Toxicologic Pathology

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Analysis of Lung Damage Induced by Trichloroethylene Inhalation in Mice Fed Diets with Low, Normal, and High Copper Content Anna Giovanetti, Luisa Rossi, Mariateresa Mancuso, Carmine C. Lombardi, Maria Rita Marasco, Fedele Manna, Pierluigi Altavista and Eddy M. Massa Toxicol Pathol 1998 26: 628 DOI: 10.1177/019262339802600506 The online version of this article can be found at: http://tpx.sagepub.com/content/26/5/628

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TOXICOLOGIC PATIIOLOGY. vol. 26, no. 5, pp. 628-635, 1998 Copyright 0 1998 by the Society of Toxicologic Pathologists

Analysis of Lung Damage Induced by Trichloroethylene Inhalation in Mice Fed Diets with Low, Normal, and High Copper Content* ANNAGIOVANE-ITI,' LUISA ROSSI,~ MARIATERESA MANCUSO,' CARMINE C. LOMBARDI,' MARIARITAMARASCO,~ FEDELE MANNA,3 PIERLUIGI ALTAVISTA,' AND EDDY M. MASSA4 'Section of Toxicology and Biomedical Sciences, ENEA CR Casaccia, Rome. Italy, *Departriieritof Biology, University "Tor Vergatn," Rome. Itafy, 'Department of Chemical arid Technological Studies of Biological Active Sirbstarices, University "La Sapienza, " Rome, Italy, and JINSIBIO, Universidad Nacional de Tircirnian, S. M. de Tircirnian, Argentina ABSTRAC~ Copper is both an essential nutrient required for the activity of several enzymes and a toxic element able to catalyze free radical formation. Trichloroethylene (TCE) is a xenobiotic that generates epoxidic intermediates by bioactivation through the cytochrome P450 system. In this study, the influence of a dietary copper imbalance on the TCE-induced lung damage was investigated. Weaning mice were fed copper-deficient, copper-sufficient. and copper-excessive diets. After 4 wk, mice were exposed for 30 min to 6,500 ppm of TCE and euthanatized 48 hr later. Lung damage in the TCE-treated mice consisted of vacuolations of Clara cells and was quantitatively evaluated by counting the vacuolated cells per micrometer of basal lamina. At the ultrastructural level, vacuolations appeared as the result of hydropic swelling of the endoplasmic reticulum cisternae. The copper-deficient mice presented the highest number of vacuolated Clara cells. These mice also showed alteration of the capillary endothelium and interstitium and decreased pulmonary copper-zinc-superoxide dismutase activity. Occurrence of oxidative stress in lungs of both copper-sufficient and coppcrdeficient mice following TCE treatment was indicated by a decrease in reduced glutathione and an increase in its oxidized form.

A'ejwmfs.

Pulmonary toxicity; dietary copper; Clara cells; electron microscopy; antioxidant defense system

