Inhibition of synaptosome membrane-bound integral enzymes by

KORPELA ~l. Inhibition of synaptosome membrane-bound integra l enzymes by organic solvents. Scand J Work Environ Health 1989;15:64-68. The possible mechanism of the depressive effect of organic sol vents on t.he central ~e~~ous system (CNS) was studied with synaptosome membranes as a model. The change.s I~ the acuvines of the. membrane-bound integral enzymes acetylcholinesterase, total ad~nos~netnphosphatase, ~nd magnesium-activated adenosinetriphosphatase were determined after treatment Withdifferent ~on.ce.ntratlons of organic solvents in vitro. Aromatic hydrocarbons and chlorinated aliphatic hydrocarbons inhibit ed all the enzyme activities concentration dependently. Alcohols had no significant effect.at the same dose levels. The results of the present study suggest that the CNS depressive effect of organic solvents may be based on their interaction with membrane integral proteins.

Organic solvents are widely used in industry (eg, in spray painting, glueing, degreasing) and in households. Their mo st important toxic effect is the depression of the acti vity of the centr al nervou s system (CNS) by a mechanism which is still unclear . The action of a wide vari ety of compounds capable of inducing anesthesia is thought to be either on lipid or on protein components of the membrane. One of the theorie s on anesthesia suggests that the potency of anesth etics is directly propo rtional to the lipid solubility of the ane sthetic in qu estion and that changes in membrane lipid s are the critica l factors leadin g to anesthesia (I). Ho wever, the action of severa l ane sthetics does not fit this theory. Another theory, which is supported by the studies of Franks & Lieb (2)(3)(4), suggests that lipid-soluble anesthetics can interact non specifically with large hydrophobic regions of membran e proteins.
In previous studies by my co-wo rkers and I, the interaction of organic solvents with integral proteins of erythrocyte membranes has been demonstrated in human s and rats at anesthetic and lower concentrations (5)(6)(7). In the present study I have investigated the chan ges in the activities of a cetylcholinesterase (AChE), total adenosinetriphosphatase (total ATPase), a nd ma gnesium-activat ed adenosinetriphosphatase (Mg 2 +--AT Pase) in rat syna ptoso me membranes expo sed to organic solvents und er in vitro conditions to evaluate further the mechani sm of the solvent-induced eNS depression on the cell membrane. I

Preparation oj synap tosomes
Male Spraque-Dawley rats (weight 200 g) were decapitat ed . The cerebrum was gently sepa rated from the cerebellum, brain stem, and meninges and immediately pla ced in ice. Th e isolation of the synaptosomes was performed by the Percoll gradient method (8). Briefly, the samples were homogenized at 800 revolutions /min with a Tefl on-gl ass homogenizer in isolat ion medium containin g 0.32 M sucrose, 5 mM N-2-hydroxyethyl-piperazine-N ' -2-ethane-ethylenediaminetet raacet ic acid (HEPES), pH 7.5 (Sigma Chemical Co , St Lou is, Missouri, Unit ed States) and 0.1 mM eth ylenediaminetetraacetate (EDTA) (Sigma Chemical Co) , and centrifuged at I 000 g for 10 min to give a pellet contain ing nuclear and cell debris and a low-speed supernatant , which was further centrifuged at 12 000 g for 20 min to produ ce the crude mito chondral pellet, which was resuspended in 3 ml of isolation medium per gram o f o rigina l wet tissue and homogenized gently.
Th e stock solution o f isosmotic Percoll (SIP) (Sigma Chemica l Co ) wa s made by the addition o f nine part s (volum e/ volum e) of the or iginal Percoll solution to one part (volume/volume) of 2.5 M sucrose, and the lower density media by the dilution of SIP with a solution containing 0.25 M sucrose, 5 mM HEPES (pH 7.2), and 0.1 mM EDTA. The pH of the gradient solutions was adjusted to 7.5. Percoll step gradients were made by the overlayering of 4 ml of 16 % Percoli solution with 4 ml of 10 % Percoll solution. The resuspended mitochondrial pellet was diluted with eight volumes of 8.5 % PercoII solution to give a final concentration of PercoII 7.5 %, and 4.5 ml of this suspension was layered onto the 10/16 0"10 Percoll gradients. After centrifugation at 15 000 g for 20 min, the synaptosomes were collected and stored in ice until used. All the procedures used for preparing the synaptosomes were carried out at 0-4°C.

Determination of acetylcholinesterase activity
In determining the AChE activity in synaptosome membranes, the method of Ellman et al (9) was used.
In the reaction mixture [10.4 ml of synaptosome suspension and 2.6 ml of 0.1 M phosphate buffer (pH 8.0)], different concentrations of organic solvents were added with a Hamilton microsyringe. After 30 min of incubation, in 3-ml, glass-stoppered tubes at 37°C with continuous shaking, the samples were transferred into a thermostated cuvette, and 100 III of 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) (Sigma Chemical Co) was added. The absorbance at 412 nm was set at zero, and the reaction was started by the addition of 20 ul of 0.075 M acetylthiocholine iodide. The change in the absorbance was measured for 5 min. The AChE activity was expressed as moles of substrate hydrolyzed per minute per gram of protein, and the activities of the solvent-treated samples were given as the percentage of the activities of the controls.

