Inter-Rater and Intra-Rater Agreement in Scoring Severity of Rodent Cardiomyopathy and Relation to Artificial Intelligence-Based Scoring.

We previously developed a computer-assisted image analysis algorithm to detect and quantify the microscopic features of rodent progressive cardiomyopathy (PCM) in rat heart histologic sections and validated the results with a panel of five veterinary toxicologic pathologists using a multinomial logistic model. In this study, we assessed both the inter-rater and intra-rater agreement of the pathologists and compared pathologists' ratings to the artificial intelligence (AI)-predicted scores. Pathologists and the AI algorithm were presented with 500 slides of rodent heart. They quantified the amount of cardiomyopathy in each slide. A total of 200 of these slides were novel to this study, whereas 100 slides were intentionally selected for repetition from the previous study. After a washout period of more than six months, the repeated slides were examined to assess intra-rater agreement among pathologists. We found the intra-rater agreement to be substantial, with weighted Cohen's kappa values ranging from k = 0.64 to 0.80. Intra-rater variability is not a concern for the deterministic AI. The inter-rater agreement across pathologists was moderate (Cohen's kappa k = 0.56). These results demonstrate the utility of AI algorithms as a tool for pathologists to increase sensitivity and specificity for the histopathologic assessment of the heart in toxicology studies.

Evaluation of the herbicide glyphosate, (aminomethyl)phosphonic acid, and glyphosate-based formulations for genotoxic activity using in vitro assays.

Glyphosate, the most heavily used herbicide world-wide, is applied to plants in complex formulations that promote absorption. The National Toxicology Program reported in 1992 that glyphosate, administered to rats and mice at doses up to 50,000 ppm in feed for 13 weeks, showed little evidence of toxicity, and no induction of micronuclei was observed in the mice in this study. Subsequently, mechanistic studies of glyphosate and glyphosate-based formulations (GBFs) that have focused on DNA damage and oxidative stress suggest that glyphosate may have genotoxic potential. However, few of these studies directly compared glyphosate to GBFs, or effects among GBFs. To address these data gaps, we tested glyphosate, glyphosate isopropylamine (IPA), and (aminomethyl)phosphonic acid (AMPA, a microbial metabolite of glyphosate), 9 high-use agricultural GBFs, 4 residential-use GBFs, and additional herbicides (metolachlor, mesotrione, and diquat dibromide) present in some of the GBFs in bacterial mutagenicity tests, and in human TK6 cells using a micronucleus assay and a multiplexed DNA damage assay. Our results showed no genotoxicity or notable cytotoxicity for glyphosate or AMPA at concentrations up to 10 mM, while all GBFs and herbicides other than glyphosate were cytotoxic, and some showed genotoxic activity. An in vitro to in vivo extrapolation of results for glyphosate suggests that it is of low toxicological concern for humans. In conclusion, these results demonstrate a lack of genotoxicity for glyphosate, consistent with observations in the NTP in vivo study, and suggest that toxicity associated with GBFs may be related to other components of these formulations.

Analysis of incidence data in developmental toxicity studies: Statistical tests to account for litter effects in fetal defect data.

BACKGROUND: When analyzing fetal defect incidence in laboratory animal studies, correlation in responses within litters (i.e., litter effects) can lead to increased false-positive rates if litter effects are not incorporated into the analysis. Studies of fetal defects require analysis methods that are robust across a broad range of defect types, including those with zero or near-zero incidence rates in control groups. METHODS: A simulation study compared power and false-positive rates for six approaches across a range of background defect rates and litter size distributions. Statistical methods evaluated included ignoring the litter effect as well as parametric and nonparametric approaches based on litter proportions, generalized linear mixed models (GLMMs), the Rao-Scott Cochran-Armitage (RSCA) trend test, and a modification to the RSCA (mRSCA) introduced here to improve estimation at low background rates. These methods were also applied to a common and a rare defect from two prenatal developmental toxicology studies conducted by the National Toxicology Program (NTP). RESULTS: At background defect rates of 1%, the mRSCA and parametric litter proportion methods provided gains in power over the nonparametric litter proportion method, the GLMM method, and the RSCA method. Simulations involving litter loss in high-dose groups showed loss of power for both litter proportion methods. CONCLUSIONS: The mRSCA test developed here compares favorably with other litter-based approaches and is robust across a range of background defect rates and litter size distributions, making it a practical choice for prenatal developmental toxicology studies involving both common and rare fetal defects.

