Catabolism Of Xenobiotics Research Articles

A plasmid-based genomic screening system for transcriptional regulators of non-adjacent xenobiotic catabolism genes.

Bacteria play an important role in the catabolism of environmental xenobiotics. The study of transcriptional regulation has greatly enhanced our understanding of the molecular mechanisms associated with these processes. However, genes encoding transcription factors (TFs) for xenobiotic catabolism are usually not located in the immediate vicinity of the catabolic genes that they regulate; therefore, functional identification of these TFs is difficult. Significantly modified from a metagenome screening method substrate-induced gene expression (SIGEX), here we propose a synthetic pSRGFP-18 plasmid-based tool as a TF reporter system. In short, two multiple cloning sites (MCS) were designed; one in front of an egfp reporter gene was constructed for promoter insertion, and the other MCS was used for shotgun cloning of genomic DNA. Based on the regulatory relationship between TFs and the promoter of their associated catabolic genes, this approach allowed us to screen for TF genes using a genome shotgun approach. This system performed well when testing the known operons. Following statistical analysis of known catabolic operons in Escherichia coli and Bacillus subtilis, the suggested region of the target promoter for this system was from - 250 to + 150. Furthermore, to broaden the applicability of this plasmid, a series of pSRGFP-18 and pBBR1-based pSRGFP-X plasmids were constructed, which had broad host ranges and contained different antibiotic markers. This study outlines a powerful tool to enable functional identification of TFs for bacterial xenobiotic catabolism.

Abstract 4946: Comprehensive molecular profiling reveals distinct immunemicroenvironment subtypes of oropharyngeal cancers

Abstract Background The oropharyngeal cancer (OPC) constitutes a distinct clinical subset of head and neck squamous cell carcinoma with high prevalence of HPV-associated tumorigenesis. However, the relationship between tumor molecular types and immune microenvironment has not been systematically delineated in OPCs. In this study, we classified the OPCs into three different molecular subtypes regarding their immunological character by comprehensive molecular profiling by RNA-seq and multiplexed immunohistochemistry (IHC). Methods The 37 surgically resected primary OPC tumors were analyzed, and the HPV status was evaluated by p16 IHC. The transcriptome profiling of tumors by RNA-seq classified OPCs into three molecular subtypes based on t-SNE plotting. The immune microenvironment of each subtype was investigated using VECTRA spectral imaging system in terms of CD8 T cell and macrophage infiltration patterns, as well as T cell exhaustion maker expressions. The 10 OPC patients treated with anti-PD-1/PD-L1 blockade were also classified into three subtypes and their therapy response was compared. Results We divided OPCs into Immune-rich (IR), mesenchymal (MS), and classical (CL) subtypes by RNA-seq clustering analysis. All IR type tumors were HPV-positive, whereas most CL types (7 out of 8) were HPV-negative. MS type showed mixed HPV status (HPV-positive in 5/14). The IR type showed favorable prognosis compared with poor prognosis of CL type, and MS type showed intermediate survival outcome. The IR type tumors showed high enrichment of adaptive immune response, T cell exhaustion, and cell cycle gene signature implying their highly immunogenic properties. The multiplexed IHC confirmed plenty of PD-1+CD8 T cells and type I macrophages infiltrating tumor nest in IR types. The MS type was characterized by upregulation of muscle, extracellular matrix, and TGF-β signatures with scant CD8 T cell signature expression. The IHC analysis of MS type showed exclusion of CD8 T cells from tumor nest that might be related to their high TGF-β-response signature (TBRS) score. CL types were related to high xenobiotic catabolism signatures, and showed low level of CD8 T cell infiltrations with focal CD73 expression. Among 10 OPC patients with anti-PD-1/PD-L1 blockade treatment, the IR type patients showed durable therapy response (3 of 4 patients), whereas CL type patients showed early progression on the treatment (3 of 4 patients). Conclusions Our comprehensive analysis reveals that OPCs can be classified into three distinct subtypes based on their immune microenvironment properties. The three molecular subtypes showed distinct therapy response on anti-PD-1/PD-L1 blockade. These findings are pertinent to develop predictive markers on immunotherapy and to design combination immunotherapy strategies in OPC patients. Citation Format: Min Hwan Kim, Jae-Hwan Kim, Dong Min Jung, Jae Woo Choi, Soo Jin Heo, Kyoung-Ho Pyo, Min Hee Hong, Byoung Chul Cho, Hye Ryun Kim. Comprehensive molecular profiling reveals distinct immunemicroenvironment subtypes of oropharyngeal cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4946.

