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Adrenalectomy and the adaptive liver response in mirex-treated rats
PESTICIDE
BIOCHEMISTRY
AND
PHYSIOLOGY
Adrenalectomy M.G. Department
of Biological
20, 330-339 (1983)
and the Adaptive Liver Response in M i rex-Treated Rats ERVINANDJAMES
Sciences,
Mississippi
State
D. YARBROUGH University,
Mississippi
State,
Mississippi
39762
Received August 2, 1982; accepted May 31, 1983 Mirex, an organochlorine compound, was administered as a single oral dose (100 mg/kg body wt) to both intact and adrenalectomized juvenile male Sprague-Dawley rats. Both mirex-treated intact and adrenalectomized animals (dosed 24 hr postsurgery) exhibited significant increases in liver weight to body weight ratios compared to controls. However, the liver weight to body weight ratios in mirex-treated intact animals were significantly greater than those observed in mirextreated adrenalectomized animals. Significant increases were observed in liver weight to body weight ratios in adrenalectomized animals treated with mirex 4 days after surgery. However, the 96-hr mortality in mirex-treated adrenalectomized animals increased from 20% (mirex dose given 1 day postadrenalectomy) to 56% (mirex dose given 4 days postadrenalectomy). Mirex treatment of intact and adrenalectomized animals had no significant effect upon either serum or hepatic activities of glutamic oxalacetic transaminase, glutamic pyruvic transaminase, sorbitol dehydrogenase, or protein concentrations. Bromsulfophthalein clearance also was not affected by mirex treatment in adrenalectomized animals. Serum glucose concentrations were significantly decreased in both intact and adrenalectomized animals by mirex treatment. Daily corticosterone supplements to adrenalectomized animals restored liver hypertrophy and serum glucose concentrations to levels observed in mirex-treated intact animals. These results suggest that mirex-induced liver enlargement may be mediated by corticosterone. INTRODUCTION
Many xenobiotic compounds induce adaptive responses within the liver (1). One such compound is the organochlorine compound mirex (1,3,4 - methenododecachlorooctahydro - 2H - cyclobuta[c,d]pentalene) which has been shown to be a potent inducer of liver enlargement in rats and mice (2-4). A single oral dose of mirex (100 mg/ kg body wt) administered to an adult rat yields a highly significant increase in the liver weight to body weight ratio (LW/BW) within 4 days (3). This enlargement is not due to an accumulation of water, lipids, or glycogen. The increase in protein necessary for cellular enlargement results from both increased protein synthesis and decreased protein catabolism (5). Following mirex treatment, liver DNA concentration decreases (3) while the rate of parenchymal DNA synthesis remains unchanged (4). Functionally, the detoxication potential of the liver is increased by mirex treatment, 330 0048-3575183 $3.00 Copyright
0 1983 by Academic
AU rights of reproduciion
Press, Inc. in my fom resewed.
as evidenced by a proliferation of the smooth endoplasmic reticulum with a concomitant increase in monooxygenase activity (6, 7). This increase in monooxygenase activity occurs even though mirex is not metabolized by mammalian systems (8). Mehendale (9) has demonstrated that mirex causes increased bile flow and decreased biliary excretion of polychlorinated biphenyls (PCBs) and their derivatives, suggesting hepatobiliary dysfunction. However, standard indicators of liver injury such as serum glutamic oxalacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), lactic dehydrogenase (LDH), and total bromsulfophthalein (BSP) clearance all remain unchanged following mirex treatment (3, 10). Acute mirex dosing of rats also yields a significant increase in serum corticosterone, the level of which remains elevated for several days (11). If the adrenal glands
ADRENALECTOMY
AND
MIREX-INDUCED
are removed prior to mirex treatment, the response in the liver becomes predominantly hyperplastic, with only a minimal increase in liver weight (4). Available evidence appears to indicate: (i) that mirex-induced liver enlargement is primarily due to cellular hypertrophy, and (ii) that this response is related to the availability of adrenal steroids. To further evaluate the relationship between the adrenal glands and the mirex-response, the effects of prior adrenalectomy upon mirex-induced liver enlargement were studied. MATERIALS
AND
METHODS
Animals and test compound. Male Sprague-Dawley rats weighing between 90 and 120g were used in all experiments. The animals were kept in rooms maintained at 24°C with a controlled 12 hr light-dark cycle and housed individually in stainlesssteel cages with wire bottoms. All animals received commercial rat chow (Purina Laboratory Rodent Chow), and tap water ad libitum, with fresh water and food supplied daily between 1:00 and 3:00 PM. Adrenalectomized (ADX) animals received 0.9% saline to drink. Adrenalectomies were performed under light ether anesthesia by means of a paravertebral dorsal approach (12). ShamADX animals were handled in exactly the same manner except that the adrenal glands were exposed, but not disturbed. Adrenalectomies were performed in a laboratory which is removed from the animal holding facilities and were completed prior to 1l:OO AM to minimize diurnal effects. The mirex used was prepared by the Mississippi State Chemical Laboratory (Mississippi State, Miss.) and was assayed to be >99% pure by capillary electron capturegas chromatography. The test dose (100 mg mirex/kg body wt) was dissolved in corn oil (20 mg/ml) and was administered by gavage, while control animals received an equivalent volume of corn oil by gavage. All dosing was completed between 8:OOand lo:@)
AM.
HEPATIC
EFFECTS
331
Animals were either ADX or sham-ADX and half of the animals in each group received a single oral dose of mirex or corn oil. All animals were dosed 24 hr after surgery and representative animals sacrificed from 1 to 7 days following dosing. Final body weights and liver weights were determined at the time of sacrifice. 96hr mortality. To examine the effect of an increase in the time interval between adrenalectomy and mirex dosing upon both the liver response and animal mortality, ADX animals received a single oral dose of mirex or corn oil at either 24, 48, 72, or 96 hr post-ADX. Animals were sacrificed 96 hr after dosing and the livers excised and weighed. The number of animals alive at 96 hr postdosing versus the total number dosed in each group was used to calculate percentage mortality. Mirex-induced hepatotoxicity. To examine the possibility that the decreased liver response and/or increased mortality observed in mirex-treated ADX animals could be related to mirex-induced hepatotoxicity, the following experiment was performed. Intact animals were randomly selected and treated with either mirex or corn oil. Sham-ADX animals were treated with either mirex or corn oil on the 4th day after surgery. ADX animals were treated with mirex or corn oil either 2,4, or 6 days after ADX. In these experiments the maximum liver response and minimal animal mortality occurred 48 hr after dosing. Therefore, 48 hr after mirex dosing the animals in each of the above groups were killed by decapitation under light ether anesthesia and exsanguinated into centrifuge tubes for preparation of serum. Serum sorbitol dehydrogenase (SDH, EC 1.1.1.14) activity was determined at 25°C by the method of Asada and Galambos (13). Glutamic oxalacetic transaminase (GOT, EC 2.6.1) and glutamic pyruvic transaminase (GPT, EC 2.6.1.2) activities were assayed at 37°C according to the methods of Karmen (14), and Time-course
experiment.
