Toxicity studies of acetoin and 2,3-pentanedione administered by inhalation to Wistar Han [Crl:WI(Han)] rats and B6C3F1/N mice.

Acetoin and 2,3-pentanedione are highly volatile components of artificial butter flavoring (ABF). Concerns over the inhalation toxicity of these compounds originate from the association between occupational exposures to ABF and adverse fibrotic lung effects, specifically obliterative bronchiolitis (OB) in the distal airways. 2,3-Pentanedione has been used as a replacement for 2,3-butanedione (diacetyl) in some ABF due to concerns about the respiratory toxicity of 2,3-butanedione. However, 2,3-pentanedione is structurally similar to 2,3-butanedione and has been shown to exhibit potency similar to 2,3-butanedione regarding airway toxicity following acute inhalation (whole-body) exposure. This report describes a series of studies to evaluate the 2-week inhalation toxicity of acetoin and the 3-month inhalation toxicity of acetoin and 2,3-pentanedione. (Abstract Abridged).

Mechanisms of Acetoin Toxicity and Adaptive Responses in an Acetoin-Producing Species, Lactococcus lactis.

Acetoin, 3-hydroxyl,2-butanone, is extensively used as a flavor additive in food products. This volatile compound is produced by the dairy bacterium Lactococcus lactis when aerobic respiration is activated by haem addition, and comprises ∼70% of carbohydrate degradation products. Here we investigate the targets of acetoin toxicity, and determine how acetoin impacts L. lactis physiology and survival. Acetoin caused damage to DNA and proteins, which related to reactivity of its keto group. Acetoin stress was reflected in proteome profiles, which revealed changes in lipid metabolic proteins. Acetoin provoked marked changes in fatty acid composition, with massive accumulation of cycC19:0 cyclopropane fatty acid at the expense of its unsaturated C18:1 fatty acid precursor. Deletion of the cfa gene, encoding the cycC19:0 synthase, sensitized cells to acetoin stress. Acetoin-resistant transposon mutagenesis revealed a hot spot in the high affinity phosphate transporter operon pstABCDEF, which is known to increase resistance to multiple stresses. This work reveals the causes and consequences of acetoin stress on L. lactis, and may facilitate control of lactic acid bacteria production in technological processes. IMPORTANCE Acetoin, 3-hydroxyl,2-butanone, has diverse uses in chemical industry, agriculture, and dairy industries as a volatile compound that generates aromas. In bacteria, it can be produced in high amount by Lactococcus lactis when it grows under aerobic respiration. However, acetoin production can be toxic and detrimental for growth and/or survival. Our results showed that it damages DNA and proteins via its keto group. We also showed that acetoin modifies membrane fatty acid composition with the production of cyclopropane C19:0 fatty acid at the expense of an unsaturated C18:1. We isolated mutants more resistant to acetoin than the wild-type strain. All of them mapped to a single locus pstABCDEF operon, suggesting a simple means to limit acetoin toxicity in dairy bacteria and to improve its production.

Flavorings-Related Lung Disease: A Brief Review and New Mechanistic Data.

Flavorings-related lung disease is a potentially disabling and sometimes fatal lung disease of workers making or using flavorings. First identified almost 20 years ago in microwave popcorn workers exposed to butter-flavoring vapors, flavorings-related lung disease remains a concern today. In some cases, workers develop bronchiolitis obliterans, a severe form of fixed airways disease. Affected workers have been reported in microwave popcorn, flavorings, and coffee production workplaces. Volatile α-dicarbonyl compounds, particularly diacetyl (2,3-butanedione) and 2,3-pentanedione, are implicated in the etiology. Published studies on diacetyl and 2,3-pentanedione document their ability to cause airway epithelial necrosis, damage biological molecules, and perturb protein homeostasis. With chronic exposure in rats, they produce airway fibrosis resembling bronchiolitis obliterans. To add to this knowledge, we recently evaluated airway toxicity of the 3-carbon α-dicarbonyl compound, methylglyoxal. Methylglyoxal inhalation causes epithelial necrosis at even lower concentrations than diacetyl. In addition, we investigated airway toxicity of mixtures of diacetyl, acetoin, and acetic acid, common volatiles in butter flavoring. At ratios comparable to workplace scenarios, the mixtures or diacetyl alone, but not acetic acid or acetoin, cause airway epithelial necrosis. These new findings add to existing data to implicate α-dicarbonyl compounds in airway injury and flavorings-related lung disease.

