Effect of exposure of various oxidants on rat liver and intestinal microsomes — A comparative study

Chem.-Biol. Interactions, 80 (1991) 135-144 Elsevier Scientific Publishers Ireland Ltd.

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E F F E C T OF EXPOSURE OF VARIOUS OXIDANTS ON RAT LIVER AND INTESTINAL MICROSOMES - - A COMPARATIVE STUDY

S. NALINI and K.A. B A L A S U B R A M A N I A N

The WeUcome Research Unit and Department of Gastroenterology, Christian Medical College Hospital, Vellore 63~ 005 (India) (Received February 13th, 1991) (Revision received June 14th, 1991) (Accepted June 27th, 1991)

SUMMARY

Rat liver and intestinal microsomes were exposed to various free radical generating systems and their effect were assessed by studying different parameters such as formation of malonaldehyde (MDA) and conjugated diene, arachidonic acid depletion and alteration in protein thiol groups and tocopherol levels. These studies revealed that liver being highly vulnerable tissue showed all the effects of free radical attack whereas intestinal microsomes were resistent to most oxidants except iron independent generation of free radicals using 2-2'-azobis (2-amidinopropane) dihydrochloride (ABAP). Intestinal microsomes were found to contain considerable amount of non-esterified fatty acids in total lipid fraction as compared to liver microsomes and iron-fatty acid complex may be incapable of participating in peroxidation. In vitro measurement of hydroxyl radical generation showed that intestinal microsomes were incapable of generating these active species. These results suggest that iron dependent free radical mediated lipid peroxidation might not occur in intestinal epithelial cells.

Key wards: Oxidants -- Peroxidation -- Liver and intestinal microsomes

INTRODUCTION

Oxygen-derived free radicals are highly reactive species produced by one electron reduction by both enzymatic and non-enzymatic pathways in biological C o ~ r ~

to: K.A. Balasubramanian, The Wellcome Research Unit and Department of

Gastroenterology, Christian Medical College Hospital, VeUore 632 004, India. 0009-2797/91/$03.50 © 1991 Elsevier Scientific Publishers Ireland Ltd.

136

systems. Involvement of free radical reactions in number of physiological and pathological events is now well recognized. One of the most extensively investigated effects of free radicals is the peroxidative breakdown of membrane polyunsaturated fatty acids. In addition to lipids, other biological compounds such as proteins and DNA also form targets for free radicals. Effect of free radicals on biological system has usually been detected by the presence of substances which give positive thiobarbituric acid reaction. Production of these substances are believed to occur through secondary reactions that parallel the general course of lipid peroxidation in vivo. Other methods of measuring peroxidation which may be considered more appropriate to study free radical damage include conjugated diene formation [1,2], fatty acid analysis [3], arachidonic acid levels [4], thiol groups [5], and alpha tocopherol levels [6,7]. Our earlier work has shown that intestinal mucosal membranes are resistant to peroxidation as judged by malonaldehyde (MDA) production and this was due to the presence of considerable amount of non-esterified fatty acids (NEFA) as part of the membrane lipids which probably form complexes with iron, making it unavailable for induction of peroxidation [8 - 10]. In the present report we have studied the extent of peroxidation in intestinal microsomes by measuring various parameters after exposure to various oxidants and compared with liver microsomes which are known to undergo peroxidation. We have also measured the capability of these microsomes to produce hydroxyl radicals. MATERIALS AND METHODS

