Harihara M. Mehendale

Tissue Repair: An Important Determinant of Final Outcome of Toxicant-Induced Injury

Tissue repair is a dynamic compensatory cell proliferation and tissue regeneration response stimulated in order to overcome acute toxicity and recover organ/tissue structure and function. Extensive evidence in rodent models using structurally and mechanistically diverse hepatotoxicants such as acetaminophen (APAP), carbon tetrachloride (CCl4), chloroform (CHCl3), thioacetamide (TA), trichloroethylene (TCE), and allyl alcohol (AA) have demonstrated that tissue repair plays a critical role in determining the final outcome of toxicity, i.e., recovery from injury and survival or progression of injury leading to liver failure and death. Tissue repair is a complex process governed by intricate cellular signaling involving a number of chemokines, cytokines, growth factors, and nuclear receptors leading to promitogenic gene expression and cell division. Tissue repair also encompasses regeneration of hepatic extracellular matrix and angiogenesis, the processes necessary to completely restore the structure and function of the liver tissue lost to toxicant-induced initiation followed by progression of injury. New insights have emerged over the last quarter century indicating that tissue repair follows a dose response. Tissue repair increases with dose until a threshold dose, beyond which it is delayed and impaired due to inhibition of cellular signaling resulting in runaway secondary events causing tissue destruction, organ failure, and death. Prompt and adequately stimulated tissue repair response to toxic injury is critical for recovery from toxic injury. Tissue repair is modulated by a variety of factors including species, strain, age, nutrition, and disease condition causing marked changes in susceptibility and toxic outcome. This review focuses on the properties of tissue repair, different factors affecting tissue repair, and the mechanisms that govern tissue repair and progression of injury. It also highlights the significance of tissue repair as a target for drug development strategies and an important consideration in the assessment of risk from exposure to toxicants.

Calpain released from dying hepatocytes mediates progression of acute liver injury induced by model hepatotoxicants

Liver injury is known to progress even after the hepatotoxicant is long gone and the mechanisms of progressive injury are not understood. We tested the hypothesis that hydrolytic enzymes such as calpain, released from dying hepatocytes, destroy the surrounding cells causing progression of injury. Calpain inhibitor, N-CBZ-VAL-PHE-methyl ester (CBZ), administered 1 h after a toxic but nonlethal dose of CCl(4) (2 ml/kg, ip) to male Sprague Dawley rats substantially mitigated the progression of liver injury (6 to 48 h) and also led to 75% protection against CCl(4)-induced lethality following a lethal dose (LD75) of CCl(4) (3 ml/kg). Calpain leakage in plasma and in the perinecrotic areas increased until 48 h and decreased from 72 h onward paralleling progression and regression of liver injury, respectively, after CCl(4) treatment. Mitigation of progressive injury was accompanied by substantially low calpain in perinecrotic areas and in plasma after CBZ treatment. Normal hepatocytes incubated with the plasma collected from CCl(4)-treated rats (collected at 12 h when most of the CCl(4) is eliminated) resulted in extensive cell death prevented by CBZ. Cell-impermeable calpain inhibitor E64 also protected against progression of CCl(4)-induced liver injury, thereby confirming the role of released calpain in progression of liver injury. Following CCl(4) treatment, calpain-specific breakdown of alpha-fodrin increased, while it was negligible in rats receiving CBZ after CCl(4). Hepatocyte cell death in incubations containing calpain was completely prevented by CBZ. Eighty percent of Swiss Webster mice receiving a lethal dose (LD80) of acetaminophen (600 mg/kg, ip) survived if CBZ was administered 1 h after acetaminophen, suggesting that calpain-mediated progression of liver injury is neither species nor chemical specific. These findings suggest the role of calpain in progression of liver injury.

Novel mechanisms in chemically induced hepatotoxicity.

