Construction of DOX/APC co-loaded BiOI@CuS NPs for safe and highly effective CT imaging and chemo-photothermal therapy of lung cancer.

Recently, a variety of nanoparticles have been widely used as imaging agents or carriers for the diagnosis and therapy of lung cancer. However, their poor imaging effect, high toxicity, pro-inflammatory effect and ineffective treatment are still a great challenge. In this work, we reported a novel kind of BiOI@CuS nanoparticle to achieve safe and effective therapy of lung cancer by co-loading hydrochloric acid doxorubicin (DOX) and aspirin phenacetin and caffeine (APC). The nanoparticles can effectively relieve inflammatory reactions induced by photo-thermal therapy (PTT). In vitro and in vivo assays showed that DOX/APC co-loaded BiOI@CuS exhibited an effective chemo-photothermal comprehensive therapy effect and good CT imaging capability. Consequently, this multifunctional nanosystem provides a versatile and promising platform in the imaging and treatment of lung cancer in further applications.

Low-diluted Phenacetinum disrupted the melanoma cancer cell migration.

Dynamic and reciprocal interactions generated by the communication between tumor cells and their matrix microenvironment, play a major role in the progression of a tumor. Indeed, the adhesion of specific sites to matrix components, associated with the repeated and coordinated formation of membrane protrusions, allow tumor cells to move along a determined pathway. Our study analyzed the mechanism of action of low-diluted Phenacetinum on murine cutaneous melanoma process in a fibronectin matrix environment. We demonstrated a reduction of dispersed cell migration, early and for as long as 24 h, by altering the formation of cell protrusions. Moreover, low-diluted Phenacetinum decreased cell stiffness highly on peripheral areas, due to a disruption of actin filaments located just under the plasma membrane. Finally, it modified the structure of the plasma membrane by accumulating large ordered lipid domains and disrupted B16 cell migration by a likely shift in the balance between ordered and disordered lipid phases. Whereas the correlation between the excess of lipid raft and cytoskeleton disrupting is not as yet established, it is clear that low-diluted Phenacetinum acts on the actin cytoskeleton organization, as confirmed by a decrease of cell stiffness affecting ultimately the establishment of an effective migration process.

3D engineered In vitro hepatospheroids for studying drug toxicity and metabolism.

Drug toxicity is one of the reasons for late stage drug attrition, because of hepatotoxicity. Various in vitro liver models like primary human hepatocytes, immortalized human hepatic cell lines, liver slices and microsomes have been used; but limited by viability, hepatic gene expression and function. The 3D-engineered construct of hepatocyte-like-cells (HLCs) differentiated from stem cells, may provide a limitless source of hepatocytes with improved reproducibility. Towards this end, we used hepatospheroids (diameter=50-80µm) differentiated from human-umbilical-cord-mesenchymal stem cells (hUC-MSCs) on 3D scaffold GEVAC (Gelatin-vinyl-acetate-copolymer) as in vitro model for studying drug metabolism/toxicity. Our data demonstrated that hUC-MSCs-derived-hepatospheroids cultured on GEVAC expressed significantly higher drug-metabolizing enzymes (CYPs) both at mRNA and activity level compared to 2D culture, using HR-LC/MS. We further showed that hepatospheroids convert phenacetin (by CYP1A2) and testosterone (by CYP3A4) to their human-specific metabolites acetaminophen and 6ß-hydroxytestosterone with a predictive clearance rate of 0.011ml/h/106 cells and 0.021ml/h/106 cells respectively, according to first-order kinetics. Hepatotoxicity was confirmed by exposing hepatospheroids to ethanol and acetaminophen; ROS generation, cell viability, cytoskeleton structure, elevation of liver function enzymes, i.e. AST and ALT, was analyzed. To the best of our knowledge, this is the first report to use hUC-MSCs-derived-hepatospheroids on GEVAC as in vitro model for drug metabolism/toxicity study; which can replace the conventional 2D-models used in drug development.

Long-term human primary hepatocyte cultures in a microfluidic liver biochip show maintenance of mRNA levels and higher drug metabolism compared with Petri cultures.

