tissue injury and repair as parallel and opposing responses to ccl4 hepatotoxicity: a novel...

ELSEVIER Toxicology 118 (1997) 181-193

Tissue injury and repair as parallel and opposing responses to Ccl, hepatotoxicity: a novel dose-response’

P.S. Rao, R.S. Mangipudy, H.M. Mehendale*

Division of Toxicology, College of Pharmacy and Health Sciences, Northeast Louisiana University, Monroe, LA 71209-0470, USA

Received 12 September 1996; accepted 12 December 1996

Abstract

Recent studies indicate that the rate and extent of tissue repair, elicited as an endogenous response to toxic insult,

are critical determinants in the ultimate outcome of hepatic injury. Therefore, the objective of this study was to

develop a dose-response relationship for Ccl, measuring liver injury and tissue repair as two simultaneous but

opposing responses. Male Sprague-Dawley rats were injected with a 40-fold dose range of Ccl, (0.1-4 ml/kg i.p.) in corn oil vehicle. Liver injury was assessed by serum enzyme elevations and histopathology, and tissue repair was

measured by [3H]thymidine incorporation into hepatonuclear DNA and proliferating cell nuclear antigen immunohis-

tochemistry over a time course of 0 to 96 h. Stimulation of cell division, evident even after a subtoxic dose of Ccl,,

increased in a dose-dependent manner until a threshold (2 ml/kg) was reached. Doses above this threshold yielded no

further increase in tissue repair. Instead, tissue repair response was significantly delayed and diminished. Injury was

markedly accelerated above the threshold indicating an unrestrained progression of injury. Although 4 ml CCl,/kg

consistently caused 80% lethality by 48 h, tissue repair response in the 20% surviving rats was increased by about

5-fold, aptly demonstrating the critical role of tissue repair in overcoming injury and enabling these animals to survive. This study suggests that, in addition to the extent of tissue repair, the time of onset of tissue repair also

determines the extent of hepatic injury and inter-individual differences in the magnitude of tissue repair may

contribute significantly to inter-individual differences in susceptibility to toxic chemicals. Thus, while dose-related and

prompt stimulation of tissue regeneration leads to recovery, delayed and attenuated repair response, occurring at

higher doses, leads to progression of injury and animal mortality. Such dose-response relationships may lead to a better understanding of the underlying cellular mechanisms of injury inflicted by chemical toxicants and aid in

fine-tuning risk assessment. 0 1997 Elsevier Science Ireland Ltd.

Abbreviations: ALT, alanine aminotransferase; CLC, colchicine; ‘H-T, tritiated thymidine; MTD, maximum tolerated dose; PCNA, proliferating cell nuclear antigen; SDH, sorbitol dehydrogenase.

* Corresponding author. Tel: + I-318-3421691; Fax: + l-318-3421686; E-mail: [email protected].

’ Preliminary findings of this study were presented at the VI Annual North American Meeting of the International Society for the

Study of Xenobiotics, Raleigh, North Carolina, October 23327, ISSX Proceedings, 6: 62, 1994.

0300-483X/97/$17.00 0 1997 Elsevier Science Ireland Ltd. All rights reserved.

PIT SO300-483X(97)03617-2

182 P.S. Rao rt al. 1 Toxicology 118 (1997) 181-193

Keywords: Dose-response; Carbon tetrachloride; Colchicine; Maximally tolerated dose; Mitosis; Necrosis; Tissue repair

1. Introduction

Several reviews describing the mechanisms of Ccl, toxicity have been published (Recknagel and Glende, 1977; Slater, 1987; Mehendale, 1989a,b, 1991). The leading theory for the mechanism of cellular damage caused by Ccl, is that the com- pound is bioactivated by Cyt P-450 to . Ccl, free radical (Koch et al., 1974; Sipes et al., 1974; Recknagel and Glende, 1977; Slater, 1987) and further converted to a peroxy radical, Ccl,0 .2 (Slater, 1987). The free radicals then react with polyunsaturated fatty acids to propagate a chain reaction leading to lipid peroxidation. The primary lesion characterized by centrilobular necrosis (Koch et al., 1974) is dependent on the dose of the chemical.

