Sex is a major effect modifier between body composition and mortality in patients with cirrhosis assessed for liver transplantation.

BACKGROUND AND AIMS: Body composition predicts mortality in patients with cirrhosis. The impact of sex on this association is unknown. We investigated the impact of sex on this association in patients with cirrhosis assessed for liver transplantation. METHODS: This single-centre retrospective cohort study included adults assessed for liver transplantation. Nutritional status was assessed using the Royal Free Hospital-Global Assessment (RFH-GA). Body composition at the third lumbar vertebrae was determined. SarcopeniaSMI was defined as Skeletal Muscle Index <50 cm2 /m2 in males and <39 cm2 /m2 in females. SarcopeniaPMI was defined as the sex-specific 25th percentile of the Psoas Muscle Index. Patients were assessed for the occurrence of liver transplantation and death. Analyses were stratified by sex. RESULTS: The cohort comprised 628 patients, including 199 females and 429 males. Both groups were similar in terms of baseline liver disease severity by Model for End-stage Liver Disease (MELD) (p = .98) and nutritional status (p = .24). SarcopeniaSMI was present in 41% of males compared to 27% of females (p < .001). In the male cohort, when adjusted for age and MELD, sarcopeniaPMI (aHR 1.74, 95% CI 1.08-2.80) and RFH-GA (aHR 1.40, 95% CI 1.03-1.90) remained independent predictors of mortality. Adipose tissue had no impact on outcomes in males. In female patients, adipose tissue (TATI or VATI depending on the multivariable model) was independently associated with mortality, whereas sarcopenia and malnutrition were not. CONCLUSIONS: This study demonstrates that male patients were susceptible to low muscle mass, whereas female patients were not. Future research in this patient population should minimize sex-related bias and present data for both groups separately.

A Case of Small Bowel Obstruction and Enterocutaneous Fistulation Resulting from a Mesenteric Haematoma following Blunt Abdominal Trauma.

A 23-year-old male with a history of previous abdominal surgery was involved in a road traffic accident. He was discharged after initial assessment but represented several days with small bowel obstruction secondary to a mesenteric haematoma. He underwent resection and recovered well but represented later on the day of discharge with a leaking surgical wound consistent with an enterocutaneous fistula. This was managed conservatively and closed spontaneously after ten days. This case serves to highlight that adhesions from previous surgery can tether the small bowel causing mesenteric injury following blunt-force trauma. It also demonstrates that postoperative ileus can result in an enterocutaneous fistula that has the appearance of an anastomotic breakdown but which resolves more rapidly.

Functional Toxicogenomic Profiling Expands Insight into Modulators of Formaldehyde Toxicity in Yeast.

Formaldehyde (FA) is a commercially important chemical with numerous and diverse uses. Accordingly, occupational and environmental exposure to FA is prevalent worldwide. Various adverse effects, including nasopharyngeal, sinonasal, and lymphohematopoietic cancers, have been linked to FA exposure, prompting designation of FA as a human carcinogen by U.S. and international scientific entities. Although the mechanism(s) of FA toxicity have been well studied, additional insight is needed in regard to the genetic requirements for FA tolerance. In this study, a functional toxicogenomics approach was utilized in the model eukaryotic yeast Saccharomyces cerevisiae to identify genes and cellular processes modulating the cellular toxicity of FA. Our results demonstrate mutant strains deficient in multiple DNA repair pathways-including homologous recombination, single strand annealing, and postreplication repair-were sensitive to FA, indicating FA may cause various forms of DNA damage in yeast. The SKI complex and its associated factors, which regulate mRNA degradation by the exosome, were also required for FA tolerance, suggesting FA may have unappreciated effects on RNA stability. Furthermore, various strains involved in osmoregulation and stress response were sensitive to FA. Together, our results are generally consistent with FA-mediated damage to both DNA and RNA. Considering DNA repair and RNA degradation pathways are evolutionarily conserved from yeast to humans, mechanisms of FA toxicity identified in yeast may be relevant to human disease and genetic susceptibility.