Evaluation of skin sensitization induced by four ionic liquids.

Ionic liquids (ILs) are synthetic solvents used as replacements for volatile organic solvents. Human exposure occurs through dermal or oral routes. In rodents, several ILs were reported to induce dermal toxicity, irritation, and sensitization. Due to the potential for occupational exposure, and industrial use as nonvolatile solvents, 1-ethyl-3-methylimidazolium chloride (EMIM, 6.25% to 50% v/v), 1-butyl-3-methylimidazolium chloride (BMIM, 3.12% to 12.5% v/v), 1-butyl-1-methylpyrrolidinium chloride (BMPY, 0.825% to 6.25% v/v), and N-butylpyridinium chloride (NBuPY, 0.825% to 12.5% v/v) were nominated to the National Toxicology Program and evaluated for skin sensitization. The test compound was applied to the ears of female BALB/c mice daily for 3 days in a primary irritancy (IRR)/local lymph node assay (LLNA). Sensitization was assessed in vitro in the direct peptide reactivity assay (DPRA), KeratinoSens™ assay, and human cell line activation test (h-CLAT). In the LLNA, the butylated ILs, BMIM, and BMPY were more potent than NBuPY (butylated) or EMIM (ethylated), which was neither an irritant nor a sensitizer. NBuPY induced skin irritation in vivo at ≥3.12% (p ≤ 0.01), and sensitization in vitro in the KeratinoSens™ assay and h-CLAT, but was negative for sensitization in vivo and in the DPRA. Although SI3 was not achieved, dermal treatment with 12.5% BMIM or 6.25% BMPY increased (p ≤ 0.01) lymph node cell proliferation in the LLNA. In vitro, BMIM was positive for sensitization in the h-CLAT, and BMPY was positive in the h-CLAT and KeratinoSens™ assay; both were negative in the DPRA. Integrated data analyses, weighted toward in vivo data, suggested that BMIM and BMPY may induce weak to mild sensitization.

The Assessment of Longitudinal Sections of Rat Female Reproductive Tissues for NTP 2-Year Toxicity and Carcinogenicity Studies.

The National Toxicology Program (NTP) now uses an extended longitudinal sectioning protocol for the uterus to better evaluate female rodent reproductive tract toxicity for all developmental and reproductive toxicology and 2-year toxicity and carcinogenicity bioassays. The previous protocol for toxicity/carcinogenicity studies involved 1 cross section midway through each uterine horn and collection of uterine cervix and vagina only if gross lesions were present. Here we compare the histological findings of the original cross sections with the additional longitudinal sections of residual uterine tissues of 7 chronic NTP rat bioassays. The goal of this study was to determine whether there might be any advantages to examining additional uterine tissue. The longitudinal protocol allowed for 10 to 20 times more uterine tissue for evaluation. Results indicate that the potential advantages of a more complete evaluation of female reproductive tract tissue include increased detection of reproductive targets, increased detection of neoplastic and nonneoplastic lesions, improved detection of tissue origin of neoplasms, less reliance on gross identification of lesions, improved accuracy in the application of severity grades, and increased detection of preneoplastic or subtle lesions.

Evaluation of 2-methoxy-4-nitroaniline (MNA) in hypersensitivity, 14-day subacute, reproductive, and genotoxicity studies.

