Induction of cytochrome P450 enzymes and over-generation of oxygen radicals in beta-carotene supplemented rats

Moreno Paolini1, Alessandra Antelli1, Laura Pozzetti1, Denisa Spetlova1, Paolo Perocco2, Luca Valgimigli3, Gian Franco Pedulli3 and Giorgio Cantelli-Forti4

1 Department of Pharmacology, Biochemical Toxicology Unit,
2 Institute of Cancerology and
3 Department of Organic Chemistry `A. Mangini', University of Bologna, Bologna, Italy


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Effects of beta-carotene (ßCT) on microsomal CYP-linked monooxygenases were investigated using both the regio- and stereo-selective hydroxylation of testosterone (as multibiomarker) and highly specific substrates as probes of various isoenzymes. CYP-catalyzed reactions were studied in the liver, kidney, lung and intestine of Sprague–Dawley rats of both sexes supplemented with 250 or 500 mg/kg body wt ßCT (per os) in a single or repeated (daily for 5 days) fashion. Generalized boosting effects (2–15-fold increases) were observed in the various tissues for carcinogen metabolizing enzymes associated with CYP1A1/2, CYP3A1/2, CYP2E1, CYP2B1/2 and CYP2C11. Induction of the most affected CYPs was corroborated by western blot linked to densitometric analyses. Measurement of reactive oxygen species (ROS) produced by subcellular preparations from either control or ßCT supplemented rats was performed by EPR detection of the nitroxide radical yielded by the reaction with ROS of the hydroxylamine spin probe bis(1-hydroxy-2,2,6,6-tetramethyl-4-piperidinyl)decandioate. Marked ROS over-generation associated with CYP induction (up to 33-fold increase in the liver) was recorded in the various organs (liver > lung > intestine > kidney). CYP and ROS induction are substantially in keeping with the concentration of ßCT accumulated in the various tissues, the liver being the most affected organ. These findings are consistent with the concept that ßCT is a pro-oxidant and potentially co-carcinogenic pro-vitamin, and may help explain why, in large quantities, it can have harmful effects in humans.

Abbreviations: APND, aminopyrine N-demethylase; ATBC, Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study; ßCT, beta-carotene; CARET, Carotene and Retinol Efficacy Trial; CYP, cytochrome P450; ECOD, ethoxycoumarin O-deethylase; EPR, electronic paramagnetic resonance; EROD, ethoxyresorufin O-deethylase; MROD, methoxyresorufin O-demethylase; OPP, oxygen partial pressure; pNFH, p-nitrophenol hydroxylase; PROD, pentoxyresorufin O-dealkylase; RAR, retinoic acid receptor; ROH, retinol; ROS, reactive oxygen species; TH, testosterone hydroxylase.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Beta-carotene (ßCT) is a violet to yellow plant pigment that can be converted into vitamin A by enzymes in the intestinal wall and liver. ßCT provides the principal dietary source of vitamin A for the majority of the world population. In the early 1980s, the public health researchers Peto et al. (1,2) hypothesized that ßCT might reduce cancer incidence, especially in the lungs. Like the other members of the carotenoid family, ßCT is indeed very effective in neutralizing singlet oxygen (1O2) and, to a lesser extent, in interrupting lipid peroxidation chain reactions. One of the well known beneficial effects of ßCT is its ability to reduce the harmful effects of solar radiation (solar eruption, urticaria solaris, etc.) on photosensitive individuals by contrasting the action of 1O2 and other reactive species, such as free radicals deriving from the excitation of protoporphyrin (3).

In recent years, many epidemiological and experimental observations have suggested that the consumption of high dietary levels of fruit and vegetables rich in carotenoids (or high serum levels of ßCT) may help prevent cancer and heart disease in humans. Following scientific and public interest in the theoretical possibility that antioxidant pro-vitamins and vitamins might have anticancer activity, randomized, controlled chemoprevention trials have tested the ability of ßCT alone or in combination with vitamins A, E or C to prevent lung cancer and other cancers. However, high intake of these micronutrients failed to reduce tumor incidence, and two intervention trials (ATBC and CARET) revealed that long-term ßCT supplementation actually increased the relative risk for lung cancer among heavy smokers and asbestos workers (412). These findings aroused widespread scientific debate, and raised the suspicion that this antioxidant may even have carcinogenic properties (1320). The possibility of any initiating (mutagenic) activity of ßCT in either in vivo or in vitro systems, where it really does seem to act as an anti-genotoxic agent, can be excluded (2123). While an exhaustive amount of data on the effects of retinol on metabolizing enzymes is reported in the literature (see for example refs 2427), contradictory experimental results on the effects of ßCT supplementation have been reported in the literature. For example, diet fed ßCT did not affect metabolizing enzymes in Wistar rats (28,29), but had an appreciable effect in Swiss mouse (30) and induced intestine aryl hydrocarbon hydroxylases in Sprague–Dawley rats (31). In the present study, an in vivo model was used to investigate whether ßCT may act by means of epigenetic mechanisms such as those associated with CYP changes (e.g. co-carcinogenesis and promotion) (32). We proposed previously that the high relative lung cancer risk recorded in the two intervention trials might be linked to phase I induction (33). Here we show that ßCT supplementation is able to induce various CYP isoforms in different rat tissues of both sexes, and that this induction generates a large amount of reactive oxygen species (ROS).


