ABSTRACT
Benzyl butyl phthalate (BBP) is a widely used plasticizer that poses various potential health hazards. Although BBP has been extensively studied, the direct mechanism underlying its toxicity in male germ cells remains unclear. Therefore, we investigated BBP-mediated male germ cell toxicity in GC-1 spermatogonia (spg), a differentiated mouse male germ cell line. This study investigated the impact of BBP on reactive oxygen species (ROS) generation, apoptosis, and autophagy regulation, as well as potential protective measures against BBP-induced toxicity. A marked dose-dependent decrease in GC-1 spg cell proliferation was observed following treatment with BBP at 12.5⯵M. Exposure to 50⯵M BBP, approximating the IC50 of 53.9⯵M, markedly increased cellular ROS generation and instigated apoptosis, as evidenced by augmented protein levels of both intrinsic and extrinsic apoptosis-related markers. An amount of 50⯵M BBP induced marked upregulation of autophagy regulator proteins, p38 MAPK, and extracellular signal-regulated kinase and substantially downregulated the phosphorylation of key kinases involved in regulating cell proliferation, including phosphoinositide 3-kinase, protein kinase B, mammalian target of rapamycin (mTOR), c-Jun N-terminal kinase. The triple combination of N-acetylcysteine, parthenolide, and 3-methyladenine markedly restored cell proliferation, decreased BBP-induced apoptosis and autophagy, and restored mTOR phosphorylation. This study provides new insights into BBP-induced male germ cell toxicity and highlights the therapeutic potential of the triple inhibitors in mitigating BBP toxicity.
Subject(s)
Acetylcysteine , Adenine , Apoptosis , Autophagy , Cell Proliferation , Phthalic Acids , Reactive Oxygen Species , Sesquiterpenes , Male , Animals , Mice , Phthalic Acids/toxicity , Autophagy/drug effects , Apoptosis/drug effects , Reactive Oxygen Species/metabolism , Sesquiterpenes/pharmacology , Acetylcysteine/pharmacology , Adenine/analogs & derivatives , Adenine/pharmacology , Adenine/toxicity , Cell Proliferation/drug effects , Cell Line , Plasticizers/toxicity , Spermatogonia/drug effectsABSTRACT
OBJECTIVE: The study's aim was to evaluate Brazilian Brown Propolis (BBP) and Artepillin C (ARC) chemopreventive action in Wistar rats' colons. METHODS: Fifty male Wistar rats were divided into ten experimental groups, including control groups, groups with and without 1,2-dimethylhydrazine (DMH) induction, and BBP, ARC, and ARC enriched fraction (EFR) treatments, for sixteen weeks. Aberrant crypt foci (ACF) were classified as hyperplastic or dysplastic, and proliferating cell nuclear antigen (PCNA) expression was quantified. RESULT: ACF amounts in experimental groups (induced or not) decreased in both colon portions, while the isolated Aberrant Crypt (AC) number increased. Experimental groups of animals showed higher hyperplasia and dysplasia amounts compared with control groups. The ACF dysplastic amount present in groups induced and treated, in both colon portions, had similar values to IDMH (DMH induction group without treatment). In addition, DMH was effective in ACF inducing and there was positive staining for PCNA in basal and upper dysplastic foci portions in all experimental groups, in the mitotic index (MI) evaluation. To conclude, considering all the experimental groups, the one treated with EFR (fraction enriched with ARC) had the lowest rates of cell proliferation. CONCLUSION: BBP and its derivatives prevented crypt cell clonal expansion.
