Differential effects of oltipraz on CYP1A and CYP2B in rat lung

Eric Le Ferrec1, Guennady Ilyin2, Karine Mahéo1, Caroline Bardiau1, Arnaud Courtois1, André Guillouzo1 and Fabrice Morel1,,3

1 INSERM U456, Faculté des Sciences Pharmaceutiques et Biologiques, Université de Rennes 1, 35043 Rennes, France and
2 INSERM U522, Hôpital Pontchaillou, 35033 Rennes, France


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Oltipraz (OPZ) is a potent chemopreventive agent against chemically-induced carcinogenesis in several animal models. It affects the expression and/or activity of xenobiotic-metabolizing enzymes and its effects are altered in the course of inflammation in liver. The present study was undertaken to analyse the effect of OPZ alone or in combination with Escherichia coli lipopolysaccharide (LPS) on the expression and activities of glutathione S-transferases (GSTs) and cytochrome P450 (CYPs) in rat lung and kidney. Male Wistar rats were fed a diet containing OPZ for 1–5 days. LPS was injected 24 h before the end of OPZ treatment (from 48 to 72 h). Total GST activity, measured using 1-chloro-2,4-dinitrobenzene as a substrate, increased slightly in both lung and kidney during OPZ treatment. As previously demonstrated in the liver, OPZ induced rat GSTP1 in both kidney and lung and this effect was totally (kidney) or partially (lung) inhibited by co-treatment with LPS. CYP1A expression and activity were strongly increased in both tissues 24 h after starting OPZ treatment and maintained for 5 days. This increase was suppressed during the acute-phase response to endotoxin. OPZ has no effect on CYP2B1 mRNA expression in the lung, but it dramatically decreased the amount and activity of the corresponding apoprotein. The OPZ-dependent decrease in the CYP2B1 apoprotein was abolished and its corresponding activity partially reversed during LPS treatment. In reconstitution experiments using cytosol from OPZ-treated or control rat lungs and microsomal fractions, CYP2B1 apoprotein was rapidly degraded in the presence of cytosol from treated rats. This effect was partially reversed in the presence of MG132, a proteasome inhibitor. These observations support the conclusion that the decrease of CYP2B1 by OPZ involves proteasome-dependent degradation and represents a new mechanism of regulation by this compound.

Abbreviations: CDNB, 1-chloro-2,4-dinitrobenzene; CYP, cytochrome P450; EROD, ethoxyresorufin O-deethylase; GST, glutathione S-transferase; LPS, lipopolysaccharide; OPZ, oltipraz; PROD, pentoxyresorufin O-dealkylase.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The dithiolethiones (constituents of cruciferous vegetables) and their synthetic analogues inhibit tumorigenesis elicited by different classes of carcinogens targeting the breast (1,2), lymph (2), forestomach and lung (3), colon (4), liver (5), skin (6), pancreas (7), trachea and bladder (1) in rodents. One mechanism proposed to account for this broad-based inhibition of carcinogenesis by dithiolethiones is induction of carcinogen-metabolizing enzymes (8). These compounds enhance the activities of glutathione S-transferase (GST), NAD(P)H quinone reductase, aflatoxin B1 aldehyde reductase and UDP-glucuronyltransferases (for a review see ref. 9). One of these chemoprotective compounds, oltipraz (OPZ), a synthetic derivative of 1,2-dithiole-3-thione, is a potent inducer of these enzymes. Recently, we demonstrated that OPZ can also affect the activity of cytochrome P450 enzymes. It has a strong inhibitory effect on CYP1A2 and 3A4 in primary human hepatocyte cultures (10) and increases the levels of CYP1A1 and CYP2B1/2 mRNA in the rat after transiently inhibiting their activities in the early stages of treatment (11). Thus both inhibition of CYPs and induction of electrophile detoxification enzymes are likely to contribute to chemoprevention by OPZ.

Although most tissues possess lower levels of carcinogen-metabolizing enzymes than the liver, it is becoming increasingly evident that extrahepatic metabolism of carcinogens can modify their bioactivity. Thus, intestinal and pulmonary detoxification enzymes could represent the first body protection against harmful agents. The kidney is also a key organ for the elimination of noxious agents, but only few data are available on the effects of dithiolethiones on renal xenobiotic-metabolizing enzymes (12). Moreover, most of these reports have focused on the effects of dithiolethiones on phase II enzymes (12), and few studies have been performed on the modulation of CYP enzymes by these compounds in kidney and lung.

The expression or activities of drug-metabolizing enzymes can be altered by environmental, genetic or physiopathological factors. Recently, we have demonstrated that the effect of OPZ is altered in the course of inflammation induced by Escherichia coli lipopolysaccharide (LPS), a classical inducer of the acute phase response (13). A strong increase in rGSTA1/2, rGSTM1, rGSTP1, CYP1A2 and CYP2B1/2 was observed in rat liver after 3 days' treatment with OPZ, but the induction of these enzymes was suppressed at the mRNA, protein and activity levels during the acute phase response to endotoxin (13). A number of physiopathological situations are known to impair the balance between activation and detoxification of carcinogens and other chemicals. Moreover, epidemiological studies indicate that chronic inflammation associated with ulcerative colitis, gastritis or hepatitis manifested during bacterial, parasitic or viral infections is linked to increased risk of development of malignant neoplasms (1416). These latter physiopathological situations have been reported to be associated with a decreased capacity to metabolize drugs, as evidenced by lower rates of elimination (17). Such situations might, therefore, interfere with the effects of OPZ, so there is a need to investigate the effect of OPZ in both normal and pathological situations.

