Activation of the Constitutive Androstane Receptor by Monophthalates.

Humans in industrialized areas are continuously exposed to phthalate plasticizers, prompting concerns of their potential toxicities. Previous studies from our laboratory and others have shown that various phthalates activate several mammalian nuclear receptors, in particular the constitutive androstane receptor (CAR), the pregnane X receptor (PXR), and the peroxisomal proliferator-activated receptors (PPARs), although often at concentration levels of questionable relevance to human exposure. We discovered that di(2-ethylhexyl) phthalate (DEHP) and di-isononyl phthalate (DiNP), two of the highest volume production agents, were potent activators of human CAR2 (hCAR2), a unique human CAR splice variant and, to a lesser degree, human PXR (hPXR). These diphthalates undergo rapid metabolism in mammalian systems, initially to their major monophthalate derivatives MEHP and MiNP. Although MEHP and MiNP are reported activators of the rodent PPARs, with lower affinities for the corresponding human PPARs, it remains unclear whether these monophthalate metabolites activate hCAR2 or hPXR. In this investigation, we assessed the relative activation potential of selected monophthalates and other low molecular weight phthalates against hCAR, the most prominent hCAR splice variants, as well as hPXR and human PPAR. Using transactivation and mammalian two-hybrid protein interaction assays, we demonstrate that these substances indeed activate hCARs and hPXR but to varying degrees. MEHP and MiNP exhibit potent activation of hCAR2 and hPXR with higher affinities for these receptors than for the hPPARs. The rank order potency for MEHP and MiNP was hCAR2 > hPXR > hPPARs. Results from primary hepatocyte experiments also reflect the MEHP and MiNP upregulation of the respective human target genes. We conclude that both di- and monophthalates are potently selective hCAR2 activators and effective hPXR activators. These results implicate these targets as important mediators of selective phthalate effects in humans. The striking differential affinities for these compounds between human and rodent nuclear receptors further implies that biological results obtained from rodent models may be of only limited relevance for interpolating phthalate-mediated effects in humans.

Proteasomal interaction as a critical activity modulator of the human constitutive androstane receptor.

The CAR (constitutive androstane receptor; NR1I3) is a critical xenobiotic sensor that regulates xenobiotic metabolism, drug clearance, energy and lipid homoeostasis, cell proliferation and development. Although constitutively active, in hepatocytes CAR is normally held quiescent through a tethering mechanism in the cytosol, anchored to a protein complex that includes several components, including heat-shock protein 90. Release and subsequent nuclear translocation of CAR is triggered through either direct binding to ligand activators such as CITCO {6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime} or through indirect chemical activation, such as with PB (phenobarbital). In the present study, we demonstrate that proteasomal inhibition markedly disrupts CAR function, repressing CAR nuclear trafficking, disrupting CAR's interaction with nuclear co-activators and inhibiting induction of CAR target gene responses in human primary hepatocytes following treatment with either PB or CITCO. Paradoxically, these effects occur following accumulation of ubiquitinated hCAR (human CAR). Furthermore, a non-proteolytic function was indicated by its interaction with a SUG1 (suppressor for Gal1), a subunit of the 26S proteasome. Taken together, these data demonstrate that the proteasome complex functions at multiple levels to regulate the functional biology of hCAR activity.

Treatment with a monoclonal antibody against methamphetamine and amphetamine reduces maternal and fetal rat brain concentrations in late pregnancy.