2-Methoxy-4-nitroaniline (MNA), an intermediate in the synthesis of azo dyes used in textiles and paints, is structurally similar to carcinogenic anilines. Human exposure occurs primarily in the occupational setting through handling of dye dust, and through use and disposal of MNA-containing products. MNA has been reported to induce contact hypersensitivity in a human, myocardial necrosis in rats, and bacterial mutagenicity. This study assessed the subacute toxicity, genotoxicity, contact hypersensitivity, and reproductive toxicity of MNA in rodents in an effort to more fully characterize its toxicological profile. B6C3F1/N mice were exposed to 0, 650, 1250, 2500, 5000, or 10,000 ppm MNA by dosed feed for 14-days to evaluate subacute toxicity and histopathological endpoints. In female mice, decreased body weight (13.5 %) and absolute kidney weight (14.8 %), compared to control, were observed at 10,000 ppm MNA; increased relative liver weight (10-12 %), compared to control, occurred at 5,000-10,000 ppm MNA. In male mice, absolute (15 %) and relative liver weights (9-13 %) were increased at 2,500-5,000 ppm and 1250-10,000 ppm MNA, compared to control, respectively. In both sexes of mice, minimal elevations of hemosiderin pigmentation (a breakdown product of erythrocytes), relative to control, were observed in the liver (10,000 ppm); minimal to moderate elevations of hemosiderin pigmentation (5,000-10,000 ppm) and minimal increases in hematopoietic cell proliferation occurred in the spleen (≥ 1250 ppm). In a reproductive toxicity study, timed-mated female Harlan Sprague Dawley rats were exposed to 0-10,000 ppm MNA by dosed feed from gestation day 6 through postnatal day (PND) 21. Decreases in mean litter weights were observed at 5000 ppm MNA, compared to control, beginning at PND1. To evaluate potential contact hypersensitivity, MNA (2.5-50 %, in dimethylformamide) was applied to the dorsa of both ears of female Balb/c mice once daily for three days. The increase observed in lymph node cell proliferation (10-50 % increase in thymidine uptake compared to control) did not reproducibly achieve the Sensitization Index (SI) 3 level, and there was no ear swelling evident following sensitization with 10-50 % MNA and challenge with 25 % MNA in the mouse ear swelling test. In bacterial mutagenicity assays, MNA (250-1000 µg/plate) induced significant increases, compared to control, in mutant colonies with and without metabolic activation enzymes in Salmonella typhimurium strains TA100 and TA98. These data indicate that MNA is genotoxic, and may induce erythrocyte damage and reactive phagocytosis by macrophages in the liver and spleen.

Quality Control of Quantitative High Throughput Screening Data.

Quantitative high throughput screening (qHTS) experiments can generate 1000s of concentration-response profiles to screen compounds for potentially adverse effects. However, potency estimates for a single compound can vary considerably in study designs incorporating multiple concentration-response profiles for each compound. We introduce an automated quality control procedure based on analysis of variance (ANOVA) to identify and filter out compounds with multiple cluster response patterns and improve potency estimation in qHTS assays. Our approach, called Cluster Analysis by Subgroups using ANOVA (CASANOVA), clusters compound-specific response patterns into statistically supported subgroups. Applying CASANOVA to 43 publicly available qHTS data sets, we found that only about 20% of compounds with response values outside of the noise band have single cluster responses. The error rates for incorrectly separating true clusters and incorrectly clumping disparate clusters were both less than 5% in extensive simulation studies. Simulation studies also showed that the bias and variance of concentration at half-maximal response (AC50 ) estimates were usually within 10-fold when using a weighted average approach for potency estimation. In short, CASANOVA effectively sorts out compounds with "inconsistent" response patterns and produces trustworthy AC50 values.

Immunotoxic and hepatotoxic effects of perfluoro-n-decanoic acid (PFDA) on female Harlan Sprague-Dawley rats and B6C3F1/N mice when administered by oral gavage for 28 days.

Poly- and perfluoroalkyl substances (PFAS) are chemically and thermally stable, hydrophobic, lipophobic compounds used in stain repellants and water and oil surfactants, and associated with immunosuppression and peroxisome proliferator activity. Perfluoro-n-decanoic acid (PFDA, (CF3(CF2)8COOH), a fluorinated straight chain fatty acid compound, is reported to induce thymic atrophy and reversible bone marrow hypocellularity in rodent models. The objective of this study was to assess potential immunotoxicity of PFDA, due to its structural similarity to other immunosuppressive PFASs. Female Harlan Sprague-Dawley rats were exposed to 0-2.0 mg PFDA/kg by oral gavage daily for 28 d. Female B6C3F1/N mice were exposed once/week to 0-5.0 mg PFDA/kg by gavage for 4 weeks. Animals were evaluated for effects on immune cell populations in spleen and bone marrow, and innate, humoral-, and cell-mediated immunity. Mice were also evaluated for resistance to Influenza virus. Treatment-related hepatocyte necrosis and hepatomegaly were observed in rats treated with 0.5 mg PFDA/kg/d. In mice, hepatomegaly (26-89%) was observed following exposure to ≥0.625 mg PFDA/kg/week, while splenic atrophy (20%) was observed at 5.0 mg PFDA/kg/week. At 5.0 mg PFDA/kg/week, total spleen cells, and Ig + and NK + cells were decreased (17.6-27%). At ≥ 1.25 mg PFDA/kg/week the numbers of splenic CD3+, CD4+, CD8+, and Mac3+ cells were decreased (10.5-39%). No changes were observed in leukocyte subpopulations in PFDA-exposed rats. Phagocytosis by fixed-tissue macrophages was decreased in liver (specific activity, 24-39%) at ≥0.25 mg PFDA/kg/d in rats. PFDA-induced effects on humoral- and cell-mediated immunity, host resistance, and bone marrow progenitor cells were limited. These data suggest that exposure to PFDA may induce adverse effects in rat liver in a manner consistent with the PFAS class, and may also alter the balance of immune cell populations in lymphoid tissues in mice.

