Stability of Hemoglobin and Albumin Adducts of Naphthalene Oxide, 1,2-Naphthoquinone, and 1,4-Naphthoquinone

Melissa A. Troester*, Andrew B. Lindstrom{dagger}, Suramya Waidyanatha*, Lawrence L. Kupper{ddagger} and Stephen M. Rappaport*,1

* Department of Environmental Sciences and Engineering, School of Public Health, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7400; {dagger} National Exposure Research Laboratory, U.S. Environmental Protection Agency, MD-44, Research Triangle Park, North Carolina 27711; and {ddagger} Department of Biostatistics, School of Public Health, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7400

Received November 15, 2001; accepted April 5, 2002


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Naphthalene is an important industrial chemical, which has recently been shown to cause tumors of the respiratory tract in rodents. It is thought that one or more reactive metabolites of naphthalene, namely, naphthalene-1,2-oxide (NPO), 1,2-naphthoquinone (1,2-NPQ), and 1,4-naphthoquinone (1,4-NPQ) contribute to the tumorigenicity of this chemical. These electrophiles are all capable of covalent binding to macromolecules including DNA and proteins. The stability of cysteinyl adducts of NPO, 1,2-NPQ, and 1,4-NPQ were investigated in both hemoglobin (Hb) and albumin (Alb) of male F344 rats following a single administration of 2 different doses (400 or 800 mg naphthalene per kg body weight). To assess the stability of Alb adducts, we compared the rates of NPO-Alb turnover (half-life of approximately 2 days) and 1,2-NPQ-Alb (half-life of approximately 1 day) to the normal turnover rate of Alb in the rat (half-life = 2.5–3 days). Based on the rapid turnover of these adducts relative to Alb itself, we concluded that they were unstable. However, the stability of Alb adducts was not affected by the dose of naphthalene administered (400 or 800 mg/kg). In contrast, NPO-Hb adducts were relatively stable (rate constant of adduct instability <= 0.01) following a 400 mg/kg dose of naphthalene, but their stability could not be estimated following an 800 mg/kg dose due to high variability among animals. The rate constants of adduct instability obtained in this study allow application of NPO and NPQ adducts to estimate the exposure to reactive electrophilic metabolites of naphthalene in the rat. In addition, some limitations of current methods for estimating adduct instability were identified.

Key Words: naphthalene-1,2-oxide; 1,2-naphthoquinone; 1,4-naphthoquinone; tumorigenicity; rat; hemoglobin; albumin.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Naphthalene is an important industrial chemical, with annual use in the United States expected to reach 1.15 x 105 metric tons in 2001 (NTP, 2000Go). Naphthalene is also a constituent of diesel fuel and jet fuel (McDougal et al., 2000Go), and is the simplest member of the family of polycyclic aromatic hydrocarbons that are formed as byproducts of combustion. Thus, there are many environmental sources of naphthalene, including fuels, cigarette smoke (Schmeltz et al., 1976Go), and vehicle exhaust (Clark et al., 1982Go).

The acute effects of human exposure to naphthalene include hemolytic anemia (Santhanakrishnan et al., 1973Go) and cataracts (van Heyningen and Pirie, 1967Go). Rodents are less susceptible to naphthalene’s hemolytic effects (Shopp et al., 1984Go), but cataracts have been seen in rats exposed to naphthalene (van Heyningen, 1979Go). Moreover, rodents are susceptible to respiratory toxicity of naphthalene, including nasal adenomas in rats (NTP, 2000Go), necrosis of pulmonary bronchiolar epithelial cells in mice (Plopper et al., 1992Go), and pulmonary alveolar/bronchiolar adenomas in female mice (Abdo et al., 1992Go).

