Excretion Of Radioactivity Research Articles

Metabolic Fate of Pitavastatin, a New Inhibitor of HMG-CoA Reductase—Effect of cMOAT Deficiency on Hepatobiliary Excretion in Rats and of mdr 1a/b Gene Disruption on Tissue Distribution in Mice

Pitavastatin is a potent competitive inhibitor of HMG-CoA reductase. In the current study, to elucidate the hepatobiliary excretion of pitavastatin, we investigated the plasma concentration and biliary excretion of (14)C-pitavastatin in EHBR. We also evaluated the distribution of pitavastatin in mdr1a/b knockout mice by whole body autoradiography and quantitative radioassay. In view of the widespread clinical use of pitavastatin and the importance of drug-drug interaction, the inhibitory effect on Pgp-mediated activation of ATPase was also investigated. No marked difference was observed in the plasma concentration and biliary excretion of radioactivity between SDR and EHBR after dosing of (14)C-pitavastatin. Little radioactive transfer into the brain was detected in mdr1a/b knockout mice and the ATPase activity of human Pgp was negligible in the presence of pitavastatin. Moreover, no inhibitory effect on the Pgp-mediated activation of ATPase by verapamil was found in the presence of pitavastatin over a wide concentration range. These results indicated that a cMOAT and Pgp-mediated transport mechanism did not play a major role in the distribution of pitavastatin.

Disability

The urinary excretion of radioactivity has been measured in man following intravenous injection of 400 mg of [8-14C]proxyphylline. Of the radioactive dose, 95% was recovered in urine 3 days after...

Metabolic fate of (-)-[4-(3)H]epigallocatechin gallate in rats after oral administration.

After oral administration of [4-(3)H]EGCg to rats, the radioactivity in blood, major tissues, urine, and feces was measured over time. The radioactivity in blood and most tissues remained low for 4 h postdose, began to increase after 8 h, peaked at 24 h, and then decreased. Major urinary excretion of radioactivity occurred in the 8-24 h period, and the cumulative radioactivity excreted by 72 h was 32.1% of the dose. The radioactivity in the feces was 35.2% of the dose within 72 h postdose. In the case of rats pretreated with antibiotics (antibiotic-pretreated rats), the radioactivity levels of the blood and urine were definitely lower than those in rats not pretreated with antibiotics (normal rats). The radioactivity recovered in the antibiotic-pretreated rat urine was estimated to be only (1)/(100) of that in the normal rat urine. These results clearly demonstrated that the radioactivity detected in the blood and urine of normal rats mostly originated from degradation products of EGCg produced by intestinal bacteria. Furthermore, a main metabolite in the normal rats was purified and identified as 5-(5'-hydroxyphenyl)-gamma-valerolactone 3'-O-beta-glucuronide (M-2). In feces of the normal rats, EGC (40.8% of the fecal radioactivity) and 5-(3',5'-dihydroxyphenyl)-gamma-valerolactone (M-1, 16.8%) were detected. These results suggested that M-1 was absorbed in the body after degradation of EGCg by intestinal bacteria, yielding M-1 with EGC as an intermediate. Furthermore, M-2 was thought to be formed from M-1 in the intestinal mucosa and/or liver, then to enter the systemic circulation, and finally to be excreted in the urine. Taking into account all of the above findings, a possible metabolic route of EGCg orally administered to rats is proposed.

The influence of co-exposure to dimethyldithiocarbamate on butadiene metabolism

Treatment of rats and mice with a single oral dose of dimethyldithiocarbamate (DMDTC; 250 mg/kg) had a marked effect on hepatic CYP2E1 and aldehyde dehydrogenase activities, measured in vitro, for up to 24 h after dosing. The same treatment did not affect CYP2A6, glutathione S-transferase, epoxide hydrolase, alcohol dehydrogenase activities or hepatic glutathione levels. As a consequence of the loss of CYP2E1 activity, butadiene metabolism in liver fractions from DMDTC treated rats and mice was markedly reduced, as was the metabolism of the mono-epoxide to the di-epoxide in mouse liver. The conversion of the mono-epoxide to the diol by epoxide hydrolases was not affected by DMDTC treatment. Urinary excretion of radioactivity, following dosing with DMDTC and exposure to 200 ppm C-14 butadiene for 6 h, was markedly reduced in rats, but increased in mice. The profiles of urinary metabolites were qualitatively similar from mice exposed to butadiene to those exposed after dosing with DMDTC. In the rat, pre-dosing with DMDTC resulted in the formation of three additional urinary metabolites following exposure to butadiene. Overall, DMDTC appears to impact qualitatively and quantitatively on the metabolism of butadiene. The nature and full significance of these changes has yet to be characterised.

