NAPHTHALENE-INDUCED RESPIRATORY TRACTTOXICITY: METABOLIC MECHANISMS OF

TOXICITY

A. Buckpitt,* B. Boland, M. Isbell, D. Morin, M. Shultz,

R. Baldwin, K. Chan, A. Karlsson, C. Lin, A. Taff, J. West,

M. Fanucchi, L. Van Winkle, and C. Plopper

Departments of Molecular Biosciences and Anatomy, Physiology, and

Cell Biology, School of Veterinary Medicine, University of California,

Davis, CA 95616

ABSTRACT

The lung, which is in intimate contact with the external environment, is

exposed to a number of toxicants both by virtue of its large surface area and

because it receives 100% of the cardiac output. Lung diseases are a major

disease entity in the U.S. population ranking third in terms of morbidity and

mortality. Despite the importance of these diseases, key issues remain to be

resolved regarding the interactions of chemicals with lung tissue and the

factors that are critical determinants of chemical-induced lung injury. The

importance of cytochrome P450 monooxygenase dependent metabolism in

chemical-induced lung injury in animal models was established over 25 years

ago with the furan, 4-ipomeanol. Since then, the significance of

biotransformation and the reasons for the high degree of pulmonary selectivity

for a myriad of different chemicals has been well documented, mainly in

rodent models. However, with many of these chemicals there are substantial

differences in the susceptibility of rats vs. mice. Even within the same species,

varied levels of the respiratory tract respond differently. Thus, key pieces of

791

DOI: 10.1081/DMR-120015694 0360-2532 (Print); 1097-9883 (Online)Copyright q 2002 by Marcel Dekker, Inc. www.dekker.com

*Corresponding author. Alan Buckpitt, Department of Molecular Biosciences, School of Veterinary

Medicine, University of California, Davis, CA 95616. E-mail: arbuckpitt@ucdavis.edu

DRUG METABOLISM REVIEWS

Vol. 34, No. 4, pp. 791–820, 2002

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data are still missing when evaluating the applicability of data generated in

rodents to primates, and as a result of this, there are substantial uncertainties

within the regulatory community with regards to assessing the risks to humans

for exposure to some of these chemicals. For example, all of the available data

suggest that the levels of cytochrome P450 monooxygenases in rodent lungs

are 10–100 times greater than those measured in the lungs of nonhuman

primates or in man. At first glance, this suggests that a significant margin of

safety exists when evaluating the applicability of rodent studies in the human,

but the issues are more complex. The intent of this review is to outline some of

the work conducted on the site and species selective toxicity and metabolism

of the volatile lung toxic aromatic hydrocarbon, naphthalene. We argue that a

complete understanding of the cellular and biochemical mechanisms by which

this and other lung toxic compounds generate their effects in rodent models

with subsequent measurement of these cellular and biochemical events in

primate and human tissues in vitro will provide a far better basis for judging

whether the results of studies done in rodent models are applicable to humans.

Key Words: Naphthalene; Clara cell toxicity; CYP2F; Covalent binding;

Naphthalene epoxide; Naphthoguinone; Lung; Human; Rodent cytochrome

P450

INTRODUCTION

The respiratory system provides a critical interface with the environment, and as

a result, it is an important portal of entry for volatile chemicals and gases. In addition,

the lung receives the entire cardiac output from the right side of the heart and thus is

exposed to chemicals via the circulation. Nononcogenic and oncogenic pulmonary

diseases are a leading cause of death and are a major factor in morbidity and disability

in the United States.[1] While cigarette smoking is an important contributor to these

diseases, other environmental factors including exposure to chemicals in the air, food,

and drinking water are likely to play a role in these disease processes. The

identification of specific factors important in the etiology of human pulmonary

diseases is a difficult and uncertain process for several reasons. The known variations

in response of different rodent species to pulmonary toxicants requiring bioactivation

results in significant uncertainties in the extrapolation of the experimental studies

conducted in animal models to the human. In addition, good methods for

distinguishing between exposures that are likely to disrupt cellular homeostasis in the

respiratory tract from those that are inconsequential are not currently available. There

are several potential approaches to address these issues all of which require a good

understanding of biochemical and cellular events that occur in target cells and that are

critical to the injury process. Once these are well understood, biomarker approaches,

which indicate that an exposure has occurred at sufficient intensity and of sufficient

duration to result in events that are likely to result in untoward effects in the cell, can

be developed and carefully validated in animal models. The overall intent would be to

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then apply these biomarkers to exposed human populations. A second approach

would involve the development of in vitro methods capable of monitoring those

biochemical/cellular events that are clearly tied to toxicity in animal models with the

intent of applying these to human or other primate tissues. Although our current

understanding of the mechanisms involved in the toxicity and potential oncogenicity

of naphthalene is not complete, there has been considerable progress in delineating

some of the key issues involved in these processes in animal models. Accordingly, the

intent of this manuscript is to outline some of these approaches and to review what is

known about the metabolic/cellular mechanisms of toxicity of naphthalene. In the

process, we will identify critical gaps in our knowledge where additional data would

be useful in refining our assessments for potential toxicity in the human.

ENVIRONMENTAL OCCURRENCE—POTENTIAL FOR HUMANEXPOSURE

Naphthalene, a slightly water soluble (32 mg/L) volatile aromatic hydrocarbon,

is present in both groundwater and air emissions from a variety of sources.[2]

Naphthalene and close structural congeners are prominent contaminants of old coal

and oil gasification sites; the concentrations of naphthalene are more than 10 fold

higher than any of the other polycyclic aromatic hydrocarbons (PAH) identified in

ambient air above the site.[3] Over 3000 such sites have been identified and many are

in heavily populated urban areas.[4] In a recent survey of hazardous waste sites,

naphthalene was listed as present in 47 of the 300 sites surveyed. Naphthalene has

been detected in urban air (average concentration of 0.9mg/m3)[5] and is also a

prominent pyrolysis product of both mainstream and side stream tobacco smoke.[6,7]

The fact that naphthalene has been detected in nearly 40% of the fat samples tested[8]

and in 75% of breast milk samples[9] obtained from humans argues that the U.S.

population is exposed to this compound. Data demonstrating the relatively rapid

metabolism of naphthalene in human liver microsomes[10] argue that the clearance of

this compound in humans is likely to be quite rapid. Despite the high fat/blood

partition ratio for naphthalene, these findings suggest that a considerable portion of

the population is likely exposed to low concentrations of this compound on a frequent

basis. Additional information on the sources of potential human exposure to

naphthalene can be obtained in a very thorough document released by the Agency for

Toxic Substances Disease Registry (ATSDR).[2]

VARIATION IN THE RESPONSE OF RODENTS TO CHEMICAL-INDUCED INJURY TO THE RESPIRATORY TRACT

A number of compounds including dichloroethylene,[11,12] methylene

chloride,[13,14] naphthalene, and close structural analogs of naphthalene (2-

methylnaphthalene),[15] when administered intraperitoneally (ip), result in highly

NAPHTHALENE-INDUCED RESPIRATORY TRACT TOXICITY 793

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selective injury to mouse but not rat lung. Again, this clearly underscores the need

to understand the mechanisms by which each of these chemicals elicits untoward

effects in the organism. For example, the selective carcinogenic actions of

methylene chloride in mouse lung have been related to the metabolic activation of

the parent compound by theta class glutathione S-transferase. The finding of very

low theta glutathione S-transferase activities in human lung[16] and that very little

of this theta class enzyme is present in the nucleus of cells in human lung[13] has

been used to argue that this compound is unlikely to be a carcinogen in human

pulmonary tissue.[13] Although these views have been challenged,[14] the fact that

the mouse is highly susceptible to methylene chloride carcinogenicity and that the

differential susceptibility between mice and rats is related to metabolism certainly

raises some concerns regarding the applicability of data derived in mouse

bioassays with methylene chloride to the human.

