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

Abstract

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.

Figure 1

Histopathological changes in nasal epithelium after PD exposure. A: Normal transition epithelium of the maxilloturbinate in an air control rat. B: Necrotizing rhinitis of the maxilloturbinate in a PD-exposed rat is characterized by necrosis of the epithelium, with few infiltrating neutrophils. C: Normal neuroepithelium of an air-exposed control rat. D: The neuroepithelium of a rat after inhaling 318-ppm PD shows loss of cells, cells with pyknotic nuclei, and foci of invagination. Scale bar = 20 μm.

Figure 2

Histopathological changes in the mainstem bronchus in a rat after inhaling 354 ppm. Changes are subtle and limited to a few cells with pyknosis or karyorrhexis (arrow) within the respiratory epithelium. Scale bar = 20 μm.

Figure 3

Luxol fast blue/PAS staining of the olfactory neuroepithelium. A: In an air-exposed control rat, the Bowman's glands contain PAS-positive secretory granules (dashed arrows), and their excretory ducts (solid arrows) traverse the basement membrane. In the lumen, small amounts of PAS-positive material are seen. B: In a PD-exposed rat from the 12-hour postexposure group, the lamina propria in this focus is devoid of PAS-positive Bowman's glands. A focus of invagination (asterisk) is associated with the greatest loss of cellularity in the olfactory neuroepithelium and lies above the excretory duct of the Bowman's gland, which traverses the basement membrane (solid arrow). In the nasal airway lumen, the amount of PAS-positive material is increased and is aggregated. Scale bar = 20 μm.

Figure 4

Immunofluorescence for activated caspase 3 (green) demonstrates cells in the execution stage of apoptosis. Immunofluorescence for OMP (red) demonstrates neurons. A: Olfactory neuroepithelium of an air-exposed control rat demonstrates numerous well-organized neuroepithelial cells (red) within the epithelium, with rare apoptotic cells (green). Beneath the neuroepithelium, olfactory nerve bundles also express OMP and stain red but show no evidence of apoptosis. B: Olfactory neuroepithelium from a rat after 6-hour inhalation exposure to 354-ppm PD. Within the neuroepithelium, the number of olfactory neurons (red) is reduced; the olfactory neurons are disorganized; and many olfactory neurons have variations in size and swelling of dendrites, consistent with degenerative changes. In the lamina propria, some olfactory nerve bundles do not contain axons with activated caspase 3, whereas other olfactory nerve bundles contain many apoptotic axons (arrows). C: A higher magnification of an olfactory nerve bundle from B showing activated caspase 3 in axons and a cell morphologically consistent with a macrophage containing apparently phagocytized cellular debris of axons with caspase 3 activation. Scale bar = 20 μm.

Figure 5

Dual-label confocal immunofluorescence for DCXR and OMP in the olfactory neuroepithelium of nasal section T2 in an air-exposed control rat. A: DCXR expression (red) is restricted to the olfactory neuroepithelium. B: Adding a second label for OMP (green) demonstrates that the OMP-positive neurons are in cells without demonstrable DCXR. C: Negative control showing low levels of autofluorescence similar to what is seen in the lamina propria and olfactory neurons in A and B. D: Higher magnification of DCXR expression in the apical cytoplasm of sustentacular cells showing a line of the diketone metabolizing enzyme at the air interface. E: OMP (green) demonstrates that the dendrites and cell bodies of neurons have little or no DCXR. F: High-magnification negative control showing low levels of autofluorescence. Scale bar = 10 μm.

Figure 6

PD-induced changes in dual-label immunofluorescence for DCXR (red) and OMP (green) in nasal section T2. A: In the air-exposed olfactory neuroepithelium, DCXR is abundant in the apical cytoplasm of sustentacular cells at the air interface. B: PD-induced cytotoxicity in the olfactory neuroepithelium disrupts the continuity of the layer of DCXR (red) at the air interface and increases exposure of neurons to the air interface. Sustentacular cells and neurons are detaching (arrows). Inset: UV fluorescence is added to demonstrate DAPI-stained nuclei in detaching cells. C: Representative focus demonstrating aggregation of DCXR (red) into vacuoles in the apical cytoplasm of sustentacular cells after inhaling 360-ppm PD. Scale bar = 20 μm.

Figure 7

Dual-label immunofluorescence for DCXR (red) and E-cadherin (green). A: In the air-exposed olfactory neuroepithelium, DCXR is abundant in the apical cytoplasm of sustentacular cells, but no detectable DCXR is in the excretory duct (arrow) of the Bowman's glands. B: In the PD-exposed neuroepithelium, a focus of severe cytotoxicity is localized to the excretory duct (arrow) of the Bowman's glands, which does not contain DCXR. Scale bar = 20 μm.

Figure 8

A: Dual-label immunofluorescence for activated caspase 3 (green) and OMP (red). Apoptosis is demonstrated by caspase 3 activation, and olfactory neurons are demonstrated by expression of OMP. In this rat from the 12-hour PD postexposure group, apoptosis and invagination of the olfactory neuroepithelium are clearly demonstrated. Olfactory nerve bundles (dashed arrows) that contain the axons of the olfactory nerves are beneath the olfactory neuroepithelium. The respiratory epithelium (solid arrows) does not contain olfactory neurons. Scale bar = 50 μm. B: Confocal microscopic image of dual-label immunofluorescence for activated caspase 3 (green) and OMP (red) in the olfactory neuroepithelium in a rat from the 12-hour PD postexposure group. Olfactory neuron dendrites are often swollen or shortened (solid arrows), and apoptosis of axons in an olfactory nerve bundle is demonstrated by expression of activated caspase 3 (dashed arrow). Scale bar = 10 µm.

Figure 9

FluoroJade B staining for degenerating neurons stains olfactory neurons of PD-exposed, but not control, rats. A: Olfactory neuroepithelium of nasal section T2 in an air-exposed control rat. B: Olfactory neuroepithelium of nasal section T2 in a PD-exposed rat. C: Olfactory neuroepithelium of nasal section T4 in a PD-exposed rat. Scale bar = 20 μm.

Figure 10

Inhalation of PD (270 ppm, 6 hours 41 minutes) alters mRNA expression in discrete brain areas. A: The inflammatory mediator IL-6 is induced in the OB, STR, HIP, and CER. B: The inflammatory mediator Nos2 is up-regulated in the OB, STR, HIP, and CER. C: Cldn1 is increased in the OB and STR, but not in the HIP or CER. D: Vegf-A expression decreases in the OB, STR, HIP, and CER. AIR denotes control group exposed to air. Asterisk denotes significant change from air-exposed controls.