Hyperthyroidism increases the risk of ozone-induced lung toxicity in rats.

The risk of lung injury from ozone exposure has been well documented. It is also known that various factors may significantly influence the susceptibility of animals to the toxic effects of ozone. In the present study, we investigated the possibility that hyperthyroidism might be associated with increases in ozone-induced pulmonary toxicity. To create a hyperthyroid condition, mature male Sprague--Dawley rats were given injections of thyroxine (dose range: 0.1 to 1 mg/kg body wt daily for 7 days). Control rats received vehicle injections. The animals were then exposed to air or ozone (dose range: 0.5 to 3 ppm for 3 h). At 18 h postexposure, bronchoalveolar lavage fluid and cells were harvested. In hyperthyroid animals, ozone exposure was associated with three- to sixfold increases in bronchoalveolar lavage fluid lactate dehydrogenase activities and albumin levels as well as the number of polymorphonuclear leukocytes harvested by bronchoalveolar lavage above levels observed in ozone-exposed control rats. Additional results from the present study suggest that these thyroid hormone-linked effects cannot be fully explained by differences in whole-body metabolic rate or changes in the inhaled dose of ozone. These findings indicate that the risk of ozone-induced lung toxicity is substantially increased in a hyperthyroid state and suggest that the susceptibility of the lung to damage from ozone exposure may be significantly influenced by individual thyroid hormone status.

Lung responses to hypothyroidism, hyperthyroidism, and lipopolysaccharide challenge in rats.

The objectives of this investigation were to study the effects of hypo- and hyperthyroidism on some factors involved in lung injury under basal conditions (air exposure) and during an inflammatory response induced by inhalation exposure to lipopolysaccharide (LPS; 100 microg/ml; 3 h) in adult rats. Thyroid status was altered by thyroidectomy or thyroxine injections for 15 d. Hyperthyroidism alone caused a greater degree of lung cell damage, an increase in the permeability of the alveolar-capillary barrier, a rise in the total number of phagocytic cells obtained by bronchoalveolar lavage (BAL), and enhanced nitric oxide (NO) release by phagocytic cells relative to that in euthyroid control animals. Hypothyroidism alone was associated with opposite effects. Exposure of animals to LPS produced inflammatory responses, which included significant increases in lung cell damage, permeability of the alveolar-capillary barrier, number of phagocytic cells obtained by BAL, and NO production by the phagocytic cells. In general, hyperthyroidism enhanced the effects of LPS, while hypothyroidism reduced LPS-induced responses. These results suggest that thyroid status alone can affect some of the factors involved in lung injury and also modulate some of the inflammatory effects of LPS. Hyperthyroidism tends to enhance lung injury, while hypothyroidism seems to reduce lung injury.

Enhancement of nitric oxide production by pulmonary cells following silica exposure.

In vivo exposure of rat lungs to crystalline silica either by intratracheal instillation or by inhalation results in an increase in mRNA levels for inducible nitric oxide synthase (iNOS) in bronchoalveolar lavage cells (BALC), elevated nitric oxide (.NO) production by BALC, and an increase in .NO-dependent chemiluminescence (CL) from alveolar macrophages (AM). Induction of iNOS message occurs in both AM and polymorphonuclear leukocytes (PMN) harvested from silica-exposed lungs but is not significantly elevated in lavaged lung tissue. In vitro exposure of AM to silica does not stimulate .NO production or enhance iNOS message. However, treatment of naive AM with conditioned media from BALC harvested from silica-exposed rats does increase iNOS message and .NO production by these AM. The potency of this conditioned medium is dependent on interaction between AM and PMN. In the rat model, a relationship exists between the ability of various dusts to cause PMN recruitment or protein leakage into the alveolar space and the induction of iNOS message in BALC, i.e., silica > coal mine dust > carbonyl iron > titanium dioxide. Similarly, a comparison of BALC from a healthy volunteer, a silica-exposed coal miner with a normal chest radiograph, and a silica-exposed coal miner with an abnormal chest radiograph shows a correlation between pathology and both the level of iNOS message in BALC and the magnitude of .NO-dependent CL from AM. These data suggest that .NO may play a role in silicosis and that human pulmonary phagocytes exhibit enhanced .NO production in response to an inflammatory insult.

