Introduction

Thioacetamide (TA), originally used as a fungicide, is a potent hepatotoxicant that has been much studied since the first report of its toxic properties (1-4). Earlier literature (5-8) suggests that an obligate intermediate metabolite of TA binds to proteins with the formation of acetylimidolysine derivatives (7) responsible for TA-induced hepatotoxic effects. Accordingly, it was reported that the hepatotoxic effects of TA are manifested only after metabolic conversion to thioacetamide-S-oxide, which undergoes further oxidative metabolism to an as yet unidentified toxic metabolite (7). TA has been reported to stimulate DNA synthesis and mitosis in the liver of rats at doses that produce limited necrosis (9-11). The rise in DNA synthesis induced by TA has been shown to follow a time course that is similar to that seen following partial hepatectomy (12). Rats treated with a single dose of 50 mg/kg TA show non-neoplastic hepatocellular proliferation indicated by ³H-thymidine (³H-T) incorporation and cell cycle progression to mitosis after administration (9,12-14).

Hepatocellular regeneration and tissue repair mechanisms have been implicated in the ultimate outcome of toxicity, after injury from a variety of structurally and mechanistically dissimilar chemicals including carbon tetrachloride (15-18), acetaminophen (14), and thioacetamide (19).

These studies have thus far illustrated two important biological events. They are the role of tissue repair in the ultimate outcome of toxicity and a dynamic coexistence of the processes of tissue repair and tissue injury as parallel but opposite biological responses and the collective influence of their interaction on the ultimate outcome of toxicity.

Intervention with the repair processes has been reported to result in unrestrained progression of hepatic injury leading to hepatic failure and animal death (20,21). Colchicine (CLC) is a widely used antimitotic agent (20-23). A single intraperitoneal (ip) administration of CLC (1 mg/kg) results in antimitosis against TA (11,13) with minimal side effects because of very rapid elimination of the compound (23-25). The objective of this study was to investigate the effect of colchicine antimitosis in a dose-response paradigm. We hypothesized that if timely and adequate tissue repair was indeed the critical underlying mechanism for the nonlethality observed with the lower six-fold dose-range of TA (19), then intervention with the repair response should convert these nonlethal doses to lethal doses. For this study we chose two doses of TA (150 and 300 mg/kg) from the 12-fold dose-range reported earlier (19). These two doses were chosen because of the accompanying high rates of tissue repair (19). After conducting lethality studies in the presence and absence of colchicine, we carried out further time course (0-96 hr) studies with the 150 mg/kg TA to quantitate tissue repair and injury. Colchicine antimitotic intervention resulted in the abolishment of the tissue repair response that led to 100% lethality from an ordinarily nonlethal dose of 150 mg/kg TA. These findings establish that the key difference between moderately toxic nonlethal doses and a lethal dose of TA is the presence or absence, respectively, of adequate and timely tissue repair response.

Materials and Methods

Male Sprague-Dawley rats (175-225 g, 7-8 weeks of age) were obtained from Harlan Sprague-Dawley, Inc. (Indianapolis, IN) and were maintained over woodchip bedding free of any chemical contaminants for 10 days on a 12-hr photoperiod in our central animal facility. A 21 1°C temperature and 50% relative humidity were maintained at all times. The animals had free access to water and normal commercial rat chow (Teklad, Madison, WI, diet #7001) ad libitum.

Thioacetamide (TA) and [³H-methyl] thymidine (³H-T, specific activity 1.7 Ci/mmol) were obtained from Sigma Chemical Co. (St. Louis, MO). The scintillation fluid (Scintiverse SX 16-4) was purchased from Fisher Scientific (Baton Rouge, LA). All other biochemicals and chemicals were obtained from Sigma Chemical Co.

