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G J Carr

Publications and source records attributed to G J Carr.

At least 19 recordsLinked to original sources
PubMed2006

Hepatic gene expression profiling using Genechips in zebrafish exposed to 17alpha-ethynylestradiol.

Genomic, proteomic, and metabolomic technologies continue to receive increasing interest from environmental toxicologists. This interest is due to the great potential of these technologies to identify detailed modes of action and to provide assistance in the evaluation of a contaminant's risk to aquatic organisms. Our experimental model is the zebrafish (Danio rerio) exposed to reference endocrine disrupting compounds in order to investigate compound-induced changes in gene transcript profiles. Adult, female zebrafish were exposed to 0, 15, 40, and 100ng/L of 17alpha-ethynylestradiol (EE2) and concentration and time-dependent changes in hepatic gene expression were examined using Affymetrix GeneChip Zebrafish Genome Microarrays. At 24, 48, and 168h, fish were sacrificed and liver mRNA was extracted for gene expression analysis (24 and 168h only). In an effort to link gene expression changes to effects on higher levels of biological organization, body and ovary weights were measured and blood was collected for measurement of plasma steroid hormones (17beta-estradiol (E2), testosterone (T)) and vitellogenin (VTG) using ELISA. EE2 exposure significantly affected gene expression, GSI, E2, T, and VTG. We observed 1622 genes that were significantly affected (p< or =0.001) in a concentration-dependent manner by EE2 exposure at either 24 or 168h. Gene ontology (GO) analysis revealed that EE2 exposure affected genes involved in hormone metabolism, vitamin A metabolism, steroid binding, sterol metabolism, and cell growth. Plasma VTG was significantly increased at 24, 48, and 168h (p< or =0.05) at 40 and 100ng/L and at 15ng/L at 168h. E2 and T were significantly reduced following EE2 exposure at 48 and 168h. GSI was decreased in a concentration-dependent manner at 168h. In this study, we identified genes involved in a variety of biological processes that have the potential to be used as markers of exposure to estrogenic substances. Future work will evaluate the use of these genes in zebrafish exposed to weak estrogens to determine if these genes are indicative of exposure to estrogens with varying potencies.

Animals↗
PubMed2002

An investigation of new toxicity test method performance in validation studies: 1. Toxicity test methods that have predictive capacity no greater than chance.

An approach commonly used to measure new toxicity test method (NTM) performance in validation studies is to divide toxicity results into positive and negative classifications, and the identify true positive (TP), true negative (TN), false positive (FP) and false negative (FN) results. After this step is completed, the contingent probability statistics (CPS), sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) are calculated. Although these statistics are widely used and often the only statistics used to assess the performance of toxicity test methods, there is little specific guidance in the validation literature on what values for these statistics indicate adequate performance. The purpose of this study was to begin developing data-based answers to this question by characterizing the CPS obtained from an NTM whose data have a completely random association with a reference test method (RTM). Determining the CPS of this worst-case scenario is useful because it provides a lower baseline from which the performance of an NTM can be judged in future validation studies. It also provides an indication of relationships in the CPS that help identify random or near-random relationships in the data. The results from this study of randomly associated tests show that the values obtained for the statistics vary significantly depending on the cut-offs chosen, that high values can be obtained for individual statistics, and that the different measures cannot be considered independently when evaluating the performance of an NTM. When the association between results of an NTM and RTM is random the sum of the complementary pairs of statistics (sensitivity + specificity, NPV + PPV) is approximately 1, and the prevalence (i.e., the proportion of toxic chemicals in the population of chemicals) and PPV are equal. Given that combinations of high sensitivity-low specificity or low specificity-high sensitivity (i.e., the sum of the sensitivity and specificity equal to approximately 1) indicate lack of predictive capacity, an NTM having these performance characteristics should be considered no better for predicting toxicity than by chance alone.

False Negative Reactions↗
PubMed2002

An investigation of new toxicity test method performance in validation studies: 2. Comparison of three measures of toxicity test performance.

