Assessment of genotoxic potential of fragrance materials in the chicken egg assays.

The genotoxic and clastogenic/aneugeneic potentials of four α,ß-unsaturated aldehydes, 2-phenyl-2-butenal, nona-2-trans-6-cis-dienal, 2-methyl-2-pentenal, and p-methoxy cinnamaldehyde, which are used as fragrance materials, were assessed using the Chicken Egg Genotoxicity Assay (CEGA) and the Hen's egg micronucleus (HET-MN) assay, respectively. Selection of materials was based on their chemical structures and the results of their previous assessment in the regulatory in vitro and/or in vivo genotoxicity test battery. Three tested materials, 2-phenyl-2-butenal, nona-2-trans-6-cis-dienal, and 2-methyl-2-pentenal, were negative in both, CEGA and HET-MN assays. These findings were congruent with the results of regulatory in vivo genotoxicity assays. In contrast, p-methoxy cinnamaldehyde, which was also negative in the in vivo genotoxicity assays, produced evidence of DNA damage, including DNA strand breaks and DNA adducts in CEGA. However, no increase in the micronucleus formation in blood was reported in the HET-MN study. Such variation in responses between the CEGA and HET-MN assay can be attributed to differences in the dosing protocols. Pretreatment with a glutathione precursor, N-acetyl cysteine, negated positive outcomes produced by p-methoxy cinnamaldehyde in CEGA, indicating that difference in response observed in the chicken egg and rodent models can be attributed to rapid glutathione depletion. Overall, our findings support the conclusion that CEGA and/or HET-MN can be considered as a potential alternative to animal testing as follow-up strategies for assessment of genotoxic potential of fragrance materials with evidence of genotoxicity in vitro.

Crotonaldehyde paralyzes arteries by inducing impairment of ion channels, vascular histiocytic injury, overproduction of reactive oxygen species, mitochondrial damage, and autophagy.

Humans are ubiquitously exposed to crotonaldehyde (CRA) endogenously and exogenously. Deeper knowledge of the pharmacological and toxicological characteristics and the mechanisms of CRA on vasculature is urgently needed for prevention of its harmfulness. The effects of acute and prolonged exposure to CRA were studied in rat isolated arteries and arterial smooth muscle cells (ASMCs). Instant exposure to CRA (1-300 µM) concentration-dependently declined the tension of pre-constricted arteries with an irreversible depression on the contractility. Prolonged exposure of rat coronary arteries (RCAs) to CRA concentration- and time-dependently depressed the arterial contractile responsiveness to various vasoconstrictors including depolarization, U46619, serotonin and Bay K8644 (an agonist of voltage-gated Ca2+ channels (VGCCs)). In fresh RCA ASMCs, CRA abated depolarization-induced elevation of intracellular Ca2+ ([Ca2+]i). Electrophysiological study revealed that acute exposure to CRA depressed the functions of Ca2+-activated Cl- channels (CaCCs), voltage-gated K+ (Kv) channels and inward rectifier K+ (Kir) channels in RCA ASMCs. Prolonged exposure of RCAs to CRA reduced the expressions of these ion channels in RCA ASMCs, disordered tissue frames, injured arterial cells, and increased autophagosomes in both ASMCs and endothelial cells. In rat aortic smooth muscle cells (A7r5), CRA exposure decreased the cell viability, elevated the intracellular levels of reactive oxygen species, reduced the mitochondrial membrane potential, and enhanced autophagy. Taken together, the present study for the first time portrays a clearer panoramic outline of the vascular effects and the mechanisms of CRA on arteries, demonstrates that CRA impairs arterial contractility, depresses VGCCs, CaCCs, Kv channels and Kir channels, reduces cell viability, and destroys the arterial histiocytes, and suggests that excessive oxidative stress, mitochondrial dysfunction and autophagy underlie these vascular damages. These findings are significant for the comprehensive evaluation of the vicious effects of CRA on arteries and suggest potential preventive strategies.

Update to RIFM fragrance ingredient safety assessment, 2-methyl-4-phenylbutyraldehyde, CAS Registry Number 40654-82-8.

