Toxicogenomic module associations with pathogenesis: a network-based approach to understanding drug toxicity.

Despite investment in toxicogenomics, nonclinical safety studies are still used to predict clinical liabilities for new drug candidates. Network-based approaches for genomic analysis help overcome challenges with whole-genome transcriptional profiling using limited numbers of treatments for phenotypes of interest. Herein, we apply co-expression network analysis to safety assessment using rat liver gene expression data to define 415 modules, exhibiting unique transcriptional control, organized in a visual representation of the transcriptome (the 'TXG-MAP'). Accounting for the overall transcriptional activity resulting from treatment, we explain mechanisms of toxicity and predict distinct toxicity phenotypes using module associations. We demonstrate that early network responses complement traditional histology-based assessment in predicting outcomes for longer studies and identify a novel mechanism of hepatotoxicity involving endoplasmic reticulum stress and Nrf2 activation. Module-based molecular subtypes of cholestatic injury derived using rat translate to human. Moreover, compared to gene-level analysis alone, combining module and gene-level analysis performed in sequence identifies significantly more phenotype-gene associations, including established and novel biomarkers of liver injury.

Do flow volume loops alter surgical management in patients with a goitre?

INTRODUCTION: Flow volume loops (FVLs) are considered part of the workup of patients with thyroid enlargement presenting to the endocrinology clinic. They are used to detect upper airway obstruction (UAO) secondary to tracheal compression (TC) from a goitre. Surgical assessment in contrast tends to focus on clinical evaluation supplemented when required by imaging. The aim of this study was to investigate whether FVLs influence the decision to operate in patients with a goitre. METHODS: We identified patients with a goitre referred by the department of endocrinology for FVLs between 2006 and 2011. The results of the FVL were collated, and their impact on patient management was assessed. RESULTS: Ninety-six patients were referred for FVL. In 38 patients, the indication was specifically to evaluate the effects of a goitre. Of these, 33 were reported as normal. Five FVLs were reported as abnormal (3 suggesting lung pathology and 2 TC). Both patients with TC on FVL presented no CT evidence of TC and underwent surgery due to abnormal cytology. Of the 33 normal FVLs, 7 underwent surgery: 2 for local compression, 4 for abnormal cytology and 1 for Graves' disease. None of the FVLs influenced the decision to operate. CONCLUSION: FVLs may detect subradiological TC, but rarely influence management in patients with a goitre. In view of this and the cost of £235 per investigation, FVL should be reserved for goitre patients with suspected primary lung pathology, where the distinction between large and small airway compression is likely to influence management.

Hepatocolonic fistula: a rare consequence of retained gallstones after laparoscopic cholecystectomy.

Spillage of gallstones during laparoscopic cholecystectomy occurs in up to 30% of cases but complications due to stone retention are less frequent. We report the first case of a hepatocolonic fistula as a consequence of a retained gallstone.

Data-driven analysis approach for biomarker discovery using molecular-profiling technologies.

High-throughput molecular-profiling technologies provide rapid, efficient and systematic approaches to search for biomarkers. Supervised learning algorithms are naturally suited to analyse a large amount of data generated using these technologies in biomarker discovery efforts. The study demonstrates with two examples a data-driven analysis approach to analysis of large complicated datasets collected in high-throughput technologies in the context of biomarker discovery. The approach consists of two analytic steps: an initial unsupervised analysis to obtain accurate knowledge about sample clustering, followed by a second supervised analysis to identify a small set of putative biomarkers for further experimental characterization. By comparing the most widely applied clustering algorithms using a leukaemia DNA microarray dataset, it was established that principal component analysis-assisted projections of samples from a high-dimensional molecular feature space into a few low dimensional subspaces provides a more effective and accurate way to explore visually and identify data structures that confirm intended experimental effects based on expected group membership. A supervised analysis method, shrunken centroid algorithm, was chosen to take knowledge of sample clustering gained or confirmed by the first step of the analysis to identify a small set of molecules as candidate biomarkers for further experimentation. The approach was applied to two molecular-profiling studies. In the first study, PCA-assisted analysis of DNA microarray data revealed that discrete data structures exist in rat liver gene expression and correlated with blood clinical chemistry and liver pathological damage in response to a chemical toxicant diethylhexylphthalate, a peroxisome-proliferator-activator receptor agonist. Sixteen genes were then identified by shrunken centroid algorithm as the best candidate biomarkers for liver damage. Functional annotations of these genes revealed roles in acute phase response, lipid and fatty acid metabolism and they are functionally relevant to the observed toxicities. In the second study, 26 urine ions identified from a GC/MS spectrum, two of which were glucose fragment ions included as positive controls, showed robust changes with the development of diabetes in Zucker diabetic fatty rats. Further experiments are needed to define their chemical identities and establish functional relevancy to disease development.

