Improving recognition and management of acute kidney injury.

Acute kidney injury (AKI) is currently suboptimally recognised and managed in the UK, despite its association with significant patient morbidity, mortality and consequent implications for healthcare economics. Our prospective study, performed in a large urban London hospital, demonstrated that the introduction of a specially designed care bundle can significantly improve documentation of baseline creatinine, assessment and optimisation of fluid status, performance of urine dip, withholding of nephrotoxic drugs, appropriate monitoring of urine output, prescription of renal drug doses, and appropriate consideration of a renal ultrasound and urinary protein-creatinine ratio. Improved compliance of appropriate investigations and initial treatments translated to decreased requirement for intensive care admission and a trend towards shorter length of stays.

Blood gene expression signatures predict exposure levels.

To respond to potential adverse exposures properly, health care providers need accurate indicators of exposure levels. The indicators are particularly important in the case of acetaminophen (APAP) intoxication, the leading cause of liver failure in the U.S. We hypothesized that gene expression patterns derived from blood cells would provide useful indicators of acute exposure levels. To test this hypothesis, we used a blood gene expression data set from rats exposed to APAP to train classifiers in two prediction algorithms and to extract patterns for prediction using a profiling algorithm. Prediction accuracy was tested on a blinded, independent rat blood test data set and ranged from 88.9% to 95.8%. Genomic markers outperformed predictions based on traditional clinical parameters. The expression profiles of the predictor genes from the patterns extracted from the blood exhibited remarkable (97% accuracy) transtissue APAP exposure prediction when liver gene expression data were used as a test set. Analysis of human samples revealed separation of APAP-intoxicated patients from control individuals based on blood expression levels of human orthologs of the rat discriminatory genes. The major biological signal in the discriminating genes was activation of an inflammatory response after exposure to toxic doses of APAP. These results support the hypothesis that gene expression data from peripheral blood cells can provide valuable information about exposure levels, well before liver damage is detected by classical parameters. It also supports the potential use of genomic markers in the blood as surrogates for clinical markers of potential acute liver damage.

Murine leukemia virus: restriction in fused permissive and nonpermissive cells.

Cultures of human cells nonpermissive for mouse leukemia virus replication could not be induced to support virus replication by homologous fusion in the presence of Moloney leukemia virus. Human cells were also fused with permissive mouse cells, and the fate of the virus in heterokaryons was determined by a simultaneous autoradiography and fluorescent antibody technique. Heterokaryons containing the full chromosome complement of both cells were likewise nonpermissive for virus synthesis, but hybrids of human and mouse cells, which lacked up to half of the human chromosome complement, were permissive for virus synthesis. The results suggest that human cell genes can direct a repressive control over mouse leukemia virus replication.

Prediction of chemical carcinogenicity in rodents from in vitro genetic toxicity assays.

Four widely used in vitro assays for genetic toxicity were evaluated for their ability to predict the carcinogenicity of selected chemicals in rodents. These assays were mutagenesis in Salmonella and mouse lymphoma cells and chromosome aberrations and sister chromatid exchanges in Chinese hamster ovary cells. Seventy-three chemicals recently tested in 2-year carcinogenicity studies conducted by the National Cancer Institute and the National Toxicology Program were used in this evaluation. Test results from the four in vitro assays did not show significant differences in individual concordance with the rodent carcinogenicity results; the concordance of each assay was approximately 60 percent. Within the limits of this study there was no evidence of complementarity among the four assays, and no battery of tests constructed from these assays improved substantially on the overall performance of the Salmonella assay. The in vitro assays which represented a range of three cell types and four end points did show substantial agreement among themselves, indicating that chemicals positive in one in vitro assay tended to be positive in the other in vitro assays.

Factors influencing hospital readmission rates after acute medical treatment.

It is a concern that increasing pressure to diagnose, treat and discharge patients rapidly is leading to unacceptably high readmission rates. Readmissions were studied over a two-month period. Patients were identified through the hospital coding system, and electronic discharge summaries provided details of each admission. In total, 69 readmissions were identified, representing 4.34% of medical admissions. Readmitted patients were older than those with single admissions (median age 75 and 71 years, respectively; p < 0.05). Initial length of stay was greater in those patients who would go on to be readmitted (median six days; single admission, two days; p < 0.0001). Seventy-one per cent of readmissions were deemed avoidable, with discharge before conclusive therapy being the leading factor implicated (56%). Readmission is more likely in older patients with complex care needs. Rapid throughput of patients is not associated with readmission. The majority of readmissions can potentially be avoided with judicious medical care.

