Redistribution of cPLA(2) in rat renal tubular cell cultures in response to PCBs.

The influence of different polychlorinated biphenyls (PCBs) upon the cytosolic phospholipase A(2) (cPLA(2)) redistribution to the particulate fraction has been investigated in rat renal proximal tubule culture cells. Treatment with Aroclor 1248 increased PLA(2) activity in the particulate fraction in a concentration-dependent manner using two radioactive substrates. However, the activity of PLA(2) in the cytosolic fraction decreased. This work also shows that 2,2',4,4',5,5'-hexachlorobiphenyl (HCB) (a di-ortho-substituted nonplanar congener) can increase the activity of PLA(2) in the particulate fraction and decrease the enzyme activity in the cytosolic fraction. The exposure of cell cultures to 3,3',4,4'-tetrachlorobiphenyl (TCB) (a non-ortho-subtituted planar congener) does not alter PLA(2) activity. These results suggest that PCBs, depending on their planar or nonplanar structures, cause a translocation of the enzyme from the cytosol to membranes. To evaluate this possibility, the contents of immunoreactive cPLA(2) were examined by immunoblot analysis in the high-speed supernatant and the particulate fraction of treated cell cultures. The increases/decreases in the amounts of cPLA(2) protein agree with the increases/decreases of PLA(2) activity previously cited. These data demonstrate that the PCB-stimulated redistribution of cPLA(2) to membranes is associated, at least in part, with the changes detected in the activity of the enzyme.

Different molecular capacity in the induction of apoptosis by polychlorinated biphenyl congeners in rat renal tubular cell cultures.

The effects of a commercial polychlorinated biphenyl (PCB) mixture (Aroclor 1248) and two individual PCB congeners were evaluated on rat renal proximal tubule culture cell viability and internucleosomal DNA fragmentation (DNA ladder) characteristic of apoptosis. Treatment with Aroclor 1248 caused the loss of cell viability and promoted apoptosis in a concentration- and time-dependent manner. The two PCB congeners assessed can also induce apoptosis. However, the extent of apoptosis generated was greater for the non-ortho-substituted planar congener (3,3',4,4-tetrachlorobiphenyl) than for the di-ortho-substituted nonplanar congener (2,2',4,4',5,5'-hexachlorobiphenyl). This correlated with the loss of cell viability since the planar compound is much more cytotoxic. The results suggest a different molecular mechanism in the induction of apoptosis by planar or nonplanar PCB congeners.

Selective fatty acid release from intracellular phospholipids caused by PCBs in rat renal tubular cell cultures.

The purpose of this study was to explore the influence of different polychlorinated biphenyls (PCBs) upon the release of oleic and palmitic acid from the intracellular lipids, which were previously labeled with [3H]oleic or [3H]palmitic acid, respectively. Studies have been realized with Aroclor 1248 (a commercial PCB mixture with 48% chlorine by weight), and two pure PCB congeners: 3,3',4, 4'-tetrachlorobiphenyl (a non-ortho-substituted planar congener) and 2,2',4,4',5,5'-hexachlorobiphenyl (a di-ortho-substituted nonplanar congener). The treatment of cells with Aroclor 1248 increased [3H]oleic acid release in a concentration-dependent manner. Our results showed that only the di-ortho-substituted congener which prefers a nonplanar configuration stimulated the release of [3H]oleic acid from the intracellular phospholipids to the culture medium, while the exposure of cell cultures to the chosen non-ortho-substituted coplanar congener did not alter the release of [3H]oleic acid to the culture medium. Finally, none of the PCBs studied could increase the release of [3H]palmitic acid from the intracellular stores significantly. The possibility that these differential alterations in the fatty acid release affect cell function during PCB exposure should therefore be postulated.

Non-muscarinic- and non-adrenergic-mediated effects of lindane on phosphoinositide hydrolysis in rat brain cortex slices.

