IL-7 inhibits tumor growth by promoting T cell-mediated antitumor immunity in Meth A model.

Immune suppression is well documented during tumor progression, which includes loss of effect of T cells and expansion of T regulatory (Treg) cells. IL-7 plays a key role in the proliferation, survival and homeostasis of T cells and displays a potent antitumor activity in vivo. In the present study, we investigated the antitumor effect of IL-7 in Meth A model. IL-7 inhibited tumor growth and prolonged the survival of tumor-bearing mice with corresponding increases in the frequency of CD4 and CD8 T cells, Th1 (CD4(+)IFN-γ(+)), Tc1 (CD8(+)IFN-γ(+)) and T cells cytolytic activity against Meth A cells. Neutralization of CD4 or CD8 T cells reversed the antitumor benefit of IL-7. Furthermore, IL-7 decreased regulatory T Foxp3 as well as cells suppressive activity with a reciprocal increase in SMAD7. In addition, we observed an increase of the serum concentrations of IL-6 and IFN-γ, and a significant decrease of TGF-ß and IL-10 after IL-7 treatment. Taken together, these results indicate that IL-7 augments T cell-mediated antitumor immunity and improves the effect of antitumor in Meth A model.

3-methylcholanthrene induces neurotoxicity in developing neurons derived from human CD34+Thy1+ stem cells by activation of aryl hydrocarbon receptor.

Developing neurons, derived from the human umbilical cord blood stem cells (hUCBSCs), were investigated for their stage-specific responses against 3-methylcholanthrene (MC), a well-known polycyclic aromatic hydrocarbon. Three-dimensional (3D) molecular docking demonstrates the strong hydrogen bonding and hydrophobic interactions of MC with amino acids of aryl hydrocarbon receptor (AHR) and aryl hydrocarbon receptor nuclear translocator (ARNT) within 4 Å and subsequent inhibition of cAMP response element-binding protein (CREB), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors. Protein-protein docking also confirms that induced levels of AHR inhibit the neurogenesis-related transcription factor (CREB) with maximum docking scores. In concurrence with in silico data, MC exposure significantly up regulates the expression and activity of AHR, CYP1A1 and glutathione S-transferase P1-1 (GSTP1-1) and down regulates the expression of CREB, AMPA and NMDA receptors in hUCBSC-derived neuronal cells at various maturity (0, 2, 4, 8 days of differentiation). MC-mediated significant down regulation in the expression of stage-specific neuronal markers (Nestin, neural cell adhesion molecule-NCAM, synaptophysin-SYP, CREB, AMPA and N-methyl-D-aspartate receptor subunit 2A-NR2A) was also noticed in cells all through the differentiation. Data identify the possible interference of MC in neuronal transmission and neurogenesis.

The effect of aryl hydrocarbon receptor ligands on the expression of polymerase (DNA directed) kappa (Polκ), polymerase RNA II (DNA directed) polypeptide A (PolR2a), CYP1B1 and CYP1A1 genes in rat liver.

The aryl hydrocarbon receptor (AhR) mediates a variety of biological responses to ubiquitous environmental pollutants. AhR is ligand activated transcription factor with high affinities for aromatic planar compounds such as ß-naphthoflavone (BNF), 3-methylcholanthrene (3-MC), benzo[a]pyrene (BaP) or dioxin (TCDD). After binding appropriate ligand, AhR trigger induction of expression of some phase I and phase II drug metabolizing genes together with numerous other genes. One of such gene appear to be polymerase (DNA directed) kappa (Polκ). Polκ gene encodes newly identified low fidelity DNA polymerase. The enzyme bypasses benzo[a]pyrene-N2-dG lesions in a mostly error free manner by incorporating predominantly dC opposite the bulky lesions. It was demonstrated that AhR activation increases expression of the mouse Polκ gene and probably human POLK gene. In this study we examined the effect of i.p. administration of different AhR ligands on the expression of Polκ, RNA polymerase II polypeptide A (PolR2a) and cytochrome P450 1B1 (CYP1B1), the genes controlled by AhR in Sprague-Dawley rat liver. Quantitative real-time RT-PCR analysis revealed significant induction in the mRNA expression levels of Polκ and PolR2a following BNF treatment. Time courses of mRNA expression after treatment with BNF were similar in both genes, with maximal increases at 8h after treatment. The maximal induction of CYP1B1 and CYP1A1 expression was observed after 24 and 8h after BNF injection, respectively. TCDD treatment caused the significant increase in the mRNA level of CYP1B1 at 72h after administration of the ligand but no effect on Polκ and PolR2a mRNA expression was observed. These results confirm connection between AhR and Polκ, and strongly suggest that AhR up-regulates the mRNA transcription of PolR2a as well. However physiological importance of AhR dependent regulation of PolR2a expression must be further elucidated.

