Disposition and metabolism of the food mutagen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in rats

The disposition and metabolism of a common food mutagen, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), was studied in rats. Five rats of both sexes were given a single oral dose of 14C-labeled MeIQx (3-4 mg/kg body wt). The male rats excreted 36% of the radioactivity and 15% of the mutagenic activity of the dose given in the urine collected during the first 24 h. In the females the corresponding urine contained 41% of the radioactivity and 12% of the mutagenicity. During the next 48 h only 1-3% of the radioactive dose was excreted in urine. The remaining dose was excreted in the feces except of less than 1% that was retained by the tissues after 72 h. The liver and kidney retained more radioactivity than other organs. In a separate study the metabolites of bile, urine and feces of both sexes were investigated. After a single oral dose of 20 mg 14C-labeled MeIQx/kg body wt, three major non-mutagenic metabolites were identified. These were 2-amino-4(or 5)-(beta-D-glucuronopyranosyloxy)-3,8-dimethylimidazo[4,5-f] quinoxaline, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxalin-4(or 5)-yl sulfate and N-(3,8-dimethylimidazo[4,5-f]quinoxalin-2-yl) sulfamate. Another two metabolites present in bile, urine and feces were 2-(beta-D-glucuronopyranosylamino)-3,8-dimethylimidazo[4,5-f ] quinoxaline and 2-amino-8-hydroxymethyl-3-methylimidazo[4,5-f]quinoxalin-4 (or 5)yl sulfate. All metabolites were essentially non-mutagenic. Most of the mutagenicity still present in bile, urine and feces could be explained by unchanged MeIQx. Unchanged MeIQx was the most abundant form excreted in urine.     

Pharmacokinetics, distribution, metabolism and excretion of [3H]UCN-01 in rats and dogs after intravenous administration

PURPOSE: To evaluate the metabolic fate of UCN-01, a signal transduction inhibitor, blood and plasma concentrations, distribution, metabolism and excretion were investigated in rats and dogs after intravenous administration of [3H]UCN-01. METHODS: The radioactivity in plasma, blood and tissues was measured after intravenous administration of UCN-01. In addition, the radioactivity excreted in bile, urine and feces was also determined. RESULTS: The radioactivity in rat and dog plasma disappeared triphasically with terminal half-lives of 21.3 and 27.2 h, respectively. The ratios of the blood-to-plasma concentrations ranged from 0.82 to 1.13 in rats and 0.81 to 1.73 in dogs. From 0.5 to 4 h after giving [3H]UCN-01 to rats, the radioactivity in all tissues except the brain and testes was higher than in plasma. The highest concentration was observed in the lungs followed by the liver and kidneys. The radioactivity was mainly excreted in feces, reaching 96.0% of the radioactivity dose in rats and 78.4% in dogs up to 168 h after injection. Since the biliary excreted radioactivity was 67.2% over 48 h in bile duct-cannulated rats, most of the radioactivity excreted in feces was from biliary radioactivity. There were several metabolites in bile samples, but little UCN-01. CONCLUSIONS: UCN-01 is mainly eliminated by the liver, and there are high concentrations of radioactivity derived from [3H]UCN-01 in all tissues except the brain and testes.     

Inhibition of growth and induction of apoptosis in human cancer cell lines by tea polyphenols

