Accumulation of Ubiquitin and Sequestosome-1 Implicate Protein Damage in Diacetyl-Induced Cytotoxicity.

Inhaled diacetyl vapors are associated with flavorings-related lung disease, a potentially fatal airway disease. The reactive α-dicarbonyl group in diacetyl causes protein damage in vitro. Dicarbonyl/l-xylulose reductase (DCXR) metabolizes diacetyl into acetoin, which lacks this α-dicarbonyl group. To investigate the hypothesis that flavorings-related lung disease is caused by in vivo protein damage, we correlated diacetyl-induced airway damage in mice with immunofluorescence for markers of protein turnover and autophagy. Western immunoblots identified shifts in ubiquitin pools. Diacetyl inhalation caused dose-dependent increases in bronchial epithelial cells with puncta of both total ubiquitin and K63-ubiquitin, central mediators of protein turnover. This response was greater in Dcxr-knockout mice than in wild-type controls inhaling 200 ppm diacetyl, further implicating the α-dicarbonyl group in protein damage. Western immunoblots demonstrated decreased free ubiquitin in airway-enriched fractions. Transmission electron microscopy and colocalization of ubiquitin-positive puncta with lysosomal-associated membrane proteins 1 and 2 and with the multifunctional scaffolding protein sequestosome-1 (SQSTM1/p62) confirmed autophagy. Surprisingly, immunoreactive SQSTM1 also accumulated in the olfactory bulb of the brain. Olfactory bulb SQSTM1 often congregated in activated microglial cells that also contained olfactory marker protein, indicating neuronophagia within the olfactory bulb. This suggests the possibility that SQSTM1 or damaged proteins may be transported from the nose to the brain. Together, these findings strongly implicate widespread protein damage in the etiology of flavorings-related lung disease.

Activation of Adenosine Monophosphate-Activated Protein Kinase Is an Additional Mechanism That Participates in Mediating Inhibitory Actions of Prostaglandin F2Alpha in Mature, but Not Developing, Bovine Corpora Lutea.

Elevated cytosolic calcium and protein kinase C are well-established mediators of luteolytic actions of prostaglandin F2alpha (PGF2alpha). The objectives of this study were to determine 1) if calcium/calmodulin-dependent kinase kinase 2 (CAMKK2) participates in mediating PGF2alpha actions in developing (Day [d]-4) and mature (d-10) bovine corpus luteum (CL), 2) distal targets of CAMKK2, 3) developmental expression of adenosine monophosphate-activated protein kinase (AMPK), and 4) effects of AMPK activation on progesterone (P4) production. Expression of AMPK increased as the CL matured. Activation of the prostaglandin receptor (FP) induced rapid phosphorylation of AMPK, which was blocked by a CAMKK2 inhibitor. Changes in basal P4 secretion in vitro were determined in response to AMPK activation via metformin (met) or 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) in d-4 and d-10 CL. Production of P4 in d-10 CL decreased with met or AICAR compared to control, similar to activation by PGF2alpha. Therefore, potential distal targets of AMPK in d-10 CL were examined during induced functional regression via exogenous PGF2alpha. Serum and luteal P4 decreased at 2 and 4 h after administration of PGF2alpha. Protein expression of LDLR decreased at 2 and 4 h, while those of ACAT1 and STAR increased 4 h after PGF2alpha. During induced regression, alterations of cholesterol transport proteins contributed to decreased luteal and serum P4. Therefore, developmental differences in signal transduction associated with FP, specifically CAMKK2 and AMPK, partially contribute to differences in the ability of PGF2alpha to induce regression in mature, but not developing, bovine CL. Multiple cholesterol transport proteins, including LDLR, were altered by PGF2alpha and could be potential AMPK targets.

Diacetyl increases sensory innervation and substance P production in rat trachea.

