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.

Aristolochic acids exposure was not the main cause of liver tumorigenesis in adulthood

Aristolochic acids (AAs) have long been considered as a potent carcinogen due to its nephrotoxicity. Aristolochic acid I (AAI) reacts with DNA to form covalent aristolactam (AL)-DNA adducts, leading to subsequent A to T transversion mutation, commonly referred as AA mutational signature. Previous research inferred that AAs were widely implicated in liver cancer throughout Asia. In this study, we explored whether AAs exposure was the main cause of liver cancer in the context of HBV infection in mainland China. Totally 1256 liver cancer samples were randomly retrieved from 3 medical centers and a refined bioanalytical method was used to detect AAI-DNA adducts. 5.10% of these samples could be identified as AAI positive exposure. Whole genome sequencing suggested 8.41% of 107 liver cancer patients exhibited the dominant AA mutational signature, indicating a relatively low overall AAI exposure rate. In animal models, long-term administration of AAI barely increased liver tumorigenesis in adult mice, opposite from its tumor-inducing role when subjected to infant mice. Furthermore, AAI induced dose-dependent accumulation of AA-DNA adduct in target organs in adult mice, with the most detected in kidney instead of liver. Taken together, our data indicate that AA exposure was not the major threat of liver cancer in adulthood.

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.

Preparation of divinylsulfone-glutathione adducts and their reactive activities with DNA in vitro / 中国药理学与毒理学杂志

OBJECTIVE To prepare the glutathione adducts of divinylsulfone (DVS), which is an important oxidative metabolism product of SM in vivo, and to investigate their reactive capability with DNA in vitro. METHODS The mustard sulfoxide (SMO) and mustard sulfone (SMO2) were prepared by oxidation reaction using HNO3 and KMnO4 as oxidants, respectively. Then, DVS was prepared through dechlorination reaction using CaCO3 under alkaline conditions. Furthermore, the DVS-GSH adduct and DVS-GSH-purine adducts were prepared and identified using ultra-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) and nuclear magnetic resonance (NMR). Finally, the adduct reac?tion process of DVS with GSH was monitored using UPLC-MS/MS. RESULTS The DVS-GSH and GSH-DVS-purine adducts were obtained through preparative HPLC and characterized using NMR and high-resolution MS. In aqueous solution, the reactive activity of DVS with GSH was significantly higher than that of SM, and the DVS-GSH adduct had high or reactive activity, which could produce a series of adducts with adenine and guanine in DNA, and the abundance of the adenine adducts was higher than that of the guanine. CONCLUSION DVS-GSH adducts still have high reactive activity with DNA, and more attention should be paid to its potential damage to DNA.

Suppression of lipid-hydroperoxide and DNA-adduct formation by isoflavone-containing soy hypocotyl tea in rats

<p><b>OBJECTIVE</b>Phytoestrogen isoflavones (IFs) are considered to suppress estrogen-related cancers through their antiestrogenic activity. The antioxidant effect of IFs, however, has not been confirmed in anin vivo system, so suppression of hydroperoxide formation and resultant DNA adduct formation were studied.</p><p><b>METHODS</b>The antioxidant effects of the soya-hypocotyl tea (SHT), which contained daidzein (14+/-1.5 mg/l) and genistein (3+/-0.5 mg/l), were examined in Wistar rats fed the AIN-76 control diet or iron deficient diet (FeD) for 4 weeks. The intake amount of the diet and IFs were measured daily. Urinary excretion of IFs was measured for 3 days before sacrifice. In addition to the serum lipid analyses, phosphatidylcholine hydroperoxide (PCOOH), and phosphatidylethanolamine hydroperoxide (PEOOH) production in red blood cells and the liver were measured as a biomarker of oxidants. Production of DNA adducts by oxidative stress was measured by the amount of 8-hydroxy-2'-deoxyguanosine (oh(8)dG) in the liver and kidney, and urine. Histological changes were checked by H&E staining and immunohistochemistry for oh(8)dG.</p><p><b>RESULTS</b>FeD rats showed anemia, growth retardation, hyperlipidemia. IFs only lowered the triacylglycerol level and did not change the cholesterol level. Rats fed the normal diet did not show suppression of PCOOH and PEOOH production in either red blood cells or the liver, while groups administered SHT showed suppressed production of PCOOH and PEOOH in the liver. The cumulative intake of daidzein, genistein and the total amount of IFs showed significant inverse associations with urinary excretion of oh(8)dG. oh(8)dG in the kidney showed an inverse association with the amount of oh(8)dG in the urine. Enzymehistochemically, a strong localization of oh(8)dG was found in the epithelial cells of the bile canaliculi and proximal tubules of the kidney.</p><p><b>CONCLUSION</b>IFs and SHT showed antioxidant effects at physiological concentrations in anin vivo system. The antioxidant effects of IFs decreased oxidation stress to the nuclear DNA, which was shown by the decreased oh(8)dG production. It is suggested that to prevent various cancers, in addition to the known antiestrogenie, antityrosin kinase, and other effects. IFs appeared to promote excretion of oh(8)dG.</p>

