Effects of perinatal exposure to benzo[a]pyrene on the expressionof PDX-1 and TFAM in pancreas and mitochondrial DNA copynumber in offspring rats / 预防医学

Objective@#To observe the effects of perinatal exposure to benzo[a]pyrene (B[a]P) on the expression of pancreatic duodenal homeobox-1 (PDX-1) and mitochondrial transcription factor A (TFAM) and mitochondrial DNA copy number in offspring mice, and to explore the role of maternal exposure to B[a]P in the pancreatic function damage of offspring mice.@*Methods@#Forty pregnant rats were randomly divided into the control group, the lowest dose group (2 μg/kg), the low dose group (200 μg/kg), medium dose group (800 μg/kg) and high dose group (1 600 μg/kg), with 8 rats in each group. From day 1 of pregnancy, each exposed group was given 0.2 mL/100 g body weight of B[a]P and corn oil mixture by gavage once a day until 3 weeks after delivery, while the control group was given the same dose of corn oil. The pancreatic tissue of three-week-old mice were collected after abdominal anesthesia for insulin immunohistochemical detection. The protein and mRNA expression levels of PDX-1 and TFAM, as well as mitochondrial DNA copy number were detected. Spearman rank correlation analysis was used to analyze the correlation between B[a]P exposure dose and the above indicators.@*Results@#The insulin-positive area ratio and average optical density of insulin in the medium and the high dose groups were significantly lower than those in the control group (all P<0.05). The insulin-positive area ratio and average optical density of insulin were negatively correlated with the B[a]P dose (rs=-0.862 and -0.858, both P<0.05). The protein expression levels of PDX-1 and TFAM in the high dose group were significantly lower than those in the control group (both P<0.05). The protein expression levels of PDX-1 and TFAM were negatively correlated with the B[a]P dose (rs=-0.756 and -0.799, both P<0.05). The mRNA expression levels of PDX-1 and mitochondrial DNA copy number in the medium and high dose groups were significantly lower than those in the control group, and the mRNA expression level of TFAM in the high dose group was significantly lower than that in the control group (all P<0.05). The mRNA expression levels of PDX-1, TFAM, and mitochondrial DNA copy number were negatively correlated with the B[a]P dose (rs=-0.722, -0.550 and -0.840, all P<0.05).@*Conclusion@#Perinatal exposure to B[a]P can induce the damage of islet β cells in offspring rats, which may be related to the decreased expression of PDX-1 and TFAM and the copy number of mitochondrial DNA.

Raman Spectroscopy Analysis of The Temporal Heterogeneity in Lung Cell Carcinogenesis Induced by Benzo(a)pyrene / 生物化学与生物物理进展

ObjectiveTemporal heterogeneity in lung cancer presents as fluctuations in the biological characteristics, genomic mutations, proliferation rates, and chemotherapeutic responses of tumor cells over time, posing a significant barrier to effective treatment. The complexity of this temporal variance, coupled with the spatial diversity of lung cancer, presents formidable challenges for research. This article will pave the way for new avenues in lung cancer research, aiding in a deeper understanding of the temporal heterogeneity of lung cancer, thereby enhancing the cure rate for lung cancer. MethodsRaman spectroscopy emerges as a powerful tool for real-time surveillance of biomolecular composition changes in lung cancer at the cellular scale, thus shedding light on the disease’s temporal heterogeneity. In our investigation, we harnessed Raman spectroscopic microscopy alongside multivariate statistical analysis to scrutinize the biomolecular alterations in human lung epithelial cells across various timeframes after benzo(a)pyrene exposure. ResultsOur findings indicated a temporal reduction in nucleic acids, lipids, proteins, and carotenoids, coinciding with a rise in glucose concentration. These patterns suggest that benzo(a)pyrene induces structural damage to the genetic material, accelerates lipid peroxidation, disrupts protein metabolism, curtails carotenoid production, and alters glucose metabolic pathways. Employing Raman spectroscopy enabled us to monitor the biomolecular dynamics within lung cancer cells in a real-time, non-invasive, and non-destructive manner, facilitating the elucidation of pivotal molecular features. ConclusionThis research enhances the comprehension of lung cancer progression and supports the development of personalized therapeutic approaches, which may improve the clinical outcomes for patients.

Map of benzoapyrene metabolites-DNA adducts in human bronchial epithelial-like cells: Based on chromatin immunoprecipitation followed by sequencing technology / 环境与职业医学

Background The active metabolite of benzo[a]pyrene (BaP), 7,8-dihydroxy-9,10-epoxybenzo[a]pyrene (BPDE), can form adducts with DNA, but the spectrum of BPDE-DNA adducts is unclear. Objective To identify the distribution of BPDE adduct sites and associated genes at the whole-genome level by chromatin immunoprecipitation followed by sequencing (ChIP-Seq), and serve as a basis for further exploring the toxicological mechanisms of BaP. Methods Human bronchial epithelial-like cells (16HBE) were cultured to the fourth generation inthe logarithmic growth phase. Cells were harvested and added to chromatin immunoprecipitation lysis buffer. The lysate was divided into experimental and control groups. The experimental group received a final concentration of 20 μmol·L−1 BPDE solution, while the control group received an equivalent volume of dimethyl sulfoxide solution. The cells were then incubated at 37 °C for 24 h. Chromatin fragments of 100-500 bp were obtained through sonication. BPDE-specific antibody (anti-BPDE 8E11) was used to enrich DNA fragments with BPDE adducts. High-throughput sequencing was conducted to detect BPDE adduct sites. The top 1000 peak sequences were subjected to motif analysis using MEME and DREME software. BPDE adduct target genes at the whole-genome level were annotated, and Gene Ontology (GO) functional analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of BPDE adduct target genes were conducted using bioinformatics techniques. Results The high-throughput sequencing detected a total of 842 BPDE binding sites, distributed across various chromosomes. BPDE covalently bound to both coding and non-coding regions of genes, with 73.9% binding sites located in intergenic regions, 19.6% in intronic regions, and smaller proportions in upstream 2 kilobase, exonic, downstream 2 kilobase, and 5' untranslated regions. Regarding the top 1000 peak sequences, four reliable motifs were identified, revealing that sites rich in adenine (A) and guanine (G) were prone to binding. Through the enrichment analysis of binding sites, a total of 199 BPDE-adduct target genes were identified, with the majority located on chromosomes 1, 5, 7, 12, 17, and X. The GO analysis indicated that these target genes were mainly enriched in nucleic acid and protein binding, participating in the regulation of catalytic activity, transport activity, translation elongation factor activity, and playing important roles in cell division, differentiation, motility, substance transport, and information transfer. The KEGG analysis revealed that these target genes were primarily enriched in pathways related to cardiovascular diseases, cancer, and immune-inflammatory responses. Conclusion Using ChIP-Seq, 199 BPDE adduct target genes at genome-wide level are identified, impacting biological functions such as cell division, differentiation, motility, substance transport, and information transfer. These genes are closely associated with cardiovascular diseases, tumors, and immune-inflammatory responses.

Element profiles of benzoapyrene malignantly transformed 16HBE cells and joint effects of copper with cisplatin or vinorelbine on cell proliferation / 中国药理学与毒理学杂志