Studies on the Metabolic Fate of M17055, a Novel Diuretic. (1): Plasma Concentration and Excretion of Radioactivity in Rat and Dog after a Single Intravenous Administration of [14C]M17055.

The plasma level and excretion of radioactivity were studied following a single intravenous administration of [14C]M17055 to male and female rats, and a female dog. 1. After a single intravenous administration to male and female rats at a dose of 0.3 mg/kg, plasma levels of radioactivity declined triphasically reaching at 48 hr the levels lower than 1/1000 of those observed at one minute after administration. Calculated CLtot, T1/2βand T1/2γ did not differ between male and female rats. But T1/2α of female rats showed lower value than that of male rats. 2. After a single intravenous administration to male rats at doses of 0.1, 1 and 3 mg/kg, plasma levels of radioactivity declined in similar manner with that at a dose of 0.3 mg/kg. Although calculated CLtot tends to decline at 1 and 3 mg/kg, the T1/2γ showed similar value at the dose range of 0.1-3 mg/kg. 3. Plasma level of radioactivity in a female dog declined triphasically reaching at 48 hr the levels 100 fold lower than those observed at 5 minutes after a single intravenous administration at a dose of 1 mg/kg. There-after, the level of radioactivity declined more slowly when compared with rats. 4. Cumulative urinary and fecal excretions of radioactivity within 168 ho urs after a single intravenous administration to male and female rats accounted for 94.10 and 97.61% of the dose, respectively. Over 90% of the dose was excreted within 48 hours after administration. Urinary excretion rate was higher in female rats than that in male rats, especially within 6 hours after administration. 5. Cumulative urinary and fecal excretions after a single intravenous administration to a female dog accounted for over 90% after administration. Total excretion of radioactivity within 168 hours accounted for 97.33% of the administered dose. 6. Biliary excretion of radioactivity after a single intravenous administration to bile-duct cannulated male rats accounted for 64.12% of the dose. About 67% of the excreted radioactivity in bile was re-absorbed in male rats. 7. The renal clearance of radioactivity after administration in male and female rats decreased and showed same level when treated with probenecid, which suggested that the sex difference of the urinary excretion may be caused by the active secretary mechanism.

Studies on the Metabolic Fate of M17055, a Novel Diuretic. (3): Plasma Concentration, Distribution and Excretion of Radioactivity in Male Rats after Repeated Daily Intravenous Administration of [14C]M17055.

The plasma levels, tissue distribution and excretion of radioactivity were studied following repeated daily intravenous administration of [14C]M17055 to male rats at a dose of 0.3 mg/kg/day. 1. The plasma levels of radioactivity at 24 hours after each of 21 day-period of repeated administration to male rats were reached the steady state (6.90-8.87 ng eq./mL) by the 8th dosing. The calculated pharmacokinetic parameters were similar as those after the single administration. 2. Although the levels of radioactivity in all tissues at 24 hours after repeated administration to male rats rose according to the times of dosing, the rising rate was slow except for blood, lung, skin, digestive organs and their contents. At 1 hour after the 21st dosing, the radioactivity distributed highly to the liver and kidney as observed after the single administration. At 168 hours after the 21st dosing, the radioactivity tended to remain in highly perfused tissues, such as liver, kidney, spleen and lung. No tissue was identified in which the radioactivity retained for long period on the whole body autoradiograms. 3. Cumulative urinary and fecal excretions of radioactivity after 21 day repeated administration accounted for 58.17 and 37.35% of the total dose, respectively. The excretion rate in urine and feces well agreed to that after the single administration. The total excretion of radioactivity, including that present in the cage washing, accounted for 97.28% of the total dose.

Metabolic Fate of Esmolol.