We hypothesized that treatment of pregnant rat dams with a dual reactive monoclonal antibody (mAb4G9) against (+)-methamphetamine [METH; equilibrium dissociation rate constant (KD) = 16 nM] and (+)-amphetamine (AMP; KD = 102 nM) could confer maternal and fetal protection from brain accumulation of both drugs of abuse. To test this hypothesis, pregnant Sprague-Dawley rats (on gestational day 21) received a 1 mg/kg i.v. METH dose, followed 30 minutes later by vehicle or mAb4G9 treatment. The mAb4G9 dose was 0.56 mole-equivalent in binding sites to the METH body burden. Pharmacokinetic analysis showed baseline METH and AMP elimination half-lives were congruent in dams and fetuses, but the METH volume of distribution in dams was nearly double the fetal values. The METH and AMP area under the serum concentration-versus-time curves from 40 minutes to 5 hours after mAb4G9 treatment increased >7000% and 2000%, respectively, in dams. Fetal METH serum did not change, but AMP decreased 23%. The increased METH and AMP concentrations in maternal serum resulted from significant increases in mAb4G9 binding. Protein binding changed from ∼15% to > 90% for METH and AMP. Fetal serum protein binding appeared to gradually increase, but the absolute fraction bound was trivial compared with the dams. mAb4G9 treatment significantly reduced METH and AMP brain values by 66% and 45% in dams and 44% and 46% in fetuses (P < 0.05), respectively. These results show anti-METH/AMP mAb4G9 therapy in dams can offer maternal and fetal brain protection from the potentially harmful effects of METH and AMP.

The orphan nuclear receptor DAX-1 functions as a potent corepressor of the constitutive androstane receptor (NR1I3).

Regulation of gene transcription is controlled in part by nuclear receptors that function coordinately with coregulator proteins. The human constitutive androstane receptor (CAR; NR1I3) is expressed primarily in liver and regulates the expression of genes involved in xenobiotic metabolism as well as hormone, energy, and lipid homeostasis. In this report, DAX-1, a nuclear receptor family member with corepressor properties, was identified as a potent CAR regulator. Results of transaction and mutational studies demonstrated that both DAX-1's downstream LXXLL and its PCFQVLP motifs were critical contributors to DAX-1's corepression activities, although two other LXXM/LL motifs located nearer the N terminus had no impact on the CAR functional interaction. Deletion of DAX-1's C-terminal transcription silencing domain restored CAR1 transactivation activity in reporter assays to approximately 90% of control, demonstrating its critical function in mediating the CAR repression activities. Furthermore, results obtained from mammalian two-hybrid experiments assessing various domain configurations of the respective receptors showed that full-length DAX-1 inhibited the CAR-SRC1 interaction by approximately 50%, whereas the same interaction was restored to 90% of control when the DAX-1 transcription silencing domain was deleted. Direct interaction between CAR and DAX-1 was demonstrated with both alpha-screen and coimmunoprecipitation experiments, and this interaction was enhanced in the presence of the CAR activator 6-(4-chlorophenyl)imidazo[2,1-b]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO). Results obtained in primary human hepatocytes further demonstrated DAX-1 inhibition of CAR-mediated CITCO induction of the CYP2B6 target gene. The results of this investigation identify DAX-1 as a novel and potent CAR corepressor and suggest that DAX-1 functions as a coordinate hepatic regulator of CAR's biological function.

The fate and function of therapeutic antiaddiction monoclonal antibodies across the reproductive cycle of rats.

During preclinical development of neuroprotective antiaddiction therapeutic monoclonal antibodies (mAbs) against phencyclidine (PCP) and (+)-methamphetamine, we discovered novel, gestation stage-specific changes in mAb disposition spanning the entire reproductive cycle of female rats. Each pharmacological change was independent of mAb dose and antigen target but was precisely coincident with transitions between the gestational trimesters, parturition, and lactation periods of the female reproductive cycle. Whereas anti-PCP mAb6B5 terminal elimination half-life (t(1/2λz)) in nonpregnant females was 6.6 ± 1.6 days, the mAb6B5 t(1/2λz) significantly changed to 3.7 ± 0.4 days, then 1.4 ± 0.1 days, then 3.0 ± 0.4 days in the second trimester, third trimester, and postpartum periods, respectively (p < 0.05 for each change). Initially, these evolving changes in mAb6B5 clearance (3.3-fold), distribution volume (1.8-fold), and elimination half-life (4.7-fold) affected our ability to sustain sufficient mAb6B5 levels to sequester PCP in the bloodstream. However, understanding the mechanisms underlying each transition allowed development of an adaptive mAb-dosing paradigm, which substantially reduced PCP levels in dam brains and fetuses throughout pregnancy. These mAb functional studies also revealed that antidrug mAbs readily cross the placenta before syncytiotrophoblast barrier maturation, demonstrating the dynamic nature of mAb pharmacokinetics in pregnancy and the importance of maintaining maternal mAb levels. These studies provide the first preclinical pregnancy model in any species for chronic mAb dosing and could have important implications for the use of antibody therapies involving blood organ barriers (such as addiction) or other chronic diseases in women of childbearing age (e.g., irritable bowel diseases, multiple sclerosis, breast cancer, rheumatoid arthritis).

