Coordinated expression of multidrug resistance-associated proteins (Mrps) in mouse liver during toxicant-induced injury.

Following acute chemical injury, hepatocytes are generally more resistant to toxicant re-exposure. Alterations in expression of hepatobiliary transport systems may contribute to this resistance by preventing accumulation of potentially toxic chemicals. Previous data demonstrate the concomitant reduction of uptake transporter and induction of efflux transporter mRNA during chemical liver injury. The present study further characterizes the expression of multidrug resistance-associated proteins 1-4 (Mrp1-4), breast cancer resistance protein (Bcrp) and sodium-taurocholate co-transporting polypeptide (Ntcp) in mouse liver following administration of the hepatotoxicants acetaminophen (APAP) and carbon tetrachloride (CCl4). Mice received hepatotoxic doses of APAP (400 mg/kg), CCl4 (10 or 25 microl/kg), or vehicle, ip. Livers were collected at 6, 24, and 48 h for Western blot quantification and immunofluorescence analysis. Protein expression of Bcrp was unchanged with treatment. Ntcp levels were preserved in APAP-exposed livers and reduced to 30-50% of control after CCl4. Conversely, Mrp1-4 expression was differentially up-regulated. CCl4 increased Mrp1 (3.5-fold), Mrp2 (1.4-fold), and Mrp4 (26-fold) while reducing Mrp3 levels to 20% of control. Administration of APAP enhanced expression of Mrp2 (1.6-fold), Mrp3 (3.5-fold), and Mrp4 (16-fold). Immunostaining of liver sections obtained 48 h after hepatotoxicant treatment confirmed expression patterns of a subset of transporters (Bcrp, Ntcp, Mrp3, and Mrp4). Double immunofluorescence imaging demonstrated the simultaneous down-regulation of Ntcp and up-regulation of Mrp4 in hepatocytes adjacent to the central vein after CCl4. Altered expression of transporters may reduce the overall chemical burden of an injured liver during recovery and contribute to the resistance of hepatocytes to subsequent toxicant exposure.     

Selective role for tumor necrosis factor-α, but not interleukin-1 or Kupffer cells, in down-regulation of CYP3A11 and CYP3A25 in livers of mice infected with a noninvasive intestinal pathogen

Hepatic cytochrome P450 (P450) gene and protein expression are modulated during inflammation and infection. Oral infection of C57BL/6 mice with Citrobacter rodentium produces mild clinical symptoms while selectively regulating hepatic P450 expression and elevating levels of proinflammatory cytokines. Here, we explored the role of cytokines in the regulation of hepatic P450 expression by orally infecting tumor necrosis factor-α (TNFα) receptor 1 null mice (TNFR1−/−), interleukin-1 (IL1) receptor null mice (IL1R1−/−), and Kupffer cell depleted mice with C. rodentium. CYP4A mRNA and protein levels and flavin monooxygenase (FMO)3 mRNA expression levels were down-regulated, while CYP2D9 and CYP4F18 mRNAs remained elevated during infection in wild-type, receptor knockout, and Kupffer cell depleted mice. CYPs 3A11 and 3A25 mRNA levels were down-regulated during infection in wild-type mice but not in TNFR1−/− mice. Consistent with this observation, CYPs 3A11 and 3A25 were potently down-regulated in mouse hepatocytes treated with TNFα. Oral infection of IL1R1−/− mice and studies with mouse hepatocytes indicated that IL1 does not directly regulate CYP3A11 or CYP3A25 expression. Uninfected mice injected with clodronate liposomes had a significantly reduced number of Kupffer cells in their livers. Infection increased the Kupffer cell count, which was attenuated by clodronate treatment. The P450 mRNA and cytokine levels in infected Kupffer cell depleted mice were comparable to those in infected mice receiving no clodronate. The results indicate that TNFα is involved in the regulation of CYPs 3A11 and 3A25, but IL1β and Kupffer cells may not be relevant to hepatic P450 regulation in oral C. rodentium infection.     

Minimal role of hepatic transporters in the hepatoprotection against LCA-induced intrahepatic cholestasis.

