Detoxification Of Polycyclic Aromatic Hydrocarbons Research Articles

Plant Defense Mechanisms against Polycyclic Aromatic Hydrocarbon Contamination: Insights into the Role of Extracellular Vesicles.

Polycyclic aromatic hydrocarbons (PAHs) are persistent organic pollutants that pose significant environmental and health risks. These compounds originate from both natural phenomena, such as volcanic activity and wildfires, and anthropogenic sources, including vehicular emissions, industrial processes, and fossil fuel combustion. Their classification as carcinogenic, mutagenic, and teratogenic substances link them to various cancers and health disorders. PAHs are categorized into low-molecular-weight (LMW) and high-molecular-weight (HMW) groups, with HMW PAHs exhibiting greater resistance to degradation and a tendency to accumulate in sediments and biological tissues. Soil serves as a primary reservoir for PAHs, particularly in areas of high emissions, creating substantial risks through ingestion, dermal contact, and inhalation. Coastal and aquatic ecosystems are especially vulnerable due to concentrated human activities, with PAH persistence disrupting microbial communities, inhibiting plant growth, and altering ecosystem functions, potentially leading to biodiversity loss. In plants, PAH contamination manifests as a form of abiotic stress, inducing oxidative stress, cellular damage, and growth inhibition. Plants respond by activating antioxidant defenses and stress-related pathways. A notable aspect of plant defense mechanisms involves plant-derived extracellular vesicles (PDEVs), which are membrane-bound nanoparticles released by plant cells. These PDEVs play a crucial role in enhancing plant resistance to PAHs by facilitating intercellular communication and coordinating defense responses. The interaction between PAHs and PDEVs, while not fully elucidated, suggests a complex interplay of cellular defense mechanisms. PDEVs may contribute to PAH detoxification through pollutant sequestration or by delivering enzymes capable of PAH degradation. Studying PDEVs provides valuable insights into plant stress resilience mechanisms and offers potential new strategies for mitigating PAH-induced stress in plants and ecosystems.

Exposure of Polycyclic Aromatic Hydrocarbons (PAHs) and Crude Oil to Atlantic Haddock (Melanogrammus aeglefinus): A Unique Snapshot of the Mercapturic Acid Pathway.

Fish exposed to xenobiotics like petroleum-derived polycyclic aromatic hydrocarbons (PAHs) will immediately initiate detoxification systems through effective biotransformation reactions. Yet, there is a discrepancy between recognized metabolic pathways and the actual metabolites detected in fish following PAH exposure like oil pollution. To deepen our understanding of PAH detoxification, we conducted experiments exposing Atlantic haddock (Melanogrammus aeglefinus) to individual PAHs or complex oil mixtures. Bile extracts, analyzed by using an ion mobility quadrupole time-of-flight mass spectrometer, revealed novel metabolites associated with the mercapturic acid pathway. A dominant spectral feature recognized as PAH thiols set the basis for a screening strategy targeting (i) glutathione-, (ii) cysteinylglycine-, (iii) cysteine-, and (iv) mercapturic acid S-conjugates. Based on controlled single-exposure experiments, we constructed an interactive library of 33 metabolites originating from 8 PAHs (anthracene, phenanthrene, 1-methylphenanthrene, 1,4-dimethylphenanthrene, chrysene, benz[a]anthracene, benzo[a]pyrene, and dibenz[a,h]anthracene). By incorporation of the library in the analysis of samples from crude oil exposed fish, PAHs conjugated with glutathione and cysteinylglycine were uncovered. This qualitative study offers an exclusive glimpse into the rarely acknowledged mercapturic acid detoxification pathway in fish. Furthermore, this furnishes evidence that this metabolic pathway also succeeds for PAHs in complex pollution sources, a notable discovery not previously reported.

Enhancement of catalytic detoxification of polycyclic aromatic hydrocarbons in fly ash from municipal solid waste incineration via magnetic hydroxyapatite-assisted hydrothermal treatment

The emission of carcinogenic, teratogenic, and mutagenic polycyclic aromatic hydrocarbons (PAHs) during municipal solid waste incineration (MSWI) of fly ash (FA) has attracted significant attention. Hydrothermal treatment (HT) has emerged as a practical approach for degrading PAHs during MSWI of FA by utilizing magnetite (Fe3O4) as a catalyst and hydrogen peroxide (H2O2) as an oxidizing agent. In this study, as an alternative to traditional hydroxyapatite (HAP), eggshell-derived magnetic hydroxyapatite (MHAP) was synthesized and applied in the hydrothermal catalytic degradation of PAHs in MSWI FA in an H2O2 system for the first time. The degradation efficiency of the PAHs is influenced not only by H2O2 but also by the choice of hydroxyapatite. Adding HAP or MHAP during hydrothermal treatment with H2O2 substantially reduced the overall PAH concentration and toxicity equivalent quantity (TEQ), superior to that without H2O2. MHAP demonstrated superior catalytic activity compared to HAP in the presence of H2O2 in the hydrothermal system. The hydrothermal detoxification of the PAHs increased with increasing MHAP dosage. By employing 0.5 mol/L H2O2 as the oxidant and 15 wt% MHAP as the catalyst, a total PAH degradation rate of 88.9 % was achieved, with a remarkable TEQ degradation rate of 98.3 %. Notably, the level of 4–6-ring PAHs, particularly benzo(a) pyrene (BaP) and dibenz(a,h)anthracene (DahA), with a TEQ of 1.0, was significantly reduced (by 69.4 % and 46.0 %, respectively). MHAP remained stable during the hydrothermal catalytic process, whereas H2O2 was effectively activated by MHAP and decomposed to produce strongly oxidizing hydroxyl (•OH) under hydrothermal conditions. •OH produced from the decomposition of H2O2 and metals on the surface of MHAP act as catalytically active centers, efficiently converting high-ring PAHs to low-ring PAHs. These findings provide valuable insights and a technological foundation for PAH detoxification in MSWI FA via hydrothermal catalytic oxidation.

