Phenylalanine treatment induces tomato resistance to Tuta absoluta via increased accumulation of benzenoid/phenylpropanoid volatiles serving as defense signals.

Tuta absoluta ("leafminer"), is a major pest of tomato crops worldwide. Controlling this insect is difficult due to its efficient infestation, rapid proliferation, and resilience to changing weather conditions. Furthermore, chemical pesticides have only a short-term effect due to rapid development of T. absoluta strains. Here, we show that a variety of tomato cultivars, treated with external phenylalanine solutions exhibit high resistance to T. absoluta, under both greenhouse and open field conditions, at different locations. A large-scale metabolomic study revealed that tomato leaves absorb and metabolize externally given Phe efficiently, resulting in a change in their volatile profile, and repellence of T. absoluta moths. The change in the volatile profile is due to an increase in three phenylalanine-derived benzenoid phenylpropanoid volatiles (BPVs), benzaldehyde, phenylacetaldehyde, and 2-phenylethanol. This treatment had no effect on terpenes and green leaf volatiles, known to contribute to the fight against insects. Phe-treated plants also increased the resistance of neighboring non-treated plants. RNAseq analysis of the neighboring non-treated plants revealed an exclusive upregulation of genes, with enrichment of genes related to the plant immune response system. Exposure of tomato plants to either benzaldehyde, phenylacetaldehyde, or 2-phenylethanol, resulted in induction of genes related to the plant immune system that were also induced due to neighboring Phe-treated plants. We suggest a novel role of phenylalanine-derived BPVs as mediators of plant-insect interactions, acting as inducers of the plant defense mechanisms.

Novel hydrocolloids synthesized by polyphenols grafted onto chitosan: A promising coating to inhibit PhIP formation during pan-frying of golden pompano fillets.

Hydrocolloids synthesized by gallic acid (GA) and ferulic acid (FA) grafting onto chitosan (CS) were characterized, and their effects on PhIP formation in pan-fried golden pompano were investigated. Spectrograms including nuclear magnetic resonance, Fourier transform infrared spectroscopy and ultraviolet-visible confirmed that GA and FA were successfully grafted onto CS via covalent bonds, with grafting degree of 97.06 ± 2.56 mg GA/g and 93.56 ± 2.76 mg FA/g, respectively. The CS-g-GA and CS-g-FA exerted better solubility and antioxidant activities than CS. For the 8-min pan-fried golden pompano fillets, CS-g-GA and CS-g-FA (0.5 %, m/v) significantly reduced the PhIP formation by 61.71 % and 81.64 %, respectively. Chemical models revealed that CS-g-GA and CS-g-FA inhibited PhIP formation mainly by decreasing the phenylacetaldehyde contents from Maillard reaction and competing with creatinine to react with phenylacetaldehyde. Therefore, it was suggested that CS-g-phenolic acids emerge as novel coating for aquatic products during processing and inhibit heterocyclic amines generation.

Synergistic Inhibitory Effects of Selected Amino Acids on the Formation of 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in both Benzaldehyde- and Phenylacetaldehyde-Creatinine Model Systems.

