Effect of polychlorinated biphenyls on the immune responses of rhesus monkeys and mice.

The relationship between lipid peroxidation, glutathione (GSH) content, and CCl₄-induced toxicity was investigated in rat hepatocytes isolated by a collagenase-perfusion technique. Two chemical initiators of lipid peroxidation, ferric ions complexed with adenosine diphosphate ($\text{ADP}\text{Fe}{}^{\mathrm{3+}}$) and diethyl maleate, were studied for comparison. CCl₄ caused a reduction of intracellular K⁺ and release of alanine aminotransferase (ALT) into the medium, but no evidence of lipid peroxidation, as measured by the absorbance of thiobarbituric acid (TBA)-reacting materials and lipid-extract diene conjugation. $\text{ADP}\text{Fe}{}^{\mathrm{3+}}$ caused lipid peroxidation, but only a small loss of K⁺. Diethyl maleate caused a greater amount of lipid peroxidation and cell damage than did $\text{ADP}\text{Fe}{}^{\mathrm{3+}}$. Neither response appeared to be related to the GSH content, which was reduced by diethyl maleate, but not by $\text{ADP}\text{Fe}{}^{\mathrm{3+}}$, and by CCl₄ only at the highest dose. The results suggest that lipid peroxidation is not a requisite step in CCl₄-induced toxicity in isolated hepatocytes.     

Inhibition of hepatic microsomal lipid peroxidation by drug substrates without drug metabolism

Experiments were performed to study the mechanism of action of drug substrates on lipid peroxidation in rat hepatic microsomes. Addition of the drug substrates, aniline, β-diethylaminoethyl diphenylpropylacetate (SKF-525A), aminopyrine, benzo[a]pyrene or ethylmorphine, to hepatic microsomes causes almost complete inhibition of NADPH-induced (enzymatic) lipid peroxidation. These substrates also produce similar inhibition of ascorbate-induced (non-enzymatic) lipid peroxidation in microsomes in which drug-metabolizing enzymes were inactivated by heat treatment. The substrate concentrations producing half-maximal inhibition ($\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ are also similar for NADPH- and ascorbate-induced lipid peroxidation. Addition of metyrapone, an inhibitor of drug metabolism, has no effect on either the $\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ values or on the maximal substrate inhibition of NADPH-induced lipid peroxidation. All five drug substrates also inhibit Fe²⁺-stimulated oxidation of linoleic acid. These results demonstrate that inhibition of lipid peroxidation in hepatic microsomes by drug substrates is independent of drug metabolism and is probably due to the antioxidant properties of the substrates.     

The effects of cadmium, manganese and aluminium on sodium-potassium-activated and magnesium-activated adenosine triphosphatase activity and choline uptake in rat brain synaptosomes

The effects of Cd²⁺, Mn²⁺ and Al³⁺ on rat brain synaptosomal sodium-potassium-activated and magnesium-activated adenosine triphosphatase (Na-K-ATPase and Mg-ATPase) activity and choline uptake were studied. All three types of metal ions inhibited Na-K-ATPase activity more markedly than Mg-ATPase activity. The rank order of inhibition of Na-K-ATPase was: $\text{Cd}{}^{\mathrm{2+}}\text{(}\text{ic}{}_{50}\text{= 5.4 μ}\text{M}\text{) >}\text{Mn}{}_{\mathrm{2+}}\text{(}\text{ic}{}_{50}\text{= 955 μ}\text{m}\text{) >}\text{Al}{}^{\mathrm{3+}}\text{(}\text{ic}{}_{50}\text{= 8.3}\text{mM}$). The rank order of inhibition of Mg- was:$\text{Cd}{}^{\mathrm{2+}}\text{(}\text{ic}{}_{50}\text{= 316 μ}\text{M}\text{>}\text{Mn}{}^{\mathrm{2+}}\text{(}\text{ic}{}_{50}\text{= 5.5}\text{mM}\text{>}\text{Al}{}^{\mathrm{3+}}\text{(}\text{ic}{}_{50}\text{= 21.9}\text{mM}\text{).}\text{Al}{}^{\mathrm{3+}}$ was most potent in inhibiting synaptosomal choline uptake ($\text{ic}{}_{50}\text{= 24μ}\text{M}$ in the absence of Ca²⁺ and 123 μ.M in the presence of 1 mM Ca²⁺). $\text{Cd}{}^{\mathrm{2+}}\text{(}\text{ic}{}_{50}\text{= 363 μ}\text{M}\text{)}$ was a more effective inhibitor of choline uptake than $\text{Mn}{}^{\mathrm{2+}}\text{(}\text{ic}{}_{50}\text{= 1.2?1.5}\text{mM}\text{)}$ . The presence of 1 mM Ca²⁺ did not alter choline uptake, nor did it antagonize the inhibitory actions of the three metals. Our observations that Cd²⁺ and Al³⁺ inhibited synaptosomal choline uptake, but did not show parallel inhibitory effects on Na-K-ATPase activity directly contradicts the ionic gradient hypothesis. These results are also discussed in relation to the in vivo neurotoxicity of cadmium, manganese and aluminium.     

