Limits...
Caenorhabditis elegans as a Model System for Studying Drug Induced Mitochondrial Toxicity.

de Boer R, Smith RL, De Vos WH, Manders EM, Brul S, van der Spek H - PLoS ONE (2015)

Bottom Line: The continuous intake of antiretroviral drugs however, leads to severe and even life-threatening side effects, supposedly by the deleterious impact of nucleoside-analogue type compounds on the functioning of the mitochondrial DNA polymerase.For detailed investigation of the yet partially understood underlying mechanisms, the availability of a versatile model system is crucial.Using a combination of molecular-biological and functional assays, combined with a quantitative analysis of mitochondrial network morphology, we conclude that anti-retroviral drugs with similar working mechanisms can be classified into distinct groups based on their effects on mitochondrial morphology and biochemistry.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biology & Microbial Food Safety, Swammerdam Institute for Life Sciences (SILS), Faculty of Science (FNWI), University of Amsterdam, Science Park 904, 1098 XH, Amsterdam, The Netherlands.

ABSTRACT
Today HIV-1 infection is recognized as a chronic disease with obligatory lifelong treatment to keep viral titers below detectable levels. The continuous intake of antiretroviral drugs however, leads to severe and even life-threatening side effects, supposedly by the deleterious impact of nucleoside-analogue type compounds on the functioning of the mitochondrial DNA polymerase. For detailed investigation of the yet partially understood underlying mechanisms, the availability of a versatile model system is crucial. We therefore set out to develop the use of Caenorhabditis elegans to study drug induced mitochondrial toxicity. Using a combination of molecular-biological and functional assays, combined with a quantitative analysis of mitochondrial network morphology, we conclude that anti-retroviral drugs with similar working mechanisms can be classified into distinct groups based on their effects on mitochondrial morphology and biochemistry. Additionally we show that mitochondrial toxicity of antiretroviral drugs cannot be exclusively attributed to interference with the mitochondrial DNA polymerase.

No MeSH data available.


Related in: MedlinePlus

Concentration dependent decrease of mtDNA.Synchronised L1 worms were put on a plate with FLT and experiments were performed after 72hrs of continuous exposure. In FLT exposed animals, the reduction of mtDNA is concentration dependent. Error bars represent 95% CI (df = 16). Significance was determined using a two-tailed student’s T test assuming unequal variances. P-value was <0.001 for all reported concentrations.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4430419&req=5

pone.0126220.g001: Concentration dependent decrease of mtDNA.Synchronised L1 worms were put on a plate with FLT and experiments were performed after 72hrs of continuous exposure. In FLT exposed animals, the reduction of mtDNA is concentration dependent. Error bars represent 95% CI (df = 16). Significance was determined using a two-tailed student’s T test assuming unequal variances. P-value was <0.001 for all reported concentrations.

Mentions: In order to validate the use of C. elegans as a model for drug induced mitochondrial dysfunction we first needed to establish whether the side effects that occur in humans can also be seen when exposing C. elegans to NRTIs, the backbone of HAART. The most obvious effect of NRTIs in patients is the reduction of mtDNA copy numbers[29]. To assess this in C. elegans we set up a quantitative PCR assay for mtDNA using the conserved mitochondrial DNA encoded COX1 gene as a target (see Materials and Methods). In a typical experiment, the nematodes were synchronized and consequently exposed to fixed concentrations of NRTIs for 72 hrs. Drugs were either mixed in with the bacterial lawn or added directly to the agar plates. Both methods were effective and are reported previously[30,31]. Since no data was available for uptake and sensitivity of C. elegans for this class of drugs we started with the NRTI 3’-deoxy-3’-Fluorothymidine (alovudine or FLT) as a benchmark. FLT is no longer used to treat HIV-1 infected individuals because of its well-known severe mitochondria related toxicity in patients[32]. Exposing the nematodes to FLT resulted in a significant decrease in mtDNA copy number (Fig 1). This decline is concentration dependent, starting from an absolute control value of 3,8x106 copies per worm. Maximal reduction to 10% of the control was reached at a concentration of 100 μM FLT. Relative quantification of mtDNA copies per nDNA (qPCR of the nuclear actin gene as described previously[33] gave identical results (S1 Table).


