Structural Requirements for the Direct and Cytochrome P450-dependent Reaction of Cyclic α,β-Unsaturated Carbonyl Compounds with Glutathione: a Study with Coumarin and Related Compounds

Abstract— The interaction of glutathione (GSH) with coumarin, or one of a series of compounds related to coumarin, was assessed in the absence and presence of liver microsomes (direct reaction and indirect reaction, respectively) to determine the structural requirements for direct and mono-oxygenase-mediated reaction of cyclic α,β-unsaturated carbonyls with GSH. Acrolein was used as a positive control for the direct reaction, and produced complete or nearly complete depletion of GSH under all assay conditions. 5,6-Dihydro-2H-pyran-2-one and 2-cyclohexen-1-one also produced substantial depletion of GSH in the direct reaction, which was not increased by the addition of liver microsomes. Coumarin, 2H-pyran-2-one and precocene I (a substituted pyran lacking the 2-one structure) were not substrates for the direct reaction but did cause depletion of GSH when incubated in the presence of rat or human liver microsomes. These depletions were dependent on a functioning mono-oxygenase system as judged by the effects of omission of cofactors, addition of competitive or inactivating inhibitors of cytochrome P450, and induction. Dihydrocoumarin, ?-valerolactone, cyclohexanone and 4H-pyran-4-one were not substrates for either the direct or indirect reaction. These findings are rationalized on the basis of a direct nucleophilic attack of GSH on the α,β-centre of the α,β-unsaturated carbonyl compounds, which is hindered by benzenoid resonance in coumarin and 2H-pyran-2-one, for which enzyme-mediated reaction with GSH, probably via a 3,4-epoxide, is the favoured mechanism.     

Shoulder instability: impact of glenohumeral arthrotomography on treatment

We used arthrotomography to study the glenoid labrum in 114 patients. Sixty-nine of the patients had anatomic instability of the shoulder (including recurrent dislocation and subluxation of the shoulder), and 45 patients had functional instability of the shoulder (denoted by chronic pain, clicking of the joint, and the sensation that an unstable condition exists without the objective signs of it). Labral tears were revealed arthrotomographically in 86% of the patients with anatomic instability, while only 40% of the patients with functional instability had labral abnormalities, and these were primarily of minor severity. Fifty-six patients (44 of whom had anatomic instability; 12, functional instability) required surgery. The surgical findings were correlated with the arthrotomographic findings, and no false-positive results were revealed. However, arthrotomography demonstrated only part of the pathologic condition of two patients. These results confirm that there is a strong correlation between labral pathologic conditions and anatomic instability of the shoulder. Arthrotomographic studies have a great impact on the selection of therapy in cases of both anatomic and functional instability of the shoulder.     

Covalent Binding of Inhaled Formaldehyde to DNA in the Nasal Mucosa of Fischer 344 Rats: Analysis of Formaldehyde and DNA by High-Performance Liquid Chromatography and Provisional Pharmacokinetic Interpretation

Covalent Binding of Inhaled Formaldehyde to DNA in the NasalMucosa of Fischer 344 Rats: Analysis of Formaldehyde and DNAby High-Performance Liquid Chromatography and Provisional PharmacokineticInterpretation. CASANOVA, M., DEYO, D. F., AND HECK, H. D'A.(1989). Fundam Appl. Toxicol. 12, 397–417. Inhalationof ³HCHO and H¹⁴CHO(6 ppm, 6 hr) resulted in the formation ofDNA-protein crosslinks in the rat nasal respiratory mucosa.The DNA was extracted and was fractionated into aqueous (AQ)and interfacial (IF) portions. AQ DNA and IF DNA were enzymaticallyhydrolyzed to deoxyribonucleosides in Tris buffer and analyzedby HPLC with liquid scintillation counting (LSC). HCHO was boundexclusively to the IF DNA, indicating that the HCHO was boundas DNA-protein crosslinks. Hydrolysis of the DNA quantitativelyreleased the HCHO; no evidence was obtained for the formationof hydroxymethyl adducts. An adduct detected previously followingincubation of mammalian cells with HCHO, N⁶-hydroxymethyldeoxyadenosine(hm6dA)[Beland F. A., Fullerton, N. F., and Heflich, R. H. (1984) J.Chromartogr. 308 121–131], was shown to be produced byreaction of HCHO with deoxyadenosine (dA) in bis-Tris bufferunder conditions similar to those used for hydrolysis of theDNA. This reaction does not occur in Tris buffer. Evidence wasobtained that most or all of the hm6dA observed can be explainedby this reaction. Based on these results, an improved methodto determine the amount of H¹⁴CHO bound to DNA was developed:the DNA is hydrolyzed in Tris buffer and analyzed by HPLC, andthe released H¹⁴CHO is derivatized with dimedone and quantitatedby LSC. Rats were exposed to a wide range of H¹⁴CHO concentrations(0.3, 0.7, 2, 6, or 10 ppm; 6 hr). DNA-protein crosslinkingoccurred at all concentrations. The formation of crosslinkswas interpreted in terms of a nonlinear pharmacokinetic modelincorporating oxidation of inhaled HCHO as a defense mechanism.The slope of the fitted concentration-response curve at 10 ppmis 7.3-fold greater than at 0.3 ppm, and the fitted detoxicationpathway is half-saturated at an airborne concentration of 2.6ppm.     

