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TOXICOLOGY

Repeated Exposures to Heroin and/or Cadmium Alter the Rate of Formation of Morphine Glucuronides in the Rat

Department of Human Physiology and Pharmacology "Vittorio Erspamer", University of Rome "La Sapienza", Rome, Italy

Received for publication June 18, 2003
Accepted August 7, 2003.

	Abstract

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After absorption, heroin is transformed into mono-acetyl-morphine and then into morphine. Morphine, in turn, is metabolized to morphine-3-glucuronide (M3G), an inactive compound, and morphine-6-glucuronide (M6G), a potent opioid agonist. Thus, changes in the rate of formation of M6G may alter the pharmacological consequences of a treatment with heroin or morphine. In this study, we investigate the effect of repeated exposures (10 daily i.p. injections) to heroin, morphine, cadmium (which has been previously shown to inhibit M3G formation in vitro), or heroin + cadmium on morphine glucuronidation both in vivo and ex vivo (i.e., microsomal preparation obtained from rats treated in vivo). Repeated heroin (2.5, 5.0, and 10 mg/kg) increased plasma levels of M6G (which was undetectable in all other groups) and reduced those of M3G. Also, the microsomal preparations obtained from the liver of repeated heroin rats, when incubated with morphine, yielded significant amounts of M6G (which was undetectable in all other groups) and decreased levels of M3G relative to the control groups. These effects were reversible upon discontinuation of heroin administration. In contrast, repeated morphine (10, 20, and 40 mg/kg) only slightly reduced M3G formation at the dose of 40 mg/kg. Repeated cadmium (5, 15, and 45 µg/kg) reduced the rate of M3G formation without inducing M6G synthesis. The effects of the repeated coadministration of heroin (10 mg/kg) and cadmium (15 µg/kg) were virtually identical to those of repeated heroin alone. In summary, repeated exposure of rats to heroin can shift morphine glucuronidation toward the formation of the active metabolite M6G.

Although there is firm evidence that morphine glucuronidation can be modulated by exposure to xenobiotics (Rane et al., 1985; Lawrence et al., 1992; Aasmundstad and Storset, 1998; Grancharov et al., 2001), it seems that morphine has little or no effect on its own glucuronidation. Even after repeated administrations, morphine pharmacokinetics remains substantially stable (Rane et al., 1985). In contrast, there is no information about the consequences of repeated administrations of heroin on the synthesis of M3G and M6G. This dearth of data is probably due to the notion that heroin is little more than a prodrug of morphine (Gutstein and Akil, 2001). There is some evidence, however, that the pharmacological profiles of heroin and morphine are not completely overlapping (Rady et al., 1991; Walker et al., 1999; Lysle and How, 2000; Haemmig and Tschacher, 2001; Tschacher et al., 2003). Particularly intriguing is the report that heroin shares with M6G the ability to bind a µ-opioid peptide receptor subtype different from the "morphine-preferring" subtype (Brown et al., 1997; Walker et al., 1999; Pasternak, 2001). Thus, it is important to ascertain whether heroin also differs from morphine in the ability to modulate the synthesis of morphine glucuronides.

The main goal of the present study was to investigate the effects of acute and repeated administrations of morphine and heroin on the rate of formation of M6G and M3G in vivo and ex vivo in the rat. We found that repeated heroin but not repeated morphine enhanced the rate of formation of M6G, whereas reducing that of M3G. To verify that the increased rate of M6G formation was not a mere consequence of the reduced synthesis of M3G, we also investigate the effects of cadmium, which has been shown to inhibit M3G formation without altering the synthesis of M6G in microsomal preparations from guinea pig liver (Lawrence et al., 1992).

	Materials and Methods

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Subjects. A total of 83 male Sprague-Dawley rats (Harlan Italy, S. Pietro al Natisone, Italy), weighing 175 to 200 g upon arrival, were used in this study. The rats were individually housed in transparent plastic hanging cages in a temperature- and humidity-controlled colony room (lights on from 7:00 AM to 7:00 PM) with ad libitum access to food and water.