Caenorhabditis elegans as a Model System for Studying Drug Induced Mitochondrial Toxicity.

de Boer R, Smith RL, De Vos WH, Manders EM, Brul S, van der Spek H - PLoS ONE (2015)

Concentration dependent decrease of mtDNA.Synchronised L1 worms were put on a plate with FLT and experiments were performed after 72hrs of continuous exposure. In FLT exposed animals, the reduction of mtDNA is concentration dependent. Error bars represent 95% CI (df = 16). Significance was determined using a two-tailed student’s T test assuming unequal variances. P-value was <0.001 for all reported concentrations.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4430419&req=5

pone.0126220.g001: Concentration dependent decrease of mtDNA.Synchronised L1 worms were put on a plate with FLT and experiments were performed after 72hrs of continuous exposure. In FLT exposed animals, the reduction of mtDNA is concentration dependent. Error bars represent 95% CI (df = 16). Significance was determined using a two-tailed student’s T test assuming unequal variances. P-value was <0.001 for all reported concentrations.
Mentions: In order to validate the use of C. elegans as a model for drug induced mitochondrial dysfunction we first needed to establish whether the side effects that occur in humans can also be seen when exposing C. elegans to NRTIs, the backbone of HAART. The most obvious effect of NRTIs in patients is the reduction of mtDNA copy numbers[29]. To assess this in C. elegans we set up a quantitative PCR assay for mtDNA using the conserved mitochondrial DNA encoded COX1 gene as a target (see Materials and Methods). In a typical experiment, the nematodes were synchronized and consequently exposed to fixed concentrations of NRTIs for 72 hrs. Drugs were either mixed in with the bacterial lawn or added directly to the agar plates. Both methods were effective and are reported previously[30,31]. Since no data was available for uptake and sensitivity of C. elegans for this class of drugs we started with the NRTI 3’-deoxy-3’-Fluorothymidine (alovudine or FLT) as a benchmark. FLT is no longer used to treat HIV-1 infected individuals because of its well-known severe mitochondria related toxicity in patients[32]. Exposing the nematodes to FLT resulted in a significant decrease in mtDNA copy number (Fig 1). This decline is concentration dependent, starting from an absolute control value of 3,8x106 copies per worm. Maximal reduction to 10% of the control was reached at a concentration of 100 μM FLT. Relative quantification of mtDNA copies per nDNA (qPCR of the nuclear actin gene as described previously[33] gave identical results (S1 Table).

Bottom Line: The continuous intake of antiretroviral drugs however, leads to severe and even life-threatening side effects, supposedly by the deleterious impact of nucleoside-analogue type compounds on the functioning of the mitochondrial DNA polymerase.For detailed investigation of the yet partially understood underlying mechanisms, the availability of a versatile model system is crucial.Using a combination of molecular-biological and functional assays, combined with a quantitative analysis of mitochondrial network morphology, we conclude that anti-retroviral drugs with similar working mechanisms can be classified into distinct groups based on their effects on mitochondrial morphology and biochemistry.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biology & Microbial Food Safety, Swammerdam Institute for Life Sciences (SILS), Faculty of Science (FNWI), University of Amsterdam, Science Park 904, 1098 XH, Amsterdam, The Netherlands.

ABSTRACT
Today HIV-1 infection is recognized as a chronic disease with obligatory lifelong treatment to keep viral titers below detectable levels. The continuous intake of antiretroviral drugs however, leads to severe and even life-threatening side effects, supposedly by the deleterious impact of nucleoside-analogue type compounds on the functioning of the mitochondrial DNA polymerase. For detailed investigation of the yet partially understood underlying mechanisms, the availability of a versatile model system is crucial. We therefore set out to develop the use of Caenorhabditis elegans to study drug induced mitochondrial toxicity. Using a combination of molecular-biological and functional assays, combined with a quantitative analysis of mitochondrial network morphology, we conclude that anti-retroviral drugs with similar working mechanisms can be classified into distinct groups based on their effects on mitochondrial morphology and biochemistry. Additionally we show that mitochondrial toxicity of antiretroviral drugs cannot be exclusively attributed to interference with the mitochondrial DNA polymerase.

No MeSH data available.


Related in: MedlinePlus