Tension- and afferent input-associated responses of neuromuscular system of rats to hindlimb unloading and/or tenotomy.

Responses of electromyogram (EMG) in soleus muscle and both afferent and efferent neurograms at the fifth lumbar (L(5)) segmental level of spinal cord were investigated during acute and chronic unloading induced by hindlimb suspension and/or tenotomy in adult rats. The soleus EMG and afferent neurogram decreased 88 and 37%, respectively, relative to those at quadrupedal posture on the floor after acute hindlimb suspension that causes passive shortening of soleus due to ankle plantarflexion. However, the afferent neurogram (P < 0.05) and soleus EMG (P > 0.05) recorded on the floor increased after tenotomy of synergists. Furthermore, the afferent input was inhibited when the soleus EMG disappeared after tenotomy of soleus. The afferent neurogram and EMG of the soleus showed correlated responses to a variety of treatments, suggesting that the afferent neurogram recorded at the L(5) segmental level reflects the neural input associated with the activity level of the soleus predominantly. The level of efferent neurogram decreased after acute hindlimb suspension but was not influenced significantly by tenotomy of synergists and/or soleus itself. The EMG and afferent neurograms remained low up to the 4th day but recovered to the preexperimental levels within 14 days, due to reorganization of sarcomere number and length, as well as the shortening of muscle fiber length and recovery of tension development. It is suggested that the levels of EMG and afferent neurogram associated with antigravity muscle are closely related to the tension development of the muscle.

Interlaboratory comparison exercise for the analysis of PCDD/Fs in samples of digested sewage sludge.

Five UK laboratories participated in a study designed to explore the principal sources of interlaboratory variation in the analysis of PCDD/Fs in sewage sludge. Samples of wet sludge, dry sludge, toluene extract of sludge and cleaned extract of sludge were prepared by an organising laboratory. The samples were analysed in duplicate by each laboratory along with a solution of PCDD/F standards and reference sediment. Mean coefficients of variation between laboratories were 45% for the wet sludge, 33% for the dry sludge, 32% for the extract of sludge, 36% for the cleaned extract of sludge, 32% for the reference sediment and 28% for the standard solution. The results were subjected to statistical analysis, which showed that there was no specific part of the analysis that introduced a dominant part of the variation. The spread of data generated from the analysis of wet sludge samples was not appreciably greater than the spread for the analysis of cleaned extracts. Thus the drying, extraction and clean up processes in the PCDD/F analysis of wet sludge did not have a dramatic effect on the interlaboratory variation.

Characterization of mediator complexes from HeLa cell nuclear extract.

A number of mammalian multiprotein complexes containing homologs of Saccharomyces cerevisiae Mediator subunits have been described recently. High-molecular-mass complexes (1 to 2 MDa) sharing several subunits but apparently differing in others include the TRAP/SMCC, NAT, DRIP, ARC, and human Mediator complexes. Smaller multiprotein complexes (approximately 500 to 700 kDa), including the murine Mediator, CRSP, and PC2, have also been described that contain subsets of subunits of the larger complexes. To evaluate whether these different multiprotein complexes exist in vivo in a single form or in multiple different forms, HeLa cell nuclear extract was directly resolved over a Superose 6 gel filtration column. Immunoblotting of column fractions using antisera specific for several Mediator subunits revealed one major size class of high-molecular-mass (approximately 2-MDa) complexes containing multiple mammalian Mediator subunits. No peak was apparent at approximately 500 to 700 kDa, indicating that either the smaller complexes reported are much less abundant than the higher-molecular-mass complexes or they are subcomplexes generated by dissociation of larger complexes during purification. Quantitative immunoblotting indicated that there are about 3 x 10(5) to 6 x 10(5) molecules of hSur2 Mediator subunit per HeLa cell, i.e., the same order of magnitude as RNA polymerase II and general transcription factors. Immunoprecipitation of the approximately 2-MDa fraction with anti-Cdk8 antibody indicated that at least two classes of Mediator complexes occur, one containing CDK8 and cyclin C and one lacking this CDK-cyclin pair. The approximately 2-MDa complexes stimulated activated transcription in vitro, whereas a 150-kDa fraction containing a subset of Mediator subunits inhibited activated transcription.

