In vitro models for biotransformation and

In Vitro Models for Biotransformation and Toxicity Studies in Farm Animals... 

In the past decades, there has been a large increase in the development and use of in vitro models, not only because of the demand of animal welkre groups to reduce the use of laboratory animals, but also because of a number of scientific and financial advantages. Especially in the case of small laboratory animals, a number of different models have become available for biotransformation and toxicity studies, e,g, perfused organs, tissue slices, isolated cells (e,g, hepatocytes), cellular fractions (e.g. microsomes), and purified enzymes. For a number of reasons (see below) isolated hepatocytes have become one of the most popular in vitro models for such studies. 

There are several ways of doing confirmatory in vivo studies. The most common involves measuring the clearance of a model substrate that is principally dependent on one CYP enzyme for its biotransformation before and after the addition of the potential inhibitor ( 37). The substrate may be given as a single test dose or dosed to steady state. Steady-state dosing is preferable because it more closely corresponds to the clinical situation. The inhibitor should be given at the clinically relevant dose and administered for a sufficient interval to assure that steady-state has been achieved, because the degree of inhibition is a function of the concentration of the inhibitor and any of its relevant metabolites, as well as their in vitro potency for inhibition. 

LC-MS-based procedures for chiral and enantioselective analysis of mammalian samples are discussed in the Sects. 3.2 and 3.2.2 [47-50], Detailed data on biotransformation in vivo especially in man are quite rare. LC-ESI MS/MS procedures are referred that allowed metabolite identification in animals in in vivo and in vitro models [51, 52],... 

Isolated renal cell components (cytosol, organelles, membranes) are also commonly used in vitro systems. Renal microsomes and cytosol are useful in examining the renal biotransformation and bioactivation of nephrotoxicants. Since mitochondria are frequently targets for nephrotoxicants, isolated renal mitochondria are also an important model system for determining the toxic mechanism(s) of some compounds. Also, the direct effects of toxicants on renal cell membranes can be studied in vesicles prepared from either the luminal (brush border) or basolateral (antiluminal, peritubular) membrane of renal cortical cells. The use of isolated cell components is helpful in answering specific questions about... 

Comparative Metabolism. Since the liver is the major organ involved in the biotransformation of xenobiotics, primary hepatocyte cultures provide an excellent model for in vitro metabolism studies. Primary hepatocyte cultures provide useful tools with which to study the comparative metabolism of xenobiotics by both humans and laboratory animals. 

Because of the possible effects of active and carrier-mediated processes and metabolic biotransformation, the issue of tissue viability is important for in vitro buccal mucosal experiments. The barrier nature of the buccal mucosa resides in the upper layers of the epithelium, where unlike in the stratum corneum, the cells contain a variety of functional organelles [119, 122, 125, 150], and so tissue viability may be an important component of the barrier function of the tissue. Various methods have been employed to assess the viability of excised buccal mucosa, including measurement of biochemical markers, microscopic methods, and linearity of transport data [42], While biochemical methods, including measurement of adenosine 5 -triphosphate (ATP) levels and utilization of glucose, provide information on the metabolic activity of the tissue, this does not necessarily relate to the barrier function of the tissue. In excised rabbit buccal mucosa, levels of ATP were measured and found to decline by 40% in 6 h, and this correlated well with transmission electron microscopic evaluation of the tissue (intact superficial cells) [32], In addition, the permeability of a model peptide was unaltered up to 6 h postmortem, but at 8 h, a significant change in permeability was observed [32], These investigators therefore claimed that excised rabbit buccal mucosa could be used for diffusion studies for 6 h. 

Calu-3 cells have shown the ability to perform fatty acid esterification of budes-onide [132], In pre-clinical studies, this esterification results in a prolonged local tissue binding and efficacy, which is not found when the esterification is inhibited [133]. The precise mechanism remains undefined in that the identity of specific enzyme(s) responsible for this metabolic reaction is unclear [134], Assessment of the potential toxicity and metabolism of pharmaceuticals and other xenobiotics using in vitro respiratory models is still at its infancy. The development of robust in vitro human models (i.e., cell lines from human pulmonary origin) has the potential to contribute significantly to better understanding the role of biotransformation enzymes in the bioactivation/detoxication processes in the lung. 

In order to use PBTK modeling in the assessment of mixtures, Cassee et al. (1998) suggest that one of the components is first modeled and regarded as the prime toxicant being modified by the other components. Based on in vitro data on the other components, effects of, e.g., inhibition or induction of specific biotransformation isoenzymes can be incorporated in the model. Effects of competition between chemicals in a mixture for the same biotransformation enzymes may also be incorporated by translating the effects into effects on the Michaelis-Menten parameters that are then incorporated into the model. 

Computer-modeling-based QSARs for analysing experimental data on biotransformation and toxicity, Toxicol, in Vitro, 15, 539-551, 2001. 

The non- and mono-ortho CBs have been quantitated accurately in the principal source, namely, commercial PCB mixtures [15, 16] additional environmental sources such as incineration have been identified [17,18] their presence in every ecosystem including the pristine polar regions has been shown [19,20] estimates of their flux in air, water, soil, and the removal mechanisms such as OH reactions in atmosphere and sediment burial in rivers and oceans have been proposed [21] their microbial degradation and biotransformation in organisms have been studied [22,23] a battery of in-vitro and in-vivo bioassays using mammalian, avian, and piscian models for the benefit of risk-assessment of these CBs have been developed [24]. Studies like these in the last decade have resulted in a new awareness of these important class of industrial contaminants. 

Often, PBPK models for toxicokinetics application require special considerations (e.g., volatile toxicants may incur tissue-air partition coefficients and alveolar elimination rates). Partition coefficients are generally obtained by measurement in the laboratory, tissue volume/blood flow data are mostly available from the scientific literature (with allometric scaling from species to species), and biotransformation data are usually obtained from in vivo and in vitro kinetic studies. Biochemical constants for metabolic pathways are captured using the maximum rate of reaction, or Vmax5 and the binding affinity of the particular substrate for the metabolizing enzyme. 

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