Burden of disease, disability-adjusted life years and frailty prevalence.

BACKGROUND: Burden of disease (BoD) using disability-adjusted life years (DALY) is a useful summary measure of population health and estimates are provided for Ireland annually. We hypothesized that BoD may be used as a predictor of frailty prevalence. AIM: To examine the correlation between frailty measured by the accumulation of deficits (frailty index, FI) and Fried frailty phenotype (FFP) classifications and BoD, in an Irish context. DESIGN: Cross-sectional secondary analysis. METHODS: Data were obtained from waves two and three of The Survey of Health, Ageing and Retirement in Europe for Irish adults aged ≥65 in 2007. Frailty was defined by a 70-item FI and the FFP. Years lived with disability (YLD), years of life lost (YLL) and DALY were calculated using adapted equations from the World Health Organization and, where possible, disability weights, sequelae and durations as in the Global BoD (GBD) project (2016). RESULTS: Of 1035 participants, 442 were ≥65 years. Mean DALY were significantly higher in those identified as frail (FI: 3.31, P < 0.0001, n = 406; FFP: 2.46, P = 0.005, n = 319). For the FI, stronger correlation was found for DALY (r = 0.5431, P < 0.0001) than for age (r = 0.275, P < 0.0001). Controlling for confounders, DALY were an independent predictor of frailty when measured with the FI (OR 1.17, 95% CI: 1.10-1.24) but not with the FFP (OR 1.079, 95%% CI 1.00-1.17). CONCLUSIONS: Frailty correlates significantly with DALY, and more so with the FI than the FFP, reaffirming that these measures are different constructs. GBD data could represent a predictor of population-level frailty estimates, facilitating improved comparisons.

Bacterial mutagenicity screening in the pharmaceutical industry.

Genetic toxicity testing is used as an early surrogate for carcinogenicity testing. Genetic toxicity testing is also required by regulatory agencies to be conducted prior to initiation of first in human clinical trials and subsequent marketing for most small molecule pharmaceutical compounds. To reduce the chances of advancing mutagenic pharmaceutical candidates through the drug discovery and development processes, companies have focused on developing testing strategies to maximize hazard identification while minimizing resource expenditure due to late stage attrition. With a large number of testing options, consensus has not been reached on the best mutagenicity platform to use or on the best time to use a specific test to aid in the selection of drug candidates for development. Most companies use a process in which compounds are initially screened for mutagenicity early in drug development using tests that require only a few milligrams of compound and then follow those studies up with a more robust mutagenicity test prior to selecting a compound for full development. This review summarizes the current applications of bacterial mutagenicity assays utilized by pharmaceutical companies in early and late discovery programs. The initial impetus for this review was derived from a workshop on bacterial mutagenicity screening in the pharmaceutical industry presented at the 40th Annual Environmental Mutagen Society Meeting held in St. Louis, MO in October, 2009. However, included in this review are succinct summaries of use and interpretation of genetic toxicity assays, several mutagenicity assays that were not presented at the meeting, and updates to testing strategies resulting in current state-of the art description of best practices. In addition, here we discuss the advantages and liabilities of many broadly used mutagenicity screening platforms and strategies used by pharmaceutical companies. The sensitivity and specificity of these early mutagenicity screening assays using proprietary compounds and their concordance (predictivity) with the regulatory bacterial mutation test are discussed.

Report of the IWGT working group on strategy/interpretation for regulatory in vivo tests II. Identification of in vivo-only positive compounds in the bone marrow micronucleus test.