Decreased incidence of retinopathy of prematurity, 1995-1997.

PURPOSE: To report a significant decrease in the incidence of retinopathy of prematurity (ROP), both in our neonatal intensive care unit (NICU) and internationally, and review factors in patient care that may be contributory. METHODS: We retrospectively reviewed the records of all neonates weighing less than 1251 g admitted to our NICU from 1995 to 1997 and evaluated the incidence and stage of ROP. These data on 191 neonates were compared with an international NICU database of 9989 similar neonates, which represents all infants who received an ophthalmologic examination in the Vermont-Oxford Network Database (VOND) in 1997, except those from our institute (the University of Kentucky). In addition to investigating the incidence of ROP, we looked at the use of antenatal corticosteroids given 1 to 7 days prepartum, the use of oxygen at 36 weeks' postconceptional age, and the use of oxygen at home upon discharge. RESULTS: In our center, we had a 36.1% incidence of ROP compared with an international incidence of 57.2% for the VOND in 1997 (P <.0001). Antenatal corticosteroids were given to 62.6% of infants in our center compared with 48.6% in the VOND (P <.005). In addition, 48.5% of our infants weighing less than 1500 g received oxygen at 36 weeks' postconceptional age versus 29.5% of the VOND infants (P <.001). Upon discharge to home, 37.5% of our infants were on oxygen compared with 15.6% of infants from all VOND centers, excluding the University of Kentucky (P <.001). CONCLUSION: The incidence of ROP in our center from 1995 to 1997 and in the VOND in 1997 show a significant decrease from the 65.8% incidence from 1986 to 1987 reported by the Multicenter Trial of Cryotherapy for ROP.

Role of mitochondrial dysfunction in S-(1,2-dichlorovinyl)-l-cysteine-induced apoptosis.

The nephrotoxicity of trichloroethylene and dichloroacetylene has previously been linked to mitochondrial dysfunction induced by the metabolite S-(1,2-dichlorovinyl)-l-cysteine (DCVC). In this study, we examined whether key biochemical steps associated with mitochondria occur in DCVC-induced apoptosis in cultured porcine proximal tubular LLC-PK1 cells. DCVC caused a decrease in mitochondrial membrane potential (mt Delta Psi) beginning at 4 h and a release of cytochrome c into the cytoplasm at 6 h. Caspase-3-like activity was detected at 6 h and extensive DNA fragmentation was observed at 8 h. Decreases in cellular ATP were not evident until 8 h and later, even though electron microscopy showed that the mitochondria were extensively swollen. Aminooxyacetic acid (AOAA), an inhibitor of cysteine-conjugate beta-lyase, protected against mitochondrial changes and apoptosis. Overexpression of the antiapoptotic Bcl-2 protein desensitized LLC-PK1 cells to DCVC-induced apoptosis. These results support the interpretation that mitochondrial release of cyt c and cyt c-dependent activation of caspase-3 could have a central role in nephrotoxicity due to haloalkene-derived cysteine S-conjugates.

Oval-shaped cornea, lens duplication, and optic nerve hypoplasia associated with myelomeningocele.

Oval-shaped cornea associated with true lens duplication and separate capsules is a rare anomaly. It can occur as an isolated finding(1,2) or be associated with other ocular and facial maldevelopments.(3-5) We report a novel association of an hourglass cornea, lens duplication, and optic nerve hypoplasia with myelomeningocele in a male infant.

Inducible P450s of the CYP9 family from larval Manduca sexta midgut.

