Effect of glutathione depletion and inhibition of glucuronidation and sulfation on 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) metabolism, PhIP-DNA adduct formation and unscheduled DNA synthesis in primary rat hepatocytes

The potent rat colon carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), unlike other food-borne heterocyclic amines, does not induce tumors in rat liver. This correlates with an extremely low level of PhIP-DNA adducts formed in this tissue, and together these observations suggest that PhIP is efficiently detoxified in the liver. In order to identify possible detoxification mechanisms, we assessed the effect of inhibition of glucuronidation, glutathione (GSH) conjugation and sulfation on PhIP metabolism and PhIP-induced DNA damage in rat hepatocytes. Hepatocytes isolated from rats pretreated with Aroclor 1254 metabolized PhIP to the same products found in vivo. N-Hydroxy-PhIP N3-glucuronide and N-hydroxy-PhIP N2-glucuronide were major and minor metabolites respectively. 32P-Postlabeling analysis of DNA from the PhIP-treated hepatocytes indicated the presence of two major adducts, one of which was identified as N-(deoxyguanosin-8-yl)-PhIP, and one minor adduct. There was no unscheduled DNA synthesis (UDS) in these cells. However, pretreatment of the hepatocytes with 1-bromoheptane and buthionine sulfoximine, which depletes GSH and prevents its resynthesis, resulted in a 15-fold increase in the formation of PhIP-DNA adducts, as well as in a high level of UDS. GSH depletion had no effect on the formation of detectable PhIP metabolites. Hepatocyte pretreatment with D-galactosamine, which inhibits glucuronidation, increased the formation of DNA adducts two-fold and UDS was increased similarly. D-Galactosamine decreased the formation of the two N-glucuronides of N-hydroxy-PhIP by 50-60%, but had no effect on other metabolites. Pentachlorophenol, which strongly inhibits sulfotransferases, decreased adduct formation slightly, but had essentially no effect on UDS or on the formation of PhIP metabolites. These results indicate that metabolic conjugation pathways involving GSH and glucuronidation may play an important role in protecting rat liver against PhIP carcinogenesis.     

Secretion and mesoderm-inducing activity of the TGF-beta-related domain of Xenopus Vg1

Vg1 is a maternal mRNA localized to the vegetal hemisphere of Xenopus embryos during blastula stages, a region responsible for the induction of mesoderm in the adjacent marginal zone. Its homology to the transforming growth factor-beta family, which includes several proteins with mesoderm-inducing activity, suggests a role for Vg1 as an endogenous mesoderm-inducing factor. However, expression of Vg1 protein in the animal hemisphere, following injection of synthetic mRNA, has no effect on development, and isolated animal caps are not mesodermalized. It is shown that Vg1 protein fails to form dimers and is not processed to release the putative bioactive domain. Furthermore it is shown that the N-terminal signal peptide of Vg1 is not cleaved following translocation into the ER, which may explain the failure of this protein to dimerize. To explore the role of Vg1 in amphibian development, a fusion protein has been made of the preproregion of Xenopus bone morphogenetic protein-4 and the putative bioactive C-terminal domain of Vg1. This fusion protein forms dimers and the C-terminal domain of Vg1 is secreted. Injection of this construct into Xenopus embryos induces the formation of a second dorsal axis and isolated animal caps are mesodermalized. The results are consistent with a role for Vg1 in mesoderm induction during Xenopus development.     

Assessing the clinical ethical competence of undergraduate medical students

At the University of Newcastle, health law and ethics is taught and assessed in each year of the five-year curriculum. However, the critical question for assessment remains: 'Does teaching ethics have a measurable effect on the clinical activity of medical students who have had such courses?' Those responsible for teaching confront this question each year they sit down to construct their assessment tools. Should they assess what the student knows? Should they assess the student's moral reasoning, that is, what decisions the student makes, and, how these decisions are justified, or should they assess what the student actually does when dealing with patients in the clinical setting, and how he or she does it? From 1982 to 1991, assessment at Newcastle was primarily aimed at determining the quality of the students' ethics knowledge base. This paper describes the strengths and limitations of a purely knowledge-based method of evaluation and why in 1992, we are now attempting to redefine and assess, what we call 'clinical ethical competence' in terms of how students actually apply this knowledge base in a controlled clinical context.     

