Styrene lung cancer risk assessment: an alternative evaluation of human lung cancer risk assuming mouse lung tumors are potentially human relevant and operating by a threshold-based non-genotoxic mode of action.

Rodent inhalation studies indicate styrene is a mouse lung-specific carcinogen. Mode-of-action (MOA) analyses indicate that the lung tumors cannot be excluded as weakly quantitatively relevant to humans due to shared oxidative metabolites detected in rodents and humans. However, styrene also is not genotoxic following in vivo dosing. The objective of this review was to characterize occupational and general population cancer risks by conservatively assuming mouse lung tumors were relevant to humans but operating by a non-genotoxic MOA. Inhalation cancer values reference concentrations for respective occupational and general population exposures (RfCcar-occup and RfCcar-genpop) were derived from initial benchmark dose (BMD) modeling of mouse inhalation tumor dose-response data. An overall lowest BMDL10 of 4.7 ppm was modeled for lung tumors, which was further duration- and dose-adjusted by physiologically based pharmacokinetic (PBPK) modeling to derive RfCcar-occup/genpop values of 6.2 ppm and 0.8 ppm, respectively. With the exception of open-mold fiber reinforced composite workers not using personal protective equipment (PPE), the RfCcar-occup/genpop values are greater than typical occupational and general population human exposures, thus indicating styrene exposures represent a low potential for human lung cancer risk. Consistent with this conclusion, a review of styrene occupational epidemiology did not support a conclusion of an association between styrene exposure and lung cancer occurrence, and further supports a conclusion that the conservatively derived RfCcar-occup is lung cancer protective.

Polymerization of Epoxides at a Static Oil-Alkaline Water Interface.

'On-water' catalysis entails the significant enhancement of a chemical reaction by water, even when those reactions are known to be water-sensitive. Here, the findings about the anionic ring opening polymerization of epoxides at the static interface between oil and alkaline water are shared. Unexpectedly, high molar mass fractions are observed with the interfacial system presented herein, albeit at very low conversions (< 5%). Styrene oxide, a notably unreactive epoxide, is chosen as the model compound to investigate the influence of several reaction parameters (i.e., pH, type of the initiator salt, polymerization time, interfacial area, solvent, shaking) on the polymerization. Poly(styrene oxide) (PSO) with an Mn of 5300 g mol-1 is observed via MALDI-ToF MS, with species of at least 8000 g mol-1. The feasibility of expanding the system to (cyclic) aliphatic and aromatic epoxides, and glycidyl ethers is also explored. The system appears to promote polymerization of epoxides that position at the interface, in such a way that initiation and propagation can occur. A mechanistic interpretation of the interfacial polymerization is suggested. The surprising results obtained in this work urge to revisit the role of water in ionic polymerizations.

Styrene Oxide Isomerase-Catalyzed Meinwald Rearrangement in Cascade Biotransformations: Synthesis of Chiral and/or Natural Chemicals.

Styrene oxide isomerase (SOI) catalyzes the Meinwald rearrangement of aryl epoxides to carbonyl compounds via a 1,2-trans-shift in a stereospecific manner. A number of cascade biotransformations with SOI-catalyzed epoxide isomerization as a key step have been developed to convert readily available substrates into valuable chiral chemicals. Cascade conversion of terminal or internal alkenes into chiral acids, alcohols or amines was achieved, which involved SOI-catalyzed enantio-retentive isomerization of terminal epoxides via 1,2-H shift, or internal epoxides via 1,2-methyl shift. SOI-involved cascades were also developed to convert racemic epoxides into chiral acids or amines via dynamic kinetic resolution. Additionally, combining SOI-catalyzed isomerization with enantioselective C-C bond forming enzymes enabled the synthesis of chiral amino acids or amino alcohols from racemic epoxides. Finally, integration of SOI-involved cascades with biosynthesis pathways allowed for the direct utilization of renewable substrates for the sustainable synthesis of high-value natural chemicals such as alcohols, acids, and esters.

Electrochemically Assisted Cycloaddition of Carbon Dioxide to Styrene Oxide on Copper/Carbon Hybrid Electrodes: Active Species and Reaction Mechanism.

