* Department of Entomology, University of California at Davis, Davis, California 95616;
Bouve College of Pharmacy and Health Sciences, Northeastern University, Boston, Massachusetts 02115; and
Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205
Received July 12, 1999; accepted September 28, 1999
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ABSTRACT |
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Key Words: 1,2-epoxynaphthalene; naphthalene; glutathione; ethyl ester glutathione; CYP1A1; Sf-21; alkaline permethylation.
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INTRODUCTION |
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It has also been proposed that conjugation with glutathione (GSH) is a detoxification route for some naphthalene metabolites (Chichester et al., 1994; Iverson et al., 1995
). In these cases, the ability of a cell to detoxify an electrophilic metabolite, along with the propensity to form it in the first place, will govern the degree of cytotoxicity. Thus, the relatively low concentrations of GSH in pulmonary tissue (Forkert, 1997
), and subsequently lessened ability to detoxify, may contribute to cytotoxicity. Some early work describes the formation of cararacts in rabbits after naphthalene administration and the mercapturic acid of naphthalene was found in the urine of rabbits and rats (Bourne and Young, 1934
; Young, 1947
). Indeed, Boyland and Sims suggest as much, though they looked at 1,2-epoxy-1,2,3,4-tetrahydroxynaphthalene, as it was more readily available (Booth et al., 1960
; Boyland and Sims, 1960
).
Inasmuch as toxicity appears to be concentrated in the lung, work on the toxicity of naphthalene metabolites has often emphasized specific CYP monooxygenases found in the lung, such as CYP2E1 (Wilson et al., 1996), CYP2F (Buckpitt et al., 1995
), and CYP2B (Van Winkle et al., 1996
). CYP1A1 is not constitutively expressed in lung tissue (McLemore et al., 1990
) nor in the liver (Omiecinski et al., 1990
; Warner et al., 1998
), hence, very little work has been done on its bioactivation of naphthalene to toxic metabolites. However, CYP1A1 is known to be induced by cigarette smoke (McLemore et al., 1990
), TCDD (Stephen et al., 1997
), phenobarbital, N-benzylimidazole (Papac and Franklin, 1988
), various pesticides including carbaryl, cypermethrin, diflubenzuron, and tetrachlorvinphos (Delescluse et al., 1998
), and various common food chemicals including flavones, indoles, and methylated xanthines (Juchau et al., 1998
). Cigarette smoke not only induces CYP1A1 but also contains naphthalene, so it is likely that smokers are exposed to CYP1A1-generated metabolites of naphthalene. The purpose of this study was to examine the cytotoxicity of CYP1A1-dependent metabolites of naphthalene.
Our study investigated naphthalene toxicity in a time- and dose-dependent manner, to insect cells expressing CYP1A1. We examined intracellular GSH levels after naphthalene intoxication and the protective effects of GSH and ethyl ester GSH, as well as the deleterious effects of GSH depletion. Using alkaline permethylation, we investigated the covalent binding of 1,2-epoxynaphthalene and its correlation with cytotoxicity.
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MATERIALS AND METHODS |
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Statistical Analyses
Student's t-test was used to determine differences in treated and control groups. Differences were considered statistically significant at p 0.05. LC50 and LT50 (time for 50% loss of viability) were determined by regression analysis. All results are mean ± standard deviation.
Toxicity and Binding Studies
Sf-21 cells were infected with the rat CYP1A1/yeast reductase fusion baculovirus or Lac Z baculovirus as control (constructed as previously described [Grant et al., 1996]) in complete medium (96% EX-Cell 401 with L-glutamine, JRH Biosciences (Lenexa, KS); 3% heat-inactivated fetal bovine serum, Intergen (Purchase, NY); and 1% Pen/Strep antibiotics, (Sigma, St. Louis, MO) at a multiplicity of infection of 0.1. One-h post-infection, 0.75 µg/ml of hemin was added to each spinner flask. At 48-h post-infection, 25 mL of cells were aliquoted into 50 mL spinner flasks, and various concentrations of naphthalene (0 mM1.5 mM) were added. At various times post-administration, aliquots were taken and viability assays were performed as described previously (Grant et al., 1996
). Briefly, cells were incubated for 2 h with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). The cells were lysed with 0.5 ml lysing solution (250 mg sodium dodecyl sulfate (SDS) in 1:1 dimethyl formamide: water, pH4.5) over 12 h in the dark to dissolve the insoluble formazan product. Aliquots were then transferred to 96-well plates and the MTT hydrolysis product quantified at 560 nm. The remainder of the cells were centrifuged at 2000 rpm for 10 min, washed with sodium phosphate buffer (0.1 M, pH 7.4) (PBS) 3 times, then lysed in 0.1% SDS. The lysate was dialyzed (3000 mw cutoff) in 0.1% SDS for 36 h at 4°C. In a separate experiment, cells were incubated with 0.5 mM [14C]-naphthalene (1.5 x 105 dpm/µl) for 24 h. Protein was precipitated with methanol, and the pellet washed 6 times with methanol. Aliquots of both pellet and wash were taken and radioactivity quantified by liquid scintillation counter. For those experiments in which the effects of GSH on toxicity were examined, cells were pretreated for 2 h either with 5-mM GSH, 5-mM ethyl ester GSH, or 0.125-mM diethylmaleate (DEM). The experiments were then performed as described above. Protein concentrations were measured using BCA reagent with bovine serum albumin as a standard. Glutathione levels were measured as free sulfhydryls using DTNB (Ellman, 1959
) and verified in several samples by HPLC, as described (Fahey and Newton, 1987
). GST levels were measured using CDNB, as described (Grant et al., 1989
).
