Impact Effect of Methyl Tertiary-Butyl Ether “Twelve Months Vapor Inhalation Study in Rats”

Simple Summary The detection of major blood-based markers for cancer requires expensive blood tests. Therefore, finding simple and effective blood-based markers is of great interest. The present results suggested that CA II, CA I, and peroxiredoxin2 could be utilized as potential biomarkers for the early detection of tracheal and lung cancer. Abstract We investigated the early risk of developing cancer by inhalation of low doses (60 µL/day) of methyl tertiary butyl ether (MTBE) vapors using protein SDS-PAGE and LC-MS/MS analysis of rat sera. Furthermore, histological alterations were assessed in the trachea and lungs of 60 adult male Wistar rats. SDS-PAGE of blood sera showed three protein bands corresponding to 29, 28, and 21 kDa. Mass spectroscopy was used to identify these three bands. The upper and middle protein bands showed homology to carbonic anhydrase 2 (CA II), whereas the lower protein band showed homology with peroxiredoxin 2. We found that exposure to MTBE resulted in histopathological alterations in the trachea and the lungs. The histological anomalies of trachea and lung showed that the lumen of trachea, bronchi, and air alveoli packed with free and necrotic epithelial cells (epithelialization). The tracheal lamina propria of lung demonstrated aggregation of lymphoid cells, lymphoid hyperplasia, hemorrhage, adenomas, fibroid degeneration, steatosis, foam cells, severe inflammatory cells with monocytic infiltration, edema, hemorrhage. Occluded, congested, and hypertrophied lung arteries in addition, degenerated thyroid follicles, were observed. The hyaline cartilage displayed degeneration, deformation, and abnormal protrusion. In conclusion, our results suggest that inhalation of very low concentrations of the gasoline additive MTBE could induce an increase in protein levels and resulted in histopathological alterations of the trachea and the lungs.


