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Effect of toxaphene on Ca2+-ATPase activity in rat brain synaptosomes was studied in vitro and in vivo. Ca2+-ATPase in calmodulin-depleted synaptosomes was inhibited in vitro to a maximum of about 50% at 150 microM toxaphene. Substrate activation kinetics of Ca2+-ATPase in synaptosomes revealed that toxaphene inhibited the enzyme activity noncompetitively by decreasing Vmax values, without affecting the enzyme-substrate affinity. Toxaphene inhibited the calmodulin activated Ca2+-ATPase activity in a concentration-dependent manner with an IC50 of 10 microM, a concentration at which no significant effect was observed on basal enzyme activity. Nuclear and P2 fraction (synaptosomes) calmodulin levels were reduced significantly in toxaphene-treated rats. The synaptosomal Ca2+-ATPase was also reduced to about 45% in toxaphene-treated rats and the activity was restored to normal levels by the exogenously added calmodulin. These results suggest that toxaphene may cause synaptic dysfunction by interfering with calmodulin and its regulation of neuronal calcium.

The effects of chlordecone, mirex, and four photoderivatives of mirex on K+-stimulated p-nitrophenyl phosphatase (KPNPPase) in rat brain synaptosomes were determined. Additionally, the effect of chlordecone on the substrate and K+ activation kinetics and the inhibition of KPNPPase at 17, 27, and 37 degrees C were determined. The sensitivity of KPNPPase to various chemicals was highly variable. Chlordecone was the most effective inhibitor with an IC50 of 6.2 X 10(-6)M. All other chemicals showed a maximum inhibition of 20% at 20 muM concentration. The order of inhibition at 20 muM concentration was chlordecone (100%) greater than 8-monohydro mirex (20%) greater than 2,8-dihydromirex (10%) greater than mirex (10%) greater than 10-monohydro mirex (9%) greater than 5,10-dihydro mirex (2%). Double reciprocal plots of the data obtained on the effect of chlordecone on the substrate and K+ activation kinetics showed that the Vmax and Km were decreased with respect to paranitrophenyl phosphate, while it was competitive inhibition with respect to K+ activation. Chlordecone inhibited KPNPPase more at 37 degrees C than at 27 degrees C and 17 degrees C suggesting a positive temperature effect. These results suggest that chlordecone is the most effective inhibitor among its structural analogs tested on KPNPPase in rat brain synaptosomes.

The effect of chlordecone on the mouse brain synaptosomal Na+-K+ ATPase, Mg2+ ATPase and p-nitrophenyl phosphatase (PNPPase) activities was determined. In addition, the effect of chlordecone on [3H] ouabain binding to synaptosomes was also investigated. The in vitro data show that chlordecone inhibits PNPPase, Na+-K+ ATPase, and Mg2+ ATPase activities with ID50 values of 4, 5 and 7 micrograms respectively. Treatment of mice with symptomatogenic doses of chlordecone resulted in a decreased synaptosomal Na+-K+ and oligomycin-sensitive (mitochondrial) Mg2+ ATPases. The decrease was dose-dependent. The oligomycin-insensitive Mg2+ ATPase activity was unaffected either in vitro or in vivo. The binding of [3H] ouabain to synaptosomal membranes was inhibited by chlordecone in a dose-dependent manner in both in vitro and in vivo experiments. The binding of [14C] chlordecone to synaptosomes occurs even at nanomolar concentrations. The marked inhibition of brain synaptosomal ATPases and ouabain binding by chlordecone suggests that chlordecone may impair active transport mechanisms in synaptosomal membranes.

Phenobarbital (PB) or SKF-525A were injected ip into adult male rats prior to administration of 1.6 microCi [14C] chlordecone (CD) by gavage. Effects of these liver function modulators on tissue distribution of CD was assessed. In all cases, animals were sacrificed at 24 h following [14C]CD gavage. The timing and number of injections of PB or of SKF-525A were varied. Doses of PB (65 mg/kg) or of SKF-525A (75 mg/kg) were used except as noted, and controls received saline. Specific radioactivities of major tissues were assayed by scintillation counting. Rats pre-treated with a single dose of SKF-525A at 6, 12, or 24 h prior to [14C]CD distribution. Similarly, PB administered at 6 h prior to [14C]CD gavage was without effect on distribution. Rats pretreated 12 or 14 h before [14C]CD with PB appeared to have increased liver specific activities and had reduced [14C]CD levels in several other tissues. These trends were more marked in a second study, in which multiple doses of PB (3 consecutive daily doses of 65 mg/kg, the final dose 24 h prior to [14C]CD or SKF-525A (1 dose of 75 mg/kg 90 min prior to [14C]CD, followed by a second dose of 38 mg/kg at 6.5 h after [14C]CD) were given. Tissue [14C]CD levels were assayed as before; urine and feces samples were also counted and reported as percent of [14C]CD administered. SKF-525A animals had significantly high [14C] levels in digestive system, while fecal and urinary levels were significantly low. No other significant alterations were observed in these animals, except that testes levels were reduced. Livers of rats receiving multiple doses of PB had significantly elevated [14C]CD levels, and all other tissues examined had levels significantly below controls. Fecal and urinary excretion of [14C]CD was significantly depressed. Studies indicate that an inducer of hepatic metabolism can dramatically alter the distribution and hence the relative toxicity of CD.

