DRE-CALUX bioassay in comparison with HRGC/MS for measurement of toxic equivalence in environmental samples.

CALUX, Chemically Activated LUciferase gene eXpression bioassay, has proven valuable for screening for and assessing toxic equivalents of dioxin-like compounds, because it detects all AhR (arylhydrocarbon receptor) ligands in a variety of sample matrices. In this study, we tried to validate DRE (dioxin-response elements)-CALUX bioassay, which has been developed by cloning mouse cyp1a1 gene in front of luciferase reporter gene. We compared DRE-CALUX bioassay with high resolution gas chromatography/mass spectrometry (HRGC/MS) for assessing environmental samples from Korea. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) dose response study showed high correlation (r2=0.89) between DRE-CALUX bioassay and EROD (ethoxyresorufin O-deethylase) bioassay, a commonly used bioassay method. The mean TEQ value of water samples was 0.57 pg-TEQ(HRGC/MS)/L and 4.97 pg-TEQ(CALUX)/L. For soil samples, HRGC/MS-TEQ values ranged from 0 to 47.18 pg-TEQ/g (dry) and correlated well (r2=0.98) with values obtained by CALUX-TEQ which ranged from 0.92 to 649.97 pg-TEQ/g (dry). The difference between the absolute TEQ values might be due to the presence of dioxin-like compounds without WHO-TEQ values rather than the difference between CALUX-REP and WHO-TEQ. Based on this study, we suggest that DRE-CALUX bioassay can serve as an alternative bioassay method for high-throughput analysis of large number of environmental samples.

Antiestrogenic potency and binding characteristics of the triphenylethylene H1285 in MCF-7 human breast cancer cells.

The antiestrogenic character and potency of 4-(N,N-diethylaminoethoxy)-4'-methoxy-alpha-(p-hydroxyphenyl)-alpha' -ethylstilbene (H1285) and its binding to estrogen receptor and to estrogen-noncompetible antiestrogen binding sites have been studied in MCF-7 human breast cancer cells. H1285 has an affinity for the estrogen receptor (Kd 0.23 nM) which is comparable to that of estradiol (Kd 0.25 nM), and the binding of these two compounds to estrogen receptor is mutually competitive. On high salt sucrose gradients, the sedimentation profiles of nuclear receptor complexes with H1285 and estradiol are different. While the sedimentation profile of the complex with estradiol varies with the buffer composition, being 4.1S in phosphate:thioglycerol: glycerol and predominantly 5.5S in Tris:EDTA buffered gradients, the H1285 receptor complex shows the same sedimentation (5.5S) regardless of the buffer composition. H1285 also binds to estrogen-noncompetable antiestrogen binding sites that are distinct from the estrogen receptor with a low affinity, only 15% that of the antiestrogen tamoxifen. The biological character and potency of H1285 were examined by determining its effects on cell proliferation, cellular progesterone receptor levels, and plasminogen activator activity. In MCF-7 cells, H1285 was a 30- to 100-fold more potent inhibitor of cell proliferation than was the antiestrogen tamoxifen, and it was approximately equipotent with the higher affinity antiestrogen trans-hydroxytamoxifen. H1285 evoked very minimal increases in cellular progesterone receptor levels, and no increase in plasminogen activator activity over a broad range of concentrations (10(-10)-10(-6)M), and it suppressed plasminogen activator activity stimulated by estradiol. Therefore, by the criteria we have used, we conclude that H1285 is a potent and very effective antiestrogen in MCF-7 cells. The ability of estradiol to reverse the suppression of cell proliferation by H1285, and the high affinity of H1285 for estrogen receptor and its low affinity for estrogen-noncompetible antiestrogen binding sites suggest that H1285 exerts its antiestrogenic effects via interaction with the estrogen receptor of these breast cancer cells.

Antiestrogen stimulation of the production of a 37,000 molecular weight secreted protein and estrogen stimulation of the production of a 32,000 molecular weight secreted protein in MCF-7 human breast cancer cells.