332
ERVIN
AND
YARBROUGH
Wroblewski and LaDue (15), respectively. Serum glucose was determined by a method based upon that of Raabo and Terkildsen (16). Protein determinations were performed by the method of Lowry et al. (17). Immediately following collection of blood, the livers were perfused in situ with 5 ml of 0.14 M KCl, excised, and weighed. A 0.5-g sample was removed and homogenized in 5 ml of 0.14 M KC1 containing 0.32 M sucrose. The homogenate was centrifuged at 600g for 15 min and the clear supernatant diluted to 0.5% with buffer and used for determination of hepatic GOT, GPT, and SDH activities and protein concentration. The remaining liver tissue was frozen at - 60°C for later determinations of total hepatic glycogen and lipids. Glycogen was extracted by the method of Hassid and Abraham (18) and glucose determined according to the method of Nelson (19). Total lipids were determined by the method of Folch et al. (20). Bromsulfophthalein clearance. To determine the effect of adrenalectomy on hepatic function in the presence of mirex, bromsulfophthalein (BSP) clearance was assessed. Three groups of animals were established, two of which were ADX and the third sham-ADX. Four days following surgery one group of ADX animals received a single oral dose of mirex while the remaining ADX and sham-ADX animals received corn oil. Forty-eight hours after mirex treatment, all animals received an intravenous (iv) injection of 75 mg BSP/kg body wt. Fifteen minutes after injection, serum BSP concentration was determined by the method of Kutob and Plaa (21). Corticosterone supplement. Animals were ADX as described above and divided into seven groups. Twenty-four hours after surgery, the animals in five groups received a single oral dose of mirex while the animals in group 6 received corn oil. Animals in group 7 received corn oil and a subcutaneous (SC)injection of soybean oil. The mirex-treated animals also received a
single, daily SCinjection of either 1, 5, 10, or 20 mg corticosterone/kg body wt dissolved in soybean oil, or soybean oil alone. The group orally dosed with corn oil also received a daily corticosterone supplement of 20 mg/kg body wt. Animals were sacrificed 96 hr after mirex or oil treatment because previous results indicated that both the liver response to mirex and the mirex-induced mortality in ADX animals were maximal while serum glucose was reduced at this time. Therefore, the effect of corticosterone supplements upon these parameters could be investigated. All animals were decapitated and exsanguinated into centrifuge tubes and the serum prepared for total glucose determinations. Liver weights and body weights were also taken. Statistics. All sample means are expressed as the mean ? standard error of the mean (x rt SEM). The data from the corticosterone supplement experiment were subjected to one-way analysis of variance followed by Duncan’s new multiple range test (DNMRT). For the remaining experiments, differences between means were determined by either the Student’s t test or by the paired sample t test (22). RESULTS
Due to the presence of significant differences (P < 0.05) in LW/BW between oil-treated intact and oil-treated ADX animals on Days 2, 5, and 6, these data were presented separately. There were significant increases (P < 0.001) in LW/BW in mirex-treated intact and ADX animals when compared to oiltreated controls on Days 1 through 7 and Days 3 and 6, respectively (Figs. IA and B). However, there was also a significant difference in LW/BW between mirextreated ADX animals and mirex-treated intact animals on Days 1 through 6: Although the maximal rate of liver enlargement occurred by Day 2 in mirex-treated intact animals there was a continued increase through Day 4 (Fig. 1A). In contrast, maxTime-course
experiment.
ADRENALECTOMY
AND ----
I
MIREX-INDUCED
HEPATIC
333
EFFECTS
SHAM + MlREX SHAM + OIL
A
‘&--&--2
-I L . 3
DAYS
6. ADX + MIREX ADX + OIL
---
4
ADX
FIG. 2. Liver weight to body weight ratios in control and mirex-treated ADX rats dosed either 1, 2, 3, or 4 days after adrenalectomy. Animals received either a single oral dose of mirex (100 mglkg body wt) in corn oil or corn oil only and were sacrificed 96 hr later. Each point represents x -t SE offrom 9 to 16 animals. Values statistically different from controls are indicated by an asterisk (*P < 0.001).
8 -
3
POST
7-
I 0
I
2
DAYS
3
4
POST MIREX
5
6
7
DOSE
FIG. 1. Liver weight to body weight ratios (L W/BW) in male rats sham adrenalectomized (Sham) or adrenalectomized (ADX) 24 hr prior to mirex treatment. Mirex was administered as a single oral dose (100 mglkg body wt), with control animals receiving an equivalent volume of corn oil. Each point represents x 2 SE of from 5 to 11 animals. LWIBW in mirextreated intact and ADX animals exhibited significant increases (P < 0.001) over their respective controls on Days 1 through 7 and Days 3 through 6, respectively. There also were significant differences (P < 0.001) in LWIBW between mirex-treated intact animals and mirex-treated ADX animals on Days 1 through 6.
and control animals. Increasing time after ADX prior to mirex treatment also increased mortality in mirex-treated ADX animals (Fig. 3). In contrast, there was no mortality in any of the control groups. Mirex-induced hepatotoxicity. Because the results obtained from the sham-ADX animals closely paralleled those in intact animals, only the intact will be reported. 1007
;
-
MIREX
----
OIL
80
z!
imal rate of liver enlargement in mirextreated ADX animals did not occur until Day 3 post-mirex dosing. Both intact and ADX animals exhibited weight gain during this 4-day period. 96hr Mortality. Although there were no differences in initial or final body weights in any of the groups tested, animals dosed with mirex 1,2, and 3 days after adrenalectomy exhibited a significant increase (P < 0.001) in LW/BW 4 days after mirex dosing (Fig. 2). However, in animals dosed 4 days after ADX there were no significant differences in LW/BW between mirex-treated
!lj
/
I
2
3
.I
4
DAYS POST AOX
3. Ninety-six-hour mortality in male adrenalectomized (ADX) rats receiving either a single oral dose of mirex (100 mglkg body wt) or corn oil at either 1, 2, 3, or 4 days following surgery. Each point denotes the percentage mor*o!ity (number deadltotal number dosed) at 96 hrfollowing mirex treatment. Each point represents from 18 to 20 animals for mirex treatment and 11 to 13 animals for controls, FIG.
334
ERVIN AND YARBROUGH
There were no differences in initial body weights between mirex-treated and control animals in any of the groups tested (intact, 2-day, 4-day, or 6-day ADX). Final body weights in intact animals were not different from initial body weights, but liver weights in mirex-treated intact animals were significantly increased (P C 0.001) over controls (Table 1). In contrast, final body weights in mirex-treated 2-day and 4-day ADX animals were significantly reduced (P < 0.05) from initial levels (Table 1). However, there were no differences in liver weights between mirex-treated and control 2-, 4-, or B-day ADX animals. Since the 2- and 4-day mirex-treated animals exhibited a loss in body weight, their LW/BW ratios were not considered valid for comparison. The fact remains that intact animals exhibited a significant increase in liver weight, whereas liver weights in mirex-treated ADX animals were not different from their respective controls. There was a significant decrease (P < 0.05) in serum protein concentration in mirex-treated animals dosed 2 days following adrenalectomy (mirex: 3.20 g/d1 +
0.07, N = 15; oil: 3.45 g/d1 rt 0.07, N = 11). There were, however, no differences in serum protein concentrations in any of the remaining test groups. Serum glucose concentrations exhibited a significant reduction (P < 0.01) in mirex-treated intact animals, and in ADX animals dosed with mirex either 2,4, or 6 days after adrenalectomy (Fig. 4). There were significant increases (P < 0.05) in hematocrit values (vol %) in mirex-treated intact (mirex: 46.28 +1.5, N = 9; control: 41.44 + 0.96, N = 9) and in both 2-day ADX animals (mirex: 52.28 + 1.65, N = 9; control: 41.21 k 1.89, N = 7) and 4-day ADX animals (mirex: 51.90 _t 3.91, N = 5; control: 41.06 +1.40, N = 9). Biochemical parameters including serum and hepatic SDH, GOT, and GFT activities were not different between mirex-treated and control animals except for a significant reduction (P < 0.05) in hepatic GPT in animals dosed with mirex 4 days after surgery (mirex: 3.94 2 0.42 mmol NADH oxidized/g tissue min-‘, N = 8; control: 5.67 + 0.37 mmol NADH oxidized/g tissue min-l, N = 9).
TABLE Body Weights and Liver Weights in M&x-Treated
1 Intact and Adrenalectomized
Body weight (g)
Intact Mirex (14) Control (12) Adrenalectomized 2 Days Mirex (15) Control (11) 4 Days Mirex (11) Control (12) 6 Days Mirex (12) Control (9)
Mule Rats”
Initial
Final
Liver weight b?)
100.71 + 3.66 95.33 + 4.62
92.31 2 3.41 99.88 + 5.23
6.93 + 0.20** 4.91 f 0.27
112.07 f 2.06 107.64 + 2.72
99.21 f 2.30* 108.24 ‘: 2.96
5.21 + 0.17 4.97 2 0.19
112.91 f 3.30 108.33 k 3.24
103.35 + 2.68* 111.89 + 2.80
5.50 Ifr 0.24 5.16 f 0.17
120.67 f 2.51 117.44 * 4.35
111.66 f 2.36 119.11 + 6.78
5.77 + 0.24 5.35 f 0.44
0 Adrenalectomized animals received mirex or corn oil either 2, 4, or 6 days following surgery. Mirex was administered as a single oral dose (100 mgikg body wt) and animals were sacrificed 48 hr after treatment. Values are expressed as x f SE with values in parentheses representing the number of animals in each group. * Final body weight significantly different from initial body weight (P < 0.05). ** Liver weight significantly different from control (P < 0.001).
ADRENALECTOMY
AND
MIREX-INDUCED
I8 IOO3 3 80 60_ ;i;.‘i I
I 0
4
2 DAYS
POST
I 6
ADX
4. Serum glucose concentrations in male rats treated with a single oral dose of mirex (100 mglkg body wt) or corn oil at either 0 days (Intact), or 2, 4, or 6 days after adrenalectomy. Each point represents x f SE of from 8 to 14 animals. All animals were sacrificed 48 hr after mirex treatment. Both mirextreated Intact and ADX animals exhibited significant decreases (P < 0.01) in serum glucose when compared to oil-treated controls. FIG.