OBJECTIVE To assess the profiles of elements in benzo[a]pyrene(BaP)induced carci-nogenesis,and explore the joint effects of copper with cisplatin or vinorelbine on cell proliferation.METHODS Forty-four elements were measured using an inductively coupled plasma mass spectrometer in 16HBE cells and BaP malignantly transformed 16HBE(T-16HBE-C1)cells.Partial least square was used to validate the robustness of cell classification of elements.Cell viability was measured by MTT assay for copper(0,237,340,487,1000 and 1432 μmol·L-1),cisplatin(0,4.4,6.1,8.6,12.0 and 16.8 μmol·L-1),and vinorelbine(0,3.8,9.8,25.0,40.0 and 64.0 μmol·L-1)to acquire their half maximal inhibitory concentra-tion(IC50).Mixtures of copper and chemotherapeutics were prepared according to the ratio of each IC50.Their joint effects on cell viability were assessed by MTT assay and isobolographic analysis.Inhibition effect of copper(0,50,100,200,400 and 800 μmol·L-1)with IC50 of cisplatin or vinorelbine on prolifera-tion of T-16HBE-C1 cells was also assessed.RESULTS A total of 29 elements were quantified in 16HBE and T-16HBE-C1 cells,among which concentrations of copper,zinc,silver,selenium and rubidium decreased(P<0.05,P<0.01),while those of molybdenum,arsenic,lithium,germanium,strontium,nickel,lanthanum,mercury,iron,and cesium increased(P<0.05,P<0.01)in T-16HBE-C1 cells.Element concen-tration could be used to distinguish T-16HBE-C1 cells from 16HBE cells.Copper concentration-dependently inhibited proliferation of both cells,with a statistically significant lower IC50 of(613±16)μmol·L-1 in 16HBE cells than(776±15)μmol·L-1 in T-16HBE-C1 cells(P<0.01).Mixtures of copper and cisplatin(1∶69.5)or vinorelbine(1∶33.4)could inhibit cell proliferation,and copper had additive effects with cisplatin or vinorelbine.When copper concentration was higher than 400 μmol·L-1,copper combined with IC50 of cisplatin or vinorelbine inhibited cell proliferation of T-16HBE-C1 cells compared with IC50 of cisplatin(11.2 μmol·L-1)or vinorelbine(23.2 μmol·L-1)alone.CONCLUSION Element profiles and correlations can change significantly after 16HBE cells are malignantly transformed by BaP.Copper could inhibit the proliferation of T-16HBE-C1 cells and have additive effects with cisplatin or vinorelbine in higher concentration.

7,8-dihydroxy-9,10-epoxybenzoapyrene induced ferroptosis in mouse hippocampal neuron HT22 cells and its mechanism / 环境与职业医学

Background Benzo[a]pyrene (BaP) has neurotoxicity, which can induce the loss of hippocampal neurons in humans and animals and lead to spatial learning and memory dysfunction, but its mechanism is still unclear. Objective To observe the ferroptosis of mouse hippocampal neuron HT22 cells induced by 7,8-dihydroxy-9,10-epoxybenzo[a]pyrene (BPDE), an active metabolite of BaP, and to explore its potential mechanism, so as to provide a basis for the study of BaP neurotoxicity mechanism. Method Mouse hippocampal neuron HT22 cells were selected and divided into four groups: solvent control group and low, medium, and high concentration BPDE exposure groups (0.25, 0.50, and 0.75 μmol·L−1). Cell survival was detected by CCK8 method. Cell morphology and ultrastructure were observed under light and electron microscopes. The levels of reactive oxygen species (ROS) and Fe2+ were detected by fluorescence probe method. Iron, 4-hydroxynonenoic acid (4-HNE), malondialdehyde (MDA), glutathione (GSH), and glutathione peroxidase (GSH-PX) levels were detected with commercial kits. The expression levels of acyl-CoA synthase long chain family member 4 (ACSL4), cyclooxygenase 2 (COX2), solute carrier family 7 member 11 (SLC7A11), and glutathione peroxidase 4 (GPX4) were detected by Western blotting. After interventions with ferroptosis inhibitors 20 μmol·L−1 deferoxamine (DFO) and 10 μmol·L−1 ethyl 3-amino-4-cyclohexylaminobenzoate (Fer-1), the cell survival rate of each BPDE exposure group and the changes of the ferroptosis characteristic indicators and protein expression levels were observed. Results With the increase of BPDE concentration, the survival rate of HT22 cells decreased gradually, and the survival rate of each BPDE group was significantly lower than that of the solvent control group (P<0.01). Under light microscope, the number of cells in the high concentration BPDE group was significantly reduced, and atrophic cells and reduced synapses were recorded. Under electron microscope, the HT22 cells in the high concentration BPDE group showed mitochondrial shrinkage, decreased crista, and increased mitochondrial membrane density. Compared with the solvent control group, the levels of intracellular lipid ROS, Fe2+, 4-HNE, and MDA significantly increased in the high concentration group (P<0.01), the GSH and GSH-PX levels were significantly decreased (P<0.01), the protein expression levels of ASCL4 and COX2 were significantly increased (P<0.01), and the protein expression levels of SCL7A11 and GPX4 were significantly decreased (P<0.01). The ferroptosis inhibitors DFO and Fer-1 significantly reversed the cell survival rate (P<0.01), the ferroptosis characteristic indicators (ROS, Fe2+, 4-HNE, MDA, GSH, and GSH-PX levels) (P<0.01), and the expression levels of ferroptosis-related proteins (ACSL4, COX2, SLC7A11, and GPX4) (P<0.01) in the high concentration BPDE group. Conclusion BPDE can induce ferroptosis in mouse hippocampal neuron HT22 cells, which may be related to the inhibition of SLC7A11/GSH/GPX4 axis and the induction of iron metabolism disorder.

Protective effect of proanthocyanidins on neurotoxicity of benzoapyrene / 环境与职业医学

Background Benzo[a]pyrene (BaP) is neurotoxic and can cause neuronal damage by oxidative stress. Proanthocyanidin (PC) has antioxidant activity, and its mechanism may related to nuclear factor-erythroid 2-related factor 2 (Nrf2)-heme oxygenase-1 (HO-1) signaling pathway. Objective To explore potential protective effect of PC on hippocampal neuron injury induced by BaP oxidative stress. Methods Hippocampal neurons of neonatal SD rats delivered within 24 h were isolated and cultured, and cell activity was detected by cell counting kit-8 (CCK-8) method. According to the pre-experimental results, a control group and three BaP groups of 10, 20 and 40 µmol·L−1 were set up for Stage I experiment. The length of neurites and number of branches of hippocampal neurons in each group were observed by immunofluorescence method. Reactive oxygen species (ROS) fluorescence probe method was used to measure ROS levels in cells. Real-time quantitative fluorescent polymerase chain reaction (qRT-PCR) and Western blotting were used to detect the mRNA and protein expression of Nrf2, Kelch-like epichlorohydrin associated protein-1 (Keap1), HO-1, B-cell lymphoma-2 (Bcl-2), and Bcl-2-associated X (Bax) in hippocampal neurons of each group, respectively. According to the results of Stage I experiment, three group were set up, including control group, BaP alone treatment group (BaP 20 µmol·L−1), and PC intervention group (BaP 20 µmol·L−1 + PC 2.5 µg·mL−1) for Stage II experiment, with the same protocol as Stage I. Results For Stage I experiment, compared with the control group, the 10, 20, and 40 µmol·L−1 BaP groups showed gradually shortened length of neurites [(177.60±3.49), (142.40±6.52), and (100.50±19.40) µm] (P<0.05) and decreased number of branches (8.00±1.00, 6.33±1.53, 4.33± 0.58) of hippocampal neurons (P<0.05); increased ROS production (2.38±0.33, 8.08±0.26, 9.86±0.19) (P<0.05); the qRT-PCR results showed that the mRNA expression levels of Nrf2 (0.35±0.03, 0.25±0.01, 0.13±0.03), Keap1 (0.70±0.01, 0.47±0.03, 0.15±0.02), HO-1 (0.77±0.02, 0.60±0.02, 0.32±0.01), and Bcl-2 (0.65±0.03, 0.47±0.02, 0.18±0.02) gradually decreased, and the mRNA expression level of Bax gradually increased (1.24±0.01, 2.25±0.15, 4.98±0.30) (P<0.05); the Western blotting results showed that the protein expression trends of Nrf2, Keap1, HO-1, Bcl-2, and Bax were consistent with the mRNA results. For Stage II experiment, compared with the BaP alone treatment group, the length of neurites in the PC intervention group became longer, (149.90±3.01) μm vs (202.00±4.45) μm (P<0.05), the number of branches increased, (4.67±0.58) vs (8.33±0.58) (P<0.05); the ROS production reduced, (10.81±0.63) vs (7.31±0.70) (P<0.05); the mRNA expression levels of Nrf2, Keap1, HO-1, and Bcl-2 increased (P<0.05), and the mRNA expression levels of Bax decreased (P<0.05); the Nrf2, Keap1, HO-1, and Bcl-2 protein expression levels increased (P<0.05), and Bax protein expression level decreased (P<0.05). Conclusion PC may exert neuroprotective effects by activating the Nrf2-HO-1 signaling pathway, inhibiting BaP-induced oxidative stress in neuronal cells, and reducing cytotoxicity.