Inhalation of diacetyl, a butter flavoring, causes airway responses potentially mediated by sensory nerves. This study examines diacetyl-induced changes in sensory nerves of tracheal epithelium. Rats (n = 6/group) inhaled 0-, 25-, 249-, or 346-ppm diacetyl for 6 hr. Tracheas and vagal ganglia were removed 1-day postexposure and labeled for substance P (SP) or protein gene product 9.5 (PGP9.5). Vagal ganglia neurons projecting to airway epithelium were identified by axonal transport of fluorescent microspheres intratracheally instilled 14 days before diacetyl inhalation. End points were SP and PGP9.5 nerve fiber density (NFD) in tracheal epithelium and SP-positive neurons projecting to the trachea. PGP9.5-immunoreactive NFD decreased in foci with denuded epithelium, suggesting loss of airway sensory innervation. However, in the intact epithelium adjacent to denuded foci, SP-immunoreactive NFD increased from 0.01 ± 0.002 in controls to 0.05 ± 0.01 after exposure to 346-ppm diacetyl. In vagal ganglia, SP-positive airway neurons increased from 3.3 ± 3.0% in controls to 25.5 ± 6.6% after inhaling 346-ppm diacetyl. Thus, diacetyl inhalation increases SP levels in sensory nerves of airway epithelium. Because SP release in airways promotes inflammation and activation of sensory nerves mediates reflexes, neural changes may contribute to flavorings-related lung disease pathogenesis.

Respiratory and olfactory cytotoxicity of inhaled 2,3-pentanedione in Sprague-Dawley rats.

Flavorings-related lung disease is a potentially disabling disease of food industry workers associated with exposure to the α-diketone butter flavoring, diacetyl (2,3-butanedione). To investigate the hypothesis that another α-diketone flavoring, 2,3-pentanedione, would cause airway damage, rats that inhaled air, 2,3-pentanedione (112, 241, 318, or 354 ppm), or diacetyl (240 ppm) for 6 hours were sacrificed the following day. Rats inhaling 2,3-pentanedione developed necrotizing rhinitis, tracheitis, and bronchitis comparable to diacetyl-induced injury. To investigate delayed toxicity, additional rats inhaled 318 (range, 317.9-318.9) ppm 2,3-pentanedione for 6 hours and were sacrificed 0 to 2, 12 to 14, or 18 to 20 hours after exposure. Respiratory epithelial injury in the upper nose involved both apoptosis and necrosis, which progressed through 12 to 14 hours after exposure. Olfactory neuroepithelial injury included loss of olfactory neurons that showed reduced expression of the 2,3-pentanedione-metabolizing enzyme, dicarbonyl/L-xylulose reductase, relative to sustentacular cells. Caspase 3 activation occasionally involved olfactory nerve bundles that synapse in the olfactory bulb (OB). An additional group of rats inhaling 270 ppm 2,3-pentanedione for 6 hours 41 minutes showed increased expression of IL-6 and nitric oxide synthase-2 and decreased expression of vascular endothelial growth factor A in the OB, striatum, hippocampus, and cerebellum using real-time PCR. Claudin-1 expression increased in the OB and striatum. We conclude that 2,3-pentanedione is a respiratory hazard that can also alter gene expression in the brain.

Nanotoxicology--a pathologist's perspective.

Advances in chemistry and engineering have created a new technology, nanotechnology, involving the tiniest known manufactured products. These products have a rapidly increasing market share and appear poised to revolutionize engineering, cosmetics, and medicine. Unfortunately, nanotoxicology, the study of nanoparticulate health effects, lags behind advances in nanotechnology. Over the past decade, existing literature on ultrafine particles and respirable durable fibers has been supplemented by studies of first-generation nanotechnology products. These studies suggest that nanosizing increases the toxicity of many particulates. First, as size decreases, surface area increases, thereby speeding up dissolution of soluble particulates and exposing more of the reactive surface of durable but reactive particulates. Second, nanosizing facilitates movement of particulates across cellular and intracellular barriers. Third, nanosizing allows particulates to interact with, and sometimes even hybridize with, subcellular structures, including in some cases microtubules and DNA. Finally, nanosizing of some particulates, increases pathologic and physiologic responses, including inflammation, fibrosis, allergic responses, genotoxicity, and carcinogenicity, and may alter cardiovascular and lymphatic function. Knowing how the size and physiochemical properties of nanoparticulates affect bioactivity is important in assuring that the exciting new products of nanotechnology are used safely. This review provides an introduction to the pathology and toxicology of nanoparticulates.

