Tissue Proteome of 2-Hydroxyacyl-CoA Lyase Deficient Mice Reveals Peroxisome Proliferation and Activation of ω-Oxidation.

Peroxisomal fatty acid α-oxidation is an essential pathway for the degradation of ß-carbon methylated fatty acids such as phytanic acid. One enzyme in this pathway is 2-hydroxyacyl CoA lyase (HACL1), which is responsible for the cleavage of 2-hydroxyphytanoyl-CoA into pristanal and formyl-CoA. Hacl1 deficient mice do not present with a severe phenotype, unlike mice deficient in other α-oxidation enzymes such as phytanoyl-CoA hydroxylase deficiency (Refsum disease) in which neuropathy and ataxia are present. Tissues from wild-type and Hacl1-/- mice fed a high phytol diet were obtained for proteomic and lipidomic analysis. There was no phenotype observed in these mice. Liver, brain, and kidney tissues underwent trypsin digestion for untargeted proteomic liquid chromatography-mass spectrometry analysis, while liver tissues also underwent fatty acid hydrolysis, extraction, and derivatisation for fatty acid gas chromatography-mass spectrometry analysis. The liver fatty acid profile demonstrated an accumulation of phytanic and 2-hydroxyphytanic acid in the Hacl1-/- liver and significant decrease in heptadecanoic acid. The liver proteome showed a significant decrease in the abundance of Hacl1 and a significant increase in the abundance of proteins involved in PPAR signalling, peroxisome proliferation, and omega oxidation, particularly Cyp4a10 and Cyp4a14. In addition, the pathway associated with arachidonic acid metabolism was affected; Cyp2c55 was upregulated and Cyp4f14 and Cyp2b9 were downregulated. The kidney proteome revealed fewer significantly upregulated peroxisomal proteins and the brain proteome was not significantly different in Hacl1-/- mice. This study demonstrates the powerful insight brought by proteomic and metabolomic profiling of Hacl1-/- mice in better understanding disease mechanism in fatty acid α-oxidation disorders.

A scoring system for the evaluation of the mutated Crb1/rd8-derived retinal lesions in C57BL/6N mice.

As part of the International Mouse Phenotyping Consortium (IMPC) programme, the MRC Harwell is conducting a large eye morphology phenotyping screen on genetically modified mice compared to the baseline phenotype observed in the background strain of C57BL/6NTac. The C57BL/6NTac strain is known to carry a spontaneous mutation in the Crb1 gene that causes retinal degeneration characterized by the presence of white spots (flecks) in the fundus. These flecks potentially represent a confounding factor, masking similar retinal phenotype abnormalities that may be detected in mutants. Therefore we investigated the frequency, position and extent of the flecks in a large population of C57BL/6NTac mice to provide the basis for evaluating the presence of flecks in mutant mice with the same genetic background. We found that in our facility males were more severely affected than females and that in both males and females the most common localisation of the flecks was in the inferior hemicycle of the fundus.

Analysis of Individual Mouse Activity in Group Housed Animals of Different Inbred Strains using a Novel Automated Home Cage Analysis System.

Central nervous system disorders such as autism as well as the range of neurodegenerative diseases such as Huntington's disease are commonly investigated using genetically altered mouse models. The current system for characterizing these mice usually involves removing the animals from their home-cage environment and placing them into novel environments where they undergo a battery of tests measuring a range of behavioral and physical phenotypes. These tests are often only conducted for short periods of times in social isolation. However, human manifestations of such disorders are often characterized by multiple phenotypes, presented over long periods of time and leading to significant social impacts. Here, we have developed a system which will allow the automated monitoring of individual mice housed socially in the cage they are reared and housed in, within established social groups and over long periods of time. We demonstrate that the system accurately reports individual locomotor behavior within the group and that the measurements taken can provide unique insights into the effects of genetic background on individual and group behavior not previously recognized.

The role of intestinal microbiota in murine models of acetaminophen-induced hepatotoxicity.

