Biomedical subjects

M E Hahn

Publications and source records attributed to M E Hahn.

At least 37 records · Page 2Linked to original sources

PubMed2000

The bioflavonoid galangin blocks aryl hydrocarbon receptor activation and polycyclic aromatic hydrocarbon-induced pre-B cell apoptosis.

Bioflavonoids are plant compounds touted for their potential to treat or prevent several diseases including cancers induced by common environmental chemicals. Much of the biologic activity of one such class of pollutants, polycyclic aromatic hydrocarbons (PAH), is mediated by the aryl hydrocarbon receptor/transcription factor (AhR). For example, the AhR regulates PAH immunotoxicity that manifests as pre-B cell apoptosis in models of B cell development. Because bioflavonoids block PAH-induced cell transformation and are structurally similar to AhR ligands, it was postulated that some of them would suppress PAH-induced, AhR-dependent immunotoxicity, possibly through a direct AhR blockade. This hypothesis was tested using a model of B cell development in which pre-B cells are cultured with and are dependent on bone marrow stromal or hepatic parenchymal cell monolayers. Of seven bioflavonoids screened, galangin (3,5,7-trihydroxyflavone) blocked PAH-induced but not C(2)-ceramide- or H(2)O(2)-induced pre-B cell apoptosis. Because galangin blocked AhR-dependent reporter gene expression, AhR complex-DNA binding, and AhR nuclear translocation, inhibition of a relatively early step in AhR signaling was implicated. This hypothesis was supported by the ability of galangin to bind the AhR and stabilize AhR-90-kDa heat shock protein complexes in the presence of AhR agonists. These studies demonstrate the utility of pre-B cell culture systems in identifying compounds capable of blocking PAH immunotoxicity, define at least one mechanism of galangin activity (i.e., repression of AhR activation), and motivate the use of this and similar dietary bioflavonoids as relatively nontoxic inhibitors of AhR agonist activity and as pharmacologic agents with which to dissect AhR signaling pathways.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1999

Identification and functional characterization of two highly divergent aryl hydrocarbon receptors (AHR1 and AHR2) in the teleost Fundulus heteroclitus. Evidence for a novel subfamily of ligand-binding basic helix loop helix-Per-ARNT-Sim (bHLH-PAS) factors.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor through which 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds cause altered gene expression and toxicity. The AHR belongs to an emerging multigene family of transcription factors possessing basic helix loop helix (bHLH) and Per-ARNT-Sim (PAS) domains. Most bHLH-PAS proteins occur as duplicates or "paralog groups" in mammals, but only a single mammalian AHR has been identified. Here we report the cDNA cloning of two distinct AHRs, designated FhAHR1 and FhAHR2, from a single vertebrate species, the teleost Fundulus heteroclitus (Atlantic killifish). Both Fundulus AHR proteins possess bHLH and PAS domains that are closely related to those of the mammalian AHR. FhAHR1 and FhAHR2 are highly divergent (40% overall amino acid identity; 61% identity in the N-terminal half), suggesting that they arose from a gene duplication predating the divergence of mammals and fish. Photoaffinity labeling with 2-azido-3-[(125)I]iodo-7, 8-dibromodibenzo-p-dioxin and velocity sedimentation analysis using 2,3,7,8-[1,6-(3)H]TCDD showed that both FhAHR1 and FhAHR2 exhibit specific, high-affinity binding of dioxins. Both AHRs also showed specific, TCDD- and ARNT-dependent interactions with a mammalian xenobiotic response element. The two Fundulus AHR genes displayed different tissue-specific patterns of expression; FhAHR1 transcripts were primarily expressed in brain, heart, ovary, and testis, while FhAHR2 transcripts were equally abundant in many tissues. Phylogenetic analysis demonstrated that Fundulus AHR1 is an ortholog of mammalian AHRs, while AHR2 forms in Fundulus and other fish are paralogous to Fundulus AHR1 and the mammalian AHRs and thus represent a novel vertebrate subfamily of ligand-binding AHRs.

Amino Acid Sequence↗

PubMed1999

The role of polycyclic aromatic hydrocarbon metabolism in dimethylbenz[a]anthracene-induced pre-B lymphocyte apoptosis.

