concerning specific chemically induced hotspots. A major mutation reporter system gene used in mammalian cells is the supF suppressor tRNA gene. The Mammalian Gene Mutation Database (MGMD) contains a considerable number of supF spontaneous mutations permitting a thorough analysis of spontaneous mutations in mammalian cell lines from different species and tissues. Analyses of spontaneous mutation spectra were performed using a range of statistical techniques. Spontaneous mutations were observed at 82.4% of the nucleotides in the supF suppressor tRNA sequence although the pattern of significant hotspots differed between cell lines. Our analyses of spontaneous mutation spectra show considerable variation both within and between cell lines for the distributions of spontaneous mutations occurring with no clear tissue or species-specific patterns emerging. In addition, spectra derived from supF recovered from liver and skin of transgenic mice, were similar to each other, but showed significant differences from many in vitro spectra. The most common base substitutions were G:C>TA transversions and G:C>A:T transitions, although levels of each type differed between cell lines. There was also variation between cell lines for the most mutable dinucleotides, however, significant hotspots were frequently observed at CpG sites and sequences containing GG/CC. We conclude that the number of varying distributions and potential hotspots for spontaneous mutations should thus be considered when comparing chemically induced mutation spectra in supF. The spectra presented here will be a useful reference for analysis and re-analysis of chemically induced spectra as well as for use in comparison with the spontaneous spectra of other gene systems.

The mammalian gene mutation database.

The Mammalian Gene Mutation Database (MGMD) is a comprehensive collection of published mutation data from the open literature on mammalian cell-based gene model mutation detection systems. The database currently contains approximately 30000 comprehensively described mutant spectra records and it is maintained and up- dated on a daily basis. The major objectives of the MGMD were (i) to provide an Internet-accessible database (http://lisntweb.swan.ac. uk/cmgt/index.htm) for chemically induced and spontaneous mutation types and spectra in selected genes; (ii) to standardize the reporting of mutations within different genes where ambiguity exists in the literature; and (iii) to provide interactive and user-friendly access to the information. A multi-option search facility has been included that allows the user to search the database for parameters such as mutagen, gene or cell type of interest. The structure of the database permits easy retrieval of specific mutation data for further analysis. Thus, the MGMD should become a useful and necessary reference source and provides an analysis tool for genetic toxicologists.

Genotypic mutation analysis in the p53 gene of benzo[a]pyrene-treated European flounder (Platichthys flesus).

We have applied a genotypic mutation detection system (the Restriction Site Mutation (RSM) assay) to detect mutations in the marine teleost flounder (Platichthys flesus). The aim of this study was to evaluate this species as an environmental indicator of genotoxic exposure. We have used the model genotoxin benzo[a]pyrene (B[a]P) to determine the limits of mutation detection in the p53 gene of flounder liver DNA. This study has revealed two important findings. Firstly, we were able to demonstrate that a polymorphism exists in the TaqI restriction site of exon 8 of the flounder p53 gene at codon 243. This polymorphic allele was present as a heterozygote at a mean frequency of 15%, whereas 85% carried the homozygous wild type sequence. Secondly, we established that B[a]P treatment resulted in specific mutational events at the adenine base of the same TaqI site, contrasting previous reports stating that there was a guanine preference for this chemical in mammalian DNA. This difference in mutation specificity may possibly be accounted for by sequence specific factors or by species differences in metabolic activation and/or DNA repair and are worthy of further study.

An assessment of the genotoxic impact of the Sea Empress oil spill by the measurement of DNA adduct levels in selected invertebrate and vertebrate species.