Chronic administration of serotonergic antidepressants attenuates the subjective effects of LSD in humans

This study investigates the possible interactions of antidepressant agents and hallucinogens in humans through structured interviews using a standardized questionnaire. Volunteer subjects recruited through announcements placed on the Internet or other sources were asked to describe the somatic, hallucinatory, and psychological effects of self-administered LSD prior to and during chronic administration of an antidepressant. Twenty-eight out of 32 subjects (88%) who had taken an antidepressant with inhibitory effects on serotonin (5-HT) reuptake (fluoxetine, paroxetine, sertraline, trazodone) for over 3 weeks had a subjective decrease or virtual elimination of their responses to LSD. An additional subject who had taken fluoxetine for only 1 week had an increased response to LSD. These data are in contrast to our previous study that reported increased responses to LSD during chronic administration of tricyclic antidepressants or lithium. Possible mechanisms of action for the effects from serotonergic antidepressants involve 5-HT2 and 5-HT1A receptors, changes in extracellular brain serotonin concentrations, and changes in brain catecholamine systems.     

Concentrations of trypsin inhibitor and immunoglobulins in colostrum of Jersey cows

Colostrum samples from 49 Jersey cows were analyzed for concentrations of trypsin inhibitor, IgG, IgM, IgA, TS, fat, specific gravity, and N fractions. Colostrum (100 ml) was sampled from each cow as soon as possible after parturition. Mean concentrations of IgG, IgM, and IgA were 84.6, 3.4, and 4.5 g/L, respectively. Mean concentration of trypsin inhibitor was 56 mg of trypsin inhibited/dl of colostrum. Concentration of trypsin inhibitor was unaffected by lactation number and averaged 60, 53, and 54 mg of trypsin inhibited/dl of colostrum for cows in first, second, and third or later lactations, respectively. Colostral trypsin inhibitor and IgG were correlated (.54), although correlations between trypsin inhibitor and IgM and IgA were not significant. Trypsin inhibitor in colostrum was also positively correlated with fat, total N, protein N, noncasein N, and TS in colostrum. Variation in concentration of trypsin inhibitor from first-milking colostrum was closely related to colostral IgG concentration and may serve to protect IgG and other proteins from proteolytic degradation in the intestine of the neonatal calf.     

Paclitaxel inhibits arterial smooth muscle cell proliferation and migration in vitro and in vivo using local drug delivery

BACKGROUND: The antineoplastic compound paclitaxel (Taxol) causes an increased assembly of extraordinarily stable microtubules. The present study was designed to characterize the effects of paclitaxel on proliferation and migration of human arterial smooth muscle cells (haSMCs) in vitro and on neointima formation in an in vivo experimental rabbit model. METHODS AND RESULTS: Both monocultures of haSMCs and cocultures with human arterial endothelial cells (haECs) were used. Cell growth after 4, 8, and 14 days was determined in the absence or presence of platelet-derived growth factor-AB (PDGF-AB), basic fibroblast growth factor (bFGF), or thrombin. Nonstop paclitaxel exposure, as well as single-dose applications of paclitaxel for 24 hours or even 20 minutes (0.1 to 10.0 micromol/L), caused a complete and prolonged inhibition of haSMC growth up to day 14, with an IC50 of 2.0 nmol/L. Mitogens or cocultures with stimulating haECs did not significantly attenuate paclitaxel-induced effects. Immunohistochemistry showed characteristic cytoskeletal changes predominantly in the microtubule network. Additionally, in 20 male New Zealand White rabbits, intimal plaques were produced by electrical stimulation. In 10 animals, paclitaxel was locally applied by use of microporous balloons. Histologically, the intima wall area, wall thickness, and degree of stenosis were reduced significantly in paclitaxel-treated animals compared with controls. CONCLUSIONS: Our data show that paclitaxel inhibits haSMC proliferation and migration in a dose-dependent manner in monocultures and cocultures even in the presence of mitogens. Furthermore, paclitaxel prevents neointima formation in rabbits after balloon angioplasty. The long-lasting effect after just several minutes' exposure time makes this lipophilic substance a promising candidate for local antiproliferative therapy of restenosis.