EXPERIMENTAL
AND
Effects
MOLECULAR
23,
PATHOLOGY
of Chlordane
144-153
(1975)
Pretreatment on the Carbon Tetrachloride’
Hepatotoxicity
of
RICHARD J. STENGER, MARK PORWAY, ELIZABETH A. JOHNSON, AND RANAJIT K. DATTA Departments and
of
Mount
Pathology, Sinai
Beth School
Israel of
Medical
Medicine
Received
of
]anuary
Center, the 21,
City
New University
York,
New of
New
York
10003
York
1975
Male albino rats weighing approximately 200 g received intraperitoneal injections of chlordane (25 mg/kg) in olive oil on each of three successive days. Controls included animals given only olive oil and untreated rats. Twenty-four hours after the last dose, augmented hepatic drug-metabolizing enzyme activity in chlordane-treated rats was reflected in uiuo by a reduction in zoxazolamine-paralysis time and in vitro by an increased hepatic microsomal cytochrome P-450 level. The insecticide-treated animals did not, however, display any increase of hepatic microsomal NADPHcytochrome c reductase activity. In chlordane-treated rats, electron microscopy revealed overt proliferation of smooth endoplasmic reticulum in the hepatocytes, particularly in those located in the central one-third to one-half of the liver lobules. Olive oil-treated controls showed no alterations in paralysis time, microsomal enzyme activity, or hepatocellular ultrastructure, when compared to the untreated animals. Identically prepared chlordane-treated and control rats then were challenged with an intraperitoneal injection of carbon tetrachloride (0.5 ml of a 25% solution of CC14 in olive oil). Some animals from each group were killed at 4 hr after the toxic challenge; and it was determined that, in each category, there was a sharp drop in hepatic microsomal cytochrome P-450 but no change in NADPH-cytochrome c reductase, as compared to prechallenge levels. The reduction in cytochrome P-450 was most striking in the insecticide-stimulated rats. The remaining animals from each CCL-injected group were sacrificed at 48 hr after the toxic challenge. Histologic slides prepared from their livers revealed more extensive hepatocellular necrosis in the chlordane-pretreated rats than was found in either the olive oil-pretreated rats or the animals with no treatment prior to CCL administration. It was concluded that chlordane can induce smooth-membrane proliferation and can enhance drug-metabolizing enzyme systems in rat liver and that these changes are associated with an enhanced hepatotoxic response to CCL administration. It was suggested that a sharp fall in hepatic microsomal cytochrome P-450 might serve as a relatively early indicator of toxic injury in an induced liver.
INTRODUCTION It is well known that many drugs stimulate proliferation of hepatocellular smooth endoplasmic reticulum and induce increased activity of hepatic microsomal drug-metabolizing enzyme systems (Conney, 1967; Conney, Davison, Gastel, and Burns, 1960; Fouts, 1963; Fouts and Rogers, 1965; Remmer and Merker, 1965). This has been thoroughly studied in the case of phenobarbital 1 This work was supported in part by U. 16774 from the National Institute of Arthritis,
S. Public Health Service Research Grant Metabolism, and Digestive Diseases. 144
Copyright 0 1975 by Academic Press.Inc. All rights of reproduction in any form reserved.
AM-
CHLORDANE
EFFECT
ON Ccl,
HEPATOTOXICITY
145
(Herdson, Garvin, and Jennings, 1964; Jones and Fawcett, 1966; Kato and Takanaka, 1968; Orrenius, 1965a, 196513;Orrenius and Ericsson, 1966; Orrenius, Ericsson, and Emster, 1965; Remmer and Merker, 1963a, 1963b; Staubli, Hess, and Weibel, 1969), but has also been explored to some extent with various insecticides, including DDT ( Ortega, 1966)) dieldrin ( Hutterer, Schaffner, Klion, and Popper, 1968)) and chlordane ( Fouts and Rogers, 1965). More recently the effect of carbon tetrachloride (CC&) upon the hepatic microsomal drug-metabolizing enzyme systems has come under scrutiny (Glende, 1972a, 197213; Recknagel and Glende, 1973; Recknagel, Glende, Ugazio, Koch, ,and Srinivasan, 1974; Slater and Sawyer, 1969), and it has been discovered that pretreatment of animals with certain inducers of these enzyme systems can considerably increase Ccl, damage to mammalian liver. Several investigative groups have explored the effects of phenobarbital in this regard (Garner and McLean, 1969; Hurwitz, 1972; Lal, Puri, and Fuller, 1970; Stenger, Miller, and Williamson, 1970; Stenger and Johnson, 1971), and an earlier study warned that pretreatment with an insecticide, viz., DDT, might also increase CCll hepatotoxicity (McLean and McLean, 1966). The major purpose of the present investigation was to determine whether pretreatment with another insecticide, viz., chlordane, would increase the hepatotoxic potential of Ccl,. MATERIALS
AND METHODS
Male albino (Holtzman) rats, weighing approximately 200 g, received intraperitoneal injections of technical chlordane 2 (25 mg/kg, prepared as a 1:30 dilution in olive oil) on each of three successive days. Control rats were injected with equivalent doses of pure olive oil; untreated animals were also used. On day 4 of the experiments, rats in each category were tested with regard to: (a) their paralysis times, (b) the status of their hepatic microsomal enzyme activities, and (c) their hepatocellular ultrastructure. For paralysis time evaluations, rats in each category were given a single intraperitoneal injection of zoxazolamine (100 mg/kg) prepared as described by Coney, Davison, Gastel, and Burns (1960). The time was measured from the point of complete immobilization to the point at which the rat moved to another location in the cage. To check the status of the enzyme systems, NADPH-cytochrome c reductase and cytochrome P-450 levels were determined for microsomal suspensions prepared from the livers of rats in each treatment category. Livers from three rats were pooled for each determination. The livers were cut into small pieces, rinsed with 1.15% KC1 solution, and homogenized in 4 vol of KCl. The homogenates were centrifuged at 12,000g for 25 min, and the resultant supernatant fractions were then centrifuged at 105,OOOgfor 60 min. The pellets thus formed were suspended in one-third the original volume of KCl, and the protein content was determined by the method of Lowry, Rosebrough, Farr, and Randall ( 1951). Aliquots of these microsomal suspensionswere diluted with 0.1 M sodium phosphate buffer, pH 7.0, to a final concentration of 1 mg protein/ml of microsomal suspension. 2 Technical chlordane, a mixture of chlorinated hydrocarbons consisting of isomers chlordane ( 1,2,4,5,6,7,8,8-octachloro-4,7-methano-3a,4,7,7a-tetrahydroindane) and closely lated compounds, was kindly provided by Velsicol Chemical Corporation, Chicago, IL,.
of
re-
146
STENGER
ET AZ,.