Efficacy of an in-home test kit in reducing dust mite allergen levels: results of a randomized controlled pilot study.

BACKGROUND: Dust mite allergens can induce allergic sensitization and exacerbate asthma symptoms. Although dust mite reduction and control strategies exist, few asthmatics employ them. OBJECTIVES: We examined whether an in-home test kit, which quantifies dust mite allergen levels, resulted in behavioral changes in implementation and maintenance of mite reduction strategies and helped reduce allergen levels in homes of dust mite-sensitive children. METHODS: We enrolled 60 households of children aged 5-15 with parent-reported dust mite allergy into a randomized controlled trial. Intervention homes (N = 30) received educational material about reducing dust mites and test kits at 1, 2, 5 and 8 months. Control homes (N = 30) received only educational material. At baseline, 6 and 12 months, study staff visited all homes, collected dust samples from three locations and obtained information about parents' mite reduction behaviors by questionnaire. Allergen concentrations (Der f 2/Der p2) in dust were assessed by immunoassays. After adjusting for visit and location, allergen concentrations in intervention and control homes were compared using mixed effects model analysis. RESULTS: In the intervention homes, allergen concentrations in the child's bedroom and living room floors were significantly reduced over time compared to control homes. Although not all location-specific differences in allergen concentrations were statistically significant, combining data across locations, there was a differential reduction in allergen concentrations in the intervention group versus the control group (p = 0.02). CONCLUSION: The use of in-home test kits along with education may beneficially influence behaviors and attitudes toward dust mite reduction strategies and help reduce residential dust mite allergen levels.

Global analysis of posttranscriptional gene expression in response to sodium arsenite.

BACKGROUND: Inorganic arsenic species are potent environmental toxins and causes of numerous health problems. Most studies have assumed that arsenic-induced changes in mRNA levels result from effects on gene transcription. OBJECTIVES: We evaluated the prevalence of changes in mRNA stability in response to sodium arsenite in human fibroblasts. METHODS: We used microarray analyses to determine changes in steady-state mRNA levels and mRNA decay rates following 24-hr exposure to noncytotoxic concentrations of sodium arsenite, and we confirmed some of these changes using real-time reverse-transcription polymerase chain reaction (RT-PCR). RESULTS: In arsenite-exposed cells, 186 probe set-identified transcripts were significantly increased and 167 were significantly decreased. When decay rates were analyzed after actinomycin D treatment, only 4,992 (9.1%) of probe set-identified transcripts decayed by > 25% after 4 hr. Of these, 70 were among the 353 whose steady-state levels were altered by arsenite, and of these, only 4 exhibited significantly different decay rates between arsenite and control treatment. Real-time RT-PCR confirmed a major, significant arsenite-induced stabilization of the mRNA encoding δ aminolevulinate synthase 1 (ALAS1), the rate-limiting enzyme in heme biosynthesis. This change presumably accounted for at least part of the 2.7-fold increase in steady-state ALAS1 mRNA levels seen after arsenite treatment. This could reflect decreases in cellular heme caused by the massive induction by arsenite of heme oxygenase mRNA (HMOX1; 68-fold increase), the rate-limiting enzyme in heme catabolism. CONCLUSIONS: We conclude that arsenite modification of mRNA stability is relatively uncommon, but in some instances can result in significant changes in gene expression.

An APOBEC cytidine deaminase mutagenesis pattern is widespread in human cancers.

