ToxSci Advance Access originally published online on December 5, 2005
Toxicological Sciences 2006 90(1):96-110; doi:10.1093/toxsci/kfj065
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Sunlight Generates Multiple Tryptophan Photoproducts Eliciting High Efficacy CYP1A Induction in Chick Hepatocytes and In Vivo
Department of Pharmacology, Weill Medical College of Cornell University, New York, New York 10021
1 To whom correspondence should be addressed at Department of Pharmacology, Weill Medical College of Cornell University, 1300 York Avenue, Room LC-401, New York, NY 10021. Fax: (212) 746–8835. E-Mail: arifkind{at}med.cornell.edu.
Received August 17, 2005; accepted November 17, 2005
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ABSTRACT |
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Photooxidized tryptophan (TRP) in tissue culture medium elicits a transient cytochrome P450 (CYP1) induction response in cultured cells. We show here that exposure of TRP to window sunlight (aTRP) greatly increased the potency, efficacy, and duration of CYP1A induction by TRP in primary chick embryo hepatocytes and in vivo. Aqueous TRP exposed to sunlight for 7 days exhibited a 100-fold or greater increase in potency over TRP in medium. The induction response was sustained for at least 48 h and was comparable in efficacy to 2,3,7,8-tetrachlorodibenzo-p-dioxin. In hepatocytes, increases in mRNAs for CYP1A4 and CYP1A5, chick orthologs of mammalian CYP1A1 and 1A2, preceded increases in CYP1A proteins and enzyme activities, 7-ethoxyresorufin deethylase (EROD) for CYP1A4 and arachidonic acid epoxygenation for CYP1A5, consistent with a transcriptional mechanism. Aryl hydrocarbon receptor (AhR) dependence was evidenced by aTRP induction of EROD in wild-type Hepa1c1c7 cells but not in AhR-defective (c35) mutants. Preparations of aTRP were stable for many months at 4°C and were relatively resistant to metabolism by hepatocytes or liver microsomes. Fractionation of aTRP by HPLC analysis coupled with EROD assays showed that aTRP contained multiple photoproducts and CYP1A inducing components, which varied in sensitivity to metabolism by hepatocytes. The previously identified TRP photoproduct, 6-formylindolo[3,2-b]carbazole (FICZ), was one component, but FICZ was not required for CYP1A induction by the aTRP mixture. These findings identify the indoor environment, and window sunlight in particular, as a new source of CYP1A inducers. Further, the evidence that biologically active metabolites of an endogenous substrate, arachidonic acid, are formed by aTRP-induced CYP1A provides a pathway by which TRP photoproducts, like toxic xenobiotics, could have significant physiologic effects.
Key Words: tryptophan photooxidation; chick embryo; CYP1A4; CYP1A5; dioxin; arachidonic acid epoxygenation; sunlight.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
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The focus of scientific interest in the function and regulation of the aryl hydrocarbon receptor (AhR) and cytochrome P450 (CYP1A) induction has shifted from the toxin 2,3,7,8 tetrachlorodibenzo-p-dioxin (dioxin, TCDD) to more common AhR ligands including indole-based compounds, related to the amino acid tryptophan (TRP) (Denison and Nagy, 2003
). This shift reflects increasing evidence that the AhR has physiologic roles (Fernandez-Salguero et al., 1995; Lahvis et al., 2000; Vasquez et al., 2003) in addition to mediating the toxicity of xenobiotics such as TCDD (Poland and Knutson, 1982).
TRP-related compounds have received particular attention because of their ubiquity in the environment. They include dietary compounds, metabolites or spontaneous breakdown products, derivatives found in vivo (Adachi et al., 2001; Bittinger et al., 2003; Bjeldanes et al., 1991), or photoproducts (Helferich and Denison, 1991; Rannug et al., 1987). Many TRP-derived products have been shown to bind to the AhR and to induce CYP1A (Adachi et al., 2004; Gillner et al., 1993; Heath-Pagliuso et al., 1998; Helferich and Denison, 1991; Jellinck et al., 1993; Lorenzen et al., 1993; Rannug et al., 1987; Sindhu et al., 2000; Spink et al., 2003).
Solutions of aqueous TRP or of TRP in peptide linkages are readily photooxidized by ultraviolet (UV) light (Tassin and Borkman, 1980). Early studies (Paine, 1976, 1977) showed that UV irradiation of tissue culture medium produced an inducer of CYP1A enzyme activity in the medium. CYP1A induction in human keratinocytes and lymphocytes by UV light has been attributed to oxidation of TRP in the medium (Wei et al., 1999), as has the induction of CYP1A by medium change in diverse cell types (Kocarek et al., 1993; Oberg et al., 2005; Segner et al., 2000; Sindhu et al., 1996, 2000; Spink et al., 2003).
There are several reports of TRP photoproducts binding to the AhR (Denison and Nagy, 2003; Heath-Pagliuso et al., 1998; Helferich and Denison, 1991; Rannug et al., 1987). Identified products include kynurenine (Denison and Nagy, 2003) and tryptamine, which are photoproducts as well as metabolites of TRP (Creed, 1984), and two photoproducts with extremely high AhR binding affinity, 6-formylindolo[3,2-b]carbazole (FICZ) and 6,12-di-formylindolo[3,2-b]carbazole (dFICZ) (Rannug et al., 1995).
Notwithstanding the high binding affinity of some TRP photoproducts, CYP1A induction responses to TRP photooxidized by UV light in human cells (Wei et al., 1998) or in response to medium change in various other cells have been reported to be transient, with maximal effects seen soon after exposure and induction no longer detectable after 24 h (Kocarek et al., 1993; Oberg et al., 2005; Segner et al., 2000; Sindhu et al., 1996, 2000; Spink et al., 2003).
