Mechanisms of Increased Liver Tissue Repair and Survival in Diet-Restricted Rats Treated with Equitoxic Doses of Thioacetamide

Udayan M. Apte*, Pallavi B. Limaye*, D. Desaiah{dagger}, Thomas J. Bucci{ddagger}, Alan Warbritton{ddagger} and Harihara M. Mehendale*,1

* Department of Toxicology, School of Pharmacy, The University of Louisiana at Monroe, Monroe, Louisiana 71209; {dagger} Department of Neurology, University of Mississippi Medical Center, Jackson, Mississippi 39216; and {ddagger} Pathology Associates–A Charles River Company, National Center for Toxicological Research, Jefferson, Arkansas 72079

Received October 17, 2002; accepted December 2, 2002


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Moderate dietary or caloric restriction (DR) modulates animal physiology in a beneficial fashion. Previously, we have reported an equitoxic dose experiment where liver injury in DR male Sprague-Dawley rats exposed to a low dose of thioacetamide (TA, 50 mg/kg) was similar to that observed in ad libitum fed (AL) rats exposed to a 12-fold higher dose (600 mg/kg). Paradoxically, the AL rats experienced 90% mortality while all of the DR rats, with the same amount of initial bioactivation-mediated liver injury, survived. The protection observed in the DR rats was due to efficient compensatory liver tissue repair, which was delayed and attenuated in the AL rats, leading to progression of liver injury. The objective of the present study was to investigate the molecular mechanisms of the enhanced tissue repair in the DR rats upon equitoxic challenge with TA. Promitogenic mechanisms and mediators such as proinflammatory cytokines (TNF-{alpha} and IL-6), growth factors (TGF-{alpha} and HGF), and inducible nitric oxide synthase (iNOS) were estimated over a time course after equitoxic challenge (50 mg/kg to DR vs. 600 mg/kg to AL rats). Except for TNF-{alpha}, all other molecules were expressed earlier and in greater amount in the DR rats. IL-6 was 10-fold greater and peaked 12 h earlier; HGF also peaked 12 h sooner in the DR rats, when it was 2.5-fold greater than the value in the AL rats. TGF-{alpha} expression in livers of DR rats increased after TA administration and peaked at 24 h. In the AL rats, it was lower and peaked at 36 h. Diet restriction alone induced iNOS 2-fold in the DR rats and remained elevated until 12 h after TA administration, then declined thereafter. The lower iNOS activity in the AL rats further decreased after TA injection. DR rats exhibited higher apoptosis after thioacetamide administration, which further increased the efficiency of tissue repair. Taken together, these data indicate that even though the liver injury is near equal in AL and DR rats, sluggish signal transduction leads to delayed liver regeneration, progression of liver injury, and death in the AL rats. The equitoxic dose experiment indicates that stimulation of tissue repair is independent of the extent of initial liver injury and is governed by physiology of diet restriction. DR stimulates promitogenic signaling leading to a quick and timely response upon liver injury, arrest of progressive injury on one hand, and recovery from injury on the other, paving the way for survival of the DR rats.

Key Words: apoptosis; HGF; IL-6; iNOS; TGF-{alpha}; tissue repair; thioacetamide; caloric restriction, cytokines, growth factors, toxic challenge.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Caloric or diet restriction (DR) is the single most effective intervention to produce extensive health benefits. Multiple studies have shown that DR increases longevity by decreasing age-related diseases including cancer, and also protects from chemical-induced toxicity (Allaben et al., 1990Go; Apte et al., 2002Go; Berg et al., 1994Go; Duffy et al., 1995Go; Keenan et al., 1995Go; Frame et al., 1998Go; Hass et al., 1996Go; Masaro, 1988; Ramaiah et al., 2000Go; Weindruch and Walford, 1992Go). A number of studies have revealed protective effects of DR against a variety of toxicants such as isoproterenol, gancyclovir, and thioacetamide (Duffy et al., 1995Go; Hass et al., 1996Go; Ramaiah et al., 2000Go). Previously, we have reported that moderate DR (65% of ad libitum [AL] for 21 days) protects male Sprague-Dawley rats from lethal challenge of the model hepatotoxicant, thioacetamide (TA) (Ramaiah et al., 1998aGo,bGo). DR rats survive in spite of 2-fold higher liver injury than AL rats, due to timely and robust liver cell proliferation and tissue repair. We have recently demonstrated that this enhanced tissue repair is due to upregulation of promitogenic signaling via IL-6-mediated and MAPK pathways (Apte et al., 2002Go).

In a low-dose study (50 mg TA/kg), DR rats exhibited 6-fold higher initial bioactivation-based liver injury. In spite of such high liver damage, DR rats survived due to enhanced tissue repair response. The increased liver injury of TA in DR rats was attributed to the induction of CYP2E1, the primary enzyme involved in bioactivation of TA (Wang et al., 2000Go; Ramaiah et al., 2001Go). To investigate whether the higher tissue repair in DR rats was stimulated by the greater injury, an equitoxic dose study was conducted. In this study DR rats were treated with a low dose of TA (50 mg/kg) and AL rats were treated with a 12-fold higher dose of TA (600 mg/kg); the two treatments produced equal initial liver injury. Even though the initial liver injury was the same, AL rats experienced a progressive increase in liver injury leading to 90% lethality, while DR rats experienced complete survival due to prompt tissue repair in the DR rats, which in case of the AL rats was delayed and inhibited. These studies indicated that the mechanism of higher survival in DR rats exposed to TA is stimulated tissue repair, and this tissue repair is not dependent on the extent of initial liver injury.

The present study is based on the hypothesis that earlier (timely) and enhanced promitogenic signaling via proinflammatory cytokines and growth factors is the mechanism behind higher tissue repair in DR rats. Literature evidence points towards TNF-{alpha} and IL-6 as major candidates involved in priming the hepatocytes to enter G1 phase from G0. Growth factors such as TGF-{alpha} and HGF further stimulate these cells to undergo mitosis. Nitric oxide is also known to prime the hepatocytes as well as stimulate them to undergo mitosis (Apte et al., 2002Go; Dalu et al., 1995Go; Diehl, 2000; Fausto et al., 1995Go; Michalopoulos and DeFrances, 1997Go; Streetz et al., 2000Go; Yamada and Fausto, 1998Go). Expression of these mitogenic factors was studied over the time course in AL and DR rats after either a low dose (50 mg TA/kg) or an equitoxic dose (50 mg/kg in DR vs. 600 mg/kg in AL). Thioacetamide is known to induce apoptosis at lower doses (Mangipudy et al., 1998Go; Witzmann et al., 1996Go). To test for the possibility of increased apoptosis in DR rats after TA administration, and to evaluate the role played by increased apoptosis in final outcome of TA-induced liver injury, the number of apoptotic cells was evaluated using TUNEL assay. DR rats did have higher apoptosis after TA administration, which further increased the efficiency of tissue repair in these rats. Here we present evidence that these promitogenic factors are expressed at earlier time points and in higher quantities in DR rats upon TA exposure. The enhanced liver regeneration in DR rats is due to the prompt promitogenic signaling stimulated by these factors in DR rats after TA-induced liver injury. Tissue repair is inhibited in AL rats because of delayed and/or inhibited mitogenic signaling. We report here that even though the initial liver injury is equal, DR rats are able to survive through timely compensatory cell division stimulated by earlier and higher expression of cytokines, nitric oxide, and growth factors.

