Toxic. in Vitro Vol. 6, No. 1, pp. 71-76, 1992 0887-2333/92$5.00+ 0.00 Printedin Great Britain.All fights reserved Copyright © 1992Pergamon Pressplc INTERACTION OF GOSSYPOL WITH DNA R. ZAIDIand S. M. HAD! Department of Biochemistry, Faculty of Life Science,Aligarh Muslim University, Aligarh-202002,India (Received 21 November 1990) Abstraet--Gossypol, a polyphenolic binaphthyl dialdehyde found in cotton seeds, is a dietary mutagen and a potential male contraceptive. Its interaction with DNA was studied using a combination of St nuclease hydrolysis and hydroxyapatite chromatography. Calf thymus DNA treated with increasing concentrations of gossypol showed a decreasing degree of St nuclease hydrolysis. An increased thermal melting temperature of treated DNA was also observed. When native DNA, treated with gossypol, was chromatographed on hydroxyapatite, it eluted with a lower molarity of phosphate buffer, indicating a lower at~nity of the DNA~lrug complex for hydroxyapatite. When gossypolbound to DNA was removed by extraction with organic solvents, the resulting DNA samples showed increased St nuclease hydrolysis and decreased thermal melting temperatures. These results indicated the formation of strand breaks in DNA. Gossypol was also shown to produce superoxide anion and singlet oxygen in aqueous solution. Strand breakage in DNA by gossypol may be caused by active oxygen species such as hydroxyl radical and singlet oxygen. INTRODUCTION Gossypol, a polyphenolic binaphthyl dialdehyde, is a mutagenic toxin present in cotton seed (Ames, 1983). In India, cotton seed is commonly used as cattle feed and elsewhere unrefined cotton seed oil is used for cooking. In addition to being a dietary mutagen, gossypol gives rise to reversible inhibition of sper- matogenesis and has been proposed as a potential male contraceptive (Qian and Wang, 1984). The genetic toxicity of gossypol in humans is therefore of special interest because the drug is intended for long-term use. It has also been tested as an anticancer agent (Srivastava and Padmanabhan, 1987) and for the treatment of certain viral infections such as herpes (Wichmann et al., 1982). Gossypoi has been reported to induce DNA strand breaks in human lymphocytes in vitro (Nordenskjold and Lambert, 1984). Above a certain threshold concentration it also has an inhibi- tory effect on DNA synthesis in mammalian cells in vitro (Wang and Rao, 1984). Such inhibition is considered to be due to its action on DNA poly- merase. Gossypol has been shown to degrade super- coiled pBR322 DNA in vitro, as revealed by the conversion of supercoiled DNA to relaxed and linear forms (Srivastava and Padmanabhan, 1987). This reaction requires the presence of a metal ion such as Fe 3+ or Co 2+, a reducing agent and oxygen. In this paper we present evidence to indicate that gossypol is able to bind to DNA and that such an interaction may lead to DNA degradation in the absence of metal ions. MATERIALS AND METHODS Calf thymus DNA (sodium salt, average mol wt 1,000,000), S~ nuclease and gossypol acetic acid Abbreviations: NBT = nitroblue tetrazolium; O2- = super- oxide radical; 022- = peroxide ion; tO2= singlet oxygen; RNO = N,N-dimethyl-p-nitrosoaniline; TNE = I0 mM- Tris-HC1, pH7.5, 10mra-NaC1 and 0.1mM-EDTA. were obtained from Sigma Chemical Co. (St Louis, MO, USA). Hydroxyapatite, DNA grade, was a product of Bio-Rad Laboratories (Richmond, CA, USA). Treatment of DNA with gossypol. A solution of ling DNA/ml was prepared in TNE (10 mi-Tris- HC1, pH 7.5, 10 mi-NaCl, 0.1 mM-EDTA). Gossypol was added from a concentrated stock solution of 10mg/ml in ethanol to obtain the desired DNA nucleotide/gossypol molar ratio. The mixture was incubated in sterile tubes for 6 hr at 37°C and subse- quently dialysed against 100 voi TNE at 4°C. The dialysed gossypol-treated DNA was used in various experiments as described below. 81 nuclease digestion and alkaline hydrolysis. St nuclease digestion of gossypol-treated DNA was carried out in 0.1 M-acetate buffer, pH 4.5, containing I mM-ZnSO4 and 20-30 units of St nuclease in a total volume of 1 ml. After incubation for 2 hr at 48°C, solutions of bovine serum albumin (I0 mg/ml; 0.2 ml) and perchloric acid (14%, v/v; I ml) were added to precipitate unhydrolysed DNA. Deoxyribonucleo- tides in the supernatant were determined either colori- metrically (Schneider, 1957) or by using absorbance at 260 nm. Alkaline hydrolysis was carried out by incubating samples of 500 #g gossypoi-treated DNA in 1 ml 0.1 M-NaOH. The degree of hydrolysis was determined as for $1 hydrolysis. Gossypol-treated DNA was also subjected to "depurination" by incubating the samples of 50°C for 6 hr prior to alkaline hydrolysis (Verly et al., 1973). Thermal denaturation of DNA as measured by the degree of $1 nuclease digestion. Melting profiles of DNA using St nuclease digestion were determined essentially as described by Case and Baker (1975). Samples containing 300 #g of native or gossypol- treated DNA were heated to the desired temperature for 8 min and quickly quenched by the addition of 2 vol ice-cold S~ nuclease standard reaction buffer. The mixture was incubated with 20-40 units of S t nuclease at 37°C for 2 hr. The reaction was stopped 71