Differential display reverse transcription-polymerase chain reaction (DDRT-PCR) was used to catalogue altered hepatic transcript expression during dimethylnitrosamine (DMN) exposure in vive. Mice were administered DMN (1.5 or 5 mg/kg) or vehicle (phosphate-buffered saline) ip once daily for up to 7 days, and livers were collected 6 h post-injection. Total RNA was reverse transcribed and eDNA subsets were selectively amplified by PCR. DDRT-PCR products were fractionated on denaturing polyacrylamide gels, and differentially expressed bands were excised, re-amplified, and subsequently cloned into a plasmid vector. This study identified 23 cDNAs that were induced and 25 cDNAs that were suppressed during DMN exposure. Altered expression during DMN exposure for cDNA clones was confirmed by Northern blotting, RNase protection, or in situ hybridization analyses. DNA sequence information indicated that four cDNAs suppressed during DMN exposure encode cytochrome P450 isoenzyme-cholesterol 7α-hydroxylase (CYP7), a monokine, a myeloid cell differentiation protein, and mouse major urinary protein (MUP). We further observed a DMN-induced increase in transcripts for complement factor 3 (C3) and serum amyloid A (SAA). In contrast, the remaining differentially expressed transcripts detected by DDRT-PCR during DMN exposure demonstrated no similarity to sequences present in Genbank, suggesting that they may encode previously unreported gene products. In situ hybridization showed MUP transcripts to be expressed by hepatic centrilobular areas that undergo necrosis during subchronic DMN exposure. Thus, the utilization of DDRT-PCR has identified several differentially expressed hepatic mRNAs associated with various doses and stages of DMN exposure.
Bibliographical noteFunding Information:
The authors acknowledge the technical assistance of M. S. Abrahamsen and A. Schroeder and thank D. Fraser and T. L. Horn for critical reading of this manuscript. This work was supported in part by National Institutes of Health Grant ES-04348.
- Cholesterol 7-α hydroxylase
- Differential display
- Major urinary protein
- Serum amyloid A