The effect of dose and enzyme inducers on the metabolism of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in rats.

Male Fischer 344 rats were given a single dose of 0.03-30 mg/kg of [2-14C]2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine ([14C]PhIP), the radioactivity in urine and feces was determined over 48 h, and the major metabolites were identified and quantified. Dose had little effect on the profile of metabolites in the urine but did influence the profile in the feces. PhIP was more efficiently metabolized at higher doses. In addition, rats were pretreated with Aroclor 1254 (PCB), 3-methylcholanthrene (MC), phenobarbital (PB), PhIP and corn oil prior to a single dose of [14C]PhIP, and compared with a control group receiving [14C]PhIP only. The major metabolites in the urine and feces were quantitated for each group, as well as PhIP binding to serum proteins, hemoglobin and selected tissues. Pretreatment with MC and PCB resulted in an increase in the amount of 4'-hydroxylation of PhIP and a decrease in the amount of N-hydroxylated metabolites in the urine. Pretreatment with PB resulted in an increase in the amount of N-hydroxylated metabolites, but a decrease in 4'-hydroxylation. Pretreatment with either MC or PCB resulted in an increase in PhIP binding to the liver and kidney, while reducing the binding in other tissues. Animals pretreated with PhIP showed few significant differences from the untreated group, while pretreatment with PB in general resulted in a decrease of PhIP binding in tissues.     

Binding of the food mutagen PhIP in pigmented tissues of mice.

The distribution of the 14C-labelled food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in the tissues of C57B1/6 and NMRI mice was studied. The results showed a high and selective binding of radioactivity in the pigment epithelium of the eye and in the fur following a single dose (0.3-4 mg/kg) of [14C]PhIP in the pigmented C57B1/6 mice whereas no such localization of radioactivity was present in the albino NMRI mice. A low but selective covalent binding of radioactivity was observed in the liver, inner cortex of the kidney and in the tracheal mucosa of [14C]PhIP-injected mice. PhIP was firmly bound to synthetic melanin pigment in vitro; only 3% was released by extraction with a phosphate buffer (pH 7) whereas 72% was released by extraction by methanol:conc. NH3 (15:1). Three hours to 7 days following a single injection of [14C]PhIP in C57B1/6 mice the radioactivity in the eye was 3- to 6-fold higher than that in the liver or kidney. Almost 60% of the radioactive material present in the pigmented epithelium of the eye 3 and 24 h following injection could be extracted by basic methanol and identified as unchanged PhIP. The residual radioactivity in the pigmented epithelium of the eyes may represent a covalent binding of [14C]PhIP metabolites to cellular constituents or to a basic methanol-resistant binding of [14C]PhIP to melanin. The results indicate that pigmented tissues may be potential target tissues for the toxic effects of PhIP and suggest that the use of hair for biological monitoring of PhIP should be examined.     

Disposition and metabolism of [14-14C] 4-demethoxydaunorubicin HCl (idarubicin) and [14-14C]daunorubicin HCl in the rat

Summary The disposition of [14-¹⁴C]4-demethoxydaunorubicin HCl ([14-¹⁴C]idarubicin HCl, [¹⁴C]IDR) and of [14-¹⁴C]daunorubicin HCl ([¹⁴C]DNR) was studied in male Sprague Dawley rats. [¹⁴C]IDR was administered either IV at 0.25 mg/kg body weight or PO at 1 mg/kg body weight, whereas [¹⁴C]DNR was dosed IV at 1 mg/kg body weight. The main elimination route for both compounds was the bile, fecal excretion representing 0.75–0.8 times the total dose at 72 h. Radioactivity due to [¹⁴C]IDR-derived species is released by the tissues at a slower rate than activity derived from [¹⁴C]DNR. After IV treatment comparable plasma levels are obtained, but tissue radioactivity is markedly lower with [¹⁴C]IDR, in keeping with the lower dosage. The ratio of plasma to tissue radioactivity is even higher in animals treated PO with [¹⁴C]IDR, because of the more extensive metabolism after this route of administration. The 13-dihydro derivatives of both [¹⁴C]IDR and [¹⁴C]DNR are the main metabolites in tissues, but in the case of the former, products of phase II reactions become more important at later times in liver and kidney and in excreta.     