The grounding of the Sea Empress oil tanker resulted in the release of 72,000 tonnes of crude oil into Milford Haven, Wales, UK. Our initial studies indicated that this contamination resulted in elevated levels of DNA adducts in one of the area's native marine species Lipophrys pholis [B.P. Lyons, J.S. Harvey, J.M. Parry, An initial assessment of the genotoxic impact of the Sea Empress oil spill by the measurement of DNA adduct levels in the intertidal teleost Lipophrys pholis, Mutat. Res. 390 (1997) 263-268]. These original studies were extended and the genotoxic impact of the oil contamination was investigated in the invertebrates Halichondria panicea and Mytilus edulis, along with the vertebrate fish species L. pholis, Pleuronectes platessa and Limanda limanda. DNA adduct levels were assessed in these species over a period of 2-17 months after the incident. The studies indicate differences in the impact of acute oil contamination upon vertebrate and invertebrate species. The oil contamination did not induce any detectable elevations in adduct levels in the invertebrate species H. panicea and M. edulis. In contrast, the oil contamination did appear to induce adducts in the vertebrate teleost species L. pholis, P. platessa and Lim. limanda. Despite some difficulties in sampling, the data obtained 12-17 months after the spill suggested that the affected species recovered from the oil contamination. While the studies indicate that the genetic impact of the oil contamination was less severe than might have been expected, it remains possible that the DNA adducts detected in the teleosts could lead to genetic changes in these species in the future.

Importance of the glycosylation and polyadenylation variants in metachromatic leukodystrophy pseudodeficiency phenotype.

Metachromatic leukodystrophy (MLD) is an inborn error of myelin metabolism caused by a deficiency of the lysosomal hydrolase, arylsulfatase A (ASA). About 1% of the normal population have ASA activity levels approximating those of MLD patients. This non-pathogenic reduction in ASA activity is caused by homozygosity for the ASA pseudodeficiency allele (ASA-PD). Although this allele contains two sequence alterations, a polyadenylation defect and an amino acid substitution (N350S), the reduction in ASA activity previously has been attributed to the polyadenylation defect which reduces the amount of ASA mRNA and hence ASA protein by approximately 90%. The identification of MLD patients who are homozygous for the ASA-PD allele has brought about the need to re-evaluate the allele in light of the possible role that it may play in the development and progression of disease. Ribonuclease protection assay analysis of ASA mRNA transcripts and an investigation into the activity and lysosomal localization of protein expressed by an ASA expression construct containing the N350S variant indicated that both the N350S and polyadenylation defects play a role in biochemically defining the ASA-PD phenotype. The combined effect of the reduction in ASA mRNA due to the polyadenylation defect and the lowering of ASA activity and aberrant targeting of the expressed N350S ASA protein to the lysosome is estimated to reduce ASA activity in pseudodeficiency homozygotes to approximately 8% of normal.

The analysis of DNA adduct formation, removal and persistence in the common mussel Mytilus edulis exposed to 4-nitroquinoline 1-oxide.

32P-postlabelling was used for the detailed analysis of 4-nitroquinoline 1-oxide (4-NQO) induced DNA adduct formation, removal and persistence in the marine shellfish Mytilus spp. The results had a number of important implications concerning the use of such DNA adducts as dosimeters of environmental genotoxin exposures. Our studies indicated that the maintenance of the Mytilus specimens under controlled laboratory conditions can result in the induction of 'stress-related adducts' seemingly related to the nature of the experimental set-up. The studies also indicated that the absorption and activation of genotoxins in this species appear to affect the rate of adduct formation, and that the maximum levels of adducts may not necessarily be induced immediately after the cessation of a genotoxin exposure. In addition, Mytilus specimens were shown to possess a significant capacity to remove these genotoxin-induced DNA adducts. The removal of these adducts appeared to be biphasic in nature, with the rapid removal of a large proportion of adducts occurring within 48 h of the cessation of the exposure, followed by a slow rate of adduct removal over the remaining period of the studies. Despite the relatively efficient removal of the majority of these genotoxin-induced DNA adducts, a proportion remained up to 56 days after the initial exposure. The persistence of these genotoxin-DNA adducts, combined with the information on the rates of adduct removal, indicated that under well-defined conditions, such adducts could serve as suitable biomarkers of environmental contamination.

The application of the 32P-postlabelling assay to aquatic biomonitoring.