Our data indicate that the extent of initial bioactivation-based injury does not govern the extent of tissue repair. The signal transduction processes modulated by physiology of diet restriction, which can operate effectively even under substantial stress of tissue injury, can stimulate higher tissue repair in DR rats independent of the dose of TA.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
All the chemicals were obtained from Sigma Chemical Co. (St. Louis, MO) unless otherwise stated and were of the highest analytical grade. Glycine, 30% acrylamide:bis acrylamide solution, Tris, Tween-20, and other materials such as filter papers, nitrocellulose membranes, etc., related to Western blotting were obtained from BioRad (Hercules, CA). Anti-TGF-{alpha} antibody was purchased from Oncogene (Cambridge, MA), anti-HGF and anti-IL-6 antibodies were purchased from Santa Cruz Biotech (Santa Cruz, CA). 3H-T was purchased from Moravek Biochemicals (Brea, CA) and 3H-arginine was obtained from New England Nuclear (Boston, MA).

Animals and treatment.
Male Sprague-Dawley rats (250–290 g) were subjected to moderate DR as described previously (Ramaiah et al., 1998bGo). Briefly, DR rats were allowed to eat 65% of the AL food consumption (Harlan Teklad Rat chow No. 7001, Madison, WI: protein 25%, fat 4.25%, fiber 4.67%, vitamins and minerals supplemented, calories 3.94 Kcal/g) and free access to water for 21 days. AL rats had free access to food and water at all times. Diet restriction was carried out for 21 days, and on day 22 both AL and DR rats were exposed to various TA treatments. All animals received humane care according to the criteria outlined in the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals.

In the low-dose studies, rats in both groups received a single nonlethal dose of TA (50 mg/kg, ip, in 6 ml saline/kg). For equitoxic dose studies, DR rats and AL rats (n = 10 per group) received 50 and 600 mg/kg TA (ip, in 6 ml saline/kg), respectively. After exposure to the equitoxic doses rats were observed for 14 days, twice daily, and signs of toxicity and deaths were recorded. For the time course studies, separate groups of AL and DR rats were treated with the either a low dose (50 mg TA/kg) or equitoxic doses (50 or 600 mg TA/kg for DR and AL, respectively) and killed under diethyl ether anesthesia at various time points. Blood was collected from individual rats in separate heparinized tubes from the dorsal aorta, and plasma was obtained by centrifugation (1000 x g for 20 min). The median lobe of the liver was fixed in 10% neutral buffered formalin and further processed for histopathological analysis. The remainder was stored at –75°C till further analysis. Neither the plasma samples nor the liver samples were pooled, and individual samples were used for further analysis.

Estimation of liver injury and cell division.
Plasma was separated by centrifugation and used for the estimation of alanine aminotransferase activity (ALT; EC 2.6.1.2.) as a marker of liver injury, using Sigma kit No. 59 UV (ALT) (Sigma Chemical Co., St Louis, MO). Liver cell division was estimated by incorporation of 3H-thymidine (3H-T) in hepatonuclear DNA as previously described (Chang and Looney, 1965Go). Rats were treated with 50 µCi 3H-T/rat (ip) 2 h prior to sacrifice at each time point. Total DNA content was measured by diphenylamine reaction.

Assessment of apoptotic cells.
Apoptotic cells were visualized and counted by TUNEL assay using ApoTag kit (Oncor, Gaithersburg, MD) according to the manufacturer’s protocol. Thin (5 µm) sections of liver from AL and DR rats, sampled at various time points (0 to 72 h) after TA administration were used. Three slides (animals)/group/time point were examined microscopically, and apoptotic cells were identified by dark brown staining. Necrotic areas were avoided while counting the apoptotic cells.

TNF-{alpha} and IL-6 ELISA.
TNF-{alpha} levels were estimated in the plasma of AL and DR rats treated with TA using a rat-specific TNF-{alpha} sandwich ELISA (Amersham Life Sciences, Piscataway, NJ) according to manufacturer’s protocol. Briefly, heparinized plasma samples (n = 4 per time point) were incubated with anti-TNF-{alpha} antibody for 1 h followed by biotinylated detection antibody. Visualization was carried out by peroxidase reaction and color intensity was measured at 450 nm in BioRad Model 550 microplate reader (BioRad, Hercules, CA). IL-6 was measured in plasma samples collected at different time points using a rat-specific IL-6 ELISA kit (R&D Systems) according to manufacturer’s protocol and similar to TNF-{alpha} ELISA.

Immunohistochemistry.
TGF-{alpha}, HGF, and IL-6 protein were evaluated immunohistochemically using paraffinized sections of liver collected over the time course. Antigen retrieval was carried out by flooding the liver sections with 0.05% saponin for 30 min, followed by treatment with TGF-{alpha} specific antibody (Ab-1, Oncogene, Cambridge, MA) at 1:500 concentration for 18 h at 4°C. For HGF, antigen retrieval was performed by citrate buffer treatment while for IL-6 no antigen retrieval was necessary. The concentrations of primary antibody were 1:200 and 1:50 for HGF and IL-6, respectively. After treatment with secondary antibody (concentration 1:10,000) and streptavidin conjugate, visualization was achieved by peroxidase reaction in all cases. Three slides were stained per time point. To get a quantitative estimate of the growth factor production from the staining intensity, a scoring scheme was devised using a scale of 0–4 staining intensity, which has been used by other investigators to get a quantitative estimate of immunohistochemical analysis (Roberts et al., 1991Go; Williams et al., 1996Go). Slides were observed under a light microscope and staining intensity was graded on coded sections (blinded), at a scale of 0 to 4 (0, none; 1, low; 2, medium; 3, high; and 4, very high staining intensity).

iNOS assay.
iNOS activity was determined according to the method of Bredt et al.(1992)Go. The iNOS assay medium (400 ml) contained 100 mM HEPES, pH 7.2; 2 mM NADPH; [3H]-arginine (0.2 µCi/ml), and 400 mg of liver cytosolic protein. The reaction mixture was incubated for 30 min at 37°C and stopped by addition of stop-buffer containing 20 mM HEPES and 10 mM EGTA at pH 5.5. The entire reaction mixture was passed through Dowex AG 50 Wx-8 resin (Na+ form) to elute the fraction containing [3H]-citrulline, which was estimated in a Beckman 6000 liquid scintillation spectrometer.