For the purpose of clarifying the pharmacokinetics of esmolol in rats, the plasma level, tissue distribution, metabolism and excretion were investigated after intravenous administration of 14C-esmolol, and the following results were obtained: 1. After intravenous administration to rats, the radioactivity in plasma decreased biphasically, and disappeared almost completely by 24 hr. The elimination half-lives were dose independent (10, 20, 40 mg/kg), and the plasma levels and AUC increased in proportion to the administered doses. The unchanged drug was not detected in plasma at 2 min after administration, but ASL-8123, the hydrolyzed metabolite of esmolol, was accounted for more than 80% of radioactivity in plasma. This indicated esmolol to be rapidly metabolized by esterases in blood. During repeated administration (for 7 days), the radioactivity in plasma showed no accumulation, and pharmacokinetic parameters after the final administration were similar to those after a single administration. 2. The radioactivity distributed rapidly and widely to tissues, and the levels were highest in kidney and lung, followed by adrenal gland, thyroid, pancreas, harderian gland, pituitary, liver and small intestine. The time course profiles in radioactivity levels in the heart, a target organ, were about 2 times higher than those in blood. In whole body autoradiograms, the transfer of radioactivity to the urine was already observed at 5 min after administration, indicating a rapid excretion of radioactivity to the urine. 3. Plasma protein binding of radioactivity in vitro was about 23%, and that in vivo (from 5 min to 8 hr after administration) accounted for 22-37%. 4. The main metabolite in plasma and urine was ASL-8123, which accounted for more than 80% of radioactivity in both samples. This indicates that enzymatic hydrolyses of esmolol in blood and the excretion of ASL-8123 into urine are rapid. Hydroxylated ASL-8123 was detected in minor quantities, but conjugates were not observed in either sample. No sex-related difference was observed in metabolite composition. 5. Radioactivity was completely excreted into feces or urine within a day, and urinary excretion (about 95% of dose) was the major elimination route. After repeated administration (for 7 days), the urine/feces ratio was not different from that after a single administration, and radioactivity was not remained. The biliary excretion of radioactivity was slight (less than 2% of dose) and the transfer into milk was observed, but the elimination of radioactivity in milk correlated with that in maternal plasma.

Disposition of propargyl alcohol in rat and mouse after intravenous, oral, dermal and inhalation exposure

1. The disposition of propargyl alcohol (PAL) radiolabelled with carbon-14 ([2,3- 14C]PAL) was determined in the F344 rat and B6C3F1 mouse following intravenous (i.v.), oral, inhalation and dermal exposure. 2. By 72 h following an i.v. (1 mg kg -1) or oral (50 mg kg-1) dose, 76-90% of the dose was excreted. Major routes of excretion by rat were urine (50-62%), CO2 (19-26%) and faeces (6-14%). Major routes of excretion by mouse were urine (30-40%), CO2 (22-26%) and faeces (10-20%). Less than 6% of the dose remained in tissues at 72 h. Biliary excretion of radioactivity by rat (62% in 4 h) was much greater than elimination in faeces (6% in 72 h), indicating that PAL metabolites underwent extensive enterohepatic recycling. 3. Dermal exposure studies demonstrated that dermal absorption of PAL was minimal due to its inherent volatility. 4. In the inhalation studies (1, 10 or 100 ppm for 6 h), 23-68% of the radioactivity to which animals were exposed was absorbed. The primary route of excretion was urine (23- 53%), and a significant portion was exhaled as volatile organics (15-30%). 5. PAL was extensively metabolized by both species. One metabolite was identified as 3,3-bis[(2-(acetylamino)-2-carboxyethyl)thio]-1-propanol, which is consistent with Banijamali et al. (1999).

UNINEPHRECTOMY INCREASES KIDNEY β2-MICROGLOBULIN: CAN IT PLAY A ROLE IN THE PROGRESSION OF KIDNEY DAMAGE?

