Biomedical subjects

G C Clark

Publications and source records attributed to G C Clark.

At least 19 recordsLinked to original sources

PubMed2001

Serum levels of TCDD and dioxin-like chemicals in Rhesus monkeys chronically exposed to dioxin: correlation of increased serum PCB levels with endometriosis.

Humans and animals are exposed daily to a complex mixture of polyhalogenated aromatic hydrocarbons (PHAHs). Previous work has shown that exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is associated with a dose-dependent increase in the incidence and severity of endometriosis in the rhesus monkey. Dioxin-like chemicals can also exert effects in combination with TCDD via the aryl hydrocarbon receptor. This study demonstrates that the serum levels of TCDD and specific dioxin-like PHAH congeners were increased in TCDD-treated animals with endometriosis 13 years after the TCDD exposure. Nine TCDD-exposed and 6 unexposed female rhesus monkeys were evaluated for serum content of relevant compounds and for endometriosis by surgical laparoscopy. Additional studies were done on 4 animals that died 7 to 11 years after exposure to TCDD and 4 lead-treated animals with no history of PHAH treatment. For TCDD-exposed and unexposed animals, TCDD exposure correlated with an increased serum TCDD concentration. Furthermore, TCDD exposure and an elevated serum TCDD concentration were associated with increased serum levels of triglycerides, 1,2,3,6,7,8-hexachlorodibenzofuran, 3,3',4,4'-tetrachlorobiphenyl (TCB) and 3,3'4,4',5-pentachlorobiphenyl (PnCB). Importantly, the animals with elevated serum levels of 3,3',4,4'-TCB, 3,3',4,4',5-PnCB and an increased total serum TEQ had a high prevalence of endometriosis, and the severity of disease correlated with the serum concentration of 3,3,',4,4'-TCB. Increased serum concentrations of coplanar PCBs were also present in lead-treated animals. Implications of these findings for human health and the prevalence of endometriosis in humans will be discussed.

Animals↗

PubMed2001

Increased tumor necrosis factor-alpha production by peripheral blood leukocytes from TCDD-exposed rhesus monkeys.

Previous work has shown that exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is associated with a dose-dependent increase in the incidence and severity of endometriosis in the rhesus monkey. Studies also suggest that immune mechanisms participate in TCDD-mediated toxicity and the pathogenesis of endometriosis. Thirteen years after TCDD treatment was terminated, we characterized the phenotypic distribution of peripheral blood mononuclear cells (PBMC) from TCDD-exposed and -unexposed rhesus monkeys and determined the ability of these cells to produce cytokines and exert cytolytic activity against NK and T-cell-sensitive cell lines. We also determined whether elevated serum levels of TCDD, dioxin-like PHAH congeners, and triglycerides correlated with changes in PBMC phenotype or function. For all animals, TCDD exposure correlated with increased PBMC tumor necrosis factor-alpha (TNF-alpha) secretion in response to stimulation by T-cell mitogen and decreased cytolytic activity against NK-sensitive target cells. Furthermore, increased production of this cytokine by PHA-stimulated leukocytes was associated with elevated serum triglyceride levels. Leukocyte TNF-alpha secretion in response to viral antigen and PBMC production of interferon gamma (IFNgamma), IL-6, and IL-10 following exposure to mitogen or antigen were unaffected by previous TCDD treatment. Although TCDD exposure was not associated with changes in PBMC surface antigen expression, elevated serum concentrations of TCDD, 1,2,3,6,7,8-hexachlorodibenzofuran and 3,3',4,4',5-pentachlorobiphenyl correlated with increased numbers of CD3+/CD25- and CD3-/CD25+ leukocytes and enhanced secretion of TNF-alpha by mitogen-stimulated PBMC. These findings indicate that TCDD-exposed rhesus monkeys with endometriosis exhibit long-term alterations in systemic immunity associated with elevated serum levels of specific PHAH congeners.

Animals↗

PubMed2001

Application of high-density array-based signature-tagged mutagenesis to discover novel Yersinia virulence-associated genes.

