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Current OSHA standards for naphthalene exposure are set at 10 ppm (time-weighted average) with a standard threshold exposure concentration of 15 ppm. While several studies have thoroughly delineated the time course and dose response of injury by naphthalene administered ip, the pattern and severity of injury by inhalation exposure are unknown. These studies compare the regiospecific and dose-dependent cytotoxicity of naphthalene after inhalation exposure. Mice and rats were exposed for 4 h to naphthalene vapor at concentrations of 0-110 ppm. In rats, no injury was observed in the lung epithelium at exposure concentrations up to 100 ppm. Exposures as low as 2 ppm produced proximal airway injury in mice, with increased severity in a concentration-dependent fashion up to 75 ppm. Terminal airways of exposed mice exhibited little or no injury at low concentrations (1-3 ppm). Exposures of 8.5 ppm or higher were required to produce injury to Clara cells in the terminal airways. In contrast, administration of naphthalene (300 mg/kg) extended the injury pattern toward the lobar bronchus. We conclude (1) the pattern of injury to naphthalene is highly dependent on the route of exposure, (2) lung injury to inhaled naphthalene is species dependent, and (3) Clara cells of mouse airways are exquisitely sensitive to inhaled naphthalene at concentrations well below the current OSHA standard for human exposure., (Copyright 2001 Academic Press.)

Glutathione S-transferases (GSTs) and epoxide hydrolases (EHs) protect cells from exogenous insult by detoxifying electrophilic compounds. Little is known about these enzyme systems during postnatal lung development. This study was designed to help establish whether the heightened neonatal susceptibility of the lung to bioactivated cytotoxicants is the result of inadequate ability to detoxify reactive intermediates. We compared the distribution of immunoreactive protein and enzymatic activity of GSTs and EHs in isolated distal airways during pre- and postnatal development in lungs of mice from 16 days gestation to 9 weeks postnatal age (adult). GST alpha, mu, and pi class protein expression in fetal and postnatal lung varied by isozyme and age. Isozymes alpha and mu are expressed at low levels before birth, high levels on postnatal day 7, low levels between postnatal days 14 and 21, high levels at postnatal day 28, and slightly lower levels in adults. Immunoreactive protein of isozyme pi has a peak expression on gestational day 18 and again on postnatal day 4, is undetectable at postnatal day 21, and is at peak levels in the adult mouse lung. GST activity in distal airways increased with age. Microsomal EH protein expression increased in intensity with age, while activity was similar in airways from all ages. We conclude that in the mouse lung (1) cellular expression of glutathione S-transferase varies by age and isozyme and does not increase with increasing age, (2) airway glutathione S-transferase activity increases with increasing age and does not correlate with immunoreactive protein expression, and (3) airway microsomal epoxide hydrolase activity does not increase, even though immunoreactive protein expression does increase with age., (Copyright 2000 Academic Press.)

The distal trachea and centriacinus of the lung are primary sites of acute injury during short-term ozone exposure; long-term exposure yields cells in these areas that are resistant to high doses of oxidant gases. Epithelial cells located in primary sites for ozone injury are also targets for chemicals that undergo cytochrome P450 (CYP)-dependent activation. These studies were designed to compare the effects of ozone exposure on pulmonary CYP2E1 in susceptible and nonsusceptible sites within the airway tree of lung. CYP2E1 activity was measured in well-defined regions of airways using p-nitrophenol, a CYP2E1-selective substrate, with HPLC/ electrochemical detection of the p-nitrocatechol. Alterations in distribution of CYP2E1 were evaluated by immunohistochemistry. CYP2E1 activities were highest in the distal bronchioles and minor daughter airways but were much lower in the lobar bronchi/ major daughter airways and trachea. Immediately after short-term ozone exposures (8 h, 1 ppm), CYP2E1 activities were elevated only in the lobar bronchi/major daughter airways. These activities remained above the filtered air control at 1 day but returned to control levels by 2 days. Immunohistochemical assessment of CYP2E1 protein in ozone and filtered air-exposed animals was consistent with the activity measurements. After long-term ozone exposures (90 days, 1 ppm), CYP2E1 activities were decreased in the major and minor daughter airways. These studies indicate that CYP2E1 activities vary substantially by airway level. However, ozone exposure only results in minimal alterations in activity with varying concentration of ozone, length of exposure, and time after exposure in any of the lung subcompartments examined.