Statistical analysis.
Group comparisons were performed using an independent t-test. A one-way analysis of variance was used to determine statistical significance that might exist between more than two distributions or sample groups. Statistical analyses were performed using SPSS 10.0 software (SPSS Inc., Chicago, IL). Statistical significance was set at p <= 0.05.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Liver injury and tissue repair after TA administration.
Liver injury was assessed by plasma ALT levels over a time course after TA administration in AL and DR rats (Fig. 1AGo). The low dose (50 mg TA/kg) produced minimal liver injury in AL rats, with the plasma ALT peaking at 36 h. DR rats exhibited massive liver injury with 6-fold higher peak plasma ALT levels in DR rats at 24 h. Injury decreased in the DR rats after 24 h, and plasma ALT levels returned to control levels by 60 h. Liver cell division was measured by pulse labeling experiments with 3H-T incorporation into hepatonuclear DNA (Fig. 1BGo). S-phase stimulation in AL rats peaked at 36 h after TA administration. In DR rats, significant increase was observed at 36 h, which further increased and peaked at 48 h. Although the 3H-T incorporation decreased after 48 h, significantly higher incorporation was observed even at 96 h after TA administration in DR rats, indicating sustained cell division.



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FIG. 1. Effect of diet restriction on hepatotoxicity of thioacetamide. (A) Plasma alanine aminotransferase (ALT) activity and (B) 3H-thymidine incorporation were measured over a time course of 0 to 120 h as markers of liver injury and tissue repair, respectively, in ad libitum and diet-restricted rats treated with thioacetamide (50 mg/kg). Results are expressed as mean ± SE (n = 4); *values significantly different from AL group at corresponding time point; !, values significantly different from DR group at 0 h; #, values significantly different from AL group at 0 h (adapted from Ramaiah et al., 1998aGo).

 
Figure 2Go represents the area under the curve (AUC) of liver injury as measured by plasma ALT (Fig. 2AGo), and tissue repair measured by 3H-T (Fig. 2BGo), after the equitoxic dose (50 mg TA/kg in DR and 600 mg TA/kg in AL). The results indicate that, after the equitoxic doses, AL and DR rats had similar liver injury until 48 h. After 48 h, liver injury in AL rats progressed further, culminating in 90% mortality, while in DR rats it significantly decreased, leading to 100% survival. The deaths in the AL group occurred after 60 h. The liver injury (plasma ALT) and tissue repair (3H-T) data, obtained at time points after 60 h, are from the surviving AL rats. This seemingly paradoxical outcome results from timely and robust tissue repair in DR rats as shown in Figure 2BGo. The AUC of 3H-T indicates that, despite equal initial liver injury, compensatory liver cell proliferation in the AL rats was inhibited until 48 h. An apparent increase in tissue repair in AL rats was observed during 60 to 120 h, but these data were obtained from very few surviving rats that had obviously survived due to stimulated repair. In DR rats, significantly higher 3H-T incorporation was evident during the first 48 h after TA administration, which further continued until 120 h. This early and sustained repair leads to survival of the DR rats.



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FIG. 2. Area under the curve (AUC) of (A) liver injury and (B) 3H-T incorporation between 0 and 48 h and 60 and 120 h after equitoxic dose of TA (600 mg TA/kg to AL vs. 50 mg TA/kg to DR produced equal initial liver injury) represented as bar diagram; *values significantly different from AL group at the corresponding time point; !, values significantly different from DR group at 0 h; #, values significantly different from AL group at 0 h (adapted from Ramaiah et al., 1998aGo).

 
TNF-{alpha} and IL-6 ELISA.
Evaluation of plasma TNF-{alpha} by ELISA after low-dose exposure indicated no significant change in AL rats (Fig. 3AGo). DR rats exhibited significantly higher plasma TNF-{alpha} at 24 h after TA treatment, which was sustained until 96 h. After the equitoxic dose exposure in AL rats, plasma TNF-{alpha} increased approximately parallel with the increase in liver injury (Fig. 3BGo). TNF-{alpha} was significantly higher in AL rats at 12 h and remained high until 72 h. No significant increase in plasma TNF-{alpha} was observed in DR rats except at 36 h after TA exposure.



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FIG. 3. Plasma TNF-{alpha} levels were measured over the time course using a rat-specific ELISA after (A) low dose of TA (50 mg/kg) and (B) equitoxic doses of TA (600 mg TA/kg to AL vs. 50 mg TA/kg to DR produced equal initial liver injury) to AL and DR rats. Values are expressed as mean ± SE (n = 4); *values significantly different from AL group at the corresponding time point; !, values significantly different from DR group at 0 h; #, values significantly different from AL group at 0 h.

 
Plasma IL-6 increased significantly in AL rats at 12 h but returned to control levels at 36 h after low dose (Fig. 4AGo). DR rats exhibited massive increase in plasma IL-6 at 12 h after TA exposure where the levels were 2.5-fold higher than AL rats at the same time point (Fig. 4BGo). This increase continued until 24 h, after which IL-6 decreased. After the equitoxic dose, AL rats had significant increase only at 24 h, which diminished thereafter. In DR rats, plasma IL-6 increased significantly and peaked 12 h earlier (12 h in DR vs. 24 h in AL) after the equitoxic dose of TA, where the values were 17-fold higher in DR rats. Immunohistochemical analysis of IL-6 further corroborated the plasma IL-6 results (Fig. 5Go). Predominant staining was in sinusoidal endothelial cells, indicating those to be the main source of IL-6 in the liver. In addition, the infiltrated monocytes, polymorphonuclear cells, and Kupffer cells were stained positive for IL-6, especially at the time points that had significant infiltration.



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FIG. 4. Plasma IL-6 levels were measured using a rat-specific ELISA after (A) low dose of TA (50 mg/kg) and (B) equitoxic doses of TA (600 mg TA/kg to AL vs. 50 mg TA/kg to DR produced equal initial liver injury) to AL and DR rats. Values are expressed as mean ± SE (n = 4). *Values significantly different from AL group at the corresponding time point; !, values significantly different from DR group at 0 h; #, values significantly different from AL group at 0 h.

 


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FIG. 5. Immunohistochemical analysis of IL-6 on liver sections of AL and DR rats after low dose of TA (50 mg/kg, upper panel) and equitoxic doses of TA (lower). Paraffin-embedded liver sections were stained with IL-6 antibody as described in Materials and Methods; the brown color (arrows) indicates IL-6 positive staining.

