Nonsterile Process for Biohydrogen Production: Recent Updates, Challenges, and Opportunities.

Hydrogen (H2), a clean and versatile energy carrier, has recently gained significant attention as a potential solution for reducing carbon emissions and promoting sustainable energy systems. The yield and efficiency of the biological H2 production process primarily depend on sterilization conditions. Various strategies, such as heat inactivation and membrane-based sterilization, have been used to achieve desirable yields via microbial fermentation. Almost every failed biotransformation process is linked to nonsterile conditions at any reaction stage. Therefore, the production of renewable biofuels as alternatives to fossil fuels is more attractive. Pure sugars have been widely documented as a costly feedstock for H2 production under sterile conditions. Biotransformation under nonsterile conditions is more desirable for stable and sustainable operation. Low-cost feeds, such as biowaste, are considered suitable alternatives, but they require appropriate sterilization to overcome the limitations of inherited or contaminating microbes during H2 production. This article describes the status of microbial fermentative processes for H2 production under nonsterile conditions and discusses strategies to improve such processes for sustainable, cleaner production.

Fugacity- and biotransformation-based mechanistic insights into the trophic transfer of organophosphate flame retardants in a subtropical coastal food web from the Northern Beibu Gulf of China.

The bioaccumulation and trophic transfer of organophosphate flame retardants (OPFRs) in marine ecosystems have attracted great attention in recent research, but our understanding of the trophic transfer mechanisms involved is limited. In this study, we investigated the trophodynamics of OPFRs and their metabolites in a subtropical coastal food web collected from the northern Beibu Gulf, China, and characterized their trophodynamics using fugacity- and biotransformation-based approaches. Eleven OPFRs and all seven metabolites were simultaneously quantified in the shellfish, crustacean, pelagic fish, and benthic fish samples, with total concentrations ranging from 164 to 4.11 × 104 and 4.56-4.28 × 103 ng/g lipid weight, respectively. Significant biomagnification was observed only for tris (phenyl) phosphate (TPHP) and tris (2-ethylhexyl) phosphate (TEHP), while other compounds except for tris(2-chloroethyl) phosphate (TCEP) displayed biomagnification trends based on Monte Carlo simulations. Using a fugacity-based approach to normalize the accumulation of OPFRs in biota to their relative biological phase composition, storage lipid is the predominant biological phase for the mass distribution of 2-ethylhexyl diphenyl phosphate (EHDPHP) and TPHP. The water content and structure protein are equally important for TCEP, whereas lipid and structure protein are the two most important phases for other OPFRs. The mass distribution of these OPFRs along with TLs can explain their trophodynamics in the food web. The organophosphate diesters (as OPFR metabolites) also displayed biomagnification trends based on bootstrapped estimation. The correlation analysis and Korganism-water results jointly suggested the metabolites accumulation in high-TL organisms was related to biotransformation processes. The metabolite-backtracked trophic magnification factors for tri-n­butyl phosphate (TNBP) and TPHP were both greater than the values that accounted for only the parent compounds. This study highlights the incorporation of fugacity and biotransformation analysis to characterize the trophodynamic processes of OPFRs and other emerging pollutants in food webs.

A novel univariate interpolation and bivariate regression hybrid method application to biodegradation of bisphenol A diglycidyl ether using laccases from Geobacillus stearothermophilus and Geobacillus thermoparafinivorans strains.

Bisphenol A diglycidyl ether (BADGE), a derivative of the well-known endocrine disruptor Bisphenol A (BPA), is a potential threat to long-term environmental health due to its prevalence as a micropollutant. This study addresses the previously unexplored area of BADGE toxicity and removal. We investigated, for the first time, the biodegradation potential of laccase isolated from Geobacillus thermophilic bacteria against BADGE. The laccase-mediated degradation process was optimized using a combination of response surface methodology (RSM) and machine learning models. Degradation of BADGE was analyzed by various techniques, including UV-Vis spectrophotometry, high-performance liquid chromatography (HPLC), Fourier transform infrared (FTIR) spectroscopy, and gas chromatography-mass spectrometry (GC-MS). Laccase from Geobacillus stearothermophilus strain MB600 achieved a degradation rate of 93.28% within 30 min, while laccase from Geobacillus thermoparafinivorans strain MB606 reached 94% degradation within 90 min. RSM analysis predicted the optimal degradation conditions to be 60 min reaction time, 80°C temperature, and pH 4.5. Furthermore, CB-Dock simulations revealed good binding interactions between laccase enzymes and BADGE, with an initial binding mode selected for a cavity size of 263 and a Vina score of -5.5, which confirmed the observed biodegradation potential of laccase. These findings highlight the biocatalytic potential of laccases derived from thermophilic Geobacillus strains, notably MB600, for enzymatic decontamination of BADGE-contaminated environments.