In order to study the biological activities of tea preparations and purified tea polyphenols, their growth inhibitory effects were investigated using four human cancer cell lines. Growth inhibition was measured by [3H]thymidine incorporation after 48 h of treatment. The green tea catechins (-)-epigallocatechin-3-gallate (EGCG) and (-)- epigallocatechin (EGC) displayed strong growth inhibitory effects against lung tumor cell lines H661 and H1299, with estimated IC50 values of 22 microM, but were less effective against lung cancer cell line H441 and colon cancer cell line HT-29 with IC50 values 2- to 3- fold higher. (-)-Epicatechin-3-gallate, had lower activities, and (-)- epicatechin was even less effective. Preparations of green tea polyphenols and theaflavins had higher activities than extracts of green tea and decaffeinated green tea. The results suggest that the growth inhibitory activity of tea extracts is caused by the activities of different tea polyphenols. Exposure of H661 cells to 30 microM EGCG, EGC or theaflavins for 24 h led to the induction of apoptosis as determined by an annexin V apoptosis assay, showing apoptosis indices of 23, 26 and 8%, respectively; with 100 microM of these compounds, the apoptosis indices were 82, 76 and 78%, respectively. Incubation of H661 cells with EGCG also induced a dose-dependent formation of H2O2. Addition of H2O2 to H661 cells caused apoptosis in a manner similar to that caused by EGCG. The EGCG-induced apoptosis in H661 cells was completely inhibited by exogenously added catalase (50 units/ml). These results suggest that tea polyphenol-induced production of H2O2 may mediate apoptosis and that this may contribute to the growth inhibitory activities of tea polyphenols in vitro.      

Mechanisms of Growth Inhibition of Human Lung Cancer Cell Line, PC-9, by Tea Polyphenols

(–)-Epigallocatechin gallate (EGCG), the main constituent of green tea, and green tea extract show growth inhibition of various cancer cell lines, such as lung, mammary, and stomach. We studied how tea polyphenols induce growth inhibition of cancer cells. Since green tea extract contains various tea polyphenols, such as EGCG, (–)-epigallocatechin (EGC), (–)-epicatechin gallate (ECG), and (–)-epicatechin (EC), the inhibitory potential of each tea polyphenol on the growth of a human lung cancer cell line, PC-9 cells, was first examined. EGC and ECG inhibited the growth of PC-9 cells as potently as did EGCG, but EC did not show significant growth inhibition. The mechanism of growth inhibition by EGCG was studied in relation to cell cycle regulation. Flow cytometric analysis revealed that treatment with 50 μM and 100 μM EGCG increased the percentages of cells in the G₂-M phase from 13.8% to 15.6% and 24.1%, respectively. The DNA histogram after treatment with 100 μM EGCG was similar to that after treatment with genistein, suggesting that EGCG induces G₂-M arrest in PC-9 cells. Moreover, we found by microautoradiography that [³H]EGCG was incorporated into the cytosol, as well as the nuclei. These results provide new insights into the mechanisms of action of EGCG and green tea extract as cancer-preventive agents in humans.     

Phase I pharmacokinetic study of tea polyphenols following single-dose administration of epigallocatechin gallate and polyphenon E.

Green tea has been shown to exhibit cancer-preventive activities in preclinical studies. Its principal active components include epigallocatechin gallate (EGCG), epigallocatechin (EGC), epicatechin (EC), and epicatechin gallate, of which EGCG is the most abundant and possesses the most potent antioxidative activity. We performed a Phase I pharmacokinetic study to determine the systemic availability of green tea catechins after single oral dose administration of EGCG and Polyphenon E (decaffeinated green tea catechin mixture). Twenty healthy subjects (five subjects/dose level) were randomly assigned to one of the dose levels (200, 400, 600, and 800 mg based on EGCG content). All subjects were randomly crossed-over to receive the two catechin formulations at the same dose level. Blood and urine samples were collected for up to 24 h after oral administration of the study medication. Tea catechin concentrations in plasma and urine samples were determined using high-performance liquid chromatography with the coulometric electrode array detection system. After EGCG versus Polyphenon E administration, the mean area under the plasma concentration-time curves (AUC) of unchanged EGCG were 22.5 versus 21.9, 35.4 versus 52.2, 101.9 versus 79.7, and 167.1 versus 161.4 min x microg/ml at the 200-, 400-, 600-, and 800-mg dose levels, respectively. EGC and EC were not detected in plasma after EGCG administration and were present at low/undetectable levels after Polyphenon E administration. High concentrations of EGC and EC glucuronide/sulfate conjugates were found in plasma and urine samples after Polyphenon E administration. There were no significant differences in the pharmacokinetic characteristics of EGCG between the two study medications. The AUC and maximum plasma concentration (Cmax) of EGCG after the 800-mg dose of EGCG were found to be significantly higher than those after the 200- and 400-mg dose. The AUC and Cmax of EGCG after the 800-mg dose of Polyphenon E were significantly higher than those after the three lower doses. We conclude that the two catechin formulations resulted in similar plasma EGCG levels. EGC and EC were present in the body after the Polyphenon E administration; however, they were present predominantly in conjugated forms. The systemic availability of EGCG increased at higher doses, possibly due to saturable presystemic elimination of orally administered green tea polyphenols.     

