Life Sciences, Vol. 34, pp. 23-29 Printed in the U.S.A.
FACTORS INFLUENCING SULFATION IN ISOLATED RAT HEPATOCYTES David W. Sundheimer and Klaus Brendel Department of Pharmacology, University of Arizona Health Sciences Center, Tucson, Arizona 85724 (Received in final form October 18, 1983) Summary Sulfation of harmol by isolated hepatocytes was dependent on an exogenous source of sulfate. Inorganic sulfate ion stimulated sulfation by over ten fold. Analysis of the stimulation of harmol sulfation by sulfate indicated a Km of 239 ~M and a Vma x of i.i ~moles harmol sulfate/min/106 cells. Cysteine also stimulated the rate of harmol sulfation but was less effective than sulfate ion. Lithium chloride inhibited harmol sulfation. Sulfation was unaffected by several metabolic alterations which inhibited harmol glucuronidation. Fasting for 24 hours, and incubation with ethanol or linoleic acid, did not influence the rate of sulfation but inhibited glucuronidation by 50 percent. Conjugation with inorganic sulfate is an important route of metabolism in mammals for a number of endogenous compounds such as catecholamines, steroids and bile acids (i). In addition, metabolism by this pathway has an important role in the elimination of several widely used therapeutic agents (2) and in the activation of N-hydroxylated aromatic amines (3-6). The recognition of the importance of sulfation in the biotransformation of these substances has led to an interest in factors which may alter the activity of this pathway. Conjugation with sulfate could be modified by several mechanisms. It has previously been widely accepted that the in vivo sulfation of exogenous substances is restricted by the limited size of endogenous sulfate pools. Recently, however, this hypothesis has been questioned (7-9). Conjugation with sulfate requires first the conversion of inorganic sulfate ion to an activated form, 3'-phosphoadenosine-5'-phosphosulfate in a process using two molecules of ATP. Thus the rate at which compounds are sulfated could also be influenced by changes in intermediary metabolism. Many substrates are simultaneously conjugated with glucuronic acid as well as sulfate (10-12), and as a result the two pathways compete for substrate. Consequently, an alteration in glucuronidation could indirectly affect conjugation with sulfate. The purposes of the present study were to investigate the dependence of sulfate conjugation by isolated rat hepatocytes on an exogenous source of sulfate and to determine the sensitivity of this pathway to alterations in intermediary metabolism. Harmol, which is both sulfated and glucuronidated, was used as a model compound in order to determine direct effects on sulfation and to monitor the interaction of the two pathways under different conditions.
0024-3205/84 $3.00 + .00 Copyright (c) 1984 Pergamon Press Ltd.
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Materials and Methods Male Sprague Dawley rats 200-300 g were fed ad libitum or, where indicated, fasted 24 hours. Hepatocytes were prepared, incubated and analyzed for harmol metabolites as previously described (13) with the following modifications. Cells were incubated at a concentration of 2 x 106 cells/ml. Viability, as determined by trypan blue exclusion, averaged 91%. Incubations contained 2.3 mM MgSO 4 except where indicated and in such cases MgCI 2 was substituted to maintain a magnesium concentration of 2.3 mM. Hepatocytes were incubated with 200 ~M harmol concentrations at which both harmol sulfation and glucuronidation proceed at maximal velocity (13). Compounds, whose effects on sulfation were studied, were added i0 minutes before the addition of harmol. Sulfate and glucuronide metabolites were measured in the same incubations, either from a disrupted cell suspension or from separated medium and cells. Results are reported as the mean of values from duplicate incubations of at least two different preparations of hepatocytes. Variability is indicated by the standard error of the mean. Results Without added sulfate, hepatocytes synthesized harmol sulfate at a rate of only 0.08 nmoles/min/106 cells. However, the addition of S04-2 to hepatocytes suspensions increased the rate of sulfation and at the highest concentration, a greater than ten-fold stimulation was observed (Fig. la). Analysis of the rate of sulfation versus the inorganic sulfate concentration indicated a K m of 239 + 42 DM for sulfate and a Vma x of i.ii + 0.22 nmoles/min/106 cells Fig. ib). T~e rate of glucuronidation (2.30 nmolesTmin/106 cells) was unaffected by the sulfate concentration (Fig. la).