Modulation of Ras signaling alters the toxicity of hydroquinone, a benzene metabolite and component of cigarette smoke.

BACKGROUND: Benzene is an established human leukemogen, with a ubiquitous environmental presence leading to significant population exposure. In a genome-wide functional screen in the yeast Saccharomyces cerevisiae, inactivation of IRA2, a yeast ortholog of the human tumor suppressor gene NF1 (Neurofibromin), enhanced sensitivity to hydroquinone, an important benzene metabolite. Increased Ras signaling is implicated as a causal factor in the increased pre-disposition to leukemia of individuals with mutations in NF1. METHODS: Growth inhibition of yeast by hydroquinone was assessed in mutant strains exhibiting varying levels of Ras activity. Subsequently, effects of hydroquinone on both genotoxicity (measured by micronucleus formation) and proliferation of WT and Nf1 null murine hematopoietic precursors were assessed. RESULTS: Here we show that the Ras status of both yeast and mammalian cells modulates hydroquinone toxicity, indicating potential synergy between Ras signaling and benzene toxicity. Specifically, enhanced Ras signaling increases both hydroquinone-mediated growth inhibition in yeast and genotoxicity in mammalian hematopoetic precursors as measured by an in vitro erythroid micronucleus assay. Hydroquinone also increases proliferation of CFU-GM progenitor cells in mice with Nf1 null bone marrow relative to WT, the same cell type associated with benzene-associated leukemia. CONCLUSIONS: Together our findings show that hydroquinone toxicity is modulated by Ras signaling. Individuals with abnormal Ras signaling could be more vulnerable to developing myeloid diseases after exposure to benzene. We note that hydroquinone is used cosmetically as a skin-bleaching agent, including by individuals with cafe-au-lait spots (which may be present in individuals with neurofibromatosis who have a mutation in NF1), which could be unadvisable given our findings.

Genome-wide functional profiling identifies genes and processes important for zinc-limited growth of Saccharomyces cerevisiae.

Zinc is an essential nutrient because it is a required cofactor for many enzymes and transcription factors. To discover genes and processes in yeast that are required for growth when zinc is limiting, we used genome-wide functional profiling. Mixed pools of ∼4,600 deletion mutants were inoculated into zinc-replete and zinc-limiting media. These cells were grown for several generations, and the prevalence of each mutant in the pool was then determined by microarray analysis. As a result, we identified more than 400 different genes required for optimal growth under zinc-limiting conditions. Among these were several targets of the Zap1 zinc-responsive transcription factor. Their importance is consistent with their up-regulation by Zap1 in low zinc. We also identified genes that implicate Zap1-independent processes as important. These include endoplasmic reticulum function, oxidative stress resistance, vesicular trafficking, peroxisome biogenesis, and chromatin modification. Our studies also indicated the critical role of macroautophagy in low zinc growth. Finally, as a result of our analysis, we discovered a previously unknown role for the ICE2 gene in maintaining ER zinc homeostasis. Thus, functional profiling has provided many new insights into genes and processes that are needed for cells to thrive under the stress of zinc deficiency.

Bone marrow genotoxicity of 2,5-dimethylfuran, a green biofuel candidate.

2,5-Dimethylfuran (DMF) is being considered as a potential green transportation biofuel, but there is limited information about its toxicity and safety. We examined DMF toxicity in the bone marrow using a murine in vitro erythropoietic micronucleus assay and found that exposure to DMF (0.1 mM, 1 hr) induced an increase in micronuclei frequency compared with controls. These data suggest that DMF may be genotoxic to hematopoietic cells and that more thorough toxicological studies on DMF should be conducted to ensure public and occupational safety before it is considered a viable biofuel and produced in mass quantities. As well as specific data on DMF, our study further validates an in vitro cell culture system that captures the essential features of the in vivo mammalian micronucleus genotoxicity assay, enabling increased throughput and controlled studies on hematopoietic DNA damage response, while reducing animal sacrifice. In vitro assays, such as the in vitro micronucleus assay, will be essential as international chemical policy is increasingly utilizing green chemistry principles that require more toxicological testing.