Acute Pulmonary Injury by Naphthalene

The pulmonary toxicity of naphthalene has been studied extensively by both

our laboratories[17 – 19] and by others.[20 – 22] Clara cells (nonciliated bronchiolar

epithelial cells) lining the airway epithelium of the mouse are the primary target

cells for naphthalene toxicity, irrespective of the route of administration. One of

the most striking features of naphthalene cytotoxicity is the high degree of tissue

and species selectivity. After parenteral administration of low doses of the

compound, the only tissue affected is the respiratory tract (Table 1). Hepatic

necrosis is not observed at any dose of naphthalene tested, while proximal tubular

cells of the kidney are injured only in some mouse strains and only at very high

doses (400 and 600 mg/kg, ip).[21] Swelling of Clara cells in terminal airways is

detected in mice at doses as low as 50 mg/kg, ip. In contrast, in rats even at LD50

doses (1600 mg/kg, ip), airway Clara cells are apparently normal. There is some

swelling of Clara cells in the hamster at the LD50 dose (800 mg/kg, ip). Thus, there

are fundamental differences between these species that can be used as a tool to

determine the relevance of particular metabolites and metabolite-macromolecular

adducts in the processes leading to cytotoxicity.[17] In all of the species tested, no

injury to the alveolar type I or II cells has been observed. In addition to the lesions

observed in the lung, the olfactory region of the nose is sensitive to naphthalene

after intraperitoneal administration[17] in both the mouse and the rat. The rat nasal

olfactory epithelium is injured at lower doses of naphthalene (200 mg/kg, ip) than

the doses required to produce injury in the nasal epithelium of the mouse

(400 mg/kg, ip) (Table 1). Finally, more recent studies investigating the sex and

strain differences in susceptibility to naphthalene toxicity indicate that the female

Swiss Webster mouse is more susceptible to the cytotoxicity of naphthalene than is

the male.[23] As discussed later, the National Toxicology Program (NTP) bioassay

investigating the possible neoplastic effects of naphthalene showed a sex

difference in susceptibility with female mice showing a slight increase in

bronchioloalveolar neoplasms over control whereas in males there was no effect.

BUCKPITT ET AL.794

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Additional work examining the acute toxicity of naphthalene administered

by inhalation has been completed.[24] Four hour exposures at concentrations as

low as 2 ppm results in detectable Clara cell injury in adult mice[24] but no injury to

Clara cells in rat lung. In mice, 4-hr exposures conducted at concentrations equal

to the current 8-hr human occupational exposure standard (10 ppm, TWA)[25]

produce substantial injury to cells in both the upper and lower respiratory tract of

the mouse (Table 1).

In addition to the species, cell, and tissue variations in susceptibility to

naphthalene induced injury, there are regional differences in susceptibility of the

airway epithelium that are dependent upon route of administration. Clara cells of

murine terminal airways are most susceptible to low doses of naphthalene

administered parenterally (Table 1).[17] Doses of 200 mg/kg and above are required

to observe toxicity in more proximal airways. Although this airway level difference

in susceptibility has been attributed to variations in rates of metabolism to the

epoxide,[26,27] data obtained from the inhalation experiments suggest that this

differential susceptibility may be due to differences in distribution of the parent

compound. After inhalation exposures, cells of the more proximal airways are

injured at lower concentrations than cells of the distal airways (bronchioles) while

higher inhaled concentrations are required to produce injury in terminal airways.

These findings are consistent with the slight water solubility of naphthalene.

While the parenteral doses used in these studies far exceed those likely to be

encountered by human populations via other routes of exposure, the fact that the

injury is localized to the epithelial cells of the respiratory tract after systemic

administration of the compound strongly supports the view that there is an

underlying biochemical basis for this injury. Numerous studies conducted over the

past 15 years have provided considerable insight regarding the initial metabolic

steps necessary for toxicity, but it is clear that additional work is needed to fully

understand the intracellular events that lead to cell death. With inhalation exposures,

Clara cell toxicity is observed in the mouse at exposure concentrations that are

plausible for human occupational exposures. Again, whether these exposure

concentrations are capable of producing any untoward effects in humans is not

known and a clear understanding of the mechanism of injury along with the

development of biomarkers that are capable of indicating exposures that are at levels

likely to result in untoward effects, is important. In addition, testing of naphthalene

for acute toxicity in animal models, which more closely resemble the human also

may provide data useful in assessing the potential toxicity of naphthalene as well as

its close structural analogs (methylnaphthalenes and nitronaphthalenes).

Tolerance to Multiple Naphthalene Exposures

In contrast to the Clara cell toxicity observed after single doses of

naphthalene, multiple daily treatments with naphthalene by both the

intraperitoneal and inhalation routes result in tolerance to high challenge doses

NAPHTHALENE-INDUCED RESPIRATORY TRACT TOXICITY 795

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Table 1. Species, Tissue, and Regional Differences in Naphthalene Toxicity

Lung Nasal Epithelium

Species Dose Route

Trachea/Lobar

Bronchus

Terminal

Bronchiole Parenchyma Olfactory Respiratory Comments

Mouse, adult male

LD50 ¼ 380 mg=kg

50 mg/kg ip 0 þ 0 0 0 No toxicity noted in liver or

kidney of male Swiss Webster

mice; ICR mice showed

lesions of proximal tubule at

highest dose (400 and

600 mg/kg)[17,21]

100 mg/kg ip 0 þþ 0 0 0

200 mg/kg ip þ /0 þþþ 0 0 0

300 mg/kg ip þþ þþþþ 0 ND ND

400 mg/kg ip þþþ þþþþ 0 þþþ 0

2–5 ppm ip þ /0 0 0 ND ND Ref. [24]

