Chemical and Colloidal Dynamics of MnO2 Nanosheets in Biological Media Relevant for Nanosafety Assessment.

Many layered crystal phases can be exfoliated or assembled into ultrathin 2D nanosheets with novel properties not achievable by particulate or fibrous nanoforms. Among these 2D materials are manganese dioxide (MnO2 ) nanosheets, which have applications in batteries, catalysts, and biomedical probes. A novel feature of MnO2 is its sensitivity to chemical reduction leading to dissolution and Mn2+ release. Biodissolution is critical for nanosafety assessment of 2D materials, but the timing and location of MnO2 biodissolution in environmental or occupational exposure scenarios are poorly understood. This work investigates the chemical and colloidal dynamics of MnO2 nanosheets in biological media for environmental and human health risk assessment. MnO2 nanosheets are insoluble in most aqueous phases, but react with strong and weak reducing agents in biological fluid environments. In vitro, reductive dissolution can be slow enough in cell culture media for MnO2 internalization by cells in the form of intact nanosheets, which localize in vacuoles, react to deplete intracellular glutathione, and induce cytotoxicity that is likely mediated by intracellular Mn2+ release. The results are used to classify MnO2 nanosheets within a new hazard screening framework for 2D materials, and the implications of MnO2 transformations for nanotoxicity testing and nanosafety assessment are discussed.

A 3D fish liver model for aquatic toxicology: Morphological changes and Cyp1a induction in PLHC-1 microtissues after repeated benzo(a)pyrene exposures.

To identify the potential environmental impacts of aquatic pollutants, rapid and sensitive screening tools are needed to assess adaptive and toxic responses. This study characterizes a novel fish liver microtissue model, produced with the cell line PLHC-1, as an in vitro aquatic toxicity testing platform. These 3D microtissues remain viable and stable throughout the 8-day testing period and relative to 2D monolayers, show increased basal expression of the xenobiotic metabolizing enzyme cytochrome P450 1A (Cyp1a). To evaluate pulsed, low-dose exposures at environmentally relevant concentrations, microtissue responsiveness to the model toxicant benzo(a)pyrene was assessed after single and repeated exposures for determination of both immediate and persistent effects. Significant induction of Cyp1a gene and protein expression was detected after a single 24h exposure to as little as 1nM benzo(a)pyrene, and after a 24h recovery period, Cyp1a expression declined in a dose-dependent manner. However, cell death continued to increase during the recovery period and alterations in microtissue architecture occurred at higher concentrations. To evaluate a pulsed or repeated exposure scenario, microtissues were exposed to benzo(a)pyrene, allowed to recover, then exposed a second time for 24h. Following pre-exposure to benzo(a)pyrene, cyp1a expression remained equally inducible and the pattern and level of Cyp1a protein response to a second exposure were comparable. However, pre-exposure to 1µM or 5µM of benzo(a)pyrene resulted in increased cell death, greater disruption of microtissue architecture, and alterations in cell morphology. Together, this study demonstrates the capabilities of this PLHC-1 microtissue model for sensitive assessment of liver toxicants over time and following single and repeated exposures.

Effects of surface-engineered nanoparticle-based dispersants for marine oil spills on the model organism Artemia franciscana.

Fine particles are under active consideration as alternatives to chemical dispersants for large-scale petroleum spills. Fine carbon particles with engineered surface chemistry have been shown to stabilize oil-in-water emulsions, but the environmental impacts of large-scale particle introduction to the marine environment are unknown. Here we study the impact of surface-engineered carbon-black materials on brine shrimp (Artemia franciscana) as a model marine microcrustacean. Mortality was characterized at 50-1000 mg/L, and levels of heat shock protein 70 (hsp70) were characterized at sublethal particle concentrations (25-50 mg/L). Functionalized carbon black (CB) nanoparticles were found to be nontoxic at all concentrations, while hydrophobic (annealed) and as-produced CB induced adverse effects at high concentrations. CB was also shown to adsorb benzene, a model hydrocarbon representing the more soluble and toxic low-molecular weight aromatic fraction of petroleum, but the extent of adsorption was insufficient to mitigate benzene toxicity to Artemia in coexposure experiments. At lower benzene concentrations (25-75 mg/L), coexposure with annealed and as-produced CB increased hsp70 protein levels. This study suggests that surface functionalization for increased hydrophilicity can not only improve the performance of CB-based dispersants but also reduce their adverse environmental impacts on marine organisms.