Regulating anaerobic metabolism and promoting myocardial ischemia-hypoxia injury by diesel particulate matter and its key component benzoapyrene via targeting oxygen sensors / 环境与职业医学

Background The exposure to diesel particulate matter (DPM) and its polycyclic aromatic hydrocarbons (PAH) is closely related to the morbidity and mortality of ischemic heart disease (IHD). However, it is unclear what key components and targets of DPM exposure involve in myocardial ischemia-hypoxia injury and associated mechanisms. Objective To identify key PAH components of DPM that act on myocardial hypoxic injury, andclarify the role of oxygen sensors-regulated anaerobic metabolism in DPM and key components-induced hypoxic injury and the targets of the key PAH components. Methods Human cardiomyocyte cell line AC16 cells were exposed to 0, 1, 5, and 10 μg·mL−1 DPM in a high glucose DMEM medium with 10% fetal bovine serum (FBS) (HGM) or low FBS (0.5%) in high glucose DMEM medium (LFM), for 12 h under 2% O2, and expression of hypoxia-inducible factor-1α (HIF-1α), Bax, and Cleaved-caspase3 was determined by Western blotting. Under normal condition, the cell viability was detected after PAH exposure for 12 h. Under the condition of ischemia-hypoxia model, cells were exposed to 0, 0.005, 0.5, and 5 µg·mL−1 PAH for 12 h, and the protein expression of HIF-1α, Bax, and Cleaved-caspase3 was determined. After exposure to DPM or PAH for 12 h, the contents of pyruvate and lactate in cells were detected. Pretreatment with glycolysis inhibitor GSK2837808A was used to explore the role of glycolysis in DPM and benzo[a]pyrene (BaP)-induced hypoxia injury. A molecular docking technique was used to analyze the binding affinity between PAH and oxygen sensors (prolyl hydroxylase domain-containing protein 2, PHD2, and factor-inhibiting hypoxia-inducible factor 1, FIH1), and the protein levels of PHD2, FIH1, and hydroxyl-HIF-1-alpha (OH-HIF-1α) after the DPM or BaP treatment were further determined. Results Under hypoxia, DPM exposure in the LFM induced the expression of HIF-1α, Bax, and Cleaved-caspase3 (P<0.01). Therefore, hypoxia and LFM were selected as the basic ischemia and hypoxia condition. Except for anthracene (Ant) (P>0.05), other PAH decreased cell viability when the concentration was above 1 μg·mL−1 (P<0.05). All concentrations of BaP induced the expression of HIF-1α protein (P<0.05), and the protein levels of Bax and Cleaved-caspase3 were up-regulated after the 0.5 and 5 µg·mL−1 BaP exposure (P<0.01). After exposure to DPM (1, 5 and 10 μg·mL−1) or BaP (0.5 and 5 μg·mL−1), the intracellular pyruvate and lactate contents increased (P<0.05). The glycolysis inhibitor co-treatment decreased the levels of HIF-1α, Bax, and Cleaved-caspase3 proteins compared with the DPM or BaP exposure group for 12 h (P＜0.05). The binding abilities of the five PAHs to the oxygen sensors PHD2 and FIH1 were strong, and BaP was the strongest. Although the DPM or BaP exposure had no effects on the protein levels of PHD2 and FIH1 in AC16 cells (P＜0.05), the protein level of OH-HIF-1α was decreased (P＜0.01). Conclusion BaP exposure can promote hypoxia and injury of myocardial cells and is the key PAH component of DPM that induces myocardial ischemia and hypoxia injury. BaP exposure inhibits the hydroxylation function of PHD2 on HIF-1α by combining with PHD2, decreases the level of OH-HIF-1α and induces HIF-1α accumulation. And then HIF-1α promotes anaerobic metabolism and accelerates ischemia and hypoxia injury of myocardial cells.

Protección del ácido clorogénico frente a lesiones inducidas por venzo(a)pireno en Saccharomyces cerevisiae / Protection of chlorogenic acid against benzo(a)pyrene-induced lesions in Saccharomyces cerevisiae / Proteção do ácido clorogênico contra lesões induzidas por benzo(a)pireno em Saccharomyces cerevisiae

El objetivo del presente estudio fue analizar el efecto del ácido clorogénico, uno de los compuestos polifenólicos con mayor concentración en la infusión de Ilex paraguariensis, sobre el daño celular y molecular inducido por el benzo(a)pireno. La infusión de Ilex paraguariensis ("mate") es bebida por la mayoría de los habitantes de Argentina, Paraguay, sur de Brasil y Uruguay. La levadura Saccharomyces cerevisiae (cepas SC7K lys2-3; SX46A y SX46Arad14() se utilizó como modelo eucariota. Las células en crecimiento exponencial se expusieron a concentraciones crecientes de benzo(a)pireno y a tratamientos combinados con una concentración de 250 ng/mL de benzo(a)pireno y ácido clorogénico a una concentración igual a la encontrada en la infusión de yerba mate. Luego de los tratamientos se determinaron fracciones de sobrevida, frecuencia mutagénica y roturas de doble cadena de ADN así como la modulación en la expresión de la proteína Rad14 a través de un análisis de Western Blot. Se observó un aumento significativo en las fracciones de sobrevida así como una disminución en la frecuencia mutagénica después de la exposición combinada con benzo(a)pireno y ácido clorogénico en comparación con los tratamientos con benzo(a)pireno como único agente. En la cepa mutante deficiente en la proteína Rad14 se observó un aumento significativo en la sensibilidad a benzo(a)pireno en comparación con la cepa SC7K lys2-3. En los tratamientos combinados de benzo(a)pireno y ácido clorogénico se observó una importante disminución de la letalidad. Con respecto a la determinación de roturas de doble cadena de ADN no se observó fraccionamiento cromosómico a la concentración de benzo(a)pireno utilizada en los experimentos. Los análisis de Western Blot mostraron un aumento en la expresión de la proteína Rad14 en las muestras tratadas con benzo(a)pireno como único agente en comparación con la muestra control. Adicionalmente se observó una disminución en la expresión de la proteína cuando en los tratamientos se utilizaron benzo(a)pireno y ácido clorogénico combinados. Los resultados indican que el ácido clorogénico disminuye significativamente la actividad mutagénica producida por el benzo(a)pireno, la cual no se encuentra relacionada con un incremento en la remoción de las lesiones inducidas por el sistema de reparación por escisión de nucleótidos.

The aim of this study was to analyze the effect of chlorogenic acid, a polyphenolic compound found at high concentrations in Ilex paraguariensis infusions, on cellular and molecular damage induced by benzo(a)pyrene. Ilex paraguariensis infusions ("mate") are consumed by most of the population in Argentina, Paraguay, southern Brazil and Uruguay. Saccharomyces cerevisiae yeast (SC7K lys2-3; SX46A and SX46Arad14( strains) were used as eukaryotic model organisms. Cells in an exponential growth phase were exposed to increasing concentrations of benzo(a)pyrene, as well as combined treatments of benzo(a)pyrene at a concentration of 250 ng/mL and chlorogenic acid at a concentration matching that which is commonly found in mate. Determinations of surviving fraction, mutagenic frequency and double strand DNA breaks induction were performed, along with the assessment of the modulation of the expression of protein Rad14 by Western Blot. A significant increase of surviving fractions and a decrease in mutagenic frequency were observed after exposure to benzo(a)pyrene plus chlorogenic acid, contrary to benzo(a)pyrene alone. A substantial increase in sensitivity to benzo(a)pyrene was observed for the Rad14 protein-deficient mutating strain when compared to the SC7K lys2-3 strain. An important decrease in lethality was observed when combined benzo(a)pyrene and chlorogenic acid treatments were applied. As for the determination of DSBs, no chromosomic fractionation was observed at the benzo(a)pyrene concentration tested in the experiments. Western Blot analysis showed an increase in the expression of protein Rad14 for samples treated with benzo(a)pyrene as a single agent when compared against the control sample. Additionally, the expression of this protein was observed to diminish when combined treatments with benzo(a)pyrene and chlorogenic acid were used. Results obtained indicate that chlorogenic acid significantly decreases the mutagenic activity of benzo(a)pyrene, which is not related to an increase in the removal of lesions induced by nucleotide excision repair system.

