Effect of exposure to nitrogen dioxide on t and b cells in mouse spleens

Effects of exposure to nitrogen dioxide (NO₂) on thymus-derived (T) and bursa-derived (B) cells were studied in albino mice. The mice were exposed continuously 24 $\text{h}\text{day}$ to 940 $\text{μg}\text{m}{}^3$ NO₂ and to 188 $\text{μg}\text{m}{}^3$ NO₂ with daily 3-h peaks 5 $\text{days}\text{week}$ of either 470, 940 or 1880 $\text{μg}\text{m}{}^3$ NO₂. After 1, 3, 6, 9 and 12 months of exposure, single-cell suspensions were prepared from spleen and incubated with phytohemagglutinin (PHA) or bacterial lipopolysaccharide (LPS). The PHA and LPS responses from mice exposed to NO₂ were generally depressed when compared with those in mice exposed for the same time periods to filtered air.     

Carbon disulphide tetratogenicity and postnatal effects in rat

Pregnant albino rats were exposed to carbon disulphide vapour in concentrations of 50, 100 or 200 $\text{mg}\text{m}{}^3$throughout gestation. Two successive generations (F₁ and F₂) were studied.Concentration levels of 100 and 200 $\text{mg}\text{m}{}^3$ produced marked dose-related impairment in the prenatal development of the F₁ progeny, with increase of early embryonal lethality, reduction in foetal weight and a high incidence of malformations affecting mostly the brain and limbs. Postnatal viability, body weight, lipid and energy metabolism and behaviour were also impaired. Behavioral deviations were observed even at 50 $\text{mg}\text{m}{}^3$.After reaching sexual maturity the F₁ rats were mated within their experimental groups, but no further carbon disulphide exposure was applied. The adverse effects on progeny were still detectable in the F₂ generation. Structural abnormalities of the same type as those found in the F₁ at 100 and 200 $\text{mg}\text{m}{}^3$ exposure were observed in their progeny and, postnatally, statistically significant behavioral changes were observed in the progeny of all test groups.     

Effect of urban ozone levels on laboratory-induced respiratory infections

The effect of the time of exposure to an aerosol of viable microorganisms on the incidence of respiratory infections associated with a 3 h exposure to ozone (O₃) was studied. The 157 and 196 $\text{μg}\text{m}{}^3$ (0.08–0.1 ppm) levels of O₃ used occur regularly in some urban communities. The studies reported here show that the susceptibility of mice to a laboratory-induced infection can be maximally enhanced if the infectious challenge is concurrent with the exposure to O₃.     

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.     

Influence of dietary polybrominated biphenyl on antibody and host defense responses in mice

Male $\text{Balb}\text{cByJ}$ mice were fed diets containing 5 or 167 ppm of polybrominated biphenyls (PBB) (Firemaster, FFl, lot No. 7042) for either 3 or 6 weeks and then evaluated for their ability to produce antibody and to resist a challenge with malaria or endotoxin. Animals which received a dietary exposure of either 5 or 167 ppm of PBB for 3 or 6 weeks manifested no alteration in their resistance to a challenge infection with Plasmodium berghei (NYU-2), a lethal murine malaria parasite. However, mice which were fed a diet containing 167 ppm of PBB for 3 or 6 weeks had a significant increase in endotoxin (LPS) sensitivity while no change in LPS sensitivity was observed in mice fed 5 ppm of PBB. Peak primary antibody production to sheep erythrocytes, expressed as $\text{PFC}\text{10}{}^{\mathrm{<mtext>6</mtext>}}$ spleen cells, was reduced almost 50% in mice fed a diet containing 167 ppm for 3 weeks, however, no change in $\text{PFC}\text{10}{}^{\mathrm{<mtext>6</mtext>}}$ cells was observed at 6 weeks. A dietary level of 5 ppm of PBB did not alter primary antibody formation at either 3 or 6 weeks. The peak secondary PFC response in both 5- and 167-ppm-treated groups was delayed by 1 day, relative to control values. $\text{PFC}\text{10}{}^{\mathrm{<mtext>6</mtext>}}$ cells, measured on the day of the peak control response, were depressed 60% at 3 weeks and over 85% at 6 weeks at both doses. Serum IgM levels in the mice receiving 167 ppm of PBB were reduced 23–63% during the I° and II° responses, serum IgG was only moderately reduced and serum IgA was unaltered throughout the study. These results suggest that PBB has a minimal effect on antibody production to T-dependent antigens or on host defense to protozoan parasites yet causes a selective depletion of serum immunoglobulin isotypes. The marked increase in LPS sensitivity suggests a compromised macrophage detoxification capability.     

Kinetic studies on the deethylation of ethoxybenzamide: A comparative study with isolated hepatocytes and liver microsomes of rat

Kinetic parameters (K_m and V_max) of ethoxybenzamide deethylation in isolated rat hepatocytes and liver microsomes were compared. Adjustment of cofactors in microsomal deethylation, such as NADPH and Mg²⁺, to give optimum conditions, and appropriate correction of the apparent kinetic parameters for nonspecific binding and microsomal yield resulted in good agreement among the kinetic parameters of isolated hepatocytes [$\text{V}{}_{\mathrm{<mtext>max</mtext>}}\text{= 0.0863 μmole · min}{}^{\mathrm{?1}}\text{· (g liver)}{}^{\mathrm{?1}}$ and K_m = 0.459 mM] and microsomes [V_max = 0.124 μmoles · min^?1 · (gliver)^?1 and K_m = 0.378 mM].     

