Huimin Ma

Effects of Benzene and Its Metabolites on Global DNA Methylation in Human Normal Hepatic L02 Cells

Benzene is an important industrial chemical that is also widely present in cigarette smoke, automobile exhaust, and gasoline. It is reported that benzene can cause hematopoietic disorders and has been recognized as a human carcinogen. However, the mechanisms by which it increases the risk of carcinogenesis are only partially understood. Aberrant DNA methylation is a major epigenetic mechanism associated with the toxicity of carcinogens. To understand the carcinogenic capacity of benzene, experiments were designed to investigate whether exposure to benzene and its metabolites would change the global DNA methylation status in human normal hepatic L02 cells and then to evaluate whether the changes would be induced by variation of DNA methyltransferase (DNMT) activity in HaeIII DNMT‐mediated methylation assay in vitro. Our results showed that hydroquinone and 1,4‐benzoquinone could induce global DNA hypomethylation with statistically significant difference from control (p < 0.05), but no significant global DNA methylation changes were observed in L02 cells with benzene, phenol, and 1,2,4‐trihydroxybenzene exposure. Benzene metabolites could not influence HaeIII DNMT activity except that 1,4‐benzoquinone shows significantly inhibiting effect on enzymatic methylation reaction at concentrations of 5 μM (p < 0.05). These results suggest that benzene metabolites, hydroquinone, and 1,4‐benzoquinone can disrupt global DNA methylation, and the potential epigenetic mechanism by which that global DNA hypomethylation induced by 1,4‐benzoquinone may work through the inhibiting effects of DNMT activity at 10 μM (p < 0.05).

Triclosan reduces the levels of global DNA methylation in HepG2 cells

Triclosan (TCS), an antibacterial agent, is widely used in a variety of personal care and industrial products. TCS is associated with the development of liver tumors in rodents and has become a concern to environmental and human health. This study is aimed at investigating whether TCS could modulate the levels of global DNA methylation (GDM) in human hepatocytes. We found that treatment with different doses (1.25-10 μM) of TCS did not affect HepG2 cell viability, but significantly reduced the levels of GDM in HepG2 cells, and inhibited DNMT1 activity. Furthermore, treatment with TCS significantly inhibited the methylated DNA-binding domain 2 (MBD2), MBD3, and MeCP2 mRNA transcription. In addition, treatment with TCS promoted the accumulation of 8-hydroxy-2-deoxyguanosine (8-OHdG) in a dose-dependent manner, which was abrogated by treatment with an antioxidant, N-acetylcysteine (NAC). Collectively, our data indicated that TCS reduced the levels of GDM and down-regulated the MBD2, MBD3, and MeCP2 gene expression by increasing 8-OHdG levels and inhibiting the DNMT1 activity in HepG2 cells.

Analysis of Global DNA Methylation Levels in Human Blood Using High-Performance Liquid Chromatography/Tandem Electrospray Ionization Mass Spectrometry

DNA methylation plays an important role in both normal physiological and disease processes. In this study, a sensitive and precise liquid chromatography-electrospray ionization tandem mass spectrometry method has been optimized for quantification of global DNA methylation levels in human peripheral blood by using multiple reaction monitoring (MRM) mode. This method was then used to analyze age-dependent association of DNA methylation. The results showed that the limit of detection was 1 pg for 5-methyl-2′-dexycytidine (5mdC) (S/N = 5.6). The linear calibration curves with correlation coefficients higher than 0.9990 were ranged 0.75–10 ppm and 0.0375–0.5 ppm for 2′-dexycytidine (dC) and 5mdC, respectively. The relative standard derivatives (RSD) of inter-day, intra-day and parallel samples were less than 5%. The analysis results of the blood samples from 60 healthy individuals showed that the child group had the highest mean 5mdC content while the adult group had the lowest values.

LINE-1 gene hypomethylation and p16 gene hypermethylation in HepG2 cells induced by low-dose and long-term triclosan exposure: The role of hydroxyl group.

