Identification of an Epoxide Metabolite of Amitriptyline In Vitro and In Vivo.

Amitriptyline (ATL), a tricyclic antidepressant, has been reported to cause various adverse effects, particularly hepatotoxicity. The mechanisms of ATL-induced hepatotoxicity remain unknown. The study was performed to identify the olefin epoxidation metabolite of ATL and determine the possible toxicity mechanism. Two glutathione (GSH) conjugates (M1 and M2) and two N-acetylcysteine (NAC) conjugates (M3 and M4) were detected in rat liver microsomal incubations supplemented with GSH and NAC, respectively. Moreover, M1/M2 and M3/M4 were respectively found in ATL-treated rat primary hepatocytes and in bile and urine of rats given ATL. Recombinant P450 enzyme incubations demonstrated that CYP3A4 was the primary enzyme involved in the olefin epoxidation of ATL. Treatment of hepatocytes with ATL resulted in significant cell death. Inhibition of CYP3A attenuated the susceptibility to the observed cytotoxicity of ATL. The metabolic activation of ATL most likely participates in the cytotoxicity of ATL.

Quantum chemical modeling of enantioselective sulfoxidation and epoxidation reactions by indole monooxygenase VpIndA1.

Indole monooxygenases (IMOs) are enzymes from the family of Group E monooxygenases, requiring flavin adenine dinucleotide (FAD) for their activities. IMOs play important roles in both sulfoxidation and epoxidation reactions. The broad substrate range and high selectivity of IMOs make them promising biocatalytic tools for synthesizing chiral compounds. In the present study, quantum chemical calculations using the cluster approach were performed to investigate the reaction mechanism and the enantioselectivity of the IMO from Variovorax paradoxus EPS (VpIndA1). The sulfoxidation of methyl phenyl sulfide (MPS) and the epoxidation of indene were chosen as the representative reactions. The calculations confirmed that the FADOOH intermediate is the catalytic species in the VpIndA1 reactions. The oxidation of MPS adopts a one-step mechanism involving the direct oxygen-transfer from FADOOH to the substrate and the proton transfer from the -OH group back to FAD, while the oxidation of indene follows a stepwise mechanism involving a carbocation intermediate. It was computationally predicted that VpIndA1 prefers the formation of (S)-product for the MPS sulfoxidation and (1S,2R)-product for the indene epoxidation, consistent with the experimental observations. Importantly, the factors controlling the stereo-preference of the two reactions are identified. The findings in the present study provide valuable insights into the VpIndA1-catalyzed reactions, which are essential for the rational design of this enzyme and other IMOs for industrial applications. It is also worth emphasizing that the quantum chemical cluster approach is again demonstrated to be powerful in studying the enantioselectivity of enzymatic reactions.

Absorption, distribution, metabolism, and elimination of epoxiconazole enantiomers in lizards (Eremias argus).

Epoxiconazole (EPX) is a world widely used chiral triazole fungicide in the agriculture field. The excessive application of this triazole may cause damage to lizards. However, limited information is known about the toxicokinetics of EPX on lizards. Our study aimed to investigate the enantioselective absorption, distribution, metabolism, and elimination (ADME) of EPX in lizards following low and high dose exposure (10 and 100 mg kg-1 bodyweitht (bw)). The results demonstrated that (+)-EPX was easier absorbed than (-)-EPX in lizard plasma. Both (+)-EPX and (-)-EPX were detected in the liver, gonad, kidney, skin, brain, and intestine, with (+)-EPX preferentially distributed in these tissues. The elimination of (-)-EPX was faster than that of (+)-EPX in lizard liver and kidney in the high dose groups. Chiral conversion was found between EPX enantiomers in lizard skin. Simultaneously, five metabolites including M2, M4, M10, M18 and M19 were detected in lizard liver and kidney after EPX enantiomers exposure. The relative concentrations of M2, M4, and M10 were higher in the liver and kidney of (-)-EPX groups than those produced from (+)-EPX groups. The metabolic enzymes CYP3A4 and SULT1A1 primarily mediated enantioselective metabolism of EPX. The conclusions drawn from this study significantly enhance our understanding of the enantioselective behaviors of chiral triazole fungicides in reptiles, offering essential guidance for assessing the risks associated with different enantiomers of triazole fungicides.

