Rapid depression of rat liver microsomal calcium pump activity after administration of carbon tetrachloride or bromotrichloromethane and lack of effect after ethanol

Calcium sequestration was studied in microsomes prepared from the livers of rats given acute doses of carbon tetrachloride (CCl₄), bromotrichloromethane (BrCCl₃), or ethanol. Hepatic mitochondrial calcium uptake was also studied in ethanol-treated animals. The effects of dose and time are reported. It was found that a dose of 6 g ethanol/kg had no effect on either microsomal or mitochondrial calcium uptake during the first 20 hr after administration. After administration of 1.0–5.0 ml CCl₄/kg, calcium uptake was reduced 85% from controls in 10 min, and 3 min after administration of 2.5 ml BrCCl₃/kg calcium uptake was reduced 90% from controls. The inhibition of microsomal calcium uptake occurred at the same time as the appearance of microsomal lipid diene conjugates and slightly after the maximal incorporation of ¹⁴C from ¹⁴CCl₄ into rat liver microsomal lipids, as reported by Rao and Recknagel, 1968, Rao and Recknagel, 1969. Exp. Mol. Pathol.10, 219–228]. Decreased microsomal calcium pumping is one of the earliest signs of CCl₄ or BrCCl₃ hepatotoxicity. This finding supports the proposal that an early disturbance of calcium uptake may contribute to the chain of events leading from localized toxigenic haloalkane metabolism to the metabolic disorganization eventuating in cell death. The complete absence of any effects of acute ethanol administration on microsomal or mitochondrial calcium pump activity suggests that liver injury by ethanol does not involve lipid peroxidation in these organelles.     

Lipid peroxidation induced by some halomethanes as measured by in vivo pentane production in the rat.

Pentane production in vivo in rats was used successfully as an index to support the concept that lipid peroxidation is involved in the toxicity of some halomethanes. The time-response relationships showed that the rats had maximum pentane production by 15–30 min following ip administration of carbon tetrachloride (CCl₄), bromotrichloromethane (BrCCl₃), and chloroform (CHCl₃). BrCCl₃ and CCl₄ caused the production of the greatest amounts of pentane, CHCl₃ induced a smaller amount of pentane production, and dichloromethane (CH₂Cl₂) did not increase pentane production over that caused by injection of the mineral oil carrier. There was a good relationship (r = 0.987; p < 0.001) between the amount of pentane produced and the bond dissociation energies of BrCCl₃, CCl₄, and CHCl₃. This finding supports the concept that trichloromethyl radicals (·CCl₃) produced by homolytic cleavage of halomethanes induce lipid peroxidation. Conjugated dienes in liver, kidney, intestine, spleen, lung, and heart were measured following administration of CCl₄ and BrCCl₃. There was a good correlation (r = 0.96; p < 0.01) between pentane production in vivo and conjugated diene formation in the liver. The liver accounted for 85 and 94% of the total conjugated dienes measured after administration of CCl₄ and BrCCl₃, respectively. The amount of pentane produced during a 30-min period following injection of 1 mmol of CCl₄ corresponded to about 0.2% of the lipid peroxides, measured as conjugated dienes, in the liver following the same time period. These results suggest that liver is also the principal site of pentane production.     

Hepatotoxicity of bromotrichloromethane--bond dissociation energy and lipoperoxidation

Bromotrichloromethane (BrCCl₃) is much more potent than CCl₄ or CHCl₃ as a liver poison. Bond dissociation energies for cleavage of H-CCl₃, Cl-CCl₃, and Br-CCl₃ are in the order H-CCl₃ > Cl-CCl₃ > Br-CCl₃. A low bond dissociation energy implies a greater tendency of the given bond to cleave homolytically. In vitro, BrCCl₃ is 200 times more potent than CCl₄ in promoting peroxidative decomposition of rat liver microsomal lipids. CHCl₃ has virtually no potency as a pro-oxidant in vitro. BrCCl₃ produces in rats about three times the degree of liver microsomal lipid peroxidation than does CCl₄, at equivalent doses. CHCl₃ does not produce lipid peroxidation in vivo. Administration of BrCCl₃ in very low doses (0.025 ml/kg of body weight) produces a dramatic fall in liver microsomal cytochrome P-450. Rats with low levels of cytochrome P-450 due to prior administration of BrCCl₃ are completely resistant to large, and otherwise lethal doses of CCl₄. A lethality study is presented which suggests that BrCCl₃ may have extrahepatic sites of action.     

