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Research ArticleArticle

Disposition and Pharmacokinetics of Phenethyl Isothiocyanate and 6-Phenylhexyl Isothiocyanate in F344 Rats

C. Clifford Conaway, Ding Jiao, Toshiyuki Kohri, Leonard Liebes and Fung-Lung Chung
Drug Metabolism and Disposition January 1999, 27 (1) 13-20;

Abstract

Naturally occurring phenethyl isothiocyanate (PEITC) and its synthetic homolog 6-phenylhexyl isothiocyanate (PHITC) are both effective inhibitors of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced lung tumor development in A/J mice and F344 rats. To help explain why PHITC is considerably more efficacious than PEITC in chemopreventive potency, comparative disposition and pharmacokinetics data for male F344 rats were obtained after a single gavage dose of 50 μmol/kg (3.71 μCi/μmol) [14C]PEITC or 50 μmol/kg (6.59 μCi/μmol) [14C]PHITC in corn oil. After [14C]PEITC dosing, whole blood 14C peaked at 2.9 h, with an elimination half-life (T1/2e) of 21.7 h; blood 14C from [14C]PHITC-treated rats peaked at 8.9 h, with an T1/2e of 20.5 h. In lungs, the target organ, the T1/2e for [14C]PHITC and its labeled metabolites were more than twice that for [14C]PEITC and its labeled metabolites. The effective dose (area under the concentration-time curve) for 14C from PHITC was greater than 2.5 times the area under the concentration-time curve of14C from PEITC in liver, lungs, and several other tissues. During 48 h, approximately 16.5% of the administered dose of [14C]PHITC was expired as [14C]CO2, more than 100 times the [14C]CO2 expired by rats treated with [14C]PEITC. In rats given [14C]PEITC, 88.7 ± 2.2% and 9.9 ± 1.9% of the dose appeared in the urine and feces, respectively, during 48 h; however, rats given [14C]PHITC excreted 7.2 ± 0.8% of the dose of14C in urine and 47.4 ± 14.0% in the feces. Higher effective doses of PHITC in the lungs and other organs may be the basis, in part, for its greater potency as a chemopreventive agent.

Phenethyl isothiocyanate (PEITC)2occurs naturally in certain cruciferous vegetables as a glucosinolate precursor (Tookey et al., 1980; Fenwick et al., 1983; Chung et al., 1992); the free isothiocyanate (ITC) is released by the enzymatic action of myrosinase when plant cells are damaged, such as in chewing. PEITC has been demonstrated to be an effective chemopreventive agent against 7,12-dimethylbenz(a)anthracene-induced tumors of the mammary gland (Wattenberg, 1977), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced tumors of the lung (Morse et al., 1989; Chung et al., 1996), andN-nitrosobenzylmethylamine-induced esophageal tumors (Stoner et al., 1991) in rats. PEITC has also inhibited the development of chemically induced tumors in mice and hamsters (Morse et al., 1991;Adam-Rodwell et al., 1993; Jiao et al., 1994a; Nishikawa et al., 1996). The synthetic homolog of PEITC, 6-phenylhexyl isothiocyanate (PHITC), is also a potent inhibitor of the development of NNK-induced lung tumors in rats and mice (Morse et al., 1991; Jiao et al., 1994a; Chung et al., 1996).

It can be generalized based on current evidence that one of the primary mechanisms by which ITCs prevent tumorigenesis is by inhibiting the microsomal metabolism of carcinogens in target tissues. ITCs act as blocking agents for NNK-induced carcinogenesis by inhibiting the microsomal metabolism of NNK to reactive species that form methyl and pyridyloxobutyl adducts in DNA (Smith et al., 1990; Hecht et al., 1996;Staretz et al., 1997). Specific cytochrome P-450 isozymes active in the metabolism of NNK by microsomes from lungs of rats are inhibited not only by PEITC and PHITC, but also by their respective glutathione, cysteine, and N-acetylcysteine conjugates and by other aryl alkyl ITCs (Guo et al., 1992; Conaway et al., 1996; Jiao et al., 1996).