This review focuses on cellular events that modulate hepatotoxicity subsequent to initial liver insult. Cellular events that determine the nature and extent of hepatotoxic injury and the ultimate outcome of that injury are also discussed. The roles of cell types other than hepatocytes, hepatocyte organelle‐spccific processes, and regeneration in progression or recovery from liver injury are emphasized. Leukocyte activities are key events in two distinct hepatotoxicities. Neutrophil‐mediated, periportal inflammation appears to play a primary role in progression of α‐naphthylisothiocyanate‐induced cholangiolitic hepatitis. However, a humorally mediated autoimmune response to protein adducts that occurs after anesthesia is critical in onset of halothane‐induced hepatitis. New insights into specific events at the hepatocyte level are also emerging. Although reducing gap junctional communication between hepatocytes can protect against progression of liver injury, down‐regulation of the subunit proteins (connexins) can isolate neoplastic cells from growth regulation. Acidic intracellular pH characteristic of hypoxia is protective against both hypoxic and toxicant‐induced cell injury In oxidative injury, a pH‐mediated mitochondrial permeability transition causes mitochondrial uncoupling and ATP loss and leads to cell death. The ultimate outcome of hepatotoxic injury depends on the extent of tissue repair. Stimulation of tissue repair after a sublethal dose of CCl4 appears to be the central mechanism in protection against death from a subsequent large dose. Taken together, these examples illustrate the importance of events subsequent to initial liver injury as determinants of extent of liver damage.—Mehendale, H. M., Roth, R. A., Gandolfi, A. J., Klaunig, J. E., Lemasters, J. J., Curtis, L. R. Novel mechanisms in chemically induced hepatotoxicity. FASEB J. 8, 1285‐1295 (1994)

Chronic ethanol intake impairs insulin signaling in rats by disrupting Akt association with the cell membrane: Role of TRB3 in inhibition of Akt/protein kinase B activation

Chronic and excessive alcohol consumption is an important and modifiable risk factor for type 2 diabetes. We previously reported elevations in hepatic Class 1 alcohol dehydrogenase (ADH) expression in ethanol-fed rats correspondent with reduced levels of mature, nuclear sterol-regulatory element-binding protein-1 (SREBP-1), an insulin-induced transcriptional repressor of the ADH gene. In this report, we have studied the effects of insulin and ethanol on ADH gene expression in a highly differentiated rat hepatoma cell line (FGC-4), as well as the in vivo effects of chronic intake of an ethanol-containing diet on hepatic insulin signaling. Insulin inhibited ADH gene expression, and this was abolished by LY294002 (a phosphatidylinositol 3-kinase inhibitor) and small interfering RNA knockdown of SREBP-1. Chronic ethanol intake led to decreased phosphorylation of Akt (protein kinase B) at Thr308, increased phosphorylation of Akt at Ser473, and decreased phosphorylation of glycogen synthase kinase-3β (a downstream effector of Akt). Hepatic membrane-associated Akt content was decreased and cytosolic Akt content was increased in rats fed an ethanol-containing diet. Thus, disruptive effects of ethanol on insulin signaling occurred via impaired phosphorylation of Akt at Thr308. TRB3, a negative regulator of Akt, was induced in liver of ethanol-fed rats. In ethanol-treated FGC-4 cells, small interfering RNA knockdown of TRB3 increased membrane-associated Akt and the phosphorylation of Akt at Thr308. Our results suggest that ethanol induces TRB3, which, through binding to the pleckstrin homology domain of Akt, prevents its plasma membrane association, Akt-Thr308 phosphorylation, and subsequent Akt-mediated signaling. Ethanol inhibition of insulin signaling reduces nuclear SREBP accumulation and results in disinhibition of Class 1 ADH transcription.

Tissue injury and repair as parallel and opposing responses to CCl4 hepatotoxicity: a novel dose-response

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 CCl4 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 CCl4 (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 immunohistochemistry over a time course of 0 to 96 h. Stimulation of cell division, evident even after a subtoxic dose of CCl4, 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 CCl4/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.

