Metabolism of chlorpromazine by pulmonary microsomal enzymes in the rat and rabbit

Pulmonary metabolism of chlorpromazine (CPZ) was compared using isolated microsomes of rat and rabbit lungs. CPZ-metabolizing activity of the rat lung was found to be 10-fold higher than that of the rabbit lung. The principal metabolic pathways were N-oxidation in the rat lung and N-demethylation in the rabbit lung. Kinetic analyses revealed that, although the values for apparent K_m were roughly similar for both pathways, V_max for N-oxidation by the rat lung was approximately ten times greater than that for N-demethylation by the rabbit lung. N-Oxidation by the rat lung had a broad range of pH optimum of 7–8, whereas N-demethylation by the rabbit lung had a pH optimum 8–9. SKF525-A, piperonyl butoxide, n-octylamine and CO did not inhibit N-oxidation by the rat lung, but inhibited N-demethylation by the rabbit lung. SKF525-A and n-octylamine stimulated the CPZ-N-oxidation by the rat lung. Hg²⁺ and Mg²⁺ inhibited N-oxidation by the rat lung. These results indicate that pulmonary metabolism of CPZ in the rat is catalyzed by a microsomal flavoprotein monooxygenase, while pulmonary metabolism in the rabbit is catalyzed by a cytochrome P-450 monooxygenase system, and that a marked species variation exists with respect to pulmonary metabolism of CPZ.     

Altered imprinting of rat liver monoamine oxidase by o,p'-DDT and methoxychlor

Neonatal administration of o,p'-DDT [1,1,1-trichloro-2-(o-chlorophenyl-2-p-chlorophenyl ethane or methoxychlor resulted in elevated levels of sex-differentiated hepatic monoamine oxidase activities in adult rats, but not in prepubertal animals. Exposure to these hormonally active xenobiotics may have changed the brain hormone environment during the critical period of development, resulting in endocrine alterations that were reflected by latent but permanent increases in hepatic monoamine oxidase activities, i.e. “altered imprinting”. Hepatic glutathione S-transferases and cytochrome P-450 content also underwent sex differentiation, but neonatal treatment with o,p'-DDT or methoxychlor did not alter levels in adult rats. However, glutathione S-transferase activities and cytochrome P-450 content were higher in prepubertal animals treated neonatally with o,p'-DDT. In contrast to monoamine oxidase, effects on glutathione S-transferase activities and cytochrome P-450 content were attributed to induction by these xenobiotics.     

Potentiation of the hepatotoxicity of carbon tetrachloride following preexposure to chlordecone (kepone) in the male rat.

The effect of carbon tetrachloride (CCl₄) challenge on several parameters of hepatic injury was determined in chlordecone (CD)-pretreated male rats. Following a 15-day feeding of 0 or 10 ppm CD, rats received a single intraperitoneal challenge of 0, 25, 50, 100, or 200 μl CCl₄/kg. Twenty-four hours after CCl₄, biliary excretion of phenolphthalein glucuronide (PG), bile secretion, and serum transaminase (SGPT, SGOT) activities were examined as functional indices of hepatic injury. Feeding of 10 ppm CD alone resulted in decreased biliary excretion of PG (59% of control) but had no effect on bile flow or on serum transaminases. Twenty-four hours after CCl₄ alone, the two high doses caused decreased biliary excretion of PG in the absence of any effect on bile flow and doubled serum transaminases at the highest dose (200 μl/kg). A single challenge of CCl₄ to CD-fed rats resulted in a dose-dependent impairment of PG excretion at a dose of 50 μl/kg and higher. Bile secretory function was severely impaired in the CD-fed animals receiving CCl₄ at the doses of 100 and 200 μl/kg. Decreased bile flow was not seen in any other groups. Greatly potentiated hepatotoxicity was reflected in the form of elevated SGPT and SGOT activities which increased in excess of 30- and 10-fold, respectively, in CD-fed rats challenged with CCl₄ at 100 and 200 μl/kg. Parenchymal liposis (cytoplasmic sudanophilic droplets) developed in all CD-fed rats receiving CCl₄, while necrosis occurred after CCl₄ at 50 μl/kg and increased in a dosedependent manner. CCl₄ controls exhibited parenchymal liposis and limited centrilobular necrosis only at the two highest doses of CCl₄. These data indicate a great potential for production of severe liver damage resulting from interactions of CCl₄ and CD exposure at levels which may be independently nontoxic.     

Acute toxicity of penitrem A in dogs.

