Acute exposure to diacetyl results in injury in the nasal passages and large airways of the rat (Hubbs et al., 2008
). Inhalation dosimetric patterns are often critical in influencing the site of injury to inspired materials (U.S. EPA, 1994
). The current study characterizes the upper airway dosimetry of diacetyl from both an experimental (uptake efficiency measurement) and a theoretical (CFD-PBPK modeling) perspective. Uptake of vapor in the airways is determined by the balance of delivery to the airway walls from the airstream and clearance from tissues via the bloodstream, metabolism, and/or direct reactivity. The potential importance of metabolism in influencing uptake has been shown by numerous studies which document that pretreatment with xenobiotic metabolizing enzyme inhibitors diminishes uptake efficiencies (Morris, 1990
, 1999; Stanek and Morris, 1999
). In general, vapors that partition extensively into tissue and/or are reactive or quickly metabolized are scrubbed from the upper airways with high efficiency. For example, weak acids (hydrofluoric, acetic, acrylic acid) are soluble and reactive in that they ionize at physiological pH. Nasal uptake efficiencies in excess of 95% are observed for such vapors (Morris and Frederick, 1995
; Morris and Smith, 1982
; Vaughan et al., 2006
). In the current study, URT uptake efficiency in excess of 90% was observed for butyric acid, a result fully consistent with the existing database. Modern modeling approaches, including hybrid CFD-PBPK models, have successfully captured uptake behavior of metabolized and/or reactive vapors and have been useful in defining and extrapolating dosimetric relationships in the rat and human.
The current studies reveal that diacetyl is a “soluble” vapor as indicated by a water:air partition coefficient (W/A) of 550; a partition coefficient that is higher than acetone and lower than ethanol vapor (Morris and Cavanagh, 1986
). Based on its partition coefficient, moderate (e.g., 25–75%) URT uptake efficiencies would be expected for diacetyl; such efficiencies were observed. Scrubbing of vapors in the nose is an important respiratory defense mechanism in that it serves to protect the lower airways. Rats are obligate nose breathers; thus, in rats the concentration of airborne vapor reaching the lungs is often much lower than the ambient air concentration. This is also true in the nose-breathing human, but during mouth breathing this defense mechanism is lost. Thus, a challenge in interpreting inhalation toxicity data is extrapolating responses in nose-breathing rodents to mouth-breathing humans.
Diacetyl was found to be metabolized in nasal and tracheal tissues via a NADPH-dependent pathway, presumably diacetyl reductase. This enzyme has previously been shown to be present in airways of the rodent and human (Nakagawa et al., 2002
); thus, its detection in nasal tissues is not unexpected. Specific activity was approximately fourfold higher in olfactory than nasal respiratory or tracheal tissues (). This is a common pattern for nasal enzymology (Dahl and Hadley, 1991
). This enzyme is thought to be a detoxification pathway for diacetyl (Nakagawa et al., 2002
). Butyric acid is a potent inhibitor of diacetyl reductase (Nakagawa et al., 2002
). By analogy to numerous previous studies (Morris, 1990
, 1999; Stanek and Morris, 1999
), inhibition of nasal metabolism of diacetyl should result in decreased scrubbing capacity. The current results indicate that coexposure to butyric acid significantly reduces nasal uptake efficiency of diacetyl but not acetone vapor. The absence of effect on acetone vapor uptake suggests that butyric acid (at this exposure level) does not cause nasal vasodilation and increased blood flow (Morris et al., 1999
). The significance of this result is threefold. First, this provides evidence that inspired diacetyl is indeed metabolized in the nasal tissues via a butyric acid–sensitive pathway, likely diacetyl reductase. Second, reduced nasal scrubbing capacity of diacetyl vapors in the presence of butyric acid or other diacetyl reductase inhibitors will enhance more distal penetration of inspired diacetyl and, therefore, places the lower airways at greater risk. Third, if diacetyl reductase is a detoxification pathway, by inhibiting this enzyme, butyric acid would enhance the toxicity of diacetyl in those tissues in which both vapors deposit. Since butyric acid is scrubbed with high efficiency in the nose, this interaction most likely occurs in the nasal airways of the rat. Under mouth-breathing conditions, the same phenomenon would be anticipated in the large airways of humans.