Previous studies have shown that PHITC is one to two orders of magnitude more potent than PEITC as an inhibitor of NNK-induced lung tumorigenesis in bioassays with A/J mice (Morse et al., 1991; Jiao et al., 1994a). The high potency of PHITC as a cancer chemopreventive agent in mice has been attributed to the increased lipophilicity and relatively low chemical reactivity toward glutathione that is imparted by the C6 alkyl chain (Jiao et al., 1994a). In a F344 rat bioassay, both PEITC and PHITC completely inhibited NNK-induced lung tumor formation at the doses used (Chung et al., 1996); hence, it was not possible to determine their relative potency in F344 rats. However, it is likely that PHITC is more potent than PEITC in F344 rats, because PHITC has been shown to be considerably more potent than PEITC as an inhibitor of rat lung and liver cytochrome P-450 isozymes, especially P-450 2B1 (Guo et al., 1993; Conaway et al., 1996; Jiao et al., 1996).

We previously reported on the tissue disposition of PEITC in mice (Eklind et al., 1991) and on the identity of urinary metabolites of PEITC in mice and humans (Eklind et al., 1991; Chung et al., 1992); however, nothing is known regarding the details of the disposition and pharmacokinetics of PEITC and PHITC. The present study compares the tissue distribution of 14C after oral administration of equivalent doses of [14C]PEITC and [14C]PHITC to male F344 rats, to provide a basis for understanding differences in the chemopreventive efficacy of PEITC and PHITC in the lungs and other organs. Single-dose pharmacokinetic parameters were calculated for whole blood and 13 organs. The excretion of radiolabel in urine, feces, and expired CO2 was also determined. This is the first comprehensive account of single-dose tissue disposition and pharmacokinetics studies of these two promising chemopreventive agents. PEITC is a candidate chemopreventive agent for lung cancer in heavy smokers, and is now undergoing a phase 1 pharmacokinetics study (National Cancer Institute, 1997). Results of the present investigation, together with our earlier work in humans (Chung et al., 1992), will provide a useful database for comparison of blood levels, rates of absorption, and rates of excretion of PEITC in rats and humans.

Materials and Methods

Chemicals.

The route of synthesis of [14C]PEITC and [14C]PHITC is outlined in Fig.1. The reaction conditions were first explored with nonradioactive material, before the procedures were used to prepare [14C]ITCs. [14C]KCN (specific activity, 57 mCi/mmol) was purchased from Moravek Biochemicals, Inc. (Brea, CA). Benzyl chloride and other unlabeled chemicals of reagent grade were bought from Aldrich (Milwaukee, WI), and unlabeled PEITC also was obtained from Aldrich. Unlabeled PHITC was synthesized and characterized in our laboratory (Morse et al., 1991;Jiao et al., 1994a). All other reagents and solvents were of analytical grade, purchased through commercial suppliers.

Figure 1
Figure 1

Synthesis scheme for [14C]PEITC and [14C]PHITC. The position of label is indicated by the asterisk.

Preparation of [14C]PhCH2 CN.

[14C]KCN (16.3 mg, 0.25 mmol) was dissolved in 0.1 ml H2O. Benzyl chloride (26.6 mg, 0.21 mmol) in 0.6 ml ethanol was added to the [14C]KCN solution with stirring. The mixture was refluxed for 3 h. After cooling to room temperature, 3 ml of methylene chloride was added. The organic phase was separated and dried over anhydrous MgSO4. After filtration, the solvent was removed with a stream of dry nitrogen. An oily liquid identified as [14C]benzyl cyanide was obtained (14.5 mg; 60% yield). Thin-layer chromatography (TLC) (CH2Cl2), Rf = 0.85, showed a single spot under UV light.1H NMR (in CDCl3), -CH2-, 2H, s, 3.80 ppm; Ar-H, 5H, m, 7.30 to 7.40 ppm.

Preparation of [14C]PhCH2CH2NH2.

[14C]benzyl cyanide, 35 mg (0.3 mmol) was added as a CH2Cl2 solution to a reaction vial, and the CH2Cl2 was removed with a stream of N2. A solution of BH3 (1.0 M) in tetrahydrofuran, 0.6 ml (0.6 mmol) was added dropwise under N2 while stirring. The mixture was refluxed for 0.5 h and then cooled to room temperature. Five normal HCl (0.2 ml) was added dropwise into the reaction mixture, which was then refluxed for another 0.5 h. The pH was adjusted to 11 to 12 with 1 N NaOH. The mixture was extracted 3 times with 2 ml of Et2O. The ether phase was dried over anhydrous MgSO4 for 2 h. The solution was filtered and the solvent removed with a stream of N2. An oily liquid was obtained (31.3 mg; 86% yield). TLC (CH2Cl2) Rf = 0.08, a single spot under UV light. 1H NMR (in CDCl3), Ph-CH2-, 2H, t, 2.80 ppm; -CH2-NH2, 2H, t, 3.00 ppm; Ar-H, 5H, m 7.15 to 7.40 ppm.