The effect of ip administered penitrem A, a tremorgenic fungal metabolite, on dogs of both sexes and mixed breeding was determined by serum tests, by observation of clinical signs and survival times, and by evaluation of gross and microscopic lesions. Alkaline phosphatase, glutamic-oxaloacetic transaminase, lactic dehydrogenase and creatinine phosphokinase activities increased; survival time varied in relation to dose of mycotoxin administered. Tremors appeared as early as 10 min after dosing and ended in clonic or tetanic convulsions in dogs receiving 0.5 mg/kg or more of penitrem A. Blood uric acid and cholesterol exceeded the normal range. Gross pathologic changes frequently observed were subserosal and submucosal hemorhages in some organs of the thoracic and peritoneal cavities. Dose-related liver damage varied from massive necrosis in dogs receiving 2.5–5.0 mg/kg of penitrem A to no pathologic alterations in dogs given 0.125 mg/kg. Treatment with pentobarbital was successful for penitrem A poisoning if administered within 1–2 hr after exposure to the mycotoxin.     

The effect of potassium dichromate on renal tubular transport processes

Several studies have been conducted to examine the effects of potassium dichromate on the morphology of renal tissue, but relatively few studies have been done on the functional aspects of the renal damage. This study was designed to examine with in vitro techniques the effects of dichromate on a number of renal tubular transport processes. The accumulation of paraaminohippurate (PAH) and of tetraethylammonium chloride (TEA) by both rat and rabbit slices was inhibited by 10^?4m potassium dichromate added to the bathing solution. On the other hand, the accumulation of the nonmetabolizable amino acid, alpha-aminoisobutyric acid (AIB), was only modestly reduced. In addition, the uptake of TEA was stimulated significantly by low concentrations (10^?6m) of potassium dichromate. Although, in general, similar results were obtained using renal cortical slices taken from pretreated animals significant differences occurred. For example, at none of the doses tested (5–20 mg/kg) was the accumulation of PAH, TEA, or AIB stimulated. This was true whether the measurements were made as soon as 0.5 hr after administration of potassium dichromate or as long as 5 days after its administration. At an intermediate dose of potassium dichromate (10 mg/kg) the lactate-stimulated accumulation of PAH was depressed, but the control or nonsubstrate-stimulated accumulation was not affected. In addition to the effects on uptake of organic compounds, potassium dichromate, whether administered to the intact animal or added to the extra-cellular fluid compartment, caused a decrease in the intracellular concentration of potassium and an increase in the intracellular concentration of sodium. From a quantitative point of view, however, the effect of the nephrotoxin administered to the intact animal was always greater on tissue electrolytes than when the nephrotoxin was added in vitro.     

Effects of rubratoxin B on the hepatic monooxygenase system in phenobarbital and 3-methylcholanthrene induced male mice

Alterations in the hepatic microsomal monooxygenase system and in the concentrations of rubratoxin B in urine and feces were examined in male mice pretreated with corn oil, phenobarbital or 3-methylcholanthrene (3MC) and then given a single i.p. dose of rubratoxin B (1 mg/2.5 ml propylene glycol/kg). Twenty-four hours later the following parameters were determined: hepatic cytochrome P-450 content, enzyme activities of NADPH-cytochrome c reductase, NADPH-dependent dehydrogenase, aniline hydroxylase and ethylmorphine N-demethylase, and hapatic microsomal protein and reduced glutathione levels. Excretion of rubratoxin B in urine and feces also was determined. Rubratoxin B reduced the elevated cytochrome P-450 (136%, 134%) and protein (128%, 112%) to control values in animals pretreated with phenobarbital or 3MC, respectively; whereas, in the corn oil pretreated group, the mycotoxin reduced cytochrome P-450 by 38%. Aniline hydroxylase activity was reduced 31% or more in all pretreated animals. Rubratoxin B did not affect ethylmorphine N-demethylase activity in mice pretreated with phenobarbital; however, the enzyme activity was decreased significantly in the 3MC group. Rubratoxin B reduced the hepatic glutathione level in animals receiving 3MC (33%) or corn oil (22%). More rubratoxin B was detected in the urine than in the feces regardless of pretreatment. Only trace amounts of toxin were detected in the feces of animals from the 3MC group. These data suggest a greater effect of rubratoxin B in the 3MC pretreated mice than in the phenobarbital animals.     