Yersinia pestis, the causative agent of plague, and the enteropathogen Yersinia pseudotuberculosis have nearly identical nucleotide similarity yet cause markedly different diseases. To investigate this conundrum and to study Yersinia pathogenicity, we developed a high-density oligonucleotide array-based modification of signature-tagged mutagenesis (STM). Y. pseudotuberculosis YPIII mutants constructed with the tagged transposons were evaluated in the murine yersiniosis infection model. The DNA tags were amplified using biotinylated primers and hybridized to high-density oligonucleotide arrays containing DNA complementary to the tags. Comparison of the hybridization signals from input pools and output pools identified a mutant whose relative abundance was significantly reduced in the output pool. Sequence data from 31 transposon insertion regions was compared to the complete Y. pestis CO92 genome sequence. The 26 genes present in both species were found to be almost identical, but five Y. pseudotuberculosis genes identified through STM did not have counterparts in the Y. pestis genome and may contribute to the different tropisms in these closely related pathogens. Potential virulence genes identified include those involved in lipopolysaccharide biosynthesis, adhesion, phospholipase activity, iron assimilation, and gene regulation. The phospholipase A (PldA) mutant exhibited reduced phospholipase activity compared to the wild-type strain and in vivo attenuation of the mutant was confirmed. The combination of optimized double tag sequences and high-density array hybridization technology offers improved performance, efficiency, and reliability over classical STM and permits quantitative analysis of data.

Animals↗

PubMed1998

A new cytochrome P450 (CYP30) family identified in the clam, Mercenaria mercenaria.

A full-length clone with sequence similarity to genes in the cytochrome P450 superfamily was isolated from a cDNA library prepared from female Mercenaria mercenaria gonadal tissue. This clone was isolated while screening an expression library with an antibody prepared against a peptide sequence within the ligand-binding region of the murine Ah receptor. Comparison of the predicted amino acid sequence of this clone to those of other cytochrome P450 genes indicated that the closest overall sequence similarity (38%) was to proteins predicted from genes in the CYP3 family. Northern blots indicated the presence of a transcript of the appropriate size (3.0 kb) with homology to the clam cytochrome P450. In vitro translation of the cDNA clone produced a 50.7-kDa protein as determined by SDS-polyacrylamide gel electrophoresis. The in vitro translated protein was not recognized on Western blots by two polyclonal antibodies specific for members of the CYP3 family. Since the degree of similarity to existing cytochrome P450 families was below the 40% level required for membership, and the expressed protein was not recognized by CYP3-specific antibodies, this clam cytochrome P450 cDNA has been placed in a new family, cytochrome P450 30 (CYP30).

Amino Acid Sequence↗

PubMed1997

Effects of TCDD on Ah receptor, ARNT, EGF, and TGF-alpha expression in embryonic mouse urinary tract.

Prenatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces hydronephrosis in C57BL/6N mice. The etiology of this response involves TCDD-induced hyperplasia of ureteric epithelium, which occludes the ureteric lumen, blocking the flow of urine. The present study localizes and examines the effects of TCDD on the expression of the Ah receptor (AhR), the Ah receptor nuclear translocator (ARNT), epidermal growth factor (EGF), and transforming growth factor-alpha (TGF-alpha) in the epithelial cells of the developing urinary tract, particularly the ureteric bud derivatives (ureter and tubules). Pregnant C57BL/6N mice were dosed on gestation day (GD) 10 with either corn oil or TCDD at 12 micrograms/kg; a dose of 24 micrograms/kg is expected to induce 100% hydronephrosis. The metanephric urinary tract is morphologically detectable as early as GD 12; thus, embryos were removed on GD 12, 13, and 14, and the lower dorsal torso was prepared for immunohistochemistry or in situ hybridization. Regardless of treatment, the expression of both AhR and ARNT increased in epithelial cells of the ureter and AhR increased in the metanephric tubules from GD 12-14. In situ hybridization localized the expression of AhR and ARNT mRNAs to these derivatives of the ureteric bud and levels of mRNA increased throughout the developmental period examined. There were no significant effects of TCDD treatment on expression of AhR, while TCDD significantly decreased levels of ARNT in tubules on GD 14. The epithelial cells of the ureter and tubules expressed TGF-alpha and EGF. EGF increased from GD 12 to 13 in the tubules and ureter, but there was no difference from GD 13 to 14. Treatment with TCDD reduced TGF-alpha significantly only in tubules on GD 13. TCDD exposure significantly decreased EGF in ureter and tubule cells on both GD 13 and 14. In summary, the epithelial cells of the embryonic mouse urinary tract expressed AhR, ARNT, EGF, and TGF-alpha in developmentally dependent patterns. These proteins are involved in the regulation of embryonic cell proliferation during normal urinary tract development and are probably involved in the hyperplastic response to TCDD.

