RESULTS AND DISCUSSION
Previous studies using the zebrafish SULTs revealed that dextrorphan may be metabolized through sulfation.16) This study aimed to investigate whether and which of the human SULT(s) is(are) capable of catalyzing the sulfation of dextrorphan. Moreover, possible sulfation of dextrorphan in cultured human cells and by human organ cytosols was evaluated.
Identification of Human SULT(s) Capable of Sulfating Dextrorphan
A panel of thirteen human SULTs was analyzed for sulfating activity with dextrorphan as a substrate. Results obtained showed that nine (SULT1A2, SULT1B1, SULT1C2, SULT1C3, SULT1E1, SULT2B1a, SULT2B1b, SULT4A1, SULT6B1) of the thirteen human SULTs displayed no detectable activities. Of the other four SULTs (SULT1A1, SULT1A3, SULT1C4, SULT2A1), SULT1A3 exhibited much stronger activities than the other three towards dextrorphan (Table 1). Based on these results, SULT1A3 is likely the major enzyme responsible for the sulfation of dextrorphan in human body. Since substrate and/or product inhibition may occur for SULTs,9,23) a concentration-dependent experiment was performed using different concentrations of dextrorphan as substrates for all four dextrorphan-sulfating SULTs. As shown in Fig. 1, no significant substrate or product inhibition was observed over the tested dextrorphan concentration ranges (up to 1 mM for SULT1A3 and SULT2A1, and up to 2 mM for SULT1A1 and SULT1C4). As shown in Table 1, in addition to dextrorphan, SULT1A3 was also capable of mediating the sulfation of dopamine (a physiological substrate), p-nitrophenol (a prototype substrate for phenol sulfotransferases), and levorphanol (a levorotatory enantiomer of dextrorphan). It is interesting to note that the sulfating activity of SULT1A3 with dextrorphan was more than 38 times higher than that with levorphanol, whereas the other three SULTs did not show similar stereoselectivity for dextrorphan and levorphanol. The sulfated product of dextrorphan generated by SULT1A3 was purified by solid-phase extraction using a Sep-Pak C18 reverse-phase cartridge and further analyzed using mass spectrometry. As shown in Fig. 2A, a prominent ion with m/z of 336, equivalent to a deprotonated mono-sulfated dextrorphan ion, was detected. This prominent ion produced a sulfonate ion with m/z of 80 and a de-sulfonated ion with m/z of 256, which is the dextrorphan ion (Fig. 2B). These results clearly indicated that the sulfation as mediated by SULT1A3 took place at 3-hydroxyl group of the aromatic ring structure of dextrorphan. Previous studies have demonstrated the expression of SULT1A3 in certain human organs including the brain, gastrointestinal tract, kidney, liver, and lung.24,25) It is possible that in these organs, SULT1A3 may serve to sulfate dextrorphan, a product generated following the O-demethylation of dextromethorphan by CYP2D6.3,4)
Table 1. Specific Activities of the Human SULTs with Dextrorphan as a Substratea)
|Specific activity (nmol/min/mg)|
a) Specific activity corresponds to nmol substrate sulfated/min/mg purified enzyme. Results shown represent the mean±standard deviation derived from three separate assays.
|Fig. 1. Concentration-Dependent Sulfation of Dextrorphan by Human SULT1A1, SULT1A3, SULT1C4, and SULT2A1|
The concentrations of dextrorphan tested were 25, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, and 1000 µM for SULT1A3 and SULT2A1, and 100, 250, 500, 750, 1000, 1250, 1500, 1750, and 2000 µM for SULT1A1 and SULT1C4.
|Fig. 2. Mass Spectrometry Analyses of Sulfated Dextrorphan|
(A) Full mass spectrum of sulfated dextrorphan isolated using a Sep-Pak C18 cartridge. (B) MS2 spectrum of 336 m/z.
It is interesting to note that while all four human dextrorphan-sulfating SULTs belong to the SULT1 family, the two zebrafish SULTs previously shown to be capable of sulfating dextrorphan are members of the SULT3 family.16) Of the two zebrafish dextrorphan-sulfating SULTs, SULT3 ST1 displayed a specific activity of 0.89 nmol/min/mg, whereas SULT3 ST3 showed a specific activity of 0.10 nmol/min/mg. In terms of the dextrorphan-sulfating activity, zebrafish SULT3 ST1 appeared to resemble more closely the major human dextrorphan-sulfating SULT, SULT1A3 (cf. Table 1). Amino acid sequence analysis, however, failed to reveal a particularly high sequence homology between human SULT1A3 and zebrafish SULT3 ST1, as compared with other zebrafish SULTs (data not shown). It thus seems that, in different species, the sulfation of dextrorphan may be mediated by SULT members that bear no direct orthologous phylogenetic relationships.
