Primary cultures of human hepatocytes were used to investigate whether the dietary isothiocyanates, sulforaphane (SFN), and phenethyl isothiocyanate (PEITC) can reduce DNA adduct formation of the hepatocarcinogen aflatoxin B(1) (AFB). Following 48 h of pretreatment, 10 and 50 microM SFN greatly decreased AFB-DNA adduct levels, whereas 25muM PEITC decreased AFB-DNA adducts in some but not all hepatocyte preparations. Microarray and quantitative reverse transcriptase (RT)-PCR analyses of gene expression in SFN and PEITC-treated hepatocytes demonstrated that SFN greatly decreased cytochrome P450 (CYP) 3A4 mRNA but did not induce the expression of either glutathione S-transferase (GST) M1 or GSTT1. The protective effects of SFN required pretreatment; cotreatment of hepatocytes with SFN and AFB in the absence of pretreatment had no effect on AFB-DNA adduct formation. When AFB-DNA adduct formation was evaluated by GST genotype, the presence of one or two functional alleles of GSTM1 was associated with a 75% reduction in AFB-DNA adducts, compared with GSTM1 null. In conclusion, these results demonstrate that the inhibition of AFB-DNA adduct formation by SFN is dependent on changes in gene expression rather than direct inhibition of catalytic activity. Transcriptional repression of genes involved in AFB bioactivation (CYP3A4 and CYP1A2), but not transcriptional activation of GSTs, may be responsible for the protective effects of SFN. Although GSTM1 expression was not induced by SFN, the presence of a functional GSTM1 allele can afford substantial protection against AFB-DNA damage in human liver. The downregulation of CYP3A4 by SFN may have important implications for drug interactions.
This study is, to the best of our knowledge, the first application of whole transcriptome sequencing (RNA-seq) to cells isolated from postmortem human brain by laser capture microdissection. We investigated the transcriptome of dentate gyrus (DG) granule cells in postmortem human hippocampus in 79 subjects with mental illness (schizophrenia, bipolar disorder, major depression) and nonpsychiatric controls. We show that the choice of normalization approach for analysis of RNA-seq data had a strong effect on results; under our experimental conditions a nonstandard normalization method gave superior results. We found evidence of disrupted signaling by miR-182 in mental illness. This was confirmed using a novel method of leveraging microRNA genetic variant information to indicate active targeting. In healthy subjects and those with bipolar disorder, carriers of a high- vs those with a low-expressing genotype of miR-182 had different levels of miR-182 target gene expression, indicating an active role of miR-182 in shaping the DG transcriptome for those subject groups. By contrast, comparing the transcriptome between carriers of different genotypes among subjects with major depression and schizophrenia suggested a loss of DG miR-182 signaling in these conditions.
To examine the character and variability of human cytochrome P450 (CYP) and microsomal epoxide hydrolase (mEH) gene expression in human blood cells, we used a highly sensitive, quantitative, competitive reverse transcriptase-coupled polymerase chain reaction (QC RT-PCR) assay to assess mRNA profiles for a battery of 8 genes, in peripheral lymphocytes isolated from 10 healthy donors. Of the genes profiled, in lymphocytes CYP2D6 was typically expressed at the highest levels (3.8 x 10(5) molecules/microg total RNA), with CYP2E1 and mEH also maintained at relatively high abundance (1.2 x 10(5) and 1.8 x 10(5) molecules/microg total RNA, respectively). CYP1A1 levels were approximately an order of magnitude lower (3.9 x 10(4) molecules/microg total RNA), followed by CYP2F1 and CYP3A levels that were near the detection limit of the assay. CYP1A2 and CYP2A6/7 mRNAs were not detected in any of the lymphocyte samples. Overall, relatively low levels of inter-individual variation (2- to 6-fold) existed among these endpoint parameters in the subjects tested. To test whether established human blood cell lines were suitable models to assess basal expression and chemical induction responsiveness of these genes, we determined that constitutive CYP and mEH mRNA profiles were essentially conserved across 4 established human blood cell lines, and highly analogous to the basal expression patterns identified in freshly isolated peripheral lymphocytes. mEH protein was detected in all of the cell lines using Western immunoblotting and chemiluminescent visualization, whereas CYP1A1, CYP2D6, CYP2E1 or CYP3A proteins were not detected in these analyses. When blood cell-derived cultures were exposed to the prototypical CYP1A and CYP3A inducers, i.e., beta-naphthoflavone (beta-NF), dexamethasone (DEX) or phenobarbital, generally little or no inductive response was manifested. Thus, the data obtained from this investigation indicate that, although human blood cell lines in general exhibit poor responsiveness to prototypical inducer exposures, the constitutive patterns of CYP and mEH expression in peripheral lymphocytes appear to exhibit relatively low levels of variation among individuals. In addition, these in vivo patterns of expression are well maintained in established cultured blood-cell lines.
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