Stereoselective determination of propranolol enantiomer in transgenic cell lines expressing human cytochrome P450 / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To establish a chiro chromatography for studying the stereoselective metabolism of propranolol (PL) in S(9) incubates prepared from transgenic cell lines expressing human cytochrome P450.</p><p><b>METHODS</b>The concentration of each enantiomer in S(9) incubates was determined through precolumn derivatization with GITC, followed by RP-HPLC assay using S-(+)-propafenone as internal standard.</p><p><b>RESULTS</b>Baseline separations among the diastereomers of S(-)-P, internal standard and R(+)-PL were achieved on Shimpack CLC C(18)ODS column, with UV detection and methanol:water:glacial acetic acid (67/33/0.05,v/v/v) as mobile phase. The assay was simple, accurate, precise and specific. The linear range was from 5 to 500 micromol/L for each enantiomer. The limit of quantitation (LOQ) for the method was 5 micromol/L for the S(-)-and R(+)-PL, respectively (n=5, RSD<10%). The analytical method afforded average recoveries of 98.7 and 98.1% for S(-)- and R(+)-PL, respectively. The reproducibility of the assay was good (RSD<10%). The time-dependent studies showed that PL had the stereoselectivity of S-(-)-isomer in metabolism via CYP2C18 and the stereoselectivity of R-(+)-isomer in metabolism via CYP2C9.</p><p><b>CONCLUSION</b>The method allows to study of stereoselective metabolism of PL in vitro.</p>

Proteomic analysis of cellular responses to low concentration of N-methyl-N'-nitro-N-nitrosoguanidine in human amnion FL cells / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the protein profile after treatment of low concentration of N- methyl-N'-nitro-N-nitrosoguanidine (MNNG) in human FL cells.</p><p><b>METHODS</b>After MNNG treatment, whole cellular proteins were separated using two-dimensional gel electrophoresis and visualized by silver staining; the digitized images were analyzed with 2D analysis software. The differentially expressed protein spots were identified by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS).</p><p><b>RESULT</b>More than 60 proteins showed significant changes in MNNG-treated cells compared to control cells (DMSO treatment). There were 18 protein spots detected only after MNNG treatment, while 13 protein spots were detected only in the control cells. Moreover, the levels of another 31 proteins were either increased or decreased in MNNG-treated FL cells. And some of the proteins were identified by MALDI-TOF-MS.</p><p><b>CONCLUSION</b>There are significant alterations of protein profile after MNNG attack.</p>

Altered of zinc finger proteins expression in FL cells following benzo a pyrene treatment / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To understand benzo[a]pyrene (B[a]P) mediated cellular responses, and to provide clues to explore molecular mechanism of mutagenesis and carcinogenesis induced by B[a]P.</p><p><b>METHODS</b>Two-dimensional electrophoresis (2-DE) was used to investigate the protein expression levels of FL cells after B[a]P exposure, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) combined with database search was applied to identify the differentially expressed proteins.</p><p><b>RESULT</b>Statistical analysis showed that the volumes of 47 protein spots were altered after B[a]P treatment (P<0.05) and 23 proteins were successfully identified, including zinc finger proteins, SWI/SNF related protein, Bromo domain containing domain and other proteins.</p><p><b>CONCLUSION</b>These affected proteins may be involved in the cellular responses to B[a]P exposure, and may mediate the B[a]P induced mutagenesis and carcinogenesis.</p>

Activation of nucleus-independent signals triggered by N-methyl-N'-nitro-N- nitrosoguanidine / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To study the effect of MNNG on inducement of non-targeted mutation and activation of several cellular signal transduction pathways, and to determine whether the activation of these signaling pathways was dependent on the DNA-damage.</p><p><b>METHODS</b>Vero cells were enucleated by discontinuous density centrifugation. The PKA activities were measured by enzyme-linked immunosorbent assay. The status of cell membrane receptors was studied with immunofluorescent staining and confocal microscopy.</p><p><b>RESULT</b>In enucleated cytoplasts, MNNG-treatment increased PKA activity for about 2.3-fold in accordance with the 2.7-fold up-regulation of PKA activity in whole vero cells exposed to MNNG. The clustering of cell surface receptors of epidermal growth factor and tumor necrosis factor alpha was also observed in cells exposed to MNNG; this phenomenon was also found in enucleated cells.</p><p><b>CONCLUSION</b>The results indicate that the initiation of signal cascades induced by low concentration of MNNG might be associated with its interaction with cell surface receptors and/or direct activation of related signal proteins but not its DNA damage.</p>