Optimization of biotransformation processes of Camarosporium laburnicola to improve production yields of potent telomerase activators.

BACKGROUND: Telomerase activators are promising agents for the healthy aging process and the treatment/prevention of short telomere-related and age-related diseases. The discovery of new telomerase activators and later optimizing their activities through chemical and biological transformations are crucial for the pharmaceutical sector. In our previous studies, several potent telomerase activators were discovered via fungal biotransformation, which in turn necessitated optimization of their production. It is practical to improve the production processes by implementing the design of experiment (DoE) strategy, leading to increased yield and productivity. In this study, we focused on optimizing biotransformation conditions utilizing Camarosporium laburnicola, a recently discovered filamentous fungus, to afford the target telomerase activators (E-CG-01, E-AG-01, and E-AG-02). RESULTS: DoE approaches were used to optimize the microbial biotransformation processes of C. laburnicola. Nine parameters were screened by Plackett-Burman Design, and three significant parameters (biotransformation time, temperature, shaking speed) were optimized using Central Composite Design. After conducting validation experiments, we were able to further enhance the production yield of target metabolites through scale-up studies in shake flasks (55.3-fold for E-AG-01, 13-fold for E-AG-02, and 1.96-fold for E-CG-01). CONCLUSION: Following a process optimization study using C. laburnicola, a significant increase was achieved in the production yields. Thus, the present study demonstrates a promising methodology to increase the production yield of potent telomerase activators. Furthermore, C. laburnicola is identified as a potential biocatalyst for further industrial utilization.

Biochemical characterization, biosynthesis mechanism, and functional evaluation of selenium-enriched Aspergillus oryzae A02.

The synthesis mechanisms and function evaluation of selenium(Se)-enriched microorganism remain relatively unexplored. This study unveils that total Se content within A. oryzae A02 mycelium soared to an impressive 8462 mg/kg DCW, surpassing Se-enriched yeast by 2-3 times. Selenium exists in two predominant forms within A. oryzae A02: selenoproteins (SeMet 32.1 %, SeCys 14.4 %) and selenium nanoparticles (SeNPs; 53.5 %). The extensive quantitative characterization of the elemental composition, surface morphology, and size of SeNPs on A. oryzae A02 mycelium significantly differs from those reported for other microorganisms. Comparative RNA-Seq analysis revealed the upregulation of functional genes implicated in selenium transformation, activating multiple potential pathways for selenium reduction. The assimilatory and dissimilatory reductions of Se oxyanions engaged numerous parallel and interconnected pathways, manifesting a harmonious equilibrium in overall Se biotransformation in A. oryzae A02. Furthermore, selenium-enriched A. oryzae A02 was observed to primarily upregulate peroxisome activity while downregulating estrogen 2-hydroxylase activity in mice hepatocytes, suggesting its potential in fortifying antioxidant physiological functions and upholding metabolic balance.

Strong heterologous electron sink outcompetes alternative electron transport pathways in photosynthesis.