The aquatic environment is known to contain a variety of natural and anthropogenic compounds that are capable of interacting with the genetic material of aquatic organisms. The increases in the levels of these anthropogenic contaminants, associated with widespread industrialisation, has led to the requirement for reliable methodologies to monitor their potential impact upon exposed aquatic organisms. Of the molecular techniques currently available, the 32P-postlabelling assay for the detection of DNA adducts offers considerable potential for the qualitative and quantitative assessment of genotoxin exposure. Here we describe several studies in which the technique was adapted for evaluation in two marine bioindicator species the common mussel Mytilus edulis and the flatfish Limanda limanda. Laboratory studies in which M. edulis specimens were exposed to 2-aminofluorene and 4-nitroquinoline 1-oxide confirmed the species' capacity to form genotoxin-related adducts. However, in further studies, no exposure-related adducts could be detected in M. edulis specimens placed in mesocosms containing environmentally realistic levels of anthropogenic contaminants. Biologically significant levels of adducts were detected in L. limanda specimens exposed to sediment bound contaminants under controlled conditions, although the levels did not appear to be statistically significant. An in situ study in which adduct levels were determined in L. limanda specimens from two sites of contrasting contamination levels proved to be more conclusive. The results were both biologically and statistically significant, suggesting that adduct levels could well be related to the levels of sediment-bound contaminants. Together the studies confirmed that the determination of the levels of DNA adducts could be used as indicators of the exposure of aquatic organisms to environmental genotoxins.

The detection of genotoxin-induced DNA adducts in the common mussel Mytilus edulis.

In order to establish the capacity of Mytilus spp. to form genotoxin-DNA adducts, a series of in vitro and in vivo studies were conducted in which tissue samples and animals were exposed to five model genotoxins. Following the in vitro characterization of the major adducts induced by the compounds, a series of in vivo studies were conducted to determine if the levels of genotoxin-DNA adduct formation followed a dose response. The results of these studies suggested that under appropriate conditions, DNA adducts in the hepatopancreas could be used as molecular dosimeters of exposure to genotoxic compounds in the species. However, these studies also revealed that the successful detection of such genotoxin-DNA adducts depends largely upon their chromatographic properties and thus the vigour of the characterization undertaken.

An initial assessment of the genotoxic impact of the Sea Empress oil spill by the measurement of DNA adduct levels in the intertidal teleost Lipophrys pholis.

The Sea Empress oil spill resulted in the release of vast quantities of potentially genotoxic contaminants into the coastal environment of the county of Pembrokeshire (UK). We are at present attempting to determine the potential genotoxic impact of the incident upon the native marine species of the area. Here we describe the levels of DNA adducts in specimens of the intertidal teleost, Lipophrys pholis, exposed to extensive oil extensive oil contamination as an indication of exposure to potential genotoxins. We detected elevated levels of adducts in L. pholis specimens from an area that underwent heavy oil contamination as compared to specimens from a clean reference area devoid of oil contamination. These preliminary studies indicated that the oil contamination induced DNA adducts in the L. pholis specimens, which could potentially cause genetic damage in this native marine species. Further studies are now required to assess the full extent of the genotoxic impact of the oil spill upon the Pembrokeshire area's native marine life.

Totally implantable venous access devices in adult patients with cystic fibrosis.

Between September 1987 and April 1995, 33 totally implantable venous access devices (TIVADs) were implanted at the Cardiff Adult Cystic Fibrosis Centre, U.K., for the purpose of intermittent antibiotic therapy, including 22 PORT-A-CATH (Simcare Ltd.) devices (PCs) to 18 patients, and 11 P.A.S.PORT (Simcare Ltd.) devices (PPs) to nine patients. There were 50 complications during 25 824 days of catheter function which were severe enough to lead to removal of the devices in eight patients (six PCs and four PPs). Overall, patients' acceptance of these devices was excellent. Despite a shorter functional time and a higher rate of complications in PPs compared with PCs, PPs were preferred by many patients for cosmetic reasons. Totally implantable venous access devices provide safe, effective and convenient means of venous access in cystic fibrosis patients requiring intermittent antibiotic therapy.

Cyclic GMP regulates activation of phosphoinositidase C by bradykinin in sensory neurons.