Antiestrogens (AE), such as tamoxifen, selectively increase the production of a secreted protein of 37,000 mol wt (Mr) in estrogen receptor-containing human breast cancer cells (MCF-7, but not in estrogen receptor-negative MDA-MB-231 cells), and the production of this protein by AE is inhibited by concomitant estradiol (E) treatment. Likewise, E increases the production of a 32,000 Mr secreted protein whose synthesis is inhibited by AE. Proteins were detected by [35S]methionine and [35S]cysteine labeling of cells and analysis of proteins by sodium dodecyl sulfate-polyacrylamide gels and fluorography. Enhanced production of the 37,000 Mr protein is observed within 6 h of AE treatment, with maximal synthesis seen at 1-2 days when this protein represents about 6% of the total radiolabeled secreted proteins. This protein is stimulated maximally (approximately 4-fold) by 10(-8) M trans-hydroxytamoxifen or LY117018 or 10(-6) M tamoxifen, and its AE specificity is seen by the fact that trans-tamoxifen increases this protein, whereas cis-tamoxifen, an estrogen, does not. In addition to stimulating the synthesis of previously identified 160,000 and 52,000 Mr secreted proteins, E increases the production of a 32,000 Mr secreted protein. When cells are grown in estrogen-free conditions, i.e. in charcoal-dextran-treated serum in medium lacking the estrogen phenol red, the basal level of the 32,000 Mr protein is extremely low, and E stimulation results in a 10-fold increase in the production of this protein, with increases observable by 6 h and maximal stimulation at 2 days. Interestingly, the basal level of synthesis of the 37,000 Mr protein is high in the absence of E and is then stimulated only minimally by the addition of AE, suggesting that this protein is clearly produced as an estrogen-antagonistic protein. Amino acid incorporation conducted in the presence of tunicamycin and endoglycosidase H indicates that both of these proteins are glycoproteins. These proteins should serve as useful markers for AE and E action and may be involved in AE and E modulation of cell proliferation and/or cell function.

An evaluation of the role of antiestrogen-binding sites in mediating the growth modulatory effects of antiestrogens: studies using t-butylphenoxyethyl diethylamine, a compound lacking affinity for the estrogen receptor.

Tert-butylphenoxyethyl diethylamine (BPEA), a compound synthesized by us, was designed to incorporate features important in binding to antiestrogen-binding sites (AEBS) while lacking features important in binding to the estrogen receptor (ER). With this compound, we have addressed the question of the role of AEBS in mediating the growth modulatory effects of antiestrogens. BPEA has an affinity for AEBS 6% that of tamoxifen and an affinity for ER less than 0.0003% that of estradiol. BPEA (10(-11)-10(-6) M) had no effect on the growth of MCF-7 breast cancer cells and no effect on inhibition of the growth of MCF-7 cells by different concentrations of the antiestrogen tamoxifen. In addition, BPEA (even at doses of 1 mg/day X 50 g rat) exhibited no uterotropic or antiuterotropic activity in immature rats and had no influence on the agonistic or antagonistic activity of varying concentrations of tamoxifen on uterine weight. Hence, we conclude that occupancy of AEBS, at least by BPEA, does not modulate growth of the uterus or breast cancer cells and does not influence the potency of tamoxifen as an antiestrogen. These findings raise serious doubts about the role of the AEBS in mediating directly the growth modulatory effects of antiestrogens.

Inhibition of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-stimulated Cyp1a1 promoter activity by hypoxic agents.

Since hypoxia-inducible factor-1alpha (HIF-1alpha) and the arylhydrocarbon receptor (AhR) shared the AhR nuclear translocator (Arnt) for hypoxia- and AhR-mediated signaling, respectively, it was possible to establish the hypothesis that hypoxia could regulate cytochrome P450 1a1 (Cyp1a1) expression. In order to test this hypothesis, we undertook to examine the effect of hypoxia on Cyp1a1 transcription in Hepa-I cells. Mouse Cyp1a1 5'-flanking DNA, 1.6 kb was cloned into pGL3 expression vector in order to construct pmCyp1a1-Luc. Hepa-I cells were transfected with pmCyp1a1-Luc and treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the presence or absence of various hypoxic agents such as 1-100 microM cobalt chloride, 1-100 microM picolinic acid, and 1-100 microM desferrioxamine. Luciferase activity of the reporter gene was measured from pmCyp1a1-Luc-transfected Hepa-I cell lysate which contains 2 microgram total protein using luciferin as a substrate. Hypoxic agents such as cobalt chloride, picolinic acid, and desferrioxamine showed inhibition of luciferase activity that was induced by 1-nM TCDD treatment in a dose-and time-dependent manner. Concomitant treatment of 150 microM ferrous sulfate with 1-100 microM desferrioxamine or 1-100 microM picolinic acid recovered luciferase activity from that inhibited by hypoxic agents or induced by TCDD. These data demonstrated that iron-chelating and hypoxic agents inhibited dioxin-induced Cyp1a1 transcription in Hepa-I cells. Thus, we might suggest that hypoxia inhibits TCDD-induced Cyp1a1 expression due to the competition between HIF-1alpha and the AhR for the Arnt in Hepa-I cells.