We report here that exposure of TRP to ordinary sunlight passing through window glass greatly increases the inducing potency of TRP. TRP exposed to sunlight for 7 days, for example, was more potent than TRP in culture medium by two to three orders of magnitude. The induction response was comparable to TCDD in efficacy and sustained for at least 48 h in cultured hepatocytes and in vivo. Sunlight-exposed TRP was found to contain a mixture of CYP1A inducers, varying in sensitivity to metabolism, of which FICZ was only one component. The induction response included enhanced formation of CYP-dependent arachidonic acid epoxides, indicating that TRP photoproducts can alter formation of biologically active endogenous substances. The results show that light in the indoor environment and, in particular, sunlight passing through window glass is a new potential source of potent and effective CYP1A inducers.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
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Reagents.
Unless otherwise indicated, chemicals were from Sigma-Aldrich (St. Louis, MO), and tissue culture reagents from GIBCO BRL Life Technologies (Gaithersburg, MD). High pressure liquid chromatography (HPLC) reagents were HPLC grade. Fertilized chicken eggs, White Leghorn strain, were obtained from Burr Farm (Hampton, CT), TCDD from the NCI Chemical Carcinogen Repository (Kansas City, MO), and FICZ from Biomol (Plymouth Meeting, PA).
Cell Cultures
Primary hepatocytes.
Livers were removed from 15- or 16-day-old chick embryos after the hearts were perfused with 5 ml of Hank's balanced salt solution (140 mM NaCl, 5.5 mM D-glucose, 5.4 mM KCl, and 0.4 mM of KH2PO4 pH 7.3) and minced and disaggregated for 20 min in 10 ml of warm Hank's solution with 0.05% collagenase. Cell suspensions were centrifuged three times at 200 x g at 4°C, each time for 7 min. The combined pellets were suspended in standard Ham's medium (Std. Ham's) with 2% fetal bovine serum (FBS). Hepatocytes were plated in Costar 24-well cell culture cluster flat-bottomed plates (Corning Inc, Corning, NY) at 0.5 x 106 cells per well in 1 ml of medium or in Costar 96-well assay plates (white-walled clear bottomed) (Corning Inc.) at 80,000 cells per well in 0.16 ml of medium and maintained in 5% CO2 in air for 48 h before medium change and further treatment. In some experiments, as indicated below, cells were plated in 6- or 10-cm culture dishes (BD Falcon, Franklin Lakes, NJ). The term "cultured hepatocytes" refers to hepatocytes after an initial 48 h in culture unless otherwise indicated.
Hepa1c1c7 and AhR mutant (c35) cells.
Mouse hepatoma Hepa1c1c7 wild type and c35 mutant cells from ATCC (Manassas, VA), were plated in 24 well dishes at 0.25 x 106 cells per well in 
MEM (minimum essential medium) with 10% FBS. Cells at semiconfluence were treated with light-exposed TRP and assayed for 7-ethoxyresorufin deethylase (EROD) (see below) after 24 h.
Tissue culture media used for primary hepatocytes.
(1) Std. Ham's—9.18 g of Basal Medium Eagle (BME) (Cellgro, Mediatech Herndon, VA) and 2.2 g of NaHCO3 were dissolved in 900 ml of distilled water. Additions were: 20 ml of 50x MEM essential amino acid solution containing 36 mM L-arginine, 10 mM L-cystine, 13.5 mM L-histidine, 20 mM L-isoleucine, 20 mM L-leucine, 25 mM L-lysine, 5 mM L-methionine, 10 mM L-phenylalanine, 20 mM L-threonine, 10 mM L-tyrosine, and 20 mM L-valine, 2.5 mM L-tryptophan, 200,000 U/penicillin-streptomycin, 20 ml of 100x MEM vitamin solution, 5 ml of 100x MEM nonessential amino acids, and 0.1 ml (10 mg/ml) of d-Biotin. pH was adjusted to 7.3–7.5, FBS added to a final concentration of 2%, and the total volume brought to 1 l with distilled water. (2) TRP-free medium—TRP-free Basal Medium Eagle (BME) was custom-prepared by Specialty Media (Phillipsburg, NJ). Additions were the same as for Std. Ham's, except that TRP was excluded from the MEM essential amino acid solution.
Photoactivation of TRP.
Aqueous TRP at 100x, unless otherwise indicated (1x is defined as the concentration of TRP in Std. Ham's (14.2 µg per ml or 69 µM)), was dissolved in 50 ml of distilled water and placed on the indoor sill of a large east-facing window in a 50-ml polypropylene tube, unless otherwise indicated. Light-exposed TRP (aTRP) was wrapped in aluminum foil and kept at 4°C before dilution in TRP-free medium for treatment of cultured hepatocytes or further analysis. TRP kept at the window wrapped in foil was a regular control.
For the dose of UVA (320 to 400 nm) received by TRP exposed to window sunlight for 7 days, an approximate dose range from 37.5 to 225 mJ/cm2 per week was calculated as follows: the average daily UV dose to the earth's surface in New York (0.63 kJ/m2 (for the 3 winter months) to 3.78 kJ/m2 (for the 3 summer months) (http://temis.nl) was multiplied by 0.17 (percent of UVA in sunlight), by 0.5 for the average transmission of light over the UVA range through the 1-in clear insulating window glass (Viracon, Owatonna, MN), and by 7 (days per week). For visible light (400 to 700 nm) an approximate dose range from 0.3 to 0.93 KJ/cm2 per week was calculated as follows: the daily dose, from 0.04 to 0.17 kJ/cm2 (Freitas, 1999), times 0.78 (percent of visible light transmitted through the glass), and multiplied by 7. The window glass screens UVB (280–315 nm) virtually entirely.
Treatment of hepatocytes.
ß-Naphthoflavone (ß-NF) in dimethylsulfoxide (DMSO) (10 mM) was diluted in TRP-free medium to 1 or 10 µM, the concentrations used in culture. TCDD (1.5 mM) in dioxane (J.T. Baker, Phillipsburg, NJ) was diluted in dioxane to 1.5 µM and further diluted in TRP-free medium to 1 nM, a maximal CYP1A-inducing concentration, for addition to hepatocytes. Equivalent amounts of DMSO or dioxane were used as controls (final concentrations <0.1%). For treatment of hepatocytes, compounds were diluted and added to cells in TRP-free medium. Triplicate wells were used for each treatment.
