Mrp1 multidrug resistance-associated protein and P-glycoprotein expression in rat brain microvessel endothelial cells

Two membrane glycoproteins acting as energy-dependent efflux pumps, mdr-encoded P-glycoprotein (P-gp) and the more recently described multidrug resistance-associated protein (MRP), are known to confer cellular resistance to many cytotoxic hydrophobic drugs. In the brain, P-gp has been shown to be expressed specifically in the capillary endothelial cells forming the blood-brain barrier, but localization of MRP has not been well characterized yet. Using RT-PCR and immunoblot analysis, we have compared the expression of P-gp and Mrp1 in homogenates, isolated capillaries, primary cultured endothelial cells, and RBE4 immortalized endothelial cells from rat brain. Whereas the mdr1a P-gp-encoding mRNA was specifically detected in brain microvessels and mdr1b mRNA in brain parenchyma, mrp1 mRNA was present both in microvessels and in parenchyma. However, Mrp1 was weakly expressed in microvessels. Mrp1 expression was higher in brain parenchyma, as well as in primary cultured brain endothelial cells and in immortalized RBE4 cells. This Mrp1 overexpression in cultured brain endothelial cells was less pronounced when the cells were cocultured with astrocytes. A low Mrp activity could be demonstrated in the endothelial cell primary monocultures, because the intracellular [3H]vincristine accumulation was increased by several MRP modulators. No Mrp activity was found in the cocultures or in the RBE4 cells. We suggest that in rat brain, Mrp1, unlike P-gp, is not predominantly expressed in the blood-brain barrier endothelial cells and that Mrp1 and the mdr1b P-gp isoform may be present in other cerebral cells.     

Inhibition of CYP1A1-dependent activity by the polynuclear aromatic hydrocarbon (PAH) fluoranthene

Polynuclear aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants, and recently bioassay-based induction studies have been used to determine exposures to complex mixtures of PAHs. Induction of CYP1A1-dependent activity in H4IIE rat hepatoma cells has been used extensively as a bioassay for halogenated aromatic hydrocarbons and more recently for PAHs. Fluoranthene (FL) is a prevalent PAH contaminant in diverse environmental samples, and FL did not induce CYP1A1-dependent ethoxyresorufin O-deethylase (EROD) activity significantly in H4IIE cells. However, in cells cotreated with 2 x 10(-5) M FL plus the potent inducers 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or benzo[k]fluoranthene (BkF) (2 x 10(-8) M), there was a significant decrease in EROD activities. Furthermore, treatment of TCDD-induced rat microsomes with FL caused an 80% decrease in EROD activity. Studies showed that FL did not affect induction of CYP1A1 protein or mRNA levels in H4IIE cells, and analysis of enzyme inhibition data using microsomal CYP1A1 indicated that FL noncompetitively inhibited CYP1A1-dependent activity. 32P-Postlabeling revealed no significant FL-DNA adduct formation in H4IIE cells treated with FL. However, in cells cotreated with FL plus BkF or benzo[a]pyrene (BaP), certain PAH-DNA adducts were induced 2-fold. This study demonstrated that FL is an inhibitor of CYP1A1-dependent enzyme activity in rat hepatoma H4IIE cells and that the genotoxic potency of some carcinogenic PAHs may be modulated by FL in mixtures containing relatively high levels of this compound.     

Co-ordinate loss of protein kinase C and multidrug resistance gene expression in revertant MCF-7/Adr breast carcinoma cells