After acclimation, rats were divided into four groups (Table 1). Groups I and II received 150 mg/kg TA ip at 0 hr. After 30 and 42 hr, Group I received control vehicle (1 ml/kg) while group II received CLC (1 mg/kg). Groups III and IV received 300 mg/kg TA at 0 hr. After 42 hr, Group III received control vehicle (1 ml/kg) while group IV received CLC (1 mg/kg). Rats of all groups were given normal diet and water ad libitum during and after treatment until termination.

One experiment was designed to determine the lethality of TA both in the presence and absence of CLC treatment in rats following the administration of each dose of TA (150, 300 mg/kg). CLC (1 mg/kg in normal saline) was administered at 30 and 42 hr (6 hr prior to peak S-phase activities at 36 and 48 hr) following the administration of 150 mg/kg TA, while CLC (1 mg/kg in normal saline) was administered 42 hr (6 hr prior to peak stimulation of cell division) following treatment with 300 mg/kg TA. The respective controls received normal saline (1 ml/kg) at the respective times. The rats were observed twice daily for 14 days and survival/lethality was recorded in each group.

Blood was collected from the dorsal aorta of rats under diethyl ether anesthesia at 0, 12, 24, 36, 48, 72, and 96 hr after TA or vehicle administration; the serum was separated for the estimation of serum enzymes alanine aminotransferase (ALT, EC 2.6. 1.2.) and sorbitol dehydrogenase (SDH, EC 1.1.1.14.) as markers of liver injury using kit # 59 UV (ALT) and kit # 50 UV (SDH), respectively, from Sigma Chemical Co.

Portions of liver from each group collected at various periods after TA treatment were washed with normal saline (0.9% NaCl), cut into small slices, and fixed in phosphate buffered 10% formaldehyde solution for 48 hr. The tissues were then transferred to 70% ethyl alcohol until they were processed. These slices were then embedded in paraffin after processing. The liver sections (5 m thick) were stained with hematoxylin-eosin (H&E) for histological examination under the light microscope.

³H-T incorporation into hepatonuclear DNA was measured using the procedure of Chang and Looney (26) as modified by Chauveau et al. (27). The DNA content of the supernatant fraction was estimated by the diphenylamine reaction (28).

The proliferating cell nuclear antigen (PCNA) study was done as described by Greenwell et al. (29) and as reported earlier (14,19).

Mean SEM were calculated for all values. Statistical differences were determined by one-way analysis of variance followed by Duncan's multiple range test to determine which means were significantly different from each other or from controls. In all cases, a p 0.05 was used as the statistical criterion to determine significant differences.

Results

All groups were observed twice a day for survival/lethality for a period of 14 days. A 100% survival was observed in the groups that did not receive CLC (groups I and III) while a 100% mortality was noted in the groups that received TA followed by CLC (groups II and IV). All the deaths occurred between 4 and 6 days.

Serum ALT and SDH were estimated as markers of liver injury over a time course of 0-96 hr after treatment with 150 mg TA/kg. Figures 1 and 2 show the ALT and SDH activities at various times after the administration of TA in the presence and the absence of CLC treatment. In the group receiving 150 mg/kg TA alone, ALT activity increased up to 48 hr and then declined to normal, indicating an initial infliction of injury followed by recovery; this is consistent with 100% animal survival. Colchicine intervention significantly altered the course of these biochemical events. Marked progression of injury, as indicated by significantly higher elevation of serum ALT, was evident from 48 hr onward. This progression of injury was associated with 100% animal mortality in this group. Sorbitol dehydrogenase elevation measured in a similar fashion exhibited the same trend (Fig. 2).

Figure 1. Serum alanine aminotransferase (ALT) was measured as a marker of liver injury over a time course (0-96 hr) following each treatment. Male Sprague-Dawley rats (175-225 g) were divided into two groups. At time zero both groups received 150/kg TA in normal saline (1 ml/kg, ip). Controls received normal saline only (1 ml/kg, ip). One group received CLC (1 mg/kg, ip) at 30 hr and 42 hr following administration of 150 mg/kg TA. The other group received normal saline (1 ml/kg) at the same time points. Results are expressed as mean SE for four rats in each group at each time point. Numbers above the error bars indicate significant differences from control (0 hr) and 150 mg/kg TA treated groups, respectively (p0.05). Control ALT value:54 U/l.

