DZF-Project: Doris Höschele

2004

Investigating Mitochondrial Toxicity of HIV-Nucleoside Reverse Transcriptase Inhibitors in ovo

Doris Höschele, M.I. Garcia Moreno, M. Wiertz, H. Enzmann
Bundesinstitut für Arzneimittel und Medizinprodukte, Kurt-Georg-Kiesinger-Allee 3, 53175 Bonn, Germany

hoeschel(at)bfarm.de

Begin of the Project: 2004

ABSTRACT
Nucleoside Reverse Transcriptase Inhibitors (NRTIs) are the cornerstone of HIV-therapy. These agents suppress HIV-replication by blocking HIV reverse transcriptase. NRTIs can also be substrates for mitochondrial (mt) DNA polymerase g leading to depletion and mutation of mtDNA with subsequent mitochondrial dysfunction.

Mitochondrial toxicity is strongly suggested to be responsible for many adverse effects, like severe lactic acidosis, hepatosteatosis, (cardio-) myopathy, pancreatitis, neuropathy, and probably lipoatrophy, observed in HIV-infected patients receiving NRTI-bound therapy. Furthermore, mitochondrial toxicity in children exposed in-utero/postnatal to NTRIs has raised concern recently.

Until now, no standardized and general accepted in vitro model exists for the investigation of mitochondrial toxicity. Currently used in vitro models have many limitations with regard to their clinical relevance. As such, in most cases results from in vitro studies have to be confirmed by in vivo studies using specialized animal models. There is a need for the development of further non-animal models of mitochondrial toxicity applicable for the investigation of NRTI-induced mitochondrial toxicity.

The aim of the project is to develop an in ovo model for the investigation of mitochondrial toxicity using different NRTIs as test substances. Mitochondrial toxicity is investigated in mitochondria from erythrocytes obtained from incubated hen’s eggs, which, in contrast to mammalian erythrocytes, are nucleated and contain mitochondria. Between days 6 to 8 of incubation, test substances are administered into egg albumen or into the air cell. At day 11, the end of the first half of the incubation period, blood is obtained from blood vessels of the chorioallantoic membrane (CAM), a non-innervated extraembryonal membrane. Quantification of mtDNA as a marker of mitochondrial toxicity is conducted by developing a new real-time PCR method. Mitochondrial function is investigated by determination of blood lactate and measurement of ATP content in erythrocytes.

The in ovo model is a simple, sensitive, and rapidly responding non-animal model fully in line with animal protection regulations and ethical aspects.