Treatment of chick embryos in ovo.
Test compounds were administered to 15-day-old embryos via injection into the fluids surrounding the embryos through a hole in the shell. After 24 or 48 h, livers were removed, and microsomes prepared (Rifkind et al., 1994).
Enzyme Assays
Assays were performed under conditions where product formation was a linear function of enzyme concentration and incubation time and substrate was not limiting. Microsomes were incubated at 37°C in a shaking water bath in air.
EROD.
(1) In cultured hepatocytes—24-well plates— Medium was removed, cells washed with phosphate buffered saline (PBS; Cellgro by Mediatech), and 0.5 ml of the EROD reaction mixture added to each well (4 µM 7-ethoxyresorufin (7-ER) and 10 µM dicumarol in Std. Ham's). After 30 min at 37°C, two 0.2-ml aliquots were removed per well, 0.25 ml of cold acetone was added to each followed by centrifugation at 1400 x g for 15 min. Resorufin fluorescence was measured in a Perkin Elmer MPF 3 spectrophotofluorimeter (Excitation (Ex) and Emission (Em) 
, 558 nm and 590 nm, respectively), using a quinine sulfate standard previously calibrated against resorufin. Emission spectra were obtained for selected samples to confirm the presence of a resorufin peak at 590 nm. Results for 24-well plates are given as pmol resorufin/well. 96-well plates—Medium was removed, cells washed with PBS, and 0.08 ml of the EROD reaction mixture described above added to each well. After 30 min at 37°C, resorufin fluorescence was read on a SpectraFluor fluorescence plate reader (Tecan, Durham, NC) at Ex and Em , 535 nm and 595 nm, respectively. A standard curve for resorufin (4.8 nM to 4.8 µM) added to wells of nontreated cultured hepatocytes immediately before reading was included in each experiment. Results for 96-well plates are given as pmol resorufin/ml. (2) In liver microsomes and hepatocyte homogenates—Reaction mixtures (0.24 ml) contained 15 to 30 µg chick embryo liver microsomal protein or 200 µg of hepatocyte homogenate protein in 0.039 M Tris-phosphate, pH 8.3, with 1.25 mM EDTA and 1 mg/ml BSA, 7-ER (4 µM unless otherwise indicated), and for hepatocyte homogenates, 10 µM dicumarol. After preincubation at 37°C for 1 min, reactions were started with 1 mM NADPH and incubated for 5 min. After adding 0.25 ml of cold acetone and centrifugation at 3,000 rpm for 15 min, resorufin was measured as above using the spectrophotofluorimeter (Rifkind et al., 1994).
Arachidonic Acid (AA) metabolism.
CYP1A5-dependent AA metabolism was measured in homogenates of chick embryo hepatocytes cultured in 10 cm BD Falcon polystyrene dishes (BD Biosciences, San Jose, CA) at 7 x 106 cells per dish or in liver microsomes (Capdevila et al., 1990; Rifkind et al., 1994), using duplicate dishes for each treatment. For each determination in hepatocytes, cells from one dish were scraped into 0.3 ml of Hank's balanced salt solution and homogenized. Reaction mixtures for hepatocytes (0.5 ml) contained 300 µg homogenate protein and for microsomes (0.25 ml), 50 µg liver microsomal protein. Both contained 30 µM 1-14C-arachidonic acid (48 mCi/mmol) (Perkin Elmer, Torrance, CA). After 2 min of preincubation at 37°C, reactions were started with 1 mM NADPH, 10 mM isocitric acid, 0.2 U isocitric dehydrogenase/ml, and 10 mM MgCl2 and incubated for 10 min at 37°C. Addition of acetic acid (0.1 or 0.2 ml for microsomes and hepatocyte homogenates, respectively) was followed by two extractions with 3 ml of ethyl acetate containing 0.005% butylated hydroxytoluene. The organic phases were pooled, dried under N2, and resuspended in 0.11 ml of 50% acetonitrile in water and 0.1% acetic acid. Products in 0.05 ml were resolved by reverse phase high pressure liquid chromatography (HPLC) using a Vydac C18 (Vydac, Hesperia, CA) column (90 Å, 5 µm particle size, 4.6 x 250 mm) and a linear gradient from 50 to 100% acetonitrile in water containing 0.1% acetic acid, at 1 ml per min for 40 min. Radioactivity was measured using a Flo-One Beta Model S radioactivity flow monitor (Packard Instrument Company, Downers Grove, IL). Products were identified by reference to HPLC retention times of pure standards (Rifkind et al., 1994).
Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) of CYP1A4 and CYP1A5.
Total RNA was extracted from cultured chick hepatocytes (4 x 106 cells per 10-cm dish) using RNA STAT-60 (Tel-Test "B," Friendswood, TX). First-strand cDNA was synthesized from the total RNA using Moloney Murine Leukemia virus reverse transcriptase (Roche, Indianapolis, IN) as directed by the manufacturer: 4 µl of 5x incubation buffer, 2 µg of total RNA, 2 µl of 10x concentrated hexamers, 1 µl of 10 mM dNTPs, 0.5 µl of 40U/µl RNase inhibitor, 1 µl of 20 U/µl of the reverse transcriptase, and DEPC-water up to 20 µl, were incubated for 2 h at 37°; all components from Roche. PCR amplification was performed using primers from chicken CYP1A4, CYP1A5, CYP2H1, or GAPDH (glyceraldehyde-3-phosphate dehydrogenase) as an internal standard (respective Gene Bank Accession Nos.: X99453, X99454, M13454, and AF047874). Primers used (Invitrogen (Carlsbad, CA)) were: for CYP1A4, 5'-GGACGGAGGCTGACAAGGTG-3' and 5'-GTGCAAACGCTGAGGGATTC-3' (Fig. 1), and 5'-TGCTGCAGGATGGTGGTGAG-3' instead of the latter in Figure 7; for CYP1A5, 5'-CTTCATGCCCTTCACCATC-3' and 5'-CAGGCCAAAAGTCATCAC-3'; for CYP2H1, 5'-CCATGGGTTATTTGTTTTGTGTT-3' and 5'-GCTGCTGCCTTGTCTGCTC-3'; for GAPDH, 5'-GGGTCTTATGACCACTGTCC-3' and 5'-GTAAGCTTCCCATTCAGCTCAG-3'. Reaction mixtures contained 2 µl of single-strand cDNA, 2 µl of the relevant sense and antisense CYP primers (final concentration in the reaction mixture, 200 nM), and 45 µl of Platinum Supermix (Invitrogen), which contains all the other components needed for the PCR reactions.
