2,3,7,8-Tetrachlorodibenzo-p-dioxin: Failure to demonstrate toxicity in twenty-three cultured cell types

doi:10.1016/0041-008X(80)90163-5

Toxicology and Applied Pharmacology

Volume 54, Issue 3, July 1980, Pages 377-383

https://doi.org/10.1016/0041-008X(80)90163-5 Get rights and content

Abstract

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), one of the most potent small molecule toxins known was investigated for its toxic effect on 23 cultured cell types, including primary cultures and cells from established and transformed cell lines. The cells, derived from tissues and/or species susceptible to TCDD toxicity in vivo, were exposed to TCDD for an extended period, usually a week or longer. Examination of the cells for altered morphology, decrease in percentage viability, or diminished growth rate revealed no toxic effect of TCDD in any cell type, including those which respond to TCDD with the induction of aryl hydrocarbon hydroxylase activity.

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Cited by (73)

In vitro toxicity and in silico docking analysis of two novel selective AH-receptor modulators
2018, Toxicology in Vitro
Citation Excerpt :
C1 and C3 were first assessed for their in vitro toxicity, in some cases in parallel with TCDD, to further compare their effects. Generally, TCDD is not overtly toxic to cultured cells in vitro, despite its drastic effects in vivo (Beatty, et al. 1975, Knutson and Poland 1980, Schecter, et al. 1987). However, the same could not be taken for granted for C1 and C3, particularly because in vivo, we had observed some effects with C2 and C4 that have not been reported with TCDD.
The mediator of dioxin toxicity, aryl hydrocarbon receptor (AHR), has also important physiological functions. Selective AHR modulators (SAHRMs) share some effects of dioxins, except for their marked toxicity. We recently characterised toxicologically two novel SAHRMs, prodrugs IMA-08401 and IMA-07101 in rats, demonstrating that they are far less deleterious than the most toxic AHR-agonist, TCDD. Here, we analysed the in vitro toxicity and in silico AHR binding of the respective active, deacetylated metabolites, IMA-06201 (N-ethyl-N-phenyl-5-chloro-1,2-dihydro-4-hydroxy-1-methyl-2-oxo-quinoline-3-carboxamide) and IMA-06504 (N-(4-trifluoromethylphenyl)-1,2-dihydro-4-hydroxy-5-methoxy-1-methyl-2-oxo-quinoline-3-carboxamide). In H4IIE rat hepatoma cells, IMA-06201 and IMA-06504 induced CYP1A1 with comparable potencies and efficacies to those of TCDD. They had little effect on cell viability as assessed by LDH leakage and MTT reduction assays, and were not mutagenic in the Ames test, but IMA-06504 elicited a maximally 2.7-fold increase in micronuclei. Molecular docking simulations showed that similar to TCDD, they occupy the central region of AHR ligand binding cavity. Hence, while showing low to negligible in vitro toxicity, these novel SAHRMs bind to the AHR qualitatively in a similar fashion to TCDD, and appear comparably powerful AHR agonists. Combined with our earlier results demonstrating that they seem considerably less toxic in vivo than TCDD, these compounds are thus highly interesting new SAHRMs.
2,3,7,8-Tetrachlorodibenzo-p-dioxin differentially suppresses angiogenic responses in human placental vein and artery endothelial cells
2015, Toxicology
Citation Excerpt :
The current observations that TCDD decreased cell proliferation and viability is the first report, as far as we are aware, to directly show the TCDD’s inhibitory effects on proliferation and viability of primary HUVECs and HUAECs. These observations are contrary to the previous studies (Beatty et al., 1975; Knutson and Poland, 1980), in which TCDD at 100 nM or 1 μM failed to affect growth and viability of 27 human and animal primary cultures and transformed cell lines (not including HUVECs and HUAECs), derived from tissues and/or species susceptible to TCDD toxicity in vivo. Nonetheless, our finding is consistent with a previous report, which showed that benzo[a]pyrene (another AhR exogenous ligand) inhibited angiogenic factors-induced neovasculogenesis and angiogenic activity of HUVECs (Li et al., 2010).