 
Immunohistochemical analysis of TGF-{alpha} and HGF.
Immunohistochemical analysis of TGF-{alpha} in livers from AL and DR rats obtained after TA administration indicated TGF-{alpha} staining primarily in the hepatocytes. The hepatocytes immediately adjacent to the necrotic cells stained positive for TGF-{alpha} (Fig. 6Go). TA produces necrosis in the centrilobular region, and healthy liver cells that stained positive for TGF-{alpha} surrounded these zones of necrotic cells. Three slides (representing three animals) per time point per group were scored using the staining intensity scoring system described in Materials and Methods (Fig. 7Go). Staining intensity scores are shown in Table 1Go for 50 mg TA/kg in AL and DR rats in comparison with the scores for AL rats receiving 600 mg TA/kg dose. The mean staining-intensity scores were used for statistical analysis and the significant differences have been shown in Fig. 7Go. TGF-{alpha} expression increased significantly at 12 h after the low dose of TA in AL rats, remained higher till 24 h, and then gradually diminished even below the control levels. DR rats displayed significantly higher expression at 12 h, which further increased and peaked at 24 h after TA administration. DR rats had significantly higher TGF-{alpha} expression at 24 to 72 h after TA administration than AL rats. After the equitoxic dose, AL rats had suppressed TGF-{alpha} expression until 36 h, where it was significantly higher than control. TGF-{alpha} further decreased in AL rats and very low expression was observed at later time points. A significant increase in TGF-{alpha} was observed in DR rats as early as 12 h after the equitoxic dose (Fig. 7BGo); it peaked at 24 h and remained significantly higher than in AL rats at 72 and 96 h.



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FIG. 6. Immunohistochemical analysis of TGF-{alpha} (upper) and HGF (lower) on liver sections of AL and DR rats after low dose of TA (50 mg/kg). Paraffin-embedded liver sections were stained with either TGF-{alpha} or HGF antibody as described in Materials and Methods. The brown color indicates TGF-{alpha}/HGF positive staining. H, TGF-{alpha}/HGF positive hepatocytes; N, necrosis; C, central vein.

 


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FIG. 7. Staining intensity scores of TGF-{alpha} immunohistochemistry after (A) low dose of TA (50 mg/kg) and (B) equitoxic doses of TA (600 mg TA/kg to AL vs. 50 mg TA/kg to DR produced equal initial liver injury) to AL and DR rats. Three slides were stained for TGF-{alpha} at various time points. Staining intensity scores were assigned in a blind fashion using a scoring scheme from 0 to 4. 0, no staining; 1, low staining; 2, medium staining; 3, high staining; 4, very high staining. Values are expressed as mean ± SE (n = 4). * = Values significantly different from AL group at the corresponding time point; !, values significantly different from DR group at 0 h; #, values significantly different from AL group at 0 h.

 

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TABLE 1 Staining Intensity Scores of TGF-{alpha} Immunohistochemistry after Low and Equitoxic Doses of Thioacetamide
 
HGF-positive staining was present in parenchymal and inflammatory cells, especially infiltrated monocytes. Similar to TGF-{alpha}, HGF expression was found in the cells immediately next to the necrotic foci (Fig. 6Go, lower panel). Staining intensity scores are shown in Table 2Go for 50 mg TA/kg in AL and DR rats, in comparison with the scores for Al rats receiving 600 mg TA/kg dose. The mean staining intensity scores were used for statistical analysis and the significant differences have been shown in Figure 8Go. Significant increase in the HGF staining was evident at 36 and 48 h in AL rats after low-dose exposure (Fig. 8AGo). DR rats exhibited slightly higher basal (zero time) HGF expression than AL rats. HGF staining increased as early as 12 h after low-dose treatment in DR rats, after which it decreased to control levels. In addition to the higher HGF at 0 h, DR rats had higher HGF than did AL rats at 12 and 72 h after the low dose of TA. After the equitoxic dose, HGF expression did not increase in AL rats until 24 h, when it peaked and then decreased to control levels by 60 h (Fig. 8BGo). In DR rats, an early increase in HGF staining was evident at 12 h but decreased to DR control levels thereafter. Their 72-h value was slightly but significantly higher than the corresponding AL value, similar to that at zero time.


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TABLE 2 Staining Intensity Scores of HGF Immunohistochemistry after Low and Equitoxic Doses of Thioacetamide
 


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FIG. 8. Staining intensity scores of HGF immunohistochemistry after (A) low dose of TA (50 mg/kg) and (B) equitoxic doses of TA (600 mg TA/kg to AL vs. 50 mg TA/kg to DR produced equal initial liver injury) to AL and DR rats. Three slides were stained for HGF. Staining intensity scores were assigned in a blind fashion using a scoring scheme from 0 to 4: 0, no staining; 1, low staining; 2, medium staining; 3, high staining; 4, very high staining. Values are expressed as mean ± SE (n = 4). *Values significantly different from AL group at the corresponding time point; !, values significantly different from DR group at 0 h; #, values significantly different from AL group at 0 h.

 
iNOS activity.
After the low dose of TA, iNOS activity increased in AL rats at 12 h, remained elevated at 24 h, but decreased even below control levels thereafter (Fig. 9AGo). DR alone induced iNOS activity almost 2-fold. DR-induced iNOS remained elevated until 12 h after which a gradual decrease in iNOS activity was observed. DR rats had significantly higher iNOS activity at 0 and 12 h compared to AL rats.



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FIG. 9. Inducible nitric oxide synthase (iNOS) activity was estimated in AL and DR rat liver homogenates after (A) low dose of TA (50 mg/kg) and (B) equitoxic doses of TA (600 mg TA/kg to AL vs. 50 mg TA/kg to DR produced equal initial liver injury) to AL and DR rats as described in Materials and Methods. Values are expressed as mean ± SE (n = 4). *Values significantly different from AL group at the corresponding time point; !, values significantly different from DR group at 0 h; #, values significantly different from AL group at 0 h.

 
AL rats exhibited an overall suppression of iNOS activity after equitoxic dose exposure. DR-induced iNOS remained elevated during the first 12 h after TA treatment and gradually decreased thereafter. DR rats had significantly higher iNOS until 48 h after the equitoxic dose of TA than AL rats (Fig. 9BGo).

Apoptosis in AL and DR rats after TA treatment.
Apoptotic cells detected by TUNEL assay were counted with a light microscope. A modest but significant increase in the number of apoptotic cells was evident in AL rats from 12 to 60 h after the low dose of TA (Fig. 10AGo). A significant increase in apoptosis in DR rats was found from 12 to 36 h and again at 60 h after the low dose. TA induced greater apoptosis in DR rats than in AL rats from 12 to 36 h and again at 60 h after low-dose exposure. After equitoxic doses, AL rats had significant increases in apoptosis at 36 h, which remained higher until 48 h after treatment (Fig. 10BGo). DR rats exhibited increased apoptosis from 12 to 36 h and again at 60 h after equitoxic doses. Although AL rats had higher apoptosis than DR rats at 36 and 48 h, DR rats exhibited increased apoptotic cells as early as 12 h, which lasted until 60 h, indicating greater total apoptosis over the time course. Although significant increases in apoptosis were found in DR rats after TA treatment, the primary type of cell death was coagulative necrosis in the centrilobular region, affecting half or more of the hepatocytes in the lobule.