O objetivo deste estudo foi analisar o efeito do ácido clorogênico, um dos compostos polifenólicos com maior concentração na infusão de Ilex paraguariensis, sobre o dano celular e molecular induzido pelo benzopireno. A infusão de Ilex paraguariensis ("mate") é consumida pela maioria dos habitantes da Argentina, Paraguai, sul do Brasil e Uruguai. A levedura Saccharomyces cerevisiae (cepas SC7K lys2-3; SX46A e SX46Arad14() foi utilizada como modelo eucariótico. Células em crescimento exponencial foram expostas a concentrações crescentes de benzopireno e tratamentos combinados foram realizados com uma concentração de 250 ng/mL de benzo(a)pireno e ácido clorogênico, igual à encontrada na infusão de erva-mate. Após os tratamentos, foram determinadas as frações de sobrevivência, frequência mutagênica e quebras de fita dupla do DNA, bem como a modulação na expressão da proteína Rad14 por meio de análise de Western Blot. Um aumento significativo nas frações de sobrevivência, bem como uma diminuição na frequência mutagênica foram observados após a exposição combinada de benzo(a)pireno e ácido clorogênico em comparação com tratamentos de agente único de benzo(a)pireno. Um aumento significativo na sensibilidade ao benzo(a)pireno foi observado na cepa mutante deficiente em proteína Rad14 em comparação com a cepa SC7K lys2-3. Nos tratamentos combinados de benzo(a)pireno e ácido clorogênico, observou-se uma diminuição significativa na letalidade. Com relação à determinação das quebras de fita dupla de DNA, não foi observado fracionamento cromossômico na concentração de benzo(a)pireno utilizada nos experimentos. A partir da análise de Western Blot, observou-se um aumento na expressão da proteína Rad14 nas amostras tratadas com benzo(a)pireno como agente único em relação à amostra controle. Além disso, uma diminuição na expressão da proteína foi observada quando combinados de benzo(a)pireno e ácido clorogênico foram usados ​​nos tratamentos. Os resultados obtidos neste trabalho indicam que o ácido clorogênico diminui significativamente a atividade mutagênica produzida pelo benzo(a)pireno, a qual não está relacionada a um aumento na remoção de lesões induzidas pelo sistema de reparo por excisão de nucleotídeos.

Effects of subchronic exposure to benzoɑpyrene on memory function and mRNA expressions of NMDA receptors in different brain regions in rats / 环境与职业医学

Background The altered expressions of hippocampal N-methyl-D-aspartate (NMDA) receptors induced by benzo[ɑ]pyrene (BaP) causes short-term spatial learning and memory impairment in humans and animals, but whether BaP causes alterations of NMDA receptor subunits in other brain regions and the associated neurotoxic mechanism is still essentially unknown. Objective To observe the mRNA expressions of NR1, NR2A, and NR2B of NMDA receptor subunits in different brain regions in SD rat model with subchronic exposure to BaP, and to provide a basis for in-depth study of the mechanism of BaP-induced neurotoxicity. Methods Forty male SD rats were selected and randomly divided into a control group and 1.00, 2.50, and 6.25 mg·kg−1 BaP exposure groups with 10 rats in each group. The exposure rats received intraperitoneal injection of BaP every other day for 90 d.The average latency to platform, the average total distance, and the duration spent in previous quadrant were measured by the Morris Water Maze. Real-time fluorescence quantitative PCR was used to detect the mRNA expressions of NR1, NR2A, and NR2B in hippocampus, cortex, cerebellum, and striatum of rats. Results The average latency to platform and the average total distance in the 2.50 and 6.25 mg·kg−1 BaP groups were significantly prolonged compared with the control group (P<0.05), and the duration that rats spent in previous quadrant in the 6.25 mg·kg−1 BaP group was significantly shortened (P<0.05). Compared with the control group, the mRNA expressions of NR1 and NR2B in the hippocampus in the 2.50 and 6.25 mg·kg−1 BaP groups were significantly reduced (P<0.05), and the NR2A mRNA expression in the hippocampus in the 6.25 mg·kg−1 BaP group was significantly reduced (P<0.05); the mRNA expressions of NR1 and NR2B in the cortical tissue in the 6.25 mg·kg−1 BaP group were significantly reduced (P<0.05), and the mRNA expression of NR2A in the cortical tissue in the 1.00 mg·kg−1 BaP group was reduced; the mRNA expression of NR2B in the cerebellar tissue in the 6.25 mg·kg−1 BaP group was significantly reduced (P<0.05); there were no differences in the mRNA expressions of NMDA receptor subunits in the striatum tissue (P>0.05). Conclusion Subchronic BaP exposure can cause short-term spatial learning and memory impairment in rats, which may be related to the down-regulation of mRNA expressions of NR1, NR2A, and NR2B in hippocampus, changes of mRNA expressions of NR1, NR2A, and NR2B in cortical area, and the down-regulation of NR2B mRNA expression in cerebellum.

Association of intrauterine benzo[a]pyrene exposure with benzo[a]pyrene diolepoxide (BPDE)-DNA adduct levels and pancreatic functional impairment in offspring rats / 预防医学

Objective@#To investigate the benzo[a]pyrene ( B[a]P ) diolepoxide ( BPDE )-DNA adduct levels in offspring rats with intrauterine exposure to B[a]P, and examine the effects of BPDE-DNA adduct levels on pancreatic functional impairment and glucose metabolism in offspring rats. @*Methods@#Forty pregnant rats were randomly divided into the blank control group, standard-dose group, low-dose group, medium-dose group and high-dose group (daily dose of 0, 2, 200, 800, 1 600 μg/kg B[a]P, respectively), of 8 animals in each group. Rats in the B[a]P treatment groups were administered by oral gavage with a mixture of B[a]P and corn oil at a dose of 0.2 mL/100 g body weight since day 1 of pregnancy until 21 days after delivery, while rats in the blank control group were given the same volume of coin oil by oral gavage. The BPDE-DNA adduct levels were measured and the pancreatic development was observed in the offspring rats 2 and 21 days and 12 weeks after birth, and the correlation between pancreas volume index and dose of exposure to B[a]P was examined using Spearman's rank correlation analysis. In addition, glucose metabolism was measured in offspring rats 12 months after birth using glucose tolerance test ( GTT ) and insulin tolerance test ( ITT ). @*Results@#There was no abnormal appearance, death, abortion or preterm birth in pregnant or offspring rats in the five groups, and no significant differences were seen in activity, diet, drinking water or mental status in rats. The greatest level of BPDE-DNA adducts was measured in offspring rats 2 days after birth, with median levels ( interquartile range ) of 1 089.60 ( 586.10 ) to 1 405.49 ( 346.47 ) pg/mL, and no BPDE-DNA adducts were found in offspring rats 12 weeks after birth. The pancreas volume index correlated negatively with the dose of exposure to B[a]P in offspring rats 2 ( rs=-0.620, P=0.001 ) and 21 days after birth ( rs=-0.801, P=0.001 ). Hypoplasia of pancreas with loose tissues was seen in offspring rats 2 days after birth, while well pancreatic development was found in offspring rats 12 weeks after birth, with tight exocrine portion. GTT showed an increase in glucose levels in offspring rats in all five groups following abdominal injection of glucose and declined 30 min post-injection ( F=365.578, P<0.001 ), and ITT showed a tendency towards a decline in glucose levels in offspring rats in all five groups ( F=461.215, P<0.001 ).@*Conclusions@#The levels of BPDE-DNA adducts in offspring rats increase with the dose of intrauterine B[a]P exposure, and insulin resistance and impaired glucose tolerance occur 12 months post-exposure to B[a]P. Intrauterine B[a]P exposure affects pancreatic development in offspring rats and causes abnormal glucose metabolism in adult offspring rats.