Animals↗

PubMed1996

Estradiol stimulates tyrosine phosphorylation of the insulin-like growth factor-1 receptor and insulin receptor substrate-1 in the uterus.

The signaling pathways associated with estrogen-induced proliferation of epithelial cells in the reproductive tract have not been defined. To identify receptor tyrosine kinases that are activated in vivo by 17 beta-estradiol (E2), uteri from ovariectomized mice were examined for enhanced tyrosine phosphorylation of various receptors and a receptor substrate following treatment with this hormone. Within 4 hr after hormone exposure, extracts showed increased phosphotyrosine (P-Tyr) immunoreactivity at several bands, including 170- and 180-kDa; these bands were still apparent at 24 hr after E2. Analysis of immunoprecipitates from uterine extracts revealed that E2 enhanced tyrosine phosphorylation of the insulin-like growth factor-1 receptor (IGF-1R) and insulin receptor substrate-1 (IRS-1) by 6 hr. Comparison of supernatants from IRS-1 and control rabbit IgG immunoprecipitates indicated that the 170-kDa P-Tyr band in extracts was equivalent to IRS-1. The receptors for epidermal growth factor, platelet-derived growth factor, and basic fibroblast growth factor did not exhibit an E2-induced increase in P-Tyr content. The nonestrogenic steroid hormones examined did not stimulate the P-Tyr content of IGF-1R or IRS-1. Immunolocalization of P-Tyr and IRS-1 revealed strong reactivity in the epithelial layer of the uterus from E2-treated mice, suggesting that the majority of P-Tyr bands observed in immunoblots originate in the epithelium. Since hormonal activation of IRS-1 is epithelial, estrogen-specific, and initiated before maximal DNA synthesis occurs following treatment with hormone, this protein, as part of the IGF-1R pathway, may be important in mediating estrogen-stimulated proliferation in the uterus.

Animals↗

PubMed1996

Isolation and characterization of a novel gene induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in rat liver.

The differential display technique was used to identify genes whose expression was regulated by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Expression of a novel sequence was up-regulated in a dose-dependent fashion in liver of Sprague-Dawley male rats exposed to both chronic and acute treatment with TCDD, as measured by densitometry of Northern blot analyses (P < 0.01). A rapid amplification of cDNA ends (RACE) procedure was used to isolate a 1.8 kb cDNA from a rat liver cDNA preparation. This cloned cDNA, called 25-Dx, was sequenced and found to encode a peptide of 223 amino acids. In control rats, the 25-Dx gene was expressed at high levels in lung and liver. A hydrophobic domain of 14 residues followed by a proline-rich domain, both located in the N-terminal region, showed 71% homology with the transmembrane domain of the precursor for the interleukin-6 receptor and a conserved consensus sequence found in the cytokine/growth factor/prolactin receptor superfamily respectively.

Amino Acid Sequence↗

PubMed1995

Identification of two binding proteins for halogenated aromatic hydrocarbons in the hard-shell clam, Mercenaria mercenaria.