Use of the γH2AX assay for assessing the genotoxicity of polycyclic aromatic hydrocarbons in human cell lines.

The development of in vitro genotoxic assays as an alternative method to animal experimentation is of growing interest in the context of the implementation of new regulations on chemicals. However, extrapolation of toxicity data from in vitro systems to in vivo models is hampered by the fact that in vitro systems vary in their capability to metabolize chemicals, and that biotransformation can greatly influence the experimental results. Therefore, much attention has to be paid to the cellular models used and experimental conditions. Polycyclic aromatic hydrocarbons (PAHs) are carcinogenic ubiquitous pollutants. Human exposure to PAHs is mainly from food origin. In this study, a detailed analysis of the biotransformation capabilities of three human cell lines commonly used for in vitro testing (HepG2, ACHN and Caco-2) was undertaken using 3 model PAHs (benzo(a)pyrene [B(a)P], fluoranthene [FLA] and 3-methylcholanthrene [3-MC]). Concomitantly the genotoxicity of these PAHs was investigated in different cell lines, using a new genotoxic assay (H2AX) in 96-well plates. The metabolic rates of B(a)P, FLA and 3-MC were similar in HepG2 and Caco-2 cell lines, respectively, though with the production of different metabolites. The ACHN cell line was shown to express very limited metabolic capabilities. We demonstrated that the PAHs having a high metabolic rate (B(a)P and 3-MC) were genotoxic from 10(-7) molar in both HepG2 and Caco-2 cells. The present study shows that H2AX measurement in human cell lines competent for the metabolism, is an efficient and sensitive genotoxic assay requiring less cells and time than other currently available tests.

Recombinant aryl hydrocarbon receptors for bioassay of aryl hydrocarbon receptor ligands in transgenic tobacco plants.

Dioxin residues widely contaminate soil and agricultural products at low concentrations and may accumulate in organisms at the top of food chains owing to their physicochemical properties. In this study, we have developed novel, dioxin-inducible, reporter gene expression systems regulated by recombinant aryl hydrocarbon receptors (AhRs). The recombinant AhRs, referred to as XDVs, consist of the DNA-binding domain of the bacterial repressor protein LexA, a 90-kDa heat shock protein- and ligand-binding regulatory domain from mouse AhR, and the transactivation domain of herpes simplex virus regulatory protein VP16. Transgenic tobacco plants carrying XDVs absorb various AhR ligands, including 3-methylcholanthrene, beta-naphthoflavone and indigo from solid medium and vermiculite, and show dose- and time-dependent expression of the beta-glucuronidase reporter gene. The results clearly suggest that XDVs are functional transcription factors that respond to AhR ligands, and that the XDV-mediated reporter gene expression system is applicable to bioassays for dioxin residues in the environment.

3-Methylcholanthrene elicits DNA adduct formation in the CYP1A1 promoter region and attenuates reporter gene expression in rat H4IIE cells.

