Assessment of the role of calcium ion in halocarbon hepatotoxicity.

Halogenated hydrocarbons (CCl4, BrCCl3, 1,1-dichloroethylene, bromobenzene) cause a wide spectrum of dysfunction and injury in liver cells. An early effect of CCl4, BrCCl3, and 1,1-dichloroethylene is destruction of the Ca2+-sequestering ability of the endoplasmic reticulum, and it has been suggested that this lesion leads to subsequent disruption of other cell functions. Work to test this hypothesis has begun in this and other laboratories. While it appears that redistribution of intracellular Ca2+ does occur following these agents, the importance of this in cell injury is not fully resolved. Current results suggest Ca2+ redistribution may be involved in some cases (e.g., surface blebbing caused by bromobenzene), but not in others (e.g., inhibition of lipid secretion by CCl4).


Introduction
Halogenated hydrocarbons such as CC14, BrCCl3, bromobenzene, 1,1-dichloroethylene, and others, are potent hepatotoxins which cause a wide spectrum of hepatocellular dysfunctions (e.g., surface blebbing, decreased lipid secretion, fatty liver, decreased protein synthesis, loss of glycogen, and ultimately, cell necrosis). It is generally considered that the hepatotoxicity of these compounds is dependent upon their metabolism by cytochrome P-450 in the mixed-function oxidase system of the liver endoplasmic reticulum (ER). In some cases (CC14, BrCC13) this enzyme catalyzes NADPH-dependent carbon-halogen bond cleavage with formation of highly reactive free radicals. It is usually considered that free radicals are too reactive to traverse significant intracellular distances, yet a number of cell functions remote from the ER are ultimately affected. Thus, it seems likely that some "toxigenic second messenger" must be formed which is capable of eliciting structural and functional abnormalities at distant loci. Considerable effort has been devoted to identification of this putative second messenger, and most investigations have focused on cytotoxic fragments derived from peroxidation of membrane lipids. However, as recently discussed (1), there is some reason to doubt that molecules of this sort are the primary mediators of haloalkane hepatotoxicity, and interest in this laboratory has turned toward another possible toxigenic second messenger, calcium ion.
The "Calcium Hypothesis" Moore et al. (2) were the first to observe that an early consequence of CC14 poisoning in rats was destruction Ca2? homeostasis in intact cells depends on complex and dynamic interactions between the plasma membrane, the mitochondria, and the ER, it is difficult to predict a priori just how loss of the Ca2 + sequestering system of the ER would affect intracellular Ca2-. One plausible hypothesis is that as the Ca2+ pool of the ER leaks into the cytoplasm, a significant increase in Ca2+ concentration in the cytoplasm would result. If so, this elevated cytosolic Ca2+ concentration might be responsible for a number of pathological developments. Alternatively, Ca2+ leaking from the ER might be accumulated by mitochondria or transported across the plasma membrane with such efficiency that cytosolic Ca2+ levels might not rise significantly. If so, the depletion of Ca2+ from the ER per se, or the overloading of Ca2+ in the mitochondria, might lead to altered functional properties of these organelles. In any event, destruction of the Ca2`sequestering activity of the ER most probably results in some redistribution of intracellular Ca2e, and it seems likely that this redistribution could have significant biological and pathological consequences.

Effect of Halocarbons on Intracellular Calcium
Despite recent advances, measurement of subcellular Ca2+ distribution and concentration is still a difficult task, and little direct experimental evidence is available regarding the effects of halocarbons on intracellular Ca"+ levels. Recent measurements in this laboratory (Brattin and Waller, in preparation) have revealed that the intracellular Ca2+ content of hepatocytes is decreased from around 3.5 nmole/mg cell protein to around 2.3 nmole/mg by a 30-min exposure to 800 ,uM CC14. This suggests that Ca2+ released from the ER is transported out of the cell by the activity of the plasma membrane Ca2+-ATPase. This observation is in contrast to several reports that CC14 administered in vivo results in a severalfold increase in liver Ca2 + content (3,4). Such an increase cannot easily be explained by an effect of CC14 on the ER, and suggests that Ca2+ balance at the plasma membrane is disturbed. Several reports of CCl4-induced inhibition of the hepatic plasmalemmal Ca2 -ATPase have appeared (5,6), but this effect does not appear to require metabolism of CCl4, and most likely is the result of "solvent effect" on the plasma membrane. In support of this, we find that high doses of CCl4 (1.5 mM or higher) do cause net accumulation of Ca2+ in isolated hepatocytes.
Of special interest is the effect of CCl4 and related compounds on the concentration of free Ca2+ in the cytoplasm of hepatoctyes. We are investigating this issue using the cytoplasmic Ca2+ indicator "quin-2" (7). Preliminary findings suggest that exposure of isolated cells to 1 mM CC14 results in a rapid (30-60 sec) increase in free cytoplasmic Ca2+. The magnitude of the increase is similar to that evoked by 10-6M phenylephrine (8). Whether this CCl4-induced increase in cytosolic Ca`is a consequence of Ca2+ release from the ER or of altered plasma membrane permeability to Ca2+ is not yet clear.

