Kristopher W. Krausz

Microbiome remodelling leads to inhibition of intestinal farnesoid X receptor signalling and decreased obesity

The antioxidant tempol reduces obesity in mice. Here we show that tempol alters the gut microbiome by preferentially reducing the genus Lactobacillus and its bile salt hydrolase (BSH) activity leading to the accumulation of intestinal tauro-β-muricholic acid (T-β-MCA). T-β-MCA is an farnesoid X receptor (FXR) nuclear receptor antagonist, which is involved in the regulation of bile acid, lipid and glucose metabolism. Its increased levels during tempol treatment inhibit FXR signalling in the intestine. High-fat diet-fed intestine-specific Fxr-null (Fxr(ΔIE)) mice show lower diet-induced obesity, similar to tempol-treated wild-type mice. Further, tempol treatment does not decrease weight gain in Fxr(ΔIE) mice, suggesting that the intestinal FXR mediates the anti-obesity effects of tempol. These studies demonstrate a biochemical link between the microbiome, nuclear receptor signalling and metabolic disorders, and suggest that inhibition of FXR in the intestine could be a target for anti-obesity drugs.

Intestinal farnesoid X receptor signaling promotes nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) is a major worldwide health problem. Recent studies suggest that the gut microbiota influences NAFLD pathogenesis. Here, a murine model of high-fat diet-induced (HFD-induced) NAFLD was used, and the effects of alterations in the gut microbiota on NAFLD were determined. Mice treated with antibiotics or tempol exhibited altered bile acid composition, with a notable increase in conjugated bile acid metabolites that inhibited intestinal farnesoid X receptor (FXR) signaling. Compared with control mice, animals with intestine-specific Fxr disruption had reduced hepatic triglyceride accumulation in response to a HFD. The decrease in hepatic triglyceride accumulation was mainly due to fewer circulating ceramides, which was in part the result of lower expression of ceramide synthesis genes. The reduction of ceramide levels in the ileum and serum in tempol- or antibiotic-treated mice fed a HFD resulted in downregulation of hepatic SREBP1C and decreased de novo lipogenesis. Administration of C16:0 ceramide to antibiotic-treated mice fed a HFD reversed hepatic steatosis. These studies demonstrate that inhibition of an intestinal FXR/ceramide axis mediates gut microbiota-associated NAFLD development, linking the microbiome, nuclear receptor signaling, and NAFLD. This work suggests that inhibition of intestinal FXR is a potential therapeutic target for NAFLD treatment.

Role of human cytochrome P450 3a4 and 3a5 in the metabolism of taxotere and its derivatives: enzyme specificity, interindividual distribution and metabolic contribution in human liver

Taxotere, a promising anticancer agent, is metabolized almost exclusively in liver and excreted from bile in all species. To determine which cytochrome P450 is involved in taxotere biotransformation, 11 cDNA-expressed human cytochrome P450s were examined for their activity in the metabolism of taxotere and its derivatives. Of all P450s, cytochrome P450 3A4 and 3A5 were the most active for the oxidation of taxotere to the primary metabolite RPR104952 and for subsequent oxidation of RPR104952 to RPR111059 and RPR111026. RP70617, an epimer of taxotere was also metabolized by both P450 3A enzymes to form metabolite XII. The activity of 3A4/5 enzymes for these substrates was 4-50-fold greater than the other P450s examined. The Kms of 3A4 and 3A5 for taxotere were 0.91 and 9.28 microM, and Vmax for the formation of RPR104952 were 1.17 and 1.36 m(-1), respectively. The contribution of the 3A enzyme complex to the metabolism of taxotere in human livers from 21 individuals was assessed with the inhibitory monoclonal antibody and ranged from 64-93%. The primary oxidative metabolism of taxotere by human liver microsomes was well correlated with 3A4-dependent reactions for testosterone 6beta-hydroxylation (r2 = 0.84), taxol aromatic hydroxylation (r2 = 0.67) and aflatoxin B1 3alpha-hydroxylation (r2 = 0.63); whereas a poor correlation was found for reactions specifically catalysed by other P450s (all r2 < or =O.17). The extent of taxotere metabolism also closely correlated with levels of 3A4 enzyme in human livers quantified with immunoblot monoclonal antibody (r2 = 0.61). These results demonstrate that the P450 3A4 and 3A5 enzymes are major determinants in taxotere oxidation and suggest that care must be taken when administering this drug with other drugs that are also substrates for these enzymes.