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Amplification conditions were for CYP1A4, 94°C x 5 min for 1 cycle, 94°C x 1 min, 59°C x 30 s, 72°C x 40 s for 30 cycles, 72°C x 10 min for 1 cycle; for CYP1A5, 94°C x 5 min for 1 cycle, 94°C x 1 min, 56°C x 30 s, 72°C x 30 s for 28 cycles, 72°C x 10 min for 1 cycle; for CYP2H1, 94°C x 5 min for 1 cycle, 94°C x 1 min, 58°C x 30 s, 72°C x 30 s for 29 cycles, 72°C x 10 min for 1 cycle. For GAPDH 1 µl of the sense and antisense GAPDH primers were added to the reaction mixture for each CYP and amplified for 23 cycles. All PCR products were obtained at cycle number below plateau levels. PCR products were separated on a 1.5% agarose gel and visualized with ethidium bromide using a UV transilluminator. Expected sizes of the PCR products were: CYP1A4, 404 bp (Fig. 1), 109 bp (Fig. 7); CYP1A5, 219 bp; CYP2H1, 286 bp; GAPDH, 170 bp. Band intensities were measured by densitometry using AlphaEaseFC software (Alpha Innotech, San Leandro, CA).
SDS–polyacrylamide gel electrophoresis/Western blotting.
Hepatocytes from triplicate wells with the same treatment on 24-well plates were scraped together, lysed in 0.15 ml of 2x sample buffer (125 mM Tris–HCl, pH 6.8, 4% SDS, 16% glycerol, 10% ß-mercaptoethanol, 0.002% bromophenol blue) and boiled for 10 min. Protein concentrations were measured (Lowry et al., 1951) on a duplicate set of three wells for each treatment, separately for each well. Identical SDS gels were prepared in each experiment and stained with Coomassie blue to confirm equal loading of lanes. Electrophoresis was at 15 mA and 30 mA through the stacking and running gels, respectively, followed by Western blotting (Kanetoshi et al., 1992; Rifkind et al., 1994). Primary antibodies were immunopurified antisera selective for CYP1A4 or CYP1A5 at 1:400 dilution or nonimmunopurified antisera recognizing both CYP1A4 and CYP1A5 at 1:3000 dilution; the secondary antibody was peroxidase-conjugated goat anti-rabbit at 1:1000 dilution. Detection was by chemiluminescence using "Western blotting detection reagent" (Amersham, Buckinghamshire, UK). Band intensities were measured by densitometry as described above.
Hydrogen peroxide (H2O2).
H2O2 was measured using an Amplex® Red H2O2/peroxidase assay kit (Molecular Probes, Eugene OR) on hepatocytes in 96-well plates (1.6 x 105 cells per well) by the manufacturer's directions.
HPLC fractionation of aTRP and TRP.
One ml of a 100x solution of aTRP exposed to sunlight for 7 days, unless otherwise indicated, or TRP not exposed to light was dried under N2 and resuspended in 0.010 ml of DMSO followed by 1 ml of 35% acetonitrile in water containing 0.1% trifluoroacetic acid (TFA). 0.225 ml (equivalent to 22.5x) were injected onto a Denali C18 monomeric, 100Å, 5 µm particle size reversed-phase column (4.6 mm x 250 mm) (Vydac) and resolved by HPLC on a linear gradient of 35% to 85% acetonitrile in water containing 0.1% TFA at 1 ml/min for 40 min. Detection was at 254 nm and in replicate samples at 386 nm using a Waters 486 UV detector (Waters Corporation, Milford, MA). The signal was transmitted from the UV detector through the Flo-One Beta detector (see above), using the auxiliary hardware option, which converts the absorbance signal to CTS as the readout. The eluate was collected in 14 fractions, which were then used for EROD testing. They were dried under N2, reconstituted in DMSO and water as above, and diluted in TRP-free medium for addition to culture. Replicate sets of fractions collected with the absorbance detector "off" to exclude any degradation effect by the UV illumination showed no differences in EROD induction. Further subfractionations were performed on a gradient of 5 to 85% acetonitrile in water with 0.1% TFA at 1 ml per min for 80 min.
Statistical evaluation.
Statistical analysis included unpaired t-tests (Graph Pad Prism 4 software, San Diego CA) for differences between group means and Pearson correlation coefficient analysis, using p < 0.05 as the criterion for significance.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
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TRP-Dependent CYP1A Induction by Medium Change in Chick Embryo Hepatocytes
In preliminary experiments (data not shown) medium change with Std. Ham's medium selectively induced mRNAs for CYP1A4 and CYP1A5 but not the AhR-independent CYP2H1 (Gilday et al., 1996
; Hobbs et al., 1986) in primary chick embryo hepatocytes, indicating selective CYP1A induction by medium change. The induction by medium change was transient, with a maximum effect at 5 h returning to basal levels by 24 h. EROD activity, which is selectively catalyzed by chick CYP1A4 (Kanetoshi et al., 1992; Rifkind et al., 1994) was also transiently increased. The induction response was entirely attributable to tryptophan (TRP) in the medium. CYP1A was not induced by spent medium or by changing serum concentrations of the medium or by TRP-free medium. The transient effect has been shown for diverse cell types including rat hepatocytes, trout liver cells, human keratinocytes, mouse hepatoma Hepa1c1c7 cells, and rat hepatoma MH1C1 cells (Kocarek et al., 1993; Oberg et al., 2005; Segner et al., 2000; Sindhu et al., 1996, 2000; Spink et al., 2003). Our preliminary findings underscore the ubiquity of the CYP1A induction response to medium change and show that it occurs in a new cell type, primary chick embryo hepatocytes.