The aim of this study was to investigate the link between protein kinase C (PKC) and multidrug resistance (mdr) phenotype. The expression of both was studied in doxorubicin-resistant MCF-7/Adr cells as they reverted to the wild-type phenotype when cultured in the absence of drug. The following parameters were measured in cells 4, 10, 15, 20 and 24 weeks after removal of doxorubicin; (1) sensitivity of the cells towards doxorubicin; (2) levels of P-glycoprotein (P-gp) and MDR1 mRNA; (3) levels and cellular localization of PKC isoenzyme proteins alpha, theta and epsilon; and (4) gene copy number of PKC-alpha and MDR1 genes. Cells lost their resistance gradually with time, so that by week 24 they had almost completely regained the drug sensitivity seen in wild-type MCF-7 cells. P-gp levels measured by Western blot mirrored the change in doxorubicin sensitivity. By week 20, P-gp had decreased to 18% of P-gp protein levels at the outset, and P-gp was not detectable at week 24. Similarly, MDR1 mRNA levels had disappeared by week 24. MCF-7/Adr cells expressed more PKCs-alpha and -theta than wild-type cells and possessed a different cellular localization of PKC-epsilon. The expression and distribution pattern of these PKCs did not change for up to 20 weeks, but reverted back to that seen in wild-type cells by week 24. MDR1 gene amplification remained unchanged until week 20, but then was lost precipitously between weeks 20 and 24. The PKC-alpha gene was not amplified in MCF-7/Adr cells. The results suggest that MCF-7/Adr cells lose MDR1 gene expression and PKC activity in a co-ordinate fashion, consistent with the existence of a mechanistic link between MDR1 and certain PKC isoenzymes.     

The multidrug resistance in human leukemias. Minireview

Leukemia/lymphoma cells, clinically refractory to therapy are often associated with expression of P-glycoprotein (P-gp), which is encoded by the multidrug resistance (MDR) gene, mdr1. Cell lines expressing mdr1 exhibit resistance to several structurally unrelated lipophilic drugs, such as anthracyclines, vinca alkaloids, and epopodophyllotoxins. This MDR can be conferred to drug-sensitive cells mdr1 cDNA transfer. In resistant cells, MDR is characterized by overexpression of P-gp and by the enhanced efflux, and P-gp fluorescence probe, rhodamine 123 (Rh 123). This can be circumvented by addition of certain non-cytotoxic drugs, such as verapamil and cyclosporin A.     

Long-term decline of atmospheric and marine pollution by polycyclic aromatic hydrocarbons (PAHS) in Germany

Concentrations of benzo[a]pyrene and benzo[e]pyrene in the atmosphere as markers for the class of PAHs decreased by about 70% within one decade in clean air as well as in industrially polluted areas of Germany when measured with passive samplers (spruce sprouts, poplar and beech leaves). The same trend has been found for East-Germany during 1991-1995. Mussels (Mytilus edulis) were found to accumulate PAHs from the aquatic environment and exhibited a seasonal periodicity of the PAH concentration. After an initial decline from 1985 to 1990, the PAH concentration remained constant until 1995 in the North Sea area investigated.     

Detoxification of optically active bay- and fjord-region polycyclic aromatic hydrocarbon dihydrodiol epoxides by human glutathione transferase P1-1 expressed in Chinese hamster V79 cells

Dihydrodiol epoxides (DEs) are important carcinogenic metabolites of polycyclic aromatic hydrocarbons (PAHs). The metabolic formation of four stereoisomeric DEs (a pair of optically active diastereomers termed as syn- and anti-form) is possible. Glutathione tranferases (GSTs) have been demonstrated to catalyze the detoxification of DEs. Purified GSTs display remarkable differences in catalytic efficiencies towards bay- and fjord-region DEs along with a high degree of regio- and stereoselectivity. Here we determined to which extent heterologously expressed human GSTP1-1, a major GST isoform in lung, affects the mutagenicity of stereoisomeric bay-region DEs of benzo[a]pyrene in Chinese hamster V79 cells. To evaluate the influence of sterical crowding in the substrate on the activity of GSTP-1, the study was extended to the strongly mutagenic fjord-region (-)-anti-DEs of benzo[c]phenanthrene and dibenzo[a,l]pyrene. GSTP1-1,reduced preferentially the mutagenicity (studied at the hprt locus) of (+)-anti and (+)-syn-DEs of benzo[a]pyrene (by 66 and 67%) as compared with the corresponding (-)-anti- and (-)-syn-enantiomers (by 15 and 13%). These results are in line with previous studies on the enantioselectivity of purified GSTP1-1 towards the DE isomers of benzo[a]pyrene and benzo[c]phenanthrene showing that enantiomers with (R)-configuration at the benzylic oxiranyl carbon are better substrates than those with (S)-configuration. Interestingly, the (-)-anti-DEs of benzo[c]phenanthrene and dibenzo[a,l]pyrene were efficiently detoxified by GSTP-1-1 in the constructed cell line (reduction of mutagenicity by 66 and 64%). This study demonstrates that differences in the caalytic activity seen for purified GST towards individual mutagens do not necessarily reflect the detoxification of DEs by the same enzyme in a living cell and provides further evidence that specific human GSTs play a role in the detoxification of DEs of PAHs.     

