Generation of 2,3,7,8-TCDD-metabolizing enzyme by modifying rat CYP1A1 through site-directed mutagenesis

Polychlorinated dibenzo-p-dioxins (PCDDs) are known as g environmental contaminants on account of the extreme toxicity. Among these compounds, 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TetraCDD) is regarded as the most toxic one. The extremely high toxicity of 2,3,7,8-TetraCDD is based on its high affinity for Ah receptor and nearly undetectable metabolism in mammalian body. Based on our previous studies, we assumed that enlarging the space of substrate-binding pocket of rat CYP1A1 might generate the catalytic activity toward 2,3,7,8-TetraCDD. Large-sized amino acid residues located at putative substrate-binding sites of rat CYP1A1 were substituted for alanine by site-directed mutagenesis. Among eight mutants examined, the mutant in the putative F-G loop, F240A, showed metabolic activity toward 2,3,7,8-TetraCDD. HPLC and GC-MS analyses strongly suggested that the metabolite was 8-hydroxy-2,3,7-TriCDD. Ah receptor assay revealed that the affinity of 8-hydroxy-2,3,7-TriCDD for Ah receptor was less than 0.01% of 2,3,7,8-TetraCDD, indicating that the F240A-dependent metabolism resulted in remarkable detoxification of 2,3,7,8-TetraCDD. The novel 2,3,7,8-TetraCDD-metabolizing enzyme could be applicable to bioremediation of contaminated soils with dioxin, elimination of dioxin from foods, and clinical treatment for people who accidentally take dioxin into their systems.     

Patent ductus venosus and dioxin resistance in mice harboring a hypomorphic Arnt allele

The Ah receptor nuclear translocator (ARNT) is the dimeric partner of hypoxia-inducible factors and thus plays a pivotal role in cellular adaptation to low oxygen environments. ARNT is also a dimeric partner for the Ah receptor (AHR), and this complex is essential in regulating the adaptive metabolic response to polycyclic aromatic hydrocarbons. Because of the essential role of ARNT in hypoxia-driven developmental events, it has been difficult to study the physiological significance of AHR.ARNT heterodimers in vivo. To address this issue, we developed a hypomorphic Arnt allele that displayed normal development and allowed the examination of the role of ARNT in AHR biology. In this regard, the AHR is also known to mediate two additional biological processes: the toxicological response to compounds such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) and the developmental closure of a fetal vascular structure known as the ductus venosus. Although the mechanism of the adaptive pathway has been well described, the mechanism of AHR-mediated signal transduction in the toxic and developmental pathways is not well understood. Liver perfusion studies demonstrated that ARNT hypomorphs have a patent ductus venosus, identical to that observed in the Ahr null mice. Parallel dioxin toxicity studies demonstrated that the ARNT hypomorphs exhibited resistance to the end points of dioxin exposure. Moreover, we observed that toxicity could be segregated from the classical adaptive responses such as P4501A induction. Taken in sum, these experiments demonstrate that ARNT is an essential component of AHR developmental signaling and shed light on the mechanism of dioxin toxicity.     

A third genetic locus affecting the Ah (dioxin) receptor

We have isolated a new benzo(a)pyrene-resistant clone, c35, of the mouse hepatoma line, Hepa-1. Cytochrome P1-450 mRNA and P1-450-dependent aryl hydrocarbon hydroxylase (AHH) activity are no longer inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin in c35. The phenotype of c35 is unstable in long-term culture. A subclone, c35-1, having partially restored AHH inducibility, was studied in detail. The concentration of dioxin required to give half-maximal induction of AHH activity was 16-fold greater in c35-1 than in Hepa-1. Scatchard analysis showed that c35-1 contains reduced levels of the Ah (dioxin) receptor, which mediates induction of P1-450, but that the affinity of the receptor for dioxin is unaltered. In vivo assays confirmed that c35-1 possesses reduced levels of receptor but showed that it is even more severely affected in nuclear translocation of the receptor. Somatic cell hybridization experiments demonstrated that c35 is recessive and belongs to a new, third complementation group of mutants defective in Ah receptor activity. We propose that c35 is mutated either in the ligand-binding Ah receptor polypeptide or in another polypeptide required for receptor function and that in c35-1 partial reversion has occurred to generate a polypeptide which is still impaired in its role in promoting nuclear translocation and/or DNA binding.     

