[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.

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.

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.

Influence of liver tumor promoters on apoptosis in rat hepatocytes induced by 2-acetylaminofluorene, ultraviolet light, or transforming growth factor beta 1.

DNA damage is recognized widely as a cause of programmed cell death (apoptosis), aimed at eliminating cells bearing genotoxic lesions. Therefore, inhibition of DNA damage-induced apoptosis may play an important role in carcinogenesis and has been suggested as a mechanism of action of tumor-promoting agents. In the present study, the effects of treatment with UV light or the carcinogenic aromatic amine 2-acetylaminofluorene (2-AAF) on apoptosis were studied in rat hepatocytes in primary culture. A significantly increased incidence of apoptotic nuclei, showing condensed or fragmented chromatin visualized with the fluorescent dye Hoechst 33258, was found after each type of treatment. After 48 h, the incidence of apoptosis had returned to the control level. When the liver tumor promoters 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and phenobarbital were added to the medium, apoptosis did not increase in UV- or 2-AAF-treated compared with untreated cultures. Furthermore, TCDD and phenobarbital suppressed internucleosomal DNA fragmentation elicited by UV irradiation. In contrast, the promoters did not suppress apoptosis induced by transforming growth factor beta 1. Immunoprecipitation of the tumor suppressor gene product p53 demonstrated that the increase in p53 observed after UV irradiation was abrogated almost completely by TCDD. Apoptosis induced in rat hepatocytes by DNA-damaging agents such as UV light or 2-AAF is suppressed by TCDD and phenobarbital. Inhibition of apoptosis allowing survival of hepatocytes bearing genotoxic lesions may be crucial for the tumor-promoting action of TCDD and phenobarbital in the liver.

Effect of phenobarbital and other liver monooxygenase modifiers on dimethylnitrosamine-induced alkylation of rat liver macromolecules.

The effects of phenobarbital (PB) and other liver monooxygenase modifiers on dimethylnitrosamine (DMN)-induced alkylation of rat liver DNA and protein were investigated at different carcinogen doses. In rats given single injections of radioactively labeled DMN, pretreatment with PB (80 mg/kg body weight, administered for 5 days) resulted in a small but significant decrease in the formation of 7-methylguanine and O6-methylguanine per mole of guanine in liver DNA associated with a decrease in the O6/N7-methylguanine ratio. The specific radioactivity of liver protein was also lowered in PB-pretreated rats. The degree of PB interference was independent of DMN dose within a carcinogen dose range of 0.5 microgram to 10 mg/kg body weight. In parallel experiments, the effects of pretreatment with PB, Aroclor 1254, pregnenolone-16 alpha-carbonitrile, butylated hydroxytoluene, beta-naphthoflavone, and ethanol on DMN-induced alkylation of liver DNA were studied at a DMN dose of 5 micrograms/kg body weight. In general, pretreatment with these modifiers of liver monooxygenase resulted in a decrease in specific alkylation of DNA and in the ratio of 7-methylguanine to guanine. If, however, 7-methylguanine levels were related to total liver DNA, these differences in DNA alkylation between controls and pretreated rats became substantially smaller, partially being negligible, since these inducers led to an increase in relative liver weight with concomitant increase in the content of liver DNA. Thus, when expressed per total liver, no significant changes in the overall extent of metabolic activation of DMN were evident. These findings are not consistent with the results of in vitro studies on DMN metabolism in microsomal systems which favored the hypothesis that changes in the metabolism of hepatocarcinogens are responsible for the reduction of liver tumor response in animals treated simultaneously with inducers of the liver monooxygenase system and hepatocarcinogens. Our findings suggest that these effects might rather be related to drug-mediated changes on the cellular level.

Persistently increased expression of a 3-methylcholanthrene-inducible phenol uridine diphosphate-glucuronosyltransferase in rat hepatocyte nodules and hepatocellular carcinomas.