The uptake efficiency observed in the rat during coexposure to diacetyl and butyric acid was consistent with a butyric acid–induced 75% reduction in metabolic capacity. This may reflect a 75% inhibition diffusely throughout the nasal airways. Alternatively, and more likely, this represents the deposition patterns of butyric acid. As evidenced by strong anterior-posterior gradients in nasal injury by weak acids (Buckley et al., 1984
; Rosenholtz et al., 1963
) in the rat, butyric acid likely only deposits most heavily in the anterior portions of the nose; thus, a greater degree of inhibition would be expected in the anterior than posterior nasal regions, and any inhibition of nasal diacetyl reductase in the rat may have been focal in nature. Irrespective of the focal versus diffuse nature of any inhibition, these studies indicate that there is a dosimetric interaction among the components of BFV and highlight the need to consider the multiple components of BFV in comprehensive safety evaluations. Our study utilized a butyric acid concentration which was 30% of the diacetyl concentration. This concentration was necessary to demonstrate inhibition of the butyric acid–sensitive diacetyl metabolic pathway in the nose, which is presumed to be diacetyl reductase. While butyric acid is a common vapor in microwave popcorn production (Boylstein et al., 2006
) and the most powerful known inhibitor of diacetyl reductase (Nakagawa et al., 2002
), its relative concentration to diacetyl in workplaces is incompletely investigated at this time.
Computational modeling has proved very useful in understanding and extrapolating inhalation dosimetry patterns for diverse vapors such as acrylic acid (Frederick et al., 1998
), ethyl acrylate (Frederick et al., 2002
), acetaldehyde (Teeguarden et al., 2008
), and acrolein (Schroeter et al., 2008
). In the current study, this modeling approach closely predicted URT uptake efficiencies of diacetyl. It is important to note that the model was not fit to the diacetyl data, rather the model was applied simply by inputting the diacetyl-specific data. Nasal injury patterns from inspired vapors are often highly localized; dosimetry models have provided data implicating regional dosage rates as being a key contributor to regionalized injury (Kimbell et al., 1997
; Schroeter et al., 2006
). Using an approach conceptualized by Kimbell (Kimbell et al., 1997
), the regional nasal injury scores in diacetyl-exposed rats were correlated with the predicted regional tissue concentrations within the nose. A strong correlation was seen. These data suggest that acute epithelial injury results when tissue concentrations exceed 2mM. The strong correlation enhances confidence in the model predictions and suggests that, as for other vapors (Kimbell et al., 1997
; Schroeter et al., 2006
), regional dosage rates are important determinants of regional nasal injury following acute diacetyl exposure. One limitation of the model predictions is that they are made assuming constant velocity unidirectional flow rather than cyclic breathing. However, it is thought that predictions under these constant velocity flow conditions are reflective of dosimetry patterns under normal respiration (Andersen and Sarangapani, 1999
An additional limitation is that our study does not model the effect of subchronic or chronic exposure to diacetyl. In mice, a 12-week exposure to 100-ppm diacetyl produces denudation, atrophy, and regeneration of bronchial epithelium with lymphocytic bronchitis seen at lower exposures, effects that may well occur when tissue concentrations are less than 2mM. It is for this reason that the 2mM value discussed in this publication should not be used for assessment of risks associated with chronic exposure scenarios. Our study does suggest the potential importance of changes seen in the nose during subchronic exposures that might affect nasal uptake of diacetyl, including chronic inflammation, squamous metaplasia, respiratory metaplasia of olfactory epithelium, and olfactory epithelial atrophy (Morgan et al., 2008
). CFD-PBPK modeling of subchronic and chronic low-level effects is a future research need. It should also be noted that measurement of URT uptake efficiencies and validation of the model for lower exposure concentrations would also represent a future research need.
In rodents, diacetyl also induces injury in the trachea and bronchi (Hubbs et al., 2008
, Morgan et al., 2008
). Both the nasal cavity and large airways are lined with a pseudostratified columnar mucociliated epithelium, but it is not known if these tissues are of equal sensitivity to diacetyl. To assess this issue, the CFD-PBPK model structure was used to extrapolate tissue concentrations in the trachea. Because there are no measures of tracheal uptake, these represent extrapolations. Extrapolated tracheal diacetyl concentrations exceed 2mM in animals exposed to 200 ppm or higher but not 100 ppm. Injury in the lower airways was not observed at the 100-ppm exposure level but was observed in rats exposed to 200 ppm and higher concentrations. Perhaps, more revealing is the relationship between the degree of tracheal injury versus the predicted tracheal epithelial concentration. These values for the trachea fell entirely within the ranges of values observed for the nasal epithelium (see ). This provides suggestive evidence that the respiratory epithelium of the nose and trachea is of similar sensitivity to diacetyl-induced injury.