Human primary hepatocytes were cultivated in a microfluidic bioreactor and in Petri dishes for 13 days. mRNA kinetics in biochips showed an increase in the levels of CYP2B6, CYP2C19, CYP2C8, CYP3A4, CYP1A2, CYP2D6, HNF4a, SULT1A1, UGT1A1 mRNA related genes when compared with post extraction levels. In addition, comparison with Petri dishes showed higher levels of CYP2B6, CYP2C19, CYP2C8, CYP3A4, CYP1A2, CYP2D6 related genes at the end of culture. Functional assays illustrated a higher urea and albumin production over the period of culture in biochips. Bioreactor drug metabolism (midazolam and phenacetin) was not superior to the Petri dish after 2 days of culture. The CYP3A4 midazolam metabolism was maintained in biochips after 13 days of culture, whereas it was almost undetectable in Petri dishes. This led to a 5000-fold higher value of the metabolic ratio in the biochips. CYP1A2 phenacetin metabolism was found to be higher in biochips after 5, 9 and 13 days of culture. Thus, a 100-fold higher metabolic ratio of APAP in biochips was measured after 13 days of perfusion. These results demonstrated functional primary human hepatocyte culture in the bioreactor in a long-term culture. Copyright © 2016 John Wiley & Sons, Ltd.

Long-term culture and cryopreservation does not affect the stability and functionality of human embryonic stem cell-derived hepatocyte-like cells.

Human embryonic stem cells (hESCs) are predicted to be an unlimited source of hepatocytes which can pave the way for applications such as cell replacement therapies or as a model of human development or even to predict the hepatotoxicity of drug compounds. We have optimized a 23-d differentiation protocol to generate hepatocyte-like cells (HLCs) from hESCs, obtaining a relatively pure population which expresses the major hepatic markers and is functional and mature. The stability of the HLCs in terms of hepato-specific marker expression and functionality was found to be intact even after an extended period of in vitro culture and cryopreservation. The hESC-derived HLCs have shown the capability to display sensitivity and an alteration in the level of CYP enzyme upon drug induction. This illustrates the potential of such assays in predicting the hepatotoxicity of a drug compound leading to advancement of pharmacology.

A sensitive and high-throughput LC-MS/MS method for inhibition assay of seven major cytochrome P450s in human liver microsomes using an in vitro cocktail of probe substrates.

A sensitive and high-throughput LC-MS/MS method was established and validated for the simultaneous quantification of seven probe substrate-derived metabolites (cocktail assay) for assessing the in vitro inhibition of cytochrome P450 (CYP) enzymes in pooled human liver microsomes. The metabolites acetaminophen (CYP1A2), hydroxy-bupropion (CYP2B6), n-desethyl-amodiaquine (CYP2C8), 4'-hydroxy-diclofenac (CYP2C9), 4'-hydroxy-mephenytoin (CYP2C19), dextrorphan (CYP2D6) and 1'-hydroxy-midazolam (CYP3A4/5), together with the internal standard verapamil, were eluted on an Agilent 1200 series liquid chromatograph in <7 min. All metabolites were detected by an Agilent 6410B tandem mass spectrometer. The concentration of each probe substrate was selected by substrate inhibition assay that reduced potential substrate interactions. CYP inhibition of seven well-known inhibitors was confirmed by comparing a single probe substrate assay with cocktail assay. The IC50 values of these inhibitors determined on this cocktail assay were highly correlated (R(2) > 0.99 for each individual probe substrate) with those on single assay. The method was selective and showed good accuracy (85.89-113.35%) and between-day (RSD <13.95%) and within-day (RSD <9.90%) precision. The sample incubation extracts were stable at 25 °C for 48 h and after three freeze-thaw cycles. This seven-CYP inhibition cocktail assay significantly increased the efficiency of accurately assessing compounds' potential inhibition of the seven major CYPs in drug development settings.

In vitro interactions of water-soluble garlic components with human cytochromes p450.

Eight water-soluble components of aged garlic extract were evaluated to assess their potential to inhibit the activity of human cytochrome-P450 (CYP) enzymes. The in vitro model consisted of human liver microsomes with index reactions chosen to profile the activity of the following six CYP isoforms: CYP1A2, 2B6, 2C9, 2C19, 2D6, and 3A. With only 2 exceptions, none of the 8 garlic components produced >50% inhibition even at high concentrations (100 micromol/L). S-methyl-L-cysteine and S-allyl-L-cysteine at 100 micromol/L produced modest inhibition of CYP3A, reducing activity to 20-40% of control. However available clinical evidence does not indicate CYP3A inhibition in vivo. The findings suggest that drug interactions involving inhibition of CYP3A enzymes by aged garlic extract are very unlikely.

