Regulation of macrophage cholesterol efflux through hydroxymethylglutaryl-CoA reductase inhibition: a role for RhoA in ABCA1-mediated cholesterol efflux

The cholesterol biosynthetic pathway produces numerous signaling molecules. Oxysterols through liver X receptor (LXR) activation regulate cholesterol efflux, whereas the non-sterol mevalonate metabolite, geranylgeranyl pyrophosphate (GGPP), was recently demonstrated to inhibit ABCA1 expression directly, through antagonism of LXR and indirectly through enhanced RhoA geranylgeranylation. We used HMG-CoA reductase inhibitors (statins) to test the hypothesis that reduced synthesis of mevalonate metabolites would enhance cholesterol efflux and attenuate foam cell formation. Preincubation of THP-1 macrophages with atorvastatin, dose dependently (1-10 microm) stimulated cholesterol efflux to apolipoprotein AI (apoAI, 10-60%, p < 0.05) and high density lipoprotein (HDL(3)) (2-50%, p < 0.05), despite a significant decrease in cholesterol synthesis (2-90%). Atorvastatin also increased ABCA1 and ABCG1 mRNA abundance (30 and 35%, p < 0.05). Addition of mevalonate, GGPP or farnesyl pyrophosphate completely blocked the statin-induced increase in ABCA1 expression and apoAI-mediated cholesterol efflux. A role for RhoA was established, because two inhibitors of Rho protein activity, a geranylgeranyl transferase inhibitor and C3 exoenzyme, increased cholesterol efflux to apoAI (20-35%, p < 0.05), and macrophage expression of dominant-negative RhoA enhanced cholesterol efflux to apoAI (20%, p < 0.05). In addition, atorvastatin increased the RhoA levels in the cytosol fraction and decreased the membrane localization of RhoA. Atorvastatin treatment activated peroxisome proliferator activated receptor gamma and increased LXR-mediated gene expression suggesting that atorvastatin induces cholesterol efflux through a molecular cascade involving inhibition of RhoA signaling, leading to increased peroxisome proliferator activated receptor gamma activity, enhanced LXR activation, increased ABCA1 expression, and cholesterol efflux. Finally, statin treatment inhibited cholesteryl ester accumulation in macrophages challenged with atherogenic hypertriglyceridemic very low density lipoproteins indicating that statins can regulate foam cell formation.     

Differential regulation of apolipoprotein B secretion from HepG2 cells by two HMG-CoA reductase inhibitors, atorvastatin and simvastatin.

The concept that hepatic cholesterol synthesis regulates hepatocyte assembly and secretion of apoB-containing lipoproteins remains controversial. The present study was carried out in HepG2 cells to examine the regulation of apoB secretion by the HMG-CoA reductase inhibitor atorvastatin. ApoB accumulation in the media was decreased by 24% and 36% at 10 microm (P < 0.02) and 20 microm (P < 0.01) of atorvastatin, respectively. Atorvastatin inhibited HepG2 cell cholesterol synthesis by up to 96% (P < 0.001) and cellular cholesteryl ester (CE) mass by 54% (P < 0.001). Another HMG-CoA reductase inhibitor, simvastatin, decreased cellular cholesterol synthesis and CE mass by up to 96% (P < 0.001) and 52% (P < 0.001), respectively. However, in contrast to atorvastatin, simvastatin had no effect on apoB secretion. To characterize the reduction in apoB secretion by atorvastatin (10 microm), pulse-chase experiments were performed and the kinetic data were analyzed by multicompartmental modeling using SAAM II. Atorvastatin had no affect on the synthesis of apoB, however, apoB secretion into the media was decreased by 44% (P = 0.048). Intracellular apoB degradation increased proportionately (P = 0.048). Simvastatin (10 microm) treatment did not significantly alter either the secretion or intracellular degradation of apoB, relative to control. The kinetics of apoB degradation were best described by a rapidly and a slowly turning over degradation compartment. The effect of atorvastatin on apoB degradation was largely confined to the rapid compartment. Neither inhibitor affected apoB mRNA concentrations, however, both significantly increased LDL receptor and HMG-CoA reductase mRNA levels. Atorvastatin treatment also decreased the mRNA for the microsomal triglyceride transfer protein (MTP) by 22% (P < 0.02). These results show that atorvastatin decreases apoB secretion, by a mechanism that results in an enhanced intracellular degradation in apoB.     

