Proof of concept for quantitative adverse outcome pathway modeling of chronic toxicity in repeated exposure.

Adverse Outcome Pathway (AOP) is a useful tool to glean mode of action (MOE) of a chemical. However, in order to use it for the purpose of risk assessment, an AOP needs to be quantified using in vitro or in vivo data. Majority of quantitative AOPs developed so far, were for single exposure to progressively higher doses. Limited attempts were made to include time in the modeling. Here as a proof-of concept, we developed a hypothetical AOP, and quantified it using a virtual dataset for six repeated exposures using a Bayesian Network Analysis (BN) framework. The virtual data was generated using realistic assumptions. Effects of each exposure were analyzed separately using a static BN model and analyzed in combination using a dynamic BN (DBN) model. Our work shows that the DBN model can be used to calculate the probability of adverse outcome when other upstream KEs were observed earlier. These probabilities can help in identification of early indicators of AO. In addition, we also developed a data driven AOP pruning technique using a lasso-based subset selection, and show that the causal structure of AOP is itself dynamic and changes over time. This proof-of-concept study revealed the possibility for expanding the applicability of the AOP framework to incorporate biological dynamism in toxicity appearance by repeated insults.

Oxidative stress-mediated epidermal growth factor receptor activation by cigarette smoke or heated tobacco aerosol in human primary bronchial epithelial cells from multiple donors.

The epidermal growth factor receptor (EGFR) signaling pathway has essential roles in maintaining homeostasis of various tissues by regulating cell proliferation and differentiation. Deregulation of the EGFR signaling pathway is associated with various chronic diseases including chronic obstructive pulmonary disease. Cigarette smoke (CS) is known to activate EGFR, which is linked to chronic obstructive pulmonary disease. The biological sequence from CS exposure to EGFR activation is initiated by oxidative stress caused by intracellular reactive oxygen species (ROS) and the depletion of glutathione, which led to EGFR ligand secretion and EGFR activation. We hypothesized that reducing exposure to CS constituents contributes to preventing CS-inducible EGFR activation. Therefore, we examined the aerosol from heated tobacco products (HTPs) because the aerosol contains fewer chemical constituents at lower levels than CS. We exposed primary human bronchial epithelial cells from four donors to the extracts of CS from a 1R6F reference cigarette or HTP aerosol from three in-market products, including our DT3.0a. The biological sequence from ROS to EGFR activation was assessed. CS induced all the tested endpoints although inter-donor differences were observed, whereas HTPs elicited most of the biological events at higher concentrations; however, EGFR phosphorylation was not observed even at fivefold higher concentration than CS. Overall, our results indicate that HTPs are less effective than CS to elicit ROS-induced EGFR activation. The reduced-risk potential of HTPs on EGFR-related diseases should be investigated further. In addition, testing with multiple donors is warranted when considering the individual differences in responses of primary cells to stimuli.

Dietary Egg Yolk Supplementation Improves Low-Protein-Diet-Induced Fatty Liver in Rats.

Egg yolk is an important source of nutrients and contains different bioactive substances. In the present study, we studied the benefits of egg yolk in preventing low-protein-diet-induced fatty liver in rats. Rats were fed the following diets, which were based on the AIN-76 formula, for 2 wk: an adequate-protein diet containing 20% casein (C), a low-protein diet containing 5% casein (LP-C), a low-protein diet supplemented with 12.5% egg yolk (LP-EY), and a low-protein diet supplemented with 4.1% egg yolk oil (LP-EYO). The low-protein diets were adjusted to contain 4.13% protein and 4.7% lipids. The LP-C diet resulted in a greater increase in the liver trigriceride (TG) and the vacuolation and a greater decrease in the serum TG and free fatty acid (FFA) than did the C diet. These deviations in the serum and liver TG, serum FFA levels and the liver histopathology were corrected in rats fed the LP-EY diet but not in those fed the LP-EYO diet. Compared to rats fed the LP-C diet, although the activities of lipogenesis-related enzymes (fatty acid synthase, glucose-6-phosphate dehydrogenase, and malic enzyme) decreased in rats fed both of the LP-EY and LP-EYO diets, the level of the microsomal TG transfer protein (MTP) increased only in rats fed the LP-EY diet. Collectively, these results suggest that dietary egg yolk supplementation decreases the LP diet-induced accumulation of TG in the liver by increasing transport of TG in the liver, and egg yolk oil alone is not sufficient enough to bring about these benefits.