The fate of [14C]streptozotocin in nicotinamide-pretreated mice: observations on pancreatic islet radioactivity and urinary N1-methyl-14C]nicotinamide-excretion.

A high labelling of the pancreatic islets was found 3 and 24 h after a diabetogenic dose of [14C]streptozotocin to mice in which the acids islet injury had been prevented by nicotinamide-pretreatment. In non-pretreated [14C]streptozotocin-injected mice, a much lower radioactivity was observed in the pancreatic islets; at 3 h and at 24 h, there was no detectable radioactivity in the islets. No evidence was found to indicate that nicotinamide-pretreatment had any marked effect on the uptake or retention of radioactivity in other tissues. N1-[methyl-14C]nicotinamide was not found in the urine of non-pretreated [14C]streptozotocin-injected mice. When the animals were pretreated with nicotinamide, N1-[methyl-14C]nicotinamide was detected in the urine, but this represented only a small fraction of the injected radioactivity and of the excreted N1-methylnicotinamide. This result does not support the hypothesis that the disturbance of the NAD-metabolism, which streptozotocin causes, is due to a methylation of nicotinamide.     

Autoradiographic distribution of [14C]-labelled pimonidazole in rhabdomyosarcoma-bearing rats and pigmented mice

Summary The hypoxic cell radiosensitizer [2-¹⁴C] pimonidazole (2-nitro--(piperidinomethyl)-l-imidazole ethanol) was injected i.p. into pigmented mice and rats bearing transplanted rhabdomyosarcoma. The injected dose level was 200 mg/kg, and the delivered activity was 96 Ci/kg. Whole-body autoradiography was carried out on all animals. We noted an extensive whole-body distribution of radioactivity. At short intervals, the autoradiograms were characterized by an accumulation of radioactivity in the metabolic and exretory organs (liver, kidney, urinary tract, and intestinal content) as well as in lymphomyeloid tissues (thyroid gland, suprarenal gland, and hypophysis) and salivary glands. In pigmented mice, the uveal and biliary tracts were the highest labelled. The liver and particularly the renal medulla were identified as sites of retention of radioactivity. In the tumor the radioactivity was detected only in peripheral regions, with higher uptake in viable zones than in necrotic islets.Abbreviations used MISO misonidazole - [¹⁴C] ou [¹⁴C] Ro, [¹⁴C] Ro 03-8799 plus Ro 03-8799-derived [¹⁴C]-labelled metabolites     

Distribution and metabolism of 1-propyl-1-nitrosourea in rats

The carcinogen 1-[14C]propyl-1-nitrosourea (PNU) was readily absorbed from the rat gut, and the radioactivity was excreted mainly in the urine and expired air. The urinary metabolites of PNU were 1-propylurea and urea. 1-Propylurea was shown to be excreted largely unchanged in the urine. Both [14C]PNU and 1-[14C]propylurea were found to be eliminated rapidly from the rat body. Besides cO2 from PNU, isopropyl alcohol was identified as a volatile metabolite in the expired air. Specific high concentrations (%/g) in main organs and tissues were not observed in adult rats 24 hr after single p.o. doses (20 mg/kg) of labeled PNU. The ureido carbon of PNU showed considerable retention in the blood, while relatively high residual levels were found in the liver for the propyl carbon. Autoradiographic studies on pregnant rats showed a uniform distribution between maternal and fetal bodies a short time after dosing. A relatively high concentration of 14C label was found in the maternal blood 24 hr after treatment with [carbonyl-14C]PNU. Localization of radioactivity in bone systems such as the fetal sterna and vertebrae was noted 6 hr after treatment with [propyl-1-14C]PNU. Metabolic pathways of PNU in the rat are proposed.     

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.     