INTRODUCTION

Trichloroethylene (TCE) is a halogenate aliphatic hydrocarbon widely employed in industry; it is also used Copper (CU) is an essential nutrient for most animals, as a solvent for active components of household products including the mouse, and is required for numerous bioand pesticides. Professional and indoor use of TCE can. chemical and physiological functions (17). Nutritional result in low-concentration long-term or high-concentracopper deficiency occurs in humans under a variety of tion acute exposures (41). TCE is metabolized by the conditions, and unrecognized or marginal deficiency may cytochrome P-450 enzymatic system, giving rise to toxic be much more common than previously realized (22). intermediates (21). In the lung, the P-450system is mainMuch of the pathology of copper deficiency may be ly located in the smooth endoplasmic reticulum (SER) of traced to metabolic defects involving various cuproenthe nonciliated Clara cells (4, 36). Clara cells have been zymes, such as cytochrome c oxidase and copper-zincdemonstrated as the target of xenobiotics requiring bioacsuperoxide dismutase, whose activities are generally detivation (5). Previous exposure to TCE vapors has propressed by copper deprivation (22). Furthermore, noncuproenzymes, such as glutathione peroxidase which has duced selective damage in mouse Clara cells, consisting an important role in removing hydrogen peroxide, are of hydropic swelling of SER cisternae and consequent known to change under conditions of dietary copper decreation of vacuoles of various sizes. Clara cells can be ficiency (28, 29, 42). so severely injured as to become necrotic and lost in the The toxic effects of copper at high levels are generally lumen. Damage was dose dependent and reached its maxattributed to its capacity for catalyzing formation of free imum 24 hr after exposure ceased. At 48 hr, repair of radicals, leading to alteration of nucleic acids, lipids, and bronchial epitheliqm was evident (40). proteins (16, 38). Copper compounds are widely used as Nothing is known about the effects of superimposing pesticides, antimicrobial sprays on agricultural crops, and the environmental stress of TCE exposure upon nutriveterinary food additives and for the preservation of nattional stress of copper imbalance. Because copper defiural and human-made materials (7, 10, 19). Toxic conciency may depress the antioxidant defense system (8, centrations of copper are found associated with certain 25, 28, 29, 42), making the cells susceptible to toxic inindustrial and agricultural discharges, which are a major termediates, a negative interaction between low-copper source of pollution (9). diet and TCE exposure could occur, especially within Clara cells as targets of xenobiotics requiring bioactiva* Address correspondence to: Dr. Anna Giovanetti. Section of Toxition. However, an excess of dietary copper usually leads cology and Biomedical Sciences, ENEA CR Casaccia sp 015. Via Anto enhanced tissue copper content and might become an guillarese 301, 1-00060 Rome, Italy; e-mail: giovanetti@casaccia. eneait. aggravating stress factor that could adversely alter the 628 0 192-623319853.OO+ $0.00 ~


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TCE, DIETARY COPPER, AND LUNG DAMAGE

effects of TCE inhalation. Thus, in this study we tested whether low or high dietary copper affects TCE toxicity in mouse lung. The analysis included morphometric evaluation of the damage and biochemical measurement of components of the antioxidant defense system, such as the copper-containing enzyme superoxide dismutase and the low-molecular-weight antioxidant glutathione. Possible variations in the content of these molecules are of particular interest in this context because both compounds are involved in antioxidant defense and in intracellular copper homeostasis (34). AND METHODS MATERIALS Aniinals. Twenty-one-day-old male weaning BC3F, mice, weighing 10.65 2 2.25 g, were obtained from the animal facility at the ENEA Research Center (Rome, Italy). They were randomly divided into 3 dietary groups (54 mice/group): copper deficient (CUD), copper sufficient (CuS), and copper excessive (CUE), and housed individually prior to and after TCE exposure in a laminar air flow system (Holten Laminair,, Allergd, Denmark). Water and food were given ad libitunz, and mice were weighed each week. The diets were powdered copperdeficient modified diet (ICN Biomedicals) supplemented with variable concentrations of cupric sulfate. Analyses by atomic absorption spectrometry (AAS) indicated that the Cu contents were 0.44 2 0.04 (CUD), 4.98 & 0.48 (CuS), and 200 2 10 (CUE) mg Cu/kg diet. TCE Exposure. After 4 wk of the respective diets, 9 animals from each dietary group were given a single nose-only exposure to 6,500 ppm TCE (Rudi pont, S. Giuliano Milanese, Italy) for 30 min in an inhalation chamber as described previously (6). For comparison, 9 other animals from each dietary group were sham-exposed to clean air. TCE vapors were generated in 2 sequential bubblers and diluted with chromatographic humidified air. Temperature was maintained at 21 t 2°C with 57 2 4% humidity. Air flow through the inhalation chamber was 5.0 Llmin. TCE concentration was regulated by controlling the bubbler temperature and air flow and was monitored every 4 min by a gas chromatograph (Varian, Sunnyvale, CA) under the following conditions: column temperature = 43°C. injector temperature = 160"C, and detector temperature = 250°C. The TCE retention time was 2.43 min. Variations of TCE concentration were less than 5 2 2%. Following exposure the animals were fed their respective diets until they were euthanatized 48 hr later for evaluation of antioxidants and morphological analysis. A 48 hr time lapse was choosen for comparison with a previous study in which TCE showed a concentration effect relationship at the same experimental point (40). Tissue Copper Level Analysis. Thirty-six unexposed mice from each dietary group were euthanatized by cervical dislocation after 4 wk of the respective diets. Lung and liver were removed immediately and stored at -30°C until analyzed for copper content by dry ashing and AAS as previously described (15). The samples were placed in quartz crucibles and dried to constant weight and then ashed in a muffle furnace at 450-500°C with a gradual increase in temperature over a period of 12 hr. The