Experiment 1. The aim of experiment 1 was to assess the effect of repeated i.p. injections of either heroin or morphine on plasma M3G and M6G, as well as on their formation in microsomal preparations. Twenty-five rats were separated into five groups. On days 1 to 9, the rats in the first three groups received a daily i.p. injection of 1 ml/kg saline (0.9% NaCl). On day 10, these rats received an injection of either saline (saline group, n = 6), 10 mg/kg morphine (morphine group, n = 6), or 10 mg/kg heroin (heroin group, n = 5). The last two groups received 10 daily injections of either 10 mg/kg morphine (repeated morphine group, n = 5) or 10 mg/kg heroin (repeated heroin group, n = 6).

Two hours after the last treatment, all rats were decapitated and their blood was collected for the quantification of morphine, heroin, 6-MAM, M3G, and M6G. Liver, brain, and kidneys were excised to obtain microsomal preparations.

Experiment 2. The aim of experiment 2 was to examine the effects of repeated administrations of different doses of heroin and morphine on the formation of M3G and M6G, as well as the reversibility of these effects upon drug discontinuation. Twenty-seven rats were separated into nine groups (n = 3 for all groups). On days 1 to 10, the rats of the first three groups received daily i.p. injections of one of three doses of morphine (10, 20, and 40 mg/kg, respectively). At the same time, the rats of other three groups received 10 injections of one of three doses of heroin (2.5, 5, and 10 mg/kg, respectively). All rats from the six groups described above were sacrificed 2 h after the last injection to obtain blood samples and microsomal preparations as described previously. Finally, the rats of the last three groups received 10 injections of 10 mg/kg heroin (days 1-10) and were sacrificed on day 11, 17, or 40, respectively (that is, after 1, 7, or 30 days of withdrawal from treatment).

Experiment 3. The aim of experiment 3 was to examine the effects of repeated administrations of different doses of cadmium on the formation of M3G and M6G by liver microsomes. Fifteen rats were separated into five groups (n = 3). On days 1 to 9, the rats of the first two groups received daily i.p. injections of 1 ml/kg saline (0.9% NaCl). On day 10, these rats received an injection of either saline (saline group) or 15 µg/kg cadmium (cadmium group). At the same time, the rats of the other three repeated cadmium groups received 10 injections of one of three doses of cadmium (5, 15, and 45 µg/kg, respectively). Two hours after the last injection all animals were sacrificed to obtain microsomal preparations as described previously.

Experiment 4. The aim of experiment 4 was to examine the effects of repeated coadministrations of cadmium and heroin on the rate of formation of M3G and M6G by liver microsomes. Twelve rats were assigned to one of four groups (n = 3 for all groups) and were administered 10 daily i.p. injections of saline, heroin (10 mg/kg), cadmium (15 µg/kg), or heroin plus cadmium (10 mg/kg + 15 µg/kg), respectively. Two hours after the last injection all animals were sacrificed to obtain blood samples and microsomal preparations as described previously.

Microsomal Preparations. Tissues were minced and rinsed in ice-cold 1.15% KCl and homogenized in 3 volumes of 100 mM phosphate buffer (pH 7.4) containing 0.25 M sucrose. The homogenate was centrifuged for 20 min at 9,000g. The supernatant was further centrifuged for 60 min at 105,000g. The resulting microsomal pellet was resuspended in 100 mM phosphate buffer containing 0.25 M sucrose, to obtain a final protein concentration of 10 mg/ml.

Glucuronidation Assay. The morphine glucuronidation assay was performed as described by Wielbo et al. (1993) under optimal conditions with respect to time and protein concentration. In detail, microsomal preparations were suspended in 100 mM phosphate buffer (pH 7.4) to a final protein concentration of 0.5 mg/ml, in the case of liver, or 1 mg/ml, in the case of kidneys and brain. Microsomes were preincubated for 20 min in 0.005% deoxycholic acid at 5°C to achieve full enzymatic activity. Morphine concentrations ranged from 0.1 to 4 mM for the calculation of M3G kinetics, and from 0.1 to 100 mM for the calculation of M6G kinetics. Incubation mixture consisted of 15 mM uridinediphosphoglucuronic acid (UDPGA), 100 mM phosphate buffer (pH 7.4), microsomes, and substrate (morphine) to a final volume of 1 ml. The reaction was started adding UDPGA. Samples and blanks (without UDPGA) were incubated in triplicates at 37°C for 30 min and the reaction was stopped with 0.5 ml of acetonitrile and centrifuged. Supernatants were then analyzed using HPLC.

