Novel approach for verification of a human PBPK modeling strategy using chimeric mice in the health risk assessment of epyrifenacil.

In the human risk assessment by physiologically based pharmacokinetic modeling (PBPK), verification of the modeling strategy and confirmation of the reliability of the output data are important when the clinical data are not available. A new herbicide, epyrifenacil, is metabolized to S-3100-CA in mammals and causes hepatotoxicity in mice. S-3100-CA is transferred to the liver by transporters and eliminated by biliary excretion and metabolism. In the previous human PBPK research, we succeeded in predicting S-3100-CA pharmacokinetics by obtaining human hepatic parameters from chimeric mice with humanized liver after we checked the model's quantitative performance using mouse experimental data. To further enhance the reliability of human PBPK data, verification of the following two points was considered effective: 1) verification of model applicability to pharmacokinetics prediction in multiple animal species, and 2) verification of the parameter acquisition methods. In this study, we applied the same modeling strategy to rats, i.e., we obtained rat hepatic parameters for PBPK from chimeric mice with rat hepatocytes, not from rats. As the simulation results, rat internal dosimetry was precisely reproduced, although it tended to be slightly overestimated by approximately two times. From the results of the sensitivity analysis, this overestimation was mainly due to hepatic parameters from chimeric mice. Therefore, it is suggested that a similar slight prediction error may occur also in human PBPK using chimeric mice, but considering the degree of error, it can be said that our modeling strategy is robust and the predicted human internal dosimetry in the previous research is reliable.

Prediction of the human pharmacokinetics of epyrifenacil and its major metabolite, S-3100-CA, by a physiologically based pharmacokinetic modeling using chimeric mice with humanized liver.

Human internal dosimetry of pesticides is essential in the risk assessment when toxicity has been confirmed in laboratory animals. While human toxicokinetics data of pesticides are hardly obtained intendedly, the use of physiologically based pharmacokinetic (PBPK) modeling has become important for predicting human internal dosimetry. Especially, when the compound exhibits complicated pharmacokinetics via active uptake, metabolism, and biliary excretion in liver, it is difficult to obtain these hepatic parameters only by the in vitro experiments. Epyrifenacil, a new herbicide, is rapidly metabolized to S-3100-CA (CA) in mammals and causes hepatotoxicity in mice. CA is eliminated from the systemic circulation by biliary excretion and metabolism in liver. Although uptake of CA by transporters is observed in mouse primary hepatocytes, significantly less of it is observed in human primary hepatocytes. In order to evaluate human internal dosimetry of CA, a precise PBPK model was developed. To obtain human hepatic parameters, i.e., hepatic elimination intrinsic clearance via biliary excretion and metabolism, we used chimeric mice with humanized liver as a model to reproduce the complicated pharmacokinetics of CA in humans. After we developed a mouse PBPK model, by replacing mouse parameters with those of humans, we calculated CA concentration in human liver. Comparing the predicted CA exposure in human liver with the measured values in mice, we demonstrated a clear interspecies difference of approximately 4 times lower Cmax and AUC in humans. This result suggested that the risk of hepatotoxicity is less in humans than in mice.

Flumioxazin metabolism in pregnant animals and cell-based protoporphyrinogen IX oxidase (PPO) inhibition assay of fetal metabolites in various animal species to elucidate the mechanism of the rat-specific developmental toxicity.

Flumioxazin, an N-phenylimide herbicide, inhibits protoporphyrinogen oxidase (PPO), a key enzyme in heme biosynthesis in mammals, and causes rat-specific developmental toxicity. The mechanism has mainly been clarified, but no research has yet focused on the contribution of its metabolites. We therefore conducted in vivo metabolism studies in pregnant rats and rabbits, and found 6 major known metabolites in excreta. There was no major rat-specific metabolite. The most abundant component in rat fetuses was APF, followed by flumioxazin and 5 identified metabolites. The concentrations of flumioxazin and these metabolites in fetuses were lower in rabbits than in rats. In vitro PPO inhibition assays with rat and human liver mitochondria showed that flumioxazin is a more potent PPO inhibitor than the metabolites. There were no species differences in relative intensity of PPO inhibition among flumioxazin and these metabolites. Based on the results of these in vivo and in vitro experiments, we concluded that flumioxazin is the causal substance of the rat-specific developmental toxicity. As a more reliable test system for research on in vitro PPO inhibition, cell-based assays with rat, rabbit, monkey, and human hepatocytes were performed. The results were consistent with those of the mitochondrial assays, and rats were more sensitive to PPO inhibition by flumioxazin than humans, while rabbits and monkeys were almost insensitive. From these results, the species difference in the developmental toxicity was concluded to be due to the difference in sensitivity of PPO to flumioxazin, and rats were confirmed to be the most sensitive of these species.