The species-specific toxicity of naphthalene has been attributed to differences in the site and rate of metabolism (O’Brien et al., 1985Go), however, the ultimate toxic metabolites have not been conclusively identified. Naphthalene is initially metabolized to naphthalene-1,2-oxide (NPO) by P450 NADP(H)-dependent oxidation. Subsequent transformation of NPO yields 1-naphthol, 1,2-dihydro-1,2-dihydroxy-naphthalene, 1,2-naphthoquinone (1,2-NPQ), 1,4-naphthoquinone (1,4-NPQ), and various sulfate, glucuronide, and glutathione conjugates (NTP, 2000Go). Although NPO has been implicated as the principal toxic metabolite in mouse lung Clara cells (Chichester et al., 1994) and rat lung (Sweeney et al., 1995Go), 1,2-NPQ is also a major metabolite bound to cysteine residues in mouse lung Clara cells in vitro (Zheng et al., 1997Go). In humans, 1,2-NPQ and 1,4-NPQ caused sister chromatid exchanges in lymphocytes and cytotoxicity to mononuclear leukocytes in vitro, but NPO did not demonstrate cytotoxic or genotoxic effects (Wilson et al., 1996Go).

It is difficult to directly measure reactive electrophilic metabolites such as NPO, 1,2-NPQ and 1,4-NPQ in vivo, so we sought assays for protein adducts of these compounds. Our laboratory has previously investigated the use of cysteinyl adducts of hemoglobin (Hb) and albumin (Alb) to assess levels of the analogous benzene metabolites, i.e., benzene oxide (BO; Lindstrom et al., 1998Go, 1999Go; Yeowell-O’Connell et al., 1996GoYeowell-O’Connell et al., 1998Go), 1,2-benzoquinone (1,2-BQ), and 1,4-benzoquinone (1,4-BQ; Waidyanatha et al., 1998Go). We extended these methods to measure cysteinyl adducts formed from reactions of NPO, 1,2-NPQ, and 1,4-NPQ with Hb and Alb (Fig. 1Go; Waidyanatha et al., in pressGo). Using this assay, we demonstrated that these Hb and Alb adducts of NPO, 1,2-NPQ, and 1,4-NPQ increased with dose following a single administration of 0–800 mg naphthalene per kg body weight in F344 rats (Waidyanatha et al., in pressGo).



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FIG. 1. Assay for measuring cysteinyl adducts formed from reaction of NPO, 1,4-NPQ, and 1,2-NPQ with Hb and Alb. Naphthalene (1) is metabolized to the electrophiles NPO (2), 1,4-NPQ (3), 1,2-NPQ (4). These electrophiles react with protein sulfhydryl groups on Hb and Alb to form adducts. Reaction of proteins with trifluoroacetic anhydride (TFAA) and methanesulfonic acid yields fluorinated derivatives NPO1-S-TFA (5), NPO2-S-TFA (6), 1,4-NPQ-S-TFA (7), and 1,2-NPQ-S-TFA (8). These derivatives can be measured using GC-MS in the negative ion chemical ionization (NICI) mode.

 
Dosimetric applications of Hb and Alb adducts require that the first-order rate constants of adduct instability be estimated in vivo (Granath et al., 1992Go). The stabilities of naphthalene-derived Hb and Alb adducts have been partially investigated in mice following ip administration of 400 mg/kg [3H]naphthalene (Cho et al., 1994Go). In that study, half-lives of 1.8 and 11.5 days were reported for uncharacterized radiolabeled adducts of Alb and Hb, respectively. However, because the stabilities of specific adducts can differ substantially (Troester et al., 2000Go), we investigated the elimination of Hb and Alb adducts formed from NPO, 1,2-NPQ, and 1,4-NPQ in F344 rats. By applying existing models for estimating the first-order rate constants of adduct instability, we also assessed how well these models describe the true behavior of the adducts.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
Ascorbic acid and hydrochloric acid (conc.) were from Fisher Scientific (Pittsburgh, PA). Acetone and hexane were from Mallinckrodt (Paris, KY). Naphthalene was from Aldrich (Milwaukee, WI). Methoxyflurane was from Pitman-Moore (Mundelein, IL). Trifluoroacetic anhydride (TFAA), obtained from Pierce (Rockford, IL), was distilled once before use. Methanesulfonic acid was from Fluka Chemical Company (Switzerland). Human Hb and Alb, used for standard curves, were from Sigma (St. Louis, MO). 1,4-NPQ-bound N-acetyl-L-cysteine (1,4-NPQ-NAC), 1,2-NPQ-bound N-acetyl-L-cysteine (1,2-NPQ-NAC), and NPO-bound N-acetyl-L-cysteine (2 isomers: NPO1-NAC with sulfur substitution at the 1 position of naphthalene and NPO2-NAC with the sulfur substitution at the 2 position of naphthalene) and deuterium-labeled cysteinyl adducts of 1,2-NPQ, 1,4-NPQ, and NPO with Alb ([2H5]1,2-NPQ-Alb, [2H5]1,4-NPQ-Alb, [2H6]NPO-Alb) were previously prepared using materials and methods described in Waidyanatha et al. (in press).