Fig. la. Effect of inorganic sulfate ion on the sulfation and glucuronidation of harmol. Hepatocytes were incubated with the indicated concentrations of S04 -2 for i0 minutes before addition of 200 ~M harmol. The rates of sulfation and glucuronidation were determined by analysis of reaction mixtures at i0 and 20 minutes after the addition of harmol.
Harmol Sulfate Harmol Glucuronide
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Fig. lb. Double reciprocal plot of harmol sulfation versus inorganic sulfate concentration (Km = 239 + 42 ~M and Vma ~ = l.ll + 0.2~ nmoles/min/10 o cells)
mM [S04-2]-1 Cysteine was also an effective sulfate donor, although equal concentrations of this compound did not stimulate the rate of sulfation as much as inorganic sulfate (Fig. 2). The rate of harmol sulfation increased with the
sulfation and glucuronidation of harmol. Preincubation with cysteine, addition of harmol and analysis of reaction mixtures were performed as described in Figure la. ~ Harmol Sulfate  Harmol Glucuronide
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eysteine concentration to a maximal rate of 0.45 nmoles/min/106 cells at 5 m M cysteine. The rate of glucuronidation (2.42 nmoles/min/106 cells) was unaffected by the presence of cysteine. Sulfation proved to be insensitive to a number of metabolic alterations which altered glucuronication (Table I). The addition of ethanol, lactate, bovine serum albumin (BSA) or BSA plus linoleic acid had no effect on the initial rate of sulfation in hepatocytes from ad libitum fed animals. However, ethanol and BSA plus linoleic acid each caused a significant inhibition of harmol glucuronidation. Hepatoeytes from 24-hour fasted rats conjugated harmol with sulfate at an initial rate similar to that observed in hepatocytes from control animals. Similarly, ethanol had no effect on the initial rate of sulfation in these cells. Fasting decreased the rate of glucuronidation and
TABLE I The Effect of Metabolic Manipulation on the Sulfation and Glucuronidation of Harmol
Hepatocyte Source ad libitum fed animals
Harmol Sul~ate nmoles/min/10 v cells
Harmol Glucu~onide nmoles/min/10 v cells
1.32 + 0.15
2.56 + 0.30
Ethanol i0 mM
1.41 + 0.20
1.34 + 0.21
Lactate i0 mM
1.25 + 0.13
2.07 + 0.20
1.36 + 0.21
2.28 + 0.15
3% BSA plus 2 m M linoleic acid
1.22 + 0.13
1.41 + 0.12
Li-Lactate i0 m M
24-hour fasted animals
, 0.56 + 0.i0
2.16 + 0.31
Li CL i0 mM
0.67 + 0.ii
2.45 + 0.27
1.04 + 0.ii
1.37 + 0.19
Ethanol i0 mM
1.23 + 0.13
0.66 + 0.15 *+
Hepatocytes were isolated from the indicated animals and preincubated with various substrates for i0 minutes. Incubations contained 2.3 mM MgSO 4. Harmol (200 ~M) was added and the initial rates of conjugation were determined. * Different from fed control p < .001. + Different from fasted control p < .01°
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the combination of fasting plus ethanol decreased the rate of glucuronidation to only 25% of that observed in control hepatocytes from ad libitum fed animals. In each case mentioned, in which an inhibition of glucuronidation was observed, a greater proportion of harmol was ultimately conjugated with sulfate (data not shown). Although the above manipulations had no effect on harmol sulfation, this reaction was inhibited by lithium ion. Initial experiments indicated that i0 mM lithium lactate reduced sulfation by over 50% (Table I). This effect proved to be an effect of lithium ion as i0 mM LiCI inhibited sulfation by 50%, but an identical concentration of lactate had no effect on the rate of sulfation. Discussion Some controversy remains regarding whether the decrease in the fraction of phenolic substrates conjugated with sulfate, which is generally observed in vivo with increasing dosage, is a result of the exhaustion of sulfate pools or the saturation of phenol sulfuryltransferase. The serum level of inorganic sulfate in the rat has been reported to be between 0.6 and 1.0 mM (7,14-16), somewhat higher than the Km for sulfate which we observed in hepatocytes. The K m for sulfate ion which we found is in reasonable agreement wi~h the value of 470 ~M in hepatocytes observed by Kolke (17), but in contrast to K m of 2.5 mM suggested by the results reported by Moldeus (18). Recent reports have indicated that serum sulfate is in rapid equilibrium with hepatic pools and that liver concentrations of sulfate are similar