The influence of lindane upon phosphatidylinositol hydrolysis in rat brain cortex slices has been investigated using anion-exchange chromatography in order to separate the water-soluble inositol metabolites. Acetylcholine, noradrenaline, and lindane induce the accumulation of myo-[2-3H]inositol as the water-soluble inositol metabolites. However, the cholinergic muscarinic antagonist atropine inhibited the stimulatory response of carbachol, but practically unmodified the effect that lindane has on inositol phosphate production. Also, prazosin anti-alpha 1 adrenoreceptors blocked noradrenaline-induced phosphoinositide hydrolysis, but had no effect on lindane-induced increase of inositol phosphate levels. The results suggest that lindane does not exert a general effect on the receptor-stimulated formation of inositol phosphates by both muscarinic and alpha 1-adrenergic agonists.

Effect of somatostatin on the mass accumulation of inositol-1,4,5-trisphosphate in rat hypothalamus, striatum, frontoparietal cortex and hippocampus.

Somatostatin-14 (SS) significantly increased inositol-1,4,5-trisphosphate (IP3) accumulation in rat hypothalamic, striatal, frontoparietal cortical and hippocampal slices. However, this stimulation of IP3 accumulation by SS was highest in the frontoparietal cortex and hippocampus. The effect was already significant with 0.01 microM in the frontoparietal cortex (P < 0.05) and hippocampus (P < 0.05) and the maximal accumulation was evident with 0.1 microM SS, in all areas studied. A concentration of 1 microM SS, lacked this effect in hypothalamus and striatum. SS rapidly increased IP3 accumulation in all brain areas studied. This effect was maximal at 15 s of incubation and decreased subsequently. At 60 s incubation, levels were still elevated in frontoparietal cortex and hippocampus but had returned to basal values in hypothalamus and striatum. Somatostatin-28 (SS-28) and the SS analogues, D-Trp8-D-Cys14 and SMS 201-995, also significantly stimulated IP3 accumulation although the effect of SMS 201-995 was greater than that of SS in the striatum in comparison with controls (P < 0.001 and P < 0.01, respectively). These results suggest that SS action at the hypothalamus, striatum, frontoparietal cortex and hippocampus is mediated at least in part by the accumulation of IP3, which may initiate intracellular processes responsible for some biological SS effects.

Hexachlorocyclohexanes affect the arachidonic acid release from phosphatidylinositol but not from other phospholipid classes in tubular cell cultures.

Gamma- and delta-isomers of hexachlorocyclohexane caused marked decreases in the levels of radioactive phospholipids, and increases in the levels of [3H]arachidonate incorporated into free fatty acids in rat renal tubular cells. The increased radioactivity of free fatty acids arises from the decrease of [3H]arachidonate incorporated into phosphatidylinositol, but not into phosphatidylcholine, phosphatidylserine or phosphatidylethanolamine. This fact suggests that phosphatidylinositol can be broken down to the fatty acid from the sn-2 position and lysophospholipid by a phospholipase activity increased by hexachlorocyclohexanes. The observed specific toxicant action could be achieved in two ways: (a) operating upon a specific phospholipase A2 that acts on phosphatidylinositol, but not on other phospholipids as substrates and/or (b) involving substrate-phospholipase A2 interactions. Interestingly, the observed effect of the delta-isomer was more pronounced than that of the gamma-one.

Increased intracellular glycerophosphoinositol and arachidonic acid are biochemical markers for lindane toxicity.

Lindane stimulates the release of both glycerophosphoinositol and arachidonic acid from phospholipids in rat renal proximal tubular cell cultures. When lindane was added to the culture medium, a correlation between the time-course profiles of glycerophosphoinositol and arachidonate release was found. This suggests a pathway in which phosphatidylinositol is not directly broken down by phospholipase C, but can instead be broken down to glycerophosphoinositol and arachidonic acid by phospholipase A enzymes. Therefore, a mechanism of action of lindane is through its effect on glycerophosphoinositol and arachidonic acid metabolism.