Caution: TFAA reacts violently with water and should only be used to derivatize proteins that are completely dry.

Animals and blood collection.
Forty-five male F344 rats (175–200 g) were obtained from Charles River Breeding Laboratories (Raleigh, NC) and were housed in polycarbonate cages on a 12-h light/dark cycle for 2 weeks before use. Food and water were provided ad libitum. Corn oil only (vehicle control) was administered by gavage to 3 rats. The remaining 42 rats were divided into 2 groups of 21 animals, and a single po dose of either 400 or 800 mg naphthalene per kg body weight was administered (in corn oil) to each animal by gavage. The 400 mg/kg dose was chosen to allow comparison with the experiments conducted at the same dose by Cho et al., 1994. The 800 mg/kg dose was chosen to be 2-fold higher, but still well below the po LD50 (12.8 g/kg) of naphthalene in the rat (Papciak and Mallory, 1990Go). Three animals from each group were sacrificed at 1, 2, 7, 14, 21, 28, and 42 days following dosing. Rats were anesthetized with methoxyflurane and blood was removed by direct cardiac puncture into a heparinized syringe. Approximately 7–9 ml of blood were collected from each animal. Blood was stored on ice immediately, and plasma and red cell fractions were separated within 2 h.

Isolation and purification of Hb and Alb.
Blood samples were centrifuged at 800 x g for 15 min and the plasma layer was removed by pipet. The red blood cell layer was washed 3 times with an equal volume of 0.9% saline. The buffy coat was removed by pipet and an equal volume of deionized water was added to the red blood cells. Samples were frozen at -20°C to lyse cells prior to isolation of Hb.

Hb and Alb were isolated according to the procedure in Rappaport et al. (Rappaport et al., 1993Go) with modifications. Briefly, Hb was obtained from thawed, lysed red cells by centrifuging at 30,000 x g at 4°C for 40 min to remove the membranes. The supernatant was dialyzed (Spectra-Pore 1, 6000-8000 MWCO) against 4 x 3.5 l of deionized water at 4°C over 24 h (rather than by Sephadex chromatography). Globin was precipitated by dropwise addition of the dialysate to cold acidified acetone (0.1% hydrochloric acid by volume). The precipitate was washed with ice-cold acetone and dried to constant weight under vacuum at 37°C.

To isolate Alb, an equal volume of saturated aqueous ammonium sulfate was added to thawed pure plasma by dropwise addition. This mixture was centrifuged at 800 x g to remove immunoglobulins. The supernatant was dialyzed (Spectra-Pore 1, 12,000 MWCO) against 4 x 3.5 l of deionized water at 4°C over 24 h. The Alb solution was lyophilized to constant weight.

Measurement of NPO, 1,2-NPQ, and 1,4-NPQ adducts with Hb and Alb.
Cysteinyl adducts of NPO, 1,2-NPQ, and 1,4-NPQ with Hb and Alb were assayed with the method of Waidyanatha et al. (in press). Briefly, 100 µl of a 50 mg/ml solution of Hb or 50 µl of a 50 mg/ml solution of Alb were added to 5 µg of deuterium-labeled protein bound internal standard ([2H5]1,2-NPQ, [2H5]1,4-NPQ, [2H6]NPO-Alb). Samples were then dried in a vacuum oven at 80°C for several h. To the dried proteins, 750 µl of TFAA and 20 µl of methanesulfonic acid were added and the proteins were incubated at 100°C for 40 min to produce 1,2-NPQ-S-TFA, 1,4-NPQ-S-TFA, and 2 structural isomers of NPO-S-TFA (NPO1-S-TFA and NPO2-S-TFA; see Fig. 1Go). The samples were then cooled to room temperature and excess TFAA was removed under a stream of nitrogen. To each sample, 1 ml of hexane was added, followed by 1 ml of 0.1 M Tris buffer (pH 7.5). The mixture was vortexed for 30 s and then centrifuged. The hexane layer was removed and washed twice with 1 ml of deionized water. Samples were then concentrated to 200 µl and transferred to vials for GC-MS analysis. Standard curves for NPO and NPQ adducts were prepared by adding various amounts of a NPO1-NAC, NPO2-NAC, 1,2-NPQ-NAC, and 1,4-NPQ-NAC to 2.5 mg portions of untreated human Alb (Sigma) or 5 mg portions of untreated human Hb (Sigma) and then performing the assay as described above for the experimental samples.

