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Nutrition and Cancer Biology Laboratory, Jean Mayer U.S. Department of Agriculture Human Nutrition Research Center on Aging at Tufts University, Boston, MA 02111
3To whom correspondence should be addressed. E-mail: xwang{at}hnrc.Tufts.edu
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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80%) inhibited by nonspecific CYP inhibitors (disulfiram and liarozole), but was partially (50%) inhibited by resveratrol (CYP1A1 inhibitor), 
-naphthoflavone (CYP1A2 inhibitor) and antibodies against CYP1A1 and CYP1A2. Cigarette smoke exposure and/or pharmacological doses of ß-carotene increased levels of CYP1A1 and 1A2 by three- to sixfold but not levels of 2E1 and 3A1 in ferret lung tissue. These findings suggest that low levels of RA in the lung of ferrets exposed to cigarette smoke and/or pharmacological doses of ß-carotene may be caused by the enhanced RA catabolism via induction of CYP, CYP1A1 and CYP1A2 in particular, which provides a possible explanation for enhanced lung carcinogenesis seen with pharmacological doses of ß-carotene supplementation in cigarette smokers.
KEY WORDS: • cigarette smoke • ß-carotene • cytochrome P450 • retinoic acid
Beneficial effects of fruits and vegetables rich in ß-carotene on risk reduction of lung cancer have been found in a number of observational epidemiological studies (1
–3
). In contrast, three large randomized clinical trials conducted to determine the effect of ß-carotene supplementation on the incidence of lung cancer indicated either no beneficial effect among apparently healthy men (4
) or a possible harmful effect among smokers or asbestos workers (5
–7
). Our recent studies, conducted in ferrets, indicate that the harmful effects of ß-carotene supplementation in smokers is associated with the dosage used at a pharmacological, but not a physiological, dose (8
,9
). One of the potential mechanisms for the results in the ferret study is that the presentation of high doses of ß-carotene via supplements to the highly oxidative environment of the lung in smokers increases the levels of oxidative metabolites of ß-carotene that may have detrimental effects (10
).
Retinoic acid (RA4), a modulator of cell proliferation and differentiation in lung epithelial cells (11
), suppresses carcinogenesis in certain epithelial tissues (12
). In our previous in vivo animal studies, we found that cigarette smoke exposure, a pharmacological dose of ß-carotene or their combined treatment for 6 mo significantly lowers RA concentrations in lung tissue of ferrets and diminishes retinoid signaling, while increasing gene expression of activating protein 1 (c-Jun and c-Fos) and cyclin D1, as well as cell proliferation (8
,9
). However, the mechanism of decreased RA concentrations in the lung as a result of the effects of smoke exposure and ß-carotene supplementation is unclear.
Cytochrome p450 enzymes (CYP) have been identified as functional enzymes in the lung and other organs, where they are inducible, and convert carcinogens present in tobacco smoke into DNA reactive metabolites (13
–15
). We proposed that the CYP induced by cigarette smoke exposure and pharmacological doses of ß-carotene could be responsible for the enhanced RA catabolism in the lung. A number of studies have demonstrated that CYP are involved in RA metabolism in many organs (16
–30
). Recent studies have confirmed the major involvement of CYP1A1, CYP1A2 and CYP3A in the oxidation of RA (26
,27
,29
,30
). Cigarette smoke has been shown to induce CYP1A1, CYP1A2, CYP2B and CYP2E1 in the lung (31
–36
); and CYP1A1, CYP1A2, CYP2B, CYP2C, CYP3A and CYP2E1 in the liver (34
,37
,38
). Several human studies have revealed the presence of CYP1A1, CYP2B6, CYP2B7, CYP2E1 and CYP3A5 in the lungs of smokers (14
) as well as the induction of CYP1A2 activity by smoking (39
). A recent study has shown that pharmacological doses of ß-carotene induced CYP1A1, CYP1A2, CYP3A1, CYP3A2, CYP2B1 and CYP2A in the lung; CYP1A1, CYP1A2, CYP3A1, CYP3A2, CYP2E1, CYP2B1 and CYP2B2 in the liver; and CYP1A1, CYP1A2, CYP3A1, CYP3A2 and CYP2E1 in the intestines of rats (40
,41
). ß-Apo-8'-carotenal, an excentric cleavage product of ß-carotene, has also been shown to induce CYP1A1 and CYP1A2 substantially (42
), which is corroborated by the results from our previous studies that the formation of ß-apo-8'-carotenal was 2.5-fold higher in lung extracts from smoke-exposed ferrets when incubated with ß-carotene than from nonsmoke-exposed ferrets (8
). Therefore, it is possible that induction of CYP by either ß-carotene oxidative cleavage products or cigarette smoke in lung tissue may have two detrimental actions: 1) bioactivating carcinogens and 2) destroying RA, thereby enhancing lung carcinogenesis.
