Quantitative analysis of gene expression changes in response to genotoxic compounds

Abstract

Techniques that quantify molecular endpoints sufficiently sensitive to identify and classify potentially toxic compounds have wide potential for high-throughput in vitro screening. Expression of three genes, RAD51C, TP53 and cystatin A (CSTA), in HEPG2 cells was measured by Q-PCR amplification. In parallel, we developed alternative assays for the same 3 gene signature based on an acridinium-ester chemiluminescent reporter molecule. HEPG2 cells were challenged with eighteen different compounds (n=18) chosen to represent compounds that are genotoxic (n=8), non-genotoxic non-carcinogenic (n=2) or have a less well defined mechanism of action with respect to genotoxicity (n=8). At least one of the three genes displayed dysregulated expression in the majority of compounds tested by Q-PCR and ten compounds changed the CSTA expression significantly. Acridinium-ester labelled probes for the three genes were synthesised and tested. Analytical sensitivity was characterised and suggested a limit of detection generally better than 0.1fmol but often 10-50 attomol. A linear amplification step was optimised and this quantitative method detected statistically significant increases in RAD51C and CSTA expression in agreement with the Q-PCR results, demonstrating the potential of this technology. The broad agreement of the amplified chemiluminescent method and Q-PCR in measuring gene expression suggests wider potential application for this chemiluminescent technology.

Evaluation and comparison of the hepatoprotective effects of trimetazidine and lovastatin against doxorubicin-induced hepatotoxicity

“…15,27 Statins, other than lovastatin, that are lipophilic are also being successfully studied for the protective benefits against DOX toxicity. 9,28 It's important to know that statin administration in cancer regimen, as is the case here, would be very low in dose and duration than for their use as anti-lipid agents. They actually appear to be related to a very low risk of liver damage.…”

“…Morphological changes in liver due to DOX have also been observed in various animal studies. 4,9 The mechanism of DOX hepatotoxicity includes the inhibition of topoisomerase II causing interference with DNA unwinding and subsequent inhibition of DNA replication. 24 Generation of oxygen free radicals is also a crucial cause of hepatocyte fragility and enzyme release.…”

“…8 Till date, several pharmacologic agents with potent antioxidants properties have been tested against the hepatotoxicity of DOX. 3,7,9 Although most of these agents were confirmed to be beneficial in the animal studies, their extrapolation in clinical trials has been implausible and none is FDA approved till date. Trimetazidine (TMZ) is an effective anti-anginal agent that improves the contractile response of chronically dysfunctional myocardium.…”