Effect on the H19 gene methylation of sperm and organs of offspring after chlorpyrifos‐methyl exposure during organogenesis period

To elucidate the effect on the H19 gene methylation of sperm and organs in offspring by chlorpyrifos‐methyl (CPM) exposure during organogenesis period, CPM was administered at doses of 4 (CPM4), 20 (CPM20), and 100 (CPM100) mg/kg bw/day from 7 days post coitum (d.p.c.) to 17 d.p.c. after mating CAST/Ei (♂) and B6 (♀). Anogenital distance (AGD) was measured at postnatal day (PND) 21. Clinical signs, body weights, feed and water consumption, organs weights, serum hormone values, and H19 methylation level of organ and sperm were measured at PND63. Body weights were significantly lower than control until PND6. AGD was significantly decreased in the CPM100 group in males and increased in the CPM20 group in females. The absolute weights of the thymus and epididymis were significantly increased for males in all of CPM treatment groups. In the CPM20 group, absolute weights of liver, kidney, heart, lung, spleen, prostate gland, and testes were significantly increased. Testosterone concentrations in serum were significantly increased by CPM treatment in males. H19 methylation level of liver and thymus showed decreased pattern in a dose‐dependent manner in males. The levels of H19 methylation in sperm were 73.76 ± 7.16% (Control), 57.84 ± 12.94% (CPM4), 64.24 ± 3.79% (CPM20), and 64.24 ± 3.79% (CPM100). Conclusively, CPM exposure during organogenesis period can disrupt H19 methylation in sperm, liver, and thymus and disturb the early development of offspring. © 2015 Wiley Periodicals, Inc. Environ Toxicol 30: 1355–1363, 2015.     

MSRE-qPCR法检测少弱精子症患者父源印记基因H19上游区域甲基化水平

目的建立精子中父源印记基因H19上游区域MSRE-qPCR检测方法,并分析男性少弱精子症患者精子父源印记基因H19上游区域甲基化水平。方法收集20例正常精液样本,同时筛选30例少弱精子症患者精液样本,应用甲基化敏感性限制性内切酶法并结合定量PCR对所有样本父源印记基因H19上游区域甲基化水平进行分析。结果正常精液样本父源印记基因H19上游区域平均甲基化率为（99.8±2.72）%,高于少弱精子症患者精液样本的（82.4±15.30）%,差异有统计学意义（P〈0.01）。结论 MSRE-qPCR法可用于父源印记基因H19上游区域甲基化水平的检测,少弱精子症者精液样本父源印记基因H19上游区域甲基化水平显著低于正常人。     

Metabolism of deltamethrin and cis- and trans-permethrin by human expressed cytochrome P450 and carboxylesterase enzymes

1. The metabolism of the pyrethroids deltamethrin (DLM), cis-permethrin (CPM) and trans-permethrin (TPM) was studied in human expressed cytochrome P450 (CYP) and carboxylesterase (CES) enzymes.

2. DLM, CPM and TPM were metabolised by human CYP2B6 and CYP2C19, with the highest apparent intrinsic clearance (CL_int) values for pyrethroid metabolism being observed with CYP2C19. Other CYP enzymes contributing to the metabolism of one or more of the three pyrethroids were CYP1A2, CYP2C8, CYP2C9*1, CYP2D6*1, CYP3A4 and CYP3A5. None of the pyrethroids were metabolised by CYP2A6, CYP2E1, CYP3A7 or CYP4A11.

3. DLM, CPM and TPM were metabolised by both human CES1 and CES2 enzymes.

4. Apparent CL_int values for pyrethroid metabolism by CYP and CES enzymes were scaled to per gram of adult human liver using abundance values for microsomal CYP enzymes and for CES enzymes in liver microsomes and cytosol. TPM had the highest and CPM the lowest apparent CL_int values for total metabolism (CYP and CES enzymes) per gram of adult human liver.

5. Due to their higher abundance, all three pyrethroids were extensively metabolised by CES enzymes in adult human liver, with CYP enzymes only accounting for 2%, 10% and 1% of total metabolism for DLM, CPM and TPM, respectively.