Aberrant Lipid Metabolism in Hepatocellular Carcinoma Revealed by Plasma Metabolomics and Lipid Profiling

There has been limited analysis of the effects of hepatocellular carcinoma (HCC) on liver metabolism and circulating endogenous metabolites. Here, we report the findings of a plasma metabolomic investigation of HCC patients by ultraperformance liquid chromatography-electrospray ionization-quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOFMS), random forests machine learning algorithm, and multivariate data analysis. Control subjects included healthy individuals as well as patients with liver cirrhosis or acute myeloid leukemia. We found that HCC was associated with increased plasma levels of glycodeoxycholate, deoxycholate 3-sulfate, and bilirubin. Accurate mass measurement also indicated upregulation of biliverdin and the fetal bile acids 7α-hydroxy-3-oxochol-4-en-24-oic acid and 3-oxochol-4,6-dien-24-oic acid in HCC patients. A quantitative lipid profiling of patient plasma was also conducted by ultraperformance liquid chromatography-electrospray ionization-triple quadrupole mass spectrometry (UPLC-ESI-TQMS). By this method, we found that HCC was also associated with reduced levels of lysophosphocholines and in 4 of 20 patients with increased levels of lysophosphatidic acid [LPA(16:0)], where it correlated with plasma α-fetoprotein levels. Interestingly, when fatty acids were quantitatively profiled by gas chromatography-mass spectrometry (GC-MS), we found that lignoceric acid (24:0) and nervonic acid (24:1) were virtually absent from HCC plasma. Overall, this investigation illustrates the power of the new discovery technologies represented in the UPLC-ESI-QTOFMS platform combined with the targeted, quantitative platforms of UPLC-ESI-TQMS and GC-MS for conducting metabolomic investigations that can engender new insights into cancer pathobiology.

Assessment of specificity of eight chemical inhibitors using cDNA-expressed cytochromes P450

1. The selectivity of eight chemical inhibitors has been extensively evaluated with 10 cDNA-expressed human cytochrome P450 isoforms (CYP). The results indicate that sulphaphenazole, quinidine and α-naphthoflavone are selective inhibitors of CYP2C9 (IC50 = 5 0.5-0.7 μM), CYP2D6 (0.3-0.4 μM) and CYP1A (0.05-5 μM) respectively on the basis of the IC50, which are much lower than those of other P450 isoforms (> 10-fold). 2. Ketoconazole exhibited potent inhibition of both CYP3A4-catalysed metabolism of phenanthrene, testosterone, diazepam (IC50 = 0.03-0.5 μM) and CYP1A1-catalysed deethylation of 7-ethoxycoumarin (0.33 μM). The selectivity of ketoconazole for other P450s was highly related to the concentration used. 3. Diethyldithiocarbamate, orphenadrine and furafylline were shown separately to be less selective inhibitors of CYP2E1, CYP2B6 and CYP1A isoforms by a broad range of IC50 that overlap those observed with other P450 isoforms. 4. Furafylline, quinidine and α-naphthoflavone activated CYP3A4-catalysed phenanthrene metabolism by 1.7-, 2- and 15-fold respectively. 5. The selectivity of orphenadrine and ketoconazole was further examined by using inhibitory monoclonal antibodies (MAb). Inhibitory MAb specific for the individual P450 isoforms may be of greater value than chemical inhibitors.