Cytochrome CYP1A (CYP1A) enzymes catalyze bioactivation of 3-methylcholanthrene (MC) to genotoxic metabolites. Here, we tested the hypothesis that CYP1A2 catalyzes formation of MC-DNA adducts that are preferentially formed in the promoter region of CYP1A1, resulting in modulation of CYP1A1 gene expression. MC bound covalently to plasmid DNA (50 micro g) containing human CYP1A1 promoter (pGL3-1A1), when incubated with wild-type (WT) liver microsomes (2 mg) and NAPPH 37 degrees C for 2h, giving rise to 9 adducts, as determined by (32)P-postlabeling. Eighty percent of adducts was located in the promoter region. Transient transfection of the adducted plasmids into rat hepatoma (H4IIE) cells for 16h, followed by MC (1 micro M) treatment for 24h inhibited reporter (luciferase) gene expression by 75%, compared to unadducted controls. Our results suggest that CYP1A2 plays a key role in sequence-specific MC-DNA adduct formation in the CYP1A1 promoter region, leading to attenuation of CYP1A1 gene expression.

Activation of the dioxin/aryl hydrocarbon receptor (AhR) modulates cell plasticity through a JNK-dependent mechanism.

Environmental chemicals such as dioxin adversely affect immune, neurological and reproductive functions and have been implicated in cancer development. However, the mechanisms responsible for dioxin toxicity are still poorly understood. Here, we show that dioxin and related pollutants trigger a marked morphological change in epithelial cells that remodel their cytoskeleton to increase interaction with extra cellular matrix while loosening cell-cell contacts. Furthermore, dioxin-treated cells show increased motility. These dioxin-mediated effects are mimicked by constitutive expression and activation of the intracellular dioxin receptor (aryl hydrocarbon receptor (AhR)). They correlate with activation of the Jun NH2-terminal kinase (JNK) and are reverted by treatment with a JNK inhibitor. Dioxin-induced effects occur 48 h post-treatment initiation, a time scale, which argues for a genomic effect of the AhR, linked to induction of target genes. This novel Ahr action on cell plasticity points to a role in cancer progression.

3-Methylcholanthrene and other aryl hydrocarbon receptor agonists directly activate estrogen receptor alpha.

3-Methylcholanthrene (3MC) is an aryl hydrocarbon receptor (AhR) agonist, and it has been reported that 3MC induces estrogenic activity through AhR-estrogen receptor alpha (ER alpha) interactions. In this study, we used 3MC and 3,3',4,4',5-pentachlorobiphenyl (PCB) as prototypical AhR ligands, and both compounds activated estrogen-responsive reporter genes/gene products (cathepsin D) in MCF-7 breast cancer cells. The estrogenic responses induced by these AhR ligands were inhibited by the antiestrogen ICI 182780 and by the transfection of a small inhibitory RNA for ER alpha but were not affected by the small inhibitory RNA for AhR. These results suggest that 3MC and PCB directly activate ER alpha, and this was confirmed in a competitive ER alpha binding assay and in a fluorescence resonance energy transfer experiment in which PCB and 3MC induced CFP-ER alpha/YFP-ER alpha interactions. In a chromatin immunoprecipitation assay, PCB and 3MC enhanced ER alpha (but not AhR) association with the estrogen-responsive region of the pS2 gene promoter. Moreover, in AhR knockout mice, 3MC increased uterine weights and induced expression of cyclin D1 mRNA levels. These results show that PCB and 3MC directly activate ER alpha-dependent transactivation and extend the number of ligands that activate both AhR and ER alpha.

Activation of p53 as a causal step for atherosclerosis induced by polycyclic aromatic hydrocarbons.

This study was performed to prove our hypothesis that the metabolite(s) of polycyclic aromatic hydrocarbons (PAHs) caused the activation or phosphorylation of p53 via DNA damage to suppress the liver X receptor (LXR)-mediated signal transductions as a probably more direct mechanism. We found that LXR-mediated trans-activation was inhibited by 3-methylchoranthrene (MC) and doxorubicin (Dox) in HepG2 cells carrying wild-type p53, but not in Hep3B cells possessing mutant p53. The exogenous expression of wild-type p53 suppressed the LXR-mediated trans-activation in Hep3B cells. The expression of mRNA for ATP binding cassette A1 was suppressed by MC and Dox in HepG2 cells. The protein expression of retinoid X receptor (RXR), a partner of LXR to form a heterodimer, was suppressed by MC and Dox in HepG2 cells.