Selective Clara cell injury produced by many bioactivated lung toxicants is thought to result from high levels of activating enzymes found in differentiated Clara cells. A recent study found an elevated susceptibility to the Clara cell toxicant 4-ipomeanol in neonatal rabbits when Clara cell P450 activity is low. To determine whether differentiating Clara cells in another species (mouse) are more susceptible to injury by a different bioactivated Clara cell toxicant (naphthalene), adult, 14-day postnatal (DPN) and 7DPN male mice were given a single intraperitoneal dose (25, 50, or 100 mg/kg) of naphthalene and killed 24 hr later. Epithelial damage, as assessed by quantitative histopathology, included cellular swelling, vacuolization, and exfoliation. In 7DPN mice, bronchiolar epithelium was severely injured at the lowest dose of naphthalene tested, 25 mg/kg. Bronchiolar epithelium in 14DPN mice was moderately injured at 25 mg/kg; injury severity was greatest at 50 and 100 mg/kg. Minimal bronchiolar epithelial injury occurred in adult mice at 50 mg/kg and moderate injury at 100 mg/kg. In proximal bronchi, epithelium of 7DPN mice showed signs of injury only at 100 mg/kg. Bronchial epithelium of adult mice was not injured at any dose. Isolated distal airways from 7DPN and 14DPN mice were more sensitive to naphthalene exposure than isolated distal airways from adult mice. Despite the low levels of P450 activity, differentiating Clara cells in neonatal mice are more susceptible to injury by the bioactivated cytotoxicant naphthalene than are differentiated Clara cells in adult mice.

Earlier studies have shown that both mainstream and sidestream cigarette smoke increase the activities of cytochrome P4501A1 and 2E1 in the lungs of adult animals; however, little information is available on the influence of ambient levels of sidestream cigarette smoke on cytochrome P450 monooxygenase activity in the developing lung. The present studies were conducted to define the developmental profiles of cytochrome P450 monooxygenases 1A1 and 2B1 in rat lung and liver and to assess the effects of aged and diluted sidestream cigarette smoke (ADSS) on the developmental profile of these two enzymes. Accordingly, pulmonary and hepatic microsomal P4501A1 and 2B1 activities were determined by measuring ethoxy- and pentoxyresorufin-O-delakylase (EROD and PROD, respectively) activity in animals exposed to filtered air or ADSS from birth to 7, 14, 21, 50, and 100 days of age. Pulmonary P4501A1 activity in control rats was not detected until 14 days of age. Activities increased threefold between 14 and 21 days of age and remained unchanged to 100 days of age. In animals exposed to ADSS from birth, pulmonary EROD activities were detected as early as 7 days postnatal and were elevated three- to fourfold above control at all other ages examined. Hepatic EROD activities were unaltered by ADSS exposure. Short-term (4-day) ADSS exposure was as effective in upregulating pulmonary microsomal EROD activities as 100-day exposures. Induction of pulmonary EROD activities and the associated increases in mRNA levels were dependent upon the particulate fraction. Stimulation of EROD activities in major and minor daughter subcompartments was three- to fourfold higher in ADSS-exposed animals compared to controls, while there was no induction in the trachea and less than a twofold increase in the parenchyma. Pulmonary PROD activities developed more slowly than EROD and did not reach adult levels until Day 50. ADSS did not alter pulmonary or hepatic PROD activities. These studies show that P4501A1 and 2B1 develop at different rates in rat lung and liver and that exposure to ADSS markedly increases P4501A1 activities in the lung at all ages examined.