Although various inhibitors have been employed to react with phenylacetaldehyde to form adducts and thus interrupt the formation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), high concentrations of PhIP remain in the final system. It remains unknown whether other critical aldehyde or ketone intermediates are involved in the generation of PhIP, and scavenging these reactive carbonyls simultaneously may achieve higher inhibitory efficiency of PhIP. In this study, reactive carbonyls in a glucose/creatinine/phenylalanine model system were first identified by gas chromatography-mass spectrometry (GC-MS), and then the single and synergistic effects of nonprecursor amino acids (cysteine, methionine, proline, histidine, arginine, and leucine) on scavenging reactive carbonyls were investigated to find out promising combination partners. The obtained results showed that the concentrations of benzaldehyde and phenylacetaldehyde in the glucose/creatinine/phenylalanine model system reached 0.49 ± 0.01 and 6.22 ± 0.21 µg/mL, respectively. Heating these carbonyl compounds in the presence of creatinine resulted in the quantity of PhIP produced increasing linearly with the added quantity of benzaldehyde (r = 0.9733, P = 0.0002) and phenylacetaldehyde (r = 0.9746, P = 0.0002), indicating that both compounds are key intermediates for PhIP generation. Among the investigated amino acids, histidine produced the maximum inhibition of PhIP formation (78-99%) in the benzaldehyde/creatinine model system, and proline produced the maximum inhibition of PhIP formation (13-97%) in the phenylacetaldehyde/creatinine model system, where both compounds decreased PhIP formation in a dose-dependent manner. Histidine in combination with proline enhanced the inhibitory effect against PhIP formation at a low addition level, where the highest inhibitory efficiency was obtained using a 1:3 mass ratio of histidine to proline (2 mg/mL in total), reducing PhIP formation by 96%. These findings suggest that histidine-proline combinations can scavenge benzaldehyde and phenylacetaldehyde simultaneously, enhancing the suppression of PhIP formation.

Glycolaldehyde induces synergistic effects on vascular inflammation in TNF-α-stimulated vascular smooth muscle cells.

Atherosclerosis is a chronic inflammatory disease that contributes to disease progression is associated with the expression of adhesion molecules in vascular smooth muscle cells (VSMCs). Glycolaldehyde (GA) has been shown to impair cellular function in various disorders, including diabetes, and renal diseases. This study investigated the effect of GA on the expression of adhesion molecules in the mouse VSMC line, MOVAS-1. Co-incubation of VSMCs with GA (25-50 µM) dose-dependently increased the protein and mRNA level of Vcam-1 and ICAM-1. Additionally, GA upregulated intracellular ROS production and phosphorylation of MAPK and NK-κB. GA also elevated TNF-α-induced PI3K-AKT activation. Furthermore, GA enhanced TNF-α-activated IκBα kinase activation, subsequent IκBα degradation, and nuclear translocation of NF-κB. These findings suggest that GA stumulated VSMC adhesive capacity and the induction of VCAM-1 and ICAM-1 in VSMCs through inhibition of MAPK and NF-κB signaling pathways, providing insights into the effect of GA to induce inflammation within atherosclerotic lesions.

Fatal metaldehyde poisoning in a dog confirmed by gas chromatography.

BACKGROUND: Metaldehyde is a toxic pesticide used mainly as a molluscicide, responsible for intoxication and deaths in both humans and animals. Accidental exposure to metaldehyde in dogs is considered rare, but severe. Data concerning clinical and veterinary forensic toxicology are largely incomplete, especially regarding case reports in dogs. The present work reports a complete and detailed description of a case from the history, clinical evolution, pathological exams and toxicological diagnosis in an accidental case of metaldehyde poisoning in dog. CASE PRESENTATION: An eleven-month-old, 3.0 kg, male German Spitz was presented for emergency care with acute vomiting and seizures 3 hours after suspected accidental ingestion of commercial molluscicide containing 3% metaldehyde (Lesmax®). The animal was in lateral recumbency and showed stuporous mentation, salivation, tonic-clonic status epilepticus, systemic tremors, bilateral miosis, absent palpebral, corneal, oculovestibular and gag reflexes, severely depressed spinal reflexes, dyspnea and tachycardia. Despite treatment, the patient progressed to comatose mentation and died. Necropsy examination revealed discrete lesions in the liver and central nervous system, while stomach examination revealed content of feed, activated charcoal and blue-green granules, compatible to the commercial formula of metaldehyde. Histology examination revealed extensive hemorrhage and severe centrolobular necrosis of the liver and tumefaction of Kupfer cells. Brain samples showed discrete hemorrhage and hyperemia. In order to confirm the diagnosis, samples from feces, stomach content, spleen, liver, heart, kidneys and brain were submitted gas chromatography analysis. Results confirmed the presence of metaldehyde in all samples. We describe clinicopathological abnormalities of a fatal case of metaldehyde poisoning in a dog, as well as postmortem diagnosis using gas chromatography. CONCLUSION: Metaldehyde poisoning is rarely reported, since the diagnosis is often difficult and the notifications scarce. To our knowledge, this is the first report describing clinical signs, pathological findings and chromatographic diagnosis. This report aims to contribute to the understanding of the pathogenesis of metaldehyde intoxication, to further explore veterinary forensic toxicology diagnosis.