Metabolism of metofluthrin in rats: I. Identification of metabolites.

1. Metofluthrin (2,3,5,6-tetrafluoro-4-(methoxymethyl)benzyl (Z/E)-(1R)-trans-2,2-dimethyl-3-(1-propenyl)-cyclopropanecarboxylate) is a novel pyrethroid insecticide, which has E/Z isomers at prop-1-enyl group. 2. Rats were orally dosed with each [14C]-labelled E/Z isomer, and the excreta were collected for isolation and identification of metabolites. Analysis of the excreta by LC/MS and NMR revealed formation of 33 and 23 (total 42) metabolites from rats dosed with Z-isomer and E-isomer, respectively. 3. Major metabolic reactions were cleavage of ester linkage, O-demethylation, hydroxylation, epoxidation or reduction of double bond, glutathione conjugation and its further metabolism, hydroxylation of epoxide and formation of lactone ring. Notably, the acid side, 2,2-dimethyl-3-(1-propenyl)-cyclopropanecarboxylic acid, was much more variously metabolised compared to chrysanthemic acid, the acid side of the known pyrethroids. 4. Major metabolites for Z-isomer mostly retained ester linkage with 1,2-dihydroxypropyl group and/or 2-methylalcohol of cyclopropane ring, while most of those for E-isomer received hydrolysis of the ester linkage without oxidation at the 1-propenyl group or the gem-methyl groups, suggesting epoxidation and hydroxylation could occur more easily on Z-isomer. 5. As the novel metabolic pathways for pyrethroids, isomerisation of ω-carboxylic acid moiety, reduction or hydration of double bond and cleavage of cyclopropane ring via epoxidation were suggested.

Metabolism of metofluthrin in rats: II. Excretion, distribution and amount of metabolites.

1. 14 C-Labelled E/Z isomers of a synthetic pyrethroid metofluthrin ((E/Z)-(1 R,3 R)-2,3,5,6-tetrafluoro-4-(methoxymethyl)benzyl 2,2-dimethyl-3-(1-propenyl)-cyclopropanecarboxylate, abbreviated as RTE/RTZ, respectively) were used for rat metabolism studies. 14 C-RTE or RTZ labelled at the carbonyl-carbon [acid-14C] or the methoxymethylbenzyl-α-carbon [alcohol-14 C] was administered orally to rats at 1 and 20 mg/kg. 2. Dosed compounds were mostly absorbed, metabolised, and rapidly excreted. Dose-related increase in blood AUC suggested no saturation of absorption at the high dose. Blood 14 C was maximal at 3-8 h and decreased with a half-life of 52-163 h. Radioactivity in tissues, blood and plasma decreased basically at the same rate and the sum fell below 0.2% of the dose at 168 h. 3. Although the major metabolic pathways of the isomers, that is, ester cleavage, O-demethylation and ω-oxidation, were similar, there was a notable difference. The RTZ double bond commonly undergoes epoxidation while RTE double bond mainly undergoes glutathione conjugation, which causes faster elimination from plasma and greater excretion into faeces on RTE. Faster urinary excretion and elimination from blood were observed for the alcohol moiety than the acid moiety. 4. In conclusion, this study described the overall metabolic profiles of metofluthrin and identified the differences in metabolic breakdown between the isomers. No marked sex-/dose-related differences were observed.

Combining Genomics To Identify the Pathways of Post-Transcriptional Nongenotoxic Signaling and Energy Homeostasis in Livers of Rats Treated with the Pregnane X Receptor Agonist, Pregnenolone Carbonitrile.