The multidrug resistance-associated proteins (Mrps) are a family of adenosine triphosphate-dependent transporters that facilitate the movement of various compounds, including bile acids, out of hepatocytes. The current study was conducted to determine whether induction of these transporters alters bile acid disposition as a means of hepatoprotection during bile acid-induced cholestasis. Lithocholic acid (LCA) was used to induce intrahepatic cholestasis. C57BL/6 mice were pretreated with corn oil (CO) or known transporter inducers, phenobarbital (PB), oltipraz (OPZ), or TCPOBOP (TC) for 3 days prior to cotreatment with LCA and inducer for 4 days. Histopathology revealed that PB and TC pretreatments provide a protective effect from LCA-induced toxicity, whereas OPZ pretreatment did not. Both PB/LCA and TC/LCA cotreatment groups also had significantly lower alanine aminotransferase values than the LCA-only group. In TC/LCA cotreated mice compared with LCA only, messenger RNA (mRNA) expression of uptake transporters Ntcp and Oatp4 was significantly increased, as were sinusoidal efflux transporters Mrp3 and Mrp4. Although in PB/LCA cotreated mice, the only significant change compared with LCA-only treatment was an increase in uptake transporter Oatp4. Oatp1 was reduced in all groups compared with CO controls. No significant changes in mRNA expression were observed in Oatp2, Bsep, Mrp2, Bcrp, Mrp1, Mrp5, or Mrp6. Mrp4 protein expression was induced in the OPZ/LCA and TC/LCA cotreated groups, whereas Mrp3 protein levels remained unchanged between groups. Protein expression of Mrp1 and Mrp5 was increased in the unprotected LCA-only and OPZ/LCA mice. Thus, transporter expression did not correlate with histologic hepatoprotection, however, there was a correlation between hepatoprotection and significantly reduced total liver bile acids in the PB/LCA and TC/LCA cotreated mice compared with LCA only. In conclusion, changes in transporter expression did not correlate with hepatoprotection, and therefore, transport may not play a critical role in the observed hepatoprotection from LCA-induced cholestasis in the C57BL/6 mouse.     

Modulation of the expression of ABC transporters in murine (J774) macrophages exposed to large concentrations of the fluoroquinolone antibiotic moxifloxacin

Long-term exposure to pharmacological agents can select for cells that overexpress efflux transporters. We previously showed that mouse J774 macrophages cultivated for a prolonged period of time with toxic concentrations of the fluoroquinolone ciprofloxacin overexpress the efflux transporter Mrp4 and display a reduced accumulation of this antibiotic, but no change in the accumulation of moxifloxacin, a closely related molecule (Antimicrob. Agents Chemother. [2006] 50, 1689-1695 and [2009] 53, 2410-2416). Because of this striking difference between the two fluoroquinolones, we have now examined the modifications in the expression of ABC efflux transporters induced by the prolonged exposure of J774 macrophages to high concentrations of moxifloxacin. The resulting cell line showed (i) no difference in the accumulation of moxifloxacin but an increased accumulation and decreased efflux of ciprofloxacin; (ii) an overexpression of the multidrug transporters Abcb1a (P-gp), Abcc2 (Mrp2) and Abcg2 (Bcrp1), and a decreased expression of Abcc4 (Mrp4). While P-gp and Bcrp1 were functional, they did not modify the cellular accumulation of fluoroquinolones. The data show that exposing cells to high concentrations of a drug that is not affected by active efflux can trigger a pleiotropic response leading to a modulation in the expression of several transporters. These changes, however, are not sufficient to protect cells against the toxicity that fluoroquinolones may exert at large concentrations. They could also cause unanticipated drug interactions in vivo, should the drug exposure grossly exceed what is anticipated from its current registered use.     

Protective role of Kupffer cells in acetaminophen-induced hepatic injury in mice

Hepatic injury induced by various toxic agents, including acetaminophen (APAP), has been attributed, in part, to the production of proinflammatory cytokines and other mediators by resident Kupffer cells within the liver. However, recent evidence from our laboratory has demonstrated that hepato-protective factors, such as interleukin (IL)-10 and cyclooxygenase-derived mediators, are also upregulated in response to hepatic damage to help protect against exacerbated injury, and Kupffer cells have been suggested to be a source of these modulatory factors. In other models, Kupffer cells also serve important regulatory functions in pathophysiological states of the liver. Therefore, we reevaluated the role of Kupffer cells in a murine model of APAP-induced liver injury using liposome-entrapped clodronate (liposome/clodronate) as an effective Kupffer cell-depleting agent. We show that in contrast to pretreatment of mice with a widely used macrophage inhibitor, gadolinium chloride, which did not deplete Kupffer cells but moderately protected against APAP-induced hepatotoxicity as reported previously, the intravenous injection of liposome/clodronate caused nearly complete elimination of Kupffer cells and significantly increased susceptibility to APAP-induced liver injury as compared with mice pretreated with empty liposomes. This increased susceptibility was apparently unrelated to the metabolism of APAP since liposome/clodronate pretreatment did not alter APAP-protein adduct levels. Instead, Kupffer cell depletion by liposome/clodronate led to significant decreases in the levels of hepatic mRNA expression of several hepato-regulatory cytokines and mediators, including IL-6, IL-10, IL-18 binding protein and complement 1q, suggesting that Kupffer cells are a significant source for production of these mediators in this model. Our findings indicate that, in addition to their protoxicant activities, Kupffer cells can also have an important protective function in the liver through the production of a variety of modulatory factors which may counteract inflammatory responses and/or stimulate liver regeneration.     

