Introduction

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Hepatocyte growth factor, a potent stimulator of DNA synthesis in hepatocytes (Matsumoto and Nakamura, 1992; Gómez-Lechón et al., 1995), was isolated from rat platelets (Nakamura et al., 1987), rabbit plasma (Zarnegar and Michalopoulos, 1989) and plasma from patients with fulminant liver failure (Gohda et al., 1988). HGF is a more effective mitogen for rat hepatocytes than EGF (Gohda et al., 1988), and it was recently shown to be a potent proliferating factor for human hepatocytes in primary culture (Gómez-Lechón et al., 1995; Strain et al., 1991). HGF is also considered to play an important role in liver regeneration in humans (Nishizaki et al., 1995). Consistent with such a role is the transient increase in plasma levels of HGF in patients after partial hepatectomy (Nishizaki et al., 1995; Selden et al., 1986), fulminant hepatic failure (Tsubouchi et al., 1989) or liver cirrhosis (Shimizu et al., 1991). HGF is now known to be expressed in a variety of tissues (Tashiro et al., 1990) and to have mitogenic activity on a wide variety of cells (Matsumoto et al., 1991; Matsumoto and Nakamura, 1993; Rubin et al., 1991). Recent experimental evidence indicates that HGF not only acts as a potent mitogen for human hepatocytes (Gómez-Lechón et al., 1995, 1996), but also has notable influence on the synthesis of plasma proteins, with effects that are just the opposite of those produced by inflammatory cytokines (Gómez-Lechón et al., 1995; Guillén et al., 1996). This indicates that HGF can regulate the expression of non-growth-related liver-specific genes. Modulation of CYP isozyme expression by other related factors such as EGF and TGF has been described previously in humans (Greuet et al., 1997) and rodent hepatocytes in primary culture (Hohne et al., 1990; Aubrecht et al., 1995; Ching et al., 1996). However, no data are available on HGF effects on the CYP system. Such information would help to determine the potential alterations of CYP isozymes and other drug-metabolizing enzymes derived from increases in serum HGF levels in pathological situations involving liver regeneration, which could modify the metabolism and elimination of drugs administered concomitantly.

In the present study, we have investigated the effects of HGF on phase I and II activities involved in xenobiotic metabolism in human hepatocytes. For phase I biotransformation, specific monooxygenase activities were used as probes of different CYP isozymes. Changes in the relative levels of specific CYP isozymes, determined by immunoblot analysis, and CYP mRNAs were also studied in the presence of HGF. GST and UGT activities were examined as representative of phase II enzymes.

	Materials and Methods

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Materials. Human recombinant HGF was obtained as described previously (Nakamura et al., 1989); 7-benzoxyresorufin, 7-ethoxyresorufin, collagenase and -glucuronidase/arylsulfatase were obtained from Boehringer Mannheim (Mannheim, Germany); MC, RIF, coumarin, 7-hydroxycoumarin, p-nitrophenol, resorufin, testosterone and 4-methylumbellipherone were purchased from Sigma (St. Louis, MO); 6-hydroxytestosterone was supplied by Steraloids Inc. (Wilton, NH); CDNB was purchased from Janssen Chimica (Beerse, Belgium); newborn calf serum was obtained from Gibco (Paisley, UK); Ham's F-12 and Leibovitz L-15 culture media were from Flow (Irvine, UK); all other reagents used in this study were of analytical grade.

