Microarray gene expression profiling of a human glioblastoma cell line exposed in vitro to a 1.9 GHz pulse-modulated radiofrequency field.

The widespread use of mobile phones has led to public concerns about the health effects associated with exposure to radiofrequency (RF) fields. The paramount concern of most persons relates to the potential of these fields to cause cancer. Unlike ionizing radiation, RF fields used for mobile telecommunications (800-1900 MHz) do not possess sufficient energy to directly damage DNA. Most rodent bioassay and in vitro genotoxicity/mutation studies have reported that RF fields at non-thermal levels have no direct mutagenic, genotoxic or carcinogenic effects. However, some evidence has suggested that RF fields may cause detectable postexposure changes in gene expression. Therefore, the purpose of this study was to assess the ability of exposure to a 1.9 GHz pulse-modulated RF field for 4 h at specific absorption rates (SARs) of 0.1, 1.0 and 10.0 W/kg to affect global gene expression in U87MG glioblastoma cells. We found no evidence that non-thermal RF fields can affect gene expression in cultured U87MG cells relative to the nonirradiated control groups, whereas exposure to heat shock at 43 degrees C for 1 h up-regulated a number of typical stress-responsive genes in the positive control group. Future studies will assess the effect of RF fields on other cell lines and on gene expression in the mouse brain after in vivo exposure.

Epithelial and fibroblast cell lines cultured from the transgenic BigBlue rat: an in vitro mutagenesis assay.

We have isolated, cultured, and immortalised three new BigBlue transgenic rat cell lines for the study of mutation induction in vitro. The two epithelial cell lines, from the mammary gland and oral cavity, were designated BBR/ME and BBR/OE, respectively, and the third is a mammary fibroblast line designated BBR/MFib. We have characterised these cell lines with respect to chromosome number and the expression of some cell-specific antigens. The clonogenic survival and cII transgene mutation induction responses of these three cell lines to N-ethyl-N-nitrosourea (ENU) treatment were determined. Both epithelial cell lines were much more sensitive to ENU toxicity than was the fibroblast cell line. However, all cell lines showed similar ENU dose-dependent increases in mutant frequency. We hope that cell lines such as these will extend the power of the BigBlue assay to in vitro studies.

Survival analysis for success of Molteno tube implants.

AIM: To apply survival analysis in assessing the long term outcome of Molteno tube implantation and to identify risk factors for failure. METHODS: A retrospective, 10 year, consecutive case series study of 119 eyes that underwent implantation of a Molteno tube. The main outcome measures considered were intraocular pressure (IOP), visual acuity, and complications. RESULTS: A 30% or greater reduction in IOP was achieved in 68.9% of cases. However, the overall, "complete success" rate (IOP <22 mm Hg with no medications) after a mean (SD) follow up period of 43 (33) months (range 6-120) was only 33.6% despite a fall in mean (SD) IOP from 38.2 (8.2) mm Hg to 20.1 (11.0) mm Hg. The "qualified success" rate (IOP <22 mm Hg with or without medications) was 60.5%. Failure was most common in the first postoperative year but could occur after several years, the survival curve having an exponential shape. The only statistically significant risk factor for failure identified was pseudophakia, although eyes with neovascular glaucoma tended to fare poorly. Postoperative IOP tended to be lower after double plate than after single plate implantation. There was no significant difference in outcome based on age, sex, race, previous penetrating keratoplasty, or previous conjunctival surgery. CONCLUSIONS: In eyes at high risk of trabeculectomy failure, implantation of an aqueous shunt device should be considered. Pseudophakia should be considered an additional risk factor for failure. Early failure appeared relatively more common but long term follow up of all cases is recommended to ensure adequate management of late failures.

IPCS guidelines for the monitoring of genotoxic effects of carcinogens in humans. International Programme on Chemical Safety.