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals
ßCT, nicotinamide adenine dinucleotide phosphate in oxidized and reduced form (NADP+ and NADPH), 7-ethoxyresorufin, p-nitrophenol, aminopyrine, ethoxycoumarin, 16{alpha}-hydroxytestosterone, corticosterone, testosterone, androst-4-ene-3,17-dione and retinol were purchased from Sigma Chemical Co. (St Louis, MO, USA); glucose 6-phosphate, glucose 6-phosphate dehydrogenase and cytochrome c from Boehringer-Mannheim (Germany); pentoxyresorufin and methoxyresorufin from Molecular Probes (Eugene, OR, USA); HPLC grade methanol, tetrahydrofuran and dichloromethane from Labscan Ltd. (Co. Dublin, Ireland); 7{alpha}-, 6ß- and 16ß-hydroxytestosterone from Steraloids (Wilton, NH); 6{alpha}-, 2{alpha}-, 2ß-hydroxytestosterone and echinenone were a generous gift from Dr P.G.Gervasi (CNR Pisa, Italy); rabbit polyclonal antibodies against purified rat liver CYPs for CYP1A1/2, CYP2B1/2, CYP2E1 and CYP3A1/2 were purchased from Chemicon International Inc. (Temecula, CA, USA); retinol acetate was a generous gift of Dr N.Tossani (University of Bologna, Italy). The hydroxylamine probe bis(1-hydroxy-2,2,6,6-tetramethyl-4-piperidinyl)decandioate was synthesized by following a previously described procedure and used as hydrochloride salt (34). All other chemicals and solvents used were of the highest purity commercially available.

Animal treatment and preparation of subcellular fractions
Male and female Sprague–Dawley rats (Harlan-Nossan, Milan, Italy), weighing 200–220 g, were housed under controlled conditions (12 h light–dark cycle, 22°C, 60% humidity). They were fed a rodent chow and had tap water ad libitum. ßCT was dissolved in corn oil and administered (250 or 500 mg/kg body wt per os) in a single or repeated (daily for 5 consecutive days) dose. Controls received vehicle only, under the same conditions. Six animals per experimental group were used. Rats were fasted 16 h prior to being killed, which occurred 24 h after the last treatment. They were killed humanely in accordance with approved Ministerial procedures appropriate to the species. Liver, kidney, lung and intestine were rapidly removed and processed separately, and the S9 fraction (9000 g) was then prepared (35). The post-mitochondrial supernatant was then centrifuged for 60 min at 105 000 g, pellet resuspended in 0.1 M K2P2O7, 1 mM EDTA (pH 7.4) and centrifuged again for 60 min at 105 000 g to give the final fraction. Washed microsomes were then resuspended with a hand-driven Potter Elvehjem homogenizer in a 10 mM Tris–HCl buffer (pH 7.4) containing 1 mM EDTA and 20% (v/v) glycerol; fractions were immediately frozen in liquid nitrogen and stored at –80°C prior to use.

Aminopyrine N-demethylase (APND) activity
Activity was determined by quantitation of CH2O release, according to Mazel (36). The total incubation volume was 3 ml, composed of 0.5 ml of a water solution of 50 mM aminopyrine and 25 mM MgCl2, 1.48 ml of a 0.60 mM NADP+, 3.33 mM G6P in 50 mM Tris–HCl buffer (pH 7.4), 0.02 ml G6PDH (0.93 U/ml) and 0.125 ml of sample (0.5 mg of protein). After 5 min of incubation at 37°C, the yellow color developed by the reaction of the released CH2O with the Nash reagent was read at 412 nm, and the molar absorptivity of 8000 used for calculation (37).

p-Nitrophenol hydroxylase (pNFH) activity
Activity was determined in a final volume of 2 ml: 2 mM p-nitrophenol in 50 mM Tris–HCl buffer (pH 7.4), 5 mM MgCl2 and a NADPH-generating system consisting of 0.4 mM NADP+, 30 mM isocitrate, 0.2 U of isocitrate dehydrogenase and 1.5 mg of proteins. After 10 min at 37°C, reaction was terminated by addition of 0.5 ml of a 0.6 N perchloric acid. Precipitated proteins were removed by centrifugation and 1 ml of resultant supernatant mixed with 1 ml 10 N NaOH. Absorbance at 546 nm was immediately measured and 4-nitrocatechol determined ({varepsilon}=10.28 mM–1 cm–1) (38).

Pentoxyresorufin O-dealkylase (PROD), ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-demethylase (MROD) activities
Reaction mixture consisted of 0.025 mM MgCl2, 200 mM pentoxyresorufin, 0.32 mg of proteins and 130 mM NADPH in 2.0 ml 0.05 M Tris–HCl buffer (pH 7.4). Resorufin formation at 37°C was calculated by comparing the rate of increase in relative fluorescence to the fluorescence of known amounts of resorufin (excitation 562 nm, emission 586 nm) (39). EROD and MROD activities were measured exactly in the same manner as described for the pentoxyresorufin assay, except that substrates concentration was 1.7 mM ethoxyresorufin and 5 mM methoxyresorufin (40).