Validated assays for human cytochrome P450 activities.

The measurement of the effect of new chemical entities on human cytochrome P450 marker activities using in vitro experimentation represents an important experimental approach in drug development. In vitro drug interaction data can be used in guiding the design of clinical drug interaction studies, or, when no effect is observed in vitro, the data can be used in place of an in vivo study to claim that no interaction will occur in vivo. To make such a claim, it must be assured that the in vitro experiments are performed with absolute confidence in the methods used and data obtained. To meet this need, 12 semiautomated assays for human P450 marker substrate activities have been developed and validated using approaches described in the GLP (good laboratory practices) as per the code of U.S. Federal Regulations. The assays that were validated are: phenacetin O-deethylase (CYP1A2), coumarin 7-hydroxylase (CYP2A6), bupropion hydroxylase (CYP2B6), amodiaquine N-deethylase (CYP2C8), diclofenac 4'-hydroxylase and tolbutamide methylhydroxylase (CYP2C9), (S)-mephenytoin 4'-hydroxylase (CYP2C19), dextromethorphan O-demethylase (CYP2D6), chlorzoxazone 6-hydroxylase (CYP2E1), felodipine dehydrogenase, testosterone 6 beta-hydroxylase, and midazolam 1'-hydroxylase (CYP3A4 and CYP3A5). High-pressure liquid chromatography-tandem mass spectrometry, using stable isotope-labeled internal standards, has been applied as the analytical method. This analytical approach, through its high sensitivity and selectivity, has permitted the use of very low incubation concentrations of microsomal protein (0.01-0.2 mg/ml). Analytical assay accuracy and precision values were excellent. Enzyme kinetic and inhibition parameters obtained using these methods demonstrated high precision and were within the range of values previously reported in the scientific literature. These methods should prove useful in the routine assessments of the potential for new drug candidates to elicit pharmacokinetic drug interactions via inhibition of cytochrome P450 activities.

Modification by analgesics of lesion development in the urinary tract and various other organs of rats pretreated with dihydroxy-di-N-propylnitrosamine and uracil.

Effects of the analgesics phenacetin, acetaminophen and antipyrine on lesion development in the urinary tract and other organs in male F344 rats were investigated. Animals were concurrently administered with 0.1% dihydroxy-di-N-propylnitrosamine (DHPN) in drinking water and 3.0% uracil in the diet for 4 weeks and then, starting 1 week after the cessation of this treatment, received basal diet or diet containing phenacetin, acetaminophen or antipyrine for 35 weeks. The occurrences of renal cell tumors were increased in the groups given phenacetin or antipyrine, as compared with the DHPN + uracil alone controls. Antipyrine, but not the two other compounds, also enhanced development of hyperplastic lesions in the renal pelvis and ureter. In the urinary bladder, phenacetin and antipyrine treatments were both associated with increased incidence of preneoplastic or neoplastic lesions. Furthermore, phenacetin alone, without the initiating agent pretreatments, induced simple hyperplasias of the urinary bladder at high incidence. Antipyrine enhanced induction of hyperplastic lesions in the ureter and was also found to increase the incidences of preneoplastic and neoplastic lesions in the liver. Although decreased incidences of tumor development of lung and thyroid were observed for the group given phenacetin, this might have been linked to the decreased weight gain. The results confirmed that combination treatment with DHPN + uracil is effective for wide-spectrum initiation of carcinogenesis in the urological tract and demonstrated significant modification potential for both phenacetin and antipyrine.

Taxol metabolism in rat hepatocytes.

Metabolism of the anticancer drug taxol was investigated in freshly isolated rat hepatocytes. Two main metabolites were separated by reversed-phase HPLC and shown by tandem mass spectrometry to be monohydroxylated metabolites. Kinetic studies revealed apparent Km values of 68 and 61 microM with identical Vmax values for the two metabolites. Verapamil and midazolam, but not phenacetin, showed concentration-dependent inhibition of taxol metabolism with both metabolites being affected equally. The IC50 was about 100 microM for verapamil and 25 microM for midazolam. These observations demonstrate for the first time in vitro metabolism of taxol and suggest that the metabolism may be subject to potentially important interactions with numerous other drugs.