Liver is one of the few adult organs demonstrating a physiological growth response (Taub, 1996). This comes from the ability of hepatocytes to undergo cell division under the action of a series of intra and extrahepatic stimuli (Pistoi and Morello, 1996). The adult hepatocytes are exquisitely sensitive to signals generated in their microenvironment and respond by modulation of many diverse liver specific functions to sustain or increase several liver specific genes while decreas- ing the expression of others (Diehl and Rai, 1996). Hepatocellular proliferation can be induced ex- perimentally in animals by chemical (Leevy et al., 1959; Nakata et al., 1985; Columbano et al., 1990) or viral agents or by partial hepatectomy (Michalopulos, 1990; Steer, 1995; Pistoi and Morello, 1996). At low doses Ccl, stimulates cell division (Soni and Mehendale, 1991a,b; Koda- vanti et al., 1992; Mehendale et al., 1994a) which protects the animal against the lethal effects of a subsequently administered high dose (Mehendale et al., 1994a). Augmented tissue repair fends off progressive loss of liver tissue and restores liver structure and function, enabling the animal to overcome massive injury from the lethal dose (Mehendale et al., 1994a,b). Autoprotection has

also been reported for dimethylnitrosamine in mice (Pound, 1975), and 2-butoxyethanol (Di- gavalli and Mehendale, 1995) and thioacetamide (Mangipudy et al., 1995a) in rats. The critical importance of stimulated tissue repair is further

illustrated by thioacetamide heteroprotection where a low dose of thioacetamide (50 mg/kg) stimulates cell division to protect the animals from the lethal effects of a high dose of acetaminophen (1800 mg/kg; Chanda et al., 1995). Phenobarbital- and isopropanol-potentiated liver injuries of Ccl, do not lead to lethality because of simultaneously stimulated tissue repair (Koda- vanti et al., 1992; Rao et al., 1996). Collectively, these studies suggest that stimulation of tissue repair is a critical response which restrains injury, irrespective of the mechanism by which it is ini- tiated. High doses of toxic chemicals inhibit stimulation of tissue repair leading to unrestrained progression of injury and animal mortality (Mehendale et al., 1994a; Mangipudy et al., 1995b) whereas at low and moderate doses, a dose-dependent stimulation of tissue repair leads to recovery from injury (Mehendale et al., 1994a; Mangipudy et al., 1995b). Therefore, the rate and extent of stimulated tissue repair appears to determine the ultimate outcome of injury by a variety of toxic chemicals (Chanda et al., 1995; Digavalli and Mehendale, 1995; Mangipudy et al., 1995b; Rao et al., 1996).

Dose-response relationship is a fundamental concept in classical pharmacology and toxicology (Klaassen and Eaton, 1988). Since tissue repair and injury are simultaneously occurring but opposing biological responses to chemicals, measuring the tissue repair response in addition to injury might increase the value of dose-response relationships in predictive toxicology. Therefore, the objective of this study was to develop a dose-response relationship for Ccl, by measuring tissue injury and repair as two parallel but opposing dynamic responses to a 40-fold dose range of Ccl, over a time course. This study was con-

P.S. Rao et al. I Toxicology 118 (1997) 181- 193 183

ducted to test the hypothesis that tissue repair increases with increasing dose until a threshold is reached. Beyond this threshold, tissue repair response is delayed and attenuated leading to unrestrained progression of injury culminating in animal death. We report here that tissue repair occurs in a dose-dependent fashion and measuring tissue repair as a compensatory response to toxic chemicals might enhance the precision and predictive value of dose-response paradigms in toxicol-

ogy.

2. Materials and methods

2.1. Chemicals

All the biochemicals and chemicals were obtained from Sigma Chemical Co. (St. Louis, MO) unless otherwise specified.

2.2. Animals

Male Sprague-Dawley rats (175-225 g, 7-8 weeks) were obtained from Harlan Sprague- Dawley Inc. (Indianapolis, IN) and were housed over sawdust bedding known to be free of any chemical contamination for 10 days in 12-h pho- toperiod, 21 k 1°C temperature and 50-80% rela- tive humidity in our central animal facility. They had free access to water and normal rodent chow (Harlan Teklad Rat Chow No. 7001, Madison, WI).