629

charred samples were wetted with deionized water, evaporated to dryness on a hot plate, and returned to the oven with a stepwise increase in temperature to 450-500°C for 2 2 hr. This procedure was repeated until the samples were completely combusted (white/gray ashes). The mineral residue was dissolved in an exact volume of 0.1 hi HNO, and diluted appropriately with deionized water for AAS determinations. Evaliiatioit of Antioxidants. Lungs were homogenized in phosphate-buffered saline (1/6, w/v) with a Potter-Elvejem glass homogenizer. Aliquots were utilized for glutathione assay. Reduced (GSH) and oxidized (GSSG) glutathione were measured by high-performance liquid chromatography as previously described (3 1). Total homogenates were then sonicated at 4°C for 3 X 30 sec at amplitude 16 pm by a Soniprep 150 MSE sonifier and centrifuged (30 min, 23,000 X g). Copper-zinc-superoxide dismutase (Cu-Zn SOD) activity was measured in supernatants by a polarographic method (33) at pH 9.6. At the conditions employed, this procedure measures only the Cu-Zn cyanide-inhibitable form of SOD. Data were expressed as micrograms per milligram of protein, with reference to purified enzyme. Protein content was assayed according to Lowry et a1 (18). Morphological Evaluation. To analyze the morphological damage, lungs were fixed in sitid by endotracheal instillation with 0.2 ml of Karnovsky fixative (0.1 M cacodylate buffer, pH 7.4, 1:2 by vol) for 10 min under deep anestehesia by sodium pentobarbital (Abbot Laboratories, North Chicago, IL) and then removed. Samples from the upper, middle, and lower portions of the left lung were postfixed in OsO, 1%. stained as a block in 0.5% uranyl acetate in 0.05 hf maleate buffer (pH 5.2), and embedded in Epon (Polysciences, Warrington, PA) (40). Two-mi-. crometer sections stained with toluidine blue were examined using light microscopy. The epithelial damage was expressed as the number of vacuolated cells per micrometer of basal lamina (V/pm). At least 1,000 Clara cells from throughout the whole bronchial tree were counted from each animal. For ultrastructural analysis, 50-nm sections selected by light microscopy were cut with a diamond knife, stained in uranyl acetate and lead citrate, and examined using an electron microscope (Zeiss EM lo). Three mice were analyzed for each experimental point. Stntistical Analysis. Data on body and organ weights, copper contents, SOD activity, and GSH and GSSG contents were analyzed for significant differences using Student's t-test. Thf: morphometric data, expressed as the number of vacuolated cells per micrometer of basal lamina (V/pm), were transformed by square root (V/pm-SR) to normalize the distribution and to stabilize the variance. To test the differences in V/pm-SR between the dietary groups and the bronchi of different diameter category (50.4 mm and >0.4 mm), the data were analyzed by a mixed model 3-way analysis of variance (ANOVA), considering the dietary groups and the bronchial diameter as fixed independent sources of variation and the individual animal as a random effect. On the basis of the results, post hoc contrasts corrected for multiple comparisons


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TABLE L-Characteristics of mice fed copper-deficient (CUD). copper-sufficient (CuS), and copper-excessive (CUE) diets. Data are given as the mean t standard deviation. n-

Characteristics

CUD

Body weight ( g ) 36 Initial 10.52 36 20.83 Final 1.26 15 Liver weight (g) 18 Lung weight (6) 0.13 Liver Cu 15 5.12 (pg/g dry wt.1 Lung Cu 18 6.08 @g/g dry wt.1 * Significantly different from control (CuS diet) at p < 0.01. ** Significantly different from control (CuS diet) at p < 0.001.

cus

f 2.16 f 2.20;; t 0.17 t 0.02

10.48 22.53 1.37 0.14

t 3.00**

13.75 t 1.65

17.45 t 2.48*

t 0.82**

9.28 t 0.50

11.46 2 0.65**

(24) between the levels of the significant factors were performed.