Mirex-treated intact animals exhibited a significant reduction (P < 0.05) in total hepatic glycogen (expressed as mg/g tissue) when compared to controls (60.5 +- 3.4, N = 5; 68.7 +. 0.5, N = 5, respectively). Mirex-treated intact animals also exhibited a significant increase (P < 0.05) in total liver lipid (mirex: 35.90 +- 2.30 mg/g tissue; control: 29.80 + 0.76 mg/g tissue). There were no differences in total liver glycogen or lipid in either control ADX or mirextreated ADX animals dosed either 2, 4, or 6 days after adrenalectomy. Hepatic glycogen levels in ADX animals were reduced (P < 0.05) from intact levels, which is consistent with known effects of adrenalectomy (23). Liver functionality. A mirex-induced increase in the rate of BSP clearance has been reported previously for intact, male Sprague-Dawley rats (10). However, no differences were detected in the rate of serum BSP clearance between intact (25.87
HEPATIC
EFFECTS
335
2 1.10, N = 6) or ADX animals receiving either mirex (31.80 + 2.26, N = 9) or corn oil (32.55 ? 2.45, N = 6). Values are expressed as mg BSPldl of serum 15 min following an iv injection of 75 mg BSP/g body wt. Corticosterone supplement. Because none of the animals included in this experiment exhibited changes in body weight, the alterations in LW/BW observed reflect actual changes in liver weight. There were no differences in LW/BW between ADX and corticosterone-supplemented (20 mg/kg body wt, SC) ADX animals 4 days after oiltreatment. ADX animals receiving mirex treatment alone, or mirex treatment and a daily SC injection of soybean oil also showed no differences in LW/BW (5.78 it 0.20 and 5.75 + 0.20, respectively). Mirextreated ADX animals receiving daily corticosterone supplements of either 1, 5, 10, or 20 mg/kg body wt exhibited significant increases in LWIBW when compared to mirex-treated ADX animals receiving a soybean oil supplement (Fig. 5). Even though corticosterone supplement did result in increased LW/BW in mirex-treated ADX animals, the response did not appear to be dose dependent. Mirex-treated ADX animals receiving either a soybean oil supplement or 1 mg/kg corticosterone in soybean oil exhibited a 33% mortality during the 96 hr following mirex treatment. Mirex-treated ADX animals receiving larger corticosterone supplements of 5, 10, or 20 mglkg corticosterone exhibited no mortality. These results, in addition to the previously described 96-hr mortality (Fig. 3), offer good evidence that the increase in mortality observed in mirextreated ADX animals is related to the absence of corticosterone in these animals. Serum glucose concentrations in mirextreated ADX animals were significantly reduced (P < 0.05) from the levels observed in control ADX animals (Fig. 5). Daily corticosterone supplements of 1 mg/kg body wt had no effect upon serum glucose concentrations in ADX mirex-treated animals
336
**** I L-i ERVIN
I
LWlBW
IO
9.
8.
MIREX (mg/kg SW) CORTICOSTERONE (mg/kg EW)
YARBROUGH
0
SERUM
GLUCOSE
* fl
I
0 12
AND
100 * 0
0
100
100
100
l
+
+
+
20
I
5
IO
150
2 F
IO0
3 8 2 ” 4 a I
50
100 +
20
FIG. 5. Liver weight to body weight ratios (LW/BW) and serum glucose concentrations in adrenalectomized (ADXI male rats receiving a single oral dose of mirex (100 mglkg body wt) or corn oil 24 hr post-ADX. All animals were sacrificed 96 hr after mirex treatment. Mirex-treated animals also received daily SC corticosterone supplements of either 0, 1, 5, 10, or 20 mg corticosteronelkg body wt. Control animals received either a corticosterone supplement of 20 mglkg body wt or soybean oil only. Values are expressed as x ? SE offrom four to six animals. Statistically signt@cant differences between mirex-treated ADX animals receiving corticosterone supplements or vehicle are indicated by an asterisk (*P < 0.05).
(Fig. 5). However, mirex-treated ADX animals receiving daily corticosterone supplements of either 5, 10, or 20 mg/kg body wt exhibited significant increases in serum glucose (Fig. 5).
adrenalectomy and mirex treatment is increased, the adrenal gland independent component appears to diminish (< 15% by Day 4) (Fig. 2). This decrease in the adrenal independent response may be related to the presence of intracellular, receptor-bound DISCUSSION corticosterone which may still be exerting The mirex-induced increases in relative an effect even though circulating corticoliver weight in intact animals observed in sterone has been abolished. Therefore, it our experiments are similar in magnitude to appears that the mirex-induced increase in values reported previously (3). Other or- liver mass observed in intact rats is preganochlorine pesticides such as DDT and dominantly dependent upon functional addieldrin have also been reported to induce renal glands. This dependence is further ilincreased liver size (24, 25). The adaptive lustrated by the fact that mirex-induced liver growth induced by mirex in intact an- liver enlargement can be restored in ADX imals represents approximately a 72% in- animals by daily corticosterone supplement crease in liver mass in 96 hr. Although a (Fig. 5). major portion of the mirex-induced liver reMirex treatment has also been shown to sponse appears to be adrenal gland depen- induce an increase in serum corticosterone dent (Fig. lA), there is a minor component concentration (11). Similar increases in which is adrenal gland independent (Fig. serum corticosterone have also been re1B). However, if the time interval between ported following treatment with DDT (24),
ADRENALECTOMY
AND MIREX-INDUCED
dieldrin (25), endrin (26), PCB’s (27), and in response to various organophosphates (28). Therefore, mirex-induced liver enlargement may be directly related to the hypertrophic potential of adrenal glucocorticoids. The ability of glucocorticoids to promote cellular hypertrophy and inhibit DNA synthesis within the liver has been well documented (29-36). It has also been demonstrated that the effect of adrenalectomy is to increase the rate of liver DNA synthesis (37), and that this response can be further stimulated by mirex (4). Thus, the increase in relative liver weight observed in ADX animals on Day 3 may represent mirex-induced cellular hyperplasia (Fig. 1). The loss of the mirex-induced growth response in the ADX animal does not appear to be due to parenchymal cell damage. In both intact and adrenalectomized animals, serum and hepatic levels of SDH, GOT, or GPT offered no evidence of mirex-induced hepatotoxicity. In addition, evaluation of hepatic functional capacity by assessment of BSP clearance rates offered no suggestion of a mirex-induced impairment of biliary excretion. Adrenalectomy prior to mirex treatment results in both a decrease in the liver response to mirex and an increase in animal mortality (Figs. 1 and 3). In addition, it appears that both of these responses are corticosterone dependent. That adrenal glucocorticoids are decisive factors in allowing an animal to cope with physical and chemical stress is well recognized (24, 28, 3840). It has been proposed that glucocorticoids are protective of an animal by “syntoxic mechanisms” which allow for increased tissue tolerance to a toxicant without increasing its metabolism (38). However, the mechanism by which these effects are exerted is unknown. The increased serum levels of corticosterone following mirex treatment (11) would be expected to yield increased serum glucose, increased hepatic gluconeogenesis and glycogenesis, a mobilization of fatty acids for energy, and a pooling of amino
HEPATIC
EFFECTS
337
acids for biosynthetic work. These responses are consistent with the known effects of chemical agents such as dieldrin, endrin, and certain organophosphates (25, 26, 28, 30). In contrast, our results indicate that mirex induces a slight hepatic glycogen depletion in intact animals and hypoglycemia in both intact and ADX animals (Fig. 4). Because mirex also induces hypoglycemia in intact animals and corticosterone supplement given to mirex-treated animals did not restore serum glucose to control levels (Fig. 5), it appears that mirex may exert a direct effect upon hepatic carbohydrate metabolism. Other chlorinated hydrocarbons (DDT and PCB’s) also effect a decrease in hepatic glycogen deposition and hypoglycemia, possibly due to an inhibition of hepatic gluconeogenesis (41,27). Garthoff et al. (27) have stated that augmented rates of gluconeogenesis are critical for normal adaptation of an animal to a variety of conditions. If increased gluconeogenesis and hyperglycemia are as important as has been suggestedin allowing an animal to cope with chemical stress, the adaptive potential of the ADX animal following mirex treatment would indeed be severely diminished. From these results, it appears that the reduction in the mirex-induced liver growth response in ADX animals may not be due to hepatic parenchymal necrosis or altered hepatic function. Instead, the response appears to be dependent upon corticosterone, and represents an indirect effect of mirex upon the liver which is mediated by the adrenal glands. However, whether the requirement for corticosterone in the mirex response is directly related to the hypertrophic potential of corticosterone or instead represents a permissive effect for some other mechanism remains to be demonstrated. ACKNOWLEDGMENT The authors would like to thank Mrs. Jo Grimley for preparation of the illustrations.