In this study, we examined the effect of OPZ on CYPs and GSTs in rat kidney and lung. We also determined whether administration of E.coli LPS could modulate constitutive and OPZ-inducible expression of these enzymes in both tissues.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals
Chromatographically pure LPS, from E.coli serotype 0127:b8, MG123, ethoxyresorufin and pentoxyresorufin were purchased from Sigma Chemical Co. (St. Louis, MO). OPZ was kindly supplied by Dr C.G. Caillard (Rhône-Poulenc Rorer, Antony, France). [{alpha}-32P]dCTP was obtained from Amersham (Arlington Heights, IL, USA). The rat CYP2B antibody, which cross-reacts with CYP2B1 and CYP2B2, was a gift from Pr Phillippe Beaune (Université Henri Descartes, Paris, France).

Animals and treatments
Male Wistar rats (~180 g) were fed for 1–5 days with a diet (AO4; Centre d'Elevage Janvier, Le Genest, France) supplemented with OPZ to a final concentration of 0.075% (w/w). Two series of experiments were performed. The first one was designed to analyse the expression and activities of CYPs and GSTs after treatment with OPZ for 1–5 days and the second one to compare the response of these enzymes to OPZ in the presence or absence of LPS. LPS was dissolved in 0.9% sterile NaCl and injected intraperitoneally at 1 mg/kg body wt, 24 h before the end of a 72 h OPZ treatment. The amount of OPZ ingested by each animal was estimated by weighing the food every day. For the first series of rats the food intake was 25 g/day. In the second series of rats the food intake of each animal was lower: 14 g/day for control and oltipraz-treated rats and 13 g/day for LPS-treated rats. All control animals received only vehicle, by the appropriate route. Four animals were used for each condition. Animals were killed by beheading. All experimental procedures were done in compliance with French laws and regulations.

RNA isolation and blot analysis
Total RNA was extracted by the method of Chirgwin et al. (18). Ten micrograms of total RNA was subjected to electrophoresis in a denaturing 6% (v/v) formaldehyde–1.2% (w/v) agarose gel and transferred on to Hybond-N nylon filters (Amersham). The integrity and relative amounts of RNA were assessed by methylene blue staining of the membrane. Prehybridization and hybridization were performed according to Church and Gilbert (19). Membranes were washed with 3xSSC/0.1% sodium dodecyl sulfate (SDS) for 30 min and then twice with 1xSSC/0.1% SDS for 10 min at 65°C. A rat GSTA1/2-specific cDNA probe was generated by reverse transcription–polymerase chain reaction (RT–PCR) using total RNA isolated from rat liver (20). The other probes used were rGSTP1 (21), CYP1A2 (22) and CYP2B (23). cDNA probes were 32P-labelled by random priming using a Rediprime labelling kit (Amersham).

An oligonucleotide specific for the 18S ribosomal RNA was 32P-labelled and used as a control, as previously described (24). Relative amounts of mRNA were determined by densitometry (Densylab; Microvision Instruments, Evry, France).

Preparation of microsomal and cytosolic fractions
After animals had been killed, the lungs and kidneys were removed, frozen in liquid nitrogen and stored at –70°C. Microsomes and cytosols were prepared by differential centrifugation and stored at –70°C prior to enzyme analysis, immunoblotting and protein degradation assays.

Enzyme assays
GST activities were determined in cytosolic fractions as described by Habig and Jakoby (25) using 1-chloro-2,4-dinitrobenzene (CDNB), a substrate for most GST subunits. This activity was related to total cellular or cytosolic proteins. Activities of ethoxyresorufin O-deethylase (EROD; associated with CYP1A1/2) and pentoxyresorufin O-dealkylase (PROD; mainly associated with CYP2B1/B2, 2C11 and 3A2) were measured as described by Burke and Mayer (26), the reaction rates were determined under linear conditions with regard to incubation time and protein concentration.

Western blot immunoassays
Microsomal proteins were diluted in 10% SDS, 1% ß-mercaptoethanol, 10 mM Tris-HCl pH 6.8 and 20% glycerol. Twenty micrograms of protein were electrophoresed in a 10% polyacrylamide slab gel and electroblotted overnight on to Hybond enhanced chemiluminescence (ECL) nitrocellulose membranes (Amersham). After blocking the filters in 3% bovine serum albumin (BSA)–Tris-buffered saline (TBS), they were incubated with rabbit anti-CYP2B antibody diluted at 1/3000 in 3% BSA–TBS–0.1% Tween 20. The filters were then washed in wash buffer (TBS, 0.1 M NaCl, 0.3% Nonidet P-40) and TBS, then incubated with peroxidase-conjugated rabbit anti-rabbit IgG. All incubations were done at room temperature for 2 h. Peroxidase activity was detected using an ECL western blotting detection system (Amersham).

In vitro assay for CYP2B1 degradation
The 105 000 x g supernatant (cytosol) was prepared by differential centrifugation from control or OPZ-treated rat lungs and used in an assay with uninduced lung microsomes. CYP2B1 degradation was determined using 50 µl of reaction mixture consisting of 50 mM Tris–HCl pH 7.9, 10 mM MgCl2, 2 mM ATP, 1 mM creatine phosphate, 40 µg/ml creatine phosphokinase, 5 mM dithiothreitol (DTT), 0.1 mg ubiquitin and 10% glycerol, in the presence of 5 µg microsomes and 200 µg cytosol. Reactions were incubated at 37°C for 1 h; the tube was then placed on ice immediately to stop the reaction. The incubation mixture was subjected to electrophoresis and probed with the anti-CYP2B antibody as outlined under `Western blot immunoassays'.