The existing information supports the use of this material as described in this safety assessment. 2-Methyl-4-phenylbutyraldehyde was evaluated for genotoxicity, repeated dose toxicity, reproductive toxicity, local respiratory toxicity, photoirritation/photoallergenicity, skin sensitization, and environmental safety. Data from read-across analog ß-methyl-benzenepentanal (CAS # 55,066-49-4) show that 2-methyl-4-phenylbutyraldehyde is not expected to be genotoxic and provide a calculated Margin of Exposure (MOE) > 100 for the repeated dose toxicity endpoint. Data on read-across analog phenylacetaldehyde (CAS # 122-78-1) provide a calculated MOE >100 for the reproductive toxicity endpoint. Data show that there are no safety concerns for 2-methyl-4-phenylbutyraldehyde for skin sensitization under the current declared levels of use. The photoirritation/photoallergenicity endpoints were evaluated based on ultraviolet/visible (UV/Vis) spectra; 2-methyl-4-phenylbutyraldehyde is not expected to be photoirritating/photoallergenic. The local respiratory toxicity endpoint was evaluated using the Threshold of Toxicological Concern (TTC) for a Cramer Class I material, and the exposure to 2-methyl-4-phenylbutyraldehyde is below the TTC (1.4 mg/day). The environmental endpoints were evaluated; 2-methyl-4-phenylbutyraldehyde was found not to be Persistent, Bioaccumulative, and Toxic (PBT) as per the International Fragrance Association (IFRA) Environmental Standards, and its risk quotients, based on its current volume of use (VoU) in Europe and North America (i.e., Predicted Environmental Concentration/Predicted No Effect Concentration [PEC/PNEC]), are <1.

Genetic architecture of the toxicological response to eucalyptol and citronellal in Drosophila melanogaster.

The excessive and indiscriminate use of synthetic insecticides has led to environmental pollution, wildlife destruction, and adverse effects on human health, while simultaneously giving rise to resistance in insect pest populations. This adaptive trait is expressed through various mechanisms, such as changes in the cuticle, heightened activities of detoxifying enzymes, and alterations in the sites of action that reduce their affinity for insecticides. In this context, we associate variation in toxicological response with genomic variation, to identify genetic polymorphisms underlying the different steps of the insect (genotype)-response (phenotype)-insecticide (environment) interaction. Under this framework, our objective was to investigate the genetic factors involved in the toxicological response of D. melanogaster lines when exposed to citronellal and eucalyptol vapors (monoterpenes of plant origin). We quantified KT50 in adult males, representing the time necessary for half of the exposed individuals to be turned upside down (unable to walk or fly). Since the genomes of all lines used are completely sequenced, we perform a Genome Wide Association Study to analyze the genetic underpinnings of the toxicological response. Our investigation enabled the identification of 656 genetic polymorphisms and 316 candidate genes responsible for the overall phenotypic variation. Among these, 162 candidate genes (77.1%) exhibited specificity to citronellal, 45 (21.4%) were specific to eucalyptol, and 3 candidate genes (1.5%) namely CG34345, robo2, and Ac13E, were implicated in the variation for both monoterpenes. These suggest a widespread adaptability in the response to insecticides, encompassing genes influenced by monoterpenes and those orchestrating resistance to the toxicity of these compounds.

Role of covalent modification by hepatic aldehydes in dictamnine-induced liver injury.

Dictamnine is a representative furan-containing hepatotoxic compound. Administration of dictamnine caused acute liver injury in mice and the metabolic activation of furan to reactive epoxy intermediate was responsible for the hepatotoxicity. This study aimed to characterize the protein adduction by endogenous hepatic aldehydes and investigate its role in dictamnine-induced hepatotoxicity. In the liver sample of dictamnine-treated mice, the protein adduction by five aldehydes was characterized as lysine residue-aldehyde adducts using high-resolution UPLC-Q/Orbitrap MS after exhaustive proteolytic digestion. The levels of protein adduct were increased at 2-3 h after the treatment with dictamnine. The formation of protein adduction increased with increasing doses of dictamnine. Inhibition of the bioactivation by CYP3A inhibitor ketoconazole prevented the protein adduction. Treatment with 2,3-dihydro-dictamnine, an analog of dictamnine that was unable to form the epoxy intermediate, did not lead to an increase in protein adduction. Application of aldehyde dehydrogenase-2 activator ALDA-1 or nucleophilic trapping reagent N-acetyl-L-lysine significantly reduced the protein adduction and attenuated dictamnine-induced liver injury without affecting the bioactivation. In conclusion, the metabolic activation of the furan ring of dictamnine resulted in the protein adduction by multiple hepatic aldehydes and the protein modification played a crucial role in dictamnine-induced liver injury.

