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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.
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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.
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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.
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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 âânos 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.
Subject(s)
Benzo(a)pyrene/pharmacology , Chlorogenic Acid/pharmacology , Cell Death/drug effects , Saccharomyces cerevisiae Proteins/adverse effects , DNA Repair Enzymes/genetics , Benzo(a)pyrene/toxicity , Mutagenesis/drug effects , Cell Death/genetics , Antimutagenic Agents/pharmacology , Saccharomyces cerevisiae Proteins/genetics , DNA Breaks, Double-Stranded/drug effects , Mutation RateABSTRACT
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.
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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.
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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.
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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.
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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.
Subject(s)
Adult , Animals , Humans , Mice , COVID-19 , Epithelial Cells , Lung , Pandemics , Peptidyl-Dipeptidase A , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Ubiquitin-Protein Ligases/geneticsABSTRACT
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
Subject(s)
Niacinamide/adverse effects , Carcinogenesis/drug effects , Lung Neoplasms/pathology , In Vitro Techniques/methods , DNA , Chemoprevention/classification , Energy Metabolism , Epithelial Cells/classificationABSTRACT
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.
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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.
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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.
Subject(s)
Animals , Humans , Mice , Benzo(a)pyrene , Brain , Dopamine , Dopaminergic Neurons , alpha-SynucleinABSTRACT
Animal models have been providing invaluable contributions to the better understanding of mechanisms of cancer (including leukaemias) development and effectiveness of most of the treatments. Chemical carcinogens are generally used to study the biology of cancers including leukaemias in many animal models, including rats and mice. The studies in most cases are aimed at the development and evaluation of cancer treatments and preventions. Some of the most common chemical carcinogens used in animal models for leukaemias include N-ethyl-N-nitrosourea (ENU), N-methyl-N-nitrosourea (MNU), dimethyl benz(a)anthracene (DMBA) and benzo(a)pyrene (BaP). This review provides highlights on different animal models of leukaemia induced by the chemical carcinogens mentioned earlier, at the same time discussing the contributions of these models to the leukaemia diagnosis in laboratory animal models for subsequent development of treatment.
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Dioxins, polybrominated diphenyl ethers, and benzo(a)pyrene are common organic pollutants in food. They have been of concern to academics and government administrations due to high residue and persistence, easy accumulation and strong harmful effects. The National Research Council of the United States of America published Toxicity Testing in the 21st Century: A Vision and Strategy in 2007, which proposed a new concept of toxicity testing that toxicity testing should take full consideration of population exposure data and base on in vitro tests, human cell lines, toxicity pathways and high-throughput screening. Meanwhile, systems biology, bioinformatics and rapid assay technologies will be used to better understand toxicity pathways—the cellular response pathways that can lead to adverse health effects when sufficient perturbing induced by chemicals exposure. The new toxicity testing strategy has changed the traditional testing pattern and has brought a wide impact on the international relevant fields. The European Union, the World Health Organization, and the United States Environmental Protection Agency, the Food and Drug Administration, and the National Center for Toxicological Research have organized relevant discussions and exploratory studies to address the new toxicity testing concept and how to evaluate and utilize the results of traditional toxicity test researches. Compared to the discussion, 'whether to do it’, ten years ago, the question, 'how to do it’, has become the concern of the current discussion. Therefore, how to respond to the concept of toxicity testing and how to effectively utilize and excavate traditional toxicity test data have been the focus of multi-disciplines and interdisciplinary academia such as toxicology, food hygiene and environmental science. Therefore, this article provides an overview of the exposure levels of dioxin, polybrominated diphenyl ethers and benzo[a]pyrene, which are typical persistent organic pollutants in food in China and the current research status of toxic pathways based on whole animal experiments. The exposure level, toxic effect and toxicity mechanism of three contaminants are analyzed and summarized in order to provide basis for future results based on the 21st century toxicity test compared with traditional tests and data mining analysis of these two kinds of data. Meanwhile, it also lays the foundation for the establishment of a toxicity testing framework based on exposure characteristics, toxic pathways, and biomarkers.
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Objective@#To explore the protective effect of Yishen Tongluo Recipe (YTR) against aberrant sperm DNA methylation in male rats exposed to benzo(a)pyrene (BaP).@*METHODS@#Thirty male SD rats of the SPF grade were randomly divided into three groups of equal number: solvent control, BaP exposure and YTR intervention. The animals of the solvent control group were injected intraperitoneally with 0.5% DMSO while those of the other two groups with BaP at 0.1 mg/kg/d, all for 60 days, and at 31 days of BaP exposure, those of the YTR group were treated intragastrically with YTR for 30 days. Then, the left epididymides were harvested from all the rats and sperm suspensions collected and centrifuged for extraction of sperm DNA. The methylated DNA immunoprecipitation sequencing (MeDIP-seq) technique was used to detect the whole-genome DNA methylation in different groups.@*RESULTS@#Exposure to BaP induced the up-regulation of 828 genes encoding mRNA in the sperm DNA, while YTR intervention produced a significant protective effect on the transforming growth factor β3 (TGF-β3), cystic fibrosis transmembrane conductance regulator (CFTR) and recombination activating gene 1 (RAG1), and down-regulated the expressions of 3 227 genes. BaP exposure also caused the up-regulation of 783 genes encoding lncRNA in the sperm DNA, and YTR treatment exhibited an evident protective effect on 62 of the up-regulated genes, induced the down-regulation of 3 378 genes, and showed a protective effect on 56 of the down-regulated genes.@*CONCLUSIONS@#YTR has a protective effect against aberrant sperm DNA methylation in male rats exposed to BaP, which may be associated with lncRNA.