Statistical analysis
Numerical values are expressed as the mean ± SD. The Student's t-test was used. Significance was set at a limit of <5% for the test. Specific sets used for comparisons are described in the figure legends.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Effects of OPZ on GST and CYP activities in rat lung and kidney
The effects of OPZ on GST activity in cytosols from kidneys and lungs of male rats over a 120 h period are shown in Figure 1A and BGo. In both tissues, CDNB activity increased slightly, but only the increases at 96 h for the lung and at 48, 72 and 96 h for the kidney were significant (P<0.05). In order to determine whether OPZ had an effect on CYP activities in rat lung and kidney, EROD and PROD activities were measured in microsomal preparations from animals treated for 1–5 days with this compound (Figure 1C and DGo). Measurement of EROD activity in lung demonstrated that CYP1A activity was significantly induced (3.5-fold, P<0.05) by OPZ after 24 h in treated rats compared with the controls and remained high until 120 h. In contrast, PROD activity (CYP2B1) was dramatically reduced (4-fold, P<0.05) after 24 h of OPZ treatment and this activity remained decreased during the 5 days of treatment. OPZ significantly increased EROD activity in rat kidney after 24 h (1.6-fold; P<0.05) and 120 h (1.7 fold, P<0.05), while CYP2B1/2 activity was undetectable in this tissue in both control and treated rats.



View larger version (35K):
[in this window]
[in a new window]
 
Fig. 1. CDNB, EROD and PROD activities in lungs and kidneys of rats treated with OPZ. Rats were maintained under control conditions or fed an OPZ-supplemented diet [final OPZ concentration 0.075% (w/w)] for 24, 48, 72, 96 or 120 h. GST activity (CDNB) was measured in cytosol prepared from lung (A) or kidney (B) samples. EROD (CYP1A) and PROD (CYP2B) activities were measured in lung (C) and kidney (D) microsomes. The values are expressed as means ± SD of four animals. Statistical analysis (Student's t-test) was performed by comparison of OPZ-treated and control rats; *P<0.05.

 
Effects of OPZ on CYP and GST mRNAs in rat lung and kidney
Steady-state mRNA levels were analysed by northern blots using specific cDNA probes for rGSTA1/2, rGSTP1, CYP1A and CYP2B. A significant increase in mRNA rGSTP1 levels (2.5-fold; P<0.001) was observed in the lungs of rats treated with OPZ for 72 h (Figure 2AGo). An increase in the amount of this transcript was also observed in rat kidney after OPZ treatment (4to 6-fold after 24 and 120 h, respectively; Figure 2BGo). While rGSTA1/2 mRNA was undetectable in rat lung, its expression in kidney was augmented by a factor of 1.5- to 2-fold after treatment with OPZ for 96 and 120 h, respectively (P<0.05; Figure 2BGo).



View larger version (37K):
[in this window]
[in a new window]
 
Fig. 2 . Expression of rGSTA1/2, rGSTP1, CYP2B and CYP1A mRNAs in lungs and kidneys of rats treated with OPZ. Rats were maintained under control conditions or fed OPZ-supplement diet for 24, 48, 72, 96 or 120 h [final concentration 0.075% (w/w)]. Total RNA was isolated from lung and kidney and subjected to northern blot analysis. (A) rGSTP1 mRNA levels in lung. (B) rGSTA1/2 and rGSTP1 mRNA levels in kidney. (C) CYP1A and CYP2B mRNA levels in lung. (D) CYP1A mRNA levels in kidney. The values are expressed as mean ± SD of four animals. mRNA levels are expressed as fold increase over the corresponding control. Statistical analysis (Student's t-test) was performed by comparison of OPZ-treated and control rats; *P<0.05; **P<0.01; ***P<0.001.

 
CYP2B steady state mRNA levels in the lungs of OPZ-treated rats were the same as those in controls (Figure 2CGo). In contrast, CYP1A mRNA expression was highly induced (8-fold; P<0.05) in this tissue after 24 h treatment with OPZ. This induction was maintained until 120 h of treatment (11-fold; P<0.01). The level of this transcript was also increased in rat kidney after 24 h of treatment with OPZ (Figure 2DGo).

Effects of OPZ on CYP2B proteins in rat lung
In order to determine whether the decreased PROD activity was a consequence of the decrease in CYP2B apoprotein content or resulted from inhibition of the corresponding activity, we performed a western blot with an anti-CYP2B antibody on microsomes isolated from control and OPZ-treated rat lungs. This analysis showed that in the lungs of treated rats, there was only 25% of the amount of CYP2B found in controls (Figure 3Go). Since CYP2B2 is not detectable in rat lung (27,28), this decrease in CYP2B apoprotein in the lungs of OPZ-treated rats probably represented a loss of constitutively expressed CYP2B1.



View larger version (31K):
[in this window]
[in a new window]
 
Fig. 3. Effect of OPZ on the expression of CYP2B in rat lung. Rats were maintained under control conditions or fed an OPZ-supplemented diet for 24, 48, 72, 96 or 120 h [final OPZ concentration 0.075% (w/w)]. Twenty micrograms of protein were electrophoresed in a 10% polyacrylamide slab gel and electroblotted overnight on to Hybond ECL nitrocellulose membranes and were incubated with rabbit anti-CYP2B antibody. (A) Western blot autoradiograms representing results from three different rats for each treatment. (B) Values are expressed as means ± SD of four animals. The CYP2B1 levels are expressed as a percentage of the control value. The statistical analysis (Student's t-test) was performed by comparison of OPZ-treated and control rats; *P<0.05.

