Novel insights into the synergetic degradation of pyrene by microbial communities from mangroves in China.

Pyrene is a high molecular weight polycyclic aromatic hydrocarbon (HMW-PAHs). It is a ubiquitous, persistent, and carcinogenic environmental contaminant that has raised concern worldwide. This research explored synergistic bacterial communities for efficient pyrene degradation in seven typical Southern China mangroves. The bacterial communities of seven typical mangroves were enriched by pyrene, and enriched bacterial communities showed an excellent pyrene degradation capacity of > 95% (except for HK mangrove and ZJ mangrove). Devosia, Hyphomicrobium, Flavobacterium, Marinobacter, Algoriphahus, and Youhaiella all have significant positive correlations with pyrene (R>0, p < 0.05) by 16SrRNA gene sequencing and metagenomics analysis, indicated that these genera play a vital role in pyrene metabolism. Meanwhile, the functional genes were involved in pyrene degradation that was enriched in the bacterial communities, including the genes of nagAa, ndoR, pcaG, etc. Furthermore, the analyses of functional genes and binning genomes demonstrated that some bacterial communities as a unique teamwork to cooperatively participate in pyrene degradation. Interestingly, the genes related to biogeochemical cycles were enriched, such as narG , soxA, and cyxJ, suggested that bacterial communities were also helpful in maintaining the stability of the ecological environment. In addition, some novel species with pyrene-degradation potential were identified in the pyrene-degrading bacterial communities, which can enrich the resource pool of pyrene-degrading strains. Overall, this study will help develop further research strategies for pollutant removal.

Organ-specific biotransformation in salmonids: Insight into intrinsic enzyme activity and biotransformation of three micropollutants.

Aquatic ecosystems continue to be threatened by chemical pollution. To what extent organisms are able to cope with chemical exposure depends on their ability to display mechanisms of defense across different organs. Among these mechanisms, biotransformation processes represent key physiological responses that facilitate detoxification and reduce the bioaccumulation potential of chemicals. Biotransformation does not only depend on the ability of different organs to display biotransformation enzymes but also on the affinity of chemicals towards these enzymes. In the present study, we explored the ability of different organs and of two freshwater fish to support biotransformation processes through the determination of in vitro phase I and II biotransformation enzyme activity, and their role in supporting intrinsic clearance and the formation of biotransformation products. Three environmentally relevant pollutants were evaluated: the polycyclic aromatic hydrocarbon (PAH) pyrene (as recommended by the OECD 319b test guideline), the fungicide azoxystrobin, and the pharmaceutical propranolol. Comparative studies using S9 sub-cellular fractions derived from the liver, intestine, gills, and brain of brown trout (Salmo trutta) and rainbow trout (Oncorhynchus mykiss) revealed significant phase I and II enzyme activity in all organs. However, organ- and species-specific differences were found. In brown trout, significant extrahepatic biotransformation was observed for pyrene but not for azoxystrobin and propranolol. In rainbow trout, the brain appeared to biotransform azoxystrobin. In this same species, propranolol appeared to be biotransformed by the intestine and gills. Biotransformation products could be detected only from hepatic biotransformation, and their profiles and formation rates displayed species-specific patterns and occurred at different magnitudes. Altogether, our findings further contribute to the current understanding of organ-specific biotransformation capacity, beyond the expression and activity of enzymes, and its dependence on specific enzyme-chemical interactions to support mechanisms of defense against exposure.

Adaptation strategies and functional transitions of microbial community in pyrene-contaminated soils promoted by lead with Pseudomonas veronii and its extracellular polymeric substances.

Pyrene was designated as a remediation target in this study, and low contamination of lead (Pb) was set to induce heavy metal stress. Pseudomonas veronii and its extracellular polymeric substances (EPSs) were chosen for biofortification, with the aim of elucidating the structural, metabolic, and functional responses of soil microbial communities. Community analysis of soil microorganisms using high-throughput sequencing showed that the co-addition of P. veronii and EPSs resulted in an increase in relative abundance of phyla associated with pyrene degradation, and formed a symbiotic system dominated by Firmicutes and Proteobacteria, which involved in pyrene metabolism. Co-occurrence network analysis revealed that the module containing P. veronii was the only one exhibiting a positive correlation between bacterial abundance and pyrene removal, indicating the potential of bioaugmentation in enriching functional taxa. Biofortification also enhanced the abundance of functional gene linked to EPS production (biofilm formation-Pseudomonas aeruginosa) and pyrene degradation. Furthermore, 17 potential functional bacteria were screened out using random forest algorithm. Lead contamination further promoted the growth of Proteobacteria, intensified cooperative associations among bacteria, and increased the abundance of bacteria with positive correlation with pyrene degradation. The results offer novel perspectives on alterations in microbial communities resulting from the synergistic impact of heavy metal stress and biofortification.