Transcription factor NRF2 regulates miR-1 and miR-206 to drive tumorigenesis

The mechanisms by which deregulated nuclear factor erythroid-2-related factor 2 (NRF2) and kelch-like ECH-associated protein 1 (KEAP1) signaling promote cellular proliferation and tumorigenesis are poorly understood. Using an integrated genomics and ¹³C-based targeted tracer fate association (TTFA) study, we found that NRF2 regulates miR-1 and miR-206 to direct carbon flux toward the pentose phosphate pathway (PPP) and the tricarboxylic acid (TCA) cycle, reprogramming glucose metabolism. Sustained activation of NRF2 signaling in cancer cells attenuated miR-1 and miR-206 expression, leading to enhanced expression of PPP genes. Conversely, overexpression of miR-1 and miR-206 decreased the expression of metabolic genes and dramatically impaired NADPH production, ribose synthesis, and in vivo tumor growth in mice. Loss of NRF2 decreased the expression of the redox-sensitive histone deacetylase, HDAC4, resulting in increased expression of miR-1 and miR-206, and not only inhibiting PPP expression and activity but functioning as a regulatory feedback loop that repressed HDAC4 expression. In primary tumor samples, the expression of miR-1 and miR-206 was inversely correlated with PPP gene expression, and increased expression of NRF2-dependent genes was associated with poor prognosis. Our results demonstrate that microRNA-dependent (miRNA-dependent) regulation of the PPP via NRF2 and HDAC4 represents a novel link between miRNA regulation, glucose metabolism, and ROS homeostasis in cancer cells.

Radiation metabolomics. 1. Identification of minimally invasive urine biomarkers for gamma-radiation exposure in mice.

Abstract Tyburski, J. B., Patterson, A. D., Krausz, K. W., Slavík, J., Fornace, A. J., Jr., Gonzalez, F. J. and Idle, J. R. Radiation Metabolomics. 1. Identification of Minimally Invasive Urine Biomarkers for Gamma-Radiation Exposure in Mice. Radiat. Res. 170, 1–14 (2008). Gamma-radiation exposure has both short- and long-term adverse health effects. The threat of modern terrorism places human populations at risk for radiological exposures, yet current medical countermeasures to radiation exposure are limited. Here we describe metabolomics for γ-radiation biodosimetry in a mouse model. Mice were γ-irradiated at doses of 0, 3 and 8 Gy (2.57 Gy/min), and urine samples collected over the first 24 h after exposure were analyzed by ultra-performance liquid chromatography–time-of-flight mass spectrometry (UPLC–TOFMS). Multivariate data were analyzed by orthogonal partial least squares (OPLS). Both 3- and 8-Gy exposures yielded distinct urine metabolomic phenotypes. The top 22 ions for 3 and 8 Gy were analyzed further, including tandem mass spectrometric comparison with authentic standards, revealing that N-hexanoylglycine and β-thymidine are urinary biomarkers of exposure to 3 and 8 Gy, 3-hydroxy-2-methylbenzoic acid 3-O-sulfate is elevated in urine of mice exposed to 3 but not 8 Gy, and taurine is elevated after 8 but not 3 Gy. Gene Expression Dynamics Inspector (GEDI) self-organizing maps showed clear dose–response relationships for subsets of the urine metabolome. This approach is useful for identifying mice exposed to γ radiation and for developing metabolomic strategies for noninvasive radiation biodosimetry in humans.

Serum Metabolomics Reveals Irreversible Inhibition of Fatty Acid β-Oxidation through the Suppression of PPARα Activation as a Contributing Mechanism of Acetaminophen-Induced Hepatotoxicity