Intensive herbicide use has selected for constitutively elevated levels of stress-responsive mRNAs and proteins in multiple herbicide-resistant Avena fatua L.

Intensive use of herbicides has led to the evolution of two multiple herbicide-resistant (MHR) Avena fatua (wild oat) populations in Montana that are resistant to members of all selective herbicide families available for A. fatua control in US small grain crops. We used transcriptome and proteome surveys to compare constitutive changes in MHR and herbicide-susceptible (HS) plants associated with non-target site resistance. Compared to HS plants, MHR plants contained constitutively elevated levels of differentially expressed genes (DEGs) with functions in xenobiotic catabolism, stress response, redox maintenance and transcriptional regulation that are similar to abiotic stress-tolerant phenotypes. Proteome comparisons identified similarly elevated proteins including biosynthetic and multifunctional enzymes in MHR plants. Of 25 DEGs validated by RT-qPCR assay, differential regulation of 21 co-segregated with flucarbazone-sodium herbicide resistance in F3 families, and a subset of 10 of these were induced or repressed in herbicide-treated HS plants. Although the individual and collective contributions of these DEGs and proteins to MHR remain to be determined, our results support the idea that intensive herbicide use has selected for MHR populations with altered, constitutively regulated patterns of gene expression that are similar to those in abiotic stress-tolerant plants. © 2017 Society of Chemical Industry.

CYP105-diverse structures, functions and roles in an intriguing family of enzymes in Streptomyces.

The cytochromes P450 (CYP or P450) are a large superfamily of haem-containing enzymes found in all domains of life. They catalyse a variety of complex reactions, predominantly mixed-function oxidations, often displaying highly regio- and/or stereospecific chemistry. In streptomycetes, they are predominantly associated with secondary metabolite biosynthetic pathways or with xenobiotic catabolism. Homologues of one family, CYP105, have been found in all Streptomyces species thus far sequenced. This review looks at the diverse biological functions of CYP105s and the biosynthetic/catabolic pathways they are associated with. Examples are presented showing a range of biotransformative abilities and different contexts. As biocatalysts capable of some remarkable chemistry, CYP105s have great biotechnological potential and merit detailed study. Recent developments in biotechnological applications which utilize CYP105s are described, alongside a brief overview of the benefits and drawbacks of using P450s in commercial applications. The role of CYP105s in vivo is in many cases undefined and provides a rich source for further investigation into the functions these enzymes fulfil and the metabolic pathways they participate in, in the natural environment.

COMMUNICATION OF NAD- AND NADP-DEPENDENT DEHYDROGENASE ACTIVITIES IN LYMPHOCYTES WITH OUTCOME OF PREVALENCE PURULENT PERITONITIS

Aim of this study was to investigate of NAD(P)-dependent dehydrogenases activity in lymphocytes at different outcomes widespread purulent peritonitis(WPP). Enzyme activity in blood lymphocytes was determined by bioluminescent method. Found that patients with a poor outcome of WPP in blood lymphocytes made more pronounced reaction of lipid anabolism and catabolism of xenobiotics. Regardless of the outcome of the disease in blood lymphocytes of WPP patients reduced levels of enzymes activity that determine the intensity of anaerobic and aerobic respiration and the level of plastic processes, but the activation reactions of lipid catabolism and transport products through glycerol-3-phosphate dehydrogenase on reactions of glycolysis. Based on the informativeness of the NAD(P)-dependent dehydrogenases activity in the classification neural network model for the disease outcome, metabolic coefficient is proposed, which has prognostic significance.