Metabolism and urinary excretion of mutagenic metabolites of benzo[a]pyrene in C57 and DBA mice strains

Groups of C57Bl/6 (B6) mice (responsive) and DBA/2 (D2) (non-responsive)mice received an i.p. dose of benzo[a]pyrene (BP) (100 mg/kgbody weight) with or without pretreatment with or without pretreatmentwith ß-naphthoflavone (BNF), an inducer of monooxygenases.The amount of mutagenic BP metabolites in the urine was determinedby the Salmonella/microsome assay (strain TA100) and excretionof BP metabolites was quantifled by h.p.l.c. After inducer treatment,the responsive mice excreted more mutagens and total oxidizedBP metabolites in the urine than did the non-responsive strain.The ratio of BP diols:total oxidized metabolites was 0.42 inB6 mice and 0.72 in D2 mice and was not affected by inducertreatment. After a dose of [¹⁴C]BP, radioactivity was measuredin the faeces for up to 7 days. The half-life of [¹⁴C]BP inthe non-responsive D2 mice was about twice that in the responsiveB6 mice. Our results agree with a previously proposed modelof the risk factors for toxic effects and/or the developmentof tumours after exposure to polycyclic aromatic hydrocarbonsin responsive and nonresponsive mouse strains.     

Disposition and metabolism of the antitumor agent pyrazine-2-diazohydroxide in mouse and beagle dog

Summary The pharmacokinetics and metabolism of pyrazine-2-diazohydroxide have been studied in the beagle dog and mouse. When pyrazine-2-diazohydroxide was administered to beagle dogs at a dose of 18.6 mg/kg (428 mg/m²) by i. v. bolus, the plasma half-life (t1/2) was 7.3 min, the apparent volume of distribution (V_d) 577 ml/kg, and the total body clearance (Cl) 55 ml/min per kg. In mice given pyrazine-2-diazohydroxide by i. v. bolus at 100 mg/kg (428 mg/m²), the t1/2 was 5.8 min, the V_d 250 ml/kg, and the Cl 30 ml/min per kg. When [2-¹⁴C]pyrazine-2-diazohydroxide was infused i. v. to mice at 100 mg/kg over 8 h, the Cl for parent drug was 122 ml/min per kg. The major product formed from pyrazine-2-diazohydroxide was 2-hydroxypyrazine, which accounted for 80% of the total radioactivity in the plasma after a 6-h drug infusion. These were three other metabolites in plasma, two more polar than pyrazine-2-diazohydroxide, which accounted for 7% of the radioactivity, and one less polar, which accounted for 5% of the radioactivity. Following an i. v. bolus dose of [2-¹⁴C]pyrazine-2-diazohydroxide, 79% of the radioactivity was excreted in the urine in 24 h, 3% in the feces, and 0.4% in the expired air; 18% remained in the carcass. The liver and kidney showed the highest tissue levels of radioactivity. 2-Hydroxypyrazine accounted for 45% of the urinary radioactivity, pyrazine-2-diazohydroxide for 14%, and a glucuronide or sulfate conjugate of 2-hydroxypyrazine for 17%. Twenty-four percent of the radioactivity eluted near the void volume on high-performance liquid chromatography and was not identified.The work reported in this paper was supported by NCI contract CM67904     

Radioactive species in rat urines and tissues after [14C] AD 32 administration

Summary [14-¹⁴C]N-Trifluoroacetyldoxorubicin-14-valerate ([¹⁴C]AD 32) was synthesized and administered IV to male rats at 9.09 mg/kg. Urinary radioactivity excreted in the 0–24 h interval was only 2.3% of the dose, N-trifluoroacetyldoxorubicin (AD 41), 13-dihydro-N-trifluoroacetyldoxorubicin (AD 92), and doxorubicin being the major urinary metabolites identified. Doxorubicin and AD 32 were the main radioactive species extracted from tissues at 24 h after treatment. The amount of doxorubicin present in the analysed tissue samples is in agreement with the relatively low toxicity of AD 32 compared with doxorubicin.     

