In vitro genotoxicity of carcinogenic asarone isomers.

The present study focused on genotoxic properties of the carcinogenic phenylpropanoids α-asarone and ß-asarone, which are found in several herbs and spices, such as Acorus calamus or Acorus gramineus. Cytotoxic and genotoxic effects were determined in human liver carinoma HepG2 cells, in hamster lung fibroblast V79 cells and in human cytochrome P450 1A2 and human sulfotransferase 1C2 transfected V79 cells (tV79). The Alamar blue assay was used to measure cytotoxicity of both isomers prior to the identification of DNA damaging properties by single cell gel electrophoresis (comet assay). Furthermore, the phosphorylation status of the histone H2AX, as a response of DNA double strand breaks, was investigated in HepG2 cells by Western blot analysis and visualized by immunofluorescence microscopy. After 24 h of incubation a significant reduction of cell viability was found. Moreover, both asarone isomers induced DNA strand breaks in V79 cells after 1 h of incubation. In tV79 cells even more pronounced DNA damaging properties were exhibited, whereas in HepG2 cells the compounds were found to be less effective. Furthermore, in tV79 cells a significant increase of formamidopyrimidine-DNA-glycosylase-sensitive sites was observed. DNA strand breaks, induced by aA, were to some extent characterized as DNA double strand breaks. In summary, asarone-induced cytotoxicity and genotoxicity is strongly influenced by the cellular metabolic enzyme status and therefore, a contribution of their respective metabolites to in vitro toxicity can be suggested.

[Lipoid Pneumonia Associated with Lipid-Containing Nasal Sprays and Nose Drops]. / Lipidpneumonie durch Lipid-haltige Nasensprays und -tropfen.

BACKGROUND: Regularly updating the German pharmacopoeia on contemporary preparations DAC/NRF, chapter "Nasal Applications" and the recommendations on "Nasal Oils" as well as "Nasal Ointments and Emulsions", the issue of the risk of lipoid pneumonia associated with the use of plant oils and when compared to mineral oils arose. MATERIAL AND METHODS: We searched different databases: the "Grosse Deutsche Arzneimittelspezialitäten-Taxe" containing all products available in German pharmacies, the Cochrane Library, the pharmacovigilance-database of the BfArM, and Medline to evaluate the benefit/risk-ratio of plant oils in nasal drops and sprays. RESULTS: In German pharmacies, a number of both, mineral oil-containing drugs for nasal application and plant oil-containing medical devices are available. The risk of lipoid pneumonia described for mineral oil-containing nasal products can not entirely be transferred to plant oil-containing products. However, evidence from the literature suggests a risk for lipoid pneumonia, which needs to be considered given the non-proven efficacy of such medical devices in the majority of proposed indications. To minimize risks, recommendations are made for patient groups that should not use lipid-containing nasal products. CONCLUSIONS: Acknowledging the potential lethal outcome of lipoid pneumonia, a demanding diagnosis, and absence of a specific therapy, lipid-containing nasal products should be used only with great caution. Based on the current knowledge, the statements regarding the risk of lipoid pneumonia for lipid-containing nasal products in the DAC/NRF should not be modified.

Detection of pyrrolizidine alkaloids in German licensed herbal medicinal teas.