Production and Release of [35S]Sulfated Dextrorphan by Cultured Caco-2 Cells and HepG2 Cells Incubated in Media Containing [35S]Sulfate and Varying Concentrations of Dextrorphan
Following the identification of human SULTs capable of sulfating dextrorphan, we were interested in finding out whether sulfation of dextrorphan may indeed occur in human cells. Cultured Caco-2 cells and HepG2 cells were used in this study. Previous studies have revealed the expression of several SULT enzymes, including SULT1A1, SULT1A2, SULT1A3, SULT1B1, SULT1C2, SULT1C4, and SULT2A1, in Caco-2 human intestinal epithelial cells.26) HepG2 human hepatoma cells, on the other hand, have been shown to express SULT1A1, SULT1A2, SULT1A3, SULT1E1, and SULT2A1.27,28) Confluent Caco-2 cells and HepG2 cells grown to confluence in different wells of a 24-well plate were incubated in sulfate-free medium containing [35S]sulfate and varying concentrations of dextrorphan. Following an 18-h incubation, the spent media collected were analyzed by the TLC procedure. As shown in Fig. 3, a significant amount of [35S]sulfated dextrorphan was detected in spent labeling medium containing as low as 5 µM of dextrorphan, which increased proportionately with increasing concentrations of dextrorphan added to the labeling media. These results showed clearly that both Caco-2 cells and HepG2 cells were capable of metabolizing dextrorphan by sulfation. As mentioned above, both HepG2 cells and Caco-2 cells have been reported to express SULT1A3, as well as SULT1A1 and SULT2A1,26–28) that are capable of sulfating dextrorphan (cf. Table 1).
|Fig. 3. Analysis of the [35S]Sulfated Dextrorphan Produced and Released by Caco-2 Human Intestinal Epithelial Cells and HepG2 Human Hepatoma Cells Labeled with [35S]Sulfate in the Presence of Dextrorphan|
Confluent Caco-2 cells and HepG2 cells were labeled with [35S]sulfate in the presence of varying concentrations (0, 5, 10, 25, 50, 100 µM) of dextrorphan. Following an 18-h incubation, spent media were collected and analyzed by TLC. The figure shows the autoradiograph taken from the TLC plate following the analysis. Lane E corresponds to [35S]sulfated dextrorphan produced enzymatically under the action of SULT1A3.
Sulfation of Dextrorphan by Human Organ Cytosols
To investigate further whether sulfation of dextrorphan may occur in human organs, enzymatic assays were performed using cytosol fractions prepared from human lung, liver, kidney or intestine. Table 2 shows the activity data obtained from these assays. Of the four human organ cytosols tested, the cytosol prepared from human intestine exhibited the highest dextrorphan-sulfating activity, followed by the cytosols prepared from human liver and lung. Whereas the cytosol prepared from kidney showed no detectable dextrorphan-sulfating activity. These results implied that intestine and liver are likely major human organs involved in the metabolism of dextrorphan through sulfation. It should be pointed out that although SULT1A3, the major dextrorphan-sulfating SULT, is known to be present at much higher level in the intestine than in the liver, the level of dextrorphan-sulfating activity detected for the liver sample amounted to more than 50% of that detected for the small intestine sample (Table 2). It is possible that the still considerable dextrorphan-sulfating activity detected for the liver sample might have been, in part, due to the presence of other dextrorphan-sulfating SULTs (cf. Table 1), particularly SULT1A1 and SULT2A1, that are known to be expressed in the liver.29,30)
Table 2. Sufating Activities of Human Lung, Liver, Kidney, Intestine Cytosol Fractions toward Dextrorphana)
|Specific activity (pmol/min/mg protein)|
a) Specific activity corresponds to pmol substrate sulfated/min/mg protein. Results shown represent the mean±standard deviation derived from three separate assays. The concentration of the substrate used in the assay mixture was 50 µM.
To sum up, the present study revealed SULT1A3 as the major human SULT enzyme capable of sulfating dextrorphan. Cell culture experiments revealed clearly the occurrence of the sulfation of dextrorphan in human cells. Moreover, human intestine, liver, and lung cytosols were shown to be capable of mediating the sulfation of dextrorphan. Collectively, the results derived from the present study provided useful information relevant to the biochemical basis for the metabolism of dextrorphan through sulfation in vitro and in vivo.