TOXICOLOGY 1 - POSTERS
TRYPTOPHAN METABOLITES, IND0LE-3-PYRUVIC ACID, DL-3INDOLELACTIC ACID, L-KYNURENINE, AND KYNURENIC ACID
ACTIVATE Ah RECEPTOR SIGNAL TRANSDUCTION
Sharon Heath-Pagliuso. Rebecca Mui, Anna Milici, Scott Giese, Regina Chin, andMichael S. Denison
Department of Environmental Toxicology, University of Califomia, Davis, CA, USA

Introduction
Many environmental pollutants have been shown to be ligands and activators of the Ah Receptor(AhR), tae most potent being the ubiquitous compound 2,3,7,8-tefrachlorodibenzo-p-dioxin(TCDD, dioxin). While tais compound is of major consequence, it is becoming evident that there
are numerous other chemicals that can bind and activate AhR . However, the endogenous
ligand(s) of AhR and tae physiological role(s) of AhR remains an enigma. Our work and the workof others have shown that the amuio acid tryptophan and several of its metabolites, as well as UV23
photoproducts of ttyptophan, are activators of the AhR signaling pathway ' . Given the large
number of indole-containing compounds and tryptophan metabolites present in biological systems,
we have begim a systematic search for other activators of AhR. Utilizing two screening assays wehave developed and optimized (gel retardation analysis (GRA) and chemically-activated luciferase
gene expression (CALUX)), we have identified several metabolites of tryptophan that are novel
activators of tae AhR signal fransduction pathway.
Materials and Methods
Gel Retardation Analysis (GRA). Male Hartley guinea pig cytosol was used as tae source of AhR.
The cytosol was incubated with DMSO (20 ul/ml), TCDD (20 nM) of tae indicated chemicals or
extracts for 2 h at 20" C followed by elecfrophoresis and autoradiography as described previously .
Nuclear extracts were obtained by treating the cells for 2 hours with the chemical prior to2

harvesting the nuclei . Quantification of tae amount of protein-DNA complex formed was canied
out using a Molecular Dynamics Phosphoimager SI.
Chemically activated luciferase expression (CALUXI bioassav. The CALUX bioassay utilizes
recombinant mouse hepatoma (HlLl.lc2) cells developed and maintained as previously
described . These cells, derived from the mouse Hepalclc7 line, contain a stably integrated DREdriven firefly luciferase reporter gene whose transcriptional activation occurs as a time and/or dose
dependent AhR activated response . For chemical treatment, HlLl.lc2 cells were grown in 6
well tissue cultare plates and incubated with DMSO, TCDD (in DMSO) or the test chemical for
four hours at 37° C After incubation the cells were lysed and luciferase activity determined usinga Dynatech ML3000 Microplate Luminometer with automatic injection of Promega luciferin .
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fluorescamine protein assay and BSA as the protein standard . Data are expressed as means andS.E. of four replicates/freatment. Comparisons between means were performed using ANOVA
and the SchefFs F test and considered significant at tae p < 0.05 level.
Test Chemicals L-kynurenine (L-K) and kynurenic acid (KA) were obtained from from ICN.
Indole-3-pymvic acid (IPA) and DL-3-indolelactic acid (ILA) were obtained from Aldrich. 3hydroxy-DL-kynurenine (3-OH-K), anthranillic acid (A-A), and kynuramine (K-NH4) wereobtained from Sigma. Chemical stocks (100 mM) were prepared as follows: L-K, ILA, and IPA
were dissolved in DMSO; KA and A-A were dissolved in glass distilled water; and, 3-OH-K (50mM) was dissolved 0.5N HCL-Tris pH8.
Results and Discussion
Incubation of the test chemicals with guinea pig cytosol shows that the indoles, ILA and IPA, arecapable of activating AhR and, surprisingly, so did L-kynurenine and kynurenic acid (Fig. 1).
Kynuramine, anthranillic acid, and 3-OH-K were inactive in this assay. The CALUX bioassay(Fig. 2) shows that tae response obtainedfromfreatingcells with 100 uM L-K and IPA was not
significantly different from 1 nM TCDD. ILA and KA were sUghtly less efficient at inducing theluciferase reporter in vivo than IPA, although in the GRA assay they appeared similar. The
differences between the two assays may be attributed to differences in solubility, uptake, and/or7
metabolism. These problems have been discussed elsewhere . K-NH4 and A-A were weak orinactive as inducers. Further evidence that these chemicals fiinction as in vivo activators of AhR
was obtained by isolating the nuclei from freated cells. To validate the in vitro assays, nuclear
exfracts of freated cells were examined. Our results from such stadies indicate that L-K, IPA,ILA, and kynumenic acid stimulate AhR nuclearfranslocalion(data not shown), indicating thatthese chemicals also activate AhR in intact cells. Interestingly, many of these same chemicals, LK and IPA, are competitive inhibitors of P4501A1-dependent EROD activity (data not shown),suggesting that they may also be subsfrates for tae AhR-induced enzyme.
Tryptophan metabolism has been actively studied because so many of the metabolites haveneurogenic activity and some are known carcinogens producing metallurgic oxidative DNA
damage . One well characterized tryptophan metabolic pathway results in the formation ofserotonin and melatonin. Anotaer branch leads to tryptamine which can easily be converted to thehallucinogenic compound N,N-dimethytryptamine. The kynurenine branch also produces
neurogenic compounds, kynurenic acid and quinolinic acid, an antagonist and agonist of the9NMDA receptor, respectively . Since many physiological processes can be affected bytryptophan metabolites; vision, well being, sleep cycles, cancer susceptibility, etc., it is not
surprising that the enzymatic reactions that determine which metabolic pathway tryptophan will
follow are highly regulated. Therefore, a back-up system to help regulate the levels of taese
chemicals at key branch points might provide an adaptive advantage. Thus, it is possible that AhR
may help regulate the levels of neurogenic or cell damaging tryptophan metabolites.
Acknowledgments
This work was supported by a grantfromthe National Institates of Environmental Health Sciences(RO1ES07865, Center Grant ES05707, and a Superfund Basic Research Grant, ES04699).
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References
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2. Heatii-Pagliuso S., Rogers W.J., Tullis K., Seidel S.D., Cenijn P., Brouwer A., Lam J., andDenison M.S. (1998) Biochemistiy 37(33), 11508-15.
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Biophys. 317,439-448.
5. Ganison P.M., Tullis K., Aarts J.M.M.J.G., Brouwer A., Giesy, J.P., and Denison, M.S. (1996)
Fund. Appl. Toxicol. 30, 194-203.
6. Lorenzen A. and Kennedy S.W.(1993) Analyt. Biochem. 214, 346-348.
7. Seidel S.D., Li V., Winter, G.M., Rogers W.J., Martinez E., Denison M.S. (2000)
Toxicological Sciences 55,107-115.
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Carcinogenesis 16(2), 349-56.
9. Stone T.W. (1993) Phannacol Rev 45(3), 309-79.

!• 1: t l ir^ I i i . l i

Fig.l. GRA of tryptophan metaboUtes.
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CALUX
500
450
LECSND400• -CMSO
b-HCL
c-K4«t4,100iM
d-3.OH-K,100iiM
e-VA. 100 gill
f-tCA,100iiM
g-lLA.t00iiM
h-L4C100i)M
i-TCDtMnM

350
300
250
200150100
SO •0Fig.2. RdalivehidQCtioncyftheluciferaseieponeTplaanidinHepal.lcellsby die indicated tiyptophan metabolites following a 4.Shr treatment IPA
Bsd L-K were not significantly different fiom TCDD.

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