Advanced Glycated apoA-IV Loses Its Ability to Prevent the LPS-Induced Reduction in Cholesterol Efflux-Related Gene Expression in Macrophages.

We addressed how advanced glycation (AGE) affects the ability of apoA-IV to impair inflammation and restore the expression of genes involved in cholesterol efflux in lipopolysaccharide- (LPS-) treated macrophages. Recombinant human apoA-IV was nonenzymatically glycated by incubation with glycolaldehyde (GAD), incubated with cholesterol-loaded bone marrow-derived macrophages (BMDMs), and then stimulated with LPS prior to measurement of proinflammatory cytokines by ELISA. Genes involved in cholesterol efflux were quantified by RT-qPCR, and cholesterol efflux was measured by liquid scintillation counting. Carboxymethyllysine (CML) and pyrraline (PYR) levels, determined by Liquid Chromatography-Mass Spectrometry (LC-MS/MS), were greater in AGE-modified apoA-IV (AGE-apoA-IV) compared to unmodified-apoA-IV. AGE-apoA-IV inhibited expression of interleukin 6 (Il6), TNF-alpha (Tnf), IL-1 beta (Il1b), toll-like receptor 4 (Tlr4), tumor necrosis factor receptor-associated factor 6 (Traf6), Janus kinase 2/signal transducer and activator of transcription 3 (Jak2/Stat3), nuclear factor kappa B (Nfkb), and AGE receptor 1 (Ddost) as well as IL-6 and TNF-alpha secretion. AGE-apoA-IV alone did not change cholesterol efflux or ABCA-1 levels but was unable to restore the LPS-induced reduction in expression of Abca1 and Abcg1. AGE-apoA-IV inhibited inflammation but lost its ability to counteract the LPS-induced changes in expression of genes involved in macrophage cholesterol efflux that may contribute to atherosclerosis.

Convenient method of producing cyclic single-chain Fv antibodies by split-intein-mediated protein ligation and chaperone co-expression.

Single-chain Fv (scFv) is a recombinant antibody in which the variable regions of the heavy chain (VH) and light chain (VL) are connected by a short flexible polypeptide linker. Compared with monoclonal antibodies, scFvs have the advantages of low-cost production using Escherichia coli and easy genetic manipulation. ScFvs are, therefore, regarded as useful modules for producing next-generation medical antibodies. The practical use of scFvs has been limited due to their aggregation propensity mediated by interchain VH-VL interactions. To overcome this problem, we recently reported a cyclic scFv whose N-terminus and C-terminus were connected by sortase A-mediated ligation. Preparation of cyclic scFv is, however, a time-consuming process. To accelerate the application study of cyclic scFv, we developed a method to produce cyclic scFv by the combined use of a protein ligation technique based on protein trans-splicing reaction (PTS) by split intein and a chaperone co-expression system. This method allows for the preparation of active cyclic scFv from the cytoplasm of E. coli. The present method was applied to the production of cyclic 73MuL9-scFv, a GA-pyridine antibody, as a kind of advanced glycation end-product. These findings are expected to evoke further application study of cyclic scFv.

Caffeic acid assists microwave heating to inhibit the formation of mutagenic and carcinogenic PhIP.