Previous studies indicated that two prototypic PAH, benzo[a]pyrene (B[a]P) and 7,12-dimethylbenz[a]anthracene (DMBA), suppress the developing immune system by inducing apoptosis in bone marrow pre-B lymphocytes. In bone marrow cultures consisting of pre-B cells growing on bone marrow stromal cell monolayers, pre-B cell apoptosis was shown to be dependent on the aryl hydrocarbon receptor/transcription factor (AhR) expressed in stromal cells. However, it was not determined if AhR activation alone is sufficient or if DMBA metabolism is required for induction of a stromal cell-derived apoptosis signal. To address these issues we assessed: 1) the ability of poorly metabolized AhR ligands to induce pre-B cell apoptosis and 2) the capacity for and the mechanism through which an early DMBA metabolite induces pre-B cell apoptosis. Three poorly metabolized AhR ligands, 2,3,7,8-tetrachlorodibenzo-p-dioxin, 3,3',4,4',5-pentachlorobiphenyl, and 3,3',4,4'-tetrachlorobiphenyl failed to induce pre-B cell apoptosis in bone marrow cultures, indicating that AhR activation alone is not sufficient to induce apoptosis and suggesting a role for PAH metabolism in induction of an apoptosis signal. Consistent with this hypothesis, DMBA-3, 4-dihydrodiol, an early DMBA metabolite, induced significant pre-B cell apoptosis. The ability of DMBA-3,4-dihydrodiol to activate the AhR, inhibition of DMBA-3,4-dihydrodiol-induced apoptosis by alpha-naphthoflavone, and the significantly lower levels of DMBA-3, 4-dihydrodiol-induced apoposis in pre-B cell populations maintained on AhR(-) stromal cells strongly support a role for the AhR in DMBA-3,4-dihydrodiol-induced apoptosis. Of two DMBA-metabolizing enzymes evaluated, CYP1A1 and CYP1B1, the latter appeared to be the more likely to play a role in DMBA-induced apoptosis. These data confirm a role for the AhR in PAH-induced pre-B cell apoptosis, indicate a role for DMBA metabolism, and suggest a feedback loop in which at least one product of DMBA metabolism augments AhR signaling, leading to induction of an apoptosis stimulus.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1999

Two forms of aryl hydrocarbon receptor type 2 in rainbow trout (Oncorhynchus mykiss). Evidence for differential expression and enhancer specificity.

Two aryl hydrocarbon receptors (AhRs), rtAhR2alpha and rtAhR2beta, were cloned from rainbow trout (rt) cDNA libraries. The distribution of sequence differences, genomic Southern blot analysis, and the presence of both transcripts in all individual rainbow trout examined suggest that the two forms of rtAhR2 are derived from separate genes. The two rtAhR2s have significant sequence similarity with AhRs cloned from mammalian species, especially in the basic helix-loop-helix and PAS functional domains located in the amino-terminal 400 amino acids of the protein. In contrast, the Gln-rich transactivation domain found in the carboxyl-terminal half of mammalian AhRs is absent from both rtAhR2s. Both clones were expressed by in vitro transcription/translation and proteins of approximately 125 kDa were produced. These proteins bind 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) and are able to bind dioxin response elements in gel shift assays. rtAhR2alpha and rtAhR2beta are expressed in a tissue-specific manner with the highest expression of rtAhR2beta in the heart. Expression of rtAhR2alpha and rtAhR2beta mRNAs is positively regulated by TCDD. Both rtAhR2alpha and rtAhR2beta produced TCDD-dependent activation of a reporter gene driven by dioxin response elements. Surprisingly, the two receptors showed distinct preferences for different enhancer sequences. These results suggest that the two receptor forms may regulate different sets of genes, and may play different roles in the toxic responses produced by AhR agonists such as TCDD.

Amino Acid Sequence↗

PubMed1999

Functional diversity of vertebrate ARNT proteins: identification of ARNT2 as the predominant form of ARNT in the marine teleost, Fundulus heteroclitus.