Análise proteômica de células BEAS-2B expostas a benzo[α]pireno e nicotinamida ribosídeo / Proteomic Analysis of BEAS-2B cells exposed to benzo[α]pyrene and nicotinamide riboside

Responsável por milhões de óbitos anuais e um grande custo para a saúde pública, o câncer é a segunda maior causa de mortes no mundo. Dentre seus diversos tipos, o câncer de pulmão, além da alta incidência, é um dos mais letais. A exposição a substâncias tóxicas provenientes da combustão de matéria orgânica, assim como o consumo de cigarro, são os principais responsáveis pela alta incidência de câncer de pulmão. Dentre estas substâncias, está o benzo[α]pireno (B[α]P), um carcinógeno completo, ou seja, capaz de iniciar e promover o processo de carcinogênese. Resultados anteriores obtidos pelo grupo demonstraram que células BEAS-2B expostas a 1 µM de B[α]P apresentaram alterações das concentrações de metabólitos intracelulares, indução de estresse redox e hipermetilação do DNA. A exposição a 1 µM de nicotinamida ribosídeo (NR), um dos precursores de NAD+, foi capaz de proteger as células BEAS-2B contra a transformação induzida por B[α]P, além de impedir totalmente que células não expostas a B[α]P formassem colônias em soft-agar. A utilização da proteômica neste trabalho permitiu verificar a abundância das proteínas nos quatro diferentes grupos de exposição: Controle, B[α]P, B[α]P + NR e NR. Após 120 h de exposição as células foram coletadas, as proteínas extraídas e preparadas para análise. Foram descobertas 3024 proteínas posteriormente analisadas com o objetivo de elucidar vias possivelmente envolvidas na proteção contra o processo de transfomação maligna. Os grupos NR e Controle demonstram ser mais parecidos em relação ao seu conteúdo, enquanto os grupos B[α]P e B[α]P + NR foram mais semelhantes entre si. A análise de proteínas exclusivas revelou menos processos relacionados ao reparo de DNA no grupo tratado apenas com B[α]P quando comparado com B[α]P + NR. A análise estatística do total de proteínas utilizando o teste ANOVA (p < 0,05, N = 5) revelou 564 proteínas diferencialmente expressas entre os grupos. A clusterização nos permitiu observar a diferença na abundância de proteínas entre os quatro tratamentos. As proteínas estão envolvidas em funções como a regulação do metabolismo, resposta a estresse, transdução de sinal, regulação de expressão gênica e morte celular. Um dos clusters (cluster 1), contendo 59 proteínas, revelou poucos processos na análise de enriquecimento, mas as proteínas contidas nele apresentam funções como controle da divisão celular, apoptose e proteção ao estresse redox. Nele podemos observar que, no geral, o tratamento com B[α]P aumentou a abundância de algumas proteínas, o que foi revertido no grupo B[α]P + NR. O tratamento apenas com NR diminuiu a abundância das proteínas contidas nesse cluster. Outro cluster (cluster 4) apresentou 51 proteínas de abundância diminuída durante a exposição ao B[α]P, o que se reverteu no grupo B[α]P + NR. As proteínas desse cluster estão envolvidas em etapas importantes da via glicolítica, de crescimento, adesão, migração e invasão celular. Apesar de ser descrito que a exposição a NR pode aumentar a eficiência do reparo de DNA, os resultados apresentados nesse trabalho indicam que o efeito protetor pode estar relacionado com a modulação do ciclo celular ou alterações na adesão celular

Responsible for millions of annual deaths and a great health expense, cancer is the second leading cause of death in the world. Among its many types, lung cancer, besides its high incidence, is also one of the most lethal. Exposure to toxic substances resulting from the combustion of organic matter, as well as cigarette consumption, are the mainly responsible for the high incidence of lung cancer. One of these substances is benzo[α]pyrene (B[α]P), a complete carcinogen, able to initiate and promote the carcinogenesis process. Results obtained previously demonstrated that BEAS-2B cells exposed to 1 µM BaP presented alterations in the levels of intracellular metabolites, induction of oxidative stress, and hypermethylation of DNA. The exposure to 1 µM nicotinamide riboside (NR), one of the precursors of NAD+, was able to protect BEAS-2B cells against the transformation induced by B[α]P, moreover, it also totally prevented the colonies formation on soft agar in cells not exposed to B[α]P. The use of proteomics allowed us to verify the abundance of proteins in the four different exposure groups: Control, B[α]P, B[α]P + NR e NR. After 120h of exposure, the cells were collected followed by the extraction of the proteins. A total of 3024 proteins were identified and analyzed aiming to elucidate possible pathways involved in the protective effect against the malignant transformation induced by B[α]P. The NR and Control groups showed to be more similar, while B[α]P and B[α]P + NR were more similar. The analysis of exclusive proteins revealed fewer processes related to DNA repair in B[α]P when compared with B[α]P + NR. The statistical analysis of the total proteins using the ANOVA test (p <0.5, N = 5) revealed 564 proteins differentially expressed between the groups. The heatmap showed the difference in protein abundance between the four treatments. Proteins are involved in functionssuch asthe regulation of metabolism, stress response, signal transduction, regulation of gene expression, and cell death. One of the clusters (cluster 1), containing 59 proteins, revealed a few processes in the enrichment analysis, but the proteins contained in it have functions such as control of cell division, apoptosis, and protection from redox stress. It is possible to observe, in general, treatment with B[α]P increased the abundance of some proteins, which was partially reversed in group B[α]P + NR. On the other hand, the NR treatment decreased the abundance of proteins contained in this cluster. Another cluster (cluster 4) showed 51 proteins of decreased abundance during exposure to B [α] P, which was partially reversed in group B[α]P + NR. The proteins in this cluster are involved in important stages of the glycolytic pathway, also in growth, adhesion, migration, and cell invasion. Although it has been described that exposure to NR can increase the efficiency of DNA repair, the results presented in this work indicate that the protective effect may be related to the modulation of the cell cycle or cell adehsion modifications

Comparative Bioaccumulation of PAH and BTEX in Malapterurus electricus (Siluriformes: Malapteruridae) and its Enteric Parasite, Electrotaenia malopteruri Sampled from Lekki Lagoon, Lagos, Nigeria / Bioacumulação comparativa de HAP e BTEX em Malapterurus electricus (Siluriformes: Malapteruridae) e seu parasita entérico, Electrotaenia malopteruri, coletado na lagoa Lekki, Lagos, Nigéria

Abstract Contamination of Lekki lagoon by petrogenic chemicals such as polycyclic aromatic hydrocarbons (PAH) and benzene, toluene, ethylbenzene, and xylene (BTEX) has been widely reported. The study was aimed at investigating the bioaccumulation of PAH and BTEX in Malapterurus electricus collected from Lekki lagoon in Lagos, Nigeria. BTEX was analyzed in the intestine, water, and sediment samples using 8260B Agilent 7890B gas chromatograph coupled to a mass spectrometer (GC-MS). While PAHs were tested in the same media using a gas chromatograph coupled to a flame ionization detector (GC-FID). Histopathological analysis of the fish intestine was conducted using haematoxylin and eosin (H&E) stains. Parasite intensity, antioxidant enzymes, and lipid peroxidation activities were investigated in the fish. The parasitic infection detected in Malapterurus electricus was Electrotaenia malopteruri. The parasite showed weak and no depurative capacities for BTEX and PAH respectively. The decrease in superoxide dismutase (SOD), catalase (CAT), and reduced glutathione (GSH) in conjunction with an increase in Malondialdehyde (MDA) characterized relatively higher susceptibility among the male fish. Varieties of tissue injuries increased with the standard length of the fish groups. Results suggest that lengthier and uninfected M. electricus were more susceptible to PAH in Lekki lagoon than the shorter and infected ones. The study demonstrated a promising tendency of the enteric parasite, E. malopteruri to depurate chlorobenzene, ethylbenzene, o-xylene, m+p-xylene, and 1,4-dichlorobenzene from the intestine of its host fish, M. electricus, while PAHs were poorly mediated by the parasite.

Resumo A contaminação da lagoa Lekki por produtos químicos petrogênicos, como hidrocarbonetos aromáticos policíclicos (HAP) e benzeno, tolueno, etilbenzeno e xileno (BTEX), foi amplamente relatada. O estudo teve como objetivo investigar a bioacumulação de HAP e BTEX em Malapterurus electricus, coletados na lagoa Lekki, em Lagos, Nigéria. O BTEX foi analisado no intestino, na água e nas amostras de sedimento usando o cromatógrafo de gás Agilent 7890B 8260B acoplado a um espectrômetro de massa (GC-MS). Já os HAP foram testados no mesmo meio utilizando cromatógrafo gasoso acoplado a detector de ionização de chama (GC-FID). A análise histopatológica do intestino do peixe foi conduzida usando colorações de hematoxilina e eosina (H&E). Intensidade de parasitas, enzimas antioxidantes e atividades de peroxidação lipídica foram investigadas nos peixes. A infecção parasitária detectada em Malapterurus electricus foi Electrotaenia malopteruri. O parasita mostrou fraca e nenhuma capacidade depurativa para BTEX e HAP, respectivamente. A diminuição da superóxido dismutase (SOD), catalase (CAT) e glutationa reduzida (GSH), em conjunto com o aumento do Malondialdeído (MDA), caracterizou a suscetibilidade relativamente maior entre os peixes machos. Variedades de lesões nos tecidos aumentaram com o comprimento padrão dos grupos de peixes. Os resultados sugerem que os M. electricus maiores e não infectados foram mais suscetíveis aos HAP na lagoa Lekki do que os menores e infectados. O estudo demonstrou uma tendência promissora do parasita entérico E. malopteruri de depurar clorobenzeno, etilbenzeno, o-xileno, m + p-xileno e 1,4-diclorobenzeno do intestino de seu peixe hospedeiro, M. electricus, enquanto os HAP foram mal mediados pelo parasita.