Quantitative assessment of estrogenic activity in the water environment of Korea by the E-SCREEN assay.

In this study, the E-SCREEN assay was optimized and validated for the sensitive quantitative determination of the total estrogenicity in river samples. River water and sediment samples were collected and analyzed with the E-SCREEN. River water (10 l) was extracted using combined solid-phase extraction in static adsorption mode with Soxhlet extraction. Estrogenic pollutants adsorbed to the XAD-4 resin were recovered with 98.24 +/- 5.90% efficiency by elution with ethyl acetate and dichloromethane (1:9). The detection limit by 17beta-estradiol equivalent concentration (EEQ) of the E-SCREEN assay was 8.03 pg EEQ/l. Among the water samples, the estrogenic activity was observed to be higher downstream of the Kumho river (7.43 ng EEQ/l) and upstream of Kum river (2.05 ng EEQ/l) than in other samples. More than 3 mg of equivalent sediment samples from the Kumho river, Kum river and Miho stream showed partial agonistic effects, and the Mankyung river showed a partial agonistic effect with only 1.5 mg of sediment. The highest value of RPE was 83.34 downstream of the Kumho river, and the lowest value of RPE was 6.52 downstream of the Miho stream. Full estrogen agonistic activities were observed downstream of the Kumho river and upstream of the Kum river. The partial agonistic activity was observed in upstream of the Kumho river, downstream of the Mankyung river, and upstream of the Miho stream, and no agonistic action was observed downstream of the Kum river or Miho stream, or upstream of the Mankyung river. The total estrogenic activity in the river water and sediment samples was between 0.50 pg/L and 7.4 ng/L, 3.39 pg/g and 10.70 pg/g.

Effects of nonylphenol, bisphenol A, and their mixture on the viviparous swordtail fish (Xiphophorus helleri).

A number of fish species have been used for studies on endocrine disrupting chemicals (EDCs). However, despite the widespread use of oviparous fish, relatively little attention has been given to viviparous species. This study investigated the effects of EDCs in a viviparous fish and examined the possible usefulness of the fish as an alternative model for the studies on EDCs. Swordtails (Xiphophorus helleri) were exposed to nonylphenol (NP), bisphenol A (BPA), and their mixture. Both short-term (3-d) and relatively long-term (60-d) exposures were carried out using adult male and 30-d-old juvenile fish, respectively. Following the short-term exposure, both NP and BPA caused vitellogenin mRNA expression. Flow cytometric analysis and terminal deoxynucleotidyl transferase assay on the testes of treated fish indicated reproductive damage. Histopathological analysis found degenerative and necrotic cells in seminiferous tubules following the exposure to 100 ppb NP. The testes with lesions were also associated with highly suppressed spermatogenesis. Following the long-term exposure, both NP and BPA exposures significantly affected the growth of swordtails. In all cases, the results showed that the mixture was always more potent than a single chemical and that swordtail fish can be a useful model for the study of endocrine disruptors.

UDPGT cDNA expression and UDPGT1 in human liver.