Origin of Chemoselectivity of Halohydrin Dehalogenase-Catalyzed Epoxide Ring-Opening Reactions.

By performing molecular dynamics (MD), quantum mechanical/molecular mechanical (QM/MM) calculations, and QM cluster calculations, the origin of chemoselectivity of halohydrin dehalogenase (HHDH)-catalyzed ring-opening reactions of epoxide with the nucleophilic reagent NO2- has been explored. Four possible chemoselective pathways were considered, and the computed results indicate that the pathway associated with the nucleophilic attack on the Cα position of epoxide by NO2- is most energetically favorable and has an energy barrier of 12.9 kcal/mol, which is close to 14.1 kcal/mol derived from experimental kinetic data. A hydrogen bonding network formed by residues Ser140, Tyr153, and Arg157 can strengthen the electrophilicity of the active site of the epoxide substrate to affect chemoselectivity. To predict the energy barrier trends of the chemoselective transition states, multiple analyses including distortion analysis and electrophilic Parr function (Pk+) analysis were carried out with or without an enzyme environment. The obtained insights should be valuable for the rational design of enzyme-catalyzed and biomimetic organocatalytic epoxide ring-opening reactions with special chemoselectivity.

Insight into the interaction of 5,6 epoxy-cholesterols with human serum albumin.

5,6-Epoxy-cholesterols has been recently revealed to control metabolic pathway in breast cancer, which makes investigating their binding interaction with human serum albumin (HSA) an attractive field of research. The main aim of this article is to examine the binding interaction of 5,6 α-epoxy-cholesterol (5,6 α EC) and 5,6 ß-epoxy-cholesterol (5,6 ß- EC) with HSA using different spectroscopic methods and molecular modeling. These compounds interact with HSA via hydrophobic interactions and hydrogen bonds with binding constants 6.3 × 105 M-1 for 5,6 α-epoxy-cholesterol and 6.9 × 105 M-1 for 5,6 ß-epoxy-cholesterol besides, the mechanism of the interaction can be attributed to static quenching. Circular dichroism data indicated that the α-helical content of HSA increased from 50.5 to 59.8 and 61.1 % after the addition of 5,6 α-ECs and 5,6 ß-EC, respectively, with a ratio of 1:2. Thermodynamic analysis revealed that binding between 5,6-epoxy-cholesterols and HSA is spontaneous and entropy-driven. The molecular docking and esterase-like activity experiments were performed to envision a link between the experimental and theoretical results. The optimal binding site of 5,6-epoxy-cholesterols with HSA was located in subdomain IIA. Moreover, theoretical calculations were performed using the B3LYP function with the 6-311++G (d,p) basis set, indicating the HOMO-LUMO energy gap of 7.874 eV for 5,6 α-epoxy-cholesterol and 7.873 eV for 5,6 ß-epoxy-cholesterol. The obtained findings are assumed to provide basic data for understanding the binding interactions of HSA with oxysterol compounds, which could help explore the pharmacokinetics and pharmacodynamics of oxysterol compounds.

Dietary lipid sensing through fatty acid oxidation and chylomicron formation in the gastrointestinal tract of rainbow trout.

