Detoxification Of Xenobiotics Research Articles

Antifungal azoles trigger a xenobiotic detoxification pathway and chitin synthesis in Neurospora crassa

The presence of antifungal drugs is prompting the fungal microorganisms to react by mechanisms broader than the resistance. The fungi evolved mechanisms, by which they respond to various stress conditions, including the presence of antifungal compounds. In this work, we studied the response of model filamentous fungus Neurospora crassa to azole antifungals in the broader context of the adaptation mechanisms. We demonstrated the increase in expression of filamentous fungi-specific genes encoding cytochrome enzymes of CYP65 clan and plasma membrane-localized ABCC transporters. Azoles appear not to conjugate with glutathione. Surprisingly, the azoles caused changes in the hyphae organization and the amount of chitin in cell wall by the same manner that was thought to be echinocandin-specific. The response to individual azoles appeared to be influenced by the structure of azole compound (prochloraz – main outlier). Taken together, these findings demonstrate the importance of study of stress response mechanisms, specifically in filamentous fungi. Many aspects of the reaction within azoles seem to be similar, though specificities are occurring.

Emerging Metabolic Profiles of Sulfonamide Antibiotics by Cytochromes P450: A Computational-Experimental Synergy Study on Emerging Pollutants.

Metabolism, especially by CYP450 enzymes, is the main reason for mediating the toxification and detoxification of xenobiotics in humans, while some uncommon metabolic pathways, especially for emerging pollutants, probably causing idiosyncratic toxicity are easily overlooked. The pollution of sulfonamide antibiotics in aqueous system has attracted increasing public attention. Hydroxylation of the central amine group can trigger a series of metabolic processes of sulfonamide antibiotics in humans; however, this work parallelly reported the coupling and fragmenting initiated by amino H-abstraction of sulfamethoxazole (SMX) catalyzed by human CYP450 enzymes. Elucidation of the emerging metabolic profiles was mapped via a multistep synergy between computations and experiments, involving preliminary DFT computations and in vitro and in vivo assays, profiling adverse effects, and rationalizing the fundamental factors via targeted computations. Especially, the confirmed SMX dimer was shown to potentially act as a metabolism disruptor in humans, while spin aromatic delocalization resulting in the low electron donor ability of amino radicals was revealed as the fundamental factor to enable coupling of sulfonamide antibiotics by CYP450 through the nonconventional nonrebound pathway. This work may further strengthen the synergistic use of computations prior to experiments to avoid wasteful experimental screening efforts in environmental chemistry and toxicology.

A Toll pathway effector protects Drosophila specifically from distinct toxins secreted by a fungus or a bacterium

The Drosophila systemic immune response against many Gram-positive bacteria and fungi is mediated by the Toll pathway. How Toll-regulated effectors actually fulfill this role remains poorly understood as the known Toll-regulated antimicrobial peptide (AMP) genes are active only against filamentous fungi and not against Gram-positive bacteria or yeasts. Besides AMPs, two families of peptides secreted in response to infectious stimuli that activate the Toll pathway have been identified, namely Bomanins and peptides derived from a polyprotein precursor known as Baramicin A (BaraA). Unexpectedly, the deletion of a cluster of 10 Bomanins phenocopies the Toll mutant phenotype of susceptibility to infections. Here, we demonstrate that BaraA is required specifically in the host defense against Enterococcus faecalis and against the entomopathogenic fungus Metarhizium robertsii, albeit the fungal burden is not altered in BaraA mutants. BaraA protects the fly from the action of distinct toxins secreted by these Gram-positive and fungal pathogens, respectively, Enterocin V and Destruxin A. The injection of Destruxin A leads to the rapid paralysis of flies, whether wild type (WT) or mutant. However, a larger fraction of wild-type than BaraA flies recovers from paralysis within 5 to 10 h. BaraAs' function in protecting the host from the deleterious action of Destruxin is required in glial cells, highlighting a resilience role for the Toll pathway in the nervous system against microbial virulence factors. Thus, in complement to the current paradigm, innate immunity can cope effectively with the effects of toxins secreted by pathogens through the secretion of dedicated peptides, independently of xenobiotics detoxification pathways.