Alkaline Permethylation of Sf-21 Cell Proteins
Alkaline permethylation of protein samples were conducted as described previously by Slaughter et al. (1993), and Slaughter and Hanzlik (1991). This method generates methylthionaphthalene derivatives that are structurally dependent on the nature of the naphthalene reactive metabolites modifying cysteine residues (Scheme 1). Briefly, the dialyzed protein samples (4.0 mL) were placed in culture tubes and mixed with 4.0 mL of 8 M NaOH, followed by addition of 1.0 mL of CH3I. The tubes were purged with nitrogen (the protein samples, NaOH solution, and CH3I had been pre-purged with nitrogen), sealed with Teflon-lined caps, and heated at 80°C for 4 h. Following the reaction, the mixture was cooled to room temperature, spiked with ethylthionaphthalene (compound 2) as an internal standard, and extracted with pentane. The pentane extracts were dehydrated with anhydrous Na2SO4, concentrated by distillation, and analyzed by GC-MS.
GC-MS Analysis
Pentane extracts obtained from alkaline permethylated protein samples were analyzed in selected ion monitoring (SIM) mode to enhance sensitivity and specificity. Molecular ions at m/z 174, 188, and 234, representing the molecular weight of compounds 1, 2, and 3, respectively, were selected to monitor methylthionaphthalene derivatives. Structural identification of methylthionaphthalene derivatives, formed in the alkaline permethylation of the protein samples, was conducted by mass matching and comparison of the relative retention times of peaks observed in the reconstructed ion chromatograph with those of the synthetic authentic standards, to the internal standard. In addition, following the first-round analysis, the remaining sample was consecutively spiked with synthetic authentic standards. The resulting spiked sample was reanalyzed by GC-MS in the same SIM mode, for further structural confirmation. Quantification was performed by integration of peak area followed by standardization in consideration of mass response factors and alkaline permethylation yields. The area of peak 2 (compound 2) was used as a quantitative internal standard.
Electrophoresis and Immunoblotting.
Sf-21 cell proteins were resolved by SDSpolyacrylamide gel electrophoresis (Mini-Protean II, Bio-Rad) as described (Laemmli, 1970) using 3.3% stacking and 12% resolving gels. Proteins were transferred to polyvinylidene difluoride (PVDF) microporous membranes (Immobilon-P transfer membranes, Millipore) by an electroblotter (LKB Novablot electrophoretic transfer kit). After 3 h, transferred (43 mA) blots were blocked by shaking overnight in 5% non-fat dry milk in phosphate buffered saline Tween (PBST) buffer. The blotted polyvinylidene difluoride membranes were incubated for 1 h with a 1/2000 dilution of primary anti-1,2-epoxynaphthalene mercapturate rabbit antiserum (Marco et al., 1993
) in PBST buffer with 3% non-fat milk. The immunoblots were incubated for 1 h with horseradish peroxidase-conjugated goat anti-rabbit IgG antibodies (1/4,000 in PBST buffer, Sigma). The blotted protein bands were detected by chemiluminescence using ECL Western blotting kits (Amersham International plc, England). Protein staining was performed using Coomassie blue.
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RESULTS |
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Toxicity of Naphthalene
Sf-21 cells expressing CYP1A1 (150 ± 15 pmol/min/mg protein) or Lac Z were incubated with various concentrations of naphthalene. Cell viability was measured using MTT. The toxicity of naphthalene to CYP1A1 expressing Sf-21 cells was dose dependent (LC50 = 0.3 ± 0.04 mM), while Lac Z expressing cells were unaffected (Fig. 1A). The 24-h time dependence (LT50 = 12 ± 1 h, time for 50% loss of viability) of 1.5 mM naphthalene toxicity to CYP1A1 expressing Sf-21 cells is described by Figure 1B
. LC50 and LT50 were determined by regression analysis. Because of the lack of toxicity to Lac Z-expressing cells, it appears that naphthalene toxicity in this system requires the presence of CYP1A1.