Introduction
Methyl tertiary-butyl ether (MTBE) is a colorless, hydrophilic, flammable liquid that is used as a fuel additive. MTBE is blended to gasoline at 15% volume to obtain a high octane rating and reduce the tailpipe emissions of carbon monoxide and other hydrocarbons [1]. The British Colombia Ministry of Environment guidelines report that the concentration of MTBE in water considered safe for human drinking, freshwater and marine life, and watering livestock ranges from 0.02 to 11 mg/L. Meanwhile, concentrations of MTBE toxic to the growth of plants and animals of the terrestrial or aquatic environment are determined as 4.8 mg/L and 2.5 to 2.9 mg/L, respectively. The LC 50 of MTBE ranges from 44 to >10,000 mg/L for the survival of invertebrates and vertebrate animals [2]. Moreover, workers at the gasoline stations are constantly exposed to MTBE vapors through direct skin contact, inhalation, or ingestion. In such individuals, MTBE has been detected in the blood, breath, and urine specimens [3]. Exposure to high levels of MTBE causes headaches, nausea, dizziness, and difficulty in breathing [4].
Yang et al. [3] reported that inhalation of MTBE (100-300 µg/mL) induced lipid disorders and liver dysfunctions in animal models. The oral LC 50 for MTBE was found to be 3866 and 4000 mg/kg for rats and mice, respectively [5]. Actually, liver adenomas were reported in female mice exposed to high doses of MTBE (3000-8000 ppm) by inhalation [6]. Meanwhile, Moser et al. [7] proposed that 8000 ppm of MTBE could cause hepatocarcinogenic effects on CD-1 and B6C3F1 mice. Similarly, Moser et al. [7] reported that MTBE promoted the formation of liver tumors in female mice. A carcinogenicity study of MTBE revealed that male Wistar rats exposed to high doses of MTBE in drinking water for 2 years developed severe and progressive nephropathy and brain astrocytomas as compared to female rats [1]. Furthermore, Clary [8] reported that rats exposed to MTBE developed severe nephropathy with hyaline droplets in the kidneys. However, renal tubular cell tumors were observed in male mice following MTBE inhalation [9]. They suggested that at exposure to high doses of MTBE, kidney toxicity evolved into nephrocarcinogenesis, suggesting the related role of alpha-2 microglobulin as a biomarker. Luo et al. [10] found some potential cancer biomarkers for MTBE-induced liver cancer; these biomarkers were separated from 800 protein spots on a 2D-PAGE gel from human sera. Moreover, lymphoimmunoblastic lymphoma in the lungs, leukemias, astrocytomas, and Leydig cell adenomas were described in rats exposed to MTBE [6]. The studies related to inhalation of high concentrations of MTBE reported the development of malignancies, such as liver, ovary, testicles, and kidney tumors, in laboratory animals. Furthermore, renal adenomas, nephropathy, tumors in testes, and toxic effects on spermatogenic cells have also been reported [11]. In addition, the effect of MTBE on blood parameters and histological alterations were reported in the bone marrow and immune system, brain, respiratory tract, and skin. Moreover, Prescott-Mathews et al. [9] suggested the role of alpha2u-N in MTBE (3000 ppm) inhalation-induced renal tumorigenesis in male rats. The early detection of cancer is important to increase the survival of patients and to avoid the progression of cancer. Knight et al. [12] reported that lung cancer was the leading cause of cancer-related deaths in the world. Gildea et al. [13] concluded that the difference between the first diagnosis of lung cancer and the final diagnosis impacted the progressive late stages of disease, with a concomitant increase in the associated treatment cost. Therefore, early diagnosis of lung cancer may decrease the resource use and costs among patients. In veterinary medicine, Tothova et al. [14] used serum proteins for the diagnosis. Naz et al. [15] detected diagnostic protein fraction (55-57 kDa) as a biomarker in benign prostatic hyperplasia. With respect to developing cancer biomarkers, Lou et al. [10] recognized an exceptional metabolic biomarker, N-acetylaspartate (NAA) in patients with lung cancer, which is present in inconspicuous levels in normal lung cells. Lou et al. [10] developed a sensitive NAA blood assay and found that elevated levels of NAA were detected in the blood, where it was detected as a metabolite in patients with lung cancer in comparison with the levels in age-matched healthy controls. Other studies indicated a close relationship between MTBE and malignancies in laboratory animals [16]. However, the detection of majority of blood-based markers for cancer requires expensive blood tests. Over the past decade, carbonic anhydrases (CAs) have been shown to be important mediators of tumor Biology 2020, 9, 2 3 of 19 cell pH by modulating the bicarbonate and proton concentrations for cell survival and proliferation. This has prompted researchers to develop methods to inhibit specific CA isoforms and use this as an anti-cancer therapeutic strategy. Of the 12 active CA isoforms, CA IX and XII, have been considered as anti-cancer targets. Other CA isoforms also show similar activity and tissue distribution in cancers; however, these have not been considered as therapeutic targets for cancer. Peroxiredoxin-2 is a protein that in humans is encoded by the PRDX2 gene. This gene encodes a member of the peroxiredoxin family of antioxidant enzymes, which reduces hydrogen peroxide and alkyl hydroperoxides. The encoded protein may play an antioxidant protective role in cells and may contribute to the antiviral activity of CD8 (+) T-cells. This protein may have a proliferative effect and play a role in cancer development or progression [17]. Studying the effect of MTBE inhalation on rats might provide evidence for cancer signs in humans. Considering this, the detection of early diagnostic biomarkers for prospective cancer has become crucial for public health assessment, especially for workers who live in contaminated regions, such as gasoline filling stations and habitations. As a consequence, the present work investigated the possibility of cancer-induced in male Wister rats exposed to low concentrations of MTBE. The objectives of the current work included: (1) to study the effect of MTBE inhalation at a low dose (60 µL/day for 3, 6, and 12 months); (2) to find cost-effective cancer biomarkers for early detection; and (3) to confirm the prospective cancer potency in the trachea and lungs.

Animals
All experiments were conducted using 60 adult male Wistar albino rats weighing 150 to 180 g. These were divided into four groups with 15 rats in each group. Rats were purchased from King Abdulaziz University, Jeddah, KSA. They allowed to adapt for one week under controlled conditions (at 22-25 • C and 50%-55% humidity) before starting experiments at the animal house of the Faculty of Applied Science, Biology Department, Umm Al-Qura University, Makkah, KSA. The Institutional Animal Care and Use Committee approved the protocols for the present study. The animals were used according to the institutional ethical guidelines (Table 1). G1: control Non exposed Non exposed 0-12 months G2: expose to 60 µL/day 3 min 0-3 months G3: expose to 0-6 months G4: expose to 0-12 months

