The effect of CMGE and caffeine on the expression levels of <i>Cyp1a1</i> and <i>Cyp1a2</i> mRNAs in rat liver.

<p>The level of mRNA expression (normalized to β-actin) is presented relative to that in animals treated with the vehicle only. Each value represents the mean ± SEM of at least three independent experiments. Statistical significance was assessed by one-way ANOVA followed by Games-Howell post test, **, p<0.01.</p

Modulation of CYP1A1 and CYP1A2 Hepatic Enzymes after Oral Administration of <i>Chios Mastic Gum</i> to Male Wistar Rats

<div><p><i>Chios mastic gum</i> (CMG), a resin derived from <i>Pistacia lentiscus</i> var. <i>chia</i>, is known since ancient times for its pharmacological activities. CYP1A1 and CYP1A2 enzymes are among the most involved in the biotransformation of chemicals and the metabolic activation of pro-carcinogens. Previous studies referring to the modulation of these enzymes by CMG have revealed findings of unclear biological and toxicological significance. For this purpose, the modulation of CYP1A1 and CYP1A2 enzymes in the liver of male Wistar rats following oral administration of CMG extract (CMGE), at the levels of mRNA and CYP1A1 enzyme activity, was compared to respective enzyme modulation following oral administration of a well-known bioactive natural product, caffeine, as control compound known to involve hepatic enzymes in its metabolism. mRNA levels of <i>Cyp1a1</i> and <i>Cyp1a2</i> were measured by reverse transcription real-time polymerase chain reaction and their relative quantification was calculated. CYP1A1 enzyme induction was measured through the activity of ethoxyresorufin-<i>O-</i>deethylase (EROD). The results indicated that administration of CMGE at the recommended pharmaceutical dose does not induce significant transcriptional modulation of <i>Cyp1a1/2</i> and subsequent enzyme activity induction of CYP1A1 while effects of the same order of magnitude were observed in the same test system following the administration of caffeine at the mean daily consumed levels. The outcome of this study further confirms the lack of any toxicological or biological significance of the specific findings on liver following the administration of CMGE.</p></div

The effect of CMGE and caffeine on the expression levels of <i>Cyp1a1</i> and <i>Cyp1a2</i> mRNAs in rat liver.

<p>The level of mRNA expression (normalized to β-actin) is presented relative to that in animals treated with the vehicle only. Each value represents the mean ± SEM of at least three independent experiments. Statistical significance was assessed by one-way ANOVA followed by Games-Howell post test, **, p<0.01.</p

The effect of CMGE and caffeine on the activity levels of CYP1A1 in rat liver.

<p>Each value represents the mean ± SEM of at least four samples. Statistical significance was assessed by Kruskal-Wallis H Test followed by Mann-Whitney U test. **:p<0.01</p

Sequence of primers used in real-time PCR, amplicon sizes and annealing temperatures.

<p>Sequence of primers used in real-time PCR, amplicon sizes and annealing temperatures.</p

Adverse Outcome Pathway-Driven Analysis of Liver Steatosis <i>in Vitro</i>: A Case Study with Cyproconazole

Adverse outcome pathways (AOPs) describe causal relationships between molecular perturbation and adverse cellular effects and are being increasingly adopted for linking <i>in vitro</i> mechanistic toxicology to <i>in vivo</i> data from regulatory toxicity studies. In this work, a case study was performed by developing a bioassay toolbox to assess key events in the recently proposed AOP for chemically induced liver steatosis. The toolbox is comprised of <i>in vitro</i> assays to measure nuclear receptor activation, gene and protein expression, lipid accumulation, mitochondrial respiration, and formation of fatty liver cells. Assay evaluation was performed in human HepaRG hepatocarcinoma cells exposed to the model compound cyproconazole, a fungicide inducing steatosis in rodents. Cyproconazole dose-dependently activated RARα and PXR, two molecular initiating events in the steatosis AOP. Moreover, cyproconazole provoked a disruption of mitochondrial functions and induced triglyceride accumulation and the formation of fatty liver cells as described in the AOP. Gene and protein expression analysis, however, showed expression changes different from those proposed in the AOP, thus suggesting that the current version of the AOP might not fully reflect the complex mechanisms linking nuclear receptor activation and liver steatosis. Our study shows that cyproconazole induces steatosis in human liver cells <i>in vitro</i> and demonstrates the utility of systems-based approaches in the mechanistic assessment of molecular and cellular key events in an AOP. AOP-driven <i>in vitro</i> testing as demonstrated can further improve existing AOPs, provide insight regarding molecular mechanisms of toxicity, and inform predictive risk assessment