Synthesis and excretion profile of 1,4- [14C]phenylenebis(methylene)selenocyanate in the rat

1,4-Phenylenebis(methylene)selenocyanate (p-XSC) inhibits chemically induced tumors in several laboratory animal models. To understand its mode of action, we synthesized p-[14C]XSC, examined its excretion pattern in female CD rats and also the nature of its metabolites. p- [14C]XSC was synthesized from alpha,alpha-dibromo-p-[ring-14C]xylene in 80% yield. The excretion profile of p-[14C]XSC (15.8 mg/kg body wt, 200 microCi/rat, oral administration, in 1 ml corn oil) in vivo was monitored by measuring radioactivity and selenium content. On the basis of radioactivity, approximately 20% of the dose was excreted in the urine and 68% in the feces over 3 days. The cumulative percentages of the dose excreted over 7 days were 24% in urine and 75% in feces, similar to excretion rates of selenium. According to selenium measurement, <1% of the dose was detected in exhaled air; radioactivity was not detected. Only 15% of the dose was extractable from the feces with EtOAc and was identified as tetraselenocyclophane (TSC). Most of the radioactivity remained tightly bound to the feces. Approximately 10% of this bound material converted to TSC on reduction with NaBH4. Organic soluble metabolites in urine did not exceed 2% of the dose; sulfate (9 % of urinary metabolites) and glucuronic acid (19.5% of urinary metabolites) conjugates were observed but their structural identification is still underway. Co-chromatography with a synthetic standard led to the detection of terephthalic acid (1,4- benzenedicarboxylic acid) as a minor metabolite. The major urinary conjugates contained selenium. Despite the low levels of selenium in the exhaled air, the reductive metabolism of p-XSC to H2Se cannot be ruled out. Identification of TSC in vivo indicates that a selenol may be a key intermediate responsible for the chemopreventive action of p- XSC.      

Excretion of DNA adducts of 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline, PhIP- dG, PhIP-DNA and DiMeIQx-DNA from the rat

The heterocyclic aromatic amines, 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP) and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx) are formed during frying of meat. PhIP and 4,8-DiMeIQx have, after metabolic activation, been shown to form adducts with DNA at the C8 of guanine both in vitro and in vivo. In order to investigate possible urinary biomarkers for estimation of the genotoxic dose of PhIP and 4,8-DiMeIQx, [3H]PhIP-dG, [3H]PhIP-DNA and [14C]4,8-DiMeIQx- DNA were injected i.p. to rats and the excretion of radioactivity in urine and faeces were measured. For all three [3H]PhIP-dG, [3H]PhIP-DNA and [14C]4,8-DiMeIQx-DNA 15-20% of the dose were excreted in the urine and 80-85% of the dose were excreted in the faeces. Urinary excretion showed maximum to 24 h (90%) with a rapid decline, 10% to 48 h and 0% to 72 h. Faecal excretion also showed maximum to 24 h (60%) with a slower decline, 30% to 48 h and 10% to 72 h. HPLC analysis of samples of urine and extracts from faeces, from rats dosed with [3H]PhIP-dG, showed that approximately 90% of the radioactivity co-eluted with PhIP- dG, indicating that PhIP-dG is excreted unmetabolized. HPLC analysis of samples of urine and extracts from faeces, from rats dosed with [3H]PhIP-DNA, showed that approximately 85% of the radioactivity co- eluted with PhIP-dG, indicating that PhIP-DNA adducts is mainly excreted as nucleoside adducts. Approximately 5% of the radioactivity excreted in the urine co-eluted with PhIP-G, indicating loss of deoxyribose. HPLC analysis of samples of urine and extracts from faeces, from rats dosed with [14C]4,8-DiMeIQx-DNA, showed that approximately 90% of the radioactivity co-eluted with 4,8-DiMeIQx-dG, indicating that 4,8-DiMeIQx-DNA adducts is mainly excreted as nucleoside adducts. Man is able to eliminate compounds of a higher mol. wt in the urine than the rat, the percentage of PhIP-dG and 4,8-DiMeIQx eliminated in the urine of man would therefore be expected to be higher than in the rat. Measurement of urinary nucleoside adducts of PhIP and 4,8-DiMeIQx could therefore provide a basis for the development of a biomonitoring strategy for the genotoxic dose of these food derived HAA.      