High-Performance Liquid Chromatography. Plasma samples underwent solid phase extraction on LiChrolut TSC (200 mg) columns (Merck, Darmstadt, Germany) following the procedures described by Wielbo et al. (1993). Columns were conditioned with methanol (3 ml) followed by water (3 ml), and phosphate buffer (0.01 M, pH 3.0). After loading the sample, the column was washed with 0.01 mM phosphate buffer (pH 3.0) and methanol. The analytes were eluted with 3 ml of NH₄OH 2% in methanol. The eluate was evaporated to dryness at 37°C under a gentle stream of nitrogen. The dry residue was dissolved in methanol and stored at 4°C until HPLC analysis was performed.

Chromatographic analyses were carried out using an HPLC system equipped with automatic sampler (model L-7250), pump (model L-7100), diode array detector (model L-7455), and fluorescence detector (model L-7480), all purchased from Merck. Data were stored and processed using appropriate software (D-7000 HPLC System Manager, version 3.1; Hitachi, Tokyo, Japan).

Separation was achieved by using columns LiChrocart-Purospher 100 RP (18.5 µm, 250 x 4 mm) (Merck) with precolumns LiChrocart-LiCrospher 100 RP (18.5 µm, 4 x 4 mm) (Merck) and following the procedures described by Huwyler et al. (1995). The mobile phase consisted of a combination of eluent A (200 mM potassium phosphate, pH 3.0) and eluent B [acetonitrile/200 mM potassium phosphate, 20:80 (v/v), pH 3.0] changing continuously along a linear gradient, so to increase the concentration of acetonitrile from 6.4 to 20% (time frame 2 to 12 min). The pump flow rate was set at 0.8 ml/min and the injection volume was 20 µl.

Morphine and its metabolites were quantified by fluorescence detection (excitation wavelength 210 nm, emission wavelength 350 nm) and UV diode array detection at 210 nm. M6G, M3G, and morphine were identified on the basis of their retention times (3.47, 5.35, and 8.67 min., respectively) and spectral data relative to reference standards (Figs. 1 and 2). To provide further evidence of the identity of M6G peak, microsomal preparations obtained from both saline and repeated heroin rats were incubated with either morphine (20 mM) or morphine plus -glucuronidase 30,000 U/ml. After 8 h of incubation, enzymatic activity was stopped with 0.5 ml of acetonitrile, and supernatants were analyzed as described above. M6G was detectable only in samples from repeated heroin rats (120.6 ± 2.0 pmol/mg proteins, n = 5). As expected, both M6G and M3G were not detectable in the samples incubated with -glucuronidase.

View larger version (23K): [in this window] [in a new window]   Fig. 1. Representative chromatograms for M3G, M6G, and morphine standards. HPLC separation was coupled with diode array (left) or fluorescence detection (right).

View larger version (23K): [in this window] [in a new window]   Fig. 2. Representative chromatograms for morphine, M6G, and M3G in samples obtained from liver microsomal preparations in rats repeatedly treated with either saline (A and B) or 10 mg/kg heroin (C and D). Samples underwent HPLC separation coupled with diode array (A and C) or fluorescence detection (B and D) detection.

Drugs. Morphine hydrochloride, heroin hydrochloride, 6-MAM, and morphine-3-glucuronide were obtained from Salars (Como, Italy). Morphine-6-glucuronide, uridinediphosphoglucuronic acid (UDPGA) and Escherichia coli-derived -glucuronidase were obtained from Sigma-Aldrich (Milan, Italy). Cadmium chloride was obtained from Merck. All drug treatments were administered by intraperitoneal injection at 12:00 PM. All solvents were HPLC grade (Merck).

Data Analysis. Statistical analyses of group differences for plasma levels (micrograms per milliliter) of heroin, 6-MAM, morphine, M3G, and M6G were conducted using one-way analyses of variance. M3G, as well as M6G formation by liver microsomes leveled off at the highest morphine concentrations. Therefore, K_m (expressed as millimolar) and V_max (expressed as nanomoles per minute per milligram of protein) of M3G and M6G formation were calculated from nonlinear Lineweaver-Burke plots derived from the Michaels-Menten equation (GraphPad Prism 3; GraphPad Software Inc., San Diego, CA). Statistical analyses of group differences for rate values were conducted using one-way analyses of variance. When appropriate, Tukey's tests were used for pairwise comparisons. Significance level was set at P < 0.05.