A survey conducted as part of an International Workshop on Genotoxicity Testing (IWGT) has identified a number of compounds that appear to be more readily detected in vivo than in vitro. The reasons for this property varies from compound to compound and includes metabolic differences; the influence of gut flora; higher exposures in vivo compared to in vitro; effects on pharmacology, in particular folate depletion or receptor kinase inhibition. It is possible that at least some of these compounds are detectable in vitro if a specific in vitro test is chosen as part of the test battery, but the 'correct' choice of test may not always be obvious when testing a compound of unknown genotoxicity. It is noted that many of the compounds identified in this study interfere with cell cycle kinetics and this can result in either aneugenicity or chromosome breakage. A decision tree is outlined as a guide for the evaluation of compounds that appear to be genotoxic agents in vivo but not in vitro. The regulatory implications of these findings are discussed.

Report of the IWGT working group on strategies and interpretation of regulatory in vivo tests I. Increases in micronucleated bone marrow cells in rodents that do not indicate genotoxic hazards.

In vivo genotoxicity tests play a pivotal role in genotoxicity testing batteries. They are used both to determine if potential genotoxicity observed in vitro is realised in vivo and to detect any genotoxic carcinogens that are poorly detected in vitro. It is recognised that individual in vivo genotoxicity tests have limited sensitivity but good specificity. Thus, a positive result from the established in vivo assays is taken as strong evidence for genotoxic carcinogenicity of the compound tested. However, there is a growing body of evidence that compound-related disturbances in the physiology of the rodents used in these assays can result in increases in micronucleated cells in the bone marrow that are not related to the intrinsic genotoxicity of the compound under test. For rodent bone marrow or peripheral blood micronucleus tests, these disturbances include changes in core body temperature (hypothermia and hyperthermia) and increases in erythropoiesis following prior toxicity to erythroblasts or by direct stimulation of cell division in these cells. This paper reviews relevant data from the literature and also previously unpublished data obtained from a questionnaire devised by the IWGT working group. Regulatory implications of these findings are discussed and flow diagrams have been provided to aid in interpretation and decision-making when such changes in physiology are suspected.

Lysis of whole blood in vitro causes DNA strand breaks in human lymphocytes.

DNA damage in lymphocytes, as measured by alkaline single cell gel electrophoresis (pH 12.7), is greatly increased by the concurrent lysis of whole blood in both freshly isolated samples and in PHA-stimulated cultures over a period of 7 days. Further, there is a marked progressive increase in DNA damage with time in PHA-stimulated lymphocytes cultured in whole blood even when the lymphocytes are separated before analysis; no such increase is seen in lymphocytes cultured alone. This indicates that there are components in whole blood that can cause DNA damage in lymphocytes, with granulocytes and lysis of red blood cells likely candidates. The DNA damage is greatly reduced in granulocyte-depleted whole blood cultures, but even in these significant increases are seen at later sampling times. Consequently, careful sample preparation is of paramount importance if the Comet assay is to be successfully used to assess DNA damage in human peripheral blood lymphocytes. Further, the progressive increase in DNA damage in whole blood cultures may influence other methods using lymphocytes for population biomonitoring and may be significant for in vitro genotoxicity testing.

Co-cultivation of CD4+ and CD8+ human T-cells leads to the appearance of CD4 cells expressing CD8 through de novo synthesis of the CD8 alpha-subunit.

The appearance of a population of dual-staining CD4+CD8+ cells in human T-lymphocyte cultures has been reported by various authors, including our own observation that they are always seen in simple phytohaemagglutinin-stimulated cultures from several different donors. The purpose of the present study was to investigate factors involved in the dual-staining (DS) phenotype, and to clarify some apparent inconsistencies between published observations. Our findings can be summarised as follows. 1. A population of DS CD4+CD8+ cells always appears in PHA-stimulated T-cell cultures if they contain both CD4 and CD8 subsets. The incidence of DS cells is related to PHA concentration, but other factors are involved since DS cells are not seen in PHA-stimulated cultures of purified CD4+ or CD8+ cells. Stimulation with PHA is not a prerequisite since very similar results are seen with ConA. 2. Direct physical contact between CD4+ and CD8+ cells is required for the appearance of the DS phenotype; soluble factors alone, including IL-4, appear nor to be responsible. 3. The DS phenotype in these conditions is always CD4+ cells weakly expressing CD8 and is a consequence of de novo synthesis of the CD8alpha molecule by the CD4+ cells.