Hypothalamic Sirt1 regulates food intake in a rodent model system.

Sirt1 is an evolutionarily conserved NAD(+) dependent deacetylase involved in a wide range of processes including cellular differentiation, apoptosis, as well as metabolism, and aging. In this study, we investigated the role of hypothalamic Sirt1 in energy balance. Pharmacological inhibition or siRNA mediated knock down of hypothalamic Sirt1 showed to decrease food intake and body weight gain. Central administration of a specific melanocortin antagonist, SHU9119, reversed the anorectic effect of hypothalamic Sirt1 inhibition, suggesting that Sirt1 regulates food intake through the central melanocortin signaling. We also showed that fasting increases hypothalamic Sirt1 expression and decreases FoxO1 (Forkhead transcription factor) acetylation suggesting that Sirt1 regulates the central melanocortin system in a FoxO1 dependent manner. In addition, hypothalamic Sirt1 showed to regulate S6K signaling such that inhibition of the fasting induced Sirt1 activity results in up-regulation of the S6K pathway. Thus, this is the first study providing a novel role for the hypothalamic Sirt1 in the regulation of food intake and body weight. Given the role of Sirt1 in several peripheral tissues and hypothalamus, potential therapies centered on Sirt1 regulation might provide promising therapies in the treatment of metabolic diseases including obesity.

Role of a pro-sequence in the secretory pathway of prothyrotropin-releasing hormone.

The biogenesis of rat thyrotropin releasing hormone (TRH) involves the processing of its precursor (proTRH) into five biologically active TRH peptides and several non-TRH peptides where two of them had been attributed potential biological functions. This process implicates 1) proper folding of proTRH in the endoplasmic reticulum after its biosynthesis and exit to the Golgi apparatus and beyond, 2) initial processing of proTRH in the trans Golgi network and, 3) sorting of proTRH-derived peptides to the regulated secretory pathway. Previous studies have focused on elucidating the processing and sorting determinants of proTRH. However, the role of protein folding in the sorting of proTRH remains unexplored. Here we have investigated the role in the secretion of proTRH of a sequence comprising 22 amino acid residues, located at the N-terminal region of proTRH, residues 31-52. Complete deletion of these 22 amino acids dramatically compromised the biosynthesis of proTRH, manifested as a severe reduction in the steady state level of proTRH in the endoplasmic reticulum. This effect was largely reproduced by the deletion of only three amino acid residues, 40PGL42, within the proTRH31-52 sequence. The decreased steady state level of the mutant DeltaPGL was due to enhanced endoplasmic reticulum-associated protein degradation. However, the remnant of DeltaPGL that escaped degradation was properly processed and sorted to secretory granules. Thus, these results suggest that the N-terminal domain within the prohormone sequence does not act as "sorting signal" in late secretion; instead, it seems to play a key role determining the proper folding pathway of the precursor and, thus, its stability.

Cold exposure increases the biosynthesis and proteolytic processing of prothyrotropin-releasing hormone in the hypothalamic paraventricular nucleus via beta-adrenoreceptors.