Low levels of flavonoids in Arabidopsis thaliana during phenanthrene exposure suggest new roles for a flavanone 3-hydroxylase

Polycyclic aromatic hydrocarbons (PAHs) contamination impacts the well-being of fauna, flora, and the environment. PAHs are a group of compounds consisting of two or more benzene rings. Due to their structure, PAHs are very stable, generally non-reactive and resistant to biodegradation; yet these compounds are cytotoxic in biological systems. In plants, PAHs induce oxidative stress and cell death, and inhibit growth. Previous PAH exposure studies from our laboratory showed an up-regulation of the Arabidopsis thaliana gene At3g51240. At3g51240 encodes a flavanone 3-hydroxylase (F3H), an enzyme catalyzing the hydroxylation of flavanones, key intermediates in the flavonoid biosynthesis pathway. Considering that flavonoids may act as antioxidants and protect cells against oxidative stress, we hypothesized that F3H was important in alleviating PAH-induced stress symptoms. Analysis of flavonoid content by high performance liquid chromatography showed low levels of flavonoids in phenanthrene-treated plants. Analysis of At3g51240 loss-of-function mutant plant lines growing on phenanthrene-containing growth media showed reduced phenotypic stress responses. Interestingly, low levels of H2O2 were observed in the mutant plants, suggesting that this putative flavanone 3-hydroxylase may be involved in PAH oxidation, with the consequent H2O2 generation. Furthermore, in vitro assays with purified recombinant 6xHis tagged AT3G51240 showed enzyme activity on the PAH when phenanthrene was used as a substrate. Together, these data suggest that F3H is involved in PAH detoxification in planta with the subsequent accumulation of toxic metabolites. This research also indicates the potentiality of using specialized metabolism enzymes such as oxidoreductase in the genetic engineering of plants to remediate PAH-contaminated soils.

Roles of Natural Phenolic Compounds in Polycyclic Aromatic Hydrocarbons Abiotic Attenuation at Soil-Air Interfaces through Oxidative Coupling Reactions.

Little information is available on the roles of natural phenolic compounds in polycyclic aromatic hydrocarbons (PAHs) attenuation at dry soil-air interfaces. The purpose of this study was to determine the roles of model phenolic constituents of soil organic matter (SOM) on the abiotic attenuation of PAHs. The phenolic compounds can significantly change the attenuation rates of PAHs, among which hydroquinone was the most effective in promoting anthracene and benzo[a]anthracene attenuation. Product identification and sequential extraction experiments revealed hydroquinone enhanced the formation of oxidative coupling products and promoted the incorporation of PAHs into humic analogues, thereby reducing potential risks to humans and ecosystems. Electron paramagnetic resonance spectroscopy analyses showed both PAHs and phenolic compounds could donate electrons to Lewis acid sites of soil minerals, resulting in the generation of persistent free radicals (PFRs). PFRs could promote the generation of ·OH to enhance PAH oxidation and could cross-couple with PAHs, resulting in high-molecular-weight oxidative coupling products. This study revealed for the first time the reaction mechanism between PAHs and phenolic components of SOM under relatively dry conditions and provided new insights into promoting PAHs detoxification in soils but also a potential strategy to increase the organic carbon sequestration.

Nitrogen addition enhanced the polycyclic aromatic hydrocarbons dissipation through increasing the abundance of related degrading genes in the soils

High concentrations of Polycyclic Aromatic Hydrocarbons (PAHs) in the soils cause significant threats to human health. Since nitrogen plays a crucial role in controlling microbial composition and functions in terrestrial ecosystems, bio-stimulation based on nitrogen has been used in PAHs contaminated environments remediation. Recent studies show that microbial community composition and organic pollutants dissipation correlate with nitrogen addition. Here, we investigated the effect of nitrogen addition on the abundance of microbial community, degrading genes, and their relationship to PAHs dissipation. After a 32-day experiment, PAHs residues in nitrogen treatment soil were reduced by 23.23%−34.21%. The application of 80 mg·kg−1 nitrate and ammonium nitrogen resulted in higher PAHs removal efficiency, and the dissipation rate of PAHs was 59.61% and 62.09%, respectively. Nitrogen application could improve the abundance and the diversity of soil microbial community. Degrading genes involved in PAH detoxification were enhanced after nitrogen addition, particularly those encoding ring-hydroxylating and catechol dioxygenases such as nahAc and nidA, thus, accelerating PAH dissipation in the soil. The results will facilitate the development of beneficial microbiome-based remediation strategies and improve agricultural production safety in PAHs-contaminated soils.