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FIG. 10. Apoptotic cells in AL and DR livers after (A) low dose of TA (50 mg/kg) and (B) equitoxic doses of TA (600 mg TA/kg to AL vs. 50 mg TA/kg to DR produced equal initial liver injury) to AL and DR rats stained by TUNEL assay as described in Materials and Methods. One thousand cells per slide were counted and the percent apoptotic cells were expressed as an index of apoptosis. Values are expressed as mean ± SE (n = 4). *Values significantly different from AL group at the corresponding time point; !, values significantly different from DR group at 0 h; #, values significantly different from AL group at 0 h.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the past two decades, extensive information has been generated on the beneficial effects of DR. These include increase in life expectancy, decrease in disease conditions including cancer, increased sensitivity of insulin to peripheral tissues and protection from chemical-induced toxicity (Allaben et al., 1990Go; Berg et al., 1994Go; Duffy et al., 1995Go; Hass et al., 1996Go; Lane et al., 1999Go; Ramaiah et al., 2000Go). Previously, we have reported that moderate DR (35% for 21 days) protects male Sprague-Dawley rats from a lethal dose of the model hepatotoxicant, TA (Ramaiah et al., 1998bGo). Seventy percent of DR rats survived as compared to 10% of the AL rats after a normally lethal challenge with TA (600 mg/kg, ip) despite a two-fold increase in bioactivation-mediated liver injury in the DR rats (Ramaiah et al., 1998bGo). Survival was due to timely and adequate compensatory tissue repair response in DR rats (Ramaiah et al., 1998aGo,bGo). Further studies revealed that IL-6, HGF, and MAPK pathways play a critical role in the timely and robust tissue repair in DR rats following TA treatment (Apte et al., 2002Go).

To assess if the extent of tissue repair after toxic challenge is governed by the extent of initial injury, an equitoxic dose experiment was conducted. In this study, DR rats treated with a 12-fold lower dose (50 mg TA/kg, ip) experienced equal liver injury to that observed in AL rats treated with 600 mg TA/kg, a lethal dose (Ramaiah et al., 1998aGo). The increased susceptibility to liver injury in DR rats was caused by prompt induction of CYP2E1, the principal enzyme involved in TA bioactivation (Ramaiah et al., 2001Go; Wang et al., 2000Go). TA is bioactivated primarily by CYP2E1 to TA-sulfoxide and TA-sulf-dioxide, which are the penultimate and ultimate reactive metabolites, respectively. Data indicated that flavin-containing monooxygenase (FMO) is not involved in bioactivation of TA. Indeed, inhibition of FMO by indole-3-carbinol in AL and DR rats lead to increase in TA-induced liver injury suggesting that FMO may detoxify TA. There was no difference in the FMO activity between AL and DR rats, ruling out the possibility of higher detoxification of TA in DR rats (Ramaiah et al., 2001Go). After the equitoxic dose, despite equal initial liver injury, 100% of the DR rats survived while only 10% of the AL rats survived. Timely and robust stimulation of liver tissue repair led to prompt recovery in DR rats (Ramaiah et al., 1998aGo).

Although initial injury is essential to trigger tissue repair, these data indicate that the regenerative response in DR rats does not depend upon the extent of initial injury. Although the initial liver injury was equal in AL and DR rats (Fig. 2AGo), the final outcome was dichotomous because of differences in liver cell division and tissue repair (Fig. 2BGo). Unlike the AL rats, the DR rats escape death after the equitoxic liver injury because of prompt and robust stimulation of cell proliferation and tissue repair, which enables them to restore the structure and function of the liver. In the 10% surviving AL rats after equitoxic doses of TA, tissue repair is very high, explaining the survival. This is also reflected in the higher tissue repair values of the AL group between 60 and 120 h in Figure 2Go (lower panel). Secondly, in DR rats, the number of apoptotic cells increases rapidly and remains high until 60 h after TA administration. This early and sustained increase in apoptosis rapidly removes damaged cells that would otherwise die later via necrosis, permitting more rapid replacement. Apoptosis has been recognized as a controlled programmed cell death leading to minimal damage to the surrounding tissue (Barros et al., 2001Go; Sartorius et al., 2001Go). The increase in apoptosis in DR rats seems to be independent of TNF-{alpha} since no substantial increase in TNF-{alpha} was observed in DR rats after TA treatment. Inhibition of compensatory cell division and apoptosis in AL rats receiving the equitoxic dose of TA leads to progression of liver injury and death. These observations suggest that the AL feeding regimen does not promote the same rate of apoptosis of weaker hepatocytes and timely compensatory tissue repair.

We hypothesized that the mechanism of timely and robust tissue repair in DR rats upon toxic challenge is a higher expression of promitogenic cytokines and growth factors accompanied by upregulated signaling. Higher hepatocyte division in caloric restriction has been demonstrated by Keenan et al.(1995)Go in both male and female Sprague-Dawley rats fed a special diet (Purina Certified Rodent Chow 200–9), which restricted the calories to 65% of AL rats. Caloric restricted (60% of AL) Fisher-344 rats also have better liver regeneration after partial hepatectomy (Chou et al., 1995Go). Cuenca and associates have recently reported that caloric restriction improves liver regeneration after partial hepatectomy, due to early mobilization of hepatocytes into S-phase (Cuenca et al., 2001Go). The priming action of proinflammatory cytokines such as TNF-{alpha}, IL-6, and nitric oxide is essential to fully activate the promitogenic stimulus of growth factors such as TGF-{alpha} and HGF (Galum et al., 2000Go; Lindros et al., 1991; Rai et al., 1998Go; Scotte et al., 1997Go; Webber et al., 1998Go). The role of growth factors such as TGF-{alpha} and HGF in stimulation of liver regeneration has been ascertained in great detail after partial hepatectomy and to some extent after chemical-induced injury (Dalu et al., 1995Go; Diehl, 2000; Fausto et al., 1995Go; Lindros et al., 1991; Soni et al., 1999Go).

The increase in cell division during the first 48 h in DR rats plays a crucial role in the final outcome of TA-induced injury. This timely and robust stimulation of tissue repair is essential for survival as illustrated in the AL rats, where the initiation of repair is delayed until after 48 h. The early upregulation of cell division by prompt and elevated expression of IL-6 and DR-induced iNOS appears to be the key for early onset of tissue repair. Both of these molecules are known to prime the hepatocytes to move from G0 to G1 phase (Diez-Fernandez et al., 1997Go; Gallucci et al., 2000Go; Galum et al., 2000Go; Hui et al., 2002Go; Rai et al., 1998Go). The primed hepatocytes are further stimulated by growth factors to progress in the cell cycle, undergo DNA synthesis, and eventually undergo mitosis. Extensive evidence suggests that stimulation by growth factors is essential for completion of the cell cycle (Boylan and Gruppusso, 1994; Burr et al., 1993Go; Factor et al., 1997Go; Kao et al., 1996Go; Seki et al., 1997Go; Webber et al., 1993Go). The role of TGF-{alpha} and HGF has been particularly well studied during regeneration after toxic insult (Dalu et al., 1995Go; Horada et al., 1999Go; Lindroos et al., 1991Go; Michalopoulos and DeFrances, 1997Go; Scotte et al., 1997Go; Tomiya et al., 1998Go).