Purification, characterization, and enzyme kinetics of a glutathione S transferase from larvae of the camel tick Hyalomma dromedarii

BackgroundGlutathione s-transferases (GSTs) perform an essential role in detoxification of xenobiotics and endogenous compounds via their conjugation to reduce glutathione. ResultsA GST enzyme, designated tick larvae glutathione S transferase (TLGST), was purified from larvae of the camel tick Hyalomma dromedarii via ammonium sulfate precipitation, glutathione-Sepharose affinity column and Sephacryl S-300 chromatography. TLGST-specific activity was found to be 1.56 Umg−1 which represents 39 folds and 32.2% recovery. The molecular weight of TLGST purified from camel tick larvae was found as 42 kDa by gel filtration. TLGST has a pI value of 6.9 and was found a heterodimeric protein of 28 and 14 kDa subunits as detected on SDS-PAGE. The Lineweaver–Burk plot calculated the km for CDNB to be 0.43 mM with Vmax value of 9.2 Umg−1. TLGST exhibited its optimal activity at pH 7.9. Co2+, Ni2+ and Mn2+ increased the activity of TLGST while Ca2+, Cu2+, Fe2+ and Zn2+ inhibited it. TLGST was inhibited by cumene hydroperoxide, p-hydroxymercuribenzoate, lithocholic acid, hematin, triphenyltin chloride, p-chloromercuribenzoic acid (pCMB), N-p-Tosyl-L-phenylalanine chloromethyl ketone (TPCK), iodoacetamide, EDTA and quercetin. pCMB inhibited TLGST competitively with Ki value of 0.3 mM. ConclusionsThese findings will help to understand the various physiologic conditions of ticks and targeting TLGST could be significant tool for development of prospective vaccines against ticks as a bio-control strategy to overcome the rapid grows in pesticide-resistant tick populations. Graphical Abstract▪

Specificity Proteins (SP) and Krüppel-like Factors (KLF) in Liver Physiology and Pathology

The liver acts as a central hub that controls several essential physiological processes ranging from metabolism to detoxification of xenobiotics. At the cellular level, these pleiotropic functions are facilitated through transcriptional regulation in hepatocytes. Defects in hepatocyte function and its transcriptional regulatory mechanisms have a detrimental influence on liver function leading to the development of hepatic diseases. In recent years, increased intake of alcohol and western diet also resulted in a significantly increasing number of people predisposed to the incidence of hepatic diseases. Liver diseases constitute one of the serious contributors to global deaths, constituting the cause of approximately two million deaths worldwide. Understanding hepatocyte transcriptional mechanisms and gene regulation is essential to delineate pathophysiology during disease progression. The current review summarizes the contribution of a family of zinc finger family transcription factors, named specificity protein (SP) and Krüppel-like factors (KLF), in physiological hepatocyte functions, as well as how they are involved in the onset and development of hepatic diseases.

New Light on the Varietal Thiols Pathway during Alcoholic Fermentation: Role of 3-S-(N-Acetyl-cysteinyl)-hexan-1-ol (NAC3SH).

For many years, knowledge on thiol precursors has been limited to S-conjugates of glutathione (G3SH), cysteine (Cys3SH), and later on the dipeptides γ-GluCys and CysGly. In this work, we took the parallel between precursor degradation and the glutathione-mediated detoxification pathway a step further by considering a new type of derivative, 3-S-(N-acetyl-l-cysteinyl)hexanol (NAC3SH). This compound was synthesized and then added to the existing liquid chromatography with tandem mass spectrometry (LC-MS/MS) method of thiol precursors. This intermediate was only identified during alcoholic fermentation in synthetic must spiked with G3SH (1 mg/L or 2.45 μmol/L) in the presence of copper with concentration above 1.25 mg/L, which demonstrates for the first time the existence of this new derivative (until 126 μg/L or 0.48 μmol/L) and the capacity of the yeast to produce such a compound. Its status as a precursor was also studied during fermentation, in which a release of 3-sulfanylhexanol was noted corresponding to a conversion yield close to 0.6%. This work completed the thiol precursor's degradation pathway in Saccharomyces cerevisiae in synthetic conditions with a new intermediate, confirming its connection with the xenobiotic detoxification pathway and giving new insights on the precursor's fate.

