No abstract

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.

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 B (NF-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 downregulation of apoptosis, altered signal transduction, and early biogenesis of critical cell components. (HEPATOLOGY 2003;38:314-325.)

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. (HEPATOLOGY 2002;36: 1098-1105.)T he liver has the capacity to regenerate after surgical or chemical removal of part of its cellular mass. During the last decade, much new information has become available on the events that may initiate liver regeneration. 1 Several converging lines of evidence have proposed that tumor necrosis factor ␣ and interleukin 6 are important components of the early signaling pathways leading to regeneration. 2-4 Hepatocytes, however, can be stimulated to proliferate by treatment with a variety of agents such as thyroid hormone, peroxisome proliferators, lead nitrate, and others that do not cause tissue injury but rather an excess of cells (primary mitogens). 5 We have previously shown that hepatocyte proliferation induced by several mitogens, especially ligands of nuclear receptors of the steroid/thyroid receptor superfamily, occurs in a tumor necrosis factor ␣-and interleukin 6 -independent pathway and in the absence of changes in immediate-early gene expression or activation of transcription factors such as nuclear factor B, STAT3, or AP-1. [6][7][8] In the same studies, it was shown that at least 3 different hepatomitogens, thyroid hormone, nafenopin, and 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), induce a very rapid increase of cyclin D1 expression and an accelerated entry of hepatocytes into S phase, suggesting that cyclin D1 may be a common critical target gene in this type of proliferation ...

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.

Background & AimVariations 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.MethodsConventionally housed C3H/HeH (CH) and C3H/HeH germ free (GF) mice were administered a 200mg/kg IP dose of acetaminophen. The severity of hepatotoxicity at 8 hours was assessed by histology and biochemical indices. A urinary metabolic profile was obtained using 1H-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.ResultsBaseline glutathione levels were significantly reduced (p=0.03) in GF mice. CYP2E1 mRNA expression and protein levels were not altered. Inter-individual 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 hours(cross-validated ANOVA p=1x10-22). Interruption of TLR4 signalling in LPS-r mice had additional protective effects.ConclusionVariations 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.

Nuclear receptors play an important role in organ physiology, linking small molecule hormones and other ligands to specific transcriptional regulatory events. More than 40 members of this receptor family have been identified in vertebrate species. 1 Two key structural elements common to all nuclear receptors include a DNA-binding domain (DBD) and a ligand or hormone-binding domain (LBD), with other regions in the N terminus and the LBD of the receptor being important mediators of the receptor's transcriptional transactivating functions. 2,3 The Retinoid X receptor (RXR) was the first orphan receptor for which a ligand was identified, 9-cis-retinoic acid, 4 and it was found that it can form heterodimers with other orphan receptors such as, for example, COUPs and HNF-4 or with a number of other members of the nuclear receptor superfamily, including the receptors for thyroid hormone (TR), androstane (CAR), all trans-retinoic acid (RAR), vitamin D (DR), farnesoids (FXR), and peroxisome proliferators (PPAR).Among the different cellular events controlled by nuclear receptors, cell proliferation is a common, although poorly understood, response. Indeed, a powerful mitogenic activity has been observed in uterine, mammary, and hepatic tissue following treatment with steroid hormones, in particular estrogens and progesterone 5,6 ; moreover, the discovery of a novel steroid hormone receptor activated by fatty acid-like chemicals, the peroxisome proliferators (PPs), 7 a class of powerful liver mitogens and rodent hepatocarcinogens, 8 has generated an increased interest for the understanding of the mechanism underlying the mitogenic effects mediated by nuclear receptors. In fact, the finding that PPs, like estrogens, do not possess any direct genotoxic property, has supported the hypothesis that oxidative stress with associated cell proliferation contributes to cancer development increasing mutational events in cells of the target organ. 9,10 Moreover, other molecules, such as T3, 9-cis retinoic acid, all trans-retinoic acids are also capable of binding and activating nuclear receptors, namely TR, RAR, RXR of the same superfamily and function as mitogens for rat liver and pancreatic cells. [11][12][13] Mice deficient in PPAR␣ (PPAR␣ Ϫ/Ϫ ) and in CAR (CAR Ϫ/Ϫ ) show a complete loss of hyperplastic response to their ligands, PPs and TCPOBOP, respectively. 14-15 These observations further sup-

Background and objectives: the increasing incidence of non-alcoholic fatty liver diseases