Study of the Distribution of Acetaminophen and Its Metabolites in Rats, from the Whole-Body to Isolated Organ Levels, by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging after On-Tissue Chemical Derivatization.

During drug development, detailed investigations of the pharmacokinetic profile of the drug are required to characterize its absorption, distribution, metabolism, and excretion properties. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is an established technique for studies of the distribution of drugs and their metabolites. It has advantages over autoradiography, which is conventionally used for distribution studies: it does not require the radiolabeling of drugs and can distinguish between the drug and its metabolites directly in the tissue. However, its lack of sensitivity in certain cases remains challenging. Novel procedures, such as on-tissue chemical derivatization (OTCD), could be developed to increase sensitivity. We used OTCD to enhance the sensitivity of MALDI-MSI for one of the most widely used drugs, acetaminophen, and to study its distribution in tissues. Without derivatization, this drug and some of its metabolites are undetectable by MALDI-MSI in the tissues of treated rats. We used 2-fluoro-1-methylpyridinium p-toluene sulfonate as a derivatization reagent, to increase the ionization yield of acetaminophen and some of its metabolites. The OTCD protocol made it possible to study the distribution of acetaminophen and its metabolites in whole-body sections at a spatial resolution of 400 µm and in complex anatomical structures, such as the testis and epididymis, at a spatial resolution <50 µm. The OTCD is also shown to be compatible with the quantification of acetaminophen by MALDI-MSI in whole-body tissues. This protocol could be applied to other molecules bearing phenol groups and presenting a low ionization efficiency.

On-tissue chemical derivatization reagents for matrix-assisted laser desorption/ionization mass spectrometry imaging.

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) is a key tool for the analysis of biological tissues. It provides spatial and quantitative information about different types of analytes within tissue sections. Despite the increasing improvements of this technique, the low detection sensitivity of some compounds remains an important challenge to overcome. Poor sensitivity is related to weak ionization efficiency, low abundance of analytes and matrix ions, or endogenous interferences. On-tissue chemical derivatization (OTCD) has proven to be an important solution to these issues and is increasingly employed in MALDI MSI studies. OTCD reagents, synthesized or commercially available, have been essentially used for the detection of small exogenous or endogenous molecules within tissues. Optimally, an OTCD reaction is performed in mild conditions, in an acceptable range of time, preserves the integrity of the tissues, and prevents the delocalization. In addition to their reactivity with a targeted chemical function, some OTCD reagents can also be used as a matrix, which simplifies the sample preparation procedure. In this review, we present an exhaustive overview of OTCD reagents and methods used in MALDI MSI studies.

PPAR agonists reduce steatosis in oleic acid-overloaded HepaRG cells.

UNLABELLED: Although non-alcoholic fatty liver disease (NAFLD) is currently the most common form of chronic liver disease there is no pharmacological agent approved for its treatment. Since peroxisome proliferator-activated receptors (PPARs) are closely associated with hepatic lipid metabolism, they seem to play important roles in NAFLD. However, the effects of PPAR agonists on steatosis that is a common pathology associated with NAFLD, remain largely controversial. In this study, the effects of various PPAR agonists, i.e. fenofibrate, bezafibrate, troglitazone, rosiglitazone, muraglitazar and tesaglitazar on oleic acid-induced steatotic HepaRG cells were investigated after a single 24-hour or 2-week repeat treatment. Lipid vesicles stained by Oil-Red O and triglycerides accumulation caused by oleic acid overload, were decreased, by up to 50%, while fatty acid oxidation was induced after 2-week co-treatment with PPAR agonists. The greatest effects on reduction of steatosis were obtained with the dual PPARα/γ agonist muraglitazar. Such improvement of steatosis was associated with up-regulation of genes related to fatty acid oxidation activity and down-regulation of many genes involved in lipogenesis. Moreover, modulation of expression of some nuclear receptor genes, such as FXR, LXRα and CAR, which are potent actors in the control of lipogenesis, was observed and might explain repression of de novo lipogenesis. CONCLUSION: Altogether, our in vitro data on steatotic HepaRG cells treated with PPAR agonists correlated well with clinical investigations, bringing a proof of concept that drug-induced reversal of steatosis in human can be evaluated in in vitro before conducting long-term and costly in vivo studies in animals and patients.

Small molecule glucokinase activators disturb lipid homeostasis and induce fatty liver in rodents: a warning for therapeutic applications in humans.