Up-regulation of Mrp4 expression in kidney of Mrp2-deficient TR- rats

Multidrug resistance-associated proteins (Mrps) are a group of ATP-dependent efflux transporters for organic anions. Mrp2 and Mrp4 are co-localized to the apical (brush-border) membrane domain of renal proximal tubules, where they may function together in the urinary excretion of organic anions. Previous reports showed that urinary excretion of some organic anions is not impaired in transport-deficient (TR-) rats, which lack Mrp2, suggesting that up-regulation of other transporter(s) may compensate for the loss of Mrp2 function. The purpose of this study was to determine whether Mrp4 expression in kidney is altered in TR- rats. Mrp4 mRNA expression was quantified using the high-throughput branched DNA signal amplification assay. Mrp4 protein expression was determined by Western blot and immunohistochemical analysis. Mrp4 mRNA in kidney of TR- rats was 100% higher than normal Wistar rats. Western blot analysis showed a 200% increase in Mrp4 protein expression in kidney of the mutant rats compared to normal rats. Immunohistochemical analysis of Mrp4 protein demonstrated apical localization of Mrp4 on renal proximal tubules, and that the immunoreactivity was more intense in kidney sections from TR- rats than those from normal rats. In summary, the results of the present study demonstrate that renal Mrp4 expression is up-regulated in TR- rats, which may explain why urinary excretion of some organic anions remains normal in the mutant rats.     

Biliary efflux transporters involved in the clearance of rosuvastatin in sandwich culture of primary rat hepatocytes

Rosuvastatin (a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor) has been shown to be excreted mostly unchanged into the bile; interactions on the level of hepatic apical efflux transporters may represent a risk of liver toxicity. So far, controversial and insufficient data are available concerning transporters involved in the elimination process. This study was designed to elucidate, which transporters take part in the biliary clearance of rosuvastatin using sandwich-cultured primary rat hepatocytes. The canalicular efflux of rosuvastatin was measured in the presence of inhibitors: Ko 134, mitoxanthrone, novobiocin for breast cancer resistance protein (Bcrp); verapamil for multidrug resistance protein (Mdr1); benzbromarone, sulfasalazine, probenecid for multidrug resistance associated protein (Mrp 2); and cyclosporine A, glibenclamide, troglitazone for bile salt export pump (Bsep). Mrp2 inhibitors decreased the biliary efflux of rosuvastatin most potently by 78.9%, 35%, 54.1%; benzbromarone, probenecid, sulfasalazine, respectively, while Bcrp and Bsep inhibitors showed much less effect (29.1%, 23.0% ,30.0%; Ko 134, mitoxanthrone, novobiocin, respectively, and 32.6%, 29.3%, 20.6%, glibenclamide, cyclosporine A, troglitazone, respectively). The marked decline of canalicular transport by Mrp2 inhibitors suggests major role of Mrp2 in this process; however, Bcrp and Bsep might also contribute to the biliary elimination of rosuvatatin in sandwich-cultured rat hepatocytes.     