The catecholamine-stimulated adenosine triphosphatase (ATP-ase) activities in mouse brain synaptosomes were inhibited by morphine both in vitro and in vivo. Morphine up to 10(-3) M had no effect on basal ATPase activities but at 10(-4) M significantly inhibited dopamine-sensitive ATPase activities in vitro. The morphine effect was antagonized by an opiate antagonist, naloxone. The catecholamine-sensitive ATPase activities were also inhibited by acute administration of morphine. The inhibition was dose-dependent. However, naloxone partially antagonized the morphine inhibition of depamine-sensitive ATPase activity but not norepinephrine-sensitive ATPase activity. A significant decrease in the sensitivity of synaptosomal ATPase to catecholamines was observed in mice rendered tolerant by morphine pellet implantation. The Na+,K+-ATPase was more affected by morphine as compared to Mg++-ATPase activity. The dopamine-sensitive Na+,K+-ATPase activity was restored by 50% in precipitated withdrawal mouse brain synaptosomes. Norepinephrine-sensitive ATPase activity was also restored partially in precipitated withdrawal animals. These results suggest that in mouse brain synaptosomes morphine may be interacting with ATPase at or near the catecholamine-active sites.

The effects of toxaphene, a chlorinated hydrocarbon pesticide, on the binding of ouabain and dopamine to rat brain synaptosomes enriched with Na+-K+ ATPase were investigated. For in vitro assessment of the effects of toxaphene, the synaptosomes prepared from normal rats were used. For in vivo effects the rats were fed on 0, 50, 100, 150 and 200 ppm toxaphene mixed in their daily ration for 8 weeks. At the end of treatment the rats were killed and synaptosomes were prepared. Toxaphene inhibited Na+-K+ and Mg2+ ATPases of synaptosomes in vitro and the inhibition was significant and concentration-dependent. The IC50 values were about 30 and 12 microM toxaphene for Na+-K+ and Mg2+ ATPases, respectively. However, much higher concentrations of toxaphene were required to inhibit the binding of [3H]ouabain and [3H]dopamine to synaptosomes. A 50% inhibition of ouabain and dopamine binding was obtained at 150 and 200 microM of toxaphene. The enzyme activities of synaptosomes in toxaphene-pretreated rats were decreased significantly. However, a dose-dependent decrease was not observed. The rats receiving dosages of 100 ppm and above showed a 30-40% decrease in enzyme activities. The binding of ouabain and dopamine to synaptosomes of toxaphene-pretreated rats showed no significant changes as compared to controls. The present in vitro results suggest that toxaphene may be an effective inhibitor of ATPases with substantial effects on the binding of ouabain and dopamine to rat brain synaptosomes. However, data obtained through in vivo studies do not support this contention. The reason for this discrepancy may be that the toxaphene is being rapidly metabolized or might not have reached the site of action.

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.

The immuno-biochemical effects of cobaltous chloride in rats receiving iron-sufficient and deficient diets were investigated. Rats receiving 100 ppm or more cobalt showed a significant reduction in thymus and body weights along with a marked decrease in hemoglobin, hematocrit, sheep agglutinins and plaque forming cells. These effects were more pronounced in rats receiving cobalt mixed with iron-deficient diet than those fed on iron-sufficient diet. The Na+-K+ and mitochondrial (Oligomycin-sensitive) Mg2+ATPase activities in brain and liver of rats fed with iron-deficient diets were decreased significantly. However, the ATPase activities in these tissues from rats fed with cobalt mixed with iron-sufficient diets were not altered.

Male Sprague-Dawley rats were administered cadmium chloride 0, 50, 100, 150 and 200 ppm for 30 days. At the end of the treatments, the body weight gains, serum glucose, serum protein, serum glutamic oxalacetic transaminase (SGOT) and serum glutamic pyruvic transaminase (SGPT) were determined. Renal and hepatic key gluconeogenic enzymes; viz., pyruvate carboxylase, phosphoenol pyruvate carboxykinase, fructose-1, 6-diphosphatase and glucose-6-phosphatase were also determined. A significant decrease in body weight gain in rats treated with cadmium was observed. The serum glucose and protein levels were increased in rats receiving cadmium through feed. All four key gluconeogenic enzymes were increased in both kidney and liver tissues of rats treated with cadmium. The present results indicate that cadmium may induce gluconeogenesis from non-carbohydrate sources.

