Introduction

Top Abstract Introduction Materials and methods Results Discussion References

Phenethyl isothiocyanate (PEITC)² occurs 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), and N-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

Top Abstract Introduction Materials and methods Results Discussion References

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.

View larger version (16K): [in this window] [in a new window]   Fig. 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), R_f = 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) R_f = 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, R_f = 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 R_f 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.

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% H₂O₂ was 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.

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  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.

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

Top Abstract Introduction Materials and methods Results Discussion References

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 of ¹⁴C in the blood after dosing with [¹⁴C]PEITC was rapid, with a half-life of absorption (T_1/2a) 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 T_1/2a 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  and . The blood two-compartment  elimination half-life for [¹⁴C]PEITC and one-compartment T_1/2e for [¹⁴C]PHITC were calculated to be 21.7 h and 20.5 h, respectively.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Time course of absorption and elimination of 14C in whole blood after administration of [14C]PEITC () and [14C]PHITC (). Pharmacokinetics program modeled fit is superimposed on data points.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Absorption and elimination of 14C in liver (A), lungs (B), and kidneys (C) after administration of [14C]PEITC and [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 and 5, and the calculated pharmacokinetic parameters for these organs are presented in Table 2. As expected, very high levels of ¹⁴C were observed at early time points in the stomach, small intestine, cecum, and colon, whereas much lower levels of ¹⁴C 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.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Absorption and elimination of 14C 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.

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Absorption and elimination of 14C 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.

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 of ¹⁴C 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.

View larger version (14K): [in this window] [in a new window]   Fig. 6.   Time course of exhaled 14CO2 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 of ¹⁴C occurred in the feces during 48 h.

View larger version (13K): [in this window] [in a new window]   Fig. 7.   Time course of cumulative excretion of 14C in urine (A) and feces (B) after oral administration of [14C]PEITC () or [14C]PHITC ().

	Discussion

Top Abstract Introduction Materials and methods Results Discussion References

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 (C_max) in liver was approximately 3.7 times the C_max in the lungs; whereas for rats in this study, the C_max 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 (T_max).

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 of ¹⁴C 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  and  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 T_1/2e 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 C_max 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 fecal ¹⁴C 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., -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) conjugatethe mercapturic acid pathwayis 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.