Enhancement of CYP1A Induction by Exposure of TRP to Sunlight; AhR Dependence of Induction by Light-Activated TRP
Aqueous solutions of TRP were exposed to window sunlight as described in "Materials and Methods" for 1 to 7 days and used to treat cultured hepatocytes at a concentration equivalent to 1x TRP, the concentration of TRP in Std. Ham's (14.2 µg/ml or 69 µM). Henceforth, unless otherwise indicated, the term "activated TRP (aTRP)" will refer to TRP exposed to sunlight for 7 days. TRP exposed to light for 1 day increased CYP1A mRNAs (Fig. 1a) and EROD (Fig. 1b) more than Std. Ham's, but the induction was still transient (lower at 24 h than 5 h). The induction response for TRP exposed to light for 7 days was sustained at 24 h, and its efficacy was comparable to TCDD. Figure 1b shows that the sustained response was achieved after 3 days of TRP exposure and that the levels were maximum starting at 5 days. In most experiments, aTRP was added to cells during medium change, but induction effects were essentially the same when aTRP was added without changing the medium. Further, photoproducts produced by exposure of TRP to sunlight at 10x had the same effects as those produced by exposure at 100x (data not shown).
Figure 1c shows that aTRP increased CYP1A4 and CYP1A5 proteins. The protein levels were elevated for at least 48 h, with the 48-h levels at 90% of the 24-h levels. After 5 h of treatment, 10x aTRP increased CYP1A protein 20% more than 1x aTRP, whereas after 24 h or 48 h of treatment, 1x and 10x were comparable in their CYP1A protein induction effects.
To determine whether CYP1A5 enzyme activity was increased by aTRP, CYP1A5-mediated arachidonic acid (AA) metabolism was measured (Kanetoshi et al., 1992; Rifkind et al., 1994) (Fig. 1d). AA epoxygenase products, epoxyeicosatrienoic acids (EETs) and EET-diols, were increased 1.6-fold after 5 h and 4.9-fold after 24 h, and monohydroxylated products, monohydroxyeicosatetraenoic acids (HETEs), were increased by 1.7-fold after 5 h and 2.8-fold after 24 h.
Figure 1e shows that the CYP1A induction by aTRP is AhR dependent. aTRP induced EROD in wild type mouse hepatoma Hepa1c1c7 cells but not in c35 cells, a mutant line derived from the wild type and expressing an AhR defective in XRE binding (Sun et al., 1997).
The inducing effect of exposure to sunlight under the conditions described above was compared to other environmental conditions in the laboratory (Table 1). TRP was activated slightly more when exposed to sunlight in polypropylene than in glass tubes. TRP wrapped in tin foil was not activated by sunlight whether kept at 4°C or at the window, indicating that sunlight was acting via a photoactivation rather than a heat effect. Exposure to UV light from a germicidal bulb in a tissue culture hood for 13 days, or to a fluorescent bulb for 7 days, produced 78% and 13% of the response to window sunlight, respectively; even ambient light elicited a small but detectable increase in EROD induction capacity. Thus, light from different sources activated TRP to varying degrees.
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UV/VIS absorption spectra of aqueous TRP shows peaks at 280 and 288 nm and a shoulder at 271 nm (Borkman et al., 1986
). The UV/VIS spectra for aTRP or TRP not exposed to light showed those same peaks, but for aTRP exposed to light for 7 days, the peak heights were reduced slightly. In separate experiments the utilization of TRP measured over a 14-day period was about 16%. aTRP also showed a small increase over baseline absorbance, declining gradually from 300 to 460 nm (Supplemental Data, item 1). The absorbance spectrum for aTRP was like to spectra reported for TRP photoactivated by UVA or UVB light (Borkman et al., 1986).
Induction of CYP1A4 and 1A5 by aTRP In Vivo
To determine if aTRP retained its inducing effect in a physiologic environment, we treated chick embryos in ovo with aTRP. Treatment with aTRP for 48 h produced a mean increase for the two concentrations tested of 7.3- fold for CYP1A protein (Fig. 2a) and of 11- and 5-fold for CYP1A4 and 1A5 enzyme activities, EROD and AA epoxygenation, respectively, compared to equivalent amount of TRP not exposed to sunlight (Fig. 2b). Effects of aTRP in vivo were dose related. The results show that the inducing compounds in aTRP were sufficiently stable in the physiologic environment to produce a substantial and sustained induction in vivo even after 48 h.
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The small increase in CYP1A induction effects at the higher dose of unexposed TRP seen in Figure 2 is probably attributable to the presence of small amounts of photoactivated TRP or other contaminants in the commercial preparations of TRP as also found by others (Heath-Pagliuso et al., 1998
; Kocarek et al., 1993; Rannug et al., 1987).