	Results

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Experiment 1. Table 1 illustrates plasma levels of morphine, heroin, 6-MAM, M3G, and M6G 2 h after an i.p. injection of heroin, or morphine in rats that had previously received nine daily injections of saline (heroin, and morphine groups), heroin (repeated heroin group), or morphine (repeated morphine group). The most striking finding was that M6G was detected only in repeated heroin rats. In contrast, M3G was detected in all groups but with significant group differences (F_3,21 = 9.5, P < 0.001). The repeated heroin group exhibited in fact a significant decrease in plasma morphine and plasma M3G relative to the heroin group (P = 0.008). Interestingly, plasma M3G levels of the heroin group were no significantly different from those of the morphine group (P = 0.81) and repeated morphine group (P = 0.99), even though it is not clear to what extent M3G levels of these groups are comparable. Obviously, heroin and 6-MAM were detected only in heroin-treated rats, with no significant differences between heroin and repeated heroin groups (P = 0.37).

The ex vivo results paralleled those obtained in vivo. Table 2 and Fig. 3 illustrate the rate of morphine glucuronidation in microsomal preparations obtained from the liver of the four groups of rats described above and from the saline group. M6G was detected only in microsomal preparations obtained from rats repeatedly exposed to heroin (Fig. 3B). The K_m and V_max for the synthesis of M6G indicate a low-affinity and low-capacity enzymatic reaction, with saturation at about 100 mM morphine. There also were significant group differences in the V_max of M3G formation (F_4,27 = 6.58, P = 0.001), with significantly lower values in the repeated heroin group relative to the saline (P = 0.005) and heroin (P = 0.01) groups (Fig. 3A). In contrast, there were no significant group differences in K_m (F_4,27 = 0.82, P = 0.52). The ratio of M3G V_max/K_m, an indicator of intrinsic clearance, was significantly affected by drug treatment (F_4,27 = 3.15, P = 0.034). In particular, this clearance was higher in the repeated heroin group with respect to the repeated morphine group (P = 0.02).

View larger version (14K): [in this window] [in a new window]   Fig. 3. Mean (±S.E.M.) rates of formation for M3G (A) and M6G (B) in microsomal preparations (0.5 mg/ml proteins) from livers excised 2 h after the last of 10 daily administrations of saline or 10 mg/kg heroin (same animals in Tables 1 and 2). Samples were incubated in triplicates at 37°C for 30 min.

As expected (Rush and Hook, 1984; Coughtrie et al., 1987; Suleman et al., 1993), the microsomal preparations obtained from brain and kidneys exhibited very low UGT activity, so that it was impossible to calculate a meaningful saturation curve (Fig. 4). However, at the two highest concentrations of morphine there were no group differences in M3G production, whereas M6G was undetectable in all treatment groups.

View larger version (15K): [in this window] [in a new window]   Fig. 4. Mean (±S.E.M.) rates of formation for M3G in microsomal preparations (1.0 mg/ml proteins) from kidneys and brains excised 2 h after the last of 10 daily administrations of saline or 10 mg/kg heroin (same animals in Fig. 3 and Tables 1 and 2). Samples were incubated in triplicates at 37°C for 30 min.

Experiment 2. Table 3 illustrates plasma levels for heroin, 6-MAM, morphine, M3G, and M6G 2 h after the last of 10 daily i.p. injections of different doses of heroin (2.5, 5, or 10 mg/kg) or morphine (10, 20, or 40 mg/kg). It can be seen that all repeated heroin groups exhibited measurable amounts of plasma M6G. In contrast, no M6G was found in the blood of rats repeatedly treated with doses of morphine as high as 40 mg/kg.

As expected, no heroin, 6-MAM, morphine, M3G, or M6G were found in the blood of three groups of rats sacrificed after 1, 7, or 30 days of withdrawal from repeated administrations of 10 mg/kg heroin. Thus, these three groups were not included in Table. 3.