Estimating the proportion of young adults on antihypertensive treatment that have been correctly diagnosed.

This paper aims to identify how many young adults on antihypertensive treatment have been misclassified as hypertensive. We identified subjects aged under 35 on antihypertensive treatment, from the Health Surveys for England, 1998-2004. Pretreatment systolic and diastolic blood pressures were calculated by adjusting on-treatment blood pressures for the effects of treatment. Treatment effects were derived from meta-analysis. Subjects were classified as hypertensive if pretreatment blood pressure was >or=160/100 mm Hg, or was >or=140/90 mm Hg in conjunction with high cardiovascular risk. We then identified the proportion of treated subjects on antihypertensive treatment who were truly eligible for treatment. From the survey data we identified 65 adults (25 men and 40 women) under 35 on diuretics, beta-blockers, angiotensin converting enzyme inhibitors, calcium blockers or other antihypertensives. Average pretreatment blood pressure was 164/100 mm Hg in those eligible for treatment, and 136/79 mm Hg in those not eligible. The analysis indicated that 29.2% of adults aged 16-34 (95% confidence interval (CI): 18.6-41.8%) were truly eligible for antihypertensive treatment: 32.0% (95% CI: 14.9-53.5%) of men and 25.0% (95% CI: 12.7-41.2%) of women. A total of 73.7% (14 of 19) of subjects eligible and 41.3% (19 of 46) of subjects not eligible for treatment either had a body mass index >30 kg m(-2) or kidney disease (chi(2)-test P=0.018). Because of biological variation in blood pressure, most young adults on treatment for hypertension have been misclassified as hypertensive. Most who have been correctly diagnosed are either clinically obese or have kidney disease.

Effect of the Fv-1 locus in vivo: host range pseudotypes of murine sarcoma virus.

Fv-1-specific host-range pseudotypes of murine sarcoma virus (MuSV) were developed by rescue from nonproducer cells with N- or B-tropic leukemia viruses. The MuSV(B) and MuSV(N) pseudotype viruses were tested in vitro and were restricted specifically by the Fv-1 gene locus. When the pseudo-type viruses were tested in vivo in mice of specific Fv-1 geno-types, tumor induction was completely restricted in nonpermissive animals, including athymic nude mice, whereas tumors grew progressively in permissive animals. All tumors regressed in adult heterozygous mice, but not in adult athymic nude mice.

Effects of nitrosocarbaryl on BALB/3T3 cells.

Carbaryl(N-methyl-1-naphthylcarbamate) and its nitrosated product, N-nitrosocarbaryl, were tested for their effects of BALB/3T3 (clone A31) cells in culture. Nitrosocarbaryl, but not carbaryl, caused transformation of the BALB/3T3 fibroblasts, but neither chemical induced the complete expression of endogenous murine leukemia virus. Transformed cells differed from the parental control cells by loss of contact inhibition, change in morphology, growth in soft agar, growth to higher saturation densities, and tumorigenicity in normal newborn and irradiated weanling mice and athymic (nude) mice. Transformed clones were found to be negative for expression of RNA tumor virus antigens, viral reverse transcriptase, and infectious virus. Thus, it appears that nitrosocarbaryl can transform BALB/3T3 cells to tumorigenic cells with altered biological properties but without complete activation of RNA tumor viruses in the transformed cells. Expression of viral antigen in the transformed cells was inducible by iododeoxyuridine, indicating that the endogenous viral genome was retained in an unexpressed state.

Genetic alterations cooperate with v-Ha-ras to accelerate multistage carcinogenesis in TG.AC transgenic mouse skin.