Digoxin up-regulates MDR1 in human colon carcinoma Caco-2 cells

Because MDR1 (P-glycoprotein) plays an important role in pharmacokinetics such as absorption and excretion of xenobiotics and multidrug resistance, an understanding of the factors regulating its function and expression is important. Here, the effects of digoxin on cell sensitivity to an anticancer drug, MDR1 function, and expression were examined by assessing the growth inhibition by paclitaxel, the transport characteristics of the MDR1 substrate Rhodamine123, and the level of MDR1 mRNA, respectively, using human colon carcinoma Caco-2 cells, which are widely used as a model of intestinal epithelial cells. The sensitivity to paclitaxel, an MDR1 substrate, in Caco-2 cells pretreated with digoxin was lower than that in non-treated cells. The accumulation of Rhodamine123 was reduced by pretreatment with digoxin and its efflux was enhanced. The level of MDR1 mRNA in Caco-2 cells was increased in a digoxin concentration-dependent manner. These results taken together suggested that digoxin up-regulates MDR1 in Caco-2 cells.     

Functionalized silicon quantum dots tailored for targeted siRNA delivery

For RNA interference (RNAi) mediated silencing of the ABCB1 gene in Caco-2 cells biocompatible luminescent silicon quantum dots (SiQDs) were developed to serve as self-tracking transfection tool for ABCB1 siRNA. While the 2-3 nm sized SiQD core exhibits green luminescence, the QD surfaces are completely saturated with covalently linked 2-vinylpyridine that may electrostatically bind siRNA. For down-regulating P-glycoprotein (Pgp) expression of the ABCB1 gene the SiQDs were complexed with siRNA. The cellular uptake and allocation of SiQD-siRNA complexes in Caco-2 cells were monitored using confocal laser scanning microscopy and transmission electron microscopy. The release of siRNA to the cytoplasm was verified through real-time PCR quantification of the reduced ABCB1 mRNA level. Additional evidence was obtained from time-resolved in situ fluorescence spectroscopic monitoring of the Pgp efflux dynamics in transfected Caco-2 cells which yielded significantly reduced transporter efficiencies for the Pgp substrate Rhodamine 123.     

Cholesterol modulates P-glycoprotein activity in human peripheral blood mononuclear cells

P-glycoprotein (P-gp) is expressed in a wide range of cell types including peripheral blood mononuclear cells (PBMCs) where it may restrict intracellular accumulation of substrates like antineoplastic agents, HIV protease inhibitors, or rhodamine123. P-gp is known to be located in membrane microdomains, whose structure and function are susceptible to cholesterol alterations. This study evaluated the effect of cholesterol alteration in human PBMCs on P-gp activity. Whereas cholesterol depletion had no effect, cholesterol repletion of depleted cells significantly decreased intracellular rhodamine123 concentrations in lymphocytes to 32.2%+/-2.7 (p<0.001) and to 41.9%+/-3.5 (p<0.001) in monocytes. After cholesterol saturation of native cells intracellular rhodamine123 fluorescence decreased to 12.4%+/-1.6 (p<0.001) in lymphocytes and 12.9%+/-3.5 (p<0.001) in monocytes. These data demonstrate that elevated cellular cholesterol levels can markedly increase P-gp activity in human PBMCs.     

Influence of IL-6 on MDR and MRP-mediated multidrug resistance in human hepatoma cells.

The objective of this study was to examine effects of interleukin-6 (IL-6) on the expression and activity of the drug resistance transporters (MDR1 and MRP) in human hepatoma cell lines. Expression and activity of MDR1 and MRP transporters were examined in IL-6-treated and control HuH 7 and HepG2 cells using semi-quantitative RT-PCR analysis and by rhodamine 123 and 5-carboxyfluorescin efflux assays. Results from RT-PCR demonstrated expression of MRP3, MRP6, and MDR1 in HuH 7 cells and expression of MRP1, MRP2, MRP3, MRP6, and MDR1 in HepG2 cells. Compared with controls, treatment of HuH 7 cells with IL-6 (10 ng/mL, 24 h) resulted in a 1.8-fold increase in MRP-mediated efflux of 5-CF with a corresponding 1.5-fold induction of MRP3 mRNA levels (p < 0.05). Similarly, in HepG2 cells, a 2-fold increase in MRP functional activity and a 1.8-fold induction of MRP1 mRNA levels were seen in the IL-6 treated cells (p < 0.05). Treatment of cells with IL-6 was also found to cause significant reductions in the expression and activity of MDR1 in HuH 7 cells, but not in HepG2 cells. Our data suggest that IL-6 induces MRP expression and activity in human hepatoma cell lines. Suppressive effects of IL-6 on MDR1 expression and activity were also observed in HuH 7 cells. This underscores the importance of examining the regulation of multiple drug resistance proteins as these proteins may have opposing regulatory mechanisms in malignant cells.     