Activation of transcription factors induced by low concentration of N-methyl-N'-nitro-N-nitrosoguanidine / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the effect of MNNG on some of the transcription factors such as NF- kappaB, CREB, AP-1 and c-Myc.</p><p><b>METHODS</b>The activities of these transcription factors were measured by transient transfection assay of SEAP vectors.</p><p><b>RESULT</b>The expressions of AP-1, CREB and NF- kappaB driven reporter genes were elevated for about 1.3, 1.4 and 1.3 times in MNNG-treated cells, respectively, as compared to untreated controls. However, the exposure of MNNG had no effect on the activity of c-Myc.</p><p><b>CONCLUSION</b>The activation of certain transcription factors might be involved in the process of untargeted mutation induced by low concentration of MNNG treatment.</p>

Cloning and bioinformatics of human REV3 gene promoter region and its response to carcinogen N-methyl-N'-nitro-N-nitrosoguanidine / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To understand the up regulatory mechanism of human REV3 gene induced by the chemical carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG).</p><p><b>METHODS</b>Bioinformatic analysis of human REV3 gene promoter region was based on BLAST alignment, promoter prediction software and recognition of transcriptional factor binding sites. Cloning of human REV3 gene promoter region was performed by nested PCR. Response of human REV3 gene promoter to the chemical carcinogen MNNG was measured by transient transfection assay based on the dual luciferase reporter assay system.</p><p><b>RESULT</b>Bioinformatic analysis showed that human REV3 gene promoter region was located on chromosome 6 PAC clone RP3-415N12, and that the hypothetical promoter region contained promoter sequences, rich CpG islands, and putative recognition sites for several transcriptional factors, including AP-1/c-Jun/c-Fos, AP-2, STAT, CREBP, and NF-kappaB. Reconstructed reporter plasmid pGL3- 2582 was established by inserting 2582 nucleotides from the promoter region into the luciferase reporter vector pGL3-Basic. Transient transfection assay showed the hypothetical REV3 promoter region had promoter function, and it responded to MNNG treatment (P<0.01).</p><p><b>CONCLUSION</b>Human mutator REV3 gene promoter region has been successfully cloned. The response of REV3 promoter region to MNNG suggests that REV3 gene can be regulated at transcriptional level under conditions of genotoxic stress.</p>

Establishment of a cell line with antisense-blocked POLH and the role of POLH in alkylating agent MNNG induced nontargeted mutagenesis / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the function of POLH(polymerase eta) through establishment of the POLH gene-blocked cell line FL-POLH(-).</p><p><b>METHODS</b>A mammalian expression vector expressing antisense POLH gene fragment pMAMneo-amp-POLHA (-) was constructed by cloning the 1473 - 2131 fragment of POLH gene into the mammalian expression vector pMAMneo-amp(-) in antisense orientation. The FL cells were transfected with this antisense RNA expressing vector and selected by G418. The mutation assay was conducted using the shuttle plasmid pZ189.</p><p><b>RESULT</b>The spontaneous mutation frequency of SupF tRNA gene in the plasmid replicated in the FL-POLH(-) was 13.5 x 10(-4), while it was 4.9x10(-4) and 3.7x10(-4) in the control cells FL and FL-M, respectively. The nontargeted mutation frequency of SupF tRNA gene decreased in the plasmid replicated in these cell lines pretreated with MNNG.</p><p><b>CONCLUSION</b>POLH plays an important role in maintenance of genetic stability and genesis of nontargeted mutation.</p>

Establishment of a HepG2 cell line stably expressing human cytochrome P450 1A2 and its metabolic activity / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To establish a HepG2 cell line stably expressing the human cytochrome P450 1A2 and to study its metabolic activity.</p><p><b>METHODS</b>The human wild-type CYP1A2 cDNA was subcloned into a mammalian expression vector pREP9. A transgenic cell line was established by transfecting the recombinant plasmid of pREP9-CYP1A2 to HepG2 cells. The expression of CYP1A2 mRNA was validated by RT PCR. The metabolic activation of HepG2 CYP1A2 cells on aflatoxin B1 (AFB1) was assayed by cytotoxicity test.</p><p><b>RESULT</b>The HepG2-CYP1A2 cells expressed CYP1A2 mRNA and could increase the cytotoxicity to AFB1 in comparison with that of wild type HepG2 cells.</p><p><b>CONCLUSION</b>The established HepG2-CYP1A2 can express the mRNA and has the metabolic activity to AFB1. The cell line may be useful for testing the toxicity and metabolism of xenobiotics, which might possibly be activated or metabolized by CYP1A2.</p>