Figure 2. Serum sorbitol dehydrogenase (SDH) was measured as a marker of liver injury over a time course (0-96 hr) following each treatment (treatment protocol as in Fig. 1). Results are expressed as mean SE for four rats in each group at each time point. Numbers above the error bars indicate significant differences from control (0 hr) and 150 mg/kg TA treated groups, respectively (p0.05). Control SDH value:78 U/ml.

The liver sections stained by H&E were examined for necrotic cells, extent of inflammation, and neutrophil infiltration. Swollen cells and a few pyknotic nuclei started appearing around the centrilobular region between 6 and 12 hr in the rats treated with 150 mg/kg TA. Pericentral hepatocellular necrosis was clearly evident at 12 hr and was progressive between 12 and 24 hr. Periportal cells remained unaffected. Moderate centrilobular necrosis observed in the 150 mg/kg TA treated group progressively declined between 24 and 48 hr. Centrilobular damage accompanied by extensive neutrophil infiltration was evident between 36 and 48 hr. In sharp contrast, CLC treatment resulted in marked centrilobular necrosis at 36 hr. This was associated with extensive neutrophil infiltration and vacoulation. Between 48 and 72 hr, progression of injury was accelerated as evidenced by widespread necrosis, crumbling centrilobular columns, and damaged portal tracts. By 96 hr, fulminant liver injury with no zonal specificity was observed, indicating complete destruction of the liver architecture.

Figure 3. Representative liver histopathology during a time course after thioacetamide treatment. Top panel represents photomicrographs of liver sections from rats receiving 150 mg/kg TA taken at 36 hr, 48 hr, and 72 hr after treatment. Bottom panel represents photomicrographs of liver sections from rats receiving 150 mg/kg TA+CLC at 36 hr, 48 hr, and 72 hr. c, central vein; p, pyknotic nuclei; v, vacuolization; n, areas of necrosis; f, fibrosis; m, mitosis.

³H-T incorporation into hepatonuclear DNA over a time course (0-96 hr) following the administration of 150 mg/kg TA alone and over 36-96 hr following treatment with TA + CLC, measured as a marker of S-phase stimulation, is represented in Figure 3. Peak ³H-T incorporation after the administration of 150 mg/kg TA occurred between 36 and 48 hr. Administration of CLC (1 mg/kg, ip) resulted in a significant reduction of S-phase stimulation (Fig. 4). This suppression sustained until 96 hr and ultimately resulted in 100% animal death. Examination of the H&E-stained sections revealed a significant mitotic activity in the rats treated with 150 mg/kg TA alone between 36 and 72 hr in sharp contrast to no mitotic activity in the rats receiving TA and CLC. Thus, timely stimulation of tissue repair was consistent with animal survival while suppression of repair activity led to a dramatic reversal of events leading to 100% animal lethality.

Figure 4. ³H-T into hepatonuclear DNA after TA treatment. ³H-T (35 Ci) was administered 2 hr prior to sacrifice at each time point. One group received CLC (1 mg/kg, ip) at 30 hr and 42 hr following administration of 150 mg/kg TA. The other group received normal saline (1 ml/kg) at the same time points. Results are expressed as mean SE for four rats at each time point. Number above error bars represents significant differences from control (0 hr) and 150 mg/kg TA treated groups, respectively (p0.05). Control ³H-T value = 150 dpm/g DNA.