GC/NICI-MS analysis.
All GC-MS analyses were conducted in negative-ion chemical ionization (NICI) mode using a Hewlett-Packard 5890 series II plus gas chromatograph equipped with a Hewlett-Packard 5989B MS engine. A DB-5 capillary column (60 m, 0.25-mm i.d, 0.25-µm phase thickness; J & W Scientific, Inc.) was used; 2-µl samples were injected in the splitless mode with a He carrier gas flow of 1.5 ml/min. Methane (source pressure of 2 Torr) was used as the chemical ionization reagent gas.

Analysis of NPO1-S-TFA and NPO2-S-TFA.
The injection port and source temperatures were 250 and 100°C, respectively. The oven temperature was held for 2 min at 75°C, then increased at 4°C/min to 160°C where it was held for 15 min, then increased at 50°C/min to 260°C where it was held for 15 min. The molecular ions of NPO1-S-TFA and NPO2-S-TFA (m/z 256) and [2H6]NPO1-S-TFA and [2H6]NPO2-S-TFA (m/z 263) were monitored using the selected ion monitoring mode as described in Waidyanatha et al. (in press).

Analysis of 1,2-NPQ-S-TFA and 1,4-NPQ-S-TFA.
The injection port and source temperatures were 250 and 150°C, respectively. For 1,2-NPQ-Alb, 1,4-NPQ-Alb and 1,4-NPQ-Hb, the oven temperature was held for 2 min at 75°C, then increased at 6°C/min to 150°C where it was held for 28 min, then increased at 50°C/min to 260°C where it was held for 15 min. For 1,2-NPQ-Hb, the oven temperature was held for 2 min at 75°C, then increased at 6°C/min to 145°C where it was held for 35 min, then increased at 50°C/min to 260°C where it was held for 10 min. The molecular ions of 1,2-NPQ-S-TFA and 1,4-NPQ-S-TFA (m/z 383) and [2H5]1,2-NPQ-S-TFA and [2H5]1,4-NPQ-S-TFA (m/z 388) were monitored using the selected ion monitoring mode.

Statistical analysis.
All statistical analyses were conducted using SAS Statistical Software (Cary, NC). Data obtained prior to peak adduct levels were excluded from linear and nonlinear regression analyses. After peak adduct levels have been obtained, it can be assumed that adduct formation does not significantly contribute to changes in adduct concentration and that the rate of change of adduct concentration is a function of adduct removal only. Standard errors were estimated for all parameters.

Estimation of rates of adduct elimination and adduct instability.
Rates of elimination and instability of Alb and Hb adducts were estimated as described in Troester et al. (2000, 2001), respectively. We define A(t) = [RY]/[Y] as the amount of adduct RY (nmol) per g of protein (i.e., NPO1-Y, NPO2-Y, 1,2-NPQ-Y, or 1,4-NPQ-Y for Y = Hb or Alb) at some time t (d) after administration of naphthalene. Because the turnover rates of Alb and Hb follow different kinetics, they were modeled differently as described below.

Alb adducts.
Following a single dose of naphthalene, Alb adducts are eliminated by first-order kinetics according to the following expression:


((1))
where A0 is the adduct concentration at t = 0 and k' (d-1) is the first-order rate constant for adduct elimination. (For a review of the derivation of this model, see Granath et al., 1992Go.) From Equation 1Go, the relationship between ln[A(t)] and t is linear with intercept ß0 = ln(A0) and slope ß1 = -k', that is,


((2))
and k' can be estimated by linear regression of ln A(t) on t. The rate of adduct elimination (k') is the sum of the species-specific rate constant of Alb turnover (kAlb in d-1) and the rate constant of adduct instability (k in d-1). That is,


((3))
For stable adducts, k = 0 and k' is equal to kAlb. Thus, adduct stability can be evaluated by comparing estimated values of k' to known or estimated values of kAlb.