Atorvastatin induces bile acid-synthetic enzyme Cyp7a1 by suppressing FXR signaling in both liver and intestine in mice

Statins are effective cholesterol-lowering drugs to treat CVDs. Bile acids (BAs), the end products of cholesterol metabolism in the liver, are important nutrient and energy regulators. The present study aims to investigate how statins affect BA homeostasis in the enterohepatic circulation. Male C57BL/6 mice were treated with atorvastatin (100 mg/kg/day po) for 1 week, followed by BA profiling by ultra-performance LC-MS/MS. Atorvastatin decreased BA pool size, mainly due to less BA in the intestine. Surprisingly, atorvastatin did not alter total BAs in the serum or liver. Atorvastatin increased the ratio of 12α-OH/non12α-OH BAs. Atorvastatin increased the mRNAs of the BA-synthetic enzymes cholesterol 7α-hydroxylase (Cyp7a1) (over 10-fold) and cytochrome P450 27a1, the BA uptake transporters Na⁺/taurocholate cotransporting polypeptide and organic anion transporting polypeptide 1b2, and the efflux transporter multidrug resistance-associated protein 2 in the liver. Noticeably, atorvastatin suppressed the expression of BA nuclear receptor farnesoid X receptor (FXR) target genes, namely small heterodimer partner (liver) and fibroblast growth factor 15 (ileum). Furthermore, atorvastatin increased the mRNAs of the organic cation uptake transporter 1 and cholesterol efflux transporters Abcg5 and Abcg8 in the liver. The increased expression of BA-synthetic enzymes and BA transporters appear to be a compensatory response to maintain BA homeostasis after atorvastatin treatment. The Cyp7a1 induction by atorvastatin appears to be due to suppressed FXR signaling in both the liver and intestine.     

Antitumor effects of atorvastatin in the chemoprevention of rat mammary carcinogenesis

Epidemiological studies indicate that 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, or statins, play a role in inhibition of several human neoplasia including breast cancer. In this study, chemopreventive effects of atorvastatin in N-methyl-N-nitrosourea-induced mammary carcinogenesis in female rats were evaluated. Atorvastatin was administered in the diet at two concentrations: 10 mg/kg (ATOR 10) and 100 mg/kg (ATOR 100). Atorvastatin treatment began 8 days prior to carcinogen administration and subsequently continued for 15 weeks till the end of the experiment. Atorvastatin at a higher dose suppressed tumor frequency by 80.5% (P = 0.0008) and tumor incidence by 49.5% (P = 0.015), and extended latency period by 14 days (P = 0.076) when compared to the control group. Atorvastatin at a lower dose did not significantly alter tumor parameters in comparison with the control group. In the specimens of mammary tumors, atorvastatin (in the ATOR 100 group) significantly decreased mRNA expression of Bcl-2 gene but non-significantly increased Bax mRNA expression compared to control group. Atorvastatin administration did not alter serum concentration of triacylglycerols, total cholesterol, and LDL cholesterol in comparison with controls. This study is the first report on tumor suppressive effect of atorvastatin in rat mammary carcinogenesis.     

PPAR gamma ligand troglitazone lowers cholesterol synthesis in HepG2 and Caco-2 cells via a reduced concentration of nuclear SREBP-2

Cholesterol synthesis in animal cells is regulated by sterol regulatory element-binding protein (SREBP)-2. The objective of this study was to investigate whether activation of peroxisome proliferator-activatedreceptor (PPAR)-gamma influences the SREBP-2 dependent cholesterol synthesis in liver and intestinal cells. Therefore, HepG2 and Caco-2 cells were incubated with and without 10 or 30 microM of troglitazone, a synthetic PPAR gamma agonist, for 4 hrs. Incubation with 10 or 30 microM of troglitazone caused a significant, dose-dependent reduction of cholesterol synthesis in both HepG2 and Caco-2 cells (P < 0.05). HepG2 and Caco-2 cells incubated with 10 or 30 microM of troglitazone had also lower mRNA concentrations and lower nuclear protein concentrations of SREBP-2 than untreated control cells (P < 0.05). mRNA concentrations of the SREBP-2 target genes HMG-CoA reductase and LDL receptor were also reduced in HepG2 and Caco-2 cells treated with 30 microM of troglitazone compared to control cells (P < 0.05). In conclusion, this study shows that PPAR gamma activation by troglitazone lowers the cholesterol synthesis in HepG2 and Caco-2 cells by reducing the concentration of nuclear SREBP-2 and successive downregulation of its target genes involved in cholesterol synthesis.     

