Rodolfo Rodriguez

Mitotic index and cell proliferation kinetics for identification of antineoplastic activity

The mitotic index (MI) and cell proliferation kinetics (CPK) of human blood lymphocyte cultures were determined to evaluate the effects of six antineoplastic drugs with well known cytostatic activity: cisplatin, melphalan, bleomycin, methotrexate, 5-fluorouracil and 6-mercaptopurine. All six drugs showed a clear effect on the inhibition of MI. The first three drugs interact directly with DNA showing a dose-related retardation of CPK. Methotrexate, 5-fluorouracil and 6-mercaptopurine, which act on ribonucleotide biosynthesis, showed no significant effects on CPK. The results suggest that CPK and MI measurements are useful for the prescreening of drugs with potential cytostatic activity.

Rapid anxiolytic activity of progesterone and pregnanolone in male rats

The effect of different doses of progesterone (1.0, 3.0, 10.0, 30.0, and 100.0 mg/kg) and pregnanolone (1.0, 3.0, 10.0, and 30.0 mg/kg) upon burying defensive and elevated plus-maze (EPM) tests was investigated in adult male rats and compared with the effects of diazepam (0.25. 0.50, 1.0, and 2.0 mg/kg). All drugs were suspended in a 0.2% methylcellulose solution and administered intraperitoneally 30 min prior to testing. Progesterone and pregnanolone were found to produce anxiolytic-like effects similar to those of diazepam. Thus, at certain doses, both drugs significantly increased the latency for burying and decreased the cumulative burying behavior, without modifying the number of shocks, and increased the time spent in the open arms of the maze, without affecting the spontaneous locomotor activity. These data clearly demonstrate that the defensive burying paradigm is useful to detect the anxiolytic-like properties of pregnanolone. An important finding was that progesterone produces significant behavioral effects 30 min after its administration. This finding suggests a rapid bioconversion of progesterone to its active ring-A reduced metabolites; however, the possibility remains that rapid behavioral effects of progesterone are due to a direct interaction with specific steroid receptors located on the plasma membrane, independently from the gamma-aminobutyric acid(A) (GABA(A)) receptor complex modulation.

PHARMACOLOGICAL CHARACTERIZATION OF OPIATE PHYSICAL DEPENDENCE IN THE ISOLATED ILEUM OF THE GUINEA-PIG

1 Physical dependence was produced in ilea from naive guinea‐pigs by exposure of the tissues to different opiates for logarithmically‐spaced periods of time (20–320 min). The responsiveness of the tissue to naloxone, as indicated by a strong contracture of the ileum, was enhanced in contrast to that found in intestines not exposed to opiates. 2 The dose‐response curves to naloxone obtained in tissues individually exposed to different opiates showed that their relative potency in increasing sensitivity to naloxone was as follows: levorphan > morphine > Met‐enkephalin > nalorphine > pentazocine. 3 The naloxone‐induced response was dose‐dependent and was directly related to the opiate concentration and length of exposure. 4 Dextrorphan, the inactive isomer of levorphan, did not increase the responsiveness of the tissues to the narcotic antagonist, indicating that the phenomenon is stereospecific. 5 The naloxone‐induced contraction in ilea exposed for 320 min to morphine (1 × 10−6 m) was not prevented or suppressed by the administration of a large dose of morphine (1 × 10 −5 m) before or immediately after the naloxone challenge. 6 The evidence presented here shows that a phenomenon resembling in vivo opiate physical dependence can be acutely produced in vitro with pharmacological characteristics similar to other naloxone‐induced abstinence effects.

Are mitotic index and lymphocyte proliferation kinetics reproducible endpoints in genetic toxicology testing

Lymphocyte proliferation kinetics is an endpoint used in genetic toxicology which has recently been proposed as an alternative for the screening of new cytostatic drugs. Although great variability for this parameter has been reported, there are few reports about the intra- and inter-individual variation of the effects of chemicals on this endpoint. For this reason, experiments were conducted to evaluate the reproducibility of the effects of a well-known cytostatic, mitomycin C (MMC), on the proliferation of PHA-stimulated human lymphocytes, both over time and among samples from several donors. Although inter-individual variability was shown in both parameters in untreated and treated cultures, this variation was not significant. Intra-individual variation was significantly detected only in cultures treated with 0.1 microM MMC.