Characterization of Dose-Response Relationships for EROD Induction by aTRP in Hepatocytes
The effect of aTRP on EROD 5 and 24 h after treatment was examined at half-log interval dilutions from 10x to below the induction threshold (about 0.001x for the preparation of aTRP shown (Fig. 3)). The results shown are representative of at least 10 other preparations of aTRP, with some variations in potency. There were four noteworthy findings: (1) Sunlight exposure greatly increased the inducing potency of TRP over the effect of Std. Ham's medium (about 330-fold for the preparation shown, based on the extrapolation that a dose halfway between 0.001x and 0.01x would have produced induction equivalent to Std. Ham's at 5 h). (2) The response to aTRP was biphasic, with 10x aTRP producing lower hepatocyte EROD levels than 1x aTRP, notwithstanding similar CYP1A4 protein levels at 1x and 10x aTRP (see Fig. 1c). (3) EROD induction was transient (lost at 24 h) for aTRP below 0.01x and became sustained (greater at 24 h than 5 h) for aTRP at 0.01x and higher. (4) aTRP produced highly effective induction. The maximum response after 24 h was about 93% of the maximum response for TCDD.
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Differences in Rates of Metabolism of Std. Ham's or aTRP
We investigated whether the loss of induction at 24 h by Std. Ham's and the more sustained effect at 24 h of aTRP were due to differences in metabolism of the inducers in each. Std. Ham's and aTRP were exposed to cultured hepatocytes for 5 or 24 h. The hepatocyte-exposed Std. Ham's or aTRP-containing medium were added to a second set of hepatocytes, and the effects on EROD examined 5 or 24 h later (Fig. 4a). The inducing capacity of Std. Ham's was totally lost after a 5-h preexposure to hepatocytes. aTRP preexposed to hepatocytes for 5 or 24 h lost 43% of its capacity to induce EROD. Thus, all of the loss for aTRP was complete by 5 h. EROD levels in the second set of hepatocytes 24 h after treatment with aTRP preexposed to hepatocytes for 5 or 24 h did not differ from EROD levels for unexposed aTRP (Fig. 4a). Thus, the ability of aTRP to elicit a sustained response at 24 h was unaffected by the partial loss of its capacity to induce at 5 h. The inset in Figure 4a shows the rapid loss of the inducing capacity of Std. Ham's by exposure to hepatocytes (t
of about 1.6 h).
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Similar results using a different approach are shown in Figure 4b. The inducing effect of Std. Ham's was completely lost after Std. Ham's was incubated with liver microsomes from DMSO- or ß-NF-treated chick embryos at 240 µg per reaction mixture. Microsomes from ß-NF- treated chick embryos at 60 µg per reaction mixture caused more inactivation than those from the DMSO-treated, suggesting that increased CYP1A enhanced the rate of metabolism of inducers in Std. Ham's. aTRP incubated with the same microsomes lost only about 35% of its inducing capacity. There were no additional losses when incubations were continued for up to 30 min. In preliminary experiments (not shown) heat-inactivated microsomes did not affect EROD induction by Std. Ham's or aTRP, and inclusion of the NADPH regenerating system was required for the metabolism.
HPLC Fractionation of aTRP
To determine whether the induction by aTRP was due to one or more components in aTRP, aTRP and TRP were subjected to reverse phase HPLC (Fig. 5a). Multiple 254 nm absorbing peaks are evident in aTRP that were missing in TRP. Detection at 386 nm (not shown) did not reveal any additional peaks. The same HPLC pattern was produced by six different preparations of aTRP exposed for 7 days at different times of the year. Figure 5b shows EROD induction for 14 fractions collected from the HPLC eluate in Figure 5a. All of the aTRP fractions exhibited greater inducing activity than the comparable fractions of nonactivated TRP, with the most induction produced by fractions 5 and 4. The results show that aTRP is a mixture of many inducing substances.
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Dose-Response Relationships for EROD Induction by HPLC-Separated Fractions of aTRP
We examined whether the aTRP fractions, which had an induction effect more than double that of the DMSO control (Fractions 1–5, 7–9, and 11), had dose effects differing from those of the unfractionated mixture (Fig. 6). The results show that the aTRP mixture contains inducing components capable on their own of induction equivalent to that in the mixture. The sum of the EROD responses for the individual fractions exceeded that of the aTRP mixture by 7-fold, 4-fold, and 1.8 fold for 1x, 0.33x, and 0.1x dilutions, respectively, while the sum approximated the induction for the mixture at 0.33x and 0.01x.
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Inducers in HPLC Fractions of aTRP Are Differently Resistant to Metabolism
As aTRP was shown to be resistant to metabolism (Fig. 4) and to be a mixture of inducers (Fig. 5), we examined whether the 14 fractions isolated from aTRP by HPLC could be separated into metabolism-sensitive or resistant groups. The 14 fractions were incubated at 1x with cultured hepatocytes for 24 h, and the medium was then transferred to a second set of cultured hepatocytes. Other hepatocytes in the second set were treated with fractions of aTRP that had not been preexposed to hepatocytes. After 24 h total RNA was extracted from the second set of hepatocytes and used to measure CYP1A4 mRNA induction by PCR. The results (Fig. 7) showed that the fractions varied in their sensitivity to metabolism. Induction by Fractions 4 and 5, the most effective inducing fractions (induction equivalent to aTRP), were not affected by preincubation with hepatocytes, indicating that the main inducers in those fractions are resistant to metabolism. Fractions 3, 6, 8, and 9, less effective inducers, were also metabolism resistant. On the other hand, Fractions 1, 2, 7, and 11–14 were sensitive to metabolism. Fraction 7 showed the greatest loss of inducing capacity after exposure to hepatocytes (44% loss); losses were 18% and 27% for Fractions 1 and 2. Fractions 11, 12, 13, and 14 essentially lost their inducing capacities after the preincubation.
Further Resolution of the More Polar HPLC Fractions of aTRP
To resolve further the components in the more polar fractions in Figure 5, which contained most of the inducing activity, Fractions 1 to 5 (F1–F5) were subjected to HPLC using a gradient of 5% to 85% acidified acetonitrile (see "Materials and Methods") (Fig. 8). UV detection at 254 nm revealed that each of those aTRP fractions contained multiple peaks. Groups of peaks or single peaks were collected and assayed for EROD (Fig. 8, right panels). The EROD-inducing capacity varied for the subfractions. Almost all of the subfractions induced EROD over the baseline (TRP-free medium). Exceptions were: subfraction b and h in F1, a and d in F2, a and h in F3, and a, b, f, g, and h in F5).
