Study on the variables affecting toxicity of hybrid toxins. The effect of different target cell receptor distribution and toxin binding chain.
Disulfide conjugates of diphtheria toxin (DT) and its fragment A (DTA) to asialoorosomucoid (ASOR) were prepared. The toxicity of the conjugates were compared with DT in isolated rat, rabbit and guinea pig hepatocytes containing different concentration of asialoglycoprotein receptors (Biochim. Biophys, Acta 942, 57, 1988). In rat hepatocytes DTA-ASOR was highly toxic with half-maximal inhibitory concentration (IC50) of protein synthesis occurring at 4 +/- 3.10(-11) M (n = 7) which was much lower than that of DT DT (7.8 +/- 9.8.10(-9) M, n = 7). In rabbit hepatocytes toxicity of the conjugate (IC50 = 5.4 +/- 4.9.10(-10) M, n = 7) was higher than that of DT (IC50 = 5 +/- 4.10(-11) M, n = 7). In guinea pig hepatocytes, DTA-ASOR was not toxic at concentration below 10(-8) M, although DT was highly toxic (IC50 = 1.8 +/- 1.4.10(-10), n = 3). In the presence of 5 microM colchicine, the toxicity of DTA-ASOR in rat and rabbit hepatocytes increased by 10-fold, while in guinea pig hepatocytes it became detectable with an IC50 of 1.2 +/- 0.8.10(-9) M (n = 3). The toxicity of DT in the rat cells was also enhanced 10-fold by colchicine, but not at all in either the rabbit or the guinea pig cells. Addition of isolated diphtheria toxin fragment B (DTB) did not affect significantly the toxicity of DTA-ASOR in all three hepatocytes and that of DT in rat hepatocytes, but reduced toxicity of DT more than 20-fold in the rabbit and guinea pig cells. Toxicity of DT-ASOR in rat hepatocytes was the same as DTA-ASOR both in the absence and presence of colchicine, and abolished completely by excess ASOR, but not by DTB. Toxicity of DT-ASOR in rabbit hepatocytes was 40-times higher than DTA-ASOR, enhanced 10-fold by cochicine and reduced more than 30-fold by excess ASOR, but only slightly by DTB. These results indicate that entry of DTA from DTA-ASOR involve a DTB-independent translocation mechanism which can be as efficient as the DTB-dependent mechanism used by DT in the rabbit and guinea pig cells. The entry of both conjugates appeared to be mediated by the asialoglycoprotein receptors. However, the DTB moiety of DT-ASOR could function only in the DT-sensitive cells indicating the lack of a DTB-mediated translocation in the DT-resistant cells.
Chang
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Biochemistry Laboratory, Isaac Gordon Center for Digestive Diseases and Nutrition, Genesee Hospital Rochester, NY 14607.
Chang
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D000818:Animals; D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins; D002470:Cell Survival; D003078:Colchicine / Q000494:pharmacology; D004167:Diphtheria Toxin / Q000008:administration & dosage / Q000494:pharmacology / Q000633:toxicity*; D004220:Disulfides; D004305:Dose-Response Relationship, Drug; D004337:Drug Carriers; D006168:Guinea Pigs; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D009961:Orosomucoid / Q000008:administration & dosage / Q000031:analogs & derivatives; D010446:Peptide Fragments / Q000008:administration & dosage / Q000494:pharmacology / Q000633:toxicity*; D011817:Rabbits; D051381:Rats; D011971:Receptors, Immunologic / Q000378:metabolism*
D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins; D004167:Diphtheria Toxin; D004220:Disulfides; D004337:Drug Carriers; D009961:Orosomucoid; D010446:Peptide Fragments; D011971:Receptors, Immunologic; C017030:asialoorosomucoid; C029795:diphtheria toxin fragment A; C039171:diphtheria toxin fragment B; D003078:Colchicine