Role of Altered Calcium Distribution in Cell Dsyfunction
Jewell et al. (9), employing a spectrophotometric assay using arsenazo III as a Ca2+ indicator, found that surface blebbing in hepatocytes following bromobenzene exposure correlated with a decrease in extramitochondrial Ca2 + (a pool which probably consists primarily of ER Ca2 ). We have examined the effects of bromobenzene administration to rats on ER calcium sequestration, and found that a dose of 800 ,uL/kg produces a 28% decrease in ER Ca2+ sequestration measured 24 hr later. Whether this decrease could account for the loss of extramitochondrial Ca2+ seen by Jewell et al. is not known. Interestingly, Jewell et al. were able to mimic bromobenzene-induced surface blebbling by incubation of hepatoctyes with A23187 in the absence but not in the presence of extracellular Ca2 . This suggests that Ca2+ depletion from some intracellular site is responsible for surface blebbling, rather than an elevation in cytosolic Ca2 .
Another prominent hepatic dysfunction following CCl4 poisoning is inhibition of VLDL secretion (10). We have sought to determine whether Ca2 + might be a mediator of this dysfunction. VLDL secretion by isolated hepatocytes is inhibited much more rapidly after CC14 treatment than is the Ca2+ sequestering activity of the ER (11). Also, the dose-response curve for CC14-induced loss of VLDL secretion is left-shifted (about 2-fold) from that of ER calcium sequestration. This lack of correlation suggests, but does not prove, that destruction of the Ca + sequestering system of the ER is not required for the CCl4-dependent inhibition of VLDL secretion. Incubation of hepatocytes with A23187 in the presence of various extracellular Ca2+ levels did not result in inhibition of VLDL secretion (11). Taken together, these observations suggest that disturbances in intracellular Ca2' may not be an important step in CCl4-induced inhibition of VLDL secretion.

Role of Extracellular Calcium
While the total Ca2 + content of liver rises significantly within 1 hr of exposure to CC14, there is a much larger rise about 12 to 24 hr after poisoning (12). This late increase in liver calcium is thought to reflect a decrease in the integrity of the hepatocyte plasma membrane with concomitant influx of Ca + from the medium. Absence of Ca2+ in the medium during this time has a protective effect, diminishing cell injury as judged by trypan blue exclusion (13). However, influx of extracellular Ca2+ does not appear to be required for the earlier injuries to the cell. For example, experiments in this laboratory have shown that CCl4-induced inhibition of VLDL secretion is essentially unaffected by the absence of extracellular Ca2+ (11). Similarly, Smith et al. (14) reported that several hepatotoxins (including CCl4 and bromobenzene) were actually more toxic to hepatocytes in the absence than in the presence of extracellular Ca2 . It should be recognized that the Ca2 + stores of the ER may be important in maintaining normal cytoplasmic Ca2+ levels in the face of reduced extracellular Ca2 +, and thus destruction of the ER Ca+ pool by halocarbon poisoning might be expected to cause increased cell sensitivity to exposure to low extracellular Ca2

Summary and Conclusions
Exposure of hepatoctyes to CCl4 and certain other halocarbons results in rapid destruction of the Ca2+ sequestering activity of the endoplasmic reticulum. A priori, it seems likely that a major redistribution of intracellular Ca2+ will occur as a consequence, and it is possible that this Ca2+ redistribution has pathological effects.
Results from this laboratory reveal that low doses of CCl4 cause Ca2+ loss from hepatoctyes, but the intracellular origin of this Ca2 + is not yet certain. Exposure of isolated cells to CCl4 also results in a rapid increase in free cytoplasmic Ca'+, but, again, the reason for this increase is not yet clear. Whether such halocarboninduced changes in cell Ca2+ are responsible for any aspect of cell injury is unknown. Regarding the phenomenon of bromobenzene-induced surface blebbing, there is suggestive evidence that Ca2 + depletion from the cell might be causal. With regard to CC14 -induced inhibition of VLDL secretion, the evidence suggests that altered Ca2+ distribution may not be essential. Influx of Ca2 + from the medium does not appear to be involved in either of these two processes, but may be important in the development of later cell pathology. Thus, Ca2+ may play different roles in the development of various aspects of cell injury, and considerable work remains to be performed in order to resolve this fascinating aspect of cellular toxicology. Work in this laboratory was supported by grant No. 9-RO1-ES01821 from the National Institute of Environmental Health Sciences, NIH, USPHS. Scot D. Pencil was supported by a National Research Service Award, Medical Scientist Training Program 2T32-GM07250.