Regulation of nitric oxide production by rat alveolar macrophages in response to silica exposure.

In the present study, it was confirmed that in vivo exposure of rats to silica significantly increases nitric oxide (NO) production by bronchoalveolar lavage cells (BALC), a population of cells that includes alveolar macrophages. Possible mechanisms whereby NO production could be upregulated by rat alveolar macrophages following silica exposure were examined to determine if there is a direct effect of silica on alveolar macrophage NO production or if other factors are involved. BALC were obtained from normal male rats and cultured for 2 h. Nonadherent cells were then removed and the enriched alveolar macrophage cell populations were exposed to test agents for 18-20 h. Media nitrate and nitrite (NOx) concentrations were used to assess NO production and, in some cases, inducible NO synthase mRNA levels were indexed. In vitro exposure to silica (0.1-100 micrograms/ml) had no significant effect on basal NO levels. Furthermore, NO generation was not additionally increased above levels induced by interferon gamma (IFN), lipopolysaccharide (LPS), or other cytokines during simultaneous incubations with silica and IFN, a 2-h pretreatment with silica followed by IFN, or preincubation with IFN, LPS, and/or other cytokines before the addition of silica. To evaluate whether cell-cell interactions might be required for the induction of NO production during silica challenge, alveolar macrophages were cultured with splenic lymphocytes or blood-derived polymorphonuclear leukocytes. Coculture of splenic lymphocytes with alveolar macrophages resulted in media NOx levels that were greater than the additive levels from each cell type. However, the presence of silica was without additional effect on NO production by either of these cell types. Furthermore, it was found that conditioned media, derived from adherent BALC following silica treatment in vivo, could induce NO production by naive alveolar macrophages. In summary, the collective results from these experiments suggest that cell-cell communication factors, involving the interaction of pneumocytes following in vivo silica exposure, are necessary for the induction of NO by alveolar macrophages.

Inhalation of toluene diisocyanate is associated with increased production of nitric oxide by rat bronchoalveolar lavage cells.

Isocyanates are used commercially, particularly in the manufacture of polyurethane coatings and foam. These compounds can pose an occupational health hazard since there is a risk of respiratory disease following isocyanate exposure. The purpose of the present study was to investigate whether a single, sublethal isocyanate inhalation is associated with increased production of the free radical nitric oxide (NO). Mature male Sprague-Dawley rats were exposed to air or toluene diisocyanate (TDI; 2 ppm) for 4 hr. Indices of pulmonary function were assessed before and after exposure to TDI fumes. At 20 hr postexposure, bronchoalveolar lavage cells (BALC) and fluid were harvested. NO synthase (NOS)-dependent reactive species production by alveolar macrophages was assessed by determining N(omega)-nitro-L-arginine methyl ester-inhibitable chemiluminescence following stimulation with unopsonized zymosan. Northern blot analysis was used to index inducible NOS mRNA levels in BALC, while nitrite and nitrate (NOx) levels were measured to determine NOx levels in the lavage fluid and the production of NO by cultured adherent BALC was indexed by measuring nitrite levels. Exposure to aerosolized TDI was associated with an increase in the number of alveolar macrophages, lymphocytes, and polymorphonuclear leukocytes harvested by bronchoalveolar lavage, relative to that from air-exposed rats. NOx levels in the lavage fluid and NOS-dependent production of reactive species by alveolar macrophages were increased following TDI exposure. In addition, inducible NO production by BALC (i.e., mRNA levels and nitrite levels in BALC conditioned media) was elevated following TDI treatment. These findings indicate that pulmonary inflammatory responses induced by TDI exposure are associated with increases in inducible NO production. Therefore, the potential role of NO in the initial pulmonary response to TDI exposure warrants further investigation.

Cytochrome P-450-dependent metabolism in alveolar type II epithelial cells: modulation by platelet-activating factor.

