David B. Mitchell

Evaluation of cytotoxicity in cultured cells by enzyme leakage

The measurement of cell injury or cytotoxicity produced by chemicals or toxicants in cell culture has routinely been evaluated by such criteria as plating efficiency, cell growth, protein or DNA synthesis, and cell viability. We describe a more sensitive and earlier index of cytotoxicity: leakage of cytoplasmic enzymes from injured cells into the culture medium.

An in vitro approach to the study of target organ toxicity of drugs and chemicals

SummaryA major goal of our laboratory has been the development of primary culture systems that retain differentiated fucntions and responses characteristic of intact tissues in vivo. Specifically, we have developed cellular models of primary cultures of rat heart, liver, and kidney cells to explore the mechanisms by which drugs or chemicals may be toxic to key organs of the body and to develop new techniques by which xenobiotics may be evaluated or identified as potential toxicants to living systems. The purpose of this paper is to describe our rationale and approach to the study of target organ toxicology with in vitro cellular systems.

Evaluation of the cytotoxicity of tricyclic antidepressants in primary cultures of rat hepatocytes.

Primary cultures of hepatocytes from postnatal Sprague-Dawley rats were grown in arginine-deficient, ornithine-supplemented medium to inhibit fibroblastic overgrowth and to selectively isolate relatively pure cultures of parenchymal hepatocytes. This system of primary cultures of rat hepatocytes was utilized to evaluate the cytotoxicity of certain tricyclic antidepressant drugs (TCAs). The compounds tested were chosen to represent two distinct chemical classifications of TCAs: the dibenzazepine derivatives, imipramine (1) and desipramine (D), and the dibenzocycloheptadiene derivatives, amitriptyline (A) and nortriptyline (N). The study also allowed direct comparison of the parent tertiary amines, A and I, and their respective demethylated pharmacologically active metabolites, N and D. The hepatotoxicity of the compounds was determined by measuring leakage of cytoplasmic enzymes, lactate dehydrogenase (LDH) and glutamic-pyruvic transaminase (GPT), into the culture medium and by assessing cell viability by the trypan blue dye exclusion test. LDH leakage was a more sensitive index of early cellular injury in this study. The compounds demonstrated a dose- and time-dependent order of toxicity; their hepatotoxicity potency was ranked as A = N greater than D greater than I.

Metabolic activation and cytotoxicity of cyclophosphamide in primary cultures of postnatal rat hepatocytes

Abstract We have developed a model of primary cultures of postnatal rat hepatocytes to characterize the metabolic activation of xenobiotics to toxic intermediates and to study the mechanism(s) by which these chemicals produce cellular injury. This model was employed to investigate the cytochrome P-450 mediated biotransformation of cyclophosphamide (CP) to cytotoxic metabolites that nonspecifically alkylate DNA and cellular proteins. The parenchymal cells were isolated by an in situ collagenase perfusion technique and cultured for 24 hr prior to drug treatment. The cultures were then exposed to CP concentrations ranging from 1 × 10 −4 M to 1 × 10 −3 M for 24 hr. Initial studies indicated minimal toxicity to non-replicating parenchymal hepatocytes maintained in ornithine-supplemented, arginine-deficient medium. The addition of arginine permitted the overgrowth of fibroblasts in the same culture system. These fibroblasts then became the target of alkylating CP metabolites produced by the par-enchymal cells. By day 3 after CP administration, cell number and total protein per dish decreased by over 40 percent. The morphology of the cultures changed dramatically because of fibroblast destruction. The cytotoxicity to dividing fibroblasts was eliminated by administering 2-diethylaminoethyl-2, 2-diphenylvalerate hydrochloride (SKF 525-A), an inhibitor of the cytochrome P-450 monooxygenase system, to the co-cultures treated with CP. The alkylating metabolites of CP produced by the parenchymal cells and released into the culture medium were quantitated by reacting aliquots of medium from CP-treated cells with 4-( p -nitrobenzyl)pyridine. These results provide both direct and indirect evidence of drug metabolism in cultured cells and suggest that this co-culture system can be utilized to evaluate the metabolic activation of xenobiotics.

The Long-Range P3 Helix of the Tetrahymena Ribozyme Is Disrupted during Folding between the Native and Misfolded Conformations

RNAs are prone to misfolding, but how misfolded structures are formed and resolved remains incompletely understood. The Tetrahymena group I intron ribozyme folds in vitro to a long-lived misfolded conformation (M) that includes extensive native structure but is proposed to differ in topology from the native state (N). A leading model predicts that exchange of the topologies requires unwinding of the long-range, core helix P3, despite the presence of P3 in both conformations. To test this model, we constructed 16 mutations to strengthen or weaken P3. Catalytic activity and in-line probing showed that nearly all of the mutants form the M state before folding to N. The P3-weakening mutations accelerated refolding from M (3- to 30-fold) and the P3-strengthening mutations slowed refolding (6- to 1400-fold), suggesting that P3 indeed unwinds transiently. Upon depletion of Mg(2+), the mutations had analogous effects on unfolding from N to intermediates that subsequently fold to M. The magnitudes for the P3-weakening mutations were larger than in refolding from M, and small-angle X-ray scattering showed that the ribozyme expands rapidly to intermediates from which P3 is disrupted subsequently. These results are consistent with previous results indicating unfolding of native peripheral structure during refolding from M, which probably permits rearrangement of the core. Together, our results demonstrate that exchange of the native and misfolded conformations requires loss of a core helix in addition to peripheral structure. Further, the results strongly suggest that misfolding arises from a topological error within the ribozyme core, and a specific topology is proposed.

Catalytic activity as a probe of native RNA folding.

As RNAs fold to functional structures, they traverse complex energy landscapes that include many partially folded and misfolded intermediates. For structured RNAs that possess catalytic activity, this activity can provide a powerful means of monitoring folding that is complementary to biophysical approaches. RNA catalysis can be used to track accumulation of the native RNA specifically and quantitatively, readily distinguishing the native structure from intermediates that resemble it and may not be differentiated by other approaches. Here, we outline how to design and interpret experiments using catalytic activity to monitor RNA folding, and we summarize adaptations of the method that have been used to probe aspects of folding well beyond determination of the folding rates.

Lack of cytotoxicity of ticrynafen in primary cultures of rat liver cells

Primary cultures of hepatocytes obtained from neonatal Sprague-Dawley rats were grown in arginine-deficient, ornithine-supplemented medium to inhibit fibroblastic overgrowth and to selectively isolate relatively pure cultures of parenchymal hepatocytes. This system of primary hepatocytes was used to study the potential cytotoxicity of ticrynafen by measuring cytoplasmic enzyme leakage, cell viability,, and total protein per culture dish. Hepatic cultures were treated with the drug in concentrations ranging from 10(-3)M to 10(-6)M and for durations from 2 to 8 h. The results of the study indicate that ticrynafen was minimally toxic to the hepatocytes.

Approaching a persistent favorskii zwitterion

Abstract 2-Phenalanone has been synthesized via a double Curtius procedure and converted to the conjugate acid of 2-phenalenylium oxide, a potential persistent Favorskii zwitterion.