H E P E S , Tris, 2-thiobarbituric acid, NADPH, ADP, bovine serum albumin, cumene hydroperoxide, 5-5'-dithiobis (2-nitrobenzoic acid), authentic fatty acids, alpha tocopherol, 1,1',3,3'-tetramethoxy propane, xanthine and xanthine oxidase were all obtained from Sigma Chemical Company. 2-2 '-azobis(2-amidinopropane)dihydrochloride (ABAP) was obtained from Polysciences Inc. U.S.A. All solvents were redistilled before use and for HPLC, special grade solvents were used. All other reagents used were analytical grade. Overnight fasted rats of body weight 2 0 0 - 2 5 0 g were killed by decapitation and liver and intestine were removed. Contents of the intestinal lumen were washed thoroughly with cold 1.15% KC1 and the mucosa scraped using a glass slide. Partially purified liver and intestinal microsomes were prepared as follows. 10% homogenate in 0.25 lVl sucrose (pH 7.4) were spun down at 1000 x g for 10 min and 12 000 × g for 20 min. Supernatant obtained was centrifuged at 105 000 x g for 60 min. This pellet which contains microsomes was washed twice with 1.15% KC1 and were stored at -70°C. When required microsomal pellet was suspended to about 20 mg protein/ml and used for further studies. Protein content was measured with bovine serum albumin used as standard [11]. In vitro lipid peroxidation was induced in both liver and intestinal microsomes by incubating with various oxidants. Microsomes corresponding to 1 mg protein in 0.1 M Tris-HC1 buffer (pH 7.1) in a total volume of 1 ml were incubated separately with the following reagents at the final concentrations stated: (a) ascorbate 500 ~M and ferrous sulphate 5 ~M; (b) NADPH 100 ~M, ADP 500 uM

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and ferrous sulphate 5 ~M; (c) cumene hydroperoxide 0.1 mM; (d) H202 10 ~M and ferrous sulphate 100 #M; (e) xanthine oxidase 10 m units and xanthine I mM; and (f) ABAP 50 raM. Control incubation had only microsomes and buffer. After incubation at 37°C for 30 rain in a shaking water bath, reaction was stopped with trichloroacetic acid and MDA in the protein free supernatant was measured using thiobarbituric acid as described [12]. MDA was calculated from standard curve prepared with 1,1',3,3'-tetramethoxy propane. For conjugated diene measurement, total lipids from the incubated microsomes were extracted according to Bligh and Dyer's method [13], dissolved in methanOl, read at 233 nm and the amount calculated using molar extinction coefficient of 2.52 x 104. Arachidonic acid level in total lipid was calculated after hydrolysis, methylation and quantification by gas chromatography. This was done using Pye Unicam PU 4550 gas chromatograph equipped with flame ionization detector and Spectraphysics PU 4811 integrator. Heptadecanoic acid was used as internal standard. Tocopherol from the incubated microsomes were extracted as described [14] and separated and quantitated by HPLC [6]. Protein bound thiol groups were assayed as described [15]. Microsomal generation of OH" or OH'-like species were determined by assaying formaldehyde production from dimethyl sulfoxide [16]. Control and peroxidized microsomes were subjected to SDS-PAGE as described [17] and the protein bands were stained with coomassie blue followed by silver nitrate. Non-esterified fatty acid content of intestinal and liver microsomes were quantified after separation of total lipids on thin layer chromatography using solvent system hexane/diethyl ether/acetic acid (80:20:1 by vol.) and further separation and quantification by gas chromatography after methylation.

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Fig. 1. Effect of oxidants on conjugated diene formation by intestinal and liver microsomes. Each value represents mean ± S.D. of four separate experiments. Incubation conditions and quantitation methods are described in the text.

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RESULTS

Figure 1 shows the amount of conjugated diene formed when liver and intestinal microsomes were exposed to various oxidants. Control intestinal microsomes formed very little conjugated diene which increased considerably when incubated with ABAP and X-Xo. In presence of other free radical generating systems intestinal microsomes did not show significant conjugated diene formation. On the other hand liver microsomes formed conjugated diene when incubated with various free radical generating systems although the amount formed was less compared to intestine using ABAP and X-Xo. Figure 2 shows decrease in arachidonic acid levels after inducing peroxidation by various systems. In intestinal microsomes, only ABAP could decrease arachidonic acid level significantly whereas in liver microsomes all free radical generating systems reduced the level of this fatty acid and ABAP showed the greatest effect. MDA estimation showed that the amount produced in intestinal microsomes was negligible as compared to that produced in liver microsomes (Fig. 3). We have earlier shown that intestinal homogenate does not have any effect on thiobarbituric acid colour reaction [9]. Incubation with ABAP alone showed an increase in MDA production by intestinal microsomes which was similar to that produced by liver microsomes although level of MDA produced was much less compared to other systems. Proteins from oxidant exposed liver and intestinal microsomes were solubilized and separated by SDS-PAGE which indicated the presence of high molecular weight proteins where peroxidation has occurred suggesting cross linking of proteins (data not shown).