The Relationship of Genetic Polymorphism Micromal Epoxide Hydrolase (EPHX1) His139Arg and Lung Cancer

Background: Microsomal epoxide hydrolase 1 (EPHX1) plays an important role in both activation and detoxification of polycyclic aromatic hydrocarbons (PAH) and aromatic amines. Polymorphism EPHX1 His139Arg in susceptibility to lung cancer has been reported with inconsistent outcomes. Aim of this study was to analyze the relationship between this polymorphism and lung cancer in smokers.Method: Consecutive sampling and case-control study was applied. Genotyping was performed by PCR-RFLP assay. The chi-square test with p<0.05 considered as significant.Results: Of all 84 subjects, in case and control groups, wild-type variant His139His were 34 (81%) and 30 (71.4%), heterozygote variant His139Arg were 8 (19%) and 12 (28.6%), there was no homozygote variant Arg139Arg identified. (p=0.36).Conclusion: The EPHX1 His139His represents a common polymorphism in both of subject groups. There is no association between His139Arg polymorphism of EPHX1 and lung cancer.

Identification of a novel CYP4V gene in the polychaete Perinereis aibuhitensis: transcriptional comparison with a CYP4B gene exposed to PAHs.

Polychaete worms can biotransform polycyclic aromatic hydrocarbons (PAHs) in environments, and the cytochrome P450 (CYP) enzyme plays an important role in this process. Herein, a novel cytochrome P450 gene was identified and characterized from the polychaete worm Perinereis aibuhitensis. The full-length cDNA, which is named CYP4V82, is 1709bp encoding a protein of 509 amino acids and has high similarity to CYP4V. The expression levels of CYP4V82 and CYP4BB4 (a CYP gene identified from P. aibuhitensis in a previous study, Chen et al. Mar Pollut Bull 64:1782-1788, 2012) exposure to various concentrations of benzo[a]pyrene (B[a]P) (0.5, 2, 4, and 8μg/L) and same mass concentrations of fluoranthene (Flu, 3.2μg/L), phenanthrene (Phe, 2.9μg/L), B[a]P (4.0μg/L) were detected to identify the function of the CYP4 family in P. aibuhitensis. Compared with CYP4BB4, CYP4V82 mRNA was minimally expressed on day 7 but highly sensitive on day 14. Notably, the expression levels of CYP4V82 and CYP4BB4 were relatively different in short-term responses to PAHs with different benzene rings of the same concentration. The expression of CYP4V82 in the B[a]P group was the highest, while that of CYP4BB4 in the Phe group was relatively higher than the two other groups. These findings suggest that PAHs are associated with the induction of CYP4V82 and CYP4BB4 expressions in P. aibuhitensis, which may have different efficiencies in the detoxification of PAHs.

Effect of magnetite on the catalytic oxidation of polycyclic aromatic hydrocarbons in fly ash from MSW incineration: A comparative study of one-step and two-step hydrothermal processes

Polycyclic aromatic hydrocarbons (PAHs), many of which are carcinogenic, teratogenic, and mutagenic, exist in fly ash (FA) produced from municipal solid waste incineration (MSWI). Hydrothermal treatment (HT) is an efficient approach to remove PAHs from MSWI FA. Here, magnetite (Fe3O4) was used as the catalyst and hydrogen peroxide (H2O2) as the oxidant for one-step and two-step catalytic hydrothermal methods. When the magnetite dosage increased to 15 wt%, the maximum degradation rates of PAHs were 84.36% and 92.51%, respectively; however, the toxicity equivalent quantity (TEQ) degradation rates of the PAHs both increased upon increasing the magnetite dose. At 20 wt% Fe3O4, the maximum TEQ degradation rates of the PAHs were 93.29% and 97.76%, respectively. The reaction between OH and PAHs is non-selective, which means that LMW, MMW, and HMW PAHs were all degraded. The decrease in TEQ was mainly due to the degradation of HMW PAHs, i.e., those with five rings. Under the same Fe3O4 dose, oxidant dose, and reaction time, the detoxification of PAHs by the two-step method was significantly better than that of the one-step method, possibly because the two-step method more effectively produced OH. The first step degraded more than 90% of PAHs, and the residual PAHs in the HT products of the first step limited the utilization of the oxidant during the second step. The minerals in the HT products implied that the two-step hydrothermal method not only produced more OH, which reacted with PAHs, but also generated metal-magnetite substitution, which affected its surface reactivity during heavy metal adsorption and catalysis. These results revealed that both magnetite and the two-step hydrothermal treatment degraded PAHs. 20 wt% magnetite was the optimal amount during the two-step hydrothermal catalytic oxidation of MSWI FA.