An area that requires further research is how best to measure test method performance in validation studies and how to set criteria that should be used to judge the adequacy of this performance. The studies reported here were designed to begin an investigation of these questions. Computer simulations were used to generate data sets similar to those that might be obtained from a large validation study. These data were then analysed using three procedures including determination of the 95% prediction interval (PI), calculation of Pearson's correlation coefficient and calculation of the contingent probability statistics (CPS), sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). The results of this work suggest that of the three approaches examined, quantitative measurements with calculation of the 95% PI provide the most information to allow discrimination between the performance of several different NTMs. The results also suggest that dividing data sets into positive and negative toxicity classifications followed by the calculation of CPS leads to considerable information loss. This loss of information may be so significant that it is not possible in certain circumstances to distinguish between NTMs that are adequate and those that are not.

Predictive Value of Tests↗
PubMed2002

An investigation of new toxicity test method performance in validation studies: 3. Sensitivity and specificity are not independent of prevalence or distribution of toxicity.

Often, the only measures of toxicity test performance provided in validation studies are the contingent probability statistics (CPS) sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). Sensitivity and specificity are generally used in preference to NPV and PPV since NPV and PPV are assumed to vary with changes in prevalence while sensitivity and specificity are assumed to be independent of changes in prevalence. The purpose of the studies reported here was to test whether or not sensitivity and specificity are actually independent of changes in prevalence. Results derived from these studies indicate that sensitivity and specificity vary significantly depending on the prevalence of toxic substances in the set of chemicals being tested. This means sensitivity and specificity should not always be considered constant indicators of toxicity test performance.

Models, Theoretical↗
PubMed1999

Quantitative measurement of acute corneal injury in rabbits with surfactants of different type and irritancy.

We have hypothesized that differences in ocular irritancy are related to differences in extent of initial injury and that, regardless of the processes leading to tissue damage, extent of injury is the primary factor that determines the final outcome of ocular irritation. In previous in vivo confocal microscopic (CM) studies we identified quantifiable differences in the extent of corneal injury occurring with four surfactants (three anionic, one cationic) known to cause different levels of ocular irritation and demonstrated that extent of initial corneal injury was related to the magnitude of cell death. The purpose of this study was to assess the applicability of this hypothesis to a broad sampling of surfactants. Specifically, initial corneal changes induced by seven different surfactants (one anionic, three cationic, three nonionic) were measured by in vivo CM and cell death was measured by an ex vivo live/dead assay. The right eye of each rabbit was treated by placing 10 microl of a surfactant directly on the cornea. Eyes were examined macroscopically and scored for irritation at 3 h and 1 day. At 3 h and 1 day, in vivo CM was used to examine the corneas and quantitate epithelial cell size, epithelial thickness, corneal thickness, and depth of stromal injury. At 3 h and/or at 1 day, corneas were removed and excised regions were placed in culture media containing 2 microM calcein AM and 4 microM ethidium homodimer. Using laser scanning CM, the number of dead epithelial and/or stromal cells in a 300 x 300 x 170-microm3 (xyz) volume of the cornea was determined. In vivo CM and live/dead assay findings revealed three surfactants to affect only the epithelium, three surfactants to affect the epithelium and superficial stroma, and one surfactant to affect the epithelium and deep stroma. Extent of initial corneal injury reflected level of ocular irritation, and magnitude of cell death was related to the extent of initial corneal injury. These findings are consistent with those for known slight, mild, and moderate to severe irritants, respectively. They suggest that our hypothesis is broadly applicable to surfactants. Additionally, we believe these surfactants should be included as part of a new "gold standard" for use in developing and validating in vitro tests to replace the use of animals in ocular irritancy testing.

Animals↗
PubMed1998

A force plate system for measuring low-magnitude reaction forces in small laboratory animals.