Following expression of UDPGTh1 and UDPGTh2 in Cos-1 cells, each isoform metabolized three types of dihydroxy- or trihydroxy-substituted ring structures, including the 3,4-catechol estrogen (4-hydroxyestrone), estriol and 17-epiestriol, and hyodeoxycholic acid (HDCA), but the UDPGTh2 isozyme was 100-fold more efficient than UDPGTh1. UDPGTh1 and UDPGTh2 are 86% identical overall (76 differences out of 528 amino acids), including 55 differences in the first 300 amino acids of the amino terminus, a domain which confers isoform substrate specificity. The data indicate a high level of conservation in the amino terminus is not required for the preservation of substrate specificity. Analysis of glucuronidation activity encoded by UDPGTh1/UDPGTh2 chimeric cDNAs constructed at their common restriction sites, Sac I (codon 279), Nco I (codon 385), and Hha I (codon 469), showed that nine amino acids between residues 385 and 469 are important for catalytic efficiency, suggesting that this region represents a domain which is critical for catalysis but distinct from that responsible for aglycon selection. Screening of leukocyte DNA cosmid library with human UDPGT-Br1 (1-470 bps) or UDPGT-Br2 (1-450 bps) resulted in three overlapping clones, which were isolated and mapped by endonucleases. Construction of subclones and DNA sequencing, Southern blot analysis revealed that a cluster of 4 exons (132, 88, 220, 1032 bps in one clone) encodes the entire region of 3' identity shared between human UDPGT-phenol, human UDPGT-Br1 and human UDPGT-Br2. A similar strategy but using probes which correspond to the unique regions of human UDPGT-Br1 and human UDPGT-Br2 showed that the exon 1 of UGT1A and UGT1D encodes the unique region of human UDPGT-Br1, and human UDPGT-Br2 and is located 5.6 and 49 Kb, respectively, upstream of the 4 common exons.

Estrogen modulation of human breast cancer cell growth.

To gain further insight into how estrogens modulate cell function, the effects of estrogen on cell proliferation were studied in human breast cancer cells. We examined the effects of estrogen on the proliferation of three human breast cancer cell lines that differed in their estrogen receptor contents. Ten nM estradiol markedly stimulated the proliferation of MCF-7 human breast cancer cells that contained high levels of estrogen receptor (1.15+/-0.03 pmole/mg protein) over that of control. In T47D cells that contained low levels of estrogen receptor (0.23+/-0.05 pmole/mg protein), Ten nM estrogen slightly stimulated the proliferation over that of control. MDA-MB-231 cells, that contained no detectable levels of estrogen receptors, had their growth unaffected by estrogen. These results showed their sensitivity to growth stimulation by estrogen correlated well with their estrogen receptor content. Also we examined the effect of estrogen on cellular progesterone receptor level as well as plasminogen activator activity in MCF-7 cells. Ten nM estradiol showed maximal stimulation of progesterone receptor level as well as plasminogen activator activity in MCF-7 cells. It is not clear whether these stimulations of progesterone receptor and plasminogen activator activity by estrogen are related to the estrogen stimulation of cell proliferation of MCF-7 cells. Studies with estrogen in human breast cancer cells in culture indicate that sensitivity to growth stimulation by estrogen correlates well with estrogen receptor contents.

Antiestrogen, trans-tamoxifen modulation of human breast cancer cell growth.

To gain further insight into how antiestrogens modulate cell function, the effects of antiestrogen on cell proliferation were studied in human breast cancer cells. We examined the effects of trans-tamoxifen on the proliferation of three human breast cancer cell lines that differed in their estrogen receptor contents. Trans-tamoxifen (1 muM) markedly inhibited the estrogen stimulated proliferation of MCF-7 human breast cancer cells that contained high levels of estrogen receptor (1.15+/-0.03 pmole/mg protein) over that of control. In T47D cells that contained low levels of estrogen receptor (0.23+/-0.05 pmole/mg protein), trans-tamoxifen (1 muM) showed minimal inhibition of estrogen stimulated cell proliferation over that of control. MDA-MB-231 cells, that contained no detectable levels of estrogen receptors, had their growth unaffected by trans-tamoxifen treatment. These results showed their sensitivity to growth inhibition by antiestrogen correlated well with their estrogen receptor content. Also we activator activity in MCF-7 cells. Trans-tamoxifen (1 muM) showed maximal inhibition of estrogen stimulated progestrone receptor level as well as plasminogen activator activity in MCF-7 cells that were stimulated by estrogen. It is not clear whether these inhibitions of progestrone receptor and plasminogen activator activity by estrogen are related to the antiestrogen inhibition of cell proliferation of MCF-7 cells. From the results of this study, it is clearly demonstrated that trans-tamoxifen is an antiestrogen in MCF-7 human breast cancer cells. Our data suggest that the biological effectiveness of trans-tamoxifen appear to result from its affinity of interaction with the estrogen receptor.

Antiestrogen interaction with estrogen receptors and additional antiestrogen binding sites in human breast cancer MCF-7 cells.