Increased UDP-glucuronosyltransferase in rat hepatocyte nodules and hepatocellular carcinomas produced by feeding 2-acetylaminofluorene or N-nitrosomorpholine was studied using isozyme-selective substrates, antibodies, and DNA probes. UDP-glucuronosyltransferase (UDP-GT) activities toward 4-methylumbelliferone, 1-naphthol, and benzo[a]pyrene-3,6-quinol were reversibly increased by short term feeding of 2-acetylaminofluorene but were persistently increased in hepatocyte nodules and differentiated hepatocellular carcinomas. Immunoblot analysis revealed that short term feeding of 2-acetylaminofluorene increased a Mr 55,000 polypeptide corresponding to the previously characterized UDP-GTI or phenol UDP-GT. However, in some hepatocyte nodules and hepatocellular carcinomas either the Mr 55,000 or a new Mr 53,000 polypeptide was preferentially increased, suggesting heterogeneous UDP-GT forms in liver nodules and carcinomas. Northern blot hybridization with a synthetic DNA probe to phenol UDP-GT demonstrated increased levels of mRNA in liver nodules. The results suggest persistently increased expression of at least two phenol UDP-GT enzyme forms in hepatocyte nodules, which may contribute to the toxin-resistance phenotype frequently observed at cancer prestages.

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.

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).

Colocalization of three types of intermediate filament proteins in perisinusoidal stellate cells: glial fibrillary acidic protein as a new cellular marker.

The presence and the colocalization of the three intermediate filament proteins, glial fibrillary acidic protein (GFAP) and the marker of mesenchymal liver cells, vimentin, were studied by an immunofluorescence double-labeling technique in cultures of isolated rat perisinusoidal stellate cells (PSC) and hepatocytes, in cocultures of isolated PSC and hepatocytes as well as in cryostat sections of rat liver. GFAP and vimentin immunoreactivities were localized in cultured PSC which were identified by the presence of the cellular marker desmin, another intermediate filament protein, or the stellate morphology to be seen after staining for one of three intermediate filament proteins. Both GFAP and vimentin were strongly expressed in the perinuclear region and the cell processes of cultured PSC. Staining for GFAP highly coincided with that for vimentin or desmin in cultured PSC and with that for vimentin in the liver sections. Desmin-positive cells were always also GFAP-positive. However, of the GFAP-positive cells only an estimated 50% were found desmin-positive. The coexpression of desmin and GFAP in the same cells appear to be unique, since apparently it has not been previously reported for any other cell type. Almost all of the vimentin-positive cells in hepatocyte culture were also expressing GFAP. Since desmin was not found in all of the cultured cells with PSC morphology, GFAP is suggested as a more reliable marker for PSC than desmin.

The influence of environmental and genetic factors on CYP2D6, CYP1A2 and UDP-glucuronosyltransferases in man using sparteine, caffeine, and paracetamol as probes.

The impact of gender, use of oral contraceptive steroids (OCS), coffee consumption and of smoking on the metabolism of sparteine, caffeine, and paracetamol was studied in 194 randomly selected subjects (98 male and 95 female). Thirty-eight of the male volunteers were cigarette smokers, 40 of the female subjects were smokers and/or users of OCS. The metabolic ratio of sparteine oxidation (MRs) showed a trimodal distribution. 7.7% of the subjects had a MRs > 20 and thus were poor metabolizers (PMs). Within the extensive metabolizer (EM) subjects, a distinct subgroup accounting for 11% was observed with 20 > MRs > 1.2. Six of the 15 phenotypical PMs were heterozygous EMs by genotyping. This indicates the existence of one or several CYP2D6 mutations which cannot be identified by the currently employed genotyping methods. In each subgroup, i.e. smokers/OCS and non-smokers/non-OCS, the cumulative frequency distribution of the heterozygous (wt/B) phenotype caused a shift to higher MRs compared with the wild-type homozygotes (wt/wt). Thus, for the in vivo activity of CYP2D6, genetic determinants prevail over environmental factors. Smoking, use of oral contraceptive steroids, caffeine consumption, or gender had no influence on sparteine metabolism. The distribution of the paracetamol glucuronide/paracetamol metabolic ratio appeared to be unimodal although skewed. Glucuronidation capacity was clearly affected by gender, OCS use and smoking. It was higher in male than in female subjects. Male smokers had the highest, and female non-smokers/non-OCS users the lowest metabolic ratio. CYP1A2 activity, as determined by a caffeine metabolic ratio ((AFMU + 1X + 1U)/1, 7U), was multimodally distributed and was clearly increased in smokers. It was significantly correlated to paracetamol glucoronidation in male heavy smokers (r=0.85), suggesting an element of co-regulation of CYP1A2 and of paracetamol conjugating UDP-glucuronosyltransferase isozymes, including UGTI.6.