Earlier studies have reported an unusually high incidence of gonadal tumors in marine bivalves in areas of potentially high exposure to herbicides including 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophe-noxyacetic acid. Some herbicides can be contaminated by halogenated aromatic compounds including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Most of the effects of planar halogenated aromatic compounds, including carcinogenic effects in vertebrates, appear to be mediated through binding to the aryl hydrocarbon receptor. The current study investigated whether halogenated aromatic hydrocarbon-binding proteins are present in the marine bivalve, Mercenaria mercenaria. We used the TCDD photoaffinity analog 2-azido-3-[125I]-iodo-7, 8-dibromodibenzo-p-dioxin to detect the presence of two proteins (28 and 39 kDa) in cytosols prepared from M. mercenaria that specifically bound this ligand. Expression of these proteins is tissue-dependent with the highest concentrations being observed in gill and gonadal tissue. Gonadal tissue also exhibited gender-specific expression with female clams exhibiting higher levels of the 39-kDa protein. Gender-and tissue-specific expression are consistent with the hypothesis that these proteins might be involved in the carcinogenic response observed in clams exposed to herbicides.

Affinity Labels↗

PubMed1995

Alterations in thyroid function in female Sprague-Dawley rats following chronic treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a multisite carcinogen. Although the hepatocarcinogenic actions of TCDD have received the most attention, it has been demonstrated in several rodent carcinogenicity bioassays that TCDD causes a dose-related increase in thyroid follicular cell adenomas and carcinomas. The purpose of the present experiment was to investigate the dose-response relationship for thyroid function alterations in female Sprague-Dawley rats following chronic treatment with TCDD. TCDD was administered via oral gavage biweekly for 30 weeks at average daily equivalent doses of 0.1-125 ng/kg/day, thereby more than encompassing the dose range historically used in previous TCDD rodent bioassays. The endpoints examined include serum levels of thyroxine (T4), triiodothyronine (T3), and thyroid-stimulating hormone (TSH). In addition, the induction of the dioxin-responsive genes UDP-glucuronosyltransferase-1 (UGT1) and cytochrome P450 1A1 (CYP1A1) in liver were measured using reverse-transcriptase-polymerase chain reaction (RT-PCR). In agreement with previous hypotheses, TCDD appears to alter thyroid function via a secondary mechanism, namely increased excretion of T4-glucuronide resulting from TCDD induction of UGT1. The observed follicular cell hyperplasia and hypertrophy are consistent with the observed elevated TSH levels and may represent the early stages in the progression of thyroid carcinogenesis. Therefore, TCDD induces alterations in thyroid hormone function, probably as a result of chronic perturbations of liver-pituitary-thyroid axis.

Animals↗

PubMed1995

TCDD reduces rat hepatic epidermal growth factor receptor: comparison of binding, immunodetection, and autophosphorylation.

Administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a potent tumor promoter, to rats resulted in a dose-dependent decrease in hepatic plasma membrane epidermal growth factor receptor (EGFR). The present study is the first to quantify and compare alterations in hepatic EGFR levels in female Sprague-Dawley rats 7 days after a single oral gavage dose of TCDD (0, 1, 5, 25, and 50 micrograms/kg) using three different techniques: (1) equilibrium receptor binding, (2) EGF induced receptor autophosphorylation, and (3) Western blot detection with a rabbit anti-rat EGFR polyclonal antibody. All three methods similarly demonstrated that the level of hepatic EGFR is significantly decreased at a dose of TCDD as low as 1 micrograms/kg. We showed that the immunoblot technique is a sensitive and quantitative alternative to radioligand binding assays. It is concluded that TCDD decreased total EGFR protein and maximum binding capacity without altering ligand binding affinity (Kd). The results demonstrated that ligand-induced autophosphorylation capacity and basal phosphotyrosine residues of plasma membrane EGFR were both decreased parallel with the decrease in EGFR protein, suggesting no TCDD-related alteration in the inherent functional ability of the receptor to undergo activation. Furthermore, it was found that the dose-response curve for EGFR protein level determined by Western blot analysis was similar for both male and female Sprague-Dawley rats.

