Abstract
The hypothesis that oxidative stress induced by acute, sublethal, gossypol treatment induces transcription of stress genes in a rodent liver cell line was tested. In northern blot analysis of gossypol-treated cells, there was a dose-dependent increase in c-fos, a component of the redox-regulated transcription factor activator protein-1 (AP-1). Induction of c-fos was biphasic. A rapid 3.33±1.37 fold induction in c-fos was detected after treatment with 5 μmol/L gossypol for 30 min. Additionally, treatment with 5 μmol/L gossypol for 12 h caused a 2.66±0.67 fold increase in c-fos expression. PCR-based subtractive hybridization was used to generate a subtracted complementary DNA (cDNA) pool representing mRNA species increased in response to gossypol exposure. Several thousand clones were grown from bacteria transformed with the subtracted cDNA pool. After screening and confirmation by northern blotting, five clones were confirmed to be induced by gossypol. Sequence analysis confirmed that four of these clones contained DNA sequences from cytochrome-c oxidase subunit II (COX II), and one contained a DNA sequence from cytochrome-c oxidase subunit I (COX I). A five-fold induction (5.13±1.39 fold) in COX II occurred after 1 h of gossypol exposure, and a three-fold induction (3.24 fold) in COX I occurred after 3 h of gossypol exposure. These studies provide further evidence that mitochondria are a major site of the cytotoxic action of gossypol based upon an adaptive response to gossypol involving the upregulation of genes from the mitochondrial genome.
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Abou-Donia MB, Othman MA, Obih P. Interspecies comparison of pharmacokinetics profile and bioavailability of (±)-gossypol in male Fischer-344 rats and male B6C3F mice. Toxicology. 1989;55:37–51.
Alford BB, Liepa GU, Vanbeber AD. Cottonseed protein: what does the future hold? Plant Foods Hum Nutr. 1996;49:1–11.
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990; 215:403–10.
Ariño J, Bosch F, Gomez-Foix AM, Guinovart JJ. Oxytocin inactivates and phosphorylates rat hepatocyte glycogen synthase. Biochem J. 1989;261:827–30.
Barhoumi R, Bailey RH, Burghardt RC. Kinetic analysis of glutathione in anchored cells with monochlorobimane. Cytometry. 1995;19:226–34.
Barhoumi R, Mouneimne Y, Phillips TD, Safe SH, Burghardt RC. Alteration of oxytocin-induced calcium oscillations in clone 9 cells by toxin exposure. Fund Appl Toxicol. 1996; 33:220–8.
Barhoumi R, Burghardt RC. Kinetic analysis of the chronology of patulin-and gossypol-induced cytotoxicity in vitro. Fund Appl Toxicol. 1996;30:290–7.
Benhaim P, Mathes SJ, Hunt TK, Scheuenstuhl H, Benz CC. Induction of neutrophil Mac-1 integrin expression and superoxide production by the medicinal plant extract gossypol. Inflammation. 1994;18:443–58.
Benz CC, Keniry MA, Ford JM et al. Biochemical correlates of the antitumor and antimitochondrial properties of gossypol enantiomers. Mol Pharmacol. 1990;37:840–7.
Benz CC, Iyer SB, Asgar, HS, Matlin SA, Aronson FR, Barchowsky A. Gossypol effects on endothelial cells and tumor blood flow. Life Sci. 1991;49:67–72.
Berardi LC, Goldblatt LA. Gossypol. In: Liener IE, ed. Toxic constituents of plant foodstuffs. 2nd ed. New York: Academic Press; 1980:183–237.
Bugeja V, Charles G, Collier D, Wilkie D. Primary mitochondrial activity of gossypol in yeast and mammalian cells. Biochem Pharmacol. 1988;37:4217–24.
Chang WY, Sugimoto Y, Shidaifat F, Kulp SK, Canatan H, Lin YC. Gossypol induces spermidine/spermine N 1-acetyltrans-ferase in canine prostate epithelial cells. Biochem Biophys Res Commun. 1997;231:383–8.
Cleeter MWJ, Cooper JM, Darley-Usmar VM, Moncada S, Schapira AHV. Reversible inhibition of cytochrome-c oxidase, the terminal enzyme of the mitochondrial respiratory chain, by nitric oxide. FEBS Lett. 1994;345:50–4.
Cotgreave IA, Moléus P. Methodologies for the application of monobromobimane to the simultaneous analysis of soluble and protein thiol components of biological systems. J Biochem Biophys Methods. 1986;13:231–49.
de Pester A, Wang YY. Genetic toxicity studies of gossypol. Mutation Res. 1993;297:293–312.