Several related cytochrome P450 cDNAs belonging to the CYP9 family have been cloned from the midgut of larval tobacco hornworms, Manduca sexta. The first P450, CYP9A2, was obtained by RT-PCR using degenerate primers. Northern blot analysis of expression in the midgut using the CYP9A2 probe revealed a significant induction by a variety of chemicals. Diets supplemented with the wild tomato compound 2-undecanone caused a dose-dependent induction which peaked after 48 h. Induction was also observed after addition to the diet of indole-3-carbinol, phenobarbital, 2-tridecanone and xanthotoxin. Neither alpha-pinene, clofibrate nor nicotine were effective inducers. The CYP9A2 probe hybridized to two mRNA species, one of 2. 0 kb and another of 4.2 kb, suggesting cross-hybridization to other P450 mRNAs. Additional P450 clones of the CYP9 family were then obtained and sequenced. Northern hybridization revealed that the 4.2 kb band also hybridized to CYP9A4 whereas the 2.0 kb hybridized to CYP9A5. Despite being 91% identical, CYP9A4 and CYP9A5 were induced differentially by clofibrate and xanthotoxin. Multiple P450 genes from various families are therefore induced in Lepidoptera in response to plant allelochemicals or xenobiotics.

Cleavage of the actin-capping protein alpha -adducin at Asp-Asp-Ser-Asp633-Ala by caspase-3 is preceded by its phosphorylation on serine 726 in cisplatin-induced apoptosis of renal epithelial cells.

Decreased phosphorylation of focal adhesion kinase and paxillin is associated with loss of focal adhesions and stress fibers and precedes the onset of apoptosis (van de Water, B., Nagelkerke, J. F., and Stevens, J. L. (1999) J. Biol. Chem. 274, 13328-13337). The cortical actin cytoskeletal network is also lost during apoptosis, yet little is known about the temporal relationship between altered phosphorylation of proteins that are critical in the regulation of this network and their potential cleavage by caspases during apoptosis. Adducins are central in the cortical actin network organization. Cisplatin caused apoptosis of renal proximal tubular epithelial cells, which was associated with the cleavage of alpha-adducin into a 74-kDa fragment; this was blocked by a general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk). Hemagglutinin-tagged human alpha-adducin was cleaved into a similar 74-kDa fragment by caspase-3 in vitro but not by caspase-6 or -7. Asp-Arg-Val-Asp(29)-Glu, Asp-Ile-Val-Asp(208)-Arg, and Asp-Asp-Ser-Asp(633)-Ala were identified as the principal caspase-3 cleavage sites; Asp-Asp-Ser-Asp(633)-Ala was key in the formation of the 74-kDa fragment. Cisplatin also caused an increased phosphorylation of alpha-adducin and gamma-adducin in the MARCKS domain that preceded alpha-adducin cleavage and was associated with loss of adducins from adherens junctions; this was not affected by z-VAD-fmk. In conclusion, the data support a model in which increased phosphorylation of alpha-adducin due to cisplatin leads to dissociation from the cytoskeleton, a situation rendered irreversible by caspase-3-mediated cleavage of alpha-adducin at Asp-Asp-Ser-Asp(633)-Ala.

In vivo behavior of peptide-specific T cells during mucosal tolerance induction: antigen introduced through the mucosa of the conjunctiva elicits prolonged antigen-specific T cell priming followed by anergy.

The mucosa of the conjunctiva is an important site of entry for environmental Ags as well as Ags emanating from the eye itself. However, very little is known about T cell recognition of Ag introduced through this important mucosal site. We have characterized the in vivo process of CD4 T cell recognition of Ag delivered via the conjunctival mucosa. Application of soluble OVA to the conjunctiva of BALB/c mice induced potent T cell tolerance. APC-presenting OVA peptide in vivo was only found in the submandibular lymph node and not in other lymph nodes, spleen, or nasal-associated lymphoid tissue. Similarly, in TCR transgenic DO11. 10 adoptive transfer mice, OVA-specific CD4+ T cell clonal expansion was only observed in the submandibular lymph node following conjunctival application of peptide. These experiments thus define a highly specific lymphatic drainage pathway from the conjunctiva. OVA-specific T cell clonal expansion peaked at day 3 following initiation of daily OVA administration and gradually declined during the 10-day treatment period, but remained elevated compared with nontreated adoptive transfer mice. During this period, the T cells expressed activation markers, and proliferated and secreted IL-2 in vitro in response to OVA stimulation. In contrast, these cells were unable to clonally expand in vivo, or proliferate in vitro following a subsequent OVA/CFA immunization. These results suggest that Ag applied to a mucosal site can be efficiently presented in a local draining lymph node, resulting in initial T cell priming and clonal expansion, followed by T cell anergy.

