Reduction in mitochondrial membrane peroxidizability index…
617
2. MATERIALS AND METHODS
Animals and diets
Six week-‐old male Wistar rats were obtained from Harlan Laboratories
(Unide, Italy). The animals were caged individually and maintained in a 12:12
(light-‐dark) cycle, 22ºC ± 2ºC and 50% ± 10% relative humidity at the animal
house of the Complutense University. Sixteen male rats were fed ad libitum with a
standard rodent diet (Panlab, Spain) and were divided into two groups of 8
animals each: CONTROL and ATENOLOL. The animals in the atenolol group had
free access to a 1 g/L atenolol solution (Sigma, A7655) in the drinking water.
Previous studies have used that dose of atenolol to cause blockade of β-‐adrenergic
receptors in rats (30). The control group had free access to the same aliquot of
drinking water without atenolol. The mean water intake per animal per day was
not significantly different between both groups. After 2 weeks of treatment, the
animals were sacrificed by decapitation. Hearts were immediately processed to
isolate functional mitochondria, which were used to measure mitochondrial
respiration and rates ROS generation rates. The remaining mitochondria were
stored at -‐80ºC for posterior analyses. These experiments in Wistar rats were
approved by the Animal Experimental Committee from the Complutense
University. The present investigation conforms to the Directive 2010/63/EU of the
European Parlament.
Isolation of functional mitochondria, oxygen consumption and ROS production
Mitochondria were obtained from fresh tissue by the procedure of Mela and
Seitz (31) with modifications. After checking the functionality and phosphorylation
capacity of the mitochondria (high respiratory control ratios) the rate of mtROSp
was measured by the fluorometric method established at our laboratory (32). The
oxygen consumption rate of heart mitochondria was measured at 37ºC in a water-‐
thermostatized incubation chamber with a computer-‐controlled Clark-‐type O2
electrode (Oxygraph, Hansatech, UK).
Oxidative damage to mtDNA (8-‐oxodG)
Isolation of mtDNA was performed by the method of Latorre and cols (33)
adapted to mammals (34)
.
The isolated mitochondrial DNA was digested to
deoxynucleoside level and the level of oxidative damage in mtDNA was estimated
by measuring the amount of 8-‐oxo-‐7,8-‐dihydro-‐2’deoxyguanosine (8-‐oxodG)
referred to that of the non-‐oxidized base (deoxyguanosine, dG) by HPLC-‐EC as
previously described (35).
Measurement of mitochondrial complexes I, II, III and IV, AIF, MnSOD, SIRT3
and SIRT5
The amounts of a) the mitochondrial respiratory chain complexes (I to IV),
the complex I regulatory factor AIF, the mitochondrial biogenesis protein