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

CCl₄, CHCl₃ 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 CCl₄ whereas CHCl₃ was approximately one-tenth as potent as CCl₄. Because neither EDC nor CHCl₃ increased lipid peroxidation in vivo, increased lipid peroxidation does not appear to be a prerequisite for inhibition of the liver-microsome calcium pump. Both CCl₄ and EDC have been shown to increase liver calcium early during intoxication, but CHCl₃ has been thought to be incapable of increasing liver calcium. Pretreatment with phenobarbital increased the CHCl₃ effect of inhibiting the liver-microsome calcium pump and resulted in a several-fold increase of liver calcium levels. CDCl₃, which is less hepatotoxic than CHCl₃, 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.     

Studies on the mechanism of carbon tetrachloride autoprotection: Effect of a protective dose of carbon tetrachloride on lipid peroxidation and glutathione peroxidase-glutathione reductase

The effect of a small protective dose of carbon tetrachloride (CCl₄) on hepatic microsomal lipid peroxidation induced by a larger dose of CCl₄ 24 h later was investigated. Endogenous hepatic microsomal lipid peroxidation as well as CCl₄-stimulated lipid peroxidation were decreased by treatment of rats with a small protective dose of CCl₄. Furthermore, glutathione peroxidase and glutathione reductase activities were not altered by single or repeated administration of CCl₄. The results suggest that destruction of cytochrome P-450 by the protective dose of CCl₄ prevents bioactivation of a subsequent dose of the hepatotoxin and, therefore, protects against lipid peroxidation and cell damage.     

Alterations in ATP-dependent calcium uptake by rat renal cortex microsomes following ochratoxin A administration in vivo or addition in vitro.

A disruption of calcium homeostasis, leading to a sustained increase in cytosolic calcium levels, has been associated with cytotoxicity in response to a variety of agents in different cell types. We have observed that administration of a single high dose or multiple lower doses of the carcinogenic nephrotoxin ochratoxin A (OTA) to rats resulted in an increase of the renal cortex endoplasmic reticulum ATP-dependent calcium pump activity. The increase was very rapid, being evident within 10 min of OTA administration and remained elevated for at least 6 hr thereafter. The increase in calcium pump activity was inconsistent with previous observations that OTA enhances lipid peroxidation (ethane exhalation) in vivo, a condition known to inhibit the calcium pump. However, no evidence of enhanced lipid peroxidation was observed in the renal cortex since levels of malondialdehyde and a variety of antioxidant enzymes including catalase, DT-diaphorase, superoxide dismutase, glutathione peroxidase, glutathione reductase and glutathione S-transferase were either unaltered or reduced. In in vitro studies, addition of OTA to cortex microsomes during calcium uptake inhibited the uptake process although the effect was reversible. Preincubation of microsomes with NADPH had a profound inhibitory effect on calcium uptake but inclusion of OTA was able to reverse the inhibition. Changes in the rates of microsomal calcium uptake correlated with changes in the steady-state levels of the phosphorylated Mg2+/Ca(2+)-ATPase intermediate, suggesting that in vivo/in vitro conditions were affecting the rate of enzyme phosphorylation.     

