Free radicals run in lizard families without (and perhaps with) mitochondrial uncoupling.

Free radicals run in lizard families without (and perhaps with) mitochondrial uncoupling Criscuolo & Bouillaud's (in press, hereafter 'CB') interesting comment on our recent papers on lizard reactive oxygen species (ROS) biology (Olsson et al. 2008a,b) is a nice demonstration of the complex mechanisms underlying the production and elimination of ROS (and other reactive molecules) and what makes them manifest at the net levels we measure. Before we respond to CB's central focus—ROS effects of mitochondrial uncouplers—we would like to point out that: (i) our paper (Olsson et al. 2008a) contains no information on mitochondrial uncoupling; it is a straightforward demonstration using flow cytome-try and fluorogenic probes that carotenoids do not depress ROS at normal electron transport chain (ETC) activity and (ii) more importantly, we demonstrate (Olsson et al. 2008b) that unmanipulated, basal level of super-oxide (i.e. net effect of production and antioxida-tion of superoxide, SO) has significant heritability (h 2 Z0.45G0.16, s.e., pZ0.012). Thus, chemical interference with ETC function is not necessary for detecting heritability for SO and, in this regard, CB's title is somewhat misleading. Considering that SO production is strongly influenced by maternally encoded mitochondrial genes (i.e. with very limited, or non-existent, recombina-tion), any other result would, in fact, have been remarkable although, to our knowledge, Olsson et al. (2008b) were the first to test this. We agree with the underlying logic of CB's take-home message—that mitochondrial uncoupling reduces SO production and prolongs lifespan (the 'uncoupling to survive' hypothesis). The biochemical mechanism by which carbonyl cyanide 3-chlorophenyl hydrazone (CCCP), usually referred to as an 'artifi-cial uncoupler', elevates ROS production was never a target in either of our studies. Furthermore, chemical uncouplers such as CCCP are known to both production in a dose-dependent and exposure-duration manner. At low doses, CCCP favours the entrance of protons into the mitochondrial matrix (uncoupling activity), whereas at high doses (more than or equal to 10 mM), CCCP inhibits respiration by acting at the Q-cycle, which triggers a higher production of SO. In yeast, another artificial uncoupler, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, increases ROS production at 5 mM with a subsequent reduction in cell lifespan (Stöckl et al. 2007). Thus, among-taxon variation in the dose-dependent effects of artificial uncouplers on ROS levels are apparent. Therefore, the concentration of CCCP that CB refers to, at which the ETC is fully uncoupled, and above which respiration starts to become inhibited, is likely to be taxon specific, and, …