NADPH-cytochrome c reductase activity was measured by a method similar to that of Mule, Redman, and Flesher ( 1967). The cuvette contained 2.8 ml of 0.1 M sodium phosphate buffer, pH 7.5, 0.025 ml of cytochrome c (from horse heart, type VI, Sigma) (20 mg/ml in 1.15% KCl), and 0.2 ml of the diluted microsomal suspension. The reaction was started by adding and mixing 0.01 ml of reduced nicotinamide adenine dinucleotide phosphate (NADPH, trisodium salt, type I, Sigma) (20 mg/ml in 4% sodium bicarbonate). The increase in optical density at 550 nm was measured for 3 min. The controls were run with 4% sodium bicarbonate (without NADPH) and their values were subtracted from the experimental values. A unit of NADPH-cytochrome c reductase activity was defined as the amount of enzyme that would cause a A OD of O.l/mg protein/ minute. One-centimeter cuvettes were used in all assayswhich were carried out in duplicate, and the mean values were accepted. To determine cytochrome P-450 content, the method of analysis was similar to that of Omura and Sato ( 1964). Portions of the aforementioned diluted microsomal suspensions were delivered into cuvettes of l-cm light path, and 10 mg of sodium hydrosulfite (dithionite) were added to both sample and reference cuvettes. Carbon monoxide was passed through a KOH trap and then bubbled through the microsomal suspension in the sample cuvette for 30 sec. Both cuvettes were capped with Teflon, and the difference in absorbance at 450 and 490 nm (A OD 450-490 nm) was determined in a Beckman model DB-G (dual-beam) spectrophotometer. Livers from rats in each category were also prepared for ultrastructural evaluation according to previously described methods (Millonig, 1961; Stenger, 1966). Finally, two studies were done to determine the extent of Ccl, toxicity in rats pretreated with chlordane, as compared to rats pretreated with olive oil alone and to rats that received no pretreatment. For these studies, on day 4 of the experiments, rats in each category received a single intraperitoneal injection of CC& (0.5 ml of a 25% solution in olive oil). Representatives from each category were killed at 4 hr after the CC14 challenge, and the status of their microsomal enzyme systems was compared to the prechallenge status. Both NADPHcytochrome c reductase and cytochrome P-450 were measured by the procedures described above. Representcative animals from each treatment category were also sacrificed at 48 hr after CC& injection, and sections from their livers were fixed in cold (4°C) 10% formalin, buffered to pH 7.0. Paraffin sections were stained with hematoxylin and eosin, and the extent of parenchymal necrosis in each liver was assessedby light-microscopic examination. RESULTS As shown in Table I, there was a highly significant reduction in zoxazolamine paralysis times for the chlordane-treated rats, as compared to either the olive oil-treated controls or the untreated animals. There was, however, no significant difference in paralysis times between the latter two groups. Table II indicates that the chlordane treatment caused a slight increase of hepatic microsomal NADPH-cyctochrome c reductase activity, but the difference from the controls was not statistically significant.
CHLORDANE
EFFECT
ON CClr HEPATOTOXICITY TABLE
147
I
EFFECT OF PRETREATMENT ON ZOXAZOLAMINE PARALYSIS TIMES Pretreatment?
No. of rats
Chlordane Olive oil None
19 17 14
a Chlordane: oil: equivalent intraperitoneally b Mean and E Comparison
Paralysis time (min) * 68.57 f 7.81 198.71 f 38.94 196.29 f 40.77
P < 0.005c P < 0.005”
25 mg/kg in olive oil, intraperitoneally, on each of three successive days. Olive amounts of pure olive oil. On day 4, all animals received zoxazolamine (100 mg/kg) as a single injection. SE are given for each category. with chlordane-treated rats by Student’s t test.
Table III reveals that the chlordane treatment did produce a highly significant increase of hepatic microsomal cytochrome P-450, when compared to either olive oil-treated or untreated animals. The chlordane injections also elicited a marked proliferation of hepatocellular smooth endoplasmic reticulum. This was particularly striking in the central one-third to one-half of the liver lobules (Fig. 1). More peripherally located hepatocytes failed to reveal any consistent differences between the chlordanetreated rats and the two control groups. Table IV demonstrates that, at 4 hr after Ccl, challenge, the hepatic microsomal NADPH-cytochrome c reductase activities were not significantly different from the prechallenge levels in any category (compare with TabIe II). On the other hand, Table V indicates that, at 4 hr after the toxic challenge, CC& administration had caused a marked and statistically significant drop in the hepatic microsomal levels of cytochrome P-450 in the chlordane-pretreated rats, as compared to the prechallenge level recorded in Table III. The reductions caused by Ccl, in the control groups, though less impressive, were also significantly below their prechallenge levels. With the light microscope, liver sections from 14 chlordane-CC&treated rats were compared with sections from 10 olive oil-CCL-treated rats and with sections from 10 animals subjected only to the CC14 challenge. As illustrated in TABLE EFFECT OF TREATMENT Treatment0 Chlordane Olive oil None
ON
II
HEPATIC MICROSOMAL NADPH-CYTOCHROME No. of determinationsb 8 8 8
c REDUCTASE
Units” 1.635 f 0.194 1.348 f 0.255d 1.445 f 0.300d
0 Chlordane: 25 mg/kg in olive oil, intraperitoneally on each of three successive days. Olive oil: equivalent amounts of pure olive oil. b In each category, livers from three rats were pooled for each determination. c Mean and SE are given. A unit of NADPH-cytochrome c reductase activity was defined as the amount of enzyme that would cause a A OD of O.l/mg protein/minute at 550 nm. d No significant difference from chlordane-treated rats by Student’s t test.