BACKGROUND & AIMS: Variations in intestinal microbiota may influence acetaminophen metabolism. This study aimed to determine whether intestinal microbiota are a source of differential susceptibility to acetaminophen-induced hepatotoxicity. METHODS: Conventionally housed C3H/HeH (CH) and C3H/HeH germ-free (GF) mice were administered a 200 mg/kg IP dose of acetaminophen. The severity of hepatotoxicity at 8 h was assessed by histology and biochemical indices. A urinary metabolic profile was obtained using (1) H-NMR. Baseline hepatic glutathione content and CYP2E1 expression were quantified. An additional group of C3H/HeJ (LPS-r) mice were assessed to determine the contribution of LPS/TLR4 signalling. RESULTS: Baseline glutathione levels were significantly reduced (P = 0.03) in GF mice. CYP2E1 mRNA expression and protein levels were not altered. Interindividual variability did not differ between GF and CH groups. No significant differences in the extent of hepatocellular injury (ALT or percentage necrosis) were demonstrated. However, a milder acute liver failure (ALF) phenotype was shown in GF compared with CH mice, with reduced plasma bilirubin and creatinine and increased blood glucose. Differential acetaminophen metabolism was demonstrated. GF mice displayed a higher urinary acetaminophen-sulphate:glucuronide ratio compared with CH (P = 0.01). Urinary analysis showed metabolic differentiation of GF and CH groups at baseline and 8 h (cross-validated anova P = 1 × 10(-22) ). Interruption of TLR4 signalling in LPS-r mice had additional protective effects. CONCLUSION: Variations in intestinal microbiota do not fully explain differential susceptibility to acetaminophen-induced hepatotoxicity. GF mice experienced some protection from secondary complications following acetaminophen overdose and this may be mediated through reduced TLR4/LPS signalling.

Multi-excitation fluorescence spectroscopy for analysis of non-alcoholic fatty liver disease.

BACKGROUND AND OBJECTIVES: The increasing incidence of non-alcoholic fatty liver diseases (NAFLD) and the consequent progression to cirrhosis is expected to become a major cause of liver transplantation. This will exacerbate the organ donor shortage and mean that 'marginal' fatty liver grafts are more frequently used. Autofluorescence spectroscopy is a fast, objective, and non-destructive method to detect change in the endogenous fluorophores distribution and could prove to be a valuable tool for NAFLD diagnosis and transplant graft assessment. MATERIALS AND METHODS: A system was constructed consisting of a fibre probe with two laser diodes that provided excitation light at 375 and 405 nm, and an imaging spectrograph system. This was used to distinguish fluorescence spectra acquired from the harvested livers from mice with NAFLD of differing severity (healthy, mild steatotic and steatohepatitic). The fluorescence data were entered into a sparse multiclass probabilistic algorithm for disease classification. Histopathology, thiobarbituric acid reactive substances (TBARS) and alanine transaminase (ALT) assays were conducted in addition to the fluorescence measurements RESULTS: TBARS and ALT assays enabled differentiation of the steatohepatitic group from the mild steatosis and control groups (P ≤ 0.028) but failed to separate the mild steatotic group from the control group. The three groups were all clearly differentiated from each other using fluorescence spectroscopy, and classification accuracy was found to be 95%. CONCLUSION: Fluorescence spectroscopy appears to be a promising approach for the analysis of diseased liver tissue.

Impact of pan-caspase inhibition in animal models of established steatosis and non-alcoholic steatohepatitis.

BACKGROUND & AIMS: Non-alcoholic fatty liver disease is a progressive condition comprising steatosis, steatohepatitis, and cirrhosis. Caspase activation mediates apoptosis and the inflammatory response. Studies demonstrate increased apoptotic activity in NASH although its pathophysiological importance is uncertain. We sought to determine the effects of irreversible pan-caspase inhibition in murine models of established steatosis (high fat diet, HFD) and steatohepatitis (methionine-choline deficient diet, MCD). METHODS: In one study arm, male C3H/HeN mice were fed HFD; in the other, Db/Db mice were fed MCD. Once disease was established, animals were randomised to receive caspase inhibitor (VX-166), TPGS/PEG vehicle or no additional therapy until the end of the study. Biochemical and histological indices were examined to determine NASH activity and tissue oxidative stress. Apoptotic activity and cell turnover were assessed immunohistochemically by staining for caspase-cleaved CK-18 and PCNA. RESULTS: MCD and HFD significantly increased apoptosis, which was reduced by VX-166 treatment. VX-166 did not reduce steatosis but reduced histological inflammation, serum ALT levels, and oxidative stress, particularly in the MCD model. TPGS/PEG vehicle also exhibited some anti-inflammatory activity. CONCLUSIONS: In both models, VX-166 inhibited apoptosis and reduced histological inflammatory infiltrate although there was a more modest impact on other indices of liver injury. In addition, TPGS/PEG vehicle also exhibited some anti-inflammatory activity, likely through the antioxidant effects of vitamin E and changes in gut flora/mucosal interactions. These data suggest that caspase inhibition may represent a valid therapeutic approach; however, further studies to assess the long-term value of more selective caspase inhibition are merited.