Effects of chronic renal failure on brain drug transporters in rats

Studies demonstrated that chronic renal failure (CRF) affects the expression and activity of intestinal, hepatic, and renal drug transporters. Such drug transporters are expressed in brain cells and at the blood-brain barrier (BBB), where they limit the entry and distribution of drugs in the brain. Perturbations in brain drug transporter equilibrium by CRF could lead to central drug toxicity. This study evaluates how CRF affects BBB drug transporters using a 5/6 nephrectomized rat model. Protein and mRNA expression of influx transporters [organic anion-transporting polypeptide (Oatp), organic anion transporter (Oat)] and efflux transporters [P-glycoprotein (P-gp), multidrug resistance-related protein (Mrp), breast cancer resistance protein (Bcrp)] were measured in CRF and control rat brain. Intracerebral accumulation of radiolabeled benzylpenicillin, digoxin, doxorubicin, and verapamil was used to evaluate BBB drug permeability. Protein expression of the transporters was evaluated in rat brain endothelial cells (RBECs) and astrocytes incubated with control and CRF rat serum. We demonstrated significant decreases (30-50%) in protein and mRNA levels of Bcrp, Mrp2 to -4, Oat3, Oatp2 and -3, and P-gp in CRF rat brain biopsies, as well as in astrocytes and RBECs incubated with CRF serum. These decreases did not correlate with in vivo changes because BBB permeability of benzylpenicillin was decreased by 30% in CRF rats, whereas digoxin, doxorubicin, and verapamil permeabilities were unchanged. It thus seems that even with decreased drug transporters, BBB integrity and function is conserved in CRF.     

Time-course activities of Oct1, Mrp3, and cytochrome P450s in cultures of cryopreserved rat hepatocytes

The organic cation transporter 1 (Oct1) has been shown to be one of the most abundant uptake transporters responsible for the uptake of xenobiotics from the sinusoidal blood across the basolateral membrane of hepatocytes. On the same membrane the multidrug resistance-associated protein 3 (Mrp3) mediates the efflux of xenobiotics or their metabolites from the hepatocytes to the blood allowing their systemic exposure. In the present study we investigated the expression and activity of Oct1 and Mrp3 in suspensions and in monolayer- and sandwich cultures, and activities of CYP2B1/2, 2D1, and 3A1 in monolayer- and sandwich cultures of cryopreserved rat hepatocytes. Oct1-mediated active uptake of 10 μM [(3)H]-1-methyl-4-phenylpyridinium (MPP+) into hepatocytes was assessed in the presence of quinidine (1 mM). The results showed the presence of active uptake of MPP+ in suspended hepatocytes (~91 pmol/min/mg protein). In hepatocytes in cultures (monolayer and sandwich) a time-dependent decrease in MPP+ uptake was observed from day 0 to 4, from 80 to 90 pmol/min/mg protein at day 0 to ca. 17 pmol/min/mg protein at day 4. Mrp3 activity in suspensions and in monolayer- and sandwich cultures were investigated by measuring the efflux of [(3)H]-taurocholate from hepatocytes in the presence of the Mrp3 inhibitor taurolithocholate-3-sulfate (TLC-S) (500μM). Cells in suspensions showed efflux of taurocholate by an active transport mechanism indicating Mrp3 activity. Experiments in monolayer- and sandwich cultures also showed Mrp3 activity at day 0 and 1 in culture whereas experiments performed at day 2-4 showed no difference in efflux of taurocholate in the presence or absence of TLC-S, suggesting an absence of Mrp3 activity. The time-dependent decrease in Oct1 activity from day 0 to day 4 in cultures was confirmed by qPCR data also showing a time-dependent decrease in mRNA expression, whereas qPCR data did not support the observed time-dependent decrease in Mrp3 activity in cultures. Time-course activities of CYP2B1/2, 2D1, and 3A1 were also investigated by using bupropion, bufuralol, and midazolam as respective substrates. Activities of CYP2D1 and 3A1 were reduced by ~75% and ~80%, respectively, from day 0 to day 4 in cultures, whereas activity of CYP2B1/2 was reduced by ~50% from day 0 to day 4.     

Lipopolysaccharide-mediated regulation of hepatic transporter mRNA levels in rats.