8.5–11.5 ppm inh þ þ /0 0 ND ND

25–31 ppm inh þþ þ 0 ND ND

72–77 ppm inh þþþ þþþ 0 ND ND

96–111 ppm inh þþþ þþþ 0 ND ND

Mouse, adult

tolerance

200 mg/kg £ 7a ip ND 0 ND ND ND Areas of bronchiolar epithelial

cell hyper-plasia observed

after 7 days, data from

Refs. [28,29]

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200 mg/kg £ 7 þ 300 mg/kg

(24 hr)

ip ND 0 ND ND ND

200 mg/kg £ 7 þ 300 mg/kg

(48 hr)

ip ND þ ND ND ND

200 mg/kg £ 7 þ 300 mg/kg

(96 hr)

ip ND þþþ ND ND ND

200 mg/kg £ 7 þ 300 mg/kg

(144 hr)

ip ND þþþþ ND ND ND

Rat, adult male

LD50 ¼ 1600 mg=kg

200 mg/kg ip 0 0 0 þþþ 0 Data from Ref. [17]

400 mg/kg ip 0 0 0 þþþ 0

800 mg/kg ip 0 0 0 þþþ 0

1600 mg/kg ip 0 0 0 þþþ 0

0–100 ppm inh 0 0 0 ND ND

Hamster, adult male

LD50 ¼ 800 mg=kg

200 mg/kg ip 0 0 0 0 0

400 mg/kg ip 0 0 0 þþþ 0

800 mg/kg ip þ 0 0 þþþ 0

ND—not determined; 0 ¼ no detectable injury; þ!þþþþ indicates increasing severity of injury as assessed by high resolution light microscopy.a Mice were treated with naphthalene at a dose of 200 mg/kg/day for 7 days and then were treated with vehicle or a challenge dose of naphthalene (300 mg/kg) at

a time after the last tolerant dose as specified in parenthesis.

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of compound.[28 – 30] As discussed earlier, substantial injury is observed in Clara

cells of mice treated with a single 200 mg/kg dose, ip. In comparison, light

microscopic and morphometric evaluation of airway epithelium from mice treated

daily with 200 mg/kg naphthalene, ip for 7 days showed only slight hyperplasia of

the epithelium in comparison to animals treated with vehicle alone[29] (Table 1). In

addition, this pretreatment regimen markedly decreased the susceptibility of the

epithelium to a large challenge dose of naphthalene (300 mg/kg, ip) when the

challenge dose was administered 24 hr after the last of the seven 200 mg/kg doses

(Table 1). As the time was extended between the last 200 mg/kg dose and the

challenge dose from 24 to 96 hr, the lungs regained a portion of their sensitivity to

the 300 mg/kg challenge dose. These data, showing that the lung becomes tolerant

to multiple doses of naphthalene at dose levels that produced substantial toxicity in

airway epithelial cells after single administration are consistent with the 14-day

and 90-day oral gavage studies. This work demonstrated no significant alterations

in serum enzyme levels, body weight, organ weight, or various indices of immune

function in CD-1 mice treated daily with doses up to 267 mg/kg (14 day) or

133 mg/kg (90 day).[31]

Carcinogenesis Studies

Chronic inhalation exposure of mice to naphthalene (10 or 30 ppm) results in

inflammation in the nose, metaplasia of the olfactory epithelium, and hyperplasia

of the respiratory epithelium.[32,33] No neoplastic effects were noted in male mice,

but a slight increase in alveolar/bronchiolar adenomas and alveolar/bronchiolar

carcinomas were noted at the highest exposure level in female mice. These results

are consistent with our findings that nasal olfactory epithelial cells and bronchiolar

epithelial cells are targets in the mouse after acute exposures to naphthalene and

that administration of the compound daily at high doses over a 7-day period results

in hyperplasia of the respiratory epithelium in the terminal airways.[29]

Recently, the NTP 2-year bioassay has been completed in rats. Animals were

exposed to vapor concentrations of 0, 10, 30, and 60 ppm. The nasal epithelium

was found to be a primary target for these exposures.[34,35] A concentration-

dependent increase in adenomas of the respiratory epithelium of the nose and of

neuroblastomas of the olfactory epithelium are considered to be significant in

these rat studies and have raised concerns about naphthalene as a potential human

carcinogen. Slight hyperplasia of the alveolar epithelium was noted but only in

female rats. Finally, inflammation of the olfactory epithelium was observed in

both males and females at all concentrations of naphthalene studied.

The species and regional differences in susceptibility to naphthalene

carcinogenicity noted in the cancer bioassays in both mice and rats correspond to

the differences noted in sensitivity during the acute toxicity studies. Mouse lung

airway epithelium is considerably more susceptible than rat lung airway

epithelium after single ip doses of naphthalene, and female mice are more

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susceptible than males.[23] The olfactory nasal epithelium of both species is highly

susceptible to naphthalene after parenteral administration of the compound with

the rat being more susceptible than the mouse. Since the regional and species

differences in susceptibility to both the cytotoxic and neoplastic effects associated

with naphthalene exposure appear to match, the underlying biochemical

mechanisms that lead to these toxicities may be similar. In the following sections,

the importance of metabolism and the role of various reactive metabolites in

cytotoxicity will be discussed.

METABOLISM AS A KEY DETERMINANT IN NAPHTHALENETOXICITY

Importance of P450-Dependent Metabolism

The Clara cell is an important target cell in the lung for cytotoxic and

genotoxic agents requiring metabolic activation. These include aromatic

hydrocarbons such as naphthalene (and several close structural analogs such as

1-nitronaphthalene), furans such as 4-ipomeanol, and nitrosamines such as 4-

(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK).[18,36,37] The sensitivity of

the Clara cell to these agents is presumed to be related to high rates of metabolic

activation catalyzed by P450 monooxygenases localized within this cell.[38]

Interaction of Reactive Metabolites with Critical Cellular Proteins as aDeterminant in Toxicity

The pioneering work of the Millers on the relationship between electrophiles

and cancer was extended to cytotoxicants by Mitchell, Jollow, and their coworkers

in their studies of the hepatotoxicants, acetaminophen and bromobenzene, in the

early 1970s.[39] Later work by Boyd and others[40] expanded these concepts to

include lung toxicants as well. All of this work has been based on the apparent

close association between overall levels of reactive metabolite binding in a

particular tissue with the incidence and severity of toxicity in that tissue. Reactive

metabolite binding does not a priori result in tissue necrosis, and there are

excellent examples, such as with 3-hydroxyacetanilide, where there is substantial

formation of reactive metabolites in the absence of any toxicity.[41]

Early in vivo studies demonstrated an interrelationship between the covalent

binding of reactive naphthalene metabolites and glutathione depletion with the

extent and severity of cytotoxicity in the lung.[42,43] These studies, like those with

acetaminophen and bromobenzene, showed that only at doses resulting in

significant glutathione depletion were significant covalent binding of reactive

metabolites and toxicity evident. All pretreatments that resulted in alterations in

the severity of bronchiolar necrosis after administration of naphthalene also

altered the levels of reactive metabolite(s) bound covalently to protein in the lung