Improvement of photosynthesis requires a thorough understanding of electron partitioning under both natural and strong electron sink conditions. We applied a wide array of state-of-the-art biophysical and biochemical techniques to thoroughly investigate the fate of photosynthetic electrons in the engineered cyanobacterium Synechocystis sp. PCC 6803, a blueprint for photosynthetic biotechnology, expressing the heterologous gene for ene-reductase, YqjM. This recombinant enzyme catalyses the reduction of an exogenously added substrate into the desired product by utilising photosynthetically produced NAD(P)H, enabling whole-cell biotransformation. Through coupling the biotransformation reaction with biophysical measurements, we demonstrated that the strong artificial electron sink, outcompetes the natural electron valves, the flavodiiron protein-driven Mehler-like reaction and cyclic electron transport. These results show that ferredoxin-NAD(P)H-oxidoreductase is the preferred route for delivering photosynthetic electrons from reduced ferredoxin and the cellular NADPH/NADP+ ratio as a key factor in orchestrating photosynthetic electron flux. These insights are crucial for understanding molecular mechanisms of photosynthetic electron transport and harnessing photosynthesis for sustainable bioproduction by engineering the cellular source/sink balance. Furthermore, we conclude that identifying the bioenergetic bottleneck of a heterologous electron sink is a crucial prerequisite for targeted engineering of photosynthetic biotransformation platforms.

Oral Bioavailability, Tissue Distribution, Metabolism, and Excretion of Panduratin A from Boesenbergia rotunda Extract in Healthy Rats.

Background: Our previous studies in vitro and in vivo have shown anti-severe acute respiratory syndrome coronavirus 2 activity of fingerroot extract (Boesenbergia rotunda) and its phytochemical panduratin A. Aim of Study: Therefore, the objective of this study was to determine the pharmacokinetic profiles of panduratin A, as a pure compound and in fingerroot extract, in rats. Materials and Methods: Male rats were randomly divided into four groups. Rats underwent intravenous administration of 4.5 mg/kg panduratin A, a single oral administration of 45 mg/kg panduratin A, or a multiple oral administration of 45 mg/kg panduratin A-consisted fingerroot extract for 7 consecutive days. The concentrations of panduratin A in plasma, tissues, and excreta were measured by using LCMS with a validated method. Results: The rats showed no change in health status after receiving all test preparations. The absolute oral bioavailability of panduratin A administered as pure panduratin A and fingerroot extract were approximately 9% and 6%, respectively. The peak concentrations for the single oral doses of 45 mg/kg panduratin A and fingerroot extract, were 4833 ± 659 and 3269 ± 819 µg/L, respectively. Panduratin A was mostly distributed in gastrointestinal organs, with the highest tissue-to-plasma ratio in the stomach. Approximately 20-30% of unchanged panduratin A from the administered dose was detected in feces while a negligible amount was found in urine. The major metabolites of administered panduratin A were identified in feces as oxidation and dioxidation products. Conclusion: Panduratin A from fingerroot extract showed low oral bioavailability, good tissue distribution, and partially biotransformed before excretion via feces. These findings will assist in developing fingerroot extract as a phytopharmaceutical product for COVID-19 treatment.

Sequential Anaerobic-Aerobic Treatment Enhances Sulfamethoxazole Removal: From Batch Cultures to Observations in a Large-Scale Wastewater Treatment Plant.

Sulfamethoxazole (SMX) passes through conventional wastewater treatment plants (WWTPs) mainly unaltered. Under anoxic conditions sulfate-reducing bacteria can transform SMX but the fate of the transformation products (TPs) and their prevalence in WWTPs remain unknown. Here, we report the anaerobic formation and aerobic degradation of SMX TPs. SMX biotransformation was observed in nitrate- and sulfate-reducing enrichment cultures. We identified 10 SMX TPs predominantly showing alterations in the heterocyclic and N4-arylamine moieties. Abiotic oxic incubation of sulfate-reducing culture filtrates led to further degradation of the major anaerobic SMX TPs. Upon reinoculation under oxic conditions, all anaerobically formed TPs, including the secondary TPs, were degraded. In samples collected at different stages of a full-scale municipal WWTP, anaerobically formed SMX TPs were detected at high concentrations in the primary clarifier and digested sludge units, where anoxic conditions were prevalent. Contrarily, their concentrations were lower in oxic zones like the biological treatment and final effluent. Our results suggest that anaerobically formed TPs were eliminated in the aerobic treatment stages, consistent with our observations in batch biotransformation experiments. More generally, our findings highlight the significance of varying redox states determining the fate of SMX and its TPs in engineered environments.

