Elsevier

Toxicology

Volume 265, Issues 1–2, 9 November 2009, Pages 1-9
Toxicology

Inflammatory and chloracne-like skin lesions in B6C3F1 mice exposed to 3,3′,4,4′-tetrachloroazobenzene for 2 years

https://doi.org/10.1016/j.tox.2009.08.017Get rights and content

Abstract

Exposure to dioxin and dioxin-like compounds (DLCs) has been connected to the induction of chloracne in humans and animals. 3,3′,4,4′-Tetrachloroazobenzene (TCAB) is an environmental contaminant that induces chloracne in humans. TCAB has been studied only to a limited extent in laboratory animals. While performing a 2-year gavage study in B6C3F1 mice to evaluate the toxic and carcinogenic effects of TCAB, we also explored potential chloracnegenic properties. Groups of 50 male and 50 female B6C3F1 mice were exposed by gavage to TCAB at dose levels of 0, 3, 10 and 30 mg/kg for 5 days a week for 2 years. The animals developed treatment-related gross inflammatory skin lesions, which were characterized histologically by inflammation, fibrosis, hyperplasia, and ulcers. Additionally, many of the animals developed follicular dilatation and sebaceous gland atrophy, consistent with chloracne-like lesions. This current 2-year study supports recently published papers showing susceptibility to chloracne in mouse strains other than hairless mice. The chloracne-like lesions were not clinically evident; therefore, our study highlights the need for careful examination of the skin in order to identify subtle lesions consistent with chloracne-like changes in rodents exposed to dioxin and DLCs. Since previous short-term studies did not demonstrate any skin lesions, we suggest that reliable assessment of all safety issues involving dioxin and DLCs requires evaluation following chronic exposure. Such studies in animal models will help to elucidate the mechanisms of dioxin-related health hazards.

Introduction

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), commonly referred to as dioxin, is one of the most toxic compounds known, presenting both carcinogenic and teratogenic properties (Johnson, 1991, Kociba and Schwetz, 1982, Safe, 1990). Certain polychlorinated dibenzodioxins, polychlorinated dibenzofurans (PCDFs), and coplanar polychlorinated biphenyls (PCBs) are commonly referred to as dioxin-like compounds (DLCs) because they have the ability to bind to the aryl hydrocarbon receptor (AhR) and exhibit biologic actions similar to those of TCDD. DLCs induce developmental, endocrine, and immunological toxicity and multi-organ carcinogenicity in animals and/or humans (ATSDR, 1998, ATSDR, 2000, Bertazzi et al., 2001, Kociba et al., 1978, Steenland et al., 2001). In addition, DLCs induce chloracne, an acneiform skin eruption.

Chloracne, one of the most common occupational dermatoses, can be extremely refractory to treatment (Taylor, 1974) and last for long periods without additional exposure to chloracnegens (Tindall, 1985). Although the clinical features of chloracne are clearly defined, the cellular and molecular mechanisms of dioxin-induced chloracne remain unknown (Panteleyev and Bickers, 2006). This condition, always a symptom of systemic poisoning and not merely a cutaneous disorder (Pastor et al., 2002), is a well-known side effect in several dioxin-exposure accidents, such as those at Seveso, Yusho, and Yu-Cheng (Guo et al., 1999, Guo et al., 2004, Schecter et al., 2006, Urabe and Asahi, 1985). Recently, deliberate poisoning with TCDD caused Ukraine president Viktor Yushchenco to develop facial chloracne during his presidential campaign (Holt, 2005, Schecter et al., 2006, Sterling and Hanke, 2005).

The National Toxicology Program (NTP) recently conducted 2-year bioassays in female Sprague–Dawley rats to evaluate the chronic toxicity and carcinogenicity induced by dioxin, structurally-related PCDFs and PCBs and mixtures of these compounds, including a mixture of TCDD, 3,3′,4,4′,5-pentachlorobiphenyl (PCB126) and 2,3,4,7,8-pentachlorodibenzofuran (PeCDF) (NTP, 2006a, NTP, 2006b, NTP, 2006c, NTP, 2006d). In these studies, increases occurred in the incidences of neoplasms and non-neoplastic lesions in several organs, notably the liver, lung, and oral mucosa (Hailey et al., 2005, Jokinen et al., 2003, NTP, 2006a, NTP, 2006b, NTP, 2006c, NTP, 2006d, Nyska et al., 2004, Nyska et al., 2005, Tani et al., 2004, Walker et al., 2005, Walker et al., 2006, Yoshizawa et al., 2005a, Yoshizawa et al., 2005b). However, none of these rat studies revealed chloracne-like skin lesions, consistent with the fact that rat species are not sensitive to dioxin-induced chloracne (Greene et al., 2003).