Conteiit of Antioxidants Table I1 summarizes the effects of copper status and TCE treatment on SOD activity and glutathione content of mouse lung. Deficiency of copper produced a significant decrease (27%) in SOD, but excess copper did not produce any change in the content of this enzyme. As far as glutathione is concerned, only CUEsamples showed a significant decrease in both GSH and GSSG. TCE exposure did not alter SOD activity in any dietary group but did affect glutathione status. GSH decreased both in CuS and CUD mouse lungs; this depletion was accompanied by an increase of GSSG. Conversely, CUEmouse lungs responded to TCE treatment by strongly increasing both GSH and GSSG content, reaching control CuS levels.

Control

CUD

1.38 2 0.17a

cus

1.88 f 0.43 (n = 6) 1.49 ? 0.25 (n = 6)

CUE

10.95 20.14 1.52 0.12

t 1.86 t 0.15

? 0.01

2 1.97 2 1.98'

t 0.11* t 0.01;

The epithelial cells lining the bronchial tree of shamexposed CuS mice consisted of ciliated and nonciliated cells. Light microscopy showed that almost all the nonciliated cells were Clara cells, characterized by protruding, strongly stained cytoplasm and the presence of small dark granules in the apical portion. Ultrastructural features of Clara cells were also observed, such as abundant SER and 2 different populations of mitochondria, 1 of large and round organelles with few cristae and the other of smaller elongated organelles with many cristae. Sham-exposed CUEmice showed no morphological alteration as compared with the CuS mice (not shown). In the CUD group, enlarged alveolar interstitium was observed with a distinctive increase of the cell number and fibers (Fig. 1) and disorganized collagen bundles (Fig. 2). Capillary endothelial cells were thickened in CUD mice (Fig. 3) but were thin and poor in cytoplasmic organelles in the CuS mice. TCE-treated animals showed selective damage of nonciliated cells consisting of various degrees of vacuolation, whereas ciliated cells were undamaged. Vacuolated cells were present throughout the bronchial tree, and the number of nonciliated vacuolated cells per unit of length of basal lamina was independent of bronchial diameter. The CUD group presented the highest number of vacuolated Clara cells per unit of length of basal lamina. V/Fm-SR relative to each animal or dietary group were submitted to factor variance analysis.

superoxide dismutase content and glutathione (GSH, GSSG) status of lungs from CUD, CuS,and CUE mice treated with ICE. Cu-Zn SOD (Irdmg pro[)

Diet

2 2.61

Morphologic Evaluation of Darnage

RESULTS Tissue Copper Concentrations Table I summarizes some characteristics of mice from each dietary group. The mean body weight gain during 4 wk of each diet was slightly but significantly lower for mice eating the CUDand CUEdiets than for those eating the CuS (control) diet. Liver and lung copper concentrations were low in mice fed the CUDdiet and elevated in mice fed the CUE diet compared to the CuS dietary group.

TABLEIL-Cu-Zn

CUE

(n = 6)

GSH

GSSG

(nmoVmg prot)

(nmoVmg prot)

TCE

Control

TCE

Control

TCE

1.49 t 0.34 (n = 6) 1.86 2 0.52 (n'= 6) 1.30 t 0.45 (n = 6)

12.28 f 1.73 (n = 4) 16.56 -t 3.21 (n = 4) 5.29 f 2.78~ (n = 5)

8.14 t 1.28" (n = 4) 8.52 t 0.45b

0.51 f 0.13< (n = 4) 1.07 2 0.21 (n = 6) 0.43 t 0.21< (n = 6 )

1.41 t 0.13" (n = 4) 2.13 t 0.32b (n = 6) 1.57 t 0.74" (n = 6 )

(n = 4)

22.19 t 7.57b (n = 6 )

~~

n = number of animals. * p < 0.05, with respect to control CuS. b p < 0.01. with respect to control of the same dietary group. c p < 0.01, with respect to control CuS.