Subject(s)
Aberrant Crypt Foci , Antineoplastic Agents , Colonic Neoplasms , Phenylpropionates , Propolis , Rats , Animals , Male , Rats, Wistar , Colonic Neoplasms/drug therapy , Proliferating Cell Nuclear Antigen/metabolism , Propolis/pharmacology , Propolis/therapeutic use , 1,2-Dimethylhydrazine/toxicity , Brazil , Aberrant Crypt Foci/drug therapy , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , CarcinogensABSTRACT
During the COVID-19 pandemic, an abundance of plastic face masks has been consumed and disposed of in the environment. In addition, substantial amounts of plastic mulch film have been used in intensive agriculture with low recovery. Butyl benzyl phthalate (BBP) and TiO2 nanomaterials (nTiO2) are widely applied in plastic products, leading to the inevitable release of BBP and nTiO2 into the soil system. However, the impact of co-exposure of BBP and nTiO2 at low concentrations on earthworms remains understudied. In the present study, transcriptomics was applied to reveal the effects of individual BBP and nTiO2 exposures at a concentration of 1 mg kg-1, along with the combined exposure of BBP and nTiO2 (1 mg kg-1 BBP + 1 mg kg-1 nTiO2 (anatase)) on Metaphire guillelmi. The result showed that BBP and nTiO2 exposures have the potential to induce neurodegeneration through glutamate accumulation, tau protein, and oxidative stress in the endoplasmic reticulum and mitochondria, as well as metabolism dysfunction. The present study contributes to our understanding of the toxic mechanisms of emerging contaminants at environmentally relevant levels and prompts consideration of the management of BBP and nTiO2 within the soil ecosystems.
Subject(s)
Nanostructures , Oligochaeta , Phthalic Acids , Animals , Humans , Oligochaeta/genetics , Ecosystem , Pandemics , Titanium , Soil , Gene Expression ProfilingABSTRACT
Beer is one of the most consumed beverages worldwide. Different materials used along its production and packaging can result in human exposure to phthalates and adipates. The aim of this study was to assess simultaneously the levels of phthalates and di-ethylhexyl adipate (DEHA) in commercial beer samples (n = 66) with a method based on DLLME and detection with GC-MS/MS, and further evaluate human exposure. Six out of seven compounds studied were found in the beers analysed, with levels ranging from 1.77 to 205.40 µg/L. The most prevalent was DEHA at 205.40 µg/L, while dimethyl phthalate (DMP) was not present in any sample. Samples with 5-6 % alcohol, packed in aluminium cans and produced in an industrial environment presented the highest level of these contaminants. Despite low-risk exposure to phthalates and adipate with beer, it is important to remember the ubiquitous nature of these compounds, which can lead to cumulative exposure.
ABSTRACT
Agricultural films are extensively utilized in high-intensity agriculture, with China's annual usage reaching 1.5 million tons. Unfortunately, the recovery rate is less than 60%, leading to an inevitable accumulation of plastic mulch in agricultural soils. This accumulation primarily introduces butyl benzyl phthalate (BBP) into soil ecosystems, whose specific effects remain largely unclear, thereby posing potential risks. The present study focuses on the exposure impact of BBP on earthworms, Metaphire guillelmi, a commonly found endogenic earthworm within real farmland, as it provides insight into the direct interaction between biota gut health and contaminants. Specifically, we studied the biomarkers related to oxidative stress, the digestive system, and neurotoxicity within the gut of Metaphire guillelmi, and the integrated biological response (IBR) index was utilized to track these markers at different timeframes after BBP exposures. Our findings indicate that BBP exposures lead to oxidative damage, digestive system inhibition, and neurotoxicity, with IBR indexes of 14.6 and 17.3 on the 14th and 28th days, respectively. Further, the underlying mechanisms at a molecular level through molecular docking were investigated. The results showed that the most unstable interaction was with the Na+K+-ATPase (binding energy: -2.25 kcal-1), while BBP displayed stable bonds with superoxide dismutase and 8-hydroxydeoxyguanosine via hydrogen bonds and hydrophobic interaction. These interactions resulted in changes in protein conformation and their normal physiological functions, offering new insights into the molecular mechanism underlying enzymatic activity changes. This study has significant implications for the prediction of toxicity, environmental risk assessment, and the establishment of regulations related to BBP.