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The time-dependent toxicity of cells intoxicated with 1.5 mM naphthalene was strongly correlated (r2: 0.85) with covalent binding (Fig. 5). Their maxima are reached at 16 h. Additionally, covalent binding was highly correlated (r2: 0.96) with the dose dependent toxicity of naphthalene (data not shown). Additionally, radioactive binding studies with 0.5 mM naphthalene revealed that 3 ± 0.2% of naphthalene is covalently bound in cells expressing CYP1A1, while no naphthalene is bound in cells expressing Lac Z. This 3% covalent binding equates to approximately 20 nmol of naphthalene equivalent bound per mg of non-dialyzable cellular protein. This value correlates well with the 28 nmol/mg naphthalene bound using the alkaline permethylation method.
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DISCUSSION |
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Because of the likelihood of CYP1A1 induction in humans and their potential exposure to naphthalene, we decided to examine the interaction of CYP1A1 and naphthalene without the confounding factors of other xenobiotic metabolizing enzymes. The Sf-21 cell-baculovirus system is ideal for that. Sf-21 cells are noctuid ovarian cells and baculoviruses are insect-specific viruses; thus, toxicity, covalent binding, and cellular co-factors can be assessed in situ, but the difficulties of working with whole animals, or mammalian cell culture, are removed. The transgenic baculovirus assay system has the disadvantages inherent to any cell-based system. The cells must be transiently transfected and there is a narrow window of time (2448 h) in which in situ toxicity assays may be done. This is also a very simple model for a very complex systema whole animal. However, when teasing apart the effects of particular metabolizing enzymes, this can be an advantage. These cells have very low levels of many endogenous enzymes involved in xenobiotic metabolism, including undetectable, or very low levels of, CYP monooxygenases, epoxide hydrolases (Grant et al., 1996), glucuronosyl transferases (Nguyen and Tukey, 1997
), and esterases (Charles et al., 1996
), among others. Thus, effects can be attributed solely to the reaction of the enzyme and the compound of interest. The baculovirus expression system is widely used in the pharmaceutical industry to express human CYP monooxygenases to generate possible metabolites of xenobiotics (Gonzalez, F.J. et al., 1991
). Grant et al. (1996) extended the concept of expression of CYP monooxygenases to the development of a simple system for examining geno- and cytotoxicity of reactive metabolites generated by CYP monooxygenases and other enzymes.
Others have seen the depletion of GSH in the presence of naphthalene metabolites such as the epoxide (Buckpitt, A. et al., 1992), especially in the presence of glutathione transferases (Chichester et al., 1994
), and the quinone (Iverson, et al., 1995
; Murty and Penning, 1992
). However, the absence of GSH depletion by lower concentrations of the epoxide in human lymphocytes has been reported as well (Wilson et al., 1996
), and cytotoxicity and GSH depletion have been attributed solely to the quinone metabolite (Wilson et al., 1996
). This confusion is compounded by the multiple enzymes and isozymes present in most cell types. Addition of GSH has been shown to protect liver microsomes from some of the toxicity of naphthalene metabolites (Tingle et al., 1993
); however, GSH cannot usually cross cell membranes, whereas ethyl ester GSH can (Nishida et al., 1996
). The levels of protection we saw with ethyl ester glutathione in Sf-21 cells were comparable to those seen by Tingle et al. (1993) using ethyl ester GSH in a microsomal system. We did see some protection by GSH, which is presumably occurring outside the cell. These data suggest that GSH protects the cells from the epoxide metabolite and depletion occurs in the absence of other clearance mechanisms, as may occur in pulmonary tissue. These data are supported by the fact that GSH depletion by DEM increased the potency of naphthalene toxicity to CYP1A1-expressing cells.
Metabolite structure was elucidated from studies of protein-bound naphthalene. This method, alkaline permethylation, detects epoxide and quinone-derived metabolites of aromatic systems covalently bound to sulfur nucleophiles of proteins (Slaughter and Hanzlik, 1991; Slaughter et al., 1993
; Zheng et al., 1997
). Alkaline permethylation of naphthalene epoxide-cysteine protein adducts would generate 2-methylthionaphthalene (compound 1) (Scheme 2), whereas, alkaline permethylation of quinone-cysteine protein adducts should produce dimethoxymethylthionaphthalenes (compound 3) resulting from either the 1,2- or the 1,4-naphthoquinone alkylation of cysteine residues of proteins (Scheme 2). The results of the alkaline permethylation of the dialysate of CYP1A1-expressing cells incubated with naphthalene show the presence of 2-methylthionaphthalene (compound 1), from the reaction of 1,2-epoxynaphthalene with a cysteine residue (see Scheme 1). Because compound 1 was not present in the alkaline permethylated dialysate of the Lac Z-expressing cells incubated with naphthalene, it must be a metabolite of CYP1A1. Additionally, compounds such as the dimethoxymethylthionaphthalenes, which would result from the alkaline permethylation of the naphthoquinones covalently bound to the sulfur of a cysteine residue, were not found under conditions where ng levels of compound could easily be distinguished from background.
