Bacterial ß-glucuronidase inhibition protects mice against enteropathy induced by indomethacin, ketoprofen or diclofenac: mode of action and pharmacokinetics.

1. We have previously demonstrated that a small molecule inhibitor of bacterial ß-glucuronidase (Inh-1; [1-((6,8-dimethyl-2-oxo-1,2-dihydroquinolin-3-yl)-3-(4-ethoxyphenyl)-1-(2-hydroxyethyl)thiourea]) protected mice against diclofenac (DCF)-induced enteropathy. Here we report that Inh-1 was equally protective against small intestinal injury induced by other carboxylic acid-containing non-steroidal anti-inflammatory drugs (NSAIDs), indomethacin (10 mg/kg, ip) and ketoprofen (100 mg/kg, ip). 2. Inh-1 provided complete protection if given prior to DCF (60 mg/kg, ip), and partial protection if administered 3-h post-DCF, suggesting that the temporal window of mucosal protection can be extended for drugs undergoing extensive enterohepatic circulation. 3. Pharmacokinetic analysis of Inh-1 revealed an absolute bioavailability (F) of 21% and a short t1/2 of <1 h. This low F was shown to be due to hepatic first-pass metabolism, as confirmed with the pan-CYP inhibitor, 1-aminobenzotriazole. 4. Using the fluorescent probe 5 (and 6)-carboxy-2',7'-dichlorofluorescein, we demonstrated that Inh-1 did not interfere with hepatobiliary export of glucuronides in gall bladder-cannulated mice. 5. These data are compatible with the hypothesis that pharmacological inhibition of bacterial ß-glucuronidase-mediated cleavage of NSAID glucuronides in the small intestinal lumen can protect against NSAID-induced enteropathy caused by locally high concentrations of NSAID aglycones.

Sp sites contribute to basal and inducible expression of the human TNIP1 (TNFα-inducible protein 3-interacting protein 1) promoter.

TNIP1 [TNFα (tumour necrosis factor α)-induced protein 3-interacting protein 1] is a co-repressor of RAR (retinoic acid receptor) and PPAR (peroxisome-proliferator-activated receptor). Additionally, it can reduce signalling stemming from cell membrane receptors such as those for TNFα and EGF (epidermal growth factor). Consequently, it influences a variety of receptor-mediated events as diverse as transcription, programmed cell death and cell cycling. Thus changes in TNIP1 expression levels are likely to affect multiple important biological end points. TNIP1 expression level changes have been linked to psoriasis and systemic sclerosis. As such, it is crucial to determine what controls its expression levels, starting with constitutive control of its promoter. Our analysis of the TNIP1 promoter revealed multiple transcription start sites in its GC-rich proximal regions along with two transcriptionally active Sp (specificity protein) sites, responsive to both Sp1 and Sp3. EMSA (electrophoretic mobility-shift assay) and ChIP (chromatin immunoprecipitation) demonstrated physical binding between Sp1 and Sp3 at these sites. A decrease in Sp1 protein levels via siRNA (short interfering RNA) or diminished Sp1 DNA binding by mithramycin decreased TNIP1 mRNA levels. This Sp-binding GC-rich region of the TNIP1 promoter also participates in transcriptional activation by ligand-bound RAR. Together, these results demonstrate newly identified regulators of TNIP1 expression and suggest possible transcription factor targets which in turn control TNIP1-related biological end points ranging from apoptosis to inflammatory diseases.

General practice encounters with Aboriginal and Torres Strait Islander people.

In the BEACH program between April 2008 and March 2013, general practitioners (GPs) recorded details of 7380 patient encounters with people who identified themselves as Aboriginal and Torres Strait Islander, which accounted for 1.5% of all BEACH encounters. The rates at which type 2 diabetes and asthma were managed at encounters with patients who identified as Aboriginal and Torres Strait Islander people were compared with those of other patients.

PPARγ and NF-κB regulate the gene promoter activity of their shared repressor, TNIP1.

