Inflammatory signaling plays a key role in tumor progression, and the pleiotropic cytokine interleukin-6 (IL-6) is an important mediator of protumorigenic properties. Activation of the aryl hydrocarbon receptor (AHR) with exogenous ligands coupled with inflammatory signals can lead to synergistic induction of IL6 expression in tumor cells. Whether there are endogenous AHR ligands that can mediate IL6 production remains to be established. The indoleamine-2,3-dioxygenase pathway is a tryptophan oxidation pathway that is involved in controlling immune tolerance, which also aids in tumor escape. We screened the metabolites of this pathway for their ability to activate the AHR; results revealed that kynurenic acid (KA) is an efficient agonist for the human AHR. Structure-activity studies further indicate that the carboxylic acid group is required for significant agonist activity. KA is capable of inducing CYP1A1 messenger RNA levels in HepG2 cells and inducing CYP1A-mediated metabolism in primary human hepatocytes. In a human dioxin response element-driven stable reporter cell line, the EC(25) was observed to be 104nM, while in a mouse stable reporter cell line, the EC(25) was 10muM. AHR ligand competition binding assays revealed that KA is a ligand for the AHR. Treatment of MCF-7 cells with interleukin-1beta and a physiologically relevant concentration of KA (e.g., 100nM) leads to induction of IL6 expression that is largely dependent on AHR expression. Our findings have established that KA is a potent AHR endogenous ligand that can induce IL6 production and xenobiotic metabolism in cells at physiologically relevant concentrations.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor involved in the regulation of multiple cellular pathways, such as xenobiotic metabolism and Th17 cell differentiation. Identification of key physiologically relevant ligand(s) that regulate AHR function remains to be accomplished. Screening of indole metabolites has identified indoxyl-3-sulfate (I3S) as a potent endogenous ligand that selectively activates the human AHR at nanomolar concentrations in primary human hepatocytes, regulating transcription of multiple genes, including: CYP1A1, CYP1A2, CYP1B1, UGT1A1, UGT1A6, IL6, and SAA1. Furthermore, I3S exhibits an ~ 500-fold greater potency in terms of transcriptional activation of the human AHR relative to the mouse AHR in cell lines. Structure-function studies reveal that the sulfate group is important determinant for efficient AHR activation. This is the first phase II enzymatic product identified that can significantly activate the AHR and ligand competition binding assays indicate that I3S is a direct AHR ligand. I3S failed to activate either CAR or PXR. The physiological importance of I3S lies in the fact that it is a key uremic toxin that accumulates to high micromolar concentrations in kidney dialysis patients, but its mechanism of action is unknown. I3S represents the first identified relatively high potency endogenous AHR ligand that plays a key role in human disease progression. These studies provide evidence that the production of I3S can lead to AHR activation and altered drug metabolism. Our results also suggest that prolonged activation of the AHR by I3S may contribute to toxicity observed in kidney dialysis patients and thus represent a possible therapeutic target.The aryl hydrocarbon receptor (AHR) 1 is a ligand-activated transcription factor that belongs to the basic helix-loop-helix/Per-Arnt-Sim (bHLH/PAS) family of transcription factors, a class of proteins that are considered environmental sensors. Prior to activation, the AHR is located in the cytoplasm of the cell, complexed with two heat-shock protein 90 molecules, X-associated