Hydrocarbons occurrence and transcriptomic response of oyster Crassostrea virginica from lagoons of the Southern Gulf of Mexico

The Gulf of Mexico is an important crude oil reserve worldwide, and the oyster Crassostrea virginica is an excellent candidate to study the oil spill impacts on marine invertebrates. In this work, the concentrations of polycyclic aromatic hydrocarbons (PAHs) and aliphatic hydrocarbons (AHs) from eight productive oyster areas in the Gulf of Mexico were measured on sediment, water, and tissues from C. virginica. In water, the highest AHs concentration was detected in Tamiahua (0.50 ng/mL), while for PAHs, the highest concentration was > 0.10 ng/mL in Tampamachoco. In sediment, Tamiahua and Tampamachoco lagoons had the highest AHs concentrations with values near 2.5 μg/g dry weight. Considering the PAHs, Tamiahua, Carmen, and Tampamachoco lagoons registered the highest levels, with values > 60 ng/g dry weight. In tissues from C. virginica, La Pesca, Cármen and Mecoacán presented the highest PAHs concentrations with values between 0.20 and 0.25 μg/g dry weight. Furthermore, from the molecular analysis of genes related with different phases of the xenobiotic detoxification process such as hypoxia inducible factor (hif-1a), cytochrome P450 10 (cyp10), flavin mono-oxygenase (fmo), glutathione S-transferase (gstΩ1), multidrug resistant protein (mdrd1), catalase (cat), among others, the differences between lagoons were significant (P< 0.05) and generally with Las Enramadas showing the highest expression levels. From genes evaluated in this study, hif-1a, cyp10, fmo, mdrd1 and cat presented the highest expression differentials between lagoons. The above allowed us to validate the utility of molecular markers in the assessment of the hydrocarbons effect on oyster under the conditions from the Southern Gulf of Mexico.

Evolution of enzyme functionality in the flavin-containing monooxygenases

Among the molecular mechanisms of adaptation in biology, enzyme functional diversification is indispensable. By allowing organisms to expand their catalytic repertoires and adopt fundamentally different chemistries, animals can harness or eliminate new-found substances and xenobiotics that they are exposed to in new environments. Here, we explore the flavin-containing monooxygenases (FMOs) that are essential for xenobiotic detoxification. Employing a paleobiochemistry approach in combination with enzymology techniques we disclose the set of historical substitutions responsible for the family’s functional diversification in tetrapods. Remarkably, a few amino acid replacements differentiate an ancestral multi-tasking FMO into a more specialized monooxygenase by modulating the oxygenating flavin intermediate. Our findings substantiate an ongoing premise that enzymatic function hinges on a subset of residues that is not limited to the active site core.

NRF2 and the Moirai: Life and Death Decisions on Cell Fates.

Significance: The transcription factor NRF2 (NF-E2-related factor 2) plays an important role as a master regulator of the cellular defense system by activating transcriptional programs of NRF2 target genes encoding multiple enzymes related to cellular redox balance and xenobiotic detoxication. Comprehensive transcriptional analyses continue to reveal an ever-broadening range of NRF2 target genes, demonstrating the sophistication and diversification of NRF2 biological signatures beyond its canonical cytoprotective roles. Recent Advances: Accumulating evidence indicates that NRF2 has a strong association with the regulation of cell fates by influencing key processes of cellular transitions in the three major phases of the life cycle of the cell (i.e., cell birth, cell differentiation, and cell death). The molecular integration of NRF2 signaling into this regulatory program occurs through a wide range of NRF2 target genes encompassing canonical functions and those manipulating cell fate pathways. Critical Issues: A singular focus on NRF2 signaling for dissecting its actions limits in-depth understanding of its intersection with the molecular machinery of cell fate determinations. Compensatory responses of downstream pathways governed by NRF2 executed by a variety of transcription factors and multifactorial signaling crosstalk require further exploration. Future Directions: Further investigations using optimized in vivo models and active engagement of overarching approaches to probe the interplay of widespread pathways are needed to study the properties and capabilities of NRF2 signaling as a part of a large network within the cell fate regulatory domain. Antioxid. Redox Signal. 38, 684-708.