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Fig. 2. Effect of oxidants on arachidonic acid depletion from the liver and intestinal microsomes. Each value represents mean ± S.D. of four separate experiments. Incubation conditions and fatty acid quantitation are described in the text.

30

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Fig. 3. Effect of oxidants on malonaldehyde formation by liver and intestinal microsomes. Each value represents mean ± S.D. of four separate experiments. Experimental details are described in the text.

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FATTY ACIDS : nmoles / mg PROTEIN Fig. 4. Total and non-esterified fatty acid composition of liver and intestinal microsomes. N E F A were separated from total lipids by thin layer chromatography and quantitated by gas chromatography. Each value represents mean ± S.D. of three separate experiments.

140 Figure 4 shows the composition of total and non-esterified fatty acids in intestinal and liver microsomes. Although both microsomes contain considerable amounts of polyunsaturated fatty acids, only intestinal microsomes had high content of non-esterified fatty acids. It is known that free radicals react with thiol groups of cellular proteins and oxidise them. Levels of protein thiol group in intestinal and liver microsomes after exposure to various free radical generating systems are shown in Fig. 5. Decrease in protein thiol groups in liver microsomes were seen with every oxidant system used whereas with intestinal microsomes, decrease had occurred only with ABAP and cumene hydroperoxide and to a lesser extent with NADPH/ADP-iron system. Membrane associated tocopherol protects from peroxidation and alteration in its level is an indication of the extent of peroxidation damage. Figure 6 shows changes in tocopherol level after exposure of liver and intestinal microsomes to various peroxidising system. Liver microsomes showed considerable decrease in the content of tocopherol after peroxidation with every oxidant system and complete disappearance was seen with ABAP and cumene hydroperoxide. On the

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Fig. 5. Effect of oxidants on microsomal protein thiol levels in liver and intestine. Each value represents mean ~: S.D. of three separate experiments.

141

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Fig. 6. Effect of oxidants on tocopherol content of liver and intestinal microsomes. Tocopherolwas quantitated by HPLC and the experimental details are given in the text. Each value represents mean + S.D. of three separate experiments.

other hand with intestinal microsomes, decrease in tocopherol level was not significant except after exposure to A B A P , X-Xo and cumene hydroperoxide which showed a decrease in its level. Incubation of microsomes with E D T A and iron, generates hydroxyl radical which can be detected by m e a s u r e m e n t of formaldehyde formed from dimethyl sulfoxide. Using this method the ability of liver

TABLE I HYDROXYL RADICAL PRODUCTIONBY LIVER AND INTESTINAL MICROSOMES Hydroxy radicals (nmol/mg protein) Liver Fe 3÷ 10 ~M EDTA 100 ~M Fe 3+ 50 ~M EDTA 500 ~M ND, Not detected.

Intestine

2.935 ± 1.1

ND

49.250 ± 6.0

ND

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and intestinal microsomes to produce hydroxyl radicals were checked. As shown in Table I, liver microsomes were capable of generating hydroxyl radicals whereas with intestinal microsomes these active species even at high concentration of iron were not detectable. Various lipids in control and peroxidized microsomes from liver and intestine were analysed and no significant alterations were seen in the content and composition of both neutral and phospholipids (data not shown). DISCUSSION