Induction of P-glycoprotein by rifampin increases intestinal secretion of talinolol in human beings: a new type of drug/drug interaction.

BACKGROUND: P-Glycoprotein is an efflux pump in many epithelial cells with excretory function. It has been demonstrated that rifampin (INN, rifampicin) induces P-glycoprotein, particularly in the gut wall. We therefore hypothesized that rifampin affects pharmacokinetics of the P-glycoprotein substrate talinolol, a beta1-blocker without appreciable metabolic disposition but intense intestinal secretion in human beings. METHODS: Pharmacokinetics of talinolol (a single dose of 30 mg administered intravenously or 100 mg administered orally for 7 days) and duodenal expression of the MDR1 gene product P-glycoprotein as assessed by reverse transcriptase-polymerase chain reaction of the MDR1-messenger ribonucleic acid, by immunohistochemistry and Western blot analysis were analyzed before and after coadministration of rifampin (600 mg per day for 9 days) in 8 male healthy volunteers (age 22 to 26 years). RESULTS: During rifampin treatment, the areas under the curve of intravenous and oral talinolol were significantly lower (21% and 35%; P < .05). Treatment with rifampin resulted in a significantly increased expression of duodenal P-glycoprotein content 4.2-fold (2.9, 6.51) (Western blot) and messenger RNA was increased in six of the eight volunteers. P-Glycoprotein expression in biopsy specimens of gut mucosa correlated significantly with the systemic clearance of intravenous talinolol (rs = 0.74; P < .001). CONCLUSIONS: Rifampin induces P-glycoprotein-mediated excretion of talinolol predominantly in the gut wall. Moreover, clearance of talinolol from the blood into the lumen of the gastrointestinal tract may be predicted by the individual intestinal P-glycoprotein expression. Thus we describe a new type of steady-state drug interaction affecting compounds that are subject to transport rather than metabolism.

Denitrosation of N-nitrosomorpholine by liver microsomes; possible role of cytochrome P-450.

Liver microsomes from male mice and rats were incubated with N-nitrosomorpholine (MoNA) and an NADPH-regenerating system. The formation of nitrite was measured after induction or inhibition of the microsomal monooxygenase system. Pretreatment of the animals with phenobarbital (PB) enhanced nitrite formation by about 200%, while 3-methylcholanthrene (3-MC)-induction showed no exceptional effects. Various specific inhibitors of the monooxygenase function including carbon monoxide decreased nitrite formation. In conjunction with results obtained by spectra studies it is suggested that N-nitrosomorpholine is denitrosated by a reduction process in which cytochrome (cyt.) P-450 seems to be involved. Nitricoxide formed is partly converted to nitrite under these conditions.

Impact of dioxin-type induction of drug-metabolizing enzymes on the metabolism of endo- and xenobiotics.

The induction of a number of drug-metabolizing enzymes is among the best-understood biochemical effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related agonists of the aryl hydrocarbon receptor (AhR). Among the cytochrome P450s (CYPs), the genes encoding CYP1A1, 1A2, and 1B1 are responsive to AhR agonists, i.e. their expression is inducible in various mammalian tissues and organs as well as in many types of cell lines and primary cells in culture. In addition, an aldehyde dehydrogenase, an NADPH-quinone-oxidoreductase, and the phase II conjugating enzymes glutathione-S-transferase (GST) Ya and UDP-glucuronosyltransferase 1A1 have been identified as responsive to AhR agonists. Induced expression of these members of the AhR gene battery is thought to be aimed at an improved elimination of the inducing agent and its metabolites. However, the identity of the physiological ligand(s) of the AhR is still obscure. The consequences of induced expression of AhR-regulated genes encoding drug-metabolizing enzymes have been investigated in human populations, e.g. in smokers, and in various experimental models. A prominent example of increased adverse effects due to the induction of CYP1A isozymes is the metabolic activation of carcinogenic aromatic amines and polycyclic aromatic hydrocarbons. An increasing amount of data is also available on the impact of dioxin-type induction on the metabolism of drugs, food constituents, and endogenous substrates. For example, the hepatic clearance of the drug theophylline, which is widely used in asthma therapy, is enhanced significantly in smokers. Increased glucuronidation of thyroxine in rats treated with TCDD or other potent AhR agonists is thought to result in hypothyroxinemia and its biological consequences, such as sustained hyperplasia of the thyroid, bearing a higher risk of thyroid cancer. The relevance of these observations for humans exposed to dioxin-type inducers is discussed.