Expression profile of a novel glutathione S-transferase gene in the marine polychaete Perinereis aibuhitensis in short-term responses to phenanthrene, fluoranthene, and benzo[α]pyrene

Polychaete worms can eliminate polycyclic aromatic hydrocarbons (PAHs) in environments through a mechanism that increases their water solubility. This detoxification starts with cytochrome P450 enzymes (CYPs) and then with glutathione S-transferases (GSTs). Here, a novel GST gene was identified and characterized from the widespread polychaete Perinereis aibuhitensis. The full-length cDNA of GST is 1544 bp and encodes 256 amino acids, belonging to the omega class. Gene expression patterns in P. aibuhitensis showed that its transcriptional level was positively correlated with the concentration of benzo[α]pyrene (0.5, 2, 4, and 8 μg/L) exposure but was negatively correlated with a PAH benzene ring after it was exposed to the same mass concentrations of fluoranthene (3.2 μg/L), phenanthrene (2.9 μg/L), and benzo[α]pyrene (4.0 μg/L) during the 14-day experimentation. These findings indicate that omega GST may play an important role in the phase II detoxification of PAHs in polychaete worms, and the persistence and bioavailability of PAHs may depend on benzene rings.

Effect of Ca–Si–Al Element Proportion on the Formation of Aluminosilicate Minerals and Detoxification of PAHs in Fly Ash from MSW Incineration during the Hydrothermal Process

Fly ash (FA) produced during municipal solid waste incineration contains hazardous polycyclic aromatic hydrocarbons (PAHs). Hydrothermal treatment (HT), a promising technology for the degradation of organic compounds, was applied in this study for hydrothermal detoxification of PAHs in FA. Due to different levels of Ca/(Si + Al) and Al/(Si + Al) molar ratios achieved by adjusting the SiO₂ and Al₂O₃ added to FA, different kinds of aluminosilicate minerals were formed and different detoxification efficiencies of PAHs were observed in HT products. For aluminosilicate minerals formed at lower (0.17) and moderate (0.51) Al/(Si + Al) ratio levels, tobermorite was obtained and it enhanced with the decrease of Ca/(Si + Al) ratio levels. At higher Al/(Al + Si) ratio levels of 0.71, tobermorite cannot be synthesized completely even when the Ca/(Si + Al) ratio for FA-30-3 (1.01) was low and in the suitable range for tobermorite formation (0.67–1.20). Compared with the Ca/(Si + Al) ratio, the Al/(Si + Al) ratio not only affected the synthesis of tobermorite but also had much more significant influence on both the removal percentage of the concentration of PAHs and the removal percentage of the toxicity equivalent quality (TEQ) of PAHs. Attributing to the fact that tobermorite cannot be synthesized at higher Al/(Al + Si) ratio levels of 0.71, the weakest detoxification efficiency of PAHs was observed. When tobermorite was effectively synthesized in FA-30-1, for which both the Ca/(Al + Si) and Al/(Al + Si) ratios were in the suitable range for tobermorite formation [Ca/(Al + Si) ratio = 0.67–1.20 and Al/(Al + Si) ratio = 0–0.22], the best detoxification efficiency of PAHs was achieved. The formation of tobermorite affected by the Ca–Si–Al element proportion during the HT process has a positive effect on the detoxification of PAHs in FA. The results of regression analysis performed for investigating the relationship between the content of minerals formed and the detoxification efficiency of PAHs showed that the formation of tobermorite is helpful to promote the detoxification of PAHs but the existence of hydrocalumite reduced the detoxification. Compared with the Ca/(Si + Al) ratio, the Al/(Si + Al) ratio has more significant influence on the formation of tobermorite and hydrocalumite.

Altered Metabolism of Polycyclic Aromatic Hydrocarbons by UDP-Glycosyltransferase 3A2 Missense Variants.

The UDP-glycosyltransferase (UGT) family of enzymes are important in the metabolism of a variety of exogenous substances including polycyclic aromatic hydrocarbons (PAHs), a potent class of environmental carcinogens. As compared to the majority of UGT enzymes, which utilize UDP-glucuronic acid as a cosubstrate, UGT3A2 utilizes alternative cosubstrates (UDP-glucose and UDP-xylose). UGT3A2 is expressed in aerodigestive tract tissues and was highly active against multiple PAHs with both cosubstrates. The goal of the present study was to assess the functional effects of UGT3A2 missense variants (MAF ≥ 0.005) on PAH metabolism and the utilization of cosubstrates. The glycosylation activity (Vmax/Km) of all variants against simple PAHs using both cosubstrates was significantly (P < 0.05) decreased by 42-100% when compared to wild-type UGT3A2. When utilizing UDP-glucose, the variant isoforms exhibited up to a 362-fold decrease in Vmax/Km when compared to wild-type UGT3A2, with a 3.1- to 14-fold decrease for D140N, A344T, and S435Y, a 24- and 43-fold decrease for A436T and R445C, respectively, and a 147- and 362-fold decrease for Y474C and Y74N, respectively. When utilizing UDP-xylose, the variants exhibited up to a 4.0-fold decrease in Vmax/Km when compared to wild-type UGT3A2; Y74N did not exhibit activity, and Y474C did not reach saturation (Km > 4000 μM). Additionally, both wild-type and variant UGT3A2 exhibited a significant (P < 0.05) difference in their utilization of UDP-glucose vs UDP-xylose as cosubstrates using 1-OH-pyrene as substrate. These data suggest that UGT3A2 missense variants decrease the detoxification of PAHs, potentially resulting in altered individual risk for PAH-related cancers.