Expression of caveolin-1 enhances cholesterol efflux in hepatic cells

HepG2 cells were stably transfected with human caveolin-1 (HepG2/cav cells). Transfection resulted in expression of caveolin-1 mRNA, a high abundance of caveolin-1 protein, and the formation of caveolae on the plasma membrane. Cholesterol efflux from HepG2/cav cells was 280 and 45% higher than that from parent HepG2 cells when human plasma and human apoA-I, respectively, were used as acceptors. The difference in efflux was eliminated by treatment of cells with progesterone. There was no difference in cholesterol efflux to cyclodextrin. Cholesterol efflux from plasma membrane vesicles was similar for the two cell types. Transfection led to a 40% increase in the amount of plasma membrane cholesterol in cholesterol-rich domains (caveolae and/or rafts) and a 67% increase in the rate of cholesterol trafficking from intracellular compartments to these domains. Cholesterol biosynthesis in HepG2/cav cells was increased by 2-fold, and cholesterol esterification was reduced by 50% compared with parent HepG2 cells. The proliferation rate of transfected cells was significantly lower than that of non-transfected cells. Transfection did not affect expression of ABCA1 or the abundance of ABCA1 protein, but decreased secretion of apoA-I. We conclude that overexpression of caveolin-1 in hepatic cells stimulates cholesterol efflux by enhancing transfer of cholesterol to cholesterol-rich domains in the plasma membrane.     

Atorvastatin reduces lipopolysaccharide-induced expression of cyclooxygenase-2 in human pulmonary epithelial cells

Objective

To explore the effects of atorvastatin on expression of cyclooxygenase-2 (COX-2) in human pulmonary epithelial cells (A549).

Methods

A549 cells were incubated in DMEM medium containing lipopolysaccharide (LPS) in the presence or absence of atorvastatin. After incubation, the medium was collected and the amount of prostaglandin E₂ (PGE₂) was measured by enzyme-linked immunosorbent assay (ELISA). The cells were harvested, and COX-2 mRNA and protein were analyzed by RT-PCR and western-blot respectively.

Results

LPS increased the expression of COX-2 mRNA and production of PGE₂ in a dose- and time-dependent manner in A549. Induction of COX-2 mRNA and protein by LPS were inhibited by atorvastatin in a dose-dependent manner. Atorvastatin also significantly decreased LPS-induced production of PGE₂. There was a positive correlation between reduced of COX-2 mRNA and decreased of PGE₂ (r = 0.947, P < 0.05).

Conclusion

Atorvastatin down-regulates LPS-induced expression of the COX-2 and consequently inhibits production of PGE₂ in cultured A549 cells.     

荷叶碱对bel-7402细胞胆固醇代谢的影响

目的:探讨荷叶主要单体成分荷叶碱对Bel-7402细胞株胆固醇吸收、合成及酯化的影响。方法:体外培养Bel-7402细胞株,待细胞长满80%后无血清培养基饥饿细胞24h,分别加入1uM、10uM、100uM的荷叶碱及10uM的阿托伐他订,实时定量PCR和Western blotting法分别检测给药24小时后细胞内低密度脂蛋白受体(LDLR)、β-羟β-甲基戊二酰辅酶A还原酶(HMGCR)、酰基辅酶A胆固醇酰基转移酶(ACAT)的基因表达和蛋白质水平。结果:与对照组相比,荷叶碱各剂量组均能明显升高LDLR、HMGCOAR基因表达及蛋白水平,且呈剂量依赖性(P<0.01),阿托伐他汀组LDLR、HMGCOAR基因表达及蛋白水平高于荷叶碱各组(P<0.01),荷叶碱、阿托伐他汀均能降低细胞内ACAT基因表达及蛋白含量,荷叶碱中剂量组ACAT基因表达及蛋白含量均高于阿托伐他汀组(P<0.01,P<0.05),荷叶碱高剂量组ACAT基因表达高于阿托伐他汀组(P<0.05),蛋白水平两组没有显著性差异。结论:荷叶碱可能是通过抑制细胞内胆固醇的合成、抑制胆固醇酯酶的活性及升高低密度脂蛋白受体的量而起到降血脂作用。     