2.3. Treatment

After an acclimation period, the rats were ran- domized into six groups and treated with Ccl, (0, 0.1, 1, 2, 3, and 4 ml/kg, i.p.) dissolved in corn oil (1 ml/kg). The control group (0 ml CC&/kg) was administered corn oil used as a vehicle. Rats of all groups were given normal diet and water ad libi- turn during and after treatment till termination.

2.4. Lethality studies

The first experiment was designed to determine the lethality following Ccl, administration in rats.

Following the administration of each dose of Ccl, (0, 0.1, 1, 2, 3, and 4 ml/kg) the rats were observed twice daily for 14 days and survival/ lethality was recorded in each group.

2.5. Plasma enzymes

Blood was collected from the dorsal aorta from four separate rats at each time point after Ccl, or vehicle administration under light diethyl ether anesthesia. Plasma was separated for the estimation of alanine aminotransferase (ALT, EC 2.6.1.2.) and sorbitol dehydrogenase (SDH, EC 1.1.1.14) as markers of liver injury, using Sigma Chemical Co. (St. Louis, MO) Kit No. 59 UV (ALT) and 50 UV (SDH).

2.6. Histopathology

After collection of blood, rats were terminated by surgically removing the liver. As indicated above, four rats were used for each time point. Portions of liver from each group collected at various periods after Ccl, treatment were washed with normal saline (0.9% NaCl), cut into small slices and fixed in 10% phosphate-buffered formaldehyde fixative solution for 48 h. The tissues were then transferred to 70% ethyl alcohol, processed, and embedded in paraffin. Liver sections (5 pm thick) were stained with hematoxylin- eosin (H&E) for histological examination under light microscope.

2.7. In vivo incorporation of t3H]thymidine into rat liver DNA

Tritiated thymidine (3H-T) incorporation into hepatonuclear DNA was measured as an index of S-phase synthesis using the procedure of Chang and Looney (1965); 35 PCi 3H-T/rat (i.p.) were administered 2 h prior to sacrifice at each time point. The DNA content of the supernatant frac- tion was estimated by the diphenylamine reaction (Burton, 1956). Since a 3H-T incorporation study may indicate DNA repair instead of DNA synthesis, a proliferating cell nuclear antigen (PCNA) assay was used to confirm S-phase stimulation and cell cycle progression.

184 P.S. Rao et al. i Toxicology 118 (1997) 181-193

2.8. PCNA assay

The PCNA assay was conducted as described by Greenwell et al. (1991). Briefly, the liver sections mounted on slides were first blocked with casein and then reacted with monoclonal antibody to PCNA (Dako Corporation, Carpentaria, CA). The antibody was then linked with biotinylated goat anti-mouse IgG antibody (Boehringer/ Mannheim, Indianapolis, IN) which was then labeled with streptavidin-conjugated peroxidase (Jackson Immunoresearch, West Grove, PA). Color was developed by exposing the peroxidase- labeled streptavidin to diaminobenzidine, which forms a brown reaction product. The sections were then counterstained with Gill’s hematoxylin. G, cells were blue and did not take the PCNA stain, G, cells were light brown in color, S-phase cell nuclei stained dark brown while the G, cells had a cytoplasmic staining with or without speckled nuclear appearance. A total of a thousand cells were counted for each liver section.

2.9. Colchicine antimitosis

If cell division and tissue repair are critical events for recovery from injury, then inhibition of mitosis should result in increased lethality, even from a moderately toxic dose of CCL, due to ineffective tissue repair. Colchicine is known to exert its antimitotic effects by inhibiting S-phase synthesis (Tsukamoto and Kojo, 1989) and by microtubular perturbation (Manfredi and Hor- witz, 1984). Colchicine (CLC, 1 mg/kg) was administered 24 h after Ccl, (1 ml/kg) administration. Plasma enzymes and ‘H-T incorporation were estimated at 36, 48, 72 and 96 h after Ccl, administration. Previous studies have shown that colchicine administered at this antimitotic dose neither affects normal liver function, nor does it interfere with Ccl, metabolism (Rao and Mehen- dale, 1991, 1993).

2.10. Statistics

Mean + S.E.M were calculated for all values. Statistical differences were determined by the General Linear Model followed by Least Square

of Means to determine the means which were significantly different from each other or from controls. In all cases, PI 0.05 was used as the statistical criterion to determine significant differences.