Determination of adenosinetriphosphatase activities
In the determination of the ATPase activities, a modification of the discontinuous method based on the determination of inorganic phosphate (10) was used.
The organic solvents were added to the 2-ml reaction mixture [50 mM tris(hydroxymethyl)aminomethanehydrochloride acid (Tris-HCl) buffer (pH 7.6), 1.5 mM adenosine-5' -triphosphate (ATP) (Sigma Chemical Co), 6.0 mM magnesium chloride, 100 mM sodium chloride, 20 mM potassium chloride, and 0.1 ml of synaptosome suspension] with a Hamilton microsyringe, and the tubes were glass-stoppered and mixed. The total ATPase activity was measured with sodium (Na" ), potassium (K "), and magnesium (Mg 2 +) ions present in the reaction mixture, and Mg 2 + -AT.Pase activity with only the magnesium ion present in the mixture. After 1 h of incubation at 37°C with continuous shaking, 2 ml of ice-cold 10 % trichloroacetic acid (TCA) was added. After centrifugation at 3 000 g for 5 min, 1 ml of the supernatant was removed for the assay, and 3 ml of 0.1 N sodium acetate, 0.4 ml of 1 % molybdate/IN sulfuric acid solu-5 tion, and 2 ml of ascorbic acid were added. The absorbances of the samples were read at 770 nrn after 25 min. The enzyme activities were expressed as moles of inorganic phosphate formed per hour per milligram of protein, and the activities of the solvent-treated samples were given as the percentage of the activities of the controls.

Protein determination
The method described by Lowry et al (11) was used for the protein determinations.

Statistical methods
All the solvent concentrations were measured in triplicate, and three independent measurement series were carried out for each solvent studied. In the statistical calculations, Student's t-test (4 degrees of freedom) was used.

Acetylcholinesterase activity
The AChE activity of the control samples was O. 10 (SE 0.05) mmol of substrate hydrolyzed per minute per gram of protein.
The aromatic hydrocarbons and chlorinated aliphatic hydrocarbons inhibited the AChE activity concentration dependently. AChE was significantly The other solvents of this group had a slighter but still significant inhibitory effect, ie, trichloroethylene and tetrachloroethylene from the concentration of 3 mM up (P < 0.0 1) and 1,1, I-trichloroethane from the concentration of 6 mM (P < 0.05) up (figure I). Of the alcohols studied, l-butanol was slightly AChE-activating at the highest concentrations (20 and 30 mM). Methanol, ethanol, and I-propanol had no effect on the enzyme activities (figure I).

Adenosinetriphosphatase activities
The control samples had a total ATPase activity of 0. ethane inhibited total ATPase even more than AChE at all the concentrations. The other chlorinated aliphatic hydrocarbons were inhibitory, too, but to a less extent ( figure 2). Alcohols had no effect on enzyme activity.
Mg 2 +-ATPase was inhibited by all the aromatic hydrocarbons and, of the chlorinated aliphatic hydrocarbons, by I, I,2,2-tetrachloroethane, tetrachloroethylene, and trichloroethylene. Alcohols had no significant effect on the Mg 2 + -A'TPase activity ( figure 3).

Discussion
The results of the present study show that the studied aromatic hydrocarbons and chlorinated aliphatic hydrocarbons inhibit AchE and ATPase activities in rat synaptosome membrane concentration dependently. These results agree with previous findings with erythrocyte membrane (5)(6)(7). Organic solvents affect both erythrocyte membrane AChE, which is situated on the outer half of the membrane, and A'TPase, the location of which is mainly on the cytoplasmic side of   the erythrocyte membrane (12). Many other compounds have also been shown to inhibit both erythrocyte and synaptosome membrane enzymes, eg, local anesthetics (13)(14)(15)(16), fatty acids (17,18), and alcohols (19)(20)(21)(22)(23). The similarity of the effects of organic solvents on synaptosome membrane and erythrocyte membrane suggests that even the erythrocyte membrane is a suitable model in studies of the mechanism of the anesthetic-like CNS effects of organic solvents. The enzyme inhibition induced by organic solvents in the present study was mainly related to the lipid solubilities of the solvents. However, I, I ,2,2-tetrachloroethane, which is the most toxic of the chlorinated hydrocarbons, was a very potent enzyme inhibitor, although its lipid solubility is low in comparison to, eg, that of the aromatic hydrocarbons.
The molecular structure of a compound may be one of the factors determining its action on biological membranes. Usually, the CNS-depressive properties of solvents are enhanced by halogenation and by an increase in the length of the carbon chain. It has also been shown that the inhibition of AChE by local anesthetics is dependent on the structure of these anesthetics (24).
In the present study, the ATPase activities were inhibited more than the AChE activity, although the location of these enzymes would indicate that AChE is more easily affected.
No significant changes were found between the total ATPase and the Mg 2 + -ATPase inhibitions. Only I, I ,2,2-tetrachloroethane inhibited the total ATPase significantly more than Mg 2 t -ATPase. Synaptosomal Na + -K t -ATPase, and Mg 2 t -ATPase also have identical sensitivities to local anesthetics (24). This phenomenon may indicate a structural relationship, similarity, or identity between these two proteins. The alcohols tested had no significant effects on the enzyme activities at the concentrations used. Higher concentrations of alcohols have been shown to inhibit synaptosomal enzymes (19)(20)(21)(22).
The present results suggest that organic solvents interact with membrane-bound integral proteins even at concentrations too low to cause any changes in the lipid components of the membrane. Not even clinical concentrations of anesthetics have been found to cause any changes in the bilayer structure in short exposures (2). The studies of Greenberg & Tsong (25,26) indicate that the local anesthetic site in the membrane is an integral protein. Thus the changes in the integral enzymes AChE and ATPase may be the critical factor which determines the anesthetic CNS effect of solvents.