Phenobarbital and propiconazole toxicogenomic profiles in mice show major similarities consistent with the key role that constitutive androstane receptor (CAR) activation plays in their mode of action.
ABSTRACT: Toxicogenomics (TGx) is employed frequently to investigate underlying molecular mechanisms of the compound of interest and, thus, has become an aid to mode of action determination. However, the results and interpretation of a TGx dataset are influenced by the experimental design and methods of analysis employed. This article describes an evaluation and reanalysis, by two independent laboratories, of previously published TGx mouse liver microarray data for a triazole fungicide, propiconazole (PPZ), and the anticonvulsant drug phenobarbital (PB). Propiconazole produced an increase incidence of liver tumors in male CD-1 mice only at a dose that exceeded the maximum tolerated dose (2500 ppm). Firstly, we illustrate how experimental design differences between two in vivo studies with PPZ and PB may impact the comparisons of TGx results. Secondly, we demonstrate that different researchers using different pathway analysis tools can come to different conclusions on specific mechanistic pathways, even when using the same datasets. Finally, despite these differences the results across three different analyses also show a striking degree of similarity observed for PPZ and PB treated livers when the expression data are viewed as major signaling pathways and cell processes affected. Additional studies described here show that the postulated key event of hepatocellular proliferation was observed in CD-1 mice for both PPZ and PB, and that PPZ is also a potent activator of the mouse CAR nuclear receptor. Thus, with regard to the events which are hallmarks of CAR-induced effects that are key events in the mode of action (MOA) of mouse liver carcinogenesis with PB, PPZ-induced tumors can be viewed as being promoted by a similar PB-like CAR-dependent MOA.
Project description:The goal of this study was to apply transcriptional analyses to hepatic tissues from mice exposed to PB, propiconazole (Pro) or triadimefon (Tri) at tumorigenic exposure levels to reveal similarities and differences in response among these treatments.The goal of this study was to apply transcriptional analyses to hepatic tissues from mice exposed to PB, propiconazole (Pro) or triadimefon (Tri) at tumorigenic exposure levels to reveal similarities and differences in response among these treatments. Experiment Overall Design: Briefly, male CD-1 mice received propiconazole (2500 ppm), tridimefon (1800 ppm), or phenobarbital (850 ppm) in the feed or, control feed for periods of 4 and 30 days. This resulted in an average daily intake of 349.6 ± 46.7 of propiconazole, 257.1 ± 33.5 of tridimefon or 97.2 ± 5.9 mg/kg/day of phenobarbital.
Project description:Characterisation of the mode of action (MOA) of constitutive androstane receptor (CAR)-mediated rodent liver tumours involves measurement 5 key events including activation of the CAR receptor, altered gene expression, hepatocellular proliferation, clonal expansion and increased hepatocellular adenomas/carcinomas. To test whether or not liver 3D microtissues (LiMTs) recapitulate CAR- mediated procarcinogenic key events in response to the prototypical CAR activator phenobarbital (PB) we performed hepatocyte proliferation (LI%) analysis in rat and human LiMTs using a microTMA technology in conjunction with integrated transcriptomics (microarray) and proteomics analysis. The rationale for this approach was that LiMTs containing parenchymal and non-parenchymal cells (NPCs) are more physiologically representative of liver and thus would generate data more relevant to the in vivo situation. Rat and human LiMTs were treated with PB over a range of concentrations (500 uM - 2000 uM) and times (24 h - 96 h) in a dose-response/time-course analysis. There was a dose-dependent induction of LI% in rat LiMTs, however there was little or no effect of PB on LI% in human LiMTs. ATP levels in the rat and human LiMTs were similar to control in all of the PB treatments. There was also a dose- and time-dependent PB-mediated RNA induction of CAR regulated genes CYP2B6/Cyp2b2, CYP3A7/Cyp3a9 and UGT1A6/Ugt1a6 in human and rat LiMTs, respectively. These CAR regulated genes were also upregulated at the protein level. Ingenuity pathways analysis (IPA) indicated that there was a significant (Z score >2.0;-log p value >) activation of CAR by PB in both human and rat LiMTs. These results indicate that human and rat LiMTs showed the expected responses at the level of PB-induced hepatocyte proliferation and enzyme induction with rat LiMTs showing significant dose-dependent effects while human LiMTs showed no proliferation response but did show dose-dependent enzyme induction at the RNA and protein levels. In conclusion LiMTs serve as a model to provide mechanistic data for 3 of the 5 key events considered necessary to establish a CAR-mediated MOA for liver tumourigenesis and thus can potentially reduce the use of animals when compiling mechanistic data packages.
