Incubation Systems

Potentially mineralizable C and N are often measured by incubating a sample of field-moist soil at a known temperature in a sealed chamber containing an alkali trap. The C02-C accumulated in the trap is measured by acid titration and this represents the quantity of C mineralized. Alternatively, C02 in the headspace of the incubation chamber can be measured using a C02 analyser. The amount of N mineralized during incubation is calculated as the difference in extractable NH4+ - andNCV-N measured in the soil before and after incubation. Mineralizable N can also be measured in an open incubation system where the soil is leached periodically and NH4+- andNCV-N in leachates is measured (Stanford 1982). 

Bartsch, H., Camus, A.-M. and Malaveille, C. (1976). Comparative mutagenicity of N-nitrosamines in a semi-solid and in a liquid incubation system in the presence of rat or human tissue fractions. Mutation Res. 37 149-162. 

Mechanism-based CYP inhibition is time dependent, as it is a consequence of metabolic activation. For the same reason, it also depends on substrate concentration. It also, usually, depends on the presence of NAD PH in the incubation system (or an NADPH regeneration system), as most of the CYP-catalyzed reactions require NADPH to occur [5],... 

Previously, hver shces were incubated in static organ cultures [1]. Hart et cd. [49] cultured rat hver slices for 24 h spread out on wet filter paper, floating on top of the incubation medium. Several slice-containing vessels were placed in a box with saturated 95% O2 and 5% CO2 at 37°C. However, the shces employed were rather thick (approximately 0.3 mm) and only the upper cell layers (0.2 mm) in the slice contained viable ceUs. Together with the introduction of the Krumdieck sheer [5,46], a new incubation technique for shces, the dynamic organ culture system (DOC), was introduced [35]. The main characteristic of this system is the intermittent exposure of the shce to incubation medium and the gas phase. The DOC is in fact a modified version of the Trowell incubation system [1]. 

Various incubation systems have also been tested using human liver shces, these inclnde the 24-well plates with magnetic stirrers [38,66,69], the six-well plates in a shaker [52] and the DOC roller system [70,71], but no direct comparison has been made as yet. Human hver slices can be cultured for 72 h in DOC and maintain their ability to respond to specific inducers of cytochrome P450 such asmethylclofenapate and Aroclor 1254 [71]. 

Finally, inter-laboratory standardization of incubation systems and culture media would increase the validity of comparisons made between results from different laboratories. 

Reiter R, Burk RF. 1988. Formation of glutathione adducts of carbon tetrachloride metabolites in a rat liver microsomal incubation system. Biochem Pharmacol 37 327-331. 

Since the introduction of the Krumdieck slicer (Krumdieck 1980) and a new incubation system for slices (Smith 1985) tissue slices are commonly used in drug metabolism and toxicity studies. 

De Kanter R, Koster HJ (1995) Cryopreservation of rat and monkey liver slices. ATLA 23 653-665 Dogterom P (1993) Development of a simple incubation system for metabolism studies with precision-cut liver slices. Drug Metab Dispos 21 699-704... 

Incorporation of ruptured erythrocytes into the incubation system, at equimolar concentrations of oxyhaemoglobin to 15-HPETE, extensively protected the membrane polyunsaturated fatty acids from lipid peroxidation... 

The complete incubation system contained isolated mitochondria resuspended in 0.25 M sucrose to a final concentration of 0.5 mg/ml protein. Light samples were exposed to an incandescent light source (a bank of 50 watt G.E. reflector bulbs) with an intensity of about 15 mW/cm for 2 hr. Six ml samples were incubated in a slowly shaking water bath at 34° C small aliquots were removed for catalase assays. Samples run in duplicate. 

Figure 9.6 The rate of dopamine formation using the homogenate of rat cerebral cortex as enzyme at 37°C. The incubation system contained 0.5 mg of rat cerebral cortex. (From Nagatsu et al., 1979.)...

Figure 9.15 The rate of adrenaline formation using a homogenate of rat hypothalamus as enzymes at 37°C. Standard incubation system containing 10 mg of tissue was used. (From Trocewicz et al., 1982.)...

Superoxide generated by xanthine oxidase or in the redox cycling of paraquat can cause the reductive release of F3 from ferritin, a process that is dependent on the activity of microsomal NADPH-cytochrome P-450 reductase [119]. Iron appears to be an essential component in the formation of reactive species such as superoxide and hydroxyl radical via redox cycling of cephaloridine. Addition of EDTA or of the specific iron chelator desferrioxamine to an incubation system containing renal cortex microsomes and cephaloridine depressed cephaloridine-induced peroxidation of microsomal lipids significantly EDTA showed a weaker effect than desferrioxamine at equimolar concentrations. By chelating F3 preferentially [120], desferrioxamine reduced the availability of F2 produced by the iron redox cycle and decreased cephaloridine-stimu-lated peroxidation of membrane lipids [36, 37]. 

Soil transformation experiments were conducted in a flow-through incubation system [7 ]. A 2-1 flask with a side-trap was connected by silicone tubing to a 250-ml... 

Olinga P, Groen K, Hof IH, De Kanter R, Koster HJ, Leeman WR, Rutten AA, Van Twillert K, Groothuis GM (1997) Comparison of five incubation systems for rat liver slices using functional and viability parameters. J Pharmacol Toxicol Methods 38(2) 59-69... 

ISO 14239 1997 Soil quality - Laboratory incubation systems for measuring the mineralization of organic chemicals in soil under aerobic conditions (available in English only). 

---