In mammals, physiological processes related to lipid metabolism, such as chylomicron synthesis or fatty acid oxidation (FAO), modulate eating, highlighting the importance of energostatic mechanisms in feeding control. This study, using rainbow trout (Oncorhynchus mykiss) as model, aimed to characterize the role of FAO and chylomicron formation as peripheral lipid sensors potentially able to modulate feeding in fish. Fish fed with either a normal- (24%) or high- (32%) fat diet were intraperitoneally injected with water alone or containing etomoxir (inhibitor of FAO rate-limiting enzyme carnitine palmitoyl-transferase 1). First, feed intake levels were recorded. We observed an etomoxir-derived decrease in feeding at short times, but a significant increase at 48 h after treatment in fish fed normal-fat diet. Then, we evaluated putative etomoxir effects on the mRNA abundance of genes related to lipid metabolism, chylomicron synthesis and appetite-regulating peptides. Etomoxir treatment upregulated mRNA levels of genes related to chylomicron assembly in proximal intestine, while opposite effects occurred in distal intestine, indicating a clear regionalization in response. Etomoxir also modulated gastrointestinal hormone mRNAs in proximal intestine, upregulating ghrl in fish fed normal-fat diet and pyy and gcg in fish fed high-fat diet. These results provide evidence for an energostatic control of feeding related to FAO and chylomicron formation at the peripheral level in fish.

Epoxide Ring-Opening Reactions for Abundant Production of Mugineic Acids and Nicotianamine Probes.

A succinct synthetic approach to mugineic acids and 2'-hydroxynicotianamine was established. Unlike all other synthetic methods, this approach utilized epoxide ring-opening reactions to form two C-N bonds and is characterized by the absence of redox reactions. Mugineic acid was synthesized from three readily available fragments on a gram scale in 6 steps. The protected 2'-hydroxynicotianamine was also synthesized in 4 steps, and the dansyl group, serving as a fluorophore, was introduced through a click reaction after propargylation of the 2'-hydroxy group. The dansyl-labeled nicotianamine (NA) iron complexes were internalized by oocytes overexpressing ZmYS1 (from maize) or PAT1 (from human) transporters, indicating successful transport of the synthesized NA-probe through these transporters.

In vitro and in vivo metabolic activation and hepatotoxicity of chlorzoxazone mediated by CYP3A.

Chlorzoxazone (CZX), a benzoxazolone derivative, has been approved for the treatment of musculoskeletal disorders to relieve localized muscle spasm. However, its idiosyncratic toxicity reported in patients brought attention, particularly for hepatotoxicity. The present study for the first time aimed at the relationship between CZX-induced hepatotoxicity and identification of oxirane intermediate resulting from metabolic activation of CZX. Two N-acetylcysteine (NAC) conjugates (namely M1 and M2) and two glutathione (GSH) conjugates (namely M3 and M4) were detected in rat & human microsomal incubations with CZX (200 µM) fortified with NAC or GSH, respectively. The formation of M1-M4 was NADPH-dependent and these metabolites were also observed in urine or bile of SD rats given CZX intragastrically at 10 mg/kg or 25 mg/kg. NAC was found to attach at C-6' of the benzo group of M1 by sufficient NMR data. CYPs3A4 and 3A5 dominated the metabolic activation of CZX. The two GSH conjugates were also observed in cultured rat primary hepatocytes after exposure to CZX. Inhibition of CYP3A attenuated the susceptibility of hepatocytes to the cytotoxicity of CZX (10-400 µM). The in vitro and in vivo studies provided solid evidence for the formation of oxirane intermediate of CZX. This would facilitate the understanding of the underlying mechanisms of toxic action of CZX.

Determinants of Meal-Induced Changes in Circulating FFA Epoxides, Diols, and Diol-to-Epoxide Ratios as Indices of Soluble Epoxide Hydrolase Activity.