Transcriptomic analysis of polyaromatic hydrocarbon degradation by the halophilic fungus Aspergillus sydowii at hypersaline conditions.

Polycyclic aromatic hydrocarbons (PAHs) are among the most persistent xenobiotic compounds, with high toxicity effects. Mycoremediation with halophilic Aspergillus sydowii was used for their removal from a hypersaline medium (1 M NaCl). A. sydowii metabolized PAHs as sole carbon sources, resulting in the removal of up to 90% for both PAHs [benzo [a] pyrene (BaP) and phenanthrene (Phe)] after 10 days. Elimination of Phe and BaP was almost exclusively due to biotransformation and not adsorption by dead mycelium and did not correlate with the activity of lignin modifying enzymes (LME). Transcriptomes of A. sydowii grown on PAHs, or on glucose as control, both at hypersaline conditions, revealed 170 upregulated and 76 downregulated genes. Upregulated genes were related to starvation, cell wall remodelling, degradation and metabolism of xenobiotics, DNA/RNA metabolism, energy generation, signalling and general stress responses. Changes of LME expression levels were not detected, while the chloroperoxidase gene, possibly related to detoxification processes in fungi, was strongly upregulated. We propose that two parallel metabolic pathways (mitochondrial and cytosolic) are involved in degradation and detoxification of PAHs in A. sydowii resulting in intracellular oxidation of PAHs. To the best of our knowledge, this is the most comprehensive transcriptomic analysis on fungal degradation of PAHs.

Abstract A41: Assessing the effect of glucobrassicin-rich Brussels sprouts on the metabolism of deuterated phenanthrene: Developing food-based chemoprevention of tobacco-related lung cancer

Abstract Introduction: Indole-3-carbinol (I3C) and 3,3-´diindolylmethane (DIM), derived from the glucosinolate glucobrassicin, are found in cruciferous vegetables. I3C and DIM possess a potent chemopreventive effect against the development of tobacco carcinogen-induced lung tumors in mice. Polycyclic aromatic hydrocarbons (PAH) are potent lung carcinogens present in high levels in tobacco smoke. An individual’s ability to metabolically activate and/or detoxify PAH may be related to lung cancer risk from smoking. We hypothesize that I3C administered by eating a rational dose of Brussels sprouts, defined in terms of glucobrassicin concentration, can decrease the metabolic activation and/or increase detoxification of PAH, represented by the noncarcinogenic PAH [D10]Phenanthrene ([D10]Phe). Use of deuterated phenanthrene eliminates confounding by environmental phenanthrene. Metabolic activation of [D10]Phe is represented by [D10]Phenanthrene tetraol ([D10]PheT), and detoxification is represented by [D10]phenanthrol. We are conducting a clinical trial to determine whether glucobrassicin-rich Brussels sprouts can favorably modify the metabolism of the [D10]Phe. Study Design: This is a single-arm clinical trial. The primary objective is to determine the effect of a 7-day course of Brussels sprout consumption on [D10]PheT. Secondary objectives include analysis of [D10]phenanthrol and the [D10]PheT:[D10]phenanthrol ratio. Exploratory objectives include determining the correlation between 24 h urinary DIM with the reduction in [D10]PheT, and determining the effect of Brussels sprout consumption on circulating immune cell composition and activity. Forty-eight generally healthy, adult current smokers and former smokers will be enrolled. Subjects are given 1 microgram of [D10]phe, and all urine is collected for 6 hours (h) afterwards to quantify baseline levels of [D10]PheT and [D10]phenanthrol. Subjects then consume 200 micromol glucobrassicin in the form of raw Brussels sprouts (~200-300 g) once daily for 7 days. Urine is collected for 24 h after vegetable consumption on days 3 ± 1 and 6 of the feeding intervention for DIM quantification. On day 7 of the feeding intervention, a second dose of 1 microgram of [D10]phe is administered at the study center 60 minutes ± 10 minutes after vegetable consumption, followed by another 6 h urine collection. [D10]pheT and [D10]phenanthrol levels are quantified using a validated tandem mass spectrometry assay. Pre- and post-[D10]PheT levels are compared using a paired t-test after log transformation. The results will also be analyzed by GSTT1 and GSTM1 genotyping. Results: Twelve subjects have completed the study. Baseline [D10]PheT levels ranged from 48.34 to 331.05 pmol/6 h. Day 7 [D10]PheT levels ranged from 26.57 to 333.35 pmol/6 h. Percent change from baseline to Day 7 [D10]PheT ranged from a 33% decrease to an 86.5% increase (mean +/- SE of 12.8 +/- 11.6%). Conclusions: The study is ongoing and more subjects are necessary to make a definitive conclusion. Citation Format: Naomi Fujioka, Michelle To, Bruce R. Lindgren, Vincent A. Fritz, Charles Rohwer, Dorothy Hatsukami, Stephen S. Hecht. Assessing the effect of glucobrassicin-rich Brussels sprouts on the metabolism of deuterated phenanthrene: Developing food-based chemoprevention of tobacco-related lung cancer [abstract]. In: Proceedings of the AACR Special Conference on Environmental Carcinogenesis: Potential Pathway to Cancer Prevention; 2019 Jun 22-24; Charlotte, NC. Philadelphia (PA): AACR; Can Prev Res 2020;13(7 Suppl): Abstract nr A41.