Independent estimates of k' were obtained for each Alb adduct in rats following administration of either 400 or 800 mg naphthalene per kg body weight. To determine whether k' differed between the 2 dose groups, a 95% confidence interval for the difference between the two estimates of k' was calculated using the multiple regression model:


((4))
, where E is the error component of the model, and Z is a dummy variable defined as 0 for adducts from the 400 mg/kg dose group and 1 for adducts from the 800 mg/kg dose group. Thus, the straight line models for estimating k' at 2 different doses are incorporated into Equation 4Go. When Z = 0 (400 mg/kg dose),


((5))
, from which k' = -ß1. When Z = 1 (800 mg/kg dose),


((6))
, from which k' = -(ß1 + ß3). The estimated difference between the two k' values is therefore given by the coefficient 3 and the significance of this difference can be judged by the 95% confidence interval for ß3.

Hb adducts.
Following a single dose of naphthalene, the elimination of Hb adducts can be described by mixed zero- and first-order kinetics according to:


((7))
, where ter (d) represents the lifetime of the erythrocyte and k (d-1) represents the rate constant of adduct instability. (For a review of the derivation of this model, see Granath et al., 1992Go and Troester et al., 2001Go.) Since Equation 7Go is nonlinear with respect to time, estimates of A0 and k were obtained by nonlinear least-squares regression of adduct concentration on time (Troester et al., 2001Go). However, preliminary inspection indicated that adduct concentrations exhibited heterogeneity of variance at the different times of sacrifice. Thus, we employed generalized least squares using a power function that models the variance of adduct concentration as a function of the mean response at each time point. (For a discussion of generalized least squares and the power function, see Davidian and Gilitinan, 1995Go.) Since generalized least squares is an iterative model-fitting process, we began by assuming that the variance of adduct concentration was constant across time and used nonlinear least-squares regression along with Equation 7Go to estimate the parameters A0 and k and to predict values of A(t). Then, we used the predicted value of adduct level at time t, Â(t), to assign a weight (wt) to each observation (at each time t) in a second iteration according to the following power function:


((8))
.

Iterations were conducted 4 times for NPO1-Hb adducts and 5 times for NPO2-Hb (with wt recalculated using Equation 8Go after each iteration); the last two iterations in each case yielded identical estimates and confidence intervals for A0 and k.

The value of ter was specified a priori using strain-specific estimates of ter for the F344 rat (Derelanko, 1987Go). The estimate of ter for F344 rats is associated with some uncertainty, so nonlinear regression using the approach above was conducted using the point estimate of ter (66 days) for the strain, as well as both the upper and lower 95% confidence interval values for ter (62 days and 70 days, respectively).

In these experiments, the concentration of Hb adducts was followed for 6 weeks following administration. As young animals increase in size, their blood volume increases proportionally and can dilute Hb adducts (Osterman-Golkar et al., 1999Go). In our study, animals increased in body weight from 261 g (SE = 2.9 g, n = 6) at dosing to 301 g (SE = 3.7 g, n = 6) 42 days after dosing. Thus, for regression of Hb adducts via Equation 7Go, the body-weight (b.w.) adjusted adduct level [A(t)adj in nmol/g] was calculated according to the following relationship (Troester et al., 2001Go):


((9))
. Then, nonlinear regression of A(t)adj on time (d) was performed according to Equation 7Go as described above.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Stability of Alb Adducts
The elimination of NPO-Alb, following administration of a single dose of naphthalene (400 mg/kg or 800 mg/kg) is shown in Figure 2Go. In both dose groups, NPO-Alb was detectable through day 14, so 4 data points (t = 1, 2, 7, 14 days) were used for regression analysis. Regression of ln A(t) on time yielded the parameter estimates summarized in Table 1Go. The estimated rate constant of adduct elimination (k') ranged from 0.30–0.38 for the structural isomers of NPO-Alb (NPO1-Alb and NPO2-Alb); these rates correspond to half-lives of approximately 2 days. There was no significant difference between dose groups for estimates of k' obtained for NPO1-Alb (p = 0.43) or NPO2-Alb (p = 0.48).