Agonist activity of naloxone benzoylhydrazone at recombinant and native opioid receptors.

1. In the present study, we examined the pharmacological activity of the putative kappa3-opioid receptor agonist naloxone benzoylhydrazone (NalBzoH) at recombinant human opioid receptors individually expressed in Chinese hamster ovary (CHO) cells and native opioid receptors present in rat striatum. 2. At the mu-opioid receptor (MOR), NalBzoH stimulated guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS) binding (pEC50=8.59) and inhibited cyclic AMP accumulation (pEC50=8.74) with maximal effects (Emax) corresponding to 55 and 65% of those obtained with the MOR agonist DAMGO, respectively. The MOR antagonist CTAP blocked the stimulatory effects of NalBzoH and DAMGO with similar potencies. 3. At the kappa-opioid receptor (KOR), NalBzoH stimulated [35S]GTPgammaS binding (pEC50=9.70) and inhibited cyclic AMP formation (pEC50=9.45) as effectively as the selective KOR agonist (-)-U-50,488. The NalBzoH effect was blocked by the KOR antagonist nor-binaltorphimine (nor-BNI) (pKi=10.30). 4. In CHO cells expressing the delta-opioid receptor (DOR), NalBzoH increased [35S]GTPgammaS binding (pEC50=8.49) and inhibited cyclic AMP formation (pEC50=8.61) almost as effectively as the DOR agonist DPDPE. Naltrindole (NTI), a selective DOR antagonist, completely blocked the response to NalBzoH (pKi of 10.40). 5. In CHO cells expressing the nociceptin/orphanin FQ (N/OFQ) receptor (NOP), NalBzoH failed to exert agonist effects and antagonized the agonist-induced receptor activation. 6. When compared to other opioid receptor ligands, NalBzoH showed an efficacy that was lower than that of morphine at MOR, but higher at KOR and DOR. 7. In rat striatum, NalBzoH enhanced [35S]GTPgammaS binding and inhibited adenylyl cyclase activity. These effects were antagonized by either CTAP, nor-BNI or NTI, each antagonist blocking a fraction of the NalBzoH response. 8. These data demonstrate that NalBzoH displays agonist activity at MOR, DOR and KOR expressed either in a heterologous cell system or in a native environment.

A common set of immediate-early response genes in liver regeneration and hyperplasia.

Partial hepatectomy (PH) and some tumor-promoting agents stimulate hepatocyte cell proliferation, but each treatment acts through distinct transcription factors. We compared mouse immediate-early gene expression changes after PH with those induced by 1,4-bis[2-(3,5-dichoropyridyloxy)]benzene (TCPOBOP), a tumor-promoting liver mitogen. PH activates nuclear factor kappa B (NF-kappa B) and Stat3, whereas TCPOBOP is a ligand for the nuclear receptor, constitutive androstane receptor (CAR). RNA from 1 and 3 hours after each treatment was hybridized to a 9,000 complementary DNA (cDNA) microarray. Of about 6,000 messenger RNAs that had detectable expression, 127 showed reproducible up-regulation or down-regulation at a significant level. The TCPOBOP response was more discrete than the PH response; they amounted to 1% and 1.9% of positive hybridizations, respectively. Twenty-three genes were regulated only by TCPOBOP, 57 only by PH, and 59 by both treatments. More detailed analysis defined 16 clusters with common patterns of expression. These patterns and quantification of hybridization levels on the array were confirmed by Northern blots. TCPOBOP selectively activated expression of a number of detoxification enzymes. In conclusion, the genes that were regulated by both treatments suggest down-regulation of apoptosis, altered signal transduction, and early biogenesis of critical cell components.

Sex difference in the proliferative response of mouse hepatocytes to treatment with the CAR ligand, TCPOBOP.