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The capacity to induce was not correlated with peak size. For F1, for example, most of the inducing activity was in the subfractions eluting after the TRP peak (i.e. fractions n–q). Subfraction q in F1, which had only some small peaks, was the most effective inducer for F1, containing about half of the inducing capacity of the unfractionated F1. For F4, the main inducing capacity resided in the smaller of the two main peaks in the fraction.
For all of the fractions except F1, the sum of the EROD values for the subfractions approximated the induction by the unfractionated fraction. For F1, the sum of the EROD values for the subfractions exceeded that of the unfractionated F1, suggesting a possible inhibitory effect among the components.
FICZ in aTRP
As FICZ has been identified as a photoproduct of TRP (Rannug et al., 1995), we investigated whether FICZ was present in the aTRP mixture (Fig. 9). Figure 9a shows that FICZ (Biomol) eluted as a single peak at about 30–32 min (the 35–85% ocatonitrile gradient), corresponding to aTRP Fraction 11 (see Fig. 5a) using the same HPLC system. There were no additional breakdown products for FICZ exposed to sunlight for 7 days (not shown). As the peak for Fraction 11 was very small even at 22.5x aTRP (Fig. 5), a larger amount of aTRP equivalent to 600x was separated on HPLC. Two peaks were evident for the elution time of Fraction 11 (Fig. 8b insert). The second peak, Fraction 11b, induced EROD (Fig. 8b), but Fraction 11a did not. Fraction 11b exhibited the same emission spectra reported for FICZ at Ex 310, 390, and 460 (Rannug et al., 1987), shown in Figure 8c for Ex 390, while Fraction 11a did not have a FICZ emission spectrum. The concentration of FICZ in Fraction 11b was measured by reference to a standard curve for the fluorescence of Biomol FICZ. The amount of FICZ in 1x aTRP was calculated to be 72 pM. To determine if FICZ was needed for the inducing effect of aTRP, the effects on EROD of combined aTRP eluates from HPLC with and without Fraction 11 included were compared (Fig. 9d). The inducing effect of aTRP was unaffected by fractionation on HPLC or by exclusion of the fraction containing FICZ from the mixture. Thus, FICZ is present in aTRP, but it was not required for the induction effect of the aTRP mixture.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
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It has been known for a long time that medium change can elicit transient CYP1 induction. Induction by medium change is of low intensity and no longer evident by 24 h. It has been attributed to photooxidized TRP in medium and was shown by Kocarek (1993)
to be elicited simply by a brief (1 h) exposure of TRP-containing medium to an ordinary 15-W fluorescent bulb. CYP1A induction by medium change is often overlooked as a confounding factor in studies of constitutive AhR expression in cultured cells, but a recent report by Oberg (2005), published while these studies were under completion, renewed attention to the phenomenon. Oberg et al. (2005) showed that medium exposed to fluorescent light in a standard culture hood for 24 h or even to a 40-W incandescent bulb produced transient CYP1A1 induction in rat hepatoma MH1C1 cells. The previously identified TRP photoproduct FICZ was detected in the light-exposed medium, although its contribution to the observed induction response was not determined. Here we report the new observation that aqueous TRP can be oxidized by sunlight to multiple photoproducts that elicit high efficacy CYP1A induction, sustained for at least 48 h in hepatocytes and in vivo and comparable in intensity to the induction by TCDD. The activation occurs simply by exposure to sunlight passing through ordinary window glass without inclusion of any additional photosensitizers such as riboflavin. Our findings show that the indoor environment is a potential source of generation of highly effective CYP1A inducers from TRP.
After 7 days of exposure to sunlight, the induction potency of TRP was increased by 100-fold or more. The photoproducts and inducing capacity of the sunlight activated TRP mixture (aTRP) were stable for months at 4°C.
HPLC analysis in combination with EROD assays showed that aTRP contained over 100 photoxidation products and over 30 inducing compounds, one of which was identified as FICZ. The evidence that FICZ was confined to only one of 14 HPLC-separated fractions of aTRP, and that its subtraction from the aTRP mixture did not affect CYP1A induction by the mixture (see Fig. 9), shows that FICZ is not critical to the inducing capacity of the aTRP mixture. The data in Figure 7 showing that F11 was metabolism sensitive are in agreement with reports that FICZ is rapidly metabolized (Wei et al., 2000) and further indicate that FICZ is not among the metabolism-resistant compounds in aTRP. The data show that aTRP contains high-efficacy inducers in addition to FICZ that remain to be identified. As shown in Figure 8, several components in aTRP, in particular subfractions q in F1, d in F4, and d in F5, were found to be particularly effective inducers, and they are leading candidates for further investigation.
As aTRP contained only small amounts of FICZ (calculated at 72 pM at 1x, for the sample in Fig. 5) which could entirely account for the induction by Fraction 11 of aTRP, the results indicate the high potency and efficacy of FICZ as a CYP1A inducer in the chick hepatocyte model. The estimate of 72 pM FICZ in 1x aTRP is reasonably consistent with the finding of Oberg et al. (2005) that 8 pM of FICZ was generated in tissue culture medium after 24 h of exposure to incandescent light, considering the differences in exposures and the difficulties in measuring the small amounts of FICZ produced.
Characterization of the inducing components in the aTRP mixture will be useful to identify the high-efficacy CYP1A-inducing photoproducts in aTRP and to understand their contribution to the induction effects of the mixture, but the mixture may in fact be more relevant than its components to human exposures, as light produces mixtures of TRP photoproducts, and human effects are likely to reflect exposures to such mixtures. The amount of TRP exposed at the window may seem high, but exposure at 10x, 1/10 of the standard levels used here (100x) produced the same effects and the same HPLC photoproducts as 100x (data not shown). Moreover, 100x TRP (6.9 mM TRP) is close to concentrations that might be expected for food products that could potentially be exposed to high-intensity light (dairy products contain up to 3.5 mM TRP (http//www.nationaldairycouncil.org)).