338
ERVIN AND YARBROUGH
REFERENCES 1. R. Schulte-Hermann and E. Schmitz, Feedback inhibition of hepatic DNA synthesis, Cell Tissue Kinet. 13, 371 (1980). 2. J. L. Byard, U. C. Koepke, R. Abraham, L. Golberg, and E Coulston, Biochemical changes in the liver of mice fed mirex, Toxicol. Appl. Pharmacol. 33, 70 (1975). 3. K. M. Robinson and J. D. Yarbrough, Liver response to oral administration of mirex in rats, Pestic. Biochem. Physiol. 8, 65 (1978). 4. P. I. Karl, “Relationship between the Adrenal Glands and Hepatic Growth Regulating Substances in Mirex-Induced Liver Growth,” Ph.D. dissertation, Mississippi State University, 1981. 5. K. M. Robinson and J. D. Yarbrough, Liver protein synthesis and catabolism in mirex-pretreated rats with enlarging livers, J. Pharmacol. Exp. Ther. 215, 82 (1980). 6. R. C. Baker, L. B. Coons, R. B. Mailman, and E. Hodgson, Induction of hepatic mixed function oxidases by the insecticide, Mirex, Environ. Res. 5, 418 (1972). 7. H. M. Mehendale, P. R. Chen, L. Fishbein, and H. B. Matthews, Effect of mirex on the activities of various hepatic mixed function oxidases, Arch. Environ. Contam. Toxicol. 1, 245 (1973). 8. J. R. Gibson, G. W. Ivie, and H. W. Dorough, Fate of mirex and its major photodecomposition product in rats, J. Agric. Food Chem. 20, 1246 (1972). 9. H. M. Mehendale, Mirex-induced suppression of biliary excretion of polychlorinated biphenyl compounds, Toxicol. Appl. Pharmacol. 36,369 (1976). 10. K. M. Robinson and J. D. Yarbrough, A study of liver function in rats with mirex-induced enlarged livers, Pestic. Biochem. Physiol. 9, 61 (1978). 11. J. D. Williams and J. D. Yarbrough, The relationship between mirex-induced liver enlargement and the adrenal glands, Pestic. Biochem. Physiol. 19, 15 (1983). 12. R. Gaunt, Adrenalectomy in the rat, Amer. J. Physiol. 103, 494 (1933). 13. M. Asada and J. T. Galambos, Sorbitol dehydrogenase and hepatocellular injury: An experimental and clinical study, Gastroenterology 44, 578 (1%3). 14. A. Karmen, A note on the spectrophotometric assay of glutamate-oxalacetic transaminase in human blood serum, J. Clin. Invest. 34, 131 (1955). 15. E Wroblewski and J. S. LaDue, Serum glutamic pyruvic transaminase in cardiac and hepaticdisease. Proc. Sot. Exp. Biol. Med. 91, 569 (1956).
16. E. Raabo and T. C. Terkildsen, On the enzymatic determination of blood glucose, Stand. J. Clin. Lab. Invest. 12, 402 (l%O). 17. 0. H. Lowry, N. J. Rosenbrough, A. L. Farr, and R. J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193, 265 (1951). 18. W. Z. Hassid and S. Abraham, Chemical procedures for analysis of polysaccharides. In Methods of Enzymology (S. Colowick and N. Kaplan, Eds.), Vol. 3, pp. 34-37, Academic Press, New York, 1957. 19. N. Nelson, A photometric adaptation of the Somogyi method for the determination of glucose, J. Biol. Chem. 153, 375 (1944). 20. J. Folch, M. Lees, and G. H. Sloan-Stanley, A simple method for the isolation and purification of total lipids from animal tissues, J. Biol. Chem. 226, 497 (1957). 21. S. D. Kutob and G. L. Plaa, Assessment of liver function in mice with bromosulphaleine, J. Appl. Physiol. 17, 123 (1962). 22. J. H. Zar, “Biostatistical Analysis,” p. 121, Prentice-Hall, Englewood Cliffs, N.J., 1974. 23. R. Steele, Influences of corticosteroids on protein and carbohydrate metabolism, in Handbook of Physiology: Endocrinology (H. Blaschko, G. Sayers, and A. D. Smith, eds.), Vol. VI, pp. 135, American Physiological Society, Washington, D.C., 1975. 24. R. J. Szot and S. D. Murphy, Phenobarbital and dexamethasone inhibition of the adrenocortical response of rats to toxic chemicals and other stresses, Toxicol. Appl. Pharmacol. 17, 761 (1970). 25. S. C. Bhatia, S. C. Sharmu, and T. A. Venkitasubramanian, Effect of dieldrin on hepatic carbohydrate metabolism and protein biosynthesis in vivo, Toxicol. Appl. Pharmacol. 24, 216 (1973). 26. K. Meena, P. K. Gupta, and S. R. Bawa, Endrininduced toxicity in normal and irradiated rats. Environ. Res. 16, 373 (1978). 27. L. H. Garthoff, E E. Cerra, and E. M. Marks, Blood chemistry alterations in rats after single and multiple gavage administration of polychlorinated biphenyl, Toxicol. Appl. Pharmacol. 60, 33 (1981). 28. S. D. Murphy and S. Porter, Effects of toxic chemicals on some adaptive liver enzymes, liver glycogen, and blood glucose in fasted rats, Biochem. Pharmacol. 15, 1665 (1966). 29. J. C. Davis and T. A. Hyde, The effect of corticosteroids and altered adrenal function on liver regeneration following chemical necrosis and partial hepatectomy, Cancer Res. 26, 217 Wm. 30. K. H. Raab and T. E. Webb, Inhibition of DNA
ADRENALECTOMY
31. 32,
33.
34. 35. 36.
AND MIREX-INDUCED
synthesis of regenerating rat liver by hydrocortisone, Experieniia 25, 1240 (1969). I. C. Henderson, R. E. Fischel, and J. N. Loeb, Suppression of liver DNA synthesis by cortisone, Endocrinology 88, 1471 (1971). J. N. Loeb, C. Borek, and L. L. Young, Suppression of DNA synthesis in hepatoma cells exposed to glucocorticoid hormone in vitro, Proc. Nat. Acad. Sci. USA 70, 3852 (1973). I. C. Henderson and J. N. Loeb, Hormone-induced changes in liver DNA synthesis: effects of glucocorticoids and growth hormone on liver growth and DNA polymerase activity, Endocrinology 94, 1637 (1974). L. Desser-Wiest, Autosynchronization of rat liver cells with endogenous corticosterone after partial hepatectomy, Cell Tiss. Kinet. 8, 1 (1975). R. Schulte-Hermann, Induction of liver growth by xenobiotic compounds and other stimuli, Crit. Rev. Toxicol. 3, 97 (1974). T. J. Castallano, R. L. Schiffman, M. C. Jacob, and J. N. Loeb, Suppression of liver cell proliferation by glucocorticoid hormone: A comparison of normally growing and regenerating
37.
38.
39.
40.
41.
HEPATIC
EFFECTS
339
tissue in the immature rat, Endocrinology 102, 1107 (1978). L. Desser-Wiest, Stimulation of DNA synthesis in rat liver by adrenalectomy, J. Endocrinol. 60, 315 (1974). P Kourounakis, H. Selye, and Y. Tache, Catatoxic steroids, in Advances in Steroid Biochemistry and Pharmacology (M. H. Brings and G. A. Christie, Eds.), Vol. 6, pp. 35-57, Academic Press, New York, 1977. W. E Bousquet, D. Rupe, and T. S. Miya, Endocrine modification of drag responses in the rat, J. Pharmacol. Exp. Ther. 147, 376 (1965). G. Weber, R. L. Singhal, S. K. Srivastava, H. J. Hird, and J. Furth, Behavior of hepatic gluconeogenic enzymes, RNA metabolism, and amino acid level in rats carrying transplantable ACTH-secreting pituitary tumors, Endocrinology 76, 902 (1965). D. L. Story and R. A. Freedland, The effect of DDT feeding on gluconeogenesis in isolated hepatocytes from starved rats, Toxicol. Appl. Pharmacol. 42, 547 (1978).
