Transcriptomic point of departure determination: a comparison of distribution-based and gene set-based approaches.

A key step in assessing the potential human and environmental health risks of industrial and agricultural chemicals is to determine the toxicity point of departure (POD), which is the highest dose level that causes no adverse effect. Transcriptomic POD (tPOD) values have been suggested to accurately estimate toxicity POD values. One step in the most common approach for tPOD determination involves mapping genes to annotated gene sets, a process that might lead to substantial information loss particularly in species with poor gene annotation. Alternatively, methods that calculate tPOD values directly from the distribution of individual gene POD values omit this mapping step. Using rat transcriptome data for 79 molecules obtained from Open TG-GATEs (Toxicogenomics Project Genomics Assisted Toxicity Evaluation System), the hypothesis was tested that methods based on the distribution of all individual gene POD values will give a similar tPOD value to that obtained via the gene set-based method. Gene set-based tPOD values using four different gene set structures were compared to tPOD values from five different individual gene distribution methods. Results revealed a high tPOD concordance for all methods tested, especially for molecules with at least 300 dose-responsive probesets: for 90% of those molecules, the tPOD values from all methods were within 4-fold of each other. In addition, random gene sets based upon the structure of biological knowledge-derived gene sets produced tPOD values with a median absolute fold change of 1.3-1.4 when compared to the original biological knowledge-derived gene set counterparts, suggesting that little biological information is used in the gene set-based tPOD generation approach. These findings indicate using individual gene distributions to calculate a tPOD is a viable and parsimonious alternative to using gene sets. Importantly, individual gene distribution-based tPOD methods do not require knowledge of biological organization and can be applied to any species including those with poorly annotated gene sets.

Evaluation of the safety and nutritional equivalency of maize grain with genetically modified event DP-Ø23211-2.

Feeding studies were conducted with rats and broiler chickens to assess the safety and nutrition of maize grain containing event DP-Ø23211-2 (DP23211), a newly developed trait-pyramid product for corn rootworm management. Diets containing 50% ground maize grain from DP23211, non-transgenic control, or non-transgenic reference hybrids (P0928, P0993, and P1105) were fed to Crl:CD®(SD) rats for 90 days. Ross 708 broilers were fed phase diets containing up to 67% maize grain from each source for 42 days. Body weight, gain, and feed conversion were determined for comparisons between animals fed DP23211 and control diets in each study. Additional measures included clinical and neurobehavioral evaluations, ophthalmology, clinical pathology, organ weights, and gross and microscopic pathology for rats, and carcass parts and select organ yields for broilers. Reference groups were included to determine if any observed significant differences between DP23211 and control groups were likely due to natural variation. No diet-related effects on mortality or evaluation measures were observed between animal fed diets produced with DP23211 maize grain and animal fed diets produced with control maize grain. These studies show that maize grain containing event DP-Ø23211-2 is as safe and nutritious as non-transgenic maize grains when fed in nutritionally adequate diets. The results are consistent with previously published studies, providing further demonstration of the absence of hazards from edible-fraction consumption of genetically modified plants.

Author Correction: Characterization of DvSSJ1 transcripts targeting the smooth septate junction (SSJ) of western corn rootworm (Diabrotica virgifera virgifera).

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Characterization of DvSSJ1 transcripts targeting the smooth septate junction (SSJ) of western corn rootworm (Diabrotica virgifera virgifera).

Transgenic maize plants expressing dsRNA targeting western corn rootworm (WCR, Diabrotica virgifera virgifera) DvSSJ1 mRNA, a Drosophila snakeskin (ssk) ortholog, show insecticidal activity and significant plant protection from WCR damage. The gene encodes a membrane protein associated with the smooth sepate junction (SSJ) which is required for intestinal barrier function. To understand the active RNA form that leads to the mortality of WCR larvae by DvSSJ1 RNA interference (RNAi), we characterized transgenic plants expressing DvSSJ1 RNA transcripts targeting WCR DvSSJ1 mRNA. The expression of the silencing cassette results in the full-length transcript of 901 nucleotides containing a 210 bp inverted fragment of the DvSSJ1 gene, the formation of a double-stranded RNA (dsRNA) transcript and siRNAs in transgenic plants. Our artificial diet-feeding study indicates that dsRNAs greater than or equal to approximately 60 base-pairs (bp) are required for DvSSJ1 insecticidal activity. Impact of specificity of dsRNA targeting DvSSJ1 mRNA on insecticidal activities was also evaluated in diet bioassay, which showed a single nucleotide mutation can have a significant impact or abolish diet activities against WCR. These results provide insights as to the functional forms of plant-delivered dsRNA for the protection of transgenic maize from WCR feeding damage and information contributing to the risk assessment of transgenic maize expressing insecticidal dsRNA.

Nutrient composition and safety evaluation of simulated isobutanol distillers dried grains with solubles and associated fermentation metabolites when fed to male Ross 708 broiler chickens (Gallus domesticus).

Saccharomyces cerevisiae genetically engineered to enhance butanol production will be used in a manufacturing process similar to that of fuel ethanol production, including co-production of distillers products for animal feed. A poultry feeding trial was conducted with simulated isobutanol-derived dried distillers grains with solubles (bDDGS), comprising non-fermentable corn solids and heat-inactivated Butamax modified yeast (BMY), to determine potential health effects. Simulated dried distillers grains were produced in 2 variants: bDDGS containing 10% (B10) or 50% (B50) BMY. The BMY concentrations were selected based on a conservative estimate from ethanol-derived distillers grains (eDDGS) approximating 2.5 and 12-fold margins of exposure. The B10 and B50 DDGS were evaluated in a 42-day feeding trial using male Ross 708 broiler chickens fed diets containing eDDGS, B50 DDGS, or B10 DDGS without or with isobutanol, 2,3-butanediol, and isobutyric acid metabolites each at target concentrations of 2 (B10-2), 5 (B10-5), or 10 (B10-10) times the anticipated specification limit in the commercial product. Diets were fed (n = 50 broilers/treatment) in 3 phases: starter phase with 8% DDGS and grower and finisher phases each with 15% DDGS. No statistically significant differences or diet-related effects on mortality, clinical pathology, or organ weights, and no microscopic observations associated with consumption of diets containing B10, B50, or B10 supplemented with metabolites at any targeted exposure level were observed. A lower (P < 0.05) mean absolute bursa of Fabricius weight in the B10-10 group compared to the B10 group was considered to be within the range of biological variability. A non-significant trend toward lower weight, gains, and feed intake, and higher feed:gain ratio was observed in the B10-10 group, and was considered a non-adverse palatability effect of consuming high concentrations of metabolites. These results demonstrate that consumption of phase diets containing simulated DDGS from a novel isobutanol production process was well-tolerated.

Comparisons of statistical models for growth curves from 90-day rat feeding studies.

The objective of this work was to compare several models of body weight data from 90-day rodent feeding trials. Polynomial and nonlinear functions relating time and weight were examined as were the use of Toeplitz error covariance structures and random coefficients. The models were evaluated by fitting them to five publicly available datasets from rat feeding studies. Model performance was assessed in terms of their ability to capture the complexity of the growth patterns, validity of necessary assumptions, and information criteria scores. The results demonstrated the importance of selecting a curve function that effectively reflects the mean response. Toeplitz error covariance structures resulted in superior model fit, while failing to address deviations from model assumptions. Models using the Richards function and random coefficients were generally superior to the other models evaluated and dramatically improved upon linear models with complex error structures.