The effect of PHA stimulation on lymphocyte sub-populations in whole-blood cultures.

Although lymphocytes in phytohaemagglutinin (PHA)-stimulated whole blood cultures are routinely used to assess genotoxin-induced chromosome damage, very little information is available on the effect of PHA on the various cell populations present, and there appear to be no data for the protocols used in routine genotoxicity assays. In this study, we used flow cytometric analysis to examine the size/complexity of the white blood cell (WBC) population and the expression of key antigenic markers by the lymphocytes over a 96-h period following PHA stimulation. The changes in the WBC population are complex, and would seem to represent different populations dying out, remaining static or starting to divide. The initial decrease seen in overall cell numbers probably reflects death of the neutrophil and monocyte populations. The subsequent increase in cell numbers appears to be due to division of the lymphocytes and, by 96 h post-stimulation, they comprise about half the total cell number and, as expected, > 90% are activated T-cells; it seems reasonable to assume that these represent the target cells in genotoxicity assays. Although we do not suggest that these findings should alter the routine conduct of clastogenicity assays using PHA-stimulated whole-blood cultures, they indicate that such tests are empirical and that closer investigation will only confirm their relatively imprecise nature.

Extended-term cultures of human T-lymphocytes: a practical alternative to primary human lymphocytes for use in genotoxicity testing.

A simplified method, using recombinant interleukin-2, foetal bovine serum and freeze-killed feeder cells, has been developed for the mass culture of T-lymphocytes derived from human peripheral blood. In this protocol, bulk cultures can be cryopreserved approximately 8 days after initiation, and subsequent mass cultures generated a further week after recovery. At the end of this period, the lymphocytes have maintained a normal karyotype and cultures from different donors are very similar in terms of rate of cell division and expression of key antigenic markers. Background micronucleus frequencies and dose-responses for micronucleus induction by a reference clastogen, hycanthone, were also very similar in all the cultures examined. Such extended-term T-lymphocyte cultures are potentially valuable in genotoxicity testing, providing cells with the normal human karyotype which can be characterised and handled with the practical convenience of established rodent cell lines.

Extended-term culture of human T lymphocytes: Material potentially useful in genotoxicity testing.

A simplified method, using recombinant interleukin-2, foetal bovine serum and freeze-killed feeder cells, has been developed for the mass culture of T lymphocytes derived from human peripheral blood. In this protocol, aliquots of bulk cultures can be cryopreserved about 8 days after initiation, and subsequent mass cultures generated a further week after recovery. At the end of this period the lymphocytes are karyotypically normal, and cultures from different donors are very similar in terms of rate of cell division and expression of key antigenic markers. Such extended-term T lymphocyte cultures are potentially valuable in genotoxicity testing, combining the practical convenience of established cell lines with the theoretical advantage of possession of the normal human karyotype.

Formaldehyde is a bacterial mutagen in a range of Salmonella and Escherichia indicator strains.

Formaldehyde was examined for bacterial mutagenicity using Escherichia coli WP2(pKM101) and WP2uvrA(pKM101), and Salmonella typhimurium TA1535, TA1537, TA1538, TA98, TA100 and TA102, in the absence of any exogenous source of metabolic activation. Using pre-incubation exposure, clear mutagenicity was seen for TA98, TA100 and TA102, and both E. coli strains. In standard plate-incorporation assays, consistent mutagenicity was seen only for TA100 and WP2uvrA(pKM101). No evidence of mutagenicity was seen for TA1535, TA1537 or TA1538 using either method of exposure. These data confirm the enhanced ability of the pre-incubation method to detect the mutagenicity of formaldehyde both quantitatively, as expressed by numbers of revertant colonies, and qualitatively, in terms of the range of indicator strains reverted. The relatively greater sensitivity of the pre-incubation assay is probably due to better containment of a volatile agent and/or lack of interaction with agar during the initial period of exposure. The findings are consistent with the suggestion that formaldehyde induces lesions in bacteria which are, at least to some extent, excision-repairable, and indicate that the presence of the R-factor plasmid may be required for the expression of its mutagenicity in excision repair-deficient Salmonella.