Caffeine, estradiol, and progesterone interact with human CYP1A1 and CYP1A2. Evidence from cDNA-directed expression in Saccharomyces cerevisiae.

Heterologous expression of cytochrome P-450 cDNAs in yeast is a potent instrument for the study of enzyme-specific parameters and can be used to answer questions with regard to substrate specificity as well as drug interaction in a background with no interfering activities. Two cDNAs of human CYP1A1 and CYP1A2 were expressed in yeast Saccharomyces cerevisiae, and microsomes of transformed strains contained substantial amounts of functional heterologous enzymes. Enzyme kinetics with 7-ethoxyresorufin as substrate resulted in KM values of 0.017 and 1.67 microM and Vmax values of 840 and 387 pmol/mg/min for CYP1A1 and CYP1A2, respectively. Both heterologous enzymes showed an overlapping substrate specificity pattern assayed with different phenoxazone ethers and caffeine. Caffeine was shown to be metabolized by CYP1A2 and CYP1A1. Both enzymes formed paraxanthine and minor amounts of theobromine; however, trimethyluric acid was exclusively formed by CYP1A1. The fact that theophylline was not formed by either enzyme anticipates the involvement of additional enzyme(s) in the primary metabolism of caffeine. Inhibition studies with caffeine, phenacetin, 17 beta-estradiol, and progesterone as inhibitors of the CYP1A1 and CYP1A2 catalyzed O-deethylation of 7-ethoxyresorufin suggest all compounds as possible substrates of CYP1A enzymes. 17 beta-estradiol inhibited CYP1A1-catalyzed paraxanthine and trimethyluric acid formation. In contrast 17 beta-estradiol did not inhibit CYP1A2-catalyzed formation of primary caffeine metabolites. These data clearly demonstrate the capacity of human CYP1A1 and CYP1A2 to metabolize caffeine. Furthermore, possible consequences of CYP1A enzyme inhibition by caffeine, phenacetin, 17 beta-estradiol, and progesterone will be discussed.

Effects of the phenacetin metabolite 4-nitrosophenetol on glycolysis and pentose phosphate pathway in human red cells.

Human erythrocytes exposed to 4-nitrosophenetol showed marked alterations of their endogenous metabolism. Rapid ferrihemoglobin formation mediated by the NADPH-dependent enzymic cycling of the nitrosoarene ("Kiese cycle") and extensive GSSG production caused an immediate drain of G-6-P into the pentose phosphate pathway at maximal flow. Despite a 2.4-fold increase in glucose phosphorylation rate and a branching ratio of 97:3 between pentose phosphate pathway and Embden-Meyerhof pathway, the G-6-P supply was obviously insufficient to meet the immense NADPH demand. Thus, a significant recycling of pentose phosphate pathway-derived F-6-P was observed in the order of 65%. Comparison of NADPH regeneration and ferrihemoglobin formation indicates the "Kiese cycle" to be a minor mechanism in ferrihemoglobin production in the case of high 4-nitrosophenetol concentrations. Most probably, reactive intermediates of 4-nitrosophenetol other than N-hydroxy-4-phenetidine, i.e. bicyclic arylamines and glutathione S-conjugates are formed which produce ferrihemoglobin without involvement of NADPH. The experiments have shown that red cells are remarkable robust to tackle the massive oxidative stress as elicited by 4-nitrosophenetol. The immediate metabolic response of the pentose phosphate pathway allows rapid regeneration of reduced glutathione. Thereby, SH-containing enzymes are effectively protected and/or regenerated and hemolysis is kept minimal. Hence, red cells are favourably suited for clearing the blood from N-oxygenated arylamines before they can reach more sensitive target organs.

Effects of the phenacetin metabolite 4-nitrosophenetol on the glutathione status and the transport of glutathione S-conjugates in human red cells.