Our data reveal earlier and elevated expression of TGF-{alpha} and earlier increase in HGF in the liver of DR rats upon equitoxic challenge. The timely increase in these growth factors coincides with the earlier increase in S-phase DNA synthesis in DR rats. These growth factors also increase in AL rats but the increase is delayed and diminishes very rapidly. This transient activity and lack of stimulation by growth factors leads to diminished cell division in the AL rats upon toxic challenge. TGF-{alpha} signals hepatocyte DNA synthesis and division via the MAPK pathway (Horada et al., 1999Go). The increase in TGF-{alpha} expression in DR rats coincides with S-phase DNA synthesis. The earlier and higher increase in TGF-{alpha} stimulates timely cell division in DR rats. The lack of timely increase in cell division in AL rats is partly explained by the delayed increase in TGF-{alpha}.

Hepatocyte growth factor is a potent mitogen for hepatocytes and is produced mainly in extrahepatic tissues. Liver cells produce HGF under certain conditions such as embryonic and fetal development, endotoxin challenge, and cirrhosis (Gao et al., 1999Go; Masson et al., 2001Go; Pediaditakis et al., 2001Go). In our study, both parenchymal and nonparenchymal cells stained positive for HGF. After equitoxic doses, the earlier increase in HGF in DR rats (at 12 h after TA treatment) correlates with the early stimulation of S-phase in the DR rats. AL rats did have increased expression of HGF at 24 to 48 h but a corresponding increase in cell division was not observed. This indicates a flaw in downstream signaling of HGF. Such downstream inhibition of signaling has been previously reported in regeneration in models of nonalcoholic fatty liver disease (Yang et al., 2001Go).

The data presented here lead to three important conclusions. (1) Tissue repair in AL rats was inhibited, in spite of equal initial liver injury as that seen. This is clearly the function of high dose and confirms the observation by our group and others that high dose leads to inhibition of repair. (2) Even more critical is the observation that in DR rats the tissue repair was not inhibited, even after massive initial liver injury, indicating that DR rats have a mechanism that can operate under extreme stress of injury to promptly stimulate the tissue repair. The signaling mechanisms in DR rats are viable, even during the massive liver injury, regardless of whether it is from a low dose due to induction of CYP2E1 or due to a high dose, thereby highlighting the benefits of diet restriction, which enable the animal to operate even under substantial stress of injury. (3) These data indicate that the stimulation of tissue repair is independent of the extent of initial liver injury. Stimulation of tissue repair is a function of physiological state of diet restriction and allows stimulation of tissue repair to occur even after exposure to a high dose, which normally inhibits it. The reason behind the prompt stimulation of tissue repair in DR, even after massive liver injury, is a timely expression of promitogenic molecules such as IL-6 and TGF-{alpha} in DR rats following TA administration. The AL rats, on the other hand, are unable to mount a tissue repair response (even though the initial liver injury is same as DR) due to inhibition of timely expression of these molecules.

Although one might surmise that the higher liver injury in DR rats receiving a normally lethal dose of TA drives the prompt and robust tissue repair, our data suggest that it is not the extent of liver injury that stimulates the vigorous tissue repair. It is the physiology of DR that leads to prompt tissue repair upon toxic challenge. Liver injury in AL rats matching that observed in DR rats failed to stimulate similar tissue repair (Fig. 11Go). This is rooted in the delay in expression of critical promitogenic molecules in AL rats. The reason the DR rats are able to mount robust tissue repair is prompt upregulation of signaling via the cytokines and growth factors. In AL rats this signaling is inhibited. Our studies reveal that physiological changes produced by DR lead to upregulation of the compensatory mitogenic signaling mechanisms and to survival after a normally lethal toxic challenge of TA.



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FIG. 11. Proposed mechanism of robust tissue repair in DR rats after toxic challenge. In AL rats, the high dose (600 mg/kg, ip) inhibits promitogenic signaling leading to inhibited repair, while in DR rats, even though liver injury inflicted by 12-fold lower dose of TA (50 mg/kg, ip) is same as in AL rats, signaling is intact which leads to prompt tissue repair and survival after a lethal dose of TA.

 


    ACKNOWLEDGMENTS
 
These studies were supported by NIEHS grant # ES-09870. This effort was supported by the Louisiana Board of Regents Support fund through The University of Louisiana, the Kitty DeGree Endowed Chair in Toxicology.


    NOTES
 
A portion of the data included in this paper was presented in the form of a poster at the 22nd Annual Meeting of the American College of Toxicology, Washington, DC, in November 2001.

1 To whom correspondence should be addressed at The University of Louisiana at Monroe, Department of Toxicology, 700 University Avenue, Sugar Hall #306B, Monroe, LA 71209-0495. Fax: (318) 342-1686. E-mail: mehendale{at}ulm.edu. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Allaben, W. T., Chou, M. W., Pegram, R. A., Leakey, J., Feuers, R. J., Duffy, P. H., Turturo, A., and Hart, R. W. (1990). Modulation of toxicity and carcinogenesis by caloric restriction. Korean J. Toxicol. 6, 167–182.

Apte, U. M., Limaye, P. B., Ramaiah, S. K., Vaidya, V. S., Bucci, T. J., Warbritton, A., and Mehendale, H. M. (2002). Upregulated promitogenic signaling via cytokines and growth factors: Potential mechanism of robust liver tissue repair in calorie-restricted rats upon toxic challenge. Toxicol. Sci. 69, 448–459.[Abstract/Free Full Text]

Barros, L. F., Hermosilla, T., and Castro, J. (2001). Necrotic volume increase and the early physiology of necrosis. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 130, 401–409.[CrossRef][ISI][Medline]

Berg, T. F., Breen, P. J., Oriku, E. T., Chen, F. X., and Hart, R. W. (1994). Acute toxicity of gancyclovir: Effect of diet restriction and chronobiology. Food Chem. Toxicol. 32, 45–50.[ISI][Medline]

Boylan, J. M., and Gruppuso, P. A. (1994). In vitro and in vivo regulation of hepatic mitogen-activated protein kinases in fetal rats. Am. J. Physiol. 267, G1078–1086.[Abstract/Free Full Text]

Bredt, D. S., Ferris, C. D., and Snyder, S. H. (1992). Nitric oxide synthase regulatory sites. Phosphorylation by cyclic AMP-dependent protein kinase, protein kinase C, and calcium/calmodulin protein kinase; identification of flavin and calmodulin binding sites. J. Biol. Chem. 267, 10976–10981.[Abstract/Free Full Text]