Blood and Tissue Samples
After the exposure period, five rats were selected randomly, sacrificed (under light ether anesthesia), and dissected. The blood samples were obtained in heparinized tubes by cardiac puncture. After centrifugation at 4000 rpm for 15 min at 4 • C, blood sera were collected and kept frozen at −80 • C until electrophoresis analysis. Simultaneously, the tracheae and lungs were extracted and processed routinely for paraffin sectioning according to the method described by Bancroft and Gamble [18,19]. After extraction, these organs were immediately washed in cold physiological saline, fixed in 10% formalin buffer for 48 h, dehydrated in an increasing concentration of ethanol, cleared in xylene, embedded in paraffin blocks, followed by cutting into 3 to 5 micron thick sections. The obtained paraffin sections were then de-paraffinized, hydrated, and stained with hematoxylin and eosin (H&E) to monitor the histopathological changes.

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis
Rat blood sera (10 µg) from each sample were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The total protein of the isolates was determined and protein analysis was performed using 10% SDS-PAGE. Protein samples were reduced by boiling for 5 min in loading buffer containing 5% β-mercaptoethanol. Next, the samples were centrifuged at 10,000× g for 3 min and directly loaded onto the gel. Protein electrophoresis was performed in vertical sub-cells (Bio-Rad, Watford, UK). Slab gels, containing 10% (w/v) resolving gel and 5% stacking gel concentrations of acrylamide, were run at a constant voltage of 80 V for 2 h. Proteins on PAGE gels were fixed in 45% methanol and 10% acetic acid in distilled water and stained in 0.25% Coomassie brilliant blue R-250 (previously dissolved in 10% acetic acid, 50% methanol, and water). The gels were destained for 3 h in 5% methanol and 7% acetic acid in distilled water to visualize protein bands. These bands were photographed in the lightbox according to the standard technique described by Laemmli [20].

LC-MS/MS Analysis
Individual gel pieces were destained and subjected to in-gel digestion using trypsin (T9600; GenDepot, Houston, TX, USA). The tryptic peptides were re-suspended in 10 µL of 5% methanol containing 0.1% formic acid solution and subjected to nanoflow LC-MS/MS analysis on a nano-LC 1000 system (Thermo Scientific) coupled to Q-Exactive Plus Hybrid mass spectrometer from Thermo Scientific. The peptides were initially loaded on a pre-column (2 cm × 100 µm), followed by a 5 cm × 150 µm-analytical columns packed with ReproSil-Pur Basic C18 equilibrated in 0.1% formic acid/water. The peptides were eluted using a 35-min discontinuous gradient of 4% to 26% acetonitrile/0.1% formic acid at a flow rate of 800 nL/min. The eluted peptides were directly electro-sprayed into the mass spectrometer operated in the data-dependent acquisition mode, thereby acquiring higher-energy collisional dissociation (HCD) fragmentation spectra of the top 20 strongest ions. The raw files with MS/MS spectra from the mass spectrometer were searched against target-decoy Rattus norvegicus reference sequence database (released January 2016, containing 60,109 entries) in Proteome Discoverer 1.4 interface (Thermo Fisher) via Mascot algorithm (Mascot 2.4, Matrix Science). The following parameters were used for the search: variable modification of oxidation on methionine and protein N-terminal acetylation; 20 ppm precursor mass tolerance; 0.5 Da fragment mass tolerance; and two missed cleavages. The peptides were identified at a 5% false discovery rate (FDR). The calculated area under the curve of peptides was used to calculate iBAQ for protein abundance using the method described by Sung et al. [21].