Project description:BACKGROUND:Yeasts, which are ubiquitous in agroecosystems, are known to degrade various xenobiotics. The aim of this study was to analyze the effect of fungicides on the abundance of natural yeast communities colonizing winter wheat leaves, to evaluate the sensitivity of yeast isolates to fungicides in vivo, and to select yeasts that degrade propiconazole. RESULTS:Fungicides applied during the growing season generally did not affect the counts of endophytic yeasts colonizing wheat leaves. Propiconazole and a commercial mixture of flusilazole and carbendazim decreased the counts of epiphytic yeasts, but the size of the yeast community was restored after 10?days. Epoxiconazole and a commercial mixture of fluoxastrobin and prothioconazole clearly stimulated epiphyte growth. The predominant species isolated from leaves were Aureobasidium pullulans and Rhodotorula glutinis. In the disk diffusion test, 14 out of 75 yeast isolates were not sensitive to any of the tested fungicides. After 48?h of incubation in an aqueous solution of propiconazole, the Rhodotorula glutinis Rg 55 isolate degraded the fungicide in 75%. Isolates Rh. glutinis Rg 92 and Rg 55 minimized the phytotoxic effects of propiconazole under greenhouse conditions. The first isolate contributed to an increase in the dry matter content of wheat seedlings, whereas the other reduced the severity of chlorosis. CONCLUSION:Not sensitivity of many yeast colonizing wheat leaves on the fungicides and the potential of isolate Rhodotorula glutinis Rg 55 to degrade of propiconazole was established. Yeast may partially eliminate the ecologically negative effect of fungicides.
Project description:The modes of triazole reproductive toxicity have been characterized by an observed increased in serum testosterone and reduced insemination and fertility indices. The key events involved in the disruption in testosterone homeostasis and reduced fertility remain unclear. Gene expression analysis was conducted on liver from Sprague Dawley rats dosed with myclobutanil (300 mg/kg/day), propiconazole (300 mg/kg/day), or triadimefon (175 mg/kg/day) for 72 hours. Pathway-based analysis highlighted key biological processes affected by all three triazoles in the liver including fatty acid catabolism, steroid metabolism, and xenobiotic metabolism. Within the pathways identified in the liver, specific genes involved in phase I-III metabolism and fatty acid metabolism were affected by all three triazoles. These modulated genes are part of a network of lipid and testosterone homeostasis pathways regulated by the constitutive androstane (CAR) and pregnane X (PXR) receptors. Gene expression profiles from this study indicate triazoles activate CAR and PXR; increase fatty acid catabolism and steroid metabolism in the liver; constituting a plausible series of key events contributing to the observed disruption in testosterone homeostasis. Experiment Overall Design: A total of 12 liver samples were analyzed. Three biological replicates each for the controls, 300 mg/kg/day myclobutanil, 300 mg/kg/day propiconazole, and 175 mg/kg/day triadimefon.