STENGER
148
TABLE EFFBCT
OFTRESTMENTON
Treatment”
HEPATIC
ET AL. III MICROSOM.IL
No. of determinations*
Chlordane Olive oil None
8 8 8
TBBLE
f 0.011 zk 0.006 f 0.006
P < 0.005d
on each
P < O.OOld
of three
successive
days.
Olive
for each determination. 1 test.
IV
Unit+
6 6 6
1.827 1.437 1.487
a Chlordane: 25 mg/kg in olive ‘oil, intraperitoneally, oil: equivalent amounts of pure olive oil. On day 4, all solution in olive oil) intraperitoneally and were killed 4 b In each category, livers from three rats were pooled c Mean and SE are given. A unit of NADPH-cytochrome amount of enzyme that would cause a A OD of O.l/mg d By Student’s t test, there is no significant difference Table II.
TABLE
Chlordane Olive oil None
nmc
No. of determinations*
Chlordane Olive oil None
Pretreatment”
A 013 450-490
ADMINISTRATION ON HEPATIC I~/~ICROSOM.~L NADPH-CYTOCHROI~IE c REDLJCF.~SE OF~RETRE.ITED AND UNTREATED R.~Ts
Pretreatment*
EFFECT
P-460
0.143 0.059 0.073
(1 Chlordane: 25 mg/kg in olive oil, intraperitoneally, oil : equivalent amounts of pure olive oil. b In each category, livers from three rats were pooled c Mean and SE are given. d Comparison with chlordane-treated rats by Student’s
EFFECTOFCCI~
CYTOCHROME
on each of three successive days. Olive animals received CC14 (0.5 ml of a 25y0 hr later. for each determination. c reductase activity was defined as the protein/minute at Fi50 nm. in any category from levels reported in
V
OF CCla ADMINISTR.~TION ON HEP~TIC ~L~ICROSOMAL P-450 OFPRETREATED AND UNTREATED RATS No. of determinations* 6 6 6
f 0.370d zk 0.20gd f 0.220d
A OD
0.049 0.036 0.043
u Chlordane: 2.5 mg/kg in olive oil, intraperitoneally, on oil: equivalent amounts of pure olive oil. On day four, all 25% solution in olive oil) intraperiotoneally and were killed * In each category, livers from three rats were pooled for c Mean and SE are given. d By Student’s t test, there is a significant difference in Table III.
* f f
CYTOCHROME
450-490
0.003 0.001 0.003
nmc
P < 0.001* P < 0.01*
P < 0.01*
each of three srxcessive animals received CC14 4 hr later. each determination. each
category
from
levels
days. Olive (0.5 ml of a
reportedin
CHLORDANE
EFFECT
ON Ccl,
HEPATOTOXICITY
149
FIG. 1. Portion of a parenchymal cell from the liver of a rat treated with chlordane for three successive days. This electron micrograph demonstrates the marked proliferation I of smooth endoplasmic reticulum that characterized many of the hepatocytes located in the central one-third to one-half of the liver lobules of such rats. The densely stained glycc bgen particles are sparsely distributed through the smooth-membrane mass. Also included in the field are intact mitochondria, several small lipid bodies (left, upper), and a portion of the cell’s nucleus (right, upper). ( X 14,350).
Fig. 2, the livers of rats pretreated with chlordane showed extensive hep;*tocellular necrosis involving approximately one-half of each liver lobule. Anin lals pretreated with olive oil and the untreated rats responded to the same dose : of
150
STENGER
ET AL.
FIG. 2. Portion of a liver lobule from a rat treated with chlordane for three successive days and then challenged with Ccl,. The animal was sacrificed 48 hr after the toxic challenge. This light micrograph illustrates the extensive parenchymal necrosis that characterized the livers of the chlordane-CCL-treated rats. At the center of the field is a central vein which is surrounded by necrotic parenchyma involving roughly one-half of the liver lobule. The necrotic tissue is demarcated from viable cells by a rim of polymorphonuclear leukocytes and hydropic hepatocytes. ( X 120).
CC& with less extensive damage, usually limited to the immediate vicinity the central vein.
of
DISCUSSION Technical chlordane, as used in this study, is a mixture of chlorinated hydrocarbons consisting predominantly of chlordane isomers. Since other halogenated hydrocarbons have been shown to induce proliferation of hepatocellular smoothsurfaced membranes, the stimulation demonstrated in the present study came as no surprise. In fact, Fouts and Rogers (1965) have previously reported such induction due to chlordane. This degree of smooth-membrane proliferation is often accompanied by enhancement of the hepatic drug-metabolizing capabilities, so the shortened zoxazolamine paralysis time and the augmented microsomal cytochrome P-450 demonstrated in this investigation can best be interpreted as evidence of such enhancement by the chlordane treatment. The failure of chlordane to cause an increase in the microsomal NADPH-cytochrome c reductase activity is somewhat perplexing, as both this enzyme and cytochrome P-450 are considered to
CHLORDANE
EFFECT
ON
Ccl,
HEPATOTOXICITY
151
function in the hepatic microsomal drug-metabolizing chain ( Orrenius, I965a). Such dissociation has, however, been observed with other inducers, particularly with certain polycyclic hydrocarbons (Baron and Tephly, 1969, 1970). The chlordane-pretreated rats suffered more extensive hepatic necrosis when challenged with Ccl, than did either of the control groups. This result also fulfilled expectations, inasmuch as other inducers of hepatic drug-metabolizing enzyme systems, e.g., phenobarbital or benzpyrene, have been reported to augment the liver-damaging effects of Ccl, (Garner and McLean, 1969; Lal, Puri, and Fuller, 1970; Pitchumoni, Stenger, Rosenthal, and Johnson, 1972; Stenger, Miller, and Williamson, 1970). Perhaps more to the point is the earlier observation of McLean and McLean (1966) that pretreatment with DDT also renders the liver more susceptible to Ccl4 injury. The prompt decrease of hepatic microsomal cytochrome P-450 after CC& challenge of the chlordane-stimulated animals was striking. Within 4 hr of the toxic challenge, a consistent and statistically significant drop was demonstrable. This observation suggests that an abrupt drop in cytochrome P-450 might serve as a relatively early indicator of haloalkane injury to the liver of a stimulated animal. Moreover, a similar, albeit less impressive, fall in the cytochrome P-450 value was observed in the unstimulated animals after Ccl* administration. Finally, this prompt effect of Ccl, administration upon cytochrome P-450 might be taken as further evidence that the locus of Ccl, metabolism is at or near the cytochrome P-450 locus on the hepatic microsomal membrane, as proposed by Garner and McLean (1969) and more recently ,advocated by Recknagel and Glende ( 1973). In this regard, it is also of interest that our studies failed to demonstrate any significant effect of either chlordane or CC& on hepatic microsomal NADPH-cytochrome c reductase activity. In the case of CC&, our findings are in accord with those reported by Glende (1972a). ACKNOWLEDGMENT Ms.