The inhibitory effect of caffeic acid on the formation of 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP) was investigated in chemical model systems under microwave heating (MW). A mechanistic study was subsequently carried out to identify the inhibitory mechanism. The results showed that both for conductive heating (CV) and MW, the inhibition of PhIP increased with the concentration of caffeic acid but decreased with the prolongation of heating time. The inhibition on PhIP under MW was always higher than under CV, which were dominated by the difference in dielectric loss (ε″). UPLC-MS analysis showed that caffeic acid releases a CO2 molecule to produce 4-vinylcatechol which can form adducts with phenylacetaldehyde, thus reducing its availability for PhIP formation. The structure of adduct was characterized as 3-(3,4-dihydroxyphenyl)-2-phenylbutanal with a molecular weight of 256. The findings indicate that trapping of phenylacetaldehyde by 4-vinylcatechol is a key mechanism of caffeic acid in inhibiting PhIP formation.

Tricyclic Nucleobase Analogs and Their Ribosides as Substrates and Inhibitors of Purine-Nucleoside Phosphorylases III. Aminopurine Derivatives.

Etheno-derivatives of 2-aminopurine, 2-aminopurine riboside, and 7-deazaadenosine (tubercidine) were prepared and purified using standard methods. 2-Aminopurine reacted with aqueous chloroacetaldehyde to give two products, both exhibiting substrate activity towards bacterial (E. coli) purine-nucleoside phosphorylase (PNP) in the reverse (synthetic) pathway. The major product of the chemical synthesis, identified as 1,N2-etheno-2-aminopurine, reacted slowly, while the second, minor, but highly fluorescent product, reacted rapidly. NMR analysis allowed identification of the minor product as N2,3-etheno-2-aminopurine, and its ribosylation product as N2,3-etheno-2-aminopurine-N2--D-riboside. Ribosylation of 1,N2-etheno-2-aminopurine led to analogous N2--d-riboside of this base. Both enzymatically produced ribosides were readily phosphorolysed by bacterial PNP to the respective bases. The reaction of 2-aminopurine-N9- -D-riboside with chloroacetaldehyde gave one major product, clearly distinct from that obtained from the enzymatic synthesis, which was not a substrate for PNP. A tri-cyclic 7-deazaadenosine (tubercidine) derivative was prepared in an analogous way and shown to be an effective inhibitor of the E. coli, but not of the mammalian enzyme. Fluorescent complexes of amino-purine analogs with E. coli PNP were observed.

Glycolaldehyde induces sensory neuron death through activation of the c-Jun N-terminal kinase and p-38 MAP kinase pathways.

Glycolaldehyde (GA) is a highly reactive hydroxyaldehyde and one of the glycolytic metabolites producing advanced glycation endproducts (AGEs), but its toxicity toward neurons and Schwann cells remains unclear. In the present study, we found that GA exhibited more potent toxicity than other AGE precursors (glyceraldehyde, glyoxal, methylglyoxal and 3-deoxyglucosone) against immortalized IFRS1 adult rat Schwann cells and ND7/23 neuroblastoma × neonatal rat dorsal root ganglion (DRG) neuron hybrid cells. GA affected adult rat DRG neurons and ND7/23 cells more severely than GA-derived AGEs, and exhibited concentration- and time-dependent toxicity toward ND7/23 cells (10 < 100 < 250 < 500 µM; 6 h < 24 h). Treatment with 500 µM GA significantly up-regulated the phosphorylation of c-jun N-terminal kinase (JNK) and p-38 mitogen-activated kinase (p-38 MAPK) in ND7/23 cells. Furthermore, GA-induced ND7/23 cell death was significantly inhibited due to co-treatment with 10 µM of the JNK inhibitor SP600125 or the p-38 MAPK inhibitor SB239063. These findings suggest the involvement of JNK and p-38 MAPK-signaling pathways in GA-induced neuronal cell death and that enhanced GA production under diabetic conditions might be involved in the pathogenesis of diabetic neuropathy.

Novel insights into the mechanism of cyclophosphamide-induced bladder toxicity: chloroacetaldehyde's contribution to urothelial dysfunction in vitro.