Platelet-activating factor (PAF), an ether lipid mediator released from activated pulmonary phagocytes, was evaluated for its ability to affect cytochrome P-450-dependent activities in isolated rat alveolar type II cells. The data indicate that at non-toxic doses, PAF caused an increase in beta-naphthoflavone (BNF) inducible/alpha-naphthoflavone (ANF) sensitive ethoxyphenoxazone deethylase (EtOPx'ase) activity. At high concentrations of PAF, inhibition of both EtOPx'ase and metyrapone (MP) sensitive benzyloxyphenoxazone debenzylase (BzOPx'ase) activities and aggregation of type II cells were observed. The PAF analogs, lyso-PAF and enantio-PAF, exhibited actions similar to those observed with PAF. PAF-induced enhancement of EtOPx'ase activity required the presence of intact cells, whereas at high PAF concentrations decreased enzyme activities were observed in both intact cell and sonicated cell preparations. The data thus suggest that xenobiotic metabolism in alveolar type II cells can be modified by an inflammatory mediator, such as PAF, produced by alveolar phagocytes.

Platelet-activating factor-induced aggregation of rat alveolar macrophages.

Isolated rat alveolar macrophages aggregate in the presence of PAF in a dose- and cell-dependent manner. Saturation was achieved at 80 microM PAF. The response was increased linearly with cell number up to a concentration of 3 x 10(6) cells/ml, but decreased at higher cell concentrations. The stereoisomer, enantio-PAF, and the C2-acetyl hydrolyzed product, lyso-PAF, each caused aggregation of isolated rat alveolar macrophages in a manner similar to that for PAF. The PAF-induced aggregation of alveolar macrophages may be mediated through non-specific binding sites and may represent a toxic response to relatively high levels of PAF that released at localized sites.

Lung cytochrome P450-dependent benzyloxyphenoxazone debenzylase and ethoxyphenoxazone deethylase activities in total microsomal and isolated alveolar type II cells: responses to changes in assay conditions with special reference to non-linear dependence at low enzyme concentration.

1. Responses of cytochrome P450-dependent ethoxyphenoxazone deethylase (EtOPx'ase) and benzyloxyphenoxazone debenzylase (BzOPx'ase) activities to changes in assay conditions were measured in total microsomal and isolated alveolar type II (tII) cells from rats pretreated with beta-naphthoflavone. 2. Whereas microsomal EtOPx'ase activity was unaffected by storage at -80 degrees C for up to 4 months, BzOPx'ase activity began to decline after only 1 month. 3. The microsomal and type II activities were unaffected by changes in pH (7.2-8.0) or salt content. 4. The type II activities increased after sonication 2.3-2.7-fold or in the presence of 10 microM dicumarol 1.7-1.9-fold. 5. Type II BzOPx'ase was sensitive to metyrapone (MP) whereas EtOPx'ase was sensitive to alpha-naphthoflavone (ANF). I50 values for the tII activities were calculated as: 0.63 microM--MP (BzOPx'ase), 80 microM--MP (EtOPx'ase), 0.024 microM--ANF (EtOPx'ase). At the highest concentration of ANF (10 microM), 50% inhibition tII BzOPx'ase was not observed. The results were similar to those obtained with the total lung microsomal fraction. 6. Microsomal and tII BzOPx'ase activities exhibited non-linear dependence at low enzyme concentration. Linearity was restored by 0.5 mM dimyristoylphosphatidylcholine.

Cytochrome P450-dependent alkoxyphenoxazone dealkylase activity in rat alveolar type II cells: effect of pretreatment with beta-naphthoflavone.