ABSTRACT
Butyl benzyl phthalate (BBP) is a common environmental pollutant, it is high in paints, adhesives and other decorative materials, food packaging bags, cleaning agents, is a plasticizer is very widely used in daily life. However, it remains unknown whether BBP causes damage to oocytes cultured in vitro and whether there is an effective rescue strategy. Here, we evaluated the effects of exposure to different concentrations of BBP (10, 50, and 100 µM) on the meiosis of porcine oocytes. The results showed that exposure to BBP (100 µM) severely impaired expansion of cumulus-oocyte complex (COCs) and PBE (control:71.6% vs 100 µM: 48.8%). Spindle conformation and chromosome alignment were also significantly abnormal (34.8% and 46.0%, respectively) compared to the control (11.1% and 17.5%, respectively), and BBP caused damage to microfilaments and cortical granules (CGs). In addition, oocyte exposure to BBP induced impaired mitochondrial function and disrupted mitochondrial integrity. Silibinin is a natural active substance isolated from the seeds of Silybum marianum (L.) Gaertneri with strong antioxidant and anti-inflammatory effects. Noteworthy, we added different concentrations of silibinin (10, 20, and 50 µM) to BBP-exposed oocytes for rescue experiments, where 50 µM effectively rescued BBP-induced meiotic failure (70.6%). It also prevented the generation of excessive autophagy and apoptosis in oocytes by inhibiting the production of ROS. In a word, our results suggest that supplementation of silibinin attenuates the impaired oocyte development caused by BBP exposureï¼which provides a potential strategy to protect oocytes from environmental pollutants.
Subject(s)
Oocytes , Oxidative Stress , Swine , Animals , Silybin/metabolism , Silybin/pharmacology , Reactive Oxygen Species/metabolism , Autophagy , Dietary SupplementsABSTRACT
The complement system is a primary component of the vertebrate innate immune system, and its activity is harmful to microorganisms and parasites. To evade complement attack, some pathogens, such as viruses, bacteria, and protozoa, can interact with complement regulatory proteins from their hosts. Our research group has described the ability of Leishmania species to bind Factor H from human serum and use it as a tool to evade the complement system. However, there is no description of the interaction of Leishmania with other complement regulatory proteins, such as the C4b-binding protein (C4bBP), a negative regulator of classical and lectins complement system pathways. The results presented in this manuscript suggest that Leishmania infantum, L. amazonensis, and L. braziliensis recruit C4bBP from human serum. The uptake of C4bBP by L. infantum was studied in detail to improve our understanding of this inhibitory mechanism. When exposed to this complement regulator, parasites with inactivated GP63 bind to C4bBP and inactivate C4b deposited on their surface after serum exposure. This inactivation occurs by the action of Factor I, a complement system protease. In addition to the C4bBP-Factor I inactivation mechanism, the surface parasite protease GP63 can also inactivate soluble C4b molecules and probably that C4b molecules deposited on the parasites surface. This manuscript shows that Leishmania has two independent strategies to inactivate C4b molecules, preventing the progress of classical and lectins pathways. The identification of the C4bBP receptor on the Leishmania membrane may provide a new vaccine target to fight leishmaniasis.
Subject(s)
Leishmania infantum , Parasites , Animals , Humans , Complement C4b-Binding Protein/metabolism , Parasites/metabolism , Leishmania infantum/metabolism , Fibrinogen , Peptide Hydrolases , LectinsABSTRACT
INTRODUCTION: The expression of miR408 is affected by copper (Cu) conditions and positively regulates anthocyanin biosynthesis in Arabidopsis. However, the underlying mechanisms by which miR408 regulates anthocyanin biosynthesis mediated by Cu homeostasis and reactive oxygen species (ROS) homeostasis remain unclear in Malus plants. OBJECTIVES: Our study aims to elucidate how miR408a and its target, basic blue protein (BBP) regulate Cu homeostasis and ROS homeostasis, and anthocyanin biosynthesis in Malus plants. METHODS: The roles of miR408a and its target BBP in regulating anthocyanin biosynthesis, Cu homeostasis, and ROS homeostasis were mainly identified in Malus plants. RESULTS: We found that the BBP protein interacted with the copper-binding proteins LAC3 (laccase) and CSD1 (Cu/Zn SOD superoxide dismutase), indicating a potential crosstalk between Cu homeostasis and ROS homeostasis might be mediated by miR408 to regulate the anthocyanin accumulation. Further studies showed that overexpressing miR408a or suppressing BBP transiently significantly increased the expression of genes related to Cu binding and Cu transport, leading to anthocyanin accumulation under light induction in apple fruit and Malus plantlets. Consistently, opposite results were obtained when repressing miR408a or overexpressing BBP. Moreover, light induction significantly increased the expression of miR408a, CSD1, and LAC3, but significantly reduced the BBP expression, resulting in increased Cu content and anthocyanin accumulation. Furthermore, excessive Cu significantly increased the anthocyanin accumulation, accompanied by reduced expression of miR408a and Cu transport genes, and upregulated expression of Cu binding proteins including BBP, LAC3, and CSD1 to maintain the Cu homeostasis and ROS homeostasis in Malus plantlets. CONCLUSION: Our findings provide new insights into the mechanism by which the miR408a-BBP-LAC3/CSD1 module perceives light and Cu signals regulating Cu and ROS homeostasis, ultimately affecting anthocyanin biosynthesis in Malus plants.