Role of lindane in membranes. Effects on membrane fluidity and activity of membrane-bound proteins.

The influence of lindane (gamma-hexachlorocyclohexane) on fluidity of plasma membranes from rat renal cortical tubules has been investigated. Preincubation with lindane increased membrane fluidity. This effect was accompanied by (i) a decrease in the transport of glucose with regard to the controls and (ii) an inhibition of the beta-adrenergic stimulatory activity upon cyclic AMP accumulation. However, a significant decrease of the membrane fluidity was found when rats were injected with lindane for 12 days. The injection of lindane exerted the opposite effect on the membrane proteins, the glucose transporter and the beta-adrenergic receptor, enhancing the glucose uptake and increasing the isoproterenol-stimulated cycle AMP accumulation. A possible explanation of the difference could involve a resistance to membrane disordering by lindane through a regulatory mechanism that would balance the activity of many lindane-sensitive proteins in insecticide-injected rats.

Lindane affects phosphoinositide turnover through a different mechanism of the phosphatidylinositol synthesis inhibition in rat renal proximal tubule cell culture.

The ability of lindane to change the metabolism of inositol phospholipids was investigated using rat renal proximal tubular cell cultures labelled with [3H]inositol. Lindane addition to the culture medium caused labelling of inositol trisphosphate, inositol bisphosphate and inositol tetrakisphosphate to decrease and that of the inositol monophosphate pool to increase. A depletion of radioactivity in phosphatidylinositols was also observed after lindane addition. Most strikingly, the addition of lindane considerably increased the levels of glycerophosphoinositol in a dose-dependent manner. The effect of lindane follows a different pattern from that of bradykinin, and it is suggested to act by stimulating phospholipase A activity(ies).

Effect of lindane on phosphatidylinositol synthesis by cerebral cortex after acute and subchronic treatment.

1. The incorporation of myo-[2-3H]inositol into phosphatidylinositols was unmodified in brain cortex miniprisms from convulsant rats. 2. However, the incorporation had increased by 300-400% in non convulsant rats which had received the same amount of lindane at a lower concentration. 3. This result suggests that phosphatidylinositols are implicated in the convulsion syndrome. 4. Experiments with lindane added in vitro were performed with both subchronically lindane intoxicated and untreated rats. 5. The results show an interesting lack of parallelism. 6. This might indicate the development of some resistance to the effects of lindane, possibly as the result of complex compensatory changes in inositol lipid biosynthesis.

Lindane decreases forskolin-stimulated cyclic AMP accumulation but does not modify Gs in rat enterocytes.

Treatment of isolated rat enterocytes with the halogenated insecticide lindane (the gamma-isomer of hexachlorocyclohexane, HCCH) did not modify the general membrane fluidity (as estimated by a fluorescence polarization technique) nor the guanine nucleotide binding regulatory protein Gs (as studied by both ADP-ribosylation of its alpha subunit by cholera toxin and Gpp[NH]p stimulation of membrane adenylate cyclase activity). However, lindane decreased in a dose-dependent manner the effect of the diterpene forskolin on direct activation of the adenylate cyclase catalytic subunit. After 5 min of cell treatment with 0.5 mM lindane, the maximal stimulatory effect of forskolin (at 100 microM) decreased by about 50%. There was a certain degree of specificity since delta-HCCH was indeed more potent, whereas dieldrin and endrin (non-lindane related halogenated compounds) behaved as lindane, and alpha- and beta-HCCH were poorly efficient on the inhibition of forskolin stimulation of adenylate cyclase activity. A similar effect of lindane was observed on receptor-stimulated cyclic AMP accumulation by using vasoactive intestinal peptide instead of forskolin. The results on a non-receptor mediated effect of lindane on the adenylate cyclase catalytic subunit itself could be related to: (i) alterations of membrane microdomains surrounding this and other integral proteins which would result in modifications of their activities; and/or (ii) a reciprocal relation between the two main routes of signal transduction so that the activation of protein kinase C (or other Ca(2+)-dependent protein kinases) by lindane would lead to phosphorylation of the adenylate cyclase catalytic subunit.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of phosphoinositide hydrolysis products induced by hexachlorocyclohexane isomers in rat brain cortex.

