A Physiologically Based Pharmacokinetic (PBPK) Modeling Framework for Mixtures of Dioxin-like Compounds.

Humans are exposed to persistent organic pollutants, such as dioxin-like compounds (DLCs), as mixtures. Understanding and predicting the toxicokinetics and thus internal burden of major constituents of a DLC mixture is important for assessing their contributions to health risks. PBPK models, including dioxin models, traditionally focus on one or a small number of compounds; developing new or extending existing models for mixtures often requires tedious, error-prone coding work. This lack of efficiency to scale up for multi-compound exposures is a major technical barrier toward large-scale mixture PBPK simulations. Congeners in the DLC family, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), share similar albeit quantitatively different toxicokinetic and toxicodynamic properties. Taking advantage of these similarities, here we reported the development of a human PBPK modeling framework for DLC mixtures that can flexibly accommodate an arbitrary number of congeners. Adapted from existing TCDD models, our mixture model contains the blood and three diffusion-limited compartments-liver, fat, and rest of the body. Depending on the number of congeners in a mixture, varying-length vectors of ordinary differential equations (ODEs) are automatically generated to track the tissue concentrations of the congeners. Shared ODEs are used to account for common variables, including the aryl hydrocarbon receptor (AHR) and CYP1A2, to which the congeners compete for binding. Binary and multi-congener mixture simulations showed that the AHR-mediated cross-induction of CYP1A2 accelerates the sequestration and metabolism of DLC congeners, resulting in consistently lower tissue burdens than in single exposure, except for the liver. Using dietary intake data to simulate lifetime exposures to DLC mixtures, the model demonstrated that the relative contributions of individual congeners to blood or tissue toxic equivalency (TEQ) values are markedly different than those to intake TEQ. In summary, we developed a mixture PBPK modeling framework for DLCs that may be utilized upon further improvement as a quantitative tool to estimate tissue dosimetry and health risks of DLC mixtures.

Genome-Wide ChIPseq Analysis of AhR, COUP-TF, and HNF4 Enrichment in TCDD-Treated Mouse Liver.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor known for mediating the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. Although the canonical mechanism of AhR activation involves heterodimerization with the aryl hydrocarbon receptor nuclear translocator, other transcriptional regulators that interact with AhR have been identified. Enrichment analysis of motifs in AhR-bound genomic regions implicated co-operation with COUP transcription factor (COUP-TF) and hepatocyte nuclear factor 4 (HNF4). The present study investigated AhR, HNF4α and COUP-TFII genomic binding and effects on gene expression associated with liver-specific function and cell differentiation in response to TCDD. Hepatic ChIPseq data from male C57BL/6 mice at 2 h after oral gavage with 30 µg/kg TCDD were integrated with bulk RNA-sequencing (RNAseq) time-course (2-72 h) and dose-response (0.01-30 µg/kg) datasets to assess putative AhR, HNF4α and COUP-TFII interactions associated with differential gene expression. Functional enrichment analysis of differentially expressed genes (DEGs) identified differential binding enrichment for AhR, COUP-TFII, and HNF4α to regions within liver-specific genes, suggesting intersections associated with the loss of liver-specific functions and hepatocyte differentiation. Analysis found that the repression of liver-specific, HNF4α target and hepatocyte differentiation genes, involved increased AhR and HNF4α binding with decreased COUP-TFII binding. Collectively, these results suggested TCDD-elicited loss of liver-specific functions and markers of hepatocyte differentiation involved interactions between AhR, COUP-TFII and HNF4α.

Single-Nuclei RNA Sequencing Assessment of the Hepatic Effects of 2,3,7,8-Tetrachlorodibenzo-p-dioxin.