To gain further insight into the mechanism of action of antiestrogens, we examined the interaction of antiestrogen with the estrogen receptor system and with estrogen- noncompetable antiestrogen binding sites. In addition to binding directly to the estrogen receptor, antiestrogens can be found associated with binding sites that are distinct from the estrogen receptor. In contrast to the restriction of estrogen receptors to estrogen target cells, such as those of uterus and mammary glands, antiestrogen binding sites are present in equal amounts in estrogen receptor-positive and-negative human breast cancer cell lines, such as MCF-7, T47D, and MDA-MB-231 that differ markedly in their sensitivity to antiestrogens. In order to gain greater insight into the role of these antiestrogen binding sites in the action of antiestrogens, we have examined the biopotency of different antiestrogens for the antiestrogen binding sites and that is CI628>tamoxifen>trans-hydroxy tamoxifen>CI628M>H1285>LY117018. This order of affinities does not parallel the affinity of these compounds for the estrogen receptor nor the potency of these compounds as antiestrogens. Indeed, compounds with high affinity for the estrogen receptor and greatest antiestrogenic potency have low affinities for these antiestrogen binding sites. Antiestrogenic potency correlates best with estrogen receptor affinity and not with affinity for antiestrogen binding sites. In summary, our findings suggested that interaction with the estrogen receptor is most likely the mechanism through which antiestrogens evoke their growth inhibitory effects.

Stimulation of troutCYP1A gene expression in mouse HEPA-1 cells by 3-methylcholanthrene.

TroutCYP1A-CAT expression construct was generated by cloning approximately 3.5 Kb 5' flanking DNA of trout liverCYP1A gene in front of CAT gene at pCAT-basic vector. Hepa 1 cells, which are known to contain a functional arylhydrocarbon receptor' were transfected with troutCYP1A-CAT using lipofectin. 3-Methylcholanthrene (1 nM) was added into hepa 1 cells in culture in order to examine if 5' flanking DNA of troutCYP1A gene could interact with mouse transactivating factors to bring about transcription of the chloramphenicol acetyltransferase(CAT) reporter gene. The level of CAT protein was measured by CAT ELISA and the level of CAT mRNA was determined by RTPCR. The treatment of 1 nM 3-methylcholanthrene resulted in two fold increases in CAT protein as well as CAT mRNA compared to untreated control hepa 1 cells. These data indicate that arylhydrocarbon receptors of mouse hepa 1 cells are functional to activate exogenously transfected troutCYP1A-CAT construct in terms of both transcription and translation of CAT. We also examined the effect of 3-methylcholanthrene on endogeneouscyp1a1 activity in hepa 1 cell. 3-Methylcholanthrene (1 nM) treatment to hepa 1 cells transfected with troutCYP1A-CAT construct stimulated the level of cyp1a1 mRNA by two folds and the activity of ethoxyresorufin-O-deethylase by two fold compared to that of control cells. In this study we reported that troutCYP1A-CAT reporter gene expression construct could be expressed by 3-methylcholanthrene treatment in mouse hepa 1 cells. Thus troutCYP1A-CAT could serve as a good model to study the mechanism of regulation ofCYP1A1 gene expression.

Cloning and expression of human liver UDP-glucuronosyltransferase cDNA, UDPGTh2.

The human liver cDNA clone UDPGTh2, encoding a liver UDP-glucuronosyltransferase (UDPGT) was isolated from a lambda gt 11 cDNA library by hybridization to mouse transferase cDNA clone, UDPGTm1. UDPGTh2 encoded a 529 amino acid protein with an amino terminus membrane-insertion signal peptide and a carboxyl terminus membrane-spanning region. There were three potential asparagine-linked glycosylation sites at residues 67, 68, and 315. In order to obtain UDPGTh2 protein encoded from cloned human liver UDP-glucuronosyltransferase cDNA, the clone was inserted into the pSVL vector (pUDPGTh2) and expressed in COS 1 cells. The presence of a transferase with Mr approximately 52,000 in transfected cells cultured in the presence of [(35)S]methionine was shown by immunocomplexed products with goat antimouse transferase IgG and protein A-Sepharose and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The expressed UDPGT was a glycoprotein as indicated by electrophoretic mobility shift in Mr approximately 3,000-4,000 when expressed in the presence of tunicamycin. The extent of glycosylation was difficult to assess, although one could assume that glycosyl structures incorporated at the level of endoplasmic reticulum were always the core oligosaccharides. Thus, it is likely that at least two moieties inserted can account for the shift of Mr approximately 3,000-4,000. This study demonstrates the cDNA and deduced amino acid sequence of human liver UDP-glucuronosyltransferase cDNA, UDPGTh2.