Preparation of Pinoresinol and Dehydrodiconiferyl Alcohol from Eucommiae Cortex Extract by Fermentation with Traditional Mucor.

Eucommiae Cortex (EC) is frequently used alone or in combination with other active ingredients to treat a range of illnesses. An efficient technical instrument for changing cheap or plentiful organic chemicals into rare or costly counterparts is biotransformation. It combines EC with biotransformation techniques with the aim of producing some novel active ingredients, using different strains of bacteria that were introduced to biotransform EC in an aseptic environment. The high-quality strains were screened for identification after the fermentation broth was found using HPLC, and the primary unidentified chemicals were separated and purified in order to be structurally identified. Strain 1 was identified as Aspergillus niger and strain 2 as Actinomucor elegans; the main transformation product A was identified as pinoresinol (Pin) and B as dehydrodiconiferyl alcohol (DA). The biotransformation of EC utilizing Aspergillus niger and Actinomucor elegans is reported for the first time in this study's conclusion, resulting in the production of Pin and DA.

Biological Activities of Novel Oleanolic Acid Derivatives from Bioconversion and Semi-Synthesis.

Oleanolic acid (OA) is a vegetable chemical that is present naturally in a number of edible and medicinal botanicals. It has been extensively studied by medicinal chemists and scientific researchers due to its biological activity against a wide range of diseases. A significant number of researchers have synthesized a variety of analogues of OA by modifying its structure with the intention of creating more potent biological agents and improving its pharmaceutical properties. In recent years, chemical and enzymatic techniques have been employed extensively to investigate and modify the chemical structure of OA. This review presents recent advancements in medical chemistry for the structural modification of OA, with a special focus on the biotransformation, semi-synthesis and relationship between the modified structures and their biopharmaceutical properties.

Monitoring iohexol and its transformation products as evidence of reclaimed water irrigation input to contiguous waterbodies.

Irrigation with reclaimed water alleviates water supply shortages, but excess application often results in impairment of contiguous waterbodies. This project investigated the potential use of iohexol, an iodinated contrast media used in medical imaging, together with its bio- and phototransformation products as unique reconnaissance markers of reclaimed water irrigation intrusion at three golf courses within the state of Florida. Inter-facility iohexol concentrations measured in reclaimed waters ranged over ~2 orders of magnitude while observed intra-facility seasonal differences were ≤1 order of magnitude. A ~50 % reduction in iohexol was observed post-disinfection for reclaimed water facilities utilizing UV light while none was observed with use of chlorine. Iohexol biotransformation products were observed to decline or shift to lower molecular weight compounds when exposed to UV light but not during disinfection using chlorine. Iohexol biotransformation products were observed in most of the samples but were more prevalent in samples collected during the dry season. Much fewer iohexol phototransformation products were observed in chlorinated reclaimed water, and they were only observed in UV light irradiated reclaimed water when the pre-disinfectant iohexol concentration was ≥5000 ng/L or from solar exposure of reclaimed water spiked with 10 µM of iohexol. For the Hillsborough golf course overlaying an aquifer, the groundwater did not contain iohexol or phototransformation products but did contain biotransformation products. It is not known if these biotransformation products are from active or historical intrusion. The additional presence of sucralose in the aquifer suggests that intrusion has occurred within the past 3 years. This study demonstrates three crucial points in attempting to utilize iohexol to denote reclaimed water intrusion from irrigation overapplication: (1) interpretable results are obtained when iohexol concentrations in the reclaimed water employed for irrigation are ≥1000 ng/L, with higher concentrations in the range of ≥5000 ng/L better able to meet analytical sensitivity requirements after further dilution or degradation in the environment; (2) it is beneficial to assess iohexol transformation products in tandem with iohexol monitoring to account for environmental transformations of iohexol during storage and transport to the receiving water of concern; and (3) inclusion of monitoring for sucralose, an artificial sweetener ubiquitous in wastewater sources that is comparatively stable in the environment, can aid in interpretating whether reclaimed water intrusion based on identification of iohexol and transformation products in the receiving water is attributable to historic or ongoing irrigation overapplications.

