Arch Toxicol (2005) 79: 74–82 DOI 10.1007/s00204-004-0590-z
M E TA B O L I C AC T I V A T I O N / I N A C T I V A T I O N
A. Nannelli Æ A. De Rubertis Æ V. Longo Æ P. G. Gervasi
Effects of dioxane on cytochrome P450 enzymes in liver, kidney, lung and nasal mucosa of rat
Received: 10 February 2004 / Accepted: 14 June 2004 / Published online: 15 October 2004 Springer-Verlag 2004
Abstract The effect of acute and chronic dioxane administration on hepatic, renal, pulmonary and nasal mucosa P450 enzymes and liver toxicity were investigated in male rats. The acute treatment consisted of two doses (2 g/kg) of dioxane given for 2 days by gavage, whereas the chronic treatment consisted of 1.5% of dioxane in drinking water for 10 days. Both the acute and chronic dioxane treatments induced cytochrome P450 2B1/2- and P450 2E1-dependent microsomal monooxygenase activities (pentoxyresorufin O-depentylase and p-nitrophenol hydroxylase) in the liver, whereas in the kidney and nasal mucosa, only the 2E1 marker activities were enhanced. In addition in the liver, an induction of 2a-testosterone hydroxylase (associated with the constitutive and hormone-dependent P450 2C11) was also revealed, whereas the hepatic P450 4Adependent x-lauric acid hydroxylase was not enhanced by any dioxane treatment. These inductions were mostly confirmed by western blot analysis of liver, kidney and nasal mucosa microsomes. In the lung, no alteration of P450 activities was observed. To assess the mechanism of 2E1 induction, the hepatic, renal and nasal mucosa 2E1 mRNA levels were also examined. Following two kinds of dioxane administration, in the liver the 2E1 induction was not accompanied by a significant alteration of 2E1 mRNA levels, while both in the kidney and nasal mucosa the 2E1 mRNA increased about 2- to 3fold, indicating an organ-specific regulation of this P450 isoform. Furthermore, dioxane was unable to alter the plasma alanine aminotransferase activity and hepatic glutathione (GSH) content, examined as an index of toxicity, when it was administered into rats with P450 2B1/2 and 2E1 preinduced by phenobarbital or fasting pretreatment. These results support the lack of or a poor formation of reactive and toxic intermediates during the A. Nannelli Æ A. De Rubertis Æ V. Longo Æ P. G. Gervasi (&) Area della Ricerca CNR, Istituto di Fisiologia Clinica, via Moruzzi 1, 56100 Pisa, Italy E-mail:
[email protected] Tel.: +0039-050-3152701
biotrasformation of this solvent, even when its metabolism was enhanced by P450 inducers. The chronic administration of dioxane was also unable to induce the palmitoyl CoA oxidase, a marker of peroxisome proliferation, excluding this as a way to explain its toxicity. Thus, although the mechanism of dioxane carcinogenicity remains unclear, the present results suggest that the induction of 2E1 following a prolonged administration of dioxane might provide oxygen radical species, and thereby contribute to its organ-specific toxicity. Keywords Dioxane Æ Cytochrome P450 induction Æ Kidney Æ Nasal mucosa
Introduction 1,4-Dioxane (dioxane) is a polar solvent widely used for detergent, ink, cosmetics and paint production but also as a preservative, fumigant and deodorant (IARC 1999). Since this compound contains no functional groups susceptible to reactivity, it is expected to be hardly hydrolysed by water or degraded by earth bacteria and, consequently, it can readily leach and accumulate into groundwater representing one possible source of contamination for humans. Studies designed to evaluate the consequences of dioxane exposure in human are limited to inhalation and dermal exposure, although investigations on risks related to drinking water exposure could be useful. The data indicated that it is irritating to mucous membranes and is able to cause hepatic and renal toxic effects at high inhalation concentrations (IARC 1999; De Rosa et al. 1996). In animals, several chronic and short-term studies have evidenced histopathological lesions and death depending on the concentration and duration of action. Moreover, long-term studies in rodents have shown that high doses of dioxane in drinking water are associated with increased incidence of renal, hepatic and nasal turbinate tumours (De Rosa et al. 1996).