We present a force plate system which measures low-magnitude vertical reaction forces generated by small laboratory animals. The force plate mechanical design minimizes radiated transverse waves, acoustic reverberation, and standing waves caused by impacts on the force plate surface. A secondary force plate and PC-based software algorithm minimize floor vibrational artifact. The force plate was used to measure function of rats during two tests: forelimb/hindlimb hopping reaction and surface righting reaction. In control rats, forelimb hopping rate exceeded hindlimb hopping rate during 16 weeks of repeated testing. Subchronic intraperitoneal (i.p.) dosing of 10 mg/kg/day acrylamide produced a selective impairment of hindlimb hopping. In contrast, single doses of haloperidol (1-5 mg/kg, i.p.) slowed the righting reaction and produced a relatively selective impairment of forelimb hopping. The force plate system presents new opportunities for performing quantitative neurological assessments of small laboratory animals when previously such tests had been performed subjectively and qualitatively.

Acrylamides↗
PubMed1998

Validation of alternative methods for toxicity testing.

Before nonanimal toxicity tests may be officially accepted by regulatory agencies, it is generally agreed that the validity of the new methods must be demonstrated in an independent, scientifically sound validation program. Validation has been defined as the demonstration of the reliability and relevance of a test method for a particular purpose. This paper provides a brief review of the development of the theoretical aspects of the validation process and updates current thinking about objectively testing the performance of an alternative method in a validation study. Validation of alternative methods for eye irritation testing is a specific example illustrating important concepts. Although discussion focuses on the validation of alternative methods intended to replace current in vivo toxicity tests, the procedures can be used to assess the performance of alternative methods intended for other uses.

Animal Testing Alternatives↗
PubMed1998

Area and depth of surfactant-induced corneal injury correlates with cell death.

PURPOSE: In previous studies in which in vivo confocal microscopy (CM) was used, quantifiable differences were identified in the corneal epithelium and stroma for surfactants producing different degrees of ocular irritation. In the present study, in vivo confocal microscopy was used to determine area and depth of the initial corneal changes, and the correlation of the data to cell death was characterized by ex vivo live-dead assay. METHODS: In four groups of rabbits (12 animals each), 10 microl surfactants known to produce slight, mild, moderate, or severe irritation was applied to the central cornea of one eye; 4 untreated rabbits served as controls. Measurements of group total mean epithelial thickness, epithelial cell area, and depth of keratocyte loss in four corneal regions were made by in vivo CM in 6 rabbits of each group and in 4 control animals at 3 hours and in the remaining rabbits at 3 hours and 1 day. Corneas were then removed and fixed for conventional histologic examination (two eyes/treatment/group), or regions were excised and placed in culture media containing 2 microM calcein-acetoxymethyl ester (calcein-AM) and 4 microM ethidium homodimer. Using laser scanning CM, the number of dead epithelial or stromal cells in a 300 x 300 x 170 microm (in the x, y, and z axes, respectively) volume of the cornea was determined. RESULTS: Confocal microscopy showed that application of the slight irritant resulted in decreased epithelial thickness at 3 hours (41.2+/-2.6 microm in treated eyes versus 43.6+/-3 microm in control eyes; n=6 and 4, respectively) and a significant decrease (P < 0.001) in epithelial cell size (630+/-203 microm2 versus 1427.2+/-90.7 microm2). On day 1, mild, moderate, and severe irritants caused complete loss of epithelium and disappearance of keratocytes to a depth of 30.8+/-10.7 microm, 47.2+/-10.4 microm, and 764.6+/-159.6 microm (n=6, 5, and 6), respectively. At 3 hours, live-dead assay detected more dead epithelial cells as a percentage of total surface cells (49.2+/-4.5% in slightly irritated eyes versus 20.9+/-3.2% in control eyes), significantly correlating with the measurement by in vivo CM of average epithelial cell size in each eye (r=-0.96; P < 0.005). On day 1, mild and moderate irritants showed increasing stromal cell death from 9.8+/-16.2 cells to 36.4+/-17.7 cells, which significantly correlated with the depth of stromal injury determined by in vivo CM (r=0.79; P < 0.00001). No surviving keratocytes were detected in severely irritated eyes. CONCLUSIONS: The data support the hypothesis that differences in surfactant-induced ocular irritation are directly related to area and depth of acute corneal injury.