PKC epsilon and an increase in intracellular calcium concentration are necessary for PGF2 alpha to inhibit LH-stimulated progesterone secretion in cultured bovine steroidogenic luteal cells.

The hypotheses that PKC epsilon is necessary for: 1) PGF2 alpha to inhibit LH-stimulated progesterone (P4) secretion, and 2) for the expression of key prostaglandin synthesizing/metabolizing enzymes were tested in bovine luteal cells in which PKC epsilon expression had been ablated using a validated siRNA protocol. Steroidogenic cells from Day -6 bovine corpus luteum (CL) were isolated and transfected to reduce PKC epsilon expression after 48, 72 and 96 h. A third tested hypothesis was that an increase in intracellular calcium concentration ([Ca(2+)]i) is the cellular mechanism through which PGF2 alpha inhibits luteal progesterone. The hypothesis was tested with two pharmacological agents. In the first test, the dose-dependent effects on raising the [Ca(2+)]i with the ionophore, A23187, on basal and LH-stimulated P4 secretion in cells collected from early (Day -4) and mid-cycle (Day -10) bovine CL was examined. In the second test, the ability of PGF2 alpha to inhibit LH-stimulated P4 secretion in Day-10 luteal cells was examined under conditions in which an elevation in [Ca(2+)]i had been buffered by means of the intracellular calcium chelator, Bapta-AM.PKC epsilon expression was reduced 65 and 75% by 72 and 96 h after transfection, respectively. In cells in which PKC epsilon expression was ablated by 75%, the inhibitory effect of PGF2 alpha on LH-stimulated P4 secretion was only 29% lower than in the LH-stimulated group. In contrast, it was reduced by 75% in the group where PKC epsilon expression had not been reduced (P < 0.05). Real time PCR analysis indicated that there were no differences in the expression of cyclooxygenase-2 (COX-2), aldoketoreductase 1B5 (AKR1B5), prostaglandin E synthase (PGES), hydroxyprostaglandin-15 dehydrogenase (PGDH) and PGE2 -9-reductase as a function of PKC epsilon down-regulation. Finally, LH stimulated secretion of P4 at each luteal stage (Day -4 and -10), and PGF2 alpha inhibited this only in Day -10 cells (P < 0.05). When A23187 was used at concentrations greater than 0.1 mumol, the induced elevation in [Ca(2+)]i inhibited the effect of LH on secretion of P4 in Day -4 and -10 cells (P < 0.05, Fig. 5). The inhibitory effect of PGF2 alpha on LH-stimulated P4 in Day -10 cells was reduced if an increase in [Ca(2+)]i was prevented with Bapta-AM. These results support the hypothesis that differential expression of PKC epsilon and an elevation of [Ca(2+)]i are important for acquisition of luteolytic response to PGF2 alpha.

Differential gene expression in the bovine corpus luteum during transition from early phase to midphase and its potential role in acquisition of luteolytic sensitivity to prostaglandin F2 alpha.