Isolation and culture of human hepatocytes. Surgical liver biopsies (1-5 g) were obtained from patients undergoing cholecystectomy after informed consent was obtained. Patients had no known liver pathology nor did they receive medication during the weeks before surgery. None of the patients were habitual consumers of alcohol or other drugs. A total of 16 liver biopsies (six males and ten females) were used. Patients' ages ranged from 26 to 71 years (table 1). Human hepatocytes were isolated by a two-step perfusion technique (Gómez-Lechón et al., 1990) and seeded on 3.5-cm-diameter dishes or 24-well plates coated with fibronectin (3.6 µg/cm²) at a density of 5 × 10⁴ cells/cm² in an appropriate volume of medium. Culture medium was Ham's F-12/Leibovitz L-15 (1:1, v/v) supplemented with 2% newborn calf serum, 5 mM glucose, 50 U/ml penicillin, 50 µg/ml streptomycin, 0.2% bovine serum albumin and 10⁸ M insulin. Medium was changed 1 hr later to remove unattached hepatocytes. By 24 hr cultures were shifted to serum-free medium containing 10⁸ M dexamethasone.

Treatment of cultures. HGF was prepared as a sterile solution in culture medium containing 2% bovine serum albumin as a stock solution of 2 ng/µl and added directly to cultures. For all the experiments, human hepatocyte cultures were incubated with 10 ng/ml (0.1 pM) HGF 24 hr after plating. This dose was chosen because it proved to be the most effective in promoting DNA synthesis and to have maximal effects on plasma protein synthesis in primary culture of human hepatocytes (Gómez-Lechón et al., 1995; Guillén et al., 1996). For CYP induction experiments, MC and RIF were dissolved in dimethylsulfoxide and added to 24-hr-old cultured human hepatocytes at a final concentration of 2 µM and 50 µM, respectively. The final concentration of solvent in culture medium was 0.5%, v/v; and the control cells were treated with the same amount of solvent. The inducers were added daily.

Biochemical determinations. EROD and BROD activities were assayed by incubating intact cultured hepatocytes with 8 µM 7-ethoxyresorufin or 15 µM 7-benzoxyresorufin, respectively; and the resorufin formed was quantified fluorimetrically as described previously (Donato et al., 1993a). CH activity was measured directly in intact cultured hepatocytes incubated with 100 µM coumarin for 30 min at 37°C. Aliquots of medium supernatants (200 µl) were incubated with 40 U of -glucuronidase and 30 U of arylsulfatase in 50 µl of 0.1 M sodium acetate buffer (pH 4.5). After 2 hr of incubation at 37°C, the samples were diluted (1:3) in 0.1 M Tris, pH 9. The 7-hydroxycoumarin formed was quantified fluorimetrically by means of a Cytofluor 2350 microplate reader (Millipore, Iberica, Barcelona, Spain) with 355 and 460 nm excitation and emission filters, respectively. PNP activity was measured directly in intact hepatocytes by incubation with 0.5 mM p-nitrophenol for 30 min (Dicker et al., 1990). 6-OHT was measured by incubating intact hepatocytes cultured for 30 min with culture medium containing 250 µM testosterone. Metabolites were extracted and analyzed by high-performance liquid chromatography as described (Donato et al., 1993b). GST activity toward CDNB was determined as described previously (Habig and Jakoby, 1981) with some modifications to adapt the technique to 96-well plates. Hepatocyte homogenates (5-10 µg of cellular protein) were transferred to a 96-well plate containing 1 mM CDNB and 0.1 M potassium phosphate buffer (pH 6.5). The final assay volume was 200 µl/well. The reaction was started by addition of 5 µl of 40 mM reduced GSH. The assay was run at 25°C, and the change in extinction at 340 nm with incubation time was measured with a microplate reader. UGT enzyme activity was determined directly in intact hepatocytes incubated with culture medium containing 100 µM 4-methylumbelliferone. After 15, 30, 45 and 60 min of incubation at 37°C, aliquots of medium (10 µl) were withdrawn and transferred to a 96-well plate, and 190 µl of 10 mM NaOH was added to each well. 4-Methylumbelliferone remaining in culture supernatants was determined fluorimetrically by means of a fluorescence microplate reader (Cytofluor 2350, Millipore) with 360 nm excitation and 450 nm emission filters. Intracellular GSH content was determined as described (Hissin and Hilf, 1976). Cellular protein was measured according to the method of Lowry et al. (1951). The rate of DNA synthesis was determined by measuring the amount of (methyl)[³H]thymidine incorporated into the trichloroacetic acid-precipitable fraction of cell homogenate as described previously (Gómez-Lechón et al., 1995). Synthesis of albumin and ₁-antichymotrypsin were measured by immunoprecipitation with specific antibodies after a pulse-labeling of 12 hr with 50 µCi/ml [³⁵S]methionine in methionine-free RPMI-1640 culture medium as described (Guillén et al., 1996).