Cytochrome P450-dependent alkoxyphenoxazone dealkylase activity was measured in alveolar type II cells from control and beta-naphthoflavone (ip) treated-rats. Type II cells were isolated from collagenase/elastase-digested lung tissue and purified by centrifugal elutriation. The specificity of the cytochrome P450-dependent activity towards four alkoxyphenoxazones (methoxy-, ethoxy-, pentoxy-, and benzyloxyphenoxazone) was measured under conditions that minimized interference by cytosolic conjugating- and NADPH-dependent quinone reductase activities. Ethoxyphenoxazone dealkylase activity was induced 17-fold following beta-naphthoflavone treatment and was further characterized by its kinetic parameters and sensitivities toward in vitro inhibitors (Km(app) = 0.20 microM, Vmax = 1.74 pmoles resorufin min-1 (10(6) cells)-1 10(6) cells; I50 (alpha-naphthoflavone) = 0.025 microM, and I50 (metyrapone) = 72 microM). beta-Naphthoflavone pretreatment of the rats did not result in statistically significant changes in methoxy-, pentoxy-, or benzyloxyphenoxazone dealkylase activity of alveolar type II cells, although, a trend towards decrease activity was observed for benzyloxyphenoxazone. beta-Naphthoflavone pretreatment had no effect on oxygen consumption or trypan blue exclusion in alveolar type II cells and macrophage ethoxyphenoxazone dealkylase and benzyloxphenoxazone dealkylase activities were not affected by the beta-naththoflavone pretreatment. The results show that exposure to beta-naphthoflavone resulted in an increase in type II cell cytochrome P450-dependent ethoxyphenoxazone dealkylase activity but not in other alveolar type II cell or macrophage alkoxyphenoxazone dealkylase activities or in parameters that monitor viability and cell wall integrity.

Nonideal behavior of alkoxyphenoxazone compounds (cytochrome P-450 substrates) in aqueous solution.

Spectral properties of the cytochrome P-450 substrates, methoxy-, ethoxy-, pentoxy-, and benzyloxyphenoxazone (MeOPx, EtOPx, PeOPx, and BzOPx, respectively) were investigated from 350 to 600 nm in ethanol and aqueous buffer. In ethanol, each alkoxyphenoxazone displayed a lambda max at 460 nm and a shoulder around 390 nm. Extinction coefficients (EmM) in ethanol were calculated as MeOPx, 20.5; EtOPx, 20.4; PeOPx, 24.7; and BzOPx, 22.4. In aqueous buffer, only MeOPx obeyed the Lambert-Beer law (lambda max = 480 nm, EmM = 22.1). Three substrates, EtOPx, PeOPx, and BzOPx, displayed anomalous behavior in aqueous solution, wherein the lambda max shifted to lower wavelengths (480-430 nm) and EmM (apparent) decreased as the alkoxyphenoxazone concentration increased. This behavior was dependent on the side chain, and the concentrations at which the spectral changes took place were estimated as: BzOPx, 2 microM; PeOPx, 5 microM; EtOPx, 17 microM; and MeOPx, greater than 20 microM. The blue shift and decreased EmM (apparent) observed for PeOPx at high concentration in aqueous buffer was reversed at high temperature. Unlike EtOPx, PeOPx, and BzOPx, and like MeOPx, hydroxyphenoxazone (resorufin) and unsubstituted phenoxazone obeyed the Lambert-Beer law in aqueous buffer and ethanol. The data suggest that the pentoxy and benzyloxy substituents facilitated a self-association process among the phenoxazones in aqueous solution. The data further show that aqueous solutions should be avoided when spectral data are used to determine alkoxyphenoxazone concentrations.

The in vitro effects of alkanes, alcohols, and ketones on rat lung cytochrome P450-dependent alkoxyphenoxazone dealkylase activities.