Subject(s)
Arabidopsis , Malus , Malus/genetics , Malus/metabolism , Copper/metabolism , Reactive Oxygen Species/metabolism , Anthocyanins/metabolism , Homeostasis , Arabidopsis/geneticsABSTRACT
Based on in vitro and in silico assays as well as proteome analysis, this study explored the nongenomic mechanism for butyl benzyl phthalate (BBP)-induced thyroid disruption. Molecular docking simulations showed that BBP could dock into the Arg-Gly-Asp (RGD) domain of integrin αvß3 and form hydrogen bonds with a docking energy of -35.80 kcal/mol. This chemical enhanced rat pituitary tumor cell (GH3) proliferation and exhibited thyroid hormone-disrupting effects at 5-10 µmol/L. Meanwhile, BBP upregulated ß3 gene expression and activated the downstream mitogen-activated protein kinase (MAPK) pathway in GH3 cells. Interestingly, GH3 cell proliferation was attenuated by integrin αvß3 inhibitor (RGD peptide) or ERK1/2 inhibitor (PD98059), suggesting that the disruptions might be partly attributed to its interaction with integrin αvß3 and activation of MAPK. Furthermore, quantitative proteomic analysis of zebrafish embryos exposed to BBP at an environmentally relevant concentration of 0.3 µmol/L revealed that BBP perturbed proteins and pathways related to cell communication (e.g., integrin binding) and signal transduction (e.g., MAPK signaling pathway). Taken together, our results supported that the biological effects of BBP-activated integrin αvß3 mediated by the nongenomic pathway play an important role in its thyroid disruption. CAPSULE: The nongenomic pathway plays a vital role in the thyroid disruption-inducing actions of BBP.
Subject(s)
Phthalic Acids , Thyroid Gland , Animals , Integrin alphaVbeta3/genetics , Integrin alphaVbeta3/metabolism , Mitogen-Activated Protein Kinases/metabolism , Molecular Docking Simulation , Phthalic Acids/metabolism , Phthalic Acids/toxicity , Proteome/metabolism , Proteomics , Rats , Zebrafish/metabolismABSTRACT
Phthalate Esters (PAEs), detected in high concentrations generally in treated wastewater discharged from wastewater treatment plants (WWTPs), are important pollutants that restrict the reuse of wastewater. Investigating the fate of these endocrine-disrupting chemicals in WWTPs is crucial in order to protect both receiving environments and ecosystems. For this purpose, di(2-ethylhexyl) phthalate (DEHP), di-n-octyl phthalate (DNOP) and benzyl butyl phthalate (BBP) in the group of PAEs were monitored in simultaneously both in wastewater and sludge lines of selected two nature-based WWTPs and one advanced biological WWTP. Although it was frequently stated that phthalates were significantly removed in WWTPs in many studies found in literature, negative removal efficiencies of selected phthalates in investigated WWTPs during the sampling period were observed generally in this study. One of the reasons for this concentration increase could be releasing of phthalates from microplastics in wastewater during the treatment process or the desorption of PAEs from treatment sludge. DNOP was the compound with the highest concentration increase at almost each treatment unit of the three WWTPs. On the other hand, total PAEs load was 1997 g d-1 in advanced biological WWTP and adsorption onto sludge of PAEs were determined as 90%. The side-stream total load returned from the decanter supernatant was 0.02% of the total PAEs load coming to advanced biological WWTP from the sewer system. As a result of detailed statistical analysis, the correlation between raw wastewater and primary clarifier (PC) effluent was determined as an increasing linear relation for DEHP and DNOP. On the other hand, moderate and strong correlations were observed both between septic tank and constructed wetland (CW) processes with raw wastewater. In the waste stabilization pond (WSP), while a significant correlation was not found between the sludge line data, homogeneous variance, strong and moderate correlations were obtained in the wastewater line data. However, while mean differences for all investigated PAEs were not significant (p > 0.05) in the wastewater line, mean differences of DEHP (p < 0.05) were significant in the sludge line according to ANOVA analysis.