Placental angiogenesis is dramatically increased during pregnancy in association with the elevated placental blood flows to support the rapidly growing fetus. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an environmental toxicant and a ligand of aryl hydrocarbon receptor (AhR). Herein, we investigated the effects of TCDD on proliferation, migration, and viability of fetoplacental endothelial cells in response to a complete growth medium which contained serum and growth supplement using human umbilical cord vein (HUVECs) and artery (HUAECs) cells as models. We found that TCDD dose- and time-dependently inhibited (p < 0.05) proliferation of HUVECs and HUAECs. Treatment with TCDD at 10 nM for 6 days inhibited (p < 0.05) migration (by ∼30%) of HUAECs, but not HUVECs. TCDD at 10 nM also decreased (p < 0.05) viability of HUVECs and HUAECs. Interestingly, specific AhR siRNA blocked (p < 0.05) the TCDD-inhibited cellular responses in HUAECs, but not HUVECs. Nonetheless, TCDD at 10 nM neither affected the cell cycle progression, nor did it induce cell apoptosis in HUVECs and HUAECs. In addition, TCDD at 10 nM also did not alter activation of ERK1/2 and AKT1 in HUVECs and HUAECs. Collectively, TCDD suppresses proliferation and/or migration (two key steps of angiogenesis) of HUVECs and HUAECs independent and dependent of AhR, respectively. These data suggest that TCDD inhibited growth of HUVECs and HUAECs via decreasing cell viability. Thus, TCDD may inhibit fetoplacental angiogenesis, leading to negative pregnancy outcomes.
Chloracne: From clinic to research
2012, Dermatologica Sinica
Citation Excerpt :
Exposure of murine fetuses in utero to TCDD led to an accelerated terminal differentiation of fetal skin.29 A survey of 23 cell lines displayed no changes in morphology, viability, or growth patterns following treatment with TCDD.30 Furthermore, TCDD caused the acceleration of differentiation of a spontaneously immortalized human keratinocyte cell line in a three-dimensional skin equivalent model as determined by morphologic characterization and by immunohistochemistry for several differentiation-specific protein markers.
Chloracne is the most sensitive and specific marker for a possible dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin) intoxication. It is clinically characterized by multiple acneiform comedone-like cystic eruptions mainly involving face in the malar, temporal, mandibular, auricular/retroauricular regions, and the genitalia, often occurring in age groups not typical for acne vulgaris. Histopathology is essential for a definite diagnosis, which exhibits atrophy or absence of sebaceous glands as well as infundibular dilatation or cystic formation of hair follicles, hyperplasia of epidermis, and hyperpigmentation of stratum corneum. The appearance of chloracne and its clinical severity does not correlate with the blood levels of dioxins. Pathogenesis of chloracne remains largely unclear. An “aryl hydrocarbon receptor”-mediated signaling pathway affecting the multipotent stem cells in the pilosebaceous units is probably the major molecular mechanism inducing chloracne. Chloracne is resistant to all the available treatment modalities used to treat acne. The aim of treatment is to lower or to eliminate the accumulated dioxins in the body at the very beginning of intoxication, e.g., by using dioxin-chelating substances such as synthetic dietary fat substitutes. The problem of dioxin contamination and its potential health hazards should be taken seriously in the wave of industrial globalization in the twenty-first century. Clinicians, especially dermatologists, are in the forefront of early diagnosis of dioxin intoxication.
2,3,7,8-Tetrachlorodibenzo-p-dioxin alters the differentiation pattern of human keratinocytes in organotypic culture
2001, Toxicology and Applied Pharmacology
Human exposure to the environmental toxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) produces a severe skin pathology known as chloracne. In these studies we employed a three-dimensional, organotypic model system to study the effects of TCDD on human skin. This model uses the spontaneously immortalized human keratinocyte cell line NIKS and recapitulates both the three-dimensional microenvironment and epithelial–mesenchymal interactions found in intact human skin. Treatment of the organotypic cultures with TCDD causes alterations in the pattern of keratinocyte terminal differentiation. Analysis at both the light and electron microscope levels reveals a fully differentiated cornified layer in TCDD-treated organotypic cultures at earlier time points than observed in vehicle (dimethyl