β2-microglobulin (β2M) is highly accumulated by the kidneys of normal rats. The aim of this study was to verify if uninephrectomy can modify the renal uptake of labeled β2M. For this purpose the radioactivity of plasma and those of the remaining kidney, liver and urine have been measured in uninephrectomized rats (NX) and in controls (C) at different times after the injection as i.v. bolus of 131I-β2M. The experiments were performed in 114 Sprague-Dawley male rats. Fifty seven animals underwent right nephrectomy, the other animals being the C. NX and their C were divided in 3 groups, studied 2, 4 and 6 weeks after nephrectomy, respectively. Part of the animals were sacrificed 12 min after the injection of labeled β2M (peak-time, i.e. time of highest kidney accumulation of 131I-β2M in the normal rat) and part 10 min later.The results demonstrate that:uninephrectomy increases plasma retention of 131I-β2Mkidney uptake (total and per gram) is always higher in NXliver uptake (much lower than that of kidney) is not influenced by uninephrectomyurine excretion of radioactivity is minimal in both NX and C.The behavior of β2M is similar to that we previously observed with α1-microglobulin and lysozyme. The higher kidney content of some low mw proteins after uninephrectomy could play a role in the progressive reduction of renal function determined by the reduction of renal mass.

Disposition of Radiolabeled FAVOR PAC in Rats

FAVOR is the name for a family of superabsorbent cross-linked sodium polyacrylate polymers developed by Stockhausen GmbH & Co KG (Krefeld, Germany) that are known for their ability to absorb and retain large volumes of fluid. The absorption, distribution, rates and routes of excretion of radiolabeled FAVOR PAC (CAS Registry No. 9003-04-7; [14C]FAVOR PAC), one member of the FAVOR family, were evaluated following a single oral administration of the compound. Male Sprague–Dawley rats were administered single doses of 26 to 39 mg/kg [14C]FAVOR PAC by gavage. Approximately 98.8% (normalized mean) of the total administered dose was excreted in the feces within 5 days, and the majority (∼88%) was excreted within the first 24 h. Urinary excretion accounted for 0.69% (normalized mean) of the total administered dose. Recovery of radioactivity in the organs, tissues, and carcass was generally less than 0.5% of the dose administered. Levels of total radioactivity in whole blood ranged from 0.75 to 1.20 μg equiv/g. Biliary elimination of total radioactivity accounted for less than 0.1% of the dose administered. The results indicate that FAVOR PAC is poorly absorbed and rapidly eliminated in feces following oral administration.

The fate of diphenyl sulphide, diphenyl sulphoxide and diphenyl sulphone in the rat.

Radiolabelled [UL-14C]-diphenyl sulphide, [UL-14C]-diphenyl sulphoxide and [UL-14C]-diphenyl sulphone were administered by gavage (1.0 mmol/kg body weight) to adult male Wistar rats following an overnight fast. For all compounds, faeces were the major route of excretion of radioactivity (50%). Urinary elimination (40%) was similar during the first (19%) and second (16%) days and a small amount of radioactivity (6%) was found within the carcass after four days. From urinary and faecal data, metabolism occurred via ring hydroxylation with subsequent conjugate formation. Oxidation of the sulphur to form the sulphoxide and sulphone also took place; a small amount of sulphoxide reduction was apparent but no sulphone reduction was found. No evidence for exclusion of the sulphur was obtained, and it appeared unlikely that extensive cleavage of the ring structures occurred.

Absorption, distribution, metabolism and excretion of intravenously and dermally administered triethanolamine in mice

Triethanolamine (TEA) is an amino alcohol having widespread applications in consumer goods and as an industrial chemical. A number of relatively high-dose dermal toxicity studies have been conducted in rats and mice reflecting the principal route of human exposure to TEA. The absorption, distribution, metabolism and excretion (ADME) of 14C-TEA derived radioactivity were determined in male C3H/HeJ mice following dermal application of 2000 mg/kg (neat) or, to characterize blood kinetics, intravenous (iv) injection of 1 mg/kg 14C-TEA. Balance and excretion data were also collected in mice utilizing several dermal dosing scenarios (1000 mg/kg in acetone, 2000 mg/kg neat, 2000 mg/kg in water) and, for comparative purposes, in male Fischer 344 rats dosed dermally with 1000 mg/kg neat 14C-TEA. Urine, feces, expired CO 2 (iv) and, where appropriate, blood were collected over a 24- or 48-hour period post-dosing. The half-life for dermal absorption of radioactivity was estimated to be 1.3 hours. Intravenously administered radioactivity was eliminated in a biphasic manner with a prominent initial phase (half-life of 0.3 hr) followed by a slower terminal phase (half-life of 10 hr). Radioactivity was excreted primarily via the urine (49–69%) as unmetabolized TEA, regardless of dosage, route or vehicle used. Fecal excretion of radioactivity comprised 16–28% of dose administered. The body burden at sacrifice (sum of liver, kidney, carcass and non-application site skin) ranged from 3 to 6% of the dose. It was concluded that TEA is absorbed extensively following dermal application to mice at dosages relevant to toxicity testing and that acetone or water vehicles do not appear to significantly alter total uptake. Significantly, the blood kinetics and ADME of TEA in mice and/or rats differs from that of a related chemical, diethanolamine, which appears to be more toxic to rodents than TEA.

