Trichloroethylene-Induced Gene Expression and DNA Methylation Changes in B6C3F1 Mouse Liver

<div><p>Trichloroethylene (TCE), widely used as an organic solvent in the industry, is a common contaminant in air, soil, and water. Chronic TCE exposure induced hepatocellular carcinoma in mice, and occupational exposure in humans was suggested to be associated with liver cancer. To understand the role of non-genotoxic mechanism(s) for TCE action, we examined the gene expression and DNA methylation changes in the liver of B6C3F1 mice orally administered with TCE (0, 100, 500 and 1000 mg/kg b.w. per day) for 5 days. After 5 days TCE treatment at a dose level of 1000 mg/kg b.w., a total of 431 differentially expressed genes were identified in mouse liver by microarray, of which 291 were up-regulated and 140 down-regulated. The expression changed genes were involved in key signal pathways including PPAR, proliferation, apoptosis and homologous recombination. Notably, the expression level of a number of vital genes involved in the regulation of DNA methylation, such as Utrf1, Tet2, DNMT1, DNMT3a and DNMT3b, were dysregulated. Although global DNA methylation change was not detected in the liver of mice exposed to TCE, the promoter regions of Cdkn1a and Ihh were found to be hypo- and hypermethylated respectively, which correlated negatively with their mRNA expression changes. Furthermore, the gene expression and DNA methylation changes induced by TCE were dose dependent. The overall data indicate that TCE exposure leads to aberrant DNA methylation changes, which might alter the expression of genes involved in the TCE-induced liver tumorgenesis.</p></div

Selected gene expression changes in the liver of mice exposed to different doses of TCE (0, 100, 500 and 1000 mg/kg b.w.) (n = 3).

<p>A) qPCR analysis of selected mRNA expression changes in mouse liver. B) Western analysis of Dnmt1 protein expression levels. Fold expression at each dose was calculated against the nonexposed samples (TCE dose level at 0 mg/kg). *, p<0.05; **, p<0.01. ***, p<0.001.</p

DNA methylation status of the promoter regions of Cdkn1a and Ihh in the liver of mice exposed to different doses of TCE (0, 100, 500 and 1000 mg/kg b.w.) (n = 3).

<p>A, D) Nucleotide sequences of Cdkn1a and Ihh promoter region fragments (upper strands) and the corresponding bisulphite-converted sequences (lower strands). CpG dinucleotides are numbered and marked in bold. The restriction enzyme cut sites are marked in italic. Primer sequences are underlined. B, E) Bisulfite sequencing of the Cdkn1a and Ihh promoter regions. Open and closed circles indicate unmethylated and methylated CpG sites respectively. Percent methylation is shown in parentheses. C) COBRA result of the promoter region of Cdkn1a at CpG4-5(CGCG) by <i>BstUI</i> 184 bp (84/100); L: Tiangen DNA ladder II; P, positive control by treating mouse genomic DNA with M.SssI. C, liver samples from mice exposed to corn oil; T, liver samples from mice exposed to TCE at 1000 mg/kg b.w. M, methylation; UM, unmethylation.</p

Global DNA methylation status of the liver of mice exposed to TCE.

<p>A–C) Bisulfite sequencing of the LINE-1, LAP-LTR and SINE B1 repetitive elements in the liver of mice treated with TCE at 0 or 1000 mg/kg b.w. Open and closed circles indicate unmethylated and methylated CpG sites respectively. Some sites are absent from the sequences in some clones due to mutations in the particular copies of the repetitive sequences. Percent methylation is shown in parentheses. D) The content of 5-mC detected by LC-MS/MS. (n = 5).</p

Comparison of mRNA expression changes detected by microarray and qPCR in mouse liver exposed to TCE at a dose level of 1000 mg/kg b.w. (n = 3).

<p>Comparison of mRNA expression changes detected by microarray and qPCR in mouse liver exposed to TCE at a dose level of 1000 mg/kg b.w. (n = 3).</p

DNA methylation status of the promoter regions of Jun and Myc in the liver from mice exposed to TCE at 0 or 1000 mg/kg b.w. (n = 3).

<p>A, D) Nucleotide sequences of Jun and Myc promoter region fragments (upper strands) and the corresponding bisulphite-converted sequences (lower strands). CpG dinucleotides are numbered and marked in bold. The restriction enzyme cut sites are marked in italic. Primer sequences are underlined. (B, E) Bisulfite sequencing of the Jun and Myc promoter regions. Open and closed circles indicate unmethylated and methylated CpG sites respectively. C) COBRA result of the promoter region of Jun at CpG3,4 (CGCG) by <i>BstUI</i> 171 bp (49/122). F) COBRA result of the promoter region of Myc at CpG4 (TCGA) by <i>TaqI</i> 132 bp (88/43). L: Tiangen DNA ladder; P, positive control by treating mouse genomic DNA with M.SssI. C, control liver samples; T, liver samples treated with TCE at 1000 mg/kg b.w. M methylation; UM, unmethylation.</p