 
Effects of LPS on CYP and GST expression and activities in lung and kidney of both control and OPZ-treated rats
LPS was injected intraperitoneally at 1 mg/kg and its effects were analysed 24 h later (between 48 and 72 h for OPZ-treated rats). Such experimental conditions have been previously shown to mimic an acute-phase response typical of bacterial infection (29). While LPS alone had no effect on basal GST activity in both lung and kidney, OPZ induction of this activity in kidney was reduced after endotoxin treatment (P<0.05) (Figure 4AGo). Under these conditions of treatment, OPZ induction of rGSTP1 mRNA in kidney was abolished 24 h after LPS injection (Figure 4BGo). In contrast, neither basal nor OPZ-induced rGSTP1 mRNA levels were affected by LPS treatment in lung (Figure 4BGo).



View larger version (29K):
[in this window]
[in a new window]
 
Fig. 4. Effects of LPS and OPZ on GST expression and activity in rat lung and kidney. Rats were maintained under control conditions or fed an OPZ-supplemented diet for 3 days at a final OPZ concentration of 0.075% (w/w). LPS was injected at 1 mg/kg 24 h before the end of OPZ treatment. (A) CDNB activities in cytosol prepared from lung and kidney samples. The values are expressed as mean ± SD (bars) of four animals. Statistical analysis (Student's t-test) was performed by comparison of OPZ-treated rats with control rats (***P<0.001), LPS-treated rats with control rats and OPZ-treated rats with OPZ/LPS-treated rats (#P<0.05). (B) Densitometric quantification of rGSTA1/2 and P1 steady state mRNA levels in rat lung and kidney after LPS and/or OPZ treatment. The values are expressed as mean ± SD (bars) of four animals. mRNA levels are expressed as a percentage of the corresponding control value. Statistical analysis (Student's t-test) was performed by comparison of OPZ-treated rats with control rats (**P<0.01; *P<0.05), OPZ-treated rats with OPZ/LPS-treated rats (##P<0.01) and OPZ/LPS-treated rats with control rats (+P<0.05).

 
Regarding CYP activities, pulmonary EROD (CYP1A) and PROD (CYP2B1) activities were reduced by 3-fold and 1.5-fold respectively compared with the controls 24 h after administration of LPS alone (Figures 5 and 6CGoGo respectively). The increase in CYP1A activity in the lung in the presence of OPZ was abolished after endotoxin treatment (Figure 5Go). The OPZ-dependent decrease in CYP2B1 apoprotein was abolished and its corresponding activity partially reversed 24 h after LPS injection in the lungs of OPZ-treated animals, while CYP2B1 transcripts remained unaffected by endotoxin treatment (Figure 6Go).



View larger version (22K):
[in this window]
[in a new window]
 
Fig. 5. EROD activity in lungs and kidneys of rats treated with OPZ and/or LPS. Rats were maintained under control conditions or fed OPZ-supplemented diet for 3 days [final OPZ concentration of 0.075% (w/w)]. LPS was injected at 1 mg/kg 24 h before the end of OPZ treatment. Microsomes were prepared from lung and kidney samples and EROD (CYP1A) activity was measured. The values are expressed as mean ± SD (bars) of four animals. Statistical analysis (Student's t- test) was performed by comparing OPZ-treated with control rats (**P<0.01) and OPZ-treated rats with OPZ/LPS-treated rats (##P<0.01).

 


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 6. Effect of OPZ and LPS on CYP2B expression and activity in rat lung. Rats were maintained under control conditions or fed an OPZ-supplemented diet for 3 days [final OPZ concentration 0.075% (w/w)]. LPS was injected at 1 mg/kg 24 h before the end of OPZ treatment. (A) CYP2B mRNA levels (determined by densitometric analysis) are expressed as a percentage of the corresponding control value after standardization to 18S RNA signals. (B) CYP2B apoprotein levels (autoradiogram and corresponding densitometric analysis) are expressed as a percentage of the corresponding control value. (C) PROD activity was measured in microsomes prepared from lung samples. All the results are expressed as mean ± SD (bars) of four animals. Statistical analysis (Student's t-test) was performed by comparing OPZ-treated rats with control rats (***P<0.001), LPS-treated rats with control rats (++P<0.01) and OPZ-treated with OPZ/LPS-treated rats (###P<0.001).

 
Cytosol-dependent proteolytic loss of lung microsomal CYP2B1
The rapid loss of lung CYP2B1 after OPZ treatment suggested a series of specific intracellular events that might target this CYP for rapid proteolysis. Recent studies have shown that the ubiquitin-ATP-dependent 26S proteasomal system is involved in degrading specific CYPs (3032). In order to determine whether this mechanism is responsible for the degradation of pulmonary CYP2B1 apoprotein in OPZ-treated rats, we incubated microsomes with cytosols isolated from both control and OPZ-treated rat lungs. Degradation of CYP2B1 apoprotein was increased when incubation was performed with OPZ-treated rat lung cytosols (Figure 7AGo). Addition of MG123, a proteasome inhibitor, partially stabilized CYP2B1 (Figure 7BGo).



View larger version (31K):
[in this window]
[in a new window]
 
Fig. 7. In vitro degradation of rat lung CYP2B1. (A) Western blot analysis of microsomes before incubation (lane 1) and microsomes incubated at 37°C for 1 h in absence (lane 2) or presence of lung cytosols prepared from three control rats (lanes 3, 4 and 5) or three OPZ-treated rats (lanes 6, 7 and 8). (B) Western blot analysis of microsomes incubated at 37°C for 1 h without (–cytosol) or with lung cytosols (+cytosol) and in presence (+I) or absence (–I) of MG132, a proteasome inhibitor. Lane 1, microsomes before incubation.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The induction of detoxication enzymes is a significant mechanism of cancer prevention through which various natural and synthetic chemoprotective agents can act (33,34). Several studies have shown that dithiolethiones can induce phase II enzymes (12,3538). However, other mechanisms may also be involved, since OPZ has a strong inhibitory effect on both human and rat hepatic CYP activities (10,11). The study reported here brings additional insights into the capacity of OPZ to modify the expression and activity of two CYPs involved in carcinogen metabolism, namely CYP2B and CYP1A, as well as rGSTA1/2 and rGSTP1 in two extrahepatic organs, lung and kidney.