The authorsthank Mr. OsmayYalis for his help in the preparationof the micrographs,and Caryl Towner for her help in the preparationof the manuscript. REFERENCES J., and TEPHLY, T. R. ( 1969). The role of hemesynthesisduring the induction of hepatic microsomalcytochrome P-450 and drug metabolismproduced by benzpyrene.
BARON,
.Biochem. Biophys. Res. Commun. 36, 526532.
J., and TEPHLY, T. R. ( 1970). Further studieson the relationshipof the stimulatory effects of phenobarbitaland 3,4-benzpyreneon hepatic hemesynthesisto their effects on hepatic microsomaldrug oxidations.Arch. Biochem. Biophys. 139, 410420. CONNEY, A. H. ( 1967). Pharmacologicalimplications of microsomalenzyme induction. BARON,
Pharmacol.
Rev. 19, 317-366.
DAVISON, C., GASTEL, R., and BURNS, J. J. (1960). Adaptive increasesin drug-metabolizingenzymesinduced by phenobarbitaland other drugs.J. Pharmacol. Erp.
CONNEY,
A. H.,
Ther. 130, l-8.
Fov~s, J. R. ( 1963). Factors influencingthe metabolismof drugs in liver microsomes. Ann. N.Y. Acad. Sci. 104, 875-880.
Fonts, J. B., and ROGERS, L. A. ( 1965). Morphologicalchangesin the liver accompanying stimulationof microsomaldrug metabolizingenzyme activity by phenobarbital,chlordane, benzpyreneor methylcholanthrenein rats. J. Pharmacol. Exp. Ther. 147, 112119.
152
STENGER
ET
AL.
I ncreased GARNER, R. C., and MCLEAN, A. E. M. ( 1969). susceptibility to carbon tetrachloride poisoning in the rat after pretreatment with oral phenobarbitone. Biochem. Pharmacol. 18, 645-650. GLENDE, E. A., JR. ( lY72a). Carbon tetrachloride-induced protection against carbon tetrachloride toxicity. The role of the liver microsomal drug-metabolizing system. Biockm. Pharmacol. 21, 1697-1702. GLENDE, E. A., JR. ( 1972b). On the mechanism of carbon tetrachloride toxicity-Coincidence of loss of drug-metabolizing activity with peroxidation of microsomal lipid. Biochem. Pharmacol. 21, 2131-2138. HERDSON, P. B., GARVIN, P. J., and JENNINGS, R. B. (1964). F ine structural changes in rat liver induced by phenobarbital. Lab. Invest. 13, 1032-1037. HURWITZ, A. (1972). Effects of microsomal enzyme inducers on animals poisoned with hepatotoxins. Toxicol. Appl. Pharmacol. 22, 339-346. HUTTERER, F., SCHAFFNER, F., KLION, F., and POPPER, H. ( 1968). Hypertrophic, hypoactive smooth endoplasmic reticulum: a sensitive indicator of hepatotoxicity exemplified by dieldrin. Science 161, 1017-1019. JONES, A. L., and FAWCETT, D. W. ( 1966). Hypertrophy of the agranular endoplasmic reticulum in hamster liver induced by phenobarbital (with a review on the functions of this organelle in liver). .I. Hi&o&em. Cytochem. 14, 215-232. KATO, R., and TAKANAKA, A. (1968). Effect of phenobarbital on electron transport system, oxidation and reduction of drugs in liver microsomes of rats of different age. J. Biochem. (Tokyo) 63, 406-408. LAL, H., Puq S. K., and FULLER, G. C. (1970). Enhanced toxicity of carbon tetrachloride inhalation after phenobarbital pretreatment. Pharmacol. Res. Commun. 2, 143-147. LO-Y, 0. H., ROSEBROUGH,N. J., FARR, A. L., and RANDALL, R. J. ( 1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. MCLEAN, A. E. M., and MCLEAN, E. K. (1969). The effect of diet and l,I,l-trichloro-2,2-bison microsomal hydroxylating enzymes and on sensitivity of (p-chlorophenyl) ethane ( DDT) rats to carbon tetrachloride poisoning. Biochem. J. 100, 564-571. MILLONIG, G. ( 1961). The advantages of a phosphate buffer for OsOI solutions in fixation. J. Appl. Phys. 32, 1637. MuL$, S. J., RED~IAN, C. M., and FLESHER, J. W. ( 1967). Intracellular disposition of H’morphine in the brain and liver of nontolerant and tolerant guinea pigs. J. Pharmacol. Exp. Ther. 157, 459471. OMURA, T., and SATO, R. ( 1964). The carbon monoxide binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. J. Biol. Chem. 239, 2370-2378. ORRENIUS, S. (1965a). On the mechanism of drug hydroxylation in rat liver microsomes. J. Cell Biol. 26, 713-723. ORRENIUS, S. ( 1965b). Further studies on the induction of the drug-hydroxylating enzyme system of liver microsomes. J, Cell Biol. 26, 725-733. ORRENIUS, S., and ERICSSON, J. L. E. ( 1966). Enzyme-membrane relationship in phenobarbital induction of synthesis of drug-metabolizing enzyme system and proliferation of endoplasmic membranes. J. Cell Biol. 28, 181-198. ORRENIUS, E., ERICSSON, J. L. E., and ERNSTER, L. (1965). Phenobarbital-induced synthesis of the microsomal drug-metabolizing enzyme system and its relationship to the proliferation of endoplasmic membranes. A morphological and biochemical study. J. Cell Biol. 25, 627639. ORTEGA, P. ( 1966). Light and electron microscopy of dichlorodiphenyltrichloroethane ( DDT) poisoning in the rat liver. Lab. Invest. 15, 657-679. PITCHUMONI, C. S., STENGER, R. J., ROSENTHAL, W. S., and JOHNSON, E. A. (1972). Effects of 3,4-benzpyrene pretreatment on the hepatotoxicity of carbon tetrachloride in rats. J. Phamacol. Exp. Ther. 181, 227-233. RECKNAGEL, R. 0. and GLENDE, E. A., JR. ( 1973). Carbon tetrachloride hepatotoxicity: An example of lethal cleavage. CRC Critical Reviews in Toxicology 2, 263-297. RECKNAGEL, R. O., GLENDE, E. A., JR., UGAZIO, G., KOCH, R. R., and SRINIVASAN, S. (1974). New data in support of the lipoperoxidation theory for carbon tetrachloride liver injury. Israel J. Med. Sci. 10, 301-311.