Investigation on the Potential Role of the AH Receptor Nuclear Translocator Protein in Vitamin D Receptor Action

Abstract

The Ah receptor nuclear translocator protein (ARNT) is required for binding of the Ah (dioxin) receptor to the xenobiotic responsive element (XRE), and is a structural component of the XRE-binding form of the Ah receptor. The vitamin D receptor requires an accessory protein for binding to the vitamin D responsive element (VDRE) in the osteocalcin gene. Since the vitamin D receptor has similarities to the Ah receptor, we investigated whether ARNT is also required for vitamin D receptor activity. Two lines of evidence demonstrate that ARNT is not required for vitamin D receptor activity, and therefore does not correspond to the vitamin D receptor accessory protein: i) Antibodies to ARNT have no effect on binding of the vitamin D receptor to the VDRE. ii) c4, a mutant of Hepa-1 cells that is defective in ARNT activity, and in which binding of the Ah receptor to the XRE does not occur, possesses a vitamin D receptor with full activity for binding the VDRE.     

Ah receptor in spleen of rodent and primate species: detection by binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin

In many species systemic toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is manifested by a generalized wasting syndrome accompanied by a variety of specific organ changes including atrophy of the thymus and spleen. TCDD toxicity in most tissues is thought to be mediated by the Ah receptor. Although the spleen is a prime target for TCDD toxicity, the possible presence of Ah receptor in the spleen has not previously been investigated. Specific binding of [3H]TCDD to Ah receptor in spleen cytosols was assessed by velocity sedimentation on sucrose gradients. Ah receptor was detected in spleen cytosols from adult Rhesus monkeys (mean +/- SEM, 36 +/- 8 fmol/mg cytosol protein), fetal Rhesus monkeys (9 +/- 6), Sprague-Dawley rats (20 +/- 5), C57BL/6J mice (18 +/- 2), New Zealand white rabbits (19 +/- 2), and Hartley guinea pigs (15 +/- 2). Ah receptor was not detectable in spleen cytosol from genetically "nonresponsive" DBA/2J mice or from Golden Syrian hamsters, a species resistant to toxicity of TCDD. Molecular properties of Ah receptor from spleen were similar to those of the receptor from liver of the same species. The high Ah receptor content in spleen cytosols from those species that are most susceptible to TCDD toxicity is consistent with the view that the Ah receptor mediates TCDD toxicity in spleen as well as in other tissues.     

Two tetratricopeptide repeat proteins facilitate human aryl hydrocarbon receptor signalling in yeast

A human aryl hydrocarbon (Ah) receptor signalling pathway was constructed in yeast and used to identify regulatory proteins that may be related to those present in mammalian cells. The sequence similarity of human hepatitis B protein X-associated protein 2 (XAP2) protein to yeast Cpr7 and Cns1 proteins suggested that these proteins might be involved in Ah receptor signalling in this model system. Ah receptor signalling from a lacZ reporter gene was reduced by approximately 60% in cells that lacked Cpr7. In vitro interaction experiments indicated that a Cpr7-GST fusion protein and Ah receptor formed a complex. Expression of Cpr7, Cns1 and the isolated tetratricopeptide repeat (TPR) region of Cpr7 from plasmids restored Ah receptor signalling function in the Cpr7-deficient strain. Thus, Cpr7 and Cns1 proteins facilitate the signalling of human Ah receptor expressed in yeast, perhaps in the same manner as the TPR-containing XAP2 protein and related chaperone proteins in mammalian cells.     