Autoradiography of [14C]N-nitrosodiethanolamine in Sprague-Dawley rats

Whole-body autoradiography in Sprague-Dawley rats injected i.v. with [14C]N-nitrosodiethanolamine ([14C]NDELA) showed a localization of tissue-bound radioactivity in the liver and the nasal olfactory mucosa. Microautoradiography of the nasal olfactory mucosa showed the highest labelling over the subepithelial glands (Bowman's glands) in the lamina propria mucosae. Experiments in vitro showed a capacity of the liver and the nasal mucosa to form 14CO2 from the [14C]NDELA. Most of the injected [14C]NDELA was recovered in the urine in a non-metabolized form. A small proportion of the dose was exhaled as 14CO2. The target tissues for the NDELA carcinogenesis in Sprague-Dawley rats are the liver and the nasal mucosa. Our results indicate that a bioactivation of the NDELA will take place in the nasal mucosa, as well as in the liver.     

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.      

In vivo binding of 3,3'-dichlorobenzidine to rat and mouse tissue DNA

The kinetics of total DNA adducts were compared in the liver, bladder epithelium and small intestinal epithelium of rats and mice following a single oral dose (100 mg/kg) of 3,3'-dichlorobenzidine [( 14C]DCB). Peak DNA binding (expressed as pmol DCB bound/mg DNA) in rat tissues was 153.5, 144.8 and 36.9 in the intestine, bladder and liver, respectively, whereas in mouse tissues, the binding was 72.5, 58.2 and 55.8, respectively. In either species, the half-life of the DNA adducts in the liver (13.5 and 13.8 days in rats and mice, respectively) was comparable to that in the bladder epithelium (14.8 and 12.7 days in rats and mice, respectively) but longer than that in the intestinal epithelium (5.9 and 4.7 days in rats and mice, respectively). Peak total DCB binding in hepatic but not intestinal or bladder epithelial DNA correlated positively with total urinary DCB metabolites. In vitro, mouse hepatic S9 was 57% more active in catalyzing the formation of DNA-binding derivatives of DCB, in parallel with the higher in in vivo maximum hepatic DNA binding in mice than in rats. Thus, a single oral dose of DCB in rats and mice leads to extensive binding of the chemical to tissue DNA, with the rate of removal of the adducts not differing between target and non-target tissues.     

Pharmacokinetic analysis and antitumor efficacy of MKT-077, a novel antitumor agent

MKT-077 (1-ethyl-2-{[3-ethyl-5-(3-methylbenzothiazolin-2-yliden)]-4-oxothiazolidin-2-ylidenemethyl} pyridinium chloride), a novel rhodacyanine dye in phase I/II clinical trials, may provide a new approach to cancer therapy based on the accumulation in the mitochondria of the cells of certain carcinomas, for example, those of the colon, breast and pancreas. To support the development of MKT-077 for clinical application as an intravenous (i.v.) therapy, we investigated the metabolic fate of [¹⁴C]MKT-077 in BDF1 mice as well as the distribution of MKT-077 in experimental LS174T tumor-bearing mice using a high-performance liquid chromatography (HPLC) method. The plasma levels of ¹⁴C after i.v. administration of [¹⁴C]MKT-077 declined in a triphasic manner. In the first distribution phase, the levels of ¹⁴C decreased with a T_1/2 of ∼5 min. In the second and terminal phase, the T_1/2 of ¹⁴C was 2.8–4.6 h and 16.2 h, respectively. C_max (1 min after injection) increased from 0.3 to 1.5 μg/ml linearly, but less than proportionately between the doses. The AUC_(0–∞) at 0.3, 1 and 3 mg/kg were 0.030 ± 0.002, 0.60 ± 0.12 and 1.73 ± 0.25 μg · h/ml, respectively. Plasma clearance was ∼1.8 l/h per kg (at doses of 1 and 3 mg/kg). The steady state volume of distribution (6.8 and 25.1 l/kg) indicated that MKT-077 distributed as a lipid-soluble molecule. The mean residence time (MRT) was 4.1 (at a dose of 1 mg/kg) and 14.1 h (at a dose of 3 mg/kg). In the first rapid phase (5 min after dosing), ¹⁴C radioactivity was detected in most of the tissues and organs, most strongly in the kidney cortex, and not in the central nervous system and testes. In the terminal phase (24 h after dosing), ¹⁴C contents increased in the intestinal tract, and in the kidney and liver were nearly to the background level. After i.v. bolus administration at a dose of 3 mg/kg of [¹⁴C]MKT-077, the predominant route of elimination of the radioactivity was via the feces, and recoveries of total radioactivity in urine and feces corresponded to 33.5% and 61.1%, respectively. More than 60% was recovered within 24 h and 95% within 1 week. MKT-077 was primarily excreted in unmetabolized form with five unidentified metabolites found in the urine and plasma. Intact MKT-077 was retained in the tumor tissue longer than in plasma and kidney in LS174T tumor-bearing mice receiving MKT-077 at an i.v. therapeutic dose (10 mg/kg). This accumulation decreased very slowly, suggesting that the high membrane potentials of tumor cell mitochondria may help retain the drug in tumors. Received: 4 February 1998 / Accepted: 29 June 1998     