The uremic toxin 3-indoxyl sulfate is a potent endogenous agonist for the human aryl hydrocarbon receptor.

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 ligands 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 approximately 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 an 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.

Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine.

Chronic or excessive (+)-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, kappa light chain, K(d) = 11 nM) and found to have similar ligand affinity (K(d) = 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 ( approximately 75%) and dimer ( approximately 25%). To test the in vivo efficacy of the scFv6H4, male Sprague-Dawley rats (n = 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(1/2lambdaz) of 5.8 min. In contrast, the larger scFv6H4 multivalent forms (dimers, trimers, etc.) showed a much longer t(1/2lambdaz) (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.

Engineering and characterization of a mouse/human chimeric anti-phencyclidine monoclonal antibody.

Previously, our laboratory produced a high affinity, anti-phencyclidine (PCP) murine monoclonal antibody (mAb6B5) that also binds other PCP-like arylcyclohexylamines. In this project, mAb6B5 is engineered into a mouse/human chimera (ch-mAb6B5) to assess the feasibility of developing it into a medication for PCP and PCP-like drug abuse. To create ch-mAb6B5, the light and heavy chain constant regions of mAb6B5 were replaced with human kappa and IgG(2) constant regions in order to decrease its potential immunogenicity in humans. To be an effective anti-PCP medication, ch-mAb6B5 must retain the critical immunochemical binding properties of mAb6B5. Expression vectors containing ch-mAb6B5 light chain and heavy chain cDNA were constructed and expressed in the murine myeloma cell line P3X63-Ag8.653. Immunoassays confirm that ch-mAb6B5 is indeed a chimera, composed of mAb6B5's PCP-binding variable domains and human kappa and IgG constant regions. Radioimmunoassays show that ch-mAb6B5 has the same drug-binding profile as mAb6B5. Ch-mAb6B5 and mAb6B5 bind PCP with a K(D) of 0.67 nM and 1.17 nM (respectively) and bind PCP-like arylcyclohexylamines 1-[1-(2-thienyl)cyclohexyl]piperidine and N-ethyl-1-phenylcyclohexylamine with similar specificity. Additionally, ch-mAb6B5 and mAb6B5 have the same calculated isoelectric points and molecular weights, critical properties in antigen-antibody interactions. These data demonstrate that mouse/human ch-mAb6B5, a "more human" version of murine mAb6B5, retains mAb6B5's unique drug-binding properties. This work supports our continued efforts to develop ch-mAb6B5 into a medication for PCP and PCP-like drug abuse - introducing the intriguing possibility of using a single therapeutic mAb for treating a class of abused drugs.

Sex differences in (+)-amphetamine- and (+)-methamphetamine-induced behavioral response in male and female Sprague-Dawley rats.

(+)-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.

Safety and efficiency of an anti-(+)-methamphetamine monoclonal antibody in the protection against cardiovascular and central nervous system effects of (+)-methamphetamine in rats.

The purpose of these studies was to determine if a high-affinity, anti-(+)-methamphetamine (METH) monoclonal antibody (mAb6H4; KD=11 nM) protects against METH-induced central nervous and cardiovascular system effects in rats. Rats (n=5 per group) received one of three anti-METH mAb6H4 doses, equal to 0.32, 0.56 or 1 times the mole equivalent (mol-eq) amount of METH in the body following a 1 mg/kg i.v. METH dose. Each rat was challenged with METH (1 mg/kg, i.v.) 1 and 4 days after the anti-METH mAb dose. The 1 mol-eq anti-METH mAb dose significantly reduced the duration of METH-induced locomotor activity (horizontal locomotion and rearing events), heart rate and blood pressure effects from 2 to 3 h to about an hour. This resulted in a significant reduction in total locomotor activity and the area under the hemodynamic effect vs. time curve for heart rate and blood pressure. In addition, the time to peak locomotor activity was decreased after the 1 mol-eq mAb dose vs. the lower doses. These changes were limited to the first METH challenge. The responses to the second METH challenge were not different from baseline. The peak hemodynamic and locomotor activity values were unchanged after both challenges. These results indicate anti-METH mAb6H4 can safely reduce the hemodynamic and locomotor effects of METH given one day after anti-METH IgG, and that the mAb is safe when administered in the absence of METH. These results are important because they indicate these antibody medications have simultaneous beneficial effects in multiple organ systems.