BIOCHEMISTRY
AND
PHYSIOLOGY
Adrenalectomy M.G. Department
of Biological
20, 330-339 (1983)
and the Adaptive Liver Response in M i rex-Treated Rats ERVINANDJAMES
Sciences,
Mississippi
State
D. YARBROUGH University,
Mississippi
State,
Mississippi
39762
Received August 2, 1982; accepted May 31, 1983 Mirex, an organochlorine compound, was administered as a single oral dose (100 mg/kg body wt) to both intact and adrenalectomized juvenile male Sprague-Dawley rats. Both mirex-treated intact and adrenalectomized animals (dosed 24 hr postsurgery) exhibited significant increases in liver weight to body weight ratios compared to controls. However, the liver weight to body weight ratios in mirex-treated intact animals were significantly greater than those observed in mirextreated adrenalectomized animals. Significant increases were observed in liver weight to body weight ratios in adrenalectomized animals treated with mirex 4 days after surgery. However, the 96-hr mortality in mirex-treated adrenalectomized animals increased from 20% (mirex dose given 1 day postadrenalectomy) to 56% (mirex dose given 4 days postadrenalectomy). Mirex treatment of intact and adrenalectomized animals had no significant effect upon either serum or hepatic activities of glutamic oxalacetic transaminase, glutamic pyruvic transaminase, sorbitol dehydrogenase, or protein concentrations. Bromsulfophthalein clearance also was not affected by mirex treatment in adrenalectomized animals. Serum glucose concentrations were significantly decreased in both intact and adrenalectomized animals by mirex treatment. Daily corticosterone supplements to adrenalectomized animals restored liver hypertrophy and serum glucose concentrations to levels observed in mirex-treated intact animals. These results suggest that mirex-induced liver enlargement may be mediated by corticosterone. INTRODUCTION
Many xenobiotic compounds induce adaptive responses within the liver (1). One such compound is the organochlorine compound mirex (1,3,4 - methenododecachlorooctahydro - 2H - cyclobuta[c,d]pentalene) which has been shown to be a potent inducer of liver enlargement in rats and mice (2-4). A single oral dose of mirex (100 mg/ kg body wt) administered to an adult rat yields a highly significant increase in the liver weight to body weight ratio (LW/BW) within 4 days (3). This enlargement is not due to an accumulation of water, lipids, or glycogen. The increase in protein necessary for cellular enlargement results from both increased protein synthesis and decreased protein catabolism (5). Following mirex treatment, liver DNA concentration decreases (3) while the rate of parenchymal DNA synthesis remains unchanged (4). Functionally, the detoxication potential of the liver is increased by mirex treatment, 330 0048-3575183 $3.00 Copyright
0 1983 by Academic
AU rights of reproduciion
Press, Inc. in my fom resewed.
as evidenced by a proliferation of the smooth endoplasmic reticulum with a concomitant increase in monooxygenase activity (6, 7). This increase in monooxygenase activity occurs even though mirex is not metabolized by mammalian systems (8). Mehendale (9) has demonstrated that mirex causes increased bile flow and decreased biliary excretion of polychlorinated biphenyls (PCBs) and their derivatives, suggesting hepatobiliary dysfunction. However, standard indicators of liver injury such as serum glutamic oxalacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), lactic dehydrogenase (LDH), and total bromsulfophthalein (BSP) clearance all remain unchanged following mirex treatment (3, 10). Acute mirex dosing of rats also yields a significant increase in serum corticosterone, the level of which remains elevated for several days (11). If the adrenal glands
ADRENALECTOMY
AND
MIREX-INDUCED
are removed prior to mirex treatment, the response in the liver becomes predominantly hyperplastic, with only a minimal increase in liver weight (4). Available evidence appears to indicate: (i) that mirex-induced liver enlargement is primarily due to cellular hypertrophy, and (ii) that this response is related to the availability of adrenal steroids. To further evaluate the relationship between the adrenal glands and the mirex-response, the effects of prior adrenalectomy upon mirex-induced liver enlargement were studied. MATERIALS
AND
METHODS
Animals and test compound. Male Sprague-Dawley rats weighing between 90 and 120g were used in all experiments. The animals were kept in rooms maintained at 24°C with a controlled 12 hr light-dark cycle and housed individually in stainlesssteel cages with wire bottoms. All animals received commercial rat chow (Purina Laboratory Rodent Chow), and tap water ad libitum, with fresh water and food supplied daily between 1:00 and 3:00 PM. Adrenalectomized (ADX) animals received 0.9% saline to drink. Adrenalectomies were performed under light ether anesthesia by means of a paravertebral dorsal approach (12). ShamADX animals were handled in exactly the same manner except that the adrenal glands were exposed, but not disturbed. Adrenalectomies were performed in a laboratory which is removed from the animal holding facilities and were completed prior to 1l:OO AM to minimize diurnal effects. The mirex used was prepared by the Mississippi State Chemical Laboratory (Mississippi State, Miss.) and was assayed to be >99% pure by capillary electron capturegas chromatography. The test dose (100 mg mirex/kg body wt) was dissolved in corn oil (20 mg/ml) and was administered by gavage, while control animals received an equivalent volume of corn oil by gavage. All dosing was completed between 8:OOand lo:@)
AM.
HEPATIC
EFFECTS
331
Animals were either ADX or sham-ADX and half of the animals in each group received a single oral dose of mirex or corn oil. All animals were dosed 24 hr after surgery and representative animals sacrificed from 1 to 7 days following dosing. Final body weights and liver weights were determined at the time of sacrifice. 96hr mortality. To examine the effect of an increase in the time interval between adrenalectomy and mirex dosing upon both the liver response and animal mortality, ADX animals received a single oral dose of mirex or corn oil at either 24, 48, 72, or 96 hr post-ADX. Animals were sacrificed 96 hr after dosing and the livers excised and weighed. The number of animals alive at 96 hr postdosing versus the total number dosed in each group was used to calculate percentage mortality. Mirex-induced hepatotoxicity. To examine the possibility that the decreased liver response and/or increased mortality observed in mirex-treated ADX animals could be related to mirex-induced hepatotoxicity, the following experiment was performed. Intact animals were randomly selected and treated with either mirex or corn oil. Sham-ADX animals were treated with either mirex or corn oil on the 4th day after surgery. ADX animals were treated with mirex or corn oil either 2,4, or 6 days after ADX. In these experiments the maximum liver response and minimal animal mortality occurred 48 hr after dosing. Therefore, 48 hr after mirex dosing the animals in each of the above groups were killed by decapitation under light ether anesthesia and exsanguinated into centrifuge tubes for preparation of serum. Serum sorbitol dehydrogenase (SDH, EC 1.1.1.14) activity was determined at 25°C by the method of Asada and Galambos (13). Glutamic oxalacetic transaminase (GOT, EC 2.6.1) and glutamic pyruvic transaminase (GPT, EC 2.6.1.2) activities were assayed at 37°C according to the methods of Karmen (14), and Time-course
experiment.