3. Results

3.1. Lethality studies

In the first experiment, six groups of rats were treated with a 40-fold dose range of Ccl, (0, 0.1, 1, 2, 3, and 4 ml/kg, i.p.) and observed for survival/lethality in each group for a period of 14 days. At the end of 14 days, 100% survival was noted in rats administered 0.1 and 1 ml/kg Ccl,, while 40, 60, and 80% mortality occurred in rats receiving 2, 3, and 4 ml CC&/kg, respectively, exhibiting a dose-dependent increase in mortality. All deaths occurred between 1 and 2 days. Doses higher than 4 ml/kg caused animal death starting from 6 h (data not shown), precluding the use of such doses for time-course studies. Hundred percent survival was noted in the vehicle control rats.

3.2. Plasma enzymes

Plasma ALT and SDH elevations were estimated as markers of liver injury over a time course (O-96 h) after Ccl, administration. Fig. 1 shows the ALT levels at various time points of the experiment after each dose. Regardless of the dose of CC&, maximum ALT elevation occurred at 36 h. In the groups receiving 0.1 and 1 ml CCl,/kg, although maximum enzyme elevations occurred at 36 h, enzyme levels declined to normal indicating recovery from injury. This decline in enzyme levels was consistent with 100% survival seen in these two groups. In groups receiving 2 ml CC&/kg, 40% (4/10) mortality was recorded. In the six rats that survived toxicity, ALT levels declined to normal at 72 h indicating full recovery. Increasing the dose to 3 ml/kg caused significant increase in enzyme elevation with 60% mortality (6/10) recorded between 18 and 48 h. Increasing the dose to 4 ml/kg caused an added increment in mortality (SOOh). Twenty percent mortality was evident

P.S. Rao et al. / Toxicology 118 (1997) 181-193 185

as early as 8 h with mortality steadily increasing to 30% (3/10) at 12 h, 60% (6/10) at 24 h, and 70% (7/10) at 36 h. Comparing the ALT values, a dose-dependent increase in injury was observed with all the doses. SDH activities exhibited a similar trend (data not shown).

3.3. Histopathology

Sections of liver stained by H&E were examined for necrotic cells, inflamed cells, and neutrophil infiltration. Fig. 2 depicts dose-related injury at 36 h (maximum injury at all doses) as observed by histopathology. Swollen cells and lipid laden cells were seen around the centrilobular zone as early as 12 h after administration of 0.1 ml CC&/kg. An increase in dose (1 ml CC&/ kg) resulted in increased inflammation and necrosis. The injury, however, was limited to the centrilobular zones. In rats receiving 2, 3, and 4 ml CC&/kg swollen, vacuolated &lls and neu- trophils were seen in the centrilobdlar region as early as 6 h. Pyknotic nuclei, inflamed cells, and

0 6 12 24 36 48 72 96

HOURS AFTER CC14ADMINISTRAT10N

Fig. 1. Male Sprague-Dawley rats (175-225 g) were divided

into four groups. At time zero, the respective groups received

intraperitoneal injections of 0.1, 1, 2, 3, and 4 ml CC&/kg in

corn oil (1 ml/kg). Controls received only the corn oil (1

ml/kg) vehicle. Plasma alanine aminotransferase (ALT) was

measured as a marker of liver injury over a time course (O-96

h) after each treatment. It should be noted that data for the 4

ml CC&/kg dose are represented by only surviving rats (30% at

36 h; 20% at 48 h and later time points). Results are expressed as mean +_ S.E.M. for four rats in each group. Numbers above error bars indicate significant differences from control (0 h),

0.1, 1, 2, and 3 ml CCl,/kg groups, respectively (PI 0.05).

Control ALT value: 54 units/l.

significant neutrophil infiltration were evident from 24 h onwards in all the dose groups. Al- though extensive necrosis was noted in the 2 ml/kg group this necrosis was limited to the centrilobular region. Extensive necrosis observed in groups receiving 3 and 4 ml CC&/kg was no longer limited to the centrilobular region but was widespread to the midzonal and periportal regions with minimal zonal differences. Maximum necrosis was observed between 24 and 48 h which was concordant with the plasma enzyme elevations (Fig. 1).