Biodegradation of pyrene by bacterial consortia: Impact of natural surfactants and iron oxide nanoparticles.

Polycyclic aromatic hydrocarbons (PAHs) are potentially hazardous compounds that could cause a severe impact on many ecosystems. They are very challenging to remove using conventional methods due to their hydrophobic nature. However, this issue can be resolved by utilizing surface-active molecules to increase their bioavailability. In this study, pyrene was chosen as the PAH compound to explore its degradability by the effect of individual bacterial strains (Pseudomonas stutzeri NA3 and Acinetobacter baumannii MN3) and mixed consortia (MC) along with natural surfactant derived from Sapindus mukorossi and iron oxide nanoparticles (NPs). Additionally, fatty acids esters, dipeptides, and sugar derivative groups were identified as potent bioactive components of natural surfactants. Various techniques, such as XRD, VSM, TEM, and FE-SEM with EDX, were utilized to characterize the pristine and Fenton-treated iron oxide NPs. The analytical results confirmed that the Fe3O4 crystal phase and spherical-shaped NPs exhibited excellent magnetic properties. The impact of natural surfactants and iron oxide NPs has significantly contributed to the biodegradation process, resulting in a prominent decrease in chemical oxygen demand (COD) levels. Gas chromatography-mass spectrometry (GC-MS) analysis showed that biodegradation systems produced primary hydrocarbon intermediates, which underwent oxidative degradation through Fenton treatment. Interestingly, synthesized iron oxide NPs effectively produced hydroxyl radical (•OH) during the Fenton reaction, which was confirmed by electron paramagnetic resonance (EPR) spectra, and the pristine iron oxide NPs underwent a material transformation observed. The study demonstrated an integrated approach for biodegradation and the Fenton reaction process to enhance the pyrene degradation efficiency (90%) compared to other systems. Using natural surfactants and iron oxide NPs in aquatic environments serves as a crucial platform at the interface of microorganisms and contaminated oil products. This interaction offers a promising solution for PAHs bioremediation.

Metagenomics reveals mechanism of pyrene degradation by an enriched bacterial consortium from a coking site contaminated with PAHs.

A bacterial consortium, termed WPB, was obtained from polycyclic aromatic hydrocarbons (PAHs) contaminated soil from a coking site. The consortium effectively degraded 100 mg L-1 pyrene by 94.8 % within 12 days. WPB was also able to degrade phenanthrene (98.3 %) and benzo[a]pyrene (24.6 %) in 12 days, while the individual isolates showed no PAHs degrading ability. Paracoccus sp. dominated the bacterial consortium (65.0-86.2 %) throughout the degradation process. Metagenomic sequencing reveals the proportion of sequences with xenobiotics biodegradation and metabolism increased throughout the degradation process indicating the great potential of WPB to degrade pollutants. The annotation of genes by metagenomic analysis help reconstruct the degradation pathways ("phthalate pathway" and "naphthalene degradation") and reveal how different bacteria contribute to the degradation process. Mycobacterium gilvum was found to carry nidAB genes that catalyze the first step of high-molecular-weight (HMW) PAHs in the degradation process despite Mycobacterium gilvum accounting for only 0.005-0.06 %. In addition, genomes of Paracoccus denitrificans and some other genera affiliated with Devosia, Pusillimonas caeni and Eoetvoesia caeni were successfully recovered and were found to carry genes responsible for the degradation of the intermediates of pyrene. These results enable further understanding of the metabolic patterns of pyrene-degrading consortia and provide direction for further cultivation and discovery of key players in complex microbial consortia.

Cometabolic degradation of pyrene with phenanthrene as substrate: assisted by halophilic Pseudomonas stutzeri DJP1.