Several studies demonstrated that rGSTA1/2 and rGSTP1 subunits are strongly induced in liver by OPZ (13,35,37,38). Analysis of OPZ-treated rat lung and kidney showed that total GST activity, measured with CDNB as a substrate, is only slightly increased in both tissues. Northern blotting showed that rGSTP1 mRNA expression is increased in both lung and kidney of treated rats, while rGSTA1/2, undetected in lung, is significantly increased in kidney only after 96 h of treatment with OPZ. A previous study investigated the effect of 1,2-dithiole-3-thione and its synthetic derivative, OPZ, on GST subunit content in Fisher rat tissues (12). An increase in rGSTP1 in both lung and kidney of treated rats was demonstrated while rGSTA1 and rGSTA2 were slightly augmented only in kidney. Our results showing the effect of OPZ on both total GST activity and mRNAs encoding rGSTP1 and rGSTA1/2 in these two tissues confirm and extend previous work of Meyer et al. (12).

Recently, we have demonstrated that the effect of OPZ on hepatic GSTs is altered in the course of LPS-induced inflammation (13,20). Indeed, OPZ induction of hepatic rGSTA1/2 and rGSTP1 expression was inhibited at the mRNA level when rats were administered LPS simultaneously. As observed in the liver, 24 h treatment with LPS reduced the effects of OPZ on both CDNB activity and rGSTP1 mRNA expression in lung and kidney, while basal activity of GST and constitutive expression of rGSTA1/2 and P1 mRNAs were not altered by the endotoxin alone. This down-regulation of OPZ-induced GSTs by LPS may involve proinflammatory cytokines, since interleukin 1, which is considered to be one of the major mediators of LPS-induced toxicity, inhibits major GSTs in primary rat hepatocytes (20). As previously observed in the liver, our results clearly demonstrate that OPZ increases the levels of GSTs in lung and kidney; this effect is completely or partially inhibited during the inflammation process in these tissues.

In contrast with GSTs, which are concomitantly induced by OPZ in lung, kidney and liver, this chemopreventive agent shows different effects on CYP1A and CYP2B depending on the target tissue. Previously, we demonstrated that OPZ is an early inhibitor of CYP1A and CYP2B activities, two enzymes involved in hepatic activation of aflatoxin B1, both in vivo and in vitro (11). However, this inhibition of CYP1A and CYP2B enzymatic activities with OPZ was followed by an induction as shown by the increased levels of their mRNAs and of caffeine metabolism in vivo after 24 h or more (11,13). In the present study we demonstrate that CYP1A steady-state mRNA levels are augmented in both lung and kidney as early as after 24 h of treatment with OPZ. The mechanism responsible for this increase of CYP1A transcript levels is not yet known. A transcriptional mechanism is probably involved since the increase in CYP1A mRNA is associated with transcriptional activation of the corresponding gene in liver of rats treated with OPZ (13). CYP1A mRNA induction was associated with an increase in the corresponding activity, namely the deethylation of resorufin. However, when animals were treated with both LPS and OPZ, induction of EROD by OPZ was completely abolished in lung. Thus, our results indicate that, as observed with GSTs, OPZ augmented CYP1A expression and activity in both lung and kidney and this increase was abolished after endotoxin treatment.

OPZ had a novel effect on pulmonary CYP2B1: we describe herein a previously unknown mechanism of OPZ. While CYP2B1 steady-state mRNA levels remained unaffected by OPZ, whatever the time of treatment, we observed a dramatic decrease in the corresponding apoprotein and activity (PROD) 24 h after OPZ treatment, suggesting the involvement of a post-transcriptional mechanism. The rapid loss of pulmonary CYP2B1 during OPZ treatment led us to further investigate the mechanism responsible for this degradation. Multiple proteolytic mechanisms for CYP degradation apparently exist, since lysosomal degradation of CYP2B1 and also CYP2E1 as well as proteolytic degradation of CYP2E1 and CYP3A by endoplasmic reticulum proteases have been reported (3942). In the context of the latter mechanism, recent findings seem to indicate that a number of endoplasmic reticulum proteins are targeted for destruction, either by ubiquitin conjugation (4345) or possibly by the proteasome (44,46). In contrast to the normal in vivo turnover of native CYP2B1, which is lysosomal, Korsmeyer et al. (32) demonstrated that heme of modification purified CYP2B1 by cumene hydroperoxide leads to the degradation of this protein and involves both ubiquitination and proteolytic degradation by the 26S proteasome. Moreover, a previous study demonstrated differences in the CYP2B1 mRNA and apoenzyme expression levels in freshly isolated and cultured Clara cells and type II alveolar cells (47), the two major cell types which express CYPs in lung. While mRNA levels remained constant throughout the culture period, the apoenzyme level of CYP2B1 decreased in culture suggesting the existence of a post-transcriptional regulatory mechanism for CYP2B1 expression in lung cells.