3.4. In vivo incorporation of [3H]thymidine into rat hepatonuclear DNA

3H-T incorporation into hepatonuclear DNA over a time course (O-96 h) following administration of each dose of Ccl, was measured as a marker of S-phase synthesis. The results are illustrated in Fig. 3. Peak S-phase synthesis after 0.1 ml CC&/kg occurred at 36 h. Administration of 1 ml CCl,/kg resulted in an increase in peak S- phase synthesis, but it occurred 12 h later at 48 h. A 20-fold increase to 2 ml CC&/kg resulted in a further delay in peak S-phase synthesis to 72 h. Increase in dose to 3 ml CC&/kg did not yield any additional increase in 3H-T incorporation but peak S-phase synthesis occurred at 48 h and was sustained up to 72 h. Thus, a dose-dependent temporal delay was observed until a threshold (2 ml/kg) was reached. Increasing the dose beyond the threshold resulted in diminished S-phase synthesis. Administration of 4 ml/kg Ccl, resulted in 80% mortality by 48 h. All rats surviving by 48 h survived thereafter. Examination of 3H-T incorporation in the surviving rats revealed an approx- imately 5-fold increase at 48 h compared to 3 ml CCl,/kg. This may explain the 20% animal survival observed consistently in this dose group.

3.5. PCNA assay

The PCNA immunohistochemical staining procedure was used to confirm the 3H-T incorporation data as well as to quantify cell cycle progression (Figs. 4 and 5). Normally, most cells are in the resting phase (G,; Fig. 4) and a rela-

186 P.S. Rao et al. / Toxicology 118 (1997) 181-193

Fig. 2. Treatment details are as described under Fig. 1. Representative liver histopathology during maximum injury (36 h) after Ccl,

treatment. Top panel represents photomicrographs of liver sections from the rats receiving 0 (A), 0.1 (B) and 2 ml CCl,/kg (C) at

36 h after TA treatment. Bottom panel represents photomicrographs of liver sections from rats receiving 2 (A), 3 (B) and 4 ml

CCl,/kg (C) at 36 h after treatment. c, central vein; p, pyknotic nuclei; v, vacuolization; n, areas of necrosis; f, fibrotic tissues; m, mitosis. Magnification, 100 x

tively small proportion of cells are in the other and 36 h. At 36 h maximum number of cells were phases of cell cycle with about 3-4% in the G, in the S-phase of cell cycle (Fig. 4) which was in phase (Fig. 4). After administration of 0.1 ml agreement with peak S-phase synthesis as evi- CCl,/kg, progression of cell cycle resulted in a dented by peak 3H-T incorporation at 36 h. At 48 large number of cells in the G, phase between 24 h most cells had progressed to G, and M phase


and by 96 h the liver returned to the normal

quiescent state. Administration of 1 ml/kg re-

sulted in a rapid progression of cells through G,

to S-phase at 36-48 h. By 48 h most cells were in

the G, and M phases, the liver returning to the normal quiescent state by 96 h. In the 2 ml/kg group, the maximum number of cells were de-

tected in S-phase at 72 h, and these cells pro-

gressed through the M phase. Increasing the dose to 3 ml/kg resulted in cells progressing to the G,

phase between 36 and 48 h. A maximum number

of S-phase cells were detected at 48 h. Progression

of cells to the M phase was seen as early as 24 h with maximum at 48 h. The high dose of 3 ml

CC&/kg resulted in a decrease in the total number of cells cycling through the different phases of the

cell cycle compared to the three lower doses (0.1, 1, and 2 ml/kg). Findings of the PCNA studies

were in agreement with 3H-T incorporation studies for all dose groups.

It should be recalled that because of 70% mortality in the 4 ml CC&/kg group, the findings at 36 h represent only 30% of the survivors. At 48 h the

findings represent only 20% of survivors (80%

mortality was noted at this time point). After administration of 4 ml CC&/kg a large number of

cells progressed through the G, phase between 36

z 14

E CClq(WW

12 I 0.1

0 6 12 24 36 48 72 06

HOURS AFTER CC14ADMINISTRATION

Fig. 3. Treatment details are as described under Fig. 1.