Different physiological conditions affect the biosynthesis and processing of hypophysiotropic proTRH in the hypothalamic paraventricular nucleus, and consequently the output of TRH. Early studies suggest that norepinephrine (NE) mediates the cold-induced activation of the hypothalamic-pituitary-thyroid axis at a central level. However, the specific role of NE on the biosynthesis and processing of proTRH has not been fully investigated. In this study, we found that NE affects gene transcription, protein biosynthesis, and secretion in TRH neurons in vitro; these changes were coupled with an up-regulation of prohormone convertase enzymes (PC) 1/3 and PC2. In vivo, NE is the main mediator of the cold-induced activation of the hypothalamic-pituitary-thyroid axis at the hypothalamic level, in which it potently stimulates the biosynthesis and proteolytic processing of proTRH through a coordinated up-regulation of the PCs. This activation occurs via beta-adrenoreceptors and phosphorylated cAMP response element binding signaling. In contrast, alpha-adrenoreceptors regulate TRH secretion but not proTRH biosynthesis and processing. Therefore, this study provides novel information on the molecular mechanisms of control of hypophysiotropic TRH biosynthesis.

Prohormone-convertase 1 processing enhances post-Golgi sorting of prothyrotropin-releasing hormone-derived peptides.

Rat prothyrotropin-releasing hormone (pro-TRH) is endoproteolyzed within the regulated secretory pathway of neuroendocrine cells yielding five TRH peptides and seven to nine other unique peptides. Endoproteolysis is performed by two prohormone convertases, PC1 and PC2. Proteolysis of pro-TRH begins in the trans-Golgi network and forms two intermediates that are then differentially processed as they exit the Golgi and are packaged into immature secretory granules. We hypothesized that this initial endoproteolysis may be necessary for downstream sorting of pro-TRH-derived peptides as it occurs before Golgi exit and thus entry into the regulated secretory pathway. We now report that when pro-TRH is transiently expressed in GH4C1 cells, a neuroendocrine cell line lacking PC1, under pulse-chase conditions release is constitutive and composed of more immature processing intermediates. This is also observed by radioimmunoassay under steady-state conditions. When a mutant form of pro-TRH, which has the dibasic sites of initial processing mutated to glycines, is expressed in AtT20 cells, a neuroendocrine cell line endogenously expressing PC1, both steady-state and pulse-chase experiments revealed that peptides derived from this mutant precursor are secreted in a constitutive fashion. A constitutively secreted form of PC1 does not target pro-TRH peptides to the constitutive secretory pathway but results in sorting to the regulated secretory pathway. These results indicated that initial processing action of PC1 on pro-TRH in the trans-Golgi network, and not a cargo-receptor relationship, is important for the downstream sorting events that result in storage of pro-TRH-derived peptides in mature secretory granules.

A mouse model recapitulating molecular features of human mesothelioma.

Malignant mesothelioma has been linked to asbestos exposure and generally has a poor prognosis because it is often diagnosed in advanced stages and is refractory to conventional therapy. Human malignant mesotheliomas accumulate multiple somatic genetic alterations, including inactivation of the NF2 and CDKN2A/ARF tumor suppressor genes. To better understand the significance of NF2 inactivation in malignant mesothelioma and identify tumor suppressor gene alterations that cooperate with NF2 loss of function in malignant mesothelioma pathogenesis, we treated Nf2 (+/-) knockout mice with asbestos to induce malignant mesotheliomas. Asbestos-exposed Nf2 (+/-) mice exhibited markedly accelerated malignant mesothelioma tumor formation compared with asbestos-treated wild-type (WT) littermates. Loss of the WT Nf2 allele, leading to biallelic inactivation, was observed in all nine asbestos-induced malignant mesotheliomas from Nf2 (+/-) mice and in 50% of malignant mesotheliomas from asbestos-exposed WT mice. For a detailed comparison with the murine model, DNA analyses were also done on a series of human malignant mesothelioma samples. Remarkably, similar to human malignant mesotheliomas, tumors from Nf2 (+/-) mice showed frequent homologous deletions of the Cdkn2a/Arf locus and adjacent Cdkn2b tumor suppressor gene, as well as reciprocal inactivation of Tp53 in a subset of tumors that retained the Arf locus. As in the human disease counterpart, malignant mesotheliomas from the Nf2 (+/-) mice also showed frequent activation of Akt kinase, which plays a central role in tumorigenesis and therapeutic resistance. Thus, this murine model of environmental carcinogenesis faithfully recapitulates many of the molecular features of human malignant mesothelioma and has significant implications for the further characterization of malignant mesothelioma pathogenesis and preclinical testing of novel therapeutic modalities.