Taken together, these observations led us to investigate whether the rapid degradation of CYP2B1 apoprotein in OPZ-treated rat lungs involves the proteasome system. Incubation of microsomes with cytosols isolated from OPZ-treated rat lungs results in a substantial CYP2B1 degradation, which is partially inhibited by MG123, a proteasome inhibitor. These observations suggest that proteasome-mediated CYP2B1 degradation occurs in OPZ-treated rat lung. Interestingly, OPZ-dependent decrease in pulmonary CYP2B1 apoprotein and activity was completely or partially reversed, respectively, in LPS-treated rats. These results demonstrate that inflammation also alters OPZ modulation of CYP2B1 in lung. LPS administration is known to result within 24 h in an acute-phase response with overexpression of proinflammatory cytokines, and recent studies have demonstrated that deubiquitinating enzymes are induced by cytokines (4850). Thus, the observation that LPS restores the level of CYP2B1 in lung of OPZ-treated rats is in line with the involvement of the ubiquitin-dependent proteasomal pathway in the degradation of CYP2B1. Since a recent study showed that OPZ can damage heme of CYPs (51), we hypothesize that the modification of CYP2B1 heme by OPZ or a metabolite could lead to the proteasomal degradation of apoprotein. Our results strongly support the view that the degradation of CYP2B1 apoprotein involves the proteasome pathway in the lung of OPZ-treated rat; however, further studies are needed to determine whether OPZ or one of its metabolites is directly responsible for this mechanism.

In conclusion, as previously shown in the liver, we have demonstrated that OPZ differentially affects xenobiotic-metabolizing enzymes, mainly CYPs, in lung and kidney. We also describe a previously unreported mechanism of regulation by OPZ. While previous studies showed an induction of expression by a transcriptional mechanism or an inhibitory effect on enzyme activity by OPZ, we have found evidence of post-translational regulation of CYP2B1 by OPZ involving the proteasome pathway. Such in vivo analyses of the effects of OPZ on carcinogen-metabolizing enzymes in different rat tissues should further improve our understanding of the chemopreventive activity of this dithiolethione.