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Chronic or excessive (ϩ)-methamphetamine (METH) use often leads to addiction and toxicity to critical organs like the brain. With medical treatment as a goal, a novel single-chain variable fragment (scFv) against METH was engineered from anti-METH monoclonal antibody mAb6H4 (IgG, light chain, K d ϭ 11 nM) and found to have similar ligand affinity (K d ϭ 10 nM) and specificity as mAb6H4. The anti-METH scFv (scFv6H4) was cloned, expressed in yeast, purified, and formulated as a naturally occurring mixture of monomer (ϳ75%) and dimer (ϳ25%). To test the in vivo efficacy of the scFv6H4, male Sprague-Dawley rats (n ϭ 5) were implanted with 3-day s.c. osmotic pumps delivering 3.2 mg/kg/day METH. After reaching steady-state METH concentrations, an i.v. dose of scFv6H4 (36.5 mg/kg, equimolar to the METH body burden) was administered along with a [ 3 H]scFv6H4 tracer. Serum pharmacokinetic analysis of METH and [3 H]scFv6H4 showed that the scFv6H4 caused an immediate 65-fold increase in the METH concentrations and a 12-fold increase in the serum METH area under the concentration-time curve from 0 to 480 min after scFv6H4 administration. The scFv6H4 monomer was quickly cleared or converted to multivalent forms with an apparent t 1/2z of 5.8 min. In contrast, the larger scFv6H4 multivalent forms (dimers, trimers, etc.) showed a much longer t 1/2z (228 min), and the significantly increased METH serum molar concentrations correlated directly with scFv6H4 serum molar concentrations. Considered together, these data suggested that the scFv6H4 multimers (and not the monomer) were responsible for the prolonged redistribution of METH into the serum.There are currently nearly 20 monoclonal antibody (mAb) medications approved by the United States Food and Drug Administration and over 20 more in early clinical or preclinical trials (Holliger and Hudson, 2005). These medications include full-length IgG mAbs, along with five mAb fragments as Fab, F(abЈ) 2 (antigen binding fragments of IgG), or singlechain variable fragment (scFv) proteins.IgG mAbs are typically chimeric, humanized, or fully human proteins and are administered to patients requiring a long-acting antagonist with minimal extravascular penetration (Bazin-Redureau et al., 1997). IgG mAb is best for this purpose because it has a terminal elimination half-life (t 1/2z ) ranging from approximately 3 to 26 days. The longest t 1/2z values are usually achieved when the antibody does not bind to tissue sites and is not prematurely cleared due to antigenicity. When a short duration of action and greater extravascular penetration are needed, a significantly smaller fragment like Fab (t 1/2z ranging from 0.5-21 h; Trang, 1992) or scFv (t 1/2z ranging from minutes to hours; Goel et al., 2000) is used. In particular, rat pharmacokinetic studies of an antianthrax toxin scFv report a t 1/2␣ of ϳ5 min (Maynard et al., 2002). In addition, PCKN studies of a scFv in mice report t 1/2␣ values of 2.7 and 1 min Willuda et al., 2001). It is possible that a short-acting scFv could be used to

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(+)-Methamphetamine (METH) and (+)-amphetamine (AMP) are structurally similar drugs that are reported to induce similar pharmacological effects in rats of the same sex. Because pharmacokinetic data suggest female rats should be more affected than males, the current studies sought to test the hypothesis that the behavioral and temporal actions of METH and AMP should be greater in female Sprague-Dawley rats than in males. Using a dosing regimen designed to reduce the possibility of tolerance and sensitization, rats were administered 1.0 and 3.0 mg/kg intravenous drug doses. Distance traveled, rearing events and focal stereotypies (e.g., head weaving, sniffing) were measured. Female rats traveled significantly greater distances and displayed a greater number of rearing events than males after both doses. Analysis of stereotypy ratings after 3.0 mg/kg revealed that focal stereotypies were more pronounced and lasted longer in females. The second study compared the potencies of METH and AMP in inducing locomotor activity and focal stereotypies in each sex. No differences in potency were found when METH and AMP effects were compared within males or females. In summary, these studies showed female rats displayed greater and longer-lasting locomotor activity and more stereotypic behaviors, supporting earlier evidence of significant sexual dimorphism in pharmacokinetics.

Phthalates and other endocrine-disruptive chemicals are manufactured in large quantities for use as plasticizers and other commercial applications, resulting in ubiquitous human exposure and thus, concern regarding their toxicity. Innate defense against small molecule exposures is controlled in large part by the constitutive androstane receptor (CAR) and the pregnane X receptor (PXR). The human CAR gene undergoes multiple alternative splicing events resulting in the CAR2 and CAR3 variant receptors. Recent studies from our laboratory show that CAR2 is potently and specifically activated by di(2-ethylhexyl) phthalate (DEHP). We hypothesized that alternative splicing is a mechanism for increasing CAR's functional diversity, broadening the human receptors' repertoire of response to environmental xenobiotics. In these studies, we examine the interaction of alternatively spliced CARs and PXR with a range of suspected endocrine disruptors, including phthalates, bisphenol A (BPA), and 4-N-nonylphenol (NP). Transactivation and two-hybrid studies in COS-1 cells revealed differential selectivity of endocrine-disrupting chemicals for the variant CAR and PXR. Ex vivo studies showed DEHP and di-isononyl phthalate potently induced CYP2B6 and CYP3A4 expression in human hepatocytes. Mutation analysis of CAR2, in silico modeling, and ligand docking studies suggested that the SPTV amino acid insertion of CAR2 creates a unique ligand-binding pocket. Alternative gene splicing results in variant CAR receptors that selectively recognize phthalates and BPA. The interaction of phthalates with CAR and PXR suggests a xenobiotic response that is complex and biologically redundant.