Metabolic bioactivation, glutathione depletion, and covalent binding are the early hallmark events after acetaminophen (APAP) overdose. However, the subsequent metabolic consequences contributing to APAP-induced hepatic necrosis and apoptosis have not been fully elucidated. In this study, serum metabolomes of control and APAP-treated wild-type and Cyp2e1-null mice were examined by liquid chromatography-mass spectrometry (LC-MS) and multivariate data analysis. A dose-response study showed that the accumulation of long-chain acylcarnitines in serum contributes to the separation of wild-type mice undergoing APAP-induced hepatotoxicity from other mouse groups in a multivariate model. This observation, in conjunction with the increase of triglycerides and free fatty acids in the serum of APAP-treated wild-type mice, suggested that APAP treatment can disrupt fatty acid beta-oxidation. A time-course study further indicated that both wild-type and Cyp2e1-null mice had their serum acylcarnitine levels markedly elevated within the early hours of APAP treatment. While remaining high in wild-type mice, serum acylcarnitine levels gradually returned to normal in Cyp2e1-null mice at the end of the 24 h treatment. Distinct from serum aminotransferase activity and hepatic glutathione levels, the pattern of serum acylcarnitine accumulation suggested that acylcarnitines can function as complementary biomarkers for monitoring the APAP-induced hepatotoxicity. An essential role for peroxisome proliferator-activated receptor alpha (PPARalpha) in the regulation of serum acylcarnitine levels was established by comparing the metabolomic responses of wild-type and Ppara-null mice to a fasting challenge. The upregulation of PPARalpha activity following APAP treatment was transient in wild-type mice but was much more prolonged in Cyp2e1-null mice. Overall, serum metabolomics of APAP-induced hepatotoxicity revealed that the CYP2E1-mediated metabolic activation and oxidative stress following APAP treatment can cause irreversible inhibition of fatty acid oxidation, potentially through suppression of PPARalpha-regulated pathways.

Intestine-selective farnesoid X receptor inhibition improves obesity-related metabolic dysfunction

The farnesoid X receptor (FXR) regulates bile acid, lipid and glucose metabolism. Here we show that treatment of mice with glycine-β-muricholic acid (Gly-MCA) inhibits FXR signalling exclusively in intestine, and improves metabolic parameters in mouse models of obesity. Gly-MCA is a selective high-affinity FXR inhibitor that can be administered orally and prevents, or reverses, high-fat diet-induced and genetic obesity, insulin resistance and hepatic steatosis in mice. The high-affinity FXR agonist GW4064 blocks Gly-MCA action in the gut, and intestine-specific Fxr-null mice are unresponsive to the beneficial effects of Gly-MCA. Mechanistically, the metabolic improvements with Gly-MCA depend on reduced biosynthesis of intestinal-derived ceramides, which directly compromise beige fat thermogenic function. Consequently, ceramide treatment reverses the action of Gly-MCA in high-fat diet-induced obese mice. We further show that FXR signalling in ileum biopsies of humans positively correlates with body mass index. These data suggest that Gly-MCA may be a candidate for the treatment of metabolic disorders.

Cytochrome P450 CYP2E1, but not nicotinamide adenine dinucleotide phosphate oxidase, is required for ethanol-induced oxidative DNA damage in rodent liver.

The occurrence of malignant tumors of the upper gastrointestinal tract and liver is, based largely on epidemiological evidence, causally related to the consumption of ethanol. It is widely recognized that oxidants play a key role in alcohol‐induced liver injury; however, it is unclear how oxidants may be involved in DNA damage. We asked whether nicotinamide adenine dinucleotide phosphate oxidase, cytochrome P450 CYP2E1, or both are responsible for the production of DNA damage. The rodent Tsukamoto‐French model of intragastric ethanol infusion was used. Wistar rats, Cyp2e1‐, p47phox‐null, and hCyp2e1 transgenic mice were used. The abundance of oxidative DNA adducts, mutagenic apurinic/apyrimidinic sites, and expression of base excision DNA repair genes was determined. In rats and wild‐type mice, ethanol treatment for 4 weeks led to an increase in oxidative DNA damage and induction of expression of the base excision DNA repair genes that are known to remove oxidative DNA lesions. No increase in either of the endpoints was observed in ethanol‐treated Cyp2e1‐null mice, whereas the magnitude of response in p47phox‐null mice and transgenic hCyp2e1 was identical to that in wild types. The increase in expression of DNA repair genes was completely abolished by treatment with the P450 inhibitor 1‐aminobenzotriazole. In conclusion, the data support the hypothesis that oxidative stress to DNA is induced in liver by ethanol. Furthermore, although it was shown that nicotinamide adenine dinucleotide phosphate oxidase‐derived oxidants are critical for the development of ethanol‐induced liver injury, CYP2E1 is required for the induction of oxidative stress to DNA, and thus may play a key role in ethanol‐associated hepatocarcinogenesis. (HEPATOLOGY 2005;41:336–344.)