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.     

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].     

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.     

Studies on the mechanism of cystamine prevention of several liver structural and biochemical alterations caused by carbon tetrachloride

Cystamine and cysteamine give type II spectral changes by interaction with microsomal suspensions while CCl₄ gives a type I spectral change. Neither cystamine nor cysteamine at saturating concentrations prevent the spectral binding of small amounts of CCl₄. Cystamine and cysteamine decelerate the reduction of cytochrome P-450 by cytochrome P-450 reductase. Cystamine administration partially prevents the irreversible binding of ¹⁴CCl₄ to microsomal lipids. This preventive effect does not significantly change when the ¹⁴CCl₄ dose is increased about 4-fold. In spite of the fact that this irreversible binding is decreased by about 60% at 6 hr by the previous cystamine administration, lipid peroxidation is only slightly prevented at 6 and 10 hr after CCl₄ and prevented to about 30% at 24 hr. Cystamine partially prevented the depression of glucose-6-phosphatase activity and the destruction of cytochrome P-450 caused by CCl₄. Cystamine pronouncedly lowers body temperature of the rats, and it is as effective in preventing CCl₄-induced necrosis and fatty liver at 24 hr as it is at 48 or 72 hr after CCl₄ administration. Cystamine stabilizes lysosomes in vitro but labilizes them when administered to rats. Cysteamine labilizes lysosomes in vitro. These results may suggest that cystamine administration partially prevents several structural and biochemical alterations caused by CCl₄ because it inhibits the CCl₄-activation step to ·CCl₃ free radical, which would take place during the reduction of the cytochrome $\text{P-450}\text{CCl}{}_4$ complex by cytochrome P-450 reductase.     

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 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.     

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.     

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.     

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.     

Effect of stannous chloride on rat femur

Oral administration of stannous chloride (SnCl₂) (1.0 mg $\text{Sn}{}^{\mathrm{2+}}\text{kg}$ body weight) to rats, twice daily for 30 or 90 days, caused a significant decrease of the calcium (Ca) content and acid phosphatase activity of the femoral epiphysis but did not reduce those of the femoral diaphysis. 1.0 mg $\text{Sn}{}^{\mathrm{2+}}\text{kg}$ for 30 days produced a significant decrease of alkaline phosphatase activity of the femoral epiphysis, but did not lower that of the femoral diaphysis. The results indicate that Sn may inhibit bone formation directly in the femoral epiphysis of rats.     

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.     

Effect of fasting,diethyl maleate,and alcohols on carbon tetrachloride-induced hepatotoxicity

The effect of fasting, diethyl maleate treatment, and methanol, ethanol, iso-propanol, or tert-butanol treatment on carbon tetrachloride (CCl₄)-induced hepatotoxicity in male Sprague-Dawley rats was studied. A 24-hr fast and the administration of diethyl maleate were found to be equipotent and not additive in potentiating CCl₄-induced hepatotoxicity; this potentiation appeared to be due to a depletion of hepatic glutathione (GSH) levels. Concomitant diurnal variations in hepatic GSH content and in CCl₄-induced hepatotoxicity also suggested hepatic GSH involvement in CCl₄-induced hepatic damage. Whereas ethanol has been reported to potentiate CCl₄-induced hepatotoxicity and to lower hepatic GSH levels, the present study suggested that these effects are due to an ethanol-induced loss of body weight. Methanol, iso-propanol, and tert-butanol, on the other hand, show the same maximal potentiation of CCl₄-induced hepatotoxicity and do so without inducing a depletion of hepatic GSH content or producing a loss of body weight. In contrast to a previous report, however, methanol was found to be markedly less effective on an equimolar basis than either iso-propanol or tert-butanol in potentiating CCl₄-induced hepatotoxicity. The study suggests that GSH is an important modulator of CCl₄-induced hepatotoxicity and also suggests that methanol, iso-propanol, and tert-butanol, but not ethanol, potentiate CCl₄-induced hepatotoxicity by a mechanism that does not involve altered hepatic GSH levels.     

Mechanisms for the biodegradation of organic mercury compounds: the action of ascorbate and of soluble proteins.

Organic mercury compounds are to varying extents degraded by ascorbate and by soluble proteins. Phenyl-, methyl-, and methoxyethyl mercury are attacked by ascorbate, provided that the solution has been exposed to air in the presence of Cu²⁺, although the reaction can subsequently proceed in the absence of air. Mercury vapour (Hg⁰) and inorganic mercury (Hg²⁺) are released, and the $\text{Hg}{}^0\text{Hg}{}^{\mathrm{2+}}$ ratio varies with the three compounds, being high with methoxyethyl-mercury and very low with methylmercury. The $\text{Hg}{}^0\text{Hg}{}^{\mathrm{2+}}$ ratio is much reduced in the presence of cysteine. It is suggested that the ascorbate free radical is responsible for the reaction. γ-Globulin can degrade phenyl-mercury, but not methyl- and methoxyethylmercury, to yield Hg²⁺. The action is much stimulated by glutathione and dithiothreitol, and the evidence suggests that these act by reducing protein disulphide groups, and that the Hg²⁺ released remains attached to reactive protein thiol groups. Serum albumin appears to have a similar action. Most of the activity of the soluble fraction of a rat liver homogenate can be accounted for by the presence of thiol-activated proteins.     