Comparison of Mycobacterium tuberculosis susceptibility testing performed with BACTEC 460TB (Becton Dickinson) and MB/BacT (Organon Teknika) systems

The recently introduced automated culture systems MB/BacT (Organon Teknika, Belgium) was compared with radiometric BACTEC 460TB (Becton Dickinson, USA) to test antimicrobial susceptibility of Mycobacterium tuberculosis to first line drugs. On 113 strains 97.5% agreement was obtained, with the difference being not significant. Concordance was practically complete for the most important drugs, isoniazid and rifampin. The two methods however significantly differed for the time needed to complete the test; in fact MB/BacT required on the average five days more than BACTEC 460TB. Despite the delay in the completion of the test, the excellent reliability along with the elimination of radioactivity and full automation make MB/BacT an attractive alternative for susceptibility testing of M. tuberculosis.

Radioiodinated N-(2-diethylaminoethyl)benzamide derivatives with high melanoma uptake: structure-affinity relationships, metabolic fate, and intracellular localization.

Several radioiodinated N-(dialkylaminoalkyl)benzamides have been used for planar scintigraphy and single-photon emission computed tomography (SPECT) of melanoma metastases. In a quest for improved melanoma uptake and tissue selectivity, structure-activity studies for N-(2-diethylaminoethyl)benzamides with variation of phenyl substituents were performed using C57Bl/6 mice bearing B16 melanoma. Compounds 2 (4-amino-5-bromo-N-(2-diethylaminoethyl)-3-[(131)I]iodo-2-methoxybenz amide) and 6 (4-acetamido-N-(2-diethylaminoethyl)-5-[(131)I]iodo-2-methoxybenzamid e) showed at 6 h post iv injection, for example, melanoma uptake of 16.6 and 23.2% ID/g, respectively (mean values, n = 3). Uptake was 3-5 times higher (P < 0.01) than observed with benzamides known from the literature and was probably facilitated by the relatively slow urinary excretion of 2 or 6. In contrast, analogues lacking either the MeO, Ac, AcNH, or Br substituents exhibited reduced tumor uptake and high urinary excretion of radioactivity in various benzamide metabolites. Uptake of radioiodinated benzamides in B16 melanoma is not mediated by a specific mechanism such as sigma-receptor binding. 2 and 6 exhibited similar melanoma uptake values but quite different sigma(1)-receptor affinities of K(i) = 0.278 +/- 0.018 and 5.19 +/- 0.40 microM, respectively. Uptake studies with IMBA (N-(2-diethylaminoethyl)-3-[(131)I]iodo-4-methoxybenzamide) or BZA (N-(2-diethylaminoethyl)-4-[(131)I]iodobenzamide) showed that with increasing dose of unlabeled compound the measured uptake of label was unchanged (IMBA) or even enhanced (BZA) while receptor binding of label decreased. Differential and equilibrium density-gradient centrifugation revealed that most of the radioactivity from labeled IMBA was associated with fractions containing melanin granules. Thus, structure-activity studies indicate that blood clearance rates and metabolic stability are the main determinants for benzamide uptake in melanoma. The high uptake and slow clearance of 6 offer considerable potential for melanoma imaging in patients, and this compound may also prove to be useful for radionuclide therapy.

Urinary excretion of metabolites following a single dermal dose of [ 14C]methyl parathion in pregnant rats