The nuclear receptor Constitutive Androstane Receptor (CAR) binds DNA as a heterodimer with the retinoic-X receptor and activates gene transcription. Previously, in vitro studies have shown that the testosterone metabolites, androstenol and androstenol, inhibit the constitutive transcriptional activity of CAR, suggesting that differences might exist in the response to CAR-mediated gene activation between different sexes. In this study, we have analyzed the response of female and male CD-1 mice to stimulation of hepatocyte proliferation caused by the CAR ligand TCPOBOP. Results showed that the labelling index of female hepatocytes at 24, 30 and 36 h after treatment was much higher than that found in males. The higher proliferative activity of female hepatocytes was associated with increased hepatic levels of cyclin D1, cyclin A, E2F and enhanced phosphorylation of pRb and p107. The increased mitogenic response of females was associated with higher mRNA levels of CYP2B10, a known target of CAR. Administration of androstenol to TCPOBOP-treated mice caused a reduction of labelling index, which was accompanied by a decrease of CYP2B10 and CAR mRNA levels. In conclusion, the results show that, in addition to microsomal detoxification, another biological response elicited by the CAR ligand TCPOBOP, namely, hepatocyte proliferation, occurs at higher levels in female than male mice, suggesting that CAR transcriptional activity in males is partially counteracted by physiological higher levels of testosterone metabolites such as androstenol and androstenol.

Different effects of the liver mitogens triiodo-thyronine and ciprofibrate on the development of rat hepatocellular carcinoma.

Previous work has shown that treatment with thyroid hormone (T3) decreased the incidence of rat hepatocellular carcinoma (HCC). The present study was designed to determine whether the inhibitory effect of T3 on HCC development was limited to early steps of the carcinogenetic process or, whether a similar effect could also be exerted by starting T3 treatment at later stages. Hepatic nodules were induced in Fischer rats by a single dose of DENA, followed by a 2-week exposure of the animals to 2-AAF and partial hepatectomy. Rats were then divided into 3 groups: group 1 was maintained on basal diet: group 2 was fed a diet containing 4 mg/kg T3 for a week, every month/7 months, starting 9 weeks after DENA administration: group 3 was exposed to cycles of T3 starting 8 months after initiation. Results demonstrate that inhibition of HCC development was essentially similar in rats exposed to T3 starting either 9 weeks or 8 months after initiation (50% inhibition compared to control rats). We have previously shown that T3-induced nodule regression and HCC inhibition occurred in spite of its mitogenic effect. Therefore, we next wished to determine whether a similar antitumoral effect could be exerted by other liver mitogens, such as peroxisome proliferators. Rats exposed to the initiation-promotion protocol described previously, were subjected to 11 cycles of a T3 or a ciprofibrate-supplemented diet, each cycle consisting of 7 days/month: the incidence of HCC and lung metastases was determined 13.5 months after initiation. Results showed that although treatment with T3 strongly inhibited HCC development (only 31% of T3+ rats showed HCC vs 91% of controls), rats given ciprofibrate developed the same number of HCC as T3-untreated rats. In conclusion, the results of this study showed that the anticarcinogenic effect of T3 is maintained also when treatment begins late in the process, and its antitumoral property appears to be specific and may not be shared by other liver mitogens.

Loss of cyclin D1 does not inhibit the proliferative response of mouse liver to mitogenic stimuli.

Cyclin D1 is considered to play a critical role in the progression from G1 to S phase of the cell cycle, and its overexpression is seen in many human tumors. However, previous studies in cell lines have shown that cyclin D1 is not sufficient to trigger cell replication. To directly test the role of cyclin D1 in the progression of the cell cycle, we have examined the proliferative response of hepatocytes to the hepatomitogen 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) in mice with homozygous disruption of the cyclin D1 gene. We found that 24 hours after administration of TCPOBOP, the number of bromodeoxyuridine (BrdU)-positive hepatocytes was significantly reduced in cyclin D1(-/-) (labeling index was 1.9% in knockout mice vs. 9.7% of heterozygous mice); however, no difference in the number of proliferating hepatocytes was found 36 or 72 hours after treatment (labeling index was 16% and 43% in cyclin D1(-/-) mice vs. 20% and 41% of heterozygous mice), indicating that lack of cyclin D1 may transiently delay entry into S phase but is not sufficient to inhibit the response of hepatocytes to mitogenic stimuli. The results also show that although there was no difference in hepatic protein levels of cyclin D2 and D3 between untreated cyclin D1(-/-) and cyclin D1(+/-) mice, messenger RNA (mRNA) and protein levels of cyclin E were much higher in the former. In conclusion, our results show that cyclin D1 is not essential for liver development and hepatocyte proliferation induced by mitogenic stimuli and suggest that overexpression of cyclin E may compensate for the lack of cyclin D1.