Identification of the Tryptophan Photoproduct 6-Formylindolo[3,2-b]carbazole, in Cell Culture Medium, as a Factor That Controls the Backgrou
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《毒物学科学杂志》
Institute of Environmental Medicine, Karolinska Institutet, SE-171 77 Stockholm, Sweden
Division of Cellular and Genetic Toxicology, Stockholm University, Stockholm, Sweden
ABSTRACT
The presence of high affinity ligands for the aryl hydrocarbon receptor (AhR) in cell culture medium has generally been overlooked. Such compounds may confound mechanistic studies of the important AhR regulatory network. Numerous reports have described that light exposed cell culture medium induces AhR-dependent activity. In this study, we aimed at identifying the causative substance(s). A three-dimensional factorial design was used to study how the background activity of CYP1A1 in a rat hepatoma cell line (MH1C1) was controlled by photoproducts formed in the medium exposed to normal laboratory light. The light induced activity was found to be tryptophan dependent, but independent of riboflavin and other components in the medium. The light exposed medium showed the same transient enzyme inducing activity in vitro as the AhR ligand 6-formylindolo[3,2-b]carbazole (FICZ). This substance, which we have previously identified as being formed in UV-exposed tryptophan solutions, is a substrate for CYP1A1 and it has a higher AhR binding affinity than TCDD. Several tryptophan related photoproducts were detected in the light-exposed medium. For the first time one of the formed photoproducts was identified as FICZ with bioassay driven fractionation coupled with HPLC/MS. These results clearly show that tryptophan derived AhR ligands, which have been suggested to be endogenous AhR ligands, influence the background levels of CYP1A1 activity in cells in culture.
Key Words: tryptophan; 6-formylindolo[3,2-b]carbazole; light; aryl hydrocarbon receptor.
INTRODUCTION
The physiological roles of the aryl hydrocarbon receptor (AhR) as well as the possible endogenous ligand(s) are still mostly unknown. The AhR belongs to the PAS family of proteins (Taylor and Zhulin, 1999). It contains a basic helix-loop-helix (bHLH) motif, but differs from most other PAS proteins by its ligand dependent activity. High-affinity ligands to AhR are lipophilic aromatic compounds that are planar or can become planar. By far the most studied AhR ligand is the persistent pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). (See Denison and Nagy, 2003, for a review.)
The AhR-ligand complex dimerizes with the AhR-nuclear-translocator (ARNT) and forms the AhR-ARNT heterodimer, which functions as an ubiquitous transcription factor and as a transcriptional coactivator (Carlson and Perdew, 2002; Ohtake et al., 2003; Puga et al., 2000). The most studied gene product and protein that is strongly induced via the liganded AhR-ARNT heterodimer is the phase I mono-oxygenase cytochrome P450 1A1 (CYP1A1). This enzyme is highly inducible in liver and in liver cell lines it is the primary enzyme that catalyzes the ethoxyresorufin-O-deethylase (EROD) activity. CYP1A1 has many endogenous and exogenous substrates and is involved in the metabolism of, e.g., polycyclic aromatic hydrocarbons into reactive and DNA-adduct forming intermediates. Because of its ubiquitous nature and evolutionary conservation, it has been hypothesized that CYP1A1 is essential for survival (Nebert and Gonzalez, 1987). However, mice with a targeted knockout of the CYP1A1 gene do not show any specific phenotype (Dalton et al., 2000).
A diurnal variation in AhR, ARNT, and CYP1A1 levels at protein or mRNA levels has been described (Huang et al., 2002; Plewka et al., 1992; Richardson et al., 1998) and in neonatal rats and in livers of mice, UV-light has been shown to increase CYP1A1 activity (Goerz et al., 1983; Mukhtar et al., 1986). In human skin, UV-B exposure results in dose-dependent induction of both CYP1A1, CYP1B1 and CYP2S1 (Katiyar et al., 2000; Smith et al., 2003). There is also a seasonal variation in CYP1A1 activity and CYP1B1 mRNA abundance in human lymphocytes, which show highest activity during the late summer and early fall (Paigen et al., 1981; Tuominen et al., 2003). In vitro, our previous results showed that the CYP1A1 mRNA levels were enhanced by UV irradiation in the presence of tryptophan (Wei et al., 1999). This result and the previous findings of high AhR binding affinity of tryptophan photoproducts (Rannug et al., 1987), indicates that tryptophan mediates UV-induced CYP1A1 expression. A specific photoproduct of tryptophan, 6-formylindolo[3,2-b]carbazole (FICZ) that can bind to and activate the AhR with a high specificity has been identified (Fig. 1) (Rannug et al., 1995).
Tryptophan is an essential amino acid present in all normal media used for cell culturing. We hypothesized that also normal laboratory light (with low energy) can cause formation of FICZ or other closely related compounds in the medium and thereby induce AhR dependent gene expression. There are several reports on CYP1A1 induction that can be explained by the formation of tryptophan oxidation products with high AhR affinity, in medium (Harvey et al., 1998; Kocarek et al., 1993; Lorenzen et al., 1993; Nemoto and Sakurai, 1991; Paine, 1976; Sadar and Andersson, 2001; Segner et al., 2000). In the medium, there are several components, beside tryptophan, that may form potent photoproducts. One well-known photosensitizer is riboflavin (vitamin B2). This substance may increase any amount of photooxidized products. Others have revealed that tryptophan in parental nutrition solutions containing riboflavin, when exposed to constant illumination, causes hepatic dysfunction in neonates that could result from the presence of photooxidized tryptophan (Bhatia and Rassin, 1985; Zaman et al., 1996).
Since the AhR is a cytosolic transcription factor that is involved in cell regulatory processes such as proliferation and differentiation, control of its constitutive activity is important in many experimental systems. As a factor that could limit the reliability of the information obtained from the extensive use of AhR dependent bio-assays it is also important to analyze if and to what extent light can influence the transcription and activity level. The aim of this project was to study and quantify the impact of light, in combination with different medium components, on the CYP1A1 activity in vitro. In addition, we aimed at identifying the active photoproduct(s).
MATERIALS AND METHODS
Chemicals.
L-tryptophan was purchased from Fluka Chemie (Switzerland, Lot no. 374921/1) and of 99.5 % purity. FICZ was synthesized and kindly provided by Dr. J. Bergman, NOVUM, Karolinska Institutet. TCDD (Dow Chemical, Midland, MI, Lot no. 851:144 II) was used under appropriate safety precautions. Riboflavin was purchased from Sigma-Aldrich (Japan) and Dimethyl sulfoxide (DMSO) from Fluka Chemie (Switzerland).
Medium and cell culturing.