Degradation of SARS-CoV-2 receptor ACE2 by the E3 ubiquitin ligase Skp2 in lung epithelial cells / 医学前沿

An unexpected observation among the COVID-19 pandemic is that smokers constituted only 1.4%-18.5% of hospitalized adults, calling for an urgent investigation to determine the role of smoking in SARS-CoV-2 infection. Here, we show that cigarette smoke extract (CSE) and carcinogen benzo(a)pyrene (BaP) increase ACE2 mRNA but trigger ACE2 protein catabolism. BaP induces an aryl hydrocarbon receptor (AhR)-dependent upregulation of the ubiquitin E3 ligase Skp2 for ACE2 ubiquitination. ACE2 in lung tissues of non-smokers is higher than in smokers, consistent with the findings that tobacco carcinogens downregulate ACE2 in mice. Tobacco carcinogens inhibit SARS-CoV-2 spike protein pseudovirions infection of the cells. Given that tobacco smoke accounts for 8 million deaths including 2.1 million cancer deaths annually and Skp2 is an oncoprotein, tobacco use should not be recommended and cessation plan should be prepared for smokers in COVID-19 pandemic.

Effects of resveratrol on expression of inflammatory cytokines and related genes in benzo (a) pyrene-induced human sebocytes / 中华皮肤科杂志

Objective:To investigate the effect of resveratrol on the expression of inflammatory cytokines and related genes in human SZ95 sebocytes induced by benzo (a) pyrene.Methods:Human SZ95 sebocytes were cultured in vitro, and divided into 4 groups: control group treated with 1‰ dimethyl sulfoxide for 27 hours, resveratrol group treated with 1 × 10 -5 mol/L resveratrol for 24 hours, benzo (a) pyrene group treated with 1 × 10 -5 mol/L benzo (a) pyrene for 3 hours, resveratrol+benzo (a) pyrene group treated with 1 × 10 -5 mol/L resveratrol for 24 hours followed by 1 × 10 -5 mol/L benzo (a) pyrene for 3 hours. Real-time fluorescence-based quantitative PCR was performed to determine the mRNA expression of interleukin (IL) -1α, IL-6, aryl hydrocarbon receptor (AhR) , cytochrome P4501A1 (CYP1A1) and cytochrome P4501B1 (CYP1B1) in SZ95 sebocytes in the above groups; Western blot analysis was conducted to determine the phosphorylation level of p38 mitogen-activated protein kinase (p38 MAPK, expressed as the ratio of phosphorylated to total p38 MAPK) and AhR protein expression; enzyme-linked immunosorbent assay (ELISA) was conducted to detect levels of IL-1α and IL-6 in the cell culture supernatant in each group. One-way analysis of variance was used for comparison of means among multiple groups, and least significant difference- t test was used for multiple comparisons. Results:The mRNA and protein expression of IL-1α in SZ95 sebocytes significantly differed among the control group, resveratrol group, benzo (a) pyrene group and resveratrol+benzo (a) pyrene group (mRNA: 2.045 ± 0.272, 2.058 ± 0.154, 3.124 ± 0.094, 2.185 ± 0.337, protein: 9.132 ± 1.181, 9.429 ± 0.771, 20.361 ± 0.907, 9.917 ± 0.897, F=14.662, 101.705, P < 0.01, < 0.001, respectively) , and were significantly lower in the resveratrol+benzo (a) pyrene group than in the benzo (a) pyrene group (both P < 0.01) . In addition, the phosphorylation level of p38 was significantly higher in the benzo (a) pyrene group than in the control group, resveratrol group and resveratrol+benzo (a) pyrene group ( F=303.129, P < 0.000 1) . The mRNA expression of AhR, CYP1A1 and CYP1B1 was significantly lower in the resveratrol+benzo (a) pyrene group than in the benzo (a) pyrene group ( t=10.64, 33.599, 18.327, respectively, all P < 0.001) . The benzo (a) pyrene group showed significantly decreased protein expression of AhR compared with the resveratrol+benzo (a) pyrene group ( P < 0.001) . Conclusion:Resveratrol can inhibit the environmental pollutant benzo (a) pyrene-induced expression of inflammatory factor IL-1α in SZ95 sebocytes, which is likely mediated by the AhR and p38MAPK pathways.

Modulação da concentração intracelular de NAD+ e seu efeito na tumorigênese induzida por benzo[a]pireno em células bronquiais epiteliais humanas / Modulation of intracellular concentration of NAD+ and its effect on benzo[a]pyrene-induced tumorigenesis in human epithelial bronchial cells

A alta incidência, prevalência e mortalidade do câncer de pulmão demonstram a necessidade de se identificar alterações moleculares envolvidas na carcinogênese pulmonar. Nesse contexto, a reprogramação do metabolismo energético é uma marca emergente do câncer. Há evidências de que benzo[a]pireno (B[a]P), um conhecido carcinógeno humano, induz alterações metabólicas via modificação da função mitocondrial tanto in vitro quanto in vivo. Uma vez que as alterações metabólicas não são somente o resultado da transformação celular, mas podem também ter papel na etiologia do câncer ao modular o epigenoma e a expressão de genes, intervir no metabolismo de células em processo de transformação pode contribuir para desvendar mecanismos de carcinogênese e revelar alvos para quimioprevenção. A fim de investigar a relação entre alterações no metabolismo celular, marcas epigenéticas e transformação celular, implementamos um modelo de tumorigênese (avaliada pela formação de colônias em soft-agar) induzida por B[a]P em células epiteliais bronquiais humanas imortalizadas (linhagem BEAS-2B) crescidas em monocamada (2D). O modelo possibilitou a observação de alterações precoces do metabolismo celular. Levando em consideração que o nucleotídeo NAD+ regula as atividades de diversas vias moleculares importantes para a sobrevivência, diferenciação, crescimento e morte celular, e que suas concentrações foram rapidamente diminuídas após exposição a B[a]P, decidimos suplementar as células BEAS-2B com nicotinamida ribosídeo (NR), um precursor intracelular de NAD+, concomitantemente à exposição a B[a]P. NR em baixa concentração no meio de cultura (1 µM) induziu estresse energético em células BEAS-2B expostas a B[a]P (1 µM) ao longo do período de uma semana de co-incubação, aumentando seletivamente a taxa de apoptose dessas células. Protegeu contra a transformação celular induzida por B[a]P e impediu completamente a formação espontânea de colônias das células controle em soft-agar. Usamos uma abordagem metabolômica direcionada a alvos específicos ("targeted metabolomics") desenvolvida no grupo para quantificar metabólitos conhecidamente alterados no câncer. Os dados indicam que NR diminui o metabolismo de glutamina nas células expostas a B[a]P, o que ocorre em paralelo com a diminuição das concentrações de citrato e aspartato, aumento da razão malato/aspartato, diminuição das razões ATP/AMP e ATP/ADP e aumento das concentrações de adenosina. As alterações se enquadram na hipótese de inibição do shuttle malato-aspartato, cuja atividade é necessária para a sobrevivência de células que sofrem o efeito Warburg (alta dependência de NADH citosólico para geração de ATP). NR adicionalmente protegeu as células contra o estresse redox, a hipermetilação do DNA e o aumento da atividade de sirtuína 1 (SIRT1) induzidos por B[a]P, além de aumentar a expressão de genes supressores tumorais (E-caderina, PTEN, semaforina 3F, p16(ink4a)) que podem ser reprimidos por CtBP (proteína ligante de NADH que atua como sensor redox e traduz a condição metabólica da célula para o controle da expressão gênica). Foi ainda observada maior atividade de PARP1 nas células expostas a B[a]P+NR em comparação aos demais grupos. Os resultados obtidos mostram que NR se contrapõe a ou exacerba alterações bioquímicas induzidas por B[a]P, diminuindo a chance de transformação carcinogênica das células BEAS-2B. Estudos em modelos mais complexos, como micro tecidos in vitro, são necessários para a confirmação do efeito quimiopreventivo da NR e alterações bioquímicas subjacentes

