Under the skin: Biotransformation of para-aminophenol and para-phenylenediamine in reconstructed human epidermis and human hepatocytes

doi:10.1016/j.toxlet.2005.03.014

Toxicology Letters

Volume 158, Issue 3, 15 September 2005, Pages 196-212

https://doi.org/10.1016/j.toxlet.2005.03.014 Get rights and content

Abstract

We investigated the biotransformation of the oxidative arylamine (AA) hair dye ingredients [¹⁴C]-para-aminophenol (PAP) and [¹⁴C]-para-phenylenediamine (PPD) in reconstructed human epidermis and human hepatocytes. Human epidermis quantitatively transformed PAP to its N-acetylated derivative (APAP), whereas hepatocytes transformed PAP to sulfate or glucuronic acid conjugates of APAP or PAP as well as free APAP. Epidermis and hepatocytes converted PPD to N-mono- (MAPPD) and N,N′-di-acetylated (DAPPD) derivatives. At higher concentrations of PPD (250–1000 μM), epidermis or hepatocytes produced more of the MAPPD, whereas concentrations below 250 μM and lower favoured formation of the DAPPD metabolite. When compared with epidermis, human hepatocytes had a three-fold or eight-fold greater capacity for generation of MAPPD or DAPPD, respectively. No evidence of transformation of PAP or PPD to N-hydroxylated derivatives was found in epidermis or hepatocytes. Our results suggest that (i) after dermal absorption of PAP or PPD, humans are systemically exposed to acetylated derivatives; (ii) current in vitro skin absorption studies may be inadapated for determination of human systemic exposure to AAs due to reduced or absent metabolic capacity of non-viable skin; (iii) due to qualitative differences between dermal and hepatic metabolism, oral toxicity studies may be unsuited for the hazard assessment of dermal exposure to AAs; and (iv) use of induced rodent liver S9 metabolic activation systems for in vitro genotoxicity studies may produce misleading results on the hazard of human dermal exposure to AAs. In conclusion, our data support the growing evidence that AAs are transformed in human skin and suggest that current practices of safety assessment of AAs should take these findings into account.

Introduction

Knowledge of pharmacokinetics and metabolism following dermal exposure are key requirements for the risk assessment of substances that come into contact with human skin. Concerning the extent of the potential human systemic exposure, current EU guidelines for the safety evaluation of hair dyes and other cosmetic ingredients generally rely on the results of in vitro skin absorption studies (SCCNFP, 2003), whereas investigation of the nature of the potential human systemic exposure agents has received little attention.

The chemical class of arylamines (AAs) include high volume chemicals that are widely used in the pharmaceutical, chemical, rubber, dye or photographic industries. Given that some AAs are also contained in oxidative hair dyes, they have considerable potential to produce skin exposure of the general population. AAs represent a large chemical class with a wide spectrum of toxicological properties. Although some low-molecular weight AAs, particularly those used in hair dyes, are non-toxic to moderately toxic and non-carcinogenic (Anonymous, 1993), their class includes some known human carcinogens, such as benzidine, 4-aminobiphenyl or 2-naphthylamine, which were recognised as early as the 19th century to produce bladder cancer in occupationally exposed workers of the German and other European dye or textile industries (Rehn, 1895, Case et al., 1954). Therefore, considerable attention has been focused over the last several decades on this class of chemicals by toxicologists and epidemiologists.

After oral administration, AAs are mainly metabolised in the liver of mammals by N-acetyltransferases that convert them into the corresponding, N-acetylated aromatic amides, which are less likely to be activated by cytochrome P-4501A2 to DNA-reactive, and potentially mutagenic and/or carcinogenic metabolites (Butler et al., 1989, Guengerich, 1992). Given that N-acetylated AAs are less reactive and more readily excreted in the urine than their parent compounds, N-acetylation of low molecular weight AAs is generally regarded as a detoxification pathway (Parkinson, 2001). The principal enzymes that catalyse acetylation of AAs are N-acetyltransferases 1 and 2 (NAT1/NAT2). NAT2 is mainly found in the liver and the gastrointestinal tract (Grant et al., 1997), whereas NAT1 is present in the mammalian liver, urinary bladder, epidermis and many other tissues (Hein, 2000, Hein, 2002). Mammalian skin has a high NAT1-mediated acetylation capacity for AAs (Kawakubo et al., 1990, Gaudet et al., 1993, Reilly et al., 2000) and is capable to transform AAs, such as para-phenylenediamine (PPD) and, possibly, para-aminophenol (PAP) to their acetylated metabolites (Kawakubo et al., 2000, Nohynek et al., 2004b; Dressler and Appleqvist, in press).