Influence of route of administration on [3H]-camptothecin distribution and tumor uptake in CASE-bearing nude mice: whole-body autoradiographic studies

 Camptothecin (CPT) inhibits the growth of a wide variety of experimental tumors. As a part of our exploration of this drug for use as a cancer chemotherapeutic agent, we studied the effect of route of administration on the absorption, distribution and tumor uptake of [³H]-CPT. The rate of disappearance of [³H]-CPT-derived radioactivity from blood during the first 48 h was highest following oral than following intravenous (i.v.) administration. Thereafter blood levels were low irrespective of route of administration. Considerable [³H]-CPT-derived radioactivity was detected in urine and feces up to 48 h after dosing. Distribution studies were conducted using quantitative whole-body autoradiography (WBA). These studies revealed that independent of the route of administration, [³H]-CPT was rapidly excreted in the bile (gallbladder) followed by elimination into the small and large intestinal tract. Levels of CPT-derived radioactivity in the kidneys were minimal and mostly localized in the renal pelvis. Hepatic concentrations of CPT were low and were almost equal to those of the tumor. The lungs of animals treated i.v. showed higher uptake of radioactivity than those treated intramuscularly or orally. Tumor/blood ratios were slightly higher following oral administration than following administration by other routes. This study indicates that CPT is primarily eliminated via the bile. The gastrointestinal tract is the major site of accumulation and excretion of CPT. Received: 13 May1994/Accepted: 18 March 1996     

Pharmacokinetics and safety of green tea polyphenols after multiple-dose administration of epigallocatechin gallate and polyphenon E in healthy individuals.

PURPOSE: Green tea and green tea polyphenols have been shown to possess cancer preventive activities in preclinical model systems. In preparation for future green tea intervention trials, we have conducted a clinical study to determine the safety and pharmacokinetics of green tea polyphenols after 4 weeks of daily p.o. administration of epigallocatechin gallate (EGCG) or Polyphenon E (a defined, decaffeinated green tea polyphenol mixture). In an exploratory fashion, we have also determined the effect of chronic green tea polyphenol administration on UV-induced erythema response. EXPERIMENTAL DESIGN: Healthy participants with Fitzpatric skin type II or III underwent a 2-week run-in period and were randomly assigned to receive one of the five treatments for 4 weeks: 800 mg EGCG once/day, 400 mg EGCG twice/day, 800 mg EGCG as Polyphenon E once/day, 400 mg EGCG as Polyphenon E twice/day, or a placebo once/day (8 subjects/group). Samples were collected and measurements performed before and after the 4-week treatment period for determination of safety, pharmacokinetics, and biological activity of green tea polyphenol treatment. RESULTS: Adverse events reported during the 4-week treatment period include excess gas, upset stomach, nausea, heartburn, stomach ache, abdominal pain, dizziness, headache, and muscle pain. All of the reported events were rated as mild events. For most events, the incidence reported in the polyphenol-treated groups was not more than that reported in the placebo group. No significant changes were observed in blood counts and blood chemistry profiles after repeated administration of green tea polyphenol products. There was a >60% increase in the area under the plasma EGCG concentration-time curve after 4 weeks of green tea polyphenol treatment at a dosing schedule of 800 mg once daily. No significant changes were observed in the pharmacokinetics of EGCG after repeated green tea polyphenol treatment at a regimen of 400 mg twice daily. The pharmacokinetics of the conjugated metabolites of epigallocatechin and epicatechin were not affected by repeated green tea polyphenol treatment. Four weeks of green tea polyphenol treatment at the selected dose and dosing schedule did not provide protection against UV-induced erythema. CONCLUSIONS: We conclude that it is safe for healthy individuals to take green tea polyphenol products in amounts equivalent to the EGCG content in 8-16 cups of green tea once a day or in divided doses twice a day for 4 weeks. There is a >60% increase in the systemic availability of free EGCG after chronic green tea polyphenol administration at a high daily bolus dose (800 mg EGCG or Polyphenon E once daily).     