The rat hepatoma cell line MH1C1 was grown in Dulbecco's Modified Eagle Medium, (DMEM) (Cat. No 41965-039, Life Technologies, Germany) supplemented with 10% fetal bovine serum, sodium pyruvate (0.6 mM), and L-glutamate (3.8 mM). This standard medium contained 16 mg tryptophan and 0.4 mg riboflavin per liter, but was also used in a tryptophan- and riboflavin-free version (Life Technologies, Germany) in experiments with variable concentrations of these constituents. Conditions in the incubator were 37°C, 95% humidified air, and 10% CO2.
Light exposure.
The medium was exposed to ordinary laboratory light either from the light source in the sterile bench (two fluorescent tubes, OSRAM L 36W/31–830) or from a normal 40 W light bulb (THORN, Frosted) at a distance of 0.5 m. The regular room lightening and daylight exposure through the windows of the laboratory (no direct sunshine) was also allowed during the light exposure periods. The ratios between UV light (250–400 nm) and visible light (400–800 nm) were 0.04 and 0.005 for the fluorescent tubes and the light bulb, respectively. The medium was exposed to light in the original plastic bottles (Life Sciences, Germany) and exposure to control bottles was avoided by covering the bottles with aluminium foil.
CYP1A1-activity.
The CYP1A1 enzyme activity was measured as EROD activity in the MH1C1 cell line essentially as described earlier (berg et al., 2001; Pohl and Fouts, 1980). Cells were seeded into 96-well plates, at a density of 20,000 cells per well, in 0.2 ml of medium. After 24 h of pre-culturing, the cells were exposed to TCDD, FICZ, or light exposed medium. TCDD, at a concentration of 155 pM (giving 100% activity), was used on every plate as a positive control and to enable expression of TCDD normalized activity. The compounds were dissolved in DMSO at a final concentration of 0.5%. After exposure, each well was washed twice; first with 1% BSA in PBS and then only with PBS. The plates were stored at –80°C until further analysis. After thawing, the formation of resorufin from ethoxyresorufin was determined fluorimetrically (544 nm/590 nm; ex/em), using wells without co-factors (NADPH, MgSO4) as blank and cells exposed to only DMSO as background (0% activity).
Experimental designs.
To study if light exposed medium could increase the background activity of CYP1A1 initial experiments were performed with a standard protocol using 24 h incubation and standard medium (16 mg tryptophan/l) exposed to light for 0, 3, or 12 h in the sterile bench (fluorescent tubes). The CYP1A1 inducing potency was measured with the EROD-assay after addition of light exposed medium directly after the light exposure and of light exposed medium that had been stored for three days in a refrigerator (4°C in the dark).
A full factorial 23 design was used to study whether variations in tryptophan and serum per se without light exposure could influence the sensitivity of the EROD-assay. Each component (tryptophan, serum, and TCDD) was tested at three dose-levels: tryptophan (0, 16, or 32 mg/l), serum (0, 5, or 10%) and TCDD (0.15, 1.5, or 155 pM).
To find the importance of tryptophan and riboflavin in combination with light exposure, a 23 full factorial design was applied, in which each component (tryptophan, riboflavin, and light exposure) was tested at three dose-levels: tryptophan (8, 16, or 24 mg/l), riboflavin (0.1, 0.4, or 0.7 mg/l), and light from fluorescent tubes (0, 12, or 24 h). A specially ordered cell culture medium without tryptophan and riboflavin was used.
In order to compare the CYP1A1-inducing capacity of FICZ and TCDD full sigmoidal dose-response and time-response curves in the EROD-assay were created in experiments in which cells were incubated with serially diluted TCDD and FICZ solutions for 3, 6, 12, and 24 h. A serial dilution of TCDD from 155 pM to 0.155 pM and of FICZ from 100,000 pM to 0.1 pM was used. Concentrations giving 50% of maximal induction (EC50) and the relative maximal inductions (Ymax) were determined.
CYP1A1 inducing potency of the light exposed medium was compared to the potency of FICZ in one experiment in which tryptophan- and riboflavin-rich medium (24 and 0.7 mg/l, respectively) was exposed to light (fluorescent tubes) in the sterile bench for 24 h. The MH1C1 cells were then incubated in the light exposed medium for 3, 6, 12, and 24 h and compared to cells incubated for the same length of time with a fixed concentration of FICZ (100 pM).
Chemical fractionation and analysis of medium.
To identify photoproduct(s) formed in the medium by exposure to ordinary laboratory light, 500 ml plastic bottles of DMEM was prepared as described above. The medium was exposed to light from a light bulb or covered with aluminium foil in the laboratory for 24 h. Serum was excluded from the medium and the tryptophan levels were fixed to either 0 or 16 mg/l. After exposing the medium to light, the medium was concentrated using Sep-Pak C18 cartridges (Waters). The cartridges were washed with water and eluted with 5 ml acetone. The extract corresponding to one light exposed bottle of medium was evaporated to dryness using a rotavapor and further dissolved in 400 μl methanol. Prior to fractionation a part of the methanol extract was diluted with H2O (1:1). Fractionations and analyses of the media extracts were performed by means of HPLC. Seven fractions were collected at different retention times (4–10 min [F1], 10–13 min [F2], 13–16 min [F3], 16–21 min [F4], 21–27 min [F5], 27–33 min [F6], and 33–40 min [F7]) and subsequently evaporated to dryness using a sped-vac centrifuge. The dried fractions were stored at –20°C and dissolved in 120 μl DMSO and tested in the EROD-assay.
HPLC analyses and fractionations were performed using a Merck Hitatchi LaChrom instrument equipped with a L-7100 pump, a L-7455 diod array detector, and a Shimadzu RF-535 fluorescence HPLC monitor, excitation 390 nm and emission 525 nm. A reverse phase Kromasil 100–5C18 column (250 x 4.6 mm) from Scantec (Sweden) was employed for analysis and separation. A 40 min linear mobile phase gradient from 5% B to 85% B (A: 0.1% TFA in H2O, B: 0.1% TFA in acetonitrile) was used at a flow rate of 1 ml/min. The column was washed with 100% B for 10 min between injections.
To confirm a formation of FICZ in the light exposed cell medium a known amount of FICZ standard solution (0.7 pmol) was added to a part (approximately 1/6) of the methanol extract of the concentrated cell medium. The enhancement in fluorescence of the peak assumed to contain FICZ was analyzed by HPLC. A further identification was performed by mass spectrometric (MS) analysis performed by adding 20 μl of acetonitrile to a dried fraction of the second peak in fraction number 7 (F7) and 10 μl of it was injected into a C-18 HPLC column (5 μm, 250 mm x 4.6 mm i.d., Alltech) using a mobile phase consisting of acetonitrile and water, both containing 1 mM of formic acid. A 40 min gradient from 35 to 85% of acetonitrile followed by 100% of acetonitrile for 10 min with a flow rate of 1.0 ml/min was supplied by two LC pumps (LC-10ADvp, Shimadzu, Japan). The flow was splitted after the HPLC column so that only 0.4 ml/min was transferred to the triple quadrupole MS detector (API 2000, Applied Biosystmes/MDS SCIEX, Canada). The operating parameters used during –ESI were: curtain gas (N2, Aquilo nitrogen generator, The Netherlands) 30 psi, gas 1 and 2 (air) 20 psi, temperature 550°C, ion spray voltage –4500 V, declustering potential –80 V, focusing potential –400 V and entrance potential –10 V. Data were collected using Analyst 1.4 from Applied Biosystems/MDS SCIEX.