Ozone-induced lung injury in rats is focal, with the primary target sites being the distal trachea and the central acinus. In both area, ozone causes cellular injury and necrosis after short-term exposures, but the areas become tolerant to further injury after long-term exposure. To investigate the role of antioxidant enzymes in the resistance of the lung to injury from long-term ozone exposure, we measured activities of three antioxidant enzymes in airway samples microdissected from specific sites within the lung: distal trachea, lobar bronchi, major daughter axial bronchi, minor daughter bronchi, distal bronchiole, and parenchyma. Fischer 344 rats were exposed to 0, 0.5, and 1 ppm ozone 6 hr/day, 5 days/week for 20 months, or to 0, 0.12, and 1 ppm for 90 days. Glutathione transferase, glutathione peroxidase, and superoxide dismutase activities were measured at the end of the exposure periods. Data were normalized for DNA content (Units/mg DNA). For both the 90-day and 20-month exposures, the activities of all three enzymes were significantly elevated in a concentration-dependent fashion in the distal bronchioles. Compared to controls, animals exposed to 1.0 ppm ozone had superoxide dismutase activities 1.6x (90 days) and 2x (20 months) greater; glutathione peroxidase had activities 1.4x (90 days) and 1.6x (20 months) greater; and glutathione S-transferase had activities 1.5x (90 days and 20 months) greater. In animals exposed for 90 days, superoxide dismutase activity was lower in major daughter bronchi and greater in minor daughter bronchi and glutathione peroxidase activity was lower in major daughter bronchi. After 20 months of exposure, superoxide dismutase activity was significantly elevated in a dose-dependent fashion in the distal trachea; glutathione peroxidase activity decreased in the major daughter bronchi and increased in the minor daughter bronchi; and glutathione S-transferase activity decreased in the major daughter bronchi. There were no changes in antioxidant enzyme levels in other subcompartments. Superoxide dismutase activity increased in a concentration-dependent fashion in the whole lung homogenate of animals exposed for 90 days, but no differences were detected in whole lung homogenates of any other exposure groups. We conclude that (1) antioxidant enzyme activities are altered on a site-specific basis in response to long-term exposure to ozone; (2) the antioxidant enzymes respond differently in different lung subcompartments; (3) activities determined for the whole lung do not reflect changes in subcompartments with variable susceptibility to injury; and (4) changes in antioxidant enzyme activities are concentration-dependent and altered by length of exposure.

The objective of this study was to characterize an in vitro model of oxidant gas toxicity, using primary cultures of alveolar type II cells maintained in serum-free medium, by evaluating (1) epithelial barrier function, (2) the stability of cellular antioxidant defenses, and (3) the response of alveolar epithelial barrier properties to ozone exposure. Antioxidant enzyme activities and glutathione levels were measured in rat type II cells that were freshly isolated, cultured for 1 day in serum-supplemented medium, and subsequently grown in serum-free nutrient medium. After measurement of peak bioelectric properties on Day 4 in primary culture, alveolar epithelial monolayers were exposed to ozone at various concentrations and lengths of exposure. Ozone-induced alterations in monolayer bioelectric properties and impairment of cellular organization were used to evaluate oxidant injury. The primary effect of ozone exposure was a dose-dependent increase in monolayer permeability, which resulted from damage to intercellular junctions and/or loss of epithelial integrity. Extensive and persistent permeability increases correlated with focal areas of epithelial degradation. The focal nature of ozone injury to alveolar epithelium in vitro suggests that individual cell susceptibility to oxidant stress may account for the overall decrement in barrier function. However, this sensitivity does not result from overall loss of antioxidant defenses associated with cell culture, as these monolayers (when cultured in serum-free medium) maintained their antioxidant enzyme activities and glutathione content at levels found in freshly isolated cells. We conclude that the sensitivity of these monolayers to ozone injury in vitro reflects a disproportionate degree of oxidant stress on cell membranes relative to intracellular antioxidant defenses, i.e., cellular susceptibility to oxidant injury may depend on the ratio of the surface area of the cell to its cytoplasmic volume.