Ethoxycoumarin O-deethylase (ECOD) activity
Activity was determined by quantitation of umbelliferone formation, according to Aitio (41). Incubation mixture consisted of 2.6 ml, composed of 1 mM ethoxycoumarin, 5 mM MgCl2, NADPH-generating system (see aminopyrine assay) and 25 ml of sample (0.1 mg of proteins). After 5 min of incubation at 37°C, reaction was stopped with 85 ml of TCA 0.31 M. The pH of the mixture was brought to about 10 by adding 0.65 ml of 1.6 M NaOH–glycine buffer (pH 10.3); amount of umbelliferone was measured fluorimetrically (excitation 390 nm; emission 440 nm).

Testosterone hydroxylase (TH) activity
Incubation and isolation.
Incubations contained liver, kidney, lung or intestine microsomes (equivalent to 1–2 mg protein), 0.6 mM NADP+, 8 mM glucose 6-phosphate, 1.4 U glucose 6-phosphate dehydrogenase and 1 mM MgCl2, in a final volume of 2 ml 0.1 M phosphate Na+/K+ buffer (pH 7.4). The mixture was pre-incubated for 5 min at 37°C. The reaction was performed at 37°C by shaking and started by the addition of 80 mM testosterone (dissolved in methanol). After 10 min, the reaction was stopped with 5 ml ice-cold dichloromethane and 12 nmol corticosterone (internal standard) in methanol. After 1 min vortexing, phases were separated by centrifugation at 2000 g for 10 min and the aqueous phase was extracted once more with 2 ml dichloromethane. The organic phase was extracted with 2 ml 0.02 N NaOH to remove lipid constituents, dried over anhydrous sodium sulphate and transferred to a small tube. Dichloromethane was evaporated at 37°C under nitrogen and the dried samples stored at –20°C. The samples were dissolved in 100 µl methanol and analyzed by HPLC (42).

HPLC separation and quantification.
Chromatographic separations were performed using a system consisting of a high-pressure pump (Waters Model 600E, Multisolvent Delivery System), a sample injection valve (Rheodyne Model 7121, Cotati, CA, USA) with a 20 µl sample loop and an ultraviolet (UV) detector (254 nm, Waters Model 486, Tunable Absorbance Detector) connected to an integrator (Millennium 2010, Chromatography Manager). For reversed-phase separation of metabolites, NOVA-PAK C18 analytical column (60 Å, 4 mm, 3.9x150 mm, Waters) was used as stationary phase. The mobile phase consisted of a mixture of solvent A [7.5% (v/v) tetrahydrofuran in water] and solvent B [7.5% (v/v) tetrahydrofuran and 60% (v/v) methanol in water] at a 1 ml/min flow rate. Metabolite separation was performed by a gradient from 30 to 100% (v/v) of solvent B over 30 min. The eluent was monitored at 254 nm and the area under the absorption band was integrated. The concentration of metabolites was determined by the ratio between respective metabolite peak areas and corticosterone (internal standard) and the calibration curves obtained with synthetic testosterone derivatives (43,44). The associated isoforms were: 6ß (CYP3A1, CYP1A1/2), 7{alpha} (CYP2A1, CYP1A1/2), 2ß (CYP3A1/2, CYP1A1), 2{alpha} (CYP2C11), 16ß (CYP2C11, CYP2B1/2), 16ß (CYP2B1/2, CYP2C11) and in position 17 (CYP3A1) (45).

Electrophoresis and western immunoblot
Liver, kidney, lung and intestine microsomal preparations obtained from both control and ßCT (500 mg/kg body wt daily for 5 consecutive days) treated male rats were solubilized in sodium dodecyl sulfate (SDS) and resolved by polyacrylamide gel electrophoresis (PAGE) according to the method of Laemmli (46) and then transferred to a nitrocellulose sheet (47). Western blot analysis using rabbit polyclonal antibodies (antiCYP2B1/2, CYP2E1, CYP1A1/2 and CYP3A1/2) raised against rat hepatic CYP subfamilies (48), was performed using microsomes (0.025 mg of microsomal protein were electrophoresed each time) and visualized with 4-chloro-1-naphthol in a 0.006% hydrogen peroxide solution. Two sets of independent experiments were set out; densitometric analysis was then performed on western blot.

Protein concentration
Protein concentration was determined according to the method described by Lowry et al. (49) and revised by Bailey (50), using bovine seric albumin as a standard and diluting samples 200 times to provide a suitable protein concentration.

EPR spin probe technique
Microsomes from male animals were incubated at 37°C directly in standard EPR capillary tubes in 0.01 M Na+/K+ phosphate buffer (pH 7.4) in the presence of 1 mM NADPH and 0.5 mM hydroxylamine probe bis(1-hydroxy-2,2,6,6-tetramethyl-4-piperidinyl)decandioate synthesized as described previously (34). After mixing, the samples were immediately placed within the spectrometer's (EPR) cavity. The nitroxide radicals generated by reaction of the probe with the ROS present in the samples were then measured by EPR on a Bruker ESP 300 spectrometer equipped with an NMR gaussmeter for field calibration, a Bruker ER 033M FF-lock and a Hewlett-Packard 5350B microwave frequency counter for the determination of the g-factor which was corrected with respect to that of perylene radical cation in concentrated H2SO4 (g = 2.00258). Spectra were recorded using the following instrumental settings: modulation amplitude = 1.0 G; conversion time = 163.84 ms; time constant = 163.84 ms; receiver gain 1.0e5; microwave power = 6.3 mW. The intensity of the first spectral line of the nitroxide was used to obtain the absolute amount of radicals per tissue milligram after calibration of the spectrometer response with a standard solution of TEMPO-choline in water, using an artificial ruby crystal as internal standard. In order to evaluate the extent of oxidation of the hydroxylamine by atmospheric oxygen under our experimental conditions, a reference sample containing only the hydroxylamine in physiologic solution was prepared for each sample and treated in the same way (51).