Kinetic studies on the metabolism of ethylmorphine by isolated hepatocytes from adult rats

Hepatocytes of adult rats were isolated by infusion of a hyaluronidase collagenase mixture. High yields of cells excluding trypan blue were obtained. These cells, in Hank's buffer containing rat serum and 0.1% glucose, $\text{N-}\text{demethylate}\text{[}{}^3\text{H-CH}{}_3\text{-N]}$ethylmorphine. The formaldehyde initially formed is further metabolized to tritiated water. Fifteen per cent of the original metabolic activity was observed after 21 hr at 37° in 5% CO₂-air, and cumulative metabolism is linear for up to 90 min under these conditions. The K_m for the N-demethylation of [³H-CH₃-N]ethylmorphine is 50 μM, 20 per cent of the value observed for this reaction by musomal preparations. An active transport of the substrate into the cell is postulated to account for this difference.     

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.     

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.     

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.     

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.     

Adenosine receptor interactions and anxiolytics

$\text{[}{}^3\text{H}\text{]-N}{}^6\text{-}\text{cyclohexyladenosine}$ and [³H]-1,3-diethyl-8-phenylxanthine label the A₁ subtype of adenosine receptor in brain membranes. The affinities of methylxanthines in competing for A₁ adenosine receptors parallel their potencies as locomotor stimulants. The adenosine agonist N⁶-(-phenylisopropyl) adenosine is a potent locomotor depressant. Both diazepam and $\text{N}{}^6\text{-(}\text{l}\text{-}\text{phenylisopropyl}\text{)}\text{adenosine}$ cause locomotor stimulation in a narrow range of subdepressant doses. Combined stimulant doses of the two agents depress motor activity, as do larger doses of either one, given separately.Evidence supporting and against the hypothesis that some of the actions of benzodiazepines are mediated via the adenosine system is reviewed. A number of compounds interact with both systems, probably because of physico-chemical similarities between adenosine and diazepam. It is concluded that of the four classic actions of benzodiazepines, the sedative and muscle relaxant (but not anxiolytic or anticonvulsant) actions could possibly be mediated by adenosine.     

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.     

Genotoxicity of N-nitrosothiazolidine in microbial and hepatocellular test systems

The genotoxicity of N-nitrosothiazolidine was studied in a microbial system and in a hepatocellular system. The compound showed positive responses in both tests, exhibiting weak mutagenicity at the lowest level tested (1 mg/plate) in the rec assay in Bacillus subtilis, and inducing statistically significant levels of DNA repair in primary hepatocyte cultures at concentrations ranging from 2 × 10^?4 to $\text{2 × 10}{}^{\mathrm{?3}}\text{m}$.     

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.     

Dansylarginine N-(3-ethyl-1,5-pentanediyl)amide: A potent and selective fluorescent inhibitor of butyrylcholinesterase

Interactions between dansylarginine N-(3-ethyl-1,5-pentanediyl)amide (DAPA) and the cholinesterases were examined by the techniques of enzyme kinetics and fluorescence spectroscopy. When tested with partially purified enzyme preparations, DAPA was a potent inhibitor of butyrylcholinesterase $\text{(}\text{IC}{}_{50}\text{= 2 × 10}{}^{\mathrm{?7}}\text{M}\text{)}$ but not of acetylcholinesterase $\text{(}\text{IC}{}_{50}\text{= 4 × 10}{}^{\mathrm{?4}}\text{M}\text{)}$. For a detailed study of the effects of DAPA on butyrylcholinesterase (BuChE), the enzyme was purified to homogeneity from horse serum, with the aid of affinity chromatography on N-methyl acridinium. The kinetics of the inhibition of purified BuChE by DAPA were complex, having both competitive and non-competitive features, and it was not possible to estimate K_i unambiguously. Spectroscopic measurements showed that the fluorescence of the dansyl moiety was strongly affected by the binding to BuChE. With excitation at 330 nm, total fluorescence emission from bound DAPA (at 450 nm and above) was 21-fold greater than from free DAPA. In a titration experiment, this enhancement of fluorescence intensity was used to calculate that each monomer of BuChE has two apparently independent DAPA-binding sites with a K_d of 4.5 × 10^?7 M. Further measurements showed that the fluorescence emission of bound DAPA was markedly blue-shifted (to 502 nm from 570 nm in free solution) and that the fluorescence lifetime of this form was greatly prolonged (to 24 nsec from 2.7 nsec). These observations indicate that the high affinity binding sites on BuChE lock DAPA in a highly non-polar environment.     

The role of lipid,free radical initiator,and oxygen on the kinetics of lipid peroxidation

The relationship between the concentration of unsaturated lipid, free radical initiator, and oxygen concentration on the kinetics of lipid peroxidation was determined. The rate of lipid peroxidation was studied with the thiobarbituric acid (TBA), diene conjugation (DC), and ferrithiocyanate (Fe-SCN) methods. The rate of peroxidation was half-order with respect to unsaturated lipid, initiator, and oxygen. The half-order relationship could be expressed as: $\text{rate}\text{= (}\text{fk}{}_1\text{k}{}_2\text{k}{}_3\text{k}{}_6{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{(}\text{azobisisobutyronitrile}\text{)}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$$\text{(}\text{RH}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{(}\text{O}{}_2\text{)}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$. The half-order relationship was found with linoleic (18:2), linolenic (18:3), and arachidonic (20:4) acids. A linear relationship existed between the logarithm of unsaturation and the rate of peroxidation. No peroxidation of linolenic acid was indicated when the DC method was employed, but was when the TBA and Fe-SCN methods were used.