CHLORDANE
EFFECT
ON
Ccl,
HEPATOTOXICITY
lTi3
REMME% H., and MERKER, H. J. ( 1963a). Enzyminduktion und vermehrung von endoplasmatischem reticulum in der leberzelle wahrend der behandlung mit phenobarbital (luminal). Klin. Wochenschr. 41, 276-283. REMMER, H., and MERKER, H. J. ( 196313). Drug-induced changes in the liver endoplasmic reticulum associated with drug-metabolizing enzymes. Science 142, 1657-1658. REMMER, H., and MERKER, H. J. ( 1965). Effect of drugs on the formation of smooth endoplasmic reticulum and drug-metabohzing enzymes. Ann. N. Y. Ad. Sci. 123, 79-96. SLATER, T. F., and SAWYER, B. C. ( 1969). The effects of carbon tetrachloride on rat liver microsomes during the first hour of poisoning in uino, and the modifying actions of promethazine. Biochem. J. 111, 317324. STXUBLI, W., HESS, It., and WEIBEL, E. R. (1969). Correlated morphometric and biochemical studies on the liver cell. II. Effects of phenobarbital on rat hepatocytes. J. Cell Biol. 42, 92-112. STENGER, R. J, ( 1966). Ultrastructural alterations within hepatic parenchymal cells after carbon tetrachloride poisoning. In “Methods and Achievements in Experimental Pathology” ( Bajusz, E., and Jasmin, G., Eds.), Vol. 1, pp. 677-700. S. Karger, Basel. STENGER, R. J., and JOHNSON, E. A. (1971). Further observations upon the effects of phenobarbital pretreatment on the hepatotoxicity of carbon tetrachloride. Erp. Mo2. Puthol. 14, 220-227. STENGER, R. J., MILLER, R. A., and WILLIAMS, J. N. ( 1970). Effects of phenobarbital pretreatment on the hepatotoxicity of carbon tetrachloride. Exp. Mol. Puthol. 13, 242-252.
AND
Effects
MOLECULAR
23,
PATHOLOGY
of Chlordane
144-153
(1975)
Pretreatment on the Carbon Tetrachloride’
Hepatotoxicity
of
RICHARD J. STENGER, MARK PORWAY, ELIZABETH A. JOHNSON, AND RANAJIT K. DATTA Departments and
of
Mount
Pathology, Sinai
Beth School
Israel of
Medical
Medicine
Received
of
]anuary
Center, the 21,
City
New University
York,
New of
New
York
10003
York
1975
Male albino rats weighing approximately 200 g received intraperitoneal injections of chlordane (25 mg/kg) in olive oil on each of three successive days. Controls included animals given only olive oil and untreated rats. Twenty-four hours after the last dose, augmented hepatic drug-metabolizing enzyme activity in chlordane-treated rats was reflected in uiuo by a reduction in zoxazolamine-paralysis time and in vitro by an increased hepatic microsomal cytochrome P-450 level. The insecticide-treated animals did not, however, display any increase of hepatic microsomal NADPHcytochrome c reductase activity. In chlordane-treated rats, electron microscopy revealed overt proliferation of smooth endoplasmic reticulum in the hepatocytes, particularly in those located in the central one-third to one-half of the liver lobules. Olive oil-treated controls showed no alterations in paralysis time, microsomal enzyme activity, or hepatocellular ultrastructure, when compared to the untreated animals. Identically prepared chlordane-treated and control rats then were challenged with an intraperitoneal injection of carbon tetrachloride (0.5 ml of a 25% solution of CC14 in olive oil). Some animals from each group were killed at 4 hr after the toxic challenge; and it was determined that, in each category, there was a sharp drop in hepatic microsomal cytochrome P-450 but no change in NADPH-cytochrome c reductase, as compared to prechallenge levels. The reduction in cytochrome P-450 was most striking in the insecticide-stimulated rats. The remaining animals from each CCL-injected group were sacrificed at 48 hr after the toxic challenge. Histologic slides prepared from their livers revealed more extensive hepatocellular necrosis in the chlordane-pretreated rats than was found in either the olive oil-pretreated rats or the animals with no treatment prior to CCL administration. It was concluded that chlordane can induce smooth-membrane proliferation and can enhance drug-metabolizing enzyme systems in rat liver and that these changes are associated with an enhanced hepatotoxic response to CCL administration. It was suggested that a sharp fall in hepatic microsomal cytochrome P-450 might serve as a relatively early indicator of toxic injury in an induced liver.
INTRODUCTION It is well known that many drugs stimulate proliferation of hepatocellular smooth endoplasmic reticulum and induce increased activity of hepatic microsomal drug-metabolizing enzyme systems (Conney, 1967; Conney, Davison, Gastel, and Burns, 1960; Fouts, 1963; Fouts and Rogers, 1965; Remmer and Merker, 1965). This has been thoroughly studied in the case of phenobarbital 1 This work was supported in part by U. 16774 from the National Institute of Arthritis,
S. Public Health Service Research Grant Metabolism, and Digestive Diseases. 144
Copyright 0 1975 by Academic Press.Inc. All rights of reproduction in any form reserved.