Water-soluble inositol metabolites were separated by anion-exchange chromatography in order to determine whether or not gamma-hexachlorocyclohexane (gamma-HCH, lindane) and related compounds affect phosphatidylinositol hydrolysis in rat brain cortex slices. Hydrolysis was increased by delta- and gamma-HCH, while alpha- and beta-HCH were inactive. Muscarinic receptor stimulation of rat cortical slices with carbachol increases inositol phosphates formation. The combined effect of carbachol and the hexachlorocyclohexane isomers together were approximately equal to the sum of the effect of each one separately. The results suggest that lindane stimulates phosphoinositide phospholipase C and/or inhibits the phosphatases implicated in dephosphorylation of inositol phosphates.

Lindane-induced modifications to membrane lipid structure: effect on membrane fluidity after subchronic treatment.

Chronic lindane intoxication by injecting subcutaneously the toxicant, resulted in an altered lipid pattern in rat ventral prostate membranes. An increase of membrane fluidity was also observed using a fluorescence polarization technique. When in vitro experiments were carried out with both treated and untreated rats, an interesting lack of parallelism was found, which could indicate the development of a resistance to membrane disordering by lindane. The observed changes in cholesterol and phospholipid composition are also consistent with the hypothesis that lindane perturbs the lipid matrix of membranes, possibly inducing complex compensatory changes in the membrane lipid composition.

The effects of different hexachlorocyclohexanes and cyclodienes on glucose uptake and inositol phospholipid synthesis in rat brain cortex.

The inositol lipids from rat brain miniprisms were deacylated and separated by anion-exchange chromatography in order to determine whether or not gamma-hexachlorocyclohexane (gamma-HCH, lindane) and related compounds affect the different phosphatidylinositols. The incorporation of myo-[2-3H]inositol into phosphatidylinositol, phosphatidylinositol monophosphate and phosphatidylinositol bisphosphate were inhibited by lindane and its delta-HCH isomer. The inhibitory effects on phosphatidylinositol synthesis are not prominent in alpha-HCH and they are not significant with the beta-HCH and cyclodienes. The results presented here indicate that the inhibitory effect of lindane and delta-HCH on the phosphatidylinositol metabolism was no exclusively due to an interference with glucose transport. Lindane-treated miniprisms showed decreased myo-[2-3H]inositol uptake and, proportionately, an even greater inhibition of inositol phospholipid synthesis. Cellular uptake can, therefore, not account for all of the lindane inhibition.

Effects of lindane on the accumulation of cyclic AMP, induced by vasoactive intestinal peptide, in isolated rat prostatic epithelial cells.

Lindane pretreatment of isolated rat prostatic epithelial cells resulted in a time- and dose-dependent impairment of the stimulation of cyclic AMP levels by vasoactive intestinal peptide (VIP); the optimal conditions for producing this impairment were found to be 5 min of cell exposure to 0.2 mm-lindane at 25 degrees C. The inhibitory effect of the insecticide was related to a decrease in VIP efficiency since the maximal cyclic AMP response (at 100 nm VIP) was about 50% of that in control cells. VIP potency was unaffected since the half-maximal cyclic AMP response was elicited at about 3 nm VIP in both the control and lindane-pretreated cells. The results indicate that lindane interferes with the cyclic AMP system in prostatic epithelium, and this could be a consequence of some interaction in the prostate gland between the two main systems for signal transduction (i.e. adenylate cyclase and phosphatidylinositol/Ca(2+)/protein kinase C).

Effects of lindane on fluidity and lipid composition in rat renal cortex membranes.