Data analysis and statistics.
Results from factorial design analyses were evaluated with Multiple Linear Regression (MLR) using the statistical package Modde 5.0 (Umetrics AB, Ume, Sweden). The goodness of fit of the models were validated by the fraction of variation of the response explained by the model (R2) and the fraction of variation of the response that can be predicted by the model (Q2). Student's t-test was used for pairwise comparisons of enzyme induction between treatment groups.
RESULTS
Ordinary medium exposed to light from the fluorescent tubes in the sterile bench induces CYP1A1 activity via the formation of stable photoproduct(s).
An increased CYP1A1 activity was observed after incubation with medium exposed to light for different length of time (Fig. 2). Already after 3 h of light exposure a significant induction corresponding to 12% of the maximal inducing effect caused by TCDD was seen. Twelve hours exposure of light increased the background CYP1A1 activity to 22% of the maximal TCDD effect. A similar effect was seen also after three days of storage in refrigerator (Fig. 2).
The response to TCDD is independent of variability in tryptophan levels without light exposure.
The CYP1A1 activity was explored and modelled in a three-dimensional space where the concentrations of tryptophan and serum were varied as well as TCDD exposure. The experiment was planned so that normal medium conditions and an activity of about 50% were in the center of the investigated space. The scaled and centered regression coefficients based on MLR analysis showed that tryptophan, with no light exposure, does not influence the CYP1A1 activity and that tryptophan does not influence the effect by TCDD (Table 1). A possible potentiation by serum was suggested (p = 0.06).
Tryptophan in combination with light controls background CYP1A1 activity.
The CYP1A1 activity was explored and modelled in a three-dimensional space where the concentrations of tryptophan and riboflavin were varied as well as light exposure. The experiment was planned so that normal medium conditions and a 12 h light exposure were in the center of the investigated space. The scaled and centered regression coefficients based on MLR analysis showed that light, tryptophan, and their interaction term explained the increased EROD activity (Table 2). Riboflavin did not influence the activity significantly; neither did any other interaction terms. After excluding the non-significant regression coefficients (related to riboflavin) a refined quantitative model was formed, describing the influence of tryptophan and light on CYP1A1 activity (Fig. 3). The goodness of fit (R2) was 0.87 and the goodness of prediction (Q2), based on internal cross validation, was 0.82.
TCDD and FICZ dose-response and time-response comparisons.
Full sigmoidal dose-response curves were generated for FICZ and TCDD. EROD activity was quantified after 3, 6, 12, and 24 h of incubation with serial dilutions of FICZ and TCDD. The results show that the EC50 value for TCDD was stable at about 16 pM, whereas the EC50 value for FICZ varied from 34 pM to 830 pM depending on the time the cells were incubated prior to enzyme activity measurement (Table 3). An average maximal induction (Ymax) of about 127% was observed for FICZ compared to TCDD.
The illuminated medium causes transient CYP1A1 induction.
Using a short incubation time (3 h), the CYP1A1-induction caused by FICZ (100 pM) and by the medium (24 mg tryptophan/l) exposed to light from fluorescent tubes (24 h) was found to be up to 50% higher than the maximal effect observed with TCDD (Table 3 and Fig. 4). The relative activity decreased with as much as 80% if the incubation time was extended to 24 h.
Several tryptophan-related photoproducts are formed.
Cell culture media with or without tryptophan (16 mg/l) were exposed to light (light bulb) for 24 h and then fractionated into seven fractions to enable an identification of the formed photoproduct(s). In parallel with fractionation, the light-exposed unfractionated medium was tested for CYP1A1 inducing activity to compare with the results obtained with the light exposure from fluorescent tubes. The result was qualitatively consistent with the results presented in Figure 3: All seven fractions were tested for CYP1A1 activity in the EROD-assay and significant tryptophan dependent effects were observed in the non-polar fractions 4–7 (Fig. 5).
Light-exposed medium with and without tryptophan was analyzed with reverse-phase HPLC using both fluorescence (390/525 nm) and UV (254 nm) detectors (Fig. 6). The large majority of the components were eluted at retention times less than 20 min. Four distinct fluorescent peaks were observed in fractions 5 to 7 [F5, 25 min; F6, 31 min; F7, 36 and 37.5 min] in the tryptophan containing and illuminated medium (Fig. 7). No such peaks were observed in fraction 4.
Identification of FICZ.
A known amount of FICZ was added to the crude methanol extract to verify if the most non-polar peak in fraction 7 was identical with FICZ (data not shown). After addition of FICZ the peak at 37.5 min was increased, confirming a formation of the tryptophan photoproduct FICZ. Compared to a known amount of FICZ, the peak area of FICZ in the concentrated cell medium was established to be 0.7 pmol and the total concentration in the cell medium was subsequently extrapolated to about 8 pM.
The presence of FICZ in the fraction 7 was further confirmed by LC/MS. A standard solution of FICZ as well as the fraction were ionized by –ESI and analyzed by SIM (Selective Ion Monitoring). Both the parent ion (m/z 283) and the fragment (m/z 255), which results from the loss of the aldehyde group were detected (Fig. 8).
DISCUSSION
Since the first observation in 1976 by Paine (Paine, 1976) light and culture-medium related induction of CYP1A1 has repeatedly been reported without any further mechanistic explanations (Harvey et al., 1998; Kocarek et al., 1993; Lorenzen et al., 1993; Nemoto and Sakurai, 1991; Paine and Francis, 1980; Sadar and Andersson, 2001; Segner et al., 2000; Sindhu et al., 1996). Others have observed that cell lines with a defective CYP1A1 gene, and thereby lacking CYP1A1-activity, express high levels of mRNA for CYP1A1 and other AhR dependent gene products even when they are grown without TCDD (Hankinson et al., 1985). One explanation for this high background CYP1A1 mRNA expression that has been put forward is that the CYP1A1 deficient cells accumulate an AhR agonist that is also a substrate for CYP1A1 (Hankinson et al., 1985; RayChaudhuri et al., 1990). The agonist was suggested to be an intermediary metabolite (i.e., an endogenous ligand) and/or a component of the medium. The present results together with our earlier published observations that the tryptophan photoproduct causes sustained CYP1A1 mRNA expression in cells deficient in CYP1A1 enzyme activity (Wei et al., 2000) support the above mentioned observations but also offer a molecular explanation for the medium component theory (cf. below).