Burr, A. W., Carpenter, M. R., Hines, J. E., Gullick, W. J., and Burt, A. D. (1993). Intrahepatic distribution of transforming growth factor {alpha} (TGF-{alpha}) during liver regeneration following carbon tetrachloride-induced necrosis. J. Pathol. 170, 95–100.[ISI][Medline]

Chang, L. O., and Looney, W. B. (1965). A biochemical and autoradioagraphy study of the in vivo utilization of tritiated thymidine in regenerating rat liver. Cancer Res. 25, 1871–1822.[ISI][Medline]

Chou, M. W., Shaddock, J. G., Kong, J., Hart, R. W., and Casciano, D. A. (1995). Effect of dietary restriction on partial hepatectomy-induced liver regeneration of aged F344 rats. Cancer Lett. 91, 191–197.[CrossRef][ISI][Medline]

Cuenca, A. G., Cress, W. D., Good, R. A., Marikar, Y., and Engelman, R. W. (2001). Calorie restriction influences cell cycle protein expression and DNA synthesis during liver regeneration. Exp. Biol. Med. 226, 1061–1067.[Abstract/Free Full Text]

Dalu, A., Cronin, G. M., Lyn-Cook, B. D., and Mehendale, H. M. (1995). Age-related differences in TGF-{alpha} and proto-oncogene expression in rat liver after a low dose of carbon tetrachloride. J. Biochem. Toxicol. 10, 259–264.[Medline]

Deihl, A. M. (2000). Cytokine regulation of liver injury and repair. Immunol. Rev.174, 160–171.[CrossRef][ISI][Medline]

Diez-Fernandez, C., Sanz, N., Bosca, L., Hortelano, S., and Cascales, M. (1997). Involvement of nitric oxide synthesis in hepatic perturbations in rat by a necrogenic dose of thioacetamide. Br. J. Pharmacol. 121, 820–826[Abstract]

Duffy, P. H., Feuers, R. J., Pipkin, J. L., Berg, T. F., Leakey, J. E. A., Turturo, A., and Hart R. W. (1995). The effect of dietary restriction and aging on the physiological response to drugs. In Dietary Restriction: Inplications for the Design and Interpretation of Toxicity and Carcinogenicuty Studies (R. W. Hart, D. A. Neumann, and R. T. Robertson, Eds.), pp. 127–140. ILSI Press, Washington, DC.

Factor, V. M., Jensen, M. R., and Thorgeirsson, S. S. (1997). Coexpression of C-myc and transforming growth factor alfa in the liver promotes early, replicative senescence and diminishes regenerative capacity after partial hepatectomy in transgenic mice. Hepatology 26, 1434–1443.[ISI][Medline]

Fausto, N., Laird, A. D., and Webber, E. M. (1995). Liver regeneration: II. Role of growth factors and cytokines in hepatic regeneration. FASEB J. 9, 1527–1536.[Abstract/Free Full Text]

Frame, L. T., Hart, R. W., and Leakey, J. E. (1998). Caloric restriction as a mechanism mediating resistance to environmental disease. Environ. Health Perspect. 106, 313–324.

Gallucci, R. M., Simeonova, P. P., Toriumi, W., and Luster, M. I. (2000). TNF-{alpha} regulates transforming growth factor alpha expression in regenerating murine liver and isolated hepatocytes. J. Immunol. 164, 872–878.[Abstract/Free Full Text]

Galum, E., Zeira, E., Pappo, O., Peters, M., and Rose-John, S. (2000). Liver regeneration induced by a designer human IL-6/sIL-6R fusion protein reverses severe hepatocellular injury. FASEB J. 14, 1979–1987.[Abstract/Free Full Text]

Gao, C., Jokerst, R., Gondipalli, P., Cai, S. R., Kennedy, S., Flye, M. W., and Ponder, K. P. (1999). Lipopolysaccharide potentiates the effects of hepatocyte growth factor on hepatocyte replication in rats by augmenting AP-1 activity. Hepatology 30, 1405–1416.[ISI][Medline]

Hass, B. S., Lewis, S. M., Duffy, P. H., Ershler, W., Feuers, R. J., Good, R. A., Ingram, D. K., Lane, M. A., Leakey, J. E. A., Lipschitz, D., Poehlman, E. T., Roth, G. S., Sprott, R. L., Sullivan, D. H., Turturro, A., Verdery, R. B., Walford, R. L., Weindruch, R., Yu, B. P., and Hart, R. W. (1996). Dietary restriction in humans: Report on the Little Rock conference on the value, feasibility, and parameters of the proposed study. Mech. Aging Dev. 91, 79–94.[CrossRef][Medline]

Horada, K., Shiota, G., and Kawasaki, H., (1999). Transforming growth factor {alpha} and epidermal growth factor receptor in chronic liver disease and hepatocellular carcinoma. Liver 19, 318–325.[ISI][Medline]

Hui, T. T., Mizuguchi, T., Sugiyama, N., Avital, I., Rozga, J., and Demetriou, A. A. (2002) Immediate early genes and p21 regulation in liver of rats with acute hepatic failure. Am. J. Surg. 183, 457–463.[CrossRef][ISI][Medline]

Kao, C., Factor, V. M., and Thorgeirsson, S. S. (1996). Reduced growth capacity from c-myc and c-myc/TGF-{alpha} transgenic mice in primary culture. Biochem. Biophys. Res. Commun. 222, 64–70.[CrossRef][ISI][Medline]

Keenan, K. P., Soper, K. A., Hertzog, P. R., Gumprecht, L. A., Smith, P. F., Mattson, B. A., Ballam, G. C., and Clark, R. L. (1995). Diet, overfeeding, and moderate dietary restriction in control Sprague-Dawley rats: II. Effects on age-related proliferative and degenerative lesions. Toxicol. Pathol. 23, 287–302.[ISI][Medline]

Lane, M. A., Ingram, D. K., and Roth, G. S. (1999). Caloric restriction in non-human primates: Effects on diabetes and cardiovascular disease risk. Toxicol. Sci. 52, 41–48.[Abstract/Free Full Text]

Lindroos, P. M., Zarneger, R., and Michalopoulos, G. K. (1991). Hepatocyte growth factor (hepatopoietin A) rapidly increases in plasma before DNA synthesis and liver regeneration stimulated by partial hepatectomy and carbon tetrachloride administration. Hepatology 13, 743–750.[ISI][Medline]

Mangipudy, R. S., Rao, P. S., Andrews, A., Bucci, T. J., Witzmann, F. A., and Mehendale, H. M. (1998). Dose-dependent modulation of cell death: Apoptosis versus necrosis in thioacetamide hepatotoxicity. Int. J. Toxicol. 17, 193–212.[CrossRef][ISI]

Masaro, E. F. (1998). Mini review-Food restriction in rodents: An evaluation of its role in aging. J. Gerontol. 43, B59–64.