[3H]Thymidine (3H-T) incorporation into hepatonuclear DNA

after CC& treatment. 3H-T was administered 2 h prior to sacrifice at each time point. Results are expressed as mean f

S.E.M. for four rats in each group. Numbers above the error bars indicate significant differences from control (0 h), 0.1, 1, 2, and 3 ml CC&/kg treated groups, respectively (P 5 0.05). Control value: 60 cpm/pg DNA x 10s.

and 48 h. A maximum number of cells were in the S-phase of the cell cycle between 36 and 48 h. The number of cells detected in the S-phase of the cell cycle were more than 20°/ higher than the number of cells detected at lower doses. A higher number of cells was also detected in the G, phase at 36 h. The maximum population of M-phase cells were detected at 36 h. These rats which survive this dose of Ccl, are able to do so as a result of a much higher capacity to stimulate higher cell division, and at an early time point. Prompt and maximal cell division seems to enable these survivors to fend off massive cell injury.

3.6. Colchicine antimitosis

The important role of cell proliferation in the outcome of Ccl, was further examined by inhibiting cell division by CLC antimitosis. Administra- tion of CLC (1 mg/kg) 24 h before maximum S-phase synthesis resulted in 40% lethality in rats receiving 1 ml CC&/kg, a dose that did not cause lethality in the absence of CLC treatment. CLC administration caused a significant inhibition of S-phase synthesis otherwise evident between 36 and 48 h for the group injected with 1 ml/kg Ccl, alone (Fig. 6). Suppression of mitosis and tissue repair were associated with a marked progression of injury as evidenced by plasma enzyme elevations (Fig. 7) consistent with increased animal mortality (40%).

4. Discussion

Compensatory cell proliferation and tissue regeneration occurs as an endogenous response following either partial hepatectomy (Steer, 1995; Pistoi and Morello, 1996) or chemical damage to the liver (Columbano et al., 1990; Chanda and Mehendale, 1996) and enable animals to overcome injury and survive (Lockard et al., 1983; Mehendale, 1991; Kodavanti et al., 1992; Rao and Mehendale, 1993; Mehendale et al., 1994a,b). Low doses of Ccl, and thioacetamide are known to stimulate cell division and tissue regeneration (Leevy et al., 1959; Nakata et al., 1985; Mehen- dale et al., 1994b; Mangipudy et al., 1995a,b).

188

Fig. 4. Treatment details are as described under Fig. 1. Graphical representation of cell cycle progression as measured by

P.S. Rao et al. i Toxicology 118 (1997) 181- 193

0 6 12 24 36 48 72 QB

0 6 12 24 36 48 72 86

60

0 6 12 24 36 48 72 es

0 6 12 24 36 48 72 96

TIME fHRS) AFTER CC& ADMlNlSTRATfON

proliferating cell nuclear antigen (PCNA) immunohistochemical procedure. Percentage of cells in each cell cycle phase was calculated

from a total of 1000 viewed cells in the liver for each animal. Each time point had four rats per group. Rats received a single dose

of 0.1, 1, 2, 3, or 4 ml CCl,/kg, i.p. Percent cells in different phases of cell cycle were then counted in liver sections obtained from

rats at O-96 h after Ccl, administration. *, indicates significant difference from control (P I 0.05). # , indicates significant from 0.1,

1, and 2 ml/kg; $, indicates significant difference from 0.1, 1, 3. and 4 ml/kg; + , indicates significant difference from 2 and 3 ml/kg.

Controls received only the vehicle (corn oil, 1 ml/kg, i.p.).

P.S Rao et al. / Toxicology 118 (1997) 181-193

CCl,,2ml/kg,48h”

189

CCId,O. 1 ml/kg,48h CC&, 1 ml/kg,48h

CCl,,3ml/kg,48h Xl,,4ml/kg,48

Fig. 5. Treatment details are as described under Fig. 1. Results of the proliferating cell nuclear antigen (PCNA) study following i.p.

treatment with Ccl,. Representative photomicrographs of liver sections from rats at 0 h (A; control), 36 h (B; 0.1 ml/kg), 48 h (C;

1 ml/kg), 72 h (D; 2 ml/kg), 48 h (E; 3 ml/kg) and 48 h (F; 4 ml/kg) after Ccl, treatment. Details of treatment are described in the text. G,: Cells with blue nuclear staining. G,: cells with light brown nuclear staining. S: cells with deep brown nuclear staining. G,:

cells with or without speckled nuclear staining and with diffused cytoplasmic staining. M: cells with diffused cytoplasmic staining

and with deep blue chromosomal staining.

