Chem.-Biol. Interactions, 19 (1977) 101--109 © Elsevier/North-Holland Scientific Publishers Ltd.
101
INHIBITION OF COLCHICINE BINDING TO RAT LIVER TUBULIN BY ALDEHYDES AND BY LINOLEIC ACID HYDROPEROXIDE
LUDOVICA GABRIEL, GABRIELLA BONELLI and MARIO U. DIANZANI Institute of General Pathology, University of Turin, Corso Raffaello 30, 10125 Turin (Italy)
(Received June 6th, 1977) (Accepted August 10th, 1977)
SUMMARY
The onset of fat accumulation within CC14 poisoned hepatocytes, occurring as early as 1 h after treatment, is known to be provoked by a block in lipoprotein secretion. Lipoprotein secretion involves the function of the microtubular system. Several data indicate that this early block in lipoprotein secretion is not primarily the consequence of impaired protein synthesis. Therefore effects of some derivatives of lipid peroxidation, i.e. aldehydes and linoleic acid hydroperoxide were investigated. The results described in this paper shown that the above mentioned lipid peroxidation derivatives inhibit, with different activities, [3H]colchicine binding to liver high-speed supernates. Percentage binding inhibition is directly related to concentrations of aldehydes or LAHPO. LAHPO is more effective than aldehydes. Among the aldehydes tested, 4-hydroxypentenal, produced during lipid peroxidation of biological materials, was the most active. The presence of thiols, added to the incubation medium, partially protects against the inhibition of [3H] colchicine binding by aldehydes. This suggests that aldehydes act by reacting with --SH groups of tubulin. The possibility that interaction between lipoperoxidation derivatives and tubulin in vivo may contribute to the onset of fat infiltration in CC14 poisoning is discussed. INTRODUCTION
It is well known that CCl4-treatment causes fat accumulation within liver cells, beginning 1 h after poisoning. CC14-induced fatty liver has been shown to be mostly due to the block in lipoprotein secretion from the liver cells Abbreviations: GSH, glutathione; GTP, guanosinetriphosphate; HPE, 4-hydroxy-2,3-transpenten-l-al; KPE, 2-ketopentanal; LAI-IPO, linoleic acid hydroperoxide; 2-Me, 2-mecaptoethanol; MGL, methylglyoxal; MLA, malealdehyde; NADPH, nicotinamide alenyl dinucleotide phosphate.
102 into the blood [ 1 ] . In the liver of treated animals, protein synthesis impairment occurs as early as 15--20 min after poisoning, so that the prevention o f apolipoprotein synthesis has long been considered the major cause for the block in lipoprotein secretion. In general terms, this mechanism is expected to cause fat infiltration only when a substantial number o f pre-existing apolipoprotein molecules have been exhausted due to their catabolism. Knowledge about the half-life of apolipoproteins derived from very low density lipoproteins, is still incomplete [2]. It is known however, that with most inhibitors of protein synthesis that are able to induce fatty liver, fat accumulation never starts before 3--6 h after treatment. The only exception to this rule are the fatty inductions caused by CC14 and CBrC13, which produce triglyceride accumulation as early as 1 h after treatment [3,4]. For this reason, the hypothesis has been advanced that initial triglyceride accumulation in haloalkane poisoning is n o t caused b y apolipoprotein synthesis impairment. The recent demonstration b y several authors [5--8] that lipoprotein secretion by the liver cells involves the microtubular system, provides a possible explanation for this derangement. The major functional protein constituting microtubules is polymerized tubulin, formed by the repetition of a heterodimer consisting of t w o different subunits. Several alkaloids, particularly colchicine, vinblastine, vincristine and podophyllotoxin, have been reported to prevent soluble tubulin polymerization by binding to specific sites. For colchicine and podophyllotoxin the sites are on the ~ subunit, while those for vinblastine and vincristine on the ~ subunit [9,10]. Prevention of the assembly of monomers into microtubules results in a block o f microtubule