Animals↗
PubMed1998

Area and depth of surfactant-induced corneal injury predicts extent of subsequent ocular responses.

PURPOSE: To correlate area and depth of initial corneal injury induced by surfactants of differing type and irritant properties with corneal responses and outcome in the same animals over time by using in vivo confocal microscopy (CM). METHODS: Six groups of six adult rabbits were treated with anionic, cationic, and nonionic surfactants that caused different levels of ocular irritation. Test materials included slight irritants: 5% sodium lauryl sulfate (SLS), polyoxyethylene glycol monoalkyl ether (POE), and 5% 3-isotridecyloxypropyl-bis(polyoxyethylene) ammonium chloride (ITDOP); mild irritants: 5% 3-decyloxypropyl-bis(polyoxyethylene) amine (DOP) and sodium linear alkylbenzene sulfonate (LAS); and a moderate irritant: a proprietary detergent (DTRGT). Ten microliters surfactant were directly applied to the cornea of one eye of each rabbit. Ten untreated rabbits served as control subjects. Area and depth of initial injury was determined by using in vivo CM to measure epithelial thickness, epithelial cell size, corneal thickness, and depth of stromal injury in four corneal regions at 3 hours and at day 1. Area and depth of corneal responses to injury were evaluated at various times from days 3 through 35 by macroscopic grading and quantitative confocal microscopy through-focusing (CMTF). RESULTS: In vivo CM revealed corneal injury with slight irritants to be restricted to the epithelium, whereas the mild and moderate irritants caused complete epithelial cell loss with increasing anterior stromal damage: DOP < LAS < DTRGT. With the slight ocular irritants there was little or no change in corneal thickness or the CMTF intensity profiles. Three hours after treatment, mild and moderate ocular irritants caused a significant increase in corneal thickness, which peaked at day 1 with DOP (483.3+/-80.1 microm) and LAS (572.3+/-60.0 microm) and day 3 with DTRGT (601.4+/-68.7 microm); returning to normal (similar to control values) by day 7 with DOP and day 35 with LAS and DTRGT. The CMTF intensity profiles also showed significant elevation over that in the anterior stroma, which peaked at day 1 with DOP (14,608+/-4,306 U [U is defined as micrometers X pixel intensity]) and day 3 with LAS and DTRGT (18,471+/-6,581 U and 22,424+/-3,704 U, respectively) and returned toward normal by day 7 with DOP and day 14 with LAS and DTRGT. Elevated CMTF profiles principally reflected the presence of hyperreflective, punctate keratocytes and inflammatory cells at days 1 and 3 and the presence of activated keratocytes at day 7. There was a significant correlation between the elevated CMTF intensity profile and the corresponding macroscopic total score in each eye (r = 0.839; P < 0.001). More important, there was a significant correlation between area and depth of initial stromal injury measured at day 1, regardless of ocular irritant and the stromal response measured by the area under the CMTF intensity profile curve in each cornea (r = 0.87; P < 0.0005). A significant correlation between the area and depth of injury and the area under the corneal thickness curve was also observed in each cornea (r = 0.75; P < 0.0005). CONCLUSIONS: In individual animals, the extent of initial stromal injury correlated with the magnitude of the corneal responses, measured by the change in corneal thickness and the CMTF depth intensity profile. These findings further support the hypothesis that area and depth of injury are the principal factors determining the early responses and eventual repair processes after accidental eye irritation. They also support the proposed use of area and depth of acute injury as a mechanistic correlate to ocular irritation in the development and validation of potential in vitro ocular irritation tests.

Animals↗
PubMed1997

Evaluation of spontaneous and chemical-induced lacI mutations in germ cells from lambda/lacI transgenic mice.

