Cytochrome P4502E1 induction increases thioacetamide liver injury in diet-restricted rats.

Earlier studies have shown highly exaggerated mechanism-based liver injury of thioacetamide (TA) in rats following moderate diet restriction (DR) and in diabetes. The objective of the present study was to investigate the mechanism of higher liver injury of TA in DR rats. Since both DR and diabetes induce CYP2E1, we hypothesized that hepatic CYP2E1 plays a major role in the bioactivation-based liver injury of TA. When male Sprague-Dawley rats (250-275 g) were maintained on diet restriction (DR, 35% of ad libitum fed rats, 21 days) the total hepatic microsomal cytochrome P450 (CYP450) was increased 2-fold along with a 4.6-fold increase in CYP2E1 protein, which corresponded with a 3-fold increase in CYP2E1 activity as measured by chlorzoxazone hydroxylation. To further test the involvement of CYP2E1, 24 and 18 h after pretreatment with pyridine (PYR) and isoniazid (INZ), specific inducers of CYP2E1, male Sprague-Dawley rats received a single administration of 50 mg of TA/kg (i.p.). TA liver injury was >2.5- and >3-fold higher at 24 h in PYR + TA and INZ + TA groups, respectively, compared with the rats receiving TA alone. Pyridine pretreatment resulted in significantly increased total CYP450 content accompanied by a 2.2-fold increase in CYP2E1 protein and 2-fold increase in enzyme activity concordant with increased liver injury of TA, suggesting mechanism-based bioactivation of TA by CYP2E1. Hepatic injury of TA in DR rats pretreated with diallyl sulfide (DAS), a well known irreversible in vivo inhibitor of CYP2E1, was significantly decreased (60%) at 24 h. CCl(4) (4 ml/kg i.p.), a known substrate of CYP2E1, caused lower liver injury and higher animal survival confirming inhibition of CYP2E1 by DAS pretreatment. The role of flavin-containing monooxygenase (FMO) in TA bioactivation implicated by previous in vitro studies, and consequent increased TA-induced liver injury in DR rats was tested in vivo with a relatively selective inhibitor of FMO, indole-3-carbinol, and then treated with 50 mg of TA/kg. FMO activity and alanine aminotransferase levels measured at different time points revealed that TA liver injury was not decreased although FMO activity was significantly decreased, suggesting that hepatic FMO is unlikely to bioactivate TA. These findings suggest induction of CYP2E1 as the primary mechanism of increased bioactivation-based liver injury of TA in DR rats.     

Toxicokinetics and toxicity of thioacetamide sulfoxide: a metabolite of thioacetamide

Thioacetamide (TA) is bioactivated by CYP2E1 to TA sulfoxide (TASO), and to the highly reactive sulfdioxide (TASO₂), which initiates hepatic necrosis by covalent binding. Previously, we have established that TA exhibits saturation toxicokinetics over a 12-fold dose range, which explains the lack of dose–response for bioactivation-based liver injury. In vivo and in vitro studies indicated that the second step (TASO → TASO₂) of TA bioactivation is less efficient than the first one (TA → TASO). The objective of the present study was to specifically test the saturation of the second step of TA bioactivation by directly administering TASO, which obviates the contribution from first step, i.e. TA → TASO. Male SD rats were injected with low (50 mg/kg, ip), medium (100 mg/kg) and high (LD₇₀, 200 mg/kg) doses of TASO. Bioactivation-mediated liver injury that occurs in the initial time points (6 and 12 h), estimated by plasma ALT, AST and liver histopathology over a time course, was not dose-proportional. Escalation of liver injury thereafter was dose dependent: low dose injury subsided; medium dose injury escalated upto 36 h before declining; high dose injury escalated from 24 h leading to 70% mortality. TASO was quantified in plasma by HPLC at various time points after administration of the three doses. With increasing dose (i.e., from 50 to 200 mg/kg), area under the curve (AUC) and C_max increased more than dose proportionately, indicating that TASO bioactivation exhibits saturable kinetics. Toxicokinetics and initiation of liver injury of TASO are similar to that of TA, although TASO-initiated injury occurs at lower doses. These findings indicate that bioactivation of TASO to its reactive metabolite is saturable in the rat as suggested by previous studies with TA.     