BACKGROUND: Because of the hepatotoxic, mutagenic, and cancerogenic effects of pyrrolizidine alkaloids (PAs) the German Federal Institute for Risk Assessment (BfR) recommends not to exceed a daily PA intake of 0.007 µg/kg body weight (0.42 µg/60 kg adult). In a recent study conducted by the BfR, up to 5647 µg PA/kg dried herbal material were detected in tea products marketed as food. PURPOSE: The present study aimed at elucidating whether medicinal teas licensed or registered as medicinal products contain PAs as well. STUDY DESIGN: One hundred sixty-nine different commercially available medicinal teas, i.e. 19 nettle (Urtica dioica L.), 12 fennel (Foeniculum vulgare Mill.), 14 chamomile (Matricaria recutita L.), 11 melissa (Melissa officinalis L.) and 4 peppermint (Mentha piperita L.) teas as well as 109 tea mixtures were analyzed for the presence of 23 commercially available PAs. METHOD: LC/MS was used for the determination of the PAs RESULTS: In general, the total PA contents ranging 0-5668 µg/kg. Thirty percent of the tested single-ingredient tea products and 56.9% of the tested medicinal tea mixtures were found to contain PA concentrations above the limit of quantification (LOQ) of 10 µg/kg. In 11 medicinal teas PA contents >300 µg/kg dry herb were determined thus exceeding the recommended limit for PA intake by BfR. In addition three products of the investigated tea mixtures revealed extremely high PA contents of 4227, 5137, and 5668 µg/kg. Generally, single-ingredient tea products contained much less or even no detectable amounts of PAs when compared to the tea mixtures. PAs in the range between 13 and 1080 µg/kg were also detected in five analyzed aqueous herbal infusions of the medicinal tea mixture products with the highest PA content. Two out of the five investigated herbal infusions exceeded the recommended BfR limit for PA intake. CONCLUSION: This study demonstrates clearly that also medicinal teas licensed as medicinal products may partly contain high amounts of PAs exceeding current recommendations. For that reason manufacturers are advised to carry out more rigorous quality control tests devoted to the detection of PAs. This is very important to minimize PAs in medicinal teas accounting for possible additional exposure of the consumer to PAs from other food sources (e.g. honey).

Mode of action and dose-response framework analysis for receptor-mediated toxicity: The aryl hydrocarbon receptor as a case study.

Dioxins and dioxin-like compounds are tumor promoters that cause liver cancer in rats and mice. The aryl hydrocarbon receptor (AHR) has been implicated as a key component in this tumor promotion response. Despite extensive knowledge of the toxicology of dioxins, no mode of action (MOA) hypothesis for their tumorigenicity has been formally documented using the Human Relevance MOA framework developed by the International Programme on Chemical Safety (IPCS). To address this information gap, an expert panel was convened as part of a workshop on receptor-mediated liver tumorigenicity. Liver tumors induced by ligands of the AHR were assessed using data for dioxins and related chemicals as a case study. The panel proposed a MOA beginning with sustained AHR activation, eventually leading to liver tumors via a number of other processes, including increased cell proliferation of previously initiated altered hepatic foci, inhibition of intrafocal apoptosis and proliferation of oval cells. These processes have been identified and grouped as three key events within the hepatocarcinogenic MOA: (1) sustained AHR activation, (2) alterations in cellular growth and homeostasis and (3) pre-neoplastic tissue changes. These key events were identified through application of the Bradford-Hill considerations in terms of both their necessity for the apical event/adverse outcome and their human relevance. The panel identified data supporting the identification and dose-response behavior of key events, alteration of the dose-response by numerous modulating factors and data gaps that potentially impact the MOA. The current effort of applying the systematic frameworks for identifying key events and assessing human relevance to the AHR activation in the tumorigenicity of dioxins and related chemicals is novel at this time. The results should help direct future regulatory efforts and research activities aimed at better understanding the potential human cancer risks associated with dioxin exposure.

Feasibility study of nonclinical safety assessments on homeopathic preparations using the example of protoanemonin in Pulsatilla pratensis L.

Homeopathy is a world-wide available form of complementary therapy, which has a tradition of 200years. Due to the long history of clinical use, i.e. reflected by the first edition of the Homeopathic Pharmacopoeia of the US of 1914, the conduct of toxicological studies is not required if the safety has been otherwise substantiated. The aim of this article is to establish a risk assessment procedure without full toxicological examination, using homeopathic preparations from Pulsatilla pratensis L. as an example. The literature review shows that protoanemonin is the most relevant constituent of these plants regarding potential toxicity. Based on structural alerts protoanemonin is classified as a Cramer class III compound with the threshold of toxicological concern (TTC) of 180µg/day in adults. Neither computer aided toxicology methods (Toxtree and Derek Nexus®) nor a literature search revealed any evidence of genotoxic, carcinogenic or teratogenic potential of protoanemonin. The protoanemonin exposure from a maximum daily dose of a typical homeopathic preparation of P. pratensis L. does not exceed the TTC. The presented method is transparent, reproducible and applicable to other homeopathic substances as a use-case scenario for computational toxicology in order to evaluate an approach for safety assessment of homeopathic medicinal products.