A novel electrochemically assisted cycloaddition process is proposed, in which highly efficient coupling of CO2 with styrene oxide (SO) can be achieved to form styrene carbonate (SC) as a high-value-added product. A series of Cu catalysts with different morphologies and chemical states were fabricated on carbon paper (CP) by using in-situ electrodeposition, and the sample with nano-dendrimer structure was found to exhibit a relatively high activity of 74.8 % SC yield with 92.7 % SO conversion under gentle reaction conditions, thus showing its potential for practical applications. The relatively high electrochemically active surface area and charge transfer ability of dendrimer-like Cu benefited the electrochemical reaction. In particular, the Cu2+ species that were formed in situ during the reaction played a vital role in enhancing the activity and selectivity of the proposed Cu/CP hybrid catalyst. Cu2+ atoms served as active sites that can not only electrochemically activate CO2 but also facilitate the ring opening of SO. Mechanistic analysis suggested that the reaction followed electrochemical and liquid-phase heterogeneous paths, which provide a new green and sustainable route for efficient utilization of CO2 resources for fine chemical electrosynthesis.

Influence of LiTaO3 (0001) and KTaO3 (001) Perovskites Structures on the Molecular Adsorption of Styrene and Styrene oxide: A Theoretical Insight by Periodic DFT Calculations.

In this research, the adsorption of styrene and styrene oxide, both biomass derivatives, on KTaO3 (001) and LiTaO3 (0001) perovskite-like structures was studied from a theoretical point of view. The study was carried out using density functional theory (DFT) calculations. The adsorption phenomenon was deeply studied by calculating the adsorption energies (Eads ), adsorbate-surface distances (Å) and evaluating the differences of charge density and charge transfer (ΔCT). For complexes adsorbed on KTaO3 (TaO2 , KO and K(OH)2 exposed layers), the highest Eads was found for styrene oxide, attributed to the oxygen reactivity of the epoxy group describing a strong interaction with the surface. However, when evaluating a K(O)2 model, a more favorable interaction of styrene with the surface is observed, resulting in a high Eads of -9.9 eV and a ΔCT of 3.1e. For LiTaO3 , more favorable interactions are found for both adsorbates compared to KTaO3 , evidenced by the higher adsorption energies and charge density differences, particularly for the styrene complex adsorbed on TaO2 exposed layer (Eads : -10.2 eV). For the LiO termination, the surface exposed oxygens are fundamental for the adsorption of styrene and styrene oxide, leading to a considerable structural distortion. The obtained results thus provide understanding of the structural features, surface reactivity and adsorption sites of LiTaO3 and KTaO3 perovskite in the context of a heterogeneous catalytic process, such as the oxidation of styrene.

Quercetin alleviates styrene oxide-induced cytotoxicity in cortical neurons in vitro via modulation of oxidative stress and apoptosis.

Styrene 7,8-oxide (SO) is the principal metabolite of styrene, an industrial neurotoxic compound which causes various neurodegenerative disorders. The present study aimed to explore the mechanisms of SO cytotoxicity (0.5 - 4 mM) in primary cortical neurons and to evaluate the neuroprotective potential of quercetin (QUER). Our results showed that exposure to SO decreased viability of cortical neurons in a concentration-dependent manner. In the presence of QUER, cell viability was increased significantly. The neuroprotective effects of QUER were associated with the reduction of intracellular Reactive Oxygen Species (ROS), the decrease in calcium overload and the restoration of mitochondrial membrane depolarization caused by SO. Additionally, to evaluate neuronal death mechanisms triggered by SO, cells were incubated with Ac-DEVD-CHO, Calpeptin and Necrostatin-1, pharmacological inhibitors of caspase-3, calpains and necroptosis respectively. The data showed that the three inhibitors reduced cell death induced by SO and suggested the implication of apoptotic, necrotic and necroptotic pathways. However, western blot analysis showed that QUER attenuated the activation of caspase-3 but did not prevent calpain activity. Taken together, these data indicated that the cytotoxicity of SO was mediated by oxidative stress and apoptosis, necrosis and necroptosis mechanisms, while the neuroprotection provided by QUER against SO depended mainly on its anti-apoptotic activity.