Prokaryotic Horizontal Gene Transfer in Freshwater Lakes: Implications of Dynamic Biogeochemical Zonation

The highly adaptive nature of prokaryotic communities in the face of changing environmental conditions reflects in part their ability to share advantageous genetic information through horizontal gene transfer (HGT). Natural freshwater lacustrine (lake) systems are a vital and finite resource, and the influence of HGT on their quality (e.g. enabling the spread of antibiotic resistance and xenobiotic catabolism genes) is likely significant. Laboratory and in situ studies indicate that the dynamic physical, chemical, and biological conditions that structure freshwater systems can influence HGT within freshwater prokaryotic communities. Thus, understanding how biogeochemical parameters impact HGT in freshwater lakes is an emerging knowledge gap with potential implications for ecosystem and human health on a global scale. In this review, we provide a general synopsis of what is known about HGT in freshwater prokaryotic communities, followed by an integrated summary of current knowledge identifying how biogeochemical factors may influence prokaryotic HGT in freshwater lacustrine systems.

Biotransformation of fluorobiphenyl by Cunninghamella elegans

The fungus Cunninghamella elegans is a useful model of human catabolism of xenobiotics. In this paper, the biotransformation of fluorinated biphenyls by C. elegans was investigated by analysis of the culture supernatants with a variety of analytical techniques. 4-Fluorobiphenyl was principally transformed to 4-fluoro-4'-hydroxybiphenyl, but other mono- and dihydroxylated compounds were detected in organic extracts by gas chromatography-mass spectrometry. Additionally, fluorinated water-soluble products were detected by (19)F NMR and were identified as sulphate and beta-glucuronide conjugates. Other fluorobiphenyls (2-fluoro-, 4,4'-difluoro- and 2,3,4,5,6-pentafluoro-biphenyl) were catabolised by C. elegans, yielding mono- and dihydroxylated products, but phase II metabolites were detected from 4,4'-difluorobiphenyl only.

Characterization of two NADPH: Cytochrome P450 reductases from cotton ( Gossypium hirsutum)

Cytochrome P450 monooxygenases (P450s) are commonly involved in biosynthesis of endogenous compounds and catabolism of xenobiotics, and their activities rely on a partner enzyme, cytochrome P450 reductase (CPR, E.C.1.6.2.4). Two CPR cDNAs, GhCPR1 and GhCPR2, were isolated from cotton ( Gossypium hirsutum). They are 71% identical to each other at the amino acid sequence level and belong to the Class I and II of dicotyledonous CPRs, respectively. The recombinant enzymes reduced cytochrome c, ferricyanide and dichlorophenolindophenol (DCPIP) in an NADPH-dependent manner, and supported the activity of CYP73A25, a cinnamate 4-hydroxylase of cotton. Both GhCPR genes were widely expressed in cotton tissues, with a reduced expression level of GhCPR2 in the glandless cotton cultivar. Expression of GhCPR2, but not GhCPR1, was inducible by mechanical wounding and elicitation, indicating that the GhCPR2 is more related to defense reactions, including biosynthesis of secondary metabolites.

The Genome of Polaromonas sp. Strain JS666: Insights into the Evolution of a Hydrocarbon- and Xenobiotic-Degrading Bacterium, and Features of Relevance to Biotechnology

Polaromonas sp. strain JS666 can grow on cis-1,2-dichloroethene (cDCE) as a sole carbon and energy source and may be useful for bioremediation of chlorinated solvent-contaminated sites. Analysis of the genome sequence of JS666 (5.9 Mb) shows a bacterium well adapted to pollution that carries many genes likely to be involved in hydrocarbon and xenobiotic catabolism and metal resistance. Clusters of genes coding for haloalkane, haloalkanoate, n-alkane, alicyclic acid, cyclic alcohol, and aromatic catabolism were analyzed in detail, and growth on acetate, catechol, chloroacetate, cyclohexane carboxylate, cyclohexanol, ferulate, heptane, 3-hydroxybenzoate, hydroxyquinol, gentisate, octane, protocatechuate, and salicylate was confirmed experimentally. Strain JS666 also harbors diverse putative mobile genetic elements, including retrons, inteins, a miniature inverted-repeat transposable element, insertion sequence transposases from 14 families, eight genomic islands, a Mu family bacteriophage, and two large (338- and 360-kb) plasmids. Both plasmids are likely to be self-transferable and carry genes for alkane, alcohol, aromatic, and haloacid metabolism. Overall, the JS666 genome sequence provides insights into the evolution of pollutant-degrading bacteria and provides a toolbox of catabolic genes with utility for biotechnology.