Also in this experiment ex vivo results were compatible with those obtained in vivo. Figures 5 and 6 show the kinetics of M6G and M3G formation in microsomal preparations from the nine groups of rats described above. It can be seen that repeated heroin altered dose dependently the V_max of M6G formation (F_2,7 = 1194.5, P < 0.001) and that this increase was reversible (F_3,10 = 90.4, P < 0.001), with return to basal levels within 30 days from the last injection of 10 mg/kg heroin. Notice that measurable amounts of M6G were also found when microsomal preparations from rats repeatedly exposed to 2.5 mg/kg heroin were incubated with 80 to 100 mM of morphine, but in this case it was impossible to determine the V_max of M6G formation. The changes in V_max of M3G formation induced by repeated heroin followed a pattern opposite to that observed for M6G. Indeed, there was a dose-dependent and reversible reduction in M3G (F_2,7 = 21.1, P = 0.004), with return to basal levels within 7 days from the last injection of heroin (F_3,10 = 78.08, P < 0.001).

View larger version (29K): [in this window] [in a new window]   Fig. 5. Mean (±S.E.M.) rates of formation for M3G (A, C, and D) and M6G (B and E) in microsomal preparations (0.5 mg/ml protein content) from livers excised 2 h, 1 day, 7 days, or 30 days after the last of 10 daily administrations of different doses of heroin or morphine (same animals in Table 3). A, B, D, and E refer to the groups treated with heroin. C refers to the groups treated with morphine. Samples were incubated in triplicates at 37°C for 30 min.

View larger version (18K): [in this window] [in a new window]   Fig. 6. Mean (±S.E.M.) Vmax for M3G and M6G in microsomal preparations from livers excised 2 h, 1 day, 7 days, or 30 days after the last of 10 daily administrations of different doses of heroin (same animals of Fig. 5 and Table 3). Samples were incubated in triplicates at 37°C for 30 min.

Figure 5 also shows that repeated morphine had little or no effect on the capacity of liver microsome to synthesize M6G and M3G. M6G was undetectable in all three repeated morphine groups. A small decrease in the V_max of M3G formation (F_2,8 = 7.97, P = 0.02) was observed in the 40 mg/kg group relative to the 10 mg/kg group (11.67 ± 0.12 versus 13.57 ± 0.56 nmol/min/mg, P = 0.022).

Experiment 3. Table 4 and Fig. 7 illustrate the V_max of M3G formation in microsomal preparation obtained from livers excised 2 h after an i.p. injection of saline or cadmium in rats that had previously received nine daily injections of saline (saline and cadmium groups) or different doses of cadmium (5, 15, or 45 µg/kg). Repeated cadmium dose dependently decreased the V_max of M3G formation (F_4,14 = 46.9, P < 0.001), with significant differences (relative to the saline group) at all doses tested (all P values < 0.05). The K_m was slightly increased at the lower doses (F_4,14 = 4.50, P = 0.024), whereas V_max/K_m ratio showed an inhibitory trend that however did not reach statistically significant levels (F_4,14 = 3.20, P = 0.062). In contrast, a single injection of 15 µg/kg cadmium had no effect on both V_max and K_m of M3G formation (P = 0.14). No detectable levels of M6G were found in any group.

View larger version (18K): [in this window] [in a new window]   Fig. 7. Mean (±S.E.M.) rates of formation for M3G in microsomal preparations (0.5 mg/ml proteins) from livers excised 2 h after the last of 10 daily administrations of saline or of different doses of cadmium (same animals in Table 4). Samples were incubated in triplicates at 37°C for 30 min.

Experiment 4. Table 5 illustrates plasma levels of heroin, 6-MAM, morphine, M3G, and M6G 2 hs after the last on ten daily i.p. injections of either heroin (10 mg/kg) or heroin plus cadmium (10 mg/kg + 15 µg/kg). There were no significant differences between the two groups (P = 0.76).

Table 6 illustrates the rate of morphine glucuronidation in microsomal preparations obtained from the two groups of rats described above as well as from the saline and repeated cadmium groups (see experiment 3). M6G was detected only in the repeated heroin and repeated heroin + cadmium groups, with no significant differences in either K_m (P = 0.11) or V_max (P = 0.16) between these two groups. There were also significant group differences in the V_max of M3G formation (F_3,11 = 487.42, P < 0.001) with significant differences between the saline group and all other groups (all P values < 0.001). V_max/K_m ratio was significantly reduced by cadmium treatment (F_3,11 = 10.07, P = 0.004; Tukey test, P = 0.003), Interestingly, the effects of repeated heroin and repeated cadmium on M3G synthesis did not seem to be additive.

