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.

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.

A case of pneumococcal typhlitis.

A 29-year-old man had abdominal pain for 24 hours. This and the results of an abdominal examination were typical of acute appendicitis. He had suffered sinusitis for two weeks. At operation, the appendix was normal; there was an abscess in the cecal wall, the exudate of which grew pneumococci. Incidental appendectomy was done and the patient was treated successfully with lincomycin hydrochloride, and later, cephalexin monohydrate. It is possible that the typhlitis was secondary to the upper respiratory infection.

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.

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.

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.

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.

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.

Dimethyl sulfoxide. Biomechanical effects on tendons.

Forty-eight mice were treated for 12 time periods from 0 days through 28 days at 24-hour intervals by rinsing the skin area of the Achilles tendon of each hindleg with 100 microliters of a solution of 70% drug grade dimethyl sulfoxide in 30% sterile water. After the treatment period the mice were killed and the Achilles tendons were subjected to separation force analysis to determine tensile strength. The mean separation force was shown to be a function of the treatment period (P = 0.0294) with a significant linear component (P = 0.0049), a significant quadratic component (P = 0.0036), and a significant cubic component (P = 0.0049). The model identified an initial area of decreasing mean separation force (0 to 7 days), an area of increasing mean separation force (7 to 22 days), and a subsequent area of decreasing mean separation force (22 to 28 days). The maximum decrease in the group mean separation force was 20.2% and was observed in the 7-day treatment group. The clinical significance of the results of this study was that the topical application of dimethyl sulfoxide was associated with periods of decreased mean separation forces in tendons. The avoidance of vigorous muscular activity is recommended during therapy with dimethyl sulfoxide.

Nutritional management of the adolescent athlete.

The difference between the adolescent athlete and his mature counterpart is that this is a period of rapid physiologic growth and change. It is important that the additional stresses of athletic competition or preparation for that competition (such as weight reduction) do not compromise these changes. Providing the proper training diet for the adolescent athlete consists of providing adequate calories in the proper balance of nutrients for the additional stress of athletics. This must also take into account the eating patterns and preferences associated with adolescence. Education of coaches, parents, and athletes is an important aspect of providing health care to an athlete or athletic team. Although it may seem more "glamorous" to work with Olympic level or elite athletes, our experience is that these individuals often already have established poor eating patterns and nutritional misinformation. Clinics or educational sessions involving younger athletes will be more successful and build a foundation for tomorrow's Olympic athletes.

Aldosterone, cortisol, and electrolyte responses to hypobaric hypoxia in moderate-altitude natives.

Serum aldosterone, cortisol, and electrolyte concentration, and urinary aldosterone and electrolyte excretion responses were examined in seven low-altitude natives (LAN) (373 m or less, aged 19-25 yr) and nine moderate-altitude natives (MAN) (1,830-2,200 m, aged 19-23 yr) for 2 d at their own residence (home) altitude (PB 740 or 585 mm Hg, respectively) and later for 2 d during decompression at a simulated altitude of 4,270 m (PB 447 mm Hg). The LAN group demonstrated higher (p less than 0.05) serum cortisol concentrations and respiration rates, and lower (p less than 0.05) serum aldosterone and potassium, and urinary aldosterone, sodium, and potassium concentrations at certain times during decompression compared to their home responses. Moderate-altitude native responses, on the other hand, were generally unchanged. Manifestations of acute mountain sickness at PB 447 mm Hg were also significantly greater in the LAN group. Thus, it appears that the MAN subjects were influenced less by the drop in ambient oxygen tension associated with PB 447 mm Hg.

How We Manage Plantar Fasciitis.

In brief: Plantar fasciitis is a prolonged overuse injury that is potentially incapacitating and causes heel or arch pain. It is common among runners and athletes who participate in jumping sports such as basketball. The onset of pain is insidious, and an athlete may tolerate it for weeks before seeking medical advice. Although few runners can remember a particular moment or event when pain began, plantar fasciitis usually occurs after sudden increases in mileage, frequency of running, or running speed. Combined therapy, including relative rest, Achilles tendon stretching, medication, and heel cups, alleviates the pain in most athletes.

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.