A biphasic CYP1A induction response to aTRP, with inhibition at 10x, shown in Figure 3, was also found for high concentrations of other indole-related inducers, indole-3-acetate and indirubin (Adachi et al., 2004; Bittinger et al., 2003). Inhibition of CYP1A activity at high concentrations of indole-related inducers could represent a means by which induction effects of such inducers could be limited in vivo. 10x aTRP did not appear to be cytotoxic, in so far as the appearance of the cells and protein measurements were unchanged and CYP levels were not depressed. Further, H2O2 levels in hepatocytes were not increased by 10x aTRP (data not shown). The evidence that the sum of EROD levels found for the components of the mixture exceeded the EROD activity of the mixture when tested at concentrations equivalent to 0.1x and higher (see Fig. 6) is compatible with competition among inducing components in the aTRP mixture for binding to the AhR or at CYP1A catalytic sites.
The rapid metabolism of the inducers in Std. Ham's by hepatocytes or liver microsomes seen in Figure 4 is consistent with the transience of the induction response to medium change with Std. Ham's. The greater resistance to metabolism of aTRP could contribute to its more sustained effects. As shown in Figure 7, the aTRP mixture contains inducers which are sensitive to metabolism, as well as inducers which are resistant. The metabolism resistance could reflect intrinsic insensitivity to metabolism, but could also be due to the amounts of the inducers in aTRP exceeding the metabolic capacity of the chick embryo hepatocytes or to the presence of inhibitors of metabolism in the aTRP fractions. Further studies will be needed to discriminate among those possibilities.
The increase in CYP-dependent AA metabolism by aTRP is significant because epoxygenase metabolites of AA have many biologic effects, including the capacity to increase intracellular calcium and alter cell proliferation and vascular function (Capdevila et al., 2000). While the ability of CYP1B1 to metabolize endogenous substrates such as estrogens is well recognized (Murray et al., 2001), CYP1A is commonly held to be involved in metabolism or activation of toxic xenobiotics. The findings show that CYP1A activated by AhR ligands, to which people are likely to be more commonly exposed than to toxic xenobiotics like TCDD, can alter production of endogenous AA metabolites.
As UVC is screened by the atmosphere, and UVB by window glass, only UVA and visible light (400–700 nm) are likely to contribute to the photoactivation by sunlight. Fluorescent bulbs emit mainly visible light with very small amounts of UVA. Even if visible light does not activate TRP directly, it could participate in the effects seen by further oxidizing TRP photoproducts initially generated by UVA (Davies and Truscott, 2001). Identification of the components in sunlight producing the photoactivation described here will require further investigation.
We show here that sunlight, even when screened by window glass, is sufficiently powerful to produce TRP photoproducts causing high-intensity CYP1A induction. Exposures for periods of weeks to such conditions can occur in the work environment. The toxicological or physiologic consequences of the generation of CYP1A inducers of TRP by light have yet to be determined. Wei et al. (1999) suggested that light may be a transducer of chemical signals involving the AhR. The AhR activation in response to such signals could be involved in regulation of normal homeostatic processes. The possibility that ingestion of food or dietary supplements containing TRP exposed to light either during or after manufacturing could lead to CYP1A induction in vivo gains credence from the evidence that aTRP was able to induce CYP1A in vivo.
Sunlight might oxidize TRP in proteins as well as in aqueous solutions, as TRP in proteins is also readily oxidized (Creed, 1984). The ability of UVA to penetrate through the epidermis and dermis to subdermal blood vessels (Moan, 2001) raises the further possibility that exposure to sunlight could lead to CYP1A induction in diverse organs via photoactivation of TRP in skin or by other routes, such as the eye (Davies and Truscott, 2001), or via the circulation. Experimental exposure to UV light can photoactivate TRP in skin (Brancaleon et al., 1999) and induce CYP1A in skin and liver in humans and animals (Goerz et al., 1996; Katiyar et al., 2000). UVA as well as UVB have been implicated in skin cancer and aging (He et al., 2004).
Seasonal changes in TRP activation by sunlight may also explain the long-known but still unexplained activation of aryl hydrocarbon hydroxylase in human lymphocytes and its seasonal variation (Paigen et al., 1981). In fact, we found that the ability of different preparations of aTRP to induce CYP1A varied seasonally (Supplemental Data, item 2). While the findings here focus on CYP1A induction effects of aTRP, a wide range of systemic effects involving TRP or its metabolites are known to be affected by sunlight, including mood and immune system function (see Allegri et al., 2003, for a comprehensive overview). These also warrant consideration as potentially significant biological consequences of TRP activation by sunlight.
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SUPPLEMENTARY DATA |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
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Supplementary data are available online at www.toxsci.oxfordjournals.org.
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ACKNOWLEDGMENTS |
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We thank Jos van Geffen at the Belgian Institute for Space Aeronomy for assistance with the calculations of solar UV and visible light, Dr. Steven Gross for use of the fluorescence plate reader, and Payal Ram for fluorescence measurements. This work was supported by NIH grant ES 03606 (to A.B.R.) and T32 CA062948 (S.D-M.).
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 S. Diani-Moore, F. Papachristou, E. Labitzke, and A. B. Rifkind INDUCTION OF CYP1A AND CYP2-MEDIATED ARACHIDONIC ACID EPOXYGENATION AND SUPPRESSION OF 20-HYDROXYEICOSATETRAENOIC ACID BY IMIDAZOLE DERIVATIVES INCLUDING THE AROMATASE INHIBITOR VOROZOLE Drug Metab. Dispos., August 1, 2006; 34(8): 1376 - 1385. [Abstract] [Full Text] [PDF]
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