Linking gene expression to mechanisms of toxicity.

Activation of gene expression is one of the earliest cellular responses to toxicity. However, our understanding of the biological and biochemical signals that activate these toxicant-responsive genes as well as the consequences of gene activation to survival of the organism remains sketchy. In this article, strategies that can be used to link changes in gene expression to biochemical mechanisms of toxicity are addressed using the hsp70 and grp78 genes as examples. The data indicate that activation of hsp70 is linked to changes in thiol-disulfide redox perturbations while grp78 activation may be caused by loss of calcium from the endoplasmic reticulum. Each gene is part of a discrete feedback regulated signaling pathway designed to protect cells against the toxic signals that activate gene expression.

The roles of caspase-3 and bcl-2 in chemically-induced apoptosis but not necrosis of renal epithelial cells.

The kidney is a target for toxicants including cisplatin and S-(1,2-dichlorovinyl)-L-cysteine (DCVC), a metabolite of the environmental contaminant, trichloroethylene. Necrosis is well characterized in kidney cells, but pathways leading to apoptosis are less clear. Cysteine conjugates are useful toxicants because they induce either necrosis or apoptosis depending on chemical structure or antioxidant status. Herein, we show that in the renal epithelial cell line LLC-PK1, activation of caspase-3 (CPP32/Yama/apopain) is crucial for apoptosis, but not necrosis. Apoptosis was blocked by zVAD.fmk, and partially by a cathepsin inhibitor. Caspase-3 activity and cleavage of poly(ADP-ribose) polymerase (PARP) was detected only during apoptosis. S-(1,1,2,2-Tetrafluoroethyl)-L-cysteine (TFEC), a metabolite of tetrafluoroethylene, kills cells only by necrosis, and did not activate caspases under any conditions. Apoptosis and activation of caspase-3 by cisplatin, but not DCVC, was prevented by bcl-2. Thus, caspase-3 activation by bcl-2-dependent and -independent mechanisms is a terminal event in chemical-apoptosis of renal epithelial cells.

Distinct endoplasmic reticulum signaling pathways regulate apoptotic and necrotic cell death following iodoacetamide treatment.

Environmental stress induces the synthesis of glucose-regulated proteins (Grps) in the endoplasmic reticulum (ER) and heat shock proteins (Hsps) in the cytoplasm. Iodoacetamide (IDAM), a prototypical alkyating agent, induces both Grp and Hsp synthesis in renal epithelial cells and causes necrosis which is prevented by prior activation of the ER stress response (pre-ER stress) [Liu, H., et al. (1997) J. Biol. Chem. 272, 21751-21759]. In this study, we examined the biochemical pathways leading to IDAM-induced apoptosis and investigated the role of the ER stress response in apoptotic cell death. The antioxidant N,N'-diphenyl-p-phenylenediamine (DPPD) prevented necrosis after IDAM treatment, but the cells went on to die with hallmarks of apoptosis, i.e., cell detachment, caspase-3 activation, cleavage of poly(ADP-ribose)polymerase (PARP), and DNA-ladder formation, all of which were blocked by the general caspase inhibitor zVAD. As with IDAM-induced necrosis, dithiothreitol protected against apoptosis, but cell permeable calcium chelators did not, suggesting that distinct biochemical pathways mediate these two forms of cell death. Pre-ER stress, but not heat shock, prevented IDAM-induced apoptosis. pkASgrp78 cells are deficient in Grp78 induction due to expression of a grp78 antisense RNA and are more sensitive to necrosis. However, these cells were resistant to IDAM-induced apoptosis and had increased basal levels of Grp94 and a KDEL-containing protein of about 50 kDa. Thus, the expression of grp78 antisense perturbs ER functions and activates expression of other ER stress genes accounting for the resistance to apoptosis. Taken together, the data describe functionally distinct signaling pathways through which the ER regulates apoptosis and necrosis caused by chemical toxicants.