Determination of ßCT and ROH levels in tissues
Extraction of ßCT and ROH. Tissue samples (300 mg) from 500 mg/kg ßCT-treated animals were homogenized in 4 ml of phospate-buffered solution (pH 7.4, 0.16 M NaCl). A sample (2 ml) of homogenate was mixed with 100 µl of internal standard (echinenone for ßCT and retinol acetate for ROH quantification), 1 ml of 0.1 M SDS and vortexed for 30 s. Ethanol (2 ml) was added and vortexed for 30 s. The mixture was extraxcted three times with 2 ml of n-hexane containing 0.025% butylated hydroxytoluene; each time it was vortexed for 2 min and then centrifuged at 4500 r.p.m. for 10 min. The n-hexane layers then were combined. After this, the n-hexane was removed under a stream of nitrogen, and 0.2 ml of mobile phase was added (52). The mixture was filtered and the solution obtained was used for HPLC analysis.

Chromatographic conditions. Chromatographic separations were performed using a system consisting of a high-pressure pump (Waters Model 600E, Multisolvent Delivery System), a sample injection valve (Rheodyne Model 7121, Cotati, CA, USA) with a 20 µl sample loop and a UV detector (Waters Model 486, Tunable Absorbance Detector) connected to an integrator (Millennium 2010, Chromatography Manager). For reversed-phase separation of metabolites, NOVA-PAK C18 analytical column (60 Å, 4 mm, 3.9x150 mm, Waters) was used as stationary phase. A guard column containing 4 µm C18 material similar to the analytical column was used to protect the column. The mobile phase for determination of ßCT consisted of a mixture (95:5) of methanol and tetrahydrofuran stabilized with butylated hydroxytoluene (0.1%). ßCT was separated at room temperature under isocratic conditions at a flow rate of 1.0 ml/min, and quantification was performed at {lambda} = 445 nm with respect to standard solution (53). For ROH, the mobile phase consisted of methanol/water (96:4) at a flow rate of 1.0 ml/min. Separation was performed at room temperature with {lambda} = 325 nm (54).

Statistics and computer analysis
Statistical analysis was performed using Wilcoxon's rank method as reported by Box and Hunter (55). The software used was Sigma Plot 5.0 and Windows 98® run on a Pentium II Celeron® IBM-compatible computer.


    Results
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 Materials and methods
 Results
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HPLC and fluorimetric-Vis analysis of microsomal monooxygenases in ßCT supplemented rats
Table IGo shows the hepatic mixed function microsomal monooxygenases measured either in control and ßCT supplemented animals using the following selected substrates as probes of different CYP isoenzymes: aminopyrine (preferential to CYP3A), p-nitrophenol (CYP2E1), ethoxycoumarin (mixed), methoxyresorufin (CYP1A2), ethoxyresorufin (CYP1A1) and pentoxyresorufin (CYP2B1). ßCT was given (per os) at 250 or 500 mg/kg body wt dose either in a single or repeated (daily for 5 consecutive days) fashion in both male and female rats. No differences in body- and organ-weights among different experimental groups were observed (data not shown).


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Table I. Effect of ßCT on rat liver microsomal mixed function oxydases
 
In the liver, the CYP2E1-linked pNPH activity was significantly (P < 0.01) increased mainly in male animals after 5 days of ßCT supplementation (2–3.6-fold increases, at 500 and 250 mg/kg body wt dose, respectively). PROD and EROD activities were induced to different extents in all situations up to ~7.6-fold in females (CYP1A1) and males (CYP2B1) in the repeated treatment. A modest increase of ECOD activity (~2-fold increase) was determined by ßCT treatment. Similarly, the CYP1A/2 associated activity was induced (up to 2.7-fold, at 250 mg/kg body wt, females) after a single ßCT dose. The regio- and stereo-selective metabolism of testosterone was used as multibiomarker of different CYPs. Table IIGo reports the effect of ßCT on TH activity in liver preparartions. The 16ß-(CYP2B1)-TH activity was induced by the pro-vitamin in all cases (ranging from ~4–11-fold, lower and higher dose, respectively, male animals) after 5 days of treatment. The 16{alpha}-(CYP2C11 and CYP2B1)-TH activity was also affected, the females being more responsive (up to 9-fold increase, at 500 mg/kg body wt ßCT, single dose). In the repeated treatment, other oxidases were significantly (P < 0.01) affected, including: 6{alpha}-TH activity (2.3–4.5-fold increase, at lower and higher dose, respectively, males); 7{alpha}-TH activity measuring CYP1A1/2 and CYP2A1 isoforms (up to 3.0-fold increase, higher dose, males); 2ß-TH activity associated to CYP3A1 and CYP1A1 (~2-fold increase, higher dose, both sexes); 6ß-TH activity testing CYP3A1 and CYP1A1/2 (up to 2-fold increase, higher dose, males); and androst-4-ene-3,17-dione linked monooxygenase (CYP3A1) activity (up to 2.6-fold increase, higher dose, females).