The purpose of these guidelines is to provide concise guidance on the planning, performing and interpretation of studies to monitor groups or individuals exposed to genotoxic agents. Most human carcinogens are genotoxic but not all genotoxic agents have been shown to be carcinogenic in humans. Although the main interest in these studies is due to the association of genotoxicity with carcinogenicity, there is also an inherent interest in monitoring human genotoxicity independently of cancer as an endpoint. The most often studied genotoxicity endpoints have been selected for inclusion in this document and they are structural and numerical chromosomal aberrations assessed using cytogenetic methods (classical chromosomal aberration analysis (CA), fluorescence in situ hybridisation (FISH), micronuclei (MN)); DNA damage (adducts, strand breaks, crosslinking, alkali-labile sites) assessed using bio-chemical/electrophoretic assays or sister chromatid exchanges (SCE); protein adducts; and hypoxanthine-guanine phosphoribosyltransferase (HPRT) mutations. The document does not consider germ cells or gene mutation assays other than HPRT or markers of oxidative stress, which have been applied on a more limited scale.

In vivo transgenic mutation assays.

Transgenic rodent gene mutation models provide quick and statistically reliable assays for mutations in the DNA from any tissue. For regulatory applications, assays should be based on neutral genes, be generally available in several laboratories, and be readily transferable. Five or fewer repeated treatments are inadequate to conclude that a compound is negative but more than 90 daily treatments may risk complications. A sampling time of 35 days is suitable for most tissues and chemicals, while shorter sampling times might be appropriate for highly proliferative tissues. For phage-based assays, 5 to 10 animals per group should be analyzed, assuming a spontaneous mutant frequency (MF) of approximately 3 x 10(-5) mutants/locus and 125,000-300,000 plaque or colony forming units (PFU or CFU) per tissue. Data should be generated for two dose groups but three should be treated, at the maximum tolerated dose (MTD), two-thirds the MTD, and one-third the MTD. Concurrent positive control animals are only necessary during validation, but positive control DNA must be included in each plating. Tissues should be processed and analyzed in a block design and the total number of PFUs or CFUs and the MF for each tissue and animal reported. Sequencing data would not normally be required but might provide useful additional information in specific circumstances. Statistical tests used should consider the animal as the experimental unit. Nonparametric statistical tests are recommended. A positive result is a statistically significant dose-response and/or statistically significant increase in any dose group compared to concurrent negative controls using an appropriate statistical model. A negative result is statistically nonsignificant with all mean MF within two standard deviations of the control.

Evidence for the lack of base-change and small-deletion mutation induction by trichloroethylene in lacZ transgenic mice.

Trichloroethylene (TCE) is a widely used industrial solvent employed mainly for degreasing and cold-cleaning metal parts. It is also used for dry cleaning, and in the production of a number of chemical products. It has been shown to induce liver and lung tumors in rodents, and have a variety of positive and negative results using in vitro and in vivo mutagenicity tests. In order to assist in the interpretation of the mechanism of carcinogenicity, TCE was tested for the ability to induce gene mutations and small deletions using the lacZ transgenic mouse model (MutaMouse). Male and female animals were exposed by inhalation to 0, 203, 1153, and 3141 ppm TCE, 6 h per day for 12 days. 14 and 60 days following the last exposure, animals were sacrificed and the mutation frequency in bone marrow, kidney, spleen, liver, lung, and testicular germ cells determined. The results of this study indicate that TCE did not induce base-change or small-deletion mutations as detected in this assay in any of the tissues examined. Environ. Mol. Mutagen. 34: 190-194, 1999. Published 1999 Wiley-Liss, Inc.

Statistical analysis of lacZ mutant frequency data from MutaMouse mutagenicity assays.

Transgenic mouse assays have provided an unprecedented opportunity to study mutagenesis in diverse rodent tissues. In this article data from MutaMouse mutagenicity assays based on the Escherichia coli gene lacZ were analyzed systematically using liver and bone marrow as potential target tissues. Sources of variation, including plates (within packaging reactions), packaging reactions (within animals) and animals, were evaluated for extra-binomial variation. Although hardly any evidence of overdispersion was detected at the plate level, limited evidence of extra-binomial variation was observed at the packaging reaction level. There was, however, much stronger evidence of overdispersion at the animal level. Statistical tests for increasing trend in mutant frequency with increasing dose were also performed at the animal level. A significant increasing trend following exposure to N-nitrosodibenzylamine was detected in liver but not in bone marrow. A logistical model was used to further describe the dose-response relationship observed in N-nitrosodibenzylamine-treated liver tissue.