Carbon tetrachloride induced liver alterations in rats pretreated with N,N'-diphenyl-p-phenylenediamine

Glucose-6-phosphatase (G6P-ase) activity of liver microsomes was decreased 15 min following carbon tetrachloride administration, but it was unaffected at 5 min. At the latter time, however, lipid peroxidation was already detectable in liver microsomal lipids. Treatment of rats with N,N′-diphenyl-p-phenylenediamine (DPPD) prior to CCl₄ poisoning did not prevent the peroxidation of microsomal lipids induced in vivo by carbon tetrachloride. Nor did it suppress the depression of G6P-ase. Malonic dialdehyde (MDA) production by liver homogenates or the microsome plus supernatant fraction was decreased by the addition of DPPD in vitro even at concentrations of 1.5 × 10^?5 and 1.5 × 10^?6 M. The in vitro pro-oxidant effect of CCl₄ was suppressed only when DPPD was present at the concentration of 1.5 × 10^?4 M. Pretreatment of rats with DPPD significantly decreased the liver steatosis induced by carbon tetrachloride but it was ineffective on the fall in the plasma triglycéride level caused by the halogenated hydrocarbon. The effect of DPPD on the liver steatosis was manifested both when DPPD was administered in oil and when it was given as an aqueous suspension. Only in the former instance was DPPD found in appreciable amounts in the microsomal lipids, while in the latter case it occurred only in traces in the microsomal fraction. It is suggested that the ability of DPPD to decrease CCl₄-induced liver triglyceride accumulation is not related to an action of DPPD on the membranes of the endoplasmic reticulum of the liver cell.     

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.     

Perturbation of liver microsomal calcium homeostasis by ochratoxin A

The effect of ochratoxin A on hepatic microsomal calcium sequestration was studied both in vivo and in vitro. The rate of ATP-dependent calcium uptake was inhibited by 42-45% in ochratoxin A intoxicated rats as compared to controls. In the presence of NADPH, addition of ochratoxin A (2.5 to 100 microM) caused a concentration-dependent inhibition of calcium uptake (28-94%) by untreated rat liver microsomes. The rate of NADPH-dependent lipid peroxidation, measured as malondialdehyde formed, was also greatly enhanced by ochratoxin A. Various agents that inhibited ochratoxin A enhanced lipid peroxidation were also able to block the destruction of calcium uptake activity. Lipid peroxidation enhanced by ochratoxin A was also accompanied by leakage of calcium from calcium-loaded microsomes. These results suggest that ochratoxin A disrupts microsomal calcium homeostasis by an impairment of the endoplasmic reticulum membrane probably via enhanced lipid peroxidation.     

Carbon tetrachloride-induced changes in adrenal microsomal mixed-function oxidases and lipid peroxidation

Studies were carried out to compare the effects of carbon tetrachloride (CCl₄) in vivo and in vitro on adrenal and hepatic microsomal metabolism in guinea pigs. CCl₄ administration in vivo decreased adrenal and hepatic microsomal cytochrome P-450 concentrations and lowered benzphetamine (BZ) demethylase and benzo[a]pyrene (BP) hydroxylase activities in both tissues. NADPH-cytochrome c reductase activity was decreased in hepatic but not in adrenal microsomes. Addition of CCl₄ to adrenal or hepatic microsomes in vitro produced a type I difference spectrum suggesting binding of CCl₄ to cytochrome(s) P-450; its magnitude was far greater in adrenal than in liver. Incubation of adrenal or hepatic microsomes in vitro with CCl₄ alone had little or no effect on mixed-function oxidase activity or on lipid peroxidation. However, when microsomes were incubated with CCl₄ + NADPH, the rates of BZ and BP metabolism were decreased, cytochrome P-450 concentrations were decreased, and lipid peroxidation was increased. The effects of CCl₄ + NADPH on enzyme activities were greater in adrenal than in hepatic microsomes. Addition of 1.0 mM EDTA or 0.1 mM MnCl₂ to the incubation medium blocked the effects of CCl₄ + NADPH on lipid peroxidation in adrenal and liver but had no effect on the decreases in mixed-function oxidase activities. The results indicate the following: (1) the adrenal cortex in the guinea pig is an active site of CCl₄ metabolism; (2) CCl₄ metabolism results in a loss of microsomal enzyme activities in the adrenal as well as liver; and (3) lipid peroxidation is not obligatory for the CCl₄-mediated destruction of microsomal enzymes.     