Environmental prognostics: an integrated model supporting lysosomal stress responses as predictive biomarkers of animal health status.

The potential prognostic use of lysosomal reactions to environmental pollutants is explored in relation to predicting animal health in marine mussels, based on diagnostic biomarker data. Cellular lysosomes are already known to accumulate many metals and organic xenobiotics and the lysosomal accumulation of the carcinogenic polycyclic aromatic hydrocarbon 3-methylcholanthrene (3-MC) is demonstrated here in the hepatopancreatic digestive cells and ovarian oocytes of the blue mussel. Lysosomal membrane integrity or stability appears to be a generic indicator of cellular well-being in eukaryotes; and in bivalve molluscs it is correlated with total oxygen and nitrogen radical scavenging capacity (TOSC), protein synthesis, scope for growth and larval viability; and inversely correlated with DNA damage (micronuclei), as well as lysosomal swelling (volume density), lipidosis and lipofuscinosis, which are all characteristic of failed or incomplete autophagy. Integration of multiple biomarker data is achieved using multivariate statistics and then mapped onto "health status space" by using lysosomal membrane stability as a measure of cellular well-being. This is viewed as a crucial step towards the derivation of explanatory frameworks for prediction of pollutant impact on animal health; and has facilitated the development of a conceptual mechanistic model linking lysosomal damage and autophagic dysfunction with injury to cells, tissues and the whole animal. This model has also complemented the creation and use of a cell-based bioenergetic computational model of molluscan hepatopancreatic cells that simulates lysosomal and cellular reactions to pollutants. More speculatively, the use of coupled empirical measurements of biomarker reactions and modelling is proposed as a practical approach to the development of an operational toolbox for predicting the health of the environment.

Retinoids repress Ah receptor CYP1A1 induction pathway through the SMRT corepressor.

CYP1A1 isoform is mainly regulated by the transcription factor AhR and to a lesser extent by the nuclear receptor RAR. The effect of a coexposure with 3MC, a AhR ligand, and RA, a RAR ligand, which are, respectively, strong and weak CYP1A1 inducers, is poorly known. We showed in Caco-2 cells that addition of RA significantly decreased 3MC-induced CYP1A1 expression by -55% for mRNA level and -30% for promoter and enzymatic activities. We further showed that RA decreased AhR protein level. Moreover, a physical interaction between AhR and the RAR-corepressor SMRT has been described in vitro. Using the corepressor inhibitor TSA, transfected-cells with SMRT cDNA, and coimmunoprecipitation experiments, we demonstrated that RA addition repressed AhR function through a marked AhR/SMRT physical interaction. This interaction explains the decrease of 3MC-induced CYP1A1 expression. This new mechanism involving the repression of AhR-induced CYP1A1 expression by retinoids allows better knowledge of the CYP1A1 regulation.

Functional analysis of xenobiotic response elements (XREs) in CYP 1A of the European Flounder (Platichthys flesus).

The induction of hepatic cytochrome P450 1A (CYP 1A) is an important step in the response to contaminants such as polycyclic aromatic hydrocarbons (PAHs), and has been used as a biomarker of exposure in fish. Several consensus response elements have been identified, including eight potential xenobiotic response elements (XREs) in the promoter region of the European flounder cytochrome P450 1A gene. However not all of these sequences are necessarily active. To help elucidate the molecular regulation of this important gene, site directed mutagenesis and dual-luciferase reporter gene assays were employed to characterize the consensus transcription factor binding sites of the CYP 1A 5' flanking region. Mutation of response elements situated -1103, -859, -709 and -172 bases upstream of the transcription start site reduced the induction to 2.75, 1.51, 3.25 and 3.05 fold, respectively, compared with the full-length promoter (4.0-fold induction) on exposure to the PAH 3-methylcholanthrene (3MC) (1.0 microM). These results indicate that four out of eight different XREs are functional in the control of CYP 1A in the flounder. The activity of these response elements adds to the evidence for considerable diversity in vertebrate CYP 1A regulation.