Subject(s)
Phthalic Acids , Water Pollutants, Chemical , Dibutyl Phthalate/analysis , Ecosystem , Environmental Monitoring , Esters/analysis , Phthalic Acids/analysis , Plastics , Ponds/analysis , Sewage/chemistry , Waste Disposal, Fluid , Wastewater/chemistry , Water Pollutants, Chemical/analysis , WetlandsABSTRACT
Benzyl butyl phthalate (BBP) has recently been implicated as an obesogen. Our recent study demonstrated that BBP can exacerbate high fat diet (HFD) induced diabesity in male mice. Here, we explored if pyrroloquinoline quinone (PQQ), a natural antioxidant andphytochemical, can attenuate metabolic aberrations induced by HFD or HFD-BBPcombination. C57Bl/6 male and female mice were fed either a chow diet (CD) or HFD with or without BBP (3 mg/kg body weight/day)and/or PQQ (20 mg/kg/day)for 16 weeks. The mice's body and tissue weight, fasting blood glucose, glucose and insulin tolerance test, and liver metabolites level weremeasured. In HFD-fed male mice, PQQ significantly attenuated the increased body weight, liver weight, fasting blood glucose, and insulin intolerance under BBP exposure.Even though female mice did show some reversal of metabolic characteristics by PQQ, the response was not similar nor consistent with the male population. Amongthe 14 hepatic metabolites that were significantly altered by HFD compared to CD, only three major metabolites (acetyl-L-carnitine, DL-stachytine, and propionylcarnitine) were decreased. These three were shown to have more reduction under BBP exposure in the presence of HFD whereas with addition of PQQ, these metabolites were restored. Pathway analysis and literature search revealed that these metabolites were negatively associated with obesity and were involved in several pathways including beta-oxidation, oxidative stress, and mitochondrial function. Overall,this finding indicated the potential use of PQQ to restore thewide range of aberrant metabolic effectinduced by an obesogen in the presence of a western diet.
Subject(s)
Liver/drug effects , Liver/metabolism , Metabolomics/methods , PQQ Cofactor/pharmacology , Phthalic Acids/toxicity , Teratogens/toxicity , Animals , Blood Glucose/drug effects , Blood Glucose/metabolism , Female , Male , Mice , Mice, Inbred C57BLABSTRACT
Butylbenzyl phthalate (BBP) has been shown to negatively affect the development of zebrafish embryos, however, its underlying mechanisms remain unclear. Therefore, our study aims to reveal the molecular mechanisms of developmental toxicity on zebrafish embryos. Zebraï¬sh embryos were exposed to BBP (0, 0.6, and 1.2 mg/L) from 4 to 72 h post-fertilization (hpf). The adverse effects on zebrafish embryos were evaluated and the transcriptional profiles of zebraï¬sh embryos were analyzed at 72hpf. Exposure to BBP decreased hatching and survival rates and induced obvious morphology abnormalities in zebraï¬sh embryos. The activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) significantly decrease and the malondialdehyde (MDA) content significantly increased with 1.2 mg/L BBP exposure. Global transcriptome profiling analysis demonstrated that 578 and 1257 genes were differentially expressed in zebrafish embryos in the 0.6 and 1.2 mg/L groups, respectively. Gene Ontology (GO) term enrichment analysis demonstrated that DEGs are related to many aspects of cell composition, biological processes, and molecular functions. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis demonstrated that 13 and 22 pathways were significantly enriched in the 0.6 and 1.2 mg/L groups, respectively. DEGs were primarily concentrated in the metabolism of the 0.6 mg/L group and in the organismal systems and particularly affected vision and digestion in the 1.2 mg/L group. Our results contribute to a better understanding of the underlying mechanisms of developmental toxicity induced by phthalates.