Effect of phenobarbital or 3-methylcholanthrene pretreatment on carbon tetrachloride-induced lipid peroxidation in rat liver.

The enhancement of carbon tetrachloride hepatotoxicity following phenobarbital pretreatment is associated with an increase in lipid peroxidation in vivo when CCl₄ is administered orally or by inhalation. However, pretreatment with 3-methylcholanthrene did not increase in vivo lipid peroxidation following CCl₄ administration by the oral or inhalation route. CCl₄ stimulated lipid peroxidation, as determined by malonaldehyde formation in vitro, and was increased by phenobarbital pretreatment but not by 3-methylcholanthrene pretreatment. These data support a relationship between microsomal drug metabolizing activity and alterations in hepatic injury and lipid peroxidation following CCl₄ administration.     

Potentiation by carbon tetrachloride of NADPH-dependent lipid peroxidation in lung microsomes.

NADPH-dependent lipid peroxidation occurs in rat lung microsomes in vitro. Expressed per wet weight of tissue we found that lung had only $\text{1}\text{100}$ the activity of liver. However, examination of the rate of malonyl dialdehyde production with different concentrations of NADPH revealed that the kinetics of lipid peroxidation in lung microsomes was indistinguishable from that of NADPH-dependent lipid peroxidation in liver microsomes. With lung microsomes supplemented with NADPH, lipid peroxidation was potentiated by CCl₄ and inhibited by EDTA, Mn²⁺, and cytochrome c.     

Influence of oxygen on the inhibition of liver microsomal activation of carbon tetrachloride by the catechol 2-hydroxyestradiol-17β

In NADPH-supplied rat liver microsomes irreversible protein and lipid binding of ¹⁴C-CCl₄-metabolites continuously increase with decreasing oxygen concentrations. In aerobic suspensions of rat liver microsomes and NADPH the catechol steroid 2-hydroxyestradiol-17β effectively inhibits not only CCl₄-initiated lipid peroxidation but also protein and lipid binding of ¹⁴C-CCl₄-metabolites. With decreasing oxygen concentrations the inhibitory potency of 2-hydroxyestradiol-17β on all three parameters connected with activation of CCl₄ is reduced. In order to obtain the same inhibitory effect more catechol compound is needed under low than under high oxygen tensions. Anaerobically, 2-hydroxyestradiol-17β was found not to inhibit irreversible protein and lipid binding and lipid peroxidation was not detectable in absence of oxygen. An oxidized catechol metabolite rather than the catechol molecule itself may be responsible for the enzymatic inhibition of the activation step of CCl₄ or may interfere with reactions of the CCl₃-radicals formed.     

Biochemical aspects of the protective action of propyl gallate on liver injury in rats poisoned with carbon tetrachloride.

Liver injury in rats intoxicated with a high dose of CCl₄ (2.5 ml/kg, po) is partially prevented by propyl gallate. The accumulation of hepatic triglycerides and the release of liver enzymes into the plasma are more effectively inhibited by this antioxidant in rats given a smaller dose of the hepatotoxin (0.25 ml/kg, po). The antioxidant activity of propyl gallate, administered in vivo, estimated by means of the production of malonyl dialdehyde in liver homogenates in vitro, has been found to decrease progressively after treatment. The concentration of nonesterified fatty acids in the serum is not affected by dosing with propyl gallate. However, propyl gallate releases the block in triglyceride secretion from the liver that occurs after administration of CCl₄. Furthermore, both the uptake of CCl₄ by the liver and the early steps of the free radical reaction (incorporation of labeled metabolites of ¹⁴CCl₄ and double bond shifting in microsomal unsaturated lipids) are unchanged by concomitant dosing with propyl gallate. This free radical scavenger, when administered in discrete doses, more markedly influences both CCl₄ liver damage and the efficiency of the hepatic drug metabolizing enzyme system. This effect in vivo is consistent with the reported in vitro interaction of propyl gallate with microsomal electron transport. These findings indicate that propyl gallate partially interferes with the enzymes bound to the endoplasmic reticulum, but affects the secondary phenomena of lipid peroxidation at the level where lipoproteins are secreted and/or the permeability of plasma membrane is altered.     