	Discussion

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The major finding reported here is that exposure to heroin can alter morphine glucuronidation, increasing the synthesis of M6G while reducing the synthesis of M3G. These changes in morphine glucuronidation required repeated administrations of heroin, as indicated by the fact that a single injection of heroin had no consequences on the synthesis of M3G and M6G. In addition they were dose related and reversible. In contrast, acute or repeated exposures to morphine had little effect on its own glucuronidation, slightly reducing the rate of M3G formation only after 10 days of exposure to the highest dose tested (40 mg/kg i.p.). This finding is in substantial agreement with a previous report (Rane et al., 1985). We also report for the first time that repeated in vivo administrations of cadmium inhibit the synthesis of M3G without eliciting M6G formation. Previous studies had shown that cadmium can produce similar effects in vitro (Lawrence et al., 1992).

Morphine glucuronidation was studied ex vivo. The microsomal preparations obtained from the livers of repeated heroin rats produced, in fact, when incubated with morphine, less M3G than those obtained from saline- or morphine-injected rats. The microsomes obtained from repeated heroin rats also yielded measurable levels of M6G, which was undetectable in all other groups. Therefore, it seems that the M6G synthetic pathway, although normally quiescent in the rat, can be induced by repeated administrations of heroin. The very low values for the V_max of M6G formation indicate a low-capacity enzymatic system. Interestingly, at the moment of sacrifice (2 h after drug dose) rats that had received repeated administrations of heroin exhibited measurable plasma levels of M6G, which was undetectable in animals treated with morphine, in agreement with the data of the literature (Milne et al., 1996). Repeated heroin rats also exhibited lower plasma M3G levels than rats that had received acute heroin, acute morphine, or repeated morphine. Thus, the results in vivo are consistent with the results of ex vivo experiments in the same rats. However, it is important to notice that plasma M6G/M3G ratio was higher than expected on the basis of the rate of formation of these two glucuronides in microsomal preparations. The reason for this apparent discrepancy is not clear and encourages to further study heroin pharmacokinetics in rats.

The fact that repeated heroin reduced the V_max but not the K_m of M3G synthesis suggests that there was a reduction in the number of UGT molecules responsible for this enzymatic pathway without changes in substrate affinity. One possibility is that heroin selectively down-regulated the expression of the UGTs involved in the synthesis of M3G. Heroin might modulate gene expression by acting either at the classical membrane opioid receptors or at the more elusive intracellular opioid receptors, the existence of which is suggested by recent reports (Belcheva et al., 1993; Ventura et al., 1998). Most interestingly, activation of the nuclear receptor, pregnane X receptor has been found to induce UGT synthesis in mouse liver (Chen et al., 2003). Whatever the binding site responsible for this hypothetical effect of heroin on gene expression, it should be selectively responsive to heroin, because morphine failed to alter the synthesis of M3G. Pasternak and colleagues have hypothesized the existence of µ-opioid peptide receptor subtypes with preferential affinity for heroin versus morphine (Pasternak, 2001). Interestingly, there is some evidence that heroin and morphine can produce opposite effects on the activity of other enzymatic systems. It has been reported in fact that heroin inhibits, whereas morphine stimulates inducible nitric-oxide-synthase (Machelska et al., 1997; Cuellar et al., 2000; Lysle and How, 2000; Lanier et al., 2002).

Alternatively, heroin might have inhibited the synthesis of M3G via direct enzymatic inhibition. This hypothesis stems from the similarities in the effects of cadmium and heroin on the synthesis of M3G. Both substances reduced the V_max of M3G formation. Furthermore, also in the case of cadmium, a single administration had no consequences on morphine glucuronidation. The ability of cadmium to alter morphine glucuronidation in vitro has been attributed to the noncompetitive inhibition of morphine UGTs via covalent binding with the enzyme (Lawrence et al., 1992). (Incidentally, this mechanism of action fits nicely with our finding that repeated administrations of 15 µg/kg cadmium in vivo were required to reduce the synthesis of M3G. It is reasonable to expect that only after progressive accumulation in the body, such low doses of cadmium can produce biologically relevant effects.) Given its molecular structure, however, it is dubious that heroin could bind covalently to the UGTs. It is also unlikely that heroin acted as a competitive inhibitor of morphine glucuronidation, because no trace of opioids was found when microsomes from heroin-treated rats were incubated in the absence of morphine. That is, the inhibitory actions of repeated heroin on morphine glucuronidation were not dependent on the presence of heroin in the microsomal preparations at the moment of testing, as entailed by a mechanism of action based on competitive inhibition. Indeed, the reported ability of codeine, flunitrazepam, chloramphenicol, probenecid, and amitriptyline to act as competitive inhibitors of morphine UGTs (Puig and Tephly, 1986; Miners et al., 1988; Thomassin and Tephly, 1990; Yue et al., 1990) requires in vitro incubation with these drugs.