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FIG. 2. Elimination of NPO1-Alb (squares) and NPO2-Alb (circles) adducts following administration of (A) 400 and (B) 800 mg naphthalene per kg of body weight. Four time points were included in the regression analysis (Equation 2Go) for NPO1-Alb and NPO2-Alb, with n = 3 for all time points except for the 14 day time point in B where n = 2. Lines represent the equations obtained from the least squares regressions of ln(NPO-Alb concentration) on time.

 

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TABLE 1 Parameter Estimates for the Elimination of NPO-Alb Adducts (Equation 2Go)
 
The Alb adducts of the NPQs were less stable than those of NPO. The turnover of NPQ-Alb is depicted in Figure 3Go. In both dose groups, 1,2-NPQ-Alb concentrations were not significantly different from the controls on day 14 (p = 0.43 and p = 0.14 for 400 mg/kg and 800 mg/kg dose groups, respectively). Thus, linear regression of ln A(t) on time was conducted using data from only 3 time points (t = 1, 2, and 7 days). The estimates of k' (0.73–0.74 d-1) obtained for 1,2-NPQ-Alb did not differ significantly by dose group (p = 0.95) with a half-life of less than 1 day in rats (Table 2Go). As shown in Figure 3Go, levels of 1,4-NPQ-Alb were indistinguishable from controls by day 7, so there were insufficient time points to estimate k' for this adduct. However, based upon the levels of 1,4-NPQ-Alb at days 1 and 2 after dosing (Table 3Go), the half-life of 1,4-NPQ-Alb would be much less than 1 day.



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FIG. 3. Elimination of 1,2-NPQ-Alb (squares) and 1,4-NPQ-Alb (circles) adducts following administration of (A) 400 and (B) 800 mg naphthalene per kg of body weight to male F344 rats. Three time points were included in the regression analysis (Equation 2Go) for 1,2-NPQ-Alb, with n = 3 at each time point. Lines represent the equations obtained from the least squares regressions of ln(1,2-NPQ-Alb concentration) on time.

 

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TABLE 2 Parameter Estimates for the Elimination of 1,2-NPQ-Alb Adducts (Equation 2Go)
 

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TABLE 3 Concentration of 1,4-NPQ-Alb at Time t Following Administration of Naphthalene to Male F344 Rats
 
Stability of Hb Adducts
The estimates Â0 and from Equation 7Go are shown in Table 4Go for body-weight adjusted NPO1-Hb and NPO2-Hb, following administration of 400 mg/kg naphthalene. For NPO1-Hb, the estimates of k were small and positive throughout the range of ter for the F344 rat. All 95% confidence intervals for k excluded 0, providing statistical evidence of adduct instability. Conversely, there was no statistical evidence of adduct instability for NPO2-Hb, since the value of ranged from negative (given ter = 62, 66 d) to positive (given ter = 70 d), with all 95% confidence intervals including 0. (A negative estimate of k would suggest that adducts decay more slowly than would be expected from simple protein turnover. However, these negative estimates of k for NPO2-Hb were extremely small and not significantly different from 0.) Figure 4Go illustrates the body weight-adjusted NPO1-Hb and NPO2-Hb concentrations for each animal, the corresponding fitted curves (Equation 7Go), and a hypothetical curve that assumes stable adducts (k = 0).


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TABLE 4 Estimated Parameters from Weighted Nonlinear Regression (Equation 7Go) of Body-Weight Adjusted NPO1-Hb and NPO2-Hb Concentration on Time following Administration of 400 mg/kg Naphthalene to F344 Rats
 


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FIG. 4. Elimination of (A) NPO1-Hb and (B) NPO2-Hb adducts following administration of 400 mg naphthalene per kg body weight to male F344 rats. Each data point represents the body-weight adjusted concentration of adducts (nmol/g Hb) for a single animal. In A, the heavy line represents the fitted nonlinear model (Equation 7Go) where ter = 66 days, = 9.6 x 10-3 d-1, and Â0 = 449.6 nmol/g; the lighter line represents a theoretical linear, zero-order (in k) model where ter = 66 days, k = 0 d-1, and A0 = 449.6 nmol/g. In B, the heavy line represents the fitted nonlinear model (Equation 7Go) where ter = 66 days, = -1.3 x 1 align="center"0-3 d-1, and Â0 = 76.8 nmol/g; the lighter line represents a theoretical linear, zero-order (in k) model where ter = 66 days, k = 0 d-1, and A0 = 76.8 nmol/g.