Carbon tetrachloride and bromotrichloromethane toxicity: Dual role of covalent binding of metabolic cleavage products and lipid peroxidation in depression of microsomal calcium sequestration

We have investigated the importance of covalent binding and lipid peroxidation on the depression of microsomal calcium sequestration associated with in vitro metabolism of ¹⁴CCl₄. Studies with CBrCl₃ are also reported. In aerobic systems, promethazine was used to block lipid peroxidation, measured as malondialdehyde (MDA) generation. Effects of low levels of lipid peroxidation were tested in Fe²⁺ -supplemented systems free of halogenated hydrocarbons. The results indicate that microsomal calcium sequestration can be depressed significantly by metabolism of either CCl₄ or CBrCl₃ in the absence of MDA generation, or by lipid peroxidation occurring in the absence of halogenated hydrocarbons.     

Evidence for carbon tetrachloride-induced lipid peroxidation in mouse liver

The involvement of lipid peroxidation in the mechanism of carbon tetrachloride-induced hepatotoxicity has been a point of controversy. Previous investigators have reported an absence of lipid peroxidative degradation products in mice after exposure to carbon tetrachloride and have used this evidence against the hypothesis that lipid peroxidation is an integral part of the events that cause tissue damage. We have compared the extent of lipid peroxidation caused by carbon tetrachloride between Sprague-Dawley rats and three strains of mice (A/J, BALB/cJ, and C57B1/6J) in in vitro and in vivo systems. Hepatic microsomes isolated from fasted mice of each strain produced more malondialdehyde (a degradation product of lipid peroxidation) per mg microsomal protein than those isolated from fasted rats at all times of incubation with CCl₄. In vivo lipid peroxidation was estimated by the lipid conjugated diene content in hepatic microsomes from the rat and three strains of mice. Increased conjugated diene formation was observed in microsomal lipids of these animals after intraperitoneal injection of CCl₄ (1 ifml/kg as a 20% solution in corn oil) when compared to animals given only corn oil, but no differences were found in the amount of conjugated dienes between mice and rats. Our observations show that the CCl₄-treated mouse undergoes hepatic lipid peroxidation at least as well as the rat, and indicate that lipid peroxidation cannot be excluded as a mechanism of carbon tetrachloride hepatotoxicity as has been claimed on the basis of its ineffectiveness in the mouse.     

The protective effects of diethyldithiocarbamate and cycloheximide on the multiple hepatic lesions induced by carbon tetrachloride in the rat.

Carbon tetrachloride-induced liver lesions in rats were studied by light and electron microscopy in relation to time and dose of administrated CCl₄ and as modified by treatment with diethyldithiocarbamate (DEDTC) or cycloheximide (CH) at different times before or after CCl₄. DEDTC prevented CCl₄-induced collapse of the membranes of the endoplasmic reticulum and necrosis in zone 3 (centrilobular). DEDTC did not prevent endoplasmic reticulum dilatation (balloon cell formation) but induced a shift in its location from zones 1 and 2 to zone 3. A similar location of balloo cells with only a rare necrotic cell was seen with very low doses of CCl₄ (2.5 to 5 μl/kg). Balloon cell formation did not precede the appearance of zone 3 necrosis with any dose of CCl₄. CH delayed the induction of CCl₄-induced necrosis for 1 day, but the development of membranelesions in the endoplasmic reticulum was unaffected. Fatty change (steatosis) was present in all three zones and was decreased but not prevented by pretreatment with either DEDTC or CH. The evidence supports the concept that liver injury induced by CCl₄ is a composite of at least three lesions: necrosis, balloon cell formation, and steatosis. Study of these three discrete lesions may help in defining the mechanism by which CCl₄ damages the liver.     