The aryl hydrocarbon receptor nuclear translocator (ARNT) is a member of the bHLH/PAS protein superfamily. ARNT dimerizes with several PAS superfamily members, including the ligand-activated aryl hydrocarbon receptor (AHR), forming a complex that alters transcription by binding specific elements within the promoters of target genes. Two genes encode different forms of the protein in rodents: ARNT1, which is widely expressed, and ARNT2, which is limited to the brain and kidneys of adults and specific neural and branchial tissues of embryos. In an effort to characterize aryl hydrocarbon signaling mechanisms in Fundulus heteroclitus, a marine teleost that can develop heritable xenobiotic resistance, we have isolated a liver cDNA encoding an ARNT homolog. The protein exhibits AHR-dependent DNA binding capability typical of other vertebrate ARNTs. Unexpectedly, phylogenetic analysis reveals that the cDNA encodes an ARNT2. This is the only detectable ARNT sequence in Fundulus liver, gill, ovary, and brain, suggesting that ARNT2 is the predominant form of ARNT in this species. Also surprising is the relative lack of sequence identity with another fish ARNT protein, rainbow trout ARNTb, which we show forms a distinct branch outside the ARNT1 and ARNT2 clades in phylogenetic analyses. Functional diversity of ARNT proteins in fish may have important implications for the assessment of aryl hydrocarbon effects on natural populations. The increasing use of fish models in developmental and toxicological studies underscores the importance of identifying taxon-specific roles of ARNT proteins and their potential dimeric partners in the PAS superfamily.

Amino Acid Sequence↗

PubMed1998

Low inducibility of CYP1A activity by polychlorinated biphenyls (PCBs) in flounder (Platichthys flesus): characterization of the Ah receptor and the role of CYP1A inhibition.

Several studies have reported a low inducibility of hepatic cytochrome P4501A (CYP1A) activity in European flounder (Platichthys flesus) following exposure to mixtures of polychlorinated biphenyls (PCBs). Here we report on mechanistic studies toward understanding this low CYP1A inducibility of flounder, involving molecular characterization of the Ah receptor (AhR) pathway as well as inhibition of the CYP1A catalytic activity by PCB congeners. Hepatic cytosolic AhR levels in flounder were determined using hydroxylapatite, protamine sulfate adsorption analysis, or velocity sedimentation on sucrose gradients. AhR levels in flounder (approximately 2-7 fmol/mg protein) were much lower than observed generally in rodents (approximately 50-300 fmol/mg protein). Molecular characterization of the flounder AhR was provided by first-strand cDNA synthesis and amplification of flounder hepatic poly(A)+ RNA using RT-PCR. A 690-bp product was found, similar in size to a Fundulus AhR cDNA. The specificity of the 690-bp band was established by Southern blotting and hybridization with a degenerate AhR oligonucleotide. The deduced amino acid sequence of the flounder AhR fragment was 59-60% identical to mammalian AhR sequences. Although the AhR is present in flounder cytosol, we were unable to demonstrate detectable amounts of inducible TCDD-AhR-DRE complex in gel-retardation assays. High induction levels of CYP1A protein and associated EROD activity have been previously found in flounder following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In contrast, the induction of CYP1A catalytic activity by PCB mixtures remains unexpectedly low. Therefore, we further characterized the inhibitory potential of PCB congeners on CYP1A activity in flounder and compared this with inhibitory effects of PCB congeners on rat CYP1A activity. Analysis in vitro demonstrated that 3,3',4,4'-tetraCB, 3,3',4,4',5-pentaCB, 2,2',4,4',5,5'-hexaCB, 3,3',4,4',5,5'-hexaCB, and the commercial PCB mixture Clophen A50 are potent competitive inhibitors of hepatic microsomal CYP1A catalytic activity in flounder and rat. The K(m) for ethoxyresorufin (0.095 microM) in flounder is strikingly close to Ki's found for the tested PCBs. This emphasizes the possible involvement of PCB congeners in inhibition of EROD activity in PHAH exposed fish. Finally, our data indicate that flounder CYP1A is more efficient in metabolizing ethoxyresorufin than that of rat CYP1A.

Amino Acid Sequence↗

PubMed1998

A fish hepatoma cell line (PLHC-1) as a tool to study cytotoxicity and CYP1A induction properties of cellulose and wood chip extracts.