Monoclonal IgG affinity and treatment time alters antagonism of (+)-methamphetamine effects in rats.

The roles of monoclonal antibody affinity and treatment time of (+)-methamphetamine-induced pharmacological effects in rats were studied using two anti-(+)-methamphetamine monoclonal antibodies. These studies tested the preclinical protective effects of monoclonal antibody antagonists in (+)-methamphetamine overdose and pretreatment scenarios. The higher affinity antibody (mAb6H4; KD=11 nM for (+)-methamphetamine) more effectively antagonized (+)-methamphetamine-induced behavioral effects (distance and rearing) than the low affinity antibody (designated mAb6H8; KD=250 nM) and had a longer duration of action. Both antibodies more effectively reduced (+)-methamphetamine effects in the overdose model than in the pretreatment model. (+)-Methamphetamine pharmacokinetic studies showed the mAb6H4 significantly reduced brain concentrations over time in both models. However, while mAb6H4 immediately reduced brain concentrations in the overdose model, it did not prevent the initial distribution of (+)-methamphetamine into the brain in the pretreatment model. Thus, anti-(+)-methamphetamine monoclonal antibody affinity and administration time (relative to (+)-methamphetamine dosing) are critical determinants of therapeutic success.

Pharmacodynamic mechanisms of monoclonal antibody-based antagonism of (+)-methamphetamine in rats.

Our studies examined pharmacokinetic mechanisms involved in high-affinity (K(d) approximately 11 nM) monoclonal antibody-based antagonism of (+)-methamphetamine-induced locomotor effects. Male rats received (+)-methamphetamine (0.3, 1, or 3 mg/kg i.v.) followed 30 min later by saline or anti-(+)-methamphetamine monoclonal antibody. All groups received a constant dose of monoclonal antibody that was equimolar in binding sites to the body burden of a 1 mg/kg i.v. (+)-methamphetamine dose 30 min after administration. The monoclonal antibody antagonized locomotor effects due to 0.3 and 1 mg/kg (+)-methamphetamine. In contrast, monoclonal antibody treatment increased locomotor activity due to 3 mg/kg (+)-methamphetamine. We also investigated the serum and brain pharmacokinetics of (+)-methamphetamine without and with the monoclonal antibody. Rats received (+)-methamphetamine (1 mg/kg i.v.) followed by saline or monoclonal antibody treatment at 30 min. The monoclonal antibody significantly increased serum methamphetamine concentrations and significantly decreased brain methamphetamine concentrations. These data indicate that anti-(+)-methamphetamine monoclonal antibody-induced pharmacodynamics are complex, but are related to time-dependent changes in (+)-methamphetamine brain distribution.

Effect of nonylphenol on serum testosterone levels and testicular steroidogenic enzyme activity in neonatal, pubertal, and adult rats.

