Histological and Ultrastructure Changes Induced by Di {2-ethylhexyl} Phthalate (dehp) in the Alveolar Tissue of Adult Albino Rats and the Possibility of Recovery

Phthalates are materials with a wide spectrum of industrial applications. High molecular weight phthalates are primarily used as plasticizers in the manufacture of flexible vinyl. Low molecular weight phthalates are used in personal-care products, as solvents for cellulose acetate, and in making lacquers, varnishes, and coatings [1]. A quarter of the plasticizers produced are made of di-(2-ethylhexyl) phthalate (DEHP). DEHP is a commonly used phthalate plasticizer in polyvinylchloride (PVC) formulations. It has a wide variety of applications from cosmetics and food packaging to medical devices, and it makes PVC tubing soft and flexible. PVC is used in a range of medical devices from intravenous (IV) fluid containers, blood bags to medical tubing (enteral feeding tubes, respiratory tubing) and catheters [2].


Introduction
Phthalates are materials with a wide spectrum of industrial applications. High molecular weight phthalates are primarily used as plasticizers in the manufacture of flexible vinyl. Low molecular weight phthalates are used in personal-care products, as solvents for cellulose acetate, and in making lacquers, varnishes, and coatings [1]. A quarter of the plasticizers produced are made of di-(2-ethylhexyl) phthalate (DEHP). DEHP is a commonly used phthalate plasticizer in polyvinylchloride (PVC) formulations. It has a wide variety of applications from cosmetics and food packaging to medical devices, and it makes PVC tubing soft and flexible. PVC is used in a range of medical devices from intravenous (IV) fluid containers, blood bags to medical tubing (enteral feeding tubes, respiratory tubing) and catheters [2].
Because Di {2-ethylhexyl} phthalate (DEHP) is not chemically bound to the polymer in a PVC medical device and it is highly lipophilic, it can be released when the device is heated or it can leach out when the device comes into contact with certain media, such as blood or other lipid-containing solutions [2,3]. The daily human exposure to DEHP worldwide is estimated to be in range from 3-30 µg/kg/day in adults, and up to 85 µg /day in children , while exposure in the medical settings (via plastic tubing, IV bags, etc.), may increase up to 2 mg/day. DEHP exposures in the medical setting are of particular concern because the amount of exposure can be substantial and because those exposed, such as premature infants and other neonates or adults with life threatening illnesses, may be particularly vulnerable to the effect of toxic chemicals [2,4].
DEHP can be absorbed from the gastrointestinal tract, the lungs and through the skin [5]. It is rapidly metabolized by pancreatic lipase to mono-2-ethylhexyl phthalate (MEHP) and 2-ethylhexanol. The MEHP is immediately further oxidized into a variety of polar products in vivo as well as in vitro [6]. The well characterized reproductive toxicity and hepatocarcinogenecity exerted by DEHP in rodents have been suggested to be caused by MEHP [7][8][9].
Pregnant female rats dosed with DEHP in their diet during their last week of pregnancy and for two days following birth had pups with a substantial decrease in the number of lung parenchymal airspaces, a significant increase in the airspace mean size, and an increase in the number of type II pneumocytes [10]. Similar "alveolar simplification" in addition to increased epithelial and mesenchymal cell proliferation was also reported in the distal lung parenchyma of rat pups treated under similar conditions and they referred these changes to unknown mechanisms [11].
As mentioned earlier, most of the available data are related to fetal lung changes due to DEHP exposure, because of that; the current study was designed to investigate the histological changes in adult respiratory alveoli induced by DEHP and the possibility of recovery after its withdrawal using light and electron microscopy.

Materials and Methods
Animals 30 adult male albino rats weighing 200-220 gm were used in this study. They were maintained at a 12-h day and 12-h night cycle. They were housed in clean properly ventilated cages under the same environmental conditions with free access to food and water for an acclimating pre-experimentation period of one week and throughout the whole period of the experiment.