At present, cometabolic degradation is an extensive method for the biological removal of high molecular weight polycyclic aromatic hydrocarbons (HMW-PAHs) in the marine environment. However, due to the refractory to degradation and high toxicity, there are few studies on pyrene (PYR) cometabolic degradation with phenanthrene (PHE) as substrate. In this study, a Pseudomonas stutzeri DJP1 strain isolated from sediments was used in the cometabolic system of PHE and PYR. The biomass and the activity of key enzymes such as dehydrogenase and catechol 12 dioxygenase of strain were improved, but the enhancement of biotoxicity resulted in the inhibition of cometabolism simultaneously. Seven metabolites were identified respectively in PYR, PHE degradation cultures. It was speculated that the cometabolism of PHE and PYR had a common phthalic acid pathway, and the degradation pathway of PHE was included in the downstream pathway of PYR. The functional genes such as PhdF, NidD and CatA involved in DJP1 degradation were revealed by Genome analysis. This study provides a reference for the biodegradation of PYR and PHE in real marine environment.

Biodegradation of polycyclic aromatic hydrocarbons (PAHs) by microbial consortium from paddy rice soil.

Polycyclic aromatic hydrocarbons (PAHs) are among the most widely spread pollutants in the environment including the agricultural soil. PAH degradation by indigenous bacteria is an effective and economical means to remove these pollutants from the environment. Here, we report a bacterial consortium (Pdy-1) isolated from paddy rice soil in northern Japan able to degrade polycyclic aromatic hydrocarbons (PAHs) at high rates. Pdy-1 was incubated with a mixture of PAH compounds (fluorene, phenanthrene, and pyrene) in Bushnell Haas Medium at a final concentration of 100 mg/L each. PDY-1 degraded 100% of fluorene, 95% of phenanthrene, and 52% of pyrene in 5 days. Phenanthrene and pyrene were completely degraded at 10 d and 15 d, respectively. Cloning of the 16S rRNA gene revealed that the consortium was composed of 40% Achromobacter and 7% each of Castelaniella, Rhodanobacter, and Hypomicrobium. Comamonas, Ferrovibrio, Terrimonas, Bordetella, Rhizobium, and Pseudonocardia were also detected. PCR-DGGE showed the dynamics of the consortium during the incubation period. Real-time PCR revealed that PAH degrading genes such as the gram-positive ring dihydroxylating genes (PAH-RDH) and pyrene dioxygenase (nidA) were most abundant at day 5 when the rapid biodegradation of the PAHs was observed. This study improves our understanding on dynamics and characteristics of an effective PAH-degrading bacterial consortium from paddy rice soil.

Evaluation of the Different Nutritional and Environmental Parameters on Microbial Pyrene Degradation by Mangrove Culturable Bacteria.

Mangrove ecosystems play curial roles in providing many ecological services and alleviating global climate change. However, they are in decline globally, mainly threatened by human activities and global warming, and organic pollutants, especially PAHs, are among the crucial reasons. Microbial remediation is a cost-effective and environmentally friendly way of alleviating PAH contamination. Therefore, understanding the effects of environmental and nutritional parameters on the biodegradation of polycyclic aromatic hydrocarbons (PAHs) is significant for the bioremediation of PAH contamination. In the present study, five bacterial strains, designated as Bp1 (Genus Rhodococcus), Sp8 (Genus Nitratireductor), Sp13 (Genus Marinobacter), Sp23 (Genus Pseudonocardia), and Sp24 (Genus Mycolicibacterium), have been isolated from mangrove sediment and their ring hydroxylating dioxygenase (RHD) genes have been successfully amplified. Afterward, their degradation abilities were comprehensively evaluated under normal cultural (monoculture and co-culture) and different nutritional (tryptone, yeast extract, peptone, glucose, sucrose, and NPK fertilizer) and environmental (cetyl trimethyl ammonium bromide (CTAB), sodium dodecyl sulfate (SDS)) parameters, as well with different co-contaminants (phenanthrene and naphthalene) and heavy metals (Cd2+, Cu2+, Fe3+, Ni2+, Mg2+, Mn2+, and Co2+). The results showed that strain Sp24 had the highest pyrene degradation rate (85%) in the monoculture experiment after being cultured for 15 days. Adding nitrogen- and carbon-rich sources, including tryptone, peptone, and yeast extract, generally endorsed pyrene degradation. In contrast, the effects of carbon sources (glucose and sucrose) on pyrene degradation were distinct for different bacterial strains. Furthermore, the addition of NPK fertilizer, SDS, Tween-80, phenanthrene, and naphthalene enhanced the bacterial abilities of pyrene removal significantly (p < 0.05). Heavy metals significantly reduced all bacterial isolates' degradation potentials (p < 0.05). The bacterial consortia containing high bio-surfactant-producing strains showed substantially higher pyrene degradation. Moreover, the consortia of three and five bacterial strains showed more degradation efficiency than those of two bacterial strains. These results provide helpful microbial resources for mangrove ecological remediation and insight into optimized culture strategies for the microbial degradation of PAHs.