Animals↗

PubMed1995

Persistence of TCDD-induced hepatic cell proliferation and growth of enzyme altered foci after chronic exposure followed by cessation of treatment in DEN initiated female rats.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent tumor promoter in two-stage models of hepatocarcinogenesis. This study focuses on the persistence or reversibility of TCDD-mediated changes in livers after 30 weeks of treatment and cessation of treatment. Diethylnitrosamine (DEN) initiated animals (175 mg/kg) were promoted bi-weekly with TCDD at a dose equivalent to 125 ng/kg/day for 30 weeks without or with a following waiting period of 32 weeks before necropsy. 2,3,7,8-Tetrachlorodibenzo-p-dioxin liver concentration decreased 300-fold above background. Induction of CYP1A1 dependent enzyme activity decreased according to TCDD tissue levels. In contrast, cell proliferation, as measured by BrdU-labeling index, was still 2.8-fold increased over controls in the TCDD group with waiting period compared to a 4-fold increase over controls at the end of the 30 week dosing period. Enzyme altered hepatic foci expressing the placental form of glutathione S-transferase decreased in number but the remaining foci were significantly increased in size and the percent of liver occupied by foci was higher at the end of the waiting period as compared to livers at the end of the dosing period. Liver tumor incidence at the end of the waiting period was 71% (5 of 7 animals) and the livers showed an increase in bile duct lesions with only mild toxicity. There was pronounced bile duct proliferation in DEN/TCDD treated animals after the waiting period with intense expression of TGF alpha in bile duct epithelial cells at detected by immunohistochemical methods. In comparison, at the end of the 30 week dosing period the livers showed more severe toxicity and only mild bile duct proliferation. Also, one small hepatocellular adenoma was observed. It is concluded that as opposed to CYP1A1 induction the more complex biological responses, cell proliferation and selective growth of certain preneoplastic foci, are persistent after prolonged TCDD treatment within the experimental framework of our study.

Animals↗

PubMed1995

Rat CYP1B1: an adrenal cytochrome P450 that exhibits sex-dependent expression in livers and kidneys of TCDD-treated animals.

The broad spectrum of biological responses associated with exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) is believed to be due to the alteration in expression of TCDD-inducible genes. The aim of this study was to investigate the effects of TCDD on the in vivo tissue-specific expression of the recently identified TCDD-inducible cytochrome P450 CYP1B1 [Sutter et al. (1994) J. Biol. Chem., 269, 13092-13099] in Sprague-Dawley rats. We cloned the 5.0 kb rat homolog of CYP1B1 from a TCDD-treated rat liver cDNA library and showed that the rat and human CYP1B1 predicted amino acid sequences are 80% identical. RNA hybridization analysis showed that CYP1B1 is constitutively expressed in the adrenal glands and also in the testes of untreated rats. This tissue distribution suggests that CYP1B1 may be a physiological steroid hydroxylase. Seventy-two hours post-administration of 25 micrograms/kg body wt TCDD by gavage, steady-state levels of the 5.1 kb CYP1B1 RNA were increased > 50-fold in liver, and to a lesser extent in kidneys, lung, heart and ovaries. Average CYP1B1 RNA levels were significantly higher in the kidneys and livers of TCDD-treated females than in those from similarly treated males. In contrast, no significant sex-difference was observed in the levels of CYP1A1 in these tissues in TCDD-treated animals. In Sprague-Dawley rats, TCDD is a more potent hepatocarcinogen in females than in males. The induction of CYP1B1 in TCDD rat liver may be a contributing factor to the carcinogenic action of this persistent environmental pollutant.

Adrenal Glands↗

PubMed1995

Expression of xenobiotic-metabolizing enzymes in cultured rat tracheal epithelial cells.