The function of hepatic transporters is to move organic substances across sinusoidal and canalicular membranes. During extrahepatic cholestasis, transporters involved in the movement of substances from blood to bile, such as sodium/taurocholate-cotransporting polypeptide (Ntcp) and multidrug resistance protein 2 (Mrp2), are down-regulated, whereas others that transport chemicals from liver to blood, such as Mrp3, are up-regulated. Unlike extrahepatic cholestasis, where transporter expression responds to the stress of accumulating bile constituents, lipopolysaccharide (LPS)-induced intrahepatic cholestasis may be directly caused by alterations in transporter expression. The aim of this study was to quantitatively determine the effect of LPS on transporter expression and study the mechanism(s) by which LPS alters mRNA levels of major hepatic transporters in Sprague-Dawley rats. Hepatic mRNA levels of Mrp2, Mrp6, multiple drug resistance protein 1a (Mdr1a), organic anion-transporting polypeptide 1 (Oatp1), Oatp2, Oatp4, Ntcp, bile salt export pump, organic cation transporter 1 (Oct1), and organic anion transporter 3 (Oat3) were dramatically decreased, beginning approximately 6 h after LPS administration, whereas Mrp5 and Oat2 levels were unchanged. In contrast, LPS increased mRNA levels of Mrp1, Mrp3, and Mdr1b concurrently with the down-regulated transporters. Pretreatment with dexamethasone, which decreases the release of cytokines, reversed the reduction of Mdr1a, Oatp1, Oatp2, Oct1, and Ntcp mRNA following LPS administration. Furthermore, dexamethasone pretreatment also prevented the LPS-mediated increase in Mrp1, Mrp3, and Mdr1b, whereas pretreatment with aminoguanidine or gadolinium chloride, an inhibitor of inducible nitric oxide synthetase and a Kupffer cell toxicant, respectively, had no effect on the LPS-induced changes. The concurrent repression and induction of various transporters, as well as dexamethasone abatement of both LPS-mediated repression and induction, indicates that these responses may be mediated through similar pathways.     

Dose-dependent localization of TCDD in isolated centrilobular and periportal hepatocytes

Dose-response relationships for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) suggest a differential sensitivity of liver cell types to the induction of cytochrome P450 gene expression, and that the induction of hepatic protein CYP1A2 causes sequestration of TCDD. In addition, immunolocalization of hepatic CYP1A1/1B1/1A2 proteins is not uniform after exposure to TCDD. The mechanism for the regio-specific induction of hepatic P450s by TCDD is unknown, but may involve the differential distribution of participants in the AhR-mediated pathway and/or regional P450 isozymes, as well as, non-uniform distribution/sequestration of TCDD. Therefore, this study examined the effects of TCDD in unfractionated, centrilobular and periportal hepatocytes isolated from female Sprague-Dawley rats acutely exposed (3 days) to a single oral dose of 0.01-10.0 microg [3H]TCDD/kg. A dose- dependent increase in concentration of TCDD was accompanied by a dose- dependent increase in CYP1A1, CYP1A2, and CYP1B1 mRNA expression and associated enzymes in all liver-cell populations. Centrilobular hepatocytes showed a 2.7- to 4.5-fold higher concentration of TCDD as compared to the periportal hepatocytes at doses up to 0.3 microg TCDD/kg. Centrilobular hepatocytes also exhibited an elevated MROD activity as compared to the periportal hepatocytes at doses up to 0.3 microg TCDD/kg. Furthermore, centrilobular hepatocytes showed an elevated concentration of induced CYP1A2 and CYP1B1 mRNA as compared to periportal hepatocytes within the 0.01- and 0.3-microg TCDD/kg- treatment groups. This is the first study to demonstrate that a dose- dependent difference in distribution of TCDD exists between centrilobular and periportal cells that might be related to regional differences in P450 induction.      

Gender-specific reduction of hepatic Mrp2 expression by high-fat diet protects female mice from ANIT toxicity

Emerging evidence suggests that feeding a high-fat diet (HFD) to rodents affects the expression of genes involved in drug transport. However, gender-specific effects of HFD on drug transport are not known. The multidrug resistance-associated protein 2 (Mrp2, Abcc2) is a transporter highly expressed in the hepatocyte canalicular membrane and is important for biliary excretion of glutathione-conjugated chemicals. The current study showed that hepatic Mrp2 expression was reduced by HFD feeding only in female, but not male, C57BL/6J mice. In order to determine whether down-regulation of Mrp2 in female mice altered chemical disposition and toxicity, the biliary excretion and hepatotoxicity of the Mrp2 substrate, α-naphthylisothiocyanate (ANIT), were assessed in male and female mice fed control diet or HFD for 4 weeks. ANIT-induced biliary injury is a commonly used model of experimental cholestasis and has been shown to be dependent upon Mrp2-mediated efflux of an ANIT glutathione conjugate that selectively injures biliary epithelial cells. Interestingly, HFD feeding significantly reduced early-phase biliary ANIT excretion in female mice and largely protected against ANIT-induced liver injury. In summary, the current study showed that, at least in mice, HFD feeding can differentially regulate Mrp2 expression and function and depending upon the chemical exposure may enhance or reduce susceptibility to toxicity. Taken together, these data provide a novel interaction between diet and gender in regulating hepatobiliary excretion and susceptibility to injury.     