Genome-Wide Functional and Stress Response Profiling Reveals Toxic Mechanism and Genes Required for Tolerance to Benzo[a]pyrene in S. cerevisiae.

Benzo[a]pyrene (BaP) is a ubiquitous, potent, and complete carcinogen resulting from incomplete organic combustion. BaP can form DNA adducts but other mechanisms may play a role in toxicity. We used a functional toxicology approach in S. cerevisiae to assess the genetic requirements for cellular resistance to BaP. In addition, we examined translational activities of key genes involved in various stress response pathways. We identified multiple genes and processes involved in modulating BaP toxicity in yeast which support DNA damage as a primary mechanism of toxicity, but also identify other potential toxicity pathways. Gene ontology enrichment analysis indicated that DNA damage and repair as well as redox homeostasis and oxidative stress are key processes in cellular response to BaP suggesting a similar mode of action of BaP in yeast and mammals. Interestingly, toxicant export is also implicated as a potential novel modulator of cellular susceptibility. In particular, we identified several transporters with human orthologs (solute carrier family 22) which may play a role in mammalian systems.

Genome-wide functional profiling reveals genes required for tolerance to benzene metabolites in yeast.

Benzene is a ubiquitous environmental contaminant and is widely used in industry. Exposure to benzene causes a number of serious health problems, including blood disorders and leukemia. Benzene undergoes complex metabolism in humans, making mechanistic determination of benzene toxicity difficult. We used a functional genomics approach to identify the genes that modulate the cellular toxicity of three of the phenolic metabolites of benzene, hydroquinone (HQ), catechol (CAT) and 1,2,4-benzenetriol (BT), in the model eukaryote Saccharomyces cerevisiae. Benzene metabolites generate oxidative and cytoskeletal stress, and tolerance requires correct regulation of iron homeostasis and the vacuolar ATPase. We have identified a conserved bZIP transcription factor, Yap3p, as important for a HQ-specific response pathway, as well as two genes that encode putative NAD(P)H:quinone oxidoreductases, PST2 and YCP4. Many of the yeast genes identified have human orthologs that may modulate human benzene toxicity in a similar manner and could play a role in benzene exposure-related disease.

Functional toxicogenomics: mechanism-centered toxicology.

Traditional toxicity testing using animal models is slow, low capacity, expensive and assesses a limited number of endpoints. Such approaches are inadequate to deal with the increasingly large number of compounds found in the environment for which there are no toxicity data. Mechanism-centered high-throughput testing represents an alternative approach to meet this pressing need but is limited by our current understanding of toxicity pathways. Functional toxicogenomics, the global study of the biological function of genes on the modulation of the toxic effect of a compound, can play an important role in identifying the essential cellular components and pathways involved in toxicity response. The combination of the identification of fundamental toxicity pathways and mechanism-centered targeted assays represents an integrated approach to advance molecular toxicology to meet the challenges of toxicity testing in the 21(st) century.

Systems biology of human benzene exposure.