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Table II. TH in liver microsomes from ßCT supplemented rats
 
In the kidney, the CYP3A-linked N-demethylation of aminopyrine was significantly (P < 0.01) induced mainly in male animals (up to ~8-fold, higher dose, both treatments) (Table IIIGo). Hydroxylation of p-nitrophenol (linked to CYP2E1) was increased (up to 2-fold, at 500 mg/kg body wt dose, repeated administration) in females only. EROD activity was also affected by single ßCT supplementation up to ~7-fold increase (males, lower dose). The other microsomal monooxygenases were not substantially changed. The effects of ßCT on testosterone metabolism are reported in Table IVGo. The 2ß-TH activity (measuring CYP1A1) was induced in female animals only (up to ~4.3-fold, single treatment, both doses). The CYP2C11-supported 2{alpha}-TH activity was induced in males only (up to 5-fold, at 500 mg/kg body wt dose, single administration). Hydroxylations in the other testosterone positions were slightly affected by ßCT intake, and in some cases (e.g. multiple treatment), a reduction of the activity (6{alpha}-, 16ß- and 2{alpha}-THs as well as androst-4-ene-3,17-dione-linked monooxygenases) was recorded.


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Table III. Effect of ßCT on rat kidney microsomal mixed function oxydases
 

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Table IV. Testosterone hydroxylase in kidney microsomes from ßCT supplemented rats
 
In the lung, after 5 days at higher ßCT doses, the CYP1A1-associated ethoxyresorufin deethylation was the most affected isoform (up to ~9.0-fold increase, males) (Table VGo). Other induced monooxygenases are reported in Table VI: 7{alpha}- (up to ~3.5-fold increase, males, higher dose, CYP3A1/2, CYP2A1), 6ß- (up to ~4-fold increase, males, higher dose, CYP1A1/2, CYP3A) and 2ß- (up to ~15-fold increase, males, higher dose, CYP3A1 and CYP1A1)-TH activities, as well as androst-4-ene-3,17-dione-linked CYP3A1 activity (up to ~9-fold increase, males, higher dose) after 5 days ßCT supplementation.


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Table V. Effect of ßCT on rat lung microsomal mixed function oxydases
 
In the intestinal microsomes, both single and repeated ßCT administration increased the CYP2E1-linked hydroxylation of p-nitrophenol (up to 6.8-fold increase, males, five lower dose administrations) and CYP1A2-linked O-demethylation of methoxyresorufin (up to 3.2-fold increase, males, single higher dose) (Table VIIGo). Five days ßCT supplementation also induced the activities APND (up to 2.0-fold increase, males) and ECOD (up to 3.0-fold increase, males). Again, only multiple ßCT treatment was able to increase the metabolism of testosterone (Table VIIIGo). The various TH activities were induced to various extents: 6{alpha}- (up to 2.0-fold, males, lower dose), 7{alpha}- (up to 2.4-fold increase, males, higher dose), 16{alpha}- (up to ~3.1-fold increase, both sexes, higher dose), 16ß- (up to ~5.6-fold increase, females, lower dose) and 2ß- (up to 5.5-fold increase, females, higher dose). Finally, the androst-4-ene-3,17-dione supported monooxygenase activity was also affected (~2.7-fold increase, both sexes, higher dose) by 5 days ßCT supplementation.


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Table VII . Effect of ßCT on rat intestine microsomal mixed function oxydases
 

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Table VIII . Testosterone hydroyxlase in intestine microsomes from ßCT supplemented rats
 
Western blot analysis
The induction of the most affected isoenzymes in the various tissues considered was sustained by means of western immunoblotting analyses, using rabbit polyclonal antibodies antiCYP3A1/2, CYP2E1, CYP1A1/2 and CYP2B1/2 (liver); antiCYP3A1/2, CYP2E1 and CYP1A1/2 (kidney); anti-CYP3A1/2 and CYP1A1/2 (lung); anti-CYP2E1, CYP3A1/2 and CYP1A1/2 (intestine). Representative immunoblotting and related quantitative densitometric analyses of typical CYP signals are shown in Figures 1–4GoGoGoGo. The induced signals observable in the various situations and corresponding to microsomes achieved from 5 days ßCT supplemented male rats (Figure 1Go: liver, a–d, lane 2; kidney, a, lane 2, b and c, lane 3; lung, a, lane 3 and b, lane 2; intestine, a, lane 3, b, lane 1 and c, lane 2) indicate that the amount of the specific CYP apoprotein is enhanced by ßCT supplementation, due to increased transcription or enzyme stabilization. Densitometric analysis was shown on western blots.