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 less-than-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 K(m) 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.     

Mechanisms of increased liver tissue repair and survival in diet-restricted rats treated with equitoxic doses of thioacetamide.

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.     

Comparative metabolism and disposition of trichloroethylene in Cyp2e1-/-and wild-type mice.

Trichloroethylene (TCE)1 is an important environmental contaminant, a well established rodent carcinogen, and a "probable human carcinogen". Metabolism of TCE occurs primarily via cytochrome P450 (P450)-dependent oxidation. In vitro studies suggested that CYP2E1 is the principal high-affinity enzyme responsible for TCE metabolism. The objective of the present work is to more directly assess the role of CYP2E1 in the metabolism and disposition of 1,2-14C-TCE administered at 250 or 1000 mg/kg (gavage) using Cyp2e1-/-[knockout (KO)] versus wild-type (WT) mice. After dosing, animals were individually placed in glass metabolism cages that allowed the collection of expired air, urine, and feces. Exhalation of TCE-derived 14CO2 increased in a dose-dependent manner in mice of both genotypes and was significantly higher in WT versus KO mice. A significantly greater percentage of the dose was exhaled in KO versus WT mice as organic volatiles (mainly as TCE). Urinary excretion was the major route of TCE metabolism in WT mice, and the percentage of dose eliminated in urine was significantly higher at the 250 versus 1000 mg/kg dose. Furthermore, urinary excretion and CO2 exhalation significantly decreased in KO versus WT mice. Pretreatment with 1-aminobenzotriazole clearly inhibited TCE metabolism as evident from increased exhalation of parent TCE, and decreased urinary excretion and CO2 exhalation in mice of both genotypes. In conclusion, these data showed that whereas CYP2E1 plays an important role in TCE metabolism and disposition, other P450s also play a significant role and may explain earlier results showing that TCE causes lung damage in KO and WT mice.     

The role of vitamin D3 upregulated protein 1 in thioacetamide-induced mouse hepatotoxicity

Thioacetamide (TA) is a commonly used drug that can trigger acute hepatic failure (AHF) through generation of oxidative stress. Vitamin D3 upregulated protein 1 (VDUP1) is an endogenous inhibitor of thioredoxin, a ubiquitous thiol oxidoreductase, that regulates cellular redox status. In this study, we investigated the role of VDUP1 in AHF using a TA-induced liver injury model. VDUP1 knockout (KO) and wild-type (WT) mice were subjected to a single intraperitoneal TA injection, and various parameters of hepatic injury were assessed. VDUP1 KO mice displayed a significantly higher survival rate, lower serum alanine aminotransferase and aspartate aminotransferase levels, and less hepatic damage, compared to WT mice. In addition, induction of apoptosis was decreased in VDUP1 KO mice, with the alteration of caspase-3 and -9 activities, Bax-to-Bcl-2 expression ratios, and mitogen activated protein kinase (MAPK) signaling pathway. Importantly, analysis of TA bioactivation revealed lower plasma clearance of TA and covalent binding of [¹⁴C]TA to liver macromolecules in VDUP1 KO mice. Furthermore, the level of oxidative stress was significantly less in VDUP1 KO mice than in their WT counterparts, as evident from lipid peroxidation assay. These results collectively indicate that VDUP1 deficiency protects against TA-induced acute liver injury via lower bioactivation of TA and antioxidant effects.     

