EFFECT OF METHYL TERTIAL BUTYL ETHER ON THE EXPRESSION OF mRNA CODING FOR 6-PHOSPHOFRUCTO-2-KINASE/FRUCTOSE-2,6-BISPHOSPHATASE-3 AND VEGF IN RAT LIVER AND LUNG

Methyl tertial butyl ether, an oxygenated compound of gasoline, is a toxic and ecolo­ gically dangerous chemical compound with an ability to introduce a variety of neurotoxic, allergic and respiratory diseases, as well as cancer and leukemia. We used RT/PCR or quan titative PCR to study the effect of methyl tertial butyl ether (everyday injection for one month) on the expression of mRNA coding for 6­phosphofructo­2­kinase/fructose­2,6­ bisphosphatase­3 (PFKFB­3) and vascular endothelial growth factor (VEGF), which are important for tumor growth and metastasis in the lung and liver. The level of PFKFB­3 and VEGF mRNA expression was increased in the liver in methyl tertial butyl ether treated rats with respect to the control animals. However, methyl tertial butyl ether had opposite ef fect on the expression of PFKFB­3 and VEGF mRNA in the lung: with strong induction of PFKFB­3 and suppression of VEGF mRNA expression. Moreover, alternative splicing of PFKFB­3 mRNA was changed in methyl tertial butyl ether treated rats in organ specific manner. Thus, methyl tertial butyl ether induces alternative splicing of PFKFB­3 mRNA accompanied by appearing of variants with longer C­terminus in the liver, and with shorter C­terminus in the lung. The results of this investigation clearly demonstrate that methyl tertial butyl ether af­ fects in organ-specific manner the expression and alternative spli cing of mRNA coding for PFKFB­3 and VEGF, key regulatory factors of glycolysis, and angiogenesis.


INTRODUCTION
Methyl tertial butyl ether is a representative of numerous oxygenated compounds which are available in gasoline and can to reduce vehicle exhaust emissions, aromatic compound content, and avoid using organolead compounds, while maintaining high oc tane number. Immediately after the introduction of methyl tertial butyl ether into gasoline, many consumers of that product in USA and other countries have been getting a variety of neurotoxic, allergic and respiratory diseases [1][2][3]. Methyl tertial butyl ether causes different cancers in rat and mice [1,3]. The precise mechanisms of methyl tertial butyl ether toxic effects leading to development of different diseases, especially cancer, re main poorly understood. Data exist that 6phosphofructo2kinase/fructose2,6bisphos phatase (PFKFB) and vascular endothelial growth factor (VEGF) are very important for tumor growth and metastasis and that methyl tertial butyl ether affects the expression of PFKFB4 mRNA and its alternative splicing, as well as the expression of Per2, BMal1, Clock, and some protein kinases responsible for regulation of key metabolic processes [4][5][6][7][8][9]. Disturbance in expression of these genes can impair cellular signaling pathways and lead to developing of different pathological processes and cancer, in particular.
An enhanced glycolysis is observed in different cells under hypoxic conditions and in malignant tumors. It is dependent on PFKFB expression and phosphorylation via hy poxia inducible factor (HIF) even at conditions of normal oxygen tension [5,15,19,20]. Transcription factor HIF plays a central role in coordinating many transcriptional adap tations to hypoxia and is an important mediator of Warburg effect in tumors [6,15,34,[39][40][41]. Many genes whose expression is regulated by hypoxia, in particular vegf and pfkfb3, are overexpressed in malignant cells and contain HIF1 binding site (hypoxia responsible element) [15,20].
Vascular endothelial growth factor (VEGF) is a very important factor of angioge nesis and is elevated in malignant tumors and different cell lines at hypoxic conditions [5,15,20,23]. Induction of its expression is tightly connected with overexpression of PFKFB isozymes and enhanced glycolysis in malignant tumors, as well as in different cells at hypoxic conditions [10,12,21,23]. Moreover, HIF, VEGF and PFKFB have a key role in the neoplastic transformation. In turn, most PFKFB isozymes are regulated by a complex network of kinases, phosphatases and metabolites [5,11,17,19].