Limettin and furocoumarins in beverages containing citrus juices or extracts.

Phototoxic and photo-genotoxic furocoumarins occur, e.g., in citrus species, parsnip, parsley, celery, and figs. They exhibit phototoxic and photo-genotoxic properties in combination with UV radiation, while less is known about the phototoxicity of the coumarin derivative limettin mainly found in limes and lemons. Risk assessment of dietary furocoumarins is based on a threshold approach and on estimates of 1.2-1.45 mg for the average daily exposure for adults via the diet in several countries. In these estimates, the major contribution to overall daily exposure has been attributed to citrus-flavored non-alcoholic beverages, in spite of a lack of analytical data for those products. Therefore, we analyzed a number of furocoumarins in a variety of citrus-containing beverages and included limettin in the pattern of analyzed constituents. Our findings provide strong evidence that grapefruit juice and not citrus-flavored non-alcoholic beverages is the major source of furocoumarin exposure in a Western diet. Based on these findings it can be assumed that the average dietary exposure to furocoumarins is about 3-fold lower than previously estimated, i.e. in the range of 548 and 2237 microg/day for the average and high consumer, respectively. The coumarin derivative limettin was mainly found in lime products.

Toxicity of green tea extracts and their constituents in rat hepatocytes in primary culture.

Recent reports on sporadic cases of liver disorders (acute hepatitis, icterus, hepatocellular necrosis) after ingestion of dietary supplements based on hydro-alcoholic extracts from green tea leaves led to restrictions of the marketing of such products in certain countries of the EU. Since green tea is considered to exert a number of beneficial health effects, and, therefore, green tea products are widely used as dietary supplements, we were interested in the possible mechanism of hepatotoxicity of green tea extracts and in the components involved in such effects. Seven hours after seeding on collagen, rat hepatocytes in primary culture were treated with various hydro-alcoholic green tea extracts (two different native 80% ethanolic dry extracts and an 80% ethanolic dry extract cleared from lipophilic compounds). Cells were washed, and reduction of resazurin, used as a viability parameter monitoring intact mitochondrial function, was determined. It was found that all seven green tea extracts examined enhanced resazurin reduction significantly at a concentration range of 100-500 microg/ml medium, while a significant decrease was observed at 1-3mg/ml medium. Decreased levels were concomitant with abundant necrosis as observed by microscopic inspection of the cultures and with increased leakage of lactate dehydrogenase activity from the cells. In a separate series of experiments, the green tea constituents (-)-epicatechin, (-)-epigallocatechin-3-gallate, caffeine and theanine were tested at concentrations reflecting their levels in a typical green tea extract. Synthetic (+)-epigallocatechin (200 microM) was used for comparison. Cytotoxicity was found with (-)-epigallocatechin-3-gallate only. The concomitant addition of 0.25 mM ascorbate/0.05 mM alpha-tocopherol had no influence on cytotoxicity. In conclusion, our results suggest that high concentrations of green tea extract can exert acute toxicity in rat liver cells. (-)-Epigallocatechin-3-gallate seems to be a key constituent responsible for this effect. The relatively low bioavailability of catechins reported after oral exposure to green tea argues, however, against a causal role of these constituents in the reported liver disorders.

[Chemical food contaminants]. / Chemische Lebensmittelkontaminanten.