A 90-Day Inhalation Toxicity Study of Raw Shale Oil in Fischer 344 Rats

A 90-Day Inhalation Toxicity Study of Raw Shale Oil in Fischer344 Rats. GORDON T., STROTHER, D. E., CRAMER, D. V., AND GOODE,J. W. (1987). Fundam. Appl. Pharmacol. 9, 287–296. Thepotential health effects of a raw shale oil were evaluated ina 90-day inhalation study in Fischer 344 rats. Groups of 15male and 15 female rats were exposed 6 hr/day, 5 days/week for13 weeks to aerosol concentrations of 0, 56, 120, or 492 mg/m³.In the high-dose group, 10 males and 7 females died prior tothe termination of the study, most within the first 5 weeksof the experiment. A dose-dependent suppression in weight gainwas seen in all of the shale oil-exposed groups. The failureto gain weight was associated with a variety of clinicopathologicabnormalities, including a dose-related decrease in red andwhite blood cells, with lowered plasma protein levels and increasedserum alkaline phosphatase, and with total bilirubin levelsin males. The exposure of the test animals to aerosolized rawshale oil was also associated with inflammatory and hyperplasticlesions in the lungs and upper respiratory tract, atrophy ofthe thyrnus and thymic-dependent portions of the peripherallymphoid system, and bone marrow. These changes demonstratethat inhalation of raw shale oil aerosol can produce major organtoxicity similar to that found after exposure to other unrefinedoil products.     

Concept Mapping: An Effective Instructional Strategy for Diet Therapy

Concept mapping is an instructional strategy that requires learners to identify, graphically display, and link key concepts in instructional reading material. Although proven effective in numerous disciplines as a means to promote critical thinking and self-directed learning, concept mapping has not been tested in diet therapy. The objective of this study was to implement concept mapping as a small-group, cooperative learning strategy in an upper-division diet therapy course and to evaluate student attitudes about the effect of concept mapping on knowledge, self-directed learning, problem-solving, and collaborative skills. Students in the first semester (n=27) initially learned course material by lecture (4 weeks) followed by an integrated mapping/lecture format (12 weeks); the second semester (n=25) used an integrated mapping lecture format for the full 16 weeks. At the end of both semesters, students completed a 10-item original survey questionnaire. Responses for first (n=25) and second (n=21) semesters were analyzed independently. Results indicated that a majority of students thought participation in concept mapping enhanced knowledge of diet therapy principles (n=19 of 25; 18 of 21), self-directed learning (n=14 of 25; 18 of 21), critical thinking (n=21 of 25; 14 of 21), problem-solving (n=22 of 25; 16 of 21), and collaboration (n=24 of 25; 20 of 21) skills. When noncooperation of teammates was a factor, concept mapping was viewed as more frustrating and time consuming than lecture. This study demonstrated concept mapping as an effective learning strategy for diet therapy; it improves students’ ability to engage in self-directed learning, critical thinking, collaboration, and creative problem solving. Results suggest that concept mapping is most effective when accompanied with comprehensive training, coordinated lectures, instructor guidance, and long-term practice. J Am Diet Assoc. 1995; 95:908–911.     

Electrotonic Inhibition and Active Facilitation of Excitability in Ventricular Muscle

Inhibition and Facilitation in Cardiac Muscle. Introduction: The effects of subthreshold electrical pulses on the response to subsequent stimulation have been described previously in experimental animal studies as well as in the human heart. In addition, previous studies in cardiac Purkinje fibers have shown that diastolic excitability may decrease after activity (active inhibition) and, to a lesser extent, following subthreshold responses (electrotonic inhibition). However, such dynamic changes in excitability have not been explored in isolated ventricular muscle, and it is uncertain whether similar phenomena may play any role in the activation pal-terns associated with propagation abnormalities in the myocardium. Methods and Results: Experiments were performed in isolated sheep Purkinje fibers and papillary muscles, and in enzymatically dissociated guinea pig ventricular myocytes. In all types of preparations introduction of a conditioning subthreshold pulse between two subthreshold pulses was followed by a transient decay in excitability (electrotonic inhibition). The degree of inhibition was directly related to the amplitude and duration of the conditioning pulse and inversely related to the postconditioning interval. Yet, inhibition could be demonstrated long after (> 1 sec) the end of the conditioning pulse. Electrotonic inhibition was found at all diastolic intervals and did not depend on the presence of a previous action potential. In Purkinje fibers, conditioning action potentials led to active inhibition of subsequent responses. In contrast, in muscle cells, such action potentials had a facilitating effect (active facilitation). Electrotonic inhibition and active facilitation were observed in both sheep ventricular muscle and guinea pig ventricular myocytes. Accordingly, during repetitive stimulation with pulses of barely threshold intensity, we observed: (1) bistability (i.e., with the same stimulating parameters, stimulus: response patterns were either 1:1 or 1:0, depending on previous history), and (2) abrupt transitions between 1:1 and 1:0 (absence of intermediate wenckebach-like patterns). Simulations utilizing an ionic model of cardiac myocytes support the hypothesis that electrotonic inhibition in well-polarized ventricular muscle is the result of partial activation of I_k following subthreshold pulses. On the other hand, active facilitation may be the result of an activity-induced decrease in the conductance of I_K1. Conclusion: Diastolic excitability of well-polarized ventricular myocardium may be transiently depressed following local responses and transiently enhanced following action potentials. On the other hand, diastolic excitability decreases during quiescence. Active facilitation and electrotonic inhibition may have an important role in determining the dynamics of excitation of the myocardium in the presence of propagation abnormalities.