Triclosan (TCS), a frequently used antimicrobial agent in pharmaceuticals and personal care products, exerts liver tumor promoter activities in mice. Previous work showed high-dose TCS (1.25-10μM) induced global DNA hypomethylation in HepG2 cells. However, whether or how tumor suppressor gene methylation changed in HepG2 cells after low-dose and long-term TCS exposure is still unknown. We investigate here the effects and mechanisms of DNA methylation of global DNA(GDM), repetitive genes, and liver tumor suppressor gene (p16) after exposing HepG2 cells to low-dose TCS (0.625-5nM)for two weeks using HPLC-MS/MS, Methylight, Q-MSP, Pyrosequencing, and Massarray methods. We found that low-dose TCS exposure decreased repetitive elements LINE-1 methylation levels, but not global DNA methylation, through down-regulating DNMT1 (DNA methyltransferase 1) and MeCP2 (methylated DNA binding domain) expression, and up-regulating 8-hydroxy-2-deoxyguanosine (8-OHdG) levels. Interestingly, low-dose TCS elevated p16 gene methylation and inhibited p16 expression, which were not observed in high-dose (10μM) group. Meanwhile, methyl-triclosan could not induce these two types of DNA methylation changes, suggesting the involvement of hydroxyl in TCS-mediated DNA methylation changes. Collectively, our results suggested low concentrations of TCS adversely affected HepG2 cells through DNA methylation dysregulation, and hydroxyl group in TCS played an important role in the effects. This study provided a better understanding on hepatotoxicity of TCS at environmentally relevant concentrations through epigenetic pathway.

Characterization of methylated and unmethylated CpG oligodeoxynucleotides by electrospray ionization tandem mass spectrometry

Methylated and unmethylated CpG oligodeoxynucleotides (ODNs) at A-rich, C-rich, G-rich and T-rich conditions were characterized by electrospray ionization tandem mass spectrometry (ESI-MS/MS). The methyalted site could be confirmed by comparison of the MS/MS spectra of methylated and unmethylated CpG-ODNs. The fragmentation patterns of the CpG-ODNs were not influenced by the presence of the methyl group but significant effects were observed for nucleobase identities and parent ion charges. The cleavage at guanine was the most facile while that at thymine was the least facile. With the increase of the parent ions charge states, the major dissociation behaviors changed from the middle to the 3′ and 5′ termini of the sequence.

Characterization of G-rich and T-rich oligonucleotides using ion-pair reversed-phase high-performance liquid chromatography/tandem electrospray ionization mass spectrometry

Characteristics of G-rich and T-rich oligonucleotides were investigated to compare their retention time, total ion current (TIC) intensity, charge-state distribution and product ions using ion-pair reversed-phase high-performance liquid chromatography/tandem electrospray ionization mass spectrometry (IP-RP-HPLC/ESI-MS) at room temperature. Three commonly used mobile phases for the analysis of oligonucleotides, triethylammonium acetate (TEAA), triethylammonium bicarbonate (TEAB) and triethylammonium hexafluoroisopropanol (HFIP) have been utilized. Retention time of G-rich and T-rich oligonucleotide was significantly different in TEAA and TEAB buffer systems, while in the HFIP buffer system it was affected more by the length of oligonucleotides. On the other hand, the ESI-MS ion abundance in the HFIP buffer system was higher than that in both TEAA and TEAB buffers. The TIC intensity of T-rich oligonucleotides was much higher than that of G-rich oligonucleotides in all mobile phases. In addition, much higher charge-state fragments were observed in the HFIP buffer system than that in the case of TEAA and TEAB buffer systems. Product ions of both G-rich and T-rich oligonucleotides were affected by the charge state of the parent ions and collision energy.

Comparison of A + T-rich oligonucleotides with and without self-complementary sequence using ion-pair reversed-phase high-performance liquid chromatography/tandem electrospray ionization mass spectrometry

Both A + T-rich oligonucleotides with and without self-complementary sequences were analyzed using ion-pair reversed-phase liquid chromatography/electrospray ionization mass spectrometry (IP-RP-HPLC/ESI-MS) by tryethylammonium acetate (TEAA) and hexafluoroisopropanol (HFIP) buffer systems to study the characteristics of their retention behavior and electrospray ionization tandem mass spectrometry (ESI-MS/MS) response. The results indicated that the chain length had the same effect on the retention of A + T-rich oligonucleotides in both of TEAA and HFIP buffer systems but the sequence had a different impact on the retention in the two buffer systems. A + T-rich oligonucleotides with a self-complementary sequence were much shorter than those without in the TEAA buffer system whereas a slight difference was observed in the HFIP buffer system. Similar total ion current (TIC) intensity was observed both in oligonucleotides with or without self-complementary sequence. The opposite trend of a change in the TIC intensities with increasing chain length were observed in both the TEAA and HFIP buffer systems. A lower charge state was predominant in the TEAA buffer system whereas a higher charge state was mainly distributed in the HFIP buffer system. The oligonucleotides without self-complementary sequences had a higher charge state than those with self-complementary sequences. A- and T- are more esily formed at a higher charge state whereas the sequence fragments will be formed more easily at a lower charge state in both A + T-rich oligonucleotides with and without self-complementary sequences.

