Inhaled diacetyl vapors are associated with flavorings-related lung disease, a potentially fatal airway disease. The reactive alpha-dicarbonyl group in diacetyl causes protein damage in vitro. Dicarbonyl/l-xylulose reductase (DCXR) metabolizes diacetyl into acetoin, which lacks this alpha-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 alpha-dicarbonyl group in the protein damage. Western immunoblots demonstrated decreased free ubiquitin in airway-enriched fractions. Transmission electron microscopy and colocalization of ubiquitin-positive puncta with lysosomal markers lysosomal-associated membrane protein 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.

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.

Flavorings-related lung disease is a potentially disabling disease of food industry workers associated with exposure to the alpha-diketone butter flavoring, diacetyl (2,3-butanedione). To investigate the hypothesis that another alpha-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.

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.