Transcriptomic, proteomic, phosphoproteomic, and metabolomic analyses were combined to determine the role of pregnane X receptor (PXR) in nongenotoxic signaling and energy homeostasis in liver after rats were repeatedly orally dosed with the PXR agonist pregnenolone carbonitrile (PCN) for 7 days. Analyses of mRNAs and proteins in the supernatant, membrane, and cytosolic fractions of enlarged liver homogenates showed diverse expression profiles. Gene set enrichment analysis showed that the synchronous increase in mRNAs and proteins involved in chemical carcinogenesis and the response to drug was possibly mediated by the PXR pathway and proteasome core complex assembly was possibly mediated by the Nrf2 pathway. In addition, levels of proteins in the endoplasmic reticulum lumen and involved in the acute-phase response showed specific increase with no change in mRNA level, and those composed of the mitochondrial inner membrane showed specific decrease. The analysis of phosphorylated peptides of poly(A) RNA binding proteins showed a decrease in phosphorylation, possibly by casein kinase 2, which may be related to the regulation of protein expression. Proteins involved in insulin signaling pathways showed an increase in phosphorylation, possibly by protein kinase A, and those involved in apoptosis showed a decrease. Metabolomic analysis suggested the activation of the pentose phosphate and anaerobic glycolysis pathways and the increase of amino acid and fatty acid levels, as occurs in the Warburg effect. In conclusion, the results of combined analyses suggest that PXR's effects are due to transcriptional and post-transcriptional regulation with alteration of nongenotoxic signaling pathways and energy homeostasis.

Identification and in silico prediction of metabolites of the model compound, tebufenozide by human CYP3A4 and CYP2C19.

The metabolites of tebufenozide, a model compound, formed by the yeast-expressed human CYP3A4 and CYP2C19 were identified to clarify the substrate recognition mechanism of the human cytochrome P450 (CYP) isozymes. We then determined whether tebufenozide metabolites may be predicted in silico. Hydrogen abstraction energies were calculated with the density functional theory method B3LYP/6-31G(∗). A docking simulation was performed using FRED software. Several alkyl sites of tebufenozide were hydroxylated by CYP3A4 whereas only one site was modified by CYP2C19. The accessibility of each site of tebufenozide to the reaction center of CYP enzymes and the susceptibility of each hydrogen atom for metabolism by CYP enzymes were evaluated by a docking simulation and hydrogen abstraction energy estimation, respectively.

Quantitative structure-activity relationship model for the fetal-maternal blood concentration ratio of chemicals in humans.

A quantitative structure-activity relationship (QSAR) model of the fetal-maternal blood concentration ratio (F/M ratio) of chemicals was developed to predict the placental transfer in humans. Data on F/M ratio of 55 compounds found in the literature were separated into training (75%, 41 compounds) and testing sets (25%, 14 compounds). The training sets were then subjected to multiple linear regression analysis using the descriptors of molecular weight (MW), topological polar surface area (TopoPSA), and maximum E-state of hydrogen atom (Hmax). Multiple linear regression analysis and a cross-validation showed a relatively high adjusted coefficient of determination (Ra(2)) (0.73) and cross-validated coefficient of determination (Q(2)) (0.71), after removing three outliers. In the external validation, R(2) for external validation (R(2)pred) was calculated to be 0.51. These results suggested that the QSAR model developed in this study can be considered reliable in terms of its robustness and predictive performance. Since it is difficult to examine the F/M ratio in humans experimentally, this QSAR model for prediction of the placental transfer of chemicals in humans could be useful in risk assessment of chemicals in humans.

Metabolism and physiologically based pharmacokinetic modeling of flumioxazin in pregnant animals.

A physiologically based pharmacokinetic (PBPK) model was developed to predict the concentration of flumioxazin, in the blood and fetus of pregnant humans during a theoretical accidental intake (1000mg/kg). The data on flumioxazin concentration in pregnant rats (30mg/kg po) was used to develop the PBPK model in pregnant rats using physiological parameters and chemical specific parameters. The rat PBPK model developed was extrapolated to a human model. Liver microsomes of female rats and a mixed gender of humans were used for the in vitro metabolism study. To determine the % of flumioxazin absorbed after administration at a dose of 1000mg/kg assuming maximum accidental intake, the biliary excretion study of [phenyl-U-(14)C]flumioxazin was conducted in bile duct-cannulated female rats (Crl:CD (SD)) to collect and analyze the bile, urine, feces, gastrointestinal tract, and residual carcass. The % of flumioxazin absorbed at a dose of 1000mg/kg in rats was low (12.3%) by summing up (14)C of the urine, bile, and residual carcass. The pregnant human model that was developed demonstrated that the maximum flumioxazin concentration in the blood and fetus of a pregnant human at a dose of 1000mg/kg po was 0.86µg/mL and 0.68µg/mL, respectively, which is much lower than Km (202.4µg/mL). Because the metabolism was not saturated and the absorption rate was low at a dose of 1000mg/kg, the calculated flumioxazin concentration in pregnant humans was thought to be relatively low, considering the flumioxazin concentration in pregnant rats at a dose of 30mg/kg. For the safety assessment of flumioxazin, these results would be useful for further in vitro toxicology experiments.