The identification and kinetics of urinary excretion of metabolites of uniformly phenyl-labeled O, O-dimethyl O-4-nitrophenyl phosphorothioate ([ 14C]methyl parathion) were carried out following a single dermal dose of 10.0 mg (10 μCi)/kg in pregnant Sprague–Dawley rats at 14–18 days of gestation. Urine was collected at each time interval of 1, 2, 4, 12, 24, 48, 72, and 96 h after dosing. Total p-nitrophenol in the conjugated and non-conjugated metabolites was measured as a marker of methyl parathion exposure. Elimination of radioactivity in the urine was rapid. Of the total 14C urinary excretion, 30% of the dose was excreted within 4 h, while 50 and 90% of the dose were recovered in the urine by 24 and 96 h, respectively. Excretion rate of total radioactivity was 60 μg methyl parathion equivalent/h (1.4 mg/day). By the end of the 96-h experiment, conjugated and non-conjugated metabolites accounted for 78.1 and 21.9%, respectively. Of the non-conjugated metabolites, p-nitrophenol and O, O-dimethyl O-4-nitrophenyl phosphate (methyl paraoxon) were identified by high performance liquid chromatography (HPLC) that accounted for 20%, and 1.9% of total urinary excretion, respectively. Appearance and disappearance rate constants of p-nitrophenol in urine were 0.12 and 0.048 μg/h, respectively. Conjugated metabolites were classified as: glucuronides 12% of urinary excretion, sulfates 3%, hot sulfuric acid hydrolysable residues 47% and 16.1% remained as unidentified water soluble metabolites. Direct hot acid hydrolysis of urine yielded 49% of extractable 14C-radioactivity compared to 62% when hot acid hydrolysis followed the enzymatic hydrolysis. The presence of the conjugated metabolites as the major class of metabolites of the total excretion indicates that determining only unbound p-nitrophenol as a biological marker for methyl parathion exposure underestimates total urinary excretion of p-nitrophenol. Sequential enzymatic and acid hydrolyses of urine prior solvent extraction are necessary for complete recovery of p-nitrophenol. The results indicate that the present method would show that the pregnant field worker or a housewife being at a greater risk than previously thought.

Fate of the Mushroom Hydrazine Agaritine in the Rat and Mouse

The fate of the mushroom hydrazine [14C]agaritine was investigated in the mouse and rat strains previously employed in carcinogenicity studies with the edible mushroom Agaricus bisporus. Agaritine was rapidly absorbed in both species, achieving higher blood levels in the mouse, but with similar area under the curve. Covalent binding of agaritine material to proteins was detected only in the liver and kidney, but the extent of binding was the same in the rat and mouse. Most of the radioactivity was excreted during the first 24 hours in both animal species: in the rat it was distributed equally between urine and feces, whereas in the mouse more of the radioactivity was excreted in the urine. No qualitative differences in the metabolic profile were evident, but quantitative differences were observed. Treatment of the urine with deconjugating enzymes did not reveal the presence of any conjugates. Agaritine, N'-acetyl-4-(hydroxymethyl)phenylhydrazine, and 4-(hydroxymethyl)benzene diazonium ion were not detected in the urine or in the plasma of either species. No mutagens or promutagens were detected by the Ames mutagenicity assay in the urine of either species after exposure to agaritine. Repeated administration of agaritine to rats and mice did not alter the urinary metabolic profile and excretion of radioactivity. Similarly, feeding mice a raw mushroom diet, according to the protocol employed in the carcinogenicity studies, did not modulate the excretion of radioactivity or the urinary metabolic pattern. No major species differences in the fate of agaritine in rat and mouse were noted that could provide a rationale for the carcinogenicity of A. bisporus in the mouse, but not in the rat.

A novel 4 S [3H]β-naphthoflavone-binding protein in liver cytosol of female Sprague‒Dawley rats treated with aryl hydrocarbon receptor agonists