     

Impact of di‐ethylhexylphthalate exposure on metabolic programming in P19 ECC‐derived cardiomyocytes

Di(2‐ethylhexyl)phthalate (DEHP) is the most common plasticizer in plastic devices of everyday use. It is a ubiquitous environmental contaminant and primarily known to impair male gonadal development and fertility. Studies concerning the long‐term effects of prenatal DEHP exposure on certain diseases [The Developmental Origins of Health and Disease paradigm (DOHaD) hypothesis] are scarce although it is proven that DEHP crosses the placenta. Rising environmental pollution during the last centuries coincides with an increasing prevalence of cardiovascular and metabolic diseases. We have investigated the effects of an early embryonic DEHP exposure at different developmental stages on cardiomyogenesis. We used an in‐vitro model, the murine P19 embryonic carcinoma cell line (P19 ECC), mimicking early embryonic stages up to differentiated beating cardiomyocytes. P19 ECC were exposed to DEHP (5, 50, 100 µg ml^–1) at the undifferentiated stage for 5 days and subsequently differentiated to beating cardiomyocytes. We analyzed the expression of metabolic (Pparg1, Fabp4 and Glut4), cardiac (Myh6, Gja1) and methylation (Dnmt1, Dnmt3a) marker genes by quantitative real‐time PCR (qRT‐PCR), beating rate and the differentiation velocity of the cells. The methylation status of Pparg1, Ppara and Glut4 was investigated by pyrosequencing. DEHP significantly altered the expression of all investigated genes. The beating rate and differentiation velocity were accelerated. Exposure to DEHP led to small but statistically significant increases in methylation of specific CpGs within Ppara and Pparg1, which otherwise were generally hypomethylated, but methylation of Glut4 was unaltered. Early DEHP exposure of P19 ECC alters the expression of genes associated with cellular metabolism and the functional features of cardiomyocytes. Copyright © 2014 John Wiley & Sons, Ltd.     

Metabolism of deltamethrin and cis- and trans-permethrin by rat and human liver microsomes,liver cytosol and plasma preparations

1. The metabolism of deltamethrin (DLM), cis-permethrin (CPM) and trans-permethrin (TPM) was studied in liver microsomes, liver cytosol and plasma from male Sprague–Dawley rats aged 15, 21 and 90 days and from adult humans.

2. DLM and CPM were metabolised by rat hepatic microsomal cytochrome P450 (CYP) enzymes and to a lesser extent by microsomal and cytosolic carboxylesterase (CES) enzymes, whereas TPM was metabolised to a greater extent by CES enzymes.

3. In human liver, DLM and TPM were mainly metabolised by CES enzymes, whereas CPM was metabolised by CYP and CES enzymes.

4. The metabolism of pyrethroids by cytosolic CES enzymes contributes to the overall hepatic clearance of these compounds.

5. DLM, CPM and TPM were metabolised by rat, but not human, plasma CES enzymes.

6. This study demonstrates that the ability of male rats to metabolise DLM, CPM and TPM by hepatic CYP and CES enzymes and plasma CES enzymes increases with age. In all instances, apparent intrinsic clearance values were lower in 15 than in 90?day old rats. As pyrethroid-induced neurotoxicity is due to the parent compound, these results suggest that DLM, CPM and TPM may be more neurotoxic to juvenile than to adult rats.

     

Expression of CYP2E1 in Human Lung and Kidney during Development and in Full-Term Placenta: A Differential Methylation of the Gene is Involved in the Regulation Process

Abstract: Methylation of dinucleotide CG residues located in the 5′end of the CYP2E1 gene has been demonstrated to play a role in the control of gene expression in the human developing liver. This study was undertaken to examine the CYP2E1 RNA content of human lung, kidney and full-term placenta and to determine whether the expression of CYP2E1 was controlled by its methylation status in these tissues. CYP2E1 was expressed at a very low level in the lung and kidney at whatever age, and at a variable level in full-term placentas. The restriction profile of genomic DNA was identical in lung and kidney and corresponded to a heavy methylation of Hpall/Mspl sites located within the promoter, the first exon and first intron of the CYP2E1 gene. A different pattern of methylation was obtained in full-term placentas, indicating that CpG residues located in the 5′end of the gene were predominantly but not fully demethylated. However, the variable level of CYP2E1 RNA in full-term placentas suggests the involvement of other elements in the regulation process of CYP2E1 in this tissue.     