 
Estimates of NPO-Hb adduct instability were not obtained following administration of 800 mg/kg naphthalene because the variability among animals at each time point was extremely large and a trend of adduct decay was not discernable. Likewise, rates of instability could not be estimated for 1,2-NPQ-Hb and 1,4-NPQ-Hb due to the low concentrations and rapid disappearance of these adducts at both doses (Table 5Go). In the 400 mg/kg dose group, the measured concentrations of 1,2-NPQ-Hb and 1,4-NPQ-Hb were significantly different from controls only on day 1 (p = 0.03 and 0.003, respectively). In the 800 mg/kg dose group, the measured concentrations of 1,2-NPQ-Hb were significantly different from controls only on days 1 (p = 0.002) and 2 (p = 0.02) and 1,4-NPQ-Hb was significantly different from controls only on day 1 (p = 0.01).


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TABLE 5 Concentrations of 1,2-NPQ-Hb and 1,4-NPQ-Hb at Time t following Administration of 400 or 800 mg Naphthalene per kg Body Weight to F344 Rats
 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The kinetic theory of Alb adducts assumes that both stable and unstable adducts are turned over according to first-order processes (Equation 1Go). However, nonlinearity was observed in the relationship between ln A(t) and time for all Alb adducts measured in this study. This is in contrast to our previous study of benzene-derived Alb adducts formed from the cysteinyl reactions of BO and 1,4-BQ with Alb (BO-Alb and 1,4-BQ-Alb, respectively), where no lack of fit was observed from the linear first-order model (Troester et al., 2000Go). Many previous studies have also observed linear first-order decay of Alb adducts (Cho et al., 1994Go; DeBord et al., 1992Go; Sabbioni et al., 1987Go; Viau et al., 1993Go), although one study of the stability of pentachlorophenol adducts on Alb did report what appeared to be biphasic decay of Alb adducts (Waidyanatha et al., 1996Go). This suggests that the elimination of some Alb adducts may involve a more complicated mechanism than permitted by current theory. Justification for an alternative model would require more time points than were available in this study, so we estimated first-order rate constants of Alb adduct instability, recognizing that this could introduce some biases. From Figures 2 and 3GoGo it appears that these biases would not be great.

The first-order rate constants (k') for adduct turnover indicate that NPO-Alb is more stable than NPQ-Alb. This is consistent with results from a similar stability study involving cysteinyl Hb and Alb adducts of BO and 1,4-BQ (only the mono-S-substituted adduct was measured), where BO-Alb was more stable than 1,4-BQ-Alb (Troester et al., 2000Go). In that study, we speculated that glutathione or other protein sulfhydryls react with the mono-S-substituted 1,4-BQ-Alb to form a multi-S-substituted adduct, accounting for the apparent instability. (Previous studies have shown that chemical oxidation of hydroquinone [HQ] in the presence of glutathione results in multi-S-substituted conjugates [Eckert et al., 1990Go, Lau et al., 1988Go], and multi-S-substituted conjugates of HQ have been identified as in vivo metabolites of HQ [Hill et al., 1993Go].)

However, if reactivity of the quinones were solely responsible for the instability of quinone-protein adducts, we would expect NPQ adducts to be more stable than the corresponding BQ adducts. That is, Michael addition occurs at the ß position of an {alpha}, ß-unsaturated ketone due to a partial positive charge at that position, which diminishes as the number of aromatic rings increases because of delocalization of electron density. The oxidation potentials of NPQs are also expected to be decreased relative to BQs, due to greater delocalization of the unpaired electron in the ortho-semiquinone radical associated with the larger ring system (McCoull et al., 1999Go). Previous studies have shown that the rate of nucleophilic addition is slower with the ortho-quinone of benzo[a]pyrene than with 1,2-NPQ (Murty and Penning, 1992Go). To the contrary, we observed a turnover rate for 1,2-NPQ-Alb (Table 2Go) more than twice that of 1,4-BQ-Alb (half-life of 2.5 days [Troester et al., 2000Go]), and the elimination of 1,4-NPQ-Alb (Fig. 3Go) appears to be even faster. At present, very little is understood about how structural characteristics of bound metabolites influence the stability of protein adducts.