Formulation of synthetic bacteria consortia for enzymatic biodegradation of polyaromatic hydrocarbons contaminated soil: soil column study.

As an efficient method to remove contaminants from highly polluted sites, enzyme biodegradation addresses unresolved issues such as bioremediation inefficiency. In this study, the key enzymes involved in PAH degradation were brought together from different arctic strains for the biodegradation of highly contaminated soil. These enzymes were produced via a multi-culture of psychrophilic Pseudomonas and Rhodococcus strains. As a result of biosurfactant production, the removal of pyrene was sufficiently prompted by Alcanivorax borkumensis. The key enzymes (e.g., naphthalene dioxygenase, pyrene dioxygenase, catechol-2,3 dioxygenase, 1-hydroxy-2-naphthoate hydroxylase, protocatechuic acid 3,4-dioxygenase) obtained via multi-culture were characterized by tandem LC-MS/MS and kinetic studies. To simulate in situ application of produced enzyme solutions, pyrene- and dilbit-contaminated soil was bioremediated in soil columns and flask tests by injecting enzyme cocktails from the most promising consortia. The enzyme cocktail contained about 35.2 U/mg protein pyrene dioxygenase, 61.4 U/mg protein naphthalene dioxygenase, 56.5 U/mg protein catechol-2,3-dioxygenase, 6.1 U/mg protein 1-hydroxy-2-naphthoate hydroxylase, and 33.5 U/mg protein protocatechuic acid (P3,4D) 3,4-dioxygenase enzymes. It was found that after 6 weeks, the average pyrene removal values showed that the enzyme solution could be effective in the soil column system (80-85% degradation of pyrene).

Whole-genome sequence analysis reveals phenanthrene and pyrene degradation pathways in newly isolated bacteria Klebsiella michiganensis EF4 and Klebsiella oxytoca ETN19.

Polycyclic aromatic hydrocarbons (PAHs) are diverse pollutants of significant environmental concerns, requiring effective biodegradation. This study used different bioinformatics tools to conduct whole-genome sequencing of two novel bacterial strains, Klebsiella michiganensis EF4 and K. oxytoca ETN19, to improve our understanding of their many genomic functions and degradation pathways of phenanthrene and pyrene. After 28 days of cultivation, strain EF4 degraded approximately 80% and 60% of phenanthrene and pyrene, respectively. However, their combinations (EF4 +ETN19) showed tremendous phenanthrene degradation efficiency, supposed to be at the first-level kinetic model with a t1/2 value of approximately 6 days. In addition, the two bacterial genomes contained carbohydrate-active enzymes and secondary metabolites biosynthetic gene clusters associated with PAHs degradation. The two genomes contained the bZIP superfamily of transcription factors, primarily the cAMP-response element-binding protein (CREB), which could regulate the expression of several PAHs degradation genes and enzymes. Interestingly, the two genomes were found to uniquely degrade phenanthrene through a putative pathway that catabolizes 2-carboxybenzalpyruvate into the TCA cycle. An operon containing multicomponent proteins, including a novel gene (JYK05_14550) that could initiate the beginning step of phenanthrene and pyrene degradation, was found in the EF4 genome. However, the degradation pathway of ETN19 showed that the yhfP gene encoding putative quinone oxidoreductase was associated with phenanthrene and pyrene catabolic processes. Furthermore, the significant expression of catechol 1,2-dioxygenase and quinone oxidoreductase genes in EF4 +ETN19 and ETN19 following the quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis confirmed the ability of the bacteria combination to degrade pyrene and phenanthrene effectively. These findings present new insight into the possible co-metabolism of the two bacterial species in the rapid biodegradation of phenanthrene and pyrene in soil environments.