In the present study, we investigated whether CYP were induced by smoke exposure and/or pharmacological doses of ß-carotene by use of Western blot analysis. Through the use of an in vitro incubation method, we conducted experiments to determine whether CYP participated in RA catabolism in the lung microsomal fractions more from smoke-exposed, ß-carotene-supplemented or smoke-exposed plus ß-carotene supplemented ferrets, compared with the controls.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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All-trans-RA, dithiothreitol, dimethyl sulfoxide, HEPES, NADPH, EDTA, resveratrol, disulfiram and retinyl acetate were purchased from Sigma Chemical (St. Louis, MO). Alpha-naphthoflavone was purchased from Aldrich (Milwaukee, WI). 4-Oxo-RA and 18-hydroxy-RA were kindly supplied by J. Sepinwall (Hoffmann-La Roche, Nutley, NJ). Liarozole was a gift from the Janssen Pharmaceutical Research Institute. Chlormethiazole was obtained from Astra Arcus (Sodertalje, Sweden). All-trans-RA was dissolved in ethanol for purification by HPLC. The peak containing RA was collected for use as a substrate. The purity of RA was 99.9%. All HPLC solvents were obtained from J. T. Baker Chemical (Philipsburg, NJ) and were filtered through a 0.45-µm membrane filter before use.
Animals, diet and study groups.
The maintenance and husbandry of ferrets were described in our previous papers (8
,9
). Twenty-four male ferrets from Marshall Farms (North Rose, NY) were randomly assigned to four groups of six animals for 6 mo as follows: 1) control; 2) cigarette smoke exposure; 3) a ß-carotene supplementation [per unit body weight, 2.4 mg/(kg·d)]; 4) cigarette smoke exposure plus ß-carotene supplementation. Ferrets were fed dry ferret food (Purina Ferret Chow, Ralston Purina, St. Louis, MO). After the 6-mo experimental period, all ferrets were killed by exsanguination from the heart under deep isoflurane anesthesia. Lung tissues collected from these ferrets were used in this current study.
Cigarette smoke exposure.
The procedure of cigarette smoke exposure in ferrets was described in our previous publications (8
,9
). The amount of smoke exposure in the ferret is similar to that found in humans smoking one and a half packages of cigarettes per day according to the concentrations of urinary cotinine equivalents (8
). The nonsmoke-exposed ferrets were housed in a separate room and underwent the exact same procedures as the smoke-exposed animals, except that they received no smoke exposure.
ß-Carotene preparation and supplementation.
The procedure of ß-carotene supplementation in ferrets was described in our previous publications (8
,9
). The average intake of ß-carotene from the basal diet during the experimental period per unit body weight was 0.16 mg/(kg·d). Because the total absorption of ß-carotene by ferrets is about 20% that in humans (43
), the ß-carotene intake from the diet per unit body weight was about 0.03 mg/(kg·d), which is equivalent to 1.8–2.1 mg of ß-carotene/d intake in a 60–70 kg person. The ß-carotene-supplemented group received, per unit body weight, 2.4 mg ß-carotene/kg·d, including the ß-carotene in the basal diet. Similarly, because the total absorption of ß-carotene by ferrets is about 20% that in humans, the actual ß-carotene intake per unit body weight was about 0.48 mg/(kg·d), which is approximately equivalent to 28.8–33.6 mg of ß-carotene/d intake in a 60–70 kg person, a dose used in smokers of the human intervention trials (7
).
Preparation of microsomal fraction from lung tissue.