Study groups
The animals were divided into three groups (10 rats each). All animals received the following via orogastric intubation once daily for 2 weeks: Group I (The control group): 2.85 ml/kg BW/day of normal saline.
Group II (The DEHP treated group): 2.85 mg/kg BW/day of DEHP [12] dissolved in normal saline (purchased from Aldrich chemical company, Germany).
Group III (The DEHP recovery group): Animals treated with 2.85 mg/kg BW/day of DEHP dissolved in normal saline for 2 weeks, and then left free without DEHP treatment for another 2 weeks.
At the end of the experiment, all rats were sacrificed by decapitation. Specimens from right lung were processed for light and transmission electron microscopes examination.

Histological study
For light microscope, the specimens were fixed in 10% formalin for 12 hours to prepare paraffin blocks. Sections (5 μm) were prepared and stained by H&E stain and Masson's trichrome [13]. For transmission electron microscope, the specimens were immediately fixed in 2.5% glutaraldhyde solution for 24 hours at 4°C, processed for preparing semithin sections (0.5 µm) to be stained by 1% toluidine blue and examined with light microscope. Then ultrathin sections (70-90 nm) were prepared and stained with lead citrate followed by uranyl acetate [14]. Stained sections were examined with a JEOL 1010 Transmission Electron Microscope in the Regional Center for Mycology and Biotechnology (RCMB) at Al-Azhar University [14].
2. Alveolar septal changes such as thickening, decrease or increase of connective tissuefibers and pulmonary interstitial changes (mononuclear cellular infiltration).
3. Vascular changes such as, congestion of pulmonary vessels and hemorrhage.

Morphometric analysis
The image analyzer (Super eye-Heidi soft) in Histology department, Faculty of Medicine, Suez Canal University was used to obtain the following morphometric data: • The mean thickness of interalveolar septa using H&E stained sections at x400 magnification.
• The mean number of type II pneumocytes/ field using oil immersion lens in H&E stained sections.
• The mean number of alveolar macrophages/ field using oil immersion lens in toluidine blue stained semithin sections.
• The mean area percentage of collagen fibers using Masson's trichrome stained sections at x400 magnification.
The previous measurements were estimated in five non-overlapping fields/section in five serial sections/rat from each animal in each group.

Statistical analysis
All statistical analysis was performed using the statistical software package SPSS 15.0 for Windows® (SPSS Inc., Chicago, IL, USA). The obtained data were expressed as mean ± standard deviation and analyzed using analysis of variance (ANOVA). Statistical significance level was defined as P<0.05.

The control group (group I)
H&E stained sections of the alveolar tissue showed normal histological architecture; numerous clear alveoli with thin interalveolar septa, and clearly seen alveolar sacs. The alveolar epithelium showed extremely flattened type I pneumocytes with densely stained flattened nuclei and very thin cytoplasm; and cuboidal type II pneumocytes with large dark stained rounded nuclei, which were commonly located near the angles between neighboring alveolar septa ( Figure 1).
Masson's trichrome stained sections revealed normal distribution of thin collagen bundles in pulmonary interstitium around alveolar sacs and in interalveolar septa ( Figure 2). Touidine blue stained semithin sections showed thin interalveolar septa that lined by alveolar epithelium which were: flattened squamous cells, type I pneumocytes with its deeply stained nuclei and type II pneumocytes with its irregular cuboidal shape, large dark stained rounded nuclei, with prominent nucleolus and vacuolated cytoplasm. Alveolar macrophages were seen with its characteristic eccentric kidney-shaped nuclei, irregular outlines and cytoplasmic granules ( Figure 3 and 4).

Transmission electron microscopic results
Ultra thin sections of alveolar tissue of the control group showed empty and clear alveolar spaces. The walls of the alveoli were lined with type I "squamous" epithelial cells and type II epithelial cells. Large elongated type I pneumocytes showed their morphologic characteristics flattened nucleus and large flat surfaces lacking microvilli. On the other hand, type II pneumocytes appeared much larger in size, with large rounded nuclei showing peripheral condensed chromatin. Their cytoplasm contained numerous dense mitochondria; membrane bounded lamellar bodies containing electron-dense secretions, surfactant. Short microvilli were seen projecting from type II penumocytes borders ( Figure 5).
Also, alveolar macrophages were evident by their characteristic indentable nuclei, short microvilli and lysosomes with different densities ( Figure 6).      Some desquamated type II pneumocytes were found in the alveolar spaces. Interstitial hemorrhage with extravasated red blood corpuscles in the alveolar spaces and in the pulmonary interstitium were also seen ( Figure 7). Comparing this group to control group, there was highly statistically significant increase in the number of type II pneumocyte and the thickness of the interalveolar septa (P < 0.001) ( Table 1).
Masson's trichrome stained sections revealed marked increase in deposition of collagen bundles in pulmonary interstitium around alveolar sacs and in interalveolar septum ( Figure 10). This was confirmed by a highly significant increase in the mean color area percentage of collagen fibers in this group compared to group I (the control group) (P<0.001) (Figure 11).