Secretory phospholipase A₂-mediated progression of hepatotoxicity initiated by acetaminophen is exacerbated in the absence of hepatic COX-2

We have previously reported that among the other death proteins, hepatic secretory phospholipase A₂ (sPLA₂) is a leading mediator of progression of liver injury initiated by CCl₄ in rats. The aim of our present study was to test the hypothesis that increased hepatic sPLA₂ released after acetaminophen (APAP) challenge mediates progression of liver injury in wild type (WT) and COX-2 knockout (KO) mice. COX-2 WT and KO mice were administered a normally non lethal dose (400 mg/kg) of acetaminophen. The COX-2 KO mice suffered 60% mortality compared to 100% survival of the WT mice, suggesting higher susceptibility of COX-2 KO mice to sPLA₂-mediated progression of acetaminophen hepatotoxicity. Liver injury was significantly higher at later time points in the KO mice compared to the WT mice indicating that the abatement of progression of injury requires the presence of COX-2. This difference in hepatotoxicity was not due to increased bioactivation of acetaminophen as indicated by unchanged cyp2E1 protein and covalently bound ¹⁴C-APAP in the livers of KO mice. Hepatic sPLA₂ activity and plasma TNF-α were significantly higher after APAP administration in the KO mice. This was accompanied by a corresponding fall in hepatic PGE₂ and lower compensatory liver regeneration and repair (³H-thymidine incorporation) in the KO mice. These results suggest that hindered compensatory tissue repair and poor resolution of inflammation for want of beneficial prostaglandins render the liver very vulnerable to sPLA₂-mediated progression of liver injury. These findings are consistent with the destructive role of sPLA₂ in the progression and expansion of tissue injury as a result of continued hydrolytic breakdown of plasma membrane phospholipids of perinecrotic hepatocytes unless mitigated by sufficient co-induction of COX-2.     

Effect of CYP1A2 deficiency on heterocyclic amine DNA adduct levels in mice.

The contribution of CYP1A2 to the formation of DNA adducts of the cooked meat-derived heterocyclic amines (HCAs) 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was examined in CYP1A2-null (knock-out, KO) and wild-type (WT) mice. IQ (25 mg and 75 mg/kg) and PhIP (150 mg/kg) were administered by gavage to mice and DNA adduct levels in liver, kidney, mammary gland and colon were examined by the 32P-postlabeling assay. Three hours after either dose of IQ, adducts levels in liver and kidney of KO mice were 20-30% of the levels in WT mice, a difference that was statistically significant (Student's t-test, P < 0.05). In the colon, adduct levels in KO mice were significantly lower than in the WT mice only at the lowest dose of IQ (1.6+/-0.6 vs 4.6+/-0.7, respectively, relative adduct labeling (RAL) x 10(8), mean+/-S.E.M., n = 3-5 mice). In the mammary gland, however, there was no difference in IQ-DNA adduct levels in KO and WT mice at either dose of IQ. Three hours after dosing with PhIP, PhIP-DNA adduct levels were statistically significantly lower in KO mice than in WT mice in all tissues examined. PhIP-DNA adducts in liver and kidney of WT mice were 9.9+/-1.1 and 22.5+/-6.9, respectively, whereas no PhIP-DNA adducts were detected in either organ of KO mice (limit of detection, 1.4-2.8 x 10(9)). PhIP-DNA adduct levels in mammary gland and colon of WT mice were 47.1+/-9.5 and 58.0+/-21.7, respectively, but accordingly only 3.8+/-0.7 and 5.4+/-0.9 in KO mice. The findings indicate that CYP1A2, responsible for IQ and PhIP N-hydroxylation, the first step in the metabolic action, significantly effects DNA adduct formation in vivo. However, the data raise the possibility that other cytochromes P450 as well as other pathways of activation potentially contribute to DNA adduct formation in specific organs, depending on the HCA substrate.     