Western blot analysis. Polyclonal antibodies against recombinant CYP1A2 and CYP3A4 were kindly provided by Dr. F. P. Guengerich (Nashville, TN). Liver S-9 fractions (30 µg protein/lane) from human cultured hepatocytes were electrophoresed in an SDS-polyacrylamide gel. Proteins were transferred to Immobilon membranes (Millipore) and sheets were incubated with rabbit antiserum raised against recombinant CYP3A4 or with goat antiserum against recombinant CYP1A2 (in the latter case, blots were then incubated with rabbit anti-goat antibody). After washing, blots were developed with horseradish peroxidase-labeled goat anti-rabbit IgG, with 0.05% diaminobenzidine (w/v) and 0.001% H₂O₂ (v/v).

Analysis of mRNA by semiquantitative RT-PCR. Total cellular RNA was extracted and reverse transcribed as described (Donato et al., 1997). For CYP3A4 cDNA amplification (Gonzalez et al., 1988) the forward primer was from 1353 to 1379 nt (5-CCT TAC ACA TAC ACA CCC TTT GGA AGT-3) and the reverse primer was from 1705 to 1734 nt (5-AGC TCA ATG CAT GTA CAG AAT CCC CGG TTA-3), and amplified a predicted 382-bp fragment. For CYP1A2 cDNA (Jaiswal et al., 1986) the forward and reverse primers, 5-AAC AAG GGA CAC AAC GCT GAA T-3 and 5-GGA AGA GAA ACA AGG GCT GAG T-3, respectively, produced a predicted 453-bp fragment between positions 1178 and 1630. For human -actin cDNA (Ponte et al., 1984) the forward primer was from 480 to 499 nt (5-CGT ACC ACT GGC ATC GTG AT-3) and the reverse primer was from 911 to 931 nt (5-GTG TTG GCG TAC AGG TCT TTG-3), and produced a 452-bp fragment. Diluted cDNA (3 µl) was amplified in 30 µl of 10 mM Tris-HCl (pH 8.8) containing 50 mM KCl, 1.5 mM MgCl₂, 0.1% Triton X-100, 60 µM each deoxynucleotide triphosphate, 1 U DNA polymerase (Dynazyme II, Finnzymes OY, Espoo, Finland and 0.2 µM each primer. Amplification was performed in a Peltier Thermal Cycler (PTC-100HB, MJ Research Inc., Watertown, MA) programmed for an initial denaturation of 4 min at 94°C, followed by 27 cycles of 45 sec at 94°C, 45 sec at 58°C and 1 min at 72°C, and a final extension of 5 min at 72°C. Appropriate dilutions were determined empirically for each cDNA to ensure that the resulting PCR products were derived only from the exponential phase of the amplification. Under these conditions, the yield of the PCR product is proportional to the input cDNA. For quantitative analysis, aliquots (25 µl) of the PCR reaction were subjected to electrophoresis on 1.2% agarose gel and the products visualized by ethidium bromide staining. The gel image was captured with a high-resolution Video-Camera (Sony CCD-IRIS), and the intensity of the bands was digitalized, analyzed and quantified with the VisiLog Software Pakage (Visilog 4)

Statistical analysis. Data were analyzed with the Student's t test. Values of P < .01 were considered significant.