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Abstract Due to the health risks for both humans and living beings caused by polycyclic aromatic hydrocarbons (PAHs), the monitoring of these compounds in environmental matrices is mandatory. This work proposes an analytical method for analyzing anthracene (AN) and benzo[a]pyrene (BaP), two of the most representative PAHs, at ultra-trace concentrations in water, employing direct injection of large volumes of samples coupled with reversed-phase high-performance liquid chromatography. For this purpose, principal component analysis was used to examine the behavior of AN and BaP within the chromatographic system. Results showed that AN and BaP chromatographic behavior can be described by three models representing their identification, the quantification of AN and that of BaP, respectively. The factors affecting the obtained models, such as the injection volume, column temperature, flow rate, strength of the mobile phase, and the excitation and emission wavelengths, were examined and optimized by means of design of experiments. Finally, the analytical method was validated, obtaining promising limits of detection and quantification. The developed analytical method was demonstrated to be useful for a sensitive analysis of the target analytes in relatively clean natural water matrices.
Resumen Los hidrocarburos aromáticos policíclicos (HAPs) causan problemas en la salud de los seres humanos y seres vivos, por lo que se requiere un monitoreo de estos compuestos en matrices ambientales. Este trabajo propone un método analítico para analizar el antraceno (AN) y el benzopireno (BAP), los hidrocarburos más representativos en concentraciones de ultra trazas en el agua, empleando inyección directa en grandes volúmenes en muestras acopladas a la fase inversa con cromatografía líquida de alto rendimiento. Por tal razón, se utilizó el análisis de componentes principales para examinar el comportamiento de AN y BAP en el sistema cromatográfico. Los resultados mostraron que el comportamiento cromatográfico de AN y BAP podría describirse por medio de tres modelos que representan su identificación, la cuantificación de AN y de BAP, respectivamente. Se examinaron los factores que afectan a los modelos obtenidos, como el volumen de inyección, la temperatura de la columna, la tasa de flujo, la fuerza de la fase móvil, y las longitudes de las ondas de excitación y emisión, y se optimizaron mediante el diseño de experimentos. Finalmente, se validó el método analítico, obteniendo límites de detección y cuantificación. Se demostró que el método analítico desarrollado fue útil para el análisis sensible de los analitos en matrices de agua natural relativamente limpia.
Resumo Devido aos riscos para a saúde tanto para humanos como para os seres vivos em geral causados pelos hidrocarbonos aromáticos policíclicos (HAPs), o monitoramento de estes compostos em matrizes ambientais é prioritário. Este trabalho propõem um método analítico para analisar antraceno (AN) e benzo[a]pireno (BaP), dois dos hidrocarbonos mais representativos, em concentrações de ultra traços em água, empregando injeções diretas de grandes volumes de amostra acoplada a cromatografia líquida de alta eficiência em fase reversa. Usando Análises por Componentes Principais e desenho experimental, foram avaliados os efeitos de diversos fatores que afetam o sistema cromatográfico, tais como o volume de injeção, a temperatura da coluna, fluxo, forca da fase móvel e comprimentos de onda de excitação e emissão. Os resultados demonstraram que o comportamento cromatográfico de AN e BaP pode ser descrito por meio de três que representam sua identificação, quantificação de AN e de BaP, respectivamente. Os resultados mostraram que o comportamento cromatográfico de NA e BAP poderia ser descrito por meio de três modelos que representam sua identificação, a quantificação de NA e de BAP, respectivamente. Examinaram-se os fatores que afetam aos modelos obtidos, como o volume de injeção, a temperatura da coluna, a taxa de fluxo, a forca da fase móvel, e as longitudes das ondas de excitação e emissão, e se otimizaram mediante o desenho experimental. Finalmente, se validou o m todo analítico, obtendo os limites de detecção e quantificação. O método analítico desenvolvido demonstrou ser útil para uma análise sensível para os compostos de interesse em matrizes de água natural relativamente limpas.