Mechanisms by which quipazine,a putative serotonin receptor agonist,alters brain 5-hydroxyindole metabolism

Quipazine (2-[1-piperazinyl] quinoline maleate) was shown to increase serotonin and decrease 5-hydroxyindoleacetic acid concentrations in whole brain, several brain regions, and the spinal cord of rats 1 hr after its administration (10 mg/kg, i.p.). In animals with transected spinal cords, quipazine induced stronger activation of extensor reflexes than 5-hydroxytryptophan, chlorimipramine, or Lilly 110140. This response could be blocked by methiothepin. In slices of rat cerebral cortex, quipazine inhibited the uptakes of [³H]-serotonin (EC₅₀ = 10^?6 M) and [³H]-norepinephrine ($\text{EC}{}_{50}\text{= 2 × 10}{}^{\mathrm{?6}}\text{m}$); it was equipotent with Lilly 110140 in inhibiting serotonin uptake, but less potent than chlorimipramine ($\text{EC}{}_{50}\text{= 10}{}^{\mathrm{?7}}\text{m}$). Quipazine administration to rats did not inhibit monoamine oxidase activity, and actually elevated brain tryptophan levels. These observations suggest that the effects of quipazine on brain serotonin and 5-hydroxyindoleacetic acid concentrations could have been caused by direct activation of central serotonin receptors (which would secondarily decrease impulse flow along serotonergic neurones), or by the inhibition of serotonin reuptake, or by both mechanisms.     

Participation of superoxide free radical and Mn2+ in sulfite oxidation.

Sulfurous acid gas is a well-known air pollutant. The participation of superoxide ($\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$), a species of activated oxygen, in sulfite oxidation was investigated in relationship to this health hazard. The reduction of nitroblue tetrazolium (NBT) was markedly accelerated in the presence of the xanthine-xanthine oxidase system (X-XO), Mn²⁺ and SO₃^2?, but not by X-XO and Mn²⁺ or X-XO and SO₃^2? alone. This accelerated NBT reduction was partially suppressed by superoxide dismutase and was completely suppressed by allopurinol. Oxygen consumption was also markedly accelerated under to condition which caused the increase in NBT reduction. Lipid peroxidation of rat liver homogenate increased in the presence of X-XO, SO₃^2?, or both. This increased lipid peroxidation was definitely suppressed by Mn²⁺. From these observations, it is suggested that chain reactions involving sulfite oxidation are initiated by O₂^? generated from X-XO, and Mn²⁺ acts as a catalyst in the process.     

Determination of extraction constant,true partition coefficient and formation constant of ion-pair complexes of quaternary ammonium salts,tetrabutylammonium bromide and isopropamide iodide.with some organic anions by a solvent extraction technique

A new method of determining the extraction constant (K_e, the true partition coefficient (TPC) and the formation constant (K_f) of ion-pairs, was developed by the solvent extraction technique. K_e and TPC were estimated from the reciprocals of the intercept and the slope of the regression line obtained by plotting

$\text{B}{}^{\mathrm{T}}{}_{\mathrm{W}}\text{APC ? dA}\text{vs}\text{B}\text{B}{}^{\mathrm{T}}{}_{\mathrm{W}}\text{dA}\text{APC ? dA}\text{+}\text{A}{}^{\mathrm{T}}{}_{\mathrm{W}}\text{+}\text{B}{}^{\mathrm{T}}{}_{\mathrm{W}}$

in the following equation.