	Results

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DNA synthesis and acute-phase protein production after HGF treatment of human hepatocytes. To evaluate the responsiveness of human hepatocytes to HGF treatment, DNA synthesis and production of acute-phase proteins were determined. Stimulation of hepatocytes with 10 ng/ml HGF for 96 hr produced a 3.5 ± 1.0-fold (n = 10) increase in DNA synthesis. HGF also influenced the synthesis of plasma proteins (determined by immunoprecipitation of secreted protein to culture medium). HGF increased the production of albumin (210 ± 40% of control, n = 3), a negative acute-phase protein, and inhibited the synthesis of the positive one, ₁-antichymotrypsin, to 42 ± 8% of control (n = 3).

Depression of the specific activity of CYP isozymes by HGF in human hepatocytes. To determine the kinetics of HGF action on the CYP system, 10 ng/ml HGF was added to cultured hepatocytes that previously had been maintained under control conditions for 24 hr or 72 hr, and CYP1A1/2 activity, assessed as EROD activity, was measured daily up to 120 hr of culture. Figure 1 shows the results obtained for culture F but is representative of the cultures from donors D and H. A time-dependent decrease in EROD activity was observed during HGF treatment. It is noteworthy that EROD reduction was slower and smaller when HGF was added at 24 hr of culture than at 72 hr. The results were similar for other CYP-dependent oxidations (data not shown). Regardless of the culture age at which exposure to HGF began, maximal activity decreases were observed by 96 hr of culture. At this time, the EROD activity of human hepatocytes exposed to HGF from 24 hr or 72 hr of culture dropped to about 60% or to 50%, respectively, of the activity of untreated cells. No toxic effects of HGF on human hepatocytes were observed (data not shown).

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Time-course effects of HGF on EROD activity. Human hepatocytes were maintained in control conditions (open circles) or exposed to 10 ng/ml HGF after 24 hr (closed circles) or 72 hr (closed squares) of culture. At the indicated times EROD was determined on cell monolayers. Values represent mean ± S.D. of three independent plates from culture F, but are representative of donors D and H. * P < .01 with respect to untreated cells.

View larger version (38K): [in this window] [in a new window]   Fig. 2.   Effects of HGF on EROD and 6-OHT activities of human hepatocytes. After 24 hr of culture, human hepatocytes from different donors were maintained in control conditions (open bars) or exposed to 10 ng/ml HGF (dark bars), and EROD (A) and 6-OHT (B) activities were measured 72 hr later (96 hr of culture). Each bar corresponds to the mean of two independent culture plates.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Effects of HGF on BROD, CH and PNP activities of human hepatocytes. After 24 hr of culture, human hepatocytes from different donors were maintained in control conditions (open bars) or exposed to 10 ng/ml HGF (dark bars), and BROD (A), CH (B) and PNP (C) activities were measured 72 hr later (96 hr of culture). Each bar corresponds to the mean of two independent culture plates.

Phase II activities and GSH levels of human hepatocytes exposed to HGF. To provide some indication of whether CYP-dependent monooxygenases were the only drug-metabolizing activities affected by HGF, the effects on UGT and GST, two phase II enzymes, were evaluated. Interindividual variations in the rates of conjugating reactions were also found (table 2). Although hepatocytes obtained from several liver donors showed small decreases in UGT (cultures G and H) and GST (cultures D, E, F and G), increases in both activities were found in cultures I and L. In contrast with the results on the CYP system, no conclusive evidence of an inhibition of phase II activities by HGF was found. Similarly, the GSH levels of human hepatocytes from three different cultures were not altered significantly by HGF treatment.