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Pharmacokinetic studies performed in humans and rats indicated that dioxane is easily absorbed after oral or inhalation exposure and it is metabolised to oxidation products rapidly excreted in the urine. In vivo investigations have identified in plasma and urine two metabolites, namely 1,4-dioxane-2-one and b-hydroxyethoxyacetic acid (HEAA), and have hypothesised an involvement of cytochrome P450 (CYP) system in this metabolism (De Rosa et al. 1996; Woo et al. 1977a; Braun and Young 1977), as shown in the Fig. 1. It has also been reported that the in vivo metabolism of dioxane is inducible by phenobarbital (Woo et al. 1977b) and that dioxane itself is able to induce its own metabolism after prolonged administration (Young et al. 1978). However, the dioxane interaction with CYP enzymes and their role in its bioactivation to reactive intermediates have not been studied. In this paper, we have investigated the influences of acute (by gavage) or chronic (in drinking water) dioxane administration on the expression of CYP isoforms in the liver, lung, kidney and nasal mucosa of rat. Moreover, we have evaluated the extent of dioxane toxicity determining the plasma alanine aminotransferase and hepatic GSH levels after its administration to rats pretreated with CYP2B1/2 or CYP2E1 inducers.
Materials and methods
anti-rat 4A1 were purchased from Gentest (Woburn, MA, USA). Goat polyclonal antibodies against rat CYP2C11 were supplied by Dr. J.B. Schenkman (Farmington, ME, USA). All other chemicals and solvents were of analytical grade and were obtained from common commercial sources. Animal treatment and preparation of microsomes This study was performed by using male SpragueDawley rats (200–250 g, Charles River, Como, Italy). In total, six different groups of animals were used. One group of rats received food and drinking water (control); a second group received dioxane in the drinking water (1.5% v/v) for 10 days (chronic treatment); a third group was treated with 2 g/kg dioxane by gavage as a 50% solution for 2 days (acute treatment); a fourth group was treated intraperitoneally (i.p.) with 500 mg/kg dioxane for 4 days; a fifth group was fasted for 2 days and treated i.p. with 2 g/kg dioxane; a sixth group was treated with PB (0.1% in drinking water for 8–10 days) and then treated i.p. with 2 g/kg dioxane. Microsomes were prepared from rat liver, kidney, lung and nasal mucosa as previously described (Menicagli et al. 1994; Longo et al. 1991) and were stored at 80C. Protein content was assayed by the method of Lowry et al. (1951).
Chemicals 1,4-Dioxane (dioxane) and phenobarbital (PB, a controlled drug available only for research purpose) were obtained from Fluka (Buchs, Switzerland). Rabbit polyclonal antibodies against rat CYP2B1 and CYP2E1 were obtained in our laboratory, as previously described by Menicagli et al. (1994), whereas anti-rat 3A2 and
Fig. 1 Metabolic pathway of dioxane metabolism (HEAA bhydroxyethoxyacetic acid)
Enzyme assays CYP content was measured by the method of Omura and Sato (1964).The hydroxylation rates of aniline (AnH) to 4-aminophenol and of p-nitrophenol (p-NPH) to 4-nitrocatechol, both markers of CYP2E1, were determined using the methods of Ko et al. (1987) and Reinke and Moyer (1985), respectively. Ethoxyresorufin O-deethylase (EROD) and pentoxyresorufin O-depentylase (PROD) activities, markers for CYP1A1/2 and CYP2B1/2, respectively, were determined by measuring the production of resorufin (Lubet et al. 1985). Erythromycin N-demethylase (ErD) activity, a marker for CYP3A1/2, was determined as described by Tu and Yang (1983). Testosterone hydroxylase activity was determined by separating the hydroxylated metabolites using a Waters 1525 high-performance liquid chromatography apparatus equipped with a Supelco LC-18 column (25x4.6 mm) as previously reported (Amato et al. 1996). KCN-insensitive palmitoyl CoA oxidation activity was determined in the whole hepatic homogenate as described by Bronfman et al. (1979). The x- and (x 1)-lauric acid hydroxylase activities were determined as previously reported (Zanelli et al. 1996). Alanine aminotransferase (ALAT) activity, a biomarker of cellular damage, was determined in serum using a commercial kit (Sclavo, Siena, Italy). The GSH content was measured following the method described by Ellman (1959).
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Immunoblot analysis Liver, kidney and nasal mucosa microsomes were subjected to sodium dodecyl sulfate-polyacylamide gel electrophoresis (SDS-PAGE) using the method of Laemmli (1970) in a Bio-Rad miniProtein II apparatus. Proteins were transferred from slab gel to nitrocellulose filters following the method of Towbin et al. (1979). Immunodetection was performed with anti-rat CYP2B1, 2E1, 2C11, 3A2 and 4A1 polyclonal antibodies. The bands on the nitrocellulose membrane were quantified by laser densitometric (Ultrascan 2202LKB). Northern blot analysis Total mRNAs were prepared from frozen hepatic, renal and nasal mucosa tissues by the method of Chomczynski and Sacchi (1987). The RNA was size-fractionated by electrophoresis in 1.25% agarose-formaldehyde gels and then transferred to nylon membranes as described by Sambrook et al. (1989). The nylon membranes were hybridised with 32P-labelled CYP2E1 rat cDNA obtained from Dr. F. Gonzales (NIH, Bethesda, MD, USA). 32 P-labelled b-actin cDNA from mouse was used as a standard during Northern blot analysis. The bands on the autoradiographs were quantified by densitometry.