101
INHIBITION OF COLCHICINE BINDING TO RAT LIVER TUBULIN BY ALDEHYDES AND BY LINOLEIC ACID HYDROPEROXIDE
LUDOVICA GABRIEL, GABRIELLA BONELLI and MARIO U. DIANZANI Institute of General Pathology, University of Turin, Corso Raffaello 30, 10125 Turin (Italy)
(Received June 6th, 1977) (Accepted August 10th, 1977)
SUMMARY
The onset of fat accumulation within CC14 poisoned hepatocytes, occurring as early as 1 h after treatment, is known to be provoked by a block in lipoprotein secretion. Lipoprotein secretion involves the function of the microtubular system. Several data indicate that this early block in lipoprotein secretion is not primarily the consequence of impaired protein synthesis. Therefore effects of some derivatives of lipid peroxidation, i.e. aldehydes and linoleic acid hydroperoxide were investigated. The results described in this paper shown that the above mentioned lipid peroxidation derivatives inhibit, with different activities, [3H]colchicine binding to liver high-speed supernates. Percentage binding inhibition is directly related to concentrations of aldehydes or LAHPO. LAHPO is more effective than aldehydes. Among the aldehydes tested, 4-hydroxypentenal, produced during lipid peroxidation of biological materials, was the most active. The presence of thiols, added to the incubation medium, partially protects against the inhibition of [3H] colchicine binding by aldehydes. This suggests that aldehydes act by reacting with --SH groups of tubulin. The possibility that interaction between lipoperoxidation derivatives and tubulin in vivo may contribute to the onset of fat infiltration in CC14 poisoning is discussed. INTRODUCTION
It is well known that CCl4-treatment causes fat accumulation within liver cells, beginning 1 h after poisoning. CC14-induced fatty liver has been shown to be mostly due to the block in lipoprotein secretion from the liver cells Abbreviations: GSH, glutathione; GTP, guanosinetriphosphate; HPE, 4-hydroxy-2,3-transpenten-l-al; KPE, 2-ketopentanal; LAI-IPO, linoleic acid hydroperoxide; 2-Me, 2-mecaptoethanol; MGL, methylglyoxal; MLA, malealdehyde; NADPH, nicotinamide alenyl dinucleotide phosphate.
102 into the blood [ 1 ] . In the liver of treated animals, protein synthesis impairment occurs as early as 15--20 min after poisoning, so that the prevention o f apolipoprotein synthesis has long been considered the major cause for the block in lipoprotein secretion. In general terms, this mechanism is expected to cause fat infiltration only when a substantial number o f pre-existing apolipoprotein molecules have been exhausted due to their catabolism. Knowledge about the half-life of apolipoproteins derived from very low density lipoproteins, is still incomplete [2]. It is known however, that with most inhibitors of protein synthesis that are able to induce fatty liver, fat accumulation never starts before 3--6 h after treatment. The only exception to this rule are the fatty inductions caused by CC14 and CBrC13, which produce triglyceride accumulation as early as 1 h after treatment [3,4]. For this reason, the hypothesis has been advanced that initial triglyceride accumulation in haloalkane poisoning is n o t caused b y apolipoprotein synthesis impairment. The recent demonstration b y several authors [5--8] that lipoprotein secretion by the liver cells involves the microtubular system, provides a possible explanation for this derangement. The major functional protein constituting microtubules is polymerized tubulin, formed by the repetition of a heterodimer consisting of t w o different subunits. Several alkaloids, particularly colchicine, vinblastine, vincristine and podophyllotoxin, have been reported to prevent soluble tubulin polymerization by binding to specific sites. For colchicine and podophyllotoxin the sites are on the ~ subunit, while those for vinblastine and vincristine on the ~ subunit [9,10]. Prevention of the assembly of monomers into microtubules results in a block o f microtubule