Soluble epoxide hydrolase (sEH) is an important enzyme for metabolic and cardiovascular health. sEH converts FFA epoxides (EpFAs), many of which are regulators of various cellular processes, to biologically less active diols. In human studies, diol (sEH product) to EpFA (sEH substrate) ratios in plasma or serum have been used as indices of sEH activity. We previously showed these ratios profoundly decreased in rats during acute feeding, possibly reflecting decreases in tissue sEH activities. The present study was designed to test which tissue(s) these measurements in the blood represent and if factors other than sEH activity, such as renal excretion or dietary intake of EpFAs and diols, significantly alter plasma EpFAs, diols, and/or their ratios. The results show that postprandial changes in EpFAs and diols and their ratios in plasma were very similar to those observed in the liver but not in other tissues, suggesting that the liver is largely responsible for these changes in plasma levels. EpFAs and diols were excreted into the urine, but their levels were not significantly altered by feeding, suggesting that renal excretion of EpFAs and diols may not play a major role in postprandial changes in circulating EpFAs, diols, or their ratios. Diet intake had significant impacts on circulating EpFA and diol levels but not on diol-to-EpFA (D-to-E) ratios, suggesting that these ratios, reflecting sEH activities, may not be significantly affected by the availability of sEH substrates (i.e., EpFAs). In conclusion, changes in FFA D-to-E ratios in plasma may reflect those in the liver, which may in turn represent sEH activities in the liver, and they may not be significantly affected by renal excretion or the dietary intake of EpFAs and diols.

Reconstructing hepatic metabolic profile and glutathione-mediated metabolic fate of acrylamide.

The toxicity of acrylamide (AA) has continuously attracted wide concerns as its extensive presence from both environmental and dietary sources. However, its hepatic metabolic transformation and metabolic fate still remain unclear. This study aims to unravel the metabolic profile and glutathione (GSH) mediated metabolic fate of AA in liver of rats under the dose-dependent exposure. We found that exposure to AA dose-dependently alters the binding of AA and GSH and the generation of mercapturic acid adducts, while liver as a target tissue bears the metabolic transformation of AA via regulating GSH synthesis and consumption pathways, in which glutamine synthase (GSS), cytochrome P450 2E1 (CYP2E1), and glutathione S-transferase P1 (GSTP1) play a key role. In response to high- and low-dose exposures to AA, there were significant differences in liver of rats, including the changes in GSH and cysteine (CYS) activities and the conversion ratio of AA to glycidamide (GA), and liver can affect the transformation of AA by regulating the GSH-mediated metabolic pathway. Low-dose exposure to AA activates GSH synthesis pathway in liver and upregulates GSS activity and CYS content with no change in γ-glutamyl transpeptidase 1 (GGT1) activity. High-dose exposure to AA activates the detoxification pathway of GSH and increases GSH consumption by upregulating GSTP1 activity. In addition, molecular docking results showed that most of the metabolic molecules transformed by AA and GA other than themselves can closely bind to GSTP1, GSS, GGT1, N-acetyltransferase 8, and dimethyl sulfide dehydrogenase 1. The binding of AA-GSH and GA-GSH to GSTP1 and CYP2E1 enzymes determine the tendentiousness between toxicity and detoxification of AA, which exerts a prospective avenue for targeting protective role of hepatic enzymes against in vivo toxicity of AA.

Triptolide induces PANoptosis in macrophages and causes organ injury in mice.

Macrophages represent the first lines of innate defense against pathogenic infections and are poised to undergo multiple forms of regulated cell death (RCD) upon infections or toxic stimuli, leading to multiple organ injury. Triptolide, an active compound isolated from Tripterygium wilfordii Hook F., possesses various pharmacological activities including anti-tumor and anti-inflammatory effects, but its applications have been hampered by toxic adverse effects. It remains unknown whether and how triptolide induces different forms of RCD in macrophages. In this study, we showed that triptolide exhibited significant cytotoxicity on cultured macrophages in vitro, which was associated with multiple forms of lytic cell death that could not be fully suppressed by any one specific inhibitor for a single form of RCD. Consistently, triptolide induced the simultaneous activation of pyroptotic, apoptotic and necroptotic hallmarks, which was accompanied by the co-localization of ASC specks respectively with RIPK3 or caspase-8 as well as their interaction with each other, indicating the formation of PANoptosome and thus the induction of PANoptosis. Triptolide-induced PANoptosis was associated with mitochondrial dysfunction and ROS production. PANoptosis was also induced by triptolide in mouse peritoneal macrophages in vivo. Furthermore, triptolide caused kidney and liver injury, which was associated with systemic inflammatory responses and the activation of hallmarks for PANoptosis in vivo. Collectively, our data reveal that triptolide induces PANoptosis in macrophages in vitro and exhibits nephrotoxicity and hepatotoxicity associated with induction of PANoptosis in vivo, suggesting a new avenue to alleviate triptolide's toxicity by harnessing PANoptosis.