    Notes
 
3 To whom correspondence should be addressed Back


    Acknowledgments
 
K.M. is a recipient of a fellowship from La Ligue Nationale Contre Le Cancer and A.C. is a recipient of a fellowship from L'Association Pour La Recherche Sur Le Cancer.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Boone,C.W., Kelloff,G.J. and Malone,W.E. (1990) Identification of candidate cancer chemopreventive agents and their evaluation in animal models and human clinical trials: a review. Cancer Res., 50, 2–9.[Abstract]
  2. Rao,C.V., Rivenson,A., Zang,E., Steele,V., Kelloff,G. and Reddy,B.S. (1996) Inhibition of 2-amino-1-methyl-6-phenylimidazo[4,5]pyridine-induced lymphoma formation by oltipraz. Cancer Res., 56, 3395–3398.[Abstract]
  3. Wattenberg,L.W. and Bueding,E. (1986) Inhibitory effects of 5-(2-pyrazinyl)-4-methyl-1,2-dithiol-3-thione (oltipraz) on carcinogenesis induced by benzo[a]pyrene, diethylnitrosamine and uracil mustard. Carcinogenesis, 7, 1379–1381.[Abstract]
  4. Rao,C.V., Tokomo,K., Kelloff,G. and Reddy,B.S. (1991) Inhibition by dietary oltipraz of experimental intestinal carcinogenesis induced by azoxymethane in male F344 rats. Carcinogenesis, 12, 1051–1055.[Abstract]
  5. Roebuck,B.D., Liu,Y.L., Rogers,A.E., Groopman,J.D. and Kensler,T.W. (1991) Protection against aflatoxin B1-induced hepatocarcinogenesis in F344 rats by 5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione (oltipraz): predictive role for short-term molecular dosimetry. Cancer Res., 51, 5501–5506.[Abstract]
  6. Surh,Y.J., Kim,S.G., Liem,A., Lee,J.W. and Miller,J.A. (1999) Inhibitory effects of isopropyl-2-(1,3-dithietane-2-ylidene)-2- [N-(4- methylthiazol-2-yl)carbamoyl]acetate (YH439) on benzo[a]pyrene-induced skin carcinogenesis and micronucleated reticulocyte formation in mice. Mutat Res., 423, 149–53.[ISI][Medline]
  7. Clapper,M.L., Wood,M., Leahy,K., Lang,D., Miknyoczki,S. and Ruggeri, B.A. (1995) Chemopreventive activity of oltipraz against N-nitrosobis(2-oxopropyl)amine (BOP)-induced ductal pancreatic carcinoma development and effects on survival of Syrian golden hamsters. Carcinogenesis, 16, 2159–2165.[Abstract]
  8. Kensler,T.W. and Helzlsouer,K.J. (1995) Oltipraz: clinical opportunities for cancer chemoprevention. J. Cell. Biochem. (Suppl.), 22, 101–107.
  9. Primiano,T., Sutter,T.R. and Kensler,T.W. (1997) Antioxidant-inducible genes. Adv Pharmacol., 38, 293–328.[Medline]
  10. Langouet,S., Coles,B., Morel,F., Becquemont,L., Beaune,P., Guengerich,F.P., Ketterer,B. and Guillouzo,A. (1995) Inhibition of CYP1A2 and CYP3A4 by oltipraz results in reduction of aflatoxin B1 metabolism in human hepatocytes in primary culture. Cancer Res., 55, 5574–5579.[Abstract]
  11. Langouet,S., Maheo,K., Berthou,F., Morel,F., Lagadic-Gossman,D., Glaise,D., Coles,B., Ketterer,B. and Guillouzo,A. (1997) Effects of administration of the chemoprotective agent oltipraz on CYP1A and CYP2B in rat liver and rat hepatocytes in culture. Carcinogenesis, 18, 1343–1349.[Abstract]
  12. Meyer,D.J., Harris,J.M., Gilmore,K.S., Coles,B., Kensler,T.W. and Ketterer,B. (1993) Quantitation of tissue- and sex-specific induction of rat GSH transferase subunits by dietary 1,2-dithiole-3-thiones. Carcinogenesis, 14, 567–572.[Abstract]
  13. Maheo,K., Morel,F., Antras-Ferry,J., Langouet,S., Desmots,F., Corcos,L. and Guillouzo,A. (1998) Endotoxin suppresses the oltipraz-mediated induction of major hepatic glutathione transferases and cytochromes P450 in the rat. Hepatology, 28, 1655–1662.[ISI][Medline]
  14. Hagen,T.M., Huang,S., Curnutte,J., Fowler,P., Martinez,V., Wehr,C.M., Ames,B.N. and Chisari,F.V. (1994) Extensive oxidative DNA damage in hepatocytes of transgenic mice with chronic active hepatitis destined to develop hepatocellular carcinoma. Proc. Natl Acad. Sci. USA, 91, 12808–12812.[Abstract/Free Full Text]
  15. Ekbom,A., Helmick,C., Zack,M. and Adami,H.O. (1990) Ulcerative colitis and colorectal cancer. A population-based study. New Engl. J. Med., 323, 1228–1233.[Abstract]
  16. Parsonnet,J., Friedman,G.D., Vandersteen,D.P., Chang,Y., Vogelman,J.H., Orentreich,N. and Sibley,R.K. (1991) Helicobacter pylori infection and the risk of gastric carcinoma. New Engl. J. Med., 325, 1127–1131.[Abstract]
  17. Renton,K.W. (1986) Factors affecting drug biotransformation. Clin. Biochem., 19, 72–75.[ISI][Medline]
  18. Chirgwin,J.M., Przybyla,A.E., MacDonald,R.J. and Rutter,W.J. (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry, 18, 5294–5299.[ISI][Medline]
  19. Church,G.M. and Gilbert,W. (1984) Genomic sequencing. Proc. Natl Acad. Sci. USA, 81, 1991–1995.[Abstract]
  20. Maheo,K., Antras-Ferry,J., Morel,F., Langouet,S. and Guillouzo,A. (1997) Modulation of glutathione S-transferase subunits A2, M1, and P1 expression by interleukin-1ß in rat hepatocytes in primary culture. J. Biol. Chem., 272, 16125–16132.[Abstract/Free Full Text]
  21. Pemble,S.E., Taylor,J.B. and Ketterer,B. (1986) Tissue distribution of rat glutathione transferase subunit 7, a hepatoma marker. Biochem. J., 240, 885–889.[ISI][Medline]
  22. Affolter,M., Labbe,D., Jean,A., Raymond,M., Noel,D., Labelle,Y., Parent-Vaugeois,C., Lambert,M., Bojanowski,R. and Anderson,A. (1986) cDNA clones for liver cytochrome P-450s from individual Aroclor-treated rats: constitutive expression of a new P-450 gene related to phenobarbital-inducible forms. DNA, 5, 209–218.[ISI][Medline]
  23. Adesnik,M., Bar-Nun,S., Maschio,F., Zunich,M., Lippman,A. and Bard,E. (1981) Mechanism of induction of cytochrome P-450 by phenobarbital. J. Biol. Chem., 256, 10340–10345.[Abstract/Free Full Text]
  24. Chan,Y.L., Gutell,R., Noller,H.F. and Wool,I.G. (1984) The nucleotide sequence of a rat 18S ribosomal ribonucleic acid gene and a proposal for the secondary structure of 18S ribosomal ribonucleic acid. J. Biol. Chem., 259, 224–230.[Abstract/Free Full Text]
  25. Habig,W.H. and Jakoby,W.B. (1981) Glutathione S-transferases (rat and human). Methods Enzymol., 77, 218–231.[Medline]