Rat tracheal epithelial (RTE) cells were cultured on membrane support with and without retinoic acid (RA). In early (6-day-old) cultures, the epithelium is a monolayer or bilayer of undifferentiated cells and secretes little mucuslike product either in the absence or presence of RA. In late (12- to 15-day-old) cultures, the epithelium differentiates as a mucociliary epithelium in the presence of RA and as a squamous epithelium in the absence of RA. The purpose of our study was to determine whether a number of xenobiotic enzymes are expressed in these cultures and whether their expression depends on the state of differentiation. Enzyme expression was characterized by electrophoresis and immunoblotting as a function of time in culture and phenotypic differentiation. Cytochrome P450 1A1 was not expressed in freshly harvested RTE cells. This isoenzyme was induced in rats by gavage with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or by exposure of early RTE cell cultures to TCDD, provided RA was also added to the cultures. Cytochrome P450 2B1 was observed in freshly isolated RTE cells, but not in early or late RTE cultures. In contrast, expression of NADPH-cytochrome P450 reductase was decreased in early cultures, but was increased in well-differentiated cultures. Flavin-containing monooxygenase was detected in lung tissue, but not in freshly harvested or cultured RTE cells. Glutathione S-transferase (GST) mu and pi were expressed in freshly harvested RTE cells. GST pi was expressed in early and late cultures, whereas GST mu was expressed in late cultures, but could not be found in early cultured RTE cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Workshop on perinatal exposure to dioxin-like compounds. VI. Role of biomarkers.

Studies of perinatal exposures to dioxin-like compounds (DLCs), coplanar polycyclic halogenated aromatics whose prototype is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), have employed a variety of outcome measures to investigate effects on the reproductive/developmental, endocrine, immune, and neurobehavioral systems. The effects include infertility, growth retardation, fetal loss, changed sexual differentiation, reduced cognitive/motor function, dermatologic and other ectodermal effects, and decreased immune response. Significant biomarkers have included sperm count; CD4/CD8 ratio; and levels of testosterone, T4, and dopamine. Using specific dioxin or PCB congeners, these and other markers were used to investigate the mechanisms of the observed effects. The DLCs, which include some PCB congeners, are characterized by high-affinity binding to the Ah receptor; most biological effects are thought to be mediated by the ligand-Ah receptor complex. Other PCB congeners have low affinity for the Ah receptor, and operate by non-Ah receptor mechanisms. The biologic activity of a PCB mixture is the sum of the agonist and antagonist activities of the different constituents in the mixture. Animal studies with specific PCB congeners can help to clarify these activities. With similar approaches, biologic markers of effect can be developed and applied in epidemiologic studies to monitor for, and predict, adverse effects in humans.

Animals↗

PubMed1995

Induced gene transcription: implications for biomarkers.

Numerous xenobiotics regulate cellular functions by altering transcription of target genes. Use of sensitive and specific biomarkers based on gene transcript levels may help clarify the shape of the dose-response curve in the low-dose region associated with human exposures to environmental concentrations of chemicals. We have quantified gene transcription induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in both animal models and humans with the use of Northern analysis and PCR-based methods. In addition, we describe a rapid and sensitive in vitro assay that we have used to screen chemicals and biological samples for their ability to alter gene transcription. Whereas some of the endpoints in our studies such as cytochrome P-450 1A1 are predictive indicators of exposure and dose, other gene responses such as growth factors are more complex and represent a critical event, progression, or adaptation to a pathological alteration. In conclusion, measurement of toxicant-induced gene transcription will contribute to the usefulness of biomarkers in addressing issues of human health and environmentally induced disease.

Animals↗

PubMed1994

Dioxin-responsive genes: examination of dose-response relationships using quantitative reverse transcriptase-polymerase chain reaction.

The purpose of the present experiments was to examine dose-response relationships for induction of hepatic mRNA following a single administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to rats. The induction of cytochrome P450-1A1 (CYP1A1) mRNA is compared to other "dioxin-responsive" genes including UDP-glucuronosyltransferase I, plasminogen activator inhibitor 2, and transforming growth factor alpha using a sensitive reverse transcriptase-polymerase chain reaction-based method. Sample-to-sample variability in amplification is a concern in using polymerase chain reaction to quantitate biological responses. However, in the present study recombinant RNA templates were synthesized to use as internal standards in both the reverse transcription and the polymerase chain reaction steps. The induction of CYP1A1 mRNA was extremely sensitive to TCDD treatment with increases observed at doses as low as 1 ng/kg body weight. The induction of CYP1A1 mRNA correlated highly (R2 > 0.90) with an increase in ethoxyresorufin-o-deethylase activity, a CYP1A1-associated enzyme activity. However, induction of CYP1A1 mRNA levels was detected at lower TCDD doses than was ethoxyresorufin-o-deethylase activity, reflecting the greater sensitivity of the reverse transcription-polymerase chain reaction approach to detect transcriptional activation of the CYP1A1 gene. UDP-glucuronosyltransferase I mRNA was increased over control (5-fold) but required 1000-times higher TCDD doses (1 microgram/kg) to result in a significant increase than did CYP1A1. Plasminogen activator inhibitor 2 and transforming growth factor alpha mRNA, both previously shown to be induced by TCDD in human keratinocytes, were not increased in rat liver. Hence, these studies reaffirm that TCDD acts through classical receptor mechanisms with gene-to-gene differences in responsiveness. The reverse transcription-polymerase chain reaction method developed to measure mRNA for dioxin-responsive genes in rat liver will allow for measuring multigene and tissue responses to TCDD and other xenobiotics with high sensitivity, reproducibility, and adaptability and should increase our understanding of various dose-response relationships.