Effect of chlordecone on pH and temperature dependent substrate activation kinetics of rat brain synaptosomal ATPases

Chlordecone, a polycyclic chlorinated insecticide known as Kepone, inhibited the activities of (Na+-K+)ATPase and Mg2+-ATPase in rat brain synaptosomes. Altered pH and specific activity curves for both enzymes demonstrated significant inhibition by chlordecone in buffered acidic, neutral and alkaline pH ranges. Noncompetitive inhibition with respect to activation by ATP in the case of (Na+-K+)ATPase was indicated by altered Vmax values with no significant change in Km values at any pH studied, except at pH 9.5. Mg2+-ATPase was inhibited uncompetitively as evidenced by altered Vmax and Km values. The activities of both ATPase were decreased in the presence of chlordecone at higher temperatures. Activation energy (delta E) values were found to be decreased significantly in the presence of chlordecone at 37 degrees. Arrhenius plots of both ATPases preincubated with chlordecone were found to be nonlinear. In the presence of chlordecone, Vmax was decreased without significant change in Km values for (Na+-K+)ATPase at all temperatures, suggesting a noncompetitive type of inhibition. In the case of Mg2+-ATPase, similar noncompetitive type inhibition was obtained at 27 degrees but not at 32 and 37 degrees. The kinetic data in general suggest that the chlordecone inhibited (Na+-K+)ATPase noncompetitively and Mg2+-ATPase uncompetitively at all pHs and temperatures studied. The present data suggest that inhibition of (Na+-K+)ATPase and Mg2+-ATPase, the two membrane-bound enzymes in synaptosomes, by chlordecone is temperature dependent and pH independent.     

Naturalistic observation of the snack-selecting behavior of obese and nonobese children

No previous study has examined the snack-selecting behavior of children although information from such a study may be useful in preventing and treating obesity or in determining its etiology. To provide data on this behavior, male (n = 190) and female (n = 166) children were unobtrusively observed purchasing snacks in either of two movie theaters. The amount of soft drinks and snack foods that were selected was recorded, and the corresponding caloric values were determined. Ratings were also made for obesity and sex. No differences for obesity or sex emerged on the measure of amount of calories selected. The implications of these results for the treatment of obesity are discussed.     

Alloxan toxicity in isolated rat hepatocytes and protection by sugars

Suspensions of isolated rat hepatocytes incubated in the presence of the diabetogenic agent alloxan exhibit time- and concentration-dependent damage. At concentrations of 3.5 mM and above, alloxan caused an increase in lactate dehydrogenase (LDH), glutamate-pyruvate transaminase (GPT) and intracellular potassium (K⁺) leakage, all of which are indices of plasma membrane damage, and decreased the intracellular reduced glutathione content (GSH) of the cells. Preincubation (10 min) in d-glucose (50 or 100 mM, but not 10 mM) partially protected the hepatocytes from LDH, GPT and K⁺ leakage and the decrease in GSH produced by alloxan (7mM) during a 60-min incubation period. Other sugars (d-galactose, 2-deoxy-d-glucose, d-fructose, d-mannoheptulose and d-mannitol) were also found to protect hepatocytes against damage caused by alloxan. d-Fructose was found to be the most potent protective sugar. These results indicate that alloxan is not selectively toxic to the pancreatic (β-cell and that sugars can protect against alloxan-induced cytotoxicity in hepatocytes.     

Mazindol: its efficacy and mode of action in generating weight loss.

The influence of the anorectic mazindol in weight loss was investigated in a double-blind protocol. Additionally, the behavioral and metabolic effects of the drug were studied in order to determine its mechanism of action. Drug eligible participants were randomly assigned to mazindol and placebo and contrasted with a group of non drug eligible controls. All subjects attended a series of 6 lectures on the modification of eating and activity. Weight loss was greater for mazindol than placebo subjects (P < 0.05). Mazindol also decreased hunger ratings and between meal snacking. No differences were observed on the metabolic measures, laboratory eating and salivary output. Subjects on mazindol exhibited a greater incidence of side-effects. It may be that the influence of mazindol, and perhaps other anorectics as well, is in part attributed to positive expectations of reduced hunger which are induced by side-effects.     

