Leon Moore

Inhibition of liver-microsome calcium pump by in vivo administration of CCl4, CHCl3 and 1,1-dichloroethylene (vinylidene chloride).

Abstract CCl 4 , CHCl 3 and 1,1-dichloroethylene (EDC) administered to rats resulted in a prompt dose-dependent inhibition of the ATP-dependent calcium pump isolated in the liver microsome fraction. EDC was slightly less potent than CCl 4 whereas CHCl 3 was approximately one-tenth as potent as CCl 4 . Because neither EDC nor CHCl 3 increased lipid peroxidation in vivo , increased lipid peroxidation does not appear to be a prerequisite for inhibition of the liver-microsome calcium pump. Both CCl 4 and EDC have been shown to increase liver calcium early during intoxication, but CHCl 3 has been thought to be incapable of increasing liver calcium. Pretreatment with phenobarbital increased the CHCl 3 effect of inhibiting the liver-microsome calcium pump and resulted in a several-fold increase of liver calcium levels. CDCl 3 , which is less hepatotoxic than CHCl 3 , did not increase liver calcium or inhibit the liver-microsome calcium pump in phenobarbital-pretreated animals. These results suggest that chlorinated hydrocarbons may increase liver calcium as a result of inhibition of a microsomal calcium pump and that this inhibition is associated with the hepatotoxicity of these compounds.

Cytosolic calcium after carbon tetrachloride, 1,1-dichloroethylene, and phenylephrine exposure: Studies in rat hepatocytes with phosphorylase a and quin2☆☆☆

Carbon tetrachloride (CCl4) and 1,1-dichloroethylene (DCE), both hepatotoxins, inhibit sequestration of Ca2+ by rat liver endoplasmic reticulum (ER) both in vivo and in vitro. It is possible that, as a result, cytosolic Ca2+ concentrations rise in liver cells. In experiments presented here, isolated hepatocytes were exposed to CCl4, DCE, and phenylephrine (PE), a non-hepatotoxic alpha 1-adrenergic agent that mobilizes Ca2+. Cytoplasmic Ca2+ concentrations were evaluated by two methods: indirectly by assaying the activity of glycogen phosphorylase a, and directly by monitoring the fluorescence of quin2. In primary hepatocyte cultures, CCl4, DCE, and PE exposure increased the activity of phosphorylase a at 5 min from 39 +/- 2 to 130 +/- 12, 80 +/- 13, and 97 +/- 10 nmoles PO4(3-)/mg protein/min respectively. In rat hepatocyte suspensions loaded with quin2 and exposed to CCl4, DCE, or PE, cytosolic Ca2+ concentrations were elevated within 20 sec to 0.83 +/- 0.13, 0.59 +/- 0.06 and 0.99 +/- 0.14 microM Ca2+ respectively. Basal Ca2+ levels in these cells averaged 0.25 +/- 0.03 microM. Thus, CCl4 and PE apparently increased cytosolic Ca2+ levels to approximately the same extent, whereas DCE was somewhat less effective. The durations of the effects of CCl4 and PE were examined further by determining their time courses of elevated phosphorylase a activity. In hepatocyte cultures, increased phosphorylase a activity persisted through at least 60 min following CCl4 exposure. In contrast, phosphorylase a activity returned to basal levels by 20 min after PE. Increases in cytoplasmic Ca2+ levels that are sustained rather than transient may distinguish these hepatotoxic chlorinated aliphatic hydrocarbons from non-toxic hormonal agents.