Chemical food contaminants are substances which are neither present naturally in the usual raw material used for food production nor are added during the regular production process. Examples are environmental pollutants or contaminants derived from agricultural production of crops or livestock or from inadequate manufacturing of the food product itself. More difficult is the classification of those compounds formed during regular manufacturing such as products of thermal processes including flavoring substances. In these cases, it is common practice to call those compounds contaminants which are known for their adverse effects such as acrylamide, whereas constituents which add to the food-specific flavor such as Maillard products formed during roasting, baking etc. are not termed contaminants. From a toxicological viewpoint this distinction is not always clear-cut. Important groups of chemical contaminants are metals such as mercury or lead, persistent organic pollutants such as polychlorinated biphenyls and related pollutants, which are regularly found in certain types of food originating from background levels of these compounds in our environment. Furthermore, natural toxins form microorganisms or plants, and compounds formed during thermal treatment of food are of major interest. In general, a scientific risk assessment has to be carried out for any known contaminant. This comprises an exposure analysis and a toxicological and epidemiological assessment. On these grounds, regulatory and/or technological measures can often improve the situation. Major conditions for a scientific risk assessment and a successful implementation of regulations are highly developed food quality control, food toxicology and nutritional epidemiology.

Tumor promoters as inhibitors of apoptosis in rat hepatocytes.

Multistage carcinogenesis in rat liver is widely used as an experimental model for the study of the critical events in tumor promotion. After an initial treatment with a genotoxic liver carcinogen ('initiation'), subsequent application of certain non-genotoxic agents can lead to the clonal expansion of putative preneoplastic cells ('promotion'). Obviously, the expansion of these clones is correlated with an increased occurrence of benign and malignant liver tumors at later time points. Since both proliferation and apoptosis were reported to be enhanced in putative preneoplastic liver foci, inhibition of apoptosis was suggested to play a critical role in tumor promotion. In rat hepatocytes in primary culture, the liver tumor promoter 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) inhibited apoptosis initiated by treatment of the cultures with UV irradiation but did not affect apoptosis in non-irradiated cultures. The suppression of apoptosis with TCDD coincided with an attenuated increase of the tumor suppressor protein p53 observed upon UV irradiation. Furthermore, TCDD treatment resulted in a marked hyperphosphorylation of p53. The fact that almost identical concentration-response curves were obtained for the phosphorylation of p53 and the induction of cytochrome P450(CYP)1A-catalyzed 7-ethoxyresorufin O-deethylase (EROD) activity indicates that p53 phosphorylation after TCDD treatment is mediated by the aryl hydrocarbon receptor (AhR) signaling cascade. With tumor-promoting 'non-dioxin-like' polychlorinated biphenyls inhibition of UV-induced apoptosis was also observed. A comparative study investigating the effects of various concentrations did not reveal, however, a clear correlation between the suppression of apoptosis and the induction of CYP2B-catalyzed 7-pentoxyresorufin O-dealkylase (PROD) activity. In summary, inhibition of UV-induced apoptosis with liver tumor promoters is observed in rat hepatocytes in culture. Hyperphosphorylation of key proteins of apoptosis including p53 seems to play a role in this effect.

Identification of human multidrug resistance protein 1 (MRP1) mutations and characterization of a G671V substitution.

The multidrug resistance protein 1 (MRP1) belonging to the ATP-binding cassette (ABC) superfamily of transport proteins can confer resistance to multiple natural product drugs and methotrexate in human tumor cells. In addition, MRP1 is expressed in normal tissues acting as an efflux pump for glutathione, glucuronate, and sulfate conjugates and may thus influence the pharmacokinetic properties of many drugs. Using polymerase chain reaction-single-strand conformation polymorphism analysis, we screened 36 Caucasian volunteers for mutations in the coding exons of the MRP1 gene, including the adjacent intron sequences. Among several mutations found, two are expected to cause amino acid substitutions. One of these mutations (G671V) was of special interest because it is located near the first nucleotide binding domain. To determine whether this mutation caused a change in the MRP1 phenotype, a mutant MRP1 expression vector was constructed and transfected into SV40-transformed human embryonic kidney cells (HEKSV293T) and the transport properties of the mutant protein were examined. Transport of the MRP1 substrates leukotriene C4, 17beta-estradiol 17beta-(D)-glucuronide, and estrone sulfate by membrane vesicles prepared from transiently transfected HEKSV293T cells was comparable to that of wild-type MRP1.