Metabolism of propyrisulfuron: 14C excretion, 14C concentration in plasma and tissues, and amount of metabolites in rats.

1. Metabolism of a novel sulfonylurea herbicide, propyrisulfuron [1-(2-chloro-6-propylimidazo[1,2-b]pyridazin-3-ylsulfonyl)-3-(4,6-dimethoxypyrimidin-2-yl)urea] labeled at the C-1 position of the propyl group and C-5 position of the pyrimidine ring with (14)C was investigated after a single oral administration in male and female rats. 2. Administered (14)C was excreted into the urine (5.7-29.8%) and feces (64.6-97.4%), respectively. (14)C concentration in plasma reached a maximum level at 4 to 12 h post-administration and then decreased rapidly with a biological half-life of approximately 23 to 32 h. Total (14)C residues in the whole body were <0.1-1.4%, suggesting that the residues were not accumulated in the tissues. 3. The amount of metabolites in urine, feces, and bile were quantified using high-performance liquid chromatography (HPLC). There were no differences in metabolites found between male and female rats. 4. The absorption for the low dose (5 mg/kg) and the high dose (1000 mg/kg) was estimated to be approximately 90% and 20%, respectively, suggesting a saturable absorption. 5. The plasma protein binding in male and female rats was ≥ 98.8%, suggesting that propyrisulfuron had a strong affinity to plasma proteins.

Metabolism of 2,6-dichloro-4-(3,3-dichloroallyloxy)phenyl 3-[5-(trifluoromethyl)-2-pyridyloxy]propyl ether (pyridalyl) in rats after repeated oral administration and a simple physiologically based pharmacokinetic modeling in brown and white adipose tissues.

Male and female Sprague-Dawley rats received repeated oral administration of 14C-2,6-dichloro-4-(3,3-dichloroallyloxy)phenyl 3- [5-(trifluoromethyl)-2-pyridyloxy]propyl ether (14C-pyridalyl) at 5 mg/kg/day for 14 consecutive days, and 14C excretion, 14C concentration in tissues, and the metabolic fate were determined. Most 14C was excreted into feces. The 14C concentrations in the blood and tissues attained steady-state levels at days 6 to 10, whereas those in white adipose tissues increased until day 14. Tissue 14C concentrations were highest in brown and white adipose tissue (38.37-57.50 ppm) but were 5.60 ppm or less in all the other tissues. Total 14C residues in blood and tissues on the 27th day after the first administration accounted for 2.6 to 3.2% of the total dose. A major fecal metabolite resulted from O-dealkylation. Analysis of metabolites in tissues revealed that the majority of 14C in perirenal adipose tissue and lungs was pyridalyl, accounting for greater than 90 and 60%, respectively, of the total, whereas a major metabolite in whole blood, kidneys, and liver was a dehalogenated metabolite. The experimental data were simulated with simple physiologically based pharmacokinetics using four-compartment models with assumption of lymphatic absorption and membrane permeability in adipose tissues. The different kinetics in brown and white adipose tissues was reasonably predicted in this model, with large distribution volume in adipose tissues and high hepatic clearance in liver. Sex-related difference of pyridalyl concentration in liver was considered to be a result of different unbound fraction times the hepatic intrinsic clearance (f x CL(int)) of 1.8 and 12 l/h for male and female, respectively.