Embryonic turkey liver: activities of biotransformation enzymes and activation of DNA-reactive carcinogens.

Avian embryos are a potential alternative model for chemical toxicity and carcinogenicity research. Because the toxic and carcinogenic effects of some chemicals depend on bioactivation, activities of biotransformation enzymes and formation of DNA adducts in embryonic turkey liver were examined. Biochemical analyses of 22-day in ovo turkey liver post-mitochondrial fractions revealed activities of the biotransformation enzymes 7-ethoxycoumarin de-ethylase (ECOD), 7-ethoxyresorufin de-ethylase (EROD), aldrin epoxidase (ALD), epoxide hydrolase (EH), glutathione S-transferase (GST), and UDP-glucuronyltransferase (GLUT). Following the administration of phenobarbital (24 mg/egg) on day 21, enzyme activities of ECOD, EROD, ALD, EH and GLUT, but not of GST, were increased by two-fold or higher levels by day 22. In contrast, acute administration of 3-methylcholanthrene (5 mg/egg) induced only ECOD and EROD activities. Bioactivation of structurally diverse pro-carcinogens was also examined using (32)P-postlabeling for DNA adducts. In ovo exposure of turkey embryos on day 20 of gestation to 2-acetylaminofluorene (AAF), 4,4'-methylenebis(2-chloroaniline) (MOCA), benzo[a]pyrene (BaP), and 2-amino-3,8-dimethylimidazo[4,5- f]quinoxaline (MeIQx) resulted in the formation of DNA adducts in livers collected by day 21. Some of the DNA adducts had (32)P-postlabeling chromatographic migration patterns similar to DNA adducts found in livers from Fischer F344 rats exposed to the same pro-carcinogens. We conclude that 21-day embryonic turkey liver is capable of chemical biotransformation and activation of genotoxic carcinogens to form DNA adducts. Thus, turkey embryos could be utilized to investigate potential chemical toxicity and carcinogenicity.

Effects of green tea on carcinogen-induced hepatic CYP1As in C57BL/6 mice.

Green tea (GT) drinking showed chemopreventive effects on various cancers. In addition, inhibition of CYP1A activity by green tea components--polyphenols--has been suggested as a chemoprevention against carcinogens that were bioactivated by CYP1As. Therefore, any changes in hepatic CYP1As may be considered as a biomarker for GT chemoprevention and clarify whether whole GT is chemopreventive for the population who are exposed to CYP1A specifically-bioactivated carcinogens. In this study, we investigated the changes in CYP1A levels by pre- and concurrent GT drinking against a CYP1A-inducing carcinogen, 3-methylcholanthrene (MC), in aryl hydrocarbon receptor responsive C57 BL/6 mice. We found that GT drinking itself induced hepatic CYP1As and enhanced MC-induced ethoxyresorufin-O-demethylase (EROD) activity (P<0.05). However, our studies of CYP1A monoclonal antibody and western blots revealed that the enhanced hepatic EROD activity by GT did not come from CYP1As. Therefore, our results suggest that GT may work to biotransform CYP1A inducing carcinogens into non-carcinogenic metabolites by modulation of other microsomal enzymes rather than CYP1As. In addition, the mechanism of GT chemoprevention may be different from that of GT components, such as polyphenols that reduce CYP1As activity.

The effect of plant phenolics on the formation of 7,12-dimethylbenz[a]anthracene-DNA adducts and TPA-stimulated polymorphonuclear neutrophils chemiluminescence in vitro.