Inhibition of apoptosis in rat hepatocytes treated with 'non-dioxin-like' polychlorinated biphenyls.

Polychlorinated biphenyls (PCBs) are among the most prominent persistent environmental pollutants exhibiting neurotoxic, teratogenic and tumour-promoting effects in experimental animals. 'Dioxin-like' properties have been assigned to a number of PCBs whereas other PCBs have been classified as 'non-dioxin-like'. Many of the latter congeners are inducers of cytochrome P450 (CYP) 2B1 and 2B2 similar to the liver tumour promoter phenobarbital. In contrast, 'dioxin-like' PCBs induce CYP1A isozymes, and other congeners have been classified as 'mixed-type' inducers. Inhibition of apoptosis of pre-neoplastic hepatocytes is thought to play a central role in tumour promotion in rat liver. We have used the inhibition of UV-induced apoptosis in rat hepatocytes in primary culture as an in vitro model for mechanistic studies on the inhibition of apoptosis. It could be shown that phenobarbital, and the 'non-dioxin-like' PCBs 28, 101 and 187 completely inhibit UV-induced apoptosis. The concentration-response curves and EC(50) values for this effect, however, were different from those of induction of CYP2B1/2B2-catalysed 7-pentoxyresorufine O-dealkylase or CYP1A-catalysed 7-ethoxyresorufine O-deethylase activities. The PCBs and phenobarbital did not affect the spontaneous incidence of apoptotic nuclei. In conclusion, 'non-dioxin-like' PCBs are likely to promote liver carcinogenesis via the suppression of apoptosis. The signaling events in rat hepatocytes leading to induction of 2B1/2B2 activity by the compounds investigated are assumed to differ from those leading to inhibition of apoptosis.

Basal expression of the rat, but not of the human, multidrug resistance protein 2 (MRP2) gene is mediated by CBF/NF-Y and Sp1 promoter-binding sites.

The most important biliary efflux transporter known so far is the multidrug resistance protein 2 (MRP2). Previously, we isolated and characterized the 5'-flanking region of the rat mrp2 gene. In the present study, we performed site-directed mutagenesis experiments indicating that both a Y-Box and a GC-Box in the rat mrp2 promoter are essential for the full basal expression of the gene, but have no significant relevance for its inducibility by the chemical carcinogen 2-acetylaminofluorene. Gel mobility shift experiments demonstrated the binding of the transcription factor CBF/NF-Y, but not of EFIA/YB-1, to the Y-Box. Site-directed mutations in the Y-Box decreasing reporter gene activity of a promoter construct prevented the binding of NF-Y. Consequently, NF-Y contributes substantially to the basal expression of the gene. A site-directed mutation in the GC-Box also reduced basal expression and resulted in a reduced complex formation with the transcription factor Sp1. The corresponding region of the human MRP2 promoter comprises no Sp1 site, but a Y-Box-like element binding YB-1 but not NF-Y, which, however, does not contribute to basal expression. In conclusion, NF-Y and Sp1 binding sites play a decisive role in the basal expression of the rat mrp2 gene, while the human MRP2 gene is regulated differently.

Inducing effects of dioxin-like polychlorinated biphenyls on CYP1A in the human hepatoblastoma cell line HepG2, the rat hepatoma cell line H4IIE, and rat primary hepatocytes: comparison of relative potencies.