Cytotoxicity and CYP1A induction properties of celluloses and wood chips were studied with a teleost liver cell line, PLHC-1. Cells were exposed to acetone extracts of celluloses produced using new bleaching techniques (elemental chlorine free, ECF; totally chlorine free, TCF) in two sulphate mills or without any bleaching (unbleached, UB) in a sulphite mill. In another set of exposures, celluloses (ECF and TCF bleached) and wood chips (from pine and birch) were collected from a sulphate mill, extracted with acetone, and the extracts used to treat the cells. After exposure, O-deethylation of 7-ethoxyresorufin (EROD, a measure of cytochrome P4501A (CYP1A) catalytic activity), and total protein content, a measure of cytotoxicity, were assayed. The presence of the CYP1A protein in the exposed cells was assessed by immunoblotting. The cellulose and wood chip extracts were able to cause both cytotoxicity and EROD induction in the PLHC-1 cells. In the exposures conducted with the material from three different mills, the celluloses made of birch were more cytotoxic and more potent inducers of EROD activity than were the celluloses of pine. Further, UB celluloses increased EROD activity and caused cytotoxicity at lower doses than material bleached with modern bleaching techniques. In the exposures made with material from one single mill, there were no clear trends between the celluloses made of pine or birch. Wood chips of pine, however, were more cytotoxic than wood chips of birch. Especially with pine wood chips, cytotoxicity interfered with the induction of EROD activity, thus complicating the evaluation of CYP1A induction. CYP1A protein content was not detected in cells exposed to extracts of celluloses or wood chips, possibly due to low amounts of protein available for the assay. Wood and pulp processing, like bleaching, may change the chemical composition of the raw material in a way that reduces the potency for biological effects of the final product, cellulose. This could explain why both UB celluloses and wood chips were more potent in the cells than ECF or TCF bleached celluloses. In this study the PLHC-1 cell line showed its potential for use in evaluating the biological activity existing in pulp and paper mill products and raw materials. The identity and source of the compounds that were able to affect the PLHC-1 cell line remain to be determined.

Animals↗

PubMed1998

Aryl hydrocarbon receptor function in early vertebrates: inducibility of cytochrome P450 1A in agnathan and elasmobranch fish.

The mammalian aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that controls the expression of cytochrome P450 1A (CYP1A) genes in response to halogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The natural ligand and normal physiologic function of this protein are as yet unknown. One approach to understanding AHR function and significance is to determine the evolutionary history of this receptor and of processes such as CYP1A induction that are controlled by the AHR in mammals. In these studies, AHR function was evaluated in representative cartilaginous fish (little skate, Raja erinacea) and jawless fish (sea lamprey, Petromyzon marinus and Atlantic hagfish, Myxine glutinosa), using CYP1A induction as a model AHR-dependent response. Treatment of skate with beta-naphthoflavone (BNF) caused an 8-fold increase in hepatic ethoxyresorufin O-deethylase (EROD) activity as well as a 37-fold increase in the content of immunodetectable CYP1A protein. Evidence of CYP1A inducibility was also obtained for another cartilaginous fish, the smooth dogfish Mustelus canis. In contrast, hepatic EROD activity was not detected in untreated lamprey nor in lamprey treated with 3,3'4,4'-tetrachlorobiphenyl (TCB), a potent AHR agonist in teleosts. A possible CYP1A homolog was detected in lamprey hepatic microsomes by one of three antibodies to teleost CYP1A, but expression of this protein was not altered by TCB treatment. CYP1A protein and catalytic activity were measurable in hagfish, but neither was induced after treatment with TCB. These results suggest that the AHR-CYP1A signal transduction pathway is highly conserved in gnathostomes, but that there may be fundamental differences in AHR signaling or AHR-CYP1A coupling in agnathan fish. Agnathan fish such as hagfish and lamprey may be interesting model species for examining possible ancestral AHR functions not related to CYP1A regulation.

Animals↗

PubMed1998

The aryl hydrocarbon receptor: a comparative perspective.

The aryl hydrocarbon receptor (Ah receptor or AHR) is a ligand-activated transcription factor involved in the regulation of several genes, including those for xenobiotic-metabolizing enzymes such as cytochrome P450 1A and 1B forms. Ligands for the AHR include a variety of aromatic hydrocarbons, including the chlorinated dioxins and related halogenated aromatic hydrocarbons whose toxicity occurs through activation of the AHR. The AHR and its dimerization partner ARNT are members of the emerging bHLH-PAS family of transcriptional regulatory proteins. In this review, our current understanding of the AHR signal transduction pathway in non-mammalian and other non-traditional species is summarized, with an emphasis on similarities and differences in comparison to the AHR pathway in rodents and humans. Evidence and prospects for the presence of a functional AHR in early vertebrates and invertebrates are also examined. An overview of the bHLH-PAS family is presented in relation to the diversity of bHLH-PAS proteins and the functional and evolutionary relationships of the AHR and ARNT to the other members of this family. Finally, some of the most promising directions for future research on the comparative biochemistry and molecular biology of the AHR and ARNT are discussed.