The extent of ferrihemoglobin formation in human erythrocytes by 4-nitrosophenetol and its metabolisation rate strongly depended on the availability of cellular GSH. Ferrihemoglobin formation rate was increased by inhibition of the red cell glutathione reductase, and 4-nitrosophenetol disappeared more slowly. When red cells were completely depleted from SH groups, ferrihemoglobin formation was retarded, despite 4-nitrosophenetol was hardly metabolized. In turn, the glutathione status of human red cells was strongly affected by 4-nitrosophenetol. GSSG, which was produced in large amounts, was reduced, as long as the reducing system was intact. The decreased total glutathione content, however, did not recover completely, indicating formation of stable glutathione S-conjugates. The active export of the stable model glutathione thioether S-(2,4-dinitrophenyl)glutathione was strongly inhibited by 4-nitrosophenetol. A Lineweaver-Burk plot of the transport data suggested a competitive inhibition mechanism, presumably caused by glutathione adducts. The results indicate that the strong pi-donor substituent in 4-nitrosophenetol enables metabolic reactions with glutathione, producing biological effects hitherto not observed with nitrosobenzene. Bicyclic arylamines and glutathione S-conjugates may cause ferrihemoglobin formation that is not brought about by the diaphorase reaction. The latter may be responsible for transport inhibition of GSSG and other glutathione S-conjugates.

Nature of cytochromes P450 involved in the 2-/4-hydroxylations of estradiol in human liver microsomes.

Kinetics of the 2- and 4-hydroxylations of estradiol (E2) by human liver microsomal samples were studied to determine the major P450 isoform involved in these endogenous reactions. Thirty human liver microsomal samples were analysed. Metabolism of 25 microM [14C]E2 produced 2-hydroxy and 4-hydroxy derivatives with a ratio of 3.2 +/- 1.5 and a great inter-individual variation. Kinetic analysis of the 2- and 4-hydroxylations of E2 exhibited a curvilinear double reciprocal plot with an apparent Km of 15 microM. Further experiments demonstrated that alpha-naphthoflavone, testosterone and progesterone increased the 2-hydroxylation activity, suggesting the involvement of a substrate activation mechanism. These two hydroxylations of E2 were shown to be catalysed by cytochrome P450 with an apparent dissociation constant Ks of 0.8 microM. These 2- and 4-hydroxylations inter-correlated significantly (r = 0.93; N = 30). The 2-hydroxylation of E2 correlated with four monooxygenase activities known to be supported by P450 3A4/3A5, namely nifedipine oxidation (r = 0.78; N = 29); erythromycin N-demethylation (r = 0.69; N = 27), testosterone 6 beta-hydroxylation (r = 0.66; N = 25) and tamoxifen N-demethylation (r = 0.64; N = 29). On the other hand, E2-hydroxylations did not correlate with activities supported by P450 1A2 and P450 2E1. Furthermore, drugs as cyclosporin, diltiazem, triacetyl-oleandomycin and 17 alpha-ethynylestradiol inhibited more than 90% of the E2-hydroxylations at concentrations < 250 microM, while weak inhibition was shown with 500 microM cimetidine and no significant inhibition with caffeine, phenacetin and omeprazole. Finally, 2- and 4-hydroxylations of E2 correlated significantly with the content of P450 3A4/3A5 immunodetected by a monoclonal antibody anti-human P450-nifedipine (r = 0.84; N = 28). E2-hydroxylation activities were inhibited by more than 80% with polyclonal anti-human anti-P450-nifedipine. Preincubation of human liver microsomes with 100 microM gestodene (a suicide substrate of P450 3A4) inactivated this P450 isoform and accordingly allowed evaluation of the contribution of other P450 isoforms to the E2 metabolism to about 21% (+/- 17%, N = 29). All these results taken together suggest that P450 3A4/3A5 are the major forms involved in the formation of catecholestrogens in the human liver microsomes.

Effects of multiple dosing of phenacetin in the micronucleus test.

As a part of the international cooperative study to identify the most sensitive regimen in the micronucleus test, phenacetin was given i.p. to male CD-1 mice at doses of 37.5, 75, 150, 300, 400, and 600 mg/kg once, twice, thrice or four times and the bone marrow cells were harvested 24 h after the final dosing. Positive responses were seen at 600 mg/kg after single and triple dosing and at 400 and 600 mg/kg after double dosing. No dose level gave a positive response after quadruple dosing. A repeated-dosing effect was detected at double and triple dosing. Although triple dosing gave the highest magnitude of micronuclei at 600 mg/kg, double dosing showed a sufficient sensitivity and was more convenient from the viewpoint of selecting a suitable test dose and carrying out the micronucleus test.