Temporal Changes in Tissue Repair Permit Survival of Diet-Restricted Rats from an Acute Lethal Dose of Thioacetamide

Although, diet restriction (DR) has been shown to substantiallyincrease longevity while reducing or delaying the onset of agerelateddiseases, little is known about the mechanisms underlying thebeneficial effects of DR on acute toxic outcomes. An earlierstudy (S. K. Ramaiah et al., 1998, Toxicol. Appl. Pharmacol.150, 12–21) revealed that a 35% DR compared to ad libitum(AL) feeding leads to a substantial increase in liver injuryof thioacetamide (TA) at a low dose (50 mg/kg, ip). Higher liverinjury was accompanied by enhanced survival. A prompt and enhancedtissue repair response in DR rats at the low dose (sixfold higherliver injury) occurred, whereas at equitoxic doses (50 mg/kgin DR and 600 mg/kg in AL rats) tissue repair in AL rats wassubstantially diminished and delayed. The extent of liver injurydid not appear to be closely related to the extent of stimulatedtissue repair response. The purpose of the present study wasto investigate the time course (0–120 h) of liver injuryand liver tissue repair at the high dose (600 mg TA/kg, ip,lethal in AL rats) in AL and DR rats. Male Sprague-Dawley rats(225–275 g) were 35% diet restricted compared to theirAL cohorts for 21 days and on day 22 they received a singledose of TA (600 mg/kg, ip). Liver injury was assessed by plasmaALT and by histopathological examination of liver sections.Tissue repair was assessed by [³H]thymidine incorporation intohepatonuclear DNA and proliferating cell nuclear antigen (PCNA)immunohistochemistry during 0–120 h after TA injection.In AL-fed rats hepatic necrosis was evident at 12 h, peakedat 60 h, and persisted thereafter until mortality (3 to 6 days).Peak liver injury was approximately twofold higher in DR ratscompared to that seen in AL rats. Hepatic necrosis was evidentat 36 h, peaked at 48 h, persisted until 96 h, and returnedto normal by 120 h. Light microscopy of liver sections revealedprogression of hepatic injury in AL rats whereas injury regressedcompletely leading to recovery of DR rats by 120 h. Progressionof injury led to 90% mortality in AL rats vs 30% mortality inDR group. In the surviving AL rats, S-phase DNA synthesis wasevident at 60 h, peaked at 72 h, and declined to base levelby 120 h, whereas in DR rats S-phase DNA synthesis was evidentat 36 h and was consistently higher until 96 h reaching controllevels by 120 h. PCNA studies showed a corresponding increasein cells in S and M phase in the AL and DR groups. DR resultedin abolition of the delay in tissue repair associated with thelethal dose of TA in ad libitum rats. Temporal changes and highertissue repair response in DR rats (earlier and prolonged) arethe conduits that allow a significant number of diet restrictedrats to escape lethal consequence.     

Streptozotocin-induced diabetic mice are resistant to lethal effects of thioacetamide hepatotoxicity

The effect of Type 1 diabetes on the toxicity of thioacetamide was investigated in a murine model. In streptozotocin-induced diabetic C57BL6 mice a LD90 dose of thioacetamide (1000 mg/kg, ip in saline) caused only 10% mortality. Alanine aminotransferase activity revealed approximately 2.7-fold less liver injury in the diabetic (DB) mice compared to the non-DB controls, at 36 h after thioacetamide (TA) administration, which was confirmed via histopathological analysis. HPLC analyses revealed lower plasma t(1/2) of TA in the DB mice. Covalent binding of [(14)C]TA to liver tissue was lower in the DB mice, suggesting lower bioactivation of TA. Compensatory hepatic S-phase stimulation as assessed by [(3)H]thymidine incorporation occurred much earlier and was substantially higher in the DB mice compared to the non-DB cohorts. Morphometric analysis of cells in various phases of cell division assessed via immunohistochemical staining for proliferating cell nuclear antigen revealed more cells in G(1), S, G(2), and M phases in the DB mice, indicating robust tissue repair in concordance with the findings of [(3)H]thymidine pulse labeling studies. The importance of tissue repair in the resistance of DB mice was further investigated by blocking cell division in the DB mice by colchicine (1 mg/kg, ip) at 40 h after TA administration, well after the bioactivation of TA. Antimitotic action of colchicine, confirmed by decreased S-phase stimulation, led to progression of liver injury and increased mortality in DB mice. These findings suggest that lower bioactivation of TA and early onset of liver tissue repair are the pivotal underpinnings for the resistance of DB mice.     