$\text{B}{}^{\mathrm{T}}{}_{\mathrm{W}}\text{APC ? dA}\text{=}\text{1}\text{K}{}_{\mathrm{e}}\text{+}\text{B}\text{B}{}^{\mathrm{T}}{}_{\mathrm{W}}\text{dA}\text{APC ? dA}\text{+}\text{A}{}^{\mathrm{T}}{}_{\mathrm{W}}\text{+}\text{B}{}^{\mathrm{T}}{}_{\mathrm{W}}\text{x}\text{1}\text{TPC}$

where [A^T_W] and [B^T_W] are the total concentrations of the cationic compound A and that of the anionic compound B in the aqueous phase respectively, APC is the apparent partition coefficient of A, dA is the partition coefficient of cation A⁺. K_f, which is expressed by K_e/TPC, was then calculated. These constants were determined for the ion-pair extraction of tetrabutylammonium bromide and isopropamide iodide with 4 organic anions, i.e. benzoic acid, p-toluenesulfonic acid, salicylic acid and taurodeoxycholic acid. This new method might be applicable to other ion-pairs without further assumptions except that the molar ratio of the ion-pair formation be 1 : 1.     

Similar activities of nerve growth factor and its homologue proinsulin in intracellular hydrogen peroxide production and metabolism in adipocytes: Transmembrane signalling relative to insulin-mimicking cellular effects

Generation of hydrogen peroxide in adipocyte plasma membrane and its intracellular metabolism and regulatory role have been shown by Mukherjee and co-workers to be a major effector system for insulin [Fedn Proc.35, 1694 (1976); Archs Biochem. Biophys.184, 69 (1977); Biochem. Pharmac.27, 2589 (1978); Fedn Proc.37, 1689 (1978); and Biochem. Pharmac.29, 1239 (1980)]. The possible involvement of this mechanism in the action of structurally similar polypeptides having some insulin-like metabolic effects was investigated. The β-subunit of nerve growth factor (2.5 S NGF, mol. wt 13,500) which has a striking structural homology with proinsulin and has been reported to exert certain insulin-like metabolic effects in its own target tissues (e.g. growing neurites and sympathetic ganglia), and the insulin-derived polypeptides, desalanine-insulin and desoctapeptide-insulin, as well as proinsulin, were examined for their effects on rat adipocytes, employing the technique of formate oxidation. Both NGF and proinsulin caused increased [¹⁴C]formate oxidation, showing similar intrinsic activities, up to a maximum of 140–160% of the basal rate; insulin increased the rate to 190–210% of the basal rate. The relative potencies of the hormones toward H₂O₂ formation and stimulation of the pentose phosphate pathway activity were: insulin $\text{(}\text{EC}{}_{50}\text{: 2.5 × 10}{}^{\mathrm{?11}}\text{M}\text{)}$, desalanine-insulin $\text{(}\text{EC}{}_{50}\text{: 2.5 × 10}{}^{\mathrm{?10}}\text{M}\text{)}$ , proinsulin $\text{(}\text{EC}{}_{50}\text{: 8 × 10}{}^{\mathrm{?9}}\text{M}\text{)}$, and NGF $\text{(}\text{EC}{}_{50}\text{: 10}{}^{\mathrm{?9}}\text{M}\text{)}$. The biologically inactive derivative, desoctapeptide-insulin, did not stimulate glucose oxidation, although it caused a small increase in formate oxidation, with an $\text{EC}{}_{50}\text{of}\text{5 × 10}{}^{\mathrm{?7}}\text{M}$, indicating a suboptimal level of H₂O₂ formation in the elevation of the hexose monophosphate shunt activity. 3-Amino-1,2,4,-triazole (50 mM), which irreversibly decomposes the peroxidatic compound II of the catalase: H₂O₂ complex, inhibited formate oxidation to a greater extent in the hormone-treated cells than in the control cells, whereas sodium azide, an inhibitor of the hemoprotein, catalase, completely inhibited it. The abilities of the polypeptides to stimulate H₂O₂ formation correlated with their abilities to promote lipogenesis from [U-¹⁴C]-D-glucose, as expected of insulin. The cellular GSH/GSSG ratio increased concomitantly with the stimulation of glucose oxidation via the shunt, indicating a tight coupling between these processes. The results confirm that the hydrogen peroxide production is a common basis of the metabolic actions of growth-promoting polypeptide hormones or mitogens beyond their respective receptors.     