Studies also indicate that the G, population of cells in the liver undergo mitosis after treatment with a low dose of hepatotoxicants (Panduro et al., 1986; Smuckler et al., 1976; Calabrese et al., 1993). New concepts emerging from these studies are that: (a) sustained and stimulated cell division

plays a critical role in the final outcome of toxic injury; (b) the biological compensatory response of tissue repair occurs in parallel with injury; (c) the net outcome of toxic injury is a result of the dynamic interaction between the two opposing forces of injury and tissue repair. Thus, it is

190 P.S. Rao et al. / Toxicology 118 (1997) 181-19.7

24 36 46 72 96

HOURS AFTER CCl4ADMINISTRATION

Fig. 6. Treatment details are as described under Methods.

[3H]Thymidine (3H-T) incorporation into hepatonuclear DNA

after CLC treatment. CLC was administered 24 h after Ccl, (1

ml/kg) treatment and 3H-T was administered 2 h prior to

sacrifice at each time point. Results are expressed as mean +

S.E.M. for four rats in each group. *, indicate significant

differences from the Ccl, + saline group.

important to consider both of these responses in

the development of dose-response relationships.

Therefore, the objective of this study was to de-

velop a dose-response for CC& measuring tissue

injury and repair as parallel but opposing biologi-

cal responses to the toxic effects of a 40-fold dose

range (0.1, 1, 2, 3, and 4 ml/kg) of Ccl, over a

time course (O-96 h). Liver injury measured by histopathology and

2000 l

1 IhI

CC14+*dine

1600 m ccl4+cLc

24 36 46 72 96

HOURS AFTER Ccl4 ADMINISTRATION

Fig. 7. Treatment details are as described under methods.

Plasma alanine aminotransferase (ALT) after CLC treatment.

CLC was administered 24 h after Ccl, (1 ml/kg) treatment and jH-T was administered 2 h prior to sacrifice at each time

point. Results are expressed as mean + S.E.M. for four rats in

each group. *, indicate significant differences from the CCI, +

saline group.

plasma enzymes elevation revealed that maximum injury occurred at 36 h, irrespective of the dose. However, the development of injury and progression or regression of that injury was dose-dependent. With 0.1 and 1 ml/kg injury was evident as early as 6 h with maximum injury at 36 h for the 1 ml CCl,/kg dose. However, with both these low doses injury was limited to the centrilobular region. At higher doses, substantial necrosis and inflammation could be detected as early as 12 h. Not only did higher injury occur at an earlier time point with higher doses (2, 3, and 4 ml/kg) but the extent of injury was also greatly magnified as evidenced by necrotic damage to the midzonal and periportal areas in addition to the centrilobular region. Above the 2 ml/kg dose the regiospe- cificity of Ccl, injury was obliterated. With 3 ml CC&/kg there was diffused necrosis throughout the lobular structure. Examination of the liver sections exposed to 4 ml/kg revealed extensive necrosis and inflammation from 12 to 36 h. Be- cause 70% of the rats receiving this dose die by 36 h, observations made at and after 36 h apply only to the surviving rats. This is also true of later time points for this group when only 20% of survivors were available for these studies.

Tissue repair was measured by assessing S- phase synthesis and PCNA. Following administration of 0.1 ml/kg, peak S-phase synthesis was observed at 36 h indicating maximum DNA synthesis at this time point. With increase in dose to 1 ml/kg, a parallel increase in the magnitude of S-phase synthesis was observed. However, peak S-phase synthesis was delayed to 48 h. With a further increase in dose to 2 ml/kg, although an increase in S-phase synthesis was observed, this response was delayed further to 72 h. An increase in dose to 3 ml/kg resulted in 60% mortality with no additional increment in S-phase synthesis. Thus, a dose-dependent increase in the tissue repair response was observed until a threshold (2 ml CCl,/kg) was reached. Because 60% of the rats receiving 3 ml CCl,/kg fail to survive at 48 h, one may only assume that S-phase stimulation in these rats was diminished and delayed. PCNA studies revealed that with each increment in dose there was an increase in the number of cells in each phase of the cell cycle indicative of increase in


proliferative activity until the threshold was reached. Beyond the threshold, although proliferation was still evident, the number of cells in each phase of the cell cycle was diminished. Similar results were reported in another study in which thioacetamide was employed as the model hepato- toxicant (Mangipudy et al., 1995a). In both these studies unrestrained progression of liver injury and consequent mortality were evident only after the failure to elicit timely and adequate tissue repair response.