Cocktail effect and synergistic mechanism of two components of Perilla frutescens essential oil, perillaldehyde and carvone, against Tribolium castaneum

Tribolium castaneum (Herbst) is one of the most harmful insects in stored products. The objective of the current study was to search for active constituents of Perilla frutescens (L.) Britt. essential oils from three localities against T. castaneum, as well as to investigate the optimal formula of active constituents and their synergistic mechanism. A total of 38 constituents were identified from the three essential oils by gas chromatography-mass spectrometry (GC-MS) according to the mass spectra and retention index values, and the relative contents of the individual components were determined by the peak normalization method. The following major compounds were identified: perillaldehyde, (R)-(-)-carvone, 2-furyl methyl ketone, d-limonene, and β-caryophyllene. Contact toxicity was assessed by topical application under room temperature, and the insects were reared in a dark incubator for 24 h at approximately 30 °C and 75 % humidity. Of these compounds, perillaldehyde (LD50 =7.71 μg/adult) and R-(-)-carvone (LD50 =10.93 μg/adult) displayed the most significant contact activities against T. castaneum. Synergistic interactions between perillaldehyde and R-(-)-carvone were evaluated using Wadley's model. All nine tested formulas displayed synergistic effects against T. castaneum, and a 1:4 (v:v) mixture exhibited an intense cocktail effect by dramatically reducing the LD50 to 2.73 μg/adult. The results of the RNA-Seq and qRT-PCR analyses suggested that the synergistic effect could be attributed to a reduced capacity for detoxification and elimination of xenobiotics. Three cytochrome P450 genes (CYP6BQ11, CYP6BQ7, and CYP6BK5) in phase I and two multidrug resistance protein genes (MRP4a and MRPX4) in phase III of the detoxification process may play essential roles. These findings provide a basis for the development of novel natural insecticides for the control of storage pests.

CCR4-NOT subunit CCF-1/CNOT7 promotes transcriptional activation to multiple stress responses in Caenorhabditis elegans.

CCR4-NOT is a versatile eukaryotic protein complex that controls multiple steps in gene expression regulation from synthesis to decay. In yeast, CCR4-NOT has been implicated in stress response regulation, though this function in other organisms remains unclear. In a genome-wide RNAi screen, we identified a subunit of the CCR4-NOT complex, ccf-1, as a requirement for the C. elegans transcriptional response to cadmium and acrylamide stress. Using whole-transcriptome RNA sequencing, we show that the knockdown of ccf-1 attenuates the activation of a broad range of stress-protective genes in response to cadmium and acrylamide, including those encoding heat shock proteins and xenobiotic detoxification. Consistently, survival assays show that the knockdown of ccf-1 decreases C. elegans stress resistance and normal lifespan. A yeast2-hybrid screen using a CCF-1 bait identified the homeobox transcription factor PAL-1 as a physical interactor. Knockdown of pal-1 inhibits the activation of ccf-1dependent stress genes and reduces C. elegans stress resistance. Gene expression analysis reveals that knockdown of ccf-1 and pal-1 attenuates the activation of elt-2 and elt-3 under stress that encode master transcriptional co-regulators of stress response in the C. elegans, and that overexpression of ELT-2 can suppress ccf-1's requirement for gene transcription in a stress-dependent manner. Our findings reveal a new role for CCR4-NOT in the environmental stress response and define its role in stress resistance and longevity in C. elegans.

The variation of a cytochrome P450 gene, CYP6G4, drives the evolution of Musca domestica L. (Diptera: Muscidae) resistance to insecticides in China

Long term and excessive insecticide use has resulted in some environmental problems and especially, insecticide resistance evolution in insect pests. The variation of cytochrome P450 monooxygenases (P450s), associated with the metabolic detoxification of toxic xenobiotics, is often involved in insecticide resistance. Here, we found that the variation in a P450 gene, CYP6G4, is the most important driver of carbamates resistance in the house fly (Musca domestica). Deciphering the detailed molecular mechanisms of the insecticide resistance is critical for performing suitable insecticide resistance management strategies. Our research results revealed that the combination of amino acid mutations (110C-330E-360N/S, 110C-330E-360S) of CYP6G4 could improve the resistance to propoxur. The nucleotide variations in the promoter region of CYP6G4 significantly increased the luciferase activity by the reporter gene assays. Additionally, miR-281-1-5p was confirmed to post-transcriptionally down-regulate the expression of CYP6G4. These findings suggest that three independent mechanisms; amino acid mutations of the P450 protein, mutations in the promoter region and low expression of post-trans-regulatory factors, as the powerful strategies for the insect resistance to toxic compounds, play a crucial role in the evolutionary processes of insecticide resistance.