Grossman LI, Shoubridge EA. Mitochondrial genetics and human disease. BioEssays. 1996;18:983–91.
Grankvist K. Gossypol-induced free radical toxicity to isolated islet cells. Int J Biochem. 1989;21:853–6.
Grune T, Reinheckel T, Joshi M, Davies KJ. Proteolysis in cultured liver epithelial cells during oxidative stress. Role of the multicatalytic proteinase complex, proteasome. J Biol Chem. 1995;270:2344–51.
Hutchinson RW, Barhoumi R, Burghardt RC. Laser cytometric analysis of gossypol-induced cytotoxicity. Tox Sub Mech. 1995;14:169–84.
Jones LA. Definition of gossypol and its prevalence in cotton seed products. In: Jones LA, Kinard DH, Mills JS, eds. Cattle research with gossypol containing feed. Memphis: National Cottonseed Products Association; 1991:1–18.
Kalla NR, Bisnooduth U, Ranga A. Response of hamster to the antifertility effect of gossypol. Acta Eur Fertil. 1990;21:25–37.
Karin M, Liu Z, Zandi E. AP-1 function and regulation. Curr Opin Cell Biol. 1997;9:240–6.
Musser SM, Stowell MHB, Chan SI. Cytochrome c oxidase: chemistry of a molecular machine. Adv Enzymol Relat Areas Mol Biol. 1995;71:79–208.
Myers BD, Thorneberry GO. Effect of gossypol on some oxidative respiratory enzymes. Plant Physiol. 1966;41:787–91.
Nu MR, Koo SK, Kim et al. In vitro inhibition of gap junctional intercellualr communication by chemical carcinogens. Toxicology. 1995;98:199–206.
Ostermeier C, Iwata S, Michel H. Cytochrome c oxidase. Curr Opin Struct Biol. 1996;6:460–6.
Risco CA, Holmberg CA, Kutches A. Effect of graded concentrations of gossypol on calf performance:toxicological and pathological considerations. J Dairy Sci. 1992;75:2787–98.
Robertson J, Bhattacharyya S, Ing N. Genbank accession number U94719; 1998.
Sen KS, Packer L. Antioxidant and redox regulation of gene transcription. FASEB J. 1996;10:709–20.
Shidaifat F, Canatan H, Kulp SK et al. Inhibition of human prostate cancer cells growth by gossypol is associated with stimulation of transforming growth factor-β. Cancer Lett. 1996;107:37–44.
Shidaifat F, Canatan H, Kulp SK et al. Gossypol arrests human benign prostatic hyperplastic cell growth at G0/G1 phase of the cell cycle. Cancer Lett. 1997;107:1003–10.
Sprecher H, Barr HM, Slotky JI, Tzukerman M, Eytan GD, Assaraf YG. Alteration of mitochondrial gene expression and disruption of respiratory function by the lipophilic antifolate pyrimethamine in mammalian cells. J Biol Chem. 1995;270:20668–76.
Takehara Y, Kanno T, Yoshioka T, Inoue M, Utsumi K. Oxygen-dependent regulation of mitochondrial energy metabolism by nitric oxide. Arch Biochem Biophys. 1995;323: 27–32.
Taanman J. Human cytochrome-c oxidase: structure, function, and deficiency. J Bioenerg Biomembr. 1997;29:151–63.
Thomas A, Crowley RS, Amico JA. Effect of progesterone on hypothalamic oxytocin messenger ribonucleic acid levels in the lactating rat. Endocrinology. 1995;136:4188–94.
Thompson DC, Barhoumi R, Burghardt RC. Comparative toxicity of eugenol and its quinone methide metabolite in cultured liver cells using kinetic fluorescence bioassays. Toxicol. Appl Pharmacol. 1998;149:55–63.
Torres J, Darley-Usmar V, Wilson MT. Inhibition of cytochrome c oxidase in turnover by nitric oxide:mechanism and implications for control of respiration. Biochem J. 1995; 312:169–73
Weinstein BI, Orenstein JM, Gebert R, Kaighn ME, Stadler VL. Growth and structural properties of epithelial cell cultures established from normal rat liver and chemically induced hepatomas. Cancer Res. 1975;35:253–63.
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Hutchinson, R., Ing, N. & Burghardt, R. Induction of c-fos, and cytochrome c oxidase subunits I and II by gossypol acetic acid in rat liver cells. Cell Biol Toxicol 14, 391–399 (1998). https://doi.org/10.1023/A:1007543510337
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DOI: https://doi.org/10.1023/A:1007543510337