Enzyme-Catalyzed Meinwald Rearrangement with an Unusual Regioselective and Stereospecific 1,2-Methyl Shift.

The Meinwald rearrangement is a synthetically useful reaction but often lacks regioselectivity and stereocontrol. A significant challenge in the Meinwald rearrangement of internal epoxides is the non-regioselective migration of different substituents to give a mixture of products. Herein, an enzyme-catalyzed regioselective and stereospecific 1,2-methyl shift in the Meinwald rearrangement of internal epoxides is reported. Styrene oxide isomerase (SOI) catalyzed the unique isomerization of internal epoxides through 1,2-methyl shift without 1,2-hydride shift to give the corresponding aldehydes and a cyclic ketone as the sole product. SOI-catalyzed isomerization showed high stereospecificity, fully retaining the stereoconfiguration. The synthetic utility of this enzymatic Meinwald rearrangement was demonstrated by its incorporation into three new types of enantioselective cascades, to convert trans-ß-methyl styrenes into the corresponding R-configured alcohols, acids, or amines in high ee and yield.

Utilization of a styrene-derived pathway for 2-phenylethanol production in budding yeast.

2-Phenylethanol (2-PE) is an important flavor ingredient and is widely applied in the fields of food, cosmetics, and pharmaceuticals. Despite that Saccharomyces cerevisiae has the ability to naturally synthesize 2-PE via the Ehrlich pathway, de novo synthesis of 2-PE in high titer still remains a huge challenge. In this study, a non-native styrene degradation pathway was introduced into S. cerevisiae, which represents the first time to demonstrate the functional expression of "styrene-derived" 2-PE synthesis in yeast. Using a host strain engineered with L-phenylalanine (L-Phe) overproduction, the heterologous 2-PE pathway coupled with endogenous Ehrlich pathway produced 233 mg/L 2-PE under shake flasks. Additionally, we further engineered the permease transporters to improve the intracellular L-Phe availability, and further improved the 2-PE titer to 680 mg/L. Taken together, our work represents one of the pioneering reports to explore "styrene-derived" pathway in S. cerevisiae. The synthetic yeast described here might be used as a platform for the future development of next-generation high-yielding 2-PE yeast strains.Key Points• A styrene-derived pathway was established in yeast for 2-phenylethanol productions; membrane-associated styrene oxide isomerase was functional in yeast.• Transporter engineering to improve the L-phenylalanine importation with enhanced 2-phenylethanol productions.

Ring-Opening Polymerization of ε-Caprolactone and Styrene Oxide-CO2 Coupling Reactions Catalyzed by Chelated Dehydroacetic Acid-Imine Aluminum Complexes.

A series of chelated dehydroacetic acid-imine-based ligands L1H~L4H was synthesized by reacting dehydroacetic acid with 2-t-butylaniline, (S)-1-phenyl-ethylamine, 4-methoxylbenzylamine, and 2-(aminoethyl)pyridine, respectively, in moderate yields. Ligands L1H~L4H reacted with AlMe3 in toluene to afford corresponding compounds AlMe2L1 (1), AlMe2L2 (2), AlMe2L3 (3), and AlMe2L4 (4). All the ligands and aluminum compounds were characterized by IR spectra, 1H and 13C NMR spectroscopy. Additionally, the ligands L1H~L4H and corresponding aluminum derivatives 1, 3, and 4 were characterized by single-crystal X-ray diffractometry. The catalytic activities using these aluminum compounds as catalysts for the ε-caprolactone ring-opening polymerization (ROP) and styrene oxide-CO2 coupling reactions were studied. The results show that increases in the reaction temperature and selective solvent intensify the conversions of ε-caprolactone to polycaprolactone. Regarding the coupling reactions of styrene oxide and CO2, the conversion rate is over 90% for a period of 12 h at 90 °C. This strategy dispenses the origination of cyclic styrene carbonates, which is an appealing concern because of the transformation of CO2 into an inexpensive, renewable and easy excess carbon feedstock.

Production of styrene oxide from styrene by a recombinant Escherichia coli with enhanced AcrAB-TolC efflux pump level in an aqueous-organic solvent two-phase system.