AM-
CHLORDANE
EFFECT
ON Ccl,
HEPATOTOXICITY
145
(Herdson, Garvin, and Jennings, 1964; Jones and Fawcett, 1966; Kato and Takanaka, 1968; Orrenius, 1965a, 196513;Orrenius and Ericsson, 1966; Orrenius, Ericsson, and Emster, 1965; Remmer and Merker, 1963a, 1963b; Staubli, Hess, and Weibel, 1969), but has also been explored to some extent with various insecticides, including DDT ( Ortega, 1966)) dieldrin ( Hutterer, Schaffner, Klion, and Popper, 1968)) and chlordane ( Fouts and Rogers, 1965). More recently the effect of carbon tetrachloride (CC&) upon the hepatic microsomal drug-metabolizing enzyme systems has come under scrutiny (Glende, 1972a, 197213; Recknagel and Glende, 1973; Recknagel, Glende, Ugazio, Koch, ,and Srinivasan, 1974; Slater and Sawyer, 1969), and it has been discovered that pretreatment of animals with certain inducers of these enzyme systems can considerably increase Ccl, damage to mammalian liver. Several investigative groups have explored the effects of phenobarbital in this regard (Garner and McLean, 1969; Hurwitz, 1972; Lal, Puri, and Fuller, 1970; Stenger, Miller, and Williamson, 1970; Stenger and Johnson, 1971), and an earlier study warned that pretreatment with an insecticide, viz., DDT, might also increase CCll hepatotoxicity (McLean and McLean, 1966). The major purpose of the present investigation was to determine whether pretreatment with another insecticide, viz., chlordane, would increase the hepatotoxic potential of Ccl,. MATERIALS
AND METHODS
Male albino (Holtzman) rats, weighing approximately 200 g, received intraperitoneal injections of technical chlordane 2 (25 mg/kg, prepared as a 1:30 dilution in olive oil) on each of three successive days. Control rats were injected with equivalent doses of pure olive oil; untreated animals were also used. On day 4 of the experiments, rats in each category were tested with regard to: (a) their paralysis times, (b) the status of their hepatic microsomal enzyme activities, and (c) their hepatocellular ultrastructure. For paralysis time evaluations, rats in each category were given a single intraperitoneal injection of zoxazolamine (100 mg/kg) prepared as described by Coney, Davison, Gastel, and Burns (1960). The time was measured from the point of complete immobilization to the point at which the rat moved to another location in the cage. To check the status of the enzyme systems, NADPH-cytochrome c reductase and cytochrome P-450 levels were determined for microsomal suspensions prepared from the livers of rats in each treatment category. Livers from three rats were pooled for each determination. The livers were cut into small pieces, rinsed with 1.15% KC1 solution, and homogenized in 4 vol of KCl. The homogenates were centrifuged at 12,000g for 25 min, and the resultant supernatant fractions were then centrifuged at 105,OOOgfor 60 min. The pellets thus formed were suspended in one-third the original volume of KCl, and the protein content was determined by the method of Lowry, Rosebrough, Farr, and Randall ( 1951). Aliquots of these microsomal suspensionswere diluted with 0.1 M sodium phosphate buffer, pH 7.0, to a final concentration of 1 mg protein/ml of microsomal suspension. 2 Technical chlordane, a mixture of chlorinated hydrocarbons consisting of isomers chlordane ( 1,2,4,5,6,7,8,8-octachloro-4,7-methano-3a,4,7,7a-tetrahydroindane) and closely lated compounds, was kindly provided by Velsicol Chemical Corporation, Chicago, IL,.
of
re-
146
STENGER
ET AZ,.
NADPH-cytochrome c reductase activity was measured by a method similar to that of Mule, Redman, and Flesher ( 1967). The cuvette contained 2.8 ml of 0.1 M sodium phosphate buffer, pH 7.5, 0.025 ml of cytochrome c (from horse heart, type VI, Sigma) (20 mg/ml in 1.15% KCl), and 0.2 ml of the diluted microsomal suspension. The reaction was started by adding and mixing 0.01 ml of reduced nicotinamide adenine dinucleotide phosphate (NADPH, trisodium salt, type I, Sigma) (20 mg/ml in 4% sodium bicarbonate). The increase in optical density at 550 nm was measured for 3 min. The controls were run with 4% sodium bicarbonate (without NADPH) and their values were subtracted from the experimental values. A unit of NADPH-cytochrome c reductase activity was defined as the amount of enzyme that would cause a A OD of O.l/mg protein/ minute. One-centimeter cuvettes were used in all assayswhich were carried out in duplicate, and the mean values were accepted. To determine cytochrome P-450 content, the method of analysis was similar to that of Omura and Sato ( 1964). Portions of the aforementioned diluted microsomal suspensions were delivered into cuvettes of l-cm light path, and 10 mg of sodium hydrosulfite (dithionite) were added to both sample and reference cuvettes. Carbon monoxide was passed through a KOH trap and then bubbled through the microsomal suspension in the sample cuvette for 30 sec. Both cuvettes were capped with Teflon, and the difference in absorbance at 450 and 490 nm (A OD 450-490 nm) was determined in a Beckman model DB-G (dual-beam) spectrophotometer. Livers from rats in each category were also prepared for ultrastructural evaluation according to previously described methods (Millonig, 1961; Stenger, 1966). Finally, two studies were done to determine the extent of Ccl, toxicity in rats pretreated with chlordane, as compared to rats pretreated with olive oil alone and to rats that received no pretreatment. For these studies, on day 4 of the experiments, rats in each category received a single intraperitoneal injection of CC& (0.5 ml of a 25% solution in olive oil). Representatives from each category were killed at 4 hr after the CC14 challenge, and the status of their microsomal enzyme systems was compared to the prechallenge status. Both NADPHcytochrome c reductase and cytochrome P-450 were measured by the procedures described above. Representcative animals from each treatment category were also sacrificed at 48 hr after CC& injection, and sections from their livers were fixed in cold (4°C) 10% formalin, buffered to pH 7.0. Paraffin sections were stained with hematoxylin and eosin, and the extent of parenchymal necrosis in each liver was assessedby light-microscopic examination. RESULTS As shown in Table I, there was a highly significant reduction in zoxazolamine paralysis times for the chlordane-treated rats, as compared to either the olive oil-treated controls or the untreated animals. There was, however, no significant difference in paralysis times between the latter two groups. Table II indicates that the chlordane treatment caused a slight increase of hepatic microsomal NADPH-cyctochrome c reductase activity, but the difference from the controls was not statistically significant.