Biotransformation of hydrocortisone succinate with whole cell cultures of Monascus purpureus and Cunninghamella echinulate.

Hydrocortisone succinate (1) is a synthetic anti-inflammatory drug and key intermediate in the synthesis of other steroidal drugs. This work is based on the fungal biotransformation of 1, using Monascus purpureus and Cunninghamella echinulata strains. Comopound 1 was transformed into four metabolites, identified as hydrocortisone (2), 11ß-hydroxyandrost-4-en-3,17-dione (3), Δ1-cortienic acid (4), and hydrocortisone-17-succinate (5), obtained through side chain cleavage, hydrolysis, dehydrogenation, and oxidation reactions. These compounds have previously been synthesized either chemically or enzymatically from different precursors. Though this is not the first report on the biotransformation of 1, but it obviously is a first, where the biotransformed products of compound 1 have been characterized structurally with the help of modern spectroscopic techniques. It is noteworthy that these products have already shown biological potential, however a more thorough investigation of the anti-inflammatory properties of these metabolites would be of high value. These results not only emphasize upon the immense potential of biotransformation in catalysis of reactions, otherwise not-achievable chemically, but also holds promise for the development of novel anti-inflammatory compounds.

D-allulose 3-epimerase for low-calorie D-allulose synthesis: microbial production, characterization, and applications.

D-allulose, an epimer of D-fructose at C-3 position, is a low-calorie rare sugar with favorable physiochemical properties and special physiological functions, which displays promising perspectives in the food and pharmaceutical industries. Currently, D-allulose is extremely sparse in nature and is predominantly biosynthesized through the isomerization of D-fructose by D-allulose 3-epimerase (DAEase). In recent years, D-allulose 3-epimerase as the key biocatalyst for D-allulose production has received increasing interest. The current review begins by providing a summary of D-allulose regarding its characteristics and applications, as well as different synthesis pathways dominated by biotransformation. Then, the research advances of D-allulose 3-epimerase are systematically reviewed, focusing on heterologous expression and biochemical characterization, crystal structure and molecular modification, and application in D-allulose production. Concerning the constraint of low yield of DAEase for industrial application, this review addresses the various attempts made to promote the production of DAEase in different expression systems. Also, various strategies have been adopted to improve its thermotolerance and catalytic activity, which is mainly based on the structure-function relationship of DAEase. The application of DAEase in D-allulose biosynthesis from D-fructose or low-cost feedstocks through single- or multi-enzymatic cascade reaction has been discussed. Finally, the prospects for related research of D-allulose 3-epimerase are also proposed, facilitating the industrialization of DAEase and more efficient and economical bioproduction of D-allulose.

Improving sunflower growth and arsenic bioremediation in polluted environments: Insights from ecotoxicology and sustainable mitigation approaches.

The issue of arsenic (As) contamination in the environment has become a critical concern, impacting both human health and ecological equilibrium. Addressing this challenge requires a comprehensive strategy encompassing water treatment technologies, regulatory measures for industrial effluents, and the implementation of sustainable agricultural practices. In this study, diverse strategies were explored to enhance As accumulation in the presence of Acinetobacter bouvetii while safeguarding the host from the toxic effects of arsenate exposure. The sunflower seedlings associated with A. bouvetii demonstrated a favorable relative growth rate (RGR) and net assimilation rate (NAR) even less than 100 ppm of As stress. Remarkably, the NAR and RGR of A. bouvetii-associated seedlings outperformed those of control seedlings cultivated without A. bouvetii in As-free conditions. Additionally, a markedly greater buildup of bio-transformed As was observed in A. bouvetii-associated seedlings (P = 0.05). An intriguing observation was the normal levels of reactive oxygen species (ROS) in A. bouvetii-associated seedlings, along with elevated activities of key enzymatic antioxidants like catalases (CAT), ascorbate peroxidase (APX), superoxide dismutase (SOD), and peroxidases (POD), along with non-enzymatic antioxidants (phenols and flavonoids). This coordinated antioxidant defense system likely contributed to the improved survival and growth of the host plant species amidst As stress. A. bouvetii not only augmented the growth of the host plants but also facilitated the uptake of bio-transformed As in the contaminated medium. The rhizobacterium's modulation of various biochemical and physiological parameters indicates its role in ensuring the better survival and progression of the host plants under As stress.