Results
Microscopic investigations revealed histopathological alterations in the trachea (Table 2) and the lungs (Table 3) exposed to low doses of MTBE (60 µL) throughout the experimental periods (3, 6, and 12 min of MTBE exposure) in comparison with the trachea and the lungs in the control rats. Tables 2 and 3 summarize the histological anomalies.  The control trachea ( Figure 1a) showed an intact inner normal mucosal layer (Mu) formed of ciliated pseudostratified columnar respiratory epithelium (RE) and lamina propria (LP). Furthermore, the control rats showed normal hyaline cartilage (HC), normal architecture of peritracheal CT containing typical blood vessels (BV) "veins and arteries" with tunica intima (TI), tunica media (TM), and tunica adventitia (TA).  observed after 12 min of exposure to MTBE (Figure 4a,c,f). Figure 4a, c illustrates hypertrophied arterial (HA) muscles "tunica media." Similarly, other blood vessels showed desquamation (DS) in tunica intima and thickening of the outer sheath "tunica adventitia" (Figure 4e). However, the lower panel of Figure 4d, e reveals fibroid changes (Fi) with fibrocytes (F), edema (e), severe emphysema (SE) with epithelialization (Epi), as well as degenerated air alveoli (Da) invaded with numerous monocytes (M).  After 3, 6, and 12 min of exposure to MTBE, the trachea showed noticeable alterations. The lining RE showed thickening (Th), deciliation (Dc), desquamation (Ds), mucosal ulceration (MUl), hyperplasia (Hp), and polyp formation (Figure 1b-d). Moreover, flattening (FE) was observed in the lining columnar epithelium similar to that present in the squamous epithelium "metaplasia" (Mp) (Figures 1b-d and 2d), as well as degenerative epithelium (DE) (Figure 2c,d). After 12 min of exposure to MTBE, the tracheal lumen (Lu) revealed the presence of eosinophilic debris formed by necrotic epithelial cells (Nc) and epithelialization (Epi) (Figure 2a,b,d-f) ( Table 2) (Figure 2f), furthermore, a small necrotic area (Ne) (Figure 2b,c), fatty degeneration (FD) (Figure 2d,e) with fibroid changes (Fi) (Figure 2a-d,f) and aggregation of plasma cells (P) (Figure 2c). Also, the LP revealed the formation of tracheal adenomas (TA) (Figure 2a,b,d,e) and some of them fused or grown to form large filled fluid large acini (Ac) (Figure 2a (Figure 2f), furthermore, a small necrotic area (Ne) (Figure 2b,c), fatty degeneration (FD) (Figure 2d,e) with fibroid changes (Fi) (Figure 2a-d,f) and aggregation of plasma cells (P) (Figure 2c). Also, the LP revealed the formation of tracheal adenomas (TA) (Figure 2a,b,d,e) and some of them fused or grown to form large filled fluid large acini (Ac) (Figure 2a  The CT of LP, after 3 min MTBE exposure, displayed congested blood vessels (CB) and blood capillaries (cc), edema (e), and infiltration of inflammatory cells and lymphoid cells (LC) (Figures 1b-f and 2a-f) with the formation of lymphoid hyperplasia (LH), focal (H), and diffuse hemorrhage (DH) (Figure 2a-f). Certain degenerative changes, such as fibroid changes (Fi), formation of lipid-laden macrophages "foam cells (FC)," small rounded and large fluid-filled acini (Ac) "tracheal adenomas (TA)," appeared along with deeply aggregated acini (Figure 2a,d-f). These structures were formed of small adenomatous corpuscles and were lined with simple glandular cuboidal epithelium (Figure 1e).
The lungs in the control rats ( Figure 3a) displayed undamaged bronchioles, blood vessels (BV), and air alveoli (AA). However, the lung tissue exposed to MTBE presented deterioration that increased with the exposure time throughout the experiment. After 3 and 6 min, the lung tissues revealed dilated blood vessels (DV), congested blood vessels (CB) and blood capillaries (cc), and dilated bronchioles (DB) (Figure 3b (Figures 3d and 4a-e).

SDS-PAGE
The total protein profile from blood sera revealed significant qualitative and quantitative differences (Table 4). Scanning analysis revealed major differences in the banding pattern of the proteins. The electrophoretic separation of blood sera collected from male rats after 3, 6, and 12 months revealed three protein bands of 29, 28, and 21 kDa, with relative mobility (RF) of 57, 0.61, and 0.72, respectively. The first, second and third fragments appeared after three months and continued to exist in the samples

SDS-PAGE
The total protein profile from blood sera revealed significant qualitative and quantitative differences ( Table 4). Scanning analysis revealed major differences in the banding pattern of the proteins. The electrophoretic separation of blood sera collected from male rats after 3, 6, and 12 months revealed three protein bands of 29, 28, and 21 kDa, with relative mobility (RF) of 57, 0.61, and 0.72, respectively. The first, second and third fragments appeared after three months and continued to exist in the samples collected at 6 and 12 months. Thus, we conclude that proteins corresponding to these bands were up-regulated following MTBE inhalation, as revealed by the intensity of the protein bands stained with Coomassie brilliant blue (Figure 5a, b and Tables 4-6).
The obtained results suggest that very low concentrations of MTBE inhalation induced the expression of three proteins in the blood sera of rats. The SDS-PAGE analysis of blood sera proteins provided useful information. However, further experiments are required to obtain a clear picture and therefore, we decided to use mass spectroscopy.    The obtained results suggest that very low concentrations of MTBE inhalation induced the expression of three proteins in the blood sera of rats. The SDS-PAGE analysis of blood sera proteins provided useful information. However, further experiments are required to obtain a clear picture and therefore, we decided to use mass spectroscopy.

