Gregory J. Carr

Community analysis by Biolog: curve integration for statistical analysis of activated sludge microbial habitats

Biolog MicroPlates are 96-well plates that contain pre-dried carbon sources and a tetrazolium violet redox dye that turns purple if added microorganisms utilize the nutrients. We have measured absorbance changes due to tetrazolium dye response for microbial communities found in activated sludge and laboratory models of these wastewater communities. To analyze the absorbance versus time data, we export and organize the data into spreadsheets and use a trapezoidal approximation to determine the area under the absorbance versus time curve for each well. The Excel-based trapezoidal approximation has been shown to be in agreement with a more complex curve fitting routine that used SAS to fit a log-logistic function to each curve, and then determined the area under each curve. This data analysis procedure has the advantage of collapsing the absorbance versus time curves down to a single value that integrates information from the entire incubation period. This single value incorporates the lag phase, the rate of development and the extent of dye development for each well. The area under the curve is then used for statistical testing to compare individual carbon source utilization, or in a multivariate pattern analysis of community metabolism of the Biolog MicroPlate carbon sources. Examples of the use of this analysis are given for microbial communities of activated sludge and laboratory models of activated sludge maintained with different types of feedstocks.

Xenobiotic metabolism gene expression in the EpiDerm™ in vitro 3D human epidermis model compared to human skin

There is an urgent need to validate in vitro human skin models for use in safety testing. An important component of validation is characterizing the metabolizing capacity of these models. We report comparison of the expression of 139 genes encoding xenobiotic metabolizing enzymes in the EpiDerm model and human skin. In microarray analysis, the expression of 87% of the genes was consistent between the EpiDerm model and human skin indicating the presence of similar metabolic pathways suggesting commonality in function. Analysis of EpiDerm models constructed from four donors showed highly comparable expression of xenobiotic metabolizing genes demonstrating reproducibility of the model. Overall, the expression of Phase II enzymes appeared to be more pronounced in human skin and the EpiDerm model than that of Phase I enzymes, consistent with the role of skin in detoxification of xenobiotics. Though the basal expression of CYPs in particular was low in EpiDerm, significant induction of CYP1A1/1B1 activity was observed following treatment with 3-methylcholanthrene. These results indicate that the xenobiotic metabolizing capacity of the EpiDerm model appears to be representative of human skin. Models such as EpiDerm provide a valuable in vitro approach for evaluation of metabolism and toxicity of cutaneous exposures to xenobiotics.

Use of fish embryo toxicity tests for the prediction of acute fish toxicity to chemicals

The fish embryo test (FET) is a potential animal alternative for the acute fish toxicity (AFT) test. A comprehensive validation program assessed 20 different chemicals to understand intra- and interlaboratory variability for the FET. The FET had sufficient reproducibility across a range of potencies and modes of action. In the present study, the suitability of the FET as an alternative model is reviewed by relating FET and AFT. In total, 985 FET studies and 1531 AFT studies were summarized. The authors performed FET-AFT regressions to understand potential relationships based on physical-chemical properties, species choices, duration of exposure, chemical classes, chemical functional uses, and modes of action. The FET-AFT relationships are very robust (slopes near 1.0, intercepts near 0) across 9 orders of magnitude in potency. A recommendation for the predictive regression relationship is based on 96-h FET and AFT data: log FET median lethal concentration (LC50) = (0.989 × log fish LC50) - 0.195; n = 72 chemicals, r = 0.95, p < 0.001, LC50 in mg/L. A similar, not statistically different regression was developed for the entire data set (n = 144 chemicals, unreliable studies deleted). The FET-AFT regressions were robust for major chemical classes with suitably large data sets. Furthermore, regressions were similar to those for large groups of functional chemical categories such as pesticides, surfactants, and industrial organics. Pharmaceutical regressions (n = 8 studies only) were directionally correct. The FET-AFT relationships were not quantitatively different from acute fish-acute fish toxicity relationships with the following species: fathead minnow, rainbow trout, bluegill sunfish, Japanese medaka, and zebrafish. The FET is scientifically supportable as a rational animal alternative model for ecotoxicological testing of acute toxicity of chemicals to fish.

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.

OECD validation study to assess intra- and inter-laboratory reproducibility of the zebrafish embryo toxicity test for acute aquatic toxicity testing

The OECD validation study of the zebrafish embryo acute toxicity test (ZFET) for acute aquatic toxicity testing evaluated the ZFET reproducibility by testing 20 chemicals at 5 different concentrations in 3 independent runs in at least 3 laboratories. Stock solutions and test concentrations were analytically confirmed for 11 chemicals. Newly fertilised zebrafish eggs (20/concentration and control) were exposed for 96h to chemicals. Four apical endpoints were recorded daily as indicators of acute lethality: coagulation of the embryo, lack of somite formation, non-detachment of the tail bud from the yolk sac and lack of heartbeat. Results (LC50 values for 48/96h exposure) show that the ZFET is a robust method with a good intra- and inter-laboratory reproducibility (CV<30%) for most chemicals and laboratories. The reproducibility was lower (CV>30%) for some very toxic or volatile chemicals, and chemicals tested close to their limit of solubility. The ZFET is now available as OECD Test Guideline 236. Considering the high predictive capacity of the ZFET demonstrated by Belanger et al. (2013) in their retrospective analysis of acute fish toxicity and fish embryo acute toxicity data, the ZFET is ready to be considered for acute fish toxicity for regulatory purposes.