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Fig. 1. (a) Western immunoblotting of male liver microsomes from positive controls (lane 3, pregnenolone 16{alpha}-carbonitrile, 100 mg/kg body wt in a single i.p. injection 24 h before being killed), negative controls (lane 1, untreated) and 500 mg/kg body wt ßCT daily for 5 days (lane 2). (b) Western immunoblotting of microsomes from positive controls (lane 1, ethanol, 15% v/v for 21 days), negative controls (lane 3, untreated) and 500 mg/kg body wt ßCT daily for 5 days (lane 2). (c) Western immunoblotting of microsomes from positive controls (lane 3, beta-naphthoflavone, 80 mg/kg in a single i.p. injection 48 h before being killed), negative controls (lane 1, untreated) and 500 mg/kg body wt ßCT daily for 5 days (lane 2). (d) Western immunoblotting of microsomes from positive controls (lane 1, sodium phenobarbital, 80 mg/kg in a single i.p. injection 24 h before being killed), negative controls (lane 2, untreated) and 500 mg/kg body wt ßCT daily for 5 days (lane 3). Twenty-five micrograms of microsomal protein was applied to the wells and probed with anti-CYP3A1/2 (a), CYP2E1 (b), CYP1A1/2 (c) and CYP2B1/2 (d). Densitometric analysis was performed on western blots; numerical values are expressed as a percentage of the negative (control) group mean.

 


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Fig. 2. (a) Western immunoblotting of male kidney microsomes from positive controls (lane 3, pregnenolone 16{alpha}-carbonitrile, 100 mg/kg body wt in a single i.p. injection 24 h before being killed), negative controls (lane 1, untreated) and 500 mg/kg body wt ßCT daily for 5 days (lane 2). (b) Western immunoblotting of microsomes from positive controls (lane 2, ethanol, 15% v/v for 21 days), negative controls (lane 1, untreated) and 500 mg/kg body wt ßCT daily for 5 days (lane 3). (c) Western immunoblotting of microsomes from positive controls (lane 2, beta-naphthoflavone, 80 mg/kg in a single i.p. injection 48 h before being killed), negative controls (lane 1, untreated) and 500 mg/kg body wt ßCT daily for 5 days (lane 3). Twenty-five micrograms of microsomal protein was applied to the wells and probed with anti-CYP3A1/2 (a), CYP2E1 (b) and CYP1A1/2 (c). Densitometric analysis was performed on western blots; numerical values are expressed as a percentage of the negative (control) group mean.

 


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Fig. 3. (a) Western immunoblotting of male lung microsomes from positive controls (lane 2, beta-naphthoflavone, 80 mg/kg in a single i.p. injection 48 h before being killed), negative controls (lane 1, untreated) and 500 mg/kg body wt ßCT daily for 5 days (lane 3). (b) Western immunoblotting of microsomes from positive controls (lane 3, pregnenolone 16{alpha}-carbonitrile, 100 mg/kg body wt in a single i.p. injection 24 h before being killed), negative controls (lane 1, untreated) and 500 mg/kg body wt ßCT daily for 5 days (lane 2). Twenty-five micrograms of microsomal protein was applied to the wells and probed with anti-CYP1A1/2 (a) and CYP3A1/2 (b). Densitometric analysis was performed on western blots; numerical values are expressed as a percentage of the negative (control) group mean.

 


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Fig. 4. (a) Western immunoblotting of male intestine microsomes from positive controls (lane 2, pregnenolone 16{alpha}-carbonitrile, 100 mg/kg body wt in a single i.p. injection 24 h before being killed), negative controls (lane 1, untreated) and 500 mg/kg body wt ßCT daily for 5 days (lane 3). (b) Western immunoblotting of microsomes from positive controls (lane 3, beta-naphthoflavone, 80 mg/kg in a single i.p. injection 48 h before being killed), negative controls (lane 2, untreated) and 500 mg/kg body wt ßCT daily for 5 days (lane 1). (c) Western immunoblotting of microsomes from positive controls (lane 3, ethanol, 15% v/v for 21 days), negative controls (lane 1, untreated) and 500 mg/kg body wt ßCT daily for 5 days (lane 2). Twenty-five micrograms of microsomal protein was applied to the wells and probed with anti-CYP3A1/2 (a), CYP1A1/2 (b) and CYP2E1 (c). Densitometric analysis was performed on western blots; numerical values are expressed as a percentage of the negative (control) group mean.

 
EPR measurements
Samples prepared by incubating ~1 mg of microsomal proteins for 1–10 min at 37°C in 0.01 M Na+/K+ phosphate buffer (pH 7.4) containing 1 mM NADPH and 0.5 mM hydroxylamine probe were analyzed by EPR spectroscopy. An intense three-line EPR spectrum attributed to the corresponding nitroxide on the basis of its spectral parameters (aN = 15.52 G, g = 2.0062) was observed. In Table IXGo, a marked and significant (P < 0.01) increase of ROS generation can be observed in all tissues considered, the liver being the most susceptible to ßCT supplementation (up to 33-fold increase with respect to controls, at the higher ßCT dose). The ranking of the relative ROS production was: liver > lung > intestine > kidney. Experiments performed in the presence of SOD (1000 U/mg) or SOD and catalase (for each 1000 U/mg) show that the formation of the nitroxide was strongly impaired, indicating that superoxide is mainly responsible for the oxidation of the hydroxylamine probe. Thus, under our experimental conditions, these enzymes reduced the amount of nitroxide detected to ~10% of the original value. However, when the microincubations were performed in the presence of SOD, which had been denatured previously by thermal shock, the EPR signal recorded ranged from 15 to 25% of that achieved in the absence of the enzyme under the same conditions. This indicates that the role of this enzyme consists mostly of a general non-specific radical scavenging activity, presumably due to the many -SH groups present in the protein itself (data not shown).