A simplified procedure for the quantitative measurement of neurological deficits after forebrain ischemia in mice.

This study describes a comprehensive method to assess neurological deficits after brain ischemia produced by sequential common carotid artery sectioning (SCAS) in aged mice, and a scale to determine the degree of functional incapacity of ischemic animals. The method involves an initial phase of undisturbed observation and a later manipulative phase during which each animal is subjected to a sequence of very simple manipulations. Sham-operated animals demonstrated 96% survival throughout the study period (72 h), whereas the 24, 48 and 72 h survival rates of SCAS-mice were 48, 38 and 36%, respectively. In the surviving SCAS-mice, we detected a total of 23 neurological alterations throughout the observation period (72 h); the most frequent alterations were: motor incoordination, abnormal body position, hypomobility, decreased body tone and muscular strength, tremor, hunched back, passivity, forelimb flexion and ataxic gait. Based on these alterations, we used a global scale that comprises 10 progressive grades beyond 0 (normal), extending to status 10 (death due to SCAS), with higher scores indicating greater deficit. The median neurological scores for sham-operated animals were 1.36, 1.48 and 1.32 at 24, 48 and 72 h, respectively, whereas total neurological scores in SCAS-mice of 6.1, 6.8 and 7.4, at 24, 48 and 72 h, respectively, were substantially greater than those observed in sham-operated animals. The simplicity of the procedure, herein described, to measure the functional neurological condition of ischemic animals, and the remarkable level of functional impairment produced by SCAS offer the possiblity to test the efficacy of putative stroke therapies and to monitor progress of deficits over time in groups of animals.

Effect of various psychotropic drugs on the performance of avoidance and escape behaviors in rats

The effect of different doses of nine psychotropic drugs upon conditioned avoidance responses (CARs) developed on a stable basis, after appropriate training, was investigated in rats and compared with their capacity to disrupt escape responses (ERs). Haloperidol (HAL), chlorpromazine (CPZ), morphine (MOR), pentobarbital (PENT), chlordiazepoxide (CDP), meprobamate (MPB), and amphetamine (AMPH) dose dependently inhibited both behaviors. Imipramine also disrupted CARs dose dependently, but did not affect ERs at maximal tolerated doses. Significant differences in the minimal effective doses, effective dose range, and time of onset and duration of action, as well as in potency, were observed. The quantitative determination of the level of selectivity, based upon the ratio ED50 escape failure/ED50 avoidance failure, indicated that all CNS depressants tested caused a selective inhibition of avoidance behavior. HAL was found to be the most specific, followed, in order, by CDP, MOR, CPZ, MPB, and PENT, whose ratio values were not significantly different. AMPH produced a nearly parallel impairment of both behaviors and quipazine only affected CARs at toxic doses. It is concluded that both neuroleptic and nonneuroleptic CNS depressant drugs have selective inhibitory effects on avoidance behavior. Data revealed differences that were more quantitative than qualitative.

Effect of electrical stimulation of the baso-lateral amygdala nucleus on defensive burying shock probe test and elevated plus maze in rats.

In the present report, the putative effect of a single electrical stimulation (75, 150 or 300 microA) to the baso-lateral amygdala (BLA) nucleus was assessed in shock probe defensive burying behavior test (DB) and elevated plus maze (EPM). These models have been used for measuring anxiety levels and screening putative anxiolytic compounds. A group of 28 rats were randomly divided for the following experimental conditions: Control-control, sham-operated, BLA stimulated groups: 75, 150 and 300 microA tested for DB. The cumulative defensive burying in a 15 min-test, the latency of burying, the number of shock received and the height of the bedding material in the probe were recorded. Another group of 28 individuals was also randomly distributed for the following experimental conditions: Control-control, sham-operated, BLA stimulated animals: 75, 150, 300 microA and tested in the EPM. The time the subjects spent in the open arms, the crosses and the faeces number excreted during the test were recorded. Decreased levels of defensive burying were observed in 75, 150 and 300 microA stimulated groups. The 150 and 300 microA groups reached statistical significance. The fact that 300 microA stimulated group showed statistically significant increase in the latency of defensive burying, in the number of shock received and decreased amount in bedding material suggests a sedative action of electrical stimulation. Increased time in the open arms and augmented number of crossings in 150 microA group was observed. No changes in the number of faeces were observed in any group. The evidence supported the notion of an inhibitory amygdaline mechanism triggered by sub-threshold electrical stimulation.