Comparison of glucuronidating activity of two human cDNAs, UDPGTh1 and UDPGTh2.

Two human liver UDP-glucuronosyltransferase cDNA clones, HLUG25 and UDPGTh2 were previously shown to encode isozymes active in the glucuronidation of hyodeoxycholic acid (HDCA) and certain estrogen derivatives (e.g., estriol and 3,4-catechol estrogens), respectively. In this study we have found that the UDPGTh-2-encoded isoform (UDPGTh2) and HLUG25-encoded isoform (UDPGTh1) have parallel aglycone specificities. When expressed in COS 1 cells, each isoform metabolized three types of dihydroxy- or trihydroxy-substituted ring structures, including the 3,4-catechol estrogen (4-hydroxyestrone), estriol, 17-epiestriol, and HDCA, but the UDPGTh2 isozyme was 100-fold more efficient than UDPGTh1. UDPGTh1 and UDPGTh2 were 86% identical overall (76 differences out of 528 amino acids), including 55 differences in the first 300 amino acids of the amino terminus, a domain which conferred the substrate specificity. The data indicated that a high level of conservation in the amino terminus was not required for the preservation of substrate selectivity. Analysis of glucuronidation activity encoded by UDPGTh1/UDPGTh2 chimeric cDNA constructed at their common restriction sites,Sac 1 (codon 297),Nco 1 (codon 385), andHha 1 (codon 469), showed that nine amino acids between residues 385 and 469 were important for catalytic efficiency, suggesting that this region represented a domain which was critical for the catalysis but distinct from that responsible for aglycone selection. These data indicate, that UDPGTh2 is a primary isoform responsible for the detoxification of the bile salt intermediate as well as the active estrogen intermediates.

Expression of human liver 3, 4-catechol estrogens UDP-Glucuronosyltransferase cDNA in COS 1 cells.

The human cDNA clone UDPGTh2, encoding a liver UDP-glucuronosyltransferase (UDPGT), was isolated from a lambdagt 11 cDNA library by hybridization to mouse transferase cDNA clone, UDPGTm1. The two clones had 74% nucleotide sequence identities in the coding region UDPGTh2 encoded a 529 amino acid protein with an amino terminus membrane-insertion signal peptide and a carboxyl terminus membrane-spanning region. In order to establish substrate specificity, the clone was inserted into the pSVL vector (pUDPGTh2) and expressed in COS 1 cells. Sixty potential substrates were tested using cells transfected with pUDPGTh2. The order of relative substrate activity, was as follows: 4-hydroxyestrone > estriol >2-hydroxyestriol > 4-hydroxyestradiol > 6alpha-hydroxyestradiol > 5alpha-androstane-3alpha, 11beta, 17beta-triol=5beta-androstane-3alpha, 11beta, 17beta-triol. There were only trace amounts of glucuronidation of 2-hydroxyestradiol and 2-hydroxyestrone, and in contrast to other cloned transferase, no gulcuronidation of either the primary estrogens and androgens (estrone, 17beta-estradiol/testosterone, androsterone) or any of the exogenous substrates tested was detected. A lineweaver-Burk plot of the effect of 4-hydroxyestrone concentration on the velocity of glucuronidation showed an apparent Km of 13 muM. The unique specificity of this transferase might play an important role in regulating the level and activity of these potent and active estrogen metabolites.

Identification of human cytochrome P450 enzymes involved in the metabolism of IN-1130, a novel activin receptor-like kinase-5 (ALK5) inhibitor.