Rhodamine 123 as a probe of transmembrane potential in isolated rat-liver mitochondria: spectral and metabolic properties

The spectral and metabolic properties of Rhodamine 123, a fluorescent cationic dye used to label mitochondria in living cells, were investigated in suspensions of isolated rat-liver mitochondria. A red shift of Rhodamine 123 absorbance and fluorescence occurred following mitochondrial energization. Fluorescence quenching of as much as 75% also occurred. The red shift and quenching varied linearly with the potassium diffusion potential, but did not respond to delta pH. These energy-linked changes were accompanied by dye uptake into the matrix space. Concentration ratios, in-to-out, approached 4000:1. A large fraction of internalized dye was bound. At concentrations higher than those needed to record these spectral changes, Rhodamine 123 inhibited ADP-stimulated (State 3) respiration of mitochondria (Ki = 12 microM) and ATPase activity of inverted inner membrane vesicles (Ki = 126 microM) and partially purified F1-ATPase (Ki = 177 microM). The smaller Ki for coupled mitochondria was accounted for by energy-dependent Rhodamine 123 uptake into the matrix. Above about 20 nmol/mg protein (10 microM), Rhodamine 123 caused rapid swelling of energized mitochondria. Effects on electron-transfer reactions and coupling were small or negligible even at the highest Rhodamine 123 concentrations employed. delta psi-dependent Rhodamine 123 uptake together with Rhodamine 123 binding account for the intense fluorescent staining of mitochondria in living cells. Inhibition of mitochondria ATPase likely accounts for the cytotoxicity of Rhodamine 123. At concentrations which do not inhibit mitochondrial function, Rhodamine 123 is a sensitive and specific probe of delta psi in isolated mitochondria.     

Euphorbia factor L1 reverses ABCB1-mediated multidrug resistance involving interaction with ABCB1 independent of ABCB1 downregualtion

Euphorbia factor L1 (EFL1) belongs to diterpenoids of genus Euphorbia. In this article, its reversal activity against ABCB1-mediated MDR in KBv200 and MCF-7/adr cells was reported. However, EFL1 did not alter the sensitivity of KB and MCF-7 cells to chemotherapeutic agents. Meanwhile, EFL1 significantly increased accumulation of doxorubicin and rhodamine 123 in KBv200 and MCF-7/adr cells, showing no significant influence on that of KB and MCF-7 cells. Furthermore, EFL1 could enhance the ATP hydrolysis activity of ABCB1 stimulated by verapamil. At the same time, EFL1 inhibited the efflux of ABCB1 in KBv200 and MCF-7/adr cells. In addition, EFL1 did not downregulate expression of ABCB1 in KBv200 and MCF-7/adr cells either in mRNA or protein level.     

ABCB4 exports phosphatidylcholine in a sphingomyelin-dependent manner

ABCB4, which is specifically expressed on the canalicular membrane of hepatocytes, exports phosphatidylcholine (PC) into bile. Because SM depletion increases cellular PC content and stimulates PC and cholesterol efflux by ABCA1, a key transporter involved in generation of HDL, we predicted that SM depletion also stimulates PC efflux through ABCB4. To test this prediction, we compared the lipid efflux activity of ABCB4 and ABCA1 under SM depletion induced by two different types of inhibitors for SM synthesis, myriocin and (1R,3S)-N-(3-hydroxy-1-hydroxymethyl-3-phenylpropyl)dodecanamide, in human embryonic kidney 293 and baby hamster kidney cells. Unexpectedly, SM depletion exerted opposite effects on ABCB4 and ABCA1, suppressing PC efflux through ABCB4 while stimulating efflux through ABCA1. Both ABCB4 and ABCA1 were recovered from Triton-X-100-soluble membranes, but ABCB4 was mainly recovered from CHAPS-insoluble SM-rich membranes, whereas ABCA1 was recovered from CHAPS-soluble membranes. These results suggest that a SM-rich membrane environment is required for ABCB4 to function. ABCB4 must have evolved to exert its maximum activity in the SM-rich membrane environment of the canalicular membrane, where it transports PC as the physiological substrate.     