Involvement of CYP3A4/5 and CYP2D6 in the metabolism of aconitine using human liver microsomes and recombinant CYP450 enzymes

Triptolide, the primary active component of Tripterygium wilfordii Hook F, has various pharmacological activities but also a narrow therapeutic window. Cytochrome P450s are proposed to be responsible for the hydroxylation of triptolide in vitro and CYP3A induction by dexamethasone can increase the metabolism of triptolide and decrease the hepatotoxicity in rat. However, triptolide-induced toxicity has not been investigated in an animal model having a suppression of P450 activities. Here we compared the toxicological effects and toxicokinetics of triptolide between liver-specific cytochrome P450 reductase (CPR) knockout (KO) mice (abolished hepatic P450 activities) and wild-type (WT) control mice after a single oral gavage of triptolide at 0.5 mg/kg or 1.0 mg/kg. A low toxic dose of triptolide at 0.5 mg/kg for WT mice resulted in severe toxicities including death in KO mice. Changes in serum biochemistry, hematology and histopathology further indicated much more severe toxicities in multiple organs in KO mice compared to WT mice after triptolide administration. The mono-hydroxylated metabolites of triptolide detected in the blood of WT mice were undetectable in KO mice, accompanied by much higher triptolide levels in the blood and tissues including the liver, kidney, and spleen determined by LC-MS/MS. Taken together, our results confirmed that inactivation of hepatic P450s abolishes the ability in metabolism of triptolide in the liver, subsequently resulting in an increase in bioavailability and toxicity of triptolide in vivo. It is suggested that P450 inhibition/inactivation might pose a significant health risk in the clinic use of triptolide.     

Breast cancer resistance protein (Bcrp/abcg2) is a major determinant of sulfasalazine absorption and elimination in the mouse

Sulfasalazine is used in the treatment of ulcerative colitis, Crohn's disease, and rheumatoid arthritis. When administered orally, sulfasalazine is poorly absorbed with an estimated bioavailability of 3-12%. Recent studies using the T-cell line (CEM) have shown that sulfasalazine is a substrate for the ATP-binding cassette (ABC) efflux pump ABCG2. ABCG2 is known to efflux a number of xenobiotics and appears to be a key determinant of efficacy and toxicity of ABCG2 substrates. To date, there has not been any systematic study on the mechanisms involved in the transport of sulfasalazine in vivo. Accordingly, we investigated whether Bcrp (abcg2) is involved in the disposition of sulfasalazine. After oral administration of 20 mg/kg sulfasalazine, the area under the plasma concentration (AUC) time profile in Bcrp1 (abcg2)-/- knockout (KO) mice was approximately 111-fold higher than that in FVB wild-type (WT) mice. After intravenous administration of 5 mg/kg sulfasalazine, the AUC in Bcrp1 (abcg2)-/- KO mice was approximately 13-fold higher than that in WT mice. Moreover, treatment of WT mice with a single oral dose of gefitinib (Iressa; 50 mg/kg), a known inhibitor of Bcrp, given 2 h prior to administering a single oral dose of sulfasalazine (20 mg/kg), resulted in a 13-fold increase in the AUC of sulfasalazine compared to the AUC in vehicle-treated mice. Since gefitinib is also an inhibitor of P-glycoprotein (P-gp), the impact of P-gp on sulfasalazine absorption in vivo was also examined. The sulfasalazine AUC in mdr1a-/- KO versus WT mice did not differ significantly after either an oral (20 mg/kg) or an intravenous dose (5 mg/kg). We conclude that Bcrp (abcg2) is an important determinant for the oral bioavailability and the elimination of sulfasalazine in the mouse, and that sulfasalazine has the potential to be utilized as a specific in vivo probe of Bcrp (abcg2).     