332
ERVIN
AND
YARBROUGH
Wroblewski and LaDue (15), respectively. Serum glucose was determined by a method based upon that of Raabo and Terkildsen (16). Protein determinations were performed by the method of Lowry et al. (17). Immediately following collection of blood, the livers were perfused in situ with 5 ml of 0.14 M KCl, excised, and weighed. A 0.5-g sample was removed and homogenized in 5 ml of 0.14 M KC1 containing 0.32 M sucrose. The homogenate was centrifuged at 600g for 15 min and the clear supernatant diluted to 0.5% with buffer and used for determination of hepatic GOT, GPT, and SDH activities and protein concentration. The remaining liver tissue was frozen at - 60°C for later determinations of total hepatic glycogen and lipids. Glycogen was extracted by the method of Hassid and Abraham (18) and glucose determined according to the method of Nelson (19). Total lipids were determined by the method of Folch et al. (20). Bromsulfophthalein clearance. To determine the effect of adrenalectomy on hepatic function in the presence of mirex, bromsulfophthalein (BSP) clearance was assessed. Three groups of animals were established, two of which were ADX and the third sham-ADX. Four days following surgery one group of ADX animals received a single oral dose of mirex while the remaining ADX and sham-ADX animals received corn oil. Forty-eight hours after mirex treatment, all animals received an intravenous (iv) injection of 75 mg BSP/kg body wt. Fifteen minutes after injection, serum BSP concentration was determined by the method of Kutob and Plaa (21). Corticosterone supplement. Animals were ADX as described above and divided into seven groups. Twenty-four hours after surgery, the animals in five groups received a single oral dose of mirex while the animals in group 6 received corn oil. Animals in group 7 received corn oil and a subcutaneous (SC)injection of soybean oil. The mirex-treated animals also received a
single, daily SCinjection of either 1, 5, 10, or 20 mg corticosterone/kg body wt dissolved in soybean oil, or soybean oil alone. The group orally dosed with corn oil also received a daily corticosterone supplement of 20 mg/kg body wt. Animals were sacrificed 96 hr after mirex or oil treatment because previous results indicated that both the liver response to mirex and the mirex-induced mortality in ADX animals were maximal while serum glucose was reduced at this time. Therefore, the effect of corticosterone supplements upon these parameters could be investigated. All animals were decapitated and exsanguinated into centrifuge tubes and the serum prepared for total glucose determinations. Liver weights and body weights were also taken. Statistics. All sample means are expressed as the mean ? standard error of the mean (x rt SEM). The data from the corticosterone supplement experiment were subjected to one-way analysis of variance followed by Duncan’s new multiple range test (DNMRT). For the remaining experiments, differences between means were determined by either the Student’s t test or by the paired sample t test (22). RESULTS
Due to the presence of significant differences (P < 0.05) in LW/BW between oil-treated intact and oil-treated ADX animals on Days 2, 5, and 6, these data were presented separately. There were significant increases (P < 0.001) in LW/BW in mirex-treated intact and ADX animals when compared to oiltreated controls on Days 1 through 7 and Days 3 and 6, respectively (Figs. IA and B). However, there was also a significant difference in LW/BW between mirextreated ADX animals and mirex-treated intact animals on Days 1 through 6: Although the maximal rate of liver enlargement occurred by Day 2 in mirex-treated intact animals there was a continued increase through Day 4 (Fig. 1A). In contrast, maxTime-course
experiment.
ADRENALECTOMY
AND ----
I
MIREX-INDUCED
HEPATIC
333
EFFECTS
SHAM + MlREX SHAM + OIL
A
‘&--&--2
-I L . 3
DAYS
6. ADX + MIREX ADX + OIL
---
4
ADX
FIG. 2. Liver weight to body weight ratios in control and mirex-treated ADX rats dosed either 1, 2, 3, or 4 days after adrenalectomy. Animals received either a single oral dose of mirex (100 mglkg body wt) in corn oil or corn oil only and were sacrificed 96 hr later. Each point represents x -t SE offrom 9 to 16 animals. Values statistically different from controls are indicated by an asterisk (*P < 0.001).
8 -
3
POST
7-
I 0
I
2
DAYS
3
4
POST MIREX
5
6
7
DOSE
FIG. 1. Liver weight to body weight ratios (L W/BW) in male rats sham adrenalectomized (Sham) or adrenalectomized (ADX) 24 hr prior to mirex treatment. Mirex was administered as a single oral dose (100 mglkg body wt), with control animals receiving an equivalent volume of corn oil. Each point represents x 2 SE of from 5 to 11 animals. LWIBW in mirextreated intact and ADX animals exhibited significant increases (P < 0.001) over their respective controls on Days 1 through 7 and Days 3 through 6, respectively. There also were significant differences (P < 0.001) in LWIBW between mirex-treated intact animals and mirex-treated ADX animals on Days 1 through 6.
and control animals. Increasing time after ADX prior to mirex treatment also increased mortality in mirex-treated ADX animals (Fig. 3). In contrast, there was no mortality in any of the control groups. Mirex-induced hepatotoxicity. Because the results obtained from the sham-ADX animals closely paralleled those in intact animals, only the intact will be reported. 1007
;
-
MIREX
----
OIL
80
z!
imal rate of liver enlargement in mirextreated ADX animals did not occur until Day 3 post-mirex dosing. Both intact and ADX animals exhibited weight gain during this 4-day period. 96hr Mortality. Although there were no differences in initial or final body weights in any of the groups tested, animals dosed with mirex 1,2, and 3 days after adrenalectomy exhibited a significant increase (P < 0.001) in LW/BW 4 days after mirex dosing (Fig. 2). However, in animals dosed 4 days after ADX there were no significant differences in LW/BW between mirex-treated
!lj
/
I
2
3
.I
4
DAYS POST AOX
3. Ninety-six-hour mortality in male adrenalectomized (ADX) rats receiving either a single oral dose of mirex (100 mglkg body wt) or corn oil at either 1, 2, 3, or 4 days following surgery. Each point denotes the percentage mor*o!ity (number deadltotal number dosed) at 96 hrfollowing mirex treatment. Each point represents from 18 to 20 animals for mirex treatment and 11 to 13 animals for controls, FIG.
334
ERVIN AND YARBROUGH
There were no differences in initial body weights between mirex-treated and control animals in any of the groups tested (intact, 2-day, 4-day, or 6-day ADX). Final body weights in intact animals were not different from initial body weights, but liver weights in mirex-treated intact animals were significantly increased (P C 0.001) over controls (Table 1). In contrast, final body weights in mirex-treated 2-day and 4-day ADX animals were significantly reduced (P < 0.05) from initial levels (Table 1). However, there were no differences in liver weights between mirex-treated and control 2-, 4-, or B-day ADX animals. Since the 2- and 4-day mirex-treated animals exhibited a loss in body weight, their LW/BW ratios were not considered valid for comparison. The fact remains that intact animals exhibited a significant increase in liver weight, whereas liver weights in mirex-treated ADX animals were not different from their respective controls. There was a significant decrease (P < 0.05) in serum protein concentration in mirex-treated animals dosed 2 days following adrenalectomy (mirex: 3.20 g/d1 +
0.07, N = 15; oil: 3.45 g/d1 rt 0.07, N = 11). There were, however, no differences in serum protein concentrations in any of the remaining test groups. Serum glucose concentrations exhibited a significant reduction (P < 0.01) in mirex-treated intact animals, and in ADX animals dosed with mirex either 2,4, or 6 days after adrenalectomy (Fig. 4). There were significant increases (P < 0.05) in hematocrit values (vol %) in mirex-treated intact (mirex: 46.28 +1.5, N = 9; control: 41.44 + 0.96, N = 9) and in both 2-day ADX animals (mirex: 52.28 + 1.65, N = 9; control: 41.21 k 1.89, N = 7) and 4-day ADX animals (mirex: 51.90 _t 3.91, N = 5; control: 41.06 +1.40, N = 9). Biochemical parameters including serum and hepatic SDH, GOT, and GFT activities were not different between mirex-treated and control animals except for a significant reduction (P < 0.05) in hepatic GPT in animals dosed with mirex 4 days after surgery (mirex: 3.94 2 0.42 mmol NADH oxidized/g tissue min-‘, N = 8; control: 5.67 + 0.37 mmol NADH oxidized/g tissue min-l, N = 9).
TABLE Body Weights and Liver Weights in M&x-Treated
1 Intact and Adrenalectomized
Body weight (g)
Intact Mirex (14) Control (12) Adrenalectomized 2 Days Mirex (15) Control (11) 4 Days Mirex (11) Control (12) 6 Days Mirex (12) Control (9)
Mule Rats”
Initial
Final
Liver weight b?)
100.71 + 3.66 95.33 + 4.62
92.31 2 3.41 99.88 + 5.23
6.93 + 0.20** 4.91 f 0.27
112.07 f 2.06 107.64 + 2.72
99.21 f 2.30* 108.24 ‘: 2.96
5.21 + 0.17 4.97 2 0.19
112.91 f 3.30 108.33 k 3.24
103.35 + 2.68* 111.89 + 2.80
5.50 Ifr 0.24 5.16 f 0.17
120.67 f 2.51 117.44 * 4.35
111.66 f 2.36 119.11 + 6.78
5.77 + 0.24 5.35 f 0.44
0 Adrenalectomized animals received mirex or corn oil either 2, 4, or 6 days following surgery. Mirex was administered as a single oral dose (100 mgikg body wt) and animals were sacrificed 48 hr after treatment. Values are expressed as x f SE with values in parentheses representing the number of animals in each group. * Final body weight significantly different from initial body weight (P < 0.05). ** Liver weight significantly different from control (P < 0.001).