Tese de DoutoradoDOIhttps://doi.org/10.11606/T.9.2021.tde-05082021-095853DocumentoTese de DoutoradoAutorCordeiro, Everson Willian Fialho (Catálogo USP)Nome completoEverson Willian Fialho CordeiroE-mailE-mailUnidade da USPFaculdade de Ciências FarmacêuticasÁrea do ConhecimentoToxicologiaData de Defesa2021-04-08ImprentaSão Paulo, 2021OrientadorLoureiro, Ana Paula de Melo (Catálogo USP) Banca examinadoraLoureiro, Ana Paula de Melo (Presidente) Àvila, Daiana Silva de Meotti, Flavia Carla Silva, Eloiza Helena Tajara da Título em portuguêsModulação da concentração intracelular de NAD+ e seu efeito na tumorigênese induzida por benzo[a]pireno em células bronquiais epiteliais humanasPalavras-chave em portuguêsBenzo[a]pireno Câncer de pulmão Metabolismo energético Nicotinamida ribosídeo Resumo em portuguêsA alta incidência, prevalência e mortalidade do câncer de pulmão demonstram a necessidade de se identificar alterações moleculares envolvidas na carcinogênese pulmonar. Nesse contexto, a reprogramação do metabolismo energético é uma marca emergente do câncer. Há evidências de que benzo[a]pireno (B[a]P), um conhecido carcinógeno humano, induz alterações metabólicas via modificação da função mitocondrial tanto in vitro quanto in vivo. Uma vez que as alterações metabólicas não são somente o resultado da transformação celular, mas podem também ter papel na etiologia do câncer ao modular o epigenoma e a expressão de genes, intervir no metabolismo de células em processo de transformação pode contribuir para desvendar mecanismos de carcinogênese e revelar alvos para quimioprevenção. A fim de investigar a relação entre alterações no metabolismo celular, marcas epigenéticas e transformação celular, implementamos um modelo de tumorigênese (avaliada pela formação de colônias em soft-agar) induzida por B[a]P em células epiteliais bronquiais humanas imortalizadas (linhagem BEAS-2B) crescidas em monocamada (2D). O modelo possibilitou a observação de alterações precoces do metabolismo celular. Levando em consideração que o nucleotídeo NAD+ regula as atividades de diversas vias moleculares importantes para a sobrevivência, diferenciação, crescimento e morte celular, e que suas concentrações foram rapidamente diminuídas após exposição a B[a]P, decidimos suplementar as células BEAS-2B com nicotinamida ribosídeo (NR), um precursor intracelular de NAD+, concomitantemente à exposição a B[a]P. NR em baixa concentração no meio de cultura (1 µM) induziu estresse energético em células BEAS-2B expostas a B[a]P (1 µM) ao longo do período de uma semana de co-incubação, aumentando seletivamente a taxa de apoptose dessas células. Protegeu contra a transformação celular induzida por B[a]P e impediu completamente a formação espontânea de colônias das células controle em soft-agar. Usamos uma abordagem metabolômica direcionada a alvos específicos ("targeted metabolomics") desenvolvida no grupo para quantificar metabólitos conhecidamente alterados no câncer. Os dados indicam que NR diminui o metabolismo de glutamina nas células expostas a B[a]P, o que ocorre em paralelo com a diminuição das concentrações de citrato e aspartato, aumento da razão malato/aspartato, diminuição das razões ATP/AMP e ATP/ADP e aumento das concentrações de adenosina. As alterações se enquadram na hipótese de inibição do shuttle malato-aspartato, cuja atividade é necessária para a sobrevivência de células que sofrem o efeito Warburg (alta dependência de NADH citosólico para geração de ATP). NR adicionalmente protegeu as células contra o estresse redox, a hipermetilação do DNA e o aumento da atividade de sirtuína 1 (SIRT1) induzidos por B[a]P, além de aumentar a expressão de genes supressores tumorais (E-caderina, PTEN, semaforina 3F, p16(ink4a)) que podem ser reprimidos por CtBP (proteína ligante de NADH que atua como sensor redox e traduz a condição metabólica da célula para o controle da expressão gênica). Foi ainda observada maior atividade de PARP1 nas células expostas a B[a]P+NR em comparação aos demais grupos. Os resultados obtidos mostram que NR se contrapõe a ou exacerba alterações bioquímicas induzidas por B[a]P, diminuindo a chance de transformação carcinogênica das células BEAS-2B. Estudos em modelos mais complexos, como micro tecidos in vitro, são necessários para a confirmação do efeito quimiopreventivo da NR e alterações bioquímicas subjacentes.Título em inglêsModulation of intracellular concentration of NAD+ and its effect on benzo[a]pyrene-induced tumorigenesis in human epithelial bronchial cellsPalavras-chave em inglêsBenzo[a]pyrene Energetic metabolism Lung cancer Nicotinamide riboside Resumo em inglêsThe high incidence, prevalence and mortality of lung cancer demonstrates the need to identify molecular changes involved in lung carcinogenesis. In this context, the reprogramming of energy metabolism is an emerging brand of cancer. There is evidence that benzo[a]pyrene (B[a]P), a known human carcinogen, induces metabolic changes via modification of mitochondrial function both in vitro and in vivo. Since metabolic changes are not only the result of cell transformation, but can also play a role in the etiology of cancer by modulating the epigenome and gene expression, intervening in the metabolism of cells in the process of transformation can contribute to unravel mechanisms of carcinogenesis and reveal targets for chemoprevention. In order to investigate the relationship between changes in cell metabolism, epigenetic marks and cell transformation, we implemented a model of tumorigenesis (assessed by the formation of colonies on soft-agar) induced by B[a]P in immortalized human bronchial epithelial cells (BEAS-2B cell line human) grown in monolayer (2D). The model enabled the observation of early changes in cell metabolism. Taking into account that the NAD+ nucleotide regulates the activities of several molecular pathways important for cell survival, differentiation, growth and death, and that their concentrations were rapidly decreased after exposure to B[a]P, we decided to supplement the BEAS-2B cells with nicotinamide riboside (NR), an intracellular precursor of NAD+, concomitantly with exposure to B[a]P. NR in low concentration in the culture medium (1 µM) induced energy stress in BEAS-2B cells exposed to B[a]P (1 µM) over the period of a week of co-incubation, selectively increasing the apoptosis rate of these cells. It protected against cell transformation induced by B[a]P and completely prevented the spontaneous formation of control cell colonies on soft-agar. We use a targeted metabolomics approach developed in the group to quantify metabolites known to be altered in cancer. The data indicate that NR decreases the glutamine metabolism in cells exposed to B[a]P, which occurs in parallel with the decrease in citrate and aspartate concentrations, increased malate/aspartate ratio, decreased ATP/AMP and ATP/ADP ratios and increased adenosine concentrations. The changes fit the hypothesis of inhibition of the malate-aspartate shuttle, whose activity is necessary for the survival of cells that suffer the Warburg effect (high dependence on cytosolic NADH for ATP generation). NR additionally protected cells against redox stress, DNA hypermethylation and increased B[a]P-induced sirtuin 1 (SIRT1) activity, in addition to increasing the expression of tumor suppressor genes (E-cadherin, PTEN, semaphorin 3F, p16 (ink4a)) that can be suppressed by CtBP (NADH-binding protein that acts as a redox sensor and translates the cell's metabolic condition to control gene expression). Higher PARP1 activity was also observed in cells exposed to B[a]P+NR compared to the other groups. The results obtained show that NR is opposed to or exacerbates biochemical changes induced by B[a]P, reducing the chance of carcinogenic transformation of BEAS-2B cells. Studies on more complex models, such as micro tissues in vitro, are necessary to confirm the chemopreventive effect of NR and underlying biochemical changes

Vanillic Acid Inhibits Lung Carcinogenesis By Modulates Glycoprotein Abnormalities, Membrane-Bound Enzymes, And Inflammatory Markers

Objective: The current plan was accompanied to explicate the possible protective role of vanillic acid (VA), on modification in lipid peroxidation, inflammatory cytokines, membrane-bound enzymes, and glycoconjugates during B(a)P induced lung cancer in Swiss albino mice. Methods: Benzo(a)pyrene was administered orally (50 mg/kg b. wt) to induce lung cancer in Swiss albino mice. lipid peroxidation, serum marker enzymes, inflammatory cytokines, membrane-bound ATPases and protein-bound carbohydrate components (Hexose, hexosamine, sialic acid and fucose) and Mast cells and PAS staining were carried out. Results: Lung cancer possessing animals exhibited increased levels of lipid peroxidation, ADA, AHH, γ-GT, 5’-NT, LDH, cytokines such as TNF-α and IL-1β, protein-bound carbohydrate components (protein-hexose, hexosamine, sialic acid, and fucose) also diminished activity of membrane-bound ATPases (Na+/K+ATPases, Ca2+ATPases, and Mg2+ATPase). Treatment with VA significantly ameliorated all these activities. Conclusion: Overall, the present study evidence to the VA has effective anti-inflammatory in addition to free radical scavenging activity for the duration of lung carcinogenesis in Swiss albino mice.