Effects of Individual Circulating FFAs on Plasma and Hepatic FFA Epoxides, Diols, and Epoxide-Diol Ratios as Indices of Soluble Epoxide Hydrolase Activity.

Oxylipins, oxidation products of unsaturated free fatty acids (FFAs), are involved in various cellular signaling systems. Among these oxylipins, FFA epoxides are associated with beneficial effects in metabolic and cardiovascular health. FFA epoxides are metabolized to diols, which are usually biologically less active, by soluble epoxide hydrolase (sEH). Plasma epoxide-diol ratios have been used as indirect measures of sEH activity. This study was designed to examine the effects of acute elevation of individual plasma FFAs on a variety of oxylipins, particularly epoxides, diols, and their ratios. We tested if FFA epoxide-diol ratios are altered by circulating FFA levels (i.e., substrate availability) independent of sEH activity. Wistar rats received a constant intravenous infusion of olive (70% oleic acid (OA)), safflower seed (72% linoleic acid (LA)), and fish oils (rich in ω-3 FFAs) as emulsions to selectively raise OA, LA, and ω-3 FFAs (eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)), respectively. As expected, olive, safflower seed, and fish oil infusions selectively raised plasma OA (57%), LA (87%), EPA (70%), and DHA (54%), respectively (p < 0.05 for all). Raising plasma FFAs exerted substrate effects to increase hepatic and plasma epoxide and diol levels. These increases in epoxides and diols occurred to similar extents, resulting in no significant changes in epoxide-diol ratios. These data suggest that epoxide-diol ratios, often used as indices of sEH activity, are not affected by substrate availability or altered plasma FFA levels and that epoxide-diol ratios may be used to compare sEH activity between conditions of different circulating FFA levels.

Targeted high-throughput mutagenesis of the human spliceosome reveals its in vivo operating principles.

The spliceosome is a staggeringly complex machine, comprising, in humans, 5 snRNAs and >150 proteins. We scaled haploid CRISPR-Cas9 base editing to target the entire human spliceosome and investigated the mutants using the U2 snRNP/SF3b inhibitor, pladienolide B. Hypersensitive substitutions define functional sites in the U1/U2-containing A complex but also in components that act as late as the second chemical step after SF3b is dissociated. Viable resistance substitutions map not only to the pladienolide B-binding site but also to the G-patch domain of SUGP1, which lacks orthologs in yeast. We used these mutants and biochemical approaches to identify the spliceosomal disassemblase DHX15/hPrp43 as the ATPase ligand for SUGP1. These and other data support a model in which SUGP1 promotes splicing fidelity by triggering early spliceosome disassembly in response to kinetic blocks. Our approach provides a template for the analysis of essential cellular machines in humans.

The effect of vitamin B12 on DNA adduction by styrene oxide, a genotoxic xenobiotic.