The sensitivities of cytochrome P450 (EC 1.14.14.1)-dependent benzyloxy- and ethoxyphenoxazone dealkylase (BzOPh'ase and EtOPh'ase, respectively) activities towards a series of aliphatic hydrocarbons were measured in the microsomal fraction of lung obtained from beta-naphthoflavone-treated rats. The unsubstituted hydrocarbons were straight-chain (n-hexane through n-undecane) and branch-chain (eight carbons). The substituted compounds were alcohols and ketones of hexane and octane. The data are expressed as I50 values, i.e. the hydrocarbon concentration required to cause 50% decrease in the rate of enzyme-catalyzed product (resorufin) formation. The unsubstituted aliphatic hydrocarbons exhibited I50 values towards BzOPh'ase from 0.76 microM (2,5-dimethylhexane) to 8.8 microM (n-hexane). The lung EtOPh'ase activity was insensitive towards the tested unsubstituted aliphatic hydrocarbons. When the alcohols and ketones of hexane and octane were tested against lung BzOPh'ase activity, I50 values ranged from 16 microM (1-octanol) to 4.8 mM (2,5-hexanedione). Lung EtOPh'ase activity exhibited some sensitivity towards the alcohols and ketones, and I50 values ranged from 0.52 mM (4-octanol) to 40.5 mM (2-hexanol). The data show rat lung BzOPh'ase and EtOPh'ase activities are differentially sensitive towards the selected unsubstituted aliphatic hydrocarbons and corresponding alcohols and ketones. The difference in sensitivities may reflect different requirements for an adventitious interaction between a hydrocarbon and enzyme active site.

The non-linear dependence of cytochrome P450 activity at low microsome concentration.

A non-linear decrease in the activity of cytochrome P450-dependent (P450) ethoxyphenoxazone deethylase was observed with intact rat liver and lung microsomal fractions, although all components of the P450 complex were present. Activity was restored by adding pre-heated microsomal membranes or synthetic phospholipid, or by concentrating the diluted preparation. Aqueous dilution of the microsomal fraction resulted in altered Vmax values, whereas Km(app) values (0.2 microM) were only slightly changed. The results are discussed in terms of the relationship between cytochrome P450 action in model systems and in native microsomal membranes.

Cytochrome P450-dependent alkoxyphenoxazone dealkylase activities in lung microsomes from untreated and beta-naphthoflavone-treated rats: effect of in vitro inhibitors.

The reactivities of four alkoxyphenoxazone derivatives toward cytochrome P450-dependent monooxygenase activity were studied in lung microsomes from control and beta-naphthoflavone (BNF)-treated rats. Ethoxyphenoxazone (EtOPh)- and methoxyphenoxazone (MeOPh)-dealkylases were induced by beta NF (16- and 3.9-fold respectively), whereas benzyloxyphenoxazone (BzOPh)- and pentoxyphenoxazone (PeOPh)-dealkylase activities were unchanged after pre-treatment with beta NF. The specific activities of BzOPh'ase from control- and beta NF-treated rats and EtOPh'ase from beta NF-treated rats were sufficiently high (214-560 pmoles resorufin per min per mg protein) for routine biochemical studies. Each differed in its sensitivity toward the in vitro inhibitors, alpha-naphthoflavone (ANF) and metyrapone (MP). In lung microsomes from beta NF-treated rats the I50 (MP) = 7.6 X 10(-5) M for EtOPh'ase and 1.0 X 10(-6) M for BzOPh'ase. I50 (ANF) = 3.8 X 10(-8) M for EtOPh'ase. I50 (ANF) for BzOPh'ase could not be determined; at the highest concentration of ANF (10(-3) M), 50% inhibition was not observed. The presence of high levels of two distinct forms of lung microsomal P450-dependent alkoxyphenoxazone dealkylases, BzOPh'ase from untreated and beta NF-treated rats and EtOPh'ase from beta NF-treated rats, will be useful for studies on specific P450-xenobiotic interactions in rat pulmonary tissue.

Influenza virus-induced alterations of cytochrome P-450 enzyme activities following exposure of mice to coal and diesel particulates.