The NTP also conducted a 2-year chronic gavage study using both rats and mice treated with 3,3′,4,4′-tetrachloroazobenzene (TCAB), a DLC formed as a byproduct during the manufacture of 3,4-dichloroaniline or its herbicidal derivatives (Bunce et al., 1979, Hill et al., 1981, Poland et al., 1976, Sundstrom et al., 1978). Environmental contamination with TCAB occurs after chloranilide herbicides are degraded in soil by soil fungi (Bartha et al., 1968, Bartha and Pramer, 1969), and by photolysis and biolysis of 3,4-dichloroaniline (Mansour et al., 1975, Miller et al., 1980). Production of TCAB is estimated to be as high as 16,000 kg in the U.S. and may account for a significant amount of dioxin-like activity in the environment (Van Birgelen et al., 1999a). Occupational exposure to TCAB may occur during the manufacture as well as the application of herbicides containing TCAB as a contaminant. There is also the potential for human exposure via the consumption of food contaminated with TCAB.

In the trans configuration, TCAB can assume a planar conformation similar to that of TCDD and, like TCDD, can induce aryl hydrocarbon hydroxylase activity in mice and chick embryos (Poland et al., 1976). In several studies conducted in animals, exposure to TCAB caused typical dioxin-like effects, including body weight loss, thymic atrophy, hepatotoxicity, anemia, developmental toxicity, and induction of cytochrome P450 1A1; an increase of porphyrins occurred in chick embryo-liver cell cultures (Hsia et al., 1980, Hsia et al., 1981, Hsia et al., 1982, Hsia and Kreamer, 1985, McMillan et al., 1990, Mensink and Strik, 1982, Schrankel et al., 1982).

Three outbreaks of chloracne have occurred among workers following exposure to TCAB (Morse and Baker, 1977, Morse et al., 1979, Scarisbrick and Martin, 1981) in chemical plants manufacturing 3,4-dichloroaniline or its derivatives. Chloracne developed in 34–61% of the exposed workers. Although chloracne is the primary adverse effect reported in humans, only one animal study revealed TCAB-induced chloracne-like lesions following painting of the inner surface of the ears of female New Zealand White rabbits with TCAB for 5 days (Hill et al., 1981). The reason for this paucity of skin effects in published animal studies is that most were conducted with rats, a species resistant to dioxin-induced chloracne (Greene et al., 2003, NTP, 2006a, NTP, 2006b, NTP, 2006c, NTP, 2006d). In a subchronic rodent toxicity study conducted, in part, to set doses for the present 2-year study, TCAB was administered by gavage to B6C3F1 mice for 13 weeks. Although the treated mice showed typical dioxin-like effects in several organs, no effect was found in the skin (Van Birgelen et al., 1999a). However, chloracne-like lesions were observed in a 13-week toxicity study of tetrachloroazoxybenzene, a structural analog of TCAB (Van Birgelen et al., 1999b).

Despite its chloracnegenic properties in humans and close structural resemblance to TCDD, TCAB has only been studied to a limited extent in laboratory animals. Results in those few short-term studies did not show marked skin effects. Therefore, while performing the 2-year chronic gavage study in B6C3F1 mice for the evaluation of toxic and carcinogenic effects, we also explored the potential chloracnegenic properties of TCAB. Oral gavage was used for these studies allowing direct comparison of the data to the other NTP DLC toxicity studies. Here we report the skin findings in B6C3F1 male and female mice following exposure to TCAB for 2 years. The other significant neoplastic and non-neoplastic effects of TCAB are described in a NTP technical report (NTP, 2009).