Subject(s)
Polycyclic Aromatic Hydrocarbons , Environmental Pollution , AnthracenesABSTRACT
Abstract Cellulosimicrobium cellulans CWS2, a novel strain capable of utilizing benzo(a)pyrene (BaP) as the sole carbon and energy source under nitrate-reducing conditions, was isolated from PAH-contaminated soil. Temperature and pH significantly affected BaP biodegradation, and the strain exhibited enhanced biodegradation ability at temperatures above 30 °C and between pH 7 and 10. The highest BaP removal rate (78.8%) was observed in 13 days when the initial BaP concentration was 10 mg/L, and the strain degraded BaP at constant rate even at a higher concentration (50 mg/L). Metal exposure experimental results illustrated that Cd(II) was the only metal ion that significantly inhibited biodegradation of BaP. The addition of 0.5 and 1.0 g/L glucose enhanced BaP biodegradation, while the addition of low-molecular-weight organic acids with stronger acidity reduced BaP removal rates during co-metabolic biodegradation. The addition of phenanthrene and pyrene, which were degraded to some extent by the strain, showed no distinct effect on BaP biodegradation. Gas chromatography-mass spectrometry (GC-MS) analysis revealed that the five rings of BaP opened, producing compounds with one to four rings which were more bioavailable. Thus, the strain exhibited strong BaP degradation capability and has great potential in the remediation of BaP-/PAH-contaminated environments.
Subject(s)
Soil Microbiology , Soil Pollutants/metabolism , Benzo(a)pyrene/metabolism , Actinobacteria/isolation & purification , Actinobacteria/metabolism , Temperature , Cadmium/metabolism , Carbon/metabolism , Carboxylic Acids/metabolism , Biotransformation , Actinobacteria/classification , Culture Media/chemistry , Enzyme Inhibitors/metabolism , Glucose/metabolism , Hydrogen-Ion Concentration , Anaerobiosis , Gas Chromatography-Mass SpectrometryABSTRACT
Abstract The purpose of our study was to divulge the antiproliferative effect of an ethanolic extract of Algerian propolis (EEP) in the human lung adenocarcinoma cell line (A549) and reveal the chemopreventive role against benzo(a)pyrene-induced lung carcinogenesis in albino Wistar rats. Cytotoxicity of EEP was evaluated using the MTT assay and cell adhesion in A549 cells. Moreover, rats were given 25 mg/kg of propolis for 5 days before induction of experimental lung cancer by a single intraperitoneal dose of 200 mg/kg benzo(a)pyrene. Body weight, lung weight, lipid peroxidation, marker enzymes, and enzymatic and non-enzymatic antioxidants were estimated. The EEP demonstrated an inhibitory effect on proliferation of A549 at 24 and 72 hours in a dose-dependent manner and blocked adhesion of the cells by fibrinogen. Moreover, EEP reduced the oxidative stress generated by benzo(a)pyrene. The pre-treatment showed that enzymatic and non-enzymatic antioxidants increased and lipid peroxidation decreased. A histological analysis further supported these findings and showed a decrease in the number of side effects. These results are particularly important for both clinical applications of propolis and the possibility for its use as a potential chemotherapeutic agent.
Subject(s)
Animals , Rats , Propolis/adverse effects , Chemoprevention/instrumentation , Lung Neoplasms/drug therapy , Antioxidants , Benzo(a)pyrene/classification , Oxidative StressABSTRACT
OBJECTIVE: To establish a method for simultaneous detection of 3 kinds of polycyclic aromatic hydrocarbons(PAHs) including phenanthrene,anthracene and 3,4-benzo( a) pyrene in workplace air by high performance liquid chromatography( HPLC). METHODS: The phenanthrene,anthracene and 3,4-benzo( a) pyrene were collected in the air of the workplace using glass fiber filter paper. The membrane sample was added with 5. 00 m L of acetone,and the samples were extracted by ultrasonic wave for 70 mintues and eluted with acetonitrile-water gradient. Liquid chromatography-UV detector with tandem fluorescence detector was used for determination. RESULTS: The good linearity rang of phenanthrene,anthracene and 3,4-benzo( a) pyrene was 0. 050-1. 000 mg/L with the correlation coefficients ≥0. 999 5; the detection limits were 0. 010,0. 007 and 0. 008 mg/L,and the minimum detectable concentrations were 0. 130,0. 090 and 0. 110μg/m3 respectively( air collection of 375 L). The desorption efficiencies were 86. 10%-99. 20% of phenanthrene,88. 60%-96. 40% of anthracene,and 84. 80%-99. 60% of 3,4-benzo( a) pyrene,respectively. The within-run relative standard deviation( RSD) were 1. 29%-3. 25%,1. 90%-3. 61% and 1. 30%-4. 82% respectively,the between-run RSD were 2. 41%-4. 07%,2. 02%-5. 12% and 2. 08%-4. 77% respectively. The standard recovery rates were 95. 00%-106. 14% of phenanthrene,93. 14%-106. 50% of anthracene, and 92. 86%-105. 50% of 3,4-benzo( a) pyrene,respectively. The samples could be stored at 4 ℃ for 7 days. CONCLUSION: This method is simple,accurate and reliable for the simultaneous detection of phenanthrene,anthracene and 3,4-benzo( a) pyrene in the air of workplace.