1. The in vitro metabolism of 3-((5-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-imidazol-2-yl)methyl)benzamide (IN-1,130), a selective activin receptor-like kinase-5 (ALK5) inhibitor and a candidate drug for fibrotic disease, was studied. 2. The cytochrome P450s (CYPs) responsible for metabolism of IN-1,130 in liver microsomes of rat, mouse, dog, monkey and human, and in human CYP supersomestrade mark, were identified using specific CYP inhibitors. The order of disappearance of IN-1,130 in various liver microsomal systems studied was as follows: monkey, mouse, rat, human, and dog. 3. Five distinct metabolites (M1-M5) were identified in all the above microsomes and their production was substantially inhibited by CYP inhibitors such as SKF-525A and ketoconazole. Among nine human CYP supersomestrade mark examined, CYP3A4, CYP2C8, CYP2D6 1, and CYP2C19 were involved in the metabolism of IN-1,130, and the production of metabolites were significantly inhibited by specific CYP inhibitors. IN-1,130 disappeared fastest in CYP2C8 supersomes. CYP3A4 produced four metabolites of IN-1,130 (M1-M4), whereas supersomes expressing human FMO cDNAs, such as FMO1, FMO3, and FMO5, produced no metabolites. 4. Hence, it is concluded that metabolism of IN-1,130 is mediated by CYP3A4, CYP2C8, CYP2D6 1, and CYP2C19.

Pharmacokinetics and tissue distribution of 3-((5-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-imidazol-2-yl)methyl)benzamide; a novel ALK5 inhibitor and a potential anti-fibrosis drug.

The authors investigated the pharmacokinetics and metabolism of 3-((5-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-imidazol-2-yl)methyl)benzamide (IN-1130), a novel ALK5 inhibitor, which suppresses renal and hepatic fibrosis, and also exerts anti-metastatic effects on breast cancer-bearing MMTV-cNeu mice model. Plasma half-lives of orally administered IN-1130 were 62.6 min in mice, 76.6 +/- 10.6 min in dogs, 156.1 +/- 19.3 min in rats, and 159.9 +/- 59.9 min in monkeys. IN-1130 showed a high apparent permeability coefficient (P(app)) of (45.0 +/- 2.3) x 10(-6) cm s(-1) in in vitro permeability tests in a Caco-2 cell monolayer model. The bioavailability of orally administered IN-1130 was 84.9% in dogs and 34.4% in monkeys (oral dose, 5.5 mg kg(-1)), 11.4% in rats and 8.95% in mice (oral dose, 50.3 mg kg(-1)), respectively. Orally given IN-1130 was readily distributed into liver, kidneys and lungs. The major metabolite of IN-1130 (M1) was detected in the systemic circulation of rat and mouse and was purified and tentatively identified as 3-((4-(3-hydroxyquinoxaline-6-yl)-5-(6-methylpyridine-2-yl)-1H-imidazol-2-yl)methyl)benzamide or 3-((4-(2-hydroxyquinoxalin-6-yl)-5-(6-methylpyridine-2-yl)-1H-imidazol-2-yl)methyl)benzamide. The highest levels of M1 were found in liver. The results of this study suggest that IN-1130 has the potential to serve as an effective oral anti-fibrotic drug.

IN-1130, a novel transforming growth factor-beta type I receptor kinase (ALK5) inhibitor, suppresses renal fibrosis in obstructive nephropathy.

The transforming growth factor-beta (TGF-beta) plays a central role in the progression of renal fibrosis. TGF-beta transduces its signal through the activin receptor-like kinase (ALK)5. IN-1130, a novel small molecule ALK5 inhibitor, inhibited the purified kinase domain of ALK5-mediated Smad3 phosphorylation with an IC(50) value of 5.3 nM. IN-1130 proved to be highly selective in a panel of 27 serine/threonine and tyrosine kinases including p38alpha mitogen-activated protein kinase. We evaluated the efficacy of IN-1130 to block renal fibrogenesis induced by unilateral ureteral obstruction (UUO) in rats. Either vehicle (saline) or IN-1130 (10 and 20 mg/kg/day) was intraperitoneally administered to UUO rats for 7 and 14 days. Phosphorylated Smad2 (pSmad2) and markers of fibrosis were analyzed in kidney tissues. In UUO control kidneys, interstitial fibrosis including tubular atrophy, loss and dilation, inflammatory cell infiltration, and fibroblast cell proliferation was prominent. These morphological changes were notably reduced by IN-1130 treatment. IN-1130 decreased levels of TGF-beta1 messenger RNA (mRNA), type I collagen mRNA, and pSmad2, compared to UUO control rats. As determined by measuring the hydroxyproline content, total kidney collagen amount was increased in UUO control kidneys, but significantly reduced by IN-1130 treatment, which was comparable to results of histochemical staining for collagen. IN-1130 also suppressed the expression of alpha-smooth muscle actin (alpha-SMA) and fibronectin in UUO kidneys. Our results show that IN-1130 suppressed the fibrogenic process of UUO, further underscoring the potential clinical benefits of IN-1130 in the treatment of renal fibrosis.