Antioxidant enzymes including catalase, superoxide dismutase, glutathione peroxidase, and glutathione S-transferases are thought to be the primary cellular defense against reactive oxygen species. Since pulmonary injury produced by oxidant air pollutants like ozone is highly focal, involving primarily the trachea and centriacinar areas of the lung, measurements of alterations in antioxidant enzyme activities in whole lung may substantially underestimate changes occurring in target areas of the respiratory tract. We have applied a technique for preparation of lung specimens from well-defined anatomic locations to determine whether the focal injury associated with ozone exposure is related to an uneven distribution of antioxidant enzyme activity in the respiratory tract. Our study compared enzyme activities in rat and monkey, species which differ considerably in sensitivity to ozone-induced injury (monkey > rat). The activities of glutathione S-transferase varied less than twofold between different airway subcompartments for both the rat and monkey. Pulmonary veins had approximately 50% of the activity of airways in both species. Glutathione peroxidase activity was slightly higher in proximal compared to distal airways of the rat but was evenly distributed at all airway levels in the monkey. In both species, activity in pulmonary veins was lower than that in airways. The activity of superoxide dismutase was similar in rat and monkey and marked differences were not observed in the various subcompartments studied. Similarly, catalase activity was relatively evenly distributed in rat airways but, in the monkey, the distal bronchiole and lobar bronchus had marginally higher activity than the trachea. We conclude that: (1) measurement of antioxidant enzyme activities in anatomic subcompartments within the lung is feasible using microdissected specimens, (2) antioxidant enzyme activity can vary in different subcompartments of the lung of the same species, (3) the pattern of variation in enzyme activity differs by the enzyme and by species, and (4) species and subcompartment differences in ozone injury are not due primarily to differences in the distribution of antioxidant enzyme activity.

Intraperitoneal administration of single doses of the volatile aromatic hydrocarbon, naphthalene, resulted in dose-dependent bronchiolar epithelial cell necrosis in mice. Twenty-four hours after a dose of 50 mg/kg, swelling of Clara cells with some exfoliation of epithelial cells was evident in half of the treated animals. At doses of 100 mg/kg small numbers of necrotic and swollen cells with pyknotic nuclei were observed. At 200 mg/kg there were substantial numbers of bronchiolar epithelial cells sloughed into the airway lumen, apical projections were virtually absent, and there were large numbers of cells with pyknotic nuclei. In contrast, bronchiolar airways from mice treated with naphthalene daily for 7 days at doses of 50, 100, or 200 mg/kg/day differed only slightly from controls. Significant protection to bronchiolar epithelial cell necrosis produced by 300 mg/kg naphthalene was afforded by seven daily injections of 200 but not 50 or 100 mg/kg naphthalene. A gradual recovery in sensitivity to the 300 mg/kg challenge dose of naphthalene was observed as the time between the last 200 mg/kg naphthalene dose increased from 24 to 144 hr. Daily administration of 200 mg/kg but not 50 or 100 mg/kg naphthalene for 7 days resulted in a selective decrease in the rate of formation of 1R,2S-naphthalene oxide by mouse lung but not liver microsomal enzymes. This selective decrease in pulmonary microsomal formation of 1R,2S-oxide continued in animals killed 48, 96, and 144 hr after the last administration of 200 mg/kg. Alterations in the rate of formation of reactive, covalently bound naphthalene metabolites in lung microsomes were not observed, nor were there any differences in the levels of covalently bound reactive metabolites in vivo between tolerant and control animals. These studies are consistent with other work showing that the lung loses susceptibility to the acute injury arising from repeated exposure to pneumotoxicants. In contrast to other studies with naphthalene where alterations in the levels of covalently bound reactive metabolites in the lung closely paralleled the extent and severity of bronchiolar injury, these studies clearly separate necrosis from covalent binding. Although the correlation was not absolute, it appears that formation of 1R,2S-oxide by microsomal enzymes in vitro is a better overall marker of decreased sensitivity to naphthalene-induced bronchiolar necrosis than is reactive metabolite binding either in vivo or in vitro.