Amino Acid Sequence↗

PubMed1998

Genetic and developmental influences on infant mouse ultrasonic calling. II. Developmental patterns in the calls of mice 2-12 days of age.

Infant house mice, Mus musculus, produce ultrasonic calls that reliably lead to retrieval by adult mice. While individual differences in calls have been demonstrated both among and within species, the influences of age and sex on call characteristics have not been systematically investigated in mice. This study examined the influences of age, sex, and genotype (inbred versus hybrid) on the rate, length, and frequency characteristics of the calls of 486 male and female mice from 2 to 12 days of age. Rate of calling followed a shallow inverted U-shaped function across days. Call lengths decreased and call frequency characteristics increased, in a linear manner, with age. Females emitted fewer calls, with a smaller bandwidth, at some ages than males. Hybrid pups produced more calls of greater length and a lower frequency than inbred pups. These results indicate the presence of cues that could allow adult mice to behave differentially toward pups as a function of their age and sex.

Age Factors↗

PubMed1997

Molecular evolution of two vertebrate aryl hydrocarbon (dioxin) receptors (AHR1 and AHR2) and the PAS family.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor through which halogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) cause altered gene expression and toxicity. The AHR belongs to the basic helix-loop-helix/Per-ARNT-Sim (bHLH-PAS) family of transcriptional regulatory proteins, whose members play key roles in development, circadian rhythmicity, and environmental homeostasis; however, the normal cellular function of the AHR is not yet known. As part of a phylogenetic approach to understanding the function and evolutionary origin of the AHR, we sequenced the PAS homology domain of AHRs from several species of early vertebrates and performed phylogenetic analyses of these AHR amino acid sequences in relation to mammalian AHRs and 24 other members of the PAS family. AHR sequences were identified in a teleost (the killifish Fundulus heteroclitus), two elasmobranch species (the skate Raja erinacea and the dogfish Mustelus canis), and a jawless fish (the lamprey Petromyzon marinus). Two putative AHR genes, designated AHR1 and AHR2, were found both in Fundulus and Mustelus. Phylogenetic analyses indicate that the AHR2 genes in these two species are orthologous, suggesting that an AHR gene duplication occurred early in vertebrate evolution and that multiple AHR genes may be present in other vertebrates. Database searches and phylogenetic analyses identified four putative PAS proteins in the nematode Caenorhabditis elegans, including possible AHR and ARNT homologs. Phylogenetic analysis of the PAS gene family reveals distinct clades containing both invertebrate and vertebrate PAS family members; the latter include paralogous sequences that we propose have arisen by gene duplication early in vertebrate evolution. Overall, our analyses indicate that the AHR is a phylogenetically ancient protein present in all living vertebrate groups (with a possible invertebrate homolog), thus providing an evolutionary perspective to the study of dioxin toxicity and AHR function.

Amino Acid Sequence↗

PubMed1997

Halogenated aromatic hydrocarbon-mediated porphyrin accumulation and induction of cytochrome P4501A in chicken embryo hepatocytes.