Phenolics, common plant constituents, form up an important part of human diet and are considered potential chemopreventive agents. In the present study, structurally diverse phenolics, such as tannic acid, protocatechuic acid, chlorogenic acid and resveratrol, were investigated for their inhibitory effects on covalent binding of 7,12-dimethylbenz[a]anthracene (DMBA) to DNA in vitro and the suppression of oxidative burst in 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated human polymorphonuclear neutrophils (PMNs). 32P-postlabeling analysis of DNA incubated with DMBA in the presence of 3-methylcholanthrene (3-MC)-induced microsomes produced three major adducts derived from anti-, syn- and anti-dihydrodiol epoxides through reactions with dGuo and dAdo, respectively. Phenolic compounds at the concentration of 150 microM reduced the levels of all DMBA-DNA adducts by 55-98%. The most dramatic effect was observed in case of tannic acid, which completely inhibited the formation of DMBA-dAdo adducts. Chlorogenic acid was the least effective inhibitor of DMBA-DNA adducts formation particularly syn-DMBADE-dAdo (20%). Human neutrophils showed a significant dose-related decrease of TPA-induced chemiluminescence after pretreatment with phenolic compounds. The most effective inhibitors were tannic acid and resveratrol with IC(50)=5.19 and 5.76 microM, respectively. These results suggest that the suppression of reactive oxygen species (ROS) and carcinogen-DNA adducts formation may be important for anticarcinogenic activity of the examined phenolics.

[Effect evaluation of three cell culture models].

Primary rat hepatocytes were cultured using three kinds of models in vitro and the enzyme leakage, albumin secretion, and cytochrome P450 1A (CYP 1A) activity were observed. The results showed that the level of LDH in the medium decreased over time in the period of culture. However, on 5 days, LDH showed a significant increase in monolayer culture (MC) while after 8 days LDH was not detected in sandwich culture (SC). The levels of AST and ALT in the medium did not change significantly over the investigated time. The basic CYP 1A activity gradually decreased with time in MC and SC. The decline of CYP 1A in rat hepatocytes was faster in MC than that in SC. This effect was partially reversed by using cytochrome P450 (CYP450) inducers such as omeprazol and 3-methylcholanthrene (3-MC) and the CYP 1A induction was always higher in MC than that in SC. Basic CYP 1A activity in bioreactor was keeped over 2 weeks and the highest albumin production was observed in bioreactor, and next were SC and MC. In conclusion, our results clearly indicated that there have some advantages and disadvantages in each of models in which can address different questions in metabolism of toxicants and drugs.

Serum increases CYP1A1 induction by 3-methylcholanthrene.

CYP1A1 is largely involved in carcinogenesis through the bioactivation of numerous procarcinogens. Exposure to environmental pollutants such as polycyclic aromatic hydrocarbons (PAHs) leads to induction of CYP1A1 via AhR pathway. We have previously demonstrated that fetal bovine serum (FBS) induces CYP1A1 gene transcription. In this work, we show evidence that the serum does not contain an AhR ligand and we evaluated the effect of a 3-methylcholanthrene (3-MC) and FBS cotreatment on CYP1A1 expression. CYP1A1 activity was potentiated by this treatment. This potentiation was at least in part associated with an increase of the CYP1A1 mRNA and gene transcription levels. FBS potentiation of CYP1A1 PAH-mediated induction was related to a significant increase of single strand breaks of DNA as compared to a single 3-MC treatment. Moreover, we demonstrated that human serum induces CYP1A1 with a high interindividual variability. The potentiation by serum of polycyclic aromatic hydrocarbon CYP1A1 induction could be involved in the etiology of some human cancers.

3-Methylcholanthrene, which binds to the arylhydrocarbon receptor, inhibits proliferation and differentiation of osteoblasts in vitro and ossification in vivo.

3-Methylcholanthrene (3MC) is a ligand for arylhydrocarbon receptor (AhR), which binds dioxin. We examined the effects of 3MC on the proliferation and differentiation of osteoblasts using cultures of rat calvarial osteoblast-like cells (ROB cells) and mouse calvarial clonal preosteoblastic cells (MC3T3-E1 cells). Analysis by RT-PCR revealed that the mRNAs for AhR and AhR nuclear translocators were expressed in both ROB and MC3T3-E1 cells. Cell proliferation and the synthesis of DNA by ROB cells and MC3T3-E1 cells were markedly inhibited on exposure of cells to 3MC. Furthermore, 3MC reduced the activity of alkaline phosphatase and the rate of deposition of calcium by cells. The level of expression of mRNA for osteocalcin, which is a marker of osteoblastic differentiation, was also depressed by 3MC. Moreover, when 3MC (1 mg/kg body weight) was administered sc to pregnant mice at 10.5, 12.5, and 14.5 d post coitus, fetuses examined subsequently at 15.5 or 17.5 d post coitus revealed evidence of inhibition of appropriate calcification of bones. The treated metacarpals showed no subperiosteal bone matrix histologically. Our findings indicate that 3MC might have critical effects on the formation of bone both in vivo and in vitro.