Polychlorinated biphenyls (PCBs) are a group of widespread environmental pollutants. Some non-ortho-substituted congeners with a high likelihood of coplanarity of both aromatic rings have been shown to act like 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as agonists of the aryl hydrocarbon receptor (AhR) subsequently leading to adverse effects, such as immunosuppression and tumor promotion. Although there is a broad base of experimental data concerning the toxicity of PCBs in laboratory animals and animal-derived primary cells and cell lines, only few experimental data are available for cells of human origin. As a parameter of AhR activation, induction of CYP1A-mediated 7-ethoxyresorufin O-deethylase (EROD) activity was determined in the human hepatoblastoma cell line HepG2 treated with the PCBs IUPAC Nos. 77, 81, 105, 114, 118, 123, 126, 156, 157, 167, 169, and 189, and with TCDD as a positive control. Compared with results in rat primary hepatocytes and the rat hepatoma cell line H4IIE, treated HepG2 cells showed lower specific EROD activities maximally inducible by TCDD and PCBs, and EC50 values were shifted to higher concentrations. Furthermore, relative potency factors (REPs) for some congeners such as PCBs 81, 126, and 169 greatly differed from those observed in cells derived from rats. Northern blot analyses showed that EROD activities run parallel to changes in CYP1A-specific mRNA contents. The considerable differences in EROD-derived REPs between cells of human and rat origin indicate the need for further investigations in experimental models from different species including humans in order to extend the database of biochemical and toxic responses to PCBs.

Malassezin--A novel agonist of the arylhydrocarbon receptor from the yeast Malassezia furfur.

The yeast Malassezia furfur converts tryptophan into several indole compounds. One of these, malassezin, was identified as 2-(1H-indol-3-ylmethyl)-1H-indole-3-carbaldehyde (1). It was synthesized from N-Boc-indole-3-carbaldehyde in five steps with 12% overall yield. The compound easily cyclizes to indolo[3,2-b]carbazole (7) which is known to interact with the arylhydrocarbon receptor (AHR). Similarly, malassezin was found to induce cytochrome P450 as an agonist of AHR (EC50 = 1.57 microM) in rat hepatocytes.

Up-regulation of transporters of the MRP family by drugs and toxins.

Expression of a variety of ABC efflux pumps including certain conjugate transporters of the multidrug resistance protein (MRP) subfamily is inducible in primate and rodent tissues, and in a variety of cell lines and primary cells in culture. In human cell lines (HepG2, MCF-7), we studied the inducibility of MRPs 1-5. Similar to the rat mrp2 gene, human mrp2 is inducible by the chemical carcinogen 2-AAF, the chemotherapeutic drug cisplatin and the barbiturate phenobarbital, as demonstrated in Northern and Western Blots. Furthermore, the antibiotic rifampicin was identified as MRP2 inducer in HepG2 cells. MRP1 and 4 mRNAs being expressed in human liver at a very low level could not be detected in HepG2 cells after treatment with various agents. However, MRP3 and 5 mRNAs were detected in addition to MRP2 and their expression was found to be increased by 2-AAF, cisplatin and rifampicin. MRP1 expression was studied in MCF-7 cells where the chemotherapeutic drug vinblastine and tert-butyl hydroquinone but not the MRP2 inducing agents described above acted as inducers.

Sequencing and tissue distribution of the canine MRP2 gene compared with MRP1 and MDR1.

The effects of xenobiotic drugs and toxic compounds depend largely on their kinetic properties, which can be influenced by transmembrane drug transporters like MDR1/P-glycoprotein and the drug-conjugate transporters multidrug resistance protein (MRP) 1 and 2. As the dog is a preferential species used in the pharmacological and toxicological evaluation of new drugs, we sequenced the canine MRP2 cDNA and investigated its expression in various canine tissues compared with the related transporters MRP1 and P-glycoprotein. The tissue distribution pattern of these ABC-transporters differs partially from the distribution described in humans. So we found relatively high renal and low hepatic canine MRP2 expression levels, relatively high hepatic canine MRP1 expression levels, and quite high levels of MRP1 and P-glycoprotein in the dog brain. The knowledge of the tissue distribution pattern of these transporters will aid to interpret pharmacokinetic and toxicokinetic data gained from dog studies and to extrapolate them to humans.