The clinical use of cyclophosphamide and ifosfamide is limited by a resultant bladder toxicity which has been attributed to the metabolite acrolein. Another metabolite chloroacetaldehyde (CAA) associated with nephrotoxicity, has not been investigated for toxicity in the bladder and this study investigates the effects of acrolein and CAA on human urothelial cells in vitro. Human urothelial cells (RT4 and T24) were treated with acrolein or CAA and changes in cell viability, reactive oxygen species, caspase-3 activity and release of urothelial mediators ATP, acetylcholine, PGE2 were measured. The protective effects of N-acetyl cysteine (NAC) were also assessed. Both metabolites were toxic to human urothelial cells, however, CAA significantly decreased cell viability at a ten-fold lower concentration (10 µM) than acrolein (100 µM). This was associated with increased ROS production and caspase-3 activity. NAC protected cells from these changes. In RT4 cells 100 µM acrolein caused a significant increase in basal and stretch-induced ATP, Ach and PGE2 release. In T24 cells chloroacetaldehyde (10 µM) increased basal and stimulated ATP and PGE2 levels. Again, NAC protected against changes in urothelial mediator release following acrolein or CAA. This study is the first to report that CAA in addition to acrolein contributes to the urotoxicity of cyclophosphamide and ifosfamide. Both metabolites altered urothelial mediator levels which could contribute to the sensory and functional bladder changes experienced by patients after treatment with cyclophosphamide or ifosfamide. Alterations in urothelial cell viability and mediator release may be causally linked to oxidative stress, with NAC providing protection against these changes.

Application of the optimized and validated LC-MS method for simultaneous quantification of tryptophan metabolites in culture medium from cancer cells.

Kynurenine pathway is the main route of tryptophan degradation generating a number of immunoregulatory compounds. Some conditions like oxidative stress, inflammatory factors might enhance tryptophan degradation. Process is active in several cells including fibroblasts, cancer cells, and immune cells, therefore it is intensively studied in context of cancer microenvironment. The validated and standardized methodology for kynurenine quantification is crucial for reliable comparison of results obtained in different studies. This paper concerns an approach for simultaneous quantification of four major tryptophan metabolites of the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in cell culture supernatants by liquid chromatography coupled with single quadrupole mass spectrometer. During development of the novel method, the principal component analysis was used to select the best mobile phase and to ensure the optimal conditions for simultaneous quantification of metabolites. The analysis involves simple protein precipitation with acidified methanol and 3-nitrotyrosine as an internal standard. The obtained limits of detection and quantification in cell culture medium were in the range of 3.31-10.80 nmol/L and 9.60-19.50 nmol/L, respectively. At the validation step, other method parameters (linearity, precision, accuracy, recovery, matrix effects) were also evaluated and satisfactory results were obtained for all target compounds. The method was applied to study tryptophan metabolites by determination of kynurenines in cell culture medium from two different human cancer cell lines (MDA-MD-231 and SK-OV-3) in context of exposure to glycation products.

Miniaturized array gas membrane separation strategy for rapid analysis of complex samples by surface-enhanced Raman scattering.

It remains a significant challenge for fast and high-throughput detection of trace analytes in complex samples with surface-enhanced Raman scattering (SERS) strategy due to the severe interference from matrices. In this work, a miniaturized array gas membrane separation (AGMS) device coupled with SERS was designed and drew up to eliminate matrix influence and improve the reproducibility of SERS signal during real sample analysis. The design of miniaturized AGMS tube was optimized based on quantitative calculation of its air permeability by computational fluid dynamics simulation. A 10 mm height tube was selected as an optimized design with a recovery of 98.3% for acetaldehyde. The practical feasibility of miniaturized AGMS was validated based on the applications in biochemical analysis and food analysis, such as albuminuria and acetaldehyde in urine sample and metaldehyde and thiram in food samples. The results showed that SERS responses of all analytes dramatically increased by eliminating sample matrices after miniaturized AGMS process. Acetaldehyde, albuminuria, metaldehyde and thiram in real samples could be accurately quantified with recoveries of 82.0-123.3%, and the analytical results were validated by corresponding standard methods with relative error ranging from -4.8% to 5.3%. Time consumption of miniaturized AGMS-SERS for one real sample analysis including sample preparation and determination was less than 20 min and could treat 96 samples with 45 min in one run. It is potential that the miniaturized AGMS technique automated by implementation with a robotic arm could greatly expand the range and accelerate the speed of SERS analysis.