The results of thymidine incorporation were further corroborated by the PCNA immunohistochemical procedure (Figs. 5, 6). Normally, most cells in the liver are in the resting phase (G₀; Fig. 5A), and a small proportion of cells are in other phases of the cell cycle. After administration of TA (150 mg/kg), cell cycle progression is stimulated; this results in a large number of cells in the G₁ phase by 24 hr and S-phase between 36 and 48 hr. The maximum number of S-phase cells seen by this method is in agreement with S-phase stimulation observed by ³H-T incorporation. Most of the cells progress to the G₂ phase between 36 and 48 hr. The maximum number of cells are seen in the M-phase at the 48 hr. By 96 hr the liver returned to normal quiescence as evidenced by the predominant number of G₀ cells. In sharp contrast, CLC intervention blocked the G₀-G₁ transition and thereafter arrested the progression of the cell cycle. S-phase synthesis and mitotic activity were ablated almost completely. Progression of the cell cycle and timely stimulation of tissue repair were consistent with animal survival, while ablation of the repair process by colchicine intervention led to unrestrained progression of injury and culminated in 100% animal mortality from an otherwise nonlethal and moderately toxic dose; this indicates that stimulated cell division and tissue repair are key events in the outcome of toxicity.

Figure 5. Results of the proliferating cell nuclear antigen study after intraperitoneal treatment with 150 mg/kg TA+vehicle and 150 mg/kg TA+CLC (1 mg/kg). Representative photomicrographs of liver sections from rats at 36 hr, 48 hr, and 72 hr after 150 mg/kg TA+vehicle treatment and 36 hr, 48 hr and 72 hr after 150 mg TA/kg+CLC treatment. G₀, cells with blue nuclear staining; G₁, cells with light-brown nuclear staining; S, cells with deep brown nuclear staining; G₂, cells with or without speckled nuclear staining and with diffused cytoplasmic staining; M, cells with diffuse cytoplasmic and deep blue chromosomal staining. Significant S-phase stimulation at 36 and 48 hr following 150 mg/kg TA is contrasted with inhibited S-phase stimulation with 150 mg/kg TA+CLC.

Discussion

Earlier studies have amply demonstrated that tissue repair is a biological response that accompanies chemical-induced injury (13-19,30). Other studies (11,13,14,22, 31-36) indicate that the G₂-subpopulation of cells in the liver undergo mitosis after treatment with low doses of toxicants such as thioacetamide, galactosamine, dichlorobenzene, CHCl₃ or CCl₄. Additionally, several investigators (37-41) have observed that newly divided cells are significantly more resistant to cytotoxic effects of a variety of toxic chemicals. Using TA as a model compound, we have also reported that the repair response can be measured in a dose-dependent and temporal manner in parallel with injury (19). The toxicodynamic interaction between tissue repair and injury in a dose-response paradigm and its effect on the ultimate outcome of toxicity has major implications. Measuring the parallel but opposing biological responses of repair and injury may provide new insights on the use of dose-response relationships in predictive toxicology and risk assessment.

The objective of this study was to subject the concept that stimulated tissue repair plays a critical role in the ultimate outcome of TA toxicity to further experimental scrutiny in a dose-response paradigm. We hypothesized that if stimulated cell division and tissue repair were indeed the mechanism of animal survival, then intervention with the repair mechanism should convert nonlethal doses into lethal doses. To test this hypothesis, we chose two doses (150 and 300 mg/kg) of TA, a model hepatotoxicant. These two doses cause moderate injury but are nonlethal by virtue of their ability to maximally stimulate hepatocellular division and tissue repair (19). CLC (1 mg/kg) was used as an antimitotic tool to block stimulated cell division and tissue repair. Earlier studies have demonstrated that CLC used in this dose does not disrupt hepatobiliary function (20,21).

A lethality study was designed (Table 1) to investigate the effect of colchicine treatment. It should be noted that 150 and 300 mg/kg TA are otherwise nonlethal in the absence of CLC treatment. While liver injury occurs, simultaneous dose-related stimulation of cell division and tissue repair allows the animals to overcome this injury and survive. However, a 100% lethality was observed in the presence of CLC treatment in both the groups. For further mechanistic inquiry, we chose the 150 mg/kg TA dose. Liver injury and tissue repair were the two parallel but opposing responses measured and were used as predictors of regression or progression of liver injury leading to animal recovery or death, respectively.