Expression of hepatic pyruvate carboxylase mRNA in C57BL/6J Ahb/b and congenic Ahd/d mice exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin

The activity and level of hepatic pyruvate carboxylase (PC) has been reported to be altered by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treatment in mice. If alteration in PC level/activity by TCDD influences TCDD toxicity, one would expect to observe an early post-exposure reduction in PC mRNA. To examine the molecular events responsible for the alteration of PC activity in livers of TCDD-treated mice, we designed a synthetic DNA oligonucleotide probe specific for PC mRNA. Northern blot analysis on RNA extracts from hepatic tissue at various times and doses post-TCDD exposure were done. Furthermore, to elucidate the role of the dioxin Ah locus on alterations of PC activity by TCDD, we utilized C57BL/6J (Ah^b/b, Ah high TCDD affinity) mice and a congenic (Ah^d/d, Ah low TCDD affinity) mouse strain. At 8 days post TCDD treatment, a dose-dependent reduction of hepatic PC mRNA levels was observed in Ah^b/b mice. The response, reduction in PC mRNA levels, in the Ah^b/b strain was about 10-fold greater than that of comparably exposed congenic Ah^d/d mice. These results indicate that previously reported reductions in PC activity/level by TCDD treatment of mice is a consequence of a reduction in PC mRNA levels and that the effect requires a competent Ah receptor. © 1996 John Wiley & Sons, Inc.     

Unique B-1 cells specific for both N-pyrrolated proteins and DNA evolve with apolipoprotein E deficiency

Lysine N-pyrrolation, a posttranslational modification, which converts lysine residues to N^ε-pyrrole-L-lysine, imparts electronegative properties to proteins, causing them to mimic DNA. Apolipoprotein E (apoE) has been identified as a soluble receptor for pyrrolated proteins (pyrP), and accelerated lysine N-pyrrolation has been observed in apoE-deficient (apoE^−/−) hyperlipidemic mice. However, the impact of pyrP accumulation consequent to apoE deficiency on the innate immune response remains unclear. Here, we investigated B-1a cells known to produce germline-encoded immunoglobulin M (IgM) from mice deficient in apoE and identified a particular cell population that specifically produces IgM antibodies against pyrP and DNA. We demonstrated an expansion of B-1a cells involved in IgM production in the peritoneal cavity of apoE^−/− mice compared with wild-type mice, consistent with a progressive increase of IgM response in the mouse sera. We found that pyrP exhibited preferential binding to B-1a cells and facilitated the production of IgM. B cell receptor analysis of pyrP-specific B-1a cells showed restricted usage of gene segments selected from the germline gene set; most sequences contained high levels of non-templated-nucleotide additions (N-additions) that could contribute to junctional diversity of B cell receptors. Finally, we report that a subset of monoclonal IgM antibodies against pyrP/DNA established from the apoE^−/− mice also contained abundant N-additions. These results suggest that the accumulation of pyrP due to apoE deficiency may influence clonal diversity in the pyrP-specific B cell repertoire. The discovery of these unique B-1a cells for pyrP/DNA provides a key link connecting covalent protein modification, lipoprotein metabolism, and innate immunity.     

Characterization of the Ah receptor from human placental tissue

The rate of thermal inactivation of the unliganded human Ah receptor, studied by sucrose density gradient centrifugation, with respect to loss of ligand binding ability, was found to be greater than those of most rodents at 20°C, but the temperature coefficient of the rate constant was much smaller than for the rodent species. This implies that the unliganded human Ah receptor would be thermally more stable than the rodent analogs at physiological temperatures. The liganded form of the human Ah receptor was found to be less stable with respect to ligand release than the rodent receptors. These differences in behavior between human and rodent Ah receptors underline the difficulties in using rodent data in the development of receptor-based models of dioxin toxicity. Attempts to develop an alternative to sucrose density gradient centrifugation, comparable with the hydroxylapatite adsorption method used to assay rodent hepatic Ah receptor, were unsuccessful.     