Tryptanthrins: a novel class of agonists of the aryl hydrocarbon receptor.

2,3,7,8-Tetrachlorodibenzo-p-dioxin and related environmental pollutants exert most of their adverse effects such as immunosuppression, induction of endocrine dysfunction, tumor promotion, and teratogenicity via the aryl hydrocarbon or dioxin receptor. While most potent agonists of the aryl hydrocarbon receptor are of synthetic origin, an increasing number of natural compounds are now recognized as receptor agonists. Our findings demonstrated that some tryptanthrin derivatives biosynthesized in incubations of Candida lipolytica with tryptophan and anthranilic acid or its derivatives were agonists of the aryl hydrocarbon receptor. The biosynthetic products 8-methyltryptanthrin, 8-chlorotryptanthrin, and 8-bromotryptanthrin induced cytochrome P4501A1 mRNA and protein in rat hepatocytes in primary culture, characteristic features of aryl hydrocarbon receptor agonists. Log-probit analysis of the catalytic activity of cytochrome P4501A1, 7-ethoxyresorufin O-deethylase (EROD), revealed EC50 induction values of 1.7, 0.25, and 0.17 microM for 8-methyltryptanthrin, 8-chlorotryptanthrin, and 8-bromotryptanthrin, respectively. Interestingly, the nonsubstituted tryptanthrin molecule, biosynthesized from the common physiological precursors tryptophan and anthranilic acid, was also active as an inducer. The specificity of the inducing effect of tryptanthrins was demonstrated in gel retardation experiments in Hepa-1 mouse hepatoma cells, showing the characteristic interaction of the activated aryl hydrocarbon receptor with an oligonucleotide containing a xenobiotic-responsive element. It is suggested that the receptor may be part of a defense system protecting higher organisms from secondary metabolites formed by the microflora of the host or its environment.

Multidrug resistance gene expression in rodents and rodent hepatocytes treated with mitoxantrone.

Overexpression of P-glycoprotein in tumor cells can represent a severe drawback for cancer chemotherapy. P-glycoprotein acts as an efflux transporter for a variety of chemotherapeutic agents. It is encoded by multidrug resistance (mdr) genes of the subfamily 1 in humans (MDR1) and rodents (mdr1a and 1b). Because mdr1 gene expression is inducible in cultured rat hepatocytes and in rat liver with chemical carcinogens such as 2-acetylaminofluorene or aflatoxin B1, which form DNA-binding electrophiles during their metabolism, we investigated whether the DNA-damaging chemotherapeutic drug mitoxantrone may induce multidrug resistance in rodents and in hepatocytes in primary culture. In H4IIE rat hepatoma cells stably transfected with a luciferase construct containing the rat mdr1b promoter, mitoxantrone caused a concentration-dependent increase in promoter activity. Mdr1 gene expression in cultured rat hepatocytes was enhanced at mitoxantrone concentrations greater than or equal to 0.1 microM and in mouse hepatocytes at 5 microM. In hepatocytes from both species, a correlation was found between mdr1 induction and the inhibition of protein synthesis. In vivo, mitoxantrone was a very powerful inducer of mdr1 gene expression in rat liver and small intestine. In rat kidney, induction of mRNA was lower, and a marginal effect was seen in lung. In contrast with rats, no significant induction of mdr1 gene expression was obtained in mouse liver. Probably as a consequence of inhibition of protein synthesis, mitoxantrone did not lead to a pronounced elevation of P-glycoprotein levels in rat liver and kidney.

Drug metabolizing enzyme activities in rat liver epithelial cell lines, hepatocytes and bile duct cells.