In Vitro-In Vivo Extrapolation of Hepatic Biotransformation Data for Fish. III. An In-depth Case Study with Pyrene.

Computational models that predict chemical bioaccumulation in fish generally account for biotransformation using an apparent first-order whole-body rate constant (kB ; d-1 ). The use of such models requires, therefore, that methods exist for estimating kB , ideally without the need to expose live animals. One promising approach for estimating kB involves the extrapolation of measured in vitro intrinsic clearance (CLIN VITRO,INT ) to the whole animal (in vitro-in vivo extrapolation, [IVIVE]). To date, however, the accuracy of such predictions has been difficult to assess due to uncertainties associated with one or more extrapolation factors and/or a mismatch between fish used to generate in vitro data and those used to conduct in vivo exposures. In the present study we employed a combined in vitro and in vivo experimental approach to evaluate the IVIVE procedure using pyrene (PYR) as a model chemical. To the extent possible, measured rates of CLIN VITRO,INT were extrapolated to estimates of kB using extrapolation factors based on measured values. In vitro material (liver S9 fraction) was obtained from fish exposed to PYR in a controlled bioconcentration study protocol. Fish from the same study were then used to estimate in vivo kB values from an analysis of chemical depuration data. Averaged across four study groups, kB values estimated by IVIVE underestimated those determined from in vivo data by 2.6-fold. This difference corresponds to a 4.1-fold underestimation of true in vivo intrinsic clearance, assuming the liver is the only site of biotransformation. These findings are consistent with previous work performed using mammals and have important implications for use of measured CLIN VITRO,INT values in bioaccumulation assessments with fish. Environ Toxicol Chem 2023;42:1501-1515. Published 2023. This article is a U.S. Government work and is in the public domain in the USA.

Changes in metabolic profiles of amphipods Allorchestes compressa after acute exposures to copper, pyrene, and their mixtures.

Amphipods are ideal indicators for biomonitoring and ecotoxicological studies of environmental contaminants because they are extensively distributed in aquatic environments, are easy to collect and are important in nutrient cycling. Marine amphipods (Allorchestes compressa) were exposed to two concentrations of copper and pyrene, and their mixtures, for 24 and 48 h. Changes in polar metabolites were assessed using Gas Chromatography Mass Spectrometry (GC-MS)-based untargeted metabolomics. Generally, limited metabolite changes were observed for copper and pyrene single exposures (eight and two significant metabolites, respectively), while 28 metabolites had changed following exposures to mixtures. Furthermore, changes were mainly observed after 24 h but had seemingly returned to control levels after 48 h. Multiple types of metabolites were affected including amino acids, Tricarboxylic acid (TCA) cycle intermediates, sugars, fatty acids, and hormones. This study highlights the sensitivity of metabolomics in assessing the impacts of low concentrations of chemicals compared to traditional ecotoxicological endpoints.

Biodegradation of benzo(a)pyrene by Pseudomonas strains, isolated from petroleum refinery effluent: Degradation, inhibition kinetics and metabolic pathway.

Benzo(a)pyrene, a five-ring polyaromatic hydrocarbon, originating from coal tar, crude oil, tobacco, grilled foods, car exhaust etc, is highly persistent in the environment. It has been classified as a Group I carcinogen, as on its ingestion in human body, diol epoxide metabolites are generated, which bind to DNA causing mutations and eventual cancer. Among various removal methods, bioremediation is most preferred as it is a sustainable approach resulting in complete mineralization of benzo(a)pyrene. Therefore, in this study, biodegradation of benzo(a)pyrene was performed by two strains of Pseudomonas, i. e WDE11 and WD23, isolated from refinery effluent. Maximum benzo(a)pyrene tolerance was 250 mg/L and 225 mg/L against Pseudomonas sp. WD23 and Pseudomonas sp. WDE11 correspondingly. Degradation rate constants varied between 0.0468 and 0.0513/day at 50 mg/L with half-life values between 13.5 and 14.3 days as per first order kinetics, while for 100 mg/L, the respective values varied between 0.006 and 0.007 L/mg. day and 15.28-16.67 days, as per second order kinetics. The maximum specific growth rate of strains WDE11 and WD23 was 0.3512/day and 0.38/day accordingly, while concentrations over 75 mg/L had an inhibitory effect on growth. Major degradation metabolites were identified as dihydroxy-pyrene, naphthalene-1,2-dicarboxylic acid, salicylic acid, and oxalic acid, indicating benzo(a)pyrene was degraded via pyrene intermediates by salicylate pathway through catechol meta-cleavage. The substantial activity of the catechol 2,3 dioxygenase enzyme was noted during the benzo(a)pyrene metabolism by both strains with minimal catechol 1,2 dioxygenase activity. This study demonstrates the exceptional potential of indigenous Pseudomonas strains in complete metabolism of benzo(a)pyrene.