Atorvastatin inhibits angiotensin-converting enzyme induction in differentiating human macrophages

Statins are effective drugs in the prevention of cardiovascular disease. Recent studies suggested that statins have additional beneficial effects on the vascular wall independent of their cholesterol-lowering effects. We investigated whether atorvastatin influences angiotensin-converting enzyme (ACE) production in differentiating human macrophages. Human peripheral blood monocytes (PBM) were isolated from fresh buffy coats. The cells were allowed to differentiate for 0-8 days in macrophage serum-free medium with 5 ng/ml granulocyte-macrophage colony-stimulating factor. Atorvastatin (0.005-0.5 microM), mevalonate (200-400 microM), geranylgeranyl pyrophosphate (1.25-2.5 microM), and/or farnesylpyrophosphate (FPP; 1.25-2.5 microM) was added on the second day of differentiation and then every other day. After incubation time, the ACE amount in intact macrophages was measured. ACE amount in PBM was low. A marked time-dependent ACE induction was noticed during differentiation of monocytes to macrophages. Atorvastatin treatment inhibited ACE induction during differentiation. In the presence of mevalonate, atorvastatin failed to downregulate ACE production. Cotreatment of the cells with atorvastatin and FPP reversed the suppressive effect of atorvastatin on ACE. In conclusion, atorvastatin inhibited ACE upregulation, normally occurring in differentiating human macrophages. This effect was mediated via the mevalonate pathway, and inhibition of FPP was probably involved. The finding that atorvastatin inhibited ACE upregulation may represent a novel pleiotropic action and an additional beneficial effect of statins in treatment of cardiovascular disease.     

Lysophosphatidylcholine promotes cholesterol efflux from mouse macrophage foam cells via PPARgamma-LXRalpha-ABCA1-dependent pathway associated with apoE

Formation of macrophage-derived foam cells is a hallmark in earlier stages of atherosclerosis (AS). Increased cholesterol efflux from macrophage foam cells promote atherosclerotic regression. In the present study, lysophosphatidylcholine (LPC) promoting cholesterol efflux from macrophage foam cells was observed, and the mechanism underlying the action was investigated. Macrophage foam cells from mice were incubated with different concentrations of LPC (10, 20, 40, 80 microM), and the free cholesterol in medium increased but total intracellular cholesterol decreased. At the same time, the expression of PPARgamma, LXRalpha, ABCA1 was enhanced in a dose-dependent manner. The treatment of macrophage foam cells with 40 microM LPC for 12, 24 and 48 h promoted cellular cholesterol efflux in a time-dependent manner, meanwhile expression of PPARgamma, LXRalpha, ABCA1 was also raised respectively. Addition of different specific inhibitors of PPARgamma (GW9662), LXRalpha (GGPP), ABCA1 (DIDS) to the foam cells significantly suppressed LPC-induced cholesterol efflux. Also treatment with specific inhibitors of PPARgamma or LXRalpha decreased ABCA1 mRNA and protein expressions. LPC (40 microM)-induced cholesterol efflux was significantly lower in macrophage foam cells from apoE deficient mice than from normal C57BL/6J mice. In contrast, 10 microg apoAI-induced cholesterol efflux from foam cells remained in apoE deficient mice. The present results indicate that LPC promotes cholesterol efflux from macrophage foam cells via a PPARgamma-LXRalpha-ABCA1-dependent pathway. Furthermore, apoE may be involved in this process.     

Stereoselective regulation of MDR1 expression in Caco-2 cells by cetirizine enantiomers

MDR1-encoded P-glycoprotein (P-gp) is a drug efflux transporter mainly expressed in liver, kidney, intestine, brain (at the level of the blood-brain barrier), and placenta. It thus plays important roles in drug absorption, distribution, and excretion. Cetirizine is a second-generation nonsedating antihistamine used to treat allergic disease of respiratory system, skin and eyes. To evaluate P-gp expression and function in Caco-2 cells pretreated with cetirizine enantiomers, we assessed the sensitivity of Caco-2 cells to paclitaxel using the MTT assay and the polarized transport of rhodamine-123 and doxorubicin across Caco-2 monolayers. RT-PCR and flow cytometry were used to assay MDR1 mRNA and P-gp protein respectively. The sensitivity of Caco-2 cells to paclitaxel decreased significantly after cells were pretreated with 100 microM R-cetirizine but increased upon treatment with S-cetirizine. The efflux of rhodamine-123 and doxorubicin was enhanced significantly after Caco-2 monolayers were pretreated with 100 microM R-cetirizine but was reduced by S-cetirizine. The MDR1 mRNA and P-gp levels in Caco-2 cells were increased by 100 microM R-cetirizine and decreased by 100 microM S-cetirizine. These results suggest that R-cetirizine up-regulates MDR1 expression while S-cetirizine down-regulates MDR1 expression.