Recent studies indicate that a subclass of APOBEC cytidine deaminases, which convert cytosine to uracil during RNA editing and retrovirus or retrotransposon restriction, may induce mutation clusters in human tumors. We show here that throughout cancer genomes APOBEC-mediated mutagenesis is pervasive and correlates with APOBEC mRNA levels. Mutation clusters in whole-genome and exome data sets conformed to the stringent criteria indicative of an APOBEC mutation pattern. Applying these criteria to 954,247 mutations in 2,680 exomes from 14 cancer types, mostly from The Cancer Genome Atlas (TCGA), showed a significant presence of the APOBEC mutation pattern in bladder, cervical, breast, head and neck, and lung cancers, reaching 68% of all mutations in some samples. Within breast cancer, the HER2-enriched subtype was clearly enriched for tumors with the APOBEC mutation pattern, suggesting that this type of mutagenesis is functionally linked with cancer development. The APOBEC mutation pattern also extended to cancer-associated genes, implying that ubiquitous APOBEC-mediated mutagenesis is carcinogenic.

Venus flytrap biomechanics: forces in the Dionaea muscipula trap.

Biomechanics of morphing structures in the Venus flytrap has attracted the attention of scientists during the last 140 years. The trap closes in a tenth of a second if a prey touches a trigger hair twice. The driving force of the closing process is most likely due to the elastic curvature energy stored and locked in the leaves, which is caused by a pressure differential between the upper and lower layers of the leaf. The trap strikes, holds and compresses the prey. We have developed new methods for measuring all these forces involved in the hunting cycle. We made precise calibration of the piezoelectric sensor and performed direct measurements of the average impact force of the trap closing using a high speed video camera for the determination of time constants. The new equation for the average impact force was derived. The impact average force between rims of two lobes in the Venus flytrap was found equal to 149 mN and the corresponding pressure between the rims was about 41 kPa. Direct measurements of the constriction force in the trap of Dionaea muscipula was performed during gelatin digestion. This force increases in the process of digestion from zero to 450 mN with maximal constriction pressure created by the lobes reaching to 9 kPa. The insects and different small prey have little chance to escape after the snap of the trap. The prey would need to overpower the "escaping" force which is very strong and can reach up to 4N.

Clustered mutations in yeast and in human cancers can arise from damaged long single-strand DNA regions.

Mutations are typically perceived as random, independent events. We describe here nonrandom clustered mutations in yeast and in human cancers. Genome sequencing of yeast grown under chronic alkylation damage identified mutation clusters that extend up to 200 kb. A predominance of "strand-coordinated" changes of either cytosines or guanines in the same strand, mutation patterns, and genetic controls indicated that simultaneous mutations were generated by base alkylation in abnormally long single-strand DNA (ssDNA) formed at double-strand breaks (DSBs) and replication forks. Significantly, we found mutation clusters with analogous features in sequenced human cancers. Strand-coordinated clusters of mutated cytosines or guanines often resided near chromosome rearrangement breakpoints and were highly enriched with a motif targeted by APOBEC family cytosine-deaminases, which strongly prefer ssDNA. These data indicate that hypermutation via multiple simultaneous changes in randomly formed ssDNA is a general phenomenon that may be an important mechanism producing rapid genetic variation.

Comparison of NTP historical control tumor incidence rates in female Harlan Sprague Dawley and Fischer 344/N Rats.

The National Toxicology Program (NTP) has historically used Fischer 344/N (F344/N) rats for the majority of its bioassays. Recently the NTP began using the Harlan Sprague Dawley (SD) as the primary rat model for NTP studies. The NTP had previously used female SD rats in nine bioassays. This article compares historical control (HC) tumor incidence rates from these nine SD rat studies with HC tumor rates from matched NTP F344/N rat bioassays to identify similarities and differences. Matching on sex, laboratory, diet, and route led to nine comparable F344/N rat studies. Our analyses revealed statistically significant strain differences, with female SD rats having lower incidence rates for clitoral gland adenoma (0.2% vs. 5.8%) and mononuclear cell leukemia (0.9% vs. 16.7%) and higher incidence rates for mammary gland fibroadenoma (67.4% vs. 48.4%), mammary gland carcinoma (10.2% vs. 2.4%), and thyroid gland C cell adenoma (25.4% vs. 13.6%) relative to female F344/N rats. These represent five of the seven most common tumor types among female SD and F344/N rats in the NTP HC database. When vehicle was included as an additional matching criterion, the number of comparable F344/N rat studies dropped to four, but similar results were obtained.