The DEHP treated group (group II)
H&E stained sections of the alveolar tissue in DEHP treated group showed marked increase in the thickness of the interalveolar septa with interstitial hemorrhage (Figure 7). Inflammatory cellular infiltration; mainly perivascular, collapse of some alveoli was also noticed (Figure 8). At the same time, there were apparent changes in type II pneumocyte; which showed necrotic changes in the form of vacuolated or deeply eosincophilic cytoplasm with pyknotic or karyorrhectic nuclei, enlarged nuclei with chromatin margination (Figure 7 and 8). Some nuclei showed mitotic figures (Figure 7 and 9).      Touidine blue stained sections showed thickened interalveolar septa, disrubted alveolar architecture, increased number of type II pneumocyte showing chromatin margination and/or karyolytic nuclei. Increased number of macrophages surrounding collapsed alveoli was evident. There was highly statistically significant increase in the number of alveolar macrophages/field when compared to group I (P<0.001); ( Figure 12) (Table 1).

Transmission electron microscopic results
Using TEM, type II pneumocytes showed cytoplasmic vacuolation and destruction of mitochondrial cristae ( Figure 13). Lamellar bodies appeared empty and the short microvilli were lost from the free surfaces of most of type II pneumocytes (Figure 12 and 14). Some nuclei were condensed with loss of normal chromatin pattern ( Figure 14); others appeared dividing (Figure 12). No changes were observed in type I pneumocytes which appeared unaffected. The alveolar macrophages were numerous and showed absence of lysosomes with loss of microvilli ( Figure 15).

The DEHP recovery group (group III)
H&E stained sections of the alveolar tissue in DEHP recovery group showed less changes compared to group II. The interalveolar septa were not as thick as in the group II. There was less inflammatory cellular infiltration ( Figure 16). Some alveoli showed extravasated blood cells in their lumen. Type II pneumocytes showed cytoplasmic degenerative changes and some necrotic type II pneumocyte were also found ( Figure  17). There was statistically significant increase in the number of type II pneumocyte and the thickness of the interalveolar septa when compared to group I (P < 0.05) and there was statistically significant decrease when compared to group II (P < 0.05); (Table 1).
Masson's trichrome stained sections revealed increased deposition of collagen bundles in the interalveolar septum and also in the wall of dilated congested pulmonary vessel wall when compared to control group ( Figure 18). There was statistical significant increase in the mean color area percentage of collagen fibers in this group compared to group I (P < 0.05) at the same time, there was statistical significant decrease when compared to group II (P<0.05); (Figure 19).
Touidine blue stained sections showed less thickened interalveolar septa with some type II pneumocyte showing chromatin margination.     Alveolar macrophages were more than that seen in control group. There was statistically significant increase in the number of alveolar macrophages/field when compared to group I (P < 0.05) and there was statistically significant decrease in their number when compared to group II (P < 0.05); (Figure 20) (Table 1).

Transmission electron microscopic results
Ultra thin sections of alveolar tissue of the DEHP recovered group showing mild improvement compared to DEHP treated group. Some of type Il pneumocytes regained their normal appearance as their nuclei showed normal pattern of chromatin distribution, and partially empty lamellar bodies (Figure 21). Some alveolar macrophages were seen in the interalveolar septa still with loss of their lysosomes and short microvilli ( Figure 11).