Isopropanol enhancement of cytochrome P-450-dependent monooxygenase activities and its effects on carbon tetrachloride intoxication

Acute or chronic treatment of rats with isopropanol caused a significant increase in hepatic cytochrome P-450 content and a two- to threefold increase in aniline hydroxylase and 7-ethoxycoumarin O-deethylase activities, but no significant change in ethylmorphine N-demethylase or benzo(a)pyrene hydroxylase activity. In rats treated with isopropanol and challenged with CCl4, liver toxicity of CCl4 was characteristically potentiated, as assessed by elevation of serum glutamic-pyruvic transaminase (SGPT) levels. Isopropanol pretreatment also potentiated CCl4-induced damage to the hepatic monooxygenase system. In addition to a decrease in cytochrome P-450, rats treated with isopropanol and challenged with CCl4 showed a nonspecific decrease not only in aniline hydroxylase and 7-ethoxycoumarin O-deethylase activities, but also in ethylmorphine N-demethylase, benzo(a)pyrene hydroxylase, and NADPH-cytochrome c reductase activities. These results were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized microsomes. The electrophoretic results showed that isopropanol pretreatment markedly potentiated the CCl4-caused destruction of cytochrome P-450 hemeproteins. The data strongly suggest that isopropanol increases one or more forms of cytochrome P-450 which selectively enhance the metabolism of CCl4 to an active metabolite. This active metabolite then causes a nonselective damage to the microsomal mixed-function oxidase system.

Stimulation of the renal endoplasmic reticulum calcium pump: a possible biomarker for platinate toxicity.

Antitumor platinum compounds such as cisplatin are frequently nephrotoxic. The mechanism of nephrotoxicity has not been determined. It has been proposed that some toxicants may act by interfering with the mechanisms that control cellular Ca2+ homeostasis. An important factor in the regulation of cytosolic Ca2+ is the endoplasmic reticulum (ER) calcium pump. The activity of this pump was determined by measuring ATP-dependent microsomal sequestration of 45Ca. Administration of nephrotoxic doses of platinum compounds to rats was associated with an increase in renal ER calcium pump activity. This was the earliest response observed after cisplatin treatment (it occurred within 4 hr) and preceded increases in blood urea nitrogen and serum creatinine by at least 1 day. The dose-response curve for the increase in renal ER calcium pump activity was similar to the increase in the number and size of smooth ER aggregates observed in the S3 segment of the proximal tubule 24 hr following cisplatin administration. Only minor morphological changes were observed at this time. There was a significant increase in calcium content of kidneys of rats 24 hr after treatment with a dose of cisplatin that caused a maximal increase in ER calcium pump activity. This indicates that a disruption of normal calcium homeostasis may occur before histological evidence of nephrotoxicity. Platinates that were not toxic to the kidney did not elevate renal ER calcium pump activity. It is suggested that the activity of the ER calcium pump may be a useful biomarker for cellular toxicity and may be a factor in the mechanism of toxicity.

Inhibition of liver endoplasmic reticulum calcium pump by CCl4 and release of a sequestered calcium pool

One of the earliest effects observed in rat liver after CCl4 administration is inhibition of an ATP-dependent calcium pump found at the endoplasmic reticulum. This report confirms that the amount of calcium associated with the microsomal fraction is reduced after CCl4 administration and, for the first time, demonstrates time-, dose-, and metabolism-dependent relationships between inhibition of the liver microsomal calcium pump and the amount of calcium found in the microsomal fraction. Furthermore, release of calcium from the endoplasmic reticulum is shown to cause activation of a cytoplasmic enzyme that responds to increases of ionized calcium, glycogen phosphorylase. This suggests that the endoplasmic reticulum calcium pump sequesters an intracellular pool of calcium within the endoplasmic reticulum. This pool of calcium may be released into the cytoplasm as a consequence of inhibition of the calcium pump by CCl4.

1,1-Dichloroethylene inhibition of liver endoplasmic reticulum calcium pump function

This study shows that 1,1-DCE promptly inhibits a calcium homeostatic function of liver ER. The correlation with GSH depletion and the effect of MFOS induction on calcium pump inhibition suggest that this is a direct effect of a 1,1-DCE metabolite on the calcium pump. As a result of calcium pump inhibition, calcium released from the ER may serve to trigger changes that result in a massive influx of extracellular calcium and, ultimately, cytotoxicity.