UDP-Glycosyltransferase 3A Metabolism of Polycyclic Aromatic Hydrocarbons: Potential Importance in Aerodigestive Tract Tissues.

Polycyclic aromatic hydrocarbons (PAHs) are potent carcinogens and are a primary risk factor for the development of lung and other aerodigestive tract cancers in smokers. The detoxification of PAHs by glucuronidation is well-characterized for the UDP-glycosyltransferase (UGT) 1A, 2A, and 2B subfamilies; however, the role of the UGT3A subfamily in PAH metabolism remains poorly understood. UGT3A enzymes are functionally distinct from other UGT subfamilies (which use UDP-glucuronic acid as a cosubstrate) due to their utilization of alternative cosubstrates (UDP-N-acetylglucosamine for UGT3A1, and UDP-glucose and UDP-xylose for UGT3A2). The goal of the present study was to characterize UGT3A glycosylation activity against PAHs and examine their expression in human aerodigestive tract tissues. In vitro metabolism assays using UGT3A2-overexpressing cell microsomes indicated that UGT3A2 exhibits glycosylation activity against all of the simple and complex PAHs tested. The Vmax/Km ratios for UGT3A2 activity with UDP-xylose versus UDP-glucose as the cosubstrate ranged from 0.65 to 4.4 for all PAHs tested, demonstrating that PAH glycosylation may be occurring at rates up to 4.4-fold higher with UDP-xylose than with UDP-glucose. Limited glycosylation activity was observed against PAHs with UGT3A1-overexpressing cell microsomes. While UGT3A2 exhibited low levels of hepatic expression, it was shown by western blot analysis to be widely expressed in aerodigestive tract tissues. Conversely, UGT3A1 exhibited the highest expression in liver with lower expression in aerodigestive tract tissues. These data suggest that UGT3A2 plays an important role in the detoxification of PAHs in aerodigestive tract tissues, and that there may be cosubstrate-dependent differences in the detoxification of PAHs by UGT3A2.SIGNIFICANCE STATEMENTUGT3A2 is highly active against PAHs with either UDP-glucose or UDP-xylose as a cosubstrate. UGT3A1 exhibited low levels of activity against PAHs. UGT3A1 is highly expressed in liver while UGT3A2 is well expressed in extrahepatic tissues. UGT3A2 may be an important detoxifier of PAHs in humans.

Glycosylation of polycyclic aromatic hydrocarbons by UDP‐glycosyltransferase 3A2 (UGT3A2) and aerodigestive tract tissues

Smoking is a primary risk factor for the development of lung and tobacco‐related cancers. There are over 70 carcinogens in tobacco smoke, and they belong to multiple chemical classes including polycyclic aromatic hydrocarbons (PAHs). The UDP‐glycosyltransferase (UGT) family of enzymes play a key role in the detoxification of several major tobacco carcinogens by glucuronidation, using UDP‐glucuronic acid as cosubstrate. While the UGT 1As, 2As, and 2Bs are well known to detoxify simple and complex PAHs, the UGT3A subfamily has been understudied in the detoxification of PAHs. UGT3A2 uses UDP‐glucose and UDP‐xylose as cosubstrates and is active against 1‐hydroxypyrene, a biomarker for PAH exposure. Our previous research has shown that UGT3A2 is expressed in the aerodigestive tract and is active against simple hydroxylated PAHs. The purpose of the present study was to determine the kinetic parameters of UGT3A2 against complex PAHs and determine glycosylation in aerodigestive tract tissues using UDP‐glucose and UDP‐xylose. Microsomes prepared from UGT3A2 overexpressing HEK293 cells exhibited glycosylation activity against complex PAHs including benzo[a]pyrene (B[a]P)‐7,8‐diol, B[a]P‐9,10‐diol, DB[a, l]P‐11,12‐diol, and 5‐methylchrysene‐1,2‐diol, with Km's ranging from 96 to 1250 μM using UDP‐glucose. The Km's were significantly lower when using UDP‐xylose, with Km's ranging from 120 to 168 μM for the same substrates. The Vmax/Km ratio of UDP‐xylose/UDP‐glucose ranged from 0.71 to 4.0. To determine the physiological significance and relevance of UDP‐xylose‐ and UDP‐glucose‐dependent glycosylation, activity assays were performed in tissues where UGT3A2 is expressed using 1‐hydroxypyrene as substrate. Using UDP‐glucose as the cosubstrate, glycosylation rates of 3.5, 5.2, and 13 pmol·min−1·mg−1 were observed for liver, intestine and kidney microsomes, respectively. The rates were higher using UDP‐xylose, with rates of 22, 9.3, and 20 pmol·min−1·mg−1 for liver, intestine and kidney, respectively. The glycosylation rate ratio for UDP‐xylose/UDP‐glucose ranged from 1.6 to 6.3 in the three tissues, with liver exhibiting the highest ratio. In addition, detectable glycosylation activity was observed in various aerodigestive tract tissues including esophagus, tonsil, bronchus, floor of mouth, tongue, trachea, and larynx, with tonsil exhibiting the highest relative activity with both UDP‐sugars. Incubations with UDP‐xylose resulted in higher levels of glycosylation activity than that observed with UDP‐glucose in esophagus, tonsil, floor of mouth, and larynx. These data suggest that UGT3A2 is playing an important role in the detoxification of PAHs in tobacco target tissues, with UDP‐xylose being the preferred cosubstrate in most tissues.Support or Funding InformationThis study is supported by the National Institute of Environmental Health Sciences (NIEHS) of the National Institutes of Health to P Lazarus (Grant R01‐ES025460), a NIEHS Research Supplement to Promote Diversity in Health‐Related Research to AG Vergara (Grant R01‐ES025460‐02S1), and a grant from the Health Sciences and Services Authority of Spokane, WA (Grant WSU002292).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Regulation of UGT2A1 by miR-196a-5p and miR-196b-5p.

