Animals were kept at room temperature of between 22 and 25 °C receiving standard diet 1324 (Altromin, Lage, Germany) and tap water ad libitum. A light and dark cycle of 12 h and a relative air humidity of between 50% and 60% were maintained in the animal room. Closed glass-spheres (63 l) were used for exposing animals to BD. The exposure system is described in detail in Filser et al. (2007). Groups of mice or rats were exposed to mean atmospheric BD concentrations (±standard deviation) of 1.0 (±0.17), 6.4 (±0.65), 11 (±1.2), 21 (±1.8), 63 (±6.8), 108 (±8.1), 311 (±24), 603 (±35), or 1180 (±101) ppm (mice) and of 1.1 (±0.20), 2.4 (±0.67), 5.6 (±1.1), 11 (±1.1), 21 (±1.1), 33 (±2.5), 62 (±8.9), 106

(±6.0), 203 (±11), 624 (±36), or 1220 (±47) ppm (rats). During the exposure experiments, Roxadustat in vitro atmospheric BD concentrations were determined at varying time periods of between 6 and 14 min and were maintained PLX-4720 mouse quasi-constant by repeatedly injecting gaseous BD (taken directly from

the gas cylinder or as a diluted gas from a storage desiccator) to replenish the losses of BD in the gas-tight spheres, which resulted from metabolic elimination and from opening the chamber for placing or removing an animal. At each exposure experiment with mice, two groups of six animals each (tail-marked by different colors) were placed with an interval of 25 min into one chamber. In experiments with rats, 4 individually tail-marked animals were successively put into one chamber at time intervals of 20 min. Rat exposures were carried out twice at BD concentrations of 1.1, 5.6, and 11 ppm. Each animal was exposed for 6.0 h. Mice were sacrificed by cervical dislocation. Using a disposable, heparin sodium-moistened syringe, up to 0.5 ml of blood was taken from the vena cava caudalis (near to the heart) of each animal of a group and injected – one after the other – in one ice-cooled 5-ml-cryotube vial (Simport, Beloeil, Quebec, Canada) that contained 40 μl of an ethanolic solution of the glutathione depleting agent DEM (515 μl DEM in 2760 μl ethanol) and 10 μl (1.0 ADP ribosylation factor and 6.4 ppm

BD) or 30 μl (11–1180 ppm BD) of the internal standard DEB-D6 (14.5 μmol/l in acetone). The vial was shaken after each blood injection. The whole procedure of pooling the blood of the 6 mice per group lasted not more than 6 min. Rats were treated according to Lee et al. (2005). Twenty minutes before sacrificing a rat, it was removed from the sphere and immediately anesthetized by injecting intraperitoneally a mixture consisting of 0.88 ml ketamine/kg body weight and 1.1 ml Rompun/kg body weight. Directly thereafter, the anesthetized animal was returned into the exposure sphere. Within 5 min, the target concentration was readjusted by compensating for the amount of BD being lost. At the end of the exposure, the anesthetized animal was removed from the sphere and sacrificed immediately.