Preparation of [14C]PEITC.

[14C]Phenethylamine, 18.4 mg, was dissolved in 1 ml of CHCl3, and 1 ml of NaOH (1 N) was added with vigorous stirring at 0°C. Using a glass syringe, 210 μl of thiophosgene solution (1.0 M in CHCl3) was added dropwise. The mixture was stirred at 0°C for 15 min, and the two phases were separated; the aqueous phase was extracted with 1.5 ml of chloroform. The organic phase was dried over anhydrous MgSO4 for 2 h. After filtration and removal of solvent, 22.5 mg of crude product was obtained, and it was purified by passing it through a silical gel column with hexane as eluant. After removing the hexane, 16.8 mg of an oily liquid (74% yield) was obtained. TLC (hexane) showed a single spot under UV light, Rf = 0.51; 1H NMR (in CDCl3), Ph-CH2, 2H, 5, 3.00 ppm; CH2-NCS, 2H, t, 3.72 ppm; Ar-H, 5H, m, 7.15–7.40 ppm. The radiochemical purity (>99%) of [14C]PEITC was confirmed by high-pressure liquid chromatography (HPLC) with unlabeled PEITC as a standard and a beta-flow detector; unlabeled PEITC was also used to dilute the product to 3.71 μCi/μmol.

Preparation of [14C]Ph(CH2)5CN.

[14C]KCN (14.5 mg–0.22 mmol) was dissolved in 0.25 ml of H2O. Ph(CH2)5Cl (34.0 mg, 0.186 mmol) was weighed into a 5-ml vial, and the [14C]KCN solution was added. The vial containing the [14C]KCN was rinsed with 1 ml dimethylformamide, and the rinse was added to the reaction mixture. After refluxing for 3 h with stirring, the reaction mixture was extracted with 3 times 1.5 ml of hexane. This fraction was dried over MgSO4 overnight, and then the solvent was removed with a stream of N2. The purity of the product was confirmed by TLC using CH2Cl2 or hexane/CH2Cl2 (4:1) as the mobile phase.

Preparation of [14C]Ph(CH2)5CH2NH2.

The [14C]Ph(CH2)5CN was transferred to a reaction vial as its Et2O solution, and the solvent was removed using a gentle stream of N2. A 1 M solution of BH3 in THF was prepared, and 0.36 ml was added dropwise with stirring. The mixture was then refluxed for 0.5 h and cooled to room temperature. After adding 0.2 ml of 5 N HCl, the mixture was refluxed again for an additional 0.5 h and cooled to room temperature. The pH was adjusted to 11 to 12 by adding 1 N NaOH. The reaction mixture was extracted 3 times with 1.5 ml of Et2O, and the solvent was removed (dry nitrogen). A single spot under UV light was seen on the TLC plate (CH3OH); it had the same Rf as an unlabeled standard.

Preparation of [14C]PHITC.

[14C]Phenylhexylamine was dissolved in 1 ml of CHCl3 and 1 ml of 1 N NaOH was then added, with stirring at 0°C. Then 280 μl of a 1 M thiophosgene solution in CHCl3 was added dropwise under nitrogen with stirring. The reaction product was extracted with 3 times with 1.5 ml of CHCl3, yielding a single spot on a TLC plate (hexane). The purity of the product was confirmed by HPLC using unlabeled PHITC as a standard and a beta-flow detector for measuring 14C. The product was diluted with unlabeled PHITC to a specific activity of 6.59 μCi/μmol.

Animals and Treatment.

Male F344 rats (Charles River, Kingston, NY) were maintained under standard conditions: 20 ± 2°C, 50 ± 10% relative humidity in a 12-h light/12-h dark cycle. The rats were provided pelleted NIH-07 diet and water ad libitum before treatment. Rats weighing 201.5 ± 7.47 g were treated by gavage with 10 μmol of [14C]PEITC (3.71 μCi/μmol) in 1 ml of corn oil per 200 g weight (50 μmol/kg). In the second experiment, rats weighing 180.4 ± 4.28 g were fasted overnight and gavaged with 10 μmol of [14C]PHITC (6.59 μCi/μmol) in 1 ml of corn oil per 200 g weight. In both experiments water was provided continually, and food was replaced 4 h after dosing. Three rats were used for each time point; the rats were sacrificed at 0.5, 1, 2, 4, 8, 24, and 48 h, respectively. Rats to be sacrificed at 24 and 48 h were placed in glass metabolism chambers so that expired radioactive organic materials, 14CO2, urine, and feces could be collected at 8, 24, and 48 h. Rats sacrificed at earlier time points were placed in stainless steel metabolism cages with wire mesh bottoms; urine and feces from these rats were collected at 2, 4, and 8 h. The urine was collected in 50-ml polypropylene centrifuge tubes immersed in dry ice and acetone; fecal pellets were collected from the bottom screen of the cage. Both urine samples and feces were stored frozen (−20°C) until analyzed.