Pathogenetic mechanisms of glaucoma not related to intraocular pressure.

The pathogenesis of the diseases termed open-angle glaucoma remains elusive but progress is being made in understanding more clearly the optic nerve head, vascular, and cellular mechanisms that are associated with them. Even though the greatest risk factors remain intraocular pressure, age, race and refractive error, the association with vascular disease continues to unfold in the literature. Clinical and laboratory studies, although not addressing mechanisms directly as far as we know, continue to probe potential pathways that will ultimately lead to subsets of open-angle glaucoma. Only when this is accomplished will we be able to focus therapy at pathogenesis rather than at risks.

Databases and software for the analysis of mutations in the human p53 gene, human hprt gene and both the lacI and lacZ gene in transgenic rodents.

We have created databases and software applications for the analysis of DNA mutations at the human p53 gene, the human hprt gene and both the rodent transgenic lacI and lacZ loci. The databases themselves are stand-alone dBASE files and the software for analysis of the databases runs on IBM-compatible computers with Microsoft Windows. Each database has a separate software analysis program. The software created for these databases permit the filtering, ordering, report generation and display of information in the database. In addition, a significant number of routines have been developed for the analysis of single base substitutions. One method of obtaining the databases and software is via the World Wide Web. Open the following home page with a Web Browser: http://sunsite.unc.edu/dnam/mainpage. html . Alternatively, the databases and programs are available via public FTP from: anonymous@sunsite.unc.edu. There is no password required to enter the system. The databases and software are found beneath the subdirectory: pub/academic/biology/dna-mutations. Two other programs are available at the site, a program for comparison of mutational spectra and a program for entry of mutational data into a relational database.

Analysis of tissue-specific lacZ mutations induced by N-nitrosodibenzylamine in transgenic mice.

N-Nitrosodibenzylamine (NDBzA) is a contaminant found frequently in rubber baby bottle nipples and pacifiers. To evaluate more fully the mutagenic potential and analyse the molecular nature of possible mutations induced in vivo, we have studied the mutagenicity of NDBzA in vivo using the MutaMouse system. NDBzA, suspended in olive oil, was administered orally once to male mice at different doses (0, 30, 100, 425 and 750 mg/kg) and the mice were killed 30 and 90 days after treatment. As a positive control, and to compare relative mutagenicity, N-nitrosodimethylamine (NDMA) was also administered to animals in the same experiment at doses of 0, 2, 6 and 10 mg/kg. Mutant frequencies were increased in both 30 and 90 day liver samples, but not in bone marrow, after both NDBzA and NDMA treatment. However, NDBzA was >100 times less mutagenic than NDMA. A total of 81 mutants obtained from liver samples of treated animals (750 mg/kg) were characterized by DNA sequencing. While spontaneous mutations in transgenic mice have been characterized previously by a preponderance of G:C-->A:T transitions, mainly at 5'-CpG-3' dinucleotide sites, the predominant type of NDBzA-induced mutation in this study was transversion, mainly G:C-->T:A changes. The molecular characteristics of mutations induced by NDBzA indicate that they may arise from specific unidentified DNA adducts and benzylation appears to be the primary mechanism involved in formation of these DNA adducts.

Automated perimetry in detecting threats to fixation.