Toxicogenomic studies, including genome-wide analyses of susceptibility genes (genomics), gene expression (transcriptomics), protein expression (proteomics), and epigenetic modifications (epigenomics), of human populations exposed to benzene are crucial to understanding gene-environment interactions, providing the ability to develop biomarkers of exposure, early effect and susceptibility. Comprehensive analysis of these toxicogenomic and epigenomic profiles by bioinformatics in the context of phenotypic endpoints, comprises systems biology, which has the potential to comprehensively define the mechanisms by which benzene causes leukemia. We have applied this approach to a molecular epidemiology study of workers exposed to benzene. Hematotoxicity, a significant decrease in almost all blood cell counts, was identified as a phenotypic effect of benzene that occurred even below 1 ppm benzene exposure. We found a significant decrease in the formation of progenitor colonies arising from bone marrow stem cells with increasing benzene exposure, showing that progenitor cells are more sensitive to the effects of benzene than mature blood cells, likely leading to the observed hematotoxicity. Analysis of transcriptomics by microarray in the peripheral blood mononuclear cells of exposed workers, identified genes and pathways (apoptosis, immune response, and inflammatory response) altered at high (>10 ppm) and low (<1 ppm) benzene levels. Serum proteomics by SELDI-TOF-MS revealed proteins consistently down-regulated in exposed workers. Preliminary epigenomics data showed effects of benzene on the DNA methylation of specific genes. Genomic screens for candidate genes involved in susceptibility to benzene toxicity are being undertaken in yeast, with subsequent confirmation by RNAi in human cells, to expand upon the findings from candidate gene analyses. Data on these and future biomarkers will be used to populate a large toxicogenomics database, to which we will apply bioinformatic approaches to understand the interactions among benzene toxicity, susceptibility genes, mRNA, and DNA methylation through a systems biology approach.

Excess single-stranded DNA inhibits meiotic double-strand break repair.

During meiosis, self-inflicted DNA double-strand breaks (DSBs) are created by the protein Spo11 and repaired by homologous recombination leading to gene conversions and crossovers. Crossover formation is vital for the segregation of homologous chromosomes during the first meiotic division and requires the RecA orthologue, Dmc1. We analyzed repair during meiosis of site-specific DSBs created by another nuclease, VMA1-derived endonuclease (VDE), in cells lacking Dmc1 strand-exchange protein. Turnover and resection of the VDE-DSBs was assessed in two different reporter cassettes that can repair using flanking direct repeat sequences, thereby obviating the need for a Dmc1-dependent DNA strand invasion step. Access of the single-strand binding complex replication protein A, which is normally used in all modes of DSB repair, was checked in chromatin immunoprecipitation experiments, using antibody against Rfa1. Repair of the VDE-DSBs was severely inhibited in dmc1Delta cells, a defect that was associated with a reduction in the long tract resection required to initiate single-strand annealing between the flanking repeat sequences. Mutants that either reduce Spo11-DSB formation or abolish resection at Spo11-DSBs rescued the repair block. We also found that a replication protein A component, Rfa1, does not accumulate to expected levels at unrepaired single-stranded DNA (ssDNA) in dmc1Delta cells. The requirement of Dmc1 for VDE-DSB repair using flanking repeats appears to be caused by the accumulation of large quantities of ssDNA that accumulate at Spo11-DSBs when Dmc1 is absent. We propose that these resected DSBs sequester both resection machinery and ssDNA binding proteins, which in wild-type cells would normally be recycled as Spo11-DSBs repair. The implication is that repair proteins are in limited supply, and this could reflect an underlying mechanism for regulating DSB repair in wild-type cells, providing protection from potentially harmful effects of overabundant repair proteins.

Methyl methanesulfonate (MMS) produces heat-labile DNA damage but no detectable in vivo DNA double-strand breaks.

Homologous recombination (HR) deficient cells are sensitive to methyl methanesulfonate (MMS). HR is usually involved in the repair of DNA double-strand breaks (DSBs) in Saccharomyces cerevisiae implying that MMS somehow induces DSBs in vivo. Indeed there is evidence, based on pulsed-field gel electrophoresis (PFGE), that MMS causes DNA fragmentation. However, the mechanism through which MMS induces DSBs has not been demonstrated. Here, we show that DNA fragmentation following MMS treatment, and detected by PFGE is not the consequence of production of cellular DSBs. Instead, DSBs seen following MMS treatment are produced during sample preparation where heat-labile methylated DNA is converted into DSBs. Furthermore, we show that the repair of MMS-induced heat-labile damage requires the base excision repair protein XRCC1, and is independent of HR in both S.cerevisiae and mammalian cells. We speculate that the reason for recombination-deficient cells being sensitive to MMS is due to the role of HR in repair of MMS-induced stalled replication forks, rather than for repair of cellular DSBs or heat-labile damage.