The comparative disposition of [14C]-fotemustine in non-tumourous and tumourous mice

Summary The distribution and excretion of radioactivity from [¹⁴C]-fotemustine was examined in mice with melanomas at different stages of development to determine whether the disease state substantially alters the disposition of the drug and its metabolites. Normal BDF₁ mice and mice that had been subcutaneously grafted with B16 melanoma either 1, 3, 7, 14 or 21 days previously were used. The animals were killed at either 5 min, or at 3, 24 or 96 h after receiving an intravenous dose of [¹⁴C]-fotemustine (20 mg/kg) and were examined either by whole-body autoradiography or by liquid scintillation counting of excreta and tissues of interest. The majority of the [¹⁴C]-fotemustine dose was excreted in the urine, with similar amounts being measured in both non-tumourous animals (61.6%±13.1%) and tumourous mice grafted 14 days previously (67.2%±5.7%). Small amounts of radioactivity, again similar in both non-tumourous and tumourous mice, were recovered in the faeces (5.4%±5.6% and 3.6%±1.8%, respectively) and as carbon dioxide (7%±3.5% and 6.4%±1%, respectively), with minimal amounts being expired as chloroethanol (<1%). When mice were examined 5 min after dosing, there was extensive tissue distribution accounting for 75%±10% of the dose. The highest concentrations determined by both whole-body autoradiography and liquid scintillation counting were measured in the excretory organs, with 33 and 28 gEq/g being found in the liver and kidney, respectively. High levels were also seen in the lung and plasma (19.8 and 19.5 gEq/g, respectively). Analysis of variance indicated that groups of tissues, such as the excretory organs, blood and plasma or the pigmented tissues, showed distinct but inconsistent patterns. Only tumours at 14 and 21 days of development were suitable for examination, and these showed levels of 12.1 gEq/g; however, the tumourto-plasma ratio increased from between approx. 0.5 and 0.6 at 5 min to approx. 2 at 96 h after dosing, suggesting retention within the melanoma, whereas the ratio for the femur remained at approx. 1. Whole-body autoradiography showed that the distribution in the tumour was not uniform, but rather was concentrated in the peripheral area (presumably viable cells) as opposed to the central necrotic region. Thus, the high and sustained concentration of radioactivity found in the active cells of the melanoma may provide an explanation for the high efficacy of the drug.     