Analysis of single nucleotide polymorphism sites in exon 4 of the p53 gene using high-performance liquid chromatography/electrospray ionization/tandem mass spectrometry

Three groups of four oligonucleotides with special single nucleotide polymorphism (SNP) sites in exon 4 of the p53 gene were analyzed with ion-pair, reversed-phase, high-performance liquid chromatography/electrospray ionization/tandem mass spectrometry. The retention order of four oligonucleotides with SNPs was C<G<A<T, regardless of whether the polymorphisms were at the 3′ end, the 5′ end, or the middle of the oligonucleotides. The charge state of the molecular ion affects the tandem mass spectra of the oligonucleotides. SNPs at the 3′ end can easily be identified from the fragmentation pattern of the 2- charge state, but not from the 3- charge state, especially from the w1 fragment. The single base may be taken as the symbol of the 5′ end SNP site derived from [M-3H]3−, but not from the [M-3H]2− charge state. The oligonucleotides with SNPs in the middle were also determined from the [M-2H]2− precursor ion.

Pet exposure in utero and postnatal decreases the effects of air pollutants on hypertension in children: A large population based cohort study

The effect of ambient air pollution exposure on childhood hypertension has emerged as a concern in China, and previous studies suggested pet ownership is associated with lower blood pressure (BP). However, limited information exists on the interactive effects pet ownership and air pollution exposure has on hypertension. We investigated the interactions between exposure to pet ownership and air pollutants on hypertension in Chinese children. 9354 students in twenty-four elementary and middle schools (aged 5-17 years) in Northeastern China were evaluated during 2012-2013. Four-year average concentrations of particulate matter with aerodynamic diameter of ≤10 μm (PM10), SO2, NO2, and O3, were collected in the 24 districts from 2009 to 2012. Hypertension was defined as average diastolic or systolic BP (three time measurements) in the 95th percentile or higher based on height, age, and sex. To examine effects, two-level regression analysis was used, controlling covariates. Consistent interactions between exposure to pet and air pollutants were observed. Compared to children exposed to pet, those not exposed exhibited consistently stronger effects of air pollution. The highest odds ratios (ORs) per 30.6 μg/m3 increase in PM10 were 1.79 (95%confidence interval [95%CI]: 1.29-2.50) in children without current pet exposure compared to 1.24 (95%CI: 0.85-1.82) in children with current pet exposure. As for BP, only O3 had an interaction for all exposure to pet ownership types, and showed lower BP in children exposed to pet. The increases in mean diastolic BP per 46.3 μg/m3 increase in O3 were 0.60  mmHg (95%CI: 0.21, 0.48) in children without pet exposure in utero compared with 0.34  mmHg (95%CI: 0.21, 0.48) in their counterparts. When stratified by age, pet exposure was more protective among younger children. In conclusion, in this large population-based cohort, pet ownership is associated with smaller associations between air pollution and hypertension in children, suggesting pet ownership reduces susceptibility to the health effects of pollutants.

Characterization of activation energy distribution for ultraviolet irradiation of T-rich oligonucleotide using high-performance liquid chromatography electrospray ionization mass spectrometry tandem mass spectrometry.

Exposure to solar ultraviolet (UV) radiation gives rise to mutations that may lead to skin cancer of human beings. A series of experiments was carried out in order to reveal activation energy distribution of deoxynucleic acid (DNA) mutation caused by UV radiation. The T-rich oligonucleotides were exposed to UV radiation with increasing intensity for different durations. Photo products of T-rich oligonucleotides were investigated using ion-pair reversed-phase high-performance liquid chromatography/tandem electrospray ionization mass spectrometry at room temperature. Two photo products of T-rich oligonucleotides were cis-syn cyclobutane pyrimidine dimmer (T[c,s]T) and the pyrimidine(6,4)pyrimidone product (T[6,4]T). Activation energy distribution of DNA mutation was calculated using a commercial kinetics analysis program by Robert L. Braun and Alan K. Burnham, Lawrence Livermore International Laboratory (version 2.4.1). To use the software for deriving the kinetics parameters, the factor T (temperature) in the software was substituted with k1R, in which k1 is a factor and R is radiation intensity. The activation energy derived ranges from 55 to 110 kJ mol−1. Using the same software, those kinetic parameters were extrapolated to a natural UV radiation process to predict the degree of DNA damage without the DNA repair process.