In order to elucidate the metabolism of AAs after human dermal exposure, we investigated the in vitro biotransformation of AAs in reconstructed human epidermis. In parallel, we studied the biotransformation of AAs in human hepatocytes in order to assess their metabolism after oral uptake and subsequent hepatic metabolism. We selected para-phenylenediamine (1,4-benzenediamine; PPD) and para-aminophenol (4-amino-1-hydroxybenzene; PAP) as model substances (Fig. 1), since human contact with these large volume and non-volatile industrial chemicals and oxidative hair dye ingredients is frequent and mainly occurs via the dermal route.

Section snippets

Biological materials

Reconstructed human epidermis (Episkin^®), a human keratinocyte tissue culture (Tinois et al., 1991), was obtained from Episkin (SMS, Lyon, France) and used on development day 13. Episkin kits consist of 12-well microplates, each containing 1.1 cm² of reconstructed epidermis on agarose gel (Fig. 2). Epidermis was transferred under sterile conditions to 12-well microplates containing William's medium. Primary human hepatocytes were obtained from BIOPREDIC (Rennes, France), generated by collagenase

Cytotoxicity

The toxicity of PAP to reconstructed human epidermis and rat hepatocytes is shown in Table 1. Over the concentration range of 25–1000 μM (2.7–109 μg/ml), PAP was non-toxic to human keratinocytes. PAP was tested in a range of 10–300 μM (1.1–33.0 μg/ml) in rat hepatocytes. Concentrations of 150 μM or above were cytotoxic to hepatocytes with an IC₅₀ value of 132 ± 2.5 μM (14.5 μg/ml). On the basis of these results, the metabolism studies in reconstructed human epidermis and hepatocytes were performed with

Discussion

Oxidative hair dyes contain PAP or PPD at typical concentrations of 10 mg/ml or less. The short duration of contact (0.5 h) of the human scalp with oxidative hair dyes and the subsequent diffusion of dye molecules through the stratum corneum into the epidermis and dermis result in a concentration gradient that produces far lower concentrations of dye molecules in the skin relative to those in the applied dye. For example, treatment of human skin in vitro with a PPD-containing oxidative hair dye

Acknowledgements

The authors gratefully acknowledge the excellent technical assistance of Frederic Cornielle. Our thanks to Drs. Henri Benech and Alain Pruvost for performance and analysis of the PAP mass spectra and Drs. Eric Dufour, Mitchell Sauerhoff, Julie Skare and David Hein for review of this manuscript and their astute and most constructive comments.

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Published by Elsevier Ireland Ltd.

Under the skin: Biotransformation of para-aminophenol and para-phenylenediamine in reconstructed human epidermis and human hepatocytes

Abstract

Introduction

Section snippets

Biological materials

Cytotoxicity

Discussion

Acknowledgements

Mutat. Res.

Food Chem. Toxicol.

Food Chem. Toxicol.

Mutat. Res.

Toxicol. Lett.

Mutat. Res.

Toxicol. Appl. Pharmacol.

Food Chem. Toxicol.

Toxicol. Appl. Pharmacol.

Food Chem. Toxicol.

Food Chem. Toxicol.

Regul. Toxicol. Pharmacol.

J. Investig. Dermatol.

Regul. Toxicol. Pharmacol.

Toxicology

J. Chromatogr.

Exp. Cell Res.

Toxicol. Appl. Pharmacol.

Toxicol. Appl. Pharmacol.

Modelling dermal pharmacokinetics using in vitro data. II. Fluazifop-butyl in man

Hum. Exp. Toxicol.

Validation of the in vivo CD1 mouse splenocyte micronucleus test

Mutagenesis

Toxicity determined in vitro by morphological alterations and neutral red absorption

Toxiocol. Lett.

Metabolic oxidation of the carcinogens 4-aminobiphenyl and 4,4′-methylene-bis(2-chloroaniline) by human hepatic microsomes and by purified rat hepatic cytochrome p-450 monooxygenases

Cancer Res.

Comparative metabolism of the designer drug 4-methylthioamphetamine by hepatocytes from man, monkey, dog, rabbit, rat and mouse

Naunyn Schmiedebergs Arch. Pharmacol.