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Table IX. ROS levels in lung, liver, kidney and intestine microsomes from ßCT supplemented rats
 
ßCT and ROH contents
After administration of 500 mg/kg body wt ßCT daily for 5 consecutive days, rats showed accumulation of various amounts of ßCT in all organs, the liver being the most affected. In contrast, the ROH concentration, as determined at the same time points, was comparable with that of the control rats (Table XGo).


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Table X. Levels of ßCT and retinol in different tissues of ßCT supplemented rats
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We report here the effects of single or repeated ßCT supplementation on the microsomal mixed function monooxygenase system of rat liver, kidney, lung and intestine in both sexes. Both testosterone (as multibioprobe) and individual specific substrates to different CYPs were used. EPR spectroscopy coupled with the spin-probe technique was also employed to measure ROS production by various subcellular preparations.

ßCT supplementation determining a substantial accumulation of ßCT itself in the considered organs, was able to induce a number of CYP isoforms in all tissues. The most affected were: CYP3A1/2, CYP2E1, CYP1A1/2 and CYP2B1/2 in the liver; CYP3A1/2, CYP2E1 and CYP1A1/2 in the kidney; CYP1A1/2 and CYP3A1/2 in the lung; and CYP3A1/2, CYP1A1/2 and CYP2E1 in the intestine. These findings were sustained by means of western blot, and the different intensities of the recorded signals were supported by densitometric analyses. Some sex differences were observed. The males were more responsive to ßCT in the lung (e.g. for CYP1A1-linked deethylation of ethoxyresorufin, androst-4-ene-3,17-dione – CYP3A1 – associated monooxygenases or CYP1A1/CYP3A1-supported testosterone 6ß-hydroxylases), whereas females were more responsive in the kidney (e.g. for CYP2E1-supported hydroxylation of p-nitrophenol, CYP2A1/CYP1A1/2-linked testosterone 7ß-hydroxylases or CYP3A1/2- CYP1A1/2-testosterone 2ß-hydroxylases).

The variations in CYP expression among the tissues could imply the existence either of different induction mechanisms, or of alterations in competing pathways involved in the disposition of compounds or metabolites, or of differences in substrate specificity. Metabolic differences in different tissues could depend on `altered' substrate specificity of the isoforms involved, along with the contribution of other CYPs in that substrate. Gender-dependent differences in enzyme induction (and to a lesser extent suppression) could be explained in terms of quantitative or qualitative differences in CYP isoenzymes under the control of sex hormones (56).

The recorded CYP induction is consistent with the concept that ßCT has co-carcinogenic potential. This may be of particular concern in the lungs of heavy smokers. Indeed, if extrapolated to humans, similar increases in CYP levels could raise the risk of lung cancer in heavy smokers, due to the immense range of tobacco-smoke pro-carcinogens (a complex mixture of more than 4000 substances, among which at least 40 have been identified as carcinogens, tumor initiators or promoters in laboratory animals) (57). In addition, many tobacco-smoke pro-carcinogens are themselves CYP inducers, and they could act in a synergic way with ßCT, thereby further contributing to the overall carcinogenic risk (58). Indeed, ferrets given high ßCT supplements and exposed to tobacco smoke had diminished retinoid signaling, resulting from the suppression of retinoic acid receptor (RAR) ß gene expression and over-expression of activator protein-1 (encoded by the c-jun and c-fos genes) (59). Increased bioactivation of pro-carcinogens to final carcinogens could facilitate lung tumorigenesis by saturating the DNA repair mechanisms and thus altering tumor suppressor genes.

EPR spectroscopy coupled with the spin-probe technique allowed us to demonstrate that the subcellular preparations from the various tissues of ßCT-supplemented rats generated large amounts of ROS. The ranking of relative ROS production was: liver > lung > intestine > kidney. The amounts of both ROS and induced CYPs in the various tissues can be explained by the accumulation of ßCT recorded in the tissues, the liver being the most susceptible organ. Considering the dosage and exposure time, the levels of ßCT observed by us were comparable with those reported elsewhere in the literature (29,60). The contribution of retinol in the CYP upregulation seems to be marginal, as the amount of vitamin A found in the various tissues was comparable with that of controls (rodent chow contains 14 000 UI/kg retinol anyway). This is not surprising, as it has been shown that carotenoids are not converted into retinol unless the need for retinol exists (61) Moreover, no strict link between the ßCT dose and its effects in different tissue types should necessarily be expected, as different induction mechanisms (for the same CYP and inducer) may exist in the various organs (62,63). The existence of NADPH-dependent production of ROS (O2·, H2O2 and HO·) by animal liver microsomes has been known for 40 years (64) and has been linked to CYP (65). Uncoupling of electron transfer and oxygen reduction from monooxygenation by CYP2B1 and CYP2E1 can result in the release of O2· and H2O2 (65,66). In 1996, we demonstrated that this is a general phenomenon involving a number of isoforms: using murine microsomes enriched in the major CYP subfamilies (including CYP2B1 and CYP2E1) such as CYP1A1, CYP1A2, CYP3A or CYP4A, we found that all the different CYPs were able to generate high amounts of ROS (47). This observation was confirmed by a recent study reporting NADPH-stimulated release of ROS by subcellular preparations enriched in specific human CYPs (67). Thus, it seems reasonable to suppose that all induced CYP isoforms could contribute to the observed increases in the amount of ROS found in microsomes from different tissues following ßCT supplementation. Furthermore, the increases in ROS could conceivably be related to the pro-oxidant effect of ßCT: the unfaltering oxidative stress derived from CYP induction per se could act synergistically with that caused by cigarette smoke containing peroxyl radicals, nitrogen dioxide, hydroquinones, etc. (13,14).