Risk reductions during pyrene biotransformation and mobilization in a model plant-bacteria-biochar system.

The productive application of motile microorganisms for degrading hydrophobic contaminants in soil is one of the most promising processes in modern remediation due to its sustainability and low cost. However, the incomplete biodegradation of the contaminants and the formation of the intermediary metabolites in the process may increase the toxicity in soil during bioremediation, and motile inoculants may mobilize the pollutants through biosorption. Therefore, controlling these factors should be a fundamental part of soil remediation approaches. The aim of this study was to evaluate the sources of risk associated with the cometabolism-based transformation of 14C-labeled pyrene by inoculated Pseudomonas putida G7 and identify ways to minimize risk. Our model scenario examined the increase in bioaccessibility to a distant source of contamination facilitated by sunflower (Helianthus annuus L.) roots. A biochar trap for mobilized pollutant metabolites and bacteria has also been employed. The experimental design consisted of pots filled with a layer of sand with 14C-labeled pyrene (88 mg kg-1) as a contamination focus located several centimeters from the inoculation point. Half of the pots included a biochar layer at the bottom. The pots were incubated in a greenhouse with sunflower plants and P. putida G7 bacteria. Pots with sunflower plants showed a higher biodegradation of pyrene, its mobilization as metabolites through the percolate and the roots, and bacterial mobilization toward the source of contamination, also resulting in increased pyrene transformation. In addition, the biochar layer efficiently reduced the concentrations of pyrene metabolites collected in the leachates. Therefore, the combination of plants, motile bacteria and biochar safely reduced the risk caused by the biological transformation of pyrene.

Revealing the key species for pyrene degradation in Vallisneria natans rhizosphere sediment via triple chamber rhizome-box experiments.

To identify key species associated with pyrene degradation in Vallisneria natans (V.natans) rhizosphere sediment, this work investigated the temporal and spatial changes in the rhizosphere microbial community and the relationship between the changes and the pyrene degradation process through a three-compartment rhizome-box experiment under pyrene stress. The degradation kinetics of pyrene showed that the order of degradation rate was rhizosphere > near-rhizosphere > non-rhizosphere. The difference in the pyrene degradation behavior in the sediments corresponded to the change in the proportions of dominant phyla (Firmicutes and Proteobacteria) and genera (g_Massilia f_Comamonadaceae, g_Sphingomonas). The symbiosis networks and hierarchical clustering analysis indicated that the more important phyla related to the pyrene degradation in the rhizosphere was Proteobacteria, while g_Sphigomonas, f_Comamonadaceae, and especially g_Massilia were the core genera. Among them, f_Comamonadaceae was the genus most affected by rhizosphere effects. These findings strengthened our understanding of the PAHs-degradation microorganisms in V.natans rhizosphere and are of great significance for enhancing phytoremediation on PAHs-contaminated sediment.

Cellular signaling crosstalk between Wnt signaling and gap junctions inbenzo[a]pyrene toxicity.

