Perturbation of calcium homeostasis by CCl4 in rats pretreated with chlordecone and phenobarbital.

Male Sprague-Dawley rats were maintained on normal powdered diet or on the same diet containing 10 ppm chlordecone (CD) or 225 ppm phenobarbital (PB) for 15 days. On day 15, they received a single IP injection of a subtoxic dose of CCl4. Induction of cytochrome P-450 was greater with phenobarbital treatment than with chlordecone, but the CCl4-induced destruction of P-450 was similar in both groups and was progressive with the dose of CCl4 and with time after CCl4 administration. CCl4 given to animals on normal diet in a dose range of 25 to 200 microL/kg did not significantly alter the P-450 levels. These findings are consistent with greater bioactivation of CCl4 after the above two pretreatments There was a massive accumulation of Ca2+ in CD- and PB-pretreated animals after CCl4 administration, CD being more effective in this regard. Elevation of cytosolic Ca2+ was progressive despite the mitochondrial and microsomal sequestration of cytosolic Ca2+ at elevated levels. This perturbation of hepatocellular Ca2+ homeostasis which occurs 3 to 6 hr after CCl4 may prevent hepatocellular repair and renovation in CD-treated animals, leading to progressive hepatic lesion, hepatic failure and animal death by 36 to 48 hr at nontoxic doses of CCl4. Neither CD nor PB nor CCl4 alone affected hepatic Ca2+. These findings suggest that excessive Ca2+ accumulation may be related to the progression of hepatotoxic response to CCl4 in CD-treated animals.


Introduction
Various chemical toxins that initiate toxic events leading to liver cell death exhibit marked alterations in intracellular Ca2+ homeostasis with excessive accumulation of Ca2+ (1,2). The intracellular Ca2 ± sequestration has been implicated as a potential mediator of toxic events which lead to hepatic cell death (3,4). Previous work from this laboratory has established the remarkable potentiation of CC14 hepatotoxicity and lethality by chlordecone (chlorinated insecticide, Kepone, CD) pretreatment in male (5,6) and female rats (7). Although an enhanced bioactivation of CC14 in CD-pretreated rats was reported (8), the quantum of increased bioactivation was considered insufficient to explain the 70-fold increase in lethality in these animals as compared to phenobarbital pretreated rats which exhibited only 2-fold increase in lethality (6).
With this background, the changes in hepatocellular Ca2+ homeostasis associated with potentiation of CC14 toxicity by CD were investigated. Also, in view of the earlier findings indicating stimulated bioactivation of CCl4 in CD-treated animals (8), it was important to determine if enhanced bioactivation of CCl4 by CD pretreatment resulted in greater destruction of cytochrome P-450. PB pretreatment was used as a positive control for the potentiation of CCl4 hepatotoxicity *Department of Pharmacology and Toxicology, The University of Mississippi Medical Center, Jackson, MS 39216.
tAuthor to whom reprint requests should be addressed.

Methods
Male Sprague-Dawley rats weighing 200 to 225 g (Charles River Breeding Laboratories, Wilmington, MA) were housed in a 12-hr photoperiod on a corn cob bedding untreated with any known inducers. The animals were maintained on normal commercial powdered rat chow (Ralston Purina Rat Chow Co., St. Louis, MO) or the diet containing 10 ppm CD or 225 ppm PB prepared as described previously (5) for 15 days. On day 15 a group of rats received a single IP injection of 100 ,uL CC14/kg in corn oil vehicle (I mL/kg) and sacrificed at 0, 0.5, 2, 6, 12, 24 and 36 hr. Hepatic microsomal cytochrome P-450 was determined by the method of Omura and Sato (9). Other groups of rats received a single IP injection of 25 to 200 FL CC14/kg and sacrificed 12 hr later. Control animals received only the vehicle. Ca2+ levels in the whole liver, mitochondria, microsomes and cytosolic fraction were determined in nitric acid-digested samples by using atomic absorption spectrophotometry.

Results and Discussion
Hepatic microsomal cytochrome P-450 levels were determined at the time the animals would have received CCl4 or at various time points after CCl4 administration. CD treatment increased the hepatic microsomal P-450 by about 60%, whereas PB almost doubled P-450 levels ( Fig. 1 rats caused a progressive and time-dependent destruction of P-450 (Fig. 1A). The percent destruction remained the same in both CD-and PB-pretreated animals, despite the unequal induction of cytochrome P-450. Administration of different doses of CC14 (25-200 ,uL/kg) caused a significant destruction of P-450 at all the doses (Fig. 1B). In the rats maintained on normal diet, these doses of CC14 did not affect P-450 levels. These data are suggestive of enhanced bioactivation of CC14 in CD and PB pretreated animals. Previous studies (8,10) have shown greater in vivo and in vitro metabolism of CC14. Since this enhanced metabolism of CC14 occurs at lesser increases in P-450 levels, these findings are consistent with induction of specific form(s) of CCl4-bioactivating hemoprotein by CD (10). However, in view of remarkable differences in the potentiation of CC14 toxicity between PB and CD treatments, it is necessary to consider factors other than just bioactivation that might be playing a role in initiating or promoting hepatic cell death due to CC14 poisoning. Dietary exposure to CD or PB did not influence whole liver or subcellular Ca2+ levels. CC14 administration at a dose of 200 FLL/kg to rats maintained on normal diet caused a significant rise in Ca2+ levels, but lower doses had no effect. Previous studies suggest that these animals recover to normal by 36 hr (11,12). A significant elevation in whole liver Ca2 + levels was evident after CC14 administration to both CDand PB-pretreated rats at all four doses used ( Fig. 2A), but the increase was much higher in CD-pretreated animals. These results are consistent with our earlier observations which indicated that animals receiving CD + 100 p.L CCl4/kg exhibit total hepatic failure with extensive hepatocellular necrosis which progresses and leads to animal death by 36 hr. In contrast, the animals receiving normal diet or PB + CC14 do not show such extensive necrosis, and these animals recover later (6,10,12). Increased Ca2 + levels after CC14 administration were readily evident in mitochondria (Fig. 2B) due to a continuous influx of extracellular Ca2 + in cytosol (Fig.  2C). Microsomes also play a role in sequestering increased cytosolic Ca2+ levels (Fig. 2D); this was especially evident at higher doses of CC14. Plasma membrane changes taking place presumably due to increased lipid peroxidation or other factors consequent to CC14 bioactivation disrupt the permeability barrier with a consequent influx of Ca2+which results in massive Ca2+ accumulation in the cell. Although, hepatic mitochondria and microsomes continue to regulate ever increasing cytosolic Ca2+ by increased sequestration, the cytosolic Ca2+ levels still remain high (Fig. 2C), leading finally to cell death. Our earlier time-course histomorphometric studies (11,12) indicate that whereas animals treated with CC14 (100 ,uL/kg) recover from liver damage by virtue of hepatocellular repair and renovation, those treated with CD + CC14 do not. Instead, 3-4 hr after CC14 when hepatocellular repair would have occurred (11,12), a progressive increase in cytosolic Ca2+ occurs in animals receiving the CD + CC14 combination treatment, suggesting a cause-effect relationship. In animals receiving CC14 alone, Ca2+ homeostasis is unperturbed, allowing the hepatocellular repair, renovation and recovery.