Section snippets

Chemical

TCAB (CAS No. 14047-09-7) was obtained from AccuStandard, Inc. (New Haven, CT) in one lot. TCAB was identified by infrared and proton nuclear magnetic resonance spectroscopy, by gas chromatography (GC) coupled with mass spectrometry, and by melting point analysis. The purity of TCAB was determined by Karl Fischer titration to determine moisture content, elemental analysis for carbon, hydrogen, nitrogen and chlorine and GC with flame ionization detection, which indicated a purity of 99.8% or

Survival

Survival of male mice exposed to 10 and 30 mg/kg/day TCAB and female mice exposed to 30 mg/kg/day TCAB was significantly less than that of the vehicle controls (NTP, 2009). All males exposed to 30 mg/kg/day TCAB died before the end of the study. The survival rates in the 0, 3, 10, and 30 mg/kg/day male dose groups were 72, 62, 11, and 0%, respectively. The last surviving male mouse in the 30 mg/kg/day dose group died during week 76. Survival rates in the 0, 3, 10, and 30 mg/kg/day female dose groups

Discussion

We have shown that administration of TCAB for 2 years induced two distinct kinds of lesions in the skin of B6C3F1 mice. Skin samples were taken from grossly apparent skin lesions in addition to apparently normal dorsal skin following standard NTP procedures. In the 13-week gavage study of TCAB, neither macroscopic nor microscopic skin lesions were observed (Van Birgelen et al., 1999a) indicating that long-term studies may be needed to detect TCAB-induced skin pathology. Incidences of

Conflict of interest statement

The authors declare they have no competing financial interests.

Acknowledgements

The authors gratefully acknowledge Ms. JoAnne Johnson, Dr. Robert Sills, and Dr. Nigel Walker from the NTP/NIEHS for their critical review of the manuscript. This research was supported [in part] by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences under Research Project Number 1 Z01 ES045004-11 BB.

References (88)

  • M. Mansour et al.

    Reaktionsverhalten von 3,4 Dichloranilin und 3,4 Dichlorphenol in Loesung, als Festkoerper und in der Gasphase bei UV-Bestrahlung

    Chemosphere

    (1975)
  • D.C. McMillan et al.

    Metabolism of the arylamide herbicide propanil. II. Effects of propanil and its derivatives on hepatic microsomal drug-metabolizing enzymes in the rat

    Toxicol. Appl. Pharmacol.

    (1990)
  • A.A. Panteleyev et al.

    2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCCD) affects keratin 1 and keratin 17 gene expression and differentially induces keratinization in hairless mouse skin

    J. Invest. Dermatol.

    (1997)
  • A. Poland et al.

    Histologic changes produced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in the skin of mice carrying mutations that affect the integument

    J. Invest. Dermatol.

    (1984)
  • C.J. Portier et al.

    Testing for increased carcinogenicity using a survival-adjusted quantal response test

    Fundam. Appl. Toxicol.

    (1989)
  • S.M. Puhvel et al.

    Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on murine skin

    J. Invest. Dermatol.

    (1988)
  • S.S. Ray et al.

    Dioxin-induced immortalization of normal human keratinocytes and silencing of p53 and p16INK4a

    J. Biol. Chem.

    (2004)
  • A. Schecter et al.

    Dioxins: an overview

    Environ. Res.

    (2006)
  • G. Sundstrom et al.

    Determination of the toxic impurities of 3,3′,4,4′-tetrachloroazobenzene and 3,3′,4,4′-tetrachloroazoxybenzene in commercial diuron, linuron and 3,4-dichloroaniline samples

    Chemosphere

    (1978)
  • J.P. Tindall

    Chloracne and chloracnegens

    J. Am. Acad. Dermatol.

    (1985)
  • A.P. Van Birgelen et al.

    Toxicity of 3,3′,4,4′-tetrachloroazobenzene in rats and mice

    Toxicol. Appl. Pharmacol.

    (1999)
  • A.P. Van Birgelen et al.

    Toxicity of 3,3′,4,4′-tetrachloroazoxybenzene in rats and mice

    Toxicol. Appl. Pharmacol.

    (1999)
  • K.J. Van den Berg et al.

    Chronic toxicity of 3,4,3′,4′-tetrachlorobiphenyl in the marmoset monkey (Callithrix jacchus)

    Toxicology

    (1988)
  • J.G. Vos et al.

    Dermal toxicity studies of technical polychlorinated biphenyls and fractions thereof in rabbits

    Toxicol. Appl. Pharmacol.

    (1971)
  • J.G. Vos et al.

    Acnegenic activity of 3-methylcholanthrene and benzo[a]pyrene, and a comparative study with 2,3,7,8-tetrachlorodibenzo-p-dioxin in the rabbit and hairless mouse

    Toxicology

    (1982)
  • O. Yamamoto et al.