~~

Vol. 26, NO. 5 , 1998


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FIG.1.-Electron micrograph of lung parenchyma of a CuS (a) and CUD(b) mouse. In the CUD mouse, alveolar interstitiurn is enlarged showing an increase in both the cellular and the matrix components as compared with the control. ALV = alveolar space; CAP = capillary lumen: EP = epithelial cell; EN = endothelial cell; FB = fibroblast. Bar = 5 pm.

The ANOVA model used, with the 3 main variables and the interaction between dietary groups and bronchial diameter, seems to fit very well with the experimental data, as can be seen by the R' value (Table 111). On the basis of the ANOVA table, only the differences between the dietary groups were significant, with p
0.4 mm.

FIG. 2.--CuD mouse. Electron micrograph of alveolar interstitium showing disorganized collagen fibers (cf). E P = epithelial cell. Bar = 1 pm.

damage was confined to Clara cells. Damage consisted of vacuolations of various degrees due to the hydropic swelling of the SER cisternae (Figs. 5 and 6). Larger vacuoles probably resulting from the coalescence of smaller vacuoles could occupy almost the whole cytoplasm. A few cells were necrotic with pyknotic nuclei. DISCUSSION The copper content of the CuS diet was adequate for the copper requirements of the mouse, and liver copper levels in animals of the CuS group were in accordance

with recommended values (32). Liver and lung copper concentrations differed significantly as dietary copper decreased (CUD group) or increased (CUE group), as compared with the CuS group. Rodents fed a copper-deficient diet for several weeks, starting at or before weaning, often have decreased body weight relative to controls (14, 20, 27). In this study, the final body weight in the CUD group was 10% lower than that in the CuS group, similar to data reported.previously (14,20). Decrease in body weight gain can occur without significant change in food consumption (14) or can parallel a decrease in food intake (20). In the present study, the daily food intake was not measured. But even if food consumption were 10% lower in the CUD group relative to the CuS group, this variation would not lead to significant deficiency in essential nutrients (other than copper) because diet formulations are based on requirements that exceed by 15% the estimated mean value for each nutrient to compensate for possible variations in require0.14

YU 2 w

0.12

-

0.10

-

0.08

-

I-

zw

u z w

2z

s

CUD FIG.3.--CuD mouse. Electron micrograph of a thicker capillary endothelial cell (EN). An erythrocyte (ER) is present in the narrowed capillary lumen. Bar = 1 pm.

cus

CUE

FIG.4.-Vacuolated Clara cells per micrometer of basal lamina (V/ pm) in the copper deficient (CUD), copper-sufficient (CuS), and copperexcessive (CUE)dietary groups. Differences between CuS and CUDand between CUD and CUE were significant 0, < 0.001).


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FIG. 5.-Electron micrograph of a bronchial tract of CUD mouse unexposed (a) and exposed (b) to TCE. Epithelium is mainly composed of ciliated and Clara cells. Clara cells (a) are clearly discernible as more electron dense with abundant endoplasmic reticulum. Other Clara cells (b) show various degrees of vacuolation. but ciliated cells are unaffected. NC = nonciliated Clara cell; CC = ciliated cell. Bar = 5 pm.

ments or consumption by individual animals (23). Therefore, the increased susceptibility to TCE of the CUD group was due to copper deficiency and not to another dietary deficiency. Decreased body weight gain in rats and mice fed high-