Polycyclic aromatic hydrocarbons as inducers of cytochrome P4501A enzyme activity in the rainbow trout liver cell line, RTL-W1, and in primary cultures of rainbow trout hepatocytes.

In order to investigate cell-specific differences in the response of in vitro models to environmental toxicants, we compared the capacity of nine polycyclic aromatic hydrocarbons (PAHs) to induce cytochrome P4501A (CYPIA) in primary rainbow trout (Oncorhynchus mykiss) hepatocytes and a rainbow trout liver cell line, RTL-W1. Induction of CYPIA was estimated from the catalytic activity of 7-ethoxyresorufin-O-deethylase (EROD) and compared by median effective concentration (EC50) values, induction spans, and benzo[a]pyrene induction equivalency factors for inducing PAHs. The influence of culture conditions was investigated with respect to the presence or absence of serum and varying exposure times. Both in vitro systems lead to an identical classification of the PAHs in noninducing (anthracene, fluoranthene, phenanthrene, and pyrene) and inducing compounds with a similar ranking of inducing PAHs. Mean EC50 values in RTL-W1 cells were, respectively, 343 and 266 nM for benzo[a]anthracene, 57 and 92 nM for BaP, 134 and 283 nM for benzo[b]fluoranthene, 455 and 270 nM for chrysene, and 98 and 116 nM for 3-methylcholanthrene. Compared to primary hepatocytes, the RTL-W1 cell line was more sensitive in its EROD response to the presence or absence of serum and to the increase in exposure time, which led to higher EC50 values.

Distribution and inducibility by 3-methylcholanthrene of family 1 UDP-glucuronosyltransferases in the rat gastrointestinal tract.

UDP-Glucuronosyltransferases (UGT) catalyze the glucuronidation of a broad spectrum of endobiotic and xenobiotic substrates. The resulting glucuronides are more hydrophilic, facilitating renal and biliary excretion. Apart from hepatic glucuronidation, high rates of gastrointestinal glucuronidation have been observed. The aim of this study was to characterize the expression of family 1 UGTs (UGT1A) in liver, kidney, and all parts of the rat gastrointestinal tract by reverse transcription polymerase reaction (RT-PCR), Northern blot, and xenobiotic induction experiments. RT-PCR experiments were performed with primers specific for all known rat UGT1A mRNAs. UGT1A1, UGT1A6, and UGT1A7 were expressed in liver, kidney, and the gastrointestinal tract. UGT1A5 transcripts were detected in liver, but not in kidney or gastrointestinal tissue. In contrast, UGT1A2 and UGT1A3 were not expressed in liver or kidney, but were detected in intestine. Low levels of UGT1A3 were detectable in duodenum and jejunum. UGT1A2 was abundantly expressed in the small intestine; expression levels in the stomach and the large intestine were low. Quantitative evaluation of RNA levels by Northern blot revealed expression in gradients, with highest UGT1A mRNA levels in duodenum and decreasing levels in the small and large intestine. Only UGT1A6 was expressed at high levels in the rectum. Rats treated with 3-methylcholanthrene (3-MC) displayed a 10-fold induction of hepatic UGT1A6 and UGT1A7 mRNAs. In gastric tissues and in intestine, induction was 4-fold and 2-fold, respectively. In contrast to the constitutive expression of UGT1A7 in kidney, UGT1A6 was inducible in the liver. Effects of 3-MC on UGT1A1 expression revealed downregulation in the liver and highly variable effects in duodenum and stomach. This study demonstrates tissue-specific expression and tissue-specific induction patterns in rat liver, kidney, and gastrointestinal tract, which may represent the physiological basis of tissue-specific glucuronidation in rats.