TG.AC transgenic mice harbor a v-Ha-ras transgene and retain two normal c-Ha-ras alleles and are susceptible to skin tumor formation by 12-O-tetradecanoylphorbol-13-acetate (TPA). To determine whether normal c-Ha-ras antagonizes the oncogenic potential of the v-Ha-ras transgene and/or whether additional non-Ha-ras 7,12-dimethylbenz(a)anthracene (DMBA) initiation target genes exist in mouse skin, which could cooperate with v-Ha-ras to increase the frequency of initiation, rate of promotion, or risk of malignant conversion, we treated TG.AC mouse skin with a single subthreshold dose of DMBA. This was followed by limited TPA or diacylglycerol promotion to select for cells with additional genetic alterations over those cells containing the v-Ha-ras transgene only. DMBA-treated/TPA-promoted TG.AC mice demonstrated a 10-fold increase in the average number of papillomas per mouse, a greater incidence of papilloma bearing-mice, and an increased papilloma growth rate when compared to acetone-treated/TPA-promoted TG.AC mice. These profound changes in papilloma frequency and growth occurred in the absence of the characteristic DMBA-induced A182-->T mutation in c-Ha-ras and immunohistochemical nuclear staining for p53 protein. DMBA-treated/acetone-promoted TG.AC mice did not develop any tumors. Limited promotion with the model diacylglycerol, sn-1,2-didecanoylglycerol, similarly produced an average of 10-fold more papillomas in DMBA-treated mice than in acetone-treated/sn-1,2-didecanoylglycerol-promoted TG.AC mice. DMBA-treated/TPA-promoted TG.AC mice developed their first malignancy by 16 weeks, and by 30 weeks, 50% of the mice developed malignancies, whereas no malignancies were observed in acetone-treated/TPA-promoted TG.AC mice. These results indicate that there exist unidentified DMBA initiation target genes in TG.AC mouse skin that cooperate with mutant Ha-ras to increase papilloma frequency, growth, and malignant conversion, and that promoter treatment can influence malignant conversion by selecting for cells with multiple genetic alterations.

Induction of retrovirus gene expression in mouse cells by some chemical mutagens.

Cell cultures derived from a variety of mouse strains were compared for their relative capacity to be induced to express endogenous retrovirus proteins by exposure to 5-iododeoxyuridine under optimized experimental conditions. Induction frequencies varied between 6.0 x 10(-1) and 1.7 x 10(-2) with AKR cells showing the highest capacity and C57BL/6 x C3H F1 cells the lowest. Virus expression was induced in AKR cells with other chemical mutagens of the polyaromatic hydrocarbon [benzo(a)pyrene and phenol, diol, and epoxide metabolites] and nucleoside analog classes, but alkylating agents were inconsistent or failed to induce. Considerable differences in the efficiency of induction were seen between various halogenated nucleosides, while under these conditions the nucleoside 5-azacytidine induced greater than 90% of AKR cells at a concentration of 2 to 4 micrograms/ml. The high frequency of induction by 5-azacytidine, relative to other nucleoside analogs, and the absence of induction by other mutagens further indicate that endogenous virus induction occurs via nonmutagenic mechanisms and that some mutagens may also affect regulatory functions independent of their mutagenic action.

In vitro transformation of Syrian hamster epidermal cells by N-methyl-N'-nitro-N-nitrosoguanidine.

The selection of Syrian hamster epidermal cells which do not terminally differentiate has provided a quantitative focus assay for in vitro chemical transformation. One-day-old Syrian hamster epidermal cells plated at 5 x 10(6)/100-mm dish were treated for 5 hr with various concentrations of N-methyl-N'-nitro-N-nitrosoguanidine. After 4 weeks, the normal epidermal cells began to terminally differentiate to keratinized squamous cells and died, but transformed epidermal colonies grew to higher cells densities and appeared as darker areas against a lightly stained normal cell background. Transformed epidermal foci were isolated and subcultured for at least 15 passages, whereas normal epidermal cells could not be subcultured under the same conditions. The transformed cells assumed the typical cobblestone-like morphology of epithelial cells, retained desmosomes and tonofilaments, and were able to use citrulline in place of arginine. Argininosuccinate synthetase (EC 6.3.4.5) activity was significantly higher in the epidermal cells than in fibroblasts. The injection of 5 x 10(6) cells of two transformed epidermal cell lines into athymic nude mice resulted in the formation of tumors which were identified as keratinizing squamous carcinomas.

Photocarcinogenesis and susceptibility to UV radiation in the v-Ha-ras transgenic Tg.AC mouse.