Three days of storage in refrigerator did not influence the effect of illuminated medium, which shows that the formed photoproduct(s) is stable in stored medium (Fig. 2). This result has implications on handling the medium since the photoproducts may be formed whenever the medium bottles are exposed to light, even long time before it is used, e.g., during production, transport, and storage. The induction of CYP1A1 activity after 3 and 12 h of incubation were equivalent to the effect obtained with about 50 and 110 pM FICZ, respectively. This means that only approx. 10–6 of the tryptophan normally available in cell culture medium (16 mg/l = 78 μM in DMEM) needs to be photooxidized to FICZ to explain the increased CYP1A1 activity.
Tryptophan did not influence the EROD-assay per se or the response to TCDD (Table 1). However, serum gave a subtle increase in the response and slightly potentiated the effect of TCDD (however without statistical significance). The observed serum effect is in accordance with previous findings by others that serum can be a CYP1A1 mRNA inducer in hepatic cells (N'Guyen et al., 2002), that the CYP1A1 induction by 3-methylcholanthrene is potentiated by serum (Guigal et al., 2001; N'Guyen et al., 2002), and that serum withdrawal leads to reduced AhR expression and loss of CYP1A inducibility (Hestermann et al., 2002). In contrast, others have reported that serum significantly lower the CYP1A1 inducing potency of TCDD as a result of decreased bioavailability in the presence of serum, effectively reducing the concentration of ligand within the cells (Hestermann et al., 2000). This was not confirmed in the present study.
The present study showed that tryptophan and light together explained 87% of the variation in background CYP1A1 activity explored and modeled in a three-dimensional space where the concentrations of tryptophan and riboflavin were varied together with light exposure.
The CYP1A1 induction caused by illuminated medium showed a maximum at short incubation times (Fig. 4). A possible explanation is metabolic breakdown of the formed photoproduct(s). We have earlier described that CYP1A1 can metabolize FICZ and that the levels of induction are dependent on the incubation time (Bergander et al., 2003, 2004; Wei et al., 2000). Others have also described UV-activated tryptophan and indolo[3,2-b]carbazole-induced CYP1A1 expression as transient (Chen et al., 1995; Kocarek et al., 1993) which is in accordance to the effects seen with other rapidly metabolized compounds (Riddick et al., 1994). It seems very plausible that the high background CYP1A1 mRNA (and lack of induction by TCDD) in CYP1A1 deficient Hepa-1 c37 cells (Hankinson et al., 1985) is caused by an inability of such mutant cells to degrade tryptophan photoproducts via CYP1A1. In the present study it was also observed that the drop in activity with time for photo-induced medium was almost identical to the drop of activity seen for FICZ, which indirectly supports the hypothesis that the induction is caused by FICZ or related compounds.
Significant tryptophan dependent effects were observed in the non-polar fractions 4 to 7. At a higher resolution, four distinct fluorescent peaks were observed in fractions 5 to 7 in the tryptophan-containing light exposed medium, which correlated with CYP1A1 activity in these fractions. In fraction 4, which also showed a significant biological activity, no tryptophan specific peaks were detected. This could be due to a high general HPLC response in this fraction that might have masked the tryptophan related peaks. However, the excitation/emission wavelengths used in this study are specific for the fluorescence properties of FICZ. Compounds exhibiting fluorescence at different wavelengths are therefore not detected in this system.
In the fluorescence chromatogram of fraction 7 of the light exposed cell medium containing tryptophan, two peaks were observed. By adding a known amount of FICZ to the crude methanol extract the most non-polar peak in fraction 7 was found to have identical retention time and fluorescence with FICZ. The identity was further confirmed by MS analysis that showed identical retention time and mass spectrum as FICZ (Fig. 8). The total concentration of FICZ in the cell medium was calculated to be about 8 pM. This concentration may explain a major part of the inducing effect of tryptophan-containing unfractionated medium exposed to light. By using a dose-response curve for FICZ, the FICZ equivalent concentration was estimated to be about 10 pM in unfractionated medium exposed to light from a light bulb for 24 h. The higher FICZ equivalent concentrations (100 pM) indicated by the model in Figure 3 might be explained by the fact that the fluorescent light tubes have a spectra with a higher portion of UV compared with the light bulb.
Tryptophan is present in biological systems both as free amino acid and in proteins. It is the precursor of several signal substances including serotonin, melatonin, tryptamine, quinolinic acid, and kynurenic acid (Hayaishi, 1993). We have hypothesized that FICZ and structurally related tryptophan photooxidation products might function as chemical messengers that activate AhR in response to light (Rannug et al., 1987; Wei et al., 2000). Other members of the PAS domain superfamily are also known to function as sensors of light and are involved in circadian rhythmicity, which corroborates a role of the AhR in light sensing (Taylor and Zhulin, 1999). Also, in support of this hypothesis, we have observed a diurnal expression of CYP1A1 and Period (Per2) genes in the posterior pituitary and the liver of untreated rats. The CYP1A1 and Per2 mRNA levels were found to be highly coordinated, indicating a possible interaction between AhR signaling and circadian response pathways (Huang et al., 2002). This indicates that the AhR may have a role in sensing light of different wavelengths. A thorough investigation of photoproduct formation at different wavelengths needs to be done. The result presented here shows that FICZ is produced not only under controlled, high energy UV-irradiation but also by ordinary laboratory light conditions. The slightly more polar fluorescent compounds seen in fractions 5, 6, and 7 have not yet been chemically identified. How much each photoproduct contributes to the inducing capacity of illuminated medium remains to be quantified.
In conclusion, this study shows that normal laboratory light can significantly induce the background CYP1A1 activity through the formation of several tryptophan related photoproducts in light exposed medium. For the first time one of the formed photoproducts was identified as FICZ. These results clearly show that tryptophan derived AhR ligands, which have been suggested to be endogenous AhR ligands, influence the constitutive levels of CYP1A1 activity in cells in culture. As a consequence, composition as well as handling of cell culture medium can cause varying levels of AhR ligands, which in turn can lead to artificial differences between experiments and cell types. Especially, in studies with short incubation times, the contribution from tryptophan-derived factors in medium may have considerable influence on the results. The presence of high affinity AhR ligands in the medium must therefore be controlled in work that aims at identifying AhR agonists and antagonists, and especially when conducting mechanistic studies of genes that are activated through the AhR or that are co-activated or co-repressed through the interaction with the AhR complex.
NOTES
1 The authors have contributed equally to this work.
ACKNOWLEDGMENTS
The authors gratefully acknowledge Nahid Amini at the Department of Analytical Chemistry, Stockholm University for performing the MS analyses. This study was performed with the financial support from Karolinska Institutet Research Funds, Swedish Fund for Research without Animal Experiments, the Swedish National Board for Laboratory Animals, and the Swedish Radiation Protection Institute.