K6-linked ubiquitylation marks formaldehyde-induced RNA-protein crosslinks for resolution.

Reactive aldehydes are produced by normal cellular metabolism or after alcohol consumption, and they accumulate in human tissues if aldehyde clearance mechanisms are impaired. Their toxicity has been attributed to the damage they cause to genomic DNA and the subsequent inhibition of transcription and replication. However, whether interference with other cellular processes contributes to aldehyde toxicity has not been investigated. We demonstrate that formaldehyde induces RNA-protein crosslinks (RPCs) that stall the ribosome and inhibit translation in human cells. RPCs in the messenger RNA (mRNA) are recognized by the translating ribosomes, marked by atypical K6-linked ubiquitylation catalyzed by the RING-in-between-RING (RBR) E3 ligase RNF14, and subsequently resolved by the ubiquitin- and ATP-dependent unfoldase VCP. Our findings uncover an evolutionary conserved formaldehyde-induced stress response pathway that protects cells against RPC accumulation in the cytoplasm, and they suggest that RPCs contribute to the cellular and tissue toxicity of reactive aldehydes.

Liver enzymes mediate the association between aldehydes co-exposure and hypertriglyceridemia.

Aldehydes are recognized environmental toxicants that may affect lipid metabolism. For instance, acrolein has been found to increase serum triglyceride (TG) levels exclusively. However, it remains unclear whether other aldehydes are also associated with hypertriglyceridemia (HTG), and what mechanisms may be involved. This cross-sectional study analyzed data from the National Health and Nutrition Examination Survey (NHANES, 2013-2014) to identify associations between serum aldehydes, liver enzymes, and HTG. Serum aldehydes included crotonaldehyde (CRAL), propanaldehyde (3AL), butyraldehyde (4AL), pentanaldehyde (5AL), isopentanaldehyde (I5AL), and heptanaldehyde (7AL). Liver enzymes included alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and gamma-glutamyltransferase (GGT). HTG was defined as fasting TG levels ≥ 1.7 mmol/L. Aldehyde co-exposure was quantified using weighted quantile sum (WQS) regression and Bayesian kernel machine regression (BKMR), while mediation analysis was performed to investigate the role of liver enzymes. Among 1474 participants (mean age 38.6 years, male 50.0%), 426 were diagnosed with HTG. 4AL, 5AL, I5AL, and 7AL were shown to be positively associated with HTG (all P values <0.05). Aldehydes co-exposure was also positively associated with HTG (OR 1.706, 95%CI 1.299-2.240), with 5AL contributing the highest weight (35.3%). Furthermore, aldehydes co-exposure showed positive associations with ALT, AST, and GGT (all P values <0.05), and all four liver enzymes were positively associated with HTG (all P values <0.05). Mediation analysis revealed that liver enzymes (ALT, AST, and GGT) may mediate the associations of 5AL and 7AL with HTG (all P values <0.05). This study identified a positive association between aldehyde co-exposure and HTG, which may be partially mediated by liver enzymes.

Rapid identification and monitoring of cooking oil fume-based toxic volatile organic aldehydes in lung tissue for predicting exposure level and cancer risks.

Cooking oil fumes (COFs) comprised of a mixture of cancer-causing volatile organic aldehydes (VOAs), particularly trans, trans-2,4-decadienal (t,t-DDE), 4-hydroxy-hexenal (4-HHE), and 4-hydroxy-nonenal (4-HNE). Monitoring toxic VOAs levels in people exposed to different cooking conditions is vital to predicting the cancer risk. For this purpose, we developed a fast tissue extraction (FaTEx) technique combined with UHPLC-MS/MS to monitor three toxic VOAs in mice lung tissue samples. FaTEx pre-treatment protocol was developed by combining two syringes for extraction and clean-up process. The various procedural steps affecting the FaTEx sample pre-treatment process were optimized to enhance the target VOAs' extraction efficiency from the sample matrix. Under the optimal experimental conditions, results exhibit good correlation coefficient values > 0.99, detection limits were between 0.5-3 ng/g, quantification limits were between 1-10 ng/g, and the matrix effect was <18.1%. Furthermore, the extraction recovery values of the spiked tissue exhibited between 88.9-109.6% with <8.6% of RSD. Cooking oil fume (containing t,t-DDE) treated mice at various time durations were sacrificed to validate the developed technique, and it was found that t,t-DDE concentrations were from 14.8 to 33.8 µg/g. The obtained results were found to be a fast, reliable, and semi-automated sample pre-treatment technique with good extraction efficiency, trace level detection limit, and less matrix effect. Therefore, this method can be applied as a potential analytical method to determine the VOAs in humans exposed to long-term cooking oil fumes.