In vitro sensitivity of rat brain and liver ATPases to pyrethroid insecticides, belonging to three categories based on the structural configuration, was studied. Rat brain and liver P2 fractions were prepared by the conventional centrifugation method, and rat brain synaptosomes were prepared by Ficoll-sucrose gradient centrifugation method. Na+, K+-ATPase and oligomycin-sensitive and -insensitive Mg2+- ATPases were determined in brain P2 fraction, whereas in liver P2 fraction only oligomycin-sensitive and -insensitive Mg2+-ATPase activities were determined. [3H]Ouabain binding studies were carried with rat brain synaptosomes. Most of the pyrethroid compounds tested inhibited brain and liver oligomycin-sensitive Mg2+-ATPases at micromolar concentrations. Type II compounds were more effective as compared to Type I compounds. Oligomycin-insensitive Mg2+-ATPase was not affected by any of the compounds tested except deltamethrin, which showed significant effect on liver enzyme. Na+, K+-ATPase of brain was less sensitive to these pyrethroids as compared to oligomycin-sensitive Mg2+-ATPase. [3H]Ouabain binding to rat brain synaptosomes was not affected significantly by these pyrethroid insecticides. These results suggest that inhibition of oligomycin-sensitive Mg2+-ATPase may be involved in the toxicity of pyrethroid compounds.

Endosulfan stereoisomer I (E-I) inhibited the activity of calmodulin-dependent Ca2+-ATPase to an extent of 15-55% in a concentration range of 1-20 microM under in vitro conditions without significantly affecting the basal enzyme activity. The less toxic isomer E-II produced no significant inhibition of Ca2+-ATPase activity up to a concentration of 40 microM. The inhibition of Ca2+-ATPase by E-I was noncompetitive with respect to substrate, free from the influence of calcium, and competitively inhibited calmodulin activation kinetics. Reconstitution with the exogenous addition of calmodulin (5 and 20 micrograms) restored the inhibited enzyme activity, indicating nonspecific binding of E-I with calmodulin. Both calmodulin-activated and basal enzyme activity was inhibited significantly in rats fed E-I (3 mg/kg body weight) for 15 d. These data suggest that endosulfan may modulate calmodulin-related events in neurons and result in its neurotoxicity.

Male Sprague-Dawley rats were treated with mirex, po, at 0, 5, 10, and 50 mg/kg/day in 0.4 ml of corn oil for 3 days. Forty-eight hours after the last treatment, the biliary excretion of exogenously provided polar metabolites of 14C-imipramine was suppressed at all levels of mirex in a dose-dependent manner. Biliary excretion of phenolphthalein glucuronide was suppressed at high doses of mirex. These effects of impaired biliary excretion were accompanied by increased bile flow. Persistence of the mirex-induced biliary excretory dysfunction toward otherwise readily excretable, preformed metabolites suggests aberration of transport of these substances from the liver to bile. Whereas mitochondrial Mg++-ATPase and microsomal Na+-K+-ATPase activities were both inhibited by exposure to mirex in a dose-dependent manner, the latter activity was consistently inhibited to a higher degree. These results are suggestive of mirex-induced interference with energy production and utilization in the manifestation of hepatobiliary dysfunction.

Effect of tricyclohexylhydroxytin (plictran) on Ca2+-ATPase activity was studied in rat brain synaptosomes under in vitro and in vivo conditions. Plictran inhibited basal Ca2+-ATPase activity with an IC50 value of 6 nM suggesting its interaction with calcium transport phenomenon. Plictran inhibited calmodulin (CaM) activated Ca2+-ATPase in a concentration-dependent manner. A complete reversal of calmodulin activation of Ca2+-ATPase was observed with 2-3 nM plictran. A 50 per cent decrease of CaM activated Ca2+-ATPase was observed with 0.5 nM plictran, a concentration at which no significant effect was observed on basal enzyme activity. Of all the brain fractions studied, calmodulin levels in P2 fractions alone were reduced significantly to about 75 per cent of control values in plictran treated rats. The synaptosomal Ca2+-ATPase was also decreased by 35 per cent, 42 per cent and 65 per cent in 10, 20 and 40 mg plictran kg-1 day-1 treated rats for 3 days respectively. The activity levels of Ca2+-ATPase in 10 and 20 mg plictran kg-1 day-1 treated rats were restored to normal level by exogenously added calmodulin. These results suggest that plictran may disrupt synaptic function by altering calcium and calmodulin regulated processes in the central nervous system.

In vivo effect of toxaphene on calcium pump activity in rat brain P2 fraction was studied. Male Sprague-Dawley rats (200-250 g) were dosed with toxaphene at 0, 25, 50, and 100 mg/kg X d for 3 d and sacrificed 24 h after last dose. Ca2+-ATPase activity and 45Ca2+ uptake were determined in brain P2 fraction. Toxaphene decreased both Ca2+-ATPase activity and 45Ca2+ uptake, and the reduction was dose-dependent. Both substrate and Ca2+ activation kinetics of Ca2+-ATPase indicated noncompetitive type of inhibition, as evidenced by decreased catalytic velocity but not enzyme-substrate affinity. The decreased Ca2+-ATPase activity and 45Ca2+ uptake were restored to normal level by exogenously added calmodulin, which increased both velocity and affinity. The inhibition of Ca2+-ATPase activity and 45Ca2+ uptake and restoration by calmodulin suggests that toxaphene may impair active calcium transport mechanisms by decreasing levels of calmodulin.