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Division of Cellular and Genetic Toxicology, Stockholm University, Stockholm, Sweden
ABSTRACT
The presence of high affinity ligands for the aryl hydrocarbon receptor (AhR) in cell culture medium has generally been overlooked. Such compounds may confound mechanistic studies of the important AhR regulatory network. Numerous reports have described that light exposed cell culture medium induces AhR-dependent activity. In this study, we aimed at identifying the causative substance(s). A three-dimensional factorial design was used to study how the background activity of CYP1A1 in a rat hepatoma cell line (MH1C1) was controlled by photoproducts formed in the medium exposed to normal laboratory light. The light induced activity was found to be tryptophan dependent, but independent of riboflavin and other components in the medium. The light exposed medium showed the same transient enzyme inducing activity in vitro as the AhR ligand 6-formylindolo[3,2-b]carbazole (FICZ). This substance, which we have previously identified as being formed in UV-exposed tryptophan solutions, is a substrate for CYP1A1 and it has a higher AhR binding affinity than TCDD. Several tryptophan related photoproducts were detected in the light-exposed medium. For the first time one of the formed photoproducts was identified as FICZ with bioassay driven fractionation coupled with HPLC/MS. These results clearly show that tryptophan derived AhR ligands, which have been suggested to be endogenous AhR ligands, influence the background levels of CYP1A1 activity in cells in culture.
Key Words: tryptophan; 6-formylindolo[3,2-b]carbazole; light; aryl hydrocarbon receptor.
INTRODUCTION
The physiological roles of the aryl hydrocarbon receptor (AhR) as well as the possible endogenous ligand(s) are still mostly unknown. The AhR belongs to the PAS family of proteins (Taylor and Zhulin, 1999). It contains a basic helix-loop-helix (bHLH) motif, but differs from most other PAS proteins by its ligand dependent activity. High-affinity ligands to AhR are lipophilic aromatic compounds that are planar or can become planar. By far the most studied AhR ligand is the persistent pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). (See Denison and Nagy, 2003, for a review.)
The AhR-ligand complex dimerizes with the AhR-nuclear-translocator (ARNT) and forms the AhR-ARNT heterodimer, which functions as an ubiquitous transcription factor and as a transcriptional coactivator (Carlson and Perdew, 2002; Ohtake et al., 2003; Puga et al., 2000). The most studied gene product and protein that is strongly induced via the liganded AhR-ARNT heterodimer is the phase I mono-oxygenase cytochrome P450 1A1 (CYP1A1). This enzyme is highly inducible in liver and in liver cell lines it is the primary enzyme that catalyzes the ethoxyresorufin-O-deethylase (EROD) activity. CYP1A1 has many endogenous and exogenous substrates and is involved in the metabolism of, e.g., polycyclic aromatic hydrocarbons into reactive and DNA-adduct forming intermediates. Because of its ubiquitous nature and evolutionary conservation, it has been hypothesized that CYP1A1 is essential for survival (Nebert and Gonzalez, 1987). However, mice with a targeted knockout of the CYP1A1 gene do not show any specific phenotype (Dalton et al., 2000).
A diurnal variation in AhR, ARNT, and CYP1A1 levels at protein or mRNA levels has been described (Huang et al., 2002; Plewka et al., 1992; Richardson et al., 1998) and in neonatal rats and in livers of mice, UV-light has been shown to increase CYP1A1 activity (Goerz et al., 1983; Mukhtar et al., 1986). In human skin, UV-B exposure results in dose-dependent induction of both CYP1A1, CYP1B1 and CYP2S1 (Katiyar et al., 2000; Smith et al., 2003). There is also a seasonal variation in CYP1A1 activity and CYP1B1 mRNA abundance in human lymphocytes, which show highest activity during the late summer and early fall (Paigen et al., 1981; Tuominen et al., 2003). In vitro, our previous results showed that the CYP1A1 mRNA levels were enhanced by UV irradiation in the presence of tryptophan (Wei et al., 1999). This result and the previous findings of high AhR binding affinity of tryptophan photoproducts (Rannug et al., 1987), indicates that tryptophan mediates UV-induced CYP1A1 expression. A specific photoproduct of tryptophan, 6-formylindolo[3,2-b]carbazole (FICZ) that can bind to and activate the AhR with a high specificity has been identified (Fig. 1) (Rannug et al., 1995).
Tryptophan is an essential amino acid present in all normal media used for cell culturing. We hypothesized that also normal laboratory light (with low energy) can cause formation of FICZ or other closely related compounds in the medium and thereby induce AhR dependent gene expression. There are several reports on CYP1A1 induction that can be explained by the formation of tryptophan oxidation products with high AhR affinity, in medium (Harvey et al., 1998; Kocarek et al., 1993; Lorenzen et al., 1993; Nemoto and Sakurai, 1991; Paine, 1976; Sadar and Andersson, 2001; Segner et al., 2000). In the medium, there are several components, beside tryptophan, that may form potent photoproducts. One well-known photosensitizer is riboflavin (vitamin B2). This substance may increase any amount of photooxidized products. Others have revealed that tryptophan in parental nutrition solutions containing riboflavin, when exposed to constant illumination, causes hepatic dysfunction in neonates that could result from the presence of photooxidized tryptophan (Bhatia and Rassin, 1985; Zaman et al., 1996).
Since the AhR is a cytosolic transcription factor that is involved in cell regulatory processes such as proliferation and differentiation, control of its constitutive activity is important in many experimental systems. As a factor that could limit the reliability of the information obtained from the extensive use of AhR dependent bio-assays it is also important to analyze if and to what extent light can influence the transcription and activity level. The aim of this project was to study and quantify the impact of light, in combination with different medium components, on the CYP1A1 activity in vitro. In addition, we aimed at identifying the active photoproduct(s).
MATERIALS AND METHODS
Chemicals.
L-tryptophan was purchased from Fluka Chemie (Switzerland, Lot no. 374921/1) and of 99.5 % purity. FICZ was synthesized and kindly provided by Dr. J. Bergman, NOVUM, Karolinska Institutet. TCDD (Dow Chemical, Midland, MI, Lot no. 851:144 II) was used under appropriate safety precautions. Riboflavin was purchased from Sigma-Aldrich (Japan) and Dimethyl sulfoxide (DMSO) from Fluka Chemie (Switzerland).
Medium and cell culturing.
The rat hepatoma cell line MH1C1 was grown in Dulbecco's Modified Eagle Medium, (DMEM) (Cat. No 41965-039, Life Technologies, Germany) supplemented with 10% fetal bovine serum, sodium pyruvate (0.6 mM), and L-glutamate (3.8 mM). This standard medium contained 16 mg tryptophan and 0.4 mg riboflavin per liter, but was also used in a tryptophan- and riboflavin-free version (Life Technologies, Germany) in experiments with variable concentrations of these constituents. Conditions in the incubator were 37°C, 95% humidified air, and 10% CO2.