Comparative evaluation of mutagenic, genotoxic, cytotoxic, and antimicrobial effects of flavour and fragrance aldehydes, ketones, oximes, and oxime ethers.

Despite the large number of odoriferous compounds available, new ones with interesting olfactory characteristics are desired due to their potentially high commercial value. Here, we report for the first time mutagenic, genotoxic, and cytotoxic effects, and antimicrobial properties of low-molecular fragrant oxime ethers, and we compare their properties with corresponding oximes and carbonyl compounds. 24 aldehydes, ketones, oximes, and oxime ethers were evaluated for mutagenic and cytotoxic effects in Ames (using Salmonella typhimurium strains TA 98 with genotype hisD3052, rfa, uvrB, pKM101, and TA100 with genotype hisG46, rfa, uvrB, pKM101, concentration range: 0.0781-40 mg/mL) and MTS (using HEK293T cell line concentration of tested substances: 0.025 mM) assays. Antimicrobial evaluation was carried out against Bacillus cereus (ATCC 10876), Staphylococcus aureus (ATCC 6538), Enterococcus hirae (ATCC 10541), Pseudomonas aeruginosa (ATCC 15442), Escherichia coli (ATCC 10536), Legionella pneumophila (ATCC 33152); Candida albicans (ATCC 10231) and Aspergillus brasiliensis (ATCC 16404) with concentration range of tested substances 9.375 - 2.400 mg/mL. Furthermore, 5 representatives of carbonyl compounds, oximes, and an oxime ether (stemone, buccoxime, citral, citral oxime, and propiophenone oxime O-ethyl ether) were evaluated for genotoxic properties in SOS-Chromotest (concentration range: 7.8·10-5 - 5·10-3 mg/mL). All of the tested compounds did not exhibit mutagenic, genotoxic, or cytotoxic effects. Oximes and oxime ethers showed relevant antimicrobial activity against pathogenic species (P. aeruginosa, S. aureus, E.coli, L. pneumophila, A. brasiliensis, C. albicans) in the MIC range 0.075 - 2.400 mg/mL compared to the common preservative methylparaben with the MIC range 0.400-3.600 mg/mL. Our study shows that oxime ethers have the potential to be used as fragrant agents in functional products.

Lipid peroxide-derived short-chain aldehydes promote programmed cell death in wheat roots under aluminum stress.

Lipid peroxidation is a primary event in plant roots exposed to aluminum (Al) toxicity, which leads to the formation of reactive aldehydes. Current evidence demonstrates that the resultant aldehydes are integrated components of cellular damage in plants. Here, we investigated the roles of aldehydes in mediating Al-induced damage, particularly cell death, using two wheat genotypes with different Al resistances. Aluminum treatment significantly induced cell death, which was accompanied by decreased root activity and cell length. Al-induced cell death displayed granular nuclei and internucleosomal fragmentation of nuclear DNA, suggesting these cells underwent programmed cell death (PCD). During this process, caspase-3-like protease activity was extensively enhanced and showed a significant difference between these two wheat genotypes. Further experiments showed that Al-induced cell death was positively correlated with aldehydes levels. Al-induced representative diagnostic markers for PCD, such as TUNEL-positive nuclei and DNA fragmentation, were further enhanced by the aldehyde donor (E)-2-hexenal, but significantly suppressed by the aldehyde scavenger carnosine. As the crucial executioner of Al-induced PCD, the activity of caspase-3-like protease was further enhanced by (E)-2-hexenal but inhibited by carnosine in wheat roots. These results suggest that reactive aldehydes sourced from lipid peroxidation mediate Al-initiated PCD probably through activating caspase-3-like protease in wheat roots.