Small molecule diketone flavorants diacetyl and 2,3-pentanedione promote neurotoxicity but inhibit amyloid ß aggregation.

We investigated the effects of the small molecule flavorants diacetyl, 2,3-pentanedione and acetoin on neuronal cell viability and ß amyloid aggregation and morphology. Two neuroblastoma cell lines, SH-SY5Y and Neuro 2a (N2a) were exposed to diacetyl, 2,3-pentanedione and acetoin, while Thioflavin T fluorescence kinetics and transmission electron microscopy were used to assess effects on Aß1-42 fibril and aggregate formation and morphology respectively. Diacetyl was intrinsically toxic to both SH-SY5Y and N2a cells, with time and concentration-dependent reductions in cell viability occurring over 24 h and 48 h incubation periods. 2.3-Pentanedione evoked a similar concentration-dependent loss of cell viability in N2a cells at 48 h, but exhibited lessened toxicity in SH-SY5Y cells over 24 h, and minimal loss of cell viability by 48 h. Diacetyl inhibited Aß1-42 aggregation kinetics, reduced aggregate and fibril density and rendered Aß1-42 into amorphous small aggregates. 2,3-Pentanedione also reduced overall aggregate formation, but to a lesser extent than diacetyl and retaining the presence of a meshwork of Aß1-42 aggregates and fibrils. Acetoin was innocuous to neuronal cells and did not alter Aß1-42 fibril density or morphology. These findings highlight the intrinsic neurotoxicity of small molecule diketone flavorants. While providing further insight into their molecular interactions with amyloidogenic proteins, the neurotoxicity of such flavorants is a significant finding and warrants further investigation.

Popcorn flavoring effects on reactivity of rat airways in vivo and in vitro.

"Popcorn workers' lung" is an obstructive pulmonary disease produced by inhalation of volatile artificial butter flavorings. In rats, inhalation of diacetyl, a major component of butter flavoring, and inhalation of a diacetyl substitute, 2,3-pentanedione, produce similar damage to airway epithelium. The effects of diacetyl and 2,3-pentanedione and mixtures of diacetyl, acetic acid, and acetoin, all components of butter flavoring, on pulmonary function and airway reactivity to methacholine (MCh) were investigated. Lung resistance (RL) and dynamic compliance (Cdyn) were negligibly changed 18 h after a 6-h inhalation exposure to diacetyl or 2,3-pentanedione (100-360 ppm). Reactivity to MCh was not markedly changed after diacetyl, but was modestly decreased after 2,3-pentanedione inhalation. Inhaled diacetyl exerted essentially no effect on reactivity to mucosally applied MCh, but 2,3-pentanedione (320 and 360 ppm) increased reactivity to MCh in the isolated, perfused trachea preparation (IPT). In IPT, diacetyl and 2,3-pentanedione (≥3 mM) applied to the serosal and mucosal surfaces of intact and epithelium-denuded tracheas initiated transient contractions followed by relaxations. Inhaled acetoin (150 ppm) exerted no effect on pulmonary function and airway reactivity in vivo; acetic acid (27 ppm) produced hyperreactivity to MCh; and exposure to diacetyl + acetoin + acetic acid (250 + 150 + 27 ppm) led to a diacetyl-like reduction in reactivity. Data suggest that the effects of 2,3-pentanedione on airway reactivity are greater than those of diacetyl, and that flavorings are airway smooth muscle relaxants and constrictors, thus indicating a complex mechanism.

Effect of some uremic toxins on oxygen consumption of rats in vivo and in vitro.

In rats, oxygen consumption is reduced by about 40-50% 24 h after bilateral nephrectomy. This is also the case when the animals are pretreated with triiodothyronine, 3 x 0.75 mg/kg body weight orally, for 2-3 days. Indole, cresol, putrescine, methylguanidine or acetoine was given intraperitoneally to normal rats at doses of between 5 and 300 mg/kg body weight. Only low single doses of indole (5 mg/kg) reduced oxygen consumption significantly. Single doses of the other substances studied were ineffective even at tenfold higher doses. Some combinations of these substances, however, (10 mg/kg each), reduced the metabolic rate significantly. In contrast to the results in vivo, plasma of uremic rats, as well as the uremic toxins, dissolved in Krebs-phosphate buffer pH 7.4 at concentrations of 30 mg/dl each, had no influence on respiration of rat diaphragma or liver slices in vitro (single substances and different combinations).