The AcrAB-TolC efflux pump is involved in the organic solvent tolerance of Escherichia coli. Most E. coli strains are highly sensitive to organic solvents such as n-hexane and cyclohexane. Here, a recombinant E. coli transformed with an expression plasmid containing acrAB and tolC became tolerant to n-hexane and cyclohexane. The levels of AcrA, AcrB, and TolC in the recombinant increased by 3- to 5-fold compared to those in the control strain without the plasmid for acrAB or tolC. To investigate the usability of the recombinant as a biocatalyst in an aqueous-organic solvent two-phase system, we further introduced xylMA xylene monooxygenase genes from Pseudomonas putida mt-2 into the recombinant and examined the production of styrene oxide from styrene. The resulting recombinant produced 1.8 mg and 1.0 mg styrene oxide mL-1 of medium in a medium overlaid with a 25% volume of n-hexane and cyclohexane containing 10% (wt vol-1) styrene, respectively.

Based on an analysis of mode of action, styrene-induced mouse lung tumors are not a human cancer concern.

Based on 13 chronic studies, styrene exposure causes lung tumors in mice, but no tumor increases in other organs in mice or rats. Extensive research into the mode of action demonstrates the key events and human relevance. Key events are: metabolism of styrene by CYP2F2 in mouse lung club cells to ring-oxidized metabolites; changes in gene expression for metabolism of lipids and lipoproteins, cell cycle and mitotic M-M/G1 phases; cytotoxicity and mitogenesis in club cells; and progression to preneoplastic/neoplastic lesions in lung. Although styrene-7,8-oxide (SO) is a common genotoxic styrene metabolite in in vitro studies, the data clearly demonstrate that SO is not the proximate toxicant and that styrene does not induce a genotoxic mode of action. Based on complete attenuation of styrene short-term and chronic toxicity in CYP2F2 knockout mice and similar attenuation in CYP2F1 (humanized) transgenic mice, limited metabolism of styrene in human lung by CYP2F1, 2 + orders of magnitude lower SO levels in human lung compared to mouse lung, and lack of styrene-related increase in lung cancer in humans, styrene does not present a risk of cancer to humans.

Enantioconvergent hydrolysis of racemic styrene oxide at high concentration by a pair of novel epoxide hydrolases into (R)-phenyl-1,2-ethanediol.

OBJECTIVES: To prepare (R)-phenyl-1,2-ethanediol ((R)-PED) with high enantiomeric excess (ee p) and yield from racemic styrene oxide (rac-SO) at high concentration by bi-enzymatic catalysis. RESULTS: The bi-enzymatic catalysis was designed for enantioconvergent hydrolysis of rac-SO by a pair of novel epoxide hydrolases (EHs), a Vigna radiata EH3 (VrEH3) and a variant (AuEH2A250I) of Aspergillus usamii EH2. The simultaneous addition mode of VrEH3 and AuEH2A250I, exhibiting the highest average turnover frequency (aTOF) of 0.12 g h-1 g-1, was selected, by which rac-SO (10 mM) was converted into (R)-PED with 92.6% ee p and 96.3% yield. Under the optimized reaction conditions: dry weight ratio 14:1 of VrEH3-expressing E. coli/vreh3 to AuEH2A250I-expressing E. coli/Aueh2 A250I and reaction at 20 °C, rac-SO (10 mM) was completely hydrolyzed in 2.3 h, affording (R)-PED with 98% ee p. At the weight ratio 0.8:1 of rac-SO to two mixed dry cells, (R)-PED with 97.4% ee p and 98.7% yield was produced from 200 mM (24 mg/ml) rac-SO in 10.5 h. CONCLUSIONS: Enantioconvergent hydrolysis of rac-SO at high concentration catalyzed by both VrEH3 and AuEH2A250I is an effective method for preparing (R)-PED with high ee p and yield.

Practical two-step synthesis of enantiopure styrene oxide through an optimized chemoenzymatic approach.