CHLORDANE
EFFECT
ON CClr HEPATOTOXICITY TABLE
147
I
EFFECT OF PRETREATMENT ON ZOXAZOLAMINE PARALYSIS TIMES Pretreatment?
No. of rats
Chlordane Olive oil None
19 17 14
a Chlordane: oil: equivalent intraperitoneally b Mean and E Comparison
Paralysis time (min) * 68.57 f 7.81 198.71 f 38.94 196.29 f 40.77
P < 0.005c P < 0.005”
25 mg/kg in olive oil, intraperitoneally, on each of three successive days. Olive amounts of pure olive oil. On day 4, all animals received zoxazolamine (100 mg/kg) as a single injection. SE are given for each category. with chlordane-treated rats by Student’s t test.
Table III reveals that the chlordane treatment did produce a highly significant increase of hepatic microsomal cytochrome P-450, when compared to either olive oil-treated or untreated animals. The chlordane injections also elicited a marked proliferation of hepatocellular smooth endoplasmic reticulum. This was particularly striking in the central one-third to one-half of the liver lobules (Fig. 1). More peripherally located hepatocytes failed to reveal any consistent differences between the chlordanetreated rats and the two control groups. Table IV demonstrates that, at 4 hr after Ccl, challenge, the hepatic microsomal NADPH-cytochrome c reductase activities were not significantly different from the prechallenge levels in any category (compare with TabIe II). On the other hand, Table V indicates that, at 4 hr after the toxic challenge, CC& administration had caused a marked and statistically significant drop in the hepatic microsomal levels of cytochrome P-450 in the chlordane-pretreated rats, as compared to the prechallenge level recorded in Table III. The reductions caused by Ccl, in the control groups, though less impressive, were also significantly below their prechallenge levels. With the light microscope, liver sections from 14 chlordane-CC&treated rats were compared with sections from 10 olive oil-CCL-treated rats and with sections from 10 animals subjected only to the CC14 challenge. As illustrated in TABLE EFFECT OF TREATMENT Treatment0 Chlordane Olive oil None
ON
II
HEPATIC MICROSOMAL NADPH-CYTOCHROME No. of determinationsb 8 8 8
c REDUCTASE
Units” 1.635 f 0.194 1.348 f 0.255d 1.445 f 0.300d
0 Chlordane: 25 mg/kg in olive oil, intraperitoneally on each of three successive days. Olive oil: equivalent amounts of pure olive oil. b In each category, livers from three rats were pooled for each determination. c Mean and SE are given. A unit of NADPH-cytochrome c reductase activity was defined as the amount of enzyme that would cause a A OD of O.l/mg protein/minute at 550 nm. d No significant difference from chlordane-treated rats by Student’s t test.
STENGER
148
TABLE EFFBCT
OFTRESTMENTON
Treatment”
HEPATIC
ET AL. III MICROSOM.IL
No. of determinations*
Chlordane Olive oil None
8 8 8
TBBLE
f 0.011 zk 0.006 f 0.006
P < 0.005d
on each
P < O.OOld
of three
successive
days.
Olive
for each determination. 1 test.
IV
Unit+
6 6 6
1.827 1.437 1.487
a Chlordane: 25 mg/kg in olive ‘oil, intraperitoneally, oil: equivalent amounts of pure olive oil. On day 4, all solution in olive oil) intraperitoneally and were killed 4 b In each category, livers from three rats were pooled c Mean and SE are given. A unit of NADPH-cytochrome amount of enzyme that would cause a A OD of O.l/mg d By Student’s t test, there is no significant difference Table II.
TABLE
Chlordane Olive oil None
nmc
No. of determinations*
Chlordane Olive oil None
Pretreatment”
A 013 450-490
ADMINISTRATION ON HEPATIC I~/~ICROSOM.~L NADPH-CYTOCHROI~IE c REDLJCF.~SE OF~RETRE.ITED AND UNTREATED R.~Ts
Pretreatment*
EFFECT
P-460
0.143 0.059 0.073
(1 Chlordane: 25 mg/kg in olive oil, intraperitoneally, oil : equivalent amounts of pure olive oil. b In each category, livers from three rats were pooled c Mean and SE are given. d Comparison with chlordane-treated rats by Student’s
EFFECTOFCCI~
CYTOCHROME
on each of three successive days. Olive animals received CC14 (0.5 ml of a 25y0 hr later. for each determination. c reductase activity was defined as the protein/minute at Fi50 nm. in any category from levels reported in
V
OF CCla ADMINISTR.~TION ON HEP~TIC ~L~ICROSOMAL P-450 OFPRETREATED AND UNTREATED RATS No. of determinations* 6 6 6
f 0.370d zk 0.20gd f 0.220d
A OD
0.049 0.036 0.043
u Chlordane: 2.5 mg/kg in olive oil, intraperitoneally, on oil: equivalent amounts of pure olive oil. On day four, all 25% solution in olive oil) intraperiotoneally and were killed * In each category, livers from three rats were pooled for c Mean and SE are given. d By Student’s t test, there is a significant difference in Table III.
* f f
CYTOCHROME
450-490
0.003 0.001 0.003
nmc
P < 0.001* P < 0.01*
P < 0.01*
each of three srxcessive animals received CC14 4 hr later. each determination. each
category
from
levels
days. Olive (0.5 ml of a
reportedin
CHLORDANE
EFFECT
ON Ccl,
HEPATOTOXICITY
149
FIG. 1. Portion of a parenchymal cell from the liver of a rat treated with chlordane for three successive days. This electron micrograph demonstrates the marked proliferation I of smooth endoplasmic reticulum that characterized many of the hepatocytes located in the central one-third to one-half of the liver lobules of such rats. The densely stained glycc bgen particles are sparsely distributed through the smooth-membrane mass. Also included in the field are intact mitochondria, several small lipid bodies (left, upper), and a portion of the cell’s nucleus (right, upper). ( X 14,350).