Uptake,distribution, and effects of carbon tetrachloride in rainbow trout (Salmo gairdneri)

Uptake, distribution, and effects of CCl₄ were studied in rainbow trout. Carbon tetrachloride (1 ml/kg, ip) produced 5- to 10-fold increases in serum GOT, GPT, and ICD activities, whereas exposure of trout to CCl₄ in the tank water (1–80 mg/liter) produced neither mortality nor significant changes in enzyme activities. CCl₄ residue(s) appeared highest in concentration in the adipose tissue, followed by liver, brain, and spleen, and was lowest in gill regardless of the administration route. The elimination rates of ¹⁴C residue(s) from the tissue samples were most rapid in muscle ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{β = 1.7}\text{hr}$) and relatively prolonged for liver ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{β = 38.9}\text{hr}$). Maximum liver concentrations of ¹⁴C residue(s) were reached at 2 hr by either ip (1 ml/kg) or water exposure (80 mg/liter) and were 4.8 μmol/g and 0.75 μmol/g, respectively. No increase in liver triglyceride (TG) concentrations were noted at liver CCl₄ concentrations that have been associated with increased TG levels in the rat. Histological examination of tissues revealed varying degrees of liver and splenic necrosis 6 hr after administration of CCl₄.     

A fluorescent analogue of methotrexate as a probe for folate antagonist molecular receptors

A dansyl-l-lysine analogue of methotrexate, $\text{N}{}^{\mathrm{\alpha }}\text{-(}\text{4-amino-4-deoxy-10-methylpteroyl}\text{)-N}{}^{\mathrm{\epsilon }}\text{-(5-[N,N-}\text{dimethylamino]-1-naphthalenesulfonyl]-1-naphthalenesulfonyl)-}\text{l}\text{-lysine}$, is a potent inhibitor of murine L1210 dihydrofolate reductase. The dansyl fluorescence emission was enhanced approximately 3-fold with a 10 nm blue shift upon binding to L1210 dihydrofolate reductase. The fluorescent analogue was only 10-fold less potent than methotrexate in inhibiting the growth of methotrexate-sensitive and -resistant L1210 cells and competes effectively for [³H]methotrexate transport with a K_i of 7.02 μM, a value virtually identical to the K_t for methotrexate in both cell lines. In addition, strong dansyl fluorescence was found to be associated with dihydrofolate reductase from methotrexate-resistant, dihydrofolate reductase-overproducing L1210 cells following incubation of viable cells with the fluorescent methotrexate analogue for 4 hr. The results demonstrate that the dansyl-l-lysine analogue of methotrexate was rapidly transported into L1210 cells where it formed a high-affinity, fluorescent complex with intracellular dihydrofolate reductase.     

A study of norepinephrine metabolism in rat brain using a double label technique

Using a double-label procedure, the incorporation of endogenously-derived ³⁵SO₄ into phenylethylene glycol sulfates (PGS) was estimated. When [³H]norepinephrine and [³⁵S]cysteine were injected concomitantly into the brain, about 30–80 percent of the tritium and about 4 percent of the ³⁵S retained in the brain 1 hr later were in PGS. In B₆-deficient rats, the proportion of ³⁵S was increased as was the ${}^{35}\text{S}{}^3\text{H}$ ratio. Probenecid caused a significant increase in the amount of PGS found in the brain, but a minimal enrichment of ³⁵S was observed, suggesting that there was little or no effect on sulfotransferases. Cysteine in high concentrations inhibits the incorporation of tritium into PGS.     

Potentiation by carbon tetrachloride of NADPH-dependent lipid peroxidation in lung microsomes.

NADPH-dependent lipid peroxidation occurs in rat lung microsomes in vitro. Expressed per wet weight of tissue we found that lung had only $\text{1}\text{100}$ the activity of liver. However, examination of the rate of malonyl dialdehyde production with different concentrations of NADPH revealed that the kinetics of lipid peroxidation in lung microsomes was indistinguishable from that of NADPH-dependent lipid peroxidation in liver microsomes. With lung microsomes supplemented with NADPH, lipid peroxidation was potentiated by CCl₄ and inhibited by EDTA, Mn²⁺, and cytochrome c.     

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.     

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.