Effect of Semecarpus anacardium Linn. nut extract on mammary and hepatic expression of xenobiotic enzymes in DMBA-induced mammary carcinoma

Breast cancer is the major cause of cancer death in women worldwide. Environmental risk factors particularly genotoxic chemicals such as polycyclic aromatic hydrocarbons (PAH) are likely to account for a much higher mortality. Xenobiotic metabolising enzymes in breast tissue are potentially important determinants in both the susceptibility to the mutagenic effects of chemical carcinogens and in the response of breast tumors to chemotherapy. The well known carcinogen 7,12-dimethylbenz( a)anthrazene of PAH family was given (25 mg/ml) orally by gastric intubation to induce mammary carcinoma in Sprague-Dawley rats. Increased level of cytochromes ( P 450, B 5), EROD, PROD activities, Phase I biotransformation enzymes (NADPH-cytochrome ( P 450) reductase, NADPH-cytochrome ( b 5) reductase, epoxide hydrolase) and expression of CYP1A1, CYP1A2 and CYP1B1 in liver and breast tissue microsome were documented in DMBA treated group. Phase II enzyme activities (glutathione- S-transferase, gluthatione peroxidase, gluatathione reductase, UDP-glucuronyl transferease) were decreased markedly in cancerous rats. The nut extract of Semecarpus anacardium was administered orally (200 mg/kg body wt/day) to the mammary carcinoma rats for 14 days. Drug treatment restored back the altered Phase I and II biotransformation enzymes thus achieving complete detoxification of the carcinogen. These findings suggest that S. anacardium can effectively modulate the catabolism of xenobiotics in rats.

Environmental genomics: exploring the unmined richness of microbes to degrade xenobiotics.

Increasing pollution of water and soils by xenobiotic compounds has led in the last few decades to an acute need for understanding the impact of toxic compounds on microbial populations, the catabolic degradation pathways of xenobiotics and the set-up and improvement of bioremediation processes. Recent advances in molecular techniques, including high-throughput approaches such as microarrays and metagenomics, have opened up new perspectives and pointed towards new opportunities in pollution abatement and environmental management. Compared with traditional molecular techniques dependent on the isolation of pure cultures in the laboratory, microarrays and metagenomics allow specific environmental questions to be answered by exploring and using the phenomenal resources of uncultivable and uncharacterized micro-organisms. This paper reviews the current potential of microarrays and metagenomics to investigate the genetic diversity of environmentally relevant micro-organisms and identify new functional genes involved in the catabolism of xenobiotics.

The heme oxygenase-carbon monoxide system: a regulator of hepatobiliary function.

Heme oxygenase (HO) degrades protoheme IX by cleaving its a-methene bridge into carbon monoxide (CO), free divalent iron (Fe21), and biliverdin-IXa.1-3 The reaction uses nicotinamide adenine dinucleotide phosphate (NADPH)– cytochrome P450 reductase as an electron donor system and molecular oxygen as the acceptor. A cytosolic enzyme, biliverdin reductase, converts biliverdin-IXa into bilirubinIXa. In humans, nearly 80% of the bilirubin in bile derives from hemoglobin heme.4 Cytochrome P450 constitutes another major source of heme undergoing degradation via HO.5 Until recently, products of the HO reaction were regarded as potentially toxic waste destined only for excretion. However, this is changing as evidence emerges for physiological roles of these compounds. CO may function as a gaseous regulator of cytochrome P450–dependent biotransformation including bile acid synthesis and xenobiotic catabolism. This article reviews the physiological and pathophysiological significance of the HO/CO system as a modulator of hepatobiliary function.

Systematic reverse genetics of transfer-DNA-tagged lines of Arabidopsis. Isolation of mutations in the cytochrome p450 gene superfamily.

We have developed an efficient reverse-genetics protocol that uses expedient pooling and hybridization strategies to identify individual transfer-DNA insertion lines from a collection of 6000 independently transformed lines in as few as 36 polymerase chain reactions. We have used this protocol to systematically isolate Arabidopsis lines containing insertional mutations in individual cytochrome P450 genes. In higher plants P450 genes encode enzymes that perform an exceptionally wide range of functions, including the biosynthesis of primary metabolites necessary for normal growth and development, the biosynthesis of secondary products, and the catabolism of xenobiotics. Despite their importance, progress in assigning enzymatic function to individual P450 gene products has been slow. Here we report the isolation of the first 12 such lines, including one (CYP83B1-1) that displays a runt phenotype (small plants with hooked leaves), and three insertions in abundantly expressed genes. The DNAs used in this study are publicly available and can be used to systematically isolate mutants in Arabidopsis.