Also, the ability of repeated heroin to increase the synthesis of M6G is not easy to explain. In the rat, there are at least two morphine UGT isoenzymes (UGT2B1 and UGT1A1) that selectively catalyze morphine glucuronidation at the 3-OH position (Ishii et al., 1994, 1997). In contrast, there is no information about the UGT isoenzyme(s) responsible for the synthesis of the minimal amount of M6G found in this species. It is possible that the same isoforms responsible for the synthesis of M3G also carry out the synthesis of M6G. Alternatively, the synthesis of M6G may depend on a specific UGT isoenzyme(s), constitutively expressed at very low levels. Thus, heroin might have selectively enhanced 6-OH glucuronidation by either up-regulating the expression of the isoenzyme(s) selective for M6G or by shifting the ratio of M6G to M3G formation within the same enzymatic system. A shift in the selectivity of the morphine UGTs might have been the result of changes in the assemblage of UGT molecules. Ishii and colleagues have found in fact that the simultaneous expression of the isoenzymes UGT2B21 and UGT2B22 in guinea pigs produces extensive M6G formation (Ishii et al., 2001). The hypothesis that heroin induced the synthesis of M6G by modulating the formation of heterooligomers of UGT is speculative but deserves to be further investigated. Finally, it is unlikely that the increased synthesis of M6G was simply the outcome of a reduced synthesis of M3G. Indeed, cadmium selectively reduced the synthesis of M3G without altering that of M6G.

Whatever the mechanisms responsible for the effects of repeated heroin on morphine glucuronidation in the rat, it is important to notice that they seem to be fully reversible, at least under the present experimental conditions. Indeed, both in vivo and ex vivo data indicated that within 7 days from the last injection of heroin, M6G and M3G levels returned to basal values.

In summary, our study suggests that a past history of heroin abuse can substantially alter the pharmacological consequences of subsequent exposures to opioids. Indeed, M6G is a potent agonist to µ-opioid peptide receptors with a long half-life and is thought to play an important role in the therapeutic and toxicological effects of morphine (Christrup, 1997; Penson et al., 2000; Ulens et al., 2001). Furthermore, it has been suggested that the pharmacological profile of M6G is closer to that of heroin than to that of morphine (Pasternak, 2001), which may contribute to the differences in the pharmacological effects of heroin and morphine observed in the clinical setting (Haemmig and Tschacher 2001; Tschacher et al., 2003). Although the metabolic pathways responsible for morphine glucuronidation in rats and humans are different, it is conceivable that our findings may have important implications for the study of heroin addiction. Fugelstad et al. (2003), for example, have hypothesized that individual variability in the activity of UGT may help explaining cases of sudden death among heroin users. Furthermore, we have recently reported that heroin addicts exhibit a higher M6G/M3G ratio than normal subjects treated with morphine (Antonilli et al., 2003).

	Footnotes

 
This study was supported by grants from the Ministry of University and of Scientific and Technological Research of Italy (Finanziamenti COFIN) and from the University of Rome "La Sapienza" (Finanziamenti per Ricerche d'Ateneo).

DOI: 10.1124/jpet.103.055467.

ABBREVIATIONS: 6-MAM, 6-monoacethylmorphine; M3G, morphine-3-glucuronide; M6G, morphine-6-glucuronide; UGT, UDP-glucuronosyltransferase; UDPGA, uridinediphosphoglucuronic acid; HPLC, high-performance liquid chromatography.

Address correspondence to: Dr. P. Nencini, Department of Human Physiology and Pharmacology "Vittorio Erspamer", University of Rome "La Sapienza", Piazzale Aldo Moro 5, I-00185, Rome, Italy. E-mail: paolo.nencini{at}uniroma1.it

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