Animals↗

PubMed1994

Accumulation of polychlorinated dibenzo-p-dioxins and dibenzofurans in liver of control laboratory rats.

Polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), and biphenyls belong to a class of compounds, the polyhalogenated aromatic hydrocarbons (PHAHs), which are ubiquitous environmental contaminants. Due to the existence of a common mechanism of action, i.e., binding to the Ah receptor, the activity of members of this class of compounds is generally expressed relative to the prototypical 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as toxic equivalency factors (TEFs). In the present studies we examined the presence of liver of untreated PCDFs in standard laboratory feed and in the liver of untreated rats at three different ages (60, 140, and 200 days) in terms of concentration and in toxic equivalents (TEQs, TEF x concentration). Feed was shown to contain trace amounts of PCDDs and PCDFs and control rat liver was shown to contain several PCDD and PCDF congeners in terms of concentration of congener and concentration of TEQs contributed by that congener. The total concentration of TEQs increased with increasing age in rat liver, going from 20 ppt TEQ at 60 days to 78 ppt TEQ at 200 days of age. This accumulation in dioxin-like activity was due primarily to PCDFs. In particular the congener 2,3,4,7,8-pentachlorodibenzofuran accrued in untreated rat liver accounting for approximately 80% of the total TEQ at 200 days of age. These studies affirm the pervasive presence of PHAHs and suggest prudence in evaluating chronic rat studies in which interference from background levels of PCDDs and PCDFs may be a factor.

Age Factors↗

PubMed1994

Lack of direct immunosuppressive effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on human peripheral blood lymphocyte subsets in vitro.

A wide variety of immunosuppressive effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in experimental animals has been documented. In contrast, the impact of dioxin on the human immune system remains controversial, although adverse health effects have been reported in humans after occupational or accidental exposure to dioxin. Recently, Neubert et al. (1991) found that a dose-dependent decrease of peripheral blood lymphocyte (PBL) subpopulations in humans and non-human primates, including helper-inducer/memory cells (CD4+CD29+) and B cells (CD20+) occurred in pokeweed mitogen (PWM) stimulated cultures at concentrations as low as 10(-12)-10(-14) M TCDD. Therefore, the direct effects of dioxin on human PBL subpopulations have been studied, in order to determine their usefulness as sensitive biomarkers for human dioxin exposure. Lymphocyte cultures from healthy individuals were treated with 10(-7) M-10(-14) M TCDD in the absence and presence of stimulation with pokeweed mitogen (PWM) or anti-CD3 monoclonal antibody (moAb; OKT3) for 3 days. Cytochrome P450 (CYP1A1) enzyme induction, one of the best studied direct biological effects of TCDD on numerous cell types, was assayed in parallel by ethoxyresorufin-O-deethylase (EROD) activity. Percentages of the different lymphocytes subsets, including CD2 (T cells); CD4; CD45 RA (suppressor-inducer/virgin T cells); CD4 CD29; CD8; CD19 (B cells) as well as interleukin 2 (IL-2) receptor (CD25) and class II antigen (HLA-DR) expression, were analyzed by flow cytometry. DNA synthesis was determined by 3H-thymidine uptake after 3 days of culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