Analysis of the Concentration of Gas-Phase and Solid-Phase Polyaromatic Hydrocarbons in Industrial Emissions from Aluminum Production

Metallurgist - Aluminum production is one source of atmospheric polycyclic aromatic hydrocarbons (PAHs), of which benzo[a]pyrene is the best-studied representative. Other high-priority PAHs are...     

cDNA cloning, sequence analysis, and induction by aryl hydrocarbons of a murine cytochrome P450 gene, Cyp1b1

C3H mouse embryo fibroblast cells, designated 10T1/2, can be transformed by physical and chemical agents including polycyclic aromatic hydrocarbons. In a previous report (Shen et al., Proc. Natl. Acad. Sci. USA 90, 11483-11487, 1993), we identified a cytochrome P450 gene induced by polycyclic aromatic hydrocarbons (PAHs) that is different from 1A1 or 1A2, and which we tentatively named P450CMEF. Here, we report the entire cDNA sequence of P450CMEF (5,128 bp) and the amino acid sequence deduced from it (543 residues). A comparison of the latter sequence with known cytochrome P450s indicates that P450CMEF is in a new subfamily of family 1 of the P450 superfamily. Accordingly, the Committee on Standardized Cytochrome P450 Nomenclature designated the gene Cyp1b1. Exposure to various aryl hydrocarbons (2.5 hr) induced Cyp1b1 mRNA in 10T1/2 cells to different degrees: 2,3,7,8-tetrachlorodibenzo-p-dioxin, 7,12-dimethylbenz[a]anthracene, benz[a]anthracene, benzo[a]pyrene, and beta-naphthoflavone were strong inducers; alpha-naphthoflavone and 3-methylcholanthrene, were moderate inducers; and benzo[e]pyrene was a weak inducer.     

Metabolism of polycyclic aromatic hydrocarbons and covalent binding of metabolites to protein in rat adrenal gland

The metabolism of the carcinogenic and adrenocorticolytic polycyclic aromatic hydrocarbon 7,12-dimethylbenz(a)anthracene in rat adrenals was investigated. Both 7,12-dimethylbenz(a)anthracene and benzo(a)pyrene, which also is a well-known carcinogen but has no short-term effects on rat adrenals, appear to be metabolized by one common type of cytochrome P-450-dependent monooxygenase localized in the endoplasmic reticulum. Studies of the kinetic properties of this cytochrome P-450 reveal that the Km values for 7,12-dimethylbenz(a)anthracene and benzo(a)pyrene are lower than 3 microM. Identification of metabolites indicates that, with both 7,12-dimethylbenz(a)anthracene and benzo(a)pyrene, phenols and diols were formed the relative rates of formation of which were markedly influenced by the expoxide hydrase inhibitor cyclohexane oxide, suggesting that epoxides are intermediate metabolites. Added or endogenous microsomal glutathione S-transferase B had little or no effect on the distribution of metabolites. A rather selective binding of metabolites of 7,12-dimethylbenz(a)anthracene to soluble and microsomal proteins was demonstrated. The adrenal cytochrome P-450 involved in the conversion of these polycyclic aromatic hydrocarbons appears to be unrelated to those responsible for the synthesis of mineralocorticoids and glucocorticoids from cholesterol. Among androgens and estrogens, estradiol proved to be the most inhibitory steroid, suggesting a role of the hydrocarbon-metabolizing cytochrome P-450 in estrogen biosynthesis. However, no such function could be demonstrated conclusively. The metabolite patterns and the effects of nonsteroid inhibitors of liver monooxygenases, e.g., alpha-naphthoflavone, SU 9055, and ellipticine, suggest that the properties of this cytochrome P-450 resemble those of the 3-methyl-cholanthrene-inducible cytochrome P-488 from rat liver.     