The spontaneous mutant frequency in germ cells isolated from seminiferous tubules of two lambda/lacI transgenic mouse strains, C57BL/6 and B6C3F1 was evaluated. At least 500 000 phage were screened for mutation at lacI for each animal using standardized assay procedures. The germ cell spontaneous lacI mutant frequency was 17.8 +/- 8.1 x 10(-6) in C57BL/6 mice and 17.0 +/- 10.0 x 10(-6) in B6C3F1 mice. The induction of germ cell mutations by three well characterized alkylating agents were also evaluated in C57BL/6 mice on day 3 after a single dose administration. The lacI mutant frequencies were significantly elevated in transgenic mice dosed with ENU at 150 mg/kg (2-fold increase above control) and iPMS at 200 mg/kg (3-fold increase above control) but not in those receiving MMS at 40 mg/kg. These findings suggest that single dose studies using the lambda/lacI transgenic system may be capable of detecting germ mutations induced by chemicals characterized either by point mutations or small, intragenic deletions but not those characterized by a predominance of multi-locus deletions.

Animals↗
PubMed1997

Evaluation of lacI mutation in germ cells and micronuclei in peripheral blood after treatment of male lacI transgenic mice with ethylnitrosourea, isopropylmethane sulfonate or methylmethane sulfonate.

Male C57B1/6 lacI transgenic mice were used to evaluate germ cell mutagenesis in vivo as part of a collaborative study. Groups of 10 mice were administered single intraperitoneal doses of ethylnitrosourea (ENU; 150 mg/kg), isopropyl methanesulfonate (IPMS; 200 mg/kg), methyl methanesulfonate (MMS; 40 mg/kg) or vehicle. Epididymal spermatozoa and testes were recovered 3 days later and DNA isolated subsequently from epididymal spermatozoa and seminiferous tubules were analyzed for lacI mutations. The mutant frequency in seminiferous tubules (average +/- SEM) increased significantly compared with untreated controls (7.2 +/- 0.7 x 10(-5) following treatment with ENU (11.7 +/- 0.8 x 10(-5), p = 0.003) or with IPMS (9.6 +/- 0.5 x 10(-5), p = 0.018) but not following treatment with MMS (8.1 +/- 0.8 x 10(-5), p = 0.213). Group mutant frequencies were not determined for epididymal spermatozoa from MMS- or IPMS-treated mice because of poor DNA recoveries. As another indicator of the genotoxicity of these alkylating agents, the frequencies of micronuclei were determined in the peripheral blood 48 h after carcinogen administration in the same transgenic mice. The micronuclei frequencies were elevated significantly (p < 0.05) by each treatment (IPMS: 1.0%; MMS: 0.94%) compared to vehicle controls (0.3%). In a separate experiment, 40 mg/kg ENU was previously found to increase the frequency of micronuclei in peripheral blood of lacI transgenic mice 48 h after treatment (3.2%; Gibson et al., 1995). These results demonstrate that the lacI transgenic mouse male germ cells are sensitive to some, but not all, mutagens under the conditions used in this experiment. Investigation of other experimental designs would offer additional perspective on the usefulness of this transgenic model for routine mutagenicity testing in germ cells.

Animals↗
PubMed1997

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.

Animals↗
PubMed1996

A comprehensive protocol for conducting the Syrian hamster embryo cell transformation assay at pH 6.70.

Studies from our laboratory have demonstrated several advantages of conducting the Syrian hamster embryo (SHE) cell transformation assay at pH 6.70 compared to that done historically at higher pH values (7.10-7.35). These include reduction of the influence of SHE cell isolates and fetal bovine serum lot variability on the assay, an increase in the frequency of chemically induced morphological transformation (MT) compared to controls, and an increased ease in scoring the MT phenotype. The purpose of this paper is to report a comprehensive protocol for conduct of the pH 6.70 SHE transformation assay including experimental procedures, a description of criteria for an acceptable assay and statistical procedures for establishing treatment-related effects. We have also identified several assay parameters in addition to pH which can affect transformation frequencies, particularly the critical role colony number per plate can have on transformation frequency. Control of this parameter, for which details are provided, can greatly increase the reproducibility and predictive value of the assay.