Suppression of Lipid-Hydroperoxide and DNA-Adduct Formation by Isoflavone-Containing Soy Hypocotyl Tea in Rats

Objective: Phytoestrogen isoflavones (IFs) are considered to suppress estrogen-related cancers through their antiestrogenic activity. The antioxidant effect of IFs, however, has not been confirmed in an in vivo system, so suppression of hydroperoxide formation and resultant DNA adduct formation were studied.Metheds: The antioxidant effects of the soya-hypocotyl tea (SHT), which contained daidzein (14+/−1.5 mg/l) and genistein (3+/−0.5 mg/l), were examined in Wistar rats fed the AIN-76 control diet or iron deficient diet (FeD) for 4 weeks. The intake amount of the diet and IFs were measured daily. Urinary excretion of IFs was measured for 3 days before sacrifice. In addition to the serum lipid analyses, phosphatidylcholine hydroperoxide (PCOOH), and phosphatidylethanolamine hydroperoxide (PEOOH) production in red blood cells and the liver were measured as a biomarker of oxidants. Production of DNA adducts by oxidative stress was measured by the amount of 8-hydroxy-2’-deoxyguanosine (oh8dG) in the liver and kidney, and urine. Histological changes were checked by H&E staining and immunohistochemistry for oh8dG.Results: FeD rats showed anemia, growth retardation, hyperlipidemia. IFs only lowered the triacylglycerol level and did not change the cholesterol level. Rats fed the normal diet did not show suppression of PCOOH and PEOOH production in either red blood cells or the liver, while groups administered SHT showed suppressed production of PCOOH and PEOOH in the liver. The cumulative intake of daidzein, genistein and the total amount of IFs showed significant inverse associations with urinary excretion of oh8dG. oh8dG in the kidney showed an inverse association with the amount of oh 8dG in the urine. Enzyme-histochemically, a strong localization of oh8dG was found in the epithelial cells of the bile canaliculi and proximal tubules of the kidney.Conclusion: IFs and SHT showed antioxidant effects at physiological concentrations in an in vivo system. The antioxidant effects of IFs decreased oxidation stress to the nuclear DNA, which was shown by the decreased oh8dG production. It is suggested that to prevent various cancers, in addition to the known antiestrogenic, antityrosin kinase, and other effects. IFs appeared to promote excretion of oh8dG.

Molecular biomarkers of occupational lung cancer

Occupational exposures to certain metals, hydrocarbons and ionizing radiation are associated with increased lung cancer in workers; because these exposures continue, lung cancer remains an important problem in industrialized nations. The gravity of the lung cancer, specifically the low cure rate associated with the disease, has forced researchers to focus efforts at developing biological indicators (biomarkers) of carcinogen exposure and early, reversible effects. This review examines critically the development of these biomarkers for occupational and environmenta exposures to polycyclic aromatic hydrocarbons (PAH), a ubiquitous class of lung carcinogens. Biomarkers of several different stages of the carcinogenic process have been proposed. Industrial hygiene and occupational health emphasize exposure and disease prevention. For this reason, biomarkers useful in industrial hygiene practice are those which measure events prior to the initiation phase of carcinogenesis; markers of later events which have a greater positive predictive value may measure irreversible effects and are more appropriate for disease screening and epidemiology. One of the strengths of biological monitoring is that exposures and effects can be measured regardless of route. Data indicates that the dermal route may be a significant pathway for delivery of PAH to the lung. This finding has important ramifications because as airborne exposure limits decrease the relative impact of dermal absorption is increased.