Human TNFAIP3 interacting protein 1 (TNIP1) has diverse functions including support of HIV replication through its interaction with viral Nef and matrix proteins, reduction of TNFα-induced signaling through its interaction with NF-κB pathway proteins, and corepression of agonist-bound retinoic acid receptors and peroxisome proliferator-activated receptors (PPAR). The wide tissue distribution of TNIP1 provides the opportunity to influence numerous cellular responses in these roles and defining control of TNIP1 expression would be central to improved understanding of its impact on cell function. We cloned 6kb of the human TNIP1 promoter and performed predictive and functional analyses to identify regulatory elements. The promoter region proximal to the transcription start site is GC-rich without a recognizable TATA box. In contrast to this proximal ~500bp region, 6kb of the promoter increased reporter construct constitutive activity over five-fold. Throughout the 6kb length, in silico analysis identified several potential binding sites for both constitutive and inducible transcription factors; among the latter were candidate NF-κB binding sequences and peroxisome proliferator response elements (PPREs). We tested NF-κB and PPAR regulation of the endogenous TNIP1 gene and cloned promoter by expression studies, electrophoretic mobility shift assays, and chromatin immunoprecipitations. We validated NF-κB sites in the TNIP1 promoter proximal and distal regions as well as one PPRE in the distal region. The ultimate control of the TNIP1 promoter is likely to be a combination of constitutive transcription factors and those subject to activation such as NF-κB and PPAR.

Work related encounters in general practice.

General practitioner participants in the BEACH (Bettering the Evaluation and Care of Health) program were asked to indicate which problems managed at the encounter were considered to be work related. From April 2007 to March 2012, at least one work related problem was managed at 2.7% of all encounters (11 429 encounters). A total of 16 045 problems were managed at these encounters, of which 11 911 (74.2%) were work related. Three-quarters (75.7%) of recorded work related encounters were claimable through workers' compensation.

Organotypic modeling of human keratinocyte response to peroxisome proliferators.

Peroxisome proliferators (PPs) are a diverse chemical group including hypolipidemic drugs and some fatty acids. Their stimulation of PP-activated receptors (PPARs) and subsequent control of gene expression regulates metabolism and differentiation in many cells. PPs have multiple opportunities to target human epidermal keratinocytes because of delivery through dietary, clinical, and/or topical exposure routes. PPAR knockout mice and PP treatment of mouse skin or human keratinocytes in monolayer culture have established some effects for PPs in cutaneous differentiation. However, incomplete epidermal maturation characteristic of monolayer keratinocytes and rodent-specific effects may limit our full understanding of human keratinocyte responses to PPs. To address these issues, we investigated PP influence on primary human keratinocytes in organotypic cultures that recapitulate biochemical markers of epidermis. We found that the PPARα agonists clofibrate, docasohexaenoic acid, and WY-14,643 produced mild to moderate keratinocyte hyperplasia, increased stratification (particularly of granular and cornified layers), and enhanced levels of the differentiation markers filaggrin, ABCA12, and phosphorylated HSP27. Several PP effects generated in the organotypic system, however, were distinct from those previously reported for rodent skin and human keratinocyte monolayer cultures, suggesting that the species and growth context of target cells can impact exposure outcomes. Given the utility of organotypic cultures for modeling the epidermis, studies in this system may bridge the gap between the rodent assays and clinical studies of human epidermal responses to PPs.

Infertility: management in Australian general practice.

From April 2007 to March 2012 in the BEACH (Bettering the Evaluation and Care of Health) program, infertility/subfertility was managed at 652 encounters with 534 general practitioners (1.3 per 1000 encounters).

Psychological encounters in general practice.

In November 2001, Focussed Psychological Strategy (FPS) Medicare item numbers were created under the Better Outcomes in Mental Health Care initiative to reimburse general practitioners trained in the provision of evidence based psychological therapies.

TNIP1 is a corepressor of agonist-bound PPARs.

Nuclear receptor (NR) coregulators include coactivators, contributing to holoreceptor transcriptional activity, and corepressors, mediating NR target gene silencing in the absence of hormone. We identified an atypical NR coregulator, TNFα-induced protein 3-interacting protein 1 (TNIP1), from a peroxisome proliferator activated receptor (PPAR) α screen of a human keratinocyte cDNA library. TNIP1's complex nomenclature parallels its additional function as an NF-κB inhibitor. Here we show TNIP1 is an atypical NR corepressor using two-hybrid systems, biochemical studies, and receptor activity assays. The requirements for TNIP1-PPAR interaction are characteristic for coactivators; however, TNIP1 partially decreases PPAR activity. TNIP1 has separable transcriptional activation and repression domains suggesting a modular nature to its overall effect. It may provide a means of lowering receptor activity in the presence of ligand without total loss of receptor function. TNIP1's multiple roles and expression in several cell types suggest its regulatory effect depends on its expression level and the expression of other regulators in NR and/or NF-κB signaling pathways. As a NR coregulator, TNIP1 targeting agonist-bound PPAR and reducing transcriptional activity offers control of receptor signaling not available from typical corepressors and may contribute to combinatorial regulation of transcription.

