Lianhui Tao

Reversed-phase high-performance liquid chromatography procedure for the simultaneous determination of S-adenosyl-L-methionine and S-adenosyl-L-homocysteine in mouse liver and the effect of methionine on their concentrations.

An improved reversed-phase high-performance liquid chromatography (HPLC) procedure with ultraviolet detection is described for the simultaneous determination of S-adenosyl-L-methionine (SAM) and S-adenosyl-L-homocysteine (SAH) in mouse tissue. The method provides rapid resolution of both compounds in a 25-microl perchloric acid extract of the tissue. The limits of detection in 25-microl injection volumes were 22 and 20 pmol for SAM and SAH, respectively. The limits of quantitation in 25-microl injection volumes were 55 and 50 pmol for SAM and SAH, respectively, with recovery consistently >98%. The assay was validated over linear ranges of 55-11000 pmol for SAM and 50-10000 pmol for SAH. The intra-day precision and accuracy were < or =6.4% relative standard deviation (RSD) and 99.9-100.0% for SAH and < or =6.7% RSD and 100.0-100.1% for SAM. The inter-day precision and accuracy were < or =5.9% RSD and 99.9-100.6% for SAH and < or =7.0% RSD and 99.5-100.1% for SAM. Compared to earlier procedures, the HPLC method demonstrated significantly better separation, detection limit and linear range for SAM and SAH determination. The assay demonstrated applicability to monitoring in mice the time-course of the effect of methionine on SAM and SAH levels in the liver. Administering methionine to mice increased by 10-fold the liver concentration of SAM and SAH within 2 h, which then rapidly decreased to the control levels by 8 h. This indicated that methionine was promptly converted to SAM and then rapidly catabolized into SAH. Thus, the metabolism of methionine to SAM should be considered in the supplementation of methionine to maintain SAM levels in the body.

Hypomethylation and overexpression of c-jun and c-myc protooncogenes and increased DNA methyltransferase activity in dichloroacetic and trichloroacetic acid-promoted mouse liver tumors

Dichloroacetic acid (DCA) and trichloroacetic acid (TCA) are mouse liver carcinogens. Methylation of the c-jun and c-myc genes, expression of both genes and DNA methyltransferase (DNA MTase) activity were determined in liver tumors initiated by N-methyl-N-nitrosourea and promoted by DCA and TCA in female B6C3F1 mice. Hypomethylated and over-expression of c-jun and c-myc genes were found in DCA- and TCA-promoted liver tumors. DNA MTase activity was increased in tumors while decreased in non-involved liver. Thus, DCA- and TCA-promoted carcinogenesis appears to include decreased methylation and increased expression of c-jun and c-myc genes in the presence of increased DNA MTase activity.

Effect of trichloroethylene on DNA methylation and expression of early‐intermediate protooncogenes in the liver of B6C3F1 mice

Trichloroethylene (TCE) is a multimedia environmental pollution that is carcinogenic in mouse liver. The ability of TCE to modulate DNA methylation and the expression of immediate‐early protooncogenes was evaluated. Female B6C3F1 mice were administered 1000 mg/kg TCE by gavage 5 days/week and killed after 5, 12, or 33 days of exposure. Methylation of DNA as 5‐methylcytosine was decreased by 5 days of treatment with TCE and remained reduced for 33 days. TCE also decreased the methylation of the promoter regions for the protooncogenes, c‐jun and c‐myc. The expression of the mRNA for the two protooncogenes was increased between 60 and 120 minutes after administering the last dose of TCE and returned to control level by 24 hours. The expression of the mRNA for c‐fos remained undetectable after administering TCE. Hence, TCE decreased the methylation both of total DNA and the promoters for the c‐jun and c‐myc genes and increased the expression of their mRNA. The decreased methylation and increased expression of the two immediate‐early protooncogenes might be associated with TCE‐induced increase in cell proliferation and promotion of tumors.

Effect of budesonide on the methylation and mRNA expression of the insulin-like growth factor 2 and c-myc genes in mouse lung tumors