An initial evaluation of the use of Euro/North American fish species for tropical effects assessments

Environmental effects and risk assessments most often employ temperate and coldwater species endemic to Europe and North America. With an increased need to assess the risk of chemicals in tropical regions, there is a concomitant need to assess the applicability of using effects assessments generated with Euro/North American species to also cover the needs of tropical species. One aspect of this need is the comparability of species sensitivities between fish from different climates. This specific need was addressed by using acute toxicity information collected from the US EPA AQUIRE database for 95 different fish species exposed to six chemicals with different modes of action. Our analysis indicates that tropical species are no more sensitive to chemicals than coldwater and temperate species. Coldwater species appear to be the most sensitive, followed by temperate and tropical, respectively. This evaluation supports the initial use of commonly tested aquatic species endemic to coldwater and temperate climates for assessing effects in tropical environs.

Ocular Irritation: Microscopic Changes Occurring Over Time in the Rat with Surfactants of Known Irritancy

The pathology of surfactant-induced ocular irritation, especially in the context of accidental human exposures and animal tests used to assess a surfactants potential ocular irritation, is not well understood. The purpose of this study was to characterize the microscopic changes in rats at 3 hr and on days 1, 2, 3, 4, 7, 14, and 35 following treatment with anionic, cationic, and nonionic surfactants of differing irritancy. The right eye of each rat was treated by placing 10 μl of a surfactant directly on the cornea. Untreated left eyes served as the controls. At each time point, eyes and eyelids were macroscopically examined and collected for microscopic examination. Macroscopically, the differing levels of irritation were characterized by differences in incidence and magnitude of scores, reflecting involvement of the cornea, conjunctiva, and iris, as well as by the incidence of neovascularization and time to recovery. Microscopically, differences in the area and depth of injury paralleled the differences seen grossly and the relative irritancies of the various surfactants. All surfactants affected the corneal and conjunctival epithelium. All surfactants, except the slightly irritating anionic surfactant, caused corneal stromal changes, with this involvement being proportional to their overall level of irritation. Corneal endothelial cell effects principally occurred with only the severely irritating cationic surfactant. Over time, responses to surfactants of differing irritancy were qualitatively and quantitatively different, and these differences correlated with the extent of initial injury. Qualitative differences in response included presence of keratocyte regeneration, corneal neovascularization, and conjunctivalization of the corneal epithelium with all of the surfactants except the slight irritant. Quantitative differences in response occurred in the extent of epithelial regeneration, edema, and inflammation for surfactants of slight to severe irritancy, and with neovascularization, keratocyte regeneration, and conjunctivalization for surfactants of mild to severe irritancy. These results suggest that by defining initial area and depth of injury associated with an ocular irritant, it may be possible to predict the subsequent response and final outcome. Such an approach would be applicable to the development of mechanistically based in vitro assays.

Impact of the phytoestrogen content of laboratory animal feed on the gene expression profile of the reproductive system in the immature female rat

The effect of the dietary background of phytoestrogens on the outcome of rodent bioassays used to identify and assess the reproductive hazard of endocrine-disrupting chemicals is controversial. Phytoestrogens, including genistein, daidzein, and coumestrol, are fairly abundant in soybeans and alfalfa, common ingredients of laboratory animal diets. These compounds are weak agonists for the estrogen receptor (ER) and, when administered at sufficient doses, elicit an estrogenic response in vivo. In this study, we assessed the potential estrogenic effects of dietary phytoestrogens at the gene expression level, together with traditional biologic end points, using estrogen-responsive tissues of the immature female rat. We compared the gene expression profile of the uterus and ovaries, as a pool, obtained using a uterotrophic assay protocol, from intact prepubertal rats fed a casein-based diet (free from soy and alfalfa) or a regular rodent diet (Purina 5001) containing soy and alfalfa. Estrogenic potency of the phytoestrogen-containing diet was determined by analyzing uterine wet weight gain, luminal epithelial cell height, and gene expression profile in the uterus and ovaries. These were compared with the same parameters evaluated in animals exposed to a low dose of a potent ER agonist [0.1 μg/kg/day 17α-ethynyl estradiol (EE) for 4 days]. Exposure to dietary phytoestrogens or to a low dose of EE did not advance vaginal opening, increase uterine wet weight, or increase luminal epithelial cell height in animals fed either diet. Although there are genes whose expression differs in animals fed the soy/alfalfa-based diet versus the casein diet, those genes are not associated with estrogenic stimulation. The expression of genes well known to be estrogen regulated, such as progesterone receptor, intestinal calcium-binding protein, and complement component 3, is not affected by consumption of the soy/alfalfa-based diet when assessed by microarray or quantitative reverse transcriptase–polymerase chain reaction analysis. Our results indicate that although diet composition has an impact on gene expression in uterus and ovaries, it does not contribute to the effects of an ER agonist.