SATURATION TOXICOKINETICS OF THIOACETAMIDE: ROLE IN INITIATION OF LIVER INJURY

Thioacetamide (TA), a potent centrilobular hepatotoxicant, undergoes a two-step bioactivation mediated by microsomal CYP2E1 to TA sulfoxide (TASO), and further to TA-S,S-dioxide (TASO2), a reactive metabolite that initiates cellular necrosis. Our earlier studies showed that bioactivation-mediated liver injury of TA is not dose-proportional. The objective of this study was to examine whether increasing doses of TA lead to enzyme saturation, thereby resulting in lack of dose-response for injury: bioactivation of TA → TASO → TASO2 may follow zero-order kinetics. A 12-fold dose range of TA (50, 300, and 600 mg/kg i.p.) was injected into male Sprague-Dawley rats. TA and TASO were quantified in plasma, liver, and urine by high-performance liquid chromatography. With increasing doses, the apparent elimination half-lives of TA and TASO increased linearly, indicating that TA bioactivation exhibits saturation kinetics. Increasing TA dose resulted in greater-than-proportional increases in plasma TA and TASO levels. The TASO/TA ratio was inversely proportional to the dose of TA. Covalent binding of 14C-TA-derived radiolabel to liver macromolecules showed a lessthan-dose-proportionate increase with a 12-fold higher dose. Less than dose-proportional covalent binding was confirmed in liver microsomal incubations with 14C-TA. Three-fold higher excretion of TASO was seen in urine at the highest dose (600 mg/kg) compared with the lowest dose (50 mg TA/kg). Incubation of TA with rat liver microsomes and purified baculovirus-expressed rat and human CYP2E1 Supersomes, over a concentration range of 0.01 to 10 mM, revealed saturation of TA conversion to TASO at and above 0.05 mM TA concentration, comparable to in vivo plasma and liver levels achieved upon administration of higher doses. Calculated Km values for TA (0.1 mM) and TASO (0.6 mM) suggest that the second step of TA bioactivation is 6-fold less efficient. Collectively, the findings indicate saturation of CYP2E1 at the first (TA to TASO) and second (TASO to TASO2) steps of TA bioactivation.

Renal and Hepatic Transporter Expression in Type 2 Diabetic Rats

Membrane transporters are critical for the uptake as well as elimination of chemicals and by-products of metabolism from the liver and kidneys. Since these proteins are important determinants of chemical disposition, changes in their expression in different disease states can modulate drug pharmacokinetics. The present study investigated alterations in the renal and hepatic expression of organic anion and cation transporters (Oats/Octs), multidrug resistance-associated proteins (Mrps), breast cancer resistance protein (Bcrp), P-glycoprotein (Pgp), and hepatic Na(+)-taurocholate cotransporting polypeptide (Ntcp) in type 2 diabetic rats. For this purpose, type 2 diabetes was induced by feeding male Sprague-Dawley rats a high fat diet followed by a single dose of streptozotocin (45 mg/kg, i.p., in 0.01 M citrate buffer pH 4.3) on day 14. Controls received normal diet and vehicle. Kidney and liver samples were collected on day 24 for generation of crude plasma membrane fractions and Western blot analysis of Oat, Oct, Mrp, Bcrp, Pgp, and Ntcp proteins. With regards to renal uptake transporters, type 2 diabetes increased levels of Oat2 (2.3-fold) and decreased levels of Oct2 to 50% of control kidneys. Conversely, efflux transporters Mrp2, Mrp4, and Bcrp were increased 5.4-fold, 2-fold, and 1.6-fold, respectively in type 2 diabetic kidneys with no change in levels of Mrp1, Mrp5, or Pgp. Studies of hepatic transporters in type 2 diabetic rats reveal that the protein level of Mrp5 was reduced to 4% of control livers with no change in levels of Bcrp, Mrp1, Mrp2, Mrp4, Ntcp, or Pgp. The changes reported in this study may have implications in type 2 diabetic patients.