PURPOSE: To study in glaucoma patients the threat to fixation on a Humphrey field analyzer program 10-2 when one of the four innermost paracentral points is defective on program 30-2. METHODS: Forty-five eyes of 45 patients with chronic open-angle glaucoma in whom at least one of the innermost four defective paracentral points was reproducibly defective on program 30-2 of the Humphrey perimeter, with a size 3 target, on two consecutive tests, were studied with program 10-2. RESULTS: Of the 45 eyes with an abnormal paracentral point of program 30-2, 30 (66%) also showed involvement of a paracentral point on program 10-2, which was considered a threat to fixation. The remaining 15 were considered not to threaten fixation imminently. CONCLUSIONS: In about one third of glaucomatous fields considered to threaten fixation on the standard programs 30-2 and 24-2, the threat was not imminent. The extra evaluation is therefore useful before making radical and precipitous changes in management of the disease.

The ability of Medmont M600 automated perimetry to detect threats to fixation.

PURPOSE: The authors studied the sensitivity and specificity of the Medmont M600 central 10 degrees program (Medmont PTY Ltd., Camberwell, Victoria, Australia) in identifying paracentral threats to fixation mapped on the Humphrey program 10-2 (Humphrey Inst. Inc., San Leandro, CA, U.S.A.). METHODS: Humphrey automated threshold perimetry (program 10-2) and Medmont M600 automated threshold perimetry (central 10 degrees program) were performed on 62 eyes of 62 patients with glaucoma, and their paracentral point defects on the field were investigated. The sensitivity and specificity of Medmont central 10 degrees program were analyzed. RESULTS: The sensitivity and specificity of Medmont M600 central 10 degrees field was 78% and 81% within 1 degree, and 95% and 83% within 3 degrees in detecting the field defects on the Humphrey program 10-2. The Medmont M600 central 10 degrees threshold visual field test took 36% of the testing time required for the Humphrey threshold 10-2 visual field examination. CONCLUSION: The Medmont perimeter seems to be efficient in its performance of central threshold testing and significantly cuts down the test time.

Toward an understanding of the use of transgenic mice for the detection of gene mutations in germ cells.

Recently-developed transgenic models have provided unprecedented access to rodent somatic and germ line tissues for the study of gene mutation in vivo. While mutations in germ cells are considered an important aspect of any regulatory assessment of the risks posed by chemicals, currently-available conventional tests, which involve the study of thousands of offspring make it impractical to test large numbers of chemicals, for the induction of inherited gene mutations. When effects in germ cells per se, rather than offspring are acceptable targets, transgenic mouse assays may provide a practical alternative. As part of an international collaborative study to begin to determine the reliability, efficacy, and role of such assays, lacZ transgenic mice (Muta Mouse) were treated with single i.p. doses of ethylnitrosourea (ENU), methyl methanesulfonate (MMS), and isopropyl methanesulfonate (iPMS), and mutant frequencies determined using phenyl-beta-D-galactoside (p-gal) positive selection. For studies using germ cells, the selection of sampling times and target cells is crucial. Spermatagonial stem cells and cells in post-spermatagonial stem cell stages are the critical target cell populations of regulatory importance. Cell populations within these categories were studied by sampling germ cells isolated from seminiferous tubules and spermatozoa from the epididymis at 91 days and 25 days after treatment. The data show that ENU and iPMS induced mutations in post-spermatagonial stem cells and spermatagonial stem cells. However, MMS did not induce mutations in either cell type, or at either sampling time, at doses approaching lethality. This result is possibly because MMS induces preferentially large lesions and chromosomal aberrations (as opposed to point mutations), which are not readily detectable with bacteriophage-based shuttle vectors. Since MMS-induced specific locus and dominant lethal mutations are induced only after the mid-spermatid stage, it is also possible that the timing used missed this effect. While the ENU and iPMS data in this study demonstrate the suitability of the lacZ male transgenic mice for the study of gene mutations in post-spermatagonial stem cells and spermatagonial stem cells by sampling cells isolated from seminiferous tubules at selected times after treatment, the MMS results do not answer fully whether transgenic mouse mutation assays can detect mutations resulting from lesions induced after the mid-spermatid stage when most cellular processing is retarded. Nevertheless, it appears clear from presently available information, that the bacteriophage-based lacZ transgenic model is suitable for the detection of gene mutations in spermatogonial stem cells, spermatocytes, and early spermatids.