Assessing the mutagenic activities of smoke from different cigarettes in direct exposure experiments using the modified Ames Salmonella assay.

The Ames assay is useful for evaluating the mutagenic potentials of chemicals, and it has been used to evaluate the mutagenic potential of cigarette smoke (CS). In vitro direct exposure systems have been developed to mimic CS exposure in the human respiratory tract, and the Ames assay has been used with such systems. Ames tests were performed using the Vitrocell(®) direct exposure system in this study. The mutagenic potentials of whole mainstream CS and gas/vapor phase fractions produced by conventional combustible cigarettes under two smoking regimens were compared. Salmonella Typhimurium TA98 and TA100 were used with and without metabolic activation, and the number of revertants induced by exposure to each CS was determined. The amount of smoke particles to which cells were exposed were also determined, and dose-response curves describing the relationships between exposure to smoke particles and the number of revertants induced were plotted. The slopes of linear regressions of the dose-response curves were determined, and the slope for each CS was used as a mutagenic activity index for that CS. A new heated cigarette was also tested and smoke from the heated cigarette had a lower mutagenic activity in TA98 and TA100 with metabolic activation than did the conventional CS. The results indicate that the direct exposure system and the Ames test can be used to determine the mutagenic potentials of CS produced by different cigarettes under different conditions (i.e., using different Salmonella Typhimurium strains with and without metabolic activation, and using different smoking conditions).

LXR agonist increases the lymph HDL transport in rats by promoting reciprocally intestinal ABCA1 and apo A-I mRNA levels.

Liver and intestine are major sites of apo A-I synthesis in mammals. ABCAI is reported to be involved in the secretion of nascent HDL from cultured intestinal cells. However, whether ABCA1 participates in the secretion of nascent HDL from the intestine has not been assessed directly in vivo. This study examined the effect of a synthetic LXR-agonist "TO" on the lymphatic transport of HDL in thoracic duct-cannulated rats. The feeding of a TO-containing diet resulted in an increased transport of cholesterol and apo A-I in the lymph d > 1.063 g/ml lipoprotein fraction than did the feeding of a control diet without TO. The transport of cholesterol in whole lymph was lower, whereas the transport of apo A-I was higher, in the TO group. The abundance of mRNAs for ABCAI and apo A-I in the intestine was increased in the TO group. Furthermore, although the TO-containing diet reportedly increased the serum HDL concentration in intact mice and rats, no such effect was observed in the cannulated rats. The LXR agonist stimulated in vivo the synthesis of nascent HDL by increasing reciprocally the mRNA for ABCAI and apo A-I in the intestine, thereby contributing to an increase in the circulating HDL.

Hypocholesterolemic effects of microbial protease-resistant fraction of Katsuobushi in ovariectomized rats depend on the both oil and undigested protein.

The purpose of this study was to examine which component in the microbial protease-resistant fraction of Katsuobushi (KBR), smoke-dried bonito, is hypocholesterolemic in ovariectomized rats (OVX-rats). KBR contains two major components: oil and protease-resistant protein. Oil extracted from KBR (EX) was rich in palmitic, oleic and docosahexaenoic acids. OVX-rats were fed one of the following diets for 28 d: diets containing casein as the protein source (C or C+EX diet), a diet containing KBR as the protein source (KBR diet) or diets containing degreased KBR as the protein source (DF/KBR or DF/KBR+EX diet). The C and DF/KBR diets contained soybean oil as the oil source. In the C+EX, KBR and DF/KBR+EX diets, soybean oil was replaced by oil extracted from KBR (EX). Plasma total- and low density lipoprotein-cholesterol concentrations in the C+EX, KBR and DF/KBR+EX groups, but not in the DF/KBR group, were significantly lower than that in the C group. Fecal bile acid excretion was significantly greater in the C+EX, KBR, DF/KBR and DF/KBR+EX groups in comparison to the C group, whereas excretion in the KBR and DF/KBR+EX groups was significantly greater than in the C+EX and DF/KBR groups. Cholesterol 7alpha-hydroxylase activity was higher in the C+EX, KBR, DF/KBR and DF/KBR+EX groups than the C group. In OVX-rats fed C, C+EX or KBR for 28 d, bile acid flux into the small intestine increased in KBR and C+EX groups in comparison to the C group. The hypocholesterolemic effect of KBR in OVX-rats reflected in increased fecal bile acid excretion may be mediated by increased bile acid flux caused by EX and the binding of bile acids by protease-resistant proteins.