BACKGROUND AND AIMS: Characterization of cell specific transcriptional responses to hepatotoxicants is lost in the averages of bulk RNA-sequencing (RNA-seq). Single-cell/nuclei RNA-seq technologies enable the transcriptomes of individual cell (sub)types to be assessed within the context of in vivo models. METHODS: Single-nuclei RNA-sequencing (snSeq) of frozen liver samples from male C57BL/6 mice gavaged with sesame oil vehicle or 30 µg/kg 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) every 4 days for 28 days was used to demonstrate the application of snSeq for the evaluation of xenobiotics. RESULTS: A total of 19,907 genes were detected across 16,015 nuclei from control and TCDD-treated livers. Eleven cell (sub)types reflected the expected cell diversity of the liver including distinct pericentral, midzonal, and periportal hepatocyte subpopulations. TCDD altered relative proportions of cell types and elicited cell-specific gene expression profiles. For example, macrophages increased from 0.5% to 24.7%, while neutrophils were only present in treated samples, consistent with histological evaluation. The number of differentially expressed genes (DEGs) in each cell type ranged from 122 (cholangiocytes) to 7625 (midcentral hepatocytes), and loosely correlated with the basal expression level of Ahr, the canonical mediator of TCDD and related compounds. In addition to the expected functions within each cell (sub)types, RAS signaling and related pathways were specifically enriched in nonparenchymal cells while metabolic process enrichment occurred primarily in hepatocytes. snSeq also identified the expansion of a Kupffer cell subtype highly expressing Gpnmb, as reported in a dietary NASH model. CONCLUSIONS: We show that snSeq of frozen liver samples can be used to assess cell-specific transcriptional changes and population shifts in models of hepatotoxicity when examining freshly isolated cells is not feasible.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) dysregulates hepatic one carbon metabolism during the progression of steatosis to steatohepatitis with fibrosis in mice.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a persistent environmental contaminant, induces steatosis that can progress to steatohepatitis with fibrosis, pathologies that parallel stages in the development of non-alcoholic fatty liver disease (NAFLD). Coincidently, one carbon metabolism (OCM) gene expression and metabolites are often altered during NAFLD progression. In this study, the time- and dose-dependent effects of TCDD were examined on hepatic OCM in mice. Despite AhR ChIP-seq enrichment at 2 h, OCM gene expression was not changed within 72 h following a bolus dose of TCDD. Dose-dependent repression of methionine adenosyltransferase 1A (Mat1a), adenosylhomocysteinase (Achy) and betaine-homocysteine S-methyltransferase (Bhmt) mRNA and protein levels following repeated treatments were greater at 28 days compared to 8 days. Accordingly, levels of methionine, betaine, and homocysteic acid were dose-dependently increased, while S-adenosylmethionine, S-adenosylhomocysteine, and cystathionine exhibited non-monotonic dose-dependent responses consistent with regulation by OCM intermediates and repression of glycine N-methyltransferase (Gnmt). However, the dose-dependent effects on SAM-dependent metabolism of polyamines and creatine could not be directly attributed to alterations in SAM levels. Collectively, these results demonstrate persistent AhR activation disrupts hepatic OCM metabolism at the transcript, protein and metabolite levels within context of TCDD-elicited progression of steatosis to steatohepatitis with fibrosis.

A toxicogenomic approach for the risk assessment of the food contaminant acetamide.

Acetamide (CAS 60-35-5) is detected in common foods. Chronic rodent bioassays led to its classification as a group 2B possible human carcinogen due to the induction of liver tumors in rats. We used a toxicogenomics approach in Wistar rats gavaged daily for 7 or 28 days at doses of 300 to 1500 mg/kg/day (mkd) to determine a point of departure (POD) and investigate its mode of action (MoA). Ki67 labeling was increased at doses ≥750 mkd up to 3.3-fold representing the most sensitive apical endpoint. Differential gene expression analysis by RNA-Seq identified 1110 and 1814 differentially expressed genes in male and female rats, respectively, following 28 days of treatment. Down-regulated genes were associated with lipid metabolism while up-regulated genes included cell signaling, immune response, and cell cycle functions. Benchmark dose (BMD) modeling of the Ki67 labeling index determined the BMD10 lower confidence limit (BMDL10) as 190 mkd. Transcriptional BMD modeling revealed excellent concordance between transcriptional POD and apical endpoints. Collectively, these results indicate that acetamide is most likely acting through a mitogenic MoA, though specific key initiating molecular events could not be elucidated. A POD value of 190 mkd determined for cell proliferation is suggested for risk assessment purposes.

