ABSTRACT
<p><b>OBJECTIVE</b>To investigate the effect of the exposure to di- (2-ethylhexyl) phthalate (DEHP) during pregnancy on the DNA methylation level of genomes in the testis of the offspring in mice.</p><p><b>METHODS</b>Pregnant KM mice were randomly divided into three groups, normal control, corn oil and DEHP-exposed. Corn oil and DEHP (500 mg/[kg x d]) were administrated respectively from gestation day 12.5 (GD 12.5) to postnatal day 3 (PND 3). The testes of the offspring were excised on PND 7, and their genomic DNA was treated with EcoR I /Msp I and EcoR I /Hpa II. The genome-wide DNA methylation patterns of the CCGG sites were detected by methylation-sensitive amplification polymorphism (MSAP). The samples were electrophoresed in the ABI 3730 DNA sequencer and the results analyzed by the Genescan3.1.</p><p><b>RESULTS</b>The average incidence of DNA methylation was (34.03 +/- 3.05)% in the DEHP-exposed mice, obviously higher than (28.37 +/- 2.37)% in the normal control and (28.58 2.45)% in the corn oil group, with statistically significant differences (P < 0.05).</p><p><b>CONCLUSION</b>Exposure to DEHP during pregnancy increases the DNA methylation level of the genome in the testis of the offspring and affects the apparent genetic modification of the genome, which may be one of the important toxicological causes of the lesion in the reproductive system.</p>
Subject(s)
Animals , Female , Male , Mice , Pregnancy , DNA Methylation , Diethylhexyl Phthalate , Pharmacology , Genome , Mice, Inbred Strains , Prenatal Exposure Delayed Effects , Random Amplified Polymorphic DNA Technique , TestisABSTRACT
<p><b>OBJECTIVE</b>Objective To construct recombinant Mycobacterium smegmatis expressing ESAT-6 of the human pathogen Mycobacterium tuberculosis.</p><p><b>METHODS</b>ESAT-6 gene was amplified from M. tuberculosis genomic DNA and inserted into an E.coli-mycobacterium shuttle vector under the control of HSP60 promoter. The recombinant vector was transformed into M. smegmatis by electroporation. To assess the ability of recombinant M. smegmatis to activate macrophage, mouse macrophage ANA-1 was cocultured with recombinant M. smegmatis. The apoptosis of ANA-1 cells was detected by flow cytometry and iNOS mRNA expression of the cells was detected by reverse transcription-polymerase chain reaction (RT-PCR). The survival of M. smegmatis strains in ANA-1 cells was evaluated.</p><p><b>RESULTS</b>The recombinant vector was verified by restriction endonuclease digestion and DNA sequencing. ESAT-6 protein was expressed in M. smegmatis in response to heat shock and the molecular weight of the expression product was identical to the expected value. The growth curve of the new recombinant M. smegmatis was consistent with that of the wild-type strain, suggesting the absence of ESAT-6 protein toxicity against M. smegmatis. The recombinant M. smegmatis did not induce significant changes in mouse macrophage ANA-1 apoptosis. Coculture of the macrophages with recombinant M. smegmatis for 4 to 24 h could induce iNOS expression in the former, and the CFU of recombination M. smegmatis grown in ANA-1 cells was much less than that of the control bacteria.</p><p><b>CONCLUSION</b>The recombinant M. smegmatis expressing M. tuberculosis ESAT-6 gene possess immunogenicity, which provides experimental evidence for the development of novel M. smegmatis-based vaccine against tuberculosis.</p>