Stepwise posttranslational processing of progrowth hormone-releasing hormone (proGHRH) polypeptide by furin and PC1.

Through a posttranslational processing mechanism, pro-growth hormone releasing hormone (proGHRH) gives rise to an amidated GHRH molecule, which in turn stimulates the synthesis and release of growth hormone. We have previously proposed a model for the biochemical processing of proGHRH [Nillni et al. (1999), Endocrinology 140, 5817-5827]. We demonstrated that the proGHRH peptide (10.5 kDa, 104 aa) is first processed to an 8.8 kDa intermediate form that is later cleaved to yield two products: the 5.2 kDa GHRH and the 3.6 kDa GHRH-RP. However, the proteolytic enzymes involved in this process are unknown. Therefore, in this study we determined which proconverting enzymes are involved in this process. We transfected different constructs in cell lines carrying different PC enzymes followed by analysis of the peptide products after metabolic labeling or Western blots. We found that in the absence of furin (LoVo cells) or CHO cells treated with BFA, only one moiety was observed, and that corresponds to the same electrophorectic mobility to the GHRH precursor. This finding strongly supports an initial role for furin in the processing of proGHRH. The results from transfections with preproGHRH alone or double or triple transfections with PC1 and PC2 in AtT-20, GH3, and GH4C1 cells indicated that PC1 is the primary enzyme involved in the generation of GHRH peptide from the 8.8 kDa intermediate form. We found that AtT-20 cells (high PC1, very low PC2) were able to generate GHRH. However, GH3 cells (high PC2, but not PC1) were able to process the 8.8 kDa peptide to GHRH only after the cotransfection with the PC1 enzyme. Transfections with preproGHRH-GFP and preproGHRH-V5 provided similar results in all the cell lines analyzed. These data support the hypothesis that proGHRH is initially cleave by furin at preproGHRH29-30, followed by a second cleavage at preproGHRH74 primarily by PC1 to generate GHRH and GHRH-RP peptides, respectively.

Accelerated progression of asbestos-induced mesotheliomas in heterozygous p53+/- mice.

Asbestos fibers produce diffuse malignant mesotheliomas in chronic rodent inhalation assays or after direct intrapleural or intraperitoneal injection. In vitro models have provided evidence that asbestos fibers are genotoxic carcinogens that can directly or indirectly generate reactive oxygen- and nitrogen-derived species that cause DNA damage. Heterozygous p53+/- mice show an increased incidence and reduced latency of malignant mesotheliomas induced by weekly intraperitoneal injections of crocidolite asbestos fibers. In this study, we investigated whether loss of heterozygosity (LOH) at the p53 tumor-suppressor gene locus contributes to accelerated tumor progression. LOH was found in 50% of the tumors produced in heterozygous p53+/- mice. In contrast to tumors that arise in p53+/+ mice or those that retained one p53 allele, LOH was associated with large tumor masses with central areas of necrosis, local invasion, and penetration of lymphatics. Increased tumor size was not associated with increased levels of cell proliferation as determined by BrdU incorporation, but it was correlated with a reduction in apoptosis as determined morphologically and by the TUNEL assay. Wild-type p53 protein is essential for cell cycle arrest in response to DNA damage and in maintenance of genomic stability. Cell lines established from tumors that showed LOH at the p53 tumor-suppressor gene locus were nearly tetraploid. These results suggest that p53 haplo-insufficiency sensitizes mice to the clastogenic or aneuploidogenic effects of crocidolite asbestos fibers, resulting in a shorter latent period. As solid tumors develop, spontaneous loss of the wild-type allele accompanied by decreased apoptosis and genetic instability is associated with accelerated tumor growth, invasion, and lymphatic dissemination.