Prasada Rao S. Kodavanti

Protection of hepatotoxic and lethal effects of CCl4 by partial hepatectomy.

CCl4 is a hepatotoxic haloalkane, capable of producing hepatocellular fatty degeneration and centrilobular necrosis. Previous reports indicate induction of liver regeneration after 36-48 hr of CCl4 treatment, which is considered as a secondary effect. The present investigation was undertaken to evaluate the primary effects of CCl4 on hepatic DNA synthesis and to correlate liver regeneration with CCl4 toxicity. These studies were conducted in normal and actively regenerating livers using male Sprague-Dawley rats undergoing sham operation (SH), or partial (70%) hepatectomy (PH). Incorporation of 3H-thymidine (3H-T) in hepatocellular nuclear DNA and autoradiographic analyses of liver sections served as indices for hepatocellular regeneration. Initial experiments established that peak regeneration occurs at 2 days post-PH (PH2) and liver regeneration phases out by 7 days post-PH (PH7). SH and PH rats were challenged with a single ip dose of either corn oil vehicle or CCl4 at either 0.1 ml/kg (to represent subtoxic dose) or 2.5 ml/kg (to represent toxic dose). The low dose of CCl4 was not toxic and did not alter 3H-T incorporation and percentage labelled cells at 6 or 24 hours after administration to SH, PH2 or PH7 groups, indicating that there was no interference with PH-stimulated hepatocellular regeneration. The high dose of CCl4 was significantly hepatotoxic and lethal in SH rats, while in PH2 rats both hepatotoxic and lethal effects were significantly decreased. 3H-T incorporation as well as percentage labelled cells, highly stimulated by PH, were significantly decreased by high dose of CCl4. However, hepatocellular regeneration in PH2 rats treated with high dose of CCl4 was still significantly higher than SH or PH, groups by virtue of the stronger stimulatory effect of PH. In PH7 rats, where hepatocellular regeneration had returned to the SH level, the hepatotoxic and lethal effects of the large dose of CCl4 were also restored. These findings show that the progressive phase of a single high dose of CCl4 injury which normally culminates in hepatotoxic and lethal effects is significantly mitigated by previously stimulated hepatocellular regeneration. High dose of CCl4 suppresses hepatocellular regeneration at early time points after administration in contrast to the smaller subtoxic dose of CCl4. By virtue of the much stronger stimulatory effect, PH results in the protection against the hepatotoxic and lethal effects of CCl4 despite the obtunding effects of the high dose on hepatocellular regeneration.

Role of hepatocellular regeneration in chlordecone potentiated hepatotoxicity of carbon tetrachloride

Previous histomorphometric studies led us to hypothesize that suppression of hepatocellular regeneration and the repair of the hepatolobular architecture was involved besides bioactivation phenomenon in the progressive and irreversible phase of toxicity resulting from CD + CCl4 interaction. We have recently observed significant protection from CD potentiated CCl4 toxicity in animals which are stimulated for active hepatocellular regeneration. The present work is an extension of our earlier histomorphometric investigation, taking 3H-thymidine (3H-T) incorporation as a biochemical parameter to assess hepatocellular regeneration followed by autoradiographic analysis of liver sections in normal (N) or chlordecone (CD) treated (10 ppm in diet for 15 days) male rats undergoing sham (SH) or partial hepatectomies (PH). Initial experiments established that in normal (N) rats, greatest 3H-T incorporation into hepatocellular nuclear DNA occurs at 2 days post-PH which returns to basal levels by 7 days. CD treatment alone did not change this phenomenon. 3H-T incorporation into nuclear DNA and the percentage of labelled cells as evidenced by autoradiography of liver sections were significantly elevated in N rats at 1–2 h after CCl4 (100 μl/kg) administration and returned to basal level by 6 h. Serum enzymes (AST and ALT) in N rats undergoing SH and PH were not altered, but were significantly elevated in CD rats following CCl4 (100 μl/kg) administration. CCl4-induced serum enzyme elevations were significantly lower in 2 days post-PH (PH2) rats when compared to SH rats or 7 days post-PH (PH7) rats maintained on CD diet, indicating that CD potentiated CCl4 hepatotoxicity is significantly reduced in livers stimulated for regenerative activity by PH. CCl4 decreased the DNA levels significantly in SH2, SH7 and PH7 rats, but not in PH2 rats receiving CD diet. 3H-T incorporation, percentage of labelled cells and number of grains per cell were significantly decreased at 2 h in PH2 rats receiving the CD + CCl4 combination treatment, reflecting the suppression of cell proliferation after CCl4 administration to CD fed rats. These results indicate that PH affords protection against CD + CCl4 interaction. The protection against hepatotoxic and lethal effects of CD + CCl4 combination by previously stimulated hepatocellular regeneration might be explained by two consequences of stimulated cell division. First, the hepatocellular architecture is renovated by the newly divided cells. Second, by virtue of the well known resistance of the newly divided cells, the progressive phase of toxicity is inhibited. These findings are supportive of our hypothesis that suppression of hepatocellular regeneration besides bioactivation phenomenon is involved in CD + CCl4 toxicity.

