Cultured cells, intact tissues and organs

To determine cellnlar H2S consnmption rates, bolns additions of known H2S concentrations were added to the respirometer chamber with or without rat aorta smooth muscle 

FIGURE 8.9 Mitochondrial O2 and H2S consumption from non-limiting O2 to anoxic conditions, (a) Isolated mitochondria were exposed to repeated bouts of 12.5 pM H2S until anoxia was achieved, (b) At higher O2 levels, both O2 and H2S consumption events are coincident, but as the O2 levels decline the events become uncoupled and O2 consumption is limited first. The multiphasic kinetics of O2 consumption may result from transient inhibition of cytochrome c oxidase by H2S. Under anoxia, H2S consumption continues at a low level (after [36] reproduced with permission of the Company of Biologists). 

Consumption rates were dependent on H2S concentration but exhibited saturation kinetics. At low H2S concentrations, cellular consumption rates were approximately tenfold greater than spontaneous oxidation rates in the absence of cells or in the presence of heat-killed cells (Fig. 8.10). Flowever, at higher FI2S concentrations, the spontaneous H2S oxidation rate approximated the measured rate with cells, indicating that H2S consumption was inhibited at higher FI2S levels. 

H2S is both produced and consumed, and consumption pathways may be coupled to mitochondrial O2 consumption. Flowever, FI2S is also toxic to mitochondria, indicating that the steady-state cellular level is under tight control. To determine the FI2S 

FIGURE 8.10 H2S consumption in rat aorta smooth muscle cells (RASMCs). Accumulated data from several experiments showing RASMC H2S consumption rates (filled circles and squares) as a function of H2S concentration, compared to H2S oxidation rates in solution without cells (open circles and squares). Heat-inactivated RASMC H2S consumption rates (open plus symbols) were equivalent to background rates without cells. Inset Representative PHSS traces showing stepwise additions of Na2S stock, at arrows, in the presence (thin line) and absence (thick line) of RASMCs (after [41]). 

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Cultured and isolated cells 8.6.13 Intact tissues and organs... 

Tissue slices and whole organ culture maintain many spatial aspects of the intact tissue and, like whole cells, maintain the linkage between Phase I and Phase II enzymes (Sipes et al., 1987). Precision-cut tissue slices are more easily and reproducibly prepared than primary hepatocytes. However, viability limitations often restrict studies to a few hours duration. Histological examination of the material after exposure to a xenobiotic is possible with tissue slices. 

A number of chapters in this volume (especially Chapters 5 and 6) provide a more thorough discussion of osmotic adjustment by intact plants and tissues in response to environmental stress and the role of osmotically active solutes in this response. The following section focuses on the role of organic osmotica in the response of plant cells to salt stress. Cultured plant cells offer the opportunity to evaluate the effect of both internally synthesised and externally administered organic osmotica. 

In group I, we considered the question of obtaining an activity from a tissue or organ, from a biological fluid, or from cultured cells. The primary task in all these samples was the separation of the extracellular and cellular compartments. Next, the problem of separation of the different cell types within the cellular compartment was considered. In the section that follows, we will open the cell for a look inside. However, let us first consider briefly the surface of the intact cell and the problems associated with the assay of any activities that might be located there. 

The use of in vivo bioassays (e.g., hyperglycemia) as a measure of insulin in an intact animal or organ is largely historic because the hormones can be measured directly, but in vitro bioassays or bioassays using endocrine responsive tissue cell cultures can be useful in screening compounds. These assays can measure changes of adenylate cyclase, intracellular calcium, and phosphoinositol metabolites. 

Besides the tissue characteristics that are manifest as a result of normal differentiation, in man and other mammals there are scores of biochemical mutations, the phenotypes of which are expressed not only in the intact organism but also at the cellular level. The properties of these markers in cell cultures have been studied and previously reviewed (Klein, 1963 Harris, 1964 Gartler and Pious, 1966 Franks, 1968 Krooth et aL, 1968). 

Infectious materials include semen, vaginal secretions, cerebrospinal fluid, synovial fluid, pleural fluid, pericardial fluid, peritoneal fluid, amniotic fluid, saliva in dental procedures, any body fluid visibly contaminated with blood, and all body fluids in situations where it is difficult or impossible to differentiate between body fluids. They also include any unfixed tissue or organ, other than intact skin, from a human (living or dead) any human immunodeficiency virus- (HIV-) containing cell or tissue cultures organ culture and HIV or hepatitis- (HBV-) containing culture media or other solutions as well as blood, organs, or other tissues from experimental animals infected with HIV or HBV. 

As a molecule passes from tissue to tissue via the blood, it is exposed to hxmdreds of possible metabolic sites it can be bound to protein, fat, or other molecules in the serum it can be sequestered in certain organs or excreted via the kidneys, the colon, or even the breath, at rates and by mechanisms that we simply do not completely xmderstand. Faced with these facts, how can anyone state dogmatically that we can replace intact animals with computers, bacteria, cell cultures, etc. ... 

In vitro systems with varying levels of complexity have been used to study xenobiotic metabolism. These include purified proteins, subcellular fractions/cell lysates, intact whole cells, tissue slices and whole organ culture. Advantages and disadvantages of the approaches are summarized below. 

The artificial milieu imposed on the tissue. These drawbacks are less important if isolated organ systems are used merely for comparing the potency of different substances. The use of isolated cells offers a further simplification of the test system. Thus, quantitation of certain drug effects can be achieved with particular ease in cell cultures. A more marked reduction consists in the use of isolated subcellular structures, such as plasma membranes, endoplasmic reticulum, or lysosomes. With increasing reduction, extrapolation to the intact organism becomes more dif cult and less certain. 

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