Oxygen uptake of isolated toad skin epithelium: micromeasurement and effect of ionic acclimation.

Oxidative metabolism of isolated toad skin epithelium (Bufo viridis) was investigated in vitro under open-circuit conditions using the spectrophotometric oxyhemoglobin micromethod. This highly sensitive technique has been adapted for studying several epithelia in parallel and for detecting possible regional variations of oxygen uptake in individual epithelium. Changes in the proportion of mitochondria-rich cells (MRC) by ionic acclimation affected oxidative metabolism under nontransporting condition. After acclimation of animals to either NaNO3 or NaCl solutions (100 mmol/l, for greater than 2 wk), the number of MRC per square millimeter in epithelia from nonacclimated and NaNO3- and NaCl-acclimated animals was 350 +/- 113, 460 +/- 196, and 107 +/- 52, respectively. O2 uptake of nonacclimated and NaNO3-acclimated epithelia was significantly higher than that of NaCl-acclimated epithelia (i.e., 0.89 and 0.90 vs. 0.57 nmol O2.h-1.mm-2, respectively). The correlation established between O2 uptake and number of MRC allowed evaluation of the respiration rate of one single MRC, i.e., approximately 1 pmol O2/h. The lowest mitochondrial oxidative activity was found in the epithelia from NaCl-acclimated toads where the uncoupler 2,4-dinitrophenol (50 mumols/l) had the highest relative stimulatory effect (+114%). Acetazolamide (50 mumols/l), a potent inhibitor of carbonic anhydrase mainly present in the MRC, reduced selectively by 31% O2 uptake of the MRC-rich epithelia (NaNO3 acclimated). O2 uptake increased significantly by approximately 80% when basolateral pH increased from 5.8 to 7.8, but did not depend on apical pH. These findings indicate that under nontransporting (open-circuit) conditions, aerobic metabolism of the isolated toad skin epithelium is related to the density and/or characteristics of the MRC.(ABSTRACT TRUNCATED AT 250 WORDS)

CO2 production of the chick embryo during the first day of post-laying development.

The carbon dioxide production of the chick embryo cultured in vitro has been determined during the first 24 h of post-laying development using a non-invasive conductometric microtechnique. The mean CO2 production of the whole blastoderm (1) increased from 16 nmol/h at laying to 231 nmol/h at early neurulation, (2) became dependent on exogenous glucose and (3) was closely linked to mechanical tension generated in the blastoderm (loosening from vitelline membrane resulted in a decrease of 56%). In our experimental conditions, no significant influence of carbonic anhydrase on the CO2 production has been detected. The value of the respiratory exchange ratio varied from about 3 at pregastrular stages to 1 at neurula stage and CO2 was produced transiently in presence of antimycin A. Such results indicate that the source of CO2 is not exclusively mitochondrial and that the relative proportions of mitochondrial and non-mitochondrial CO2 productions might vary significantly throughout the early development. Our findings confirm that the metabolism of the chick embryo becomes more and more oxidative from laying onwards and suggest that the modifications of metabolism observed during the studied period of development could be associated with functional differentiation.

Mitochondrial uncoupling as a regulator of life-history trajectories in birds: an experimental study in the zebra finch.

Mitochondria have a fundamental role in the transduction of energy from food into ATP. The coupling between food oxidation and ATP production is never perfect, but may nevertheless be of evolutionary significance. The 'uncoupling to survive' hypothesis suggests that 'mild' mitochondrial uncoupling evolved as a protective mechanism against the excessive production of damaging reactive oxygen species (ROS). Because resource allocation and ROS production are thought to shape animal life histories, alternative life-history trajectories might be driven by individual variation in the degree of mitochondrial uncoupling. We tested this hypothesis in a small bird species, the zebra finch (Taeniopygia guttata), by treating adults with the artificial mitochondrial uncoupler 2,4-dinitrophenol (DNP) over a 32-month period. In agreement with our expectations, the uncoupling treatment increased metabolic rate. However, we found no evidence that treated birds enjoyed lower oxidative stress levels or greater survival rates, in contrast to previous results in other taxa. In vitro experiments revealed lower sensitivity of ROS production to DNP in mitochondria isolated from skeletal muscles of zebra finch than mouse. In addition, we found significant reductions in the number of eggs laid and in the inflammatory immune response in treated birds. Altogether, our data suggest that the 'uncoupling to survive' hypothesis may not be applicable for zebra finches, presumably because of lower effects of mitochondrial uncoupling on mitochondrial ROS production in birds than in mammals. Nevertheless, mitochondrial uncoupling appeared to be a potential life-history regulator of traits such as fecundity and immunity at adulthood, even with food supplied ad libitum.

Cost-effective experimental design to support modeling of concentration-response functions

Modeling concentration-response function became extremely popular in ecotoxicology during the last decade. Indeed, modeling allows determining the total response pattern of a given substance. However, reliable modeling is consuming in term of data, which is in contradiction with the current trend in ecotoxicology, which aims to reduce, for cost and ethical reasons, the number of data produced during an experiment. It is therefore crucial to determine experimental design in a cost-effective manner. In this paper, we propose to use the theory of locally D-optimal designs to determine the set of concentrations to be tested so that the parameters of the concentration-response function can be estimated with high precision. We illustrated this approach by determining the locally D-optimal designs to estimate the toxicity of the herbicide dinoseb on daphnids and algae. The results show that the number of concentrations to be tested is often equal to the number of parameters and often related to the their meaning, i.e. they are located close to the parameters. Furthermore, the results show that the locally D-optimal design often has the minimal number of support points and is not much sensitive to small changes in nominal values of the parameters. In order to reduce the experimental cost and the use of test organisms, especially in case of long-term studies, reliable nominal values may therefore be fixed based on prior knowledge and literature research instead of on preliminary experiments