Possible role of newly synthesized heme in signaling under stress agents action Barannik Tatyana, Strel’chenko Ekaterina, Inshina Natalya, Nikitchenko Iryna, Kaliman Pavel a.

Kharkov National University, Biochemistry Department, 4, Svobody Sq., Ukraine, 61077

E-mail: tbarannik@univer.kharkov.ua Introduction

Being lipophylic prooxidant heme molecule takes an active part in signaling mechanisms under physiological and stress conditions. Heme is a key molecule both in mediating the effects of oxygen on various cellular processes and in the responses to environmental stress including xenobiotics, radiation and bacterial toxins [1]. Heme is known to induce expression of genes encoding erythropoietin and heme oxygenase-1 (stress protein HSP32, the inducible form of key enzyme of heme degradation) [2]. Heme-regulated motif was revealed in transcriptional repressor Bach1, heme oxygenase-2, precursor of key enzyme of heme biosynthesis 5-aminolevulinate synthase (ALAS), glutathione-S-transfe­rases and some others.

Under various stress conditions the increase of heme content observed in mammals’ tissues, especially in liver, is considered to be due to exogenous heme transport from blood stream (under erythrocytes lysis) to sites of its degradation in liver [3]. The other source of heme is the newly synthesized one, taking into account the induction of key enzyme of heme biosynthesis pathway (ALAS) under various stress conditions [4]. The increase of the flow of newly synthesized heme to microsomes revealed under stress conditions may point at the enhanced utilisation of it in heme oxygenase reaction [5].

One of the indexes of free heme content in rat liver cytosol is heme saturation of TDO, which in the rat liver exists in form of holoenzyme (hemoprotein) and apoprotein able to bind free heme [6]. One of the proteins possibly involved in new heme transport from mitochondria is GST [7]. Thus we investigated heme metabolism and distribution in the rat liver under Hg²⁺ and Cd²⁺ ions action after -tocoferol pretreatment and under severe heme accumulation in serum in glycerol model in vivo

Materials and methods

Metal salts dissolved in 0,9% NaCl were injected to 3-months male Wistar rats. CdCl₂ was injected s.cc at the dose of 14 mg and HgCl₂ i.p. 7 mg per 1 kg b.w. Control animals were injected with the corresponding volume of 0,9% NaCl. -Tocoferol acetate (10%) was injected i.m. in dose 50 mg per 1 kg b.w. 2 hrs before metal salts. In glycerol model 50% solution of glycerol was injected in dose 7,5 ml/kg (Ѕ of dose per each thigh muscle). Cycloheximide was injected s.c. in dose 2 mg/ 1 kg b.w. 0,5 hrs before glycerol injection. Blood was collected to obtain the serum. The liver was perfused with cooled physiological saline in situ. Subcellular fractions were prepared by differential centrifugation at 2^oC. In In vitro experiments liver homogenate (25%) in sucrose-containing buffer was incubated at 37^oC with free access to air oxygen for 5 min (control without additions) in presence of GSH (final concentration 1.2 mM), -tocopherol acetate (0.6 mM), H₂O₂ (2 mM). After incubation homogenate was twice diluted and used for preparation of subcellular fraction (at 2^oC) which were frozen-melt before determination of heme content and GST activity. ALAS (EC 2.3.1.37) activity was determined by the amount of -ALA synthesized during incubation with corresponding substrates as described [8] and expressed in nmol 5-ALA/min per mg protein. HO (EC 1.14.99.3) activity was determined by bilirubin formation [9] and expressed in nmol bilirubin/min per mg protein. Total heme content was determined by absorbance of complex of heme with pyridine and expressed in nmol/mg of protein [10]. Accumulation of hemolysis products in serum was measured by the difference in optical absorbance in the Soret region (390-450 nm) as described in [11] and expressed in A/mg protein. Holoensyme and total activity of tryptophan-2,3-dioxygenase (TDO, EC 1.13.11.11) were determined by kinurenine formation [6] and expressed in nmol kinurenine/min per mg protein. Heme saturation of TDO (TDO%) was calculated as a ratio of holoenzyme to total activity and expressed in %. GSH content assayed by absorbance of GSH complex with alloxane as described [11] and expressed in moles/g tissue. GST (EC 2.5.1.18) activity was determined by increase of absorbance of GSH complex with CDNB and expressed in nmol CDNB/ min per mg of protein [12]. Protein content was determined by Lowry method modified by Miller.