Upregulated promitogenic signaling via cytokines and growth factors: potential mechanism of robust liver tissue repair in calorie-restricted rats upon toxic challenge.

Previously we reported that moderate calorie restriction or diet restriction (DR, calories reduced by 35% for 21 days) in male Sprague-Dawley rats protects from a lethal dose of thioacetamide (TA). DR rats had 70% survival compared with 10% in rats fed ad libitum (AL) because of timely and adequate compensatory liver cell division and tissue repair in the DR rats. Further investigation of the mechanisms indicate that enhanced promitogenic signaling plays a critical role in this stimulated tissue repair. Expression of stimulators of promitogenic signaling interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS), hepatocyte growth factor (HGF), transforming growth factor-alpha (TGF-alpha), and epidermal growth factor receptor (EGFR) were studied during liver tissue repair after TA-induced liver injury. Plasma IL-6 was significantly higher in the DR rats, with 6-fold higher expression at 48 h after TA administration. Immunohistochemical localization revealed significantly higher expression of IL-6 in the hepatic sinusoidal endothelium of DR rats. Expression of TGF-alpha and HGF was consistently higher in the livers of DR rats from 36 to 72 h. EGFR, which serves as a receptor for TGF-alpha, was higher in DR rats before TA administration and remained higher till 48 h after TA intoxication. DR-induced 2-fold increase in hepatic iNOS activity is consistent with early cell division in DR rats after TA challenge. These data suggest that the reason behind the higher liver tissue repair after TA-induced hepatotoxicity in DR rats is timely and higher expression of the growth stimulatory cytokines and growth factors. It appears that the physiological effects of DR make the liver cells vigilant and prime the liver tissue promptly for liver regeneration through promitogenic signaling upon toxic challenge.     

Propiconazole-induced cytochrome P450 gene expression and enzymatic activities in rat and mouse liver

Propiconazole is a N-substituted triazole used as a fungicide on fruits, grains, seeds, hardwoods, and conifers. In the present study, propiconazole was examined for its effects on the expression of hepatic cytochrome P450 genes and on the activities of P450 enzymes in male Sprague-Dawley rats and male CD-1 mice. Rats and mice were administered propiconazole by gavage daily for 14 days at doses of 10, 75, and 150 mg/kg body weight/day. Quantitative real time RT-PCR assays of rat hepatic RNA samples from animals treated at the 150 mg/kg body weight/day dose revealed significant mRNA overexpression of the following genes compared to control: CYP1A2 (1.62-fold), CYP2B1 (10.8-fold), CYP3A1/CYP3A23 (2.78-fold), and CYP3A2 (1.84-fold). In mouse liver, propiconazole produced mRNA overexpression of Cyp2b10 (2.39-fold) and Cyp3a11 (5.19-fold). mRNA expression of CYP1A1 was not detected in liver tissues from treated or controls animals from either species. Propiconazole significantly induced both pentoxyresorufin O-dealkylation (PROD) and methoxyresorufin O-dealkylation (MROD) activities in both rat and mouse liver at the 150 mg/kg body weight/day and 75 mg/kg body weight/day doses. In summary, these results indicated that propiconazole induced CYP1A2 in rat liver and CYP2B and CYP3A families of isoforms in rat and mouse liver.     