Analysis of Correlated Gene Expression Data on Ordered Categories.

A bootstrap based methodology is introduced for analyzing repeated measures/longitudinal microarray gene expression data over ordered categories. The proposed non-parametric procedure uses order-restricted inference to compare gene expressions among ordered experimental conditions. The null distribution for determining significance is derived by suitably bootstrapping the residuals. The procedure addresses two potential sources of correlation in the data, namely, (a) correlations among genes within a chip ("intra-chip" correlation), and (b) correlation within subject due to repeated/longitudinal measurements ("temporal" correlation). To make the procedure computationally efficient, the adaptive bootstrap methodology of Guo and Peddada (2008) is implemented such that the resulting procedure controls the false discovery rate (FDR) at the desired nominal level.

A response to information criterion-based clustering with order-restricted candidate profiles in short time-course microarray experiments.

BACKGROUND: For gene expression data obtained from a time-course microarray experiment, Liu et al. developed a new algorithm for clustering genes with similar expression profiles over time. Performance of their proposal was compared with three other methods including the order-restricted inference based methodology of Peddada et al. In this note we point out several inaccuracies in Liu et al. and conclude that the order-restricted inference based methodology of Peddada et al. (programmed in the software ORIOGEN) indeed operates at the desired nominal Type 1 error level, an important feature of a statistical decision rule, while being computationally substantially faster than indicated by Liu et al. RESULTS: Application of ORIOGEN to the well-known breast cancer cell line data of Lobenhofer et al. revealed that ORIOGEN software took only 21 minutes to run (using 100,000 bootstraps with p = 0.0025), substantially faster than the 72 hours found by Liu et al. using Matlab. Also, based on a data simulated according to the model and parameters of simulation 1 (sigma2 = 1, M = 5) in [1] we found that ORIOGEN took less than 30 seconds to run in stark contrast to Liu et al. who reported that their implementation of the same algorithm in R took 2979.29 seconds. Furthermore, for the simulation studies reported in [1], unlike the claims made by Liu et al., ORIOGEN always maintained the desired false positive rate. According to Figure three in Liu et al. their algorithm had a false positive rate ranging approximately from 0.20 to 0.70 for the scenarios that they simulated. CONCLUSIONS: Our comparisons of run times indicate that the implementations of ORIOGEN's algorithm in Matlab and R by Liu et al. is inefficient compared to the publicly available JAVA implementation. Our results on the false positive rate of ORIOGEN suggest some error in Figure three of Liu et al., perhaps due to a programming error.

NBME Obstetrics and Gynecology clerkship final examination scores: predictive value of standardized tests and demographic factors.

OBJECTIVE: To assess the predictive value of standardized tests and demographic factors on performance on the National Board of Medical Examiners (NBME) Obstetrics and Gynecology (OBGYN) examination. STUDY DESIGN: The study included 171 students who rotated through obstetrics and gynecology from 1992 to 2001. Correlations between NBME OBGYN scores and United States Medical Licensing Examination (USMLE) step 1, MCAT and GPA scores, and temporal and demographic factors were analyzed. RESULTS: The mean Medical College Admission Test (MCAT), NBME and USMLE step 1 scores were 24.03, 67.47 and 194.53, respectively. Significant correlations with NMBE OBGYN were USMLE step 1 scores (r = .517, p < 0.001), MCAT scores (r = .481, p < 0.001), faculty evaluation grade (r=.223, p <0.01), OBGYN residency interest (r= .179, p < 0.05) and female gender (r=.157, p< 0.05) with a significant negative correlation with clerkship duration (r = -.208, p < 0. 01). On logistic regression analysis, USMLE, MCAT, academic year and faculty evaluation grade were independently predictive of NBME OBGYN scores. All preclinical standardized tests showed positive correlation with progressive academic years and decreasing student age. and decreasing student age. CONCLUSION: USMLE step 1, MCAT scores and faculty evaluation grades were predictive of NMBE OBGYN scores. They may be helpful in assessing third-year medical students in need of special supervision and assistance.