The v-Ha-ras transgenic Tg.AC mouse line has proven to be a useful model for the study of chemical carcinogenic potential. We undertook experiments designed to study the effect of the physical carcinogen, UV radiation, on tumorigenesis in this mouse strain. Following a total of three exposures on alternating days to a radiation source covering a cumulative UVR exposure range of 2.6-42.6 kJ per m2, squamous papillomas developed by 4 wk after initial exposure in a dose-dependent manner. Malignancies developed within 18-30 wk following the initial UVR exposure and were all diagnosed as squamous cell carcinoma or spindle cell tumors. In contrast to other mouse stains used in photocarcinogenesis studies, few p53 mutations were found in Tg.AC malignancies upon polymerase chain reaction-single stranded conformational polymorphism analysis of exons 4-8 followed by sequencing of suspicious bands; however, all tumors analyzed by in situ hybridization expressed the v-Ha-ras transgene. Immunohistochemical analysis of UVR-exposed skin taken 24 h after the last of three exposures (13.1 kJ per m2 total UVR) showed expression of p53 in hair follicles and in interfollicular epidermis, which indicates that the gene was functional. Thus, although there are some differences between the Tg.AC and other mouse models, these results suggest that the Tg.AC mouse may be a useful model for the study of acute exposure photocarcinogenesis.

Immunofluorescent analysis of murine leukemia virus-infected cells by flow microfluorometry.

Flow microfluorometric assay with a Cytofluorograf model 4801 in combination with immunofluorescence was applied to the quantitative assay of cells exogenously infected with mouse leukemia viruses and to the chemical induction of virus in AKR cells. The Cytofluorograf provides sensitivity equal to the visual method and is capable of assaying up to 5000 cells/sec with specificity equivalent to that of the direct visual immunofluorescence assay, thereby providing a large, statistically valid sampling in a fraction of the time required by visual counting. Flow microfluorometry also provides a method of quantitatively resolving fluorescent cell populations on the basis of relative size and degree of fluorescence.

In vitro quantitation of lethal and physiologic effects of total body irradiation on stromal and hematopoietic stem cells in continuous bone marrow cultures from Rf mice.

The role of stromal-supportive cells in hematopoietic stem cell responses to irradiation is poorly understood. The effects of in vivo total body irradiation (TBI) and interval from TBI to explant of marrow on: stromal cell proliferation in vitro; stromal cell support of hematopoiesis in continuous bone marrow culture; and generation of WEHI-3 growth factor (GF)-dependent lines of hematopoietic progenitor cells were evaluated. Continuous marrow cultures from non-irradiated control RfM/UN, C57BL/6J, C3H/HeJ, and N:NIH (Swiss) mice generated pluripotential hematopoietic stem cells (CFUs) and committed granulocyte-macrophage progenitor cells (GM-CFUc) for over 20 weeks. Explant of marrow at 2, 4, 5, or 6 months after single fraction TBI (300-800 rad) was associated with decreased longevity of hemopoiesis (2-12 weeks), and a decrease in the proliferative capacity of fibroblastic adherent-stromal colony forming cells (CFUf) as measured by colony size at 14 days and number of colonies per 10(6) cells plated. In contrast, explant of marrow 8 to 24 months after TBI produced cultures with longevity that was indistinguishable from age-matched control cultures (19-24 weeks). Marrow from irradiated first and second generation recipients of serially transferred marrow demonstrated a similar 7-month in vivo recovery period; however, the plateau maximum duration of hemopoiesis did not return to control levels. Purified stromal cell cultures were prepared by corticosteroid-deprivation of explanted marrow for 28 days and were then engrafted in vitro with marrow from C57BL/6J or RfM/UN mice that had been irradiated 1 month previously. Hemopoiesis in these cultures was restored, and they produced GM-CFUc and granulocytes for 15-24 weeks. Thus, healthy stroma supported growth of recently irradiated hemopoietic cells in vitro. Nonadherent cells removed from the above continuous marrow cultures generated clonal non-leukemogenic WEHI-3 GF-dependent hemopoietic progenitor cell lines with a frequency concordant with radiation effects on culture longevity, and this was increased by the presence of purified healthy stromal cultures. Indirect effects of x-irradiation on hemopoietic stem cells through damage and repair in the stromal cell compartment can be effectively studied with the present bone marrow culture system.

Morphological transformation of Syrian hamster embryo cells by mezerein.

Mezerein (MEZ) has been described as a weak complete tumor promoter but an effective stage II promoter in the mouse skin initiation-promotion tumor model. In this study MEZ produced a strong transformation response when tested under code in the Syrian hamster embryo (SHE) clonal morphological transformation assay. Using a standard 7-day exposure protocol designed to detect complete carcinogens, MEZ was active at non-toxic concentrations, producing a linear response between 0.3-10 ng/ml in a log-log plot of transformation activity versus concentration. These concentrations were in the same range as those which had been shown to elicit promotion activity in several in vitro cell culture systems. Our data suggest that the SHE assay has the ability to detect some tumor promoters under the same conditions used to test for complete carcinogens. The possibility that MEZ may possess in vivo carcinogenic activity cannot be excluded.