Glutathione metabolism and utilization of external thiols by cigarette smoke-challenged, isolated rat and rabbit lungs☆

The purpose of the present investigation was to understand the acute effects of cigarette smoke on glutathione (GSH) metabolism and on utilization of external thiols by cigarette smoke-exposed, perfused rat and rabbit lungs. Most of the experiments were carried out using freshly drawn cigarette smoke. However, cigarette smoke condensate was used in some perfusions for the comparison of the effects between the types of exposures on utilization of external thiols. Cigarette smoke decreased GSH levels significantly (50%) without any increase in glutathione disulfide (GSSG) in both rabbit and rat lungs. In smoke-exposed rabbit lungs, protein thiol groups (protein-SH) decreased significantly (17%) without a significant change in protein-GSH mixed disulfides. However, in the rat lungs, cigarette smoke did not decrease protein-SH and protein-GSH mixed disulfides, indicating species variation in the effect of cigarette smoke. Cigarette smoke inhibited selenium-dependent and -independent GSH peroxidase activities in the rat lung (33%), but not in the rabbit lung. GSH S-transferase and GSSG reductase activities were not altered in cigarette smoke-challenged rabbit and rat lungs. gamma-Glutamylcysteine synthetase and glucose-6-phosphate dehydrogenase activities were significantly lower in smoke-exposed rat lungs as against control lungs, indicating that rat lung enzymes were more susceptible to the effects of cigarette smoke when compared to those of rabbits. N-Acetylcysteine, but not GSH, added to the perfusate significantly protected rabbit lung from smoke-induced GSH depletion. Smoke condensate added to the perfusate also caused GSH depletion in rabbit lung, and GSH or N-acetylcysteine added to the perfusion medium protected the lung indicating that GSH in the media directly interacts with condensate in the media before coming in contact with cellular GSH. These results indicate that acute smoke inhalation decreases pulmonary GSH and that the decreased GSH was not related to disulfide formation. Inhibited GSH synthesis in rat lung could account for the loss of GSH in part after exposure to cigarette smoke. The alternative pathway of GSH utilization could be conjugation with electrophilic smoke components. Thiols, like N-acetylcysteine, were protective against cigarette smoke-induced damage to the rabbit lung. The mechanism could be either by the increased GSH synthesis or by the direct delivery of sulfhydryls from N-acetylcysteine.

In vivo effects of triorganotins on calmodulin activity in rat brain

We have recently reported that the triorganotins are effective inhibitors of calmodulin (CaM) activity in vitro. The present experiments were designed to investigate the in vivo effects of triorganotins, that is, tributyltin (TBT), triethyltin (TET), and trimethyltin (TMT) on rat brain CaM activity. Male Sprague-Dawley rats were treated orally with TET (0.5, 1.0, and 1.5 mg/kg/d), TMT (0.75, 1.50, and 2.50 mg/kg/d), and TBT (0.75, 1.50, and 2.50 mg/kg/d) for 6 d and they were sacrificed 24 h after the last dose. There was significant loss of body weight in the high-dose group of the organotin treated rats. Ca(2+)-ATPase activity was determined in rat brain synaptic membranes. TET and TMT inhibited Ca(2+)-ATPase in a dose-dependent manner but TBT exhibited its inhibitory effect only at the highest dose (2.5 mg/kg/d). The inhibition of Ca(2+)-ATPase by these triorganotin compounds was reversed to control levels by the addition of CaM (5-10 micrograms) exogenously. The CaM levels of the synaptic membranes of the organotin-treated rats were not significantly changed. The data presented in this paper demonstrate that triorganotins impair the Ca(2+)-pump activity by interacting with CaM, which is a regulatory protein of Ca(2+)-ATPase. The present in vivo data and our previously reported in vitro data together indicate that triorganotins associated neurotoxicity may be due to an altered CaM activity in brain.