The discovery that ßCT supplementation can actually be inversely linked to the prevention of cancer in humans has prompted much speculation. One of the most interesting hypotheses is that while ßCT may itself act as an anticarcinogen (on the basis of known antioxidant properties), its oxidized products, which are particularly high in the lung fluids of smokers, can facilitate carcinogenesis (68). Our findings are in keeping with this concept, since the oxidative stress produced by ROS over-generation following ßCT supplementation could contribute, in synergy, with cigarette smoke to reduce the levels of unoxidized ßCT (i.e. the protective form of ßCT) and to increase the mixture of oxidation products (68). Interestingly, the antioxidant properties of ßCT are strictly dependent on oxygen partial pressure (OPP). In vitro experiments at different OPP, have unambiguously demonstrated that ßCT behaves as an antioxidant at OPP that are significantly less than the oxygen pressure found in normal air (69). At higher OPP, ßCT was found to lose its antioxidant activity and actually shows a pro-oxidant effect. This unusual behaviour has been explained in terms of the high reactivity of ßCT towards peroxy radicals (equation 1) and, to the reversibility of the reaction between the ßCT radical and molecular oxygen (equation 2).


(The formation of the ßCT· radical could depend either on the radical adduct of the peroxy radical from the ROO· oxidizable substrate to ßCT or on hydrogen abstraction from ßCT.)






At high OPP, the equilibrium of equation 2 shifts to the right, i.e. towards the peroxyl radical ßCTOO· which propagates the autoxidative chain (equation 3). At low OPP, on the other hand, the equilibrium shifts towards the ßCT· radical, which behaves as a trap for ROO· (equation 4) and can, thus, interrupt the radical chain reaction. This could explain the ability of ßCT to behave as an antioxidant or pro-oxidant depending on the OPP. A number of reports have now confirmed this phenomenon in purified systems (70), microsomes (68), cell lines (71) and bacteria (72). The pro-oxidant effects of ßCT have also been found to increase with higher concentrations and longer reaction times (ßCT is thus slightly autocatalytic) (73). The OPP of most normal tissues is lower than that of atmospheric air, whereas the cells lining the outer surface of the lung are exposed to considerably higher OPP. This might make these cells especially subject to the pro-oxidant effect of ßCT.

Another possible explanation that has been advanced for the surprising clinical findings regarding ßCT supplementation springs from the observation that many of the individuals who developed lung cancer in these trials were also heavy consumers of alcohol (74), which is itself an inducer of carcinogen metabolizing enzymes (75). Alcohol and ßCT may, thus, act synergistically in boosting phase I reactions (76). In addition, vitamin A and/or ßCT could potentiate the hepatotoxicity of ethanol, and vice versa (76,77).

More generally, oxidative stress is thought to play a major contributory role in the pathogenesis of many degenerative or chronic diseases including cancer, but it should not be forgotten that many other determinants (e.g. environmental carcinogens, genetic predisposition, virus) also play pivotal roles in the development of these diseases. It could, thus, be naive to think that cancer incidence can simply be controlled by dietary supplementation of antioxidant vitamins, even though the radical-trapping ability of ßCT (and probably of other carotenoids) may theoretically suggest a potential role in the prevention or reduction of cancer incidence in humans (7886). The intervention trials indicating that straightforward ßCT supplements are at best of limited value and may even be deleterious have tempered some of the premature enthusiasm for mass chemopreventive micronutrient supplementation. Bearing in mind the limitation of the rat model, the results of the present study may help deepen our understanding of the causes behind the harmful outcomes associated with ßCT supplementation. In humans, corresponding increases in CYP isoforms (co-carcinogenesis) and in ROS levels (which can act at all levels of the multi-step carcinogenesis process) could both increase cancer susceptibility, particularly in heavy smokers. These findings may, thus, be of relevance to public health policy and should be considered as further evidence of the potentially harmful effects of recommending supplementations with these micronutrients on a large scale.


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Table VI. TH in lung microsomes from ßCT supplemented rats
 

    Notes
 
4 To whom correspondence should be addressedEmail: paolini{at}biocfarm.unibo.it Back


    Acknowledgments
 
This work was supported by the University of Bologna and MURST (Ministry of the University and of Technological and Scientific Research) 40 and 60% grants. We thank Dr Emanuela Marchesi for her excellent technical assistance. Denisa Spetlova's stay in our Labs was made possible by grant from European Union's Erasmus Program. We are grateful to Robin M.T.Cooke for editing.


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 Top
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 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received September 18, 2000; revised April 9, 2001; accepted May 22, 2001.