The hybrid CFD-PBPK modeling approach was developed to facilitate rodent-to-man extrapolations (Frederick et al., 1998
). This is accomplished by using the human-specific parameters in the model. This approach was used to predict nasal uptake efficiencies for vinyl acetate which were subsequently confirmed by experimentation (Hinderliter et al., 2005
). Because there is a strong scientific foundation for these models, the output represents informed estimates of human inhalation dosimetric relationships. The model predicts that diacetyl will be scrubbed with less efficiency in the upper airways in the human than in the rodent. In this regard, diacetyl appears similar to acrylic acid, vinyl acetate, and ethyl acrylate (Frederick et al., 1998
; Hinderliter et al., 2005
). From an inhalation risk perspective, reduced scrubbing capacity in the upper airways of the human correlates with increased delivery of material to potentially more sensitive lower airway sites.
The sensitivity analysis of the CFD-PBPK provides insights into important pathways for diacetyl uptake. In general terms, in both the rat and the human uptake efficiency was moderately dependent on metabolism rate (as measured by Vmax
) and more strongly sensitive to partition coefficient, tissue depth, tissue vapor diffusivity, and blood flow rates. These observations suggest that partitioning into tissue and removal via the circulation is the predominant pathway responsible for diacetyl uptake. Since the depth of the nasal mucosa in the human is greater than in the rat, uptake via this pathway in the human would be expected to be less effective than in the rat. This may be the primary basis for the lower predicted uptake efficiency in the human. URT uptake efficiency in the human appears to be more sensitive than in the rat to the partition coefficient, tissue diffusivity, and tissue depth; similar observations have been made in modeling efforts on acrolein (Schroeter et al., 2008
) but not acetaldehyde (Teeguarden et al., 2008
). Thus, these relationships may be vapor specific. Predicted URT uptake efficiency in the rat was insensitive to changes in the air-phase mass transfer coefficient suggesting that a minimal air-phase diffusion limitation exists in this species. Previous modeling efforts have reached a similar conclusion (Bush et al., 1998
; Frederick et al., 1998
). Although on an absolute basis human nasal cavity uptake was not particularly sensitive to air-phase mass transfer coefficient, it was considerably more sensitive than the rat. This is particularly evident when comparing the effect of reducing the mass transfer coefficient by 10-fold. This produced less than a 1% change in predicted diacetyl uptake in the rat compared to a 30% reduction in the human, results very similar to those for acrylic acid vapor (Frederick et al., 1998
). In toto
, these results suggest that there is a proportionately greater air-phase resistance to uptake in the human than in the rat URT.
Clinical findings in individuals occupationally exposed to high concentrations of BFV indicate bronchiolar injury, data supported by the presence of constrictive bronchiolitis in two of the three patients with lung biopsies (Akpinar-Elci et al., 2004
). However, nasal and tracheal/bronchial injury predominates in rodents exposed to BFV or diacetyl (Akpinar-Elci et al., 2004
; Hubbs et al., 2002
; Kreiss et al., 2002
; Van Rooy et al., 2007
). This species difference in regional respiratory tract injury is not uncommon. For example, hydrogen fluoride has long been known to be a lower respiratory tract irritant in the human (Dalbey et al., 1998
), yet in the rodent it is scrubbed with high efficiency in the nose and produces injury only in that site (Dalbey et al., 1998
; Morris and Smith, 1982
; Rosenholtz et al., 1963
; Stavert et al., 1991
). Formaldehyde injury and DNA-protein cross-link levels are elevated in the anterior portion of the nose of rodents but are observed in the trachea and major bronchi as well as the nose in the monkey; decreased scrubbing capacity of the upper airways of the primate compared to the rodent is thought to be a critical factor in this species difference (Casanova et al., 1991
The current model projects less-efficient airway scrubbing of diacetyl in the nose-breathing human than in the rat. It is important to recognize that in mouth-breathing humans, the nasal passages are bypassed and delivery to the lower airways is enhanced over that in nose breathing. The concentration of diacetyl in air penetrating to the bronchi in nose-breathing rats exposed to 100 ppm was predicted to be 62 ppm; in contrast, in mouth-breathing humans this concentration was predicted to be 97 ppm, roughly 1.5-fold higher, suggesting that the delivered dose to intrapulmonary airways in the human may be much greater than that in the rat. In addition to these factors, the dosimetric interaction between diacetyl and butyric acid may also be important in regional airway injury in humans exposed to butter flavor vapors. The current results indicate that butyric acid diminishes upper airway scrubbing of diacetyl and enhances its penetration to the lower airways. This was shown for the nose but would likely also occur in the large airways during mouth breathing. Thus, on the basis of these dosimetric relationships, it would appear that nasal and tracheal airway injury in the rodent exposed to diacetyl may well be predictive of injury in the intrapulmonary airways of mouth-breathing humans exposed to airborne BFV.