LC-MS/MS Analysis
The most abundant proteins from upper, middle, and lower bands ( Figure 5) were identified as carbonic anhydrase 2 and carbonic anhydrase 1; carbonic anhydrase 2; and peroxiredoxin 2, respectively, by mass spectrometry (Table 6).

Discussion
The present study performed a preliminary analysis of the differences in the protein profiles obtained from the blood sera collected at different time points from male rats exposed to low concentrations of MTBE using the polyacrylamide gel electrophoresis technique. SDS-PAGE has been previously and successfully used to detect a novel, blood-based prostate cancer marker [22,23]. Carbonic anhydrases (CAs) have been studied for over 90 years [24]. Since their discovery in 1933, CAs have been at the forefront of scientific studies, ranging from basic enzymology to the application of structural biology and in silico approaches to study protein dynamics [25,26]. Of the 15 isoforms of CAs expressed in humans, only CA IX and CA XII have been implicated in cancer. These enzymes are transmembrane proteins in which their extracellular domains contain the catalytic activity; these are involved in regulating the tumor microenvironment [3,27]. The CA family has been shown to create and maintain the pH differential in tumor cells [28][29][30][31][32][33]. This suggests that CA I could potentially serve as a plasma biomarker [34]. Recently, CA1 gene amplification was detected in approximately 25% of breast cancer studies [32]. Similar to CA1, the most common gene alterations in CA 2 were amplifications, especially in neuroendocrine prostate cancer, breast cancer, prostate adenocarcinomas, and metastatic cancers followed by mutations [32,33]. Thus, both CA I and II may be potential targets for treating several cell-type-specific cancers. The microscopic investigation verified the deteriorating histopathological alterations in rat trachea and lungs of rats at 3, 6, and 12 months after MTBE exposure. Conspicuous histopathological alterations were reported. The lining epithelia of trachea and lung bronchioles showed ulceration, desquamation, hyperplasia, and polyp formation. However, lymphoid hyperplasia, tracheal and lung adenoma, edema, and monocytic infiltration in the tracheal LP and in the perivascular and peribronchiolar CT of bronchioles of lungs, as well as a focal abscess in the lung parenchyma were observed. The reason for the observed hydrops in the epithelial cells could be related to the absorption of water. The fibroid degeneration could be explained by the degeneration of lung parenchyma, activation of fibroblasts with subsequent fibrosis, and defectiveness in the smooth muscle fibers of interstitial CT as a benign smooth muscle tumor "leiomyoma". The observed lung leiomyoma might be a consequence of collapsed air alveoli and occlusion of some bronchioles. Similarly, Nuovo and Schmittgen [34] suggested that leiomyomas signified independent and multifocal smooth muscle proliferation. Besides, the observed hyaline cartilage deformation, thyroid follicles degeneration, and lung abscess were not reported earlier following MTBE exposure. With respect to the mentioned adenoma in the trachea and lung tissues, the present investigation suggested that it may be an early stage of adenocarcinoma. In rats and mice, Bird et al. [35] observed hepatocellular adenomas in females, and interstitial cell adenomas of the testes were observed in males exposed to 3000 and 8000 ppm of MTBE. Moreover, testicular and renal adenomas, in addition to tubular carcinoma, were reported in male rats exposed to MTBE. Narrowing of the vascular lumen induces vascular occlusion in the adjacent regions, whereas the hypertrophy of lung arteries may be induced by hyperplastic and hypertrophied smooth muscle fibers of tunica media and increased number of interstitial elastic fibers. The present investigation suggested that the hypertrophy of tunica media could be a result of pulmonary hypertension, inflammation, and partial obstruction or interruption of the blood flow. Structurally, the enlargement of muscle cells could be attributed to the decrease in the nucleo-cytoplasmic ratio induced by increasing the density of sarcoplasmic reticulum and mitochondria. Moreover, Gabella [36] added that the observed hypertrophy could be due to the fact that muscle cells may be dividing longitudinally. With respect to the monocytic infiltration observed in the present study, Li et al. [37] proved that radiolabeled monocytes migrated into the acute inflamed rat lungs. However, Azoulay et al. [38] concluded leukemic pulmonary infiltration to be the first manifestation of acute monocytic