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in vivo. Specifically, inhibition of the cytochrome P450 monooxygenases

decreased binding and toxicity while glutathione depletion increased toxicity and

covalent binding of reactive metabolites.[42] Moreover, the rates of metabolic

activation of naphthalene to covalently bound metabolites are substantially higher

in preparations of target cells or in isolated tracheobronchial airways from the

mouse than they are in mouse hepatocytes or in rat airways.[44] Likewise, the

extent of glutathione depletion in the lung in vivo and the rates of metabolism of

naphthalene to reactive metabolites in lung microsomal preparations in vitro are

substantially greater in the mouse than in the rat.[21] The importance of glutathione

as a detoxication pathway for naphthalene metabolism is supported by recent

studies showing that increased glutathione synthesis is one of the underlying

reasons why mice treated with high doses of naphthalene daily become tolerant to

large challenge doses of this compound.[30] However, the tissue selective toxicity

of naphthalene is not reflected by tissue selective binding of reactive metabolites

in vivo.[42] The levels of bound metabolite in the liver and kidney were as high as

in the lung. There are several possibilities to explain these data which are

consistent with a role of reactive metabolites in lung injury including: (1) there are

several different reactive metabolites generated from naphthalene and differences

in target and nontarget tissue are related to the chemical nature of bound

metabolites; (2) binding only occurs in a small population of lung cells and thus

measurements of the level of bound metabolites in vivo considerably

underestimate the levels of reactive metabolite bound in target cell populations;

and (3) macromolecules adducted by reactive metabolites in target and nontarget

tissues differ. The corollary to this third possibility is that a protein that is adducted

in the lung but not the liver, is key to the loss of homeostasis in the lung. We have

recently identified several proteins adducted by reactive naphthalene metabolites

and are in the process of determining both the chemical nature of the intermediate

bound and the peptide residues adducted (Isbell et al., unpublished). It is clear

from the work that we have done thus far that there is a high degree of selectivity

for reactive metabolite binding. Although some of the proteins adducted by

reactive metabolites of naphthalene are high abundance proteins, others, which are

present at very low levels in the cell, are also highly labeled.

In summary, a clear understanding of the catalytic activities of the enzymes

involved in both the metabolic activation of naphthalene and detoxification of

naphthalene metabolites in both rodents and in primates will provide a far better

basis for assessing the potential risks of exposure to naphthalene in the human.

Moreover, delineation of the roles of reactive metabolites in processes that lead to

toxicity should provide targets for the development of biomarkers that can be

validated in rodent models and subsequently applied in humans. In the following,

the enzymes responsible for the formation of both the primary and several of the

secondary metabolites of naphthalene will be discussed individually along with

what is known about their potential to result in cytotoxicity. In each case, the

current state of knowledge of species differences in these processes will be

discussed.

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NAPHTHALENE METABOLISM

Overview

The initial and obligate first reaction in naphthalene metabolism involves the

formation of an unstable 1,2-epoxide[45] (Fig. 1). In buffer at pH 7.4, this

metabolite has a half-life of 2–3 min[46] whereas in solutions of albumin the half-

life is considerably greater (11 min).[47] A number of further metabolites can be

generated directly from the epoxide including 1-naphthol (nonenzymatic),

naphthalene dihydrodiol (microsomal epoxide hydrolase), naphthalene diep-

oxides,[48] and glutathione conjugates (glutathione S-transferase).[45] Each of these

secondary metabolites can undergo further biotransformation, and in two of three

cases (1-naphthol and naphthalene dihydrodiol), these biotransformations can

result in more reactive metabolites that have been implicated in both the cytotoxic

and the potential oncogenic actions associated with naphthalene exposure in

animals. The potential for each of these metabolites to produce cytotoxic and/or

oncogenic effects will be discussed in additional detail along with their further

metabolism.

Naphthalene-1,2-epoxide

Recognition of the importance of regio- and stereochemistry in the formation

and toxicology of epoxides and diol epoxides of larger PAH led to development of

methods to assess both the regio- and stereochemistry of formation of these

unstable chemical entities.[49] As with the larger PAH, some of the cytochrome

P450 monooxygenases show remarkable stereoselectivity in the formation of

naphthalene epoxides.[46,50] Cytochrome P450 2B shows a slight preference for

the formation of the (1S,2R )-naphthalene epoxide (74%) whereas cytochrome

P450 1A1 preferentially generates (1R,2S )-epoxide (73–95%) (Fig. 2). Studies

showing marked differences in the formation of glutathione conjugates in lung vs.

liver microsomal incubations containing naphthalene, glutathione, and glutathione

transferases suggested that there might be differences in the stereoselectivity of

naphthalene epoxide formation,[51] but definitive demonstration of this required

additional structural information.[52] Further studies showed the preferential

formation of (1R,2S )-naphthalene epoxide at high rates in lung microsomes and in

isolated dissected airways of the mouse in comparison to the rat[27,53] and

suggested that the stereochemistry of epoxidation might be important in the target

tissue and species selectivity of naphthalene toxicity (Table 2). Although it is

possible that the toxicologic potency of the naphthalene epoxide enantiomers

differ, we felt it was far more likely that differential susceptibility was related to

differences in the rates of formation of the dihydrodiol or glutathione conjugates

(i.e., possible detoxication reactions), which, in turn, would control the residence

time of the epoxide in the cell. Work conducted in isolated hepatocytes appeared

to support this view. Addition of (1S,2R )-naphthalene epoxide to isolated murine

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hepatocytes resulted in significant losses in cell viability while addition of the

(1R,2S )-naphthalene epoxide enantiomer did not.[54] (1R,2S )-Naphthalene

epoxide was metabolized to the dihydrodiol in hepatocytes at much higher rates

than the (1S,2R )-enantiomer suggesting that the longer cellular half-life of the

(1S,2R )-naphthalene epoxide contributes to the higher cytotoxicity observed with

this enantiomer. Definitive analysis of the importance of the stereochemistry of

epoxidation of naphthalene in the lung is problematic because of the instability of

the epoxide. However, short incubations of racemic naphthalene epoxide with

dissected airways of both the rat and mouse and in proximal vs. distal airways

Figure 1. Overview of naphthalene metabolism showing the formation of multiple, reactive

metabolites.

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showed very little difference in the rates of formation of glutathione conjugates or

in the diastereomers produced. Likewise, there were no discernable differences in

the rates of dihydrodiol production between rat and mouse airways that appeared

to relate to the species differences in response to naphthalene.[27] Thus, it appeared

that the primary difference between species might have less to do with the

stereochemistry of epoxide formation than to the rates at which this intermediate

was generated.