Effect of benzo(a)pyrene on dopaminergic neurons and α-synuclein in brain and its mechanism involved / 北京大学学报(医学版)

OBJECTIVE@#To analyze the effect of benzopyrene on the decrease of dopaminergic neurons, and the increase and aggregation of α-synuclein, which are the pathological features of Parkinson's disease, and to explore its possible mechanisms.@*METHODS@#Eight-month-old transgenic mice with human SNCA gene were randomly divided into a BaP-exposed group and a control group. BaP and solvent corn oil were injected intraperitoneally to BaP-exposed group and control group respectively, once a day for 60 days. The motor dysfunction of mice was tested by rotarod test. The effects of BaP on the decrease of dopaminergic neurons and increase and aggregation of α-synuclein were observed by immunohistochemistry and Western blot experiments respectively, and the expression of related mRNA was detected by quantitative real-time PCR (qRT-PCR). Twenty genes were tested in the study, mainly related to neurotransmitter transporter (2 genes), neurotransmitter receptor function (10 genes), cellular autophagy (5 genes), and α-synuclein aggregation and degradation (3 genes).@*RESULTS@#After BaP exposure, the movement time of the mice in the rotarod test was significantly reduced (P<0.05). The substantia nigra dopami-nergic neurons in the mice were significantly reduced, which was 62% of the control group (P<0.05), and the expression of α-synuclein in the midbrain increased, which was 1.36 times that of the control group (P<0.05). After BaP exposure, mRNA expressions of 14 genes in the midbrain of the mice were significantly down-regulated (P<0.05). Alpha-synuclein degradation and cell autophagy (5 genes), neuron transporters (2 genes), and neurotransmitter receptor functions (5 genes) were involved. The expression of one gene, Synphilin-1, was significantly up-regulated (P<0.01), which was related to α-synuclein aggregation.@*CONCLUSION@#BaP exposure not only inhibited function of neurotransmitter receptor and dopamine transporter, but also interfered cell autophagy, thereby hindering the degradation of α-synuclein, which could lead to decrease of dopaminergic neurons in substantia nigra and increase and aggregation of α-synuclein in midbrain, as the significant pathology of Parkinson's disease. Therefore, BaP exposure may increase the risk of Parkinson's disease.

A pyrene-based ratiometric fluorescent probe with a large Stokes shift for selective detection of hydrogen peroxide in living cells / 药物分析学报

Hydrogen peroxide (H2O2) plays a significant role in regulating a variety of biological processes. Dys-regulation of H2O2 can lead to various diseases. Although numerous fluorescent imaging probes for H2O2 have been reported, the development of H2O2 ratiometric fluorescent probe with large Stokes shift re-mains rather limited. Such probes have shown distinct advantages, such as minimized interference from environment and improved signal-to noise ratio. In this work, we reported a new pyrene-based com-pound Py-VPB as H2O2 fluorescent probe in vitro. The probe demonstrated ratiometric detection behavior, large Stokes shift and large emission shift. In addition, the probe showed high sensitivity and selectivity towards H2O2 in vitro. Based on these excellent properties, we successfully applied Py-VPB to the visualization of exogenous and endogenous H2O2 in living cells. Cell imaging study also showed that our probe was localized in the mitochondria. We envision that the probe can provide a useful tool for unmasking the biological roles of mitochondrial H2O2 in living systems.

The molecular mechanism of the effect of benzoαpyrene on autophagy of molecular chaperones under simulated hypoxia / 药学学报

In this study, the effect of benzo[α]pyrene (BaP) on chaperone-mediated autophagy (CMA) in a simulated hypoxia environment was observed and the relationship to heat shock protein 90 (HSP90) was clarified. With HSP90 inhibitor geldanamycin (GA) and HSP90α silenced, the mRNA and protein expression of hypoxia-inducible factor-1α (HIF-1α), HSP90, heat shock cognate protein 70 (HSC70), and lysosomal associated protein 2A (LAMP-2A) of A549 cells on hypoxic environment by BaP were tested. Alkaline comet experiment, immunofluorescence γ-H2AX focus experiment, quantitative real-time PCR (qPCR), and Western blot analyses were used to clarify the relationship between the DNA damage of different concentrations of BaP in A549 cells and the mRNA and protein expression of CMA-related factors. The results show that hypoxia can promote the expression of mRNA and protein of CMA-related factors in A549 cells. This study found that BaP has an inhibitory effect on CMA under the hypoxic environment. The inhibition or silencing of HSP90 will enhance the inhibitory effect of BaP on CMA. In a normoxic environment, BaP causes DNA damage and promotes CMA.

Role of telomere shortening and p53-p21- related pathway protein in learning and memory impairment induced by benzoapyrene in mice / 解剖学报

Objective To observe the changes of telomere length and p53 and p21 protein expression levels in mice exposed to different time and doses, and to explore the role of related pathways protein in benzo[a]pyrene in learning and memory impairment. Methods Ninety healthy male C57 mice, 30 in each batch, were exposed for 1 month, 2 months and 3 months respectively. Each batch was set with blank control group, solvent control group, 1. 00 mg/ kg, 2. 50 mg/ kg and 6. 25 mg/ kg benzo[a]pyrene-treated groups, 6 in each group, inoculated intraperitoneally every other day. Learning and memory ability was detected by Morris water maze test, ELISA was used to detect the content of benzo[a] pyrene-7, 8-dihydrodiol-9,10-epoxide-DNA(BPDE-DNA) adduct in mice plasma. There was no significant difference in the relative telomere length of the mice at 1 mouth of each group. The length in mice after treated for 2 and 3 months were significantly shorter than that of the non-infected group (P<0. 05). And the plasma BPDE-DNA adduct content in each exposed group was negatively correlated with the relative telomere length. Western blotting was used to detect the expression of p53-p21 related pathway protein and neural stem cell proliferation and differentiation related proteins Nestin and βⅢ-tubulin in mouse hippocampus. Relative telomere length was detected by Real-time PCR. Results The escape latency of mice in each exposed group was significantly prolonged, and the times of crossing platforms was significantly reduced. BPDE-DNA adduct was not detected in the non-dose group and in the exposed group showed an upward trend (P<0. 05). Compared with the non-dose group, the expression levels of p53 and p21 in the exposed group increase with the dose of the drug (P<0. 05). When the dose was 1. 0 mg/ kg, the expression level of Nestin did not significantly increase. The expression level of Nestin increased in the remaining exposure groups (P<0. 05), and the expression level of Nestin was lower than that in the 1. 0 mg/ kg and 2. 5 mg/ kg groups at the dose of 6. 25 mg/ kg for 3 months (P<0. 05). At 1 month, there was no significant difference in the expression of βⅢ-tubulin between the exposed and non-infected groups. At 2 and 3 months, the expression of βⅢ-tubulin in the exposed group decreased (P<0. 05). Conclusion Intraperitoneal injection of benzo[a] pyrene can induce DNA damage and telomere shortening due to BPDE-DNA adduct formation, which leads to activation of p53-p21 signaling pathway. Telomere shortening and p53-p21 signaling pathway activation-mediated DNA damage response may be important mechanisms for inhibiting proliferation and differentiation of hippocampal neurons.