Previous dose range-finding studies with nonylphenol (NP) administered to rats in a soy- and alfalfa-free diet showed apparent feminization of several endpoints in male rats at doses of 25 ppm and above. One possible mechanism contributing to these effects is a reduction of testosterone at critical developmental periods. The present study was conducted as an adjunct to a multigeneration study and was designed to examine the effect of NP on testosterone production. Male rats in the F1 and F2 generations were exposed through their dams or directly to various dietary doses of NP (0, 25, 200 and 750 ppm) throughout gestation and until sacrifice at either postnatal day 2 (PND2), PND50, or PND140. Male pups in the F3 generation were examined only on PND2. At PND2, serum testosterone levels were significantly decreased in all groups exposed to NP in the F1 generation, but not in the F2 or F3 generations. The activity of 17alpha-hydroxylase/C17, 20 lyase (P450c17) in PND2 testicular homogenates was not affected by NP treatment. In F1 and F2 PND50 and PND140 rats, NP treatment did not affect serum testosterone levels. The absolute dorsolateral prostate weight was increased in the 200 and 750 ppm dose groups only in the F1 PND50 rats, however, no significant effects were observed in other male reproductive organs. NP treatment did not affect P450c17 activity in microsomes prepared from testes of F1 PND50 or PND140 rats. However, P450c17 activity was significantly decreased in testicular microsomes of F(2) PND50 (200 and 750 ppm dose groups) and PND140 (25, 200, and 750 ppm dose groups) rats. A decrease in testicular beta-nicotinamide adenine dinucleotide phosphate (NADPH) P450 reductase was also observed in all PND50 and PND140 NP-exposed rats of the F1 and F2 generations. The ability of NP to directly inhibit P450c17 activity in vitro at concentrations of 1-100 microM was also demonstrated. These results indicate that NP can inhibit the activity of enzymes involved in testosterone synthesis, but suggest minimal effects on testosterone or testosterone-dependent endpoints via this mechanism.

(+)-Methamphetamine-induced spontaneous behavior in rats depends on route of (+)METH administration.

These studies examined the role of (+)-methamphetamine ((+)METH) administration route on spontaneous behavioral activity vs. time relationships, and pharmacokinetic mechanisms for differences in effects. Male Sprague-Dawley rats (n=6 per administration route) received saline and three doses (0.3, 1.0 and 3.0 mg/kg) of (+)METH in a mixed-sequence design by intravenous (iv), subcutaneous (sc) or intraperitoneal (ip) administration. Locomotion and stereotypy were quantified by video-tracking analysis. The effects of (+)METH on spontaneous behavior were dose- and route-dependent. In particular, total locomotor activity was greatest following 3.0 mg/kg intraperitoneally (P<0.05) and stereotypy ratings were greatest following 3.0 mg/kg subcutaneously (P<0.05). In addition, the duration of locomotor effects was greatest after 3.0 mg/kg subcutaneously (P<0.05). Serum pharmacokinetic parameters were determined in separate rats given 3.0 mg/kg by subcutaneous and intraperitoneal administration (n=4 per administration route). The (+)METH elimination half-life was not different between the routes, but the (+)METH AUC was greater (P<0.05), and the (+)METH and (+)-amphetamine (AMP) maximum concentrations occurred later following subcutaneous than after intraperitoneal dosing (P<0.05), increasing and prolonging drug exposure. In conclusion, the overall pattern of (+)METH effects on locomotor activity depend on dose and the route of administration, which affects serum concentration and the time course of behavioral effects.

Pharmacological effects of two anti-methamphetamine monoclonal antibodies. Supporting data for lead candidate selection for clinical development.

This lead candidate selection study compared two anti-(+)-methamphetamine (METH) monoclonal antibodies (mAb) to determine their ability to reduce METH-induced locomotor effects and redistribute METH and (+)-amphetamine (AMP) in a preclinical overdose model. Both mAbs have high affinity for METH, but mAb4G9 has moderate and mAb7F9 has low affinity for AMP. In the placebo-controlled behavioral experiment, the effects of each mAb on the locomotor response to a single 1 mg/kg intravenous (IV) METH dose were determined in rats. The doses of mAb binding sites were administered such that they equaled 1, 0.56, 0.32, and 0.1 times the molar equivalent (mol-eq) of METH in the body 30 min after the METH dose. METH disposition was determined in separate animals that similarly received either a 1 or 0.32 mol-eq dose of mAb binding sites 30 min after a 1 mg/kg METH dose. Total METH-induced distance traveled was significantly reduced in rats that received the highest three doses of each mAb compared with saline. The duration of METH effects was also significantly reduced by mAb7F9 at the highest dose. The disposition of METH was altered dose-dependently by both mAbs as shown in reductions of volume of distribution and total clearance, and increases in elimination half-life. These data indicate that both mAbs are effective at reducing METH-induced behavior and favorably altering METH disposition. Both were therefore suitable for further preclinical testing as potential human medications for treating METH use; however, due to results reported here and in later studies, mAb7F9 was selected for clinical development.