Automated dose-response analysis of the relative hepatic gene expression potency of TCDF in C57BL/6 mice.

Toxic equivalency factors (TEFs) are assigned to dioxin-like chemicals based on relative potency (REP) values of individual adaptive and toxic responses compared to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Agilent 4x44K oligonucleotide microarrays were used to examine the hepatic gene expression potency of 2,3,7,8-tetrachlorodibenzofuran (TCDF), relative to TCDD with complementary histopathology, TCDD and TCDF tissue level analysis, and ethoxyresorufin-O-deethylase (EROD) assay data. Immature ovariectomized C57BL/6 mice were gavaged with 0.03, 0.1, 0.3, 1, 3, 10, 30, or 100 microg/kg TCDD, the World Health Organization TEF-adjusted doses (10 x TCDD dose) of TCDF (0.3, 1, 3, 10, 30, 100, or 300 microg/kg), or sesame oil vehicle and killed at 72 h. Two thousand two hundred eighty-eight and 1347 genes were differentially expressed (P1(t) > 0.90) at one or more doses by TCDD and TCDF, respectively. Automated dose-response modeling (ToxResponse Modeler) identified a total of 1027 and 837 genes with either a sigmoidal, exponential, linear, Gaussian, or quadratic dose-response relationship 72 h after treatment in TCDD and TCDF, respectively. Two hundred seventy genes exhibited a sigmoidal TCDD-induced dose-response (ED(50s) from 0.08 to 42.2 microg/kg) compared to only 179 sigmoidal responsive genes (ED(50s) from 0.74 to 299.9 microg/kg) elicited by TCDF. Of the 1027 TCDD dose-responsive genes, 654 were not examined further due to the lack of a dose response elicited by TCDF. Of the 373 genes that exhibited a TCDD and TCDF dose response, REPs were calculated for the 83 genes that exhibited comparable sigmoidal curve shapes and slopes. The median REP for these 83 genes was 0.10, with a maximum REP of 0.56 and a minimum of 0.01. REPs of 0.04 were also calculated for EROD and increase in relative liver weight (RLW) at 72 h. Collectively, the lower number of TCDF-induced genes compared to TCDD and the 0.04 REPs for EROD activity and increased RLW are not consistent with the TEF of 0.10 for the hepatotoxicity of TCDF in C57BL/6 mice at 72 h.

Use of Xenopus laevis as a model for investigating in vitro and in vivo endocrine disruption in amphibians.

The estrogenic activity of 17beta-estradiol (E2), alpha-zearalenol (alpha-ZEA), genistein (GEN), and 4-t-octylphenol (4-t-OP) was investigated using Xenopus laevis-based assays. All test compounds competed with [3H]E2 for binding to a recombinant Xenopus estrogen receptor (xER) with the following relative affinities: E2 > alpha-ZEA > 4-t-OP > GEN. The ability of these compounds to induce xER-mediated reporter gene expression was then assessed in MCF-7 human breast cancer cells cotransfected with a Gal4-xERdef chimeric estrogen receptor and a Gal4-regulated luciferase reporter gene. Luciferase activity was increased 30- to 50-fold by 10 nM E2 relative to that in solvent control. Maximal reporter gene activity induced by 10 nM alpha-ZEA was 54% of that induced by E2; however, the activity did not increase following doses of up to 10 microM. A dose of 1 microM 4-t-OP induced 23% of the maximal reporter gene activity induced by E2, whereas 10 microM GEN induced activity to the same level as E2. A dose-dependent increase in vitellogenin (VTG) mRNA expression was observed in Xenopus treated intraperitoneally with E2 at 0.05 to 5 mg/kg/d for three consecutive days, with the maximal induction observed in the group receiving 1 mg/kg/d. The alpha-ZEA, GEN, and 4-t-OP also significantly induced VTG mRNA expression, although at higher doses. These results demonstrate the utility of X. laevis as an amphibian model to assess the estrogenic activity of endocrine disruptors.