Considerable progress has been made in the past 10 years in understanding

the importance of specific pulmonary cytochrome P450 monooxygenase isoforms

in the metabolic activation of lung toxicants. The contribution of each of these

proteins to this process is dependent not only on the amounts of protein present but

also on the catalytic activities of each of the proteins. Unfortunately, quantifying

the amounts of each of the cytochrome P450 isoforms present in various

subcompartments of the lung is difficult and in only a few cases has purified

Figure 2. Stereochemistry of naphthalene epoxide formation and elimination of thioethers in

mouse urine.

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Table 2. Species Comparisons in the Rates of Conversion of Naphthalene to Naphthalene Oxides: Pulmonary and Nasal Tissue

Lung Microsomes Species

Rate of Metabolism (nmol/-

min/mg Protein)

Stereoselectivity Ratio of

(1R,2S ) to (1S,2R )

Vmaxa

(nmol/mg/min)

Kma

(mM )

Diol as a %

Total References

Mouse 13.8b 10 (at saturating substrate

concentrations); 30 at low

concentrations

14 40 8 53

Rat 1.69b 1.3 or less 0.88 10 5 53

Hamster 5.12b 0.5–0.9 1.45 20 25 53

Rhesus

macaque

0.15 0.12 ND ND 21 53

Human 0.12 0.85 ND ND n/ac 86

Nasal post mitochondrial

supernatant (olfactory)

Mouse 87 12.7 ND ND 7.4 53

Rat 43.5 RELSP , 36 ND ND 4.1 53

Hamster 3.9 ND ND ND 7.8 53

ND—not determined.a Apparent Michaelis constants were calculated by using the total rate of formation of diol plus glutathione conjugates.b Incubations were conducted at the same time for comparative purposes and contained microsomes, 0.5 mM naphthalene, 5 mM glutathione, glutathione S-

transferase, and NADPH regenerating system.c The amount of diol generated cannot be calculated because cyclohexene oxide was added to the incubations as an inhibitor of epoxide hydrolase.

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protein been available as standard.[55] More information is available on the

catalytic properties of some of the P450 monooxygenases through the use of

recombinant proteins. Since most toxicant exposure is unintended, toxicants (as

compared to most drug entities) are present in very small quantities. We argue that

a P450 monooxygenase with a Km for a protoxicant in the mM range is unlikely to

contribute to the metabolism of these substrates in vivo, and therefore,

understanding the catalytic efficiencies of these proteins with different substrates

is an important part of understanding the potential for these toxicants to result in

cellular injury. Accordingly, in the following sections we provide a description of

what is known about the catalytic activities of the proteins thought to be important

in the formation of naphthalene epoxides with particular emphasis on the

differences in activity among orthologues across species (Table 3).

Enzymology of Naphthalene Epoxide Formation

CYP2F1 (Human)

Nagata and his coworkers[56] purified a cytochrome P450 monooxygenase

from mouse liver that metabolized naphthalene rapidly and with high

stereoselectivity. Antibodies raised to this protein were used to clone and

sequence the cDNA coding for a 50-kDa protein that was present at high levels in

mouse lung and liver and which had 82% sequence homology[57] to a cDNA that

had been cloned earlier from human lung. The human (CYP2F1) and mouse

(CYP2F2) proteins were expressed in HEPG2 cells and yeast, respectively.

Recombinant CYP2F1 metabolized a number of substrates including ethoxycou-

marin and propoxycoumarin thus indicating that the expressed protein was

catalytically active.[58] Other studies in which CYP2F1 was expressed in

lymphoblastoid cells[59] demonstrated a naphthalene turnover (,0.035 nmol

conjugate/min/nmol P450) that was less than 0.1% the rate of metabolism

observed with the mouse orthologue.[60] The recombinant human CYP2F1 showed

slight stereopreference in the generation of (1S,2R )-naphthalene epoxide. In

contrast, 3-methylindole, another substrate requiring metabolic activation for

pulmonary toxicity, was metabolized by CYP2F1 with relatively high catalytic

efficiency (Vmax 1.3 nmol/nmol P450/min; Km 60mM ) indicating that the protein

is fully functional. Since the amounts of CYP2F1 are limited, kinetic studies have

not been conducted with naphthalene.

CYP2F2 (Mouse)

Naphthalene is metabolized with a high degree of stereoselectivity by

recombinant mouse CYP2F2 expressed in either yeast[57] or in SF-21 insect

cells.[60] A very high Vmax (107 nmol product/nmol P450/min) and low Km (3mM )

for the metabolism of naphthalene by recombinant CYP2F2 is consistent with the

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Table 3. Species Comparisons in the Rates of Conversion of Naphthalene to Naphthalene Oxides: Recombinant Proteins

Recombinant

Enzymes Species

% Sequence

Homology

(with Human)

Rate of Metabolism

(nmol/min/nmol P450

at concentration)

Stereoselectivity

(1R,2S ):(1S,2R )

Vmax

(nmol/min/mol

P450) Km (mM ) References

CYP2F1 Human 100 0.045 at 0.5 mM

naphthalene

0.13:1 ND ND Ref. 58 (gene

sequence);

Ref. 59 (activity)

CYP2F2 Mouse 82 107 at 0.5 mM 66 107 3 60

CYP2F3 Goat 84 Active but rate

not specified

High ND ND 85

CYP2F4 Rat 83 ˆ—————————— Data not available ——————————! Baldwin, unpublished

ND—not determined.

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importance of this protein in the metabolic activation and toxicity of naphthalene

in mouse lung. Previous work from our laboratories demonstrated a lung:blood

partition coefficient of 2.25 (unpublished observations). The levels of naphthalene

in the blood after a 7-hr inhalation exposure at 10 ppm in mice are 0.59 and

0.27mg/mL in male and female mice, respectively.[35] A 3mM Km is equivalent to

0.38mg/mL, and this suggests that the levels of naphthalene in target tissues easily

exceed the Km of CYP2F2 (mouse). Although the recently published

physiologically based pharmacokinetic (PBPK) model suggested that the lung

levels of naphthalene would be slightly lower (0.1mg/mL), these levels are still

within a range where CYP2F2 would be catalytically efficient. Similarly, in a

limited unpublished study (Cho and Buckpitt), unchanged naphthalene, measured

in whole lungs of mice treated with naphthalene (200 mg/kg) by gavage was 8.2

and 3.9mg/g at 30 and 60 min after administration, respectively. These

concentrations far exceed the Km of CYP2F2.

CYP2F4 (Rat)

Earlier work[57] failed to demonstrate the presence of transcript for CYP2F in

rat lung. In addition, while immunocytochemistry with antibodies generated to the

mouse 2F showed high staining intensities for this protein in mouse lung airway

epithelial cells, no detectable staining was observed in either the rat or primate.[27]

This suggested that the differences observed in susceptibility between these two

species might simply be related to the presence of a P450 isoform with high

catalytic activities toward naphthalene and similar substrates[61] in sensitive

species that are either not present or are present at low concentrations in resistant

species. Later studies demonstrated significant levels of CYP2F transcript by

Northern blot analysis, and a CYP has been cloned and sequenced from the rat

(Baldwin, NM_019303). The rat sequence has 93% identity with the mouse and

83% with the human (Baldwin, unpublished). Characterization of the catalytic

activities of the expressed protein and determination of the quantitative

distribution of this protein in mouse vs. rat lungs may indicate whether the

mouse/rat differences in susceptibility are related to lower protein levels or to

altered catalytic activities of the protein.