Concentrations of nitrate in normal human urine and the effect of nitrate ingestion

A method suitable for the analysis of nitrate in human urine was developed. Normal urinary concentrations of nitrate in urine of human volunteers in Dade County, Florida, where the drinking water contains negligible amounts of nitrate, averaged 47.6 ppm of $\text{NO}{}_3{}^{\mathrm{?}}$ (SD = 17.3). On a vegetable and preserved-meat-free diet, the nitrate concentration was reduced (10 to 30 ppm of $\text{NO}{}_3{}^{\mathrm{?}}$), but, on nitrate-supplemented drinking water, the urinary concentration rose to a range of 34–87 ppm of $\text{NO}{}_3{}^{\mathrm{?}}$. A high vegetable diet resulted in peak urinary nitrate concentrations of 270–425 ppm. These results indicated that nitrate in drinking water is a factor in determining urinary nitrate concentration, but that vegetable ingestion is of greater significance.     

The effect of bencyclane on the oxidative phosphorylation in isolated mitochondria of heart and liver

Our experiments were designed to localize the inhibitory influence of bencyclane² on the process of oxidative phosphorylation in isolated heart and liver mitochondria. The following results were obtained: (1) The state-3-respiration of rat liver and rabbit heart mitochondria was inhibited by bencyclane. This inhibition was dependent on the substrate used as energy donator, being much more pronounced with glutamate $\text{(}\text{ed}{}_{50}\text{= 3.17 × 10}{}^{\mathrm{?8}}\text{or}\text{1.85 × 10}{}^{\mathrm{?7}}\text{moles/mg}$ of protein, respectively) than with succinate $\text{(}\text{ed}{}_{50}\text{= 3.4 × 10}{}^{\mathrm{?7}}\text{or}\text{4.78 × 10}{}^{\mathrm{?7}}\text{moles/mg}$ of protein, respectively). Since the 2,4-dinitrophenol stimulated respiration was equally inhibited, and glutamate transfer through the mitochondrial membrane not influenced, we assume the NADH-coenzyme-Q-reductase to be the site of interaction at the molecular level. (2) Bencyclane stimulates the state-4-respiration of isolated mitochondria with $\text{concentrations}\text{\$}\text{?}\text{= 10}{}^{\mathrm{?5}}\text{M}$. This effect depends on the molar bencyclane concentration of the incubation medium, and is not abolished by the addition of atractyloside, oligomycin or ruthenium red. Therefore, it is suggested that uncoupling of oxidative phosphorylation is the reason for this bencyclane effect. Theoretically, both of the described effects result in a reduction of the amount of ATP in the living cell. Possible consequences on myocardial function and the cardiovascular system are discussed in terms of previously published data in this field.     

Placental transfer and tissue localization of digoxin in the pregnant rat

Serum and tissue digoxin concentrations were estimated by radioimmunoassay in maternal and fetal animals at various times following the injection of pregnant rats, on Days 19 or 20 of gestation, with digoxin (0.1 mg/kg iv). Maternal serum digoxin concentrations were 3.5 to 5 times greater than corresponding fetal concentrations during the early period of sampling (2–10 min) and ranged from 1.8- to 2.7-fold higher for the remainder of each experiment. The elimination of digoxin from maternal serum followed a biexponential pattern ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{α = 0.36}\text{hr}\text{; t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{β = 3.52}\text{hr}\text{; K}{}_{\mathrm{<mtext>el</mtext>}}\text{= 0.39}\text{hr}{}^{\mathrm{?1}}$) and was similar to that observed in placental tissue and the fetal circulation. Maternal tissue concentrations of digoxin were highest in the liver followed in descending order by the skeletal muscle, heart, and kidney. The distribution pattern in fetal tissues differed significantly as indicated: heart > kidney > liver. These data have demonstrated that digoxin is rapidly transferred across the placenta of the pregnant rat. Differences in the fetal/maternal tissue distribution patterns of the drug may reflect developmental influences upon tissue binding and/or regional perfusion.     