At the termination of the experiment, each rat was anesthetized with CO2, and blood (3–6 ml) was taken by cardiac puncture using 7-ml heparinized Vacutainer tubes (Becton Dickinson, Rutherford, NJ). The rat was then decapitated, and tissues (brain, nasal mucosa, esophagus, stomach, pancreas, heart, spleen, lungs, liver, kidneys, small intestines, cecum, and colon) were immediately removed, rinsed in ice-cold saline, wrapped in aluminum foil, and frozen in liquid nitrogen. Flash-frozen tissues were transferred to a −20°C freezer until analyzed. The tubes of blood were kept on ice; after mixing, two 200-μl aliquots were taken for analysis of radioactivity.

Analysis of 14C in Blood and Tissues, Carbon Dioxide, Expired Air, and Excreta. 14C in Tissues and Blood.

Each tissue or organ was thawed, rinsed in ice-cold saline and patted dry, placed in a tared 20-ml scintillation vial, and weighed. Two or three volumes of 0.05 M Tris-HCl, pH 7.4, were added, and the tared weight recorded. The tissue was then homogenized using an IKA Ultra-Turrax T25 tissue-mizer (IKA-Works, Inc., Cincinnati, OH) with the exception of liver, kidneys, and lungs (see below). Duplicate 400-μl aliquots of homogenate were pipeted into scintillation vials and weighed, and then 1 ml of Solvable (Packard Instruments, Meriden, CT) was added. The vial was capped and incubated for 3 h at 50°C to solubilize the tissue; 100 μl of 30% H2O2was then added, and the loosely capped vial was allowed to stand at room temperature for 1 h to decolorize the sample (for blood, 0.5 ml 0.5 N HCl was added to further decolorize the sample and to inhibit chemiluminescence). Before scintillation counting, Atomlight cocktail (9 ml) (Packard Instruments) was added. Background-corrected disintegrations per minute per milligram wet tissue weights were calculated and expressed as picomoles per milligram wet tissue weight on the basis of the specific activity of 14C.

Livers, kidneys, and lungs were rinsed in ice-cold saline, patted dry, weighed, and homogenized (1:4, w/v) in 0.25 M sucrose, 0.05 M Tris-HCl, pH 7.4 (4°C), using a Potter-Elvejhem (Teflon/glass) homogenizer to preserve the integrity of subcellular organelles. The homogenate fractions were weighed and then weighed 300 to 400-μl aliquots were taken for determination of 14C using the methods described above.

Exhaled Radioactivity.

Rats in groups 6 and 7 (sacrificed at 24 and 48 h, respectively) were placed in glass metabolism chambers (Bio-Serve, Frenchtown, NJ), and constant air flow through the chambers was maintained. The air flow exiting the chambers was scrubbed through 200 ml of absolute ethanol to remove exhaled organics, followed in series by a gas washing bottle containing 200 ml of 4 N NaOH to collect expired CO2(Ioannou et al., 1984). Gas washing bottles were replaced at 8 and 24 h, and duplicate samples of 1 ml of ethanol plus 10 ml of Atomlight scintillation cocktail (Packard Instruments, Meriden, CT), or duplicate samples of 0.5 ml of NaOH solution and 20 ml of Atomlight were pipeted into scintillation vials, mixed, and allowed to stand in the dark for 30 min before counting. Quench-corrected β activity was measured using a LC 6800 liquid scintillation spectrometer (Beckman Instruments, Irvine, CA), background was subtracted, and the recovered 14C per fraction was calculated.

Analysis of 14C in Urine and Feces.

Urine fractions from each rat were thawed, quantitatively transferred to a tared vial, and weighed. Weighed aliquots of vortexed urine (200–400 μl) were then placed in scintillation vials and analyzed for 14C (method described above). The amount of excreted radioactivity at each time point was calculated and expressed as cumulative percentage of administered dose.