UDP-glucuronosyltransferase (UGT) 2A1 is an important enzyme in the detoxification of polycyclic aromatic hydrocarbons found in cigarette smoke. This enzyme is expressed in aerodigestive tract tissues including lung as both its wild-type and exon 4-deleted splice variant isoforms, with the latter acting as a negative regulator of wild-type UGT2A1 activity. UGT2A1 regulation may also be mediated by microRNA (miRNA). To identify miRNA important in the regulation of UGT2A1, expression analysis in tandem with in silico analysis suggested miR-196a-5p and miR-196b-5p as potential top candidates. Significant reductions in firefly luciferase activity were observed in human embryonic kidney cell line 293 cells cotransfected with the wild-type UGT2A1 3′-untranslated region (UTR)-containing luciferase plasmid and either miR-196a-5p (62%, P = 0.00080) or miR-196b-5p (60%, P = 0.00030) mimics. In pull-down assays, there was a 3.4- and 5.2-fold increase in miR-196a-5p (P = 0.054) and miR-196b-5p (P = 0.035), respectively, using the UGT2A1 3′-UTR biotinylated mRNA probe as compared with the β-actin coding region control mRNA probe. UGT2A1 mRNA was reduced by 25% (P = 0.058) and 35% (P = 0.023) in H146 and H1944 cells, respectively, after overexpression of the miR196a-5p mimic. A similar 32% (P = 0.030) and 41% (P = 0.016) reduction was observed after over-expression of the miR-196b-5p mimic. In H146 cells transfected with miRNA mimic together with a small interfering RNA (siRNA) specific for the UGT2A1 splice variant, a significant reduction in 3-hydroxy-benzo[a]pyrene-glucuronide formation was observed. The miR-196a-5p- and miR-196b-55p-treated cells exhibited reductions of 35% (P = 0.047) and 44% (P = 0.0063), respectively. These data suggest that miR-196a-5p and miR-196b-5p play an important role in UGT2A1 regulation within the lung and potentially other aerodigestive tract tissues.

Detoxification of Polycyclic Aromatic Hydrocarbons (PAHs) by UDP‐glycosyltransferase 3A2 (UGT3A2) Variants using Alternative Sugars