Animals↗
PubMed1996

Mutational spectra in transgenic animal research: data analysis and study design based upon the mutant or mutation frequency.

Understanding chemically induced changes in mutational spectra can aid in deciphering mechanisms of mutagenesis. In this paper, we propose the use of statistical methods that are based upon the mutation frequency, rather than simple mutant counts which have no relationship to the mutation frequency. These methods have a number of advantages over the current standard analysis: an improved means of identifying those classes/sites of mutation which have treatment-related induction, greater sensitivity to localized differences in spectra (e.g., limited to a single base pair), one-sided tests for induction of mutations, tests of dose-response, and a framework for sample-size estimation in terms of the number of mutants to sequence. As examples, the methods are applied to data from transgenic mutation assays.

Animals↗
PubMed1995

Statistical design and analysis of mutation studies in transgenic mice.

We have been working on identifying sources of variability in data from transgenic mouse mutation assays in order to develop appropriate statistical methods and designs for routine studies. Data from our lab and elsewhere point to the presence of significant animal-to-animal variability, which must be taken into account in statistical hypothesis tests. Here, the usual Cochran-Armitage (CA) test for trend in mutant frequencies, which takes the transgene as the experimental unit, and a generalized Cochran-Armitage test (GCA), which takes the animal as the experimental unit, are contrasted in computer simulations that help to quantify the differences between these statistical tests. The simulations report the statistical power of each test to detect treatment group differences, and their type I error rates. We find in general that the GCA test performs poorly compared to the CA test when it is appropriate to take the transgene as the experimental unit, and the study also uses a small number of animals. However, the CA test performs poorly in small group-size studies when the animal is the appropriate experimental unit. Extensions of the computer simulations allow for identification of cost-effective experimental designs. The results emphasize that the benefits of using additional animals in these mutation studies can be realized without substantial increases in costs. Here we illustrate the methods for liver studies in our lab. These methods can be used to derive optimal experimental designs for any combination of spontaneous mutant frequency and animal-to-animal variability.

Animals↗
PubMed1995

Detection of aneuploidy-inducing carcinogens in the Syrian hamster embryo (SHE) cell transformation assay.

As evidenced by the recent report of the Commission of the European Communities (CEEC) project (Detection of Aneugenic Chemicals-CEEC project, 1993), there currently is a great deal of effort towards developing and validating assays to detect aneuploidy-inducing chemicals. In this report, we describe the utility of the Syrian hamster embryo (SHE) cell transformation assay for detecting carcinogens with known or suspected aneuploidy-inducing activity. The following carcinogens were tested: asbestos, benomyl, cadmium chloride, chloral hydrate, diethylstilbestrol dipropionate, and griseofulvin. Thiabendazole, a noncarcinogen, was also tested. Chemicals of unknown or inconclusive carcinogenicity data, colcemid, diazepam, econazole nitrate, and pyrimethamine were also evaluated. All of the above chemicals except thiabendazole induced a significant increase in morphological transformation (MT) in SHE cells. Based on these results as well as those published in the literature previously, the SHE cell transformation assay appears to have utility for detecting carcinogens with known or suspected aneuploidy-inducing ability.

Aneuploidy↗
PubMed1994

Sources of variability in data from a lacI transgenic mouse mutation assay.

Experimental features of a transgenic mouse mutation assay based on a lacI target transgene from Escherichia coli are considered in detail. 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 fractions. The sources include plate-to-plate (within packages), package-to-package (within animals), and animal-to-animal (within study) variability. Data from two laboratories are evaluated, using various statistical methods to identify excess variability. Results suggest only scattered patterns of excess variability, except possibly in those cases where genomic DNA from test animals is stored for extended periods (e.g., > 90 days) after isolation from tissues. Further study is encouraged to examine the validity and implications of this time/storage-related effect.

9,10-Dimethyl-1,2-benzanthracene↗