In vitro release testing method development for ophthalmic ointments.

It is essential as well as challenging to develop a reliable in vitro release testing method for determining whether differences in release profiles exist between qualitatively and quantitatively equivalent ophthalmic ointment formulations. There is a lack of regulatory guidance on in vitro release testing methods for ophthalmic formulations. Three different in vitro release testing methods 1) USP apparatus 4 with semisolid adapters; 2) USP apparatus 2 with enhancer cells; and 3) Franz diffusion cells were investigated. Qualitatively and quantitatively equivalent ointments were prepared via hot melting and simple mixing methods using four different sources of excipients (i.e. white petrolatum). The ointment formulations were characterized for content uniformity, particle size, and rheological parameters. All the formulations showed adequate content uniformity and similar particle size. The ointments prepared via the hot melting processes showed higher rheological parameters, as did the ointments prepared using 'white' petrolatum that exhibited a yellowish color. The three in vitro release testing methods were compared and evaluated for reproducibility, discriminatory capability, and correlation with the rheological parameters. Compared with the compendial methods, the non-compendial method (Franz diffusion cells) showed poorer reproducibility. All three methods possessed the ability to discriminate between the ophthalmic ointments with manufacturing differences. However, the USP apparatus 4 method displayed the largest margin of discrimination between the release profiles of the different ophthalmic ointments. In addition, the in vitro release rate obtained using the USP apparatus 4 method showed the strongest logarithmic linear correlation with the rheological parameters (Power law consistency index (K value) and crossover modulus) compared to the other two methods.

Mechanisms of cell death pathway activation following drug-induced inhibition of mitochondrial complex I.

Respiratory complex I inhibition by drugs and other chemicals has been implicated as a frequent mode of mitochondria-mediated cell injury. However, the exact mechanisms leading to the activation of cell death pathways are incompletely understood. This study was designed to explore the relative contributions to cell injury of three distinct consequences of complex I inhibition, i.e., impairment of ATP biosynthesis, increased formation of superoxide and, hence, peroxynitrite, and inhibition of the mitochondrial protein deacetylase, Sirt3, due to imbalance of the NADH/NAD(+) ratio. We used the antiviral drug efavirenz (EFV) to model drug-induced complex I inhibition. Exposure of cultured mouse hepatocytes to EFV resulted in a rapid onset of cell injury, featuring a no-effect level at 30µM EFV and submaximal effects at 50µM EFV. EFV caused a concentration-dependent decrease in cellular ATP levels. Furthermore, EFV resulted in increased formation of peroxynitrite and oxidation of mitochondrial protein thiols, including cyclophilin D (CypD). This was prevented by the superoxide scavenger, Fe-TCP, or the peroxynitrite decomposition catalyst, Fe-TMPyP. Both ferroporphyrins completely protected from EFV-induced cell injury, suggesting that peroxynitrite contributed to the cell injury. Finally, EFV increased the NADH/NAD(+) ratio, inhibited Sirt3 activity, and led to hyperacetylated lysine residues, including those in CypD. However, hepatocytes isolated from Sirt3-null mice were protected against 40µM EFV as compared to their wild-type controls. In conclusion, these data are compatible with the concept that chemical inhibition of complex I activates multiple pathways leading to cell injury; among these, peroxynitrite formation may be the most critical.

Human TNFα-induced protein 3-interacting protein 1 (TNIP1) promoter activation is regulated by retinoic acid receptors.

Coregulator proteins play key roles in transcriptional control by members of the nuclear receptor superfamily. Previously, we demonstrated that tumor necrosis factor α (TNFα)-induced protein 3-interacting protein 1 (TNIP1) is a corepressor of agonist-bound retinoic acid receptors (RARs). Additionally, TNIP1 has been shown to repress peroxisome proliferator-activated receptors (PPAR) and NF-κB activity and interact with HIV proteins nef and matrix. TNIP1 transcriptional regulation, however, is under studied. Here we show that under permissive epigenetic conditions, TNIP1 expression is induced by all trans retinoic acid (ATRA). Within a 6000 bp region of the human TNIP1 promoter we cloned, both proximal and distal promoter regions are RAR responsive with the latter having RA response elements (RAREs) recognizable by their sequence and functionality in native promoter and synthetic RARE luciferase constructs, EMSA, and ChIP assays. These findings suggest a feedback loop whereby RARs activate expression of TNIP1, which then attenuates their activity. Together with anticipated constitutive transcription factors and the previously described NF-κB-responsiveness of the proximal TNIP1 promoter, the expected combinatorial control of TNIP1 expression could likely modulate TNIP1's impact in any of its target pathways. The degree of control by RARs or other transcription factors would in turn depend on their cell-specific level of expression and/or activation from signals in the environment such as ATRA and TNFα.