Synthesis of deuterium- and tritium-labeled 4-hydroxyandrostene-3,17-dione,an aromatase inhibitor,and its metabolism in vitro and in vivo in the rat

The metabolism of the aromatase inhibitor 4-hydroxyandrostenedione (4-OHA) was studied in vitro and in vivo in the rat. To accomplish this, deuterium- and tritium-labeled 4-OHA were prepared from 4-hydroxyandrosta-4,6-diene-3,17-dione. The latter was synthesized from 4-androstene-3,17-dione. Using deuterated 4-OHA in in vitro incubations of rat ovarian microsomes, 4-hydroxytesterone (4-OHT) was identified by gas chromatography/mass spectroscopy as the major metabolite. 4-OHT constituted approximately 20% of the total radioactivity from [6,7-³H]-4-OHA in the ovarian microsomal incubations. Conversion of [6,7-³H]-4-OHA to 4-hydroxyestrone was approximately 0.1%. The major metabolite of [6,7-³H]-4-OHA in vivo identified in the free, neutral fraction of rat blood was 3β-hydroxyandrostane-4,17-dione. This metabolite accounted for approximately 5% of the total radioactivity in the blood, whereas 4-OHT accounted for only 0.1%. 4-OHT inhibited in vitro ovarian aromatization by 59%, but 3β-hydroxyandrostane-4,17-dione had little effect. It was concluded that the in vivo effects of 4-OHA previously reported are largely due to its own activity although additional effects of its metabolic products cannot be excluded.     

Delayed neurotoxic, late acute and cholinergic effects of S,S,S-tributyl phosphorotrithioate (DEF): subchronic (90 days) administration in hens

Subchdronic administration of S,S,S-tributyl phosphorotrithioate (DEF) caused 3 toxicologic effects in hens, depending upon route of administration. Small delay oral doses (0.5--20 mg/kg) of DEF produced ataxia, which progressed to paralysis and death in some birds. Large daily oral doses (40 and 80 mg/kg) caused a 'late acute' effect 4 days after administration. The clinical signs of the late acute effect were identical to those produced by n-butyl mercaptan (nBM), a hydrolytic product of DEF, and were not relieved by atropine sulfate. The late acute effect of DEF overlapped with the clinical signs of delayed neurotoxicity. These hens died early, and while one hen showed histopathological lesions in peripheral nerves, another showed unequivocal lesions in the central nervous system. Topical application of daily doses of DEF consistently produced delayed neurotoxicity in the absence of late acute poisonining and was characterized by degeneration of the central and peripheral nerve tissues. Orally administered DEF was rapidly metabolized in the gastrointestinal tract to nBM, which apparently caused the late acute toxic effect. Topically administered DEF, which was not subjected to gastrointestinal tract hydrolysis, caused delayed neurotoxicity but did not produce a late acute effect.     

Differential metabolism of O-ethyl O-4-nitrophenyl phenylphosphonothioate by rat and chicken hepatic microsomes

The in vitro hepatic metabolism of O-ethyl O-4-nitrophenyl phenylphosphonothioate (EPN) was investigated in the hen (a species that is sensitive to EPN delayed neurotoxicity) and the rat (an insensitive species). EPN, which produced a Type I binding spectrum on incubation with cytochrome P-450, was converted by liver microsomes from both species to its oxygen analog, O-ethyl O-4-nitrophenyl phenylphosphonate (EPNO), and to p-nitrophenol (PNP). The formation of EPNO and PNP was dependent on the presence of NADPH in the reaction mixture and could be inhibited by either SKF-525A or by anaerobic conditions. The rates of EPNO and PNP formation by rat liver microsomes were, however, 3- and 20-fold higher, respectively, than the rates of formation by chicken liver microsomes. There was also a 4-fold difference in the cytochrome P-450 contents of the liver microsomes. The EPNO-hydrolyzing activity of rat liver microsomes was much greater than that of chicken liver microsomes. EPNO metabolism, in contrast to EPN metabolism, did not require NAPDH nor was it inhibited by SKF-525A or by anaerobic conditions. Prior exposure of rats to phenobarbital (PB) or Arochlor 1254 resulted in an increase in hepatic microsomal EPN metabolism and cytochrome P-450 content. On the other hand, 3-methylcholanthrene (3-MC) treatment elevated microsomal cytochrome P-450 but did not increase EPNO or PNP formation. Pretreatment with EPN did not alter either microsomal EPN metabolism or cytochrome P-450 levels. In chickens, prior exposure to PB, 3-MC or 100 mg/kg EPN increased EPNO and PNP formation by liver microsomes as well as cytochrome P-450 levels; prior exposure of chickens to 15 mg/kg EPN did not alter these variables. The λ_max Soret bands of the reduced hepatic cytochrome P-450 complexes from these animals differed as follows (rat then chicken): untreated, 450 vs 452 nm; PB-treated, 450 vs 451 nm; and 3-MC-treated, 448 vs 449 nm. None of the above treatments had an effect on EPNO metabolism by liver microsomes.     