Ah receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin. Codistribution of unoccupied receptor with cytosolic marker enzymes during fractionation of mouse liver, rat liver and cultured Hepa-1c1 cells

In early experiments Ah receptor appeared to be localized in cytosol when in its unoccupied state and it was thought that the receptor translocated into nuclei only when occupied by its ligands. However, a recent report [Whitlock and Galeazzi (1984) J. Biol. Chem. 259, 980-985] concluded that unoccupied Ah receptor in the intact cell was primarily located within the nucleus and that apparent 'cytosolic' Ah receptor was a redistribution artifact caused by fractionation of cells in large volumes of buffer. We examined the effect of buffer volume and ionic strength on apparent 'cytosolic' versus 'nuclear' distribution of unoccupied Ah receptor in liver from C57BL/6J mice and Sprague-Dawley rats as well as Hepa-1c1c9 cells in culture. In all three systems the Ah receptor appears to shift out of the nuclear fraction and into the cytosolic fraction as the volume of buffer is increased or when the ionic strength of the buffer is increased. In each system, however, the distribution of the Ah receptor was identical to the distribution of each of three standard cytosolic marker enzymes: aldolase, glucose-6-phosphate dehydrogenase and lactate dehydrogenase. Co-distribution of unoccupied Ah receptor with these cytosolic marker enzymes during fractionation at varied buffer volumes and ionic strengths makes it seem unlikely that the unoccupied receptor is predominantly a 'nuclear' component in intact cells. Marker enzyme data favor an interpretation that unoccupied Ah receptor is primarily cytoplasmic or that this soluble protein is in equilibrium between cytoplasm and nucleus.     

Association of fertility, fitness and longevity with the murine Ah locus among (C57BL/6N) (C3H/HeN) recombinant inbred lines

The Ah locus encodes a cytosolic receptor which controls the induction of enzymes that metabolize drugs, chemical carcinogens, and other environmental pollutants. B6NXC3N recombinant inbred lines have been developed from the progenitors C57BL/6N and C3H/HeN inbred mouse strains. Ah phenotyping at each generation has resulted in the establishment of some lines containing high levels of the high-affinity Ah receptor; other lines, very low levels. A genetic model involving two unlinked loci is offered to explain the distribution of Ah receptor levels among (C57BL/6N) (C3H/HeN)F2 individuals. Between generations 7 and 13, individual females and males from the B6NXC3N recombinant inbred lines were crossed with DBA/2N males and females. Presence of high levels of the high-affinity Ah receptor in both female and male B6NXC3N mice was found to be associated with greater fertility, fitness, and longer life span. The data suggest that these parameters are correlated with the Ah locus or a closely segregating gene.     

Superantigen-based tumor therapy: in vivo activation of cytotoxic T cells

We have recently demonstrated that the superantigen staphylococcal enterotoxin A (SEA) targets in vitro activated cytotoxic T lymphocytes against tumor cells expressing major histocompatibility complex (MHC) class II antigens. In this report we analyze the use of SEA as an immunoactivator in vivo. Treatment of mice with SEA activated a fraction of CD3⁺ T cells apparently as a function of their T cell receptor V expression. SEA induced interleukin-2 receptor expression and proliferation in both CD4⁺ and CD8⁺ T cells. This proliferative response was dose-dependent (0.1 – 100 µg/mouse), peaked during day 1 after treatment and declined to background levels within 4 days. The cytotoxic response, measured as cytotoxicity to SEA-coated MHC class II⁺ target cells (staphylococcal-enterotoxin-dependent cell-mediated cytotoxicity, SDCC), was maximal at a dosage of 1 µg SEA/mouse. The SDCC was confined to the CD8⁺ T cell compartment, peaked 2 days after treatment and declined to background levels within 4 days. A second injection of SEA on day 5 after the first SEA treatment resulted in SDCC function with kinetics and magnitude identical to that seen after one injection. These results pave the way for the use of SEA in the treatment of MHC class II⁺ tumors.     