633

copper diets was attributed to reduced food consumption (13). Such a decrease in body weight gain was also noted in our CUE group, although we did not measure the food intake. The small increase in liver weight in the CUE group could be related to the histological changes reported in copper-loaded rat liver (12). The morphologic effects of copper deficiency on the lung of young animals, characterized by structural deterioration of the alveolar wall, have been attributed to a deficit in the copper-dependent enzyme lysyl oxidase and a resultant inability to cross-link connective tissue proteins (37). Akers and Saari (2), using transmission electron microscopy, showed that CUD rat lungs had thicker endothelial and basement membrane components, and exposure of the CUD animals to hyperoxia caused both enhanced disruption and thickening of the blood-air barrier. These authors concluded that copper deficiency enhances the damage caused by oxygen toxicity, an effect that may be a consequence of reduced antioxidant status. In the present study, we observed in both the TCE-treated and untreated CUD mice a thickening of the capillary endothelium. The endothelial cells, which are normally characterized by their extreme thinness and the scarcity of cytoplasmic organelles, were so enlarged and endowed with organelles in the CUDmice as to fill the whole capillary lumen. However, we did not observe any change in the basement membrane. Alteration in the copper status of the animals can result in changes of the activity of cuproenzymes such as Cu-Zn SOD and noncuproenzymes such as glutathione peroxidase, which are involved in the antioxidant defense (7, 8,25,28,29,42). Evidence is accumulating that indicates that copper-deficient animals are prone to oxidative damage, including the enhanced formation of products of lipid peroxidation (30). Low levels of dietary copper have been reported to compromise the structural integrity of vessel walls by reducing the activity of the enzyme SOD, with subsequent increase of lipid peroxides (2). The typical diet in the USA is low in copper, and this finding has been related to the occurrence of cardiovascular diseases (1). In the present study, a significant decrease of Cu-Zn SOD was observed in lungs of CUD mice. This decrease may lead to increased flux of reactive oxygen species and oxidative stress, thus explaining the enhanced susceptibility of CUD lungs to TCE. This hypothesis is supported by the finding that TCE treatment both in CuS and CUDdietary groups led to a decrease of reduced glutathione content, with concomitant increase of oxidized glutathione, which indicates that oxidative stress was occurring. However, toxicity was observed only in the CUD group, probably because of strengthening of the effect by contemporary decrease of SOD. Recently, enhancement of oxidative damage by the concurrent presence of peroxide and bromotrichloromethane was demonstrated (35). It seems likely that the halocarbon toxicity may be amplified by elevated levels of lipid peroxide, and alkoxyl radicals appear to be closely involved in the mechanism of damage. Thus, the increased number of damaged Clara cells observed in the TCE-treated CUD group might result from the combination of halocarbon and higher levels of lipid peroxides.


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FIG.6.-Higher magnification of 1 nonciliated Clan cell of a TCE-treated mouse. Vacuolations consisted of dilatation of SER cisternae (arrows). Bar = 1 pm.

Particularly interesting are the results on glutathione status obtained for the CUEdietary group, where reduced glutathione decreased in lungs under control conditions. This finding might be explained on the basis of the assumption that copper binds reduced glutathione to form stable complexes (1 1). Because TCE-treatment fully restored glutathione content, it might interfere with copper intracellular trafficking. The maintenance of a correct antioxidant system in CUE lungs may account for the absence of morphological changes following TCE treatment. Selective injury to Clara cells in the pulmonary epithelium following TCE inhalation was probably related to their high numbers of enzymes devoted to halocarbon bioactivation. These cells show a marked activity of P450 isoenzymes bound to the endoplasmic reticulum membranes (28,42). TCE metabolism gives rise to electrophilic epoxides (3) that bind to membrane macromolecules, inducing a disarrangement of membrane structure (39). Thus, the hydropic swelling of SER cisternae that we observed in the TCE-treated mice could be caused by an increase in the SER membrane permeability induced by epoxide generation. In the present study, mice were euthanatized 48 hr after the TCE treatment. In this amount of time, reparative mechanisms are operating, and most of the Clara cells from mice fed a standard diet (with normal Cu content) are restored (40). Therefore, the persistence of vacuolated cells observed in this study in mice fed the CUD diet could be due to a failure in the removal of toxic intermediates or in the reparative mechanisms. In summary, the results of this study 1) confirm that

low dietary copper leads to alterations in the vessel walls and 2) show the influence of copper deficiency on the cellular damage induced by the xenobiotic TCE. In particular, the number of vacuolated Clara cells induced in CUD mice by TCE inhalation increased relative to the CuS mice 48 hr after TCE exposure. The mechanism of this effect has not been established, but alterations in the antioxidant system appear to be involved. A systematic study of xenobiotic metabolism in dietary copper imbalance is required, with emphasis on the number of toxic intermediates generated and possible variations in both the bioactivating and detoxifying enzymes. ACKNOWLEDGMENTS We acknowledge Dr. Stefan0 Rufini and Dr. Maria Balduzzi for their critical review of the manuscript and Mr. Luigi Grisorio for his excellent technical assistance. This work was partially supported by the CNR special project “Glutatione e enzimi antiossidanti.”

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