Identification of Differentially Expressed Genes in Hepatic HepG2 Cells Treated with Acetaminophen Using Suppression Subtractive Hybridization

Abstract

Acetaminophen (N-acetyl-p-aminophenol; paracetamol; APAP) is a widely used drug for the treatment of pain and fever, but overdose produces severe hepatotoxicity in humans and experimental animals.1,2) APAP hepatotoxicity is due to its biotransformation to a reactive metabolite, N-acetyl-pbenzoquinone imine (NAPQI) by cytochrome P450 (CYP), leading to cellular glutathione depletion by NAPQI conjugation followed by covalent binding of NAPQI to cellular proteins.3,4) APAP toxicity was not observed in CYP1A2 and CYP2E1 double-null mice, indicating that the formation of the metabolites of APAP by the CYPs is a critical step in the initiation phase of the hepatotoxicity. 5) Thus, it is indisputable that the reactive metabolite of APAP plays an essential role for the initiation phase of APAP-induced hepatotoxicity. However, in the progression phase following initiation, there is almost no information besides the fact that chemokines are associated with hepatocytes regeneration following toxicity. 6)Since drug metabolism is impaired in patients with liver disease, the total amount of non-metabolized drug in the serum is increased in the patients with damaged liver. 7,8) In the case of hepatotoxicity induced by APAP, high level of non-metabolized APAP in the serum of the patients has been observed. 9) Therefore, evaluation of the effect of non-metabolized APAP on hepatocytes is required for full understanding of the progression phase of APAP-induced hepatotoxicity.There are several studies on the gene expression profiles associated with hepatotoxicity in vitro and in vivo. [10][11][12] These reports reveal the specific gene expression profiles from several types of hepatotoxins using microarray technology. The DNA microarray is a useful technology that was developed as a way to simultaneously evaluate the relative levels of large numbers of mRNA gene transcripts. However, the technology is limited by its insensitivity to transcripts of low abundance.13) In another experimental approach, suppression subtractive hybridization (SSH) was also employed to identify differentially regulated genes. This method makes it possible to detect some low abundance transcripts because of a normalization step to equalize the abundance of target in the subtracted cDNA population. 14,15) In the present study, to clarify the effect of APAP on hepatic cells, we investigated the gene increased in APAPtreated HepG2 cells, which show low metabolic activity for APAP. The identification of the differentially expressed genes was performed with the SSH technique and two genes that had changed their expression levels in APAP-treated HepG2 cells were determined. MATERIALS AND METHODSMaterials APAP was purchased from Merck Hoei (Osaka, Japan). Polymerase chain reaction (PCR) primers were purchased from Sigma Genosys (Ishikari, Japan). All other chemicals were of analytical grade or equivalent, and purchased commercially.Cell Culture Human hepatic HepG2 cells were purchased from American Type Tissue Collection (ATCC, Rockvill, MD, U.S.A.), and cultured ...

Identification of Differentially Expressed Genes Induced by Angiotensin Ii in Rat Cardiac Fibroblasts

“…The suppression PCR effect has been described as long, inverted terminal repeats, which can selectively suppress amplification of undesirable sequences in PCR procedures when attached to DNA fragments. Since its description by Diatchenko et al , 21 the SSH method has frequently been applied to identify differentially expressed genes in disease‐associated cells or conditions and to study gene expression in response to various cell culture conditions 23–27 . We have applied this technique to obtain differentially expressed cDNAs in rat cardiac fibroblasts induced by AngII.…”

“…Since its description by Diatchenko et al, 21 the SSH method has frequently been applied to identify differentially expressed genes in disease-associated cells or conditions and to study gene expression in response to various cell culture conditions. [23][24][25][26][27] We have applied this technique to obtain differentially expressed cDNAs in rat cardiac fibroblasts induced by AngII. Then, the differentially expressed cDNAs were cloned to create a rat cardiac fibroblast subtraction cDNA library.…”

Up-regulation of the lysyl hydroxylase 2 gene by acetaminophen and isoniazid is modulated by transcription factor c-Myb

“…Although researchers have reported on the pathogenic mechanism of addictive hepatic impairment, problems at the genetic, protein and cellular signalling levels have not been solved. We have previously reported that mRNA levels of lysyl hydroxylase 2 (LH2), thymidine synthetase and several genes involved in the mechanism of hepatic fibrosis are increased or decreased in HepG2 cells treated with acetaminophen, one of the drugs inducing hepatic impairment [2] . Solving common problems in hepatic fibrosis caused by drugs would be useful not only for the prevention of hepatic fibrosis in clinical therapy but also for developing a drug in the pre‐clinical stage.…”

“…found, in the case of hepatotoxicity induced by acetaminophen, high levels of non‐metabolized acetaminophen in the serum of the patients [4] . We therefore used HepG2 cells in which metabolic activation of drugs was reported to be generally weaker than that in normal liver cells [5] and it was previously found that the expression level of LH2 mRNA increased in HepG2 cells treated with acetaminophen [2] …”

Avian influenza virus infection induces differential expression of genes in chicken kidney

“…A PCR-based cDNA subtraction technique, named as suppression subtractive hybridization (SSH), has been successfully applied as a powerful tool to identify differentially expressed genes in many areas (Zhao et al, 2005;He et al, 2005;Iguchi et al, 2005). To examine whether HPAIV infection could induce differential expression of kidney genes, we constructed two subtractive cDNA libraries from uninfected and infected kidneys using SSH, and characterized their gene expressions by dot blot and DNA sequencing.…”