Disposition and metabolism of KW-2149, a novel anticancer agent

KW-2149 is a new derivative of mitomycin C (MMC). The plasma concentrations, distribution, metabolism, and excretion of [³H]-KW-2149 in normal and tumor-bearing mice after i. v. administration of 16.6 mg/kg were investigated. The plasma radioactivity decreased biexponentially after i. v. administration in normal mice. However, the unchanged drug disappeared rapidly, showing a half-life (t _1/2) of 9.7 min, which was shorter than MMC's (18 min). The radioactivity was excreted in mouse urine (33%) and feces (58%) within 144 h. High radioactivity was distributed in the gallbladder, liver, kidney, pancreas, and lung at 1 h after i. v. administration to normal mice. The tumor concentration was lower than the plasma or blood concentration. The lowest radioactivity was observed in the brain. The metabolic rate of KW-2149 was very rapid. The methyl sulfide form (M-16), the symmetrical disulfide dimer (M-18), and the albumin conjugate were detected in plasma, which possessed anticellular activity. The specific anticellular activity of these compounds against uterine carcinoma (HeLa S3) was 1/100, 1, and 1/20 respectively, as compared with that of KW-2149.Abbreviations MMC mitomycin C - LD₁₀ 10% lethal dose - HPLC high-performance liquid chromatography - AUC area under the concentration-time curve - t _1/2 half-life - Vd_ss volume of distribution at steady state - Cl_tot total clearance     

Pharmacokinetics of deguelin, a cancer chemopreventive agent in rats

PURPOSE: To study the pharmacokinetics of deguelin, a naturally occurring potential cancer chemopreventive agent, in rats. METHODS: [3H]Deguelin was administered intravenously (i.v.) under anesthesia, and blood samples were collected over 24 h. [3H]Deguelin and metabolites were extracted from plasma with ethyl acetate, and quantified by HPLC. Data were analyzed with the WinNolin pharmacokinetic software package to determine pharmacokinetic parameters. A three-compartment first-order elimination model was used to fit the plasma concentration-time curve. In addition, deguelin concentrations in tissues after i.v. and intragastric (i.g.) administration were determined by HPLC, and excretion (feces and urine) was evaluated over a 5-day period after i.g. administration. RESULTS: Deguelin exhibited a mean residence time (MRT) of 6.98 h and terminal half-life (t1/2(gamma)) of 9.26 h. The area under the curve (AUC) and total clearance (Cl) were 57.3 ng.h/ml and 4.37 l/h per kg, respectively, with an apparent volume of distribution (V) and volume of distribution at steady-state (Vss) of 3.421 l/kg and 30.46 l/kg, respectively. Following i.v. administration, the relative levels of tissue distribution were as follows: heart > fat > mammary gland > colon > liver > kidney > brain > lung. Following i.g. administration, the relative levels of tissue distribution were as follows: perirenal fat > heart > mammary gland > colon > kidney > liver > lung > brain > skin. Within 5 days of i.g. administration, about 58.1% of the [3H]deguelin was eliminated via the feces and 14.4% via the urine. Approximately 1.7% of unchanged deguelin was found in the feces, and 0.4% in the urine. CONCLUSIONS: An initial pharmacokinetic investigation of deguelin showed that this rotenoid has a relatively long MRT and half-life in plasma in the rat. The compound distributed in the tissues and excreted as metabolites, mainly via the feces.     