Induction of cytochrome P-450 in congenic C57BL/6J mice by isosafrole: lack of correlation with the Ah locus

Isosafrole induction of cytochrome P-450 was compared in congenic strains of C57BL/6J mice, one of which expresses normal levels of the Ah receptor [B6(Ahb)], and another that does not contain a measurable receptor concentration [B6(Ahd)]. Using sucrose gradient analysis of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) binding, an Ah receptor concentration of 69.1 +/- 3.8 fmol/mg protein was measured in the hepatic cytosol from B6(Ahb) mice, while no receptor could be detected in the cytosol from B6(Ahd) mice. Isosafrole treatment (75 mg/kg X 3 days) increased the total hepatic microsomal cytochrome P-450 content to the same extent in the two congenic strains. The level of microsomal monooxygenase induction in the isosafrole-treated B6(Ahd) mice was greater than that of B6(Ahb) mice for ethylmorphine N-demethylase and isosafrole metabolite-complex formation, the latter a measure of cytochrome P2-450. In the case of 7-ethoxycoumarin O-deethylase only the isosafrole-treated B6(Ahd) mice had elevated microsomal activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) also revealed a similar induction pattern for the two congenic strains, following isosafrole treatment. Thus, the isosafrole treated B6(Ahd) mice produced an equivalent or slightly larger induction of cytochrome P-450 than the B6(Ahb) mice, suggesting that there is no direct role for the Ah receptor in the regulation of these cytochrome P-450 monooxygenase activities by isosafrole.     

Dietary Nucleotides Increase the Proportion of a TCRγδ+ Subset of Intraepithelial Lymphocytes (IEL) and IL-7 Production by Intestinal Epithelial Cells (IEC); Implications for Modification of Cellular and Molecular Cross-talk between IEL and IEC by Dietary Nucleotides

We have investigated the effects of dietary nucleotides on intraepithelial lymphocytes (IEL) and intestinal epithelial cells (IEC) in weanling mice. The proportion of T-cell receptor (TCR) γδ⁺ IEL in BALB/c mice fed a diet supplemented with nucleotides (NT(+) diet) was significantly higher than that in mice fed the nucleotide-free diet, while the proportion of TCRαβ⁺ IEL in NT(+) diet-fed mice was significantly decreased. The change of the TCRαβ⁺/TCRγδ⁺ ratio was mainly observed in a CD8αα⁺ subset of IEL. IEC from NT(+) diet-fed mice produced a higher level of IL-7, which is important in the development of TCRγδ⁺ IEL, than those from control diet-fed mice. The expression levels of IL-7 and IL-2 receptors on IEL were not different between the two dietary groups. Our findings suggest that the increased population of a TCRγδ⁺ IEL subset by feeding nucleotides may be caused by the increased production of IL-7 by IEC.     

Membrane effects of the polyene antibiotic amphotericin B and of some of its derivatives on lymphocytes

Amphotericin B (AmB) exhibits immunomodulating properties in mice.In vitro studies on lymphocytes, in relation with these properties, are reported here with AmB and two of its derivatives: the N-Fructosyl (N-Fru AmB) and the N-thiopropionyl (AmBSH) derivatives. Interactions of these molecules with thymocytes, a sensitive cell type, demonstrated that the extent of binding is not a toxicity parameter. In contrast, membrane fludity changes have been observed and appeared to be related to toxicity.Experiments performed with normal B lymphocytes have shown that Amphotericin B derivatives were more potent polyclonal B cell activators than the parent compound. To go further in the understanding of these events, we have investigated in a B cell line WEHI 231, the changes in intracellular Ca²⁺ and membrane potential induced by AmB and AmBSH. The two polyenes were shown to induce membrane depolarization but no intracellular Ca²⁺ increase.