The expression of PFKFB3 and VEGF mRNA and its splice variants in different organs of methyl tertial butyl ether treated rats was not studied yet, although significance of PFKFB3 and VEGF in the regulation of glycolysis, angiogenesis and malignant transformation is well known. The main goal of this study was to investigate the effect of methyl tertial butyl ether on the expression of mRNA coding for PFKFB3 and VEGF, as well as the expresssion of its alternative splice variants in rat liver and lung. That is necessary for identifiation of possible target sites of methyl tertial butyl ether which is considered to be an ecologically dangerous chemical compound.

Animals.
Male Wistar rats (initial body weight 180-200 g) were treated with methyl tertial butyl ether (500 mg/kg) ones per day for a month in cold stress conditions. In one month, control and methyl tertial butyl ether treated animals were killed and their tissues were frozen in liquid nitrogen before RNA extraction.
RNA isolation. RNA was isolated from the liver and lung by using acid guanidi niumphenolchlorophorm extraction method of Chomczynski and Sacchi, as descri bed [10].
Expression of PFKFB-3 and VEGF mRNA. The expression of PFKFB3 mRNA was examined by polymerase chain reaction (PCR) of corresponding cDNA. Super Script II Reverse Transcriptase, ("Invitrogen", USA) and oligo(dT) were used for cDNA synthesis. Reverse transcription was performed according to the manufacturer's proto col, by using 0.4 mg of total RNA. The amplification of PFKFB-3 cDNA (1 ml of reverse transcription product that corresponds 20 ng of total RNA was used for reverse trans cription) was performed with HotStarTaq Master Mix Kit ("QIAGEN", USA) and "Master Cycler Personal" ("Eppendorf", Germany).
PCR amplification of VEGF was performed by using forward primer 5′AACCAT GAACTTTCTGCTCTC3′ and reverse primer 3′GACAAGCCAAGGCGGTGAGCC5′. These oligonucleotides correspond to sequences 1011-1031 and 1642-1662 of the pub lished rat VEGF cDNA (GenBank accession number NM_031836). PCR amplification of alternative splice variant of VEGF (GenBank accession number AY702972) was per formed by using forward primer 5′TGCCCCTAATGCGGTGTGCG3′ and reverse primer 5′GTGGTCACTTACTTTTCTGGC3′. These oligonucleotides correspond to sequences 230-249 and 411-431 of the published rat VEGF cDNA of this unique alternatively splice variant. The primers were purchased from "Sigma".
bactin mRNA expression was used as a loading control of analyzed RNA [23]. The polymerase chain reaction products were analyzed by electrophoresis in agarose gel and stained with the ethidium bromide.
Quantitative polymerase chain reaction was performed on "Stratagene Mx 3000P cycler", using SYBRGreen Mix and specific sets of primers for PFKFB3 (forward -5′TGGACGACTTCATGAAGAG3' and reverse -5′GCATTGGCGAACTTCTTGC3′; the nuc leo tide sequences of these primers correspond to sequences 956-974 and 1247-1229 of rat PFKFB3 cDNA; GenBank accession number NM_057135) and VEGF, as described previously [23]. Reaction was performed in triplicate. Analysis of quantitative PCR was performed by using special computer program "Differential ex pression calculator" and statistical analysis in the Excel program.

RESULTS AND DISCUSSION
As shown in Fig. 1, the levels of mRNA coding for PFKFB-3 and VEGF, are signifi cantly increased in liver of methyl tertial butyl ether treated rats with respect to the control animals. However, methyl tertial butyl ether had opposite effect towards the expression of PFKFB3 and VEGF mRNA in the lung: strong induction of PFKFB3 (+119%) and suppression of VEGF (25%) mRNA. Besides, we have studied the expression of alter native splice variants and two sets of primers specific for 3′coding region of PFKFB3 mRNA. Thus, our results suggest that methyl tertial butyl ether enhances glycolysis via indu cing PFKFB3 expression. These results agree with data about key role of PFKFB3 in enhancing glycolysis [14,15,17,19,20]. Moreover, methyl tertial butyl ether strongly induces expression of GAPDH mRNA, both in liver and lung (Fig. 2), that is also induced at conditions of enhanced glycolysis [15]. Fig. 2 shows two main bands of PFKFB3 cDNA in the liver and lung of control and methyl tertial butyl ether treated rats revegled with M3 and M5 primers. Methyl tertial butyl ether increases the expression of PFKFB3 mRNA variants of upper band in the liver and lower band -in the lung. Fig. 3 demonstrates similar results with another set of primers (M4 and M5), however, two additional bands of PFKFB-3 cDNA were identified. Alternative splice variants of PFKFB3 mRNA differ in the exon structure of Cterminal regulatory region (Fig. 4). Two bigger (NM_057135 and D87241-D87243) and three smal ler (D87244, D87245 and EU034674) alternative splice variants were revealed. One can see two bands of PFKFB3 mRNA splice variants: the upper band contains four bigger vari ants, while the lower band -only two smaller variants (D87244 and D87245). In Fig. 3, one can see four bands. Band 1 contains the biggest alternative splice variants of rat PFKFB3 mRNA (NM_057135) which has a full set of known exons and additional exons (13a and 15a) of Cterminal regulatory region. Band 2 contains three bigger variant of rat PFKFB3 mRNA (D87241-D87243). Two of them contain 13 th additional exon and one -15 th addi tional exon (Fig. 4). Two smaller variants which do not have those additional exons are lo cated in the band 3 (Fig. 3). Band 4 of PFKFB3 mRNA (Fig. 3) contains the smallest splice variant which does not have 14 th and 15 th exons, as well as 13 th and 15 th additional exons. There are three stop codons for different alternative splice variants of PFKFB3 mRNA (Fig.  4). They are resulting in three different types of Cterminus amino acid sequence (Fig. 5).