d-Amphetamine and gamma-butyrolactone alteration of dopamine synthesis in the terminals of nigrostriatal and mesolimbic neurons. Possible role of various autoreceptor sensitivities

The rate of synthesis of dopamine (DA) was estimated in vivo in terminals of nigrostriatal and mesolimbic neurons by measuring the accumulation of dihydroxyphenylalanine (DOPA_ in the striatum and in the nucleus accumbens and olfactory tubercle of male rats following the administration of an inhibitor of aromatic L-amino acid decarboxylase. Low to moderate doses of d-amphetamine (0.5 to 1.0 mg/kg) increased DA synthesis in the striatum but were without effect in the nucleus accumbens and olfactory tubercle; a higher dose (5.0 mg/kg) did not alter DA synthesis in any of the brain regions. Despite the lack of effect of d-amphetamine per se in the nucleus accumbens and olfactory tubercle, in both of these regions, as well as in the striatum, this drug was effective in reversing the decreased DA synthesis induced by a monoamine oxidase inhibitor (nialamide) or a DA agonist (apomorphine). γ-Butyrolactone (GBL) increased DA synthesis in the striatum, nucleus accumbens and olfactory tubercle, but it was less potent, less effective, and had a shorter duration of action in the latter two regions. d-Amphetamine, like GBL, may exert part of its effect on DA synthesis by reducing impulse flow in ascending DA neurons, since a low dose of d-amphetamine (0.125 mg/kg) potentiated the GBL-induced increase in DA synthesis in striatum, nucleus accumbens and olfactory tubercle. Regional differences in the actions of GBL and d-amphetamine on DA synthesis may be related to differences in receptor-mediated mechanisms that regulate the synthesis of this amine in neurons terminating in the striatum versus those terminating in nucleus accumbens and olfactory tubercle. At least one difference appears to be in the sensitivity of DA autoreceptors in these regions. The dose of apomorphine needed to reverse the GLB-induced increase of DA synthesis in nucleus accumbens and olfactory tubercle was approximately 1/10 that necessary to cause the same effect in the striatum. Pretreatment with a DA antagonist (haloperidol) potentiated the GBL-induced stimulation of DA synthesis in nucleus accumbens and olfactory tubercle, but not in the striatum. These results suggest that the reduced abilities of GBL and amphetamine to increase DA synthesis in nucleus accumbens and olfactory tubercle may result from a greater sensitivity of autoreceptors on mesolimbic DA neurons permitting a tighter regulatory control over DA synthesis in these neurons.     

Tolerance, dependence and lethality in morphine-dependent mice after repeated oral administration of methadone

Mice were rendered tolerant to and dependent on morphine via a morphine pellet implantation. Three days later methadone hydrochloride was administered at a dose of 100mg/kg per os 3 hours after pellet removal and then daily for a total of 5--6 days. This dose of methadone was shown to exhibit a high efficacy for the blockade of morphine abrupt withdrawal jumping and only minimal toxicity. Under these conditions, the level of analgetic tolerance with respect to morphine and methadone and the level of dependence as measured by the naloxone ED50 were initially elevated by the morphine treatment. However, upon substitution with oral methadone these levels declined with time at a rate which did not differ from that of a group of mice receiving only water after morphine pellet removal. Despite these findings, the methadone treatment was associated with an increasing tolerance to methadone lethality during the administration of this narcotic which was nearly double that of a similarly treated water control group by the sixth day. This observation could not be explained by an elevation in the level of cellular tolerance rendered by the methadone treatment since the morphine LD50 was not elevated following identical treatment with morphine and then methadone. The significance of these results is discussed with respect to the role of methadone administration and its metabolism in the modification of tolerance and dependence.     

Benzodiazepine induced state-dependent learning: A correlative of abuse potential?

Using a two-way avoidance procedure, the two major benzodiadepines, diazepam and chlordiazepoxide, were tested for state-dependent learning effects in rats. Results showed that compared to saline control animals, subjects receiving diazepam or chlordiazepoxide during extinction displayed significantly more rapid extinction but a significantly greater amount of spontaneous recovery of the conditioned avoidance response. No significant dose-response differences were found. These findings are consistent with Overton's hypothesis that drug state discriminability and state-dependent learning parallel a compound's abuse potential. Drug state discriminability vs state-dependent learning is discussed and a potential relationship between state dependent properties and physical dependence is suggested.