Effects of HGF on induced CYP1A1/2 and CYP3A4 activities in human hepatocytes. We next investigated the effects of HGF on the inducibility of CYP isozymes. Of the five different CYP isozymes studied, CYP1A1/2 and CYP3A4 are known to be highly inducible by xenobiotics. MC (2 µM) and RIF (50 µM) were selected as specific inducers for CYP1A1/2 and CYP3A4, respectively. After 24 hr of culture, cells were exposed to the inducers in the presence or absence of HGF, and specific CYP activities were measured at 96 hr of culture. Figure 4A shows EROD activity levels in MC-induced human hepatocytes from three different donors. Exposure of human hepatocytes to MC for 72 hr resulted in a large increase (8- to 11-fold greater than untreated cells) in EROD activity. HGF produced a significant reduction in EROD activity induced by MC to 52 to 81% of that of hepatocytes treated with MC alone. A 72-hr treatment with RIF increased the 6-OHT activity of human hepatocytes by a factor of approximately 2 (fig. 4B). Again, reductions in RIF-induced CYP3A4 activity ranging from 30 to 70% were observed in hepatocytes exposed to HGF. Comparison of these results with those obtained for MC-induced EROD activity suggested that the effects of HGF on CYP3A4 induction are greater than on CYP1A1/2.

View larger version (36K): [in this window] [in a new window]   Fig. 4.   Effects of HGF on the induction of EROD and 6-OHT activities. (A) After 24 hr of culture, human hepatocytes were maintained in control medium (shaded bars) or exposed to 2 µM MC (open bars) or to 2 µM MC and 10 ng/ml HGF (dotted bars), and EROD activity was determined by 96 hr of culture. (B) After 24 hr of culture, human hepatocytes were maintained in control medium (shaded bars) or exposed to 50 µM RIF (open bars) or to 50 µM RIF and 10 ng/ml HGF (dotted bars), and 6-OHT activity was determined by 96 hr of culture. Each bar corresponds to the mean ± S.D. of three independent plates. * P < .01 with respect to hepatocytes treated with MC or RIF.

Effects of HGF on basal and induced CYP1A2 and CYP3A4 protein and mRNA levels in human hepatocytes. Based on the profound alterations observed in specific activities of CYP isozymes because of HGF, changes in CYP protein levels produced by HGF treatment were investigated. Individual CYP1A2 and CYP3A4 levels were analyzed by blotting of the cell lysates and immunodetection with specific polyclonal antibodies. Western blots obtained from culture P, but representative of liver samples N and O, are shown in figure 5. CYP1A2 levels increased after a 72-hr treatment (from 24 to 96 hr of culture) of human hepatocytes with MC, as expected, and HGF was able to reduce accumulation of both constitutive and induced CYP1A2 apoprotein. On the other hand, immunoblot analysis revealed that RIF clearly induced CYP3A4 levels of human hepatocytes. Again, accumulation of both basal and RIF-induced CYP3A4 protein was markedly reduced in the presence of HGF.

View larger version (32K): [in this window] [in a new window]   Fig. 5.   Effects of HGF on basal and induced CYP1A2 and CYP3A4 protein levels. After 24 hr of culture, human hepatocytes was maintained in control conditions or exposed to 2 µM MC, 50 µM RIF and/or 10 ng/ml HGF for an additional 72 hr. At 96 hr of culture, cellular lysates (30 µg protein/lane) were analyzed by Western blot with anti-CYP1A2 or anti-CYP3A4 polyclonal antibodies. The results correspond to culture P but are representative of cultures from donors N and O. Lane 1, control; lane 2, HGF; lane 3, MC; lane 4, MC + HGF; line 5, RIF; line 6, RIF + HGF.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   Effects of HGF on basal and induced CYP1A2 and CYP3A4 mRNA levels of human hepatocytes. (A) After 24 hr of culture, human hepatocytes were maintained in control medium or exposed to 2 µM MC in the absence (open bars) or the presence (dark bars) of 10 ng/ml HGF. (B) After 24 hr of culture, human hepatocytes were maintained in control medium or exposed to 50 µM RIF in the absence (open bars) or the presence (solid bars) of 10 ng/ml HGF. CYP1A2 and CYP3A4 mRNAs were quantified by RT-PCR analysis 48 hr later. Values were calculated from confirmed 3-4 RT-PCR bands and are expressed as percentage of CYP mRNA/mRNA -actin. Each bar corresponds to the mean of two independent determinations from a representative culture (donor I).