Enantiopure styrene oxide (SO) and its derivatives are important building blocks for chiral synthesis. In this study, we developed an attractive "1-pot, 2-step" chemoenzymatic approach for producing enantiopure SO with 100 % theoretical yield. This approach involved asymmetric reduction of α-chloroacetophenone by an alcohol dehydrogenase (ADH; step 1), followed by base-induced ring closure (epoxidation) of enantiopure 2-chloro-1-phenylethanol produced by the ADH (step 2). By-product formation during epoxidation was suppressed to <1 % by adding methyl tert-butyl ether (MTBE) as the second phase. Therefore, with this optimized approach, ADH from Lactobacillus kefir (LkDH) successfully produced 1 M (S)-SO, with 99 % analytical yield and 97.8 % enantiomeric excess (ee). In the preparation of (R)-SO, a semi-rational strategy of active pocket iterative saturation mutagenesis (ISM) was successfully used to inverse the enantioselectivity of LkDH (muDH2, F147L/Y190P/A202F/M206H/V196L/S96D/K97V), which produced the opposite enantiomer (R)-2-chloro-1-phenylethanol. Through the optimized chemoenzymatic approach, muDH2 was successfully used to prepare 1 M (R)-SO, with 98.1 % ee and 99.0 % analytical yield. Our results indicated that this optimized chemoenzymatic approach could be used to produce both enantiomers of SO at concentrations as high as 120 g/L within 14 h, which is the highest concentration as far as we know. MuDH2 obtained through ISM also showed reversed enantioselectivity toward another 13 aromatic ketones, compared with wild-type (WT) LkDH. Furthermore, a molecular docking experiment demonstrated that muDH2 inverted the binding orientation of the substrate, which may be the reason for its inverse enantioselectivity.

Efficient biosynthesis of (R)-mandelic acid from styrene oxide by an adaptive evolutionary Gluconobacter oxydans STA.

BACKGROUND: (R)-mandelic acid (R-MA) is a highly valuable hydroxyl acid in the pharmaceutical industry. However, biosynthesis of optically pure R-MA remains significant challenges, including the lack of suitable catalysts and high toxicity to host strains. Adaptive laboratory evolution (ALE) was a promising and powerful strategy to obtain specially evolved strains. RESULTS: Herein, we report a new cell factory of the Gluconobacter oxydans to biocatalytic styrene oxide into R-MA by utilizing the G. oxydans endogenous efficiently incomplete oxidization and the epoxide hydrolase (SpEH) heterologous expressed in G. oxydans. With a new screened strong endogenous promoter P12780, the production of R-MA was improved to 10.26 g/L compared to 7.36 g/L of using Plac. As R-MA showed great inhibition for the reaction and toxicity to cell growth, adaptive laboratory evolution (ALE) strategy was introduced to improve the cellular R-MA tolerance. The adapted strain that can tolerate 6 g/L R-MA was isolated (named G. oxydans STA), while the wild-type strain cannot grow under this stress. The conversion rate was increased from 0.366 g/L/h of wild type to 0.703 g/L/h by the recombinant STA, and the final R-MA titer reached 14.06 g/L. Whole-genome sequencing revealed multiple gene-mutations in STA, in combination with transcriptome analysis under R-MA stress condition, we identified five critical genes that were associated with R-MA tolerance, among which AcrA overexpression could further improve R-MA titer to 15.70 g/L, the highest titer reported from bulk styrene oxide substrate. CONCLUSIONS: The microbial engineering with systematic combination of static regulation, ALE, and transcriptome analysis strategy provides valuable solutions for high-efficient chemical biosynthesis, and our evolved G. oxydans would be better to serve as a chassis cell for hydroxyl acid production.

Evaluation of DNA adduct damage using G-quadruplex-based DNAzyme.

Toxicity assessment is a major problem in pharmaceutical candidates and industry chemicals development. However, due to the lack of practical analytical methods for DNA adduct analysis, the safety evaluation of drug and industry chemicals was severely limited. Here, we develop a DNAzyme-based method to detect DNA adduct damage for toxicity assessment of drugs and chemicals. Among 18 structural variants of G4 DNAzyme, EA2 DNAzyme exhibits an obvious DNA damaging effect of styrene oxide (SO) due to its unstable structure. The covalent binding of SO to DNAzyme disrupts the Hoogsteen hydrogen bonding sites of G-plane guanines and affects the formation of the G4 quadruplex. DNA damage chemicals reduce the peroxidase activity of the G4 DNAzyme to monitor the DNA adduct damage by disrupting the structural integrity of the G4 DNAzyme. Our method for genotoxic assessment of pharmaceutical candidates and industrial chemicals can elucidate the complex chemical pathways leading to toxicity, predict toxic effects of chemicals, and evaluate possible risks to human health.