Serum enzyme (ALT, SDH) elevation and liver histopathology (Figs. 1, 2, 3) were used as indices of liver injury following the administration of 150 mg/kg TA both in the presence and absence of CLC intervention over a time course of 0-96 hr and 36-96 hr, respectively. In the absence of CLC treatment, a moderate elevation of serum enzyme was observed between 24 and 48 hr. Thereafter, the animals recovered from liver injury, as evidenced by the prompt decline of enzyme activity between 72 and 96 hr to control level (Figs. 1, 2). In contrast, CLC treatment resulted in a dramatic progression of injury as indicated by progressive elevation of serum enzyme activity between 48 and 96 hr. Under a light microscope, liver sections revealed identical injury in both groups at 36 hr (Fig. 3). Thereafter, injury progressed remarkably between 36 and 96 hr in the rats receiving CLC after 150 mg/kg TA, as evidenced by severe centrilobular necrosis, vacuolation, and congestion; injury regressed in the rats receiving 150 mg/kg TA alone. An interesting observation here is that the 36 hr injury was not significantly different between the CLC-treated and CLC-nontreated groups. This indicates that the inflictive phase of liver injury from TA was not affected by CLC treatment.

Figure 6. Graphical representation of cell cycle progression as measured by proliferating cell nuclear antigen immunohistochemical procedure. Percentage was calculated from a total of 1000 viewed cells in the centrilobular region of the liver for each animal. Each time point had four rats per group. Rats received a single dose of 150 mg/kg TA or 150 mg/kg TA+CLC, intraperitoneally. Percent cells in different phases of cell cycle were then counted during a time course of 0-96 hr. ^*, significant difference from control; Ý, significant difference between groups (p0.05). Control rats received vehicle only (normal saline, 1 ml/kg, intraperitoneally).

Tissue repair indicated by the S-phase DNA synthesis and PCNA assay (Figs. 4, 5, 6) explains the dichotomy between the outcomes of TA treatment with or without CLC. Following the administration of the 150 mg/kg TA alone, there is a peak and sustained ³H-T incorporation into hepatonuclear DNA between 36 and 48 hr (Fig. 4). These rapidly dividing cells form new resilient hepatic parenchymal cells that continuously replace the dead cells, thereby restoring hepatolobular architecture and function. Consequently, injury is not only restrained from progression, liver structure and function are restored which eliminates liver injury altogether between 48 and 72 hr. In contrast, CLC treatment results in an ablation of this compensatory mechanism as shown by blocked S-phase stimulation, as well as cell cycle progression. Failure in the timely appearance of an adequate number of new cells allows unrestrained progression of injury on the one hand and deteriorating hepatic structure and function on the other, leading to lethality from an otherwise nonlethal dose. It should be noted that, only after failure of cell division to occur, the progressive phase of injury flares in the group that receives CLC.

These findings have the potential to affect current risk assessment and predictive toxicology methods. A knowledge of the mechanisms of infliction of injury by toxic chemicals has given us the ability to predict with a degree of confidence that a particular chemical or physical agent will or will not inflict tissue injury under a given set of exposure circumstances. However, that the ultimate outcome of the injury is determined by the initiation of timely and adequate tissue repair calls for a clearer understanding of the underlying biology to attain greater precision in predictive toxicology.

These findings have two broad implications. First, an increased understanding of the biological events that follow infliction of injury (toxicodynamic phase) helps to bridge the link between mechanisms responsible for the injury (Stage I) and the ultimate outcome of that injury (Stage II) (42). Secondly, the finding that the repair response can be manipulated and that such manipulation leads to a complete reversal in the outcome of toxic injury opens up avenues for therapeutic applications in the realm of biomedicine.