Vitamin B12 (cyano- or hydroxo-cobalamin) acts, via its coenzymes, methyl- and adenosyl-cobalamin, as a partner for enzymatic reactions in humans catalysed by methionine synthase and methylmalonyl-CoA mutase. As well as its association with pernicious anaemia, human B12 deficiency may also be a risk factor for neurological illnesses, heart disease and cancer. In the present work the effect of vitamin B12 (hydroxocobalamin) on the formation of DNA adducts by the epoxide phenyloxirane (styrene oxide), a genotoxic metabolite of phenylethene (styrene), has been studied using an in vitro model system. Styrene was converted to its major metabolite styrene oxide as a mixture of enantiomers using a microsomal fraction from the livers of Sprague-Dawley rats with concomitant inhibition of epoxide hydrolase. However, microsomal oxidation of styrene in the presence of vitamin B12 gave diastereoisomeric 2-hydroxy-2-phenylcobalamins. The quantitative formation of styrene oxide-DNA adducts was investigated using 2-deoxyguanosine or calf thymus DNA in the presence or absence of vitamin B12. Microsomal incubations containing either deoxyguanosine or DNA in the absence of vitamin B12 gave 2-amino-7-(2-hydroxy-1-phenylethyl)-1,7-dihydro-6H-purin-6-one [N7-(2-hydroxy-1-phenylethyl)-guanine], and 2-amino-7-(2-hydroxy-2-phenylethyl)-1,7-dihydro-6H-purin-6-one [N7-(2-hydroxy-2-phenylethyl)guanine] as the principal adducts. With deoxyguanosine the level of formation of guanine adducts was ca. 150 adducts/106 unmodified nucleoside. With DNA the adduct level was 36 pmol/mg DNA (ca. 1 adduct/0.83 × 105 nucleotides). Styrene oxide adducts from deoxyguanosine or DNA were not detected in microsomal incubations of styrene in the presence of vitamin B12. These results suggest that vitamin B12 could protect DNA against genotoxicity due to styrene oxide and other xenobiotic metabolites. However, this potential defence mechanism requires that the 2-hydroxyalkylcobalamins derived from epoxides are not 'anti-vitamins' and ideally liberate, and therefore, recycle vitamin B12. Otherwise, depletion of vitamin B12 leading to human deficiency could increase the risk of carcinogenesis initiated by genotoxic epoxides.

Lipid mediators generated by the cytochrome P450-Epoxide hydrolase pathway.

The cytochrome P450 (CYP) soluble epoxide hydrolase (sEH) pathway generates a large number of biologically active epoxides and diols from a range of ω-3 and ω-6 polyunsaturated fatty acids (PUFAs). While epoxides of arachidonic acid or epoxyeicosatrienoic acids are probably the best studied of these mediators, epoxides of linoleic acid as well as the fish oils; docosahexaenoic acid and eicosapentaenoic acid have also been attributed signaling actions. Cell and tissue levels of the PUFA epoxides are largely determined by the sEH and in many cases inflammation and chronic diseases, e.g., cardiovascular disease, diabetes and Alzheimer's disease, have been associated with increased sEH expression and the accelerated conversion of PUFA epoxides to their corresponding diols. In low concentrations, the diols act to influence stem and progenitor cells as well as brown adipose tissue but in high concentrations, they tend to have pro-inflammatory and cytotoxic effects that promote disease progression. This review outlines some of the actions to the PUFA epoxides and diols in physiology and pathophysiology as well as the beneficial effects associates with sEH inhibition.

Relationship between acrylamide and glycidamide hemoglobin adduct levels and osteoarthritis: a NHANES analysis.