Clearly, further characterization of the catalytic activities of human CYP2F1

and possibly nonhuman primate orthologues will be essential to understand the

potential importance of these proteins in the metabolic activation of low molecular

weight aromatic hydrocarbons. In our view, it is important to note that the cellular

concentration of each of these P450 proteins will be a key factor in determining the

outcome of exposure. High concentrations of protein in a few cells will likely have

far greater chances of resulting in deleterious effects in response to exposure than

low concentrations of protein spread over many cells. Thus, any assessment of the

importance of these isoforms will have to take into account the localization of the

protein.

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Overall, these studies indicate that there may be major catalytic differences in

CYP2F proteins in human vs. mouse lungs. These differences, if well established

experimentally, could have considerable influence on our assessments of the

likelihood that naphthalene produces lung injury in the human. What has not been

determined at this stage is whether CYP2F is responsible for catalyzing the metabolic

activation of naphthalene in the nasal epithelium. The data in Table 3 shows that the

apparent turnover of naphthalene in postmitochondrial supernatants prepared from

olfactory nasal mucosa are much higher in the mouse and rat than they are in the

hamster. This corresponds with the differences noted in susceptibility of the olfactory

epithelium in these 3 rodent species to naphthalene toxicity. There are several

cytochrome P450 isoforms present in the nasal mucosa including CYP1A2, 2A, 2B,

2C, 2G1, 2J, and 3A,[62,63] but to date, the catalytic activities with naphthalene have

not been established. The critical issues that need to be addressed in order to better

understand the potential for naphthalene to cause nasal epithelial cell toxicity in the

human are the same as those for the lung. The catalytic efficiencies of each of the

P450s in naphthalene metabolism along with an assessment of the amounts of P450

protein present in nasal mucosa will need to be determined.

Role of Naphthalene Oxide in the Toxicity of Naphthalene and as anIntermediate Which Binds Covalently to Cellular Proteins

Work conducted in both the isolated perfused mouse lung and in isolated

murine Clara cells indicates that of the naphthalene metabolites tested,

naphthalene oxide produces Clara cell necrosis at the lowest concentrations of

any of the metabolites. Perfusion of isolated murine lungs with 0.25–1mmol

naphthalene oxide over a period of 1 hr produced a dose-dependent decrease in the

mass of airway epithelial cells (volume/surface area), a decrease in the mass of

nonciliated cells, and an increase in the mass of vacuolated cells.[47] The

magnitude of changes in airway epithelial cell morphometry elicited by perfusion

of the lung with 1mmol racemic naphthalene oxide were similar to those observed

after perfusion with 10mmol naphthalene. In comparison, 10mmol 1,2-

naphthoquinone resulted in only slight increases in vacuolated epithelial cell

mass and in no significant changes in either total cell mass or in nonciliated cell

mass. No alterations in bronchiolar epithelial cell morphology were observed in

lungs perfused with 6.6mmol 1-naphthol or 10mmol 1,4-naphthoquinone over a

period of 1 hr. While these data are consistent with the view that naphthalene oxide

mediates the Clara cell toxicity of naphthalene, contributions to this toxicity from

the quinones cannot be ruled out. The further conversion of naphthalene oxide to

other metabolites was not measured directly, and it is possible that some of the

epoxide is converted to quinone. Pulmonary glutathione was depleted by perfusion

with naphthalene oxide indicating that the epoxide was capable of reaching

intracellular compartments. However, the loss of glutathione only accounted for

approximately half of the naphthalene oxide entering the lung. The quinones are

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chemically unstable, and it is not clear how much of these metabolites actually

reached the epithelial cells of the lung. It is also possible that the toxicity of

naphthalene is mediated by both naphthalene oxide and naphthoquinones.

Naphthalene oxide depletes glutathione thus enhancing the susceptibility of target

cells to small amounts of naphthoquinone which either bind covalently to critical

macromolecules or which redox cycle leading to the generation of reactive oxygen

species eventually leading to cell necrosis. Studies that are currently underway are

designed to identify those metabolites that are bound covalently to proteins.

In other studies using isolated murine Clara cells, Chichester and her

coworkers[64] demonstrated not only that naphthalene resulted in concentration-

and time-dependent losses in cell viability but that naphthalene oxide was the most

potent of the toxicants added to the incubation. Nearly complete loss of Clara cell

viability was noted after 4-hr incubations where naphthalene oxide (0.5 mM final

concentration) was added slowly over a period of 60 min. Addition of 1,4-

naphthoquinone (0.5 mM ) to isolated Clara cells resulted in similar losses of

viability while the 1,2-quinone, 1-naphthol, and naphthalene dihydrodiol were

considerably less cytotoxic. Again, these data must be interpreted cautiously

because there may be dramatic differences in the amounts of each of these

metabolites that reach the intracellular compartment of the Clara cell.

Data showing that the cytochrome P450 monooxygenase inhibitor, piperonyl

butoxide, blocks the loss of cell viability caused by naphthalene but not

naphthalene oxide, further supported the view that naphthalene oxide plays a

major role in naphthalene-induced Clara cell necrosis. Again, these studies cannot

rule out the possibility that the cytotoxicity of naphthalene represents the

combined actions of naphthalene oxide thus making the cell considerably more

vulnerable to 1,2-naphthoquinone that could be generated from the dihydrodiol

through dihydrodiol dehydrogenase.

Finally, additional data that support the importance of the epoxide in

mediating both the toxicity and the protein binding of reactive metabolites comes

from work in isolated murine hepatocytes.[54] Incubation of isolated hepatocytes

with (1S,2R )-naphthalene oxide resulted in nearly complete loss of cell viability

whereas incubation with the (1R,2S )-epoxide enantiomer caused no loss in trypan

blue dye exclusion. These differences have been attributed to the fact that the

(1R,2S )-epoxide is converted much more rapidly to the dihydrodiol than the

(1S,2R )-enantiomer presumably resulting in higher intracellular concentrations of

(1S,2R )-epoxide. Addition of radiolabeled naphthalene oxide to isolated

hepatocytes resulted in time-dependent covalent binding of radiolabel to proteins

which increased rapidly from 1 to 5 min and slowed considerably thereafter. The

fact that radioactivity from [3H]-naphthalene oxide rapidly bound to proteins with

no apparent lag period and that the rate of increase dropped dramatically after

5 min (at a time when most of the naphthalene oxide would have either been

biotransformed or would have undergone spontaneous hydration) argues against

the involvement of secondary metabolites, arising from either further metabolism

of 1-naphthol or the dihydrodiol, in this process.