Subject(s)
Embryo, Nonmammalian/drug effects , Gene Expression Regulation, Developmental , Phthalic Acids/toxicity , Transcriptome , Zebrafish/embryology , Zebrafish/metabolism , Animals , Catalase/metabolism , Glutathione Peroxidase/metabolism , Malondialdehyde/metabolism , Superoxide Dismutase/metabolismABSTRACT
Left bundle branch pacing (LBBP) is effective in patients with heart failure, left ventricular ejection fraction (LVEF) of ≤35%, and a widened QRS complex. LBBP leads to iatrogenic incomplete right bundle branch block (iRBBB). Bi-bundle pacing can resolve iRBBB, further narrowing the QRS duration, and may improve LVEF. (Level of Difficulty: Advanced.).
ABSTRACT
BACKGROUND: Biogeochemical-Argo floats are collecting an unprecedented number of profiles of optical backscattering measurements in the global ocean. Backscattering (BBP) data are crucial to understanding ocean particle dynamics and the biological carbon pump. Yet, so far, no procedures have been agreed upon to quality control BBP data in real time. METHODS: Here, we present a new suite of real-time quality-control tests and apply them to the current global BBP Argo dataset. The tests were developed by expert BBP users and Argo data managers and have been implemented on a snapshot of the entire Argo dataset. RESULTS: The new tests are able to automatically flag most of the "bad" BBP profiles from the raw dataset. CONCLUSIONS: The proposed tests have been approved by the Biogeochemical-Argo Data Management Team and will be implemented by the Argo Data Assembly Centres to deliver real-time quality-controlled profiles of optical backscattering. Provided they reach a pressure of about 1000 dbar, these tests could also be applied to BBP profiles collected by other platforms.
ABSTRACT
Hypospadias is a defect in penile urethral closure that occurs in approximately 1/150 live male births in developed nations, making it one of the most common congenital abnormalities worldwide. Alarmingly, the frequency of hypospadias has increased rapidly over recent decades and is continuing to rise. Recent research reviewed herein suggests that the rise in hypospadias rates can be directly linked to our increasing exposure to endocrine disrupting chemicals (EDCs), especially those that affect estrogen and androgen signalling. Understanding the mechanistic links between endocrine disruptors and hypospadias requires toxicologists and developmental biologists to define exposures and biological impacts on penis development. In this review we examine recent insights from toxicological, developmental and epidemiological studies on the hormonal control of normal penis development and describe the rationale and evidence for EDC exposures that impact these pathways to cause hypospadias. Continued collaboration across these fields is imperative to understand the full impact of endocrine disrupting chemicals on the increasing rates of hypospadias.
ABSTRACT
We study the pricing strategies of supply chains of green products under behaviour-based pricing. Considering consumer preferences for green product functional attributes and environmental attributes, we construct a two-stage supply chain. The optimal behaviour pricing of green products is solved, and the effects of green sensitivity and the cost coefficient on the optimal price are analysed. We find that when consumers are less sensitive to the greenness, with the increase in the market share of green products, green product retailers will increase the loyalty price. An increase in greenness sensitivity and a decrease in the greenness cost coefficient will increase the wholesale prices and retail prices of green products. Consumer attention to the greenness and a decrease in the initial market share of green products will be conducive to promoting the greenness and improving the environment. Consumers' emphasis on the greenness of their products will lead to higher profits for the manufacturers and retailers of green products.
Subject(s)
Commerce , Consumer Behavior , Costs and Cost AnalysisABSTRACT
Lyme disease is the most widespread vector-transmitted disease in North America and Europe, caused by infection with Borrelia burgdorferi sensu lato complex spirochetes. We report the solution NMR structure of the B. burgdorferi outer surface lipoprotein BBP28, a member of the multicopy lipoprotein (mlp) family. The structure comprises a tether peptide, five α-helices and an extended C-terminal loop. The fold is similar to that of Borrelia turicatae outer surface protein BTA121, which is known to bind lipids. These results contribute to the understanding of Lyme disease pathogenesis by revealing the molecular structure of a protein from the widely found mlp family.