Gap junctional intercellular communication (GJIC) is considered a key biological mechanism to maintain homeostasis in cell differentiation and growth. In addition, as another major signaling pathway associated with cell proliferation and differentiation, Wnt/ß-catenin signaling appears to trigger several cellular responses against injury. The purpose of the present study was to investigate the effects of a known toxic agent, benzo[a]pyrene (BaP), on the regulation and interaction between GJIC and Wnt/ß-catenin signaling. BaP treatment resulted in GJIC inhibition and decreases the major GJIC protein connexin 43 (Cx43) in WB-F344 rat liver epithelial cells. We also found BaP-mediated downregulation of Wnt/ß-catenin signaling related to the PI3K-Akt pathway. To identify the relationship between GJIC and Wnt/ß-catenin signaling, we treated WB-F344 cells with the Wnt agonist CHIR99021 and found that it inhibited GJIC while causing a significant reduction in Cx43 expression at both the mRNA and protein levels, through the repression of promoter activity. This Wnt agonist-mediated GJIC inhibition was confirmed using a small interfering RNA directed against the Wnt antagonist Dact2, indicating that Wnt/ß-catenin signaling negatively regulates GJIC. Despite the inverse correlation between Wnt/ß-catenin signaling and Cx43 promoter activation as indicated by downregulation of ß-catenin nuclear translocation and upregulation of Cx43 promoter activation involving HNF3ß, BaP treatment decreased the Cx43 protein expression, which was associated with protein degradation, possibly through protein kinase C activation. In conclusion, our results revealed the mechanism of BaP-induced inhibition of GJIC and Wnt/ß-catenin signaling. More importantly, linking Wnt/ß-catenin signaling to Cx protein expression will have profound implications in understanding the relationships among different major signaling pathways associated with cell proliferation and differentiation in toxicity.

Urinary PAHs metabolites in Karakoram Highway's heavy traffic vehicle (HTV) drivers: evidence of exposure and health risk.

The current study features PAHs exposure on Karakoram Highway, a route of utmost importance in Pakistan. The drivers of heavy traffic vehicles (HTV) on Karakoram Highway spend long hours amid dense traffic and therefore, inevitably inhale huge amount of PAH carcinogens. The urinary metabolites of PAHs in such drivers (meeting selection criteria n = 48) and a control group (n = 49) were comparatively profiled. The higher urinary biomarkers among ninety-six percent HTV drivers were evident of PAHs exposure. We observed elevated concentrations of urinary benzo[a]pyrene metabolites (3-OH-BaP = 3.53 ± 0.62 ng g-1 creatinine and 9-OH-BaP = 3.69 ± 0.74 ng g-1 creatinine) in HTV driver's samples compared to controls (0.85 ± 0.08 and 0.31 ± 0.03 ng g-1 creatinine, respectively). Interestingly, urinary benzo[a]pyrene metabolites were detected in almost similar amount among HTV drivers irrespective of their working hours. A distinct smoking effect was manifested with rising urinary levels of 1-hydroxypyrene, 2-hydroxyphenanthrene, and 3-hydroxybenzo[a]pyrene with corresponding increase in driving hours per day. These metabolites exhibited characteristic exposures to low molecular weight volatile PAHs that are commonly found in vehicular exhaust. The elevated PAH body burden was directly linked to the nature of their job and the route-long environmental pollution on Karakoram Highway. Additionally, the poor economic status and smoking also increased HTV driver's health vulnerability and significantly declined their health capacity. There was conclusive evidence that HTV drivers were exposed to PAHs during a ride on Karakoram Highway, back and forth, an aspect not reported earlier.

Diminishing toxicity of pyrene on photosynthetic performance of soybean using Bacillus subtilis (NCIM 5594).

Polycyclic aromatic hydrocarbons are persistent organic pollutants causing serious environmental problems, being toxic to plants and difficult to remediate. Pyrene is one such extremely dangerous compound that is toxic for the environment. This study suggests the use of Bacillus subtilis (National Collection of Industrial Microorganisms [NCIM] 5594) to overcome inhibitory effects of pyrene on soybean photosynthesis. The toxicity of pyrene to soybean was evident from a significant decrease in seed germination parameters, photosynthetic performance and biomass during growth of soybean in pyrene contaminated soil. Efficiency of performance index, light absorption, trapping and electron transport were reduced in plants grown in pyrene contaminated soil while significant recovery in these parameters was observed in plants grown in pyrene+B. subtilis treated soil. Activity levels of dehydrogenase and lipase enzymes significantly recovered in pyrene+B. subtilis treated soil. After extraction of pyrene from soil and soybean plant, concentration of pyrene was lowered in pyrene+B. subtilis treated soil and plants. These findings suggest efficient degradation of pyrene by B. subtilis . About 70% degradation of pyrene was achieved in soil using B. subtilis ; thus it is a useful strain for crop improvement in pyrene polluted soil.