Masson, S., Daveu, M., Francois, A., Bodenant, C., Hiron, M., Teniere, P., Salier, J. P., and Scotte, M. (2001). Up-regulated expression of HGF in rat liver cells after experimental endotoxemia: A potential pathway for enhancement of liver regeneration. Growth Factors 18, 237–250.[ISI][Medline]

Michalopoulos, G. K., and DeFrances, M. C. (1997). Liver regeneration. Science 276, 60–66.[Abstract/Free Full Text]

Pediaditakis, P., Lopez-Talavera, J. C., Petersen, B., Monga, S. P., and Michalopoulos, G. K. (2001). The processing and utilization of hepatocyte growth factor/scatter factor following partial hepatectomy in rats. Hepatology 34, 688–693.[CrossRef][ISI][Medline]

Rai, R. M., Lee, F. Y., Rosen, A., Yang, S. Q., Lin, H. Z., Koteish, A., Liew, F. Y., Zaragoza, C., Lowenstein, C., and Diehl, A. M. (1998). Impaired liver regeneration in inducible nitric oxide synthase deficient mice. Proc. Natl. Acad. Sci. U.S.A. 95, 13829–13834.[Abstract/Free Full Text]

Ramaiah, S. K., Apte, U. M., and Mehendale H. M. (2000). Diet restriction as a protective mechanism in non-cancer toxicity outcomes: A review. Int. J. Toxicol. 19, 1–13.[CrossRef]

Ramaiah, S. K., Apte, U. M., and Mehendale, H. M. (2001). CYP2E1 induction increases thioacetamide-liver injury in diet restricted rats. Drug Metab. Dispos. 29, 1088–1095.[Abstract/Free Full Text]

Ramaiah, S. K., Bucci, T. J., Warbritton, A., Soni, M. G., and Mehendale, H. M. (1998a). Temporal changes in tissue repair permit survival of diet-restricted rats from acute lethal dose of thioacetamide. Toxicol. Sci. 45, 233–241.[Abstract]

Ramaiah, S. K., Soni, M. G., Bucci, T. J., and Mehendale, H. M. (1998b). Diet restriction enhanced compensatory liver tissue repair and survival following administration of lethal dose of thioacetamide. Toxicol. Appl. Pharmacol. 150, 12–21.[CrossRef][ISI][Medline]

Roberts, D. W., Bucci, T. J., Benson, R. W., Warbritton, A. R., McRae, T. A., Pumford, N. R., and Hinson, J. A. (1991). Immunohistochemical localization and quantification of the 3-(cystein-S-yl)-acetaminophen protein adduct in acetaminophen hepatotoxicity. Am. J. Pathol. 138, 359–371.[Abstract]

Sartorius, U., Schmitz, I., and Krammer, P. H. (2001). Molecular mechanisms of death receptor-mediated apoptosis. Chembiochem. 2, 20–29.[CrossRef][ISI][Medline]

Scotte, M., Laquerriere, A., Masson, S., Hiron, M., Teniere, P., Hemet, J., Lebreton, J. P., and Daveau, M. (1997). Transforming growth factor {alpha} (TGF-{alpha}) expression correlates with DNA replication in regenerating rat liver, whatever the hepatectomy extent. Liver 17, 171–176.[ISI][Medline]

Seki, S., Sakai, Y., Kitada, T., Kawakita, N., Yanai, A., Tsutsui, H., Sakaguchi, H., Kuroki, T., and Monna, T. (1997). Induction of apoptosis in a human hepatocellular carcinoma cell line by a neutralizing antibody to transforming growth factor-{alpha}. Virchows Arch. 430, 29–35.[ISI][Medline]

Soni, M. G., Ramaiah, S. K., Mumtaz, M. M., Clewell, H., and Mehendale, H. M. (1999). Toxicant-inflicted injury and stimulated tissue repair are opposing toxicodynamic forces in predictive toxicology. Regul. Toxicol. Pharmacol. 29, 165–174.[CrossRef][ISI][Medline]

Streetz, K. L., Luedde, T., Manns, M. P., and Trautwein, C. (2000). Interleukin-6 and liver regeneration. Gut 47, 309–312.[Free Full Text]

Tomiya, T., Ogata, I., and Fujiwara, K. (1998). Transforming growth factor {alpha} levels in liver and blood correlate better than hepatocyte growth factor with hepatocyte proliferation during liver regeneration. Am. J. Pathol. 153, 955–961.[Abstract/Free Full Text]

Wang, T., Shankar, K., Ronis, M. J., and Mehendale, H. M. (2000). Potentiation of thioacetamide liver injury in diabetic rats is due to induced CYP2E1. J. Pharmacol. Exp. Ther. 294, 473–479.[Abstract/Free Full Text]

Webber, E. M., Bruix, J., Pierce, R. H., and Fausto, N. (1998). Tumor necrosis factor primes hepatocytes for DNA replication in the rat. Hepatology 28, 1226–1234.[ISI][Medline]

Webber, E. M., Fitzgerald, M. J., Brown, P. I., Bartlett, M. H., and Fausto, N. (1993). Transforming growth factor-{alpha} expression during liver regeneration after partial hepatectomy and toxic injury and potential interaction between transforming growth factor-{alpha} and hepatocyte growth factor. Hepatology 18, 1422–1431.[CrossRef][ISI][Medline]

Weindruch, R., and Walford, R. L. (1992). Dietary restriction in mice beginning at one year of age: Effect on life span and spontaneous cancer incidence. Science 215, 1415–1418.

Williams, R. H., Stapleton, A. M., Yang, G., Truong, L. D., Rogers, E., Timme, T. L., Wheeler, T. M., Scardino, P. T., and Thompson, T. C. (1996). Reduced levels of transforming growth factor ß receptor type II in human prostate cancer: An immunohistochemical study. Clin. Cancer. Res. 2, 635–640.[Abstract]

Witzmann, F. A, Fultz, C. D., Mangipudy, R. S., and Mehendale HM (1996). Two-dimensional electrophoretic analysis of compartment-specific hepatic protein charge modification induced by thioacetamide exposure in rats. Fundam. Appl. Toxicol. 31, 124–132.[CrossRef][ISI][Medline]

Yamada, Y., and Fausto, N. (1998). Deficient liver regeneration after carbon tetrachloride injury in mice lacking type 1 but not type 2 tumor necrosis factor receptor. Am. J. Pathol. 152, 1577–1589.[Abstract]

Yang, S. Q., Lin, H. Z., Mandal, A. K., Huang, J., and Diehl, A. M. (2001). Disrupted signaling and inhibited regeneration in obese mice with fatty livers: Implications for nonalcoholic fatty liver disease pathophysiology. Hepatology 34, 694–706.[CrossRef][ISI][Medline]