Prenatal exposure to ethanol: a specific effect on the H19 gene in sperm

Alcohol exposure during pregnancy induces a range of disorders in the offspring. Methylation changes in imprinted genes may play a role in the teratogenic effects of alcohol. We evaluated the possible effects of alcohol administration in pregnant mice on the methylation pattern of 5 imprinted genes (H19, Gtl2, Peg1, Snrpn and Peg3) in somatic and sperm cell DNAs of the male offspring. The effects observed were a 3% (p < 0.005) decrease in the number of methylated CpGs of H19 in the F1 offspring sperm, a 4% (p < 0.005) decrease in the number of methylated CpGs of H19 in the F2 offspring brain and a 26% (p < 0.05) decrease in the mean sperm concentration. CpGs 1 and 2 of the H19 CTCF-binding site 2 exhibited significant methylation percentage losses. H19 CTCF-binding sites are important for the regulation of Igf2 gene expression. The hypomethylation of H19 may contribute to the decreased spermatogenesis in the offspring.     

Pharmacokinetics and tissue distribution of chlorpheniramine in rabbits after intravenous administration

Intravenous studies of chlorpheniramine (CPM) were conducted in six New Zealand White male rabbits (mean wt. 3.88 kg). CPM and its two demethylated metabolites in arterial serum and urine were assayed by HPLC. Triexponential equations were needed to fit the i.V. CPM serum data in three rabbits, while biexponential equations were required in the other three rabbits. Harmonic mean of V₁, V_ss, V _area , CL,and terminal t _1/2 were 2.84, 10.8, and 15.5 liters/kg, and 4.14 liters/kg/hr and 2.57 hr, respectively. The average serum protein binding was 44%. The average blood to plasma concentration ratio was 1.85. Estimated mean hepatic blood extraction ratio based on i.v. studies was 0.88. Tissue distribution studies showed rapid and extensive uptake of CPM by various organs such as lung, kidneys, and brain after i.v. bolus injection, as their concentrations were 160-, 80-, and 31- fold higher than the plasma level. The amount of CPM in the muscle was calculated to represent about 50% of CPM present in the body near the steady state. Variation in plasma protein and tissue binding was postulated to be an important factor for the observed marked interspecies difference in the apparent volume of distribution of CPM. Only 2% of the dose was excreted unchanged in the urine.     

Gestational exposure to low‐dose zearalenone disrupting offspring spermatogenesis might be through epigenetic modifications

Zearalenone (ZEA), a F‐2 mycotoxin produced by Fusarium, has been found to be an endocrine disruptor through oestrogen receptor signalling pathway to impair spermatogenesis. The disruption on reproductive systems by ZEA exposure might be transgenerational. In our previous report, we have found that low dose (lower than no‐observed effect level, NOEL) of ZEA impaired mouse spermatogenesis and decreased mouse semen quality. The purpose of the current investigation was to explore the impacts of low‐dose ZEA on spermatogenesis in the offspring after prenatal exposure and the underlying mechanisms. And it demonstrated that prenatal low‐dose ZEA exposure disrupted the meiosis process to inhibit the spermatogenesis in offspring and even to diminish the semen quality by the decrease in spermatozoa motility and concentration. The DNA methylation marker 5hmC was decreased, the histone methylation markers H3K9 and H3K27 were elevated, and oestrogen receptor alpha was reduced in the offspring testis after prenatal low‐dose ZEA exposure. The data suggest that the disruption in spermatogenesis by prenatal low‐dose ZEA exposure may be through the modifications on epigenetic pathways (DNA methylation and histone methylation) and the interactions with oestrogen receptor signalling pathway. Moreover, in the current study, the male offspring were indirectly exposed to low‐dose ZEA through placenta and the spermatogenesis in offspring was disrupted which suggested that the toxicity of ZEA on reproductive systems was very severe. Therefore, we strongly recommend that greater attention should be paid to this mycotoxin to minimize its adverse impact on human spermatogenesis.     

Sodium fluoride disrupts DNA methylation of H19 and Peg3 imprinted genes during the early development of mouse embryo

Sodium fluoride (NaF) is associated with embryonic and fetal development abnormalities, but the mechanism by which this occurs is unclear. DNA methylation, an important epigenetic reprogramming mechanism, is essential for normal embryonic development. Thus, we investigated the effect of NaF on DNA methylation in early mouse embryos, as well as mouse sperm and liver using bisulfite sequencing and ELISA. Data indicate that H19, a paternally imprinted gene, compared to control embryos, was less methylated in 8-cell embryos from pregnant mice treated with NaF (100 mg/l) in drinking water for 48 h. Peg3, a maternally imprinted gene, and the Line1 repeated sequence were similarly methylated in NaF-treated and control embryos. Oral ingestion of NaF for 35 days did not significantly change Line1 and genomic global DNA methylation in the liver. H19, Rasgrf1, Line1, and genomic global DNA methylation were also similar in NaF-treated and control sperm. Female mice mated with NaF-treated male mice (35 days) had less methylated H19, but Peg3 was significantly more methylated. Line1 was similarly methylated in treated 8-cell embryos, compared to control embryos. NaF treatment of male mice before copulation significantly increased the expression of H19 in blastocysts, whereas H19 expression was not detected in 8-cell embryos. Data suggest that NaF may interact directly with the embryo to disrupt the maintenance of normal gene imprinting during pregnancy. Long-term NaF exposure of males may not directly affect DNA methylation of the sperm and liver, but the sperm may signal to early embryos with abnormal gene imprinting.     