Project description:Rodent carcinogenicity studies are useful for screening for human carcinogens but they are not perfect. Some modes of action (MOAs) lead to cancers in both experimental rodents and humans, but others that lead to cancers in rodents do not do so in humans. Therefore, analysing the MOAs by which chemicals produce tumours in rodents and determining the relevance of such tumour data for human risk are critical. Recently, experimental data were obtained as case examples of an evaluation of the human relevance of pyrethroid (metofluthrin and momfluorothrin)- and pyrethrins-induced liver tumours in rats based on MOA. The MOA analysis, based on the International Programme on Chemical Safety (IPCS) framework, concluded that experimental data strongly support that the postulated MOA for metofluthrin-, momfluorothrin- and pyrethrins-produced rat hepatocellular tumours is mediated by constitutive androstane receptor (CAR) activation. Since metofluthrin and momfluorothrin are close structural analogues, reproducible outcomes for both chemicals provide confidence in the MOA findings. Furthermore, cultured human hepatocyte studies and humanized chimeric mouse liver studies demonstrated species difference between human hepatocytes (refractory to the mitogenic effects of these compounds) and rat hepatocytes (sensitive to their mitogenic effects). These data strongly support the hypothesis that the CAR-mediated MOA for liver tumorigenesis is of low carcinogenic risk for humans. In this research, in addition to cultured human hepatocyte studies, the usefulness of the humanized chimeric liver mouse models was clearly demonstrated. These data substantially influenced decisions in regulatory toxicology. In this review I comprehensively discuss the human relevance of the CAR-mediated MOA for rodent liver tumorigenesis based on published information, including our recent molecular research on CAR-mediated MOA.
Project description:The goal of this study was to apply transcriptional analyses to hepatic tissues from mice exposed to PB, propiconazole (Pro) or triadimefon (Tri) at tumorigenic exposure levels to reveal similarities and differences in response among these treatments.The goal of this study was to apply transcriptional analyses to hepatic tissues from mice exposed to PB, propiconazole (Pro) or triadimefon (Tri) at tumorigenic exposure levels to reveal similarities and differences in response among these treatments. Overall design: Briefly, male CD-1 mice received propiconazole (2500 ppm), tridimefon (1800 ppm), or phenobarbital (850 ppm) in the feed or, control feed for periods of 4 and 30 days. This resulted in an average daily intake of 349.6 ± 46.7 of propiconazole, 257.1 ± 33.5 of tridimefon or 97.2 ± 5.9 mg/kg/day of phenobarbital.
Project description:High doses of sodium phenobarbital (NaPB), a constitutive androstane receptor (CAR) activator, have been shown to produce hepatocellular tumors in rodents by a mitogenic mode of action (MOA) involving CAR activation. The effect of 1 week dietary treatment with NaPB on liver weight and histopathology, hepatic CYP2B enzyme activity and CYP2B/3A mRNA expression, replicative DNA synthesis and selected genes related to cell proliferation and functional transcriptomic and metabolomic analyses was studied in male CD-1 mice, Wistar Hannover (WH) rats and chimeric mice with human hepatocytes. The treatment of chimeric mice with 1000-1500 ppm NaPB resulted in plasma levels around 3-5 fold higher than those observed in human subjects given therapeutic doses of NaPB. NaPB produced dose-dependent increases in hepatic CYP2B activity and CYP2B/3A mRNA levels in all animal models. Integrated functional metabolomic and transcriptomic analyses demonstrated the responses to NaPB in human liver were clearly different from those in rodents. While NaPB produced a dose-dependent increase in hepatocyte replicative DNA synthesis in CD-1 mice and WH rats, no increase in replicative DNA synthesis was observed in human hepatocyte-originated areas of chimeric mice. In addition, treatment with NaPB had no effect on Ki-67, PCNA, GADD45β, and MDM2 mRNA expression in chimeric mice, whereas significant increases were observed in CD-1 mice and/or WH rats. Thus, while NaPB could activate CAR in rodent and human hepatocytes, NaPB did not increase replicative DNA synthesis in human hepatocytes of chimeric mice, whereas it was mitogenic to rat and mouse hepatocytes. As human hepatocytes are refractory to the mitogenic effects of NaPB, the MOA for NaPB-induced rodent liver tumor formation is thus not relevant for humans. Male CD-1 mice were fed diets containing 0 (control) or 2500 ppm NaPB for 7 days. Liver samples were used for gene expression analysis.
Project description:The conclusions of EFSA following the peer review of the initial risk assessments carried out by the competent authorities of the rapporteur Member State, Finland, and co-rapporteur Member State, the United Kingdom, for the pesticide active substance propiconazole are reported. The context of the peer review was that required by Commission Implementing Regulation (EU) No 844/2012. The conclusions were reached on the basis of the evaluation of the representative use of propiconazole as a fungicide on wheat and barley. The reliable end points, appropriate for use in regulatory risk assessment are presented. Missing information identified as being required by the regulatory framework is listed. Concerns are identified.