Dopaminergic effects of buspirone,a novel anxiolytic agent

The novel anxiolytic drug buspirone raised striatal levels of the dopamine metabolites homovanillic acid (HVA) and dihydroxyphenylacetic acid (DOPAC) 1 hr after oral administration. This effect was dose-dependent with a peak at 60 min. No changes were observed in the levels of 3-methyxytyramine (3MT), the extraneuronal metabolite of dopamine. Noradrenaline, serotonin and its metabolite 5-hydroxyindoleacetic acid (5HIAA) were not affected. Buspirone displaced [³H]spiroperidol from striatal binding sites, with an ic₅₀ (1.8 × 10^?7 M), comparable to that of clozapine ($\text{ic}{}_{50}\text{= 1.4 × 10}{}^{\mathrm{?7}}\text{M}$) but considerably lower than that of haloperidol (4.7 × 10^?9 M). Buspirone was only a weak inhibitor of dopamine-stimulated adenyl cyclase. Buspirone was not active on the binding of trifluoperazine to calmodulin and did not modify calmodulin-induced activation of phosphodiesterase (PDE). Repeated administration of buspirone did not increase the number of DA receptors. These data show that, although buspirone has antidopaminergic activity, it can hardly be classified as a classic neuroleptic agent.     

Tissue distribution of histamine in a mutant mouse deficient in mast cells: Clear evidence for the presence of non-mast-cell histamine

The contents of histamine in various tissues of mutant mice deficient in mast cells ($\text{W}\text{W}{}^{\mathrm{v}}$) and in congenic normal mice (+/+) were determined by high-performance liquid chromatography and were compared. In spite of the absence of mast cells in $\text{W}\text{W}{}^{\mathrm{v}}$ mice, the histamine content of their whole bodies was about 5–10% of that of +/+ mice. The skin, heart and lungs of $\text{W}\text{W}{}^{\mathrm{v}}$ mice contained negligible amounts of histamine (about 2% of that in +/+ mice), but the liver, kidneys and spleen contained appreciable histamine (8–15% of that in +/+ mice), and the brain and stomach contained much histamine (45 and 34%, respectively, of that in +/+ mice). These results indicate the presence of non-mast-cell histamine, especially in the brain and stomach, where it may play important physiological roles.     

Effect of diphenylhydantoin on the uptake and catabolism of l-[3H]norepinephrine in vitro in rat cerebral cortex tissue

The effect of diphenylhydantoin on the accumulation of [³H]norepinephrine in vitro was examined in brain slices prepared from rat cerebral cortex. High concentrations of diphenylhydantoin (10^?3 M) caused a significant reduction in the 5-min accumulation of [³H]norepinephrine. On the other hand, 10^?5–10^?4 M diphenylhydantoin facilitated the 20-min accumulation of [³H]norepinephrine. This facilitative action of diphenylhydantoin was (1) associated with a reduction in oxidative catabolism of [³H]norepinephrine and (2) abolished by the 2-hr pretreatment of rats with 100 mg/kg of nialamide (i.p.). The inhibitory action of diphenylhydantoin on the oxidative catabolism of [³H]norepinephrine was observed in both whole and lyzed crude synaptosomal preparations. When diphenylhydantoin and pargyline were compared, it was found that pargyline $\text{(}\text{id}{}_{50}\text{= 1.5 × 10}{}^{\mathrm{?6}}\text{M}\text{)}$ was 37 times more effective than diphenylhydantoin $\text{(}\text{id}{}_{50}\text{= 5.5 × 10}{}^{\mathrm{?5}}\text{M}\text{)}$ in inhibiting the oxidative deamination of [³H]norepinephrine. These results suggest that diphenylhydantoin alters norepinephrine metabolism in cerebral cortex slices by an inhibitory action on (1) monoamine oxidase activity and (2) the neuronal uptake system.     