Aberrant methylation‐induced dysfunction of p16 is associated with osteoblast activation caused by fluoride

Chronic exposure to fluoride continues to be a public health problem worldwide, affecting thousands of people. Fluoride can cause abnormal proliferation and activation of osteoblast and osteoclast, leading to skeletal fluorosis that can cause pain and harm to joints and bones and even lead to permanent disability. Nevertheless, there is no recognized mechanism to explain the bone lesions of fluorosis. In this work, we performed a population study and in vitro experiments to investigate the pathogenic mechanism of skeletal fluorosis in relation to methylation of the promoter of p16. The protein coded by the p16 gene inhibits cdk (cyclin‐dependent kinase) 4/cdk6‐mediated phosphorylation4 of retinoblastoma gene product and induces cell cycle arrest. The results showed that hypermethylation of p16 and reduced gene expression was evident in peripheral blood mononuclear cells of patients with fluorosis and correlated with the level of fluoride exposure. Studies with cell cultures of osteoblasts revealed in response to sodium fluoride (NaF) treatment, there was an induction of p16 hypermethylation and decreased expression, leading to increased cell proliferation, a longer S‐phase of the cell cycle, and development of skeletal fluorosis. Further, the methylation inhibitor, 5‐aza‐2‐deoxycytidine, reversed the p16 hypermethylation and expression in response to NaF. These results reveal a regulatory role of p16 gene methylation on osteoblasts activation during the development of skeletal fluorosis.     

Analysis of Hepatic Transport of Cefpiramide in Rats with Obstructive Jaundice by Using Isolated Hepatocytes

The mechanism of the diminished biliary clearance of cefpiramide (CPM) in rats with obstructive jaundice (OJ) was investigated by using isolated hepatocytes. The kinetics of CPM uptake by hepatocytes isolated from normal rats and rats with OJ could be explained by the combination of saturable carrier-mediated and nonsaturable first-order rate processes. The maximum uptake rate (V _max) of the carrier-mediated process was significantly decreased in OJ, compared with normal hepatocytes, while the Michaelis constant (K _m) and the first-order rate constant (k _d) were not significantly different. This result indicated that the number of CPM transport carriers was decreased in OJ hepatocytes. Further, no CPM uptake occurred from the serum of OJ rats into normal hepatocytes. Partial recovery of CPM uptake after treatment of OJ serum with activated charcoal suggested the accumulation of inhibitors of CPM uptake in OJ serum.     

血根碱对脂多糖致H9c2细胞氧化损伤的改善作用

目的 观察血根碱（sanguinarine,SAN）对脂多糖（lipopolysaccharide,LPS）诱导的H9c2细胞氧化应激的影响,并对其与HO-1/NOX2途径之间的关系进行探讨。方法 不同因素干预H9c2细胞后,采用细胞活性试剂盒检测不同浓度SAN和/或LPS对H9c2细胞生存的影响;酶标仪及荧光显微镜检测SAN对LPS诱导细胞产生活性氧（reactive oxygen species,ROS）的影响; Real time RT-PCR检测SAN对LPS诱导的NOX2、P47PHOX、HO-1、SOD1、SOD2基因表达的影响; MDA检测试剂盒检测MDA的变化。结果 单用SAN对H9c2细胞活性无影响,LPS可显著降低细胞活性,而SAN与LPS共同干预则可呈浓度依赖性地提高细胞活性,降低ROS产生。LPS处理12 h上调H9c2细胞NOX2、p47phox mRNA表达、下调HO-1 mRNA表达; SAN预处理后,细胞内NOX2、p47phox mRNA下调、HO-1 mRNA上调,而SAN与锌原卟啉IX预处理则可逆转这种现象。SAN上调SOD1及SOD2 mRNA表达,降低MDA产生。结论 SAN可通过活化HO-1/NOX2途径,抑制ROS的产生,从而使H9c2细胞免受LPS介导的损伤,提高细胞活力。     