An improved procedure for the in vitro expansion of human T-lymphocyte clones for mutant analysis.

Assays detecting mutants present in human peripheral blood T-lymphocytes have been developed to monitor the population for somatic mutations. In order to investigate the nature of the mutations, colonies are further expanded in vitro by repeated lectin stimulation. To characterise fully each mutant clone, sufficient cells (approximately 10(7)) must be available for several molecular and biochemical techniques to be employed. These techniques, and their importance to the assay for population monitoring, are discussed briefly. We report here that the expansion of mutant colonies to approximately 10(7) cells by repeated lectin stimulation is not effective for all T-cell clones but that an alternative "lectin free" expansion method has enabled us to expand all the clones tested from a variety of normal donors and other individuals of interest.

Sensitivity of Salmonella typhimurium TA97a to the type of agar used for preparation of Vogel-Bonner plates.

Recent problems with the supply of Difco bacto agar have forced some laboratories to evaluate alternative agars for use in the Salmonella/microsome assay. This led to the independent observation in two laboratories (Boots and Glaxo) that Salmonella typhimurium TA97a is sensitive to certain types of agar that may be used to prepare Vogel-Bonner minimal medium plates. A programme of work was, therefore, undertaken to investigate this phenomenon; 9-aminoacridine hydrochloride (at Boots) and 4-nitro-o-phenylenediamine (at Glaxo) were tested against TA1537 and TA97a using Vogel-Bonner plates prepared with a number of different agars. Three agars (Lab M, Difco Bi-tek and Beckton Dickinson granulated) were identified which, although supporting normal growth of TA1537 revertant colonies, gave much reduced control counts and responses to the mutagens with TA97a. One agar, Becton Dickinson grade A, gave poor responses with TA1537 but produced satisfactory results with TA97a. In contrast to the Vogel-Bonner plates, varying the type of agar used in the top agar overlays had little effect on the responses obtained. On the basis of these comparisons, Becton Dickinson purified agar was selected as a suitable alternative to Difco bacto and it was concluded that laboratories using agars other than these, or purchasing pre-poured plates without specifying the type of agar, should be made aware of potential problems with TA97a.

Heat shock proteins in cultured human keratinocytes and fibroblasts.

Heat shock induces in cells the synthesis of specific proteins called heat-shock proteins. We have compared the induction of these proteins in human keratinocytes, skin fibroblasts, and a human epithelial tumor cell line following exposure to weak and strong inducing agents (heat, cadmium sulphate, and sodium arsenite). The induction of heat shock proteins was measured in cells by one-dimensional gel electrophoresis of [35S] methionine-labeled proteins and by immunofluorescence using a specific HSP72 monoclonal antibody. Both HSP90 and HSP116 were constitutively expressed in these cell types. Exposure of these cells to weak inducing agents such as heat or cadmium sulphate resulted in the synthesis of HSP72 and HSP90, whereas HSP28 and HSP116 synthesis was detected in keratinocytes and fibroblasts following exposure to the strong inducing agent sodium arsenite. In addition, sodium arsenite induced the synthesis of HSP46 in human keratinocytes. Immunofluorescence demonstrated a rapid and reversible accumulation of the 72-kD heat shock protein within the nucleolus of heat-stressed human keratinocytes and fibroblasts.

In vitro reconstruction of human skin: The use of skin equivalents as potential indicators of cutaneous toxicity.