Fecal samples were weighed in tared 20-ml vials, and 9 volumes of 50 mM potassium phosphate buffer, pH 7.4, was added. Each sample plus buffer was weighed, capped, and the feces were subsequently allowed to soften for several hours. The sample was then thoroughly homogenized and vortexed, and weighed aliquots (200–400 μl) were pipeted into scintillation vials. Radioactivity was determined by digestion in 1 ml of Solvable, addition of H2O2 to decolorize the sample, and scintillation counting as above. Total fecal radioactivity at each time point was calculated for each rat and was expressed as cumulative percentage of the administered dose.

Pharmacokinetic Evaluation of Analytical Data.

Using the specific activity of administered [14C]PEITC and [14C]PHITC, respectively, ITC concentrations in tissues were calculated and expressed as picomoles per milligram wet weight, whereas the 14C in whole blood was expressed in terms of picomoles per microliter. The mean values at each time point were used for the plots in the figures and for the pharmacokinetic parameter estimates, except when the coefficient of variation exceeded 30%; an outlying value was then excluded from the mean. The pharmacokinetic parameter estimates were derived from single- or two-compartment models available in WinNonlin Version 1.5 (Scientific Consulting, Inc., Apex, NC).

Results

Tissue Distribution and Pharmacokinetics of 14C in Whole Blood, Liver, Lungs, and Kidneys.

The time course of 14C absorption and elimination in whole blood after administration of [14C]PEITC or [14C]PHITC is presented in Fig.2. The rate of appearance of14C in the blood after dosing with [14C]PEITC was rapid, with a half-life of absorption (T1/2a) of 1.3 h and peak concentration of 18.8 pmol (pmol/μl = nmol/ml) occurring at 2.9 h (Table 1). The rate of absorption of [14C]PHITC in the bloodstream was somewhat slower, with a T1/2a of 2.7 h and a peak concentration of 7.4 pmol/μl at 8.9 h. A two-compartment model was used for calculating pharmacokinetic parameters for [14C]PEITC-derived 14C for whole blood, because it more closely approximated the data points than a single compartment model. A one-compartment model was used, however, for analysis of whole blood data after administration of [14C]PHITC and for all other tissues, because the number of data points was insufficient to accurately define any differences between α and β. The blood two-compartment β elimination half-life for [14C]PEITC and one-compartment T1/2e for [14C]PHITC were calculated to be 21.7 h and 20.5 h, respectively.

Figure 2
Figure 2

Time course of absorption and elimination of14C in whole blood after administration of [14C]PEITC (•) and [14C]PHITC (■). Pharmacokinetics program modeled fit is superimposed on data points.

View this table:
Table 1

Pharmacokinetic parameters for whole blood, liver, lungs, and kidneys after oral administration of [14C]PEITC and [14C]PHITC

The time courses of absorption and elimination of 14C in liver, lungs, and kidneys after administration of the labeled ITCs are shown in Fig. 3, whereas the respective calculated pharmacokinetic parameters are also presented in Table 1. Maximum levels of 14C in the liver were reached a few minutes before those in blood, and normalized levels of ITCs (picomoles per disintegration per minute times disintegrations per minute per milligram tissue) in the liver after administration of [14C]PHITC were considerably higher than for [14C]PEITC over extended periods. The computer-generated elimination curves from which pharmacokinetic parameters were derived (not shown except for whole blood) were, in most instances, very similar to the respective plots of mean [14C]ITC concentrations for each of the organs and tissues. A comparison of effective dose [area under the curve (AUC) in picomoles per milligrams times hours] shows that PHITC and its metabolites are cleared from the liver much more slowly (>2.6-fold) than PEITC and its metabolites (Table 1).

Figure 3
Figure 3

Absorption and elimination of14C in liver (A), lungs (B), and kidneys (C) after administration of [14C]PEITC and [14C]PHITC.

In lungs, maximum levels of PEITC and PHITC were similar, but the elimination of 14C from [14C]PHITC was 2.2-fold slower. At 48 h, the concentration of [14C]PHITC and its labeled metabolites in the lung was about twice that of [14C]PEITC and its labeled metabolites (Fig. 3B); AUC calculations (Table 1) indicate that the effective dose of [14C]PHITC and its metabolites was approximately 2.6-fold greater in the lungs than that of [14C]PEITC and its metabolites.

Curves for the absorption and elimination of 14C in the kidneys after dosing with [14C]PEITC or [14C]PHITC are presented in Fig. 3C. The shapes of the curves are similar to those observed for whole blood. Unlike in liver, in kidneys the peak concentration for [14C]PEITC and its labeled metabolites was higher than that of [14C]PHITC and labeled metabolites. On the basis of the respective AUCs, the effective dose of PHITC and its labeled metabolites delivered to the kidneys was 1.3 times greater than the effective dose of PEITC and its labeled metabolites. The half-lives of absorption and elimination for [14C]PEITC and [14C]PHITC in kidneys parallel those observed for whole blood, but with a two-fold extended T1/2e for [14C]PHITC.