Gene expression network related to DNA methylation and miRNA regulation during the process of aflatoxin B1-induced malignant transformation of L02 cells

Aflatoxin is a secondary metabolite secreted by Aspergillus flavus, parasitic Aspergillus, and other fungi through the polyketone pathway, and it can be detected in many foods. Aflatoxin has strong toxicity and carcinogenicity, and many studies have shown that aflatoxin is highly associated with liver cancer. In the present study, malignant transformation of L02 cells was induced by aflatoxin B1 (AFB1), and the gene expression, miRNA expression, and methylation level were detected by high-throughput sequencing. The gene and miRNA expression results showed that 2547 genes and 315 miRNAs were changed in the AFB1-treated group compared with the DMSO group. Among them, RSAD2 and SCIN were significantly upregulated, whereas TRAPPC3L and UBE2L6 were significantly downregulated. Has-miR-33b-3p was significantly upregulated, whereas Has-miR-3613-5p was significantly downregulated. The methylation results showed that 2832 CpG sites were methylated on the promoter or coding DNA sequence (CDS) of the gene, whereas the expression of DNMT3a and DNMT3b was significantly upregulated. Moreover, hypermethylation occurred in TRAPPC3L, CDH13, and SPINK13. The results of GO and KEGG pathway analyses showed that significantly changed genes and miRNAs were mainly involved in tumor formation, proliferation, invasion, and migration. The results of network map analysis showed that Hsa-miR-3613-5p, Hsa-miR-615-5p, Hsa-miR-615-3p, and Hsa-miR-3158-3p were the key miRNAs for malignant transformation of L02 cells induced by AFB1. In addition, the expression of ONECUT2, RAP1GAP2, and FSTL4 was regulated by DNA methylation and miRNAs. These results suggested that the gene expression network regulated by DNA methylation and miRNAs may play a vital role in AFB1-induced hepatocellular carcinoma.     

Distribution of Pentazocine in Foetal and Maternal Mouse Tissues and of Radioactivity along the Gut of Mice Dosed with [3H]Pentazocine

Abstract

1. The concn. of pentazocine in the maternal and foetal tissues of the pregnant mouse was measured.

2. One hour after intra-muscular injection of pentazocine to the 19-day pregnant mouse, the levels of pentazocine were similar in maternal and foetal muscle (0.55 and 0.62 μg/g resp.). Corresponding values for maternal and foetal liver were 0.67 and 0.46 μg/g. The level of pentazocine in the placenta was 1.5 μg g.

3. Mice were killed at intervals after intra-muscular injection of [³H]pentazocine and the distribution of radioactivity along the alimentary tracts was demonstrated.

4. Bile and gut-contents of dosed mice contained conjugates of pentazocine and norpentazocine with small amounts of the conjugated pentazocine trans-alcohol. Traces of pentazocine, norpentazocine and of pentazocine trans-acid were detected in bile; in the gut contents small amounts of pentazocine and norpentazocine were detected with trace amounts of the trans-acid.     

Short Report: omeprazole-tetracycline combinations are inadequate as therapy for Helicobacter pylori infection

Background: Current triple antimicrobial therapies cure Helicobacter pylori infection in 60–90% of cases but are cumbersome. Addition of omeprazole to amoxycillin has been shown to enhance effectiveness when compared to amoxycillin alone Method: We studied omeprazole 20 mg t.d.s. plus tetracycline 500 mg q.d.s. for 14 days (OMP/TCN) and omeprazole 40 mg in the morning plus tetracycline 500 mg q.d.s. along with bismuth subsalicylate tablets 2 q.d.s. (OMP/TCN/BSS) for 14 days. Forty-four patients (19 OMP/TCN, 25 OMP/TCN/BSS) with H. pylori peptic ulcer disease were studied. H. pylori status was evaluated at least 4 weeks after ending antimicrobial therapy. Results: In the OMP/TCN group cure of H. pylori infection was achieved in 5/19 (26%). Adding bismuth to the regimen improved the results; 4 weeks after ending therapy cure of H. pylori infection was achieved in 12/25 (48%). Conclusions: Neither regimen can be recommended for routine cure of H. pylori infection. Although one cannot predict which antimicrobial therapies will be enhanced by the addition of omeprazole, these data suggest that future studies should evaluate drugs whose effectiveness is compromised by low pH.