We have investigated a relationship between two detoxication systems, metabolic detoxication through the cytochrome P-450 (P-450) pathway and resistance to infection through interferon (IFN), in mice infected with influenza virus following exposure to coal dust (CD) and diesel exhaust (DE) particulates. Mice were exposed by inhalation to filtered air (FA; control), CD, or DE for 1 month and then inoculated intranasally (IN) with influenza virus. During infection, 7-ethoxycoumarin deethylase (7ECdeEt'ase) and ethylmorphine demethylase (EMdeMe'ase) (monooxygenases), and NADPH cytochrome c reductase (NADPH c red'ase) were measured in liver microsomes. Temporal patterns of enzyme activities were observed with control animals. EMdeMe'ase and NADPH c red'ase exhibited peak values at Day 4 postinfection (27.6 and 482 nmole/min/mg protein, respectively), compared to initial activities (9.1 and 307 nmole/min/mg protein, respectively). 7ECdeEt'ase activity decreased between Days 1-3 postvirus infection and thereafter returned to the original value (1.7 nmole/min/mg protein). When the mice were first exposed to CD or DE particulates for 1 month prior to influenza infection, changes in enzyme temporal patterns were observed. The increased EMdeMe'ase activity at Day 4 was not observed in mice exposed to CD and was reduced in mice exposed to DE. Preexposure to either particulate resulted in the abolition of the increased Day 4 activity of NADPH c red'ase. The 7ECdeEt'ase postinfection temporal pattern was not affected by a preexposure to either particulate. Estimates of the enzyme activities after the 1-month exposure to FA, CD, or DE but before virus infection indicated no changes due to particulate exposure alone. Under these conditions of particulate exposure and virus infection, serum IFN levels in the mice used in this study peaked at Days 4-5 and were unaffected by the 1-month preexposure to CD or DE (Hahon et al., (1985). The data suggest the relationship that exists between metabolic detoxication and resistance to infection in normal mice was altered during a short-term preexposure to CD or DE.

In vitro effects of straight-chain alkanes (n-hexane through n-dodecane) on rat liver and lung cytochrome P-450.

To evaluate the effect of straight-chain alkanes on normal detoxication reactions, we studied the in vitro effect of the homologous series n-hexane through n-dodecane on two cytochrome P-450 (EC 1.14.14.1) enzyme activities. Benzo[a]pyrene hydroxylase (BaPOHase) and 7-ethoxycoumarin deethylase activities were measured in liver and lung microsomes of control and beta-naphthoflavone-treated rats. In the presence of 2 mM n-hexane through n-dodecane, liver BaPOHase activity decreased from 67% of control with n-dodecane to 21% of control with octane. Lung benzo[a]pyrene hydroxylase was insensitive to all tested alkanes at 2 mM. In the presence of 2 mM alkanes, liver 7-ethoxycoumarin deethylase activity decreased from 73% of control with n-octane to 28% with n-octane. Lung 7-ethoxycoumarin deethylase was also sensitive to the alkane series. In the presence of 2 mM alkane the greatest effect was obtained with n-octane and represented a 56% loss in activity. Alkane concentration-dependence measurements showed 0.02-0.20 mM as the sensitive region of the curve for n-octane with maximal loss of activity achieved at 0.20 mM. Liver ethoxycoumarin deethylase activity from beta-naphthoflavone-treated rats was less sensitive towards the reactive alkane, n-octane, than the activity from control rats. Double-reciprocal-plot analysis revealed the maximal velocity (Vmax) was decreased in the presence of 0.2 mM n-octane. Hence this hydrocarbon did not exert its effect solely as an alternate substrate. The data show the n-alkanes, n-hexane through n-dodecane, interfered with a normal detoxication pathway in a manner that was chainlength-dependent, tissue-specific, and dependent on the preexposure history of the animal.

Relative effects of asbestos and wollastonite on alveolar macrophages.

Rabbit alveolar macrophages were exposed in culture to chrysotile asbestos, wollastonite, or latex, and the effects on various biochemical and physiological parameters related to cellular viability and fibrogenicity were determined. Exposure of alveolar macrophages to asbestos, wollastonite, or latex for 3 d has no effect on oxygen consumption or cellular volume. However, treatment of alveolar macrophages with as little as 25 micrograms asbestos/ml for 1 d increases lysosomal enzyme release and decreases membrane integrity, i.e., decreases trypan blue exclusion and increases leakage of cytosolic enzymes. In contrast, exposure of alveolar macrophages to wollastonite or latex at 250 micrograms/ml does not induce lysosomal enzyme release or alter membrane integrity even after 3 d of exposure in culture. These data suggest that chrysotile asbestos damages rabbit alveolar macrophages, while wollastonite, a potential substitute for asbestos, is far less cytotoxic.