Isoniazid-induced cell death is precipitated by underlying mitochondrial complex I dysfunction in mouse hepatocytes.

Isoniazid (INH) is an antituberculosis drug that has been associated with idiosyncratic liver injury in susceptible patients. The underlying mechanisms are still unclear, but there is growing evidence that INH and/or its major metabolite, hydrazine, may interfere with mitochondrial function. However, hepatic mitochondria have a large reserve capacity, and minor disruption of energy homeostasis does not necessarily induce cell death. We explored whether pharmacologic or genetic impairment of mitochondrial complex I may amplify mitochondrial dysfunction and precipitate INH-induced hepatocellular injury. We found that INH (≤ 3000 µM) did not induce cell injury in cultured mouse hepatocytes, although it decreased hepatocellular respiration and ATP levels in a concentration-dependent fashion. However, coexposure of hepatocytes to INH and nontoxic concentrations of the complex I inhibitors rotenone (3 µM) or piericidin A (30 nM) resulted in massive ATP depletion and cell death. Although both rotenone and piericidin A increased MitoSox-reactive fluorescence, Mito-TEMPO or N-acetylcysteine did not attenuate the extent of cytotoxicity. However, preincubation of cells with the acylamidase inhibitor bis-p-nitrophenol phosphate provided protection from hepatocyte injury induced by rotenone/INH (but not rotenone/hydrazine), suggesting that hydrazine was the cell-damaging species. Indeed, we found that hydrazine directly inhibited the activity of solubilized complex II. Hepatocytes isolated from mutant Ndufs4(+/-) mice, although featuring moderately lower protein expression levels of this complex I subunit in liver mitochondria, exhibited unchanged hepatic complex I activity and were therefore not sensitized to INH. These data indicate that underlying inhibition of complex I, which alone is not acutely toxic, can trigger INH-induced hepatocellular injury.

TNIP1, a retinoic acid receptor corepressor and A20-binding inhibitor of NF-κB, distributes to both nuclear and cytoplasmic locations.

An increasingly wide range of functions, from repression of NF-κB signaling to protection from apoptosis, is being recognized for tumor necrosis factor α-induced protein 3-interacting protein 1 (TNIP1). The authors recently demonstrated TNIP1 interaction with and repression of liganded retinoic acid receptors, distinguishing it from the more typical NCoR and SMRT corepressors, which function only in the absence of ligand. To improve their understanding of TNIP1's roles in physiologic and pathologic events, the authors examined its distribution in normal and malignant human tissues and cultured cells. They found cytoplasmic and nuclear TNIP1 in normal skin keratinocytes as it colocalized with retinoic acid receptor α, one of the nuclear receptors it corepresses. Nuclear and cytoplasmic TNIP1 was also found in the malignant keratinocytes of squamous cell carcinomas. Compared to adjacent normal tissues of other organs, TNIP1 staining and distribution varied with increased levels in esophageal cancer and marked decreases in prostate cancer. The varying levels and distribution of TNIP1 in normal and disease state tissues could be expected to affect processes in which TNIP1 is involved, such as NF-κB and nuclear receptor signaling, possibly contributing to the disease course or response to therapies targeting these key players of cell growth and differentiation.

Scanning for transcription factor binding by a variant EMSA.

Detection of in vitro protein-DNA interaction is one of many investigational analyses for transcription factor regulation of gene promoters. The electrophoretic mobility shift assay (EMSA) has proven widely popular in this respect by integrating individual techniques (protein isolation, nucleic acid radiolabeling, and gel electrophoresis) into one protocol. However, relatively short DNA oligomers are often used which in many cases presupposes what one sequence out of a promoter of possibly thousands of base pairs is the candidate region interacting with a transcription factor. This can be an experimentally distressing situation when multiple putative binding sites of less than perfect consensus may be present making selection of any one or even a few potential sites uncertain or when one is seeking improved throughput as opposed to a one factor:one oligomer approach for in vitro testing of algorithm-predicted binding sites. We describe here our use and refinement of a variant EMSA that can employ multiple and relatively long (up to 1000 bp) probes of promoter sequence in one binding reaction for interaction with nuclear proteins in general and individual transcription factors in particular. We provide labeling and electrophoresis methods suitable for such probes and also highlight the mobility shift differences one can expect with the variant probe method.