The microsomal fraction of lung tissue was prepared at 4°C by differential centrifugation according to the procedures described previously, with minor modification (44
–46
). Briefly, lung tissue was homogenized (weight:volume = 1:4) in a Brinkmann Polytron homogenizer (Westbury, NY) in buffer [Hepes, 10 mmol/L; EDTA, 1 mmol/L; DTT, 2 mmol/L; phenylmethylsulfonyl fluoride, 1 mmol/L; leupeptin, 0.5 mg/L; and aprotinin, 0.5 mg/L (pH 7.35)]. The homogenate was centrifuged in a Sorval RT6000 refrigerated centrifuge (Du Pont, Newtown, CT) at 800 x g at 4°C for 30 min. The collected supernatant fractions were centrifuged at 28,000 x g for 15 min, and the resulting supernatant fractions were centrifuged again at 105,000 x g for 1 h. A microsomal pellet was obtained after rehomogenization in buffer and centrifugation for 1 h at 105,000 x g. The microsomes were stored at -80°C.
Incubation and extraction procedure.
To study whether the catabolism of RA is related to CYP induced by cigarette smoke exposure and/or pharmacological doses of ß-carotene, incubations were done using lung microsomal fractions for three ferrets in each group and RA concentrations (2 to 10 µmol/L) with or without CYP enzyme inhibitors and CYP antibodies against CYP1A1, CYP1A2, CYP3A1 and CYP2E1, as described in previous studies, with minor modification (46
–48
). RA concentrations used in the present study were within the range of the previous studies (27
,29
,30
,46
–48
), which allowed us to detect sufficient amounts of RA metabolites for evaluation of RA catabolism.
Briefly, 1 mg of microsomal protein was incubated under red light with the following additions in glass vials at 37°C in a shaking water bath for a 3-min preincubation: buffer [Hepes, 20 mmol/L; KCl, 150 mmol/L; MgCl2, 5 mmol/L; EDTA, 1 mmol/L (pH, 7.35)] and substrate (RA dissolved in dimethyl sulfoxide, 10 µL) in a final volume of 1 mL. The incubation mixtures were preincubated with either CYP antibodies or CYP inhibitors [resveratrol for CYP1A1 (49
–51
), -naphthoflavone for CYP1A2 (52
), chlormethiazole for CYP2E1 (53
,54
), as well as liarozole (55
) and disulfiram (56
), two nonspecific CYP inhibitors] for 10 min before the incubation. After preincubation, the reaction was started by adding NADPH (1 mmol/L) and the mixtures were incubated for 10 min at 37°C. Two control vials were run lacking either substrate or microsomes. The vials were uncovered and the incubation mixtures were exposed to room air as the gas phase.
After incubation, RA and its polar metabolites were extracted and analyzed by the method described previously with minor modification (45
,46
,57
). Briefly, 50 µL of an ethanolic solution of KOH (1 mol/L), 200 µL of ethanol and 0.5 mL of H2O were added to 1.0 mL of incubation mixture, followed by the addition of the internal standard, retinyl acetate, in 50 µL of ethanol. The mixture was extracted by adding 3 mL of hexane, vortexing, and then centrifuging for 10 min at 800 x g at 4°C. The hexane layer was removed. The residue was then acidified by adding 100 µL of HCl (6 mol/L). A second extraction was performed with 3 mL of hexane. The two extractions were pooled, dried under N2 and resuspended in 50 µL of ethanol for injection into the HPLC system as described below.
HPLC analysis for 4-oxo-RA, 18-hydroxy-RA and RA.
A gradient reverse phase HPLC was used for the analysis of RA and its polar metabolites as described previously, with minor modification (46
). Briefly, the gradient reverse phase HPLC system consisted of a Waters 616 Pump (Waters Chromatography Division/Millipore, Medford, MA), a Waters 600s controller, a Waters 717 Plus autosampler, a Waters 996 photodiode array detector, a Vydac 201TP54 C18 4.6 x 25-cm reverse phase column (Vydac, Hesperia, CA) and a Waters 840-Digital 350 data station. The HPLC mobile phase was CH3OH:H2O (75:25, v/v, 1% ammonium acetate in H2O, solvent A) and CH3OH (solvent B). The gradient procedure (at a flow rate of 1.3 mL/min) was as follows: 100% solvent A was used for 8 min followed by a 10-min linear gradient to 20% solvent A and 80% solvent B, then a 10-min linear gradient to 100% solvent B, then 100% solvent B kept for 5 min, then a 2-min gradient back to 100% solvent A. In this HPLC system, 4-oxo-RA, 18-hydroxy-RA, RA and retinyl acetate (internal standard) eluted at 4.5, 5.6, 17.2 and 24.1 min, respectively. Individual retinoids (4-oxo-RA, 18-hydroxy-RA, RA) were identified by coelution with standards and absorption spectrum analysis. Retinoids were quantified relative to the internal standard (retinyl acetate), by determining peak areas calibrated against known amounts of standards.