Cytotoxicity of a Novel Compound Produced in Foods via the Reaction of Amino Acids with Acrolein along with Formaldehyde.

Acrolein (ACR) and formaldehyde (FA) are toxic aldehydes co-produced in foods. This work found that amino acids, the nucleophiles ubiquitously existing in foods, can react simultaneously with them. Six amino acids, including γ-aminobutyric acid (GABA), glycine, alanine, serine, threonine, and glutamine, can scavenge ACR and FA at 37, 85, and 160 °C. GABA had the highest scavenging capacity for ACR and FA, by 79 and 13% at 37 °C for 2 h, and 99 and 48% at 160 °C for 30 min, respectively. Moreover, a new type of compound with a basic structure of 5-formyl-3-methylene-3,6-dihydropyridin was identified in all reactions and formed by 1 molecule of FA and amino acid and 2 molecules of ACR. The content of this compound was higher than that of free ACR in typical thermally processed foods. Moreover, the compounds produced from different amino acids showed different cytotoxicity values. In gastric epithelial and human intestinal epithelial cell lines, the cytotoxicity values of serine-sourced and threonine-sourced products were lower than that of ACR but higher than that of FA, whereas others had less toxicity compared with the two aldehydes. Considering that the content of serine-sourced products was the highest in almost all tested foods, their safety needs to be evaluated.

Electronic cigarette vaping with aged coils causes acute lung injury in mice.

Electronic cigarettes (e-cigarettes) have been used widely as an alternative to conventional cigarettes and have become particularly popular among young adults. A growing body of evidence has shown that e-cigarettes are associated with acute lung injury and adverse effects in multiple other organs. Previous studies showed that high emissions of aldehydes (formaldehyde and acetaldehyde) in aerosols were associated with increased usage of the same e-cigarette coils. However, the impact on lung function of using aged coils has not been reported. We investigated the relationship between coil age and acute lung injury in mice exposed to experimental vaping for 1 h (2 puffs/min, 100 ml/puff). The e-liquid contains propylene glycol and vegetable glycerin (50:50, vol) only. The concentrations of formaldehyde and acetaldehyde in the vaping aerosols increased with age of the nichrome coils starting at 1200 puffs. Mice exposed to e-cigarette aerosols produced from 1800, but not 0 or 900, puff-aged coils caused acute lung injury, increased lung wet/dry weight ratio, and induced lung inflammation (IL-6, TNF-α, IL-1ß, MIP-2). Exposure to vaping aerosols from 1800 puff-aged coils decreased heart rate, respiratory rate, and oxygen saturation in mice compared to mice exposed to air or aerosols from new coils. In conclusion, we observed that the concentration of aldehydes (formaldehyde and acetaldehyde) increased with repeated and prolonged usage of e-cigarette coils. Exposure to high levels of aldehyde in vaping aerosol was associated with acute lung injury in mice. These findings show significant risk of lung injury associated with prolonged use of e-cigarette devices.

In vitro and in vivo low-dose exposure of simulated cooking oil fumes to assess adverse biological effects.

Cooking oil fumes (COFs) represent a major indoor environmental pollutant and exhibit potent mutagenic or carcinogenic health effects caused by containing various heterocyclic aromatic amines (HAAs) and long-chain aldehydes. Despite some evaluation of the cumulative exposure of COFs to cancer cells under high concentration were evaluated, their biological adverse effects with low-dose exposure to healthy cells had been inadequately investigated. Herein, we firstly scrutinized the three selected typically toxic compounds of heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 3,8-dimethylammidazo[4,5-f]quinoxalin-2-amine (MeIQx) and trans, trans-2,4-decadienal (TDA)) emitted from COFs. In vitro studies revealed that the PhIP, MeIQx and TDA aerosol particles were negligible toxicity to cancer cells (A549 and HepG-2) but strong cytotoxicity to normal healthy cells (HelF and L02) under 0.5-4 µg/mL low dose exposure based on the reactive oxygen species (ROS) mechanism. In vivo studies demonstrated that PhIP caused significant lung and liver damage after exposure to PhIP for 30 days with mice. These results indicated the direct proof of healthy cell damage even at low-dose exposure to HAAs and aldehydes.