  26. Burke,M.D. and Mayer,R.T. (1983) Differential effects of phenobarbitone and 3-methylcholanthrene induction on the hepatic microsomal metabolism and cytochrome P-450-binding of phenoxazone and a homologous series of its N-alkyl ethers (alkoxyresorufins). Chem. Biol. Interact., 45, 243–258.[ISI][Medline]
  27. Paolini,M., Mesirca,R., Pozzetti,L., Sapone,A. and Cantelli-Forti,G. (1995) Induction of CYP2B1 mediated pentoxyresorufin O-dealkylase activity in different species, sex and tissue by prototype 2B1-inducers. Chem.-Biol. Interact., 95, 127–139.[ISI][Medline]
  28. Omiecinski,C.J. (1986) Tissue-specific expression of rat mRNAs homologous to cytochromes P-450b and P-450e. Nucleic Acids Res., 14, 1525–1539.[Abstract]
  29. Baumann,H. and Gauldie,J. (1994) The acute phase response. Immunol. Today, 15, 74–80.[ISI][Medline]
  30. Yang,M.X. and Cederbaum,A.I. (1996) Role of the proteasome complex in degradation of human CYP2E1 in transfected HepG2 cells. Biochem. Biophys. Res. Commun., 226, 711–716.[ISI][Medline]
  31. Wang,H.F., Figueiredo Pereira,M.E. and Correia,M.A. (1999) Cytochrome P450 3A degradation in isolated rat hepatocytes: 26S proteasome inhibitors as probes. Arch. Biochem. Biophys., 365, 45–53.[ISI][Medline]
  32. Korsmeyer,K.K., Davoll,S., Figueiredo-Pereira,M.E. and Correia,M.A. (1999) Proteolytic degradation of heme-modified hepatic cytochromes P450: A role for phosphorylation, ubiquitination, and the 26S proteasome? Arch. Biochem. Biophys., 365, 31–44.[ISI][Medline]
  33. Prochaska,H.J., Santamaria,A.B. and Talalay,P. (1992) Rapid detection of inducers of enzymes that protect against carcinogens. Proc. Natl Acad. Sci., USA, 89, 2394–2398.[Abstract]
  34. Wilkinson,J.T. and Clapper,M.L. (1997) Detoxication enzymes and chemoprevention. Proc. Soc. Exp. Biol. Med., 216, 192–200.[Abstract]
  35. Langouet,S., Morel,F., Meyer,D.J., Fardel,O., Corcos,L., Ketterer,B. and Guillouzo,A. (1996) A comparison of the effect of inducers on the expression of glutathione-S-transferases in the liver of the intact rat and in hepatocytes in primary culture. Hepatology, 23, 881–887.[ISI][Medline]
  36. Egner,P.A., Kensler,T.W., Prestera,T., Talalay,P., Libby,A.H., Joyner,H.H. and Curphey,T.J. (1994) Regulation of phase 2 enzyme induction by oltipraz and other dithiolethiones. Carcinogenesis, 15, 177–181.[Abstract]
  37. Davidson,N.E., Egner,P.A. and Kensler,T.W. (1990) Transcriptional control of glutathione S-transferase gene expression by the chemoprotective agent 5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3- thione (oltipraz) in rat liver. Cancer Res., 50, 2251–2255.[Abstract]
  38. Buetler,T.M., Gallagher,E.P., Wang,C., Stahl,D.L., Hayes,J.D. and Eaton,D.L. (1995) Induction of phase I and phase II drug-metabolizing enzyme mRNA, protein, and activity by BHA, ethoxyquin, and oltipraz. Toxicol Appl. Pharmacol., 135, 45–57.[ISI][Medline]
  39. Masaki,R., Yamamoto,A. and Tashiro,Y. (1987) Cytochrome P-450 and NADPH-cytochrome P-450 reductase are degraded in the autolysosomes in rat liver. J. Cell. Biol., 104, 1207–1215.[Abstract]
  40. Eliasson,E., Mkrtchian,S. and Ingelman-Sundberg,M. (1992) Hormone and substrate-regulated intracellular degradation of cytochrome P450 (2E1) involving MgATP-activated rapid proteolysis in the endoplasmic reticulum membranes. J. Biol. Chem., 267, 15765–15769.[Abstract/Free Full Text]
  41. Ronis,M.J., Johansson,I., Hultenby,K., Lagercrantz,J., Glaumann,H. and Ingelman-Sundberg,M. (1991) Acetone-regulated synthesis and degradation of cytochrome P450E1 and cytochrome P4502B1 in rat liver. Eur. J. Biochem., 198, 383–389.[Abstract]
  42. Zhukov,A., Werlinder,V. and Ingelman-Sundberg,M. (1993) Purification and characterization of two membrane bound serine proteinases from rat liver microsomes active in degradation of cytochrome P450. Biochem. Biophys. Res. Commun., 197, 221–228.[ISI][Medline]
  43. Roberts,B.J., Song,B.J., Soh,Y., Park,S.S. and Shoaf,S.E. (1995) Ethanol induces CYP2E1 by protein stabilization. Role of ubiquitin conjugation in the rapid degradation of CYP2E1. J. Biol. Chem., 270, 29632–29635.[Abstract/Free Full Text]
  44. Jensen,T.J., Loo,M.A., Pind,S., Williams,D.B., Goldberg,A.L. and Riordan,J.R. (1995) Multiple proteolytic systems, including the proteasome, contribute to CFTR processing. Cell, 83, 129–135.[ISI][Medline]
  45. Correia,M.A., Davoll,S.H., Wrighton,S.A. and Thomas,P.E. (1992) Degradation of rat liver cytochromes P450 3A after their inactivation by 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine: characterization of the proteolytic system. Arch. Biochem. Biophys., 297, 228–238.[ISI][Medline]
  46. McGee,T.P., Cheng,H.H., Kumagai,H., Omura,S. and Simoni,R.D. (1996) Degradation of 3-hydroxy-3-methylglutaryl-CoA reductase in endoplasmic reticulum membranes is accelerated as a result of increased susceptibility to proteolysis. J. Biol. Chem., 271, 25630–25638.[Abstract/Free Full Text]
  47. Lag,M., Becher,R., Samuelsen,J.T., Wiger,R., Refsnes,M., Huitfeldt,H.S. and Schwarze,P.E. (1996) Expression of CYP2B1 in freshly isolated and proliferating cultures of epithelial rat lung cells. Exp. Lung Res., 22, 627–649.[ISI][Medline]
  48. Zhu,Y., Lambert,K., Corless,C., Copeland,N.G., Gilbert,D.J., Jenkins,N.A. and D'Andrea,A.D. (1997) DUB-2 is a member of a novel family of cytokine-inducible deubiquitinating enzymes. J. Biol. Chem., 272, 51–57.[Abstract/Free Full Text]
  49. Zhu,Y., Carroll,M., Papa,F.R., Hochstrasser,M. and D'Andrea,A.D. (1996) DUB-1, a deubiquitinating enzyme with growth-suppressing activity. Proc. Natl Acad. Sci. USA, 93, 3275–3279.[Abstract/Free Full Text]
  50. D'Andrea,A. and Pellman,D. (1998) Deubiquitinating enzymes: a new class of biological regulators. Crit. Rev. Biochem. Mol. Biol., 33, 337–352.[Abstract]
  51. Langouet,S., Furge,L.L., Kerriguy,N., Nakamura,K., Guillouzo,A. and Guengerich,F.P. (2000) Inhibition of human cytochrome P450 enzymes by 1,2-dithiole-3-thione, oltipraz and its derivatives, and sulforaphane. Chem. Res. Toxicol., 13, 245–252.[ISI][Medline]
Received June 22, 2000; revised September 26, 2000; accepted October 2, 2000.