Distribution and Pharmacokinetics of [14C]ITCs and Metabolites in Other Organs.

The tissue distribution of 14C in nasal mucosa, esophagus, stomach, small intestine, cecum, colon, pancreas, spleen, heart, and brain are depicted in Figs. 4 and5, and the calculated pharmacokinetic parameters for these organs are presented in Table2. As expected, very high levels of14C were observed at early time points in the stomach, small intestine, cecum, and colon, whereas much lower levels of14C were consistently found in the esophagus and nasal mucosa. The distribution of radiolabel in the alimentary tract reflects the method of administration, in which the ITC, dissolved in corn oil vehicle, was injected directly into the stomach. Table 2 shows that in the [14C]PHITC experiment, relatively high amounts of radioactivity were retained in the cecum, as compared with [14C]PEITC; however, small but significant amounts of [14C]PEITC and its metabolites remained in the cecum over the course of 48 h. In both studies, intermediate concentrations of 14C were detected in the pancreas and spleen, and lower concentrations occurred in the heart and brain. In both experiments, the initially high levels of 14C in the alimentary tract were greatly reduced by 24 h. Consistent with findings in blood, liver, and lung, the retention of 14C in other major organs was generally longer after administration of [14C]PHITC than after [14C]PEITC.

Figure 4
Figure 4

Absorption and elimination of14C in organs from male rats treated with [14C]PEITC. A, Nasal mucosa, esophagus, and stomach. B, Small intestine, cecum, and colon. C, Pancreas, spleen, heart, and brain.

Figure 5
Figure 5

Absorption and elimination of14C in organs from male rats treated with [14C]PHITC. A, Nasal mucosa, esophagus, and stomach. B, Small intestine, cecum, and colon. C, Pancreas, spleen, heart, and brain.

View this table:
Table 2

Pharmacokinetic parameters for selected organs after oral administration of [14C]PEITC or [14C]PHITC

Exhaled Radioactivity.

Radioactivity from both [14C]PEITC and [14C]PHITC was exhaled mainly as [14C]CO2 (Fig.6). After 24 h, the amount of [14C]CO2 from [14C]PEITC-treated rats in the traps was 0.039 ± 0.007 μCi (0.1% of applied dose), compared with 8.18 ± 0.67 μCi (13.8% of applied dose) in NaOH traps after administration of [14C]PHITC. Considerably less 14C was exhaled after 24 h (24–48 h) in both studies. Very small amounts of14C were exhaled as ethanol-soluble organic compounds; the cumulative 14C measured in ethanol traps 24 h after administration of [14C]PEITC was 0.0021 ± 0.0014 μCi (0.006% of applied dose), compared with 0.0040 ± 0.0016 μCi (0.007% of applied dose) in ethanol traps 24 h after administration of [14C]PHITC.

Figure 6
Figure 6

Time course of exhaled14CO2 expressed as cumulative percentage of applied dose after oral administration of [14C]PEITC (•) or [14C]PHITC (■).

Radioactivity in Urine and Feces.

The cumulative excretion of 14C in urine and feces is shown in Fig 7. For rats given [14C]PEITC, 88.7 ± 2.2% and 9.9 ± 1.9% of the radioactivity appeared in the urine and feces, respectively, after 48 h, and most of the urinary excretion of 14C had occurred within the first 24 h after dosing. When rats were given [14C]PHITC, however, 7.2 ± 0.8% of the dose was excreted in urine, but the major route of elimination was fecal excretion: 47.4 ± 14.0% of the administered dose of14C occurred in the feces during 48 h.

Figure 7
Figure 7

Time course of cumulative excretion of14C in urine (A) and feces (B) after oral administration of [14C]PEITC (•) or [14C]PHITC (■).

Discussion

Data for disposition and excretion of [14C]PEITC were previously established for female A/J mice (Eklind et al., 1991). In that study, 5 μmol of [14C]PEITC (2 μCi) was administered by gavage in corn oil to unfasted mice (∼20 g weight). Peak levels of 14C occurred in the heart, liver, and lungs within 4 to 8 h. In kidneys, tissue levels of 14C were roughly constant during the first 8-h period and declined thereafter. Brain and spleen 14C peaked at 1 h; the earliest time point measured. In mice, the maximal ITC concentration in tissue (Cmax) in liver was approximately 3.7 times the Cmax in the lungs; whereas for rats in this study, the Cmax in liver was approximately 4 times greater than that in the lungs. Thus, the data for rats are similar to those previously reported for mice, although the rate of absorption of 14C in rats appears to have been more rapid on the basis of time at maximum concentration (Tmax).