Colchicine Antimitosis Abolishes CCl4 Autoprotection

A subtoxic dose of CCl 4 is known to destroy liver microsomal cytochrome P-450 and this is widely accepted as the mechanism of CCl4 autoprotection. Circumstantial evidence suggests that while cytochrome P-450 is significantly decreased, this mechanism alone cannot explain the phenomenon of autoprotection. Previous studies have established that hepatocellular regeneration is stimulated as early as 6 hr after the administration of a low dose of CC14. If the early phase stimulation of hepatocellular regeneration by the protective dose is indeed the mechanism of autoprotection, then ablation of this early phase of tissue healing by colchicine should result in an abolishment of autoprotection. Present studies were conducted to test this conceptual premise. The protection afforded by a low dose of CCl4 (LCCl4, 100 μl/kg, ip) on the toxic effects of a subsequently administered moderately toxic dose of CCl4 (HCCl4, 2.5 ml/kg, ip) was established in male Sprague-Dawley rats. The protective dose provided 100% protection, whereas only 62.5% survival was observed when the corn oil vehicle was administered instead of the protective dose of LCCl4. Colchicine administration (1 mg/kg, ip in saline) 2 hr prior to the injection of LCCl4 led to a complete loss of autoprotection resulting in 100% mortality in rats given the HCCl4. Earlier studies have established that colchicine selectively suppresses the early phase of hepatocellular regeneration at 6 hr without influencing the second phase at 36-48 hr. The consequence of colchicine antimitosis on the toxicological endpoints of liver injury was evaluated by serum enzyme elevations and by histopathological examination of the liver during a time course of 6, 24, 48, 72, and 96 hr after the administration of HCCl4. In the autoprotection regime, after only a transient and modest elevation of serum alanine and aspartate transaminases, complete recovery occurred by 96 hr. Hepatocellular necrosis was consistently lower compared to all other groups. Colchicine preadministration in the autoprotection regime resulted in significantly greater and progressive elevation of the serum enzymes and a correspondingly commensurate progression of hepatic lesion. Toxic effects of HCC14 alone were more rapidly and maximally augmented by colchicine preadministration. The role of hepatocellular regeneration in autoprotection was evaluated by 3H-thymidine incorporation in hepatocellular nuclear DNA and by morphometric estimation of mitotic index. While HCCl4 alone resulted in some stimulation of 3H-thymidine incorporation and mitosis, the regenerative activity observed with prior LCCl4 administration was remarkably greater, particularly at 48 hr. Colchicine preadministration in either of these 2 protocols decisively obtunded the stimulated regenerative activities essentially abolishing the tissue healing mechanisms. These results indicate that hepatocellular regeneration stimulated by the LCCl4 not only enhances recovery from limited liver injury but also augments tissue repair process after massive liver injury is elicited by the subsequent HCCl 4 dose. Selective ablation of the early phase hepatocellular regeneration stimulated by the protective low dose of CCl4, by colchicine antimitosis, results in abolishment of CCl4 autoprotection. Furthermore, in the absence of the early phase stimulation of tissue healing mechanisms, toxicity of a moderately toxic dose of CCl4 is further augmented. These findings indicate the critical importance of the early stimulation of hepatocellular regeneration and tissue healing processes in the hepatotoxicity of CCl4.