Trichloroethylene-Induced DNA Methylation Changes in Male F344 Rat Liver

Trichloroethylene (TCE), a common environmental contaminant, causes hepatocellular carcinoma in mice but not in rats. To understand the mechanisms of the species-specific hepatocarcinogenecity of TCE, we examined the methylation status of DNA in the liver of rats exposed to TCE at 0 or 1000 mg/kg b.w. for 5 days using MeDIP-chip, bisulfite sequencing, COBRA, and LC-MS/MS. The related mRNA expression levels were measured by qPCR. Although no global DNA methylation change was detected, 806 genes were hypermethylated and 186 genes were hypomethylated. The genes with hypermethylated DNA were enriched in endocytosis, MAPK, and cAMP signaling pathways. We further confirmed the hypermethylation of Uhrf2 DNA and the hypomethylation of Hadhb DNA, which were negatively correlated with their mRNA expression levels. The transcriptional levels of Jun, Ihh, and Tet2 were significantly downregulated, whereas Cdkn1a was overexpressed. No mRNA expression change was found for Mki67, Myc, Uhrf1, and Dnmt1. In conclusion, TCE-induced DNA methylation changes in rats appear to suppress instead of promote hepatocarcinogenesis, which might play a role in the species-specific hepatocarcinogenecity of TCE

Broad-Specificity Chemiluminescence Enzyme Immunoassay for (Fluoro)quinolones: Hapten Design and Molecular Modeling Study of Antibody Recognition

On the basis of the structural features of (fluoro)­quinolones (FQs), pazufloxacin was first used as a generic immunizing hapten to raise a broad-specificity antibody. The obtained polyclonal antibody exhibited broad cross-reactivity ranging from 5.19% to 478.77% with 21 FQs. Furthermore, the antibody was able to recognize these FQs below their maximum residue limits (MRLs) in an indirect competitive chemiluminescence enzyme immunoassay (ic-CLEIA), with the limit of detection (LOD) ranging from 0.10 to 33.83 ng/mL. For simply pretreated milk samples with spiked FQs, the ic-CLEIA exhibited an excellent recovery with a range of 84.6–106.9% and an acceptable coefficient of variation below 15%, suggesting its suitability and reliability for the use of a promising tool to detect FQs. Meanwhile, comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) models, with statistically significant correlation coefficients (<i>q</i>²_CoMFA = 0.559, <i>r</i>²_CoMFA = 0.999; <i>q</i>²_CoMSIA = 0.559, <i>r</i>²_CoMSIA = 0.994), were established to investigate the antibody recognition mechanism. These two models revealed that in the antibody, the active cavity binding FQs’ 7-position substituents worked together with another cavity (binding FQs’ 1-position groups) to crucially endow the high cross-reactivity. This investigation will be significant for better exploring the recognition mechanism and for designing new haptens

Investigation of an Immunoassay with Broad Specificity to Quinolone Drugs by Genetic Algorithm with Linear Assignment of Hypermolecular Alignment of Data Sets and Advanced Quantitative Structure–Activity Relationship Analysis

A polyclonal antibody against the quinolone drug pazufloxacin (PAZ) but with surprisingly broad specificity was raised to simultaneously detect 24 quinolones (QNs). The developed competitive indirect enzyme-linked immunosorbent assay (ciELISA) exhibited limits of detection (LODs) for the 24 QNs ranging from 0.45 to 15.16 ng/mL, below the maximum residue levels (MRLs). To better understand the obtained broad specificity, a genetic algorithm with linear assignment of hypermolecular alignment of data sets (GALAHAD) was used to generate the desired pharmacophore model and superimpose the QNs, and then advanced comparative molecular field analysis (CoMFA) and advanced comparative molecular similarity indices analysis (CoMSIA) models were employed to study the three-dimensional quantitative structure–activity relationship (3D QSAR) between QNs and the antibody. It was found that the QNs could interact with the antibody with different binding poses, and cross-reactivity was mainly positively correlated with the bulky substructure containing electronegative atom at the 7-position, while it was negatively associated with the large bulky substructure at the 1-position of QNs

Four Specific Hapten Conformations Dominating Antibody Specificity: Quantitative Structure–Activity Relationship Analysis for Quinolone Immunoassay

Antibody-based immunoassay methods have been important tools for monitoring drug residues in animal foods. However, because of limited knowledge about the quantitative structure–activity relationships between a hapten and its resultant antibody specificity, antibody production with the desired specificity is still a huge challenge. In this study, the three-dimensional quantitative structure–activity relationship (3D QSAR) was analyzed in accordance with the cross-reactivity of quinolone drugs reacting with the antibody raised by pipemidic acid as the immunizing hapten and compared with the reported cross-reactivity data and their hapten structures. It was found that the specificity of a quinolone antibody was strongly related to the conformation of the hapten used and that hapten conformations shaped like the letters “I”, “P”, and “Φ” were essential for the desired high specificity with low cross-reactivity, but that the hapten conformation shaped like the letter “Y” led to an antibody with broad specificity and high cross-reactivity. Almost all of the antibodies against quinolones could result from these four hapten conformations. It was first found that the concrete conformations dominated the specificity of the antibody to quinolone, which will be of significance for the accurate hapten design, predictable antibody specificity, and better understanding the recognition mechanism between haptens and the antibodies for immunoassays