Is styrene competitive for dopamine receptor binding?

The potential role of styrene oxide in altering the dopaminergic pathway in the ear is investigated by means of molecular docking and molecular dynamics simulations. We estimate the binding affinity of both styrene oxide and dopamine to the dopaminergic receptor DrD2 by computing the free-energy difference, ∆G, between the configuration where the ligand is bound to the receptor and the situation in which it is "infinitely" far away from it. The results show that the styrene oxide has a somewhat lower affinity for binding with respect to dopamine, which, however, may not be enough to prevent exogenous high concentration styrene oxide to compete with endogenous dopamine for DrD2 binding.

High-Performance Styrene Epoxidation with Vacancy-Defect Cobalt Single-Atom Catalysts.

Exploring highly active and cost-effective catalysts for styrene epoxidation is of great significance, but it remains challenging to simultaneously achieve excellent conversion and selectivity toward styrene oxide. In this work, the structures and performance of Co, Fe, and Cu single-atom catalysts (SACs) in styrene epoxidation with tert-butyl hydroperoxide (TBHP) are predicted using density functional theory (DFT) calculations. The results reveal that the Co-N structure prefers that of styrene oxide over Fe-N and Cu-N structures. This predicted result is verified via catalytic evaluations, where the Co SACs displayed significantly higher styrene oxide selectivity than Fe and Cu SACs. Moreover, the activity of Co SAC can be further improved by the construction of unsaturated vacancy-defect cobalt single sites. As a result, excellent performance with styrene conversion of 99.9% and styrene oxide selectivity of 71% is achieved after a reaction time of 8 h on the optimal Co SAC.

Engineering a Synthetic Pathway for Tyrosol Synthesis in Escherichia coli.

Tyrosol is an aromatic compound with great value that is widely used in the food and pharmaceutical industry. In this study, we reported a synthetic pathway for converting p-coumaric acid (p-CA) into tyrosol in Escherichia coli. We found that the enzyme cascade comprising ferulic acid decarboxylase (FDC1) from Saccharomyces cerevisiae, styrene monooxygenase (SMO), styrene oxide isomerase (SOI) from Pseudomonas putida, and phenylacetaldehyde reductase (PAR) from Solanum lycopersicum could efficiently synthesize tyrosol from p-CA with a conversion rate over 90%. To further expand the range of substrates, we also introduced tyrosine ammonia-lyase (TAL) from Flavobacterium johnsoniae to connect the synthetic pathway with the endogenous l-tyrosine metabolism. We found that tyrosol could be efficiently produced from glycerol, reaching 545.51 mg/L tyrosol in a tyrosine-overproducing strain under shake flasks. In summary, we have established alternative routes for tyrosol synthesis from p-CA (a potential lignin-derived biomass), glucose, and glycerol.

Promotional effect of Mn-doping on the catalytic performance of NiO sheets for the selective oxidation of styrene.

The direct oxidation of styrene into high-value chemicals under mild reaction conditions remains a great challenge in both academia and industry. Herein, we report a successful electronic structure modulation of intrinsic NiO sheets via Mn-doping towards the oxidation of styrene. By doping NiO with only a small content of Mn (Mn/Ni atomic ratio of 0.030), a 75.0% yield of STO can be achieved under the optimized reaction conditions, which is 2.13 times higher than that of the pure NiO. In addition, the catalyst exhibits robust stability and good recycling performance. The performance enhancement originates from the synergistic effect regarding the abundant Ni(II) species, the rich oxygen vacancy sites and the large amount of surface redox centers. This work provides new findings of the elemental-doping-induced multifunctionality in designing powerful catalysts for the efficient and selective oxidation of styrene and beyond.