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1-Naphthol: A Possible Precursor for Reactive and Cytotoxic

Metabolites

A number of studies suggested that phenols that are products generated

spontaneously from the precursor aromatic hydrocarbon epoxides, could undergo

further metabolism to reactive, cytotoxic metabolites. For example, Hesse and

Metzger[65] demonstrated not only that 1-naphthol was efficiently metabolized by

rat liver microsomes to reactive metabolites but that 1-naphthol was likely a key

intermediate in the formation of covalently bound metabolites from naphthalene.

The evidence for this included the fact that inhibition of epoxide hydrolase did not

increase reactive metabolite binding in microsomal incubations with naphthalene,

addition of UDP glucuronic acid decreased binding, and addition of unlabeled 1-

naphthol to microsomal incubations containing [14C]-naphthalene decreased the

overall amounts of radiolabel covalently bound to microsomal proteins. Further

work in hepatocyte incubations confirmed these data showing that inhibitors of

either sulfation or glucuronidation markedly increased reactive metabolite binding

to proteins.[66] Later studies, which compared the rates of metabolism of

naphthalene to 1-naphthol and to covalently bound metabolites in murine lung vs.

liver microsomal incubations, demonstrated that the rates of 1-naphthol formation

were much higher in lung than in liver but that the rates of generation of reactive

metabolites in microsomal incubations from these two tissues were nearly

identical.[67] Addition of an epoxide hydrolase inhibitor to co-incubations of

human liver microsomes with peripheral mononuclear leukocytes increased both

the cytotoxicity and formation of covalent protein adducts.[10] In vivo, reactive

metabolites from [14C]-1-naphthol become bound covalently to proteins in lung,

liver, and kidney, but there was virtually no difference in the amounts of reactive

metabolite bound after administration of 1-naphthol compared to naphthalene.[67]

Since only a portion of the administered naphthalene would be expected to be

converted to 1-naphthol, we felt that this argued against a role of this metabolite in

covalent binding to proteins in vivo. Additional studies by O’Brien and her

coworkers[21] and Buckpitt and his colleagues[67] showed that while 1-naphthol

has a lower LD50 than naphthalene, it did not result in injury in any of the tissues

studied.

More recent work has again raised the issue of the importance of secondary

metabolites in naphthalene toxicity. Human peripheral mononuclear cells were

considerably less sensitive to the cytotoxic effects of naphthalene oxide than to 1-

naphthol (in the presence of human liver microsomes) and both 1,2- and 1,4-

naphthoquinone.[68] Finally, alkaline permethylation techniques developed for

examining the nature of reactive bromobenzene metabolites were applied to

studies in which murine Clara cells were incubated with naphthalene.[69] The

ratios of adducts generated from the 1,2-quinone vs. the 1,2-epoxide were 32:1,

and there was no evidence for the formation of 1,4-naphthoquinone. The results of

these studies are not in agreement with earlier work published by Doherty and her

coworkers.[70] These investigators showed that reactive metabolites generated in

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microsomal incubations from 1-naphthol were not trapped with ethylene diamine,

a compound that reacts rapidly with 1,2-naphthoquinone. Based on these results,

1,4-naphthoquinone was the likely precursor to reactive metabolites generated

from 1-naphthol.

1,2-Dihydroxy-1,2-dihydronaphthalene (Naphthalene Dihydrodiol)

Although the dihydrodiols generated from aromatic hydrocarbon epoxides

are generally less reactive than the parent epoxide, these metabolites are the

precursors to the diol epoxide metabolites thought to be the penultimate

carcinogens from numerous PAH. Strong support for the in vivo formation of the

diol epoxide and/or diepoxide has been obtained from identification of urinary

naphthalene metabolites. A number of trihydroxytetrahydromethylthio deriva-

tives[48] and a trihydroxytetrahydromercapturate[71] have been recovered from the

urine of mice and rats treated with naphthalene. To our knowledge, there is no

quantitative information available on the rates of formation of these metabolites in

target tissues of susceptible and nonsusceptible species; thus, their role in the

toxicity of naphthalene is not known.

In addition to the reactions described earlier, naphthalene dihydrodiol can

undergo further biotransformation by cytosolic dihydrodiol dehydrogenase to

generate the catechol (1,2-dihydroxynaphthalene) that is easily oxidized to 1,2-

naphthoquinone, a compound that is both reactive and will undergo redox

cycling.[72,73] As discussed earlier, work in isolated Clara cells indicated that the

1,2-quinone was the major species bound to proteins covalently.[69] Antibodies to

the 1,2-naphthoquinone adducts have been utilized in a Western blot assay to show

that there are numerous protein adducts generated from the quinone in mouse liver

homogenate incubations.[74] 1,2-Naphthoquinone is mutagenic in the Ames

assay[73] and has been shown to form N-7 adducts with deoxyguanosine in

vitro.[75] These interactions could lead to depurination although this was not

demonstrated with 1,2-naphthoquinone. In addition, these adducts have not been

demonstrated in cell-based systems or in vivo. It is important to note that if 1,2-

naphthoquinone is shown to be an important intermediate in naphthalene toxicity,

the human could actually be more susceptible than some of the rodent models that

have been studied. This is based on the fact that both epoxide hydrolase[76] and

dihydrodiol dehydrogenase[72] have been shown to be highly active in human

tissues in comparison to rodents.

BIOMARKERS OF EXPOSURE AND EFFECT

As stated earlier, one of the approaches that can be used to provide further

information regarding rodent-to-human comparisons in terms of their response to

a chemical can be to develop biomarkers that not only indicate an exposure has

NAPHTHALENE-INDUCED RESPIRATORY TRACT TOXICITY 811

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occurred but also that the exposure has occurred at a level sufficient to cause

toxicity. Although biomarkers that are capable of providing this information for

naphthalene have not been developed and validated in animal models, there are

two urinary metabolites, which have been used to demonstrate that an exposure

has occurred and that at least the initial step in the metabolism of naphthalene has

taken place. These biomarkers include the measurement of mercapturic acid

metabolites and conjugates of naphthol in the urine.

Mercapturic acids were identified as urinary metabolites of naphthalene

more than 40 years ago.[77] Although the methodology used to examine the

amounts of mercapturate in urine of a number of rodent species and man was

semiquantitative (staining intensities of spots isolated by paper chromatog-

raphy), Boyland and Sims[77] presented convincing evidence that all species

treated with naphthalene including rodents and humans were capable of

generating and eliminating these metabolites in the urine. Rodent species were

treated with doses that varied from 200 to nearly 1000 mg/kg, ip while human

volunteers took doses of 500 mg orally (,10 mg/kg). Subsequent work, by

Summer, Rozman and their colleagues,[78,79] showing no detectable increase in

urinary thioethers in either the chimpanzee or Rhesus macaque after

administration of naphthalene at doses as high as 200 mg/kg are at variance

with the findings in man. However, these studies involved the alkaline

hydrolysis of the urine containing mercapturate followed by measurement of

the free thiols using Ellman’s reagent, and it is not clear whether these assays

yield reliable data since standards were not available.