to those in the serum (9,19). Our results indicate that a 30-40 percent decrease in serum sulfate concentration would cause a moderate depression in the rate of sulfation and that a greater decrease in serum levels as observed after the administration of 1 nmole/Kg acetaminophen to rats (14,20) would severely decrease sulfation capacity. This dose of acetaminophen reduces serum sulfate levels by over 80 percent to well within the K m range for sulfate which we observed in hepatocytes. Our results and those of Koike together with these previous observations illustrate the manner by which a large reduction in serum sulfate can influence the production of sulfate conjugates. On the other hand, decreases in the relative formation of sulfate conjugates observed in vivo without appreciable reduction in serum sulfate levels (7) and in vitro in the presence of excess sulfate ion (21,9) are more likely explained by the saturation of phenol sulfuryltransferase with substrate. Recently, the facilitated transfer of inorganic sulfate into hepatocytes by a carrier-mediated mechanism has been observed (22). The Km of 239 ~M for sulfate stimulation of harmol sulfation which we observed in hepatocytes is much less than the K m of 6.5 mM observed for sulfate uptake by hepatocytes. Similarly, the Vm~ x for harmol sulfation of 1.12 nmoles/min/106 cells is considerably lower t~an the Vmax of 4.8 nmoles/min/106 cells reported for sulfate transfer. The Km value which we found is, however, in reasonable agreement with the K m value of i00 ~M for sulfate observed for ATP sulfurylase (23). These reports suggest that the Km which we observed reflects the saturation of hepatocyte ATP sulfurylase rather than saturation of carrier-facilitated sulfate uptake. Sodium stimulated carrier-mediated transport of sulfate by membrane vesicles from rat ileum (24) and kidney (25) has also recently been reported. Lithium is known to inhibit sodium-dependent membrane transfer. It is possible that the reduction of harmol sulfation which we observed by lithium was a result of the inhibition of sulfate uptake by this agent.
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The lesser ability of equal amounts of cysteine to support harmol sulfation suggest that some step in the conversion of cysteine to sulfate is rate limiting in the overall process. Cysteine is transferred into hepatocytes by a carrier-mediated process (26) and thereafter undergoes a sequence of reactions leading to the formation of inorganic sulfate (27). The rate of harmol sulfation probably reflects the steady state levels of sulfate generated from cysteine. Sulfation and glucuronidation by isolated hepatocytes have been shown to be decreased by the metabolic inhibitors, rotenone and 2,4 dinitrophenol. Both pathways seemed equally sensitive, although depending upon the substrate and conditions, there was variation in the relative degree to which each of the pathways was inhibited (28). Our results, however, indicate a difference in the sensitivity of sulfation and glucuronidation to more physiological changes in metabolic state. Unlike glucuronidation, the initial rate of sulfation was not markedly affected by any of the alterations of metabolism studied. The activation of sulfate requires ATP and, thus, could be sensitive to changes in this substrate. Cytoplasmic ATP levels decrease moderately after fasting (29). These changes, however, are apparently not sufficient to cause more than a slight decrease in the rate of sulfation. Glucuronidation which requires not only an adequate supply of ATP for synthesis of UTP, also requires the conversion of UDP-glucose to UDP-glucuronic acid by a NADHlinked dehydrogenase, was inhibited by conditions or substrates which are known to increase the cytoplasmic redox potential. The differences in the response of the two pathways to metabolic changes could be of significance. The insensitivity of sulfation to conditions which decreased glucuronidation could result in the conversion of a greater fraction of a given compound to the sulfate conjugate. This might alter the toxicity of N-hydroxyl arylamines, such as N-acetyl aminofluorene, whose conjugates with sulfate and glucuronic acid exhibit different degrees of reactivity (30). Acknowledsement The authors acknowledge
support from NIH Training Grant T-32-ESO7091.
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