Altered hepatic energy status in chlordecone (kepone®)-potentiated ccl4 hepatotoxicity

Previous studies have demonstrated that increased intracellular calcium, depletion of glycogen, and suppressed hepatocellular division resulting in progression of hepatic lesion without recovery are associated with chlordecone (CD)-potentiated CCl4 hepatotoxicity. Since these phenomena are indicative of compromised hepatic energy status, the present studies were designed to investigate this possibility. Neither hepatic ATP content nor mitochondrial Mg2(+)-ATPase was altered significantly in rats maintained on diets contaminated with either CD (10 ppm), or phenobarbital (PB; 225 ppm) alone for 15 days. Similarly, CCl4 (100 microL/kg) administration alone did not alter hepatic ATP levels or mitochondrial Mg2(+)-ATPase activity in rats maintained on a normal diet. However, CCl4 administration to CD pretreated rats resulted in significantly decreased hepatic ATP content as early as 1 hr (36%), and this decrease was irreversibly progressive with time (81% at 6 hr). Oligomycin-sensitive Mg2(+)-ATPase was decreased significantly only starting at 6 hr (21%) after CCl4 administration, indicating that depletion of ATP at early time points was most likely due to rapid utilization consequent to toxic events. CCl4 administration to mirex or PB pretreated rats resulted in a smaller decrease in ATP levels (18-24%) only at 24 hr, returning to normal levels by 36-48 hr, in accord with rapid recovery from limited liver injury. These findings indicate that CCl4 administration to CD but not to PB or mirex pretreated rats results in a severely compromised energy status of the liver. The progressive and early depletion of liver ATP and the inhibition of Mg2(+)-ATPase in CD + CCl4 treated rats indicate the association of compromised energy status with altered Ca2+ homeostasis, depletion of glycogen, and suppressed cell division in CD-potentiated CCl4 toxicity.

Inhibition of Ca2+ transport associated with cAMP-dependent protein phosphorylation in rat cardiac sarcoplasmic reticulum by triorganotins

Organotin compounds have been shown to interfere with cardiovascular system. We have studied the in vitro and in vivo effects of tributyltin bromide (TBT), triethyltin bromide (TET) and trimethyltin chloride (TMT) on the cardiac SR Ca2+ pump, as well as on protein phosphorylation of SR proteins, in order to understand the relative potency of these tin compounds. All the three tin compounds inhibited cardiac SR45Ca uptake and Ca2+-ATPase in vitro in a concentration-dependent manner. The order of potency for Ca2+-ATPase as determined by IC50, is TBT (2 μM) > TET (63 μM) > TMT (280 μM). For45Ca uptake, it followed the same order i.e., TBT (0.35 μM) > TET (10 μM) > TMT (440 μM). In agreement with the in vitro results, both SR Ca2+-ATPase and45Ca uptake were significantly inhibited in rats treated with these tin compounds, indicating that these tin compounds inhibit cardiac SR Ca2+ transport. cAMP significantly elevated (70–80%) the32P-binding to SR proteins in vitro in the absence of any organotin. In the presence of organotins, cAMP-stimulated32P-binding to proteins was significantly reduced, but the decrease was concentration dependent only at lower concentrations. The order of potency is TBT > TET > TMT. In agreement with in vitro studies, cAMP-dependent32P bound to proteins was significantly reduced in rats treated with TBT, TET and TMT. SDS-polyacrylamide gel electrophoresis of the cardiac SR revealed at least 30 Coomassie blue stainable bands ranging from 9 to 120 kDa. Autoradiographs from samples incubated in the presence of cAMP indicated32P incorporation in seven bands. Of these, the band corresponding to about 24 kDa molecular weight protein decreased in its intensity with the treatment of organotins. These results suggest that triorganotins may be affecting Ca2+ pumping mechanisms through the alteration of phosphorylation of specific proteins in rat cardiac SR.