Resveratrol ameliorates carbon tetrachloride-induced acute liver injury in mice

Present investigation aimed to evaluate the hepatoprotective potential of resveratrol (30 mg/kg, po) in mice following two different routes (po and sc) of exposure to carbon tetrachloride (CCl₄, 1.0 ml/kg). Administration of CCl₄ caused significant increase in the release of transaminases, alkaline phosphatase, lactate dehydrogenase, γ-glutamyl transpeptidase, creatinine kinase, total bilirubin, urea and uric acid in serum. Significantly enhanced hepatic lipid peroxidation and oxidized glutathione with marked depletion in reduced glutathione were observed after CCl₄ intoxication. It was also found that CCl₄ administration caused severe alterations in liver histology. Hepatic injury was more severe in those animals who received CCl₄ by oral route than those who exposed to CCl₄ subcutaneously. Resveratrol treatment was able to mitigate hepatic damage induced by acute intoxication of CCl₄ and showed pronounced curative effect against lipid peroxidation and deviated serum enzymatic variables as well as maintained glutathione status toward control. Treatment of resveratrol lessened CCl₄ induced damage in liver. The results of the present study suggest that resveratrol has potential to exert curative effects against liver injury.     

Carbon tetrachloride-induced lipid peroxidaton: Simultaneous in vivo measurements of pentane and chloroform exhaled by the rat

Simultaneous measurements of pentane, an index of lipid peroxidation, and chloroform (CHC4), an index of carbon tetrachloride (CCl₄) metabolism, were made on samples of breath from rats injected with 30μl CCl₄/100 g body wt. In the first 3 h after administration of CCl₄, rats fasted overnight metabolized less CCl₄ and exhaled less pentane than did fed rats. Multiple injections of CCl₄ decreased both the metabolism of CCl₄ to CHCl₃ and the level of in vivo lipid peroxidation following administration of a subsequent dose of CCl₄. Dietary vitamin E provided limited protection from CCl₄-induced lipid peroxidation and had no effect on the rate of CCl₄ metabolism to CHCl₃.     

Liver membrane calcium transport in diquat-induced oxidative stress in vivo

Hepatic necrosis is produced rapidly by 0.1 mmol/kg diquat in male Fischer-344 rats but not Sprague-Dawley rats, yet massive oxidant stress is caused by diquat in both strains of rat. Liver plasma membrane calcium uptake was unaltered by diquat treatment in either strain. However, diquat inhibited ATP-dependent calcium sequestration by hepatic microsomes from Fischer rats by 33% (33 +/- 2 versus 50 +/- 2 nmol/mg/20 min), whereas liver microsomal calcium uptake in Sprague-Dawley rats was not decreased by diquat treatment. Microsomes of diquat-treated Fischer rats showed marked increases in calcium efflux versus controls (k efflux = 0.115 +/- 0.027 versus 0.051 +/- 0.005 min-1; p less than 0.025), but microsomes of diquat-treated Sprague-Dawley rats exhibited no significant change in efflux rate. Calcium uptake by the endoplasmic reticulum of saponin-permeabilized isolated hepatocytes was diminished in parallel with diquat cytotoxicity. Significant increases in 11-, 12-, and 15-hydroxy 20:4 fatty acids were found in liver microsomes isolated after diquat treatment in vivo and administration of desferrioxamine (0.24 mmol/kg, intraperitoneally) administered before diquat significantly protected against the inhibition of microsomal calcium uptake. These data suggest a possible role for Fenton chemistry and lipid peroxidation in this feature of diquat-generated hepatic damage in vivo.