Strain-dependent differences in the metabolism of 3-methylcholanthrene by maternal, placental, and fetal tissues of C57BL/6J and DBA/2J mice

C57BL/6J (B6) and DBA/2J (D2) mice have different susceptibilities to developmental toxicity and transplacental carcinogenesis induced by in utero exposure to polycyclic aromatic hydrocarbons, which has been associated with polycyclic aromatic hydrocarbon metabolism and inducibility at the Ah locus. The distribution of total 3-methylcholanthrene (3-MC)-associated radioactivity in maternal, placental, and fetal tissues of beta-naphthoflavone-pretreated pregnant B6 and D2 mice was determined up to 12 h after p.o. exposure to [6-14C]-3-MC (63 mg/kg, 20 mu Ci) on gestational day 17. 3-MC-associated radioactivity in maternal plasma was not significantly different in the two strains. However, D2 tissue homogenates had consistently higher levels of 3-MC-associated radioactivity, which included both bound and free parent compound and metabolites. Increased metabolism of 3-MC by B6 maternal liver was suggested by the induced levels of aryl hydrocarbon hydroxylase activity in that tissue and by the observation that levels of total radioactivity decreased more rapidly in B6 tissues than in D2 tissues. The D2 fetal lung, the target tissue for 3-MC-induced transplacental carcinogenesis, appeared to accumulate 3-MC-associated radioactivity for a longer period of time than either the D2 fetal liver or the B6 fetal tissues. This study suggests that the genetic differences in fetal susceptibility to the developmental toxicity and transplacental carcinogenesis of 3-MC may be related to the presystemic elimination of the compound from both maternal and fetal tissues.     

Tumor-initiating potency of a novel heterocyclic amine, aminophenylnorharman in mouse colonic carcinogenesis model

A novel heterocyclic amine, 9-(4'-aminophenyl)-9H-pyrido[3,4-b]indole (aminophenylnorharman, APNH), which is formed from nonmutagenic 9H-pyrido[3,4-b]indole (norharman) and aniline, is mutagenic to bacteria and mammalian cells and potently carcinogenic in rats. APNH is detected in human urine samples, suggesting that humans are continuously exposed to APNH. In the present study, (32)P-postlabelin analysis revealed that the levels of APNH-DNA adduct 24 hr after the treatment with APNH (1, 5 and 20 mg/kg body weight) in male ICR mice were increased in a dose-dependent manner in the colon and liver. Based on these findings, we determined the tumor-initiating potency of APNH in an inflammation-related and two-stage mouse colon carcinogenesis model. Male Crj: CD-1 (ICR) mice were given a single intragastric administration (1, 2, 5 or 10 mg/kg body weight) of APNH and subsequent 1-week oral exposure to dextran sodium sulfate (DSS, 2% in drinking water). Treatment with APNH and DSS resulted in numerous colon tumor development: the incidence and multiplicity of the tumors were the highest in the mice received 10 mg/kg body weight of APNH and followed by DSS. Development of colon tumors was dose-dependent of APNH. Seven of 9 (77.8%) colonic adenocarcinomas developed in mice treated with APNH (10 mg/kg body weight) and DSS had beta-catenin gene mutations at codons 32 and 37, being predominantly transversion. These findings indicate that APNH has an initiating activity in inflamed mouse colon and the APNH-DNA adduct formation correlates with its tumorigenic potential.     

A mass balance and disposition study of the DNA methyltransferase inhibitor zebularine (NSC 309132) and three of its metabolites in mice.