    Photocontact dermatitis and chloracne: two major occupational and environmental skin diseases induced by different actions of halogenated chemicals

    J. Dermatol. Sci.

    (2003)
  • Agency for Toxic Substances and Disease Registry (ATSDR), 1998. Toxicological Profile for Chlorinated...
  • Agency for Toxic Substances and Disease Registry (ATSDR), 2000. Toxicological Profile for Polychlorinated Biphenyls...
  • C. Anderson et al.

    Metabolic requirements for induction of contact hypersensitivity to immunotoxic polyaromatic hydrocarbons

    J. Immunol.

    (1995)
  • A.J. Bailer et al.

    Effects of treatment-induced mortality and tumor-induced mortality on tests for carcinogenicity in small samples

    Biometrics

    (1988)
  • R. Bartha et al.

    Pesticide transformations: production of chloroazobenzenes from chloroanilines

    Science

    (1968)
  • R. Bartha et al.

    Transformation of the herbicide methyl-N-(3,4-dichlorophenyl)-carbamate (Swep) in soil

    Bull. Environ. Contam. Toxicol.

    (1969)
  • P.A. Bertazzi et al.

    Health effects of dioxin exposure: a 20-year mortality study

    Am. J. Epidemiol.

    (2001)
  • V. Bonvallot et al.

    Organic compounds from diesel exhaust particles elicit a proinflammatory response in human airway epithelial cells and induce cytochrome p450 1A1 expression

    Am. J. Respir. Cell Mol. Biol.

    (2001)
  • G.A. Boorman et al.

    Quality assurance in pathology for rodent carcinogenicity studies

  • M.B. Cachon-Gonzalez et al.

    Structure and expression of the hairless gene of mice

    Proc. Natl. Acad. Sci. U.S.A.

    (1994)
  • D.R. Davila et al.

    Role of alterations in Ca(2+)-associated signaling pathways in the immunotoxicity of polycyclic aromatic hydrocarbons

    J. Toxicol. Environ. Health

    (1995)
  • P. Fernandez-Salguero et al.

    Immune system impairment and hepatic fibrosis in mice lacking the dioxin-binding Ah receptor

    Science

    (1995)
  • Food and Drug Administration, 1987. Good Laboratory Regulations. Final Rule. 21 CFR 58. Fed. Reg. 52,...
  • J.F. Greene et al.

    Basis for a proposed reference dose (RfD) for dioxin of 1–10 pg/kg-day: a weight of evidence evaluation of the human and animal studies

    J. Toxicol. Environ. Health B: Crit. Rev.

    (2003)
  • Y.L. Guo et al.

    Chloracne, goiter, arthritis, and anemia after polychlorinated biphenyl poisoning: 14-year follow-up of the Taiwan Yucheng cohort

    Environ. Health Perspect.

    (1999)
  • Y.L. Guo et al.

    Yucheng: health effects of prenatal exposure to polychlorinated biphenyls and dibenzofurans

    Int. Arch. Occup. Environ. Health

    (2004)
  • J.R. Hailey et al.

    Classification of proliferative hepatocellular lesions in Harlan Sprague–Dawley rats chronically exposed to dioxin-like compounds

    Toxicol. Pathol.

    (2005)
  • G.W. Hambrick

    The effect of substituted naphthalenes on the pilosebaceous apparatus of rabbit and man

    J. Invest. Dermatol.

    (1957)
  • Cited by (17)

    • DNA damage by 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced p53-mediated apoptosis through activation of cytochrome P450/aryl hydrocarbon receptor

      2017, Environmental Toxicology and Pharmacology
      Citation Excerpt :

      The poisoning is often associated with skin lesions and diverse skin reactions. Chloracne is the most consistent manifestation of TCDD intoxication, and considered to be its reliable “hallmarker” (Ramot et al., 2009). In our present study, we investigated the mechanism of DNA damage-induced apoptosis by TCDD on human keratinocyte cells (HaCaT).

    • AhR signalling and dioxin toxicity

      2014, Toxicology Letters
    • Integument

      2013, Haschek and Rousseaux's Handbook of Toxicologic Pathology
    • Integument

      2013, Haschek and Rousseaux's Handbook of Toxicologic Pathology, Third Edition: Volume 1-3
    View all citing articles on Scopus
    View full text