Role of Hepatocellular Regeneration in CCl4 Autoprotection

The destruction of liver microsomal cytochromes P450 by a previously administered low dose of CCl4 has been widely accepted as the mechanism of CCl4 autoprotection. However, circumstantial evidence suggests that this mechanism cannot completely explain the phenomenon of autoprotection. The protective effect of a low dose of CCl4 (0.3 ml/kg, po) on the lethal effect of a subsequently administered high dose (5 ml/kg, po) was established in male Sprague Dawley rats. The protective dose permitted 100% survival, whereas only 15% survival was observed without it. Hepatotoxicity, measured by serum enzyme elevations (aspartate transaminase, alanine transaminase, and sorbitol dehydrogenase) and histopathological changes 24 hr after the treatment with high dose, was similar in both the groups, even though the protective dose had significantly decreased liver microsomal cytochromes P450 (to 62% of normal) and associated enzymes, aminopyrine demethylase and aniline hydroxylase. Rats pretreated with CoCl2 to decrease hepatic microsomal cytochrome P450 to 44% of normal levels did not show a significant protection from the hepatotoxicity of high dose of CCl4. Previous studies have established that hepatocellular regeneration is stimulated within 6 hr after the administration of a low dose of CCl4. Based on this observation, a premise that autoprotection results from augmented recovery from injury rather than decreased injury appears likely. Hence, the role of hepatocellular regeneration was evaluated by following 3H-thymidine incorporation in hepatocellular nuclear DNA, labelling index by autoradiography, and by morphometric estimation of mitotic index. After administration of the protective dose of CCl4, stimulated nuclear DNA synthesis measured by 3H-thymidine incorporation into nuclear DNA was increased and this remained high even after subsequent administration of high dose of CCl4. Forty-eight hr after the administration of a lethal dose of CCl4 alone (5 ml/kg, po), labelling index was slightly increased, but mitotic index was not increased. In the surviving rats (15%), both labelling index and mitotic index were significantly elevated after an additional 24 hr. In rats receiving the protective dose, a significantly greater elevation of labelling index as well as mitotic index occurred 48 hr after the administration of the same lethal dose of CCl4. These results suggest that hepatocellular regeneration stimulated by the protective dose, as a biological response recruited to overcome the accompanying limited injury, may augment and sustain tissue repair processes to permit tissue restoration even after the massive liver injury elicited by the subsequent large dose of CCl4.

Protection of hepatotoxic and lethal effects of CCl4 by partial hepatectomy.

CCl4 is a hepatotoxic haloalkane, capable of producing hepatocellular fatty degeneration and centrilobular necrosis. Previous reports indicate induction of liver regeneration after 36-48 hr of CCl4 treatment, which is considered as a secondary effect. The present investigation was undertaken to evaluate the primary effects of CCl4 on hepatic DNA synthesis and to correlate liver regeneration with CCl4 toxicity. These studies were conducted in normal and actively regenerating livers using male Sprague-Dawley rats undergoing sham operation (SH), or partial (70%) hepatectomy (PH). Incorporation of 3H-thymidine (3H-T) in hepatocellular nuclear DNA and autoradiographic analyses of liver sections served as indices for hepatocellular regeneration. Initial experiments established that peak regeneration occurs at 2 days post-PH (PH2) and liver regeneration phases out by 7 days post-PH (PH7). SH and PH rats were challenged with a single ip dose of either corn oil vehicle or CCl4 at either 0.1 ml/kg (to represent subtoxic dose) or 2.5 ml/kg (to represent toxic dose). The low dose of CCl4 was not toxic and did not alter 3H-T incorporation and percentage labelled cells at 6 or 24 hours after administration to SH, PH2 or PH7 groups, indicating that there was no interference with PH-stimulated hepatocellular regeneration. The high dose of CCl4 was significantly hepatotoxic and lethal in SH rats, while in PH2 rats both hepatotoxic and lethal effects were significantly decreased. 3H-T incorporation as well as percentage labelled cells, highly stimulated by PH, were significantly decreased by high dose of CCl4. However, hepatocellular regeneration in PH2 rats treated with high dose of CCl4 was still significantly higher than SH or PH, groups by virtue of the stronger stimulatory effect of PH. In PH7 rats, where hepatocellular regeneration had returned to the SH level, the hepatotoxic and lethal effects of the large dose of CCl4 were also restored. These findings show that the progressive phase of a single high dose of CCl4 injury which normally culminates in hepatotoxic and lethal effects is significantly mitigated by previously stimulated hepatocellular regeneration. High dose of CCl4 suppresses hepatocellular regeneration at early time points after administration in contrast to the smaller subtoxic dose of CCl4. By virtue of the much stronger stimulatory effect, PH results in the protection against the hepatotoxic and lethal effects of CCl4 despite the obtunding effects of the high dose on hepatocellular regeneration.