PURPOSE: To elucidate the in vivo metabolic fate of zebularine (NSC 309132), a DNA methyltransferase inhibitor proposed for clinical evaluation in the treatment of cancer. EXPERIMENTAL DESIGN: Male, CD(2)F(1) mice were dosed i.v. with 100 mg/kg 2-[(14)C]zebularine. At specified times between 5 and 1,440 minutes, mice were euthanized. Plasma, organs, carcass, urine, and feces were collected and assayed for total radioactivity. Plasma and urine were also analyzed for zebularine and its metabolites with a previously validated high-pressure liquid chromatography assay. A similar experiment was done with 2-[(14)C]uridine, the proposed primary metabolite of zebularine. RESULTS: Maximum plasma concentrations were 462, 306, 33.6, 21.7, and 11.5 mumol/L for total radioactivity, zebularine, uridine, uracil (each at 5 minutes), and dihydrouracil (at 15 minutes), respectively. Total radioactivity, zebularine, uridine, uracil, and dihydrouracil were rapidly eliminated from plasma, and after 45 minutes, none of the individual compounds could be quantitated by high-pressure liquid chromatography. Plasma data were consistent with sequential conversion of zebularine to uridine, uracil, and dihydrouracil. 2-Pyrimidinone was not observed. Prolonged retention of radioactivity, at concentrations higher than in plasma, was observed in tissues. Recovery of given radioactivity in urine (30.3% of dose), feces (0.4% of dose), cage wash (7.9% of dose), and tissues and carcass (6.1% of dose) after 24 hours implied that up to 55% of radioactivity was expired as (14)CO(2). Comparison of zebularine and uridine pharmacokinetic data indicated that approximately 40% of the zebularine dose was converted to uridine. CONCLUSIONS: Zebularine is extensively and rapidly metabolized into endogenous compounds that are unlikely to have effects at the concentrations observed.     

Uptake and specific binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin in the olfactory mucosa of mice and rats

Whole-body autoradiography of mice and rats after i.v. administration of 2,3,7,8-[14C]tetrachlorodibenzo-p-dioxin ([14C]TCDD) showed a selective localization of radioactivity in the liver and nasal olfactory mucosa. In microautoradiograms of solvent extracted sections of the skulls of mice given injections of [3H]TCDD, no radioactivity was observed in the olfactory mucosa, suggesting that TCDD is not covalently bound in this tissue. The amount of specific [3H]TCDD binding sites in cytosol from the ethmoturbinates of rats (33 fmol/mg cytosolic protein) was comparable to that of the liver cytosol as estimated by electrophoresis in polyacrylamide concentration gradient gel, and therefore probably too low to explain the retention of radioactivity in the olfactory mucosa. The specific TCDD binding species in the mucosa of the ethmoturbinates exhibited a similar binding affinity for [3H]TCDD, ligand specificity, and molecular weight as the TCDD receptor from rat liver. The 7-ethoxyresorufin O-deethylase activity of the mucosa of the ethmoturbinates was induced less than twice by administration of the TCDD receptor ligand beta-naphthoflavone (5,6-benzoflavone) 40 h before killing. By administration of beta-naphthoflavone (5,6-benzoflavone) 16 h before killing, mRNA coding for cytochrome P-450d but not for cytochrome P-450c was induced to detectable levels in the mucosa of the ethmoturbinal tissue of the rat. The basal activity of 7-ethoxyresorufin O-deethylation of the mucosa of the ethmoturbinates of the rat was comparable to the corresponding activity of the liver. This basal metabolic activity of the ethmoturbinal tissue was only marginally inhibited by antibodies raised against beta-naphthoflavone (5,6-benzoflavone) induced hepatic cytochrome P-450s. Thus, enzymes other than cytochrome P-450c may possibly account for a part of the basal 7-ethoxyresorufin O-deethylase activity in the rodent olfactory mucosa.     

Binding to cellular macromolecules as a possible mechanism for the cytotoxicity of misonidazole

Reduction of the nitro group occurred when [14C]misonidazole was treated with zinc dust in aqueous solution in the presence of ammonium chloride. When the reduction mixture was allowed to react with calf thymus DNA or bovine albumin, radioactivity was bound to both DNA and protein. Under the same conditions, misonidazole did not bind to these macromolecules. Analysis of the reduction mixture indicated that the hydroxylamine, amine, and hydrazo derivatives of mizonidazole were the major products. In a number of tissues of C3H mice after administration of [14C]misonidazole, radioactivity was detected in the DNA, RNA, and protein fractions. Similar results were also obtained with Chinese hamster ovary cells incubated with the drug in the absence of oxygen. It is postulated that nitroreduction and binding of the nitroreduction products to macromolecules is a probable mechanism for the mutagenic and cytotoxic properties of misonidazole.