The conserved microRNA-229 family controls low-insulin signaling and dietary restriction induced longevity through interactions with SKN-1/NRF2.

Several microRNAs have emerged as regulators of pathways that control aging. For example, miR-228 is required for normal lifespan and dietary restriction (DR) mediated longevity through interaction with PHA-4 and SKN-1 transcription factors in Caenorhabditis elegans. miR-229,64,65, and 66, a cluster of microRNAs located adjacent to each other on chromosome III, are in the same family as miR-228, albeit with slight differences in the miR-228 seed sequence. We demonstrate that, in contrast to the anti-longevity role of miR-228, the miR-229-66 cluster is required for normal C. elegans lifespan and for the longevity observed in mir-228 mutants. miR-229-66 is also critical for lifespan extension observed under DR and reduced insulin signaling (IIS) and by constitutive nuclear SKN-1. Both DR and low-IIS upregulate the expression of the miRNA cluster, which is dependent on transcription factors PHA-4, SKN-1, and DAF-16. In turn, the expression of SKN-1 and DAF-16 requires mir-229,64,65,66. miR-229-66 targets the odd-skipped-related transcription factor, odd-2 to regulate lifespan. Knockdown of odd-2 increases lifespan, suppresses the short lifespan of mir-229,64,65,66(nDf63) III mutants, and alters levels of SKN-1 in the ASI neurons. Together with SKN-1, the miRNA cluster also indirectly regulates several genes in the xenobiotic detoxification pathway which increases wild-type lifespan and significantly rescues the short lifespan of mir-229,64,65,66(nDf63) III mutants. Thus, by interacting with SKN-1, miR-229-66 transduces the effects of DR and low-IIS in lifespan extension in C. elegans. Given that this pathway is conserved, it is possible that a similar mechanism regulates aging in more complex organisms.

Cap 'n' Collar C and Aryl Hydrocarbon Receptor Nuclear Translocator Facilitate the Expression of Glutathione S-Transferases Conferring Adaptation to Tannic Acid and Quercetin in Micromelalopha troglodyta (Graeser) (Lepidoptera: Notodontidae).

Micromelalopha troglodyta (Graeser) (Lepidoptera: Notodontidae) is a notorious pest of poplar. Coevolution with poplars rich in plant secondary metabolites prompts M. troglodyta to expand effective detoxification mechanisms against toxic plant secondary metabolites. Although glutathione S-transferases (GSTs) play an important role in xenobiotic detoxification in M. troglodyta, it is unclear how GSTs act in response to toxic secondary metabolites in poplar. In this study, five GST gene core promoters were accurately identified by a 5' loss luciferase reporter assay, and the core promoters were significantly induced by two plant secondary metabolites in vitro. Two transcription factors, cap 'n' collar C (CncC) and aryl hydrocarbon receptor nuclear translocator (ARNT), were cloned in M. troglodyta. MtCncC and MtARNT clustered well with other insect CncCs and ARNTs, respectively. In addition, MtCncC and MtARNT could bind the MtGSTt1 promoter and strongly improve transcriptional activity, respectively. However, MtCncC and MtARNT had no regulatory function on the MtGSTz1 promoter. Our findings revealed the molecular mechanisms of the transcription factors MtCncC and MtARNT in regulating the GST genes of M. troglodyta. These results provide useful information for the control of M. troglodyta.

The complete genome assemblies of 19 insect pests of worldwide importance to agriculture