Interaction of [3H]flunitrazepam with the benzodiazepine receptor: Evidence for a ligand-induced conformation change

The interaction of [³H]flunitrazepam with benzodiazepine receptors in rat brain homogenates was studied in the presence of 2 μM endogenous GABA at 0° at pH 7.2. Equilibrium binding experiments showed a dominant component of high affinity with an equilibrium dissociation constant K = 0.86 ± 0.07 nM which accounted for 75% of total binding and another component of lower affinity (K ? 30 nM). The dissociation kinetics of the [³H]flunitrazepam complex at the high affinity site were strictly monophasic with a rate constant k_off = (7.7 ± 0.3) × 10^?4/sec. The association kinetics with the high affinity sites were studied with ligand concentrations [L]₀ in large excess over binding sites. The kinetics were in accordance with a single exponential with a reaction rate τ^?1. In the higher concentration range [L]₀ ? 10 nM, τ^?1 as a function of [L]₀ deviated from linearity and started to level off. The data are compatible with a two-step mechanism where R and L rapidly combine to form a pre-complex RL which then slowly isomerizes to the final complex C:

where $\text{K}{}_1\text{= (}\text{[}\text{R}\text{][}\text{L}\text{]}\text{([}\text{RL}\text{]}\text{)}$ and $\text{[}\text{RL}\text{]}\text{[}\text{C}\text{]}\text{=}\text{k}{}_{\mathrm{?2}}\text{k}{}_2\text{= k}{}_2$. Nonlinear parameter estimation yielded K₁24.2 ± 7.1 nM, k₂ = (2.8 ± 0.5) × 10^?2/sec and k_?2 = (9 ± 2) × 10^?4/sec. The isomerization step might reflect a ligand-induced conformation change of the high affinity site which is involved in the potentiation of GABA-ergic transmission produced by the benzodiazepines.     

Toxicology of helium diving environments: protective effects of raised He pressure on cholinergic activation in ganglia

The effect of helium gas in $\text{He}\text{O}{}_2$ breathing gas mixtures on excitability of the cholinergic component of ganglionic transmission has been studied in the cat superior cervical ganglion preparation (SCG) under hyperbaric conditions. The depression of SCG sensitivity to acetylcholine (ACh⁺) stimulation seen at 0.80 atm pressure of He (in 1 atm $\text{He}\text{O}{}_2$, relative to 1 atm $\text{N}{}_2\text{O}{}_2$) is fully reversed when the partial pressure of He is raised to 14.4 atm. Additionally, high pressure He stabilizes the amplitude of ACh⁺-stimulated SCG response over a 2-hr interval, reversing the temporal decay pattern seen in 1-atm experiments with $\text{He}\text{O}{}_2$ or $\text{N}{}_2\text{O}{}_2$.     

Correlation of biologic data with physico-chemical properties among the vinca alkaloids and their congeners.

Displacement of [³H]vinblastine binding to tubulin by other Vinca alkaloid derivatives has been demonstrated to be a competitive process, allowing for determination of the association constant of each drug. Correlation of LD₅₀ data and anti-P-388 activity was found with log P and log K_a, according to the equations: $\text{log}\text{LD}{}_{50}\text{= 0.129 (}\text{log}\text{P)}{}^2\text{? 0.522}\text{log}\text{P ? 0.479}\text{log}\text{K}{}_{\mathrm{a}}\text{+ 4.652}\text{log}\text{P ? 388 = 0.222 (}\text{log}\text{P)}{}^2\text{? 1.059}\text{log}\text{P ? 0.520}\text{log}\text{K}{}_{\mathrm{a}}\text{+ 5.366}$. Vincristine and desacetylvinblastine were the two most active agents in this series. That the latter drug had significant biologic activity was of considerable interest, since it is known to be a human metabolite.