The response of Escherichia coli to the alkylating agents chloroacetaldehyde and styrene oxide.

DNA damage is ubiquitous and can arise from endogenous or exogenous sources. DNA-damaging alkylating agents are present in environmental toxicants as well as in cancer chemotherapy drugs and are a constant threat, which can lead to mutations or cell death. All organisms have multiple DNA repair and DNA damage tolerance pathways to resist the potentially negative effects of exposure to alkylating agents. In bacteria, many of the genes in these pathways are regulated as part of the SOS reponse or the adaptive response. In this work, we probed the cellular responses to the alkylating agents chloroacetaldehyde (CAA), which is a metabolite of 1,2-dichloroethane used to produce polyvinyl chloride, and styrene oxide (SO), a major metabolite of styrene used in the production of polystyrene and other polymers. Vinyl chloride and styrene are produced on an industrial scale of billions of kilograms annually and thus have a high potential for environmental exposure. To identify stress response genes in E. coli that are responsible for tolerance to the reactive metabolites CAA and SO, we used libraries of transcriptional reporters and gene deletion strains. In response to both alkylating agents, genes associated with several different stress pathways were upregulated, including protein, membrane, and oxidative stress, as well as DNA damage. E. coli strains lacking genes involved in base excision repair and nucleotide excision repair were sensitive to SO, whereas strains lacking recA and the SOS gene ybfE were sensitive to both alkylating agents tested. This work indicates the varied systems involved in cellular responses to alkylating agents, and highlights the specific DNA repair genes involved in the responses.

Site-specific glycations of apolipoprotein A-I lead to differentiated functional effects on lipid-binding and on glucose metabolism.

Prolonged hyperglycemia in poorly controlled diabetes leads to an increase in reactive glucose metabolites that covalently modify proteins by non-enzymatic glycation reactions. Apolipoprotein A-I (apoA-I) of high-density lipoprotein (HDL) is one of the proteins that becomes glycated in hyperglycemia. The impact of glycation on apoA-I protein structure and function in lipid and glucose metabolism were investigated. ApoA-I was chemically glycated by two different glucose metabolites (methylglyoxal and glycolaldehyde). Synchrotron radiation and conventional circular dichroism spectroscopy were used to study apoA-I structure and stability. The ability to bind lipids was measured by lipid-clearance assay and native gel analysis, and cholesterol efflux was measured by using lipid-laden J774 macrophages. Diet induced obese mice with established insulin resistance, L6 rat and C2C12 mouse myocytes, as well as INS-1E rat insulinoma cells, were used to determine in vivo and in vitro glucose uptake and insulin secretion. Site-specific, covalent modifications of apoA-I (lysines or arginines) led to altered protein structure, reduced lipid binding capability and a reduced ability to catalyze cholesterol efflux from macrophages, partly in a modification-specific manner. The stimulatory effects of apoA-I on the in vivo glucose clearance were negatively affected when apoA-I was modified with methylglyoxal, but not with glycolaldehyde. The in vitro data showed that both glucose uptake in muscle cells and insulin secretion from beta cells were affected. Taken together, glycation modifications impair the apoA-I protein functionality in lipid and glucose metabolism, which is expected to have implications for diabetes patients with poorly controlled blood glucose.

Receptor for advanced glycation end products (RAGE)-mediated cytotoxicity of 3-hydroxypyridinium derivatives.