The UDP‐glycosyltransferase (UGT) family of enzymes detoxify endogenous and exogenous substances by conjugating lipophilic chemicals with a UDP‐sugar, thereby making them more hydrophilic, water‐soluble and ultimately more readily excreted from the body. Examples of such chemicals are steroids, drugs, and a variety of carcinogens including polycyclic aromatic hydrocarbons (PAHs), a class of procarcinogenic chemicals produced by incomplete combustion such as during tobacco smoking. The UGT1A/2A/2B subfamily of enzymes are known to detoxify PAHs; however, the UGT3A subfamily has been understudied in the detoxification of tobacco carcinogens, although they have been shown to detoxify 1‐hydroxypyrene (1‐HP), a PAH metabolite. Additionally, the UGT3A subfamily also uses different sugars than the UGT1A and UGT2 enzymes, which use UDP‐glucuronic acid. The objective of the present study is to characterize the UGT3A2‐mediated detoxification of PAHs using the alternative sugars UDP‐glucose and UDP‐xylose and to assess the potential functional role of UGT3A variants in tobacco‐related cancer susceptibility. Microsomes prepared from UGT3A2‐overexpressing HEK293 cells exhibited significant glycosylation activity against a variety of PAHs including 1‐HP, 1‐hydroxybenzo(a)pyrene (1‐OH‐B(a)P), 3‐OH‐B(a)P, 7‐OH‐B(a)P, 8‐OH‐B(a)P, 9‐OH‐B( a)P, benzo(a)pyrene‐7,8‐diol, and dibenzo(a,l)pyrene‐11,12‐diol, using both UDP‐glucose and UDP‐xylose as the co‐substrate. Using UDP‐glucose, UGT3A2 exhibited Km ‘s ranging from 3.3 – 11.8 μM for 1‐HP, 1‐OH‐B(a)P, 3‐OH‐B(a)P, 7‐OH‐B(a)P, and 9‐OH‐B(a)P. The Km's were comparable when using UDP‐xylose, with Km's ranging from 1.2 – 9.6 μM for the same substrates. The Vmax/Km ratio of UDP‐xylose/UDP‐glucose ranged from 1.0 – 2.1, demonstrating that PAH glycosylation may be occurring at rates up to two‐fold higher with UDP‐xylose as the co‐substrate. To assess the potential functional role of co‐substrate selectivity for UGT3A2 genetic variants (MAF ≥ 0.5%), site‐directed mutagenesis was performed on the wild‐type UGT3A2 plasmid to create seven variants that were also overexpressed in HEK293 cells. Microsomes from UGT3A2D140N overexpressing cells exhibited up to a two‐fold higher level of glycosylation activity with UDP‐xylose than UDP‐glucose. All variants had decreased glycosylation activity (Vmax/Km) than microsomes from wild‐type UGT3A2‐overexpressing cells. These data suggest there may be differences in co‐substrate‐dependent detoxification of PAHs by UGT3A2 and UGT3A2 variants may play a role in lung and other tobacco‐related cancers.Support or Funding InformationThis study is supported by the National Institute of Environmental Health Sciences (NIEHS) of the National Institutes of Health to P Lazarus (Grant R01‐ES025460), a NIEHS Research Supplement to Promote Diversity in Health‐Related Research to AG Vergara (Grant R01‐ES025460‐02S1), and a grant from the Health Sciences and Services Authority of Spokane, WA (Grant WSU002292).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Seasonal variations in fine particle composition from Beijing prompt oxidative stress response in mouse lung and liver

Exposure to air pollution can induce oxidative stress, inflammation and adverse health effects. To understand how seasonal and chemical variations drive health impacts, we investigated indications for oxidative stress and inflammation in mice exposed to water and organic extracts from urban fine particles/PM2.5 (particles with aerodynamic diameter ≤ 2.5 μm) collected in Beijing, China. Higher levels of pollution components were detected in heating season (HS, winter and part of spring) PM2.5 than in the non-heating season (NHS, summer and part of spring and autumn) PM2.5. HS samples were high in metals for the water extraction and high in polycyclic aromatic hydrocarbons (PAHs) for the organic extraction compared to their controls. An increased inflammatory response was detected in the lung and liver following exposure to the organic extracts compared to the water extracts, and mostly in the HS PM2.5. While reduced antioxidant response was observed in the lung, it was activated in the liver, again, more in the HS extracts. Nrf2 transcription factor, a master regulator of stress response that controls the basal oxidative capacity and induces the expression of antioxidant response, and its related genes were induced. In the liver, elevated levels of lipid peroxidation adducts were measured, correlated with histologic analysis that revealed morphologic features of cell damage and proliferation, indicating oxidative and toxic damage. In addition, expression of genes related to detoxification of PAHs was observed. Altogether, the study suggests that the acute effects of PM2.5 can vary seasonally with stronger health effects in the HS than in the NHS in Beijing, China and that some secondary organs may be susceptible for the exposure damage. Specifically, the liver is a potential organ influenced by exposure to organic components such as PAHs from coal or biomass burning and heating.

Detoxification of polycyclic aromatic hydrocarbons (PAHs) in Arabidopsis thaliana involves a putative flavonol synthase

Polycyclic aromatic hydrocarbons (PAHs) are environmental contaminants with cytotoxic, teratogenic and carcinogenic properties. Bioremediation studies with bacteria have led to the identification of dioxygenases (DOXs) in the first step to degrade these recalcitrant compounds. In this study, we characterized the role of the Arabidopsis thaliana AT5G05600, a putative DOX of the flavonol synthase family, in the transformation of PAHs. Phenotypic analysis of loss-of-function mutant lines showed that these plant lines were less sensitive to the toxic effects of phenanthrene, suggesting possible roles of this gene in PAH degradation in vivo. Interestingly, these mutant lines showed less accumulation of H2O2 after PAH exposure. Transgenic lines over-expressing At5g05600 showed a hypersensitive response and more oxidative stress after phenanthrene treatments. Moreover, fluorescence spectra results of biochemical assays with the recombinant His-tagged protein AT5G05600 detected chemical modifications of phenanthrene. Taken together, these results support the hypothesis that AT5G05600 is involved in the catabolism of PAHs and the accumulation of toxic intermediates during PAH biotransformation in plants. This research represents the first step in the design of transgenic plants with the potential to degrade PAHs, leading to the development of vigorous plant varieties that can reduce the levels of these pollutants in the environment.