Molecular dissection of dimethylnitrosamine (DMN)-induced hepatotoxicity by mRNA differential display.

Differential display reverse transcription-polymerase chain reaction (DDRT-PCR) was used to catalogue altered hepatic transcript expression during dimethylnitrosamine (DMN) exposure in vivo. Mice were administered DMN (1.5 or 5 mg/kg) or vehicle (phosphate-buffered saline) i.p. once daily for up to 7 days, and livers were collected 6 h post-injection. Total RNA was reverse transcribed and cDNA subsets were selectively amplified by PCR. DDRT-PCR products were fractionated on denaturing polyacrylamide gels, and differentially expressed bands were excised, reamplified, 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 7 alpha-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.