Advanced glycation end products (AGEs) formed from glyceraldehyde (Gcer) and glycolaldehyde (Gcol) are involved in the pathogenesis of diabetic complications, via interactions with a receptor for AGEs (RAGE). In this study, we aimed to elucidate the RAGE-binding structure in Gcer and Gcol-derived AGEs and identify the minimal moiety recognized by RAGE. Among Gcer and Gcol-derived AGEs, GLAP (glyceraldehyde-derived pyridinium) and GA-pyridine elicited toxicity in PC12 neuronal cells. The toxic effects of GLAP and GA-pyridine were suppressed in the presence of anti-RAGE antibody or the soluble form of RAGE protein. Furthermore, the cytotoxicity test using GLAP analog compounds indicated that the 3-hydroxypyridinium (3-HP) structure is sufficient for RAGE-dependent toxicity. Surface plasmon resonance analysis showed that 3-HP derivatives directly interact with RAGE. These results indicate that GLAP and GA-pyridine are RAGE-binding epitopes, and that 3-HP, a common moiety of GLAP and GA-pyridine, is essential for the interaction with RAGE.

Immunization with gingipain A hemagglutinin domain of Porphyromonas gingivalis induces IgM antibodies binding to malondialdehyde-acetaldehyde modified low-density lipoprotein.

Treatment of periodontitis has beneficial effects on systemic inflammation markers that relate to progression of atherosclerosis. We aimed to investigate whether immunization with A hemagglutinin domain (Rgp44) of Porphyromonas gingivalis (Pg), a major etiologic agent of periodontitis, would lead to an antibody response cross-reacting with oxidized low-density lipoprotein (OxLDL) and how it would affect the progression of atherosclerosis in low-density lipoprotein receptor-deficient (LDLR-/-) mice. The data revealed a prominent IgM but not IgG response to malondialdehyde-acetaldehyde modified LDL (MAA-LDL) after Rgp44 and Pg immunizations, implying that Rgp44/Pg and MAA adducts may share cross-reactive epitopes that prompt IgM antibody production and consequently confer atheroprotection. A significant negative association was observed between atherosclerotic lesion and plasma IgA to Rgp44 in Rgp44 immunized mice, supporting further the anti-atherogenic effect of Rgp44 immunization. Plasma IgA levels to Rgp44 and to Pg in both Rgp44- and Pg-immunized mice were significantly higher than those in saline control, suggesting that IgA to Rgp44 could be a surrogate marker of immunization in Pg-immunized mice. Distinct antibody responses in plasma IgA levels to MAA-LDL, to Pg lipopolysaccharides (Pg-LPS), and to phosphocholine (PCho) were observed after Rgp44 and Pg immunizations, indicating that different immunogenic components between Rpg44 and Pg may behave differently in regard of their roles in the development of atherosclerosis. Immunization with Rgp44 also displayed atheroprotective features in modulation of plaque size through association with plasma levels of IL-1α whereas whole Pg bacteria achieved through regulation of anti-inflammatory cytokine levels of IL-5 and IL-10. The present study may contribute to refining therapeutic approaches aiming to modulate immune responses and inflammatory/anti-inflammatory processes in atherosclerosis.

Schisandra chinensis extract decreases chloroacetaldehyde production in rats and attenuates cyclophosphamide toxicity in liver, kidney and brain.