Light exposure.
The medium was exposed to ordinary laboratory light either from the light source in the sterile bench (two fluorescent tubes, OSRAM L 36W/31–830) or from a normal 40 W light bulb (THORN, Frosted) at a distance of 0.5 m. The regular room lightening and daylight exposure through the windows of the laboratory (no direct sunshine) was also allowed during the light exposure periods. The ratios between UV light (250–400 nm) and visible light (400–800 nm) were 0.04 and 0.005 for the fluorescent tubes and the light bulb, respectively. The medium was exposed to light in the original plastic bottles (Life Sciences, Germany) and exposure to control bottles was avoided by covering the bottles with aluminium foil.
CYP1A1-activity.
The CYP1A1 enzyme activity was measured as EROD activity in the MH1C1 cell line essentially as described earlier (berg et al., 2001; Pohl and Fouts, 1980). Cells were seeded into 96-well plates, at a density of 20,000 cells per well, in 0.2 ml of medium. After 24 h of pre-culturing, the cells were exposed to TCDD, FICZ, or light exposed medium. TCDD, at a concentration of 155 pM (giving 100% activity), was used on every plate as a positive control and to enable expression of TCDD normalized activity. The compounds were dissolved in DMSO at a final concentration of 0.5%. After exposure, each well was washed twice; first with 1% BSA in PBS and then only with PBS. The plates were stored at –80°C until further analysis. After thawing, the formation of resorufin from ethoxyresorufin was determined fluorimetrically (544 nm/590 nm; ex/em), using wells without co-factors (NADPH, MgSO4) as blank and cells exposed to only DMSO as background (0% activity).
Experimental designs.
To study if light exposed medium could increase the background activity of CYP1A1 initial experiments were performed with a standard protocol using 24 h incubation and standard medium (16 mg tryptophan/l) exposed to light for 0, 3, or 12 h in the sterile bench (fluorescent tubes). The CYP1A1 inducing potency was measured with the EROD-assay after addition of light exposed medium directly after the light exposure and of light exposed medium that had been stored for three days in a refrigerator (4°C in the dark).
A full factorial 23 design was used to study whether variations in tryptophan and serum per se without light exposure could influence the sensitivity of the EROD-assay. Each component (tryptophan, serum, and TCDD) was tested at three dose-levels: tryptophan (0, 16, or 32 mg/l), serum (0, 5, or 10%) and TCDD (0.15, 1.5, or 155 pM).
To find the importance of tryptophan and riboflavin in combination with light exposure, a 23 full factorial design was applied, in which each component (tryptophan, riboflavin, and light exposure) was tested at three dose-levels: tryptophan (8, 16, or 24 mg/l), riboflavin (0.1, 0.4, or 0.7 mg/l), and light from fluorescent tubes (0, 12, or 24 h). A specially ordered cell culture medium without tryptophan and riboflavin was used.
In order to compare the CYP1A1-inducing capacity of FICZ and TCDD full sigmoidal dose-response and time-response curves in the EROD-assay were created in experiments in which cells were incubated with serially diluted TCDD and FICZ solutions for 3, 6, 12, and 24 h. A serial dilution of TCDD from 155 pM to 0.155 pM and of FICZ from 100,000 pM to 0.1 pM was used. Concentrations giving 50% of maximal induction (EC50) and the relative maximal inductions (Ymax) were determined.
CYP1A1 inducing potency of the light exposed medium was compared to the potency of FICZ in one experiment in which tryptophan- and riboflavin-rich medium (24 and 0.7 mg/l, respectively) was exposed to light (fluorescent tubes) in the sterile bench for 24 h. The MH1C1 cells were then incubated in the light exposed medium for 3, 6, 12, and 24 h and compared to cells incubated for the same length of time with a fixed concentration of FICZ (100 pM).
Chemical fractionation and analysis of medium.
To identify photoproduct(s) formed in the medium by exposure to ordinary laboratory light, 500 ml plastic bottles of DMEM was prepared as described above. The medium was exposed to light from a light bulb or covered with aluminium foil in the laboratory for 24 h. Serum was excluded from the medium and the tryptophan levels were fixed to either 0 or 16 mg/l. After exposing the medium to light, the medium was concentrated using Sep-Pak C18 cartridges (Waters). The cartridges were washed with water and eluted with 5 ml acetone. The extract corresponding to one light exposed bottle of medium was evaporated to dryness using a rotavapor and further dissolved in 400 μl methanol. Prior to fractionation a part of the methanol extract was diluted with H2O (1:1). Fractionations and analyses of the media extracts were performed by means of HPLC. Seven fractions were collected at different retention times (4–10 min [F1], 10–13 min [F2], 13–16 min [F3], 16–21 min [F4], 21–27 min [F5], 27–33 min [F6], and 33–40 min [F7]) and subsequently evaporated to dryness using a sped-vac centrifuge. The dried fractions were stored at –20°C and dissolved in 120 μl DMSO and tested in the EROD-assay.
HPLC analyses and fractionations were performed using a Merck Hitatchi LaChrom instrument equipped with a L-7100 pump, a L-7455 diod array detector, and a Shimadzu RF-535 fluorescence HPLC monitor, excitation 390 nm and emission 525 nm. A reverse phase Kromasil 100–5C18 column (250 x 4.6 mm) from Scantec (Sweden) was employed for analysis and separation. A 40 min linear mobile phase gradient from 5% B to 85% B (A: 0.1% TFA in H2O, B: 0.1% TFA in acetonitrile) was used at a flow rate of 1 ml/min. The column was washed with 100% B for 10 min between injections.
To confirm a formation of FICZ in the light exposed cell medium a known amount of FICZ standard solution (0.7 pmol) was added to a part (approximately 1/6) of the methanol extract of the concentrated cell medium. The enhancement in fluorescence of the peak assumed to contain FICZ was analyzed by HPLC. A further identification was performed by mass spectrometric (MS) analysis performed by adding 20 μl of acetonitrile to a dried fraction of the second peak in fraction number 7 (F7) and 10 μl of it was injected into a C-18 HPLC column (5 μm, 250 mm x 4.6 mm i.d., Alltech) using a mobile phase consisting of acetonitrile and water, both containing 1 mM of formic acid. A 40 min gradient from 35 to 85% of acetonitrile followed by 100% of acetonitrile for 10 min with a flow rate of 1.0 ml/min was supplied by two LC pumps (LC-10ADvp, Shimadzu, Japan). The flow was splitted after the HPLC column so that only 0.4 ml/min was transferred to the triple quadrupole MS detector (API 2000, Applied Biosystmes/MDS SCIEX, Canada). The operating parameters used during –ESI were: curtain gas (N2, Aquilo nitrogen generator, The Netherlands) 30 psi, gas 1 and 2 (air) 20 psi, temperature 550°C, ion spray voltage –4500 V, declustering potential –80 V, focusing potential –400 V and entrance potential –10 V. Data were collected using Analyst 1.4 from Applied Biosystems/MDS SCIEX.