Carbon disulfide hepatoxicity and inhibition of liver microsome calcium pump

This work has shown that CS2 promptly inhibits the liver ER calcium pump only in those animals that subsequently develop hepatic necrosis. In this respect, inhibition of the ER calcium pump by CS2 resembles the actions of chlorinated hydrocarbon hepatotoxins. This lends further support to the suggestion that disruption of calcium homeostasis is an important early step in the action of at least some hepatotoxins [1-5]. CS2 appears to be the first example of a hepatotoxin other than chlorinated hydrocarbons that inhibit the liver ER calcium pump early in the course of intoxication. Finally, studies by others [17,18] suggest a mechanism by which CS2 can interact with and inhibit the liver ER calcium pump.

Halocarbon hepatotoxicity is not initiated by Ca2+-stimulated endonuclease activation

Previous studies from this laboratory have demonstrated that cytosolic Ca2+ rapidly rises to supraphysiologic levels in liver cells exposed to the hepatotoxins carbon tetrachloride (CCl4) and 1,1-dichloroethylene (DCE) in vivo and in vitro. The present study examines whether this increase in intracellular Ca2+ activates endonucleases that could initiate or contribute to the ensuing hepatotoxic events. Initial experiments demonstrated that there was no generalized breakdown of hepatic DNA in intact rats exposed to CCl4 and DCE, as assessed by the appearance of nucleosomal fragments in liver nuclear DNA separated on agarose gels. Nor was generalized fragmentation observed in DNA isolated from primary hepatocyte cultures exposed to halocarbons, except at very late times following loss of plasma membrane integrity. Endonuclease activation was further examined at a more sensitive level by specifically monitoring hypersensitive sites (HSS) in serum albumin gene. Actively transcribed genes, such as albumin in liver tissue, are extremely sensitive to attack by exogenous nucleolytic enzymes at discrete sites. We speculated that subtle halocarbon-induced endonuclease activation would first become evident at these sites. To locate HSS, DNA was digested with restriction enzymes Eco R1 or Hind III, electrophoresed on agarose gels, blotted onto nitrocellulose, and hybridized to a 32P-labeled 1400 bp rat albumin genomic clone. No cleavage at hypersensitive sites was detected in DNA isolated from rat liver or hepatocyte DNA at early times when elevations of Ca2+ were developing. Thus, these data indicate that endonuclease activation by intracellular Ca2+ and resultant nucleolytic destruction of DNA is not an early event in the hepatotoxicity produced by halocarbons.

Reversible inhibition of renal microsome calcium pump by furosemide

Abstract Renal microsomes have been shown to have an energy-dependent calcium pump activity. We now demonstrate that three diuretic compounds inhibit this calcium pump in vitro . Characterization of furosemide inhibition demonstrates that this agent is a reversible, non-competitive inhibitor of the renal microsome calcium pump. Furosemide, under similar conditions does not inhibit Na/K-ATPase activity in the renal microsome fraction. Furosemide may be useful to define function of the microsomal calcium pump in non-muscle cells.

Enhanced inhibition of hepatic microsomal calcium pump activity by CCl4 treatment of isopropanol-pretreated rats

Pretreatment of rats with isopropanol enhanced both hepatotoxicity and calcium pump inhibition after CCl4 exposure in vivo or in vitro. Animals were given isopropanol (1.25 ml/kg) 18 hr before CCl4 (0.01 to 1.0 ml/kg). CCl4 hepatotoxicity, judged as increased appearance of glutamic-pyruvate transaminase in serum, was enhanced by isopropanol pretreatment. Pretreatment of rats with isopropanol made CCl4 as much as 20- to 30-fold more potent as an inhibitor of the calcium pump. Inhibition of another endoplasmic reticulum enzyme, glucose 6-phosphatase, was also enhanced by isopropanol pretreatment. In contrast to the effect of CCl4 in control animals, in isopropanol-pretreated rats given CCl4, depletion of liver glutathione was observed. Altered CCl4 metabolism in isopropanol-pretreated animals may result in production of increased amounts of phosgene (or other metabolites) responsible for inhibition of the liver microsome calcium pump and glutathione depletion.