P450-dependent mono-oxygenase and conjugating enzyme activities were studied in rat liver epithelial cells (RLEs) and compared to those in hepatocytes and bile duct cells. Various RLE cell lines were investigated since (a) they are suspected to be derived from cells in the lineage from putative pluripotent stem cells to either hepatocytes or bile duct cells, and (b) they may represent targets of chemical carcinogens. Despite considerable variation between lines, common features were recognized. P450-dependent monooxygenase activities (7-ethoxyresorufin O-deethylase and 7-ethoxycoumarin O-deethylase) were undetectable in all RLEs and bile duct cells, and were uninducible by benz(a)anthracene. In contrast, glucuronosyltransferase (GT), sulfotransferase and GSH transferase activities were clearly detectable. Conjugating enzyme activities increased until confluency of the cell cultures was reached. Under the latter conditions, GT activities towards 4-methylumbelliferone or benzo(a)pyrene-3,6-quinol (substrates of a 3-methylcholanthrene-inducible phenol GT) were similar to those found in hepatocytes or bile duct cells. Using a selective cDNA probe, phenol GT mRNA was clearly detectable in RLE1. In contrast, GT activity towards 4-hydroxybiphenyl was much lower than in hepatocytes or bile duct cells (0.04- and 0.03-fold). Sulfotransferase and GSH transferase activities were also roughly comparable to those found in hepatocytes and in bile duct cells. The results suggest that RLEs and bile duct cells exhibit both high conjugating enzyme activities and a lack of P450-dependent mono-oxygenase activities, a pattern resembling the 'toxin-resistance phenotype' found in putative preneoplastic hepatocyte foci and nodules.

Requirement for metabolic activation of acetylaminofluorene to induce multidrug gene expression.

Previously we have demonstrated that several xenobiotics can induce multidrug (mdr) gene expression in cultures of primary isolated hepatocytes. One of the best of these xenobiotic inducers in rat hepatocytes is 2-acetylaminofluorene (2-AAF), which induces mdr expression by an enhancement of mdr gene transcription. In all species studied to date, AAF is extensively and variously metabolized. In this study we have sought to determine if AAF per se or a metabolite is responsible for mediating the increase in mdr gene transcription and expression. This study demonstrates that AAF per se is not active, but that the effect of AAF we have observed on mdr gene transcription and expression in the rat is due to the formation of a reactive metabolite(s). Our data indicate that this reactive metabolite is probably N-acetoxy-2-aminofluorene or the sulfate ester of N-hydroxy-AAF. The requirement for the formation of one of these metabolites may explain the differences in species response to AAF, in terms of mdr gene expression, that we have observed. We hypothesize that the mechanism by which mdr gene transcription is increased in response to AAF involves a covalent interaction between a reactive metabolite and an mdr gene regulatory protein. Our current work is concerned with the exploration of this hypothesis.

Dioxinlike components in incinerator fly ash: a comparison between chemical analysis data and results from a cell culture bioassay.

Potent polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and dioxinlike polychlorinated biphenyls (PCBs) are among the most relevant toxic emissions from incinerators. Induction of cytochrome P450 1A1-catalyzed 7-ethoxyresorufin O-deethylase (EROD) activity in mammalian cell culture (EROD bioassay) is thought to be a selective and sensitive parameter used for the quantification of dioxinlike compounds. Fly ash extracts from municipal waste incinerators (MWI), a crematorium, wood combustors, and a noble metal recycling facility were analyzed in the EROD bioassay using rat hepatocytes in primary culture. Fractions containing 2,3,7,8-substituted PCDDs/PCDFs, dioxinlike PCBs, and 16 major polycyclic aromatic hydrocarbons (PAHs) were isolated from the extract and analyzed by gas chromatography-mass spectrometry (GC-MS) and by the EROD bioassay. It was found that with MWI samples the bioassay of the extract resulted in a two- to fivefold higher estimate of TCDD equivalents (TEQ) than the chemical analysis of PCDDs/PCDFs and PCBs. However, the outcome of both methods was significantly correlated, making the bioassay useful as a rough estimate for the sum of potent PCDDs/PCDFs and dioxinlike PCBs in extracts from MWI fly ash samples and in a fly ash sample from a crematorium. In noble metal recycling facility and wood combustor samples, higher amounts of PAHs were found, contributing to more pronounced differences between the results of both methods. The remaining unexplained inducing potency in fly ash samples probably results from additional dioxinlike components including certain PAHs not analyzed in this study. The hypothesis that emissions from MWI of hitherto unidentified dioxinlike compounds are higher by orders of magnitude than emissions of potent PCDDs/PCDFs and dioxinlike PCBs could not be confirmed. We found no indication for a marked synergistic interaction of dioxinlike fly ash components in the bioassay.