More recent work has investigated the elimination of diastereomeric

mercapturates as a means of measuring the rates and formation of naphthalene

epoxides in mice compared to rats after administration of naphthalene by both

parenteral and inhalation routes.[71] These studies showed that there were no

significant differences observed in the percentage of the dose eliminated as

mercapturate in urine between mice (25–34%) and rats (24–35%) and that the

amount of mercapturate eliminated as a percentage of dose did not vary after ip

administration. The amounts of urinary mercapturate eliminated following 4-hr

inhalation exposures were substantially greater in mice than rats on a per gram

body weight basis, and this is consistent with the higher rates of epoxide formation

in lung microsomes and in dissected airways from mice compared to rats.[27,52]

These studies also explored the possibility that measurement of diastereomeric

mercapturates could be used to assess the stereochemistry of epoxidation in vivo.

Earlier studies had demonstrated that relatively consistent amounts of the 2-

glutathionyl adducts derived from both the (1R,2S )- and the (1S,2R )-naphthalene

epoxides were eliminated in the urine as the corresponding mercapturic acids after

intravenous administration of diastereomeric glutathione conjugates.[80] However,

the adduct formed by glutathione conjugation at C-1 underwent significant

metabolism to the pyruvic acid derivative. Nevertheless, measurement of the

ratios of mercapturates derived from thioether formation at C-2 would provide a

means for measuring the stereochemistry of naphthalene oxide metabolism in

BUCKPITT ET AL.812

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vivo. After parenteral administration of naphthalene to mice, the ratio of

diastereomeric urinary mercapturates derived from the (1R,2S )- to (1S,2R )-

epoxide was 1:1 at low doses (1–3 mg/kg), increased to 3:1 at intermediate doses

(50 mg/kg), and decreased to 2:1 at high doses (100/200 mg/kg). In rats, these

ratios remained less than 1:1 at all doses.[71] After inhalation exposures, ratios of

mercapturates derived from the (1R,2S )-epoxide were 5–6:1 at low concen-

trations (less than 15 ppm) and decreased to 3:1 at higher concentrations (15–

100 ppm) in mice, while in rats, the ratios were 1:1 or less for all concentrations.

Again these data are consistent with the ratios of naphthalene epoxide enantiomers

generated in vitro.

Measurement of conjugates of 1-naphthol in the urine has been shown to be

well correlated with naphthalene exposures in workplace settings (naphthalene

and tar distillation, creosote impregnation) even at low exposure concen-

trations.[81,82] Unfortunately, neither of these studies controlled for smoking, and it

is clear that this is a major contributor to 1-naphthol levels in the urine. Recent

work showed that urinary 1-naphthol levels were three fold higher in smokers than

in nonsmokers.[83] The other consideration necessary in monitoring urinary 1-

naphthol is that this is a key metabolite of the insecticide carbaryl.[84]

Although the presence of either the mercapturic acid or 1-naphthol in the

urine correlate with exposure to naphthalene and the presence of either metabolite

is indicative that P450-dependent metabolism of naphthalene has occurred, neither

provides much information with respect to events that are closely tied to toxicity.

It is possible that once the importance of various protein adducts in events that lead

to cellular injury is well understood, and the pathways by which these adducts are

eliminated from the organism are delineated that biomarkers based on adducts

with peptides derived from the target proteins would be excellent markers of both

exposure and effect.

FUTURE DIRECTIONS

Key issues remain to be resolved in assessing the potential for

naphthalene to result in toxicity or oncogenicity in humans. As discussed in

this review, there are substantial differences in the metabolism of naphthalene

among rodent species, and these variations appear to be tied to the extent and

severity of toxicity. It is clear from the work conducted to date that the rates of

metabolism of naphthalene in the lung are substantially different in rodents,

humans, and nonhuman primates. Preliminary studies appear to indicate that

differences in the catalytic activities of the proteins involved in naphthalene

metabolism as well as the amounts of protein present might account for these

differences. Although these data suggest that the respiratory tract of the human

would be far less susceptible to naphthalene than the respiratory tissue of

rodents, careful studies are needed to define the localization of the primary

metabolic enzymes involved in both the activation and detoxication of

NAPHTHALENE-INDUCED RESPIRATORY TRACT TOXICITY 813

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naphthalene. As pointed out in this review, measurement of rates of

metabolism in homogenate of a very heterogenous tissue such as the lung can

yield data that are not informative with respect to reactions taking place in a

very small proportion of target cells. Thus, additional efforts are underway to

localize some of the proteins in the respiratory tract and to study metabolism

in target cell populations of both humans and nonhuman primates. Since the

NTP bioassays have identified the nasal epithelium as an important target for

naphthalene, additional work is needed to examine rates of metabolism of

naphthalene in this portion of the respiratory tract.

Although many of the areas of the respiratory tract targeted acutely by

naphthalene are the same as those where tumors develop, there are some

important differences, and the two responses, namely cytotoxicity and

carcinogenicity, might be totally separate. Additional work examining the

importance of specific protein adducts in cytotoxicity and potentially

carcinogenicity (alteration of DNA repair enzymes for example) could be

very helpful in development of biomarkers that would clearly test the possibility

that humans are susceptible to naphthalene and/or close structural analogs.

Identification of the precursor reactive metabolite responsible for the covalent

binding would help focus the research on those enzyme pathways responsible

for generating that metabolite as well as those that prevent the formation or

detoxify it once it is formed.

More work is needed to identify which of the cytochrome P450

monooxygenases are responsible for the initial step in naphthalene metabolism

in the nasal epithelium, and work comparing the catalytic efficiencies and

measurement of amounts of CYP2F in rodent and primate nasal epithelium needs

to be completed. Finally, the development of in vitro approaches that can be used

to determine the susceptibility of target and nontarget regions of rodent respiratory

tract along with extension to the respiratory system of nonhuman primates or

tissue from humans, would provide more definitive information regarding cross

species comparisons for cytotoxicity. The availability of these data should be

helpful not only in determining whether naphthalene exposures represent possible

health risks in the human but also, on a larger scale, should be useful in

determining whether the mouse, which is far more susceptible to a number of

respiratory tract toxicants than other rodents, is a good experimental model for

assessing the potential deleterious effects of these agents in the human.

ACKNOWLEDGMENTS

Work from the authors laboratories has been supported by funding from the National

Institutes of Environmental Health Sciences (ES 04311, ES 04699, ES 08408, ES 09681).

Support for core facilities used in this work has come from the Center for Environmental

Health (ES 05707). We gratefully acknowledge the American Chemical Council’s

Naphthalene and Hydrocarbon Solvents Panels for financial support for the preparation of

this review.

BUCKPITT ET AL.814

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BUCKPITT ET AL.820

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