Circulating natural antibodies against 3'-sialyllactose complement the diagnostic performance of CA19-9 for the early detection of pancreatic ductal adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma is a devastating malignancy with an extremely poor prognosis. Although the most widely used biomarker for pancreatic cancer is carbohydrate antigen CA19-9, it is elevated mainly in the late stage of pancreatic cancer. Some serum natural antibodies against carbohydrates have been shown to be possible diagnostic markers for cancer. OBJECTIVE: This study was conducted to determine whether the level of natural antibodies against carbohydrates fluctuates in pancreatic ductal adenocarcinoma. METHODS: Serum from pancreatic cancer subjects (n= 55) and 43 subjects free of malignant disease were studied. The contents of natural antibodies against sialyl glycans and CA19-9 in serum were determined by enzyme-linked immunosorbent assay. RESULTS: The level of serum anti-3'-sialyllactose antibodies in pancreatic cancer subjects was significantly lower than that in healthy controls. In contrast, the amounts of serum antibodies against other sialyl glycans were comparable between the two groups. Concentration of serum anti-3'-sialyllactose IgG provided excellent AUC of 0.86, with sensitivity 82%, specificity 81%, and accuracy 82%. The combination of serum anti-3'-sialyllactose IgG with CA19-9 improved the sensitivity of pancreatic cancer detection at an early stage. CONCLUSIONS: Natural antibodies against 3'-sialyllactose constitute a promising biomarker for pancreatic cancer detection. The measurement of serum anti-3'-sialyllactose antibodies could play a supportive role in diagnostics and complement the performance of CA19-9 for the early detection of pancreatic ductal adenocarcinoma.

Metabolism of pyridalyl in rats.

Metabolism of pyridalyl [2,6-dichloro-4-(3,3-dichloroallyloxy)phenyl 3-[5-(trifluoromethyl)-2-pyridyloxy]propyl ether] was examined in male and female Sprague-Dawley rats. After a single oral administration of [dichlorophenyl-(14)C]pyridalyl at 5 or 500 mg/kg, the (14)C concentration in blood reached maxima at 2 to 10 h and then decreased rapidly with a biological half-life of approximately 11 to 12 h. (14)C concentrations in liver, fat, adrenal gland, and spleen were relatively high at a low dose, reaching 2.3 to 2.7, 1.9 to 2.3, 1.1 to 1.9, and 1.4 ppm, respectively, in these tissues at 2 to 24 h after administration. Although (14)C elimination from fat and hair and skin was relatively slow compared with that from other tissues, the total residue on the 7th day was low, in the range of 1.3 to 2.3% of the dose. The (14)C distribution in tissues with a high dose, as examined by whole-body autoradiography, was similar to that observed for the low dose. Results revealed that more than 88% of the dosed radiocarbon was excreted within 1 day after administration, with cumulative (14)C excretion into urine and feces 7 days after administration of 1.7 to 2.6 and 98.7 to 101.7%, respectively. One urinary and fecal major metabolite (resulting from O-dealkylation) and two minor metabolites were identified by NMR and mass spectrometry. Residual (14)C in fat was extracted, and analysis by thin-layer chromatography showed it to be due to pyridalyl itself. No marked sex-related differences were observed in (14)C elimination, (14)C distribution, and metabolites.

Comparison of the effects of the synthetic pyrethroid Metofluthrin and phenobarbital on CYP2B form induction and replicative DNA synthesis in cultured rat and human hepatocytes.

High doses of Metofluthrin (MTF) have been shown to produce liver tumours in rats by a mode of action (MOA) involving activation of the constitutive androstane receptor leading to liver hypertrophy, induction of cytochrome P450 (CYP) forms and increased cell proliferation. The aim of this study was to compare the effects of MTF with those of the known rodent liver tumour promoter phenobarbital (PB) on the induction CYP2B forms and replicative DNA synthesis in cultured rat and human hepatocytes. Treatment with 50 microM MTF and 50 microM PB for 72 h increased CYP2B1 mRNA levels in male Wistar rat hepatocytes and CYP2B6 mRNA levels in human hepatocytes. Replicative DNA synthesis was determined by incorporation of 5-bromo-2'-deoxyuridine over the last 24 h of a 48 h treatment period. Treatment with 10-1000 microM MTF and 100-500 microM PB resulted in significant increases in replicative DNA synthesis in rat hepatocytes. While replicative DNA synthesis was increased in human hepatocytes treated with 5-50 ng/ml epidermal growth factor or 5-100 ng/ml hepatocyte growth factor, treatment with MTF and PB had no effect. These results demonstrate that while both MTF and PB induce CYP2B forms in both species, MTF and PB only induced replicative DNA synthesis in rat and not in human hepatocytes. These results provide further evidence that the MOA for MTF-induced rat liver tumour formation is similar to that of PB and some other non-genotoxic CYP2B form inducers and that the key event of increased cell proliferation would not occur in human liver.

Involvement of Peroxisome Proliferator-Activated Receptor-Alpha in Liver Tumor Production by Permethrin in the Female Mouse.