Results Effects of dioxane administration on hepatic P450 activities Results of studies on the effects of dioxane administration on hepatic CYP-dependent monooxygenase activities are shown in Table 1 and Fig. 2. Dioxane was administered to rats either by gavage at a dose of 2 g/kg for 2 days (acute treatment) or in drinking water (1.5% v/v, corresponding at about 0.4 g/kg per day) for 10 days (chronic treatment). Both 1,4-dioxane treatments were unable to alter significantly the CYP content, or EROD and x-lauric acid hydroxylase activities but increased markedly the 2B1-dependent PROD activity (Lubet et al. 1985) and the AnH, p-NPH and (x 1)-lauric acid hydroxylase activities, all of which are linked to the CYP2E1 (Reinke Table 1 Monooxygenase activities in liver microsomes from control rats and rats treated with dioxane by gavage or in drinking water. Values are mean ±SD of three experiments; each experiment used liver microsomes pooled from three or four control or dioxane-treated rats. Enzyme activities are expressed as nmol/min
et al. 1985; Reinke and Meyer 1985; Amet et al. 1994). On the other hand, the ErD activity, dependent on the CYP3A isoform (Arlotto et al. 1987), was induced (about 2-fold) only by the gavage treatment. To further investigate the effects of dioxane, the microsomal metabolism of testosterone, an endogenous substrate selectively oxidised by many CYPs, (Amato et al. 1996) was studied (Fig. 2). Both dioxane treatments induced several fold (about 18–20) 2B1/2-dependent 16b-testosterone hydroxylase, but only the acute dioxane administration was able to induce, although weakly, CYP3A-linked 6b-testosterone hydroxylase (Platt et al. 1989), in keeping with the ErD data shown in Table 1. In addition, to our surprise, it was also observed that both 1,4-dioxane treatments induced 17OT-, 16a- and particularly 2a-testosterone hydroxylases linked to CYP2C11 (Platt et al. 1989), the major P450 isoform constitutively present in the liver (Fujita et al. 1989). To confirm these inductions, hepatic microsomal proteins from control and dioxane-treated rats were analysed by western blotting using polyclonal antibodies raised against rat CYP2B1, 2C11, 2E1, 3A2, and 4A1. As illustrated in Fig. 3A, in control microsomes antiCYP2E1 recognised a protein band, in agreement with the constitutive presence of 2E1 in rat liver (Amato et al. 1996). In microsomes from acute and chronic dioxanetreated rats, the 2E1 apoprotein content, as determined by densitometry, increased to about 420% and 670% of control value, respectively. When anti-CYP2B1 was used (Fig. 3B), two apparent protein bands were recognised that were ascribable to 2B1 and 2B2 in microsomes from both acute and chronic dioxane-treated rats, whereas only very faint bands were visible in control microsomes, in keeping with the very low constitutive level of 2B1 and 2B2 in rat liver (Imaoka et al. 1990). Anti-CYP3A2 recognised in control microsomes (Fig. 3C) a single band ascribable to 3A1 and 3A2, being immunochemically indistinguishable and with the same molecular mass (Gemzik et al. 1992). This protein band was found to be enhanced (to about 150% of control) only in microsomes from rats acutely treated with dioxane. On the other hand, the protein band recognised by anti-CYP2C11 in control microsomes was not found to be significantly enhanced in microsomes from dioxane-treated rats however treated (Fig. 3D), although an increasing trend of the 2C11 apoprotein was visible in microsomes from rats chronically treated with dioxane. per mg protein (EROD ethoxyresorufin O-deethylase, AnH aniline hydroxylation, p-NPH p-nitrophenol hydroxylation, ErD erythromycin N-demethylase, PROD pentoxyresorufin O-depentylase, Lauric H. lauric acid hydroxylase)
Treatment
EROD
AnH
p-NPH
ErD
PROD
x-Lauric H.
(x 1)-Lauric H.
Control Dioxane (gavage) Dioxane (drinking water)
0.03±0.008 0.03±0.007 0.04±0.009
0.71±0.07 2.95±0.21** 3.04±0.43**
0.95±0.06 2.78±0.61** 5.09±0.36**
0.57±0.04 1.35±0.18** 0.64±0.24
0.01±0.003 0.10±0.03** 0.06±0.015**
0.8±0.2 1±0.3 0.9±0.3
0.6±0.2 1.3±0.4* 1.5±0.3**
*p