β-Naphthoflavone (β-NF) is a widely used inducer of phase-I and phase-II enzymes controlled by aryl hydrocarbon receptor (AhR). Studies of competitive binding with 3H-labelled 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3-methylcholanthrene (3-MC) and benzo[a]pyrene (B[a]P) have shown that β-NF is a high-affinity ligand for AhR and also for polycyclic aromatic hydrocarbon (PAH)-binding protein, both soluble proteins of rat liver in 8 S and 4 S fractions, respectively, of sucrose gradients. This study examined binding of [3H]β-NF to liver cytosolic proteins of female Sprague-Dawley rats. Treatment of rats with β-NF, 3-MC, TCDD or α-naphthoflavone (α-NF) increased the specific [3H]β-NF binding to liver cytosol up to 125-fold that of vehicle (corn oil)-treated rats (< 100 fmol/mg of protein). Sucrose gradients revealed a large 4 S and a small 8 S peak of radioactivity from [3H]β-NF binding to cytosols of β-NF-, 3-MC-, TCDD- or α-NF-treated rats. Whereas co-incubation with the unlabelled β-NF eliminated both peaks, co-incubation with 2,3,7,8-tetrachlorodibenzofuran (TCDF) eliminated only the 8 S peak. The sucrose density gradient from [3H]TCDD binding to cytosol of β-NF- or TCDD-treated rats yielded a small 4 S and a larger 8 S peak; only the latter was abolished by co-incubation with TCDF. Thus, the patterns of sedimentation, distribution and elimination of radioactivity from the 8 S fraction of the liver cytosols from β-NF-, 3-MC-, TCDD- or α-NF-treated rats were characteristic for the AhR, whereas those from the 4 S fraction appeared specific for [3H]β-NF binding. The data indicate that potent AhR agonists, TCDD, 3-MC and β-NF, and to a lesser extent α-NF, a weak AhR agonist, induce a 4 S [3H]β-NF-binding protein in liver cytosol of female rats. α-NF, β-NF and 3-MC were effective competitors (80-85% inhibition) of the [3H]β-NF-specific binding to the β-NF-, 3 MC- or TCDD-induced 4 S protein, whereas several PAHs including B[a]P and benzo[e]pyrene were only weak competitors. The increased [3H]β-NF binding was not associated with glycine N-methyltransferase activity. Hence, the 4 S [3H]β-NF-binding protein described herein differs from the constitutive 4 S PAH-binding protein of rat liver cytosols in the inducibility by β-NF and 3-MC, ligand-binding characteristics, and lack of glycine N-methyltransferase activity. Gel filtration on Sephacryl of liver cytosols from β-NF-treated rats indicated a molecular mass of ≈ 42 kDa for [3H]β-NF-bound protein and suggested that it was derived from a large mass component that before the radioligand binding was eluted with the void volume of the gel and sedimented in a 7 S fraction of the sucrose gradient. The [3H]β-NF binding activity was not eluted with glutathione S-transferase Ya, aldehyde-3-dehydrogenase or DT-diaphorase [NAD(P)H: quinone oxidoreductase] activities, which are AhR-controlled and β-NF-inducible. Further studies are needed to determine the identity and function of this novel protein which may be involved either directly or indirectly (as a carrier protein) in xenobiotic metabolism in vivo.

A novel 4 S [3H]β-naphthoflavone-binding protein in liver cytosol of female Sprague–Dawley rats treated with aryl hydrocarbon receptor agonists

beta-Naphthoflavone (beta-NF) is a widely used inducer of phase-I and phase-II enzymes controlled by aryl hydrocarbon receptor (AhR). Studies of competitive binding with (3)H-labelled 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD), 3-methylcholanthrene (3-MC) and benzo[a]pyrene (B[a]P) have shown that beta-NF is a high-affinity ligand for AhR and also for polycyclic aromatic hydrocarbon (PAH)-binding protein, both soluble proteins of rat liver in 8 S and 4 S fractions, respectively, of sucrose gradients. This study examined binding of [(3)H]beta-NF to liver cytosolic proteins of female Sprague-Dawley rats. Treatment of rats with beta-NF, 3-MC, TCDD or alpha-naphthoflavone (alpha-NF) increased the specific [(3)H]beta-NF binding to liver cytosol up to 125-fold that of vehicle (corn oil)-treated rats (<100 fmol/mg of protein). Sucrose gradients revealed a large 4 S and a small 8 S peak of radioactivity from [(3)H]beta-NF binding to cytosols of beta-NF-, 3-MC-, TCDD- or alpha-NF-treated rats. Whereas co-incubation with the unlabelled beta-NF eliminated both peaks, co-incubation with 2,3, 7,8-tetrachlorodibenzofuran (TCDF) eliminated only the 8 S peak. The sucrose density gradient from [(3)H]TCDD binding to cytosol of beta-NF- or TCDD-treated rats yielded a small 4 S and a larger 8 S peak; only the latter was abolished by co-incubation with TCDF. Thus, the patterns of sedimentation, distribution and elimination of radioactivity from the 8 S fraction of the liver cytosols from beta-NF-, 3-MC-, TCDD- or alpha-NF-treated rats were characteristic for the AhR, whereas those from the 4 S fraction appeared specific for [(3)H]beta-NF binding. The data indicate that potent AhR agonists, TCDD, 3-MC and beta-NF, and to a lesser extent alpha-NF, a weak AhR agonist, induce a 4 S [(3)H]beta-NF-binding protein in liver cytosol of female rats. alpha-NF, beta-NF and 3-MC were effective competitors (80-85% inhibition) of the [(3)H]beta-NF-specific binding to the beta-NF-, 3 MC- or TCDD-induced 4 S protein, whereas several PAHs including B[a]P and benzo[e]pyrene were only weak competitors. The increased [(3)H]beta-NF binding was not associated with glycine N-methyltransferase activity. Hence, the 4 S [(3)H]beta-NF-binding protein described herein differs from the constitutive 4 S PAH-binding protein of rat liver cytosols in the inducibility by beta-NF and 3-MC, ligand-binding characteristics, and lack of glycine N-methyltransferase activity. Gel filtration on Sephacryl of liver cytosols from beta-NF-treated rats indicated a molecular mass of approximately 42 kDa for [(3)H]beta-NF-bound protein and suggested that it was derived from a large mass component that before the radioligand binding was eluted with the void volume of the gel and sedimented in a 7 S fraction of the sucrose gradient. The [(3)H]beta-NF binding activity was not eluted with glutathione S-transferase Ya, aldehyde-3-dehydrogenase or DT-diaphorase [NAD(P)H: quinone oxidoreductase] activities, which are AhR-controlled and beta-NF-inducible. Further studies are needed to determine the identity and function of this novel protein which may be involved either directly or indirectly (as a carrier protein) in xenobiotic metabolism in vivo.