The use of surrogate end‐point biomarkers could help in the development of chemopreventive agents. To define potential surrogate end‐point biomarkers, the ability of budesonide to decrease mRNA expression of the insulin‐like growth factor‐2 (Igf‐II) and c‐myc genes and to cause the remethylation of the genes was investigated in lung tumors. Lung tumors were induced in female strain A mice by administering i.p. 16 mg/kg vinyl carbamate for 2 consecutive wk or by a single dose of 100 mg/kg benzo[a]pyrene (B[a]P). Thirty‐four weeks later, the mice given vinyl carbamate received budesonide (0.6 or 2.4 mg/kg diet) for 7 d and then were killed. Mice were killed 24 wk after administration of B[a]P. The mRNA expression of the Igf‐II and c‐myc genes was increased in lung tumors relative to normal lung tissue. Budesonide decreased mRNA expression of both genes in tumors. The methylation status of 27 CpG sites in the differentially methylated region 2 in the Igf‐II gene was determined with the bisulfite‐treated DNA‐sequencing procedure. The numbers of methylated CpG sites were 17–21 in normal lung (70.4 ± 2.6%); 0–2, and 1–2 in lung tumors induced by vinyl carbamate and B[a]P (4.9 ± 1.2% and 4.6 ± 1.2%, respectively); and 4–5 or 7–16 in tumors after treatment with 0.6 or 2.4 mg/kg budesonide (16.0 ± 1.2% and 46.2 ± 5.1%, respectively). Thus, lung tumors had strikingly less methylated CpG sites than normal lung tissue, while even limited treatment with budesonide resulted in remethylation of the CpG sites in tumors. With HpaII digestion followed by Southern blot analysis, the internal cytosine of CCGG sites in the c‐myc gene was found to be methylated in normal lung tissue, whereas some of the sites were unmethylated in lung tumors. Treatment for 7 d with budesonide resulted in the remethylation of these sites. In conclusion, mouse lung tumors showed decreased methylation of the Igf‐II and c‐myc genes that was associated with increased expression of these genes. Budesonide treatment caused remethylation and decreased expression of both genes. The results support the possibility of using decreased mRNA expression and remethylation of the Igf‐II and c‐myc genes as biomarkers for the efficacy of budesonide.

Loss of heterozygosity on chromosome 6 in dichloroacetic acid and trichloroacetic acid-induced liver tumors in female B6C3F1 mice

Dichloroacetic acid (DCA) and trichloroacetic acid (TCA) are major metabolites of tetrachloroethylene (PCE) and trichloroethylene (TCE) and are found in chlorinated drinking water. All four chlorinated compounds are liver carcinogens in B6C3F1 mice. It has previously been reported that approximately 20% of hepatic tumors induced by PCE exhibited loss of heterozygosity (LOH) on chromosome 6, suggesting the presence of a tumor suppressor gene. In the current investigation, we determined whether TCA or DCA also induced LOH on chromosome 6. Liver tumors were initiated in 15 day old female B6C3F1 mice with N-methyl-N-nitrosourea (MNU) and promoted with 20 mmol/l DCA or TCA in their drinking water. Twenty-four and thirty-seven liver tumors promoted by DCA and TCA, respectively, were examined for LOH using 4 polymorphic loci on chromosome 6. Ten of 37 (27%) tumors (7 of 27 carcinomas and 3 of 10 adenomas) promoted by TCA exhibited LOH at least for two loci on chromosome 6. All 10 tumors that exhibited LOH, lost the C57BL/6J allele at both the D6mit9 loci, while two also lost at least one of the C3H/HeJ alleles. No LOH on chromosome 6 was observed in the 24 liver tumors promoted by DCA. The LOH on chromosome 6 in TCA but not in DCA-promoted tumors supports it as an active metabolite of PCE and demonstrates different pathogenesis at least for some of the DCA and TCA-promoted liver cancer.

Quantification of carvone, cineole, perillaldehyde, perillyl alcohol and sobrerol by isocratic high-performance liquid chromatography

A simple and rapid isocratic HPLC assay is presented for the analysis and quantification of monoterpenes, i.e., carvone, cineole, perillaldehyde, perillyl alcohol and sobrerol in the diet of laboratory animals. The monoterpenes were extracted from the diet using 90% methanol in water. The analysis for the monoterpenes was performed by HPLC using a Whatman PartiSphere C18 column with UV detection. The mobile phase was isocratic methanol-water (72:28, v/v) for carvone, cineole, perillaldehyde and perillyl alcohol and methanol-water (65:35, v/v) for sobrerol. The method was simple with good repeatability (R.S.D. = 4.2-16.1%), reproducibility (R.S.D. = 3.7-19%), and accuracy (R.S.D. = 5.5-23.3%). The detection limit of the monoterpenes extracted from the diet was 2 micrograms/g for carvone, perillaldehyde and sobrerol, 20 micrograms/g for perillyl alcohol and 100 micrograms/g for cineole. Excellent resolution was achieved between the monoterpenes and the constituents of the diet. The method demonstrated applicability to monitoring the formulation of monoterpenes in the diet of laboratory animals.