sulfoxide)-treated controls. Furthermore, TCDD-treated organotypic cultures exhibit aberrant distribution of several differentiation-specific protein markers. Basal cells in TCDD- and DMSO-treated organotypic cultures show no differences in proliferation as measured by quantification of 5-bromo-2′-deoxyuridine (BrdU)-positive nuclei. No aberrant BrdU uptake was detected outside of the basal layer. Neither TUNEL labeling nor immunohistochemical staining with an antibody to active caspase-3 revealed increased apoptosis in TCDD-treated organotypic cultures relative to controls. These data clearly indicate that TCDD modulates homeostasis in a model of human stratifying epithelium independent of changes in proliferation and apoptosis, exclusively by impacting keratinocyte terminal differentiation. This TCDD-induced effect on differentiation-specific proteins results in profound changes in the tissue architecture.
The Ah receptor is not involved in 2,3,7,8-tetrachlorodibenzo-p-dioxin- mediated apoptosis in human leukemic T cell lines
1998, Journal of Biological Chemistry
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a common environmental pollutant causing public concern. Its toxic effects include disruption of the immune, endocrine, and reproductive systems, impairment of fetal development, carcinogenicity, and lethality in rodents. Here, we report that TCDD induces apoptosis in two cultured human leukemic lymphoblastic T cell lines. This cell death was found not to be dependent on an aryl hydrocarbon receptor and to be inhibited by the inhibitor of tyrosine kinases and caspases. Apoptosis-linked c-Jun N-terminal kinase is rapidly activated in these cells by the treatment with TCDD. A dominant-negative mutant of c-Jun N-terminal kinase prevented cell death in the treatment with TCDD. Furthermore, TCDD decreases the Bcl-2 protein level in these cell lines. These findings will help in the understanding of the molecular mechanism underlying TCDD-mediated immunotoxicity.
Induction of mono-oxygenase activity in prostaglandin H synthase-competent ovine seminal vesicle cell cultures by 2,3,7,8-tetrachlorodibenzo-p-dioxin
1996, Toxicology in Vitro
Cell cultures derived from ovine seminal vesicles (OSV cells) express high levels of prostaglandin H synthase (PHS) and have been found to be a suitablein vitro model for studies on PHS-mediated bioactivation of certain xenobiotics The extrahepatic tissue of origin apparently lacks constitutively expressed mono-oxygenase (MFO) activity such as cytochrome P-450 (CYP1A1)-dependent ethoxyresorufin-O-deethylase (EROD). However, treatment of OSV cell cultures with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 0.001–10 nm) resulted in a dose-dependent induction of CYP1A1-associated EROD activity. Subconfluent OSV cells exposed to 100 pm TCDD for various lengths of time express the highest EROD activity after 48 hr of treatment. Benz[a]anthracene also induced EROD activity in OSV cells, but, in accordance with its low affinity for the Ah receptor at much higher concentrations (1–10 μm) than TCDD. In line with the observed functional response (EROD induction) the presence of Ah receptor was confirmed by biochemical analysis: on incubation with [³H]TCDD 5 fmol ligand/mg nuclear protein were specifically bound to Ah receptor after incubation (2 hr) of OSV cells with [³H] TCDD. TCDD was not cytotoxic for OSV cells up to 10 nm as judged by growth curves; rather, their growth was moderately stimulated by TCDD, significantly at 1 nm (a concentration which also induced EROD activity). Despite a clear dose-related response of OSV cells to known inducers of CYP1A1, the induced EROD activity levels (0.4−0.7pmol/min× 10⁶cells) are very low compared with values reported for hepatic cells in culture and lower than EROD activity induced in colon tumour cells. Thus, and since OSV cells do not constitutively express appreciable MFO activity, it is suggested that only PHS-mediated oxidation reactions contribute to the bioactivation of xenobiotics in this model. In conclusion, although OSV cells clearly respond to Ah receptor ligands by EROD induction, they are of limited value for bioassay of TCDD and related compounds in environmental samples. However, PHS-competent OSV cells are an interesting model for further studies of TCDD-related effects on PHS activity/expression, because of the functional Ah receptor.