Normal mammary gland morphology in pubertal female mice following in utero and lactational exposure to genistein at levels comparable to human dietary exposure.

The objective of the study was to determine the effect of in utero and lactational exposure to genistein (0, 0.1, 0.5, 2.5 and 10 mg/kg/day) on mammary gland morphology in female B6D2F1 mice at levels comparable to or greater than human exposures. The effect of diethylstilbestrol (DES; 0, 0.1, 1, 10 microg/kg/day) on the mammary gland was also examined as a positive estrogenic control. Pregnant females were treated by daily gavage from gestational day 12 to postnatal day (PND) 20. Female offspring were weaned on PND21 and mammary gland whole mounts were examined for growth (length and area of the epithelial tree), proliferation (number of terminal end buds (TEBs)), and differentiation (density of alveolar buds (ABs)) on PND49. The highest dose of DES induced a significant increase in mammary gland growth (P<0.05) and also decreased the number of TEBs (P<0.06). The density of ABs was not significantly affected by DES. By contrast to DES, genistein had no effect on mammary gland morphology at any dose. These results suggest that in utero and lactational exposure to genistein at levels comparable to or greater than human exposures do not adversely affect mammary gland development in pubertal female B6D2F1 mice.

Gestational and lactational exposure of male mice to diethylstilbestrol causes long-term effects on the testis, sperm fertilizing ability in vitro, and testicular gene expression.

The objective of the study was to determine the long-term effects of gestational and lactational exposure to diethylstilbestrol (DES; 0, 0.1, 1, and 10 microg/kg maternal body weight) on mouse testicular growth, epididymal sperm count, in vitro fertilizing ability, and testicular gene expression using cDNA microarrays and real-time PCR in mice on postnatal day (PND) 21, 105, and 315. In the high dose group there was a persistent decrease in the number of Sertoli cells, and sperm count was decreased on PND315 (P < 0.05). Sperm motion was unaffected; however, the in vitro fertilizing ability of epididymal sperm was decreased in the high dose group on both PND105 (P < 0.001) and PND315 (P < 0.05). Early and latent alterations in the expression of genes involved in estrogen signaling (estrogen receptor alpha), steroidogenesis (steroidogenic factor 1, 17alpha-hydroxylase/C17,20-lyase, P450 side chain cleavage, steroidogenic acute regulatory protein, and scavenger receptor class B1), lysosomal function (LGP85 and prosaposin), and regulation of testicular development (testicular receptor 2, inhibin/activin beta C, and Hoxa10) were confirmed by real-time PCR. The results demonstrate that early exposure to DES causes long-term adverse effects on testicular development and sperm function, and these effects are associated with changes in testicular gene expression, even long after the cessation of DES exposure.

In silico approaches to mechanistic and predictive toxicology: an introduction to bioinformatics for toxicologists.

Bioinformatics, or in silico biology, is a rapidly growing field that encompasses the theory and application of computational approaches to model, predict, and explain biological function at the molecular level. This information rich field requires new skills and new understanding of genome-scale studies in order to take advantage of the rapidly increasing amount of sequence, expression, and structure information in public and private databases. Toxicologists are poised to take advantage of the large public databases in an effort to decipher the molecular basis of toxicity. With the advent of high-throughput sequencing and computational methodologies, expressed sequences can be rapidly detected and quantitated in target tissues by database searching. Novel genes can also be isolated in silico, while their function can be predicted and characterized by virtue of sequence homology to other known proteins. Genomic DNA sequence data can be exploited to predict target genes and their modes of regulation, as well as identify susceptible genotypes based on single nucleotide polymorphism data. In addition, highly parallel gene expression profiling technologies will allow toxicologists to mine large databases of gene expression data to discover molecular biomarkers and other diagnostic and prognostic genes or expression profiles. This review serves to introduce to toxicologists the concepts of in silico biology most relevant to mechanistic and predictive toxicology, while highlighting the applicability of in silico methods using select examples.