In this system, thiol linked adducts of naphthalene epoxide are thought to be the major covalent adducts. The alkaline permethylation only detects thiol ether-linked protein adducts and estimates of covalent binding based on 14[C] experiments are quantitatively very similar to results from GC-MS, as shown in the results section. In addition, adducts are detected on Western blot to naphthalene adducts using antibodies highly selective for thiol adducts of naphthalene 1,2-oxide. Antibodies sensitive for other forms of protein-bound naphthalene did not detect protein adducts from this system.
Covalent binding was highly correlated with both dose- and time-dependent toxicity. The Lac Z-expressing cells showed no evidence of covalent binding of any naphthalene metabolites to cysteine residues, nor did they show any toxicity due to naphthalene at or up to 1 mM. This result further emphasizes that the toxicity of naphthalene in this system is due to enzymatic activation by CYP1A1, followed by protein binding. The dose-dependent binding and toxicity curves displayed a threshold effect. This was also seen in the Western blot. This is what one would expect to see if GSH were protective in this system.
It is not clear to what protein naphthalene is covalently bound. The data from the SDSPAGE and Western blot suggest that it might simply be bound to albumin. However, the antibody used recognizes the mercapturic acid metabolite of naphthalene but does not cross-react or reacts very weakly (<5%) with a variety of other naphthalene derivatives (Marco et al., 1993). Most importantly, 1,2-epoxynaphthalene does not detectably react with albumin (Cho et al., 1994b
). It is notable, however, that recent work in mammalian systems has detected acetaminophen-binding proteins at 44 kDa, identified as a subunit of glutamine synthetase (Bulera et al., 1995
), and at 54 kDa, identified as aldehyde dehydrogenase (Landin et al., 1996
). However, 58 kDa and 56 kDa proteins were most strongly bound by acetaminophen and have been identified as acetaminophen-binding protein and selenium-binding protein (Bartolone et al., 1992
; Cohen et al., 1997
; Pumford et al., 1992
).
Zheng et al. (1997) found both dimethoxymethylthionaphthalenes and methylthionaphthalene after alkaline permethylation of the dialysate of Clara cells incubated with naphthalene, indicating the presence of both the 1,2-naphthoquinone and 1,2-epoxynaphthalene as reactive metabolites. Clara cells are known to express multiple CYP monooxygenases (Buckpitt et al., 1995). Since we saw only saw a derivative of the epoxide metabolite, we propose that the naphthoquinone does not spontaneously generate under these conditions; enzymes needed for its production were absent and it was not formed by CYP1A1.
These data lead us to speculate that 1,2-epoxynaphthalene is short-lived due to reaction with water, soluble epoxide hydrolase, and other enzymes. When enzyme systems are present that generate longer-lived quinone metabolites, Michael reactions with quinones predominate in cytotoxicity and in depleting GSH. Thus, we have used the baculovirus-Sf-21 cell system to isolate continuous in situ production of 1,2-epoxynaphthalene from other reactions, and demonstrate that the epoxide itself is capable of cytotoxicity and GSH depletion. This further validates the baculovirus-Sf-21 cell system.
In conclusion, humans are exposed to naphthalene, which is metabolized by many CYP monooxygenases. In mice, selective pulmonary lesions appear to lead to death following naphthalene exposure (Buckpitt et al., 1995; Cho et al., 1994a
). However, we do not know the primary intracellular target of naphthalene nor how this binding initiates a series of events leading to cell death. While constitutively expressed CYP monooxygenases are capable of metabolizing naphthalene, induction of CYP1A1 in exposed individuals may enhance risk. Therefore, using a baculovirus expression system, we have shown that CYP1A1 expression dramatically increases cell death, glutathione depletion, and the formation of covalent adducts of naphthalene linked to protein through a thioether in position 2. The toxicity and covalent binding of naphthalene adducts is especially important when considering the exposure of smokers with induced lung CYP1A1 exposed to naphthalene in cigarette smoke.
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ACKNOWLEDGMENTS |
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NOTES |
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