There are many insect pests worldwide that damage agricultural crop and reduce yield either by direct feeding or by the transmission of plant diseases. To date, control of pest insects has been achieved largely by applying synthetic insecticides. However, insecticide use can be seriously impacted by legislation that limits their use or by the evolution of resistance in the target pest. Thus, there is a move towards less use of insecticides and increased adoption of integrated pest management strategies using a wide range of non-chemical and chemical control methods. For good pest control there is a need to understand the mode of action and selectivity of insecticides, the life cycles of the pests and their biology and behaviours, all of which can benefit from good quality genome data. Here we present the complete assembled (chromosome level) genomes (incl. mtDNA) of 19 insect pests, Agriotes lineatus (click beetle/wireworm), Aphis gossypii (melon/cotton aphid), Bemisia tabaci (cotton whitefly), Brassicogethes aeneus (pollen beetle), Ceutorhynchus obstrictus (seedpod weevil), Chilo suppressalis (striped rice stem borer), Chrysodeixis includens (soybean looper), Diabrotica balteata (cucumber beetle), Diatraea saccharalis (sugar cane borer), Nezara viridula (green stink bug), Nilaparvata lugens (brown plant hopper), Phaedon cochleariae (mustard beetle), Phyllotreta striolata (striped flea beetle), Psylliodes chrysocephala (cabbage stem flea beetle), Spodoptera exigua (beet army worm), Spodoptera littoralis (cotton leaf worm), Diabrotica virgifera (western corn root worm), Euschistus heros (brown stink bug) and Phyllotreta cruciferae (crucifer flea beetle). For the first 15 of these we also present the annotation of genes encoding potential xenobiotic detoxification enzymes. This public resource will aid in the elucidation and monitoring of resistance mechanisms, the development of highly selective chemistry and potential techniques to disrupt behaviour in a way that limits the effect of the pests.

First genome-scale insights into the virulence of the snow mold causal fungus Microdochium nivale

Pink snow mold, caused by a phytopathogenic and psychrotolerant fungus, Microdochium nivale, is a severe disease of winter cereals and grasses that predominantly occurs under snow cover or shortly after its melt. Snow mold has significantly progressed during the past decade, often reaching epiphytotic levels in northern countries and resulting in dramatic yield losses. In addition, M. nivale gradually adapts to a warmer climate, spreading to less snowy territories and causing different types of plant diseases throughout the growing period. Despite its great economic importance, M. nivale is poorly investigated; its genome has not been sequenced and its crucial virulence determinants have not been identified or even predicted. In our study, we applied a hybrid assembly based on Oxford Nanopore and Illumina reads to obtain the first genome sequence of M. nivale. 11,973 genes (including 11,789 protein-encoding genes) have been revealed in the genome assembly. To better understand the genetic potential of M. nivale and to obtain a convenient reference for transcriptomic studies on this species, the identified genes were annotated and split into hierarchical three-level functional categories. A file with functionally classified M. nivale genes is presented in our study for general use. M. nivale gene products that best meet the criteria for virulence factors have been identified. The genetic potential to synthesize human-dangerous mycotoxins (fumonisin, ochratoxin B, aflatoxin, and gliotoxin) has been revealed for M. nivale. The transcriptome analysis combined with the assays for extracellular enzymatic activities (conventional virulence factors of many phytopathogens) was carried out to assess the effect of host plant (rye) metabolites on the M. nivale phenotype. In addition to disclosing plant-metabolite-upregulated M. nivale functional gene groups (including those related to host plant protein destruction and amino acid metabolism, xenobiotic detoxication (including phytoalexins benzoxazinoids), cellulose destruction (cellulose monooxygenases), iron transport, etc.), the performed analysis pointed to a crucial role of host plant lipid destruction and fungal lipid metabolism modulation in plant-M. nivale interactions.

Both age and social environment shape the phenotype of ant workers

Position within the social group has consequences on individual lifespans in diverse taxa. This is especially obvious in eusocial insects, where workers differ in both the tasks they perform and their aging rates. However, in eusocial wasps, bees and ants, the performed task usually depends strongly on age. As such, untangling the effects of social role and age on worker physiology is a key step towards understanding the coevolution of sociality and aging. We performed an experimental protocol that allowed a separate analysis of these two factors using four groups of black garden ant (Lasius niger) workers: young foragers, old foragers, young nest workers, and old nest workers. We highlighted age-related differences in the proteome and metabolome of workers that were primarily related to worker subcaste and only secondarily to age. The relative abundance of proteins and metabolites suggests an improved xenobiotic detoxification, and a fuel metabolism based more on lipid use than carbohydrate use in young ants, regardless of their social role. Regardless of age, proteins related to the digestive function were more abundant in nest workers than in foragers. Old foragers were mostly characterized by weak abundances of molecules with an antibiotic activity or involved in chemical communication. Finally, our results suggest that even in tiny insects, extended lifespan may require to mitigate cancer risks. This is consistent with results found in eusocial rodents and thus opens up the discussion of shared mechanisms among distant taxa and the influence of sociality on life history traits such as longevity.