ADRENALECTOMY
AND
MIREX-INDUCED
I8 IOO3 3 80 60_ ;i;.‘i I
I 0
4
2 DAYS
POST
I 6
ADX
4. Serum glucose concentrations in male rats treated with a single oral dose of mirex (100 mglkg body wt) or corn oil at either 0 days (Intact), or 2, 4, or 6 days after adrenalectomy. Each point represents x f SE of from 8 to 14 animals. All animals were sacrificed 48 hr after mirex treatment. Both mirextreated Intact and ADX animals exhibited significant decreases (P < 0.01) in serum glucose when compared to oil-treated controls. FIG.
Mirex-treated intact animals exhibited a significant reduction (P < 0.05) in total hepatic glycogen (expressed as mg/g tissue) when compared to controls (60.5 +- 3.4, N = 5; 68.7 +. 0.5, N = 5, respectively). Mirex-treated intact animals also exhibited a significant increase (P < 0.05) in total liver lipid (mirex: 35.90 +- 2.30 mg/g tissue; control: 29.80 + 0.76 mg/g tissue). There were no differences in total liver glycogen or lipid in either control ADX or mirextreated ADX animals dosed either 2, 4, or 6 days after adrenalectomy. Hepatic glycogen levels in ADX animals were reduced (P < 0.05) from intact levels, which is consistent with known effects of adrenalectomy (23). Liver functionality. A mirex-induced increase in the rate of BSP clearance has been reported previously for intact, male Sprague-Dawley rats (10). However, no differences were detected in the rate of serum BSP clearance between intact (25.87
HEPATIC
EFFECTS
335
2 1.10, N = 6) or ADX animals receiving either mirex (31.80 + 2.26, N = 9) or corn oil (32.55 ? 2.45, N = 6). Values are expressed as mg BSPldl of serum 15 min following an iv injection of 75 mg BSP/g body wt. Corticosterone supplement. Because none of the animals included in this experiment exhibited changes in body weight, the alterations in LW/BW observed reflect actual changes in liver weight. There were no differences in LW/BW between ADX and corticosterone-supplemented (20 mg/kg body wt, SC) ADX animals 4 days after oiltreatment. ADX animals receiving mirex treatment alone, or mirex treatment and a daily SC injection of soybean oil also showed no differences in LW/BW (5.78 it 0.20 and 5.75 + 0.20, respectively). Mirextreated ADX animals receiving daily corticosterone supplements of either 1, 5, 10, or 20 mg/kg body wt exhibited significant increases in LWIBW when compared to mirex-treated ADX animals receiving a soybean oil supplement (Fig. 5). Even though corticosterone supplement did result in increased LW/BW in mirex-treated ADX animals, the response did not appear to be dose dependent. Mirex-treated ADX animals receiving either a soybean oil supplement or 1 mg/kg corticosterone in soybean oil exhibited a 33% mortality during the 96 hr following mirex treatment. Mirex-treated ADX animals receiving larger corticosterone supplements of 5, 10, or 20 mglkg corticosterone exhibited no mortality. These results, in addition to the previously described 96-hr mortality (Fig. 3), offer good evidence that the increase in mortality observed in mirextreated ADX animals is related to the absence of corticosterone in these animals. Serum glucose concentrations in mirextreated ADX animals were significantly reduced (P < 0.05) from the levels observed in control ADX animals (Fig. 5). Daily corticosterone supplements of 1 mg/kg body wt had no effect upon serum glucose concentrations in ADX mirex-treated animals
336
**** I L-i ERVIN
I
LWlBW
IO
9.
8.
MIREX (mg/kg SW) CORTICOSTERONE (mg/kg EW)
YARBROUGH
0
SERUM
GLUCOSE
* fl
I
0 12
AND
100 * 0
0
100
100
100
l
+
+
+
20
I
5
IO
150
2 F
IO0
3 8 2 ” 4 a I
50
100 +
20
FIG. 5. Liver weight to body weight ratios (LW/BW) and serum glucose concentrations in adrenalectomized (ADXI male rats receiving a single oral dose of mirex (100 mglkg body wt) or corn oil 24 hr post-ADX. All animals were sacrificed 96 hr after mirex treatment. Mirex-treated animals also received daily SC corticosterone supplements of either 0, 1, 5, 10, or 20 mg corticosteronelkg body wt. Control animals received either a corticosterone supplement of 20 mglkg body wt or soybean oil only. Values are expressed as x ? SE offrom four to six animals. Statistically signt@cant differences between mirex-treated ADX animals receiving corticosterone supplements or vehicle are indicated by an asterisk (*P < 0.05).
(Fig. 5). However, mirex-treated ADX animals receiving daily corticosterone supplements of either 5, 10, or 20 mg/kg body wt exhibited significant increases in serum glucose (Fig. 5).
adrenalectomy and mirex treatment is increased, the adrenal gland independent component appears to diminish (< 15% by Day 4) (Fig. 2). This decrease in the adrenal independent response may be related to the presence of intracellular, receptor-bound DISCUSSION corticosterone which may still be exerting The mirex-induced increases in relative an effect even though circulating corticoliver weight in intact animals observed in sterone has been abolished. Therefore, it our experiments are similar in magnitude to appears that the mirex-induced increase in values reported previously (3). Other or- liver mass observed in intact rats is preganochlorine pesticides such as DDT and dominantly dependent upon functional addieldrin have also been reported to induce renal glands. This dependence is further ilincreased liver size (24, 25). The adaptive lustrated by the fact that mirex-induced liver growth induced by mirex in intact an- liver enlargement can be restored in ADX imals represents approximately a 72% in- animals by daily corticosterone supplement crease in liver mass in 96 hr. Although a (Fig. 5). major portion of the mirex-induced liver reMirex treatment has also been shown to sponse appears to be adrenal gland depen- induce an increase in serum corticosterone dent (Fig. lA), there is a minor component concentration (11). Similar increases in which is adrenal gland independent (Fig. serum corticosterone have also been re1B). However, if the time interval between ported following treatment with DDT (24),
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dieldrin (25), endrin (26), PCB’s (27), and in response to various organophosphates (28). Therefore, mirex-induced liver enlargement may be directly related to the hypertrophic potential of adrenal glucocorticoids. The ability of glucocorticoids to promote cellular hypertrophy and inhibit DNA synthesis within the liver has been well documented (29-36). It has also been demonstrated that the effect of adrenalectomy is to increase the rate of liver DNA synthesis (37), and that this response can be further stimulated by mirex (4). Thus, the increase in relative liver weight observed in ADX animals on Day 3 may represent mirex-induced cellular hyperplasia (Fig. 1). The loss of the mirex-induced growth response in the ADX animal does not appear to be due to parenchymal cell damage. In both intact and adrenalectomized animals, serum and hepatic levels of SDH, GOT, or GPT offered no evidence of mirex-induced hepatotoxicity. In addition, evaluation of hepatic functional capacity by assessment of BSP clearance rates offered no suggestion of a mirex-induced impairment of biliary excretion. Adrenalectomy prior to mirex treatment results in both a decrease in the liver response to mirex and an increase in animal mortality (Figs. 1 and 3). In addition, it appears that both of these responses are corticosterone dependent. That adrenal glucocorticoids are decisive factors in allowing an animal to cope with physical and chemical stress is well recognized (24, 28, 3840). It has been proposed that glucocorticoids are protective of an animal by “syntoxic mechanisms” which allow for increased tissue tolerance to a toxicant without increasing its metabolism (38). However, the mechanism by which these effects are exerted is unknown. The increased serum levels of corticosterone following mirex treatment (11) would be expected to yield increased serum glucose, increased hepatic gluconeogenesis and glycogenesis, a mobilization of fatty acids for energy, and a pooling of amino
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acids for biosynthetic work. These responses are consistent with the known effects of chemical agents such as dieldrin, endrin, and certain organophosphates (25, 26, 28, 30). In contrast, our results indicate that mirex induces a slight hepatic glycogen depletion in intact animals and hypoglycemia in both intact and ADX animals (Fig. 4). Because mirex also induces hypoglycemia in intact animals and corticosterone supplement given to mirex-treated animals did not restore serum glucose to control levels (Fig. 5), it appears that mirex may exert a direct effect upon hepatic carbohydrate metabolism. Other chlorinated hydrocarbons (DDT and PCB’s) also effect a decrease in hepatic glycogen deposition and hypoglycemia, possibly due to an inhibition of hepatic gluconeogenesis (41,27). Garthoff et al. (27) have stated that augmented rates of gluconeogenesis are critical for normal adaptation of an animal to a variety of conditions. If increased gluconeogenesis and hyperglycemia are as important as has been suggestedin allowing an animal to cope with chemical stress, the adaptive potential of the ADX animal following mirex treatment would indeed be severely diminished. From these results, it appears that the reduction in the mirex-induced liver growth response in ADX animals may not be due to hepatic parenchymal necrosis or altered hepatic function. Instead, the response appears to be dependent upon corticosterone, and represents an indirect effect of mirex upon the liver which is mediated by the adrenal glands. However, whether the requirement for corticosterone in the mirex response is directly related to the hypertrophic potential of corticosterone or instead represents a permissive effect for some other mechanism remains to be demonstrated. ACKNOWLEDGMENT The authors would like to thank Mrs. Jo Grimley for preparation of the illustrations.