The influence of lindane upon dynamic properties of plasma membranes from rat renal cortex has been investigated using a fluorescence polarization technique. Preincubation with lindane increased membrane fluidity in a manner that is dose-dependent. This increase was higher in brush border membranes than in basolateral membranes. However, a significant decrease of the membrane fluidity was found in brush border membranes when rats were injected with lindane for 12 days. A possible solution to this difference could involve a resistance to membrane disordering by lindane through a regulatory mechanism that would balance the amount of cholesterol and phospholipid classes in the renal cortex membranes of lindane-injected rats.

Influence of lindane on the fluidity of the rat ventral prostate membranes.

The influence of lindane upon the dynamic properties of plasma membranes from rat ventral prostate has been investigated using a fluorescence polarization technique. Preincubation with lindane decreased the fluorescence polarization in a dose dependent manner. This effect, which is associated with an increased membrane fluidity, occurred in a very short period of time. Lindane also provoked a number of changes in lipid biosynthesis from acetate in the membrane. Less [1-14C]acetate was incorporated into cholesterol and more into phospholipids when this liposoluble toxicant was added to the preincubation medium. However, not all phospholipid classes were equally increased, because while the rate of acetate incorporation was greater into choline glycerophospholipids than into ethanolamine glycerophospholipids, both were higher than the rates of acetate incorporation into serine glycerophospholipids and sphingomyelin.

Effect of lindane upon the beta-adrenergic stimulation of cyclic AMP accumulation in rat renal cortical tubules caused by alterations in membrane fluidity.

The influence of lindane (gamma-hexachlorocyclohexane) on fluidity and lipid composition in rat renal cortical tubules has been investigated. Lindane increased membrane fluidity as measured by a fluorescence polarization technique using the probe diphenylhexatriene. This effect was dose-dependent and was accompanied by a 70% inhibition of the beta-adrenergic stimulatory activity upon cyclic AMP accumulation after 30 min of preincubation with lindane at 25 degrees C. Experiments with increasing concentrations of isoproterenol indicated that the efficacy, but not the potency, of the beta-adrenergic effect upon cyclic AMP accumulation was affected by lindane. Lindane toxicity could also be associated with variations in the incorporation of acetate into various lipid classes. Lindane increased acetate incorporation into phospholipids and decreased that into cholesterol.

Comparison of linoleate, palmitate and acetate metabolism in rat ventral prostate.

Rat ventral prostate incorporated (1-14C)acetate, (1-14C)palmitate and (1-14C)linoleate into different phospholipids in a time-dependent process. The rate of incorporation into total phospholipids was higher with linoleate (10.0 nmol/g) than with either palmitate (5.8 nmol/g) or acetate (4.7 nmol/g). Predominant labelling with all the radioactive substrates assayed was found in choline glycerophospholipids (PC). The radioactive profiles for linoleate in the other ventral prostate phospholipids differed from those obtained with palmitate and acetate. Specifically linoleate was incorporated into inositol glycerophospholipids plus lysoethanolamine glycerophospholipids (PI + LPE) and not into sphingomyelin (SM), while palmitate and acetate incorporated into SM but not into PI + LPE. Acetate showed the highest oxidation to CO2 whereas no differences were observed in the radioactivity incorporated into CO2 from a saturated (palmitate) or an essential unsaturated fatty acid (linoleate). These studies also show zinc-dependence by the acetate to CO2 oxidation.

Effects of lindane on the glucose metabolism in rat brain cortex cells.

The influence of 0.5 mM gamma-hexachlorocyclohexane (gamma-HCH, lindane) on glucose transport has been investigated using the analog 3-O-methyl-D-(U-14C)glucose. The glucose uptake was lineal for at least 10 sec. Preincubation of dissociated brain cortex cells with lindane decreased the transport of glucose with respect to the controls. The treatment of brain cortex cells with other organochlorine compounds indicated that the alpha-, delta-HCH isomers and dieldrin reproduced the same inhibitory pattern, while beta-HCH and endrin were inactive. The total radioactivity incorporated into CO2 from (U-14C) glucose in the cerebral cortex is also inhibited by lindane in a time dependent manner.