Amiodarone and desethylamiodarone increase intrasynaptosomal free calcium through receptor mediated channel

SummaryLong term amiodarone (AM) therapy has been associated with several side effects including neurotoxicity. Since AM alters Ca2+ regulated events, we have studied its effects on the compartmentation of free Ca2+ in the synaptosomes as an attempt to understand the mechanism of AM and its metabolite, desethylamiodarone (DEA)-induced neurotoxicity. Intact brain synaptosomes were prepared from male Sprague-Dawley rats. Both AM and DEA produced a concentration dependent increase in intrasynaptosomal free Ca2+ concentration ([Ca2+]i) to micromolar levels. The increase in [Ca2+]i was not transient and a steady rise was observed with time. Omission of Ca2+ from the external medium prevented the AM- and DEA-induced rise in [Ca2+]i suggesting that AM and DEA increased the intracellular [Ca2+]i due to increased influx of Ca2+ from external medium. AM- and DEA-induced increase in intrasynaptosomal [Ca2+]i was neither inhibited by a calcium channel blocker, verapamil, nor with a Na+ channel blocker, tetrodotoxin. However, the blockade of [Ca2+]i rise by AM and DEA was observed with MK-801, a receptor antagonist indicating that AM and DEA induced rise in [Ca2+]i is through receptor mediated channel. Both AM and DEA also inhibited N-methyl-D-aspartic acid (NMDA)-receptor binding in synaptic membranes in a concentration dependent manner, DEA being more effective, indicating that AM and DEA compete for the same site as that of NMDA and confirm the observation that these drugs increase intrasynaptosomal [Ca2+]i through receptor mediated channel. 45Ca accumulation into brain microsomes and mitochondria was significantly inhibited by AM and DEA, but without any effect on the Ca2+ release from these intracellular organelles. Also, both these drugs did not interfere with inositol 1,4,5-trisphosphate induced Ca2+ release from microsomes even at 10 μM concentration. These results clearly indicate that both AM and DEA increase intrasynaptosomal [Ca2+]i by an action on receptor mediated channel in plasma membrane, but not due to the release of Ca2+ from intracellular storage sites. This initial rise in [Ca2+]i, together with other changes in Ca2+ homeostasis, might be responsible for AM and DEA-induced neurotoxicity.

Effect of selected insecticides on rat brain synaptosomal adenylate cyclase and phosphodiesterase.

Previous reports from our laboratory and others clearly indicated that organochlorine insecticides such as chlordecone and DDT are potent inhibitors of ATPases involved in active ion transport. The present studies were initiated to study the effect of plictran, chlordecone, toxaphene, aldrin, dieldrin, endrin, isodrin, and telodrin on enzymes involved in cyclic AMP metabolism. Rat brain synaptosomes were prepared by Ficoll-sucrose gradient centrifugation method. Adenylate cyclase activity, which is involved in anabolism of cAMP, was determined using the radioactive method by measuring [32P]cAMP formed during hydrolysis of [32P]ATP. Phosphodiesterase activity, which is involved in the catabolism of cAMP, was estimated by measuring [3H]adenosine formed using [3H]cAMP as a substrate. Synaptosomal adenylate cyclase activity was inhibited significantly by plictran with an IC50 of 25 microM, and a maximum inhibition of 30% was observed with 50 microM chlordecone. Toxaphene, aldrin, dieldrin, endrin, isodrin, and telodrin did not affect the adenylate cyclase activity. Similarly, none of the insecticides studied inhibit the activity levels of synaptosomal phosphodiesterase. The significant inhibition of adenylate cyclase observed with plictran might be due to the tin component, since several heavy metals affect cAMP metabolism. The lack of inhibition of adenylate cyclase and phosphodiesterase with other compounds tested clearly supports our postulation that these organochlorine insecticides exert their neurotoxic action by the selective inhibition of ATPases in synaptosomes.