In this study, male F344 rats gavaged with [14C]PEITC were not fasted before dosing, whereas rats given [14C]PHITC were fasted overnight; in both experiments food was reintroduced 4 h after dosing. Food in the stomach tends, as a general principle, to retard absorption of drugs and xenobiotics; therefore, rates of single-dose absorption as indicated by the half-life of absorption of [14C]PEITC compared with that of [14C]PHITC in blood, liver, lungs, kidneys, nasal mucosa, spleen, and heart would probably have been even faster if the rats had also been fasted in the [14C]PEITC experiment. Tissues of the alimentary tract, pancreas, and heart did not demonstrate significant differences in the rates of absorption of14C in the two experiments (Table 2). In the instance of the alimentary tract, for both experiments, the time of the peak concentrations of 14C appeared to be strongly dependent on the rate of intestinal passage of the dose, as shown in Figs. 4 and 5.

Pharmacokinetic parameters for absorption and elimination of [14C]PEITC and its labeled metabolites for whole blood were calculated using a two-compartment model, but because too few time points were available for a reliable estimation of the parameters α and β in all other instances, including whole blood [14C]PHITC, a single-compartment model was used. Whole blood rather than plasma was used to monitor radioactivity, because PEITC and PHITC both bind readily to proteins and other cellular nucleophiles (our unpublished data).

The longer alkyl chain length (C6) of PHITC imparts greater lipophilicity than the structural features of PEITC (Morse et al., 1991; Jiao et al., 1994a); this property probably accounts for the consistently slower appearance of [14C]PHITC in the bloodstream and in major organs. The higher lipophilicity of PHITC may also account for a decrease in the rate of elimination compared with PEITC. PHITC in the intestinal lumen would have to pass through several lipid bilayer membranes during the processes of absorption from the gut and distribution to the tissues with their various cell types; one would therefore expect that PHITC would have been more readily sequestered in lipids during these processes. In this study, the T1/2e values calculated from the tissue distribution data were consistently longer for PHITC than for PEITC. An indeterminate fraction of the [14C]PHITC may have been deposited in the esophagus rather than in the stomach during gavage dosing of some rats at early time points; this may account for the high esophageal tissue concentrations of 14C at 0.5 to 2 h.

A very large proportion of the dose of [14C]PHITC absorbed from the gut is evidently retained in liver tissue during the initial pass of blood through the liver, thus decreasing the blood-borne concentrations available for distribution to other organs (Fig. 3A). Slower rates of absorption and elimination of [14C]PHITC and its labeled metabolites would largely account for the higher AUC values observed for liver, lungs, kidneys, and other organs, especially when Cmax values were considerably lower for [14C]PHITC than for [14C]PEITC. Significant amounts of radioactivity remained in the tissues of rats treated with [14C]PHITC at the 48-h time point, much more so than in rats treated with [14C]PEITC.

The much higher proportion of administered 14C in the feces after dosing with [14C]PHITC (Fig. 7B) could indicate a slower rate of absorption from the intestinal tract; however, the pharmacokinetics data for the liver (Table 1), the high lipophilicity of PHITC, and several large unknown 14C metabolite peaks, with minimal amounts of [14C]PHITC in HPLC chromatograms of organic extracts of feces (Conaway et al., 1998), suggest that biliary excretion of 14C metabolites, possibly glucuronides, may account for a significant portion of fecal14C from [14C]PHITC. Identification of fecal metabolites of [14C]PEITC and [14C]PHITC is presently underway, and studies designed to measure biliary excretion are planned.

The chemical structures of PEITC and PHITC and the observed differences in the tissue disposition and excretion may explain differences in the metabolism of these ITCs. Because a much greater proportion of the administered dose of [14C]PHITC was retained in the liver, a greater extent of oxidative metabolism of [14C]PHITC by liver enzymes could have been anticipated, e.g., β-oxidation of alkyl acid moieties from PHITC with ultimate formation of [14C]CO2. [14C]CO2 exhaled by rats treated with [14C]PHITC exceeded that by [14C]PEITC-treated rats more than a 100-fold.