Effects of motorcycle exhaust on cytochrome P-450-dependent monooxygenases and glutathione S-transferase in rat tissues

The effects of motorcycle exhaust (ME) on cytochrome P-450 (P-450)-dependent monooxygenases were determined using rats exposed to the exhaust by either inhalation, intratracheal, or intraperitoneal administration. A 4-wk ME inhalation significantly increased benzo[a]pyrene hydroxylation, 7-ethoxyresorufin O-deethylation, and NADPH-cytochrome c reductase activities in liver, kidney, and lung microsomes. Intratracheal instillation of organic extracts of ME particulate (MEP) caused a dose- and time-dependent significant increase of monooxygenase activity. Intratracheal treatment with 0.1 g MEP extract/kg markedly elevated benzo[a]pyrene hydroxylation and 7-ethoxyresorufin O-deethylation activities in the rat tissues 24 h following treatment. Intraperitoneal treatment with 0.5 g MEP extract/kg/d for 4 d resulted in significant increases of P-450 and cytochrome b5 contents and NADPH-cytochrome c reductase activity in liver microsomes. The intraperitoneal treatment also markedly increased monooxygenases activities toward methoxyresorufin, aniline, benzphetamine, and erythromycin in liver and benzo[a]pyrene and 7-ethoxyresorufin in liver, kidney, and lung. Immunoblotting analyses of microsomal proteins using a mouse monoclonal antibody (Mab) 1-12-3 against rat P-450 1A1 revealed that ME inhalation, MEP intratracheal, or MEP intraperitoneal treatment increased a P-450 1A protein in the hepatic and extrahepatic tissues. Protein blots analyzed using antibodies to P-450 enzymes showed that MEP intraperitoneal treatment caused increases of P-450 2B, 2E, and 3A subfamily proteins in the liver. The ME inhalation, MEP intratracheal, or MEP intraperitoneal treatment resulted in significant increases in glutathione S-transferase activity in liver cytosols. The present study shows that ME and MEP extract contain substances that can induce multiple forms of P-450 and glutathione S-transferase activity in the rat.     

Effect of polycyclic aromatic hydrocarbons on the elimination kinetics of pyrene and the urinary excretion profile of 1-hydroxypyrene in the rat

Pyrene was chosen as a noncarcinogen model of polycyclic aromatic hydrocarbons (PAHs). Groups of male Wistar rats were dosed with pyrene and with mixture of pyrene and fluoranthene, pyrene and benz[a]anthracene, or pyrene, fluoranthene, and benz[a]anthracene at 20 mg/kg by intravenous or oral routes. Blood samples were taken at 0.25, 0.5, 1, 2, 3, 4, and 5 h after administration. The concentration of pyrene was determined by gas chromatography. The toxicokinetic parameters for pyrene were determined from the time course of blood concentration. A significant increase in the bioavailability of pyrene after treatment with other PAHs was observed. Urinary 1-hydroxypyrene excretion was analyzed after pretreatment with acenaphthene, naphthalene, chrysene, phenanthrene, benz[a]anthracene, and benzo[a]pyrene. The urine from rats was collected for 3 d and the concentration of 1-hydroxypyrene was determined using high-performance liquid chromatography (HPLC). Most compounds examined caused a decrease in the urinary excretion of the metabolite of pyrene.     

Ascorbic acid deficiency reduces the level of mRNA for cytochrome P-450 on the induction by polychlorinated biphenyls

Ascorbic acid (AsA) deficiency causes a decrease in hepatic concentration of cytochrome P-450 and a decrease in hepatic activity of drug-metabolizing enzymes in rats unable to synthesize AsA (ODS rats). To study the mechanism of the decrease in hepatic concentration of cytochrome P-450 isozymes by AsA deficiency, we chose the xenobiotics-inducible cytochrome P-450 and performed the experiments indicated below. AsA-deficient rats were fed polychlorinated biphenyls (PCB) which markedly induce both CYP1A subfamily and several isozymes in CYP2B subfamily. First, we assayed the activities of two drug-metabolizing enzymes so that one could be functionally distinguished from another. AsA deficiency significantly reduced the hepatic activity of aminopyrine-N-demethylase in ODS rats with and without dietary PCB, but had no effect on benzo(a)pyrene hydroxylase activity. Secondly, quantitative immunoblot analyses demonstrated that the levels of CYP2B1/2B2 and CYP1A1 in the AsA-deficiency rats fed PCB were approximately 60 and 80% lower than those found in rats fed AsA-supplemented diet. The degree of reduction in CYP2B1/2B2 was greater than CYP1A1. Thirdly, AsA deficiency caused a decrease in hepatic abundance of CYP2B1/2B2 mRNA, whereas it had no effect on the levels of CYP1A1 and 1A2 mRNA. These results indicated that dietary AsA selectively affects the levels of CYP2B1/2B2 mRNA among cytochrome P-450 induced by PCB and plays important roles for optimum induction of drug-inducible cytochrome P-450. We concluded that AsA deficiency decreases specific froms of drug-inducible cytochrome P-450, especially CYP2B1/2B2 and that the reduction of CYP2B1/2B2 mRNA level in AsA-deficient rats caused a decrease in cytochrome P-450 concentration and hepatic activity of drug-metabolizing enzymes.     