BACKGROUND AND PURPOSE: Small-molecule glucokinase activators (GKAs) are currently being investigated as therapeutic options for the treatment of type 2 diabetes (T2D). Because liver overexpression of glucokinase is thought to be associated with altered lipid profiles, this study aimed at assessing the potential lipogenic risks linked to oral GKA administration. EXPERIMENTAL APPROACH: Nine GKA candidates were qualified for their ability to activate recombinant glucokinase and to stimulate glycogen synthesis in rat hepatocytes and insulin secretion in rat INS-1E cells. In vivo activity was monitored by plasma glucose and HbA1c measurements after oral administration in rodents. Risk-associated effects were assessed by measuring hepatic and plasma triglycerides and free fatty acids, as well as plasma aminotransferases, and alkaline phosphatase. KEY RESULTS: GKAs, while efficiently decreasing glycaemia in acute conditions and HbA1c levels after chronic administration in hyperglycemic db/db mice, were potent inducers of hepatic steatosis. This adverse outcome appeared as soon as 4 days after daily oral administration at pharmacological doses and was not transient. GKA treatment similarly increased hepatic triglycerides in diabetic and normoglycaemic rats, together with a pattern of metabolic phenotypes including different combinations of increased plasma triglycerides, free fatty acids, alanine and aspartyl aminotransferases, and alkaline phosphatase. GKAs belonging to three distinct structural families induced hepatic steatosis in db/db mice, arguing in favour of a target-mediated, rather than a chemical class-mediated, effect. CONCLUSION AND IMPLICATIONS: Given the risks associated with fatty liver disease in the general population and furthermore in patients with T2D, these findings represent a serious warning for the use of GKAs in humans. LINKED ARTICLE: This article is commented on by Rees and Gloyn, pp. 335-338 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2012.02201.x.

Coordinate Transcriptomic and Metabolomic Effects of the Insulin Sensitizer Rosiglitazone on Fundamental Metabolic Pathways in Liver, Soleus Muscle, and Adipose Tissue in Diabetic db/db Mice.

Rosiglitazone (RSG), developed for the treatment of type 2 diabetes mellitus, is known to have potent effects on carbohydrate and lipid metabolism leading to the improvement of insulin sensitivity in target tissues. To further assess the capacity of RSG to normalize gene expression in insulin-sensitive tissues, we compared groups of 18-day-treated db/db mice with increasing oral doses of RSG (10, 30, and 100 mg/kg/d) with untreated non-diabetic littermates (db/+). For this aim, transcriptional changes were measured in liver, inguinal adipose tissue (IAT) and soleus muscle using microarrays and real-time PCR. In parallel, targeted metabolomic assessment of lipids (triglycerides (TGs) and free fatty acids (FFAs)) in plasma and tissues was performed by UPLC-MS methods. Multivariate analyses revealed a relationship between the differential gene expressions in liver and liver trioleate content and between blood glucose levels and a combination of differentially expressed genes measured in liver, IAT, and muscle. In summary, we have integrated gene expression and targeted metabolomic data to present a comprehensive overview of RSG-induced changes in a diabetes mouse model and improved the molecular understanding of how RSG ameliorates diabetes through its effect on the major insulin-sensitive tissues.

Metabonomic studies on human hepatocyte in primary culture.

Mechanisms involved in induction processes have been investigated using fresh human hepatocytes in culture as a cellular model and using mass spectrometry-based metabonomics as a global investigation tool. Sample preparation to data analysis have been detailed in an approach enabling to separate drug-induced (endogenous metabolites) and drug-related (drug metabolites) biomarkers for reference inducers. Rifampicin, a nuclear pregnane X receptor (PXR) ligand; CITCO, a nuclear constitutive androstane receptor (CAR) ligand; and phenobarbital, which activates both CAR and PXR, have been used. Specific intra-cellular metabolites have been isolated for rifampicin and CITCO as potential endogenous biomarkers of their respective induction mechanism. A mixture of these two types of biomarkers modified in the same way after treatment with either rifampicin or CITCO on the one hand and with phenobarbital on the other hand has been found.

Integrated comparison of drug-related and drug-induced ultra performance liquid chromatography/mass spectrometry metabonomic profiles using human hepatocyte cultures.