Conjugation of ITCs with glutathione (GSH) and subsequent excretion in the urine as the N-acetylcysteine (NAC) conjugate—the mercapturic acid pathway—is the major route of metabolism for naturally occurring benzyl ITC, allyl ITC, and other ITCs in rats and humans (Brüsewitz et al., 1977; Mennicke et al., 1983, 1988;Ioannou et al., 1984; Chung et al., 1992; Jiao et al., 1994b). In these experiments, more than 90% of radioactivity in organic extracts of urine from [14C]PEITC-treated rats was identified as [14C]PEITC-NAC by cochromatography of authentic standard and subsequent confirmation by mass spectroscopy. In contrast, [14C]PHITC-NAC was not detected in urine from [14C]PHITC-treated rats (Conaway et al., 1998). The rate of conjugation of PEITC with GSH is approximately 3 times faster than the reaction of PHITC with GSH (Jiao et al., 1994a). Therefore, other pathways, including oxidation of the alkyl side chain, possible conjugation of [14C]PHITC with glucuronic acid in the liver and biliary excretion, and ultimate appearance of radiolabel in the feces may substantially account for the observed differences in the patterns of excretion of [14C].

During 72 h following dosing of female A/J mice, the cumulative urinary excretion of [14C]PEITC-derived radioactivity was 55.2%, whereas 23.3% of 14C activity appeared in the feces (Eklind et al., 1991). The higher dose per kilogram of body weight of PEITC in the A/J mouse study may have influenced the relative proportion of administered dose that was absorbed from the gut. The major urinary metabolites in mice were PEITC-NAC (17.4–27.6% of urinary 14C) and a cyclic pyruvic acid conjugate of PEITC (46.8–58.0% of urinary 14C). After administration of watercress to humans, which is a dietary source of PEITC, PEITC-NAC was the only major metabolite observed in the urine (Chung et al., 1992), suggesting that the metabolic pathways in humans for PEITC and perhaps other ITCs are similar to those occurring in rats.

On the basis of the data for male rats given identical doses of ITCs, the effective concentration of [14C]PHITC and/or its metabolites in the lungs was considerably higher than that of [14C]PEITC and/or its labeled metabolites; the lung AUC for [14C]PHITC and its 14C metabolites was more than 2.6 times greater than that for [14C]PEITC and its 14C metabolites. This observation, and the evidence for higher potency of PHITC as an inhibitor of microsomal P-450s that metabolize the tobacco-specific nitrosamine NNK in the lung (Guo et al., 1993), supports the notion that, as in A/J mice, the chemopreventive efficacy of PHITC in rat lungs would be greater than that of PEITC. Lower doses of PEITC and PHITC than were used in the previous bioassay (Chung et al., 1996) would be necessary, however, to conclusively differentiate their respective efficacies as chemopreventive agents for NNK-induced lung tumors in F344 rats.

Acknowledgments

The animal treatments in these studies were performed by the Research Animal Facility of the American Health Foundation under the supervision of Joel Reinhardt. We thank Ilse Hoffman for critically reading the manuscript.

Footnotes

  • Send reprint requests to: Dr. C. C. Conaway, Division of Carcinogenesis and Molecular Epidemiology, American Health Foundation, 1 Dana Road, Valhalla, NY 10595.

  • ↵1 Present address: Mitsui Norin Co., Ltd., Food Research Laboratories, 233 Miyabara, Fujieda City, Shizouka 426-01, Japan.

  • This study was supported by Grant CA46535 from the National Cancer Institute. This article is number 29 in the series “Dietary Inhibitors of Chemical Carcinogenesis”.

  • Abbreviations used are::
    PEITC
    phenethyl isothiocyanate
    ITC
    isothiocyanate
    PHITC
    6-phenylhexyl isothiocyanate
    PEITC-NAC
    N-acetylcysteine conjugate of PEITC
    PHITC-NAC
    N-acetylcysteine conjugate of PHITC
    GSH
    glutathione
    NNK
    4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone
    AUC
    area under the concentration-time curve
    T1/2a
    half-life of absorption
    T1/2e
    half-life of elimination
    Tmax
    time at maximum concentration
    Cmax
    maximum ITC concentration in tissue
    KCN
    potassium cyanide
    TLC
    thin layer chromatography
    HPLC
    high-pressure liquid chromatography

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Disposition and Pharmacokinetics of Phenethyl Isothiocyanate and 6-Phenylhexyl Isothiocyanate in F344 Rats

C. Clifford Conaway, Ding Jiao, Toshiyuki Kohri, Leonard Liebes and Fung-Lung Chung
Drug Metabolism and Disposition January 1, 1999, 27 (1) 13-20;
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