Morphine dependence in the isolated guinea-pig ileum and its modification by p-chlorophenylalanine

Abstract Experiments were performed to quantitatively determine morphine physical dependence in the isolated guinea-pig ileum and to assess the influence of p-chlorophenylalanine (PCPA) on its development. Ileum segments taken from animals treated with 10 s.c. injections of 100 mg/kg of morphine, given at intervals of 8 hr without interruption, responded with intense, prolonged, dose-dependent contractions to the in vitro administration of naloxone, although contractions guinea-pigs also responded to naloxone, although contractions were smaller and of short duration. The sensitivity to naloxone on segments isolated from morphinized animals was compared to that of controls. Ilea from morphine-treated guinea-pigs were 8 to 32 times more sensitive to naloxone, as determined by a shift in the naloxone concentration-response curve to the left. There was also a three-fold increase in the maximum response. This phenomenon was taken as evidence of narcotic dependence. PCPA, given before morphine administration, at doses producing only a slight (11%) decrease in intestinal serotonin (5-HT) levels, partially reduced the sensitivity of the morphine-treated ileum to naloxone. However, high doses of PCPA, decreasing intestinal 5-HT by 40%, enhanced the abstinence-like effects of naloxone in the morphine pretreated ileum. PCPA by itself changed the responsiveness of the non-morphinized ileum to naloxone. The direction and magnitude of the change produced by PCPA alone was roughly equivalent to that produced by the serotonin depletor in the morphinized ileum. This finding indicates that PCPA has no effect upon the development of physical dependence in the isolated ileum. It remains to be determined whether or not the increased sensitivity to naloxone induced by high doses of PCPA has something in common with the changes in responsiveness to the antagonist induced by narcotics.

Altered responsiveness of the guinea-pig isolated ileum to smooth muscle stimulants and to electrical stimulation after in situ ischemia.

We evaluated changes in contractility of the guinea‐pig isolated ileum, using intact segments and myenteric plexus‐longitudinal muscle (MPLM) preparations, after several times (5–160 min) of ischemia in situ. Intestinal ischemia was produced by clamping the superior mesenteric artery. Ischemic and nonischemic segments, obtained from the same guinea‐pig, were mounted in organ baths containing Krebs‐bicarbonate (K‐B) solution, maintained at 37°C and gassed with 95% O2/5% CO2. The preparations were allowed to equilibrate for 60 min under continuous superfusion of warm K‐B solution and then electrically stimulated at 40 V (0.3 Hz, 3.0 ms). Thereafter, complete noncumulative concentration–response curves were constructed for acetylcholine (ACh), histamine (HIS), potassium chloride (KCl), and barium chloride (BaCl2). Mean Emax (maximal response) values were calculated for each drug. Our study shows that alterations of chemically and electrically evoked contractions are dependent on ischemic periods. It also demonstrates that contractile responses of ischemic tissues to neurogenic stimulation decreases earlier and to a significantly greater extent than the non‐nerve mediated responses of the intestinal smooth muscle. Contractile responses to smooth muscle stimulants were all similarly affected by ischemia. Electron microscopy images indicated necrotic neuronal death. The decrease in reactivity of ischemic tissues to electrical stimulation was ameliorated by dexrazoxane, an antioxidant agent. We consider the guinea‐pig isolated ileum as a useful model system to study the processes involved in neuronal ischemia, and we propose that the reduction in maximal responses to electrical stimulation is a useful parameter to study neuroprotection.

Changing the countenance of pharmacology courses in medical schools

Pharmacology, the science of drugs,is a very complex discipline. In gen-eral, it is the study of the interactionbetween chemical substances andliving matter and its consequences.In particular, it attempts to apply theknowledge and laws derived fromsuch interactions to solve specificproblems. Emerging as a product ofthis discipline is a body of funda-mental knowledge with applicationin many other sciences, primarilymedicine. For the clinician and medical science student, the scope ofpharmacology is, at first glance, lessextensive. The clinician is particu-larly concerned with chemical en-tities that provoke human illness andthose that possess selective biologi-cal activity useful for the treatmentof disease. The medical student isinterested in learning the pharmaco-logical basis of therapeutics.The first pharmacology courses in medical schools were orientedtowards a materia medica content,dealing with drugs of plant or animal origin, and pharmacy