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REFERENCES 1. R. Schulte-Hermann and E. Schmitz, Feedback inhibition of hepatic DNA synthesis, Cell Tissue Kinet. 13, 371 (1980). 2. J. L. Byard, U. C. Koepke, R. Abraham, L. Golberg, and E Coulston, Biochemical changes in the liver of mice fed mirex, Toxicol. Appl. Pharmacol. 33, 70 (1975). 3. K. M. Robinson and J. D. Yarbrough, Liver response to oral administration of mirex in rats, Pestic. Biochem. Physiol. 8, 65 (1978). 4. P. I. Karl, “Relationship between the Adrenal Glands and Hepatic Growth Regulating Substances in Mirex-Induced Liver Growth,” Ph.D. dissertation, Mississippi State University, 1981. 5. K. M. Robinson and J. D. Yarbrough, Liver protein synthesis and catabolism in mirex-pretreated rats with enlarging livers, J. Pharmacol. Exp. Ther. 215, 82 (1980). 6. R. C. Baker, L. B. Coons, R. B. Mailman, and E. Hodgson, Induction of hepatic mixed function oxidases by the insecticide, Mirex, Environ. Res. 5, 418 (1972). 7. H. M. Mehendale, P. R. Chen, L. Fishbein, and H. B. Matthews, Effect of mirex on the activities of various hepatic mixed function oxidases, Arch. Environ. Contam. Toxicol. 1, 245 (1973). 8. J. R. Gibson, G. W. Ivie, and H. W. Dorough, Fate of mirex and its major photodecomposition product in rats, J. Agric. Food Chem. 20, 1246 (1972). 9. H. M. Mehendale, Mirex-induced suppression of biliary excretion of polychlorinated biphenyl compounds, Toxicol. Appl. Pharmacol. 36,369 (1976). 10. K. M. Robinson and J. D. Yarbrough, A study of liver function in rats with mirex-induced enlarged livers, Pestic. Biochem. Physiol. 9, 61 (1978). 11. J. D. Williams and J. D. Yarbrough, The relationship between mirex-induced liver enlargement and the adrenal glands, Pestic. Biochem. Physiol. 19, 15 (1983). 12. R. Gaunt, Adrenalectomy in the rat, Amer. J. Physiol. 103, 494 (1933). 13. M. Asada and J. T. Galambos, Sorbitol dehydrogenase and hepatocellular injury: An experimental and clinical study, Gastroenterology 44, 578 (1%3). 14. A. Karmen, A note on the spectrophotometric assay of glutamate-oxalacetic transaminase in human blood serum, J. Clin. Invest. 34, 131 (1955). 15. E Wroblewski and J. S. LaDue, Serum glutamic pyruvic transaminase in cardiac and hepaticdisease. Proc. Sot. Exp. Biol. Med. 91, 569 (1956).
16. E. Raabo and T. C. Terkildsen, On the enzymatic determination of blood glucose, Stand. J. Clin. Lab. Invest. 12, 402 (l%O). 17. 0. H. Lowry, N. J. Rosenbrough, A. L. Farr, and R. J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193, 265 (1951). 18. W. Z. Hassid and S. Abraham, Chemical procedures for analysis of polysaccharides. In Methods of Enzymology (S. Colowick and N. Kaplan, Eds.), Vol. 3, pp. 34-37, Academic Press, New York, 1957. 19. N. Nelson, A photometric adaptation of the Somogyi method for the determination of glucose, J. Biol. Chem. 153, 375 (1944). 20. J. Folch, M. Lees, and G. H. Sloan-Stanley, A simple method for the isolation and purification of total lipids from animal tissues, J. Biol. Chem. 226, 497 (1957). 21. S. D. Kutob and G. L. Plaa, Assessment of liver function in mice with bromosulphaleine, J. Appl. Physiol. 17, 123 (1962). 22. J. H. Zar, “Biostatistical Analysis,” p. 121, Prentice-Hall, Englewood Cliffs, N.J., 1974. 23. R. Steele, Influences of corticosteroids on protein and carbohydrate metabolism, in Handbook of Physiology: Endocrinology (H. Blaschko, G. Sayers, and A. D. Smith, eds.), Vol. VI, pp. 135, American Physiological Society, Washington, D.C., 1975. 24. R. J. Szot and S. D. Murphy, Phenobarbital and dexamethasone inhibition of the adrenocortical response of rats to toxic chemicals and other stresses, Toxicol. Appl. Pharmacol. 17, 761 (1970). 25. S. C. Bhatia, S. C. Sharmu, and T. A. Venkitasubramanian, Effect of dieldrin on hepatic carbohydrate metabolism and protein biosynthesis in vivo, Toxicol. Appl. Pharmacol. 24, 216 (1973). 26. K. Meena, P. K. Gupta, and S. R. Bawa, Endrininduced toxicity in normal and irradiated rats. Environ. Res. 16, 373 (1978). 27. L. H. Garthoff, E E. Cerra, and E. M. Marks, Blood chemistry alterations in rats after single and multiple gavage administration of polychlorinated biphenyl, Toxicol. Appl. Pharmacol. 60, 33 (1981). 28. S. D. Murphy and S. Porter, Effects of toxic chemicals on some adaptive liver enzymes, liver glycogen, and blood glucose in fasted rats, Biochem. Pharmacol. 15, 1665 (1966). 29. J. C. Davis and T. A. Hyde, The effect of corticosteroids and altered adrenal function on liver regeneration following chemical necrosis and partial hepatectomy, Cancer Res. 26, 217 Wm. 30. K. H. Raab and T. E. Webb, Inhibition of DNA
ADRENALECTOMY
31. 32,
33.
34. 35. 36.
AND MIREX-INDUCED
synthesis of regenerating rat liver by hydrocortisone, Experieniia 25, 1240 (1969). I. C. Henderson, R. E. Fischel, and J. N. Loeb, Suppression of liver DNA synthesis by cortisone, Endocrinology 88, 1471 (1971). J. N. Loeb, C. Borek, and L. L. Young, Suppression of DNA synthesis in hepatoma cells exposed to glucocorticoid hormone in vitro, Proc. Nat. Acad. Sci. USA 70, 3852 (1973). I. C. Henderson and J. N. Loeb, Hormone-induced changes in liver DNA synthesis: effects of glucocorticoids and growth hormone on liver growth and DNA polymerase activity, Endocrinology 94, 1637 (1974). L. Desser-Wiest, Autosynchronization of rat liver cells with endogenous corticosterone after partial hepatectomy, Cell Tiss. Kinet. 8, 1 (1975). R. Schulte-Hermann, Induction of liver growth by xenobiotic compounds and other stimuli, Crit. Rev. Toxicol. 3, 97 (1974). T. J. Castallano, R. L. Schiffman, M. C. Jacob, and J. N. Loeb, Suppression of liver cell proliferation by glucocorticoid hormone: A comparison of normally growing and regenerating
37.
38.
39.
40.
41.
HEPATIC
EFFECTS
339
tissue in the immature rat, Endocrinology 102, 1107 (1978). L. Desser-Wiest, Stimulation of DNA synthesis in rat liver by adrenalectomy, J. Endocrinol. 60, 315 (1974). P Kourounakis, H. Selye, and Y. Tache, Catatoxic steroids, in Advances in Steroid Biochemistry and Pharmacology (M. H. Brings and G. A. Christie, Eds.), Vol. 6, pp. 35-57, Academic Press, New York, 1977. W. E Bousquet, D. Rupe, and T. S. Miya, Endocrine modification of drag responses in the rat, J. Pharmacol. Exp. Ther. 147, 376 (1965). G. Weber, R. L. Singhal, S. K. Srivastava, H. J. Hird, and J. Furth, Behavior of hepatic gluconeogenic enzymes, RNA metabolism, and amino acid level in rats carrying transplantable ACTH-secreting pituitary tumors, Endocrinology 76, 902 (1965). D. L. Story and R. A. Freedland, The effect of DDT feeding on gluconeogenesis in isolated hepatocytes from starved rats, Toxicol. Appl. Pharmacol. 42, 547 (1978).