Metabolism and disposition of bladder carcinogens in rat and guinea pig: possible mechanism of guinea pig resistance to bladder cancer

The metabolism and disposition of N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT) and 2-amino-4-(5-nitro-2-furyl)thiazole (ANFT) were studied in rat and guinea pig. Rat is susceptible whereas guinea pig is resistant to FANFT-induced bladder cancer. Rats and guinea pigs were p.o. administered either 2-[14C]ANFT or 2-[14C]FANFT (100 mg/kg), and 18-h urine and feces were collected. Tissue distribution of radiolabel was determined. In both species, the highest concentrations of radioactivity expressed as nmol/g tissue were observed in the urine and intestines. Urinary metabolites were separated by high-performance liquid chromatography and radioactivity determined by radioanalytical detection. FANFT was not detected in urine from either species under any experimental condition. More ANFT was observed in urine following FANFT than ANFT administration. This deformylation-dependent excretion of FANFT was demonstrated in both species and has been previously described as renal metabolic/excretory coupling. Less ANFT, the carcinogen more proximate than FANFT, is excreted in guinea pigs compared with rats. A unique ANFT metabolite was identified in guinea pig but not rat urine. This metabolite represented 80 and 18% of radioactivity recovered in guinea pig urine following ANFT and FANFT administration, respectively. A metabolite produced by guinea pig liver and kidney microsomes in the presence of uridine-5'-diphosphoglucuronic acid coeluted with this unique metabolite. The urinary metabolite was characterized using hydrolytic enzymes, acid hydrolysis, and mass spectrometry and identified as an ANFT-N-glucuronide. A unique UDP-glucuronosyl-transferase appears to be responsible, at least in part, for the reduced amount of free ANFT excreted by guinea pigs compared with rats. Reduced levels of urinary ANFT observed in guinea pigs may partially explain the resistance of this species to FANFT-induced bladder cancer.     

Clinical pharmacokinetics of 5-fluorouracil and its metabolites in plasma, urine, and bile

Kinetics of 5-fluorouracil (FUra) and FUra metabolites in plasma and urine were investigated in 10 cancer patients following i.v. bolus administration of 500 mg/m2 FUra with 600 microCi of [6-3H]FUra. Biliary excretion was examined in two patients with external biliary catheters. Quantitation of unchanged drug and metabolites was assessed by a highly specific high-performance liquid chromatographic method. FUra plasma levels declined rapidly with an apparent elimination half-life of 12.9 +/- 7.3 min. Dihydrofluorouracil was detected within 5 min in most patients, demonstrating rapid catabolism and reached maximum peak levels of 23.7 +/- 9.9 microM at approximately 60 min. The apparent elimination half-life of dihydrofluorouracil (61.9 +/- 39.0 min) was consistently greater than that of the unchanged drug. The apparent elimination half-lives of the subsequent metabolites alpha-fluoro-beta-ureidopropionic acid and alpha-fluoro-beta-alanine were prolonged with values of 238.9 +/- 175.4 min and 1976 +/- 358 min, respectively. Approximately 60-90% of the administered dose was excreted in urine within 24 h, primarily as alpha-fluoro-beta-alanine. Biliary excretion accounted for 2-3% of total administered radioactivity. The major fraction of this radioactivity eluted on high-performance liquid chromatography as a previously unrecognized FUra metabolite. Analysis of its structure is currently ongoing in our laboratory. In conclusion, this study provides the first comprehensive analysis of the formation and excretion of FUra metabolites in plasma, urine, and bile following i.v. bolus administration of FUra in humans.     