Evaluation and validation issues in the development of transgenic mouse carcinogenicity bioassays.

Transgenic mouse models have emerged as plausible alternatives to long-term bioassays for carcinogenicity. Three transgenic lines evaluated to date have shown a clear capability to discriminate between carcinogens and noncarcinogens, using long-term bioassay results as the standard. The data also suggest that the transgenic lines will not fully duplicate long-term bioassay results. It is proposed that these models do not respond to chemicals that have induced highly restricted species or strain-specific tumor responses in mice or rats. Rather, the value of the transgenic models is predicated on a preferential response to transspecies carcinogens (i.e., those positive in both rats and mice, often including tumors in the same tissues). Thus, although results in transgenic models may not be completely concordant with long-term bioassays, the data can be used effectively in chemical and drug safety assessments. Further, it is proposed that validation of the models is readily achievable via ongoing studies. Validation of any alternative model is best achieved by sufficient mechanistic understanding of the model to reasonably predict the outcome of bioassays conducted in the models and use all available information on the drug or chemical. This goal can now be met with the transgenic mouse lines.

The genetic toxicity database of the National Toxicology Program: evaluation of the relationships between genetic toxicity and carcinogenicity.

The database of the U.S. National Toxicology Program has been developed over approximately two decades, principally focused on substances evaluated for carcinogenicity in rodent bioassays. These assays generally provide data on the relative toxicity and carcinogenicity of chemicals based upon discrete subchronic (13 week) and chronic (104 week) exposures. A major value of these data are that the assay protocols, rodent strains, and technical methodologies have been generally consistent, thus permitting comparisons between assays and chemicals. The genotoxicity data for many of the same chemicals have been developed also using standardized biological systems and protocols. Data for assays including mutagenicity in Salmonella and mouse lymphoma cells, chromosomal aberrations, and sister chromatid exchange in Chinese hamster ovary cells, transformation of Balb/c 3T3 cells, and in vivo cytogenetic effects in rodents have been compiled for many chemicals. The results of all of these assays provide a substantial database for evaluating chemical effects and for defining the complex relationships between mutagenicity and carcinogenicity.

A perspective on nonmutagenic mechanisms in carcinogenesis.

Although there is compelling evidence for multiple mutagenic events in the induction of cancers, there is also substantial evidence in support of nonmutagenic mechanisms. It is proposed that the genetic basis of noninduced or spontaneous tumors, as well as cancers induced by nonmutagens, involves heritable changes in the regulation of gene expression.

Predictions for the outcome of rodent carcinogenicity bioassays: identification of trans-species carcinogens and noncarcinogens.

Thirty chemicals or substances currently undergoing long-term carcinogenicity bioassays in rodents have been used in a project to further evaluate methods and information that may have the capability of predicting potential carcinogens. In our predictions the principal information used includes structural alerts and in vitro test results for Salmonella mutagenicity, relative subchronic toxicity, and the sites and types of pathology found in subchronic (90-day) studies. This group of chemicals differs significantly from those used previously to evaluate predictive methods in that 23 of 30 are defined as nonmutagenic by conventional criteria. The goal of this predictive effort is to identify categorically the chemicals that have the capacity to induce cancers in both rats and mice (trans-species carcinogens) and those that are not carcinogenic in either rats or mice. Chemicals that show properties that may be associated with tumor induction in either species, i.e., species-specific cancers, are categorized as being of "uncertain predictability." This category includes chemicals believed to have limited carcinogenic potential that is manifested principally as a consequence of the genetic background of the test strain of inbred rodent.

Genetic toxicology: current status of methods of carcinogen identification.

A critical aspect of the efforts to relate the results of short-term genetic toxicity tests with those from long-term rodent tests for carcinogens is the quality and consistency of the studies conducted by the National Toxicology Program. Analysis of the results in relationship to chemical structure has shown that mutagenic potential is a primary risk factor for carcinogen identification. Chemicals positive in the Salmonella assay-generally possess "structural alerts" for electrophilic interactions, are predominantly represented among chemicals producing trans-species carcinogenic effects in rodents, and among those identified as carcinogenic to humans. Current efforts are aimed at defining toxicological, structural, and mechanistic properties of nonmutagens that are carcinogenic in rodents.