Transcriptional Regulation on Aneuploid Chromosomes in Divers Candida albicans Mutants.

Candida albicans is a diploid fungus and a predominant opportunistic human pathogen. Notably, C. albicans employs reversible chromosomal aneuploidies as a means of survival in adverse environments. We previously characterized transcription on the monosomic chromosome 5 (Ch5) that arises with adaptation to growth on the toxic sugar sorbose in the mutant Sor125(55). We now extend this analysis to the trisomic hybrid Ch4/7 within Sor125(55) and a diverse group of three mutants harboring a single Ch5. We find a similar pattern of transcriptional changes on either type of aneuploid chromosome within these mutants wherein expression of many genes follows chromosome ploidy, consistent with a direct mechanism to regulate genes important for adaptation to growth. In contrast, a significant number of genes are expressed at the disomic level, implying distinct mechanisms compensating for gene dose on monosomic or trisomic chromosomes consistent with maintaining cell homeostasis. Finally, we find evidence for an additional mechanism that elevates expression of genes on normal disomic Ch4 and Ch7 in mutants to levels commensurate with that found on the trisomic Ch4/7b in Sor125(55). Several of these genes are similarly differentially regulated among mutants, suggesting they play key functions in either maintaining aneuploidy or adaptation to growth conditions.

Toxicological effects of pet food ingredients on canine bone marrow-derived mesenchymal stem cells and enterocyte-like cells.

We developed an in vitro method to assess pet food ingredients safety. Canine bone marrow-derived mesenchymal stem cells (BMSC) were differentiated into enterocyte-like cells (ELC) to assess toxicity in cells representing similar patterns of exposure in vivo. The toxicological profile of clove leave oil, eugenol, guanosine monophosphate (GMP), GMP + inosine monophosphate, sorbose, ginger root extract, cinnamon bark oil, cinnamaldehyde, thyme oil, thymol and citric acid was assessed in BMSC and ELC. The LC50 for GMP + inosine monophosphate was 59.42 ± 0.90 and 56.7 ± 3.5 mg ml(-1) for BMSC and ELC; 56.84 ± 0.95 and 53.66 ± 1.36 mg ml(-1) for GMP; 0.02 ± 0.001 and 1.25 ± 0.47 mg ml(-1) for citric acid; 0.077 ± 0.002 and 0.037 ± 0.01 mg ml(-1) for cinnamaldehyde; 0.002 ± 0.0001 and 0.002 ± 0.0008 mg ml(-1) for thymol; 0.080 ± 0.003 and 0.059 ± 0.001 mg ml(-1) for thyme oil; 0.111 ± 0.002 and 0.054 ± 0.01 mg ml(-1) for cinnamon bark oil; 0.119 ± 0.0004 and 0.099 ± 0.011 mg ml(-1) for clove leave oil; 0.04 ± 0.001 and 0.028 ± 0.002 mg ml(-1) for eugenol; 2.80 ± 0.11 and 1.75 ± 0.51 mg ml(-1) for ginger root extract; > 200 and 116.78 ± 7.35 mg ml(-1) for sorbose. Lemon grass oil was evaluated at 0.003-0.9 in BMSC and .03-0.9 mg ml(-1) in ELC and its mechanistic effect was investigated. The gene toxicology studies showed regulation of 61% genes in CYP450 pathway, 37% in cholestasis and 33% in immunotoxicity pathways for BMSC. For ELC, 80% for heat shock response, 69% for beta-oxidation and 65% for mitochondrial energy metabolism. In conclusion, these studies provide a baseline against which differential toxicity of dietary feed ingredients can be assessed in vitro for direct effects on canine cells and demonstrate differential toxicity in differentiated cells that represent gastrointestinal epithelial cells.

In vitro safety assessment of food ingredients in canine renal proximal tubule cells.

In vitro models are useful tools to initially assess the toxicological safety hazards of food ingredients. Toxicities of cinnamaldehyde (CINA), cinnamon bark oil, lemongrass oil (LGO), thymol, thyme oil (TO), clove leaf oil, eugenol, ginger root extract (GRE), citric acid, guanosine monophosphate, inosine monophosphate and sorbose (SORB) were assessed in canine renal proximal tubule cells (CPTC) using viability assay and renal injury markers. At LC50, CINA was the most toxic (0.012mg/ml), while SORB the least toxic (>100mg/ml). Toxicities (LC50) of positive controls were as follows: 4-aminophenol (0.15mg/ml in CPTC and 0.083mg/ml in human PTC), neomycin (28.6mg/ml in CPTC and 27.1mg/ml in human PTC). XYL displayed lowest cytotoxic potency (LC50=82.7mg/ml in CPTC). In vivo renal injury markers in CPTC were not significantly different from controls. The LGO toxicity mechanism was analyzed using qPCR and electron microscopy. Out of 370 genes, 57 genes (15.4%) were significantly up (34, 9.1%) or down (23, 6.2%) regulated, with the most upregulated gene gsta3 (∼200-fold) and the most affected pathway being oxidative stress. LGO induced damage of mitochondria, phospholipid accumulation and lack of a brush border. Viability assays along with mechanistic studies in the CPTC model may serve as a valuable in vitro toxicity screening tool.

Adaptation of Candida albicans to growth on sorbose via monosomy of chromosome 5 accompanied by duplication of another chromosome carrying a gene responsible for sorbose utilization.

Candida albicans, a fungus that normally inhabits the digestive tract and other mucosal surfaces, can become a pathogen in immunocompromised individuals, causing severe or even fatal infection. Mechanisms by which C. albicans can evade commonly used antifungal agents are not fully understood. We are studying a model system involving growth of C. albicans on toxic sugar sorbose, which represses synthesis of cell wall glucan and, as a result, kills fungi in a manner similar to drugs from the echinocandins class. Adaptation to sorbose occurs predominantly due to reversible loss of one homolog of chromosome 5 (Ch5), which results in upregulation of the metabolic gene SOU1 (SOrbose Utilization) on Ch4. Here, we show that growth on sorbose due to Ch5 monosomy can involve a facultative trisomy of a hybrid Ch4/7 that serves to increase copy number of the SOU1 gene. This shows that control of expression of SOU1 can involve multiple mechanisms; in this case, negative regulation and increase in gene copy number operating simultaneously in cell.

L-Sorbose but not D-tagatose induces hemolysis of dog erythrocytes in vitro.

Previous investigations have demonstrated that L-sorbose induces hemolysis of dog erythrocytes. This effect is probably the consequence of an ATP depletion of the red blood cells subsequent to inhibition of hexokinase, and thus the glycolytic pathway, by sorbose 1-phosphate. In the present study, the susceptibility of dog erythrocytes to D-tagatose, a stereoisomer of L-sorbose, was examined. Washed dog erythrocytes were suspended in Hanks' balanced salt solution (HBSS, containing 5.6 mM glucose) with or without the addition of 0.6, 6, and 60 mM L-sorbose or D-tagatose, or in HBSS with total glucose concentrations of 5.6, 6 and 60 mM D-glucose. After incubation for 24 h at 34 degrees C, the suspensions were centrifuged, and the percentage of hemolysis was determined by measuring the hemoglobin in the sediment and the supernatant. The amount of hemoglobin released in the medium did not differ significantly between the control (HBSS) and the test incubations with glucose or D-tagatose supplementation. In contrast, the addition of 6 and 60 mM L-sorbose resulted in significant hemolysis. At the low dose (0.6 mM), L-sorbose did not have an adverse effect. It is concluded that D-tagatose, unlike L-sorbose, does not have a hemolytic effect on canine erythrocytes.