Comparing the effects of exposure of liver and intestinal microsomes to oxidants as assessed by peroxidation products and alterations in certain constituents showed that there is marked difference in susceptibility to oxidants between these two tissues. Liver microsomes were able to produce considerable amount of MDA when exposed to different oxidants whereas only ABAP exposure could produce comparable MDA in intestinal microsomes. Exposure of intestinal microsomes to ABAP and X-Xo resulted in formation of conjugated dienes which was even more than that produced by liver microsomes whereas other oxidants did not have much effect. Conjugated dienes are considered as the initial products of peroxidation whereas MDA is known to be the end product. It is not clear why X-Xo exposures of intestinal microsomes show differences in MDA and conjugated diene formation. It is likely that the organisation of membrane lipids and the differential rate of degradation of dienes to malonaldehyde might be responsible for this. Arachidonic acid measurement showed that ABAP exposure could decrease this fatty acid in both liver and intestinal microsomes whereas other oxidants had significant effect only on liver microsomes. ABAP is a water soluble free radical generator which undergoes spontaneous thermal decomposition to form two carbon centered radicals and this process does not require iron [18,19]. Intestinal microsomes were not peroxidised by cumene hydroperoxide which requires cytochrome P-450 to catalyse the reaction. It is known that intestinal microsomes have negligible amount of cytochrome P-450 [20]. Free radical exposure could bring about oxidation of protein thiol groups and the membrane antioxidant, alpha-tocopherol. Measurement of protein thiol and tocopherol levels of intestinal and liver microsomes after exposure to various oxidants showed a decrease in their contents which was more pronounced in liver microsomes as compared to intestine. ABAP and cumene hydroperoxide exposure of intestinal microsomes reduced the level of protein thiol and tocopherol and other systems did not have significant effect. It is possible that cumene hydroperoxide being a strong oxidant may directly oxidize thiols and tocopherol and the observed effect may not be mediated by free radicals. Intestinal microsomes were found to be susceptible to X-Xo system as indicated by conjugated diene formation, tocopherol depletion and partial reduction in the level of arachidonic acid and protein thiol. The X-Xo system prod/Uces relatively unreactive superoxide anion radical and H202. H202 is relatiw~ly innocuous to membranes except where iron is present. Recently it has been suggested that X-

143

Xo could directly produce hydroxyl radicals [21], which has been questioned [22]. It is likely that the type of oxidizing species generated may differ from system to system and the effect of these oxidising species and the product formed may differ in different tissues. Free iron is essential for generation of hydroxyl radicals through Fenton reaction and these are the active species involved in damage to biological system. Comparison of hydroxyl radical generation by liver and intestinal microsomes clearly showed that intestinal microsomes were not capable of generating these species. This reaction might require cytochrome P-450 which is lacking in intestinal microsomes. Recent observation on the effect of iron chelator desferrioxamine on H202 induced mucosal permeability suggests that OH" formation was not responsible for altered intestinal mucosal permeability [23]. Resistance of intestinal microsomes to iron dependent lipid peroxidation and their inability to generate hydroxyl radicals suggest that this tissue is protected from lipid peroxidation damage. This protection is probably offered by the large amount of non-esterified fatty acids in this tissue and probably the fatty acid-iron complex is incapable of participating in Fenton reaction. This is very much relevant to the in vivo conditions prevailing in the gastrointestinal lumen where free iron is available in considerable amount for absorption and iron is known to be absorbed in the ferrous form [24,25]. Hence this tissue is continuously exposed to components of free radical generating system which probably is prevented by formation of iron-fatty acid complex. It has been suggested that iron forms complex with fatty acids and the large amount of nonesterified fatty acids present in intestinal brush border membrane is involved in iron transport across the membrane [26,27]. Recently it has been shown that tobacco smoke contains free fatty acid which facilitates the transport of iron across the cell membrane (J.W. Eaton, pers. commun.). The present data using various parameters has indicated that possibly intestinal mucosal membranes are resistant to normal process of iron induced lipid peroxidation. ACKNOWLEDGEMENT

The Wellcome Research Unit is supported by The Wellcome Trust, London. Financial assistance from the Department of Science and Technology, Government of India, through a research grant is acknowledged. The authors are thankful to Professor V.I. Mathan for his keen interest in this work. REFERENCES 1 B.O.Watson, R. Busto, J. Goldberd, M. Santiso, S. Yoshida and M.D. Ginsberg, Lipid peroxidation in vivo induced by reversal global ischemia in rat brain, J. Neurochem., 42 (1984) 268- 274. 2 J. Hogberg, S. Orrenius and R.E. Larson, Lipid peroxidation in isolated hepatocytes, Eur. J. Biochem., 50 (1975) 595-602. 3 M.T. Curtis, D. Gilfor and J.L. Farber, Lipid peroxidation increases the molecular order of microsomal membranes, Arch. Biochem. Biophys., 235 (1984) 644-649. 4 P.B. McCay, D.D. Gibson, K.L. Fong and K.R. Hornbrook, Effect of glutathione peroxidase activity in lipid peroxidation in biological membranes, Biochim. Biophys. Acta, 431 (1976) 459 - 468.

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