Nitric oxide inhibits dioxin action for the stimulation of Cyp1a1 promoter activity.

Since it is known that hypoxia increases inducible nitric oxide synthase (iNOS) gene expression through the hypoxia responsive element, it was hypothesized that nitric oxide could be a mediator of hypoxia to inhibit Cyp1a1 promoter activity. In order to test this hypothesis, we have undertaken a study to examine the effects of hypoxia and nitric oxide on Cyp1a1 promoter activity in Hepa I cells. Mouse Cyp1a1 5' flanking DNA, 1.6kb, was cloned into pGL3 expression vector in order to construct pmCyp1a1-Luc. Hepa I cells were transfected with pmCyp1a1-Luc and were treated with 10(-9)M 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the presence or absence of various hypoxic agents such as 10(-6)-10(-4) cobalt chloride or 10(-6)-10(-4)M picolinic acid or 10(-6)-10(-4) M desferrioxamine. The luciferase activity of the reporter gene was measured from pmCyp1a1-Luc transfected Hepa I cell lysate which contains 2 microg total protein using luciferin as a substrate. Hypoxic agents such as cobalt chloride, picolinic acid, and desferrioxamine showed inhibition of luciferase activity that was induced by 10(-9) M TCDD treatment in a dose dependent manner. Concomitant treatment of 1 mM N(G)-nitro-l-arginine with 10(-6)-10(-4) M cobalt chloride or 10(-6)-10(-4) M desferrioxamine or 10(-6)-10(-4) M picolinic acid or 10(-6)-10(-4) M sodium nitroprusside recovered luciferase activity from the TCDD induced luciferase activity that was inhibited by hypoxic agents. These data demonstrated that nitric oxide might be a mediator of iron chelating agents and hypoxic agents to inhibit dioxin induced Cyp1a1 promoter activity in Hepa I cells.

Cloning and expression of human liver UDP-glucuronosyltransferase in COS-1 cells. 3,4-catechol estrogens and estriol as primary substrates.

The human cDNA clone, UDPGTh-2, encoding a liver UDP-glucuronosyltransferase (transferase) was isolated from a lambda gt11 cDNA library by hybridization to the mouse transferase cDNA clone, UDPGTm-1 (Kimura, T., and Owens, I. S. (1987) Eur. J. Biochem.168, 515-521). The two clones have nucleotide sequence identities in the coding region of 74%. UDPGTh-2 encodes a 529-amino acid protein with an NH2 terminus membrane-insertion signal peptide and a carboxyl terminus membrane-spanning region. There are three potential asparagine-linked glycosylation sites at residues 67, 68, and 315. In order to establish substrate specificity, the clone was inserted into the pSVL vector (pUDPGTh-2) and expressed in COS-1 cells. The presence of a transferase with Mr congruent to 52,000 in transfected cells cultured in the presence of [35S]methionine was shown by immunocomplexed products with goat antimouse transferase IgG (Mackenzie, P. I., Hjelmeland, L. H., and Owens, I. S. (1984) Arch. Biochem. Biophys. 231, 487-497) and protein A-Sepharose and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The transferase is a glycoprotein as indicated by a shift in Mr congruent to 3000-4000 when expressed in the presence of tunicamycin. Sixty potential substrates were tested using cells transfected with pUDPGTh-2. The order of relative substrate activity was as follows: 4-hydroxyestrone greater than estriol greater than 2-hydroxyestriol greater than 4-hydroxyestradiol greater than 6 alpha-hydroxyestriol greater than 5 alpha-androstane-3 alpha,11 beta,17 beta-triol = 5 beta-androstane-3 alpha,11 beta,17 beta-triol. There were only trace amounts of glucuronidation of 2-hydroxyestrone and 2-hydroxyestradiol, and, in contrast to other cloned transferases, no glucuronidation of either the primary estrogens/androgens (estrone, 17 beta-estradiol/testosterone, androsterone) or any of the exogenous substrates tested. A Lineweaver-Burk plot of the effect of 4-hydroxyestrone concentration on the velocity of glucuronidation shows an apparent Km of 13 microM. The unique specificity of this transferase for 3,4-catechol estrogens and estriol suggests it may play an important role in regulating the level and activity of these potent and active estrogen metabolites.