Study of the ability of phenacetin, acetaminophen, and aspirin to induce cytotoxicity, mutation, and morphological transformation in C3H/10T1/2 clone 8 mouse embryo cells.

Use of the analgesic compounds acetylsalicylic acid (aspirin), phenacetin, and acetaminophen has been correlated with increased risk of renal cancer in humans. Hence, we studied these compounds for ability to induce cytotoxicity, mutation to ouabain resistance, and morphological transformation in cultured C3H/10T1/2 clone 8 (10T1/2) mouse embryo cells. All three compounds were cytotoxic from 0.5-mg/ml to 2-mg/ml concentrations as evidenced by decreased plating efficiency. None of the compounds induced detectable base substitution mutations to ouabain resistance even at cytotoxic concentrations. Aspirin did not induce morphological transformation. Both phenacetin and acetaminophen induced low but concentration-dependent numbers of atypical, weak type II morphologically transformed foci; at equimolar concentrations, phenacetin was 1.1- to 3.0-fold more active in inducing these foci. Neither phenacetin nor acetaminophen was cotransforming with 3-methylcholanthrene, and neither compound promoted cell transformation when added to 3-methylcholanthrene-initiated 10T1/2 cells. The focus-inducing potency of both compounds was increased by addition of an Arochlor-induced hamster liver S9 fraction as an exogenous metabolizing system. However, seven putative metabolites of phenacetin and acetaminophen that were tested--N-hydroxyphenacetin, p-phenetidine, p-aminophenol, p-nitrosophenol, benzoquinone, acetamide, and N-acetyl-p-benzoquinoneimine--were inactive in transformation assays at the concentrations reducing plating efficiency of treated cells to 50% of the plating efficiency of nontreated (control) cells. Several acetaminophen- and phenacetin-induced foci were cloned, expanded into cell lines, and characterized. These cell lines stably formed type II foci when maintained at confluence for 2 to 4 wk in reconstruction experiments with nontransformed 10T1/2 cells; however, they did not exhibit significantly increased saturation density compared to 10T1/2 cells, and they did not grow in soft agarose. These results suggest that metabolic intermediates of high concentrations of phenacetin and acetaminophen induce a low frequency of nonneoplastic morphological transformation of 10T1/2 mouse embryo cells.

The effects of acetaminophen, antipyrine and phenacetin on rat urothelial cell proliferation.

Abuse of combination analgesics containing phenacetin, antipyrine (phenazone) and caffeine have been associated with urinary tract tumors. Phenacetin and antipyrine have been shown to be promoters of urinary tract carcinogenesis and antipyrine is also a weak urinary tract carcinogen. Acetaminophen, the main metabolite of phenacetin, is one of the most commonly used analgesics in the USA. In the present study, the dose-related effect on the cell proliferation of the urothelium was evaluated in male Sprague-Dawley rats by autoradiography. Nine groups of twenty, 6-week old rats were treated with 0.5%, 1.0% or 1.5% of acetaminophen, antipyrine or phenacetin in the diet. A tenth group of rats received control diet without added chemicals. Ten rats from each group were killed after each of 6 and 12 weeks of feeding. There was a dose-related increase in the labeling index in the urothelium of the bladder and kidney, particularly after 6 weeks of drug administration. In particular, the 1.0% and 1.5% dose levels of antipyrine and phenacetin showed a marked proliferative effect on the urothelium. In the bladder after 6 weeks, the labeling indices were significantly increased. After 12 weeks, although numerically increased, the indices were not statistically significant. In the renal pelvic urothelium the labeling index was significantly increased in antipyrine and phenacetin treated rats at doses of 1.0% and 1.5%. After 12 weeks the majority of rats treated with 1.5% antipyrine and phenacetin had labeling indices greater than or equal to 2-fold than the control rats both in the kidney and bladder. The increased labeling indices were associated with urothelial hyperplasia, in particular after 6 weeks. In the rats treated with antipyrine there were significant degenerative changes in the urothelial cells expressed as marked vacuolization. The vacuolization is considered to be a toxic effect and the beginning of cell death. Thus cell death with regeneration may be responsible for the increased labeling index in the antipyrine groups. High doses of antipyrine were also associated with renal papillary necrosis in 50% of the rats.