Prior exposure of cultured neonatal rat dorsal root ganglion (DRG) neurons to bradykinin resulted in marked attenuation of bradykinin-induced activation of phosphoinositidase C (PIC). The (logconcentration)-response curve for bradykinin-induced [3H]inositol trisphosphate ([3H]IP3) formation was shifted to the right and the maximum response was reduced. Bradykinin increases cyclic GMP (cGMP) in DRG neurons [Burgess, Mullaney, McNeill, Coote, Minhas and Wood (1989) J. Neurochem. 53, 1212-1218] and treatment of the neurons with dibutyryl cGMP (dbcGMP) had a similar, inhibitory, effect on bradykinin-induced [3H]IP3 formation. NG-Nitro-L-arginine (LNNA) blocked bradykinin-induced formation of cGMP. It prevented the functional uncoupling induced by pretreatment with bradykinin, but not the inhibitory effect of dbcGMP on [3H]IP3 formation. The ability of LNNA to prevent desensitization was reversed by excess L-arginine, indicating that its actions were mediated through inhibition of nitric oxide synthase. In addition to functional desensitization, exposure to bradykinin reduced the number of cell-surface receptors detected with [3H]bradykinin, without affecting its KD value for the remaining sites. In contrast to bradykinin, pretreatment with dbcGMP had no effect on either the KD or B(max) for [3H]bradykinin binding. This implies that the inhibitory effect of dbcGMP was down-stream from the binding of bradykinin to its receptor and upstream of IP3 formation. The lack of effect of dbcGMP on [3H]bradykinin binding suggests that the decrease in receptor number induced by bradykinin was mediated by a different mechanism and was not a key factor in the rapid phase of desensitization in these cells.

Activation of protein kinase C by the capsaicin analogue resiniferatoxin in sensory neurones.

Resiniferatoxin and capsaicin are sensory neurone-specific excitotoxins that operate a common cation channel in nociceptors. Resiniferatoxin is structurally similar to capsaicin and to phorbol esters. Specific [3H]-resiniferatoxin binding, which was detected in the membrane (KD value 1.8 +/- 0.2 nM) but not cytosolic fraction of rat dorsal root ganglia, could not be displaced by phorbol 12,13-dibutyrate. Conversely, resiniferatoxin did not displace [3H]phorbol 12,13-dibutyrate binding in either the cytosolic or membrane fraction. Resiniferatoxin and capsaicin both caused translocation of protein kinase C in dorsal root ganglion neurones (EC50 value 18 +/- 3 nM). This translocation was greatly reduced but not abolished, in the absence of external Ca2+, suggesting that it was secondary to Ca2+ entry. Resiniferatoxin also caused direct activation of a Ca(2+)- and lipid-dependent kinase (or kinases) in the cytosolic fraction of dorsal root ganglia, at concentrations (100 nM to 10 microM) higher than required for displacement of [3H]resiniferatoxin binding or translocation of protein kinase C. Capsaicin (up to 10 microM) was unable to mimic this effect. These data imply that although resiniferatoxin-induced translocation of protein kinase C in dorsal root ganglion neurones was mainly indirect, it also caused direct activation of a protein kinase C-like kinase in these cells.

Effect of ilioinguinal and iliohypogastric nerve block and wound infiltration with 0.5% bupivacaine on postoperative pain after hernia repair.

We have compared, in 40 adult males, the effect on pain in the first 24 h after herniorrhaphy of preincisional ilioinguinal and iliohypogastric nerve block and wound infiltration with 0.5% bupivacaine or saline. After operation, patients received morphine i.v. via a patient-controlled analgesia machine and visual analogue pain scores (VAS) at rest and on movement were recorded. The bupivacaine group consumed less morphine in the first 6 h after operation. There was no difference in morphine consumption between the two groups in the next 18 h. The time to first analgesia was delayed in the bupivacaine group and was not followed by a rebound increase in requirement for analgesia. There was no significant difference in VAS scores at rest but there was a significantly higher pain score with movement in the saline group. We have shown that the combination of nerve block and wound infiltration reduces consumption of morphine in the first 24 h after herniorrhaphy. We have failed to show any effect of 0.5% bupivacaine beyond the first 6 h after operation.