	Discussion

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The present results provide the first evidence of the repressive effects of HGF on the regulation of human hepatic drug-metabolizing activities. The data clearly show that treatment of human hepatocytes with HGF produces profound alterations in the activity of specific CYP isozymes, but no effects on the activity of phase II enzymes, UGT and GST, and GSH levels were observed. HGF action on the CYP system did not seem to be specific to a particular isozyme. A general down-regulation of the functionality of all the CYPs studied, namely CYP1A1/2 (assessed by EROD activity; Sandhu et al., 1994), CYP2A6 (CH; Yun et al., 1991), CYP2B6 (BROD; Waxman et al., 1991), CYP2E1 (PNP; Patten et al., 1992) and CYP3A4 (6-OHT; Waxman et al., 1991) was produced (figs. 1-3). HGF not only produces alterations in the basal CYP system but also clear reductions in inducible CYP isozymes. The observed increase in the expression of CYP1A2 and CYP3A4 isozymes in cultured human hepatocytes produced by MC and RIF, respectively, coincides with previous reports (Pichard et al., 1992; Schuetz et al., 1993; Abdel-Razzak et al., 1994). The mechanisms by which HGF produces a negative control of CYP isozymes remain unknown, but it seems likely that mechanisms common to different CYP isozymes are involved. Treatment of human cultured hepatocytes with HGF led to a reduction in specific CYP apoprotein and mRNA levels, which could explain the decreases observed in CYP activities. The reductions in mRNA levels may be caused by the HGF interference with the transcriptional activation of CYP genes or to an increased rate of mRNA degradation. Further research should be done to clarify the intracellular signaling events involved in HGF down-regulation of CYP gene expression.

Increased protein degradation after HGF treatment could also contribute to the observed decrease in CYP activity. Phosphorylation of CYPs by a cAMP-dependent protein kinase has emerged as a potential regulatory event of catalytic activity and degradation of hepatic CYPs (Koch and Waxman, 1991). However, we previously showed that HGF induces an early increase in intracellular calcium in human hepatocytes, but no changes in cAMP were observed (Gómez-Lechón et al., 1996). This suggests that CYP repression by HGF is not mediated by cAMP-dependent protein kinase A. On the other hand, nitric oxide might play a role in controlling early responses after partial hepatectomy (Obolenskaya et al., 1994; Hortelano et al., 1995). Growth factors and cytokines involved in the regenerative process may potentially induce nitric oxide synthase expression and be responsible for the increased nitric oxide levels found in regenerative liver. Recent studies indicate that the inhibition of CYP activities produced by cytokines in rat (Stadler et al., 1994) and human (Donato et al., 1997) hepatocytes are mediated by nitric oxide. However, the fact that nitric oxide release into culture medium did not increase during treatment with HGF (data not shown) suggests that the mechanism of action of HGF on hepatocellular function in cultured hepatocytes is not mediated by nitric oxide.