The accumulation and efflux of lead partly depend on ATP-dependent efflux pump–multidrug resistance protein 1 and glutathione in testis Sertoli cells

Since lead accumulation is toxic to cells, its excretion is crucial for organisms to survive the toxicity. In this study, mouse testis sertoli (TM4) and Mrp1 lower-expression TM4-sh cells were used to explore the lead accumulation characteristics, and the role of ATP-dependent efflux pump–multidrug resistance protein 1 (Mrp1) in lead excretion. TM4 cells possess Mrp-like transport activity. The expression levels of mrp1 mRNA and Mrp1 increased after lead treatments at first and then decreased. The maximum difference of relative mRNA expression reached 10 times. In the presence of lead acetate, the amount of cumulative lead in TM4-sh was much higher than that in TM4. After the treatment with lead acetate at 10–40 μM for 12 h or 24 h, the differences were about 2–8 times. After with the switch to lead-free medium, the cellular lead content in TM4-sh remains higher than that in TM4 cells at 1,3, 6, and 9 h time points (P < 0.01). Energy inhibitor sodium azide, Mrp inhibitors MK571 and glutathione (GSH) biosynthesis inhibitor BSO could block lead efflux from TM4 cells significantly. These results indicate that lead excretion may be mediated by Mrp1 and GSH in TM4 cells. Mrp1 could be one of the important intervention points for lead detoxification.     

Ischemia-reperfusion of rat livers decreases liver and increases kidney multidrug resistance associated protein 2 (Mrp2).

Hepatic ischemia-reperfusion (IR) injury during liver transplantation can lead to cholestasis and remote organ dysfunction. Multidrug resistance-associated proteins (Mrps) are efflux transporters known to transport a diverse set of substrates, such as amphipathic chemicals, organic anions, and endogenous molecules. The purpose of this study was to determine the effect of hepatic IR injury on the expression of Mrps in rat liver and kidney. Male Sprague-Dawley rats were subjected to 60 min of partial hepatic ischemia. At various times after reperfusion (0, 3, 6, 24, and 48 h), the ischemic lobes were harvested as well as kidneys. RNA and protein expression of Mrps in livers and kidneys were determined by the branched DNA method, Western blot analysis, and tissue immunofluorescence. Mrp2 mRNA and protein expression in livers decreased after IR. Conversely, Mrp2 mRNA and protein expression in kidneys increased after IR. Mrp3 mRNA expression, and Mrp4 mRNA and protein expression in kidneys transiently increased after IR. The intensity of immunofluorescent staining of Mrp2 corresponded to changes in Mrp2 expression in livers and kidneys after IR as detected by Western blot analysis and was localized to the apical membrane domain in both tissues. These results demonstrate that after hepatic IR, downregulation of hepatic Mrp2 and upregulation of renal Mrp2 occur. These decreases in hepatic Mrp2 may contribute to cholestasis, yet increases in kidney may protect from oxidative stress and/or inflammation after hepatic IR.     

A histochemical and pathological study on the interrelationship between TCDD-induced AhR expression, AhR activation, and hepatotoxicity in mice

The objective of this study was to establish a correlation and interrelationship between aryl hydrocarbon receptor (AhR) and hepatotoxicity induced by TCDD. Young male ICR mice were exposed to TCDD via dermal exposure at doses of 0, 2.5, 25, and 125 ng TCDD twice weekly for 20 wk. Histopathological examination revealed a classic pattern and dose-dependent pathological changes in sinusoidal dilatations, hepatocellular swelling and degeneration, fatty infiltration, and hepatocellular necrosis. Immunochemical staining for AhR also demonstrated that the AhR-positive hepatocytes were centrilobular in location, especially with those cells cuffing the central vein. With exposures to TCDD, the number of AhR-positive cells increased with dose. Furthermore, we also demonstrated all the hepatocytes that exhibited pathological changes (e.g., fatty infiltration or necrosis) were AhR-positive. Depletion in AhR (AhR removal after activation) in many centrilobular hepatocytes, with disappearance of the AhR positive cuffing, was observed in the high TCDD exposed animals. AhR activation was also evident by the increase in CYP 1A2 expression in many centrilobular hepatocytes, especially those exposed to high doses of TCDD. Studies in the past, with experiments performed separately, could only suggest the association of AhR expression and hepatocellular toxicity. By examining the AhR expression, AhR activation (CYP 1A2 expression upregulation), and hepatocellular pathology together, it was possible to correlate these factors in the same animal and even in the same cells. Our finding provided direct evidence on the interaction and causal relationship between AhR activation and hepatic toxicity.