Western blot analysis.
Western blot analysis was carried out using the microsomal fraction of lung tissue from six ferrets in each group as described previously (46
). Briefly, 20 µg of microsomal protein was added to a solution containing HEPES, 50 mmol/L (pH 7.4); NaCl, 150 mmol/L; MgCl2, 1.5 mmol/L; EDTA, 5 mmol/L; 1% Triton X-100; 10% glycerol; and a mixture of protease inhibitors, and boiled for 6 min before being run on 10% SDS-PAGE. Protein concentrations were determined using a Coomassie Plus protein assay kit (Pierce, Rockford, IL). The protein was transferred to nitrocellulose membranes, which were then blocked overnight in 5% nonfat milk in Tris-buffered saline containing 0.05% Tween 20 (TBS-T). Antibody against CYP was diluted 1:1000 in TBS-T, and membranes were incubated at room temperature for 1 h. The blots were rinsed and washed five times in TBS-T for 30 min, and then incubated at room temperature for 30 min in TBS-T containing a 1:5000 dilution of the horseradish peroxidase-labeled secondary antibody (Bio-Rad, Hercules, CA). After the final wash with TBS-T, the blots were developed using the ECL Western blotting system (Amersham, Arlington Heights, IL) and analyzed by densitometry (Model 710; Bio-Rad).
Antibodies against CYP1A1 and CYP1A2 were purchased from Affinity BioReagents (Golden, CO). Antibodies against CYP2E1 and CYP3A1 were purchased from Chemicon International (Temecula, CA).
Statistical analysis.
Results are expressed as means ± SD and data were analyzed by one- or two-way ANOVA followed by Tukey’s Honest test. Differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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4-Oxo-RA and 18-hydroxy-RA were produced after incubation of RA with lung microsomal fractions of control ferrets, ferrets exposed to smoke exposure, ferrets supplemented with a pharmacological dose of ß-carotene and ferrets with the combined treatments (Fig. 1
). The retention time (RT) of the 4-oxo-RA (RT = 4.5 min) and 18-hydroxy-RA (RT = 5.6 min) matched exactly the RT of the authentic standards of 4-oxo-RA and 18-hydroxy-RA. The absorption maximum of the 4-oxo-RA was 358 nm, which matched that of the authentic standard of 4-oxo-RA, whereas the 18-hydroxy-RA showed an absorption maximum of 338 nm, which matched that of the authentic standard of 18-hydroxy-RA. Neither 4-oxo-RA nor 18-hydroxy-RA was detected in two additional control incubations done without RA or without lung microsomal fractions (data not shown).
|
. Among the three treatment groups, the production of 4-oxo-RA and 18-hydroxy-RA was the highest in ferrets exposed to both cigarette smoke and ß-carotene, intermediate in ferrets exposed to cigarette smoke alone and, although not significantly different from those exposed to cigarette smoke, numerically lower in ferrets supplemented with ß-carotene alone (Fig. 1)
. The differences in production of 4-oxo-RA and 18-hydroxy-RA between ferrets exposed to both cigarette smoke and ß-carotene and those exposed to either cigarette smoke or ß-carotene were significant at all RA doses, except for 2 µmol/L. In addition, the production of 4-oxo-RA and 18-hydroxy-RA from RA increased significantly with increasing concentrations of RA in a dose-dependent manner up to 5 µmol/L within each group (Fig. 1)
. Expression of CYP1A1, CYP1A2, CYP3A1 and CYP2E1 in ferret lung microsomes.