Data analysis and statistics.
Results from factorial design analyses were evaluated with Multiple Linear Regression (MLR) using the statistical package Modde 5.0 (Umetrics AB, Ume, Sweden). The goodness of fit of the models were validated by the fraction of variation of the response explained by the model (R2) and the fraction of variation of the response that can be predicted by the model (Q2). Student's t-test was used for pairwise comparisons of enzyme induction between treatment groups.
RESULTS
Ordinary medium exposed to light from the fluorescent tubes in the sterile bench induces CYP1A1 activity via the formation of stable photoproduct(s).
An increased CYP1A1 activity was observed after incubation with medium exposed to light for different length of time (Fig. 2). Already after 3 h of light exposure a significant induction corresponding to 12% of the maximal inducing effect caused by TCDD was seen. Twelve hours exposure of light increased the background CYP1A1 activity to 22% of the maximal TCDD effect. A similar effect was seen also after three days of storage in refrigerator (Fig. 2).
The response to TCDD is independent of variability in tryptophan levels without light exposure.
The CYP1A1 activity was explored and modelled in a three-dimensional space where the concentrations of tryptophan and serum were varied as well as TCDD exposure. The experiment was planned so that normal medium conditions and an activity of about 50% were in the center of the investigated space. The scaled and centered regression coefficients based on MLR analysis showed that tryptophan, with no light exposure, does not influence the CYP1A1 activity and that tryptophan does not influence the effect by TCDD (Table 1). A possible potentiation by serum was suggested (p = 0.06).
Tryptophan in combination with light controls background CYP1A1 activity.
The CYP1A1 activity was explored and modelled in a three-dimensional space where the concentrations of tryptophan and riboflavin were varied as well as light exposure. The experiment was planned so that normal medium conditions and a 12 h light exposure were in the center of the investigated space. The scaled and centered regression coefficients based on MLR analysis showed that light, tryptophan, and their interaction term explained the increased EROD activity (Table 2). Riboflavin did not influence the activity significantly; neither did any other interaction terms. After excluding the non-significant regression coefficients (related to riboflavin) a refined quantitative model was formed, describing the influence of tryptophan and light on CYP1A1 activity (Fig. 3). The goodness of fit (R2) was 0.87 and the goodness of prediction (Q2), based on internal cross validation, was 0.82.
TCDD and FICZ dose-response and time-response comparisons.
Full sigmoidal dose-response curves were generated for FICZ and TCDD. EROD activity was quantified after 3, 6, 12, and 24 h of incubation with serial dilutions of FICZ and TCDD. The results show that the EC50 value for TCDD was stable at about 16 pM, whereas the EC50 value for FICZ varied from 34 pM to 830 pM depending on the time the cells were incubated prior to enzyme activity measurement (Table 3). An average maximal induction (Ymax) of about 127% was observed for FICZ compared to TCDD.
The illuminated medium causes transient CYP1A1 induction.
Using a short incubation time (3 h), the CYP1A1-induction caused by FICZ (100 pM) and by the medium (24 mg tryptophan/l) exposed to light from fluorescent tubes (24 h) was found to be up to 50% higher than the maximal effect observed with TCDD (Table 3 and Fig. 4). The relative activity decreased with as much as 80% if the incubation time was extended to 24 h.
Several tryptophan-related photoproducts are formed.
Cell culture media with or without tryptophan (16 mg/l) were exposed to light (light bulb) for 24 h and then fractionated into seven fractions to enable an identification of the formed photoproduct(s). In parallel with fractionation, the light-exposed unfractionated medium was tested for CYP1A1 inducing activity to compare with the results obtained with the light exposure from fluorescent tubes. The result was qualitatively consistent with the results presented in Figure 3: All seven fractions were tested for CYP1A1 activity in the EROD-assay and significant tryptophan dependent effects were observed in the non-polar fractions 4–7 (Fig. 5).
Light-exposed medium with and without tryptophan was analyzed with reverse-phase HPLC using both fluorescence (390/525 nm) and UV (254 nm) detectors (Fig. 6). The large majority of the components were eluted at retention times less than 20 min. Four distinct fluorescent peaks were observed in fractions 5 to 7 [F5, 25 min; F6, 31 min; F7, 36 and 37.5 min] in the tryptophan containing and illuminated medium (Fig. 7). No such peaks were observed in fraction 4.
Identification of FICZ.
A known amount of FICZ was added to the crude methanol extract to verify if the most non-polar peak in fraction 7 was identical with FICZ (data not shown). After addition of FICZ the peak at 37.5 min was increased, confirming a formation of the tryptophan photoproduct FICZ. Compared to a known amount of FICZ, the peak area of FICZ in the concentrated cell medium was established to be 0.7 pmol and the total concentration in the cell medium was subsequently extrapolated to about 8 pM.
The presence of FICZ in the fraction 7 was further confirmed by LC/MS. A standard solution of FICZ as well as the fraction were ionized by –ESI and analyzed by SIM (Selective Ion Monitoring). Both the parent ion (m/z 283) and the fragment (m/z 255), which results from the loss of the aldehyde group were detected (Fig. 8).
DISCUSSION
Since the first observation in 1976 by Paine (Paine, 1976) light and culture-medium related induction of CYP1A1 has repeatedly been reported without any further mechanistic explanations (Harvey et al., 1998; Kocarek et al., 1993; Lorenzen et al., 1993; Nemoto and Sakurai, 1991; Paine and Francis, 1980; Sadar and Andersson, 2001; Segner et al., 2000; Sindhu et al., 1996). Others have observed that cell lines with a defective CYP1A1 gene, and thereby lacking CYP1A1-activity, express high levels of mRNA for CYP1A1 and other AhR dependent gene products even when they are grown without TCDD (Hankinson et al., 1985). One explanation for this high background CYP1A1 mRNA expression that has been put forward is that the CYP1A1 deficient cells accumulate an AhR agonist that is also a substrate for CYP1A1 (Hankinson et al., 1985; RayChaudhuri et al., 1990). The agonist was suggested to be an intermediary metabolite (i.e., an endogenous ligand) and/or a component of the medium. The present results together with our earlier published observations that the tryptophan photoproduct causes sustained CYP1A1 mRNA expression in cells deficient in CYP1A1 enzyme activity (Wei et al., 2000) support the above mentioned observations but also offer a molecular explanation for the medium component theory (cf. below).