HGF is a potent inducer of expression of early response genes such as c-fos, c-jun and c-myc, and stimulates DNA synthesis in primary culture of human hepatocytes (Gómez-Lechón et al., 1996). Highly proliferating systems such as regenerating liver after partial hepatectomy (Marie et al., 1988; Habib et al., 1994), fetal liver (Hakkola et al., 1994), hepatomas (Degawa et al., 1995) or liver carcinoma-derived cell lines (Grant et al., 1988; Donato et al., 1994) show depressed hepatic CYP-dependent activities. However, a direct influence of the state of hepatocyte proliferation on the repression of CYP gene expression has not been demonstrated, and it was reported previously that the repression of the CYP system produced by specific growth factors in mouse hepatocytes is not linked to cell cycle progression or stimulation of DNA synthesis per se (Hohne et al., 1990; Aubrecht et al., 1995). Greuet et al. (1997) emphasize that the loss of cell-cell contacts in the subconfluent cultures with respect to confluent (nonproliferative) ones, rather than the proliferative status of cells per se, is responsible for the dramatic decrease in the inducible and constitutive expression of CYP genes, and that the presence of EGF only produces a minor contribution to the decrease in the expression of these genes. These authors suggest that the inhibition of CYP expression occurs during the priming period (progression of cells through G1 phase to a restriction point) that renders hepatocytes competent to respond to growth factors (Fausto et al., 1995). Previous results from our laboratory suggested that not only the duration but particularly the timing of HGF stimulation determines the extent of DNA synthesis and the entry of hepatocytes into the S phase (Gómez-Lechón et al., 1996). In fact, stimulation of hepatocytes from 20 to 72 hr had almost no effect (priming period), but incubation of cells with HGF for the same period of time but from 72 to 120 hr resulted in maximal DNA synthesis. HGF seems to be more effective in inhibiting CYP1A1/2 activity when hepatocytes are treated in the 72- to 120-hr period (fig. 1), which indicates that the inhibition of CYP expression is even greater after the cells cross the restriction point.

The decrease in the detoxication capacity of the regenerating liver after partial hepatectomy observed in rats is attributable to reduced apoprotein amounts of various CYP forms (Marie et al., 1988; Ronis et al., 1992; Trautwein et al., 1997), but the mechanism of this down-regulation remains to be determined. Recently, it has been shown that EGF and TGF decrease the expression of CYP isozymes in rodent hepatocytes (Hohne et al., 1990; Aubrecht et al., 1995; Ching et al., 1996), and a possible role of these growth factors in the suppression of hepatic CYP expression that occurs during liver regeneration has been postulated. HGF plays an important role in the initiation of DNA synthesis in the regenerating liver (Nishizaki et al., 1995; Lindross et al., 1991). The clinical significance of changes in serum HGF in patients undergoing hepatectomy remains unclear, but it has been suggested that these changes constitute an indicator of hepatic regeneration. When HGF effects on human hepatocytes are considered in the context of the events that occur during liver regeneration, it is conceivable that HGF also plays a key role in controlling the hepatic CYP system. Down-regulation of CYP expression observed during regeneration after hepatectomy could then be triggered by the increases observed in HGF serum levels.

Because CYP isozymes belonging to families 1 through 3 are mainly involved in xenobiotic metabolism (Gonzalez, 1989), our findings on CYP1A1/2, CYP2A6, CYP2B6, CYP2E1 and CYP3A4 isozymes could be used directly as an indication of the general effects of HGF on drug metabolism by human liver. This is of concern because of the potential therapeutic use of HGF in the future in certain situations involving liver regeneration or repair (Matsumoto and Nakamura, 1996). It can reasonably be predicted from our study that the metabolism of therapeutic agents administered during the course of certain pathological states (e.g., toxic hepatitis, chronic viral hepatitis or cirrhosis), in which liver regeneration takes place, may be reduced. The pharmacological and toxicological consequences of this effect can be clinically relevant. Impaired biotransformation prolongs the duration and intensity of the action of drugs, and potential toxicity, as a consequence of xenobiotic accumulation, can appear. Although biotransformation generally parallels a detoxication process, drug-metabolizing enzymes, and particularly CYPs, can generate metabolites that are more toxic and reactive than the original compound (Gonzalez and Gelboin, 1994). Ultimately it is the balance among bioactivation, detoxication and defense mechanisms that determines the susceptibility of the organism to chemicals. The degree of HGF influence on chemical detoxication depends not only on the response observed in CYP enzymes, but also on the effects produced on phase II detoxifying enzymes. In this context, HGF only suppressed CYP expression in human hepatocytes, and it did not down-regulate UGT and GST activities and GSH levels, which have general protective effects against reactive species mostly generated by CYP-dependent oxidations.