The NPO-Alb adducts also appeared to be less stable than BO-Alb adducts (' = 0.225 d-1, 1/2 = 3.1 d [Troester et al., 2000Go]). NPO-Alb adducts were turned over with first-order rate constants that ranged from 0.30 to 0.38 d-1 (Table 1Go). Assuming that the BO-Alb represents a stable adduct, then Alb = 0.23 d-1 in the particular F344 rats used in our studies, from which we estimate 0.07 <= <= 0.15 d-1 for NPO-Alb (Equation 3Go). Interestingly, this range of for NPO-Alb is not consistent with values predicted for NPO-Hb (Table 4Go), which indicated no evidence of instability in vivo. This is not the first study to demonstrate a lack of agreement between the stability of Hb and Alb adducts (e.g. ortho-toluidine [DeBord et al., 1992Go], benzo[a]pyrenediolepoxide [Viau et al., 1993Go], and naphthalene [Cho et al., 1994Go]), although these previous studies did not characterize metabolite- and amino acid-specific adducts.

To our knowledge, the dose-dependence of protein adduct stability has not been tested previously. In this study, the dose of naphthalene did not appear to have a significant effect on the stability of naphthalene-derived Alb adducts. Statistical comparisons of the rate constants obtained for NPO-Alb, 1,2-NPQ-Alb, and 1,4-NPQ-Alb at 2 different doses (400 mg/kg and 800 mg/kg) demonstrated no significant difference in adduct stability.

NPO1-Hb adducts were only slightly unstable and NPO2-Hb adducts were stable following administration of 400 mg/kg naphthalene. However, the stability of NPO-Hb could not be investigated following an 800 mg/kg dose of naphthalene due to high variability in adduct concentration among animals at each time point. We suspect that the variability that is apparent in Hb adduct concentration, but not Alb adduct concentration, is a result of the greater toxicity of naphthalene on the hematopoeitic (site of Hb production) system relative to the hepatic system (site of Alb production). Hematopoietic effects are common following administration of a wide variety of chemicals due to the involvement of several organs (including bone marrow, thymus, lymph nodes, spleen, liver, stomach, intestines, and kidney among others) in the production of red blood cells. Toxicity to any of these organs could alter the concentration of Hb or the lifetime of the red blood cell (ter), thereby increasing variability in adduct levels. In fact, naphthalene has been shown to cause hemolytic anemia in humans, though rodents may be less susceptible to these effects (NTP, 2000Go). Therefore, in spite of our inability to document instability of NPO-Hb following an 800 mg/kg dose, we suspect that these adducts will not be useful indicators of exposure at doses of naphthalene that induce acute toxicity.

It was once believed that only stable Hb and Alb adducts were useful for exposure assessment. More recently, relationships between unstable adducts and blood concentrations of electrophiles have been thoroughly described and equations have been published that require estimates of k to calculate the mean daily blood concentration of electrophile (Granath et al., 1992Go, Troester et al., 2001Go). These equations account for differences in kAlb and ter between species, but the validity of these equations ultimately depends upon the assumption that k is similar across species. Direct estimates of k in humans are seldom available. In spite of the instability observed for some of the naphthalene-derived adducts, these adducts are still more persistent than the reactive electrophiles from which they are formed and are useful in estimating the systemic doses of these metabolites.


    ACKNOWLEDGMENTS
 
This work was supported in part by the National Institute of Environmental Health Sciences through grants P42ES05948, T32ES07018, and P30ES10126. The research described in this document was also supported in part by the U.S. Environmental Protection Agency.


    NOTES
 
1 To whom correspondence should be addressed. Fax: (919) 966-0521. E-mail: stephen_rappaport{at}unc.edu. Back

The research described in this document was supported in part by the U.S. Environmental Protection Agency. It has been subjected to Agency review and approved for publication. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.


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