Allochthonous arbuscular mycorrhizal fungi promote Salix viminalis L.-mediated phytoremediation of polycyclic aromatic hydrocarbons characterized by increasing the release of organic acids and enzymes in soils.

Polycyclic aromatic hydrocarbons (PAHs) are well known persistent organic pollutants that have carcinogenic, teratogenic, and mutagenic effects on humans and animals. Arbuscular mycorrhizal fungi (AMF) that can infest plant hosts and form symbioses may help plants to enhance potential rhizosphere effects, thus contributing to the rhizodegradation of PAH-contaminated soils. The present study aimed to assess the effectiveness of AMF on enhancing Salix viminalis-mediated phytoremediation of PAH-polluted soil and clarify the plant enzymatic and organic acid mechanisms induced by AMF. Natural attenuation (NA), phytoremediation (P, Salix viminalis), S. viminalis-AMF combined remediation using willow inoculated with Funneliformis mosseae (PM), Laroideoglomus etunicatum (PE), and Rhizophagus intraradices (PI) were used as strategies for the remediation of PAH-polluted soils. The results showed that AMF inoculation contributed to the dissipation of the high-molecular-weight PAH benzo (α) pyrene that had concentrations in PM, PE, and PI treatments of 40.1 %, 24.49 %, and 36.28 % of the level in the NA treatment, and 62.32 %, 38.05 %, and 56.38 % of the level in the P treatment after 90 days. The mycorrhizal treatment also improved the removal efficiency of phenanthrene and pyrene, as their concentrations were sharply decreased after 30 days compared to the NA and P treatments. The research further clarified the changes in rhizosphere substances induced by AMF. Organic acids including arachidonic acid, octadecanedioic acid, α-linolenic acid, 10,12,14-octadecarachidonic acid and 5-methoxysalicylic acid that can act as co-metabolic substrates for certain microbial species to metabolize PAHs were significantly increased in AMF-inoculated treatments. AMF inoculation also elevated the levels of polyphenol oxidase, laccase, and dehydrogenase, that played crucial roles in PAHs biodegradation. These findings provide an effective strategy for using AMF-assisted S. viminalis to remediate PAH-polluted soils, and the results have confirmed the key roles of organic acids and soil enzymes in plant-AMF combined remediation of PAHs.

Contribution of dicarboxylic acids to pyrene biodegradation and transcriptomic responses of Enterobacter sp. PRd5.

The colonization of degrading endophytic bacteria is an effective means to reduce the residues of polycyclic aromatic hydrocarbons (PAHs) in crops. Dicarboxylic acids, as the main active components in crops, can affect the physiological activities of endophytic bacteria and alter the biodegradation process of PAHs in crops. In this study, malonic acid and succinic acid were selected as the representatives to investigate the contribution of dicarboxylic acids to pyrene biodegradation by endophytic Enterobacter sp. PRd5 in vitro. The results showed that dicarboxylic acids improved the biodegradation of pyrene and altered the expression of the functional gene of strain PRd5. Malonic acid and succinic acid reduced the half-life of pyrene by 20.0% and 27.8%, respectively. The degrading enzyme activities were significantly stimulated by dicarboxylic acids. There were 386 genes up-regulated and 430 genes down-regulated in strain PRd5 with malonic acid, while 293 genes up-regulated and 340 genes down-regulated with succinic acid. Those up-regulated genes were distributed in the functional classification of signal transduction, membrane transport, energy metabolism, carbohydrate metabolism, and amino acid metabolism. Malonic acid mainly enhanced the central carbon metabolism, cell proliferation, and cell activity. Succinic acid mainly improved the expression of degrading gene. Overall, the findings of this study provide new insights into the regulation and control of PAH stress by crops. KEY POINTS: • Dicarboxylic acids improved the biodegradation of pyrene by Enterobacter sp. PRd5. • The degrading enzyme activities were stimulated by dicarboxylic acids. • There are different facilitation mechanisms between malonic acid and succinic acid.