Project description:Yeast-like symbiotes (YLS), harbored in the abdomen fat-body cells of the rice brown planthopper (BPH), Nilaparvata lugens Stål (Hemiptera: Delphacidae), are vital to the growth and reproduction of their host. It is feasible to manipulate BPH infestation on rice by inhibiting YLS using fungicide. In this study, the fungicide propiconazole was injected into the hemolymph of BPH thorax via microinjection to investigate its effect on YLS, especially the dominant species, Hypomyces chrysospermus, and their host BPH. Propiconazole markedly reduced the total number of YLS and H. chrysospermus in BPH hemolymph and fat body, thereby leading to an obvious higher mortality and lower fecundity of BPH than the negative control (PBS, phosphate buffer solution). After microinjecting propiconazole, the survival rate of BPH nymphs at the 5th instar was significantly lower than that obtained after PBS treatment. Eight days after propiconazole microinjection, the BPH survival rate dropped to 40%, only half of BPH survival rate treated with PBS microinjection. For female adults (1-day-old), there were significant differences in the survival rates between BPHs treated with propiconazole and those treated with PBS at days 5-8. The fecundity of BPH decreased significantly by microinjecting propiconazole and averaged only 229 eggs per female, which was 20% less than that of the negative control. Furthermore, we reared BPH on the susceptible variety TN1 sprayed with propiconazole to prove the feasibility manipulating field occurrence of BPH by inhibiting YLS using fungicides. The number of YLS and H. chrysospermus in BPH obviously declined. Subsequently, the survival rate and fecundity of BPH significantly decreased after feeding on rice treated with propiconazole. Meanwhile, the propiconazole residue was detected in the hemolymph and gut of BPH by HPLC analysis within 1 day of feeding. Inhibiting YLS using fungicides was a novel and effective way to control BPH infestation.
Project description:The triazole antifungals myclobutanil (MYC), propiconazole (PPZ) and triadimefon (TDF) [Propiconazole CASNR 60207-90-1; Triadimefon CASNR 43121-43-3; Myclobutanil CASNR 88671-89-0] all disrupt steroid hormone homeostasis and cause varying degrees of hepatic toxicity. To identify biological pathways consistently activated across various study designs, gene expression profiling was conducted on livers from rats following acute, repeated dose, or prenatal to adult exposures. To explore conservation of responses across species, gene expression from these rat in vivo studies were also compared to in vitro data from rat and human primary hepatocytes exposed to MYC, PPZ, or TDF. Pathway and gene level analyses across time of exposure, dose, and species identified patterns of expression common to all three triazoles, which were also conserved between rodents and humans. Pathways affected included androgen and estrogen metabolism, xenobiotic metabolism signaling through CAR and PXR, and CYP mediated metabolism. Many of the differentially expressed genes are regulated by the nuclear receptors CAR, PPAR alpha and PXR, including ABC transporter genes (Abcb1 and MDR1), genes significant to xenobiotic, fatty acid, sterol and steroid metabolism (Cyp2b2 and CYP2B6; Cyp3a1 and CYP3A4; Cyp4a22 and CYP4A11) and xxx (Ugt1a1 and UGT1A1). Modulation of hepatic sterol and steroid metabolism is a plausible mechanism for triazole induced increases in serum testosterone. The gene expression changes caused by all three triazoles appear to focus on pathways regulating lipid and testosterone homeostasis, identifying potential common mechanisms of triazole hepatotoxicity that are conserved between rodents and humans. Keywords: dose response, time course, comparative toxicogenomics Overall design: A total of 43 samples were analyzed. Four biological replicates for the controls (DMSO 0.1%), 2 biological replicates for positive control Rifampicin CASNR 13292-46-1, 4 biological replicates for positive control Phenobarbital sodium CASNR 57-30-7, 3 biological replicates for low dose myclobutanil, 3 biological replicates for mid dose myclobutanil, 4 biological replicates for high dose myclobutanil. Four biological replicates each for low, mid, and high dose propiconazole, and 4, 4, and 3 biological replicates each for low, mid, and high dose triadimefon, respectively.