The survivors of the 4 ml/kg group aptly demonstrate that prompt and exacting tissue repair occurring in a timely fashion decisively influ- ences the final outcome of liver injury. Administration of 4 ml CC&/kg consistently resulted in 80% mortality. Mortality, recorded as early as 8 h (20%) steadily increased with time. Examination of 3H-T incorporation in the 20% rats surviving at 48 h revealed a massive increase (5-fold) in S-phase synthesis at this time point which was sustained until 72 h. These findings were corroborated by PCNA studies which revealed a commensurate increase in the number of cells progressing through cell division cycle compared to the lower doses. This timely and highly stimulated tissue repair appears to restrain the progression of liver injury by replacing the dead cells by newly divided, resilient cells resulting in restoration of the hepatic lobular structure. Newly divided cells are resilient to toxic injury (Farber et al., 1976; Chang et al., 1985; Ruth et al., 1986; Digavalli and Mehendale, 1995) and it is this resiliency which allows the new cells to restore liver function and restrain the progression of injury. Although only a few (20%) rats survive this highly toxic dose, this number of rats consistently survive raising the issue of genetic variation and the influence of genetic selection in the outcome of hepatic injury. Studies examining species (Cai and Mehendale, 1990, 1991) and strain (Kulkarni et al., 1996) differences in susceptibility to toxic chemicals demonstrate possible genetic differences in tissue repair responses. Marked inter-individual differences in human sensitivity to toxic chemicals may also be related to the rates of this compensatory tissue repair response.

S-phase synthesis, measured as an index of tissue repair, was significantly diminished after CLC administration (Fig. 6), and hepatic injury (ALT elevation and necrosis) was significantly increased indicating that in the absence of prompt stimulation of cell division liver injury inflicted by Ccl, progresses in an unrestrained manner. Thus, interference with tissue repair resulted in the con- version of a sublethal dose (1 ml/kg) to a lethal dose. In another study by Mangipudy et al. (1996) interference with cell proliferation via CLC (1 mg/kg) antimitosis resulted in marked progression of liver injury leading to 100% mortality after administration of otherwise non-lethal, moderately toxic doses of thioacetamide. Our findings are in agreement with earlier findings of Calabrese et al. (1994) where interference with cell division by CLC administration led to an increase in hepatotoxicity and lethality after administration of a subtoxic dose of Ccl,.

This study establishes that acute exposure to increased doses of Ccl, stimulates cell proliferation as a compensatory mechanism in a dose-dependent manner until a threshold is reached. This is essentially a mechanism that permits survival at maximally tolerated doses (MTDs) employed in long-term chronic and cancer bioassays. Under these conditions, repeated exposure to toxicants has two important implications. First, higher cell proliferation lends to greater errors in DNA repli- cation. Faust0 and coworkers have shown increased cell proliferation and high cell turnover to cause hepatocyte dysplasia, abnormal morphol- ogy, and increased incidence of hepatic tumors in transgenic mice expressing constitutive overex- pression of TGFa: (Faust0 et al., 1995). Second, repeated exposure may alter the regulatory mechanisms of cell proliferation. For example, chronic ethanol feeding was found to desensitize the hepatocytes to the trophic effects of hormones and disrupt G-protein mediated signalling to impair wound healing in ethanol-induced liver injury in rats (Diehl et al., 1992; Ackerman et al., 1993). Recent studies indicate that repeated exposure to low levels of thioacetamide (50 mg/kg) diminishes tissue repair resulting in sustained mild injury (Mangipudy et al., 1995~).

192 P.S. Rao et al. / Toxicology 118 (1997) 181-193

Based on these observations the present study helps us to understand mechanisms of stimulated cell proliferation as a compensatory response to liver injury in a dose-response paradigm. It also provides a scientific rationale for measuring tissue repair as a parallel but opposing biological response in addition to measuring injury in affected tissues and organs. Further, measuring these two responses will aid in fine-tuning risk assessment while allowing evaluation of the use of MTDs and better interpretation of the outcomes of cancer bioassays.

Acknowledgements

This study was partly supported by a starter grant from The Burroughs Wellcome Fund.

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