Osteoarthritis (OA) is the most prevalent degenerative joint disease, and acrylamide is a chemical produced when foods are processed at high temperatures. Recent epidemiological research linked acrylamide exposure from the diet and environment to a number of medical disorders. However, whether acrylamide exposure is associated with OA is still uncertain. This study was aimed at assessing the relationship between OA and hemoglobin adducts of acrylamide and its metabolite glycidamide (HbAA and HbGA). Data were taken from four cycles of the US NHANES database (2003-2004, 2005-2006, 2013-2014, 2015-2016). Individuals aged between 40 and 84 years who had complete information on arthritic status as well as HbAA and HbGA levels were eligible for inclusion. Univariate and multivariate logistic regression analysis s was performed to determine associations between study variables and OA. Restricted cubic splines (RCS) were used to examine non-linear associations between the acrylamide hemoglobin biomarkers and prevalent OA. A total of 5314 individuals were included and 954 (18%) had OA. After adjusting for relevant confounders, the highest quartiles (vs. lowest) of HbAA (adjusted odds ratio (aOR) = 0.87, 95% confidence interval (CI), 0.63-1.21), HbGA (aOR = 0.82, 95% CI, 0.60-1.12), HbAA + HbGA (aOR = 0.86, 95% CI, 0.63-1.19), and HbGA/HbAA (aOR = 0.88, 95% CI, 0.63--1.25) were not significantly associated with greater odds for OA. RCS analysis revealed that HbAA, HbGA, and HbAA + HbGA levels were non-linearly and inversely associated with OA (p for non-linearity < 0.001). However, the HbGA/HbAA ratio displayed a U-shaped relationship with prevalent OA. In conclusion, acrylamide hemoglobin biomarkers are non-linearly associated with prevalent OA in a general US population. These findings implicate ongoing public health concerns for widespread exposure to acrylamide. Further studies are still warranted to address the causality and biologic mechanisms underlying the association.

Tandemly duplicated CYP82Ds catalyze 14-hydroxylation in triptolide biosynthesis and precursor production in Saccharomyces cerevisiae.

Triptolide is a valuable multipotent antitumor diterpenoid in Tripterygium wilfordii, and its C-14 hydroxyl group is often selected for modification to enhance both the bioavailability and antitumor efficacy. However, the mechanism for 14-hydroxylation formation remains unknown. Here, we discover 133 kb of tandem duplicated CYP82Ds encoding 11 genes on chromosome 12 and characterize CYP82D274 and CYP82D263 as 14-hydroxylases that catalyze the metabolic grid in triptolide biosynthesis. The two CYP82Ds catalyze the aromatization of miltiradiene, which has been repeatedly reported to be a spontaneous process. In vivo assays and evaluations of the kinetic parameters of CYP82Ds indicate the most significant affinity to dehydroabietic acid among multiple intermediates. The precursor 14-hydroxy-dehydroabietic acid is successfully produced by engineered Saccharomyces cerevisiae. Our study provides genetic elements for further elucidation of the downstream biosynthetic pathways and heterologous production of triptolide and of the currently intractable biosynthesis of other 14-hydroxyl labdane-type secondary metabolites.

Rice peroxygenase catalyzes lipoxygenase-dependent regiospecific epoxidation of lipid peroxides in the response to abiotic stressors.

The peroxygenase pathway plays pivotal roles in plant responses to oxidative stress and other environmental stressors. Analysis of a network of co-expressed stress-regulated rice genes demonstrated that expression of OsPXG9 is negatively correlated with expression of genes involved in jasmonic acid biosynthesis. DNA sequence analysis and structure/function studies reveal that OsPXG9 is a caleosin-like peroxygenase with amphipathic α-helices that localizes to lipid droplets in rice cells. Enzymatic studies demonstrate that 12-epoxidation is slightly more favorable with 9(S)-hydroperoxyoctadecatrienoic acid than with 9(S)-hydroperoxyoctadecadienoic acid as substrate. The products of 12-epoxidation are labile, and the epoxide ring is hydrolytically cleaved into corresponding trihydroxy compounds. On the other hand, OsPXG9 catalyzed 15-epoxidation of 13(S)-hydroperoxyoctadecatrienoic acid generates a relatively stable epoxide product. Therefore, the regiospecific 12- or 15-epoxidation catalyzed by OsPXG9 strongly depends on activation of the 9- or 13- peroxygenase reaction pathways, with their respective preferred substrates. The relative abundance of products in the 9-PXG and 13-PXG pathways suggest that the 12-epoxidation involves intramolecular oxygen transfer while the 15-epoxidation can proceed via intramolecular or intermolecular oxygen transfer. Expression of OsPXG9 is up-regulated by abiotic stimuli such as drought and salt stress, but it is down-regulated by biotic stimuli such as flagellin 22 and salicylic acid. The results suggest that the primary function of OsPXG9 is to modulate the level of lipid peroxides to facilitate effective defense responses to abiotic and biotic stressors.