leukemia. The hydropic degeneration and lipid-laden macrophages "foam cells" detected in the present work could be explained by the work of Romero et al. [39], who reported that foam cells developed from neighboring alveolar epithelial type II cells, which responded to the injury by accumulating lipids into the distal airspaces of the lungs. Moreover, MTBE inhalation induced histopathological hazards in the brain, liver, spleen, lungs, kidney epididymis, and gonads, as well as led to renal and gonadal tumors in males [40,41]. Furthermore, other reports concluded that MTBE administered in drinking water or by inhalation induced chronic progressive nephropathy of minimal to mild severity in male Wistar rats and Fischer rats "F344". However, Kenneth et al. [11] reported that MTBE gavage in rat and mouse inhalation elevated tumor rates in the kidneys and Leydig interstitial cells of male rats and leukemia/lymphomas in female rats. They believed that MTBE or its metabolites are cytotoxic, tumorigenic, and could damage DNA, which may increase the cancer potency, unless at a very low dose of 0.003 mg/kg b.wt. Moreover, Belpoggi et al. [42] reported that MTBE exposure induced an increase in dysplasias and immunoblastic lymphomas among male and female rats. In addition, it resulted in reactive hyperplasia of the peribronchial lymphoplasma cellular tissue, also found to be the most frequent alteration in the lungs. Besides, lymphoid hyperplasias were actually explained as reactive marginal zone B-cell lymphomas [42]. Moreover, Schoeb et al. [42] reported that rats exposed to MTBE developed lympho-immunoblastic lymphoma. Recently, Schoeb et al. [42] confirmed that MTBE induced several biochemical and physiological dysfunctions. She suggested that MTBE generated reactive oxygen species with subsequent harmful effects on different body systems and organs as well as on the normal body physiology, including liver, kidneys, blood, bone marrow, and the nervous, respiratory, reproductive, and immune systems. The present investigation demonstrated that inhalation of MTBE at low doses induced a noteworthy difference in the pattern of protein bands in the rat sera. The effect of MTBE has been investigated in blood samples, breath, and urine specimens [18]. Unfortunately, no data have been reported on the effects of MTBE exposure on electrophoretic protein profile of rat blood sera. The present electrophoretic protein profile of blood sera in male Wister rats exposed to MTBE showed the presence of three protein bands. Some of these proteins are identified using mass spectroscopy in direct or indirect association with cancer potency. Numerous investigations revealed that a high dose of MTBE significantly exerted toxicity on white blood cell count, including lymphocytes, granulocytes, eosinophils, as well as on hemoglobin. Moreover, the biochemical parameters, such as triglycerides, cholesterol, and transaminases (ALT and AST), were increased in the sera of treated rabbits [12]. In the literature, there are no reports on the abnormal electrophoretic protein profile of rat blood sera after MTBE exposure to detect early bio-indicators for carcinogenicity in mammalian models. The electrophoretic profile obtained in the present study revealed three abnormal protein bands containing numerous types of proteins. Some of these proteins were identified to be associated with several carcinomas. In conclusion, the present study identified specific protein bands that could be used to develop early, serum-based, cost-effective, diagnostic biomarkers for carcinogenicity. We believe that these biomarkers would assist in early health assessment of workers in environments such as gas stations using gasoline mixed with MTBE and the residents exposed to car fumes. We considered all isoforms of CAs and their possible roles in tumorigenesis as well as their potential as targets for cancer therapy.

Conclusions
Collectively, our results suggest that SDS-PAGE and mass spectroscopy could serve as potential techniques for analyzing blood-based early diagnostic markers following exposure to very low concentrations of MTBE. These could serve as promising tools for assessing inhalation toxicity and carcinogenicity associated with MTBE vapor inhalation effects. Whether our detected and identified early markers are valid in human blood plasma or not will be the matter of our next investigation. Based on the findings of the present study, we conclude that inhalation of very low concentrations of the gasoline additive MTBE could induce an increase in protein levels much before the first appearance of histopathological effects on male rats.