Quantitative whole-body radioluminography-future strategy for balance and tissue distribution studies.

The present routine to conduct balance and/or tissue dissection distribution studies has now and then been questioned, because the way they are generally conducted does not produce information in proportion to the spending of animal and personnel resources. Usually only total radioactivity is measured and due considerations are not always taken to the metabolic fate of the label. In this study a different strategy is presented-integrating quantitative whole-body radioluminography and different chromatographic methods on extracts of tissue pieces punched from the whole-body sections. In addition to the saving in cost and time, the proposed integrated whole-body radioluminographic/metabolic profile protocol will provide (i) a detailed picture of the distribution of radioactivity at selected dose levels and time points in male, female, and pregnant animals; (ii) the time course of radioactivity in blood/plasma and tissues selected from the images (approximate half-life and AUC); (iii) accumulated urinary and fecal excretion of radioactivity and an estimate of the proportion of radioactive metabolites; (iv) tissue information about the proportion of parent drug versus metabolites of pieces punched from the whole-body sections; and (v) indications of possible tissue binding.

Pharmacokinetics of [14C]Genistein in the Rat: Gender-Related Differences, Potential Mechanisms of Biological Action, and Implications for Human Health

Mass balance, plasma pharmacokinetics, tissue distribution, and metabolism of [14C]genistein were investigated in male and female rats (n = 5) following an oral dose of [14C]genistein (4 mg kg−1) to determine potential sites and mechanisms of biological action. Mean total excretion of radioactivity in urine and feces for both sexes was 66 and 33% of the dose respectively at 166 h after administration. Mean and maximal concentrations of radioactivity in plasma were significantly (p < 0.02) higher in male than female rats, with half-lives of 12.4 and 8.5 h, respectively. The concentration of radioactivity was significantly (p < 0.002) higher in liver from females than males and in reproductive (vagina, uterus, ovary, and prostate) compared with other peripheral organs. Analysis of plasma extracts by radio-HPLC-MS indicated that the predominant metabolites were genistein glucuronides, 4-hydroxyphenyl-2-propionic acid, and trace amounts of parent compound (<5%). Radioactive residues in uterus and prostate were predominantly parent compound and 4-hydroxyphenyl-2-propionic acid, respectively. Significantly (p < 0.05) increased retention of [14C]genistein or metabolites was associated with reproductive organs, such as vagina, uterus, ovary, and prostate, likely to contain relatively high concentrations of estrogen receptors or binding proteins compared with other peripheral tissues. Factors liable to influence bioavailability of biologically active genistein or metabolites, such as dietary intake, warrant further investigation to determine the risks or benefits for different consumer groups of phytoestrogen-containing foods.