The biochemical variations induced in human primary hepatocyte cultures by reference activators of xenoreceptor CAR (NR1I3) and PXR (NR1I2), i.e., rifampicin, phenobarbital, and 6-(4-chlorophenyl)imidazo[2,1-b] [1,3]thiazole-5-carbaldehyde O-3,4-dichlorobenzyl) oxime (CITCO), were investigated using a global metabonomics approach. Cultured human hepatocytes were treated with the three drugs before analysis of intracellular and extracellular media by ultra performance liquid chromatography/time-of-flight-mass spectrometry (UPLC/TOF-MS) technique, in order to list endogenous compounds potentially related to a PXR or CAR induction mechanism and to identify drug metabolites related to each treatment. The emphasis was put on the quality of the analytical data (dilution/filtration strategy before data processing) and on the appropriate pattern recognition techniques. In cellular media, the most significant variations seen in the data are not related to the treatments but to the source of hepatocytes, illustrating the importance of the genetic and/or environmental background in human liver experiments. However when applying classical multivariate statistical approaches (principal component analysis (PCA) and orthogonal partial least squares (O-PLS)), the statistical weight due to drug metabolites, present only in the treated groups, hinders the interpretation because of their predominance compared to most of the changes seen in endogenous metabolites. A new statistical approach, called shared and unique structure (SUS) plot, enabling the comparison of different treatments having the same control has been applied, allowing separation of clearly exogenous variables (drug metabolites) from endogenous biomarkers. Endogenous variables (either up- or down-regulated) have been attributed specifically to the impact of rifampicin (PXR ligand), CITCO (CAR ligand), and phenobarbital (CAR and PXR activator) on the biological regulation pathways of the hepatocytes. This global approach coupled to a statistical pretreatment of the data, enabling the separate capture of both drug related and drug induced biomarkers, represents a powerful technique for future mechanistic studies using cellular tools.

A novel pregnane X receptor and S14-mediated lipogenic pathway in human hepatocyte.

The pregnane X receptor (PXR) initially isolated as a nuclear receptor regulating xenobiotic and drug metabolism and elimination, seems to play an endobiotic role by affecting lipid homeostasis. In mice, PXR affects lipid homeostasis and increases hepatic deposit of triglycerides. In this study, we show that, in human hepatocyte, PXR activation induces an increase of de novo lipogenesis through the up-regulation of S14. S14 was first identified as a thyroid-responsive gene and is known to transduce hormone-related and nutrient-related signals to genes involved in lipogenesis through a molecular mechanism not yet elucidated. We demonstrate that S14 is a novel transcriptional target of PXR. In addition, we report an increase of fatty acid synthase (FASN) and adenosine triphosphate citrate lyase genes expression after PXR activation in human hepatocyte, leading to an increase of fatty acids accumulation and de novo lipogenesis. RNA interference of the expression of S14 proportionally decreases the FASN induction, whereas S14 overexpression in human hepatic cells provokes an increase of fatty acids accumulation and lipogenesis. These results demonstrate for the first time that xenobiotic or drug-activated PXR promote aberrant hepatic de novo lipogenesis via activation of the nonclassical S14 pathway. In addition, these data suggest that the up-regulation of S14 by PXR may promote aberrant hepatic lipogenesis and hepatic steatosis in human hepatocytes.

Mass spectrometry-based metabolomics: accelerating the characterization of discriminating signals by combining statistical correlations and ultrahigh resolution.

A strategy combining autocorrelation matrices and ultrahigh resolution mass spectrometry (MS) was developed to optimize the characterization of discriminating ions highlighted by metabolomics. As an example, urine samples from rats treated with phenobarbital (PB) were analyzed by ultrahigh-pressure chromatography with two different eluting conditions coupled to time-of-flight mass spectrometric detection in both the positive and negative electrospray ionization modes. Multivariate data analyses were performed to highlight discriminating variables from several thousand detected signals: a few hundred signals were found to be affected by PB, whereas a few tenths of them were linked to its metabolism. Autocorrelation matrices were then applied to eliminate adduct and fragment ions. Finally, the characterization of the ions of interest was performed with ultrahigh-resolution mass spectrometry and sequential MS(n) experiments, by using a LC-LTQ-Orbitrap system. The use of different eluting conditions was shown to drastically impact on the chromatographic retention and ionization of compounds, thus providing a way to obtain more exhaustive metabolic fingerprints, whereas autocorrelation matrices allowed one to focus the identification work on the most relevant ions. By using such an approach, 14 PB metabolites were characterized in rat urines, some of which have not been reported in the literature.