Concentration-dependent induction of cytochrome P4501A (CYP1A) and intracellular porphyrin accumulation were observed following treatment of chicken embryo hepatocyte (CEH) cultures with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 3,3',4,4'-tetrachlorobiphenyl (PCB 77, IUPAC nomenclature), 2,3',4,4',5-pentachlorobiphenyl (PCB 118), 3,3',4,4',5-pentachlorobiphenyl (PCB 126), 3,3',4,4',5,5'-hexachlorobiphenyl (PCB 169), and a commercial mixture of PCBs (Aroclor 1254). For these halogenated aromatic hydrocarbons (HAHs), or mixture, maximal CYP1A activity [measured as ethoxyresorufin-O-deethylase (EROD) activity] and immunodetectable protein were observed at concentrations just prior to, or coincident with, the concentrations at which porphyrin accumulation became evident. Both immunodetectable CYP1A protein and catalytic activity decreased at high concentrations of these compounds, but the rate and extent of decrease of immunodetectable CYP1A protein varied. Time-course studies with PCB 77 indicated a decrease in potency and an increase in maximal CYP1A induction between 24 and 48 hr of exposure which may indicate in vitro metabolism of this HAH. Intracellular accumulation of total porphyrins without CYP1A induction, was observed for 2,2',5,5'-tetrachlorobiphenyl (PCB 52), 2,2',6,6'-tetrachlorobiphenyl (PCB 54), 2,2',3,5',6-pentachlorobiphenyl (PCB 95), 2,2',4,5,5'-pentachlorobiphenyl (PCB 101), 2,2',3,3',6,6'-hexachlorobiphenyl (PCB 136), and 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153). Overall, these results are consistent with a role for CYP1A induction and/or Ah receptor activation in porphyrin accumulation mediated by HAHs with a planar configuration, whereas those that are not planar may mediate porphyrin accumulation by a mechanism not involving induction of CYP1A.

Animals↗

PubMed1997

Genetic and developmental influences on infant mouse ultrasonic calling. I. A diallel analysis of the calls of 3-day olds.

Ultrasonic calls produced by young mice reliably elicit investigation and retrieval by adults. While there are large individual differences in the characteristics of these calls, little work has been done to partition that variation. We completed a 4 x 4 diallel cross and Hayman analyses on several characteristics of these cries. The major result was the detection of directional dominance toward a higher rate of calling, longer calls, and calls of lower overall frequency with a greater bandwidth. Within the context of biometrical genetic theory, we conclude that calls with such characteristics may have important fitness value. Extending this idea, we propose that within the population sampled for this study (the animals of the four inbred strains and 12 F1 hybrid groups), the calls most effectively eliciting investigation and retrieval would be calls with the average hybrid values of the diallel cross.

Animals↗

PubMed1996

Uroporphyrin accumulation associated with cytochrome P4501A induction in fish hepatoma cells exposed to aryl hydrocarbon receptor agonists, including 2,3,7,8-tetrachlorodibenzo-p-dioxin and planar chlorobiphenyls.

Hepatic uroporphyria is a well-known effect of halo- genated aromatic hydrocarbons in mammalian and avian systems, including primary cell cultures, but attempts to produce uroporphyria in vertebrate (mammalian) hepatoma lines have been unsuccessful. In this study, the ability of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), and selected chlorobiphenyl congeners to cause uroporphyria was examined in a fish hepatoma cell line (PLHC-1) that expresses aryl hydrocarbon (Ah) receptors and an inducible cytochrome P4501A (CYP1A). Dose-dependent accumulation of porphyrins was observed in cells treated for 48 h with TCDD or 3,3',4,4'-tetrachlorobiphenyl (3,3',4,4'-TCB; IUPAC 77) when the heme precursor delta-aminolevulinic acid (ALA) was present during the last 5 h of treatment. HPLC analysis identified the porphyrins as uroporphyrin (approximately 80%) and heptacarboxylporphyrin (approximately 20%). Uroporphyria did not occur in cells treated with TCDD or 3,3',4,4'-TCB in the absence of added ALA. ALA-dependent porphyrin accumulation was also seen following treatment of PLHC-1 cells with TCDF or with the non-ortho-substituted chlorobiphenyls 3,4,4',5-tetrachlorobiphenyl (IUPAC 81) and 3,3',4,4',5-pentachlorobiphenyl (IUPAC 126). Neither of the mono-ortho-substituted chlorobiphenyls 2,3,3',4,4'-pentachlorobiphenyl (IUPAC 105) or 2,3',4,4'5-pentachlorobiphenyl (IUPAC 118) increased the porphyrin content of PLHC-1 cells. The ability of the PCB congeners to cause porphyria correlated with their ability to induce the CYP1A catalytic activity ethoxyresorufin 0-deethylase (EROD) and immunodetectable CYP1A protein in these cells, suggesting direct or indirect regulation of porphyrin accumulation via the Ah receptor and/or the induced CYP1A. Induction of EROD activity by TCDD, TCDF, and the planar polychlorinated biphenyls was biphasic, with increases at lower concentrations of inducer followed by decreased induction at higher concentrations, as seen previously. EC50 values for porphyrin accumulation were similar to, or slightly higher than, the concentrations at which peak EROD activities were obtained, suggesting a relationship between the decline in EROD activity and enhanced porphyrin accumulation. alpha-Naphthoflavone inhibited TCDD-induced EROD activity and porphyrin accumulation, providing further evidence for the involvement of a fish CYP1A in the mechanism of this prophyria. Addition of 3,3',4,4'-TCB to TCDD-treated cells also inhibited EROD activity, but enhanced porphyrin accumulation, suggesting that an interaction between the halogenated inducer and the induced CYP1A is necessary for the porphyrogenic response. PLHC-1 cells grown in medium supplemented with ALA may be a useful model System for studying mechanisms of chemical uroporphyria induced by Ah receptor agonists.