Three days of storage in refrigerator did not influence the effect of illuminated medium, which shows that the formed photoproduct(s) is stable in stored medium (Fig. 2). This result has implications on handling the medium since the photoproducts may be formed whenever the medium bottles are exposed to light, even long time before it is used, e.g., during production, transport, and storage. The induction of CYP1A1 activity after 3 and 12 h of incubation were equivalent to the effect obtained with about 50 and 110 pM FICZ, respectively. This means that only approx. 10–6 of the tryptophan normally available in cell culture medium (16 mg/l = 78 μM in DMEM) needs to be photooxidized to FICZ to explain the increased CYP1A1 activity.
Tryptophan did not influence the EROD-assay per se or the response to TCDD (Table 1). However, serum gave a subtle increase in the response and slightly potentiated the effect of TCDD (however without statistical significance). The observed serum effect is in accordance with previous findings by others that serum can be a CYP1A1 mRNA inducer in hepatic cells (N'Guyen et al., 2002), that the CYP1A1 induction by 3-methylcholanthrene is potentiated by serum (Guigal et al., 2001; N'Guyen et al., 2002), and that serum withdrawal leads to reduced AhR expression and loss of CYP1A inducibility (Hestermann et al., 2002). In contrast, others have reported that serum significantly lower the CYP1A1 inducing potency of TCDD as a result of decreased bioavailability in the presence of serum, effectively reducing the concentration of ligand within the cells (Hestermann et al., 2000). This was not confirmed in the present study.
The present study showed that tryptophan and light together explained 87% of the variation in background CYP1A1 activity explored and modeled in a three-dimensional space where the concentrations of tryptophan and riboflavin were varied together with light exposure.
The CYP1A1 induction caused by illuminated medium showed a maximum at short incubation times (Fig. 4). A possible explanation is metabolic breakdown of the formed photoproduct(s). We have earlier described that CYP1A1 can metabolize FICZ and that the levels of induction are dependent on the incubation time (Bergander et al., 2003, 2004; Wei et al., 2000). Others have also described UV-activated tryptophan and indolo[3,2-b]carbazole-induced CYP1A1 expression as transient (Chen et al., 1995; Kocarek et al., 1993) which is in accordance to the effects seen with other rapidly metabolized compounds (Riddick et al., 1994). It seems very plausible that the high background CYP1A1 mRNA (and lack of induction by TCDD) in CYP1A1 deficient Hepa-1 c37 cells (Hankinson et al., 1985) is caused by an inability of such mutant cells to degrade tryptophan photoproducts via CYP1A1. In the present study it was also observed that the drop in activity with time for photo-induced medium was almost identical to the drop of activity seen for FICZ, which indirectly supports the hypothesis that the induction is caused by FICZ or related compounds.
Significant tryptophan dependent effects were observed in the non-polar fractions 4 to 7. At a higher resolution, four distinct fluorescent peaks were observed in fractions 5 to 7 in the tryptophan-containing light exposed medium, which correlated with CYP1A1 activity in these fractions. In fraction 4, which also showed a significant biological activity, no tryptophan specific peaks were detected. This could be due to a high general HPLC response in this fraction that might have masked the tryptophan related peaks. However, the excitation/emission wavelengths used in this study are specific for the fluorescence properties of FICZ. Compounds exhibiting fluorescence at different wavelengths are therefore not detected in this system.
In the fluorescence chromatogram of fraction 7 of the light exposed cell medium containing tryptophan, two peaks were observed. By adding a known amount of FICZ to the crude methanol extract the most non-polar peak in fraction 7 was found to have identical retention time and fluorescence with FICZ. The identity was further confirmed by MS analysis that showed identical retention time and mass spectrum as FICZ (Fig. 8). The total concentration of FICZ in the cell medium was calculated to be about 8 pM. This concentration may explain a major part of the inducing effect of tryptophan-containing unfractionated medium exposed to light. By using a dose-response curve for FICZ, the FICZ equivalent concentration was estimated to be about 10 pM in unfractionated medium exposed to light from a light bulb for 24 h. The higher FICZ equivalent concentrations (100 pM) indicated by the model in Figure 3 might be explained by the fact that the fluorescent light tubes have a spectra with a higher portion of UV compared with the light bulb.
Tryptophan is present in biological systems both as free amino acid and in proteins. It is the precursor of several signal substances including serotonin, melatonin, tryptamine, quinolinic acid, and kynurenic acid (Hayaishi, 1993). We have hypothesized that FICZ and structurally related tryptophan photooxidation products might function as chemical messengers that activate AhR in response to light (Rannug et al., 1987; Wei et al., 2000). Other members of the PAS domain superfamily are also known to function as sensors of light and are involved in circadian rhythmicity, which corroborates a role of the AhR in light sensing (Taylor and Zhulin, 1999). Also, in support of this hypothesis, we have observed a diurnal expression of CYP1A1 and Period (Per2) genes in the posterior pituitary and the liver of untreated rats. The CYP1A1 and Per2 mRNA levels were found to be highly coordinated, indicating a possible interaction between AhR signaling and circadian response pathways (Huang et al., 2002). This indicates that the AhR may have a role in sensing light of different wavelengths. A thorough investigation of photoproduct formation at different wavelengths needs to be done. The result presented here shows that FICZ is produced not only under controlled, high energy UV-irradiation but also by ordinary laboratory light conditions. The slightly more polar fluorescent compounds seen in fractions 5, 6, and 7 have not yet been chemically identified. How much each photoproduct contributes to the inducing capacity of illuminated medium remains to be quantified.
In conclusion, this study shows that normal laboratory light can significantly induce the background CYP1A1 activity through the formation of several tryptophan related photoproducts in light exposed medium. For the first time one of the formed photoproducts was identified as FICZ. These results clearly show that tryptophan derived AhR ligands, which have been suggested to be endogenous AhR ligands, influence the constitutive levels of CYP1A1 activity in cells in culture. As a consequence, composition as well as handling of cell culture medium can cause varying levels of AhR ligands, which in turn can lead to artificial differences between experiments and cell types. Especially, in studies with short incubation times, the contribution from tryptophan-derived factors in medium may have considerable influence on the results. The presence of high affinity AhR ligands in the medium must therefore be controlled in work that aims at identifying AhR agonists and antagonists, and especially when conducting mechanistic studies of genes that are activated through the AhR or that are co-activated or co-repressed through the interaction with the AhR complex.
NOTES
1 The authors have contributed equally to this work.
ACKNOWLEDGMENTS
The authors gratefully acknowledge Nahid Amini at the Department of Analytical Chemistry, Stockholm University for performing the MS analyses. This study was performed with the financial support from Karolinska Institutet Research Funds, Swedish Fund for Research without Animal Experiments, the Swedish National Board for Laboratory Animals, and the Swedish Radiation Protection Institute.
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