Sources of variability in data from a positive selection lacZ transgenic mouse mutation assay: an interlaboratory study.

Experimental features of a positive selection transgenic mouse mutation assay based on a lambda lacZ transgene are considered in detail, with emphasis on results using germ cells as the target tissue. Sources of variability in the experimental protocol that can affect the statistical nature of the observations are examined, with the goal of identifying sources of excess variation in the observed mutant frequencies. The sources include plate-to-plate (within packages), package-to-package (within animals), and animal-to-animal variability. Data from five laboratories are evaluated in detail. Results suggest only scattered patterns of excess variability below the animal-to-animal level, but, generally, significant excess variability at the animal-to-animal level. Using source of variability analyses to guide the choice of statistical methods, control-vs-treatment comparisons are performed for assessing the male germ cell mutagenicity of ethylnitrosourea (ENU), isopropyl methanesulfonate (iPMS), and methyl methanesulfonate (MMS). Results on male germ cell mutagenesis of ethyl methanesulfonate (EMS) and methylnitrosourea (MNU) are also reported.

Databases and software for the analysis of mutations in the human p53 gene, the human hprt gene and both the lacI and lacZ gene in transgenic rodents.

We have created databases and software applications for the analysis of DNA mutations at the humanp53gene, the humanhprtgene and both the rodent transgeniclacIandlacZlocus. The databases themselves are stand-alone dBASE files and the software for analysis of the databases runs on IBM-compatible computers. Each database has a separate software analysis program. The software created for these databases permit the filtering, ordering, report generation and display of information in the database. In addition, a significant number of routines have been developed for the analysis of single base substitutions. One method of obtaining the databases and software is via the World Wide Web (WWW). Open the following home page with a Web Browser: http://sunsite.unc.edu/dnam/mainpage.ht ml . Alternatively, the databases and programs are available via public FTP from: anonymous@sunsite.unc.edu . There is no password required to enter the system. The databases and software are found beneath the subdirectory: pub/academic/biology/dna-mutations. Two other programs are available at the site-a program for comparison of mutational spectra and a program for entry of mutational data into a relational database.

Comparison of psychophysical and electrophysiological testing in early glaucoma.

PURPOSE: To compare the sensitivity and specificity of a wide range of psychophysical and electrophysiological tests in the detection of early glaucomatous damage. METHODS: Forty-three normals and 43 patients with early glaucoma, some still without field defects, were tested with differential light threshold perimetry, short-wavelength automated perimetry, high-pass resolution perimetry, motion detection, flicker contrast sensitivity, flickering and isoluminantly matched letter tests, and pattern and flash electroretinography, including photopic, scotopic, oscillatory potentials, and 30 Hz flicker. Receiver operating characteristic analysis was applied to continuous variables derived from each of the tests. RESULTS: Most parameters reflected glaucomatous loss to some degree, even though only single variables were analyzed separately in the receiver operating characteristic analysis. The pattern electroretinogram and some of the letter acuity tests had the best sensitivity and specificity, followed by short-wavelength automated perimetry and high-pass resolution-perimetry. Motion detection, flicker contrast, and flash electroretinogram parameters scored poorly. Six patients with normal results on the Humphrey field test had abnormal results on many of the other tests. CONCLUSIONS: Applying different psychophysical and electrophysiological tests may add to our ability to detect early glaucomatous damage.

Comparison of the molecular characteristics of lacZ transgenic mouse mutations detected by visual and positive selection.

lacZ gene mutations in the transgenic Muta Mmouse can be detected by two different selection systems. While mutant frequencies recovered by phenyl-beta-D-galactoside (P-gal) selection are comparable with those obtained using 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside (X-gal) as substrate for beta-galactosidase, there may still be differences at the molecular level in the mutations detected by these two methods. Accordingly, we have examined a spectrum of mutants recovered from the X-gal system by regrowing these mutants on the P-gal plates. All colourless X-gal mutants grew normally on the P-gal plates. However, 11 out of 53 single light blue mutants, which express partial beta-galactosidase activity, produce few or no plaques on the P-gal plates, indicating the possible loss of some mutations using the positive selection system. Further analysis of mutant phenotypes and base changes indicates that such loss is not mutation-type specific. Our data suggest that the positive selection method can detect the majority of lacZ mutations detectable by visual selection and is more efficient at detecting mutants within a reduced range of beta-galactosidase activity.