Distribution and metabolism of the natural anticarcinogen phenethyl isothiocyanate in A/J mice

The distribution and metabolism of phenethyl isothiocyanate (PEITC), a naturally occurring anticarcinogen, was investigated in A/J mice. Mice were administered 5 mumol of [14C]PEITC (2 microCi/mouse) by gavage and killed at 1, 2, 4, 8, 24, 48 or 72 h after dosing. Radioactivity present in the spleen, heart, liver, lung, kidney, brain, urine and feces was measured. Lung, the target tissue of PEITC inhibition of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) lung tumorigenesis, showed maximum radioactivity between 4 and 8 h after dosing, suggesting this time period would be optimal for maximal inhibition by PEITC in A/J mice. Approximately 50% of the total radioactivity was excreted within 24 h after dosing with nearly 80% of radioactivity found in urine and feces at 72 h. Two metabolites were isolated by reverse-phase HPLC from urine of mice treated with PEITC. The identities of these metabolites were determined by comparison with synthetic standards and by NMR and MS. The major metabolite was a cyclic mercaptopyruvic acid conjugate, whereas the minor metabolite was an N-acetylcysteine conjugate. Approximately 25% of the administered dose of PEITC was excreted as the cyclic mercaptopyruvic acid conjugate and 10% as the N-acetylcysteine conjugate. These results suggest that urinary metabolites of PEITC may provide potentially useful dosimeters for this natural anticarcinogen.     

trans-4-Hydroxytamoxifen concentration and metabolism after local percutaneous administration to human breast

trans-4-Hydroxytamoxifen (4-OHTAM), a very active metabolite of the antiestrogen tamoxifen, was percutaneously administered to the affected breast of nine patients before surgery for breast cancer in order to evaluate 4-OHTAM absorption through the skin and its subcellular localization and metabolism. After percutaneous administration of 80 muCi, [3H]-4-OHTAM was detected in breast tissue. It was especially concentrated in tumor tissue and nuclear and cytosolic fractions, in which it remained unmetabolized except for limited isomerization from the trans to the cis form. In contrast to breast tissue, concentrations of radioactivity remained low in plasma but with a high proportion of metabolites. In another experiment [3H]tamoxifen was percutaneously administered over the breast of 3 patients, resulting in tissue retention weaker and shorter than after [3H]-4-OHTAM. In addition [3H]-4-OHTAM was administered to either breast or abdominal skin; the appearance of radioactivity in plasma and urine was delayed after administration to the breast in comparison with administration to the abdomen. It therefore appears that 4-OHTAM passes through the skin and is concentrated in receptor structures of breast tissue, thus avoiding the hepatic metabolism subsequent to p.o. administration. We suggest that local percutaneous administration of this active antiestrogen could be useful in the treatment of hormone-dependent benign breast diseases.     

Anticancer agents coupled to N-(2-hydroxypropyl)methacrylamide copolymers. II. Evaluation of daunomycin conjugates in vivo against L1210 leukaemia

DBA2 mice were inoculated i.p. with 10(5)L1210 cells. Animals subsequently treated with daunomycin (single i.p. dose, 0.25-5.0 mg kg-1) all died. The maximum increase in mean survival time observed was approximately 135%. Animals treated with N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers conjugated to daunomycin (DNM) showed a significant increase in mean survival time when the polymer-drug linkage was biodegradable (i.e., Gly-Phe-Leu-Gly). Such treatment also produced a number of long term survivors (greater than 50 days). In contrast, HPMA copolymer conjugated to DNM via a non-degradable linkage (Gly-Gly) produced no increase in survival time relative to untreated control animals. The effect observed with biodegradable HPMA copolymer-DNM conjugates was dependent on the concentration of conjugated drug administered (optimum greater than 5 mg kg-1); the frequency of administration (multiple doses were more effective than single); the timing of administration (single doses given on days 1 and 3 were most effective); and the site of tumour inoculation and route of drug administration. Biodegradable HPMA copolymer-DNM conjugates administered i.p. were active against L1210 inoculated s.c. at higher doses than required to curb a peritoneal tumour. Under certain experimental conditions polymer-DNM conjugates containing fucosylamine or galactosamine proved more active than conjugates without the carbohydrate moeity. The mechanism of drug-conjugate action in vivo is at present unclear. Radioiodination of polymer showed approximately 75% of polymer-drug conjugate to be excreted 24 h after i.p. administration. Synthesis of HPMA conjugates containing [3H]DNM showed that polymer containing Gly-Gly-[3H]DNM was excreted (60% of radioactivity in the urine, 24 h) in macromolecular form. In contrast polymer containing Gly-Phe-Leu-Gly-[3H]DNM was largely excreted in the form of low molecular weight species.     