The protein levels of CYP1A1 and CYP1A2 were significantly higher (three- to sixfold) in the lung tissue of ferrets exposed to cigarette smoke, a pharmacological dose of ß-carotene or the combination compared with those in control ferrets (Fig. 2
). However, the amounts of CYP3A1 and CYP2E1 in the three treatment groups were not different from those of the control ferrets (Fig. 2)
.
|
Liarozole (nonspecific CYP inhibitor) significantly inhibited the production of 4-oxo-RA and 18-hydroxy-RA from RA in the three treatment groups (Fig. 3
). Five different doses of liarozole (1, 5, 10, 20 and 50 µmol/L) were used to further evaluate whether the inhibition of production of 4-oxo-RA and 18-hydroxy-RA from RA by liarozole is dose dependent (Fig. 4
). In smoke-exposed ferrets (Fig. 4
, top panels) and ß-carotene-supplemented ferrets (Fig. 4
, bottom panels), significant dose-dependent reductions in the formation of 4-oxo-RA and 18-hydroxy-RA from RA were observed up to doses of 10 µmol/L.
|
|
in the lung tissue of ferrets exposed to cigarette smoke or pharmacological doses of ß-carotene is involved in RA catabolism, we used resveratrol (CYP1A1 inhibitor) and -naphthoflavone (CYP1A2 inhibitor) in our incubation experiments. In contrast to the effect of nonspecific CYP inhibitors [liarozole and disulfiram, which caused a nearly 80% reduction in the oxidation of RA into 4-oxo-RA and 18-hydroxy-RA in both smoke-exposed ferrets (Table 1
) and ß-carotene-supplemented ferrets (Table 2
)], resveratrol or -naphthoflavone resulted in a nearly 50% reduction in the oxidation of RA into 4-oxo-RA and 18-hydroxy-RA in both smoke-exposed (Table 1)
and ß-carotene-supplemented ferrets (Table 2)
. Similar to the effects of the specific inhibitors, pretreatment with CYP1A1 or CYP1A2 antibodies in the incubation mixture using lung microsomes from either smoke-exposed (Table 1)
or ß-carotene-supplemented (Table 2)
ferrets also reduced the formation of 4-oxo-RA and 18-hydroxy-RA from RA. However, preincubation with chlormethiazole (CYP2E1 inhibitor) or antibodies against CYP2E1 or CYP3A1 with lung microsomes from either smoke-exposed or ß-carotene-supplemented ferrets did not inhibit the formation of 4-oxo-RA and 18-hydroxy-RA from RA (Tables 1
and 2)
.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
. Elevated expressions of CYP1A1 and CYP1A2 in the lungs exposed to cigarette smoke in our study are similar to previous reports of smoke exposure on CYP induction (35
,36
,39
). Consistent with a study conduced in mice exposed to smoke (35
), we also observed no increase in the expression of CYP3A1 in smoke-exposed ferrets. However, the elevated expression of CYP2E1, observed in mice exposed to smoke by others (34
,35
), was not evident in our study. This could be attributable to the differences in animal species or smoking models that were used in these studies.
The observation of increased expressions of CYP1A1 and CYP1A2 induced by pharmacological doses of ß-carotene in our study is consistent with the findings from Paolini et al. (40
,41
). However, we observed no elevation in CYP3A1 expression reported by Paolini et al. (40
,41
). This could be explained by the different doses and duration of ß-carotene treatment used and/or different animal species. In the present study, we gave ferrets a pharmacological dose of ß-carotene [per unit body weight, 2.4 mg/(kg·d)], equivalent to 30 mg/d for a 70 kg person for 6 mo, which mimics the ß-carotene supplementation levels used in human clinical trials (4
,5
,7
). However, Paolini et al. (40
,41
) fed rats extremely high doses of ß-carotene [per unit body weight, 250 or 500 mg/(kg·d)] in a single or repeated (5 consecutive days) dose.