Fig. 2, the livers of rats pretreated with chlordane showed extensive hep;*tocellular necrosis involving approximately one-half of each liver lobule. Anin lals pretreated with olive oil and the untreated rats responded to the same dose : of
150
STENGER
ET AL.
FIG. 2. Portion of a liver lobule from a rat treated with chlordane for three successive days and then challenged with Ccl,. The animal was sacrificed 48 hr after the toxic challenge. This light micrograph illustrates the extensive parenchymal necrosis that characterized the livers of the chlordane-CCL-treated rats. At the center of the field is a central vein which is surrounded by necrotic parenchyma involving roughly one-half of the liver lobule. The necrotic tissue is demarcated from viable cells by a rim of polymorphonuclear leukocytes and hydropic hepatocytes. ( X 120).
CC& with less extensive damage, usually limited to the immediate vicinity the central vein.
of
DISCUSSION Technical chlordane, as used in this study, is a mixture of chlorinated hydrocarbons consisting predominantly of chlordane isomers. Since other halogenated hydrocarbons have been shown to induce proliferation of hepatocellular smoothsurfaced membranes, the stimulation demonstrated in the present study came as no surprise. In fact, Fouts and Rogers (1965) have previously reported such induction due to chlordane. This degree of smooth-membrane proliferation is often accompanied by enhancement of the hepatic drug-metabolizing capabilities, so the shortened zoxazolamine paralysis time and the augmented microsomal cytochrome P-450 demonstrated in this investigation can best be interpreted as evidence of such enhancement by the chlordane treatment. The failure of chlordane to cause an increase in the microsomal NADPH-cytochrome c reductase activity is somewhat perplexing, as both this enzyme and cytochrome P-450 are considered to
CHLORDANE
EFFECT
ON
Ccl,
HEPATOTOXICITY
151
function in the hepatic microsomal drug-metabolizing chain ( Orrenius, I965a). Such dissociation has, however, been observed with other inducers, particularly with certain polycyclic hydrocarbons (Baron and Tephly, 1969, 1970). The chlordane-pretreated rats suffered more extensive hepatic necrosis when challenged with Ccl, than did either of the control groups. This result also fulfilled expectations, inasmuch as other inducers of hepatic drug-metabolizing enzyme systems, e.g., phenobarbital or benzpyrene, have been reported to augment the liver-damaging effects of Ccl, (Garner and McLean, 1969; Lal, Puri, and Fuller, 1970; Pitchumoni, Stenger, Rosenthal, and Johnson, 1972; Stenger, Miller, and Williamson, 1970). Perhaps more to the point is the earlier observation of McLean and McLean (1966) that pretreatment with DDT also renders the liver more susceptible to Ccl4 injury. The prompt decrease of hepatic microsomal cytochrome P-450 after CC& challenge of the chlordane-stimulated animals was striking. Within 4 hr of the toxic challenge, a consistent and statistically significant drop was demonstrable. This observation suggests that an abrupt drop in cytochrome P-450 might serve as a relatively early indicator of haloalkane injury to the liver of a stimulated animal. Moreover, a similar, albeit less impressive, fall in the cytochrome P-450 value was observed in the unstimulated animals after Ccl* administration. Finally, this prompt effect of Ccl, administration upon cytochrome P-450 might be taken as further evidence that the locus of Ccl, metabolism is at or near the cytochrome P-450 locus on the hepatic microsomal membrane, as proposed by Garner and McLean (1969) and more recently ,advocated by Recknagel and Glende ( 1973). In this regard, it is also of interest that our studies failed to demonstrate any significant effect of either chlordane or CC& on hepatic microsomal NADPH-cytochrome c reductase activity. In the case of CC&, our findings are in accord with those reported by Glende (1972a). ACKNOWLEDGMENT Ms.
The authorsthank Mr. OsmayYalis for his help in the preparationof the micrographs,and Caryl Towner for her help in the preparationof the manuscript. REFERENCES J., and TEPHLY, T. R. ( 1969). The role of hemesynthesisduring the induction of hepatic microsomalcytochrome P-450 and drug metabolismproduced by benzpyrene.
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EFFECT
ON
Ccl,
HEPATOTOXICITY
lTi3
REMME% H., and MERKER, H. J. ( 1963a). Enzyminduktion und vermehrung von endoplasmatischem reticulum in der leberzelle wahrend der behandlung mit phenobarbital (luminal). Klin. Wochenschr. 41, 276-283. REMMER, H., and MERKER, H. J. ( 196313). Drug-induced changes in the liver endoplasmic reticulum associated with drug-metabolizing enzymes. Science 142, 1657-1658. REMMER, H., and MERKER, H. J. ( 1965). Effect of drugs on the formation of smooth endoplasmic reticulum and drug-metabohzing enzymes. Ann. N. Y. Ad. Sci. 123, 79-96. SLATER, T. F., and SAWYER, B. C. ( 1969). The effects of carbon tetrachloride on rat liver microsomes during the first hour of poisoning in uino, and the modifying actions of promethazine. Biochem. J. 111, 317324. STXUBLI, W., HESS, It., and WEIBEL, E. R. (1969). Correlated morphometric and biochemical studies on the liver cell. II. Effects of phenobarbital on rat hepatocytes. J. Cell Biol. 42, 92-112. STENGER, R. J, ( 1966). Ultrastructural alterations within hepatic parenchymal cells after carbon tetrachloride poisoning. In “Methods and Achievements in Experimental Pathology” ( Bajusz, E., and Jasmin, G., Eds.), Vol. 1, pp. 677-700. S. Karger, Basel. STENGER, R. J., and JOHNSON, E. A. (1971). Further observations upon the effects of phenobarbital pretreatment on the hepatotoxicity of carbon tetrachloride. Erp. Mo2. Puthol. 14, 220-227. STENGER, R. J., MILLER, R. A., and WILLIAMS, J. N. ( 1970). Effects of phenobarbital pretreatment on the hepatotoxicity of carbon tetrachloride. Exp. Mol. Puthol. 13, 242-252.