Inhibition of activator protein 1 activity and cell growth by purified green tea and black tea polyphenols in H-ras-transformed cells: structure-activity relationship and mechanisms involved.

ras gene mutation, which perpetually turns on the growth signal transduction pathway, occurs frequently in many cancer types. The mouse epidermal JB6 cell line has been transfected with a mutant H-ras gene to mimic carcinogenesis in vitro. These transformed cells (30.7b Ras 12) are able to grow in soft agar, exhibiting anchorage independence and high endogenous activator protein 1 (AP-1) activity, which can be detected by a stable AP-1 luciferase reporter. The present study investigated the ability of different pure green and black tea polyphenols to inhibit this ras signaling pathway. The major green tea polyphenols (catechins), (-)-epigallocatechin-3-gallate (EGCG), (-)-epigallocatechin, (-)-epicatechin-3-gallate, (-)-epicatechin, and their epimers, and black tea polyphenols, theaflavin, theaflavin-3-gallate, theaflavin-3'-gallate, and theaflavin-3,3'-digallate (TFdiG), were compared with respect to their ability to inhibit the growth of 30.7b Ras 12 cells and AP-1 activity. All of the tea polyphenols except (-)-epicatechin showed strong inhibition of cell growth and AP-1 activity. Among the catechins, both the galloyl structure on the B ring and the gallate moiety contributed to the growth inhibition and AP-1 activity; the galloyl structure appeared to have a stronger effect on the inhibitory action than the gallate moiety. The epimers of the catechins showed similar inhibitory effects on AP-1 activity. The addition of catalase to the incubation of the cells with EGCG or TFdiG did not prevent the inhibitory effect on AP-1 activity, suggesting that H2O2 does not play a significant role in the inhibition by tea polyphenols. Both EGCG and TFdiG inhibited the phosphorylation of p44/42 (extracellular signal-regulated kinase 1 and 2) and c-jun without affecting the levels of phosphorylated-c-jun-NH2-terminal kinase. TFdiG inhibited the phosphorylation of p38, but EGCG did not. EGCG lowered the level of c-jun, whereas TFdiG decreased the level of fra-1. These results suggest that tea polyphenols inhibited AP-1 activity and the mitogen-activated protein kinase pathway, which contributed to the growth inhibition; however, different mechanisms may be involved in the inhibition by catechins and theaflavins.     

Benzo(a)pyrene disposition and metabolism in rats following intratracheal instillation

[3H]Benzo(a)pyrene [B(a)P] disposition and metabolism were investigated in male Sprague-Dawley rats. [3H]B(a)P, in a vehicle of triethylene glycol, was administered by intratracheal instillation (1 microgram/kg body weight), and the amount of radioactivity in various organs was determined at timed intervals between 5 and 360 min. Elimination of radioactivity from lungs was biphasic with half-lives of 5 and 116 min. Radioactivity in liver increased rapidly, reaching a maximum of 21% of the dose within 10 min after instillation and decreasing thereafter until less than 5% of the dose was detected at 360 min after instillation. The carcass accounted for 15-30% of the dose within the time intervals investigated. Toxicokinetic parameters to describe elimination of unmetabolized B(a)P from blood following intratracheal administration were found to be very similar to those calculated following i.v. administration. B(a)P metabolites in lung, liver, and intestinal contents were identified. Notably, quinones were at highest concentrations in both lung and liver 5 min after instillation, accounting for 12 and 7% of organic extractable material, respectively. B(a)P disposition was also investigated in animals with and without biliary cannulas. Distribution patterns among organs were similar though the amount excreted in bile and intestinal contents was 74 and 40% of the dose, respectively. Types of metabolites in bile and intestinal contents were identified and compared. Lower fractions of the administered dose were detected as thioether and glucuronic acid conjugates in intestinal contents than in bile, indicating that enterohepatic circulation of B(a)P metabolites was occurring.