Animals↗

PubMed1996

Cytochromes P450 (CYP) in the Poeciliopsis lucida hepatocellular carcinoma cell line (PLHC-1): dose- and time-dependent glucocorticoid potentiation of CYP1A induction without induction of CYP3A.

Glucocorticoids are being found to influence expression of cytochrome P450 (CYP) genes in multiple subfamilies in mammals (J.S. Sidhu, and C.J. Omiecinski (1995) Pharmacogenetics 5, 24--36). In the present study we investigated CYP1A and CYP3A expression in the fish Poeciliopsis lucida hepatocellular carcinoma cell line (PLHC-1) after coadministration of CYP1A and CYP3A inducers, including glucocorticoids. A putative CYP3A protein is expressed in PLHC-1 cells but its content was not altered by exposure of cultures to the prototypical mammalian CYP3A inducers dexamethasone (DEX), pregnenolone-16 alpha-carbonitrile (PCN), or rifampicin (RIF). However, when coadministered with 3,3', 4,4'-tetrachlorobiphenyl or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), DEX but not PCN or RIF caused increases in the degree of CYP1A induction by these aryl hydrocarbon receptor (AHR) agonists. This increase was seen both in CYP1A protein content and rates of ethoxyresorufin-O-deethylase (EROD) activity. DEX alone caused no induction of CYP1A, indicating that the enhancement of CYP1A induction caused by DEX + AHR agonists was not an additive effect but rather a potentiation. The dose of DEX required for maximal potentiation was three orders of magnitude greater at 48 h than the dose required at 24 h. Moreover, the degree of potentiation of CYP1A induction was much greater at the lower doses than at the highest doses of TCDD. There was up to 20-fold potentiation of EROD induction in cultures exposed to 0.1 nM TCDD. Two other glucocorticoid receptor (GR) agonists, cortisol and prednisone, also produced a strong potentiation of CYP1A induction, but other mammalian CYP3A inducers that are not GR agonists, such as the anti-glucocorticoid PCN, the anti-mineralocorticoid spironolactone, or the macrolide antibiotics RIF and troleandomycin, did not potentiate the CYP1A induction in PLHC-1 cells. Addition of the mammalian GR antagonists PCN or RU 38486 reduced the DEX-mediated potentiation of CYP1A induction, whereas spironolactone had no effect on the potentiation. RU 38486 also potentiated the induction of EROD activity by the TCDD, which suggests that RU 38486 acts as a partial GR agonist in PLHC-1 cells. These results suggest that potentiation of CYP1A induction in this nonmammalian cell line proceeds by a classical GR-mediated pathway, independently of the expression of CYP3A. However, the complex interaction between doses of both GR and AHR agonists and duration of exposure, suggests that additional processes influence this potentiation. The unusually strong potentiation at lower doses of TCDD may make PLHC-1 cells particularly suitable in exploring further the consequences of this potentiation.

Animals↗

PubMed1996

Cytochrome P4501A induction in avian hepatocyte cultures: a promising approach for predicting the sensitivity of avian species to toxic effects of halogenated aromatic hydrocarbons.