Acrylamide and Glycidamide Hemoglobin Adduct Levels and Breast Cancer Risk in Japanese Women: A Nested Case-Control Study in the JPHC.

BACKGROUND: Acrylamide (AA) is classified as "probably carcinogenic to humans (class 2A)" by the International Agency for Research on Cancer. AA causes cancer owing to its mutagenic and genotoxic metabolite, glycidamide (GA), and its effects on sex hormones. Both AA and GA can interact with hemoglobin to hemoglobin adducts (HbAA and HbGA, respectively), which are considered appropriate biomarkers of internal exposure of AA. However, few epidemiologic studies reported an association of HbAA and HbGA with breast cancer. METHODS: We conducted a nested case-control study within the Japan Public Health Center-based Prospective Study cohort (125 cases and 250 controls). Cases and controls were categorized into tertiles (lowest, middle, and highest) using the distribution of HbAA or HbGA levels in the control group and estimated ORs and 95% confidence intervals (CI) using conditional logistic regression, adjusting for potential confounders. RESULTS: No association was observed between HbAA (ORHighestvs.Lowest, 1.34; 95% CI, 0.69-2.59), HbGA (ORHighest vs. Lowest, 1.46; 95% CI, 0.79-2.69), their sum HbAA+HbGA (ORHighest vs. Lowest, 1.36; 95% CI, 0.72-2.58) and breast cancer; however, some evidence of positive association was observed between their ratio, HbGA/HbAA, and breast cancer (ORHighest vs. Lowest, 2.19; 95% CI, 1.11-4.31). CONCLUSIONS: There was no association between biomarkers of AA and breast cancer. IMPACT: It is unlikely that AA increases breast cancer risk; however, the association of AA with breast cancer may need to be evaluated, with a focus not only on the absolute amount of HbAA or HbGA but also on HbGA/HbAA and the activity of metabolic genes.

Wastewater-derived organic contaminants in fresh produce: Dietary exposure and human health concerns.

Irrigation with reclaimed wastewater is a growing practice aimed at conserving freshwater sources, especially in arid and semiarid regions. Despite the apparent advantages to water management, the practice of irrigation with reclaimed wastewater exposes the agroenvironment to contaminants of emerging concern (CECs). In this report, we estimated the unintentional dietary exposure of the Israeli population (2808 participants) to CECs from consumption of produce irrigated with reclaimed wastewater using detailed dietary data obtained from a National Health and Nutrition Survey (Rav Mabat adults; 2014-2016). Human health risk analyses were conducted based on acceptable daily intake (ADI) and threshold of toxicological concern (TTC) approaches. The highest unintentional exposure to wastewater-borne CECs was found to occur through the consumption of leafy vegetables. All analyzed CECs exhibited hazard quotients <1 for the mean- and high-exposure scenarios, indicating no human health concerns. However, for the extreme exposure scenario, the anticonvulsant agents lamotrigine and carbamazepine, and the carbamazepine metabolite epoxide-carbamazepine exhibited the highest exposure levels of 29,100, 27,200, and 19,500 ng/person (70 kg) per day, respectively. These exposure levels exceeded the TTC of lamotrigine and the metabolite epoxide-carbamazepine, and the ADI of carbamazepine, resulting in hazard quotients of 2.8, 1.1, and 1.9, respectively. According to the extreme estimated scenario, consumption of produce irrigated with reclaimed wastewater (leafy vegetables in particular) may pose a threat to human health. Minimizing irrigation of leafy vegetables using reclaimed wastewater and/or improving the quality of the reclaimed wastewater using an advanced treatment would significantly reduce human dietary exposure to CECs.