The nongenotoxic pyrethroid insecticide permethrin produced hepatocellular tumors in CD-1 mice but not in Wistar rats. Recently, based on findings of a Pathology Working Group involving an expert panel of pathologists, it was concluded that permethrin increased liver tumors at 2500 and 5000 ppm in female mice, but no treatment-related tumorigenic response occurred in male mice at dose levels examined in the 2-year bioassay. To evaluate a possible mode of action (MOA) for the permethrin female CD-1 mouse hepatocellular tumors, a number of investigative studies were conducted. In time-course studies in female CD-1 mice, permethrin increased relative liver weight and enhanced hepatocyte proliferation within 1 week. Treatment with permethrin resulted in marked increases in CYP4A enzyme activities and mRNA levels, but only slightly increased CYP2B markers, suggesting that permethrin primarily activates the peroxisome proliferator-activated receptor alpha (PPARα) and to a much lesser extent the constitutive androstane receptor. The effects of permethrin on relative liver weight, hepatocyte proliferation and CYP4A enzyme activities and mRNA levels were dose-dependent and were reversible within 5 weeks after cessation of treatment. The hepatic effects of permethrin observed in wild-type female mice were markedly reduced in PPARα knockout female mice. These results demonstrate that the MOA for hepatocellular tumor formation by permethrin in female mice involves activation of PPARα resulting in a mitogenic effect. The MOA for permethrin-induced mouse liver tumor formation due to PPARα activation is considered to be not plausible for humans. This conclusion is strongly supported by available epidemiological data for permethrin.

Species differences in the developmental toxicity of procymidone-Placental transfer of procymidone in pregnant rats, rabbits, and monkeys.

To clarify species differences in the developmental toxicity of procymidone (Sumilex®, a fungicide for agricultural use), placental transfer studies were conducted using 14C-labeled procymidone in pregnant rats, rabbits, and monkeys. These studies demonstrated that maternal-to-fetal transfer of the parent compound and its hydroxylated metabolite, which are both weak anti-androgenic agents, occurred more easily than that of other metabolites, with much higher absolute concentrations achieved in the fetal circulation of rats than of rabbits or monkeys. Notably, in rats, the fetal plasma concentration of the hydroxylated metabolite was higher than that of procymidone, especially after repeated oral administration of procymidone. These results suggest that the hydroxylated metabolite is the most relevant metabolite involved in teratogenic activity in rats.

Editor's Highlight: Development of Novel Neural Embryonic Stem CellTests for High-Throughput Screening of Embryotoxic Chemicals.

There is a great demand for appropriate alternative methods to rapidly evaluate the developmental and reproductive toxicity of a wide variety of chemicals. We used the differentiation of mouse embryonic stem cells (mESCs) into cardiomyocytes as a basis for establishing a rapid and highly reproducible invitro embryotoxicity test known as the Hand1-Luc Embryonic Stem Cell Test (Hand1-Luc EST). In this study, we developed novel neural-Luc ESTs using two marker genes for neural development, tubulin beta-3 (Tubb3) and Reelin (Reln), and evaluated the capacity of these tests to predict developmental toxicity. In addition, we tested whether an integrated approach (a combination of neural-Luc ESTs and the Hand1-Luc EST) improved developmental toxicant detection. To perform our neural-Luc ESTs, we needed to generate stable transgenic mESCs with individual promoters linked to the luciferase gene, and to establish that similar changes in promoter activities and mRNA expression levels occur during neural differentiation. Based on the concentration-response curves of 15 developmental toxicants and 17 non-developmental toxic chemicals, we derived a prediction formula and assessed the capacity of this formula to predict developmental toxicity. Although both were highly sensitive and specific for predicting developmental toxicity, neural-Luc ESTs had similar predictive capacities. In contrast, neural-Luc ESTs and Hand1-Luc EST had significantly different predictive powers. As expected, the combination of these ESTs increased the sensitivity of developmental toxicant detection. These results demonstrate the convenience and the usefulness of this combination of ESTs as an alternative assay system for future toxicological and mechanistic studies of developmental toxicity.

Editor's Highlight: Mode of Action Analysis for Rat Hepatocellular Tumors Produced by the Synthetic Pyrethroid Momfluorothrin: Evidence for Activation of the Constitutive Androstane Receptor and Mitogenicity in Rat Hepatocytes.