Evaluation of a vincristine resistant Caco-2 cell line for use in a calcein AM extrusion screening assay for P-glycoprotein interaction.

AIM: To develop a fast fluorometric screening assay based on vincristine resistant Caco-2 cells (Caco-2VCR) in order to elucidate potential P-glycoprotein (Pgp) interactions of compounds, and to characterise Caco-2VCR cells with regard to their expression of the efflux transporters Pgp, MRP1 and MRP2. METHODS: We applied the Caco-2VCR cells to a 96-well plate-based calcein AM extrusion assay. The Caco-2VCR cells were cultured as monolayers and incubated with calcein AM with/without addition of Pgp modulators. Fourteen known Pgp modulators were tested in the assay (chloropromazine, cyclosporin A, domperidone, digoxin, ivermectin, ketoconazole, loperamide, metoprolol, propranolol, progesterone, quinidine, quinine, verapamil and vincristine). For each compound an EC50 value was calculated. Protein and mRNA levels of the efflux transporters were analysed by Western blot and polymerase chain reaction techniques. RESULTS: All compounds with the exception of digoxin displayed increased calcein levels. Protein and mRNA analysis showed increased levels of Pgp after vincristine exposure, while expression of the efflux transporters MRP1 and MRP2 remained unchanged. CONCLUSIONS: The calcein AM extrusion assay applied to Caco-2VCR cells can be a valuable tool as a screening assay for new compounds and their potential interaction with P-glycoprotein.

Genomic structure of the human Ah receptor nuclear translocator gene (hARNT).

The vertebrate basic helix-loop-helix/Per-ARNT-Sim (bHLH/PAS protein) ARNT (aryl hydrocarbon receptor nuclear translocator) plays a crucial role in transcriptional regulation as the common subunit of a number of transcriptionally active complexes. Several studies indicate that ARNT might be involved in the pathogenesis of various genetic diseases. In this study we provide the first report on the genomic structure of the human ARNT gene (hARNT). Human BAC and PAC libraries were screened, and clones positive for ARNT were mapped, subcloned, and sequenced. The gene has an overall size of about 65 kb and consists of 22 exons varying in size from 25 to 214 bp. Splice junctions follow the GT/AG consensus except for intron 11 starting with GC at its 5' end. Knowledge of exon-intron boundaries and intronic sequences neighboring the exons allows the extended search for polymorphisms and variants in human genomic DNA. The exonintron arrangement is highly similar to the murine arnt gene (marnt) except for a slight shift in the last three exons. 5' RACE indicated several transcription start sites, one of them identical with the major transcription start site of marnt.

The effect of rifampin treatment on intestinal expression of human MRP transporters.

The importance of the ATP-dependent transporter P-glycoprotein, which is expressed in the brush border membrane of enterocytes and in other tissues with excretory function, for overall drug disposition is well recognized. For example, induction of intestinal P-glycoprotein by rifampin appears to be the underlying mechanism of decreased plasma concentrations of P-glycoprotein substrates such as digoxin with concomitant rifampin therapy. The contribution of transporter proteins other than P-glycoprotein to drug interactions in humans has not been elucidated. Therefore, we tested in this study the hypothesis whether the conjugate export pump MRP2 (cMOAT), which is another member of the ABC transporter family, is inducible by rifampin in humans. Duodenal biopsies were obtained from 16 healthy subjects before and after nine days of oral treatment with 600 mg rifampin/day. MRP2 mRNA and protein were determined by reverse transcription-polymerase chain reaction and immunohistochemistry. Rifampin induced duodenal MRP2 mRNA in 14 out of 16 individuals. Moreover, MRP2 protein, which was expressed in the apical membrane of enterocytes, was significantly induced by rifampin in 10 out of 16 subjects. In summary, rifampin induces MRP2 mRNA and protein in human duodenum. Increased elimination of MRP2 substrates (eg, drug conjugates) into the lumen of the gastrointestinal tract during treatment with rifampin could be a new mechanism of drug interactions.