Combined effects of carbon tetrachloride and chlordecone on calmodulin activity in gerbil brain.

The potentiation of carbon tetrachloride (CCl4) toxicity by chlordecone (CD) pretreatment in different animal models is well established. However, these studies have only dealt with hepatotoxicity. The present study was initiated to determine whether CD preexposure potentiates CCl4 neurotoxicity in gerbils. Gerbils were chosen for the reason that the metabolism of CD in gerbil is similar to that of humans. Gerbils (50-80 g), fed on diet without or with CD (10 ppm) for 15 d, were challenged with a single dose of CCl4 (15 microliters, ip). Ca(2+)-ATPase and calmodulin (CaM) activities were determined in gerbil brain P2 fraction and cytosol, respectively, at intervals of 0.5, 2, 6, 12, and 24 h after CCl4 administration. Ca(2+)-ATPase and CaM activities were decreased at 0.5 and 2 h in both CD-preexposed and CCl4-treated gerbils. However, CaM activity returned to normal levels after 6 h and Ca(2+)-ATPase activity showed 80% recovery after 2 h. In vitro experiments showed that CCl4 alone at 5 microM concentration inhibited Ca(2+)-ATPase activity up to 50%. Combination of CD (0.5 microM) and CCl4 (1 and 5 microM) on Ca(2+)-ATPase activity showed no additive effect in vitro. Interaction between CCl4 and CaM was studied in the presence and absence of CD by monitoring NPN fluorescence. The decrease in NPN fluorescence observed with CCl4 was not potentiated by CD preincubation. These data suggest that CD does not enhance CCl4-induced alterations of Ca(2+)-ATPase and CaM activities in gerbil brain.

In vivo effects of toxaphene on calmodulin-regulated calcium-pump activity in rat brain.

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.

Toxaphene inhibition of calmodulin-dependent calcium ATPase activity in rat brain synaptosomes.

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.

Effect of tricyclohexylhydroxytin on synaptosomal Ca2+-dependent ATP hydrolysis and rat brain subcellular calmodulin.

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 vitro effects of toxaphene on mitochondrial calcium ATPase and calcium uptake in selected rat tissues.

In vitro effects of toxaphene on Ca2+-ATPase activity and 45Ca2+-uptake were studied in mitochondrial fractions of heart, kidney and liver tissues of rat. Mitochondrial fractions were prepared by the conventional centrifugation method. Ca2+-ATPase activity was determined by measuring the inorganic phosphate liberated during ATP hydrolysis. Toxaphene inhibited Ca2+-ATPase in a concentration dependent manner in all the three tissues. Substrate activation kinetics, with heart, kidney and liver tissue fractions, revealed that toxaphene inhibited Ca2+-ATPase activity non-competitively by decreasing the maximum velocity of the enzyme without affecting the enzyme-substrate affinity. Toxaphene also inhibited mitochondrial 45Ca2+-uptake in the three selected tissues in a concentration dependent manner. These results indicate that toxaphene is an inhibitor of mitochondrial Ca2+-ATPase and calcium transport in heart, kidney and liver tissues of rat.

Effect of toxaphene on the binding of 3H-labeled ouabain and dopamine to rat brain synaptosomes.

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.

Induction of rat hepatic microsomal enzymes by toxaphene pretreatment.

The effects of pretreatment of rats with toxaphene on hepatic drug metabolizing enzymes and several other parameters of the mixed function oxidase system were investigated. Adult male Sprague-Dawley rats were fed diets containing 0, 50, 100, 150 and 200 ppm of toxaphene for 14 days. The body weight gain was unaltered as well as the food consumption in all the toxaphene fed groups. There was no change in the weights of brain, kidney, heart, and testes but the liver weight was significantly increased. The thymus weight in all the toxaphene fed grups was decreased. Hydroxylation of pentobarbital and aniline was significantly enhanced in rats exposed to toxaphene. Ethylmorphine-N-demethylase activity in the toxaphene treated rats was also elevated. Enhanced hydroxylation of pentobarbital was also evident from the decreased sleeping time following pentobarbital administration. Exposure to toxaphene increased cytochrome P-450, NADPH-cytochrome c-reductase and dehydrogenase in hepatic microsomal fractions. The binding of aniline and hexobarbital to microsomes was also enhanced, suggesting that the intermediate steps in the electron-transfer system were increased. In conclusion, pretreatment of rats with toxaphene for fourteen days resulted in the induction of the hepatic mixed function oxidase system.

Adenosine triphosphatase activities in brain, kidney and liver of mice treated with toxaphene.

The sensitivity of Na+-K+ and Mg++ Adenosine Triphosphatases (ATPases) in mouse tissues to toxaphene, a highly chlorinated camphene, was determined both in vivo and in vitro. The brain and kidney Na+-K+ ATPase activities were significantly inhibited in vitro by toxaphene. Interestingly, the inhibition was not significantly increased with an increase in the concentration of toxaphene. The oligomycin-sensitive (mitochondrial Mg++ ATPase activities in mouse brain, kidney and liver fractions were significantly inhibited by toxaphene in a concentration-dependent fashion. The oligomycin-insensitive Mg++ ATPase in all tissues examined was also inhibited but less sensitive to toxaphene than mitochondrial Mg++ ATPase. In contrast to in vitro response, the brain ATPases were not altered in mice fed toxaphene by oral intubation for three days. The renal and hepatic ATPase activities were significantly decreased in toxaphene treated mice with the oligomycin-insensitive Mg++ ATPase activity being only slightly altered.