Pirarubicin nuclear uptake does not correlate with its induced cell death effect during reversal of multidrug resistance by quinine in human K562 and CEM leukemic cells

A number of small and lipophilic cations are able to reverse in vitro the resistance to anthracyclines and other natural products through their interaction with P-glycoprotein or P-gp. However, some modulators do not interact with P-gp. We have demonstrated in a previous a work, using confocal laser microspectrofluorometry, that quinine does not increase nuclear anthracycline uptake in multidrug-resistant Chinese hamster ovary LR73 cells. In this case the LR73 cells were transfected with the mdr1 gene. Moreover, quinine induced in these cells an increase of mdr1 gene expression. In the present study, we investigated verapamil and quinine for their ability to increase nuclear pirarubicin uptake in multidrug-resistant K562R and CEMR human leukemic cell lines. These two cell lines resist, respectively, to doxorubicin and vinblastine and both overexpress the P-gp. Verapamil was able to restore nuclear pirarubicin in both cell lines. On the other hand, quinine was unable to significantly increase nuclear pirarubicin uptake. Both modulators were able to restore pirarubicin sensitivity in both resistant cell lines. After treatment with quinine, mdr1 gene and P-gp expression was not significantly altered as observed previously in the LR73 cells. This suggest that the effect of quinine on mdr1 gene expression is dependent on the cell line studied. These data suggest that quinine could modify the molecular environment of anthracyclines and/or its binding to a possible cytoplasmic target, and that the mechanisms by which anthracyclines induce cell death, and ways by which chemotherapy fails in multidrug-resistant leukemic cells remain complex and are related to more than one target.     

Increasing incidence of childhood class V lupus nephritis

1. Homozygously mdr1a gene disrupted mice (mdr1a(-/-) mice) and wild type mice (mdr1a(+/+) mice) were used to develop a method for P-glycoprotein (P-gp) function imaging non-invasively and to study the effect of a P-gp reversal agent on its function in vivo. 2. [11C]verapamil (0.1 mg/kg) was administered and the changes in tissue concentrations were determined ex vivo by organ extirpation and in vivo with PET. To block P-gp function, cyclosporin A was administered. 3. Biodistribution studies revealed 9.5-fold (P < 0.001) and 3.4-fold (P < 0.001) higher [11C]verapamil in the brain and testes of mdr1a(-/-) mice than in mdr1a(+/+) mice. Cyclosporin A (25 mg/kg) increased [11C]verapamil levels in the brain and testes of mdr1a(+/+) mice in both cases 3.3-fold (P < 0.01 (brain); P < 0.001 (testes)). Fifty mg/kg cyclosporin A increased [11C]verapamil in the brain 10.6-fold (P < 0.01) and in the testes 4.1-fold (P < 0.001). No increases were found in the mdr1a(-/-) mice. This indicates complete inhibition of P-gp mediated [11C]verapamil efflux. 4. Positron camera data showed lower [11C]verapamil levels in the brain of mdr1a(+/+) mice compared to those in mdr1a(-/-) mice. [11C]verapamil accumulation in the brain of mdr1a(+/+) mice was increased by cyclosporin A to levels comparable with those in mdr1a(-/-) mice, indicating that reversal of P-gp mediated efflux can be monitored by PET. 5. We conclude that cyclosporin A can fully block the P-gp function in the blood brain barrier and the testes and that PET enables the in vivo measurement of P-gp function and reversal of its function non-invasively.