Prior administration of a low dose of thioacetamide protects type 1 diabetic rats from subsequent administration of lethal dose of thioacetamide

Previously, we reported that an ordinarily non-lethal dose of thioacetamide (TA, 300 mg/kg) causes 90% mortality in type 1 diabetic rats due to inhibited liver tissue repair, whereas 30 mg TA/kg allows 100% survival due to stimulated although delayed tissue repair. Objective of this investigation was to test whether prior administration of a low dose of TA (30 mg/kg) would lead to sustainable stimulation of liver tissue repair in type 1 diabetic rats sufficient to protect from a subsequently administered lethal dose of TA. Therefore, in the present study, the hypothesis that preplacement of tissue repair by a low dose of TA (30 mg TA/kg, ip) can reverse the hepatotoxicant sensitivity (autoprotection) in type 1 diabetic rats was tested. Preliminary studies revealed that a single intraperitoneal (ip) administration of TA causes 90% mortality in diabetic rats with as low as 75 mg/kg. To establish an autoprotection model in diabetic condition, diabetic rats were treated with 30 mg TA/kg (priming dose). Administration of priming dose stimulated tissue repair that peaked at 72h, at which time these rats were treated with a single ip dose of 75 mg TA/kg. Our results show that tissue repair stimulated by the priming dose enabled diabetic rats to overexpress, calpastatin, endogenous inhibitor of calpain, to inhibit calpain-mediated progression of liver injury induced by the subsequent administration of lethal dose, resulting in 100% survival. Further investigation revealed that protection observed in these rats is not due to decreased bioactivation. These studies underscore the importance of stimulation of tissue repair in the final outcome of liver injury (survival/death) after hepatotoxicant challenge. Furthermore, these results also suggest that it is possible to stimulate tissue repair in diabetics to overcome the enhanced sensitivity of hepatotoxicants.     

P-glycoprotein has differential effects on the disposition of statin acid and lactone forms in mdr1a/b knockout and wild-type mice.

In the present study we examined the disposition of atorvastatin, lovastatin, and simvastatin in acid and lactone forms and pravastatin in acid form in multidrug-resistant gene (mdr1a/b) knockout (KO), and wild-type (WT) mice. Each statin was administered s.c. to mdr1a/b KO and WT mice at 3.0 mg/kg (n > or = 3 mice/time point). Blood, brain, and liver samples were harvested at 0, 0.5, 1.5, and 3 h postdose. Plasma and tissue concentrations of the acid and lactone (only the acid form was determined for pravastatin) were determined using a liquid chromatography-mass spectrometry method. Both lactone and acid were observed in plasma when lactones were administered, but only acids were detected when the acid forms were administered. The plasma and liver concentrations of acid or lactone were similar between the KO and WT mice. Two- to 23-fold higher concentrations were observed in liver than in plasma, suggesting potential uptake transporters involved. A significantly higher (p < 0.05) brain penetration in the KO compared with the WT mice was observed for lovastatin acid (but the brain/plasma ratio was low for both KO and WT mice) and lactone and simvastatin lactone but not for atorvastatin or pravastatin. The present results suggest that mouse P-glycoprotein does not affect the lactone-acid interconversion or liver-plasma distribution. Furthermore, P-glycoprotein plays a limited role in restricting the brain penetration of the acid forms of atorvastatin, pravastatin, simvastatin, lovastatin, and atorvastatin lactone but may limit the brain availability of the lactone forms of simvastatin and lovastatin.     

Nonalcoholic steatohepatitic (NASH) mice are protected from higher hepatotoxicity of acetaminophen upon induction of PPARalpha with clofibrate