High dietary levels of momfluorothrin, a nongenotoxic synthetic pyrethroid, induced hepatocellular tumors in male and female Wistar rats in a 2-year bioassay. The mode of action (MOA) for rat hepatocellular tumors was postulated to occur via activation of the constitutive androstane receptor (CAR), as momfluorothrin is a close structural analogue of the pyrethroid metofluthrin, which is known to produce rat liver tumors through a CAR-mediated MOA. To elucidate the MOA for rat hepatocellular tumor formation by momfluorothrin, this study was conducted to examine effects on key and associative events of the CAR-mediated MOA for phenobarbital based on the International Programme on Chemical Safety framework. A 2-week in vivo study in Wistar rats revealed that momfluorothrin induced CYP2B activities, increased liver weights, produced hepatocyte hypertrophy and increased hepatocyte replicative DNA synthesis. These effects correlated with the dose-response relationship for liver tumor formation and also showed reversibility upon cessation of treatment. Moreover, momfluorothrin did not increase CYP2B1/2 mRNA expression and hepatocyte replicative DNA synthesis in CAR knockout rats. Using cultured Wistar rat hepatocytes and the RNA interference technique, knockdown of CAR resulted in a suppression of induction of CYP2B1/2 mRNA levels by momfluorothrin. Alternative MOAs for liver tumor formation were excluded. A global gene expression profile analysis of the liver of male Wistar rats treated with momfluorothrin for 2 weeks also showed similarity to the prototypic CAR activator phenobarbital. Overall, these data strongly support that the postulated MOA for momfluorothrin-induced rat hepatocellular tumors as being mediated by CAR activation.

Prediction of in vivo developmental toxicity by combination of Hand1-Luc embryonic stem cell test and metabolic stability test with clarification of metabolically inapplicable candidates.

Hand1-Luc Embryonic Stem Cell Test (Hand1-Luc EST) is a promising alternative method for evaluation of developmental toxicity. However, the problems of predictivity have remained due to appropriateness of the solubility, metabolic system, and prediction model. Therefore, we assessed the usefulness of rat liver S9 metabolic stability test using LC-MS/MS to develop new prediction model. A total of 71 chemicals were analyzed by measuring cytotoxicity and differentiation toxicity, and highly reproducible (CV=20%) results were obtained. The first prediction model was developed by discriminant analysis performed on a full dataset using Hand1-Luc EST, and 66.2% of the chemicals were correctly classified by the cross-validated classification. A second model was developed with additional descriptors obtained from the metabolic stability test to calculate hepatic availability, and an accuracy of 83.3% was obtained with applicability domain of 50.7% (=36/71) after exclusion of 22 metabolically inapplicable candidates, which potentially have a metabolic activation property. A step-wise prediction scheme with combination of Hand1-Luc EST and metabolic stability test was therefore proposed. The current results provide a promising in vitro test method for accurately predicting in vivo developmental toxicity.

Hand1-Luc embryonic stem cell test (Hand1-Luc EST): a novel rapid and highly reproducible in vitro test for embryotoxicity by measuring cytotoxicity and differentiation toxicity using engineered mouse ES cells.

The embryonic stem cell test (EST) is a promising alternative method for evaluating embryotoxicity of test chemicals by measuring cytotoxicity and differentiation toxicity using mouse ES cells. Differentiation toxicity is analyzed by microscopically counting the beating of embryonic bodies after 10 days of culture. However, improvements are necessary to reduce the laborious manipulations involved and the time required to obtain results. We have previously reported the successful stable transfection of ES cells (ES-D3) with the heart and neural crest derivatives expressed transcript 1 (Hand1) gene and the establishment of a 96-well multi-plate-based new EST with luciferase reporter assay 6 days after treatment with test chemicals. Now, we propose an even more rapid and easier EST, named Hand1-Luc EST. We established another cell line to monitor the Hand1 gene expression via a luciferase reporter gene. By mRNA analysis and luciferase assay, we examined in detail the luciferase activity during cell differentiation, which allowed us to reduce the time of measurement from day 6 to day 5 (120 hr). Furthermore, the protocol was improved, with, among others, the measurement of cytotoxicity and differentiation toxicity taking place in the same 96-well round bottom plate instead of two different plates. With the positive control, 5-fluorouracil (5-FU), and 9 test chemicals, data with high reproducibility and very low variation (CV < 50%) in the relevant endpoints were obtained. This study shows that the Hand1-Luc EST could provide an accurate and sensitive short-term test for prediction of embryotoxicants by measuring cytotoxicity and differentiation toxicity from the same sample.