IPCS harmonization of methods for the prediction and quantification of human carcinogenic/mutagenic hazard, and for indicating the probable mechanism of action of carcinogens.

A flow chart is presented as a recommended sequence of tests to predict the carcinogenic hazard, and to predict and quantify the mutagenic hazard to germ cells of chemicals to humans. Ten associated principles of testing for these endpoints are also suggested. These recommendations are the result of a meeting convened under the auspices of the International Programme on Chemical Safety (IPCS), as part of their project on 'Harmonization of Approaches to the Assessment of Risk from Exposure to Chemicals'. The meeting was held at Carshalton, Surrey, from 13-17 February 1995.

Database and software for the analysis of mutations at the lacZ locus in transgenic rodents.

The use of transgenic rodents is becoming increasingly widespread in genetic toxicology. In an effort to centralize and standardize the information regarding mutations in rodents bearing the lacZ transgene, we have created a computerized database that contains published information about DNA sequence alterations on over 100 mutants. Information on the literature citation, mutagenic conditions, organs from specific animals, mutation frequency in each organ, specific mutation, amino acid change, and other data are provided for each mutant. We have also produced a software package for the analysis of the lacZ database. Routines have been developed for the analysis of single base substitutions, including programs to 1) determine whether two mutational spectra are statistically different, 2) determine whether mutations show a DNA strand bias, 3) determine the frequency of transitions and transversions, 4) display the number and kind of mutations observed at each base in the coding region, 5) perform nearest-neighbor analysis, and 6) display mutable amino acids in the lacZ protein. The software runs only on IBM-compatible machines running Microsoft Windows. The software and lacZ database are freely available via the Internet (http:@sunsite.unc.edu/dnam/mainpage.ht ml). These programs simplify the analysis of the rapidly increasing information about lacZ mutation. The programs permit the facile comparison between different lacZ data sets as well as the identification of mutational patterns that may be of importance to experimenters studying the mechanisms of mutation and mutational spectra in transgenic animals.

Temporal and molecular characteristics of lacZ mutations in somatic tissues of transgenic mice.

In order to help establish criteria for optimizing protocols for in vivo mutation studies, lacZ transgenic mice (Muta mouse) were treated with five consecutive daily doses of ethylnitrosourea (50 mg/kg), sampled at times up to 55 days after treatment, and mutant frequencies and DNA sequences determined for liver and bone marrow. In the bone marrow, the mutant frequency rose very rapidly in the first 5 days after treatment to 34 times the control frequency. Subsequently, there was a brood peak where the mutant frequency did not vary significantly, although it did appear to begin to decline after 45 days. In contrast, in the liver, the peak mutant frequency (11 times the control frequency) was not achieved until 35 days, after which there appeared to be a slow decline up to 55 days, which was not statistically significant. Once the maximum mutant frequency was reached, the mutation spectra in the two tissues were indistinguishable. In contrast to the G:C-->A:T transitions in 5'-CpG sites characteristic of untreated mice, A:T-->T:A transversions and A:T-->G:C transitions were prominent in both liver and bone marrow of ENU-treated mice, suggesting the involvement of unrepaired O2- and O4-ethylthymine adducts. In addition, G:C-->T:A transversions were induced in liver. This study demonstrates the possibility that although tissues may have different mutation fixation times, a single mutation fixation time equal to the longest time may be appropriate for in vivo mutation studies, provided that the mutation frequency does not decline appreciably after the peak is reached. This study also illustrates the necessity of ensuring that mutation characteristics are determined after optimal fixation has occurred.