Comet assay in reconstructed 3D human epidermal skin models – investigation of intra- and inter-laboratory reproducibility with coded chemicals

Reconstructed 3D human epidermal skin models are being used increasingly for safety testing of chemicals. Based on EpiDerm™ tissues, an assay was developed in which the tissues were topically exposed to test chemicals for 3h followed by cell isolation and assessment of DNA damage using the comet assay. Inter-laboratory reproducibility of the 3D skin comet assay was initially demonstrated using two model genotoxic carcinogens, methyl methane sulfonate (MMS) and 4-nitroquinoline-n-oxide, and the results showed good concordance among three different laboratories and with in vivo data. In Phase 2 of the project, intra- and inter-laboratory reproducibility was investigated with five coded compounds with different genotoxicity liability tested at three different laboratories. For the genotoxic carcinogens MMS and N-ethyl-N-nitrosourea, all laboratories reported a dose-related and statistically significant increase (P < 0.05) in DNA damage in every experiment. For the genotoxic carcinogen, 2,4-diaminotoluene, the overall result from all laboratories showed a smaller, but significant genotoxic response (P < 0.05). For cyclohexanone (CHN) (non-genotoxic in vitro and in vivo, and non-carcinogenic), an increase compared to the solvent control acetone was observed only in one laboratory. However, the response was not dose related and CHN was judged negative overall, as was p-nitrophenol (p-NP) (genotoxic in vitro but not in vivo and non-carcinogenic), which was the only compound showing clear cytotoxic effects. For p-NP, significant DNA damage generally occurred only at doses that were substantially cytotoxic (>30% cell loss), and the overall response was comparable in all laboratories despite some differences in doses tested. The results of the collaborative study for the coded compounds were generally reproducible among the laboratories involved and intra-laboratory reproducibility was also good. These data indicate that the comet assay in EpiDerm™ skin models is a promising model for the safety assessment of compounds with a dermal route of exposure.

Pathology of Ocular Irritation with Bleaching Agents in the Rabbit Low-Volume Eye Test

Despite differences in the processes leading to tissue damage, the ocular irritation response to various surfactants, two concentrations of an acid and an alkali, and an acetone, alcohol, aromatic amine, and aldehyde has been shown to depend on the extent of initial injury. The purpose of this study was to assess the extent to which this fundamental relationship exists for bleaching agents in the rabbit low-volume eye test. Ten μl of sodium perborate monohydrate (NaBO 3), sodium hypochlorite (NaOCl), 10% hydrogen peroxide (H2O 2), and 15% H2O2 was applied directly to the cornea of the right eye of each rabbit. Macroscopic assessments for irritation were made 3 hours after dosing and periodically until 35 days. Light microscopic examinations were conducted on tissues obtained at 3 hr and on 1, 3, and 35 days. In vivo confocal microscopy (CM) and measurements of dead corneal epithelial cells and keratocytes at 3 hours and 1 day were used to characterize quantitatively initial corneal injury, while in vivo CM performed at 3 hours and 1, 3, 7, 14, and 35 days was used to characterize quantitatively the corneal changes over time. The changes with NaBO3 and NaOCl were consistent with mild irritancy. For both, corneal injury was limited to the epithelium and superficial stroma. The changes with 10% H2O2 and 15% H2O2 were consistent with severe irritation. Both concentrations affected the epithelium and deep stroma, with 15% H2O 2 also at times affecting the endothelium. However, unlike other irritants previously studied, with 10% H2O2 and 15% H2O 2 there was an incongruity between the extent of epithelial and stromal injury, with stromal injury being more extensive than epithelial injury. A similar, although less dramatic, effect was observed with NaBO3. Additionally, there was still significant keratocyte loss at 35 days with 10% H2O2 and 15% H2O2 even though the eyes at times were considered to be macroscopically normal. These observations highlight the need to include both epithelial and stromal components in an ex vivo or in vitro alternative assay. In conclusion, these results continue to support and extend our hypothesis that ocular irritation is principally defined by the extent of initial injury despite clear differences in the means by which irritants cause tissue damage. Importantly, we have identified unique differences in the ocular injury and responses occurring with bleaching agents that are important to consider in the development and validation of alternative ocular irritation tests to characterize a broad range of materials differing in type and irritancy.