Investigating the biochemical signatures and physiological roles of the FMO family using molecular phylogeny

Group B flavin-dependent monooxygenases are employed in swathes of different physiological functions. Despite their collectively large substrate profile, they all harness a flavin-based C4a-(hydro)peroxy intermediate for function. Within this class are the flavin-containing monooxygenases (FMOs), representing an integral component within the secondary metabolism of all living things – xenobiotic detoxification. Their broad substrate profile makes them ideal candidates for detoxifying procedures as they can tackle a range of compounds. Recent studies have illustrated that several FMOs, however, have unique substrate profiles and differing physiological functions that implicate new roles within secondary and primary metabolism. Herein this article, by employing phylogenetic approaches, and inspecting structures of AlphaFold generated models, we have constructed a biochemical blueprint of the FMO family. FMOs are clustered in four distinct groups, with two being predominantly dedicated to xenobiotic detoxification. Furthermore, we observe that differing enzymatic activities are not constricted to a ‘golden’ set of residues but instead an intricate constellation of primary and secondary sphere residues. We believe that this work delineates the core phylogeny of the Group B monooxygenases and will prove useful for classifying newly sequenced genes and provide directions to future biochemical investigations.

Evaluation of total glutathione-s-transferase levels in serum of patients with oral malignancy.

Squamous cell carcinoma-related antigen is not always sensitive enough for the early detection of oral cancer which is why a new marker has been desired as a substitute to be applied for serum diagnosis of oral cancer. Reactive oxygen species (ROS) has been known to play an important role in carcinogenesis. Glutathione-s-transferases (GSTs) are a family of eukaryotic and prokaryotic phase-II metabolic isoenzymes involved in xenobiotic detoxification. This correlation of the ROS species function and their role in initiation and progression of cancer could be exploited as of diagnostic value. The biologic function of the GSTs in human head-and-neck squamous cell carcinomas has been studied by researchers at gross as well as molecular levels. Taking into consideration this scientific background, future scope and perspectives, we initiated this study. This study was performed as a prospective case-control in vitro analytical study with subjects (n = 40) fulfilling the prerequisite conditions and were compliant. The case group (n = 20) was subjects with histopathologically proven cases of oral malignancy and age- and sex-matched control group (n = 20). The enzyme GST was evaluated in sera of all participants and then comparison was done between two groups as well as correlation with histopathologic grading for oral malignancy. The mean serum GST activity in oral cancer patients was significantly higher than that of the control group. The present study has compared the alterations of enzyme in relation to histopathological grading of oral malignancy and found increased serum GST activity of well-differentiated and moderately differentiated carcinomas than the poorly differentiated carcinoma in terms of mean. Increased expression of the enzyme, as reported in the present study, can be due to tumor burden which attributes to overproduction of GST by cancer cells. The major clinical significance of the present study is that it gives important information regarding a new tumor progression and prognosis marker.

The antioxidant glutathione.

Reduced glutathione (GSH) is an essential non-enzymatic antioxidant in mammalian cells. GSH can act directly as an antioxidant to protect cells against free radicals and pro-oxidants, and as a cofactor for antioxidant and detoxification enzymes such as glutathione peroxidases, glutathione S-transferases, and glyoxalases. Glutathione peroxidases detoxify peroxides by a reaction that is coupled to GSH oxidation to glutathione disulfide (GSSG). GSSG is converted back to GSH by glutathione reductase and cofactor NADPH. GSH can regenerate vitamin E following detoxification reactions of vitamin E with lipid peroxyl radicals (LOO). GSH is a cofactor for GST during detoxification of electrophilic substances and xenobiotics. Dicarbonyl stress induced by methylglyoxal and glyoxal is alleviated by glyoxalase enzymes and GSH. GSH regulates redox signaling through reversible oxidation of critical protein cysteine residues by S-glutathionylation. GSH is involved in other cellular processes such as protein folding, protecting protein thiols from oxidation and crosslinking, degradation of proteins with disulfide bonds, cell cycle regulation and proliferation, ascorbate metabolism, apoptosis and ferroptosis.