Prostaglandin F2 alpha (PGF(2alpha)) brings about regression of the bovine corpus luteum (CL). This luteolytic property of PGF(2alpha) is used in beef and dairy cattle to synchronize estrus. A limitation of this protocol is insensitivity of the early CL to luteolytic actions of PGF(2alpha). The mechanisms underlying this differential luteal sensitivity are poorly understood. The developing CL has a maximum number of PGF(2alpha) receptors; therefore, differences in signaling events may be responsible for luteal insensitivity. Hence, differential gene expression at two developmental stages of CL, Day 4 (D-4) and D-10 after estrus, might account for differences in signal transduction pathways associated with luteal sensitivity. This possibility was examined in these studies. Microarray analysis (n = 3 cows per stage) identified 167 genes that were differentially expressed (P < 0.05). These were categorized into genes involved in protein biosynthesis and modification (18.5%), transcription regulation and DNA biosynthesis (18.5%), miscellaneous (17.0%), cell signaling (12.0%), steroidogenesis and metabolism (10.2%), extracellular matrix and cytoskeletal proteins (9.5%), unknown functions (6.0%), protein degradation (5.3%), and antioxidant property (3.0%). Real-time PCR confirmed the differential expression of nine selected genes, including tyrosine 3-monooxygenase/tryptophan 5-monooxygense activation protein zeta polypeptide (YWHAZ) and regulator of G protein signaling 2 24-kDa (RGS2), observed in microarray. Furthermore, the in vivo effect of exogenous PGF(2alpha) (n = 3 cows per stage) on selected genes that were found to be differentially expressed during this developmental transition was examined. PGF(2alpha) increased the expression of a guanine nucleotide-binding protein (G protein) beta polypeptide 1 (GNB1) in D-4 CL and calcium/calmodulin-dependent kinase kinase 2 beta (CAMKK2) in D-10 CL. Therefore, GNB1, CAMKK2, YWHAZ, and RGS2 are candidate genes that may have a significant role in acquisition of luteal sensitivity to PGF(2alpha). Additional evidence supporting the significance of the microarray data was obtained from the observation that the amount of CAMKK2 paralleled the differential mRNA expression observed for this gene when examined by microarray analysis and by real-time RT-PCR. Furthermore, the two types of luteal steroidogenic cells known to be targets for PGF(2alpha) actions were demonstrated to be a cellular source for CAMKK2.

Effects of endothelin receptor type-A and type-B antagonists on prostaglandin F2alpha-induced luteolysis of the sheep corpus luteum.

Three experiments were designed to examine the mechanisms that govern prostaglandin (PGF2alpha)-induced regression of the sheep corpus luteum. Evidence is presented supporting the involvement of endothelin 1 (EDN1) in PGF2alpha-induced luteolysis. Experiment 1 measured effects of PGF2alpha when actions of EDN1 were blocked by sustained administration of a type-A endothelin (EDNRA) or type-B endothelin (EDNRB) antagonist in vivo. Experiment 2 examined antisteroidogenic actions of PGF2alpha and EDN1 in the presence of an EDNRA or EDNRB antagonist in Day-8 luteal minces. In experiment 3, luteal cellular expression of EDNRA and EDNRB was determined immunohistochemically. Relative gene expression of EDNRA and EDNRB receptors was examined by real-time RT-PCR in Day-8 sheep corpora lutea. EDNRA, but not EDNRB, participated in antisteroidogenic actions of EDN1. During the first 12 h after PGF2alpha-induced luteolysis, EDNRA antagonist did not prevent a decline in serum progesterone concentrations. Early actions of PGF2alpha are either direct or mediated by something other than EDN1. However, beyond 12 h after PGF2alpha, serum progesterone concentrations increased in EDNRA antagonist-treated animals until they were the same as saline-treated controls, whereas an EDNRB antagonist had no effect in vivo or in vitro. The EDNRA antagonist negated the antisteroidogenic actions of EDN1 but only partially abolished the actions of PGF2alpha in vitro. In contrast, the EDNRB antagonist was ineffective in abolishing antisteroidogenic actions of EDN1 and PGF2alpha. Whereas real-time RT-PCR demonstrated high expression of EDNRA and low expression of EDNRB, immunohistochemically, only EDNRA was located in small steroidogenic, endothelial, and smooth muscle cells. In summary, studies in ovine corpora lutea provided strong evidence that: 1) EDNRA, but not EDNRB, mediates antisteroidogenic actions of EDN1, 2) actions of PGF2alpha are both independent of and dependent upon mediation by EDN1, and 3) small steroidogenic cells are targets for antisteroidogenic effects of EDN1. Furthermore, the results from experiment 1 suggest that the intermediary role of EDN1 may be more important in later stages of luteal regression.