More important, we established links between CYP induction and RA catabolism by cigarette smoke exposure and/or pharmacological doses of ß-carotene supplementation in lung. Consistent with previous findings (16
,46
,47
), we confirmed 4-oxo-RA and 18-hydroxy-RA as the principal metabolites of RA after incubation of RA with lung microsomal fractions from smoke-exposed ferrets and/or ß-carotene-supplemented ferrets. We did not detect 4-hydroxy-RA because 4-hydroxy-RA is a transient metabolite in the pathway of oxidation of RA to 4-oxo-RA (47
). We observed that the production of 4-oxo-RA and 18-hydroxy-RA was increased substantially (seven- to 10-fold) in a dose-dependent manner after incubation of RA with lung microsomes from ferrets exposed to cigarette smoke, a pharmacological dose of ß-carotene or the combination (Fig. 1)
. Furthermore, the production of 4-oxo-RA and 18-hydroxy-RA was significantly inhibited (80%) by adding two nonspecific CYP inhibitors, liarozole (55
) and disulfiram (56
) (Tables 1
and 2)
. Using the inhibitory property of resveratrol on CYP1A1 (49
–51
) and -naphthoflavone on CYP1A2 (52
) and antibodies against CYP1A1 or CYP1A2, we observed a nearly 50% reduction in the formation of 4-oxo-RA and 18-hydroxy-RA (Tables 1
and 2)
. These findings are consistent with the results from the Western blot analysis that the expressions of CYP1A1 and CYP1A2 were markedly elevated in lung tissue of ferrets exposed to cigarette smoke and pharmacological dose of ß-carotene than those in control ferrets.
In the present study, CYP1A1 and CYP1A2, not CYP2E1 and CYP3A, are the major CYP induced by cigarette smoke or ß-carotene supplementation. In addition, we observed no inhibitory effect on the formation of 4-oxo-RA and 18-hydroxy-RA by addition of chlormethiazole, an inhibitor of CYP2E1 (53
,54
), and antibodies against CYP2E1 or CYP3A1. These results strongly indicate that cigarette smoke and a pharmacological dose of ß-carotene can enhance the oxidation of RA into polar metabolites through induction of CYP1A1 and CYP1A2 in the lung. Because neither specific CYP inhibitors nor nonspecific CYP inhibitors completely inhibited the enhanced RA catabolism arising from smoke exposure or pharmacological doses of ß-carotene, other CYP enzymes also might be involved in RA metabolism, such as CYP26 (23
), CPYf (18
) and CYP2C8 (19
,25
,27
,29
,30
).
The induction of CYP by cigarette smoke exposure in lung tissue has been attributed to the bioactivation of carcinogens and the enhancement of lung carcinogenesis (31
). Because RA acts on normal bronchial epithelium by blocking squamous metaplasia (12
,58
–60
), which occurs during the early stages of lung carcinogenesis, our findings that cigarette smoke exposure enhanced RA catabolism via induction of CYP indicate that alteration of RA metabolism may also contribute to lung carcinogenesis. This notion was supported by our previous observations that low RA levels were accompanied by appearances of squamous metaplasia in the lungs of ferrets exposed to cigarette smoke and/or a pharmacological dose of ß-carotene (8
,9
). In contrast, ß-carotene, when given at a low dose, could act to supply adequate RA to alleviate the squamous metaplasia in the lung tissue of smoke-exposed ferrets (9
).
In this study, we demonstrated that a pharmacological dose of ß-carotene alone induced CYP and enhanced RA catabolism (Fig. 1
, Table 2
). Of note, the Physicians’ Health Study trial (4
) did not find increased lung cancer with ß-carotene supplementation. This may be because only few participants were exposed to cigarette smoke that contains carcinogens, even the CYP that may have been induced by a high dose of ß-carotene in this population. Because there was no assessment of either premalignant lesions or CYP induction in this trial, further follow-up of this population is warranted.
In summary the findings from the present study offer a biochemical mechanism for the reduction in lung RA concentrations seen in the smoke-exposed ferrets, with or without a pharmacological dose of ß-carotene, and also provide a possible explanation for the enhancement of lung carcinogenesis by ß-carotene supplementation in cigarette smokers. Future studies should evaluate whether restoration of lung RA homeostasis by RA supplementation or by inhibiting CYP-enhanced RA catabolism can have chemopreventive effects against lung carcinogenesis.
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FOOTNOTES |
|---|
2 Supported by the American Cancer Society (grant RSG-01-030-01-CNE), BASF Inc. (Basel, Switzerland) and the U.S. Department of Agriculture (under agreement no. 1950-51000-048-01A). Any opinions, findings, conclusions or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the views of the U.S. Department of Agriculture.
4 Abbreviations used: CYP, cytochrome P450 enzymes; LMF, lung microsomal fraction; RA, retinoic acid.
Manuscript received 13 August 2002. Initial review completed 5 September 2002. Revision accepted 30 September 2002.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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