As shown in Fig. 5, different alternative splice variants of PFKFB3 mRNA have diffe rent quantity and position of serine residues (underlined) in the Cterminus that could have certain significance in regulation of enzymatic activity through serine phosphorylation [11,18]. Moreover, three bigger splice variants of PFKFB3 mRNA (NM_057135, D87241 and D87242) contain an additional exon 13a (29 amino acid segment), which has six ser Fig. 2. Analysis of PFKFB3 and GAPDH mRNA expression in the liver and lung of control (C) and animals treated with methyl ter tial butyl ether (M), using RTPCR. Ampli fication of PFKFB-3 mRNA was carried out using M3 forward and M5 reverse primers. Amplified products were ana lyzed by electrophoresis in the agarose gel. The bactin mRNA expression was used to standardize loading RNA quanti ty. A representative (of four) gel electro phoresis is shown Fig. 3. Analysis of PFKFB3 mRNA expression in the liver and lung of control (C) and animals treated with methyl tertial butyl ether (M), using RT-PCR. Amplification of PFKFB3 mRNA was carried out by using M4 forward and M5 reverse pri mers. Amplified products were analyzed by electrophoresis in the agarose gel. The bactin mRNA expression was used to standardize loading RNA quantity. A representative (of four) gel electro phoresis is shown ine residues, however its significance in the regulation of PFKFB-3 activity via phosphory lation has not been studied yet. There are data that phosphorylation of Ser 490 is responsible for activation of glycolysis in human cancer [18]. As shown in Fig. 3 the expression of smallest isoform of alternative splicing variant of PFKFB3 mRNA is decreased in the liver and increased in the lung of methyl tertial butyl ether treated rats with respect to the control animals. Recently, it was shown that expression of this splicing variant is decreased in the heart and lung at diabetes [24]. Thus, methyl tertial butyl ether affects alternative splicing of PFKFB3 mRNA in organ specific manner. The results presented in Fig. 6, clearly demonstrated the existence of additional band of VEGF cDNA in the liver and lung of methyl tertial butyl ether treated animals containing several shorter alternative splicing variants of VEGF mRNA [23]. The level of expression of these alternative splicing isoforms of VEGF mRNA were increased in the liver and lung in rats treated with methyl tertial butyl ether, with respect to the control animals. cDNA of that band was cloned and new alternative splicing variant of VEGF mRNA with 14 bases insert after 5 th exon was identified (GenBank accession number AY702972). As shown in Fig. 7, the level of expression of this alternative splicing variant of VEGF mRNA was increased in the liver and lung in rats treated with methyl tertial butyl ether, with respect to the control animals. If shouled be noted that in the liver this induction was much stronger.
Thus, the methyl tertial butyl ether, toxic and ecologically dangerous chemical com pound affects the expression of PFKFB3 and VEGF mRNA that can be important regula tory mechanism controlling cell glycolysis and angiogenesis. Besides, the expression of PFKFB3 and VEGF mRNA alternative splicing variants can be used as sensitive test for detection of toxic action of ecologically dangerous chemical compounds. More focused studies are needed for better understanding mechanisms by which aliphatic ethers, such as methyl tertial butyl ether, can affect health and environment.