The objective was to investigate if the hepatotoxic sensitivity in nonalcoholic steatohepatitic mice to acetaminophen (APAP) is due to downregulation of nuclear receptor PPARalpha via lower cell division and tissue repair. Male Swiss Webster mice fed methionine and choline deficient diet for 31 days exhibited NASH. On the 32nd day, a marginally toxic dose of APAP (360 mg/kg, ip) yielded 70% mortality in steatohepatitic mice, while all non steatohepatitic mice receiving the same dose survived. (14)C-APAP covalent binding, CYP2E1 protein, and enzyme activity did not differ from the controls, obviating increased APAP bioactivation as the cause of amplified APAP hepatotoxicity. Liver injury progressed only in steatohepatitic livers between 6 and 24 h. Cell division and tissue repair assessed by (3)H-thymidine incorporation and PCNA were inhibited only in the steatohepatitic mice given APAP suggesting that higher sensitivity of NASH liver to APAP-induced hepatotoxicity was due to lower tissue repair. The hypothesis that impeded liver tissue repair in steatohepatitic mice was due to downregulation of PPARalpha was tested. PPARalpha was downregulated in NASH. To investigate whether downregulation of PPARalpha in NASH is the critical mechanism of compromised liver tissue repair, PPARalpha was induced in steatohepatitic mice with clofibrate (250 mg/kg for 3 days, ip) before injecting APAP. All clofibrate pretreated steatohepatitic mice receiving APAP exhibited lower liver injury, which did not progress and the mice survived. The protection was not due to lower bioactivation of APAP but due to higher liver tissue repair. These findings suggest that inadequate PPARalpha expression in steatohepatitic mice sensitizes them to APAP hepatotoxicity.     

Dose–response involvement of constitutive androstane receptor in mouse liver hypertrophy induced by triazole fungicides

To clarify the dose–response relationship between constitutive androstane receptor (CAR) activity and induction of cytochrome P450 2B (CYP2B) expression and hypertrophy by triazole fungicides in mouse liver, three dose levels of cyproconazole (Cypro), tebuconazole (Teb), fluconazole (Flu), and phenobarbital (PB), a typical CYP2B inducer, were administrated in diet to male wild-type (WT) and CAR-knockout (CARKO) mice for one week. In WT mice, all compounds dose-dependently induced liver weight increases and hepatocellular hypertrophy accompanied by CYP2B expression. In CARKO mice, these effects were not induced by PB, while Cypro or Flu induced these effects only at the highest dose. Dose-dependent liver hypertrophy was detected in CARKO mice treated with Teb, but at the lowest dose the intensity was weakened compared to WT mice. The present results indicate that Cypro and Flu mainly induced CAR-mediated liver hypertrophy, while Teb slightly involved CAR. The involvement of CAR in triazole-induced liver hypertrophy was dose-responsive. In addition, all three triazoles have non-CAR-mediated liver hypertrophy pathways, indicating that the hypertrophy induced by these triazoles differs from that of PB.     

Hepatoprotective role of endogenous interleukin-13 in a murine model of acetaminophen-induced liver disease

Recent evidence suggests that a deficiency in one or more hepatoprotective regulatory mechanisms may contribute to determining susceptibility in drug-induced liver disease. In the present study, we investigated the role of interleukin (IL)-13 in acetaminophen (APAP)-induced liver disease (AILD). Following APAP (200 mg/kg) administration to male C57BL/6 wild-type (WT) mice, hepatotoxicity developed up to 24 h post-APAP, with a concomitant increase in serum IL-13 concentration. Pretreatment of these mice with an IL-13-neutralizing antibody exacerbated liver injury, as did APAP administration to IL-13 knockout (KO) mice in comparison to WT mice. No difference was observed in either overall APAP-protein adduct formation or liver glutathione levels between KO and WT mice following APAP administration, suggesting that the increased susceptibility of IL-13 KO mice to AILD was not due to enhanced APAP bioactivation but rather injurious downstream events. In this regard, multiplex antibody arrays were used to identify potential IL-13-regulated biomarkers, including various cytokines and chemokines, as well as nitric oxide (NO), associated with AILD that were present at higher concentrations in the sera of APAP-treated IL-13 KO mice than in WT mice. Subsequent inhibition studies determined interferon-gamma, NO, neutrophils, natural killer cells, and natural killer cells with T-cell receptors had pathologic roles in AILD in IL-13 KO mice. Taken together, these results suggest that IL-13 is a critical hepatoprotective factor modulating the susceptibility to AILD and may provide hepatoprotection, in part, by down-regulating protoxicant factors and cells associated with the innate immune system.