Concentration-dependent effects of halogenated aromatic hydrocarbons (HAHs) on cytochrome P4501A (CYP1A) induction in primary hepatocyte cultures prepared from embryos of chickens (four breeds), pheasants, turkeys, ducks (three breeds), and herring gulls were determined. CYP1A activity was estimated by measuring ethoxyresorufin O-deethylase (EROD) activity and the concentration of immunodetectable CYP1A was estimated using mouse monoclonal antibody 1-12-3 that was prepared against scup (Stenotomus chrysops) CYP1A1. The HAHs studies were 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 3,3',4,4'-tetrachlorobiphenyl (PCB 77, IUPAC nomenclature), 3,4,4',5-tetrachlorobiphenyl (PCB 81), 3,3',4,4',5-pentachlorobiphenyl (PCB 126), 3,3',4,4',5,5'-hexachlorobiphenyl (PCB 169), 2,3,3',4,4'-pentachlorobiphenyl (PCB 105), and 2,3',4,4',5-pentachlorobiphenyl (PCB 118). Two general types of comparisons were made: (1) relative potencies of compounds within a species (expressed relative to TCDD as induction equivalency factors, IEFs) and (2) relative sensitivity of each species to EROD induction by each compound. Three methods for estimating potency were compared. These were: (1) the concentration of inducer that produced a half-maximal (EC50) EROD response, (2) the concentration producing a response equivalent to 10% of the maximal response produced by TCDD (ECTCDD 10%), and (3) a slope ratio method. For each method, the rank order in potency was TCDD > or = TCDF > PCB 126 > PCB 81 > PCB 77 > PCB 169 in chicken, pheasant, and turkey hepatocytes. The rank order was similar in duck and herring gull hepatocytes with the following exceptions: TCDF was approximately 2- to 4-fold more potent than TCDD in duck hepatocytes; PCB 169 induced EROD in gulls, but PCB 77 had no measurable effect in this species. PCB 118 was a relatively weak EROD inducer in most species/breeds, but it did not induce EROD in Pekin ducks or gulls. PCB 105 was a weak inducer in White Leghorn chicken and turkey hepatocytes, but it did not induce EROD in other species. The EC50, ECTCDD10% and slope ratio methods for estimating potencies generally gave similar IEFs for compounds that produced a maximal response that was at least 60% of the maximal response produced by TCDD. For compounds that caused a response that was 50% or lower than that produced by TCDD, EC50-based IEFs were greater (10- to 100-fold) than ECTCDD10%-based IEFs or slope-ratio-based IEFs. Among species, the rank order in sensitivity to EROD induction was chicken > pheasant > turkey > or = duck > or = herring gull. The relative sensitivity of avian hepatocyte cultures to EROD induction by PCB 77 was similar to the relative sensitivity of these species (reported elsewhere) to lethality after in ovo injection of PCB 77. Chicken hepatocyte cultures were 5-10 times more sensitive to EROD induction by TCDD than were pheasant hepatocyte cultures, which is identical to the difference in sensitivity of these species to the lethal effect of TCDD after in ovo injection. Measuring the sensitivity of hepatocyte cultures to EROD induction might be useful for estimating the sensitivity of avian species (including rare or endangered species, where it is impossible to conduct in vivo studies) to the embryotoxic effects of TCDD, non-ortho substituted PCBs, and other aryl hydrocarbon receptor agonists.

Animals↗

PubMed1996

Issues in the genetics of social behavior: revisited.

Though social behavior has not been overlooked by behavior geneticists, the number of studies is small when compared to those on individual traits. One reason for the neglect may be the difficulty of making connections between genes and social behaviors, which by definition involve the interaction of two or more organisms. Fuller and Hahn (1976) addressed this issue and described three means of establishing social groups that would facilitate genetic analysis. We survey the literature on agonistic behavior in mice from 1976 through 1994 and describe interesting uses of those three methods. One of those methods (the standard tester design) often employs a "noninteractive" social partner. We present data showing that the standard tester design may be more valuable when using an evocative and interactive standard tester.

Aggression↗

PubMed1995

Evolutionary conservation of the vertebrate Ah (dioxin) receptor: amplification and sequencing of the PAS domain of a teleost Ah receptor cDNA.

The PAS domain of a teleost Ah receptor was amplified using reverse transcription-PCR with degenerate primers containing inosine. The deduced amino acid sequence of the amplified cDNA fragment was 62-64% identical with the PAS domains of mammalian Ah receptors. These data demonstrate the homology of Ah receptors in mammals and fish, and reveal regions of this protein that are highly conserved between these diverse vertebrate groups.

Amino Acid Sequence↗