ETHNOPHARMACOLOGICAL RELEVANCE: Schisandra chinensis (Turcz.) Baill (S. chinensis) has been used for thousands years in China, and is usually applied in treatment of urinary tract disorders and liver injury. S. chinensis extract (SCE) has board protective effects on liver, kidney and nervous system. Schisandra lignans are generally considered as the bioactive components of SCE. AIM OF THE STUDY: To investigate the pharmacokinetic herb-drug interactions (HDIs) between SCE and cyclophosphamide (CTX). To evaluate the protective effects of SCE against CTX induced damage in rat liver, kidney and brain. MATERIALS AND METHODS: The pharmacokinetic HDIs between SCE and CTX were investigated by determining plasma concentrations of CTX and three metabolites, namely 4-ketocyclophosphamide (4-Keto), 2-dechloroethylcyclophosphamide (DCCTX) and carboxyphosphamide (CPM) using a previously developed UPLC-MS/MS method. To evaluate the protective effects of SCE pretreatment, toxicity and oxidation stress assessments along with histology investigations were carried out in rat liver, kidney and brain. RESULTS: The equimolar produced metabolite DCCTX was chosen to reflect chloroacetaldehyde (CAA, a toxic metabolite of CTX) production in rats. Single-dose pretreatment of SCE significantly reduced CAA production and decreased the Cmax and AUC0-24h of DCCTX by 69% and 49% respectively (P < 0.05). After pretreated with SCE for 7 consecutive days, the Cmax and AUC0-24h of DCCTX were still decreased (-25% and -37%, P < 0.05) when compared with CTX alone group. Parallel toxicity and oxidation stress investigations showed that single-dose SCE pretreatment significantly decreased plasma BUN and Cr levels (-12% and -46%, respectively) and reduced liver AST activity (-32%). Moreover, SCE pretreatment potently increased the brain GSH content by 7.8-fold, and reduced MDA levels in rat liver, kidney and brain by 39%, 28% and 31%, respectively (compared with CTX alone group). The protective effects of SCE were also supported by histological observations. CONCLUSION: Our experiment results suggest that S. chinensis may find use as a complementary medicine in CTX treatment.

Effects of ketoconazole on cyclophosphamide metabolism: evaluation of CYP3A4 inhibition effect using the in vitro and in vivo models.

Cyclophosphamide (CP) is widely used in anticancer therapy regimens and 2-dechloroethylcyclophosphamide (DECP) is its side-chain dechloroethylated metabolite. N-dechloroethylation of CP mediated by the enzyme CYP3A4 yields nephrotoxic and neurotoxic chloroacetaldehyde (CAA) in equimolar amount to DECP. This study aimed to evaluate the inhibitory effect of ketoconazole (KTZ) on CP metabolism through in vitro and in vivo drug-drug interaction (DDI) research. Long-term treatment of KTZ induces hepatic injury; thus single doses of KTZ at low, middle, and high levels (10, 20, and 40 mg/kg) were investigated for pharmacokinetic DDI with CP. Our in vitro human liver microsome modeling approach suggested that KTZ inhibited CYP3A4 activity and then decreased DECP exposure. In addition, an UHPLC-MS/MS method for quantifying CP, DECP, and KTZ in rat plasma was developed and fully validated with a 4 min analysis coupled with a simple and reproducible one-step protein precipitation. A further in vivo pharmacokinetic study demonstrated that combination use of CP (10 mg/kg) and KTZ (10, 20, and 40 mg/kg) in rats caused a KTZ dose-dependent decrease in main parameters of DECP (Cmax, Tmax, and AUC0-∞) and provided magnitude exposure of DECP (more than a 50% AUC decrease) as a consequence of CYP3A inhibition but had only a small effect on the CP plasma concentration. Our results suggested that combination usage of a CYP3A4 inhibitor like KTZ may decrease CAA exposure and thus intervene against CAA-induced adverse effects in CP clinical treatment.

Amino Acids Inhibitory Effects and Mechanism on 2-Amino-1-Methyl-6-Phenylimidazo [4,5-b]Pyridine (PhIP) Formation in the Maillard Reaction Model Systems.

This study was to investigate the inhibitory effects of amino acids (AAs) on the formation of 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) and to evaluate the inhibition mechanism of PhIP in Maillard model systems. Different AAs were individually added into model systems heat-treated at 180 °C/1 h. The PhIP, phenylacetaldehyde (PheAce), and pyrazines derivatives were determined using HPLC and GC-MS. AAs significantly reduced (P < 0.05) PhIP levels in a dose-dependent response, ranking as: Trp = Lys > Pro > Leu > Met > Val > Ile > Thr > Phe > Asp, at the highest molar ratio. The PheAce content was gradually reduced with increasing AAs levels, suggesting that AAs may inhibit PhIP formation through scavenging the available PheAce. A correlation between PhIP inhibition and PheAce-scavenging activity of AAs was observed when PheAce and AAs were heated. The variety and quantity of pyrazines formed are highly depending on the type of AAs.