Evaluation and classification of pediatric ocular trauma.

PURPOSE: To offer to the pediatric emergency physician consistent and unambiguous terms for the description of pediatric ocular trauma, based upon an adapted version of a standardized classification system. To show the potential effect of this reclassification system in a tertiary care emergency department. METHODS: The authors reviewed a new classification system of ocular trauma and adapted it for use by pediatric emergency physicians. In addition, a retrospective analysis of the records of pediatric patients presenting over a 2-year period to a tertiary emergency department with ocular complaints was performed. The diagnoses related to ocular trauma were reclassified according to the new classification system. RESULTS: Over a 2-year period, 117 pediatric patients were evaluated for ophthalmic complaints. Sixty-seven (57%) of these cases involved an ocular contusion or ruptured globe; however, six disparate diagnoses were given. The cases were reclassified into an adapted, unambiguous, classification system. In some cases, the reclassification altered the indication for immediate ophthalmologic referral. CONCLUSION: There is currently no standardized system of terminology to describe pediatric ocular trauma. This may lead to confusion in communication among the pediatric emergency physician, the pediatrician, and the ophthalmologist. Consistent, unambiguous, terminology will assist in this communication, facilitate the writing of peer-reviewed articles and case reports, and increase the level of accurate documentation in the medical record.

Hepatocyte growth factor induces tubulogenesis of primary renal proximal tubular epithelial cells.

Hepatocyte growth factor (HGF)-induced tubulogenesis has been demonstrated with renal epithelial cell lines grown in collagen gels but not with primary cultured renal proximal tubular epithelial cells (RPTEs). We show that HGF selectively induces proliferation and branching morphogenesis of primary cultured rat RPTEs. Additional growth factors including fibroblast growth factor (FGF)-1, epidermal growth factor (EGF), FGF-7, or insulin-like growth factor-1 (IGF-1) did not selectively induce tubulogenesis. However, when administered in combination, these factors initiated branching morphogenesis comparable to HGF alone and greatly augmented HGF-induced proliferation and branching. Microscopic analysis revealed that branching RPTEs were undergoing tubulogenesis and formed a polarized epithelium. TGF-beta1 blocked HGF- or growth factor cocktail (GFC; HGF, FGF-1, EGF, IGF-1)-induced proliferation and branching morphogenesis. Adding TGF-beta1 after GFC-induced tubulogenesis had occurred caused a progressive regression of the tubular structures, a response associated with an increase in apoptosis of the RPTEs. Primary cultured RPTEs are capable of undergoing HGF-induced tubulogenesis. Unlike cell lines, combinations of growth factors differentially augment the response.

Dephosphorylation of focal adhesion kinase (FAK) and loss of focal contacts precede caspase-mediated cleavage of FAK during apoptosis in renal epithelial cells.

The relationship between focal adhesion protein (FAK) activity and loss of cell-matrix contact during apoptosis is not entirely clear nor has the role of FAK in chemically induced apoptosis been studied. We investigated the status of FAK phosphorylation and cleavage in renal epithelial cells during apoptosis caused by the nephrotoxicant dichlorovinylcysteine (DCVC). DCVC treatment caused a loss of cell-matrix contact which was preceded by a dissociation of FAK from the focal adhesions and tyrosine dephosphorylation of FAK. Paxillin was also dephosphorylated at tyrosine. DCVC treatment activated caspase-3 which was associated with cleavage of FAK. However, FAK cleavage occurred after cells had already lost focal adhesions indicating that cleavage of FAK by caspases is not responsible for loss of FAK from focal adhesions. Accordingly, although inhibition of caspase activity with zVAD-fmk blocked activation of caspase-3, FAK cleavage, and apoptosis, it neither affected dephosphorylation nor translocation of FAK or paxillin. However, zVAD-fmk completely blocked the cell detachment caused by DCVC treatment. Orthovanadate prevented DCVC-induced tyrosine dephosphorylation of both FAK and paxillin; however, it did not inhibit DCVC-induced apoptosis and actually potentiated focal adhesion disorganization and cell detachment. Thus, FAK dephosphorylation and loss of focal adhesions are not due to caspase activation; however, caspases are required for FAK proteolysis and cell detachment.