Multi-species analyses of direct activators of the constitutive androstane receptor.

The constitutive androstane receptor (CAR; NR1I3) is a member of the nuclear receptor superfamily and functions as an important xenochemical sensor and transcriptional modulator in mammalian cells. Upon chemical activation, CAR undergoes nuclear translocation and heterodimerization with the retinoid X receptor subsequent to its DNA target interaction. CAR is unusual among nuclear receptors in that it possesses a high level of constitutive activity in cell-based assays, obscuring the detection of ligand activators. However, a human splice variant of CAR, termed CAR3, exhibits negligible constitutive activity. In addition, CAR3 is activated by ligands with similar specificity as the reference form of the receptor. In this study, we hypothesized that similar CAR3 receptors could be constructed across various mammalian species' forms of CAR that would preserve species-specific ligand responses, thus enabling a more sensitive and differential screening assessment of CAR response among animal models. A battery of CAR3 receptors was produced in mouse, rat, and dog and comparatively evaluated with selected ligands together with human CAR1 and CAR3 in mammalian cell reporter assays. The results demonstrate that the 5-amino acid insertion that typifies human CAR3 also imparts ligand-activated receptor function in other species' CAR while maintaining signature responses in each species to select CAR ligands. These variant constructs permit in vitro evaluation of differential chemical effector responses across species and coupled with in vivo assays, the species-selective contributions of CAR in normal physiology and in disease processes such as hepatocarcinogenesis.

Selective phthalate activation of naturally occurring human constitutive androstane receptor splice variants and the pregnane X receptor.

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.

Hepatocyte differentiation.

Increasingly, research suggests that for certain systems, animal models are insufficient for human toxicology testing. The development of robust, in vitro models of human toxicity is required to decrease our dependence on potentially misleading in vivo animal studies. A critical development in human toxicology testing is the use of human primary hepatocytes to model processes that occur in the intact liver. However, in order to serve as an appropriate model, primary hepatocytes must be maintained in such a way that they persist in their differentiated state. While many hepatocyte culture methods exist, the two-dimensional collagen "sandwich" system combined with a serum-free medium, supplemented with physiological glucocorticoid concentrations, appears to robustly maintain hepatocyte character. Studies in rat and human hepatocytes have shown that when cultured under these conditions, hepatocytes maintain many markers of differentiation including morphology, expression of plasma proteins, hepatic nuclear factors, phase I and II metabolic enzymes. Functionally, these culture conditions also preserve hepatic stress response pathways, such as the SAPK and MAPK pathways, as well as prototypical xenobiotic induction responses. This chapter will briefly review culture methodologies but will primarily focus on hallmark hepatocyte structural, expression and functional markers that characterize the differentiation status of the hepatocyte.

Kynurenic acid is a potent endogenous aryl hydrocarbon receptor ligand that synergistically induces interleukin-6 in the presence of inflammatory signaling.

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.

Functional and biological determinants affecting the duration of action and efficacy of anti-(+)-methamphetamine monoclonal antibodies in rats.

These studies examined the in vivo pharmacokinetics and efficacy of five anti-methamphetamine monoclonal antibodies (mAbs, K(D) values from 11 to 250 nM) in rats. While no substantive differences in mAb systemic clearance (t(1/2)=6.1-6.9 days) were found, in vivo function was significantly reduced within 1-3 days for four of the five mAbs. Only mAb4G9 was capable of prolonged efficacy, as judged by prolonged high methamphetamine serum concentrations. MAb4G9 also maintained high amphetamine serum concentrations, along with reductions in methamphetamine and amphetamine brain concentrations, indicating neuroprotection. The combination of broad specificity for methamphetamine-like drugs, high affinity, and prolonged action in vivo suggests mAb4G9 is a potentially efficacious medication for treating human methamphetamine-related medical diseases.