Structural diversification of vitamin D using microbial biotransformations.

Vitamin D deficiencies are linked to multiple human diseases. Optimizing its synthesis, physicochemical properties, and delivery systems while minimizing side effects is of clinical relevance and is of great medical and industrial interest. Biotechnological techniques may render new modified forms of vitamin D that may exhibit improved absorption, stability, or targeted physiological effects. Novel modified vitamin D derivatives hold promise for developing future therapeutic approaches and addressing specific health concerns related to vitamin D deficiency or impaired metabolism, such as avoiding hypercalcemic effects. Identifying and engineering key enzymes and biosynthetic pathways involved, as well as developing efficient cultures, are therefore of outmost importance and subject of intense research. Moreover, we elaborate on the critical role that microbial bioconversions might play in the a la carte design, synthesis, and production of novel, more efficient, and safer forms of vitamin D and its analogs. In summary, the novelty of this work resides in the detailed description of the physiological, medical, biochemical, and epidemiological aspects of vitamin D supplementation and the steps towards the enhanced and simplified industrial production of this family of bioactives relying on microbial enzymes. KEY POINTS: • Liver or kidney pathologies may hamper vitamin D biosynthesis • Actinomycetes are able to carry out 1α- or 25-hydroxylation on vitamin D precursors.

Utilization of diverse probiotics to create human health promoting fatty acids: A review.

Many probiotics produce functional lipids with health-promoting properties, such as short-chain fatty acids, linoleic acid and omega-3 fatty acids. They have been shown to maintain gut health, strengthen the intestinal barrier, and have anti-inflammatory and antioxidant effects. In this article, we provide an up-to-date review of the various functional lipids produced by probiotics. These probiotics can be incorporated into foods, supplements, or pharmaceuticals to produce these functional lipids in the human colon, or they can be used in industrial biotechnology processes to generate functional lipids, which are then isolated and used as ingredients. We then highlight the different physiological functions for which they may be beneficial to human health, in addition to discussing some of the challenges of incorporating probiotics into commercial products and some potential solutions to address these challenges. Finally, we highlight the importance of testing the efficacy and safety of the new generation of probiotic-enhanced products, as well as the great potential for the marketization of related products.

Exploring the interactions of JAK inhibitor and S1P receptor modulator drugs with the human gut microbiome: Implications for colonic drug delivery and inflammatory bowel disease.

The gut microbiota is a complex ecosystem, home to hundreds of bacterial species and a vast repository of enzymes capable of metabolising a wide range of pharmaceuticals. Several drugs have been shown to affect negatively the composition and function of the gut microbial ecosystem. Janus Kinase inhibitors and Sphingosine-1-phosphate receptor modulators are drugs recently approved for inflammatory bowel disease through an immediate release formulation and would potentially benefit from targeted colonic targeted delivery to enhance the local drug concentration at the diseased site. However, their impact on the human gut microbiota and susceptibility to bacterial metabolism remain unexplored. With the use of calorimetric, optical density measurements, and metagenomics next-generation sequencing, we show that JAK inhibitors have a minor impact on the composition of the human gut microbiota, while ozanimod exerts a significant antimicrobial effect, leading to a prevalence of the Enterococcus genus and a markedly different metabolic landscape when compared to the untreated microbiota. Moreover, ozanimod is the only drug subject to enzymatic degradation by the human gut microbiota sourced from six healthy donors.. Overall, given the crucial role of the gut microbiome in health, screening assays to investigate the interaction of drugs with the microbiome should be encouraged for the pharmaceutical industry as a standard in the drug discovery and development process.

Resistance towards and biotransformation of a Pseudomonas-produced secondary metabolite during community invasion.