A three-dimensional reconstruction of living skin-the skin equivalent-has been modified to accept materials that can be applied topically to the skin. Using an irritant gel containing 10% benzoyl peroxide, changes in the skin equivalent model were investigated. Histologically, epidermal necrosis and vacuolar change were observed within 4-6 hr after topical application. Using skin equivalents prelabelled with [(3)H]arachidonic acid, studies involving direct measurement using HPLC and radioimmunoassay have indicated the release of potent lipoxygenase products, such as leukotriene B(4) and 15-hydroxyeicosa-tetraenoic acid. These preliminary results suggest that the skin equivalent may prove to be a useful in vitro model for the prediction of cutaneous toxicity of topically applied substances.

1,8-Dinitropyrene: comparative mutagenicity in Chinese hamster V79 and CHO cells.

1,8-Dinitropyrene (1,8-DNP) was clearly mutagenic at the hprt locus in CHO cells, but not detectably mutagenic in V79 cells, following a 3-h treatment period. Preliminary data indicate that CHO, but not V79, cells have measurable levels of N-acetyltransferase activity, and this may contribute to the differential sensitivity of the two cell lines to the mutagenicity of 1,8-DNP.

The comparative responses of Salmonella typhimurium TA1537 and TA97a to a range of reference mutagens and novel compounds.

Salmonella typhimurium TA97a was added to the set of indicator strains routinely used for the Ames test in this laboratory (TA1535, TA1537, TA1538, TA98 and TA100) for a trial period during which a total of approximately 40 reference mutagens and novel pharmaceutical compounds were examined. The conclusions from this trial were as follows: (i) there are agents mutagenic for TA1537 which are not detected by TA97a. (ii) except for agents requiring the R factor plasmid, TA97a shows no increased sensitivity to mutagens when compared with TA1537 and (iii) nearly all the limited published database is for the original isolate, TA97, rather than TA97a, and the results obtained here indicate significant differences in response between the two; TA97a remains, therefore, essentially unvalidated.

DNA alkylations and mutation after exposure to ethyl methanesulphonate in mammalian cell lines routinely used in mutagenicity testing.

DNA ethylations were measured in four mammalian cell lines, Chinese hamster ovary CHO, mouse lymphoma L5178Y t k+/-, human lymphoblastoid TK6 and Chinese hamster V79, following exposure to [3H]ethyl methanesulphonate. Concurrent estimates of cytotoxicity and gene mutation were also carried out. Total DNA-binding and relative levels of ethylation at N-7 guanine (N-7G), O6 guanine (O6G) and N-3 adenine (N-3A) were essentially the same in all four cell lines. Differences in response to EMS between the cell lines, namely the greater cytotoxicity in TK6 cells, would therefore appear to reflect subsequent handling of the DNA lesions, rather than different levels of DNA ethylation in the cell lines.

Determination of benzidine--DNA adduct formation in CHO, HeLa, L5178Y, TK6 and V79 cells.

Using 32P-postlabelling, evidence of DNA adduct formation was sought in six mammalian cell lines, namely Chinese hamster ovary (CHO), human cervical carcinoma (HeLa S3), mouse lymphoma L5178Y tk +/- and L5178Y wild-type, human lymphoblastoid TK6 and Chinese hamster V79, following treatment with benzidine (BZD) in the presence of S-9. Adduct formation was also determined in calf thymus DNA reacted in vitro with N-hydroxy-N'-acetyl-BZD, and in liver DNA from mice given a single intraperitoneal injection of BZD. DNA adducts were detected in the calf thymus DNA sample and in mouse liver DNA, but not in DNA from any of the six cell lines. The absence of adduct formation is consistent with the lack of mutagenicity of BZD in CHO, and V79 and in L5178Y cells at the hprt locus, and in TK6 cells at the tk and hprt loci. These results also suggest that the observed mutagenicity of BZD at the tk locus in L5178Y cells may be due to a mechanism(s) not involving covalent binding to DNA.