Terrestrial biotechnology

Ecophysiological and molecular biological investigations of soil microorganisms have developed well, including critical studies of the fate and transport of genetically-modified microorganisms. Such studies will make contributions to areas ranging from the understanding of global change to the catabolism of xenobiotics in soil. Inundative inoculation procedures have not yet sustained their early promise. Greater opportunity appears to lie in the management of indigenous or intrinsic biota. For the protection of human health and the environment, environmental diagnostics, which will include the use of novel biosensors, would seem to offer great prospects

Biodegradation of halogenated organic compounds

In this review we discuss the degradation of chlorinated hydrocarbons by microorganisms, emphasizing the physiological, biochemical, and genetic basis of the biodegradation of aliphatic, aromatic, and polycyclic compounds. Many environmentally important xenobiotics are halogenated, especially chlorinated. These compounds are manufactured and used as pesticides, plasticizers, paint and printing-ink components, adhesives, flame retardants, hydraulic and heat transfer fluids, refrigerants, solvents, additives for cutting oils, and textile auxiliaries. The hazardous chemicals enter the environment through production, commercial application, and waste. As a result of bioaccumulation in the food chain and groundwater contamination, they pose public health problems because many of them are toxic, mutagenic, or carcinogenic. Although synthetic chemicals are usually recalcitrant to biodegradation, microorganisms have evolved an extensive range of enzymes, pathways, and control mechanisms that are responsible for catabolism of a wide variety of such compounds. Thus, such biological degradation can be exploited to alleviate environmental pollution problems. The pathways by which a given compound is degraded are determined by the physical, chemical, and microbiological aspects of a particular environment. By understanding the genetic basis of catabolism of xenobiotics, it is possible to improve the efficacy of naturally occurring microorganisms or construct new microorganisms capable of degrading pollutants in soil and aquatic environments more efficiently. Recently a number of genes whose enzyme products have a broader substrate specificity for the degradation of aromatic compounds have been cloned and attempts have been made to construct gene cassettes or synthetic operons comprising these degradative genes. Such gene cassettes or operons can be transferred into suitable microbial hosts for extending and custom designing the pathways for rapid degradation of recalcitrant compounds. Recent developments in designing recombinant microorganisms and hybrid metabolic pathways are discussed.

Biodegradation of halogenated organic compounds.

In this review we discuss the degradation of chlorinated hydrocarbons by microorganisms, emphasizing the physiological, biochemical, and genetic basis of the biodegradation of aliphatic, aromatic, and polycyclic compounds. Many environmentally important xenobiotics are halogenated, especially chlorinated. These compounds are manufactured and used as pesticides, plasticizers, paint and printing-ink components, adhesives, flame retardants, hydraulic and heat transfer fluids, refrigerants, solvents, additives for cutting oils, and textile auxiliaries. The hazardous chemicals enter the environment through production, commercial application, and waste. As a result of bioaccumulation in the food chain and groundwater contamination, they pose public health problems because many of them are toxic, mutagenic, or carcinogenic. Although synthetic chemicals are usually recalcitrant to biodegradation, microorganisms have evolved an extensive range of enzymes, pathways, and control mechanisms that are responsible for catabolism of a wide variety of such compounds. Thus, such biological degradation can be exploited to alleviate environmental pollution problems. The pathways by which a given compound is degraded are determined by the physical, chemical, and microbiological aspects of a particular environment. By understanding the genetic basis of catabolism of xenobiotics, it is possible to improve the efficacy of naturally occurring microorganisms or construct new microorganisms capable of degrading pollutants in soil and aquatic environments more efficiently. Recently a number of genes whose enzyme products have a broader substrate specificity for the degradation of aromatic compounds have been cloned and attempts have been made to construct gene cassettes or synthetic operons comprising these degradative genes. Such gene cassettes or operons can be transferred into suitable microbial hosts for extending and custom designing the pathways for rapid degradation of recalcitrant compounds. Recent developments in designing recombinant microorganisms and hybrid metabolic pathways are discussed.