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^☆: Supported in part by National Institute of Environmental Health Science Grant 1-R01-ES-00965 and National Cancer Institute Core Grant CA-07175.

²: Recipient of Research Career Development Award K-04 ES-0017.

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Toxicology and Applied Pharmacology

Abstract

References (31)

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on mammalian cells in tissue culture

Toxicol. Appl. Pharmacol.

The induction in vitro of the synthesis of γ-aminolevulinic acid synthetase in chemical porphyria: A response to certain drugs, sex hormones, and foreign chemicals

J. Biol. Chem.

Mouse myelomas and lymphomas in culture

Exp. Cell Res.

Teratocarcinoma de la Souris Isolement, Culture et Properietes de Cellules a Potentialites Multiples

Ann. Microbiol. (Paris)

Aryl hydrocarbon hydroxylase induction in human lymphocyte cultures by 2,3,7,8-tetrachlorodibenzo-p-dioxin

Life Sci.

Protein measurement with the folin phenol reagent

J. Biol. Chem.

The comparative toxicity of chlorinated dibenzo-p-dioxins in mice and guinea pigs

Toxicol. Appl. Pharmacol.

Toxicological assessment of hexachlorobiphenyl isomers and 2,3,7,8-tetrachlorodibenzofuran in chicks

Toxicol. Appl. Pharmacol.

Stereospecific, high affinity binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin by hepatic cytosol

J. Biol. Chem.

Isolation of lymphoma cell variants resistant to killing by glucocorticoids

Cell

Two new rat hepatoma cell lines for studying the unbalanced blocked ontogeny hypothesis

Detection of chlorinated dioxins: Induction of aryl hydrocarbon hydroxylase activity in rat hepatoma cell culture

Toxicol. Appl. Pharmacol.

Aryl hydrocarbon hydroxylase activity of twenty-three halogenated dibenzo-p-dioxins

Toxicol. Appl. Pharmacol.

Pathologic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin in laboratory animals

Environ. Health Perspect.

An ascites tumor derived from early splenic lesion of Friend's disease: A preliminary report

GANN

Cited by (73)

In vitro toxicity and in silico docking analysis of two novel selective AH-receptor modulators

2,3,7,8-Tetrachlorodibenzo-p-dioxin differentially suppresses angiogenic responses in human placental vein and artery endothelial cells

Chloracne: From clinic to research

2,3,7,8-Tetrachlorodibenzo-p-dioxin alters the differentiation pattern of human keratinocytes in organotypic culture

The Ah receptor is not involved in 2,3,7,8-tetrachlorodibenzo-p-dioxin- mediated apoptosis in human leukemic T cell lines

Induction of mono-oxygenase activity in prostaglandin H synthase-competent ovine seminal vesicle cell cultures by 2,3,7,8-tetrachlorodibenzo-p-dioxin

2,3,7,8-Tetrachlorodibenzo-p-dioxin: Failure to demonstrate toxicity in twenty-three cultured cell types☆

Abstract

Toxicol. Appl. Pharmacol.

J. Biol. Chem.

Exp. Cell Res.

Ann. Microbiol. (Paris)

Life Sci.

J. Biol. Chem.

Toxicol. Appl. Pharmacol.

Toxicol. Appl. Pharmacol.

J. Biol. Chem.

Cell

Two new rat hepatoma cell lines for studying the unbalanced blocked ontogeny hypothesis

Detection of chlorinated dioxins: Induction of aryl hydrocarbon hydroxylase activity in rat hepatoma cell culture

Toxicol. Appl. Pharmacol.

Aryl hydrocarbon hydroxylase activity of twenty-three halogenated dibenzo-p-dioxins

Toxicol. Appl. Pharmacol.

Pathologic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin in laboratory animals

Environ. Health Perspect.

An ascites tumor derived from early splenic lesion of Friend's disease: A preliminary report

GANN