Discussion
DEHP is known to be released with time into the biological fluids and redistributed into various tissues causing a harmful effect on the health [10,15].In laboratory animals, DEHP administration showed toxic effect on several organs, particularly the lung [11]. The present study examined the histological changes in rat`s lung treated with di(2ethyl-hexyl) phthalate (DEHP) .
In agreement with the data obtained from several studies [16,17], our results showed that the lung of rats was found to be sensible to DEHP toxicity. The alveolar tissues from DEHP-treated rats revealed significant increase in the mean thickness of alveolar septa (31.24 **± 3.322), the number of macrophages (9.5**± 1.581), type II pneumocyte (9.1**± 1.3) and the mean area percentage of collagen fibers deposition; when compared with the normal animal tissues. Also, the alveolar tissue from DEHP-treated rats showed variation of the alveolar shape and size, where some were obliterated and others were dilated; but all revealed thickened wall, infiltrated with more inflammatory cells. Also, inflammatory cellular infiltration had been noticed around the thickened pulmonary blood vessels. These changes are commonly described with severe airway injury, fibrosis and chronic inflammatory lung diseases [18][19][20]. This may be the result of phthalate-induced increases in oxidative stress or inflammation in animal tissues. Oxidative stress and inflammation are known to be interrelated, and oxidative stress-stimulated inflammation has been proposed to be part of the etiologic pathway for DEHP-induced tumorigenesis [21]. However, DEHP is supposed to act through peroxisome proliferatoractivated receptors (PPARs) that were over-expressed in cultured type II pneumocytes. Overexpression of peroxisome proliferator-activated receptor-γ (PPARγ) in type II pneumocytes induces inflammatory reaction, inflammatory cell infiltration and T-cell suppression in the lung tissue [17].
Our results also indicated increase in the number of type II pneumocytes in treated animals with disturbed lamellar bodies which was also found in a similar study [10]. Surprisingly, a similar increase in type II pneumocytes has occasionally seen in children affected with hyaline membrane disease [18], a pathology to which DEHP leakage from PVC tubes has been suggested to contribute [22].The increased number of type II pneumocytes might depend on delayed differentiation    into flattened type I pneumocytes, or might due to cellular proliferation. However, in this study we have noticed many mitotic figures in type II pneumocytes that could due to the effect of peroxisomal proliferation [17]. Also, chronic inflammatory process induces conversion of bone marrow mesenchymal stem cells into type II pneumocytes [23], leading to increase their number. Additionally, PPARs help in increasing translocation of triglycerides to type II pneumocytes and keep them in the surfactant secreting phenotype and preventing these cells from migration and conversion into flattened type I pneumocytes [10].
On the other hand, our electron microscopic results showed many morphological changes in type II pneumocytes in animals treated with DEHP. The changes included nuclear chromatin margination and condensation, cytoplasmic vaculation and disturbed mitochondrial cristae. These changes in type II pneumocytes are similar to the apoptotic and necrotic picture that was described in many studies [24][25][26] and was contributed due to the toxic effect of DEHP. Yao et al. [25] demonstrated that exposure to DEHP results in the enhanced production of Tumor Necrosis Factor-A (TNFA) and its consequent initiation of cell apoptosis through the activation of the FASL/FAS signaling pathway [25].
The disturbed lamellar bodies that we had noticed in this study might affect the surfactant production, secretion and/or readsorption by type II pneumocytes [21].Also, our result showed loss of lysosomes from macrophages, which is in agreement with others, where they have reported that DEHP-induced release of lysosomal enzymes from cultured alveolar macrophages associated with constriction and edema of pulmonary vessels [27,28].
After stopping DEHP administration for 2 weeks, the lung alveolar tissues showed mild histological and ultrastructure improvement, but complete recovery was not achieved. This was evident by the reduction of the number of type II pneumocyte, alveolar macrophages and decreased thickness of interalveolar septa in this group when compared to DEHP treated group. The absence of normal alveolar tissue architecture indicates that the DEHP induced chronic lung toxicity that seems to be duration or dose dependant [29].
Unfortunately, there was little information about the parameters determining the degree by which DEHP migrates from medical devices as temperature, surface-to-volume ratios, nature of surrounding media and its agitation, storage time, thermodynamic properties of DEHP as its vapor-pressure and degree of PVC degradation. The influence of these parameters on the leaching of DEHP is important in assessing the best condition for use of medical devices to reduce the release of the plasticizer and, as a consequence, its absorption from the patients. Other points which are unclear; the DEHP metabolizing rates across species and ages, actual toxic concentrations of DEHP and/ or its metabolites, and the different administration routes in order to minimize the harmful effect of DEHP.