Subject(s)
Bacterial Outer Membrane Proteins/chemistry , Borrelia burgdorferi/metabolism , Lipoproteins/chemistry , Amino Acid Sequence , Bacterial Outer Membrane Proteins/genetics , Bacterial Outer Membrane Proteins/metabolism , Borrelia/chemistry , Borrelia/metabolism , Borrelia burgdorferi/chemistry , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Humans , Lipoproteins/genetics , Lipoproteins/metabolism , Lyme Disease/microbiology , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
In this study, an anticalin that could specifically bind paraquat (PQ), a quaternary nitrogen herbicide, as a new set of engineered receptor protein with antibody-like properties was generated to detect PQ concentration. To this end, a native and random library was constructed and engineered to allow in vitro transcription and translation using an Escherichia coli lysate system. Meanwhile, a PQ derivative that carries an active aliphatic carboxylate group at the end of an aliphatic spacer arm was synthesized. Then, this compound was coupled covalently to the carrier protein bovine serum albumin/ovalbumin and amino-functionalized paramagnetic beads. Alternating selection in solution and immobilization in microtiter wells were used to pan mRNA-ribosome-antibody complexes. After several rounds of ribosome display, three variants were selected from a random library of the bilin-binding protein. The variants that could bind complex PQ with high affinity and exhibit IC50 values as low as 14.039 ± 0.970 ng/mL were identified. Moreover, the limits of detection reached 0.083 ± 0.011 ng/mL. Our data suggest that the generation of anticalins may provide a promising alternative to recombinant antibody fragments to create a stable receptor protein against hapten with bioanalytical relevance.
Subject(s)
Lipocalins/chemistry , Ovalbumin/chemistry , Paraquat/chemistry , Ribosomes/chemistry , Serum Albumin, Bovine/chemistry , Animals , Cattle , Magnetic Phenomena , Molecular Structure , Peptide LibraryABSTRACT
A comprehensive study on packaging used in commercially available milk products from Spanish markets has been presented. Concentrations of four phthalates, seven parabens and BPA were determined in forty-two milk products. Eleven brands and five types of packaging (metallic aluminium bag, carton, high-density polyethylene, metal pail and polyethylene terephthalate) were included in the study. BPA showed the lowest concentrations (8.3 pg/g f.w.), far below those of phthalates (6431 pg/g f.w.) and parabens (6234 pg/g f.w.). Metallic aluminium bags were the least migrating packaging (considering plasticisers and monomers) followed by HDPE bottles, in the case of phthalates. Parabens showed their highest concentrations for fresh-milk samples. Levels found were far below the specific migration limits established by the EU and the cumulative hazard index was lower than 1, indicating that adverse health effects were not expected. In general, the results found in Spanish samples were lower than those reported in other countries.
Subject(s)
Milk/chemistry , Plasticizers/analysis , Preservatives, Pharmaceutical/analysis , Animals , Benzhydryl Compounds/analysis , Humans , Limit of Detection , Parabens/analysis , Phenols/analysis , Phthalic Acids/analysis , SpainABSTRACT
Exposure to endocrine disrupting chemicals (EDCs) used in plastic manufacturing processes may be contributing to the current increase in metabolic disorders. Here, we determined that benzyl butyl phthalate (BBP), a common EDC and food packaging plasticizer, mixed into chow diet (CD) and high fat diets (HFD) at varying concentrations (4 µg/kg body weight (bw)/day, 169 µg/kg bw/day, 3 mg/kg bw/day, 50 mg/kg bw/day) produced a number of detrimental and sex-specific metabolic effects in C57BL/6 male and female mice after 16 weeks. Male mice exposed to moderate (3 mg/kg bw/day) concentrations of BBP in an HFD were especially affected, with significant increases in body weight due to significant increases in weight of liver and adipose tissue. Other doses did not show any significant changes when compared to only CD or HFD alone. HFD in the presence of 3 mg/kg bw/day BBP showed significant increases in fasting blood glucose, glucose intolerance, and insulin intolerance when compared to HFD alone. Furthermore, this group significantly alters transcriptional regulators involved in hepatic lipid synthesis and its downstream pathway. Interestingly, most of the BBP doses had no phenotypic effect when mixed with CD and compared to CD alone. The female mice did not show a similar response as the male population even though they consumed a similar amount of food. Overall, these data establish a dose which can be used for a BBP-induced metabolic research model and suggest that a moderate dosage level of EDC exposure can contribute to widely ranging metabolic effects.