Metabolic activation of phenacetin in rat nasal mucosa: dose-dependent binding to the glands of Bowman.

The metabolism and binding of the analgetic drug [ring-3H]phenacetin in the nasal mucosa were studied in vitro and in vivo in male Sprague-Dawley rats. As shown by whole-body and light microscopic autoradiography there was an irreversible binding of metabolites to the glands of Bowman in the olfactory mucosa after high but not after low doses of [3H]phenacetin. In the other tissues, the distribution of radioactivity was not changed when the dose was increased. Autoradiography of [3H]-acetaminophen showed no preferential uptake of radioactivity in the olfactory mucosa. At incubation of nasal septa with [3H]phenacetin in vitro, a binding of metabolites to the glands of Bowman was observed indicating that the metabolism occurred in situ. In rats, glutathione (GSH) depleted by pretreatment with phorone, there was a binding to the glands of Bowman in the olfactory mucosa also after a trace dose of [3H]phenacetin. Addition of GSH decreased the irreversible binding of [3H]phenacetin metabolites that occurred in 9000 X g nasal mucosa supernatants incubated with [3H]phenacetin. There was a moderate decrease in the level of nonprotein sulfhydryl groups, mainly GSH, in the olfactory mucosa after administration of 100-300 mg/kg phenacetin. Collectively, these data suggest that phenacetin is metabolized and subsequent to GSH depletion, bound preferentially in the glands of Bowman. The data also suggest that in situ metabolic activation and binding of phenacetin in the rat nasal mucosa at high doses may play a role in the pathogenesis of the nasal tumors induced by high doses of phenacetin in the rat.

Effect of phenacetin and caffeine on N-butyl-N-(4-hydroxybutyl)-nitrosamine-initiated urothelial carcinogenesis in rats.

The present experimental study was undertaken to clarify whether phenacetin and caffeine exert a cocarcinogenic and/or promoting effect on N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN)-initiated urothelial carcinogenesis. BBN was initially administered to female Wistar rats by gavage in 3 consecutive fractionated doses of 100 mg/kg body weight each at 24-hour intervals. Phenacetin was continuously fed at a daily dose of 500 mg/kg body weight, and caffeine was given in the drinking water at a dose of 110 mg/kg body weight/day throughout the experiment. After an experimental period of 21 months the incidence of BBN-induced tumors in the urinary bladder (number of rats with a bladder tumor) had not increased following additional administration of phenacetin alone (47%) or in combination with caffeine (48%) compared with the control group, the animals of which received exclusively BBN (44%). However, there was a significant enhancement of a multifocal tumor development (number of rats with more than 1 tumor in the bladder), when additionally phenacetin was fed alone (44% of the tumor-bearing animals) or in combination with caffeine (47%) compared with the control rats treated with BBN alone which showed only solitary tumors. Similarly, the incidence of a multicentric tumor development had increased, although not significantly, following administration of phenacetin alone or simultaneously with caffeine for 15 months. Caffeine revealed no complete initiating carcinogenic potential for the resting as well as the regenerating bladder urothelium stimulated to proliferate by either a partial cystectomy or cyclophosphamide. Furthermore, no cocarcinogenic and/or promoting activity of caffeine on BBN-initiated bladder tumor development was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of DNA, RNA and protein synthesis and chromatin alteration by N-hydroxyphenacetin.

The effects of N-hydroxyphenacetin on DNA function and structure were investigated to elucidate the involvement of phenacetin in analgesic nephropathy and transitional cell carcinoma. N-Hydroxyphenacetin or a metabolite inhibited synthesis of DNA, RNA and protein; DNA inhibition was greater at higher pH. No single-strand breaks were detectable in DNA after N-hydroxyphenacetin treatment and no appreciable effect on cell viability was observed at concentrations up to 5 mM. N-Hydroxyphenacetin-induced alteration to chromatin structure was detected using nucleoid sedimentation analysis. Direct binding to plasmid DNA was not observed. These observations are consistent with a role for phenacetin metabolites in renal disease.