The role of antagonistic secondary metabolites produced by Pseudomonas protegens in suppression of soil-borne phytopathogens has been clearly documented. However, their contribution to the ability of P. protegens to establish in soil and rhizosphere microbiomes remains less clear. Here, we use a four-species synthetic community (SynCom) in which individual members are sensitive towards key P. protegens antimicrobial metabolites (DAPG, pyoluteorin, and orfamide A) to determine how antibiotic production contributes to P. protegens community invasion and to identify community traits that counteract the antimicrobial effects. We show that P. protegens readily invades and alters the SynCom composition over time, and that P. protegens establishment requires production of DAPG and pyoluteorin. An orfamide A-deficient mutant of P. protegens invades the community as efficiently as wildtype, and both cause similar perturbations to community composition. Here, we identify the microbial interactions underlying the absence of an orfamide A mediated impact on the otherwise antibiotic-sensitive SynCom member, and show that the cyclic lipopeptide is inactivated and degraded by the combined action of Rhodococcus globerulus D757 and Stenotrophomonas indicatrix D763. Altogether, the demonstration that the synthetic community constrains P. protegens invasion by detoxifying its antibiotics may provide a mechanistic explanation to inconsistencies in biocontrol effectiveness in situ.

Mass spectrometry-based metabolomics study of nicotine exposure in THP-1 monocytes.

The tobacco alkaloid nicotine is known for its activation of neuronal nicotinic acetylcholine receptors. Nicotine is consumed in different ways such as through conventional smoking, e-cigarettes, snuff or nicotine pouches. The use of snuff has been associated with several adverse health effects, such as inflammatory reactions of the oral mucosa and oral cavity cancer. We performed a metabolomic analysis of nicotine-exposed THP-1 human monocytes. Cells were exposed to 5 mM of the alkaloid for up to 4 h, and cell extracts and medium subjected to untargeted liquid chromatography high-resolution mass spectrometry. Raw data processing revealed 17 nicotine biotransformation products. Among these, cotinine and nornicotine were identified as the two major cellular biotransformation products. The application of multi- and univariate statistical analyses resulted in the annotation, up to a certain level of identification, of 12 compounds in the cell extracts and 13 compounds in the medium that were altered by nicotine exposure. Of these, four were verified as methylthioadenosine, cytosine, uric acid, and L-glutamate. Methylthioadenosine levels were affected in both cells and the medium, while cytosine, uric acid, and L-glutamate levels were affected in the medium only. The effects of smoking on the pathways involving these metabolites have been previously demonstrated in humans. Most of the other discriminating compounds, which were merely tentatively or not fully identified, were amino acids or amino acid derivatives. In conclusion, our preliminary data suggest that some of the potentially adverse effects related to smoking may also be expected when nicotine is consumed via snuff or nicotine pouches.

Isolated and mixed effects of pure and formulated abamectin and difenoconazole on biochemical biomarkers of the gills of Danio rerio.

Pesticides are released into the environment daily, and their effects on nontarget species in aquatic ecosystems have been widely reported. To evaluate the adverse effects caused in adults of Danio rerio species exposed to the pesticides abamectin, difenoconazole, and their commercial formulations (Kraft 36EC® and Score 250EC®), both isolated and in mixtures, biochemical biomarkers were analyzed in the gills of organisms exposed to sublethal concentrations. To this end, the activities of the enzymes 7-ethoxyresorufin-O-deethylase (EROD), glucuronosyltransferase (UDPGT), glutathione-S-transferase (GST), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), lipid hydroperoxide (LH), and malondialdehyde (MDA), which are indicative of oxidative stress, were measured after 48 h of exposure to the different pesticide treatments. The results showed a significant increase in EROD activity and MDA levels in the gills of fish exposed to the commercial formulation of abamectin. When the fish were exposed to difenoconazole and its commercial formulation, an increase in GST, GPx, and MDA levels and a decrease in GR activity were observed in the gills. Furthermore, the responses of the biomarkers were more pronounced in organisms exposed to mixtures of both active ingredients and commercial formulations. It is concluded that the commercial formulations Kraft 36EC® and Score 250EC® and their mixtures cause significant alterations in the detoxification metabolism of exposed organisms and induce oxidative stress in fish.